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.     

Increasing ovulation rate and lambing percentage by active immunization against androstenedione in dairy sheep breeds

A total of 1200 ewes, two hundred from each of six dairy breeds-the Karagouniko (K), the Serres (S), the Vlachiko (V), the Florinis (F), the Kimis (Km) and the Chios (Ch)-were used to examine the efficacy of a new immunogen (androstenedione-7a-HSA + DEAE-dextran) in improving reproductive performance. Ewes of each breed were randomly allotted to two groups, the control and the treated. Treated ewes were given two injections of 2 ml immunogen three weeks apart, while the control ewes were untreated. Rams were introduced to all ewes after the second injection. Twenty ewes in each group for each breed were laparotimized 7 to 10 days post-mating. Incidence of estrus and mating, the ovulation rate (OR), lambing percentage (Lp. 100), litter size (LS), and lamb (LbW) and litter weight (LW) were recorded. The OR was increased in immunized ewes compared to controls from 1.60, 1.46, 1.50, 2.50, 2.50 and 2.90 to 2.50, 2.06, 2.20, 3.20 (P<0.05), 4.20 and 4.80 (P<0.01), respectively, in K, S, V, F, Km and Ch breeds. No significant difference on Lp. 100 was observed between control and treated ewes of all breeds. The LS was increased in immunized ewes by 17, 20, 18, 28, 57 and 0 lambs compared to control ewes in K, S, V, F, Km and Ch breeds, respectively; however, the increase was statistically significant (P<0.05) only in Km and F breeds. The LbW was decreased in immunized ewes compared to controls; however, the decrease was statistically significant (P<0.05) only in the V breed. When compared to control ewes the LW was greater in immunized ewes of S, K, Km and F breeds while in Ch and V breeds LW was smaller. It was concluded that an increase in ovulation rate by active immunization against androstenedione may not be reflected in an increase of litter size in dairy sheep. Breed differences must be considered when producers intend to use this technique to improve litter size.     

Effects of passive immunization against inhibin on ovulation rate and embryo recovery in holstein heifers

The effects of acute neutralization of endogenous inhibin on ovulation rate and circulating FSH levels were investigated. Nine or ten days after estrus, 5 heifers were given a single injection of 75 ml iv inhibin antiserum produced in a castrated male goat, while another 5 were given the same amount of a castrated male goat serum. All heifers were given injections of PGF2alpha im at 48 h and 60 h after the serum injection. Those exhibiting an estrus were artificially inseminated with frozen-thawed semen. Seven or eight days after the insemination, ova or embryos were collected using a non-surgical method. Administration of inhibin antiserum resulted in a significant increase in the number of medium-sized follicles compared with the number in the control animals. The number of large follicles in the inhibin-neutralized animals was 4.8 +/- 2.4 (mean +/- SEM; n = 5) on the day of estrus, while there was a single large follicles in the ovaries of control animals. Seven or eight days after estrus, 3 to 16 ova or embryos were recovered from 4 of 5 animals, and 64 % of the total ova/embryos were transferable. Administration of inhibin antiserum produced a significant increase in the concentrations of plasma FSH from 12 to 72 h after the serum injection compared with the levels in the control animals (P < 0.05). After the onset of estrus, preovulatory LH and FSH surges were noted in inhibin-neutralized animals and magnitude of the rise in each hormone was similar to the control animals. The present study demonstrates that a single injection of the inhibin antiserum induces multiple ovulations probably by enhancing FSH secretion, and that recovery of embryos is equal to that observation after an ordinary FSH treatment.     

Effect of unilateral ovariectomy, gonadotropin stimulation and immunization against a synthetic peptide of the inhibin alpha-subunit on follicular development and oocyte recovery in cattle

Nulliparous Holstein cows were randomly distributed among 4 treatment groups to test the effects of treatments, including unilateral ovariectomy, anti-inhibin immunization and gonadotropin stimulation on ovarian follicle population and oocyte recovery. The Control treatment consisted of intact cows (I-Control). Unilaterally ovariectomized cows were included in the 3 remaining treatments consisting of ovariectomy alone (U-Control), cows immunized against a synthetic peptide of the alpha(c)-subunit of bovine inhibin (alpha(c)I; U-IH), and cows stimulated with FSH (Super-Ov; 75 units/female/week) and also immunized with alpha(c)I as in the previous treatment (U-IH/FSH). Oocytes were collected by transvaginal ultrasound-guided aspiration on a weekly basis from cows in each treatment for 5 consecutive weeks. Intact Control cows had a greater (P<0.05) number of follicles > or = 3 mm per female (4.7) than the U-Control and U-IH cows (2.6 and 2.9, respectively), and had a similar number of follicles as the U-IH/FSH treatment group (3.5). The numbers of follicles aspirated (2.7 to 3.6) and oocytes recovered/cow (1.6 to 2.6) were similar for cows in the I-Control, U-IH and U-IH/FSH treatment groups. Cows in the U-Control treatment group had a lower (P<0.05) number of aspirated follicles (2.0) and recovered oocytes (1.1) than the I-Control cows. Cows in the U-IH/FSH and U-IH treatments had follicles with larger (P<0.01) diameters (8.7 and 8.2 mm, respectively) than cows in the I-Control (6.6 mm) and U-Control (5.7 mm) treatments. In conclusion, unilateral ovariectomy did not result in compensatory increase of follicle number or size in the intact ovary; cows in the U-IH/FSH treatment group had a greater number of follicles aspirated than the U-Control cows. In addition, the anti-alpha(c)I immunization may have played a role in increasing the number and diameter of the follicles. None of the treatments evaluated in this study improved oocyte retrieval over that of the intact, nontreated cows.     

Effects of bovine follicular fluid inhibin on serum gonadotrophin concentrations in ewes during oestrus

Experiments were conducted with ewes to investigate the effects of an enriched bovine follicular fluid inhibin preparation (INH) on gonadotrophin secretion after the onset of oestrus. Administration of INH (10 mg) 1 h after the onset of oestrus did not significantly alter the preovulatory FSH and LH surges or the second FSH peak. To determine the effects of INH on the second FSH surge, ewes were treated with saline (N = 7) or INH (N = 10) at 4 h (10 mg) and 24 h (5 mg) after the peak of the preovulatory LH surge. The second FSH surge was delayed about 24 h (P less than 0.05) in ewes treated with INH; however, the delay did not alter the interval to the next oestrus. In a third experiment, 16 ewes were assigned to 4 groups in a 2 x 2 factorial with the main effects being ovariectomy at 4 h and INH treatment (10 mg) at 4, 20 and 36 h after the peak of the LH surge. Controls received sham ovariectomy and saline injection as appropriate. Ovariectomy resulted in a rapid increase in serum FSH but not LH and this was delayed (P less than 0.05) by INH treatment. These results indicate that inhibin has a selective inhibitory action on FSH secretion in ewes and suggests that the second FSH surge results from increased basal FSH secretion due to decreased endogenous inhibin levels.     

Measurement of dimeric inhibins and effects of active immunization against inhibin alpha-subunit on plasma hormones and testis morphology in the developing cockerel

Inhibins and activins are implicated as endocrine regulators of follicle-stimulating hormone production and of testicular steroidogenesis and spermatogenesis in mammals. The potential involvement of these proteins in cockerels was investigated by measurement of circulating inhibin A, inhibin B, total inhibin alpha-subunit immunoreactivity (ir-alpha), activin A, LH, FSH, and testosterone from the juvenile state through to sexual maturity. Plasma inhibin A remained low between 6 to 12 wk of age and increased approximately threefold (P < 0.05) to a prepubertal peak between Weeks 14 to 18, followed by a gradual decline to the end of the study (Week 24). Although plasma FSH levels were not correlated to inhibin A before Week 16 (r = -0.17), they were negatively correlated from Week 18 (r = -0.49; P < 0.005). Inhibin B levels were below the assay detection limit until 16 wk of age but thereafter rose steadily in parallel with FSH (r = 0.27; P < 0.02) and testosterone (r = 0.35; P < 0.005). Thus, inhibins A and B showed divergent profiles during sexual maturation. Plasma ir-alpha levels were much higher than dimeric inhibin levels throughout, although the relative difference varied with age. Plasma activin A levels were below the assay detection at all times. Juvenile cockerels were actively immunized against a synthetic chicken inhibin alpha-subunit peptide conjugate to determine effects on plasma hormones and on testicular weight, morphology, and activin A content. Immunization generated circulating antibodies that bound (125)I-bovine 32-kDa inhibin but did not affect plasma FSH or testosterone levels at any stage of development. However, immunization reduced postpubertal plasma LH levels (P < 0.05) and promoted increased testicular weight (24%; P < 0.01) and total testicular activin A content (42%; P < 0.001) at 24 wk. Testis weight of immunized birds was positively correlated with inhibin antibody titer (r = 0.61; P < 0.05). Live weight gain was not affected by immunization. Morphometric analysis of testis sections showed that inhibin immunization had no effect on the fractional volume of the seminiferous tubule wall, seminiferous tubule lumen, or interstitial tissue area. Likewise, seminiferous tubule surface area and surface area:volume ratios were not different from controls. These findings support differential roles for inhibins A and B in regulating the pituitary-testicular axis during sexual maturation in the cockerel but highlight the need for more detailed studies to distinguish between potential endocrine and local intragonadal roles of inhibin-related peptides and to elucidate the mechanism by which immunization against inhibin alpha-subunit promotes testis enlargement without raising plasma FSH.     

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.     

Gonadotrophin concentrations and ovulation rates in Suffolk ewes actively or passively immunized against inhibin alpha

Mature Suffolk ewes were either actively or passively immunized against the synthetic fragment of porcine inhibin alpha, pI alpha(1-30), to determine the effects on gonadotrophin secretion and ovulation rate. Thirteen control ewes were immunized against human serum albumin, 12 ewes were actively immunized against pI alpha(1-30) and 36 ewes were passively immunized with pI alpha(1-30) antiserum. Blood samples were collected at 4-h intervals for 72 h from oestrus-synchronized ewes following the withdrawal of the progestagen pessaries. Mean gonadotrophin concentrations measured during the oestrous cycle of control ewes, ewes actively immunized against pI alpha(1-30) and ewes passively immunized against pI alpha(1-30) were similar, but their secretory profiles differed. Serum concentrations of follicle-stimulating hormone (FSH) were highest in ewes which had received antiserum at the time of pessary withdrawal; FSH concentrations did not decrease during the follicular phase of the oestrous cycle in ewes given antiserum 24 h after pessary withdrawal. Subtle but significant increments in serum FSH concentrations were observed in all passively immunized ewes in which sampling commenced at the time of treatment. The amplitude of the preovulatory luteinizing hormone (LH) peak, but not of the FSH peak, and the postovulatory secondary rise in FSH were lower (P less than 0.05) in actively immunized ewes than in control ewes. The mean (+/- s.e.) ovulation rate for actively immunized ewes (6.6 +/- 1.0) was 3 times higher (P less than 0.05) than that for control ewes (2.0 +/- 0.2), but was unaffected by passive immunization (range, 1.8-2.3).(ABSTRACT TRUNCATED AT 250 WORDS)     

Increase in ovulation rate after immunization of Merino ewes with a fraction of bovine follicular fluid containing inhibin activity

Affinity chromatography of bovine follicular fluid using Matrix gel Red A resulted in a 20-fold increase in inhibin-like specific activity assessed in a mouse ovulation inhibition test system. When this material was emulsified with Freund's adjuvant and injected into adult Merino ewes their mean ovulation rate was increased from 1.2 to 2.3 (P less than 0.01). Follicles of diameter greater than or equal to 3.5 mm and 2-3.4 mm were also increased (4.33 vs 2.25 and 5.39 vs 2.44 per ewe respectively). The ovulation rate response was variable and transient. Length of oestrous cycles, number of granulosa cells per follicle and seasonal oestrous patterns were not affected. Plasma from the immunized ewes contained antibodies to the immunogen and reversed the ovulation-inhibiting effects of bovine follicular fluid in mice.     

Changes in plasma inhibin A levels during sexual maturation in the female chicken and the effects of active immunization against inhibin alpha-subunit on reproductive hormone profiles and ovarian function

Inhibins and activins are firmly implicated in the control of pituitary FSH secretion and ovarian follicular development in mammals. As in mammals, inhibin A and activin A are expressed in the preovulatory follicles of birds, and a defined ovulation cycle for inhibin A has recently been demonstrated in the laying hen. To investigate further the role of inhibin-related proteins in developing pullets, circulating concentrations of inhibin A, inhibin B, total immunoreactive inhibin alpha-subunit (ir-alpha), activin A, LH, FSH, and progesterone were measured from the juvenile state through to sexual maturity in 22 birds. In the 11 birds assigned to control groups, plasma inhibin A levels were low from 7 to 13 wk of age rising about threefold to a peak at Week 19 after which levels fell slightly to a plateau level characteristic of adult hens. Plasma inhibin A levels were negatively correlated with FSH (r = -0. 33; P: < 0.001) and positively correlated with progesterone (r = 0. 67; P: < 0.001) and ir-alpha (r = 0.53; P: < 0.001). Plasma ir-alpha levels were much higher than inhibin A levels although the relative differences varied with age. Plasma levels of inhibin B and activin A were below assay detection limits at all times. The remaining group of 11 birds was actively immunized (IMM) against a synthetic chicken inhibin alpha-subunit peptide (amino acids 1-26). The IMM generated circulating antibodies that bound native bovine inhibin A but altered neither plasma FSH nor progesterone levels relative to control birds at any stage of development nor the timing of first oviposition in week 19. Apart from a transient decline 1 wk after primary IMM, plasma LH concentrations did not differ from controls. Comparison of the numbers and size-class distribution of ovarian follicles at 29 wk showed an approximate twofold increase in the number of 8- to 9.9-mm-diameter follicles (control; 1.82 +/- 0.44 vs. IMM; 3.91 +/- 0.89; P: < 0.05), a size class that corresponds to follicles that have just joined the preovulatory hierarchy. The numbers of growing follicles in other size-classes and the sizes of hierarchical F(1)-F(7) follicles were not altered by IMM. However, the number of postovulatory follicles increased (control 3.73 +/- 0. 20 vs. IMM 5.55 +/- 0.28; P: < 0.01), and significantly more (P: < 0. 02) immunized hens laid two eggs within a 24-h period on at least one occasion (control 1 of 11 vs. IMM 9 of 11). The IMM increased (P: < 0.05) activin A content of F(1) and F(2) theca layers and decreased (P: < 0.05) activin A content in F(3) and F(4) granulosa layers, raising the possibility of a local intraovarian role of activin in mediating the response to IMM. These findings support a role for inhibin A in regulating the entry of follicles into the preovulatory hierarchy in the chicken, although further studies are required to establish the mechanism by which inhibin IMM increases the rate of follicle selection and ovulation without raising plasma FSH.     

Effect of active immunization against inhibin on hormonal concentrations and semen characteristics in Shiba bucks

Active immunization against inhibin increased ovulation rate in females; in males, the effects of active immunization against inhibin on hormonal concentrations and sperm production need more investigation. To test the hypothesis that active immunization against inhibin increases FSH secretion and sperm output, the present study was undertaken to determine the effects of active immunization against inhibin on hormonal profile and sperm production in Shiba bucks. The bucks were actively immunized against inhibin alpha-subunit (immunized group, n=6) or Freund adjuvant (control group, n=5) four times, at 5-weeks intervals. Blood samples were collected twice-weekly and two successive ejaculates of semen were collected (with an artificial vagina) once-weekly. Plasma concentrations of FSH, LH and testosterone were measured by radioimmunoassay (RIA) and sperm motility characteristics were measured by computer-assisted sperm analysis (CASA). All inhibin-immunized bucks produced antibodies against inhibin. Relative to control bucks, in immunized bucks there were significant increases in plasma FSH concentrations and in sperm concentrations from 5 to 9 weeks and from 8 to 11 weeks, respectively, after primary immunization. However, plasma concentrations of LH and testosterone, semen volume, percentage of motile spermatozoa and motility parameters (straight-line velocity, curvilinear velocity and linearity index) were similar in both groups. In conclusion, active immunization against inhibin alpha-subunit increased FSH secretions and enhanced sperm production in bucks, whereas LH and testosterone concentrations, semen volume and sperm motility parameters were unaffected. Active immunization against inhibin could be used to improve fertility in Shiba bucks.     

Effects of immunization against bone morphogenetic protein 15 and growth differentiation factor 9 on ovulation rate, fertilization, and pregnancy in ewes

Immunization of ewes against growth differentiation factor 9 (GDF9) or bone morphogenetic protein 15 (BMP15) can lead to an increased ovulation rate; however, it is not known whether normal pregnancies occur following such treatments. The aims of the present study were to determine the effects of a short-term immunization regimen against BMP15 and GDF9 on ovulation rate, fertilization of released oocytes, the ability of fertilized oocytes to undergo normal fetal development, and the ability of immunized ewes to carry a pregnancy to term. Ewes were given a primary and booster immunization against keyhole limpet hemocyanin (KLH; control, n = 50), a GDF9-specific peptide conjugated to KLH (GDF9, n = 30), or a BMP15-specific peptide conjugated to KLH (BMP15, n = 30). The estrous cycles of all ewes were synchronized, and ewes were joined with fertile rams approximately 14 days after the booster immunization. The number of corpora lutea was determined by laparoscopy 3-4 days following mating. Subsequently, about one-half of the ewes in each group underwent an embryo transfer procedure 4-6 days following mating, with the embryos being transferred to synchronized, nonimmunized recipients. The remaining ewes were allowed to carry their pregnancies to term. Short-term immunization against either BMP15 or GDF9 peptides resulted in an increase in ovulation rate with no apparent detrimental affects on fertilization of released oocytes, the ability of fertilized oocytes to undergo normal fetal development, or the ability of the immunized ewes to carry a pregnancy to term. Therefore, regulation of BMP15, GDF9, or both is potentially a new technique to enhance fecundity in some mammals.     

Effects of active immunization against GnRH on serum LH,inhibin A,sexual development and growth rate in Chinese female pigs

Surgical castration of young female pigs is common practice in Chinese pig farming today. The purpose of the present study is to investigate anti-GnRH immunization as a practical alternative to surgical castration for female pigs. Thirty-six Chinese female crossbred pigs (Chinese Yanan x Yorkshire) were selected from 12 litters, three pigs from each litter, at the age of 10-13 weeks. One pig from each litter was immunized with 62.5 microg D-Lys6-GnRH-tandem-dimer peptide conjugated to ovalbumin in Specol adjuvant at Week 0 (0 week post-vaccination, wpv), and a booster vaccination was given 8 weeks later (8 wpv). Its intact and castrate littermates (surgically castrated at the time of weaning, i.e. at 6 weeks of age) were administered the vehicle and served as controls. Antibody titers, serum LH and inhibin A were determined at the day of first vaccination, every 4 weeks thereafter and at the day of slaughter (18 wpv). At slaughter, ovaries were inspected for the presence of follicles and corpora lutea, and ovarian and uterine weights were recorded. Ten of twelve immunized pigs responded well to the immunization (immunocastrated animals), while the remaining two pigs responded poorly (nonresponders). Antibody titres in immunocastrated animals steadily increased after immunization, became maximal at 12 wpv and remained high until slaughter. Serum LH levels were reduced (P < 0.05) in immunocastrated pigs as compared to intact controls and surgical castrates. Serum inhibin A levels decreased after vaccination, and equaled surgical castrate levels from 8 wpv until the end of the experiment. Ovarian and uterine weights (1.3 +/- 0.2 and 43.9 +/- 11.4 g, respectively; mean +/- S.E.M.) were significantly lower (P < 0.05) in immunocastrates than in intact controls (9.4 +/- 1.1 and 390.9 +/- 67.2 g, respectively). Antibody titers were significantly lower (P < 0.05) in nonresponders than in immunocastrated pigs from 12 wpv to slaughter. Ovarian and uterine weights were similar in nonresponders and in intact controls. Macroscopically, no follicular structures were found in ovaries of immunocastrated pigs, while large follicles or corpora lutea were observed in the ovaries of both nonresponders and intact controls. Although not significant, immunocastrates had a numerically higher average daily gain than surgical castrates and intact controls (0.74 +/- 0.04 versus 0.66 +/- 0.04 versus 0.66 +/- 0.03 kg per day, respectively; mean +/- S.E.M., P = 0.09). Results obtained in the present study demonstrate that anti-GnRH immunization can be an attractive alternative to surgical castration for Chinese crossbred female pigs. Our results also question the beneficial effect of surgical castration on growth as compared to intact controls.     

Active and passive immunoneutralization of inhibin increases follicle-stimulating hormone levels and ovulation rate in ewes.

Two experiments were conducted to determine effects of active and passive immunoneutralization of inhibin on FSH secretion and ovulation rate. A synthetic peptide (alpha-IF) matching the N-terminus of the alpha-subunit of ovine inhibin was coupled to human alpha-globulin (h alpha-G) and used as an immunogen. In experiment 1, estrus was synchronized in 10 sheep that had been actively immunized against alpha-IF-h alpha-G or h alpha-G. Plasma FSH levels were similar in the two groups of ewes at -52 and -48 h (0 h = onset of estrus). In alpha-IF-h alpha-G-immunized ewes, FSH increased from -48 to -44 h (18.8-22.1 ng/ml), and then fell to 16.2 ng/ml by 0 h. In h alpha-G-immunized ewes, FSH decreased from -48 to 0 h (17.6-7.2 ng/ml). Ovulation rate was higher in alpha-IF-h alpha-G- than h alpha-G-immunized ewes (9.4 vs. 2.4). In experiment 2, antibodies (Ab) were extracted from sera obtained from experiment 1 ewes and then were injected i.v. into 12 other ewes. Estrus was synchronized twice during the breeding season using progesterone-releasing pessaries (CIDR-G). One day before CIDR-G withdrawal, alpha-IF-h alpha-G and h alpha-G Ab were administered in a crossover design. After injection of Ab against alpha-IF-h alpha-G, plasma FSH increased from 0 to 24 h post-injection (10.9-21.5 ng/ml), after which levels fell to 14.2 ng/ml by onset of the preovulatory LH surge.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of mouse epidermal growth factor on plasma concentrations of FSH, LH and progesterone and on oestrus, ovulation and ovulation rate in merino ewes

The 24 h i.v. infusion of Merino ewes with 60 or 100 microgram mouse epidermal growth factor (EGF)/kg body weight on Days 4, 9 or 14 of the oestrous cycle decreased the strength of wool attachment and caused marked changes in subsequent reproductive performance. In ovaries removed 2 days after EGF treatment all follicles greater than or equal to 0.6 mm diameter were atretic. After 7 days either a normal pattern of atresia or no atresia was evident while after 12 days the pattern of follicular atresia was similar to that in controls. Irrespective of stage of cycle EGF caused dose-dependent increases in plasma FSH concentrations that persisted for up to 14 days. Changes in plasma LH concentrations were generally similar after infusion on Days 4 and 14, but were smaller and shorter-lived after infusion on Day 9. Irrespective of dose, the infusion of EGF on Days 4 and 14 caused immediate luteolysis then the formation of a luteinized follicle in many ewes. Most ewes treated on Day 4 returned to oestrus between Days 17 and 21 with the same ovulation rate (1.3) as the controls. Of those infused on Day 14 oestrus occurred about a cycle length later than expected and their ovulation rate then (1.9) was also similar to that of the controls (1.7). Luteal function was not affected in ewes infused on Day 9, and most returned to oestrus between Days 17 and 20 with an ovulation rate of 3.2. Fertile rams were not placed with the ewes until after the differences in ovulation rate had been observed. Mating occurred generally 2-4 weeks after treatment, and there were no differences between EGF-treated and control ewes in fertility or fecundity. The results are interpreted as indicating that mouse EGF induces ovarian follicular atresia but has differential effects on luteal function according to the stage of the oestrous cycle at which it is given. As a consequence of these two effects, which lead to differential changes in gonadotrophin secretion, ovarian function may be temporarily impaired, little affected or improved.     

Increased ovulation rate in androstenedione-immune ewes is not due to elevated plasma concentrations of FSH

Two experiments were undertaken to determine the hormonal response of Merino ewes to immunization against androstenedione (Fecundin). In Exp. 1 peripheral concentrations of LH, FSH and progesterone were monitored in spontaneously cycling ewes (20 immunized and 21 controls). In Exp. 2 (10 immunized and 10 controls) the same hormones were measured in ewes before and after prostaglandin (PG)-induced luteolysis and, in addition, the pattern of pulsatile LH secretion was determined during the luteal (PG + 12 days), early follicular (PG + 24 h) and late follicular (PG + 40 h) phase of the oestrous cycle. Ovulation rates were measured in both experiments. The results of these experiments indicate that androstenedione-immune animals have elevated ovulation rates (0.6-0.7 greater than control animals; P less than 0.05) associated with elevated plasma concentrations of LH and progesterone. The magnitude of the increase in plasma progesterone was correlated with androstenedione antibody titre (r = 0.6, P less than 0.001). LH pulse frequency of androstenedione-immune ewes tended to be higher at all stages of the oestrous cycle, but this difference was only significant (P less than 0.05) during the luteal phase. Mean plasma concentrations of FSH did not differ significantly between immunized and control ewes at any stage of the cycle. Analysis of periodic fluctuations in FSH during the luteal phase revealed that androstenedione-immune animals had a similar number of fluctuations of a similar amplitude to those of control animals, but the nadir of these fluctuations was lower (P less than 0.05) in immunized animals. A significant (P less than 0.05) negative correlation existed between androstenedione antibody titre and the interval between FSH peaks (r = -0.49) and androstenedione antibody titre and FSH nadir concentrations (r = -0.46). It is concluded that plasma FSH concentrations are not a determinant of ovulation rate in androstenedione-immune ewes and that increased LH concentrations, or perturbation of normal intraovarian mechanisms, may be responsible for the increase in ovulation rate observed in ewes immunized against androstenedione.     

Effect of passive immunization against inhibin on FSH secretion, folliculogenesis and ovulation rate during the follicular phase of the estrous cycle in mares

Physiological roles of inhibin in mares were investigated by means of passive immunization using an antiserum to inhibin that had been raised in a castrated goat. Eight mares were given an intravenous injection of either 100 mL (n = 4) or 200 mL (n = 4) of inhibin antiserum 4 d after a single intramuscular injection of PGF2 alpha on Day 8 after ovulation, 4 control mares were treated with 100 mL castrated goat serum in the same manner. Jugular vein blood samples were collected after treatment with the serum until 192 h post treatment. Follicular growth and ovulations were monitored by ultrasound examination at 24-h intervals. The ability of the inhibin antiserum to neutralize the bioactivity of equine inhibin was examined in vitro using a rat pituitary cell culture system. Suppression of secretion of FSH from cultured rat pituitary cells by equine follicular fluid was reversed by the addition of increasing doses of the inhibin antiserum, thereby indicating its bioactivity. Plasma levels of FSH and estradiol-17 beta were higher in mares treated with the inhibin antiserum. The ovulation rate was significantly higher in mares treated with antiserum (100 mL = 3.75 +/- 0.63; 200 mL = 4.50 +/- 0.65) than in control mares (1.25 +/- 0.25). These results demonstrate that inhibin is important in regulating FSH secretion and folliculogenesis in mares. They also show that neutralization of the bioactivity of inhibin may become a new method for the control of folliculogenesis and ovulation rate in mares.     

Changes in plasma concentrations of immunoreactive inhibin,progesterone, and bioactive gonadotrophin during pregnancy in the marmoset monkey

This study describes the peripheral concentrations of immunoreactive (ir) inhibin, progesterone, and bioactive gonadotrophin during pregnancy in the marmoset monkey, a New World primate. Blood samples were taken every two weeks from six animals from ovulation to parturition. Plasma ir-inhibin concentrations were measured by inhibin α-subunit-directed radioimmunoassay (RIA) and a recently developed two-site immunoradiometric assay (IRMA) which is specific for inhibin αβ dimer. Concentrations of α-inhibin increased (P < 0.001) during early pregnancy to reach a peak on week 12 of pregnancy and showed a positive correlation with bioassayable gonadotrophin concentrations (r = 0.5, n = 64; P < 0.001). The concentrations of both α-inhibin and gonadotrophin showed no further peaks and declined (P < 0.001) to low levels prior to birth. Concentrations of dimeric inhibin were substantially lower than those measured by RIA and did not vary significantly during pregnancy. Progesterone concentrations remained at luteal-phase levels during the first half of pregnancy and increased (P < 0.05) during the second half to reach maximum concentrations just prior to birth. The relationship between α-inhibin, gonadotrophin, and progesterone suggests that the increase in α-inhibin may be luteal in source and under the control of gonadotrophin. The absence of a second increase in α-inhibin later in pregnancy and the finding that lategestation placenta contained very little α-inhibin differs from observations in Old World primates studied and suggests that the placenta may not be a source of inhibin during pregnancy in the marmoset. The finding of high levels of α-inhibin, but not dimeric inhibin, suggests that inhibin-related molecules may have a role other than suppression of pituitary folliclestimulating hormone. © 1994 Wiley-Liss, Inc.     

Oestrus, time of ovulation, ovulation rate and conception rate in progestagen-treated ewes given Gn-RH, Gn-TH analogues and gonadotrophins.

The influence of Gn-RH, hCG and a PMSG-hCG mixture (PG600) on the time of ovulation, ovulation rate and on the occurrence of oestrus in ewes treated with progestagen-impregnated sponges for 12 days examined. The effects of Gn-RH analogues on plasma LH, oestrus, ovulation and conception rate were also investigated. Six separate experiments were carried out. When 50 micrograms Gn-RH were given 24 h after sponge removal ovulation occurred in 44--46% of ewes within 24 h and in all ewes by 34 h. Gn-RH was a more potent ovulation synchronizer than hCG. Both hCG and PG600 reduced the incidence of overt oestrus. Gn-RH also had this effect in ewes treated during February and May but not in August and September. Gn-RH analogues given 2 days before sponge removal significantly increased ovulation rate. The display of oestrus was not affected in ewes treated 2 days before sponge removal but was suppressed in 43-69% of ewes treated with an analogue at the time of sponge removal. Ovulation occurred in 50-62% of ewes within 30-35 h of injection of Gn-RH analogues, regardless of the time of their administration. The release of LH in response to one analogue was not influenced by the presence of the progestagen-impregnated sponge in the vagina. When given a Gn-RH analogue 2 days before sponge removal or at the time of sponge removal 63 and 62% of mated ewes became pregnant compared with 70% of control ewes.