DOMESTICANIMAL ENDOCRINOLOGY
Vol. 8(2):209-221, 1991
DYNAMICS OF OVARIAN FOLLICULAR DEVELOPMENT IN CATTLE DURING THE ESTROUS CYCLE, EARLY PREGNANCY AND IN RESPONSE TO PMSG M.A. Driancourt*, W.W. Thatcher**, M. Terqui* and D. Andrieu* *Laboratoire de la Reproduction Institut National Recherche Agronomique Nouzilly 37380, France **Dairy Science Department University of Florida Gainesville, Florida 32611-0701, USA Received August 27, 1990
ABSTRACT Ovarian follicular dynamics of cattle were examined during the estrous cycle, early pregnancy and in response to PMSG. Number and size of follicles were monitored by ultrasonographic examinations. During the estmus cycle, distinct periods of follicular dominance (measured by the increase in difference in size between the largest and second largest follicle) occurred in both the luteal (Days 6-8) and proestrus (18-22) phases of the estrous cycle (two follicular waves). Associated with the well timed development of the first dominant follicle was a change in distribution of follicle numbers in small (< 5 mm; increased on Days 2-4), medium (6-8 mm; increased on Days 3-5) and large (_>9 mm; increased on Days 6-9) follicular size classes. Follicular development was greater on the ovary bearing the CL for the period that the CL was present. The dominant follicle formed during the fn'st follicular wave was capable of ovulating (6 of 8 heifers) following an injection of a synthetic analogue of prostaglandin F2et on Day 9 of the estrous cycle. During early pregnancy (Days 6-34), follicular development (size of largest follicle, number of follicles and total accumulated size of all follicles) on the ovary bearing the CL was suppressed between Days 24 and 34 of pregnancy. This was a local effect in that follicular development was sustained on the contralateral ovary. Therefore, the CL or conceptus may be regulating follicular development in a manner to help prevent luteolysis. Associated with the injection of PMSG was an initial increase in the number of small follicles followed by their recruitment into medium and large size classes leading to ovulation. Number of follicles > 5 nun on the Day of estms was related (r = .97) to the number of subsequent embryos and oocytes collected. Ultrasonography is a valuable technique to monitor ovarian follicular dynamics in cattle, and can thereby be used to infer changes in physiological and endocrine states. INTRODUCTION Despite extensive studies, the processes contributing to the differentiation of a single follicle (in natural cycles) or of several follicles (in PMSG/FSH stimulated cycles) are still far from clear in cattle. A major limitation of most studies, using slaughterhouse material or histological examination of the ovaries (1,2), is that information on the dynamics of follicular growth is provided by different animals. High between-animal variability in ovarian follicular populations (1,3) obscures precise physiological changes. A major limitation of the study of follicular growth by monitoring the levels of estradiol 17-B in peripheral blood (4,5) is that the ovarian origin of the steroid measured is not assured. Techniques partly overcoming these problems have been developed. The marking of individual follicles at repeated laparotomies provided basic information on follicular turnover throughout the estrous cycle (6,7). Insights into the mechanisms by which PMSG/FSH trigger superovulation have been obtained by comparing the population of ovarian follicles in one ovary sampled before stimulation and the number of ovulations in the contralateral ovary after PMSG administration (3). However, a number of points still need to be clarified. While there is wide agreement that there are discrete periods of growth of large follicles ("waves") during the estmus cycle, the number of Copyright © 1991 by Dornendo, Inc.
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such waves (two: 1,8,9; v s . three: 7) and follicular dynamics during these waves still deserves further investigation. In PMSG/FSH stimulated cycles, increased ovulation rate could be due to recruitment of small follicles (3), increased growth rate, blockade or reversal of atresia. However, their respective contributions are unknown. With recent progress in ultrasound technology, ultrasonographic examination of the ovaries, previously limited to mares (10,11), has been extended to cows (9,12, 13). As monitoring of the daily changes in follicular populations is now feasible, objectives of this study were to: 1) establish the pattern of growth of large follicles throughout the estrous cycle; 2) examine how this pattern is altered during early pregnancy, since an altered follicular turnover during early (14) or mid (15) pregnancy has been presented; 3) follow the differentiation of the ovulatory follicles in PMSG stimulated cycles.
MATERIALS AND METHODS Validation of ultrasonic measurements of follicles. During the hours (2 to 12 hr) preceding slaughter, ovaries of 27 cows were examined by ultrasonography, and the number and size of all follicles > 3 mm in diameter were determined. Ovaries were collected at slaughter, follicular fluid was aspirated from specific follicles and size deduced from the amount of follicular fluid aspirated. Diameter of a follicle was calculated assuming the follicle was a sphere ( V= 4/3 ~ [D/213; V=volume, D=diameter ). Ultrasound examinations. Ultrasound examinations were made with a linear array scanner equipped with a 7.5 MHZ transducer designed for intrarectal insertion in horses (Toshiba SAL 32B; Tokyo, Japan). All the gain settings, together with the brightness and contrast settings, were maintained constant for the duration of the experiments. Fecal material was removed, genital tract was localized and ovaries were positioned along the wall of the rectum. Serial planes of scanning for each ovary were conducted in order to visualize all follicles. This was necessary; otherwise small follicles could be masked by larger follicles or corpus luteum tissue. Brightness of the ultrasound images is related to tissue density. Fluid is nonechogenic, and therefore follicles look like black spheres. Solid tissue reflects sound waves, causing corpora lutea to appear as bright grey areas, distinguishable from ovarian stroma because of its grainy appearance. Follicle size was measured with built-in calipers after freezing the ultrasound image on the console video screen, and follicles were thereafter ranked into three size classes: < 5 mm, 6-8 mm and _>9 mm in diameter. This classification system was selected due to its physiological value. Follicles that subsequently ovulate are usually at least 5 mm in diameter at the time of luteolysis (7,16,17). Bovine follicles acquire a high content of intrafollicular estradiol 17-g and start to develop LH receptors on the granulosa cells when they reach 9 mm (18). Heifers were exanuned for estrus twice daily with a bull that had a surgically deflected penis. Ovulation was determined by the disappearance of one (natural cycles) or several (PMSG treated cycles) large follicles during the course of ultrasound examinations. EXPERIMENT I: FOLLICULAR GROWTH THROUGHOUT THE ESTROUS C Y C L E AND EARLY PREGNANCY. The estrous cycles of eight nulliparous HolsteinFriesian heifers were synchronized using a 9-d treatment with a progestogen implant and prostaglandin (42). Starting on the day of the synchronized estrus following implant removal, heifers underwent ultrasound examinations 6 days a week until the following ovulation. On Day 9 of the subsequent cycle (Day 0 = estrus), the eight heifers were injected with 2 ml of a prostaglandin analogue (Prosolvin, Intervet, France) to induce CL regression and test the ovulatory potential of the mid cycle follicle. Ultrasound examination began again on the day of prostaglandin injection and continued until the day of ovulation. Heifers observed in estrus were bred by artificial insemination, and ultrasound examinations resumed at Day 6 post es-
FOLLICULAR DEVELOPMENT IN CA'I-i'LE
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tins until Day 34 (for the pregnant heifers) or return to estrus (Day 22 for nonpregnant heifers). Size of largest and second largest follicle, number of follicles in each class and other follicular responses were evaluated by least squares analysis of variance (50) during the three physiological periods (estrous cycle, following prostaglandin injection and pregnancy). The overall mathematical model included cow, ovary with and without a corpus luteum, day, ovary by day interaction and residual error. EXPERIMENT H: DIFFERENTIATION OF LARGE FOLLICLES F O L L O W I N G PMSG INJECTION. To monitor ovarian response to PMSG, seven crossbred heifers, 1834 months old, and eight parous Holstein Friesian cows were treated with PMSG as described by Philippo & Rowson (19). On Days 8-14 following estrus, 2500 IU (heifers) or 3000 IU (cows) of PMSG (Chronogest, Intervet, France) were injected once. Forty-eight hr later, 4 ml of a prostaglandin analogue (Prosolvin, Intervet, France) were injected; on the day estrus was f'n'st detected, 1.5 ml of a PMSG antiserum (produced by J. Saumande) were injected intravenously. Follicular growth was monitored daily from the day of PMSG injection until two days after estrus. Ovulatory response to PMSG injection was measured either by counting the luteal structures at laparoscopy (heifers) or by embryo collection (embryos + unfertilized eggs) at Day 8. Such a procedure provides reliable information on the number of ovulations where the number of ovulations is not extremely high (3). Size of largest follicle and number of follicles within classes were analysed by least squares analysis of variance. The mathematical model included age (cow v s . heifer), animal nested in age, day, age by day and residual error. RESULTS Validation of ultrasonic measurements. There was a linear relationship between follicle diameter measured by ultrasound (y) and follicle diameter (mm) measured at slaughter (x) that was best described by the regression equation (y = -0.04 + 0.80 x (R2 = 0.94). Ultrasonography provides reliable but slightly underestimated estimates of follicle size. A linear relationship between the estimates of follicle numbers at ultrasound (y) and at slaughter (x) was also found (y = 0.83 + 0.86 x, R 2 = 0.58) demonstrating that follicle numbers are properly estimated by ultrasonography. Follicular dynamics throughout the estrous cycle. The dynamic changes in ovarian follicles for one representative heifer during the entire experimental period that encompassed the synchronized estrous cycle, the prostaglandin-induced ovulation, and during the 34 days of pregnancy are depicted in Figure I. During the synchronized estrous cycle of this heifer, follicular development was greater on the CL-bearing ovary (left ovary), and the ensuing ovulation occurred on the contralateral ovary. The large mid-cycle follicle on the right ovary of the second cycle ovulated following injection of prostaglandin. During pregnancy, early follicular development was greater on the CL bearing ovary. However, follicular development was clearly diminished by 25 days of pregnancy (experimental Day 60) on the CLbearing ovary or the ovary ipsilateral to the uterine horn with a conceptus, whereas follicular development was sustained on the contralateral ovary. For the synchronized estrous cycle of all heifers, there was no effect of day (P < .07), or ovary x day interaction (P >. I0) for the total number of follicles seen at ultrasonography. In contrast, there was a significant ovary x day interaction when diameters of the largest (P < .01) and second largest (P < .01) follicles were studied (Table I). The maximum size of the largest follicle was reached at Days 7-9 (12 ram), Days 12-13 (II ram) and Days 20-22 (or Day 0=-12 ram), while maximum size for diameter of the second largest follicle occurred on Days 8-10 (7.0 ram) and Days 11-12 (8.2 turn). Differences between the diameters of largest and second largest follicles were low on Days 2-4 and high on Days 6-8 and 18-22. This suggests that during the estrous cycle there were two periods or waves of follicular growth cul-
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Fig. 1. Distribution of ovarian follicles in a heifer during the estrous cycle, after an injection of a prostaglandin analogue (PGF), and during early pregnancy (Days 6 to 34) following detection of estrus (E) and artificial insemination (AI). Follicles connected by a line represent changes in diameter of the same follicle among days.
minating in a follicle of ovulatory size in this population of heifers. There was no statistically significant difference between growth rate of the large follicle developing during the luteal (1.8 + .35 mm/day) and follicular ( 1.5 +. 18 mm/day) phases. Variability in size of these follicles on specific days before they reach maximum diameter also was similar between follicular and luteal phases (Table 1). The distribution of heifers relative to the days prior to ovulation when the ovulatory follicle could be identified as the largest follicle was: 2, 1, 3 and 2 heifers on Days -2, -3, -4 and -5, respectively. There also were significant differential day effects for the number of follicles in the three size classes (< 5 mm, 6-8 mm and > 9 mm; Table 2). The first follicle wave following ovulation was well synchronized in all heifers. Increases in the number of small follicles were noted on Days 2-4. One to 2 days later an increase in the number of medium size follicles (Days 3-5) occurred, and this was followed by an increase in large follicles between days 6-9. This growth of follicles did not occur randomly between ovaries (with or without a CL) as evidenced by significant ovary (medium and large follicular classes) and ovary by day inter-
FOLLICULAR DEVELOPMENT IN CATTLE
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TABLE 1. LEASTSQUARESMEANSFORDtAM~F.nS (MM)OF LARGESTFOLtJCLE, SECONDLARO~STFOLtJCLE, ACCUMtm~TEDFOLLICLEDIA~-r~S Am) NUMBEROF FOLtaCLESON Tt.mOvAaY W r m (+) oR W r m o u z (-) A CORPUSLUTEUMFOR EIGHTHotsrE~-FRIF,SL~q HEroins DUR~6 THE ESTROUSCYCLE. Day of Cycle 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 21 22 Standard Error of Mean
Largest" Follicle(ram)
Second" Largest FoUicle(mm)
+CL
-CL
+CL
-CL
+CL
-CL
+CL
-CL
+CL
-CL
12.2 7.1 2.7 3.8 5.8 9.5 9.4 11.5 12.0 11.5 10.6 7.7 10.5 9.8 8.8 9.1 9.1 7.4 8.7 8.0 6.4 6.4 9.3
2,1 2,5 3,4 4.4 3.8 4,8 5.8 5.9 5,1 5.2 5.0 6.5 4.5 4.4 3.8 4.1 5.1 6.0 6.9 9.0 7.9 8.2 7.0
4.0 2.6 2.1 2.8 4.5 5.5 5.9 5.5 7.0 6.8 6.8 5.8 8.2 6.4 6.5 5.5 5.6 4.4 4.8 2.7 4.5 3.3 4.2
.9 i.6 2.9 2.8 2.7 2.2 2.5 2.3 2.1 2.8 3.0 2.0 2.5 1.8 1.3 2.8 2.1 3.2 3.0 2.6 2.5 1.5 1.2
8.3 4.5 .6 1.0 1.3 4.0 3.5 6.0 5~ 4.8 3.8 1.8 2.3 3.4 2.3 3.6 3.5 3.0 3.8 5.3 1.9 3.0 5.1
1.3 .9 .5 1.6 1.1 2.6 3.3 3.6 3.0 2.5 2.0 4.4 2.0 2.6 2.5 1.4 3.0 2.8 3.8 6.3 5.4 6.6 5.8
1.8 2.1 2.3 2.0 3.3 2.7 2.1 2.8 2.5 2.5 2.6 2.6 2.8 2.4 2.3 2.3 2.3 2.2 2.4 2.3 2.2 2.5 2.3
.8 1.4 2.9 2.6 2.0 2.5 1.6 1.6 1.4 1.5 2.0 1.2 1.5 1.1 1.0 1.6 1.8 2.4 2.2 2.2 1.8 1.3 1.3
16.3 11.9 7.6 9.2 15.4 17.5 17.1 ~.3 ~.9 20.6 ~.6 17.4 23.2 18.6 18.1 17.4 16.1 15.2 16.6 13.0 10.8 12.6 14.5
3.0 5.0 9.4 10.4 9.4 9.8 8.8 8.5 7~8 8.4 9.4 9.6 6.8 6.5 5.0 7.4 7.8 10.4 11.2 12.8 11.1 10.0 9.2
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• P 5 ram) detected (X) and embryos plus oocytes (Y) collected (r = .95; Y = .38 + .72 X; P < .01).
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DRIANCOURT ET AL
25
NUMBER OF FOLLICLES
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Fig. 2. Distributionof ovarian folliclesamong small (3-5 mm), medium(6-8 mm) and large (> 9 mm) follicular classes followinginjectionof PregnantMares Serum Gonadotrophin(PMSG) in animalsclassifiedas responders (2A; > 20 follicles9 mm or largerat Day 5 after PMSG)or nonresponders(2B; < 5 follicles9 mm or largerat Day 5 after PMSG). DISCUSSION The main conclusions of this study are: 1) several periods of follicle turnover occur at discrete periods throughout the estrous cycle with similar rates of follicular enlargement occurring during these follicular waves; 2) local regulatory roles of the CL during the estrous cycle and of the CL-conceptus complex during early pregnancy are evident, and 3) several follicular regulatory systems are involved in the ovarian response to PMSG (e.g., smaller lbllicle sizes at the times of follicular recruitment and ovulation, and increased follicular growth rate). Ultrasonography is a reliable technique to examine and monitor changes in the population of follicles > 3 mm which form the population of gonadotrophic dependent follicles (20)
FOLLICULAR DEVELOPMENT IN CATTLE
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and, hence, which are involved in the development of the ovulatory follicle as demonstrated by follicle cautery (7,16) or ultrasonography (17). Active follicular turnover occurs throughout the estrous cycle. This conclusion is in agreement with previous reports obtained by ink marking (cattle: 7; sheep: 21) or by ultrasonography (cattle: 9,12,13). Evidence gathered in the present study leads to the following scenario of follicle development. Starting on Days 24 post estrus, a group of follicles initiates growth. On approximately Day 5, growth of usually only one follicle is sustained while growth rate of other follicles declines. From Day 5 to Day 9, this mid-cycle follicle is considered dominant. Dominance is characterized by: the large difference in diameters between the largest and the second largest follicles seen in this study; the decrease in the number of follicles smaller than 8 mm in this study; the increased growth rate of follicles following cautery of the mid-cycle follicle (7), and reduced ovarian response to PMSG when it is injected early (Days 6-8) in the cycle (19,22). Blockade of growth of the smaller follicles, due to atresia and reduced response to gonadotrophins, also is a feature associated with follicular dominance in sheep (23) and primates (24,25). Functionally, this mid-cycle follicle is fully differentiated and its size is similar to an ovulatory follicle (12). It has the ability to produce large amounts of estradiol (18), and contains functional LH receptors on the granulosa cells (18). The competence of this follicle is confLrmed by it's ability to ovulate following prostaglandin injection on Day 9 and the subsequent fertility of the ovum. Noteworthy is the observation that growth and differentiation of this mid-cycle follicle proceeded during a time reported to have a low frequency of LH pulses compared to the follicular phase (26,47) and just following the post ovulatory FSH surge (27). Despite this, follicular growth and differentiation was very similar for the mid-cycle and ovulatory follicles. Possibly gonadotrophins may only play a permissive role on follicular growth once their level exceeds a threshold as previously suggested in sheep (21) and/or gonadotrophins may modulate follicular growth indirectly, through secretion of growth factors as shown in vitro (28). Although previously described in sheep (29) and mares (30), it is unclear how and why this mid-cycle follicle managed to remain at a steady size and functional for several days. Its competence is verified by its ability to ovulate following prostaglandin induced CL regression at Day 9. The duration in survival of the mid-cycle follicle and lifespan of the CL are the main factors modulating the number of follicular waves during the estrous cycle and could explain discrepancies between reports in the number of such waves at histological examination (1,8), after ultrasound monitoring (9,31,48,49) or following peripheral estradiol measurement (4,5). Mean size of the ovulatory follicle for the synchronized estrous cycle was 12.2 mm, and no follicle smaller than l0 nun ovulated. This is in agreement with data showing the presence of LH receptors on bovine granulosa cells only when follicles reach l0 mm (32). The day in which the ovulatory follicle could be identified prior to estrus was quite variable among the heifers. Therefore identification of the ovulatory follicle prior to 2 to 3 days before estrus is unreliable. This conclusion agrees with that of Quirk et al. (17) and Dufour et al. (6). The unreliability is linked to the high variability in the size of the ovulatory follicle on given days before estrus and the large overlap of the size of ovulatory and non-ovulatory follicles (cattle: 17; sheep: 23). A stimulatory effect of the CL on follicular growth during the synchronized estrous cycle was detected in this study as evidenced by a preferential location of the midcycle follicle on the CL-bearing ovary, together with higher follicle numbers and larger size of these follicles. Such a stimulatory effect is in agreement with studies comparing follicle numbers on ovaries with or without a CL by ultrasonography (13) or at slaughter (7,51). However, this effect was not detected in some studies (34). Data supporting a stimulatory action of the CL have been obtained in sheep where follicular growth was increased to a larger extent on the CL-bearing ovary following follicle cautery (33). The factors involved in this stimulatory effect are unknown. Progesterone is unlikely to be involved as most authors
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DRIANCOURT ET AL
agree that it is mostly inhibitory (35,36). Production by the CL of angiogenic factors (37) could be responsible for the higher blood flow to the CL bearing ovary (38) and to individual follicles, thus promoting their growth. Interactions between the CL- and non CL-bearing ovaries and uterine horus of pregnancy (ipsilateral and contralateral to the conceptus) have been demonstrated previously by observations of ovaries during early pregnancy (14,52), mid-pregnancy (15) or in the early postpartum period (39). Reduction of follicular growth on the ovary containing the CL and/or ipsilateral to the conceptus also agrees with the finding of a preferential resumption of ovulation during the early postpartum period on the ovary contralateral to the side of pregnancy (40,41). The present experiment confirmed this CL/conceptus effect and showed that it became ultrasonically detectable at Day 24 post-estrus, and affected follicle size as well as follicle numbers. Origin and identification of the factors involved in this reduction of follicular growth require further investigation. Understanding this process could be of importance in preventing luteolysis following conception, since estradiol plays a key role in production of prostaglandins by the uterus (42,43). Whether this localized decrease in follicle development is due to the CL, as suggested by Pierson and Ginther (52), or due to the conceptus warrants additional investigation. Administration of exogenous gonadotrophins also markedly altered follicular growth. Preovulatory follicles of PMSG-treated animals were smaller (9-10 mm v s . 12 mm in diameter for the estrous cycle). This feature was not noted previously in a small sample of FSH-treated heifers (9). This suggests that PMSG affects functional differentiation of the follicles, inducing the appearance of LH receptors in smaller follicles in PMSG treated animals than in cycling animals (18,32). The population of follicles involved in the ovarian response to PMSG could not be visualized at ultrasound examination at the beginning of the stimulation. Increases in the number of follicles > 5 mm were first detected on Day 2 following PMSG injection, demonstrating that PMSG mobilizes follicles much smaller than in the normal cycle. The follicles mobilized by PMSG are thought to be > 1.7 mm in diameter (3), while those undergoing growth during the cycle are > 5 mm (7,t6,17 and this study). These small follicles, once recruited for growth by PMSG grow at a slightly faster rate than during the normal cycle. The mean growth rate of stimulated follicles was 3 mm/day v s . 1.5 mm/day for the ovulatory follicles in cyclic animals. The increased growth rate found in the present study agrees with the findings of Lussier and coworkers (44). In the later study, 4 days were required for gonadotropin-stimulated follicles to grow from 1.7 to 9 mm in diameter, while 8 days were required for follicles in normally-cycling cows to enlarge from 3.7 to 8.6 mm in diameter. This enhanced growth rate is unlikely to be the consequence of granulosa cell division, since the mitotic index of the granulosa cells is unaffected by PMSG injection (3). The enhanced growth may therefore be the result of increased fluid accumulation in the antrum. Hence, the smaller follicles induced to grow towards ovulation by PMSG are likely to be deficient in their content of granulosa cells, and those follicles ovulating are likely to be highly variable in their number of granulosa cells. The relationship between number of granulosa cells of such follicles and oocyte quality is unknown, as is the relationship between granulosa cell numbers and timing of ovulation. Timing of ovulation occurred over a 48-hr period (this study and 45). Highly variable responses to PMSG were noted among individual animals classified as either cows or heifers. This variability, which is commonly found in the superovulatory responses (46), was detectable in the form of reduced and delayed accumulation of follicles as the time after PMSG elapsed. Monniaux, Chupin & Saumande (3) also demonstrated a reduced population of follicles > 1.7 mm in diameter for low responders. On the day of estrus, ultrasonographic estimates of follicle numbers were related closely to actual numbers of subsequent oocytes and embryos. Thus ultrasound monitoring can be used to measure the likely ovarian response before performing artificial insemination.
FOLLICULAR DEVELOPMENT IN CATTLE
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It can be concluded that, while gonadotrophins can alter growth of large follicles in cattle, local factors such as those produced by the CL or the conceptus also modulate follicular growth. ACKNOWLEDGEMENTS/FOOTNOTES Journal series no. R-01190 of the Florida AgriculturalExperimentalStation. 1This work was partially funded by a research grant to W.W. Tha~:her from the Institut National Recherche Agronomiqueof France; the donationof antiPMSGserumby J. Saumande,INRA, Nouzilly,France is deeply appreciated. 2Request for reprints to W.W. Thatcher, Dairy Science Department, IFAS-0701, University of Florida, Gainesville,FL 32611-0701
REFERENCES I. Rajakoski E. Ovarian follicular system in sexually mature heifers with special reference to seasonal, cyclical and left-right variations. Acta Endocrinol [Suppl] (Copenh) 52:1-68, I%0. 2. Choundary JB, Geir HT, Marion GB. Cyclic changes in bovine vesicular follicles. J Anita Sci 27:468-471, 1968. 3. Monniaux D, Chupin D, Saumande J. Superovulatory responses of cattle. Thefiogenology 19:5581, 1983. 4. Glencross RG, Munro IB, Seuror BE, Pope GS. Concentrations of oestradiol 17B, oestron¢, and progesterone in jugular venous plasma of cows during the ocstrous cycle and early pregnancy. Acta Endocrino173:374-384, 1973. 5. Shemesh M, Ayalon N, Lindner HR. Ocstradiol levels in the peripheral blood of cows during the oestrous cycle. J Endocrino155:73-78, 1972. 6. Dufour JJ, Whitmore I-IL, Ginther OJ, Casida LE. Identification of the ovulatory follicle by its size on different days of the estrous cycle in heifers. J Anim Sci 34:85-87, 1972. 7. Matron P, Adelakoun V, Couture Y, Dufour JJ. Growth and replacement of the bovine ovarian follicles during the estrous cycle. J Anita Sci 52:813-820, 1981. 8. Moor RM, Kruip TH AM, Green D. Extra-ovarian control of folliculogenesis: limits of superovulation? Theriogenology 21:103-115, 1984. 9. Pierson RA, Ginther OJ. Ultrasonography of the bovine ovary. Theriogenology 21:495-504, 1984. I0. Palmer E, Driancourt MA. Use of ultrasonic echography in equine gynecology. Theriogenology 13:203-210, 1980. 1I. Ginther OJ, Pierson RA. Ultrasonic anatomy of equine ovaries. Theriogenology 21:471-483, 1984. 12. Pierson RA, Ginther OJ. Follicular populations during oestrous cycle in heifers. I. Influence of day. Anita Reprod Sci 14:165-176, 1987a. 13. Pierson RA, Ginther OJ. Follicular populations during oestrous cycle in heifers. II. Influence of right and left sides and intraovarian effect of the corpus luteum. Anim Reprod Sci 14:177-186, 1987b. 14. Guilbault LA, Dufour JJ, Thatcher WW. Ovarian follicular development during early pregnancy in cattle. J Reprod Ferti178:127-135, 1986. 15. Rexroad CE, Casida LE. Ovarian follicular development in cows, sows, and ewes in different stages of pregnancy as affected by number of corpora lutea in the same ovary. J Anim Sci 41:10901097, 1975. 16. Chupin D, Saumande J. Effect of exogenous prostaglandin and/or estrogen on luteolysis after electrocauterization of the largest follicle at the end of the bovine estrous cycle. Theriogenology 16: 497-504, 1981. 17. Quirk SM, Hickey GJ, Fortune JE. Growth and regression of ovarian follicles during the follicular phase of the estrous cycle in heifers undergoing spontaneous and PGF-2~ induced luteolysis. J Reprod Ferti177:21 I-219, 1986. 18. Ireland JJ, Roche JF. Development of non-ovulatory antral follicles in heifers: changes in steroid in follicular fluid and receptors for gonadotrophins. Endocrinology 112:150-156, 1983. 19. Phillipo M, Rowson LEA. Prostaglandin and superovulation in the bovine. An Biol Anim Bioch Biophys 15:233-240, 1975. 20. Miller KF, Critser JK, Rowe RF, Ginther OJ. Ovarian effects of bovine follicular fluid treatment in sheep and cattle. Biol Reprod 21:537-544, 1979. 21. Driancourt MA, Philipon P, Locotelli A, Jacques E, Webb R. Are differences in FSH concentrations involved in the control of ovulation rate in Romanov and ll¢-de-France ewes? J Reprod Fertil 83:509-516, 1988.
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22. Sreenan JM, Beeham D, Gosling JE Ovarian responses in relation to endocrine status following PMSG stimulation in the cow. In "Control of reproduction in the cow", Ed JM Sreenan, Martinus Nijhoff, The Hague, pp 144-158, 1978. 23. Driancourt MA, Cahill LE. Preovulatory follicular events in sheep. J Reprod Fertil 71:205-211, 1984. 24. DiZerega GS, Hodgen GD. The primate ovarian cycle: suppression of human menopausal gonadotrophin induced follicular growth in the presence of the dominant follicle. J Clin Endocrinol Metab 50:819-825, 1980. 25. Gougeon A, Lefevre B, Testart J. Recrutement et selection du follicule dominant pendant le cycle menstruel spontane ou stimule chez la femme. In "Periode Peri-ovulatoire". Eds Salat-Baroux J, Thibanlt C; Publ Masson Paris, pp l-I l, 1984. 26. Schallenberger E, Schams D, Butterman B, Walter DL. Puisatile secretion of gonadotrophins, ovarian steroids, and ovarian oxytocin during prostaglandin induced regression of the corpus luteum in the cow. J Reprod Fertil 71:493-501, 1984. 27. Dobson H. Plasma gonadotropins and oestradiol during oestrus in the cow. J Reprod Fertil 52:5156, 1978. 28. Hammond PM, English HE Regulation of desoxyribonucleic acid synthesis in cultured porcine granulosa cells by growth factors and hormones. Endocrinology 120:1039-1048, 1987. 29. Driancourt MA, Cahill LP, Bindon BM. Ovarian follicular population and preovulatory enlargement in Booroola and control Merino ewes. J Reprod Fertil 73:93-107, 1985. 30. Driancourt MA, Palmer E, Bour B. Croissance du follicule preovulatoire chez la jument, in "periode peri-ovulatorie". Eds Salat-Baroux J., Thibault C. publ Masson Paris. pp l-1 l, 1984. 3 I. Sirois J, Fortune JE. Ovarian follicular dynamics during the estrous cycle in heifers monitored by real-time ultrasonography. Biol Reprod 39:308-317, ! 988. 32. Ireland JJ, Roche IF. Development of non-ovulatory antral follicles in cattle after prostaglandin induced luteolysis: changes in serum hormones, steroids in follicular fluid and gonadotropin receptors. Endocrinology 111:2077-2086, 1982. 33. Dufour JJ, Ginther OJ, Casida LE. Corpus luteum action on ovarian follicular development after destruction of macroscopically visible follicles in ewes. Proc Soc Exp Biol Med 138:475-478, 1971. 34. Ireland JJ, Coulson PB, Murphee RL. Follicular development during four stages of the estrous cycle of beef cattle. J Anim Sci 49:1261-1269, 1979. 35. Fukuda M, Katayama K, Tojo S. Inhibitory effect of progesterone on follicular growth and induced superovulation in the rat. Arch Gynecol 230:77-87, 1980. 36. Fortune JE, Vincent SE. Progesterone inhibits the induction of aromatase activity in rat granulosa cells in vitro. Biol Reprod 28:1078-1089, 1983. 37. Gospodarowicz D, Thrakal KK. Production of a corpus luteum angiogenic factor responsible for proliferation of capillaries and neovascularization of the corpus luteum. Proc Nail Acad Sci USA 75:847-851, 1978. 38. Wise TH, Caton D, Thatcher WW, Barron DH, Fields MJ. Ovarian function during the estrous cycle of the cow: ovarian blood flow and progesterone release rate. J Anim Sci 55:627-636, i 982. 39. Dufour JJ, Roy GL. Distribution of ovarian follicular populations in the dairy cow within 35 days after parturition. J Reprod Fertil 75:229-235, 1985. 40. Saiduddin S, Riesen JW, Tyler WJ, Casida LE. Some carry over effects of pregnancy on post-parturn ovarian function in the cow. J Dairy Sci 41:1090-1097, 1967. 41. Lewis GS, Thatcher WW, Bliss EL, Drost M, Collier RJ. Effects of heat stress during pregnancy on postpartum reproductive changes in Holstein cows. J Anim Sci 58:174-186, 1984. 42. Thatcher VOW,Terqui M, Thimonier J, Mauleon E Effect of estradiol-1713 on peripheral plasma concentration of 15-keto-13,14-dihydro PGF2ct and luteolysis in cyclic cattle. Prostaglandins 31:745-756, 1986. 43. Fogwell RL, Cowley JL, Wortman JA, Ames NK, Ireland JJ. Luteal function in cows following destruction of ovarian follicles at midcycle. Theriogenoiogy 23:389-398, 1985. 44. Lussier JG, Matton P, Dufour JJ. Growth rates of follicles in the ovary of the cow. J Reprod Fertil 81:301-307, 1987. 45. Moncada Angel, AH. Endoscopic examination of cattle with particular reference to superovulation. Anim Breed Abst 48:5935, 1979. 46. Saumande J, Chupin D, Mariana J-C, Ortavant R, Mauleon E Factor affecting the variability of ovulation rates after PMSG stimulation. In "Control of reproduction in the cow", Ed JM Sreenan, Martinus Nijhoff, The Hague, pp 195-224, 1978. 47. Rahe CH, Owens RE, Fleeger, JL, Newton HJ, Harms PG. Pattern of plasma luteinizing hormone in the cyclic cow: dependence upon the period of the cycle. Endocrinology 107:498-503, 1980.
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48. Savio. JD, Keenan L, Boland MP, Roche JF. Pattern of growth of dominant follicles during the oestrous cycle of heifers. J Reprod Ferti183:663-671, 1988. 49. Knopf L, Kastelic JP, SchaUenberger E, Ginther OJ. Ovarian follicular dynamics in heifers: test of two-wave hypothesis by ultrasonically monitoring individual follicles. Domest Anim Endocrinol 6:111-119, 1989. 50. SAS User's Guide. Statistics. SAS Institute Inc. Cary NC, 1985. 51. Maurasse C, Matron P, Dufour JJ. Ovarian follicular populations at two stages of an estrous cycle in heifers given high energy diets. J Anita Sci 61:1194-1210, 1985. 52. Pierson RA and Ginther OJ. Intraovarian effect of the corpus luteum on ovarian follicles during early pregnancy in heifers. Anim Reprod Sci 15:53-60, 1987.
Vol. 8(2):209-221, 1991
DYNAMICS OF OVARIAN FOLLICULAR DEVELOPMENT IN CATTLE DURING THE ESTROUS CYCLE, EARLY PREGNANCY AND IN RESPONSE TO PMSG M.A. Driancourt*, W.W. Thatcher**, M. Terqui* and D. Andrieu* *Laboratoire de la Reproduction Institut National Recherche Agronomique Nouzilly 37380, France **Dairy Science Department University of Florida Gainesville, Florida 32611-0701, USA Received August 27, 1990
ABSTRACT Ovarian follicular dynamics of cattle were examined during the estrous cycle, early pregnancy and in response to PMSG. Number and size of follicles were monitored by ultrasonographic examinations. During the estmus cycle, distinct periods of follicular dominance (measured by the increase in difference in size between the largest and second largest follicle) occurred in both the luteal (Days 6-8) and proestrus (18-22) phases of the estrous cycle (two follicular waves). Associated with the well timed development of the first dominant follicle was a change in distribution of follicle numbers in small (< 5 mm; increased on Days 2-4), medium (6-8 mm; increased on Days 3-5) and large (_>9 mm; increased on Days 6-9) follicular size classes. Follicular development was greater on the ovary bearing the CL for the period that the CL was present. The dominant follicle formed during the fn'st follicular wave was capable of ovulating (6 of 8 heifers) following an injection of a synthetic analogue of prostaglandin F2et on Day 9 of the estrous cycle. During early pregnancy (Days 6-34), follicular development (size of largest follicle, number of follicles and total accumulated size of all follicles) on the ovary bearing the CL was suppressed between Days 24 and 34 of pregnancy. This was a local effect in that follicular development was sustained on the contralateral ovary. Therefore, the CL or conceptus may be regulating follicular development in a manner to help prevent luteolysis. Associated with the injection of PMSG was an initial increase in the number of small follicles followed by their recruitment into medium and large size classes leading to ovulation. Number of follicles > 5 nun on the Day of estms was related (r = .97) to the number of subsequent embryos and oocytes collected. Ultrasonography is a valuable technique to monitor ovarian follicular dynamics in cattle, and can thereby be used to infer changes in physiological and endocrine states. INTRODUCTION Despite extensive studies, the processes contributing to the differentiation of a single follicle (in natural cycles) or of several follicles (in PMSG/FSH stimulated cycles) are still far from clear in cattle. A major limitation of most studies, using slaughterhouse material or histological examination of the ovaries (1,2), is that information on the dynamics of follicular growth is provided by different animals. High between-animal variability in ovarian follicular populations (1,3) obscures precise physiological changes. A major limitation of the study of follicular growth by monitoring the levels of estradiol 17-B in peripheral blood (4,5) is that the ovarian origin of the steroid measured is not assured. Techniques partly overcoming these problems have been developed. The marking of individual follicles at repeated laparotomies provided basic information on follicular turnover throughout the estrous cycle (6,7). Insights into the mechanisms by which PMSG/FSH trigger superovulation have been obtained by comparing the population of ovarian follicles in one ovary sampled before stimulation and the number of ovulations in the contralateral ovary after PMSG administration (3). However, a number of points still need to be clarified. While there is wide agreement that there are discrete periods of growth of large follicles ("waves") during the estmus cycle, the number of Copyright © 1991 by Dornendo, Inc.
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such waves (two: 1,8,9; v s . three: 7) and follicular dynamics during these waves still deserves further investigation. In PMSG/FSH stimulated cycles, increased ovulation rate could be due to recruitment of small follicles (3), increased growth rate, blockade or reversal of atresia. However, their respective contributions are unknown. With recent progress in ultrasound technology, ultrasonographic examination of the ovaries, previously limited to mares (10,11), has been extended to cows (9,12, 13). As monitoring of the daily changes in follicular populations is now feasible, objectives of this study were to: 1) establish the pattern of growth of large follicles throughout the estrous cycle; 2) examine how this pattern is altered during early pregnancy, since an altered follicular turnover during early (14) or mid (15) pregnancy has been presented; 3) follow the differentiation of the ovulatory follicles in PMSG stimulated cycles.
MATERIALS AND METHODS Validation of ultrasonic measurements of follicles. During the hours (2 to 12 hr) preceding slaughter, ovaries of 27 cows were examined by ultrasonography, and the number and size of all follicles > 3 mm in diameter were determined. Ovaries were collected at slaughter, follicular fluid was aspirated from specific follicles and size deduced from the amount of follicular fluid aspirated. Diameter of a follicle was calculated assuming the follicle was a sphere ( V= 4/3 ~ [D/213; V=volume, D=diameter ). Ultrasound examinations. Ultrasound examinations were made with a linear array scanner equipped with a 7.5 MHZ transducer designed for intrarectal insertion in horses (Toshiba SAL 32B; Tokyo, Japan). All the gain settings, together with the brightness and contrast settings, were maintained constant for the duration of the experiments. Fecal material was removed, genital tract was localized and ovaries were positioned along the wall of the rectum. Serial planes of scanning for each ovary were conducted in order to visualize all follicles. This was necessary; otherwise small follicles could be masked by larger follicles or corpus luteum tissue. Brightness of the ultrasound images is related to tissue density. Fluid is nonechogenic, and therefore follicles look like black spheres. Solid tissue reflects sound waves, causing corpora lutea to appear as bright grey areas, distinguishable from ovarian stroma because of its grainy appearance. Follicle size was measured with built-in calipers after freezing the ultrasound image on the console video screen, and follicles were thereafter ranked into three size classes: < 5 mm, 6-8 mm and _>9 mm in diameter. This classification system was selected due to its physiological value. Follicles that subsequently ovulate are usually at least 5 mm in diameter at the time of luteolysis (7,16,17). Bovine follicles acquire a high content of intrafollicular estradiol 17-g and start to develop LH receptors on the granulosa cells when they reach 9 mm (18). Heifers were exanuned for estrus twice daily with a bull that had a surgically deflected penis. Ovulation was determined by the disappearance of one (natural cycles) or several (PMSG treated cycles) large follicles during the course of ultrasound examinations. EXPERIMENT I: FOLLICULAR GROWTH THROUGHOUT THE ESTROUS C Y C L E AND EARLY PREGNANCY. The estrous cycles of eight nulliparous HolsteinFriesian heifers were synchronized using a 9-d treatment with a progestogen implant and prostaglandin (42). Starting on the day of the synchronized estrus following implant removal, heifers underwent ultrasound examinations 6 days a week until the following ovulation. On Day 9 of the subsequent cycle (Day 0 = estrus), the eight heifers were injected with 2 ml of a prostaglandin analogue (Prosolvin, Intervet, France) to induce CL regression and test the ovulatory potential of the mid cycle follicle. Ultrasound examination began again on the day of prostaglandin injection and continued until the day of ovulation. Heifers observed in estrus were bred by artificial insemination, and ultrasound examinations resumed at Day 6 post es-
FOLLICULAR DEVELOPMENT IN CA'I-i'LE
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tins until Day 34 (for the pregnant heifers) or return to estrus (Day 22 for nonpregnant heifers). Size of largest and second largest follicle, number of follicles in each class and other follicular responses were evaluated by least squares analysis of variance (50) during the three physiological periods (estrous cycle, following prostaglandin injection and pregnancy). The overall mathematical model included cow, ovary with and without a corpus luteum, day, ovary by day interaction and residual error. EXPERIMENT H: DIFFERENTIATION OF LARGE FOLLICLES F O L L O W I N G PMSG INJECTION. To monitor ovarian response to PMSG, seven crossbred heifers, 1834 months old, and eight parous Holstein Friesian cows were treated with PMSG as described by Philippo & Rowson (19). On Days 8-14 following estrus, 2500 IU (heifers) or 3000 IU (cows) of PMSG (Chronogest, Intervet, France) were injected once. Forty-eight hr later, 4 ml of a prostaglandin analogue (Prosolvin, Intervet, France) were injected; on the day estrus was f'n'st detected, 1.5 ml of a PMSG antiserum (produced by J. Saumande) were injected intravenously. Follicular growth was monitored daily from the day of PMSG injection until two days after estrus. Ovulatory response to PMSG injection was measured either by counting the luteal structures at laparoscopy (heifers) or by embryo collection (embryos + unfertilized eggs) at Day 8. Such a procedure provides reliable information on the number of ovulations where the number of ovulations is not extremely high (3). Size of largest follicle and number of follicles within classes were analysed by least squares analysis of variance. The mathematical model included age (cow v s . heifer), animal nested in age, day, age by day and residual error. RESULTS Validation of ultrasonic measurements. There was a linear relationship between follicle diameter measured by ultrasound (y) and follicle diameter (mm) measured at slaughter (x) that was best described by the regression equation (y = -0.04 + 0.80 x (R2 = 0.94). Ultrasonography provides reliable but slightly underestimated estimates of follicle size. A linear relationship between the estimates of follicle numbers at ultrasound (y) and at slaughter (x) was also found (y = 0.83 + 0.86 x, R 2 = 0.58) demonstrating that follicle numbers are properly estimated by ultrasonography. Follicular dynamics throughout the estrous cycle. The dynamic changes in ovarian follicles for one representative heifer during the entire experimental period that encompassed the synchronized estrous cycle, the prostaglandin-induced ovulation, and during the 34 days of pregnancy are depicted in Figure I. During the synchronized estrous cycle of this heifer, follicular development was greater on the CL-bearing ovary (left ovary), and the ensuing ovulation occurred on the contralateral ovary. The large mid-cycle follicle on the right ovary of the second cycle ovulated following injection of prostaglandin. During pregnancy, early follicular development was greater on the CL bearing ovary. However, follicular development was clearly diminished by 25 days of pregnancy (experimental Day 60) on the CLbearing ovary or the ovary ipsilateral to the uterine horn with a conceptus, whereas follicular development was sustained on the contralateral ovary. For the synchronized estrous cycle of all heifers, there was no effect of day (P < .07), or ovary x day interaction (P >. I0) for the total number of follicles seen at ultrasonography. In contrast, there was a significant ovary x day interaction when diameters of the largest (P < .01) and second largest (P < .01) follicles were studied (Table I). The maximum size of the largest follicle was reached at Days 7-9 (12 ram), Days 12-13 (II ram) and Days 20-22 (or Day 0=-12 ram), while maximum size for diameter of the second largest follicle occurred on Days 8-10 (7.0 ram) and Days 11-12 (8.2 turn). Differences between the diameters of largest and second largest follicles were low on Days 2-4 and high on Days 6-8 and 18-22. This suggests that during the estrous cycle there were two periods or waves of follicular growth cul-
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minating in a follicle of ovulatory size in this population of heifers. There was no statistically significant difference between growth rate of the large follicle developing during the luteal (1.8 + .35 mm/day) and follicular ( 1.5 +. 18 mm/day) phases. Variability in size of these follicles on specific days before they reach maximum diameter also was similar between follicular and luteal phases (Table 1). The distribution of heifers relative to the days prior to ovulation when the ovulatory follicle could be identified as the largest follicle was: 2, 1, 3 and 2 heifers on Days -2, -3, -4 and -5, respectively. There also were significant differential day effects for the number of follicles in the three size classes (< 5 mm, 6-8 mm and > 9 mm; Table 2). The first follicle wave following ovulation was well synchronized in all heifers. Increases in the number of small follicles were noted on Days 2-4. One to 2 days later an increase in the number of medium size follicles (Days 3-5) occurred, and this was followed by an increase in large follicles between days 6-9. This growth of follicles did not occur randomly between ovaries (with or without a CL) as evidenced by significant ovary (medium and large follicular classes) and ovary by day inter-
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TABLE 1. LEASTSQUARESMEANSFORDtAM~F.nS (MM)OF LARGESTFOLtJCLE, SECONDLARO~STFOLtJCLE, ACCUMtm~TEDFOLLICLEDIA~-r~S Am) NUMBEROF FOLtaCLESON Tt.mOvAaY W r m (+) oR W r m o u z (-) A CORPUSLUTEUMFOR EIGHTHotsrE~-FRIF,SL~q HEroins DUR~6 THE ESTROUSCYCLE. Day of Cycle 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 21 22 Standard Error of Mean
Largest" Follicle(ram)
Second" Largest FoUicle(mm)
+CL
-CL
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12.2 7.1 2.7 3.8 5.8 9.5 9.4 11.5 12.0 11.5 10.6 7.7 10.5 9.8 8.8 9.1 9.1 7.4 8.7 8.0 6.4 6.4 9.3
2,1 2,5 3,4 4.4 3.8 4,8 5.8 5.9 5,1 5.2 5.0 6.5 4.5 4.4 3.8 4.1 5.1 6.0 6.9 9.0 7.9 8.2 7.0
4.0 2.6 2.1 2.8 4.5 5.5 5.9 5.5 7.0 6.8 6.8 5.8 8.2 6.4 6.5 5.5 5.6 4.4 4.8 2.7 4.5 3.3 4.2
.9 i.6 2.9 2.8 2.7 2.2 2.5 2.3 2.1 2.8 3.0 2.0 2.5 1.8 1.3 2.8 2.1 3.2 3.0 2.6 2.5 1.5 1.2
8.3 4.5 .6 1.0 1.3 4.0 3.5 6.0 5~ 4.8 3.8 1.8 2.3 3.4 2.3 3.6 3.5 3.0 3.8 5.3 1.9 3.0 5.1
1.3 .9 .5 1.6 1.1 2.6 3.3 3.6 3.0 2.5 2.0 4.4 2.0 2.6 2.5 1.4 3.0 2.8 3.8 6.3 5.4 6.6 5.8
1.8 2.1 2.3 2.0 3.3 2.7 2.1 2.8 2.5 2.5 2.6 2.6 2.8 2.4 2.3 2.3 2.3 2.2 2.4 2.3 2.2 2.5 2.3
.8 1.4 2.9 2.6 2.0 2.5 1.6 1.6 1.4 1.5 2.0 1.2 1.5 1.1 1.0 1.6 1.8 2.4 2.2 2.2 1.8 1.3 1.3
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3.0 5.0 9.4 10.4 9.4 9.8 8.8 8.5 7~8 8.4 9.4 9.6 6.8 6.5 5.0 7.4 7.8 10.4 11.2 12.8 11.1 10.0 9.2
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• P 5 ram) detected (X) and embryos plus oocytes (Y) collected (r = .95; Y = .38 + .72 X; P < .01).
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25
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Fig. 2. Distributionof ovarian folliclesamong small (3-5 mm), medium(6-8 mm) and large (> 9 mm) follicular classes followinginjectionof PregnantMares Serum Gonadotrophin(PMSG) in animalsclassifiedas responders (2A; > 20 follicles9 mm or largerat Day 5 after PMSG)or nonresponders(2B; < 5 follicles9 mm or largerat Day 5 after PMSG). DISCUSSION The main conclusions of this study are: 1) several periods of follicle turnover occur at discrete periods throughout the estrous cycle with similar rates of follicular enlargement occurring during these follicular waves; 2) local regulatory roles of the CL during the estrous cycle and of the CL-conceptus complex during early pregnancy are evident, and 3) several follicular regulatory systems are involved in the ovarian response to PMSG (e.g., smaller lbllicle sizes at the times of follicular recruitment and ovulation, and increased follicular growth rate). Ultrasonography is a reliable technique to examine and monitor changes in the population of follicles > 3 mm which form the population of gonadotrophic dependent follicles (20)
FOLLICULAR DEVELOPMENT IN CATTLE
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and, hence, which are involved in the development of the ovulatory follicle as demonstrated by follicle cautery (7,16) or ultrasonography (17). Active follicular turnover occurs throughout the estrous cycle. This conclusion is in agreement with previous reports obtained by ink marking (cattle: 7; sheep: 21) or by ultrasonography (cattle: 9,12,13). Evidence gathered in the present study leads to the following scenario of follicle development. Starting on Days 24 post estrus, a group of follicles initiates growth. On approximately Day 5, growth of usually only one follicle is sustained while growth rate of other follicles declines. From Day 5 to Day 9, this mid-cycle follicle is considered dominant. Dominance is characterized by: the large difference in diameters between the largest and the second largest follicles seen in this study; the decrease in the number of follicles smaller than 8 mm in this study; the increased growth rate of follicles following cautery of the mid-cycle follicle (7), and reduced ovarian response to PMSG when it is injected early (Days 6-8) in the cycle (19,22). Blockade of growth of the smaller follicles, due to atresia and reduced response to gonadotrophins, also is a feature associated with follicular dominance in sheep (23) and primates (24,25). Functionally, this mid-cycle follicle is fully differentiated and its size is similar to an ovulatory follicle (12). It has the ability to produce large amounts of estradiol (18), and contains functional LH receptors on the granulosa cells (18). The competence of this follicle is confLrmed by it's ability to ovulate following prostaglandin injection on Day 9 and the subsequent fertility of the ovum. Noteworthy is the observation that growth and differentiation of this mid-cycle follicle proceeded during a time reported to have a low frequency of LH pulses compared to the follicular phase (26,47) and just following the post ovulatory FSH surge (27). Despite this, follicular growth and differentiation was very similar for the mid-cycle and ovulatory follicles. Possibly gonadotrophins may only play a permissive role on follicular growth once their level exceeds a threshold as previously suggested in sheep (21) and/or gonadotrophins may modulate follicular growth indirectly, through secretion of growth factors as shown in vitro (28). Although previously described in sheep (29) and mares (30), it is unclear how and why this mid-cycle follicle managed to remain at a steady size and functional for several days. Its competence is verified by its ability to ovulate following prostaglandin induced CL regression at Day 9. The duration in survival of the mid-cycle follicle and lifespan of the CL are the main factors modulating the number of follicular waves during the estrous cycle and could explain discrepancies between reports in the number of such waves at histological examination (1,8), after ultrasound monitoring (9,31,48,49) or following peripheral estradiol measurement (4,5). Mean size of the ovulatory follicle for the synchronized estrous cycle was 12.2 mm, and no follicle smaller than l0 nun ovulated. This is in agreement with data showing the presence of LH receptors on bovine granulosa cells only when follicles reach l0 mm (32). The day in which the ovulatory follicle could be identified prior to estrus was quite variable among the heifers. Therefore identification of the ovulatory follicle prior to 2 to 3 days before estrus is unreliable. This conclusion agrees with that of Quirk et al. (17) and Dufour et al. (6). The unreliability is linked to the high variability in the size of the ovulatory follicle on given days before estrus and the large overlap of the size of ovulatory and non-ovulatory follicles (cattle: 17; sheep: 23). A stimulatory effect of the CL on follicular growth during the synchronized estrous cycle was detected in this study as evidenced by a preferential location of the midcycle follicle on the CL-bearing ovary, together with higher follicle numbers and larger size of these follicles. Such a stimulatory effect is in agreement with studies comparing follicle numbers on ovaries with or without a CL by ultrasonography (13) or at slaughter (7,51). However, this effect was not detected in some studies (34). Data supporting a stimulatory action of the CL have been obtained in sheep where follicular growth was increased to a larger extent on the CL-bearing ovary following follicle cautery (33). The factors involved in this stimulatory effect are unknown. Progesterone is unlikely to be involved as most authors
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agree that it is mostly inhibitory (35,36). Production by the CL of angiogenic factors (37) could be responsible for the higher blood flow to the CL bearing ovary (38) and to individual follicles, thus promoting their growth. Interactions between the CL- and non CL-bearing ovaries and uterine horus of pregnancy (ipsilateral and contralateral to the conceptus) have been demonstrated previously by observations of ovaries during early pregnancy (14,52), mid-pregnancy (15) or in the early postpartum period (39). Reduction of follicular growth on the ovary containing the CL and/or ipsilateral to the conceptus also agrees with the finding of a preferential resumption of ovulation during the early postpartum period on the ovary contralateral to the side of pregnancy (40,41). The present experiment confirmed this CL/conceptus effect and showed that it became ultrasonically detectable at Day 24 post-estrus, and affected follicle size as well as follicle numbers. Origin and identification of the factors involved in this reduction of follicular growth require further investigation. Understanding this process could be of importance in preventing luteolysis following conception, since estradiol plays a key role in production of prostaglandins by the uterus (42,43). Whether this localized decrease in follicle development is due to the CL, as suggested by Pierson and Ginther (52), or due to the conceptus warrants additional investigation. Administration of exogenous gonadotrophins also markedly altered follicular growth. Preovulatory follicles of PMSG-treated animals were smaller (9-10 mm v s . 12 mm in diameter for the estrous cycle). This feature was not noted previously in a small sample of FSH-treated heifers (9). This suggests that PMSG affects functional differentiation of the follicles, inducing the appearance of LH receptors in smaller follicles in PMSG treated animals than in cycling animals (18,32). The population of follicles involved in the ovarian response to PMSG could not be visualized at ultrasound examination at the beginning of the stimulation. Increases in the number of follicles > 5 mm were first detected on Day 2 following PMSG injection, demonstrating that PMSG mobilizes follicles much smaller than in the normal cycle. The follicles mobilized by PMSG are thought to be > 1.7 mm in diameter (3), while those undergoing growth during the cycle are > 5 mm (7,t6,17 and this study). These small follicles, once recruited for growth by PMSG grow at a slightly faster rate than during the normal cycle. The mean growth rate of stimulated follicles was 3 mm/day v s . 1.5 mm/day for the ovulatory follicles in cyclic animals. The increased growth rate found in the present study agrees with the findings of Lussier and coworkers (44). In the later study, 4 days were required for gonadotropin-stimulated follicles to grow from 1.7 to 9 mm in diameter, while 8 days were required for follicles in normally-cycling cows to enlarge from 3.7 to 8.6 mm in diameter. This enhanced growth rate is unlikely to be the consequence of granulosa cell division, since the mitotic index of the granulosa cells is unaffected by PMSG injection (3). The enhanced growth may therefore be the result of increased fluid accumulation in the antrum. Hence, the smaller follicles induced to grow towards ovulation by PMSG are likely to be deficient in their content of granulosa cells, and those follicles ovulating are likely to be highly variable in their number of granulosa cells. The relationship between number of granulosa cells of such follicles and oocyte quality is unknown, as is the relationship between granulosa cell numbers and timing of ovulation. Timing of ovulation occurred over a 48-hr period (this study and 45). Highly variable responses to PMSG were noted among individual animals classified as either cows or heifers. This variability, which is commonly found in the superovulatory responses (46), was detectable in the form of reduced and delayed accumulation of follicles as the time after PMSG elapsed. Monniaux, Chupin & Saumande (3) also demonstrated a reduced population of follicles > 1.7 mm in diameter for low responders. On the day of estrus, ultrasonographic estimates of follicle numbers were related closely to actual numbers of subsequent oocytes and embryos. Thus ultrasound monitoring can be used to measure the likely ovarian response before performing artificial insemination.
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It can be concluded that, while gonadotrophins can alter growth of large follicles in cattle, local factors such as those produced by the CL or the conceptus also modulate follicular growth. ACKNOWLEDGEMENTS/FOOTNOTES Journal series no. R-01190 of the Florida AgriculturalExperimentalStation. 1This work was partially funded by a research grant to W.W. Tha~:her from the Institut National Recherche Agronomiqueof France; the donationof antiPMSGserumby J. Saumande,INRA, Nouzilly,France is deeply appreciated. 2Request for reprints to W.W. Thatcher, Dairy Science Department, IFAS-0701, University of Florida, Gainesville,FL 32611-0701
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