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Practical sulphur-selenium metabolic interactions in dairy cattle D’Aleo, Lillian Marie Anna 1984

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PRACTICAL SULPHUR-SELENIUM METABOLIC INTERACTIONS IN DAIRY CATTLE by LILLIAN MARIE ANNA D'ALEO B.Sc, The University of Manitoba A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Animal Science) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June 1984 © L i l l i a n Marie Anna D'Aleo, 1984 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f A n i m a l S c i e n c e The U n i v e r s i t y o f B r i t i s h C olumbia 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3 Date June 28, 19"84 DE-6 (3/81) 0 - i i -ABSTRACT Seventy-one dairy c a t t l e were used to study p r a c t i c a l sulphur-selenium metabolic i n t e r a c t i o n s . Upon calving, cows were randomly assigned to one of the following four treatment groups: control (0.35% S, 0.31 mg/kg Se), S supplemented (0.15% S, 0.31 rag/kg Se), Se supplemented (0.35% S, 0.72 mg/kg Se), and S & Se supplemented (0.50% S, 0.72 mg/kg Se). The basal r a t i o n was made up of 55% forage as a l f a l f a cubes, 35% dairy concentrate, and 10% beet pulp. Cows were fed th e i r respective experimental diets u n t i l dry o f f . During the dry period, cows were maintained on a dry cow r a t i o n low i n Se (0.07 mg/kg Se) for a minimum of 45 days. Two weeks pr i o r to p a r t u r i t i o n , cows were again assigned to t h e i r respective treatment groups. S and Se were supplemented as sodium selenate and elemental sulpher at l e v e l s of 0.40 mg/kg Se and 0.15% S, re s p e c t i v e l y , to y i e l d the f i n a l dietary concentrations given above. Milk samples were c o l l e c t e d on alternate days from a l l cows for milk progesterone a n a l y s i s . Monthly blood samples were taken for plasma Se a n a l y s i s . Cows from treatments 1 - 3 were sampled at estrus for plasma LH a n a l y s i s . Treatment did not influence milk or fat y i e l d . The t o t a l d a i l y consumption of a l f a l f a cubes, 14% dairy concentrate and complete rati o n was found to be s i g n i f i c a n t l y greater during the second l a c t a t i o n for most treatment groups. Beet pulp consumption was not s i g n i f i c a n t l y a l t e r e d i n the subsequent l a c t a t i o n or among treatment groups. Response curves for plasma Se response to treatment were found to be s i g n f i c a n t l y d i f f e r e n t among treatment groups during the increasing - i i i -plasma Se phase (months 1 - 4 ) and the plateau phase (months 4 - 10). Slope analysis indicated that slopes were s i m i l a r among treatment groups during both phases of the Se response curve. Regression equations, however, during both phases were d i f f e r e n t among treatment groups. The Y-intercept for treatment groups 1 - 4 were 0.08 ± 0.02 (s . e . ) , 0.07 ± 0.02 ( s . e . ) , 0.09 ±0.02 ( s . e . ) , and 0.08 ± 0.03 (s.e.) mg/kg Se, re s p e c t i v e l y , with group 3 being s i g n i f i c a n t l y higher than treatment groups 1 and 2. During month 4 - 1 0 , the Se response curve of treatment group 4 was s i g n i f i c a n t l y d i f f e r e n t (p _< 0.05) from a l l other groups. The Y-intercept during the plateau phase for treatments 1 - 4 was 0.10 ± 0.02 ( s . e . ) , 0.11 ± 0.02 ( s . e . ) , 0.09 ± 0.02 ( s . e . ) , and 0.13 ± 0.02 (s.e.) mg/kg Se, r e s p e c t i v e l y . Dry cow plasma Se le v e l s of animals on treatment groups 1 - 4 were 0.06, 0.06, 0.05, and 0.07 ± 0.04 mg/kg Se (p £ 0 . 0 5 ) . Calf plasma Se l e v e l s were 0.04, 0.05, 0.04, and .06 ± 0.02 mg/kg Se for calves whose dams were on treatment groups 1 - 4 , resp e c t i v e l y (p >^  0.05). Days to f i r s t service was not influenced by breed or treatment. Days open, calving i n t e r v a l , services/confirmed conception, and ca l f b i r t h weights were s i g n i f i c a n t l y greater for breeds, but these parameters were not affected by treatment group. Cows entering t h e i r second l a c t a t i o n were assigned to the same experimental r a t i o n as the previous l a c t a t i o n , and were designated as being on experimental treatment groups 5, 6, 7, and 8. Milk progesterone concentrations during periods 0 and 2 of the estrous cycle were not d i f f e r e n t among treatment groups (p >^  0.05). - i v -During period 1, milk progesterone concentrations of treatment group 6 were s i g n i f i c a n t l y higher than the control group of the f i r s t l a c t a t i o n (2.42 and 2.06 ± 0.61 ng/ml, r e s p e c t i v e l y ) . During period 3 of the estrous c y c l e , treatment groups 2, 4, 6, 7, and 8 had s i g n i f i c a n t l y higher milk progesterone concentrations than the control group ( f i r s t l a c t a t i o n ) . During period 4 of the estrous cycle, milk progesterone concentrations of cows on treatment group 6 were higher than those of cows on treatment group 1 (9.42 and 6.56 ± 2.25 ng/ml, r e s p e c t i v e l y ) . Also cows on treatment group 2 had s i g n i f i c a n t l y lower milk progesterone concentrations than cows on treatment groups 4 and 5 (8.43 and 8.45 ± 2.25, r e s p e c t i v e l y ) . . Period lengths of the estrous cycle were not influenced by treatment. Both milk progesterone concentrations and period length (days) were found to be d i f f e r e n t among the various cycle types i d e n t i f i e d . Mean cycle length among treatment groups was found to be s i m i l a r . However, during the second l a c t a t i o n , the frequency of long estrous cycles was 15.6% in both the control and S supplemented group, 3.1% i n the Se supplemented group and 12.5% i n the S and Se supplemented group. The Incidence of abortion during the second l a c t a t i o n (based on milk progesterone concentrations) was 21.4% i n both the control and S supplemented groups, while no Incidence of abortion occurred i n the two Se supplemented groups. Milk progesterone concentrations at breeding time were s i g n i f i c a n t l y higher for Holsteins than for Ayrshires (2.00 and 1.76 ± - v -8.81 ng/ml, r e s p e c t i v e l y ) . Also, milk progesterone concentrations were s i g n i f i c a n t l y higher for cows on treatment group 8 in comparison to control cows (2.73 and 1.53 ± 8.81 ng/ml, r e s p e c t i v e l y ) . Plasma Se concentrations at breeding were also s i g n i f i c a n t l y lower i n control cows i n comparison to cows on treatment group 4 (0.09 and 0.12 mg/kg Se, r e s p e c t i v e l y ) . A s i g n i f i c a n t treatment by (un)successful breeding i n t e r a c t i o n for milk progesterone concentrations was observed. Mean milk progesterone l e v e l s for cows on treatment group 8 i n the unsuccessful breeding group were s i g n i f i c a n t l y higher than mean milk progesterone concentration recorded i n a l l other treatment groups. Basal plasma LH l e v e l s were s i g n i f i c a n t l y lower for Ayrshires in comparison to Holsteins (0.64 and 0.78 ± 0.49 ng/ml). Treatment did not a f f e c t LH lag time or basal LH but the preovulatory LH peak was s i g n i f i c a n t l y greater in the control grup (50.93 ± 13.09 ng/ml) than i n either the S or Se supplemented groups (23.05 and 14.70 ± 13.09 ng/ml, r e s p e c t i v e l y ) . The incidence of retained placenta during the second l a c t a t i o n for treatment group 5 - 8 was 22.4%, 1.1%, 0.0%, and 0.0%, r e s p e c t i v e l y . The incidence of abnormal uterus was 18.8%, 10.0%, 14.3%, and 0.0% for treatment groups 5 - 8 . Calving ease was not dependent on treatment group. However, the c o r r e l a t i o n between calving ease and dam plasma Se (r = -0.6164, n = 12) was found to be s i g n i f i c a n t (p - 0.05), i n d i c a t i n g a reduction i n the degree of calving d i f f i c u l t i e s with increasing plasma Se l e v e l s . The incidence of m a s t i t i s was found to be dependent on treatment group during the f i r s t l a c t a t i o n with the highest incidence of m a s t i t i s 1 - v i -occurring i n the Se supplemented group. Somatic c e l l counts were not d i f f e r e n t among treatment groups. Other health parameters monitored were not dependent on treatment group. In summary, S does not appear to Inh i b i t Se metabolism as r e f l e c t e d by plasma Se l e v e l s i n response to treatment and i n view of the response i n the productive, reproductive and health parameters monitored. - v i i -TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS v i i LIST OF TABLES i x LIST OF FIGURES x i i ACKNOWLEDGEMENTS x i i i INTRODUCTION 1 LITERATURE REVIEW 4 Selenium .' 4 Reproduction 27 MATERIALS AND METHODS 49 Animal Management.... 50 A n a l y t i c a l Procedures 51 Experimental Design 57 S t a t i s t i c a l Analysis 63 RESULTS 65 Ration Selenium Levels 65 Production T r a i t s 71 Plasma Selenium Reponse to Treatment 73 Reproductive T r a i t s 80 Progesterone Data 83 Plasma Selenium Levels and Milk Progesterone Concentrations at Breeding Time 98 Plasma LH T r a i t s 105 Uterine and Ovarian T r a i t s 110 Calving Ease T r a i t s 113 Health Problems 116 Correlations and Regressions 123 DISCUSSION 140 Ration Selenium Levels 140 Cow and Calf Plasma Selenium Levels 141 Production 149 Reproduction • 149 Health Data 173 - v i i i -Page CONCLUSION 174 BIBLIOGRAPHY 179 APPENDICES 188 - i x -LIST OF TABLES Table Page 1 Selenium Levels (ppb) i n Feed Sources (DM basis) 66 2 Sulphur and Selenium Composition of Experimental Rations (DM basis) 67 3 E f f e c t of Breed on Daily Feed Intake (kg DM Basis) and Production T r a i t s (BCA for Milk Y i e l d and % F a t ) . . 69 4 E f f e c t of Treatment on Daily Feed Intake (kg DM Basis) and Production T r a i t s (BCA for Milk Y i e l d and kg Fat) 70 5 Selenium Content of Weighback Samples 72 6 Regression Analyses for the E f f e c t of Time on Selenium Response for Breeds 75 7 E f f e c t of Breed on Cow and Calf Plasma Selenium Levels (ppb) 76 8 Regression Analyses for the E f f e c t of Time on Selenium Response for Treatment Groups 78 9 E f f e c t of Treatment on Cow and Calf Plasma Selenium Levels (ppb) 79 10 E f f e c t of Breed on Productive T r a i t s 82 11 E f f e c t of Treatment on Reproductive T r a i t s 84 12 E f f e c t of Breed on Milk Progesterone Concentrations During Periods of the Estrous Cycle 86 13 E f f e c t of Treatment on Milk Progesterone Concentrations During Periods of the Estrous Cycle.... 87 14 E f f e c t of Type of Cycle on Milk Progesterone Concentration During Periods of the Estrous Cycle 90 15 E f f e c t of Breeds on Period Length (Days) 91 16 E f f e c t of Treatment on Period Length (Days) 92 17 E f f e c t of Type of Cycle on Period Length (days) 94 18 E f f e c t of Breed on Cycle Length 96 - x -Table Page 19 E f f e c t of Treatment on Cycle Length 97 20 E f f e c t of Breed on Plasma Selenium and Milk Progesterone Concentrations at Time of Breeding 99 21 E f f e c t of Treatment on Plasma Se (mg/kg) and Milk Progesterone (ng/ml) at Breeding 101 22 E f f e c t of Plasma Selenium and Milk Progesterone Concentrations on Success of Breeding 102 23 Milk Progesterone Concentrations for Treatment by (Un) successful Breeding Interaction 104 24 E f f e c t of Breed on Plasma LH Lag Time (days) and Basal LH Levels (ng/ml) 107 25 E f f e c t of Treatment on LH Lag Time (Days), Basal LH Levels (ng/ml) and Peak LH During Pre-Ovulatory . Surge (ng/ml) 109 26 Treatment by Breed E f f e c t on Basal LH Levels (ng/ml).. I l l 27 The Incidence of Ovarian, Vaginal, and Uterine Abnormalities and Retained Placenta..... 112 28 E f f e c t of Treatment on the Observed Incidence of Reproductive T r a i t s 114 29 The E f f e c t of Breed on Ease of Calving and Calf M o r t a l i t y 117 30 E f f e c t of Treatment on Ease of Calving 118 31 E f f e c t of Breed on Health T r a i t s 119 32 E f f e c t of Breed on Somatic C e l l Count 121 33 E f f e c t of Treatment on the Observed Incidence of Health Problems 122 34 E f f e c t on Treatment of Somatic C e l l Count 124 35 Correlations Between Dam Plasma Selenium (mg/kg) and Calf B i r t h Weight (kg) and Ease of Calving 125 36 C o r r e l a t i o n Between Calf Plasma Se (mg/kg) and Calf B i r t h Weight (kg) 127 - x i -Table Page 37 Correlations Between Peak Milk Progesterone Concentration During the Estrous Cycle and LH Lag Time (days) and Basal Plasma LH (ng/ml) 129 38 Correlations Between Cow Plasma Selenium (mg/kg) and LH Lag Time (days) and Basal Plasma LH (ng/ml) 130 39 Correlations Between Milk Progesterone Levels (ng/ml) and Plasma Selenium Levels (mg/kg) at time of Breeding.. 132 40 Correlations Between Milk Progesterone (ng/ml) and Plasma Selenium (mg/kg) l e v e l s for Treatment by ( U n s u c c e s s f u l Breeding Group..... 134 41 Correlations Between Treatment Groups and the Incidence of Reproductive Problems 135 42 Correlations Between Calving Ease Categories and Treatment Groups 137 43 Correlations Between the Incidence of Health Problems and Treatment Group 139 - x i i -7 LIST OF FIGURES P a g e Figure 1: Influence of breed on mean plasma selenium l e v e l s during the treatment period (months 1-10) and the dry period (month 12) 74 Figure 2: E f f e c t of treatment group on plasma selenium l e v e l s during the treatment period (months 1-10) and the dry period (month 12) 8 1 Figure 3: D e f i n i t i o n s of the d i v i s i o n of estrous cycles into periods on the basis of milk, progesterone concentrations 85 Figure A: C y c l i c a l milk progesterone a c t i v i t y (ng/ml) and i t s r e l a t i o n s h i p to plasma LH (ng/ml) at estrus 1° 6 Figure 5 : Incidence (%) of retained placenta and abnormal u t e r i i (with normal ovaries) for treatment groups during the second l a c t a t i o n 115 - x i i i -ACKNOWLEDGEMENT I would l i k e to acknowledge the e f f o r t s of a l l those involved i n the research and preparation of t h i s t h e s i s . I would e s p e c i a l l y l i k e to thank Dr. J.A. Shelford, Department of Animal Sciences, U.B.C. with whom many hours were spent b a t t l i n g uncooperative cows and computers; whose supportive supervision and advice were greatly appreciated and whose friendship I have come to greatly value. Thanks to L.J. Fisher, Research Station, Aggasiz for his suggestions and input as well as Dr. R.G. Peterson and Mrs. M. S t r i k e r for t h e i r advice and assistance i n the data a n a l y s i s . I would also l i k e to thank the technicians In the Department of Animal Science, U.B.C. for the analysis of milk samples. Les Szabo and William Slack of the Dairy Research Unit, U.B.C. deserve many thanks for th e i r help i n taking blood samples. F i n a l l y , thanks are due to Agriculture Canada who provided funds enabling the conduction of t h i s study. - 1 -INTRODUCTION THE SELENIUM PROBLEM IN BRITISH COLUMBIA Selenium responsive diseases prevalent i n c a t t l e and poultry have been i d e n t i f i e d i n southern B r i t i s h Columbia (Miltimore 1974). Other reports ( c i t e d by Miltimore 1974), though unpublished, suggest that Se def i c i e n c y may be a problem i n l i v e s t o c k throughout extensive areas of the remainder of B.C. as w e l l . Problems were encountered when f i s h meal was eliminated from poultry rations due to i t s high cost i n B.C. (Jenkins and Hidiroglou 1972). Selenium supplementation prevented poor reproductive performance and loss of replacement p u l l e t s i n breeder turkeys. Miltimore ( c i t e d by Hoffman et a l 1973) indicated that a high proportion of B.C. grains and forages have low Se concentrations and that Se l e v e l s can be var i a b l e even when the same material was grown i n adjacent areas. Selenium i n forages ranged from 0.06 to 1.56 mg/kg: oats from 0.01 to 0.09 mg/kg; barley from 0.06 to 0.53 mg/kg, and wheat from 0.04 to 2.17 mg/kg. Feed grains produced i n Peace River which are used extensively for feeding l i v e s t o c k and poultry in the Fraser Valley were low i n Se (Fenimore et a l 1983). In the Windemere Valley of Southeastern B.C., marginally d e f i c i e n t sera Se l e v e l s i n beef c a t t l e were related to the moderate to very low Se le v e l s i n hay. C l i n i c a l problems r e f l e c t i v e of t h i s marginal deficiency included weak calves, neonatal diarrhea, pnemonia, poor growth, poor reproductive - 2 -performance, and retained placenta (Fenimore et a l 1983). White Muscle Disease was uncommon, however. Upon supplementation or i n j e c t i o n with selenium/vitamin E, the incidence of these c l i n i c a l symptoms was s i g n i f i c a n t l y reduced. Miltimore et a l (1974) conducted an extensive study to determine differences in Se feed concentrations between geographic areas of B.C. In t h i s study the province was divided into eleven regions based on cl i m a t i c regions and major farming areas. The data indicated that i n B.C. there were no large areas producing high Se feeds. Selenium concentrations in samples of legume, grass and corn silage from the coastal region were approximately half of the Se concentrations i n respective samples from the Thompson region. The Se concentrations of sedge hay had s i m i l a r Se concentrations i n a l l regions. In general, grasses and legumes had lower Se l e v e l s than wheat. It was estimated that based on the NRC recommendation of 0.1 mg/kg Se for c a t t l e , 76% of the c a t t l e fed mainly corn s i l a g e and 46% of the c a t t l e fed sedge hay may have inadequate Se intakes and therefore maybe under varying degrees of n u t r i t i o n a l s t r e s s . Stress due to i n s u f f i c i e n t dietary Se would only be expected i n 20% of the c a t t l e fed primarily grass and legumes. The degree of stress placed on l i v e s t o c k would l a r g e l y depend on the region of o r i g i n of the feeds as well as the type of grain supplement fed. Sulphur has for many years been suspected of i n t e r f e r i n g with Se metabolism. At what l e v e l ( s ) the antagonism occurs i s not known, but i t has been suggested that S may be responsible for the v a r i a b l e responses to dietary selenium. Also, some studies have shown that reducing 75 dietary S r e s u l t s i n increased blood Se (Pope et a l . , 1979). - 3 -Many areas of B.C. are developing S d e f i c i e n t s o i l s and consequently S f e r t i l i z a t i o n i s being used to increase y i e l d and protein content of forages and grains. As a r e s u l t of S f e r t i l i z a t i o n to s o i l s , forage S l e v e l s increase, and subsequently, so too does the intake of S by ruminants (Massey 1973). Consequently, concern has arisen over the p o t e n t i a l of excess dietary S i n augmenting Se deficiency problems i n dairy c a t t l e . I t i s , therefore, the objective of t h i s study to determine the p r a c t i c a l s i g n i f i c a n c e of S-Se metabolic in t e r a c t i o n s on s p e c i f i c reproductive, productive, and health parameters. - 4 -LITERATURE REVIEW SELENIUM After i t s discovery i n 1818 by Berzelius selenium (Se) was v i r t u a l l y ignored u n t i l the early 1900's, when i t was i d e n t i f i e d as a toxic element ( O l d f i e l d 1978). The term " a l k a l i disease" referred to chronic selenium poisoning of l i v e s t o c k , and u n t i l 1956, Se was known only for i t s t o x i c i t y . Klaus Schwarz proved the e s s e n t i a l i t y of Se by demonstrating that trace l e v e l s of Se had protective biochemical a c t i v i t y independent of vitamin E. These findings led to the i d e n t i f i c a t i o n of several "selenium-responsive" diseases i n both l i v e s t o c k and humans ( O l d f i e l d 1980) . Selenium deficiences are well documented i n animals and f o r t y species have been i d e n t i f i e d which demonstrate Se responsive diseases. These diseases include 1) aspermia and growth f a i l u r e i n rats (Herrick 1975), 2) White Muscle Disease (WMD) i n ruminants ( O l d f i e l d 1981 Schubert et a l 1961, and Ekermans & Schneider 1982), 3) exudative di a t h e s i s i n chicks (Nesheim & Scott 1961), 4) i n f e r t i l i t y i n ra t s , chicks, and ruminants (Jenkins & Hidiroglou 1972, and Nesheim & Scott 1961), 5) blockage of embryogenesis i n cows ( M i l l e r 1981), 6) pancreatic necrosis and f i b r i o s i s i n chicks (Herrick 1975 and Nesheim & Scott 1961), and Keshan's Disease (a f a t a l d i l a t e d cardiomyopathy) i n humans (Anono 1980, Schroeder et a l 1970, Thompson & Robinson 1980, and Young 1981) . - 5 -Since 1956, two l i n e s of thinking emerged, as Se had been both condemned as a carcinogen and hailed as an anticarcinogen ( O l d f i e l d 1978). Nelson and other researchers ( c i t e d by O l d f i e l d 1978) fed die t s containing Se as either selenized grain or ammonium potassium selenide to r a t s , inducing l i v e r c i r r h o s i s . O l d f i e l d (1978) points out that severe lesions i n the damaged l i v e r s of the rats were referred to as 'low grade carcinoma', even though i t was possible the lesions may have been representative not of hepatic neoplasia, but of hepatic regeneration. Nelson's ( c i t e d by O l d f i e l d 1978) re s u l t s led to the l i s t i n g of Se as a carcinogen i n the Food Additive Amendment of 1958. Selenium i s now known to be an e s s e n t i a l trace element i n both animal and human n u t r i t i o n and i s no longer considered a carcinogen (Frost 1981, O l d f i e l d 1980, and O l d f i e l d 1978). Total tumor incidence of chemically induced epidermal, hepatic, colon, and mammary cancer In rodents was reduced by Se supplementation ( P o i r i e r & Milner 1983). Also, i t was recently suggested that Se a l t e r s or i n h i b i t s v i r a l l y induced cancer as Se was found to s i g n i f i c a n t l y decrease the occurrence of spontaneous mammary tumors ( P o i r i e r & Milner 1983). Mice were fed tor u l a yeast d i e t s supplemented with 2.5 or 5.0 mg/kg Se as sodium se l e n i t e (Na2Se03) or by administration of 2.0 yg Se i n t r a p e r i t o n e a l l y . Both ascit e s and s o l i d E h r l i c h tumors were s i g n i f i c a n t l y i n h i b i t e d by both modes of Se administration. Furthermore, the su r v i v a l time of E h r l i c h a s c i t e s tumor bearing mice was also Increased and It was suggested that the antitumerogenic properties of selenium may r e s u l t from the e f f e c t of some intermediate i n the d e t o x i f i c a t i o n pathway of Se. - 6 -Several studies have attempted to determine possible responses to Se supplementation. Results have often been contradictory and v a r i a b l e . Oh et a l ( c i t e d by Ekermans & Shneider 1982) found improved weight gains i n 18 week old lambs supplemented with Se, whereas Peter (1980) found no s i g n i f i c a n t response i n either f e r t i l i t y or lamb growth. Byers and Moxon (1980) indicated that when growth rates i n c a t t l e are high, and the d i e t i s either d e f i c i e n t or marginal i n Se, c a t t l e w i l l respond p o s i t i v e l y to increased Se supplementation (by i n j e c t i o n ) . The requirement for Se i s the greatest when the requirement for protein i s the greatest. Also, when protein i n feed i s d e f i c i e n t , the response to supplemental Se i s the greatest (Ekermans & Shneider 1982, Moxon 1981, Byers & Moxon 1980). Several researchers have found that the incidence of retained placenta decreased with increasing Se l e v e l s i n the d i e t (Ekermans & Schneider 1982, and J u l i e n et a l 1976). Contrary to t h i s , others have found that the incidence of retained placenta did not respond to selenium/vitamin E treatment (Fenimore et a l 1983). Metabolism of Selenium Selenium Absorption Selenium appears to be absorbed i n a s i m i l a r fashion In both ruminants and non-ruminants. Radio-tracer studies have shown that e s s e n t i a l l y no ^^Se administered intraruminally as selenomethionine i s absorbed i n the rumen of sheep. Three hours a f t e r dosing, 0.05%, 2.0%, 0.14%, and 0.05% of the t o t a l administered dose was found i n the t o t a l blood volume, the t o t a l muscle mass, the l i v e r , and the kidneys, - 7 -res p e c t i v e l y (Ammerman & M i l l e r 1974). Other studies with '-"Se have indicated that only s l i g h t absorption occurs i n the abomasum, and that -^*Se i s secreted into the duodenum and jejunum, with net absorption occurring i n the posterior portion of the small i n t e s t i n e (Ammerman & M i l l e r 1974). Conversely, other studies (NRC 1980) indicate that the duodenum i s the main s i t e of absorption. In studies with organic s a l t s , Hidiroglou and Jenkins (1973) found a s i m i l a r pattern i n the l i q u i d phase of digesta i n sheep af t e r administering o r a l and intravenous doses 7 5 of Se-selenonomethionine. It appears that a major part of the Se i n the l i q u i d phase i s protein bound. When the microbial c e l l protein i s hydrolyzed, Se i s absorbed i n the form of Se containing free amino acids (Ammerman & M i l l e r 1974 and Hidiroglou & Jenkins 1973). At six hours post dosing, the rumen b a c t e r i a l protein contained 50% of the 75g e (Ammerman & M i l l e r 1974 and Hidiroglou & Jenkins 1973). Hidiroglou and Jenkins (1973) also found that bacteria can metabolize selenomethionine to form selenocystine, both compounds being incorporated into b a c t e r i a l p r o t e i n . It appears that both absorption and excretion of Se i s responsive to tissue needs (Church et a l 1971). Selenium Excretion Selenium may be excreted v i a the lungs, feces, and urine and the r e l a t i v e amounts of Se excreted by each route w i l l depend on the mode of Se administration as well as Se status and species (Church et a l 1971). Under normal conditions, o r a l Se i s predominantly excreted i n the feces of ruminants and in the urine of non-ruminants (Church et a l 1971 - 8 -and Ammerraan & M i l l e r 1974). In ruminants, the major route of excretion at low dietary l e v e l s i s f e c a l because a f r a c t i o n of the ingested Se becomes incorporated into the microbial mass (Ammerman & M i l l e r 1974). Although unabsorbed Se constitutes a major portion of f e c a l Se, Se from b i l e , pancreatic ducts, and i n t e s t i n a l mucosal c e l l s i s also incorporated i n the feces (Church et a l 1971). As o r a l administration of Se increases, expired Se increases and f e c a l loss remains f a i r l y constant (Church et a l 1971). Urinary Se tends to r i s e at moderate Se intakes, but then declines as Se intake continues to r i s e (Church et a l 1971). The amount and proportion of Se excreted v i a the feces, urine or expired a i r depends l a r g e l y on the l e v e l of intake, the form of Se i n the d i e t , the nature of the diet and the species of animal (Ekermans & Schneider 1974). Expired Se becomes s i g n i f i c a n t only i n cases of t o x i c i t y (Ekermans and Schneider 1974). Injected Se i n sheep i s excreted proportionately to the injected dose and excreted predominantly i n the urine, while f e c a l excretion of Se i s small and remains constant with dosage l e v e l . At high dosage l e v e l s , expired Se w i l l exceed f e c a l loss by several times (Church et a l 1971). Dietary Selenium Transport Following absorption, Se i s f i r m l y bound to many proteins (Ammerraan & Schneider 1974). It i s unknown whether protein synthesis i s a p r e r e q u i s i t e of Se attachment to proteins, but incorporation of Se amino acids into animal proteins i s an active process and i s presumed by some to occur by s i m i l a r enzymic reactions as for the corresponding - 9 -sulphur (S) containing amino acids (Ammerman & Schneider 1974, and NRC 1971). Other evidence tends to suggest that S and Se may share the same metabolic pathways since Se appears to be c l o s e l y associated with many S-containing constituents (McConnell et a l 1974, and Ammerman & Schneider 1974). It i s believed that the metabolism of Se involves the incorporation of Se Into isologues of S-containing amino acids, p a r t i c u l a r i l y methionine (Ammerman & Schneider 1974). Se metabolism and transport may be summarized, i n the following sequence of events: Se i s taken up by the erythrocytes a f t e r entering portal blood, and i s then metabolized to i t s reduced form (X-Se) before being released into plasma. In the plasma, Se becomes protein bound (Pp-x-Se). As portal blood passes through the l i v e r , the hepatocytes absorb the erythrocyte metabolite of Se, and further metabolize i t to a d i f f e r e n t metabolic form (X-'Se) before releasing i t to the plasma (Hidiroglou 1982). Equilibrium between the predominant protein bound form (Pp-x-Se) and the free Se form (X'-Se) i s established i n the plasma. Thus Se i s c i r c u l a t e d to, and deposited i n , a l l peripheral tissues (Hidiroglou 1982 and NRC 1980). Selenium Incorporation Into Proteins The process by which selenoamino acids are incoporated into proteins i s not known (McConnell et a l 1971). In polypeptide synthesis, activated amino acids react only with t h e i r s p e c i f i c tRNA(s). McConnell et a l (1971) found that an amino acid analogue (such as selenomethion-ine) , capable of deceiving the a c t i v a t i o n enzyme would be incorporated into the protein instead of the natural amino acid (such as methionine). - 10 -The raethionyl-t-RNA synthetase of 15. Colt aminoacylates methionine t-RNA with both methionine and selenomethionine, and there e x i s t s a competitive I n h i b i t i o n between the two (McConnell et a l 1974, and McConnell et a l 1979). These findings further support the hypothesis that Se metabolism may u t i l i z e , i n part, S metabolic pathways. Forms o f S e l e n i u m Selenium i s usually found i n only minute quantities due to the r e l a t i v e l y low l e v e l s of Se In s o i l and due to the active conversion of Se to organic forms i n plant tissue (NRC 1971). Selenium i n crop and forages are predominantly found as an i n t e g r a l component of the plant proteins, mainly i n analogues of some S-amino acids (predominantly selenocysteine, selenocystine, selenomethionine, and selenolcystathionine) (NRC 1971). Various inorganic and organic forms of Se appear to have s i m i l a r b i o l o g i c a l a c t i v i t y for animals (NRC 1971). Schwarz and F o l t z ( c i t e d in NRC 1971) found that selenic a c i d , selenate, selenium dioxide, s e l e n i t e , and selenocynate are r e l a t i v e l y s i m i l a r i n a c t i v i t y to various organic selenides as well as selenocystine, selenocytathionine, and seleno-methionine. However, elemental selenium i s i n a c t i v e b i o l o g i c a l l y (NRC 1971). Sulphur and Se have s i m i l a r chemical and physical properties. The atoms of the two elements are s i m i l a r in s i z e , both in the ion i c and covalent states, and t h e i r respective valence s h e l l s have si m i l a r e l e c t r o n i c configurations. Selenium can exist in the -2 (hydrogen - 11 -s e l e n i d e ) , 0 (elemental selenium), +4 (inorganic s e l e n i t e s ) , and +6 (selenate) oxidation states (NRC 1980). Hydrogen selenide i s a highly toxic gas and i n the presence of oxygen w i l l decompose r a p i d l y to y i e l d elemental selenium and water. Organic selenides are extremely v o l a t i l e and several have been i d e n t i f i e d i n b i o l o g i c a l materials. The heavy metal selenides are i n s o l u b l e . Elemental Se i s f a i r l y stable i n nature as i t i s neither oxidized nor reduced e a s i l y i n nature (NRC 1980). The soluble inorganic s e l e n i t e s are highly t o x i c . Selenite forms stable adsorption complexes In s o i l with iron and aluminum sesquioxides (NRC 1980). Selenite, when added to a c i d i c s o i l s w i l l reduce to the elemental form and consequently w i l l become unavailable to plants. A l k a l i n e and o x i d i z i n g conditions i n s o i l s w i l l favor stable selenate formation. Selenates are soluble and loosely complexed by sesquioxides and consequently highly a v a i l a b l e to plants and e a s i l y leached from s o i l s . T o x i c i t y studies with rats indicated that selenides and element-a l selenium are poorly absotbed. Selenium from seleniferous grains i s very well absorbed and i s better absorbed than sele n i t e s and selenates. F u n c t i o n s o f S e l e n i u m In 1973, Hoekstra and co-workers ( c i t e d by O l d f i e l d 1980) at the U n i v e r s i t y of Wisconson demonstrated that enzyme preparations of red blood c e l l s from Se d e f i c i e n t animals were low i n glutathione peroxidase (GSH-Px). Flohe et a l ( c i t e d by Diplock 1981) concurrently contributed further to the understanding of the fundamental metabolic function of Se by demonstrating that GSH-Px contained 4 gram atoms of Se per mole of - 12 -the enzyme, or 1 gram atom of Se i n each of the enzyme's 4 molecular sub-units. As a component of GSH-Px, Se functions i n maintaining the i n t e g r i t y of c e l l u l a r membranes v i a the breakdown of peroxides a r i s i n g from tissue l i p i d peroxidation (Church et a l 1971 and Ammerraan & M i l l e r 1974). Selenium exerts an e f f e c t on pancreatic l i p a s e production (required for g a s t r o i n t e s t i n a l absorption of l i p i d s and tocopherols) by maintaining normal pancreatic morphology (Church et a l 1971). Whether selenium's involvement i n pancreatic function Is simply a manifestation of i t s r e l a t i o n s h i p to GSH-Px i s not yet known. Other possible functions of Se yet to be f u l l y investigated have been proposed. Diplock and Lucy ( c i t e d i n NRC 1980) have suggested that selenide may possibly be contained at the active s i t e of non-heme proteins. Levander ( c i t e d i n NRC 1980) has suggested that selenium i s somehow involved i n the electron transport chain, supported by Whanger et a l ' s ( c i t e d i n NRC 1980) discovery i n lamb muscle of the existence of a seleno-protein with a heme group also found i n cytochrome C. There i s s t i l l much to be learned concerning the s p e c i f i c functions of selenium as i s evidenced by the numerous and disparate d e f i c i e n c y symptoms seen in animals (Church 1971). Selenium and Vitamin E Biochemistry Molecular Oxygen Reduction Although O2 i s an e s s e n t i a l component for maintaining l i f e , i t also can be highly toxic to a l l forms of l i f e . Both vitamin E and Se - 13 -are involved i n l i m i t i n g the harmful e f f e c t s of O2 (Diplock 1980). The process by which O2 can become toxic to l i v i n g tissues involves the following sequence of events: 0 2 ^ O2 ^ 0 2 2 = = H 0 2 = H 2 0 2 ^ OH" + 0H« 5 20H~ where sing l e electrons are sequentially added to molecular oxygen (Diplock 1980). Superoxide dismutase (SOD) w i l l catalyze the decomposition of 0 2~ to H 20 2 and 0 2, while peroxidases and catalases w i l l catalyze the decomposition of 2H 20 2 to 2H 20 and 0 2, rendering these anions harmless. However, depending on the microenvironment ( i e , pH and metal ions) i n which 0 2~ and H 2 0 2 are generated and subsequent s t a b i l i t y or i n s t a b i l i t y , further reactions can p o t e n t i a l l y produce hydroxyl r a d i c a l s («0H), which are highly destructive (Diplock 1980). The Haber-Weiss reaction explains the i n t e r a c t i o n : 0 2~ + H 2 0 2 * O2 + 0H~ + «0H The reaction may be catalyzed by a redox active metal such as ir o n as i n the following: F e + + + + 0 2~ F e + + + 0 2 Other powerful oxidants may be produced as reaction products of 0 2~, for example: F e ^ + 0 2~ * [Fe0 2] + 0 - 14 -Working simultaneously, SOD, peroxidases, and catalase destroy O2 and H2O2, thus preventing any further reactions forming destructive species (Diplock 1980). Added to t h i s , a-tocopherol functions to prevent the development of peroxidative chain reactions among polyunsaturated f a t t y acids (PUFA) (Diplock 1980). Protective Mechanisms of a-Tocoperol and SE Containing GSH-PX The following discussion w i l l involve only the mechanisms of a-tocopherol and Se containing GSH-Px i n the protection of b i o l o g i c a l membranes against l i p i d peroxidation and the subsequent by-products. Vitamin E Function I t has been suggested by Lucy ( c i t e d by Diplock 1980) that vitamin E functions i n a physiochemical role to s t a b i l i z e b i o l o g i c a l membranes rather than as an antioxidant protecting unsaturated l i p i d s from oxidation as was i n i t i a l l y suggested by Tappel (1978). The hypothesis developed by Diplock and Lucy (1973) i s now the most widely accepted theory regarding the i n t e r a c t i o n of vitamin E and Se (Hoekstra 1975). Diplock and Lucy (1973) suggest that ot-tocopherol s t a b i l i z e s membranes with high PUFA content by means of l i p i d - l i p i d Interactions between the unsaturated f a t t y acids and vitamin E. More s p e c i f i c a l l y , there e x i s t s a s p e c i f i c physiochemical i n t e r a c t i o n between the f a t t y acyl chains of polyunsaturated phospholipids and the phytyl side-chain of vitamin E forming a complex with the following functional properties: 1. oxidative destruction of polyunsaturated f a t t y acids and c e l l u l a r membranes i s i n h i b i t e d , - 15 -2. b i o l o g i c a l membranes of high PUFA content are less permeable, and 3. endogenous membrane bound phospholipases are i n h i b i t e d from degrading membrane phospholipids which would prevent non-haem ir o n proteins situated at the membrane surface from becoming exposed to molecular oxygen (Diplock & Lucy 1973). Therefore vitamin E prevents the production of oxygen r a d i c a l s which could p o t e n t i a l l y damage membrane bound proteins Furthermore, Diplock and Lucy (1973) suggest that, i n vivo, a primary function of vitamin E i s the i n h i b i t i o n of oxidation of proteins containing selenide located i n the mitochondria and smooth endoplasmic reticulum. Functions of Glutathione Peroxidase Glutathione peroxidase, the Se-containing enzyme i s primarily associated with the aqueous phase of the cytosol whereas vitamin E i s located i n the membrane i t s e l f (Combs et a l 1975). Glutathione peroxidase destroys H 20 2 and f a t t y acid peroxides that would otherwise cause oxidative degradation of c e l l u l a r components by u t i l i z i n g reducing equivalents from glutathione (GSH). Thus, peroxidative damage of erythrocyte plasma membranes w i l l be dependent on Se l e v e l s i n the d i e t , as GSH-PX i s dependent on dietary Se (Combs et a l 1975). The following discussion i s focused on the functions of GSH-Px in blood and and l i v e r . - 16 -(l)GSH-Px i n Blood Due to t h e i r high content of PUFAs and to the i r d i r e c t exposure to molecular oxygen, erythrocyte plasma membranes are very l a b i l e to l i p i d peroxidation (Combs et a l 1975). The red blood c e l l may be protected from oxidative damage (due to hydrogen peroxides) by a glutathione dependent pathway, i l l u s t r a t e d as follows (Ganther et a l 1976): Glucose + Glucose-6-phosphate NADP+ 2GSH ROOH + E:Hexokinase / \/ \/ 'D GSSG-R GSH-Px (or 6-PGD) / \ S V y \ / \ / \ £ NADPH GSSG R0H + H 2 6 Phosphogluconate £ NADPH GSSG R0H + H 20 (or ribulose 5-phosphate) +H+ (or 2H2O) where: G-6PD = 6-phosphodehydrogenase 6-PGD » 6-phosphogluconate dehydrogenase GSSG-R - glutathione reductase GSH-Px = glutathione peroxidase Glutathione i s p r e f e r e n t i a l l y oxidized by peroxides and the reac-t i o n i s catalyzed by GSH-Px (Ganther et a l 1976). Glutathione reductase then catalyzes the reduction of the oxidized GSH by reduced NADPH generated i n the hexose monophosphate shunt pathway. Oxidative damage to hemoglobin could be expected i f any one of the four enzymes in the protective pathway were to develop decreased a c t i v i t y or reduced synthe-s i s due to, for example, dietary d e f i c i e n c i e s of S-amino acids or Se. Noguchi et a l (1973) demonstrated that plasma GSH-Px f e l l to almost zero l e v e l s just p r i o r to the onset of exudative diathesis i n chicks. Noguchi et a l (1973) proposed, therefore, that a primary function of plasma GSH-Px was to prevent the peroxidation of the plasma membrane of c a p i l l a r y endothelial c e l l . - 17 -GSH-Px I n L i v e r In the l i v e r , the primary role of GSH-Px may be to catalyze the decomposition of l i p i d hydroperoxides rather than hydrogen peroxide (Ganther et a l 1976). Catalase has no a c t i v i t y toward l i p i d peroxides whereas GSH-Px reacts with several organic peroxides with l i t t l e s p e c i f i c i t y . In the l i v e r , high microsomal enzyme a c t i v i t y produce l i p i d peroxides. Oxidation of PUFA located in the 0-acyl p o s i t i o n of phosphatidyl choline and phosphatildy ethanolamine r e s u l t s i n membrane fragmentation accompanied by the d i s a s s o c i a t i o n of proteins from the endoplasmic reticulum. Consequently, membrane bound enzyme a c t i v i t y i s a l t e r e d . The a b i l i t y of dietary Se to prevent l i v e r necrosis i n rats fed inadequate l e v e l s of vitamin D and S-containing amino acids supports the preceeding hypothesis. Ganther et a l ( c i t e d by Kice 1980) have proposed a scheme elucida t i n g a mechanism for GSH-Px based on Se functioning at the active s i t e of the enzyme. The mechanism i s as follows: (l) +R00H E-SeH (selenol) ^E-Se-OH (s e l e n i n i c acid) (selenosulfide) STEP (1): Selenol at the active s i t e of GSH-Px undergoes oxidation to form s e l e n i n i c a c i d . - 18 -STEP (2): Seleninic acid (oxidized form of GSH-Px) reacts with a GSH to y i e l d the mixed selenosulfide and water. STEP (3): A second molecule of GSH cleaves the selenosulfide linkage producing oxidized GSH and restoring GSH-Px to the selenol form (reduced form of GSH-Px) (Kice 1980, and Ganther et a l 1976). The reaction can be taken further to include selenium c y c l i n g between selenenic acid and s e l e n i n i c acid as follows (Kice 1980): R00H ROH R00H ROH E-Se-SG E-Se-SG 0 Selenium-Vitamin E Inte r a c t i o n The i n t e r a c t i o n between Se and vitamin E in preventing oxidative damage can be summarized schematically as follows: Enzyme Systems such as xanthine oxidase, amino acid oxidase, e tc. OXIDANT STRESSORS H20 + 1/2 0 2 catalase H,0 2" 2 Chemical damage to c r i t i c a l SH proteins ^ 2H 20 2GSH 2 GSSG UNSATURATED LIPIDS ROOH + l i p i d peroxidation malonic diald'ehyde, etc., C e l l damage ROH + H 20 - 19 -In the above diagram, vitamin E "blocks" reaction (1), and Se as a component of GSH-PX, catalyzes reaction (2) (Leach 1975). Tissue D i s t r i b u t i o n of Se Selenium i s found i n a l l c e l l s and tissues of the body and concentrations of the element vary among the various tissues with varying l e v e l s of Se i n the die t (Underwood 1971).- The highest concentration of Se i s found i n the kidney ( p a r t i c u l a r i l y the kidney cortex) followed by glandular t i s s u e s , such as the pancreas, p i t u i t a r y , and l i v e r . The next tissue i n the descending order of Se concentration i s cardiac muscle, which has cons i s t e n t l y higher Se concentrations than s k e l e t a l muscle. Skeletal muscle, bone, and blood Se le v e l s are r e l a t i v e l y low compared to those tissues mentioned above and adipose tissue i s very low i n Se. Selenium concentrations of the l i v e r or kidney are the most r e f l e c t i v e of the true Se status of an animal as these tissues are the most s e n s i t i v e to changes i n dietary Se. Rats, swine, sheep, and c a t t l e have very s i m i l a r tissue Se concentrations. Mean tissue Se values (mg/kg wet weight) i n sheep reported by Godwin ( c i t e d by Church 1971) are as follows: kidney cortex, 1.38 - 1.46; l i v e r , 0.48 - 0.97; cardiac muscle, 0.15 - 0.20; s k e l e t a l muscle, 0.04 - 0.06; pancreas, 0.34 - 0.44; ovary, 0.19 - 0.25; cerebrum, 0.07 -0.09; and wool, 0.21 - 0.49. Toxic Se l e v e l s fed to c a t t l e resulted i n l i v e r and kidney Se concentrations as high as 5 - 7 mg/kg and muscle Se concentrations of 1 - 2 mg/kg. Hair Se l e v e l s In c a t t l e are normally i n the range of 1 -- 20 -4 mg/kg, however, c a t t l e on seleniferous range had hair Se l e v e l s averaging over 10 mg/kg. Selenium i n Reproduction Selenium i s required for optimal reproductive performance i n dairy c a t t l e , and Se d e f i c i e n t d i e t s can r e s u l t i n reproductive i n e f f i c i e n c y manifested by longer calving i n t e r v a l s (Maas 1982 and Chancellor 1980). However, evidence for Se involvement i n reproduction has often been contradictory. In New Zealand and A u s t r a l i a Se-responsive i n f e r t i l i t y i n ewes occurs in areas where there i s also a high incidence of N u t r i t i o a n a l Muscular Dystrophy i n lambs (Maas 1982). Hartley (1963) indicated that Se treatment two to four weeks pr i o r to mating greatly reduced i n f e r t i l i t y a t t r i b u t a b l e to embryonic death. Wilkins and Kilgour (1982) conducted a study to assess the e f f e c t of Se before mating on the proportion of ewes lambing. They found that 46% of the untreated group did not return to service, yet f a i l e d to lamb, whereas t h i s occured in only 5% of the treated group. The difference was highly s i g n i f i c a n t and the authors interpreted the r e s u l t s as i n d i c a t i v e of a high f a i l u r e rate of the embryo or fetus occurring 13 days after f e r t i l i z a t i o n . Other studies have shown that when Se was administered o r a l l y to ewes one month p r i o r to mating, with subsequent monthly doses throughout gestation, lambing percentages increased (Hidiroglou 1979). Early embryonic mortality between 20 to 30 days gestation in a l a t e r study was at t r i b u t e d to be the cause of reduced f e r t i l i t y ( c i t e d by M i t c h e l l et a l 1975). However, in M i t c h e l l et a l ' s study (1975), the - 21 -o v e r a l l f e r t i l i t y between Se-deficient and Se-suppleraented ewes was not d i f f e r e n t . Furthermore, M i t c h e l l et a l (1975) suggested that reduced f e r t i l i t y may be a r e s u l t of f e r t i l i z a t i o n f a i l u r e rather than as a r e s u l t of embryonic mortality because a large proportion of ova i n utero-tubal flushings were u n f e r t i l i z e d . Reduced twinning rate has also been observed i n Se-deficient ewes ( M i t c h e l l et a l 1975). Other studies have shown no s i g n i f i c a n t d i f f e r e n c e between Se-deficient and Se-suppleraented ewes with respect to o v e r a l l f e r t i l i t y , i ncluding ovulation rates and embryonic loss (Hidiroglou 1979). The Se requirement for optimal reproduction i s also contradictory for c a t t l e . Low Se l e v e l s (0.05ppm) i n some Uganda dairy herds i s responsible i n part, for i n f e r t i l i t y . However, other reports have indicated no b e n e f i c i a l e f f e c t of t r e a t i n g beef cows with Se p r i o r to breeding i n improving subnormal conception rates (Hidiroglou 1979). Segerson et a l (1977) conducted a study i n which Se/vitamin E supplemented and non-supplemented beef cows were maintained on either an adequate plane of n u t r i t i o n (APN) or inadequate plane of n u t r i t i o n (IPN). Their r e s u l t s Indicate that the f e r t i l i z a t i o n rate (number of f e r t i l e ova) of cows on an APN increased when supplemented with Se/vitamin E, however, Se or vitamin E alone had no e f f e c t on % f e r t i l i z a t i o n . They suggested the reduced f e r t i l i t y rates may be due to a f a i l u r e of f e r t i l i z a t i o n r e s u l t i n g from a lack of sperm transport to the s i t e of f e r t i l i z a t i o n based on the fact that no sperm was found i n and around the zona p e l l u c i d a of the u n f e r t i l i z e d ova. Another l a t e r study by Segerson et a l (1981) demonstrated that f e r t i l i t y tends to be greater i n Se/vitamin E treated ewes on an APN as opposed to an INP. - 22 -Both studies (Segerson et a l 1981, and Segerson et a l 1977) support Segerson's hypothesis which proposes that Se and/or vitamin E may influence uterine muscular function In such a way as to increase uterine contractions. Another study by Segerson et a l (1980) demonstrated that Se-deficient ewes supplemented with Se had a s i g n i f i c a n t l y increased v e l o c i t y of uterine contractions (18.4 mm/sec) compared to Se-deficient ewes not supplemented (9.5 mm/sec). It was concluded that poor f e r t i l i t y observed In Se d e f i c i e n t ewes was due to impaired uterine muscular function, where the uterus was not capable of producing the contractions required to f a c i l i t a t e sperm transport to the f e r t i l i z a t i o n s i t e (Segerson et a l 1980). Hartley (1963), however, was unable to detect any benefit of Se supplementation to Se-deficient ewes i n the f e r t i l i t y rate of ova. Retained placenta i s a condition where the f e t a l placenta f a i l s to separate from the maternal placentome and normally occurs i n approximately 10% (or less) of parturient dairy cows ( J u l i e n & Conrad 1976a). Trinder et a l (1973) demonstrated the r e l a t i o n s h i p between low blood Se l e v e l s and a high frequency of retained placenta. In an e a r l i e r study, Trinder et a l (1969) had found that Se and vitamin E administered together was more e f f e c t i v e i n preventing retained placenta than either substance injected alone. J u l i e n and Conrad (1976a) were able to reduce the Incidence of retained placenta from 38% In control cows to 0% i n Se treated cows (regardless of mode of supplementation). J u l i e n et a l (1976b) further demonstrated a reduction i n retained placenta from 51.2% to 8.8% following Se i n j e c t i o n 20 days p r i o r to c a l v i n g . Consequently, J u l i e n et a l (1976b) suggested that Se - 23 -d e f i c i e n c y may be expressed c l i n i c a l l y as retained placenta i n the mature dairy cow, though no mechanism by which retained placenta i s manifested was elucidated. Contrary to the r e s u l t s reported by Trinder et a l (1973) and J u l i e n et a l (1976a and 1976b), Gwazdauskas et a l (1978) found that the incidence of retained placenta was not reduced by Se/vltamin E treatment. Gwazdauskas et a l (1978) also found no s i g n i f i c a n t r e l a t i o n s h i p to e x i s t between Se/vitamin E treatment and number of v i s i b l e heats p r i o r to f i r s t breeding, days open, and services per confirmed conception. In agreement with Gwazdauskas et a l (1978), Shingoethe et a l (1982) could find no benefit i n supplementing Se to cows already comsuming adequate amounts of Se i n reducing the frequency of retained placenta. Segerson and Ganapathy (1981) demonstrated that a single dose of Se/vitamin E was not e f f e c t i v e i n reducing retained placenta i n severly Se d e f i c i e n t cows but the dose of Se/vitamin E was e f f e c t i v e i n marginally Se d e f i c i e n t cows. This finding may explain the discrepancies previously observed of the e f f e c t of Se/vitamin E treatment as prophylaxis against retained placenta (Hidiroglou 1982). In 1980., Buck et a l (1980) conducted a study to determine a s p e c i f i c role for selenium i n normal ruminant reproductive physiology. Their r e s u l t s using l a b e l l e d indicated that the corpus luteum, p i t u i t a r y , non-luteal ovarian t i s s u e , dam placentae, f e t a l p i t u i t a r y , and adrenal glands have a requirement for Se. Other i s o t o p i c studies have demonstrated that the adrenal gland, p i t u i t a r y , and ovary have high 75 concentrations of Se ( c i t e d by Buck et a l 1980). - 24 -In a study by Conrad et a l ( c i t e d by Gwazdauskas et a l 1979), pre-partal i n j e c t i o n of 100 pg Se/Kg body weight (as sodium sele n i t e ) and adequate vitamin E given to dairy cows (of which 46% had previously experienced c y s t i c ovaries) prevented ovarian cysts post c a l v i n g . These findings tend to suggest that d e f i c i e n t l e v e l s of Se could lead to reproductive dysfunctions manifested by reduced f e r t i l i t y (Buck et a l 1980). It has recently been suggested that GSH-Px may be involved i n ste r o i d synthesis, s p e c i f i c a l l y prostaglandin synthesis (Diplock 1980 and Hoekstra 1975). Arachadonic acid, upon release from membrane phospholipids would require protection by vitamin E and GSH-Px mechanisms against non-specific attack by oxygen (Diplock 1980) . Glutathione peroxidase, though not obligatory, can convert prostaglanin G 2 to prostaglandin H2. It has been suggested that GSH-Px may function i n the modulation of substrates flowing through the prostaglandin synthesis pathways by exerting i t s e f f e c t on substrates i n h i b i t o r y to those pathways (Diplock 1980). Sulphur—Selenium Interactions It i s well documented that the response to dietary Se l e v e l s i s v a r i a b l e . It has been suggested that the v a r i a b i l i t y i n response to Se may be due, i n part at l e a s t , to the antagonism of other minerals at either the s o i l / p l a n t or plant/animal l e v e l (Massey 1973). One such mineral i s sulphur (Massey 1973). Evidence suggests that S and Se may share some of the same metabolic pathways (Ammerman & M i l l e r 1974, and McConnell et a l 1974). - 25 -I t i s believed that the metabolism of Se involves the incorporation of Se into isologues of S-containing amino acids, p a r t i c u l a r i l y methionine and cystine i n protein biosynthesis (McConnell et a l 1974). Methionine and selenomethionine are very s i m i l a r chemically as Is evidenced by the fa c t that selenomethionyl t-RNA and methionyl t-RNA are involved i n polypeptide synthesis much to the same extent. Also i t has been suggested by McConnel et a l (1974) that selenomethionine and methionine may share the same transport system as the conditions for active transport for the two amino acids are nearly i d e n t i c a l i n the hamster i n t e s t i n e . There e x i s t s a competitive i n h i b i t i o n between selenomethion-ine as would be expected i n an active transport system (McConnell et a l 1974). The a v a i l a b l e l i t e r a t u r e on sulphur-selenium i n t e r a c t i o n s i n b i o l o g i c a l systems has been to a large extent inconclusive and disparate. Pope et a l (1979), found that ruminant animals on the lowest dietary S regime (0.05%) i n the t r i a l had s i g n i f i c a n t l y elevated blood Se and a l t e r a t i o n s i n ruminal Se metabolism i n response to S was suggested by rumen and f e c a l data. Pope et a l (1979) suggest that i t i s l i k e l y that increasing S intake would increase the numbers of Desulphovibrio bacteria in the rumen. Consequently, more Se would be reduced enzymatically to H2Se u t i l i z i n g pathways p a r a l l e l i n g those for sulphate reduction. h^Se being less stable than H2S would dissociate to form elemental selenium (which could then be reused by the rumen bacteria) or would form insoluble metal selenides such as CuSe, thus rendering a large proportion of Se unavailable for adsorption. Pope et a l (1979) concluded that more Se would be incorporated i n t o , and absorbed as, microbial amino acids when S i s l i m i t i n g metabolism. Whanger et a l (1969) found that s u l f a t e did not increase the incidence of WMD and i n fact t h e i r r e s u l t s indicate that s u l f a t e added to low selenium diets tended to s l i g h t l y reduce the incidence of WMD. In a further study, Whanger et a l (1970) found that s u l f a t e a c t u a l l y delayed the onset of WMD, based on serum glutamic-oxaloacetic transaminase (GOT), l a c t i c dehydrogenase (LDH), and malic dehydrogenase (MDH) as in d i c a t o r s of WMD. However, s u l f a t e did s i g n i f i c a n t l y increase the number of lambs with degenerative heart l e s i o n s . Whanger (1970) suggested that the c o n f l i c t i n g reports on the i n t e r a c t i o n of S and Se could be explained by the S status of an animal possibly influencing Se metabolism. Hintz and Hogue (1964) demonstrated that organic S had no s i g n i f i c a n t e f f e c t on the c l i n i c a l incidence of WMD, but substantial amounts of inorganic S added to the di e t increased the incidence of WMD. - 27 -REPRODUCTION The normally c y c l i n g cow i s polyestrus and c y c l i c a l a c t i v i t y throughout the year i s recurrent and regular (Sorenson 1979). At six to ten months of age, hei f e r s reach puberty and have regular c y c l e s . The average estrous cycle i s 21 days long and estrus i s 16 - 18 hours In duration (Sorenson 1979). Ovulation occurs 12 - 14 hours a f t e r the end of estrus (Laing 1979). The endocrine glands and t h e i r respective hormones control reproduction i n mammals to a large extent, and a l l phases of reproduc-ti o n are influenced (Roberts 1971). The hormones involved i n the regulation of reproduction may either have stimulatory or i n h i b i t o r y e f f e c t s on each other and on the reproductive organs. Reproduction i s under neuroendocrine control and nervous mechanisms are responsible for ovulation i n species where ovulation i s not spontaneous (Roberts 1979). The hypothalamic control of the p i t u i t a r y and i t s secretions, as well as the involvement of the ovaries, w i l l be discussed, as the hormones produced by these two structures are those primarily concerned with reproduction (Sorenson 1979). Hypothalamus The hypothalamus i s located at the base of the brain and i s composed of several b i l a t e r a l l y paired n u c l e i (Hafez 1980). There e x i s t s between the hypothalamus and the p i t u i t a r y a unique vascular connection known as the hypothalamo-hypophyseal portal system (Cole & Cupps 1977 and - 28 -Hafez 1980). This i s a true portal system i n that i t begins and ends i n c a p i l l a r i e s without passing through the heart and a d i r e c t vascular pathway exists between the hypothalamus and the anterior p i t u i t a r y gland (Cole & Cupps 1977). A r t e r i a l blood flows into the p i t u i t a r y v i a the superior and i n f e r i o r hypophyseal a r t e r i e s . At the median eminence and pars nervosa, c a p i l l a r y loops are formed from which the blood may flow i n t o the hypothalamo-hypophyseal portal v e s s e l s . The blood then flows through the p i t u i t a r y s t a l k to terminate In c a p i l l a r i e s i n the anterior p i t u i t a r y (Hafez 1980). The i n f e r i o r hypophyseal artery also provides blood to the anterior p i t u i t a r y as well as the posterior p i t u i t a r y . Because some of the venous outflow from the anterior p i t u i t a r y flows back to the hypothalamus, the hypothalamus i s exposed to various concentrations of p i t u i t a r y hormones i n the retrograde flow of blood. Thus, there i s an associated negative feedback regulation by p i t u i t a r y hormones on the hypothalamus known as the short-loop feed back system There are various hormones produced by the hypothalamus that are involved i n c o n t r o l l i n g p i t u i t a r y function. These include l e u t i n i z i n g hormone releasing hormone (LHRH), thyrotropin releasing hormone (TRH), and p r o l a c t i n releasing hormone (PRH). The following discussion w i l l be l i m i t e d to only those hormones involved i n reproduction. Hypothalamic Hormones LHRH i s secreted by the hypothalamus and i s responsible for the release of f o l l i c l e stimulating hormone (FSH) and l e u t i n i z i n g hormone (LH) d i r e c t l y from the anterior p i t u i t a r y i n the cow (Convey 1973 and Sorenson 1979). - 29 -An t e r i o r P i t u i t a r y Hormones The gonadotropins i n the female cause ovarian stimulation to produce the following sequence of events: (1) growth of f o l l i c l e s i n the ovary, (2) growth and maturation of the oocytes i n the f o l l i c l e s , (3) production and secretion of estrogen i n the f o l l i c l e s by the c e l l u l a r components, (4) ovulation, (5) corpus luteum (CL) development, and (6) CL secretion of progesterone (Hafez 1980). FSH Is a gonadotropin released by the basophilic c e l l s of the anterior p i t u i t a r y and functions to stimulate f o l l i c u l a r growth i n the female (Sorenson 1979). FSH i s active early i n f o l l i c u l a r development and Is d e f i n i t e l y required for f o l l i c l e antrum formation (Hafez 1980). FSH also acts s y n e r g i s t i c a l l y with estrogen r e s u l t i n g i n the formation of FSH and LH receptors located i n the f o l l i c u l a r granulosa c e l l s . LH i s produced by the basophils of the anterior p i t u i t a r y and func-tions to stimulate ovulation and growth of the l u t e a l c e l l s of the CL a f t e r ovulation has occured i n the female (Sorenson 1979). LH Is also involved i n the a b i l i t y of the granulosa c e l l s to secrete progesterone, and may be involved i n regulating the blood flow to the ovaries (Hafez 1980) . Gonadal Hormones The ovarian hormones are the estrogens and progestins. The estro-genic hormones include estrone and e s t r a d i o l ( n a t u r a l l y occurring) - 30 -produced by the granulosa c e l l s and theca interna of the ovarian f o l l i c l e . The other estrogen i s e s t r i o l , an excretory product (Sorenson 1979). Estrogens have a wide range of ph y s i o l o g i c a l functions and are necessary for the psychological display of estrus (Hafez 1980). Estrogens are also involved i n the regulation of the release of the p i t u i t a r y hormones, as well as aiding i n the implantation process and potentiating oxytocin and prostaglandin e f f e c t s on uterine contractions (Hafez 1980). The progestins include the hormones progesterone and pregnenediol. The ovary l u t e a l c e l l s produce progesterone and the excretory product i s pregnendiol (Hafez 1980). Progesterone synergizes with estrogen i n the growth of the uterine and mammary glands and several other p h y s i o l o g i c a l functions. At least for the f i r s t t h i r d of pregnancy i n the cow, progesterone produced by the CL i s necessary for the continued mainten-ance of pregnancy. Progesterone i s extremely important i n regulating the estrous cycle as high l e v e l s of th i s hormone w i l l I n h i b i t estrus and the ovulatory surge of LH. F i n a l l y , uterine contractions are i n h i b i t e d by progesterone while the endometrial glands are stimulated to secrete endometrial f l u i d which i s e s s e n t i a l to the pre-implantation blastocyst as a source of nourishment (Hafez 1980). As mentioned previously, LHRH stimulates the anterior p i t u i t a r y d i r e c t l y to release FSH and LH (Convey 1973 and Hafez 1980). Zolman ( c i t e d by Convey 1973) found a dose related increase i n LH release when the bovine p i t u i t a r y tissue was exposed to 1 or 4 ng of porcine p u r i f i e d LH. Only a b r i e f exposure of the p i t u i t a r y c e l l s to LHRH was needed to increase the LH l e v e l s which persisted beyond the exposure period. The - 31 -fa c t that the induced LH increase persisted even when LHRH was removed indicated that LHRH was bound to the p i t u i t a r y c e l l s (Zolman c i t e d by Convey 1973). The degree to which the p i t u i t a r y i s capable of responding to LHRH i s greatly affected by the reproductive state of the animal (Convey 1973). This i s evidenced by the varying response observed by several researchers. In Zolmans study ( c i t e d by Convey 1973), he i f e r s i n the l u t e a l phase of the estrous cycle that were subjected to intravenously administered doses of LHRH showed a peak LH response that was best characterized as a quadratic function. Increasing the dose of LHRH beyond 80 ug In th i s study caused no further release of LH (Zolman, c i t e d by Convey 1973). In yet another study by Swanson and Hafs ( c i t e d by Convey 1973), to e l i c i t an increased LH output, the maximum l e v e l of LHRH used during the pre-ovulatory surge was twice the maximum LHRH l e v e l used i n the Zolman study ( c i t e d by Convey 1973). Also, the increased release of LH during pro-estrus and the simultaneous f a l l i n the hypothalamic content of LHRH was consistent with the theory that LHRH tr i g g e r s the pre-ovulatory release of LH by the p i t u i t a r y that i n turn t r i g g e r s ovulation (Swanson & Hafs, cited by Convey 1973 and Lamming & Amoroso 1966). The enhanced release of LHRH i s thought to be a consequence of the greater l e v e l s of c i r c u l a t i n g estrogen and progesterone produced by the maturing ovarian f o l l i c l e s (Lamming & Amoroso 1966). However, the high l e v e l s of progesterone produced by the mid-cycle f o l l i c l e seem to i n h i b i t LHRH and consequently i n h i b i t the release of LH and possibly FSH (Hackett & Hafs 1969). The depressed FSH release may a l t e r n a t i v e l y be caused by the release of only marginal l e v e l s of estrogen from the - 32 -mid-cycle f o l l i c l e . Increased l e v e l s of LHRH are therefore associated with the release of LH and consequent ovulation and l u t e a l growth. However, during the period of maximum progesterone production, there i s a reduction i n the l e v e l of LHRH. Thus, a f t e r day 18 of the estrous c y c l e , CL regression occurs, releasing the hypothalamus from progesterone i n h i b i t i o n . The hypothalamus i s then able to release LHRH, r e s u l t i n g i n the release of FSH and LH which stimulate rapid f o l l i c u l a r development, ovulation, and f i n a l l y , rapid l u t e a l growth r e s u l t i n g i n the onset of another period of prolonged progesterone blockage of the hypothalamus. The repressive action of progesterone released by the mid-cycle f o l l i c l e may be mediated through the hypothalamus or may be exerted d i r e c t l y on the f o l l i c l e . The phenomenon of LHRH self-priming has been observed i n the bovine (Padmanabhan et a l 1981). LHRH self-priming refers to the capacity of the p i t u i t a r y to secrete increased LH i n response to LHRH i n vivo when the gland has previously been exposed to small quantities of LHRH. Results from a study conducted by Padmanabhan et a l (1981) indicates that the LHRH observed i n c a t t l e i n vivo i s due, i n part at l e a s t , to a d i r e c t e f f e c t of LHRH on the adenohypophysis. Although the mechanism by which LHRH self-priming occurs i s not yet understood,, the degree to which i t occurs could depend on the i n t e r v a l between LHRH challenge as evidenced by Padmanabhan et al' s (1981) study. In t h i s study, LHRH self-priming occured when the i n t e r v a l between exposures was 40, 80, or 160 minutes, but i t did not occur when the i n t e r v a l was extended to 320 minutes. Thus, the phenomenon of LHRH self-priming may be a s i g n i f i c a n t - 33 -f a c t o r i n the p i t u i t a r y ' s capacity to release gonadotropins i n response to LHRH during estrus and p r i o r to ovulation. The Estrous Cycle At b i r t h , the bovine as well as other mammals possess th e i r entire complement of oocytes (Sorenson 1979). Twenty-four thousand oocytes have been reported to be present i n 14 to 15 year old cows. The p i t u i t a r y and hypothalamus control ovarian functions which include f o l l i c l e growth, estrogen secretion, ovulation, CL formation, progesterone secretion, and CL regression (Hansel & Snook 1970). F o l l i c l e Stimulating Hormone (FSH) as well as r e l a t i v e l y small amounts of LH are e s s e n t i a l for f o l l i c l e maturation and estrogen sec r e t i o n . Ovulation occurs as a r e s u l t of an ovulatory surge of LH (possibly synergizing with FSH) early i n estrus. LH has been l o c a l i z e d i n the d e l t a c e l l s of the p i t u i t a r y by Simmons and associates ( c i t e d by Hansel & Snook 1970). The rapid degranulation of periodic a c i d - S c h i f f e (PAS) p o s i t i v e delta c e l l s which occurs at the beginning of estrus apparently represents LH release (the ovulatory surge) i n i t i a t e d by LHRH. P i t u i t a r y LH continually increased from day 2 to 11 of the estrous cyc l e , but on day 18, a s i g n i f i c a n t decrease occurred, p r i o r to the ovulatory peak seen on day 20 (Hackett & Hafs 1969). Hackett and Hafs (1969) also noted that the pre-ovulatory release of LH was preceded by a pre-ovulatory release of FSH by approximately 2 days. In a study by Sinha and Tucker ( c i t e d by Hansel & Snook 1970), the p i t u i t a r y l u t e o t r o p i c hormone ( p r o l a c t i n ) l e v e l s decreased quickly between days 0 and 2. From these studies, i t i s apparent that FSH, LH, and p r o l a c t i n - 34 -are released sequentially to produce f o l l i c u l a r maturation, ovulation, and the onset of l u t e i n i z a t i o n , r e s p e c t i v e l y (Hansel & Snook 1970). For every one f o l l i c l e that ovulates i n the cow, there are about twelve f o l l i c l e s that die during the maturation process (Sorenson 1979). The primary f o l l i c l e i s a developing^oocyte with a single layer of c e l l s surrounding i t . The primary f o l l i c l e develops into a secondary f o l l i c l e when the f o l l i c u l a r c e l l s multiply to form two c e l l layers surrounding the oocyte. As f o l l i c u l a r c e l l s continue to multiply, the structure i s known as the growing f o l l i c l e . In the growing f o l l i c l e , f l u i d f i l l e d areas are formed by the separation of c e l l s . The antrum of the f o l l i c l e i s formed when the f l u i d - f i l l e d spaces are united. At th i s point, the oocyte Is known as the primary oocyte (Hansel & Snook 1970). The mature f o l l i c l e i s the res u l t of further growth and organization of the granulosa and stroma c e l l s . During the maturation process of the f o l l i c l e , the ovum insi d e the f o l l i c l e i s developing as well (Sorenson 1979). The primary oocyte divides m e i o t i c a l l y i n an unequal fashion r e s u l t i n g i n the production of the secondary oocyte and the f i r s t polar body. The secondary oocyte remains as such u n t i l ovulation. There are a number of f o l l i c l e s which begin to grow every day, and th e i r growth Is controlled by, as yet, some unknown intraovarian factor (Hafez 1980). The formation of the antrum and f o l l i c u l a r growth to ovulatory size i s FSH and LH dependent and the f i r s t phase i s shorter than the second phase of f o l l i c l e growth. The pattern of f o l l i c u l a r development during the estrous cycle i n the bovine has not yet been conclusively demonstrated as many studies have yielded c o n f l i c t i n g evidence. Rajakoski (1960) proposed that there - 35 -are i n fact two waves of f o l l i c u l a r growth. The f i r s t wave of f o l l i c u l a r growth appears to culminate at about day 12, whereupon the f o l l i c l e s become a t r e t i c . The other wave of f o l l i c u l a r growth culminates at estrus (Matton et a l 1981 and Rajakoski 1960). Other researchers have proposed that there e x i s t s c e r t a i n periods during the estrous cycle when f o l l i c u l a r growth begins and accelerates (Matton et a l 1981). S t i l l others claim that the growth of f o l l i c l e s i s continuous and not dependent on the phase of the c y c l e . Matton et a l (1981) presented r e s u l t s which tend to support the theory that the largest f o l l i c l e has an intraovarian i n h i b i t o r y action on smaller f o l l i c l e s present. They further suggested that one of many medium sized f o l l i c l e s becomes dominant, thus developing a new hierchy while the other remain behind i n t h e i r growth. The new dominant f o l l i c l e then, may i n h i b i t the growth of the smaller f o l l i c l e s . The CL i s thought to be involved i n t h i s process by reducing both the rate of growth and a t r e s i a . The f o l l i c l e of the ovary functions i n : 1) gameteogenesis ( c y c l i c a l l y producing f e r t i l i z a b l e ova), and 2) steroidogenesis (the production of steroid hormones i n a balanced r a t i o for the development and maintenance of the ge n i t a l t r a c t for f a c i l i t a t i n g the migration of the early embryo and ensuring implantation and uterine development (Hafez 1980). In domestic species that have reached puberty, the estrous cycle i s composed of 2 phases that blend into each other. The two phases are the f o l l i c u l a r phase and the l u t e a l phase (Roberts 1971). - 36 -During pro-estrus of the f o l l i c u l a r phase, the graafian f o l l i c l e i s stimulated by FSH to grow and produce e s t r a d i o l (Roberts 1971). Antrum formation and f o l l i c u l a r growth are dependent e n t i r e l y on FSH and LH (Hafez 1980). E s t r a d i o l i s capable of enhancing the mito t i c e f f e c t of FSH. Apparently, FSH functions to stimulate the granulosa c e l l s through membrane receptors. The number of receptor s i t e s remains the same during the ent i r e period of f o l l i c u l a r growth. FSH also Increases the number of LH receptors, thereby inducing granulosa c e l l s e n s i t i v i t y to LH. The increase of LH receptors prepares the granulosa c e l l s for l u t e i n i z a t i o n by LH during the LH ovulatory surge (Merz et a l 1981 and Hafez 1980). FSH and LH stimulating the granulosa and theca c e l l s , r e s p e c t i v e l y , are responsible for the steroidogenic production by the f o l l i c l e , that i s , the production of e s t r a d i o l 17-3 (primarily) as well as progestins and androgens (Hafez 1980). The presence of e s t r a d i o l or i t s synthesis in the f o l l i c l e may be an e s s e n t i a l part of f o l l i c u l a r a c t i v i t y (Merz et a l 1981). It i s thought that e s t r a d i o l may, f i r s t , be d i r e c t l y involved i n i n h i b i t i n g f o l l i c u l a r a t r e s i a of the pre-ovulatory f o l l i c l e and secondly, may play a part i n ensuring the f o l l i c l e responds to the LH ovulatory surge. A two c e l l mechanism has been proposed for estrogen production i n the bovine f o l l i c l e . The testosterone produced by the theca c e l l s i n response to the binding of LH, i s transported to the granulosa c e l l s . In the granulosa c e l l s , FSH binds to testosterone, thus inducing i t s aromatization to e s t r a d i o l . Because FSH and LH are capable of stimulating d i f f e r e n t c e l l s of the f o l l i c l e , the r a t i o of FSH - 37 -to LH may be an important parameter for monitoring normal ste r o i d production i n the ovaries (Hafez 1980). FSH and LH bind to c e l l membrane receptors, and consequently stimulate adenylcyclase a c t i v i t y which i n turn r e s u l t s i n increases of i n t r a c e l l u l a r cAMP (Hafez, 1980). S p e c i f i c enzyme a c t i v i t y of the s t e r o i d producing pathway i s augmented by protein kinase (mechanism unknown) which i s activated by the increased cAMP l e v e l s . It i s now generally accepted that FSH i s responsible for aromatase a c t i v i t y whereas LH i s responsible for the formation of pregnenedone and progesterone from c h o l e s t e r o l or 20 a-hydroxycholesterol side cleavage (Hafez 1980 and M e l l i n and Erb 1965). As the cow approaches the estrus state, there Is an increased concentration of plasma estrogen (Henricks et a l 1971). A study by Henricks et a l (1971) indicated that an increase in estrogen occured 3 to 15 hours p r i o r to an increase i n serum LH. These findings are i n agreement with the theory that the Increased estrogen l e v e l s may be responsible for the release of LH. In addition, progesterone was found to be at very low l e v e l s 1 - 3 days before estrus which supports the b e l i e f that before estrogen concentrations can begin to r i s e , progester-one must f a l l to low l e v e l s . Furthermore, several hours before the release of LH occurs, estrogen concentrations must reach a peak l e v e l . Estrogen i s e s s e n t i a l then, for the release of LH by the adenohypophysis while progesterone i n h i b i t s the e f f e c t s of estrogen. - 38 -F o l l i c u l a r Growth During F o l l i c u l a r and Luteal Phases of the Estrous  Cycle The f o l l i c u l a r phase of the estrous cycle spans the period from the regression of the CL of the previous cycle to the following ovulation and i s about 4 - 5 days long i n the cow (Hafez 1980). However, there are also a n t r a l f o l l i c l e s present during the l u t e a l phase which indicates that the true f o l l i c u l a r phase may extend beyond 4 or 5 days. The l u t e a l phase therefore p a r t i a l l y overlaps the true f o l l i c u l a r phase which may cause basal FSH and LH l e v e l s i n plasma and t h e i r r e l a t i o n -ships to f o l l i c u l a r growth to become unclear. Domestic animals appear to have a second r i s e of FSH which occurs 20 to 30 hours af t e r the ovulatory surge of LH and FSH. The second post-ovulatory peak of FSH stimulates antrum formation i n f o l l i c l e s including those destined to ovulate i n the next one or two c y c l e s . Therefore, the time spanning a n t r a l formation and ovulation may be e i t h e r 17 or 34 days. The extended f o l l i c u l a r phase of the domestic animal may be due to the e f f e c t s exerted by progesterone from the CL which acts to slow down f o l l i c u l a r growth. There i s an immediate acceleration of f o l l i c u l a r growth when progesterone l e v e l s are reduced by CL enucleation or prostaglandin l u t e o l y s i s . The cycle i s shortened and ovulation occurs i n about 3 days following these treatments and i s i n fact the basis for estrus synchronization. Endocrine Factors i n Relation to Ovulation Snook et a l (1971) and other researchers have found that at the onset of estrus, the major peak of LH occurred. E s t r a d i o l acts both at - 39 -the p i t u i t a r y and the hypothalamic l e v e l (Hafez 1980). E s t r a d i o l acts to further s e n s i t i z e the gonadotropin producing c e l l s of the p i t u i t a r y to the LHRH released by the hypothalamus. Thus, the same amount of LHRH released by the hypothalamus r e s u l t s i n an increased LH release by the p i t u i t a r y near the end of the estrous cycle when e s t r a d i o l l e v e l s have reached a maximum (Hafez 1980). Savard (1973) found that the plasma LH l e v e l s during the pre-ovulatory LH surge increased ten f o l d the tonic plasma LH l e v e l s while Mori et a l (1974) found the increased plasma LH to be twenty-six times higher than observed at other times during the estrous c y c l e . As mentioned e a r l i e r , d e c l i n i n g progesterone l e v e l s may be a major factor responsible for the onset of the pre-ovulatory LH surge (Black & Hansel 1972). The exact time at which the preciptuous decline i n plasma progesterone l e v e l s occurs i s i n d i v i d u a l l y v a r i a b l e among animals (Kazama & Hansel 1970). Some animals show the decline as early as day 16 of the estrous cycle and others as l a t e as day 19 ( i n normally c y c l i n g cows). Corpus luteum secretory a c t i v i t y and peripheral blood progesterone l e v e l s are c l o s e l y r e l a t e d , therefore plasma porgesterone l e v e l s may be monitored to indicate CL a c t i v i t y (Heap & Holdsworth 1981). The l e v e l of progesterone i n milk also r e f l e c t s CL secretory a c t i v i t y because progesterone i s transferred into milk. The advantage of progesterone analysis of milk samples over plasma samples i s that the former i s r e a d i l y a v a i l a b l e and a highly s e n s i t i v e radio-immunoassay technique for milk progesterone concentrations has been developed. - 40 -Other researchers have found that the reduction of the t o t a l progestin content including progesterone occurs approximately 24 to 28 hours p r i o r to the rapid regression of the CL ju s t before estrus (Kazama & Hansel 1970). In f a c t , the cow d i f f e r s from several other species i n that l i t t l e progesterone i s secreted p r i o r to ovulation (Kazama & Hansel 1970). The ovulatory surge of LH induces l u t e i n i z a t i o n , the rapid growth and formation of the CL, and ovulation (Savard 1973). It has been suggested that the process of l u t e i n i z a t i o n of the granulosa c e l l s i n response to the ovulatory surge of LH may be mediated by cAMP. This suggestion arose from the fac t that cAMP mirrors the l u t e i n i z a t i o n process because of i t s increased concentration i n cultured granulosa c e l l s although the mechanism involved i s not understood. LH i s the major l u t e o t r o p i c factor i n c a t t l e , however, the p o s s i b i l i t y that LH may synergize with several other hormones to e l i c i t a l u t e o t r o p i c response does ex i s t (Hansel & Snook 1970). Between days 3 and 15 of the bovine estrous cy c l e , there i s a s i g n i f i c a n t c o r r e l a t i o n between peripheral progesterone and LH l e v e l s (Snook et a l 1971). During t h i s time, the CL i s growing and a c t i v e l y producing progesterone and i t appears that only small amounts of LH are required since LH l e v e l s are low. The low l e v e l s of LH during the early l u t e a l phase may also indicate that some other factor i s involved for CL development and progesterone secretion (Snook et a l 1971). LH i s thought to stimulate the progesterone synthesis i n the bovine CL mainly by an increased conversion rate of cho l e s t e r o l to progesterone (Hansel & Snook 1970). Hansel and Snook (1970) suggest that newly synthesized cholesterol which - 41 -apparently f a i l s to become e q u i l i b r i a t e d with the t o t a l i n t r a c e l l u l a r pool, i s p r e f e r e n t i a l l y u t i l i z e d . Furthermore, i t i s the i n a v a i l a b i l i t y of the newly formed ch o l e s t e r o l which i n some way may i n i t i a t e the l u t e o l y t i c process. Ovulation P r i o r to ovulation, the pre-ovulatory f o l l i c l e must undergo some major changes which includes the maturation of the ooctye (cytoplasmic and nuclear), the d i s r u p t i o n i n the granulosa layer of the cumulus c e l l cohesiveness, and the thinning and subsequent rupture of the external wall of the f o l l i c l e (Hafez 1980). Which f o l l i c l e w i l l eventually ovulate i s a subject which i s l i t t l e understood. However, i t appears that the f o l l i c l e destined to ovulate receives the greatest volume of blood as well as having more permeable c a p i l l a r i e s than a t r e t i c f o l l i c l e s . This suggests that part of the e f f e c t of LH on the f o l l i c l e s may be to enhance the v a s c u l a r i t y to the f o l l i c l e s i n response to LH stimulation and the greater metabolic requirements of the f o l l i c l e s following gonadotropin stimulation. In the mammalian ovary ovulation i s able to occur at any point on i t s surface with the exception of the h i l u s . Ovulation occurs randomly in the cow with respect to the ovary containing the former CL. However, the cow tends to ovulate from the r i g h t ovary 60% of the time. Separating the oocyte and the exterior f o l l i c l e are several tissue layers which must be broken down before ovulation can occur (Hafez 1980). Changes i n the granulosa and theca c e l l r e l a t i o n s h i p during pre-ovulatory growth r e s u l t s In an increased f o l l i c u l a r e l a s t i c i t y . The - 42 -f o l l i c l u l a r surface v a s c u l a r i t y Increases (except at the center) as the f o l l i c l e enlarges and begins to protrude from the surface of the ovary. The avascular area at the center of the f o l l i c l e w i l l eventually be the point of rupture. The granulosa c e l l s and the cumulus c e l l s of the growing graafian f o l l i c l e s are indis t i n g u i s h a b l e c y t o l o g i c a l l y . The cumulus c e l l s develop close contact with the c e l l membrane of the oocyte. Pr i o r to ovulation, the cumulus mass and c e l l s begin to separate from each other u n t i l c a v i t i e s appear (Hafez 1980). Eventually, only the zona p e l l u c i d a and the cumulus c e l l s located therein are l e f t surrounding the oocyte, to form the corona r a d i a t a . The oocyte i s freed from the granulosa layer when the cumulus c e l l s d i s s o c i a t e . Approximately three hours a f t e r the gonadotropin surge, meiosis resumes (nuclear maturation) and continues u n t i l about one hour p r i o r to ovulation when the f i r s t polar body i s expelled. A viscous mass which i s composed of glycoproteins secreted by the cumulus c e l l s i s formed around the oocyte and corona. Following the rupture of trie f o l l i c l e , the viscous mass f a c i l i t a t e s the transport of the oocyte to the Umbrae. In the cow, j u s t p r i o r to, or during ovulation, the secondary oocyte begins to develop and passes through the prophase and metaphase stages where development ceases u n t i l f e r t i l i z a t i o n occurs (Sorenson 1979). The secondary oocyte then resumes i t s meiotic d i v i s i o n once f e r t i l i z a t i o n has occurred. Extrusion of the second polar body occurs and the f e r t i l i z e d ovum r e s u l t s . Thus the 2N complement of the primary oocyte i s reduced to IN i n the ovum. The secondary oocyte begins to deteriorate i n the metaphase stage i f f e r t i l i z a t i o n f a i l s to occur and - 43 -the structure i s absorbed by the reproductive t r a c t wall, or i t may pass to the e x t e r i o r . F e r t i l i z a t i o n For f e r t i l i z a t i o n to occur, three prerequisites are necessary: sperm transport, capacitation, and oocyte maturation (previously discussed) (Baker & Polge 1976). Soon a f t e r ovulation, f e r t i l i z a t i o n occurs i n the ampullary region of the oviduct. Embryologically, the spermatozoon entry into the ovum activates the ovum, while g e n e t i c a l l y , paternal chromosomes and c e n t r i o l e are added (involved i n subsequent c e l l d i v i s i o n ) and a d i p l o i d state i s established. At th i s time also, the sex of the embryo i s established. Spermatozoa are usually ejaculated into the vagina or c e r v i c a l lumen of the cow, but i t must be transported from the s i t e of deposition to encounter the ovum i n the ampullary region of the oviduct (Baker & Polge 1976). Uterine contractions and sperm m o t i l i t y and t h e i r importance i n transport of bovine spermatozoa i n the cow's g e n i t a l t r a c t i s s t i l l unclear. However, i t has been shown that the s t i m u l i of both Al and natural mating causes contractions of the bovine uterus and consequently increased sperm transport. Also, l i v e spermatozoa were transported through the reproductive t r a c t considerably f a s t e r than dead spermatozoa. It has been suggested that because dead spermatozoa show an Increased s t i c k i n e s s , they may be more e a s i l y removed from the cow's luminal f l u i d . The hormonal state of the female at insemination also appears to e f f e c t the number of spermatozoa reaching the oviduct. It - 44 -appears that b u l l spermatozoa Is more e a s i l y transported i n cows i n the f o l l i c u l a r phase of the estrous cycle as opposed to the l u t e a l phase. Certain changes must be undergone by the spermatozoa while i n the female g e n i t a l t r a c t (capacitation) (Baker & Polge 1976). Although the process of capacitation i s not f u l l y understood, i t appears that the spermatozoa must spend a c e r t a i n period of time i n the female g e n i t a l t r a c t under c e r t a i n conditions before penetration of the ova can occur (Hafez 1980, Baker & Polge 1976, and Sorenson 1979). The time required f o r b u l l spermatozoa capacitation i s s t i l l not known (Baker & Polge 1976). One aspect of capacitation involves removing the seminal plasma components from the sperm surface. This process may act u a l l y make receptor s i t e s a v a i l a b l e which are required for the i n t e r a c t i o n of sperm and ova. It has also been shown that attaching seminal plasma proteins to sperm can r e s u l t i n decapacitation. While i n the uterus, the outer plasma membrane and the acrosomal membrane of the spermatozoa i s a l t e r e d . This appears to be a preparatory step for the acrosome reaction which occurs i n the oviduct near the cumulus mass (Baker & Polge 1976). The acrosomal membrane fuses with the plasma membrane i n the acrosomal reaction and openings are developed i n th i s fused membrane through which p r o t e o l y t i c enzymes can leak. The fused membrane i s l o s t s h o r t l y a f t e r fusion occurs. The p r o t e o l y t i c enzymes released are required for the penetration of the zona p e l l u c i d a by the sperm. There are four acrosomal enzymes: 1. hyaluronidase, which digests hyaluronic acid between cumulus c e l l s , - 45 -2. corona-penetrating enzyme, which appears to digest the adhesive matter between corona c e l l s , 3. acrosin, which digests the zona p e l l u c i d a , and 4. sperm neuraminidase (SN) whose enzymatic a c t i v i t y i s not known, though i t i s thought to prevent polyspermy because i t i n h i b i t s the entry of sperm through the zona p e l l u c i d a . Thus i t seems that enzymatic digestion of a channel through the zona p e l l u c i d a allows for penetration (Baker & Polge 1976). There are varying theories pertaining to the events which occur during the process, however. The most widely accepted theory i s that i n the cow, the sperm head attaches to the v i t t i l i n e membrane immediately following zona penetration. This i s followed by the fusion of the plasma membrane of the ovum and sperm, with the points of contact being the m i c r o v i l l i on the ovum surface and the posterior portion of the sperm head. The c e l l s become continuous with each other and the contact progressively enlarges u n t i l eventually ooplasm surrounds the ent i r e sperm head and t a i l . At th i s point, the spermatozoa and ovum constitute one c e l l as they are i n the same plasma membrane. Af t e r the second meiotic d i v i s i o n resumes i n the ovum and the second polar body i s expelled, the female pronucleus formed moves towards the center of the ooplasm as does the male pronucleus (Baker & Polge 1976, and Hafez 1980). The head of the f e r t i l i z i n g spermatozoa begins to swell and the paternal chromatin decondenses following the spermatozoans entry Into the ooplasm. With the remains of the sperm t a i l s t i l l attached, the now round male pronucleus moves to the center of the ooplasm. The union of the two pronuclei occurs only a few hours - 46 -a f t e r penetration. Fourteen to sixteen hours a f t e r penetration, pronucei membrane loss occurs and the paternal and maternal chromatin unite (syngamy)s This stage of development appears to be very short. Implantation In cows, i t has been shown that maternal recognition of pregnancy (prevention of l u t e a l regression) occurs 15 to 17 days after f e r t i l i z a -t i o n (Beal et a l 1981, and Bazer et a l 1979). The actual mechanisms which control the maintenance of the CL and synthesis of progesterone i n cows during early pregnancy are s t i l l e s s e n t i a l l y unknown. Beal et a l (1981) suggest that 18-day old bovine blastocysts contain molecules or fragments of larger molecules which are able to d i r e c t l y stimulate the bovine CL to secrete progesterone. Furthermore, they suggest that prostaglandins or steroids may also be involved. Cook and Hunter (1978) claim that although steroidogenesis by the cow blastocyst appears to occur p r i o r to, and following attachment, products appear to be natural steroids and estrogen production i s low or absent. The steroids produced may act c e n t r a l l y as pre-hormones and serve as a signal to the dam of the presence of the uterine embryo. Normal embryo development w i l l occur only i f a threshold l e v e l of l u t e a l progesterone production i s attained or surpassed. The threshold l e v e l necessary for embryo s u r v i v a l by day 15 i s thought to be in the area of 100/ug progesterone per ml of plasma (Staples & Hansel 1961a, and Staples et a l 1961b). The present a v a i l a b l e evidence suggests that i t i s maternal progesterone which induces the synthesis of uterine secretions and i t s components (Cook & Hunter 1978). - 47 -In the cow, the embryo enters the uterus from the f a l l o p i a n tubes approximately 3 days af t e r f e r t i l i z a t i o n (Cook & Hunter 1978). The ovarian s t e r o i d hormones control the transport of the embryo along the oviduct into the uterus. The timing of the embryo entry into the uterus i s c r i t i c a l to normal embryonic development and to a successful pregnancy. There are 4 pre-implantation features which apply not only to c a t t l e , but to sheep and pigs as w e l l . The features are as follows: 1. Shortly a f t e r the trophoblast hatches from the zona p e l l u c i d a , i t p r o l i f e r a t e s extensively, r e s u l t i n g i n the notable expansion of the b l a s t o c y s t . 2. Before the embryo implants, or more accurately, attaches, the cow embryo Is i n the uterine lumen i n a f r e e - l i v i n g stage for a prolonged period of time. 3. Implantation i n cows i s ce n t r a l and i t can be termed s u p e r f i c i a l because the r e s u l t i n g placenta formed Is of the syndesmo-chorial type where the conceptus remains i n the uterine lumen. 4. Intrauterine migration of the embryo, though of low incidence i n c a t t l e , s t i l l does occur. Cook and Hunter (1978) indicate that attachment begins at about day 30 i n the cow. At t h i s time, v i l l i of the trophoblast make a connection i n the crypts of the caruncles (maternal tissue) and the functional placentome i s eventually formed. However, King et a l (1980) indic a t e that apposition and adhesion between the trophoblast and maternal epithelium was detectable as early as day 20. At day 29, the - 48 -Intimacy of attachment had increased as had the size of the placentorae, although no v i l l i or crypts had formed (King et a l 1980). In order for the embryo to survive and mature into a fetus, the regression of the CL must be prevented and uterine formation of histotrophe (uterine f l u i d ) must occur (Cook & Hunter 1978). A few generalizations can be drawn from the available information on the events occuring during implantation (Enders 1976). Before adhesion can begin, apposition of the endometrium to the blastocyst Is absolutely e s s e n t i a l (Enders 1976). In the cow, where the blastocyst i s large, apposition r e s u l t s as the blastocyst grows and expands, thus bringing i t s surface into contact with endometrial lumen e p i t h e l i a l c e l l s (Enders 1976). R e l a t i v e l y l i t t l e i s known about the adhesive process. The regions of adhesion are areas where the trophoblast and uterine c e l l membranes are associated c l o s e l y i n a p a r a l l e l fashion and the distance between the two i s less than the normal i n t e r c e l l u l a r distance. Therefore, some reduction i n the c e l l coats or i n t h e i r constituents should occur (Enders 1976). Before adhesion can become e f f e c t i v e , there must be changes i n the surface coats and i n the shape of either one or both c e l l surfaces. This would e f f e c t i v e l y increase the areas of the c e l l membranes which are i n apposition (Enders,1976) . Adhesion i s thought to Involve both the development of adhesive groups on the f e t a l surface as well as some a l t e r a t i o n s to the maternal surface. In species such as the cow where attachment i s e s s e n t i a l l y s u p e r f i c i a l , the most intimate connection formed during the attachment process may in fact be the adhesion of the trophoblast to the uterine epithelium (Enders 1976). - 49 -MATERIALS AND METHODS The main objective of the study was to investigate metabolic S -Se in t e r a c t i o n s on s p e c i f i c productive, reproductive and health parameters. The data used i n t h i s study was co l l e c t e d e n t i r e l y from the University of B r i t i s h Columbia dairy research herd. Since the beginning of the study i n October 1981 u n t i l i t s conclusion i n December, 1983, 37 Holstein-Friesen and 34 Ayrshire cows were randomly assigned to one of four treatment groups i n order to a t t a i n an unbiased test of treatment and breed e f f e c t s and experimental error as well as to maximize p r e c i s i o n . The treatment groups were: (1) basal di e t ; no sulphur or selenium supplementation, (2) basal diet; supplemented with 0.15% sulphur only, (3) basal diet supplemented with 0.400 mg/kg selenium, and (4) basal diet supplemented with 0.15% sulphur and 0.400 mg/kg selenium. The experiment spanned a period of 26 months; i n th i s time period the majority of cows had completed t h e i r f i r s t l a c t a t i o n on treatment and were eit h e r fi n i s h e d or at various stages of t h e i r second l a c t a t i o n . Individual cows were put on the t r i a l at calving and fed the same dietary r a t i o n over 2 l a c t a t i o n s . During the dry period, cows were maintained on a normal, a l l forage dry cow r a t i o n . Se status of cows - 50 -was monitored throughout l a c t a t i o n on the basis of monthly blood c o l l e c t i o n s for plasma Se a n a l y s i s . Animal Management Cows were housed i n a free s t a l l barn during the l a c t a t i o n period and fed i n d i v i d u a l l y twice d a i l y using Calan e l e c t r o n i c doors. After completing t h e i r f i r s t l a c t a t i o n , the dry cows were housed separately i n open pens. During this time the cows were maintained on a normal dry cow feeding schedule consisting of an a l l forage rat i o n for a minimum of 45 days. One to two weeks pr i o r to calv i n g , the cows were moved to box s t a l l s and adapted to the forage-concentrate d i e t . Postpartum cows rejoined the milking herd 5 days after calving at which point cows were put onto the same treatment as i n the previous l a c t a t i o n . Cows were maintained on t h e i r respective treatments from 5 days post calving to the dry off period. The r a t i o n fed consisted of forage as a l f a l f a cubes (free choice), beet pulp (25% of concentrate), and a commercial dairy concentrate*with no added selenium (fed at a l e v e l of 1 kg concentrate/3 kg mil k ) . Sulphur, as elemental sulphur, and selenium, as sodium selenate, were supplemented as top-dressing. Sulphur was fed as elemental sulphur as i s currently suggested by the feed industry. Water and iodized s a l t were av a i l a b l e ad l i b i t u m . A l l animals on treatment were sampled for milk progesterone analyses while a random sample of cows from treatment groups 1 - 3 were sampled for plasma LH analyses. Milk sampling for progesterone determination began immediately a f t e r calving and continued u n t i l confirmed conception (by r e c t a l •Supplemented with 6.6 I.U. vitamin E per kilogram concentrate (D.M. b a s i s ) . - 51 -palpation) or u n t i l the animal was cu l l e d from the herd. Blood sampling for the pre-ovulatory LH surge during estrus was based primarily on milk progesterone p r o f i l e s for i n d i v i d u a l cows. Blood sampling began 2-3 days af t e r the pre-estrus decline In milk progesterone concentrations occurred. The time period between previously observed behavioral estruses for i n d i v i d u a l cows (when avail a b l e ) was also a h e l p f u l aid in determining time of sampling. Blood samples for plasma Se determination were taken from a l l cows on a monthly basis throughout the experimental period beginning one month aft e r the onset of the experiment. Blood samples for Se determination were also taken randomly from calves whose dams had completed one f u l l l a c t a t i o n on treatment. Calf weights were obtained immediately a f t e r b i r t h . A n a l y t i c a l Procedures 1. Blood Sampling Blood samples obtained for plasma Se analyses were a l l c o l l e c t e d by jugular venipuncture using 20 1/2 guage needles and 15 ml heparinized vacutainer tubes. Two 15 ml samples of blood were c o l l e c t e d from each cow at monthly i n t e r v a l s throughout the duration of the experiment. Se status of dairy c a t t l e may be estimated by selenium concentrations i n serum, l i v e r and whole blood and by the a c t i v i t y of glutathione peroxidase i n serum and erythrocytes (Thompson et a l . , 1980). Schalz et a l (1979) demonstrated that blood Se concentration and blood GSH-Px a c t i v i t y are highly correlated (r = 0.958). However, i n - 52 -view of recent reports of the presence of non-selenium dependent GSH-Px i n various tissues, which could increase the experimental error, plasma Se was selected as the i n d i c a t o r of Se status. Plasma LH samples were taken from cows i n treatment groups 1-3 only because i t was assumed that plasma Se l e v e l s would be adequate i n treatment group 3 and marginal to d e f i c i e n t i n treatment groups 1 and 2. Also, i n view of the intense sampling schedule (blood samples for plasma LH analysis were taken every 4-6 hours for 2-4 days during e s t r u s ) , i t was f e l t that a more complete sampling of three treatment groups would be advantageous. Five to ten mis of blood for plasma LH analyses were co l l e c t e d from cows on treatment groups 1 - 3 using 20 1/2 guage needles and heparinized 10 ml vacutainer tubes. Both jugular and coccygeal venipuncture techniques were used to c o l l e c t blood samples. Blood samples were taken at 4 - 6 hour i n t e r v a l s beginning j u s t p r i o r to the expected onset of estrus u n t i l standing heat was observed (usually consisted of a 2 - 4 day sampling period). Blood samples were immediately centrifuged for 10 minutes or stored i n 4°C r e f r i g e r a t o r overnight and centrifuged the following day. Plasma was f i l t e r e d from the centrifuged samples, stored i n vacutainer tubes and frozen u n t i l time of a n a l y s i s . 2. Se Analysis Plasma and feed samples were analyzed by hydride generation atomic absorption spectrometry as described by Tam and Lacroix (1982) - 53 -with only minor modifications to the procedure. The modifications include 1) an increased ashing temperature for feed samples (150°C instead of 100°C) and 2) 3 - 5 mis of methanol was used to wet feed samples p r i o r to ashing. The analysis was c a l i b r a t e d using the National Bureau of Standards C e r t i f i c a t e of Analysis (Standard Reference Material 1577 for Bovine L i v e r ) . Plasma and feed samples for Se determination were cone i n duplicates; Se l e v e l s reported here are average values. 3 . LH Analysis Frozen plasma samples were analyzed for LH by Reproductive Endocrine Assay Systems (REAS) (The Reproductive Endocrine Laboratories, Department of P h y s i o l o g i c a l Sciences, College of Veterinary Medicine, Un i v e r s i t y of Saskatoon, Saskatoon, Saskatchewan, S7N 0W0). V a l i d a t i o n of the assay and references to the a n a l y t i c a l procedure are given i n Appendix 1. 4. Milk Sampling and Milk Progesterone Analysis Milk samples (as strippings) for milk progesterone analysis by radio-immuno assay (RIA) were taken at the afternoon milking on alternate days for each cow and were placed i n 6 oz. whirl pak bags and preserved with 40 mg potassium dichromate t a b l e t s . Samples were stored i n a 4°C r e f r i g e r a t o r u n t i l a n a l y s i s . Milk progesterone was analyzed by an RIA technique described by Shelford et a l (1979). The technique i s a modification of that i n i t i a l l y described by Heap et a l (1973). - 54 -An enzyme linked immunosorbent assay (ELISA) for milk progesterone developed by Helix Biotech Ltd. (217-7080 River Road, Richmond, B r i t i s h Columbia) was used for immediate milk progesterone determination for estrus detection. Milk strippings for the ELISA procedure were handled i n a s i m i l a r manner as those used for RIA except the preservative was not added. Fresh or day old milk samples were assayed using the ELISA procedure and r e s u l t s were obtained within two to three hours. Standards, pre-coated tubes, and activated s t i c k s were obtained from Helix Biotech. The procedure was as follows: 0.02 mis of milk standard of known progesterone concentration (0, 5.0 and 10.0 ng/ml) or 0.20 mis of milk sample of unknown progesterone concentration were placed i n pre-coated tubes. One pre-activated s t i c k was dropped into each tube and incubated at room temperature for one hour. The s t i c k s were removed from the tubes and the contents discarded. Tubes were then completely f i l l e d with an ELISA wash solution (1% NaHC03, 0.05% Tween 20, and 0.02% Na-azide) and l e t to stand for 5 minutes before discarding the contents. The rinse procedure with the ELISA wash solution was repeated 3 times. One ml of substrate (1% Diethanolamine, 2 mM MgCl2> 0.02% Na-azide, and 1 mg/ml P-nitrophosphenyl orthophosphate) was added to a l l tubes and incubated at 37°C for 1 - 2 hours. The reaction was terminated by adding 0.1 ml of 5 N NaOH. The tubes were compared v i s u a l l y to the standards and categorized as 0 - 5, 5 - 10, or >10 ng progesterone per ml of milk. The advantage of the ELISA over the RIA i s the swiftness of the procedure. Using the ELISA procedure, the analysis - 55 -of samples was accomplished a l l i n one day. With the RIA technique, several days elapsed between when the assay was done and when r e s u l t s from that assay were obtained. During the estrous cycle, the time between the pre-estrus progesterone drop and the onset of estrus i s generally one to three days. It i s therefore e s s e n t i a l to detect the pre-estrus drop i n progesterone as soon as po s s i b l e . c 5 . Feed S a m p l i n g Samples of a l l batches of forages, concentrates, and mineral supplements used i n the study were taken for Se a n a l y s i s . Weighback samples were c o l l e c t e d weekly and composited into monthly samples. Monthly composites of weighbacks for each treatment group were randomly selected for Se a n a l y s i s . 6 . D a t a C o l l e c t i o n I n d i v i d u a l cow summary information concerning reproduction, production, and health data was obtained from the University of B r i t i s h Columbia research record keeping system. More s p e c i f i c a l l y information concerning d a i l y intake, milk and fat y i e l d s , reproductive parameters incl u d i n g days to f i r s t s e rvice, days open, calving i n t e r v a l (days), services/confirmed conception, c a l f b i r t h weight, milk progesterone concentrations during estrous cycles, calving ease data, reproductive health problems and health problems other than those associated with reproduction were obtained. Milk y i e l d s on the herd were obtained from l a c t a t i o n records, and - 56 -milk f a t composition was determined by B.C.M.A. Dairy Laboratory (Burnaby, B.C.) using i n f r a r e d milk a n a l y s i s . 7 . BCA's f o r Milk and Fat Y i e l d Incomplete l a c t a t i o n s were extended to 305-day l a c t a t i o n s for milk y i e l d and kg fat by the M u l t i p l i c a t i v e Factor method using cumulative y i e l d (MLC) as described by Batra and Lee ( i n press). The MLC equation used i s as follows: TL = ci x CLi where TL = t o t a l l a c t a t i o n y i e l d , CLi = the cumulative l a c t a t i o n up to the I*-*1 day of l a c t a t i o n , and c^ = the r a t i o factor of the t o t a l on cumulative y i e l d . Complete and extended l a c t a t i o n y i e l d s and kg fat were converted to BCA values using the following formula: TL/BCA fac t o r * x 100 = BCA value The BCA values calculated were introduced into the analysis of variance as dependent v a r i a b l e s . *BCA factors used were the Agriculture Canada O f f i c i a l Breed Class Average Factors (1977) for Hosteins and Ayrshires. - 5 7 -Experimental Design Analysis of Variance This study was conducted to test the following n u l l hypotheses: 1. Excess dietary S does not enhance Se defi c i e n c y i n l a c t a t i n g dairy c a t t l e . 2. Treatment group does not influence production, reproduction, and feed intake t r a i t s . The following independent variables were introduced into the analysis of variance: Treatment Group: At calvi n g , each cow was randomly assigned to one of the four experimental d i e t s , that i s , the co n t r o l , the S supplemented, Se supplemented, or S and Se supplemented groups. In several analyses, cows on treatments 1, 2, 3, and 4 entering th e i r 2nd l a c t a t i o n on treatment were designated as being on treatment 5, 6, 7 and 8 res p e c t i v e l y and included i n the ANOVA as such. Where empty c e l l s occurred using this method, data over 2 l a c t a t i o n s were pooled, thus, only the 4 treatment groups were incuded i n the ANOVA. Breed: Since breed may influence production, c a l f weights, and poss i b l y reproductive performance as well as other parameters, t h i s factor was Included i n the analysis of variance models. The complete l i n e a r model used to test the n u l l hypotheses was as follows: - 58 -'ijk where Y the dependent variable T u i the o v e r a l l mean of samples the e f f e c t of the i t n treatment B. J the e f f e c t of the j th breed the i n t e r a c t i o n of the i t h treatment with the j t h breed E the unexplained residual error associated with each sample This model was condensed by eliminating non s i g n i f i c a n t i n t e r a c t i o n s when required. The main e f f e c t breed was also deleted when not a p p l i c a b l e . The following t r a i t s were analyzed using t h i s l i n e a r model: BCA's for 305 day milk and f a t y i e l d , days to f i r s t service a f t e r p a r t u r i t i o n , days open, calving i n t e r v a l , number of services/conception, somatic c e l l counts, LH lag time, magnitude of the pre-ovulatory plasma LH surge, basal plasma l e v e l s , estrous cycle lengths, c a l f weights. For the analysis of plasma selenium l e v e l s and milk progesterone concentrations at the time of breeding, (un)successful breeding was introduced into the l i n e a r model as an independent v a r i a b l e . Milk progesterone and plasma Se concentrations at breeding were analyzed with the following complete l i n e a r model (non-significant i n t e r a c t i o n s deleted when required: Y i j k = u + T i + Bj + C k + T i C k + B j C k + E i j k - 59 -where Y . = the dependent variable milk progesterone or plasma Se at time of breeding u = the o v e r a l l mean of a l l samples = the e f f e c t of the i t h treatment Bj = the e f f e c t of the j t h breed = the e f f e c t of the kth conception T\C, = the i n t e r a c t i o n of the i t h treatment with the k t n i k conception B.C. = the i n t e r a c t i o n of the j t h breed with the k t h i k J conception E^.^ = the unexplained residual error associted with each sample Estrous cycles were broken down into periods (0-4) where periods were defined by increases or decreases i n progesterone concentrations (above or below pre-set l i m i t s ) . Estrous cycles were then c l a s s i f i e d into 1 of 13 cycle types (described i n the following discussion) based on the occurrence and duration of s p e c i f i c periods within s p e c i f i c time i n t e r v a l s . Each period was then analyzed respectively for progesterone concentration and period duration among breeds, treatments and cycle types. The following gives a b r i e f d e s c r i p t i o n of the 5 periods tested. The postpartum quiescence period (period 0) was defined by progesterone l e v e l s maintained below 4 ng/ml from freshening u n t i l the f i r s t r i s e above 2 ng/ml leading to a subsequent r i s e above 4 ng/ml. Cycle length was defined as the time period from a r i s e i n milk - 60 -progesterone concentrations from below 4 ng/ml, to a concentration above 12 ng/ml, following by a subsequent decline to below 4 ng/ml. Estrus detected by milk progesterone concentrations was defined by a f a l l i n milk progesterone concentrations to below 4 ng/ml af t e r a previous r i s e above 12 ng/ml. Cycles were categorized as one of the following cycle types based on milk progesterone concentrations. Cycle Type 0 - period 0 only (quiescence) . Cycle Type 1 - post-quiescence: progesterone r i s e s from < 4 ng/ml to > 5 ng/ml, then f a l l s back to < 4 ng/ml i n 18 days. Cycle Type 2 - post quiescence: progesterone r i s e s from < 4 ng/ml to > 12 ng/ml, then f a l l s back to < 4 ng/ml i n 18-24 days. Cycle Type 3 - post quiescence: progesterone r i s e s from < 4 ng/ml to > 12 ng/ml, f a l l s to < 4 ng/ml i n > 24 days. Cycle Type 4 - normal cycle, progesterone r i s e s from < 4 ng/ml to > 12 ng/ml; f a l l s to < 4 ng/ml i n 18-24 days. Cycle Type 5 - long cycle: progesterone r i s e s from < 4 ng/ml to > 12 ng/ml; f a l l s to < 4 ng/ml i n 25-30 days. Cycle Type 6 - short cycle: progesterone r i s e s from < 4 ng/ml to > 12 ng/ml; f a l l s to < 4 ng/ml i n less than 17 days. - 61 -Cycle Type 7 - confirmed pregnant cycle: progesterone > 12 ng/ml for 25 days; with breeding followed by a po s i t i v e vet check. Cycle Type 8 - unconfirmed pregnancy: progesterone > 12 ng/ml for 25 days; breeding but no vet check. Cycle Type 9 - embryonic mortality predicted; progesterone > 12 ng/ml for 24 days; then f a l l s to < 4 ng/ml, with breeding. Cycle Type 10 - l u t e a l cyst: during period 3, progesterone > 12 ng/ml for more than 24 days; no breeding. Cycle Type 11 - f o l l i c u l a r cyst: period 1 longer than 12 days, and period 3 shorter than 12 days. Cycle Type 12 - f o l l i c u l a r cyst followed by l u t e a l cyst: period 1 longer than 12 days and period 3 longer than 12 days. Cycle Type 13 - a l l other c y c l e s . For the analysis of milk progesterone concentrations during the d i f f e r e n t periods of the estrous cycle and the duration of the periods i n estrous cycles, cycle type was Introduced Into the l i n e a r model as an independent v a r i a b l e . The s i m p l i f i e d l i n e a r model was as follows: Y l j k = u + T ± + Bj + C k + E. I j k where Y i j k = the independent variables mentioned above u = the o v e r a l l mean of a l l samples = the e f f e c t of the i t n treatment - 62 -Bj = the e f f e c t of the j t h breed = the e f f e c t of the k t n cycle type T± x B i x Cfc = the e f f e c t of the i t h treatment with the j t h breed and the k t h c y c l e . E^.j^ = the unexplained residual error associated with each sample Plasma Selenium T r a i t s Plasma selenium response over time was analyzed among breeds and treatments with the following simple l i n e a r regression model: Y = (m)x + b where Y = the dependent variable plasma Se concentration x = the independent v a r i a b l e time (days) b = the y intercept m = a constant term. Chi-square Test C r i t e r i o n The e f f e c t of S-Se in t e r a c t i o n s on the observed frequency of s p e c i f i c health, vet and ease of calving parameters as well as the observed frequency of cycle types was analyzed for independence of vari a b l e s using the X test c r i t e r i o n for di s c r e t e data (Steel & To r r i e 1980). The test c r i t e r i o n used was: 2 ^2 _ £ (expected - observed) expected Two-way contingency tables were produced containing the dependent v a r i a b l e s mentioned above c l a s s i f i e d by treatment group. - 63 -S t a t i s t i c a l Analysis The General Linear Hypothesis package program UBC BMD:10V (1975) was used for the analysis of variance and covariance. F-tests for test of s i g n i f i c a n c e were based on a Type I error p r o b a b i l i t y of p < 0.05. Mean separation was done by the program using the Student-Newman-Keuls multiple range t e s t . ANOVA with BMD:10V enabled the testing of the following A. p r i o r i . n u l l hypotheses: (1) Se treated cows do not d i f f e r from non-selenium treated cows. UBC S t a t i s t i c a l Package for the Social Sciences (SPSS:9) (1983) was used to produce two-way contingency tables for health, vet, calving ease, and estrous cycle type data. The tables were constructed to include row, column and t o t a l percentage of frequency d i s t r i b u t i o n s for each v a r i a b l e tested. The tables produced were analyzed with the test for independence. (For 2 x 2 contingency tables with less than 21 cases, a Fisher's test was used; for a l l other 2 x 2 tables, a Yates' corrected chi-square was used). The program also gave Pearson r c o r r e l a t i o n c o e f f i c i e n t s between the v a r i a b l e and treatment group. The UBC Triangular Regression Package (TRP) (1978) was used to perform simple regression analyses and to produce simple l i n e a r regression equations i n the form y = ax + b. Scattergrams were produced using UBC:TRP. The UBC:TRP program was also used to compute Pearson product moment c o e f f i c i e n t s of c o r r e l a t i o n (a measure of the l i n e a r c o r r e l a t i o n between v a r i a b l e s ) . Regression analyses were done on, and c o r r e l a t i o n c o e f f i c i e n t s , - 64 -and regression equations produced f o r , the following data sets: 1. The e f f e c t of plasma selenium on milk progesterone concentrations at breeding, c l a s s i f i e d by treatment group, and by conception/non-conception. 2. The e f f e c t of dam plasma Se and c a l f plasma Se l e v e l s on c a l f b i r t h weights. 3. The e f f e c t of dam plasma Se on calving ease. 4. The e f f e c t of time on plasma Se l e v e l s i n response to treatment. 5. The e f f e c t of cow plasma Se and milk progesterone concentrations at breeding on basal LH and LH lagtime. UBC:SLTEST (1984), an equality of slope test was used to test d i f f e r e n c e s i n the regression analyses for the variables mentioned in the preceeding se c t i o n . The program also computed regression equations and c o e f f i c i e n t s . The regression c o e f f i c i e n t s were tested with an F-test to determine whether differences were due to sampling errors or due to r e a l differences between treatment groups. - 65 -RESULTS Ration Selenium Levels Selenium content of a l l feed sources used i n the t r i a l as determined by hydride generation atomic absorption spectrometry are given i n Table 1. Appendix 2 gives the mineral content of the mineral mix added to a l l d i e t s . Vitamin E lev e l s i n a l f a l f a cubes range from 15-26 mg/kg (NRC 1971). Barley contains approximtely 14 mg/kg vitamin E. In addition, 6.6 I.U./kg vitamin E was added to the mineral mix. The dairy c a t t l e requirement of vitamin E in rations i s considered to be 10 mg/kg feed (DM b a s i s ) . Therefore, the vitamin E lev e l s fed in th i s study were well above adequate. It has been observed that c e r t a i n Se and or vitamin E def i c i e n c y diseases w i l l respond to supplementation of Se or vitamin E or both. In the context of th i s study, dietary feed l e v e l s were sim i l a r among treatment groups. Because dietary vitamin E lev e l s were well above adequate i n this study, responses which are s p e c i f i c a l l y Se related would be expected i n cases where a Se d e f i c i e n t state i s achieved. However, i t i s possible that the adequacy of vitamin E may mask a Se def i c i e n c y to a ce r t a i n extent. Table 2 gives selenium and sulphur concentrations achieved i n the t o t a l r a t i o n a f t e r supplementation f o r the four treatment groups. Intake o Means and standard deviations for d a i l y intakes (DM basis) of - 66 -Table 1 Selenium Levels (ppb) i n Feed Sources (DM basis) Selenium Content (DM basis) (mg/kg) Feed Source X s .d n Range A l f a l f a Cubes 0.289 0.043 9 0.074 - 0.474 14% Dairy Concentrate 0.371 0.025 12 0.220 - 0.523 Beet Pulp 0.250 - 1 -Control Mineral Mix 0.0 - 1 -Sulphur Mineral Mix 0.759 - 1 -Selenium Mineral Mix 1 x 10 mg/kg - 1 -Local Hay (Dry Cow Ration) 0.075 0.040 2 0.047 - 0.120 - 67 -Table 2 Sulphur and Selenium Composition of Experimental Rations (DM basis) Treatment Feed Component Sulphur (%) Selenium (mg/kg) Control S supplemental Se supplemental S + Se supplemental 0.35 0.50 0.35 0.50 0.310 0.311 0.721 0.722 - 68 -forage, dairy concentrate, beet pulp, and t o t a l feed intake analyzed by breed are given i n Table 3. Breed E f f e c t Breed differences were s i g n i f i c a n t at p < 0.05 for the d a i l y intake of dairy concentrate and t o t a l d a i l y intake. In both cases, the Holstein breed consumed more of the rat i o n than the Ayrshire breed. The consumption of a l f a l f a cubes and beet pulp was not s i g n i f i c a n t l y d i f f e r e n t between the breeds, although s i g n i f i c a n c e was approached (p = 0.06). Treatment E f f e c t When feed intake data (DM basis) was analyzed by treatment group (Table 4) s i g n i f i c a n t treatment differences were found for the d a i l y consumption of a l f a l f a cubes, dairy concentrate and t o t a l intake. However, there was no difference among treatments i n the consumption of beet pulp. Cows on treatment group 5 consumed s i g n i f i c a n t l y more a l f a l f a cubes (8.80 ± 1.59 kg/day) than cows on treatment group 3 (7.32 ± 1.59 kg/day) (p <^  0.05). The d a i l y consumption of dairy concentrate was greater for cows on treatment groups 6, 7, and 8 (7.09, 6.67, and 6.60 ± 1.05 kg/day, respectively) than for cows on treatment groups 2, . 3, and 4 (5.82, 5.95,and 5.71 kg/day, respectively) (p<_0.01). The difference i n t o t a l d a i l y intake between treatment groups was highly s i g n i f i c a n t , and mean separation was s i m i l a r to that observed for concentrate intake. Cows on treatment groups 6, 7, and 8 consumed Table 3 E f f e c t of Breed on Daily Feed Intake (kg DM Basis) and Production T r a i t s (BCA for Milk Y i e l d and kg Fat) Daily Feed Feed Intake (kg DM) and Production T r a i t s Holstein X Ayrshire X s.d. Sign i f i c a n c e A l f a l f a Cubes 8.10 8.11 1.59 n.s. 15% Dairy Concentrate 7.04 5.32 1.05 ** Beetpulp 3.36 3.22 0.48 n.s. Total 18.45 16.09 2.09 ** Milk Y i e l d (BCA Index) 163.80 154.00 28.39 n.s. kg Milk Fat (BCA Index) 107.20 117.20 21.51 n.s. n 32 38 n.s. = not s i g n i f i c a n t ** = s i g n i f i c a n t l y d i f f e r e n t at p < 0.01 Table 4 Ef f e c t of Treatment on kgs Daily Feed Intake (DM Basis) and Production T r a i t s (BCA for Milk Y i e l d and kg Fat) Treatment 1 2 3 4 5 6 7 8 X X X X X X X X s . d . A l f a l f a Cubes 8.01 a b 7.42 a b 7.32 a 7.49 a b 8.80 b 8.34 a b 7.65 a b 8.45 a b 1.59 (kg DM) 14% Dairy Concentrate 6.38 X y 5.82 X ,5.95 X 5.71 x 6.22 x y 7.09 y 6.67 y 6.60 y 1.05 (kg DM) Beet Pulp (kg DM) 3.28 3.27 3.28 3.18 3.48 3.18 3.15 3.40 0.48 Total Daily 17.61 x y 16.45 X 16.51 X 16.34 X 18.45 y 18.55 y 17.42 x y 18.39 y 2.09 Intake (kg DM) n (for above 18 12 13 15 8 5 , 1 2 variables Milk Y i e l d (BCA) 162.4 158.6 150.5 158.5 150.4 180.7 155.0 169.0 28.39 kg Milk Fat (BCA) 117.1 109.1 112.2 108.4 111.1 114.2 128.0 126.0 21.51 n ( f o r Milk Y i e l d 19 15 18 19 14 7 4 6 and kg Milk Fat) a»b superscripts denoting d i f f e r e n t homogeneous subsets i n the same hori z o n t a l row at p < 0.05. x > v superscripts denoting d i f f e r e n t homogeneous subsets i n the same ho r i z o n t a l row at p <_0.01. - 71 -18.55, 17.42, and 18.39 ± 2.09 kg of feed/day, res p e c t i v e l y whereas cows on treatments 2, 3, and 4 had s i g n i f i c a n t l y lower (p <^  0.01) feed intakes of 16.45, 16.51, and 16.34 ± 2.09 Kg of feed/day, r e s p e c t i v e l y . Mean selenium concentrations of weighback samples analyzed by treatment group (Table 5) were found to be s i g n i f i c a n t l y d i f f e r e n t (p <^  0.001). The control and sulphur supplemented diets had selenium l e v e l s of 0.557 and 0.524 ± 0.929 mg/kg, respectively, which were s i g n i f i c a n t l y lower (p <_ 0.001) than the selenium l e v e l s i n weighbacks from treatment groups 3 and 4 (2.257, and 3.312 ± 0.929 mg/kg, re s p e c t i v e l y . Production T r a i t s Breed E f f e c t Mean milk y i e l d (BCA) and kg fat (BCA) analyzed by breed and treatment group are given i n Tables 3 and 4, r e s p e c t i v e l y . The Holstein breed had a greater mean BCA milk y i e l d (163.0 ± 28.39) than the Ayrshire breed (154.0 ± 28.39). Although the difference i n milk y i e l d was not s i g n i f i c a n t l y d i f f e r e n t , s i g n i f i c a n c e was approached at p = 0.14. In contrast, the Ayrshire breed had a mean BCA for kg milk fat of 117.2 ± 21.51 which tended to be greater than that of the Holstein breed (107.2 ± 21.51). As with milk y i e l d , the dif f e r e n c e between breeds for kg milk fat did approach s i g n i f i c a n c e (p = 0.06). Treatment E f f e c t Treatment group had no influence on BCA milk y i e l d or BCA kg milk Table 5 Selenium Content of Weighback Samples (mg/kg) Treatment 1 . 2 3 4 X n X n X n X n s'.d. Weighback Se 556.7 X 6 0.524x 6 2.257 y 6 3.312 y 5 0.929 Content (mg/kg) x,y = superscripts denoting d i f f e r e n t homogeneous subsets i n the same row at p < 0.001. - 73 -f a t . The differences between means for both BCA milk y i e l d and kg milk fat among treatment groups 1 through 8 were not found to be s i g n i f i c a n t . Plasma Selenium Response to Treatment Data represented i n Tables 6 and 7, and Figures 1 and 2, was co l l e c t e d over 2 l a c t a t i o n s . Breed E f f e c t Figure 1 i l l u s t r a t e s the plasma selenium response over time between breeds. It was observed that plasma Se l e v e l s rose within the f i r s t 4 months of treatment before plateauing u n t i l the dry period, a f t e r which a sharp decline i n plasma selenium le v e l s occurred. Slopes and l i n e a r regression equations were compared between breeds. Regression equations and standard error of Y between plasma Se and time analysed by breed are given i n Table 6. During the f i r s t 4 months, plasma Se l e v e l s were found to be s i g n i f i c a n t l y correlated to time for both breeds. The regression equations derived for breeds were found to have s i m i l a r slopes, and equations. Using the regression equations, projected plasma Se concentrations at day 60 (mid point of increasing plasma Se phase during months 1-4) and day 210 (mid-point of plasma Se concentrations during plateau phase) were calculated and are given i n Table 7. Mean dry cow plasma Se l e v e l s and c a l f plasma Se l e v e l s are also given i n Table 7. Breed did not influence plasma Se l e v e l s during the f i r s t 4 months of treatment, the plateau phase, or the dry period. -74-TIME (months) F i g u r e 1: I n f l u e n c e o f b r e e d on mean plasma s e l e n i u m l e v e l s d u r i n g t h e t r e a t m e n t p e r i o d (months 1 - 1 0 ) and t h e d r y p e r i o d (month 12) . - 75 -Table 6 Regression Analyses for the E f f e c t of Time on selenium Response for Breeds Breed Regression Equation SE(Y) Significance Months 1-4 Holsteins Y = 0.085 + 0.18 X 0.023 ** Ayrshires Y = 0.084 + 0.24 X 0.022 ** Months 4-10 Holsteins Y = 0.101 + 0.12 X 10" 1 X 0.020 n.s. Ayrshires Y = 0.105 + 0.10 X 10" 1 X 0.020 n.s. * * S i g n i f i c a n c e at p £ 0.01 n.s. - not s i g n i f i c a n t - 76 -Table 7 E f f e c t of Breed on Cow and Calf Plasma Selenium Levels (mg/kg) Holstein Ayrshire Plasma Se (mg/kg) X n X n s .d. S i g n i f . Cow Plasma Se* (Month 1-4) 0.096 128 0.097 107 0.024 n.s. Cow Plasma Se** (Month 4-10) 0.103 137 0.107 69 0.201 n.s. Dry Cow Plasma Se 0.064 10 0.057 7 0.036 n.s. Calf Plasma Se 0.043 9 0.054 9 0.022 n.s. n.s. = not s i g n i f i c a n t •Projected means at day 60 (mid- point of increasing plasma Se phase). **Projected means at day 210 (mid -point of plasma Se plateau phase). - 77 -Treatment E f f e c t Regression analyses between plasma Se and time were done among treatment groups 1-4. Table 8 gives the equations and S.E. of Y for months 1-4 (increasing plasma Se phase) and 4-10 (plasma Se plateau phase). Equality of slope tests indicated that a l l slopes were s i m i l a r during both phases. S i m i l a r i l y , during both phases equations were found to be s i g n i f i c a n t l y d i f f e r e n t . Table 9 gives the projected plasma Se concentrations at day 60 and 210 (using the appropriate regression equation). During months 1-4, plasma Se concentrations for treatment group 3 were s i g n i f i c a n t l y higher (p <^  0.05) than treatment groups 1 and 2. During the plateau phase, however, treatment group 4 had s i g n i f i c a n t l y higher plasma Se l e v e l s than groups 1, 2, and 3. Cow plasma Se l e v e l differences between treatments during the plateau phase (months 4 - 10) were highly s i g n i f i c a n t (p <^  0.001). Cows on treatment groups 1, 2, 3, and 4 had mean plasma Se l e v e l s during the plateau phase of 0.098, 0.101, 0.104, and 0.116 ± 0.019 mg/kg, re s p e c t i v e l y . In the plateau phase alone, cows on treatment group 1, 2, and 3 had s i m i l a r plasma Se l e v e l s and were s i g n i f i c a n l y lower (p j< 0.05) than plasma Se l e v e l s of cows on treatment group 4. Calf plasma Se l e v e l s did not vary between treatments (p j< 0.05) (Table 7). However, when dam selenium was used as a covariable i t was found to be s i g n i f i c a n t at p < 0.01. This i s i n d i c a t i v e of dam Se being a s i g n i f i c a n t factor i n the v a r i a b i l i t y observed i n c a l f plasma Se l e v e l s . The adjusted means for c a l f plasma Se l e v e l s were 0.040, 0.050, - 78 -Table 8 Regression Analyses for the E f f e c t of Time on selenium Response for Treatment Groups Treatment Regression Equation S.E.(Y) Significance Months 1-4 1 2 3 4 Y = 0.085 + 0.14 X Y = 0.074 + 0.30 X Y = 0.089 + 0.19 X Y = 0.084 + 0.25 X 0.021 0.021 0.020 0.025 ** ** Months 4-10 1 Y 2 Y 3 Y 4 Y = 0.097 + 0.12 x 10 X = 0.0110 - 0.62 x 10" 1 X = 0.093 + 0.59 x 10 X = 0.125 - 0.51 x 1 0 - 1 X 0.019 0.018 0.021 0.018 n.s. n.s. n.s. n.s. • S i g n i f i c a n c e at p < 0.05 ••S i g n i f i c a n c e at p < 0.01 n.s. - not s i g n i f i c a n t Table 9 E f f e c t of Treatment on Cow and Calf Plasma Selenium Levels (mg/kg) Treatment 1 2 3 4 Plasma Se (mg/kg) X n X n X n X n s .d. Cow Plasma Se* (months 1-4) 0.093 X 77 0.092 X 56 0.101 7 47 0.099 x y 55 0.024 Cow Plasma Se** (months 4-10) 0.098X 73 - 47 0.105 X 38 - 48 0.020 Dry Cow Plasma Se 0.055 5 0.064 5 0.048 2 0.068 5 0.036 Calf Plasma Se 0.040 5 0.050 6 0.037 1 0.058 6 0.022 x» vsuperscripts denote d i f f e r e n t homogeneous subsets i n same row at p ^ O . 0 1 . *Projected mean at day 60 (mid-point of increasing plasma Se phase). **Projected mean at day 210 (mid-point of plasma Se plateau phase). - 80 -0.037, and 0.058 ± 0.022 mg/kg, for calves produced from dams on treatments 1, 2, 3, and 4 r e s p e c t i v e l y . Figure 2 depicts the cow plasma Se response curve for treatment groups. The pattern of plasma Se l e v e l s a s s i m i l a t i o n i s s i m i l a r to that observed for breeds. Plasma Se l e v e l s generally appear to increase u n t i l approximately month 4 of treatment ap p l i c a t i o n a f t e r which a plateau appears to be maintained u n t i l month 10. After the 10th month, plasma Se l e v e l s begin to decrease, coinciding with cows entering the dry period and being maintained on Se d e f i c i e n t rations (normal dry cow ration) (p <^  0.001) . Reproductive T r a i t s  Breed E f f e c t There was no difference between breeds i n the number of days to f i r s t service (p ^ 0 . 0 5 ) . Days to f i r s t service for the Holstein and Ayrshire breeds (Table 10) were 92.9, and 90.5 ± 27.13 days, r e s p e c t i v e l y . Table 10 also contains means and standard deviations for days open and services per confirmed conception. Breed differences were s i g n i f i c a n t (p <^  0.01), however, for the number of days open. The Ayrshire breed had a s i g n i f i c a n t l y shorter Interval from calving to conception than the Holstein breed (101.6 and 132.5 ± 43.84 days, r e s p e c t i v e l y ) . Consistent with t h i s f i n d i n g , the Holstein breed required s i g n i f i c a n t l y more (p <^  0.05) services per conception than the Ayrshire breed(1.81 and 1.38 ± 0.82 services/confirmed conception, r e s p e c t i v e l y ) . Also consistent with the above observations, the -81-0 1 2 3 4 5 6 7 8 9 10 11 12 TIME (months) F i g u r e 2: E f f e c t o f t r e a t m e n t group on plasma s e l e n i u m l e v e l s d u r i n g t h e t r e a t m e n t p e r i o d (months 1-10) and t h e d r y p e r i o d (month 12) . - 82 -Table 10 E f f e c t of Breed on Reproductive T r a i t s Holstein Ayrshire • T r a i t X n X n s.d. Significance Days to 1st Service 92.89 53 . 90.54 54 27.13 n.s. Days Open 132.50 46 101.60 49 43.84 ** Calving Interval (Days) 416.40 21 375.90 23 39.38 * Services/ Confirmed Conception 1.81 42 1.38 -47 0.82 ** Calf B i r t h Weight (kg) 42.16 21 33.22 20 5.72 ** n.s. = Not s i g n i f i c a n t * = Significance d i f f e r e n c e at p £ 0.05. ** = Significance difference at p < 0.01. - 83 -difference i n calving i n t e r v a l between breeds was highly s i g n i f i c a n t (p £ 0.01). The calving i n t e r v a l was 416.40 and 375.90 ± 39.38 days for Holsteins and Ayrshires r e s p e c t i v e l y . Treatment E f f e c t Table 11 gives means and standard deviations for cows on treatment groups 1 - 4 for days to f i r s t service, number of days open, calving i n t e r v a l and breeding per confirmed conception. Treatment had no influence on any of these t r a i t s as differences between treatment groups were non-significant (p >^  0.05) i n a l l cases. Progesterone Data Figure 3 i l l u s t r a t e s the characterization of estrous cycles into periods on the basis of milk progesterone concentrations. Milk progesterone concentrations for periods 0, 1, 2, 3, and 4 were subjected to analysis of variance. For the analysis of period 3 among breeds and treatments, cycle types 7 (confirmed pregnancy), 8 (unconfirmed pregnancy), 9 (embryonic mortality predicted), 10 ( c y s t i c CL), 12 ( c y s t i c f o l l i c l e and c y s t i c CL), and 13 ( a l l other cycles) were detected from the analysis as these cycles would be expected to have an extended period 3 (or abnormal as i n the case of cycle type 13) which could mask any true breed or treatment e f f e c t . Means and standard deviations for the influence of breed and treatment on milk progesterone concentrations i n periods 0 - 4 are given i n Table 12 and 13, r e s p e c t i v e l y . Table 11 Eff e c t of Treatment on Reproductive T r a i t s Treatment 1 2 3 4 T r a i t X n X n X n X n s.d Days to 1st Service 87.94 31 93.57 23 86.28 25 100.6 24 27.13 n.s. Days Open 115.0 30 118.2 22 112.6 23 121.7 20 43.84 n.s. Calving Interval (days) 386.3 16 388.6 11 392.1 7 418.9 10 39.38 n.s. Services/Confirmed Conception 1.73 30 1.50 20 1.43 21 1.60 18 0.82 n.s. Calf B i r t h Weight (kg) 36.31a 15 39.15 10 38.40 7 38.3 9 5.72 n.s. n.s. = not s i g n i f i c a n t -85-10 30 40 50 DAYS POST PARTUM 60 70 A—B: period 0 (quiescence); progesterone maintained < 4 ng/ml u n t i l the f i r s t rise above 2 ng/ml leading to a rise above 4 ng/ml. B—C : duration of the f i r s t estrous cycle (cycle type 1). C—D: period 1 of the second estrous cycle; progesterone levels are < 4 ng/ml. D—E : period 2 of the second estrous cycle; progesterone levels are <4 ng/ml and > 12 ng/ml. E—F : period 3 of the second estrous cycle; progesterone levels are > 12 ng/ml. F — G : period 4 of the second estrous cycle; progesterone levels f a l l from >12 ng/ml to < 4 ng/ml. H : estrus predicted; progesterone f a l l s below 4 ng/ml after a rise above 12 ng/ml. Figure 3 : D e f i n i t i o n s of the d i v i s i o n of estrous cycles into periods on the basis of milk progesterone concentrations. - 86 -Table 12 E f f e c t of Breed on Milk Progesterone Concentrations During Periods of the Estrous Cycle Milk Progesterone Concentration (ng/ml) Holstein Ayrshire Period X n X n s.d. Sig n i f i c a n c e 0 1.82 46 1.79 48 0.67 n.s. 1 2.25 314 2.70 231 0.61 n.s. 2 7.09 249 7.07 195 1.86 n.s. 3 24.40 208 25.59 143 9.18 n.s. 4 7.71 78 7.81 55 2.25 n.s. n.s. = Not s i g n i f i c a n t Table 13 Treatment Eff e c t of Treatment on Milk Progesterone Concentrations During Periods of the Estrous Cycle Milk Progesterone Concentration (ng/ml) Period 1 1.59 17 2.06 a 97 7.05 90 21.39 3 62 6.56 f g 28 2 1.66 10 2.26 a b 63 7.11 51 26.27 b 42 6.18f 11 3 1.88 15 2.34 a b 96 6.91 86 23.62 a b 63 7.81 f g h 25 4 1.56 12 2.14 a b 107 7.12 91 27.24 b 78 8.43 g h 34 5 2.12 15 2.37 a b 70 7.10 53 2 4 . l 5 a b 43 8.45 g h 14 6 2.09 10 2.42 b 49 7.32 37 25.62 b 27 9.42 h 12 7 2.06 7 2.37 a b 30 7.14 13 26.97 b 18 7.67 f g h 3 8 1.49 8 2.35 a b 33 7.15 23 26.45 b 18 7.17 f g h 6 s.d. 0.67 0.61 1.86 9.18 2.25 ^ " s u p e r s c r i p t s denoting dif f e r e n t homogenous subsets In same column at p <^0.01. x»ysuperscripts denoting different homogenous subsets In same column at p ^  0.001. f>S.^superscripts denoting d i f f e r e n t homogenous subsets In same column at p <^0.05. - 88 -Breed E f f e c t H o l s t e i n and Ayrshires had s i m i l a r milk progesterone concentrations for periods 0, 1, 2, and 4, as differences were not s i g n i f i c a n t (p >^  0.05). However, mean milk progesterone l e v e l s during period 3 tended to be higher for the Ayrshires than the Holsteins (24.40 and 25.59 ±9.18 ng/ml, r e s p e c t i v e l y ) , although the difference was not s i g n i f i c a n t . Treatment E f f e c t The influence of treatment on milk progesterone concentrations was found to be s i g n i f i c a n t for 3 of the 5 periods tested. Differences between milk progesterone concentrations among treatments was not found to be s i g n i f i c a n t for periods 0 and 2. S i g n i f i c a n t differences i n milk progesterone concentrations among treatments did occur, however, for periods 1, 3, and 4. During period 1, mean milk progesterone concentrations were s i g n i f i c a n t l y greater for cows on treatment group 6 as compared to cows on treatment group 1 (2.42 and 2.06 ± 0.61 ng/ml, respectively) (p <^  0.01). During period 3, cow milk progesterone concentrations on treatment group 1 were s i g n i f i c a n t l y lower than progesterone concentrations recorded for cows on treatments 2, 4, 6, 7, and 8. During period 4, progesterone concentrations recorded for cows on treatment group 2 were s i g n i f i c a n t l y lower than those recorded for cows on treatment groups 4, 5, and 6. Also cows on treatment group 1 had - 89 -lower mean progesterone concentrations than cows on treatment 7. C y c l e Type E f f e c t Table 14 gives mean milk progesterone concentrations and standard deviations recorded for periods 1 - 4 among the d i f f e r e n t cycle types previously described i n Materials and Methods. Cycle type 0 and period 0 were not included i n this analysis as cycle type 0 was comprised of period 0 only. Differences i n milk progesterone concentrations during both periods 2 and 4 were not s i g n i f i c a n t among the d i f f e r e n t cycle types. However, differences between progesterone concentrations during periods 1, and 3 were found to be highly s i g n i f i c a n t (p <^  0.001). During period 1, mean milk progesterone concentrations recorded for cycle type 8 (unconfirmed pregnancy) was s i g n i f i c a n t l y lower than the mean recorded for cycle types 1 (short f i r s t c y c l e ) , 2 (normal f i r s t c y c l e ) , 6 (short cycle) and 10 ( c y s t i c CL) (p £ 0.001). Mean milk progesterone concentrations during period 3 were found to be s i g n i f i c a n t l y lower (p <_ 0.001) for cycle type 1 (short f i r s t cycle) compared to cycle types 7 (confirmed pregnancy), 8 (unconfirmed pregnancy), 9 (abortion) and 10 ( c y s t i c CL). Table 15 and 16 gives means and standard deviations for period length (days) analyzed by breed and treatment, r e s p e c t i v e l y . As in the analysis of milk progesterone concentrations, cycle types 7, 8, 9, 10, 12, and 13 were not included i n the analysis for the same reasons given previously. T a b l e 14 E f f e c t o f T y p e o f C y c l e o n M i l k P r o g e s t e r o n e C o n c e n t r a t i o n D u r i n g P e r i o d s o f t h e E s t r o u s C y c l e M i l k P r o g e s t e r o n e C o n c e n t r a t i o n ( n g / m l )  P e r i o d i e T y p e X n X n X n X n 1 2 . 7 2 b 37 6 . 7 0 5 6 1 9 . 9 3 3 3 7 8 . 7 9 12 2 2 . 7 7 b 13 7 . 7 2 10 2 4 . 4 3 a b 13 8 . 0 0 5 3 2 . 4 5 a b 2 8 . 8 0 2 1 9 . 4 0 a b 2 1 1 . 1 0 2 4 2 . 1 3 a b 2 2 0 7 . 2 4 1 6 9 2 6 . 1 9 b 2 2 0 7 . 6 8 7 5 5 2 . 1 7 a b 3 2 7 . 4 3 2 5 2 5 . 5 2 a b 3 2 6 . 4 0 16 6 2 . 4 8 b 3 5 7 . 5 5 2 2 2 2 . 8 9 a b 3 5 8 . 0 5 6 7 2 . 0 5 a b 4 3 6 . 8 8 34 3 5 . 5 8 b 4 3 - -8 1 . 8 3 a 11 7 . 3 9 10 4 0 . 7 1 b 11 - -9 2 . 3 8 a b 15 7 . 3 6 11 3 0 . 4 0 b 15 8 . 7 0 3 1 0 2 . 6 0 b 31 7 . 4 5 17 4 0 . 5 4 b 3 8 8 . 1 0 3 11 2 . 3 8 a b 17 6 . 1 8 12 2 1 . 6 5 a b 1 2 7 . 0 5 2 12 1 . 9 0 a b 3 2 - - 2 6 . 5 2 a b 3 2 7 . 8 0 2 13 2 . 2 5 a b 8 5 6 . 6 0 7 2 6 . 8 5 a b 3 4 7 . 8 4 7 s . d . 0 . 6 1 1 . 8 6 1 0 . 4 2 7 . 2 5 , b s u p e r s c r i p t s d e n o t i n g d i f f e r e n t h o m o g e n o u s s u b s e t s I n s a m e c o l u m n a t p < 0 . 0 0 1 . - 91 -Table 15 Effect of Breeds on Period Length (Days) Holstein Ayrshire Period X n X n s.d. Significance 0 27.41 46 30.17 48 12.12 n.s. 1 8.14 314 6.76 231 6.98 * 2 4.63 249 4.09 195 3.87 n.s. 3 10.17 208 10.17 143 21.68 n.s. 4 3.21 78 3.91 55 2.89 n.s. •Significant difference at p < 0.05 n.s. = not significant. Table 16 E f f e c t of Treatment on Period Length (Days) Period Length (Days) Period 1 2 3 4 Treatment X n X n X n X n 1 6.81 97 4.32 90 9.57 62 3.57 28 2 6.94 63 3.96 51 9.60 42 . 2.73 11 3 7.60 96 3.95 86 10.02 63 2.76 25 4 8.64 107 4.62 91 9.86 78 2.91 34 5 7.27 70 4.17 53 10.65 43 4.71 14 6 5.74 49 4.43 37 11.41 27 3.92 12 7 8.27 30 4.54 13 12.22 18 7.67 3 8 9.94 33 6.83 23 10.50 18 5.17 6 s .d. 6.98 3.87 21.68 2.89 Significance n.s. n.s. n.s. n.s. n.s. not s i g n i f i c a n t . - 93 -Breed E f f e c t The duration of period 1 was found to be s i g n i f i c a n t l y longer (p £ 0.05) i n Holsteins than i n Ayrshires (8.14 and 6.76 ± 6.98 days, r e s p e c t i v e l y ) . Period 2 tended to be longer i n Holsteins than In Ayrshires as well (4.63 and 4.09 ± 3.87 days, r e s p e c t i v e l y ) , however, s i g n i f i c a n c e was not achieved (p £ 0.05). Duration of periods 0, 3, and 4, were not s i g n i f i c a n t l y d i f f e r e n t among breeds. Treatment E f f e c t Differences a t t r i b u t a b l e to the influence of treatment were found to be non-significant (p >^  0.05) for a l l periods tested. Cycle Type E f f e c t Means and Standard deviations for the d i f f e r e n t cycle types are given i n Table 17. Differences i n period length among a l l cycle types were found to be highly s i g n i f i c a n t (p <^  0.001). The duration of period 1 was found to be s i g n i f i c a n t l y shorter for cycle types 1 (short f i r s t c y c l e ) , 2 (normal f i r s t c y c l e ) , and 6 (short c y c l e ) , and 10 ( c y s t i c CL) in comparison to cycle types 11 ( f o l l i c u l a r c y s t ) , 12 ( f o l l i c u l a r and l u t e a l c y s t ) , and 13 ( a l l other c y c l e s ) . For the duration of period 2, although the difference among cycle types was found to be highly s i g n i f i c a n t , mean separation indicated that only one homogeneous subset existed. This finding indicates that the v a r i a t i o n i n period length was too great to allow for separation among - 94 -Table 17 E f f e c t of Type of Cycle on Period Length (days) Period Cycle Type 1 X n 2 X n 3 X n 4 X n 1 3.43 a 37 4.30 56 5.92 a 37 3.00 a b 12 2 4.31 a 13 3.60 10 12.08 a 13 2.80 a 5 3 4.00 a b 2 2.00 2 3 5 . 0 a b c 2 4.0 a b 2 4 6.71 a b 220 3.52 169 11.05 3 220 2.613 a 133 5 4.31 a b 32 4.28 25 12.06 3 32 5.81 a b 16 6 4.00 a 35 2.73 22 6.20 a 35 3.33 a 6 7 7.02 a b 43 3.94 34 82.72 c 43 — 8 6.73 a b 11 4.80 10 69.64 c 11 — 9 7.20 a b " 15 3.64 11 92.60 C 15 8.00 a b 3 10 5.16 a 31 4.88 17 57.0 b c 38 2.00 a b 3 11 20.76 b 17 5.42 12 7.58 a 12 4.00 a 2 12 19.00 b 4 — 21.50 a b 4 10.50 b 2 , 13 11.47 b 85 7.0 7 12.41 a 34 5.57 a b 7 s .d. 6.98 3.87 28.10 2.89 a » b » c » superscripts denoting d i f f e r e n t homogeneous subsets i n same column at p < 0.05 Note: For period 2, p <_ 0.001; however separation of means was not pos s i b l e . - 95 -means . F o r p e r i o d 3 , c y c l e type 1 ( s h o r t f i r s t c y c l e ) , 2 (norma l f i r s t c y c l e ) , and 4 (norma l c y c l e ) were s i g n i f i c a n t l y (p <_ 0 .001) s h o r t e r than p e r i o d l e n g t h s of c y c l e t y pe s 7 ( c o n f i r m e d p r e g n a n c y ) , 8 ( u n c o n f i r m e d p r e g n a n c y ) , 9 ( a b o r t i o n ) , and 10 ( c y s t i c C L ) . These r e s u l t s a r e not u n e x p e c t e d as p r o l o n g e d p e r i o d s of h i g h p r o g e s t e r o n e l e v e l s a r e c h a r a c t e r i s t i c o f p r e g n a n t and c y s t i c c y c l e s . P e r i o d 4 was found to be s i g n i f i c a n t l y l o n g e r (p <^  0 .001) f o r c y c l e t y p e 12 ( c y s t i c f o l l i c l e and c y s t i c CL) i n c o m p a r i s o n to c y c l e t y p e s 2 ( n o r m a l f i r s t c y c l e ) , 4 ( n o r m a l c y c l e ) , 6 ( s h o r t c y c l e ) , and 11 ( c y s t i c f o l l i c l e ) . A g a i n t h e s e r e s u l t s were e x p e c t e d as c y s t i c o v a r i e s i n the CL s t a g e would not r e g r e s s n o r m a l l y r e s u l t i n g i n i n c r a s e d p r o g e s t e r o n e c o n c e n t r a t i o n s . Frequency of Cycle Types The f r e q u e n c y (%) of the v a r i o u s c y c l e t ypes o b s e r v e d among t r e a t m e n t s i s g i v e n i n Append ix 3 . A X^ t e s t c r i t e r i o n was used to t e s t f o r i ndependence o f v a r i a b l e s . Based on the r e s u l t s (p _> 0 . 0 5 ) t h e r e was not e v i d e n c e to i n d i c a t e dependence of v a r i a b l e s . Cycle Lengths C o m p a r i s o n s o f c y c l e l e n g t h s between b r e e d s and t r e a t m e n t s a r e g i v e n i n T a b l e s 18 and 19, r e s p e c t i v e l y . C y c l e t y p e s o m i t t e d from the a n a l y s i s i n c l u d e c y c l e s w i t h p r e g n a n c i e s ( c o n f i r m e d and p r e d i c t e d ) , c y c l e s w i t h l u t e a l c y s t s , s h o r t f i r s t c y c l e s , and o t h e r abnormal Table 18 E f f e c t of Breed on Cycle Length Holstein Ayrshire X n X n s.d. Sig n i f i c a n c e Cycle Length 20.91 126 21.33 79 3.60 n.s. n.s. = not s i g n i f i c a n t . Table 19 E f f e c t of Treatment on Cycle Length Treatment 1 2 3 4 5 6 7 8 s . d . Significance X X X X X X X X Cycle Length 21.53 19.52 20.84 21.98 21.48 19.36 21.25 21.14 3.60 n.s. n 38 27 37 51 23 14 8 7 n.s. = not s i g n i f i c a n t - 98 -cy c l e s . Estrous cycles longer than 30 days were also omitted as such cycles were not perceived as being c h a r a c t e r i s t i c of normally c y c l i n g cows, and could introduce unexplained v a r i a t i o n i n the analysis of variance. Breed did not influence cycle length (p >^  0.05). The mean cycle length for the Holstein breed was 20.91 ± 3.60 days, and was si m i l a r for the Ayrshire breed (21.33 ± 3.60 days). S I m i l a r i l y , differences i n cycle length among treatments were not found to be s i g n i f i c a n t l y d i f f e r e n t (p _> 0.05) . Mean cycle lengths analyzed by both breed and treatments were within the range of 19 - 23 days considered to be the duration of a normal estrous cycle i n cows. Plasma Selenium Levels and Milk Progesterone Concentrations at Breeding  Time Plasma Se le v e l s and milk progesterone concentrations at time of breeding were compared between breeds, treatments, and successful vs unsuccessful breedings. Treatments 1, 2, 3, and 4 i n the f i r s t l a c t a t i o n were repeated for a second l a c t a t i o n and designated as treatments 5, 6, 7, and 8, r e s p e c t i v e l y . This was done because a t r e a t -ment by successful/unsuccesful breeding i n t e r a c t i o n for the progesterone data was found to exi s t when the data was handled i n th i s manner. Breed E f f e c t Table 20 gives plasma Se le v e l s and milk progesterone concentra-- 99 -Table 20 E f f e c t of Breed on Plasma Selenium and Milk Progesterone Concentrations at Time of Breeding Holstein Ayrshire T r a i t at Breeding X n s.d. Significance Plasma Se (mg/kg) 0.103 84 0.105 56 0.023 n.s. Milk Progesterone 2.00 . 100 (ng/ml) 1.76 62 8.81 n.s. = Not s i g n i f i c a n t * = S i g n i f i c a n t difference at p < 0.05, - 100 -tions at breeding time for breeds. The plasma Se le v e l s for Holsteins and Ayrshires were 0.103 and 0.105 ± 0.023 mg/kg, r e s p e c t i v e l y . Although s i g n i f i c a n c e for the difference between plasma Se l e v e l s among breeds was approached, It was not achieved (p = 0.163). The difference in milk progesterone concentrations among the two breeds, however, was found to be s i g n i f i c a n t (p £ 0 . 0 5 ) . The Ayrshire cows tended to have lower milk progesterone concentrations at breeding time than did the Holstein cows (1.76 and 2.0 ± 8.81 ng/ml). Treatment E f f e c t Mean plasma Se le v e l s at breeding and standard deviations are l i s t e d i n Table 21. Analysis of variance indicated that the difference between treatment groups for plasma Se l e v e l s at breeding time was highly s i g n i f i c a n t (p <_0.01). Mean plasma Se l e v e l s for cows on t r e a t -ment group 1 were s i g n i f i c a n t l y lower than cows on treatment group 4 (0.091 and 0.120 ± 0.023 mg/kg). S i m i l a r i l y , the difference i n mean milk progesterone concentra-tions were s i g n i f i c a n t l y d i f f e r e n t (p <_ 0.05) between treatment groups (Table 21). The mean milk progesterone concentration for control cows was s i g n i f i c a n t l y lower than the mean milk progesterone concentration recorded for cows on treatment group 8 (1.53 and 2.73 ± 8.81 ng/ml). E f f e c t of Plasma Selenium and Milk Progesterone Concentrations on  Conception Table 22 gives the means and standard deviations of plasma Se - 101 -Table 21 E f f e c t of Treatment on Plasma Se (mg/kg) and Milk Progesterone (ng/ml) at Breeding Plasma Se Level (kg/mg) Milk Progesterone Concentration (ng/ml) Treatment X n X n 1 0.091 3 28 1.53 a 36 2 0.100 b a 17 1.89 a b 21 3 0.106 b a 22 7.99 a b 32 4 0.120 b 25 1.98 a b 3k0 5 0.101 b a 20 1.91 a b 18 6 0.101 b a 10 1.78 a b 10 7 0.096 b a 8 2.33 a b 6 8 0.116 b a 10 2.73 b 9 s.d. 0.023 8.81 a » D superscripts denoting d i f f e r e n t homogeneous subsets i n the same column at p £ 0.05. - 102 -Table 22 E f f e c t of Plasma Selenium and Milk Progesterone Concentrations on Success of Breeding Unsuccessful Breeding Successful Breeding T r a i t at Breeding X n X n s.d. Significance Plasma Se (mg/kg) 0.106 72 0.102 68 0.022 n.s. Milk Progesterone (ng/ml) 1.86 85 1.96 77 8.81 n.s. n.s. = not s i g n i f i c a n t - 103 -l e v e l s and milk progesterone concentrations at time of breeding for successful and unsuccessful breedings. Plasma Se le v e l s at breeding time for successful breedings was not d i f f e r e n t (p >_ 0.05) from those for unsuccessful breedings (0.102 and 0.106 ± 0.022 mg/kg, r e s p e c t i v e l y ) . Neither was there a difference i n milk progesterone concentrations at breeding time between successful and unsuccessful breedings (1.96 and 1.86 ± 8.81 ng/ml, r e s p e c t i v e l y ) . S i g n i f i c a n t interactions w i l l be discussed below. Interactions A s i g n i f i c a n t i n t e r a c t i o n was found to exi s t when an analysis of treatment group by breeding success (+/-) was used. Table 23 gives means and standard deviations of milk progesterone concentrations for the group by (un)successful breeding i n t e r a c t i o n . Within the successful breeding group, treatment group did not influence milk progesterone concentrations. However, i n the unsuccessful breeding group, milk progesterone concentrations were s i g n i f i c a n t l y higher (p <^  0.05) for cows on treatment group 8 than for those cows on treatment groups 1 - 6 . It was observed that generally milk progesterone concentrations at time of breeding were s l i g h t l y lower i n the unsuccessful breeding group than i n the successful breeding group, though differences were not s i g n i f i c a n t with one exception. Cows on treatment group 8 had s i g n i f i c a n t l y higher mean milk progesterone concentrations i n the unsuccessful breeding group than cows i n the successful breeding group (3.78 and 1.90 ± 8.81 ng/ml, r e s p e c t i v e l y ) . - 104 -Table 23 Milk Progesterone Concentrations for Treatment by (Un)successful Breeding Interaction Unsuccessful Breeding Successful Breeding Treatment X n X n 1 1.54a 20 1.53 a 16 2 1.96a 9 1.84a 12 3 1.74a 17 2.28 a b 15 4 1.97 a 21 2.01 a b 9 5 1.71 a 7 2.03 a b 11 6 1.46a 5 2.10 a b 5 7 2.10 a b 2 2.45 a b 4 8 3.78 b 4 1.90 a 5 s.d. • 8.81 a>b superscripts denoting d i f f e r e n t homogeneous subsets at p _< 0.05 - 105 -Also, mean milk progesterone concentrations tended to increase from the f i r s t l a c t a t i o n (treatments 1 - 4) to the subsequent l a c t a t i o n (treatments 5 - 8 ) . The only s i g n i f i c a n t increase i n milk progesterone concentrations between l a c t a t i o n s occurred in the unsuccessful breeding group; milk progesterone concentrations increase from 1.97 ± 8.81 ng/ml for cows on treatment group 4 ( l a c t a t i o n 1) to 3.78 ± 8.81 ng/ml i n group 8 ( l a c t a t i o n 2). Plasma LH T r a i t s Figure 4 gives a diagramatic representation of milk progesterone concentrations i n r e l a t i o n to plasma LH concentrations. LH lag time was defined as the time i n t e r v a l between the I n i t i a l pre-estrus drop i n milk progesterone concentration to the i n i t i a l r i s e In plasma LH concentrations of the preovulatory LH surge. Basal plasma LH was defined as base l e v e l LH concentrations exclusive of the preovulatory LH surge and p u l s a t i l e LH surges. Plasma LH concentrations below 1.5 ng/ml were included i n basal plasma LH computations. Plasma LH l e v e l s above 1.5 ng/ml and below 5 ng/ml were considered to be i n d i c a t i v e of p u l s a t i l e LH surges. Plasma LH l e v e l s r i s i n g above 5ng/ml were i n d i c a t i v e of the preovulatory LH surge. Breed E f f e c t The plasma LH lag time (days) for Holsteins and Ayrshires was 3.49 and 2.13 ± 2.07 ng/ml (Table 24). Significance between breed di f f e r e n c e s was approached (p = 0.127) but was not achieved. -106-DAYS OF THE ESTROUS CYCLE F i g r u e 4 : C y c l i c a l m i l k progesterone a c t i v i t y (ng/ml) and i t s r e l a t i o n s h i p t o plasma LH (ng/ml) a t e s t r u s . - 107 -Table 24 E f f e c t of Breed on Plasma LH Lag Time Basal LH Levels (ng/ml) (days) and Holstein Ayrshire Plasma LH T r a i t X n X n s.d. Significance LH Lag Time (days) 3.49 9 2.13 6 2.07 n.s. Basal LH (ng/ml) 0.78 55 0.64 62 0.49 * • S i g n i f i c a n t difference at p <_ 0.05. n.s = not s i g n i f i c a n t - 108 -As well as having a greater plasma LH lag time, the Holstein breed had s i g n i f i c a n t l y higher plasma basal LH concentrations than the Ayrshire breed (0.78 and 0.64 ± 0.49 ng/ml, r e s p e c t i v e l y ) . Treatment E f f e c t The differences i n mean plasma LH lag time between cows on treatment groups 1 - 3 were not s i g n i f i c a n t (p >^  0.05). The mean plasma LH lag time of cows on treatment groups 2 and 3 were 2.62 and 2.50 ± 2 . 7 days, re s p e c t i v e l y , whereas the LH lag time for the control group was 3.72 ± 2.07 days. Although the differences between groups were not s i g n i f i c a n t , i t was observed that the mean LH lag time for cows in the control group was one f u l l day longer than cows i n either of the two other groups (Table 25). There was also no difference among treatments for basal LH le v e l s recorded. Cows on treatment groups 1, 2, and 3 had mean basal LH l e v e l s of 0.68, 0.67, and 0.77 ± 0.49 ng/ml, r e s p e c t i v e l y . The peak plasma LH concentrations recorded during the preovulatory surge was found to be s i g n i f i c a n t l y d i f f e r e n t (P £ 0.05) among treatment groups (Table 25). Cows in the control group had considerably higher mean plasma peak LH concentrations during the preovulatory LH surge than cows in either group 2 or group 3 (50.93, 23.05, and 14.70 ± 13.09 ng/ml, resp e c t i v e l y ) . Interactions A breed by treatment i n t e r a c t i o n was found to be highly - 109 -T a b l e 25 E f f e c t o f T rea tment on LH Lag Time ( d a y s ) , B a s a l LH L e v e l s (ng/ml) and Peak LH D u r i n g the P r e - o v u l a t o r y LH Surge (ng/ml) T r e a t m e n t 1 2 3 X n X n X n s . d . LH l a g t ime ( d a y s ) 3 .72 5 2.62 5 2.50 5 2.07 B a s a l LH (ng/ml) 0 .68 46 0 .67 29 0 .77 42 0 .49 Peak LH d u r i n g p r e - o v u l a t o r y s u r g e (ng/ml) 5 0 . 9 3 3 3 2 3 . 0 5 b 4 1 4 . 7 0 b 2 13.09 a » D s u p e r s c r i p t s d e n o t i n g d i f f e r e n t homogeneous s u b s e t s i n same row at p < 0 . 0 5 . - 110 -s i g n i f i c a n t (p <_ 0.001) for basal plasma LH concentrations. Table 26 gives mean plasma basal LH l e v e l s classed by treatment group and breed. The s i g n i f i c a n t i n t e r a c t i o n indicates that the two d i f f e r e n t breeds reacted d i f f e r e n t l y to the ap p l i c a t i o n of the d i f f e r e n t treatments. In both treatment groups 1 and 3, the Holstein breed had lower mean basal LH l e v e l s than the Ayrshire breed. However, the s i t u a t i o n i s reversed for treatment group 2, with the Holstein breed having a higher basal plasma LH concentration than the Ayrshire breed. Uterine and Ovarian T r a i t s  Breed E f f e c t Table 27 gives the incidence (%) of uterine and ovarian abnormalities among breeds for l a c t a t i o n 1 (treatments 1 - 4 ) and l a c t a t i o n 2 (treatments 5 - 8 ) . Data for the incidence of m e t r i t i s , v a g i n i t i s , and retained p l a c e n t a 1 among breeds i s also given i n Table 27. Differences between breeds were found to be non-significant (p >_ 0.05) for a l l ovarian and uterine t r a i t s l i s t e d i n Table 27 over both l a c t a t i o n s . S i m i l a r i l y , differences i n the incidence of m e t r i t i s , v a g i n i t i s , and retained placenta observed ( f o r both la c t a t i o n s ) was not s i g n i f i c a n t l y d i f f e r e n t among breeds. For both sets of data, a Yates corrected Chi-square analysis (employing an ar c - s i n transformation) was used, as sample sizes were greater than 21. Retained placenta for th i s study was defined as a f a i l u r e of the f e t a l placenta to separate from the maternal placentome within 24 hours of p a r t u r i t i o n . - I l l -Table 26 Treatment by Breed E f f e c t on Basal LH Levels (ng/ml) Breed Holstein Ayrshire Treatment X n X n s.d. 1 0.58 a , b 16 0.73 b 30 2 1.31 C 11 0.28 a 18 0.49 3 0.69 b 28 0.93 b c 14 a,b,c = superscripts denoting d i f f e r e n t homogeneous subsets at p <_0.05. - 112 -Table 27 The Incidence of Ovarian, Vaginal, and Uterine Abnormalities and Retained Placenta Lactation 1 Lactation 2 T r a i t Holsteins Ayrshires S i g . 1 Holsteins Ayrshire S i g . Normal uterus Inactive Ovaries (%) 0.0 2.9 n.s. 0.0 4.3 n.s. Abnormal Ovaries <%) 5.4 2.9 n.s. 5.0 8.7 n.s. Abnormal Uterus Normal Ovaries (%) 10.8 8.8 n.s. 15.0 8.7 n.s. Cysti c F o l l i c l e (%) - - 5.0 4.3 n.s. Cystic CL (%) - - 5.0 0.0 n.s. Abnormal Uterus Inactive Ovaries (%) _ — 0.0 4.3 n.s. n 37 34 20 23 M e t r i t i s (%) 0.0 5.9 n.s. 9.5 4.3 n.s. V a g i n i t i s (%) - - 4.8 0.0 n.s. Retained Placenta (%) - - 4.8 8.7 n.s. n 37 34 21 23 S i g . = Sig n i f i c a n c e - 113 -Treatment E f f e c t The observed incidence (%) of uterine and ovarian abnormalities for treatments 1 - 4 ( l a c t a t i o n 1) and 5 - 8 ( l a c t a t i o n 2) are given i n Table 28. Data for the incidence of m e t r i t i s , v a g i n i t i s , and retained placenta for treatments i s also given i n Table 18. None of the comparisons were found to be s i g n i f i c a n t l y d i f f e r e n t (p >^  0.05) between treatment groups (as determined by a Fisher's Chi-square test c r i t e r i o n ) probably due to the small sample size of some treatment groups used i n the a n a l y s i s . Although s i g n i f i c a n c e between treatments was not achieved f o r any of the t r a i t s l i s t e d i n Table 28, some trends are apparent. In the second l a c t a t i o n , the incidence of abnormal uterus (with normal ovaries) i n treatment groups 5, 6, 7, and 8 was 18.8%, 10.0%, 14.3%, and 0.0%, r e s p e c t i v e l y , whereas the Incidence of retained placenta was 22.4%, 11.1%, 0.0%, and 0.0%, r e s p e c t i v e l y . In both cases, the greatest incidence of uterine problems occurred i n the control group (Figure 5). Calving Ease T r a i t s Ease of calving was categorized as one of the following: 1) normal presentation, no assistance required, 2) normal presentation, easy p u l l required, 3) normal presentation, hard p u l l required, 4) mal presentation, easy p u l l required, 5) mal presentation, hard p u l l required, and 6) unobserved. T a b l e 2 8 E f f e c t o f T r e a t m e n t o n t h e O b s e r v e d I n c i d e n c e o f R e p r o d u c t i v e H e a l t h T r a i t s T r e a t m e n t T r a i t ( Z ) ( l a c t a t i o n 1 ) ( l a c t a t i o n 2 ) 4 S i g n i f i c a n c e 5 8 S i g n i f i c a n c e I n a c t i v e 0 . 0 O v a r i e s A b n o r m a l 0 . 0 O v a r i e s A b n o r m a l 5 . 3 U t e r u s C y s t i c 0 . 0 F o l l l c e s C y s t i c C L 0 . 0 A b n o r m a l U t e r u s 0 . 0 ( w i t h I n a c t i v e o v a r i e s ) M e t r i t i s 0 . 0 V a g i n i t i s 0 . 0 R e t a i n e d P l a c e n t a n 19 0 . 0 0 . 0 0 . 0 1 1 . 1 5 . 6 n . s . 0 . 0 0 . 0 0 . 0 0 . 0 6 . 3 0 . 0 0 . 0 5 . 6 n . s . 1 2 . 5 1 0 . 0 0 . 0 0 . 0 5 . 6 2 2 . 2 n . s . 0 . 0 n . s . 0 . 0 0 . 0 0 . 0 n . s . 0 . 0 0 . 0 0 . 0 n . a . 0 . 0 0 . 0 1 1 . 1 n . s . 0 . 0 0 . 0 0 . 0 n . s . 1 8 . 8 1 0 . 0 1 4 . 3 0 . 0 16 18 18 5 . 6 0 . 0 2 2 . 4 18 0 . 0 2 8 . 6 0 . 0 0 . 0 1 1 . 1 1 4 . 3 0 . 0 0 . 0 7 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 1 0 . 0 6 . 3 0 . 0 0 . 0 0 . 0 1 0 . 0 1 1 . 1 0 . 0 10 n . s . n . s . n . s . n . s . n . s . n . s . n . s . n . s . n . s . " n o t s i g n i f i c a n t -115-F i g u r e 5: I n c i d e n c e (%) o f r e t a i n e d p l a c e n t a and a b n o r m a l u t e r i i ( w i t h n o r m a l o v a r i e s ) f o r t r e a t m e n t g r o u p s d u r i n g t h e s e c o n d l a c t a t i o n . - 116 -Only calves whose dams had completed one f u l l l a c t a t i o n on treatment were included in the a n a l y s i s . Breed and Treatment E f f e c t Each calving was designated to one of the categories of calving ease discussed above. The calving ease data c l a s s i f i e d by breed and ease of calving i s given i n Table 29. Based on a Yates' corrected chi-square test c r i t e r i o n , calving ease was independent (p >_ 0.05) of breed for the various calving ease categories tested. The incidence of c a l f m o r tality was also found to be independent of breed (Table 29). Table 30 gives the observed incidence of calving ease categories c l a s s i f i e d by treatment. As with breeds, calving ease was found to be independent of treatment group. S i m i l a r i l y , c a l f mortality was Independent of treatment group (Table 30). Health Problems Breed E f f e c t Table 31 gives the incidence of health problems (other than reproductive problems) c l a s s i f i e d by breed. M a s t i t i s appeared to be more prevalent i n the Holstein breed than i n the Ayrshire breed, though not s i g n i f i c a n t l y so. During the second l a c t a t i o n , the incidence of m a s t i t i s for the Holsteins was 38.1% whereas the incidence was less than half for the Ayrshire breed (13.0%). Although s i g n i f i c a n c e for dependence between ma s t i t i s and treatment group was approached, i t was not achieved (p = 0.117). In view of the r e l a t i v e l y high proportion of - 117 -Table 29 The E f f e c t of Breed on Ease of Calving and Calf M o r t a l i t y Calving T r a i t Holstein Ayrshire Significance Normal Presentation (no assistance) (%) 38.1 31.8 n.s. Normal Presentation (easy p u l l ) (%) 19.0 22.7 n.s. Normal Presentation (hard p u l l ) (%) 14.3 0.0 n.s. Mall Presentation (easy p u l l ) (%) 4.8 4.5 n.s. Mall Presentation (hard p u l l ) (%) 4.8 0.0 n.s. Unobserved (%) 19.0 41.0 n.s. Calf M o r t a l i t y (%) 4.8 4.5 n.s. n 21 22 n.s. = not s i g n i f i c a n t - 118 -T a b l e 3 0 E f f e c t o f T r e a t m e n t o n E a s e o f C a l v i n g T r e a t m e n t ( 2 n d l a c t a t i o n o n l y ) T r a i t (%) 1 2 3 4 S i g n i f i c a n c e N o r m a l P r e s e n t a t i o n ( n o a s s i s t a n c e ) 5 0 . 0 3 0 . 0 0 . 0 4 0 . 0 n . s . N o r m a l P r e s e n t a t i o n ( e a s y p u l l ) 6 . 3 2 0 . 0 4 2 . 9 3 0 . 0 n . s . N o r m a l P r e s e n t a t i o n ( h a r d p u l l ) 1 2 . 5 0 . 0 1 4 . 3 0 . 0 n . s . M A L p r e s e n t a t i o n ( e a s y p u l l ) 6 . 3 1 0 . 0 0 . 0 0 . 0 n . s . M A L p r e s e n t a t i o n ( h a r d p u l l ) 0 . 0 0 . 0 1 4 . 3 3 0 . 0 n . s . U n o b s e r v e d 2 5 . 0 4 0 . 0 2 8 . 6 0 . 0 C a l f m o r t a l i t y 6 . 0 0 . 0 1 4 . 3 1 0 . 0 n . s . n 16 1 0 7 10 n . s . •=• n o t s i g n i f i c a n t - 119 -Table 31 E f f e c t of Breed on Health T r a i t s Lactation 1 Lactation 2 Holsteins Ayrshires S i g . 1 Holsteins Ayrshires S i g . M a s t i t i s (%) 18.9 8.9 n.s. 38.1 13.0 n.s. Milk Fever (%) 0.0 11.8 n.s. 0.0 4.3 n.s. Displaced Abomasum (%) 5.4 0.0 n.s. - -Injury Feet and Legs (%) 2.7 2.9 n.s. - -Foot Rot (%) 5.4 2.9 n.s. - -n 37 34 21 23 Sig. = S i g n i f i c a n c e - 120 -m a s t i t i s observed i n the herd, an analysis of variance on somatic c e l l data was performed. Table 32 gives the means and standard deviations of somatic c e l l data analyzed among breeds. The somatic c e l l count of Holsteins and Ayrshires was 219.40 and 165.50 ± 275.19 Ct/1000, r e s p e c t i v e l y . Although s i g n i f i c a n c e was approached, i t was not achieved (p = 0.07). The incidence of displaced abomasum, injury to feet and legs, and foot rot were a l l s t a t i s t i c a l l y independent of breed in the f i r s t l a c t a t i o n (none of the health problems occurred during the second l a c t a t i o n ) . S i m i l a r i l y , the incidence of milk fever was found to be independent of breed (Table 31). Treatment E f f e c t The incidence of health problems other than reproductive problems analyzed by treatment group over two l a c t a t i o n s are given in Table 33. During the f i r s t l a c t a t i o n , the incidence of m a s t i t i s was 5.3%, 12.5%, 33.3%, and 5.6% i n treatment groups 1 to 4, r e s p e c t i v e l y , and was s i g n i f i c a n t at p £ 0 . 0 5 . In the second l a c t a t i o n , however, the incidence of mas t i t i s was independent of treatment group. It was observed that the incidence of mas t i t i s increased dramatically from the f i r s t l a c t a t i o n to the next for treatment groups 1, 2, and 4 (16.7%, 33.3%, and 40.0%, r e s p e c t i v e l y ) . However, for treatment group 3, the incidence of mastitis i n the second l a c t a t i o n a c t u a l l y decreased from 33.3% to 14.3%. In view of the high proportion of m a s t i t i s observed over both l a c t a t i o n s , an analysis of variance was performed on somatic - 121 -Table 32 E f f e c t of Breed on Somatic C e l l Count Holstein Ayrshire X n X n s.d. Significance Somatic C e l l 219.4 124 165.53 132 275.19 n.s. Count (xlOOO) n.s. = not s i g n i f i c a n t . Table 33 Eff e c t of Treatment on the Observed Incidence of Health Problems Treatment ( l a c t a t i o n 1) ( l a c t a t i o n 2) T r a i t (%) 1 2 3 4 Significance 5 6 7 8 Sign i f i c a n c e M a s t i t i s 5.3 12.5 33.3 5.6 * 16.7 33.3 14.3 40.0 n.s. Milk, fever 10.5 0.0 5.6 5.6 n.s. 0.0 11.1 0.0 0.0 n.s. Displaced Abomasum 0.0 0.0 5.6 5.6 n.s. - - - - -Injury to Feet and Legs 5.3 6.3 0.0 0.0 n.s. - - - - -Foot rot 5.3 6.3 0.0 11.1 n.s. - - - - -n 19 16 18 18 18 9 7 10 n.s. = not s i g n i f i c a n t * = s i g n i f i c a n t at p £ 0.05 - 123 -c e l l count data for treatments 1 to 8 (Table 34). Mean somatic c e l l counts were not s i g n i f i c a n t l y d i f f e r e n t among treatment groups, nor were mean c e l l counts abnormally high, which i s not consistent with the incidence of m a s t i t i s observed. The incidence of milk fever, displaced abomasum, injury to feet and legs, and foot rot was not dependent on treatment (p >^  0.05). Correlations and Regressions Co r r e l a t i o n s Between Dam and C a l f plasma Selenium Levels and  Calving T r a i t s Dam plasma Se at calving was correlated with c a l f weights at b i r t h and with ease of calving category for treatments 1, 2, 3, and 4 and over a l l treatment groups ( i e , treatments one to four, c o l l e c t i v e l y ) (Table 35). Dam plasma selenium was not found to be s i g n i f i c a n t l y correlated to c a l f b i r t h weight (p >^  0.05) among treatments or over a l l treatment groups. However, the c o r r e l a t i o n (r = 0.6626, n = 8) between dam plasma Se and c a l f weight for group 2 did approach s i g n i f i c a n c e with p = 0.073. This finding would suggest that with increasing dam plasma selenium, c a l f b i r t h weights increase as w e l l , which may be i n d i c a t i v e of improved c a l f health. Regression equations for dam plasma selenium versus c a l f b i r t h weight are given i n Appendix 4. An equality of slope test with dam plasma selenium as the continuous independent v a r i a b l e Indicated s i m i l a r i t y of slopes and equations (p _> 0.05) between regression equations for treatments 1 to 4. The common regression equation was determined to be Y = 37.44 ± 0.014X. Table 34 E f f e c t on Treatment of Somatic C e l l Count Treatment 1 2 3 4 5 6 7 8 X X X X X X X X s.d. Significance Somatic C e l l 243.1 112.8 210.6 164.2 224.7 150.6 58.67 305.0 275.19 n.s. Count (x 1000) n 56 31 50 56 30 " 25 3 5 n.s. = not s i g n i f i c a n t Table 35 Correlations Between Dam Plasma Selenium and Calf Birth Weight and Ease of Calving Dam Plasma Selenium Treatment 1 2 3 4 Overall r r r r r Calf Weight 0.1372 0.6626 -0.4049 0.0410 0.0612 n 11 8 6 9 Ease of Calving -0.6164* -0.0730 0.0936 -0.2362 -0.3090 n 12 8 6 10 36 * s i g n i f i c a n t at p < 0.05 - 126 -For regression analysis, ease of calving categories were assigned increasing d i s c r e t e numerical values with increasing d i f f i c u l t y of c a l v i n g . Dam plasma Se was correlated to calving ease for treatments 1, 2, 3, and 4, and over a l l treatments combined. The c o r r e l a t i o n c o e f f i c i e n t (r = -.3090, n = 36) for dam plasma Se versus calving ease over a l l treatments approached s i g n i f i c a n c e with p = 0.067. When broken down into treatment groups 1 - 4 , the c o r r e l a t i o n (r = -0.6164, n = 12) between dam plasma Se and ease of calving was found to be s i g n i f i c a n t (p £ 0.05) for treatment group 1 only. The c o r r e l a t i o n c o e f f i c i e n t s for treatment groups 2, 3, and 4 were not s i g n i f i c a n t . This f i n d i n g indicates that i n the control group, as dam plasma Se le v e l s increased, calving d i f f i c u l t i e s decreased. Equality of slopes test indicated common slopes and regression equations (p >^  0.05) for treatment groups 1 - 4 (Appendix 4). The common regression equation was Y = 16.07 ± 0.1074X. Table 36 gives c o r r e l a t i o n c o e f f i c i e n t s between c a l f plasma Se l e v e l s and c a l f b i r t h weight for treatments 1 - 4 and over a l l treatments i n c l u s i v e l y are given. Calf plasma Se and c a l f b i r t h weights were not s i g n i f i c a n t l y correlated for the four treatment groups or over a l l treatments c o l l e c t i v e l y . S i m i l a r i l y , slopes and regression equations for treatment groups were not d i f f e r e n t . The common equation was determined to be Y = 42.3 ± 0.08X. Table 36 Correlation Between Calf Plasma Se and Calf B i r t h Weight Calf Se (mg/kg) Treatment 1 2 3 4 Overall r r r r r Calf Weight 0.1226 -0.4379 -0.8218 -0.4191 -0.316 (kg) n 9 7 3 5 24 - 128 -Correlations Between Plasma LH Data , Milk Progesterone Peak and Plasma  Selenium Concentrations Table 37 gives c o r r e l a t i o n c o e f f i c i e n t s and si g n i f i c a n c e between peak milk progesterone concentrations (ng/ml) during the estrous cycle and basal plasma LH (ng/ml) and plasma LH lag time (days). Peak milk progesterone concentration was not s i g n i f i c a n t l y correlated to either basal plasma LH or LH lag time ( f o r treatments 1 - 3 and over a l l treatments taken c o l l e c t i v e l y ) . Regression equations for peak progesterone (independent variable) versus basal plasma LH l e v e l s and LH lag time are given i n Appendix 5. Equality of slope tests indicate that slopes and regression equations are common for peak progesterone concentrations regressed against both basal plasma LH and LH lag time among treatment groups; Common equations for basal plasma LH and LH lag time regressed against peak progesterone concentrations were Y = 0.8042 ± 0.1189 x 10 _ 3X and Y = 2.773 + o.4811 x 10 - 2X, r e s p e c t i v e l y . C o r r e l a t i o n s Between Cow Plasma Selenium, Basal Plasma LH Levels, and LH Lag Time Co r r e l a t i o n c o e f f i c i e n t s and si g n i f i c a n c e for cow plasma Se correlated with basal plasma LH l e v e l s and LH lag time are given i n Table 38. Co r r e l a t i o n c o e f f i c i e n t s between cow plasma Se and LH lag time were high for treatment groups 1, 2, and 3 (r = 0.7572, -0.5004, and 0.4104, r e s p e c t i v e l y ) , however, s i g n i f i c a n c e was not achieved (p >_ 0.05). Significance may have been achieved had sample sizes been l a r g e r . Cow plasma Se l e v e l s and LH lag time over a l l treatments taken Table 37 Correlations Between Peak Milk Progesterone Concentration During the Estrus Cycle and LH Lag Time (days) and Basal Plasma LH (ng/ml) Milk Progesterone Peak (mg/ml) Treatment 1 2 3 Overall r r r r Basal LH ng/ml -0.1918 0.1692 -0.1002 0.0043 n 5 5 7 16 LH lag time (days) -0.5264 0.6180 -0.0825 0.0351 n 5 4 5 15 Table 38 Correlations Between Cow Plasma Selenium and LH Lag Time and Basal Plasma LH Cow Plasma Selenium (mg/kg) Treatment 1 2 3 Overall r r r r Basal plasma LH (ng/ml) -0.6156 -0.0734 -0.7904 -0.438 n 5 4 6 LH lag time (days) 0.7572 -0.5004 0.4104 0.195 n 4 5 5 - 131 -c o l l e c t i v e l y were not s i g n i f i c a n t l y correlated (p = 0.05). Also, cow plasma Se was not s i g n i f i c a n t l y correlated with basal plasma LH l e v e l s over a l l treatments i n c l u s i v e l y , although s i g n i f i c a n c e was approached (p = 0.102). Correlation c o e f f i c i e n t s for treatment groups 1, 2, and 3 were -0.6156, -0.0734, and -0.7904, resp e c t i v e l y and were not s i g n i f i c a n t . However, the c o r r e l a t i o n between cow plasma Se and basal plasma LH l e v e l s for treatment group 3 did approach s i g n i f i c a n c e (p = 0.061). This c o r r e l a t i o n indicates that as cow plasma Se increased, basal plasma LH l e v e l s decreased. Regression equations between cow plasma Se and both basal plasma LH l e v e l s and LH lag time for treatments 1 - 3 are given i n Appendix 5. Equality of slope tests indicated common slopes and equations (p >_ 0.05) for cow plasma Se regressed with basal plasma LH and regressed with LH lag time. Common equations were Y = 1.176 - 0.819 x 10"4X and Y = 1.31 ± 0.012X, r e s p e c t i v e l y . C o r r e l a t i o n s Between Plasma Se and Milk Progesterone Concentrations at Breeding Cor r e l a t i o n c o e f f i c i e n t s and si g n i f i c a n c e for plasma Se lev e l s (mg/kg) correlated with milk progesterone concentrations (ng/ml) at time of breeding (for treatments 1 - 4 and for a l l treatments taken c o l l e c t i v e l y ) are given i n Table 39. The c o r r e l a t i o n between plasma Se and milk progesterone for treatment group 2 (r = -0.5706, n = 15) was the only s i g n i f i c a n t c o r r e l a t i o n observed. The negative c o r r e l a t i o n suggests that within this treatment group, as plasma Se l e v e l s increased, milk progesterone le v e l s decreased. - 132 -Table 39 Correlations Between Milk Progesterone Levels (ng/ml) and Plasma Selenium Levels (mg/kg) at time of Breeding Treatment Milk Progesterone (ng/ml) r n Significance 1 -0.1699 28 n.s. 2 -0.5706* 15 . 0.03 3 -0.1262 22 n.s. 4 0.0801 22 n.s. 5 0.0943 18 n.s. 6 -0.0217 9 n.s. 7 -0.0439 6 n.s. 8 0.4286 9 n.s. (1-8 i n c l u s i v e ) 0.0251 129 n.s. *p < 0.05 n.s. = not s i g n i f i c a n t - 133 -Table 40 gives c o r r e l a t i o n c o e f f i c i e n t s and si g n i f i c a n c e for plasma Se correlated with milk progesterone concentrations c l a s s i f i e d by treatment group and (un)successful breeding group. In the unsuccessful breeding group, plasma Se was s i g n i f i c a n t l y correlated (p £ 0.05) to milk progesterone concentrations for treatment group 2 (r = -0.7332, n = 8) and treatment group 8 (r = 0.9967, n = 4) only. For treatment group 2, plasma Se was negatively correlated to milk progesterone concentration, whereas i n treatment group 8 the reverse s i t u a t i o n ( i . e . , p o s i t i v e c o r r e l a t i o n between variables) was observed. This indicates that for treatment group 2, as plasma Se l e v e l s increased, milk progesterone concentrations decreased; for treatment group 8, as plasma Se l e v e l s increased, milk progesterone concentrations increased. In the successful breeding group, plasma Se was not found to be correlated to milk progesterone concentrations (p >^  0.05). Regression equations for plasma Se le v e l s vs milk progesterone concentrations analyzed by treatment group and treatment by (un)successful breeding group are given i n Appendix 6. Equality of slope test indicated that slopes and equations were common between treatment groups and between successful and unsuccessful breedings for treatments. The common regression equation for plasma Se le v e l s versus milk progesterone concentrations for treatment group and breeding success was Y = 1.854 ± 0.002X. Correlations and Regressions f o r Reproductive Health Data Table 41 gives the c o r r e l a t i o n c o e f f i c i e n t s and s i g n i f i c a n c e of Table 40 Correlations Between Milk Progesterone (ng/ml) and Plasma Selenium (mg/kg) levels for Treatment by (un)successful Breeding Group Nonconception Conception Treatment r n Significance r n Significance 1 0.4135 12 n.s. -0.4581 16 n.s. 2 -0.7332* 8 0.04 -0.2704 7 n.s. 3 0.2823 11 n.s. -0.3144 11 n.s. 4 0.1384 17 n.s. 0.0005 5 n.s. 5 -0.1941 7 n.s. -0.0007 11 n.s. 6 0.3636 4 n.s. -0.2673 5 n.s. 7 -1.0000 2 n.s. 0.3249 4 n.s. 8 0.9967* 4 n.s. -0.4509 5 n.s. (1-8) 0.1237 65 n.s. -0.0163 64 n.s. *p < 0.05 n.s. = not s i g n i f i c a n t - 135 -Table 41 Correlations Between Treatment Groups and the Incidence of Reproductive Problems Treatment Lactation 1 Lactation 2 r Significance r S i gnificance Normal Uterus (Inactive Ovaries) 0.158 0.227 n.s. Normal Uterus (Abnormal Ovaries) 0.155 -0.214 n.s. Abnormal Uterus (Normal Ovaries) 0.189 -0.201 n.s. Abnormal Uterus (Inactive Ovaries) - -0.164 n.s. Normal Uterus (C y s t i c F o l l i c l e s ) - 0.139 n.s. Abnormal Ovaries (Cystic CL) - 0.227 n.s. M e t r i t i s 0.226* 0.03 0.104 n.s. V a g i n i t i s - 0.229 n.s. Retained Placenta - -0.300* 0.02 n 71 43 *p _< 0.05 n.s. = not s i g n i f i c a n t - 136 -ovarian and uterine data correlated with treatment group. Several of the c o r r e l a t i o n s approached s i g n i f i c a n c e , however, none was achieved (p >^  0.05). It i s i n t e r e s t i n g to note that the incidence of abnormal ovaries and abnormal uterus was p o s i t i v e l y correlated to treatment group during the f i r s t l a c t a t i o n and negatively correlated to treatment group i n the second l a c t a t i o n . Correlation c o e f f i c i e n t s and s i g n i f i c a n c e between treatments 1 - 4 ( l a c t a t i o n 1) and treatments 5 - 8 ( l a c t a t i o n 2) and the incidence of m e t r i t i s , v a g i n i t i s and retained placenta are also given i n Table 41. M e t r i t i s was s i g n i f i c a n t l y correlated (r = 0.226, n = 71) to treatment group in l a c t a t i o n 1. This finding indicates that the incidence of m e t r i t i s during l a c t a c t i o n 1 increased as treatment group number increased from one to four (with increasing d i e t a r y and plasma Se l e v e l s ) . This f i n d i n g was consistent with the incidence of abnormal uterus approaching s i g n i f i c a n c e (p = 0.057) and being p o s i t i v e l y correlated with treatment group (r = 0.189, n = 71). In the second l a c t a t i o n , however, no s i g n i f i c a n t c o r r e l a t i o n existed between the incidence of m e t r i t i s and treatment group. Retained placenta was also s i g n i f i c a n t l y correlated (r = -0.30, n = 43) with treatment group (p _< 0.05), i n d i c a t i n g that the incidence of retained placenta decreased with Increasing treatment group number ( i . e . , treatments 5 - 8). C o r r e l a t i o n s Between Treatment Group and Ease of Calving Category Ease of calving scores were correlated with treatment groups (Table 42). There was no s i g n i f i c a n t c o r r e l a t i o n s between d i f f i c u l t y of c a l v i n g and treatment groups (p _> 0.05). For i n d i v i d u a l categories of - 137 -Table 42 Correlations Between Calving Ease Categories and Treatment Groups Treatment r n Normal Presentation (no assistance) -0.1583 14 Normal Presentation (easy p u l l ) 0.2753* 9 Normal Presentation (hard p u l l ) -0.1364 3 Mal Presentation (easy p u l l ) -0.1411 2 Mal Presentation (hard p u l l ) 0.0971 1 For A l l Calving Ease Categories 0.0062 43 * S i g n i f i c a n t c o r r e l a t i o n of p <_ 0.05. Note: Unobserved calvings are not l i s t e d . - 138 -calving ease correlated with treatment group, only one calving ease category was found to be s i g n i f i c a n t l y correlated with treatment group. Treatment group was p o s i t i v e l y correlated (r = 0.2753, n = 9) to the calving ease category of normal presentation requiring an easy p u l l . This c o r r e l a t i o n indicates that the need for only minor assistance at calving occurred more frequently i n the Se supplemented groups. A l l other categories of calving ease were not found to be correlated with treatment group. Correlations Between Treatment Group and Health Problems Table 43 gives c o r r e l a t i o n c o e f i c i e n t s and s i g n i f i c a n c e for health problems (other than reproductive t r a i t s ) correlated to treatment group. None of the co r r e l a t i o n s tested were found to be s i g n i f i c a n t . - 139 -Table 43 Correlations Between the Incidence of Treatment Group Health Problems and Treatment Lacta t i o n 1 Lactation 2 r S i g n i f i c a n c e r S i g n i f i c a n c e M a s t i t i s 0.074 n.s. 0.164 n.s. Milk. Fever -0.052 n.s. -0.026 n.s. Displaced Abomasum 0.151 n.s. -Injury to Feet and-Legs -0.149 n.s. -Foot Rot 0.094 n.s. -n 71 43 n.s. = not s i g n i f i c a n t - 140 -DISCUSSION Ration Selenium Levels Perry et a l (1978), Maus et a l (1980), and others have demonstrated that blood serum selenium l e v e l s respond to supplemental di e t a r y Se and may r e f l e c t tissue storage le v e l s i n cases of dietary Se de f i c i e n c y . NRC (1980) recommendations for adequate dietary l e v e l s of Se for l a c t a t i n g dairy c a t t l e range from 0.100 to 0.300 mg/kg (DM b a s i s ) . However, there i s controversy as to whether these le v e l s of dietary Se are indeed s u f f i c i e n t , e s p e c i a l l y for high producing animals such as dairy c a t t l e (Church, 1971). Maus et a l (1980) indicate that cows consuming 0.15 mg/kg Se had plasma Se le v e l s ranging from 0.082 to 0.092 mg/kg, which are i n d i c a t i v e of marginally d e f i c i e n t Se status conditions. Marginal and severly d e f i c i e n t dietary Se l e v e l s are considered to f a l l within a range of 0.050 0.100 mg/kg Se and less than 0.050 mg/kg, re s p e c t i v e l y (Rickaby, 1980). In the present study, Se and S le v e l s i n feeds prior to supplementation were hoped to be approximately 0.050 mg/kg and .15%, re s p e c t i v e l y . With the exception of the dry cow r a t i o n , the lev e l s of sulphur and selenium i n the feeds used i n this study were much higher than a n t i c i p a t e d . Consequently, the average l e v e l s of selenium and sulphur of the experimental rations were higher than o r i g i n a l l y planned. The l e v e l of Se i n the basal r a t i o n were above l e v e l s considered to be adequate (0.100 - 0.300 mg/kg Se), however the lev e l s were within - 141 -the ranges considered safe (0.100 - 2.000 mg/kg) and well below toxic l e v e l s (5.000 mg/kg Se) (NRC 1980). Also, although dietary Se l e v e l s were higher than i n i t i a l l y planned, the l e v e l s are comparable to p r a c t i c a l l e v e l s currently being used i n the Dairy Feed industry. L o c a l l y grown forages are low i n Selenium (0.020 - 0.120 mg/kg Se, Cathcart et a l . , 1978). Concentrates which are imported into B r i t i s h Columbia are generally much higher i n Se. Further, selenium i s rou t i n e l y added to the concentrate mix at a l e v e l of 0.200 mg/kg, y i e l d i n g a selenium concentration between 0.400 to 0.700 mg/kg Se. Additional Se i s added to some Dairy concentrates v i a veterinary p r e s c r i p t i o n . F i n a l l y , cows may be consuming addit i o n a l Se from trace mineralized s a l t (0.010 mg/kgSe). The experimental diets fed in t h i s study then, f a l l within the upper and lower ranges used on dairy farms in B.C. Cow and C a l f Plasma Selenium Levels Evidence for the existence of a sulphur - selenium i n t e r a c t i o n at the metabolic l e v e l i n dairy c a t t l e has been both sparse and c o n f l i c t i n g . The present theory i s that S and Se, being chemically s i m i l a r , i n t e r a c t at a b i o l o g i c a l l e v e l through a competitive transport mechanism (Whanger et a l 1969, and Bonhorst & Palmer 1957). Pope et al (1979) found that with increased dietary S concentrations i n sheep, the urinary excretion of ^ S e increased, as was evidenced by reduced plasma r a d i o a c t i v i t y as well as reduced apparent selenium retention. Only those animals on the lowest dietary S regime (0.05% S) i n the t r i a l - 142 -had s i g n i f i c a n t l y elevated blood ° S e (Pope et a l 1979). Blood '-'Se l e v e l s changed most dramatically when dietary S increased from 0.05 to 0.10%. However, no change i n blood ?5g e occurred when dietary S was increased from 0.16 to 0.20% (Pope et a l 1979). Similar findings were reported by White and Sommers (1977) who observed reduced blood Se le v e l s when dietary S concentrations rose from 0.07% to 0.20%. In contrast, Allaway and Hodgson (1964) could find no consistency i n the e f f e c t of high S forage rations i n increasing Se defi c i e n c y i n l i v e s t o c k . Acuff and Smith (1981) demonstrated that rats fed 0.0002% (low), 0.02% (optimal), and 0.42% (high) S as inorganic sulphate (S0 4) had absorbed 9.7%, 16.5%, and 13.2% 7 5 S e (0.75 mg Se/ml d i s t i l l e d H 20 administered v i a a stomach tube), r e s p e c t i v e l y . Rats fed the lowest l e v e l of dietary s u l f a t e (0.0002%) had the highest l e v e l of 7 5 unabsorbed Se. Maus et a l (1980) fed four le v e l s of dietary Se (as sodium se l e n i t e ) ranging from 0.300 to 0.700 mg/kg, comparable to the dietary l e v e l s used i n the present study. For a l l d i e t s , Maus et a l (1980) found that plasma Se le v e l s were s i m i l a r among a l l treatment groups. Contrary to t h i s , i n the present study, differences i n plasma Se l e v e l s were found to be s i g n i f i c a n t l y d i f f e r e n t among treatment groups. During the increasing plasma Se phase (months 1-4), regression equation and slopes of those equations were compared. Differences between slopes were not d i f f e r e n t i n d i c a t i n g s i m i l a r response to diet i n terms of Se a s s i m i l a t i o n . However, treatment group 3 had s i g n i f i c a n t l y d i f f e r e n t regression equation than treatment groups 1 and 2, i n d i c a t i n g that the y-intercept were s i g n i f i c a n t l y higher i n th i s group. - 143 -Generally In the present study, plasma Se l e v e l s increased from the onset of the t r i a l u n t i l approximately month 4 of treatment, at which point plasma Se lev e l s reached a plateau. This pattern of Increased plasma Se i n response to increased dietary l e v e l s of Se over several months, indicates that Se accomodation i s slow and latent i n agreement with others (Maus et a l 1980) who have made s i m i l a r observations. In the present study, plasma Se l e v e l s of cows during the dry period ( p r i o r to treatment) were found to be s i g n i f i c a n t l y lower than plasma Se l e v e l s of samples taken at the f i r s t and subsequent months of treatment. Maus et a l (1980) found that plasma Se l e v e l s of Se supplemented animals i n week 7 were increased s i g n i f i c a n t l y from those i n week 1. In Maus et al's study (1980), a l l but the highest Se supplemented group (0.700 mg/kg Se) plateaued at 7 weeks of treatment. Plasma Se l e v e l s of the group supplemented with 0.700 mg/kg continued to increase beyond week 7 (though not s i g n i f i c a n t l y so) and plateaued at 13 weeks (Maus et a l 1980). In the present study, a l l treatment groups demonstrated increasing plasma Se l e v e l s u n t i l month 4 of treatment, except the Se supplemented group. Group 3 reached a high plasma Se l e v e l i n month 3 of treatment p r i o r to a subsequent decline i n plasma Se l e v e l s . The decline i n plasma Se l e v e l s observed i n cows on treatment group 3 during the fourth and f i f t h month of treatment was unexpected. During months 4 and 5 of treatment, cows on treatment group 3 had plasma Se l e v e l s ranging from 0.064 - 0.125 mg/kg Se (n = 8) and 0.073 - 0.115 mg/kg Se (n = 7), r e s p e c t i v e l y . - 144 -Maus et a l (1980) found plasma Se l e v e l s plateaued at 0.120 mg/kg Se and that there was no difference between treatments. In the present study, however, plateau Se l e v e l s were found to be s i g n i f i c a n t l y d i f f e r e n t among treatment groups. Slope analysis indicated no di f f e r e n c e among treatments during the plateau phase, as expected. However, the regression equation derived for group 4 was found to be s i g n i f i c a n t l y d i f f e r e n t from a l l other treatment groups. This finding indicates that the S and Se supplemented group had s i g n i f i c a n t l y elevated plasma Se l e v e l s during the plateau phase above a l l other treatment groups. These re s u l t s would indicate that when both S and Se l e v e l s are f a i r l y high i n the d i e t , S a c t u a l l y enhances the uptake of Se as r e f l e c t e d by plasma Se concentrations. The r e s u l t s reported here support work by Paulson et a l ( c i t e d by Church 1971) who demonstrated that Se uptake by the small i n t e s t i n e was increased on high S di e t s (0.62%). Hintz and Hogue (1964) however, demonstrated that inorganic sulphate (Na2S0tt) fed i n conjunction with sodium selenate (Na2Se03) reduced the effectiveness of selenium absorption i n sheep. In contrast, r e s u l t s from the present study i n d i c a t e that t h i s form of S a c t u a l l y appears to enhance the uptake of Se. The plasma Se le v e l s recorded i n the present study were somewhat lower than those reported by Maus et a l (1980) for s i m i l a r dietary Se l e v e l s . Therefore, i t appears that adequate plasma Se l e v e l s were attained at the dietary Se le v e l s fed In t h i s study. However, c e r t a i n responses to varying dietary Se l e v e l s were observed. It Is f e l t that - 145 -had a deficiency state been Induced i n cows with respect to Se, responses i n the monitored t r a i t s may have yielded more conclusive evidence. In f a c t , the r e l a t i v e non-response to treatment observed i n t h i s study was probably due to plasma Se l e v e l s of Se unsupplemented groups being j u s t adequate. Weighback Se analysis demonstrated that the weighbacks from groups supplemented with Se had at least three f o l d the actual dietary l e v e l of Se i n the r a t i o n . Weighbacks for the four treatment groups contained 0.551, 0.524, 2.257 and 3.312 ± 0.929 mg/kg Se, r e s p e c t i v e l y . These findings may have been the r e s u l t of supplemental Se having been applied as top dressing. The Se supplement used was i n a powder form, which may have s i f t e d out through the feed to the bottom of the feed bins, mixing with fines and consequently being l e f t , as cows tend to avoid eating fi n e material. As a r e s u l t , some portion of the supplement topdressing was not consumed. However, even i n the most extreme cases, the weighbacks represented less than 5% of the supplemental selenium, and therefore the error involved i s very small. Daily feed intake of cows on treatment groups 2 and 4 increased s i g n i f i c a n t l y from the f i r s t l a c t a t i o n to the next. Although feed intake increased for cows on treatment groups 1 and 3 as w e l l , the increase was not found to be s i g n i f i c a n t for these two treatment groups. Concentrate intake of animals on treatments 2, 3, and 4 also increased s i g n i f i c a n t l y from one l a c t a t i o n to the next, whereas control cows maintained a f a i r l y constant intake of concentrate. These r e s u l t s may r e f l e c t an age e f f e c t on consumption. Many cows entering the t r i a l - 146 -were f i r s t c a l f h e i f e r s and i t i s therefore not sur p r i s i n g that as these animals matured from th e i r f i r s t l a c t a t i o n to the second l a c t a t i o n , feed consumption tended to increase. The only s i g n i f i c a n t difference i n a l f a l f a cube consumption observed was the lower consumption of cubes by cows on treatment group 3 than control cows i n the second l a c t a t i o n . The intake r e s u l t s reported here are not i n d i c a t i v e of any clear trend, nor does there appear to be any single explanation for the observed differences i n d a i l y feed intakes. Perry et a l (1978) demonstrated that an eight week period was necessary to cause a s i g n i f i c a n t decline i n the serum selenium l e v e l s of Holstein calves fed low l e v e l s of dietary selenium (0.025 mg/kg Se). In yet another study by Perry et a l ( c i t e d by Perry et a l 1978), brood cows required 60 to 90 days on low Se di e t s to cause s i g n i f i c a n t declines i n serum Se l e v e l s . Fenimore (1983) reported a case i n which mean serum Se l e v e l s of 0.080 mg/kg Se i n c a t t l e f e l l to. a mean of 0.015 mg/kg Se aft e r the c a t t l e had been on f e r t i l i z e d and i r r i g a t e d pastures for a period of 6 weeks. J u l i e n et a l (1976a) conducted a study in which cows were maintained on a dry cow r a t i o n extremely low i n Se (0.020 - 0.040 mg/kg Se). After at least 40 days on the t r i a l plasma Se l e v e l s ranged from 0.020 to 0.040 mg/kg Se, although i n i t i a l or p r e - t r i a l plasma Se le v e l s were not given. In the present study, during the dry period, cows plasma Se l e v e l s declined to s i g n i f i c a n t l y lower concentrations In a 1 -2 month period than l e v e l s observed during the treatment period ( i . e . , months 1 - 10). However, blood samples from dry cows were taken at - 147 -random time periods. It was, therefore, not possible to assess the depletion rate of plasma Se in th i s study. It can only be stated that plasma Se l e v e l s f e l l s i g n i f i c a n t l y between the end of l a c t a t i o n and ju s t p r i o r to p a r t u r i t i o n . Dry cows i n this study were maintained on the dry cow r a t i o n (0.075 mg/kg Se) for a minimum of 45 days. Plasma Se le v e l s for a l l cows ranged from 0.025 to 0.020 mg/kg Se (n = 17). At both the low and high l e v e l s of dietary Se fed In the present study, groups supplemented with S had s l i g h t l y higher plasma Se l e v e l s than t h e i r unsupplemented counterparts, although differences were not s i g n i f i c a n t . However, dry cow plasma Se l e v e l s were s i g n i f i c a n t l y lower than plasma Se l e v e l s of cows on treatment. Dry cow plasma Se l e v e l s were much higher than those observed by J u l i e n et a l (1976a), but t h i s i s most probably due to differences i n Se l e v e l s of dry rations fed. The dry r a t i o n i n this study was below adequate while the dry rations used by J u l i e n et a l (1976a) could be considered well below adequate to severly d e f i c i e n t . Perry et a l (1978) observed that cows fed higher l e v e l s of Se (5mg/head/day) pr i o r to discontinued supplementation, displayed s i g n i f i c a n g l y slower depletion rates. He concluded, therefore, that plasma Se l e v e l s may r e f l e c t tissue storage as well as dietary l e v e l s of Se (Perry et a l 1978). In contrast to Perry et al's (1978) find i n g s , dry cow plasma Se lev e l s i n this t r i a l were not d i f f e r e n t among the higher or lower dietary Se groups. Perry et a l (1978) demonstrated that c a l f plasma Se lev e l s were greater for those calves whose dams had dietary Se supplementation than for those calves whose dams were given no dietary Se supplementation. - 148 -Calf plasma Se l e v e l s i n Perry et al's (1978) study ranged from 0.020 -0.048 mg/kg Se. The mean c a l f plasma Se l e v e l s from the present study tended to f a l l i n the upper end of the range given by Perry et a l (1978) and i n some cases, exceeded that range. Calves from dams on treatments 1 - 4 had plasma Se le v e l s of 0.040, 0.050, 0.037, and 0.058 mg/kg Se, re s p e c t i v e l y . Calves from dams on treatment groups 4 had plasma Se le v e l s beyond those reported by Perry et a l (1978), but th i s may be due to the r e l a t i v e l y higher dietary Se lev e l s fed the dams ( p r i o r to the dry period) i n th i s study. The low c a l f plasma Se l e v e l s recorded i n t h i s study may be r e f l e c t i v e of the d e f i c i e n t plasma Se l e v e l s observed i n cows maintained for an extended period ( 1 - 2 months or greater) on dry cow rations d e f i c i e n t i n Se. Maus et a l (1980) advises that c a l f plasma Se le v e l s shoud be maintained at a minimum of 0.050 mg/kg Se i n order to avoid problems with muscular dystrophy. It becomes apparent that the present practice of feeding unsupplemented rations (with respect to Se) to dry cows may not be b e n e f i c i a l and should be re-evaluated. Research in the area of breed responses to Se or S and Se supplementation i s lacking . Mean plasma Se le v e l s of Holsteins and Ayrshires over the entire experimental period were not d i f f e r e n t (0.097 and 0.098 ± 0.021 mg/kg Se, r e s p e c t i v e l y ) . Neither was there any diff e r e n c e i n plateau plasma Se l e v e l s between the two breeds; Holsteins had mean plasma Se l e v e l s of 0.103 and 0.106 ± 0.019 mg/kg Se, re s p e c t i v e l y . Also, the pattern of plasma Se re p l e t i o n during treatment and depletion during the dry period was v i r t u a l l y i d e n t i c a l for the two - 149 -breeds. It can be concluded that breed differences do not account for any appreciable v a r i a t i o n i n plasma Se response to Se and S supplementation. Production Gwazdauskas et a l (1979) investigated the e f f e c t of selenium/vitamin E supplementation by i n j e c t i o n on milk y i e l d (305 day-2x mature equivalent) and found that treatment did not influence milk y i e l d . Larson et a l (1980) also concluded that serum Se concentrations were not related to milk y i e l d or milk f a t . In agreement with these r e s u l t s , BCA milk y i e l d s and kg milk fat were not s i g n i f i c a n t l y d i f f e r e n t between treatment groups i n the present study. Although Holsteins had a higher mean 305-day BCA index for milk y i e l d (163.8 ± 28.39) than the Ayrshire breed (154.0 ± 28.39), differences were not s i g n i f i c a n t . S i m i l a r i l y , breed did not influence kg milk f a t , although s i g n i f i c a n c e was approached (p = 0.06). Reproduction Days Open, Days to F i r s t Service, Calving Intervals and Services  per Conception Information on r e l a t i o n s h i p s between Se status of cows and reproductive paramters such as number of days to f i r s t service, days from p a r t u r i t i o n to conception, calving i n t e r v a l s , and services per confirmed conception i s l i m i t e d . A study by Larson et a l (1980) examined the r e l a t i o n s h i p between reproductive performance and blood - 150 -composition. Of a l l the components analysed ( c e l l volume, hemoglobin, t o t a l protein, calcium, phosphorus, magnesium, copper, zinc, and selenium), only serum Se was found to have the most important r e l a t i o n s h i p with services per conception and days open. Larson et a l (1980) found that Se was p o s i t i v e l y correlated to services/conception and days open, but was not related to days to f i r s t s e r v i c e . Quadratic, e f f e c t s were found to be s i g n i f i c a n t for Se for both services/conception and days open (Larson et a l 1980). Larson et al's (1980) study suggested that there e x i s t s an optimal concentration for serum Se and that l e v e l s in excess of, or less than, the optimal Se concentration, may be detrimental. They demonstrated that Se l e v e l s i n i t i a l l y f e l l quickly and then declined more slowly as days to f i r s t service and days open increased. Serum Se l e v e l s reached a minimum at 104 days and 95 days for days to f i r s t service, and days open, r e s p e c t i v e l y . In contrast, Gwazdauskas et a l (1979) found no influence of breed or treatment on either days open or services/conception for cows treated with a 10 ml i n j e c t i o n of selenium/vitamin E (2.19 mg sodium s e l e n i t e + 50 mg vitamin E/ml) 28 to 30 days prepartum versus cows that were not treated. In agreement with Gwazdauskas et a l (1979), the r e s u l t s from the present study indicate that treatment did not influence days open or services per conception. Cows on treatments 1, 2, 3, and 4 i n this study required 1.73, 1.50, 1.43 and 1.60 ± 0.82 services/conception, r e s p e c t i v e l y , which i s somewhat lower than mean sevices/conception of In contrast, Gwazdauskas et a l (1979) found no influence of breed or treatment on either days open or services/conception for cows treated - 151 -composition. Of a l l the components analysed ( c e l l volume, hemoglobin, t o t a l protein, calcium, phosphorus, magnesium, copper, zinc, and selenium), only serum Se was found to have the most important r e l a t i o n s h i p with services per conception and days open. Larson et a l (1980) found that Se was p o s i t i v e l y correlated to services/conception and days open, but was not related to days to f i r s t s e r v i c e . Quadratic e f f e c t s were found to be s i g n i f i c a n t for Se for both services/conception and days open (Larson et a l 1980). Larson et a l ' s (1980) study suggested that there e x i s t s an optimal concentration for serum Se and that l e v e l s in excess of, or less than, the optimal Se concentration, may be detrimental. They demonstrated that Se l e v e l s i n i t i a l l y f e l l quickly and then declined more slowly as days to f i r s t service and days open increased. Serum Se l e v e l s reached a minimum at 104 days and 95 days for days to f i r s t s e rvice, and days open, r e s p e c t i v e l y . In contrast, Gwazdauskas et a l (1979) found no influence of breed or treatment on either days open or services/conception for cows treated with a 10 ml i n j e c t i o n of selenium/vitamin E (2.19 mg sodium s e l e n i t e + 50 mg vitamin E/ml) 28 to 30 days prepartum versus cows that were not treated. In agreement with Gwazdauskas et a l (1979), the r e s u l t s from the present study indicate that treatment did not influence days open or services per conception. Cows on treatments 1, 2, 3, and 4 i n this study required 1.73, 1.50, 1.43 and 1.60 ±0.82 services/conception, r e s p e c t i v e l y , which i s somewhat lower than mean sevices/conception of 1.9 (treated) and 2.0 (control) ±0.12 reported by Gwazdauskas et a l (1979) and 1.92 ± 0.11 reported by Larson et a l (1980). - 152 -In the present study, the number of days to conception was 115.0, 118.2, 112.6, and 121.7 ± 43.8 for cows on treatments 1 - 4 , r e s p e c t i v e l y . The mean number of days to conception was much greater i n t h i s study i n comparison to those reported by Gwazdauskas et a l (1981) (treated: 101.7 ± 38.5 days, untreated: 109.0 ± 43.7 days) and Larson et a l (1980) (106.5 ± 4 . 0 [s.e.] days). The fact that fewer breedings were required to achieve conception, along with a longer i n t e r v a l from p a r t u r i t i o n to conception i n t h i s t r i a l indicates that cows were bred l a t e r (probably due to missed heats), with a high rate of success. In the present study, treatment did not influence days to f i r s t s e r v ice which i s i n agreement with Larson et a l ' s (1980) observation that serum Se was not related to days to f i r s t s e r v i c e . The mean number of days to f i r s t service for cows on treatment groups 1, 2, 3, and 4 was 87.94, 93.57, 86.28, and 100.60 ± 27.13 days, r e s p e c t i v e l y . Although differences between treatment groups i n days to f i r s t service were not s t a t i s t i c a l l y s i g n i f i c a n t , i t i s apparent the mean i n t e r v a l from p a r t u r i t i o n to f i r s t service was somewhat longer for cows on treatment group 4. Since the number of services/conception was s i m i l a r for cows on a l l treatments, i t must be assumed that heat detection was more d i f f i c u l t or less e f f i c i e n t i n t h i s group. Calving i n t e r v a l i n response to Se supplementation has not been previously investigated. In order to maintain a 12 month calving i n t e r v a l , Louca and Legates (1968) suggest that conception must occur by day 85 post partum. In a l l treatments, a 365 day calving i n t e r v a l was exceeded (treatment did not influence calving i n t e r v a l ) corresponding to - 153 -the r e l a t i v e l y longer period between p a r t u r i t i o n and conception. O v e r a l l , the mean values for the t r a i t s discussed above were not abnormal. For example, the response to Se supplementation may have been greater had there been a poorer conception rate i n the herd. Gwazdauskas et a l (1979) found no breed differences between Holsteins, Jersey, Guernsey, and Ayrshire cows on days open and services per conception for Se treated and control cows. In contrast, i n the present study breed differences were found to be s i g n i f i c a n t for the e f f e c t of Se supplementation on days open, calving i n t e r v a l , and services/conception with the Holsteins having longer i n t e r v a l s than Ayrshires for a l l three t r a i t s . Calf B i r t h Weights Holstein calves at b i r t h were s i g n i f i c a n t l y heavier than Ayrshire calves (42.16 and 33.22 ± 5.72 Kg). These r e s u l t s were not unexpected as the Holstein breed i s a larger breed than the Ayrshire breed. Perry et a l (1978) found no difference In b i r t h weight of beef calves whose dams were fed 0, 1, or 5 mg Se/head/day beginning 90 days pre-partura. S i m i l a r i l y i n studies with sheep, Whanger et a l (1977) reported that mean b i r t h weights between lambs whose dams were injec t e d with varying l e v e l s of Se and /or vitamin E were not d i f f e r e n t . In agreement with Perry et a l (1978) and Whanger et a l (1977), Moxon (1981) also found no difference i n c a l f b i r t h weights for calves whose dams were fed 0.048 or 0.132 mg/kg Se in the r a t i o n . The calves i n the present study had mean b i r t h weights of 36.31, 39.15, 38.40, and 38.30 ± - 154 -5.72 kg, whose dams were on the c o n t r o l , S supplemented, Se supplemented, and S Se Se supplemented, r e s p e c t i v e l y . Differences between c a l f b i r t h weights were not s i g n i f i c i a n t l y d i f f e r e n t between treatment groups, agreeing with the r e s u l t s of Perry et a l (1978), Whanger et a l (1977), and Moxon (1981). Correlations between dam plasma Se and c a l f b i r t h weight for the four treatment groups was not s i g n i f i c a n t . However, the c o r r e l a t i o n between dam plasma Se le v e l s and c a l f b i r t h weight for treatment group 2 was f a i r l y high (r = 0.6626, n = 8) and did approach s i g n i f i c a n c e (p = 0.07). The c o r r e l a t i o n suggests that with increasing dam plasma Se, c a l f b i r t h weights tend to increase which may r e f l e c t improved c a l f health. The fact that the c o r r e l a t i o n approached s i g n i f i c a n c e i n the lower plasma Se group and not i n the higher plasma Se groups (groups 3 and 4) may indicate that dam plasma Se becomes a c r i t i c a l factor i n c a l f health when average dam plasma Se l e v e l s throughout the e n t i r e gestation period are less than adequate. Retained Placenta, Calving D i f f i c u l t i e s , Uterine and Ovarian  Problems Langlands et a l (1982) demonstrated that Se supplementation increased uterine Se content in non-pregnant ewes. Trinder et a l (1973, 1969) have demonstrated that vitamin E and Se administration together was more e f f e c t i v e i n preventing retained placenta than either substance injec t e d alone. A l a t e r study demonstrated by J u l i e n et a l (1976a) demonstrated a r e l a t i o n s h i p between low blood Se l e v e l s and a high incidence of retained placenta. J u l i e n et a l (1976a) have shown that - 155 -regardless of mode of supplementation, Se reduced the incidence of retained placenta from 38% (control cows) to 0% (Se treated cows) and was not dependent on vitamin E. In yet another study by J u l i e n et a l (1976b) Se i n j e c t i o n 20 days p r i o r to calving reduced placental retention from 51.2% to 8.8%. Plasma Se l e v e l s ranged from 20.0 - 50.0 ppb Se i n control animals and 80.0 - 100.0 ppb i n the treated group ( J u l i e n et a l 1976b). These r e s u l t s led J u l i e n et a l (1976a) to suggest that i n the mature dairy cow, Se and/or vitamin E deficiency may be related to poorer uterine health, consequently Se d e f i c i e n c y may be expressed c l i n i c a l l y as retained placenta. In the present study, the Incidence of retained placenta was only considered for the second l a c t a t i o n as those occurring i n the f i r s t l a c t a t i o n would not r e f l e c t the influence of treatment with dietary Se supplementation. Retained placenta occurred with a frequency of 22.4% i n the control group, 11.1% i n S-supplemented group, and 0.0% in both groups with Se supplementation. It has been noted that retained placenta normally occurs i n approximately 10% or less of parturient dairy cows ( J u l i e n et a l , 1976a). However, Hidiroglou (1982) points out that i f the incidence of retained placenta occurs with a frequency of 15% or more, the problem of retained placentae may be n u t r i t i o n a l l y induced. The r e s u l t s of t h i s study with respect to retained placenta appears to r e l a t e well with Hidiroglou's (1982) statement and the findings of J u l i e n et a l (1967a) and Trinder et a l (1973, 1969). The control group had what could be considered an abnormally high incidence of retained placenta with borderline Se d e f i c i e n c y , whereas, the Se - 156 -supplemented groups ( i e , 3 and 4) with adequate plasma Se l e v e l s had no 1-incidence of retained placenta. In contrast to these findings, Gwazdauskas et a l (1978) reported that Se/vitamin E treatment did not reduce the Incidence of retained placenta. Schingoethe et a l (1982) also, concluded from t h e i r studies that Se supplementation did not reduce the incidence of retained placenta when cows were already consuming adequate amounts of Se. In the present study, a l l treatment groups were consuming what i s considered 'adequate' dietary Se, yet a d d i t i o n a l dietary Se supplementation did reduce the incidence of retained placenta. In t h i s study, the incidence of abnormal uterus during the f i r s t l a c t a t i o n period was 5.3%, 6.3%, 5.6%, 22.2% for treatment groups 1 - 4 , r e s p e c t i v e l y . In the second l a c t a t i o n , however, the incidence increased to 18.8%, 10.0%, 14.3% for treatment groups 1 - 3 , but decreased dramatically to 0.0% for treatment group 4. It appears then, that supplemental dietary Se l e v e l s may have a r e s i d u a l e f f e c t as (with the exception of group 3) the incidence of abnormal u t e r i ! became more pronounced for cows with marginally d e f i c i e n t plasma Se l e v e l s and less pronounced for cows with adequate plasma Se l e v e l s in the subsequent l a c t a t i o n . The incidence of m e t r i t i s and v a g i n i t i s were not abnormally high i n either the f i r s t or second l a c t a t i o n for any of the experimental groups. Retained placenta was negatively correlated (r = -0.300) to treatment group (p <_ 0.05) i n d i c a t i n g that as dietary Se (and consequently plasma Se) increased, the incidence of retained placenta - 157 -decreased. S i m i l a r i l y , the incidence of abnormal u t e r i i tended to be negatively correlated (r = -0.201) with treatment group, though t h i s c o r r e l a t i o n was not s i g n i f i c a n t . This r e l a t i o n s h i p also suggests a reduced incidence of abnormal u t e r i i with increasing dietary and plasma Se. During the second l a c t a t i o n , the highest incidence of retained placenta and abnormal u t e r i i occurred with the greatest frequency i n the control group while no occurrence of either retained placenta or abnormal u t e r i i was observed i n treatment group 4. Treatment group 3 also appeared to have a somewhat higher incidence of abnormal u t e r i i r e l a t i v e to other groups, but the Incidence was not abnormally high and i n t h i s group as well, retained placenta f a i l e d to occur. Treatment group 2 had a s i m i l a r incidence of abnormal u t e r i i and retained placenta (10.0% and 11.1%, r e s p e c t i v e l y ) , although plasma Se l e v e l s were s i m i l a r to those of the control group. The r e s u l t s may be explained by taking into consideration the calving ease r e s u l t s i n conjunction with the retained placenta data. Treatment group 1 appeared to have a greater proportion of calving d i f f i c u l t i e s than group 2 which may explain the higher incidence of uterine problems i n th i s group. A l l categories of calving ease were not correlated to plasma Se l e v e l s with the exception of calving c l a s s i f i e d as normal presentation with an easy p u l l required (r = 0.2753) (p < 0.037). This c o r r e l a t i o n 6 0 indicates that normal c a l f presentations requiring an easy p u l l occurred more frequently for cows with increased dietary and plasma Se l e v e l s , which may indicate a benefical e f f e c t of Se in f a c i l i t a t i n g c a l v i n g . The increased occurrence of less d i f f i c u l t calvings, may in turn contribute - 158 -to a lower Incidence of retained placenta. Cows on treatment groups 3 and 4 also had a fair proportion of calving d i f f i c u l t i e s with an incidence of calf mal presentation (requiring hard pull) of 14.3%, and 30.0, respectively, yet no Incidence of retained placenta occurred in either group. It appears then, that cows with an adequate Se status have healthier u t e r i i , as demonstrated by the fact that the problem of retained placenta can be avoided, even with d i f f i c u l t births. These results indicate that adequate Se status in cows is a prophylactic against retained placenta. Furthermore, adequate Se status of dams becomes essential in reducing the occurrence of retained placenta in herds in which dystocia, either due to oversized calves, abnormal presentation, or other factors, is a problem. Buck et al (1980), using labelled 7 5Se, determined that the corpus luteum, pituitary, non-luteal ovarian tissues, and dam placentae require Se. Buck et al (1980) cites other isotopic studies which demonstrate that the pituitary, ovary, and adrenal glands contained high 7 5 amount of Se. A 100 ug injection of Se/kg body weight (as sodium selenite with adequate vitamin E) administered to dairy cows prevented post calving ovarian cysts in cows previously experiencing cystic ovaries. In this study, the incidence of inactive ovaries (with normal uterus) occurred only in treatment group 4 in both lactation 1 (5.6%) and lactation 2 (10.0%). This level of Incidence cannot be considered abnormally high. Also the incidence of inactive ovaries was not found to be dependent on treatment group. Similarily, the Incidence of - 159 -i n a c t i v e ovaries (with abnormal u t e r i i ) and corpora lutea- was not abnormally high for any one treatment, and these t r a i t s were found to be independent of treatment group. Cystic f o l l i c l e s did not occur i n any treatment groups during the f i r s t l a c t a t i o n . During the second l a c t a t i o n , however, there was a high occurrence of c y s t i c f o l l i c l e s (28.6%) in treatment group 3, though t h i s t r a i t was found to be independent of treatment group. There appears to be no explanation for t h i s observation. The incidence of abnormal ovaries was 0.0% for cows on both treatment groups 1 and 2, 11.1% for cows of treatment 3, and 5.6% for cows on trlu0?ent group 4 during the f i r s t l a c t a t i o n . However, i n the second l a c t a t i o n , the s i t u a t i o n was reversed. Control and group 2 cows had 12.5% and 10.0% incidence of abnormal ovaries, whereas the occurrence of abnormal ovaries i n cows on treatment groups 3 and 4 was not observed. In both l a c t a t i o n s , again, the frequency of abnormal ovaries diagnosed could not be considered abnormally high. O v e r a l l , ovarian problems seem to have occurred randomly and the inconsistency of the r e s u l t s with respect to Se status tends to suggest that in t h i s experiment, Se status may not be associated with the ovarian problems monitored. Correlations between a l l ovarian problems discussed and treatment groups were n o n - s i g n i f i c a n t . Hidiroglou (1982) c i t e s Libby and Segerson's (1981) r e s u l t s i n d i c a t i n g that low Se status was not associated with i n f e r t i l i t y (determined by f e r t i l e ova collected) however, a greater number of sperm per f e r t i l i z e d ovum was c o l l e c t e d from Se/vitamin E treated cows supporting the hypothesis that poor f e r t i l i t y i n Se - 160 -d e f i c i e n t animals i s the r e s u l t of reduced sperm transport rather than ova i n f e r t i l i t y . The r e s u l t s from t h i s study lends support to t h i s hypothesis. Breeding Data An extensive l i t e r a t u r e search on the topic of Se status of cows and possible e f f e c t s on l e v e l s of reproductive hormones (with s p e c i f i c i n t e r e s t i n progesterone and LH) proved f r u i t l e s s and indicates a need for research i n t h i s area. Recently, i t was discovered that GSH-Px can convert prostaglandin G2 to prostaglandin H 2, and that GSH-Px functions by exerting i t s e f f e c t on substrates Inhibitory to prostaglandin synthesis pathway (Diplock 1980). Prostaglandin H 2 i s the precursor to prostaglandins E2 and ?2a (prostaglandin F 2 a i s responsible for the l y s i s of the funcional corpora lutea and i s therefore i n d i r e c t l y responsible for the preciptuous pre-estrus decline In progesterone concentrations, by removing the progesterone hypothalamal "bloc k " . r e s u l t i n g in the pre-ovulatory LH surge and o v u l a t i o n ) . This work (Diplock 1980) may also be s i g n i f i c a n t i n that GSH-Px, a Se containing enzyme, may possibly exert some e f f e c t on the metabolism of other s t e r o i d hormones as w e l l . This area, however, requires more extensive research. Plasma Se l e v e l s between breeds at time of breeding were s i m i l a r . Milk progesterone concentrations, however, were s i g n i f i c a n t l y lower (p £ 0 . 0 5 ) for Ayrshires than for Holsteins (1.76 and 2.00 ± 8.81 ng/ml, respectively) at time of breeding. - 161 -Cows on treatment group 1 had s i g n i f i c a n t l y lower (p £ 0.01) plasma Se l e v e l s than cows on treatment group 4 (0.091 and 0.120 ± 0.023 mg/kg, r e s p e c t i v e l y ) . During the second l a c t a t i o n , however, differences i n plasma Se l e v e l s at breeding were not s i g n i f i c a n t . I t was observed that, o v e r a l l , plasma Se l e v e l s of cows on Se supplemented die t s tended to be higher i n l a c t a t i o n 1 than i n l a c t a t i o n 2, though not s i g n i f i c a n t l y so. Conversely cows on treatment 1 ( no Se or S supplementation) tended to have s l i g h t l y higher plasma Se l e v e l s i n the second l a c t a t i o n (0.091 and 0.101 ± 0.023 mg/kg Se, respectively) (p >^  0.05). Plasma Se l e v e l s of cows with S supplementation alone remained constant from one l a c t a t i o n to the next (0.100 and 0.101 ± 0.023 mg/kg Se, r e s p e c t i v e l y ) . The fact that i n t h i s group, plasma Se l e v e l s remained constant over both l a c t a t i o n s at l e v e l s considered to r e f l e c t adequate Se status (0.100 mg/kg Se) for l a c t a t i n g dairy c a t t l e , t h i s l e v e l of plasma Se may indeed be a b i o l o g i c a l l y optimal l e v e l . This theory i s further substantiated by the fact that cows with plasma Se l e v e l s less than adequate tended to have increased plasma Se l e v e l s i n subsequent l a c t a t i o n s while cows with plasma Se l e v e l s greater than 0.10 mg/kg i n the f i r s t l a c t a t i o n , tended to have reduced plasma Se i n the subsequent l a c t a t i o n . The Increasing and decreasing trends i n plasma Se observed i n the subsequent l a c t a t i o n i s probably due to a l t e r a t i o n s i n Se metabolism as the same diets were fed in the second l a c t a t i o n . Therefore, the altered plasma Se l e v e l s observed i n the subsequent l a c t a t i o n were not of dietary o r i g i n , e s p e c i a l l y since cows were phased on to the study at d i f f e r e n t time periods. Since Se - 162 -absorption and excretion i s responsive to tissue needs (Church et a l 1971), i t i s conceivable that cows i n the control group may have been absorbing and/or u t i l i z i n g dietary Se more e f f i c i e n t l y i n the subsequent l a c t a t i o n r e f l e c t e d by increased plasma Se l e v e l s . Conversely, reduced plasma Se l e v e l s observed i n cows on treatment 4 may be r e f l e c t i v e of increased excretion or reduced absorption of excess dietary selenium i n the body's attempt to achieve an optimal Se balance. Although plasma Se l e v e l s of cows on treatment 3 were reduced i n the second l a c t a t i o n as well, the plasma Se l e v e l s a c t u a l l y f e l l below the adequate l e v e l . Plasma Se l e v e l s decreased from 0.106 ± 0.023 mg/kg i n the f i r s t l a c t a t i o n to 0.096 ± 0.022 mg/kg i n the subsequent l a c t a t i o n , though differences were not s i g n i f i c a n t . The reason(s) for t h i s finding i s not c l e a r . Milk progesterone l e v e l s at breeding of cows in the control group were s i g n i f i c a n t l y lower than those of cows on treatment group 8 (1.53 and 2.73 ± 8.81 ng/ml, respectively) (p £ 0.05). Mean milk progesterone concentrations between a l l other treatment groups were not found to be d i f f e r e n t , however. O v e r a l l , progesterone values at breeding tended to be higher i n the second l a c t a t i o n than i n the f i r s t . Correlations between plasma Se and milk progesterone were found to be s i g n i f i c a n t for treatment group 2 only (r = -0.5706, n = 15) (p £ 0 . 0 5 ) . The s i g n i f i c a n t c o r r e l a t i o n found i n group 2 suggests that as plasma Se l e v e l s increased, milk progesterone concentrations decreased at time of breeding. Comparisons between plasma Se l e v e l s and milk progesterone - 163 -l e v e l s were done among successful and unsuccessful breedings. Plasma Se l e v e l s for successful (0.102 ± 0.022 mg/kg Se) and unsuccessful (0.106 ± 0.022 mg/kg Se) breedings was not found to be s i g n i f i c a n t l y d i f f e r e n t . S i m i l a r i l y , milk progesterone l e v e l s at breeding for successful (1.96 ± 8.81 ng/ml) and unsuccessful (1.86 ± 8.81 ng/ml) breedings were not found to be d i f f e r e n t . However, milk progesterone l e v e l s i n the unsuccessful breeding group were found to be s i g n i f i c a n t l y higher i n the Se and S supplemented group (second l a c t a t i o n ) than a l l treatment groups during the f i r s t l a c t a t i o n , and the control and S supplemented groups i n the second l a c t a t i o n . It was observed that the mean milk progesterone concentration of group 8 at breeding was higher than a l l other means calcula t e d , for both the successful and unsuccessful breeding groups. Shemesh et a l (1983) indicates that mean progesterone l e v e l s i n unextracted fore milk from cows during estrus were maintained below 1 ng/ml. Shelford et a l (1979) found that the mean milk progesterone concentration at day 0 of the estrous cycle i n samples of milk strippings was 2.8 ± 1.1 ng/ml. The r e l a t i v e l y high mean milk progesterone concentrations of cows on treatment group 8 with unsuccessful breedings may have resulted i n f e r t i l i z a t i o n f a i l u r e . Three of the four cows on treatment group 8 had milk progesterone concentrations of below 2.0 ng/ml when bred, while the remaining cow was observed to have a milk progesterone concentration of 7.7 ng/ml at the time of breeding. This may r e f l e c t a treatment e f f e c t where high progesterone concentrations at breeding either delayed or prevented ovulation. Behavioral estrus w i l l occur only when progesterone l e v e l s - 164 -have f a l l e n to very low l e v e l s (below 2 ng/ml), allowing for LH stimulation of the f o l l i c l e leading to f o l l i c u l a r growth and estrogen production (estrogen produced by the growing f o l l i c l e i s responsible for behavioral e s t r u s ) . In view of the evidence presented by Diplock (1980) that Se may a c t u a l l y enhance protaglandin synthesis, i t i s fe a s i b l e that inadequate protaglandin ?2a m a v r e s u l t i n incomplete l u t e o l y s i s of the corpus luteum. Consequently, progesterone l e v e l s would not f a l l to extremely low l e v e l s , thereby prevening or delaying estrus. It i s l i k e l y that the r e l a t i v e l y high milk progesterone concentrations of cows on treatment group 8 with unsuccessful breeding may have resulted i n f e r t i l i z a t i o n f a i l u r e . The high mean milk progesterone concentration observed was more l i k e l y due to la t e inseminations (post ovulation with CL formation and increasing progesterone production by the CL) rather than to a true treatment e f f e c t . Progesterone Concentrations During the Estrous Cycle Laing and Heap ( c i t e d by Booth 1979) demonstrated that milk progesterone i n the bovine was highly correlated to CL a c t i v i t y . Further, Hoffman and Hamburger ( c i t e d by Booth 1979) depicted varying milk progesterone l e v e l s throughout the estrous cycle with milk progesterone l e v e l s increasing i n early pregnancy. Researchers have found that milk progesterone concentrations are s i m i l a r to, or higher than, plasma progesterone concetrations (Heap et a l 1973). Milk progesterone a n a l y s i s , therefore, can be e f f i c i e n t l y u t i l i z e d to characterize reproductive disorders of i n d i v i d u a l cows as - 165 -well as being an e f f e c t i v e diagnostic tool i n pregnancy determination (Heap et a l 1973, Lamming 1966, and Claus et a l 1983). Whole milk or any f r a c t i o n thereof can be used for progesterone concentration a n a l y s i s . In t h i s study milk progesterone analysis was done on milk strippings for the following reasons: 1. ease of obtaining samples, and 2. milk strippings contain the greatest proportion of f a t as compared to either fore milk or composite milk samples (Shelford et a l 1979). As milk fat contains the greatest proportion of progesterone than other f r a c t i o n s of milk, the use of milk fat for milk progesterone determinations i s superior because the method i s both highly se n s i t i v e and repeatable (Claus et a l 1983).' . The use of milk strippings in determining milk progesterone concentrations, therefore, gives clear discriminatory l e v e l s , whereas with foremilk samples (not corrected for f a t ) , progesterone concentrations at estrus and at 24 days (pregnant) overlapped (Shelford et a l 1979, and Claus et a l 1983). By using milk strippings then, the p r o b a b i l i t y of making a f a l s e diagnosis of pregnancy i s greatly reduced (Shelford et a l 1979). Throughout the estrous cycle, milk progesterone concentrations exhibit c y c l i c v a r i a t i o n s r e f l e c t i v e of c y c l i c ovarian functioning (Hoffman and Hamburger, c i t e d by Booth 1979). In a t y p i c a l estrous cycle, milk progesterone concentrations are very low (close to 0) during estru s . Milk progesterone concentrations begin to r i s e slowly at day 4 - 166 -and peak between 12 and 16 days of the estrous c y c l e . Between day 17 and 20 of a normal 21-day estrous cycle, milk progesterone concentrations decline rapidly (Booth 1979). In t h i s study, d i f f e r e n t cycle types were i d e n t i f i e d and characterized by periods (defined by s p e c i f i c parameters set for milk progesterone concentrations described in Materials and Methods) and the duration of the periods observed. Overall the mean length of estrous cycles (excluding a l l cycles > 30 days long) was s i m i l a r between breeds and treatment groups. Mean estrous cycle lengths observed in t h i s study were well within the normal range of 19 - 23 days quoted by B r i t t (1980). Also, mean cycle lengths were very s i m i l a r to those reported by B r i t t (1980) of 21.1 and 21.4 days. Milk progesterone concentrations of period 1, 3, and 4 were s i g n i f i c a n t l y influenced by treatment group. For the period of time when milk progesterone concentrations remained below 4 ng/ml ( i . e . , period 1), cows on treatment group 1 exhibited s i g n i f i c a n t l y lower (p £ 0.05) milk progesterone concentrations than cows on treatment group 6 (2.06 and 2.42 ± 0.61 ng/ml, r e s p e c t i v e l y ) . Milk progesterone concentrations during period 3 (milk progesterone concentrations > 12 ng/ml) were also s i g n i f i c a n t l y lower (p <_ 0.01) for cows on treatment group 1 in comparison to cows on treatment groups 2, 4, 6, 7, and 8. In both cases, the control group had s i g n i f i c a n t l y lowered milk progester-one concentrations than groups with Se and/or S supplementation. At what l e v e l increased dietary Se (and consequently increased plasma Se) - 167 -may function to e l i c i t the observed responses i n reproductive hormone balance mechanisms can not be ascertained from t h i s study. It was also observed that during period 4, S supplemented animals (treatment group 2) i n the f i r s t l a c t a t i o n had s i g n i f i c a n t l y lowered milk progesterone concentrations i n comparison to S and Se supplemented animals (treatment group 4). Milk progesterone concentrations also were higher i n the control and S supplemented groups i n the second l a c t a t i o n i n comparison to the S supplemented group of the f i r s t l a c t a t i o n . Mean milk progesterone concentrations of the d i f f e r e n t periods among the various cycle types defined were found to be s i g n i f i c a n t l y a l t e r e d during periods 1 and 3, corresponding to the f o l l i c u l a r and l u t e a l phases of the estrous cycle, r e s p e c t i v e l y . Cycle type 1 (short f i r s t c y c l e ) , 2 (normal f i r s t c y c l e ) , 10 ( l u t e a l c y s t ) , and 6 (short cycle) had s i g n i f i c a n t l y higher mean milk progesterone concentrations than cycle type 8 (unconfirmed pregnancy) during period 1. Also period 1 was shorter i n duration for a l l these cycle types i n comparison to cycles with f o l l i c u l a r cysts. The very low progesterone concentration during period 1 achieved by cows ex h i b i t i n g cycle type 8 may indicate that pregnancy may r e s u l t only If progesterone l e v e l s reach extremely low l e v e l s . Concentrations above a c e r t a i n l e v e l during t h i s period may be i n d i c a t i v e of abnormal ovarian functioning and suggests that the increased l e v e l s of progesterone do not provide optimum conditions, whether due to progesterone alone, or in r e l a t i o n to other reproductive hormones. Abnormal ovarian functioning during f i r s t cycles post parturn i s not s u r p r i s i n g as i t i s well documented that during the f i r s t and - 168 -possibly second estrus cycles post partum, reproductive hormonal production i s reduced. Mather et a l (1978) and Peters and R i l e y (1982) observed that, generally, progesterone concentrations during the f i r s t detected estrous cycle were lowered. These studies, however, did not take into account the varous phases of the estrous cycle as described i n the present study. In t h i s study, progesterone l e v e l s were generally observed to be lower throughout the entire cycle ( f o r cycle type 1), however, during the f o l l i c u l a r phase of the cycle (period 1) progesterone concentrations were a c t u a l l y higher than i s normally observed. In the present study i t was observed that cycles i n which c y s t i c corpora lutea w i l l develop have increased progesterone concentrations as e a r l y as period 1 and that period 1 i s reduced i n duration. This may indicate that f o l l i c l e s which w i l l eventually develop into c y s t i c CL's are abnormal at the f o l l i c u l a r stage (possibly due to early l u t e i n i z a t i o n ) , and i t may therefore be possible to detect such f o l l i c l e s early i n the estrous c y c l e . Short cycles (<17 days long) exhibited higher progesterone concentrations during period one. Period one was also s i g n i f i c a n t l y shorter for cycle type 6 (long cycle) which may indicate abnormal f o l l i c u l a r development and early l u t e i n i z a t i o n . From these observations, i t becomes apparent that i t i s possible to d i s t i n g u i s h between f o l l i c l e s destined to become c y s t i c f o l l i c l e s or c y s t i c corpora lutea based on both milk progesterone concentrations and period length (as defined i n t h i s study) within the f i r s t week of the estrous c y c l e . - 169 -During period 3, mean milk progesterone concentrations were s i g n i f i c a n t l y higher for pregnant cycles, abortions, and cycles with c y s t i c corpora lutea i n comparison to f i r s t cycles (short i n duration) post partum. Period 3 of the pregnant, abortive, and c y s t i c cycles were also s i g n i f i c a n t l y longer than several of the other cycles defined. These r e s u l t s indicate that milk progesterone concentrations during period 3 of the estrous cycle can also be used to monitor normal or abnormal ovarian a c t i v i t y ( f o r example with c y s t i c corpora lutea when no breeding has occurred) or used i n pregnancy diagnosis e a r l i e r than 21 days (based on increased progesterone concentrations during period 3. Other s i g n i f i c a n t differences i n period length among cycle types were observed for both periods 2 and 4. Although the differences among cycle types were highly s i g n i f i c a n t , the v a r i a b i l i t y observed i n the duration of period 2 was so great that separation of means was not possible and drawing conclusions from such information was not possib l e . In cycle type 3 (long f i r s t cycle) a somewhat longer period 4 was observed compared to other post quiescent cycles monitored, though the differences were not s i g n i f i c a n t . This observation suggests that the transient l u t e o l y t i c stage i s i n someway impaired, perhaps due to i n s u f f i c i e n t prostaglandin F201 stimulation. However, t h i s i n t e r p r e t a t i o n i s highly speculative and further research i s required i n th i s area. Although cycle type was found to be independent of treatment group, c e r t a i n trends were observed i n the frequency d i s t r i b u t i o n of cycle types among treatments. Based on milk progesterone data, during the second l a c t a t i o n , embryonic mortality was observed to occur with a - 170 -r e a l a t i v e l y high frequency i n both control and S supplemented cows (21.4%), whereas cows with Se supplementation had no incidence of the problem. Other i n t e r e s t i n g observations include the greater frequency of long f i r s t cycles post quiescent (cycle type 1) and long cycles ( c y c l e type 6) i n comparison to Se supplemented cows. These r e s u l t s lend support to findings of other researchers that indicates Se i s required for optimal reproductive performance. L u t e i n i z i n g Hormone Data L u t e i n i z i n g hormone i s the major hormone responsible for CL growth and maintenance and progesterone secretion i n the cow (Hansel & Snook. 1970). Snook et a l (1971) found a p o s i t i v e c o r r e l a t i o n between plasma LH and progesterone l e v e l s from day 3 to day 15 of the estrous c y c l e . However, during the l a s t part of the estrous cycle, LH and progesterone are negatively correlated (Snook et a l 1971). Furthermore, i t has been demonstrated that progesterone exerts an i n h i b i t o r y e f f e c t on LH secretion by the p i t u i t a r y (Hansel & Snook 1970). The rapid decline of progesterone p r i o r to estrus usually occurs on day 17 or 18 of the estrous cycle, but can occur as l a t e as day 20, even in cows with normal 21 day estrous cycle (Hansel & Snook 1970). S i m i l a r i l y , the occurrence of the LH preovulatory peak i s v a r i a b l e i n terms of whether i t appears j u s t p r i o r to, or j u s t a f t e r the onset of estrus (Hansel & Snook 1970). Furthermore, Hansel and Snook (1970) noted that the large pre-ovulatory surge never occurred u n t i l the i n i t i a t i o n of the preciptuous decline i n progesterone. This observation - 171 -i n d i c a t e s a negative feedback mechanism whereby progesterone "blocks" p i t u i t a r y secretion of LH u n t i l about the 1 7 t h day of the estrous c y c l e . When the p i t u i t a r y i s released from the progesterone feedback (caused by the l u t e o l y t i c e f f e c t s of prostaglandin F201), LH l e v e l s increase r a p i d l y r e s u l t i n g i n ovulation. Therefore, the i n t e r v a l between the i n i t i a l decline in plasma progesterone and the onset of the LH pre-ovulatory surge was measured i n t h i s study. This i n t e r v a l was found to be 3.49 and 2.13 ± 2.07 days for Holsteins and Ayrshires, r e s p e c t i v e l y . Though the diffe r e n c e was not s i g n i f i c a n t between breeds, i t must be noted that on average, the i n t e r v a l observed for the Holstein breed was at l e a s t one f u l l day longer i n duration. S i m i l a r i l y , although treatment group had no s i g n i f i c a n t influence on t h i s i n t e r v a l , cows i n the control group had a mean i n t e r v a l time of 3.72 ± 2.07 as compared to cows on treatment groups 2 and 3 which had an LH lag time of 2.62 and 2.50 ± 2.07. The longer i n t e r v a l s could t h e o r e t i c a l l y delay ovulation, and possibly delay the optimum breeding time. Also the longer i n t e r v a l may r e s u l t from reduced prostaglandin F 2 a synthesis, which could r e s u l t i n a delay i n the l u t e o l y s l s of the CL and prolonged elevated progesterone concentrations. Snook et a l (1971) Indicated that the preovulatory LH surge varied from 7 to 50 ng/ml. In a study by Schams et a l (1978) the mean plasma LH peak during the pre-ovulatory surge i n a normal cycle was 13.5 ±9.9 ng/ml. In the present study, treatment did s i g n i f i c a n t l y influence the magnitude of the peak value recorded during the preovulatory LH surge, with control group cows ex h i b i t i n g much higher - 172 -peak LH l e v e l s than either cows on treatment groups 2 or 3. The mechanism(s) responsible for the increased l e v e l s of plasma LH during the preovulatroy LH surge observed i n the control group cannot be explained within the scope of the study. However, i t can be stated that Se, i n some way, i s involved i n the balanced production of LH. Whether Se exerts i t s e f f e c t at the l e v e l of the hypothalamus, p i t u i t a r y , or the ovary cannot be ascertained by th i s study, and further research i s required i n t h i s area. However, i t can be speculated that increased l e v e l s of LH during the preovulatory surge could r e s u l t i n the increased production of progesterone beyond that which i s normal. It i s known that increased progesterone l e v e l s can ac t u a l l y i n h i b i t or i n t e r f e r e with reproductive functions (Hansel & Snook 1970). However, progesterone concentrations i n the present study were actually found to be s i g n i f i c a n t l y lower at breeding In the control group than i n other treatment groups. This would tend to agree with the findings c i t e d by Hansel and Snook (1970) that a negative r e l a t i o n s h i p exists between LH and progesterone concentrations at estrus. Schams et a l (1978) indicated that mean basal l e v e l s of LH were below 1 ng/ml. The r e s u l t s from the present study agree with those of Schams et a l (1978). Mean plasma basal LH l e v e l s observed i n the present study were below 1 ng/ml. However, breed differences were s i g n i f i c a n t (p £ 0.05) as Holsteins had plasma LH l e v e l s of 0.78 ± 0.49-ng/ml while the Ayrshires had mean basal l e v e l s of plasma LH of 0.64 ± 0.49 ng LH/ral. Although treatment did not influence basal LH l e v e l s , a breed - treatment i n t e r a c t i o n indicated that the two breeds responded - 173 -d i f f e r e n t l y to treatments. The Holstein breed had much higher basal LH l e v e l s than the Ayrshires in treatment group 2, whereas i n treatment group 3, the Ayrshire cows exhibited much higher basal LH l e v e l s . The reason for these findings, however, i s not r e a d i l y apparent. Health Data Health problems i n dai r y cows i n r e l a t i o n to Se other than those associated with reproduction have not been investigated to any great extent. Schingoethe et a l (1982) conducted a study to determine the e f f e c t s of supplementing cows consuming adequate Se (0.30 mg/kg Se) with Se and vitamin E pre-partum. Schingoethe et a l (1982) reported that the incidence of milk fever between control and treated animals was s i m i l a r . In the present study, the incidence of milk fever in both l a c t a t i o n s was not found to be dependent on treatment group. S i m i l a r i l y , the incidence of displaced abomasum, i n j u r i e s to feet and legs, and foot rot during the f i r s t l a c t a t i o n were not d i f f e r e n t between treatment groups, nor was the incidence exceptionaly high i n any group. Breed differences were not s i g n i f i c a n t for any of the parameters mentioned e i t h e r . The incidence of milk fever, however did approach s i g n i f i c a n c e (p = 0.10) during the f i r s t l a c t a t i o n as Ayrshires tended to have a higher incidence of milk fever (11.8%) than the Holsteins (0.0%). During the f i r s t l a c t a t i o n , m a s t i t i s was observed with a frequency of 5.3%, 12.5%, 33.3%, and 5.6% in treatment groups 1 - 4 , r e s p e c t i v e l y . The high occurrence of the problem in treatment group 3 - 174 -was found to be dependent on treatment group (p £ 0.05). In the second l a c t a t i o n , the incidence of m a s t i t i s had increased to 16.7%, 33.3%, and 40.0% for treatment groups 1, 2, and 4, r e s p e c t i v e l y , whereas the incidence declined to 14.3% for treatment group 3. However, occurrence of m a s t i t i s i n l a c t a t i o n 2 were independent of treatment group. Neither was there any s i g n i f i c a n t c o r r e l a t i o n between treatment group and the incidence of m a s t i t i s during the second l a c t a t i o n . O v e r a l l , the incidence of m a s t i t i s across a l l treatment groups ( i . e . , over two l a c t a t i o n s ) was 25% and was higher than d e s i r a b l e . Somatic c e l l counts were not d i f f e r e n t among treatment groups and further did not appear to be disproportionately high as would be expected with the high l e v e l of m a s t i t i s observed. The reason for t h i s finding may be that c e l l counts were done once per month throughout l a c t a t i o n , which did not necess a r i l y coincide with the occurrence of m a s t i t i s i n any one cow. Somatic c e l l data perhaps may have yielded more information had c e l l counts been done on cows at the time of contracting m a s t i t i s i n order to give some i n d i c a t i o n of the se v e r i t y of the disease. Therefore, there was no obvious or apparent reason for such a high frequency of the problem. I t can only be speculated that perhaps herd management may have had some bearing on the frequency with which mast i t i s was observed i n the UBC dairy herd. - 175 -CONCLUSION This study was conducted to determine the e f f e c t of a d d i t i o n a l d i e t a r y sulphur on the selenium response i n dairy cows, and consequent ra m i f i c a t i o n s on s p e c i f i c productive, reproductive and health parameters. Seventy-one l a c t a t i n g dairy c a t t l e were assigned to one of four dietary treatment groups: control (0.310 mg/kg Se, 0.35% S), S supplemented (0.311 mg/kg Se, 0.50% S), se supplemented (0.721 mg/kg Se, 0.35% S), and S & Se supplemented (0.722 mg/kg Se, 0.50% S). Monthly blood samples taken from a l l cows were used to monitor Se status i n response to treatment. The r e s u l t s from t h i s study indicate that plasma Se l e v e l s increased u n t i l approximately month four of treatment at which point plasma Se l e v e l s plateaued. During the f i r s t A months on treatment, cows supplemented with Se only had s i g n i f i c a n t l y d i f f e r e n t response curves than control or S supplemented cows, although slopes were s i m i l a r . Sulphur supplementation did not a l t e r plasma Se l e v e l s during t h i s time period. During the plateau phase, however, S & Se supplemented animals had s i g n i f i c a n t l y elevated plasma Se l e v e l s i n comparison to a l l other groups (based on comparison of equations). It may therefore be concluded that at higher dietary l e v e l s of S and Se, S enhances Se uptake or u t i l i z a t i o n . At lower l e v e l s of dietary Se, S did not appear to a l t e r Se metabolism. Dry cows consuming a normal dry cow r a t i o n which was found to be - 176 -d e f i c i e n t i n Se ( 0 . 0 7 5 mg/kg Se) had s i g n i f i c a n t l y r e d u c e d p lasma Se l e v e l s . Mean p lasma Se l e v e l s r e c o r d e d f o r d r y cows were found to be w e l l be low adequate p lasma Se l e v e l s . Breed and t r e a t m e n t were found to be s i g n i f i c a n t i n f l u e n c e s on t h e d a i l y i n t a k e o f f o r a g e , c o n c e n t r a t e , and t o t a l r a t i o n . The H o l s t e i n b r e e d consumed s i g n i f i c a n t l y more o f a l l t h r e e f e e d components t h a n the A y r s h i r e b r e e d . G e n e r a l l y , f e e d c o n s u m p t i o n I n c r e a s e d from the f i r s t l a c t a t i o n to the subsequent l a c t a t i o n , wh ich was p r o b a b l y due to cows m a t u r i n g f rom one l a c t a t i o n to the n e x t . M i l k y i e l d and kg m i l k f a t y i e l d were not i n f l u e n c e d by t r e a t m e n t . Dam p lasma Se was a s i g n i f i c a n t f a c t o r i n the v a r i a b i l i t y o b s e r v e d i n c a l f p lasma Se c o n c e n t r a t i o n s r e c o r d e d . No d i f f e r e n c e s among t r e a t m e n t g roups were o b s e r v e d i n days to f i r s t s e r v i c e , days o p e n , c a l v i n g i n t e r v a l and b r e e d i n g s / c o n f i r m e d c o n c e p t i o n . B r e e d , however , d i d i n f l u e n c e days o p e n , as the H o l s t e i n b r e e d had a s i g n i f i c a n t l y l o n g e r i n t e r v a l f rom c a l v i n g to c o n c e p t i o n than the A y r s h i r e b r e e d . P lasma Se l e v e l s a t b r e e d i n g were found to be d i f f e r e n t between b r e e d s . M i l k p r o g e s t e r o n e c o n c e n t r a t i o n s r e c o r d e d f o r A y r s h i r e s at b r e e d i n g t ime were s i g n i f i c a n t l y l ower than t h o s e r e c o r d e d f o r H o l s t e i n s . P lasma Se l e v e l s a t b r e e d i n g were s i g n i f i c a n t l y l ower i n c o n t r o l cows than f o r cows w i t h S & Se s u p p l e m e n t a t i o n . S i m i l a r i l y , m i l k p r o g e s t e r o n e c o n c e n t r a t i o n s at b r e e d i n g were s i g n i f i c n t l y l ower i n c o n t r o l cows i n c o m p a r i s o n to S & Se supp lemented c o w s . 177 -Plasma Se l e v e l s and milk progesterone concentrations at breeding time were not d i f f e r e n t for successful and unsuccessful breedings. A s i g n i f i c a n t i n t e r a c t i o n was found to ex i s t between treatments and (un)successful breedings. Milk progesterone concentrations were s i g n i f i c a n t l y higher for cows i n the unsuccessful breeding group (during the second l a c t a t i o n ) on the S & Se supplemented d i e t i n comparison to a l l treatment groups ( f i r s t l a c t a t i o n ) and the control and S supplemnted groups (second l a c t a t i o n ) . In the successful breeding group, milk progesterone concentrations were not d i f f e r e n t among treatments. Estrous cycles were characteized into d i f f e r e n t cycle types based on the occurrence of periods of s p e c i f i c progesterone concentrations. O v e r a l l , cycle lengths were not influenced by breed or treatment, however, i t was observed that during the second l a c t a t i o n , abnormally long cycles and abortions were observed more frequently i n the control and S supplemented group than i n either group with Se supplementation. Milk progesterone concentrations during s p e c i f i c periods of the estrous cycle were infuenced by treatment group. Cows on the control group exhibited s i g n i f i c a n t l y lower milk progesterone concentrations during periods 1 and 3 i n comparison to groups with Se and/or S supplementation. During period 4, mean milk progesterone concentrations were reduced i n the S supplemented i n comparison to the Se and S & Se supplemented groups. Progesterone concentrations during period 1 and 3, corresponding to the f o l l i c u l a r and l u t e a l phases of the estrous c y c l e , were found to be s i g n i f i c a n t l y altered among the various cycle types tested, - 178 -i n d i c a t i n g that progesterone concentrations during these periods can be used to monitor (ab)normal ovarian functioning. Holsteins had s i g n i f i c a n t l y higher basal plasma LH concentrations than Ayrshires, but breed did not influence LH lag time. Control cows had s i g n i f i c a n t l y elevated plasma LH concentrations during the preovulatory LH surge. Treatment did not influence plasma basal LH concentrations or LH lag time. However, i t was observed that control animals had an LH lag time one f u l l day longer than either S or Se supplemented animals. A s i g n i f i c a n t breed treatment i n t e r a c t i o n existed for plasma basal LH concentrations, i n which Holsteins reacted d i f f e r e n t l y to S supplementation than Ayrshires. A l l ovarian, uterine, and reproductive health t r a i t s were found to be independent of treatment, however, i t must be noted that the incidence of retained placenta i n the control and S supplemented groups during the second l a c t a t i o n was higher than i n eit h e r Se supplemented groups. Corresponding to these findings, the incidence of abnormal u t e r i i was also much higher i n those groups not supplemented with Se as opposed to those that were supplemented with Se. M a s t i t i s was found to be dependent on treatment group during the f i r s t l a c t a t i o n only. During the second l a c t a t i o n , however, the incidence of m a s t i t i s was not found to be dependent on treatment group. A l l other health t r a i t s tested were found to be independent of treatment group. - 179 -I n c o n c l u s i o n : 1 . S s u p p l e m e n t a t i o n o f d i e t s does not i n t e r f e r e w i t h Se m e t a b o l i s m , r a t h e r i t would appear t h a t when S and Se l e v e l s a r e f a i r l y h i g h In the d i e t , S a c t u a l l y enhances Se up take o r u t i l i z a t i o n . 2. Se s u p p l e m e n t a t i o n does no t i n f l u e n c e p r o d u c t i o n . 3 . Dam p lasma Se i s a s i g n i f i c a n t f a c t o r i n c a l f p lasma Se c o n c e n t r a t i o n s . A . Se s u p p l e m e n t a t i o n does i n f l u e n c e the c o n c e n t r a t i o n s o f p r o g e s t e r o n e ( m i l k ) and LH ( p l a s m a ) . 5 . M i l k p r o g e s t e r o n e c o n c e n t r a t i o n s d u r i n g the f o l l i c u l a r and l u t e a l phases o f the e s t r o u s c y c l e may be used to m o n i t o r n o r m a l o v a r i a n a c t i v i t y . 6 . 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Whanger, P.D., P.H. Weswig, O.H. Muth, and J.E. O l d f i e l d (1970). Selenium and white muscle disease; e f f e c t of su l f a t e and energy l e v e l s on plasma Se enzymes and ruminal microbes. - 189 -Whanger, P.D., P.H. Weswlg, J.A. Schultz and J.E. O l d f i e l d . (1977). E f f e c t s of selenium and vitamin E d e f i c i e n c i e s on reproduction, growth blood components and tissue lesions i n sheep fed p u r i f i e d d i e t s . J . N u t r i t i o n 107: 1288-1297. White, C.L., and Sommers, M. (1977). Sulphur selenium studies i n sheep. 1. The e f f e c t s of varying dietary sulphate and selenomethionine on sulpher, nitrogen, and selenium metabolism i n sheep. Aust. J . B i o l . S c i . 30: 47-56. Wilkins, J.F., and R.J. K i l g o u r . (1982). Production responses i n selenium supplemented sheep i n northern New South Wales. 1. I n j e r t i l i t y i n ewes and associated production. Aust. journal Exp. A g r i c . Antra. Husb. 22: 18-23. Young, V.R. (1981). Selenium; A case for i t s e s s e n t i a l i t y i n man. New England J . Med. 304(2): 1228-1230. - 190 -APPENDICES - 191 -APPENDIX 1 REFERENCES VALIDATING THE LEUTINIZING HORMONE ASSAY 1 . Kennedy, Richard I., and Norman C. Rawlings. (1984). Administration of constant low doses of androgens to steers by s i l a s t i c Implant: Suppression of gonadrotropins and peripheral conversion of androgens. Submitted to Journal of Andrology, March/April e d i t i o n . 2. Cook, S.J., N.C. Rawlings, and R.I. Kennedy 1983. Quantitation of si x androgens by combined high performance l i q u i d chromatography and radioimmunoassay. Submitted for p u b l i c a t i o n , Steroids. 3. Niswender, G.D., Reichert, L.E. J r . , Midgley, A.R. J r . , and Nalbandov, A.V. (1969). Radioimmunoassay for bovine and ovine l u t e n i n i z i n g hormone. Endocrinology, 1969; 84: 1166-1173. - 192 -APPENDIX 2 VITAMIN AND MINERAL COMPOSITION OF LOW-CAL MINERAL MIX Mineral Mix Component Level i n Mix (DM basis) Phosphorus 22% Iron 13,000 mg/kg Iodine 100 mg/kg Zinc 10,000 mg/kg Manganese 5,000 mg/kg Cobalt 50 mg/kg Copper 2,500 mg/kg Vitamin A 500,000 I.U./kg Vitamin D 50,000 I.U./kg Vitamin E (added) 6.6 I.U./kg APPENDIX 3 FREQUENCY (Z) OF TYPES OF CYCLES OBSERVED AMONG TREATMENT GROUPS Incidence of Observed Frequency (%) Cycle Types Treatment 0 1 2 3 4 5 6 7 8 9 10 11 12 13 1 18.1 20.0 15.4 0.0 19.2 18.8 14.3 28.6 0.0 7.1 21.1 11.8 0.0 15.0 2 10.6 10.0 7.7 0.0 11.9 6.3 22.9 11.9 9.1 14.3 7.9 11.8 25.0 10.0 3 16.0 18.6 15.4 0.0 2.05 12.5 8.6 14.3 27.3 7.1 15.8 23.5 0.0 15.0 4 12.8 18.6 7.7 0.0 25.6 15.6 11.4 14.3 18.2 28.6 18.4 23.5 0.0 20.0 5 16.0 10.0 15.4 50.0 9.6 15.6 22.9 14.3 9.1 21.4 15.8 23.5 0.0 14.0 6 10.6 10.0 7.7 50.0 5.0 15.6 14.3 11.9 0.0 21.4 5.3 0.0 25.0 11.0 7 7.4 4.3 23.1 0.0 5.5 3.1 0.0 2.4 18.2 0.0 10.5 0.0 25.0 5.0 8 8.05 8.6 7.7 0.0 2.7 12.5 5.7 2.4 18.4 0.0 5.3 5.9 25.0 10.0 194 -APPENDIX 4 REGRESSION EQUATIONS FOR DAM PLASMA SELENIUM VERSUS CALF BIRTH WEIGHT AND CALVING EASE Regression Equation Regression Equation Treatment (Calf Weight) (Calving Ease) 1 Y = 34.20 + 0.0338x n = 11 Y = 33.58 - 0.2895x n = 9 2 Y = 25.42 + 0.2192x n = 8 Y = 33.58 - 0.2895x n = 7 3 Y = 50.89 - 0.1308x n = 6 Y = 2.779 + 0.0397x n = 3 4 Y = 32.61 + 0.0092x n = 9 Y = 11.21 - 0.0639x n = 5 - 195 -APPENDIX 5 REGRESSION EQUATIONS FOR PEAK PROGESTERONE CONCENTRATIONS VERSUS BASAL PLASMA LH LEVELS AND LH LAG TIME T r e a t m e n t B a s a l LH vs Peak P r o g e s t e r o n e 1 Y = 0.8853 - 0 .3885 x 1 0 " 2 x n = 5 2 Y = 0.5357 + 0.5427 x 1 0 " 2 x n = 4 3 Y - 0 .9863 - 0 .3078 x 10~ 2 x n - 7 T r e a t m e n t LH Lag Time v s Peak P r o g e s t e r o n e 1 Y = 6.751 - 0.9621 x 1 0 " ^ n = 5 2 Y = 0.5672 + 0.8043 x 1 0 - 1 x n = 5 3 Y = 2.817 - 0 .8736 x 10~ 2 x n = 5 

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