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Estimation of genetic and environmental parameters of some blood serum components in dairy cattle Basuthakur, Arun Kumar 1973

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ESTIMATION OF GENETIC AND ENVIRONMENTAL PARAMETERS OF SOME BLOOD SERUM COMPONENTS IN DAIRY CATTLE by ARUN KUMAR BASUTHAKUR B. Sc. (Cal.University India), B.V.SC. & A.H. (Cal.University India) Diploma Sheep & Wool (I.C.A. R.India), M.S.(Montana State University U.S.A.) A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of ANIMAL SCIENCE We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1973 i i In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Animal Science' The University of British Columbia Vancouver 8, Canada D a t e June , 1973 ABSTRACT The va r i a t i o n s due to environmental and genetic causes i n the blood serum components i n dairy c a t t l e were estimated from blood samples c o l l e c t e d from 226 Holstein and Holstein-Ayrshire cross bred female calves and cows, at the University of B r i t i s h Columbia, Oyster River Research Farm number two. The blood serum samples were analyzed for p r o t e i n , albumin, g l o b u l i n , calcium, inorganic phosphorus, c h o l e s t e r o l , blood urea nitrogen (B.U.N.), u r i c a c i d , c r e a t i n i n e , a l k a l i n e phosphatase, and serum glutamic oxaloacetic transaminase (S.G.O.T.), by SSM 12/60 multi-channel sequential analyzer. The albumin And globulins c t ^ , a2, 3^ , a n <* Y l e v e l s i n the blood were estimated using t h i n plate agarose gel electrophoresis. The age i n days as a source of v a r i a t i o n ( f i t t e d within p h y s i o l o g i c a l groups) s i g n i f i c a n t l y affected most of the blood serum components i n the young female calves and h e i f e r s , bred h e i f e r , l a c t a t i n g non-pregnant, l a c t a t i n g pregnant and dry animal groups. The days pregnant was a s i g n i f i c a n t source of v a r i a t i o n i n the d i f f e r e n t types of serum pro t e i n s , B.U.N, and a l k a l i n e phosphatase i n the present study. The days l a c t a t i n g f i t t e d as a source of v a r i a t i o n i n the l a c t a t i n g non-pregnant animals s i g n i f i c a n t l y affected the blood serum iv components-cholesterol, B.U.N., globulin and T n e days lactating f i t t e d as a source of variation in lactating pregnant animals accounted for a significant amount of variation only in cholesterol. The lactation t r a i t s (Kg. milk, Kg. milk f a t , Kg. milk protein and Kg. lactose produced by the animal in the milking prior to collection of blood sample) as sources of variation in the milking animals significantly affected the blood serum components cholesterol, uric acid, globulins and The physiological groups (young animals, bred heifers, lactating non-pregnant group, lactating pregnant group and dry animal group) accounted for the major portion of the variation in almost a l l the blood serum components in the present study. The linear function of age when fitted across groups was important and accounted for a significant amount of variation in almost a l l of the blood serum components. Breed groups f i t t e d as percentage Holstein ( I . Holstein, I I . 75 to 62 percent Holstein, I I I . 50 to 25 percent Holstein) accounted for significant amounts of variation only in three blood serum components, uric acid, globulin a9 and globulin $,. V The mean and standard deviation of each of the blood serum components and the product moment c o r r e l a t i o n between the blood serum components, between the sources of v a r i a t i o n , and between the blood serum components and sources of var i a t i o n s within p h y s i o l o g i c a l groups were also computed. The h e r i t a b i l i t i e s , genetic, phenotypic, and environmental co r r e l a t i o n s were calculated using the paternal h a l f sib i n t r a class c o r r e l a t i o n method. The h e r i t a b i l i t y estimates for creatinine and S.G.O.T. were r e l a t i v e l y high, 0.77 and 0.46 r e s p e c t i v e l y . The h e r i t a b i l i t y estimates for other blood serum components were; B.U.N. 0.27; glo b u l i n - 3X 0.12; a l k a l i n e phosphatase 0.11; u r i c acid 0.10; albumin 0.07; gl o b u l i n $2 0.06; glo b u l i n 0.03; chol e s t e r o l 0.02. Twelve animals showed improper separation of globulins - 8-^  and 62 a t t n e t r a n s f e r r i n zone" i n agarose gel electrophoresis. These animals were compared with respect to t h e i r t r a n s f e r r i n and y- g l o b u l i n f r a c t i o n s with a group of twelve randomly drawn animals having regular separation of globulins 8-^  and 82 a t t n e t r a n s f e r r i n zone. These groups were found to be s i g n i f i c a n t l y d i f f e r e n t v i with respect to t h e i r mean l e v e l of y-globulin, t r a n s f e r r i n and t r a n s f e r r i n y g l o b u l i n r a t i o . The twelve animals with improper separation of globulins 8^ and B 2 a t t n e t r a n s f e r r i n zone were also observed to be from the higher producing animals i n the herd. A l l twelve animals had one Holstein s i r e as a common ancestor. The s i g n i f i c a n t sources of environmental v a r i a t i o n i n the present study were due primarily, to the physiological status of the animals during sampling. Genetic v a r i a t i o n was present i n some of the blood serum constituents. The environmental and genotypic relationships between the blood serum constituents and production t r a i t s require further investigation and may lead to improved selection techniques. TABLE OF CONTENTS Page ABSTRACT i i i CONTENTS v i i LIST OF TABLES x i LIST OF FIGURES x i v LIST OF PLATES x v i ACKNOWLEDGEMENTS x v i i INTRODUCTION 1 REVIEW OF LITERATURE 4 I Estimates of blood components .... 4 II Measurement of blood serum components 8 MATERIALS AND METHODS 15 I Experimental animals 15 II Blood sampling 16 III The SMA 12/60 Multi-Channel analyzer 18 IV Electrophoresis of proteins 18 V Measurement of the graphs 19 VI Separation of serum t r a n s f e r r i n . 19 VII Milk samples 20 VIII The sample s i z e for d i f f e r e n t analysis 20 IX S t a t i s t i c a l technique 21 v i i i Page A. Estimation of Environmental and Genetic Components of V a r i a t i o n 21 1. The l i n e a r model for the young female animal group.. 2 3 2. The l i n e a r model for the bred h e i f e r 24 3. The l i n e a r model for the l a c t a t i n g non-pregnant animals 25 4. The l i n e a r model for the l a c t a t i n g pregnant cows.... 27 5. The l i n e a r model for the dry animals 28 B. Estimation of Sire Component of Variance for Genetic Parameters 34 H e r i t a b i l i t y 38 Genetic C o r r e l a t i o n 39 Environmental C o r r e l a t i o n . . . . 40 Phenotypic C o r r e l a t i o n between t r a i t s 41 RESULT AND DISCUSSION 42 I THE EFFECT OF TIME. LAPSE BETWEEN FEEDING OR FEEDING AND MILKING AND COLLECTION OF BLOOD SAMPLES ON EACH BLOOD SERUM COMPONENT WITHIN EACH MANAGEMENT GROUP.. 42 II ANALYSIS OF DIFFERENT PHYSIOLOGICAL EFFECTS ON THE BLOOD SERUM COMPONENTS 51 A. Linear Regression Analysis.... 51 Total blood serum protein 51 Blood serum albumin. 53 Blood serum g l o h u l i n 53 ix Page Blood serum c a l c i u m 53 Blood serum i n o r g a n i c phosphorus 53 Blood serum c h o l e s t e r o l 54 Blood urea n i t r o g e n 54 Blood serum u r i c a c i d 54 Blood serum c r e a t i n i n e 54 Blood serum a l k a l i n e phosphatase 55 Blood serum o x a l o a c e t i c t r a n s a -minase 55 (S.G.O.T.) Blood serum albumin separated by e l e c t r o p h o r e s i s 55 Blood serum g l o b u l i n ct^ 55 Blood serum g l o b u l i n o.^ 55 Blood serum g l o b u l i n 8^ 55 Blood serum g l o b u l i n 82 56 Blood serum g l o b u l i n Y 56 Age of animals 66 Days pregnant 69 Days l a c t a t i n g 70 The Kg. of m i l k , Kg. of m i l k F a t , Kg. of m i l k p r o t e i n and Kg. l a c t o s e 71 B. The Product Moment C o r r e l a t i o n W i t h i n the P h y s i o l o g i c a l Groups 73 (1) Young female animal group 73 (2) Bred h e i f e r group 74 (3) L a c t a t i n g non-pregnant group and X Page (4) L a c t a t i n g pregnant group 78 (5) Dry animal group 85 I I I ADJUSTMENT OF DATA 85 IV THE ANALYSIS OF GROUP AND BREED EFFECT 92 A. A n a l y s i s o f . V a r i a n c e F i t t i n g F i x e d Model 92 B. Comparison of Group Means of the Blood Serum Components. 99 Breed Groups 100 V TO ESTIMATE GENETIC .COMPONENTS OF VARIANCE 126 A. The Mixed Model to E s t i m a t e G e n e t i c Parameters 126 B. Genetic Parameters 130 The h e r i t a b i l i t y e s t i m a t e s 137 The g e n e t i c c o r r e l a t i o n s . . 140 The environmental c o r r e l a t i o n s 143 The ph e n o t y p i c c o r r e l a t i o n s 143 VI ANIMALS WITH MISSING IVth PEAK 147 SUMMARY AND CONCLUSION 162 REFERENCES 170 APPENDIX 176 CONTENTS 177 x i LIST OF TABLES TABLE Page 1 THE MEAN (S.D.) FOR SOME HUMAN BLOOD COMPONENTS OBTAINED AFTER ANALYSIS OF SMA 12/60 AND HAND METHOD AS REPORTED BY LASZLO MAKK, ET AL. , (1969) 11 2 NUMBER OF ANIMALS IN EACH PERCENTAGE HOLSTEIN CATEGORY 16 3 THE REGRESSION COEFFICIENTS FOR THE BLOOD SERUM COMPONENTS AFTER REGRESSING ON TIME LAPSE BETWEEN FEEDING OR FEEDING TO PRODUCTION WITHIN SIX MANAGEMENT GROUPS 43 4 MEAN AND STANDARD DEVIATION (S.D.) FOR TIME LAPSE (IN HOURS) BETWEEN FEEDING OR FEEDING AND PRODUCTION AND COLLECTION OF BLOOD SAMPLES FOR THE SIX MANAGEMENT GROUPS 50 5 THE PHYSIOLOGICAL GROUPS AND THE VARIABLES MEASURED WITHIN GROUPS 52 6 THE SIMPLE REGRESSION COEFFICIENTS AND THE PROPORTION OF VARIATION (R2) FITTING MODEL -2 WITHIN YOUNG FEMALES AND PARTIAL REGRESSION COEFFICIENTS AND R2 FITTING MODELS 3 AND 6 WITHIN BRED HEIFER AND DRY ANIMALS, RESPECTIVELY, FOR ALL THE BLOOD SERUM COMPONENTS... 57 7 THE PROPORTION OF VARIATION (R2) FOR THE MODEL -4 AND PARTIAL ? REGRESSION COEFFICIENTS AND R FOR EACH SOURCES FITTED FOR THE BLOOD SERUM COMPONENTS WITHIN THE LACTATING NON PREGNANT GROUP 60 8 THE PROPORTION OF VARIATION (R2) FOR MODEL -5 AND PARTIAL REGRESSION COEFFICIENTS AND R2 FOR EACH SOURCES FITTED FOR THE BLOOD SERUM COMPONENTS WITHIN THE LACTATING PREGNANT GROUP 63 PRODUCT MOMENT : CORRELATION BETWEEN ALL CONTINUOUS VARIABLES MEASURED IN GROUP I (YOUNG FEMALE ANIMALS) BASED ON 51 PAIRED OBSERVATIONS PRODUCT MOMENT CORRELATION BETWEEN ALL CONTINUOUS VARIABLES MEASURED IN GROUP I I (BRED HEIFER) BASED ON 39 PAIRED OBSERVATIONS PRODUCT MOMENT CORRELATION BETWEEN ALL CONTINUOUS VARIABLES MEASURED IN GROUP I I I (LACTATING AND OPEN COWS) BASED ON 54 PAIRS OF OBSERVATIONS.. PRODUCT MOMENT CORRELATION BETWEEN ALL CONTINUOUS VARIABLES MEASURED IN GROUP IV (LACTATING PREGNANT) BASED ON 53 PAIRS OF OBSERVATIONS PRODUCT MOMENT CORRELATION BETWEEN ALL CONTINUOUS VARIABLES MEASURED IN GROUP V (DRY ANIMALS) BASED ON 14 PAIRED OBSERVATIONS SIMPLE REGRESSION COEFFICIENTS ? AND THE PORTION OF VARIATION (R ) ACCOUNTED FOR IN THE BLOOD SERUM COMPONENTS REGRESSED ON AGE (IN DAYS) WITHIN EACH PHYSIOLOGICAL GROUP SIGNIFICANT REGRESSION COEFFICIENTS WHICH WERE USED TO ADJUST DATA WITHIN THE PHYSIOLOGICAL GROUPS FOR THE HERD ANALYSIS OF VARIANCE FITTING FIXED EFFECT MODEL (7) TO ESTIMATE THE BREED GROUPS, PHSYIOLOGICAL GROUPS AND AGE EFFECT FOR EACH BLOOD SERUM COMPONENT USING DATA ADJUSTED ACCORDING TO MODEL (8) x i i i TABLE Page 17 ANALYSIS OF PARTITIONED SUMS OF SQUARES OF BREED EFFECT FROM THE FIXED EFFECT MODEL (7) FOR THE URIC ACID, GLOBULIN a AND GLOBULIN ^ . 101 18 LEAST SQUARESCONSTANTS FOR THE DEPENDENT VARIABLES AFTER FITTING GROUPS AND BREEDS AS FIXED EFFECT AND AGE AS COVARIABLE (MODEL 7) 104 19 THE EXPECTED COMPONENTS OF VARIANCE FOR TWO BLOOD SERUM COMPONENTS FITTING THE SAME MODEL (10) AND THE EXPECTED COMPONENT OF CO-VARIANCE 127 20 THE INDEPENDENT VARIABLES FITTED FOR EACH BLOOD COMPONENT WITHIN GROUPS AND THE SIGNIFICANT COEFFICIENTS USED FOR CORRECTING THE COMPONENTS WITHIN PHYSIOLOGICAL GROUPS: FOR 158 DAUGHTERS OF THE 15 HOLSTEIN BULLS 129 21 ANALYSIS OF VARIANCE FITTING MIXED EFFECT MODEL (10) TO ESTIMATE THE SIRE COMPONENT OF VARIANCE FOR THE BLOOD SERUM COMPONENTS, USING DATA ADJUSTED ACCORDING TO MODEL (8)... 131 22 HERITABILITY AND STANDARD ERRORS FOR TEN BLOOD SERUM COMPONENTS 139 23 GENETIC CORRELATIONS BETWEEN THE TEN BLOOD COMPONENTS AND THEIR STANDARD ERRORS 141 24 ENVIRONMENTAL AND PHEUOTYPIC CORRELATIONS FOR TEN BLOOD COMPONENTS 144 25 MEAN + STANDARD DEVIATION (S.D.) FOR THE MISSING 4TH PEAK GROUP AND GROUP WITH NORMAL PEAKS 158 26 PRODUCTION DETAIL OF THE 12 ANIMALS WITH MISSING 4TH PEAK 160 xiv LIST OF FIGURES FIGURE Page 1 ANALYSIS OF BREED GROUP MEANS FITTING ORTHOGONAL CONTRAST FOR URIC ACID, GLOBULIN a AND GLOBULIN & ? 102 2 ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST. DEPENDENT VARIABLE ... PROTEIN 10.5 3 ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST. DEPENDENT VARIABLE. ..ALBUMIN 106 4 ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST. DEPENDENT VARIABLE ...GLOBULIN 107 5 ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST. DEPENDENT VARIABLE...CALCIUM 109 6 ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST. DEPENDENT VARIABLE...INORGANIC PHOSPHORUS I l l 7 ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST. DEPENDENT VARIABLE...CHOLESTEROL.. 112 8 ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST. DEPENDENT VARIABLE...BLOOD UREA NITROGEN 114 9 ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST. DEPENDENT VARIABLE...URIC ACID 115 XV FIGURE Page 10 ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST. DEPENDENT VARIABLE ...CREATININE... 116 11 ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST. DEPENDENT VARIABLE...ALKALINE PHOSOPHOTASE 119 12 ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST. DEPENDENT VARIABLE...SERUM GLUTAMIC OXALO ACETICTRANSAMINASE 120 13 ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST. DEPENDENT VARIABLE ...GLOBULIN a . . 122 14 ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST. DEPENDENT VARIABLE ...GLOBULIN c ^ . . 123 15 ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST. DEPENDENT VARIABLE ... GLOBULIN 3 ^ . 124 16 ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST. DEPENDENT VARIABLE...GLOBULIN y . . 125 x v i LIST OF PLATES PLATE Page I SAMPLE NUMBERS 103, 103-D AND 92, 9 2-D WITH MISSING 4TH PEAK ALL PROCESSED ON SEPARATE PLATES 148 II ELECTROPHORETICALLY SEPARATED SERUM PROTEIN COLUMNS AND THEIR DUPLICATES 150 I I I TYPICAL SERUM PROTEIN COLUMNS SEPARATED BY ELECTROPHORESIS ON THIN AGAROSE GEL PLATE 151 IV DENSITOMETER TRACING OF SAMPLE NUMBER 92 WITH MISSING 4TH PEAK... 152 V THE TYPICAL DENSITOMETER TRACING OF BLOOD SERUM PROTEIN COLUMNS FOR HOLSTEIN SEPARATED BY ELECTRO-PHORESIS ON THIN PLATE AGAROSE GEL TECHNIQUE IN THE PRESENT STUDY 153 VI SAMPLE NUMBERS 200, 46, 28 ARE SAMPLES WITH ILL-DEFINED 4TH COLUMN SAMPLE NUMBERS 94, 54 AND 1 WITH PROPERLY DEFINED SIX COLUMNS 154 VII RIVONAL-LACTATE TREATED SAMPLES... 156 V I I I DENSITOMETER TRACING AFTER SEPARATION OF TRANSFERRIN AND y-GLOBULIN FROM OTHER SERUM PROTEINS 157 x v i i ACKNOWLEDGEMENTS I am g r a t e f u l t o Dr. Raymond G. P e t e r s o n f o r h i s keen i n t e r e s t and s u p e r v i s i o n o f the p r o j e c t i n a d d i t i o n to a l l the f a c i l i t i e s he p r o v i d e d f o r the work. I am a l s o g r a t e f u l to Dr. W. D. K i t t s , Chairman o f the department f o r h i s h e l p and s u g g e s t i o n s . I wish to express my g r a t i t u d e to Dr. C. W. R o b e r t s , Dr. C.R. K r i s h n a m u r t i , Dr. C. A. Hornby and Dr. A. Kozak the members o f my committee f o r t h e i r h e l p and s u g g e s t i o n s . I am t h a n k f u l to Dr. R.. J . Hudson f o r h i s i n t e r e s t and s u g g e s t i o n s r e g a r d i n g the l a b o r a t o r y t e c h n i q u e s used under t h i s p r o j e c t . My s i n c e r e thanks are a l s o to Mr. C. J . W i l l i a m s , Mr. L. Dunn, Mr. J . S h e l f o r d and Mr. S. Masson f o r t h e i r h e l p and i n t e r e s t i n many ways f o r t h i s p r o j e c t . My thanks are a l s o to Mr. Leo Kansky, manager, U n i v e r s i t y of B r i t i s h Columbia O y s t e r R i v e r Research Farm Number Two and the students w i t h summer job a t the Farm f o r t h e i r h e l p d u r i n g the sample c o l l e c t i o n . My thanks are a l s o to Mrs. G. Huchelaga, o t h e r o f f i c e s t a f f and x v i i i l a b o r a t o r y s t a f f f o r t h e i r h e l p and i n t e r e s t d u r i n g the p e r i o d . I am t h a n k f u l t o my w i f e Shyamali f o r t y p i n g the f i r s t d r a f t o f t h i s t h e s i s . I am a l s o g r a t e ' f u l t o her and my son Babua f o r t h e encouragement and t h e i r u n d e r s t a n d i n g and c o n t i n u i n g p a t i e n c e d u r i n g the p r o j e c t . 1 INTRODUCTION The animals of d i f f e r e n t s p e c i e s possess d i s t i n c t i v e m e t a b o l i c systems, which are r e l a t e d to the a b i l i t y of the animal to cope w i t h i t s environment to u t i l i z e a v a i l a b l e n u t r i e n t s and perform v a r i o u s p r o d u c t i o n f u n c t i o n s . W i t h i n a s p e c i e s the i n h e r i t e d d i f f e r e n c e s i n the p h y s i o l o g i c a l make-up between i n d i v i d u a l s i s r e s p o n s i b l e , a t l e a s t i n p a r t , f o r the v a r i a t i o n between i n d i v i d u a l s i n a dapting t o the a v a i l a b l e environment. U l t i m a t e l y t h i s v a r i a t i o n i n the p h y s i o l o g i c a l system i s r e s p o n s i b l e f o r the v a r i a t i o n i n v a r i o u s p r o d u c t i o n f u n c t i o n s . S i n c e b l o o d i s the v i t a l f l u i d i n mammals, the study o f the b lood components would seem t o m a n i f e s t the i n h e r i t e d p h y s i o l o g i c a l d i f f e r e n c e s i f i n f a c t these d i f f e r e n c e s are h e r i t a b l e , i n the domestic animals o f mammalian o r d e r . In domestic animals the f i r s t major study i n d i c a t i n g a g e n e t i c b a s i s f o r the v a r i a t i o n i n a p h y s i o -l o g i c a l t r a i t was r e p o r t e d by S m i t h i e s and Hickman (1958) w i t h serum p r o t e i n separated by e l e c t r o p h o r e s i s . S i m i l a r s t u d i e s have subsequently been r e p o r t e d by o t h e r workers with v a r i o u s domestic a n i m a l s . These s t u d i e s have been ce n t e r e d e s p e c i a l l y on the t r a n s f e r r i n or i r o n bound 3-globulins i n b lood serum. The polymorphism o f t r a n s f e r r i n 2 i n those domestic animals s t u d i e d were h y p o t h e s i z e d by the authors to be due t o m u l t i p l e a l l e l i c systems. Very few s t u d i e s have been p u b l i s h e d d e a l i n g w i t h the continuous v a r i a t i o n of blood serum p r o t e i n s or non p r o t e i n b lood serum c o n s t i t u e n t s . A study of the sources of v a r i a t i o n and the p a t t e r n of i n h e r i t a n c e of these b l o o d components may produce i n f o r m a t i o n u s e f u l to p h y s i o l o g i c a l r e s e a r c h and f u r t h e r i n v e s t i g a t i o n may l e a d t o the use of these b l o o d serum component t r a i t s as t o o l s i n s e l e c t i o n programmes f o r gross p r o d u c t i o n t r a i t s o f s u p e r i o r a n i m a l s . To t h i s end b l o o d samples were analyzed t o e s t i m a t e the e x t e n t to which v a r i o u s environmental f a c t o r s i n f l u e n c e the e x p r e s s i o n o f the b l o o d serum i n g r e d i e n t s i n c a t t l e and t o determine i f the q u a n t i t a t i v e v a r i a t i o n i n the c o n s t i t u e n t s have a g e n e t i c b a s i s . Relevant g e n e t i c parameters were estimated from the H o l s t e i n and H o l s t e i n - A y r s h i r e c r o s s d a i r y c a t t l e of the Oyster R i v e r Research U n i t - 2 o f the U n i v e r s i t y of B r i t i s h Columbia. The blood serum components s t u d i e d were t o t a l p r o t e i n , albumin, g l o b u l i n , c a l c i u m , i n o r g a n i c phosphorus, . c h o l e s t e r o l , b lood u r e a n i t r o g e n , c r e a t i n i n e , b i l i r u b i n a l k a l i n e phosphatase, l a c t i c dehydrogenase, serum g l u t a m i c oxalo a c e t i c t r a n s a m i n a s e , and p a r t s of serum p r o t e i n s 3 s eparated by e l e c t r o p h o r e s i s albumin, g l o b u l i n a^, g l o b u l i n c ^ / g l o b u l i n g l o b u l i n (i, a n <^ y ~ g l o b u l i n . 4 REVIEW OF LITERATURE I ESTIMATES OF BLOOD COMPONENTS In l i v e s t o c k the s t a t i s t i c a l e s t i m a t e s of p r o d u c t i o n o r performance parameters has been p r i m a r i l y concerned with gross p r o d u c t i o n f u n c t i o n s such as average d a i l y g a i n i n the feed l o t , m i l k y i e l d , wool y i e l d , egg y i e l d , e t c . There are many w e l l documented e s t i m a t e s o f the h e r i t a b i l i t i e s and g e n e t i c c o r r e l a t i o n s of such t r a i t s , under v a r i o u s environmental c o n d i t i o n s . Very few e s t i m a t e s of g e n e t i c or environmental parameters f o r blood c o n s t i t u e n t s are r e p o r t e d i n the l i t e r a t u r e . S t u f f l e b e a m e t al.,(1966) w h i l e s t u d y i n g the r e l a t i v e v a l u e o f c e r t a i n economic and blood component t r a i t s i n s e l e c t i o n i n d i c e s r e p o r t e d e s t i m a t e s o f h e r i t a b i l i t y f o r plasma u r i c a c i d (0.20), plasma c r e a t i n i n e (0.41), t o t a l serum p r o t e i n (0.18), t o t a l serum c h o l e s t e r o l (0.55), serum c a l c i u m (0.30), serum phosphorus (0.39), i n H e r e f o r d b u l l c a l v e s . S i x t y animals were chosen a t random i n 1961 and 1962 from the H e r e f o r d b u l l c a l f crop a t the U n i v e r s i t y Research Centre a t Weldon S p r i n g s , M i s s o u r i . The randomly chosen b u l l c a l v e s were r a i s e d i n a s p e c i a l f e e d - l o t f o r 182 days i n 1961 and 135 days i n 1962 p r i o r t o the c o l l e c t i o n of bl o o d samples. The h e r i t a b i l i t y e s t i m a t e s were determined by h a l f - s i b 5 analyses within years. The r e l a t i o n s h i p s between subsequent weight of the carcass and the blood components c r e a t i n , c r e a t i n - c r e a t i n i n e r a t i o , t o t a l serum protein and serum sodium l e v e l at weaning was observed to be high by these workers. Stufflebeam and Lasely (1970) reported estimates of h e r i t a b i l i t y f o r serum c h o l e s t e r o l (0.80+0.42) i n Hereford c a t t l e . 143 young Hereford b u l l s were chosen at random from the University of Missouri herd during a 2-year period and raised i n a feed-lot for at l e a s t 140 days pr i o r to sampling of blood. The h e r i t a b i l i t y estimates were calculated within years using the paternal h a l f - s i b method of ana l y s i s . The genetic and phenotypic c o r r e l a t i o n s between cholesterol and post weaning growth were computed using the variance-covariance method. A s i g n i f i c a n t genetic c o r r e l a t i o n between serum cholest e r o l and post weaning growth rate of 0.63 and a s i g n i f i c a n t phenotypic corre-l a t i o n between serum ch o l e s t e r o l and growth rate of 0.15 was reported by these workers. Roubicek and Ray (19 72) reported a study of t o t a l serum protein, albumin and gl o b u l i n f r a c t i o n s a , 3 / and y obtained by electrophoresis i n a Hereford herd over a period of six years. The progeny were sampled at mean ages of 235, 340, 600 and 710 days and included 700 to 980 progeny each year. The various main e f f e c t s 6 f i t t e d i n the model assumed by them were y e a r s , s i r e s , sex ( b u l l s , h e i f e r s ) , age of dam c l a s s e s (3 and 4 , 5 - 8 , 9 and olde r ) and a l l t w o - f a c t o r i n t e r a c t i o n s i n v o l v i n g y e a r s , sex and age of dam. The date of b i r t h (age) was i n c l u d e d i n the model as a c o v a r i a b l e . The a n a l y s i s was conducted w i t h i n the sampled age groups (as mentioned above 235, 3 40, 600 and 710 days) o f the progeny. A t o t a l o f 12 t o 28 s i r e s were r e p r e s e n t e d i n the v a r i o u s age groups s t u d i e d and the estimate o f h e r i t a b i l i t y were o b t a i n e d u s i n g p a t e r n a l h a l f - s i b c o v a r i a t i o n s . The year of sampling accounted f o r the major p o r t i o n o f the t o t a l v a r i a b i l i t y o f the serum p r o t e i n . The year e f f e c t s f o r a l l the serum p r o t e i n s a t a l l the mean age groups of progeny were h i g h l y s i g n i f i c a n t i n t h e i r s t u d y . The s i r e e f f e c t s were s i g n i f i c a n t f o r serum albumin and gamma g l o b u l i n i n a l l f o u r age groups, however, b e t a g l o b u l i n was e f f e c t e d by s i r e s o n l y i n the weaning age group. The year by sex i n t e r a c t i o n appeared important a t the 600 to 710 day age but not younger age. The ot h e r two f a c t o r i n t e r a c t i o n s i n c l u d e d i n the model were s t a t i s t i c a l l y n o n - s i g n i f i c a n t . The h e r i t a b i l i t y e s t i m a t e s ranged from 0.22 to 0.30 f o r gamma g l o b u l i n , and 0.12 to 0.20 f o r albumin. The h e r i t a b i l i t y e s t i m a t e s f o r a l pha and beta g l o b u l i n f r a c t i o n s were low and i n c o n s i s t e n t . The h e r i t a b i l i t y e s t i m a t e s f o r t o t a l serum p r o t e i n ranged from 7 0.20 to 0.30 excepting for the 340 day age group which was 0.06. The simple c o r r e l a t i o n s among serum proteins reported were low. The c o r r e l a t i o n between these serum proteins and the subsequent body weights reported were observed to be extremely v a r i a b l e . The study of v a r i a t i o n i n serum protein was f i r s t i n i t i a t e d because of reported v a r i a t i o n of normal human serum by Smithies (1955). Smithies and Hickman (1957) f i r s t reported the inherited v a r i a t i o n s i n the serum proteins of Ayrshire and Holstein c a t t l e . The v a r i a t i o n of serum protein detected by electrophoresis was hypothesized by Smithies and Hickman to be due to a genetic mechanism involving three a l l e l e s at a locus. Subsequently, Ashton (1957), reported the inherited v a r i a t i o n i n the serum proteins separated by electrophoresis i n starch gels following Smithies method on cows, pigs, horses and dogs. Ashton and McDougall (1958) reported a study of beta-globulin polymorphism i n c a t t l e , sheep and goats. They recognized the B-globulin phenotypes and indicated these to be under the genetic control of three, f i v e , two and three allelomorphs respectively at a locus. The locus was designated by 8 and the beta-globulin bands separated by electrophoresis as A, B, C, D, and E i n decreasing 8 order o f e l e c t r o p h o r i t i c m o b i l i t y . G i b l e t e t al.,(1964) presented evidence t h a t 8 - g l o b u l i n e s i n man and c a t t l e are i r o n - b i n d i n g serum p r o t e i n s and hence were c a l l e d t r a n s f e r r i n s T h i s l e d to the change i n the name of the l o c u s to Tf i n s t e a d o f 8. Ashton and Hewetson (19 69) r e p o r t e d t h a t w i t h d a i r y c a t t l e a p o s i t i v e e s t i m a t e o f the e f f e c t o f r e p l a c i n g a TfA by a T f D2 gene was 170 pounds more m i l k and 6.5 days lon g e r l a c t a t i o n . G a l l and Berg (1964) s t u d i e d the mechanism o f i n h e r i t a n c e o f bovine serum t r a n s f e r r i n s i n one H e r e f o r d herd and s i x h y b r i d herds comprised o f Gallway, Aberdeen, Angus and C h a r o l a i s b r e e d s . T h e i r f i n d i n g s a l s o supported the t h r e e - a l l e l e theory o f i n h e r i t a n c e o f the polymorphisms o f t r a n s f e r r i n bands. I I MEASUREMENT OF BLOOD SERUM COMPONENTS The s i m p l e procedure o f s e p a r a t i n g serum p r o t e i n s was f i r s t d e s c r i b e d by Kunkel e t al.,(1952) by f i l t e r paper e l e c t r o p h o r e s i s . S m i t h i e s (1955) came up with zone e l e c t r o p h o r e s i s combining the advantages o f the low a b s o r p t i o n c h a r a c t e r i s t i c s o f the s t a r c h g r a i n and d e t e c t i o n o f p r o t e i n by s t a i n i n g . S i n c e then d i f f e r e n t medias f o r b e t t e r s e p a r a t i o n o f p r o t e i n s were 9 t r i e d . E l e v i t c h e t a l . , (1966) r e p o r t e d a study comparing d i f f e r e n t media and t h e i r e f f i c i e n c y i n s e p a r a t i n g d i f f e r e n t kinds of p r o t e i n s . The d i f f e r e n t media s t u d i e d by them were t h i n agarose g e l , paper, a g a r , c e l l u l o s e a c e t a t e , s t a r c h b l o c k , a c r y l a m i d e g e l e t c . In subsequent y e a r s due to the importance of the t r a n s f e r r i n p a r t o f the serum p r o t e i n i n r e l a t i o n t o m i l k p r o d u c t i o n and Y - g l o b u l i n f r a c t i o n i n r e l a t i o n t o antibody p r o d u c t i o n , methods were developed to s e p a r a t e them from o t h e r serum p r o t e i n s b e f o r e e l e c t r o p h o r e s i s . Krawczynska et a l . , (1967) and Segan (1968) used r i v a n o l (2, 5 diamino -7- e t h o x y a c i d i n e ) l a c t a t e t o separate t r a n s f e r r i n and Y - g l o b u l i n from o t h e r p r o t e i n s i n human serum. Hudson et a l . , (1970) used the r i v a n o l technique to s e p a r a t e serum immunoglobulins i n sheep. The a n a l y s i s of s e v e r a l o t h e r n o n - p r o t e i n b l o o d components a t a time from a composite sample o f animal o r i g i n i s a l s o a r e c e n t development. The hand method to a n a l y s e one b l o o d component a t a time i n v o l v e d time and l a b o u r . The s i n g l e channel a u t o a n a l y s i s was the f i r s t t e c h n i c a l .advance to reduce l a b o u r and time and t h i s was subsequently a m p l i f i e d to a m u l t i - c h a n n e l a n a l y z e r . Skeggs (1964) gave the f i r s t r e p o r t on the use of the e i g h t channel m u l t i p l e automatic a n a l y z e r system 10 developed by the Technicon Instrument Corp. New York at the Department of Medicine and Surgery, Veterans Administration Hospital and the Department of Pathology, Western Reserve University of Cleveland, Ohio. Adam Couts et a l . , (1967) reports the construction of the 16 channel sequential multiple auto analyzer (SMA) for the Los Angeles V.A. Center, by the Technicon Instrument Corp. New York. As the use of SMA machines increased, i t was necessary to evaluate the accuracy of the equipment for diagnosis and also quantitative recovery of various blood components. Laszlo Makk, et a l . , (1969) compared the re s u l t s obtained from SMA 12/60 (12-channel) instrument with that of manual techniques. They studied 1000 abnormal and 500 normal human blood constituent and concluded that SMA 12/60 re s u l t s were highly accurate provided proper controls were maintained. The means and standard d e v i a t i o n s of some human blood components obtained by SMA 12/60 and hand method as reported by them are given i n table 1 for comparison. 11 TABLE 1. THE MEAN (S.D.) FOR SOME HUMAN BLOOD COMPONENTS OBTAINED AFTER ANALYSIS OF SMA 12/60 AND HAND METHOD AS REPORTED BY LASZLO MAKK, ET AL.f(1969) SOME HUMAN BLOOD COMPONENTS MEAN (S.D.) SMA 12/60 ,MANUAL Calcium 8.7 mg. % (0. 15 mg. %) 8.7 mg. % (0. 21 mg. %) In. Phosphorus 3.7 mg. % (0. 13 mg. %) 3.5 mg. % (0. 3 mg. %) Glucose 109 mg. % (4. 1 mg. %) 110 mg. % (4. 0 mg. %) Blood Urea Nitrogen 11 mg. % (0. 15 mg. %) l i : 2 mg. % (0. 3 mg. %) Uric Acid 5.3 mg. % (0. 16 mg. %) 5.8 mg. % (0. 12 mg. %) Cholesterol 195 mg. % (4. 8 mg. %) 194 mg. % (6. 6 mg. %) Total Protein 6.15 gia. % (0. 11 gm. %) 6.7 gm. % (0. 23 gm. %) Albumin 3.65 gm. % (0. 11 gm. %•) 3.9 gm. % (0. 2 gm. %) B i l i r u b i n 3.3 mg. % (0. 06 mg. %) 3.7 mg. % (0. 3 mg. %) Alk a l i n e Phosphatase 40 mu/ml (1. La c t i c Dihydrogenase 105 mu/ml(2, Serum Glutamic Oxaloacetic Transaminase 30 mu/ml(2. 5 mu/ml) 1.25 25 mu/ml) 250 46 mu/ml) 22.5 (B-L Units) (0.124) (Warb unit) (11.5) (R & F Unit) (2.7) 12 F i n l y e t a l . , (19 69) attempted to use the SMA 12/60 machine f o r a c c u r a t e chemical a n a l y s i s i n the c l i n i c a l c h e m i s t r y l a b o r a t o r y . They s t u d i e d the p r e c i s i o n of measurements w i t h i n run and random d u p l i c a t e s w i t h i n and between r u n , on the same day and c o n s e c u t i v e days. These authors concluded t h a t the p r e c i s i o n and ac c u r a c y o f the SMA 12/60 was as good as hand methods and s i n g l e channel auto a n a l y z e r technique and i n some cases s l i g h t l y more a c c u r a t e . However, the r e p e a t a b i l i t y ( w i t h i n s i n g l e run) was b e t t e r than r e p r o d u c i b i l i t y ( w i t h i n b a t c h o f between d a y s ) . Broughton e t a l . , (1968) r e p o r t e d an assessment on the accuracy o f the measurement o f b l o o d components by SMA 12 ( h o s p i t a l model) from B r i t a i n . The p r e c i s i o n o f the machine compared s a t i s f a c t o r i l y w i t h the r o u t i n e d i a g n o s t i c l a b o r a t o r i e s . I n s t r u m e n t a l d r i f t and the i n t e r a c t i o n between s u c c e s s i v e samples on some channels were o c c a s i o n a l l y marked. The n e c e s s i t y f o r a s e q u e n t i a l m u l t i - c h a n n e l a n a l y z e r , f i r s t arose from c l i n i c a l uses t o reduce c o s t and f a c i l i t a t e d i a g n o s i s . The methods o f a n a l y s i s i n i t i a l l y u t i l i z e d were a d a p t a t i o n s o f the hand methods c u r r e n t l y i n use. L a t e r r e s e a r c h however, was d i r e c t e d towards improvement of the c u r r e n t t e c h n i q u e s , as w e l l a s , development o f new techniques s u i t a b l e f o r the automated 13 a n a l y s e s . In subsequent years t h i s need of a d a p t a t i o n of chemical a n a l y s i s to automation ( i n a d d i t i o n t o o t h e r t h i n g s ) l e d to the d i s c o v e r y of s e v e r a l methods f o r e s t i m a t i n g the l e v e l o f one b l o o d component. In the p r e s e n t s t u d y , p r o t e i n s o b t a i n e d by SMA 12/60 and e l e c t r o p h o r e s i s , was of p a r t i c u l a r i n t e r e s t as both methods were used to estimate these b l o o d serum components. Goodwin e t a l . , (1965) compared the p r o t e i n e s t i m a t i o n method by b i u r e t r e a c t i o n a f t e r s e p a r a t i n g albumin and g l o b u l i n by s a l t f r a c t i o n a t i n g t e c h n i q u e , and s e p a r a t i n g albumin from g l o b u l i n by methyl or e t h y l a l c o h o l i n a c i d media and zone e l e c t r o p h o r e t i c t e c h n i q u e . The o b j e c t i o n s suggested by them wit h r e g a r d to e l e c t r o p h o r e t i c technique were: 1. D i f f e r e n c e s i n the s t a i n i n g c h a r a c t e r i s t i c s o f the v a r i o u s p r o t e i n s . 2. A b s o l u t e e r r o r s a s s o c i a t e d w i t h albumin t r a i l . T h i s phenomenon causes the p r o t e i n to be d i s p r o p o r t i o n a t e l y contaminated. The albumin a n a l y z e s low, f o r i t i s the source of the t r a i l ; and a and B - g l o b u l i n s a n a l y z e h i g h f o r they are superimposed on the albumin t r a i l , whereas the Y - g l o b u l i n s remain uncontaminated by the t r a i l as they move o p p o s i t e to the albumin. 14 3. Differences i n staining c h a r a c t e r i s t i c s due to protein-bound components, such as l i p i d s , b i l i r u b i n , hemo-globin, carbohydrates, etc., In addition, these compounds associated with i n d i v i d u a l proteins are disproportionately bound. In conclusion the authors supported the auto-analyzer method over electrophoresis for protein and albumin determination. Kaplan et a l . , (1957), Brooksby (1947), Malkay et a l . , (1957) Lippman and :£anovitz. (1952) also compared proteins obtained by d i f f e r e n t methods including protein separated by electrophoresis and state that i t i s very d i f f i c u l t to compare the proteins obtained by electrophoresis and other chemical methods. Jager et a l . , (1950) studied s i x methods for the comparative analysis of serum albumin. He reported very l i t t l e agreement between serum albumins obtained by d i f f e r -ent methods. In s p i t e of a l l the c r i t i c i s m against separation of serum protein by electrophoresis, Dimopoullos (1963) suggested i t has been observed to be consistent, popular and inexpensive means of analyzing the plasma proteins. 15 MATERIALS AND METHODS I EXPERIMENTAL ANIMALS The experimental animals c o n s i s t e d o f 226 d a i r y c a t t l e from the U n i v e r s i t y o f B r i t i s h Columbia Farm No. 2 a t Oyster R i v e r and i n c l u d e d h e i f e r c a l v e s , h e i f e r s , l a c t a t i n g cows and dry a n i m a l s . The herd was under a normal management system s i m i l a r to t h a t o f a commercial d a i r y farm. The animals used i n t h i s experiment were from the c r o s s b r e e d i n g study a n a l y z e d by W i l l i a m s (1971) and i n c l u d e d animals s i r e d by H o l s t e i n , A y r s h i r e and H o l s t e i n X A y r s h i r e c r o s s b u l l s . The animals were c a t e g o r i z e d i n t o H o l s t e i n breed groups based on p e r c e n t H o l s t e i n . T a b l e 2 g i v e s the a c t u a l number.of animals i n each c a t e g o r y . Because of the s m a l l numbers i n some of these c a t e g o r i e s , they were pooled to form t h r e e breed groups as shown i n the l a s t column o f the t a b l e 2. 16 TABLE 2 Number of Animals i n Each Percentage H o l s t e i n Category Percent H o l s t e i n Breed Number of Animals f o r Each Category 1. 100% 126 H 2. 75% 27 3. 69% 2 4. 63% 5 5. 62% 11 . 6. 50% 23 7. 38% 4 8. 37% 12 9. 31% 2 10. 25% 14 Grouping o f C a t e g o r i e s I = 126 animals II = 45 animals (75% - 62%) I I I = 55 animals (50% - 25%) I I BLOOD SAMPLING Blood samples were taken from a l l female animals on the farm w i t h sampling groups d i c t a t e d by the management system. These management groups were as f o l l o w s : 1. H e i f e r s i n the b r e e d i n g group which i n c l u d e d both pregnant and open h e i f e r s . These animals were b e i n g f e d green chopped g r a s s , hay and some g r a i n . 2. Dry cows on p a s t u r e w i t h supplemental g r a i n and hay f e e d i n g . .3. H e i f e r c a l v e s which were b e i n g f e d hay. and g r a i n . 4. Bred h e i f e r s on pas t u r e and some dry cows which r e c e i v e d no supplemental f e e d i n g . 5. Open h e i f e r s on p a s t u r e r e c e i v i n g no supplemental f e e d i n g . 6. M i l k i n g herd on p a s t u r e w i t h supplemental f e e d i n g o f hay and g r a i n . 1 7 The time l a p s e between the time of b l e e d i n g and the time o f m i l k i n g was measured f o r the l a c t a t i n g animals as the d i f f e r e n c e between the time the animal was b l e d and the time the animal e n t e r e d the m i l k i n g p a r l o u r p r e v i o u s to sampling i n minutes. S i n c e g r a i n was f e d i n the m i l k i n g p a r l o u r and hay was a v a i l a b l e on l e a v i n g the p a r l o u r the time l a p s e between m i l k i n g and b l e e d i n g and between f e e d i n g and b l e e d i n g were c o n s i d e r e d to be the same. The time i n t e r v a l f o r the n o n - l a c t a t i n g groups on dry feed was the l a p s e i n minutes between the l a s t f e e d i n g and the a c t u a l b l e e d i n g o f each i n d i v i d u a l a n i m a l . The time l a p s e f o r animals on p a s t u r e was measured as the d i f f e r e n c e between the time the group was c o n f i n e d p r i o r t o b l e e d i n g and the a c t u a l time the i n d i v i d u a l animal was b l e d . The b l o o d was c o l l e c t e d from the j u g u l a r v e i n i n 20 cc s i l i c o n i z e d p l a i n v a c u t a i n e r tubes (without a n t i -coagulant) f i t t e d w i t h a d i s p o s a b l e n e e d l e . The t e s t tubes w i t h the b l o o d samples ( a f t e r l o o s e n i n g the stopper) were kept i n an u p r i g h t p o s i t i o n f o r approximately two hours to a l l o w f o r c o a g u l a t i o n . The samples were then c e n t r i f u g e d a t 3,000 RPM f o r twenty minutes. The serum was p i p e t t e d out by p a s t u r e p i p e t t e to form t h r e e sub-samples. The sub-samples were s e a l e d and f r o z e n immediately u s i n g dry i c e and were kept f r o z e n u n t i l the samples were a n a l y z e d . 18 One set of b l o o d serum samples was a n a l y z e d by the SSM 12/60 m u l t i c h a n n e l s e q u e n t i a l a n a l y z e r . The second s e t was analyzed u s i n g e l e c t r o p h o r e s i s technique to c h a r a c t e r i z e the serum p r o t e i n s . I I I THE SMA 12/60 MULTI-CHANNEL ANALYZER The m u l t i - c h a n n e l a n a l y z e r was used to analyze f o r t o t a l p r o t e i n , albumin, c a l c i u m , i n o r g a n i c phosphorus, c h o l e s t e r o l , blood urea n i t r o g e n , u r i c a c i d , c r e a t i n i n e , t o t a l b i l i r u b i n , a l k a l i n e phosphatase, l a c t i c dehydrogenase and serum g l u t a m i c o x a l o a c e t i c transaminase (S.G.O.T.). The a n a l y s e s were done by a commercial l a b o r a t o r y , the M e t r o p o l i t a n B i o c h e m i c a l Lab o f Vancouver. The equipment used was a 12 channel model 60 s e q u e n t i a l m u l t i p l e a n a l y s e r (SMA) which was s t a n d a r d i z e d w i t h normal bovine serum. The p r i n c i p a l method o f a n a l y s i s f o r each component i s g i v e n i n the appendix. IV ELECTROPHORESIS OF PROTEINS The e l e c t r o p h o r e s i s of the serum samples was done wit h "Agarose f i l m " u s i n g the c a s s e t t e system as s u p p l i e d by the " A n a l y t i c a l Chemists, I n c . , P a l o A l t a , C a l i f . 94303." The c o l o r i n t e n s i t y was e v a l u a t e d by a p h o t o v o l t which produced a g r a p h i c r e l a t i o n s h i p between the p r o t e i n s 19 where the separated p r o t e i n bands appear as peaks. The D e n s i c o r d Model 542A of P h o t o v o l t 1115 Broadway, N.Y. 10010 was used f o r t h i s study and the peaks were marked f o r the s p e c i f i c p r o t e i n s as shown by the " A n a l y t i c a l Chemists, I n c . , Palo A l t o , C a l i f . " The d e t a i l of the procedure f o r e l e c t r o p h o r e s i s f o l l o w e d i s g i v e n i n the appendix. V MEASUREMENT OF THE GRAPHS The h e i g h t o f the peaks r e l a t i v e to the base l i n e was taken t o be the r e l a t i v e amount of each serum p r o t e i n and f o r a n a l y t i c a l purposes were expressed as the f r a c t i o n o f the t o t a l . In g e n e r a l , t h e r e were s i x peaks - Albumin, G l o b u l i n a G l o b u l i n a_, G l o b u l i n 8 , _L ^ X G l o b u l i n B^' a n <* G l o b u l i n y. The computarized g r a p h i c to d i g i t a l c o n v e r t e r method was used t o measure these peaks. VI SEPARATION OF SERUM TRANSFERRIN Twelve serum samples f a i l e d to show s e p a r a t i o n of the 4th and 5th peak. These twelve samples were drawn from the r e s e r v e sub-set mentioned e a r l i e r and were analyzed t o sepa r a t e the t r a n s f e r r i n ( 8^ and fc^) a m ^ Y f r a c t i o n o f the g l o b u l i n from the remaining serum p r o t e i n s . 20 The method used was t h a t g i v e n by Segan (1968) and Hudson e t a l . , (1970) with m o d i f i c a t i o n s as g i v e n i n the appendix. The methods of e l e c t r o p h o r e s i s , densitometer scanning and d i g i t i z a t i o n as d e s c r i b e d f o r p r o t e i n f r a c t i o n s were f o l l o w e d w i t h the e x c e p t i o n s mentioned i n the appendix. VII MILK SAMPLES M i l k samples from l a c t a t i n g animals were c o l l e c t e d a f t e r the morning m i l k i n g on the day the b l o o d samples were c o l l e c t e d . The samples were sent t o the P r o v i n c i a l Government Branch L a b o r a t o r y i n Vancouver, B.C. f o r a n a l y s i s o f m i l k f a t , p r o t e i n and l a c t o s e c o ntent u s i n g an (IRMA) i n f r a - r e d m i l k a n a l y s e r (Grubb-Parsons, MKII). V I I I THE SAMPLE SIZE FOR DIFFERENT ANALYSIS Two b l o o d serum components were excluded from the p r e s e n t a n a l y s i s . B i l i r u b i n was d i s t r i b u t e d i n o n l y a few c l a s s e s which d i d not l e a d to a s a t i s f a c t o r y a n a l y s i s . L a c t i c dehyrogenase o b s e r v a t i o n s were ve r y h i g h i n the o r d e r of 600mu/100 ml blood which i s beyond the p h y s i o l o g i c a l e x p e c t a t i o n . S i m i l a r r e s u l t was a l s o observed by Spate e t a l . , (1970) w h i l e a n a l y s i n g f o r l a c t i c dehydrogenase from f r i d g e d r i e d bovine b l o o d serum samples. From the 21 t o t a l sample o f 226 a n i m a l s , one animal showed abnormal va l u e f o r p r o t e i n and two animals abnormal v a l u e s f o r c h o l e s t e r o l . These t h r e e animals were excluded from a l l a n a l y s e s . The 12 animals which showed m i s s i n g 4th peak d u r i n g densitometer scanning of e l e c t r o p h o r e t i c a l l y s eparated serum p r o t e i n s were t r e a t e d s e p a r a t e l y f o r a n a l y t i c a l purposes. Hence, the g e n e r a l a n a l y s i s of data c o n s i s t s of 211 a n i m a l s . (See a p p e n d i x ) . The d i s t r i b u t i o n o f the raw data f o r each b l o o d serum component i s g i v e n i n the appendix. IX STATISTICAL TECHNIQUE The a n a l y t i c a l technique used i n the p r e s e n t study was designed to account f o r as many sources of v a r i a t i o n s as p o s s i b l e f o r each b l o o d component. The causes of v a r i a t i o n s were c l a s s i f i e d i n t o two c a t e g o r i e s ; environmental and g e n e t i c . A f t e r a c c o u n t i n g f o r a l l p o s s i b l e sources of environmental v a r i a t i o n s , the v a r i a t i o n due to g e n e t i c causes were enumerated and g e n e t i c parameters f o r each o f these b l o o d components were e s t i m a t e d . A. E s t i m a t i o n of Environmental and G e n e t i c Components of  V a r i a t i o n : Blood being the main t r a n s p o r t a t i o n medium of the animal body, i t was expected t h a t the feed absorbed by the system and t r a n s p o r t e d by blood or the p r o d u c t i o n 22 s y n t h e s i z e d by the animal body from blood are the important and immediate sources of environmental e f f e c t c a u s i n g v a r i a t i o n , i n the bl o o d serum components. In view o f the above, each b l o o d serum component was r e g r e s s e d s e p a r a t e l y w i t h i n each o f the s i x management groups on the time l a p s e d between f e e d i n g or f e e d i n g and p r o d u c t i o n and c o l l e c t i o n o f b l o o d samples from the a n i m a l s . The f o l l o w i n g simple l i n e a r model was assumed o f o r each o f the 17 blood components w i t h i n each o f the s i x management groups. Yi = 3o+ 3 l X i + £ l (1) * Where: y. = observed v a l u e o f the bl o o d component on the x"^1 a n i m a l , X ^ = time l a p s e d i n minutes between f e e d i n g (or f e e d i n g and pr o d u c t i o n ) t i l and c o l l e c t i o n o f b l o o d f o r the i a n i m a l . 8 = s c a l a r c o n s t a n t , o Bi = the r e g r e s s i o n c o e f f i c i e n t f o r the X -i independent v a r i a b l e , * R e f e r r e d i n t e x t as model numbers 23 and e. = the d e v i a t i o n o f the y. val u e from the r e g r e s s i o n l i n e ; r a n d o m . e r r o r , 'NID (0, a2 ) Age o f the a n i m a l , the c o n d i t i o n o f l a c t a t i o n and/or pregnancy and the d u r a t i o n of l a c t a t i o n and pregnancy were c o n s i d e r e d t o be the other p o s s i b l e sources of v a r i a t i o n s i n the v a r i o u s b l o o d serum c o n s t i t u e n t s . Animals s u b j e c t e d t o the common cause or causes o f v a r i a t i o n s were grouped t o g e t h e r f o r the a n a l y t i c a l purpose. The groups were d e f i n e d as young a n i m a l s , bred h e i f e r s , l a c t a t i n g non pregnant a n i m a l s , l a c t a t i n g and pregnant animals and dry a n i m a l s . The mean and standard e r r o r (S.E.) f o r the sources o f v a r i a t i o n s w i t h i n each group are g i v e n i n t a b l e no. 15. Each group was a n a l y s e d u s i n g a s p e c i f i c l i n e a r r e g r e s s i o n model designed to estimate the e f f e c t s unique t o t h a t group. 1. The l i n e a r model f o r the young female animal group. T h i s group i n c l u d e d animals from c a l v e s and to y e a r l i n g h e i f e r s which had not been bred and confirmed pregnant. y± = 80 + B r X i + e ± (2) 24 Where: y. = the observed v a l u e of the b l o o d th serum component on the i a n i m a l , X^ = age i n days ( b i r t h o f the animal to the th day of b l o o d sampling) o f the i a n i m a l , 80 = s c a l a r c o n s t a n t , 81 = the r e g r e s s i o n c o e f f i c i e n t f o r the X^ independent v a r i a b l e , and e.. = the d e v i a t i o n o f the y. v a l u e from the 1 1 2 r e g r e s s i o n l i n e . Random e r r o r , NID (0 , a ') 2. The l i n e a r model f o r the bred h e i f e r : T h i s i n c l u d e s o n l y those h e i f e r s which were confirmed pregnant. y± = B o + 81X iL + 82X 2 i + e ± > (3) Where: y. = the observed v a l u e o f the b l o o d serum J 1 component on the i * " *1 a n i m a l , ( i = 1, 2, n) , 25 X ^ = days pregnant (day l a s t bred to the day o f b l o o d sampling) f o r the . t h • -m 1 a n i m a l , X2 i = a^e ^n ^a^s ( b i r t h o f the animal to the days o f b l o o d sampling) o f .th , the 1 a n i m a l , Bo = s c a l a r c o n s t a n t , Bi '82 = r e g r e s s i o n c o e f f i c i e n t f o r and X^ r e s p e c t i v e l y , and = the d e v i a t i o n o f the y^ v a l u e from the r e g r e s s i o n s u r f a c e . Random 2 e r r o r , NID (0, a ) 3. The l i n e a r Model f o r the L a c t a t i n g N o n p r e g n a n t Animals I n c l u d e s animals l a c t a t i n g but confirmed not pregnant the day b l o o d samples were c o l l e c t e d . ^1 = B0 + * ixl i + B2x2. + B3X3. + B4x4. + 35X5 i + B6X6 i + £i ( 4 ) Where: y^ = the observed v a l u e o f the b l o o d serum component on the i * " *1 a n i m a l , ( i = 1, 2, n) 26 l i = days l a c t a t i n g (the day of l a s t p a r t u r i t i o n to the day of blood t h sampling) f o r the i a n i m a l , X2 i = k i l o g r a m o f m i l k produced by the th i animal i n the morning m i l k i n g the day the b l o o d sample was c o l l e c t e d , X^j^ = k i l o g r a m o f f a t produced i n the t h morning m i l k i n g by the i a n i m a l , X ^ = k i l o g r a m o f p r o t e i n produced i n t h the morning m i l k i n g by the i a n i m a l , X,.^  = k i l o g r a m o f l a c t o s e produced i n the morning m i l k i n g by the i a n i m a l , Xg^ = age i n days ( b i r t h to the day o f 4-Vi b l o o d sampling) f o r the i animal, 8 Q = s c a l a r c o n s t a n t , Ba/ 8 2 , 8 3 * 8i»/ 8 s » 8 6 = r e g r e s s i o n c o e f f i c i e n t s f o r the independent v a r i a b l e s X^, X^, X^, Xj., Xg r e s p e c t i v e l y , 27 and e. = the d e v i a t i o n of the y. value from 1 1 x the r e g r e s s i o n s u r f a c e . Random 2 e r r o r , NID (0, a ) • 4. The l i n e a r model f o r the l a c t a t i n g pregnant cows: T h i s group i n c l u d e s o n l y those l a c t a t i n g animals which were confirmed pregnant. Y± = 6o +3iX 1 ; L + 82x2;L + 63x3;L + 3-X^ + B5X5 i + 86X6 i + 67X7i + e i (5) Where: y. = the observed v a l u e o f the b l o o d th serum component on the i a n i m a l , ( i = 1, 2, n) X ^ = days l a c t a t i n g (the day of l a s t p a r t u r i t i o n to the day o f b l o o d t h sampling) f o r the i a n i m a l , X2 i= ^a¥s Pregnant (day l a s t bred to the day o f bl o o d sampling) f o r . th . . the x a n i m a l , ^2±= k i l o g r a m o f m i l k produced by the th i animal i n the morning m i l k i n g the day the bl o o d sample was c o l l e c t e d , 28 X.. = k i l o g r a m of f a t produced i n the morning 4 i th m i l k i n g by the i a n i m a l , X,.^  = k i l o g r a m of p r o t e i n produced i n th the morning m i l k i n g by the i a n i m a l , Xg^ = k i l o g r a m of l a c t o s e produced i n the t h morning m i l k i n g by the i a n i m a l , X^^ = age i n days ( b i r t h to the day o f t h b l o o d sampling) f o r the i a n i m a l , 8 = s c a l a r c o n s t a n t , o 81,82, 83,84,85,86,87 = r e g r e s s i o n c o e f f i c i e n t s f o r the independent v a r i a b l e s X^, X^, X^/ X^, Xj-, Xg, X^ r e s p e c t i v e l y , and e. = the d e v i a t i o n o f the y. v a l u e from the r e g r e s s i o n s u r f a c e . Random e r r o r , NID (0, a 2) . 5. The l i n e a r model f o r the dry animals: T h i s i n c l u d e d those animals which have had a t l e a s t one l a c t a t i o n and a l l were confirmed pregnant. y = 8Q + 81 X 1 ± + 82 X 2 j L + e ± (6) 29 Where: = ^he observed v a l u e o f the b l o o d serum t h component of the i a n i m a l . ( i = 1, 2 n) X ^ = days pregnant (day l a s t bred to the day t h of b l o o d sampling) f o r the i a n i m a l . X2 i = a^e days ( b i r t h to the day of b l o o d t h sampling) f o r the i a n i m a l . Bo = s c a l a r c o n s t a n t . Bi# B2 = r e g r e s s i o n c o e f f i c i e n t f o r the independent v a r i a b l e s X^ and X2 r e s p e c t i v e l y . e. = the d e v i a t i o n o f y. v a l u e from the x 2 x r e g r e s s i o n s u r f a c e . Random e r r o r NID (0, a2) Breeds and s i r e s were c o n s i d e r e d to be the p r i n c i p l e causes o f g e n e t i c v a r i a t i o n i n the p r e s e n t s t u d y . S i n c e the animals i n t h i s study were e i t h e r H o l s t e i n o r H o l s t e i n - A y r s h i r e c r o s s b r e e d s . The breed c o m p o s i t i o n based on percentage of H o l s t e i n , was c o n s i d e r e d . The breed groups and number of animals i n each group are g i v e n i n Table 2 . 30 The p h y s i o l o g i c a l groups, as mentioned e a r l i e r , and breed group were c o n s i d e r e d t o be p o s s i b l e cause of v a r i a t i o n . The a d j u s t e d d a t a f o r f i x e d e f f e c t s unique t o each p h y s i o l o g i c a l group was analyse d w i t h the f o l l o w i n g model: Y. .. = a + G. + B .+b. (A) . + e . .. (7) l j k 1 j I l j k l j k Where i j k Y = the k*"*1 o b s e r v a t i o n i n the i * " *1 t h group and j breed group, a = the o v e r a l l mean when A.. i s equal i i M t o z e r o , t h G^ = the p h y s i o l o g i c a l group o f the i animal under s i m i l a r cause or causes as e x p l a i n e d , i = 1, 2, 5; Bj = breed groups grouped as per the percentage of H o l s t e i n breed w i t h A y r s h i r e , j = 1, 2, 3; bj = r e g r e s s i o n of the dependent v a r i a b l e Y. ., on the independent continuous v a r i a b l e A.., each time h o l d i n g the 13k ^ 3 1 f i t t e d d i s c r e t e v a r i a b l e s , groups (G.) and breeds (B.) c o n s t a n t , x j A^j^. = Age i n days f o r the c o r r e s p o n d i n g Yi j k ' 2 and £ . ., = random e r r o r , NID (0, cx ) (K = l,2,..n) The model was analyzed by the l e a s t squares technique f o r unequal s u b c l a s s numbers as g i v e n by Harvey (1960). Age was a v a i l a b l e f o r a l l the groups and hence was f i t t e d i n the model as a c o v a r i a b l e . Sources o f v a r i a t i o n such as l a c t a t i o n , pregnancy, e t c . were not common f o r a l l the p h y s i o l o g i c a l groups and were e v a l u a t e d by groups u s i n g models ( 2 ) , ( 3 ) , ( 4 ) , (5) and ( 6 ) . To combine the d a t a o f the p h y s i o l o g i c a l groups the o b s e r v a t i o n s were a d j u s t e d f o r s i g n i f i c a n t sources o f v a r i a t i o n unique to each p h y s i o l o g i c a l group u s i n g r e g r e s s i o n t e c h n i q u e s . For the young female animal group, no c o r r e c t i o n was used as age was the o n l y environmental source o f v a r i a t i o n f i t t e d . For the bred h e i f e r and dry animal groups the blood serum components were r e g r e s s e d s e p a r a t e l y 32 within each group on the days pregnant. The blood serum component of the l a c t a t i n g non pregnant and l a c t a t i n g pregnant groups were f i t t e d i n a stepwise regression model within groups. The independent variables considered are shown i n models (4) and (5)excluding age i n both the cases. The stepwise regression technique followed were b a s i c a l l y those described by Draper et a l . , (1966). Each blood component within each group was corrected for only the s i g n i f i c a n t regression c o e f f i c i e n t s , as suggested by Kempthorne (1966). The model used for the correction of the each blood serum component within each group was as follows: *ik = Yi k ' bl k ( Xi ik - 3 fi k) + b2k( X2ik"X"2k) + b £ k ( X£ i k- X £ k ) ( 8 ) Where: i = 1, 2, n. k = 1, 2, 4. (The 4 p h y s i o l o g i c a l groups excluding young female animals). I = 1, 2, 7. y± = calculated value y^ = observed value of the blood serum th component for the i animal. th X „ . , = the measurement of the % independent Jlik variable (days pregnant, kilogram milk e t c . as explained i n models (4) and (5) on the i t * 1 animal i n group of animals. X = the mean of the i^ 1 independent variable £k for the k'*"*1 group. t i l b. = the regression c o e f f i c i e n t for the SL £k t h independent v a r i a b l e i n the k group. The blood serum components for each animal a f t e r c o r r e c t i o n within groups were analyzed by the l i n e a r model - 7 to estimate the e f f e c t of p h y s i o l o g i c a l groups and breeds. S i g n i f i c a n t breed group e f f e c t s were evaluated using orthogonal contrasts. The comparison chosen were Holstein vs. the two cross bred groups and one crossbred group vs. the other crossbred group. The orthogonal c o e f f i c i e n t s which define the desired contrasts between the breeds are: Br. 1 Br. 2 Br. 3 2 -1 -1 0 1 - 1 — ( 9 ) Where: Br. 1 = 100 per cent Holstein, Br. 2 = 75 per cent to 62 per cent Holstein and Br. 3 = 50 per cent to 25 per cent Holstein.,. 34 The s i g n i f i c a n c e o f the mean squares f o r these i n d i v i d u a l degrees of freedom were t e s t e d as shown by Harvey (1960). S i g n i f i c a n t p h y s i o l o g i c a l group e f f e c t s were e v a l u a t e d u s i n g the Duncan's new m u l t i p l e range t e s t as shown by Kramer (1957). B. Est i m a t e o f S i r e Component o f . V a r i a n c e f o r Gen e t i c Parameters: To e s t i m a t e the g e n e t i c v a r i a t i o n due to the s i r e s o n l y the H o l s t e i n s i r e s w i t h t h r e e o r more progeny were c o n s i d e r e d . Only f i v e A y r s h i r e s i r e s were r e p r e s e n t e d i n the t o t a l sample of which o n l y t h r e e had t h r e e o r more progeny. In a d d i t i o n , no pure A y r s h i r e progeny were a v a i l a b l e . In view o f the above o n l y the progeny o f the H o l s t e i n s i r e s were c o n s i d e r e d i n the e s t i m a t i o n o f s i r e components o f v a r i a n c e . T h i s reduced the sample s i z e o f 211 to 158 a n i m a l s . The da t a were c o r r e c t e d by the r e g r e s s i o n c o e f f i c i e n t s as d e s c r i b e d f o r the t o t a l sample s t a r t i n g from the o r i g i n a l data w i t h i n the groups d e f i n e d f o r these 158 a n i m a l s . The b l o o d serum components under each group which were a f f e c t e d by the d i f f e r e n t independent v a r i a b l e s f i t t e d are g i v e n i n box number one and the a c t u a l c o e f f i c i e n t s used to c o r r e c t the data i n box number two o f the appendix. 35 Estimation of the s i r e component of v a r i a t i o n was based on a mixed model with groups as fi x e d e f f e c t , s i r e as random e f f e c t , and age l i n e a r and quadratic as covariables. The l i n e a r mixed model assumed was as follows: Y. = a + G. + S . + (A) . .. + b 2 ( A ) 2 i j k + e i j k (10) Where Y. ., = observation of blood serum i j k th component on the k progeny .th , . .th . i n 1 group s i r e d by j s i r e , a = the o v e r a l l mean when (A) . ., and I J K 2 (A) . ., are equal to zero I J K G^ = the ph y s i o l o g i c a l groups as defined by l a c t a t i o n stage, pregnancy, etc., i = 1/ 2, 5; Sj = s i r e s , j = 1, 2, 15; th (A) . ., = age of the k animal i n days I J K (birth to the day of blood sampling); 2 ( A ) i j k = ( A ) i j k s c 3 u a r e d ' b l & b2 = P a r t ^ a l regression of the 36 and dependent v a r i a b l e on the independent continuous v a r i a b l e s (A) and ( A )2 h o l d i n g Gi and S.. c o n s t a n t , 2 e... = random e r r o r , NID (0 , o ) (K=l, 2 ,. . .n). 1 J K The model (10) was a n a l y s e d by the l e a s t - s q u a r e s technique as mentioned e a r l i e r . The d e s i g n of a n a l y s i s o f v a r i a n c e and expected mean squares f o r the model was as f o l l o w s : AN OVA (11) Sources o f Sums o f Mean V a r i a t i o n D.F. Squares Squares E (M.S.) Groups (G) (G-l) S SG MSG a 2 + K„0 e z S i r e s (S) (S-l) s s s MSS _ 2 ° e + K, s 2 Age (A) 2 S SA MSA R e s i d u a l n-G-S-L S SR MSR a2 e T o t a l n-1 37 T  2 0 = i a i (Fixed effect) G-l K l ' K2 = c o e ^ ^ i c i e n t s X = function of ( 8's) 2 a e was the t e s t i n g term for a l l the sources of v a r i a t i o n f i t t e d . Age l i n e a r and age quadratic was f i t t e d 2 together a e + X( B^, B 2 ) w ; i - t h t w o degrees of freedom and then the l i n e a r and quadratic a f t e r l i n e a r were computed. The s i r e component of v a r i a t i o n was estimated from the present model as follows: -2 ( c j 2e + K l 0 2 ) - (a 2e) °s = (12) Kl which i s 0 MS - MS„ ^ — S Iv K l k^ = weighted number of progeny per s i r e . . The k^ value was calculated as shown by Cunningham (1969) and Searle (1971) f o r models having j u s t one random e f f e c t factor i n a mixed model [tr(K) + Sm (K)] (13) Where: Sm(K) = 1* K l , the sum of the elements of K tr(K) = trace of K 38 The g e n e t i c parameters f o r the d i f f e r e n t b l o o d components were c a l c u l a t e d as f o l l o w s from the above model. H e r i t a b i l i t y : The h e r i t a b i l i t y was c a l c u l a t e d from the p a t e r n a l h a l f s i b i n t r a c l a s s c o r r e l a t i o n s . ~ 2 2 4 (  0 s} as hZ = — (14) "2 "2 a + a e s 2 Where h = h e r i t a b i l i t y m narrow sense, "2 a e = component of e r r o r v a r i a n c e , "2 and o s = s i r e component of v a r i a n c e , e s t i m a t e s 1/4 o f the a d d i t i v e g e n e t i c v a r i a n c e , 1/16 of a d d i t i v e x a d d i t i v e g e n e t i c v a r i a n c e . 2 The v a r i a n c e component a e c o n s i s t s o f t o t a l 2 2 v a r i a n c e (a T) - Cov HS. Where o"T i s the t o t a l v a r i a n c e . The Cov HS = l/4Va + l/16Vaa + l/64Vaaa + . . . — (15) ~2 T h i s a e a f t e r removing group e f f e c t s e s t i m a t e s a l l the environmental v a r i a n c e p l u s the remainder of the a d d i t i v e and non a d d i t i v e g e n e t i c v a r i a n c e . Hence ^2 ^ 2 4 times the ( a d d i t i v e v a r i a n c e ) d i v i d e d by a + a e 2 (pheonotype v a r i a n c e ) i s h i n narrow sense. 39 2 The standard error of h was calculated as shown by Swiger et a l . , (1964) adopted from Fisher's (1925) i n t r a class c o r r e l a t i o n . S.E. (h2) = 2^n-l) (1-t)) 2 [l + K-Dtf K x 2 £(N-S) (S-D)] (16) 2 V7here a s t = 2 2 a + a e s n = number of observation ., s = number of s i r e s ., and k^ = c o e f f i c i e n t . Genetic Correlation; The formula as given by Falconar (1961) to estimate genetic c o r r e l a t i o n was used. cT AB rG = \Jl\ (A)]. H ( Bg (17) 2 Where o (A) = s i r e component for t r i a t A. 2 Q (B) = s i r e component for t r i a t B. 0A B = covariance of t r i a t s A and B. The covariance of t r a i t s A and B was obtained as follows: 40 E AB =JZ(A+B)2 - EA2 - ZB2]/2 Where A = the o b s e r v a t i o n o f one b l o o d component. B = the o b s e r v a t i o n o f the o t h e r b l o o d component. The standard e r r o r (S.E.) o f g e n e t i c c o r r e l a t i o n was c a l c u l a t e d as shown by Reevs (1955) and Robertson (1959) S.E.(rG) E" (rG>l| S.E.h2(A) . S.E.h2(B) 2(h2(A) • hZ (B) (19) Environmental C o r r e l a t i o n : The c o r r e l a t i o n due to environment between two t r a i t s was c a l c u l a t e d as f o l l o w s : a| ~a2e ( A ) . (B)j \J Q2e(A ) j ^2e (B) ] rE = (20) Where a j~a 2e (A) . (B)j = c o v a r i a n c e o f r e s i d u a l v a r i a n c e o f t r a i t A and B. 41 2 2 CT e(A)&a e(B) = r e s i d u a l varxance of t r a x t A and t r a i t B r e s p e c t i v e l y . Phenotypic C o r r e l a t i o n between the T r a i t s : The formula as shown by P i r c h n e r (1968) was used to estimate the phenotypic c o r r e l a t i o n s between the t r a i t s . r p = rG \jh2A . \Jh2B + r£ \J(l-h2A) ( l - h2B ) — (21) 42 RESULTS AND DISCUSSION I THE EFFECT OF THE TIME LAPSE BETWEEN FEEDING OR FEEDING AND MILKING AND COLLECTION OF BLOOD SAMPLES ON EACH BLOOD SERUM COMPONENT WITHIN EACH MANAGEMENT GROUP As mentioned p r e v i o u s l y animals from which b l o o d samples were c o l l e c t e d r e p r e s e n t e d s i x groups based on the p r e v a l e n t management system. The time l a p s e from f e e d i n g or f e e d i n g and p r o d u c t i o n to t a k i n g the bl o o d sample was c o n s i d e r e d t o be a p o s s i b l e source o f v a r i a t i o n a f f e c t i n g the l e v e l o f v a r i o u s b l o o d c o n s t i t u e n t s (Model - 1 ) . Sin c e the e f f e c t of l a p s e d time was unique f o r each management group; each b l o o d serum component was r e g r e s s e d on l a p s e time w i t h i n groups. The l i n e a r c o e f f i c i e n t s , means and standard d e v i a t i o n s (S.D.), f o r each b l o o d serum component w i t h i n each management group are g i v e n i n t a b l e number 3. The time l a p s e was a s i g n i f i c a n t source o f v a r i a t i o n o n l y i n t o t a l p r o t e i n i n management group f i v e (open h e i f e r s on p a s t u r e ) ; c a l c i u m i n management group s i x ( m i l k i n g h e r d ) ; i n o r g a n i c phosphorus i n management groups t h r e e and f o u r (young female c a l v e s , pregnant h e i f e r s , and dry cows), and c r e a t i n i n e i n management group t h r e e . TABLE NO. 3 THE REGRESSION COEFFICIENTS FOR THE BLOOD SERUM COMPONENTS AFTER REGRESSING ON TIME LAPSE BETWEEN FEEDING OR FEEDING AND PRODUCTION WITHIN SIX MANAGEMENT GROUPS Dependent V a r i a b l e s P r o t e i n Albumin G l o b u l i n Management Groups Management Groups Management Groups 1 2 3 4 5 6 1 2 CN 00 r~ r- m CO *y CN) CN IT) o o\ rH o O VO i H rH in o ro o rH o O O o o o o C o e f f i c i e n t s in VO CN co rH (Ti m vo co CN CN vo o CO m CN o o o VO rH rH o o o o o o o o O O o Mean-(S.D.) F o r (Ti co rH 00 CTl in t> in rH CN CTl 00 o vo co ro VO CTl rH o rH rH CN CN CO vo co CN r - 00 the Dependent o o o o o o o o o o O O O o o O o o +l +1 +l +1 + i +1 +1 + i +1 +1 +1 -H +1 +i +1 + 1 -H +l V a r i a b l e s rH VO CO r- m o rH m rH t> co o CN VD a\ 00 CN r~- vo rH rH 00 O CN rH r- vo CO in vo r» VO vo CN CN rH CN CN CN in in in a = x 10~ 4 * S i g n i f i c a n t 'F' P < 0.05 Continued. TABLE NO. 3 THE REGRESSION COEFFICIENTS FOR THE BLOOD SERUM COMPONENTS CONTINUED Dependent Variables Calcium In. Phosphorus Cholesterol Management 0 Groups Management Groups Management Groups 1 2 3 4 5 6 1 2 3 4 5 6 * * vo VO CN r-CO ro CN CO CO in VO o H ro o vo co cn o in H CM CN o o vo o r- vo o o 1 1 I CN rH CN • CN ,—. ^ . • VO H co VO VO —. . , — CO C\ o VO in CO co o ro m • . . O rH o o o CN CN CN CN o O —' — ' — ' - — s — *—' — ' - — CO ro VO * ^ v • '—' + 1 + 1 + 1 +i + l +1 + 1 +1 + i + 1 +1 + 1 in CO co o rH CN rH o r~- rH r-CO CN O CN co o rH rH CO •<* vo vo CO VO in in ^ cn CT\ CO ro rH rH rH rH CN rH 00 LH CA rH OT\ C o e f f i c i e n t s £ S £ £ 2 3 o o o o o o Mean (S.D.)For ro cn cn ,-H rH co * ' in CN m m vo the Dependent Variables £ £ £ S £ £ + 1 HH +1 +1 +1 +1 o r~ cn r~ CN cn vo r- rH o vo r-cn ffi m o o cn a = xlO " 4 * S i g n i f i c a n t 'F' P < 0.05 Continued. i t -TABLE NO. 3 THE REGRESSION COEFFICIENTS FOR THE BLOOD SERUM COMPONENTS CONTINUED. Dependent V a r i a b l e s B.U.N. U r i c A c i d C r e a t i n i n e Management Groups Management Groups Management Groups 1 2 3 4 5 6 1 2 3 4 5 6 o in <N in ro vo * L O ro vo . r - o cn rH V O vo rH ro C O r- C O ro C o e f f i c i e n t s r- r~- vo • JOO C N cn ro m C N t> co rH V O C N o C N rH C N rH rH o o rH ro o o o L O C N rH o o 1 l 1 1 l o O 1 O o O 1 o o l o 1 o 1 o 1 o o Mean (S.D.) For ID oo rH rH vo •^ r ro i C N rH i cn rH rH C N rH rH . o ro C O o C M rH C N rH H C N C N C N rH o rH rH the Dependent * C N ro • C N • rH • rH • o O O o o O O O O o o o — v — *•—• m ro cr\ r  in cn ro C O r- rH cn O tn C N in V a r i a b l e s vo ro rH ro C O r» 00 cs r- C O cn cn C N cn cn rH C O V O r- o cn cn o O o o o o o rH o o rH o a = x 10 4 * S i g n i f i c a n t 'F1 P < 0.05 Continued. U l TABLE NO .3 THE REGRESSION COEFFICIENTS FOR THE BLOOD SERUM COMPONENTS CONTINUED Dependent Variables Alk. Phosphatase S.G.O.T. E. Albumin Management Groups 1 2 3 4 5 6 Management Groups 1 2 3 4 5 6 Management Groups 1 2 3 4 5 6 Co e f f i c i e n t s Mean (S.D.) For the Dependent Variables m VO ro CTl o r-ro co co co v o o in co CN co o cn CO CN co CO in CN CN CO VO in o rH rH CO 00 in in r> oo co r~ rd CO rH CO rH r-CO CO o r- O r— CO co CO VO O CN ro CTl co in o o in CN CN o o rH rH • • • • * • o o o o o o o I O o 1 r- co rH rH rH CN CN co co rH r~ r- r- CM o CN rH CN O (Tl rH O ro in CM CN •=3< o •<? o co co rH vo vo 00 o I o I o .1 o I m o m o co CM o CM co CM CN rH cn co CM CM co CTl CM CTl CM CO rH CTl ro o tO ro (0 CN [— rH CN in CN CO CN CM O O rH O O O I I «3* O O O r*» o cn CN ro CN a = x I O - 4 * S i g n i f i c a n t 'F' P <_ 0.05 Continued. CTl TABLE NO -3 THE REGRESSION COEFFICIENTS FOR THE BLOOD SERUM COMPONENTS CONTINUED Dependent Variables Globulin * 1 Globulin « 2 Globulin 8 X Managment Management Management Groups Groups Groups Co e f f i c i e n t s 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 rd rd rd rd rd rd rd rd rd rd rd rd rd rd rd rd rd rd in ro vo r- cn in CN vo rH CN rH m CN in o rH rH rH rH VO o rH o CO o CN ro oo vo CN CN rH rH CO CO o CO CN CN CN CN r- ro rH o o CN H O CM o o rH o O CN o O o CN O vo o o rH o 1 o 1 O o l o l o 1 o 1 O 1 O o 1 O 1 o i O 1 o 1 o o 1 o 1 o M p a r l (a T)\ Pnr l N l N C N r - I C N r - l . r - l r - 1 C N r H r - f r H C N r H . - H r H . - l r H m e a n l o . u j r o r o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o the Dependent — — w ^ ^ s _ y ^ ^ ^ - ^ ^ CN o ro CN CN CN CN CN rH CN CN CN in VO in in Variables rH rH rH rH rH rH rH rH rH rH H rH rH rH rH rH rH rH o o o o o o o o o o O o o O o o o o a = x 10" 4 * S i g n i f i c a n t 'F' P < 0.05 TABLE NO. 3 THE REGRESSION COEFFICIENTS FOR THE BLOOD SERUM COMPONENTS CONTINUED Dependent V a r i a b l e s G l o b u l i n 3 Management Groups G l o b u l i n Y Management Groups 1 2 3 4 5 6 1 2 3 AC 4 5 6 rd cd cd cd rd <d cd rd rd rd rd ro rH in r- rH rH o o VO rH in in rH in o o CM o CM o CM CM CM o rH CM rH rH rH in rH rH o O o co CM rH in rH rH CM C o e f f i c i e n t s Mean (S.D.) For the Dependent V a r i a b l e s o o i .—. CM rH O rH rH rH rH CM CM CM rH CM CM o • O O O O O O O O O O • O * • • • o ' — o o o o O O O o O O t> 00 IO l> r- r- CO r- CM CM CO VO rH rH rH rH rH rH rH rH rH rH rH rH O O O O O O o o o O O o a= x 10-4 * S i g n i f i c a n t 'F' P < 0.05 4 9 I t was concluded t h a t time l a p s e d between f e e d i n g or f e e d i n g and p r o d u c t i o n and c o l l e c t i o n o f the bl o o d sample was not g e n e r a l l y an important source of v a r i a t i o n i n the p r e s e n t s t u d y . The apparent absence o f a c o n s t a n t time l a p s e e f f e c t may have been i n p a r t due to the l e n g t h of time between f e e d i n g to sample c o l l e c t i o n , as w e l l a s , the l a c k of i n f o r m a t i o n on the a c t u a l i n t a k e by the i n d i v i d u a l a n i m a l . The mean time l a p s e d and S.D. f o r each management group i s g i v e n i n t a b l e number 4. The s h o r t e s t mean l a p s e d time f o r a group was 2.56 hours f o r group two and the l a p s e d time f o r i n d i v i d u a l animals exceeded two hours i n a l l c a s e s . The l a p s e d time f o r l a c t a t i n g animals was a j o i n t e f f e c t o f f e e d i n g and m i l k s e c r e t i o n s i n c e the animals were f e d c o n c e n t r a t e s i n the p a r l o u r d u r i n g m i l k i n g . The time l a p s e d was a s i g n i f i c a n t source o f v a r i a t i o n o n l y f o r bl o o d serum c a l c i u m i n t h i s management group. The reason f o r p o s i t i v e r e g r e s s i o n c o e f f i c i e n t (0.109) of b l o o d serum c a l c i u m on l a p s e d time f o r the l a c t a t i n g animals was not c l e a r . The i n c r e a s i n g l e v e l o f c a l c i u m i n the b l o o d , as time l a p s e i n c r e a s e d , may r e f l e c t e i t h e r r e c ouping o f i t s normal b l o o d c a l c i u m l e v e l d r a i n e d o f f d u r i n g m i l k i n g or be due to "the i n t a k e o f feed i n the p a r l o u r , p r i o r to the c o l l e c t i o n o f b l o o d samples. TABLE NO 4 MEAN AND STANDARD DEVIATION (S.D.) FOR TIME LAPSE IN HOURS BETWEEN FEEDING OR FEEDING AND PRODUCTION AND COLLECTION OF BLOOD SAMPLES FOR THE SIX MANAGEMENT GROUPS. MANAGEMENT GROUPS DESCRIPTION OF ANIMALS KIND OF FEED SUPPLIED DURING SAMPLING LAPS TIME BETWEEN FEEDING OR FEEDING AND MILKING... TO COLLECTION OF BLOOD SAMPLES - MEAN (S.D.) 1 i) Pregnant h e i f e r s i i ) Open he i f e r s i) Hay & ( i i ) Grain 3.13 (0.12) 2 i) Dry animals i) Pasture ( i i ) Grain i i i ) Hay 2.56 (0.57) 3 i) Heifer calves i) Hay & ( i i ) Grain 3.00 (0.29) 4 i) Pregnant h e i f e r s i i ) Dry animals i) Only on pasture 3.58 (0.18) 5 i) Open he i f e r s i) Only on pasture 4.37 (0.51) 6 i) Lactating animals (a) Pregnant (b) Open i) Pasture ( i i ) Grain i i i ) Hay 3.15 (1.11) 51 II ANALYSIS OF DIFFERENT PHYSIOLOGICAL EFFECTS OF THE BLOOD SERUM COMPONENTS To estimate the effects of age, pregnancy, stage of lactation and level of production, the animals were grouped into physiological groups, for analytical purposes. The analysis of each blood serum component was done on a within group basis. The physiological groups and the sources of va r i a b i l i t y which were measured for each group are given in table number 5. A. Linear Regression Analysis The sources of variations f i t t e d within each group were unique for the respective groups, as mentioned previously and the effect of these potential sources of variation within a group were not consistent for a l l blood serum component-studies as would be expected. The blood components within each physiological group were analyzed according to the linear models 2, 3, 4, 5 and 6. See table 6, 7 and 8. Total blood serum protein: The Model - 2 used for the young animal group accounted for 31% of the variation in the total blood serum protein which was significant. The Model - 4, fi t t e d within the lactating non-pregnant group accounted for 33% of variation in serum 52 TABLE NO. 5 THE PHYSIOLOGICAL GROUPS AND THE VARIABLES MEASURED WITHIN GROUPS Group The P h y s i o l o g i c a l The Sources o f V a r i a t i o n s Nos Groups Assumed Measured w i t h i n each group 1. Young female animals (1) Age (120 days and above, not y e t d e c l a r e d pregnant) 2. Pregnant or Bred (1) Age H e i f e r s . (Declared (2) Days Pregnant f i r s t time pregnant up to the day b e f o r e gave b i r t h to c a l f . ) 3. L a c t a t i n g and non- (1) Age pregnant a n i m a l s . (2) Days l a c t a t i n g (The day the animal (3) The amount o f m i l k gave b i r t h to c a l f t o produced p r i o r to the day b e f o r e the sampling animal d e c l a r e d (4) F a t pregnant.) (5) P r o t e i n (6) L a c t o s e , produced i n the m i l k i n g p r i o r t o s ampling. 4. L a c t a t i n g and pregnant (1). Age a n i m a l s . (The day the (2) Days l a c t a t i n g l a c t a t i n g animal (3) Days pregnant d e c l a r e d pregnant to (4) The amount of m i l k the day b e f o r e the produced p r i o r t o animals d e c l a r e d dry.) sampling (5) F a t (6) P r o t e i n and (7) L a c t o s e produced i n the m i l k i n g p r i o r t o sampling 5. Dry animals (The (1) Age animals who have a t (2) Days pregnant l e a s t one l a c t a t i o n and now have been stopped m i l k i n g due to the d r y i n g stage o f l a c t a t i o n c y c l e . A l l pregnant.) 53 p r o t e i n . The o t h e r t h r e e models f i t t e d w i t h i n the bred h e i f e r group, (Model - 3 ) , l a c t a t i n g pregnant group (Model - 5 ) , and dry animal group (Model - 6 ) f a i l e d to account f o r s i g n i f i c a n t amount of v a r i a t i o n i n t o t a l b l o od serum p r o t e i n i n the p r e s e n t s t u d y . Blood serum albumin: The Model - 2 f o r young animal and Model - 3 f o r bred h e i f e r groups s i g n i f i c a n t l y accounted f o r 34% and 18% of the v a r i a t i o n i n b l o o d serum albumin r e s p e c t i v e l y . Blood serum g l o b u l i n : Model - 2 f o r the young animal group and Model - 4 f o r the l a c t a t i n g non-pregnant group accounted f o r s i g n i f i c a n t amounts o f the v a r i a t i o n i n b l o o d serum g l o b u l i n , 14% and 28% r e s p e c t i v e l y . Blood serum c a l c i u m : Model - 2 f o r the young animal group accounted f o r 26% of v a r i a t i o n and Model - 3 f o r the bred h e i f e r group accounted f o r 13% o f v a r i a t i o n . Blood serum i n o r g a n i c phosphorus: Model - 2 and Model - 4 f o r the young animal group and f o r the l a c t a t i n g non-pregnant group, accounted f o r 35% of the v a r i a t i o n and 39% o f v a r i a t i o n , i n the b l o o d serum i n o r g a n i c phosphorus r e s p e c t i v e l y . 54 Blood serum c h o l e s t e r o l : A l l the models f i t t e d accounted f o r s i g n i f i c a n t amount of v a r i a t i o n i n blood serum c h o l e s t e r o l e x c e p t i n g Model - 3 i n bred h e i f e r group. Model - 2 i n young animal group accounted f o r 37%; Model - 4 i n l a c t a t i n g non-pregnant group accounted f o r 43%; Model - 5 i n l a c t a t i n g pregnant group accounted f o r 24% and Model - 6 i n dry animal group accounted f o r 63% o f the v a r i a t i o n i n b l o o d serum c h o l e s t e r o l . Blood urea n i t r o g e n (B.U.N.): Model - 4 f o r the l a c t a t i n g non-pregnant group accounted f o r 24%; Model - 5 f o r the l a c t a t i n g pregnant group accounted f o r 29% and Model - 6 f o r the dry animal group accounted f o r 47% of the v a r i a t i o n i n B.U.N. A l l o f these models accounted f o r s i g n i f i c a n t amounts o f v a r i a t i o n . Blood serum u r i c a c i d : Model - 2 f o r the young animal group accounted f o r 24% and Model - 5 f o r the l a c t a t i n g pregnant group accounted f o r 29% o f v a r i a t i o n i n b l o o d serum u r i c a c i d . Blood serum c r e a t i n i n e : Model - 2 f o r the young animal group accounted f o r 11% and Model - 4 f o r the l a c t a t i n g pregnant group accounted f o r 23% o f v a r i a t i o n i n the b l o o d serum c r e a t i n i n e . 55 Blood serum A l k a l i n e phosphatase: Model-2 f o r the young animal group accounted f o r 10%, Model-5 f o r the l a c t a t i n g pregnant group accounted f o r 29% and Model-6 f o r the dry animal group accounted f o r 67% o f the v a r i a t i o n i n blood serum a l k a l i n e phosphatase f o r the r e s p e c t i v e groups. Blood Serum g l u t a m i c o x a l o a c e t i c transaminase (S.G.O.T.): Model-2 f o r the young animal group and Model-6 f o r the dry animal group s i g n i f i c a n t l y accounted f o r 46% and 56% o f the v a r i a t i o n r e s p e c t i v e l y i n S.G.O.T. Blood serum albumin separated by e l e c t r o p h o r e s i s : Model-5 f o r the l a c t a t i n g pregnant group s i g n i f i c a n t l y accounted f o r 26% o f the v a r i a t i o n i n the b l o o d serum albumin separated by e l e c t r o p h o r e s i s . Blood serum g l o b u l i n OJT, : Model-2 f o r the young animal group accounted f o r 13%, Model-4 f o r the l a c t a t i n g non pregnant group accounted f o r 27% and Model-5 f o r the l a c t a t i n g pregnant group accounted f o r 58% o f the v a r i a t i o n , i n the b l o o d serum g l o b u l i n a i _ f w i t h i n the r e s p e c t i v e groups. Blood serum g l o b u l i n a?: Model-5 f o r the l a c t a t i n g pregnant group accounted f o r 32% o f the v a r i a t i o n i n the blood serum g l o b u l i n a.2-Blood serum g l o b u l i n • - g-[ : Model-2 f o r the young animal group accounted f o r 39% and Model -4 f o r the l a c t a t i n g non pregnant group accounted f o r 24% o f the v a r i a t i o n i n b l o o d serum g l o b u l i n 56 El o o d serum g l o b u l i n Model - 4 f o r the l a c t a t i n g non-pregnant group s i g n i f i c a n t l y accounted f o r 38% and Model - 5 f o r the l a c t a t i n g pregnant group accounted f o r 24% of the v a r i a t i o n i n the b l o o d serum g l o b u l i n . Blood serum g l o b u l i n y: Model - 2, f o r the young animal group accounted f o r 10% and Model - 5 f o r the l a c t a t i n g pregnant group accounted f o r 33% of v a r i a t i o n i n b l ood serum.y g l o b u l i n . See t a b l e s 6, 7, and 8. TABLE NO -6 THE SIMPLE REGRESSION COEFFICIENTS AND THE PROPORTION OF VARIATION (R2) FITTING MODEL (2) WITHIN YOUNG FEMALES AND PARTIAL REGRESSION COEFFICIENTS AND R2 FITTING MOGELS 3 AND 6 WITHIN BRED HEIFER AND DRV ANIMALS, RESPECTIVELY, FOR ALL THE BLOOD SERUM COMPONENTS Young Females Bred Heifer Dry animals Blood Serum Components Age i n days R2 f o r Days Pregnant Age i n days R or Days Pregnant Age i n days C o e f f i c i e n t s R2 complete Model ( ) C o e f f i c i e n t R2 C o e f f i c i e n t R2 complete Model ( ) C o e f f i c i e n t R2 C o e f f i c i e n t R2 T o t a l P r o t e i n 1.940(a)* (gm/100 ml. blood) 0.31 0.07 -4.464(a) 0.07 3. .000(a) 0 .06 0.31 -4.218(a) 0. 11 0. 315(a) 0 .10 Albumin (gm/loo ml.. 0.899 (a)* blood) 0.34 0.18* -1.785(a) 0.11 * 1. 619(a) 0 .17* 0.13 0.524(a) 0. 05 0. 070(a) 0 .12 Glo b u l i n (gn/loo ml. 1.040 (a)* blood) 0.14 0.03 -2.679(a) 0.03 1. 400 (a) 0 .02 0.30 -4.742 (a) 0^ 15 0. 244 (a) 0 .06 Calcium (rr.g./loo ml. 2.822 (a)* blood) 0.26 0.13* -3.966(a) 0.05 4. 255 (a) 0 .11* 0.06 -1.297(a) 0. 05 -0. 074 (a) 0 .02 Inorganic Phosphorus -5.818 (a) 0.35 0.07 4.313(a) 0.04 -4. 032 0 .07 0.27 0.010 0. 25 -0. 064 (a) 0 .00 (mg/100 ml. blood) * R i s s i g n i f i c a n t a t P £ 0.05 on 'F' d i s t r i b u t i o n , (a) - X10"3 Continued... TABLE NO . 6 THE SIMPLE REGRESSION COEFFICIENT ETC. FOR MODELS 2, 3 and . 6 Blood Serum Ccnpor.ents Young Females . .... Age in days R2 for Days Pregnant Age in days R2 Days Pregnant Age in days Coefficients R2 Complete Coefficxent R2 Coefficient R2 For Complete : ~ Model ( 3 ) Model ( 6) Coefficient R2 Coefficient R2 Bred Heifer Dry Animals Cholesterol 0.283* 0.37 0.10 (mg/100 ml.blood) B.U.N. -3.493(a) 0.02 0.01 (mg/100 ml. blood) Uric Acid -0.872(a)* 0.24 0.07 (mg/loo ml. blood) Creatinine -0.282 (a)* 0.11 0.10 (mg/100 ml. blood) Alk. Phosphatase -0.094* 0.10 0.38* (milimicron/ml.blood) S.G.O.T. 0.108*' 0.46 0.02 (milimcron/ml. blood) -0.246 0.07 0.204 0.10 0.63* 6.004(a) 0.00-0.282(a) 0.00 0.47* 1.263(a) 0.05-1.081(a) 0.07 0.27 0.734 (a) 0.04-0.164(a) 0.00 0.04. -0.187 0.05 0.042 0.01 0.67* -0.028 0.01 -0.021 0.01 0.56* -0.371 0.37* .-0.034 0.46* 0.070 0.43* 0.147(a)0.00 0.984 (a) 0.23 0.592 (a) 0.03 -0.294 0.19* 7.671(a) 0.00' 0.053(a)0.10 .0.048 (a)0.03 -.0.043 0.63* 0.011 0.50* * R i s significant at P < 0.05 on 'F' distribution, (a) " X10"3 Continued... TABLE NO . 6 THE SIMPLE REGRESSION COEFFICIENT ETC. FOR MODELS 2, 3 and 6. CONTINUED. B r » d H q - ( , f p r Dry Animals Blood Serum Young Females R* for Days Pregnant Age in days R2D a y s Pregnant Age in days Components Age in days Complete — — — — — - po r complete Coefficients R2 Model (3 ) Coefficient R2 Coefficient R2 Model ( 5) Coefficient R2 Coefficient R2 E. Albumin -0.086(a) 0.05 0.00 0.012(a) 0.00 0.015(a) 0.00 0.07 0.222(a) 0.05 -0.005(a) 0.00 (Part of the total Peaks) Glboulin - 0.1 0.058 (a)* 0.13 0.02 0.017(a) 0.00-0.031(a) 0.01 0.24 -0.025(a) 0.01 -0.011(a) 0.23 (Part of the total Peaks) Globulin - a2 0.016(a) 0.03 0.01 0.021(a) 0.00-0.020(a) 0.01 0.03 -0.023(a) 0.01 0.002(a) 0 01 (Part of the total peaks) -Globulin - B, 0.067(a)* 0.39 0.09 -0.061(a) 0.02 0.004(a) 0.00 0.03 -0.033(a) 0.02 -0.003(a) 0.02 (Part of the total peaks) I Glboulin - B, 0.003(a) 0.00 0.02 -0.026(a) 0.01 0.029(a) 0.01 0.17 -0.105(a) 0.09, 0.005(a) 0.03 (Part of the total • | peaks) ~ " r  Y-globulin -0.057(a)* 0.10 0.02 0.034(a) 0.00 0.004(a) 0.00 0.30 -0.034 (a) O.Ol! 0.013(a) 0.23 (Part of the total j peaks) *R2 i s significant at P £ 0.05 on 'F' distribution, (a) r XIO-3 <J3 TABLE NO: 7 THE PROPORTION OF VARIATION (R2) FOR THE MODEL (4) AND PARTIAL REGRESSION COEFFICIENTS AND R2 FOR EACH SOURCES FITTED FOR THE BLOOD SERUM COMPONENTS WITHIN THE LACTATING NON PREGNANT GROUP Blood Serum R2 Days La c t a t i j i g Kg. of Kg. of Kg. of Milk Kg. of Age i n days Components For the Milk Milk Fat L- P r o t e i n Lactose Complete Model ( 4) C o e f f i c i e n t R2 C o e f f i c i e n t R2 C o e f f i c i e n t R2 C o e f f i c i e n t R2 C o e f f i c i e n t R2 C o e f f i c i e n t R2 T o t a l P r o t e i n 0.33* -0.056(a) 0.00 0.257 0.05* -1.552 0.04-2.465 0.05 -3.704 0.03 0.516(a) 0.19* (gm/100 ml.blood) Albumin 0.11 0.554(a) 0.03 -0.012 0.00 0.274 0.01 -0.103 0.00 0.487 0.01 0.034(a) 0.01 (gm/100 ml. blood) G l o b u l i n 0.28* -0.610(a) 0.00 0.268 0.05 -1.826 0.05 -2.373 0.05 -4.190 0.04 0.482(a) 0.15* (gm/100 ml. blood) Calcium 0.12 -0.386(a) 0.00 -0.085 0.00 0.304 0.00 -1.493 0.2 2.095 0.01 -0.312(a) 0.05 (mg/100 ml. blood) Inorganic Phosphorus ,4 0.39 (mg/100 ml. blood) 1.109(a) 0.01-0.185 0.01 -0.425 0.00 0.491 0.00 2.337 0.01-0.663 0.15* * The R2 s i g n i f i c a n t a t P-<_ 0.05 on 'F* d i s t r i b u t i o n (a) - X10"3 Continued.-TABLE NO. 7 THE PROPORTION VARIATION <R2) for THE MODEL (4)..AND REGRESSION COEFFICIENTS ETC. CONTINUED Blood Serum R2 Days Lactating Kg. of Kg. of Kg. of Milk Kg. of Age in days Components For the ' Milk Milk Fat ~Protein Lactose Complete . , Model ( 4) Coefficient R2 Coefficient R2 Coefficient R2 Coefficient R2 Coefficient R2 Coefficient R' Cholesterol 0.4 3* (mg/100 ml. bipod) 0.372 0.17* -3.673 0.00 7.181 0.00 -325.5 0.10* 287.4 0.00 0.014 0.00 B.U.N. 0.24* (mg/100 ml. blood) 0.016 0.06* -0.532 0.01 4.985 0.01 12.580 0.03 16.07 0.01 -1.851(a) 0.05* Uric Acid 0.15 (mg/100 ml. blood) -0.208(a) 0.00 0.014 0.00 -0.503 0.03 - 0.893 0.05 0.420 0.00 -0.017 0.00 Creatinine 0.23* (mg/100 ml. blood) 0.020(a) 0.00 -0.017 0.01 0.063 0.00 0.399 0.06 0.011 0.00 0.064(a)0.12* Alk. Phosphatase 0.09 (milimicron/ml.blood) 0.020 0.00 -0.207 0.00 16.01 0.00 0.169 0.00 -13.34 0.00 -8.419(a)0.04 S.G.O.T. 0.10 (milirr.icron/ml .blood) -0.049 0.02 1.498 0.00 -12.11 0.00 - 74.16 0.04 22.56 0.00 -6.611 (a)0.03 * The R2 significant at P < O.05 on *F! distribution (a) - X 10"3 Continued. TABLE NO. 7 THE PROPORTION VARIATION (R2) FOR THE MODEL (4) AND REGRESSION COEFFICIENTS ETC. CONTINUED Blood Serum R2 Days Lactating Kg. of Kg. of Kg. of Milk Kg. of Age in days Components For the Milk Milk Fat Protein Lactose Complete Model ( 4) Coefficient R2 Coefficient R2 Coefficient R2 Coefficient R2 Coefficient R2 Coefficient R2 E. Albumin 0.11 -0.111(a) 0.04 9.719(a) 0.02 -0.048 0.01-0.032 0.00 -0.147 0.02 -0.004 0.00 (Part of the total peak heights) Globulin - a. 0.27* 0.053(a) 0.08*-2.708(a) 0.01 0.013 0.01 2.850(a) 0.00 0.052 0.02 -0.008(a) 0.12* (Part of the total peak heights) Globulin - a2 0.17 -0.014(a) 0.01 -1.201(a) 0.00 -4.257(a) 0.00 0.044 0.06 -0.015 0.00 -0.002(a) 0.01 (Part of the total peak heights) Globulin -61 0.24* 0.018(a) 0.01 4.280(a0 0.05 7.660(a) 0.00 0.038 0.04 0.041 0.01 0.001(a) 0.00 (Part of the total peak heights) Globulin $2 (Part of the peak heights) Y-globulin (Part of the peak heights) b 2 0.38* 0.101(a) 0.11*-0.149(a) 0.00 0.015 0.01.-0.048 0.08* 0.028 0.01 . 0.005(a) 0.06* total 0.12 -0.010(a) 0.00 -1.133(a) 0.00 0.014 0.00 -5.182(a) 0.00 0.036 0.00 0.011(a) 0.05 total * The R2 is significant at P < 0.05 on T" distribution. E» Albumin obtained by electrophoresis, (a)- X10"3 Continued.. rO TABLE NO. 8 THE PROPORTION OF VARIATION (R2) FOR MODEL (S) ANIl PARTIAL REGRESSION COEFFICIENTS, AND R2 POR EACH SOURCES FITTED FOR THE BLOOD SERUM COIIPONENTS WITHIN THE LACTATING PRGENANT GROUP. Blood Serum R2 Days Pregnant Days La c t a t i n g kg. of Kg. milk Kg. Milk Kg.Lactose • Age i n Components For the milk produce Fat Protein . Days  Complete - - ^ . - . • Model (5) C o e f f i c i e n t Rz C o e f f i c i e n t Rz C o e f f i c i e n t R2 C o e f f i c i e n t R2 C o e f f i c i e n t R2 C o e f f i c i e n t R2 Coefficient R2 T o t a l P r o t e i n 0.09 4.61(a) 0.02 >0.103(a) 0.00 -0.516 0.03 -2.335 0.01 1.918 0.00 10.25 0.05 0.101(a) 0.00 (gm/100 ml. blood) Albumin 0.08 -0.598(a) 0,01 -0..401(a) 0.01 25.8(a) 0.00 -0.367 0.01 0.544 0.01 -0.815 . 0.01 -0.030 (a) 0.01 (gm/100 ml.blood) Gl o b u l i n 0.12 4.443(a) 0.02 0.334(a) 0.00 -0.447 0.04 -1.098 0.00 0.053 0.00 9.584 0.06 0.254(a) 0.02 (gm/100 ml.blood) Calcium 0.07 1.177(a) 0.00 -1.430(a) 0.01 -21.083(a) 0.00 1.068 0.01 -0.368 0.00-0.776 0.00 -0.037(a) 0.00 (mg/100 ml. blood) Inorganic Phosphorus 0.21 -2.518(a) 0.02 1.147(a) 0.01 0.104 0.00 -0.343 0.00 0.519 0.00 -3.695 0.02 -0.212(a) 0.04 (mg/100 ml. blood) * R2 s i g n i f i c a n t a t P <, 0.05 on 'F' d i s t r i b u t i o n , (a) - X I O- 3 cn co 2 TABLE NO. 8 THE PROPORTION OF VARIATION (R ) FOR MODEL (5) REGRESSION COEFFICIENT ETC. CONTINUED. Blood Serum R2 Days Pregnant Days Lactat i n g Kg. of Kg. Milk Eg. Milk Kg. Lactose Age i n days Components For the Milk Produce Fat Pro t e i n Complete Model ( <-5) C o e f f i c i e n t R2 C o e f f i c i e n t R2 C o e f f i c i e n t R2 C o e f f i c i e n t R2 C o e f f i c i e n t R2 C o e f f i c i e n t R2 c o e f f i c - R2  l e n t  C h o l e s t e r o l 0.24* 0.031 0.00 -0.180 0.02 28.64 0.06 235.9 0.07* -103.5 0.00 -540.2 0.07* -0.026 0.11* (mg/100 ml. blood) B.UvN. C.29* -6.689(a) 0.01 5.830(a) 0.01 0.475 0.01 8.171 0.03 1.657 0.00 - 5.631 0.00 -1.522 0.11* (mg/100 ml.blood) (a) U r i c Acid 0.29* 0.418(a) 0.00 -0.242(a) O.OO 0.120 0.05 -0.125 0.00 0.991 0.02 - 2.691 0.08* 0.072 0.04 (mg/100 ml. blood) C r e a t i n i n e 0.14 (mg/100 ml. blood) Alk.Phosphatase 0, (milimcron/ml.blood) S.G.O.T. 0.06 (milimcron/ml.blood) (a) 0.209(a) 0.01 0.525(a) 0.05 14.62(a)0.00 -0.221 0.02 - 0.281 0.01 41.691(a)0.00 0.012 0.01 (a) .29* -0.145 0.03 0.130 0.04 -0.168 0.00 -13.63 0.00 -81.33 0.01 11.62 0.00 -10.49 0.06* (a) 0.107 0.03 -6.774(a) 0.00 3.771 0.01 27.20 0.01 -12.80 0.00 -55.04 0.00 -3.54 0.01 * R2 s i g n i f i c a n t a t P <_ 0.05 on 'F' d i s t r i b u t i o n , (a) - X10"3 Continued... cn TABLE NO. 8. THE PROPROTION OP VARIATION (R2) CONTINUED.. Blood Serum R2 Days Pregnant Days L a c t a t i n g Kg. of Kg. Milk Kg. Milk Kg. Lactose. Age i n da y s Components For the Milk Produce Fat Protein Complete • —- _ _ _ _ _ _ _ _ Model (3 ) C o e f f i c i e n t R2 C o e f f i c i e n t Rz C o e f f i c i e n t R2 C o e f f i c i e n t R2 C o e f f i c i e n t R2 C o e f f i c i e n t R2 C o e f f i c i e n t R E. Albumin 0.26* -0.132(a) 0.08* 0.022(a) 0.00 12.45(a) 0.02 -0.116 0.03 0.211 0.03 -0.311 0.04 -0.019(a) 0.10* (Part of the t o t a l peak hights) G l o b u l i n -a^ (Part of the peak height) G l o b u l i n -<i2 (Part of the peak height! G l o b u l i n - 8 (Part of the peak height) G l o b u l i n - 6 (Part of the peak height) Y-globulin (Part of the peak height) a1 0.58* -0.132(a) 0.11* -0.048(a) 0.02 -4.563(a) 0 . 0 2 - 0 . 0 7 4 0.10*-0.011 0.00 0.068 . 0.02 -0.005(a) 0.06* t o t a l - a20.32* -0.084(a) 0.09* -0.036(a) 0.02 -2.364(a) 0.01 0.020 0.01-0.019 0.00 0.034 0.01 -0.001(a) 0.00 t o t a l pBlO.16 -0.031(a) 0.01 -0.046(a) 0.05 -1.811(a) 0.01 0.017 0.01 0.110 0.01 8.911(a) 0.00 0.001(a) 0.00 t o t a l 20.24* -0.038 (a) 0.01 0.057(a) 0.04 -2.164(a) 0.01 -6.120(a)0.00-0.054 . 0.02 0.075 0.03 o.001(a) 0.20* t o t a l 0.33* -0.031(a) 0.00 0.048(a) 0.01 -1.869(a) 0.00 0.013 0.00-0.134 0.05 0.127 0.03 0.017(a) 0.27* t o t a l * The R2 s i g n i f i c a n t a t P < o.05 on 'F? d i s t r i b u t i o n (a') - SUO-"* CTl 6 6 Age o f the animals expressed as number of days from b i r t h t o day the blood sample was c o l l e c t e d was the o n l y source o f v a r i a t i o n common to a l l the p h y s i o l o g i c a l groups and consequently was i n c l u d e d as a source of v a r i a t i o n i n a l l the l i n e a r r e g r e s s i o n models. Age i n days was the o n l y source of v a r i a t i o n f i t t e d i n Model-2, f o r the young animal group w h i l e the models f o r a l l o t h e r groups i n c l u d e d a d d i t i o n a l e f f e c t s . Age i n days was found t o be an important source of the v a r i a t i o n i n most of the b l o o d serum c o n s t i t u e n t s i n the young animal group, the l a c t a t i n g non-pregnant group and the l a c t a t i n g pregnant group. The age e f f e c t was a s i g n i f i c a n t source of v a r i a t i o n f o r a l l the bl o o d serum components i n the young animal group w i t h the e x c e p t i o n o f B.U.N., albumin o b t a i n e d by e l e c t r o p h o r e s i s , g l o b u l i n 0.2 and 82- g l o b u l i n . (See t a b l e . 6 ) . The age of the animal i n the l a c t a t i n g non-pregnant group was a s i g n i f i c a n t source o f v a r i a t i o n i n bl o o d serum p r o t e i n , g l o b u l i n , i n o r g a n i c phosphorus, B.U.N., c r e a t i n i n e , g l o b u l i n -ct]_ and g l o b u l i n 8 2 * T n e l i n e a r model f i t t e d to analyse t h i s group (Model-4) accounted f o r 33% o f the v a r i a t i o n i n the blood serum p r o t e i n , w i t h age a f t e r f i t t i n g a l l o t h e r sources a c c o u n t i n g f o r the major p o r t i o n (19%). S i m i l a r r e s u l t s were found w i t h g l o b u l i n where the t o t a l l i n e a r model accounted 28% of the v a r i a t i o n and age a f t e r f i t t i n g a l l other sources accounted f o r 15%; the l i n e a r model 67 accounted for 39% of the v a r i a t i o n i n inorganic phosphorus and age a f t e r f i t t i n g a l l other sources accounted for 15% of the v a r i a t i o n . In c r e a t i n i n e the l i n e a r model accounted for 23% of the v a r i a t i o n and age a f t e r f i t t i n g a l l other sources accounted for 12% of the v a r i a t i o n . The p a r t i a l regression c o e f f i c i e n t s f o r the blood serum components, t o t a l protein (b =0.516X10 - 3), glob u l i n (b=0.482 X I O - 3 ) , c r e a t i n i n e (b=0.064 X IO - 3) and g l o b u l i n 8 2 (b=0.005 X IO" 3) on age suggested an increasing l e v e l of these blood serum components with age i n the l a c t a t i n g non-pregnant group. The l e v e l of inorganic phosphorus (b= -0.663), ch o l e s t e r o l (b= -1.851X 10"^) and g l o b u l i n aT_(b=-0.008 X 10 - 3) i n t h i s group was found to decrease with each u n i t increase i n age of t h i s group. Age s i g n i f i c a n t l y effected several blood serum components i n the l a c t a t i n g pregnant group. These included, c h o l e s t e r o l , B.U.N., al k a l i n e phosphatase, albumin separated by electrophoresis, g l o b u l i n cxi_, g l o b u l i n 8 2 a n d y - g l o b u l i n . The l i n e a r model f i t t e d to analyse t h i s group (Model-5) accounted 24% of the v a r i a t i o n i n g l o b u l i n 8 2 with age a f t e r f i t t i n g a l l other sources accounting for the major portion 20%. S i m i l a r l y the l i n e a r model accounted for 33% of v a r i a t i o n i n y-globulin and age a f t e r f i t t i n g a l l other *b = p a r t i a l regression c o e f f i c i e n t 68 sources accounted for 27%. Age a f t e r f i t t i n g a l l other sources accounted for 11% of the v a r i a t i o n i n cho l e s t e r o l , 11% of the v a r i a t i o n i n B.U.N., 6% of the va r i a t i o n i n a l k a l i n e phosphatase, 10% of the v a r i a t i o n i n albumin obtained by electrophoresis and 6% of the v a r i a t i o n i n g l o b u l i n a ^. A l l of the s i g n i f i c a n t p a r t i a l regression c o e f f i c i e n t s associated with the various blood serum components and age i n the l a c t a t i n g pregnant group were negative with the exception of glo b u l i n 82 (b=0.001 X 10~ 3) and y - g l o b u l i n (b=0.017 X 10~ 3) (see table 8). This suggested a decreasing trend of c h o l e s t e r o l , B.U.N, a l k a l i n e phosphatase, albumin obtained by e l e c t r o -phoresis and gl o b u l i n a^, and an increasing trend i n globul i n f r a c t i o n s 32 and y with the increasing age of the animal within t h i s group. Age i n days was a s i g n i f i c a n t source of the v a r i a t i o n i n albumin and calcium i n the bred h e i f e r group and i n ch o l e s t e r o l , a l k a l i n e phosphatase and S.G.O.T. i n the dry animal group. Other blood serum components were not effected by age within these two ph y s i o l o g i c a l groups. The p a r t i a l regression c o e f f i c i e n t s for albumin and calcium on age i n bred h e i f e r group were p o s i t i v e (albumin = 1.619 X -3 -3 10 and calcium = 4.255 X 10 ) in d i c a t i n g that an increase i n blood serum albumin and calcium was associated with increasing age within t h i s group. The p a r t i a l regression c o e f f i c i e n t s for c h o l e s t e r o l (b =-0.034) 69 and a l k a l i n e phosphatase (b=-0.043)on age were negative and S.G.O.T. (b=0.11) p o s i t i v e i n dry animal group suggesting decreasing l e v e l of the former two blood serum components and increasing l e v e l of the l a t t e r blood serum component with increasing age i n the group. See Table 6. Days pregnant was a po t e n t i a l source of v a r i a t i o n i n the various blood serum components with three ph y s i o l o g i c a l groups; the bred h e i f e r group, the l a c t a t i n g pregnant group and the dry animal group. See Models 3, 5 and 6 respe c t i v e l y . Days pregnant a f t e r f i t t i n g a l l other sources accounted for 11% of v a r i a t i o n i n blood serum albumin i n the bred h e i f e r group, and 37% of v a r i a t i o n i n cho l e s t e r o l , 43% of v a r i a t i o n i n B.U.N., and 19% of v a r i a t i o n i n a l k a l i n e phosphatase i n the dry animal group. The p a r t i a l regression c o e f f i c i e n t for albumin (b=1.785 -3 X 10 ) on days pregnant i n the bred h e i f e r group suggested a decreasing l e v e l of albumin with advancing days of pregnancy. In the dry animals, ch o l e s t e r o l (b=-0.294) l e v e l s decreased but the B.U.N, l e v e l (b=0.070) increased with advancing pregnancy. See table 6. Days pregnantwasa s i g n i f i c a n t a f f e c t i n albumin separated by electrophoresis, g l o b u l i n c t ^ , g l o b u l i n - a 2 within the l a c t a t i n g pregnant group. Days pregnant a f t e r f i t t i n g a l l other sources accounted for 8% of v a r i a t i o n i n albumin separated by electrophoresis, 11% of v a r i a t i o n i n 70 glo b u l i n - a-^  and 9% of v a r i a t i o n i n gl o b u l i n -a. ^  i n t h i s group. The p a r t i a l regression c o e f f i c i e n t s within t h i s group suggested that the albumin obtained by e l e c t r o -_ 3 phoresis (b=0.312 X 10 ) increased and glo b u l i n a-^  (b=-0.132 X 10~ 3) and gl o b u l i n a 2 (b=-0.084 X 10~ 3) decreased with increasing days of pregnancy. See table 8. Days l a c t a t i n g as a source of v a r i a t i o n was f i t t e d using the Models - 4 and 5 for the l a c t a t i n g non-pregnant and the l a c t a t i n g pregnant groups r e s p e c t i v e l y . Days l a c t a t i n g a f t e r f i t t i n g a l l other sources accounted for 17% of the v a r i a t i o n i n c h o l e s t e r o l , 6% of the va r i a t i o n i n B.U.N., 8% of the v a r i a t i o n i n gl o b u l i n and 11% of the v a r i a t i o n i n g l o b u l i n 82' i n the l a c t a t i n g non-pregnant group. The p a r t i a l regression c o e f f i c i e n t s for these blood serum components regressed on days l a c t a t i n g were ch o l e s t e r o l (b=0.372), B.U.N. (b_0.016), glo b u l i n a ± (b=0.053 X IO - 3) , g l o b u l i n 8-, (b=0.101 X IO - 3) a l l i n d i c a t i n g an increasing l e v e l of these blood serum components with increasing days i n l a c t a t i o n i n the l a c t a t i n g non-pregnant group. None of the blood serum components were found to be effected s i g n i f i c a n t l y by the days l a c t a t i n g i n the l a c t a t i n g pregnant group i n the present study. See tables 7 and 8. 71 The Kg. of m i l k , Kg. of m i l k f a t , Kg. o f  m i l k p r o t e i n and Kg. l a c t o s e produced a t the morning m i l k i n g p r i o r to sampling were c o n s i d e r e d to be p o s s i b l e sources of v a r i a t i o n s , a f f e c t i n g the l e v e l of v a r i o u s b l o o d c o n s t i t u e n t s i n the m i l k i n g a n i m a l s . These sources of v a r i a t i o n s were f i t t e d i n a d d i t i o n to o t h e r e f f e c t s f o r the l a c t a t i n g non-pregnant and l a c t a t i n g pregant group as e x p l a i n e d i n Model - 4 and Model - 5. The l e v e l o f m i l k p r o t e i n produced a t the p re-sampling m i l k i n g had a s i g n i f i c a n t e f f e c t on c h o l e s t e r o l and g l o b u l i n B_, i n the l a c t a t i n g pregnant group and accounted f o r 10% of the v a r i a t i o n i n b l o o d serum c h o l e s t e r o l and 8% of the v a r i a t i o n i n g l o b u l i n a f t e r o t h e r p r o d u c t i o n t r a i t s , days pregnant and age were f i t t e d . The l e v e l of p r o d u c t i o n of the pre-sampling m i l k i n g had the f o l l o w i n g e f f e c t s on the b l o o d serum components i n the l a c t a t i n g non-pregnant group. Kg. m i l k f a t was a s i g n i f i c a n t e f f e c t and accounted f o r 7% o f the v a r i a t i o n i n c h o l e s t e r o l and 10% o f the v a r i a t i o n i n g l o b u l i n c t ^ . The l e v e l of l a c t o s e p r o d u c t i o n was a s i g n i f i c a n t source o f v a r i a t i o n and accounted f o r 7% o f the v a r i a t i o n i n c h o l e s t e r o l and 8% o f v a r i a t i o n i n u r i c a c i d i n the l a c t a t i n g pregant group. 72 The p a r t i a l regression c o e f f i c i e n t s for the cholest e r o l and g l o b u l i n on Kg. milk protein, (b = -325 and b = -0.048, respectively) suggested a decrease i n the l e v e l of blood serum ch o l e s t e r o l and g l o b u l i n 82 with the increasing production of milk protein i n the l a c t a t i n g pregnant group. The p a r t i a l regression c o e f f i c i e n t s for the c h o l e s t e r o l (b = 235.9) and g l o b u l i n (b = 0.074) on Kg. milk f a t suggested an increasing l e v e l of these blood serum components with increasing f a t i n milk i n the l a c t a t i n g pregnant group. Whereas cho l e s t e r o l (b = -540.2) and u r i c acid (b = -2.691) on Kg. lactose i n the l a c t a t i n g pregnant group indicates a decreasing l e v e l of blood serum cho l e s t e r o l and u r i c a c i d with increasing l e v e l of lactose i n milk. In general the amount of milk f a t , milk protein and lactose i n milk are p o s i t i v e l y c o r r e l a t e d . Under the present study i t was d i f f i c u l t to explain the d i r e c t i o n of the l e v e l of cholest e r o l with milk protein and lactose which was the same, but opposite with the milk f a t . See table 8. The present study indicates that the e f f e c t s of the production t r a i t s as measured by Kg. of milk, Kg. of milk protein and Kg. of lactose produced p r i o r to sampling influenced only the blood serum c h o l e s t e r o l , u r i c acid, g l o b u l i n a 1 and g l o b u l i n 8 0 (see tables 7 and 8). 73 B: The Product Moment Cor r e l a t i o n Within The Phy s i o l o g i c a l  Groups: The product moment c o r r e l a t i o n between the sources f i t t e d , between the sources and blood serum components and between the blood components within each p h y s i o l o g i c a l group were compiled to indicate the degree to which these variables vary together. (1) Young female animal group;-Age i n days was the only source of v a r i a t i o n measured within t h i s group as mentioned e a r l i e r and i t was s i g n i f i c a n t l y c orrelated with most of the blood serum components, evaluated. Protein, albumin, g l o b u l i n and glo b u l i n f r a c t i o n s c t ^ , ar^ 8^ and B 2 were p o s i t i v e l y correlated with age. The protein metabolic end products B.U.N, u r i c acid and creatinine were negatively correlated with age, which may be due to the e f f e c t of ac t i v e growth i n these young females. The rate of protein biosynthesis i n the body of the young animals i s expected to be more than the rate of protein catabolism and these c o r r e l a t i o n indicate that as age, within the group increased"the protein metabolic end products decreased. Dimopoullus (1963) while discussing plasma proteins; suggested that plasma or serum protein concentration represents the balance between the process of biosynthesis and catabolism. 74 The n e g a t i v e c o r r e l a t i o n o f y - g l o b u l i n (-0.31) w i t h age w i t h i n t h i s group may be due to the h i g h e r d e c r e a s i n g r a t e o f maternal Y- g l o b u l i n r e c e i v e d w i t h c o l o s t r u m and m i l k than t h a t of the r a t e o f b i o s y n t h e s i s of Y- g l o b u l i n i n these animals from the r e t i c u l o endothe-l i a l system. Cal c i u m was p o s i t i v e l y c o r r e l a t e d w i t h age-(0.51) and n e g a t i v e l y c o r r e l a t e d w i t h i n o r g a n i c phosphorus (-0.59). T h i s was expected due to the demand of c a l c i u m f o r bone f o r m a t i o n i n these growing animals and i t s p h y s i o -l o g i c a l r e l a t i o n with i n o r g a n i c phosphorus. A l k a l i n e phosphatase was n e g a t i v e l y c o r r e l a t e d w i t h age. A l c r o f t e t a l . , (1941) a l s o r e p o r t e d t h a t i n c a t t l e serum a l k a l i n e phosphatase a c t i v i t y p r o g r e s s i v e l y decreased w i t h advancing age. See t a b l e 9. (2) Bred h e i f e r group:-Days pregnant and age w i t h i n t h i s group was p o s i t i v e l y c o r r e l a t e d (0.87), which was expected s i n c e age i s used as the c r i t e r i o n f o r the i n i t i a t i o n o f b r e e d i n g . Age was n e g a t i v e l y c o r r e l a t e d with the a l k a l i n e phosphatase i n the bred h e i f e r group (-0.57) which agrees w i t h A l c r o f t e t a l . , (1941) as a l r e a d y mentioned under the TABLE NO .9 PRODUCT MOMENT CORRELATION BETWEEN ALL CONTINUOUS VARIABLES MEASURED IN GROUP I (YOUNG FEMALE ANIMALS) BASED ON 51 PAIRED OBSERVATIONSi Age 2 (1) 0.55* Total Protein i •>: 3 0,58* 0.63* Album (3) 4 0.37* 0.91* 0.24 S 0.51* 0.57* 0.59* 6 -0.59* -0.39* -0.41* 7 0.60* 0.52* 0.57* 8 -0.15 -0.32* -0.11 9 -0.49* -0.20 -0.34* 10 - -0.31* 0.11 0.04 11 -0.32* -0.19 -0.15 12 0.68* 0.48* 0.46* 13 ' -0.22 -0.35* -0.12 14 0.36* 0.07 0.21 15 0,19 0.09 0.22 16 0.63* 0.40* 0.35* 17 0.03 0.53* 0.06 18 -0.31* o.ii- -0.25 Globulin (4) 0.39* Calcium Inorganic -0.26 -0.66*Phosphorus . 0.34* 0.64* -06.6S* C h °l e s t e r a l -0.35* -0.39* 0.57* -0.51* h'V. £ ' -0.07 -0.28* 0.53* -0.45* 0.18 Uri<j9Aci<J 0.11 0.18 -0.23 0.08 -0.39* 0.14 C r e atini'>e u <" Alkaline -0.16 -0.08 0.07 -0.11 -0.10 0.00 0.23 Phosphatase 0.34* 0.52* -0.53*- 0.63° -0.27* -0.45* -0.11 -o!lO 8 , <( _ _ )T' -0.37* -0.10 0.26 -0.23 0.38* 0.08 -0.14 0.03 -0.25 Albumin -0.02 0.21 -0.41* 0.53* -0.38* -0.29 * 0.08 0.07 0.41 * -oill * G 1 < J ? ^ i n ° 1 1 ' Globulin a, -0.01 -0.05 -0.11 0.15 -0.16 -0.12 -0.08 0.02 0.18 -0.73 * 0.52 * (15) 2 Globulin Bl 0.31* 0.30* -0.37* 0.48* -0.19 -0.27 * -0.05 0.00 0.48 * -0.50 * 0.49 * 0.33! (16) Globulin 0 0.63* 0.10 -0.06 -0.02 -0.21 0.12 0.15 -0.14 0.03 -0.64* -0.14 0.26 0.16 (17) 2 1 'Globulin y 0.27* -0.18 0.11 -0.31* -0.17 0.23 0.21 -0.06 -0.21 -0.62* -0.14 0.27 * -0.19 0.69 * ( 1 8 ) E. • Cosfficients _>0.27 are significantly different from '0' at P <_ 0.05 on *r' table'. E. - separated by electrophoresis 76 young animal groups. But under the present study the days pregnant was also negatively correlated (-0.61) with the alkaline phosphatase which does not agree with Alcroft's report of increase of alkaline phosphatase level during pregnancy. See table 10. Total protein, albumin, and globulin were positively correlated to calcium and cholesterol and negatively correlated to inorganic phosphorus in the bred heifer group. Calcium and inorganic phosphorus was negatively correlated (-0.54) which was expected due to the physiological relation between these elements in catt l e . The non-diffusible part of calcium remains bound to protein in blood serum, therefore, both calcium and protein 10.58) was negatively correlated with inorganic phosphorus (-0.54 and -0.37 respectively). This negative trend of relationship of calcium and inorganic phosphorus was also found with cholesterol and B.U.N, in the present study within the bred heifer group. Calcium was positively correlated with cholesterol (0.49) and negatively correlated with B.U.N. (-0.52), whereas inorganic phosphorus was negatively correlated with cholesterol (-0.48) and positively correlated with B.U.N. TABLE KO. 10 PRODUCT HOME!IT CORRELATION BETWEN ALL CONTINUOUS VARIABLES MEASURED IN GROUP IKBRED HEIFERS) BASED ON 39 PAIRED OBSERVATIONS. 1 Pregnant A?e In Days Protein Albumin Globulin Calcium Inorganic Phosphorus Cholesterol B.U.N. Uric Acid Creatinine Alkaline .Phosphatase S.G.O.T. E. Albunin Globulin oi 1 2 3 4 5 6 7 8 9 10 i l 12 13 14 15 0.87* -0.11 0.04 0.07 0.26 0.4C* -0.13 -0.C5 0.95* 0.16 0.14 0.29 0.58* 0.71* 0.40* -0.06 -0.1S -0.37* -0.45* -0.25 -0.S4* 0.01 0.16 0.48* 0.56* 0.33* 0.49* -0.48* 0.11 0.10 -0.32* -0.21 -0.28 -0.52* 0.61* -0.37* -0.02 • -CIS -0.23 -0.12 -0.21 -0.21 0.03 -0.12 0.06 0. 20* 0.23 -0.06 0.21 -0.14 0.19 -0.21 -0.11 -0.09 0.11 -0.61* -0.57* 0.12 0.C8 0.11 0.02 -0.00 0.25 -0.18 -0.31* -0.34* -0.C2 o.c; 0.11 0.09 0.08 -0.08 0.07 0.16 0.29 0.02 -0.15 0.20 0.05 0.C5 -0.21 0.11 -0.26 -0.20 0.08 -o:o3 0.34* -0.05 0.05 0.06 0.10 -C.ll -0.13 -0.35* -0.18 -0.33* -0.15 0.21 -0.21 -0.03 0.17 -0.03 0.03 -0.09 -0.64* -0.C3 • -0.07 -0.35* . -0.12 -0.35* -0.02 0.08 -0.14 -0.12 0.25 0.09 -0.08 -0.01 -0.6S* 0.76* -0.29 -0.25 -0.12 0.07 -0.15 0.11 -0.12 0.07 -0.32* 0.18 -0.09 0.19 0.08 -0.60* 0.56-0.06 0.10 0.50* -0.04 0.56* 0.33* -0.21 0.25 -0.30* -0.11 -0.11 -0.13 -0.11 -0.73* 0.08 0.13 0.12 0.63* -0.07 0.72* 0.29 -0.14 0.19 -0.26 -0.21 -0.02 -0.16 -0.14 -0.54* -0.11 _22_ Globulir. Cict'J h i. 17 18 0.72* 0.14 -0.11 0.21 -0.21 *Coef Jicients > 0.30 aro aignificantly different from *0* at P < 0.05 on 'r' tablo. 2. . Obtained by electrophoresis 78 (0.61). The o t h e r c o r r e l a t i o n s between the b l o o d serum components f o r the bred h e i f e r group are g i v e n i n t a b l e 10. (3) L a c t a t i n g non-pregnant group and (4) L a c t a t i n g pregnant group: In the l a c t a t i n g non-pregnant group, days l a c t a t i n g was observed to be n e g a t i v e l y c o r r e l a t e d w i t h the amount of mi l k (-0.39), p r o t e i n (-0.44) and l a c t o s e (-0.36) produced a t the m i l k i n g j u s t p r i o r to bl o o d s a m p l i n g . These p r o d u c t i o n t r a i t s were p o s i t i v e l y c o r r e l a t e d w i t h b l o o d serum c h o l e s t e r o l (0.52) and g l o b u l i n (0.44). Kg. f a t i n m i l k was p o s i t i v e l y c o r r e l a t e d w i t h age (0.36), and n e g a t i v e l y c o r r e l a t e d w i t h i n o r g a n i c phosphorus (-0.32). M i l k p r o t e i n y i e l d was p o s i t i v e l y c o r r e l a t e d w i t h l a c t o s e y i e l d (0.91) i n the same m i l k , but n e g a t i v e l y c o r r e l a t e d w i t h the i n o r g a n i c phosphorus (-0.33), B.U.N. (-0.30), g l o b u l i n 8T_ (-0.29) and g l o b u l i n £2 (-0.26) i n bl o o d serum. L a c t o s e , on the o t h e r hand, showed n e g a t i v e c o r r e l a t i o n o n l y w i t h i n o r g a n i c phosphorus (-0.33) and g l o b u l i n 3]_ (-0.39) i n the b l o o d serum. Age o f the animals w i t h i n the l a c t a t i n g non pregnant group was p o s i t i v e l y c o r r e l a t e d w i t h serum p r o t e i n (0.46) g l o b u l i n (0.38), c r e a t i n i n e (0.35), g l o b u l i n f r a c t i o n 62 (0.29) and Y(0.31), and n e g a t i v e l y c o r r e l a t e d w i t h c a l c i u m , i n o r g a n i c phosphorus, B.U.N, and g l o b u l i n aj_# i n the blood serum. See t a b l e 11. TABLE NO. 11. ^ ^ ^ ^ " ^ ^ ^ ^ T I O ^ B E r W E N ALL CONTINUOUS VARIABLES MEASURED IB GROUP III (LACTATING AND OPEN COWS) Days :ctating Kg of Kg. Hi lk Fat *9. Milk Protein Kq Lactose Age in Days Protein Albumin Globulin Calcium Inorganic Phosphorus Choles-terol 1 2 3 4 5 6 7 8 9 10 11 12 -0.39* -0.21 0. 13 -C.44* 0. 90* 0.12 -0.35* 0. 99* 0.03 0.91* -0.C3 0. 27* 0.36* 0.22 0.21 O.C? c. C3 0.C4 -0.10 -0.01 0.46* o!:i 0. 20 0.11. 0.16 0.20 0.18 0.05 c a -0. C3 0.00 -0.14 -0.07 0.38* 0.95* -0.27* O.C 5 -0. 17 -0.12 -0.21 -0.14 -0.30* 0.12 0.40* -0.01 0.22 -0. 39* -0.32* -0.33* -0.33* -0.55* -0.16 -0.20 -0.09 0.25 0.52* -0. 05 -0.13 -0.22 -0.01 0.12 0.21 0.37* 0.09 0.26*. 0.05 0.43* 0.35- -0. 24 -0.12 -0.30* -0.21 -0.28* -0.21 0.23 -0.26* 0.25 0.25 -0.02 0. 14 -0.25 0.04 0.17 -0.09 0.08 -0.03 0.09 0-.S2 0.34* 0.17 0.52 -0. 11 0.13 0.01 -0.12 0.35* 0.02 0.02 0.01 -0.14 -0.07 -0.20 0.12 -0. 23 -0.03 -0.21 -0.23 -0.23 -0.03 -0.07 -0.01 0.33* 0.18 0.15 -C.I4 0. 10 -0.10 0.02 0.10 -0.17 0.02 -0.1,2 0.05 0.38* 0.30* 0.10 -e.25* c. 13 -0.C2 0.16 0.17 -0.03 -0.09 0.15 -0.13 0.29* -0.04 -0.03 C.33« -c. 19 -C.19 -C.13 -C.14 -0.41* -0.29* 0.10 -0.31* 0.08 0.29* 0.33* >-0.C5 -0. 25 -C.07 -0.10 -0.24 -0.19 -0.22 -0.10 -0.19 -0.11 0.22 -0.22 0.24 -0. 41 -0.07 -0.29* -0.39* -0.09 -0.30* 0.07 -0.31* -0.09 0.14 0.04 0.44* -0. 12 0.10 -0.26* -0.12 0.29* 0.26* -0.08 0.27* ' -0.14 -0.15-. 0.26* -0.39 0. 21 0.16 0.1S 0.21 0.31* 0.49* -0.26* 0.55* -0.36* -0.24 -0.19 Alkaline 0, a, s, B.U.N. Uric Creat- Phospha- S.G.O.T. E. ' 1 Acid inine tase Alburr.in Globulin Globulin Globulin 16 0.08 0.06 -0.06 -0.02 0.03 -0.21 0.11 0.40* -0.14 0.08 0.31* -0.13 0.22 -0.11 -0.04 -0.13 -0.21 0.07 0.11 -0.11 0.05 0.11 -0.12 0.22 -0.31*-9.24 0.11 0.27* 0.04 0.29* 0.08 -0.11 -0.64* 0.05 -0.10 -0.58* 0.51* 0.03 -0.31* -0.37* 0.36* 0.04 -0.11 -0.53* 0.13 -0.19 -0.15 -0.53* -0.05 0.52* -0.17 0.07 -0.04 -0.44" 0.36* * Coefficients > 0.26 are significantly different from '0' at P < 0.05! on 'r' table. E» Albumin obtained of electrophoresis. vo 80 The number of days l a c t a t i n g w i t h the l a c t a t i n g non-pregnant group was n e g a t i v e l y c o r r e l a t e d w i t h m i l k p r o t e i n and l a c t o s e produced on the day of b l o o d sampling. The s i g n i f i c a n t c o r r e l a t i o n c o e f f i c i e n t s between the b l o o d serum components and the l a c t a t i n g t r a i t s were a l l n e g a t i v e . The blood serum components r e l a t e d to l a c t a t i n g t r a i t s were i n o r g a n i c phosphorus, B.U.N., and g l o b u l i n $1 and 32 f r a c t i o n s . The n e g a t i v e r e l a t i o n s h i p between the i n o r g a n i c phosphorus and the v a r i o u s p r o d u c t i o n t r a i t s may be due to the i n c r e a s e d carbohydrate metabolism d u r i n g l a c t a t i o n , as suggested by Simesen (1963) who i n d i c a t e d t h a t i n a n i m a l s , h i g h e r carbohydrate metabolism was a s s o c i a t e d with.lower l e v e l of i n o r g a n i c phosphorus i n the b l o o d . In a d d i t i o n , i n o r g a n i c phosphorus i s a l s o b eing s e c r e t e d w i t h the m i l k . The c o r r e l a t i o n s between the b l o o d serum components w i t h i n the l a c t a t i n g non pregnant group were as f o l l o w s : t o t a l p r o t e i n was p o s i t i v e l y c o r r e l a t e d w i t h t o t a l g l o b u l i n (0.95) and n e g a t i v e l y c o r r e l a t e d w i t h the albumin (-0.27). The p r i n c i p l e g l o b u l i n f r a c t i o n s c o n t r i b u t i n g t o t h i s p o s i t i v e c o r r e l a t i o n seemed to be $2 and Y"*§l°bulins, s i n c e , o n l y the 32 and y f r a c t i o n s of g l o b u l i n were found to be p o s i t i v e l y c o r r e l a t e d w i t h the t o t a l p r o t e i n and a^, c ^ , 8]_ were n e g a t i v e l y c o r r e l a t e d w i t h the t o t a l p r o t e i n . Calcium was p o s i t i v e l y c o r r e l a t e d w i t h albumin (0.40), c h o l e s t e r o l (0.26), 81 alkaline phosphatase (0.33), S.G.O.T. (0.38),albumin separated by electrophoresis (0.29) and negatively correlated with y-globulin (-0.36). Other important blood serum components showing significant correlations were cholesterol, positively correlated with B.U.N. (0.43), globulin (0.33) and &^  (0.26); B.U.N, with y-globulin (-0.31); uric acid with S.G.O.T. (0.40); alkaline phosphatase with S.G.O.T. (0.27); and albumin separated by electrophoresis with S.G.O.T. (0.29). As expected, the albumin obtained by electrophoresis was negatively correlated with a l l the fractions of globulin. See table 11. In the lactating and pregnant group the days pregnant was positively correlated with days lactating (0.66), creatinine (0.26), and albumin separated by electro-phoresis (0.33), and negatively correlated with globulin (-0.58), a2 (-0.49), &1 (-0.31). The number of days lactating was negatively correlated with kg. of milk and lactose produced (-0.36) and (-0.35) respectively similar to the relationship found in the lactating non-pregnant group. The kg. of protein in milk was not significantly correlated to the number of days lactating unlike the lactating non-pregnant group. The correlation of days lactating with the blood serum components was observed to be exactly the same as that for days pregnant, i.e., positively correlated with 82 c r e a t i n i n e (0.29) and albumin o b t a i n e d by e l e c t r o p h o r e s i s (0.26), and n e g a t i v e l y c o r r e l a t e d w i t h g l o b u l i n f r a c t i o n s a 1 (-0.44), a 2 (-0.39), and 6-^  (-0.33). The days l a c t a t i n g f o r the l a c t a t i n g pregnant group and f o r the l a c t a t i n g non pregnant group, i n r e s p e c t to t h e i r c o r r e l a t i o n w i t h the b l o o d serum components, was found t o behave d i f f e r e n t l y under the p r e s e n t s t u d y . The days l a c t a t i n g i n the l a c t a t i n g pregnant group was confounded w i t h the days pregnant, which may e x p l a i n t h i s d i f f e r e n c e . Blood serum, i n o r g a n i c phosphorus, was n e g a t i v e l y c o r r e l a t e d w i t h a l l the l a c t a t i n g t r a i t s i n both l a c t a t i n g groups. A l k a l i n e phosphatase showed a n e g a t i v e c o r r e l a t i o n w i t h a l l the l a c t a t i n g t r a i t s i n the l a c t a t i n g pregnant group. But a l k a l i n e phosphatase f a i l e d to show any c o r r e l a t i o n w i t h the l a c t a t i n g t r a i t s i n the l a c t a t i n g non pregnant a n i m a l s , which suggested t h a t the n e g a t i v e c o r r e l a t i o n o f a l k a l i n e phosphatase w i t h the l a c t a t i n g t r a i t s found i n l a c t a t i n g pregnant cows may be due to the e f f e c t o f pregnancy, or the combined e f f e c t s o f pregnancy and l a c t a t i o n . A l k a l i n e phosphatase was a l s o found t o be n e g a t i v e l y c o r r e l a t e d w i t h age i n days i n the l a c t a t i n g pregnant group. B.U.N, was p o s i t i v e l y c o r r e l a t e d with a l l the l a c t a t i n g t r a i t s i n the l a c t a t i n g pregnant group but n e g a t i v e l y c o r r e l a t e d w i t h the l a c t a t i n g t r a i t s i n the .22 m m mm 000001300 lit I oooooooooo OOOOOOOOOOOOOOO „„ooooooooo?oooo= .OOOOOOOOOOOOOOOOO 83 84 l a c t a t i n g non pregnant a n i m a l s . The days l a c t a t i n g on the ot h e r hand was p o s i t i v e l y c o r r e l a t e d w i t h B.U.N, i n the l a c t a t i n g non pregnant animals but a n e g a t i v e tendency i n the case o f the l a c t a t i n g pregnant animals (although non-s i g n i f i c a n t i n the l a t t e r c a s e ) . T h i s c o n t r a d i c t o r y t r e n d of r e l a t i o n s h i p between these two p r o d u c t i o n groups, w i t h r e g a r d to the days l a c t a t i n g and l a c t a t i n g t r a i t s w i t h B.U.N, was d i f f i c u l t to e x p l a i n . P r e w i t t e t a l . , (1971)in an experiment w i t h t h r e e groups o f l a c t a t i n g Guernsey cows r e p o r t e d an i n c r e a s i n g l e v e l o f mean B.U.N, f o r e i g h t weeks and a d e c l i n e i n mean weekly y i e l d o f m i l k and m i l k p r o t e i n w i t h the animals who were s t a r t e d on feed w i t h low p r o t e i n l e v e l s . The B.U.N, i n the l a c t a t i n g non pregnant animals showed a s i m i l a r t r e n d i n the p r e s e n t s t u d y . T h i s may i n d i c a t e l a c t a t i n g s t r e s s a s s o c i a t e d w i t h f i r s t stage o f l a c t a t i o n when i n c r e a s e d amount o f p r o t e i n i s b e i n g m e t a b o l i s e d f o r energy. The B.U.N, i n l a c t a t i n g pregnant a n i m a l s , was n e g a t i v e l y c o r r e l a t e d w i t h the days pregnant, which may i n d i c a t e d e c r e a s i n g e f f e c t o f l a c t a t i o n s t r e s s w i t h the i n c r e a s i n g days o f pregnancy when l e s s and l e s s p r o t e i n i s being m e t a b o l i s e d . Lane e t a l . , (1966) a l s o r e p o r t e d a s i m i l a r d e c l i n e o f the mean B.U.N, l e v e l i n Guernsey c a t t l e w i t h i n c r e a s i n g p e r i o d s o f pregnancy. 85 (5) Dry animal group:-In the dry animal group, i n o r g a n i c phosphorus and B.U.N, were observed t o be p o s i t i v e l y c o r r e l a t e d w i t h days pregnant (0.51 and 0.68 r e s p e c t i v e l y ) . Age i n days was n e g a t i v e l y c o r r e l a t e d w i t h c h o l e s t e r o l (-0.51), a l k a l i n e phosphatase (-0.69), and S.G.O.T. (-0.76).- The c o r r e l a t i o n between the b l o o d components i n the dry group were i n g e n e r a l agreement wi t h the bred h e i f e r group. I n o r g a n i c phosphorus, which was observed t o be p o s i t i v e l y c o r r e l a t e d w i t h c a l c i u m i n both l a c t a t i n g groups was n e g a t i v e l y c o r r e l a t e d w i t h c a l c i u m (-0.66), l i k e t h a t o f bred h e i f e r groups. See t a b l e 13.' I l l ADJUSTMENT OF DATA To e v a l u a t e sources o f v a r i a t i o n s common to a l l the groups i t was necessary to a d j u s t the raw o b s e r v a t i o n of each b l o o d serum c o n s t i t u e n t f o r the s i g n i f i c a n t sources o f v a r i a t i o n s unique t o each group. These sources o f v a r i a t i o n s were d i s c u s s e d p r e v i o u s l y and t a b u l a t e d i n t a b l e 5. The data was not a d j u s t e d f o r age as t h i s source o f v a r i a t i o n was common to a l l groups and was i n c l u d e d i n the combined group a n a l y s i s as a c o v a r i a b l e . The e f f e c t o f age w i t h i n each group on each b l o o d serum component was a l s o e v a l u a t e d u s i n g simple r e g r e s s i o n technique and. these r e s u l t s a re g i v e n i n t a b l e 14. TA8L3 X0.13 PRODUCT XOHZNT CORRELATION BETWEEN ALL CONTINUOUS VARIABLES MEASURED IN OROCP V (DRY. ANIMALS) BASED ON 14.PAIRED OBSERVATIONS. Days Pregnant Age In Days Protein Albumin Globulin Calcium Inorganic, phosphorus Cholesterol B.U.N. Uric Acid .Creatinine Alkaline Ph03phatace S.G.O.T. E. Albumin Globulin "1 Globulin "2 2 3 4 5 « ' , 9 9 10 11 12 13 14 15 16 J -0.32 3 •0.4« 0.44 4 0.11 0.31 0.19 5 -3.4 9 0.39 0.98* -0.01 { -0.19 -0.10 0.43 0.01 0.43 7 0.51* -0.13 -0.57" 0.02 -0.59* -0.66* -0.41 -0.51' 0.1S -0.23 0.21 0.43 -0.57 9 0.68* -0.21 -0.52* 0.04 -0.54° -0.54* 0.62* -0.25 10 0.40 0.18 0.01 0.31 -0.05 • -0.09 0.41 -0.31 0.31 11 0.12 0.12 -0.39 -0.27 -0.34 -0.03 0.34 , -0.31 0.08 0.00 12 - o . : o -0.69* -C.13 -C.52* -0.03 0.14 -0.33 0.64* -0.23 -0.49 -0.24 13 0.23 -0.76* -0.34 -0.15 -0.32 0.00 0.07 0.50 0.47 -0.20 -0.22 0.44 14 0.26 -0.13 -0.43 -0.10 -0.4 2 -0.55* 0.50 -0.07 . 0.59* o.2e 0.17 -0.15 0.25 13 0.07 -0.4 8 -0.26 0.11 -0.29 0.24 -0.34 0.35 -0.13 -0.47' -0.21 0.54* 0.39 -0.54* 16 -0.15 0.14 -0.14 0.33 -0.21 0.10 -0.13 -0.03 -0.19 -0.11 -0.16 -0.11 -0.23 -0.58* 0.53* 17 -0.11 -0.10 0.19 0.18 0.16 0.52* -0.39 0.11 -0.36 -0.33 -0.24 0.28 0.01 -0.89* 0.71* 0.60* 19 -0.37 0.28 0.65* -0.07 0.67* 0.59* -0.51* 0.06 -0.64* -0.21 ' -0.06 0.12 -0.3« -0.90 0.22 0.25 19 -0.27 0.54* 0,76* -0.06 0.79* 0.37' ' -0.29 -0.22 -0.57* 0.11 0.04 -0.31 -0.59* -0.54*' -0.33 -0.04 0.71* 0.18 •Coefficient > 0.51 . are significantly different from ' 0 ' at » < 0.05 <"> 'r' table E. - Obtained by electrophoresis OO TABLE NO. 14 SIMPLE REGRESSION COEFFICIENTS AND THE PORTION OF VARIATION (R2)ACCOUNTED FOR IN THE BLOOD w m ! M > n MD™ ™ , e ON AGE (IN DAYS) WITHIN EACH PHYSIOLOGICAL GROUPS: T K E B L O 0 D S E R U M COMPONENTS REGRESSED Blood Serum Young Female Animals Bred Heif e r s Lactating non-Pregnant, Lactating Pregnant . Dry Animals Components C o e f f i c i e n t s R2 C o e f f i c i e n t s 11- C o e f f i c i e n t R-i C o e f f i c i e n t R<! C o e f f i c i e n t R-< T o t a l * P r o t e i n 1.9400(a) 0.31 0.2169(a) 0.00 0.4795(a) 0.21 -0.0281(a) 0.00 0.4229 (a) 0.19 Albumin 0.8993(a) 0.34 0.4871(a) 0.07 0.0624 (a) 0.03 -0.0264(a) 0.00 0.0569(a) 0.09 G l o b u l i n 1.0400 (a) 0.14 -0.2701(a) * 0.00 0.4171(a) 0.15 0.1499 (a) 0.01 0.3661 (a) 0.15 Calcium 2.8220(a) 0.26 1.782(a) 0.08 -0.3615(a) 0.09 -0.0295(a) 0.00 -0.0404(a) 0.01 Inorganic * Phosphorus -5..8180(a) 0.35 -1.342(a) 0.03 -0.8334(a) 0.30 -0.2118(a) 0.05 -0.1989 (a) 0.02 C h o l e s t e r o l 0, .2825 0.37 0.0506 ' 0.02 0.0121 0.01 -0.0151 0.05 * -0. 0241 0 .26 B.U.N. -3. • 4930 (a) 0.02 3.462(a) 0.00 * -1.980 (a) 0.08 * -1.227 (a) 0.10 -1. 650(a) 0 .04 U.Acid -0, • 8720(a) 0.24 -0.2938(a) 0.02 -0.0357(a) 0.00 * 0.1343 (a) 0.17 0. 0277(a) 0 .03 Cr e a t i n i n e -0, .2818(a) 0.11 0.2934 (a) 0.06 * 0.0571(a) 0.12 0.0124(a) . 0.01 0. 0323 (a) 0 .13 A l k . * * . * Phosphatase-0. .0935 0.10 -0.1S87 0.33 -8.874(a) 0.05 -0.0141 0.15 -0. 0355 0 .47 * The c o e f f i c i e n t accounted f o r the s i g n i f i c a n t amount of v a r i a t i o n at p _ 0.05 on 'F' d i s t r i b u t i o n (a) - X10"3 Continued... TABLE NO . 14 SIMPLE REGRESSION COEFFICIENTS CONTINUED Blood Serum Young Female Animals Bred Heif e r s Lactating non-pregnant Lactating Pregnant Dry Animals Components C o e f f i c i e n t s R^  C o e f f i c i e n t s C o e f f i c i e n t R- C o e f f i c i e n t R* C o e f f i c i e n t R~ S.G.O.T. * 0.1076 0.45 3.408(a) 0.00 -6.434(a) 0.03 -0.6998(a) 0.00 * -0.0107 0.57 E l . Albumin •0.0856 (a) 0.05 0.0223(a) 0.00 -0.0019(a) 0.00 -0.0088(a) 0.03 -0.0103(a) 0.02 G l o b u l i n °1-* 0.0582(a) 0.13 -0.0192(a) 0.02 * -0.0083(a) 0.16 * -0.0075(a) 0.16 -0.0108(a) 0.23 Gl o b u l i n -2. 0.0158 (a) 0.03 -0.0078(a) ' 0.00 -0.0034 0.04 -0.0028(a) 0.04 0.0021(a) 0.02 Glo b u l i n 61-* 0.0666 (a) 0.39 -0.0333(a) 0.06 -0.0016(a) 0.01 -0.0003 0.00 -0.0019(a) 0.01 G l o b u l i n 62. 0.0031(a) 0.00 0.0126 Ja) 0.01 * 0.0049(a) 0.08 * 0.0061 0.15 0.0075 0.08 * * * " * * G l o b u l i n y.-0.0574(a) 0.10 0.0254(a) 0.01 0.0103(a) 0.09 0.0135(a) 0.24 0.0139(a) 0.29 Mean ± S.D. of Ag w i t h i Group °Lw?e _ 348.53 ± 110 705.85 + 110.13 1649.65 ± 627.58 1725.17 ± 765.61 2079.86 + 707.02 n each — Number of Observations 51 39 ' 5 4 53 14 * The c o e f f i c i e n t accounted f o r the s i g n i f i c a n t amount of v a r i a t i o n at p < on 'F' d i s t r i b u t i o n (a) = X10"3 89 Each blood serum component within each group was adjusted for a l l s i g n i f i c a n t sources of v a r i a t i o n s using regression c o e f f i c i e n t s obtained by an i n t e r a t i v e or stepwise regression technique. The i n i t i a l regression model used for the groups were those already discussed under l i n e a r regression an a l y s i s . The l i n e a r models 3, 4, 5 and 6 for bred h e i f e r group, l a c t a t i n g non pregnant group, l a c t a t i n g pregnant group and dry animal group respectively were used except age was eliminated from the model i n each case. This reduced models 3 and 6 to regression of blood serum components on days pregnant i n the bred h e i f e r and dry animal groups. Models 4 and 5 used for the l a c t a t i n g non pregnant and l a c t a t i n g pregnant groups included l a c t a t i o n t r a i t s and l a c t a t i o n t r a i t s and days pregnant resp e c t i v e l y . Since age was the only e f f e c t evaluated i n the young animal group these data were not adjusted. The method of adjusting the raw data for s i g n i -f i c a n t sources of variati o n s i s given i n model 8. The s i g n i f i c a n t c o e f f i c i e n t s used to adjust the raw data i n the present study are given i n table 15. The frequency d i s t r i b u t i o n of each blood serum component - raw data -, and change i n frequency d i s t r i b u t i o n due to adjustment within p h y s i o l o g i c a l groups are shown i n appendix under d i s t r i b u t i o n curves. TABLE NO. 15 SIGNIFICANT REGRESSION COEFFICIENTS WHICH WERE USED TO ADJUST DATA WITHIN THE PHYSIOLOGICAL GROUPS, FOR THE HERD. •. Groups Independent Variables Mean + S.E. for Independent Significant Coefficients for the Blood Corrponents ^ ^ ^ j ^ ^ i s ) ' Variables Alkaline Phosphatase Bred Heifer Days Pregnant 137.74+12.61 -0.2383 Inorganic , , . _ TT „ E. Globulin Globulin Globulin „, \ Cholesterol B.U.N. ... . D „ Phosphorus Albumin a i 31 B 2 Lactating Ncn-pregnant Anijrals Days Lactating Kg .Milk Produced Kg.Fat Kg .Protein 87.61+10.88 16.18+ 0.60 0.37+ 0.02 0.55+ 0.02 0.4090 0.0199 -0.1159(a)0.0536{a) • 0.0584(a) -0.4229 -1.073(a) Kg.Lactose 0.81+ 0.03 6.542 Continued a = X10"3 Table No. 15 (Continued) Groups Independent IX-san + S.E. for Independent Variables Significant Coefficients for the Blood Ccnponents ^^?ajvLgS) Variables Inorganic _. „ .T Creat- _,, . E. Globulin Globulin Globulin „ a. B.U.N. . . Phospha- • n Phosphorus mine a.... Albumin ai a2 PI "case Days Pregnant 96.77+6.82 0.2639(a) -0.1699 ( a ) -0.1023(a) -0.0462(a' Days Lactating 220.55+8.88 0.4610(a) Lactating and Kg. milk Produced 11.69+0.42 Pregnant Aniraals Kg.Fat 0.34+0.01 0.0365 Kg.Protein 0.41-0.01 -99.040 Kg.Lactose 0.56-0.02 -1.5950 6.6111 B.U.N. Dry Animals Days Pregnant 224.93-15.34 0.0695 a = 10 92 IV. THE ANALYSIS OF GROUP AND BREED EFFECT A: Analysis of Variance F i t t i n g Fixed Model: The e f f e c t s of p h y s i o l o g i c a l groups and breed groups were evaluated using the l i n e a r model 7. The phys i o l o g i c a l groups and breed groups were considered to be fixed e f f e c t and age was used as a covariable. The 2 mean squares (M.S.) and the portion of sum of squares (R ) accounted for by each sources f i t t e d i n each blood serum components are given i n table 16. The degrees of freedom for the residual mean squares i n each blood serum component depends on the number of corrections made on the raw data. One degree of freedom was subtracted from the residual degrees of freedom for each s i g n i f i c a n t source of v a r i a t i o n used to adjust the raw data. The p h y s i o l o g i c a l group and age of the animal accounted for the largest portion of the v a r i a b i l i t y i n most of the blood serum constituents studied. Physiological groups accounted for 9% and age 2% of the v a r i a b i l i t y of t o t a l protein. For albumin the ph y s i o l o g i c a l group accounted for 18% of v a r i a b i l i t y and age was not s i g n i f i c a n t . In g l o b u l i n s i g n i f i c a n t amount of v a r i a t i o n were accounted by age 7% and by groups 3%. The p h y s i o l o g i c a l groups accounted 13% of the v a r i a t i o n TABLE NO. 16 ANALYSIS OF VARIANCE FITTING FIXED EFFECT MODEL (7) TO ESTIMATE THE BREED GROUPS, PHYSIOLOGICAL GROUPS AND AGE EFFECT FOR EACH BLOOD SERUM COMPONENT USING DATA ADJUSTED ACCORDING TO MODEL (8) DEPENDENT VARIABLES Sources of Var i a t i o n Protein Albumin Globulin D.F. M.S. R2 D.F. M.S. R 2 D.F. M.S. R2 Breeds 2 0.0748 0.001 2 0.0836 0 .015. 2 0 .0826 0 .001 Groups 4 3.9328* 0 .0 86 4 0.4974* 0 .176 4 2 .5184* 0.068 Age 1 2.8612* 0 .016 1 0.0319 0 .003 1 4. 4259* 0 .031 Residual (b) 203 0.6493 0 . 718 203 0 .0422 0 . 761 203 0 .5101 0 .700 Total Sums of Squares 210 183.64 210 11.28 210 147.90 * S i g n i f i c a n t source at P 0.05 on 'F 1 d i s t r i b u t i o n . (b) = Appropriate degrees of freedom were deducted from the Residual degrees of freedom for the number of corrections done for the blood component. TABLE NO. 16 ANALYSIS OF VARIANCE CONTINUED DEPENDENT VARIABLES Sources of Va r i a t i o n Calcium Inorganic Phosphorus Cholesterol D.F. M.S . R 2 D.F. M.S . R 2 D.F . M-'. S . R 2 Breeds 2 0 .7714 0 .015 2 1.3022 0 .005 2 4504 ' 0 .011 Groups 4 3 . 3634* 0 .127 4 1 5 . 7 9 2 * 0 .133 4 541* . , 0 . 254 Age 1 0 .7880 0 .007 1 7 . 1 9 7 8 * 0 .015 1 300 0 .00? Residual (b) 203 0 .4388 0 . 842 201 1.5680 0 .664 202 2505 0 .593 Total Sums of S q u a r e s 210 105 .74 210 475 .28 210 853122 * S i g n i f i c a n t source at P < 0.05 on 'F' d i s t r i b u t i o n . (b) = Appropriate degrees of freedom were deducted from the Residual degrees of freedom for the number of corrections done for the blood component. TABLE NO. 16 ANALYSIS OF VARIANCE CONTINUED DEPENDENT VARIABLES Sources of Variations B.U.N. Uric Acid Creatinine D.F M.S. R 2 D.F M.S. R 2 D.F. M.S. R 2 Breeds 2 30 . 84 0.011 2 0.1941* 0.030 2 0.0262 0 .010 Groups 4 598.65* 0 .418 4 0.3669* 0.115 4 0 .6847* 0 .484 Age 1 100.57* 0 .018 1 0.1583 0.012 1 0.0364 0.006 Residual (b) 200 12.22 0. 427 203 0.0502 0.797 202 0 .0137 0 .489 Total Sums of Squares 210 5723.29 210 12.78 210 5.66 * S i g n i f i c a n t source at P <0. 05 on 'F 1 d i s t r i b u t i o n . (b) » Appropriate degrees of freedom were deducted from the Residual degrees of freedom for the number of corrections done for the blood component. vo TABLE NO. 16 ANALYSIS OF VARIANCE CONTINUED DEPENDENT VARIABLES Sources of Variations A l k a l i n e Phosphatase S.G.O.T. E. Albumin D.F. M.S. R 2 D.F. M.S. R 2 D.F. M.S. # R 2 Breeds 2 499 0 .002 2 475.3 0 .011 2 4.4795 0.023 Groups 4 9978 * 0.096 4 1169.4* 0 .055 4 0.8110 0 .008 Age 1 16961* 0 .041 1 351.1 0.004 1 6.5002 0 .016 Residual (b) 200 1018 0.488 203 392 .3 0.937 201 1.8430 0 .904 Total Sum of Scmares 210 417576 210 85030.9 210 396.55 S i g n i f i c a n t source at P <_ 0.05 on 'F' d i s t r i b u t i o n . (b) = Appropriate degrees of freedom were deducted from the Residual degrees of freedom for the number of corrections done for the blood component. # = X10" 3 vo TABLE NO . 16 ANALYSIS OF VARIANCE CONTINUED DEPENDENT VARIABLES Sources of Var i a t i o n Globulin Globulin Globulin 8 l D.F. M.S. # R 2 D.F. M.S. # R 2 D.F. M.S. # R2 2 0.3779 0.014 2 0.4658* 0 .041 2 0. 7185* 0 .039 4 1.1871 * 0 .091 4 0 .2054*' 0.036 4 1.2240* 0 .132 1 2.1130* 0 .040 1 0 .0811 0.004 1 0.1400 0.004 201 0.2163 0 . 829 202 0.1056 0.931 201 0.1333 0. 724 Breeds Groups Age Residual (b) Total Sums of 210 52.42 210 22.91 210 37.02 Squares • * S i g n i f i c a n t source at P < 0.05 on 'F* d i s t r i b u t i o n . (b) = Appropriate degrees of freedom were deducted from the Residual degrees of freedom for the number of corrections done for the blood component. # = X10"3 TABLE NO -16 ANALYSIS OF VARIANCE CONTINUED DEPENDENT VARIABLES Sources of Va r i a t i o n Globulin D.F. M.S. * R 2 D.F. M.S. # R 2 Breeds 2 0.1432 0.008 2 0 .5952 0.008 Groups 4 0.13 33 0 .014 4 2 .0177* 0.057 Age 1 2.1085* 0 .057 1 7.6281* 0.054 Residual (b) 202 0.1431 0.782 203 0.4323 0 .620 Total Sums of 210 36.93 210 141.55 S i g n i f i c a n t source at P < 0.05 on 'F' d i s t r i b u t i o n . (b) = Appropriate degrees of freedom were deducted from the Residual degrees of freedom for the number of corrections done for the blood component. # = X10"3 99 i n both c a l c i u m and i n o r g a n i c phosphorus. Whereas age was not a s i g n i f i c a n t source o f v a r i a t i o n i n c a l c i u m and accounted f o r o n l y 2% o f v a r i a t i o n i n i n o r g a n i c phosphorus. The p h y s i o l o g i c a l groups were s i g n i f i c a n t source o f 2 v a r i a t i o n i n the f o l l o w i n g blood c o n s t i t u e n t s . The R * va l u e s a re g i v e n i n p a r e n t h e s i s . C h o l e s t e r o l ( 2 5 % ) , B.U.N. (42%), U r i c A c i d ( 1 2 % ) , C r e a t i n i n e (48%), A l k a l i n e Phosphatase (10%), S.G.O.T. ( 6 % ) , G l o b u l i n B (13%). Age accounted f o r a s i g n i f i c a n t amount of v a r i a t i o n i n B.U.N. ( 2 % ) , A l k a l i n e Phosphatase ( 4 % ) , G l o b u l i n ct ( 4 % ) , G l o b u l i n ft^ a n d Y - g l o b u l i n ( 6 % ) , i n a d d i t i o n t o t o t a l p r o t e i n and g l o b u l i n . Breed group accounted f o r a s i g n i f i c a n t amount of v a r i a t i o n o n l y i n t h r e e b l o o d serum components; u r i c a c i d ( 3 % ) , g l o b u l i n (4%) and g l o b u l i n 3£ ( 4 % ) . See t a b l e 16. B: Comparison o f Group Means o f the Blood Serum Components The b l o o d serum components which were found to be s i g n i f i c a n t l y e f f e c t e d by groups ( p h y s i o l o g i c a l or breed) i n the f i x e d model 7 were f u r t h e r a n a l y z e d t o enumerate and compare the group means w i t h i n each b l o o d serum component. Breed Groups:- the s i g n i f i c a n t e f f e c t s o f breed on u r i c a c i d , g l o b u l i n and g l o b u l i n 3-^  were e v a l u a t e d u s i n g o r t h o g o n a l c o n t r a s t . The t h r e e breed groups were, £ : R = The p r o p o r t i o n o f v a r i a t i o n accounted by the source f i t t e d . 100 (1) H o l s t e i n , (2) H o l s t e i n - A y r s h i r e c r o s s bred w i t h 75% to 62% of H o l s t e i n b l o o d and (3) H o l s t e i n - A y r s h i r e c r o s s bred w i t h 50% H o l s t e i n o r l e s s . (See t a b l e 2). The comparison chosen were the H o l s t e i n v e r s u s the two c r o s s b r e d groups t o determine i f these breed e f f e c t s were d i f f e r e n t between H o l s t e i n and A y r s h i r e and c r o s s b r e d 1 v s . c r o s s b r e d 2 to determine i f d i f f e r e n c e e x i s t e d between c r o s s b r e d groups. (See model 9). The mean squares f o r each s i n g l e degree o f freedom c o n t r a s t and the a p p r o p r i a t e . r e s i d u a l mean squares are g i v e n i n t a b l e 17. The b l o o d serum components o f the H o l s t e i n group was s i g n i f i c a n t l y lower i n u r i c a c i d ( H o l s t e i n = 0.857 c r o s s b r e d = 0.942), g l o b u l i n a2 ( H o l s t e i n = 0.118 c r o s s b r e d = 0.123) and g l o b u l i n 8^ ( H o l s t e i n = 0.145 c r o s s b r e d = 0.150) than the combined c r o s s b r e d groups. No d i f f e r e n c e was found between the two c r o s s b r e d groups f o r any o f these t h r e e b l o o d serum components, (see f i g u r e 1). T h i s i n d i c a t e s t h a t the d i f f e r e n c e between the mean l e v e l i n these t h r e e b l o o d serum components i s due to breed d i f f e r e n c e between H o l s t e i n and A y r s h i r e . P h y s i o l o g i c a l Groups:- The p h y s i o l o g i c a l group as a source o f v a r i a t i o n was s i g n i f i c a n t f o r a l l the bl o o d serum components except albumin o b t a i n e d by e l e c t r o p h o r e s i s , and TABLE NO. 17 ANALYSIS OF PARTITIONED SUMS OF SQUARES OF SREED EFFECT FROM THE FIXED EFFECT MODEL (7) FOR URIC ACID, GLOBULIN' a2 AND GLOBULIN B Blood Serum Components FROM FIXED EFFECT MODEL (7) THE ORTHOGONAL CONTRAST GROUPS For Breed effect Residual Mean and Variance For the Single degree Holstein VS Crossbred Between Cross bred Sums of Squares R2 D.F. M.S. Group 1 . Group 2&3 Group 2 Group .3 Uric Acid 0.38812 0.030 203 0.0502 Mean M.S. R2 0.857 - 0.942 0.2042 * 0.016 0.897 - 0.986 0.1375 0.012 Globulin 0 2 0.9316(a) 0.041 202 (a) 0.1056 Mean M.S. R2 0.118 - 0.123 0.6826 (a) * 0.031 0.124 - 0.121 0.1311(a) 0.006 (a) Globulin 6^  . 1.4369(a) 0.039 202 0.1333 Mean 0.145 - 0.150 0.152 - 0.148 M.S. 0.9686(a)* 0.2771(a) R2 0.026 0.007 (a) = Significant 'F.' at P£ 0.05 = X10-3 102 FIGURE 1. ANALYSIS OF BREED GROUP MEANS FITTING ORTHOGONAL CONTRAST FOR URIC ACID, GLOBULIN - (*2 AND GLOBULIN - g. URIC ACID . X = 0.910 GLOBULIN -X = 0.120 Breed Groups * 1 2 I <*2 o GLOBULIN - g ' X = 0.150 1 % I Breed Grups * S i g n i f i c a n t l e v e l P _ 0.05 Means u n d e r l i n e d by the same l i n e are not s i g n i f i c a n t l y d i f f e r e n t . — = O v e r a l l mean * = Breed Groups = (1) H o l s t e i n , (2) 75 to 62 p e r c e n t H o l s t e i n , (3) 50 p e r c e n t and below H o l s t e i n 103 g l o b u l i n ft^- T n e group means f o r each b l o o d serum components were t e s t e d w i t h the Duncan's new m u l t i p l e range t e s t (Kramer, 1957). The r e s u l t s o f these t e s t s and the p h y s i o l o g i c a l group means d e v i a t i o n ' from the o v e r a l l mean are shown i n the f i g u r e s 2 to 16 and i n t a b l e 18. The mean v a l u e f o r t o t a l p r o t e i n i n the young animal group (6.78 gm/100 n l . blood) was found t o be s i g n i f i c a n t l y lower than a l l o t h e r groups. The mean v a l u e f o r the bred h e i f e r group (7.38 gm/100 m l . blood) was not s i g n i f i c a n t l y d i f f e r e n t from the l a c t a t i n g non pregnant and l a c t a t i n g pregnant group, however, the mean f o r the l a c t a t i n g non-pregnant group (7.72 gm/100 m l . b l o o d was s i g n i f i c a n t l y h i g h e r than the group mean of l a c t a t i n g pregnant animals (7.36 gm/100 m l . blood) and lower than the dry a n i m a l s . The mean t o t a l p r o t e i n f o r the dry animals was s i g n i f i c a n t l y h i g h e r (7.7 6 gm/100 m l . blood) than the other groups i n the pre s e n t study. See F i g u r e 2. The mean va l u e o f albumin and g l o b u l i n , i n the young animal group (1.98 and 4.88 gm/100 m l . blood r e s p e c t i v e l y ) was lower than the o t h e r groups, but s i g n i f i -c a n t l y lower o n l y from the bred h e i f e r group and l a c t a t i n g pregnant group f o r albumin and l a c t a t i n g non-pregnant group and dry animal group f o r g l o b u l i n (see f i g u r e s 3 and 4 ) . The mean v a l u e o f albumin i n dry animals (2.13 gm/100 ml. blood) was the same as the other groups and g e n e r a l l y \?U NO. 18 Variables LEAST SQUARE CONSTANTS FOR - THE DEPENDENT VARIABILES AFTER FITTING GROUfS AND BREEDS AS FIXED EFFECT AND AGE AS CO VARIABLE - (MODEL 7) BLOOD SERUM COMPONENTS K-fiber of Total Observation* Protein Albunin Olobulln Calclu, IXll&l. ChQle.t.r.1. B . , . „ . ™ g Cre-tlnine , . ;«.B.Q.,. . . ^ i n .. ^ U " . . . ° ^ U " . " • J . " " 211 7.41 (9) 2.12 (9) 5.29 9.90 5.75 195.0B (mg) 18.11 tmg) 0.91 0.97 (ug) 72.93 (mu) ' ). 86.2 (flu) 0/30 I 0.12 I 0.12 r 0.15 I 0.17 0.14 rc-'r-..-* icrale Aninals (Croup 1) ferei Hellers :G-2-;p 3! :a:*.s*.lr^ rrecr.ant X.-.ir>jls (Croup 4) ?rv xnirals I3rou? J! 39 5* S3 14 -0.(214* -0.1436* -0.41C8* -0.1459* -0.1239 -0.0153 0.3178* -0.0415 0.3(04 -0.0517 0.3328* 0.0425 -0.0824 0.00(9 0.2S42 0.3939* -0.1761 • -0.0467 0.1763 1.1781* -47.51 -4.705* -0.0169 -0.0313 30.69* -0.4144 -20.29* -4.841* -0.0478 0.1430* 12.08* -0.3975* 38.97* 3.454* 0.1621* -0.1555* -18.86* -0.3190 53.74* 5.934* 0.0307 -0.1271* -15.37--0.02(7 -24.92 0.158 -0.1280* 0.1708* - 8.54 ,(e) -2.394 2.382 ( a' -1.421<a> ,!«) 8.346* -7.2691 6.394 1 , te)* 8.574 7.768 -7.196 («)• 2.377(a> -7.701 <«>• 0.103'"'-10.074l -1.5361*' e.l43(*>' 4.958 -2.189 (a) 6.216 (a)« 1.458 " " -5.144 (a)* 2.1521*1 -2.856 (a) 6.845 0 . 5 0 8 ( a l - 1 1 . 2 0 8 { a ) * -0 . 4 8 6 ( a > -4 .001 ( < l 1 1 . 6 5 s ' * ' ,(a)' !3) 5C< or.4 Belcv Kolstein 120 40 0.0393 0.0262 0.0210 • 0.1267 -0.0091 -0.0461 0.0361 -0.0400 -0.0302 0.0211 -0.0559 -0.0866 (a>* 0.0234(a) 0.2757(a)*-0.1163 (a) E. - Mbualn obtained by electrophoresis. Ku - Miliaicron /1C0 n l . blood. ( . pa« ol the l i s ol a l l the six peaks 0.1080 -0.1413 -0.3516la>* - 1.62 -0.446 -0.0562* 0.0191 1.49 i-10.59 -0.633 -0.0164 -0.0218 - 3.80 12.21 1.07B* 0.0727* 0.0027 2.31 -1.856 6.239<a) -2.799* -2.362 -10.569(a)* 1.072(a) ,(a)* 4.218 4.330<a) -0.033, a» -0.417,a' -2.261(a) -4.331 -(a)* 0.492(a) 2.t£2( 1.769(a) 1.4S91 , (a) - 2.27<a>-1.314ta)*0.0521ta!0.0250 l a ) -17.07<«>' -2.456 ( alo. 016 <«>*-0.006 ( a' * -0.001(a» 0.002(a» 0.006(a)* <g) * gm/100 nil. blood <ng> - mg/1 a - X10" * Constanta significantly different from zero at 't1. probablillty <_ 0.05. (rtg) • m 00 ml. blood -3 O FIGURE 2 . ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST DEPENDENT VARIABLE ... PROTEIN. 3 6 0 o o 4-0 o tn 20 J 1 U GROUPS' Significant Level P 0.05. Means Underlined By The Same Line Are.Not Significantly Differe X = Least Square Mean (Overall). * 1. Young females; 2. Bred h e i f e r - v ' 3 . L a c t a t i n g non-pregnant, 4. L a c t a t i n g pregnant, 5. Dry a n i m a l s . FIGURE 3. 106 ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST DEPENDENT VARIABLE . . . ALBUMIN. o o o 30 20 io I I 1 GROUPS* Significant Level P 0.05. Means Underlined By The Same Line Are Not Significantly Different. X = Least Square Mean (Overall). * 1. Young females, 2. Bred h e i f e r , 3. Lactating non-pregnant, 4. Lactating pregnant, 5. Dry animals. FIGURE 4. 107 ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST DEPENDENT VARIABLE GLOBULIN. 6-o o o H XI o o H > Cn 20 5" 4 1 GROUPS* Significant Level P 0.05. Means Underlined By The Same Line Are Not Significantly Different. X = Least Square Mean (Overall), * 1. Young females, 2. Bred h e i f e r , 3. Lactating non-pregnant, 4. Lactating pregnant, 5. Dry animals. 108 e q u i v a l e n t t o the o v e r a l l mean. The mean albumin l e v e l f o r the l a c t a t i n g pregnant animal group (2.16 gm/100 m l . blood) was lower than the bred h e i f e r group (2.27 gm/100 ml. b l o o d ) . No d i f f e r e n c e f o r g l o b u l i n was observed between the mean of the l a c t a t i n g non-pregnant (5.62 gm/100 ml. blood) and dry animal groups; between dry animals (5.57 gm/100 m l . b l o o d ) , l a c t a t i n g pregnant animals (5.21 gm/100 ml. b l o o d ) , l a c t a t i n g pregnant animals (5.21 gm/100 ml. blood) and bred h e i f e r s (5.17 gm/100 ml. blood) and between the l a c t a t i n g pregnant, bred h e i f e r s and young a n i m a l s . The o r d e r o f the groups f o r g l o b u l i n r e v e r s e d from t h a t for, albumin, w i t h the ex c e p t i o n o f the young animal group. T h i s was due, a t l e a s t i n p a r t , t o the way the g l o b u l i n was c a l c u l a t e d , from t o t a l p r o t e i n by s u b t r a c t i n g albumin. The mean serum p r o t e i n l e v e l s ( t o t a l p r o t e i n , albumin and g l o b u l i n ) o f animals i n p r o d u c t i o n were found t o be s i g n i f i c a n t l y h i g h e r than groups which had not y e t begun t h e i r p r o d u c t i o n . T h i s may be due to the i n c r e a s e d m o b i l i z a t i o n o f s t o r e d p r o t e i n when the animals are under p r o d u c t i o n s t r e s s . The mean va l u e o f b l o o d serum c a l c i u m f o r the bred h e i f e r group (10.29 mg/100 m l . blood) was found t o be s i g n i f i c a n t l y h i g h e r than a l l other groups except the dry animal group (10.1 mg/100 ml. b l o o d ) . These two groups are the pregnant groups not i n f l u e n c e d by the e f f e c t o f l a c t a t i o n . In c o n t r a s t , the l a c t a t i n g non-pregnant and the young animal groups FIGURE 5. 109 ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST DEPENDENT VARIABLE ... CALCIUM. o O rH Xi o o rH /2of I OS. SO 6-oV X - q-°o. Z r H- 3 1 J GROUPS'1 I 1 Significant Level P 0.05. Means Underlined By The Same Line Are Not Significantly Different, X = Least Square Mean (Overall). * 1. Young females, 2. Bred h e i f e r , 3. Lactating non-pregnant, 4. Lactating pregnant, 3. Dry animals. 110 were below the o v e r a l l mean. T h i s suggested i n the p r e s e n t a n a l y s i s , the mean c a l c i u m l e v e l below the o v e r a l l mean were a s s o c i a t e d w i t h e a r l y l a c t a t i o n and growth. The e f f e c t o f pregnancy and mid to l a t e l a c t a t i o n were found not to depress the c a l c i u m l e v e l i n the p r e s e n t s t u d y , (see f i g u r e 5) . The s i g n i f i c a n t group e f f e c t i n i n o r g a n i c phosphorus i n the a n a l y s i s of v a r i a n c e was a t t r i b u t e d t o the d i f f e r e n c e s between growing animals and a l l o t h e r groups i n the p r e s e n t s t u d y . Young animals were s i g n i f i c a n t l y h i g h e r i n i n o r g a n i c phosphorus l e v e l than a l l o t h e r groups and no d i f f e r e n c e s were found between the o t h e r 4 group means. (See f i g u r e 6). The group mean v a l u e s of c h o l e s t e r o l f o r the l a c t a t i n g non-pregnant (234 mg/100 m l . blood) and the l a c t a t i n g pregnant (248 mg/100 ml. blood) were not d i f f e r e n t from each o t h e r but both were s i g n i f i c a n t l y h i g h e r than a l l o t h e r p h y s i o l o g i c a l g roups. No d i f f e r e n c e was observed between the mean v a l u e s o f bred h e i f e r s (179 mg/100 m l . b l o o d ) , and dry animal (170 mg/100 m l . blood) groups or between dry animal and young animal (147 mg/100 m l . blood) groups, however, bred h e i f e r s were s i g n i f i c a n t l y d i f f e r e n t from t h a t of young animals ( f i g u r e 7.) These r e s u l t s suggested t h a t the c h o l e s t e r o l l e v e l i s not e f f e c t e d by pregnancy but a s s o c i a t e d w i t h the e f f e c t s o f l a c t a t i o n . FIGURE 6. I l l ANALYSIS O F GROUP MEANS BY DUNCAN'S NEW M U L T I P L E RANGE T E S T D E P E N D E N T V A R I A B L E . . . INORGANIC PHOSPHORUS. 8e>f o 4o o o 6 i 5* i GROUPS* Significant Level P 0. 05, Means Underlined by the Same Line Are Not Significantly Different; X - Least Square Mean (Overall). * 1. Young females, 2. Bred h e i f e r , 3 L a c t a t - i ™ „ pregnant, 4. L a c t a t i n g pregnant, 5 Dr? animals ^ FIGURE 7. 112 ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST DEPENDENT VARIABLE ... CHOLESTEROL. 3oo 25o 0 0 i H Xi 2.00 • rH g 150 O O rH \ too g 5-0 1 X 8. 3 5 5~ 1 1 I 1 \ -I GROUPS* Significant Level P 0.05. Means Underlined By The Same Line Are Not Significantly Different X = Least Square Mean (Overall). * 1. Young females, 2. Bred h e i f e r , 3. Lactating non-pregnant, 4. Lactating pregnant, 5. Dry animals. 113 No s i g n i f i c a n t d i f f e r e n c e between the young animals (11.37 mg/100 ml. blood) and bred h e i f e r s (11.24 mg/100 ml. blood) was found f o r B.U.N., but other p h y s i o l o g i c a l groups were s i g n i f i c a n t l y d i f f e r e n t from one another (see f i g u r e 8 ) . The mean l e v e l o f u r i c a c i d f o r the l a c t a t i n g non pregnant group (1.11 mg/100 ml. blood) i n the pres e n t study was found to be hi g h e r and s i g n i f i c a n t l y d i f f e r e n t from the other groups. No d i f f e r e n c e was observed between the l a c t a t i n g pregnant, young animals and bred h e i f e r s and between young a n i m a l s , bred h e i f e r s and dry a n i m a l s , (see f i g u r e 9 ) . No d i f f e r e n c e i n the l e v e l o f c r e a t i n i n e was found between the dry animal (1.14 mg/100 ml. blood) and bred h e i f e r groups (1.10 mg/100 ml. blood) and between the l a c t a t i n g non-pregnant (0.80 mg/100 ml. blood) and l a c t a t i n g pregnant (0.84 mg/100 ml. blood) groups. The young animal group (0.90 mg/100 ml. blood) was i n t e r -mediate and s i g n i f i c a n t l y d i f f e r e n t from a l l o t h e r groups, (see f i g u r e 10). The young animal group mean va l u e f o r c r e a t i n i n e was ver y near the o v e r a l l mean v a l u e . The l a c t a t i n g pregnant and the l a c t a t i n g non-pregnant group mean v a l u e s were s i g n i f i c a n t l y below and the dry animals and the bred h e i f e r s groups were s i g n i f i c a n t l y h i g h e r than the o v e r a l l mean. T h i s suggested t h a t the low mean va l u e s 114 FIGURE 8. A N A L Y S I S O F G R O U P M E A N S B Y D U N C A N ' S N E W M U L T I P L E R A N G E T E S T D E P E N D E N T V A R I A B L E . . . B L O O D U R E A N I T R O G E N . 30 13 O o I—I -43 UQ ~ o o IS-tT> 10 & 5" • GROUPS* Significant L e v e l P 0.05. Means Underl ined By The Same Line A r e Not Significantly Different X = Least Square Mean (Overall) . * 1. Young females, 2. Bred h e i f e r , 3. L a c t a t i n g non-pregnant, 4. L a c t a t i n g pregnant, 5. Dry a n i m a l s . FIGURE 9, 115 ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST ! DEPENDENT VARIABLE ... URIC ACID. - • • • , .1. IS xi o o • H o o s 3 4- 1 2 | | GROUPS* 1 J Significant Level P 0.05. Means Underlined By The Same Line Are Not Significantly Different, X = Least Square Mean (Overall! * 1. Young females, 2. Bred h e i f e r , 3. Lactating non-pregnant, 4. Lactating pregnant, 5. Dry animals. 116 FIGURE 10. . ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST DEPENDENT VARIABLE . . . CREATININE, I'X 8 W Xi • OS o o ob' rH \ o 5" 2 I 4 1 1 I— GROUPS* Significant Level P 0.05. Means Underlined By The Same Line Are Not Significantly Different X = Least Square Mean (Overall). * 1. Young females, 2. Bred h e i f e r , 3. Lactating non-pregnant, 4. Lactating pregnant, 5. Dry animals. 117 f o r the c r e a t i n i n e may be due to the i n f l u e n c e of l a c t a t i o n i n the l a c t a t i n g non-pregnant and l a c t a t i n g pregnant groups, under the present s t u d y . No evidence i n the l i t e r a t u r e was a v a i l a b l e to compare the p r e s e n t t r e n d of blood serum c r e a t i n i n e . C r e a t i n i n e i s r e p o r t e d to remain r e l a t i v e l y c o n s t a n t i r r e s p e c t i v e o f n u t r i t i o n , e x e r c i s e , e t c . , i n blood (Gans; 19 70) and the e x c r e t i o n of c r e a t i n i n e i n the u r i n e i s not g r e a t l y a f f e c t e d by exogenous sources of f e e d or by e x e r c i s e ( A l l e n ; 1970). However, other workers Walker et a l . , (1962); Walker, (1971), F i t c h (1961), S r i v a s t a v a e t a l . , (1965), F i s h e r (1965) r e p o r t e d t h a t the c r e a t i n i n e e x c r e t i o n f l u c t u a t e s a p p r e c i a b l y with d i e t a r y c o n d i t i o n s . C r e a t i n i n e i s the metabolic end product o f c r e a t i n e . C r e a t i n e on the o t h e r hand i s s y n t h e s i z e d from amino a c i d through two s t e p s , c a t a l y z e d by g l y c i n e - a r g i n i n e transamidinase and glycocyamine-methionine m e t h y l t r a n s -f e r a s e . I t i s c o n j e c t u r e d , i n the p r e s e n t study t h a t l a c t a t i n g animals need these amino a c i d s f o r m i l k p r o t e i n s y n t h e s i s , probably a small amount of the amino a c i d s are a v a i l a b l e from the amino a c i d p o o l which i s used f o r c r e a t i n e s y n t h e s i s . As a r e s u l t i t i s hypothesized t h a t c r e a t i n e metabolism goes down g i v i n g r i s e to a lower l e v e l o f c r e a t i n i n e i n the blood serum. 118 The mean l e v e l of a l k a l i n e phosphatase i n the young animal group (10 3.62 mu/100 ml. blood) was found to be s i g n i f i c a n t l y higher than a l l other group mean values, (see figure 11). No difference was observed between the bred h e i f e r group (85.11 mu/100 ml. blood) and the dry animal group (64.39 mu/100 ml. blood). Lactating non-pregnant and l a c t a t i n g pregnant groups were s i g n i f i c a n t l y lower than the bred h e i f e r group, but not d i f f e r e n t from the dry animal group. A l l c r o f t et a l . , (1941) also report-ed a higher blood serum a l k a l i n e phosphatase i n young animals than older animals which corresponds with the present study. The mean value for S.G.O.T. i n the l a c t a t i n g non-pregnant animals (95.17 mu/100 ml. blood) was s i g n i f i c a n t l y higher than a l l other group mean values (see figure 12) except the l a c t a t i n g pregnant group. The l a c t a t i n g pregnant group, however, was not d i f f e r e n t from the young animal, bred h e i f e r , or dry animal groups. The group e f f e c t for blood serum proteins separated by electrophoresis, was s i g n i f i c a n t f or glob u l i n f r a c t i o n s o ^ , a^, 8 1 and y. No difference was observed for g l o b u l i n a ^ , between the mean values of young animals (0.12 par t ) , bred h e i f e r (0.12 pa r t ) , l a c t a t i n g non-pregnant (0.13 part) and l a c t a t i n g pregnant (0.13 part) groups. The young animal and bred h e i f e r groups were not 119 FIGURE 11. ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST DEPENDENT VARIABLE . . . ALKALINE PHOSPHATASE. ISO TJ O O £ o o 100 50 X= 7^  93. 1 % I 5" * 1 GROUPS* Significant Level P 0.05. Means Underlined By The Same Line Are Not Significantly Different X = Least Square Mean (Overall). * 1. Young females, 2. Bred h e i f e r , 3. Lactating non-pregnant, 4. Lactating pregnant, 5. Dry animals. 120 FIGURE 12. ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST DEPENDENT VARIABLE . . . SERUM GLUTAMIC OXALOACETICTRANSAMINASE. l%0 loo-o o rH •° 60 rH S o kO o 0 Y GROUPS* Significant Level P 0. 05. Means Underlined By The Same Line Are Not Significantly Different, X = Least Square Mean (Overall). * 1. Young females, 2. Bred h e i f e r , 3. Lactating non-pregnant, 4. Lactating pregnant, 5. Dry animals. 121 d i f f e r e n t s i g n i f i c a n t l y from the dry animal (0.11 part) group. (see figure 13). Dry animals were s i g n i f i c a n t l y lower than the l a c t a t i n g non-pregnant and l a c t a t i n g pregnant animal groups. The glo b u l i n l e v e l s i n the young animal group (0.11 part) was s i g n i f i c a n t l y lower than the bred h e i f e r group (0.12 part) (figure 14). The mean l e v e l of serum gl o b u l i n f r a c t i o n with the young animal (0.16 part) and bred h e i f e r (0.16 part) groups was found to be s i g n i f i c a n t l y higher than the la c t a t i n g non-pregnant (0.14 par t ) , l a c t a t i n g pregnant (0.14 part) and the dry animal (0.14 part) groups. The mean l e v e l of the y-globulin f r a c t i o n with the l a c t a t i n g non-pregnant (0.14 part) and the dry animal (0.15 part) groups were s i g n i f i c a n t l y higher than a l l other group mean values. The dry animal group consists of the animals with higher average age under the present study and as reported by other workers (Dimopoullos; 1963) th i s high l e v e l of y-globulin i s expected to be more due to age than that of other e f f e c t s of pregnancy or l a c t a t i o n . FIGURE 13. 122 ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST DEPENDENT VARIABLE . . . GLOBULIN - £ co M rd CD th vo m o a CO CD -P m o +> rd P4 <5o i5 •/0 '0 6" %^ 013.1. 1 I 1 GROUPS* Significant Level P 0.05. Means Underlined By The Same Line Are Not Significantly Different X = Least Square Mean (Qverall). * 1. Young f e m a l e s . 2. Bred h e i f e r , 3. L a c t a t i n g non-pregnant, 4. L a c t a t i n g pregnant, 5. Dry a n i m a l s . FIGURE 14. 123 ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW-MULTIPLE RANGE T E S T D E P E N D E N T V A R I A B L E ... GLOBULIN - £ 01 M rd <u CU vo m o CO '15 | -to o U 05 X - 0-121. GROUPS* Significant Level P 0.05. Means Underlined By The Same Line Are Not Significantly Different X = Least Square Mean (Overall). * 1. Young females, 2. Bred h e i f e r , 3. L a c t a t i n g non-pregnant, 4. L a c t a t i n g pregnant, 5. Dry a n i m a l s . FIGURE 15. 124 ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST DEPENDENT VARIABLE ... GLOBULIN - /3, peaks VO VM *o o -e 1 15 -w CD Xi » 10 o — u OS (0 0 1 1 5" 1 4 3 GROUPS* J Significant Level P 0.05. Means Underlined By The Same Line Are Not Significantly Different. X = Least Square Mean (Overall). * 1. Young females, 2. Bred h e i f e r , 3. Lactating non-pregnant, 4. Lactating pregnant, 4. Dry animals. FIGURE 16. 125 ANALYSIS OF GROUP MEANS BY DUNCAN'S NEW MULTIPLE RANGE TEST DEPENDENT VARIABLE „ . . GLOBULIN w ro" a vo W OJ xi •P o -p u rrj •if -10 05" X = o i * 7 . 5- 3 A 1 SL GROUPS* Significant Level P 0.05. Means Underlined By The Same Line Are Not Significantly Different. X = Least Square Mean (Overall) . * 1. Young females, 2. Bred h e i f e r , 3. Lactating non-pregnant, 4. Lactating pregnant, 5. Dry animals. 126 V. TO ESTIMATE GENETIC COMPONENT OF.VARIANCE To estimate the g e n e t i c parameters o f the b l o o d serum c o n s t i t u e n t s i t was necessary t o remove environmental sources o f v a r i a t i o n by a p p l y i n g the same s t a t i s t i c a l technique used f o r the a n a l y s i s o f p h y s i o l o g i c a l and breed e f f e c t s . The g e n e t i c c o v a r i a n c e between two bl o o d serum components was e s t i m a t e d from the a n a l y s i s o f c o v a r i a n c e . The c o v a r i a n c e i n terms o f the expected c o v a r i a n c e components corresponds t o the expected v a r i a n c e components when the l i n e a r model used to analyze the two v a r i a b l e s i s : the same. S i n c e e s t i m a t e s o f a l l p o s s i b l e c o v a r i a n c e s were d e s i r e d the l i n e a r models used f o r a l l b l o o d c o n s t i t u e n t s were i d e n t i c a l . The expected v a l u e s o f the v a r i a n c e and c o v a r i a n c e components u s i n g model -10 are g i v e n i n Table 19. The c o v a r i a n c e components "between s i r e s " have a l r e a d y been shown, under s t a t i s t i c a l techniques i n terms of expected g e n e t i c components, (Model 1 5 ) . The v a r i a n c e component between s i r e s w i l l y i e l d the h e r i t a b i l i t y i n the narrow sense and the r a t i o between the s i r e c o v a r i a n c e component, and the geometric mean o f the s i r e v a r i a n c e components w i l l y i e l d the g e n e t i c c o r r e l a t i o n . A: The Mixed Model to E s t i m a t e G e n e t i c Parameters The model f i t t e d f o r e s t i m a t i n g g e n e t i c parameters f o r the TABLE NO. 19 THE E X P E C T E D COMPONENTS OF VARIANCE FOR TWO BLOOD SERUM COMPONENTS F I T T I N G THE SAME MODEL (10) AND THE E X P E C T E D COMPONENT OF COVARIANCE SOURCES D.F. Expected Component of Variance for the Blood Serum Constituent (1) Expected Component of Variance for the Blood Serum Constituent (2) Expected Component of Covariance between the Blood Serum Components (1) and (2) GROUPS G-l <j2ei + K26! d 2e 2 + K2 e2 aei.e 2 + K2 (o6i . 82) SIRES S-l 02e2 + K162S2 ae1.e2 + Kj. <aS1#s2) AGE 2 a2ex + XX ( Bllf 6l 2) tf2e2 + A2 ( 321 ' e22> ae!.e2 + X3 ( B11 , B12f 621 . *2 2 Y l 2 ) RESIDUAL n- f(G-l) „ 2 +(S-lj+2J i. ° e2 0 e r e2 2 S-i a i (G-l) = Function of ( B's ) for blood serum components one and two respectively = Correlation between the two blood serum components. = Function of (6's &Yi2) 1' .Y12 *3 128 a n a l y s i s o f v a r i a n c e i s shown i n model-10 under s t a t i s t i c a l t e c h n i q u e s . The sources f i t t e d were groups, s i r e s and the c o v a r i a b l e age w i t h two degrees of freedom as age l i n e a r and q u a d r a t i c . The expected component o f v a r i a n c e f o r the model i s g i v e n i n ANOVA Model - 11. Only progeny o f H o l s t e i n s i r e s were c o n s i d e r e d f o r the g e n e t i c a n a l y s i s . The 46 animals which were s i r e d by f i v e A y r s h i r e b u l l s and one H o l s t e i n by A y r s h i r e c r o s s b u l l were excluded from t h i s a n a l y s i s . In a d d i t i o n , H o l s t e i n s i r e progeny groups o f t h r e e o r l e s s were not i n c l u d e d i n the a n a l y s i s as mentioned e a r l i e r . Due to the above r e s t r i c t i o n s , the sample number f o r g e n e t i c a n a l y s i s was 158 animals s i r e d by 15 H o l s t e i n s i r e s (see the a p p e n d i x ) . The b l o o d serum components raw d a t a f o r these 158 animals were a d j u s t e d f o r e f f e c t s unique to each p h y s i o l o g i c a l group u s i n g the same methods as used i n a d j u s t i n g the raw d a t a to f i t to the model-7. The c l a s s i f i c a t i o n o f p h y s i o l o g i c a l groups and the sources measured w i t h i n groups were the same used i n the a n a l y s i s o f group and breed e f f e c t s . The c o e f f i c i e n t s used f o r the adjustment are shown i n the appendix (box number two.) The e f f e c t s o f the v a r i o u s sources o f v a r i a t i o n w i t h i n each p h y s i o l o g i c a l group was s i m i l a r to those o b t a i n e d w i t h the t o t a l d a t a . See t a b l e 20. T A B L E NO • 20 T H E I N D E P E N D E N T V A R I A B L E S F I T T E D F O R ' E A C H B L O O D C O M P O N E N T W I T H I N G R O U P S A N D T H E S I G N I F I C A N T C O E F F I C I E N T S U S E D F O R C O R R E C T I N G T H E C O M P O N E N T S W I T H I N P H Y S I O L O G I C A L G R O U P S : F O R X58 D A U G H T E R S O F T H E 15 H O L S T E I N B U L L S . * Groups Independent V a r i a b l e s F i t t e d C o e f f i c i e n t s of S i g n i f i c a n t Blood Components (Dependent Variables) Pregnant Heifers Days Pregnant A l k a l i n e Phosphatase -0.2136 Lactating Non-Pregnant Animals Days L a c t a t i n g Kg. Milk Fat P r o t e i n Lactose C h o l e s t e r o l B.U.N. G l o b u l i n Globulin 8 2 rhosphorUc G l o b u l i n a2 G l o b u l i n S i Globulin—y Acid 0.5593 0.0224 0.0781(a) 0.0681 (a) -0.0609 -2.3530 (a) -1.2270 (a) -1.3391(a) 3.2654 0.0512 0.3386 Pregnant t L a c t a t i n g Animals Days Pregnant Days L a c t a t i n g Kg '. Milk Fat P r o t e i n Inorganic U r i c Globulin E . Albumin G l o b u l i n Globulin $2 Globulin Bi Creatine Phosphorus Ch o l e s t e r o l Acid 4.4341 (a) 0.2859(a)fO.1464(a) -0.1129(a) -0.0615 0.9043(a) 0.0260 -0.0993 37.7525 -0.4767 0.7363 -563.6670 Dry Animals Days Pregnant Glo b u l i n B.U.N. 6.2510(a) 0.06634 t o v o * Vide Box No. 2 of Appendix (a) -X 10 130 The expected components of v a r i a t i o n are g i v e n i n model 11, i n the s t a t i s t i c a l a n a l y s i s . The mean squares and a s s o c i a t e d v a r i a n c e s f o r each source f i t t e d are g i v e n i n t a b l e 21. The two degrees of freedom f i t t e d f o r age were separated i n t o the l i n e a r and q u a d r a t i c a f t e r l i n e a r . I t was found t h a t the l i n e a r f u n c t i o n of age accounted f o r the major p o r t i o n of the v a r i a t i o n i n a l l the b l o o d components. B: G e n e t i c Parameters The expected (E) s i r e component of v a r i a n c e 2 a as mentioned e a r l i e r i s s E ( a2 ) = Ca2e + K± ^a2s) - ( a2 e) ( f r Q m ^ 1 model) where a2 = r e s i d u a l or e r r o r v a r i a n c e e and = weighted number of progeny per s i r e . Due to sampling e r r o r and some unknown causes o f v a r i a t i o n s which c o u l d not be removed from the e r r o r term, 2 (the ag ) the b l o o d serum components, (1) t o t a l p r o t e i n (2) c a l c i u m , (3) i n o r g a n i c phosphorus, (4) albumin o b t a i n e d by e l e c t r o p h o r e s i s and the g l o b u l i n f r a c t i o n s (5) a^, (6) a2 and (7) y, y i e l d e d n e g a t i v e s i r e component o f v a r i a n c e and hence were not c o n s i d e r e d f o r e s t i m a t i n g TABLE NO. 21 ANALYSIS OF VARIANCE FITTING MIXED EFFECT MODEL (10) TO ESTIMATE THE SIRE COMPONENT OF VARIANCE FOR THE BLOOD SERUM COMPONENTS, USING DATA ADJUSTED ACCORDING TO MODEL (8) T o t a l P r o t e i n Albumin G l o b u l i n Sources o f V a r i a t i o n s D .F . M. S. R2 D.F. M .S. R2 D.F. M. S . R2 Groups 4 0. 594 0 .027 4 0 .212 * 0.092 4 0. 586 0 .028 S i r e s 14 0 . 208 0 .033 14 0 .048 0.078 14 0 . 359 0 .059 Age C u r v i l i n e a r 2 0. 658 0 .015 2 1 .189 0.274* 2 2 . 649 0 .063 * (i) Age L i n e a r ( 1) 1. 316 0 .015 ( 1) 2 .302 0.266 * ( 1 ) 5. 154 0.061* ( i i ) Age Q u a d r a t i c a f t e r L i n e a r • ( 1 ) 0. 763 0 .000 ( 1 ) 0 .076 - 0.009 ( 1 ) 0 . 144 3 .002 R e s i d u a l (b) 137 0 . 322 0 .497 137 0 .04 2 0.671 135 0 . 337 0 .539 T o t a l Sums o f Squares, 157 88. 77 157 8 .6567 157 84. 49 * S i g n i f i c a n t source a t P £ 0.05 on *F' d i s t r i b u t i o n . b = A p p r o p r i a t e degrees of freedom were deducted from the r e s i d u a l degrees of freedom f o r the number of c o r r e c t i o n s done to the blood serum component. TABLE NO.21 ANALYSIS OF VARIANCE CONTINUED. Calcium Inorganic Phosphorus C h o l e s t e r o l Sources o f V a r i a t i o n s D.F. M .S. R2 D.F. M • S. R2 D.F. M.S . R 2 Groups 4 0 .9447 0.045 4 3 .04 * 0. 047 4 11923* 0 .088 S i r e s 14 0 .3557 0 .060 14 0 .82 0 . 045 14 2082 0 .054 Age C u r v i l i n e a r 2 0 .1483 0 .002 2 36 .82 0 . 285* 2 55231 0 .204* (i) Age L i n e a r ( 1 ) 0 .1379 0 .002 ( 1 ) 71 .52 0 . * 2 77 ( 1 ) 106885 0 .19 7* ( i i ) Age Q u a d r a t i c a f t e r L i n e a r ( 1 ) 0 .0104 0 .000 ( 1 ) 2 .11 0 . 008 ( 1 ) 3577 0 .007 R e s i d u a l (b) 137 0 .4811 0.783 134 . 0 . 89 0. 474 134 2016 0 . 499 T o t a l Sum of Squares;157 83 .99 157 257 .9 157 542170 * S i g n i f i c a n t source a t P <_ 0.05 on 'F1 d i s t r i b u t i o n . (b) = A p p r o p r i a t e degrees of freedom were deducted from the r e s i d u a l degrees of freedom f o r the number of c o r r e c t i o n s done to the blood serum component. u> TABLE NO . 3,1 ANALYSIS OF VARIANCE CONTINUED. B.U.N. U r i c A c i d C r e a t i n i n e Sources of V a r i a t i o n s D.F. M.S • R2 D.F. M .S. R2 D.F. M . s . R2 Groups 4 206 .1* 0 .202 4 0 .387 * 0.182 4 0 .5226* 0 . 436 S i r e s 14 19 .64* 0 .067 14 0 .053 0.0 88 14 0 .0347* 0 . 101 Age C u r v i l i n e a r 2 46 .56 0 .024 2 1 .247 0.294* 2 0 .0880 0. 037 (i) Age L i n e a r (1 ) 92 .96 0.023 (1) 2 .422 0 .285* (1) 0 .5167 0 . 036 ( i i ) Age Q u a d r a t i c a f t e r L i n e a r (1) 0 .16 0 .000 (D 0 .073 0 .009 (1) 0 .1709 0. 001 R e s i d u a l (b) 135 12 .32 0 .40 8 135 0 .045 0 .708 134 0 .0118 0. 330 T o t a l Sums of Squaresl57 4078 157 8 .499 157 4 . 797 * S i g n i f i c a n t source a t P < 0.05 on 'F* d i s t r i b u t i o n . (b) = A p p r o p r i a t e degrees of freedom were deducted from the r e s i d u a l degrees of freedom f o r the number of c o r r e c t i o n s done to the blood serum component. TABLE NO. 21 ANALYSIS OF VARIANCE CONTINUED. A l k a l i n e Phosphatase S.G.O.T. E. Albumin Sources o f V a r i a t i o n s D.F. M.S. R2 D.F. M.S. R2 D.F. M .S. R2 Groups 4 1037 0 .018 4 547. 0 .032 4 0 . 35 7 0 .004 S i r e s 14 811 0 .049 14 785 0 .162* 14 1 .112 0.047 Age C u r v i l i n e a r 2 36859 0.319* 2 2499 0 .735* 2 4 .051 0 .024 (i) Age L i n e a r ( 1 ) 72102 0.313* ( 1 ) 47832 0 .703* ( 1 ) 7 .690a 0.023 ( i i ) Age Q u a d r a t i c a f t e r L i n e a r ( 1 ) 1617 0.007 ( 1 ) 2154 0 .032 ( 1 ) 0 .321 0 .009 R e s i d u a l (b) 136 662 0.391 137 380 0.766 136 2 .098( a ) 0 . 867 T o t a l Sums o f Squaresl57 230641 157 68003 157 0 . 3288 * S i g n i f i c a n t source a t P £ 0.05 on 'F' d i s t r i b t i o n . (a) = X 1 0- 3 (b) = A p p r o p r i a t e degrees of freedom were deducted from the r e s i d u a l degrees of freedom f o r the number of c o r r e c t i o n s done to the blood serum component. co TABLE NO. 21 ANALYSIS OF VARIANCE CONTINUED. G l o b u l i n G l o b u l i n Q-? G l o b u l i n $]_ Sources of V a r i a t i o n s D.F. M.S. R2 D.F. M.S. R D.F. M.S. R Groups 4 0 .618 ,(a)0 .065 4 0 . 0551(a)0 .010 4 0 .6684* (a) 0 .105 S i r e s 14 0 .134 (a)0 .049 14 0 .0784 (a)0 .052 14 0 .1745 (a) 0 .091 Age C u r v i l i n e a r 2 0 .925 (a)0 .049 2 2 .13(a) 0 .202* 2 5 .9172 (a) 0 .440* (i) Age L i n e a r (1 ) 1 . 740 (a)o .046 (1) 4 .09 (a)o .194* (1 ) 0 .0115 0 .430* ( i i ) Age Q u a d r a t i c a f t e r L i n e a r (1 ) 0 .10 6 (a)0 .003 (1 ) 0 .169 (a)0 .008 (1 ) 0 .3094 (a) 0 .012 R e s i d u a l (b) 135 0 .220 0 .781 134 0 .142(a)0 .907 135 0 .1398 (a) 0 .703 ?otal Sums of Squares 157 0 .038 157 0 .0211 157 0 .0269 * S i g n i f i c a n t source a t P <_ 0.05 on 'F' d i s t r i b u t i o n . (a) = X.10"3 (b) A p p r o p r i a t e degrees of freedom were deducted from the r e s i d u a l degrees of freedom f o r the number of c o r r e c t i o n s done to the blood serum component. t —1 TABLE NO. 21 ANALYSIS OF VARIANCE CONTINUED. G l o b u l i n '82 G l o b u l i n Y Sources of V a r i a t i o n s D.F. M . s . R2 D.F. M. s. R2 Groups 4 0 .1451 (a) 0.022 4 1. 652* (a) 0.071 S i r e s 14 0 .1576 (a) 0 .082 14 0 . 274-. 0.042 Age C u r v i l i n e a r 2 0 .0321 (a) 0.002 2 0. 001' 0.039 (i) Age L i n e a r U> 0 . 3432 (a) 0 .001 (1) 3. 579 ;(a) 0 .038 ( i i ) Age Q u a d r a t i c a f t e r L i n e a r (1) 0 .0297 (a) 0 .001 (1) 0. 061 (a) 0.001 R e s i d u a l (b) 136 0 .1397 (a) 0 . 707 136 0 . 393 (a) 0 .579 T o t a l Sums of Squares 157 0 .0269 157 0 . 0925 * S i g n i f i c a n t source a t P < 0.05 on *F' d i s t r i b u t i o n . (a) = X10~3 (b) = A p p r o p r i a t e degrees of freedom were deducted from the r e s i d u a l degrees of freedom f o r the number of c o r r e c t i o n s done to the b l o o d serum component. 137 genetic parameters. The analysis of variance for these blood serum components are given i n table 21. The ten blood serum components with p o s i t i v e s i r e components of variance used for estimating the genetic parameters are: (1) albumin, (2) gl o b u l i n , (3) c h o l e s t e r o l , (4) blood urea nitrogen (B.U.N.), (5) u r i c acid, (6) creatinine, (7) a l k a l i n e phosphatase, (8) S.G.O.T., (9) globul i n 8" and (10) gl o b u l i n 8„. The analysis of 1 ^ variance of these ten blood components are given i n table 21. The h e r i t a b i l i t y estimates were ca l c u l a t e d according to model 16 and these values are tabulated i n table 22. The h e r i t a b i l i t y estimates for cr e a t i n i n e and S.G.O.T. was found to be comparatively high, 0.77 and 0.46 respe c t i v e l y . The h e r i t a b i l i t y estimates for B.U.N, was 0.27, a l k a l i n e phosphatase 0.11, u r i c acid 0.10 and globul i n fc^ 0.12. The h e r i t a b i l i t y estimates for the albumin 0.07, gl o b u l i n 0.03, cho l e s t e r o l 0.02 and globul i n 0.0 6 were found to be comparatively low i n the present study. Stuffelbeam et a l . , (1966)/in a study of the r e l a t i v e value of c e r t a i n economic t r a i t s and blood components for 138 s e l e c t i o n i n d i c e s i n H e r e f o r d b u l l calves' i n the f e e d l o t , r e p o r t e d h e r i t a b i l i t y e s t i m a t e s f o r plasma c r e a t i n i n e of 0.41, u r i c a c i d 0.20, t o t a l serum p r o t e i n 0.18 and c h o l e s t e r o l 0.55. T h e i r sample c o n s i s t e d of 60 randomly s e l e c t e d H e r e f o r d b u l l c a l v e s i n 1961 and 1962, and r a i s e d on a s p e c i a l f e e d f o r 135 and 182 days r e s p e c t i v e l y p r i o r to s a mpling. The animals i n c l u d i n g both the years were rep r e s e n t e d by 13 s i r e groups. S t u f f e l b e a m e t a l . , (1970), i n a s i m i l a r study w i t h 143 young H e r e f o r d b u l l c a l v e s d u r i n g a 2 year p e r i o d r e p r e s e n t e d by 14 s i r e groups, r e p o r t e d h e r i t a b i l i t y e s t i m a t e f o r b l o o d serum c h o l e s t e r o l of 0.80. The c o m p a r a t i v e l y h i g h h e r i t a b i l i t y o f c h o l e s t e r o l as observed by them i n Herefords did not agree w i t h the low h e r i t a b i l i t y e s t i m a t e (0.02) o b t a i n e d f o r H o l s t e i n , i n . , the p r e s e n t s t u d y . However, the c o m p a r a t i v e l y h i g h e s t i m a t e of h e r i t a b i l i t y - ( a f t e r c h o l e s t e r o l ) f o r plasma c r e a t i n i n e r e p o r t e d by them i n H e r e f o r d b u l l c a l v e s was a l s o i n agree-ment w i t h the c o m p a r a t i v e l y h i g h h e r i t a b i l i t y e s t i m a t e (0.77) o b t a i n e d f o r b l o o d serum c r e a t i n i n e w i t h the p r e s e n t s t u d y . 139 Table No. 22 HERITABILITY AND STANDARD ERRORS FOR TEN BLOOD SERUM COMPONENTS. Blood Components H e r i t a b i l i t y S.E. Albumin 0.07 0.22 G l o b u l i n 0.04 0.20 C h o l e s t e r o l 0.02 0.20 Blood Urea N i t r o g e n 0.28 0.27 U r i c A c i d 0.10 0.22 C r e a t i n i n e 0.77 0.37 A l k a l i n e Phosphatase 0.11 0.23 S.G.O.T. 0.46 0.32 G l o b u l i n „ 0.12 0.23 G l o b u l i n n B2 0.06 0.21 Numbers o f s i r e = 15.0 K = Weiahted Number o f Progeny Per S i r e = 8.11 140 Roubicek e t a l . , (1972) w h i l e a n a l y z i n g serum p r o t e i n s i n a H e r e f o r d herd i n A r i z o n a r e p o r t e d h e r i t a b i l i t y e s t i mate f o r serum p r o t e i n o f 0.30, albumin 0.20, g l o b u l i n a 0.16, 8 0.03, and y 0.27. T h e i r sample c o n s i s t e d of 700 to 9 80 progeny a t each sampling p e r i o d over 6 y e a r s . The progeny were sampled a t mean ages of 235, 340, 600 and 710 days. A t o t a l o f 12 t o 28 s i r e s were r e p r e s e n t e d i n the v a r i o u s age c l a s s i f i c a t i o n s . The h e r i t a b i l i t y e s t i m a t e s o b t a i n e d f o r g l o b u l i n f r a c t i o n s 8-^  and 82 (0.12 and 0.06 r e s p e c t i v e l y ) i n H o l s t e i n s w i t h the p r e s e n t study was co m p a r a t i v e l y h i g h e r than t h a t of the h e r i t a b i l i t y e s t i m a t e of the g l o b u l i n f r a c t i o n of 8 (0.03) r e p o r t e d by them. The h e r i t a b i l i t y e s t i m a t e f o r b l o o d serum albumin of 0.07 i n H o l s t e i n s o b t a i n e d w i t h the p r e s e n t study was lower than the h e r i t a b i l i t y e s t imate o f serum albumin o f 0.20 i n He r e f o r d b u l l s r e p o r t e d by them. No r e f e r e n c e s were found i n the l i t e r a t u r e w i t h o t h e r b l o o d serum components e v a l u a t e d i n the pr e s e n t study f o r H o l s t e i n s . The g e n e t i c c o r r e l a t i o n s were c a l c u l a t e d a c c o r d i n g t o the model 17 and are t a b u l a t e d i n t a b l e 23. C r e a t i n i n e showed a n e g a t i v e c o r r e l a t i o n w i t h g l o b u l i n (-0.58) but p o s i t i v e w i t h B.U.N. (0.18). A l k a l i n e phosphatase was p o s i t i v e l y c o r r e l a t e d to albumin (0.45), but n e g a t i v e l y w i t h B.U.N. (-0.21). S.G.O.T. was found to be n e g a t i v e l y TABLE NO. 23 GENETIC CORRELATION BETWEEN THE TEN BLOOD COMPONENTS AND THEIR STANDARD ERRORS. Albumin Globulin Cholesterol B.U.N. Uric Acid Creatinine pn o s p h a t a s e S.G.O.T. Globulin ^ Globulin - 2.86 Cholesterol 5.56 - 2.49 B.U.N. 3.09 2.41 2.06 Uric Acid 3.45 - 1.74 - 4.52 0.01 1.08 Creatinine 1.32 - 0.58 0.83 3.07 1.51 0.18 0.47 -0.21 2.01 Alkaline 0.45 3.33 . - 2.84 1.17 Phosphatase 1.45 0.97 S.G.O.T. 0.93 1.46 2.15 1.49 - 0.01 -0.26 0.14 0.89 0.38 Globulin 1.17 0.88 0.58 - 1.35 0.55 0.69 - 0.14 1.47 0.15 0.67 Globulin £L 0.42 1.92 1.10 - 4.13 1.00 - 0.64 1.19 0.38 0.78 -1.01 -1.77 -0.16 1.85 0.58 0.54 0.24 1.02 1.96 N.B. The standard errors are given below the respective genetic c o r r e l a t i o n s . 14 2 c o r r e l a t e d w i t h albumin (-0.93) and c r e a t i n i n e (-0.26). G l o b u l i n 3^ was p o s i t i v e l y c o r r e l a t e d w i t h g l o b u l i n (0.88), B.U.N. (0.55), c r e a t i n i n e (0.15), S.G.O.T. (0.58) but n e g a t i v e l y c o r r e l a t e d w i t h u r i c a c i d (-0.44). G l o b u l i n was p o s i t i v e l y c o r r e l a t e d w i t h albumin (0.42), c r e a t i n i n e (0.38) and S.G.O.T. (0.24), but n e g a t i v e l y c o r r e l a t e d w i t h u r i c a c i d (-0.64) and a l k a l i n e phosphatase (-0.16). B.U.N, was p o s i t i v e l y c o r r e l a t e d to u r i c a c i d . In the p r e s e n t study the p r o t e i n m e t a b o l i c end p r o d u c t , c r e a t i n i n e , was p o s i t i v e l y c o r r e l a t e d w i t h B.U.N, and B.U.N, w i t h u r i c a c i d . Whereas the serum p r o t e i n s g l o b u l i n 6^ and 62 though p o s i t i v e l y c o r r e l a t e d w i t h c r e a t i n i n e and B.U.N, were n e g a t i v e l y c o r r e l a t e d w i t h u r i c a c i d . T h i s suggested the genes r e s p o n s i b l e f o r the i n c r e a s i n g l e v e l o f b l o o d serum g l o b u l i n s 3^ and are a l s o r e s p o n s i b l e f o r the h i g h e r m e t a b o l i c r a t e o f the p r o t e i n s p r o d u c i n g c r e a t i n i n e and B.U.N, but probab l y suppresses the m e t a b o l i c r a t e o f n u c l e o p r o t e i n s whose m e t a b o l i c end product i s u r i c a c i d . However, as the c r e a t i n i n e i s p o s i t i v e l y c o r r e l a t e d w i t h B.U.N, and B.U.N, w i t h u r i c a c i d t h e r e must be a s e t o f genes r e s p o n s i b l e f o r the t o t a l metabolism o f p r o t e i n i n the animal body which i n c r e a s e s the r a t e o f a l l p r o t e i n metabolism a t the same t i m e . No r e c o r d o f g e n e t i c c o r r e l a t i o n s f o r these blood components i n c a t t l e were 1 4 3 a v a i l a b l e i n the l i t e r a t u r e to compare too the p r e s e n t f i n d i n g s . S e v e r a l e s t i m a t e s o f g e n e t i c c o r r e l a t i o n s i n the p r e s e n t study r e s u l t e d i n v a l u e s above one. These e s t i m a t e s , o f c o u r s e , are not v a l i d but do i n d i c a t e the d i r e c t i o n o f the r e l a t i o n s h i p . The environmental and phenotypic c o r r e l a t i o n s were c a l c u l a t e d a c c o r d i n g t o the models 20 and 21 r e s p e c t i v e l y and are g i v e n i n t a b l e 24. The environmental c o r r e l a t i o n s . Albumin was n e g a t i v e l y c o r r e l a t e d w i t h g l o b u l i n (-0.17) and p o s i t i v e l y c o r r e l a t e d w i t h c h o l e s t e r o l (0.36) and B.U.N. (0.28). G l o b u l i n was n e g a t i v e l y c o r r e l a t e d w i t h B.U.N. (-.26) and p o s i t i v e l y c o r r e l a t e d w i t h g l o b u l i n 3^ (0.45). C h o l e s t e r o l was p o s i t i v e l y c o r r e l a t e d w i t h u r i c a c i d (0.18) a l k a l i n e phosphatase (0.16) and S.G.O.T. (0.18). B.U.N. was n e g a t i v e l y c o r r e l a t e d w i t h both g l o b u l i n f r a c t i o n s 3^ (-0.16) and fc^ (-0.17). A l k a l i n e phosphatase was p o s i t i v e l y c o r r e l a t e d w i t h S.G.O.T. (0.21) and g l o b u l i n was p o s i t i v e l y c o r r e l a t e d w i t h g l o b u l i n ^ (0.26). The phenotypic c o r r e l a t i o n . Albumin was n e g a t i v e l y c o r r e l a t e d w i t h g l o b u l i n (-0.29), B.U.N. (-0.23), u r i c a c i d (-0.31), c r e a t i n i n e (-0.28), and S.G.O.T. (-0.19). G l o b u l i n TABLE NO. 24 ENVIRONMENTAL AND PHENOTYPIC CORRELATIONS FOR TEN BLOOD COMPONENTS. Albumin Globulin Cholesteral B.U.N. Uric Acid Creatinine S^1^? S.G.O.T: G1 0^1 1 1 Phosphatase B ^  -0.17* -0.29* 0.36* 0.13 0.15 0.19* Globulin Cholesterol B.U.N. Uric Acid Creatinine Alkaline Phosphatase S.G.O.T. Globulin B ^  , , .. -0.21* 0.45* -0.04 -0.17* 0.05 . -0.05 0.09 -0.03 0.26* Glo£>u±inp2 _0 > 1 5 0 > 4 ? A -0.17* -0.01 -0.00 0.06 0.07 0.02 0.40* 0.28* -0.26* 0.01 -0.23* 0.04 0.14 -0.04 0.08 0.18* 0.06 -0.31* -0.03 -0.01 0.05 0.11 -0.14 -0.14 0.44 -0.11 -0.28* -0.12 -0.21* 0.09 0.52* 0.04 0.04 0.16* -0.09 -0.03 -0.04 0.07 0.21* 0.28* -0.11 -0.32* -0.35* -0.05 0.02 0.18* 0.08 0.12 0.01 0.21* -0.19* 0.19* 0.28* 0.57* 0.06 -0.16* -0.12 -0.22 -0.01 0.04 -0.16* 0.01 -0.16* -0.03 -0.09 0.09 0.05 -0.03 -0.03 -0.01 -0.02 -0.23* 0.08 .04 05  -0.05 09 .03 -0.15 0. 7* -0.17* -0.01 -0.00 0.06 0.07 0* S i g n i f i c a n t at P <_ 0.05 Upper l i n e - Environmental C o r r e l a t i o n s (r) Lower l i n e - Phenotypic C o r r e l a t i o n s (r) 145 was p o s i t i v e l y c o r r e l a t e d w i t h C h o l e s t e r o l (0.19), a l k a l i n e phosphatase (0.21), S.G.O.T. (0.19), and 62 (0.47). C h o l e s t e r o l was found t o be n e g a t i v e l y c o r r e l a t e d w i t h c r e a t i n i n e (-0.21) and g l o b u l i n B2 (-0.11) and p o s i t i v e l y c o r r e l a t e d w i t h a l k a l i n e phosphatse (0.28) and S.G.O.T. (0.28). B.U.N, was found t o be p o s i t i v e l y c o r r e l a t e d w i t h S.G.O.T. (0.57). U r i c a c i d was p o s i t i v e l y c o r r e l a t e d w i t h c r e a t i n i n e (0.52) and n e g a t i v e l y c o r r e l a t e d w i t h a l k l a i n e phosphatase (-0.32). C r e a t i n i n e showed a neg a t i v e c o r r e l a t i o n w i t h a l k a l i n e phosphatase (-0.35). G l o b u l i n $^ showed a n e g a t i v e c o r r e l a t i o n w i t h a l k a l i n e phosphatase (-0.23) and as expected a p o s i t i v e c o r r e l a t i o n w i t h the g l o b u l i n 32 (0.40). See Table 24. 2 The amount of v a r i a t i o n accounted f o r (R ) by the s t a t i s t i c a l model (model 10) was i n most of the cases 40% to 50% of v a r i a t i o n i n the blood serum components. Even though v a r i a t i o n accounted f o r by the model f i t t e d i n the blood serum p r o t e i n was 50.30% the s i r e component was n e g a t i v e . I n o r g a n i c phosphorus gave s i m i l a r r e s u l t s . 146 P o s s i b l e reasons f o r these n e g a t i v e e s t i m a t e s of s i r e components i n c l u d e sampling e r r o r , undetected confounding o f s i r e and environment and i n e f f i c i e n t e s t i m a t e s due to d i s p r o p o r t i o n a t e s u b c l a s s numbers. E s p e c i a l l y i n the case of b l o o d serum components, i t can be s a i d t h a t i n a d d i t i o n t o the known e x t r i n s i c f a c t o r s which have been f i t t e d , t h e r e are a l s o i n t r i n s i c causes o f v a r i a t i o n s which c o u l d not be removed under the pr e s e n t study and i s i n c l u d e d i n the r e s i d u a l v a r i a n c e . One of such i n t r i n s i c f a c t o r s i n the b l o o d may be t h a t the l e v e l of a p a r t i c u l a r b l o o d serum component i s governed by the l e v e l o f the o t h e r bood serum components. However, i t might be of i n t e r e s t t o see i f the l e v e l o f some of the components are i n c l u d e d i n the model as c o v a r i a b l e f o r another component and the r e s i d u a l v a r i a n c e i s d e c r e a s e d . The environmental c o r r e l a t i o n was computed from the r e s i d u a l s of the a n a l y s i s of v a r i a n c e and c o v a r i a n c e ; as the c o v a r i a n c e between the two t r a i t s d i v i d e d by the geometric mean of the v a r i a n c e s (model-20). The phenotypic c o r r e l a t i o n i s the product moment c o r r e l a t i o n between the t r a i t s on a w i t h i n s u b c l a s s b a s i s or i t may be c a l c u l a t e d u s i n g the r e l a t i o n s h i p between the g e n e t i c environmental and phenotypic c o r r e l a t i o n s . 147 In the present study the environmental correlations between some blood serum components were negative as the phenotypic correlations/genetic correlations were low in addition to the low h e r i t a b i l i t i e s . VI. ANIMALS WITH MISSING IV PEAK The blood serum samples of twelve animals failed to show the fourth peak in separation of the blood serum proteins by electrophoresis. Two of these animals were also included in the samples which were randomly drawn for duplicate runs and no discrepancy between the two runs was observed. Plate I shows the two samples 92 and 103 with absence of the 4th peak and the corresponding duplicates marked as 9 2-D, and 103-D. The overlapping near the globulin 3 and y region is well marked. Samples No. 10 and No. 165 on Plate I show the normal separation of bands at that region. As mentioned in the appendix the samples were analyzed in groups of eight on a single agarose plate. 14 PLATE I T Sa 2 9 * A SAMPLE NUMBERS 103, 103-D AND 92, 9 2-D WITH MISSING 4TH PEAK WERE ALL PROCESSED ON SEPARATE PLATES. 1. SAMPLE NUMBER 165 WITH SIX PEAKS WAS PROCESSED WITH 10 3-D (WITH MISSING 4TH PEAK) IN THE SAME PLATE. 2. SAMPLE NUMBER 10 WITH ALL SIX PEAKS WAS PROCESSED WITH NUMBER 92 (MISSING 4TH PEAK) 149 Samples 92, 92-D, 103, 103-D were a l l run on d i f f e r e n t p l a t e s . But sample no. 10 was on the same p l a t e as sample 92 and sample 165 was on the same p l a t e as no. 103-D. Samples 10 and 165 have normal s e p a r a t i o n but not samples 92 and 103. T h i s e l i m i n a t e s any p o s s i b l e e r r o r due t o the p l a t e s . The normal appearance o f the s e p a r a t i o n columns and t h e i r d u p l i c a t e s are a l s o shown on P l a t e s I I and I I I , and the d i f f e r e n c e can be e a s i l y d i s c e r n e d . P l a t e No. IV shows the densitometer graph o b t a i n e d f o r sample 92. T h i s was the t y p i c a l graph p a t t e r n o b t a i n e d f o r a l l the 12 samples without a f o u r t h peak. A normal s e p a r a t i o n o f the s i x peaks i s g i v e n on p l a t e no. V f o r comparative purposes. In view of the above i t was d e c i d e d to compare these 12 samples w i t h 12 samples drawn randomly from the group which have a l l t h e i r 6 peaks d i f f e r e n t i a t e d . P l a t e VI shows a few w i t h m i s s i n g IV peak and a few o f the randomly drawn samples from the normal group. The t r a n s -f e r r i n and y g l o b u l i n zone were separated from the o t h e r p a r t s o f p r o t e i n and compared between the two groups (normal and abnormal) i n a d d i t i o n t o the blood components. The method used f o r s e p a r a t i o n as mentioned e a r l i e r , was by r i v o n a l l a c t a t e . (see the a p p e n d i x ) . P l a t e No. V I I shows 150 PLATE I I r 1 I S ! ! ^ ^ ^ %u ELECTROPHORETICALLY SEPARATED SERUM PROTEIN COLUMNS AND THEIR DUPLICATES. PLATE I I I TYPICAL SERUM PROTEIN COLUMNS SEPARATED BY ELECTROPHORESIS ON THIN AGAROSE GEL PLATE. 152 PLATE IV DENSITOMETER TRACING OF SAMPLE NUMBER 92 WITH MISSING 4TH PEAK. THE ARROW SHOWS THE REGION OF 4TH PEAK. 153 PLATE V | i : s : £/? K J THE TYPICAL DENSITOMETER TRACING OF BLOOD SERUM PROTEIN COLUMNS FOR HOLSTEIN SEPARATED BY ELECTROPHORESIS ON THIN PLATE AGAROSE GEL TECHNIQUE IN THE PRESENT STUDY. 154 PLATE VI r ^ • c B • H I | 6 11 o SAMPLE NUMBERS 200, 46, 28 ARE SAMPLES WITH ILL-DEFINED 4TH COLUMN (MISSING 4TH PEAK) SAMPLE NUMBERS 94, 54 and 1 WITH PROPERLY DEFINED SIX COLUMNS (WITH ALL SIX PEAKS) 155 the two groups a f t e r e l e c t r o p h o r e s i s . Group I r e p r e s e n t s the samples which had m i s s i n g f o u r t h peaks and Group I I r e p r e s e n t s the t h r e e randomly drawn samples from the 6 peaks group. Marked d i f f e r e n c e s w i t h r e g a r d t o the t r a n s f e r r i n were n o t i c e d . P l a t e No. V I I I -shows the densitometer graph of t r a n s f e r r i n and y g l o b u l i n . Graph I I I i s f o r sample 92 which was m i s s i n g the 4th peak, and Graphs I and I I show normal samples. A ' t ' t e s t was run to t e s t the d i f f e r e n c e between the mean o f these two groups w i t h r e g a r d t o t r a n s f e r r i n , Y g l o b u l i n , t r a n s f e r r i n and Y g l o b u l i n r a t i o w i t h a l l o t h e r b l o o d components. The v a l u e s are t a b u l a t e d i n t a b l e 25. The mean d i f f e r e n c e o f t r a n s f e r r i n , y g l o b u l i n and t h e i r r a t i o was observed to be s t a t i s t i c a l l y s i g n i f i c a n t but a l l o t h e r s were n o n - s i g n i f i c a n t . When the a c t u a l c o r r e c t e d l a c t a t i o n y i e l d o f these animals were compared i n the h e r d , n e a r l y a l l o f them were above the herd average. See Table 26. A c t u a l l y sample no. 3 f o r cow no. 60110; sample no. 58, cow no. 66117; and sample no. 86, cow no. 67114 produced the h i g h e s t amount of p r o t e i n (more than 850 l b s . i n one l a c t a t i o n ) i n the PLATE VII / " \ r 9. U 4 I • ) B 8 B RIVANOL - LACTATE TREATED SAMPLES. I . SAMPLES 3, 92, 28 HAVING MISSING 4TH PEAK IN ORIGINAL SEPARATION I I . SAMPLES 37, 15, 269 HAVING ALL SIX PEAKS DEFINED FOR ORIGINAL SEPARATION. 157 PLATE V I I I T 1 I — — - • ZOr -! | -20T— ' A\ _0-l . j / \ 7 l O - i -V-/ 1 % • - • 1 DENSITOMETER TRACING AFTER SEPARATION OF TRANSFERRIN AND y -GLOBULIN FROM OTHER SERUM PROTEINS. GRAPHS I AND I I FOR SAMPLES WITH WELL DEFINED SIX PROTEIN COLUMNS AND GRAPH I I I SAMPLE NUMBER 92 WITH ILL-DEFINED 4TH COLUMN IN ORIGINAL SAMPLE BEFORE SAMPLES WERE TREATED FOR SEPARATION OF TRANSFERRIN AND y- GLOBULIN 158 •TABLE NO 25: MEAN + STANDARD DEVIATION (S.D.) FOR THE MISSING 4TH PEAK •GROUP AND GROUP WITH NORMAL PEAKS Group - 1 Group - 2 vComponents Animals w i t h Animals w i t h M i s s i n g Peak a l l the Peaks n = 12 n = 12 Mean S • D. Mean S .D. - T r a n s f e r r i n •0 .3822+ 0 .107* 0 .4903+ 0 .115 .Y - G l o b u l i n 0 .6178+ 0 .107* 0 .5097+ 0 .115 T r a n s f e r r i n Y - G l o b u l i n R a t i o 0 .6677+ 0 .311* 1 .057 + 0 .470 T o t a l P r o t e i n 8 .1833+ 0 .804 7 .7333+ 0 .761 Albumin 2 .1167+ 0 .153 2 .1000+ 0 .204 G l o b u l i n 6 .0667+ 0 .789 5 .633 + 6 .778 Calcium 9 .9000+ 0 .497 10 .025 + 0 .538 In o r g a n i c Phosphorus 5 .3333+ 0 .942 5 .583 + 1 .373 C h o l e s t e r o l 206 .583 +51 .685 195 .417 +71 .616 B.U.N. 18 .000 + 6 .410 15 .083 + 5 .838 U r i c A c i d 0 .9417+ 0 .202 0 .8667+ 0 .231 C r e a t i n i n e 0 .9083+ 0 .131 1 .0417+ 0 .211 A l k a l i n e Phosphatase 48 .250 +40 .645 67 .583 + 44 .057 S.G.O.T. 87 .583 +26 .325 83 .333 + 20 .259 The p a i r o f means f o r the groups s i g n i f i c a n t l y d i f f e r e n t a t *p' <: 0.05 on ' t ' d i s t r i b u t i o n . 159 h e r d . Only sample no. 100, cow no. 67144 was below average. There i s a s i g n i f i c a n t d i f f e r e n c e between these two p r o t e i n s and t h e i r r a t i o between the two groups although a p p a r e n t l y a l l o t h e r b l o o d c o n s t i t u e n t s are g e n e r a l l y the same. Group I (with m i s s i n g f o u r t h peak) a c t u a l l y showed l e s s t r a n s f e r r i n and h i g h e r y g l o b u l i n and low t r a n s f e r r i n to y g l o b u l i n r a t i o i n comparison to group 2 (with a l l 6 p e a k s ) . A d d i t i o n a l l y group 1 turned out to have h i g h e r m i l k l a c t a t i o n p r o d u c t i o n . (see t a b l e 2 6 ) . Ashton (1958) r e p o r t e d the l a c k of slow - a p r o t e i n s i n two out o f 2,500 Guernsey c a t t l e serum examined. He a l s o observed one o f those two (the o t h e r was y e t to s t a r t p r o d u c t i o n ) which had been i n p r o d u c t i o n f o r the l a s t two years had the b e s t i n d i v i d u a l m i l k y i e l d r e c o r d i n the m i l k - r e c o r d i n g Guernsey herds i n S t a f f o r d s h i r e . He used s t a r c h g e l f o r e l e c t r o p h o r e s i s . In the p r e s e n t case agarose g e l was u sed. In s t a r c h g e l the y g l o b u l i n m i g r a t e s c o m p l e t e l y o p p o s i t e to o t h e r p r o t e i n s from the p l a c e of a p p l i c a t i o n ( w e l l ) . But i n case of a g a r o s e , the y g l o b u l i n TABLE 26. PRODUCTION DETAIL OF THE 12 ANIMALS WITH MISSING 4TH PEAK MILK FAT PROTEIN Cow Number Number of Records Mean Y i e l d Deviation from Herd Mean Mean Fat Deviation % Mean Protein Deviation % 60110 3 20,505 +4,284 731 +182 3 .56 690 +160 3.37 61129 4 18,966 +2,745 624 + 75 3 .29 616 + 86 3.25 65128 3 17,328 +1,107 647 + 98 3 .73 573 + 43 3.30 65132 4 17,819 +1,598 629 + 80 3 .53 555 + 25 3.11 65133 4 17,053 +0,832 559 + 10 3 .28 556 + 26 3.26 66117 3 21,708 +5,487 683 +134 3 .15 703 +173 3.2 4 67114 2 21,992 +5,771 666 +117 3 .03 710 +180 3.23 67122 2 18 ,000 +1,779 526 - 23 2 .92 570 + 40 3 .17 67144 3 14,809 -1,412 461 - 88 3 .11 486 - 44 3 .28 67147 2 16,228 +0,007 662 +113 4 .08 563 + 33 3.47 69102 1 17,338 +1,117 608 + 59 3 .51 576 + 46 3.3 2. 70112 Not i n -Production Herd Mean on ' 21st Feb.1972 on 119 animals ad adjusted 16,221 549 3.39 530 3.26 161 migrates to both s i d e s of the p o i n t of a p p l i c a t i o n ( p o s i t i v e as w e l l as n e g a t i v e s i d e s ) . T h i s can be observed i n p l a t e V I I I where no p r o t e i n s o t h e r than t r a n s f e r r i n and g l o b u l i n are p r e s e n t . The d e p r e s s i o n on the agarose p l a t e s where the samples were a p p l i e d appears w e l l above on the curve f o r the y g l o b u l i n i n a l l the t h r e e samples. On s t a r c h g e l the slow a p r o t e i n (which i s r e f e r r e d t o as g l o b u l i n i n the p r e s e n t s t u d y ) , mentioned by Ashton, 1958a, appears j u s t above the w e l l below the t r a n s f e r r i n zone. In agarose p l a t e s , m i grates below the band s i m i l a r t o paper e l e c t r o p h o r e s i s . Due to the d i f f e r e n c e s observed i n these 12 a n i m a l s , i t was d e c i d e d to t r a c e the p e d i g r e e o f these animals and i t was observed t h a t a l l o f the animals r e l a t e back to a s i n g l e H o l s t e i n b u l l . The p e d i g r e e s are g i v e n i n the appendix. 162 SUMMARY AND CONCLUSION The animals used i n t h i s experiment i n c l u d e d 226 H o l s t e i n and H o l s t e i n - A y r s h i r e c r o s s bred h e i f e r s and cows. The animals were c a t e g o r i z e d a c c o r d i n g to the p e r c e n t H o l s t e i n and were separated i n t o H o l s t e i n , 75 -62% H o l s t e i n and 50 - 25% H o l s t e i n . The herd was under a normal management system s i m i l a r to t h a t of a commercial d a i r y farm. The b l o o d t i s s u e i s the major v e h i c u l a r system i n the animal body and i t i s expected t h a t feed absorbed by the animal and/or the m i l k s y n t h e s i z e d by the l a c t a t i n g animal are probably sources of v a r i a t i o n s a f f e c t i n g the l e v e l o f the b l o o d serum components. For t h i s reason the time l a p s e i n minutes between the f e e d i n g or f e e d i n g and m i l k i n g o f the animal and blood sampling was r e c o r d e d to study these e f f e c t s . Each blood serum component was r e g r e s s e d on the time l a p s e between f e e d i n g or f e e d i n g and m i l k i n g w i t h i n the groups of animals under s i m i l a r management s i t u a t i o n s . 163 The time l a p s e between f e e d i n g or f e e d i n g and m i l k i n g , and sampling was not found to be a generally•• important source of v a r i a t i o n i n the p r e s e n t s t u d y . The apparent absence of a c o n s t a n t time l a p s e e f f e c t was l i k e l y due, i n l a r g e measure, to the l e n g t h o f time between f e e d i n g and sample c o l l e c t i o n . The s h o r t e s t mean la p s e d time f o r a group was 2.56 h o u r s . Sources of v a r i a t i o n s such as pregnancy, days l a c t a t i n g , e t c . , were unique f o r p a r t i c u l a r groups o f a n i m a l s . These d i f f e r e n c e s i n p h y s i o l o g i c a l s t a t e were used to s u b d i v i d e the animals i n t o f i v e groups f o r a n a l y t i c a l p u rposes. The groups were, (1) young female a n i m a l s , (2) bred h e i f e r s , (3) l a c t a t i n g non pregnant a n i m a l s , (4) l a c t a t i n g pregnant animals and (5) dry a n i m a l s . They were g e n e r a l l y r e f e r r e d t o as p h y s i o l o g i c a l groups i n the p r e s e n t s t u d y . The sources o f v a r i a t i o n s measured w i t h i n each p h y s i o l o g i c a l group were, age i n young animals; days pregnant and age i n bred h e i f e r and dry animals; days l a c t a t i n g , l a c t a t i o n t r a i t s (kg. o f m i l k , m i l k f a t , m i l k p r o t e i n and l a c t o s e produced b e f o r e sampling) and age i n the l a c t a t i n g non pregnant animals and days pregnant, days l a c t a t i n g and l a c t a t i o n t r a i t s i n a d d i t i o n t o age i n the l a c t a t i n g pregnant a n i m a l s . The e f f e c t s o f these 164 sources w i t h i n each group on each b l o o d serum component was analyzed u s i n g l i n e a r r e g r e s s i o n t e c h n i q u e s . A simple l i n e a r r e g r e s s i o n model wi t h age as a source of v a r i a t i o n was f i t t e d i n the young animal group. M u l t i p l e r e g r e s s i o n models w i t h sources o f v a r i a t i o n s mentioned p r e v i o u s l y were f i t t e d i n the othe r f o u r p h y s i o l o g i c a l groups. Age was found t o account f o r a s i g n i f i c a n t amount of v a r i a t i o n i n the young animal group f o r a l l o f the bl o o d serum components except B.U.N., albumin separated by e l e c t r o p h o r e s i s , g l o b u l i n and g l o b u l i n y. In the ot h e r p h y s i o l o g i c a l groups, age, independent o f o t h e r sources f i t t e d , accounted f o r the major p o r t i o n o f the v a r i a t i o n i n most o f the blood serum components. The number of days pregnant i n the pregnant animals (bred h e i f e r , l a c t a t i n g pregnant and dry animals) i n g e n e r a l a f f e c t e d the v a r i o u s types o f serum p r o t e i n s , B.U.N. and a l k a l i n e phosphatase. The number of days l a c t a t i n g was found to be important o n l y i n the l a c t a t i n g non pregnant group. Days l a c t a t i n g a f t e r f i t t i n g a l l o t h e r sources accounted f o r s i g n i f i c a n t amount o f v a r i a t i o n i n blood serum c h o l e s t e r o l , B.U.N., g l o b u l i n a, and g l o b u l i n 8 0 . None o f the blood serum 165 components were a f f e c t e d by days l a c t a t i n g i n the l a c t a t i n g pregnant group. The l a c t a t i o n t r a i t s , Kg. m i l k , Kg. m i l k f a t , Kg. m i l k p r o t e i n and Kg. l a c t o s e produced j u s t p r i o r to t a k i n g the blo o d samples, o n l y a f f e c t e d the blood serum c h o l e s t e r o l , u r i c a c i d , g l o b u l i n ot^ and g l o b u l i n 3^  l e v e l s . The product moment c o r r e l a t i o n s between the sources f i t t e d , between the sources and b l o o d serum components and between the bl o o d components w i t h i n each p h y s i o l o g i c a l group were c a l c u l a t e d to est i m a t e the degree t o which these v a r i a b l e s vary t o g e t h e r and a l s o to compare the prese n t d a t a w i t h the s i m i l a r c o r r e l a t i o n s r e p o r t e d i n the l i t e r a t u r e . The mean, standard d e v i a t i o n and the c o r r e l a t i o n c o e f f i c i e n t s o f the b l o o d serum components i n the p r e s e n t study are t a b u l a t e d and were g e n e r a l l y i n agreement w i t h o t h e r workers. (Altman e t a l . , 1961; D i m p o u l l o s , 1963; Simesen, 1963; Roubicek, e t a l . , 1972). To e s t i m a t e the sources o f v a r i a t i o n s common to a l l the p h y s i o l o g i c a l groups, the dat a was a d j u s t e d f o r s i g n i f i c a n t continuous v a r i a b l e s unique to each group and each blood serum component. 166 The e f f e c t o f the p h y s i o l o g i c a l groups and breed groups on each b l o o d serum component was a n a l y s e d u s i n g l e a s t squares technique f o r unequal numbers (Harvey 1960) assuming a f i x e d model; f i t t i n g both the p h y s i o l o g i c a l and breed groups as f i x e d e f f e c t s and age as a c o v a r i a b l e . P h y s i o l o g i c a l groups i n the model accounted f o r the major p o r t i o n o f the v a r i a b i l i t y f o r most o f the b l o o d serum components and age was g e n e r a l l y next i n importance. The breed groups f i t t e d as a percentage o f H o l s t e i n breed forming t h r e e breed groups was s i g n i f i c a n t source o n l y i n the b l o o d pserum u r i c a c i d , g l o b u l i n a2 and g l o b u l i n g^. The breed group means f o r these t h r e e b l o o d serum components were s t u d i e d f i t t i n g o r t h o g o n a l c o n t r a s t . The mean l e v e l o f the b l o o d serum components, u r i c a c i d , g l o b u l i n Q%2 and g l o b u l i n g^ were found t o be s i g n i f i c a n t l y lower i n H o l s t e i n group than t h a t o f both the c r o s s b r e d groups. The i n c r e a s e d mean l e v e l w i t h these t h r e e b l o o d serum components was concluded as A y r s h i r e breed e f f e c t w i t h the p r e s e n t s t u d y . D i f f e r e n c e s between the f i v e p h y s i o l o g i c a l groups f o r each b l o o d serum component was t e s t e d u s i n g Duncan's new m u l t i p l e range t e s t as shown by Kramar (1957). The s i g n i f i c a n t d i f f e r e n c e s between the p h y s i o l o g i c a l groups were e x p l a i n e d , i n p a r t s by the p h y s i o l o g i c a l 167 The blood serum components were ana l y z e d by the SSM 12/60 m u l t i - c h a n n e l s e q u e n t i a l a n a l y z e r . The p r o t e i n c o n s t i t u e n t s of the serum were a l s o separated i n t o albumin and g l o b u l i n f r a c t i o n s a^, a^r B2 an& Y < by e l e c t r o p h o r e s i s on agarose t h i n g e l p l a t e s . While s e p a r a t i n g the serum p r o t e i n s by e l e c t r o p h o r e s i s , twelve samples behaved d i f f e r e n t l y from the o t h e r s as the g l o b u l i n 0^ and B2 f a i l e d to s e p a r a t e . The t r a n s f e r r i n s ( g l o b u l i n B-^  and B2) and y - g l o b u l i n of these animals were compared w i t h those of twelve o t h e r a n i m a l s , w i t h normal serum p r o t e i n s s e p a r a t i o n . A marked d i f f e r e n c e between the two groups was observed w i t h r e g a r d to t r a n s f e r r i n , y - g l o b u l i n , and t r a n s f e r r i n and y - g l o b u l i n r a t i o . The twelve animals w i t h improper d i f f e r e n t i a t i o n o f g l o b u l i n 0^ and 02 were observed to be i n good h e a l t h and were among the h i g h e r p r o d u c i n g animals i n the h e r d . The p e d i g r e e of these twelve animals were t r a c e d back to a s i n g l e H o l s t e i n b u l l . These r e s u l t s may l e a d to new areas o f r e s e a r c h not covered i n depth by the p r e s e n t s t u d y . The g e n e t i c parameters were c a l c u l a t e d from a d j u s t e d data on 158 daughters of 15 H o l s t e i n s i r e s . The mixed model used to e s t i m a t e environmental and a d d i t i v e 168 g e n e t i c p o r t i o n s of v a r i a t i o n s i n each b l o o d serum component c o n s i s t e d of p h y s i o l o g i c a l groups, s i r e s , and age c u r v i l i n e a r as a c o v a r i a b l e . As expected groups accounted f o r the major p o r t i o n o f the v a r i a t i o n i n most of the b l o o d serum components. Age as a l i n e a r f u n c t i o n a c r o s s groups accounted f o r a s i g n i f i c a n t amount of v a r i a t i o n i n a l l of the b l o o d serum components. The q u a d r a t i c e f f e c t of age i n the pr e s e n t study was not impo r t a n t . H e r i t a b i l i t y e s t i m a t e s were c a l c u l a t e d from the p a t e r n a l h a l f - s i b c o r r e l a t i o n s . The g e n e t i c , phenotypic and environmental c o r r e l a t i o n s were computed u s i n g the v a r i a n c e - c o v a r i a n c e method. The h e r i t a b i l i t y e stimates f o r the b l o o d serum components i n the p r e s e n t study were; c r e a t i n i n e 0.77, S.G.O.T. 0.46, B.U.N. 0.28, g l o b u l i n &1 0.12, a l k a l i n e phosphatase 0.11, u r i c a c i d 0.10, albumin 0.07, g l o b u l i n 62 0.06, g l o b u l i n 0.04 and c h o l e s t e r o l 0.02. The s i r e components f o r b l o o d serum t o t a l p r o t e i n , c a l c i u m , i n o r g a n i c phosphorus, albumin s e p a r a t e d by e l e c t r o p h o r e s i s , g l o b u l i n f r a c t i o n s a^, a^r and y was found to be n e g a t i v e probably due to sampling e r r o r and as such t h e i r g e n e t i c parameters c o u l d not be e s t i m a t e d . 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Analysis of the y i e l d of milk, milk components and energy i n a Holstein, Ayrshire and Holstein-Ayrshire cross dairy c a t t l e population. M. Sc. Di s s e r t a t i o n . University of B r i t i s h Columbia. 176 A P P E N D I X 177 CONTENTS IN APPENDIX Page BOX I BLOOD COMPONENTS AFFECTED BY INDEPENDENT VARIABLES FITTED WITHIN EACH GROUP OF ANIMALS, ARRANGED IN INCREASING NUMBERS OF EFFECTS FOR 158 DAUGHTERS OF 15 HOLSTEIN SIRES 180 BOX I I METHOD USED FOR CORRECTING EACH BLOOD COMPONENT FOR EACH SAMPLE WITH COEFFICIENTS AND THE DEVIATION OF EACH INDEPENDENT VARIABLE FROM ITS GROUP MEAN, FOR THE 158 DAUGHTERS OF 15 HOLSTEIN SIRES . 181 TABLE NO. 27. MEAN AND STANDARD DEVIATION (S.D.) OF THE FINAL GROUPS AND THE COMPLETE SAMPLE. (UNADJUSTED DATA) 182 THE FREQUENCY DISTRIBUTION OF RAW DATA FOR BLOOD SERUM COMPONENTS INCLUDING DISTRIBUTION OF BLOOD SERUM COMPONENTS WHICH WERE ADJUSTED WITH THE SIGNIFICANT REGRESSION COEFFICIENTS WITHIN GROUPS. 183 PEDIGREE OF 12 ANIMALS WITH MISSING 4TH PEAK DUE TO IMPROPER SEPARATION OF PROTEIN AT TRANSFERRIN Y-GLOBULIN ZONE IN THIN PLATE AGAROSE GEL ELECTROPHORESIS 211 THE SCHEMATIC DIAGRAM PRESENTING THE ULTIMATE ANALYSIS OF THE 226 BLOOD SERUM SAMPLES COLLECTED FOR THE PRESENT STUDY 212 THE SSM 12/60 FOR THE ANALYSIS OF BLOOD COMPONENTS 213 178 Page THE TEST OF REPRODUCIBILITY OF THE SSM 12/60 MACHINE USED FOR THE PRESENT STUDY 219 TABLE NO. 28 THE ANALYSIS OF HUMAN BLOOD SERUM DATA TO TEST THE REPRODUCIBILITY OF THE SSM 12/60 MACHINE WHICH WAS USED TO ANALYZE THE BOVINE BLOOD SERUM COMPONENTS IN THE PRESENT STUDY 220 THE ELECTROPHORESIS OF SERUM PROTEINS 221 PREPARATION OF SAMPLES FOR ELECTROPHORESIS 225 TABLE NO. 29 COMPARISON OF THE SAMPLE MEANS OF ELECTROPHORETICALLY SEPARATED SERUM PROTEINS AND THE DUPLICATES" FOR EACH DAY OF ELECTROPHORESIS 227 COMPARISON OF BLOOD SERUM ALBUMIN OBTAINED BY SSM 12/60 AND ELECTROPHORESIS 228 ANALYSIS OF MISSING 4TH PEAK ANIMALS 230 179 Page SEPARATION OF SERUM TRANSFERRIN 232 180 BOX NO. I BLOOD COMPONENTS AFFECTED BY INDEPENDENT VARIABLES FITTED WITHIN EACH GROUP OF ANIMALS, ARRANGED IN INCREASING NUMBER OF EFFECTS FOR 158 DAUGHTERS OF 15 HOLSTEIN SIRES. Blood Components Eff e c t e d by 1. E. Albumin Days pregnant (P.L.) 2. Globulin - 82 Days l a c t a t i n g (L.O.) 3. Globulin - y Milk (L.O.) 4. Al k a l i n e Phosphatase Days pregnant (B.H.) 5. Globulin Days pregnant (D.) + Days pregnant (P.L.) 6. B.U.N. Days pregnant (D.) + Days l a c t a t i n g (L.O.) 7. Uric Acid Protein (L.O.) + Protein (P.L.) 8. Globulin ai Days pregnant (P.L.) + Days l a c t a t i n g (L.O.) 9 . Globulin 8 1 Days pregnant (P.L.) + Milk (L.O.) 10. Inorganic Phosphorus Days pregnant (P.L.) + Milk (L.O.) + Fat (L.O.) 11. Cholesterol Days l a c t a t i n g (L.O.) + Milk (P.L.) + Lactose (P.L.) 12. Creatinine Days l a c t a t i n g (P.L.) + Milk (P.L.) + Fat (P.L.) 13. Globulin 0 2 Days pregnant (P.L.) + Milk (L.O.) + Protein (L.O.) 2 P.L. = Pregnant and l a c t a t i n g group. 3 L.O. = Lactating non-pregnant group. 4 B.H. = Pregnant Heifer group. 5 D. = Dry pregnant group. BOX NO. II METHOD USED FOR CORRECTING EACH BLOOD COMPONENT FOR EACH SAMPLE WITH COEFFICIENTS AND THE DEVIATION OF EACH INDEPENDENT VARIABLE FROM ITS GROUP MEAN, FOR THE 158 DAUGHTERS OF 15 HOLSTEIN SIRES I PREGNANT HEIFERS. A l k a l i n e Phosphatase - A l k a l i n e Phosphatase-(-0.2136*pays Pregnant)-132.69)) II LACTATING NON-PREGNANT. ANIMALS Inorganic Phosphorus - Inorganic Phosphorus-Ch o l e s t e r o l Blood Urea Nitrogen U r i c A c i d G l o b u l i n a i Glob u l i n a: Gl o b u l i n Si Glob u l i n B2 G l o b u l i n y Cholesterol Blood Urea Nitrogen U r i c Acid G l o b u l i n exi Glob u l i n aj Globulin Si Globulin 6j Globulin Y •((-0.0609*(K.Milk-16.1059))+(3.2654* (Fat-0.370612)) ) (0.5593* ((Days Lactating)-75.4082)) (0 .0224* ((Days Lactating)-75.4082) ) (0.3386*(Protein-0.553673)) (0.0000779*((Days Lactating)-75.4082)) ( (-0.0002353*(K.Milk-16.1059)) +(0.0512*(Protein-0.553673)) (-0.001227*(K.Milk-16.1059)) (0.00006808* ((Days Lactating)-75.4082)) •(0.0013 39*(K.Milk-16.1059)) III LACTATING AND PREGNANT ANIMALS Glob u l i n Inorganic Phosphorus C h o l e s t e r o l U r i c A c i d C r e a t i n i n e G l o b u l i n Inorganic Phosphorus Cho l e s t e r o l U r i c Acid C r e a t i n i n e E.Albumin Globulin G l o b u l i n , Glob u l i n 8i E.Albumin Globulin Globulin Globulin di Si IV DRY AND PREGNANT ANIMALS Glob u l i n = Blood Urea Nitrogen = Globulin Blood Urea Nitrogen -(0.004 434* ((Days Pregnant)-99 .1395)) •(0.0993*(K.Milk-11.8090)) -((37.7525*(K.Milk-11.8090)) +(-563.6670* (Lactose-0.57186)) -(0.7363*(Protein-0.411628)) -( 0) .0009043* ((Days Lactating)-226.14)) + (0.0260*(K.Milk-11.809)) + (-0.4767* (Fat-0.333721)))) •(0.00C2859*((Days Pregnant)-99.1395) ) -(-0.0001464*((Days Pregnant)-99.1395)) -(-0.0001129*((Days Pregnant)-99.1395)) •(-0.00006154*((Days Pregnant)-99.1395)) •(0.006251*(Days Pregnant)-222.08)) •(0.06634*(Days Pregnant)-222.08)) Explanation: 1 . Days La c t a t i n g s 2 . Days Pregnant = 3. K.Milk O 4. Fat ta 5 . P r o t e i n rz 6 . Lactose -7. E. 8. * Number of days i n l a c t a t i o n (Day bled-Day the l a s t c a l f born). Number of days confirmed pregnant (Day bled-Day bred l a s t which has been confirmed f o r pregnancy) Kilogram of milk produced i n the milking before taking blood sample. K i l o of f a t i n the above milk K i l o of pro t e i n i n the above milk K i l o of lactose i n the above milk Electrophoresis M u l t i p l i c a t i o n Table No. 29 Mean and Standard Deviations (S.D.) of the Final Groups and the Complete Sample. (Unadjusted Data) Pregnant Pregnant Blood Young Animals Bred Heifers N o n Rotating lactating Animals For the Sample r__ r >_, r. 0_x. e Animals Animals J ~ usrnponercs ^ = 5 1 ) ( N = 3 9 ) ^ = 5 4 ) ( N = g 3 ) ^ = 1 4 ) ( N = 2 n ) Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D. 'ibtal Protein 6.5999 0.417 7.297 0.652 7.814 Albumin 1.953 0.183 2.264 0.205 3.079 _Globulin 4.647 0.333 5.033 0.587 5.736 "Calcium 9.672 0.662 10.413 0.679 9.729 Inorganic Phosphorus 7.241 1.173 5.482 0.815 5.198 Cholesterol 143.451 55.429 175.769 34.911 226.696 B.U.N. 12.392 2.808 11.744 4.024 18.607 Uric Acid 0.841 0.213. 0.815 0.218 1.049 Creatinine 0.914 0.108 1.105 0.138 0.837 Alkaline Phosphatase 118.235 34.985 94.436 30.596 46.089 S.G.O.T. 84.392 18.874 84.974 9.221 92.768 E . Albumin 0.316 0.0453 0.311 0.0497 0.291 Globulin 0.126 0.0191 0.122 0.016 0.124 Globulin 0.118 0.010 0.123 0.0118 0.121 Globulin 0.155 0.013 0.153 0.015 0.140 Globulin 0.160 0.013 0.165 0.013 0.169 Globulin 0.124 0.022 0.126 0.0235 0.156 0.669 7.498 1.249 7.939 0.675 7.361 0.931 0.216 2.179 0.232 2.147 0.130 2.112 0.231 0.685 5.362 1.065 5.793 0.661 5.261 0.834 0.745 9.855 0.639 9.979 0.371 9.889 0.711 0.969 5.223 0.702 5.827 1.904 5.787 1.318 65.438 245.849 53.574 161.067 37.011 198.911 66.294 4.438 21.075 3.031 14.800 5.634 16.221 5.370 0.250 0.942 0.252 0.807 0.109 0.914 0.246 0.109 0.-58 0.103 1.173 0.194 0.933 0.166 24.449 49.038 27.990 49.13 35.169 73.037 42,763 22.698 89.377 24.162 76.200 9.748 .87.351 20.030 0.035 0.294 0.040 0.290 0.053 0.301 0.044 0.013 0.123 0.014 0.104 0.153 0.122 0.016 0.011 0.121 0.010 0.119 0.010 0.120 0.108 0.011 0.142 0.009 0.144 0.013 0.147 0.013 0.011 0.173 0.0121 0.176 0.019 0.168 0.013 0.021 0.146 0.021 0.167 0.019 0.141 0.026 a = gm./lOO ml blood b = mg./lOO ml blood c = milimicron ./100 ml blood d = Part of the sum of the six peak heights. THE FREQUENCY DISTRIBUTION OF RAW DATA FOR BLOOD SERUM COMPONENTS INCLUDING DISTRIBUTION OF BLOOD SERUM COMPONENTS WHICH WERE ALSO ADJUSTED WITH THE SIGNIFICANT REGRESSION COEFFICIENTS WITHIN GROUPS. Explanation of abbreviations used i n the figures 1. FRQ. = Frequency 2. G. = Phy s i o l o g i c a l groups. (1) The number of observations and mean (S.D.) for the raw data are tabulated i n l a s t column of table No. 27. (2) The c o e f f i c i e n t s used to adjust the raw data and the group means (S.D.) of the sources of v a r i a t i o n are given i n table No. 15. 184 DflTfl SET 1 FRO.DISTRIBUTION FOR PROTEIN (UNRDJUSTED DRTfl). 185 DflTfl SET 2 FRQ.DISTRIBUTION FOR ALBUMIN (UNADJUSTED DflTfl). 186 OPJR SET 3 FRQ DISTRIBUTION FOR GLOBULIN (UNADJUSTED DRTR) 187 DRTfl SET 4 FRQ DISTRIBUTION FOR CRLCIUM (UNADJUSTED DRTR) 188 DflTR SET 5 FRO DISTRIBUTION FOR IN-PHOSPHORUS (UNADJUSTED DflTR) 189 190 191 192 DflTR SET 7 FRO DISTRIBUTION FOR B.U.N. (UNADJUSTED DflTR) 193 DRTR SET 7 FRQ.DISTRIBUTION OF B.U.N. (RDJUSTED DRTR WITHIN G.) 194 DflTR SET 8 FRQ DISTRIBUTION FOR URIC-RCID (UNADJUSTED DflTR) 195 196 ORTR SET 9 FRO.DISTRIBUTION OF CREATININE • (ADJUSTED DATA WITHIN G} 197 DRTR SET 10 FRO DISTRIBUTION FOR RL.PHOSPHRTRSE (UNROJUSTED DRTR) 198 DRTR SET 10 FRQ.DISTRIBUTION OF RLK.PHOSPHATASE (ADJUSTED DATA WITHIN G) 199 200 DRTR SET 1 2 FRO DISTRIBUTION FOR EP.BLBUMIN (UNRDJUSTED DRTR) 201 DflTfl SET 1 2 FRQ.DISTRIBUTION OF ELECT.RLBUMIN (ADJUSTED DRTR WITHIN G) 202 DATA SET 13 FRO DISTRIBUTION FOR ALPHA—1 (UNADJUSTED DATB) 203 DRTR SET 13 FRO.DISTRIBUTION OF GLOBULIN-RLPHR-1 IADJUSTED DATA WITHIN G) 204 DRTR SET 1 4 FRO DISTRIBUTION FOR RLPHR—2 (UNRDJUSTED DRTR) 205 DRTR SET 14 FRQ.DISTRIBUTION OF GLOBULIN-RLPHR-2 (RDJUSTED DRTR WITHIN G.) 206 207 208 DRTR SET 16 FRO DISTRIBUTION FOR BETR--2 (UNADJUSTED DRTR) DRTR SET 16 FRQ.DISTRIBUTION OF GLOBULIN -BETR-2 (ADJUSTED DflTR WITHIN G.) 210 IIGREE 0? 12 ANIMALS WITH MISSING «th PEAK DUE TO IMPROPER SEPARATION OF PROTEIN AT TRANSFERRIN Y - GLOBULIN ZONE IN THIN PLATE AGAROSE GEL ELECTROPHORESIS. Sandra (51129) < Ubyssey Erperor BAB's^-(57111) Ester \ Ubyssey Emperor C o r a ^ — (54122) (58105) O M i l l i e (67122) 7i*ta (oil4.) K - g (67114) Erilen (60110) Fleur (65133) Orchid (69102) !taty ^ Hemlock ^ — (70112) ^ (67149) Hs-da ^ (67147) x Hawthorn^— (57144) Lis* ^ Ubvssey Valeboy «7ane^-(6S117) (64106) -.Tar-ay ^ — • (62124)C  J 9 -Ubyssey Kagic Elsie (60102) O -Gilfp.ore Emperor Hangerveld^-(905) -Medov Fraser Snowball^— (53104). O Orna ^  (63133)^) J -UbyBsey Valboy^-1 (6101) -Ubyssey Valiant Felice (61106) ^ 4 Ubyssey Final Tribute^-. (Fi'L, ^ jT _Agnes Riverdene Magic <e_ (1298779) (j. -Paulholm"Eir.presa^-La Vata Hazel (514774) -Paulholm Emperor (111768) g» -Carnation Inka <£_ Eirp. (682072,^ -Gilmore Or-msby-^-Snowball (229339)--Carnation Skipper^--Ubyscey 3eauty Comet (5801) t C,1,8658, g* lilmore Sis Sharon Ornsby •Carnation Governor Imperial g* (205749) g* 7> Gilmore Beauty » Shcror. (86844)C» -Robridge Beauty s J Vole (1117922) 9 -Gilmore^— Valiant ? (219120)0 Governor of Carnation Carnations-Skylark Kagic (219115)^, V -Carnation Black Magic "mircore Magic Blue Eoy-, (FPL, CT S.B. Only those ancestors are shown i n the pedigree which traces back to a single bull for a l l 12 anteals. THE SCHEMATIC DIAGRAM PRESENTING THE ULTIMATE ANALYSIS OF THE 226 BLOOD SERUM SAMPLES COLLECTED FOR THE PRESENT STUDY: Main Sample: 226 ^ ^ D a i r y C a t t l e : 1. H o l s t e i n 2 . H o l s t e i n - A y r s h i r e c r o s s . 12 with m i s s i n g 4 t h .Peak \/_ 1. Pedigree 2. P r o d u c t i o n 211 Qq Sample used to estimate G e n e t i c and Environmental v a r i a t i o n s A n a l y t i c a l study o f v a r i a t i o n due t o d i f f e r e n t s o u r c e s . 158 Daughters s i r e d by 15 H o l s t e i n s i r e s For g e n e t i c a n a l y s i s Randotti sample of 12. With a l l Peaks,. i 12 P a i r s - t o compare b l o o d serum components by 11 * t e s t . Study o f : 1. H e r i t a b i l i t y 2. Genetic c o r r e l a t i o n s 3. Phenotypic c o r r e l a t i o n s 4. Environmental c o r r e l a t i o n s 213 The SSM 12/60 for the Analysis of Blood Components The p r i n c i p l e of methods followed for the analysis of blood components i n the SMA 12/60 multichannel analyzer was as follows: 1. Total Protein. The method consisted of a modified b i u r e t reaction. Copper i n a l k a l i n e solution, forms a purple complex with the peptide linkage of amino acids i n protein. The protein stream i s mixed with the b i u r e t reagent and the absorbance of the sample a n a l y t i c a l stream i s measured at 550 n m i n a 15 mm l g by 1.5 mm ID. The serum blank i s determined by d i l u t i n g the samples with a l k a l i n e iodide s o l u t i o n and measuring the absorbance of the blank a n a l y t i c a l stream at 550 n m l g by 1.5 mm ID f l o w c e l l . The blank i s automatically subtracted by d i f f e r e n t i a l colorimetry. 2. Albumin. The method was based on the quantitative binding of the anionic dye, 2-(4-hydroxyazo-benzene) benzoic acid (H A B A), s p e c i f i c a l l y -to serum albumin. However, a phosphate buffer PH 6.50 (0.27 MPO 3) was used to increase the 214 H A B A dye concentration. The absorbance of the sample a n a l y t i c a l stream i s measured at 505 n m i n a 15 mm l g by 1.5 mm ID flow c e l l . The serum blank i s determined by d i l u t i n g the sample with phosphate buffer and then measuring the absorbance as mentioned above.(Dean et a l . , (1969). 3. Calcium. The d i l u t e serum sample i s mixed with 0.3 N hydrochloric acid containing 8-hydroxy-quinoline to release protein - bound calcium and combine with the magnesium. This i s dialyzed into the a n a l y t i c a l stream of cresolphathaleen complexone, again containing 7-hydroxyquinoline. A colored complex between calcium and dye i s formed upon the addition of diethylamine. The absorbance of the a n a l y t i c a l stream i s measured at 570 n m i n a 15 mm l g by 1.5 ID flow c e l l . 4. Inorganic Phosphate. Serum i s mixed with and also dialyzed into IY. H 2S0 4. The dialy z a b l e phosphate i s then mixed with an a c i d i c s o l u t i o n of ammonium molybdate with the formation of phosphomolybdic acid and t h i s i s immediately reduced by stannous chloride - hydrazine. The absorbance of the a n a l y t i c a l stream i s measured at 660 n m i n a 15 mm ID flow c e l l . 215 5. Cholesteral. The method i s based on a modification of the Lieberman - Burchard reagent for use i n the d i r e c t determination of serum c h o l e s t e r o l . Diluted serum i s mixed with a stable reagent comprised of g l a c i a l a c e t i c acid, acetic anhydride, and s u l f u r i c e a c i d . The absorbance of the a n a l y t i c a l stream i s measured at 630 n m i n a 15 mm ID flow c e l l . 6. Blood Urea Nitrogen (B.U.N.) Urea i n r e l a t i v e l y weak acid s o l u t i o n , reacts with d i a c e t y l -monoxine. The presence of thiosemicarbazide and f e r r i c ion i n t e n s i f i e s the color of the reaction. The mixture was heated to 90°C f o r color development and the absorbance of the a n a l y t i c a l stream was measured at 520 n m i n a 15 mm l g by 1.5 mm ID flow c e l l . 7. Uric Acid. Serum was d i l u t e d with NACL and then dialyzed against a sodium tungstate - hydroxylamine so l u t i o n . Phosphotungstic acid was added and the absorbance of the color produced i n the a n a l y t i c a l stream was measured at 660 n m i n a 15 mm l g by 1.5 mm ID flow c e l l . 8. Total B i l i r u b i n . Serum b i l i r u b i n was reacted with diazotized s u l p h o n i l i c acid i n the presence of caffine-sodium benzoate reagent to form 216 colored o z o b i l i r u b i n . The absorbance of the a l k a l i n e o z o b i l i r u b i n i n the sample stream i s measured at 600 n m i n a 15 mm l g by 1.5 mm ID flow c e l l . 9. Creatinine. The sample stream segmented with a i r , was d i l u t e d with 1.8 / NaCI. This d i l u t e d stream then entered the doner side of the d i a l y z e r . The a n a l y t i c a l stream consists of water segmented with a i r . A f t e r emerging from d i a l y z e r the a n a l y t i c a l stream was joined with 0.5 N NaOH These two components were mixed and then joined with saturated p i c r i c a c i d . The three components were mixed and phased to the c o l o r i -meter. The absorbance was measured at 505 n m i n a 15 mm l g by 1.5 flow c e l l . 10. A l k a l i n e Phosphatase. The procedure was based on the enzymatic hydrolysis of P-nitrophenyl phosphate during incubation at 37.5-c. Following incubation, the free P-nitrophenyl i s dialyzed into a 2-amino -2-methel-l-propanol (AMP) buffer a n a l y t i c a l stream. The dialyzed P-nitrophenyl i s intensely colored under a l k a l i n e conditions and thereby provides i t s own chromagen. D i a l y s i s eliminates the i n t e r -ference of b i l i r u b i n and the need f o r blank 217 correction. The absorbance i s measured at 410 n m i n a 15 mm l g by 2.0 mm ID flow c e l l . 11. L a c t i c Dehydrogenase. L a c t i c dehydrogenase catatyzes the oxidation of l a c t a t e to pyruvate i n the presence of NAD. The reaction i s as follows. Lactate + NAD LDH Pyruvate + NADH The enzymatic a c t i v i t y i s proportional to the amount of NADH produced. The absorbance of the product produced i s measured at 340 n m i n a 15 mm l g by 2.0 mm ID flow c e l l . 12. Serum Glutamic-Oxalacetic Transaminase. The method consists of two steps. (a) Serum i s d i l u t e d with a substrate and incubated at 37.5°C f o r about 6.1 minutes to produce the reaction as follows: ASPERTIC ACID + ALPHA-KETOGLUTAMIC ACID SGOT OXALACETIC ACID + GLUTAMIC ACID. The concentration of products formed under t h i s reaction within a given period of time i s proportional to the SGOT a c t i v i t y i n the serum. (b) The products dialyze through the d i a l y z e r membrane and the oxalacetate reacts with NADH i n presence of the enzyme malate dehydrogenase 218 (MDH) to form malate. OXALACETE = NADH (absorbent) MALATE +NAD (non-absorbent) The NADH absorbs l i g h t a t 340 n m, NAD does not absorb a t 340 n m. Thus the enzyme a c t i v i t y i s i n v e r s e l y p r o p o r t i o n a l to the change i n the absorbance. T h i s change i n absorbance i s measured a t 340 n m i n a 15 mm 1 g by 2.0 mm ID flow c e l l s . The L a c t i c dehydrogenase i n the machine showed some absnormal v a l u e s and hence was r e j e c t e d and not i n c l u d e d f o r a n a l y s i s . G l o b u l i n . The t o t a l g l o b u l i n i s simply the d i f f e r e n c e o f albumin s u b t r a c t e d from t o t a l p r o t e i n . 219 THE TEST OF REPRODUCIBILITY OF THE SSM 12/60 MACHINE USED FOR THE PRESENT STUDY: In the present case, two analyses done on two d i f f e r e n t days during the month of July 1971 were compared to estimate the range of p r e c i s i o n . The reference data was the data f o r analyzing blood one week before the current data. Both the data were on human blood. See table 27. The c o e f f i c i e n t of v a r i a t i o n shows very l i t t l e change and the machine seemed quite r e l i a b l e . The machine was monitored during the operation a f t e r each 15 samples to check i t s d r i f t and r e p e a t a b i l i t y , to ensure proper control as suggested by other workers. 220 TABLE NO. 28 THE ANALYSIS OF HUMAN BLOOD SERUM DATA TO TEST THE REPRODUCIBILITY OF THE SSM 12/60 MACHINE WHICH WAS USED TO ANALYZE THE BOVINE BLOOD SERUM COMPONENTS UNDER THE PRESENT STUDY* Blood Components DATA I DATA I I Mean ( N ) S . D . C.V.% Mean S.D. C.V.% Alk a l i n e Phosphatase 201 .5 (22)9 .02 4. 48 212 .74(23) 34 .44 16 .19 S.G.O.T.(a) 204 .27 (23)8 .71 4. 27 213 .00(23) 8 .80 4 .13 Cholesterol 156 .91 (23)8 .98 5. 73 154 .71(21) 4 .57 2 .96 B.U.N.(b) 40 ,52(23)0 .67 1. 64 41 .00(21) 0 .00 0 .00 Calcium 12 .09 (23)0 .14 1. 18 12 .10 (23) 0 .22 1 .85 Inorganic Phosphorus 5 .25 (23)0 .06 1. 13 5 .24(23) 0 .08 1 .61 Total Protein 5 ,76 (22)0 .09 1. 57 5 .81(22) 0 .11 1 .91 Albumin 3 .24(20)0 .07 2. 12 3 .33(23) 0 .08 2 .44 B i l i r u b i n 4 .21(23)0 .06 1. 49 4 .28 (23) 0 .15 3 .42 Creatinine 3 .54 (23)0 .10 2. 92 3 .49(24) 0 .04 1 .17 U r i c Acid 9 .19(22)0 .11 1. 21 9 .17.(23) 0 .11 1 .16 * = Data supplied by the Metropolitan Biomedical Laboratory owner of the SSM 12/60 Machine a = Serum glutamic oxaloacetic transminase b = Blood Urea Nitrogen Data I = The recent data Data I I = The data analyzed one week before the recent data. 221 The Electrophoresis of Serum Proteins The cassette system consisted of (a) the c e l l and power supply components, (b) a quantitative m i c r o l i t e r sample dispenser with disposable t i p s , (c) s t i r - s t a i n dishes, (d) Universal electrophoresis f i l m , (e) prepackaged stains and (f) prepackaged b u f f e r . The cassette electrophoresis c e l l was composed of the parts (1) c e l l base and (2) c e l l cover. The c e l l base was attached with the power supply by banana plugs which are joined with the two carbon electrods of the two chambers of the c e l l . The c e l l cover which f i t s around the c e l l has a "U" shaped holder inside to hold the agarose f i l m . Each agarose f i l m consisted of eight wells for eight samples to be analyzed at a time. The prepackaged buffer and stains were prepared as d i r e c t e d by the supplier. The procedure f o r electrophoresis was as follows: 1. Each compartment of the c e l l was f i l l e d with 100 c c . buffer. To ensure equal amounts i n each compartment a f t e r keeping the c e l l on the even surface, one end was 222 t i l t e d so that the f l u i d from each chamber joins above the p a r t i t i o n i n g l i n e and there i s no. flow from one to the other. Any l i q u i d on the separating wall was soaked o f f . Each time fresh buffer s o l u t i o n was used a f t e r washing the c e l l with deionized water and wiping dry with kimwipe. 2. The power cord of the power u n i t was then plugged into the 110 V, 50/60 Hz, wall o u t l e t . The power unit remains attached with the c e l l as described before. 3. The agarose f i l m s t r i p fixed on the p l a s t i c p late container was then separated out of i t s p l a s t i c base and placed evenly on the table with i t s p l a s t i c backing. The agarose layer facing upward. The plates were preferred on a black surface as i t gave better v i s i b i l i t y of the wells on the agarose l a y e r . 4. The m i c r o l i t e r dispenser using fresh disposable t i p s each time was used to dispense the samples i n the wells. The dispenser was adjusted for 0.9 u l . The care was taken so that the t i p s of the disposable piston was pushed, the sample 223 formed a small drop at the end. The well was touched with the drop. The sample was taken i n by surface tension. 5. Each plate contains 8 wells and hence when a l l the eight wells have been completed the f i l m was l i f t e d by its corner with the help of the p l a s t i c backing. The cover was then kept i n p o s i t i o n and the f i l m slipped into the "U" shaped holder. The agarose faces up and the wells are on the l e f t of the operator. 6. The c e l l cover i s then put on the c e l l c a r e f u l l y so that the cover s k i r t edge i s r e s t i n g evenly on the c e l l base and the red l i g h t of the power unit i s l i g h t e d ensuring power supply. The power supply i s f i x e d at 90 v o l t s r e c t i f i e d d i r e c t current. 7. The electrophoresis was done for 35 minutes at each run. 8. At the end of the run the plate was immersed i n the amino black s t a i n i n a s t a i n i n g dish for 15 minutes. 224 9. Then oven dried at 80 C. Care was taken so that the agarose i s thoroughly dried to prevent the loss of agarose during the d i s -tai n i n g procedure. 10. The agarose films were then washed with two changes of 5% a c e t i c a c i d for 2 minutes each and then washed by immersing i n c l e a r deionized water and oven dried at 80°C. 11. The films thus prepared were f i x e d back again on t h e i r o r i g i n a l p l a s t i c protective covers with sample numbers and kept. To ensure proper holding, a latex rubber band was slipped on each pl a t e . 225 Preparation of Samples for Electrophoresis:- The set of samples for electrophoresis were i n a separate container. I t took 4 days to run a l l the samples. The number of samples were adjusted so that the minimum agarose plate space i s wasted and i t i s fe a s i b l e to run a l l the samples on that day. The container containing the freeze dried samples was brought to the top of the r e f r i g e r a t o r and the desired number of samples were quickly drawn at random and the container returned to the p o s i t i o n i n the r e f r i g e r a t o r , each morning. This was done to avoid thawing of any of the samples again and again. The samples f o r the day were then arranged on a t e s t tube rack. Ten blank t e s t tubes were kept ready at hand. From the f i r s t t e s t tube rack, ten samples were drawn at random and placed on a second t e s t tube rack. Then ten blank t e s t tubes were taken and sample numbers of each one of the second rack were entered to keep record of the duplicate samples. A l l the samples and blank test tubes were then put at random on a big t e s t tube rack. Eight t e s t tubes at random were then picked up and bundled together. This was done as the plates have eight wells and only eight samples are to be run each time. In t h i s way the duplicates appeared at random i n the bundles. 226 The mean difference between the o r i g i n a l and duplicates for the f i v e days were tested with * t ' test and as no difference was observed the o r i g i n a l s only were considered f o r the general analysis. See table 29. TABLE 29 COMPARISON OF THE SAMPLE MEANS OF ELECTROPHORETICALLY SEPARATED SERUM PROTEINS AND THE DUPLICATES FOR EACH DAY OF ELETROPHORESIS. SERUM PROTEINS SEPARATED BY ELECTRO-PHORESIS DAY OF ELECTROPHORESIS _I II III IV  Mean of Mean of Mean of Mean of Mean of Mean of Mean of Mean of 10 , the 10 10 O r i - the 10 the 10 the 10 10 the 10 O r i g i n a l Dupli- g i n a l Dupli- O r i g i n a l Dupli- O r i g i n a l Dupli-Samples cates .Samples. gates... Samples cates Samples cates Albumin 0.303 0.303 0.310 0.313 0.313 0.303 0.315 0.295 Globulin 0.122 0.121 0.122 0.122 0.121 0.121 0.125 0.123 Globulin a 2 0.121 0.120 0.122 0.123 0.121 0.120 0.119 0.121 Globulin Bx 0.147 0.135 0.144 0.145 0.143 0.147 0.155 0.142 Globulin 02 0.168 0.161 0.160 0.166 0.168 0.166 0.160 0.163 Globulin Y 0.141 0.144 0.143 0.144 0.144 0.144 0.134 0.143 N.B. The differ e n c e between the p a i r of means f o r each serum protein for each day tested by ' t ' te s t at P <• 0 . 0 5 . A l l p a i r s found n o n - s i g n i f i c a n t . 228 COMPARISON OF BLOOD SERUM ALBUMIN OBTAINED BY SSM 12/60 AND ELECTROPHORESIS:  To t e s t whether i t w i l l be possible to work out each protein f r a c t i o n from the t o t a l protein obtained by SSM 12/60, the albumin part of electrophoresis column was multipled with the SMA 12/60 t o t a l protein and compared with the SMA 12/60 albumin. Their mean and standard deviations for 211 animals were as follows: Mean SSM 12/60 T. protein x E. Albumin 2.2 351 SSM 12/60 Albumin 2.11378 As both the methods are assumed to measure the same component i t was expected that the c o r r e l a t i o n between the two w i l l be high, but the c o r r e l a t i o n c o e f f i c i e n t obtained was 0.2574. Albumin f r a c t i o n of electrophoresis (SSM 12/60 T. protein x E. A-bumin) when regressed on albumin from SSM 12/60 showed a s i g n i f i c a n t equation with constant = 1.421 c o e f f i c i e n t = 0.3853 and R 2 = 0.06 In view of these low c o r r e l a t i o n c o e f f i c i e n t s and 2 R the t o t a l protein, albumin and gl o b u l i n (obtained by subtracting albumin from t o t a l protein) of SSM 12/60 and the protein f r a c t i o n s from electrophoresis were a l l treated separately. S.D. 0.3409 0.2278 229 Similar r e s u l t s while comparing serum proteins obtained by d i f f e r e n t methods were also reported by other workers (Kaplan et a l . , 1937; Booksby, 1947; Malkay et a l . , 1957; Lippman et a l . , 1952; Jager et a l . , 1950; Goodwin et a l . , 1965). 230 Analysis of Missing .4th Peak Animals; To compare and analyze the 12 samples with missing 4th peak, 12 samples at random from those animals which do not have the 4th peak missing were drawn from the t h i r d set of freeze-dried samples. The samples with proper i d e n t i f i c a t i o n marks were treated for separation of t r a n s f e r r i n and y- g l o b u l i n . The methods of electrophoresis, d i g i t i z a t i o n , etc. were done as described before. The two peaks f o r t r a n s f e r r i n and y-globulin was added and the actual peak height divided by t h i s sum. The same method was followed as for the 6 peaks before. T G t t+g t+g Where T = T r a n s f e r r i n G = y-gl o b u l i n t = D i g i t i z e d height of t r a n s f e r r i n peak, and g = D i g i t i z e d height of y- g l o b u l i n peak. The ratio;. R t. G for t r a n s f e r r i n with y-globulin was calculated as: R t : G » t/t+g g/t+g The eleven blood components and these three new factors, t r a n s f e r r i n , Y - g l o b u l i n and Rt:G f o r t n e s e 24 animals, (12 missing peak; 12 samples drawn) were separated out and t h e i r mean for each component was tested with ' t' t e s t . See table 2*". 232 S e p a r a t i o n o f Serum T r a n s f e r r i n ; The R i v a n o l as (2.5 d i a m i n o - 7 - ethoxyaeridine) 1.5% e t h a c r i d i n e l a c t a t e i n water 0.5 cc with serum 0.5 cc were g e n t l y mixed and i n c u b a t e d a t 2 2 c ° f o r two h o u r s . The e t h a c r i d i n e l a c t a t e was then p r e c i p i t a t e d w i t h 5% (W/V) N a c l . The p r e c i p i t a t e was separated out by c e n t r i -f u g i n g a t R.P.M. 3,000 f o r 10 m i n u t e s . The c l e a r s o l u t i o n was then d i a l y z e d a g a i n s t d i s t i l l e d water o v e r - n i g h t to get r i d o f any N a c l l e f t . The water was changed every f o u r h o u r s . The samples then p r o p e r l y numbered were f r e e z e d r i e d , and kept f o r e l e c t r o p h o r e s i s . The same method o f e l e c t r o p h o r e s i s , d e n s i t o meter scanning and d i g i t i z a t i o n as d e s c r i b e d f o r p r o t e i n f r a c t i o n s was f o l l o w e d w i t h the f o l l o w i n g e x c e p t i o n s . 1. The amount added to agarose p l a t e was d o u b l e d . The same d i s p o s a b l e t i p s and m i c r o d i s p e n s o r was used, but each sample was a p p l i e d twice i n each w e l l . The c a r e was taken so t h a t the w e l l s do not o v e r f l o w . 2. The e l e c t r o p h o r e s i s run was i n c r e a s e d t o one h o ur. 3. The d i g i t i z e r program was changed to r e c o r d two peaks o n l y , t r a n s f e r r i n and y - g l o b u l i n . 

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