Effect of LH or GnRH on the dominant follicle of the first follicular wave in beef heifers

A study was designed to characterise ovarian follicular dynamics in heifers treated with porcine luteinizing hormone (pLH) or gonadotropin releasing hormone (GnRH) on days 3, 6 or 9 (ovulation = day 0), corresponding to the growing, early-static, and late-static phases of the first follicular wave. Following ovulation, 65 beef heifers were assigned, by replicate, to the following seven treatment groups: 25 mg im of pLH on days 3, 6 or 9 (n = 9 per group); 100 microg im of GnRH on days 3, 6 or 9 (n = 9 per group); or controls (no treatment; n = 11). Ovulation occurred within 36 h in 67%, 100% and 67% of heifers treated with pLH and in 89%, 56% and 22% of heifers treated with GnRH on days 3, 6 or 9, respectively (treatment-by-day interaction, P < 0.09). Combined for all treatment days, ovulation rates were 78% and 56% in pLH- and GnRH-treated groups, respectively (P < 0.09). Overall, mean day (+/- SD) of emergence of the second follicular wave in heifers that ovulated was different from that in controls or in heifers that did not ovulate (P < 0.05). Mean (+/- SD) day of emergence of the second wave occurred earlier (day 5.6+/-1.2; P < 0.05) in heifers that ovulated after treatment on day 3 (n = 14) than in controls (day 8.7+/-1.6; n = 11); however, wave emergence in all heifers treated on day 6 (day 8.1+/-0.5; n = 18) did not differ from controls, regardless of whether or not ovulation occurred. In the heifers that ovulated in response to treatment on day 9 (n = 8), the emergence of the second follicular wave was delayed (day 10.9+/-0.4; P < 0.05). The day of emergence of the second wave in the 14 treated heifers that failed to ovulate, irrespective of the day of treatment (day 8.9+/-1.4) did not differ from control heifers. The emergence of the second wave was more synchronous in day 6 heifers (regardless of whether they ovulated) and in day 9 heifers that ovulated compared to control heifers (P < 0.05). Results did not support the hypothesis that the administration of pLH or GnRH at known stages of the follicular wave in cycling heifers would consistently induce ovulation or atresia and, thereby, induce emergence of a new follicular wave at a predictable interval. New wave emergence was induced consistently (1.3 days post-treatment) only in those animals that ovulated in response to treatment. However, 22% of LH-treated heifers and 44% of GnRH-treated heifers failed to ovulate. Treatments did not induce atresia of the dominant follicle or alter the interval to new wave emergence in animals that did not ovulate in response to treatment.     

Follicular and hormonal dynamics during the first follicular wave in heifers

A few days after the first follicular wave emerges as 4-mm follicles, follicular deviation occurs wherein 1 follicle of the wave continues to grow (dominant follicle) while the others regress. The objectives of this study were to characterize follicle growth and associated changes in systemic concentrations of gonadotropins and estradiol at 8-h intervals encompassing the time of follicle deviation. Blood samples from heifers (n = 11) were collected and the ovaries scanned by ultrasound every 8 h from 48 h before to 112 h after the maximal value for the preovulatory LH surge. The follicular wave emerged at 5.8 +/- 5.5 h (mean +/- SEM) after the LH surge, and at this time the future dominant follicle (4.2 +/- 0.8 mm) was larger (P < 0.001) than the future largest subordinate follicle (3.6 +/- 0.1 mm). There was no difference in growth rates between the 2 follicles from emergence to the beginning of the deviation (0.5 mm/8 h for each follicle), indicating that, on average, the future dominant follicle maintained a size advantage over the future subordinate follicle. Deviation occurred when the 2 largest follicles were 8.3 +/- 0.2 and 7.8 +/- 0.2 mm in diameter, at 61.0 +/- 3.7 h after wave emergence. Diameter deviation was manifested between 2 adjacent examinations at 8-h intervals. Mean concentrations of FSH decreased, while mean concentrations of LH increased 24 and 32 h before deviation, respectively, and remained constant (no significant differences) for several 8-h intervals encompassing deviation. In addition to the increase and decrease in circulating estradiol concentrations associated with the preovulatory LH surge, an increase (P < 0.05) occurred between the beginning of deviation and 32 h after deviation. The results supported the hypotheses that deviation occurs rapidly (within 8 h), that elevated systemic LH concentrations are present during deviation, and that deviation is not preceded by an increase in systemic estradiol.     

Hormone concentrations temporally associated with contralateral and ipsilateral relationships between the CL and preovulatory follicle during the third follicular wave in heifers

Concentrations of circulating hormones after Day 14 (Day 0 = ovulation) were determined daily in 87 interovulatory intervals (IOIs) in heifers. The IOIs were grouped into four permutations according to an ipsilateral (Ipsi) or contralateral (Contra) relationship between the CL and the preovulatory follicle and two (2W) or three (3W) follicular waves per IOI. The number of IOIs per group differed (P < 0.005) from equality among the Ipsi-2W (n = 27), Contra-2W (n = 31), Ipsi-3W (n = 9), and Contra-3W (n = 20) groups. A continuous decrease in progesterone (luteolysis) began later (P < 0.05) in the Contra-3W group (Day 18.0 ± 0.4) than in each of the Ipsi-2W (15.4 ± 0.2), Contra-2W (15.6 ± 0.2), and Ipsi-3W (16.2 ± 0.5) groups. Concentrations of LH and estradiol began to increase near the beginning of luteolysis in each group. A minor FSH surge that did not stimulate a major follicular wave developed in about 50% of the IOIs in each group, except that none were detected in the Ipsi-3W group. The minor FSH surge reached a peak about 4 days before ovulation and several days after wave 3 had emerged. The hypothesis that luteolysis begins earliest in two-wave IOIs, intermediate in three-wave IOIs with an ipsilateral CL/follicle relationship, and latest in three-wave IOIs with a contralateral relationship was supported. The hypothesis that a minor FSH surge occurs most frequently in association with three follicular waves was not supported.     

Changes in concentrations of follicular fluid factors during follicle selection in mares

The temporal relationships in the changes in concentrations of follicular fluid factors during follicle selection were characterized in mares. All follicles > or =5 mm were ablated 10 days after ovulation, followed by follicular fluid collection from the three largest follicles (F1, F2, and F3) when F1 of the new wave reached a diameter of 8.0-11.9, 12.0-15.9, 16.0-19.9, 20.0-23.9, 24.0-27.9, or 28.0-31.9 mm (n = 4-8 mares/range). Diameter deviation between F1 and F2 began during the 20.0- to 23.9-mm range, as indicated by a greater difference in diameter between the two follicles at the 24.0- to 27.9-mm range than at the 20.0- to 23.9-mm range. Androstenedione concentrations increased in F1, F2, and F3 between the 16.0- to 19.9- and 20.0- to 23.9-mm ranges. In contrast, estradiol, free insulin-like growth factor (IGF)-1, activin-A, and inhibin-A concentrations increased only in F1 beginning at the 16.0- to 19.9-mm range. As a result, the concentrations of all four factors were higher in F1 than in F2 and F3 at all the later ranges, including the 20.0- to 23.9-mm range (beginning of diameter deviation). Concentrations of progesterone differentially increased in F1, concentrations of androstenedione and IGF-binding protein (IGFBP)-2 increased only in F2 and F3, and concentrations of inhibin-B differentially decreased in F2 and F3 simultaneous with the beginning of deviation. Concentrations of FSH, LH, pro-alphaC inhibin, and total inhibin did not change differentially among follicles. Results indicated that, on a temporal basis, estradiol, free IGF-1, activin-A, and inhibin-A may have played a role in the initiation of follicle deviation. In addition, these four factors as well as progesterone, androstenedione, IGFBP-2, and inhibin-B may have been involved in the subsequent differential development of the follicles.     

The effects of dose and duration of administration of pFSH during the first follicular wave on the ovulation rate of beef heifers

Four experiments were carried out to examine the effects of administration of pFSH (Vetrepharm) from Day 3 of the estrous cycle in conjunction with PG on Day 5 on follicular populations and ovulation rate in heifers. In Experiment 1, 47 heifers were allocated to 1 of 4 treatment groups (n = 11 to 12 per group): a) control, b) 1.5 mg pFSH, c) 2.0 mg pFSH or d) 2.5 mg pFSH until estrus. Heifers assigned to the 3 treatments had a higher ovulation rate than the controls (P < 0.05). In Experiment 2, 45 heifers were allocated to 1 of 5 treatment groups (n = 8 to 10 per group): a) control, b) 1.0 mg pFSH until PG, c) 1.0 mg pFSH until estrus, d) 1.5 mg pFSH until PG or e) 1.5 mg pFSH until estrus. From Day 5, heifers assigned to pFSH treatments had more large follicles than the controls (P < 0.05). There was no effect of treatment on the incidence of twin ovulations. In Experiment 3, 43 heifers were assigned to 1 of 3 groups (n = 11 to 16 per group): a) control, b) 1.0 mg pFSH until estrus or c) 1.5 mg pFSH until estrus. At slaughter, 14 d after administration of PG, the incidence of twin ovulations was 0/11, 7/16 and 8/16 for Groups a, b and c, respectively (P = 0.011). In Experiment 4, pFSH (1.5 mg) was administered to 3 groups during the development of the first dominant follicle: a) growth phase (n = 19); b) static phase (n = 17); and c) decline phase (n = 17). All pFSH-treated heifers had a higher ovulation rate than the controls (P < 0.05); heifers assigned to Group c had a higher ovulation rate than those in Groups a or b (P < 0.05). More heifers assigned to Group c (7/17) superovulated than in the other 2 groups (P < 0.05). In conclusion, administration of 1.0 or 1.5 mg pFSH twice daily beginning at Day 3 of the estrous cycle in association with the induction of luteolysis increased the ovulation rate significantly when pFSH treatment was continued to onset of estrus. The ovulation rate and the occurrence of multiple ovulations were significantly higher when pFSH was administered at the time that the first dominant follicle was in decline.     

Follicular dynamics and plasma FSH and progesterone concentrations during follicular deviation in the first post-ovulatory wave in Nelore (Bos indicus) heifers

The objective of the present study was to characterize ovarian follicular dynamics and hormone concentrations during follicular deviation in the first wave after ovulation in Nelore (Bos indicus) heifers. Ultrasonographic exams were performed and blood samples were collected every 12h from the day of estrus until 120-144 h after ovulation in seven females. Deviation was defined as the point at which the growth rate of the dominant follicle became greater than the growth rate of the largest subordinate follicle. Deviation occurred approximately 65 h after ovulation. Growth rate of the dominant follicle increased (P<0.05) after deviation, while growth rate of the subordinate follicle decreased (P<0.05). Diameter of the dominant follicle did not differ from the subordinate follicle at deviation (approximately 5.4mm). The dominant follicle (7.6mm) was larger (P<0.05) than the subordinate follicle (5.3mm) 96 h after ovulation or 24h after deviation. Plasma FSH concentrations did not change significantly during the post-ovulatory period. The first significant increase in mean plasma progesterone concentration occurred on the day of follicular deviation. In conclusion, the interval from ovulation to follicular deviation (2.7 days) was similar to that previously reported in B. taurus females, but follicles were smaller. Diameters of the dominant follicle and subordinate follicle did not differ before deviation and deviation was characterized by an increase in dominant follicle and decrease in subordinate follicle growth rate. Variations in FSH concentrations within 12-h intervals were not involved in follicular deviation in Nelore heifers.     

Intraovarian effect of dominant follicle and corpus luteum on number of follicles during a follicular wave in heifers

The intraovarian relationships among dominant follicle (DF), corpus luteum (CL), and number of follicles between Days 0 to 5 (Day 0 = ovulation) in wave 1 (n = 65 waves) and Days 9 to 13 in wave 2 (n = 62) were analyzed in separate experiments in Bos taurus heifers. Ovaries were grouped into intraovarian patterns of DF–CL, DF alone, CL alone, and neither DF nor CL. In wave 1, the pattern frequencies of DF–CL or neither DF nor CL (34% each) were greater (P < 0.0004) than for DF alone or CL alone (16% each). The number of growing follicles ≥5.0 mm, was greater (P < 0.0001) in ovaries with the DF, even when the DF was removed from the tally (P < 0.03). In a factorial analysis of wave 1, there was a positive main effect of DF (3.9 ± 0.2 vs. 2.2 ± 0.2 follicles; P < 0.0001), but the main effect of CL and the interaction of DF and CL were not significant. In a factorial analysis of wave 2, there were more (P < 0.0001) follicles greater than 6 mm in ovaries with a DF when the DF was included and an approaching difference (P < 0.09) when the DF was excluded. The main effect of CL and the interaction of DF and CL were not significant. The hypothesis that both the DF and CL have a positive intraovarian effect on number of follicles in waves 1 and 2 was only partly supported; the DF, but not the CL, had an effect in the factorial analyses. Previous reports in cattle and sheep of a positive intraovarian effect of CL on number of follicles are questionable in that location of the DF was not considered.     

Effect of bovine follicular fluid administration during or after norgestomet treatment on the fate of the proestrus dominant follicle in heifers

The objective was to examine the influence of follicle stimulating hormone (FSH) on the maintenance and ovulation of the proestrus dominant follicle (DF) in cattle. This was investigated by subjecting a proestrus DF, maintained by a single norgestomet (N) implant, to bovine follicular fluid (BFF) injections during N treatment and immediately after its removal. Earlier, we demonstrated that with an insertion of a single N implant the proestrus DF could be maintained for 9 days without affecting its ovulatory capacity. Eighteen cycling Holstein heifers were treated with a N implant at proestrus. The day of implant insertion was designated day 1 of the implant period and the implant was retained for 9 days. Heifers (n = 6 per group) were randomly allocated to receive saline for 4 days from day 5 to day 8 of the implant period and for 4 days from the day of implant removal to day 3 after removal (CONTROL) or BFF from day 5 to day 8 of implant period (BFF-DURING) or BFF from the day of implant removal to day 3 after implant removal (BFF-AFTER). Injections (10 ml) were given i.v. twice daily and the ovaries monitored by ultrasonography daily, throughout and after the implant period. All CONTROL heifers maintained the DF during treatment and ovulated following implant withdrawal. In all BFF-DURING heifers, the BFF injections caused regression of the DF and its disappearance. In three of the BFF-AFTER heifers, BFF injections caused regression of the DF. In the remaining three BFF-AFTER heifers, the DF ovulated. Mean plasma FSH concentrations did not differ (P > 0.05) between the CONTROL and BFF-DURING heifers. However, the mean plasma FSH concentrations were lower in BFF-AFTER heifers compared with CONTROLS (P < 0.05). Mean plasma LH concentrations did not differ among treatment groups (P > 0.05). In summary, BFF treatment caused atresia of the proestrus DF when maintained by N and this was not associated with suppression of circulating FSH. Administration of BFF after implant removal resulted in an equal chance of ovulation or regression of DF. Regression was associated with suppression of FSH and LH.     

Effects of a dominant follicle on ovarian follicular dynamics during the oestrous cycle in heifers

Two hypotheses were tested: (1) a dominant follicle causes regression of its subordinate follicles, and (2) a dominant follicle during its growing phase suppresses the emergence of the next wave. Cyclic heifers were randomly assigned to one of four groups (6 heifers/group): cauterization of the dominant follicle of Wave 1 or sham surgery (control) on Day 3 or Day 5 (day of ovulation = Day 0). Ultrasonic monitoring of individually identified follicles was done once daily throughout the interovulatory interval. The onset of regression (decreasing diameter) of the largest subordinate follicle of Wave 1 was delayed (P less than 0.01) by cauterization of the dominant follicle of Wave 1 on Day 3 compared to controls (mean onset of regression, Days 10.8 +/- 2.1 vs 4.3 +/- 0.4). Cauterization of the dominant follicle of Wave 1 on Days 3 or 5 caused early emergence (P less than 0.01) of Wave 2 when compared to controls (Day-3 groups: Days 5.5 +/- 0.4 vs 9.6 +/- 0.7; Day-5 groups: Days 7.0 +/- 0.3 vs 9.1 +/- 0.4). The results supported the two hypotheses. In addition, cauterization of the dominant follicle of Wave 1 on Days 3 or 5 increased the incidence of 3-wave interovulatory intervals.     

Alterations in follicular fluid steroids and follicular hCG and FSH binding during atresia in hamster

Preovulatory follicles from hamsters treated on proestrus for 1-3 days with phenobarbital sodium exhibited early signs of atresia after 2-3 days of ovulatory delay. A significant increase in follicular fluid progesterone was evident by Day 1 of delay. Concentrations of androstenedione in follicular fluid were unaffected by ovulatory delay. Follicular fluid levels of estradiol in delayed follicles were either higher than proestrous values after 1 day of delay or lower after 2 and 3 days of delay. hCG binding was slightly higher than proestrous controls after ovulatory delay whereas FSH binding was significantly lower than controls after 2 and 3 days of ovulatory delay. These results indicate that in the barbiturate-treated hamster the elevated follicular fluid levels of progesterone precede by 1-2 days the previously reported increase in steroidogenic capability of delayed follicles to produce progesterone in vitro; this correlated with an increase in the ratio of hCG:FSH binding and this was mostly due to a decrease in FSH binding to whole follicles.     

Effect of dominant follicle persistence on follicular fluid oestradiol and inhibin and on oocyte maturation in heifers

The aim of the present study was to characterize in detail the cytoplasmic and nuclear morphology of cattle oocytes recovered from follicles that are dominant for more than 9 days (with low fertility after ovulation), and to relate morphological changes to intrafollicular markers of follicle health. Beef heifers received prostaglandin F2 alpha and a synthetic progestagen (3 mg Norgestomet) for 2 or 10 days on the first day of dominance of the second dominant follicle (DF2) of the oestrous cycle, to give a 4 day (n = 19; N2) or 12 day (n = 21; N10) duration of dominance of the dominant follicle at ovariectomy 18 h after implant removal and before the predicted gonadotrophin surge. Ultrasound scanning determined emergence of a new wave of follicles in five N10 heifers the day before (n = 1) or day of ovariectomy (n = 4) (N10-NonDom). Dominant follicles from the remaining N10 heifers (N10-Dom) were larger (P < 0.05) on the day of ovariectomy (17.8 +/- 0.6 mm) than those from N2 heifers (13.6 +/- 0.4 mm). The oestradiol:progesterone ratio of follicular fluid from N10-Dom heifers was reduced (21.7 +/- 3.1 versus 34.1 +/- 4.4; P < 0.05), while inhibin A (as measured by immunoradiometric assay) was increased (12.7 +/- 1.0 versus 9.0 +/- 0.7 micrograms ml-1; P < 0.05) compared with N2 heifers. Eleven of twelve N2 oocytes demonstrated nuclear activation without germinal vesicle breakdown, while seven of eight N10-Dom oocytes had undergone germinal vesicle breakdown and had progressed to metaphase I (6/8) or II (1/8). In contrast to N2 oocytes, N10-Dom oocytes showed a larger perivitelline space containing more cumulus cell process endings, vacuoles, irregular vesicles, and more mitochrondia and lipid droplets throughout the ooplasm, yet the degree of cumulus cell expansion and atresia was similar. Thus, final oocyte maturation leading to metaphase I is initiated in most dominant follicles with a dominance period of > 9 days before the gonadotrophin surge and is associated with a reduction in dominant follicle health. However, ovulatory ability is maintained and will lead to the ovulation of aged oocytes, markedly reducing subsequent pregnancy rates.     

Alterations in intrafollicular regulatory factors and apoptosis during selection of follicles in the first follicular wave of the bovine estrous cycle

Changes in follicular fluid (FF) concentrations of estradiol, inhibin forms, and insulin-like growth factor binding proteins (IGFBPs), percentage of apoptotic granulosa cells (%A), and follicular size for individual follicles in a growing cohort were determined throughout the first wave of follicular development during the bovine estrous cycle and related to FSH decline. Four groups of heifers (n = 31) were ovariectomized between Days 1.5 and 4.5 of the estrous cycle at 5 +/- 1, 33 +/- 2, 53 +/- 1, and 84 +/- 2 h after the periovulatory peak in FSH concentrations. Follicles > or = 2.5 mm were dissected, measured, and FF aspirated. The five largest follicles were ranked based on their diameter (F1 to F5). Diameters of F1 to F5 were positively correlated with interval from FSH peak (r > or = 0.6, P < 0.05). Five hours after the FSH peak, follicular diameter and FF concentrations of estradiol, inhibins, and IGFBPs were similar for F1 to F5. From 5 to 33 h, amounts of the six precursor inhibin forms (> or = 48 kDa) increased (P < 0.05) in F1 follicles. The IGFBPs in F1 follicles remained low at all time periods. At 33 h, amounts of IGFBP-4 and -5 were higher (P < 0.05) in F4 and F5 compared with F1 follicles. At 84 h, IGFBP-2, -4, and -5 were increased (P < 0.05) in F3, F4, and F5 compared with F1. At 5, 33, or 53 h, %A was not different between follicles in any size class. At 84 h %A was increased (P < 0.05) in follicles <6 mm in diameter. However, at that time, %A did not differ between the selected DF and the largest subordinate follicle. For individual heifers, the selected DF at 84 h was largest in size, highest in estradiol, and lowest in IGFBP-2 and -4. The F1 follicle had highest estradiol in 23 of 27 heifers irrespective of stage of the wave and lowest IGFBP-4 in 19 of 21 heifers from 33 h. We concluded that the earliest intrafollicular changes that differentiate a dominant-like follicle from the growing cohort are enhanced capacity to produce estradiol and maintenance of low levels of IGFBPs.     

Blood flow to follicles and CL during development of the periovulatory follicular wave in heifers

The hemodynamics of the developing CL and the future dominant follicle (DF) was studied in 22 heifers during wave 1 on Days 0 to 5 (Day 0 = ovulation). Color-Doppler ultrasonography was used to determine the resistance index (RI) at the most prominent Doppler signal in an ovarian arterial branch before entry into the ovary; a decrease in RI indicates a downstream increase in vascular perfusion. The RI for each of four intraovarian patterns averaged over days was different (P < 0.05) from each of the other patterns as follows: DF–CL (DF and CL in the same ovary), 0.52 ± 0.02; CL alone, 0.60 ± 0.01; DF alone, 0.67 ± 0.01; neither DF nor CL, 0.78 ± 0.01. The differences in RI among intraovarian patterns began on Day 0 or 1, indicating that the extent of vascular perfusion on Days 0 to 5 for the various patterns may have been influenced by events that occurred before ovulation. The percentage of the DF wall with color-flow signals was greater (P < 0.05) in the DF–CL pattern than in the DF pattern on each of Days 2 to 5 and was greater (P < 0.0001) in the DF–CL pattern when the DF was adjacent to the CL (40.2 ± 2.0%) than when separated (24.5 ± 1.9%). Dimensions of DF (P < 0.01) and CL (P < 0.02) were greater when adjacent to each other. The results supported the hypotheses for wave 1 that (1) vascular perfusion is greater for the DF–CL intraovarian pattern than for the DF or CL pattern and (2) the extent of blood-flow Doppler signals in the wall of the developing DF is greater for the DF–CL pattern than for the DF pattern. Our preferred interpretation is that a change in vascular perfusion of the CL is accompanied by a similar change in perfusion of the DF when the two structures are in the same ovary especially adjacent.     

Relationship between plasma beta-carotene concentrations during the peripartum period and ovulation in the first follicular wave postpartum in dairy cows

Beta-carotene functions independently of vitamin A in the reproductive performance of dairy cows. The concentrations of beta-carotene in plasma decrease during the dry period, and reach a nadir in about the first week postpartum. This coincides with a negative energy balance, which affects the onset of the first ovulation in early postpartum cows. Thus, we hypothesised that plasma beta-carotene concentrations during the peripartum period may affect ovulation in the first follicular wave postpartum in dairy cows. The aim of the present study was to investigate changes in the profiles of plasma beta-carotene concentrations during the peripartum period in ovulatory and anovulatory cows during the first follicular wave postpartum. We used 22 multiparous Holstein cows, which were fed a total mixed ration consisting of grass, corn silage and concentrate, and collected blood samples for beta-carotene and progesterone analysis from week 3 prepartum to week 3 postpartum when the period of day 0-6 after parturition was regarded as the parturient week (week 0). The first ovulation was confirmed using the profile of plasma progesterone concentrations and colour Doppler ultrasound. Thirteen cows ovulated during the first postpartum follicular wave. Parity, the dry-off period, calving interval, mastitis episodes, and actual 305 days' milk yield during the previous lactation, and milk composition in the last month during the previous lactation in this study did not differ between ovulatory and anovulatory cows. Differences in the plasma beta-carotene profile were observed between ovulatory and anovulatory cows. Plasma beta-carotene concentrations at week 3 prepartum were greater in ovulatory cows (2.97+/-0.24 mg/L) than in anovulatory cows (1.53+/-0.14 mg/L; P<0.001), after that its concentrations in ovulatory cows decreased and reached the lowest level at week 1 postpartum, although its concentrations in anovulatory cows remained unchanged. No differences in plasma beta-carotene concentrations between the two groups were observed postpartum. The present study indicates for the first time that the lower beta-carotene concentrations in plasma during the prepartum period is associated with anovulation during the first follicular wave postpartum.     

Selection of a dominant follicle and suppression of follicular growth in heifers

The following aspects of follicle-stimulating hormone (FSH)-follicular relationships were studied in heifers: (1) the role of the decline in circulating levels of FSH in selection of a dominant follicle of a follicular wave; (2) the relationship of an FSH nadir (low levels between surges) to the absence of development of new follicles of a detectable diameter during the interim between the emergence of successive waves. A recombinant DNA-derived bovine FSH was used. Administration of bovine follicle-stimulating hormone (bFSH) for two days before the time of selection of the dominant follicle of the first post-ovulatory follicular wave delayed the time of divergence of the follicles into dominant and subordinates (first significant divergence: bFSH treatment before selection, Day 4.0; bFSH treatment after selection, Day 2.5; controls, Day 2.5: ovulation, Day 0). Significantly greater growth of the first and second largest subordinates occurred in the pre-selection group. A superovulatory dose of bFSH for 4 days with PGF₂-induction of luteolysis resulted in multiple ovulations when begun on Day 1 (before the expected time of follicle divergence; mean 2.8 ovulations per heifer) than when begun on Day 5 (after divergence; mean 1.0 ovulation per heifer). Administration of bFSH during the expected time (Days 5 and 6) of an FSH nadir did not alter the day of detectable emergence of the next follicular wave. Results supported the following hypotheses: (1) a decline in the wave-stimulating FSH surge is an integral component of the selection mechanism that results in the divergence into dominant and subordinate follicles; (2) the nadir between FSH surges does not account directly for the absence of the development of new follicles between the emergence of waves.     

Ovarian follicular fluid eicosanoid concentrations during the pre-ovulatory period in humans

Prostaglandins are involved in ovulation and in every mammal studied so far, ovulation has been inhibited by prostaglandin inhibition. Information regarding the role of leukotrienes and thromboxanes in ovulation is more limited. In order to study the production of eicosanoids in human pre-ovulatory follicular fluid, follicular aspiration was timed by means of serial ultrasound scans and human chorionic gonadotrophin (hCG) to be immediately pre-ovulatory. 11 women were studied and the eicosanoids measured by radioimmunoassay (RIA). The follicular fluid was found to contain leukotrienes (LT) B4, LTC4 (D4, E4), prostaglandin (PG) E2, PGF2 alpha 6 keto PGF1 alpha k and thromboxane (TX) B2. This is the first published report of leukotrienes in human follicular fluid in spontaneous cycles, and is one of the few reports showing prostaglandins and thromboxanes. The significance of demonstrating leukotrienes in human follicular fluid is discussed as is the correlation between individual eicosanoids in the human ovary.     

Minor FSH surge,minor follicular wave,and resurgence of preovulatory follicle several days before ovulation in heifers

Blood samples were collected and follicle diameters were determined daily beginning on Day 12 (Day 0 = ovulation) in 35 interovulatory intervals (IOIs) in heifers. A minor follicular wave with maximal diameter (6.0 ± 0.3 mm) on Day −4 was detected in six of seven IOIs that were scanned for follicles 4 mm or greater. The number of IOIs with a CV-identified minor FSH surge toward the end of the IOI was greater (P < 0.03) in two-wave IOIs (10/17) than in three-wave IOIs (4/18). The 17 two-wave IOIs were used for study of the temporal relationships among preovulatory follicle, FSH, LH, and estradiol. Daily growth rate of the preovulatory follicle was maximum on Days −11 to −7, minimum (P < 0.05) on Days −7 to −4, and increased (resurged, P < 0.05) on Days −4 to −3. A transient increase in FSH was maximum on mean Day −4, and the peak of a minor FSH surge occurred on Day −4.5 ± 0.2. Concentration of LH and estradiol increased between Days −5 and −4. Results demonstrated resurgence of the preovulatory follicle apparently for the first time in any species. Resurgence seemed more related temporally to the minor FSH surge than to the LH increase, but further study is needed. Results supported the novel hypotheses that a minor FSH surge near the end of the IOI is temporally associated with (1) the emergence of a minor follicular wave and (2) the resurgence in growth rate of the preovulatory follicle.     

Alterations in follicular fluid estradiol, progesterone and insulin concentrations during ovarian acyclicity in water buffalo (Bubalus bubalis)

Ovarian acyclicity is one of the most important causes of infertility in water buffalo. Recent studies have indicated alterations in the composition of follicular fluid during the condition. The aim of this study was to determine the changes in follicular fluid concentrations of estradiol, progesterone and insulin during ovarian acyclicity in water buffalo. Ovaries were collected from 50 acyclic and 95 cyclic (control) buffaloes and follicular fluid was aspirated from small (5.0-6.9 mm), medium (7.0-9.9 mm) and large (≥10.0 mm) sized follicles. Estradiol concentration was lower (P<0.0001) in acyclic (1.4 ± 0.09 ng/ml) than in cyclic (3.3 ± 0.18 ng/ml) buffaloes. Regardless of the ovarian cyclic status, there was an increase (P<0.01) in estradiol concentration with the increase in follicle size; the mean concentrations were 2.4 ± 0.16 ng/ml, 2.8 ± 0.29 ng/ml and 3.5 ± 0.41 ng/ml in small, medium and large follicles, respectively. A higher (P<0.001) progesterone concentration was recorded in acyclic (24.3 ± 2.61 ng/ml) compared to the cyclic (7.6 ± 0.79 ng/ml) group. Furthermore, acyclic buffaloes had a lower (P<0.05) concentration of insulin in the follicular fluid than that of cyclic buffaloes (15.2 ± 1.55 μIU/ml versus 25.9 ± 2.78 μIU/ml, respectively). In conclusion, acyclic buffaloes have lower concentrations of estradiol and insulin concurrent with higher concentrations of progesterone in the follicular fluid. These hormonal changes in the follicular microenvironment are possibly a manifestation of the disturbances in the normal follicular development leading to anovulation and anestrus in acyclic buffaloes.     

Acute fasting in heifers as a model for assessing the relationship between plasma and follicular fluid NEFA concentrations

It is known from epidemiological studies that negative energy balance in early lactating dairy cows is related to a depression in reproductive performance. Elevated plasma concentrations of non-esterified fatty acids (NEFA) are a typical metabolic characteristic of these animals and are proposed as the possible link. The suggestion is that NEFA might have a direct effect on the ovary, by affecting development of the oocyte or the granulosa cells. However, no data is available concerning the relationship between the concentration of NEFA in follicular fluid and plasma. Therefore, a cross-over study with 10 heifers around 15 months of age was conducted to analyze this relationship and examine the suggested effects of the negative energy balance on follicular growth. Investigation of these effects was performed on fasted heifers. The experimental treatment consisted of feeding hay with a subsequent period of fasting, to induce elevated plasma NEFA concentrations. Sampling of follicular fluid was performed using transvaginal aspiration of follicles, which were standardized using a synchronization protocol. In addition, concentrations of glucose, insulin, NEFA, and estradiol were measured in plasma. Follicular estradiol and progesterone concentrations were also measured to assess the quality of the dominant follicle. Fasting resulted in significantly lower plasma glucose (P=0.0006) and plasma insulin (P<0.0001) concentrations, higher plasma estradiol (P=0.008) and higher NEFA (P<0.001) concentrations, and smaller follicles (P=0.04) with lower estradiol:progesterone (E/P) ratios (P=0.05). Concentrations of NEFA in follicular fluid and plasma were closely related. Given this close relationship, we concluded that the presence of high plasma NEFA concentrations might link energy metabolism in early lactation with fertility.     

Plasma inhibin A in heifers: relationship with follicle dynamics, gonadotropins, and steroids during the estrous cycle and after treatment with bovine follicular fluid

The relationship between follicle growth and plasma inhibin A, FSH, LH, estradiol (E), and progesterone was investigated during the normal bovine estrous cycle and after treatment with steroid-free bovine follicular fluid (bFF) to arrest follicle development. In the first study, four heifers were monitored over three prostaglandin (PG)-synchronized cycles. Blood was collected every 2-8 h, and ovaries were examined daily by ultrasonography. Inhibin A was measured using a modified enzyme-linked immunosorbent assay that employed a new monoclonal antibody against the alpha subunit of bovine inhibin. Plasma inhibin A ( approximately 50 pg/ml before luteolysis) rose steadily during the induced follicular phase (P < 0.05) to a peak ( approximately 125 pg/ml) coincident with the preovulatory E/LH/FSH surge. After ovulation, inhibin A fell sharply (P < 0.05) to a nadir ( approximately 55 pg/ml) coincident with the secondary FSH rise. During the next 3 days, inhibin A increased to approximately 90 pg/ml in association with growth of the new dominant follicle (DF). Plasma E also rose twofold during this period, whereas FSH fell by approximately 50%. Inhibin A was negatively correlated with FSH (r = -0.37, P < 0.001) and positively correlated with E (r = 0.49, P < 0.0001). Observations on eight cycles (two cycles/heifer), in which growth of the ovulatory DF was monitored from emergence to ovulation, showed that the first-wave DF (DF1) ovulated in three cycles and the second-wave DF (DF2) in five cycles. After PG, plasma inhibin A and E increased similarly in both groups, with concomitant falls in FSH. In the former group, the restricted ability of DF1 to secrete both inhibin A and E was restored after luteolysis. Results indicate that dynamic changes in the secretion of both E and inhibin A from the DF contribute to the fall in FSH during the follicular phase and to the generation and termination of the secondary FSH surge, both of which play a key role in follicle selection. In the second study, bFF (two dose levels) was administered to heifers (n = 3-4) for 60 h starting from the time of DF1 emergence. Both doses suppressed FSH (P < 0.05) and blocked DF1 growth to the same extent (P < 0.01), although inhibin A levels were only marginally raised by the lower dose (not significant compared to controls). The high bFF dose raised (P < 0.001) inhibin A to supraphysiological levels ( approximately 1 ng/ml). A large "rebound" rise in FSH occurred within 1 day of stopping both treatments, even though the inhibin A level in the high-dose bFF group was still approximately threefold higher than that in controls. This indicates that desensitization of gonadotropes to inhibin negative feedback is a contributory factor, together with reduced ovarian output of E, in generation of the post-bFF rebound in FSH.