Gonadotrophin responsiveness, aromatase activity and insulin-like growth factor binding protein content of bovine ovarian follicles during the first follicular wave

The aim of this study was to examine the function of granulosa cells and hormone concentrations in follicular fluid in bovine ovarian follicles during selection of the first dominant follicle. Ovaries were obtained from beef heifers on days 1-5 after ovulation: follicles > 4 mm in diameter were dissected and follicular fluid and granulosa cells were collected from individual follicles. Oestradiol production by granulosa cells after culture with testosterone was used to determine aromatase activity and responsiveness to gonadotrophins was determined by cAMP production after culture with FSH or LH. Concentrations of oestradiol, progesterone and insulin-like growth factor binding proteins (IGFBPs)-4 and -5 were measured in follicular fluid. Follicles were classified as largest or smaller (days 1 and 2), or dominant or subordinate (days 3-5). Aromatase activity was greater in granulosa cells from the largest follicle than in granulosa cells from smaller follicles on days 1, 3, 4 and 5 (P < 0.05). Responsiveness to LH was not detected in granulosa cells on day 1, but from day 2 to day 5 cells from the largest follicle were significantly more responsive than cells from smaller follicles (P < 0.05). Responsiveness to FSH was detected in granulosa cells from all follicles from day 1 onwards and did not differ between cells from the largest follicle or smaller follicles on any day. Follicular fluid concentrations of oestradiol and the ratio of oestradiol:progesterone were greater and concentrations of IGFBP-4 and -5 were lower in the largest follicle than in smaller follicles from day 2 to day 5 (P < 0.05). In conclusion, selection of the dominant follicle is associated with increased granulosa cell aromatase activity followed by increased cAMP response to LH and follicular fluid oestradiol concentrations, and decreased follicular fluid concentrations of IGFBP-4 and -5 within 2 days after ovulation.     

Follicular waves and circulating concentrations of gonadotrophins,inhibin and oestradiol during the anovulatory season in mares

Follicular waves and associated circulating hormone concentrations were studied during the anovulatory season in pony mares (n=8). Follicles were monitored by ultrasonography and a blood sample was taken daily from 29 January until ovulation (mean, 28 April). A mid-anovulatory period (largest follicle, 16.0+/-0.5 mm in diameter) and transitional period (largest follicle, 22.4+/-0.5 mm) were distinctive in each mare. The two periods were delineated by an increase in the diameter of the largest follicle to >/=21.0 mm. Follicular waves, identified by significant increases in the mean diameter of the second to sixth largest follicles, were detected during both the mid-anovulatory and transitional periods. The mean number of follicles >/=15.0 mm in diameter and the diameter of the second to sixth largest follicles increased in association with statistically identified FSH surges. The pattern of the FSH concentration changes during surges did not change during the mid-anovulatory and transitional periods. During the declining portion of the FSH surge, follicle growth continued and circulating total inhibin increased, indicating suppression of FSH by inhibin from the growing follicles. Circulating oestradiol or LH did not change relative to wave emergence. Results indicated that follicular waves occurred during the second-half of the anovulatory season, even during the period of lowest follicular activity. On a temporal basis, follicular wave emergence was stimulated by surges in circulating FSH. However, the increase in follicle growth to >/=21.0 mm in diameter for the wave at the beginning of the transitional period and for the subsequent waves was not attributable to a change in the characteristics of the associated FSH surges.     

Hormonal mechanism of follicle deviation as indicated by major versus minor follicular waves during the transition into the anovulatory season in mares

Follicle growth and circulating hormone concentrations were compared between an interovulatory interval and the first 60 days of the anovulatory season in pony mares. Daily observations were made from November of three groups: (i) ablation of follicles of >/=6 mm in diameter at day 10 after an ovulation that initiated an interovulatory interval, as determined retrospectively (ovulatory group, n=8), (ii) ablation at day 10 after the last ovulation of the year (anovulatory-10 group, n=6); and (iii) ablation at day 60 after the last ovulation of the year (anovulatory-60 group, n=6). Follicular waves were defined as major (dominant follicle) and minor (no dominant follicle). The percentage of mares with major waves after ablation for the ovulatory, anovulatory-10 and anovulatory-60 groups was 100, 33 and 0%, respectively, and the percentage with minor waves was 0, 67 and 100%, respectively. Minor waves were also detected in 83% of anovulatory mares between day 20 and day 60. Growth of the largest follicle was similar for major waves and minor waves but only until the beginning of deviation in the major waves. FSH surges after ablation were similar for all groups and for surges detected during days 20-60. Concentrations of LH were greater in association with major waves than with minor waves. Both diameter of the largest follicle and LH concentrations for minor waves were greater after ablation at day 10 after the last ovulation of the year than after ablation at day 60. The results of this study indicate that major follicular waves developed in some mares early in the anovulatory season and that minor waves developed throughout the first 2 months. Despite similarities in the wave-stimulating FSH surge, differences in follicle growth occurred and were attributable, on a temporal basis, to differences in LH concentrations. A minor wave developed into a major wave when the largest follicle reached a diameter characteristic of the beginning of deviation in the presence of an adequate LH stimulus for continued growth of a dominant follicle.     

Effect of number and diameter of follicles on plasma concentrations of inhibin and FSH in mares

The role of the number of follicles and circulating immunoreactive inhibin in the decrease in plasma FSH concentrations that occurs during development of a follicular wave was studied in mares. All follicles > or = 6 mm in diameter were ablated by ultrasound-guided transvaginal aspiration of follicular fluid on day 10 after ovulation. During the subsequent wave, all follicles, the three largest follicles (three follicle group), the largest follicle (single follicle group) or no follicles were retained and the remaining follicles were ablated before they reached > 10 mm in diameter (n = 10-11 mares per group). Ablation of new follicles was continued until the day on which the largest follicle of the new wave reached 25 mm in diameter (day 18 after ovulation in the 'no follicle' group). Diameters of retained follicles were measured once a day by transrectal ultrasonography. Plasma samples were taken once a day and analysed by radioimmunoassay for concentrations of FSH and immunoreactive inhibin (includes dimeric inhibin as well as free alpha-subunit forms). Data were normalized to the day of the expected start of the decrease in plasma FSH concentrations (day 0: largest follicle 13 mm in diameter in the follicle-retained groups). A simultaneous increase in circulating concentrations of FSH (P < 0.05) and immunoreactive inhibin (P < 0.05) occurred before the largest follicle reached 13 mm in diameter, which indicates that immunoreactive inhibin produced by follicles < 13 mm in diameter did not suppress FSH. Plasma concentrations of FSH decreased (P < 0.05) and immunoreactive inhibin concentrations increased (P < 0.05) after day 0 in the follicle-retained groups. A slower decrease in FSH concentrations was associated temporally with a delay in the increase in immunoreactive inhibin concentrations in the 'single follicle' group relative to the 'three follicle' and 'all follicle' groups. All follicle-retained groups had similar plasma concentrations of FSH and immunoreactive inhibin after the expected beginning of deviation in growth rates between the two largest follicles (largest follicle 22-23 mm in diameter). These results indicated that the decrease in plasma FSH concentrations from the start of the decrease until the expected day of deviation was a function of multiple follicles of a wave and was attributable to the secretion of inhibin. Thereafter, the largest follicle alone accounted for the continued FSH suppression.     

Development and validation of a short-term, serum-free culture system for bovine granulosa cells: evaluation of the effects of somatotropin and growth hormone-releasing factor on estradiol production

The objective of this study was to develop and validate a short-term, serum-free culture system to determine whether recombinant bovine somatotropin (rbST) or recombinant bovine growth hormone-releasing factor (rbGRF) altered the estradiol-producing capacity of bovine granulosa cells isolated from dominant or subordinate follicles of the first follicular wave. Thus, ovaries were obtained at an abattoir from cows that were between d 2 to 5 or 6 to 10 of the estrous cycle. Three size classes of follicles were isolated from each cow's ovaries: small (2 to 5 mm in diameter), medium (6 to 14 mm), or the largest (6 to 19 mm). In vivo steroid-producing capacity of follicles was assessed by measuring concentration of estradiol, progesterone, androstenedione and 5alpha-dihydrotestosterone in each follicle. In vitro steroid-producing capacity was assessed by culturing granulosa cells from the different follicle sizes for 48 h in serum-free media with 19-OH androstenedione and measuring the estradiol and progesterone concentrations in media at the end of culture. The effect of different doses of FSH, rbST, or rbGRF on estradiol and progesterone production by granulosa cells from each follicle size class during d 2 to 5 or 6 to 10 was also evaluated. A high percentage (91.7%) of the largest follicles obtained on d 2 to 5 was estrogen-active (estradiol > progesterone) compared with other follicle classifications (d 2 to 5, small = 0%, medium = 13.8%; d 6 to 10, small = 0%, medium = 3.3%, largest = 33.3%). Estradiol was highest (P < 0.05) in the largest follicle on d 2 to 5 and correlated positively with follicle diameter. The pattern of in vitro production of estradiol by granulosa cells from the different follicle size classes reflected the original in vivo capacity of follicles to produce estradiol. However, only granulosa cells from the largest estrogen-active follicle on d 2 to 5 produced more estradiol than progesterone in vitro. Progesterone production by granulosa cells from all follicle classifications was increased by FSH, but FSH only enhanced estradiol production by granulosa cells from the largest estrogen-active follicles on d 2 to 5. Recombinant bST blocked the FSH-induced increase in estradiol by granulosa cells from the largest estrogen-active follicles on d 2 to 5, whereas rbGRF had no effect on steroid production. Based on these results, we concluded that short-term, serum-free culture of bovine granulosal cells obtained from first-wave follicles at an abattoir could be used to reflect reliably the original in vivo estradiol-producing capacity of granulosal cells, and that neither rbST nor rbGRF enhance basal or FSH-induced estradiol production by bovine granulosa cells from first-wave follicles.     

Effect of elevating luteinizing hormone action using low doses of human chorionic gonadotropin on double ovulation,follicle dynamics,and circulating follicle-stimulating hormone in lactating dairy cows

Double ovulation and twin pregnancy are undesirable traits in dairy cattle. Based on previous physiological observations, we tested the hypothesis that increased LH action [low-dose human chorionic gonadotropin (hCG)] before the expected time of diameter deviation would change circulating FSH concentrations, maximum size of the second largest (F2) and third largest (F3) follicles, and frequency of multiple ovulations in lactating dairy cows with minimal progesterone (P4) concentrations. In replicate 1, multiparous, nonbred lactating Holstein dairy cows (n = 18) had ovulation synchronized. On d 5 after ovulation, all cows had their corpus luteum regressed and were submitted to follicle (≥3 mm) aspiration 24 h later to induce emergence of a new follicular wave. Cows were then randomized to NoP4 (untreated) and NoP4+hCG (100 IU of hCG every 24 h for 4 d after follicle aspiration). Ultrasound evaluations and blood sample collections were performed every 12 h for 7 d after follicle aspiration. All cows were then treated with 200 μg of GnRH to induce ovulation. In replicate 2, cows (n = 16) were resubmitted to similar procedures (i.e., corpus luteum regression, follicle aspiration, randomization, ultrasound evaluations every 12 h, GnRH 7 d after aspiration). However, cows in replicate 2 received an intravaginal P4 device that had been previously used (～18 d). Only cows with single (n = 15) and double (n = 16) ovulations were used in the analysis. No significant differences were detected for frequency of double ovulation, follicle sizes, and FSH concentrations across replicates (NoP4 vs. LowP4 and NoP4+hCG vs. LowP4+hCG), so data were combined. Double ovulation was 40% for control cows with no hCG (CONT) and 62.5% with hCG (hCG). Double ovulation increased as the maximum size of F2 increased: <9.5 mm and 9.5–11.5 mm (7.7%) and ≥11.5 mm (94.1%). The hCG group had more cows with F2 > 11.5 (69%) than with 9.5 ≥ F2 ≤ 11.5 (25%) and F2 < 9.5 (6%). In agreement, F2 and F3 maximum size were larger in the hCG group, but FSH concentrations were lower after F1 > 8.5 mm compared with CONT. In contrast, FSH concentrations were greater before deviation (F1 closest value to 8.5 mm) in cows with double ovulations than in those with single ovulations, regardless of hCG treatment. In addition, time from aspiration to deviation was shorter in cows with double rather than single ovulation and in cows treated with hCG as a result of faster F1, F2, and F3 growth rates before diameter deviation. In conclusion, greater FSH and follicle growth before deviation seems to be a primary driver of greater frequency of double ovulation in lactating cows with low circulating P4. Moreover, the increase in follicle growth before deviation and in the maximum size of F2 during hCG treatment suggests that increased LH may also have a role in stimulating double ovulation.     

Comparison of ovarian function and circulating steroids in estrous cycles of Holstein heifers and lactating cows

Ovarian function was compared between nulliparous heifers (n = 29; 10 to 16 mo old) and lactating Holstein cows (n = 31; 55.9 +/- 3.5 d postpartum). Follicular dynamics, corpus luteum growth, and regression, and serum steroid concentrations were evaluated through ultrasonography and daily blood sampling. Most heifers (27 of 29) but only 14 of 31 cows had typical spontaneous estrous cycles after cycles were initiated. Twelve cows had atypical cycles, and 5 became anovulatory during the study. The 12 cows with atypical estrous cycles had low serum estradiol after luteolysis and failed to ovulate the dominant follicle present at luteolysis. Heifers and cows with typical cycles were compared directly. Interovulatory intervals were similar between heifers (22.0 +/- 0.4 d) and cows (22.9 +/- 0.7 d). Those animals had estrous cycles with either 2 (15 heifers; 11 cows), 3 (9 heifers; 2 cows), or 4 follicular waves (3 heifers; 1 cow). Cows ovulated later after luteolysis than heifers (5.2 +/- 0.2 vs. 4.6 +/- 0.1 d, respectively), and had more multiple ovulations (17.9 vs. 1.9%). Maximal serum estradiol concentration preceding ovulation was lower in cows than in heifers (7.9 +/- 0.8 vs. 11.3 +/- 0.6 pg/mL) even though ovulatory follicles were larger in cows (16.8 +/- 0.5 vs. 14.9 +/- 0.2 mm). Similarly, maximal serum progesterone concentration was lower for cows (5.6 +/- 0.5 vs. 7.3 +/- 0.4 ng/mL), whereas maximal volume of luteal tissue was larger for cows than heifers (11,120 +/- 678 vs. 7303 +/- 308 mm3). Thus, higher incidence of reproductive anomalies in lactating cows, such as low conception rate, ovulation failure, delayed ovulation, and multiple ovulations, may be due to lower circulating steroid concentrations in spite of larger ovulatory follicles and luteal structures.     

Extending the postpartum anovulatory period in dairy cattle with estradiol cypionate

Estradiol cypionate (ECP), a long-acting estrogen, has been used therapeutically in early postpartum (PP) dairy cows. In experiment 1, effects of ECP on circulating reproductive hormones, cyclicity, and ovarian function in early PP dairy cows were investigated. Lactating Holsteins received 10 mg of ECP (ECP; n = 17) or placebo (CON; n = 16) on d 7 PP. Serum and ultrasound data were acquired from 5 to 90 d of lactation. Compared to CON, ECP cows had greater serum estradiol for 10 d and lower serum FSH for 15 d posttreatment. After ECP, the appearance of follicles > or = 10 mm and time to first ovulation were delayed. Nevertheless, by 90 d PP, normal estrous cycles were found in only 50% of CON versus 88% of ECP cows. Primiparous, but not multiparous, cows receiving ECP had higher milk yields. Experiment 2 investigated effects of ECP on reproduction and milk production on a commercial dairy. Cows were blocked by parity and randomized to three treatments: 0 (n = 85), 4 (n = 85), or 10 (n = 86) mg of ECP on d 5 to 8 PP. Data included cycling status (two ultrasound examinations at 30 to 33 d PP and 7 d later), individual reproduction records, and daily milk yields from 10 to 90 d PP. In primiparous cows, ECP had no significant effects on ovulatory status or milk yields. By 40 d PP, a greater percentage of multiparous cows receiving 10 mg of ECP remained anovulatory compared with those receiving 0 or 4 mg. Milk yields were highest for multiparous cows receiving 4 mg of ECP, intermediate for the 10-mg dose, and lowest for controls. Lower conception was observed in multiparous cows receiving 4 mg of ECP. In summary, ECP delayed time to first ovulation particularly in multiparous cows, an effect associated with observed inhibition of circulating FSH. Milk yield responses to ECP were inconsistent within parity groups across the two experiments.     

Akt and Erk signal transduction pathways are early markers of differentiation in dominant and subordinate ovarian follicles in cattle

Dominant follicles are those that continue to develop and have the potential to ovulate while subordinate follicles regress. Characteristics of dominant follicles include a larger diameter, higher intrafollicular estradiol, and lower IGF-binding protein (IGFBP)-4 concentrations compared with other cohort follicles. Follicle development is regulated by endocrine hormones that act via intracellular signaling pathways. Here, we show the differences in Akt, Erk, c-Jun N-terminal protein kinase, and p-38 signaling pathways between dominant and subordinate follicles at the dominance stage of the follicle wave. However, earlier in the follicle wave (dominant follicle selection), there were only differences in the levels of Akt and Erk signal transduction proteins among dominant and subordinate follicles. Using this profile of Akt and Erk protein expression in granulosa and theca cells of selected dominant follicles compared with subordinate follicles, we suggest a predictive model to identify future dominant and subordinate follicles from the pool of otherwise similar cohort follicles at the time of follicle wave emergence. We conclude that the Erk and Akt signal transduction pathways are important for dominant follicle selection and development and, furthermore, that the observed differences in these pathways mark the future dominant follicle from subordinate follicles before differences in follicular diameter, follicular fluid estradiol, and IGFBP-4 concentrations are apparent.     

Influence of uterine bacterial contamination after parturition on ovarian dominant follicle selection and follicle growth and function in cattle

First postpartum dominant follicles are preferentially selected in the ovary contralateral to the previously gravid uterine horn. The aim of the present study was to test the hypothesis that uterine bacterial contamination alters the location of ovarian follicle emergence and selection, and inhibits follicle growth and function. Swabs were collected from the uterine body lumen of cattle on days 7, 14, 21 and 28 after parturition. Bacteria were identified by aerobic and anaerobic culture; bacterial growth was scored semiquantitatively and animals were categorized into standard or high bacterial contamination categories on the basis of the number of colonies detected. Follicular growth and function were monitored by daily transrectal ultrasonography, and estimation of plasma FSH, oestradiol and progesterone concentrations. There was no effect of bacterial contamination on plasma FSH concentration profiles or emergence of the ovarian follicle wave. When uterine bacterial growth scores were high on day 7 or day 21 after parturition, fewer first (1/20 versus 15/50; P < 0.05) or second (1/11 versus 13/32; P < 0.05) dominant follicles were selected in the ipsilateral compared with the contralateral ovary, respectively. The diameter of the first dominant follicle was smaller in animals with a high day 7 bacterial score (P < 0.001), dominant follicle growth was slower (P < 0.05) and oestradiol secretion was decreased (P < 0.05). The present study provides evidence for an effect of the uterus on the ovary after parturition, whereby uterine bacteria have a contemporaneous localized effect on ovarian follicle selection and subsequent growth and function, but not on initial emergence.     

Survival factors regulating ovarian apoptosis -- dependence on follicle differentiation

Only a minute fraction of the ovarian follicles present in a fetal ovary will complete the path to ovulation. Most of the follicles will undergo atresia, a hormonally controlled apoptotic process. Apoptosis occurs at each stage of follicular development and there is a marked reduction in the number of follicles present at birth. Stage-dependent mechanisms of follicle survival can be postulated to achieve co-ordinated development, leading to ovulation of a small fraction of follicles. Indeed, hormone and growth factor regulation of follicular atresia is stage-specific. This short review considers the factors that promote survival of ovarian follicles throughout development, including endocrine, locally produced and intracellular mediators, as exemplified mainly by follicular development in rodents. In primordial follicles, oocyte apoptosis is considered to be the cause of subsequent follicle degeneration. In slow-growing preantral follicles, FSH is not a survival factor, but some locally produced growth factors are. Progression to the antral follicle stage is probably the most critical stage of follicle development in vivo, and FSH is a major survival factor at this stage. In addition, insulin-like growth factor I and interleukin 1beta are potent survival factors for cultured rat follicles at the antral stage. Preovulatory follicles express receptors for LH, and both of the gonadotrophins are survival factors at this stage. Relatively little is known about the period between the LH surge and ovulation; however, it has been suggested that at this stage progesterone acts as a survival factor.     

Follicle selection in cattle and horses: role of intrafollicular factors

The eminent event in follicle selection during a follicular wave in monovular species is diameter deviation, wherein one follicle continues to grow (developing dominant) and other follicles (subordinates) begin to regress. In cattle, the IGF system, oestradiol and LH receptors are involved in the intrafollicular events initiating deviation as indicated by the following: (1) concentrations of free IGF-I and oestradiol in the follicular fluid and number of LH receptors in the follicular wall increase more dramatically in the future dominant follicle than in the future subordinate follicles before the beginning of deviation and (2) ablation of the largest follicle (LF) or injection of recombinant human IGF (rhIGF)-I into the second LF at the expected beginning of deviation increases the concentrations of oestradiol in second LF before the expected beginning of deviation between second LF and third LF. In horses, an increase in free IGF-I, oestradiol, inhibin-A and activin-A is greater in the future dominant follicle than in other follicles before the beginning of deviation. However, free IGF-I is the only one of these four factors needed for the initiation of deviation in horses as indicated by the following: (1) ablation of LF at the expected beginning of deviation increases the concentrations of free IGF-I in second LF before the beginning of deviation between second LF and third LF but does not increase the other factors; (2) injection of rhIGF-I into second LF at the expected beginning of deviation causes second LF to continue to grow and become a codominant follicle and (3) injection of IGF-binding protein-3 into LF at the expected beginning of deviation causes LF to regress and second LF to become dominant. Thus, the dramatic changes in the IGF system in LF compared to other follicles before the beginning of deviation play a crucial role in the events that lead to the beginning of diameter deviation in both cattle and horses. Oestradiol and LH receptors also play a role in cattle. These intrafollicular events prepare the selected follicle for the decreasing availability of FSH and increasing availability of LH. The other follicles of the wave have the same future capability but do not have adequate time to attain a similar preparatory stage.     

The absence of corpus luteum formation alters the endocrine profile and affects follicular development during the first follicular wave in cattle

We previously established a bovine experimental model showing that the corpus luteum (CL) does not appear following aspiration of the preovulatory follicle before the onset of LH surge. Using this model, the present study aimed to determine the profile of follicular development and the endocrinological environment in the absence of CL with variable nadir circulating progesterone (P(4)) concentrations during the oestrous cycle in cattle. Luteolysis was induced in heifers and cows and they were assigned either to have the dominant follicle aspirated (CL-absent) or ovulation induced (CL-present). Ultrasound scanning to observe the diameter of each follicle and blood collection was performed from the day of follicular aspiration or ovulation and continued for 6 days. The CL-absent cattle maintained nadir circulating P(4) throughout the experimental period and showed a similar diameter between the largest and second largest follicle, resulting in co-dominant follicles. Oestradiol (E(2)) concentrations were greater in the CL-absent cows than in the CL-present cows at day -1, day 1 and day 2 from follicular deviation. The CL-absent cows had a higher basal concentration, area under the curve (AUC), pulse amplitude and pulse frequency of LH than the CL-present cows. After follicular deviation, the CL-absent cows showed a greater basal concentration, AUC and pulse amplitude of growth hormone (GH) than the CL-present cows. These results suggest that the absence of CL accompanying nadir circulating P(4) induces an enhancement of LH pulses, which involves the growth of the co-dominant follicles. Our results also suggest that circulating levels of P(4) and E(2) affect pulsatile GH secretion in cattle.     

Interactions of follicular factors and season in the regulation of circulating concentrations of gonadotrophins in mares

Follicle growth and associated changes in circulating hormone concentrations were evaluated after follicle ablation in mares (n = 13) during four 13 day periods beginning at means of -98.5, -61.4, -26.0 and 10 days from the first ovulation and corresponding to mid-anovulatory, early-transitional, late-transitional and ovulatory periods, respectively. During each period, all follicles > 5 mm in diameter were ablated (day 0) followed by no subsequent ablations (all-follicle group) or the ablation of all follicles > 5 mm when a new follicle reached > 10 mm (0-follicle group). A follicular wave emerged at means of days 1.5-2.5 in all mares of the all-follicle group during each period. Follicle activity increased between the mid-anovulatory and the transitional periods, as indicated by increases in the diameter of the largest follicle and the number of follicles in the all-follicle groups; nonetheless, there were no differences in the FSH concentrations of the wave-stimulating surge throughout the study. Circulating total inhibin increased between day 3 and day 6 in the all-follicle groups but not in the 0-follicle groups, and was higher during the late-transitional and ovulatory periods than during earlier periods in the all-follicle groups. In the all-follicle groups, circulating FSH decreased between day 3 and day 6. In the 0-follicle groups, FSH did not decrease and was higher during the late-transitional and ovulatory periods than during the mid-anovulatory period. Circulating LH was higher during the late-transitional and ovulatory periods than during earlier periods and was not different between the all-follicle and 0-follicle groups. On the basis of temporal relationships, it was concluded that the follicles of a wave secreted inhibin during all periods and the follicular inhibin suppressed circulating FSH. An increase in circulating inhibin induced by the growth of follicles > 21 mm in diameter during the late-transitional and ovulatory periods counteracted a stimulatory effect on FSH mediated by season.     

Steroidogenesis in bovine granulosa cells: the effect of short-term changes in dietary intake

The nutritional status of a cow is a key factor in the regulation of both follicle growth and oocyte quality. In this study, the effect of diets designed to increase circulating insulin and insulin-like growth factor I (IGF-I) concentrations on steroid production by granulosa cells in vitro was examined to analyse the mechanisms through which these changes occur. Hereford x Friesian heifers (n = 24) were offered maintenance or twice maintenance diets during the experimental period (17 days). Circulating concentrations of FSH did not differ between the two dietary groups, whereas insulin and IGF-I concentrations showed significant diet x day of oestrous cycle interactions. Ovaries were collected on day 3 of the first follicle wave after synchronization of oestrus. Granulosa cells were isolated from small (1-4 mm) and medium-sized (4-8 mm) follicles and cultured in the presence of long R3-IGF-I or bFSH or both. After 4 days in culture, granulosa cells isolated from small follicles, but not medium-sized follicles, collected from cattle offered the twice maintenance diet secreted significantly higher (P < 0.05) amounts of oestradiol compared with granulosa cells collected from cattle offered the maintenance diet. The effect was apparent in either the presence or absence of FSH and long R3-IGF-I. This nutritional effect on aromatase activity in granulosa cells was not apparent after day 6 of culture. There was no effect of diet on progesterone production by granulosa cells after 4 or 6 days of culture. These results support the hypothesis that dietary-induced changes in circulating insulin and IGF-I concentrations have a direct effect on the steroidogenic potential of bovine granulosa cells from small follicles. The dietary-induced increases in aromatase activity in small follicles combined with the increased concentration of metabolic hormones are possible mechanisms through which short-term changes in nutrition may affect follicle dynamics.     

The FecB (Booroola) gene acts at the ovary: in vivo evidence

The aim of this study was to differentiate between pituitary and ovarian actions of the FecB gene by measuring the ovarian response to a standardized treatment with gonadotrophins designed to mimic the changes in FSH and LH that occur in the follicular phase of the ovarian cycle in ewes, with (Fec(B/-), n=6) and without (Fec(+/+), n=9) the gene, that were rendered hypogonadotrophic by pretreatment with a potent antagonist of GnRH. Ewes with ovarian autotransplants were used to facilitate the assessment of follicular function by the collection of ovarian venous blood and ultrasonography. The gonadotrophin regimen resulted in concentrations of FSH and LH that were similar to concentrations found in a normal cycle and did not differ between genotypes. Follicular development and ovulation occurred in all animals, and patterns of secretion of oestradiol, androstenedione and inhibin A were normal. Despite these endocrine similarities, the antral follicle population stimulated by FSH infusion retained the characteristic genotypic difference with the ovaries of Fec(+/+) animals containing a range of follicle sizes with decreasing proportions of small (<3.5 mm in diameter) and medium (3.5-4.5 mm in diameter) follicles as well as large follicles (> or =4.5 mm in diameter), whereas the ovaries of Fec(B/-) ewes contained no follicles of >4.5 mm in diameter. This genotypic difference was retained after ovulation with gene carriers having more preovulatory follicles/corpora lutea (3.8+/-0.3) of a smaller diameter (5.3+/-0.3 mm) than did non-gene carriers (1.7+/-0.3; 11.4+/-0.9 mm; P<0.05). As ewes carrying the FecB gene mutation were able to ovulate more follicles than non-gene carriers, despite identical concentrations and patterns of FSH and LH stimulation, the results of this study support the hypothesis that the FecB gene acts at the ovary to enhance ovarian sensitivity to gonadotrophic stimulation.     

Analysis of the variations of follicular fluid composition during follicular growth and maturation in the mare using proton nuclear magnetic resonance (1H NMR)

Follicular development and ovulatory processes in mammals involve local biochemical changes as a result of substantial modifications in cellular metabolism, the most well known of which is steroid variation. In the present study, the intrafollicular variation of several other components was studied using proton nuclear magnetic resonance ((1)H NMR). This approach made it possible to demonstrate that the intrafollicular biochemical content changes during follicular growth and maturation. Follicular fluid was aspirated by ovarian puncture of the dominant follicle at various physiological stages of its development: early dominant, late dominant and preovulatory. Serum samples were collected during each puncture session. (1)H NMR was used to evaluate intrafollicular and circulating glycoconjugates (sugar chains and N-acetyl groups), lipoproteins (CH(3) and CH(2) groups), glucose metabolites (trimethylamines, acetate and lactate), amino acids (glutamine/glutamate and alanine), creatine/creatinine and polyamines. Follicular fluids were assayed by radioimmunoassay for oestradiol and progesterone contents. The intrafollicular contents of alanine and lipoproteins (CH(3) groups) decreased in the dominant follicle during growth, whereas concentrations of progesterone and oestradiol increased significantly. After injection of gonadotrophin to induce ovulation, follicular maturation was characterized by a decrease in glycoconjugates (sugar chains), trimethylamines and acetate, a decrease in oestradiol concentration, and a further increase in CH(3) groups of lipoproteins and progesterone. The results from the present study showed a clear correlation between the intrafollicular content of alanine and that of oestradiol. A correlation between progesterone and glycoconjugates (sugar chains) was also observed. Therefore, (1)H NMR was shown to be effective for studying specific changes in the biochemical composition of the follicular fluid that occur during follicular development. For the first time, the variation of several compounds (glycoconjugates, lipoproteins, glucose metabolites, amino acids and polyamines) in relation to growth and maturation was demonstrated. Some of these changes could be of crucial importance for follicular maturation and ovulation as well as for oocyte maturation and further fertilization.     

Endocrine milieu and developmental dynamics of ovarian cysts and persistent follicles in postpartum dairy cows

Ovarian follicular cysts and persistent follicles are follicular pathologies involved in reduced fertility of dairy cows. Two separate experiments were performed on high-yielding Holstein cows to characterize ovarian cyclicity and evaluate the developmental dynamics of follicle pathologies postpartum. In experiment 1, 58 cows were monitored by ultrasonography twice weekly from d 18±1 to 69±2 postpartum. First ovulation occurred 38±3, 27±2, 20±1, and 25±3 d postpartum in cows with 1 cycle (n=11), 2 cycles (n=21), 3 cycles (n=13), and 4 cycles (n=7), respectively. Follicular pathologies were developed in cows that were either acyclic (n=6) or had 1 or 2 cycles, but not in cows with more than 2 cycles. In experiment 2, 47 cows were monitored twice weekly from 10 d postpartum to second ovulation. Follicles ≥17 mm in diameter in 2 consecutive scans were aspirated, and concentrations of various hormones were measured. Cows were defined as cyclic (n=30; 64%) or with the potential to develop follicular pathology (n=17; 36%). Aspirated follicles (n=27) were classified into 3 main groups based on follicular growth rate, follicular diameter, and ovarian activity before and after follicular aspiration. Dominant follicles (n=4) were defined as large follicles (20 mm in diameter) with growth rate ≤1 mm/d and normal ovarian activity. Persistent follicles (n=6) had the same growth rate and diameter as the dominant follicles, but persisted at the same diameter for ≥10 d. Ovarian cysts (n=17) were defined as the largest follicular structures (19 to 32 mm in diameter), with abnormal growth rate (>1 mm/d) and abnormal ovarian activity. Single or turnover cysts did not differ in their growth parameters and were therefore combined and further classified according to follicular-fluid hormone concentrations. Estradiol-dominant cysts (n=7) were characterized by normal estradiol (284 to 659 ng/mL) and progesterone (20 to 113 ng/mL) concentrations, similar to those of the dominant follicle (554 to 993 ng/mL and 44 to 106 ng/mL, respectively). Progesterone-dominant cysts (n=5) were characterized by low estradiol (0.06 to 330 ng/mL) and high progesterone (586 to 3,288 ng/mL) concentrations. Low-steroidogenic active cysts (n=5) were characterized by low concentrations of both estradiol (23 to 61 ng/mL) and progesterone (17 to 205 ng/mL). Characterization of spontaneously forming cysts might enable definition of the formation of ovarian follicular pathologies in postpartum cows.     

Ovarian follicular activity in lactating Holstein cows supplemented with monensin

The objective of this study was to determine effects of monensin on ovarian follicular development and reproductive performance in postpartum dairy cows. Forty-eight multiparous Holstein cows were randomly assigned to receive either a control total mixed ration (n = 24) or the same diet plus 22 mg of monensin/kg (n = 24) from 21 d before anticipated calving until cows were either confirmed pregnant or were >180 d postpartum. Monensin had no effect on development of the first dominant follicle postpartum or the numbers of class 1 (3 to 5 mm), 2 (6 to 9 mm), or 3 (10 to 15 mm) follicles. Control cows had more class 4 (>15 mm) follicles at 10 to 13 d postpartum than cows in the monensin group. The first dominant follicle postpartum ovulated, regressed, or became cystic unrelated to differences between diets. However, the first ovulation postpartum occurred earlier in monensin-fed cows than in the control group (27.2 +/- 2.1 d vs. 32.4 +/- 1.5 d), with no dietary effects on the diameter of the ovulating follicle. Similarly, treatments did not differ in the proportion of cows with 2 or 3 waves of ovarian follicular development per cycle, nor in the number of follicles of all classes during the breeding period. Times of ovulation following treatment with prostaglandin F2alpha were not different between dietary groups. Pregnancy rates after timed artificial insemination were similar between diets. Supplementation with monensin resulted in a shorter postpartum interval to first ovulation but did not affect other reproductive measures in healthy, lactating dairy cows.