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.     

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.     

Influence of CIDR treatment during superovulation on embryo production and hormonal patterns in cattle

One of the major sources of success in embryo transfer is timing of AI relative to the LH surge and ovulation. The aim of this study was to compare the embryo production following superovulation during a PGF2alpha (control cycle) or a CIDR-B synchronized cycle (CIDR-B cycle). CIDR-B (CIDR-B ND, Virbac, Carros, France) was inserted on Day 11 of a previously synchronized cycle and left for 5 days. A total dose of 350 microg FSH was administered (eight injections i.m. for 4 days; first on Day 13, decreasing doses) and PGFalpha analog (750 microg i.m.: Uniandine ND, Schering-Plough, Levallois-Perret, France) injected at the time of third FSH injection. Artificial inseminations were performed 12 and 24 h after standing estrus (Day 0). Embryos were collected on Day 7. Luteinizing hormone was measured by EIA (Reprokit Sanofi, Libourne, France) from blood samples collected every 3 h for 36 h, starting 24 h after PGF2alpha (control cycle) or 12 h after CIDR-B removal (CIDR-B cycle). The effects of treatment group and interval between the LH peak and AI (two classes, < 10 and > or = 10 h) on embryo production and quality were analyzed by ANOVA. No effect of treatment was observed on embryo production variables. The intervals between the end of treatment and onset of estrus and between end of treatment and LH surge were greater in heifers treated during a control than a CIDR-B cycle, respectively (45.5 +/- 1.4 versus 31.9 +/- 0.7; 42.0 +/- 1.6 versus 31.0 +/- 1.5; P < 0.05), but maximal LH and estradiol concentrations, at the preovulatory surge were similar in control and CIDR-B synchronized heifers. The numbers of viable and Grade I embryos were significantly increased (P < 0.01) when animals had an interval from LH peak to first AI > or = 10 h (7.2 +/- 0.9 and 3.5 +/- 0.6) when compared to shorter intervals (4.2 +/- 1.1 and 2.0 +/- 0.7) whereas total number of embryos was unchanged (11.8 +/- 1.4 versus 10.3 +/- 1.8). It is concluded that late occurrence of LH peaks in relation to estrous behavior is associated with a lower embryo quality when first AIs are performed systematically 12 h after standing estrus. Further studies are needed to know if results may be improved when making AI at a later time after standing estrus or if LH assays are useful to better monitor AI time.     

Improved in vitro embryo development using in vivo matured oocytes from heifers superovulated with a controlled preovulatory LH surge

In bovine in vitro embryo production, the IVM step is rather successful with 80% of the oocytes reaching the MII stage. However, the extent to which the process limits the yield of viable embryos is still largely unknown. Therefore, we compared embryonic developmental capacity during IVC of IVF oocytes which had been matured in vitro with those matured in vivo. In vitro maturation was carried out for 22 h using oocytes (n = 417) obtained from 2- to 8-mm follicles of ovaries collected from a slaughterhouse in M199 with 10% fetal calf serum (FCS), 0.01 IU/mL LH, and 0.01 IU/mL FSH. In vivo matured oocytes (n = 219) were aspirated from preovulatory follicles in eCG/PG/anti-eCG-superovulated heifers 22 h after a fixed time GnRH-induced LH surge; endogenous release of the LH surge was suppressed by a Norgestomet ear implant. This system allowed for the synchronization of the in vitro and in vivo maturation processes and thus for simultaneous IVF of both groups of oocytes. The in vitro developmental potential of in vivo matured oocytes was twice as high (P < 0.01) as that of in vitro matured oocytes, with blastocyst formation and hatching rates 11 d after IVC of 49.3 +/- 6.1 (SEM; n = 10 heifers) vs 26.4 +/- 1.0% (n = 2 replicates), and 39.1 +/- 5.1% vs 20.6 +/- 1.4%, respectively. It is concluded that IVM is a major factor limiting in the in vitro production of viable embryos, although factors such as the lack of normal preovulatory development of IVM oocytes contributed to the observed differences.     

Differential requirement for pulsatile LH during the follicular phase and exposure to the preovulatory LH surge for oocyte fertilization and embryo development in cattle

The requirement for pulsatile LH and the LH surge for the acquisition of oocyte fertilizing potential and embryo developmental competency was examined in Zebu heifers. Follicular growth was superstimulated using the GnRH agonist-LH protocol in which pulsatile LH and the preovulatory LH surge are blocked. In experiment 1, heifers were assigned on Day 7 of the estrous cycle to receive: group 1A (n = 5), 1.5 mg norgestomet (NOR) implant; group 1B (n = 5), GnRH agonist implant. Follicular growth was superstimulated with 2x daily injections of FSH from Day 10 (a.m.) to Day 13 (p.m.), with PGF2alpha injection on Day 12 (a.m.). Heifers were ovariectomized on Day 15 (a.m.) and oocytes were placed immediately into fertilization, without 24 h maturation. Respective cleavage and blastocyst development rates were: group 1A, 0/64 oocytes (0%) and 0/64 (0%); group 1B, 34/70 oocytes (48.6%) and 2/70 (2.9%). In experiment 2, heifers were assigned on Day 7 of the estrous cycle to receive: group 2A (n = 10), 1.5 mg NOR implant; group 2B (n = 10), GnRH agonist implant; group 2C (n = 10), GnRH agonist implant. Follicular growth was superstimulated as in experiment 1 above. Heifers in groups 2A and 2B received an injection of 25 mg LH on Day 14 (p.m.) and all heifers were ovariectomized on Day 15 (a.m.); oocytes were placed immediately into fertilization without 24 h maturation. Cleavage rates were similar for heifers in group 2A (84/175 oocytes, 48.0%), group 2B (61/112 oocytes, 54.5%) and group 2C (69/163, 42.3%). Blastocyst development rates were similar for heifers in group 2A (22/175 oocytes, 12.6%) and group 2B (25/112 oocytes, 22.3%) and lower (P < 0.05) for heifers in group 2C (9/163 oocytes, 5.5%). Oocytes obtained from heifers treated with GnRH agonist, without injection of exogenous LH, underwent cleavage indicating that neither pulsatile LH nor the preovulatory LH surge are obligatory for nuclear maturation in cattle oocytes. Exposure to a surge-like increase in plasma LH increased embryo developmental competency indicating that the preovulatory LH surge promotes cytoplasmic maturation. The findings have important implications for controlling the in vivo maturation of oocytes before in vitro procedures including nuclear transfer.     

Temporal relationships among estrus, body temperature, milk yield, progesterone and luteinizing hormone levels, and ovulation in dairy cows

Estrous cycles of 10 postpartum cyclic Holstein cows were synchronized using prostaglandin f(2alpha) (PGF(2alpha)) given twice 12 d apart to study the relationship of the onset of estrus, body temperature, milk yield, luteinizing hormone (LH) and progesterone concentration to ovulation. Blood samples and body temperatures (vaginal and rectal) were taken every 4 h until ovulation, starting 4 h prior to the second PGF(2alpha) treatment. All cows were observed for estrus following the second administration of PGF(2alpha). Ultrasound scanning of the ovaries commenced at standing estrus and thereafter every 2 h until the disappearance of the fluid filled preovulatory follicle (ovulation). Two cows failed to ovulate and became cystic following the second PGF(2alpha) treatment. The remaining eight cows exhibited a decline in progesterone to <1.0 ng/ml within 28 h, standing estrus and a measurable rise (> 1.0 degrees C) in vaginal but not rectal temperature, and ovulated 90 +/- 10 h after the second PGF(2alpha) treatment. Onset of standing estrus, LH peak and vaginal temperature were highly correlated (P<0.05) with time of ovulation (0.82, 0.81 and 0.74, respectively). Intervals to ovulation tended to depend upon parity. Pluriparous (n = 4) and biparous (n = 4) cows ovulated within 24 and 30 +/- 3 h from the onset of standing estrus; 22 and 31 +/- 2 h from the LH peak; and 22 and 27 +/- 3 h from peak vaginal temperature (mean +/- standard error of the mean), respectively. The results indicated that the onset of standing estrus and rise in vaginal temperature are good practical parameters for predicting ovulation time in dairy cattle.     

Gamete recovery and follicular transfer (graft) using transvaginal ultrasonography in cattle

Current in vitro culture systems may not be adequate to support maturation, fertilization and embryo development of calf oocytes. Thus, we initiated a study to investigate an alternative method of assessing oocyte competence in vivo, initially using oocytes from adults. Experiment 1 was done to determine if follicle puncture would alter subsequent follicle development, ovulation and CL formation. In control (no follicle puncture, n = 3) and treated (follicle puncture, n = 3) heifers, ultrasound-guided transvaginal follicle aspiration was used to ablate all follicles > or = 5 mm at random stages of the estrous cycle to induce synchronous follicular wave emergence among heifers; PGF2 alpha was given 4 d later. Three days after PGF2 alpha, the preovulatory follicle in treated heifers was punctured with a 25-g needle between the exposed and nonexposed portions of the follicular wall, and 200 microL of PBS were infused into the antrum. There was no significant difference between control and treated heifers for mean diameter of the dominant follicle prior to ovulation, the interval to ovulation following PGF2 alpha, or first detection and diameter of the CL. Experiment 2 was designed to assess multiple embryo production following interfollicular transfer of oocytes (i.e., transfer of multiple oocytes from donor follicles to a single recipient preovulatory follicle). Follicular wave emergence was synchronized among control (no follicle puncture, n = 5), oocyte recipient (n = 7) and oocyte donor (n = 5) heifers as in Experiment 1. In control and oocyte recipient heifers, a norgestomet ear implant was placed at the time of ablation and removed 4 d later, at the second PGF2 alpha treatment. In oocyte donor heifers, FSH was given the day after ablation, and, 4 d later, oocytes were collected by transvaginal follicle aspiration, pooled and placed in holding medium. Five or 6 oocytes were loaded into the 25-g needle of the follicle infusion apparatus with < or = 200 microL of transfer medium. Puncture of the preovulatory follicle of recipient heifers was done as in Experiment 1. Immediately thereafter, LH was given to control and oocyte recipient heifers, but only the recipients were inseminated. Ovarian function was assessed by transrectal ultrasonography and control and oocyte recipient heifers were sent to the abattoir 2 or 3 d after ovulation, where excised oviducts were flushed. The interval between LH administration and ovulation (33 to 36 h) was highly synchronous within and among control and oocyte recipient heifers. Four of 5 (80%) ova were collected from controls and 16 of a potential 43 (37%) ova/embryos were recovered from oocyte recipients; 8 embryos from 3 heifers. Thus, the gamete recovery and follicular transfer procedure (GRAFT) did not alter ovulation or subsequent CL formation, and resulted in the recovery of multiple ova/embryos in which a total of 19 oocytes yielded as many as 8 early embryos, a 42% embryo production rate.     

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)     

Effects of copulation on timing of the LH surge following synchronization of estrus in the bovine

Forty-four crossbred postpubertal bovine females were used to study how mating with a bull affected estradiol-17beta (E(2)) secretion and timing of the preovulatory LH surge. Estrous cycles were synchronized with two injections of prostaglandin-F(2alpha) (PGF(2alpha)) 11 d apart. Females were either isolated from males (NE) or exposed to epididectomized bulls (BE) after the second PGF(2alpha) injection. Females exposed to bulls were allowed to mate once and then were separated from the bull. Blood samples were collected at 2-h intervals from the second PGF(2alpha) injection until 12-h post injection to monitor progesterone (P(4)) and luteinizing hormone (LH) concentrations and at hourly intervals from 12 h to 60 h post-injection to monitor LH secretion and timing of the preovulatory LH surge. Samples were also collected at 4-h intervals until 60 h post-injection to monitor estrogen (E(2)) secretion. LH surges were detected in 16 and 14 of 22 females from the BE and NE groups, respectively, during the 60-h period after PGF(2alpha) injection Mean P(4) concentrations and time of P(4) decline to <1 ng/ml were not different between the two treatment groups (P>0.30). Mean E(2) concentration during the 60-h sampling period was different (P<0.003) between BE and NE groups, and a significant treatment effect (P<0.002) occurred 48 h, 52 h and 60 h after the second PGF(2alpha) injection. However, mean LH concentration before the LH surge, duration of the LH surge and peak LH concentration during the surge were not different between the BE and NE groups (P>0.40). Mean time for the second PGF(2alpha) injection to the beginning of the LH surge was 51.6 +/- 1.5 h (X +/- S E) for the females not exposed to bulls and 48.5 +/- 1.4 h for females exposed to bulls (P>0.14). In this study, the presence of and/or mating by a bull did not affect LH secretion or timing of the preovulatory LH surge after PGF(2alpha) administration.     

Use of a GnRH agonist to prevent the endogenous LH surge and injection of exogenous LH to induce ovulation in heifers superstimulated with FSH: a new model for superovulation

A new protocol for superovulating cattle which allows for control of the timing of ovulation after superstimulation with FSH was developed. The preovulatory LH surge was blocked with the GnRH agonist deslorelin, and ovulation was induced by injection of LH. In Experiment 1, heifers (3-yr-old) were assigned to a control group (Group 1A, n = 4) or a group with deslorelin implants (Group 1B, n = 5). On Day -7, heifers in Group 1A received a progestagen CIDR-B((R))device, while heifers in Group 1B received a CIDR-B((R))device + deslorelin implants. Both groups were superstimulated with twice daily injections of FSH (Folltropin((R))-V): Day 0, 40 mg (80 mg total dose on Day 0); Day 1, 30 mg; Day 2, 20 mg; Day 3, 10 mg. On Day 2, heifers were given PGF (a.m.) and CIDR-B((R)) devices were removed (p.m.). Three heifers in Group 1A had a LH surge and ovulated, whereas neither of these events occurred in Group 1B (with deslorelin implants) heifers. In Experiment 2, heifers (3-yr-old) were assigned to 1 of 4 equal groups (n = 6). On Day -7, heifers in Group 2A received a norgestomet implant, while heifers in Groups 2B, 2C and 2D received norgestomet + deslorelin implants. Heifers were superstimulated with FSH starting on Day 0 as in Experiment 1. On Day 2, heifers were given PGF (a.m.) and norgestomet implants were removed (p.m.). Heifers in Groups 2B to 2D were given 25 mg LH (Lutropin((R))): Group 2B, Day 4 (a.m.); Group 2C, Day 4 (p.m.); Group 2D, Day 5 (a.m.). Heifers in Group 2A were inseminated at estrus and 12 and 24 h later, while heifers in Groups 2B to 2D were inseminated at the time of respective LH injection and 12 and 24 h later. Injection of LH induced ovulation in heifers in Groups 2B to 2D. Heifers in Group 2C had similar total ova and embryos (15.2 +/- 1.4) as heifers in Group 2A (11.0 +/- 2.8) but greater (P < 0.05) numbers than heifers in Group 2B (7.0 +/- 2.3) and Group 2D (6.3 +/- 2.0). The number of transferable embryos was similar for heifers in Group 2A (5.8 +/- 1.8) and Group 2C (7.3 +/- 2.1) but lower (P < 0.05) for heifers in Group 2B (1.2 +/- 0.8) and Group 2D (1.3 +/- 1.0). The new GnRH agonist-LH protocol does not require observation of estrus, and induces ovulation in superstimulated heifers that would not have an endogenous LH surge.     

Comparison of two MGA-PGF2alpha systems for synchronization of estrus in beef heifers

Yearling beef heifers (n = 193) were used to evaluate reproductive performance attained with 2 MGA-PGF(2)alpha synchronization systems. These treatments were compared with an untreated control group. The 14-d MGA heifers were synchronized by feeding 0.5 mg MGA/h/d for 14 d. At 17 d after the last MGA feeding, these heifers were injected with PGF(2)alpha (25 mg, im). Heifers in the 7-d MGA treatment group were fed 0.5 mg MGA/h/d for 7 d and received a 25-mg, im injection of PGF(2)alpha on the last day of the MGA feeding period. Heifers in all 3 treatment groups were observed for estrus every 12 h for 7 d beginning 24 h after the PGF(2)alpha injection. Heifers observed in estrus during this 7-d period were artificially inseminated approximately 12 h after the onset of estrus. The percentages of heifers in estrus during the 7-d synchronized period were 75.4, 56.3 and 17.2% for the 14-d MGA, 7-d MGA and control groups, respectively. The estrous responses were significantly different in each treatment. The percentage of heifers in estrus during the peak 24-h period was higher (P < 0.05) in heifers synchronized with the 14-d MGA system than in heifers synchronized with the 7-d MGA system (75.5 vs 50.0%). The synchronized conception rate of the 14-d MGA heifers was significantly higher (65.3%) than that of both the 7-d MGA (41.7%) and control (45.4%) heifers. Synchronized conception rates were similar (P = 0.79) in the 7-d MGA and control treatments. Synchronized pregnancy rates were 55.2, 32.4 and 15.2% for the 14-d MGA, 7-d MGA and control groups, respectively. Both synchronization treatments resulted in significantly higher synchronized pregnancy rates compared with that of the controls. The synchronized pregnancy rate was higher (P < 0.05) in the 14-d MGA group than it was in the 7-d MGA group. The mean day of conception within the breeding season was 11.5 and 9.3 d shorter in the 14-d MGA heifers than in the 7-d MGA and control heifers, respectively. Our results indicate that using the 14-d MGA system to synchronize estrus in beef heifers results in better reproductive performance than that attained in heifers synchronized with the 7-d MGA system or in control heifers.     

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.     

Comparison of oocyte developmental competence and follicular steroid content of nulliparous heifers and cows at different stages of lactation

Reduced reproductive performance and lower conception rates of lactating cows are closely associated with genetic progress for high milk production. In contrast, the fertility of nulliparous Holstein heifers has remained fairly stable over the years and appears to be markedly higher than that of mature lactating cows. Possible differences in oocyte quality and follicular steroid levels, which could be associated with the low fertility of high-lactating cows, were examined in 13-month-old heifers, cows around the time of first AI (60-95 d post-partum, yielding 49+/-2.4 kg/d) and cows at mid-lactation (120-225 d post-partum, yielding 37+/-2.1 kg/d). Estrus was synchronized by two doses of PGF2alpha and follicles (5-8 mm) were aspirated on days 4, 8, 11 and 15 of the cycle by an ultrasound-guided procedure. Oocytes were morphologically examined, matured in vitro, chemically activated and cultured for 8d. Cleavage rate and the proportion of developing parthenogenetic blastocysts were determined on days 3 and 8 post-activation, respectively. On day 17, heifers and cows received additional PGF2alpha and follicular fluids from preovulatory follicles were collected on day 19 perior to the expected estrus. Follicular-fluid volumes were similar in cows and heifers, as were estradiol, progesterone and androstenedione concentrations in the follicular fluid. Percentages of high-grade oocytes, proportions of cleaved oocytes and developed blastocysts did not differ between the groups. Results suggest that the fertility gap between nulliparous heifers and high-lactating cows is not directly related to steroid content in the preovulatory follicular fluid or oocyte developmental competence.     

Timing of the onset and duration of ovulation in superovulated beef heifers

Thirty-two beef heifers were induced to superovulate by the administration of follicle stimulating hormone-porcine (FSH-P). All heifers received 32 mg FSH-P (total dose) which was injected twice daily in decreasing amounts for 4 d commencing on Days 8 to 10 of the estrous cycle. Cloprostenol was administered at 60 and 72 h after the first injection of FSH-P. Heifers were observed for estrus every 6 h and were slaughtered at known times between 48 to 100 h after the first cloprostenol treatment. The populations of ovulated and nonovulated follicles in the ovaries were quantified immediately after slaughter. Blood samples were taken at 2-h intervals from six heifers from 24 h after cloprostenol treatment until slaughter and the plasma was assayed for luteinizing hormone (LH) concentrations. The interval from cloprostenol injection to the onset of estrus was 41.3 +/- 1.25 h (n = 20). The interval from cloprostenol injection to the preovulatory peak of LH was 43.3 +/- 1.69 h (n = 6). No ovulations were observed in animals slaughtered prior to 64.5 h after cloprostenol (n = 12). After 64.5 h, ovulation had commenced in all animals except in one animal slaughtered at 65.5 h. The ovulation rate varied from 4 to 50 ovulations. Approximately 80% of large follicles (> 10 mm diameter) had ovulated within 12 h of the onset of ovulation. Onset of ovulation was followed by a dramatic decrease in the number of large follicles (> 10 mm) and an increase in the number of small follicles (相似文献   

Pulsatile secretion of gonadotrophins, ovarian steroids and ovarian oxytocin during prostaglandin-induced regression of the corpus luteum in the cow

A luteolytic dose (500 micrograms) of cloprostenol was given on Day 12 of the oestrous cycle to 5 heifers. Blood samples were collected simultaneously from the caudal vena cava and jugular vein at 5-20-min intervals from -6 to 0 (control period), 0 to 12 and 24 to 36 h after PG injection. Pulses of LH were secreted concomitantly with pulses of FSH during all sampling periods. However, during the control period separate FSH pulses were detected resulting in a shorter (P less than 0.01) interpulse interval for FSH than LH (93 versus 248 min). LH and FSH pulse frequencies increased (P less than 0.01) beginning 1-3 h after PG to interpulse intervals of 59 and 63 min, respectively, and continued to be maintained 24-36 h after PG. Concomitantly there was a 2-3-fold increase (P less than 0.01) in basal concentrations and pulse amplitude for LH (but not FSH). FSH basal concentrations and pulse amplitudes decreased (P less than 0.05) in 3 heifers 24-36 h after PG. Pulsatile secretion of oestradiol was observed at frequencies similar to LH during the periods 4-12 h (3 heifers) and 24-36 h (2 heifers) after PG, respectively, resulting in higher (P less than 0.05) mean oestradiol concentrations. Progesterone concentrations in the vena cava increased (P less than 0.01) 5-10 min after PG but decreased (P less than 0.01) 67% by 20 min after PG. This decrease was followed by a rise (P less than 0.05) beginning 2-3 h after PG and lasting for an average of 3.3 h.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of brief postponement of the preovulatory LH surge on ovulation rates and embryo formation in eCG/prostaglandin-treated heifers

The aim of this study was to investigate whether prolongation of the period of preovulatory follicular development after superovulation reduces heterogeneity of oocytes of stimulated follicles with respect to the potential to mature, to ovulate, to be fertilized and to develop into embryos. Heifers were treated with eCG on Day 10 and prostaglandin (PG) 48 h later. At the time of eCG administration some of the heifers received a norgestomet implant (N) to suppress the LH surge. After 96 to 104 h, N was removed and an LH surge was induced with GnRH (G) (N/G); the other animals served as controls. Matured oocytes (Experiment A: n=9, 139 [N/G] and 11, 125 [Control] heifers, oocytes), zygotes and oviducts (Experiment B: n=8, 44 [N/G] and 9, 72 [Control] heifers, zygotes) and embryos (Experiment C: n=11, 205 [N/G] and 11, 165 [Control] heifers, embryos) were collected at 22 to 26 h, 38 to 52 h and 7 days after the LH surge, respectively. Hatched blastocyst formation of matured oocytes (Experiment A) was analyzed after 11 days of IVC after IVF. In vivo fertilization rate of zygotes, the presence of periodic acid-Schiff (PAS) positive granules in the oviduct (Experiment B) and stage of development of embryos (Experiment C) were analyzed stereomicroscopically. The mean interval between PG and the LH surge was 53.8+/-3 (SD) (N/G) vs. 42.4+/-4 h (Control). The maximum peripheral estradiol-17beta concentration (529+/-36 [SEM] [N/G] vs. 403+/-17 pmol/L [Control]) and the response to superovulation (25.4+/-2 [N/G] vs. 18.7+/-2 [Control]) were higher in N/G than in Control heifers. Hatched blastocyst formation rate (37.4 [N/G] vs. 33.6% [Control]), in vivo fertilization rate (69.0+/-14 [N/G] vs. 73.0+/-10% [Control]) and the yield of total embryos (3.8+/-1 [N/G] vs. 5.6+/-2 [Control]) did not differ between groups. The percentage of heifers with abundant PAS-positive granules in the distal ampulla (0 [N/G] vs. 31% [Control]) was reduced after N/G treatment. Prolongation of the period of preovulatory follicular development increased the number of mature follicles and ovulations but did not result in higher embryo yield, possibly because of an impaired oviductal environment.     

Follicular growth, endocrine response and embryo yields in sheep superovulated with FSH after pretreatment with a single short-acting dose of GnRH antagonist

The objective of this study was to characterize follicular development, onset of oestrus and preovulatory LH surge, and in vivo embryo yields of sheep superovulated after treatment with a single dose of 1.5mg of GnRH antagonist (GnRHa). At first FSH dose, ewes treated with GnRH antagonist (n=12) showed a higher number of gonadotrophin-responsive follicles, 2-3mm, than control ewes (n=9, 13.5+/-3.8 versus 5.3+/-0.3, P<0.05). Administration of FSH increased the number of >or=4mm follicles at sponge removal in both groups (19.3+/-3.8, P<0.0005 for treated ewes and 12.7+/-5.4, P<0.01 for controls). Thereafter, a 25% of the GnRHa-treated sheep did not show oestrous behaviour whilst none control sheep failed (P=0.06). The preovulatory LH surge was detected in an 88.9% of control ewes and 66.7% of GnRHa-treated sheep. A 77.8% of control females showed ovulation with a mean of 9.6+/-0.9 CL and 3.3+/-0.7 viable embryos, while ewes treated with GnRHa and showing an LH surge exhibited a bimodal distribution of response; 50% showed no ovulatory response and 50% superovulated with a mean of 12.2+/-1.1 CL and 7.3+/-1.1 viable embryos. In conclusion, a single dose of GnRHa enhances the number of gonadotrophin-dependent follicles able to grow to preovulatory sizes in response to an FSH supply. However, LH secretion may be altered in some females, which can affect the preovulatory LH surge and/or can weak the terminal maturation of ovulatory follicles.     

Prostaglandins and preovulatory follicular maturation in mice

Experiments have been carried out in an effort to reverse the indomethacin-induced inhibition of preovulatory follicular development in immature superovulated mice utilizing prostaglandins E2 and F2 alpha. All mice were primed with 5 IU pregnant mare's serum gonadotropin followed 40 h later by 80 IU luteinizing hormone (LH). Animals were sacrificed 10 1/2 or 11 1/2-12 h post-LH, at which time ovaries were fixed and prepared for microscopic observation. Control mice receiving both indomethacin and prostaglandin (PG) vehicles averaged 92% germinal vesicle breakdown, and 82% of maturing oocytes were surrounded by an expanded cumulus oophorus. Ovarian weight increased by 29% and the apical walls of preovulatory follicles demonstrated appreciable thinning following LH administration. In mice receiving indomethacin plus PG vehicle, follicular maturation was suppressed in a dose-dependent manner; in mice receiving 10 mg/kg, less than 50% of the oocytes resumed meiosis and, of these, only 9% were accompanied by cumulus expansion. Ovarian weight gain was also inhibited, and the apical follicle wall exhibited few signs of preovulatory thinning. PGE2 and PGF2 alpha both reversed the inhibition of cumulus and oocyte maturation induced by indomethacin, though PGE2 was more effective. Only PGF2 alpha promoted apical follicular thinning, and neither PG had a significant effect on ovarian weight. We conclude that, in mice, PGs may play an integral role during preovulatory maturation of the oocyte and cumulus, as well as thinning of the apical wall.     

Efficiency of single-sire mating programs with beef bulls mated to estrus synchronized females

Efficiency in reference to pregnancy rates of breeding beef bulls with estrus synchronized cows and heifers was tested. Most bulls (104 of 112) were given a breeding soundness examination and two 10-min libido/serving capacity tests. Females received either Syncro-Mate-B (SMB) or two injections of Prostaglandin F(2)alpha (PGF) to synchronize estrus. They were assigned to single-sire breeding groups with bull-to-female ratios ranging from 1:7 to 1:51. Control groups consisted of untreated females maintained in single-sire breeding pastures with ratios from 1:24 to 1:37. Continuous observations of sexual activity were made for 30 h (SMB) and 48 h (PGF). After the 120-h posttreatment breeding period, females were placed in breeding pastures. During the synchronized breeding period the percentage of pregnant cattle of total treated was 43.5 +/- 1.7% compared (P < 0.01) with 58.9 +/- 3.3% for the control group after 23 d of breeding. At end of 28-d (treated) and 46-d (control) period, the percentage of pregnant females was 75.0 +/- 2.4 and 79.6 +/- 4.7, respectively (P > 0.05). In SMB trials, the percentages of females exhibiting estrus, those serviced at estrus and those pregnant following service during the synchronized breeding period were 90.8 +/- 1.5, 73.3 +/- 4.5 and 56.4 +/- 5.6%, respectively. In PGF trials, the means for these same factors were 78.3 +/- 2.4, 70.4 +/- 5.9 and 56.1 +/- 6.5%, respectively.     

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.