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Ovarian follicular populations in buffaloes and cows
Animal Reproduction Science, 19 {1989) 171-178
171
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
Ovarian Follicular P o p u l a t i o n s in Buffaloes and Cows LE VAN TY*, D. CHUPIN and M.A. DRIANCOURT**
INRA, Reproductive Physiology, 37 380 Monnaie (France) (Accepted 19 January 1989)
ABSTRACT Le Van Ty, Chupin, D. and Driancourt, M.A., 1989. Ovarian follicular populations in buffaloes and cows. Anita. Reprod. Sci., 19: 171-178. Follicular populations and the main parameters of follicular development were compared in the ()varies of eight swamp buffaloes and four cows from Indonesia and Vietnam. The ovarian follicles were counted, measured, ranked in non-atretic and atretic (more than five pyknotic bodies) and grouped into six classes according to diameter. The overall population of antral follicles in buffaloes was only 20% of that of cows (47.5 _+23.8 vs 233.0 _+95.8, P < 0.002), and this difference was apparent in every size class. In contrast, the extent of atresia was similar in the two species. There were also no differences in the processes of oocyte growth, granulosa cell multiplication and antrum development between buffaloes and cows. It is likely that the poor follicular population found in buffalo ovaries is responsible for the limited response to superovulation of this species.
INTRODUCTION
In many developing countries, buffaloes are used as an important source of milk and meat and as draught animals. However, their productivity is still very limited. One of the possibilities of enhancing their production efficiency is genetic improvement of the existing populations. This involves identification of the animals with high producing abilities (i.e. high milk production, high growth rate, short calving intervals or early first calving) followed by their multiplication to supply breeding stock to local farmers. As embryo transfer is feasible in buffaloes (Drost et al., 1983), superovulation and embryo transfer can be used to achieve this. However, the ovarian response of buffaloes to superovulation is low, with mean ovulation rates ranging from 2 to 5 following *Present address: Laboratory of Biology of Reproduction, Dalat Center, National Institute of Scientific Research of Vietnam, Republic of Vietnam. **To whom reprint requests should be addressed.
0378-4320/89/$03.50
© 1989 Elsevier Science Publishers B.V.
172 administration of 3000 IU PMSG or 40-50 mg pFSH (Sharifuddin and Jainudeen, 1984; Drost et al., 1986; Karaivanov, 1986). As clear relationships have been demonstrated in cattle (Monniaux et al., 1983) and sheep (Driancourt, 1987) between the populations of ovarian follicles and ovarian response to exogenous gonadotrophins, the aim of this study was to compare follicle numbers and dynamics in buffalo versus cow ovaries. MATERIALSAND METHODS
Animals Eight swamp buffaloes and four cows, 4-8 years old, originating from Vietnam (six buffaloes) or Indonesia (two buffaloes, four cows) were used in this study. Ovaries were obtained either at slaughter of cycling animals at unspecified stages of the oestrous cycle (pairs of ovaries from three buffaloes and four cows) or at hemiovariectomy on the penultimate day of a synchronization treatment (Chupin and Saumande, 1979) consisting of a 5-mg oestradiol valerate injection followed by a Norgestomet implant for 9 days (single ovaries of five buffaloes).
Histological techniques The ovaries were immediately fixed in Bouin Holland's solution, embedded in paraffin wax and serially sectioned at 10 pm. One section out of six was mounted, stained with haematoxylin and examined microscopically. All antral follicles (i.e. those exhibiting a cavity larger than 1000 pm 2) were counted and measured using the oocyte as a marker to avoid counting follicles twice. The area of each follicle was determined by tracing the follicle at the basement membrane with an electronic planimeter through a drawing tube fixed to the microscope. The antrum and, when its nucleus was visible, the oocyte were similarly measured. All areas measured in ttm 2 were converted to diameters, assuming that the follicles and oocytes were spherical. Follicles were then ranked in the size classes previously defined by Dufour and Roy (1985): 0.160.28 mm, 0.29-0.67 mm, 0.68-1.57 mm, 1.58-3.67 mm, 3.68-8.56 mm and > 8.56 mm. A follicle was considered to be atretic when there were more than five pyknotic bodies in the section studied (Driancourt et al., 1987).
Statistical methods Owing to the lack of homogeneity in the variances of follicle numbers in buffalo and cow ovaries and to the small sample size, non-parametric testing (i.e. Mann-Whitney test to compare means and Wilcoxon test to compare
17~
paired means) was used (Siegel, 1956). Between-breed differences in a n t r u m and oocyte growth were detected by calculating the regression lines linking a n t r u m and follicle sizes or oocyte and follicle sizes and comparing the slopes and intercepts by the M a n n - W h i t n e y test. N u m b e r of granulosa cells per section and per follicle were calculated according to Gougeon (1982). Mitotic indices (i.e. the ratio of the number of mitotic figures to the number of granulosa cells in the section studied) were compared by a two-way ANOVA after arcsine transformation of the data. RESULTS
Follicular populations When the right and left ovaries from three buffaloes were compared, they contained similar numbers of follicles (i.e. 11, 38 and 57 follicles in the right and 11, 29 and 75 follicles in the left ovaries, respectively). Their size distribution was also similar (Fig. 1 ). Hence, in the remainder of the study, a single ovary was used. There were also similar numbers of follicles in Indonesian (50 and 96 antral follicles) and Vietnamese buffaloes (19 to 59 antral follicles). Hence buffaloes originating from both countries were pooled to be compared to cows.
There were highly significant differences in the total population of antral follicles ( 47.5 + 23.8 vs 233.0 _+95.8 in buffaloes vs cows, respectively; P < 0.002 ) ( m e a n + s . d . ) , in the population of non-atretic follicles (38.9+16.6 vs 207.3 + 70.0 in buffaloes vs cows, respectively; P < 0.002 ) and in the population of follicles in each of the first four size classes (P < 0.02, P < 0.002, P < 0.002, P < 0.002, respectively) (Fig. 2). However, despite the low numbers of antral follicles in buffaloes, their size distribution was similar to that found in cows (Fig. 2. ). The number of non-atretic follicles > 1.7 m m in diameter, which are the follicles involved in the ovulatory ovarian response to exogenous gonadotrophins according to Monniaux et al. (1983), ranged from 1 to 5 (mean 2.9) in buffaloes as opposed to a range of 17 to 32 (mean 22.1 ) in cows.
Follicular growth There were no species differences in the slopes and intercepts of the regression lines linking oocyte and follicle size during the period of rapid oocyte growth (classes 1 and 2). The overall regression lines were y = 4 5 4 8 x - 1 7 4 1 2 and y = 4 2 4 9 x - 15601 for buffaloes and cows respectively (y being oocyte size and x being log of follicle size). Furthermore, the maximal size of the oocyte observed in the largest follicle was also similar in buffaloes (98.6/~m in diameter t and cows (100.8/~m in diameter). There were also no differences related to species in the slopes and intercepts
174 40
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Fig. 1. Size distribution of the antral follicles in the left and right ovaries of 3 buffaloes.
of the regression lines linking antrum size (y = log of antrum size ) and follicle size (x = log of follicle size ). The respective regression lines are y = 1.43 x - 2.81 and y = 1.45 x - 2 . 9 1 for buffaloes and cows. The increase in the number of granulosa cells with size was also identical in the two breeds as evidenced by the following regression lines linking these two parameters: y = 1.12 x - 1 . 6 4 8 and y = 1.31 x - 1.635 for buffaloes and cows, respectively, with y being the log of the number of granulosa cells and x the log of follicle size. Finally, there were no species or follicle size effects nor any species-by-follicle-size interactions on the changes in the mitotic index of the granulosa cells with size (Fig. 3 ). In both species, the mitotic index was low in classes 1 and 4 and slightly higher in classes 2 and 3. There were not enough healthy follicles in classes 5 and 6 to include them in the study.
175 follicle number 100
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40
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0.16
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Fig. 2. Size distribution of the antral follicles in the ovaries of 8 buffaloes ( • ) and 4 cows ( [] ). m i t o t i c index of the
granules
cells (%)
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Fig. 3. Changes ( + s.d. ) in the mitotic index (%) of the granulosa cells with increasing follicle size in buffaloes ( • ) and cows ( • ). DISCUSSION T h e m a i n c o n c l u s i o n of t h i s s t u d y is t h a t , while t h e d e v e l o p m e n t a l p r o c e s s e s are s i m i l a r in follicles of b u f f a l o e s a n d cows, t h e n u m b e r of a n t r a l follicles f o u n d in b u f f a l o o v a r i e s is o n l y 20% of t h a t of cow ovaries.
176 The estimates of follicular population in cows obtained in this study are in good agreement with previous reports in a wide range of breeds (Scaramuzzi et al., 1980; Maurasse et al., 1985; Dufour and Roy, 1985). This study is, however, the first report on follicular populations in buffaloes. It is in good agreement with the finding, in an abattoir survey, that the proportion of ovaries devoid of surface follicles was much higher in buffaloes than in cattle (Dobson and Kamonpatana, 1986). Possible reasons for the poor follicular population of buffaloes are a low exit of the reserve of primordial follicles and/or a reduced growth rate of the growing follicles and/or a high extent of atresia (Mauleon and Mariana, 1977 ). As it was demonstrated that neither growth rate nor atresia differed between cows and buffaloes, it is likely that the limited number of growing follicles in buffaloes is linked to a limited initiation of follicular growth from the primordial reserve. The factors causing this require further investigation. They could be either the small size of the reserve of primordial follicles, a feature which is associated in mares with poor folliculogenesis (Driancourt et al., 1982), or a small percentage of easily mobilizable primordial follicles amongst the reserve (Mariana, 1978). Furthermore, if it is assumed that, as demonstrated in cows (Monniaux et al., 1983), the number of ovulations following gonadotrophin administration is highly related to the number of healthy follicles > 1.7 mm in diameter, it is not surprising that buffaloes with about 2.9 such follicles per ovary had a much smaller ovulatory response to PMSG than cows with about 22.1 such follicles per ovary. It is interesting to note that mares, which have a folliculogenesis numerically similar to that of buffaloes (Driancourt et al., 1982), also exhibit a limited response to superovulation (Woods et al., 1982). Mobilization of a higher number of follicles over 1.7 mm in diameter before treatment is therefore probably necessary to increase the ovarian response to PMSG in buffaloes. This could be achieved either by a pretreatment with gonadotrophins (Ware et al., 1987), although its efficiency in cattle is still debated (Monniaux et al., 1983; Guilbault et al., 1988 ), or with gonadotrophin releasing hormone agonist, a procedure efficient in humans (Neveu et al., 1987). However, while these pretreatments will have to be long enough to cover the time (40 days) required for the transit of follicles from antrum formation to large size (Lussier et al., 1987), they may also quickly exhaust the reserve of primordial follicles. Strategies which are less follicle consuming may have to be devised. As it has been demonstrated in rats (De Reviers and Mauleon, 1979), sheep (Sonjaya and Driancourt, 1987) and pigs (M.A. Driancourt, unpublished results) that massive follicular growth compared to adult numbers occurs in prepubertal animals, and as it is feasible to superovulate cattle as soon as 1-2 months of age (Seidel et al., 1971 ), a possibility of improving the ovarian response to exogenous gonadotrophins in buffaloes would be to superovulate prepubertal buffaloes.
177 ACKNOWLEDGEMENTS
Mr. Le Van Ty was supported by CIES during his stay in France. The authors are grateful to Dr. H. Sonjaya for provision of some of the ovaries and to Mr. R. Procureur for collection of the remainder of the ovaries.
REFERENCES Chupin, D. and Saumande, J., 1979. New attemps to decrease the variability of ovarian response to PMSG in cattle. Ann. Biol. Anim. Biochim. Biophys., 19: 1489-1498. De Reviers, M.M. and Mauleon, P., 1979. Evolution of ovarian follicular population and serum gonadotrophins in the prepubertal period of two strains of rats. Ann. Biol. Anita. Biochim. Biophys., 19: 1745-1756. Dobson, H. and Kamonpatana, M., 1986. A review of female cattle reproduction with special reference to a comparison between buffaloes, cows and zebu. J. Reprod. Fertil., 77: 1-36. Driancourt, M.A., 1987. Ovarian features contributing to the variability of PMSG induced ow~lation rate in sheep. J. Reprod. Fertil., 80: 207-212. Driancourt, M.A., Paris, A., Roux, C., Mariana, J.C. and Palmer, E., 1982. Ovarian tbllicular populations in pony and saddle type mares. Reprod. Nutr. Dev., 22: 1035-1047. Driancourt, M.A., Fry, R.C., Clarke, I.J. and Cahill, L.P., 1987. Follicular growth and regression during the 8 days after hypophysectomy in sheep. J. Reprod. Fertil., 79: 635-641. Drost, M., 1983. Reciprocal embryo transfer between water buffaloes and cattle. Proc. Annu. Meet. Soc. Theriogenology, Nashville, TN, pp. 63-70. Drost, M., Wright, J.M. and Elsden, R.P., 1986. Intergeneric embryo transfer between water buffalo and domestic cattle. Theriogenology, 25: 13-23. Dufour, J.J. and Roy, G.L., 1985. Distribution of ovarian follicular populations in the dairy cow within 35 days after parturition. J. Reprod. Fertil., 73: 229-235. Gougeon, A., 1982. Cindtique de la croissance et de l'dvolution des follicules ovariens pendant le cycle menstruel chez la femme. Th~se D. Sc. Paris VI. Guilbault, L.A., Roy, G.L., Grasso, F., Menard, D.P. and Bousquet, D., 1988. Ovarian tbllicular dynamics in superovulated heifers pretreated with FSH-P at the beginning of the oestrous cycle. An ultrasonographic approach. Theriogenology, 29:257 (abstr.). Karaivanov, C., 1986. Comparative studies on the superovulatory effect of PMSG and FSH in water buffalo (Bubalus bubalis). Theriogenology, 26:51 60. Lussier, J.G., Matton, P. and Dufour, J.J., 1987. Growth rates of follicles in the ovary of the cow. J. Reprod. Fertil., 81; 301-307. Mariana, J.C., 1978. Analyse biom&rique de l'index de marquage des cellules folliculeuses et de la taille des ovocytes des follicules primordiaux d'ovaire de ratte adulte cyclique. Ann. Biol. Anim. Biochim. Biophys., 18: 1333-1342. Mauleon, P. and Mariana, J.C., 1977. Oogenesis and folliculogenesis. In: H.H. Cole. and P.T. Cupps t Editors), Reproduction in Domestic Animals. Academic Press, London, pp. 175 202. Maurasse, C., Matton, P. and Dufour, J.J., 1985. Ovarian follicular populations at two stages of an oestrous cycle in heifers given high energy diets. J. Anim. Sci., 61: 1194-1200. Monniaux, D., Chupin, D. and Saumande, J., 1983. Superovulatory responses of cattle. Theriogenology, 19: 55-81. Neveu, S., Hedon, B., Bringer, J., Chinchole, J.M., Arnal, F., Humeau, C., Cristol, P. and Viala, J.L., 1987. Ovarian stimulation by a combination of a gonadotrophin releasing hormone agonist and gonadotrophins for in vitro fertilization. Fertil. Steril., 47:639 643.
178 Scaramuzzi, R.J., Turnbull, K.E. and Nancarrow, C.P., 1980. Growth of Graafian follicles in cows following luteolysis induced by the prostaglandin F2-alpha analogue cloprostenol. Aust. J. Biol. Sci., 33: 63-69. Seidel, G.E., Larson, L.L. and Foote, R.H., 1971. Effects of age and gonadotrophin treatment on superovulation in the calf. J. Anim. Sci., 33: 617-622. Sharifuddin, W. and Jainudeen, M.R., 1984. Superovulation and non surgical collection of ova in the water buffalo (Bubalus bubalis). Proc. 10th Int. Congr. Anim. Reprod. and AI, Urbana, pp. 240-242. Siegel, S., 1956. Non-parametric Statistics for the Behavioral Sciences. McGraw-Hill, New York, pp. 68-125. Sonjaya, H. and Driancourt, M.A., 1987. Ovarian follicles during infancy in Romanov and Ile de France ewe lambs. J. Reprod. Fertil., 81: 241-248. Ware, C.B., Northley, D.L. and First, N.L., 1987. Effect of administration of FSH at the beginning of the cycle on the subsequent response to superovulation treatment in heifers. Theriogenology, 27:292 (abstr.). Woods, G.L., Scraba, S.T. and Ginther, O.J., 1982. Prospects for induction of multiple ovulations and collection of multiple embryos in the mare. Theriogenology, 17: 61-72.
171
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
Ovarian Follicular P o p u l a t i o n s in Buffaloes and Cows LE VAN TY*, D. CHUPIN and M.A. DRIANCOURT**
INRA, Reproductive Physiology, 37 380 Monnaie (France) (Accepted 19 January 1989)
ABSTRACT Le Van Ty, Chupin, D. and Driancourt, M.A., 1989. Ovarian follicular populations in buffaloes and cows. Anita. Reprod. Sci., 19: 171-178. Follicular populations and the main parameters of follicular development were compared in the ()varies of eight swamp buffaloes and four cows from Indonesia and Vietnam. The ovarian follicles were counted, measured, ranked in non-atretic and atretic (more than five pyknotic bodies) and grouped into six classes according to diameter. The overall population of antral follicles in buffaloes was only 20% of that of cows (47.5 _+23.8 vs 233.0 _+95.8, P < 0.002), and this difference was apparent in every size class. In contrast, the extent of atresia was similar in the two species. There were also no differences in the processes of oocyte growth, granulosa cell multiplication and antrum development between buffaloes and cows. It is likely that the poor follicular population found in buffalo ovaries is responsible for the limited response to superovulation of this species.
INTRODUCTION
In many developing countries, buffaloes are used as an important source of milk and meat and as draught animals. However, their productivity is still very limited. One of the possibilities of enhancing their production efficiency is genetic improvement of the existing populations. This involves identification of the animals with high producing abilities (i.e. high milk production, high growth rate, short calving intervals or early first calving) followed by their multiplication to supply breeding stock to local farmers. As embryo transfer is feasible in buffaloes (Drost et al., 1983), superovulation and embryo transfer can be used to achieve this. However, the ovarian response of buffaloes to superovulation is low, with mean ovulation rates ranging from 2 to 5 following *Present address: Laboratory of Biology of Reproduction, Dalat Center, National Institute of Scientific Research of Vietnam, Republic of Vietnam. **To whom reprint requests should be addressed.
0378-4320/89/$03.50
© 1989 Elsevier Science Publishers B.V.
172 administration of 3000 IU PMSG or 40-50 mg pFSH (Sharifuddin and Jainudeen, 1984; Drost et al., 1986; Karaivanov, 1986). As clear relationships have been demonstrated in cattle (Monniaux et al., 1983) and sheep (Driancourt, 1987) between the populations of ovarian follicles and ovarian response to exogenous gonadotrophins, the aim of this study was to compare follicle numbers and dynamics in buffalo versus cow ovaries. MATERIALSAND METHODS
Animals Eight swamp buffaloes and four cows, 4-8 years old, originating from Vietnam (six buffaloes) or Indonesia (two buffaloes, four cows) were used in this study. Ovaries were obtained either at slaughter of cycling animals at unspecified stages of the oestrous cycle (pairs of ovaries from three buffaloes and four cows) or at hemiovariectomy on the penultimate day of a synchronization treatment (Chupin and Saumande, 1979) consisting of a 5-mg oestradiol valerate injection followed by a Norgestomet implant for 9 days (single ovaries of five buffaloes).
Histological techniques The ovaries were immediately fixed in Bouin Holland's solution, embedded in paraffin wax and serially sectioned at 10 pm. One section out of six was mounted, stained with haematoxylin and examined microscopically. All antral follicles (i.e. those exhibiting a cavity larger than 1000 pm 2) were counted and measured using the oocyte as a marker to avoid counting follicles twice. The area of each follicle was determined by tracing the follicle at the basement membrane with an electronic planimeter through a drawing tube fixed to the microscope. The antrum and, when its nucleus was visible, the oocyte were similarly measured. All areas measured in ttm 2 were converted to diameters, assuming that the follicles and oocytes were spherical. Follicles were then ranked in the size classes previously defined by Dufour and Roy (1985): 0.160.28 mm, 0.29-0.67 mm, 0.68-1.57 mm, 1.58-3.67 mm, 3.68-8.56 mm and > 8.56 mm. A follicle was considered to be atretic when there were more than five pyknotic bodies in the section studied (Driancourt et al., 1987).
Statistical methods Owing to the lack of homogeneity in the variances of follicle numbers in buffalo and cow ovaries and to the small sample size, non-parametric testing (i.e. Mann-Whitney test to compare means and Wilcoxon test to compare
17~
paired means) was used (Siegel, 1956). Between-breed differences in a n t r u m and oocyte growth were detected by calculating the regression lines linking a n t r u m and follicle sizes or oocyte and follicle sizes and comparing the slopes and intercepts by the M a n n - W h i t n e y test. N u m b e r of granulosa cells per section and per follicle were calculated according to Gougeon (1982). Mitotic indices (i.e. the ratio of the number of mitotic figures to the number of granulosa cells in the section studied) were compared by a two-way ANOVA after arcsine transformation of the data. RESULTS
Follicular populations When the right and left ovaries from three buffaloes were compared, they contained similar numbers of follicles (i.e. 11, 38 and 57 follicles in the right and 11, 29 and 75 follicles in the left ovaries, respectively). Their size distribution was also similar (Fig. 1 ). Hence, in the remainder of the study, a single ovary was used. There were also similar numbers of follicles in Indonesian (50 and 96 antral follicles) and Vietnamese buffaloes (19 to 59 antral follicles). Hence buffaloes originating from both countries were pooled to be compared to cows.
There were highly significant differences in the total population of antral follicles ( 47.5 + 23.8 vs 233.0 _+95.8 in buffaloes vs cows, respectively; P < 0.002 ) ( m e a n + s . d . ) , in the population of non-atretic follicles (38.9+16.6 vs 207.3 + 70.0 in buffaloes vs cows, respectively; P < 0.002 ) and in the population of follicles in each of the first four size classes (P < 0.02, P < 0.002, P < 0.002, P < 0.002, respectively) (Fig. 2). However, despite the low numbers of antral follicles in buffaloes, their size distribution was similar to that found in cows (Fig. 2. ). The number of non-atretic follicles > 1.7 m m in diameter, which are the follicles involved in the ovulatory ovarian response to exogenous gonadotrophins according to Monniaux et al. (1983), ranged from 1 to 5 (mean 2.9) in buffaloes as opposed to a range of 17 to 32 (mean 22.1 ) in cows.
Follicular growth There were no species differences in the slopes and intercepts of the regression lines linking oocyte and follicle size during the period of rapid oocyte growth (classes 1 and 2). The overall regression lines were y = 4 5 4 8 x - 1 7 4 1 2 and y = 4 2 4 9 x - 15601 for buffaloes and cows respectively (y being oocyte size and x being log of follicle size). Furthermore, the maximal size of the oocyte observed in the largest follicle was also similar in buffaloes (98.6/~m in diameter t and cows (100.8/~m in diameter). There were also no differences related to species in the slopes and intercepts
174 40
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Fig. 1. Size distribution of the antral follicles in the left and right ovaries of 3 buffaloes.
of the regression lines linking antrum size (y = log of antrum size ) and follicle size (x = log of follicle size ). The respective regression lines are y = 1.43 x - 2.81 and y = 1.45 x - 2 . 9 1 for buffaloes and cows. The increase in the number of granulosa cells with size was also identical in the two breeds as evidenced by the following regression lines linking these two parameters: y = 1.12 x - 1 . 6 4 8 and y = 1.31 x - 1.635 for buffaloes and cows, respectively, with y being the log of the number of granulosa cells and x the log of follicle size. Finally, there were no species or follicle size effects nor any species-by-follicle-size interactions on the changes in the mitotic index of the granulosa cells with size (Fig. 3 ). In both species, the mitotic index was low in classes 1 and 4 and slightly higher in classes 2 and 3. There were not enough healthy follicles in classes 5 and 6 to include them in the study.
175 follicle number 100
80
60
40
. . . . . o$oeG e e o e l
0.16
e o e e e
0.28
0.67
1.57
3.67
diameter (ram)
Fig. 2. Size distribution of the antral follicles in the ovaries of 8 buffaloes ( • ) and 4 cows ( [] ). m i t o t i c index of the
granules
cells (%)
0.3
I
0.2
-~
0.1 , 1
0.16
2
0.28
, 3
0.67
4
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3.67
d i a m e t e r (mrn)
Fig. 3. Changes ( + s.d. ) in the mitotic index (%) of the granulosa cells with increasing follicle size in buffaloes ( • ) and cows ( • ). DISCUSSION T h e m a i n c o n c l u s i o n of t h i s s t u d y is t h a t , while t h e d e v e l o p m e n t a l p r o c e s s e s are s i m i l a r in follicles of b u f f a l o e s a n d cows, t h e n u m b e r of a n t r a l follicles f o u n d in b u f f a l o o v a r i e s is o n l y 20% of t h a t of cow ovaries.
176 The estimates of follicular population in cows obtained in this study are in good agreement with previous reports in a wide range of breeds (Scaramuzzi et al., 1980; Maurasse et al., 1985; Dufour and Roy, 1985). This study is, however, the first report on follicular populations in buffaloes. It is in good agreement with the finding, in an abattoir survey, that the proportion of ovaries devoid of surface follicles was much higher in buffaloes than in cattle (Dobson and Kamonpatana, 1986). Possible reasons for the poor follicular population of buffaloes are a low exit of the reserve of primordial follicles and/or a reduced growth rate of the growing follicles and/or a high extent of atresia (Mauleon and Mariana, 1977 ). As it was demonstrated that neither growth rate nor atresia differed between cows and buffaloes, it is likely that the limited number of growing follicles in buffaloes is linked to a limited initiation of follicular growth from the primordial reserve. The factors causing this require further investigation. They could be either the small size of the reserve of primordial follicles, a feature which is associated in mares with poor folliculogenesis (Driancourt et al., 1982), or a small percentage of easily mobilizable primordial follicles amongst the reserve (Mariana, 1978). Furthermore, if it is assumed that, as demonstrated in cows (Monniaux et al., 1983), the number of ovulations following gonadotrophin administration is highly related to the number of healthy follicles > 1.7 mm in diameter, it is not surprising that buffaloes with about 2.9 such follicles per ovary had a much smaller ovulatory response to PMSG than cows with about 22.1 such follicles per ovary. It is interesting to note that mares, which have a folliculogenesis numerically similar to that of buffaloes (Driancourt et al., 1982), also exhibit a limited response to superovulation (Woods et al., 1982). Mobilization of a higher number of follicles over 1.7 mm in diameter before treatment is therefore probably necessary to increase the ovarian response to PMSG in buffaloes. This could be achieved either by a pretreatment with gonadotrophins (Ware et al., 1987), although its efficiency in cattle is still debated (Monniaux et al., 1983; Guilbault et al., 1988 ), or with gonadotrophin releasing hormone agonist, a procedure efficient in humans (Neveu et al., 1987). However, while these pretreatments will have to be long enough to cover the time (40 days) required for the transit of follicles from antrum formation to large size (Lussier et al., 1987), they may also quickly exhaust the reserve of primordial follicles. Strategies which are less follicle consuming may have to be devised. As it has been demonstrated in rats (De Reviers and Mauleon, 1979), sheep (Sonjaya and Driancourt, 1987) and pigs (M.A. Driancourt, unpublished results) that massive follicular growth compared to adult numbers occurs in prepubertal animals, and as it is feasible to superovulate cattle as soon as 1-2 months of age (Seidel et al., 1971 ), a possibility of improving the ovarian response to exogenous gonadotrophins in buffaloes would be to superovulate prepubertal buffaloes.
177 ACKNOWLEDGEMENTS
Mr. Le Van Ty was supported by CIES during his stay in France. The authors are grateful to Dr. H. Sonjaya for provision of some of the ovaries and to Mr. R. Procureur for collection of the remainder of the ovaries.
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