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Timing of emergence of ovulatory follicles in polyovulatory goats
Animal Reproduction Science 91 (2006) 275–284
Timing of emergence of ovulatory follicles in polyovulatory goats M. Cueto a,∗ , A. Gibbons a , R. Alberio b , H. Taddeo a , A. Gonzalez-Bulnes c b
a Reproducci´ on & Gen´etica, INTA Bariloche, CC 277, 8400 Bariloche, Argentina Biotecnolog´ıa de la Reproducci´on, INTA Balcarce, CC 276, 7620 Balcarce, Argentina c Dpto. Reproducci´ on Animal y Conservaci´on de Recursos Zoogen´eticos, SGIT-INIA, Avda., Puerta de Hierro km 5.9, 28040 Madrid, Spain
Received 20 January 2005; received in revised form 16 February 2005; accepted 21 April 2005 Available online 6 June 2005
Abstract The current study characterized the timing of emergence of ovulatory follicles during the follicular phase of the estrous cycle in polyovulatory does and assessed whether selection may influence ovulation rate through differences in ovarian follicular dynamics, by characterizing preovulatory follicular emergence and growth in two ecotypes of Neuquen-Criollo Argentinean goats (Short-Hair, n = 11 and Long-Hair, n = 9). During the breeding season, the time of estrus was synchronized in all does with two doses of a prostaglandin analogue. Ovarian laparoscopies were performed on days 17 and 19 after the induced estrus (day 0) and 7–15 h after the beginning of the subsequent estrus. Results indicate that both ecotypes of goats have common features in the ovarian follicular population and in the patterns of preovulatory follicular enlargement. In all the goats, most of the preovulatory follicles arose from the pool of follicles present in the ovary between days 17 and 19 of the estrous cycle. These follicles were all larger than 2 mm at emergence, being the largest growing follicle present in the ovaries on days 17 and 19 in 56.5 and 78.6% of the does, respectively. The appearance of new follicles remained unaffected, while the mean number of small growing follicles decreased (P < 0.05) during the follicular phase, indicating that preovulatory follicles do not suppress the emergence of new follicles but inhibit the growth of small follicles. A separate analysis of single and double ovulating does showed that 75% of the second ovulatory follicles in polyovulatory goats was present on the ovarian surface between days 17 and 19 of the estrous cycle, but appeared later in the other 25% of ∗
Corresponding author. Tel.: +54 2944 422731; fax: +54 2944 424991. E-mail address: [email protected] (M. Cueto).
0378-4320/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2005.04.007
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the estrous cycles. These findings support the hypothesis that follicular dominance effects are exerted during the preovulatory period, when the growth of follicles other than the ovulatory is inhibited, and that increases in ovulation rate in small ruminants are related to a reduced incidence of follicular atresia and an extended period of ovulatory follicle recruitment. © 2005 Elsevier B.V. All rights reserved. Keywords: Goat; Follicular dominance; Folliculogenesis; Luteolysis; Ovulation rate
1. Introduction In small ruminants, prolificacy is essentially determined by ovulation rate, and ovulation rate is determined by preovulatory ovarian follicular development. Increases in ovulation rate have been related to an extended period of ovulatory follicle recruitment, both in sheep (Bartlewsky et al., 1999) and goats (Ginther and Kot, 1994). Thereafter, selected follicles reach the dominant stage and exert an inhibitory effect on growth of the remaining follicles. In small ruminants, even in monovular females, dominance is less significant than in heifers (Fortune, 1994). The dominant follicle suppresses but not totally so the emergence of new follicles, although it inhibits subsequent growth to larger sizes (Gonzalez-Bulnes et al., 2001). In goats, the greater incidence of polyovulatory cycles as compared with cattle led to the introduction of the concept of co-dominance to explain the presence of two large follicles in each wave of ovarian follicular development (Rubianes and Menchaca, 2003). However, there are estrous cycles where some ovulatory follicles emerge later, grow and there are subsequent ovulation from these follicles even in the presence of ovulatory follicles that had previously emerged (Ginther and Kot, 1994; Gonzalez-Bulnes et al., 2005). This finding is inconsistent with the concept of follicular dominance that exists in cattle. The first objective of current study was to characterize the timing of emergence of ovulatory follicles during the follicular phase of the estrous cycle in polyovulatory goats. Does from the Neuquen-Criollo breed, that originated in the North Region of the Patagonia in Argentine were used for the study. Different environmental conditions and artificial selection resulted in two different ecotypes of the Neuquen-Criollo breed, mainly characterized by their coat length, the Shortand the Long-Hair goats (Lanari et al., 2003). The Short-Hair goats, traditionally selected for meat, are mostly distributed in the ecological sub-area of Barrancas, a mountainous zone (1200–2400 m) with considerable annual rainfall (1000 mm) and snowfall from April to December. The Long-Hair goats were selected for shearing and are reared in A˜nelo subarea, a lowland (<300–600 m) and arid territory (130 mm annual rainfall). Reproductive performance between ecotypes in their regional sub-areas is also different with the ShortHair goats being more prolific than Long-Hair (1.7 versus 1.3; Lanari, 2003). In the current study, we have used females from both strains and compared the main features of ovarian follicular populations around the time of onset of estrus. The reproductive activity of animals is determined by a complex adaptation to environmental cues; with those that are most important being photoperiod, nutrition and sociosexual signals (Martin et al., 2004). Breeding patterns have evolved so that environmental factors
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are optimal for reproduction to occur in the two ecotypes. Domestication and selection processes have increased the ovulation rate of domestic sheep and goats when compared with their wild ancestors, either by artificial selection or, indirectly, though enhanced management and nutrition conditions as genetic selection has occurred. The second objective of current work was to determine whether the background of selection has induced differences in the preovulatory ovarian follicular dynamics of Shortand Long-Hair goats by rearing and evaluating the animals in a common habitat.
2. Material and methods 2.1. Animals and experimental procedure Eleven Short-Hair (SH) and nine Long-Hair (LH) Neuquen-Criollo goats 4–5 years of age were used. Animals were kept outdoors, under extensive management conditions, at the experimental farm of the INTA Bariloche, Argentine, at 41◦ S and under natural daylength. In the breeding season (April–May), time of estrus was synchronized in all the does by using two i.m. doses of 125 g of a prostaglandin analogue (cloprostenol, Estroplan® , Lab. Syntex, Argentina) 11 days apart. Detection of estrus was performed twice daily with three vasectomized bucks starting 24 h after the second prostaglandin injection until the onset of the subsequent estrus. Presence of preovulatory follicles and characteristics of remaining follicular population were assessed by laparoscopy on days 17 and 19 after the induced estrus (day 0) and 7–15 h after the beginning of the next spontaneous estrus. Ovulation rate was determined by laparoscopic examination of the corpora lutea present in the ovaries on day 6 after the onset of estrus. Laparoscopies were performed with a 4 mm endoscope (Richard Wolf, Knittlingen, West Germany). Local anaesthesia (1 ml of 2% xylocaine) was injected s.c. 3–4 cm anterior to the udder, 4–5 cm on each side of the mid-ventral area. The largest diameter of all follicles ≥2 mm in size was measured with a manipulation probe engraved with a millimeter scale. The relative position of each follicle to other follicles and/or luteal structures was recorded in a diagram of the ovaries to evaluate development in successive observations. During the laparoscopies, serial images of the ovaries were recorded with a digital camera allowing for the possibility of subsequent analysis. On days 17 and 19 of the estrous cycle, blood samples (5 ml) were collected by jugular venopuncture just prior laparoscopies and serum was stored at −20 ◦ C until assayed for progesterone. Plasma progesterone concentrations were measured by direct solidphase radioimmunoassay (DPC, Diagnostic Products Co., Los Angeles, CA), as described (Rubianes and Ungerfeld, 1993). The intra-assay coefficients of variation were 4.5% for values >0.1 ng/ml and 11.8% for values around 0.1 ng/ml, which was the detection limit for the technique. 2.2. Statistical analysis of experimental data Data obtained were summarized to characterize follicular development and relationship among follicles of different sizes; the model included time, ecotype and ovulation rate.
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Initially, diameters of ovulatory follicles were examined to characterize the day of detection and both initial and maximum diameters. After this, all follicles recorded were classified as total (≥2 mm), large (≥6 mm), medium (4–5 mm) or small follicles (2–3 mm). Thereafter, follicles were classified as being in the growing phase (those that increased in size between two successive laparoscopies), static phase (those that maintained their diameter), regressing phase (those that decreased in size between two successive laparoscopies) and new follicles (not previously detected). Follicle sizes (small, medium and large) were considered in such classification. Four types of statistical analyses were conducted in data assessment. The first analysis evaluated the effect of genotype and ovulation rate on number of follicles of different sizes (small, medium, large and total follicles) and their dynamics (number of growing, static, decreasing and new follicles for all follicle sizes) by analysis of variance (ANOVA). Interactions were not included in the model. The second analysis tested the effect of day on follicle sizes and dynamics by repeated measurement ANOVA, followed by Fischer’s protected least significance as a multiple comparison test for significant effects (Statview, 1986). In the third analysis, effects of genotype and ovulation rate on the percentage of follicles present in the ovaries and on the largest growing follicle on days 17 and 19 of the estrous cycle were evaluated by contigency tables using the Chi square test criterion. Lastly, rates of growth and regression of ovarian follicles were examined by paired ttest for differences between ovulatory and non-ovulatory follicles in both ecotypes. Mean diameters of ovulatory follicles at days 17 and 19 after the induced estrus and at the onset of the surveyed estrus were compared between ecotypes, monovular and polyovulatory cycles and between the first and the second ovulatory follicles in double ovulations by paired t-test. The SAS Package (Statistical Analysis System, 1992) was used to perform the analyses. Results were expressed as means ± S.E.M. and statistical significance was accepted from P < 0.05.
3. Results All the goats showed estrus behavior, with a mean inter-estrus interval of 20.9 ± 0.1 days both for Short- and Long-Hair goats. Ovulation rates were also similar for both ecotypes (1.6 ± 0.15 for SH and 1.6 ± 0.18 for LH). Serum concentrations of progesterone decreased between days 17 and 19 in both ecotypes. On day 17 of the estrous cycle, none of the LH does and only 27% of SH does had progesterone concentrations below 0.5 ng/ml, while on day 19, 67% of LH does and 64% of SH does had basal concentrations of progesterone (<0.5 ng/ml). During the follicular phase, changes in the frequency of follicular size (mean number of small, medium, large and total follicles) or in follicular dynamics (growing, static, regressing and new follicles for all follicle sizes) were similar between both ecotypes. Analysis of follicle frequency for all size classes during the observation period showed that the mean number of ≥2 mm follicles was affected by day of estrous cycle in both groups (Fig. 1) with a decrease between day 19 and onset of estrus behavior (P < 0.05). This decrease was mainly caused by a decline in the mean number of small (2–3 mm) follicles
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Fig. 1. Frequency size distribution (mean ± S.E.M.) of total, small (2–3 mm), medium (4–5 mm) and large follicles (≥6 mm) in Short- and Long-Hair Neuquen-Criollo goats between day 17 of the estrous cycle and the onset of estrus. Different superscripts indicate statistically significant differences between days (P < 0.05).
(P < 0.05) because number of medium (4–5 mm) follicles did not change during this period. Conversely, the mean number of large (≥6 mm) follicles increased from day 17 to the onset of signs of estrus (P < 0.05) with this increment being greater in does with double than single ovulations (P < 0.05). During the follicular phase, assessment of pattern of follicular development indicated the number of small growing follicles decreased between day 19 of the estrous cycle and time of detection of behavioural estrus (P < 0.05), while the number of large growing follicles increased (P < 0.05) with time (Fig. 2). This increase in large follicles was greater in polyovulatory than monovular estrous cycles (P < 0.05). No significant differences were observed, however, in the number of medium growing, static, regressing or newly detected follicles for all size classes during the time of observation (P > 0.05). In both ecotypes, most of the preovulatory follicles arose from the pool of antral follicles present in the ovary between days 17 and 19 of the estrous cycle (Table 1) with a diameter ranging from 2 to 6 mm on day 17. On day 19, the mean size ranged between 3 and 7 and Table 1 Effects of ecotypes (Short-Hair vs. Long-Hair) and ovulation rate (monovular vs. polyovulatory) on percentage of preovulatory follicles present in the ovaries and percentage of preovulatory follicles being the largest on days 17 and 19 of the estrous cycle in Neuquen-Criollo goats Day 17 of the estrous cycle
Day 19 of the estrous cycle
Follicles present in the ovaries
Largest growing Follicle
Follicles present in the ovaries
Largest growing Follicle
Ecotypes Short-Hair Long-Hair
67% (12/18) 71% (10/14)
69% (9/13) 40% (4/10)
89% (16/18) 86% (12/14)
81% (13/16) 75% (9/12)
Cycles Monovular Polyovulatory
88% (7/8) 63% (15/24)
57% (4/7) 56% (9/16)
88% (7/8) 88% (21/24)
100% (7/7) 71% (15/21)
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Fig. 2. Frequency distribution (mean ± S.E.M.) of: (a) growing, (b) static, (c) decreasing and (d) new follicles for follicular size classes (small, 2–3 mm; medium, 4–5 mm; large, ≥6 mm) in (1) Short-Hair and (2) LongHair Neuquen-Criollo goats at day 19 and at the onset of estrus. Statistically significant differences in follicular populations between day 19 and estrus are indicated with different letters (P < 0.05).
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Table 2 Rates of growth and regression between days 17 and 19 of the estrous cycle and day 19 and the onset of estrus of ovulatory and non-ovulatory follicles from Short- and Long-Hair Neuquen-Criollo goats Ecotypes
Days 17–19 Ovulatory follicles
Day 19—onset of estrus signs Non-ovulatory follicles
Ovulatory follicles
Non-ovulatory follicles
Growth rate (mm/day) Short-Hair 0.77 ± 0.16a Long-Hair 1.00 ± 0.19a
0.52 ± 0.01b 0.51 ± 0.06b
0.81 ± 0.24a 1.08 ± 0.30a
0.71 ± 0.08b 0.59 ± 0.04b
Regression rate (mm/day) Short-Hair Long-Hair
1.16 ± 0.09 1.05 ± 0.09
1.17 ± 0.06 1.11 ± 0.07
All values are means ± S.E.M. Values with different superscripts (a and b) within rows are significantly different (P < 0.05).
between 3 and 6 mm for SH and LH goats, respectively. However, there were 11–14% of the ovulatory follicles that emerged after day 19. A total of 69% of the follicles from which ovulation occurred were the largest present in the ovaries on day 17 in the SH goats, with a mean size of 4.1 ± 0.1 mm. The percentage was 40% in LH goats, with a mean diameter of 3.1 ± 0.4 mm. On day 19 of the estrous cycle, 81% of the follicles from which ovulation occurred were the largest in the ovaries of SH does, with a mean size of 4.8 ± 0.3 mm, and 75% were the largest in LH goats, being 4.4 ± 0.3 mm in diameter. When single and double ovulatory estrous cycles were considered separately, 63 and 88% of the ovulatory follicles from estrous cycles with double ovulations were present on days 17 and 19, respectively. These proportions were similar to monovular cycles (88 and 88% for days 17 and 19 of estrus, respectively). In 5 of 12 polyovulatory cycles (41.7%), the second ovulatory follicle was present on the ovarian surface on day 17; in 4 of 12 (33.3%) emergence was on day 19, while in 25% emergence was between day 19 and the onset of estrus. In polyovulatory cycles, 56 and 71% of the ovulatory follicles were largest on days 17 and 19 of the estrous cycle, respectively. In monovular cycles, 57% of the ovulatory follicles were largest on day 17, whereas all the ovulatory follicles were the largest follicle on day 19. In all the goats, the mean diameter of the preovulatory follicle increased at a rate around 1 mm/day (Table 2), at a rate significantly greater than in non-ovulatory follicles (P < 0.05). At the onset of estrus behavior, ovulatory follicles reached a mean diameter of 6.1 ± 0.3 mm in SH goats and 6.5 ± 0.4 mm in LH goats. Mean diameters of ovulatory follicles at days 17 and 19 of the estrous cycle after the induced estrus, and at the onset of estrus were similar (P > 0.05) between ecotypes (monovular and polyovulatory cycles) and between the first and the second ovulatory follicles when double ovulations occurred.
4. Discussion The present study indicates that both ecotypes of Neuquen-Criollo goats have common features in the ovarian follicular population and in the patterns of preovulatory follicular
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development. This finding suggests that influence from artificial selection on reproductive characteristics of domestic Short- and Long-Hair goats have not affected ovarian activity during the estrous cycle, in the manner that has been previously reported in sheep (GonzalezBulnes et al., 2001). Our results showed that, in monovular goats, the preovulatory follicle was the largest growing follicle present in both ovaries at time of first observation, around 2–4 days before the onset of behavioural estrus. Some of the follicles from which ovulation occurred were visible on the ovarian surface for at least 5 days before the onset of signs of behavioural estrus, coincidentally with observations in Saanen goats (de Castro et al., 1999) and Murciana goats (Gonzalez-Bulnes et al., 1999b). The ovulatory follicles increased in size at a growth rate of 1.1 mm/day, which is similar to that previously described (Ginther and Kot, 1994; Gonzalez-Bulnes et al., 1999a,b; Schwarz and Wierzchos, 2000; Pinczak et al., 2001) reaching a maximum diameter of 6.1 mm in Short-Hair and 6.5 mm in Long-Hair goats. This finding agrees with prior data in different goat breeds (de Castro et al., 1999; GonzalezBulnes et al., 1999b; Schwarz and Wierzchos, 2000), although Ginther and Kot (1994) reported a greater diameter, 9.7 mm, in Saanen breed. When all information is assessed, the data confirm the previous hypothesis (Rubianes and Menchaca, 2003): (1) permanent availability of one or more follicles that have developed to the extent that ovulation can occur from the follicles at the time of luteolysis; (2) follicles that can be selected from which ovulation can occur from the largest follicles present in the ovary; (3) a large variability in size of ovulatory follicle emergence, from 2 to 7 mm; (4) all follicles larger than 2 mm can develop to the extent that ovulation can occur from them at time of luteal regression. Current data also indicate dominance effects of the largest follicle during the preovulatory period, when the growth of follicles other than the ovulatory follicle is inhibited as previously described (Menchaca et al., 2002). These results give new evidence indicating the existence, in goats, of follicular dominance during the final growth of ovulatory follicles (de Castro et al., 1999), rather than in the luteal phase (Menchaca and Rubianes, 2002; GonzalezBulnes et al., 2005). The data for the present study also supports the notion of dominance mechanisms differing from that reported in cows (Ginther et al., 1989). In these species, the dominant follicle has an active role both inhibiting the growth of the other follicles in the cohort and inhibiting the emergence of new large follicles (Fortune, 1994). Dominance during follicular development in goats would be more similar to sheep (Gonzalez-Bulnes et al., 2001) in which the dominant follicle inhibits the growth but does not suppress the emergence of new follicles, although it also inhibits their subsequent growth to larger sizes. Ovulation rate in monovular goats would be limited by large amounts of follicular atresia rather than by lesser recruitment of follicles into the growing pool. This would be consistent with similar hypothesis developed in sheep (Driancourt et al., 1990; Lopez-Sebastian et al., 1997) and confirmed thereafter (Gonzalez-Bulnes et al., 2004). In most of the goats with double ovulation, one of the ovulatory follicles was the largest growing follicle present in both ovaries at time of first observation. However, a second ovulatory follicle was present on the same day; which is consistent with the concept of co-dominance (Rubianes and Menchaca, 2003) to explain the presence of two large follicles in each wave. However, there were some estrous cycles where the second ovulatory follicles emerged later, grew, and ovulation occurred from them in the presence of the first ovulatory follicle. This finding, confirmed previous descriptions (Ginther and Kot, 1994;
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Gonzalez-Bulnes et al., 2005), and reinforces the idea of a dominance effect similar to that described for ewes, that is weaker and easier to overcome than in heifers. Furthermore, this provides additional evidence for the hypothesis that increases in ovulation rate are not related to greater follicle recruitment, but instead to an extended period of ovulatory follicle recruitment (Ginther and Kot, 1994). In conclusion, the current work does not provide evidence of any significant influence of the genetic selection background on preovulatory follicular dynamics in domestic NeuquenCriollo goats. The ovulatory follicle in monovular goats, and one of the ovulatory follicles in polyovulatory goats, was the largest growing follicle present in both ovaries at the time of luteolysis. The second ovulatory follicle in polyovulatory goats emerged coincidentally with the first one in most of the estrous cycles, but appeared later in some does. This feature, which is inconsistent with the dominance effect, supports the hypothesis that increases in ovulation rate in small ruminants are related to a reduced incidence of follicular atresia and an extended period of ovulatory follicle recruitment.
Acknowledgements The authors thank to Jos´e Mar´ıa Garramu˜no and the staff of the experimental farm of INTA Bariloche, to Marcelo Aba, director of the Laboratory of Endocrinology of the Faculty of Veterinary Sciences of Tandil (Argentine) for radioimmunoassays of progesterone. Financial support was received from FonCyT (National Fund of Science and Technology, Argentine). References Bartlewsky, P.M., Beard, A.P., Cook, S.J., Chandolia, R.K., Nonaramooz, A., Rawlings, N.C., 1999. Ovarian antral follicular dynamics and their relationship with endocrine variables throughout the oestrous cycle in breeds of sheep differing in prolificacy. J. Reprod. Fertil. 115, 111–124. de Castro, T., Rubianes, E., Menchaca, A., Rivero, A., 1999. Ovarian dynamics, serum estradiol and progesterone concentrations during the interovulatory interval in goats. Theriogenology 52, 399–411. Driancourt, M.A., Castonguay, F., Bindon, B.M., Piper, L.R., Quirke, J.F., Hanrahan, J.P., 1990. Ovarian follicular dynamics in lines of sheep (Finn, Merinos) selected on ovulation rate. J. Anim. Sci. 68, 2034–2041. Fortune, J.E., 1994. Ovarian follicular growth and development in mammals. Biol. Reprod. 50, 225–232. Ginther, O.J., Kot, K., 1994. Follicular dynamics during the ovulatory season in goats. Theriogenology 42, 987–1001. Ginther, O.J., Knopf, L., Kastelic, L.P., 1989. Temporal associations among ovarian events in cattle during estrous cycles with two and three follicular waves. J. Reprod. Fertil. 87, 223–230. Gonzalez-Bulnes, A., Osoro, K., Lopez-Sebastian, A., 1999a. Ultrasonic assessment of the ovarian response in eCG-treated goats. Small Rumin. Res. 34, 65–69. Gonzalez-Bulnes, A., Santiago Moreno, J., Gomez-Brunet, A., Inskeep, E.K., Townsend, E.M., Lopez-Sebastian, A., 1999b. Follicular dynamics during the oestrous cycle in dairy goats. Anim. Sci. 68, 547–554. Gonzalez-Bulnes, A., Santiago-Moreno, J., Garcia-Garcia, R.M., del Campo, A., Gomez-Brunet, A., LopezSebastian, A., 2001. Origin of the preovulatory follicle in Mouflon sheep (Ovis gmelini musimon) and effect on the growth of remaining follicles during the follicular phase of oestrous cycle. Anim. Reprod. Sci. 65, 265–272. Gonzalez-Bulnes, A., Souza, C.J.H., Campbell, B.K., Baird, D.T., 2004. Effect of ageing on hormone secretion and follicular dynamics in sheep with and without the Booroola gene (FecB). Endocrinology 145, 2858– 2864.
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Gonzalez-Bulnes, A., Diaz-Delfa, C., Garcia-Garcia, R.M., Urrutia, B., Carrizosa, J.A., Lopez-Sebastian, A., 2005. Origin and fate of preovulatory follicles after induced luteolysis at different stages of the luteal phase of the oestrous cycle in goats. Anim. Reprod. Sci. 86, 237–245. Lanari, M.R., 2003. Variaci´on y diferenciaci´on gen´etica y fenot´ıpica de la Cabra Criolla Neuquina en relaci´on con su sistema rural campesino. Tesis Doctoral. Universidad del Comahue, Centro Regional Universitario Bariloche, Argentina. Lanari, M.R., Taddeo, H., Domingo, E., P´erez Centeno, M., Gallo, L., 2003. Phenotypic differentiation of local Criollo Goat Population in Patagonia (Argentina). Archiv Fur Tierzucht. Arch. Anim. Breed. 46, 347–356. Lopez-Sebastian, A., Gonzalez-Bulnes, A., Santiago-Moreno, J., Gomez-Brunet, A., Townsend, E.C., Inskeep, E.K., 1997. Patterns of follicular development during the oestrous cycle in monovular Merino del Pais ewes. Anim. Reprod. Sci. 48, 279–291. Martin, G.B., Rodger, J., Blache, D., 2004. Nutritional and environmental effects on reproduction in small ruminants. Reprod. Fertil. Dev. 16, 491–501. Menchaca, A., Rubianes, E., 2002. Relation between progesterone concentrations during the early luteal phase and follicular dynamic in goats. Theriogenology 57, 1411–1419. Menchaca, A., Pinczak, A., Rubianes, E., 2002. Follicular recruitment and ovulatory response to FSH treatment initiated on day 0 or day 3 post-ovulation in goats. Theriogenology 58, 1713–1721. Pinczak, A., Menchaca, A., Rubianes, E., 2001. Seguimiento ultrasonogr´afico ov´arico y uterino durante la gestaci´on temprana de la cabra (Ovarian and uterine scanning during the early pregnancy in goats). In: Proceedings of the IV International Symposium on Animal Reproduction, C´ordoba, Argentina, p. 298 (Abstract). Rubianes, E., Menchaca, A., 2003. The pattern and manipulation of ovarian follicular growth in goats. Anim. Reprod. Sci. 78, 271–287. Rubianes, E., Ungerfeld, R., 1993. Uterine involution and ovarian changes during early postpartum of autumnlambing Corriedale ewes. Theriogenology 40, 365–372. SAS, 1992. SAS User’s Guide: Statistics (Version 6.08). SAS Institute, Cary, NC. Schwarz, T., Wierzchos, E., 2000. Relationship between FSH and ovarian follicular dynamics in goats during the estrous cycle. Theriogenology 53, 381 (Abstract). Statview, 1986. Abacus Concepts Inc., Brain Power Inc., Calabsas, CA.
Timing of emergence of ovulatory follicles in polyovulatory goats M. Cueto a,∗ , A. Gibbons a , R. Alberio b , H. Taddeo a , A. Gonzalez-Bulnes c b
a Reproducci´ on & Gen´etica, INTA Bariloche, CC 277, 8400 Bariloche, Argentina Biotecnolog´ıa de la Reproducci´on, INTA Balcarce, CC 276, 7620 Balcarce, Argentina c Dpto. Reproducci´ on Animal y Conservaci´on de Recursos Zoogen´eticos, SGIT-INIA, Avda., Puerta de Hierro km 5.9, 28040 Madrid, Spain
Received 20 January 2005; received in revised form 16 February 2005; accepted 21 April 2005 Available online 6 June 2005
Abstract The current study characterized the timing of emergence of ovulatory follicles during the follicular phase of the estrous cycle in polyovulatory does and assessed whether selection may influence ovulation rate through differences in ovarian follicular dynamics, by characterizing preovulatory follicular emergence and growth in two ecotypes of Neuquen-Criollo Argentinean goats (Short-Hair, n = 11 and Long-Hair, n = 9). During the breeding season, the time of estrus was synchronized in all does with two doses of a prostaglandin analogue. Ovarian laparoscopies were performed on days 17 and 19 after the induced estrus (day 0) and 7–15 h after the beginning of the subsequent estrus. Results indicate that both ecotypes of goats have common features in the ovarian follicular population and in the patterns of preovulatory follicular enlargement. In all the goats, most of the preovulatory follicles arose from the pool of follicles present in the ovary between days 17 and 19 of the estrous cycle. These follicles were all larger than 2 mm at emergence, being the largest growing follicle present in the ovaries on days 17 and 19 in 56.5 and 78.6% of the does, respectively. The appearance of new follicles remained unaffected, while the mean number of small growing follicles decreased (P < 0.05) during the follicular phase, indicating that preovulatory follicles do not suppress the emergence of new follicles but inhibit the growth of small follicles. A separate analysis of single and double ovulating does showed that 75% of the second ovulatory follicles in polyovulatory goats was present on the ovarian surface between days 17 and 19 of the estrous cycle, but appeared later in the other 25% of ∗
Corresponding author. Tel.: +54 2944 422731; fax: +54 2944 424991. E-mail address: [email protected] (M. Cueto).
0378-4320/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2005.04.007
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the estrous cycles. These findings support the hypothesis that follicular dominance effects are exerted during the preovulatory period, when the growth of follicles other than the ovulatory is inhibited, and that increases in ovulation rate in small ruminants are related to a reduced incidence of follicular atresia and an extended period of ovulatory follicle recruitment. © 2005 Elsevier B.V. All rights reserved. Keywords: Goat; Follicular dominance; Folliculogenesis; Luteolysis; Ovulation rate
1. Introduction In small ruminants, prolificacy is essentially determined by ovulation rate, and ovulation rate is determined by preovulatory ovarian follicular development. Increases in ovulation rate have been related to an extended period of ovulatory follicle recruitment, both in sheep (Bartlewsky et al., 1999) and goats (Ginther and Kot, 1994). Thereafter, selected follicles reach the dominant stage and exert an inhibitory effect on growth of the remaining follicles. In small ruminants, even in monovular females, dominance is less significant than in heifers (Fortune, 1994). The dominant follicle suppresses but not totally so the emergence of new follicles, although it inhibits subsequent growth to larger sizes (Gonzalez-Bulnes et al., 2001). In goats, the greater incidence of polyovulatory cycles as compared with cattle led to the introduction of the concept of co-dominance to explain the presence of two large follicles in each wave of ovarian follicular development (Rubianes and Menchaca, 2003). However, there are estrous cycles where some ovulatory follicles emerge later, grow and there are subsequent ovulation from these follicles even in the presence of ovulatory follicles that had previously emerged (Ginther and Kot, 1994; Gonzalez-Bulnes et al., 2005). This finding is inconsistent with the concept of follicular dominance that exists in cattle. The first objective of current study was to characterize the timing of emergence of ovulatory follicles during the follicular phase of the estrous cycle in polyovulatory goats. Does from the Neuquen-Criollo breed, that originated in the North Region of the Patagonia in Argentine were used for the study. Different environmental conditions and artificial selection resulted in two different ecotypes of the Neuquen-Criollo breed, mainly characterized by their coat length, the Shortand the Long-Hair goats (Lanari et al., 2003). The Short-Hair goats, traditionally selected for meat, are mostly distributed in the ecological sub-area of Barrancas, a mountainous zone (1200–2400 m) with considerable annual rainfall (1000 mm) and snowfall from April to December. The Long-Hair goats were selected for shearing and are reared in A˜nelo subarea, a lowland (<300–600 m) and arid territory (130 mm annual rainfall). Reproductive performance between ecotypes in their regional sub-areas is also different with the ShortHair goats being more prolific than Long-Hair (1.7 versus 1.3; Lanari, 2003). In the current study, we have used females from both strains and compared the main features of ovarian follicular populations around the time of onset of estrus. The reproductive activity of animals is determined by a complex adaptation to environmental cues; with those that are most important being photoperiod, nutrition and sociosexual signals (Martin et al., 2004). Breeding patterns have evolved so that environmental factors
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are optimal for reproduction to occur in the two ecotypes. Domestication and selection processes have increased the ovulation rate of domestic sheep and goats when compared with their wild ancestors, either by artificial selection or, indirectly, though enhanced management and nutrition conditions as genetic selection has occurred. The second objective of current work was to determine whether the background of selection has induced differences in the preovulatory ovarian follicular dynamics of Shortand Long-Hair goats by rearing and evaluating the animals in a common habitat.
2. Material and methods 2.1. Animals and experimental procedure Eleven Short-Hair (SH) and nine Long-Hair (LH) Neuquen-Criollo goats 4–5 years of age were used. Animals were kept outdoors, under extensive management conditions, at the experimental farm of the INTA Bariloche, Argentine, at 41◦ S and under natural daylength. In the breeding season (April–May), time of estrus was synchronized in all the does by using two i.m. doses of 125 g of a prostaglandin analogue (cloprostenol, Estroplan® , Lab. Syntex, Argentina) 11 days apart. Detection of estrus was performed twice daily with three vasectomized bucks starting 24 h after the second prostaglandin injection until the onset of the subsequent estrus. Presence of preovulatory follicles and characteristics of remaining follicular population were assessed by laparoscopy on days 17 and 19 after the induced estrus (day 0) and 7–15 h after the beginning of the next spontaneous estrus. Ovulation rate was determined by laparoscopic examination of the corpora lutea present in the ovaries on day 6 after the onset of estrus. Laparoscopies were performed with a 4 mm endoscope (Richard Wolf, Knittlingen, West Germany). Local anaesthesia (1 ml of 2% xylocaine) was injected s.c. 3–4 cm anterior to the udder, 4–5 cm on each side of the mid-ventral area. The largest diameter of all follicles ≥2 mm in size was measured with a manipulation probe engraved with a millimeter scale. The relative position of each follicle to other follicles and/or luteal structures was recorded in a diagram of the ovaries to evaluate development in successive observations. During the laparoscopies, serial images of the ovaries were recorded with a digital camera allowing for the possibility of subsequent analysis. On days 17 and 19 of the estrous cycle, blood samples (5 ml) were collected by jugular venopuncture just prior laparoscopies and serum was stored at −20 ◦ C until assayed for progesterone. Plasma progesterone concentrations were measured by direct solidphase radioimmunoassay (DPC, Diagnostic Products Co., Los Angeles, CA), as described (Rubianes and Ungerfeld, 1993). The intra-assay coefficients of variation were 4.5% for values >0.1 ng/ml and 11.8% for values around 0.1 ng/ml, which was the detection limit for the technique. 2.2. Statistical analysis of experimental data Data obtained were summarized to characterize follicular development and relationship among follicles of different sizes; the model included time, ecotype and ovulation rate.
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Initially, diameters of ovulatory follicles were examined to characterize the day of detection and both initial and maximum diameters. After this, all follicles recorded were classified as total (≥2 mm), large (≥6 mm), medium (4–5 mm) or small follicles (2–3 mm). Thereafter, follicles were classified as being in the growing phase (those that increased in size between two successive laparoscopies), static phase (those that maintained their diameter), regressing phase (those that decreased in size between two successive laparoscopies) and new follicles (not previously detected). Follicle sizes (small, medium and large) were considered in such classification. Four types of statistical analyses were conducted in data assessment. The first analysis evaluated the effect of genotype and ovulation rate on number of follicles of different sizes (small, medium, large and total follicles) and their dynamics (number of growing, static, decreasing and new follicles for all follicle sizes) by analysis of variance (ANOVA). Interactions were not included in the model. The second analysis tested the effect of day on follicle sizes and dynamics by repeated measurement ANOVA, followed by Fischer’s protected least significance as a multiple comparison test for significant effects (Statview, 1986). In the third analysis, effects of genotype and ovulation rate on the percentage of follicles present in the ovaries and on the largest growing follicle on days 17 and 19 of the estrous cycle were evaluated by contigency tables using the Chi square test criterion. Lastly, rates of growth and regression of ovarian follicles were examined by paired ttest for differences between ovulatory and non-ovulatory follicles in both ecotypes. Mean diameters of ovulatory follicles at days 17 and 19 after the induced estrus and at the onset of the surveyed estrus were compared between ecotypes, monovular and polyovulatory cycles and between the first and the second ovulatory follicles in double ovulations by paired t-test. The SAS Package (Statistical Analysis System, 1992) was used to perform the analyses. Results were expressed as means ± S.E.M. and statistical significance was accepted from P < 0.05.
3. Results All the goats showed estrus behavior, with a mean inter-estrus interval of 20.9 ± 0.1 days both for Short- and Long-Hair goats. Ovulation rates were also similar for both ecotypes (1.6 ± 0.15 for SH and 1.6 ± 0.18 for LH). Serum concentrations of progesterone decreased between days 17 and 19 in both ecotypes. On day 17 of the estrous cycle, none of the LH does and only 27% of SH does had progesterone concentrations below 0.5 ng/ml, while on day 19, 67% of LH does and 64% of SH does had basal concentrations of progesterone (<0.5 ng/ml). During the follicular phase, changes in the frequency of follicular size (mean number of small, medium, large and total follicles) or in follicular dynamics (growing, static, regressing and new follicles for all follicle sizes) were similar between both ecotypes. Analysis of follicle frequency for all size classes during the observation period showed that the mean number of ≥2 mm follicles was affected by day of estrous cycle in both groups (Fig. 1) with a decrease between day 19 and onset of estrus behavior (P < 0.05). This decrease was mainly caused by a decline in the mean number of small (2–3 mm) follicles
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Fig. 1. Frequency size distribution (mean ± S.E.M.) of total, small (2–3 mm), medium (4–5 mm) and large follicles (≥6 mm) in Short- and Long-Hair Neuquen-Criollo goats between day 17 of the estrous cycle and the onset of estrus. Different superscripts indicate statistically significant differences between days (P < 0.05).
(P < 0.05) because number of medium (4–5 mm) follicles did not change during this period. Conversely, the mean number of large (≥6 mm) follicles increased from day 17 to the onset of signs of estrus (P < 0.05) with this increment being greater in does with double than single ovulations (P < 0.05). During the follicular phase, assessment of pattern of follicular development indicated the number of small growing follicles decreased between day 19 of the estrous cycle and time of detection of behavioural estrus (P < 0.05), while the number of large growing follicles increased (P < 0.05) with time (Fig. 2). This increase in large follicles was greater in polyovulatory than monovular estrous cycles (P < 0.05). No significant differences were observed, however, in the number of medium growing, static, regressing or newly detected follicles for all size classes during the time of observation (P > 0.05). In both ecotypes, most of the preovulatory follicles arose from the pool of antral follicles present in the ovary between days 17 and 19 of the estrous cycle (Table 1) with a diameter ranging from 2 to 6 mm on day 17. On day 19, the mean size ranged between 3 and 7 and Table 1 Effects of ecotypes (Short-Hair vs. Long-Hair) and ovulation rate (monovular vs. polyovulatory) on percentage of preovulatory follicles present in the ovaries and percentage of preovulatory follicles being the largest on days 17 and 19 of the estrous cycle in Neuquen-Criollo goats Day 17 of the estrous cycle
Day 19 of the estrous cycle
Follicles present in the ovaries
Largest growing Follicle
Follicles present in the ovaries
Largest growing Follicle
Ecotypes Short-Hair Long-Hair
67% (12/18) 71% (10/14)
69% (9/13) 40% (4/10)
89% (16/18) 86% (12/14)
81% (13/16) 75% (9/12)
Cycles Monovular Polyovulatory
88% (7/8) 63% (15/24)
57% (4/7) 56% (9/16)
88% (7/8) 88% (21/24)
100% (7/7) 71% (15/21)
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Fig. 2. Frequency distribution (mean ± S.E.M.) of: (a) growing, (b) static, (c) decreasing and (d) new follicles for follicular size classes (small, 2–3 mm; medium, 4–5 mm; large, ≥6 mm) in (1) Short-Hair and (2) LongHair Neuquen-Criollo goats at day 19 and at the onset of estrus. Statistically significant differences in follicular populations between day 19 and estrus are indicated with different letters (P < 0.05).
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Table 2 Rates of growth and regression between days 17 and 19 of the estrous cycle and day 19 and the onset of estrus of ovulatory and non-ovulatory follicles from Short- and Long-Hair Neuquen-Criollo goats Ecotypes
Days 17–19 Ovulatory follicles
Day 19—onset of estrus signs Non-ovulatory follicles
Ovulatory follicles
Non-ovulatory follicles
Growth rate (mm/day) Short-Hair 0.77 ± 0.16a Long-Hair 1.00 ± 0.19a
0.52 ± 0.01b 0.51 ± 0.06b
0.81 ± 0.24a 1.08 ± 0.30a
0.71 ± 0.08b 0.59 ± 0.04b
Regression rate (mm/day) Short-Hair Long-Hair
1.16 ± 0.09 1.05 ± 0.09
1.17 ± 0.06 1.11 ± 0.07
All values are means ± S.E.M. Values with different superscripts (a and b) within rows are significantly different (P < 0.05).
between 3 and 6 mm for SH and LH goats, respectively. However, there were 11–14% of the ovulatory follicles that emerged after day 19. A total of 69% of the follicles from which ovulation occurred were the largest present in the ovaries on day 17 in the SH goats, with a mean size of 4.1 ± 0.1 mm. The percentage was 40% in LH goats, with a mean diameter of 3.1 ± 0.4 mm. On day 19 of the estrous cycle, 81% of the follicles from which ovulation occurred were the largest in the ovaries of SH does, with a mean size of 4.8 ± 0.3 mm, and 75% were the largest in LH goats, being 4.4 ± 0.3 mm in diameter. When single and double ovulatory estrous cycles were considered separately, 63 and 88% of the ovulatory follicles from estrous cycles with double ovulations were present on days 17 and 19, respectively. These proportions were similar to monovular cycles (88 and 88% for days 17 and 19 of estrus, respectively). In 5 of 12 polyovulatory cycles (41.7%), the second ovulatory follicle was present on the ovarian surface on day 17; in 4 of 12 (33.3%) emergence was on day 19, while in 25% emergence was between day 19 and the onset of estrus. In polyovulatory cycles, 56 and 71% of the ovulatory follicles were largest on days 17 and 19 of the estrous cycle, respectively. In monovular cycles, 57% of the ovulatory follicles were largest on day 17, whereas all the ovulatory follicles were the largest follicle on day 19. In all the goats, the mean diameter of the preovulatory follicle increased at a rate around 1 mm/day (Table 2), at a rate significantly greater than in non-ovulatory follicles (P < 0.05). At the onset of estrus behavior, ovulatory follicles reached a mean diameter of 6.1 ± 0.3 mm in SH goats and 6.5 ± 0.4 mm in LH goats. Mean diameters of ovulatory follicles at days 17 and 19 of the estrous cycle after the induced estrus, and at the onset of estrus were similar (P > 0.05) between ecotypes (monovular and polyovulatory cycles) and between the first and the second ovulatory follicles when double ovulations occurred.
4. Discussion The present study indicates that both ecotypes of Neuquen-Criollo goats have common features in the ovarian follicular population and in the patterns of preovulatory follicular
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development. This finding suggests that influence from artificial selection on reproductive characteristics of domestic Short- and Long-Hair goats have not affected ovarian activity during the estrous cycle, in the manner that has been previously reported in sheep (GonzalezBulnes et al., 2001). Our results showed that, in monovular goats, the preovulatory follicle was the largest growing follicle present in both ovaries at time of first observation, around 2–4 days before the onset of behavioural estrus. Some of the follicles from which ovulation occurred were visible on the ovarian surface for at least 5 days before the onset of signs of behavioural estrus, coincidentally with observations in Saanen goats (de Castro et al., 1999) and Murciana goats (Gonzalez-Bulnes et al., 1999b). The ovulatory follicles increased in size at a growth rate of 1.1 mm/day, which is similar to that previously described (Ginther and Kot, 1994; Gonzalez-Bulnes et al., 1999a,b; Schwarz and Wierzchos, 2000; Pinczak et al., 2001) reaching a maximum diameter of 6.1 mm in Short-Hair and 6.5 mm in Long-Hair goats. This finding agrees with prior data in different goat breeds (de Castro et al., 1999; GonzalezBulnes et al., 1999b; Schwarz and Wierzchos, 2000), although Ginther and Kot (1994) reported a greater diameter, 9.7 mm, in Saanen breed. When all information is assessed, the data confirm the previous hypothesis (Rubianes and Menchaca, 2003): (1) permanent availability of one or more follicles that have developed to the extent that ovulation can occur from the follicles at the time of luteolysis; (2) follicles that can be selected from which ovulation can occur from the largest follicles present in the ovary; (3) a large variability in size of ovulatory follicle emergence, from 2 to 7 mm; (4) all follicles larger than 2 mm can develop to the extent that ovulation can occur from them at time of luteal regression. Current data also indicate dominance effects of the largest follicle during the preovulatory period, when the growth of follicles other than the ovulatory follicle is inhibited as previously described (Menchaca et al., 2002). These results give new evidence indicating the existence, in goats, of follicular dominance during the final growth of ovulatory follicles (de Castro et al., 1999), rather than in the luteal phase (Menchaca and Rubianes, 2002; GonzalezBulnes et al., 2005). The data for the present study also supports the notion of dominance mechanisms differing from that reported in cows (Ginther et al., 1989). In these species, the dominant follicle has an active role both inhibiting the growth of the other follicles in the cohort and inhibiting the emergence of new large follicles (Fortune, 1994). Dominance during follicular development in goats would be more similar to sheep (Gonzalez-Bulnes et al., 2001) in which the dominant follicle inhibits the growth but does not suppress the emergence of new follicles, although it also inhibits their subsequent growth to larger sizes. Ovulation rate in monovular goats would be limited by large amounts of follicular atresia rather than by lesser recruitment of follicles into the growing pool. This would be consistent with similar hypothesis developed in sheep (Driancourt et al., 1990; Lopez-Sebastian et al., 1997) and confirmed thereafter (Gonzalez-Bulnes et al., 2004). In most of the goats with double ovulation, one of the ovulatory follicles was the largest growing follicle present in both ovaries at time of first observation. However, a second ovulatory follicle was present on the same day; which is consistent with the concept of co-dominance (Rubianes and Menchaca, 2003) to explain the presence of two large follicles in each wave. However, there were some estrous cycles where the second ovulatory follicles emerged later, grew, and ovulation occurred from them in the presence of the first ovulatory follicle. This finding, confirmed previous descriptions (Ginther and Kot, 1994;
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Gonzalez-Bulnes et al., 2005), and reinforces the idea of a dominance effect similar to that described for ewes, that is weaker and easier to overcome than in heifers. Furthermore, this provides additional evidence for the hypothesis that increases in ovulation rate are not related to greater follicle recruitment, but instead to an extended period of ovulatory follicle recruitment (Ginther and Kot, 1994). In conclusion, the current work does not provide evidence of any significant influence of the genetic selection background on preovulatory follicular dynamics in domestic NeuquenCriollo goats. The ovulatory follicle in monovular goats, and one of the ovulatory follicles in polyovulatory goats, was the largest growing follicle present in both ovaries at the time of luteolysis. The second ovulatory follicle in polyovulatory goats emerged coincidentally with the first one in most of the estrous cycles, but appeared later in some does. This feature, which is inconsistent with the dominance effect, supports the hypothesis that increases in ovulation rate in small ruminants are related to a reduced incidence of follicular atresia and an extended period of ovulatory follicle recruitment.
Acknowledgements The authors thank to Jos´e Mar´ıa Garramu˜no and the staff of the experimental farm of INTA Bariloche, to Marcelo Aba, director of the Laboratory of Endocrinology of the Faculty of Veterinary Sciences of Tandil (Argentine) for radioimmunoassays of progesterone. Financial support was received from FonCyT (National Fund of Science and Technology, Argentine). References Bartlewsky, P.M., Beard, A.P., Cook, S.J., Chandolia, R.K., Nonaramooz, A., Rawlings, N.C., 1999. Ovarian antral follicular dynamics and their relationship with endocrine variables throughout the oestrous cycle in breeds of sheep differing in prolificacy. J. Reprod. Fertil. 115, 111–124. de Castro, T., Rubianes, E., Menchaca, A., Rivero, A., 1999. Ovarian dynamics, serum estradiol and progesterone concentrations during the interovulatory interval in goats. Theriogenology 52, 399–411. Driancourt, M.A., Castonguay, F., Bindon, B.M., Piper, L.R., Quirke, J.F., Hanrahan, J.P., 1990. Ovarian follicular dynamics in lines of sheep (Finn, Merinos) selected on ovulation rate. J. Anim. Sci. 68, 2034–2041. Fortune, J.E., 1994. Ovarian follicular growth and development in mammals. Biol. Reprod. 50, 225–232. Ginther, O.J., Kot, K., 1994. Follicular dynamics during the ovulatory season in goats. Theriogenology 42, 987–1001. Ginther, O.J., Knopf, L., Kastelic, L.P., 1989. Temporal associations among ovarian events in cattle during estrous cycles with two and three follicular waves. J. Reprod. Fertil. 87, 223–230. Gonzalez-Bulnes, A., Osoro, K., Lopez-Sebastian, A., 1999a. Ultrasonic assessment of the ovarian response in eCG-treated goats. Small Rumin. Res. 34, 65–69. Gonzalez-Bulnes, A., Santiago Moreno, J., Gomez-Brunet, A., Inskeep, E.K., Townsend, E.M., Lopez-Sebastian, A., 1999b. Follicular dynamics during the oestrous cycle in dairy goats. Anim. Sci. 68, 547–554. Gonzalez-Bulnes, A., Santiago-Moreno, J., Garcia-Garcia, R.M., del Campo, A., Gomez-Brunet, A., LopezSebastian, A., 2001. Origin of the preovulatory follicle in Mouflon sheep (Ovis gmelini musimon) and effect on the growth of remaining follicles during the follicular phase of oestrous cycle. Anim. Reprod. Sci. 65, 265–272. Gonzalez-Bulnes, A., Souza, C.J.H., Campbell, B.K., Baird, D.T., 2004. Effect of ageing on hormone secretion and follicular dynamics in sheep with and without the Booroola gene (FecB). Endocrinology 145, 2858– 2864.
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