Williams, Richard David
(2004)
Immunomodulation of reproductive function in domestic ruminants.
PhD thesis, University of Nottingham.
Abstract
Active immunisation against GnRH inhibits reproductive function by inducing a hypogonadotropic condition associated with gonadal atrophy. Despite economic, ethical and environmental advantages of GnRH immunisation in cattle over conventional castration methods, the technology has not yet been commercially adopted. Primarily because of the requirement for numerous booster vaccinations because of the reversibility of physiological effects, the commercial efficacy of immunocastration is currently poor. However, neonatal GnRH immunisation in sheep can result in a permanent suppression of reproduction (Brown et al., 1994; 1995; Clarke et al., 1998). These findings and a study in pigs (Molenaar et al., 1993) indicate that, the hypothalamic/pituitary gland unit (HPU) may be particularly susceptible to GnRH antibodies during a specific window of development in the pre-pubertal animal, but no long-term studies in cattle have been conducted. Therefore the primary objective of this project was to determine the effect of neonatal immunisation against GnRH in cattle. Beef cross bull (n=9; Chapter 3) and heifer calves (n=9; Chapter 4) were vaccinated against a newly developed (Pfizer®) GnRH construct vaccine at -2, 6 and 13 weeks of age. Nine calves of each sex served as negative controls, receiving saline injections only. The GnRH vaccine had proved effective (Dr. A.R. Peters, personnel communication 2000) in inducing immune responses and reducing variation between animals in unpublished industrial studies, compared to earlier vaccines, and hence was reasoned to be capable of raising GnRH antibodies despite the relative immaturity of the neonatal immune system. Following vaccination, circulating GnRH antibodies and reproductive hormones, such as FSH (Chapters 3 and 4), testosterone (Chapter 3), progesterone (to assess onset of puberty) and oestradiol (Chapter 4) were measured and additional intensive serial bleeds were carried out to assess LH parameters up to and beyond puberty (puberty defined by testes circumference in bulls). Gonadal (antral follicles and testes growth) and accessory gland development was quantified throughout the trial using ultrasound scanning. Sexual behaviour (Chapter 3) was studied from 38 weeks of age, while an assessment of sperm quality (Chapter 3), and anabolic response to vaccination was also performed post-mortem (Chapters 3 and 4).
GnRH immunisation in neonatal calves did not permanently impair reproduction. A temporary suppression in reproductive function was evident through the disruption of pituitary gland function, as indicated by a reduction of LH pulse amplitude and mean plasma LH concentrations (Chapters 3 and 4). In addition, a reduction in medium- sized follicle numbers, testes growth, plasma testosterone concentration, vesicular gland length and juvenile aggression occurred. Some beneficial anabolic effects were observed e.g., carcass composition grades. Changes all occurred subsequent to increased GnRH antibody titres in immunised cattle. Despite some evidence of prolonged effects on LH amplitude and circulating testosterone after anti-GnRH titres had dissipated, all inhibited parameters, except carcass quality, returned to levels comparable to control animals by 72 weeks of age. No treatment effects on FSH concentrations, large follicle numbers, reproductive tracts (post mortem) or peri- and post-pubertal behaviours were observed following treatment. Sperm morphological abnormalities tended to be more prevalent in GnRH immunised bulls.
A significant increase in GnRH antibody titres occurred at -23 weeks of age (Chapter 4), this may have been a rebound in antibody titre, possibly caused by an anti-idiotype immune response (antibody response to GnRH antibodies), or due to significant maturational changes in immune function at this time causing a delayed response to vaccination. Alternatively a novel "auto-immune" response may have been detected, which if confirmed/repeatable might be incorporated into an immunisation protocol to act as a "self-booster". However, no previous reports of such an event have been published and further investigation is urgently required.
A more prolonged or permanent suppression of reproductive function may be possible following an earlier, greater and more sustained elevation of antibody titres during the neonatal period. Further development of GnRH vaccines and/or protocols (prime-boost, cytokine modulation vaccines, concomitant passive and active immunisation and pregnant cow GnRH vaccination), and studies of performance and GnRH antibody mechanism(s) of action in cattle are required. Chapters 3 and 4 provide a comprehensive study on pubertal development and neonatal GnRH vaccination, thus contributing significantly to knowledge in these fields. Currently, the vaccine used in this trial may be used to delay puberty in older calves or transiently suppress reproductive function to aid management.
The economical viability of animal production systems such as beef and lamb are closely related to rates of reproduction. The Fec B gene in ewes increases ovulation rate and litter size, possibly through the development of precocious follicles, which can switch their primary dependence from FSH to LH. As a result, more follicles are selected to continue growth to an ovulatory size. The precise mechanisms by which these processes occur have recently been shown to involve oocyte follicle interactions (see section 1.1.5). Follicle development is modulated by GHIIGF and inhibin, however attempts to increase follicular development and ovulation through active inhibin immunisation alone have been variable and hence not commercially attractive. To develop successful protocols to induce twin ovulations in cows· and ewes, without superovulation, a clearer, more details understanding of follicullogenesis is required. The objective of the current study was to better understand these mechanisms through investigating interactions of GH/GF and inhibin in the ovary, follicle development, steroidogenesis, and receptor populations using an anoestrous sheep model.
Spring born Mule x Charolais ewe lambs were actively immunised (n=8) against porcine inhibin α-C 1-26 peptide conjugated to KLH in NUFCA (primary and 3 boosters (NUFA», while 8 served as negative controls. Seven days following the final booster, the ewes were subdivided to give four groups: (1) controls + saline (n=4); (2) controls + rbGH (4ml s.c; 1mg. mr1; n=4); (3) inhibin immunised + saline (n=4); and (4) immunised + rbGH (n=4). Recombinant bovine growth hormone (rbGH) was given (Lm.) for 6 days. On day 4 GnRH (Receptal®; 1 ml) was injected s.c, to all animals to initiate the beginning of a new follicular wave. Blood samples were collected fortnightly to measure inhibin antibody titres, IGF-I, FSH and steroids. On the seventh day ensuing slaughter serum antibodies and ovaries were harvested. Left ovaries were intended for ISH (mRNA for P450arom) and/or immunohistochemical analysis. Follicles from right ovaries were dissected out, counted, measured and cultured in M199 at 37°C for 2 hours. Culture media was then assayed for oestradiol. Follicle shells were stored at -180°C for LH receptor binding studies.
This work reports on the influence of different treatments on follicle populations. All immunised animals produced antibodies, which bound to 1251-inhibin. Using ANOVA to compare treatments it was observed that, Inhibin immunisation significantly (P<O.05) increased the number of follicles >3.5mm in diameter, but did not affect the smaller <3.5mm population. In contrast, rbGH administration led to a significant (P<O.05) elevation of follicles <3.5mm, without increasing the >3.5mm follicle numbers. These findings are in agreement with previous research. The molecular studies of left ovaries are not presented herein as due to time constraints the work was not completed and is currently on going.
In conclusion, additional results of this study are required to meet the objectives of the experiment. Further research is required on dominant follicle selection if superovulatory programmes in both livestock and humans are to be more precisely controlled and readily accepted.
An increased twinning rate in beef suckler cows is acknowledged as an optimal means of improving production efficiency (Diskin et al, 1987), and preliminary observations (Campbell, Webb and Gong, unpublished) suggests that following oestrous cycle synchronisation, a passive inhibin immunisation protocol gives a more predictable twin ovulation response than active inhibin vaccination. Therefore, using inhibin antiserum raised in ewes (Chapter 5), the final thesis objective was to investigate the efficacy of a bolus and booster antiserum administration protocol in heifers and the effects of passive inhibin immunisation on hormone parameters (FSH and progesterone), folliculogenesis (in vivo) ovulation rate and conception.
Beef cross heifers (n=18; used earlier in Chapter 4 study) were randomly allocated to receive inhibin antiserum or NSS (controls). Following the Chapter 4 study, and 4 months summer break at pasture, heifers were considered to be exhibiting typical cyclic reproductive function based on hormone measurements and scanning of ovaries. Oestrous cycles' were synchronised using 2 injections (Lm.) of PG 11 days apart and antiserum 'bolus' (108ml) was injected into the jugular vein 4 days after 2nd PG treatment. Subsequently, at 24h intervals for 5 days 14ml booster infusions were administered. Five days after ovulation, as confirmed by twice daily scanning, a 3rd PG was given and 72 and 96h later, heifers were A.1. Blood samples were collected every 8 hours from 2nd PG to 2nd A.1.
Dominant follicle number and ovulation rate was unaffected by treatment due to inadequate inhibin antibodies in antiserum compared with the earlier trial (Campbell, Webb and Gong, unpublished), however, recruited follicle numbers were enhanced in inhibin immunised heifers. In addition, further evidence that inhibin antibodies act at the ovarian level to enhance recruitment, or reduce atresia was provided, as FSH concentrations were similar between groups. Higher inhibin antibody titre may be necessary to over-ride the strong dominant follicle selection mechanism in heifers. In addition, the results of this and a previous trial demonstrate that a threshold inhibin antibody titre range is required to obtain a reproducible increase in ovulation rate on which future passive inhibin immunisation studies may be based. Finally, this approach of a bolus followed by subsequent small antiserum injections has successfully been shown to give a steady and consistent concentration of circulating inhibin antibody titres.
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