GnRH improves the recovery rate and the in vitro developmental competence of oocytes obtained by transvaginal follicular aspiration from superstimulated heifers

In this study we assessed the effect of GnRH on the recovery rate, meiotic synchronization and in vitro developmental competence of oocytes recovered close to the expected time of ovulation. Twenty-three heifers were superstimulated with FSH, and luteolysis was induced by PGF(2alpha) injection 48 h after the start of treatment Twelve heifers received 200 microg GnRH at 34 h after PGF(2alpha) treatment, Blood samples were collected between 35 to 47 h after PGF(2alpha) administration to determine the time of the LH surge. Transvaginal follicular aspiration was performed at 60 h after PGF(2alpha), and the recovered oocytes were fertilized or fixed either immediately or after 24 h of maturation in vitro. GnRH-treated heifers showed an LH surge within 3 h after treatment, while only 4 of the 10 heifers in the control group exhibited an LH surge by 47 h after treatment with PGF(2alpha). The average number of large follicles (> 10 mm) was 21.3 +/- 2.3 and 19.3 +/- 2.4 for GnRH-treated and control heifers, respectively. The oocyte recovery rate was 87.7 and 63.1% (P < 0.05), respectively, and most of the cumulus-oocyte-complexes (COC) recovered from the 2 groups had an expanded cumulus (80.4 and 80.5%, respectively). Oocytes with an expanded cumulus from the GnRH group had completed meiotic maturation at higher rate than the controls (97 vs 20%;P < 0.05). In vitro development to the blastocyst stage of cumulus-expanded oocytes fertilized immediately after recovery was higher in GnRH-treated than in control heifers (60.3 vs 40.0%; P < 0.05). No difference was observed when oocytes with compact or expanded cumulus were matured in vitro for 24 h before fertilization. These results indicate that GnRH injections improve the oocyte recovery rate and that oocytes have a higher development competence than those obtained from non-GnRH-treated animals. We propose that this higher in vitro developmental competence may result from a more synchronous or further advanced meiotic maturation. However, due to the small number of oocytes in our study, we must emphasize that our findings on meiotic resumption are of preliminary nature.     

Preovulatory endocrinology and oocyte maturation in superovulated cattle

Preovulatory follicular and oocyte nuclear maturation was studied in donor cattle induced to superovulate with a PMSG- or FSH-prostaglandin regimen. Plasma concentrations of progesterone (P4) and luteinizing hormone (LH) and follicular fluid levels of progesterone and estradiol-17β were measured and related to the oocyte nuclear maturation stages. The oocyte donors could be divided into two distinct groups. Group I had entirely normal periovulatory P4 and LH concentrations, and the majority of follicles and oocytes followed a characteristic pattern, clearly time-related to the LH peak. Group II had deviating levels of P4 and/or LH in the pre- and periovulatory period, and a majority of their follicles and oocytes had disturbed maturation, such as abnormal P4:E2 ratio in follicles and prematurely activated or meiotically arrested oocytes. It is concluded that a certain proportion of superovulated cows and heifers develop abnormal follicular/oocyte maturation and constitute poor oocyte, and probably also embryo, donors.     

Concentrations of steroids and gonadotropins in follicular fluid from normal heifers and heifers primed for superovulation

The concentrations of six steroids and of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured in follicular fluid from preovulatory and large atretic follicles of normal Holstein heifers and from preovulatory follicles of heifers treated with a hormonal regimen that induces superovulation. Follicular fluid from preovulatory follicles of normal animals obtained prior to the LH surge contained extremely high concentrations of estradiol (1.1 +/- 0.06 micrograms/ml), with estrone concentrations about 20-fold less. Androstenedione was the predominant aromatizable androgen (278 +/- 44 ng/ml; testosterone = 150 +/- 39 ng/ml). Pregnenolone (40 +/- 3 ng/ml) was consistently higher than progesterone (25 +/- 3 ng/ml). In fluid obtained at 15 and 24 h after the onset of estrus, estradiol concentrations had declined 6- and 12-fold, respectively; androgen concentrations had decreased 10- to 20-fold; and progesterone concentrations were increased, whereas pregnenolone concentrations had declined. Concentrations of LH and FSH in these follicles were similar to plasma levels of these hormones before and after the gonadotropin surges. The most striking difference between mean steroid levels in large atretic follicles (greater than 1 cm in diameter) and preovulatory follicles obtained before the LH surge was that estradiol concentrations were about 150 times lower in atretic follicles. Atretic follicles also had much lower concentrations of LH and slightly lower concentrations of FSH than preovulatory follicles. Hormone concentrations in follicles obtained at 12 h after the onset of estrus from heifers primed for superovulation were similar to those observed in normal preovulatory follicles at estrus + 15 h, except that estrogen concentrations were about 6-40 times lower and there was more variability among animals for both steroid and gonadotropin concentrations. Variability in the concentrations of reproductive hormones in fluid from heifers primed for superovulation suggests that the variations in numbers of normal embryos obtained with this treatment may be due, at least in part, to abnormal follicular steroidogenesis.     

Roles of the dominant follicle and the pattern of oestradiol in induction of preovulatory surges of LH and FSH in prepubertal heifers by pulsatile low doses of LH

Prepubertal crossbred beef heifers were injected (i.v.) with 50 micrograms bovine LH every 2 h for 48 h (first injection at 0 h). At 28 h, number and diameter of ovarian follicles were determined by ultrasonic scanning, and unilateral removal of either the ovary bearing the largest follicle (Group UL, N = 5) or the opposite ovary (Group UO, N = 4) was performed; control animals remained intact (Group I, N = 5). Blood samples were taken every 2 h (starting at 0 h) for a 60-h period to assess concentrations of gonadotrophins and oestradiol. Preovulatory-like surges of LH occurred in 0/5, 4/4 and 5/5 heifers for Groups UL, UO and I respectively; the time of the LH surge did not differ between animals in Groups I and UO (mean = 40 h). FSH in Group UL heifers rose to a plateau immediately after unilateral ovariectomy; this pattern was not observed in the other two groups (P less than 0.01). The area under the curve for FSH was significantly different (P less than 0.05) among groups after 28 h. Preovulatory-like surges of FSH occurred coincidently with those of LH, except for one Group I heifer. An increase in the concentrations of oestradiol between 0 and 28 h was detected in all animals. Profiles of oestradiol during this period did not differ between heifers that had an LH surge (Group UO and I) and those that did not (Group UL).(ABSTRACT TRUNCATED AT 250 WORDS)     

Estrogen - Induced release of luteinizing hormone in prepubertal and postpubertal heifers

This experiment was designed to determine the age at which estradiol-17beta (E(2)) first induces a preovulatory-like surge of luteinizing hormone (LH) in prepubertal heifers. Responses of prepubertal animals 3 to 4 and 5 to 6 months of age were compared with those of postpubertal heifers that received 25 mg prostaglandin F(2)alpha at 0800 hr on day 15 of the estrous cycle. E(2) (500mug) induced surges of LH in 1 5 heifers 3 to 4 months of age, 3 3 heifers 5 to 6 months of age and 5 5 postpubertal heifers. Duration of response and interval between E(2) injection and peak of the response were longer in postpubertal heifers than in those 5 to 6 months old (P<0.10). Peak response and total amount of LH released were greater in animals 5 to 6 months old (P<0.10). Only one prepubertal heifer had elevated concentrations of progesterone following an LH surge. Four of 5 postpubertal heifers receiving E(2) and 3 of 4 postpubertal heifers receiving corn oil had corpora lutea and similar patterns of progesterone concentrations. We conclude that ability to release an LH surge in response to E(2) develops in heifers between 3 and 5 months of age, but that this induced surge does not cause ovulation.     

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.     

The acute effects of oestradiol-17beta and synthetic LH-RH on plasma LH levels in freemartin heifers.

Injection of oestradiol was followed by a surge of plasma LH within 24 h in only 7 of 12 freemartins. Elevations of plasma LH were less than those reported for normal non-cyclic heifers, but some freemartins showed a delayed, or more prolonged, LH response. Responsiveness to oestradiol was not related to degree of chimaerism or plasma androstenedione level, and most of the animals responded similarly in two trials carried out 4 months apart, during which time plasma androstenedione levels had more than doubled. Freemartins which showed an LH surge after oestradiol treatment released greater amounts of LH after the injection of LH-RH than did non-responders.     

Temporal and endocrine sequelae of aspirating follicular contents in rhesus monkeys

Aspiration of ovarian follicular contents in humans is a well-established procedure used to obtain oocytes for fertilization in vitro (IVF). However, the effects of aspiration on the menstrual cycle and resulting luteal function have been incompletely characterized. The present study was designed to investigate alterations in the temporal and endocrine characteristics of menstrual cycles following aspiration of contents of the dominant preovulatory follicle (DF) on day 10 of the cycle in normal rhesus monkeys. When aspiration was performed prior to the preovulatory surge of luteinizing hormone (LH), cycle length was extended to 38.6 ± 8.6 [15] (x days ± SD, [n]), as compared to 29.5 ± 5.7 [8] days when the surge occurred before the time of aspiration. Mean and maximal amounts of progesterone (P) in the luteal phase and the number of days in which P-values were > 1 ng/ml were significantly greater when aspiration was performed prior to the surge of LH than for aspiration after this event. Laparoscopic observations made in the midluteal phase in animals of the former group demonstrated that the corpus luteum (CL was derived from a follicle other than the original DF which had been aspirated on day 10 of the menstrual cycle; observations in the latter group of animals indicated that the CL was derived from the DF.     

Effects of an LHRH antagonist on the time of occurrence and amplitude of the preovulatory LH surge, progesterone and estradiol secretion, and ovulation in superovulated Holstein heifers

Beginning on Day 10 or 11 of the estrous cycle, mature Holstein heifers were given a superovulatory regimen of twice-daily injections of porcine FSH, together with injections of PG with the fifth and sixth FSH injections. Every 12 h from 24 to 60 h after PG administration, the animals received im injections of different doses of the LH releasing hormone antagonist [N-Ac-D-Nal(2)(1), D-pCl-Phe(2), D-Trp(3), D-Arg(6), D-Ala(10)]-LHRH or vehicle. Follicular development was monitored by transrectal ultrasonography every 12 h from 24 to 120 h after PG administration. All animals were given hCG at 72 h after PG injection, and were artificially inseminated. At Day 7 of gestation, the corpora lutea were counted by ultrasonography, and embryos were collected by nonsurgical flushing of the uterus. Treatment with the antagonist resulted in a dose-dependent decrease in the amplitude of the LH surge and in delays in the time of occurrence of the LH surge, ovulation and the shift from estradiol to progesterone production. These results indicate that LHRH antagonists can be used to delay the LH surge and ovulation in superovulated heifers. This finding may be beneficial to studies in the superovulation of cattle.     

Effects of unilateral ovariectomy on follicular development and ovulation in cattle

In a study of 4 cyclic dry cows (Trial I) and 6 cyclic puberal heifers (Trial II), unilateral ovariectomy increased the number of ovulatory follicles, did not alter the hormone profile, cycle length or the number of follicular waves. Ovarian follicular development in all 4 cows was monitored daily using transrectal ultrasonography until the day of ovulation, during which period daily blood samples were also taken from the tail vein for determination of plasma FSH, LH and P4 concentrations. Unilateral ovariectomy was performed on the day after ovulation and ovarian activity was again monitored daily (ultrasonography and blood sampling for FSH, LH and P4) for 2 consecutive cycles (8 cycles in all). Estrus in all 6 heifers was synchronized using 2 injections of PGF2 alpha given 12 d apart. Similarly, ovarian activity in the 6 puberal heifers was monitored daily using ultrasonography and blood sampling for 1 complete control cycle. Following estrus and ovulation the left ovary was removed in all the animals, and thereafter 1 complete cycle was followed. Mean cycle length, FSH, LH and P4 concentrations before and after unilateral ovariectomy were compared using paired sample t-test. The results show that unilateral ovariectomy neither altered the cycle length nor the number of follicular waves in the cows, but it increased the number of ovulatory follicles (2 follicles developed and ovulated in 6 of the 8 cycles). The mean diameter of the largest follicle was 16.1 +/- 0.9 mm and the second largest 12.5 +/- 0.9 mm. No significant (P > 0.05) differences were observed in FSH (0.72 +/- 0.09 vs 0.71 +/- 0.07), LH (0.42 +/- 0.1 vs 0.37 +/- 0.07) and P4 (2.8 +/- 0.6 vs 2.6 +/- 0.4) levels before and after unilateral ovariectomy. Of the 6 heifers, 5 had 2 waves and 1 heifer had 3 waves of follicular growth during the control cycle, and this pattern did not change after the procedure. Mean cycle length (20.7 +/- 0.9 vs 21 +/- 0.9) did not differ before and after unilateral ovariectomy, and 4 of the 6 heifers ovulated twin follicles following ovariectomy. The mean diameter of the largest follicle was 14.5 +/- 0.7 mm and second largest measured 12.1 +/- 0.8 mm. No significant (P > 0.05) differences were observed in FSH (0.16 +/- 0.09 vs 0.21 +/- 0.07), LH (0.11 +/- 0.1 vs 0.15 +/- 0.07) and P4 levels (3.6 +/- 0.26 vs 3.8 +/- 0.29) before and after unilateral ovariectomy. Based on these results, we conclude that unilateral ovariectomy is an ideal method for obtaining twin ovulations in cows and heifers.     

Characterization of thymosin alpha 1 and beta 4 during the bovine estrual period: effects of elevated estradiol and progestin

At present, there is a renewed interest in thymic function and its secretions in relation to endocrine control and reproductive function. In an initial experiment, 60 crossbred heifers (18-20 mo) were detected in estrus and assigned to control or FSH superovulatory groups. On Days 7-14 of the subsequent estrous cycle, FSH was administered for 5 days and prostaglandin F2 alpha (PGF2 alpha) was administered at 48 and 60 h after the initial FSH injection. Control animals received only PGF2 alpha injections between Days 9 and 15 of the cycle. Blood samples were collected from all animals at the time of PGF2 alpha injection and every 12 h thereafter to 72 h post PGF2 alpha injection. In a subsequent experiment, 103 crossbred heifers (16-18 mo) were superovulated with FSH and synchronized to estrus with PGF2 alpha administered 60 h after the initial FSH injection. Twenty-eight of the heifers received Norgestomet implants 12 h prior to the initial PGF2 alpha injection to inhibit the LH surge. Blood samples were collected from animals at 12-h intervals until the PGF2 alpha injection and every 6 h thereafter until 108 h post PGF2 alpha treatment. Although thymosin beta 4 concentrations did change over the estrual period, no differences were noted between control and superovulatory animals in the initial experiment even though estradiol concentrations were increased tenfold from the FSH stimulated ovary. In the second experiment, thymosin beta 4 and alpha 1 increased as the estrual period progressed and decreased (p less than 0.05) subsequent to the LH surge. (ABSTRACT TRUNCATED AT 250 WORDS)     

Oocyte structure and follicular steroid concentrations in superovulated versus unstimulated heifers

A highly variable yield of viable embryos in superovulated cattle is a major hindrance to the embryo transfer industry. To trace the cause of this problem, investigations were carried out on the intrafollicular steroids and structure of oocytes originating from follicles of follicular stimulating hormone (FSH)-stimulated (superovulated) and unstimulated heifers. Unstimulated heifers were slaughtered at midcycle, or administered cloprostenol (PG) at midcycle and slaughtered after 24, 48, or 72 hr, while superovulated heifers were administered 4 injections of pFSH (2 injections per day) and slaughtered 12 hr later, or administered 6, 7, or 8 injections of FSH in combination with PG at the 5th and 6th injection, and slaughtered 24, 36, or 60 hr, respectively, after the first PG injection. The follicular fluid from the largest (presumptive dominant) follicle of the unstimulated heifers and from potentially ovulatory follicles (≥8 mm in diameter) of the superovulated heifers were assayed for estradiol-17β (E2) and progesterone (P4), while the oocyte cumulus complexes from such follicles were processed for transmission electron microscopy. The mean E2 and especially P4 concentrations of the potentially ovulatory follicles of the superovulated heifers were lower than similar follicles of the unstimulated animals (83.7 ± 76.7 ng/ml vs. 208.1 ± 357.0 ng/ml, P > 0.05 and 31.1 ± 38.7 ng/ml vs. 150.3 ± 202, P < 0.05, respectively). The unstimulated oocytes had, in general, spherical oocyte nuclei and compact nucleoli before PG administration, while after PG, undulation of the nuclear envelope and nucleolus vacuolization was characteristic. The superovulated oocytes, in comparison, displayed the following deviations: premature perivitelline space formation, lack of nucleolar vacuolization, reduced amount of lipid droplets and lack of lipid-mitochondria association, enlarged rough endoplasmic reticulum compartment, and increased condensation of chromatin and elongation, i.e., expansion of some cumulus cells. Degenerative oocytes were only found in the superovulated group. It is concluded that FSH-stimulation is associated with reduced intrafollicular E2 and P4 concentrations and subcellular deviations in the oocytes that are established early in the superovulatory process. These deviations may contribute to the reduced developmental competence of superovulated oocytes. © 1994 Wiley-Liss, Inc.     

Ultrasonography and hormone profiles of adrenocorticotrophic hormone (ACTH)-induced persistent ovarian follicles (cysts) in cattle

The objective of this study was to develop a model for the study of abnormal ovarian follicles in cattle by treating heifers with adrenocorticotrophic hormone (ACTH) (100 iu at 12 h intervals for 7 days, beginning on day 15 of the oestrous cycle). Cortisol concentrations increased (P < 0.05) within 24 h after beginning ACTH treatment and cortisol and progesterone concentrations remained elevated after cessation of ACTH treatment for 8 and 4 days, respectively. The pulses and surges of LH decreased during ACTH treatment, but FSH profiles were similar to those in controls and persistent or prolonged follicles were eventually observed in all heifers. In five heifers, prolonged dominant follicles ovulated after 10 days, whereas in six heifers, persistent follicular structures were present for 20 days, but ceased to secrete oestradiol after approximately 12 days. In the heifers with persistent follicular structures, new follicles emerged when the persistent follicle became non-oestrogenic. During the last 2 days of normal follicular growth, the concentration of oestradiol was greater than it was during prolonged or persistent follicle development (P < 0.05). There were no differences in the growth rates or maximum diameters of abnormal follicles that had different outcomes, but oestradiol concentrations were greater in prolonged follicles that ovulated compared with those follicles that persisted (P = 0.06). In conclusion, stimulation with ACTH resulted in a marked deviance from normal follicular activity. The aberrations were probably caused by the interruption of pulsatile secretion of LH (but not FSH) leading to decreased but prolonged oestradiol secretion.     

Active immunization of the cow against oestradiol-17beta

Six beef heifers were immunized over a 4-month period with an oestradiol-17beta-BSA conjugate in Freund's adjuvant. There was an interference with oestrus in the treated heifers; 2 ceased to exhibit oestrus, one exhibited one oestrus and three exhibited oestrus after Day 47 of treatment. The control heifers treated with Freund's adjuvant had normal oestrous cycles. The antiserum titre rose in all treated heifers and attained its highest level in the 2 animals in which oestrus did not recur. The temporal changes in plasma LH, progesterone and oestradiol were normal during the pretreatment period, but became abnormal during the 120 days after immunization. Although plasma oestradiol-17beta rose at the expected time of oestrus after treatment, it was apparently effectively neutralized by the antiserum induced by treatment as evidenced by the absence of an LH surge. Plasma progesterone levels fell to baseline and remained low, indicating lack of formation of corpora lutea.     

Oocyte morphology in dominant and subordinate follicles

The structure of oocytes aspirated from the dominant and its subordinate follicles was investigated from the achievement of follicular dominance to ovulation. Ovulation was induced in 18 heifers and 5 cows by injection of cloprostenol at days 8–14 (day 0 = day of ovulation), and follicular development was monitored by ultrasonography. The animals were slaughtered at days 3–11, but animals slaughtered on days 8–11 were given a second injection of cloprostenol at day 7 to allow ovulation of the dominant follicle of the first follicular wave. Oocytes were aspirated from the dominant (largest) and two largest subordinat efollicles and processed for transmission electron microscopy, whereas the follicular fluids were analyzed for concentrations of estradiol-17β (E2) and progesterone (P4). Dominant follicular growth was associated with increase in the concentration of E2 and P4 in the follicular fluid, which was E2-dominated. From days 3–7, the dominant oocytes had pronounced junctional contacts with the cumulus cells and a nonundulating nuclear envelope but showed an increase in the number of lipid droplets and a decrease in the size of Golgi complexes, the size of cortical granule clusters, and the number of microvilli stacks. After cloprostenol injection on day 7, but before the anticipated LH surge, the dominant oocytes showed a reduced oocyte cumulus contact, vacuolization of the nucleolus, undulation of the nuclear envelope, and dispersal of the mitochondrial clusters. The morphological alterations occurring in the dominant oocytes before the anticipated LH surge are suggested to be a prerequisite for the oocyte to achieve the competence to undergo final maturation. Subordinate follicles ceased growing at about days 3–4 and their follicular fluid had low E2:P4 ratio or was P4-dominated. Subordinate oocytes displayed degenerative features in their cumulus investment and nuclear activation and maturation especially after day 5. The structural changes associated with oocyte degeneration showed similarities with the processes seen before and during final maturation of the dominant oocytes. © 1994 Wiley-Liss, Inc.     

Opioid modulation of LH secretion during the oestrous cycle of heifers

Injections of an opioid agonist (bremazocine) and/or an antagonist (quadazocine) were given to heifers during the luteal or follicular phase of the oestrous cycle. Quadazocine was injected (210 mg/injection) three times at 2-h intervals, and bremazocine was injected (0.45 mg/injection) every 15 min for 6 h. Blood samples were taken every 15 min beginning 6 h before treatments started and continued for 18 h. LH secretion patterns were not affected by quadazocine in the luteal-phase heifers, but quadazocine and bremazocine had marked effects during the follicular phase. Quadazocine increased LH secretion by increasing peak height but not peak frequency. Bremazocine decreased LH secretion through both peak height and frequency. This decrease was of greater magnitude than the increase due to quadazocine. When quadazocine and bremazocine were given together, these effects were cancelled and none of the effects carried over into the bleeding period after treatments stopped. No apparent interruption of follicular maturation was detected since all follicular-phase heifers were detected in oestrus at normal intervals. We conclude that heifers in this experiment did not have an opioid-mediated mechanism for progesterone suppression of LH but that an opioid mechanism for modulating LH does exist during the follicular phase.     

Superstimulation of ovarian follicular growth with FSH oocyte recovery, and embryo production from Zebu (Bos indicus) calves: effects of treatment with a GnRH agonist or antagonist

The capacity of heifer calves of a late sexually maturing Zebu (Bos indicus) genotype to respond to superstimulation with FSH at a young age and in vitro oocyte development were examined. Some calves were treated with a GnRH agonist (deslorelin) or antagonist (cetrorelix) to determine whether altering plasma concentrations of LH would influence follicular responses to FSH and oocyte developmental competency. Brahman calves (3-mo-old; 140 +/- 3 kg) were randomly assigned to 3 groups: control (n = 10); deslorelin treatment from Day -8 to 3 (n = 10); and cetrorelix treatment from Day -3 to 2 (n = 10). All calves were stimulated with FSH from Day 0 to 2, and were ovariectomized on Day 3 to determine follicular responses to FSH and to recover oocytes for in vitro procedures. Before treatment with FSH, heifers receiving deslorelin had greater (P < 0.001) plasma LH (0.30 +/- 0.01 ng/ml) than control heifers (0.17 +/- 0.02 ng/ml), while plasma LH was reduced (P < 0.05) in heifers treated with cetrorelix (0.13 +/- 0.01 ng/ml). Control heifers had a surge release of LH during treatment with FSH, but this did not occur in heifers treated with deslorelin or cetrorelix. All heifers had large numbers of follicles > or = 2 mm (approximately 60 follicles) after superstimulation with FSH, and there were no differences (P > 0.10) between groups. Total numbers of oocytes recovered and cultured also did not differ (P > 0.05) for control heifers and heifers treated with deslorelin or cetrorelix. Fertilization and cleavage rates were similar for the 3 groups, and developmental rates to blastocysts were also similar. Zebu heifers respond well to superstimulation with FSH at a young age, and their oocytes are developmentally competent.     

Effect of GnRH antagonist-induced prolonged follicular phase on follicular atresia and oocyte developmental competence in vitro in superovulated heifers

A GnRH antagonist (Antarelix) was used to suppress endogenous pulsatile secretion of LH and delay the preovulatory LH surge in superovulated heifers to study the effect of a prolonged follicular phase on both follicle and oocyte quality. Oestrous cycles were synchronized in 12 heifers with progestagen (norgestomet) implants for 10 days. On day 4 (day 0 = day of oestrus), heifers were stimulated with 24 mg pFSH for 4 days and luteolysis was induced at day 6 with PGF2 alpha (2 ml Estrumate). Animals in the control group (n = 4) were killed 24 h after the last FSH injection. At this time, heifers in group A36h (n = 4) and group A60h (n = 4) were treated with 1.6 mg of Antarelix every 12 h for 36 and 60 h, respectively, and then killed. After dissection of ovarian follicles, oocytes were collected for individual in vitro maturation, fertilization and culture; follicular fluid was collected for determination of steroid concentrations, and granulosa cells were smeared, fixed and stained for evaluation of pycnosis rates. Granulosa cell smears showed that 90% of follicles were healthy in the control group. In contrast, 36 and 58% of the follicles in group A36h showed signs of early or advanced atresia, respectively, while 90% of the follicles in group A60h showed signs of late atresia. Intrafollicular concentrations of oestradiol decreased (P < 0.0001) from healthy follicles (799.14 +/- 40.65 ng ml-1) to late atretic follicles (3.96 +/- 0.59 ng ml-1). Progesterone concentrations were higher (P < 0.0001) in healthy follicles compared with atretic follicles, irrespective of degree of atresia. Oestradiol:progesterone ratios decreased (P < 0.0001) from healthy (4.58 +/- 0.25) to late atretic follicles (0.07 +/- 0.009). The intrafollicular concentrations of oestradiol and progesterone were significantly higher (P < 0.0001) in the control than in the treated groups. The oestradiol:progesterone ratio was higher (P < 0.0001) in the control (4.55 +/- 0.25) than in the A36h (0.40 +/- 0.05) and A60h (0.07 +/- 0.009) groups. Unexpectedly, the cleavage rate of fertilized oocytes, blastocyst rate and number of cells per blastocyst were not significantly different among control (85%, 41% and 95 +/- 8), A36h (86%, 56% and 93 +/- 5) and A60h (88%, 58% and 79 +/- 4) groups. In addition, there were no significant differences in the blastocyst rates from oocytes derived from healthy (45%), early atretic (54%), advanced atretic (57%) and late atretic follicles (53%). In conclusion, the maintenance of the preovulatory follicles in superovulated heifers with a GnRH antagonist induced more atresia and a decrease in oestradiol and progesterone concentrations. However, the developmental potential in vitro to day 8 of the oocytes recovered from these atretic follicles was not affected.     

Studies of the pathogenesis of bovine pestivirus-induced ovarian dysfunction in superovulated dairy cattle

Two experiments (Experiment I, n=12 Holstein-Friesian heifers; Experiment II, n=8 Jersey cows) were conducted to investigate the pathogenesis of bovine pestivirus-induced ovarian dysfunction in cattle. In both experiments the cattle were superovulated with twice daily injections of a porcine pituitary extract preparation of follicle stimulating hormone (FSH-P), for 4 days commencing on Day 10+/-2 after a presynchronised oestrus. The heifers received a total dose of 30 mg and the cows 32 mg of FSH-P. Prostaglandin F(2alpha) (PGF(2alpha)) was administered 48 h after commencement of superovulation and all cattle were artificially inseminated (AI) between 48 and 66h after PGF(2alpha) treatment. In both experiments bovine pestivirus seronegative cattle (Experiment I, n=6; Experiment II, n=4) were inoculated intranasally with an Australian strain of non-cytopathogenic bovine pestivirus (bovine viral diarrhoea virus Type 1) 9 days prior to AI. Bovine pestivirus infection was confirmed by seroconversion and/or virus isolation in all of the inoculated cattle, consistent with a viremia occurring approximately between Day 5 prior to AI and the day of AI. Ovarian function was monitored in both experiments by daily transrectal ultrasonography and strategic blood sampling to determine progesterone, oestradiol-17beta, luteinising hormone (LH) and cortisol profiles. Non-surgical ova/embryo recovery was performed on Day 7 after AI. In Experiment II half the cattle were slaughtered on Day 2 and the remainder on Day 8 after AI, and the ovaries submitted for gross and histopathological examination including immunohistochemistry to demonstrate the presence of bovine pestivirus antigen. In both studies, comparisons were made between infected and confirmed uninfected (control) animals. Overall the bovine pestivirus infected cattle had significantly lower (P<0.05) ova/embryo recovery rates compared to the control cattle. There was evidence of either an absence (partial or complete) of a preovulatory LH surge or delay in timing of the LH peak in the majority (90%) of infected heifers and cows, and histologically, there was evidence of non-suppurative oophoritis with necrosis of granulosa cells and the oocyte in follicles from the infected cows. By contrast only 20% of the control heifers and cows had evidence of absence of a pre-ovulatory LH surge. These experiments collectively demonstrate that bovine pestivirus infection during the period of final growth of preovulatory follicles may result in varying degrees of necrosis of the granulosa cells with subsequent negative effects on oestradiol-17beta secretion which in turn negatively affects the magnitude and/or timing of the preovulatory LH surge.     

The variability in the interval between estrus and ovulation in cattle and its determinants

Fertility of Holstein cows has been decreasing for years and, to a lesser extent, the fertility of heifers too but more recently. A hypothesis to explain this phenomenon may be that the chronology of events leading to ovulation is different for those animals bred nowadays when compared to what was reported previously; this would result in an inappropriate time of insemination. Therefore, two experiments were designed to investigate the relationships among estrus behavior, follicular growth, hormonal events and time of ovulation in Holstein cows and heifers. In the first experiment, the onset of estrus, follicular growth, patterns of estradiol-17beta, progesterone and LH, and the time of ovulation were studied in 12 cyclic Holstein heifers that had their estrus synchronized using the Crestar method; this was done twice, 3 weeks apart. The intervals between estrus and ovulation, estrus and the LH peak, and between the LH peak and ovulation were, respectively, 38.5 h +/-3.0, 9.1 +/- 2.0 and 29.4 h +/-1.5 (mean+/- S.E.M). The variation in the interval between estrus and the LH peak explained 80.6% of the variation in the interval between estrus and ovulation. The intervals between estrus and the LH peak, and estrus and ovulation were correlated with estradiol-17beta peak value (r=-0.423, P <0.04 and r=-0.467, P<0.02, respectively). Positive correlation coefficients for the number of follicle larger than 5 mm, and negative correlation coefficients for the size of the preovulatory follicle with the intervals between estrus and LH peak, LH peak and ovulation, and estrus and ovulation suggest an ovarian control of these intervals. In respect to its role to explain the variation in the interval between estrus and ovulation, the variation in the interval between estrus and the LH peak was evaluated further in a second set of experiments utilizing 12 pubertal Holstein heifers and 35 Holstein cows. The duration of the interval between the beginning of estrus and the LH peak was longer in heifers than in cows (4.15 h versus -1.0 h; P <0.002); the variation for this interval was higher in cows than in heifers (S.E.M.= 1.2 h versus 0.8 h; P=0.01). According to the results of these studies it can be proposed that estradiol and other product(s) of ovarian origin regulate not only the duration of intervals between the onset of estrus and the LH surge but also between the LH surge and ovulation. From the results obtained in the first experiment, it may be postulated that differences observed between cows and heifers for the duration of the interval between onset of estrus and the LH surge as well as for the variation of this interval would be observed also for the interval between the onset of estrus and ovulation. Therefore, on a practical point of view, the long interval between the onset of estrus and ovulation and the high variation of this interval, especially in cows, may be a source of low fertility and should be considered when analysing reproductive disorders.