Quantitative determination of follicle size distribution in the guinea pig ovary after hemicastration and PMSG treatment

In order to investigate the action point of intraphysiological or supraphysiological elevation of FSH during the preovulatory period on follicular development, adult guinea pigs underwent unilateral ovariectomy on days 10, 12 and 14 of the estrous cycle (N = 6 each group). Thereafter, guinea pigs were injected twice daily with either vehicle or pregnant mare's serum gonadotropin (PMS). After 2 days, the remaining ovaries were removed. The resected ovaries were fixed, embedded in paraffin, serially sectioned (7 microns) and stained with Azan. All follicles greater than 70 microns were classified by size and atretic stage. The follicular size distribution was not affected by hemicastration at day 10, although the ratio of atretic follicles (greater than 400 microns) decreased from 51% to 32% (P less than 0.01). Hemicastration at day 12 increased the largest nonatretic population (70-99 microns group) from 17% to 26%, and the ratio of atretic follicles (greater than 400 microns) decreased from 35% to 23%. The peak size distribution of follicles was shifted from 70-99 microns to 200-299 microns by PMS, and follicles 600-899 microns in size contained an increased percentage of atresia, which resulted in the bimodal distribution of viable follicles greater than 400 microns. These data suggest that 2 day hemicastration promotes an influx of primordial follicles into growing follicles and suppresses the atretic process by a different mechanism depending on the date of hemicastration in the estrous cycle. Conversely, hemicastration + PMS accelerated viable follicle growth to increase the percentage of atresia.     

Estradiol-induced blockade of ovulation in the cow: effects on luteinizing hormone release and follicular fluid steroids

Administration of 10 mg estradiol valerate (EV) to nonlactating Holstein cows on Days 16 of the estrous cycle prevented ovulation in 7 of 8 cows for 14 days post-injection. In these 7 cows, the timing of luteolysis and the luteinizing hormone (LH) surge was variable but within the normal range. At 14 days post-treatment, each of these cows had a large (greater than 10 mm) follicle, with 558 +/- 98 ng/ml estradiol-17 beta, 120 +/- 31 ng/ml testosterone, and 31 +/- 2 ng/ml progesterone in follicular fluid (means +/- SE). A second group of animals was then either treated with EV as before (n = 22), or not injected (control, n = 17) and ovariectomized on either Day 17, Day 18.5, Day 20, or Day 21.5 (24, 60, 96, or 132 h post-EV). Treatment with EV did not influence the timing of luteolysis, but surges of LH occurred earlier (59 +/- 8 h post-EV vs. 100 +/- 11 h in controls). The interval from luteolysis to LH peak was reduced from 44 +/- 6 h (controls) to 6.9 +/- 1.5 h (treated). Histologically, the largest follicle in controls tended to be atretic before luteolysis, but nonatretic afterwards, whereas the largest follicle in treated animals always tended to be atretic. Nonatretic follicles contained high concentrations of estradiol (408 +/- 59 ng/ml) and moderate amounts of testosterone (107 +/- 33 ng/ml) and progesterone (101 +/- 21 ng/ml), whereas atretic follicles contained low concentrations of estradiol (8 +/- 4 ng/ml) and testosterone (12 +/- 4 ng/ml), and either low (56 +/- 24 ng/ml) or very high (602 +/- 344 ng/ml) concentrations of progesterone. This study suggests that EV prevents ovulation by inducing atresia of the potential preovulatory follicle, which is replaced by a healthy large follicle by 14 days post-treatment.     

Endocrine and ovarian responses associated with the first-wave dominant follicle in cattle.

To examine endocrine and biochemical differences between dominant and subordinate follicles and how the dominant follicle affects the hypothalamic-pituitary-ovarian axis in Holstein cows, the ovary bearing the dominant follicle was unilaterally removed on Day 5 (n = 8), 8 (n = 8), or 12 (n = 8) of synchronized estrous cycles. Follicular development was followed daily by ultrasonography from the day of detected estrus (Day 0) until 5 days after ovariectomy. Aromatase activity and steroid concentrations in first-wave dominant and subordinate follicles were measured. Intact dominant and subordinate follicles were cultured in 4 ml Minimum Essential Medium supplemented with 100 microCi 3H-leucine to evaluate de novo protein synthesis. Five days after unilateral ovariectomy, cows were resynchronized and the experiment was repeated. Follicular growth was characterized by the development of single large dominant follicles, which was associated with suppression of other follicles. Concentrations of estradiol-17 beta (E2) in follicular fluid and aromatase activity of follicular walls were higher in dominant follicles (438.9 +/- 45.5 ng/ml; 875.4 +/- 68.2 pg E2/follicle) compared to subordinate follicles (40.6 +/- 69.4 ng/ml; 99.4 +/- 104.2 pg E2/follicle). Aromatase activity in first-wave dominant follicles was higher at Days 5 (1147.1 +/- 118.1 pg E2/follicle) and 8 (1028.2 +/- 118.1 pg E2/follicle) compared to Day 12 (450.7 +/- 118.1 pg E2/follicle). Concentrations of E2 and androstenedione in first-wave dominant follicles were higher at Day 5 (983.2 +/- 78.2 and 89.5 +/- 15.7 ng/ml) compared to Days 8 (225.1 +/- 78.6 and 5.9 +/- 14.8 ng/ml) and 12 (108.5 +/- 78.6 and 13.0 +/- 14.8 ng/ml). Concentrations of progesterone in subordinate follicles increased linearly between Days 5 and 12 of the estrous cycle. Plasma concentrations of FSH increased from 17.9 +/- 1.4 to 32.5 +/- 1.4 ng/ml between 0 and 32 h following unilateral removal of the ovary with the first-wave dominant follicle. Increases in plasma FSH were associated with increased numbers of class 1 (3-4 mm) follicles in cows that were ovariectomized at Day 5 or 8 of the cycle. Unilateral ovariectomy had no effects on plasma concentrations of LH when a CL was present on the remaining ovary. First-wave dominant follicles incorporated more 3H-leucine into macromolecules and secreted high (90,000-120,000) and low (20,000-23,000) molecular weight proteins that were not as evident for subordinate follicles at Days 8 and 12.(ABSTRACT TRUNCATED AT 400 WORDS)     

An enzymatic method for dissociation of intact follicles from the hamster ovary: histological and quantitative aspects

An enzymatic method was developed to collect intact follicles at different stages of development from cyclic hamsters to study ovarian folliculogenesis under various circumstances. Ovaries from 6 adult hamsters on each day of the cycle (Day 1 = ovulation) were collected, corpora lutea and large preantral and antral follicles were dissected, and follicles saved. Minced ovaries were then incubated with a mixture of collagenase, DNAse and pronase at 37 degrees C for 20 min to disperse intact follicles. Histological studies with 2191 isolated follicles revealed 10 different stages of follicular development (depending on the number of granulosa cell layers surrounding the oocyte and development of the antrum). Of the total follicular population, 14% showed signs of atresia, with 50% of those having 1-3 layers of granulosa cells (Stages 1-3); a second peak of 18% was observed in antral follicles (Stages 8-10). No signs of thecal cells were evident until the follicles reached Stage 6 (7-8 layers of granulosa cells), which possibly accounts for reduced atresia in this class and beyond. Ultrastructural study revealed that there were no signs of morphological damage to the basement membrane or to other subcellular organelles in the small preantral follicles. The presence of subnuclear lipid droplets in follicles with 3 layers of granulosa cells provided evidence for potential steroidogenesis by small follicles. The number of Stage 1-10 follicles was remarkably constant throughout the estrous cycle (460 +/- 34 per animal on Day 1 vs. 492 +/- 66 on Day 4). The usefulness of this method in analyzing follicular kinetics is illustrated in experiments involving hypophysectomy and the effects of unilateral ovariectomy. This procedure offers an improved method to study the factors responsible for the growth and the differentiation of small preantral follicles in the mammalian ovary.     

The effect of a GnRH analogue on the dynamics of follicular development and synchronization of estrus in lactating cyclic dairy cows

A GnRH analogue was used to synchronize ovarian follicular development prior to an injection of PGF(2alpha) for the synchronization of estrus in lactating Holstein cows. On Day 12 (estrus = Day 0) of the experimental cycle, cows (n = 8) were injected with 8 mug Buserelin (BUS group), followed by 25 mg PGF(2alpha) 7 d later (Day 19). Control cows (n = 7) received PGF(2alpha) on Day 12 (PGF group). Ovaries were scanned daily via ultrasonography, and plasma progesterone and estradiol concentrations were determined. Sizes of all visible follicles were recorded. Follicles were classified as small (3 to 5 mm), medium (6 to 9 mm), or large (>/= 10 mm). Between Days 12 and 16 of the cycle, the number of large follicles in PGF cows remained unchanged (1.2), whereas in the BUS group, the number of large follicles decreased from 1.3 on Day 12 to 0.5 on Day 15. Only 4 of 7 PGF cows ovulated a second-wave dominant follicle. In the BUS group, 7 of 8 cows ovulated a GnRH analogue induced dominant follicle that was first identified on Day 15. During the follicular phase (last 5 d prior to estrus), plasma progesterone declined in association with CL regression in both groups, and estradiol concentrations increased, reaching higher (P<.0.05) preovulatory peak concentration in BUS cows than in PGF cows (14.0 +/- 1.0 vs 10.4 +/- 1.1 pg/ml). The number of medium-size follicles was smaller and the number of small-size follicles tended to be higher in BUS cows than in the PGF-treated group. On the day of estrus, the size of the ovulatory follicle (16.1 vs 13.3 mm) and the size difference between the ovulatory and second largest follicle (11.4 vs 6.2 mm) were both larger in BUS cows than in PGF-treated cows, suggesting a more potent dominance effect of the ovulatory follicle in the BUS cows. This study suggests that a GnRH analogue can alter follicular development prior to synchronization of estrus with an injection of PGF(2alpha) in lactating dairy cows.     

Alterations in follicular estradiol and gonadotropin receptors during development of bovine antral follicles

It was hypothesized that growth divergence of dominant and subordinate follicles during Wave 1 and growth termination of the dominant follicle would be associated with changes in the number of gonadotropin receptors on granulosa cells and estradiol in follicular fluid. To test this hypothesis, follicular development of 16 Holstein heifers was monitored by ultrasound, and follicles were collected on Days 2,4,6 and 10 (Day 0 = ovulation). Dominant follicles were compared across days, whereas dominant and largest subordinate follicles were compared on Days 2 and 4 only. The numbers of LH and FSH receptors on the granulosa cells of dominant follicles did not differ significantly over Days 2, 4, 6 and 10. In contrast, concentrations of estradiol in follicular fluid decreased (P < 0.05) from Days 2 to 10 (373 +/- 150 to 42 +/- 12 ng/ml) and concentrations of progesterone in follicular fluid increased (P < 0.05) from Days 2 to 10 (12.2 +/- 2.3 to 24.4 +/- 4.8 ng/ml). Correspondingly, the ratio of estradiol:progesterone in the dominant follicles decreased (P < 0.05) from Days 2 to 10. Comparisons between dominant and subordinate follicles indicated greater (P < 0.05) estradiol concentrations in the dominant follicle on Day 2, but the number of gonadotropin receptors was not different until Day 4. Thus, differences in concentrations of follicular fluid estradiol, but not numbers of granulosa cell gonadotropin receptors, were associated with the early growth divergence of dominant and subordinate follicles (Day 2) and the eventual growth termination of the dominant follicle (Day 10). Late divergence (Day 4) was associated with higher gonadotropin receptor numbers and follicular estradiol concentrations in the dominant than in the subordinate follicles. These results indicate that an increase in estradiol productivity of the selected dominant follicle occurred before an increase in the number of gonadotropin receptors.     

Effect of clomiphene citrate on the ovary of a wild rat, Bandicota bengalensis

The effects of clomiphene citrate (0.3 or 3.0 mg/kg body weight/day) for 10 consecutive days on the ovary of a wild rat, Bandicota bengalensis, were studied. The low dose of clomiphene decreased the number of nonatretic follicles larger than 400 microns in diameter, increased atresia in follicles smaller than 200 microns, inhibited granulosal mitosis in follicles less than 200 microns and between 401 and 600 microns in diameter and inhibited thecal mitosis in follicles smaller than 400 microns and larger than 600 microns. The high dose of clomiphene increased the number of follicles between 201 and 400 microns, decreased the number of follicles larger than 600 microns, increased atresia in follicles of 51-400 microns and increased granulosal mitosis in follicles of 201-400 microns diameter. In both the doses, clomiphene inhibited the ovulation rate (p less than 0.005), with 25 and 35% of the rats being anovulatory in low and high doses, respectively. In addition, clomiphene caused irregularity in the estrous cycles associated with increased cycle length. These results suggest that the clomiphene-induced partial inhibition of ovulation is possibly through its action on follicular growth and atresia mainly in nonantral (less than 200 microns) and mature follicles (401-600 microns).     

Suppression of the secondary FSH surge with bovine follicular fluid is associated with delayed ovarian follicular development in heifers

Holstein heifers were given 5 injections (twice/day) of 10 ml charcoal-extracted bovine follicular fluid (bFF; N = 6) or 10 ml saline (N = 5) beginning 12 h after the onset of oestrus. Blood samples were collected for determination of plasma concentrations of FSH, LH, progesterone and oestradiol-17 beta. Treatment with bFF suppressed the secondary FSH surge (P less than 0.01). Cessation of bFF injections was followed by a rebound period during which FSH was elevated compared with controls (P less than 0.01). Daily ultrasonographic examinations revealed that follicular growth occurred in waves, with 4 of 5 control heifers exhibiting 3 waves and the other 2 waves. In contrast, 5 of 6 bFF-treated animals exhibited 2 waves and the other 3 waves. Appearance of follicles in the first wave was delayed in bFF-treated heifers (Day 3.3 +/- 0.3 compared with Day 1.4 +/- 0.2; P less than 0.0001) and appearance of the dominant follicle of the first wave was delayed (Day 4.5 +/- 0.3 compared with Day 1.8 +/- 0.2; P less than 0.0001). Follicles in the second wave appeared later in animals treated with bFF (Day 12.7 +/- 0.4 compared with Day 10.4 +/- 0.6; P less than 0.01), and the dominant follicle of this wave also appeared later (Day 13.0 +/- 0.5 compared with Day 10.6 +/- 0.5; P less than 0.01). Oestradiol-17 beta increased during the early luteal phase, but this increase occurred later in heifers treated with bFF (peak concentrations on Day 6.3 +/- 0.6 compared with Day 4.2 +/- 0.2; P less than 0.05). LH, progesterone and cycle length were not affected by bFF. Delayed follicular growth associated with suppression of FSH suggests that the secondary FSH surge is important in the initiation of follicular development early in the bovine oestrous cycle, and thus may play a role in the regulation of ovarian follicular dynamics.     

Role of intrafollicular regulators and FSH in growth and development of large antral follicles in sheep

Manipulation of circulating concentrations of hormones and ovarian follicle status was carried out on Day 11-12 of the oestrous cycle in sheep. All follicles visible on the ovary were ablated by cautery and ewes were treated with oestradiol or ovine follicular fluid (oFF) to suppress FSH or with PMSG to increase circulating gonadotrophic activity. One group underwent unilateral ovariectomy which greatly increased endogenous FSH and was the only treatment which significantly affected LH pulse frequency. The size distribution of antral follicles, the extent of atresia and the mitotic index of granulosa cells of follicles on Day 15 showed that (a) treatment with oFF inhibited the growth of follicles beyond 2 mm diameter by suppressing the mitotic index of the granulosa cells and (b) the concentration of FSH in peripheral plasma was related to the ability of small antral follicles to grow during the late luteal-early follicular phase of the cycle. Subsequently, it was demonstrated that oFF inhibits, in a dose-dependent manner, folliculogenesis sustained by PMSG in ewes on Days 12-15. Inhibition of folliculogenesis was represented by a decrease in those follicles greater than 4 mm, an increase in the relative proportion of follicles less than 2 mm, and minimal change in the average number of follicles visible on the ovarian surface, and a decrease in the mitotic index of granulosa cells of follicles less than 2 mm. There was no change in the extent of atresia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synchronization of ovulation in beef cows (Bos indicus) using GnRH, PGF2alpha and estradiol benzoate

The objective of this study was to evaluate protocols for synchronizing ovulation in beef cattle. In Experiment 1, Nelore cows (Bos indicus) at random stages of the estrous cycle were assigned to 1 of the following treatments: Group GP controls (nonlactating, n=7) received GnRH agonist (Day 0) and PGF2alpha (Day 7); while Groups GPG (nonlactating, n=8) and GPG-L (lactating, n=9) cows were given GnRH (Day 0), PGF2alpha (Day 7) and GnRH again (Day 8, 30 h after PGF2alpha). A new follicular wave was observed 1.79+/-0.34 d after GnRH in 19/24 cows. After PGF2alpha, ovulation occurred in 19/24 cows (6/7 GP, 6/8 GPG, 7/9 GPG-L). Most cows (83.3%) exhibited a dominant follicle just before PGF2alpha, and 17/19 ovulatory follicles were from a new follicular wave. There was a more precise synchrony of ovulation (within 12 h) in cows that received a second dose of GnRH (GPG and GPG-L) than controls (GP, ovulation within 48 h; P<0.01). In Experiment 2, lactating Nelore cows with a visible corpus luteum (CL) by ultrasonography were allocated to 2 treatments: Group GPE (n=10) received GnRH agonist (Day 0), PGF2alpha (Day 7) and estradiol benzoate (EB; Day 8, 24 h after PGF2alpha); while Group EPE (n=11), received EB (Day 0), PGF2alpha (Day 9) and EB (Day 10, 24 h after PGF2alpha). Emergence of a new follicular wave was observed 1.6+/-0.31 d after GnRH (Group GPE). After EB injection (Day 8) ovulation was observed at 45.38+/-2.03 h in 7/10 cows within 12 h. In Group EPE the emergence of a new follicular wave was observed later (4.36+/-0.31 d) than in Group GEP (1.6+/-0.31 d; P<0.001). After the second EB injection (Day 10) ovulation was observed at 44.16+/-2.21 h within 12 (7/11 cows) or 18 h (8/11 cows). All 3 treatments were effective in synchronizing ovulation in beef cows. However, GPE and, particularly, EPE treatments offer a promising alternative to the GPG protocol in timed artificial insemination of beef cattle, due to the low cost of EB compared with GnRH agonists.     

Changes in gonadotrophin secretion and ovarian antral follicular activity in seasonally breeding sheep throughout the year

Overall, significantly more antral follicles greater than or equal to 1 mm diameter were present in Romney ewes during anoestrus than in the breeding season (anoestrus, 35 +/- 3 (mean +/- s.e.m.) follicles per ewe, 23 sheep; Day 9-10 of oestrous cycle, 24 +/- 1 follicles per ewe, 22 sheep; P less than 0.01), although the mean numbers of preovulatory-sized follicles (greater than or equal to 5 mm diam.) were similar (anoestrus, 1.3 +/- 0.2 per ewe; oestrous cycle, 1.0 +/- 0.1 per ewe). The ability of ovarian follicles to synthesize oestradiol did not differ between anoestrus and the breeding season as assessed from the levels of extant aromatase enzyme activity in granulosa cells and steroid concentrations in follicular fluid. Although the mean plasma concentration of LH did not differ between anoestrus and the luteal phase of the breeding season, the pattern of LH secretion differed markedly; on Day 9-10 of the oestrous cycle there were significantly more (P less than 0.001) high-amplitude LH peaks (i.e. greater than or equal to 1 ng/ml) in plasma and significantly fewer (P less than 0.001) low amplitude peaks (less than 1 ng/ml) than in anoestrous ewes. Moreover, the mean concentrations of FSH and prolactin were significantly lower during the luteal phase of the cycle than during anoestrus (FSH, P less than 0.05, prolactin, P less than 0.001). It is concluded that, in Romney ewes, the levels of antral follicular activity change throughout the year in synchrony with the circannual patterns of prolactin and day-length. Also, these data support the notion that anovulation during seasonal anoestrus is due to a reduced frequency of high-amplitude LH discharges from the pituitary gland.     

Short-term effects of FSH in vitro on granulosa cells of individual sheep follicles

In Romanov ewes at Day 13 or 14 of the cycle, granulosa cells originating from individual follicles were studied in short-term incubations for aromatase activity and thymidine incorporation. The study was performed on 76 follicles of different sizes (2-7 mm diameter) and degree of atresia, as assessed by histological examination of smears of granulosa cells. As atresia progressed, the labelling index and aromatase activity of granulosa cells decreased. In normal follicles, when follicular diameter increased, the labelling index decreased, while aromatase activity of granulosa cells and oestradiol-17 beta concentration in follicular fluid increased. There was a negative relationship between oestradiol concentration in follicular fluid and the labelling index of granulosa cells in vitro (rs = -0.75; P less than 0.01), suggesting an inverse relationship between growth and differentiation of granulosa cells in normal sheep follicles. In normal small and medium-sized follicles (2-6 mm), incubation with FSH (100 ng/ml) for 2 h increased significantly the labelling index of granulosa cells. In normal medium-sized follicles (4-6 mm), incubation with FSH (50 ng/ml) for 1 h decreased the aromatase activity of granulosa cells. From these results, it is suggested that FSH acts mainly on cells in the G1 phase of the cell cycle, which are steroidogenically active, and makes them move into the S phase where their steroidogenic activity is temporarily inhibited.     

Expression and function of Fas antigen vary in bovine granulosa and theca cells during ovarian follicular development and atresia

Fas antigen is a receptor that triggers apoptosis when bound by Fas ligand (FasL). A role for Fas antigen in follicular atresia was studied in follicles obtained during the first wave of follicular development during the bovine estrous cycle (estrus is Day 0). Granulosa and theca cells were isolated from healthy dominant follicles and the two largest atretic subordinate follicles on Day 5, atretic dominant follicles on Days 10-12, and preovulatory follicles on Day 1. Fas antigen mRNA levels were highest in granulosa cells from subordinate as compared to other follicles, and lowest in theca cells from healthy Day 5 dominant as compared to other follicles. FasL alone had no effect on viability of granulosa or theca cells but became cytotoxic in the presence of interferon-gamma (IFN). IFN has been shown to induce responsiveness to Fas antigen-mediated apoptosis in other cell types. In the presence of IFN, killing of granulosa cells by FasL was greater in subordinate compared to healthy dominant follicles on Day 5, did not differ between healthy and atretic dominant follicles, and was similar in theca among all follicles. Granulosa cells from preovulatory follicles, which had been exposed to the LH surge in vivo, were completely resistant to FasL-induced killing. In summary, Fas antigen expression, and responsiveness to Fas antigen-mediated apoptosis, vary during follicular development.     

Long-term follicular dynamics and biochemical characteristics of dominant follicles in dairy cows subjected to acute heat stress

The objective of this study was to examine the quality of successive dominant follicles (DFs) after induced heat stress. Non-lactating dairy cows expressing estrus at normal intervals were allocated randomly to heat stress (HS; n=8) and control (C; n=8) groups. Cows received GnRH (100 microg, i.m.) on Day 0, a progesterone CIDR-B device on Day 4 and prostaglandin (PGF(2alpha); 25mg, i.m.) on Day 7 upon removal of the CIDR device. The DF and follicles >5mm were aspirated on Day 8, and GnRH (100 microg) injected following aspiration, to initiate a new follicular wave. In this manner, a DF was aspirated every 8 days (one "follicular cycle") for 10 cycles. After the first follicular cycle, HS cows were placed in environmental chambers for 7 days during the second follicular cycle (8h per day at 43.3 degrees C set point and 16h per day at 24 degrees C for 4 days, and 8h per day at 43.3 degrees C set point and 16h per day at 32.2 degrees C set point for 3 days; relative humidity, 40%) and thereafter maintained outdoors with control cows at a mean ambient temperature (18.5 degrees C; range 12.7-26 degrees C). Rectal temperature increased (P<0.001) in HS as compared with C cows (39.28+/-0.01 degrees C versus 38.78+/-0.01 degrees C). Concentrations of estradiol (E(2); 1662+/-189 versus 1493+/-188ng/ml) and progesterone (P(4); 44.7+/-5 versus 54.1+/-5.1ng/ml) in follicular fluid (FF) of DF did not differ between C and HS treatments, respectively. Total FF protein concentration was greater (P<0.05) in HS (99.7+/-2.3mg/ml) than in C (92.7+/-2.3mg/ml). Heat shock protein 90 (Hsp 90) in FF was not altered by heat stress. IGF-II ligand blots were conducted with FF samples (n=79) from four HS and four C cows. There was a predominance of IGFBP-3 in 76 of 79 FF samples, indicating healthy follicular status, and only three FF samples had the lower molecular weight IGFBP-2 indicative of a poor quality follicle. Plasma P(4) and E(2) concentrations did not differ between C and HS groups. The number of class 1 and 3 follicles increased during and just after heat stress, but the number of class 2 follicles did not differ between C and HS cows. Heat stress appeared to induce a decrease in follicular dominance, but GnRH-induced follicular cycles resulted in development of healthy preovulatory follicles in both groups.     

Milk yield, energy balance, hormone, follicular and oocyte measures in early and mid-lactation Holstein cows

The purpose of the study was to determine the influence of energy status on metabolic and endocrine measures, follicular development, and the quality of oocytes obtained from cows during early and mid-lactation (ML). We selected Holstein cows at calving to be assigned to the early lactation (EL) group (n = 8), while we assigned cows at about day 90 postpartum to the ML group (n = 7). We obtained blood samples twice weekly from 4 weeks before aspiration to the aspiration periods for metabolite and hormone determinations. We performed ultrasound-guided transvaginal follicular aspiration (TVFA) twice weekly on all cows for a 10-week period. We obtained follicular fluid from the largest follicle > 10 mm in diameter for hormone determinations. We analyzed data by ANOVA, using the general linear model (GLM) procedures. Energy balance was positive (2.43 +/- 0.32 Mcal/kg) for ML cows and negative (-1.55 +/- 0.33 Mcal/kg) for EL cows. Serum progesterone (P4) for ML cows decreased rapidly from the first aspiration session (2.7 +/- 0.1 ng/ml) and reached a nadir at Week 8 (0.33 +/- 0.1 ng/ml), while follicular fluid P4 increased from 0.9 +/- 0.5 to 5.6 +/- 0.05 ng/ml. Serum and follicular fluid P4 remained relatively constant over the entire aspiration period for EL cows. Follicular fluid insulin-like growth factor I (TGF-I) concentrations increased linearly for EL and ML cows, but the increase was more rapid (159 +/- 36 to 200 +/- 36 ng/ml) for ML cows than for EL cows (145 +/- 36 to 164 +/- 36 ng/ml). Serum IGF-I followed the same pattern for ML cows but declined for EL cows. Early lactation cows experienced a rapid decrease in serum nonesterified fatty acids (NEFA; 0.32 +/- 0.2 to 0.22 +/- 0.2 meq/l), while serum NEFA concentrations were relatively stable (0.19 +/- 0.2 to 0.22 +/- 0.2 meq/l) for ML cows over the aspiration period. The number of follicles obtained from the twice weekly aspiration sessions increased linearly for both EL and ML cows (P < 0.05) over the 10-week period. However, the number of follicles increased from 14.2 +/- 0.5 (Day 119) to 18.1 +/- 0.5 (Day 190) in the ML cows, compared to the changes from 14.9 +/- 0.3 (Day 32) to 15.7 +/- 0.5 (Day 90) for the EL cows. These results indicate that cows are physiologically under more production stress during EL, but increasing follicular fluid and serum IGF-I throughout ML may reflect potential differences in follicle and oocyte measures, compared to cows in EL.     

Morphological and functional characterization of large antral follicles in three breeds of sheep with different ovulation rates

Morphological and functional features of large ovarian follicles from three breeds of sheep, with different ovulation rates (Finnish Landrace N = 12, Finnish Landrace X Scottish Blackface N = 16, Merino X Scottish Blackface N = 16) were compared by integrating three techniques; ink labelling, in-vitro oestradiol production and morphological classification. The follicles were removed at two stages of the follicular phase, 1 (PG + 1) or 2 (PG + 2) days after PGF-2 alpha treatment and compared after monitoring their rates of growth with the use of ink labelling. After ovariectomy all follicles greater than or equal to 1 mm in diameter were dissected, and the 8 largest were incubated individually for 2 h to assess their ability to secrete oestradiol and testosterone. After incubation the follicles were processed for histological examination and checked for atresia. An analysis of the follicle population was based on in-vitro oestradiol secretion rates in all three breeds; an oestrogen-active population producing 500-8100 pg oestradiol/ml/h and an oestrogen-inactive population producing 0-499 pg oestradiol/ml/h. A comparison of the 3 approaches demonstrated agreement on 94.3 +/- 1.2% of occasions. Ink-labelling demonstrated that all follicles identified as oestrogen-active were increasing in size. Within oestrogen-active follicles significant correlations were detected between oestradiol production and testosterone production (r = 0.42), oestradiol production and granulosa cell number (r = 0.45) and between oestradiol production and mitotic index (r = -0.38). A regression model fitting breed, stage of atresia, granulosa cell number, in-vitro testosterone production and mitotic index demonstrated that granulosa cell number is a characteristic which contributes significantly to the variation of in-vitro oestradiol production in oestrogen-active and oestrogen-inactive follicles. There was no significant difference between breeds in the mean number of ink-labelled follicles growing from Day PG - 1 to Day PG + 1. There was a significant difference between the breeds in the number of ink-labelled follicles growing between Days PG + 1 and PG + 2 (Days 1 and 2 of the follicular phase), the number being similar to the ovulation rate for the breed. The majority of the oestrogen-active follicles had been recruited by Day PG - 1, although in the Finnish Landrace genotypes more than 30% were recruited on or after Day PG + 1 compared to less than 10% in Merino x Scottish Blackface ewes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transvaginal ultrasound guided follicular aspiration of bovine oocytes

A transvaginal ultrasound guided follicular aspiration technique was developed for the repeated collection of bovine oocytes from natural cycling cows. In addition, the feasibility of using this method for collecting immature oocytes for in vitro embryo production was also evaluated. Puncturing of visible follicles for ovum pick-up was performed in 21 cows over a three month period. All visible follicles larger than 3 mm were punctured and aspirated three times during the estrous cycle on Day 3 or 4, Day 9 or 10 and Day 15 or 16. The mean (+/- SEM) estrous cycle length after repeated follicle puncture was 22.2 +/- 0.3 days. The mean total number of punctured follicles per estrous cycle was 12.6 +/- 0.3. The largest (P<0.05) number of follicles punctured (5.1 +/- 0.3) for ovum pick-up was on Day 3 or 4 of the estrous cycle. The overall recovery rate of 541 punctured follicles was 55%. Most oocytes (P<0.05) were aspirated from follicles smaller than 10 mm. Following in vitro maturation and fertilization (IVM/IVF), 104 oocytes were transferred to sheep oviducts. Six days later, 75 ova/embryos were recovered, after flushing the oviduct of the sheep, of which 24% developed into transferable morulae and blastocysts. In this study, a reliable nonsurgical, follicular aspiration procedure was used for the repeated collection of immature oocytes which could be used successfully for in vitro production of embryos. This procedure offers a competitive alternative to conventional superovulation/embryo collection procedures.     

Are differences in FSH concentrations involved in the control of ovulation rate in Romanov and Ile-de-France ewes?

Despite differences in FSH concentrations ranging from 1.5 ng/ml (Romanov ewes) to 4 ng/ml (Ile-de-France ewes) between the follicular and luteal phases, follicular growth (numbers of follicles growing, growth rates, maximum size reached) was morphologically similar between the two stages of the cycle. Injection of 750 i.u. hCG at Day 6 or 16 of the cycle triggered ovulation of 4.1 +/- 0.7 and 4.0 +/- 1.3 follicles in Romanov and 2.2 +/- 0.5 and 1.7 +/- 0.5 follicles in Ile-de-France ewes, respectively, demonstrating that functional differentiation was similar between the two stages of the cycle. As gonadotrophin environment differs between these two stages of the cycle, this suggests that there is a wide flexibility in the amount of gonadotrophins required to trigger terminal follicular growth and that ovarian requirements for gonadotrophins might work through thresholds. When Romanov and Ile-de-France ewes were given similar amounts of exogenous gonadotrophins (1250 i.u. PMSG, 750 i.u. hCG) after hypophysectomy, ovulation rates were close to the usual values (Romanov, 5.5 +/- 3.9; Ile-de-France, 1.4 +/- 0.5), demonstrating that differences in gonadotrophin concentrations during the follicular phase do not play a major role in the high ovulation of the Romanov compared to the Ile-de-France ewes.     

Development of dominant follicles and length of ovarian cycles in post-partum dairy cows

The resumption of ovarian activity after normal calvings was studied in 18 lactating Friesian cows. Since, in 17 cows, first post-partum ovulation occurred without overt oestrous behaviour being detected, the resultant cycles were called 'ovarian cycles'. The mean (+/- s.d.) length of the ovarian cycles was 21.0 +/- 8.7 days. The duration of cycles tended to be normal (18-24 days) or long (greater than or equal to 25 days) when the ovulatory dominant follicles were identified before Day 10 post partum; they were consistently short (9-13 days) when dominant follicles identified after Day 20 post partum ovulated. When such follicles were detected between Days 10 and 20 post partum, long, normal and short ovarian cycles were detected. The number of waves of follicular growth with associated dominant follicles observed during the ovarian cycles tended to be related to cycle length; short cycles had 1 dominant follicle, normal cycles predominantly 2, and long cycles mostly 3 dominant follicles. The mean (+/- s.d.) duration of 13 oestrous cycles studied was 23.1 +/- 2.1 days. Of these cycles, 7 had 3 and 6 had 2 dominant follicles. The oestrous cycles with 3 dominant follicles had a mean (+/- s.d.) duration of 24.0 +/- 1.2 days and the respective dominant non-ovulatory follicles reached maximum sizes on Days 8 and 18, respectively; oestrous cycles with 2 dominant follicles were 22.2 +/- 2.6 days in duration, and the dominant non-ovulatory follicle reached maximum size by Day 8. Ovarian follicular development during the first 45 days of pregnancy was characterized by the growth and regression of successive dominant follicles, each lasting 10-12 days. These results show that the first ovarian cycle was predominantly short when the ovulatory dominant follicle was first detected after Day 20 post partum.     

Characteristics of bovine estrous cycles during repeated transvaginal, ultrasound-guided puncturing of follicles for ovum pick-up

Repeated transvaginal ultrasound guided puncturing of visible follicles was performed for ovum pick-up (OPU) during Periods A and B, each of which lasted 3 mo. During Period A, 10 cows (A) were used in the study. Period B commenced 1 mo after Period A and two groups of animals were used. The first group (B1) consisted of 9 of 10 cows from Group A. The second experimental group of animals in Period B consisted of 11 cows (B2) which had not been submitted to previous puncture. During the study, all visible follicles larger than 3 mm were punctured and aspirated three times, on Day 3 or 4, Day 9 or 10 and Day 15 or 16 of the estrous cycle. The mean estrous cycle length (+/- SEM) after repeated follicle puncture did not differ among the three groups and was 22.3 +/- 0.4, 22.5 +/- 0.4 and 22.1 +/- 0.3 d for groups A, B1 and B2, respectively. The mean total number (+/- SEM) of punctured follicles per estrous cycle in Group A (13.1 +/- 0.5) was significantly larger than in Groups B1 (11.2 +/- 0.4) and B2 (11.6 +/- 0.4). The largest number of follicles punctured for ovum pick-up in all three groups was always on Day 3 or 4 of the estrous cycle: 4.9 +/- 0.3 follicles; the mean (+/- SEM) number of punctured follicles on Day 9 or 10 and Day 15 or 16 was significantly (P<0.05) lower: 3.4 +/- 0.2 and 3.9 +/- 0.2, respectively. In Period A, primarily 3- to 5-mm follicles were punctured per estrous cycle, while 6- to 10-mm follicles were predominantly punctured in Period B (P<0.05). Recovery rate of oocytes on Day 3 or 4, Day 9 or 10 and Day 15 or 16 were 53, 50 and 52%, respectively. Most oocytes (P<0.05) were aspirated from follicles smaller than 10 mm.