Ovarian dynamics, serum estradiol and progesterone concentrations during the interovulatory interval in goats

ELSEVIER

OVARIAN DYNAMICS, SERUM ESTRADIOL AND PROGESTERONE CONCENTRATIONS DURING THE INTEROVULATORY INTERVAL M GOATS T de Castro,’ E Rubianes, A Menchaca and A River0 Department of Physiology, Faculty of Veterinary, Lasplaces 1550, Montevideo, Received for publication: Accepted:

Uruguay

4 June 1998 1 February 1999

ABSTRACT Ovarian changes determined by daily transrectal ultrasonic scanning, and its correlation with serum progesterone (P4) and estradiol (E2) concentrations were studied in seven cyclic Saanen goats, Estrous cycles were synchronized with 2 injections of a PGF2, analogue 9 d apart. All follicles 22 mm in diameter and CL were measured each day. One goat showed a longer interestrous interval, associated with development of a cystic-luteinized structure. The mean interovulatory interval for the other 6 goats was 20.8f0.4 d. The incidence of goats with 4, 3, and 2 follicular waves was 3, 1 and 2 respectively; follicular waves emerged on Days 0.5f0.6, 7.2fO 7, 10.7M.5 and 13.7fo.8 for Wave I, 2, 3 and the Ovulatory wave, respectively. The largest follicle of Wave 2 was smaller (4.9+0. I mm) than the largest follicles of Wave 3 (6.2rtO. I mm; P10.01) and of the Ovulatory wave (7.0+0.5 mm; P
Science

lnc

Key words: estrogen, progesterone, follicular wave, ultrasound, goat

Acknowledgments We thank W. W Thatcher for critical revision of the manuscript; A. Meikle and the technical staff of the Laboratory of Clinical Chemistry, Uppsala, Sweden, for E2 assay; A. Salina for animal handling; and Universal Lab for Glandinex Supported by CSIC. ‘Correspondence and reprint requests. [email protected] Theriogenology 52:3QQ-411, 0 1999 by Eleevier Science

1999 Inc.

0095691w9Qi$-eee tront PII SOO93-691X(99)00136-7

matter

400 TNTRODUCTION To date reports of ovarian activity during the caprine estrous cycle are limited. Early studies using serial laparoscopies (8) or ovaries from the slaughterhouse (6) could not consistently characterize follicular development during the caprine cycle. It was found that both tertiary (I 5 mm) and preovulatory follicles were present throughout the cycle, with a relatively constant number of both follicle categories observed from the early through the late luteal phases Investigators postulated that the large follicles observed in the mid to late luteal phases were not the follicles which ovulated at the subsequent estrus and that the follicle(s) selected for ovulation developed sometime after Day 17 of the cycle (8). Transrectal ultrasonic imaging provided a means for repeated, direct, noninvasive monitoring and measuring of follicles regardless of their depth within the ovary (15). The wide use of daily ovarian uhrasonography associated with hormonal studies in cattle during the last 10 yr have shown clear wave patterns of growth and regression of large antral follicles (with identified phases of recruitment, selection, dominance and atresia) and have expanded the comprehension of the mechanisms associated with growth and development of large preovulatory follicles (14). Nevertheless, comparable experimental approaches to characterize follicular dynamics during the estrous cycle in goats are scarce (I 3). Endocrine events in peripheral blood during the estrous cycle have been studied in detail in ruminants (goats: 10; cattle: 38; ewes: 9). In cattle, a high correlation between ultrasonic assessment of the corpus luteum (CL) and peripheral progesterone levels has been found (21, 37) However, timctional demise of the CL precedes physical regression by 1 to 2 d (21). Several studies in goats describing progesterone profiles throughout the estrous cycle are available (10. 20), but the correlation between CL assessed by ultrasound, serum progesterone concentrations and follicular dynamics are not available. During the bovine and ovine ovulatory cycle, in addition to the preovulatory peak in plasma estradiol concentration at the onset of estrus, there occurs a second peak of lower magnitude at 4 to 6 d later (4, 16. 18, 40). In goats, results are controversial about this phenomenon Abeyawardene et al. (1) reported a preovulatory peak of estradiol occurring after the onset of estrus and another peak of less magnitude between Days 4 and 7 of the estrous cycle. LeyvaOcariz et al. (23) failed to detect significant changes in estradiol concentrations during the luteal phase Furthermore, the correlation between changes of estradiol concentrations and ovarian dynamics during the caprine estrous cycle has not been evaluated. The aims of the present study were to characterize ovarian changes during the interovulatory interval in goats by daily ultrasonic scanning and to correlate ultrasonic data with serum concentrations of ovarian steroids. MATERIALS

AND METHODS

Animals and Equipment Seven nulliparous Saanen goats (20 month old, weighing 39.7k1.7 kg, mean+SEM) were used during the breeding season (May) at the experimental laboratory of the Department of

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Physiology, Montevideo, Uruguay (35” SL). The animals were fed alfalfa hay and pellets, and water was available ad libitum. The goats were housed outdoors in a sheltered pen (40 m x 40 m). and indoor box stalls (3 m x 3 m) were used for ultrasonic examinations. The study was conducted during the breeding season and included I interovulatory interval for each goat. Estrous cycles were synchronized with 2 injections of a PGFz,, analogue ( I60 pg. Glandinex, Universal Lab, Montevideo, Uruguay) 9 d apart. Estrous behavior was checked twice a day with a vasectomized buck throughout the experimental period. Estrus was defined as the moment when the goat stand to be mounted by the teaser buck The interovulatory interval studied (analyses of data) was the one right after the second PGFl,, injection Ovarian images were obtained with a B-mode scanner equipped with a 7 5 MHz linear array transducer (Scanner 480, Pie Medical, Maastricht, Netherlands). A slightly arched plastic open tube (diameter, 30 mm; length, 40 cm) was fastened to the transducer with duct tape so that the probe could be manipulated externally into the rectum (13) Daily blood samples were obtained by jugular venipuncture into vacuttainer tubes Samples were permitted to clot at room temperature and were centrifuged within 2 h after collection Serum was stored frozen at -20°C until assayed for hormones. Ultrasonic Evaluations Ovarian ultrasonic examinations were carried out daily by the same operator between 0800 and 1000 h This operator had been scanning the same animals for 2 mo prior to the commencement of the study. Goats were restrained in a standing position in a wooden chute designed for that purpose. Fecal pellets were removed digitally, and carbxymethylcellulose gel (SO mL) was introduced with a syringe into the rectum (33). Procedure to locate the ovaries was the same as described by Ginther and Kot (I 3). All follicles observed were recorded, and antral diameters of follicles >2 mm were measured Diameter, position and characteristics of the CL as well as luteal cavities were registered. The location of follicles was sketched relative to each other and to the CL AfIer registering the locations and diameters, the sketch was compared with that of the previous day’s Examinations were also recorded by video tape, one tape per goat (Sony. Tokyo, Japan), for further clarification of data Hormone Analysis Concentrations of progesterone were measured by an lmmuChemr” Progesterone “‘1 RIA kit (ICN Pharmaceuticals, Inc., Diagnostics Division, Cosa Mesa. CA, USA). Sensitivity was 0 I ng/mL, the intrassay and interassay coefficients of variation were IO and 14%, respectively Samples for estradiol-17P were determined in duplicate by a ‘25I RIA, using DPC RIA kits (Estradiol double antibody, KESTRADIOL 17BD, Diagnostics Products Corporation, Los Angeles. CA. USA) previously validated for ovine/bovine serum (26, 38) The detection of the assay was 0 8 pg/mL The intra-assay coefficients of variation were 21% for I I pg/mL and decreased below 10% for concentrations between 3 and 49 pg/mL The inter-assay coefficients

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of variation for 3 control samples were 24% (1.4 pg/mL), 8.2% (11.5 pg/mL) and 9 1% (19 Pg/nU Follicle Data Analysis The total number of follicles 22 mm in diameter were assessed on each day. When an identified follicle was first detected at 4 mm, that follicle was presumed to have been 3 mm on the previous day. The day of emergence of a follicle was the day that a follicle was 3 mm in diameter followed by an increase in diameter to t4 on the following day. Term wave was defined as a group of follicles that gave origin to 1 or more follicles 25 mm in diameter. Day of emergence of the wave was the day of emergence of the largest follicle of that wave, and more than 1 d was allowed for all the follicles of a wave to emerge (13). Duration of growth of a follicle was the time taken by that follicle to grow from 3 mm in diameter (emergence) to its maximum diameter, and growth rate was calculated as (maximum diameter - 3 mm)/duration of growth (33). Inter-wave interval was the time between the emergence of 2 successive waves. Mean diameter of the 2 largest follicles and diameter of the CL were analyzed for each day of the cycle The occurrence of ovulation was detected by collapse of a large follicle (usually greater than 5 mm in diameter). Statistical Analyses Mean diameters of largest follicles, diameters of the CL, mean total number of follicles 22 mm in diameter, and mean daily concentrations of progesterone and estradiol were analyzed by the general linear model (GLM) procedure of the Statistical Analysis System (35) that examined effects of goat and day. Characteristics of growth and regression of the largest and second largest follicles were compared by ANOVA. RESULTS Follicle Dynamics One goat showed a longer interestrous interval associated with development of a cysticluteinized structure. Data from this goat is presented separately. The mean (fSEM) interovulatory interval for the other 6 goats was 20.8~tO.4 d, mean interestrous interval was 20.9M.4 d, and the interval between the onset of estrus and ovulation was 1.4M.2 d. Three goats had 4 follicular waves, 1 goat had 3 waves, and 2 had 2 waves during the interovulatory interval (Figure 1). Some overlapping between follicles of different waves was sometimes found. Data of days of wave emergence and of maximum diameters attained by the largest follicles are shown on Table 1. Growth rates for the largest follicles of the successive waves were not different (0.7ti.l mm/d [n=6]; 0.5ti.l mm/d [r&l; 0.7N.O mm/d [n=3]; and 0.6+0.1 mm/d [n=6], for first. second, third and ovulatory wave follicles, respectively).

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Wave

0

2

4

1

6

Wave3

8

10

12

14

16

O”

18

20

22

Days

Figure 1 Follicular growth (Ov =ovulation)

profiles of goats with 2 (a), 3 (b) and 4 (c) follicular

waves

Table 1 Day of wave emergence (DE) and day of maximum diameter attained by the largest follicle (DM) during the interovulatory interval (Mean k SEM)

Goats with 2 Waves(n=2) 3 Waves(n=li 4 Waves\~l=?j ____-(range)

Wave 1 DE DM I.Sf0.5 6.Oti.O 0 O.O+l

I (-2 to 2)

Anovulatory Waves Wave 2 DE DM

5 53403

7 6.720.9

(5 to 6)

(5 to 8)

Ovulatory Wave Wave 3 DE DM

10 10.3+1.8 (8 14) to

10.7kO.5 ( 10 to 12)

15.7M.7

DE DM 11.5+0 5 21.sio.5 14 15 Ok1.2

19 21.0+0

6

(14 to 17) (13 to 17) (20 to 22)

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404

The largest follicle of Wave 2 was smaller (4.9&O. 1 mm) than the largest follicles of Wave 3 (6.2-tO. 1 mm; PO. 1). The profile of the diameter of the largest follicle recorded each day was bimodal, being minimal on Days 0 and 11 and maximal on Days 6 and -1 [the day before ovulation). The mean diameters of the largest and second hugest Eotlicles for the interovulatory interval are depicted (Figure 2a). The differences in mean diameters of the largest and second largest follictes were significant (P10.01) on Days 3, 15 and 16 post ovulation, and tended to be significant (Pi0 09) on Days 1, 6, 12 and 13 post ovulation. For goats with single ovulations (n=5), retrospective analysis of the growth profiles of the 2 largest folhcles showed that 2 d before ovulation the diameter of rhe ovulatory follicle was larger (P& 01) than first subordinate follicle (Figure 2b).

(b)

(a) 8

fl Larggt Fdllde

a

8 /

-6

-5

-4

-3

-2

-1

0

Days previous to ovulation 0

2

4

6

8

10

12 Davs

14

16

23

4

-2

Figure 2. (a) Mean (GEM) diameters of largest and second largest follicles throughout the interovulatory interval in cyclic Saanen goats. Data were normalized to day of ovulation (Day 0). Differences in size between largest and second largest folhcles a Pi0 01, b. PcO.09 (b) Mean (+SEM) diameter of the ovulatory follicle (0) and the first subordinate follicle (+) relative to day of ovulation (Day 0). An asterisk indicates the first day on which a pair of dominant and subordinate follicles differed significantly (P
405

lhefiogenology

some isolated fluctuations, and augmented sharply in coincidence with progesterone declination (luteolysis) around Day 16. Concentrations of estradiol peaked (16 92.4 pg/mL) 2 d before ovulation (Figure 3 a). The early luteal rise was associated with the growth of the largest follicle of Wave 1 from 3 to 4-5 mm. As the dominant follicle continued to grow, estradiol concentrations fell to basal levels and progesterone concentrations increased (Figure 3 b).

6)

(4 20 I

-

2 0 -7

0

2

4

6

a

10 12 14

"6-4-2

0

21 -6 -4 -2 ‘0’ ‘2 4 6 8 Days

0

Days

Figure 3 (a) Mean (*SEM) diameter of the CL (m), estradiol (e) and progesterone (4) concentrations during the interovulatory interval in Saanen goats (n=6). Data were normalized to the day of ovulation (b) Mean (+ SEM) diameter of the Dominant Follicle of Wave I (0) and mean concentrations of estradiol (0) in cyclic Saanen goats. Data were normalized to the time when the dominant follicle attained its maximum diameter (Day 0). Progesterone and Corpus Luteum The functional CL was detected ultrasonically on Day 3 post ovulation The newly forming CL was less echogenic (more shades of dark gray) than at later stages The mature CL was observed as a gray echogenic structure with marked boundaries that attained a mean maximum diameter of 13.5t0.8 mm between Days 8 and 14 (Figure 3 a). Two goats showed CL with anechoic central luteal cavities (fluid-filled). The presence of these cavities showed no relationship with progesterone concentrations. Luteolysis was observed ultrasonically as a decrease in definition of luteal tissue, mainly of the CL boundaries on Day 16.3~0.3. This occurred coincidently with the first day of a significant decrease in progesterone concentrations (6.6M.5 ng.‘mL vs 3.7+0 6 ng/mL for Days 15 and 17, respectively; P60.01, Figure 3 a). Kegressing CL remained detectable but progressively IOSI definition on the remaining days of the cycle Mean progesterone concentrations are depicted in Figure 3a. When progesterone profiles during the seven normal interovulatory intervals were grouped according to the number of waves

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observed in each goat, 2 different patterns were emerged. In 4 goats that had 4 follicular waves, progesterone concentrations rose continously from Day 1 and peaked on Day 10. In the other 3 goats, a biphasic pattern was observed, with a gradual rise in progesterone concentrations from Day 1 to Day 7 and then a sharp rise around Days 8 to 10. Mean serum progesterone concentrations were higher in the 4-wave goats between Day 5 and Day 10 (5.9H.4 ng/mL) than in the other goats (3.9M.3 ng/mL; P10.01). In all animals, serum concentrations of progesterone started to decline on Day 15 and attained basal levels on Day 19 of the interovulatory interval. One goat showed incomplete estrous behavior after the second PGFza injection, failed to ovulate within the expected time, and developed an ovarian structure characterized by a thick outer wall with a fluid-filled cavity of 8.5 mm in diameter (cyst). Six days after the incomplete estrous behavior another follicle ovulated and formed a normal CL. This ovulation was not associated with estrous behavior. Coincidentally with this late ovulation the original cyst luteinized and resembled the characteristics of a normal CL at ultrasonography. The following interovulatory interval was of 20 d and 4 follicular waves developed (Figure 4).

(4 13 -c E $7 E g

11

Q 5 3 1 -8 -6 -4

-2

0

2

4

6

8

10 12 14 16 18 20

(b)

-8

-6

-4 -2

0

2

4

6

8

10 12 14 16 16 20

Days

Figure 4. Ovarian dynamics (a) and serum estradiol(0) and progesterone (+) concentrations (b) of one goat with an incomplete estrous behavior and formation of a cyst followed by a normal interovulatory interval. The black barr means time of estrus. DISCUSSION The results of daily ultrasonography in this study indicate that the interovulatory interval in goats is characterized by a wave-like pattern of follicular development, This is similar to previous findings inwhich daily ultrasonic scanning was used in other ruminant species (cattle:

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24, 28, 39; sheep: 12, 29; deer: 3) and confirms previous observations reported in goats (13). Ginther and Kot (15) using a similar experimental approach and the same breed, found a predominant wave pattern of 4 waves, emerging, respectively, around Days -1,4, 8 and 13 of the interovulatory interval. In our study, the wave pattern found ranged between 2 and 4 follicular waves, The day of emergence of Wave 1 and the Ovulatory wave for goats that had 4 waves was between the range reported in the previous work (13). However, emergence of Wave 2 (Day 7) and Wave 3 (Day 11) occurred, on average, 3 d later. The interval between emergence of Wave 1 and Wave 2 averaged 7 d, but the following interwave intervals were shorter (Wave 2 to Wave 3: 4 d; Wave 3 to the Ovulatory wave: 4 d). The mean interval between the emergence of the ovulatory follicle and the time of ovulation was 6 d. These different speed of wave turnover could be related to different endocrine enviroments throughout the estrous cycle. Several studies completed in the cow (2, 36) and in the ewe (33) report the effect of plasma progesterone levels on follicular development. In cows progesterone affects dominant follicle development in a dose-dependent manner (2). Higher (supraluteal) levels shortened the interval to the emergence of the following wave (2). The mechanism by which progesterone inhibits follicular growth is through suppression of LH pulse frequency which is critical for continued growth of large follicles (36). Moreover, in the ewe, a negative correlation was found between high progesterone levels and the diameter attained by the largest follicle during the early luteal phase (33). Therefore, luteal progesterone concentrations, by the suppression of LH pulse frequency, may contribute to follicular turnover (7). Hence, the smaller diameter reached by the largest follicle of Wave 2 in our study could be attributed to the negative influence of increased progesterone levels during its growing phase as was demonstrated in cow (11, 39). Collectively, these observations suggest that as the luteal phase progresses. follicular turnover increases, and the interwave intervals are shortened in goats. In cattle it has been described widely that each wave has identified phases of emergence, recruitment, selection, dominance and regression (39). The indicators of follicular dominance are 1) a deviation on growth profiles between the largest and the second largest follicles of a wave. and 2) regression of small follicles in concert with continued growth of the largest follicle (11). According to our results, functional dominance would be present during the follicular phase and during the early luteal phase in the cyclic goat, The ovulatory wave emerges around Day 14, and 3 to 4 d prior to ovulation a deviation between the growth of the selected follicle and the first subordinate follicle was observed. In addition, the diameter profiles of the 2 largest follicles showed a different slope between Days 0 and 6 post ovulation (Figure 2). The small number of goats used and the smaller size differences between follicles of the goat could explain only a statistical tendency for differences between dominant and subordinate follicles in the early luteal phase. Nevertheless, after an initial increase (associated with Wave 1 emergence) the number of small-medium follicles declined and remained low in association with the growth of the largest follicle. These 2 observations support the concept that during the early luteal phase dominance is also operative in goats as was postulated previously (13). Ginther and Kot (13) observed that dominance was more common in Waves l and 4 in the goat, but suggested that this phenomenon was ditTicult to assess in this species because of the common occurrence of 2 dominant follicles per wave Although most of the goats studied were monovulatory, and this condition could permit better documentation of follicular dominance, the above mentioned indicators of dominance can not be firmly documented when an active CL is present, suggesting that dominance is not operative during the mid-late luteal phase.

400

Theriogenology

The estradiol profiles observed in the present work characterized by 2 peaks, one during the follicular phase and the other, a smaller peak, during the early luteal phase agree with a previous report (1). During the luteal phase, the changes in serum estradiol concentration were observed only with development of Wave 1. Early works in the cow (4, 19) and sheep (25) show that estrogens are mainly produced by the dominant follicle of a wave (4, 25). Subordinate follicles contribute with less than 10% of the ovarian estradiol production (4, 25). Our present study shows that during the luteal phase estradiol was produced only by the dominant follicle of Wave 1. However, this secretion was limited to a short period related to the initial and mid growing phase of dominant follicle of Wave 1. During the late growing phase, the production of estradiol was stopped prior to the time when follicle attained its maximum diameter (Figure 6). A similar relationship was documented in the cow (16). Estradiol concentrations were greatest during the growing phase of the first dominant follicle and were significantly reduced when the dominant follicle attained its largest diameter (30). Frequency of LH pulses and mean concentrations of LH were greatest during the growing phase of the first dominant follicle, when secretion of estradiol was greatest and peripheral concentrations of progesterone were cl.0 ng/mL (30). Thereafter, concentrations of LH decreased during the plateau phase in association with the decrease in the frequency of pulses of LH, probably owing to increased peripheral progesterone concentrations, In our work, the dissociation between estradiol levels and the growth profile of the dominant follicle was related with the increase of progesterone levels, a finding also documented in ewes (40). In this species, the secretion of ovarian steroids declined before any significant change in the diameter of the dominant follicle of Wave 1 (40). Moreover. it was found that dominant follicle of Wave 2 did not respond to LH as the dominant follicle of Wave 1 (40). The estrogen output into the ovarian vein was lower after an LH pulse, and this could be interpreted as a loss of aromatase activity, since androgen production (estrogen precursor) was similar from Wave 1 and Wave 2 (41). The loss of aromatase activity represents one of the initial signs of atresia preceding morphological changes (41) and this could explain the deviation between estrogen production and the growth profile of the dominant follicle. The ovarian follicles secreting estradiol at a high rate, and the sudden inhibition of this by the rising concentration of plasma progesterone, probably due in part, to the reduction of plasma LH pulse frequency to a level at which the high level of follicular estradiol secretion is no longer supported (5). The development of a cystic structure observed in 1 goat after an ovulatory failure could be discussed in relation to the short cycles commonly observed in seasonal anestrous goats during the transition to the breeding season (22) or the lowered fertility observed after traditional estrus synchronization treatments (27). The structure was correlated with a ‘Iuteal” pattern of progesterone secretion, indicating that it was at least in part luteinized. A similar observation has been reported recently in the red deer (3). The use of ultrasonography will clarify this phenomenon. since hormonal and/or behavioral approaches alone could not provide consistent interpretations regarding this alteration. Cystic condition has been studied in detail in cattle (17). and several factors have been implicated. Some authors have suggested an inadequate LH release and/or a failure of estrogen positive feedback to induce a LH surge (42). but the status of the dominant follicle when the LH surge is elicited can be also one of the factors implicated (32. 34) For example. an increase in LH receptors is necessary for complete follicular development and ovulation, and LH receptor number increases as follicles grow and decreases as follicles start to undergo atresia (3 1)

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Theriogenology

In conclusion, the ultrasonographic study found that follicular dynamics of cycling goats is characterized by a wave-like pattern, Occurrence of follicular dominance and of estrogen production differ among the follicular waves. The first follicular wave and the ovulatory wave are active producers of estradiol and there large follicles exert dominance on other follicles of the cohort In contrast, waves that emerge in the mid luteal phase were not associated with changes in estradiol concentrations in serum and dominance could not be documented. These different patterns can be related with differences in progesterone concentrations as the waves develop. Increased progesterone concentrations promote follicular turnover, and attenuate growth of large follicles which advances emergence of the successive wave. This effect of progesterone could be exerted via negative feed back on the hypothalamus-pituitary axis controlling LH secretion (36) and/or directly on the dominant follicle controlling gonadotropin action (i.e., affecting gonadotropin thecal and /or granulosa cells receptors; 36). At the time of luteolysis the effect of progesterone is removed, and the large follicle will produce a larger quantity of estradiol and exerts dominance over the subordinate cohort of follicles, REFERENCES 1 Abeyawardene SA, Pope GS. Concentrations of oestradiol 17B in plasma and milk and progesterone in plasma during the oestrous cycle and in early pregnancy in goats. Br Vet J 1990; 146:101-105. 2. 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 1992; 95:627-640. 3 Asher GW, Scott IC, O’Neill KT, Smith .JF, Inskeep EK, Towsend EC. Ultrasonographic monitoring of antral follicle development in red deer (Cervus elauhus). J Reprod Fertil 1997; 111: 91-99 4 Badinga L, Driancourt MA, Savio JD. Wolfenson D, Drost M, de la Sota RL, Thatcher WW Endocrine and ovarian responses associates with the first-wave dominant follicle in cattle Biol Reprod 1992; 47: 871-883. 5. Baird DT, Scaramuzzi RJ. The source of ovarian oestradiol and androstenedione in the sheep during the luteal phase. Acta Endocrinol 1976;83:402-406. 6. Batista M, Gonzalez F, Gracia A. Distribution de la poblacion folicular en ovarios caprinos (Distribution of follicular populations in caprine ovaries). Proc 5” Cong on Anim Sci, ITEA, 1993; 42-44 abstr. 7. Bodensteiner KJ, Wiltbank MC, Bergfelt DR, Ginther OJ. Alterations in follicular estradiol and gonadotropin receptors during development of bovine antral follicles, Theriogenology 1996; 45:499-5 12. 8 Camp JC, Wildt DE, Howard PK, Stuart LD, Chakraborty PK. Ovarian activity during normal and abnormal lenght estrous cycles in the goat, Biol Reprod 1983; 28:673-681, 9. Campbell BK, Mann GE, MC Neilly AS, Baird DT. The pattern of ovarian inhibin , estradiol and androstenedione secretion during the estrous cycle in the ewe. Endocrinology 1990; 127, 227-235. 10 Chemineau P, Gauthier D, Poirier JC, Saumande J Plasma levels of LF, FSH, prolactin, oestradiol 17b and progesterone during natural and induced oestrus in the dairy goat. Theriogenology 1982; 17:3 13-323 11. Ginther OJ, Knopf L, Kastelic JP. Temporal associations among ovarian events during

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bovine oestrous cycles with two and three follicular waves. J Reprod Fertil 1989; 87:223-230 12. Ginther OJ, Kot K, Wiltbank MC. Associations between emergence of follicular waves and fluctuations in FSH concentrations during the estrous cycle in ewes. Theriogenology 1995; 43 :689-703. 13. Ginther OJ, Kot K. Follicular dynamics during the ovulatory season in goats. Theriogenology 1994; 42987-1001. 14. Ginther OJ, Wiltbank MC. Fricke PM, Gibbons nl Kot K. Selection of the dominant follicle in cattle. Minireview. Biol Reprod 1996; 55: 1187-l 194. 15. Grifftn PG, Ginther OJ. Ressearch applications of ultrasonic imaging in reproductive biology. J Anim Sci 1992; 70: 953-972. 16. Guibault LA, Bolamba D, Desaulniers DM and Lussier JG. Follicular and hormonal events associated with the transient increase in estradiol concentrations during the first wave of follicular development in cattle. Theriogenology 1993; 39:228. 17. Hamilton SA, Garverick HA, Keisler DH, Xu ZZ, Loos K, Youngquist RS. Sallfen, BE. Characterization of ovarian follicular cysts and associated endocrine profiles in dairy cows. Biol Reprod 1995; 53: 890-898. 18 Hay MF and Moor RM Functional and structural relationships in the Graatian follicle population of the sheep ovary. J Reprod Fert 1975; 45:583-593. 19 Ireland JJ, Fogwell RL, Oxender WD, Ames K and Cowley JL. Production of estradiol by each ovary during the estrous cycle of cows. J of Anim Sci 1984; 59:764-771. 20. Jones DE, Knifton A. Progesterone concentration in the peripheral plasma of goat during the estrous cycle. Res Vet Sci 1972; 13: 193-195. 2 I. Kastelic JP, Bergfelt DR and Ginther OJ. Relationship between ultrasonic assessment of the corpus luteum and plasma progesterone concentration in heifers. Theriogenology 1990; 33:1269-1278. 22. Lassoued N, Khaldi G, Cognit Y, Chemineau P, Thimonier J. Effet de la progeterone sur le taux d’ovulation et la duree du cycle ovarien induits par effet male chez la brebis Barbarine et la chevre locale tunisienne. Reprod Nutr Dev 1995; 35: 4 15-426. 23. Leyva-Ocaritz H, Munro C, Stabenfeldt GH. Serum LH, FSH, estradiol-170 and progesterone profiles of native and crossbred goats in a tropical semiarid zone of Venezuela during the estrous cycle. Anim Reprod Sci 1995; 39:49-55. 24. Lucy MC, Savio JD, Badinga L, De La Sota RL, Thatcher WW. Factors that affect follicular dynamics in cattle. J Anim Sci 1992; 70:3615-3626. 25 Mann GE, McNeilly AS, Baird DT. Hormone production in vivo and in vitro from follicles at different stages of the estrous cycle in sheep. J Endocrinol 1992; 132: 225-234. 26. Meikle A, Tasende C. Rodniguez M and Garofalo EG. Effects of estradiol and progesterone on the reproductive tract and on uterine sex steroid receptors in female lamb. Theriogenology 1997;48:1105-1113. 27. Moore NW. Eppleston J. The control of oestrous, ovulation and fertility in relation to artificial insemination in the Angora goat. Aust J Agric Res 1979; 30: 965-972. 28. Pierson RA, Ginther OJ. Follicular populations during the estrous cycle in heifers. I. Influence of day. Anim Reprod Sci 1987; 14: 165- 176. 29 Ravindra JP, Rawlings NC, Evans ACO, Adams GP. Ultrasonographic study of ovarian follicular dynamics in ewes during the oestrus cycle. J Reprod Fertil 1994; 101:501-509. 30. Rhodes FM, Fitzpatrick LA, Entwistle KW, Kinder JE Hormone concentrations in the caudal vena cava during the first ovarian follicular wave of the oestrous cycle in heifers, J Reprod Fertil 1995; 104:33-39.

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