Effect of inhibin immunisation using different synthetic peptide fragments of the bovine αc-subunit on plasma anti-inhibin titres,plasma FSH concentrations and the incidence of multiple ovulation in heifers

In an effort to improve the effectiveness of inhibin immunisation in promoting multiple ovulation in cattle and to clarify the mechanism(s) involved, heifers (n = 5 per group) were immunised against ovalbumin conjugates of different synthetic peptide sequences of the α_c-subunit of bovine inhibin (bIα) selected using antigenic prediction methods. Plasma inhibin antibody titre (percentage binding of ¹²⁵I-labelled M_r 32 000 native bovine inhibin), plasma follicle-stimulating hormone (FSH) concentration and ovulatory response (number of corpora lutea observed by transrectal ovarian ultrasonography) were recorded over a 16 week period.Heifers immunised against the bIα1–29 and 63–72 peptides (alone or in combination), had relatively high anti-inhibin titres (over 7.5% binding) and showed a significantly (P < 0.05) increased incidence of multiple ovulations (18–65%) compared with ovalbumin-immunised controls. However, immunisation against the 1–16 and 108–123 peptides was relatively ineffective in generating antibodies reactive with native inhibin (less than 7.5% binding) and gave little or no increase in incidence of multiple ovulations (0–10%).Analysis of results for all 33 heifers revealed a significant linear relationship between mean inhibin antibody titre and mean plasma FSH concentration (r = 0.42; P < 0.02) and between mean inhibin antibody titre and incidence of multiple ovulation (r = 0.89; P < 0.0001). A significant quadratic relationship existed between mean inhibin antibody titre and the mean number of ovulations per cycle (r = 0.88; P < 0.0001). However, partial correlation analysis showed a highly significant association between anti-inhibin titre and ovulatory response which was independent of changes in mean plasma FSH concentrations.These results extend previous studies involving inhibin peptide-immunised cattle by showing that the magnitude of the ovulatory response is directly related to the prevailing titre of antibodies reactive with native inhibin. However, they do not support the hypothesis that the ovulatory response is mediated solely by a rise in FSH secretion.     

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.     

Immunization against recombinant bovine inhibin alpha subunit causes increased ovulation rates in gilts

Immunization of gilts in a commercial piggery against a fusion protein of the alpha subunit of bovine inhibin, produced by recombinant DNA methods, resulted in mean ovulation rate increases of 35% at the oestrus at which, under the piggery's management practices, they would have been mated. Sera from two immunized groups showed mean binding of 6.6% and 4.9% when assayed, at 1:800 final dilution, against iodinated bovine inhibin (Mr 31,000). Ovulation rates of immunized gilts were highly correlated with the ability of serum to bind iodinated native inhibin (r = 0.62; P less than 0.001), particularly when weight and age were included in the correlation (r = 0.72; P = 0.001), and inhibin binding accounted for 38% of the total variation in ovulation rate. Immunization caused no deleterious effects on growth rate or onset of oestrus. These results demonstrate the potential for use of such immunization to increase prolificacy in gilts and young sows.     

Effects of active immunization of prepubertal heifers against prostaglandin F2α, on the onset of puberty and subsequent ovarian function

The objective of these two experiments was to determine the effect of immunization of prepubertal heifers against prostaglandin F_2α conjugated to human serum albumin (PGF-HSA) on the onset of puberty and subsequent ovarian function. Heifers that were 2–3 months of age (n=34; mean ± standard error of the mean (SEM) bodyweight 75 ± 1.4 kg) were given a primary immunization (Day 0) and a booster 183 days later using one of two adjuvants; non-ulcerative Freund's adjuvant (NUFA) and diethylaminoethyl-dextran (DEAF-dextran) and one of two doses of PGF-HSA as immunogen (1 and 10 mg), with six to seven heifers per treatment; control heifers (n=7) were untreated. The experiment lasted up to 562 days. Plasma antibody titres every 2–3 weeks and progesterone concentrations two to three times weekly were determined. Oestrous behaviour was observed twice daily. In addition, the follicular status of ovaries of cyclic (n=4), prepubertal (n=6) or prolonged luteal phase ( > 200 days, induced by immunization against PGF) heifers (n=5) were monitored daily for 23 consecutive days by ultrasound examination. Antibody titres were higher (P < 0.05) immediately following booster immunization in heifers immunized with DEAF-dextran than with NUFA. High titres were maintained for longer (P < 0.05) in heifers immunized with NUFA than with DEAF-dextran. The mean number of oestrous periods was decreased (P < 0.05) in PGF-immunized heifers in comparison with control heifers following booster immunization (3.1, 0.5, 0.9, 1.5, 1.1 in control heifers and in heifers immunized with NUFA-1 mg, NUFA-10 mg, DEAE-dextran-1 mg and DEAE-dextran-10 mg, respectively. Puberty was delayed (P < 0.05), relative to control heifers, by 185 and 122 days (pooled standard error of the difference, 89) in heifers immunized with NUFA-1 mg and DEAE-dextran-10 mg, respectively. After first ovulation at puberty, persistent corpora lutea (CL) formed in two of six heifers immunized with NUFA-1 mg, five of seven immunized with NUFA-10 mg, two of seven immunized with DEAE-dextran-1 mg and one of seven immunized with DEAE-dextran-10 mg. The duration of persistence was 103 ± 19, 301 ± 33, 124 ± 23 and 100 days, respectively. The mean (±SEM) number of dominant follicles was not different (P > 0.05) for the three groups of heifers that were examined by ultrasound (3.7 ± 0.2, 3.8 ± 0.3 and 3.2 ± 0.3 for cyclic, prepubertal and persistent CL, respectively); neither was their rate of growth (1.8 ± 0.2, 1.5 ± 0.1 and 1.5±0.2 mm day⁻¹, respectively), rate of atresia (1.3 ± 0.1, 1.6 ± 0.1 and 1.8 ± 0.2 mm day⁻¹, respectively) or the maximum size of dominant follicles (11.2 ± 0.4, 9.6 ± 0.3 and 12.4 ± 1.8 mm, respectively). In summary, immunization of prepubertal heifers against PGF delayed the onset of puberty and reduced subsequent oestrous behaviour due to the formation of persistent CL following first ovulation and there was a similar growth pattern of dominant follicles before first ovulation in prepubertal heifers, cyclic heifers and heifers which have long-term persistent CL.     

Active immunization against inhibin-based peptides to increase ovulation rate in non-prolific Malpura ewes

The aim of this study was to explore the possibility of increasing the ovulation rate of Malpura, a non-prolific tropical breed of sheep by immunization against inhibin-based peptide immunogens. Ewes were divided into three groups (n = 5 each) and actively immunized against the synthetic peptides from the α_C [bIα(1–29)-Tyr³⁰] or α_N [bI-₄₃-Tyr¹⁵²(153–167)Cys¹⁶⁸] area of the bovine inhibin α-subunit conjugated to ovalbumin or against ovalbumin (control). Each ewe received a primary immunization of 400 μg immunogen and 3 boosters, each of 200 μg immunogen at 4-week intervals. Estrus was synchronized using a double PGF₂α injection schedule and laparoscopy was performed after each estrus to determine the ovulation response. Immunization against both the peptides did not affect the interval from PGF treatment to the onset of estrus, the duration of estrus and the number of large unovulated follicles. In contrast to the complete absence of multiple ovulations in the controls, all the ewes immunized against α_C or α_N peptides showed multiple ovulations (range 2–7) in all the three estrous cycles evaluated, except for one ewe immunized against the α_N peptide, which exhibited multiple ovulations in only 1 out of the 3 estrous cycles. Compared to that of the controls (1.0 ± 0.9, 1.0 ± 0.0 and 0.6 ± 0.2, respectively), the mean ovulation rate was higher (P < 0.01) in the ewes immunized against the αC (4.8 ± 1.02, 5.0 ± 1.05 and 5.0 ± 0.45, respectively) or against αN (4.5 ± 1.19, 2.5 ± 0.87 and 2.7 ± 0.75, respectively, P < 0.05) peptide in estrous cycles numbers 1, 2 and 3. These results show that active immunization against inhibin-based peptide immunogens is effective in increasing ovulation rate in Malpura, a non-prolific breed of sheep and that it may be an alternative to conventional superovulation regimes.     

Active immunization with a synthetic fragment of pig inhibin alpha-subunit increases ovulation rate and embryo production in superovulated ewes but season affects its efficiency.

Two experiments were designed to determine the effects of active immunization against one of two synthetic peptides from humans (inhibin-like peptide) or pigs (inhibin alpha-subunit) on antibody titres, ovulation rate and embryo production in ewes superovulated with 16 U ovine FSH. In Expt 1, during the breeding season, 30 ewes were subdivided into three groups: group I served as the non-immunized control; group II was immunized against inhibin-like peptide (100 micrograms inhibin-like peptide equivalent, followed by three booster injections); group III was immunized against pig inhibin alpha-subunit conjugated to human serum albumin (96 micrograms for the primary administration and 46 micrograms for the booster). In Expt 2, the efficiency of immunization against pig inhibin alpha-subunit on ovarian response and embryo production was evaluated during the non-breeding season in two groups of ewes (n = 12): group IV was a non-immunized control; Group V was immunized against pig inhibin alpha-subunit. During the breeding season, the ewes immunized against pig inhibin alpha-subunit showed higher antibody titres compared with the group immunized against inhibin-like peptide (P < 0.01) and a significant increase in ovulation rate (12.1) compared with both the control (5.0; P < 0.05) and the inhibin-like peptide-immunized group (3.1; P < 0.01). Immunization against pig inhibin alpha-subunit increased transferable embryo yield 4.5-fold (6.7 versus 1.5; P < 0.01) and improved embryo quality (94.6 versus 40.6%; P < 0.01). During the non-breeding season, immunization against pig inhibin alpha-subunit enhanced ovulation rate from 2.6 in the controls to 9.4 (P < 0.01) but did not affect transferable embryo production (3.9 versus 2.1; P > 0.05) and significantly lowered their quality (54.1 versus 100%; P < 0.01). In conclusion, active immunization against pig inhibin alpha-subunit can improve superovulatory response during the breeding season, while it appears to be unable to increase embryo yield during the seasonal anoestrus.     

Increase in ovulation rate by active immunization against bovine inhibin-based synthetic peptides in a non-prolific sheep breed

The objective of this study was to explore the possibility of a recurrent increase in the ovulation rate of Malpura sheep, a non-prolific breed, by immunization against inhibin-based peptide immunogens over a period of 3 years. Adult ewes (4–7 years of age) and weighing between 28 and 38 kg were randomly allocated equally to three treatment groups. The immunization of the ewes was initiated during the autumn breeding season. Ewes were divided into three groups (n = 5 ewes/group) and actively immunized against the synthetic peptides from the α_C [bIα(1–29)-Tyr³⁰] (Group I) or α_N [bI-₄₃-Tyr¹⁵²(153-167)Cys¹⁶⁸] (Group II) area of the bovine inhibin α-subunit, conjugated to ovalbumin or against ovalbumin (control). Each ewe received a primary immunization of 400 μg immunogen and 3 booster injections, 200 μg immunogen each at 4-week intervals. Estrous was synchronized in all the ewes by administering two doses of PGF₂α at 10-day intervals for three consecutive years. Ovaries of ewes were examined each year between days 4 and 6 of the synchronized cycle, with the aid of the laparoscope to determine the ovulation rate. Active immunization significantly (p < 0.05) increased the ovulation rate. The overall ovulation rate, irrespective of the treatment period was 5.2 ± 0.44, 2.3 ± 0.38 and 0.9 ± 0.11 in Group I, Group II and the control, respectively. Although the beneficial effect of immunization on ovulation rate persisted for the entire period of the study, the interaction between immunization treatment and the time period was non-significant. The results clearly indicate that the active immunization against inhibin peptides can induce multiple ovulations in Malpura ewes and its effect on multiple ovulations is sustained for a prolonged period of time after the initial immunization.     

Active immunization of post-pubertal heifers against prostaglandin F2α to suppress oestrous behaviour: effect of adjuvant and dose of immunogen

Two experiments were performed to develop an effective prostaglandin F_2α immunization protocol to suppress oestrous behaviour in beef heifers. In Experiment 1, a 3 × 2 factorial plan (n=4–5 per treatment) was used to test three doses (3.3, 10 and 30 mg) of a prostaglandin F_2α- human serum albumin (PGF-HSA) conjugate as the immunogen and two adjuvants, GNE (proprietary product; Intervet, The Netherlands) and diethylaminoethyl (DEAE)-dextran. Heifers (n=5) in a control group were untreated. Booster immunizations were given on Days 42 and 145 after the primary immunization (Day 0) and data collection for statistical purposes ended on Day 297. After Day 42 the incidence of oestrous behaviour was: (1) greater (P < 0.05) for control than immunized heifers (4.3 and 2.2, respectively), (2) greater (P < 0.05) for heifers immunized using GNE than for heifers immunized using DEAE-dextran (2.6 and 1.9, respectively), and (3) greater for heifers immunized with 30 mg of immunogen than for those immunized with either 3.3 or 10 mg (3.1, 1.7 and 1.9, respectively). Suppression of oestrous behaviour was accompanied by formation of a persistent corpus luteum (CL). Persistent CL were formed in ten of the 28 immunized heifers and the mean (± standard error of the mean) duration of persistence was 397 ± 85 days. In Experiment 2, a 2 × 2 factorial plan (n=6–7 per treatment) was used to test two doses (1 and 10 mg) of the PGF-HSA conjugate as the immunogen and two adjuvants, non-ulcerative Freund's adjuvant (NUFA) and DEAE-dextran. A control group was untreated (n=6). Booster immunization was given on Day 183 after the primary immunization (Day 0) and the experiment finished on Day 384. Antibody titres were higher (P < 0.05) in NUFA-treated heifers than in DEAE-dextran-treated (1 mg) heifers in the 183- to 283-day period. After Day 183, oestrous behaviour was suppressed in 26 out of the 27 immunized heifers. Persistent CL were maintained for longer (P < 0.05) in NUFA-treated heifers (245 days) than in DEAE-dextran-treated heifers (166 days) but there was no difference due to dose of immunogen (208 and 203 days, 1 and 10 mg, respectively). It is concluded that immunization against PGF-HSA results in suppression of oestrous behaviour in heifers due to prolongation of the life-span of the CL; however, efficacy of response is dependent on the immunization regime used.     

Advancement of puberty in ewe lambs by active immunisation against inhibin early in life

Active immunisation of lambs early in life with inhibin can advance puberty and increase ovulation rate but these effects appear not to be mediated through changes in FSH concentrations. The aims of this study were to advance puberty in ewe lambs and determine if increased plasma concentrations of gonadotropins are responsible for the advancement of puberty. Ewe lambs were immunised at 3, 7 and 15 weeks of age against either a synthetic inhibin alpha subunit peptide 1–32 conjugated to human serum albumin (HSA), or an inhibin preparation purified from porcine follicular fluid (porcine monoclonal purified inhibin; pMPI), or HSA alone (control immunogen).Immunisation with inhibin alpha peptide 1–32 produced antibodies which bound iodinated native bovine inhibin and advanced puberty (time of first ovulation) and increased ovulation rate but did not significantly increase plasma FSH concentrations, although LH concentrations were lower (P < 0.01) on a number of occasions. In contrast, immunisation with pMPI significantly (P < 0.01) increased FSH and LH concentrations following the first booster immunisation, although FSH was only transiently elevated. Despite these increases in gonadotropins, no advancement of puberty was observed in PMPI immunised ewe lambs.This study confirms that active immunisation of ewe lambs early in life against inhibin advances puberty via a mechanism which does not significantly increase plasma gonadotrophin concentrations. Immunisation to advance puberty also results in persistent increases in ovulation rates in later breeding seasons.     

Effects of active immunization against a synthetic peptide sequence of the inhibin alpha-subunit on plasma gonadotrophin concentrations, ovulation rate and lambing rate in ewes.

Thirty adult Mule (Blue-faced Leicester x Swaledale) ewes were actively immunized against a synthetically produced peptide corresponding to the N-terminus of the alpha-subunit of bovine inhibin conjugated to tuberculin purified protein derivative (PPD). Primary immunization in the late anoestrous period was followed by two booster injections at 5 week intervals. Control groups were either not immunized (n = 15) or received PPD only (n = 15). Ten days after the second booster, oestrus was synchronized using progestagen sponges and ovulation rate was assessed by laparoscopy on days 9-10 of the cycle. Blood samples were taken at the time of each immunization and immediately before laparoscopy. Ewes were mated with fertile rams in mid-November and the resulting conception, pregnancy and lambing rates monitored. All inhibin-immunized ewes generated antibodies that bound 125I-labelled native bovine inhibin (M(r) 32,000), and their plasma follicle-stimulating hormone (FSH) concentrations after the second booster were significantly higher than the preimmunization values (30%; P less than 0.001) and the corresponding value in the controls (25%; P less than 0.025). Inhibin immunization was associated with a 90% increase in ovulation rate (P less than 0.005) and had no adverse effect on conception rate (100%), pregnancy rate (100%) or length of gestation (146 days). However, only a 37% increase (P less than 0.05) in lambing rate was recorded for inhibin-immunized ewes, indicating a higher incidence of wastage of ova, or embryos, or both, in these ewes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunization against inhibin enhances both embryo quantity and quality in Holstein heifers after superovulation and insemination with sex-sorted semen

The objective was to investigate the feasibility of improving embryo yield in superovulated cows following insemination with sex-sorted semen by prior immunization against inhibin. Twenty-eight heifers were allocated into three groups: High (n = 10), Low (n = 10), and Control (n = 8). The High group received one primary (1 mg) and two booster (0.5 mg) vaccinations (28-d intervals) with a recombinant inhibin α-subunit in 1 mL of white oil adjuvant, whereas the Low group received half that dose, and the Control group received only adjuvant. After the last immunization, all heifers underwent a standard superovulation treatment (decreasing doses of pFSH for 4 d), followed by two AI with 2 × 10⁶ sex-sorted semen after the onset of estrus. Inhibin-immunized heifers had higher (P < 0.01) plasma antibody titres, and an earlier onset of estrus (P < 0.05) than Control heifers. The total number of embryo/ova, transferable, and grade 1 embryos in the High group (15.4 ± 1.9, 5.7 ± 0.7, and 3.8 ± 1.0, respectively) was significantly greater than that of the Control group (9.1 ± 1.2, 3.1 ± 0.5, and 0.6 ± 0.2), but was intermediate (P > 0.05) in the Low group (13.0 ± 2.3, 4.4 ± 0.7, and 1.2 ± 0.3). There were no significant differences among groups in number of unfertilized ova and degenerated embryos. The High group also had higher (P > 0.05) plasma progesterone concentrations on the day of embryo collection. In conclusion, immunization against inhibin improved both embryo quantity and quality following superovulation and insemination with sex-sorted semen.     

Effect of immunizing prepuberal lambs of low and high ovulation rate genotypes with inhibin partially purified from bovine follicular fluid

Active immunization of prepuberal lambs with a partially purified inhibin preparation, isolated from bovine follicular fluid, increased the ovulation rate. In ewe lambs of a low fecundity breed (Suffolk x Galway), the ovulation rate rose from 1.15 to 1.95 (P<0.05) compared with that of the controls. An ovulation rate of 3.38 was recorded for immunized ewe lambs of a high fecundity breed (Finn x Dorset Horn), while the rate for mature ewes from the same flock was 2.29. Immunization did not affect the time of onset of puberty or estrous cycle length. Following immunization, antibodies were produced that bound to a pure preparation of 68kDa bovine inhibin. This report demonstrates the production of antibody to a 68kDa preparation of inhibin following active immunization of sheep using a partially purified preparation. It was concluded that the increased ovulation rate was due to the production of antibodies to inhibin, which may have reduced its negative feedback effect of FSH secretion.     

Modifying the double-Ovsynch protocol to include human chorionic gonadotropin to synchronize ovulation in dairy cattle

The objectives were to determine whether rates of conception, ovulation, presynchronization, or follicle and CL characteristics were altered after modifying the Double-Ovsynch (DO) protocol to include hCG compared with the DO protocol. Primiparous and multiparous lactating dairy cows (N = 183), and nulliparous dairy heifers (N = 51) were used. Cows were blocked by parity and heifers were stratified by age and breed before being randomly assigned to one of two treatments. All females received either 100 μg GnRH or 2000 IU hCG im, at initiation of the Pre-Ovsynch (PO) portion of the DO protocol (PO: GnRH/hCG, 7 days PGF_2α and 3 days GnRH). After 7 days, females started the Breeding-Ovsynch portion of the DO protocol (Breeding-Ovsynch: GnRH, 7 days, PGF_2α, 48 or 56 h and GnRH 16 hours timed artificial insemination with sex-sorted semen). Transrectal ultrasonography and blood samples were used to assess ovarian structures, ovulation, pregnancy diagnosis, and concentration of progesterone in plasma. Conception rates were similar in females treated with GnRH or hCG in cows (32.2 and 25.0%) and in heifers (30.8 and 36.0%). Ovulation rates in cows at the onset of PO were increased with hCG compared to GnRH (77.2 vs. 62.2%, P < 0.05). Concentrations of progesterone 7 days post-hCG or GnRH were greater in cows treated with hCG compared with GnRH (least significant mean ± SEM; 4.3 ± 0.3 and 3.0 ± 0.3 ng/mL, P < 0.01), but did not differ in heifers (4.5 ± 0.9 and 2.9 ± 0.9 ng/mL). More cows ovulated within 7 days post-hCG and a greater proportion of these cows tended to have failed luteal regression by Day 3 post-PGF_2α compared with cows that had ovulated to GnRH (29.6 vs. 16.1%, P ≤ 0.10). The overall percentage of females which were synchronized to PO did not differ between GnRH- or hCG-treated cows (61.5% and 52.2%) and heifers (42.3% and 40.0%). In conclusion, no overall improvement in fertility was achieved by replacing the first injection of GnRH in the DO protocol with hCG.     

Effect of active immunization against androstenedione on ovarian function in dairy cows and prepubertal gilts

Two experiments were performed to assess the effects of immunization against androstenedione on ovarian function in cows during the first 100 days after calving and in gilts at the onset of puberty.Seven Holstein-Friesian cows were immunized against androstenedione-11α-human serum albumin (AND-HSA) at 3 weeks before and at 3 weekly intervals after calving and compared with ten cows immunized against HSA. Titres against androstenedione were significant but low and variable (1: 280-1: 4). Two cows with the highest titres exhibited abnormal ovarian function which could be attributable to treatment. One cow developed a large follicular cyst and nymphomania. The other cow exhibited mild super ovulation in her first cycle after calving and multiple ovulations during later cycles. Other cows in the AND-HSA group also had a tendency to develop larger than normal follicles and follicular cysts.Groups of twelve prepubertal gilts were immunized against either androstenedione 11α-hemisuccinyl bovine serum albumin (AND-BSA) or BSA at 100, 121 and 142 days of age. Titres against AND were significant but were low and variable (1: 2500-1: 10). There was no effect of immunization against AND on onset of ovulation, puberty or ovulation rate in the first or second ovulatory cycles.     

Ovulation and embryo recovery rates following immunization of mares against an inhibin alpha-subunit fragment

Six normally cycling mares were immunized 5 times at 3-week intervals with a synthetic porcine inhibin alpha-subunit fragment which had been conjugated to bovine serum albumin and emulsified in Freund's incomplete adjuvant. Immunized mares ovulated a significantly larger (P < 0.01) number of follicles per estrous cycle (2.8 +/- 1.1; range 1 to 8 ovulations) than 14 nonimmunized control mares (1.1 +/- 0.1; range 1 to 2 ovulations). Day-7 embryo recovery rates tended to be higher (P < 0.1) in immunized mares (1.6 +/- 0.5 embryos per flush) than in control mares (0.7 +/- 0.2 embryos per flush). No differences in interovulatory intervals were found between the 2 groups. These results indicate that immunization against inhibin may be useful in inducing development and ovulation of multiple follicles for embryo transfer in the mare.     

Bioactivity of ovulation inducing factor (or nerve growth factor) in bovine seminal plasma and its effects on ovarian function in cattle

To understand the role of ovulation-inducing factor (or nerve growth factor) (OIF [NGF]) in bovine seminal plasma, we (1) used an in vivo llama bioassay to test the hypothesis that bovine seminal plasma induces ovulation and CL development in llamas similar to that of llama seminal plasma when the dose of seminal plasma is adjusted to ovulation-inducing factor content (experiment 1) and (2) determined the effect of bovine seminal plasma on the interval to ovulation and luteal development in heifers (experiment 2). Within species, seminal plasma was pooled (n = 160 bulls, n = 4 llamas), and the volume of seminal plasma used for treatment was adjusted to a total dose of 250 μg of ovulation-inducing factor. In experiment 1, mature female llamas were assigned randomly to four groups and treated intramuscularly with either 10 mL of PBS (negative control, n = 5), 50-μg GnRH (positive control, n = 5), 6-mL of llama seminal plasma (n = 6), or 12 mL of bull seminal plasma (n = 6). Ovulation and CL development were monitored by transrectal ultrasonography. In experiment 2, beef heifers were given a luteolytic dose of prostaglandin followed by 25-mg porcine LH (pLH) 12 hours later to induce ovulation. Heifers were assigned randomly to three groups and given 12 mL bovine seminal plasma intramuscularly 12 hours after pLH treatment (n = 10), within 4 hours after ovulation (n = 9), or no treatment (control, n = 10). Ovulation was monitored by ultrasonography every 4 hours, and the CL development was monitored daily until the next ovulation. In experiment 1, ovulation was detected in 0/5, 4/5, 4/6, 4/6 llamas in the PBS, GnRH, llama seminal plasma, and bovine seminal plasma groups, respectively (P < 0.05). Luteal development was not different among groups. In experiment 2, the interval to ovulation was more synchronous (range: 4 vs. 22 hours; P < 0.0001) in heifers treated with seminal plasma before ovulation compared with the other groups. Luteal development was not different among groups; however, plasma progesterone concentrations tended to be greater in the postovulation treatment group compared with other groups. In summary, results confirmed the presence of bioactive ovulation-inducing factor in bull seminal plasma and supported the hypothesis that bovine and llama seminal plasma have similar ovulatory effects, using a llama bioassay. Treatment with bovine seminal plasma resulted in greater synchrony of ovulation in heifers pretreated with pLH. Plasma progesterone concentration tended to be higher in heifers given bovine seminal plasma within 4 hours after ovulation, suggesting that bovine ovulation-inducing factor is luteotrophic.     

Growth differentiation factor 9 (GDF9) suppresses follistatin and follistatin-like 3 production in human granulosa-lutein cells

Background

We have demonstrated that growth differentiation factor 9 (GDF9) enhances activin A-induced inhibin β _B-subunit mRNA levels in human granulosa-lutein (hGL) cells by regulating receptors and key intracellular components of the activin signaling pathway. However, we could not exclude its effects on follistatin (FST) and follistatin-like 3 (FSTL3), well recognized extracellular inhibitors of activin A.

Methodology

hGL cells from women undergoing in vitro fertilization (IVF) treatment were cultured with and without siRNA transfection of FST, FSTL3 or GDF9 and then treated with GDF9, activin A, FST, FSTL3 or combinations. FST, FSTL3 and inhibin β _B-subunit mRNA, and FST, FSTL3 and inhibin B protein levels were assessed with real-time RT-PCR and ELISA, respectively. Data were log transformed before ANOVA followed by Tukey''s test.

Principal Findings

GDF9 suppressed basal FST and FSTL3 mRNA and protein levels in a time- and dose-dependent manner and inhibited activin A-induced FST and FSTL3 mRNA and protein expression, effects attenuated by BMPR2 extracellular domain (BMPR2 ECD), a GDF9 antagonist. After GDF9 siRNA transfection, basal and activin A-induced FST and FSTL3 mRNA and protein levels increased, but changes were reversed by adding GDF9. Reduced endogenous FST or FSTL3 expression with corresponding siRNA transfection augmented activin A-induced inhibin β _B-subunit mRNA levels as well as inhibin B levels (P values all <0.05). Furthermore, the enhancing effects of GDF9 in activin A-induced inhibin β _B-subunit mRNA and inhibin B production were attenuated by adding FST.

Conclusion

GDF9 decreases basal and activin A-induced FST and FSTL3 expression, and this explains, in part, its enhancing effects on activin A-induced inhibin β _B-subunit mRNA expression and inhibin B production in hGL cells.     

Influence of perioestrous suprabasal progesterone levels on cycle length,oestrous behaviour and ovulation in heifers

In order to induce suprabasal plasma concentrations of progesterone after luteolysis and to determine their effect on oestrous behaviour and ovulation, heifers subcutaneously received silicone implants containing 2.5 (n = 4), 5 (n = 4), 6 (n = 3), 7.5 (n = 3) or 10 (n = 4) g of progesterone, or an empty implant (controls, n = 5) between days 8 and 25 of the cycle (ovulation designated Day 0). Growth of dominant follicles and time of ovulation were determined by ultrasound, and signs of oestrus were recorded and scored. Blood was collected at 2–4 h intervals from Days 15 to 27 and assayed for progesterone concentration. In all heifers, plasma concentrations of progesterone sharply decreased during Days 16–18. Control heifers had their lowest progesterone levels on Days 20.5 and 21, standing oestrus on Day 19.5 ± 0.4 (mean ± SEM), and ovulated on Day 20.7 ± 0.4. A similar pattern was observed in heifers treated with 2.5 and 5 g progesterone. Heifers treated with 6, 7.5 and 10 g of progesterone showed an extended (P < 0.05) interovulatory interval. Onset of prooestrus and time of maximum expression of signs of oestrus were not significantly different from those in controls. However, there was an absence of standing oestrus in most of the cases, signs of oestrus lasted longer (P < 0.05) and were weaker in intensity when doses increased. In these groups, the lowest progesterone concentrations were attained shortly after implant removal. Some heifers treated with 6 and 7.5 g of progesterone had standing oestrus and post oestrous bleeding as seen in the controls but ovulation occurred from Days 24.5 to 27. When plasma progesterone concentrations were over 1 nmol 1⁻¹, disturbed oestrus and delayed ovulation occurred. The extended period of prooestrus and oestrus and delayed ovulation were similar to that described in cases of repeat breeding. It is suggested that suprabasal plasma concentrations of progesterone, after luteolysis, may lead to asynchrony between onset of oestrus and ovulation and consequently be a cause of repeat breeding in cattle.     

Multiple ovulation resulting from low level administration of PMSG to cattle at different stages of the oestrous cycle

Two experiments were carried out to determine whether differences in sensitivity to exogenous gonadotrophin which exist during the oestrous cycle can be used effectively in the induction of multiple pregnancy in cattle. In Experiment I, Hereford heifers and cows were injected with 800 IU pregnant mare serum gonadotrophin (PMSG) on approximately day 10 of the oestrous cycle, followed 48 h later by 30 mg prostaglandin F_2α (PGF_2α). Controls were treated with PGF_2α alone. Mean ovulation rates based on rectal palpation were 1.33 ± 0.10 (range: 1–2) and 3.05 ± 0.68 (range: 1–13) for 21 control and 21 treated animals, respectively (P < 0.02). In Experiment II, Hereford cows were treated with 800 IU PMSG on either day 5 (14 cows) or day 10 (12 cows) of the oestrous cycle, followed 48 h later by PGF_2α. Mean numbers of ovulations for animals that became pregnant were 1.50 ± 0.26 (range 1–3) and 3.80 ± 0.74 (range 1–7), respectively (P < 0.02). A high incidence of embryonic wastage occurred by 120 days of gestation in animals treated on day 10. The results of this study indicate that taking advantage of an animal's reduced responsiveness to exogenous gonadotrophin during the early portion of the oestrous cycle may be useful in developing methods for inducing multiple births with exogenous gonadotrophins.     

Detection of estrous behavior in buffalo heifers by radiotelemetry following PGF2α administration during the early or late luteal phase

This study examined the usefulness of radiotelemetry for estrous detection in buffalo heifers and the impact of prostaglandin F_2α (PGF_2α) administration during the early or late luteal phase on estrous behavior and ovulatory follicle variables. Induction of estrus with PGF_2α at a random stage of the estrous cycle was followed by the arbitrary division of heifers into groups receiving a second dose of PGF_2α during either the early (n = 33) or late (n = 17) luteal phase (6–9 or 11–14 days after estrus, respectively) for the induction of synchronized estrus. The electronic detection of synchronized estrus by radiotelemetry was confirmed using ultrasonography every 6 h until ovulation. Radiotelemetry was 90% efficient and 100% accurate for estrous detection. Intervals between the PGF_2α dose and the beginning of synchronized estrus (40.7 ± 10.9 vs. 56.7 ± 12.8 h) or ovulation (70.0 ± 11.3 vs. 85.6 ± 12.5 h) were shorter (P < 0.05) for heifers receiving PGF_2α during the early luteal phase. PGF_2α administration during the early or late luteal phase produced similar (P > 0.05) results for the duration of estrus, the intervals from the beginning or end of estrus to ovulation, the number and duration of mounts per estrus, the duration of mounts, the diameter of the ovulatory follicle and the luteal profile on day 5 after estrus. In conclusion, radiotelemetry was a suitable tool for the efficient and accurate detection of estrus in buffalo heifers. Treatment with PGF_2α during the early luteal phase had a shorter interval to synchronized estrus and ovulation; however, estrous behavior, ovulatory follicle dynamics and subsequent luteal activity were similar following PGF_2α administration during the early or late luteal phase.