- Email: [email protected]
Follicular atresia and LH concentrations during the follicular phase of the estrous cycle in the goat (Capra hircus)
Animal Reproduction Science 51 Ž1998. 23–30
Follicular atresia and LH concentrations during the follicular phase of the estrous cycle in the goat ž Capra hircus / ) F. Gonzalez-Valle , M. Batista-Arteaga, A. Gracia-Molina ´ Departamento de Patologıa ´ Animal, Facultad de Veterinaria, UniÕersidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain Accepted 25 June 1997
Abstract The objective of the study was to identify the effects of LH on the final follicle maturation process as well as the incidence of atresia during the follicular phase of the goat’s estrous cycle. In Experiment 1, concentrations of the LH were measured during the follicular phase of a synchronized cycle in 8 Canary goats. In Experiment 2, the same animals were synchronized again. On each day of a 4-day experimental period Žday 0 s day of sponges withdrawal., 2 of the goats were bilaterally ovariectomized. Follicles with a diameter ) 1 mm were dissected out to obtain qualitative histological data in normal, early atretic I, early atretic II, advanced atretic I and advanced atretic II follicles. The total interval from sponge withdrawal to LH peak was 77.5 " 9.8 h. LH peak concentration averaged 44 " 5.3 ngrml and the mean length of the preovulatory surge Žamounts over 10 ngrml. was 8.9 " 0.9 h. During the total follicular phase, there were more atretic follicles than normal follicles Ž58 vs. 30, P - 0.05.. The number of early and advanced atretic follicles was similar. There were more early atresia I than early atresia II follicles Ž23 vs. 6, P - 0.05.. On day 2, the number of advanced atretic follicles was greater than early atretic follicles Ž10 vs. 4, P - 0.05.. There was an increase in the number of early atretic follicles from day 2 to day 4 Ž4 vs. 9, P - 0.05., which was consistent with the effects of the preovulatory LH surge. q 1998 Elsevier Science B.V. Keywords: Goat; Follicular dynamics; LH; Follicular atresia
) Corresponding author. Departamento de Patologıa ´ Animal, Facultad de Veterinaria, cr Francisco Inglot Artiles 12, 35016 Las Palmas de Gran Canaria, Spain. Tel.: q34 28 454346; fax: q34 28 45430; e-mail: [email protected]
0378-4320r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 7 8 - 4 3 2 0 Ž 9 7 . 0 0 0 5 5 - 9
24
F. Gonzalez-Valle et al.r Animal Reproduction Science 51 (1998) 23–30 ´
1. Introduction Several recent studies in sheep have shown that pituitary gonadotrophins influence the growth of ovarian follicles and that suppression of this influence is associated with atresia in antral follicles ŽDufour et al., 1979; Baird and McNeilly, 1981; Greenwald and Terranova, 1988.. Follicles greater than 1 mm in diameter respond positively to a modest gonadotrophic stimulus, but follicles greater than 2.5 mm in diameter are totally dependent on adequate amounts of gonadotrophins ŽScaramuzzi et al., 1993.. Serum concentrations of LH and its pulsatile frequency increase considerably during the follicular phase of the estrous cycle. These increments may contribute to preovulatory follicle selection by differentially altering the responsiveness of follicle growth to the stimulus of FSH ŽMcNeilly et al., 1991.. Atresia is a complex process occurring at any stage of follicular growth ŽMariana et al., 1991.. The earliest signs of atresia in antral follicles are defined by degenerative changes that involve granulosa cells, both in sheep ŽMurdoch, 1992. and in goats ŽSharma et al., 1992.. Many features of terminal follicle growth remain unknown, but the degree of atresia is a determining factor for this growth. In the goat, data about the factors involved in terminal follicle growth are sparse. The aim of the following experiment was to contribute to the knowledge of those factors, by recording the changes in LH levels towards the preovulatory peak and measuring the incidence of atresia during the follicular phase of the estrous cycle of goats.
2. Material and methods 2.1. Experimental animals Eight Canary dairy goats Ž Capra hircus ., 2–4 years old and weighing 48 " 3 kg were used in this experiment. They had experienced regular 20–22 days estrous cycles before the experiment, and were monitored twice daily for estrus. They were kept in a covered pen at the Experimental Farm of the Gran Canaria Council ŽCanary Islands. throughout the experiment, and were fed with green forage plus concentrates. Water was always available. 2.2. Experiment 1 Estrous cycles were synchronized by insertion of progestagen-impregnated vaginal sponges Ž45 mg fluorogestone acetate, Chrono-gest w , Intervet. for 11 days during the breeding season ŽNovember.. Each goat received an injection of luprostiol, a potent analogue of prostaglandin F-2 a ŽProsolvin w , Intervet. in order to assure luteal regression at sponge withdrawal. Blood samples were collected from the jugular vein at intervals of 4 h beginning at 30 h after sponge removal until ovulation. Ovulation was detected by daily laparoscopic explorations. Blood samples were placed in heparinized tubes and were immediately centrifuged at 2000 g for 15 min. The plasma was
F. Gonzalez-Valle et al.r Animal Reproduction Science 51 (1998) 23–30 ´
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removed, labelled and stored frozen Žy708C. until assayed for LH. Concentrations of LH were measured in duplicate at the Station de Physiologie de la Reproduction des Mammiferes Domestiques INRA ŽNouzilly, France., using the double radioimmunoassay decribed previously in the ewe by Pelletier et al. Ž1968., and validated for the goat by Chemineau et al. Ž1982.. Purified ovine LH, CY1086 ŽDr. Y. Combarnous, INRA, Nouzilly, France; equivalent to 3.5 = NIH-oLH-S1. was used as standard reference and also for preparation of tracer. The tracer was iodinated using 125 I ŽAmersham-France.. The specific activity of the tracer was 73,7 m Cirm g, corresponding to an average incorporation of one 125 I atom per molecule of oLH. Rabbit anti-ovine LH antiserum Žanti-oLH-L 3 . was supplied by Dr. M.P. Dubois, INRA, Nouzilly, France. The second antibody was anti-rabbit globulin serum obtained by immunization of a mare. The minimal detectable level was 1 ngrml Žmeasured at BrB 0 s 80%.. Intra-assay coefficients of variation were 14.4%, 3.6% and 11.9% in the low, medium and high level, respectively. 2.3. Experiment 2 The same goats were resynchronized 50 days later using the previously described treatment. On each day of a 4-day experimental period Ži.e. days 1, 2, 3 and 4; day 0 s day of sponges withdrawal., 2 goats selected at random were bilaterally ovariectomized. The animals were sedated with xylazine ŽIM. ŽRompun ´ w , Bayer. and anesthetized locally by paravertebral, bilateral lumbar infiltration of lidocaine. The ovaries were then surgically removed by a medial abdominal laparotomy. Immediately after removal, the ovaries were placed in a 10% solution of formaldehyde. Later, each follicle with a diameter) 1 mm was carefully dissected out. Each dissected follicle was embedded in paraffin and sectioned at a thickness of 3–4m. One of every 10 sections were mounted and stained using Harris’ hematoxilyn–eosin method ŽLuna, 1960.. To
Fig. 1. Plasma concentrations of LH Žmean"SEM..
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F. Gonzalez-Valle et al.r Animal Reproduction Science 51 (1998) 23–30 ´
establish follicular quality, 2–3 sections of the mid-zone of each follicle were microscopically observed, according to the classification established by Webb et al. Ž1989.: Ø Normal: less than 5 pycnotic bodies in the granulosa cell layer of the section studied. Ø Early atresia I: 5–100 pycnotic bodies in the granulosa cell layer of the section studied. Ø Early atresia II: 100–200 pycnotic bodies in the granulosa cell layer of the section studied. Ø Advanced atresia I: numerous pycnotic bodies, although still a distinct granulosa cell layer was observed. Ø Advanced atresia II: numerous pycnotic bodies, but no distinct granulosa cell layer was observed. 2.4. Statistics The differences between the number of normal and atretic follicles, and between various degrees of atresia were statistically analyzed using a chi-square test. The results of LH measurements are shown as means" standard errors of the mean ŽSEM.. The differences were considered significant at P - 0.05. 3. Results 3.1. Experiment 1 The mean interval from the synchronization treatment to LH peak was 77.5 " 9.8 h. Fig. 1 shows the pattern of LH mean levels adjusted to the peak. Maximal levels of LH averaged 44 " 5.3 ngrml and the length of preovulatory surge Žamounts over 10 ngrml. averaged 8.9 " 0.9 h. 3.2. Experiment 2 The number of follicles evaluated each day during the period of study was 25 " 3.7 Žmean " SEM.. Table 1 shows the proportional incidence of atresia during the experimental period. Over the entire study period, the number of atretic follicles was greater than the number of normal follicles. This preponderance of atretic follicles respect to normal follicles was statistically significant Ž P - 0.05. on days 1 and 4.
Table 1 Number of normal and atretic follicles throughout the experimental period Experimental period Day 1 Normal follicles Atretic follicles
a
7 18 b
Day 2 9 14
Day 3 9 13
Day 4 a
5 13 b
Numbers in the same column with distinct superscripts differ significantly Ž P - 0.05..
Total 30 a 58 b
F. Gonzalez-Valle et al.r Animal Reproduction Science 51 (1998) 23–30 ´
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Table 2 Number of follicles with various degree of atresia throughout the experimental period Experimental period Day 1
Day 2
Day 3
Day 4
Total
Total atresia Early atresia Advanced atresia
9 9
4a 10 b
7 6
9c 4d
29 29
Early atresia Type I Type II
7 2
4 y
5 2
7 2
23 a 6b
AdÕanced atresia Type I Type II
7 2
7 3
1 5
4 y
19 10
Within the same group ŽTotal, Early and Advanced atresia., numbers in rows or columns with distinct superscripts differ significantly Ž P - 0.05..
The evolution of the degree of atresia throughout the experimental period is shown in Table 2. On day 2, there were more advanced atretic follicles than early atretic follicles. By contrast, on day 4, there were more early atretic follicles than advanced atretic follicles. On the other hand, from day 2 to day 4, there was a significant increase in the number of early atretic follicles and the corresponding fall in the number of advanced atretic follicles. Over the entire study period, there was a significant preponderance of early atretic type I follicles over early atretic type II follicles. However, no difference was found between the numbers of advanced atretic type I follicles and advanced atretic type II follicles.
4. Discussion The mean interval between PGF-2 a administration and LH peak in Canary goat Ž77.5 h. was somewhat longer than intervals obtained in studies carried out with other breeds Ž55–65 h: Ott et al., 1980; Mori and Kano, 1984; Bretzlaff et al., 1988; Greyling and van Niekerk, 1990.. On this basis, Llewelyn et al. Ž1993a. suggested that terminal follicle growth takes longer in goat breeds in the tropics than in breeds in a more temperate climate. In our experiment, the mean length of the LH preovulatory surge in the Canary goat Ž8.9 h. was similar to the result obtained by Llewelyn et al. Ž1993b. in the British White goat Ž10 h., utilizing the same criterion to determine the length of the LH surge. In the Alpine goat, Chemineau et al. Ž1982. obtained a length of 8 h, while Bono et al. Ž1983. reported 16 h in the same breed, and Mori and Kano Ž1984. 7.6 h in Shiba goat. Blood sampling regimens differed: 2-h intervals in Llewelyn et al. Ž1993b., Mori and Kano Ž1984. and Chemineau et al. Ž1982., 8-h intervals in Bono et al. Ž1983. and 4-h intervals in our study. These differences in the length of the LH preovulatory surge could have
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F. Gonzalez-Valle et al.r Animal Reproduction Science 51 (1998) 23–30 ´
been caused by time of sampling and by different methods used in establishing the start and finish of the LH preovulatory discharge. In our study, the percentage of atretic follicles in the total experimental period was identical to that obtained by Brand and de Jong Ž1973. in Texel sheep sampled at the same days and follicular size. Moreover, the percentage of atretic follicles was similar on each day in both studies. Our study allowed for a detailed evaluation of atretic processes and their timing during follicular phase. On day 2 of the follicular phase, a significant prevalence of advanced atresia was recorded. This fact suggests that some follicles that presented early atresia on day 1, had already shown advanced atresia on day 2. From day 2 on, there was an inversion in the degree of atresia, as early atresia began to rise until it reached a maximum on day 4. This increase seems to reflect an extensive degeneration of normal follicles. Until day 2, this level of degeneration was not so evident, since the proportion of early atresia was falling. Therefore, it seemed that from day 2 on, there were some changes in the physiology of the follicular development that accelerated the rate and the incidence of atresia. Several successive waves of follicular growth have been described both in the goat ŽGinther and Kot, 1994. and in the sheep ŽNoel ¨ et al., 1993; Ravindra et al., 1994. with only one wave associated with ovulation. In our observations, on days 1 and 4, there was a significative prevalence of atretic follicles respect to normal follicles, and the proportion of atretic follicles was similar in both days. However, on day 4, there was a significant prevalence of early atretic follicles, while on day 1, the number of early and advanced atretic follicles was the same. The difference between days 1 and 4 in the proportion of early and advanced atresia could be due to the fact that day 1 relates to the wave of follicular growth in which the ovulation takes place, while day 4 relates to the emergence of the next wave of follicular growth. Otherwise, if our observations on days 1 and 4 are related to the same wave of follicular development, the difference in the proportion of early and advanced atresia, could be due to the presence of changing physiological factors that generate atresia. Moor et al. Ž1975. noted that in sheep, there were structural changes in granulosa cells about 12 h after the LH peak, such as cellular dissociation and rupture of the basal lamina. In the same vain, Scaramuzzi et al. Ž1993. have shown that complete atresia of all gonadotrophin-dependent follicles was assured by the detrimental effects of the high amounts of LH in the preovulatory discharge. In the Canary goat, preovulatory LH surge takes place by the beginning of day 3. Therefore, these detrimental effects will appear in the follicles by the end of preovulatory period. This is consistent with the increase of early atresia registered on day 4.
Acknowledgements The authors wish to gratefully acknowledge the following contributions: Department of Morphology, University of Las Palmas Gran Canaria and Experimental Farm of the Gran Canaria Council for the use of their equipment, Station de Physiologie de la Reproduction des Mammiferes Domestiques INRA, Nouzilly for the measurement of
F. Gonzalez-Valle et al.r Animal Reproduction Science 51 (1998) 23–30 ´
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LH, and Dr. J.L. Alabart for his assistance in hormone assays. This research was partially supported by DGA, Diputacion ´ General de Aragon ´ ŽProject 19r91.. References Baird, D.T., McNeilly, A.S., 1981. Gonadotrophic control of follicular development and function during the estrous cycle of the ewe. J. Reprod. Fertil. Suppl. 30, 119–133. Bono, G., Cairoli, F., Tamanini, C., Abrate, L., 1983. Progesterone, estrogen, LH, FSH, and prolactin concentrations in plasma during the estrous cycle in goat. Reprod. Nutr. Dev. 23, 217–222. Brand, A., de Jong, W.H.R., 1973. Qualitative and quantitative micromorphological investigations of the tertiary follicle population during the estrous cycle in sheep. J. Reprod. Fertil. 33, 431–439. Bretzlaff, K.N., Weston, P.G., Hixon, J.E., Ott, R.S., 1988. Plasma luteinizing hormone and progesterone concentrations in goats with estrous cycles of normal or short duration after prostaglandin F2 a administration during diestrus or pregnancy. Am. J. Vet. Res. 49, 939–943. Chemineau, P., Gauthier, D., Poirier, J., Saumande, J., 1982. Plasma levels of LH, FSH, prolactin, estradiol 17-8 and progesterone during natural and induced estrus in the dairy goat. Theriogenology 17, 313–323. Dufour, J., Cahill, L.P., MaulJon, P., 1979. Short and long-term effects of hypophysectomy and unilateral ovariectomy on ovarian follicular populations in sheep. J. Reprod. Fertil. 57, 301–309. Ginther, O.J., Kot, K., 1994. Follicular dynamics during the ovulatory season in goats. Theriogenology 42, 987–1001. Greenwald, G.S., Terranova, P.F., 1988. Follicular selection and its control. In: Knobil, E., Neill, J.D. ŽEds.., The Physiology of Reproduction. Raven Press, NY, pp. 387–445. Greyling, J.P.C., van Niekerk, C.H., 1990. Effect of pregnant mare serum gonadotrophin ŽPMSG. and route of administration after progestagen treatment on estrus and LH secretion in the Boer goat. Small Rum. Res. 3, 511–516. Llewelyn, C.A., Ogaa, J.S., Obwolo, M.J., 1993a. Plasma progesterone profiles and variation in cyclic ovarian activity throughout the year in indigenous goats in Zimbabwe. Anim. Reprod. Sci. 30, 301–311. Llewelyn, C.A., Perrie, J., Luckins, A.G., Munro, C.D., 1993b. Estrus in the British White goat: timing of plasma luteinizing hormone surge and changes in behavioral and vaginal traits in relationship to onset of estrus. Br. Vet. J. 149, 171–182. Luna, L.G., 1960. Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. McGraw-Hill, NY. Mariana, J.C., Monniaux, D., Driancourt, M.A., MaulJon, P., 1991. Folliculogenesis. In: Cupps, P.T. ŽEd.., Reproduction of Domestic Animals. Academic Press, San Diego, pp. 119–171. McNeilly, A.S., Picton, H.M., Campbell, B.K., Baird, D.T., 1991. Gonadotrophic control of follicle growth in the ewe. J. Reprod. Fertil. 43, 177–186. Moor, R.H., Hay, M.F., Seamark, R.F., 1975. The sheep ovary: regulation of steroidogenic, haemodynamic and structural changes in the largest follicle and adjacent tissue before ovulation. J. Reprod. Fertil. 45, 595–604. Mori, Y., Kano, Y., 1984. Changes in plasma concentrations of LH, progesterone and estradiol in relation to the occurrence of luteolysis, estrus and time of ovulation in the Shiba goat Ž Capra hircus .. J. Reprod. Fertil. 72, 223–230. Murdoch, W.J., 1992. Comparative morphometry and steroidogenic function of antral ovine follicles destined for ovulation or atresia. Domest. Anim. Endocrinol. 9, 219–224. Noel, ¨ B., Bister, J.L., Paquay, R., 1993. Ovarian follicular dynamics in Suffolk ewes at different periods of the year. J. Reprod. Fertil. 99, 695–700. Ott, R.S., Nelson, D.R., Hixon, J.E., 1980. Peripheral serum progesterone and luteinizing hormone concentrations of goats during synchronization of estrus and ovulation with prostaglandin F-2 a . Am. J. Vet. Res. 41, 1432–1434. Pelletier, J., Kann, G., Dolais, J., Rosselin, G., 1968. Dosage radio-immunologique de l’hormone lutJinisante plasmatique chez le mouton. Mise au point de la technique de dosage. C.R. Acad. Sci. Paris, t. 266, Serie ´ D: 2291–2294.
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Ravindra, J.P., Rawlings, N.C., Evans, A.C.O., Adams, G.P., 1994. Ultrasonographic study of ovarian follicular dynamics in ewes during the estrous cycle. J. Reprod. Fertil. 101, 501–509. Scaramuzzi, R.J., Adams, N.R., Baird, D.T., Campbell, B.K., Downing, J.A., Findlay, J.K., Henderson, K.M., Martin, G.B., Mcnatty, K.P., Mcneilly, A.S., Tsonis, C.G., 1993. A model for follicle selection and the determination of ovulation rate in the ewe. Reprod. Fertil. Dev. 5, 459–478. Sharma, R.K., Khajuria, M., Guraya, S.S., 1992. Morphology of normal and atretic follicles of goat ovary during anestrous. Int. J. Anim. Sci. 7, 81–84. Webb, R., Gauld, I.K., Driancourt, M.A., 1989. Morphological and functional characterization of large antral follicles in three breeds of sheep with different ovulation rates. J. Reprod. Fertil. 87, 243–255.
Follicular atresia and LH concentrations during the follicular phase of the estrous cycle in the goat ž Capra hircus / ) F. Gonzalez-Valle , M. Batista-Arteaga, A. Gracia-Molina ´ Departamento de Patologıa ´ Animal, Facultad de Veterinaria, UniÕersidad de Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain Accepted 25 June 1997
Abstract The objective of the study was to identify the effects of LH on the final follicle maturation process as well as the incidence of atresia during the follicular phase of the goat’s estrous cycle. In Experiment 1, concentrations of the LH were measured during the follicular phase of a synchronized cycle in 8 Canary goats. In Experiment 2, the same animals were synchronized again. On each day of a 4-day experimental period Žday 0 s day of sponges withdrawal., 2 of the goats were bilaterally ovariectomized. Follicles with a diameter ) 1 mm were dissected out to obtain qualitative histological data in normal, early atretic I, early atretic II, advanced atretic I and advanced atretic II follicles. The total interval from sponge withdrawal to LH peak was 77.5 " 9.8 h. LH peak concentration averaged 44 " 5.3 ngrml and the mean length of the preovulatory surge Žamounts over 10 ngrml. was 8.9 " 0.9 h. During the total follicular phase, there were more atretic follicles than normal follicles Ž58 vs. 30, P - 0.05.. The number of early and advanced atretic follicles was similar. There were more early atresia I than early atresia II follicles Ž23 vs. 6, P - 0.05.. On day 2, the number of advanced atretic follicles was greater than early atretic follicles Ž10 vs. 4, P - 0.05.. There was an increase in the number of early atretic follicles from day 2 to day 4 Ž4 vs. 9, P - 0.05., which was consistent with the effects of the preovulatory LH surge. q 1998 Elsevier Science B.V. Keywords: Goat; Follicular dynamics; LH; Follicular atresia
) Corresponding author. Departamento de Patologıa ´ Animal, Facultad de Veterinaria, cr Francisco Inglot Artiles 12, 35016 Las Palmas de Gran Canaria, Spain. Tel.: q34 28 454346; fax: q34 28 45430; e-mail: [email protected]
0378-4320r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 7 8 - 4 3 2 0 Ž 9 7 . 0 0 0 5 5 - 9
24
F. Gonzalez-Valle et al.r Animal Reproduction Science 51 (1998) 23–30 ´
1. Introduction Several recent studies in sheep have shown that pituitary gonadotrophins influence the growth of ovarian follicles and that suppression of this influence is associated with atresia in antral follicles ŽDufour et al., 1979; Baird and McNeilly, 1981; Greenwald and Terranova, 1988.. Follicles greater than 1 mm in diameter respond positively to a modest gonadotrophic stimulus, but follicles greater than 2.5 mm in diameter are totally dependent on adequate amounts of gonadotrophins ŽScaramuzzi et al., 1993.. Serum concentrations of LH and its pulsatile frequency increase considerably during the follicular phase of the estrous cycle. These increments may contribute to preovulatory follicle selection by differentially altering the responsiveness of follicle growth to the stimulus of FSH ŽMcNeilly et al., 1991.. Atresia is a complex process occurring at any stage of follicular growth ŽMariana et al., 1991.. The earliest signs of atresia in antral follicles are defined by degenerative changes that involve granulosa cells, both in sheep ŽMurdoch, 1992. and in goats ŽSharma et al., 1992.. Many features of terminal follicle growth remain unknown, but the degree of atresia is a determining factor for this growth. In the goat, data about the factors involved in terminal follicle growth are sparse. The aim of the following experiment was to contribute to the knowledge of those factors, by recording the changes in LH levels towards the preovulatory peak and measuring the incidence of atresia during the follicular phase of the estrous cycle of goats.
2. Material and methods 2.1. Experimental animals Eight Canary dairy goats Ž Capra hircus ., 2–4 years old and weighing 48 " 3 kg were used in this experiment. They had experienced regular 20–22 days estrous cycles before the experiment, and were monitored twice daily for estrus. They were kept in a covered pen at the Experimental Farm of the Gran Canaria Council ŽCanary Islands. throughout the experiment, and were fed with green forage plus concentrates. Water was always available. 2.2. Experiment 1 Estrous cycles were synchronized by insertion of progestagen-impregnated vaginal sponges Ž45 mg fluorogestone acetate, Chrono-gest w , Intervet. for 11 days during the breeding season ŽNovember.. Each goat received an injection of luprostiol, a potent analogue of prostaglandin F-2 a ŽProsolvin w , Intervet. in order to assure luteal regression at sponge withdrawal. Blood samples were collected from the jugular vein at intervals of 4 h beginning at 30 h after sponge removal until ovulation. Ovulation was detected by daily laparoscopic explorations. Blood samples were placed in heparinized tubes and were immediately centrifuged at 2000 g for 15 min. The plasma was
F. Gonzalez-Valle et al.r Animal Reproduction Science 51 (1998) 23–30 ´
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removed, labelled and stored frozen Žy708C. until assayed for LH. Concentrations of LH were measured in duplicate at the Station de Physiologie de la Reproduction des Mammiferes Domestiques INRA ŽNouzilly, France., using the double radioimmunoassay decribed previously in the ewe by Pelletier et al. Ž1968., and validated for the goat by Chemineau et al. Ž1982.. Purified ovine LH, CY1086 ŽDr. Y. Combarnous, INRA, Nouzilly, France; equivalent to 3.5 = NIH-oLH-S1. was used as standard reference and also for preparation of tracer. The tracer was iodinated using 125 I ŽAmersham-France.. The specific activity of the tracer was 73,7 m Cirm g, corresponding to an average incorporation of one 125 I atom per molecule of oLH. Rabbit anti-ovine LH antiserum Žanti-oLH-L 3 . was supplied by Dr. M.P. Dubois, INRA, Nouzilly, France. The second antibody was anti-rabbit globulin serum obtained by immunization of a mare. The minimal detectable level was 1 ngrml Žmeasured at BrB 0 s 80%.. Intra-assay coefficients of variation were 14.4%, 3.6% and 11.9% in the low, medium and high level, respectively. 2.3. Experiment 2 The same goats were resynchronized 50 days later using the previously described treatment. On each day of a 4-day experimental period Ži.e. days 1, 2, 3 and 4; day 0 s day of sponges withdrawal., 2 goats selected at random were bilaterally ovariectomized. The animals were sedated with xylazine ŽIM. ŽRompun ´ w , Bayer. and anesthetized locally by paravertebral, bilateral lumbar infiltration of lidocaine. The ovaries were then surgically removed by a medial abdominal laparotomy. Immediately after removal, the ovaries were placed in a 10% solution of formaldehyde. Later, each follicle with a diameter) 1 mm was carefully dissected out. Each dissected follicle was embedded in paraffin and sectioned at a thickness of 3–4m. One of every 10 sections were mounted and stained using Harris’ hematoxilyn–eosin method ŽLuna, 1960.. To
Fig. 1. Plasma concentrations of LH Žmean"SEM..
26
F. Gonzalez-Valle et al.r Animal Reproduction Science 51 (1998) 23–30 ´
establish follicular quality, 2–3 sections of the mid-zone of each follicle were microscopically observed, according to the classification established by Webb et al. Ž1989.: Ø Normal: less than 5 pycnotic bodies in the granulosa cell layer of the section studied. Ø Early atresia I: 5–100 pycnotic bodies in the granulosa cell layer of the section studied. Ø Early atresia II: 100–200 pycnotic bodies in the granulosa cell layer of the section studied. Ø Advanced atresia I: numerous pycnotic bodies, although still a distinct granulosa cell layer was observed. Ø Advanced atresia II: numerous pycnotic bodies, but no distinct granulosa cell layer was observed. 2.4. Statistics The differences between the number of normal and atretic follicles, and between various degrees of atresia were statistically analyzed using a chi-square test. The results of LH measurements are shown as means" standard errors of the mean ŽSEM.. The differences were considered significant at P - 0.05. 3. Results 3.1. Experiment 1 The mean interval from the synchronization treatment to LH peak was 77.5 " 9.8 h. Fig. 1 shows the pattern of LH mean levels adjusted to the peak. Maximal levels of LH averaged 44 " 5.3 ngrml and the length of preovulatory surge Žamounts over 10 ngrml. averaged 8.9 " 0.9 h. 3.2. Experiment 2 The number of follicles evaluated each day during the period of study was 25 " 3.7 Žmean " SEM.. Table 1 shows the proportional incidence of atresia during the experimental period. Over the entire study period, the number of atretic follicles was greater than the number of normal follicles. This preponderance of atretic follicles respect to normal follicles was statistically significant Ž P - 0.05. on days 1 and 4.
Table 1 Number of normal and atretic follicles throughout the experimental period Experimental period Day 1 Normal follicles Atretic follicles
a
7 18 b
Day 2 9 14
Day 3 9 13
Day 4 a
5 13 b
Numbers in the same column with distinct superscripts differ significantly Ž P - 0.05..
Total 30 a 58 b
F. Gonzalez-Valle et al.r Animal Reproduction Science 51 (1998) 23–30 ´
27
Table 2 Number of follicles with various degree of atresia throughout the experimental period Experimental period Day 1
Day 2
Day 3
Day 4
Total
Total atresia Early atresia Advanced atresia
9 9
4a 10 b
7 6
9c 4d
29 29
Early atresia Type I Type II
7 2
4 y
5 2
7 2
23 a 6b
AdÕanced atresia Type I Type II
7 2
7 3
1 5
4 y
19 10
Within the same group ŽTotal, Early and Advanced atresia., numbers in rows or columns with distinct superscripts differ significantly Ž P - 0.05..
The evolution of the degree of atresia throughout the experimental period is shown in Table 2. On day 2, there were more advanced atretic follicles than early atretic follicles. By contrast, on day 4, there were more early atretic follicles than advanced atretic follicles. On the other hand, from day 2 to day 4, there was a significant increase in the number of early atretic follicles and the corresponding fall in the number of advanced atretic follicles. Over the entire study period, there was a significant preponderance of early atretic type I follicles over early atretic type II follicles. However, no difference was found between the numbers of advanced atretic type I follicles and advanced atretic type II follicles.
4. Discussion The mean interval between PGF-2 a administration and LH peak in Canary goat Ž77.5 h. was somewhat longer than intervals obtained in studies carried out with other breeds Ž55–65 h: Ott et al., 1980; Mori and Kano, 1984; Bretzlaff et al., 1988; Greyling and van Niekerk, 1990.. On this basis, Llewelyn et al. Ž1993a. suggested that terminal follicle growth takes longer in goat breeds in the tropics than in breeds in a more temperate climate. In our experiment, the mean length of the LH preovulatory surge in the Canary goat Ž8.9 h. was similar to the result obtained by Llewelyn et al. Ž1993b. in the British White goat Ž10 h., utilizing the same criterion to determine the length of the LH surge. In the Alpine goat, Chemineau et al. Ž1982. obtained a length of 8 h, while Bono et al. Ž1983. reported 16 h in the same breed, and Mori and Kano Ž1984. 7.6 h in Shiba goat. Blood sampling regimens differed: 2-h intervals in Llewelyn et al. Ž1993b., Mori and Kano Ž1984. and Chemineau et al. Ž1982., 8-h intervals in Bono et al. Ž1983. and 4-h intervals in our study. These differences in the length of the LH preovulatory surge could have
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been caused by time of sampling and by different methods used in establishing the start and finish of the LH preovulatory discharge. In our study, the percentage of atretic follicles in the total experimental period was identical to that obtained by Brand and de Jong Ž1973. in Texel sheep sampled at the same days and follicular size. Moreover, the percentage of atretic follicles was similar on each day in both studies. Our study allowed for a detailed evaluation of atretic processes and their timing during follicular phase. On day 2 of the follicular phase, a significant prevalence of advanced atresia was recorded. This fact suggests that some follicles that presented early atresia on day 1, had already shown advanced atresia on day 2. From day 2 on, there was an inversion in the degree of atresia, as early atresia began to rise until it reached a maximum on day 4. This increase seems to reflect an extensive degeneration of normal follicles. Until day 2, this level of degeneration was not so evident, since the proportion of early atresia was falling. Therefore, it seemed that from day 2 on, there were some changes in the physiology of the follicular development that accelerated the rate and the incidence of atresia. Several successive waves of follicular growth have been described both in the goat ŽGinther and Kot, 1994. and in the sheep ŽNoel ¨ et al., 1993; Ravindra et al., 1994. with only one wave associated with ovulation. In our observations, on days 1 and 4, there was a significative prevalence of atretic follicles respect to normal follicles, and the proportion of atretic follicles was similar in both days. However, on day 4, there was a significant prevalence of early atretic follicles, while on day 1, the number of early and advanced atretic follicles was the same. The difference between days 1 and 4 in the proportion of early and advanced atresia could be due to the fact that day 1 relates to the wave of follicular growth in which the ovulation takes place, while day 4 relates to the emergence of the next wave of follicular growth. Otherwise, if our observations on days 1 and 4 are related to the same wave of follicular development, the difference in the proportion of early and advanced atresia, could be due to the presence of changing physiological factors that generate atresia. Moor et al. Ž1975. noted that in sheep, there were structural changes in granulosa cells about 12 h after the LH peak, such as cellular dissociation and rupture of the basal lamina. In the same vain, Scaramuzzi et al. Ž1993. have shown that complete atresia of all gonadotrophin-dependent follicles was assured by the detrimental effects of the high amounts of LH in the preovulatory discharge. In the Canary goat, preovulatory LH surge takes place by the beginning of day 3. Therefore, these detrimental effects will appear in the follicles by the end of preovulatory period. This is consistent with the increase of early atresia registered on day 4.
Acknowledgements The authors wish to gratefully acknowledge the following contributions: Department of Morphology, University of Las Palmas Gran Canaria and Experimental Farm of the Gran Canaria Council for the use of their equipment, Station de Physiologie de la Reproduction des Mammiferes Domestiques INRA, Nouzilly for the measurement of
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LH, and Dr. J.L. Alabart for his assistance in hormone assays. This research was partially supported by DGA, Diputacion ´ General de Aragon ´ ŽProject 19r91.. References Baird, D.T., McNeilly, A.S., 1981. Gonadotrophic control of follicular development and function during the estrous cycle of the ewe. J. Reprod. Fertil. Suppl. 30, 119–133. Bono, G., Cairoli, F., Tamanini, C., Abrate, L., 1983. Progesterone, estrogen, LH, FSH, and prolactin concentrations in plasma during the estrous cycle in goat. Reprod. Nutr. Dev. 23, 217–222. Brand, A., de Jong, W.H.R., 1973. Qualitative and quantitative micromorphological investigations of the tertiary follicle population during the estrous cycle in sheep. J. Reprod. Fertil. 33, 431–439. Bretzlaff, K.N., Weston, P.G., Hixon, J.E., Ott, R.S., 1988. Plasma luteinizing hormone and progesterone concentrations in goats with estrous cycles of normal or short duration after prostaglandin F2 a administration during diestrus or pregnancy. Am. J. Vet. Res. 49, 939–943. Chemineau, P., Gauthier, D., Poirier, J., Saumande, J., 1982. Plasma levels of LH, FSH, prolactin, estradiol 17-8 and progesterone during natural and induced estrus in the dairy goat. Theriogenology 17, 313–323. Dufour, J., Cahill, L.P., MaulJon, P., 1979. Short and long-term effects of hypophysectomy and unilateral ovariectomy on ovarian follicular populations in sheep. J. Reprod. Fertil. 57, 301–309. Ginther, O.J., Kot, K., 1994. Follicular dynamics during the ovulatory season in goats. Theriogenology 42, 987–1001. Greenwald, G.S., Terranova, P.F., 1988. Follicular selection and its control. In: Knobil, E., Neill, J.D. ŽEds.., The Physiology of Reproduction. Raven Press, NY, pp. 387–445. Greyling, J.P.C., van Niekerk, C.H., 1990. Effect of pregnant mare serum gonadotrophin ŽPMSG. and route of administration after progestagen treatment on estrus and LH secretion in the Boer goat. Small Rum. Res. 3, 511–516. Llewelyn, C.A., Ogaa, J.S., Obwolo, M.J., 1993a. Plasma progesterone profiles and variation in cyclic ovarian activity throughout the year in indigenous goats in Zimbabwe. Anim. Reprod. Sci. 30, 301–311. Llewelyn, C.A., Perrie, J., Luckins, A.G., Munro, C.D., 1993b. Estrus in the British White goat: timing of plasma luteinizing hormone surge and changes in behavioral and vaginal traits in relationship to onset of estrus. Br. Vet. J. 149, 171–182. Luna, L.G., 1960. Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. McGraw-Hill, NY. Mariana, J.C., Monniaux, D., Driancourt, M.A., MaulJon, P., 1991. Folliculogenesis. In: Cupps, P.T. ŽEd.., Reproduction of Domestic Animals. Academic Press, San Diego, pp. 119–171. McNeilly, A.S., Picton, H.M., Campbell, B.K., Baird, D.T., 1991. Gonadotrophic control of follicle growth in the ewe. J. Reprod. Fertil. 43, 177–186. Moor, R.H., Hay, M.F., Seamark, R.F., 1975. The sheep ovary: regulation of steroidogenic, haemodynamic and structural changes in the largest follicle and adjacent tissue before ovulation. J. Reprod. Fertil. 45, 595–604. Mori, Y., Kano, Y., 1984. Changes in plasma concentrations of LH, progesterone and estradiol in relation to the occurrence of luteolysis, estrus and time of ovulation in the Shiba goat Ž Capra hircus .. J. Reprod. Fertil. 72, 223–230. Murdoch, W.J., 1992. Comparative morphometry and steroidogenic function of antral ovine follicles destined for ovulation or atresia. Domest. Anim. Endocrinol. 9, 219–224. Noel, ¨ B., Bister, J.L., Paquay, R., 1993. Ovarian follicular dynamics in Suffolk ewes at different periods of the year. J. Reprod. Fertil. 99, 695–700. Ott, R.S., Nelson, D.R., Hixon, J.E., 1980. Peripheral serum progesterone and luteinizing hormone concentrations of goats during synchronization of estrus and ovulation with prostaglandin F-2 a . Am. J. Vet. Res. 41, 1432–1434. Pelletier, J., Kann, G., Dolais, J., Rosselin, G., 1968. Dosage radio-immunologique de l’hormone lutJinisante plasmatique chez le mouton. Mise au point de la technique de dosage. C.R. Acad. Sci. Paris, t. 266, Serie ´ D: 2291–2294.
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