- Email: [email protected]
Superovulatory response in anestrous ewes is affected by the presence of a large follicle
SUPEROVULATORY RESPONSE THE PRESENCE E. Rubianes’,
D. Ibarra,
EWES IS AFFECTED IN ANESTROUS OF A LARGE FOLLICLE
R. Ungerfeld,
B. Carbajal
BY
and T. de Castro
Departamento de Fisiologia, Fact&ad de Veterinaria Lasplaces 1550. Montevideo, Uruguay Received
for publication: Accepted:
February 18, 1994 November 4, 1994
ABSTRACT Superovulatory response to conventional treatment with eCG (1200 IU) and progestagen sponges (MAP, n=9: FGA, n=9; or controls without sponge, n=6) was studied in Corriedale anestrous ewes. The follicular population just before the administration of eCG and the total ovarian response (large anovulatory follicles plus normal CL and prematurly regressing CL) to treatment were determined after laparotomy. Pretreatment with progestagen did not modify the number or class of follicles greater than 1 mm observed on the ovarian surface at the time of eCG administration (19 + 2.2 follicles vs 19 t 2.9 follicles, for pooled progestagen-treated groups and control groups, respectively; mean f SEM) but significantly decreased the number of large anovulatory follicles (4.7 f 1.0 vs 10.2 + 2.6; Ps 0.01) observed following treatment. Progestagen-treated animals were classified according to the presence (n=13) or absence (n=S) of a large follicle (LF: t 4 mm diameter) on the ovarian surface at the time of eCG treatment; a qualitatively better superovulatory response was observed in ewes without large follicle (large anovulatory follicles: 1.6 f 0.7 vs 5.X f 1.3, P< 0.05; normal CL: 7.0 f 1.4 vs 3.X f 1.0, PS 0.1; normal CL/total ovarian response: 7X.7 + 10.1 % vs 34.9 f X.2 %, P < 0.01; for ewes without LF and ewes with 1 to 2 LF respectively). No differences were observed in the individual ovulatory response when comparing ovaries ipsilateral or contralateral to LF in a same animal, indicating that the effect of LF on the superovulatory response would be fundamentally systemic. This work shows that, similar to what occurs in cows, the presence of a large follicle at the time of gonadotropin administration decreases the superovulatory response in anestrous ewes. Key words:
superovulation,
follicular
dominance,
ewe, seasonal
anestrus,
gonadotropin
Acknowledgments The authors thank Professor R. Tagle and his collaborators for the progesterone assay; Kmaid and BSc. C. Vifioles for their helpful assistance during the experiment; Ms. R. for her assistance with the bibliography and Ms. R. Gonzalez and Mr. A. Salinas for laboratory assistance. This research was supported by the Faculty of Veterinary and ‘Correspondence: fax (598-2) 680130.
Theriogenology 43:465-472,1995 0 1995 by Elaevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
Dr. S. Vilaro their INIA.
0093-691X/95/$1 0.00 SSDI 0093-691X(94)00039-W
INTRODUCTION Superovulation is currently the major limiting factor for embryo transfer programs in ruminants. The results of superovulatory treatments have been characterized by a great variability in the ovulatory rate, partly due to the lack of knowledge regarding the factors regulating ovarian dynamics, particularly recruitment, selection and follicular dominance mechanisms. The study of ovarian physiology by means of transrectal ulnasonography in cows shows that superovulatory response is affected if a dominant follicle is present at the time of commencing gonadotropin treatment (5,6,11,12,14). Similarly, it has been demonstrated that the removal of a dominant follicle in the cow is followed by immediate resumption of growth of small follicles, indicating that the single large follicle inhibits the development of smaller follicles (X). Some studies indicate that in ewes, as in cows, the presence of a large follicle impinges on the superovulatory response. Cognie (2) obtained a higher ovulatory rate following the production of a complete depletion of large follicles (2 3 mm) from the ovarian surface by infusion of a GnRH agonist over 2 wk. Nevertheless, the presence of dominance by the preovulatory follicle over the remaining follicular population has been debated in this species. Driancourt et al. (4) found that the response to the administration of eCG (PMSG) before or after the emergence of the preovulatory follicle during the follicular phase was no different, and that gonadotropic treatment during Day 6 of the cycle induced a similar ovulatory rate in animals with cauterized healthy large follicles and in sham-treated animals. The researchers suggest that dominance is probably not operative in sheep. In this paper we present a study of the ovarian response to superovulatory treatment as a function of the conditions present (progestagen priming, presence of a large follicle) at the beginning of superovulatory treatment during the seasonal anestrous, i.e., when changes in ovarian dynamics are less dramatic, thus not allowing for spontaneous ovulation. MATERIALS AND METHODS The experiment was carried out during the deep seasonal anestrous (early spring) at the experimental laboratory of the Department of Physiology, Montevideo, Uruguay. Multiparous Corriedale ewes, a single ovulator breed, weighting around 40 kgs were used. The animals were fed with alfalfa grass and pellets ad libitum and housed outdoors in sheltered pens (15 m X 15 m) and indoor box stalls (3 m X 3 m) were used when handling required it. At the beginning of the experiment 10 ewes received a sponge with 60 mg of medroxyprogesterone acetate (MAP group) and 10 other ewes received a sponge with 30 mg of flurogestone acetate (FGA group). All ewes remained 13 d with the sponges. At 4X h before sponge withdrawal, all the animals were submitted to a fust laparotomy under local and regional anesthesia, and the structures in both ovaries were recorded. lmmediately following surgery, all the animals received an intramuscular injection of 1200 IU of eCG (Folligon, Intervet, Holland).
467
Theriogenology
Following sponge withdrawal, the animals were tested every 12 h with a vasectomized ram. When the estrus was detected, the ewes were placed 24 h with a ram. Five days after mating, all ewes underwent a second laparotomy to establish the number of corpora lutea and follicles larger than 6 mm present on the surface of both ovaries. Daily blood samples were collected through jugular venipuncture after sponge withdrawal and up to the day of the second laparotomy. The blood serum was separated by centrifugation within the first 2 h, and was frozen until the time of processing for progesterone determination by RIA. Another 6 ewes were submitted to similar treatment with the exception of the intravaginal implant of progestagen (control group). Three of these ewes and 2 of the ewes treated with progestagens did not show estrus, and the second laparotomy was performed Y d aftergonadotropin treatment in these 5 ewes. The average time between these laparatomies was the same as that of the estrous ewes. The follicular structures observed on the ovarian surface during laparotomy were divided into 3 different classes: small follicles (1 mm to < 2 mm), medium follicles (2 mm to < 4 mm) and large follicles (2 4 mm). Anovulatory follicles larger than 6 mm, the normal corpora lutea and prematurly regressing corpora lutea were recorded during the second laparotomy. A corpus luteum was considered as a prematurly regressing corpus luteum when it presented a pale appearance, was smaller in size than normal CL (< 6 mm) and did not protrude (1.5). The study of circulating concentrations of progesterone allowed for the corroboration of this definition since the prematurly regressing corpora lutea are accompanied by a decrease of that hormone below 1 @ml within the 4X to Yh h postestrus. The total ovarian response was defined as the sum of large anovulatory follicles, normal CL and prematurly regressing CL present in the ovaries during the second laparotomy. Progesterone measurement was carried out by RIA according to a previously described technique (13). One-way ANOVA after checking for homogeneity of the variance by the test of Bartlett (when variance was heterogenous a square root transformation was performed) and Fisher’s Exact Probability Test were used for statistical analysis (16). RESULTS Ovarian
Follicle
Population
at eCG Administration
Two of the 20 ewes treated with laparotomy (Day 11) and were therefore
progestagens presented a corpus excluded from the experiment.
luteum
at the first
As may be observed in Table 1, treatment with MAP or FGA during 1 I days did not alter the mean number of the 3 follicular classes, or the average size of the larger follicle observed on the ovarian surface. No differences were observed either between the follicular population present in the right and left ovaries (small follicles + medium follicles: X.2 + 1.1 vs X.Y ir 1.2, for right and left ovaries, respectively; mean ? SEM).
468
Theriogenology
Table 1. Ovarian follicular population of ewes in seasonal anestrus with or without 1 l-day progestagen treatment (X f SEM) Treatment
Small follicles
Medium follicles
Large follicles
MAP (n=Y) FGA (n=Y)
12.7 f 2.5
3.1 f 0.‘)
0.9 f 0.3
3.x f 0.3
15.x f 2.6
4.2 f 0.6
1.3 f 0.3
4.4 f 0.3
14.2 f 1.9
3.7 f 0.6
1.1 f 0.2
4.2 f 0.3
12.0 f 2.0
6.0 f 1.2
1.0 f 0.2
4.3 f 0.3
Control (n=6)
Largest follicle diameter (mm)
Table 2. Effect of the presence of a large follicle (LF) at time of eCG administration on superovulatory responses (X f SEM) Ewes
n
LAF
PRCL
NCL
**
Without LF
5
1.6 f 0.7
With lto2LF
13
5.x f 1.3
TOR
NCL/TOR %
* o*o
1.5 f 0.7
***
7.0 f 1.4
8.6 f 0.‘)
7x.7 f 10.1
3.8 f I.0
11.0 f 1.4
34.Yf 8.2
*= P< 0.1; **= P< 0.05; ***= P< 0.01. LAF: large anovulatory follicle; PRCL: prematurly regressing corpora lutea; NCL: normal corpora lutea; TOR: total ovarian response. Five of the 18 ewes (3 of the MAP group and 2 of the FGA group) did not present large follicles in either ovary. The remaining 13 had 1 (n= 6) or 2 (n= 7) large follicles. Of these 7 ewes 5 had both follicles in the same ovary. Estrous Responses to Treatment Sixteen of the 1X ewes treated with progestagen (Y/Yin the MAP group and 7/Y in the FGA group) showed estrus, while only 3 of the 6 controls exhibited estrus (PI 0.05). The interval between progestagen withdrawal and estrous behavior was longer in the FGA group than in the MAP group (35.7 f 3.9 h vs 22.5, f 2.5 h; PI 0.05).
lheriogenology
469
Table 3. Ovarian response according to the presence or absence of a large follicle (LF) at time of eCG administration (X f SEM) superovulatory
Ewes
response2
LAF
NCL
TOR
NCL/TOR %
9.5 f 1.6
2.5 f 0.6
1.x zk0.6
5.2 f 0.X
31.2 + x.x
8.7 k 1.7
3.0 * 0.Y
1.x f 0.5
5.4 I? 0.x
3x.5 Ik I 1.6
9.1 f 1.1
2.8 f 0.5 a
1.x + 0.4 :’
5.3 f 0.6
34.7 * 7.1 d
7.7 f 1.0
0.x f 0.3 b
3.4 f 0.7 ”
4.3 f 0.7
73.Y + 10.6 c
S’+M
With LF Ovaries without LF (n=ll) Ovaries with LF (n=ll)
Without LF Ovaries (n= 10)
a,‘.’ Columns with different superscripts differ (a vs b: P< 0.05; a vs c: P< 0.01). ’ Total small (1 to 2 mm) ~1~s medium (2 to 4 mm) follicles present in each ovary at time of eCG administration. LAF: large anovulatory follicles; NCL: normal corpora lutea; TOR: total ovarian response.
Superovulatory Response The total ovarian response (total corpora lutea plus large anovulatory follicles) to treatment with eCG of control ewes (no progestagen pretreatment) was similar to the total ovarian response of ewes treated with progestagen (10.4 f 1.0 vs 12.7 f 3.0 for ewes with and without sponges, respectively) but the quality of the response was very different. With one exception, the control animals did not ovulate, and their ovaries presented a high number of large anovulatory follicles (IO.2 f 2.6 vs 4.7 f 1.0 for control and sponges-treated ewes respectively; P<: 0.01). The mean number of large anovulatory follicles (4.4 f 1.6 vs 4.8 f 1.4), the mean number of CL (4.0 f 0.8 vs 5.3 f 1.6) and the percentage of normal CL of the total ovarian response (52.3 rt 10.9 % vs 41.8 f 12.0 %) did not differ between animals treated with MAP or FGA, respectively. Prematurly regressing corpora lutea occurred on an all or none basis within a ewe. The study of serum profiles of progesterone allowed for the corroboration of the observation carried out during the second laparotomy regarding the existence of prematurly regressing corpora lutea. Progesterone values were similar at Day 1 after estrus (3.4 + 1.6 vs 3.X f 2.Y
Theriogenology
470
@ml for ewes with prematurly regressing corpora lutea and normal corpora lutea, respectively) but were significantly different at Day 5 postestrus (0.7 f 0.2 vs 34.7 f 8.6 n&ml for ewes with prematurly regressing corpora lutea and normal corpora lutea, respectively; P< 0.01). A greater frequency of ewes with prematurly regressing corpora lutea was observed in the group treated with FGA (4/9 in the FGA group vs O/9 in the MAP group; P< 0.05). No prematurly regressing corpora lutea were observed in the control group. Independent of the progestagen used, no differences were observed between the right and left ovaries in response to superovulatory treatment. The mean number of large anovulatory follicles was 2.3 f. 0.6 and 2.1 f 0.5, and the mean normal CL was 2.0 f 0.5 and 2.3 f 0.4 corpora lutea for the right and left ovary, respectively. Presence of Large Follicles and Superovulatory Response Differences in the response to superovulatory treatment were observed when the animals were grouped according to the presence (n= 13) or absence (n= 5) of large follicles the day of eCG administration. As is shown in Table 2, the mean number of anovulatory follicles larger than 6 mm observed in the second laparotomy was significantly greater (PI 0.05) and the mean number of normal corpora lutea tended to be smaller (PC 0.1) in animals with at least one large follicle the day eCG was administered. The quality of the superovulatory response measured as the percentage of normal corpora lutea in the total ovarian response was clearly larger in animals that did not have large follicles at the time of eCG administration (78.7 % vs 34.9 %; P< 0.01). There were no difference in the superovulatory response between the ovary ipsilateral or contralateral of a large follicle at the time eCG was administered (Table 3).
DISCUSSION Pretreatment with progestagen increased the induction of estrous and improved the quality of the superovulatory response to eCG treatment in anestrous ewes. These results are not related to differences in the follicular population observed at the time eCG was injected because neither the total number nor the proportion of follicular classes present on the ovarian surface were different in the animals treated with progestagens over 11 d compare with that of the controls. These results may be interpreted in the light of earlier work by Cran (3) who observed that eCG administration on Days 2 to 3 of the cycle (low levels of circulating progesterone) produced an ovarian response characterized by a high percentage of cystic follicles larger than 6 mm that tended to luteinize without ovulating. Nevertheless, if the treatment is carried out on Days 5 to 7 of the cycle, (i.e., when progesterone levels have risen) an ovulatory response could be elicited together with a decrease in the percentage of cystic follicles observed. This effect of progestagen priming on the superovulatory response may be exerted at central level, producing a better synchronization between follicular growth and the gonadotropin surge (17) and/or at local level by somehow facilitating the maturation process of preovulatory follicles (9).
471
Theriogenology
The greater frequency of premature luteal regression observed in the ewes treated with FGA as compared to ewes treated with MAP could be linked to differences in steroidogenesis induced in preovulatory follicles by both progestagens. The formation of subnormal corpora lutea is preceded by an alteration in the steroidogenic function of preovulatory follicles (IX) while the greater progestagen withdrawal to estrus interval observed in the FGA group could be reflecting a decreased steroidogenic function. Another explanation involves continued secretion of PGF initiated by the presence of sponges for 13 d or premature secretion of PGF as a result of inadequate priming with progestagens (7,lO). Our present results show that, as occurs in the cow (5,6,X, 12,14), the presence of a large follicle at the time of gonadotropin administration affects the superovulatory response in the ewe. Similarly, we have found that the aforementioned effect is fundamentally systemic as no different responses were observed in a same animal in the ipsilateral and contralateral ovary in relation to the large follicle. This is in agreement with Adams et al. (1), who demonstrated that the dominant effect of the large follicle during the estrous cycle in the cow is exerted through a systemic pathway by means of follicular factors such as inhibin and/or estrogens. When we administer gonadotropic factors (eCG) we are eliminating the element of passive dominance (capacity of the dominant follicle to decrease FSH levels), which suggests that active dominance is present in seasonal anestrus in the ewe. The differences between our results and those obtained by Driancourt et al. (4) could be due to the fact that the relationships between the large and the smaller follicles during the estrous cycles are different from those observed with superovulatory treatment or that other factors improve the inhibitory action of dominant follicle products during the non-reproductive seasonal phase. New trials, particularly with a less stressing experimental approach, as transrectal ultrasonography, are required in order to give forth further light on these aspects.
REFERENCES 1. Adams GP. Matteri RL, Ginther OJ. Effect of progesterone on ovarian follicles, emergence of follicular waves and circulating follicle-stimulating hormone in heifers. J Reprod Fertil lYY2;95:627-640. 2. Cognie Y. Progress in reproduction techniques in sheep. World Sheep and Wool Congress lYY2;C:7:1-16. 3. Cran DG. Follicular development in the sheep after priming with PMSG. J Reprod Fertil lYX3;67:415-423. 4. Driancourt MA, Webb R, Fry RC. Does follicular dominance occur in ewes’? J Reprod Fertil 1YYl;Y3:h3-70. 5. Guibault LA, Grass0 F, Lussier JG, Rouillier P, Matton P. Decreased superovulatory responses in heifers superovulated in the presence of a dominant follicle. J Reprod Fertil 199 1;Y1:Xl-X9. 6. Huhtinen M, Raino V, Aalto J, Bredbacka P, Maki-Tanila A. Increased ovarian responses in the absence of a dominant follicle in superovulated cows. Theriogenology lYY2;37:457-463. 7. Hunter MG, Ayad VJ, Gilbert CL, Southee JA, Wathes DC. Role of prostaglandin F-2 alpha and oxytocin in the regression of GnRH-induced abnormal corpora lutea in anestrous ewes. J Reprod Fertil IYXY;XS: 551-561.
472
Theriogenology
X. Ko JCH, Kastelic JP, Del Campo MR, Ginther OJ. Effects of a dominant follicle on ovarian follicular dynamics during oestrus cycle in heifers. J Reprod Fertil 1991;91:511-519. 9. McLeod BJ, Haresign W. Evidence that progesterone may influence subsequent luteal function in the ewe by modulating preovulatory follicle development. J Reprod Fertil 1984;71:381-386. 10. Ottobre JS, Vincent DL, Silvia WJ, Inskeep EK. Aspects of regulation of uterine secretion of prostaglandins during the oestrous cycle and early pregnancy. An Rep Sci 1984;7:75-100. 11. Pierson RA, Ginther OJ. Follicular populations during the estrous cycle in heifers. III. Time of selection of the ovulatory follicle. An Rep Sci 1988;16:81-95. 12. Rouillier P, Matton P, Guilbault L, Grass0 F, Lussier J. Influence of a dominant follicle atresia and estradiol release by ovarian follicles during superovulation in cattle. Theriogenology 1990;33:313 abstr. 13. Rubianes E, Ungerfeld R. Uterine involution and ovarian changes during early post partum in autumn-lambing Corriedale ewes. Theriogenology 1993;40:365-372. 14. Savio JD, Bongers H, Drost M, Lucy MC, Thatcher WW. Follicular dynamics and superovulatory response in Holstein cows treated with FSH-P in different endocrine states. Theriogenology 1991;35:915-929. 15. Schiewe MC, Fitz TA, Brown JL, Stuart LD, Wildt DE. Relationship of oestrus synchronization method, circulating hormones, luteinizing hormone and prostaglandin F-2 alfa receptors and luteal progesterone concentration to premature luteal regression in superovulated sheep. J Reprod Fertil 1991;93:1Y-30. 16. Snedecor GW, Cochran WC. Statistical Methods. The Iowa State University Press, Ames, IA, 1980. 17. Southee JA, Hunter MG, Haresign W. Function of abnormal corpora lutea in vivo after GnRH-induced ovulation in the anestrous ewe. J Reprod Fertil 1988;84:131-137. 1X. White LM, Keisler DH, Dailey RA, Inskeep EK. Characterization of ovine follicles destined to form subfunctional corpora lutea. J Anim Sci 1987;65:1595-1601.
D. Ibarra,
EWES IS AFFECTED IN ANESTROUS OF A LARGE FOLLICLE
R. Ungerfeld,
B. Carbajal
BY
and T. de Castro
Departamento de Fisiologia, Fact&ad de Veterinaria Lasplaces 1550. Montevideo, Uruguay Received
for publication: Accepted:
February 18, 1994 November 4, 1994
ABSTRACT Superovulatory response to conventional treatment with eCG (1200 IU) and progestagen sponges (MAP, n=9: FGA, n=9; or controls without sponge, n=6) was studied in Corriedale anestrous ewes. The follicular population just before the administration of eCG and the total ovarian response (large anovulatory follicles plus normal CL and prematurly regressing CL) to treatment were determined after laparotomy. Pretreatment with progestagen did not modify the number or class of follicles greater than 1 mm observed on the ovarian surface at the time of eCG administration (19 + 2.2 follicles vs 19 t 2.9 follicles, for pooled progestagen-treated groups and control groups, respectively; mean f SEM) but significantly decreased the number of large anovulatory follicles (4.7 f 1.0 vs 10.2 + 2.6; Ps 0.01) observed following treatment. Progestagen-treated animals were classified according to the presence (n=13) or absence (n=S) of a large follicle (LF: t 4 mm diameter) on the ovarian surface at the time of eCG treatment; a qualitatively better superovulatory response was observed in ewes without large follicle (large anovulatory follicles: 1.6 f 0.7 vs 5.X f 1.3, P< 0.05; normal CL: 7.0 f 1.4 vs 3.X f 1.0, PS 0.1; normal CL/total ovarian response: 7X.7 + 10.1 % vs 34.9 f X.2 %, P < 0.01; for ewes without LF and ewes with 1 to 2 LF respectively). No differences were observed in the individual ovulatory response when comparing ovaries ipsilateral or contralateral to LF in a same animal, indicating that the effect of LF on the superovulatory response would be fundamentally systemic. This work shows that, similar to what occurs in cows, the presence of a large follicle at the time of gonadotropin administration decreases the superovulatory response in anestrous ewes. Key words:
superovulation,
follicular
dominance,
ewe, seasonal
anestrus,
gonadotropin
Acknowledgments The authors thank Professor R. Tagle and his collaborators for the progesterone assay; Kmaid and BSc. C. Vifioles for their helpful assistance during the experiment; Ms. R. for her assistance with the bibliography and Ms. R. Gonzalez and Mr. A. Salinas for laboratory assistance. This research was supported by the Faculty of Veterinary and ‘Correspondence: fax (598-2) 680130.
Theriogenology 43:465-472,1995 0 1995 by Elaevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
Dr. S. Vilaro their INIA.
0093-691X/95/$1 0.00 SSDI 0093-691X(94)00039-W
INTRODUCTION Superovulation is currently the major limiting factor for embryo transfer programs in ruminants. The results of superovulatory treatments have been characterized by a great variability in the ovulatory rate, partly due to the lack of knowledge regarding the factors regulating ovarian dynamics, particularly recruitment, selection and follicular dominance mechanisms. The study of ovarian physiology by means of transrectal ulnasonography in cows shows that superovulatory response is affected if a dominant follicle is present at the time of commencing gonadotropin treatment (5,6,11,12,14). Similarly, it has been demonstrated that the removal of a dominant follicle in the cow is followed by immediate resumption of growth of small follicles, indicating that the single large follicle inhibits the development of smaller follicles (X). Some studies indicate that in ewes, as in cows, the presence of a large follicle impinges on the superovulatory response. Cognie (2) obtained a higher ovulatory rate following the production of a complete depletion of large follicles (2 3 mm) from the ovarian surface by infusion of a GnRH agonist over 2 wk. Nevertheless, the presence of dominance by the preovulatory follicle over the remaining follicular population has been debated in this species. Driancourt et al. (4) found that the response to the administration of eCG (PMSG) before or after the emergence of the preovulatory follicle during the follicular phase was no different, and that gonadotropic treatment during Day 6 of the cycle induced a similar ovulatory rate in animals with cauterized healthy large follicles and in sham-treated animals. The researchers suggest that dominance is probably not operative in sheep. In this paper we present a study of the ovarian response to superovulatory treatment as a function of the conditions present (progestagen priming, presence of a large follicle) at the beginning of superovulatory treatment during the seasonal anestrous, i.e., when changes in ovarian dynamics are less dramatic, thus not allowing for spontaneous ovulation. MATERIALS AND METHODS The experiment was carried out during the deep seasonal anestrous (early spring) at the experimental laboratory of the Department of Physiology, Montevideo, Uruguay. Multiparous Corriedale ewes, a single ovulator breed, weighting around 40 kgs were used. The animals were fed with alfalfa grass and pellets ad libitum and housed outdoors in sheltered pens (15 m X 15 m) and indoor box stalls (3 m X 3 m) were used when handling required it. At the beginning of the experiment 10 ewes received a sponge with 60 mg of medroxyprogesterone acetate (MAP group) and 10 other ewes received a sponge with 30 mg of flurogestone acetate (FGA group). All ewes remained 13 d with the sponges. At 4X h before sponge withdrawal, all the animals were submitted to a fust laparotomy under local and regional anesthesia, and the structures in both ovaries were recorded. lmmediately following surgery, all the animals received an intramuscular injection of 1200 IU of eCG (Folligon, Intervet, Holland).
467
Theriogenology
Following sponge withdrawal, the animals were tested every 12 h with a vasectomized ram. When the estrus was detected, the ewes were placed 24 h with a ram. Five days after mating, all ewes underwent a second laparotomy to establish the number of corpora lutea and follicles larger than 6 mm present on the surface of both ovaries. Daily blood samples were collected through jugular venipuncture after sponge withdrawal and up to the day of the second laparotomy. The blood serum was separated by centrifugation within the first 2 h, and was frozen until the time of processing for progesterone determination by RIA. Another 6 ewes were submitted to similar treatment with the exception of the intravaginal implant of progestagen (control group). Three of these ewes and 2 of the ewes treated with progestagens did not show estrus, and the second laparotomy was performed Y d aftergonadotropin treatment in these 5 ewes. The average time between these laparatomies was the same as that of the estrous ewes. The follicular structures observed on the ovarian surface during laparotomy were divided into 3 different classes: small follicles (1 mm to < 2 mm), medium follicles (2 mm to < 4 mm) and large follicles (2 4 mm). Anovulatory follicles larger than 6 mm, the normal corpora lutea and prematurly regressing corpora lutea were recorded during the second laparotomy. A corpus luteum was considered as a prematurly regressing corpus luteum when it presented a pale appearance, was smaller in size than normal CL (< 6 mm) and did not protrude (1.5). The study of circulating concentrations of progesterone allowed for the corroboration of this definition since the prematurly regressing corpora lutea are accompanied by a decrease of that hormone below 1 @ml within the 4X to Yh h postestrus. The total ovarian response was defined as the sum of large anovulatory follicles, normal CL and prematurly regressing CL present in the ovaries during the second laparotomy. Progesterone measurement was carried out by RIA according to a previously described technique (13). One-way ANOVA after checking for homogeneity of the variance by the test of Bartlett (when variance was heterogenous a square root transformation was performed) and Fisher’s Exact Probability Test were used for statistical analysis (16). RESULTS Ovarian
Follicle
Population
at eCG Administration
Two of the 20 ewes treated with laparotomy (Day 11) and were therefore
progestagens presented a corpus excluded from the experiment.
luteum
at the first
As may be observed in Table 1, treatment with MAP or FGA during 1 I days did not alter the mean number of the 3 follicular classes, or the average size of the larger follicle observed on the ovarian surface. No differences were observed either between the follicular population present in the right and left ovaries (small follicles + medium follicles: X.2 + 1.1 vs X.Y ir 1.2, for right and left ovaries, respectively; mean ? SEM).
468
Theriogenology
Table 1. Ovarian follicular population of ewes in seasonal anestrus with or without 1 l-day progestagen treatment (X f SEM) Treatment
Small follicles
Medium follicles
Large follicles
MAP (n=Y) FGA (n=Y)
12.7 f 2.5
3.1 f 0.‘)
0.9 f 0.3
3.x f 0.3
15.x f 2.6
4.2 f 0.6
1.3 f 0.3
4.4 f 0.3
14.2 f 1.9
3.7 f 0.6
1.1 f 0.2
4.2 f 0.3
12.0 f 2.0
6.0 f 1.2
1.0 f 0.2
4.3 f 0.3
Control (n=6)
Largest follicle diameter (mm)
Table 2. Effect of the presence of a large follicle (LF) at time of eCG administration on superovulatory responses (X f SEM) Ewes
n
LAF
PRCL
NCL
**
Without LF
5
1.6 f 0.7
With lto2LF
13
5.x f 1.3
TOR
NCL/TOR %
* o*o
1.5 f 0.7
***
7.0 f 1.4
8.6 f 0.‘)
7x.7 f 10.1
3.8 f I.0
11.0 f 1.4
34.Yf 8.2
*= P< 0.1; **= P< 0.05; ***= P< 0.01. LAF: large anovulatory follicle; PRCL: prematurly regressing corpora lutea; NCL: normal corpora lutea; TOR: total ovarian response. Five of the 18 ewes (3 of the MAP group and 2 of the FGA group) did not present large follicles in either ovary. The remaining 13 had 1 (n= 6) or 2 (n= 7) large follicles. Of these 7 ewes 5 had both follicles in the same ovary. Estrous Responses to Treatment Sixteen of the 1X ewes treated with progestagen (Y/Yin the MAP group and 7/Y in the FGA group) showed estrus, while only 3 of the 6 controls exhibited estrus (PI 0.05). The interval between progestagen withdrawal and estrous behavior was longer in the FGA group than in the MAP group (35.7 f 3.9 h vs 22.5, f 2.5 h; PI 0.05).
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Table 3. Ovarian response according to the presence or absence of a large follicle (LF) at time of eCG administration (X f SEM) superovulatory
Ewes
response2
LAF
NCL
TOR
NCL/TOR %
9.5 f 1.6
2.5 f 0.6
1.x zk0.6
5.2 f 0.X
31.2 + x.x
8.7 k 1.7
3.0 * 0.Y
1.x f 0.5
5.4 I? 0.x
3x.5 Ik I 1.6
9.1 f 1.1
2.8 f 0.5 a
1.x + 0.4 :’
5.3 f 0.6
34.7 * 7.1 d
7.7 f 1.0
0.x f 0.3 b
3.4 f 0.7 ”
4.3 f 0.7
73.Y + 10.6 c
S’+M
With LF Ovaries without LF (n=ll) Ovaries with LF (n=ll)
Without LF Ovaries (n= 10)
a,‘.’ Columns with different superscripts differ (a vs b: P< 0.05; a vs c: P< 0.01). ’ Total small (1 to 2 mm) ~1~s medium (2 to 4 mm) follicles present in each ovary at time of eCG administration. LAF: large anovulatory follicles; NCL: normal corpora lutea; TOR: total ovarian response.
Superovulatory Response The total ovarian response (total corpora lutea plus large anovulatory follicles) to treatment with eCG of control ewes (no progestagen pretreatment) was similar to the total ovarian response of ewes treated with progestagen (10.4 f 1.0 vs 12.7 f 3.0 for ewes with and without sponges, respectively) but the quality of the response was very different. With one exception, the control animals did not ovulate, and their ovaries presented a high number of large anovulatory follicles (IO.2 f 2.6 vs 4.7 f 1.0 for control and sponges-treated ewes respectively; P<: 0.01). The mean number of large anovulatory follicles (4.4 f 1.6 vs 4.8 f 1.4), the mean number of CL (4.0 f 0.8 vs 5.3 f 1.6) and the percentage of normal CL of the total ovarian response (52.3 rt 10.9 % vs 41.8 f 12.0 %) did not differ between animals treated with MAP or FGA, respectively. Prematurly regressing corpora lutea occurred on an all or none basis within a ewe. The study of serum profiles of progesterone allowed for the corroboration of the observation carried out during the second laparotomy regarding the existence of prematurly regressing corpora lutea. Progesterone values were similar at Day 1 after estrus (3.4 + 1.6 vs 3.X f 2.Y
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@ml for ewes with prematurly regressing corpora lutea and normal corpora lutea, respectively) but were significantly different at Day 5 postestrus (0.7 f 0.2 vs 34.7 f 8.6 n&ml for ewes with prematurly regressing corpora lutea and normal corpora lutea, respectively; P< 0.01). A greater frequency of ewes with prematurly regressing corpora lutea was observed in the group treated with FGA (4/9 in the FGA group vs O/9 in the MAP group; P< 0.05). No prematurly regressing corpora lutea were observed in the control group. Independent of the progestagen used, no differences were observed between the right and left ovaries in response to superovulatory treatment. The mean number of large anovulatory follicles was 2.3 f. 0.6 and 2.1 f 0.5, and the mean normal CL was 2.0 f 0.5 and 2.3 f 0.4 corpora lutea for the right and left ovary, respectively. Presence of Large Follicles and Superovulatory Response Differences in the response to superovulatory treatment were observed when the animals were grouped according to the presence (n= 13) or absence (n= 5) of large follicles the day of eCG administration. As is shown in Table 2, the mean number of anovulatory follicles larger than 6 mm observed in the second laparotomy was significantly greater (PI 0.05) and the mean number of normal corpora lutea tended to be smaller (PC 0.1) in animals with at least one large follicle the day eCG was administered. The quality of the superovulatory response measured as the percentage of normal corpora lutea in the total ovarian response was clearly larger in animals that did not have large follicles at the time of eCG administration (78.7 % vs 34.9 %; P< 0.01). There were no difference in the superovulatory response between the ovary ipsilateral or contralateral of a large follicle at the time eCG was administered (Table 3).
DISCUSSION Pretreatment with progestagen increased the induction of estrous and improved the quality of the superovulatory response to eCG treatment in anestrous ewes. These results are not related to differences in the follicular population observed at the time eCG was injected because neither the total number nor the proportion of follicular classes present on the ovarian surface were different in the animals treated with progestagens over 11 d compare with that of the controls. These results may be interpreted in the light of earlier work by Cran (3) who observed that eCG administration on Days 2 to 3 of the cycle (low levels of circulating progesterone) produced an ovarian response characterized by a high percentage of cystic follicles larger than 6 mm that tended to luteinize without ovulating. Nevertheless, if the treatment is carried out on Days 5 to 7 of the cycle, (i.e., when progesterone levels have risen) an ovulatory response could be elicited together with a decrease in the percentage of cystic follicles observed. This effect of progestagen priming on the superovulatory response may be exerted at central level, producing a better synchronization between follicular growth and the gonadotropin surge (17) and/or at local level by somehow facilitating the maturation process of preovulatory follicles (9).
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The greater frequency of premature luteal regression observed in the ewes treated with FGA as compared to ewes treated with MAP could be linked to differences in steroidogenesis induced in preovulatory follicles by both progestagens. The formation of subnormal corpora lutea is preceded by an alteration in the steroidogenic function of preovulatory follicles (IX) while the greater progestagen withdrawal to estrus interval observed in the FGA group could be reflecting a decreased steroidogenic function. Another explanation involves continued secretion of PGF initiated by the presence of sponges for 13 d or premature secretion of PGF as a result of inadequate priming with progestagens (7,lO). Our present results show that, as occurs in the cow (5,6,X, 12,14), the presence of a large follicle at the time of gonadotropin administration affects the superovulatory response in the ewe. Similarly, we have found that the aforementioned effect is fundamentally systemic as no different responses were observed in a same animal in the ipsilateral and contralateral ovary in relation to the large follicle. This is in agreement with Adams et al. (1), who demonstrated that the dominant effect of the large follicle during the estrous cycle in the cow is exerted through a systemic pathway by means of follicular factors such as inhibin and/or estrogens. When we administer gonadotropic factors (eCG) we are eliminating the element of passive dominance (capacity of the dominant follicle to decrease FSH levels), which suggests that active dominance is present in seasonal anestrus in the ewe. The differences between our results and those obtained by Driancourt et al. (4) could be due to the fact that the relationships between the large and the smaller follicles during the estrous cycles are different from those observed with superovulatory treatment or that other factors improve the inhibitory action of dominant follicle products during the non-reproductive seasonal phase. New trials, particularly with a less stressing experimental approach, as transrectal ultrasonography, are required in order to give forth further light on these aspects.
REFERENCES 1. Adams GP. Matteri RL, Ginther OJ. Effect of progesterone on ovarian follicles, emergence of follicular waves and circulating follicle-stimulating hormone in heifers. J Reprod Fertil lYY2;95:627-640. 2. Cognie Y. Progress in reproduction techniques in sheep. World Sheep and Wool Congress lYY2;C:7:1-16. 3. Cran DG. Follicular development in the sheep after priming with PMSG. J Reprod Fertil lYX3;67:415-423. 4. Driancourt MA, Webb R, Fry RC. Does follicular dominance occur in ewes’? J Reprod Fertil 1YYl;Y3:h3-70. 5. Guibault LA, Grass0 F, Lussier JG, Rouillier P, Matton P. Decreased superovulatory responses in heifers superovulated in the presence of a dominant follicle. J Reprod Fertil 199 1;Y1:Xl-X9. 6. Huhtinen M, Raino V, Aalto J, Bredbacka P, Maki-Tanila A. Increased ovarian responses in the absence of a dominant follicle in superovulated cows. Theriogenology lYY2;37:457-463. 7. Hunter MG, Ayad VJ, Gilbert CL, Southee JA, Wathes DC. Role of prostaglandin F-2 alpha and oxytocin in the regression of GnRH-induced abnormal corpora lutea in anestrous ewes. J Reprod Fertil IYXY;XS: 551-561.
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X. Ko JCH, Kastelic JP, Del Campo MR, Ginther OJ. Effects of a dominant follicle on ovarian follicular dynamics during oestrus cycle in heifers. J Reprod Fertil 1991;91:511-519. 9. McLeod BJ, Haresign W. Evidence that progesterone may influence subsequent luteal function in the ewe by modulating preovulatory follicle development. J Reprod Fertil 1984;71:381-386. 10. Ottobre JS, Vincent DL, Silvia WJ, Inskeep EK. Aspects of regulation of uterine secretion of prostaglandins during the oestrous cycle and early pregnancy. An Rep Sci 1984;7:75-100. 11. Pierson RA, Ginther OJ. Follicular populations during the estrous cycle in heifers. III. Time of selection of the ovulatory follicle. An Rep Sci 1988;16:81-95. 12. Rouillier P, Matton P, Guilbault L, Grass0 F, Lussier J. Influence of a dominant follicle atresia and estradiol release by ovarian follicles during superovulation in cattle. Theriogenology 1990;33:313 abstr. 13. Rubianes E, Ungerfeld R. Uterine involution and ovarian changes during early post partum in autumn-lambing Corriedale ewes. Theriogenology 1993;40:365-372. 14. Savio JD, Bongers H, Drost M, Lucy MC, Thatcher WW. Follicular dynamics and superovulatory response in Holstein cows treated with FSH-P in different endocrine states. Theriogenology 1991;35:915-929. 15. Schiewe MC, Fitz TA, Brown JL, Stuart LD, Wildt DE. Relationship of oestrus synchronization method, circulating hormones, luteinizing hormone and prostaglandin F-2 alfa receptors and luteal progesterone concentration to premature luteal regression in superovulated sheep. J Reprod Fertil 1991;93:1Y-30. 16. Snedecor GW, Cochran WC. Statistical Methods. The Iowa State University Press, Ames, IA, 1980. 17. Southee JA, Hunter MG, Haresign W. Function of abnormal corpora lutea in vivo after GnRH-induced ovulation in the anestrous ewe. J Reprod Fertil 1988;84:131-137. 1X. White LM, Keisler DH, Dailey RA, Inskeep EK. Characterization of ovine follicles destined to form subfunctional corpora lutea. J Anim Sci 1987;65:1595-1601.