Oestrous behaviour and development of preovulatory follicles in goats induced to ovulate using the male effect with and without progesterone priming

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1 CSIRO PUBLISHING Reproduction, Fertility and Development,,, Oestrous behaviour and development of preovulatory follicles in goats induced to ovulate using the male effect with and without progesterone priming A. Gonzalez-Bulnes A,C, J. A. Carrizosa B, B. Urrutia B and A. Lopez-Sebastian A A Instituto Nacional de Investigación y Tecnologia Agraria y Alimentaria, Departamento de Reproduccion Animal, Avda, Puerta de Hierro s/n, -Madrid, Spain. B Instituto Murciano de Investigacion y Desarrollo Agrario y Alimentario, Consejeria de Agricultura, Agua y Medio Ambiente, -La Alberca, Murcia, Spain. C Corresponding author. bulnes@inia.es Abstract. The objective of the current study was to examine follicular dynamics in goats induced to ovulate by using the male effect with or without progesterone pretreatment. Ten Murciano Granadina goats were treated with a single dose of mg of progesterone in olive oil on the day of male effect induction (Day ), whereas ten does remained untreated. Changes in the ovarian follicular population and onset of oestrous behaviour were checked daily from Day to the day on which ovulation was detected by ultrasonography. All of the treated goats and only % of control goats (P <.) showed oestrous behaviour between and h after the introduction of bucks. Differences in follicle growth were found between the groups. The control goats exhibiting oestrous behaviour during the first days of ovarian screening and all of the goats treated with progesterone had their preovulatory follicles in growing phase. However, the largest follicles in control does not showing oestrus in these first days were in regressing phase, being necessary for the emergence of new preovulatory follicles from a new follicular wave between Days and, for exhibition of oestrous behaviour and subsequent ovulation. In conclusion, the appearance of short luteal phases and delayed ovulation in goats treated with the male effect during seasonal anoestrous depends on the stage of follicle development at the time of buck introduction. A single injection of progesterone stimulates the emergence of a new follicular wave, able to grow and give adequate ovulation after induction of the male effect. Extra keywords: folliculogenesis, oestrus, ovulation. Introduction The male effect, which is the induction of fertile reproductive activity during seasonal anoestrous in small ruminant females by the introduction of males after a previous isolation period, although a well-known practice in Europe from ancient times, was first described for goats in a scientific journal in (Shelton ). The exposition of does to bucks induces oestrous behaviour, followed by a preovulatory luteinising hormone (LH) surge and ovulation, from to h after buck introduction (Chemineau ); although this can be delayed for several days in some cases (Ott et al. ). The quality of these first ovulations is very poor and most of them are short cycles with low luteal activity and fertility. Several studies in sheep have shown that short cycles can be avoided by combining the male effect with pharmacological treatments, i.e. by the administration of mg of progesterone in a single intramuscular dose (Oldham et al. ). Most of the females treated with progesterone show oestrous behaviour and ovulation with luteal phases of normal length; moreover, the interval from male introduction to fertile oestrus is reduced, allowing a better synchronization of mates and births. The response to the male effect has been described accurately in small ruminants (for reviews, see Martin et al. for sheep and Walkden-Brown and Restall for goats). Follicle growth patterns after male introduction have been determined in sheep (for review, see Ungerfeld et al. ), but there are scarce studies explaining the patterns of follicular development in response to the buck effect in goats. The objective of the current study was to study follicular dynamics, using transrectal ultrasonography, in goats induced to ovulate by using the male effect with or without progesterone pretreatment. Such data would allow determination of whether previously reported differences in fertility between does treated or not treated with progesterone can be caused by differences in the dynamics of follicle growth. CSIRO./RD -//

2 Reproduction, Fertility and Development A. Gonzalez-Bulnes et al. Materials and methods Animals and experimental design The study was developed in the non-breeding season (April) using multiparous Murciano Granadina goats aged between and years. The does were maintained outdoors in sand-floor stables with access to indoor facilities at the experimental farm of the Instituto Murciano de Investigacion y Desarrollo Agrario y Alimentario-Murcia in Spain (latitude N). All of the females were separated from any contact with males, isolated in another indoor facility at a distance of m, for at least weeks before the start of the experiment. Three blood samples ( ml) were drawn for determination of progesterone (i.e. ovulatory activity) at and days before, and on the same day that bucks were introduced (Day ). Blood samples were obtained using vacuum blood evacuation tubes containing heparin (Vacutainer Systems Europe; Becton Dickinson, Meylan Cedex, France). At introduction of the males, half of the animals (n = ) were treated with a single dose of mg of progesterone (-pregneno-,-diona; Sigma Chemical Co., St Louis, MO, USA) in olive oil, whereas the other half (n = ) were injected only with olive oil and acted as a control. The onset of oestrous behaviour, determined by the acceptance of copula with bucks different than those used for induction of the male effect, and any change in the follicular population of the ovaries were checked daily from the day of introduction of the bucks (Day ) to the day on which first ovulation was detected by ultrasonography. Progesterone was also determined daily from Days to and twice weekly from Days to to evidence the appearance of short cycles. Assessment of follicular development and ovulation The number and size of all follicles mm and occurrence of ovulation was determined daily by.-mhz transrectal ultrasonography (Aloka SSD-; Ecotron, Madrid, Spain), as described previously (Gonzalez- Bulnes et al., ). In brief, observations were conducted with the goat placed in dorsal recumbence on a metallic cradle as used for laparoscopy.after introducing a hydrosoluble contact gel into the rectum to enhance the ultrasound transmission, the probe was placed in the rectum with the transducer orientated perpendicularly to the abdomen wall. When the urinary bladder was surpassed and the uterine horns were located, the probe was rotated laterally clockwise and counter-clockwise to observe ovaries and their structures. Progesterone assays Blood samples were centrifuged at g for min. Thereafter, the plasma was stored at C until assayed for determination of progesterone levels. Progesterone was measured in duplicate using a previously described double-antibody radioimmunoassay (Lopez-Sebastian et al. ), with a sensitivity of. ng ml and intra- and inter-assay coefficients of variation of.% and.%, respectively. Statistical analysis Ultrasonographic data were summarised to characterise patterns of follicular development. All follicles recorded by ultrasonography were characterised by their day of detection (or day of emergence), initial and maximum diameters and interval from first detection to disappearance (follicle lifespan). Follicles in synchronous recruitment and growth were considered part of a wave. In a second analysis, follicles were classified by their largest diameter as total ( mm), small (. mm), medium (.. mm) and large (>. mm) follicles. After this, the number of new follicles (not detected previously), growing follicles (those that had increased in diameter compared to the previous day) and regressing follicles (those that had decreased in diameter or disappeared compared to the previous day) were also considered. Finally, in agreement with previous studies (Lopez-Sebastian et al. ), the size of the largest follicle (LF) and the second and third largest follicles (LF and LF, respectively) were determined without regard to their history and the fate of these follicles. When identifying the largest follicle, the first such follicle recorded took precedence when another reached the same size but did not become larger than the first follicle. The effects of treatment and day on follicular population, the percentage of animals displaying oestrus signs, the timing of oestrus detection and the frequency of does presenting with normal or short-length cycles were tested by analysis of variance (ANOVA) after transformation of individual percentages to the arcsine. Pearson correlation analysis and linear regression procedures were carried out to assess possible correlation of the number of follicles of various size categories to hours after treatment. Data were expressed as mean ± s.e.m. and differences were considered to be statistically significant at P<.. Results The appearance of oestrus and ovulation was detected earlier in does treated with progesterone. All of the females from this group showed signs of oestrous behaviour during the first days after induction of the buck effect, whereas only % of control goats developed behavioural oestrus during this period, at Day after buck introduction (P <.), as summarised in Table. Ultrasonographic screening was then stopped in these goats and continued in the remaining females. The two control does showing signs of oestrus during the first days of the buck effect returned to oestrus on Days and after introduction of the males, whereas the remaining eight control does showed oestrus between Days and after introduction of the males; so the mean time for onset of oestrus was around h for this group, which was significantly higher than in the treated group (P <.). However, only three does showed a short length cycle and returned to oestrus or days after the first detection (%) in the group treated with progesterone, a lower percentage than in the control group (%, P<.). The ultrasonographic screening carried out during the first days after male introduction in both groups showed no differences in the total number of follicles present in the ovaries, neither between groups nor between hours in the same treatment group or between goats showing oestrus or not (Fig. a), but substantial differences were found when the development of every follicle was considered. New follicles entered into Table. Highlights of the main reproductive events in response to the induction of male effect in control goats and in does pretreated with a single intramuscular progesterone dose at buck introduction End point Treated goats Control goats P-value (n = ) (n = ) Goats showing oestrous <. behaviour Goats ovulating <. Goats responding with <. short cycles Follicles in growing phase <. at buck effect

3 Buck effect with or without progesterone treatment Reproduction, Fertility and Development (a) (b) (c) (d ) Fig.. Mean numbers of (a) total, (b) newly detected, (c) size-regressing and (d ) growing follicles during the first wave of follicular development after induction of the male effect in control goats not showing oestrus during the first days after male introduction (left hand panel), control goats showing oestrus (middle panel) and does pretreated with a single intramuscular progesterone dose (right hand panel). The white section of the bar indicates the mean number of small follicles (. mm), the grey section represents the mean number of medium follicles (.. mm) and the black section indicates the mean number of large follicles (. mm).

4 Reproduction, Fertility and Development A. Gonzalez-Bulnes et al. (a) (b) (c) Number of follicles Number of follicles Time after introduction of the males (h) Fig.. Mean diameters of the largest follicle (LF) and the second and third largest follicles (LF and LF) during the first wave of follicular development after induction of the male effect in control goats not showing oestrus during the first days after male introduction (a), control goats showing oestrus (b) and does pretreated with a single intramuscular progesterone dose (c). the growing pool on every day of the ultrasonographic screening (Fig. b), this number being higher for the first h and decreasing thereafter between and h in both groups (P =. for control does and P<. for progesteronetreated does). There were no significant differences between treatment groups, except for the first h when the number of new follicles was found to be higher in goats treated with progesterone (. ±. v.. ±., P<.). The number of new follicles was higher in control does with heat signs when compared to goats not showing oestrous behaviour in the first days, although differences did not reach statistical significance. The mean number of follicles that regressed in diameter (Fig. c) increased with time in all of the animals (. ±. to. ±. in control and. ±. to. ±. in progesterone-treated does, P<. for both groups). However, the pattern of regression was affected by treatment and the presence or absence of heat signs in the first days. In the goats treated with progesterone and in control does showing oestrus during this period, the highest differences between hours were found between and h (. ±. v.. ±., P<. and. ±. v.. ±., respectively). In control does failing to display oestrous signs during the first days, the highest differences between hours were found between and h (. ±. v.. ±., P<.). These patterns of recruitment and regression of follicles caused a significant decrease in the number of follicles in growing phase during the first h after induction of the male effect in both treatment groups (. ±. to. ±., P<. for control and. ±. to. ±., P<. for progesterone-treated does) (Fig. d). This decline was related to a significant decrease in the number of small (. ±. to. ±, P<. for control and. ±. to. ±., P<. for progesterone group) and medium (. ±. to. ± and. ± to.±., P<. for both groups) growing follicles. In control does showing signs of oestrus, this decrease was stopped at h due to the fact that oestrus was displayed at h. Together with the number of growing follicles in the ovaries, the diameter of the largest follicles and their evolution during the first days of observation were also affected by progesterone treatment. Figure illustrates the differences between groups in the relationship between the largest follicle on each day (LF) and the second and third largest follicles (LF and LF). In control does without oestrous signs during the first days, LF increased between and h (. ±. to. ±. mm, P<.), remained static between and h, and tended to decrease between and h (. ±. to. ±. mm, P =.). The mean size of LF and LF decreased. In progesterone-treated does, both LF and LF grew during the h (. ±. to. ±. mm, P<. for LF and. ±. to. ±. mm, P<. for LF). In contrast, LF remained stable, so difference in diameters between LF and LF increased with time (P <.). The mean diameters of LF and LF at h were higher in the progesterone-treated group than in control does without oestrous signs during the first days (P <.). The evolution of LF, LF and LF in control does bearing heat in such period was similar to progesterone-treated does, growing during the h (. ±. to. ±. mm for LF,. ±. to. ±. mm for LF and. ±. to. ±. mm for LF). The analysis of the development of individual follicles (Fig. ) showed that the two control goats exhibiting oestrous behaviour during the period of ovarian screening and all of the goats treated with progesterone had their largest follicles (the preovulatory follicles) in growing phase. However, the largest follicles in control does not showing oestrus in these first days entered into regressing phase during the period of ultrasonographic screening. The emergence of a new follicular wave and new preovulatory follicles, on Days

5 Buck effect with or without progesterone treatment Reproduction, Fertility and Development Diameter (mm) (a) Diameter (mm) (b) (c) Time after introduction of the males (h) Fig.. Pattern of growth and regression of follicles that reached mm during the period of development of ovulatory follicles in response to induction of the male effect in three representative goats. The first showed oestrus at Day after buck introduction without progesterone priming (a), the second showed oestrus at Day after buck introduction without progesterone priming (b) and the third showed oestrus at Day after buck introduction with progesterone priming (c). and after buck introduction, were necessary to develop oestrous behaviour and subsequent ovulation. During this period, the number of new follicles entering into the growing pool increased on Day (. ±.) but decreased slightly thereafter (. ±. on Day ), whereas the number of follicles regressing in diameter increased to. ±. on Day ; so the final number of growing follicles increased to. ±. on Day but dropped to. ±. on Day (P <.). The mean diameter of the three largest follicles remained stable or decreased from Day to Day (. ±. to. ±. mm, P<. for LF,. ±. to. ±. mm for LF and. ±. to. ±. mm for LF), and grew from Day to (. ±. mm, P<. for LF,. ±. mm, P<. for LF and. ±. mm, P<. for LF). Discussion The concepts that arise from the current experiment, at first glance, are the existence of short luteal phases and delayed ovulation in goats treated with the male effect during seasonal anoestrus, and the beneficial effect of the use of progesterone priming on such response. Both facts are well known from the s. The third, and novel, result is the finding that delays in the onset of oestrous behaviour and ovulation in response to the buck effect are determined by the existence of preovulatory follicles in a stage inappropriate for ovulation. These follicles enter into static or atretic phases shortly after male introduction, being necessary the development of a second wave of preovulatory follicles. The use of a single dose of progesterone avoids such problems and allows the appearance of oestrous signs and subsequent ovulation. However, this effect was not related to a delay in the day of appearance of oestrus and ovulation, but to differences in patterns of follicular growth compared with females without progesterone priming. In this way, administration of progesterone stimulates the emergence of a new follicular wave, able to grow and give adequate ovulation after induction of the male effect. In the present study, in goats induced to resume reproductive activity using the male effect without progesterone priming, although hormonal status (oestradiol and inhibin A secretion) of large follicles could not be determined, the ultrasonographic scanning gave evidence that follicles stimulated to grow were in atresia.thus, the development of a new wave, from which ovulatory follicles arose, was necessary, which was related to delayed oestrus and ovulation in % of the does. However, in those animals with large oestrogenic follicles able to induce oestrous behaviour and ovulation in the first wave of development (% of the goats), the ovulation was associated with short cycles. In all of the goats induced to resume reproductive activity using the male effect with progesterone priming, the growth of preovulatory follicles was similar to that described previously during oestrous cycles in

6 Reproduction, Fertility and Development A. Gonzalez-Bulnes et al. the breeding season (Ginther and Kot ; de Castro et al. ; Gonzalez-Bulnes et al. ), leading to a normal duration cycles in % of the does. Early studies indicate that resumption of normal function of the hypothalamo pituitary axis may require the stimulus of a small quantity of progesterone from a first ovulation or luteinisation in non-primed does, the ovulation rate being significantly greater at the second ovulation and similar to cyclic females (Chemineau et al. ).This may be related to the findings of the current study, where the two largest follicles grew after progesterone priming. However, although LH patterns could not be determined, these effects were not entirely related to a delay in the day of appearance of oestrus, the preovulatory LH surge or ovulation after the buck effect, because the oestrus and ovulation were detected earlier than in does not treated with progesterone. The effect of progesterone seemed to be related to a better pattern of growth of preovulatory follicles, coincidentally with a previous hypothesis in sheep (Martin et al. ). In goats, Walkden-Brown and Restall () consider a similar situation, reinforced by the fact that progesterone priming at a neural level is not required for the expression of oestrus in the doe (Sutherland and Lindsay ); however, the influence of LH in such an effect may not be discarded as in all mammals, LH has a key role in the final maturation and establishment of oestradiol secretory patterns of the preovulatory follicles (Baird ; Campbell et al. ). In conclusion, our current results support previous reports regarding the existence of short luteal phases and delayed ovulation in goats treated with the male effect during seasonal anoestrus. The stage of follicle development at the time of buck introduction may play a key role in the quality of ovulation. A single injection of progesterone homogenises the response to the buck effect as it stimulates the emergence of a new follicular wave. Thus, the young follicles will grow and ovulate under the effect of the increased LH pulse frequency induced by the male. Acknowledgments The authors gratefully thank A. Exposito, A. Rabadan, A. Lopez and M. A. Caja for skilled technical assistance, and B. Cabellos and A. Gomez-Brunet for hormone assays. This work was supported by funds from Instituto Nacional de Investigación y Tecnologia Agraria y Alimentaria (RTA - ) and Ministerio de Ciencia ytecnología (AGB: Programa Ramon y Cajal). References Baird, D. T. (). Factors regulating the growth of the preovulatory follicle in sheep and human. J. Reprod. Fertil.,. Campbell, B. K., Scaramuzzi, R. J., and Webb, R. (). Control of antral follicle development and selection in sheep and cattle. J. Reprod. Fertil. Suppl.,. Chemineau, P. (). Effect on oestrus and ovulation of exposing creole goats to the male at three times of the year. J. Reprod. Fertil.,. Chemineau, P., Baril, G., Leboeuf, B., Maurel, M. C., Roy, F., Pellicer- Rubio, M., Malpaux, B., and Cognie, Y. (). Implications of recent advances in reproductive physiology for reproductive management of goats. J. Reprod. Fertil. Suppl.,. de Castro, T., Rubianes, E., Menchaca, A., and Rivero, A. (). Ovarian dynamics, serum estradiol and progesterone concentrations during the interovulatory interval in goats. Theriogenology,. doi:./s-x()- Ginther, O. J., and Kot, K. (). Follicular dynamics during the ovulatory season in goats. Theriogenology,. doi:./-x()-x Gonzalez-Bulnes, A., Santiago Moreno, J., Gomez-Brunet, A., Inskeep, E. K., Townsend, E. M., and Lopez-Sebastian, A. (). Follicular dynamics during the oestrous cycle in dairy goats. Anim. Sci.,. Gonzalez-Bulnes, A., Diaz-Delfa, C., Carrizosa, J. A., and Lopez- Sebastian, A. (). 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Effect of the presence of the male on initiation of estrous cycle activity of goats. Theriogenology,. doi:./-x()- Shelton, M. (). Influence of the presence of a male goat on the initiation of estrous cycling and ovulation of Angora does. J. Anim. Sci.,. Sutherland, S. R. D., and Lindsay, D. R. (). Ovariectomized does do not require progesterone priming for oestrous behaviour. Reprod. Fertil. Dev.,. doi:./rd Ungerfeld, R., Forsberg, M., and Rubianes, E. (). Overview of the response of anoestrous ewes to the ram effect. Reprod. Fertil. Dev.,. doi:./rd Walkden-Brown, S. W., and Restall, B. J. (). Environmental and maternal factors affecting reproduction. In Proceedings of the VI International Conference on Goats. pp.. (International Academic Publishers: Beijing, China.) Manuscript received January ; revised and accepted June.