The effects of once or twice daily injections of pFSH on superovulatory response in heifers

Follicle stimulating hormone (FSH) is a glycoprotein hormone with a short half-life and has to be given twice daily for 3-4 days to induce superovulation in heifers. Since such a regimen is time consuming we compared the ovulatory response and yield of embryos in heifers following superovulation with either once or twice daily injections of pFSH for 4 days during the mid-luteal phase of a synchronized estrous cycle or during a prolonged luteal phase in heifers which had been immunized against prostaglandin F2alpha (PG). In Experiment 1, crossbred heifers (n = 42) previously actively immunized against a PG immunogen were superovulated in a 2 (cyclic or persistent corpus luteum) x 2 (once or twice daily injection) factorial plan. The heifers were superovulated with 75 units pFSH, which was injected subcutaneously once (22.5, 22.5, 15 and 15 units per day) or twice daily (9.3 units per injection) for 4 days. In Experiment 2, cyclic crossbred beef heifers (n = 80) were superovulated using pFSH which was given randomly to heifers once daily subcutaneously (T1) or twice daily intramuscularly (T2) using the same daily dose of 9, 7, 5, and 3 mg per day. Estrus was induced in all heifers in both experiments using 500 mug and 250 mug Cloprostenol 12 hours apart on the third day of pFSH injections. All heifers were inseminated twice with frozen-thawed semen at 12 and 24 hours after the onset of standing estrus or at 56 and 72 hours after the first PG if estrus was not observed. Embryos were recovered at slaughter and graded on a scale of 1 to 5 (1 = excellent, 5 = degenerated). Data were recorded for the number of corpora lutea (CL), large (>/=10 mm) and medium (5-9 mm) follicles, number of embryos recovered and embryo morphology. Data were analyzed by least squares analysis of variance procedures. In Experiment 1, there was no difference in ovulation rate between main effects. Fewer embryos were recovered from heifers with a persistent corpus luteum (pCL) and injected once daily (1.71+/-.75 vs 5.75+/-1.27) than from any other group. Heifers with pCL yielded lower (P < 0.05) numbers of freezable embryos than cyclic animals, regardless of injection regimen. In Experiment 2, T2 heifers had a significantly higher number of CL (16.4+/-1.7 vs 7.7+/-1.7; P = 0.0003), large follicles (4.1+/-0.5 vs 2.8+/-0.5; P = 0.04), medium follicles (6.4+/-0.7 vs 4.4+/-0.7; P = 0.04), embryos recovered (9.6+/-1.1 vs 4.9+/-1.1; P = 0.0025) and freezable embryos (4.7+/-0.7 vs 2.1+/-0.7; P = 0.014) than T1 heifers. It is concluded that a single daily subcutaneous injection of pFSH results in a lower superovulatory response than the twice daily regimen in heifers.     

Changes in peripheral inhibin levels and follicular development following treatment of buffalo with PMSG and Neutra-PMSG for superovulation

Fourteen buffalo were synchronized by administration of a prostaglandin (PG) salt Lutalyse in a double injection schedule, with a single intramuscular (im) injection of 25 mg at Day -13, followed by 30 mg and 20 mg im 12 h apart on Day 0 of the experiment. The 30-mg PG injection was designated as 0 h of the experiment. Group I animals (n = 4) received saline and served as the controls, while animals in Groups II and III (n = 5 each) received PMSG (2500 IU im at -48 h. Group III animals were administered 5 ml Neutra-PMSG intravenously at 60 h. Blood samples were collected every 48 h from Day -12 to Day -4, every 24 h from Day -4 to Day 0, every 3 h from Day 1 to Day 4 and every 24 h from Day 5 to Day 10 of experiment for the measurement of peripheral plasma inhibin concentrations by RIA. The number of large follicles (> 10 mm diameter) in animals of Groups II and III was assessed by ultrasonography on Days -2, -1, 0, 1, 2, 5 and 7 of the experiment. Treatment with PMSG of Group II animals resulted in a significant increase (P < 0.05) in plasma inhibin concentrations over that of control animals of Group I at 24 to 99 h, with a peak inhibin concentration of 1.01 +/- 0.31 ng/ml at 48 h. Treatment with Neutra-PMSG in Group III animals caused a significant reduction (P < 0.05) in the peripheral inhibin concentrations at 84 to 120 h and in the number of large unovulated follicles at 168 h compared with that in Group II animals. Peripheral inhibin levels in Group III animals came down to those of Group I after 21 h of Neutra-PMSG treatment. These results suggest that treatment of buffalo with PMSG for superovulation causes a marked rise in peripheral inhibin concentrations. Administration of Neutra-PMSG after PG treatment reduces the peripheral inhibin concentrations and the number of large unovulated follicles.     

Effects of dose and route of administration of cloprostenol on luteolysis,estrus and ovulation in beef heifers

Four experiments were conducted (with crossbred beef heifers) to determine the effects of dose and route of administration of cloprostenol on luteolysis, estrus and ovulation. In Experiment 1, 19 heifers with a CL > or = 17 mm in diameter were randomly allocated to receive cloprostenol as follows: 100 microg s.c., 250 microg s.c., or 500 microg i.m. Heifers given 100 microg s.c. had a longer (P<0.03) interval (120.0 h+/-10.7 h; mean+/-S.E.M.) from treatment to ovulation than those given either 250 microg s.c. or 500 microg i.m. (92.0 h+/-7.4 h and 84.0 h+/-8.2 h, respectively). In Experiment 2, 28 heifers were given porcine LH (pLH), followed in 7 days by cloprostenol (same doses and routes as in Experiment 1), and a second dose of pLH 48 h after cloprostenol. Luteolysis occurred in all heifers, and no difference was detected among treatment groups in the interval from cloprostenol treatment to ovulation (mean, 101 h; P<0.9). In Experiment 3, 38 heifers at random stages of the estrous cycle (but with plasma progesterone concentrations > or =1.0 ng/ml) received 500 or 125 microg cloprostenol by either i.m. or s.c. injection (2/2 factorial design). There was no difference (P<0.4) among groups in the proportions of heifers that were detected in estrus or that ovulated. However, the interval from cloprostenol treatment to estrus was shorter (P<0.02) in the group that received 500 microg i.m. (58.5h) than in the other three groups (500 microg s.c., 75.0 h; 125 microg i.m., 78.0 h; and 125 microg s.c., 82.3h). In Experiment 4, 36 heifers were treated (as in Experiment 3) on Day 7 after ovulation. The proportions of heifers detected in estrus and ovulating after 125 microg s.c. (33 and 44%, respectively) or 125 microg i.m. (55 and 55%) were lower (P<0.05) than in those that received 500 microg s.c. (100 and 100%), but not different from those receiving 500 microg i.m. (78 and 89%, respectively). Overall, ovulation was detected in 9/18 heifers given 125 microg and 17/18 heifers given 500 microg of cloprostenol, on Day 7 (P<0.01) and was detected in 17/20 heifers given 125 microg and 18/18 heifers given 500 microg of cloprostenol, at random stages of the estrous cycle (P>0.05). Although there was no significant difference in luteolytic efficacy between i.m. and s.c. injections of the recommended dose (500 microg) of cloprostenol, variability in responsiveness to a reduced dose depended upon CL sensitivity, therefore, reduced doses cannot be recommended for routine use.     

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

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

Effect of season and gonadotropins on the superovulatory response in camel (Camelus dromedarius)

The purpose of the present investigation was to study the extent to which season and the gonadotropin preparation interferes with the superovulatory response in the dromedary. Adult camels were treated for superovulation during the breeding (November to April) and non-breeding season (May to October). Animals were synchronized by daily i.m. injections of progesterone (125 mg/animal/day, Jurox, UK) for 10 to 14 days. Superovulation was induced by 400mg pFSH alone (Follitropin V, Vetrepharm, Canada) administered in eight descending doses at 12h intervals or a combination of PMSG (2000IU, Folligon, Intervet, The Netherlands), injected with last injection of progesterone and 400mg pFSH in eight descending doses. The follicular development was daily assessed by ultrasonography of the ovaries. The donors were classified as per their response to the superovulatory treatment into very good (>10 follicles), good (5-10 follicle), poor (2-4 follicles) or no response (1 or no follicle) on each ovary. Ovulation was induced by injecting 3000 IU hCG (Chorulon, Intervet) at the time of first mating. The donors were mated twice at an interval of 12h when all or most of the follicles reached to a size of about 1.0-1.7 cm. Camels were flushed non-surgically on Day 6 or 7 after the ovulation. The proportion of camels showing very good response during the breeding as well as non-breeding season was higher (P<0.05) when a combination of pFSH and eCG was used compared with pFSH only. There was no difference (P>0.05) in the proportion of donors flushed successfully (embryos recovered) when treated either with a combination of pFSH and eCG or pFSH alone during the breeding and non-breeding season. The rate of recovery of ova/embryos and proportion of transferable embryos was higher (P<0.05) when donors were treated with pFSH+eCG compared with pFSH only during the breeding as well as non-breeding season. The results may indicate that ova/embryo recovery rate of the dromedary is influenced by the gonadotropin preparation but is not appreciably affected by the season.     

Superovulatory response following transvaginal follicle ablation in cattle

A study was designed to compare superovulatory responses in cattle when gonadotropin treatment followed 1 of 3 different treatments to synchronize follicular wave emergence. Animals at unknown stages of the estrous cycle were randomly assigned to 3 groups: ablation of the 2 largest follicles per pair of ovaries (n = 21); ablation of all follicles > or = 5 mm (n = 19); or intramuscular administration of 5 mg estradiol-17beta plus 100 mg progesterone (n = 23). All animals were given a CIDR-B intravaginally at the time of the respective treatments. Gonadotropin treatment, initiated 1 d after follicle ablation or 4 d after estradiol plus progesterone treatment, in the respective groups, consisted of 200 mg of pFSH divided in decreasing doses twice daily over 4 d. Cloprostenol (500 microg) was given at 48 and 60 h after the first pFSH treatment; CIDR-B devices were removed at the time of the second cloprostenol treatment. Ovarian ultrasonography was done on the days of CIDR-B insertion, first gonadotropin treatment, and at 36 and 72 h after CIDR-B removal. Cattle were inseminated twice, at 60 and 72 h after the first injection of cloprostenol. Ovarian and ova/embryo data were collected at slaughter 5, 6 or 7 d after insemination. No differences were detected among groups in the number of follicles > or = 8 mm at the time of first insemination (20.4 +/- 1.7 vs 16.6 +/- 2.0 vs 19.9 +/- 2.3; P > 0.05). At slaughter, no differences were detected among groups in the numbers of CL (23.3 +/- 1.9 vs 17.9 +/- 1.9 vs 20.1 +/- 2.6; P < 0.05), unovulated follicles > or = 8 mm (2.2 +/- 0.5 vs 2.1 +/- 0.3 vs 3.7 +/- 0.9; P < 0.05), ova/embryos (11.0 +/- 1.4 vs 12.2 +/- 1.3 vs 8.5 +/- 1.3; P < 0.05), fertilized ova (9.4 +/- 1.3 vs 10.1 +/- 1.2 vs 7.5 +/- 1.1; P < 0.05) or transferable embryos (8.2 +/- 1.2 vs 8.4 +/- 1.3 vs 6.5 +/- 0.9; P < 0.05). Variation in the numbers of CL (P = 0.1) and unovulated follicles > or = 8 mm (P < 0.01) was lower in the ablation groups than in the steroid-treated group. Results suggest that follicle ablation is as effective as estradiol plus progesterone in synchronizing follicular wave emergence for superstimulation in cattle, and that ablation of the 2 largest follicles is as efficacious as ablating all follicles > or = 5 mm.     

Failure of the LH-releasing hormone agonist, deslorelin, to prevent development of a persistent follicle in heifers synchronized with norgestomet

The use of exogenous progestagens for estrus synchronization in cattle can result in a persistent dominant follicle which is associated with reduced fertility. We examined whether the LHRH agonist, deslorelin, would prevent the formation of a persistent follicle in heifers synchronized with norgestomet. The estrous cycles of heifers were synchronized with cloprostenol, and on Day 7 of the ensuing cycle the heifers received one of the following treatments for 10 d: Group C (n = 5), untreated control; Group N (n = 6), injection of a luteolytic dose of cloprostenol on Days 7 and 8 and implant of norgestomet from Day 7 to Day 17 (i.e. typical 10-day norgestomet implant period); Group D (n = 6), injection of cloprostenol on Days 7 and 8 and implants of deslorelin from Day 7 to Day 17; Group ND (n = 6), injections of cloprostenol and both norgestomet and deslorelin implants as above. Follicle growth was monitored using ultrasonography. Group-N heifers showed continued follicle growth and had larger follicles on Day 17 of the cycle than Group-C heifers (16.8 +/- 1.6 and 10.4 +/- 1.6 mm). Follicle growth for Group-D and ND heifers was similar and variable, and seemed to depend on follicle status at the initiation of treatment. Heifers with follicles of 5 to 10 mm (n = 9) in diameter either showed no follicle growth (2 9 ) or developed large follicles (7 9 ), while heifers with follicles approximately 12 mm (n = 3) in diameter showed follicle atresia with no further significant growth. On Day 17, size of the largest follicle was similar for Group-ND (14.3 +/- 2.9) and Group-D (16.8 +/- 1.6) heifers. Heifers in Group N showed estrous behavior 1.8 +/- 0.2 d after treatment, whereas heifers in Groups D and ND did not show estrus for 2 to 4 wk. The results show that combined treatment with progestagen and an LHRH agonist does not consistently prevent the development of a persistent dominant follicle and that return to estrus can be delayed after treatment with an LHRH agonist.     

Embryo production and endocrine response in ewes superovulated with PMSG, with or without monoclonal anti-PMSG administered at different times

Mature nonlactating Altamurana ewes (n = 168) were synchronized in the seasonal anestrus period with FGA-impregnated intravaginal pessaries for 12 d. In Experiment 1, 48 ewes were divided into a 3 x 4 factorial design for anti-PMSG monoclonal antibody (AP) bioassay test. Concomitant injections of PMSG (1000, 1500, 2000 IU) and AP (0, 1, 2, 3 microl/IU PMSG) were given, and ovarian response was evaluated by laparoscopy. In Experiment 2, 120 ewes were divided into 8 experimental groups (n = 15 per group). The ewes treated with 1000 or 1500 IU PMSG at -24 h from sponge removal were given AP intravenously at 50 h after pessary withdrawal, 12 or 24 h after the onset of estrus, while the controls did not receive AP. Blood samples were collected from ewes (n = 6) treated with 1500 IU PMSG with or without anti-PMSG. Ovarian response and embryo production were evaluated on Day 7 after sponge removal upon laparotomy. It was found that 1 microl AP was effective in neutralizing 1 IU PMSG. No significant differences in serum concentrations of progesterone were observed among the groups of superovulated ewes. Estradiol-17 beta levels were reduced following AP treatment 12 h after the onset of estrus. At a lower dosage of superovulatory treatment (1000 IU PMSG), AP injected at 12 or 24 h after the onset of estrus significantly lowered large follicles (P < 0.01) and increased the rate of ovulation (P < 0.05). Moreover, embryo production showed a more than two-fold increase (P < 0.01) of viable embryos following AP injection at 12 or 24 h after the onset of estrus (3.2 to 3.3 vs 1.3, with vs without anti-PMSG). It is concluded that superovulatory treatment with 1000 IU PMSG plus AP administered at a fixed time after the onset of estrus may improve ovarian response and the yield of viable embryos in ewes.     

Superovulation in heifers given FSH initiated either at day 2 or day 10 of the estrous cycle

The aim of this study was to determine if initiation of superovulation in heifers during the time of development of the first dominant follicle (Days 2 to 6) would give equivalent ovulation and embryo production rates as treatment initiated at mid-cycle. Estrus was synchronized in 60 beef heifers using luprostiol (PG) and they were randomly allocated to treatment with 4.5, 3.5, 2.5 and 1.5 mg of porcine follicle stimulating hormone (FSH) administered twice daily, either on Days 2, 4, 5 and 6 (Day-2 group), respectively, or with similar doses at four consecutive days during mid-cycle (Day-10 group, initiation on Day 9 to 11). All heifers received 500 mug cloprostenol at the fifth FSH injection and 250 mug at the sixth injection. Blood samples for progesterone determination were collected at the time of FSH injections. Heifers were slaughtered 7 d post estrus, and the number of ovulations and large follicles (>/=10mm) were determined on visual inspection of the ovary. Following flushing of the uterine horns the quality of embryos and the fertilization rate were determined. Significant differences between treatments were determined using a two-sided t-test, and frequency distributions were compared using Chi-square tests. The mean number (+/-SEM) of ovulations for heifers in the Day-10 group was 12.9+/-1.0, and 8.5+/-0.9 embryos were recovered. Both the number of ovulations (6.7+/-0.8) and embryos recovered (4.1+/-0.6) were lower (P=0.0001) in heifers in the Day-2 group. Following grading based on a morphological basis, a higher number (P=0.002) of embryos was categorized as Grades 1 and 2 (4.1+/-0.6) and Grade 3 (2.1+/-0.4) in Day-10 heifers than in the Day-2 group (Grade 1 and 2, 1.9+/-0.3; Grade 3, 0.7+/-0.2). The number of Grade 4 and 5 embryos (Day 10, 1.6+/-0.2; Day 2, 1.4+/-0.2) and the number of unfertilized ova (Day 10, 0.7+/-0.4; Day 2, 0.2+/-0.1) did not differ between treatments. Progesterone concentrations were lower (P=0.0001) in Day-2 heifers at FSH treatment prior to prostaglandin, and the decline was more rapid following prostaglandin injection at Day 5 (P=0.02). Results of this study indicate that the number of ovulations and embryos recovered was lower in heifers when FSH treatment was initiated on Day 2 compared with Day 10 of the estrous cycle.     

Effects of follicle-stimulating hormone with and without luteinizing hormone on serum hormone concentrations, follicle growth, and intrafollicular estradiol and aromatase activity in gonadotropin-releasing hormone-immunized heifers

To evaluate the roles of FSH and LH in follicular growth, GnRH-immunized anestrous heifers (n = 17) were randomly assigned (Day 0) to one of three groups (n = 5 or 6). Group 1 received i.m. injections of 1.5 mg porcine FSH (pFSH) 4 times/day for 2 days; group 2 received i.v. injections of 150 microg pLH 6 times/day for 6 days; group 3 received both pFSH and pLH as described for groups 1 and 2. After slaughter on Day 6, measurements were made of follicle number and size, and follicular fluid concentrations of progesterone (P(4)), estradiol (E(2)), and aromatase activity. Injection of pFSH increased (P: < 0.01) the serum concentrations of FSH between 12 and 54 h. Infusion of pLH increased (P: < 0.05) mean and basal concentrations of LH and LH pulse frequency. Serum E(2) concentrations were higher (P: < 0.05) for heifers given pFSH + pLH than those given either pFSH or pLH alone. There was no difference (P: > or = 0.24) between treatments in the number of small follicles (<5 mm). Heifers given pFSH or pFSH + pLH had more (P: < or = 0.02) medium follicles (5.0-9.5 mm) than those that were given pLH alone (none present). Heifers given pFSH + pLH had more (P: = 0.04) large follicles (> or =10 mm) than those given either pLH or pFSH alone (none present). Overall, only 1 of 35 small follicles and 2 of 96 medium follicles were E(2)-active (i.e., E(2):P(4) >1.0), whereas 18 of 21 large follicles (all in the pFSH + pLH treatment) were E(2)-active; of these, 8 of 18 had aromatase activity. Concentrations of E(2) and E(2) activity in follicular fluid were correlated (r > or = 0.57; P: < 0.0001) with aromatase activity in heifers given pLH + pFSH. In conclusion, pLH failed to stimulate follicle growth greater than 5 mm; pFSH stimulated growth of medium follicles that were E(2)-inactive at slaughter and failed to increase serum E(2) concentrations; whereas pFSH + pLH stimulated growth of medium follicles and E(2)-active large follicles, and a 10- to 14-fold increase in serum E(2) concentrations.     

Ovarian superstimulatory response relative to follicular wave emergence in heifers

Two experiments were designed to evaluate the responsiveness of beef heifers to superstimulatory treatments administered during the first follicular wave. Heifers were examined daily (Experiment 1) or twice daily (Experiment 2) by ultrasonography to determine the status of follicular wave development and the day of initiation of superstimulatory treatment. Heifers in both experiments were superstimulated with a total dose of 10 ml Folltropin (equivalent to 200 mg of NIH-FSH-P1), divided into 10 equal intramuscular injections over 5 days. On the last day of treatment, heifers received 500 mug of cloprostenol after each injection of Folltropin to induce luteolysis. In the respective groups, superstimulatory treatments were initiated on Day -1, Day 0 (day of ovulation) or Day +1 for Experiment 1, and on Day -1, Day 0, Day +1 or Day +2 for Experiment 2. In Experiment 1, the number of ovulations in each ovary was assessed by ultrasonography and by counting the number of corpora lutea (CL) in each ovary at slaughter. The correlation between both techniques for assessing ovulatory response was high (r= 0.98; P< 0.0001), and there was no significant difference in the mean number of ovulations detected by ultrasound (5.7+/-1.1) versus the mean number of CL counted at slaughter (6.2+/-1.2). In Experiment 1, the mean (+/- SEM) number of CL counted at slaughter in heifers treated on Day -1 (9.4+/-3.8) and Day 0 (7.3+/-1.6) was higher (P< 0.05) than that of heifers treated on Day +1 (0.7+/-0.3). The mean number of follicles >/=7 mm in diameter on the last day of treatment was also higher (P<0.05) in the Day -1 group compared with the Day +1 group; the Day 0 group was intermediate. In Experiment 2, the mean number of ovulations was higher (P< 0.05) in the Day 0 group (18.4+/-3.4) than the Day -1 (9.5+/-2.3), Day +1 (6.7+/-2.2) or Day +2 (6.5+/-2.3) groups. Heifers in the Day -1, and Day 0 groups had more (P< 0.05) follicles >/=7 mm at the end of treatment compared with heifers in the Day +1 or the Day +2 group. The stated hypothesis was supported: exogenous FSH treatment initiated at the time of wave emergence, near the expected time of the endogenous wave-eliciting FSH surge, has a positive effect on the superstimulatory response. A higher superstimulatory response was elicited when treatments were initiated on the day of, or the day before, wave emergence compared with that of later treatments.     

Superovulatory response of ovarian follicles of Wave 1 versus Wave 2 in heifers

Experiments were designed to test the hypotheses that ovarian follicular response to superstimulatory treatment initiated during Wave 1 is equivalent to that of Wave 2, and recovery rate and quality of ova embryos derived from follicles of Wave 1 are equivalent to those derived from follicles of Wave 2. In a preliminary experiment (Experiment 1), heifers were given Folltropin-V (20 mg NIH-FSH-P1, im, bid for 5 d) beginning the day after emergence of the first (n=10) or second (n=10) follicular wave of the estrous cycle, equivalent to approximately Day 1 and Day 10, respectively (Day 0=ovulation). Luteolysis was induced with cloprostenol (500 mug im, bid) on the fourth day of treatment. Fewer (P<0.05) ovulations per heifer were induced in the Wave 1 group than in the Wave 2 group (4.6+/-1.0 vs 9.1+/-1.3). However, the interval from wave emergence to initiation of treatment was found, in retrospect, to have been longer (P<0.05) in the Wave 1 group, i.e., treatment was initiated relatively later with respect to wave emergence. Experiment 2 was designed to correct this disparity and to initiate the same treatment protocol on the day of wave emergence rather than the day after (n=21 per Wave group). There was no difference between Wave 1 and Wave 2 groups in the interval from wave emergence to initiation of treatment (0.4+/-0.1 d), the number of ovulations detected by ultrasonography (6.6+/-1.0 vs 8.2+/-1.7), the number of CL detected at slaughter (6.5+/-0.9 vs 8.1+/-1.8), the total number of ova embryos recovered (5.2+/-0.7 vs 5.1+/-0.8), or the number of fertilized embryos collected (2.8+/-0.6 vs 3.0+/-0.6). In addition, there was no difference between groups in the proportion of heifers that ovulated in either experiment; collectively, luteolysis and ovulation was induced in 58 of 60 heifers. The results supported the general hypothesis that follicles and oocytes of the first and second follicular waves are equivalent in the response to superstimulatory treatment. Regardless of which follicular wave, initiation of treatment near the time of wave emergence appears critical for maximal superovulatory response. Because of the consistency in the time of emergence of Wave 1 (day of ovulation) and equivalence in superovulatory response, use of Wave 1 rather than subsequent follicular waves may be more convenient and time-sparing in superovulation programs; the day of estrus (day before ovulation) may be used as a consistent point of reference for the start of treatment.     

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.     

The effect of immunization of heifers against alpha 1-26 inhibin conjugate on the superovulatory response to pFSH

A total of 121 heifers was blocked by time and diet and then randomly assigned, within block, to an inhibin-immunized (I) or a control (C) group. Immunized heifers (n = 61) received a primary immunization (Day 0) with 0.33 mg of an alpha 1-26 bovine inhibin fragment-human serum albumin (HSA) conjugate injected with non-ulcerative Freund's and DEAE-dextran adjuvants. Booster injections were given on Days 28 and 56. Control heifers (n = 60) received HSA and adjuvants. On Days 56 and 83 the ovaries of heifers were examined by ultrasound to determine the ovulation rate, and blood samples were collected for antibody titer determination. On Day 84, 61 heifers (C, n = 30; I, n = 31) received a total of 24 mg of porcine follicle stimulating hormone (pFSH), while 60 heifers (C, n = 30; I, n = 30) received 12 mg im pFSH, which was administered twice daily for 4 d in decreasing doses during the mid-luteal phase of the estrous cycle. Luteolysis was induced with prostaglandin F(2alpha) analog. The heifers were artificially inseminated and were slaughtered 7 d after estrus. Embryos were recovered and morphologically graded on a scale of 1 to 5 (1 = excellent; 5 = degenerated). Antibody titers (percentage binding at 1:125 serum dilution) differed (P < 0.01) between Group C and I heifers at Days 56 (0.1 vs 30%) and 83 (0.2 vs 37%), and 26% of Group I and 1% of Group C heifers (P < 0.01) had twin ovulations on Day 83. The mean number of embryos recovered was reduced (P = 0.02) in Group I heifers (8.9 +/- 1.2) compared with C heifers (12.1 +/- 1.1); however, the mean number of freezable embryos (Grades 1 and 2) was not affected (P = 0.61) by immunization, and there was no interaction with pFSH (P = 0.36). Ovulation rate as well as embryo yield and quality were not different (P > 0.10) between Group C and I heifers when 12 mg pFSH were administered; however, immunization decreased the superovulatory response to 24 mg of pFSH.     

Superovulatory response and quality of embryos recovered from anestrus ewes after a single injection of porcine FSH dissolved in polyvinylpyrrolidone

The effects of a single injection of porcine FSH (pFSH) administered in long acting vehicle on the superovulatory response of milk (Sarda breed) sheep were determined during the anestrous season. The sheep (n=42), synchronized with intravaginal sponges (40 mg fluorogestone acetate -FGA- for 14 d) were submitted 24 h before sponge removal to three different superovulatory treatments. Group 1 (n=16) was treated with a single intramuscular (im) injection of 16 mg of pFHS dissolved in 30 % polyvinylpyrrolidone (PVP); Group 2 (n=12) was injected im with 6, 5, 3 and 2 mg of pFSH every 12 h over 2 d; Group 3 (n=14) was given 800 IU of PMSG and 12 mg of pFSH. All sheep were mated with a fertile ram. Embryos were recovered surgically at Day 7 of sponge removal and graded for the quality according to their morphology. The percentage of good quality embryos recovered was 84% in Group 1, 68% in Group 2 and 77% in Group 3. Data for the onset of estrus, number of corpora lutea (CL), number of unovulated follicles, embryo recovery rate, embryo quality and fertilization rate were recorded for the 3 groups. The onset of estrus, number of CL, number of unovulated follicles, fertilization rate and number of good quality embryos did not differ significantly among the 3 groups. The embryo recovery rate was significantly lower in the group treated with PMSG-FSH (Group 3) than in the 2 other groups. It is concluded that during the anestrous season a single injection im of pFSH results on average in a superovulatory response as good as the more traditional treatments like multiple injections of pFSH and PMSG-pFSH combined.     

A histological study of corpora lutea from superovulated beef heifers

There is great variability between animals in the number of viable embryos produced following different superovulation regimens. It is not clear if all the follicles that ovulate produce healthy oocytes and form normal corpora lutea (CL) following superovulation. The objective of this study was to assess and compare CL from heifers undergoing three superovulatory regimes with CL from unstimulated heifers on the basis of morphology and morphometric analysis of luteal cells.Beef heifers were superovulated using either: (a) 24 mg porcine follicle stimulating hormone (pFSH) given twice daily over a 4 day period in decreasing doses commencing on day 10 of the oestrous cycle; (b) a single injection of 2000 IU pregnant mare serum gonadotrophin (PMSG) given on day 10 of the cycle; (c) as in (b) but followed by 2000 IU anti-PMSG (IgG to neutralise endogenous PMSG) at the time of the first insemination which was 12–18 h after the onset of oestrus (n = 33 per treatment). Luteolysis was induced 48 h after initial gonadotrophin administration and CL were collected on day 7 of the subsequent cycle and from ten unstimulated heifers (controls) at the same stage of the oestrous cycle. CL morphology was studied at light and electron microscopy levels. Morphometric analysis was performed on luteal cells. Subcellular morphology was similar in heifers from all groups. However, CL from superovulated heifers had more connective tissue than CL from control heifers; the connective tissue content of CL in the anti-PMSG-treated group was particularly marked. Both large and small luteal cells in the heifers receiving anti-PMSG had significantly smaller (P < 0.001) area and sphere volume than similar cells from CL of heifers in the other groups.     

Superovulation of Holstein heifers by a single subcutaneous injection of FSH dissolved in polyvinylpyrrolidone

This study was undertaken to determine whether a single injection of porcine FSH (pFSH) would induce a superovulatory response in cattle. Holstein heifers were given a single injection of pFSH (30 mg, s.c.) dissolved in saline (Group 1, n = 5); 50% polyvinylpyrrolidone (PVP; Group 2, n = 5); or 25% PVP (Group 3, n = 4). Group-4 heifers (n = 5) were given multiple intramuscular injections of pFSH every 12 h for 3 d at decreasing doses, for a total of 30 mg. All animals received a single injection of 750 microg PGF2 alpha 48 h after the initiation of pFSH treatment. Animals exhibiting estrus were artificially inseminated twice throughout estrus. Ova and embryos were recovered nonsurgically. Ovaries were examined by transrectal ultrasonography or by palpation per rectum on Day 7 or 8 of estrus. Plasma concentrations of pFSH, bovine FSH progesterone, estradiol-17 beta and inhibin were determined by specific radioimmunoassays. The number of corpora lutea (CL) and the numbers of total and transferable embryos which were detected and recovered in Groups 2 and 3 were equivalent to the numbers detected and recovered in Group 4. In Group 1, however, only 1 of 5 animals ovulated even a single oocyte. The present study demonstrated that only a single injection of pFSH dissolved in PVP was capable of inducing a superovulatory response by maintaining a high plasma FSH concentration to allow for the recovery of a sufficient number of embryos for transplantation.     

Effects of progesterone pretreatment on fertility of gilts mated at an induced pubertal estrus

The effects of progesterone (100 mg/d, im) on pubertal fertility were examined in 247 gilts over 3 experiments. In the first experiment, 128 gilts were exposed to progesterone for 0, 2, 4 or 8 d before receiving PMSG (750 IU) 1 d later. The number of large (>4mm) follicles or corpora lutea (CL) were determined on the day of PMSG injection, Day 0 (onset of estrus), Day 1 or Day 10 (n=8). In the second experiment, embryonic survival was observed in 68 gilts after induction of estrus with PG600 (400 IU PMSG, 200 IU hCG). Vehicle or progesterone was previously administered for 2 d to these gilts, and they were allowed 1, 2, or 3 d between the last progesterone injection and PG600. In Experiment 3, a field trial was conducted in which 51 gilts received vehicle or progesterone for 2 d, followed by a 3-d interval before injection of PG600 to induce estrus. The gilts were allowed to farrow. Treatment with progesterone 1 d before PMSG increased (P<0.05) the number and size of preovulatory follicles and increased (P<0.05) the number of corpora lutea. However, the percentage of gilts pregnant by Day 10, the number of embryos recovered per gilt and embryonic survival were reduced (P<0.05) with progesterone pretreatment. Utilizing a smaller dose of PMSG (750 vs 400 IU) with PG600 negated the effects of progesterone pretreatment on ovulation rate. When the interval between progesterone treatment and PG600 was lengthened to 3 d embryonic survival to Day 30 improved but was similar to that of the vehicle/PG600 treated gilts. Fertility, as defined as conception rate and litter size, was similar between gilts exposed to vehicle or progesterone. These results indicate that pretreatment with progesterone up to the day before PMSG might improve follicular development and ovulation rate at the pubertal estrus with a dose of 750 IU of PMSG but not with the 400 IU (PG600). Reducing the dose of PMSG to 400 IU and allowing for 3 d between progesterone and gonadotropin treatment reduced the incidence of uterine infections but resulted in a fertility rate similar to that of gilts receiving PG600 alone.     

Ovarian follicular superstimulation and oocyte maturation in the anoestrous southern hairy-nosed wombat, Lasiorhinus latifrons

This study investigates the effect of three exogenous gonadotrophin regimens on ovarian follicular development in southern hairy-nosed wombats during the non-breeding season. Females were given either porcine follicle stimulating hormone (pFSH; total of 200 mg at 12 h intervals over 7 (Group 1), or 4 days (Group 2)), or pregnant mares' serum gonadotrophin (PMSG; single dose of 150 I.U. (Group 3)). In all treatment groups 25 mg of porcine luteinising hormone (pLH) was used to trigger maturation; Groups 1 and 2 received pLH 12 h after the final pFSH injection and Group 3 received pLH 72 h after PMSG. The results showed Group 1 produced significantly more follicles per ovary (5.91+/-1.28) than Group 2 (1.67+/-0.62), or Group 3 (2.17+/-1.16) at p<0.05. Control females received saline injections concurrently with the three treatment groups (n=6; 2 control animals for each treatment group). No follicular development occurred in any control female. Analysis of oocyte nuclear status revealed that while oocytes from all three treatment groups had resumed meiosis, only those in Group 1 (7-day pFSH/pLH treatment) progressed to metaphase II. These results have implications for the development of assisted breeding strategies in this species.     

Ultrasonographic determination of ovarian follicular. Development in superovulated heifers pretreated with FSH-P at the beginning of the estrous cycle

On Day 3 of the estrous cycle (estrus = Day 0), dairy heifers were given either 10 mg i.m. FSH-P (FSH-P primed; n = 9) or a saline vehicle (saline primed; n = 9). On Day 10, all heifers were superovulated with FSH-P (total = 27.7 mg i.m.) in declining doses over 5 d. Heifers were inseminated artificially at estrus. From Day 2 until estrus, the number and size of follicles >2 mm were monitored daily by ultrasonography. The mean (+/- SEM) number of corpora lutea (CL) (6.2 +/- 1.5 vs 10.7 +/- 0.9; P<0.05) and the mean number of recovered embryos and unfertilized ova (3.6 +/- 1.7 vs 8.4 +/- 2.2; P<0.05) were lower in FSH-P-primed than in saline-primed heifers. Prior to initiation of superovulation, follicles >10 mm appeared on Days 6 to 7 in saline-primed heifers but only on Days 8 to 10 in FSH-P-primed heifers (P<0.05). Also, until Day 10, the mean number of follicles 4 to 6 mm and 7 to 10 mm was higher (P<0.05) in FSH-P-primed than in saline-primed heifers. After initiation of the superovulatory treatment (Day 10 to estrus), saline-primed heifers had a greater and faster increase in the mean number of follicles >10 mm (P<0.02) than FSH-P-primed heifers did. Depletion in the number of follicles 2 to 3 mm (P<0.001) between Day 10 and estrus and in the number of follicles 4 to 6 mm (P<0.05) between Day 12 and estrus occurred in both groups of heifers. Decreased superovulatory response and embryo recovery in FSH-P-primed heifers may have been due to the presence of large follicles (>10 mm) prior to the initiation of the superovulatory treatment which reduced the ability of small follicles to grow into larger size classes during superovulatory treatment.