Bull calves were immunized with GnRH analogue–human serum albumin (HSA-Cys-Gly-GnRH) conjugate to determine the effects of dose, adjuvant type and interval between primary and booster injections on plasma testosterone and LH concentrations, and testes and body growth. Friesian bull calves aged between 8 and 10 weeks (n = 72) were blocked according to age and weight and, within block, randomly assigned to 12 treatment combinations (n = 6 per treatment combination) in a 3 × 2 × 2 factorial plan. Main effects were (i) conjugate dose (0.0, 0.1 or 1.0 mg HSA-Cys-Gly-GnRH), (ii) adjuvant (diethylaminoethyl-dextran or non-ulcerative Freund's adjuvant), and (iii) interval between primary (day 0) injection and booster injection (day 28 or 56). Plasma testosterone and LH concentrations and antibody titres were determined in blood samples collected at 14 day intervals during the experiment (140 days). Testicular measurements were taken in situ every 28 days. Antibody titres (% binding at 1:160 dilution) were ≥ 10% 28 days after booster injection and remained high for 140 days in 47 of 48 GnRH-immunized bulls. The mean titre was higher (P < 0.05) in response to the 1.0 mg dose compared with the 0.1 mg dose (37.7% versus 29.6% binding, respectively; pooled SED 2.55%). Mean LH and testosterone concentrations were reduced (P < 0.05) in immunized animals compared with controls. However, the 1.0 mg dose decreased mean testosterone concentrations by a greater extent (P< 0.001) than either the 0.1 mg or 0.0 mg doses. Testes length and depth, and scrotal circumference were decreased (P< 0.001) in immunized animals compared with controls; however, the 1.0 mg dose decreased (P< 0.001) testes parameters to the greatest extent. There was no effect of conjugate dose on average daily gain in body mass. It is concluded that (i) dose of conjugate, type of adjuvant and interval between primary and booster injections affected antibody titres, (ii) the use of 0.1 or 1.0 mg of HSA-Cys-Gly-GnRH decreased LH and testosterone concentrations, and testicular development throughout the experiment, without adversely affecting body growth, and (iii) an effective protocol is 1.0 mg GnRH–HSA conjugate, given in the adjuvant diethylaminoethyl dextran, with a primary–booster interval of 56 days.
M. Finnerty, W. J. Enright, C. A. Morrison and J. F. Roche
D. G. Morris, M. G. McDermott, M. G. Diskin, C. A. Morrison, P. J. Swift and J. M. Sreenan
Three peptide sequences from the bovine inhibin α-subunit (P1: 18–30; P2: 63–72 and P3: 107–122) were synthesized and conjugated to human serum albumin (HSA). Hereford crossheifers (n = 5 per group) were injected with 3 mg of one of the peptide conjugates, followed by three booster injections at intervals of 11 weeks. Control heifers (n = 5) were injected with HSA only. Antibodies recognizing both the individual peptides and 32 kDa bovine inhibin were generated and ovulation rate was increased in peptide immunized heifers. In group P1, 1 of 5 heifers responded with an increased ovulation rate whereas in groups P2 and P3, 5 of 5 and 4 of 5 heifers, respectively, had an increased ovulation rate. In group P2, in the first oestrous cycle following booster injections 2 and 3, 4 of 5 and 3 of 5 heifers, respectively, responded with twin ovulations, whereas a fourth heifer had three ovulations following booster injection 3. After breeding following booster injection 3, 3 of 5 heifers in group P2 and 1 of 5 in group P3 gave birth to twin calves. This study demonstrates the potential of immunizing against synthetic peptide sequences of the α-subunit of bovine inhibin to increase ovulation and twinning rates in cattle.
D. G. Morris, M. G. McDermott, M. Grealy, M. G. Diskin, C. A. Morrison, P. J. Swift and J. M. Sreenan
The effects of immunizing cattle against either of two peptides from the amino terminal peptide (αN) of the α43-subunit of bovine inhibin on ovulation rate, gonadotrophin concentration and fertility were investigated. Two peptide sequences from the αN-subunit of bovine inhibin (P1N, bIα-(8-20) and P2N, bIα-(153-167)) were synthesized and conjugated to human serum albumin (HSA). Hereford-cross heifers (n = 5 per group) were given an initial injection of 3 mg of one of the peptide conjugates, followed by three booster injections (1.5 mg) at intervals of 11 weeks. Control heifers (n = 5) were injected with HSA only. Blood samples were taken once a week to measure antibody titre and every hour at about the time of the first oestrus and during the mid-luteal phase after the second booster injection, to measure FSH and LH concentrations. Ovulation rate was measured by ultrasonography. Gonadotrophin concentrations were analysed for four periods relative to the peak (time = 0 h) of the preovulatory LH surge as follows: pre-surge: −16 to −5 h; surge: −4 to 4 h; post-surge: 5 to 16 h and a period of 12 h during the mid-luteal (days 10–12) phase. Antibodies that bound to the individual peptides were generated and the ovulation rate increased (P < 0.05) in immunized heifers. Control heifers had one ovulation at all ovulatory cycles monitored. In group P1N, one heifer had two ovulations at each of the six cycles monitored, while another heifer had two ovulations at one cycle. Three heifers in group P2N had more than one ovulation; one heifer had a superovulation response on seven occasions and another heifer on three occasions while the third heifer had two ovulations at one cycle. The superovulation responses ranged from three to eight ovulations. There was no evidence of a significant correlation between peptide antibody titre, ovulation rate and FSH at any of the periods studied. After mating following the third booster injection, nine of the ten immunized and all five control heifers calved. One heifer from group P1N and two heifers from group P2N gave birth to twin calves. These data show that in cattle immunization against the αN-subunit of bovine inhibin significantly disrupts the mechanism(s) controlling ovulation rate but does not impair fertility.
K. P. McNatty, D. A. Heath, K. M. Henderson, S. Lun, P. R. Hurst, L. M. Ellis, G. W. Montgomery, L. Morrison and D. C. Thurley
Summary. The patterns of ovarian follicular development and the steroidogenic properties of individual follicles (≥2 mm diam.) were assessed in Angus cows from Day − 5 until Day + 1 of the oestrous cycle (oestrus = Day 0). Individual follicles were judged to be healthy or atretic using a new classification system incorporating assessments of thecal vascularity and colour, the number of granulosa cells, the presence or absence of debris in follicular fluid and the status of the oocyte.
The results suggest that the theca interna of small antral follicles (< 5 mm diam.) responds to LH and synthesizes androstenedione before the granulosa cells develop an appreciable ability to metabolize androgen to oestrogen. Regardless of follicle size, the output of thecal androstenedione per unit mass of tissue remained unchanged in healthy but not in atretic follicles. On a per cell basis, aromatase activity increased in granulosa cells from healthy but not from atretic follicles with increasing follicle size. Peak levels of aromatizing activity were consistently observed in dominant oestrogenenriched follicles on Day 0 although similar activity was also observed in some healthy follicles (≥8 mm diam.) on other days of the cycle. Early atresia in bovine follicles was characterized by an absence or lowering of aromatase activity in granulosa cells which always preceded any reduction in the thecal steroidogenic response to LH.
It was estimated that between 20 and 60 antral follicles (≥2 mm diam.) per cow may respond to LH by synthesizing androgen whereas only 1-3 follicles (>5 mm diam.) have granulosa cells capable of metabolizing androstenedione or testosterone to oestradiol.