@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix skos: . vivo:departmentOrSchool "Land and Food Systems, Faculty of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "Taylor, Christopher C."@en ; dcterms:issued "2010-10-07T19:44:43Z"@en, "1990"@en ; vivo:relatedDegree "Master of Science - MSc"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description """Studies to monitor bovine ovarian function with regard to follicular growth and turnover, and corpus luteum (CL) growth and function, were carried out during three different reproductive states: the postpartum anestrus period, early pregnancy and during the artificial control of the estrous cycle with the synthetic progestin norgestomet. Ovarian function was monitored using a combination of ultrasound imaging and progesterone (P₄) profiling. Growth of large antral follicles (> 10mm) was found to commence very early in the postpartum period and ovulation occurred as early as the first week postpartum. Short first postpartum estrous cycles (< 18 days) were observed in a minority of the animals studied (4/10) and the occurance of a short first cycle was not associated with an early ovulation following parturition. Growth of large antral follicles occurred in a wave-like pattern during the postpartum estrous cycles with most cycles being composed of two waves of growth, the second wave resulting in the growth of the ovulatory follicle. A wave-like pattern of growth of large dominant follicles was also seen through the first 60 days of pregnancy. There was no difference between pregnant and non pregnant cows in the size of the dominant follicle found on day 20. In addition no effect of the CL could be found on the side on which the dominant follicle was found, it was as likely to be on the ipsilateral ovary to the CL as on the contra lateral. The gonadotrophin ihibitor norgestomet did not effect follicular dynamics in the presence of the CL, however in the absence of a CL the dominant follicle present was maintained for the duration of the norgestomet treatment and then went on to ovulate upon norgestomet removal. In addition there was no new growth of antral follicles in the absence of a CL. Norgestomet did not effect the temporal relationship between the onset of standing estrus, the LH surge and ovulation. The results of the three studies suggest that a wavelike pattern of growth of large antral follicles is a characteristic of the bovine ovary regardless of the reproductive state."""@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/28994?expand=metadata"@en ; skos:note "OVARIAN ACTIVITY IN POSTPARTUM, EARLY PREGNANT AND NORGESTOMET SYNCHRONIZED DAIRY CATTLE By CHRISTOPHER C. TAYLOR B . S c , The U n i v e r s i t y o f B r i t i s h Columbia, 1987 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF ANIMAL SCIENCE We accept t h i s t h e s i s as conforming t o the standard THE UNIVERSITY OF BRITISH COLUMBIA FEBRUARY, 1990 © C h r i s t o p h e r C. T a y l o r 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 it 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 or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT S t u d i e s t o monitor bovine o v a r i a n f u n c t i o n w i t h r e g a r d to f o l l i c u l a r growth and t u r n o v e r , and corpus luteum (CL) growth and f u n c t i o n , were c a r r i e d out d u r i n g t h r e e d i f f e r e n t r e p r o d u c t i v e s t a t e s : the postpartum a n e s t r u s p e r i o d , e a r l y pregnancy and d u r i n g the a r t i f i c i a l c o n t r o l o f the e s t r o u s c y c l e w i t h the s y n t h e t i c p r o g e s t i n norgestomet. O v a r i a n f u n c t i o n was monitored u s i n g a combination o f u l t r a s o u n d imaging and progesterone (P4) p r o f i l i n g . Growth of l a r g e a n t r a l f o l l i c l e s (> 10mm) was found t o commence v e r y e a r l y i n the postpartum p e r i o d and o v u l a t i o n o c c u r r e d as e a r l y as the f i r s t week postpartum. S h o r t f i r s t postpartum e s t r o u s c y c l e s (< 18 days) were observed i n a m i n o r i t y o f the animals s t u d i e d (4/10) and the occurance of a s h o r t f i r s t c y c l e was not a s s o c i a t e d w i t h an e a r l y o v u l a t i o n f o l l o w i n g p a r t u r i t i o n . Growth of l a r g e a n t r a l f o l l i c l e s o c c u r r e d i n a wave-like p a t t e r n d u r i n g the postpartum e s t r o u s c y c l e s w i t h most c y c l e s b e i n g composed o f two waves of growth, the second wave r e s u l t i n g i n the growth of the o v u l a t o r y f o l l i c l e . A w a v e - l i k e p a t t e r n o f growth of l a r g e dominant f o l -l i c l e s was a l s o seen through the f i r s t 60 days o f pregnancy. There was no d i f f e r e n c e between pregnant and non pregnant cows i n the s i z e of the dominant f o l l i c l e found on day 20. In i i a d d i t i o n no e f f e c t of the CL c o u l d be found on the s i d e on which the dominant f o l l i c l e was found, i t was as l i k e l y t o be on the i p s i l a t e r a l ovary t o the CL as on the c o n t r a l a t e r a l . The gonadotrophin i h i b i t o r norgestomet d i d not e f f e c t f o l l i c u l a r dynamics i n the presence of the CL, however i n the absence of a CL the dominant f o l l i c l e p r e s e n t was maintained f o r the d u r a t i o n of the norgestomet treatment and then went on t o o v u l a t e upon norgestomet removal. In a d d i t i o n t h e r e was no new growth of a n t r a l f o l l i c l e s i n the absence of a CL. Norgestomet d i d not e f f e c t the temporal r e l a t i o n s h i p between the onset of s t a n d i n g e s t r u s , the LH surge and o v u l a t i o n . The r e s u l t s of the t h r e e s t u d i e s suggest t h a t a wave-l i k e p a t t e r n of growth of l a r g e a n t r a l f o l l i c l e s i s a c h a r a c -t e r i s t i c o f the bovine ovary r e g a r d l e s s of the r e p r o d u c t i v e s t a t e . i i i TABLE OF CONTENTS L i s t of T a b l e s v i L i s t of F i g u r e s v i i i L i s t of P l a t e s x Acknowledgements x i Forward x i i L i t e r a t u r e Review 1 Ovarian Dynamics and the Resumption of E s t r o u s C y c l e s i n Postpartum D a i r y cows 16 F o l l i c u l a r Dynamics and Corpus Luteum Growth and F u n c t i o n During the F i r s t 60 Days of Pregnancy i n D a i r y Cows 3 9 F o l l i c u l a r Dynamics and Temporal R e l a t i o n s h i p s Between Body Temperature, Standing E s t r u s , L u t e i n i z i n g Hormone and O v u l a t i o n i n Dairy H e i f e r s Synchronized With the P r o g e s t i n Norgestomet 62 i v T a b l e of c o n t e n t s cont. Concluding D i s c u s s i o n 79 References 87 v LIST OP TABLES T a b l e 2.1. Mean i n t e r v a l i n days ( ± S.D.) from c a l v i n g t o f i r s t o v u l a t i o n , f i r s t e s t r u s , u t e r i n e i n v o l u t i o n , l e n g t h o f the f i r s t and second postpartum e s t r o u s c y c l e s and maximum progesterone (P4) c o n c e n t r a t i o n (ng/ml) i n d a i r y cows 2 2 Tabl e 3.1. I n t r a u t e r i n e s t r u c t u r e s v i s u a l i z e d on d i f f e r e n t days p o s t b r e e d i n g u s i n g u l t r a -sound imaging i n d a i r y c a t t l e 47 Ta b l e 3.2. Comparison of the diameter (± S.E.M.) of the l a r g e s t f o l l i c l e p r e s e n t on the o v a r i e s d u r i n g the f i r s t 24 days of e a r l y pregnant and non pregnant d a i r y cows 51 Table 3.3. Comparison o f the t o t a l number (± S.E.M.) of v i s i b l e f o l l i c l e s on both o v a r i e s d u r i n g the f i r s t 24 days between e a r l y pregnant and non pregnant d a i r y cows 52 v i L i s t of t a b l e s cont. T a b l e 3.4. Comparison of the diameter (mm ± S.E.M.) of the corpus luteum d u r i n g the f i r s t 24 days p o s t b r e e d i n g o f e a r l y pregnant and non pregnant d a i r y cows Tabl e 4.1. Mean i n t e r v a l i n hours (± S.D.) from norgestomet implant removal t o s t a n d i n g e s t r u s , temperature peaks, LH peak and o v u l a t i o n , and c o r r e l a t i o n c o e f f i c i e n t s (r) with the time t o o v u l a t i o n T a b l e 4.2. Pregnancy r a t e s i n . d a i r y cows s y n c h r o n i z e d w i t h norgestomet ear implants... v i i LIST OF FIGURES F i g . 2 . 1 . C o r r e l a t i o n between corpus luteum diameter and c i r c u l a t i n g progesterone i n postpartum d a i r y c a t t l e 27 F i g . 2.2. P a t t e r n s of r e t u r n t o r e p r o d u c t i v e c y c l e s i n postpartum d a i r y c a t t l e 28 F i g . 2.3. Growth of dominant f o l l i c l e s i n t h r e e r e p r e s e n t a t i v e postpartum d a i r y cows 3 0 F i g . 2.4. Comparison between plasma and m i l k progesterone i n postpartum d a i r y c a t t l e 31 F i g . 3.1. Diameter of the l a r g e s t f o l l i c l e on the r i g h t and l e f t o v a r i e s of a pregnant d a i r y cow (82019) 49 F i g . 3.2. T o t a l number of f o l l i c l e s on the CL b e a r i n g and c o n t r a l a t e r a l o v a r i e s of e a r l y pregnant d a i r y cows 50 v i i i L i s t o f f i g u r e s c o n t . F i g . 3 . 3 . D i a m e t e r o f t h e c o r p u s l u t e u m i n e a r l y p r e g n a n t a n d n o n p r e g n a n t d a i r y cows a s d e t e r m i n e d b y u l t r a s o u n d i m a g i n g 55 F i g . 3 . 4 . C i r c u l a t i n g p l a s m a p r o g e s t e r o n e i n e a r l y p r e g n a n t a n d n o n p r e g n a n t d a i r y cows 56 F i g . 4 . 1 . Day o f o n s e t o f a t r e s i a o f m i d c y c l e d o m i n a n t f o l l i c l e s a n d l u t e o l y s i s i n n o r g e s t o m e t s y n c h r o n i z e d h e i f e r s 69 F i g . 4 . 2 . P l a s m a p r o g e s t e r o n e p r o f i l e s i n h e i f e r s i m p l a n t e d w i t h n o r g e s t o m e t 70 F i g . 4 . 3 . T i m e t o e s t r u s , LH p e a k a n d o v u l a t i o n a f t e r n o r g e s t o m e t i m p l a n t r e m o v a l i n d a i r y h e i f e r s 74 i x LIST OP PLATES P l a t e 2.1. U l t r a s o u n d images of the o v a r i e s of postpartum d a i r y cows 2 4 P l a t e 2.2. U l t r a s o u n d images of a l a r g e p e r s i s t e n t f o l l i c l e i n a postpartum d a i r y cow 25 P l a t e 3.1. U l t r a s o u n d images of the CL b e a r i n g ovary and the g r a v i d u t e r i n e horn i n a pregnant d a i r y cow 45 P l a t e 3.2. U l t r a s o u n d images of the CL b e a r i n g ovary and the g r a v i d u t e r i n e horn i n a pregnant d a i r y cow 4 6 P l a t e 4.1. U l t r a s o u n d images of the l e f t and r i g h t ovary of a h e i f e r r e c e i v i n g a norgestomet ear implant d u r i n g p r o e s t r u s 71 x ACKNOWLEDGMENTS I would s i n c e r e l y l i k e t o thank Dr. R. Rajamahendran f o r h i s guidance and support through t h e two y e a r s t h a t have gone i n t o t h i s work. H i s humour and enthusiasm f o r the work has made the time a j o y . I would a l s o l i k e t o thank him f o r the leeway and encouragement he p r o v i d e d f o r the development of my own i d e a s . I would l i k e t o express my g r a t i t u d e t o the people a t the South Campus d a i r y farm, e s p e c i a l l y Paul W i l l i n g and the m i l k e r s , Les Szabo and Hans Pederson, f o r t h e i r c o o p e r a t i o n and p a t i e n c e d u r i n g my i n t e r f e r e n c e w i t h t h e i r m i l k i n g r o u t i n e . F i n a l l y , and most i m p o r t a n t l y , I wish t o thank Matthew T a y l o r , who gave me cause. xi FORWARD The hormonal factors involved i n the growth of ovarian f o l l i c l e s and growth and regression of the corpus luteum i n the bovine are quite well documented, however, the actual changes i n f o l l i c u l a r populations on d i f f e r e n t days of the estrous cycle have only recently been reported. The use of ultrasound imaging i n recent years has allowed the continual monitoring of i n d i v i d u a l animals over a period of time, thus permitting actual changes i n f o l l i c u l a r populations and the growth and regression of the corpus luteum to be v i s u a l i z e d . This t h e s i s describes the f o l l i c u l a r dynamics and corpus luteum growth and function during three d i f f e r e n t physiologic states: the postpartum anestrus period, early pregnancy and during the exogenous control of the estrous cycle with the progestin norgestomet. Each chapter i s treated as a seperate paper with an introduction, materials and methods, r e s u l t s and discussion. A general discussion of the relevance of each study to the other two and the possible implications and future researh which the present studies suggest i s presented i n the f i n a l chapter. x i i LIT REVIEW CHAPTER 1 LITERATURE REVIEW One o f the main c h a r a c t e r i s t i c s o f a f u n c t i o n a l ovary i n s e x u a l l y mature mammals i s the growth and m a t u r a t i o n o f f o l -l i c l e s , t he r u p t u r e o f a s e l e c t few i n the p r o c e s s o f o v u l a -t i o n and a t r e s i a o f the v a s t m a j o r i t y . T h i s p r o c e s s begins e a r l y i n embryonic and f e t a l l i f e w i t h the d i f f e r e n t i a t i o n of the female ovary, the fo r m a t i o n o f a p r i m o r d i a l f o l l i c l e r e s e r v e and the subsequent growth and d i f f e r e n t i a t i o n o f f o l l i c l e s . FORMATION OF THE GONAD The ovary d i f f e r e n t i a t e s from the embryonic gonad which develops as a t h i c k e n i n g along the v e n t r a l c r a n i a l area of the mesonephros, the p r i m o r d i a l kidney ( W i t s c h i , 1951; Hamilton and Mossman 1972). There have been s e v e r a l d i f f e r e n t t h e o r i e s as t o the o r i g i n o f gonadal c e l l s . In s t u d i e s of amphibian gonads, W i t s c h i (1948) concluded t h a t the gonad was composed of two types of t i s s u e s : the c o r t e x , d e r i v e d from coelomic e p i t h e l i u m , s e c r e t e s c o r t e x i n ; and the medulla, de-r i v e d from mesonephros, s e c r e t e s m e d u l l a r i n . He proposed t h a t these s e c r e t o r y products a c t a n t a g o n i s t i c a l l y and t h a t an 1 LIT REVIEW excess o f one product causes the atrophy o f the opposing t i s -sue type. In the female excess c o r t e x i n causes the degenera-t i o n o f the medulla and l e a d s t o the development of the ovary. Burns (1961) proposed another model i n which primary sex cords p r o l i f e r a t e from germinal e p i t h e l i u m and form the med-u l l a r y c o r d s . In t h i s model an ovary develops as the r e s u l t o f a second p r o l i f e r a t i o n o f germinal e p i t h e l i u m forming the c o r t i c a l c o r d s . The med u l l a r y cords which would form the t e s t e s i n a male are pushed i n t o the medulla where they de-generate. A t h i r d model based on s t u d i e s of f e t a l human (P i n k e r t o n e t a l . , 1961) and bovine (Gropp and Ohno 1966) o v a r i e s sug-g e s t t h a t gonadal blastema o r i g i n a t e s from mesonephros i n much the same manner as t h a t d e s c r i b e d by W i t s c h i (1951) . Gropp and Ohno (1966) found t h a t i n the bovine f e t u s the mesonephric t i s s u e extends from the gl o m e r u l a r t u f t of the d e v e l o p i n g kidney t o the gonadal r i d g e where they mix with mesenchyme and germ c e l l s . In an e x t e n s i v e review on the development of the mammal-i a n gonad, Byskov (1986) suggests t h a t the primary t i s s u e type i v o l v e d i n gonad f o r m a t i o n i s the mesonephros wi t h some ce l o m i c e p i t h e l i u m , however t h e r e are no primary o r secondary p r o l i f e r a t i o n s . In the cow formation of the g e n i t a l r i d g e o c u r r s a t about 30 days of g e s t a t i o n (Byskov, 1986). 2 LIT REVIEW PRIMORDIAL GERM CELLS: ORIGIN AND MIGRATION P r i m o r d i a l germ c e l l s (PGCs) are the germ c e l l s of both the male and female t h a t o r i g i n a t e e x t r a g o n a d a l l y and migrate t o the u n d i f f e r e n t i a t e d gonad. In what i s now a c l a s s i c ex-periment, E v e r e t t (1943) t r a n s p l a n t e d embryonic mouse g e n i t a l r i d g e s a t d i f f e r e n t stages of development-under the kidney c a p s u l e o f a d u l t mice. Examination two weeks l a t e r r e v e a l e d t h a t t r a n s p l a n t s d e r i v e d from 9.5 t o 10 day f e t u s e s c o n t a i n e d no PGCs whereas t r a n s p l a n t s from 11 t o 14 day o l d f e t u s e s d i d . E v e r e t t concluded t h a t germ c e l l s were extragonadal i n o r i g i n and t h a t they invaded the g e n i t a l r i d g e a f t e r day 10 i n the f e t a l mouse. In t h e i n t e r v e n i n g y e a r s many s t u d i e s have been done i n an e f f o r t t o t r a c e the c e l l l i n e from which the PGCs a r i s e . D e s p i t e these e f f o r t s i t i s s t i l l not p o s s i b l e t o determine when, and from where, these c e l l s a r i s e (Byskov, 1986). How-ever, s e v e r a l c h a r a c t e r i s t i c s of these c e l l s have shed some l i g h t on t h e i r o r i g i n . The p e r i p h e r a l cytoplasm has been found t o have h i g h a l k a l i n e phosphatase a c t i v i t y (McKay e t a l . , 1953). T h i s c h a r a c t e r i s t i c has been used t o e s t a b l i s h the m i g r a t o r y pathway from the y o l k sac to the g e n i t a l r i d g e i n the mouse embryo (Chiquoine, 1954). The mechanisms i n v o l v e d i n the movement of the PGCs from the y o l k sac t o the d e v e l o p i n g gonads are a l s o not c l e a r . S e v e r a l hypotheses have been proposed and i t i s l i k e l y t h a t 3 LIT REVIEW t r a n s l o c a t i o n i n v o l v e s a combination of mechanisms. Ps e u d o p o d i a - l i k e s t r u c t u r e s have been r e p o r t e d on the s u r f a c e of PGCs i n s e v e r a l s p e c i e s i n c l u d i n g the mouse ( C l a r k and Eddy, 1975), r a t (Eddy, 1974) and human (Kuwana and Fujimoto, 1983). I t has a l s o been r e p o r t e d t h a t morphogenic movements of the t i s s u e s i n which the PGCs are embedded h e l p t o t r a n s -p o r t the c e l l s (Jeon and Kennedy, 1973; Snow, 1981). F i n a l l y , W i t s c h i (1948) suggested t h a t PGCs were d i r e c t e d t o t h e i r d e s t i n a t i o n by chemoattractants produced i n the gonadal r i d g e . Once the PGCs a r r i v e a t the gonadal r i d g e they are q u i c k l y e n c l o s e d i n germ c e l l compartments where they p r o l i f -e r a t e and d i f f e r e n t i a t e under the r e g u l a t i o n of the surround-i n g somatic c e l l s . DIFFERENTIATION OF THE OVARY Development up t o the p o i n t where the PGCs have come t o r e s t i n the g e n i t a l r i d g e i s i d e n t i c a l i n both male and female embryos. A l f r e d J o s t (1959) suggested t h a t i t i s a t t h i s stage t h a t d i f f e r e n t i a t i o n t o e i t h e r t e s t e s or o v a r i e s takes p l a c e . He proposed t h a t i n the absence o f s e c r e t i o n s from p r i m o r d i a l t e s t e s the r e p r o d u c t i v e t r a c t would develop along the female l i n e . T h i s has s i n c e become the c e n t r a l paradigm of sexual development (Wilson, 1985). 4 LIT REVIEW One o f the f i r s t s i g n s o f d i f f e r e n t i a t i o n of the ovary i s the o b s e r v a t i o n t h a t germ c e l l s are e n t e r i n g m e i o s i s ( P e t e r s , 1970). However, i n some s p e c i e s such as the sheep, cow, p i g and r a b b i t m e i o s i s i s d e l a y e d w h i l e the germ c e l l s become e n c l o s e d i n germ c e l l cords (Mauleon, 1969) which then procede t o break up by approximately 1/2 term. The f i r s t c e l l s t o e n t e r m e i o s i s are found i n the c e n t r a l portion.,, of the gonad a d j a c e n t t o the mesonephric c o n n e c t i o n . T h i s i s t r u e whether m e i o s i s i s immediate or d e l a y e d (Byskov, 1986). The c o n n e c t i o n of m e i o t i c c e l l s w i t h the mesonephros has l e d t o the p r o p o s a l t h a t the mesonephros i s r e s p o n s i b l e f o r i n i t i a t i n g m e i o s i s (Byskov, 1975) and t h a t i n t e r c e l l u l a r b r i d g e s h e l p s y n c h r o n i z e d i v i s i o n s . S p e c i e s w i t h a d e l a y p e r i o d p r i o r t o the i n i t i a t i o n of m e i o s i s e x h i b i t a t r a n s -i t o r y s e c r e t i o n of sex s t e r o i d s which are thought t o i n h i b i t m e i o s i s . Once the s e c r e t i o n s have ceased m e i o s i s i s i n i t i a t e d (Byskov, 1986). M e o i s i s i s a r r e s t e d i n the l a s t phase of the m e i o t i c prophase, the d i p l o t e n e stage, when the oocyte becomes surrounded by g r a n u l o s a c e l l s and a b a s a l lamina t o form a f o l l i c l e ( P e t e r s , 1978). As w i t h m e i o s i s , f o l l i c l e f o rmation always s t a r t s i n the i n n e r p o r t i o n of the ovary immediately a f t e r the f i r s t o ocytes r e a c h the d i p l o t e n e stage (Mossman and Duke, 197 3). Granulosa c e l l s a r i s e from mesonephric d e r i v e d c e l l s (Byskov and Lintern-Moore, 1973) and are i n i t i a l l y connected t o the r e t e c o r d s . Formation of the b a s a l lamina breaks t h i s connec-5 LIT REVIEW t i o n and thus f o l l i c l e s become independent u n i t s (Hashimoto and E g u c h i , 1955). The s t i m u l u s f o r f o l l i c l e f o r m a t i o n i s not known. Granu-l o s a c e l l p r o l i f e r a t i o n o c c u r s i n v i t r o without hormones (Baker and Neal, 1973; C h a l l o n e r , 1975). In a d d i t i o n , t r e a t -ment of a d u l t mice w i t h a n t i - g o n a d o t r o p h i n s does not i n t e r u p t e a r l y f o l l i c u l a r growth (Nakano e t a l . , 197-5), although go-nadotrophins can a c c e l e r a t e the r a t e of growth of p r e - a n t r a l f o l l i c l e s (Hansel and Convey, 1983) . I t has l o n g been h e l d t h a t f u r t h e r growth of f o l l i c l e s t o the a n t r a l stage r e q u i r e s s t i m u l a t i o n by gonadotrophins: hypophysectomy r e s u l t s i n the a r r e s t of a n t r a l f o l l i c u l a r growth and atrophy of the r e p r o d u c t i v e t r a c t due t o the decrease i n c i r c u l a t i n g sex s t e r i o d s n o r m a l l y produced by a n t r a l f o l l i c l e s (Gulyas e t a l . , 1977). Treatment of r a t s w i t h a n t i b o d i e s f o r f o l l i c l e s t i m u l a t i n g hormone (FSH) b l o c k s the development of the p o o l of o v u l a t o r y f o l l i c l e s (Welschen and D u l l a a r t , 1976). In a d d i t i o n , a d m i n i s t r a t i o n of exogenous gonadotrophins, namely (FSH), pregnant mares serum gonadotrophin (PMSG) or t h e i r analogues, s t i m u l a t e s a n t r a l f o l l i c u l a r growth i n p r e p u b e r t a l h e i f e r s and induces a s u p e r o v u l a t o r y response i n h e i f e r s and mature cows (Casida e t a l . , 1943; Onuma e t a l . , 1970; S e i d e l et a l . , 1971). 6 LIT REVIEW THE WAVE PATTERN HYPOTHESIS OF FOLLICULAR GROWTH The bovine ovary c o n t a i n s , on average, over 100,000 f o l -l i c l e s a t b i r t h ( E r i c k s o n , 1966). Throughout l i f e , p r i m o r d i a l f o l l i c l e s e n t e r a po o l o f growing f o l l i c l e s , w i t h a s e l e c t few g o i n g on t o o v u l a t i o n , w h i l e most r e g r e s s and become a t r e t i c . Smeaton and Robertson (1971), marking i n d i v i d u a l f o l l i c l e s w i t h I n d i a i n k showed t h a t a sequence o f f o l l i c l e s grow, r e g r e s s and are then r e p l a c e d by othe r f o l l i c l e s d u r i n g a s i n g l e e s t r o u s c y c l e i n the sheep. S i m i l a r f i n d i n g s have been r e p o r t e d i n cows (Matton e t a l . , 1981). R a j a k o s k i (1960) put f o r t h the h y p o t h e s i s t h a t f o l l i c u l a r growth i n the cow takes p l a c e i n a wave-like p a t t e r n w i t h a s i n g l e c y c l e c o n s i s t i n g of two waves. The f i r s t wave of growth o c c u r i n g between day 1 and day 12 of the c y c l e (day 1 = day of e s t r u s ) and the second wave of growth commencing on day 13 and c u l m i n a t i n g i n the s e l e c t i o n and o v u l a t i o n o f a s i n g l e f o l l i c l e a t the subsequent e s t r u s . A s i m i l a r p a t t e r n of f o l -l i c u l a r growth has been proposed i n sheep (Brand and DeJong, 1973). Although t h i s h y p o t h e s i s has come under some c r i t i s i s m ( S p i c e r and Ekternkamp, 1986) the idea has p e r s i s t e d . One problem has been t h a t u n t i l r e c e n t l y most s t u d i e s on f o l l i c u l a r dynamics i n the bovine have been r e s t r i c t e d t o sla u g h t e r h o u s e s t u d i e s or s t u d i e s i n v o l v i n g r e c t a l p a l p a t i o n . Both o f th e s e methods have t h e i r l i m i t a t i o n s . The major l i m i t a t i o n w i t h sampling from a slaughterhouse i s t h a t i t 7 LIT REVIEW p r e c l u d e s m o n i t o r i n g i n d i v i d u a l animals over a p e r i o d of time perhaps o b s c u r i n g the dynamic nature of f o l l i c u l a r growth. In the p a s t decade u l t r a s o u n d imaging has been used i n numerous s t u d i e s of f o l l i c u l a r growth and a t r e s i a . P i e r s o n and G i n t h e r (1987a) d e s c r i b e d a two wave e s t r o u s c y c l e i n h e i f e r s , w h i l e S i r o i s and Fortune (1988) suggest t h a t the m a j o r i t y of h e i f e r s s t u d i e d have t h r e e waves of f o l l i c u l a r growth d u r i n g a s i n g l e c y c l e . A t h i r d study found both two and t h r e e waves of growth i n h e i f e r s (Savio e t a l . , 1988). One common theme i n a l l of the s t u d i e s u s i n g u l t r a s o u n d imaging i s t h a t t h e r e i s a wave-like p a t t e r n of growth of l a r g e dominant f o l l i c l e s d u r i n g t h e c y c l e and t h a t the dominant f o l l i c l e a t the time of l u t e o l y s i s i s the f o l l i c l e t h a t o v u l a t e s . SELECTION OF THE OVULATORY FOLLICLE The p r o c e s s by which the o v u l a t o r y f o l l i c l e i s s e l e c t e d i s as y e t unknown. Removal of the corpus luteum (CL) i n cows (Hammond and Bhattacharya, 1944) and sheep (Smeaton and Robertson, 1971) r e s u l t s i n o v u l a t i o n w i t h i n 48 t o 72 hours. However i t i s not the dominant f o l l i c l e t h a t i s p r e s e n t a t the time of CL removal (Smeaton and Robertson, 1971) or p r o s t a g l a n d i n (PGF20;) induced l u t e o l y s i s ( I r e l a n d and Roche, 1982) which o v u l a t e s , a new f o l l i c l e o r f o l l i c l e s grow while the o l d ones become a t r e t i c . These r e s u l t s suggest t h a t 8 LIT REVIEW s e l e c t i o n of the o v u l a t o r y f o l l i c l e has o c c u r r e d p r i o r t o the induced l u t e o l y s i s or CL removal, t h a t i s s e l e c t i o n takes p l a c e e a r l y i n the c y c l e or even d u r i n g the p r e v i o u s c y c l e . However, c o n f l i c t i n g evidence would suggest t h a t s e l e c t i o n of the o v u l a t o r y f o l l i c l e t a k e s p l a c e r e l a t i v e l y l a t e i n the c y c l e . Removal o f f o l l i c l e s by c a u t e r y ( T s o n i s e t a l . , 1982) or x - i r r a d i a t i o n ( D r a i n c o u r t and Mariana, 1982) does not r e s u l t i n a l e n g t h e n i n g of the f o l l i c u l a r phase. However i t i s p o s s i b l e t h a t s e l e c t i o n of f o l l i c l e s from the p o o l of p r e a n t r a l f o l l i c l e s has a l r e a d y taken p l a c e and t h a t the o v u l a t o r y f o l l i c l e s undergo r a p i d growth once l u t e o l y s i s commences. OVULATION O v u l a t i o n o c c u r s as the r e s u l t of a complex mechanism i n v o l v i n g changes i n end o c r i n e p a t t e r n s , b i o c h e m i c a l changes w i t h i n the o v u l a t o r y f o l l i c l e and neuromuscular mechanisms. A gonadotropin surge t r i g g e r s the onset of o v u l a t i o n and r e s u l t s from the decrease i n c i r c u l a t i n g p r o g e s t e r o n e ( P 4 ) a f t e r l u t e o l y s i s and an i n c r e a s e i n c i r c u l a t i n g estradiol-17/3 ( E 2 ) from the o v u l a t o r y f o l l i c l e . - P e l l e t i e r and Thimonier (1975), i n experiments wi t h c a s t r a t e d female r a t s , demon-s t r a t e d t h a t E 2 treatment i n i t i a l l y r e s u l t s i n a decrease i n l u t e i n i z i n g hormone (LH) due t o n e g a t i v e feedback, but w i t h 9 LIT REVIEW c o n t i n u e d treatment t h e r e i s a s w i t c h t o p o s i t i v e feedback r e s u l t i n g i n the LH surge p r e c e d i n g o v u l a t i o n . Simultaneous a d m i n i s t r a t i o n of P4 supresses t h e E 2 p o s i t i v e feedback. The i n c r e a s e d LH s e c r e t i o n enhances s t e r o i d s y n t h e s i s and s e c r e t i o n by the o v u l a t o r y f o l l i c l e and decreases the E 2 : P 4 r a t i o i n the f o l l i c u l a r f l u i d as a r e s u l t o f a sw i t c h from E 2 p r o d u c t i o n t o P4 p r o d u c t i o n (Gerard e t a l . , 1979). L i p n e r and Green (1971) found t h a t i n h i b i t i o n o f P4 s y n t h e s i s b l o c k e d o v u l a t i o n . I t appears t h a t P4 a c t s t o s t i m u l a t e c o l l a g e n a s e a c t i v i t y i n the f o l l i c u l a r w a l l ( R o n d e l l , 1970). In c r e a s e s i n p r o s t a g l a n d i n s ( P G F 2 A and P G E 2 ) are seen somewhat l a t e r than the i n c r e a s e i n s t e r i o d s . Ainsworth e t a l . (1975) showed t h a t p r o s t a g l a n d i n s do not s t a r t t o i n -crease i n f o l l i c u l a r f l u i d u n t i l about 30 hours a f t e r the LH surge i n the p i g and don't reach t h e i r peak u n t i l about 4 0 hours. I n h i b i t i o n o f i n t r a f o l l i c u l a r p r o s t a g l a n d i n s y n t h e s i s has been shown t o b l o c k o v u l a t i o n i n r a b b i t s and sows (Armstrong, 1975). I t i s b e l i e v e d t h a t P G F 2 A i s i n v o l v e d i n f o l l i c u l a r r u p t u r e v i a i t s r u p t u r i n g e f f e c t s on lysosomal membranes (Weiner and Kaley, 1972). F i n a l l y neuromuscular systems appear t o be i n v o l v e d i n the r u p t u r i n g of the o v u l a t o r y f o l l i c l e . I n h i b i t i o n of /3-and-r e n e r g i c r e c e p t o r s d e l a y s o v u l a t i o n and reduces the o v u l a t i o n r a t e i n r a b b i t s (Virutamasen e t a l . , 1976). 10 LIT REVIEW FORMATION AND FUNCTION OF THE CORPUS LUTEUM A f t e r o v u l a t i o n the r u p t u r e d f o l l i c l e r e o r g a n i z e s t o form a t r a n s i t o r y gland, the CL. The main l u t e o t r o p h i n i n most domestic farm s p e c i e s i s LH (Hansel and Convey, 1983). The CL has been found t o be composed of two d i f f e r e n t c e l l t ypes i n the bovine, ovine and i n swine: s m a l l t h e c a l d e r i v e d c e l l s and l a r g e c e l l s p r i m a r i l y d e r i v e d from g r a n u l o s a c e l l s (Hansel, 1988). The s m a l l t h e c a l d e r i v e d c e l l s make up approximately 95% of the c e l l s of the corpus luteum found d u r i n g a normal e s t r o u s c y c l e and are v e r y r e s p o n s i v e t o LH and cAMP modulators. A d d i t i o n a l l y , these s m a l l c e l l s i n c r e a s e both b a s a l and LH s t i m u l a t e d P4 s y n t h e s i s i n response t o P G F 2 a c h a l l e n g e s w h i l e the l a r g e c e l l s d ecrease LH s t i m u l a t e d s y n t h e s i s i n response t o the same c h a l l e n g e , l e a d i n g t o the b e l i e f t h a t the l u t e o t r o p h i c e f f e c t s o f P G F 2 a m a Y D e mediated v i a the l a r g e c e l l s (Hansel 1988) . LUTEOLYSIS In normal r e p e t e t i v e e s t r o u s c y c l e s i t i s necessary f o r the CL t o undergo l u t e o l y s i s c a u s i n g the f a l l i n c i c u l a t i n g P4 and a l l o w i n g the animal t o come i n t o e s t r u s . A l a r g e body of evidence has shown t h a t the l u t e o l y s i n i s P G F 2 a of u t e r i n e 11 LIT REVIEW o r i g i n (Thatcher e t a l . , 1984). Removal of the u t e r u s extends the l i f e s p a n of the CL (Ginther e t a l . , 1967). S e c t i o n i n g of the broad ligament i p s i l a t e r a l t o the CL has the same e f f e c t (Hixon and Hansel, 1974). F i n a l l y , a d m i n i s t r a t i o n of exoge-nous P G F 2 a induces l u t e o l y s i s . A complete mechanism f o r the i n d u c t i o n of l u t e o l y s i s i n the nonpregnant cow has been out-l i n e d - (Thatcher e t a l . , 1988). E2 from a mid c y c l e dominant f o l l i c l e induces the formation of e n d o m e t r i a l o x y t o c i n r e c e p t o r s , o x y t o c i n s y n t h e s i z e d and s e c r e t e d by the CL then b i n d s t o these r e c e p t o r s and s t i m u l a t e s the endometrium t o s e c r e t e PGF20; which then causes l u t e o l y s i s . In the case of pregnancy i t i s e s s e n t i a l t h a t the CL be maintained beyond i t s normal e s t r o u s c y c l e l i f e s p a n . In the bovine and ovine, conceptus s e c r e t o r y p r o d u c t s which can prevent the s e c r e t i o n of endometrial PGF20: have been i s l o a t e d (Thatcher e t a l . , 1989). I t i s suggested t h a t these tropho-b l a s t i c p r o t e i n s a c t by s t i m u l a t i n g the s y n t h e s i s of an endo-m e t r i a l p r o s t a g l a n d i n synthetase i n h i b i t o r , thus s u p r e s s i n g the s y n t h e s i s and s e c r e t i o n of PGF20: a n d p r e v e n t i n g l u t e o -l y s i s . There i s a l s o evidence t o suggest t h a t s e c r e t i o n of any P G F 2 a t h a t i s s y n t h e s i z e d i s i n an e x o c r i n e d i r e c t i o n i n t o the lumen of the u t e r u s i n pregnant animals as opposed t o the s e c r e t i o n i n an endocrine d i r e c t i o n i n c y c l i n g animals (Thatcher e t a l . 1984). 12 LIT REVIEW ULTRASOUND IMAGING In the p a s t most s t u d i e s on f o l l i c u l a r dynamics and corpus luteum growth have had t o r e l y on s l a u g h t e r h o u s e m a t e r i a l or r e c t a l palpation,however i n the l a s t decade the use of u l t r a s o u n d imaging has become prominant i n the study of o v a r i a n f u n c t i o n . There are b a s i c a l l y two types of u l t r a s o u n d scanners used f o r anatomic v i s u a l i z a t i o n : s e c t o r scanners, which use a s i n g l e p i z o e l e c t r i c c r y s t a l which r o t a t e s and produces a p i e shaped image, and l i n e a r a r r a y scanners, which use a l i n e a r row of c h r y s t a l s and produce a square image. The main f e a t u r e of each type i s the probe which c o n t a i n s the c h r y s t a l or c r y s t a l s . These p i z o e l e c t r i c c r y s t a l s expand and c o n t r a c t r a p i d l y when e x c i t e d by e l e c t r i c c u r r e n t . The expansion and c o n t r a c t i o n d i s p l a c e s molecules and i n t h i s way the waves are propogated through the t i s s u e s . At t i s s u e i n t e r f a c e s the sound i s r e f l e c t e d back and r e c e i v e d by the probe, a g a i n ex-c i t i n g the p i z o e l e c t r i c c r y s t a l s which then c o n v e r t the energy back t o e l e c t r i c i t y . The frequency o f t h e e l e c t r i c impulse i s then converted i n t o v a r y i n g shades of grey by the u l t r a s o u n d imager p r o d u c i n g an image on the s c r e e n . The d e n s i t y of the t i s s u e determines the frequency a t which the wave impulse i s r e f l e c t e d , f l u i d absorbs the waves and t h e r e f o r e sends back no impulse, p r o d u c i n g a b l a c k area i n the image. Very dense t i s s u e such as c a r t i l a g e or bone 13 LIT REVIEW sends back a h i g h frequency impulse p r o d u c i n g a v e r y b r i g h t or hyperehogenic image, o t h e r t i s s u e s produce images of v a r y -i n g shades of grey and i n t h i s way the image can be analysed and s t r u c t u r e s v i s u a l i z e d . The frequency of the probe determines the depth t o which the sound waves w i l l p e n e t r a t e and the l e v e l o f r e s o l u t i o n of the image. A h i g h frequency probe w i l l p r o v i d e the b e s t r e s o -l u t i o n but w i l l not p e n e t r a t e v e r y deeply. A low frequency probe w i l l p e n e t r a t e deeper a t the expense of r e s o l u t i o n . The frequency of the probes range between 2 and 9 megahertz (mHz). Most p r e v i o u s s t u d i e s examining the r e p r o d u c t i v e t r a c t of the bovine have used l i n e a r a r r a y scanners equipped w i t h r e c t a l probes. The e n t i r e probe can be i n s e r t e d i n t o the rectum and l a y e d over the u t e r u s or o v a r i e s f o r examination. In a d d i t i o n most modern machines can f r e e z e the image and s t r u c t u r e s can be measured u s i n g c a l l i p e r s b u i l t i n t o the system. C a l l i b r a t i o n of the c a l i p e r s can be checked u s i n g a phantom. P r e v i o u s s t u d i e s i n the bovine have a l s o v a l i d a t e d the measurement of s t r u c t u r e s by measurent o f t h e same s t r u c -t u r e s a f t e r s l a u g h t e r ( P i e r s o n and G i n t h e r , 1987a). The p r e s e n t t h e s i s i n v o l v e s u l t r a s o u n d imaging used i n c o n j u n c t i o n w i t h progesterone p r o f i l e s t o study f o l l i c u l a r dynamics and corpus luteum growth and f u n c t i o n d u r i n g t h r e e d i f f e r e n t r e p r o d u c t i v e s t a t e s : d u r i n g the postpartum p e r i o d , 14 L I T R E V I E W a time where v e r y l i t t l e work has been done on f o l l i c u l a r dynamics; d u r i n g e a r l y pregnancy, when i t has been hypothes-i z e d t h a t the i n f l u e n c e of the corpus luteum on f o l l i c u l a r growth on the i p s i l a t e r a l ovary changes from p o s i t i v e t o neg-a t i v e ; and f i n a l l y under the i n f l u e n c e o f the norgestomet, a p r o g e s t i n which i s used as an e s t r o u s s y n c h r o n i z i n g agent i n beef c a t t l e . 15 OVARIAN ACTIVITY IN POSTPARTUM COWS CHAPTER 2 OVARIAN DYNAMICS AND THE RESUMPTION OF ESTROUS CYCLES IN POSTPARTUM DAIRY COWS ABSTRACT Ten postpartum d a i r y cows were monitored f o r o v a r i a n a c t i v i t y by u s i n g a combination of u l t r a s o u n d imaging and prog e s t e r o n e p r o f i l e s . Blood and m i l k samples were c o l l e c t e d and o v a r i e s scanned r e c t a l l y s t a r t i n g 2 and 14 days p o s t -partum, r e s p e c t i v e l y , and continued u n t i l t he end of the second e s t r o u s c y c l e . U l t r a s o u n d imaging r e v e a l e d the presence o f a corpus luteum i n fo u r of te n animals by 14 days postpartum and i n e i g h t of ten by 25 days. T h i s was l a t e r c o n f i r m e d by progesterone d e t e r m i n a t i o n . There were no d i f f e r e n c e s between p r i m i p a r o u s and p l u r i p a r o u s animals w i t h r e g a r d t o time t o f i r s t postpartum o v u l a t i o n , d u r a t i o n o f 1st e s t r o u s c y c l e , peak plasma progesterone l e v e l s o r time t o u t e r i n e i n v o l u t i o n . Time t o f i r s t observed e s t r u s was g r e a t e r (P<0.1) i n pri m i p a r o u s than p l u r i p a r o u s cows. The second postpartum e s t r o u s c y c l e was s i g n i f i c a n t l y l o n g e r (P<0.05) w i t h g r e a t e r plasma progesterone c o n c e n t r a t i o n s than the f i r s t c y c l e . Maximum corpus luteum diameter as measured by u l t r a s o u n d imaging was not c o r r e l a t e d t o the l e n g t h of the 16 OVARIAN ACTIVITY IN POSTPARTUM COWS 1st or 2nd postpartum e s t r o u s c y c l e s or t o the maximum plasma progesterone i n the 1st e s t r o u s c y c l e but was p o s i t i v e l y c o r r e l a t e d t o the maximum plasma progesterone i n the 2nd postpartum e s t r o u s c y c l e . F o l l i c u l a r growth o c c u r r e d i n two waves i n most of the postpartum e s t r o u s c y c l e s . Whole m i l k p r o g e s t e r o n e p r o f i l e s were s i m i l a r i n p a t t e r n t o t h e plasma p r o f i l e s . INTRODUCTION O v a r i a n f o l l i c u l a r dynamics and the r e t u r n t o normal c y c l i c a c t i v i t y i n postpartum d a i r y c a t t l e are not w e l l understood. The postpartum p e r i o d i s c h a r a c t e r i z e d by a b e h a v i o r a l a n e s t r u s l a s t i n g from 20 t o 70 days ( C a l l a h a n e t a l . , 1971; Schams e t a l . , 1978). The f i r s t b e h a v i o r a l e s t r u s i s o f t e n preceded by a s h o r t e s t r o u s c y c l e c h a r a c t e r i z e d by an absence of o v e r t s i g n s o f e s t r u s and a s h o r t l u t e a l phase w i t h subnormal progesterone (P 4) b l o o d c o n c e n t r a t i o n s (Robertson, 9172; Edgerton and Hafs, 1973; Manns e t a l . , 1973). The cause of these \" s h o r t \" f i r s t c y c l e s i s not c e r t a i n but s e v e r a l i d e a s have been put f o r t h i n c l u d i n g FSH d e f i c i e n c y (Ramirez-Godinez e t a l . , 1982) and premature l u t e o l y s i s by p r o s t a g l a n d i n F20: (pGF2o:) from the i n v o l u t i n g u t e r u s ( T r o x e l e t a l . , 1980; Hanzen, 1986). Another f a c t o r r e p o r t e d t o i n f l u e n c e the l e n g t h of the a n e s t r o u s p e r i o d i s 17 OVARIAN ACTIVITY IN POSTPARTUM COWS p a r i t y ; printiparous cows have l o n g e r c a l v i n g t o f i r s t e s t r u s i n t e r v a l s than p l u r i p a r o u s cows (King and McLeod, 1983). P r e v i o u s s t u d i e s have r e l i e d e i t h e r on s l a u g h t e r h o u s e m a t e r i a l (Morrow e t a l . , 1966) which p r e c l u d e s m o n i t o r i n g i n d i v i d u a l animals over a p e r i o d of time o r on r e c t a l p a l p a t i o n (Saiduddin e t a l . , 1968) i n which a measure of accu r a c y and p r e c i s i o n i s l o s t (Landsverk and K a l b e r g , 1988). U l t r a s o u n d imaging has been used e f f e c t i v e l y t o i d e n t i f y o v a r i a n f o l l i c l e s and c o r p o r a l u t e a i n c y c l i n g h e i f e r s ( P i e r s o n and G i n t h e r , 1987) and cows (Rajamahendran and Walton, 1988) on a continuous b a s i s , however, t h e r e appear t o be no r e p o r t s on the use o f u l t r a s o u n d imaging i n the study of o v a r i a n f u n c t i o n i n the postpartum cow. The purpose of the p r e s e n t study i s t o u t i l i z e both u l t r a s o u n d imaging and P4 hormone p r o f i l e s f o r the m o n i t o r i n g of o v a r i a n a c t i v i t y i n postpartum d a i r y cows, and t o e v a l u a t e the r e l i a b i l i t y o f whole m i l k progesterone c o n c e n t r a t i o n s as a means o f a s s e s s i n g postpartum o v a r i a n a c t i v i t y . MATERIALS AND METHODS Ten postpartum d a i r y cows (8 H o l s t e i n s , 2 A y r s h i r e s ) were s e l e c t e d a t random from the U n i v e r s i t y of B r i t i s h Columbia d a i r y herd between October 1987 and January 1988. The cows ranged from 2 t o 6 y e a r s of age. Three animals were 18 OVARIAN ACTIVITY IN POSTPARTUM COWS f i r s t c a l v e r s . C alves were weaned w i t h i n 24 hours o f b i r t h . Animals were kept i n s e p a r a t e c a l v i n g pens away from the herd f o r a minimum of two weeks f o l l o w i n g p a r t u r i t i o n w i t h the f i r s t c a l v e r s g e n e r a l l y remaining i n t h e pens l o n g e r than the p l u r i p a r o u s cows. Cows were checked f o r s i g n s o f e s t r u s a t both a.m. and p.m. m i l k i n g s . A l l cows r e c e i v e d a mix of a l f a l f a -cubes and c o n c e n t r a t e i n equal p r o p o r t i o n s s t a r t i n g a t 4 kg per day and i n c r e a s i n g w i t h i n c r e a s e d m i l k p r o d u c t i o n . Animals were scanned t h r e e times per week u s i n g a r e a l -time l i n e a r a r r a y u l t r a s o u n d scanner (Tokyo K e i k i LS300, Tokyo K e i k i Co. L t d . , Tokyo, Japan) and a 5 MHz r e c t a l t r a n s -ducer s t a r t i n g 14 days postpartum and c o n t i n u i n g u n t i l the end of t h e second postpartum e s t r o u s c y c l e . O v a r i e s were scanned i n s e v e r a l p l a n e s t o i d e n t i f y a l l f o l l i c l e s and to determine t h e presence of a corpus luteum. Landmarks on the ovary such as a corpus luteum, o t h e r f o l l i c l e s and the p o l e s of the ovary as w e l l as t h e o r i e n t a t i o n of the ovary were used t o i d e n t i f y and monitor i n d i v i d u a l f o l l i c l e s . The image was f r o z e n and the l a r g e s t f o l l i c l e on each ovary and the corpus luteum, i f p r e s e n t , were measured u s i n g a b u i l t - i n c a l i p e r system. P r i n t s of the image were made u s i n g a v i d e o p r o c e s s i n g u n i t ( M i t s u b i s h i E l e c t r o n i c s Co. L t d . , Tokyo, Japan.). U t e r i n e i n v o l u t i o n was determined by r e c t a l p a l p a -t i o n . The p o i n t a t which the u t e r u s stopped d e c r e a s i n g i n s i z e and had r e g a i n e d i t s tone was c o n s i d e r e d the p o i n t at 19 OVARIAN ACTIVITY IN POSTPARTUM COWS which u t e r i n e i n v o l u t i o n was complete. Blood samples were. c o l l e c t e d from a t a i l v e i n t h r e e s t a r t i n g 2 days postpartum. Samples were c e n t r i f u g e d , plasma was s e p e r a t e d and then s t o r e d a t -20°C u n t i l a n a l y s i s . Whole m i l k samples were c o l l e c t e d a t the a f t e r n o o n m i l k i n g on the same days as the plasma and p r e s e r v e d w i t h potassium dichromate t a b l e t s a t 5'C u n t i l a n a l y s i s . Plasma and m i l k samples were a n a l y s e d f o r p4 u s i n g a coated tube radioimmunoassay k i t (Coat-A-Count, D i a g n o s t i c Products Corp., Los Angeles, CA) . Plasma, m i l k or r e f e r e n c e standard (0.1 ml) was added t o tubes coated w i t h a s p e c i f i c a ntibody. Reference standards c o n t a i n e d between 0 and 4 0 ng/ml P 4 . B u f f e r e d i l 2 5 p 4 ( i m i ) w a s added to a l l tubes, v o r t e x e d and l e f t t o i n c u b a t e f o r t h r e e hours. Tubes were decanted a f t e r the i n c u b a t i o n p e r i o d and c l e a n e d with a c o t t o n swab above the 1 ml mark t o remove excess- plasma, m i l k or t r a c e r . Tubes were counted f o r 1 minute u s i n g a gamma counter (Packard Auto-Gamma 500). C o e f f i c i e n t s of v a r i a t i o n w i t h i n and between plasma assays were 7.7 and 8.3% r e s p e c t i v e l y . The l i m i t of assay s e n s i t i v i t y was 0.05 ng/ml. The c o e f f i c i e n t s of v a r i a t i o n f o r the m i l k assays were 9.2 and 12.4% and the l i m i t of s e n s i t i v i t y was 0.05 ng/ml. P r i m i p a r o u s and p l u r i p a r o u s animals were compared u s i n g a Student's t t e s t f o r time t o f i r s t postpartum o v u l a t i o n , time t o f i r s t observed e s t r u s , d u r a t i o n of 1st and 2nd e s t r u s c y c l e s , peak plasma p4 and time to u t e r i n e i n v o l u t i o n . A 20 OVARIAN ACTIVITY IN POSTPARTUM COWS Student's t t e s t was a l s o used t o compare c h a r a c t e r i s t i c s of the 1st and 2nd postpartum e s t r o u s c y c l e s among a l l animals. C o r r e l a t i o n s between maximum CL diameter, peak P4 concent-r a t i o n s and d u r a t i o n of the e s t r o u s c y c l e s were determined by l e a s t squares l i n e a r r e g r e s s i o n and Pearson r c o r r e l a t i o n . RESULTS The time t o f i r s t o v u l a t i o n a f t e r p a r t u r i t i o n ranged from 10 t o 55 days (Table 2.1). There was no d i f f e r e n c e between p r i m i p a r o u s and p l u r i p a r o u s animals w i t h r e g a r d t o r e t u r n t o c y c l i c a c t i v i t y . The i n t e r v a l from c a l v i n g t o f i r s t observed e s t r u s was h i g h l y v a r i a b l e r a n g i n g from 17 t o 139 days postpartum. Primiparous animals tended (P<0.1) t o have a l o n g e r b e h a v i o r a l a n e s t r u s p e r i o d than p l u r i p a r o u s animals (Table 2.1). U t e r i n e i n v o l u t i o n was g e n e r a l l y complete by 4 weeks postpartum and t h e r e was no s i g n i f i c a n t d i f f e r e n c e between p r i m i p a r o u s and p l u r i p a r o u s animals. U l t r a s o u n d imaging r e v e a l e d the o v a r i e s t o be a c t i v e w i t h r e g a r d t o f o l l i c u l a r growth d u r i n g the a n o v u l a t o r y p e r i o d i n a l l animals. O v a r i e s were c h a r a c t e r i z e d by s e v e r a l s m a l l (3-8mm) and medium (9-15mm) s i z e d f o l l i c l e s d u r i n g t h i s time ( P l a t e 2.1). One of two animals (84001) w i t h a prolonged a n o v u l a t o r y p e r i o d developed a l a r g e p e r s i s t e n t f o l l i c l e on the l e f t ovary ( P l a t e 2.2) a t t a i n i n g a maximum diameter of 21 OVARIAN ACTIVITY IN POSTPARTUM COWS T a b l e 2.1. Mean i n t e r v a l i n days (± S.D.) from c a l v i n g t o f i r s t o v u l a t i o n , f i r s t e s t r u s , u t e r i n e i n v o l u t i o n , l e n g t h o f the 1st and 2nd postpartum e s t r o u s c y c l e s and maximum p r o -ogesterone ( P 4 ) c o n c e n t r a t i o n (ng/ml) i n d a i r y cows (n = 10). O v e r a l l P rimiparous P l u r i p a r o u s 1st o v u l a t i o n 21 ± 14 16 ± 7 24 ± 17 1st e s t r u s 59 ± 38 89 ± 55 46 ± 23 U t e r i n e i n v o l u t i o n 27 ± 5 2 5 + 1 27 ± 6 1st c y c l e l e n g t h 17 ± 7 a 15 ± 7 18 ± 8 2nd c y c l e l e n g t h 23 ± 5 a 21 ± 1 24 ± 6 P4 1st c y c l e 3.1 ± 1.2 a 3.1 ± 1.2 3.0 ± 1.7 P4 2nd c y c l e 4.4 ± 1.0 a 4.7 ± 0.8 4.3 ± 1.0 a d i f f e r e n c e w i t h i n groups p<0.05 22 OVARIAN ACTIVITY IN POSTPARTUM COWS 47mm by 3 6 days postpartum. The r i g h t ovary remained r e l a t i v e l y i n a c t i v e w i t h o n l y s m a l l f o l l i c l e s (<4mm) being observed w h i l e the p e r s i s t a n t f o l l i c l e developed. S h o r t l y a f t e r t he p e r s i s t a n t f o l l i c l e a t t a i n e d i t s maximum diameter l a r g e r f o l l i c l e s s t a r t e d t o develop on t h e c o n t r a l a t e r a l ovary w i t h one f i n a l l y o v u l a t i n g 42 days postpartum. The p e r s i s t a n t f o l l i c l e had begun t o r e g r e s s a t t h i s p o i n t but was s t i l l a pproximately 4 0mm i n diameter. The o t h e r animal w i t h an extended a n o v u l a t o r y p e r i o d ap-peared t o have normal f o l l i c u l a r development and tu r n o v e r . One f o l l i c l e grew t o a diameter of 20mm by 24 days p o s t -partum but f a i l e d t o o v u l a t e . There was a c o n t i n u a l t u r n o v e r of f o l l i c l e s u n t i l one f i n a l l y o v u l a t e d a t 55 days p o s t -partum. There was no d i f f e r e n c e between p r i m i p a r o u s and p l u r i -parous animals w i t h r e g a r d t o d u r a t i o n o f t h e 1st or 2nd es t r o u s c y c l e s o r peak plasma P4 l e v e l s (Table 2.1). Four of ten animals had a f i r s t postpartum c y c l e s h o r t e r than 14 days, f i v e o f t e n had a c y c l e o f 18-21 days and one animal had a 1 s t c y c l e g r e a t e r than 21 days i n d u r a t i o n . The s i d e of pregnancy appeared t o have no e f f e c t on the s i d e o f f i r s t postpartum o v u l a t i o n : f o u r o f t e n animals had t h e i r f i r s t o v u l a t i o n on the same s i d e as the p r e v i o u s pregnancy. The second postpartum c y c l e was s i g n i f i c a n t l y l o n g e r (P<0.05) and had h i g h e r peak progesterone l e v e l s (P<0.05) than the f i r s t postpartum c y c l e . 23 OVARIAN ACTIVITY IN POSTPARTUM COWS P l a t e 2.1. ULTRASOUND IMAGES OF THE OVARIES IN POSTPARTUM DAIRY C A T T L E Picture taken 14 days PP. Note small f o l l i c l e s on l e f t and r i g h t ovaries. Image of ovaries 48 days PP. Note medium and small f o l l i c l e s on l e f t and small f o l l i c l e s on r i g h t ovary. Picture taken 7 days aft e r the f i r s t PP ovulation. Note CL on l e f t and 19m» f o l l i c l e on rig h t . F t b / 6 3 / 8 8 1 4 : 4 1 I D : 85614 Probe: 5 M H z Distance! Area: Fek /9 1 / 88 1 4 : 5 0 I D : 83002 Probe: 5MHz Distance: 1 7 mm P r e * : F ^ L / 8 3 / 8 8 1 5 : 8 5 I D : 8 4 8 2 9 Probe: 5MHz Distance: 25M P r e * : 24 OVARIAN ACTIVITY IN POSTPARTUM COWS P l a t e 2.2. ULTRASOUND IMAGES OF THE OVARIES IN POSTPARTUM DAIRY C A T T L E Ft t , /8 1 / 8 8 1 5 : 82 I D : 83018 Probe: 5MHz Distance: 29 M Area: _ J ^ ^ 0 3 / 8 8 1 4 : 44 1 I D : 1 85814 Probe: 1 ' 5HHz Distance: 1 • 4 1 firea: • • F * b / -05 /88 1 4 : 22 I D : 84881 Probe: 5MHz Distance: 2 1 mm Area: Ovaries 8 days af t e r f i r s t PP ovulation. Note large CL (L) and 2 medium sized mid cycle f o l l i c l e s (R), Ultrasound image of a large f o l l i c l e (L) and a CL with a ce n t r a l cavity (R) af t e r spontaneous ovulation. Picture of above two days l a t e r . Note decrease i n f o l l i c l e s i z e (L) and CL cavity (1 25 OVARIAN ACTIVITY IN POSTPARTUM COWS There was no c o r r e l a t i o n between maximum corpus luteum diameter and maximum plasma P 4 c o n c e n t r a t i o n i n the f i r s t postpartum e s t r o u s c y c l e however t h e r e a s t r o n g c o r r e l a t i o n (r=0.88) i n the second c y c l e ( F i g . 2.1). D u r a t i o n of the f i r s t o r second postpartum e s t r o u s c y c l e s were not c o r -r e l a t e d t o the maximum diameter of the corpus luteum. The r e t u r n t o r e p r o d u c t i v e c y c l e s f o l l o w e d one of two d i s t i n c t p a t t e r n s ( F i g 2.2): a v e r y s h o r t a n o v u l a t o r y p e r i o d of.two weeks or l e s s , f o l l o w e d by a normal l e n g t h e d e s t r o u s c y c l e w i t h an adequate l u t e a l phase and a normal P4 p r o f i l e ( e a r l y ) , or, a prolonged a n o v u l a t o r y p e r i o d of t h r e e weeks or g r e a t e r , f o l l o w e d by a s h o r t e s t r o u s c y c l e w i t h an inadequate l u t e a l phase w i t h a subnormal P4 p r o f i l e ( d e l a y e d ) . There were g e n e r a l l y two waves of f o l l i c u l a r growth d u r i n g an e s t r o u s c y c l e , each wave r e s u l t i n g i n a l a r g e dominant f o l l i c l e , as r e v e a l e d by u l t r a s o u n d imaging; o n l y t h r e e of twenty-one c y c l e s had t h r e e waves, a l l o t h e r s had two waves, i n c l u d i n g s h o r t c y c l e s ( F i g . 2.3a, b & c) . T y p i c a l l y the f i r s t wave of growth commenced on day 2.5 ± 0.9 of normal lengthed c y c l e s (day 0 = e s t r u s ) and r e s u l t e d i n a l a r g e mid c y c l e f o l l i c l e o f 16.5 ± 2.5 mm i n diameter. T h i s f o l l i c l e was maintained u n t i l day 14.1 ± 1.0 of the c y c l e when i t began t o r e g r e s s . The second wave of f o l l i c u l a r growth commenced on day 15.8 ± 1.8 and r e s u l t e d i n a l a r g e o v u l a t o r y f o l l i c l e (20.9 ± 3.9 mm). E a r l y d u r i n g each wave of 26 OVARIAN ACTIVITY IN POSTPARTUM COWS Fig 2.1 Correlation between corpus luteum diameter and circulating progesterone in postpartum dairy cattle I | , 25 30 35 40 Corpus Luteum Diameter (mm) 27 O V A R I A N A C T I V I T Y I N P O S T P A R T U M C O W S Fig 2.2 Patterns of return to reproductive cycles in postpartum dairy cattle 28 OVARIAN ACTIVITY IN POSTPARTUM COWS growth a single f o l l i c l e appeared to become dominant and continued growing while other v i s i b l e f o l l i c l e s on each ovary began to decrease i n s i z e . The P4 p r o f i l e s from the whole milk samples followed a s i m i l a r pattern to those from the plasma samples (Fig. 2.4). Milk progesterone concentrations tended to s t a r t increasing and generally peaked on the same days as d i d the plasma conc-entrations, however, peak milk P4 concentrations tended to be much more var i a b l e and much higher than the plasma concentrations. 2 9 OVARIAN ACT IV ITY IN POSTPARTUM COWS F i g . 2.3. Growth of dominant f o l l i c l e s i n 3 r e p r e s e n t a t i v e postpartum d a i r y cows. 45 - i 40 -D 35 -a 30 -m e 25 -t e 20 -r 15 -m 10 -m 5-0 -Animal 85013 (Early) Ovulation 10 45 - i 40 -D 35 -a 30 -m e 25 -t e 20 -r 15 -m 10 -m 5 -0 -Animal 32031 (Medium) Ovulation Estrus Estrus 10 45 - i 40 -D i 35 -a 30 -m e 25 -t e 20 -r 15 -m 10 -m 5 -o -• Animal 83002 (Prolonged) Ovulation E3trus j v + * i * * , 4 1 1 r i 1 1 1 1 1 1 10 15 2 0 25 30 3 5 40 4 5 50 5 5 60 65 70 7 5 80 85 Days Postpartum F o l l i c l e Le f t O v a r y C L 1st C y c l e ••*•• Fo l l i c le R ight O v a r y - * > _ C L 2nd C y c l e 3 0 OVARIAN ACTIVITY IN POSTPARTUM COWS Fig . 2.4 Compar ison between p lasma and milk progesterone in postpartum dairy cat t le 10 9 -8 -7 -P 4 6 -5 10 15 20 Days From Est rus (estrus * day 0) plasma progesterone IMMimilk progesterone 31 OVARIAN ACTIVITY IN POSTPARTUM COWS DISCUSSION The average time t o the f i r s t postpartum o v u l a t i o n of 21 days and the f i n d i n g t h a t 8 of 10 animals o v u l a t e d by 25 days postpartum agree w e l l w i t h e a r l i e r f i n d i n g s based on slaughterhouse and hormonal s t u d i e s showing l u t e a l a c t i v i t y i n 88% o f animals by 35 days a f t e r c a l v i n g ( B a l l and Lamming, 1983) and 95% by 50 days (Morrow e t a l . , 1966). U l t r a s o u n d imaging r e v e a l e d by the presence of a corpus luteum-that f o u r animals had o v u l a t e d by 14 days postpartum. T h i s was l a t e r c onfirmed by the P 4 assays which showed an i n c r e a s e i n c i r c u l a t i n g P 4 i n these animals. Scanning p r i o r t o 14 days postpartum was not p o s s i b l e due t o the s i z e of the u t e r u s . O v a r i a n f o l l i c l e s have been d e t e c t e d e a r l y i n t h e second week postpartum by r e c t a l p a l p a t i o n (Saiduddin e t a l . , 1968) and s l a u g h t e r h o u s e s t u d i e s have found o v a r i a n changes due t o f o l l i c l e growth 5 t o 7 days postpartum (Morrow e t a l . , 1966). The i n t e r v a l from c a l v i n g t o the f i r s t observed e s t r u s was g e n e r a l l y l o n g e r and much more v a r i a b l e . Only one animal of the t e n was observed i n e s t r u s a t the f i r s t postpartum o v u l a t i o n . During the p r e s e n t study p r i m i p a r o u s animals tended (P<0.1) t o take l o n g e r t o show t h e i r f i r s t postpartum e s t r u s than p l u r i p a r o u s animals. T h i s agrees w i t h s t u d i e s i n beef c a t t l e (King and McLoed, 1984). However, time t o f i r s t observed e s t r u s must be looked a t with c a u t i o n , i t depends g r e a t l y on the e f f i c i e n c y of e s t r u s d e t e c t i o n . Primiparous 32 OVARIAN ACTIVITY IN POSTPARTUM COWS animals were housed i n c a l v i n g pens i s o l a t e d from the main herd f o r a l o n g e r p e r i o d than p l u r i p a r o u s animals. A d d i t i o n -a l l y , animals housed i n c a l v i n g pens were kept i n a sep e r a t e h o l d i n g pen d u r i n g m i l k i n g and t h e r e f o r e t h e i r exposure t o oth e r animals i n e s t r u s was minimal. These p r a c t i c e s may c o n t r i b u t e t o the a p p a r e n t l y l o n g e r p e r i o d o f a n e s t r u s seen i n t he f i r s t c a l v e r s i n the U n i v e r s i t y o f B r i t i s h Columbia herd. Another c o n t r i b u t i n g f a c t o r may be the b e h a v i o r o f the animals. F i r s t c a l v i n g h e i f e r s u s u a l l y a re s m a l l e r and l e s s a g g r e s s i v e which may be expressed as l e s s mounting b e h a v i o r than n o r m a l l y seen i n p l u r i p a r o u s cows, however few s t u d i e s have looked a t t h i s . Only two animals (83002 & 84001) had pr o l o n g e d anovu-l a t o r y p e r i o d s g r e a t e r than 25 days, one of which (84001) developed a l a r g e p e r s i s t a n t f o l l i c l e on the l e f t ovary. The spontaneous o v u l a t i o n o f a 26mm f o l l i c l e on the c o n t r a l a t e r a l ovary corresponded w i t h the r e g r e s s i o n o f the p e r s i s t e n t f o l l i c l e . The f a c t t h a t the l a r g e f o l l i c l e had begun t o re g r e s s as i n d i c a t e d by u l t r a s o n o g r a p h i c imaging suggests t h a t i t had stopped s e c r e t i n g l a r g e amounts o f e s t r o g e n s . I t a l s o suggests t h a t i t had not become l u t e i n i z e d and was not s e c r e t i n g P 4 . T h i s was confirmed by the P4 assays which showed the animal t o have b a s a l P4 l e v e l s u n t i l a f t e r o v u l a t i o n . The o t h e r animal (83002) wi t h an extended a n o v u l a t o r y p e r i o d appeared t o have normal f o l l i c u l a r development and 33 OVARIAN ACTIVITY IN POSTPARTUM COWS t u r n o v e r but f a i l e d t o o v u l a t e e a r l y i n t h e postpartum p e r i o d . The reason f o r t h i s f a i l u r e i s not known but may i n c l u d e i n s u f f i c i e n t e s t r a d i o l s e c r e t i o n t o produce an LH surge, a prolonged postpartum r e f r a c t o r y p e r i o d o f the h y p o t h a l a m u s - p i t u i t a r y a x i s , inadequate LH a v a i l a b l e t o produce a surge o r i n s u f f i c i e n t LH r e c e p t o r s on the p r e -o v u l a t o r y f o l l i c l e . The f i r s t p o s t p a r t u m e s t r o u s c y c l e i n t h i s cow was c h a r a c t e r i z e d by a s m a l l corpus luteum o f 23mm wit h a 5 day l i f e span and v e r y low P4 s e c r e t i o n s u p p o r t i n g the p o s s i b i l i t y o f an LH d e f i c i e n c y . Hypophysectomy i n the f i r s t t h r e e days f o l l o w i n g o v u l a t i o n r e s u l t s i n e a r l y r e g r e s s i o n o f the CL (Niswender e t a l . , 1986). The second postpartum c y c l e was of normal d u r a t i o n w i t h normal plasma P4 c o n c e n t a t i o n s . O v e r a l l t he f i r s t postpartum e s t r o u s c y c l e was found t o be s i g n i f i c a n t l y s h o r t e r w i t h lower peak P4 l e v e l s than the second c y c l e , thus c o n f i r m i n g p r e v i o u s r e p o r t s (Robertson, 1972; Edgerton and Hafs, 1973; Manns e t a l . , 1983). However, l o o k i n g a t i n d i v i d u a l animals i t was found t h a t t he decrease i n f i r s t c y c l e l e n g t h was due t o a g r e a t l y reduced l u t e a l phase i n f o u r o f the t e n cows s t u d i e d , the remaining s i x cows a l l had c y c l e s which f e l l i n the normal range o f 18-22 days and normal P4 p r o f i l e s . The cause of the s h o r t l u t e a l phase i s not known. Maximum diameter of the corpus luteum i n the f i r s t postpartum l u t e a l phase c o u l d not be c o r r e l a t e d t o i t s d u r a t i o n . A d d i t i o n a l l y , one animal with a c y c l e l e s s than 14 34 OVARIAN ACTIVITY IN POSTPARTUM COWS days had a corpus luteum of g r e a t e r than 3 0mm s u g g e s t i n g t h a t diameter o f t h e corpus luteum i s not the l i m i t i n g f a c t o r i n the f i r s t postpartum c y c l e . Plasma P4 was lower i n the f i r s t c y c l e and was not c o r r e l a t e d t o the diameter of the corpus luteum. I t i s l i k e l y t h a t c o r p o r a l u t e a of c y c l e s l e s s than 14 days i n l e n g t h begin t o r e g r e s s b e f o r e they reach t h e i r peak P4 production... There has been the s u g g e s t i o n t h a t PGF2a s e c r e t e d d u r i n g u t e r i n e i n v o l u t i o n may be r e s p o n s i b l e f o r premature l u t e o -l y s i s e a r l y i n the postpartum p e r i o d (Hanzen, 1986) but s e v e r a l o b s e r v a t i o n s i n the p r e s e n t study appear t o c o n t r a -d i c t t h i s p o s s i b i l i t y . Two of the f o u r animals which had f i r s t postpartum c y c l e s l e s s than 14 days i n d u r a t i o n were the two animals w i t h prolonged anovulatory p e r i o d s . P r e v i o u s s t u d i e s suggest t h a t PGF2a l e v e l s have r e t u r n e d t o b a s a l c o n c e n t r a t i o n s by t h r e e weeks pos t c a l v i n g ( L i n d e l l e t a l . , 1983) and t h e r e f o r e p r o s t a g l a n d i n should not have been a f a c t o r i n s h o r t e n i n g the c y c l e l e n g t h i n these two animals. The remaining two animals w i t h s h o r t f i r s t postpartum e s t r o u s c y c l e s (82031 & 85018) o v u l a t e d on day 22 and day 23 p o s t -partum. U t e r i n e r e g r e s s i o n i n both animals was complete by 26 days postpartum s u g g e s t i n g t h a t PGF2a l e v e l s would have been e l e v a t e d e a r l y i n the c y c l e s , a time when PGF2 a has no e f f e c t on c y c l e l e n g t h ( F r e d r i k s s o n e t a l . , 1988). These o b e r v a t i o n s suggest t h a t f a c t o r s o t h e r than PGF2a may be i n v o l v e d i n d e t e r m i n i n g the d u r a t i o n of the f i r s t postpartum e s t r o u s 35 OVARIAN ACTIVITY IN POSTPARTUM COWS c y c l e . T h e p r e s e n t s t u d y w o u l d n o t a p p e a r t o s u p p o r t t h e h y p o t h e s i s o f i n a d e q u a t e F S H ( R a m i r e z - G o d i n e z e t a l . , 1982) a s a l l a n i m a l s h a d g r o w t h o f a n t r a l f o l l i c l e s s t a r t i n g e a r l y i n t h e - p o s t p a r t u m p e r i o d . I t i s p o s s i b l e t h a t i n a d e q u a t e L H s u p p o r t d u r i n g t h e l u t e a l p h a s e o r p o o r r e s p o n s e t o L H d u e t o i n s u f f i c i e n t r e c e p t o r s may b e t h e c a u s e o f t h e i n a d e q u a t e l u t e a l p h a s e , h o w e v e r i t i s i m p o s s i b l e t o t e l l f r o m t h e p r e s e n t s t u d y . S e v e r a l r e p o r t s s u g g e s t t h a t t h e c o r p u s l u t e u m o f t h e j u s t t e r m i n a t e d p r e g a n c y may h a v e a n e g a t i v e e f f e c t o n f o l -l i c u l a r g r o w t h i n t h a t o v a r y ( D u f o u r a n d R o y , 1985) . T h e f i r s t p o s t p a r t u m o v u l a t i o n i n t h e p r e s e n t s t u d y was u n a f f e c t e d b y t h e s i d e o f p r e g n a n c y , f o u r o f t e n a n i m a l s h a d t h e i r f i r s t o v u l a t i o n o n t h e same s i d e a s t h e p r e v i o u s p r e g n a n c y . H o w e v e r , two o f t h e s e a n i m a l s w e r e a n i m a l s w i t h a n a n o v u l a t o r y p e r i o d o f g r e a t e r t h a n 28 d a y s . F o l l i c u l a r g r o w t h may h a v e b e e n i n f l u e n c e d b y t h e s i d e o f p r e g n a n c y , a t 14 d a y s p o s t p a r t u m t h e l a r g e s t f o l l i c l e was o n t h e same s i d e a s t h e p r e v i o u s p r e g n a n c y i n o n l y two a n i m a l s l e n d i n g some s u p p o r t t o t h e p o s s i b l e n e g a t i v e i n f l u e n c e o f t h e c o r p u s l u t e u m o f p r e g n a n c y o n f o l l i c u l a r g r o w t h e a r l y i n t h e p o s t p a r t u m p e r i o d . T h e s e c o n d p o s t p a r t u m c y c l e was g e n e r a l l y much l e s s v a r i a b l e i n l e n g t h t h a n t h e f i r s t c y c l e , a l t h o u g h o n e a n i m a l d i d h a v e a p r o l o n g e d c y c l e o f 36 d a y s . A s w i t h t h e f i r s t c y c l e t h e d u r a t i o n o f t h e s e c o n d e s t r u s c y c l e was n o t c o r -36 OVARIAN ACTIVITY IN POSTPARTUM COWS r e l a t e d t o t h e maximum d i a m e t e r o f t h e c o r p u s l u t e u m , h o w -e v e r , t h e r e was a s t r o n g c o r r e l a t i o n ( r = 0 . 8 8 ) b e t w e e n maximum c o r p u s l u t e u m d i a m e t e r a n d p e a k p l a s m a P4 c o n c e n t r a t i o n s . I t seems r e a s o n a b l e t o s u s p e c t t h a t t h e d i a m e t e r o f t h e o v u l a t o r y f o l l i c l e i s p o s i t i v e l y c o r r e l a t e d t o t h e maximum c o r p u s l u t e u m d i a m e t e r , h o w e v e r t h e p r e s e n t s t u d y was u n a b l e t o c o n f i r m t h i s . A n i m a l s w e r e s c a n n e d o n l y t h r e e t i m e s p e r w e e k s o n o t a l l a n i m a l s w e r e s c a n n e d o n t h e d a y o f e s t r u s o r o v u l a t i o n a n d t h e r e f o r e t h e maximum d i a m e t e r o f t h e o v u l a t o r y f o l l i c l e was n o t known i n a l l a n i m a l s . U l t r a s o u n d i m a g i n g r e v e a l e d t h a t f o l l i c u l a r g r o w t h o c c u r r e d i n two w a v e s i n t h e v a s t m a j o r i t y o f c y c l e s . T h i s p a t t e r n o f g r o w t h s u p p o r t s t h e f i n d i n g s o f P e i r s o n a n d G i n t h e r (1987) i n n o r m a l l y c y c l i n g h e i f e r s . S h o r t c y c l e s a l s o a p p e a r e d t o h a v e two w a v e s o f f o l l i c l e g r o w t h h o w e v e r t h e r e s u l t i n g m i d c y c l e f o l l i c l e was n o t m a i n t a i n e d b u t r a t h e r b e g a n t o r e g r e s s i m m e d i a t e l y . O t h e r s h a v e r e p o r t e d t h a t t h e m a j o r i t y o f a n i m a l s s t u d i e d h a d t h r e e w a v e s o f g r o w t h ( S i r o i s a n d F o r t u n e , 1 9 8 8 ; S a v i o e t a l . , 1 9 8 8 ) . I n t h e p r e s e n t s t u d y o n l y 3 o f 21 c y c l e s f o l l o w e d h a d t h r e e w a v e s a n d o f t h o s e t h r e e , t w o w e r e c y c l e s o f g r e a t e r t h a n 2 2 d a y s . T h e P4 p r o f i l e s f r o m w h o l e m i l k s a m p l e s f o l l o w e d t h e p l a s m a p r o f i l e s c l o s e l y w i t h r e g a r d t o t h e p a t t e r n o f s e c r e -t i o n a n d c o n f i r m e d c o n c l u s i o n s d r a w n f r o m t h e u l t r a s o u n d i m a g e s . A s h a s b e e n p r e v i o u s l y r e p o r t e d ( M a t h e r e t a l . , 1 9 8 8 ; K a s s a e t a l . , 1986) m i l k P4 l e v e l s a p p e a r t o p r o v i d e a 37 OVARIAN ACTIVITY IN POSTPARTUM COWS r e l i a b l e means of a s s e s s i n g l u t e a l f u n c t i o n i n the postpartum d a i r y cow and can now be taken advantage o f by the commercial d a i r y farmer u s i n g a cowside progesterone k i t t o i d e n t i f y t r u l y a n e s t r u s / a n o v u l a t o r y animals. U l t r a s o u n d imaging was u s e f u l f o r m o n i t o r i n g ovarian, a c t i v i t y i n postpartum animals. I t s non i n v a s i v e nature a l l o w s animals t o be monitored r e p e t i t i v e l y so growth of o v a r i a n s t u c t u r e s can be f o l l o w e d i n a s i n g l e animal. 38 FOLLICULAR AND CL GROWTH IN PREGNANT COWS CHAPTER 3 FOLLICULAR DYNAMICS AND CORPUS LUTEUM GROWTH AND FUNCTION DURING THE FIRST 60 DAYS OF PREGNANCY IN DAIRY CATTLE ABSTRACT T h e e f f e c t s o f p r e g n a n c y o n f o l l i c u l a r d y n a m i c s , a n d c o r p u s l u t e u m g r o w t h a n d f u n c t i o n a r e s o m e w h a t c o n t e n t i o u s . I n a n e f f o r t t o l e a r n m o r e a b o u t t h e e f f e c t s o f p r e g n a n c y , t h e r e p r o d u c t i v e t r a c t s o f 16 b r e d d a i r y cows w e r e m o n i t o r e d u s i n g a l i n e a r a r r a y u l t r a s o u n d d e v i c e e q u i p p e d w i t h a r e c t a l p r o b e t h r o u g h t h e f i r s t 60 d a y s o f p r e g n a n c y o r a r e t u r n t o e s t r u s . I n a d d i t i o n , b l o o d s a m p l e s w e r e c o l l e c t e d f o r p r o g e s t e r o n e d e t e r m i n a t i o n . O f t h e s i x t e e n c o w s , e l e v e n w e r e d i a g n o s e d p r e g n a n t o n t h e b a s i s o f v i s u a l i z a t i o n o f a n e m b r y o w i t h a h e a r t b e a t b y 25 d a y s p o s t a r t i f i c i a l i n s e m i n a t i o n . T h e r e w e r e two e m b r y o n i c m o r t a l i t i e s b e t w e e n 28 a n d 32 d a y s . A two w a v e p a t t e r n o f f o l l i c u l a r g r o w t h a n d a t r e s i a , w i t h e a c h wave r e s u l t i n g i n a l a r g e d o m i n a n t f o l l i c l e , was s e e n i n b o t h p r e g n a n t a n d n o n p r e g n a n t c o w s . P r e g n a n t cows h a d s i g n i f i c a n t l y m o r e f o l l i c l e s (P<0 .05) t h a n n o n p r e g n a n t c o w s ; h o w e v e r t h e r e was no d i f f e r e n c e i n t h e s i z e o f t h e l a r g e s t f o l l i c l e p r e s e n t o n t h e o v a r i e s . A w a v e - l i k e p a t t e r n o f g r o w t h o f l a r g e f o l l i c l e s c o n t i n u e d t h r o u g h o u t t h e s t u d y 39 F O L L I C U L A R AND C L GROWTH I N PREGNANT COWS p e r i o d i n t h e p r e g n a n t c o w s . P r o g e s t e r o n e p r o f i l e s f o r t h e f i r s t 18 d a y s , c o r p u s l u t e u m g r o w t h r a t e a n d maximum s i z e o f t h e c o r p u s l u t e u m w e r e s i m i l a r i n b o t h p r e g n a n t a n d n o n p r e g n a n t c o w s . I t i s c o n c l u d e d t h a t p r e g n a n c y h a s l i t t l e e f f e c t o n t h e g r o w t h o f d o m i n a n t f o l l i c l e s d u r i n g t h e f i r s t 60 d a y s o f p r e g n a n c y a n d h y p o t h e s i z e d t h a t l a r g e f o l l i c l e s s e r v e a s a s a f e g u a r d t o i n s u r e a \" q u i c k r e t u r n t o e s t r u s i n t h e e v e n t o f e m b r y o n i c m o r t a l i t y . I t i s a l s o c o n c l u d e d t h a t p r e g n a n c y h a s l i t t l e e f f e c t o n c o r p u s l u t e u m g r o w t h a n d f u n c t i o n f o r t h e f i r s t 60 d a y s o t h e r t h a n m a i n t e n a n c e b e y o n d i t s n o r m a l l i f e s p a n . I N T R O D U C T I O N C y c l i n g cows g e n e r a l l y h a v e two o r t h r e e w a v e s o f f o l -l i c u l a r g r o w t h d u r i n g a n o r m a l e s t r o u s c y c l e ( R a j a m a h e n d r a n a n d T a y l o r , 1 9 8 8 ; S i r o i s a n d F o r t u n e , 1 9 8 8 ; P e i r s o n a n d G i n t h e r , 1987) e a c h wave c o n s i s t i n g o f a p o o l o f s m a l l f o l l i c l e s a t t h e s t a r t o f t h e w a v e , f o l l o w e d b y a p e r i o d o f g r o w t h a n d e n d i n g w i t h t h e g r o w t h o f a l a r g e d o m i n a n t f o l l i c l e w h i l e t h e o t h e r s b e c o m e a t r e t i c . I t i s u n c e r t a i n w h e t h e r t h i s w a v e - l i k e p a t t e r n o f f o l l i c u l a r g r o w t h a n d a t r e s i a o c c u r s i n p r e g n a n t a n i m a l s . E a r l i e r s t u d i e s ( P e i r s o n a n d G i n t h e r , 1 9 8 7 ; R e x r o a d a n d C a s i d a , 1978) s h o w e d t h a t a f t e r t h e f i r s t t w e n t y d a y s o f p r e g n a n c y f o l l i c u l a r g r o w t h 4 0 FOLLICULAR AND CL GROWTH IN PREGNANT COWS s l o w s , t h e s i z e o f t h e l a r g e s t f o l l i c l e d e c r e a s e s a n d t h e c o r p u s l u t e u m (CL) h a s a n e g a t i v e e f f e c t o n f o l l i c u l a r g r o w t h o n t h e i p s i l a t e r a l o v a r y . One s t u d y ( G u i l b a u l t e t a l . , 1986) f o u n d t h a t b y 17 d a y s p o s t e s t r u s p r e g n a n t c o w s h a d f e w e r a t r e t i c f o l l i c l e s i n t h e s m a l l e r s i z e c l a s s e s w h i l e l a r g e r s i z e c l a s s e s h a d a h i g h e r p r o p o r t i o n o f a t r e t i c f o l l i c l e s t h a n n o n p r e g n a n t c o w s . I t was c o n c l u d e d t h a t t h e p r e s e n c e o f a c o n c e p t u s p r o m o t e s a m o r e r a p i d t u r n o v e r o f s m a l l f o l l i c l e s t o m e d i u m s i z e d o n e s a n d t h e n l i m i t s f u r t h e r g r o w t h b y i n c r e a s i n g a t r e s i a . I n a d d i t i o n , i t h a s b e e n s u g g e s t e d t h a t o n e o f t h e m a j o r d i f f e r e n c e s b e t w e e n p r e g n a n t a n d n o n p r e g n a n t cows w i t h r e g a r d t o o v a r i a n f u n c t i o n i s t h e a b s e n c e o f a l a r g e o v u l a t o r y f o l l i c l e i n p r e g n a n t a n i m a l s t h u s r e m o v i n g t h e l u t e o l y t i c e f f e c t s o f e s t r a d i o l - 1 7 / 3 ( E 2 ) ( T h a t c h e r e t a l . , 1 9 8 8 ) . C o n c e p t u s r e m o v a l s t u d i e s a n d t h e t r a n s f e r o f e m b r y o s t o n o n b r e d cows ( B e t t e r i d g e e t a l . , 1 9 8 0 ; N o r t h e y a n d F r e n c h , 1980) h a v e s h o w n t h a t t h e c o n c e p t u s t r a n s m i t s a s i g n a l t o t h e m a t e r n a l c o m p a r t m e n t a p p r o x i m a t e l y 15 d a y s a f t e r f e r t i l i z -a t i o n t o p r e v e n t l u t e o l y s i s b y p r o s t a g l a n d i n F 2 A ( P G F 2 A ) . W h e t h e r t h i s s i g n a l i n c l u d e s a l u t e o t r o p h i c c o m p o n e n t h a s n o t b e e n s h o w n , a l t h o u g h some r e p o r t s s u g g e s t t h a t p r e g n a n t cows h a v e h i g h e r p l a s m a ( L u k a s z e w s k a a n d H a n s e l , 1980) a n d m i l k ( L a m m i n g e t a l . , 1 9 8 9 ; B u l m a n n a n d L a m m i n g , 1978) p r o g e s t e r -t e r o n e ( P 4 ) c o n c e n t r a t i o n s t h a n n o n p r e g n a n t a n i m a l s a s e a r l y a s 10 d a y s p o s t e s t r u s . 41 FOLLICULAR AND CL GROWTH IN PREGNANT COWS The aim of the pr e s e n t study was t o f u r t h e r e l u c i d a t e the e f f e c t s o f the e a r l y conceptus on f o l l i c u l a r dynamics, and CL growth and f u n c t i o n . U l t r a s o u n d imaging was used t o monitor o v a r i a n dynamics w i t h r e g a r d t o f o l l i c u l a r growth and a t r e s i a , CL growth and i d e n t i f i c a t i o n o f embryonic v e s i c l e s and the embryo proper. The i n c i d e n c e o f embryonic m o r t a l i t y and plasma P4 were a l s o measured. M a t e r i a l s and Methods S i x t e e n postpartum d a i r y cows r a n g i n g from 2k t o 8 years of age were s e l e c t e d a t random from the U n i v e r s i t y o f B r i t i s h Columbia d a i r y herd. A l l cows were housed i n a c l o s e d f r e e s t a l l barn and r e c e i v e d a mix of a l f a l f a cubes and co n c e n t r a t e i n equal p r o p o r t i o n s . T o t a l r a t i o n was a d j u s t e d a c c o r d i n g t o the cows m i l k p r o d u c t i o n . A l l cows were bred ap-pr o x i m a t e l y 12 hours a f t e r the onset o f s t a n d i n g e s t r u s between 60 and 90 days po s t c a l v i n g . U l t r a s o u n d imaging commenced one day p o s t A l . Cows were scanned d a i l y u n t i l 20 days and then t h r i c e weekly u n t i l 60 days o f pregnancy o r a r e t u r n t o e s t r u s . U l t r a s o u n d examinations were performed u s i n g a l i n e a r a r r a y r e a l - t i m e u l t r a s o u n d d e v i c e (Tokyo K e i k i LS3 00, Tokyo K e i k i Co. L t d . Tokyo, Japan) equipped with a 5 MHz r e c t a l probe. A f t e r the ev a c u a t i o n o f f e c e s , the probe was p l a c e d i n t r a r e c t a l l y and 42 FOLLICULAR AND CL GROWTH IN PREGNANT COWS the r e p r o d u c t i v e t r a c t examined. The o v a r i e s were scanned i n s e v e r a l p l a n e s t o i d e n t i f y a l l f o l l i c l e s > 2mm i n diameter and t o p r o v i d e an image o f the CL w i t h i t s g r e a t e s t c r o s s s e c t i o n a l a r e a . D e s i r e d images were f r o z e n on the screen, measurements taken u s i n g a b u i l t - i n c a l i p e r system and a hard copy made u s i n g a v i d e o p r o c e s s i n g u n i t ( M i t s u b i s h i E l e c t r o n i c s Co. L t d . , Tokyo, Japan). The u t e r i n e horns and body were scanned over t h e i r e n t i r e l e n g t h and i n s e v e r a l p l a n e s i n o r d e r t o examine u t e r i n e c o n t e n t s , and t o i d e n t i f y embryonic v e s i c l e s and the embryo proper. As wit h the o v a r i e s , d e s i r e d images were f r o z e n and hard c o p i e s made. Blood samples were c o l l e c t e d from a t a i l v e i n i n t o hep-a r i n i z e d tubes p r i o r t o each u l t r a s o u n d examination. Plasma was s e p a r a t e d and s t o r e d a t -20°C f o r P4 a n a l y s i s . P4 was measured by a commercially a v a i l a b l e s o l i d phase RIA system ( D i a g n o s t i c s Corp., Los Angeles, C a l i f o r n i a ) . Plasma or r e f e r e n c e s t a n d a r d (0.1 ml) was added t o tubes coated w i t h a s p e c i f i c a n t i b o d y . Reference standards c o n t a i n e d between 0 and 40 ng/ml P 4. B u f f e r e d T.125 p 4 ( 1 . 0 ml) was added t o a l l tubes, v o r t e x e d and l e f t t o incubate f o r t h r e e hours. Tubes were decanted a f t e r the i n c u b a t i o n p e r i o d and c l e a n e d w i t h a c o t t o n swab above the 1 ml mark t o remove excess plasma or t r a c e r . Tubes were counted f o r 1 minute i n a gamma counter (Packard Auto-Gamma 500). I n t r a and i n t e r assay c o e f f i c i e n t s of v a r i a t i o n were 6.2% and 9.8%, r e s p e c t i v e l y and the l i m i t of d e t e c t i o n was 0.05 ng/ml. 43 FOLLICULAR AND CL GROWTH IN PREGNANT COWS A n i m a l s r e t u r n i n g t o e s t r u s p r i o r t o t h e i d e n t i f i c a t i o n o f a n e m b r y o p r o p e r w e r e deemed t o b e n o n p r e g n a n t . T o t a l n u m b e r o f f o l l i c l e s , d i a m e t e r o f t h e l a r g e s t f o l l i c l e p r e s e n t , d i a m e t e r o f t h e C L a n d P4 c o n c e n t r a t i o n s o n s p e c i f i c d a y s p o s t b r e e d i n g w e r e c o m p a r e d b e t w e e n p r e g n a n t a n d n o n p r e g n a n t c o w s u s i n g a n a n a l y s i s o f v a r i a n c e f o r r e p e a t e d m e a s u r e s ( S A S , 1 9 8 5 ) . T o t a l n u m b e r o f f o l l i c l e s o n t h e C L b e a r i n g o v a r y a n d o n t h e c o n t r a l a t e r a l o v a r y w e r e a l s o c o m p a r e d u s i n g a n ANOVA f o r r e p e a t e d m e a s u r e s . RESULTS C h a n g e s i n t h e a p p e a r a n c e o f t h e u t e r u s , d e v e l o p m e n t o f t h e f e t u s a n d i m a g e s o f t h e C L b e a r i n g o v a r y i n a r e p r e s e n t -a t i v e p r e g n a n t cow a r e shown i n P l a t e s 3 . 1 & 3 . 2 . P r e s u m p t i v e e m b r y o n i c v e s i c l e s w e r e s e e n i n 14 o f t h e 16 c o w s e x a m i n e d ( T a b l e 3 . 1 ) ; h o w e v e r o n l y 11 a n i m a l s w e r e d i a g n o s e d p r e g n a n t b a s e d o n v i s u a l i z a t i o n o f a n e m b r y o p r o p e r w i t h a h e a r t b e a t . T h e r e w e r e two e m b r y o n i c m o r t a l i t i e s , a s d i a g n o s e d b y t h e c e s s a t i o n o f t h e e m b r y o n i c h e a r t b e a t , o n e b e t w e e n 28 a n d 30 d a y s p o s t b r e e d i n g a n d t h e o t h e r b e t w e e n d a y 30 a n d 3 2 . T h e s e two c o w s r e t u r n e d t o e s t r u s 4 0 a n d 63 d a y s p o s t b r e e d i n g , r e s p e c t i v e l y , s h o r t l y a f t e r l u t e o l y s i s h a d t a k e n p l a c e . I n t h e f i r s t 24 d a y s f o l l o w i n g b r e e d i n g , a l l a n i m a l s h a d two w a v e s o f f o l l i c u l a r g r o w t h . T h e f i r s t wave r e s u l t e d i n a F O L L I C U L A R A N D C L G R O W T H I N P R E G N A N T C O W S P l a t e 3 . 1 . U l t r a s o u n d images of the CL b e a r i n g ovary and the g r a v i d u t e r i n e horn i n a pregnant d a i r y cow. J u l / 0 6 ' 8 8 l l : l 4 I D : 82919 Probe: •5 MHz Distance: area: C i r c : Page 1 19 D ft V S POST P I HORN • N I C L L I C L E S r f c U I T H 1 0 P R O P E R l Note large mid-cycle fo l l ic le (short arrow) and CL (long arrow). Note embryonic vesicle (long arrow) now visible 1n the uterus. M1d-cycle fo l l ic le 1s now decreasing 1n diameter. Embryo proper 1s clearly visible (long arrow). M1d-cycle fo l l ic le 1s no longer visible. No change 1n diameter of CL. Jul/ 1 1 ^ 8 8 1 3 : 2 ? ID: 32019 Probe: 5 MHz Distance: Area: C i r c : Page 1 2 4 D ft V S P O S T H i ITH!^GW-\"H13^' B O T H f s W * H<3k< R I G H T C V R R V 5MM EWBRVO * ~ 3 4 X 21WBL'- ' -P O S S I B L E HEORTI 1 0 M M F D L L I C L E 45 FOLLICULAR AND CL GROWTH IN PREGNANT COWS P l a t e 3.2. U l t r a s o u n d images o f the CL b e a r i n g ovary and the g r a v i d u t e r i n e horn i n a pregnant d a i r y cow. Jul • 2 2/-8 8 1 1 : 5 9 ID : 82919 Probe: 5 MHz Distance: 1 8 mm Area: Pa*e 1 35 DPVS P O S T fl I 16MM E M B * ^ Q » ~ H E A R T B E A T \" OBSEI RIGHT OUflRV 30X25MM CL 7MM F O L L I C J - E _ Fetal limb buds are now visible (short arrow). Also note presence of the amnion (long arrow). Aug' 0 2 / 8 8 1 3 : 1 0 I D: 82019 Probe: 5MHz Distance: Page 2 4 5 D A V S POST PI 25MM E M B R ^ t K ^ R^OURRV * w f r t ^ f f t t L i re L I Note fetal heart surrounded by pericardium (short arrow). Also note new dominant fo l l ic le on the CL bearing ovary (long arrow). Fetal limbs are clearly visible (short arrows). Also note ossification of the skul1 (long arrow). Rug/ 1 7 / 8 8 1 2 : 4 3 ID : 82919 Probe: 5MHz Distance: Rrea: C i r c : Page 2 60 DRVS P O S T RI 46 FOLLICULAR AND CL GROWTH IN PREGNANT COWS T a b l e 3.1. I n t r a u t e r i n e s t r u c t u r e s v i s u a l i z e d on d i f f e r e n t days p o s t b r e e d i n g u s i n g u l t r a s o u n d imaging i n d a i r y c a t t l e . S t u c t u r e s v i s u a l i z e d % Animals Days p o s t b r e e d i n g Presumptive Embryonic V e s i c l e s 88% (14/16) 15.4 + 2.7 Embryo Proper 69% (11/16) 23.9 + 1.5 Embryonic Heartbeat 69% (11/16) 25.0 + 1.1 Embryonic Heartbeat 56% (9/16) 60 47 FOLLICULAR AND CL GROWTH IN PREGNANT COWS m i d c y c l e f o l l i c l e w h i c h a t t a i n e d i t s maximum s i z e 8 . 8 ± 1 . 5 d a y s p o s t b r e e d i n g a n d p e r s i s t e d u n t i l d a y 1 6 . 1 ± 2 . 0 when i t was r e p l a c e d b y a s e c o n d d o m i n a n t f o l l i c l e f r o m t h e s e c o n d w a v e o f f o l l i c u l a r g r o w t h . T h e r e was no d i f f e r e n c e b e t w e e n p r e g n a n t a n d n o n p r e g n a n t cows i n t h e maximum d i a m e t e r a t t a i n e d b y e i t h e r o f t h e d o m i n a n t f o l l i c l e s ( T a b l e 3 . 2 ) , h o w e v e r , t h e s e c o n d d o m i n a n t f o l l i c l e r e s u l t e d i n o v u l a t i o n i n n o n p r e g n a n t cows w h e r e a s i n p r e g n a n t a n i m a l s t h i s f o l l i c l e d e c r e a s e d g r a d u a l l y i n s i z e . P r e g n a n t cows h a d s i g n i f i c a n t l y (P<0 .05) m o r e v i s i b l e f o l l i c l e s o n d a y s 8 t o 1 2 , 15 t o 18 a n d 19 t o 24 t h a n n o n p r e g n a n t c o w s ( T a b l e 3 . 3 ) . T h e d i f f e r e n c e t e n d e d (P<0 .1) t o b e g r e a t e r o n d a y s 15 t o 18 a n d 19 t o 24 t h a n o n d a y s 8 t o 1 2 . I n p r e g n a n t c o w s , t h e w a v e - l i k e p a t t e r n o f f o l l i c u l a r g r o w t h c o n t i n u e d t h r o u g h t h e f i r s t s i x t y d a y s o f p r e g n a n c y ( F i g . 3 . 1 ) , e a c h wave r e s u l t i n g i n a l a r g e d o m i n a n t f o l l i c l e c o r r e s p o n d i n g t o t h e m i d c y c l e a n d o v u l a t o r y f o l l i c l e s o f a n o r m a l e s t r o u s c y c l e . T h e r e was no d i f f e r e n c e i n d i a m e t e r o f t h e s e l a t e r f o l l i c l e s when c o m p a r e d t o t h e c o r r e s p o n d i n g t y p e i n a n o r m a l c y c l e , a l t h o u g h t h e r e was a t e n d e n c y (P<0 .1 ) f o r t h e d o m i n a n t f o l l i c l e f o u n d b e t w e e n d a y 56 a n d 60 t o b e s m a l l e r t h a n i t s c o r r e s p o n d i n g m i d c y c l e f o l l i c l e f o u n d b e t w e e n d a y 8 a n d 12 ( 1 4 . 7 ± 3.0mm v s 1 7 . 1 ± 0 . 8 m m ) . No i n t e r a c t i o n b e t w e e n t h e s i d e o f t h e C L a n d s i d e o f t h e d o m -i n a n t f o l l i c l e o r t o t a l n u m b e r o f f o l l i c l e s o n t h e i p s i l a t -e r a l o r c o n t r a l a t e r a l o v a r y c o u l d b e e s t a b l i s h e d , ( F i g . 3 . 2 ) . 48 FOLLICULAR AND CL GROWTH IN PREGNANT COWS Fig . 3.1 Diameter of the largest fol l ic le on the right and left ovaries of pregnant dairy cow (82019) 25 i 0 n r i i i i i i i i 1 i 0 5 10 15 20 25 30 35 40 45 50 55 60 Days Post Breeding* • Fol l ic le left ovary 0 Fol l ic le right ovary I • D a y 0 • D a y o f I n s e m i n a t i o n 49 FOLLICULAR AND CL GROWTH IN PREGNANT COWS Fig . 3.2 Total number of fo l l ic les on the CL bearing and contra latera l ovar ies of ear ly pregnant dairy cows 12 N u 10 m b 8 -2 -0 M I M I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I M ! I I I I I I I I I I I I I I ! I I I I I 0 5 10 15 20 25 30 35 40 45 50 55 Day Post Breeding I S E M N o n C L b e a r i n g o v a r y 0 C L b e a r i n g o v a r y D a y 0 » d a y o f b r e e d i n g 5 0 FOLLICULAR AND CL GROWTH IN PREGNANT COWS T a b l e 3 .2 . Comparison o f the diameter ( ± SEM) o f the l a r g e s t f o l l i c l e present on t h e o v a r i e s d u r i n g the f i r s t 24 days o f e a r l y pregnant and non pregnant d a i r y cows. Pregnancy s t a t u s a t day 25 based on ul t rasonograghy Diameter o f l a r g e s t f o l l i c l e (mm) Days p o s t b r e e d i n g 8 - 1 2 15 - 18 19 - 24 Pregnant (n=ll) 16.6 ± 0 . 8 a 15.6 ± 0 . 5 a 17.1 ± 0 . 8 a Non pregnant (n=5) 16.8 ± 0 . 8 a 16.6 ± 0 . 6 a 17.6 ± 0 . 8 a V a l u e s w i t h i n columns w i t h d i f f e r e n t s u p e r s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t (P < 0 .05) . 51 FOLLICULAR AND CL GROWTH IN PREGNANT COWS T a b l e 3 .3 . Comparison o f the t o t a l number (± SEM) o f v i s i b l e f o l l i c l e s on both o v a r i e s d u r i n g the f i r s t 24 days between e a r l y pregnant and non pregnant d a i r y cows. Pregnancy s t a t u s a t day 25 based on ul t rasonograghy Number o f f o l l i c l e s Days post breeding 8 - 1 2 15 - 18 19 - 24 Pregnant (n=ll) 15.5 ± 0 . 4 a 16.7 ± 0 . 5 a 16.0 ± 0 . 4 a Non pregnant (n=5) 12.0 ± 0 . 6 b 11.0 ± 0 . 7 b 10.3 ± V a l u e s w i t h i n columns w i t h d i f f e r e n t s u p e r s c r i p t s are s i g n i f i c a n t l y d i f f e r e n t (P < 0 .05) . 52 FOLLICULAR AND CL GROWTH IN PREGNANT COWS T h e C L a c h i e v e d i t s maximum c r o s s s e c t i o n a l d i a m e t e r b y 9 . 2 ± 1 . 7 d a y s p o s t b r e e d i n g i n b o t h g r o u p s o f a n i m a l s . T h e r e was no d i f f e r e n c e b e t w e e n p r e g n a n t a n d n o n p r e g n a n t c o w s i n t h e maximum a r e a o f t h e C L ( T a b l e 3 . 4 ) . No i n c r e a s e i n t h e s i z e o f t h e C L was o b s e r v e d a t a n y s t a g e o f t h e s t u d y p e r i o d a f t e r d a y 10 i n p r e g n a n t a n i m a l s ( F i g . 3 . 3 ) . C L r e g r e s s i o n commenced 3 . 2 ± 1 . 8 d a y s p r i o r t o t h e o n s e t o f e s t r u s i n n o n p r e g n a n t c o w s . C i r c u l a t i n g P4 r o s e f r o m b a s a l c o n c e n t r a t i o n s 3 . 7 ± 1 . 1 d a y s p o s t b r e e d i n g a n d p e a k e d o n d a y 1 3 . 2 ± 2 . 0 ( F i g . 3 . 4 ) . T h e r e was n o d i f f e r e n c e b e t w e e n p r e g n a n t a n d n o n p r e g n a n t cows i n t h e p e a k l e v e l s o f c i r c u l a t i n g P4 a c h i e v e d ( 4 . 8 ± 1 . 1 n g / m l f o r p r e g n a n t v s 5 . 3 ± 0 . 9 n g / m l f o r n o n p r e g n a n t c o w s ) . H o w e v e r i n n o n p r e g n a n t cows P 4 b e g a n t o d e c l i n e 4 . 6 ± 1 . 1 d a y s p r i o r t o e s t r u s i n c o n t r a s t t o p r e g n a n t c o w s i n w h i c h P4 l e v e l s r e m a i n e d r e l a t i v e l y c o n s t a n t t h r o u g h o u t t h e r e m a i n d e r o f t h e s t u d y p e r i o d . 53 FOLLICULAR AND CL GROWTH IN PREGNANT COWS T a b l e 3 .4 . Comparison o f the diameter (mm ± SEM) o f t h e corpus luteum d u r i n g the f i r s t 24 days p o s t b r e e d i n g o f e a r l y pregnant and non pregnant d a i r y cows. Pregnancy s t a t u s a t day 25 based on ul t rasonograghy Diameter o f the corpus luteum (mm) Days post breeding: 8 - 1 2 15 - 18 19 - 24 Pregnant (n=ll) 31.1 ± 0 . 7 a 31.3 ± 1 . 0 a 30.2 ± 1 . 6 a Non pregnant (n=5) 29.2 ± 1 . 0 a 30.2 ± 1 . 5 a 23.8 ± 2 . 4 b Values w i t h i n columns w i t h d i f f e r e n t s u p e r s c r i p t s a r e s i g n i f i c a n t l y d i f f e r e n t (P < 0 .05) . 54 FOLLICULAR AND CL GROWTH IN PREGNANT COWS Fig . 3.3 Diameter of the corpus luteum in ear ly pregnant and non pregnant dai ry cows as determined by ul t rasound imaging Day 0 « day of breeding 55 FOLLICULAR AND CL GROWTH IN PREGNANT COWS Fig. 3.4 Circulat ing plasma progesterone in ear ly pregnant and non pregnant dairy cows I SEM Pregnant cows 0 Non pregnant cows •Day 0 - day of breeding 56 FOLLICULAR AND CL GROWTH IN PREGNANT COWS DISCUSSION T h e p r e s e n c e o f p r e s u m p t i v e e m b r y o n i c v e s i c l e s i n 88% o f o b s e r v e d c o w s , a n e m b r y o p r o p e r i n 69% a n d a f i n a l p r e g n a n c y r a t e o f 56% a g r e e s w e l l w i t h r e p o r t s i n d i c a t i n g f e r t i l i z a t i o n r a t e s i n d a i r y c a t t l e a s h i g h a s 90% b u t f i n a l p r e g n a n c y r a t e s i n t h e 50 t o 60% r a n g e a f t e r e m b r y o n i c m o r t a l i t y ( H u n t e r , 1 9 8 4 ) . T h e m a j o r i t y o f e m b r y o n i c m o r t a l i t y t a k e s p l a c e b e t w e e n 14 a n d 17 d a y s o f g e s t a t i o n , a p p r o x i m a t e l y t h e t i m e when t h e e m b r y o t r a n s m i t s a s i g n a l f o r t h e m a t e r n a l r e c o g n i t i o n o f p r e g n a n c y ( B e t t e r i d g e e t a l . , 1 9 8 0 ; N o r t h e y a n d F r e n c h , 1 9 8 0 ) . A n o t h e r c r i t i c a l p e r i o d a p p e a r s t o b e a r o u n d 3 0 d a y s o f g e s t a t i o n o r when p l a c e n t o m e f o r m a t i o n i s t a k i n g p l a c e . D a t a f r o m t h e p r e s e n t s t u d y a g r e e s w e l l w i t h t h e t i m i n g o f e m b r y o n i c l o s s a n d f i n a l p r e g n a n c y r a t e s . One c o n c e r n i s t h a t s t r u c t u r e s v i s u a l i z e d p r i o r t o t h e i d e n t -i f i c a t i o n o f t h e e m b r y o p r o p e r may n o t b e c o n c e p t u s s t r u c t u r e s . N o r m a l u t e r i n e s e c r e t i o n s c o u l d b e c o n f u s e d f o r e m b r y o n i c v e s i c l e s . H o w e v e r , i n t h e p r e s e n t s t u d y v e s i c l e s w e r e j u d g e d t o b e o f e m b r y o n i c o r i g i n b a s e d o n t h r e e c r i t e r i a : s i z e , 2 t o 3mm i n d i a m e t e r ; l o c a t i o n , i n i t i a l l y o n l y i n t h e u t e r i n e h o r n i p s i l a t e r a l t o t h e C L , t h e n e l o n g a t i n g a n d s p r e a d i n g t o b o t h h o r n s b y 19 d a y s p o s t b r e e d i n g ; a n d d e f i n i t i o n , w e l l d e f i n e d s t r u c t u r e s w i t h d i s t i n c t b o r d e r s . U t e r i n e s e c r e t i o n s t e n d t o b e much l e s s 57 FOLLICULAR AND CL GROWTH IN PREGNANT COWS w e l l d e f i n e d a n d v e r y d i f f u s e t h r o u g h o u t t h e u t e r u s a s v i s u a l i z e d b y u l t r a s o n o g r a p h y . C o n t r a r y t o p r e v i o u s r e p o r t s , ( P e i r s o n a n d G i n t h e r , 1 9 8 7 ; R e x r o a d a n d C a s i d a , 1975) cows i n t h e p r e s e n t s t u d y c o n t i n u e d t o h a v e a r e g u l a r p a t t e r n o f f o l l i c u l a r g r o w t h t h r o u g h t h e f i r s t 60 d a y s o f p r e g n a n c y . L a r g e d o m i n a n t f o l l i c l e s w e r e p r e s e n t a t p e r i o d s c o r r e s p o n d i n g t o t h e m i d c y c l e a n d o v u l a t o r y p e r i o d s . T h e C L d i d n o t i n f l u e n c e t h e s i d e - o n w h i c h t h e l a r g e s t f o l l i c l e s w e r e l o c a t e d , t h e y w e r e j u s t a s l i k e l y t o b e o n t h e i p s i l a t e r a l o v a r y a s o n t h e c o n t r a l a t e r a l o v a r y t o t h e C L . T h e r e was n o c h a n g e i n t h e t o t a l n u m b e r o f f o l l i c l e s t h r o u g h t h e s t u d y p e r i o d i n p r e g n a n t c o w s . T h e p r o p o r t i o n o f l a r g e (> 10mm) a n d m e d i u m s i z e d (7 t o 10mm) f o l l i c l e s was s i m i l a r t o c o r r e s p o n d i n g p e r i o d s o f a n o r m a l e s t r o u s c y c l e . T h i s i s i n c o n t r a s t t o r e p o r t s s u g g e s t i n g a n i n c r e a s e d r a t e o f a t r e s i a i n t h e l a r g e r s i z e c l a s s e s o f f o l l i c l e s d u r i n g p r e g n a n c y ( G u i l b a u l t e t a l . , 1 9 8 6 ) . A l t h o u g h a t r e s i a c a n n o t b e m e a s u r e d u s i n g u l t r a s o u n d i m a g i n g , t h e a b s e n c e o f a n y d e c r e a s e i n t h e p r o p o r t i o n o f l a r g e a n d m e d i u m s i z e d f o l l i c l e s w o u l d a r g u e a g a i n s t a n y i n c r e a s e i n a t r e s i a . One w o u l d e x p e c t a d e c r e a s e i n t h e n u m b e r o f f o l l i c l e s i n t h e l a r g e r s i z e c l a s s e s a n d a n i n c r e a s e i n t h e n u m b e r o f f o l l i c l e s i n t h e s m a l l e r s i z e c l a s s e s i f t h e r a t e o f a t r e s i a w e r e l e s s i n s m a l l f o l l i c l e s a n d g r e a t e r i n m e d i u m s i z e d f o l l i c l e s . T h e p h y s i o l o g i c a l s i g n i f i c a n c e o f t h e l a r g e f o l l i c l e s i s 58 FOLLICULAR AND CL GROWTH IN PREGNANT COWS n o t i m m e d i a t e l y c l e a r . A b o v i n e c h o r i o n i c g o n a d o t r o p h i n a n a l -o g o u s t o p r e g n a n t m a r e s s e r u m g o n a d o t r o p h i n h a s b e e n h y p o t h -e s i z e d ( T h a t c h e r e t a l . , 1988) b u t w h e t h e r l a r g e f o l l i c l e s b e c o m e l u t e i n i z e d a s i n t h e h o r s e h a s n o t b e e n s h o w n . One s t u d y s h o w e d t h a t l a r g e f o l l i c l e s f r o m p r e g n a n t cows h a d h i g h e r P 4 : E 2 r a t i o s t h a n f o l l i c l e s o f t h e same s i z e i n c y c l i n g c o w s ( C h o u d a r y e t a l . , 1 9 6 8 ) ; h o w e v e r t h i s i s u s u a l l y t a k e n a s a m e a s u r e o f a t r e s i a n o t l u t e i n i z a t i o n . I t i s p o s s i b l e t h a t l a r g e f o l l i c l e s s e c r e t e E2 e v e n d u r i n g p r e g n a n c y . I t i s h y p o t h e s i z e d t h a t E2 f r o m t h e m i d c y c l e d o m i n a n t f o l l i c l e s t i m u l a t e s t h e s y n t h e s i s o f e n d o m e t r i a l o x y t o c i n r e c e p t o r s a n d t h a t o x y t o c i n p r o d u c e d b y t h e C L t h e n s t i m u l a t e s t h e s y n t h e s i s a n d s e c r e t i o n o f P G F 2 a b y t h e e n d o -m e t r i u m a n d r e s u l t s i n l u t e o l y s i s ( H a n s e l , 1 9 8 8 ) . H o w e v e r , i t a p p e a r s t h a t t h e E2 - o x y t o c i n - P G F 2 a p a t h w a y f o r l u t e o l y s i s i s u n c o u p l e d a t t h e l e v e l o f t h e u t e r u s ( T h a t c h e r e t a l . , 1988) a n d t h e r e f o r e p h y s i o l o g i c a l l e v e l s o f E2 w o u l d n o t b e l u t e o l y t i c . I n c r e a s e s i n E2 a r o u n d d a y 20 i n p r e g n a n t cows h a v e n o t b e e n r e p o r t e d a n d i n c r e a s e s a f t e r w a r d s a r e u s u a l l y a t t r i b u t e d t o b e o f c o n c e p t u s o r i g i n ( R o b e r t s o n a n d K i n g 1 9 7 9 ) . T h e e m b r y o n i c m o r t a l i t i e s s u g g e s t a n i n t r i g u i n g p o s s i b -i l i t y , l a r g e f o l l i c l e s may h a v e no s i g n i f i c a n t p h y s i o l o g i c a l r o l e f o r p r e g n a n c y , b u t a r e p r e s e n t a s a s a f e g u a r d i n t h e e v e n t o f e a r l y e m b r y o n i c m o r t a l i t y . T h e p r e s e n c e o f l a r g e f o l l i c l e s e n s u r e s a r e l a t i v e l y f a s t r e t u r n t o e s t r u s i n two 59 FOLLICULAR AND CL GROWTH IN PREGNANT COWS w a y s : F i r s t , a l a r g e f o l l i c l e w i l l i n i t i a t e l u t e o l y s i s o n c e t h e i n f l u e n c e o f t h e c o n c e p t u s i s r e m o v e d , s e c o n d l y , a s u b -s e q u e n t f o l l i c l e w i l l d e v e l o p a n d b e a v a i l a b l e f o r o v u l a t i o n o n c e p r o g e s t e r o n e h a s r e t u r n e d t o b a s a l l e v e l s , t h u s p r e v e n t i n g a p r o l o n g e d a n e s t r u s p e r i o d f o l l o w i n g e m b r y o n i c m o r t a l i t y . T h e f i n d i n g t h a t p r e g n a n t cows h a d m o r e f o l l i c l e s t h a n c o w s s u b s e q u e n t l y d i a g n o s e d n o n p r e g n a n t i s d i f f i c u l t t o r e c o n c i l e . T o t a l n u m b e r s o f v i s i b l e f o l l i c l e s w e r e s i m i l a r u n t i l a p p r o x i m a t e l y d a y 7 t o 8 p o s t b r e e d i n g . T h e p r e g n a n c y x p e r i o d t r e n d w o u l d s u g g e s t t h i s was a r e a l p h e n o m e n o n a n d n o t a n e x p e r i m e n t a l a r t i f a c t , h o w e v e r i t i s i m p o s s i b l e t o a t t r i b -u t e a c a u s a l e f f e c t o f p r e g n a n c y , i t i s j u s t a s l i k e l y t h a t cows w i t h m o r e f o l l i c l e s t e n d t o become p r e g n a n t o r m a i n t a i n t h e i r p r e g n a n c y when c o m p a r e d t o cows w i t h f e w e r f o l l i c l e s . F u r t h e r s t u d i e s f o r c o n f i r m a t i o n a r e r e q u i r e d . R e s u l t s o f t h e p r e s e n t s t u d y d o n o t l e n d s u p p o r t t o t h e c o n c e p t o f a n e a r l y p r e g n a n c y l u t e o t r o p h i c f a c t o r . P4 l e v e l s , C L g r o w t h r a t e a n d maximum d i a m e t e r w e r e s i m i l a r i n b o t h p r e g n a n t a n d n o n p r e g n a n t cows u n t i l a b o u t 18 d a y s p o s t b r e e d i n g when t h e d e c l i n e o f P4 a n d d e c r e a s e i n C L s i z e c o m m e n c e d d u e t o l u t e o l y s i s i n t h e n o n p r e g n a n t g r o u p . A f t e r a p p r o x i m a t e l y d a y 10 , t h e s i z e o f t h e C L r e m a i n e d c o n s t a n t f o r t h e d u r a t i o n o f t h e s t u d y p e r i o d i n p r e g n a n t c o w s . T h i s a g r e e s w i t h r e p o r t s o f no i n c r e a s e i n C L w e i g h t o r c e l l n u m b e r a f t e r d a y 12 i n p r e g n a n t cows ( H a n s e l , 1 9 8 8 ) . T h e r e 60 FOLLICULAR AND CL GROWTH IN PREGNANT COWS does appear t o be a s l i g h t i n c r e a s e i n c i r c u l a t i n g P 4, although not s i g n i f i c a n t , a f t e r a pproximately 30 days o f pregnancy but the o r i g i n o f t h i s cannot be concl u d e d from the pr e s e n t study. A study i n v o l v i n g a l a r g e number of bred d a i r y cows found t h a t pregnant cows tended t o have h i g h e r m i l k P 4 l e v e l s as e a r l y as 10 days p o s t e s t r u s compared t o cows t h a t subsequently r e t u r n e d t o e s t r u s (Lamming e t a l . , 1989), however the authors o f t h a t r e p o r t suggest t h a t t he smooth p r o g r e s s i v e i n c r e a s e of P 4 i n the pregnant cows does not l e n d support t o an embryo d e r i v e d l u t e o t r o p h i c f a c t o r . In c o n c l u s i o n , i t appears t h a t f o l l i c u l a r dynamics undergo v e r y l i t t l e change over the f i r s t 60 days o f p r e g -nancy. I t i s hy p o t h e s i z e d t h a t the growth o f l a r g e f o l l i c l e s s e r v e s mainly as a safe g u a r d i n the event o f e a r l y embryonic m o r t a l i t y and may have l i t t l e p h y s i o l o g i c s i g n i f i c a n c e f o r pregnancy i t s e l f . There was no evidence f o r an e a r l y l u t e o -t r o p h i c f a c t o r as no i n c r e a s e i n P 4 or CL s i z e c o u l d be found over nonpregnant cows. 61 TIMING OF ESTRUS EVENTS DURING ESTRUS SYNCHRONIZATION CHAPTER 4 FOLLICULAR DYNAMICS AMD TEMPORAL RELATIONSHIPS BETWEEN BODY TEMPERATURE, STANDING ESTRUS, LUTEINIZING HORMONE AND OVULATION IN DAIRY HEIFERS SYCHRONIZED WITH THE PROGESTIN NORGESTOMET ABSTRACT T h e e f f e c t s o f n o r g e s t o m e t i m p l a n t t r e a t m e n t o n f o l l i c -u l a r d y n a m i c s , c o r p u s l u t e u m g r o w t h a n d f u n c t i o n a s w e l l a s t h e t e m p o r a l r e l a t i o n s h i p s b e t w e e n b o d y t e m p e r a t u r e , s t a n d i n g e s t r u s , L H s u r g e a n d o v u l a t i o n f o l l o w i n g i m p l a n t r e m o v a l w e r e s t u d i e d i n 16 c y c l i n g H o l s t e i n h e i f e r s . E s t r o u s c y c l e s o f t h e h e i f e r s w e r e s y n c h r o n i z e d w i t h two i n j e c t i o n s o f p r o s t a g l a n -d i n F 2 a ( P G F 2 Q ; ) a n d t h e n h e i f e r s w e r e i m p l a n t e d w i t h a 9 d a y n o r g e s t o m e t e a r i m p l a n t e i t h e r a t t h e m i d d l e o f t h e s y n c h r o -n i z e d c y c l e ( d i e s t r u s ) o r a t t h e e n d o f t h e s y n c h r o n i z e d c y c l e ( p r o e s t r u s ) . F o l l i c u l a r d y n a m i c s , c o r p u s l u t e u m g r o w t h a n d r e g r e s s i o n , a n d c i r c u l a t i n g p r o g e s t e r o n e w e r e n o t a f f e c -t e d b y n o r g e s t o m e t t r e a t m e n t . T h e d o m i n a n t f o l l i c l e p r e s e n t a t t h e t i m e o f n o r g e s t o m e t i m p l a n t a t i o n i n t h e p r o e s t r u s g r o u p was m a i n t a i n e d d u r i n g t h e 9 d a y i m p l a n t p e r i o d i n 6 o f 8 h e i f e r s a n d o v u l a t e d a f t e r i m p l a n t r e m o v a l . T i m e t o s t a n d i n g e s t r u s a n d t i m e t o L H p e a k f o l l o w i n g i m p l a n t r e m o v a l 62 TIMING OF ESTRUS EVENTS DURING ESTRUS SYNCHRONIZATION w e r e v e r y h i g h l y c o r r e l a t e d w i t h t h e t i m e o f o v u l a t i o n ( 0 . 9 2 a n d 0 . 9 6 , r e s p e c t i v e l y ) . O n s e t o f s t a n d i n g e s t r u s a n d t h e L H p e a k a n d t h e o n s e t o f s t a n d i n g e s t r u s a n d p e a k v a g i n a l a n d r e c t a l t e m p e r a t u r e s w e r e a l s o h i g h l y c o r r e l a t e d ( 0 . 9 6 , 0 . 8 2 a n d 0 . 8 1 , r e s p e c t i v e l y ) . I t i s c o n c l u d e d t h a t a n y d e c r e a s e i n p r e g n a n c y r a t e s w i t h n o r g e s t o m e t i s n o t d u e t o a s y n c h r o n y b e t w e e n e s t r u s , t h e L H s u r g e a n d o v u l a t i o n b u t may b e d u e t o a g i n g o f t h e o v u l a t o r y f o l l i c l e . INTRODUCTION T h e u s e o f a r t i f i c i a l i n s e m i n a t i o n a n d e m b r y o t r a n s f e r i n b o t h d a i r y a n d b e e f o p e r a t i o n s h a s l e d t o t h e w i d e s p r e a d u s e o f e s t r o u s s y n c h r o n i z a t i o n a s a r e p r o d u c t i v e management t o o l . T h e r e a r e two p r e d o m i n a n t m e t h o d s o f s y n c h r o n i z a t i o n . T h e f i r s t m e t h o d i s b a s e d o n t h e l u t e o l y t i c a c t i v i t y o f p r o s t a g l a n d i n ^2a ( P G F 2 a ) a n d i t s a n a l o g u e s , h o w e v e r S m i t h e t a l . (1985) r e p o r t e d t h a t t h e P G F 2 a a n a l o g u e , A l f a p r o s t o l , d i d n o t c o n s i s t e n t l y i n d u c e l u t e o l y s i s u n t i l a f t e r 5 d a y s p o s t e s t r u s i n n o n l a c t a t i n g H e r e f o r d c o w s . A s y s t e m i n v o l v i n g two i n j e c t i o n s o f P G F 2 a o r i t s a n a l o g u e s a d m i n i s t e r e d 1 0 - 1 2 d a y s a p a r t h a s a l s o b e e n u s e d . I n t h i s s y s t e m a l l t r e a t e d cows s h o u l d h a v e a r e s p o n s i v e c o r p u s l u t e u m (CL) a t t h e t i m e o f t h e s e c o n d i n j e c t i o n a n d come i n t o e s t r u s w i t h i n 72 - 96 h o u r s . T h i s m e t h o d h a s r e s u l t e d i n p r e g n a n c y r a t e s e q u a l t o 63 TIMING OP ESTRUS EVENTS DURING ESTRUS SYNCHRONIZATION c o n t r o l s i n some s t u d i e s b u t n o t o t h e r s ( H a n s e l a n d C o n v e y , 1 9 8 3 ) . T h e s e c o n d m e t h o d o f e s t r o u s s y n c h r o n i z a t i o n i s b a s e d o n t h e u s e o f p r o g e s t i n s t o b l o c k t h e l u t e i n i z i n g h o r m o n e (LH) s u r g e f o l l o w i n g n a t u r a l o r i n d u c e d l u t e o l y s i s , a n d t h e r e f o r e s u p p r e s s e s t r u s a n d o v u l a t i o n . W i t h d r a w l o f t h e p r o g e s t i n r e m o v e s t h e g o n a d o t r o p h i n i n h i b i t i o n a n d r e s u l t s i n e s t r u s w i t h i n 72 - 96 h o u r s . 1 7 a - a c e t o x y - l l / ? - m e t h y l - 1 9 - n o r - p r e g - 4 -e n e - 3 , 1 0 - d i o n e ( N o r g e s t o m e t ) h a s b e e n f o u n d t o b e a v e r y p o t e n t p r o g e s t i n c a p a b l e o f s u p p r e s s i n g b o t h e s t r u s a n d o v u l a t i o n a t v e r y l o w c o n c e n t r a t i o n s ( W i s h a r t , 1972) . T h e S y n c r o - m a t e B s y s t e m , w h i c h i n v o l v e s n o r g e s t o m e t t h a t h a s b e e n i n c o r p o r a t e d i n a h y d r o p h y l i c p o l y m e r e a r i m p l a n t a n d u s e d i n c o n j u n c t i o n w i t h a n i n j e c t i o n o f e s t r a d i o l v a l e r a t e a d m i n i s t e r e d o n t h e d a y o f i m p l a n t t o i n d u c e l u t e o l y s i s , i s u s e d f o r e s t r o u s s y n c h r o n i z a t i o n i n t h e b e e f i n d u s t r y , h o w e v e r , l o w e r e d p r e g n a n c y r a t e s h a v e b e e n r e p o r t e d i n n u m e r o u s s t u d i e s ( W a l t e r s e t a l . , 1 9 8 4 ; R e n t f r o w e t a l . , 1 9 8 7 ; W i l l i a m s a n d K o v a c i k , 1 9 8 7 ; B r o w n e t a l . , 1 9 8 8 ; M i k e s k a a n d W i l l i a m s , 1 9 8 8 ) . I t h a s b e e n s u g g e s t e d t h a t t h e l o w e r e d f e r t i l i t y r a t e s may b e d u e t o e i t h e r a s y n c h r o n y b e t w e e n e s t r u s , t h e L H s u r g e a n d o v u l a t i o n ( R e n t f r o w e t a l . , 1987) o r a d e l a y i n t h e s e l e c t i o n o f t h e o v u l a t o r y f o l l i c l e ( M i k e s k a a n d W i l l i a m s , 1 9 8 8 ) . R a j a m a h e n d r a n e t a l . (1989) r e p o r t e d a s t r o n g c o r r e l a t i o n b e t w e e n t h e o n s e t o f s t a n d i n g e s t r u s , t h e L H s u r g e a n d o v u l a t i o n i n d a i r y cows s y n c h r o n i z e d w i t h 2 64 TIMING OF ESTRUS EVENTS DURING ESTRUS SYNCHRONIZATION i n j e c t i o n s o f P G F 2 t t g i v e n 12 d a y s a p a r t . S i m i l a r s t u d i e s i n c a t t l e s y n c h r o n i z e d w i t h n o r g e s t o m e t h a v e n o t b e e n r e p o r t e d . T h e p r e s e n t s t u d y was c o n d u c t e d t o m o n i t o r t h e f o l l i c u -l a r d y n a m i c s a n d C L g r o w t h a n d f u n c t i o n d u r i n g n o r g e s t o m e t t r e a t m e n t a n d t o d e t e r m i n e t e m p o r a l r e l a t i o n s h i p s b e t w e e n c h a n g e s i n b o d y t e m p e r a t u r e , t h e o n s e t o f s t a n d i n g e s t r u s , t h e L H s u r g e a n d o v u l a t i o n f o l l o w i n g n o r g e s t o m e t i m p l a n t r e m o v a l . MATERIALS AND METHODS S i x t e e n c y c l i n g h o l s t e i n h e i f e r s w e r e s e l e c t e d f r o m t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a d a i r y h e r d . T h e h e i f e r s w e r e i n i t i a l l y s c a n n e d w i t h a l i n e a r a r r a y u l t r a s o u n d d e v i c e ( T o k y o K e k e i L S 3 0 0 , T o k y o J a p a n ) e q u i p p e d w i t h a r e c t a l p r o b e t o d e t e r m i n e t h e r e p r o d u c t i v e s t a t u s o f t h e a n i m a l s . A n i m a l s w e r e t h e n s y c h r o n i z e d w i t h 2 i n j e c t i o n s o f P G F 2 a ( L u t a l y s e , U p j o h n C o . , K a l a m a z o o , MI) a d m i n i s t e r e d 11 d a y s a p a r t a n d r a n d o m l y a s s i g n e d t o o n e o f two t r e a t m e n t s . H e i f e r s i n t r e a t m e n t 1 r e c e i v e d a 6 mg n o r g e s t o m e t e a r i m p l a n t ( S a n o f i C a n a d a I n c . , V i c t o r i a v i l l e , Q u e b e c ) b e t w e e n d a y 9 a n d d a y 11 [ d a y 0 = d a y o f e s t r u s f o l l o w i n g P G F 2 a s y n c h r o n i z a t i o n (PGF e s t r u s ) ] a n d h e i f e r s i n t r e a t m e n t 2 r e c e i v e d t h e e a r i m p l a n t o n c e a n o v u l a t o r y f o l l i c l e was a p p a r e n t a n d C L r e -g r e s s i o n was u n d e r w a y a c c o r d i n g t o t h e u l t r a s o u n d i m a g i n g . 65 TIMING OF ESTRUS EVENTS DURING ESTRUS SYNCHRONIZATION T h e i m p l a n t was l e f t i n p l a c e f o r 9 d a y s i n a l l a n i m a l s a n d a l l h e i f e r s r e c e i v e d 5 mg o f P G F 2 a o n e d a y b e f o r e i m p l a n t r e m o v a l . J u g u l a r b l o o d s a m p l e s w e r e c o l l e c t e d d a i l y f r o m d a y 1 p o s t PGF e s t r u s u n t i l i m p l a n t r e m o v a l a n d e v e r y 4 h t h e r e a f t e r u n t i l t h e s u b s e q u e n t o v u l a t i o n . P l a s m a was s e p -a r a t e d a n d s t o r e d a t - 2 0 \" C u n t i l a n a l y s i s f o r L H a n d P4 u s i n g r a d i o i m m u n o a s s a y p r o c e d u r e s d e s c i b e d e a r l i e r ( V o s t e r m a n s a n d W a l t o n , . 1 9 8 5 ; R a j m a h e n d r a n a n d T a y l o r , 1 9 9 0 ) . T h e . , a s s a y s t a n d a r d f o r t h e L H d e t e r m i n a t i o n was N A I M M D - b L H - 4 ( 2 . 2 x N I H - L H - B 1 ) . A l l r e s u l t s w e r e t h e n c o r r e c t e d t o N I H - L H - B 1 . T h e i n t e r a n d i n t r a a s s a y c o e f f i c i e n t s o f v a r i a t i o n w e r e 19% a n d 6%, r e s p e c t i v e l y , f o r L H a n d 13% a n d 7% f o r P4 . T h e s e n s i t i v i t i e s o f t h e L H a n d P4 a s s a y s w e r e 0 . 1 a n d 0 . 0 5 n g / m l , r e s p e c t i v e l y . V a g i n a l a n d r e c t a l t e m p e r a t u r e s w e r e t a k e n e v e r y 4 h f r o m i m p l a n t r e m o v a l u n t i l o v u l a t i o n . A n i m a l s w e r e o b s e r v e d f o r e s t r u s d u r i n g a n d b e t w e e n t h e s a m p l i n g p e r i o d s a f t e r i m -p l a n t r e m o v a l . O n s e t o f s t a n d i n g e s t r u s was d e f i n e d a s t h e t i m e f r o m i m p l a n t r e m o v a l u n t i l t h e f i r s t o b s e r v a t i o n o f t h e h e i f e r s t a n d i n g t o b e m o u n t e d . T h e o v a r i e s w e r e s c a n n e d b y l i n e a r a r r a y u l t r a s o n o g r a p h y d a i l y f r o m d a y 1 p o s t PGF e s t r u s u n t i l t h e o n s e t o f s t a n d i n g e s t r u s a f t e r i m p l a n t r e m o v a l a n d t h e n e v e r y 2 h u n t i l o v u l a t i o n w h i c h was d e t e r m i n e d b y t h e a c u t e d i s a p p e a r a n c e o f t h e d o m i n a n t f o l l i c l e ( R a j a m a h e n d r a n e t a l . , 1 9 8 9 ) . T h e 66 TIMING OF ESTRUS EVENTS DURING ESTRUS SYNCHRONIZATION ovaries were scanned i n several planes to i d e n t i f y a l l v i s i b l e f o l l i c e s and the CL. Landmarks such as a CL, other f o l l i c l e s , the poles of the ovary and the o r i e n t a t i o n of the ovary were used to i d e n t i f y i n d i v i d u a l f o l l i c l e s . Appropriate images were frozen and structures measured using a b u i l t - i n c a l i p e r system and then hard copies were made for a permanent record using a video processing uni t (Mitsubishi Electronics Co. Ltd., Tokyo, Japan). Heifers were bred approximately 12- h a f t e r the onset of standing estrus. Heifers not returning to estrus before 28 days post insemination were scanned using the ultrasound device f o r confirmation of pregnancy. Pregnancy was diagnosed on the basis of v i s u a l i z a t i o n of the embryo proper with a heartbeat. Correlations between the onset of standing estrus, peak vaginal and r e c t a l temperatures, time to peak LH and ovula-t i o n were made using Pearson r c o r r e l a t i o n s . Comparisons between the two treatment groups with, regard to the time to standing estrus and the time to ovulation were made using a Student's t t e s t . In addition, timing of the onset of a t r e s i a of dominant f o l l i c l e s was compared between the two treatments using a t t e s t . Heifers .receiving the norgestomet implant during proestrus served as controls for the h e i f e r s receiving the implant during diestrus with regard to growth and a t r e s i a of dominant f o l l i c l e s and the onset of l u t e o l y s i s . 67 TIMING OF ESTRUS EVENTS DURING ESTRUS SYNCHRONIZATION RESULTS A l l h e i f e r s h a d two o r t h r e e w a v e s o f f o l l i c u l a r g r o w t h f o l l o w i n g t h e P G F 2 a s y n c h r o n i z e d e s t r u s . A l l h e i f e r s r e -c e i v i n g a n o r g e s t o m e t i m p l a n t d u r i n g d i e s t r u s h a d a f u n c t i o n a l C L (P4 > l n g / m l ) a t t h e t i m e o f i m p l a n t . T h e t i m i n g o f a t r e s i a o f m i d c y c l e f o l l i c l e s , t h e t i m i n g o f t h e o n s e t o f l u t e o l y s i s ( F i g . 4 . 1 ) a n d c i r c u l a t i n g c o n c e n t r a t i o n s o f P4 ( F i g . 4 . 2 ) w e r e n o t a f f e c t e d b y n o r g e s t o m e t t r e a t m e n t d u r i n g d i e s t r u s . U l t r a s o u n d i m a g i n g s h o w e d t h a t C L r e g r e s s i o n h a d c o m m e n c e d a t t h e t i m e o f t h e P G F 2 a i n j e c t i o n a d m i n i s t e r e d t h e d a y b e f o r e i m p l a n t r e m o v a l i n h e i f e r s r e c e i v i n g t h e i m p l a n t d u r i n g d i e s t r u s , a n d t h i s was l a t e r c o n f i r m e d b y P4 a n a l y s i s . T h e d o m i n a n t f o l l i c l e a t t h e t i m e o f C L r e g r e s s i o n w e n t o n t o o v u l a t e i n a l l h e i f e r s r e g a r d l e s s o f t r e a t m e n t . I n h e i f e r s r e c e i v i n g a n i m p l a n t d u r i n g p r o e s t r u s , C L r e g r e s s i o n was w e l l u n d e r w a y a t t h e t i m e o f i m p l a n t i n s e r t i o n a c c o r d i n g t o b o t h t h e u l t r a s o u n d i m a g e s a n d P4 a n a l y s i s ( F i g . 4 . 2 ) . T h e d o m i n a n t f o l l i c l e a t t h e t i m e o f i m p l a n t i n s e r t i o n o v u l a t e d i n 2 o f t h e 8 h e i f e r s w i t h i n 48 h o f t h e i n s e r t i o n . I n t h e r e m a i n i n g 6 h e i f e r s t h e d o m i n a n t f o l l i c l e was m a i n t a i n e d f o r t h e e n t i r e 9 d a y t r e a t m e n t p e r i o d a n d w e n t o n t o o v u l a t e f o l l o w i n g i m p l a n t r e m o v a l ( P l a t e 4 . 1 ) . A l l h e i f e r s r e c e i v i n g t h e n o r g e s t o m e t i m p l a n t d u r i n g d i e s t r u s w e r e o b s e r v e d i n s t a n d i n g e s t r u s w i t h i n 54 h o f t h e 68 TIMING OF ESTRUS EVENTS DURING ESTRUS SYNCHRONIZATION F ig . 4.1 Day of onset of a t res ia of mid cyc le dominant fo l l ic les and lu teo lys is in norgestomet synchron ized hei fers Day of cyc le* 25 - i 20 -Diestrus P roes t rus D ies t rus P roes t rus D ies t rus P roe s t ru s Time of Implant I S.D. i l l Atresia 1st fo l l ic le [ZD Atres ia 2nd fol l ic le • ! Luteo lys i s •Day 0 - day of es t rus 69 TIMING OF ESTRUS EVENTS DURING ESTRUS SYNCHRONIZATION Fig 4.2 P lasma progesterone prof i les in hei fers implanted wi th norgestomet 6 i 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 Day of Cycle (day 0 • day of es t rus) I S E M - Q - D iestrus implant 0 Proestrus implant 7 0 TIMING OP ESTRUS EVENTS DURING ESTRUS SYNCHRONIZATION Plate 4 . 1 . Ultrasound images of the l e f t and r i g h t ovary of a h e i f e r r e c i e v i n g a norgestomet ear implant during pro-estrus. • 0 9 / 8 9 11:02 88«2 r \"hp; 5 M H z i l a n c e : Urn/ 1 5 / 8 9 1 0 : 5 3 C88«2 i '.'be: 5 M H z i : t a n c e : Image of left and right ovary on day of norgestomet implantation. Note presence of large dominant fo l l i c l e on left ovary (long arrow) and many small follicles on both ovaries (short arrows). Image taken 6 days after norgestomet inplantation. The dominant follicle persists, however note the absence of small follicles. 2 2 hours after norgestomet implant removal the dominant fo l l i c l e has yet to ovulate. Note the appearance of a new pool of small antral follicles (short arrow). 5 3 hours following norgestomet implant removal the dominant fo l l i c l e has ovulated. 71 TIMING OP ESTRUS EVENTS DURING ESTRUS SYNCHRONIZATION norgestomet implant removal. Two h e i f e r s r e c e i v i n g the implant d u r i n g p r o e s t r u s o v u l a t e d s h o r t l y a f t e r implant i n -s e r t i o n and one o t h e r showed no s i g n s of e s t r u s f o l l o w i n g implant removal and t h e r e f o r e are not i n c l u d e d i n the temper-a t u r e , LH and o v u l a t i o n data. The remaining 5 h e i f e r s t h a t r e c e i v e d the norgestomet implant d u r i n g p r o e s t r u s were a l l observed i n s t a n d i n g e s t r u s w i t h i n 54 h of the implant removal. Times t o s t a n d i n g e s t r u s , LH peak, temperature peaks and o v u l a t i o n are summarized i n Table 4.1. Standing e s t r u s and the LH peak and s t a n d i n g e s t r u s and peak v a g i n a l and r e c t a l temperatures were h i g h l y c o r r e l a t e d (0.96, 0.82 and 0.81, r e s p e c t i v e l y ) . Cows t h a t r e c e i v e d the norgestomet implant d u r i n g p r o -e s t r u s g e n e r a l l y came i n t o e s t r u s f a s t e r and o v u l a t e d e a r l i e r than cows r e c e i v i n g the implant d u r i n g d i e s t r u s ( F i g 4.3) a l -though t h e r e was no s i g n i f i c a n t d i f f e r e n c e (P < 0.05). Pregnancy r a t e s as diagnosed by u l t r a s o u n d imaging a t 28 days po s t b r e e d i n g are summarized i n t a b l e 4.2. The o v e r a l l pregnancy r a t e was 61.5% and t h e r e was no d i f f e r e n c e between treatments i n the pregnancy r a t e . 72 TIMING OF ESTRUS EVENTS DURING ESTRUS SYNCHRONIZATION T a b l e 4 . 1 . Mean i n t e r v a l i n h o u r s ( ± SD) f r o m n o r g e s t o m e t i m p l a n t r e m o v a l t o s t a n d i n g e s t r u s , t e m p e r a t u r e p e a k s , L H p e a k a n d o v u l a t i o n , a n d c o r r e l a t i o n c o e f f i c i e n t s ( r ) w i t h t h e t i m e t o o v u l a t i o n (n = 1 3 ) . P a r a m e t e r Mean SD r S t a n d i n g E s t r u s 3 0 . 7 9 . 8 0 . 9 2 * V a g i n a l T e m p e r a t u r e 3 6 .4 9 . 9 0 . 5 1 R e c t a l T e m p e r a t u r e 3 6 . 8 8 . 6 0 . 4 4 L H P e a k 3 4 . 2 8 . 8 0 . 9 6 * O v u l a t i o n 6 0 . 1 1 1 . 4 1 . 0 0 * S i g n i f i c a n t l y c o r r e l a t e d (P < 0 . 0 1 ) 73 TIMING OF ESTRUS EVENTS DURING ESTRUS SYNCHRONIZATION Fig 4.3 Time to estrus, LH peak and ovulation after norgestomet implant removal in dairy heifers 80 -70 -DiestrusProestrusDiestrusProestrusDiestrusPro est rus Time of Implant I S.D. HHl Standing es t rus I 1 LH peak MS Ovulation TIMING OF ESTRUS EVENTS DURING ESTRUS SYNCHRONIZATION T a b l e 4.2. Number of pre'gnancies i n d a i r y cows s y n c h r o n i z e d w i t h norgestomet ear imp l a n t s . Treatment # Bred # Pregnant a t 28 days D i e s t r u s implant P r o e s t r u s implant T o t a l 13 7 5 TIMING OF ESTRUS EVENTS DURING ESTRUS SYNCHRONIZATION DISCUSSION Data i n the present study do not support the contention that there i s .asynchrony between estrus, the LH surge and ovulation i n cows synchronized with norgestomet (Rentfrow et al-.'> - 1987) . The cor r e l a t i o n s between the onset of standing estrus and the LH surge, standing estrus and ovulation, and the LH surge and ovulation were very high. Although both r e c t a l and vaginal temperatures were correlated with standing estrus, the c o r r e l a t i o n with ovulation was not s i g n i f i c a n t . The s i n g l e best parameter for basing the time of insemination s t i l l appears to be the onset of standing estrus. The temporal re l a t i o n s h i p s between standing estrus, the LH surge and ovulation are very much i n agreement with those reported i n cows synchronized with P G F 2 a (Rajamahendran et a l . 1989). The present study further confirms that young animals ovulate l a t e r i n r e l a t i o n to estrus and the LH surge than mature cows (Rajamahendran et a l . , 1989; Schams et a l . , 1977; Christenson et a l . , 1975). Heifers receiving the implant during diestrus generally came into estrus l a t e r and had greater v a r i a b i l i t y than animals r e c e i v i n g the implant during proestrus. This may be due to c i r c u l a t i n g concentrations of P4 greater than basal at the time of implant removal i n the he i f e r s treated during diestrus whereas P4 was basal at the time of removal i n 76 TIMING OF ESTRUS EVENTS DURING ESTRUS SYNCHRONIZATION h e i f e r s r e c e i v i n g t h e i m p l a n t d u r i n g p r o e s t r u s . T h e e l e v a t e d P4 w o u l d c a u s e n e g a t i v e f e e d b a c k o n t h e h y p o t h a l a m i c -p i t u a t a r y a x i s , t h u s p r o l o n g i n g t h e t i m e t o e s t r u s , t h e L H p e a k a n d o v u l a t i o n a n d i n c r e a s i n g t h e v a r i a t i o n i n h e i f e r s r e c e i v i n g t h e i m p l a n t d u r i n g d i e s t r u s . A d m i n i s t r a t i o n o f t h e P G F 2 a t w o d a y s p r i o r t o i m p l a n t r e m o v a l r a t h e r t h a n t h e d a y p r e c e d i n g r e m o v a l s h o u l d t i g h t e n t h e s y n c h r o n i z a t i o n . T h e n o r m a l S y n c r o - m a t e B p l a n r e c o m m e n d s a n i n j e c t i o n o f e s t r a d i o l v a l e r a t e t o i n d u c e l u t e o l y s i s o n t h e d a y o f i m p l a n t r a t h e r t h a n P G F 2 a p r i o r t o r e m o v a l . G u i l b a u l t e t a l . (1989) r e p o r t e d t h a t e s t r a d i o l v a l e r a t e may n o t a l w a y s b e e f f e c t i v e i n i n i t i a t i n g l u t e o l y s i s a n d t h a t t h i s f a i l u r e r a t e i n c r e a s e s when g i v e n t o cows w i t h a d e v e l o p i n g C L . P o s s i b l e r e a s o n s f o r a d e c r e a s e d f e r t i l i t y r a t e i n cows s y n c h r o n i z e d w i t h n o r g e s t o m e t may l i e i n t h e f o l l i c u l a r d y n a m i c s o f a n i m a l s s y n c h r o n i z e d w i t h t h e S y c h r o m a t e B m e t h o d . M i k e s k a a n d W i l l i a m s (1988) s u g g e s t e d t h a t r e t a r d e d s e l e c t i o n o r m a t u r a t i o n o f t h e o v u l a t o r y f o l l i c l e a f t e r i m p l a n t r e m o v a l may d e c r e a s e c o n c e p t i o n r a t e s i n t i m e d -i n s e m i n a t e d f e m a l e s . T h e p r e s e n t s t u d y w o u l d s u g g e s t t h a t s e l e c t i o n o f t h e o v u l a t o r y f o l l i c l e i s n o t a f f e c t e d b y n o r g -e s t o m e t t r e a t m e n t , h o w e v e r i n t h e a b s e n c e o f t h e c o r p u s l u t e u m t h e s e l e c t e d f o l l i c l e i s m a i n t a i n e d f o r a p r o l o n g e d p e r i o d o f t i m e , p o s s i b l y l e a d i n g t o o v e r m a t u r a t i o n . W i t h l u t e o l y s i s o c c u r i n g e a r l y a f t e r n o r g e s t o m e t i m p l a n t a t i o n , d u e t o t h e e s t r a d i o l v a l e r a t e i n j e c t i o n i n t h e n o r m a l S y n c r o - m a t e 77 TIMING OF ESTRUS EVENTS DURING ESTRUS SYNCHRONIZATION B protocol, the ovulatory f o l l i c l e would be selected and maintained for the remainder of the norgestomet treatment period. Maintenance of the selected f o l l i c l e f o r a nine day period may e f f e c t i t s f e r t i l i t y . Pregnancy rates i n the present study do not r e f l e c t any decrease i n f e r t i l i t y i n h e i f e r s where the ovulatory f o l l i c l e had been maintained for the 9 day norgestomet treatment, however the number of animals i s very small and a much larger number of animals would be \"required to r e f l e c t a difference. In conclusion, i t would appear that norgestomet t r e a t -ment i s an e f f e c t i v e method of estrus synchronization with no e f f e c t on the r e l a t i o n s h i p between the onset of standing estrus, the LH surge and ovulation following implant removal. I t would appear that any decrease i n pregnancy rate i s not due to asynchrony between the above parameters, but may be due to a combination of the early l u t e o l y s i s induced by the e s t r a d i o l valerate i n j e c t i o n at the beginning of the treatment period, as used i n the normal Syncro-mate B plan, and the prolonged maintenance of the ovulatory f o l l i c l e i n the absence of the corpus luteum. The use of PGF2a t o induce l u t e o l y s i s towards the end of the treatment period rather than e s t r a d i o l valerate at the onset of treatment would seem to be a more e f f e c t i v e protocol as i t minimizes the chance of the ovulatory f o l l i c l e growing and being maintained for a prolonged period of time before ovulation. 78 DISCUSSION CHAPTER 5 GENERAL DISCUSSION The ^previous three chapters have described three indep-endent studies looking into f o l l i c u l a r dynamics and corpus luteum function i n dairy c a t t l e during d i f f e r e n t repro-ductive states. Although these were independent studies several d i f f e r e n t concepts, themes and trends emerge. Many, i f not a l l of these are based on circumstantial or secondary observations and for t h i s reason the concepts are put fort h merely as ideas f o r discussion, not hard bound conclusions. The present chapter w i l l probably leave more questions asked than answered, however studies designed to shed some l i g h t on some of the questions raised w i l l be proposed. GROWTH OF DOMINANT FOLLICLES From the three studies described i t would appear that the wave-like pattern of growth of large dominant f o l l i c l e s i s a c h a r a c t e r i s t i c of the bovine ovary during several d i f -ferent reproductive states. Waves of growth of large dominant f o l l i c l e s are seen i n the anestrous period following partur-i t i o n , during the f i r s t 60 days of pregnancy and even during treatment with a gonadotropin i n h i b i t o r , such as norgestomet, 79 DISCUSSION as long as there i s a corpus luteum present. Hansel and Convey (1983) point out that the pattern of secretion of gonadotrophins do not adequately explain the pattern of growth and regression of f o l l i c l e s . The observation that the pattern of growth of large dominant f o l l i c l e s i s unaltered during the f i r s t 60 days of pregnancy, a period of low gonad-otrophin a c t i v i t y , and under gonadotrophin i n h i b i t i o n i n the presence of a CL serve to underline t h i s f a c t . Hansel and Convey (1983) suggest an intraovarian l e v e l of regulation of f o l l i c u l a r growth, perhaps v i a the CL. The study using norgestomet for estrus synchronization may pro-vide some support for a r o l e of the CL i n the regulation of f o l l i c u l a r growth, however i t would appear to be an endocrine e f f e c t as opposed to a s t r i c t l y paracrine e f f e c t . In the presence of the corpus luteum norgestomet had no e f f e c t on the growth of large antral f o l l i c l e s , there continued to be the normal wave-like pattern of growth and t h i s growth was independent of the side of the CL. However, once l u t e o l y s i s had commenced there was a marked absence of turnover of large a n t r a l f o l l i c l e s . Moreover there was e s s e n t i a l l y no new growth of medium (7 to 10mm) or small (2 to 6mm) sized f o l -l i c l e s . Maintenance of the dominant f o l l i c l e i n the absence of the CL and continued turnover i n the presence of a CL suggest a r o l e f o r the CL i n a t r e s i a of large dominant f o l l i c l e s . I t i s not possible from the present work to speculate on the 80 DISCUSSION mechanism by which the CL acts but work elsewhere point to P4 as a possible e f f e c t o r . Fortune (personal communication) ad-minstered P4 to dairy cows once l u t e o l y s i s had commenced i n order to t e s t i f there would be a continued pattern of growth and a t r e s i a of f o l l i c l e s . When P4 was administered at doses below a normal mid l u t e a l concentration the dominant f o l l i c l e at the time of l u t e o l y s i s was maintained. When mid l u t e a l doses were administered the wave-like pattern of growth and a t r e s i a was restored. These r e s u l t s combined with the norg-estomet synchronization r e s u l t s (chapter 4) would suggest that mid l u t e a l concentrations of progesterone may i n i t i a t e a t r e s i a of large dominant f o l l i c l e s . I t i s i n t e r e s t i n g that the f i r s t dominant f o l l i c l e of an estrous cycle i n cows reaches i t s maximum diameter between day 8 and 9 of the cycle and i s maintained u n t i l day 13 to 15 before i t begins to decrease i n s i z e whereas i n cows with 3 waves of growth the 2nd mid cycle f o l l i c l e i s not maintained for any length of time. Timing of the onset of the decrease i n s i z e of the f i r s t dominant f o l l i c l e corresponds c l o s e l y with the time at which P4 reaches i t s maximum c i r c u l a t i n g concentrations. In addition, h e i f e r s with three waves of f o l -l i c u l a r growth i n a single estrous cycle have s i g n i f i c a n t l y longer l u t e a l phases than h e i f e r s with 2 waves (Ginther et a l . , 1989). I t i s possible that i n cows with 2 waves of growth l u t e o l y s i s has already commenced, and therefore P4 i s f a l l i n g , before the dominant f o l l i c l e of the second wave has 81 DISCUSSION reached i t s maximum diameter. In cows with three waves the CL i s s t i l l secreting large amounts of P4 when the second dominant f o l l i c l e reaches i t s maximum s i z e and i t therefore becomes a t r e t i c and permits a t h i r d wave of growth. In mature l a c t a t i n g cows the vast majority of cycles studied had two waves of f o l l i c u l a r growth. In contrast, h e i f e r s generally had three waves of growth, and yet when the duration of the l u t e a l phases are compared ( f i g . 3.4 and f i g . 4 . 3 , respectively) there i s no d i f f e r e n c e . I f one accepts the hypothesis that P4 may play a r o l e i n a t r e s i a then the difference between h e i f e r s and mature cows could be explained on the basis of the s i z e of the dominant f o l l i c l e s . The dominant f o l l i c l e s i n h e i f e r s are generally smaller than those seen i n mature cows while growth rates of the f o l l i c l e s would appear to be s i m i l a r . The second dominant f o l l i c l e achieves i t s maximum siz e e a r l i e r i n h e i f e r s than i n mature cows and therefore i s subject to high l e v e l s of progesterone leading to a t r e s i a and a t h i r d wave. SHORT LUTEAL PHASE The postpartum study described i n chapter 2 found that on average the f i r s t postpatum estrous cycle was shorter than subsequent cycles and t h i s was mainly due to a shortened l u t -eal phase. However, less than h a l f of the cows studied actu-82 DISCUSS ION a l l y had short f i r s t cycles while the remaining cows a l l had normal lengthed cycles. The i n t e r e s t i n g finding was that the short cycles were not associated with the e a r l i e s t ovulations but were i n fac t associated with a delay i n the return to estrus. I t was concluded that the timing was not r i g h t for short cycles to be at t r i b u t e d to the secretion of P G F 2 t t from the i n v o l u t i n g uterus and that the short cycles were more probably due to an LH deficiency or a deficiency i n LH receptors. A r e s u l t i n the norgestomet synchronization study would appear to support a r o l e f o r an LH deficiency. Two of the h e i f e r s implanted during proestrus went on to ovulate despite the norgestomet implant. Both of these h e i f e r s experienced short cycles and the P4 p r o f i l e s c l o s e l y re-sembled those seen i n the postpartum cows with short cycles. I t i s hypothesised that the norgestomet implant was put i n place too l a t e to block the LH surge but soon enough to block any post surge LH support that the CL may require for a normal estrous cycle l i f e s p a n . A simple experiment using norgestomet implanted so as to allow the LH surge but block any post surge release and LH or hCG replacement would help to show whether post surge LH support i s indeed required for a normal l i v e d CL. A more puzzling question i s why are short postpartum estrous cycles associated with a delay i n the return to c y c l -ing a c t i v i t y ? Growth of large dominant f o l l i c l e s appears to begin very shortly following p a r t u r i t i o n , i f not even before. 8 3 D I S C U S S I O N F o l l i c l e s reach ovulatory s i z e early i n the postpartum period even i n cows with a prolonged anestrus. This would indicate that the a v a i l a b i l i t y of large sized f o l l i c l e s i s not the l i m i t i n g factor i n the duration of the postpartum anestrous. Ovulatory sized f o l l i c l e s are available but f o r some reason f a i l to ovulate. This would again suggest e i t h e r an LH d e f i -ciency or an LH receptor deficiency. An LH d e f i c i e n c y would help explain the association between the delay i n return to estrus and short cycles. An abnormally high LH pulse frequency during the post-partum period could be an explaination. LH \"leakage\" due to an increased LH pulse frequency could r e s u l t i n inadequate LH stores thus preventing an LH surge, estrus and ovulation. As LH stores slowly b u i l d , a point i s reached at which there i s enough to produce an LH surge but an i n s u f f i c i e n t amount to provide any post surge support thus r e s u l t i n g i n an ovulation followed by a short l i v e d CL. Although there i s no d i r e c t evidence for such a mechanism there are some secondary observations which may support such a hypothesis. Treatment of anestrous cows and c y s t i c cows with hCG w i l l induce ovulation suggesting that the problem may not be i n the receptor content. A d d i t i o n a l l y , norgestomet has been used i n beef c a t t l e to decrease the incedence of postpartum short cycles. Since norgestomet acts by blocking LH i t i s possible that i t allows stores to be r e b u i l t so there i s an adequate amount present for post surge support. F i n a l l y , Soules et a l . 84 DISCUSSION (1987) were able to induce short l i v e d corpora lutea i n women by increasing LH pulse frequency i n the f o l l i c u l a r phase. This resulted i n a decrease i n post surge LH pulse frequency, short l i v e d CLs and inadequate P 4 . Experiments designed to show that there i s an abnormally high LH pulse frequency i n cows with a prolonged anestrous period are easy to conceive but d i f f i c u l t to execute. Freq-uent sampling of LH during the postpartum period should pro-vide the answer, however samples must be taken on the order of every 10 minutes for several hours i n order to determine pulse frequency. This and the fact that i t i s not possible to predict which animals w i l l ovulate early and which w i l l not make t h i s a cumbersome study. LOCAL EFFECT OF THE CORPUS LUTEUM ON FOLLICULAR GROWTH Several reports suggest that the corpus luteum may have a l o c a l e f f e c t on f o l l i c u l a r growth. Peirson and Ginther (1987b) suggest that the CL has a p o s i t i v e e f f e c t on f o l -l i c u l a r growth on the i p s i l a t e r a l ovary during r e p e t i t i v e estrous cycles but a f t e r approximately 20 days of pregnancy the CL may have a negative influence. Guilbault et a l . (1986) also suggest that during pregnancy the CL may increase f o l -l i c u l a r turnover in the i p s i l a t e r a l ovary. No p o s i t i v e or negative influence of the CL could be established i n any of 85 D I S C U S S I O N the three studies presented i n the present t h e s i s . Total number of f o l l i c l e s were s i m i l a r on both the CL bearing ovary and the c o n t r a l a t e r a l ovary. In addition the l a r g e s t f o l l i c l e was j u s t as l i k e l y to be on the CL bearing ovary as the c o n t a l a t e r a l ovary i n a l l three studies. I t does appear that the CL may have an endocrine e f f e c t on f o l l i c u l a r growth, perhaps i n i t i a t i n g turnover of f o l -l i c l e s as dicussed above. Further studies are required to better elucidate the r e l a t i o n s h i p between the CL and f o l l i c -u l a r growth. Overall ultrasound imaging proved to be a u s e f u l l t o o l i n the study of f o l l i c u l a r dynamics and corpus luteum growth and regression. I t s r e l a t i v e l y non invasive nature and i t s capacity to allow for the monitoring of i n d i v i d u a l animals over a period of time have helped i n our understanding of the dynamic nature of f o l l i c u l a r growth i n the bovine ovary. 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