Effects of immunization against alpha-inhibin using two adjuvants on daily sperm production and hormone concentrations in ram lambs.

Twenty-five ram lambs were immunized against alpha-inhibin peptide emulsified in Freund's adjuvant (FRA), Emulsigen (EML) containing an oligodeoxynucleotide as an immunostimulant, or adjuvant without alpha-inhibin antigen (control). Four immunizations were administered during an 85-d period, after which testes were obtained for determination of daily sperm production (DSP) and histological evaluation. alpha-Inhibin antibody (Ab) titers were 70-fold greater in lambs treated with FRA than in EML-treated ram lambs. alpha-Inhibin immunization had no effect on testes weight or on plasma concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone. Mean DSP/g tended (P=0.1) to be greater in alpha-inhibin-immunized (EML=17.6x10(6); FRA=15.8x10(6)) ram lambs than in control animals (14.4x10(6)). One of the 8 control ram lambs had an elevated DSP/g, which was a statistical outlier. Without data from this lamb, DSP/g was increased (P<0.01) in alpha-inhibin-immunized ram lambs by 28% over controls. No association was found between the titer of alpha-inhibin Ab developed and DSP/g. Histologically, the percentage of testicular area occupied by seminiferous tubules differed (P=0.01) by treatment and was greatest (82%) in EML-treated ram alpha-inhibin-immunized lambs and lowest (74%) in control animals. Percentage tubular area and DSP/g were correlated (r=0.57, P=0.003). Findings show that (1) the extent of the increase in DSP/g is not dependent on the titer of alpha-inhibin Ab; (2) the increase in DSP/g is achieved through an increase in the mass of seminiferous tubules; and (3) FRA elicits a greater alpha-inhibin Ab titer than EML containing an oligodeoxynucleotide.

Effects of immunization against two inhibin antigens on daily sperm production and hormone concentrations in ram lambs.

The gonadal hormone inhibin regulates daily sperm production (DSP) indirectly through negative feedback control of FSH secretion and may also affect DSP via direct actions within the testis. Studies attempting to increase DSP through the immunization against inhibin have yielded equivocal results. The current study compared 2 inhibin antigens for effects on DSP and hormone secretion. Hampshire ram lambs (BW = 42 +/- 2 kg; age = 113 +/- 3 d) were assigned randomly to 3 groups: 1) control (n = 4); 2) alpha-peptide conjugate (PTC, n = 6); and 3) alpha-subunit (SUB, n = 6). Antigen PTC consisted of an alpha-inhibin, N-terminal, 25-amino acid peptide conjugated to ovalbumin. Antigen SUB was the complete inhibin alpha-subunit. Lambs were immunized on d 0 (June 19, 2006), 18, 38, and 63. Body weight was recorded on immunization days and scrotal circumference on d 63. Blood samples were collected on d 0, 7, 14, 18, 28, 35, 38, 49, 56, 63, and 70. Rams were slaughtered on d 71. Testes were weighed, and parenchyma was obtained for DSP determination. Plasma alpha-inhibin antibody titer and LH, FSH, and testosterone concentrations were measured. alpha-Inhibin antibody titer was first detectable on d 14 in both PTC- and SUB-immunized ram lambs and generally increased thereafter. Mean DSP per gram of testis (DSP/g) was increased (P < 0.01) 26% in PTC- and SUB-immunized ram lambs over that in control ram lambs. Total DSP per ram lamb and testes weight did not differ among the 3 treatment groups. Variation in DSP per ram lamb and testes weight were greater (P = 0.05) in PTC- and SUB-immunized ram lambs than in control ram lambs. Plasma FSH concentrations were similar in PTC- and SUB-immunized ram lambs. Immunization against either alpha-inhibin antigen did not alter LH, testosterone, BW, or scrotal circumference. Findings indicate that 1) the 2 alpha-inhibin antigens increase DSP/g to similar extents; 2) alpha-inhibin antibody may act at least in part through an intratesticular mechanism because DSP/g was increased in some animals without concomitant increases in FSH; and 3) immunization against alpha-inhibin may affect testes weight by actions independent of those that regulate DSP/g.

Induction of cyclicity in postpartum anestrous beef cows using progesterone, GnRH and estradiol cypionate (ECP).

The objective of the present study was to determine whether treatment of postpartum multiparous and primiparous anestrous beef cows with an intravaginal progesterone-releasing insert (CIDR) and PGF(2alpha), with and without the addition of GnRH or estradiol cypionate (ECP) at the time of CIDR insertion, is effective in stimulating onset of estrous cycles. Postpartum lactating Angus primiparous (n=47, 2 years of age, 495+/-6 kg) and multiparous (n=76, >or=3 years of age, 553+/-9 kg) cows were assigned by calving date to four blocks spaced 21-day apart. Cows were assigned sequentially by calving date to four treatment groups: (1) PGF(2alpha) (n=30), (2) CIDR-PGF(2alpha) (n=30), (3) GnRH-CIDR-PGF(2alpha) (n=33), and (4) ECP-CIDR-PGF(2alpha) (n=27). Intravaginal CIDR inserts were in place from days -7 to 0. A single 100 microg injection of GnRH or 2 mg ECP were administered on day -7, and 25mg PGF(2alpha) was administered on day 0. Day 0 averaged 38+/-1 day postpartum. Blood samples were collected on days -19, -9, 0, 5, 9, 12, 16, 19, 23, 26, and 30 for determination of plasma progesterone concentrations. Pre-treatment luteal activity (progesterone>or=1 ng/ml) was detected in 19% of primiparous and 8% of multiparous cows. Progesterone concentrations on day 0 were greater (P<0.001) in primiparous (3.2+/-0.3 ng/ml) than multiparous (2.0+/-0.2 ng/ml) cows. Following CIDR withdrawal, progesterone concentrations from days 5 to 30 were used to categorize response profiles as either: (1) treatment-induced onset of estrous cycles, (2) continued anestrus, or (3) spontaneous ovulation and subsequent formation of a CL. Incidence of treatment-induced onset of estrous cycles, which was defined as progesterone concentrations >or=1 ng/ml in three or more consecutive samples from days 9 to 19, was influenced by treatment and parity. Percentages of cows initiating estrous cycles were greater (P<0.001) in the three CIDR-treated groups than in the PGF(2alpha) group (55 and 8%, respectively). Percentages of cows initiating estrous cycles in the CIDR-PGF(2alpha), GnRH-CIDR-PGF(2alpha), and ECP-CIDR-PGF(2alpha) groups were 55, 58, and 52%, respectively. Incidence of treatment-induced estrous cycles in the three CIDR-treated groups of cows was greater (P=0.008) in primiparous (76%) than multiparous (43%) cows. Treatment of postpartum anestrous primiparous and multiparous beef cows with CIDR-PGF(2alpha) approximately 40-day postpartum provides an approach to increase the percentage of cows that have reinitiated estrous cycles by the start of the breeding season.

Changes in spermatogenesis and endocrine function in the ram testis due to irradiation and active immunization against luteinizing hormone-releasing hormone.

Spermatogonial stem cell transplantation is a technique that has potential in livestock to enhance genetic gain and generate transgenic offspring through the male germ line. A means for depletion of endogenous germ cells in a recipient's seminiferous tubules is necessary for this technology to be applied. The objectives of this study were to evaluate several methods for depletion of endogenous germ cells in the testes of adult rams and to evaluate ultrasound-guided injections into the rete testes as a means for infusing a suspension into the seminiferous tubules. Sixteen adult rams were randomly divided into 4 treatment groups (n = 4 per group). Treatments consisted of active immunization against LHRH (IMM), localized testicular irradiation (IR), LHRH immunization + irradiation (IMM+IR), and untreated control. Serial bleedings were conducted pretreatment and monthly after treatment for 4 mo, at which time all rams were castrated. Both IMM and IMM+IR rams received exogenous gonadotropin in the form of Perganol weekly for 8 wk before castration to bypass the immunization. All rams also received an ultrasound-guided injection of PBS containing 0.4% trypan blue into the rete testis of one testicle before castration. Rams receiving IMM and IMM+IR treatments had higher (P < 0.05) average percentages of seminiferous tubule cross sections with depleted germ cells compared with controls. Serum testosterone was decreased (P < 0.05) in IMM and IMM+IR rams 1 mo after treatment and throughout the remainder of the study compared with controls and IR rams, which were not different from each other. Serum inhibin concentration was unchanged in all rams following treatment indicating that Sertoli cell function was unaltered. A greater (P < 0.05) average percentage of the total testicular area could be filled with the trypan blue solution by rete testis injection in IMM and IMM+IR rams. These data demonstrate the depletion of endogenous germ cells in adult ram testes without alteration of Sertoli cell viability and function that have potential as methods for preparing recipient animals for germ cell transplantation.

Plasma LH, FSH, testosterone, and age at puberty in ram lambs actively immunized against an inhibin alpha-subunit peptide.

Active immunization against inhibin has been shown to advance puberty and increase ovulation rate in ewe lambs; but in ram lambs, effects on puberty and sperm production are equivocal. The objective of the present study was to determine whether active immunization against an inhibin alpha-subunit peptide advances the onset of puberty in ram lambs. St. Croix hair sheep ram lambs were assigned to inhibin-immunized (n = 7) and control (n = 8) treatment groups. Lambs in the inhibin-immunized group were immunized against a synthetic peptide-carrier protein conjugate, alpha-(1-25)-human alpha-globulin (halpha-G), and control lambs were immunized against halpha-G. Lambs were immunized at 3, 7, 13, 19, 25, 31, and 37 weeks of age. On the day of immunization a blood sample was collected and lambs were weighed. Another blood sample was collected 1 week following each immunization. At 20 weeks of age additional blood samples were collected at 20 min intervals for 8h. Beginning at 20 weeks of age and at weekly intervals thereafter, scrotal circumference (SC) was measured and semen was collected using electroejaculation. A subsequent ejaculate was collected 1 week following onset of puberty, which was defined as the week of age when an ejaculate first contained > or =50 x 10(6) sperm cells. In control lambs, plasma alpha-(1-25)-antibody (Ab) was nondetectable. In inhibin-immunized lambs, alpha-(1-25)-Ab titer increased from 7 to 25 weeks of age and then plateaued at a level that varied (P<0.001) among animals. Body weight and SC of control and inhibin-immunized lambs were similar at the onset of puberty. At pubertal onset inhibin-immunized lambs were older than control lambs (31.9+/-0.5 vs. 29.5+/-0.7 weeks of age, P<0.05). Plasma FSH concentrations were similar in control and inhibin-immunized lambs from 3 to 38 weeks of age. Plasma LH levels were lower (P<0.01) in inhibin-immunized than control lambs. During the 8-h blood sampling period at 20 weeks of age, LH and testosterone concentrations were lower (P<0.05) in inhibin-immunized than control ram lambs, and the LH pulse frequency was similar in the two groups of animals. The decreased LH secretion is consistent with the immunoneutralization of a putative inhibin alpha-subunit-related peptide that stimulates LH secretion in ram lambs. Present findings show that active immunization against an inhibin alpha-peptide delays rather than advances puberty in ram lambs.

Induction of ovarian cystic follicles in sheep.

Cystic follicles are a significant cause of infertility in women, dairy cattle and sheep. Sheep were used as a model to identify factors that may elicit formation of cystic follicles. Insulin resistance and elevated LH activity were tested in overweight ewes because of associations among these factors and the formation of cystic follicles. Sheep were synchronized using a progesterone-releasing pessary and insulin resistance was induced during the synchronization period through administration of bovine somatotropin. Following removal of pessaries follicular growth was stimulated by treatment with eCG or eCG and hCG (PG-600). Follicular growth was monitored via daily transrectal ultrasonography and blood samples were collected for hormonal analyses. Six of 18 ewes had a subnormal or absent preovulatory gonadotropin surge and developed cystic follicles. Neither insulin resistance nor elevated LH activity were associated with formation of cystic follicles. Ewes that developed cystic follicles were heavier (93 +/- 4 kg) than ewes that ovulated (81 +/- 3 kg; P = 0.02). Furthermore, following pessary removal and initiation of daily ultrasonography, ewes that developed cystic follicles lost body weight (-3 +/- 1%), while ovulatory ewes continued to gain body weight (1 +/- 1%; P = 0.005). It is speculated that in heavy ewes metabolic factors associated with acute body weight loss inhibit the positive feedback of estradiol and thereby suppress the preovulatory gonadotropin surge leading to formation of cystic follicles.

Effects of dietary lysine intake during lactation on blood metabolites, hormones, and reproductive performance in primiparous sows.

Effects of three dietary lysine (protein) concentrations during lactation on metabolic state, protein metabolism, reproductive hormones, and performance were investigated in 36 primiparous sows. Sows were assigned randomly to one of three diets containing .4% (low lysine, LL), 1.0% (medium lysine, ML), or 1.6% (high lysine, HL) total lysine from intact protein sources. All diets contained 2.1 Mcal NE/kg and exceeded the recommended requirements for all other nutrients. Actual lysine intakes over an 18-d lactation were 16, 36, and 56 g/d for sows fed LL, ML, and HL, respectively. Fractional breakdown rate of muscle was determined on d 4 and 15 of lactation by using a three-compartment kinetic model of 3-methylhistidine metabolism. Increasing lysine intake during lactation did not affect fractional breakdown rate of muscle on d 4 of lactation but decreased it on d 15 (P < .05). Sows fed LL had a reduced number of LH pulses on d 12 and 18 (P < .05) and reduced serum estradiol (E2) concentration on d 18 of lactation compared with sows fed ML and HL treatments. However, LH pulses and E2 concentrations were similar between ML and HL treatments (P > .35). Increasing lysine intake increased serum urea nitrogen (SUN) and postprandial insulin concentrations (P < .05) during lactation but had no effect on plasma glucose concentrations (P > .20). Sows fed HL had greater serum IGF-I on d 6 and 18 than sows fed ML (P < .05). Number of LH peaks was correlated with serum insulin concentration 25 min after feeding on d 6 and 18 (r = .31 to .41; P < .1) and pre- (r = .33 to .46) and postprandial (r = .30 to .58) SUN concentrations (P < .05) during different stages of lactation. Results indicate that, compared with medium lysine intake, low lysine intake increased muscle protein degradation and decreased concentrations of insulin, SUN, and estradiol and LH pulsatility. In contrast, high lysine (protein) intake increased SUN, insulin, and IGF-I, but did not increase secretion of estradiol and LH compared with medium lysine intake. Furthermore, nutritional impacts on reproduction may be mediated in part through associated effects on circulating insulin concentration.

Inhibin localization in equine granulosa-theca cell tumours and inhibin forms in tumour fluid.

The aim of this study was to examine inhibin production in granulosa-theca cell tumours (GTCT). The experimental aims were: (i) to determine GTCT cell types that produce inhibin alpha- and betaA-subunits; (ii) to determine whether alpha- and betaA-subunit forms differ in GTCT fluid and normal equine follicular fluid (eFF); and (iii) to determine whether dimeric inhibin (alpha betaA) is present in GTCT plasma and tumour fluid. Plasma, tumour fluid and tumour tissue were collected from mares (n=6) with GTCT. Plasma and eFF were collected during the follicular phase from mares (n=4) undergoing normal cycles. Immunohistochemical examination of GTCT tumour sections showed strong inhibin alpha- and betaA-Subunit immunostaining in granulosa cells and polyhedral-shaped cells in the thecal-stromal layer. The presence of polyhedral-shaped cells was related to testosterone concentration in tumour fluid. Low molecular weight alpha-subunit forms were less abundant in tumour fluid than in eFF, whereas the amounts of betaA-subunit forms were similar in tumour fluid and eFF. Concentrations of betaA were increased in plasma from mares with GTCT and similar in tumour fluid and eFF. In summary, lower molecular weight alpha-subunit forms were less prominent in GTCT fluid than in eFF and concentrations of betaA were higher in GTCT plasma than in control plasma.

Dimeric inhibin concentrations in mares with granulosa-theca cell tumors.

OBJECTIVE: To determine whether concentrations of dimeric inhibin (CaCA) are greater in plasma and tumor fluid from mares with granulosa-theca cell tumors (GTCT), compared with concentrations in plasma and equine follicular fluid (eFF) from control mares. ANIMALS: 6 mares with GTCT and 12 clinically normal mares. PROCEDURE: The alphabetaA immunoradiometric assay used 2 antibodies, one against each subunit of inhibin (alpha and betaA subunits). Tumor tissue, tumor fluid, and a single blood sample were collected at the time of surgical removal of the GTCT. A single blood sample was collected from 7 control mares during various stages of the estrous cycle. Five other control mares were ovariectomized when their ovaries contained growing follicles of 25 to 35 mm in diameter. A blood sample and eFF from the largest follicle were collected at the time of ovariectomy. RESULTS: Mares with GTCT had significantly greater plasma concentrations of betabetaA (mean +/- SEM, 0.86 +/- 0.53 ng of recombinant human-alphabetaA/ml), compared with control mares (0.14+/-0.02 ng/ml). Concentrations of alphabetaA in tumor fluid and eFF were similar. Concentrations of alphabetaA were significantly lower after ovariectomy. CONCLUSIONS AND CLINICAL RELEVANCE: Dimeric inhibin concentration was higher in plasma from mares with GTCT than in plasma from control mares. Increased granulosa cell mass and loss of mechanisms regulating alphabetaA release in mares with GTCT likely accounted for the increase in plasma concentrations. Measurement of alphabetaA concentrations may be useful for identifying mares with GTCT.

Estrus synchronization and artificial insemination of hair sheep ewes in the tropics

Hair sheep ewes (St. Croix White and Barbados Blackbelly) were used to evaluate 3 methods of estrus synchronization for use with transcervical artificial insemination (TAl). To synchronize estrus, ewes (n=18) were treated with PGF2alpha (15 mg, im) 10 d apart, with controlled internal drug release (CIDR) devices containing 300 mg progesterone for 12 d (n=18), or with intravaginal sponges containing 500 mg progesterone for 12 d (n=18). On the day of the second PGF2alpha injection or at CIDR or sponge removal, sterile rams were placed with the ewes. Jugular blood samples were collected from the ewes at 6-h intervals until the time of ovulation, and daily for 16 d after estrus (Day 0). Plasma was harvested and stored at -20 degrees C until LH, and progesterone concentrations were determined by RIA. There was no difference (P>0.10) in time to estrus among the CIDR-, PGF2 alpha- or sponge-treated ewes. All of the ewes in the CIDR group and 94.4 percent of the sponge treated ewes exhibited estrus by 36 h after ram introduction, while only 72.2 percent of PGF2alpha-treated ewes showed signs of estrus by this time (P<0.06). The time from ram introduction to ovulation was not different (P>0.10) among CIDR, PGF2alpha- or sponge-treated ewes. The time to preovulatory LH surge was similar (P>0.10) among treatment groups. Hair sheep ewes (n=23) were synchronized using PGF2alpha and bred by TAI using frozen-thawed semen 48 h after the second injection. The conception rate to TAI was 2/23 (8.7 percent) and produced 3 ram lambs. In a subsequent trial, 17 ewes were synchronized with CIDR devices and bred by TAI using frozen-thawed semen 48 h after CIDR removal, resulting in a conception rate of 52.9 percent (9/17). It is possible to synchronize estrus in hair sheep using either CIDRs, sponges or PGF2alpha. Even though there were no significant differences in the timing of ovulation or the LH surge among the treatment groups, a higher concentration rate was achieved in ewes synchronized with CIDR devices during the second trial. This may reflect an increase in the skill level of the TAI technician.(AU)

Estrus synchronization and artificial insemination of hair sheep ewes in the tropics.

Hair sheep ewes (St. Croix White and Barbados Blackbelly) were used to evaluate 3 methods of estrus synchronization for use with transcervical artificial insemination (TAI). To synchronize estrus, ewes (n = 18) were treated with PGF2alpha (15 mg, im) 10 d apart, with controlled internal drug release (CIDR) devices containing 300 mg progesterone for 12 d (n = 18), or with intravaginal sponges containing 500 mg progesterone for 12 d (n = 18). On the day of the second PGF2alpha injection or at CIDR or sponge removal, sterile rams were placed with the ewes. Jugular blood samples were collected from the ewes at 6-h intervals until the time of ovulation, and daily for 16 d after estrus (Day 0). Plasma was harvested and stored at -20 degrees C until LH, and progesterone concentrations were determined by RIA. There was no difference (P>0.10) in time to estrus among the CIDR-, PGF2alpha- or sponge-treated ewes. All of the ewes in the CIDR group and 94.4% of the sponge treated ewes exhibited estrus by 36 h after ram introduction, while only 72.2% of PGF2alpha-treated ewes showed signs of estrus by this time (P<0.06). The time from ram introduction to ovulation was not different (P>0.10) among the CIDR-, PGF2alpha- or sponge-treated ewes. The time to the preovulatory LH surge was similar (P>0.10) among CIDR, PGF2alpha and sponge treated ewes. Progesterone levels through Day 16 after the synchronized estrus were not different (P>0.10) among treatment groups. Hair sheep ewes (n = 23) were synchronized using PGF2alpha and bred by TAI using frozen-thawed semen 48 h after the second injection. The conception rate to TAI was 2/23 (8.7%) and produced 3 ram lambs. In a subsequent trial, 17 ewes were synchronized with CIDR devices and bred by TAI using frozen-thawed semen 48 h after CIDR removal, resulting in a conception rate of 52.9% (9/17). It is possible to synchronize estrus in hair sheep using either CIDRs, sponges or PGF2alpha. Even though there were no significant differences in the timing of ovulation or the LH surge among the treatment groups, a higher conception rate was achieved in ewes synchronized with CIDR devices during the second trial. This may reflect an increase in the skill level of the TAI technician.

Effects of passive immunization using antibody against an alpha-inhibin peptide on follicle-stimulating hormone concentrations and litter size in sows.

The objective of the experiment was to determine whether passive immunization against inhibin at weaning would increase FSH secretion and thereby influence postweaning reproductive performance in sows. Commercial Yorkshire sows (n = 173) were assigned within parity to 5 alpha-inhibin fragment antibody (alpha-IF-Ab) dosage groups: 0 (control), 3.25, 6.5, 13 and 26 RP-2 kU/kg alpha-IF-Ab. Antibody had been semipurified from ovine antisera raised against alpha-IF, a peptide that mimicked the N-terminal region of inhibin's alpha-subunit. A RP-2 U refers to a laboratory reference preparation. Sows were administered a single intramuscular injection of control solution or alpha-IF-Ab just before 21-d-old piglets were weaned. Blood samples were taken immediately before immunization and 24 h later. Sows were bred upon expression of estrus. Serum alpha-IF-Ab titers in sows 24 h following passive immunization increased (P < 0.001) with dosage. In control sows serum FSH concentrations decreased 24% by 24 h postweaning (P < 0.001). The decrease was diminished or prevented by alpha-IF-Ab treatment in a dose-responsive manner (P < 0.001). Most (167/173) sows were bred within 10 d postweaning, and wean-to-service intervals tended (P < 0.1) to be shorter in the 13 and 26 RP-2 kU/kg alpha-IF-Ab dosage groups. Farrowing rate was 72% (124/173) and was similar among sows in the alpha-IF-Ab dosage groups. Litter size, expressed as total or live piglets born per sow or per sow farrowed, was unchanged by alpha-IF-Ab treatment. Results demonstrate that 1) inhibin plays a key role in regulating FSH secretion at weaning, and 2) blocking the acute postweaning drop in FSH secretion has little if any effect on subsequent reproductive performance.

Effect of eCG on the pregnancy rate of ewes transcervically inseminated with frozen-thawed semen outside the breeding season.

An experiment was conducted to determine whether factors affecting pregnancy rate out-of-season are associated more with transcervical artificial insemination (T-AI) procedures or with the reproductive state of the ewe. Twenty Finncross ewes were treated with progesterone sponges, and at sponge removal (0 h) 10 ewes were treated with eCG. Blood samples were collected for LH and progesterone analyses, and follicular development was monitored using ultrasonography. Ewes were inseminated from 48 to 52 h with 200 million motile frozen-thawed spermatozoa. The incidence of estrus, LH surges and ovulation was greater (P < 0.01) and intervals to these responses were shorter (P < 0.01) in the eCG-treated ewes. The number of follicles > 5 mm was higher (P < 0.05) in eCG-treated than control ewes. Progesterone concentrations increased and remained elevated through Day 19 in 7 eCG-treated and in 1 control ewe, and these ewes were pregnant based upon ultrasonographic examination. The results demonstrate that the T-AI technique using frozen-thawed semen produces a relatively high (70%) pregnancy rate out-of-season. The pregnancy rate was found to reflect primarily the reproductive condition of the ewe.

Effects of passive immunization against inhibin-peptide on secretion of follicle-stimulating hormone and ovulation rate in ewes carrying the Booroola fecundity gene.

Objectives of the study were to determine whether 1) inhibin negative feedback regulation of FSH secretion is diminished in ewes carrying a copy of the Booroola fecundity (FecB) gene and 2) differential FSH secretion is obligatory for expression of gene-specific differences in ovulation rate (OR). The approach was to compare FSH and ovulatory responses to passive immunoneutralization of inhibin in ewes with and without a copy of the FecB gene. Twenty-eight 2- to 3-yr-old ewes were assigned within genotype to antibody (alpha-IF-Ab) or control groups. Genotypes consisted of 3/4 Rambouillet x 1/4 Booroola ewes with one copy of the FecB gene (FecB+; 57 kg) and 3/4 Rambouillet x 1/4 Booroola ewes without the FecB gene (++; 59 kg). Estrus was synchronized during the breeding season using progesterone-releasing pessaries (CIDR-G). Pessaries were removed at 0 h. A single injection of alpha-IF-Ab or control solution was given at -48 h. Alpha-IF-Ab had been developed against a synthetic inhibin fragment matching the N-terminal region of ovine inhibin's alpha subunit. For injection, alpha-IF-Ab had been precipitated from ovine immune sera and concentrated. Blood samples were collected at 6-h intervals from -48 to 48 h, and laparoscopy was performed 14 days after CIDR-G withdrawal. All ewes exhibited estrus and ovulated. Genotype and alpha-IF-Ab treatment were without effect on intervals to estrus. Both factors affected OR (p < or = 0.001). Mean OR in control ++ and FecB+ ewes were 1.6 and 2.7, respectively; mean OR in alpha-IF-Ab-treated ++ and FecB+ ewes were 2.5 and 4.6, respectively. Following injection of alpha-IF-Ab, FSH concentrations increased within 6 h, peaked 12-18 h later, and then declined. Magnitude of FSH increases was similar in ++ and FecB+ ewes (70% and 85% over control values, respectively). Results demonstrate that 1) inhibin negative feedback regulation of FSH secretion is not a site of FecB gene action and 2) the mechanism by which the FecB gene increases OR does not necessarily involve increased FSH secretion during the period of preovulatory follicular development.

Measurement of dimeric inhibin in porcine serum: evidence for low concentrations and existence of binding proteins.

An immunoradiometric assay and serum extraction procedure were developed to measure dimeric inhibin in porcine serum with minimal interference by putative inhibin-binding proteins. Assay sensitivity was 50 pg/tube, and it incorporated antibodies against the N-terminal region of inhibin's alpha-subunit, alpha-(1-25)-Ab, and against the C-terminal region of inhibin's beta A-subunit. To determine whether inhibin-binding proteins were present in porcine serum, serum was incubated with [125I]-recombinant human (rh)-inhibin and then chromatographed by gel filtration. Radioiodinated rh-inhibin was associated with protein(s) > 600 kDa. Radioiodinated rh-inhibin also was incubated with alpha 2-macroglobulin, an inhibin-binding protein in human and rat serum. Elution profiles were similar for serum and alpha 2-macroglobulin. Serum- and alpha 2-macroglobulin-[125I]rh-inhibin complexes dissociated upon exposure to 8 M urea. Porcine serum was treated with urea, after which inhibin was isolated and concentrated. The recovery of rh-inhibin added to starting serum was 28%. Concentrations of endogenous dimeric inhibin were < 28 pg/ml in serum collected from sows at random stages of the estrous cycle and were < 21 pg/ml in serum collected from sows 2 d postweaning. Results demonstrate that 1) concentrations of dimeric inhibin are low in porcine serum, and 2) an inhibin-binding protein(s), consistent with alpha 2-macroglobulin, is present in porcine serum.

Effect of concentrate source and amount in diets on plasma hormone concentrations of prepubertal heifers.

Effects of concentrate source (corn or soybean hulls) and amount (40 or 80% of DMI) on plasma hormone concentrations were evaluated for 32 prepubertal Holstein heifers in a 2 x 2 factorial design. Blood samples were collected every other day from d -4 through d 28 of the experiment and were analyzed for growth hormone, prolactin, IGF-1, and glucose. Ruminal samples were collected by esophageal tube on d 0, 7, 14, 21, and 28 and were analyzed for VFA content. Serial blood samples were collected on either d 25 or 26 at 20-min intervals for 8 h and were used for profile analysis of growth hormone and prolactin. Heifers fed 80% concentrate had higher IGF-1 concentrations in serum (158.1 vs. 131.9 ng/ml) than did heifers fed 40% concentrate, and heifers fed corn tended to have higher IGF-1 concentrations than heifers fed soybean hulls. Prolactin concentrations in samples that were collected every other day were higher for heifers fed the 80% soybean hull diet than for those fed 40% concentrate diets or the 80% corn diet. Effects of source and amount of concentrate on plasma prolactin concentrations were not evident in serial samples. Concentrate source or amount had no effect on plasma growth hormone or glucose. At high concentrate intakes, the source (soybean hulls vs. corn) affected intake parameters, ruminal VFA content, and prolactin concentrations.

Passive immunization of ewes against an inhibin-like peptide increases follicle-stimulating hormone concentrations, ovulation rate, and prolificacy in spring-mated ewes.

The objective of this experiment was to determine whether passive immunization against a synthetic inhibin-like peptide (alpha-IF) increases FSH secretion and reproductive performance of ewes bred in the spring. Crossbred ewes were assigned to three treatment groups of 10 ewes each. Progesterone-releasing pessaries (CIDR-G) were inserted and left in place for 12 d. The CIDR-G were removed on April 7 (0 h), and three rams were introduced. A single 1.8-mL i.m. injection containing 0, 315-, or 630-reference preparation-2 (RP-2) kU semipurified alpha-IF-antibody (Ab) was given at -48 h. Antibody solution was prepared by ammonium sulfate precipitation and concentration of ovine immune sera that had been generated against alpha-IF-human-alpha-globulin. After the alpha-IF-Ab injection, plasma alpha-IF-Ab titers and FSH concentrations increased within 6 h, peaked 12 to 24 h later, and then decreased. At 12 h after injection, FSH concentrations were increased (P = .05) 28 and 42% over control values in ewes injected with 315- and 630-RP-2 kU alpha-IF-Ab, respectively. All ewes expressed estrus and ovulated, except one control that did not ovulate. Ovulation rate (OR) was increased (P < .0001) by alpha-IF-Ab treatment. Mean OR was 1.0 in control ewes, 1.7 in ewes given 315-RP-2 kU, and 2.5 in ewes injected with 630-RP-2 kU. Fertility (ewes lambing/ewes exposed) from breeding at the induced-synchronized estrus tended (P = .09) to be greater for ewes injected with 630-RP-2 kU alpha-IF-Ab.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of passive immunization of ewes against an inhibin-peptide on gonadotropin levels, ovulation rate, and prolificacy.

The experimental objectives were to determine whether injection of semi-purified (sp; ammonium sulfate-precipitated) and highly purified (hp; immunoaffinity-purified) ovine antibody (Ab) against an inhibin-peptide fragment (alpha-IF) before the preovulatory period would 1) stimulate FSH secretion in a dose-response manner, 2) induce an increase in ovulation rate, and 3) affect pregnancy rate and prolificacy (lambs born alive per ewe lambing). During the early breeding season, estrus was synchronized in 30 2-yr-old crossbred ewes through use of progesterone-releasing pessaries (CIDR-G). Two doses (330 and 660 laboratory reference preparation [RP-2] kU) of sp- and hp-alpha-IF-Ab were injected i.m. 48 h before CIDR-G removal (6 ewes per group). Six other ewes received control solution. Plasma alpha-IF-Ab titers peaked at 12 h postinjection. Plasma FSH levels were higher (p < 0.02) in alpha-IF-Ab-treated ewes than in control ewes from 12 to 24 h postimmunization. Magnitudes of FSH increases were similar in ewes administered sp- and hp-alpha-IF-Ab and were greater (p < 0.05) in ewes receiving 660 than in those receiving 330 RP-2 kU. Compared to control values, the higher alpha-IF-Ab dose increased FSH levels by 44 +/- 5% and the lower dose increased the levels by 22 +/- 3%. Plasma LH levels were similar among passively immunized and control sheep. Ovulation rate was increased (p < 0.0005) by alpha-IF-Ab treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Estrous cycle characteristics of postpubertal gilts approaching anestrus due to limited dietary energy intake.

Twenty-nine Duroc x (Yorkshire x Landrace) gilts, approximately 9 mo of age and each having experienced at least three estrous cycles, were allotted randomly to five dietary treatments. Each treatment provided a different daily intake of metabolizable energy: .25, .5, .75, 1.0 Mcal (severely restrictive [RES]; chosen to cause cessation of estrous cycles) or 3.4 Mcal (control; designed for maintenance of estrous cyclicity). Blood samples were drawn by venipuncture every 3 d and sera were assayed for progesterone (P4) concentration. Estrous cycle length was estimated by defining d 0 as the last day when serum P4 concentrations were < 1 ng/mL before increasing to > 1 ng/mL. Gilts were considered acyclic when serum P4 levels remained < 1 ng/mL for four consecutive samples (9 d). Anestrus occurred after 78.7 +/- 6.1 d in all RES gilts (n = 23) and in one control on d 24. Compared with the control treatment, RES treatments resulted in longer (P < .05) average estrous cycles (21.0 +/- .2 vs 20.2 +/- .3 d) and greater (P < .05) mean serum P4 concentrations (13.7 +/- .4 vs 11.4 +/- .8 ng/mL). There were no differences in P4 or cycle length measurements among RES treatments. Cycle duration and serum P4 concentrations of the final estrous cycle before anestrus were similar to those of previous cycles. It is concluded that severe energy restriction caused elevated serum P4 concentrations. Neither cycle length nor serum P4 concentration was notably altered as gilts neared anestrus.

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)