IDENTIFICATION OF OVARIAN FOLLICLES AT ESTRUS AND DEVELOPMENT OF THEIR ENSUING CORPORA LUTEA IN SINGLE AND MULTIPLE OVULATING EWES ON TWO FEEDING REGIMES J. J. DUFOURTand P. MATTON'z lresearch Station, Agriculture Canada, Lennoxville, Quebec JtM 123 and2department Biotogy, Sherbrooke University, Sherbrooke, Que. JIK2RI. Received24 Mar. 1977, acceptetl 27 July 1917. DuFoun, J. J. eno MrrroN, P. 1977. Identification ovarian s at estrus and development their ensuing corpora lutea in single and multiple ovulating ewes on two feeding regimes. Can.J. Anim. Sci. 57: 64j-652. Twelve hours after standing estrus was first detected, 30 ewes had their four largest s identified and marked with India ink. Seven days later, ovulation rates and origin the corpora lutea (CL) from previously marked fotlicles were determined. Feeding a high energy ration from day l0 to estrus did not change the ovulation rate as compared to feeding a low energy ration. Its only effect was to increase the diameter the third and fourth largest s. The diameter the largest was identical in single and multiple ovulators. However, in multiple ovulators, the diameter the second largest was larger than in single ovulators. The largest identified at estrus developed into a CL that was l5%o heavier in singte than in multiple ovulators. In multiple ovulators, the second largest developed into a CL weight similar to that from the largest. on a identifi6 et marqu6 i I'encre de Chine, les 'quatre plus gros s de 30 brebis, 12 h aprbs le d6but de I'oestrus. Septjours plus tard on a abattu les brebis et d6termin6 I'origine et le taux d'ovulation. L'imposition d'un r6gime h haute teneur 6nerg6tique pendant une p6riode de 6 jours, h partir du l0ibme jour du cycle jusqu'au d6but de I'oestrus n'a pas chang6 le taux d'ovulation. Son seul effet a 6t6 d'accroitre le diambtre des 3ibme et 4idme plus gros follicules pr6sents au temps du marquage. Aprds avoir class6 les brebis selon le taux d'ovulation observ6 aprbs 1e marquage, nous avons constat6 que les brebis i ovulation multiple avaient, au temps du marquage, un deuxibme plus gros follicule plus grand que les brebis ir ovulation simple. Le plus grand follicule identifi6 au temps de l'oestrus 6tait de mome grandeur chez toutes les brebis mais chez les brebis ir ovulation simple i1 se d6veloppait en des corps jaunes qui 6taient de 157o plus lourds que ceux des brebis ir ovulation multiple. Chez les brebis h ovulation multiple, il y avait une diff6rence de I. 8 mm entre le diambtre des deux plus gros follicules. I1s ont produit cependant des corps j aunes de poids indentiques. In the single ovulating cow and ewe, the The purpose the present experiment largest identified 3 days prior to was to determine the diameter ovarian estrus is the one destined to ovulate (Dufour s present at estrus in sheep that etal. 1972; Bh6rer etal. 1976).In multiple would ovulate one or more s. Some ovulators, however, it is not possible to ewes received additional energy in the week predict the origin the two s preceding estrus in an atternpl to produce ovulated solely on the basis their size. differentovulationrates. Bh6rer et al. (1976) in sheep marked the two largest s one or more days prior MATERTALS AND METHODS to estrus and showed that while the largest Thirty DLS ewes (fz Dorser, l/a Leicester, and ovulated, the second ovulation 1/a Suffott; were used in this study. All had occurred mostly from an unidentified. lambed twice before in 2 consecutive years. Can. J. Anim. Sci. s7:647-6s2(dec. r9z7) During 2 mo starting on 5 Aug. 1976, the ewes 647
648 CANADIAN JOURh\AL OF ANINlAL SCIENCE were fed a medium quality hay ad libitum. The experimental period began on l-5 Oct., when the ewes were checked daily for estrus. On day l0 an estrous cycle, the ewes were divided at random into two groups. The first group u'as fed a lorv energy ration a medium quality hay ad libitum. The feeding the second group was supplemented with 1.0 kg a grain mixture. Following distribution to the two feeding regimes, the ewes were checked for estrus twice daily at l2-h intervats. They were laparotomized l2 h after standing heat was first detected, while the ewes were under general anesthesia (pentobarbitone sodium). The diameter the four largest s was determined using a Vernier caliper, and marked. Marking u'as done by injecting "dots" lndia ink with a hypodelmic syringe around the periphery the s just beneath the ovarian tunic. Location and diameter the four largest marked s were recorded. After surgery all ewes were fed medium quality hay ad libitum until autopsied. Duling autopsy, performed 7 days after laparotomy, the reproductive organs were removed and the ovaries examined. A marked was considered to have ovulated if the CL was completely outlined by dots India ink. The origin the CL was noted and each luteal structure weighed. Diameters marked s still present were also recorded. Data were analyzed by analysis variance. RESULTS Ovarian Follicles at Estrus Among the four largest s measured at estrus, the sizes the third and the fourth largest s were significantly influenced by the levels energy intake (Table 1). They averaged, respectively,0.t and 0.4 mm larger in diameter (P < 0.05) in ewes Levels energy -- High Low fed the high energy, as compared to those fed the low energy ration. The average diameters the largest and the second largest were similar in size for the ewes fed the two levels energy. All 30 ewes had ovulated at least one when autopsied 7 days after identification the four largest s present at estrus. Ten ewes, five on each energy ration, ovulated one marked. Among the 20 remaining ewes, 9 on the high and 10 on the low energy rations ovulated two s. Only one ewe on the high energy ration ovulated three s. To determine the size ovarian s present at estrus in ewes which ovulated one or more than one marked, the ewes were classified as single or multiple ovulators and the diameters the two largest marked s determined accordingly. In multiple ovulators, the size the second largest averaged 5.4 mm, 1.5 mm larger in diameter (P < 0.001) than in single ovulators (Table 2). The minimum size the second largest recorded at estrus that ovulated was 3.8 mm in twin ovulators. The difference in size the largest for the two groups ewes was not statistically significant (P > 0.05). The minimum size the largest that ovulated was 5.0 and 5.5 mm in single and twin ovulators, respectively. Development CorPora Lutea The development CL from s identified at estrus was observed 7 days later and determined according to the ovulation rates. Since the feeding regimes, Table 1. Effects level energy in the rations ewes on the follicular development observed at estrus Number ewes l5 t) Largest Diameter s (mm) Second largest '7.2+.Oi a 4.8!1.3 a 6.7 + 0.9 a 4.9+ l.l a Third largest Fourth largest 4.0+1.0 a 3.3+O.'/ a 3.3+0.4 b 2.91:0.3 b imean t standard deviation. a,b: Means in vertical columns followed by different letters are significantly different atp < 0.05
DUFOUR AND MATTON-OVARIAN FOLLICLES AT ESTRUS 649 Table 2. Characteristics the two largest s present at cstrus in ewes with single or multiple ovulations and development their ensuing corpora lutea (CL) Ovulation rates 1 CL (10 ewes) 2 CL (19 ewes)t Items Average diameter al estrus (mm) Average weight CL originating from marked s { mg)$ Largest 6.6+0.71 a 512+ 139 2nd largest 3.9i0.8 -t Largest 7.2+ 1.0 a 488+lll 2nd largest 5.4+ 1.0 y 461+ 125 fdata from one ewe with three ovulations were not included *Mean + standard deviation. $All differences were not significant..z Differencebetweensingleandmultipleovulatorsintheirlargestwasnotsignificant. x-y Differences between single and multiple ovulators in their second largest was significant atp < 0.001. alone or in combination with the ovulation rates did not affect the weight the CL, it was ignored in the analysis. In single ovulators, the largest present at estrus developed into CL averaging 512 mg, 75Vo heavier than CL developing from the largest present in multiple ovulators (0.10 > P > 0.05, Table 2). In multiple ovulators, the largest two s developed into CL averaging 488 mg and 461 mg in that order. Fate the "Non-ovulating" Marked Follicles Eighteen (627o) the ewes had at least one marked present on the ovary I wk following ovulation (Table 3).In 1 these l8 ewes the marked s had increased Ovulation rates in size, while in the remaining ll it had decreased. The increase or decrease was generally small, yet in some ewes the increase was as high as 6.2 mm and the decrease as high as 2.4 mm. DISCUSSION No stimulatory effect the feeding regime was observed on the ovulation rate. It is known in pigs that the stimulatory effect the feeding regime is mediated by a stimulation the follicular development, since high energy regime increases the number s ovulatory size at estrus (Dailey et al. 1972). In the present experiment, only the third and fourth largest s appeared to be stimulated by the high energy ration. It is then possible that Table 3. Fate marked non-ovulated s in ewes with single or multiple ovulations No. No. ewes ewes Number marked s still present in a ewe at autopsy Change in diameter (mm) Change in diameter (mm) No. No. ewes Avg Range ewes Avg Range One marked ovulated Two marked s ovulated l0 +O.62 5.56 to 1.1 l9-0.33 4.0 to - 1.9 + 1.35 0.3 to 2.4 +0.58 6.2to 1.9
650 CANADIAN JOURNAL OF ANIMAL SCIENCE the absence the expected increase in ovulation rate could result from the failure the high energy ration to stimulate the development the second largest, if one assumes that the effect the feeding reigme is similar in the pig and the sheep. It is not known, however, whether or not the length the high energy feeding period was optimal in bringing a positive follicular development, but results obtained in pigs have indicated that a 6-day period is sufficient to bring about a stimulation follicular development (Dailey et al 1972). The effect the high energy level on ovulation rates and presumably on follicular development would appear to be influenced by the time at which the treatment is imposed during the breeding season. Dufour and Wolynetz (1971) observed a significant increase in the proportion ewes with multiple ovulation only when the effect feeding was registered at the third and fourth estrus the breeding season. At the first and second estrus the beginning the breeding season, they could not find an effect feeding a high energy ration. Likewise, Hulet et al. (1968) could not modify the ovulation rate by changing the level nutrition during the peak the breeding season. It would therefore appear that the sensitivity the ovary and/or the strength the ovulating stimulus go through various phases, being refractory during the early part and the peak the breeding season. The only difference between these two phases would be that during the early part the breeding season, the ovulation rate remains low, while during the peak the breeding season, it is high. All ovulations observed in the ewes studied originated from marked s. By estrus, it became possible to predict the origin any number ovulations in the ewe on the basis follicular size. In twin ovulators, in addition to the first ovulations originating from the largest s, all second ovulations came from the second largest. Bh6rer et al. (1976) were not able to predict the origin the two ovulated s when they were identified one or more days prior to estrus. The reason for their inability to predict the origin the second ovulation was probably that, when measured, the second largest was in a state atresia and by the time estrus another had grown to the stage the second largest. In single or multiple ovulators, the diameter the largest was not significantly different in size when measured at estrus. However, the other large s, and especially the second largest, were significantly larger in multiple ovulators. Since in cases double ovulations the second ovulation always came from the identified at estrus as the second largest, it appears that the size the second largest at estrus is very important in determining the rate ovulation. The mechanism by which double ovulations occur in sheep appears to be an increase in the number large preovulatory s present at estrus. This was demonstrated following PMSG injection (Ellington et al. 1970; Hulet et al. 1969; Shelton and Moore 1967). Because Thimonier and Pelletier (l9ll') could not detect a difference in the magnitude LH surge by single and multiple ovulators at estrus, the level LH may not be responsible for the rupture an increased number s in multiple ovulators. The present results on the fate marked non-ovulated s are consistent with the concept that s are in a state continuous growth and atresia. These results extend the work Smeaton and Robertson (1971) who observed that s marked twice at 7-day intervals had regressed in all seven treated ewes. In the present study, all marked s had regressed in only 38Vo treated ewes. For the other ewes, at least one was still present within 7 days. The difference between the two studies could be due to the types s studied and/or the
DUFOUR AND MATTON-OVARIAN FOLLICLES AT ESTRUS 651 techniques used. Smeaton and Robertson (1971) followed up only the largest using the technique puncturing each for injection carbon. In the present study, only the ovarian stroma surrounding each was pricked and marked with India ink. The destiny the four largest s was then followed up. Puncturing ovarian s directly may have damaged the integrity the foitlctes. Indeed Hunter and Baker (1915) using this technique interfered with ovulation in pigs. Smeaton and Robertson (1971) could only predict the ovulating when marking was done at or just before estrus. At that time the ovulating had probably already received its signal and puncture the would not interfere with ovulation. Using the present technique, s destined to ovulate have been determined as long as 3 days prior to estrus (Dufour et al. 1972; Bh6rer et al. 197 6). The results observed on the development CL showed that there was no direct relationship between the diameter the s ovulated and the weight the CL formed from them. In multiple ovulators the CL originating from the largest and the second largest were similar in weight in spite the fact that the diameter the largest was 1.8 mm larger than that the second largest. To explain the difference observed between the weight the CL originating from the largest s in single and multiple ovulators, it may be suggested that the growth immediately preceding ovulation is more rapid in single ovulators than in multiple ovulators. In multiple ovulators, however, this hypothesis cannot explain the recovery in size the second largest prior to ovulation, if the two largest s were ovulated at the same time. Another probable explanation is that the size the preovulatory had no effect on the weight the CL, but rather that the development the CL was influenced during the days following ovulation by the amount available gonadotropins. Kaltenbach et al. (1968) demonstrated the need a hypophysial luteotrophic support for CL formation and maintenance in hypophysectomized ewes. Thus the hypothesis would imply that the LH secretion was similar in amount in multiple and single ovulators and the two CL present in multiple ovulators would receive an equal amount LH and attain the same development. In contrast, for single ovulators, all the LH is directed to the development the only one CL leading to a heavier structure. ACKNOWLEDGMENTS The authors wish to thank A. Bouchard and Y. Vilandr6 for technical assistance during surgery and processing the reproductive organs, and D. Pitmann and M. Perreault for animal care and data collection. BHERER, J., DUFOUR, J. and MATTON, P. 1976. Destin6e des deux plus gros follicules des ovaires de brebis d la suite de la destruction du plus gros follicule et/ou de I'enlbvement du corps jaune ) deux moments du cycle. Can. J. Physiol. Pharmacol. 54: 7 - ll. DAILEY, R. A., CLARK, J. R., FIRST, N. L., CHAPMAN, A. B. and CASIDA, L. E. 1972. Effects high and low feeding at two stages the estrous cycle on follicular development in gilts from four genetics groups. J. Anim. Sci. 35: l21o-1215. DUFOUR, J., WHITMORE, H. L., GINTHER, O.J. and CASIDA, L. E. l9t2.identification the ovulating by its size on different days the estrous cycle in heifers. J. Anim. Sci. 34: 85-87. DUFOUR, J. J. and WOLYNETZ, M. 1977. Effects energy levels imposed before or during the estrous season on rates and locations ovulation in sheep. Can. J. Anim. Sci. 57: r69-t7 6. ELLINGTON, E. F., KNIGHT, A. D. and FOX, D. W. 1970. Reproductive performance ewes in an early weaning controlled-breeding program. J. Anim. Sci.3l: 927-932. HULET, C. V., PRICE, D. A. and FOOTE, W. C. 1968. Effects variation in light, month year and nutrient intake on reproductive phenomena in ewes during the breeding season. J. Anim. Sci. 27: 684-690
652 canadian JoURNAL OF ANIMAL SCIENCE HULET, C. V., FOOTE, W. C. and PRICE, D. A. 1969. Ovulation rate and subsequent lamb production in the nulliparous and prinriparous ewe. J. Anim. Sci.28: 512-516. HUNTER, R. H. F. and BAKER, T. G. 1975. Development and fate porcine Graafian s identified at different stages the estrouscycle. J. Reprod. Fertil.43: 193-196. KALTENBACH, C. C., GRAPER, J. W., NISEWENDER, G. D. and NALBANDOV, A. V. 1968. Luteotrophic properties some pituitary hormones in non-pregnant hypophysectomized ewes. Endocrinolosv 82: 8 I 8-824. SHELTON, J. N. and MOORE, N. W. 1967. Response the ewe to pregnant mare serum and to horse anterior pituitary extract. J. Reprod. Fertil. 14: 175-1'7'7. SMEATON, T. C. and ROBERTSON, H. A. 1971. Studies on the growth and atresia Graafian s in the ovary the sheep. J. Reprod. Fertil. 25: 243-252. THIMONIER, J. and PELLETIER, J. 1971. Diff6rence g6n6tique dans la d6charge ovulante (LH) chez des brebis de race Ile-de-France; relations avec le nombre d'ovulations. Ann. Biol. Anim. Biochim. Biophvs. ll:559-567.