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The effects of calcium carbonate on the apparent digestibility, serum concentration and apparent retention.. 1981

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THE EFFECTS OF CALCIUM CARBONATE ON THE APPARENT DIGESTIBILITY, SERUM CONCENTRATION AND APPARENT RETENTION OF DIETARY MINERALS IN DAIRY CATTLE by EDWARD BYRON CATHCART B. Sc. (Agr.), University of B r i t i s h Columbia, 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Animal Science) We accept t h i s t h e s i s as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA August, 1981 (c) Edward Byron Cathcart, 1981 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 the 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 r\ n Sc. The 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 2075 Wesbrook P l a c e V ancouver, Canada V6T 1W5 - i i - ABSTRACT The e f f e c t of increasing the calcium content of a hay-grain d i e t fed to postparturient d a i r y c a t t l e was studied. Mineral apparent d i g e s t i b i l i t i e s , serum concentrations and apparent mineral retentions were monitored while the animals were under the stress of peak l a c t a t i o n . Nine Holstein and seven Ayrshire cows were randomly assigned to the con t r o l (0.7% Ca) or the Ca-treatment (1.5% Ca) based on c a l v i n g order. The die t s otherwise contained adequate n u t r i e n t s . Each animal was fed to appetite f o r 80 days with no difference (p > . 05) occurring between treatments i n intake when expessed as a percen- tage of body weight. D a i l y milk production (4% FCM) was higher (p < .05) as was the average body weight (p<.01) for the control animals r e f l e c t i n g the disproportionate number of young animals on the calcium treatment. A f t e r a minimum of 60 days on t r i a l , 5 cows from each t r e a t - ment were exposed to a 5 day d i g e s t i b i l i t y c o l l e c t i o n period. No change i n organic matter or nitrogen apparent d i g e s t i b i l i t y occurred (p>.05) but there were higher (p<.05) l e v e l s of calcium and i r o n , increased (p<.01) l e v e l s of copper, and lower (p<.05) zinc and molybdenum apparent d i g e s t i b i l i t i e s f o r animals on the Ca-treatment. Fecal pH was higher (p<.05) i n the calcium treated cows i n d i c a t i n g a b u f f e r i n g e f f e c t occurred as a r e s u l t of the addition of the calcium carbonate. No change (p>.05) was evident i n the secretion of minerals into the milk but urinary phosphorus excretion was s i g n i f i c a n t l y - i i i - higher (p<.05) i n the control group. Milk progesterone was analyzed to correspond blood samples (average of 14 per animal) to s p e c i f i c regions of the estrus c y c l e . Serum phosphorus, i r o n , copper and zinc varied with reproductive c y c l i n g as phosphorus dropped (p<.01) at the onset of regular estrus while the other minerals fluctuated with the cycle (copper and zinc (p< .05), i r o n (p<.01)). In the serum of Ca-treated animals, calcium and zinc concentrations were higher (p<.01), copper increased (p<.05), and phosphorus was lower (p<.01) than the l e v e l s f o r the control ani- mals. Breed e f f e c t s were apparent as both phosphorus and copper were higher (p< .01) i n the serum of Ayrshires than of Holsteins. Plasma glucose concentrations proved not to be d i f f e r e n t (p>.05) between treatments. Calcium supplementation of the d i e t allowed the animals to go from a negative to a p o s i t i v e calcium balance (p<.01). I t also increased (p<.05) the amount of phosphorus apparent re t e n t i o n . In a l l , 6 e s s e n t i a l minerals had a l t e r e d apparent d i g e s t i b i l i t i e s and/or serum concentrations with possible long term e f f e c t s on animal metabo- l i s m . - iv - TABLE OF CONTENTS Abstract i i Li s t of Tables v Acknowledgements • v i Introduction 1 Literature Review 3 Materials and Methods 17 a) Data Collection and Analysis 17 b) St a t i s t i c a l Analysis 22 Results and Discussion 26 Summary and Conclusions 70 Bibliography 75 Appendix 86 - V - LIST OF TABLES Table 1 Nutrient Content of Rations Table 2 Other Nutrients in Rations Table 3 Performance Parameters Table 4 Summary of Percentage Apparent Digestibilities Table 5 Original Milk Progesterone Category Descriptions Table 6 Additional Hypotheses, ANOVA #1 and #2 Table 7 Combined Milk Progesterone Categories Table 8 Additional Hypotheses, ANOVA #3 Table 9 Summary of Serum Constituents Table 10 Total Daily Output of the Macro Elements in the Milk and Urine Table 11 Apparent Percentage Retention of the Macro Elements Table 12 Summary of the Effects of Calcium Carbonate on the Apparent Digestibility, Serum Concentration and Apparent Retention of Dietary Minerals 27 28 30 36 49 49 51 51 54 61 62 73 - v i - ACKNOWLEDGEMENTS The author would like to extend appreciation towards a l l those who offered guidance throughout the preparation of this thesis. To Dr. J.A. Shelford and Dr. R.G. Peterson who were freely open to consultation, I thank you for your stimulating thoughts and discussions. To the other members of my committee, I thank you for your encouragement. For technical assistance I would like to thank Mabel Striker for her thoughts on computer analyses and Gilles Galzy for his expertise in the f i e l d of atomic absorption. Also, I extend my appreciation to Evelyn Raine for typing this document. Finally, I could not express adequately the support I received from my loving wife Adrienne. - 1 - INTRODUCTION Calcium has long been recognized as an e s s e n t i a l component i n d a i r y c a t t l e n u t r i t i o n . I t s functions include s k e l e t a l growth and maintenance as well as many metabolic a c t i v i t i e s . In dairy c a t t l e , milk production and f e t a l growth are of prime importance; both of which depend on an adequate supply of calcium. The problem most often encountered i n high milk producing cows i s the increased need for calcium can not be met during the peak l a c t a t i o n period following par- t u r i t i o n . The maximum absorption rate of di e t a r y calcium i s insuf- f i c i e n t to meet the drain of calcium into the milk r e s u l t i n g i n withdrawal of calcium from the s k e l e t a l stores. I f t h i s i s only a temporary condition then the animal can recover and replace the l o s t calcium when milk production decreases. However, i f i t i s prolonged or i f the demand i s too rapid to enable the mobilization procedure to i n i t i a t e , then serious health problems occur. The minimum l e v e l of d i e t a r y calcium recommended by NRC (1978) has i n the past not allowed s u f f i c i e n t uptake of calcium to prevent a negative calcium balance during e a r l y l a c t a t i o n (unpublished data). The apparent d i g e s t i b i l i t y of t h i s calcium was low when com- pared with the optimum l e v e l suggested by Ramberg et a l . (1974). As the r e l a t i o n s h i p was dependent on di e t a r y intake l e v e l s , a study was proposed to investigate the p o s s i b i l i t y of increasing the amount of calcium absorbed by feeding higher l e v e l s of calcium to postparturient - 2 - da i r y cows. Factors other than calcium absorption may be affected when increased quantities of calcium are fed. Interactions with dietary ingredients within the dige s t i v e t r a c t , competition for absorption s i t e s and a l t e r a t i o n s i n storage and metabolism of several minerals can occur. This presents a major d i f f i c u l t y i n e s t a b l i s h i n g minimum requirements f o r di e t a r y nutrients as these influences can be quite v a r i a b l e . As we l l , homeostatic control of many of the body minerals complicates any i n v e s t i g a t i o n of metabolic i n t e r a c t i o n s . Whether these i n t e r a c t i o n s manifest within the range of increased calcium intake necessary to allow greater absorption of calcium has not yet been demonstrated. Several non-dietary f a c t o r s may also a f f e c t digestion and metabolism of the minerals. The p h y s i o l o g i c a l state of the animal has a major impact. In a t r i a l such as t h i s , the stage of l a c t a t i o n and the milk y i e l d would be responsible f o r the calcium drain on the ani- mals. Age, pregnancy, reproductive c y c l i n g and animal health have also been implicated i n al t e r e d digestion and metabolism (Thompson et a l . , 1978). Such factors must be considered before any meaningful r e l a t i o n s h i p s can be established. I t was the prime purpose of t h i s study to investigate the p o s s i b i l i t y of r a i s i n g the amount of calcium absorbed by animals under the stress of peak l a c t a t i o n . A l t e r a t i o n s i n apparent uptake and serum concentrations of the re l a t e d d i e t a r y nutrients were observed. Al s o , an i n v e s t i g a t i o n was undertaken of some of the non-dietary fac- t o r s r e l a t e d to absorption and retention of the minerals. - 3 - LITERATURE REVIEW The r o l e of calcium i n animal n u t r i t i o n has long been studied and there are many well established f a c t s . There are also many theories that remain unsubstantiated due to a lack of documentation or to ambiguities from s i m i l a r experiments providing d i f f e r i n g conclu- s i o n s . In t h i s study, the e f f e c t of increased d i e t a r y calcium on mineral absorption and the resultant concentrations of these minerals i n the serum were of prime i n t e r e s t . Unfortunately, r a r e l y does an experimentor i n n u t r i t i o n deal with a basic c a u s a l - e f f e c t r e l a - t i o n s h i p . Many fa c t o r s that may appear i n c i d e n t a l to the imposed treatment often influence the outcome of an experiment. In the pre- sent case these factors may be associated with the feedstuff; such as the p h y s i c a l form, the corresponding dietary components, or the bypro- ducts of di g e s t i o n . They could also be p h y s i o l o g i c a l parameters such as age and production stresses or they could r e s u l t from environmental conditions. The objective of the following section i s to i d e n t i f y some of the secondary i n t e r a c t i o n s that occur with calcium and to gain i n s i g h t i n t o how these might a l t e r the digestion and metabolism of d i e t a r y calcium. The addition of calcium i n the form of carbonate may r e s u l t i n an a l t e r a t i o n i n the pH i n the d i g e s t i v e t r a c t . Wheeler and Noller (1977) reported highly s i g n i f i c a n t differences (p<.01) between the pH l e v e l s within the small i n t e s t i n e , colon and feces of c a t t l e fed supplemental calcium carbonate versus those on unsupplemented d i e t s . - 4 - Such an a l t e r a t i o n i n the acid/base balance could have profound e f f e c t s on the a v a i l a b i l i t y of many of the dietary minerals. For example, Ender and Dishington (1970) proposed the a l k a l i n i t y of the d i e t was the o v e r r i d i n g factor i n determining calcium a v a i l a b i l i t y . According to Smith and McAllan (1966) a p r e c i p i t a t i o n of calcium phosphate occurred i n the small i n t e s t i n e of ruminants. The degree of p r e c i p i t a t i o n increased slowly with r i s i n g pH to the l e v e l of about 6.5 where i t then advanced r a p i d l y . The authors also showed an increased nonphosphate binding of calcium and magnesium i n the i l e a l e f f l u e n t with increasing pH. The exact nature of the organic c o n s t i - tuents involved i n the binding was unknown. Grace et a l . (1977) were able to separate out a d i l u t e a l k a l i - i n s o l u b l e f r a c t i o n associated with magnesium binding. This f r a c t i o n contained p e c t i n , which Molloy and Richards (1971) observed to be able to bind varying amounts of magnesium and calcium. Smith and McAllan (1966) observed a com- p e t i t i o n between magnesium and calcium for part of t h i s binding material of which calcium was generally bound more e f f i c i e n t l y at pH 5.5 and above. Substantial pH dependent binding of cations by the n o n - c e l l u l o s i c polysaccharides of the plant c e l l wall has been demonstrated by Branch et a l . (1975). These authors suggested the i o n i z a t i o n of the carboxyl groups of uronic acid might be responsible f o r binding appreciable amounts of d i e t a r y calcium. Other factors which may a l t e r calcium and magnesium a v a i l a b i - l i t y are also i n t e r r e l a t e d with pH change. Decreasing the a c i d i t y of the abomasal digesta would cause greater d i s s o c i a t i o n of proteins, - 5 - carbohydrates and other such organic molecules. This would allow increased opportunity f o r the binding of calcium and magnesium ions by e l e c t r o s t a t i c a t t r a c t i o n (Storry, 1961b). Nucleic acids have been shown by Chang and Carr (1968, c i t e d by Braithwaite, 1976) to be potent calcium binding agents down to pH 5.5. Also, p r e c i p i t a t i o n of calcium and magnesium soaps has occurred at higher pH l e v e l s espe- c i a l l y when f a t was added to the d i e t (Storry, 1961b; Tillman and Brethour, 1958). E a r l y work done by Talapatra et a l . (1948) discounted the influence of known calcium binding agents, such as oxa- l a t e and phytate, i n ruminants. This pH dependent p r e c i p i t a t i o n apparently did not occur as these complexes were metabolized by rumen microorganisms. Whether the e f f e c t of d i e t a r y protein on calcium absorption i s only pH r e l a t e d i s inconclusive. Braithwaite (1978) suggested decreased calcium retention i n protein d e f i c i e n t sheep may p o s s i b l y be due to decreased calcium binding p r o t e i n , hence a decreased rate of calcium absorption. A v a i l a b i l i t y of calcium from inorganic sources tended to be higher than that from organic sources (Hansard et a l . , 1957). The authors suggested decreased l e v e l s of d i e t a r y protein may be p a r t i a l l y responsible for the increased a v a i l a b i l i t y . Perhaps up to a c e r t a i n amount of d i e t a r y protein i s required for optimum absorp- t i o n of calcium but further amounts tend to decrease the a v a i l a b i l i t y of the calcium depending on the pH of the digesta. A change i n pH within the i n t e s t i n a l t r a c t may a l t e r the a v a i l a b i l i t y of calcium yet have no e f f e c t on the amount absorbed. - 6 - This could happen i f the a l t e r a t i o n i n a c i d i t y occurred a f t e r the area where most calcium was absorbed. Several authors (Ben-Ghedalia et a l . , 1975; Grace et a l . , 1974; Klooster, 1976) reported the major s i t e of calcium absorption i n ruminants to be the upper small i n t e s t i n e . Other workers showed s i g n i f i c a n t calcium absorption p r i o r to t h i s area. For example, Cragle (1973) proposed a net absorption of calcium from the abomasum. Leibholz (1974) indicated as much as 50% of calcium absorption occurred before the digesta reached the duodenum i n sheep that were i n p o s i t i v e calcium balance. In the c a l f , the aboma- sum a c t i v e l y p a r t i c i p a t e d i n calcium uptake (Yang and Thomas, 1965). The p r e c i p i t a t i o n of i n s o l u b l e calcium phosphate, according to Smith and McAllan (1966), was not appreciable u n t i l the pH exceeded about 6.5. Most reports indicated the pH of the material flowing through the upper sections of ruminant's small i n t e s t i n e remained a c i d i c (Ben-Ghedalia et a l . , 1975; Kay, 1969; wheeler and N o l l e r , 1977; Wheeler, 1980; Yano et a l . , 1979). Only as the digesta advanced along the i n t e s t i n e d i d the pH r i s e above 6.5 and a concomitant decrease i n calcium s o l u b i l i t y occurred. This information would therefore suggest the calcium a v a i l a b i l i t y i n the prime areas f o r absorption would not be influenced by the increase i n pH r e s u l t i n g from the a d d i t i o n a l d i e t a r y calcium carbonate. Similar occurrences could benefit or be detrimental to other mineral a v a i l a b i l i t i e s depending on t h e i r s i t e s of absorption. Magnesium and phosphorus are the minerals most often associated with calcium i n terms of a v a i l a b i l i t y . Phosphorus appears to be absorbed - 7 - in the same region as does calcium (Ben-Ghedalia et a l . , 1975; Grace et a l . , 1974; Klooster, 1976) with some net absorption occurring within the reticulo-rumen (Cragle, 1973). Reports on magnesium absorption are c o n f l i c t i n g . Cragle (1973) and Smith and McAllan (1966) claimed the main s i t e to be the upper small i n t e s t i n e while Ben-Ghedalia et a l . (1975) and Grace et a l . (1974; 1977) proposed absorption to take place p r i m a r i l y i n the forestomach and colon areas. Both magnesium and phosphorus have been reported to decrease i n solu- b i l i t y past the midpoint of the small i n t e s t i n e (Ben-Ghedalia et a l . , 1975). Like calcium, phosphorus absorption should be l i t t l e affected by a pH change i n the lower small i n t e s t i n e . Depending on the true s i t e of absorption of magnesium, a r i s e i n a l k a l i n i t y may or may not have a profound i n t e r a c t i o n . The modifying influence of the r a t i o of concentrations of d i e t a r y calcium to phosphorus on absorption and metabolism of each of these minerals i s often c i t e d i n the l i t e r a t u r e . Various dietary r a t i o s of calcium to phosphorus have been fed to ruminants, ranging from le s s than 1:1 to greater than 10:1. There i s general agreement that r a t i o s lower than 1:1 decreased calcium absorption (Manston, 1967; Wise et a l . , 1963) but r a t i o s between 1:1 and 7:1 produced no a l t e r e d absorption (Lomba et a l . , 1969; Manston, 1967; Wise et a l . , 1963). These same authors concluded absorption of phosphorus was unaffected by a l t e r i n g d i e t a r y r a t i o s . Lueker and Lofgreen (1961) observed no e f f e c t of the r a t i o - 8 - (even up to 10:1) on absorption but did f i n d an influence upon the excretion of metabolic f e c a l phosphorus. The phosphorus appeared to be absorbed i n d i r e c t r e l a t i o n to d i e t a r y phosphorus intake, whereas calcium was more influenced by p h y s i o l o g i c a l parameters. In an experiment with calcium d e f i c i e n t sheep, Braithwaite (1975) summarized i n that instance, phosphorus retention was c o n t r o l l e d by the rate of calcium r e t e n t i o n . I t therefore appears that within a wide range of calcium to phosphorus r a t i o s , neither mineral w i l l be d i f f e r e n t i a l l y absorbed but phosphorus excretion may be a l t e r e d . This wide v a r i a b i l i t y i n the p r a c t i c a l dietary r a t i o s of calcium to phosphorus l e v e l s i n ruminant feedstuffs i s i n contrast to the narrow proportions acceptable f or monogastric animals. Most single stomached animals require the calcium and phosphorus i n the d i e t to be t i g h t l y regulated around 2:1, the r a t i o of calcium to phosphorus i n the bone. Smith and McAllan (1966) demonstrated a d i r e c t r e l a t i o n s h i p between the concentration of inorganic phosphate and that of bound calcium i n the i n t e s t i n a l digesta. This was dependent on the calcium to phosphorus r a t i o and the i n t e s t i n a l pH l e v e l . A l t e r i n g the calcium to phosphorus r a t i o i n monogastric di e t s would have con- s i d e r a b l y greater e f f e c t on the calcium and phosphorus a v a i l a b i l i t y as the pH of the duodenal and i l e a l contents i s higher i n monogastric animals than i t i s i n ruminants (Wheeler, 1980). Through recent advances i n ra d i o a c t i v e tracer techniques some of the mechanisms of calcium absorption have been elucidated. To gain - 9 - i n s i g h t i n t o the i n t e r a c t i o n s of increased calcium i n the d i e t with other d i e t a r y minerals i t i s necessary to appreciate the complexities of t h i s mineral's absorption process. The method of calcium absorp- t i o n i n non-ruminants proposed by Wasserman and Taylor (1973, c i t e d by Braithwaite, 1974) was used by the author to explain the adaptations by sheep to changes i n calcium intake. This concept involved two pro- cesses. The f i r s t was a non-saturable, d i f f u s i o n a l one r e l a t e d to the concentration of calcium i n the i n t e s t i n e . The second was a saturable, a c t i v e process r e l a t e d to body needs. Braithwaite (1976) postulated the a c t i v e absorption method to be the major control mecha- nism for calcium absorption and stated that i t probably was dependent on bone calcium reserves. E a r l i e r work by Scott (1965) and Storry (1961a) suggested only a small f r a c t i o n of the calcium i n the sheeps i n t e s t i n e was absorbed by simple d i f f u s i o n of the free ion. The a c t i v e absorption of calcium has been observed by DeLuca (1976) to be dependent on vitamin D. The metabolite 1, 25-dihydroxy- c h o l e c a l c i f e r o l (D.C.C.) as well as vitamin D-induced calcium - binding proteins have been implicated i n the process. I t i s therefore understood that depending on an adequate supply of vitamin D, the absorption of calcium from the d i e t s fed to ruminants can be strongly influenced by t h e i r metabolic requirements for calcium. Another possible complication when considering mineral i n t e r a c t i o n s with calcium i s the excretion of metabolic calcium into the d i g e s t i v e t r a c t . Braithwaite (1976) summarized the main losses of calcium and phosphorus i n t o the i n t e s t i n e as that due to the d i g e s t i v e - 10 - j u i c e s . However, the amount of endogenous calcium excreted appears to be r e l a t i v e l y stable over varied conditions. Lueker and Lofgreen (1961) reported e s s e n t i a l l y constant excretion of calcium from diets with a h i g h l y v a r i a b l e calcium content and a wide range of calcium to phosphorus rations (0.8:1 to 6.0:1). Similar r e s u l t s were produced by Hansard et a l . (1954; 1957) and Lengemann (1965). The l a t t e r author suggested the amount of endogenous calcium l o s t into the d i g e s t i v e t r a c t of d a i r y c a t t l e was dependent on the l e v e l of calcium i n the serum. This dependency was previously reported to occur i n non- ruminants by Gran (1960, c i t e d by Braithwaite, 1976). Ramberg et a l . (1970) indicated a bias may e x i s t such that metabolic calcium excreted i n t o the i n t e s t i n e may be p r e f e r e n t i a l l y absorbed over dietary calcium. When comparing absorption rates from diets varying i n calcium content, A l i and Evans (1967) i n f e r r e d part of the reason for lower absorption from diets supplying l i t t l e calcium would be due to lower calcium i n the duodenal secretions and hence les s a v a i l a b l e calcium. Visek et a l . (1953) determined a lack of e f f e c t on calcium excretion due to increasing age when the rates were corrected for bodyweight. This information was used by Hansard et a l . (1954) to i n f e r i t was possible to measure the u t i l i z a t i o n of calcium from various d i e t a r y sources providing there was a constancy within groups of animals of known age and n u t r i t i o n a l status. Absorption measured by apparent d i g e s t i b i l i t y assumes constant endogenous excretion. Ramberg et a l . (1974) concluded t h i s assumption met i f the f e c a l data were averaged over a week or more. - 11 - Many of the above mentioned factors may act separately or i n combination such that the type of feedstuff influences the quantity of calcium absorbed. Calcium i n milk was shown to be almost t o t a l l y a v a i l a b l e to ruminants by Hansard et a l . (1954). However, i n forage d i e t s , l e s s than 40% a v a i l a b i l i t y i s generally reported (Braithwaite, 1975; Braithwaite and Riazuddin, 1971; Paquay et a l . , 1968). When grain was added to an a l l forage d i e t , Conrad and Hibbs (1973) i n d i - cated an improved calcium a v a i l a b i l i t y occurred. Stevenson and Unsworth (1978) concluded the type of roughage-cereal d i e t fed to sheep had a s i g n i f i c a n t e f f e c t on the apparent a v a i l a b i l i t y and reten- t i o n of calcium. Calcium from inorganic sources has been demonstrated to be s l i g h t l y more a v a i l a b l e than from organic sources (Hansard et a l . , 1957). Such r e s u l t s would make i t necessary to u t i l i z e v i r t u a l l y i d e n t i c a l d i e t s , varying only i n calcium content, to determine any true e f f e c t s of calcium supplementation. U t i l i z i n g blood mineral l e v e l s to indicate the concentration of those minerals within the body can lead to gross inaccuracies. Most minerals e x i s t i n separate forms r e f e r r e d to as pools or compart- ments within the body. For example, Braithwaite et a l . (1969) pro- posed the existence of s i x compartments of calcium i n sheep, one of which was serum. The concentration of calcium i n any of these pools depended on metabolic needs as well as di e t a r y intake. The main storage compartment appeared to be the bone which was under p o s i t i v e hormonal c o n t r o l . Copp (1969) indicated the parathyroid hormone increased the rate of calcium absorption into the bone while thyro- - 12 - c a l c i t o n i n caused a release of calcium from the bone. The intermediate pools a l l acted to buffer the system such that the serum most l i k e l y d i d not r e f l e c t t o t a l body storage of calcium. Ramberg et a l . (1976) concluded the concentration of calcium i n the serum remained remarkably constant over wide ranges of calcium intake which r e f l e c t e d the effectiveness of the homeostatic c o n t r o l . Blood values do however, show enough v a r i a b i l i t y to provide a reasonable estimation of d i e t a r y intake (McDonald and Belonje, 1975). One of the more profound factors which may exert an influence on the imposed treatments of an experiment dealing with mineral balance i s the p h y s i o l o g i c a l state of the animals. Thompson et a l . (1978) stated that a meaningful quantitative r e l a t i o n s h i p between d i e t and blood could only be determined when allowances were made for non- d i e t a r y f a c t o r s which a f f e c t the blood constituents. Some of these considerations were age, stage of l a c t a t i o n , milk production, gestation and animal health. Braithwaite (1975) reported the e f f i c i e n c y of absorption of d i e t a r y calcium to be high and r e l a t i v e l y constant over wide ranges of calcium intakes i n young growing ruminants. However, t h i s e f f i c i e n c y decreased with increasing calcium intake i n older animals and generally decreased with age (Braithwaite, 1976). The author stated that i n young animals, "the rate of absorption i s l i m i t e d by the a v a i l a b i l i t y of d i e t a r y calcium," whereas i n older animals, "the e f f i c i e n c y of absorption does not to any s i g n i f i c a n t degree r e f l e c t the a v a i l a b i l i t y of d i e t a r y calcim." Here, the decrease i n e f f i c i e n c y of absorption - 13 - with increasing age was postulated to r e f l e c t decreased calcium requirements. This f l u c t u a t i o n , as well as that of other minerals, has been monitored i n the blood by workers attempting to diagnose mineral dis o r d e r s . Thompson et a l . (1978) indicated a general decrease i n calcium, phosphorus, sodium, potassium and copper with increasing age. Inorganic phosphorus had the most dramatic decline from the under three year group to the f i v e to s i x year group. Not a l l researchers drew the same conclusions. Tumbleson et a l . (1973) produced s i m i l a r r e s u l t s f o r calcium and inorganic phosphorus but reported no e f f e c t of age on sodium or potassium. Kitchenham et a l . (1975) indicated no. s i g n i f i c a n t r e l a t i o n s h i p existed between calcium, sodium, potassium, copper or i r o n and age. These authors also stated that adjustment of inorganic phosphorus concentrations f o r age was perhaps u n j u s t i f i e d due to the small si z e of the reduction and the r e l a t i v e inconsistency of the measurement among herds. However, incorporating the e f f e c t of age into a study on mineral absorption and blood composition was con- sidered necessary. During the period of high calcium demand following the onset of l a c t a t i o n , calcium absorption has been shown by Symonds et a l . (1966) to increase. The authors concluded however, t h i s increase was i n s u f f i c i e n t to supply the needs of l a c t a t i o n and the animal was soon thrust i n t o negative calcium balance. Ramberg et a l . (1970) also demonstrated a r i s e i n calcium absorption from the gut e a r l y a f t e r the onset of l a c t a t i o n which was independent of the d i e t a r y calcium intake. - 14 - This increase i n calcium absorption a f t e r c a l v i n g has been described by Braithwaite et a l . (1972) and Klooster (1976) to be p r i m a r i l y an increase i n e f f i c i e n c y of absorption along with some decrease i n f e c a l endogenous calcium. This r e l a t i o n s h i p was apparent i n mature animals as w e l l , which temporarily reversed the decreased e f f i c i e n c y of absorp- t i o n due to age. Braithwaite (1976) used t h i s information to further elucidate on the theory of decreased absorption being due to decreased requirements i n older animals. Once l a c t a t i o n ceased and body stores were regenerated, the e f f i c i e n c y of absorption returned to i t s nor- mally low l e v e l . In studying the metabolic p r o f i l e of dai r y c a t t l e , Manston and Rowlands (1973) reported the v a r i a t i o n i n plasma inorganic c o n s t i - tuents among cows within the same milk y i e l d group to be les s than that among a l l cows within the herd. Rowlands and Pocock (1976) suggested the cows should be selected by t h e i r stage of l a c t a t i o n rather than t h e i r milk y i e l d . This increased the accuracy of the te s t as the blood constituents appeared to be more v a r i a b l e the closer the date of sampling was to c a l v i n g . I t was therefore i n f e r r e d that both stage of l a c t a t i o n and milk y i e l d should be considered i n any com- parisons of blood constituents. Work done on blood mineral concentrations with r e l a t i o n s h i p to estrus c y c l i n g has led to implications that reproductive hormones may influence serum mineral l e v e l s . Bach and Messervey (1969) reported possible s u b c l i n i c a l hypocalcemia a f t e r the estrus period i n d a i r y c a t t l e . The authors noted a drop i n the d i f f u s i b l e calcium - I n - f r a c t i o n over estrus and postulated that estrogen enhanced production of the calcium binding p r o t e i n . Braithwaite et a l . (1972) demonstrated that estrogen administered to sheep increased the rate of calcium absorption, retention and bone accretion. Plasma copper i n r a t s changed as plasma estrogen varied during the estrus cycle (Sato and Henkin, 1973). The authors considered t h i s to be due to estrogen stimulated ceruloplasmin synthesis. However, Money et a l . (1967) reported no e f f e c t on blood or l i v e r copper l e v e l s when estrogens were administered to sheep. Plasma zinc concentrations varied l i t t l e i n h e i f e r s taken at various stages of the estrus cycle (Dufty et a l . , 1977). According to Underwood (1977), manganese metabolism was influenced by estrogenic hormones. Any influence of gestation i n e a r l y pregnancy would most l i k e l y be due to an a l t e r a t i o n i n hormone l e v e l s rather than mineral demand for f e t a l growth. Breed e f f e c t s have been reported i n two papers. Serum calcium, potassium and magnesium were analyzed by Kitchenham and Rowlands (1976) to be s i g n i f i c a n t l y d i f f e r e n t between Holsteins and Ayr s h i r e s . South A f r i c a n c a t t l e breeds were shown by Heynes (1971) to have d i f f e r e n t blood mineral l e v e l s . While there i s r e l a t i v e l y l i t t l e information on t h i s parameter, these differences warrent further study. Seasonal f l u c t u a t i o n s i n blood mineral l e v e l s have been t i e d to v a r i a t i o n s i n d i e t s due to a l t e r e d climate, s o i l conditions and methods of management (Manston and Rowlands, 1973; Rowlands et a l . , 1979; Thompson et a l . , 1978). Such environmental factors can be excluded from a c o n t r o l l e d experimental s i t u a t i o n . - 16 - Blood sampling times and methods were also demonstrated to a f f e c t blood parameters. Plasma volume was influenced by water intake or by feeding on die t s containing high water content (Thompson et a l . , 1978). This i n turn had a d i l u t i n g e f f e c t on component l e v e l s . The constituents returned to t h e i r normal l e v e l s within four to six hours a f t e r feeding. Dougherty (1970) concluded venipuncture could cause excitement i n the animal r e s u l t i n g i n al t e r e d glucose, calcium and p o s s i b l y other blood constituents. Considering these f a c t o r s , every attempt should be made to keep blood sampling times and methods as consistent as possible between sample periods. To conclude, i t i s evident that there are many factors which might a f f e c t the r e s u l t s of the present experiment. These concerns range from the type of feedstuff fed to the p h y s i o l o g i c a l state of the animals used on the t r i a l s . To minimize any confounding i n t e r a c t i o n s , the experimental design should include animal parameters such as age, stage of l a c t a c t i o n , milk production, reproductive c y c l i n g and breed. Treatment rations should be as chemically and p h y s i c a l l y simi- l a r as pos s i b l e containing s u f f i c i e n t nutrients to allow optimum healt h , growth and production. Uncontrollable parameters such as a l t e r e d g a s t r o - i n t e s t i n a l a c i d i t y should be monitored as c l o s e l y as po s s i b l e to gain inference to any r e s u l t i n g complications. F i n a l l y , blood sampling procedures should be consistent throughout the t r i a l . - ] 7 - MATERIALS AND METHODS a) Data C o l l e c t i o n and A n a l y s i s : The experiment was c a r r i e d out between September 1979 and A p r i l 1980 on the U.B.C. campus dairy herd. Both Holstein and Ayrshire cows due to calve during t h i s period were used. There were approximately equal numbers of each breed. One t h i r d of the av a i l a b l e animals were h e i f e r s , the oldest cows were between 6 and 7 years at c a l v i n g and the average age was 42 months. The animals from each breed were randomized across 2 treatments depending on order of c a l v i n g . Of the 22 animals expected to calve, 2 did not calve during the t r i a l period and 4 others were removed from the t r i a l due to problems associated with abnormal c a l v i n g . This r e s u l t e d i n 8 cows per treatment, equal numbers of each breed on the control d i e t but 5 Holsteins and 3 Ayshires on the calcium treatment. Each animal remained on the t r i a l f o r 80 days postpartum. This period was chosen to enhance any i n t e r a c t i o n s due to production s t r e s s . The d i e t s were formulated to include recommended minimun l e v e l s of a l l nutrients as suggested by the National Research Council (1978). A commercial dairy concentrate containing 14% crude protein (as fed basis) and a vitamin-mineral pre-mix was fed along with chopped, orchardgrass hay at a r a t i o of 60% hay:40% grain. A g r i c u l t u r a l grade limestone (CaCOj) was added to the grain f r a c t i o n of the calcium treatment to ra i s e the concentration of calcium from 0.7% to 1.5% of the d i e t . A l l components of the die t s were c o l l e c t e d i n advance of the t r i a l to reduce the v a r i a b i l i t y i n the ingredients. - 18 - Loose cobalt-iodized salt was made available to the cows. Dry cows were brought into maternity s t a l l s approximately one week prior to calving where the orchardgrass hay was introduced to their diet. After parturition, the t r i a l rations were fed in increasing quantities until ad libitum intakes occurred. Feed intakes reached upwards of 3.5% of body weight per day for most cows by one week postpartum. The animals were moved to a free-stall barn where each cow was fed individually using the Calan door system. Feed levels were adjusted periodically to ensure the animals consumed a l l that was weighed out for them. Small quantities of unspoiled feed l e f t in a bunk was removed and mixed with the next feed. Weighbacks were collected and stored for analyses when larger volumes of feedstuffs remained. Occasional checks of intake levels were made by removing some of the animals from the free-stall area and placing them in stanchions where closer observations were made. Throughout the t r i a l , feeding occurred twice daily coinciding with milking. Daily records were kept of individual cow feed intakes and milk production. Once the milk was analyzed for milk fat percentage +, the total milk production was adjusted to the standard 4% fat corrected milk. Water intakes were not monitored although samples were collected periodically for mineral analyses. Weekly feed samples were collected +Infra-Red Milk Analysis, Provincial Government Dairy Testing Laboratory, Burnaby, B.C. - 19 - and stored f o r future t e s t i n g . From approximately 10 days a f t e r c a l v i n g , milk s t r i p p i n g samples were taken every second morning. Progesterone l e v e l s were determined on these samples by the method of Shelford et a l . (1979). Based on observed heats and progesterone predicted heats, blood samples were taken from the jugular vein at various stages of the reproductive c y c l e . The average number of samplings per cow was 14 with a range of 9 to 16. Blood samples were c o l l e c t e d between the afternoon milking period and the subsequent feeding period. A l l cows were weighed a f t e r they were bled. Approximately 30 ml blood was drawn f o r serum mineral analy- ses as well as 10 ml for plasma glucose determination. Hematocrit readings were undertaken on random samples to ensure plasma volume did not a l t e r g r e a t l y . A f t e r coagulation, the samples were centrifuged then f i l t e r separated. Any samples which exhibited v i s i b l e hemolysis were noted as t h i s condition can grossly influence serum magnesium and potassium l e v e l s ( C h r i s t i a n and Feldman, 1970). The serum was divided o i n t o 2 storage v i a l s which were kept frozen (-18 C) u n t i l the end of the t r i a l . F i v e cows from each treatment were confined i n stanchions where data was c o l l e c t e d to determine apparent d i g e s t i b i l i t i e s of the feed minerals. The animals chosen f o r t h i s t r i a l were ones that had been consuming the experimental r a t i o n f o r a minimum 60 days and who were not expected to be i n estrus during the c o l l e c t i o n period. Each - 20 - cow was placed i n a stanchion f o r one week to allow i t to become fami- l i a r with the area. Feed intakes were c l o s e l y monitored over t h i s period to ensure near t o t a l consumption. During the 5 day c o l l e c t i o n , feed intakes, f e c a l and urinary excretions, milk production and body weights were recorded f o r each animal. The cows were confined t o t a l l y to t h e i r stanchions and were milked with portable milking u n i t s . Feed samples c o l l e c t e d from each feeding were mixed together then subsampled f o r future chemical analyses. Fecal grab samples were composited and subsamples were taken f o r dry matter determinations. Values f o r pH were measured on random fresh f e c a l samples. Urine, c o l l e c t e d v i a a catheter, was not sampled u n t i l the f i n a l 2 days of the c o l l e c t i o n period due to blood c l o t s appearing during the i n i t i a l p a r t of the t r i a l . As suggested by C h r i s t i a n and Feldman (1970), the urine was made a c i d i c by adding 1 to 2 ml g l a c i a l a c e t i c a c i d per 100 ml. This was done to prevent the p r e c i p i t a t i o n of calcium phosphate which could entrap other mineral ions as w e l l . Milk samples from the evening milking period were kept separate from those of the morning. Blood samples were c o l l e c t e d twice during the d i g e s t i b i l i t y t r i a l . Rumen f l u i d was sampled by means of a hose connected to a vacuum pump. A l l samples were stored frozen (-18 C) for future analyses. Samples of the feedstuffs and feces were dried f o r 48 hrs. at 65°C i n a mechanical convection oven then were ground to pass through a 0.8 mm mesh s t a i n l e s s s t e e l screen. These were subsampled and stored i n polyethylene bags. Digestion involved the wet acid - 2 1 - technique of Parkinson and Al l e n (1975). Nitrogen and phosphorus were subsequently analyzed on a Technicon AutoAnalyzer I I . Calcium, potassium and magnesium were read on a Perkin Elmer 560 Atomic Absorption Spectrophotometer. Iron, copper, manganese and zinc were also read by atomic absorption spectroscopy but a f t e r a dry ash method as described by Heckman (1967). Dry ashing also yielded the t o t a l organic matter by d i f f e r e n c e . The turbidimetric method of Ferrara et a l . (1965) was used to measure s u l f u r while organic extraction was required f o r both molybdenum (Stupar et a l . , 1974) and selenium (Michie et a l . , 1978). Most serum minerals were analyzed by atomic absorption spectroscopy. The calcium method was that of C a l i et a l . (1973), magnesium and potassium were read i n d i l u t e d samples. Copper and zinc were measured by the method of Parker et a l . (1967), i r o n by Olson and Hamlin's (1969) technique and manganese was read by methods of addi- t i o n s as suggested by Mahoney et a l . (1969). Colorimetric deter- mination of phosphorus ( L i t t l e et a l . , 1971), the turbidimetric measurement of s u l f u r (Kennedy and M i l l i g a n , 1978) and the florometric a n a l y s i s of selenium (Michie et a l . , 1978) were used. Molybdenum was not determined due to the low l e v e l s i n the serum and the inadequacies of the present techniques. The plasma was analyzed f o r glucose using the Sigma Technical method No. 510 (1978). The minerals i n urine and milk were analyzed using s i m i l a r methods as those f o r serum. Milk samples were f i r s t deproteinized according to the technique of Brooks et a l . (1970). - 22 - Three other organic constituents of the feedstuffs were measured as w e l l . The f i r s t was d i g e s t i b l e energy, the difference between the gross energy of the feed and that of the feces, as deter- mined on the Gallenkamp Adiabatic Bomb Calorimeter. Acid detergent f i b r e was analyzed using the modified method of Waldern (1971). L a s t l y , ether extract was measured a f t e r extraction on a Goldfisch u n i t (Labconco). (b) S t a t i s t i c a l A n a l y s i s : The treatment e f f e c t was of prime i n t e r e s t i n t h i s experiment but there were other parameters which also had to be accounted f o r . The e f f e c t of breeds on the variables measured was unknown. Work done by Kitchenham and Rowlands (1976) was of s u f f i c i e n t i n t e r e s t to include breeds i n the model. Both treatments and breeds were viewed as f i x e d e f f e c t s i n a 2 x 2 f a c t o r i a l experiment. Four covariables were i n i t i a l l y considered important. These were; feed intakes expressed as a percentage of body weight, age at ca l v i n g (months), d a i l y milk production (kg 4%FCM) and days fr e s h . The f i r s t three applied to the apparent d i g e s t i b i l i t y data while a l l but the f i r s t one were used i n the analysis of the serum constituents. The incor- poration of categories of reproductive c y c l i n g into the model for the ana l y s i s of serum t r a i t s resulted i n these being nested within the fac- t o r i a l arrangement of the feeding experiment. - 23 - The model used to determine the effects on apparent digesti- b i l i t y was: where Yijk = u + Ti + Bj + TBij + I i j k + Aijk + Mijk + Eijk u = overall mean common to a l l samples. Ti = the effect of the i t h treatment. Bj = the effect of the jth breed. TBij = the interaction of the jth breed within the ith treatment I i j k = the covariable feed intake expressed as a precen- tage of body weight. Aijk = the covariable age at calving (months). Mijk = the covariable daily milk production recorded in kg 4%FCM. Eijk = the unexplained residual error associated with each sample. - 24 - The model used to determine the effects on serum constituents was: Y i j k l = u + Ti + Bj + Ck + TBij + TCik + A i j k l + Mijkl + F i j k l + E i j k l where u = overall mean common to a l l samples. Ti = the effect of the ith treatment. Bj = the effect of the jth breed. Ck = the effect of the kth category of reproductive cycle. TBij = the interaction of the jth breed within the ith treatment. TCik = the interaction of the kth category with in the i t h treatment. A i j k l = the covariable age at calving (months). Mijkl = the covariable daily milk production recorded in kg 4%FCM. F i j k l = the covariable days fresh or number of days from onset of lactation. E i j k l = the unexplained residual error associated with each sample. - 25 - Analyses were done by least-squares techniques using UBC BMD 10V for ANOVA with unequal numbers of observations per c e l l . The Newman-Keuls test was used for the comparison of means. Other factors related to the total feed and energy intakes were analyzed with the package program MFAV, using the following model: where Yijk = u + Ti + Bj + TBij + Aijk + Fijk + Wijk + Eijk u = overall mean common to a l l samples. Ti = the effect of the i t h treatment. Bj = the effect of the jth breed. TBij = the interaction of the jth breed within the ith treatment• Aijk = the covariable age at calving (months). Fijk = the covariable days fresh or number of days from the onset of lactation. Wijk = the covariable body weight (kg). Eijk = the unexplained residual error associated with each sample. The parameters measured were feed intakes, milk production, body weight change and plasma glucose concentrations. - 26 - RESULTS AND DISCUSSIONS Chemical analyses of the feedstuffs were undertaken on samples c o l l e c t e d over the period of the t r i a l . Results f o r the com- ponents of prime i n t e r e s t are presented i n Table 1 along with the minimum l e v e l s suggested by NRC (1978). Nutrients of secondary impor- tance to t h i s experiment but ones which could a f f e c t the performance of the cows, appear i n Table 2. As can be seen, a l l nutrients except p o s s i b l y energy, were suf- f i c i e n t to meet the requirements of the l a c t a t i n g dairy c a t t l e . Potassium, i r o n and manganese were considerably higher i n con- centration than the recommended minimum l e v e l s . While both i r o n and manganese were well within the safe l i m i t s f o r consumption (Underwood, 1977), the high l e v e l s of potassium may have interacted to depress magnesium absorption (Suttle and F i e l d , 1967). Also, due to con- tamination of the limestone used to supplement the treatment d i e t , the concentrations of i r o n and copper were higher i n the treatment r a t i o n than i n the con t r o l one. These factors w i l l be dealt with l a t e r i n the d i s c u s s i o n . Loose, cobalt-iodized s a l t was offered free-choice throughout the experiment. The amount of s a l t intake was not monitored and therefore any pos s i b l e e f f e c t of increased dietary calcium carbonate on sodium, cobalt and iodine remain undetermined. According to NRC (1978), the l e v e l of sodium required by milking dairy c a t t l e i s 0.18%. While the amount present i n the t r i a l rations was higher than t h i s , i t was judged best not to l i m i t the amount of s a l t a v a i l a b l e to the animals. - 27 - TABLE 1 Nutrient Content of Rations Component O.M.% N % Ca % P % K % Mg % S % Control 90.31 2.72 0.72 0.50 2.70 0.30 0.35 Ca-Treatment 90.31 2.72 1.50 0.50 2.70 0.30 0.35 NRC (1978 ) u minimum l e v e l s 2.08 0.43 0.31 2.56 0.60 0.40 0.80 0.20 0.20 Fe ppm Cu ppm Mn ppm Zn ppm 248 13.2 110 66 275 15.6 110 66 50 10 40 40 Mo ppb Se ppb 672 169 672 169 100 Ca:P 1.4: 1 3: 1 1.4:1 - 1.5:1 f o r l a c t a t i n g dairy c a t t l e between 400 and 600 kg producing between 8 and 36 kg milk d a i l y . - 28 - TABLE 2 Other Nutrients i n Rations Component D i g e s t i b l e Energy Mcal/kg A c i d Detergent F i b r e % Ether Extract % Na % NaCl % Co ppm I ppm Vitamin A I.U./kg Vitamin D I.U./kg Tot a l Diet (both Treatments) 3. 08 21.8 3.5 0.27 ad l i b 0 ad l i b 0 ad l i b 0 3425 c 310 c NRC (1978)° minimum l e v e l s 2.78 - 3.31 21 2 0. 18 0.46 0. 10 0.50 3200 300 f o r l a c t a t i n g dairy c a t t l e between 400 and 600 kg producing bet- ween 8 and 36 kg milk d a i l y . ad l i b i t u m consumption of cobalt-iodized s a l t , c a l c u l a t e d from grain source only. - 29 - Four parameters were measured to ensure adequate intakes of nu t r i e n t s occurred. These were feed intakes, milk production (4%FCM), body weight and plasma glucose concentrations (Table 3). Feed intakes, considered as a percentage of body weight, did not d i f f e r (p>.05) between treatments. The mean value for animals on the co n t r o l r a t i o n was 3.68% (dry matter b a s i s ) . Those cows on the calcium treatment consumed an average of 3.61% of t h e i r body weight i n feed per day. The range of intakes of a l l animals once adapted to the d i e t was from 3.16% to 4.22% which i s high when compared to the suggested maximum dry matter intakes of the NRC (1978) of 2.5% to 3.5% of body weight. The energy content of the d i e t (Table 2) did not meet the requirements of high milk y i e l d i n g cows (NRC, 1978). However, the higher than average feed intakes would p a r t i a l l y balance any energy d e f i c i t r e s u l t i n g i n near optimum engery intakes. The second v a r i a b l e measured was d a i l y milk production. Once standardized to the 4% f a t corrected milk l e v e l , the mean production of 24.49 kg and 23.71 kg for the co n t r o l and treatment animals respec- t i v e l y were demonstrated 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<.05). This r e f l e c t e d the disproportionate number of young animals on the calcium treatment. In comparison with past records and those of contemporary herd mates, milk production d i d not appear to be affected by either treatment. Body weight measurements were taken frequently throughout the t r i a l corresponding with blood sampling times. Due to i n d i v i d u a l ani- - 30 - TABLE 3 Performance Parameters Parameter Control C a-T reatment Mean1, Std. Dev. Mean Std. Dev. Feed Intake % of body weight 3.68 0.14 D a i l y Milk Production kg 4% FCM 24.49* 7.03 Body Weight kg 522.9** 45.8 Plasma Glucose mg/100 ml 53.18 8.06 Body Weight Change (slope) 0.036 3.61 23.71* 51.35 0. 16 5.86 492.5** 33.7 10.68 0.063 S i g n i f i c a n t l y d i f f e r e n t *(p<.05), **(p<.01) d n = 8 - 31 - mal v a r i a b i l i t y , an analysis of covariance was performed comparing the slopes of the l i n e s associated with body weight change f o r cows on each treatment. No s i g n i f i c a n t difference (p>.05) occurred as most animals, once age was accounted f o r , remained a constant weight (Control slope = 0.036, Ca-Treatment slope = 0.063). The mean weight of 492.5 Kg for the treatment animals was s i g n i f i c a n t l y lower (p<.01) than the average weight of 522.9 Kg for the control cows. Plasma glucose has been reported to be a stable, r e l i a b l e diagnostic a i d for assessing metabolic carbohydrate disturbances i n da i r y c a t t l e (Athanasiou and P h i l l i p s , 1978). No treatment e f f e c t s (p>.05) were observed for plasma glucose concentrations. The mean glu- cose l e v e l f o r the control animals was 53.18 mg/100 ml. A value of 51.32 mg/100 ml was determined i n the calcium treated animals. Other reports of glucose concentrations i n dairy c a t t l e have a range of means from 36.3 to 69.9 mg/100 ml (Manston and Rowlands, 1973; Rowlands et a l . , 1977; Thompson et a l . , 1978) with a grand mean near 45 mg/100 ml. Hunter (1977) suggested the glucose content of plasma less than 25 mg/100 ml generally was i n d i c a t i v e of i n s u f f i c i e n t energy supply which resulted i n lower non-return r a t e s . In the current study, no sample was analyzed to have les s than 38 mg/100 ml glucose concentration. Parker and Blowey (1976) rel a t e d plasma glucose to the stage of l a c t a t i o n . Concentrations of glucose increased from calving to nine weeks postpartum then leve l e d o f f at approximately 60mg/100 ml. While most animals i n t h i s t r i a l had lower glucose concentrations e a r l y i n - 32 - t h e i r l a c t a t i o n , not a l l animals d i d . This v a r i a b i l i t y r esulted i n a la c k of s i g n i f i c a n c e (p>.05) between the glucose concentration and the covariable days f r e s h . Hewett (1974) also reported no s i g n i f i c a n t e f f e c t of stage of l a c t a t i o n . Time of sampling appears to be r e l a t i - v e l y unimportant when u t i l i z i n g the glucose concentration to deter- mine metabolic carbohydrate disturbances, e s p e c i a l l y a f t e r the f i r s t few weeks postpartum. Considering the t o t a l nutrients i n the d i e t as well as the above parameters, i t i s apparent that adequate l e v e l s of dietary nutrients were ingested by both groups of animals. In an attempt to i d e n t i f y any a l t e r a t i o n of the pH l e v e l within the d i g e s t i v e t r a c t due to the a d d i t i o n a l calcium carbonate, samples of rumen f l u i d and feces were analyzed. Values for the rumen f l u i d ranged from a minimum of 7.5 to as high as pH 9.0. Heavy con- tamination with s a l i v a and unknown sample s i t e s within the rumen contributed to these high, random values. These data were considered unrepresentative and were therefore not recorded. Fecal pH values were more b e f i t t i n g those expected. The mean f o r the c o n t r o l group was 7.35 with a standard deviation of 0.16 while that f o r the calcium treated animals was 7.60 ± 0.13. A s i g n i f i c a n t d i f f e r e n c e (p<.05) existed between the two treatments implying calcium carbonate did indeed r a i s e the pH i n the d i g e s t i v e t r a c t . Wheeler and Noller (1977) stated, "that a pH measurement made on a grab sample of feces i s an excellant i n d i c a t o r of pH i n the small - 33 - i n t e s t i n e , " as there was no s i g n i f i c a n t difference between the average pH of the digesta within the small i n t e s t i n e and that of the feces. Mean values f o r the small i n t e s t i n e contents and feces of steers fed a high concentrate d i e t were about 5.8 pH u n i t s . The pH l e v e l rose to 6.8 when calcium carbonate was added to the d i e t . Russell and co-workers (1980) also recorded increased f e c a l pH i n steers fed high grain d i e t s supplemented with limestone. This r e l a t i o n s h i p was not consistent within sheep (Yano et a l . , 1979). No s i g n i f i c a n t d i f - ference i n pH was indicated as the calcium carbonate additions to a basal r a t i o n r a i s e d the calcium content from 0.1% to 1.2%. The f e c a l pH mean value was 8.3 while that i n the small i n t e s t i n e was 5.4, 7.4 and 8.5 r e s p e c t i v e l y f o r the upper, middle and lower portions. I t i s suggested that i n the present experiment the difference i n pH i n the feces was representative of differences within the d i g e s t i v e t r a c t . Unfortunately, i t cannot be known whether t h i s a l t e r a t i o n i n a c i d i t y i t s e l f was s u f f i c i e n t to cause modified dietary mineral a v a i l a b i l i t y . In f i r s t performing the s t a t i s t i c a l analysis of the d i g e s t i - b i l i t y t r i a l data, three covariables were included i n the model. These were; feed intakes expressed as a percentage of body weight, age at c a l v i n g (months) and d a i l y milk production (4% FCM). Each of these f a c t o r s have been reported to a f f e c t some mineral absorptions, however, none of the covariables proved to be s i g n i f i c a n t f o r any of the v a r i a b l e s . This can be explained by the control imposed on the experiment. F i r s t l y , the feed intakes were maintained at or near aid - 34 - l i b i t u m and when compared on a basis of body weight percentage, were discovered to be unaffected by treatment. High intakes of feed by a l l animals would produce s i m i l a r rates of passage through the dige s t i v e t r a c t . Considerable v a r i a t i o n i n digesta passage rate could r e s u l t i n a l t e r e d uptakes of many of the die t a r y nutrients i n c l u d i n g the minerals (Underwood, 1977). The second covariable, age, had r e l a t i - v e l y l i t t l e v a r i a t i o n . The youngest animal was twenty-four months at c a l v i n g and the oldest was sixt y - f o u r months. Most reports on e f f e c t s of age on a l t e r e d mineral absorptions ind i c a t e these e f f e c t s occur i n very young or very o l d animals. For example, obvious decreased absorption of calcium with increasing age may not appear u n t i l the animals are over ten years o l d (Hansard et a l . , 1954). Other mineral absorptions are eit h e r unaffected by age or the e f f e c t s are n e g l i g i b l e as to be masked by the v a r i a b i l i t y among animals (Scott, 1965). While the average d a i l y milk production varied widely between animals, each animal was i n i t s peak l a c t a t i o n over the t r i a l . I t can therefore be argued that the nutrient requirements would be at t h e i r highest during t h i s term. For example, even i f calcium absorption were at a maximum i t may not be able to supply the t o t a l amount of calcium required for the milk (Symonds et a l . , 1966). The v a r i a b i l i t y i n milk production during t h i s phase would have a lesser e f f e c t than i t would at a l a t e r stage of the l a c t a t i o n curve. Due to the lack of s i g n i f i c a n c e of the covariables, a second an a l y s i s of variance was undertaken deleting the covariables. This made the t e s t more robust by increasing the degrees of freedom i n - 35 - the error term which decreased the error mean square. The mean values and standard deviations of the apparent d i g e s t i b i l i t i e s of organic matter, nitrogen and eleven minerals are reported i n Table 4. Measurement of organic matter and nitrogen apparent d i g e s t i - b i l i t i e s were included to ensure two things. F i r s t of a l l , the e f f e c t of d i e t a r y calcium, or i n d i r e c t l y that of d i e t a r y calcium carbonate, on the organic constituents was assessed. I t was also necessary to ensure there was no obvious d e f i c i e n c y i n the organic content which might have influenced the inorganic component apparent d i g e s t i b i l i - t i e s . Both organic matter and nitrogen apparent d i g e s t i b i l i t i e s were shown to be unaffected (p>.05) by the increased calcium con- c e n t r a t i o n . The mean values f o r the organic matter were 75.41% and 75.88% for the con t r o l group and calcium treated cows re s p e c t i v e l y . The nitrogen values were even closer together at 74.79% for animals on the c o n t r o l r a t i o n and 74.76% for those on the calcium treatment. Smith et a l . (1966) fed al t e r e d q u a n t i t i e s and r a t i o s of calcium and phosphorus (1:1 to 4:1, 8:1 to 8:8) to d a i r y steers and noted no s i g n i f i c a n t change i n organic matter or nitrogen apparent d i g e s t i b i l i - t i e s . The mean organic matter d i g e s t i b i l i t y ranged from 67% to 71% while the means for nitrogen varied from 57% to 64%. Varner and Woods (1972) recorded digestion c o e f f i c i e n t s f o r organic matter of 74.2% and f o r p r o t e i n of 71.0% for a calcium carbonate supplemented r a t i o n fed to steers. Therefore, i t can be assumed there was adequate dietary organic matter and nitrogen present and that calcium did not a l t e r the - 36 - TABLE 4 Summary of Percentage Apparent D i g e s t i b i l i t i e s Component Control Ca-Treatment (%) Mean e Std.Dev. Mean e Std.Dev. O.M. 75.41 2.34 75.88 3.49 N 74.79 3.25 74.76 2.57 Ca 5.57* 4.53 26.60* 12.13 P 33.87 7.15 36.89 10.39 K 93.73 1.78 93.41 2.24 Mg 28.02 11.13 31.18 5.80 S 61.58 2.65 61.80 4.67 Fe -13.61* 10.55 2.98* 12.04 Cu 15.00** 5.43 34.98** 2.70 Zn 27.46* 9.40 10.40* 4.61 Mn 13.77 9.58 11.32 5.33 Mo 20.83* 6.05 9.32* 10.44 Se 45.49 8.09 43.66 9.01 S i g n i f i c a n t l y d i f f e r e n t *(p<.05), **(p<.01) e n = 5 - 37 - a v a i l a b i l i t y of these components. The apparent d i g e s t i b i l i t i e s of calcium associated with the two treatments did prove to be significantly different (p< . 0 5 ) . The level of 26.60% for animals on the calcium treatment approached the upper limits for apparent absorption from adequate diets according to Braithwaite et a l . (1969). U t i l i z i n g radioactive labling procedures, Braithwaite (1974) and Braithwaite and Riazuddin (1971) indicated the true absorption of calcium in sheep to be 37% to 40%. The 10% to 13% difference between the true calcium ava i l a b i l i t y and that which was apparent was due to endogenous excretion. Ramberg et a l . (1974) stated that 28% was about maximum apparent absorption but at lower intakes the absorption was less. This relationship was reported to be linear down to low intakes where i t became curvilinear. As the mean value of apparent d i g e s t i b i l i t y of the control ration calcium was only 5.57%, this would imply the ration contained less than optimum levels of calcium. The only explanation for this decreased absorption on the control ration would be due to lower calcium availability. Hansard et a l . (1957) reported the calcium ava i l a b i l i t y from ground limestone to be only marginally higher than that from orchardgrass hay. If such was the case then the increased availability of calcium probably was not due to the source. Additional limestone was thought to have adjusted the pH throughout the digestive tract as evidenced by the rise in fecal pH. However, this would be expected to have an adverse effect - 38 - by increasing the p r e c i p i t a t i o n of calcium phosphate (Smith and McAllan, 1966). If t h i s change i n a c i d i t y did occur, i t must not have been s u f f i c i e n t to cause p r e c i p i t a t i o n i n the f i r s t h a l f of the small i n t e s t i n e where calcium i s reportedly absorbed. I t therefore appears necessary to have a c e r t a i n precentage calcium i n the d i e t to overcome the calcium binding factors present i n the digesta. Under the d i e t a r y conditions of t h i s experiment, i t i s suggested the NRC (1978) recommended l e v e l s of 0.43 to 0.60% calcium would be too low to supply adequate calcium. Considering the needs of postparturient d a i r y c a t t l e , two to three times the recommended l e v e l s would be required to reach maximum calcium absorption r a t e s . To further the arguement that the increased d i e t a r y calcium carbonate did not appreciably a l t e r the pH l e v e l i n the upper i n t e s t i n e , there was no s i g n i f i c a n t difference (p>.05) i n phosphorus apparent d i g e s t i b i l i t i e s . Had the p r e c i p i t a t i o n of calcium phosphate occurred within the area of absorption there would have been a decreased d i g e s t i b i l i t y of both calcium and phosphorus for animals on the calcium treatment. This lack of difference agrees with Manstons' (1967) findings of no a f f e c t on the absorption of phosphorus by v a r i a t i o n i n calcium intake by d a i r y c a t t l e . T o o t h i l l (1963) reported a s i g n i f i c a n t decrease i n the percentage of phosphorus absorbed with increased calcium l e v e l s i n the d i e t s of r a t s . This species d i f f e r e n - t i a t i o n may be due to the higher pH l e v e l of the material flowing through the upper section of the monogastric i n t e s t i n e (Ben-Ghedalia - 39 - et a l . , 1975). The mean values for phosphorus apparent d i g e s t i b i l i t y of 33.87% and 36.89% for the control and calcium treatments r e s p e c t i v e l y , f a l l within the range reported by other workers. Lomba et a l . (1969) in d i c a t e d phosphorus d i g e s t i b i l i t i e s to be between 30 and 45% while Grace et a l . (1977) stated apparent absorption of phosphorus from many d i e t s fed to ruminants was u s u a l l y less than 30%. As the animals on the calcium treatment absorbed c o n s i s t e n t l y high l e v e l s of phosphorus and much more calcium than those on the control r a t i o n , the r a t i o of calcium to phosphorus of 3:1 appears to be more b e n e f i c i a l than that of 1.4:1. Magnesium absorption was also unaffected (p>.05) by the change i n calcium l e v e l s i n the d i e t . The apparent d i g e s t i b i l i t i e s were 28.02% for the control and 31.18% for the calcium treated cows. These are close to the average of 27.8% reported by Lomba et a l . (1968) for l a c t a t i n g d a i r y c a t t l e . i There i s evidence i n mongastric species which r e l a t e s magne- sium u t i l i z a t i o n to the l e v e l of d i e t a r y calcium. For example, Nugara and Edwards (1963) indicated high l e v e l s of calcium i n the d i e t of the chick prevented the deposition of magnesium i n the bone. The authors concluded t h i s to be due to a common mechanism of absorption for diva- l e n t ions. T o o t h i l l (1963) noted a s i m i l a r r e s u l t i n the r a t where add i t i o n of calcium chloride to the water supply s i g n i f i c a n t l y reduced the absorption of magnesium as indicated by increased f e c a l magnesium. Such an i n t e r a c t i o n was also evidenced i n guinea p i g s . Morris and - 40 - O'Dell (1963) postulated the d i r e c t p h y s i o l o g i c a l antagonism between calcium and magnesium to be responsible for the decreased absorption of magnesium. Whether t h i s i n t e r a c t i o n o s t e n s i b l y manifests i t s e l f i n rumi- nants i s undetermined. Increased calcium concentrations i n the digesta reduced absorption of magnesium i n calves (Smith and McAllan, 1967). This was i r r e s p e c t i v e of phosphate concentrations and pH l e v e l within the d i g e s t i v e t r a c t which implies magnesium absorption occurred p r i o r to the middle to lower small i n t e s t i n e . The above two factors exhibited marked reduction i n magnesium u l t r a f i l t r a b i l i t y i n the lower sections of the small i n t e s t i n e (Smith and McAllan, 1966). Scott (1965) claimed, "that any competition between calcium and magnesium i n absorption i n the sheep i s s u f f i c i e n t l y small to be concealed i n the v a r i a b i l i t y which e x i s t s between experiments." Smith (1969) suggested magnesium absorption across the i n t e s t i n a l wall was passive. Assuming t h i s and presuming a consistent flow of digesta, any a l t e r a t i o n i n the e f f i c i e n c y of absorption of magnesium would be due to a l t e r e d a v a i l a b i l i t y . As t h i s did not occur i n the present experiment, i t must be considered that any antagonism between calcium and magnesium i n the gut i s of a c u r v i l i n e a r r e l a t i o n s h i p . The change i n calcium content of the treatment d i e t was not s u f f i c i e n t to cause a change i n the a v a i l a b i l i t y of magnesium. The high potassium concentration i n the d i e t s was of concern as to i t s e f f e c t on magnesium absorption. Suttle and F i e l d (1967) - 41 - concluded high potassium intakes depressed magnesium absorption i n sheep. Because the apparent d i g e s t i b i l i t i e s of magnesium were r e l a t i v e to the range reported by other workers, i t i s suggested that the high potassium content did not appreciably a f f e c t magnesium a v a i l a b i l i t y . The apparent d i g e s t i b i l i t y of potassium bore no s i g n i f i c a n t r e l a t i o n s h i p (p>.05) with that of calcium. Both the apparent and true d i g e s t i b i l i t i e s of potassium are reported to be very high, unre- l a t e d to potassium intakes and f a i r l y constant (Paquay et a l . , 1969). These authors were unable to show a s i g n i f i c a n t c o r r e l a t i o n between the fate of potassium and that of calcium, magnesium or phosphorus i n d a i r y c a t t l e . The work reported here i s i n agreement with these con- cepts as the mean apparent d i g e s t i b i l i t y f o r animals on the control r a t i o n was 93.73% and for those on the calcium teatment was 93.41%. The apparent d i g e s t i b i l i t i e s of s u l f u r , copper and molybdenum w i l l be discussed together due to t h e i r reported i n t e r a c t i o n s (Underwood, 1977). In the review a r t i c l e of Huisingh et a l . (1973), the mechanisms involved i n the i n t e r a c t i o n s were explained as an anta- gonism between s u l f a t e and molybdate f o r transport systems and between copper, s u l f a t e and molybdenum as the formation of unavailable cupric thiomolybdate. In the present study, there was no s i g n i f i c a n t difference (p>.05) between s u l f u r d i g e s t i b i l i t i e s yet there was a hig h l y s i g n i f i c a n t d i f f e r e n c e (p<.01) for copper and a s i g n i f i c a n t d i f f e r e n c e (p<.05) f o r molybdenum apparent d i g e s t i b i l i t i e s . The value f o r copper - 42 - apparent d i g e s t i b i l i t y rose from 15.00% on the control r a t i o n to 34.98% for animals on the calcium treatment. The su l f u r d i g e s t i b i l i - t i e s were very constant with l e v e l s of 61.58% and 61.80% for the two treatments. Molybdenum decreased i n apparent d i g e s t i b i l i t y as the calcium l e v e l s increased. The control r a t i o n mean was 20.83% while that of the calcium treatment was only 9.32%. Because the di e t a r y concentration of s u l f u r did not vary nor di d the apparent d i g e s t i b i l i t y , i t i s suggested the antagonism between molybdenum and s u l f u r was not the reason f o r decreased molybdenum d i g e s t i b i l i t y . The other two p o s s i b i l i t i e s e x i s t i n g are a decreased molybdenum a v a i l a b i l i t y allowed an increased copper absorption or an increased copper a v a i l a b i l i t y caused the concomitant decrease i n molybdenum apparent d i g e s t i b i l i t y . Dick (1954) indicated very high intakes of calcium carbonate (90 g/day) l i m i t e d the storage of copper i n the l i v e r of adult sheep. However, feeding lower l e v e l s (35 g/day) to sheep did not a f f e c t the l i v e r copper concentration (Hemingway et a l . , 1962). Kirchgessner (1965) reported a drop i n copper retention i n sheep and dair y h e i f e r s when increased l e v e l s of calcium were fed. The author recorded no e f f e c t on the copper absorption by pigs and concluded t h i s may be due to pH differences i n the dig e s t i v e t r a c t of the two species. These reports are contrary to the r e s u l t s of the present t r i a l and as there are no reports on the e f f e c t of calcium on molybdenum a v a i l a b i l i t y , no d e f i n i t i v e conclusion i s p o s s i b l e . This author does favour the concept of the increased copper a v a i l a b i l i t y with increased d i e t a r y calcium carbonate intake. The - 43 - f i r s t of two reasons f o r t h i s opposing view concerns the amount and form of copper i n the calcium treatment d i e t . There was a copper con- tamination of the limestone r e s u l t i n g i n a s l i g h t increase i n the copper l e v e l s i n the treatment #2 (13.2 ppm vs 15.6 ppm). While t h i s may be small, i t could p o s s i b l y have had a s i g n i f i c a n t e f f e c t on the a v a i l a b i l i t y of the copper. The form of t h i s extra copper was pro- bably as carbonate as i t was associated with calcium carbonate. Underwood (1977) indicated the copper i n carbonate form was more e a s i l y d i g e s t i b l e than from any other form i n the d i e t . As a r e s u l t of the contaminated limestone, the copper l e v e l i n the supplemented grain portion of treatment #2 was 22% greater than i n the unsupple- mented grain portion of the control r a t i o n . This could have been the main reason f o r the increased copper apparent absorption. To expand on the second l i n e of reasoning regarding possible increased copper d i g e s t i b i l i t y requires i n c l u s i o n of another f a c t o r , d i e t a r y z i n c . The amount of zinc apparent d i g e s t i b i l i t y dropped s i g n i - f i c a n t l y (p<.05) from 27.46% in animals on the control d i e t to 10.40% f o r cows consuming the extra calcium carbonate. Zinc absorption i s reported to vary from a low l e v e l to greater than 80% of d i e t a r y intake depending on the source of the d i e t and other r e l a t e d factors ( M i l l e r , 1973). One of these factors was d i e t a r y calcium. Newland et a l . (1958) surmised that increased calcium uptake would i n t e r f e r e with zinc retention i n growing pigs but Suttle and M i l l s (1966) claimed t h i s to be due to a lower zinc a v a i l a b i l i t y i n calcium supplemented - 44 - r a t i o n s . Other authors have concluded while there appeared to be lower a v a i l a b i l i t i e s of zinc when high l e v e l s of calcium were present i n the d i e t , there was a paradoxical increase i n zinc retention (Kirchgessner, 1965; M i l l e r , 1973; M i l l s and Williams, 1971). According to O'Dell (1969), d i e t a r y phytate reacted to decrease the s o l u b i l i t y of z i n c . This process was accelerated upon addition of excess calcium both by mass action formation of calcium zinc phytate as well as by elevating i n t e s t i n a l pH. Zinc phytate was l e a s t soluble at pH 6 and above. However, M i l l e r (1975) indicated the phytate interac- t i o n with zinc was not evident i n ruminants. If high calcium diets lower zinc a v a i l a b i l i t y , there should be an increase i n copper a v a i l a - b i l i t y as both copper and zinc compete for the protein metal-binding s i t e s i n the i n t e s t i n e (Underwood, 1977). Unfortunately, the depth of t h i s experiment w i l l not allow a conclusion to be drawn. Dietary calcium may have increased the a v a i l a b i l i t y of d i e t a r y copper causing an interference i n zinc and molybdenum absorption. I t may also have decreased the a v a i l a b i l i t y of d i e t a r y zinc and molybdenum r e s u l t i n g i n increased copper a v a i l a b i l i t y . A l t e r n a t i v e l y , calcium carbonate may simply have acted as a c a r r i e r f o r a more highly a v a i l a b l e form of contaminant copper which i n turn interacted to reduce the l e v e l of zinc and molybdenum absorption. The e f f e c t of a l t e r e d pH can not be ruled out as the a v a i l a b i l i t y of copper and zinc were gr e a t l y influenced by the pH i n the g a s t r o - i n t e s t i n a l t r a c t (Kirchgessner et a l . , 1977). Further work i s necessary i n t h i s area. - 45 - The i n t e r p r e t a t i o n of the apparent d i g e s t i b i l i t y of i r o n i s confusing. A s i g n i f i c a n t difference (p<.05) existed between the means of the two treatments yet the mean value for the animals on the co n t r o l r a t i o n was negative (-13.61%). I t i s generally agreed that except through bleeding, i r o n excretion i n the gut of dairy c a t t l e i s very l i m i t e d (Underwood, 1977). Because of t h i s , a l t e r a t i o n s i n b o d i l y i r o n contents occurs through adjustment i n absorption rates. The author recorded i r o n absorption to be n i l when body stores were adequate and only during periods of need was i r o n absorbed across the i n t e s t i o n a l mucosa. The low mean value for apparent absorption of i r o n by animals on the calcium treatment (2.98%) insinuates these a n i - mals had adequate body stores of i r o n . Some of these animals also were i n negative absorption states as indicated by the high standard deviation (12.04%) associated with the mean. As to why the control group's mean i r o n d i g e s t i b i l i t y value proved to be negative can only be speculated upon. Negative i r o n retention has been demonstrated to occur i n horses by Spais et a l . (1977). The values ranged from -34.9% to 59.8% with a mean retention of 9.5%. These r e s u l t s were influenced by copper concentration but not by zinc or manganese l e v e l s . Possible forms of contamination were removed from the area where the animals were housed and less than 1% of the t o t a l d a i l y intake of i r o n could have found i t s way in t o the animals through ingestion of water. I t was therefore assumed there was net excretion of i r o n into the gut. Als o , t h i s excretion was being af f e c t e d by one or more factors that - 46 - became l i m i t e d when a higher l e v e l of calcium carbonate was added to the d i e t . The d i f f e r e n c e between the two i r o n d i g e s t i b i l i t y means may be due to s i m i l a r reasons as those expressed for copper. L i t t l e i s known about the a v a i l a b i l i t y of i r o n from various sources. The f a c t there was a higher concentration of i r o n i n the calcium treatment d i e t due to contamination of the calcium carbonate cannot be ignored. M i l l s and Williams (1971) implied i r o n absorption was adversely modified by calcium. Also, as the a v a i l a b i l i t y of i r o n i s reported to be g r e a t l y influenced by the pH i n the g a s t r o i n t e s t i n a l t r a c t (Kirchgessner et a l . , 1977), the calcium treatment should have lowered the d i g e s t i b i l i t y of i r o n due to the t a c i t increase i n pH. Copper, zinc and manganese have a l l been associated with lowered i r o n absorp- t i o n through d i r e c t competition f o r absorption s i t e s (Underwood, 1977). Zinc also i n t e r f e r e d with the incorporation into and the release of i r o n from f e r r i t i n . If copper was competing for absorption s i t e s then there should have been a drop not a r i s e i n i r o n absorption on the calcium treatment. It i s therefore suggested the decreased zinc a v a i l a b i l i t y i n the calcium supplemented r a t i o n probably was i n f l u e n t i a l i n bringing about the increase i n apparent d i g e s t i b i l i t y of i r o n . The mean value for manganese apparent d i g e s t i b i l i t y f o r those animals on the control treatment did not d i f f e r (p>.05) from that for cows on the calcium treatment. The l e v e l s were 13.77% and 11.32% - 47 - r e s p e c t i v e l y . In r a t s , L a s s i t e r et a l . (1972) reported a major e f f e c t of low d i e t a r y calcium on manganese metabolism both at the tissue l e v e l and i n the gut. The l e v e l s of calcium i n the above experiment were 0.1% and 0.6% with the r e s u l t i n g manganese d i g e s t i b i l i t i e s of 6.3% for the low calcium r a t i o n and 18.6% for the high calcium d i e t . I t i s therefore understood that i f t h i s r e l a t i o n s h i p does manifest i t s e l f i n d a i r y c a t t l e , the concentration of calcium i n the control d i e t was not low enough to a l t e r manganese metabolism. High dietary calcium and phosphorus appeared to increase the requirement for manganese i n c a t t l e by a f f e c t i n g absorption i n the small i n t e s t i n e as well as r e t e n t i o n i n the ti s s u e (Vagg and Payne, 1971). Again, the calcium l e v e l required to achieve t h i s r e s u l t was outside the range of that imposed on the present experiment. This was e s p e c i a l l y true of the phosphorus l e v e l , while there may be a r e l a t i o n s h i p between the amount of calcium i n the d i e t and manganese absorption and retention, the moderate concentrations of d i e t a r y calcium appear to bear no consequence. Selenium concentration i n the feces was between 54 and 57% of th a t i n the d i e t f or both treatments. The mean apparent d i g e s t i b i l i t y f o r the c o n t r o l animals was 45.49% which was not s i g n i f i c a n t l y d i f - f e r e n t (p>.05) from that of animals on the calcium treatment (43.66%). A report of selenium d i g e s t i b i l i t y i n hoggets (Cousins and Cairney, 1961 ) concluded the feces contained roughly 40% of the quan- t i t y of selenium fed. Butler and Peterson (1961) suggested t h i s l e v e l - 48 - was low f o r sheep as t h e i r r e s u l t s showed greater than 50% dietary selenium appeared i n the feces. These values are higher than those for monogastrics whose main route of selenium excretion i s v i a the urine. Formation of i n s o l u b l e , unavailable forms of selenium occur i n the rumen under hi g h l y reducing conditions (Underwood, 1977) which explains t h i s contrast i n methods of excretion. There are no reports of calcium a l t e r i n g the a v a i l a b i l i t y or absorption of d i e t a r y selenium i n ruminants. Milk progesterone l e v e l s were analyzed to i d e n t i f y the blood sampling times with s p e c i f i c regions of the reproductive c y c l e . The actual progesterone value was assigned to an i n t e g r a l category depending on i t s association with respect to the samples taken before and a f t e r i t . O r i g i n a l l y , there were nine such categories which are defined i n Table 5. Of the eight animals on each treatment, two did not begin c y c l i n g during the eighty day t r i a l . These four animals were diagnosed as having c y s t i c ovaries, which when treated resulted i n normal c y c l i n g . A l l other animals varied i n length of time between c a l v i n g and the onset of estrus. This ranged from 32 to 48 days. One complete reproductive cycle occurred before insemination was attempted and a l l cows exhibited at l e a s t one complete cycle within the t r i a l p e r i o d . To determine the e f f e c t of the reproductive cycle on the serum constituents, two separate analyses of variance were undertaken. The f i r s t u t i l i z e d the data of a l l cows while the second combined only - 49 - TABLE 5 O r i g i n a l Milk Progesterone Category Descriptions Category Des c r i p t i o n 1 Before any a c t i v i t y . 2 Random peak not associated with c y c l i n g . 3 F i r s t Proestrus 4 Estrus 5 Metestrus 6 Metestrus to Diestrus 7 Diestrus 8 Diestrus to Proestrus 9 Proestrus Maximum determinable l e v e l = 50 ng/ml TABLE 6 Add i t i o n a l Hypotheses ANOVA #1 and #2 Ho: u l = u ° c d Ho: u 3 = u 5 e e e Ho: U 6 - u ? = u 8 Progesterone Levels (ng/ml) 1 - 3 7 - 1 5 7 - 1 5 1 - 3 10 - 35 35 - 50 50 50 - 35 35 - 10 a, b, c, d, e Means i n the same l i n e with d i f f e r e n t superscript l e t t e r s d i f f e r s i g n i f i c a n t l y (p<.05). - 50 - those animals that cycled. Treatment and breed e f f e c t s were con- sidered as were the covariables of age at c a l v i n g , d a i l y milk produc- t i o n and the number of days since c a l v i n g . The f i r s t a nalysis of variance yielded several treatment by category i n t e r a c t i o n s whereas the second did not. This was i n t e r p r e t e d to mean the noncycling animals were in f l u e n c i n g the r e s u l t s due to having a l l t h e i r sample values placed i n the f i r s t category and should therefore not be included when attempting to determine the e f f e c t s of the reproductive c y c l e . The second analysis showed no dif f e r e n c e (p>.05) between many of the categories when the a d d i t i o n a l hypotheses l i s t e d i n Table 6 were tested. I t was decided to combine some of the categories to increase the number of observations i n each one. These new categories are described i n Table 7. The data was re-analyzed using the a d d i t i o n a l hypotheses i n Table 8. Serum inorganic phosphorus had a highly s i g n i f i c a n t difference (p<.01) i n category e f f e c t s . The values associated with precycling (category P) were higher than those of the other three categories. The predicted means were 6.42 mg/100 ml for category P and 5.13, 5.34, and 5.70 f o r categories E, T and D r e s p e c t i v e l y . None of the covariables exerted a s i g n i f i c a n t e f f e c t (p>.05) on the serum t r a i t . I t therefore was concluded reproductive c y c l i n g caused a depression i n the serum inorganic phosphorus l e v e l s . Serum i r o n proved to be highly s i g n i f i c a n t l y d i f f e r e n t (p<.01) when comparing categories P and E with categories T and D. I t also approached s i g n i f i c a n c e (p = 0.06) when category P was com- - 5 1 - TABLE 7 Combined Milk Progesterone Categories D e s c r i p t i o n Pre Cycling Estrus T r a n s i t i o n - Includes Proestrus and Metestrus Diestrus Previous Progesterone Categories 1, 2, 3 4 5, 9 6, 7, 8 Observations per category 47 28 38 49 TABLE 8 A d d i t i o n a l Hypotheses, ANOVA #3 u t i l i z i n g combined milk progesterone categories Ho Ho Ho Ho 3 u p - u E U T " U D u + u _ - u _ - u = 0 P E T D 2u u T - u D = 0 u ^ - u = 0 T D - 52 - pared with the others and when category E was contrasted with T and D. The only r e a l lack of difference occurred between category T and D. Because of the d e f i n i t e difference between values associated with low progesterone concentration and those with high progesterone con- c e n t r a t i o n , i t can be stated that the reproductive hormones exerted an e f f e c t on serum i r o n concentrations. In t h i s case the predicted means being tested were 1.22 mg/1 for category P and 1.19 mg/1 for category E versus 1.12 mg/1 for category T and 1.09 mg/1 for category D. Both serum copper and serum zinc showed a s i g n i f i c a n t d i f - ference (p<.05) when the mean of category T was contrasted with the other category means. The predicted means tested f or the four cate- gories f o r copper were 0.68, 0.70, 0.67 and 0.71 mg/1 and for zinc were 0.96, 0.93, 0.89 and 0.98 mg/1. As category T contained data from proestrus and metestrus there could p o s s i b l y have been an e f f e c t of the t r a n s i t i o n i n hormone l e v e l s on serum copper and serum z i n c . The serum inorganic s u l f a t e l e v e l s i n category P were s i g n i - f i c a n t l y d i f f e r e n t (p<.05) from those i n categories E, T and D. However, there was also a highly s i g n i f i c a n t (p<.01) e f f e c t of the cov a r i a b l e , days f r e s h . This category difference was dismissed due to the coincidence of category P being present only i n the e a r l y post- p a r t u r i e n t stage. No other serum components were found to vary with reproduc- t i v e c y c l i n g . The report of Bach and Messervey (1969) regarding the p o s s i b l e influence of estrogen on serum calcium could not be substan- t i a t e d here as the d i f f u s a b l e calcium f r a c t i o n was not measured. - 53 - Two of the serum minerals exhibited s i g n i f c a n t l y higher (p<.01) concentration i n the Ayrshires than i n the Holsteins. These constituents were phosphorus and copper. The mean phosphorus con- c e n t r a t i o n f or the Ayrshires was 6.29 mg/100 ml with a standard de v i a t i o n of 1.30 mg/100 ml. The Holstein serum contained an average of 5.19 mg/100 ml phosphourus with a standard deviation of 1.41 mg/100 ml. Copper average values were 0.74 mg/1 for the Ayrshires and 0.65 mg/1 for the Holsteins with standard deviations of 0.09 mg/1 for each group. As there was no breed e f f e c t i n apparent d i g e s t i b i l i t i e s of any of the minerals, these differences must be metabolically c o n t r o l l e d . Kitchenham and Rowlands (1976) did not study copper l e v e l s but did consider phosphorus. These authors reported no s i g n i - f i c a n t d i f f e r e n c e i n phosphorus concentrations between Ayrshires and Holsteins but did f i n d lower l e v e l s of calcium, magnesium and potassium i n the blood of the Ayrshires. I t i s assumed there was a n u t r i t i o n a l e f f e c t as well which confounded the comparison of the pre- sent study with that of Kitchenham and Rowlands (1976). A summary of the mean and standard deviation values for the serum constituents f o r the animals of each treatment appear i n Table 9. Indications of the covariable e f f e c t s on the serum components are presented i n Table A of the appendix. Serum calcium l e v e l s showed a highly s i g n i f i c a n t (p<.01) response, increasing from 9.87 mg/100 ml for animals on the control - 54 - TABLE 9 Summary of Serum Constituents Component Control Mean 9 s td.Dev. Ca-Treatment Mean1 Std.Dev. Ca mg/100 ml P mg/100 ml K meq/1 Mg mg/100 ml S mg/100 ml Fe mg/1 Cu mg/1 Zn mg/1 Se ug/1 9.87** 5.79** 5.36 2.39 47.2 1.16 0.67* 0.90** 66. 1* 0.80 1.46 0.94 0.35 6.3 0.23 0. 10 0.21 19.4 10.22** 5.42** 5.26 2.41 48. 1 1. 16 0.69* 1.02** 70.7* 0.70 1.46 0.80 0.22 7. 1 0. 19 0. 10 0.20 20.0 S i g n i f i c a n t l y d i f f e r e n t *(p<.05), **(p<.01) 9 n = 78 h n = 84 Mo - not analyzed Mn - grand mean 12 ug/1 - 55 - ra t i o n to 10.22 mg/100 ml for those animals r e c e i v i n g a d d i t i o n a l calcium. While the l e v e l of the serum calcium i n the control animals was well within the p h y s i o l o g i c a l range expected for dair y c a t t l e (8.78 - 10.38 mg/100 ml : Rowlands et a l . , 1977), i t was apparently being l i m i t e d by the concentration of calcium i n the d i e t and the a v a i l a b i l i t y of such. This r i s e corresponds to the findings of other authors that serum calcium shows a small e f f e c t of di e t a r y conditions (McDonald and Belonje, 1975; Payne et a l . , 1970; Thompson et a l . , 1978). The change i n calcium l e v e l s i n the serum was i n v e r s e l y r e l a t e d to the change i n serum inorganic phosphorus (p<.01). The mean for the control group (5.79 mg/100 ml) was higher than that for the calcium treatment (5.42 mg/100 ml). In s i m i l a r work reported by Bushman et a l . (1965), Hoar et a l . (1970) and Smith et a l . (1966), the l e v e l of phosphorus i n serum dropped when the calcium content of the d i e t was ra i s e d i n r e l a t i o n to the constant amount of die t a r y phosphorus. Smith et a l . (1966) described that a l t e r i n g the calcium to phosphorus r a t i o i n the feedstuff from 1:1 to 2:1 to 4:1 lowered the serum phosphorus but the authors reported i t , "did not, at any time, drop below the range normally expected for dair y cows." This range i s generally agreed to be between 4.6 and 7.8 mg/100 ml (Rowlands et a l . , 1977) with a mean near 5.5 mg/100 ml (Hunter, 1977, based on 1157 ana- lyses from l a c t a t i n g dairy c a t t l e . ) The calcium to phosphorus r a t i o change d i d not c o n s i s t e n t l y produce a change i n plasma inorganic phosphorus l e v e l s i n other studies such as those by Parker and Blowey - 56 - (1976) and Swenson et a l . (1962). Several authors have reported the phosphorus l e v e l i n the blood to be d i r e c t l y r e l a t e d to phosphorus intake but not to the phosphorus l e v e l s i n the body (McDonald and Belonje, 1975; Payne et a l . , 1970; Wise et a l . , 1963). As there was no s i g n i f i c a n t difference (p>.05) between the two treatments f o r apparent d i g e s t i b i l i t y of phosphorus, the increased calcium can be assumed to have had an e f f e c t on phosphorus metabolism. Serum potassium l e v e l s were not s i g n i f i c a n t l y d i f f e r e n t (p>.05) between the two treatments. The respective values for the co n t r o l and the calcium treated animals were 5.36 and 5.26 meg/1. Rowlands et a l . (1977) reported the range of serum potassium l e v e l s of 351 l a c t a t i n g d a i r y cows between 40 and 100 days post-calving to be 4.11 t o 6.11 meg/1 with a mean of 5.06 meq/1. Magnesium l e v e l s i n the serum were also unaffected by increased calcium intakes (p>.05). The mean values of 2.39 mg/100 ml f o r the con t r o l group and 2.41 mg/100 ml for the calcium treatment animals correspond to the published range of 2.28 to 3.08 mg/100 ml with a mean of 2.63 mg/100 ml (Rowlands et a l . , 1977). Parker and Blowey (1976) reported serum mangesium l e v e l s i n animals fed diets with calcium : phosphorus r a t i o s more c l o s e l y aligned with those of t h i s t r i a l . Their mean values were 2.29 to 2.45 mg/100 ml with no s i g n i f i - cant d i f f e r e n c e due to the calcium : phosphorus r a t i o . There are reports (Bushman et a l . , 1965; Hoar et a l . , 1970) i n d i c a t i n g serum magnesium was adversely a f f e c t e d by increased d i e t a r y calcium. - 57 - However, t h i s occurred only when magnesium d i g e s t i b i l i t y was lowered due to an i n t e r a c t i o n i n the gut with the increased calcium. As there was no change i n the apparent d i g e s t i b i l i t y of magnesium i n t h i s experiment, and no difference i n serum magnesium l e v e l s , i t can be concluded there was no s i g n i f i c a n t physical or metabolic i n t e r a c t i o n between calcium and magnesium. Kennedy and M i l l i g a n (1978) monitored the l e v e l of serum inorganic s u l f a t e i n sheep. The values ranged from 32.5 to 60.0 mg/1 with a mean of 45.0 mg/1. The mean values reported here are 47.2 mg/1 f o r the co n t r o l group and 48.1 mg/1 for the animals r e c e i v i n g extra calcium. These means were not s i g n i f i c a n t l y d i f f e r e n t (p>.05) and as there was no change i n apparent d i g e s t i b i l i t y of s u l f u r , there are no obvious i n t e r a c t i o n s between s u l f u r and calcium. Serum i r o n l e v e l s were apparently equal f o r animals on either treatment. The mean values were the same with only a s l i g h t d i f - ference i n the standard deviations (1.16 ± 0.23 mg/1 for the co n t r o l versus 1.16 ± 0.19 mg/1 for the calcium treatment). The widest range i n values reported i n the l i t e r a t u r e was that of Stout et a l . (1976), being 0.37 to 3.21 mg/1 with a mean of 1.72 mg/1. The change i n apparent absorption of i r o n when calcium was added to the d i e t did not follow through to the serum. Copper l e v e l s i n the serum of the animals on the calcium treatment were s i g n i f i c a n t l y higher (p<.05) than those on the control treatment. The means of 0.67 mg/1 and 0.69 mg/1 were quite s i m i l a r and considering the standard deviation of 0.10 mg/1 i n each case, i t - 58 - i s questionable as to whether these means are indeed d i f f e r e n t . Copper i n serum i s metabolically c o n t r o l l e d by the l i v e r and except i n the case of de f i c i e n c y or t o x i c i t y , should be l i t t l e a f fected (Underwood, 1977). However, as there was a highly s i g n i f i c a n t increase i n copper apparent d i g e s t i b i l i t i e s i n the calcium treated animals, i t i s possible t h i s increased copper could have contributed to t h i s r i s e i n serum copper. Hemingway et a l . (1962) reported a s l i g h t but s i g n i f i c a n t reduction i n serum copper l e v e l s i n sheep drenched d a i l y with calcium carbonate. The authors did not mention any information regarding d i g e s t i b i l i t y of copper so i t i s impossible to conclude as to whether t h i s e f f e c t was due to an interference i n absorption or metabolism. The range of serum values f o r l a c t a t i n g d a i r y c a t t l e reported by Rowlands et a l . (1977) was from 0.56 to 0.96 mg/1. The mean value was stated to be 0.73 mg/1. Increased d i e t a r y calcium resulted i n a s i g n i f i c a n t (p<.05) decrease i n zinc absorption but concomitantly caused a highly s i g n i f i - cant (p<.01) increase i n serum zin c . This paradox was f i r s t reported by Kirchgessner et a l . (1960; c i t e d by M i l l s and Williams, 1971). The mean value of serum zinc i n the animals on the control r a t i o n was 0.90 mg/1 whereby that from the animals on the high calcium d i e t was 1.02 mg/1. Depending on the stage of estrus, Dufty et a l . (1977) reported mean serum zinc values of 1.01 ± 0.16 mg/1 to 1.15 ± 0.27 mg/1. One mechanism that could p o s s i b l y account for t h i s increase i n serum zinc would be a metabolic i n t e r a c t i o n between calcium and z i n c . I t i s suggested increased l e v e l s of calcium i n the serum caused an increased - 59 - release of zinc from the tissues to the serum or a blockage i n the deposition of z i n c . Serum manganese l e v e l s were analyzed but due to l i m i t a t i o n s i n the techniques a v a i l a b l e , only an estimate could be reported. The amount present i n the serum of the cows on both treatments varied about the mean of 12 ug/1. The technique was not s u f f i c i e n t l y accurate to enable a difference to be determined between the t r e a t - ments. As to the possible e f f e c t of calcium on manganese, L a s s i t e r et a l . (1972) u t i l i z e d the isotope Mn to determine a major change i n manganese metabolism. This occurred both i n the tissue and within the i n t e s t i n e s when d e f i c i e n t l e v e l s of calcium were fed to r a t s . There i s widespread disagreement i n the l i t e r a t u r e as to the exact range of manganese i n serum (Underwood, 1977). U n t i l further methods become a v a i l a b l e , any e f f e c t of calcium on serum manganese i n dai r y c a t t l e w i l l remain unknown. Serum molybdenum could not be analyzed due to the low l e v e l s expected and the present inadequacies of equipment. There was a s i g n i f i c a n t increase (p<.05) in the l e v e l of serum selenium i n the animals exposed to a higher l e v e l of dietary calcium. However, there was also a s i g n i f i c a n t (p<.05) treatment : breed i n t e r a c t i o n . On examination of the data i t appeared that the low serum selenium values associated with one animal unduly affected the mean. This s i g n i f i c a n t difference must therefore be rejected as a r e a l d i f f e r e n c e due to treatment. Removing the data of t h i s one ani- mal from the analysis deleted the s i g n i f i c a n t d i f f e r e n c e . The mean - 60 - values of the remaining animals were 72 + 20 ug/1 whereas the l e v e l of serum selenium i n the cow i n question was only 4 4 + 1 3 ug/1. Underwood (1977) indicated the mean value of whole bood selenium i n ruminants was 80 ug/1 with a range of 40 to 200 ug/1. No explanation can be o f f e r e d as to why the selenium l e v e l i n the one animal was so low. M i l k and urine samples were analyzed to enable estimation of the percentage retention of the various minerals within the c a t t l e on each treatment. Due to the very low l e v e l s of trace elements i n the milk and urine and the d i f f i c u l t y i n assessing these concentrations accurately, a delineation of less than 3% of intake values was assumed to be n e g l i g i b l e . As a r e s u l t , only the macro mineral l e v e l s were used i n the estimation of the retention r a t e . The assumption there- fore was the apparent d i g e s t i b i l i t i e s of the trace elements repre- sented c l o s e l y enough the percentage of the mineral intake retained. Table 10 provides the means and standard deviations for the t o t a l d a i l y output of macro elements i n the milk and urine of the animals on the two treatments. The apparent percentage retention for these same minerals are presented i n Table 11. In ruminants, the urinary excretion of calcium has been reported to be both marginal and stable over wide ranges i n intakes (Braithwaite, 1974; Braithwaite, 1975, Ramberg et a l . , 1974). These losses were not s i g n i f i c a n t l y a f f e c t e d by changes i n absorption by age (Braithwaite and Riazuddin, 1971). Ramberg et a l . (1976) stated that i n general, the small v a r i a t i o n s observed i n urinary calcium could have - 61 - TABLE 10 To t a l Daily Output of the Macro Elements i n the Milk and Urine MILK Component (g/day) Ca P K Mg S Control Ca-Treatment Mean 1 27.51 15.32 32.09 2.56 6. 15 Std.Dev. 7. 06 3.50 9.83 0.67 3. 18 Mean 1 27.34 14.31 33.20 2.51 6.33 Std.Dev. 1.99 3.45 5.56 0.25 2.81 URINE Ca P K Mg S 0.34 8.84* 247.40 3.96 3.26 0. 18 3.54 87.39 1.92 1.82 0.57 6.53* 256.20 3.44 4. 18 0. 30 1. 15 85.29 2.24 2.33 S i g n i f i c a n t l y d i f f e r e n t * (p<.05) - 62 - TABLE 11 Apparent Percentage Retention of the Macro Elements Component (%) Ca P K Mg S Control Mean' Std.Dev. -18.6** 6.0 3.1* 10.4 27.3 23.4 14.1 9.0 13.4 6.7 Ca-Treatment MeanJ Std.Dev. 14.5** 12.8 10.3* 6.0 25.7 20.1 18.7 5.0 14.0 8.1 S i g n i f i c a n t l y d i f f e r e n t * (p<.05), ** (p<.01) j n = 5 - 63 - only a t r i v i a l e f f e c t on calcium metabolism. Such was the case in the present study where there was a s l i g h t but nonsignificant (p>.05) di f f e r e n c e between the means of the two treatments. The mean calcium excretion i n the urine for the control animals (0.34 g/day) repre- sented 0.31% of the average d a i l y intake of calcium while that for the calcium treatment group (0.57 g/day) comprised 0.23%. Paquay et a l . (1968) determined a highly s i g n i f i c a n t c o r r e l a t i o n between d i g e s t i b l e calcium and the calcium i n the milk. No such r e l a t i o n s h i p existed i n t h i s t r i a l as there was no s i g n i f i c a n t difference (p>.05) between treatments for calcium contents of the milk yet there was for apparent d i g e s t i b i l i t i e s . The l e v e l s of calcium i n the milk of the animals on both treatments were consistent with the mean value reported by Ramberg et a l . (1974) of 1.2 g/1. Converting the values i n Table 10 r e s u l t e d i n means of 1.13 g/1 and 1.15 g/1 r e s p e c t i v e l y f o r the c o n t r o l and calcium treated groups. Phosphorus i n the urine has been reported to decrease as calcium intake increases (Braithwaite, 1975; Bushman et a l . , 1965). The former author described the hypothetical mechanism for such action i n the following manner. "When the calcium intake was increased, more calcium was absorbed and bone resorption decreased. The supply of phosphorus from bone was reduced and urinary excretion of phosphorus decreased." This would explain the s i g n i f i c a n t l y lower (p<.05) phosphorus excretion i n the urine of animals fed supplemental calcium when compared with the cows on the control r a t i o n . The range of phosphorus excretion determined by Lomba et a l . (1969) was from 0.3 - 64 - to 3 0.5 g/day with a mean of 6.6 g/day i n the l a c t a t i n g cow. The phosphorus mean excretion rates of the control group (8.84 g/day) and the calcium treated animals (6.53 g/day), were well within the normal range. There was no s i g n i f i c a n t difference (p>.05) between groups f o r phosphorus secretion into the milk. The average amount secreted by the con t r o l group was 15.32 g/day and that of the calcium treated group was 14.31 g/day. The mean value (10.4 g/day) presented by Lomba et a l . (1969) was low but the range (3.2 to 21.4 g/day) covered the l e v e l s encountered i n the present t r i a l . The potassium l e v e l s i n the milk and urine varied widely within treatments but there was no difference (p>.05) in the mean qu a n t i t i e s of the two treatments. The range of milk potassium losses quoted by Paquay et a l . (1969) vary from 10.9 to 33.0 g/day with a mean of 23.7 g/day. The average potassium losses of the control and calcium treated animals, 32.09 and 33.20 g/day res p e c t i v e l y , were at the high end of the range most l i k e l y due to the high dietary content of potassium. As the urine i s the primary route f or potassium excre- t i o n ( M i l l e r , 1975) i t was not s u r p r i s i n g that almost 60% of the d i e t a r y potassium intake appeared i n the urine. The average d a i l y excretion of potassium by animals on the control d i e t was 247.40g (59.18% of intake) and the amount for the calcium treated cows was 256.20 g (59.82% of in t a k e ) . The average d a i l y secretion of magnesium into the milk f or the co n t r o l animals (2.56 g/day) did not d i f f e r (p>.05) from the amount secreted by the animals on the calcium treatment (2.51 g/day). - 65 - The range of values reported by Lomba et a l . (1968) for magnesium losses to the milk was 0.62 to 2.83 g/day with a mean of 1.85 g/day. While these authors indicated there was a nonsignificant c o r r e l a t i o n between d i g e s t i b l e magnesium and milk magnesium, they implied a s h i f t i n magnesium intakes most l i k e l y would influence the milk magnesium content. The mean intake l e v e l s that were reported were h a l f of the average f o r animals on t h i s experiment which would j u s t i f y the added secr e t i o n into the milk. The urinary magnesium was also reported to be s i g n i f i c a n t l y correlated with magnesium intake. Again, the mean d a i l y urinary excretions of magnesium for the animals on the present t r i a l (3.96 g/day f o r the c o n t r o l , 3.44 g/day f o r the calcium treatment) were s l i g h t l y higher than that derived by Lomba et a l . (1968) of 2.9 g/day. There was no difference (p>.05) in milk or urine sulfur con- tents when expressed as a t o t a l d a i l y output. Jenny and O'Dell (1979) demonstrated the urinary s u l f u r excretion i n dairy steers to be bet- ween 13.4 and 15.1% of the d i e t a r y intake l e v e l s . The r e s u l t s recorded i n Table 10 amount to approximately 14% of the dietary intake f o r the animals on both treatments. The major difference appears to be i n the amount of s u l f u r absorbed as there was about 11/2 times greater absorption of s u l f u r by the l a c t a t i n g dairy cows than by the d a i r y steers studied by Jenny and O'Dell (1979). This s u l f u r was most l i k e l y deposited i n the milk. Feeding higher l e v e l s of calcium to postpartum dairy c a t t l e - 66 - r e s u l t e d i n a highly s i g n i f i c a n t (p<.01) increase i n the percentage calcium retained. The animals on the control r a t i o n were i n a d e f i - c i t calcium balance r e q u i r i n g an average of 18.6% more calcium to meet a l l t h e i r p h y s i o l o g i c a l requirements. Presumably, these animals would a t t a i n a p o s i t i v e balance once the heavy l a c t a t i o n stress was removed. Braithwaite (1974) indicated calcium retention would be increased for up to f i f t e e n weeks a f t e r animals were able to absorb adequate calcium such that body stores could be replenished. After t h i s period, reten- t i o n returned to near zero. The calcium treated cows were i n a p o s i - t i v e balance (14.5% apparent retention) which would i n f e r these animals were stressed e a r l y i n t h e i r l a c t a t i o n s and were i n the pro- cess of r e b u i l d i n g body stores. The phosphorus apparent retention also increased from a mean of 3. 1% to a mean of 10.3% (p<.05) with the addition of calcium to the f e e d s t u f f . As the apparent absorption was not influenced, but the serum phosphorus l e v e l s and urinary phosphorus l e v e l s were lower, t h i s would support the hypothesis of Braithwaite (1975). I t i s evident the increased d i e t a r y calcium was s u f f i c i e n t to cause a net uptake of calcium into the bone which prevented the release of phosphorus. Potassium, magnesium and s u l f u r did not vary i n apparent absorption, milk secretion or urine excretion l e v e l s so the apparent r e t e n t i o n i t s e l f also did not change (p>.05). In t h i s case, increased dietary calcium had no e f f e c t on these mineral elements. Interp r e t a t i o n of the trace element apparent retention values remain gaurded as the concentrations i n the milk and urine were - 67 - unattainable. M i l l s and Williams (1971) stated that copper, zinc and manganese urinary output represents only 1 - 2% of the t o t a l of each mineral excreted. Kirchgessner (1965) claimed the milk secretion rates were only 1 - 3% of the d i e t a r y intake and the above minerals were retained at a rate of 10 to 20%. Iron losses to the milk and urine are both le s s than 0.5% of d i e t a r y l e v e l s (Underwood, 1977). This author also quoted the majority of molybdenum and selenium excre- t i o n i n ruminants appeared i n the feces unlike monogastrics whose main route of excretion for these two minerals was the urine. While the apparent d i g e s t i b i l i t y rates of the trace minerals are an overestima- t i o n of the retention of these elements, they most l i k e l y r e f l e c t a trend. The implications from such a study as t h i s can be extended towards d a i r y c a t t l e p r o d u c t i v i t y . Besides obvious parameters which a f f e c t growth, maintenance, milk production and f e r t i l i t y , many fac- t o r s have more subtle e f f e c t s depending on t h e i r association with other d i e t a r y ingredients. The s l i g h t a l t e r a t i o n s i n some of the minerals apparent absorptions and serum concentration would most l i k e l y have t h e i r greatest influence on f e r t i l i t y . Valyuskin (1974, c i t e d by Hideroglou, 1979) monitored feedstuffs fed to cows over a period of months. The author noted trace element l e v e l s i n the feed altered those i n the blood which caused a response i n f e r t i l i t y . Few reports consider the e f f e c t of increased calcium on reproduction. Swenson et a l . (1962) added excess calcium to non-legume d i e t s fed to d a i r y c a t t l e and noticed i n h i b i t e d ovulation and estrus - 68 - c y c l i n g . By adding trace elements to the d i e t , f e r t i l i t y improved. The calcium to phosphorus r a t i o has been studied by L i t t l e j o h n and Lewis (1960) and Parker and Blowey (1976), both of whom concluded that over a wide range the r a t i o appeared not to a f f e c t f e r t i l i t y . Moderately low (40 ppm) dietary manganese had no obvious e f f e c t on f e r - t i l i t y unless combined with a high calcium to phosphorus r a t i o whereby f e r t i l i t y was depressed (Hignett, 1960). No d e f i n i t e conclusions can be drawn from t h i s experiment as to the e f f e c t s of increased calcium intakes on f e r t i l i t y . Several inferences can be made on the possible a l t e r a t i o n s i n f e r t i l i t y brought about with the change i n dietary calcium l e v e l s . Hunter (1977) noted changes i n the plasma inorganic phosphate and blood glucose concentrations around mating time appeared to produce the greatest e f f e c t on f e r t i l i t y . With lower serum phosphorus con- centrations i n animals on the calcium treatment, the p o s s i b i l i t y of t h i s occurring would be greater. Both serum copper and serum zinc l e v e l s were increased with the addition of calcium carbonate. Copper l e v e l s i n the blood were studied by Hignett (1960) and L i t t l e j o h n and Lewis (1960). No s i g n i f i c a n t r e l a t i o n s h i p was apparent between copper i n the blood and reproductive performance. However, low f e r t i l i t y i n dairy c a t t l e i n d i c a t i v e of delayed or depressed estrus has been shown to be due to copper de f i c i e n c y (Underwood, 1977). Manickam (1977, c i t e d by Hideroglou, 1979) reported s i g n i f i - c a t l y higher sera l e v e l s of copper and zinc as well as i r o n and manga- nese i n regular breeder cows versus repeat breeders. These r e s u l t s - 69 - favour the animals on the calcium treatment. Whether the lack of a d d i t i o n a l calcium carbonate had a true influence on the l e v e l of serum selenium i s unknown. Such a d e f i c i e n c y has resulted in impaired reproductive performance i n a l l species studied (Underwood, 1977). Increasing the recommended calcium content of d a i r y c a t t l e r a t i o n s from 0.7% to 1.5% by adding supplemental calcium carbonate may improve the f e r t i l i t y of the animals. This may be brought about by increasing the l e v e l of copper and zinc and p o s s i b l y selenium in the serum. The higher l e v e l of dietary calcium would also adjust the calcium to phosphorus r a t i o closer to the range Kendall et a l . (1970) recommended when considering the needs of p a r t u r i e n t cows. - 70 - SUMMARY AND CONCLUSIONS This study was i n i t i a t e d to determine i f i t was possible to increase the amount of calcium absorbed by postparturient d a i r y c a t t l e under the stress of peak l a c t a t i o n . I t was also of prime i n t e r e s t to discover any detrimental e f f e c t s of such an increase. I t was deter- mined that by increasing the calcium content of the d i e t to approxima- t e l y twice the recommended l e v e l , the apparent absorption of calcium rose from a low l e v e l to near the maximum po s s i b l e . This led to an increase i n calcium apparent retention such that the animals went from a negative to a p o s i t i v e calcium balance. The dietary i n t e r a c t i o n s were confounded due to the form of the calcium added to the d i e t . However, i n many cases i t was possible to separate out the e f f e c t s of the a d d i t i o n a l calcium such that conclusions could be drawn. F i r s t l y , there was no a l t e r a t i o n i n the ad l i b i t u m intakes of the d i e t s or were there any change i n the d i g e s t i b i l i t i e s of the orga- n i c constituents. Neither milk production, body weight change nor plasma glucose were affected by the treatment. Addition of calcium had no gross physical or p h y s i o l o g i c a l e f f e c t s on the cows over the duration of the t r i a l . The one minor phy s i c a l v a r i a t i o n which occurred as a r e s u l t of the supplemental calcium carbonate was the i n f e r r e d increase i n i n t e s t i n a l pH. Of the si x minerals which exhibited differences i n apparent absorption and/or serum concentration, three agree well with the documen- t a t i o n i n the l i t e r a t u r e . The increased calcium intakes were r e f l e c t e d by a r i s e i n serum calcium l e v e l s . The zinc d i g e s t i b i l i t i e s - 71 - were lower due to an i n t e r a c t i o n with calcium in the i n t e s t i n e . However, the serum values of zinc were higher most l i k e l y because of a metabolic i n t e r a c t i o n with the calcium at the ti s s u e l e v e l . Although phosphorus d i g e s t i b i l i t i e s were apparently unaffected by the extra calcium, the serum concentrations were lower as was the urinary excre- t i o n r a t e . This was a r e s u l t of increased deposition of calcium which l e d to a lower mobi l i z a t i o n of phosphorus from the bone. The remaining element i n t e r a c t i o n s are less c l e a r l y defined. The e f f e c t on copper d i g e s t i b i l i t y was opposite that reported by other workers. The apparent d i g e s t i b i l i t y increased probably due to the contamination of the limestone with copper. I t i s possible part of t h i s increase was due to the decreased a v a i l a b i l i t y of the z i n c . The increased copper d i g e s t i b i l i t y most l i k e l y was responsible for the r i s e i n the serum copper l e v e l s . S i m i l a r l y , i r o n apparent d i g e s t i b i - l i t y may have been affected by the contaminant i r o n i n the limestone or by the decreased zinc a v a i l a b i l i t y . The e f f e c t s of the treatment on molybdenum remains undetermined as i t i s unknown whether calcium or copper was responsible for the decrease i n apparent d i g e s t i b i l i t y . Further work i s required to elucidate these matters. Equally as important to the evaluation of the main e f f e c t are the minerals which were unaffected by the treatment. Both phosphorus and magnesium absorptions remained constant despite reports of these elements c l o s e l y i n t e r a c t i n g with calcium i n the d i g e s t i v e t r a c t . I t was concluded the main s i t e s of absorption of these two minerals must be p r i o r to the middle to lower small i n t e s t i n e otherwise p r e c i p i t a - - 72 - t i o n of the calcium and magnesium phosphates would have resulted i n lower apparent absorptions. Also, the implied a l t e r a t i o n i n the pH l e v e l of the digesta probably was not a major factor governing the a v a i l a b i l i t i e s of calcium, magnesium or phosphorus. The increased calcium to phosphorus r a t i o appeared not to influence the absorption of the minerals but may have been responsible for the decrease in phosphorus excretion into the urine and the lower serum phosphorus l e v e l s . The increase i n d i e t a r y calcium had no e f f e c t on potassium, magnesium or s u l f u r . Serum selenium concentrations were higher i n the animals on the treatment d i e t but i t i s doubtful whether calcium was responsible. The e f f e c t s of calcium carbonate on the apparent d i g e s t i b i l i t y , serum concentration and apparent retention of dietary minerals i n the present experiment are summarized i n Table 12. Two secondary e f f e c t s were studied as w e l l . These were the influence of breeds and of reproductive c y c l i n g on serum constituent l e v e l s . Phosphorus and copper were i n higher concentration in the serum of Ayrshire cows than i n that of the Holsteins. These two minerals were also shown to fluctuate with the reproductive hormones as were zinc and i r o n . Any study dealing with comparisions of serum mineral l e v e l s should account for these f a c t o r s . The long term e f f e c t s of such a treatment as t h i s can only be postulated. Calcium supplementation r e s u l t e d i n i n t e r a c t i o n s with both macro and trace minerals. As a r e s u l t , i t would be expected that a l t e r e d metabolism may eventually show up most l i k e l y manifesting i t s e l f i n the f e r t i l i t y and reproduction of the cows. A study on t h i s - 73 - Table 12 Summary of the E f f e c t s of Calcium Carbonate on the Apparent D i s g e s t i b i l i t y , Serum Concentration and Apparent Retention of Dietary Minerals Apparent Serum Apparent Mineral D i g e s t i b i l i t y Concentration Retention Ca + ++ ++ P 0 + K 0 0 0 Mg 0 0 0 S 0 0 0 Fe + 0 N.A. Cu ++ + N.A. Zn - ++ N.A. Mn 0 N.A. N.A. Mo - N.A. N.A. Se 0 + N.A. 0 Unaffected + Rose S i g n i f i c a n t l y (p<.05) ++ Rose Highly S i g n i f i c a n t l y (p<.01) - Dropped S i g n i f i c a n t l y (p<.05) Dropped Highly S i g n i f i c a n t l y (p < .01) N.A. Not Analyzed - 74 - aspect should be i n i t i a t e d to determine whether the long term e f f e c t s are b e n e f i c i a l or detrimental. This study has i d e n t i f i e d the f a c t that the NRC (1978) recom- mended d i e t a r y calcium l e v e l s were too low to maintain postparturient d a i r y c a t t l e i n p o s i t i v e calcium balance. I t has also exhibited i t p o s s i b l e to increase the l e v e l of calcium absorbed by r a i s i n g the calcium content of the d i e t by 2 to 3 times the recommended rate. A suggested more b e n e f i c i a l l e v e l of calcium in the r a t i o n would be 1.5% with a calcium to phosphorus r a t i o of 3:1. The long term e f f e c t s of such a treatment require further research. BIBLIOGRAPHY A l i , R. and J.L. Evans, 1967. E f f e c t of d i e t a r y calcium, b u f f e r i n g capacity, lactose and EDTA on pH and calcium absorption from g a s t r o n i n t e s t i n a l segments i n the growing r a t . J . Nutr. 93:273 - 279. Athanasiou, V.N. and R.W. P h i l l i p s , 1978. S t a b i l i t y of plasma metabo- l i t e s and hormones i n parturient d a i r y cows. Amer. J . Vet. Res. 39:953 - 956. Bach, S.J. and A. Messervey, 1969. Observations on the d i f f u s i b l e calcium f r a c t i o n i n the serum of the cow during oestrus and during p a r t u r i t i o n . Vet. Rec. 84:210 - 213. Ben-Ghedalia, D., H. Tagari, S. Zamwel, and A. Bondi, 1975. S o l u b i l i t y and net exchange of calcium, magnesium and phosphorus i n digesta flowing along the gut of the sheep. Br. J. Nutr. 33:87 - 94. Braithwaite, G.D., R.F. Glascock and Sh. Riazuddin, 1969. Calcium metabolism i n l a c t a t i n g ewes. Br. J. Nutr. 23:827 -834. Braithwaite, G.D. and Sh. Riazuddin, 1971. The e f f e c t of age and l e v e l of di e t a r y calcium intake on calcium metabolism i n sheep. Br. J. Nutr. 26:215 - 225. Braithwaite, G.D., R.F. Glascock and Sh. Riazuddin, 1972. The e f f e c t of hexoestrol on calcium metabolism i n the sheep. Br. J . Nutr. 28:269 -273. Braithwaite, G.D., 1974. The e f f e c t of changes of di e t a r y calcium concentration on calcium metabolism i n sheep. Br. J . Nutr. 31:319 -331. Braithwaite, G.D., 1975. Studies on the absorption and retention of calcium and phosphorus by young and mature Ca-deficient sheep. Br. J. Nutr. 34:311 - 324. Braithwaite, G.D., 1976. Calcium and phosphorus metabolism i n rumi- nants with s p e c i a l reference to parturient p a r e s i s . J . Dairy Res. 43:501 - 520. Braithwaite, G.D., 1978. The e f f e c t of di e t a r y protein intake on calcium metabolism of the pregnant ewe. Br. J. Nutr. 40:505 - 507. Branch, W.J., D.A.T. Southgate and W.P.T. James, 1975. Binding of calcium by di e t a r y f i b r e : i t s r e l a t i o n s h i p to unsubsti- tuted uronic acids. Proc. Nutr. Soc. 34:120A. - 76 - Brooks, I.D., G.A. Luster and D.G. Easterly, 1970. A procedure for the rapid determination of the major cations i n milk by atomic absorption spectrophotometry. At. Absorp. Newsl. 9:93-94. Bushman, D.H., R.J. Emeric and L.B. Embry, 1965. Experimentally induced ovine phosphatic u r o l i t h i a s i s : Relationships i n v o l v i n g dietary calcium, phosphorus and magnesium. J . Nutr. 87:499 - 503. Bu t l e r , G.W. and P.J. Peterson, 1961. Aspects of the faecal excretion of selenium by sheep. N.Z.J. A g r i c . Res. 4:481 - 491. C a l i , J.P., G.N. Bowers and D.S. Young, 1973. A referee method for the determination of t o t a l calcium i n serum. C l i n . Chem. 19:1208 - 1213. C h r i s t i a n , G.D. and F.J. Feldman, 1970. Atomic Absorption Spectroscopy. Applications i n Agr i c u l t u r e , Biology and Medicine. Wiley - Interscience. New York, London, Sydney, Toronto. Conrad, H.R. and J.W. Hibbs, 1973. Changes i n ab s o r b a b i l i t y of calcium and phosphorus associated with d i e t a r y ingredients. J . Dairy S c i . 56:646. Copp, D.H., 1969. Endocrine control of calcium homeostasis: A Review. J . Endocr. 43:137 - 161. Cousins, F.B. and I.M. Cairney, 1961. Some aspects of selenium meta- bolism i n sheep. A u s t r a l . J. A g r i c . Res. 12:927 - 942. Cragle, R.G., 1973. Dynamics of mineral elements i n the dige s t i v e t r a c t of ruminants. Fed. Proc. 32:1910 - 1914. DeLuca, D.F., 1976. Metabolism of vitamin D, current status. Amer. J . C l i n . Nutr. 29:1255 - 1274. Dick, A.T., 1954. Studies on the a s s i m i l a t i o n and storage of copper i n crossbred sheep. A u s t r a l . J . Agr i c . Res. 5:511 - 544. Dougherty, R.P., 1970. Discussion on "Hypocalcaemia and hypomagnesaemia" In. Physiology of Digestion and Metabolism i n the Ruminant. Proceedings of the Third International Symposium, Cambridge, England. Ed. A.T. P h i l l i p s o n , O r i e l Press, Newcastle. Dufty, J.H., J.B. Birigley and L.Y. Cove, 1977. The plasma zinc con- centration of nonpregnant, pregnant and parturient Hereford c a t t l e . A u s t r a l . Vet. J . 53:519 - 522. - 77 - Ender, F. and I.W. Dishington, 1970. E t i o l o g y and prevention of pare- s i s puerperalis i n d a i r y cows. In. Parturient Hypocalcaemia. Ed. J.J.B. Anderson, Academic Press, New York. F e r r a r a , I.W., R.S. Floyd and R.W. Blanchar, 1965. Turbidimetric determination of s u l f a t e by the autoanalyzer: Sulfur i n plant materials by digestion with n i t r i c and p e r c h l o r i c a c i d . In. Automation i n A n a l y t i c a l Chemistry. Mediad Inc. White P l a i n s , New York. Grace, N.D., M.J. Ulyatt and J.C. Macrae, 1974. Quantitative di g e s t i o n of fresh herbage by sheep. I I I . The movement of Mg, Ca, P, K and Na i n the d i g e s t i v e t r a c t . J . A g r i c . S c i . , Camb. 82:321 - 330. Grace, N.D., E. Davies and J . Monro, 1977. Association of Mg, Ca, P and K with various f r a c t i o n s i n the d i e t , digesta and faeces of sheep fed fresh pasture. N.Z.J. Ag r i c . Res. 20:441 - 448. Hansard, S.L., C.L. Comar and M.P. Plumber, 1954. The e f f e c t s of age upon calcium u t i l i z a t i o n and maintenance requirements i n the Bovine. J . Anim. S c i . 13:25 - 36. Hansard, S.L., H.M. Crowder and W.A. Lyke, 1957. The b i o l o g i c a l a v a i l a b i l i t y of calcium i n feeds for c a t t l e . J . Anim. S c i . 16:437 - 443. Heckman, M., 1967. Minerals i n feeds by atomic absorption spectrophotometry. J.A.O.A.C. 50:45-50. Hemingway, R.G., N.A. Brown and J.S.S. I n g l i s , 1962. The e f f e c t s of calcium carbonate, lead acetate and copper supplements on blood and l i v e r copper concentrations of young sheep. Res. Vet. S c i . 3:348 - 356. Hewett, C., 1974. On the causes and e f f e c t s of v a r i a t i o n s i n the blood p r o f i l e of Swedish dai r y c a t t l e . Acta. Vet. Scand. Supp. 50:1 - 152. Heynes, H. 1971. The e f f e c t of breed on the composition of blood. J . A g r i c . S c i . , Camb. 76:563 - 565. H i d i r o g l o u , M., 1979. Trace element d e f i c i e n c i e s and f e r t i l i t y i n ruminants: a review. J . Dairy S c i . 62:1195 - 1206. Hignett, S.L., 1960. The influence of n u t r i t i o n on female f e r t i l i t y i n some of the large domestic animals. Proc. Nutr. Soc. 19:8 - 14. - 78 - Hoar, D.W., R.J. Emeric and L.B. Embry, 1970. Potassium, phosphorus and calcium and i n t e r r e l a t i o n s h i p s i n f l u e n c i n g feedlot performance and phosphatic u r o l i t h i a s i s i n lambs. J . Anim. S c i . 30:597 - 600. Huisingh, J . , G.G. Gomez and G. Matrone, 1973. Interactions of copper, molybdenum and s u l f a t e i n ruminant n u t r i t i o n . Fed. Proc. 32:1921 - 1924. Hunter, A.P., 1977. Some n u t r i t i o n a l factors a f f e c t i n g the f e r t i l i t y of d a i r y c a t t l e . N.Z. Vet. J . 25:305 - 307. Jenny, B.F. and G.D. O'Dell, 1979. Ration d i g e s t i b i l i t y and mineral balance i n steers fed a complete e n s i l e d d i e t . J . Anim S c i . 48:1525 - 1529. Kay, R.N.B., 1969. Digestion of protein i n the i n t e s t i n e s of adult ruminants. Proc. Nutr. Soc. 28:140 - 150. Kendall, K.A., K.E. Harshbarger, R.L. Hays, E.E. Ormiston and S.L. Spahr, 1970. Responses of dair y cows to diets con- t a i n i n g varied l e v e l s of calcium and phosphorus. J . Dairy S c i . 53:681 - 682. Kennedy, P.M. and L.P. M i l l i g a n , 1978. Quantitative aspects of trans- formations of sulphur i n sheep. Br. J. Nutr. 39:65 - 83. Kirchgessner, M., 1965. A v a i l a b i l i t y of some trace elements. Proc. Nutr. Soc. 24:89 - 99. Kirchgessner, M., H.-P. Roth, R. Spoerl, A. Schnegg, R.J. Kellner and E. weigand, 1977. A comparitive view on trace elements and growth. Nutr. Metab. 21:119 - 143. Kitchenham, B.A., G.J. Rowlands and H. Shorbagi, 1975. Relationships of concentrations of c e r t a i n blood constituents with milk y i e l d and age of cows i n dair y herds. Res. Vet. S c i . 18:249 - 252. Kitchenham, B.A. and G.J. Rowlands, 1976. Differences i n the con- centrations of c e r t a i n blood constituents among cows i n a dairy herd. J . A g r i c . S c i . , Camb. 86:171 - 179. Klooster, A. Th. van't., 1976. Adaption of calcium absorption from the small i n t e s t i n e of dair y cows to changes i n the di e t a r y calcium intake and at the onset of l a c t a t i o n . Z. T i e r p h y s i o l . , Tierernahrg. u. Futtermittelkde. 37: 169 - 182. - 79 - L a s s i t e r , J.W., W.J. M i l l e r , F.M. Pate and R.P. Gentry, 1972. E f f e c t of d i e t a r y calcium and phosphorus on 5 4Mn metabolism following s i n g l e tracer i n t r a p e r i t o n e a l and o r a l doses i n rats (36140). Soc. Expt. B i o l . and Med. 139:345 - 348. Leibholz, J . , 1974. The flow of calcium and phosphorus i n the di g e s t i v e t r a c t of sheep. A u s t r a l . J. Agr i c . Res. 25:147 - 154. Lengemann, F.W., 1965. Lack of e f f e c t of l e v e l of dietary calcium upon f e c a l endogenous calcium. J . Dairy S c i . 48:1718 - 1719. L i t t l e , D.A. P.J. Robinson, M.J. Playne and K.P. Haydock, 1971. Factors a f f e c t i n g blood inorganic phosphorus deter- minations i n c a t t l e . A u s t r a l . Vet. J . 47:153 - 156. L i t t l e j o h n , A.I. and G. Lewis, 1960. Experimental studies of the r e l a t i o n s h i p between the calcium - phosphorus r a t i o n of the d i e t and f e r t i l i t y i n h e i f e r s : A preliminary report. Vet. Rec. 72:1137 - 1144. Lomba, F., R. Paquay, V. Bienfet and A. Lousse, 1968. S t a t i s t i c a l research on the fate of di e t a r y mineral elements i n dry and l a c t a t i n g cows. I I . Magnesium. J. Agri c . S c i . , Camb. 71:181 - 188. Lomba, F., R. Paquay, V. Bienfet and A. Lousse, 1969. S t a t i s t i c a l research on the fate of di e t a r y mineral elements i n dry and l a c t a t i n g cows. I I I . Phosphorus. J. Agr i c . S c i . , Camb. 73:215 - 222. Lueker, C E . and G.P. Lofgreen, 1961. E f f e c t s of intake and calcium to phosphorus r a t i o on absorption of these elements by sheep. J . Nutr. 74:233 - 238. Mahoney, J.P., K. Sargent, M. Greland and W. Small, 1969. Studies on manganese. I. Determination i n serum by atomic absorp- t i o n spectrophotometry. C l i n . Chem. 15:312 - 322. Manston, R., 1967. The influence of di e t a r y calcium and phosphorus concentration on t h e i r absorption i n the cow. J . Agri c . S c i . , Camb. 68:263 - 268. Manston, R. and G.J. Rowlands, 1973. A n a l y t i c a l v a r i a t i o n i n metabo- l i c p r o f i l e t e s t i n g . J . Dairy Res. 40:85 - 92. McDonald, N. and P.C. Belonje, 1975. The e f f e c t of d i f f e r e n t dietary l e v e l s of Ca and P on the plasma Ca, inorganic P and Mg as well as the ash, Ca, P and Mg content of c e r v i c a l - 80 - vertebrae and t o t a l body of sheep. J.S. M r . Vet. Ass. 46:331 - 334. Michie, N.D., J.E. Dixon and N.G. Bunton, 1978. C r i t i c a l review of A.O.A.C. fluorometric method for determining selenium i n foods. J.A.O.A.C. 61:48 - 51. M i l l e r , W.J., 1973. Dynamics of absorption rates, endogenous excretion, tissue turnover, and homeostatic control mechanisms of zin c , cadmium, manganese and n i c k e l i n ruminants. Fed. Proc. 32:1915 - 1920. M i l l e r , W.J., 1975. New concepts and developments i n metabolism and homeostasis of inorganic elements i n dai r y c a t t l e . A review. J . Dairy S c i . 58:1549 - 1560. M i l l s , C.F. and R.B. Williams, 1971. Problems i n the determination of trace element requirements of animals. Proc. Nutr. Soc. 30:83-91. Molloy, L.F. and E.L. Richards, 1971. Complexing of calcium and magnesium by organic constituents of Yorkshire Fog (Holcus lanatus). J . S c i . Fd. A g r i c . 22:397 - 402. Money, D.F.L., K.G. Hogan and S.M. Hare, 1967. The e f f e c t s of an oestrogenic preparation on blood and l i v e r copper con- centrations and teat length of sheep. N.Z.J. Agric. Res. 10:345 - 349. Morris, E.R. and B.L. O'Dell, 1963. Relationship of excess calcium and phosphorus to magnesium requirement and t o x i c i t y i n Guinea Pigs. J . Nutr. 81:175 - 181. NRC, 1978. Nutrient requirements of dai r y c a t t l e . F i f t h revised ed. National Academy of Sciences - National Research Council, Washington, D.C. Newland, H.W., D.E. U l l r e y , J.A. Hoefer and R.W. Luecke, 1958. The r e l a t i o n s h i p of d i e t a r y calcium to zinc metabolism i n p i g s . J . Anim. S c i . 17:886 - 892. Nugara, D. and H.M. Edwards, J r . , 1963. Influence of d i e t a r y Ca and P l e v e l s on the Mg requirement of the chick. J . Nutr. 80: 181 - 184. O'Dell, B.L., 1969. E f f e c t of d i e t a r y components upon zinc a v a i l a b i l i t y . Amer. J. C l i n . Nutr. 22:1315 - 1322. Olson, A.D. and W.B. Hamlin, 1969. A new method for serum iron and t o t a l iron-binding capacity by atomic absorption spectrophotometry. C l i n . Chem. 15:438 - 444. - 81 - Paquay, R., F. Lomba, A. Lousse and V. Bienfet, 1968. S t a t i s t i c a l research on the fate of dietary mineral elements i n dry and l a c t a t i n g cows. I. Calcium. J . Agr i c . S c i . , Camb. 71:173 - 180. Paquay, R., F. Lomba, A. Lousse and V. Bienfet, 1969. S t a t i s t i c a l research on the fate of dietary mineral elements i n dry and l a c t a t i n g cows. V. Potassium. J . Ag r i c . S c i . , Camb. 73:445 - 452. Parker, B.N.J, and R.W. Blowey, 1976. Investigations into the r e l a - t i o n s h i p of selected blood components to n u t r i t i o n and f e r t i l i t y of the dairy cows under commercial farm conditions. Vet Rec. 98:394 - 404. Parker, M.M., F.L. Humoller and D.J. Mahler, 1967. Determination of copper and zinc i n b i o l o g i c a l material. C l i n . Chem. 13:40 - 48. Parkinson, J.A. and S.E. A l l e n , 1975. A wet oxidation procedure s u i t a b l e for the determination of nitrogen and mineral nutrients i n b i o l o g i c a l m a t e r i a l . Commun. S o i l S c i . Plant Anal. 6:1 - 11. Payne, J.M., S.M. Dew, R. Manston and M. Faulks, 1970. The use of a metabolic p r o f i l e t e s t i n dairy herds. Vet. Rec. 87:150 - 158. Ramberg, C.F., J r . , G.P. Mayer, D.S. Kronfeld, J.M. Phang and M. Berman, 1970. Calcium k i n e t i c s i n cows during late pregnancy, p a r t u r i t i o n , and e a r l y l a c a t i o n . Amer. J. P h y s i o l . 219:1166 - 1177. Ramberg, C.F., J r . , D.S. Kronfeld and G.D.A. Wilson, 1974. Regulation of calcium metabolism i n c a t t l e during growth, gestation, l a c t a t i o n and changes i n d i e t . In. Digestion and Metabolism i n the Ruminant. Ed. I.W. McDonald and A.C.I. Warner, The University of New England publishing u n i t , Armidale, A u s t r a l i a . Ramberg, C.F., J r . , G.P. Mayer, D.S. Kronfeld and J.T. Potts, J r . , 1976. Dietary calcium, calcium k i n e t i c s and plasma parathyroid hormone concentration i n cows. J . Nutr. 106:671 -679. Rowlands, G.J. and R.M. Pocock, 1976. S t a t i s t i c a l basis of the Compton metabolic p r o f i l e t e s t . Vet. Rec. 98:333 - 338. Rowlands, G.J., W. L i t t l e and B.A. Kitchenham, 1977. Relationships between blood composition and f e r t i l i t y i n dairy cows - a f i e l d study. J . Dairy Res. 44:1 - 7. - 82 - Rowlands, G.J., W. L i t t l e , A.J. Stark and R. Manston, 1979. The blood composition of cows i n commercial dair y herds and i t s r e l a t i o n s h i p s with season and l a c t a t i o n . Br. Vet J. 135:64 - 73. R u s s e l l , J.R., A.W. Young and N.A. Jorgensen, 1980. E f f e c t of sodium bicarbonate and limestone additions to high grain diets on feedlot performance and ruminal and f e c a l parameters i n f i n i s h i n g steers. J . Anim. S c i . 51:996 - 1002. Sato, N. and R.I. Henkin, 1973. Pituitary-gonadal regulation of copper and zinc metabolism i n the female r a t . Amer. J. P h y s i o l . 225:508 - 512. Scott, D., 1965. Factors i n f l u e n c i n g the secretion and absorption of calcium and magnesium i n the small i n t e s t i n e of the sheep. Quart. J. Expt. P h y s i o l . 50:312 - 329. Shelford, J.A., T. Grisenthwaite, S. Barrington, R.G. Peterson and L.J. Fisher, 1979. Milk sampling methods for a pro- gesterone assay f o r e a r l y pregnancy diagnosis. Can. J . Anim. S c i . 59:77 - 82. Sigma Technical B u l l e t i n #510, 1978. The enzymatic c o l o r i m e t r i c deter- mination of glucose i n whole blood, plasma or serum at 425 - 475 nm. Sigma Chemical Company, St. Louis, M i s s o u r i . Smith, A.M., G.L. Hoick and H.B. Spafford, 1966. I. Calcium, phosphorus and vitamin D. J . Dairy S c i . 49:239 - 243. Smith, R.H. and A.B. McAllan, 1966. Binding of magnesium and calcium i n the contents of the small i n t e s t i n e of the c a l f . Br. J . Nutr. 20:703 - 717. Smith, R.H. and A.B. McAllan, 1967. P r e c i p i t a t i o n of magnesium i n ass o c i a t i o n with phosphate under the conditions obtaining i n the c a l f ileum. Proc. Nutr. Soc. 26:xxxii. Smith, R.H., 1969. Absorption of major minerals i n the small i n t e s t i - nes of the ruminant. Proc. Nutr. Soc. 28:151 - 160. Spais, A.G., A. Papasteriadis, N. Roubles, A. Agiannidis, N. Yantzis and S. Argyroudis, 1977. Studies on i r o n , manganese, z i n c , copper and selenium retention and i n t e r a c t i o n i n horses. In. Trace Element Metabolism i n Man and Animals, Proceedings of the 3rd International Symposium, F r e i s i n g . Ed. M. Kirchgessner. Stevenson, M.H. and E.F. Unsworth, 1978. Studies on the absorption of calcium, phosphorus, magnesium, copper and zinc by sheep - 83 - fed on roughage-cereal d i e t s . Br. J. Nutr. 40:491 - 496. Storry, J.E., 1961a. Studies on calcium and magnesium i n the alimen- t a r y t r a c t of sheep. I. The d i s t r i b u t i o n of calcium and magnesium i n the contents taken from various parts of the alimentary t r a c t . J . A g r i c . S c i . , Camb. 57:97 - 102. Storry, J.E., 1961b. Studies on calcium and magnesium i n the alimen- t a r y t r a c t of sheep. I I . The e f f e c t of reducing the a c i d i t y of abomasal digesta i n v i t r o on the d i s t r i b u t i o n of calcium and magnesium. J . A g r i c . S c i . , Camb. 57:103 - 109. Stout> W.L., D.C. Kradel, G.A. Jung and C.G. Smiley, 1976. Blood com- p o s i t i o n of well-managed high-producing Holstein cows i n Pennsylvania. Penn. State. U. Prog. Rep. 38. Stupar, J., F. Dolinsek, M. Spenko and J . Furlan, 1974. Determination of molybdenum i n plant material by atomic absorption flame spectroscopy. Landwirtsch Forsch. 27:51 - 61. S u t t l e , N.F. and C.F. M i l l s , 1966. Studies on the t o x i c i t y of copper to p i g s . 2. E f f e c t of protein source and other dietary components on the response to high and moderate intakes of copper. Br. J. Nutr. 20:149 - 162. S u t t l e , N.F. and A.C. F i e l d , 1967. Studies on magnesium i n ruminant n u t r i t i o n . 8. E f f e c t of increased intakes of potassium and water on the metabolism of magnesium, phosphorus, sodium, potassium and calcium i n sheep. Br. J. Nutr. 21:819 - 831. Swenson, M.J., D.D. Goetsch and G.K.L. Underbjerg, 1962. E f f e c t s of d i e t a r y trace minerals, excess calcium and various roughages on the hemogram, t i s s u e s , and estrous cycles of Hereford h e i f e r s . Amer. J. Vet. Res. 23:803 - 808. Symonds, H.W., R. Manston, J.M. Payne and B.F. Sarisom, 1966. Changes i n the calcium and phosphorus requirements of the dairy cow at p a r t u r i t i o n with p a r t i c u l a r reference to the amounts supplied to the foetus i n utero. Br. Vet. J. 122:196 - 200. Talapatra, S.K., S.C. Ray and K.C. Sen, 1948. Calcium a s s i m i l a t i o n i n ruminants on o x a l a t e - r i c h d i e t . J . A g r i c . S c i . , Camb. 38: 163 - 166. Thompson, J.K., D.C. Macdonald and R.W. Warren, 1978. Multiple blood analyses of dairy cows as a management a i d . The North - 84 - of Scotland College of A g r i c u l t u r e . Waverley Press Ltd., Aberdeen, Scotland. Tillman, A.D. and J.R. Brethour, 1958. U t i l i z a t i o n of phytin phosphorus by sheep. J . Anim. S c i . 17:104 - 112. T o o t h i l l , J ., 1963. The e f f e c t of c e r t a i n dietary factors on the apparent absorption of magnesium by the r a t . Br. J . Nutr. 17: 125 -134. Tumbleson, M.E., W.E. Wingfield, H.D. Johnson, J.R. Campbell and C C . Middleton, 1973. Serum e l e c t r o l y t e concentrations as a function of age, i n female dair y c a t t l e . C o r n e l l Vet. 63:58 - 64. Underwood, E.J., 1977. Trace Elements i n Human and Animal N u t r i t i o n . Fourth Ed. Academic Press, New York, San Fransico, London. Vagg, M.J. and J.M. Payne, 1971• E f f e c t of r a i s e d dietary calcium on the g a s t r o i n t e s t i n a l absorption and rate of excretion of manganese by dairy cows. In. Mineral Studies with Isotopes i n Domestic Animals. International Atomic Energy Agency, Vienna. Varner, L.W. and W. Woods, 1972. E f f e c t of calcium and starch addi- t i o n s upon r a t i o n d i g e s t i b i l i t y by steers. J . Anim S c i . 35:410. Visek, W.J., R.A. Monroe, E.W. Swanson and C.L. Comar, 1953. Calcium metabolism i n d a i r y cows as studied with 4 5 C a . J . Dairy S c i . 36:373 - 384. Waldern, D.E., 1971. A rapid micro-digestion procedure for neutral and acid detergent f i b r e . Can. J . Anim. S c i . 51:67 - 71. Wheeler, W.E. and C.H. N o l l e r , 1977. G a s t r o i n t e s t i n a l t r a c t pH and starch i n feces of ruminants. J . Anim. S c i . 44:131 - 135. Wheeler, W.E., 1980. G a s t r o i n t e s t i n a l t r a c t pH environment and the influence of b u f f e r i n g materials on the performanc of ruminants. J . Anim. S c i . 51:224 - 235. Wise, M.B., A.L. Ordoveza and E.R. Barrick, 1963. Influence of v a r i a t i o n s i n dietary calcium: phosphorus r a t i o on per- formance and blood constituents i n calves. J . Nutr. 79:79 - 84. - 85 - Yang, M.G.. and J.W. Thomas, 1965. Absorption and secretion of some organic and inorganic constituents and the d i s t r i b u t i o n of these c o n s i t i t u e n t s throughout the alimentary t r a c t of young calves. J . Nutr. 87:444. Yano, H., H. Matsui and R. Kawashima, 1979. E f f e c t s of dietary calcium l e v e l s on concentration and s o l u b i l i t y of macro minerals i n the dig e s t i v e t r a c t of sheep. J . Anim. S c i . 48:954 - 960. APPEND IX T a b l e A • F ' V a l u e s o f S e r u m C o n s t i t u e n t s f o r R e l a t e d ANOVA M o d e l T e r m s + Y i j k l T B ^ ( d . f . ) ( 1 ) ( 1 ) ( 3 ) ( 1 ) C a 2 1 . 2 * * 1.2 1 . 2 1 .9 P 6 . 8 * * 6 . 8 * * 4 . 9 * 0 K 0 . 3 0 . 2 0 1 . 3 Mg 2 . 8 1 .0 1 .7 0 S 2 . 6 0 2 . 0 0 . 3 F e 0 0 2 . 6 0 . 6 C u 4 . 2 * 9 . 5 * * 1 .7 0 . 4 Z n 2 3 . 4 * * 1.5 1 . 5 1 .7 Se 4 . 2 * 1 3 . 9 * * 0 . 2 5 . 5 * + S e e p a g e 2 4 f o r e x p l a n a t i o n o f t e r m s . S i g n i f i c a n t l y d i f f e r e n t * ( p < . 0 5 ) , * * ( p < . 0 1 ) A i j k l M i j k l F i j k l ( 3 ) ( 1 ) ( 1 ) ( 1 ) 0 . 2 0 . 9 3 . 8 * 0 . 4 0 . 2 0 1 . 5 2 . 8 0 . 8 3 . 0 0 . 3 4 . 9 * 2 . 4 6 . 2 * 0 . 2 7 . 4 * * 0 . 2 0 0 . 1 1 2 . 2 * * 1 . 3 8 . 2 * * 4 . 2 * 0 . 4 0 . 4 1 2 . 4 * * 4 . 6 * 3 0 . 2 * * 2 . 0 1 . 3 0 . 8 4 . 5 * 1 . 6 6 . 3 * 7 . 3 * * 1.1

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