112 Follicular dynamics and oestrus response of Alpine goats with oestrus/ovulation synchronized during the early transitional reproductive phase using gonadotrophin given early or late

Oestrus and ovulation synchronization (E/OS) regimens for fixed-timed breeding are useful when consistently eliciting ovulation. Early synchronization in the reproductive season promotes unreliable oestrus behavioural and physiologic response due to insufficient ovarian priming. In ruminants, equine (eCG) or human chorionic gonadotrophin (hCG) has FSH bioactivity or elicits ovulation, correspondingly. Hence, 120 and 60IU of eCG and hCG, respectively, are included in goat E/OS regimes. This experiment addresses the time when eCG/hCG (CG) is given relative to progestagen (P4) withdrawal and its effect on oestrus and ovulation. Fourteen non-lactating, Alpine breed goats, ranging from 1 to 6 kiddings, averaging±s.d. 3.9±2.0 years of age, and 56.3±5.0kg of bodyweight (BW), and body condition score (BCS) of 2.6±0.2, were evaluated early in the transitional reproductive phase (12h daylight:12h darkness). E/OS was accomplished with 12 day of P4 (200 mg) exposure by intravaginal insert. Three treatments were evaluated: Traditional (T; n=4) did not receive CG and served as the control group; early (E; n=5) received CG 24h before P4 removal; and reverse (R; n=5) received PGF2α 24h before P4 withdrawal as well as CG concurrent with P4 withdrawal. Oestrus response to E/OS was evaluated 24h after breeding using epididymectomized bucks. Ovaries were scanned by transrectal ultrasound (Aloka SSD-500V/7.5-MHz linear array probe) for 4 consecutive days starting with the first i.m. injection of 1.0mL of PGF2α or 1.5mL of CG. Images were digitized (MediCapture™) for later morphometry. The absence of effect (P>0.05) from concomitant variables age, BW, BCS, and parity was ascertained using a logistic regression model (JMP/SAS v15; SAS Institute Inc.). Ovulation, defined as the disappearance of the largest (>7mm) preovulatory follicle on a subsequent observation, was 100, 80, and 100%; and the average±s.e. number of ovulations per goat was 2±0.41, 1±0.32, and 1.3±0.49 (P>0.05). Table 1 summarises follicular size documented of 122 observations of ovulatory areas. Graafian follicle location was not different for left or right ovaries (P>0.05). Oestrus behavioural response to each E/OS treatment (T, E, or L) up to 24h after P4 removal was 50, 80, and 80% (P>0.05), respectively. In summary, oestrus response and ovulation were not affected by the variables studied. In this experience the timing of CG was not relevant. Table 1. Mean diameter (±SE; mm) of nonovulatory and preovulatory follicles on left (L) and right (R) ovaries E/OS protocol Non- and preovulatory Nonovulatory Preovulatory L R L R L R Traditional 6.4±0.6 5.5±0.6 4.8±0.4 4.5±0.4 7.7±0.4 8.9±0.3 Early 5.2±0.3 5.5±0.4 4.9±0.2 4.4±0.3 8.4±1.2 8.1±0.2 Reverse 6.6±0.5 5.3±0.6 5.1±0.4 4.4±0.5 8.7±0.3 8.1±0.2 Overall 5.7±0.19 4.7±0.14 8.3±0.14

Identification of amino-acids in the alpha-subunit first and third loops that are crucial for the heterospecific follicle-stimulating hormone activity of equid luteinizing hormone/choriogonadotropin

OBJECTIVE: To identify amino-acids in the alpha-subunit important for expression of heterospecific FSH activity of horse (e) LH/choriogonadotropin (CG) (eLH) and donkey (dk) LH/CG (dkLH) (FSH/LH ratio ten times higher for eLH than for dkLH); this FSH activity absolutely requires an equid (donkey or horse) alpha-subunit combined with an equid beta-LH subunit. DESIGN: Chimeric alpha-subunits possessing the first 63 amino-acids of the porcine (p) and the last 33 amino-acids of the donkey alpha-subunit (alphap-dk) and the inverse (alphadk-p) were constructed. Porcine-specific amino-acids were introduced by mutagenesis in donkey alpha-subunit at positions 70, 85, 89, 93 and 96 (alphadk5xmut), 18 (alphadkK18E) or 78 (alphadkI78A). METHODS: These different alpha-subunits were co-transfected in COS-7 cells with beta-eLH, beta-dkLH and beta-eFSH. The LH and FSH bioactivities of the dimers were then assessed in two heterologous in vitro bioassays. RESULTS: alphap-dk or alphadk-p exhibited FSH activity when co-expressed with beta-eLH but not with beta-dkLH. alphadkK18E or alphadkI78A gave hybrids with no FSH activity and important LH activity when expressed with beta-dkLH. alphadkI78A/betaeLH displayed an FSH/LH ratio as low as that of dkLH. However, mutation at 78 in alpha-dk had no effect on FSH bioactivity when co-expressed with beta-eFSH. CONCLUSIONS: Amino-acids present in both the first two-thirds and the last third of the alpha-subunit of equid LHs are involved in their heterologous biospecificity. Ile alpha78 exerts as strong an influence on it as the beta102-103 residues. By contrast, this residue plays no role in the FSH specificity of eFSH.

Antiequine Chorionic Gonadotropin (eCG) Antibodies Generated in Goats Treated with eCG for the Induction of Ovulation Modulate the Luteinizing Hormone and Follicle-Stimulating Hormone Bioactivities of eCG Differently

In dairy goats, treatments associating a progestogen and the equine chorionic gonadotropin (eCG) are the easiest way to induce and synchronize estrus and ovulation and to permit artificial insemination (AI) and/or out of season breeding. From the first treatment, the injection of eCG induces, in some females, the production of anti-eCG antibodies (Abs) that will interfere with the effectiveness of subsequent treatments. These anti-eCG Abs delay the preovulatory LH surge and the ovulation time, leading to poor fertility of the treated females. In this study, by in vitro bioassays, we show that anti-eCG Abs can positively or negatively modulate the LH and/or FSH bioactivities of eCG. Moreover, the modulation level of eCG bioactivity does not depend on the anti-eCG Ab affinity for eCG, as shown by surface plasmon resonance technology. The specificity of anti-eCG Abs tested by competitive ELISA highlighted the importance of a glycan environment in the recognition mechanism, especially the sialic acids specific to eCG. The different effects of anti-eCG Abs on eCG bioactivities could be explained by two hypotheses. First, steric hindrance preventing the interaction of eCG with its receptors would explain the inhibitory effect of some anti-eCG Abs; second, a conformational change in eCG by anti-eCG Abs could induce inhibition or potentiation of eCG bioactivities. It is significant that these modulations of eCG bioactivities by anti-eCG Abs impact mainly on the FSH bioactivity of eCG, which is essential for ovarian stimulation and subsequent fertility after treatment and AI, and to a lesser extent on LH bioactivity.

Enhancement of FSH bioactivity in vivo using site-specific antisera

The ability of site-specific antipeptide antisera to enhance the biological activity of ovine FSH (oFSH) in vivo was investigated using hypopituitary Snell dwarf mice. These animals were shown to respond to increasing doses of oFSH (3·3–90 μg/day), administered in two daily injections over a 5-day treatment period, in a highly significant dose-dependent fashion. The responses measured were increases in uterine weight, ovarian weight and the index of keratinisation in vaginal smears. The dose-dependent response to oFSH confirmed the suitability of this animal model for these investigations and suggested the suboptimal dose of oFSH (20 μg/day) for use in enhancement studies. Five peptides derived from the β subunit of bovine FSH (bFSH) (A, residues 33–47; B, 40–51; C, 69–80; D, 83–94; E, 27–39) were used to generate polyclonal antipeptide antisera. Of these peptides, only A and B produced an antiserum (raised in sheep) capable of recognising 125I-bFSH in a liquid phase RIA. Antisera prepared against peptide A or peptide B were found to significantly enhance the biological activity of 20 μg oFSH/day over a 5-day treatment period. The response to antipeptide antisera alone did not differ significantly from that observed in PBS-injected control animals, neither did the response to FSH alone differ from that observed in animals treated with FSH plus preimmune serum. Thus the enhanced responses are dependent upon the presence of FSH plus antipeptide antiserum. Peptides A and B are located in a region thought to be involved in receptor recognition, this may have implications for the mechanism underlying this phenomenon and/or the structure/function relationships of FSH. That FSH-enhancing antisera can be generated by immunisation of animals with peptides A and B suggests that it may be possible to develop these peptides as vaccines capable of increasing reproductive performance, such as ovulation rate. The high degree of sequence homology between ovine, bovine and porcine (and to a lesser extent human and equine) FSH in the region covered by peptides A and B suggests that these peptides could also be used to promote and regulate ovarian function in all of these species. Journal of Endocrinology (1997) 152, 355–363

Pharmacokinetics and pharmacodynamics of recombinant and urinary human FSH in the male monkey (Macaca fascicularis)

A dose-finding study was performed in adult male monkeys (Macaca fascicularis) to evaluate the pharmacokinetics and pharmacodynamics of a recombinant human FSH preparation (rhFSH). Groups of five monkeys were randomly assigned to receive single i.m. injections of 0·9% (w/v) NaCl (diluent), 6, 12 or 24 IU rhFSH/kg or 24 IU urinary human FSH/kg (uhFSH). The doses were based on an in vivo ovarian weight gain assay. Blood samples were collected 24 h before and immediately prior to injections, and 4, 8, 12, 24, 72 and 96 h after injections for determination of serum levels of immunoactive FSH by fluoroimmunoassay, bioactive FSH by an in vitro Sertoli cell assay, and inhibin and testosterone by radioimmunoassay. Inhibin was chosen as a marker for in vivo hFSH activity, since the secretion of inhibin in male monkeys is under the control of FSH. Administration of hFSH resulted in dose-related increases in serum hFSH concentrations. rhFSH and uhFSH exhibited similar pharmacokinetics. Comparable findings were obtained when serum samples were analysed for in vitro FSH bioactivity. Maximum serum hFSH levels were obtained 4–6 h after administration and the elimination half-life of hFSH was on average 18–22 h. The serum pharmacokinetics of rhFSH were linear within the dose range explored. Baseline inhibin concentrations varied significantly between groups. However, when the changes in inhibin concentrations were normalized to the baseline values (per cent change, area under curve and maximum inhibin level), a dose-dependent stimulatory effect of rhFSH on serum inhibin was evident. This effect attained statistical significance with doses of 24 IU rhFSH/kg and 24 IU uhFSH/kg, and the serum inhibin responses to rhFSH and uhFSH were not significantly different. No significant differences were observed with regard to the serum concentrations of testosterone between the diluentand hFSH-treated groups. It was concluded that rhFSH is bioactive in terms of stimulating testicular inhibin production in the male monkey and that the pharmacokinetic properties of rhFSH and uhFSH are similar. Journal of Endocrinology (1994) 141, 113–121