Summary. The role of the embryo in promoting increased plasma concentrations of immunoreactive inhibin after conception in the marmoset monkey was determined by flushing embryos from the uterus between days 5 and 9 after ovulation (implantation commences on days 11–12). Blood samples were taken from each animal (three times a week) after ovulation until the end of the luteal phase. Plasma inhibin concentrations were measured using a radioimmunoassay based on antisera against a synthetic fragment of the α-subunit of human inhibin. When embryos were flushed on days 5 and 6 (n = 6) after ovulation inhibin concentrations did not exceed 250 ng ml⁻¹ for the duration of the luteal phase. In contrast when embryos were flushed on days 7 (n = 4), 8 (n = 4) and 9 (n = 3) maximum concentrations of inhibin always exceeded 250 ng ml⁻¹, reaching > 400 ng ml⁻¹ when embryos were flushed on days 8 and 9. Inhibin concentrations remained high for the duration of the luteal phase, which varied in length between 20 and 32 days. Significantly (P < 0·01) higher mean plasma concentrations of immunoreactive inhibin were first recorded on days 7–8 after ovulation in animals that had embryos flushed on days 7, 8 and 9 compared with concentrations in animals that had embryos flushed on days 5 and 6. Inhibin could not be detected in the medium of embryos cultured for up to 2 weeks. In two control animals, inhibin concentrations exceeded 250 ng ml⁻¹ after sham operation on day 6, whereas inhibin did not exceed 250 ng ml⁻¹ when unfertilized eggs were flushed from the uterus of an animal on day 8 after ovulation. Progesterone concentrations did not differ significantly among animals irrespective of the day of embryo flushing. The results suggest that the marmoset pre-implantation embryo provides a signal on days 7–8 which triggers an increase in luteal inhibin production. The continued presence of this putative signal was not necessary for maintaining increased inhibin production.

Keywords: embryo; inhibin; pregnancy; marmoset monkey

Changes in intrafollicular concentrations of different forms of inhibin (free α-subunits and αβ dimers) occur during follicle development and may influence the oocyte maturation process. The aim of this study was to investigate the effects of inhibin A and free α-subunit (pro-αC) isolated from bovine follicular fluid on maturation of bovine cumulus–oocyte complexes, as reflected by their competence for embryo development after in vitro fertilization. Bovine cumulus–oocyte complexes were isolated from ovaries obtained from an abattoir and were cultured for 22–24 h at 38.5°C in TCM-199 medium supplemented with 10% oestrous cow serum, pregnant mares' serum gonadotrophin (2.5 iu ml⁻¹) and either inhibin A (0, 0.2 and 1.0 μg ml⁻¹) or pro-αC (0, 2 and 10 μg ml⁻¹). Neither inhibin A nor free α-subunit affected the cleavage rate of cumulus–oocyte complexes after fertilization (approximately 60%). Inhibin A reduced the proportion of cleaved oocytes reaching the eight-cell stage by 19% (P < 0.05), but did not affect the yield of blastocysts. However, pro-αC decreased the proportion of cleaved oocytes that reached the eight-cell (25%; P < 0.05) and blastocyst (28%; P < 0.05) stages. In addition, a negative correlation (r = −0.55, P < 0.001) was found between concentrations of total immunoreactive (ir) α-inhibin (measured by radioimmunoassay) produced by untreated control cumulus–oocyte complexes and their post-cleavage development to the blastocyst stage. In a second experiment, mouse monoclonal antibodies (20 μg ml⁻¹) against two different regions of the inhibin α-subunit precursor (pro-region and αC fragment) were tested for their ability to neutralize endogenous inhibin α-subunit-related molecules produced by cumulus cells; control cumulus–oocyte complexes were treated with normal mouse IgG (20 μg ml⁻¹). Although the cleavage rate was not affected, the yield of blastocysts was significantly higher in the presence of mouse monoclonal antibodies to both pro-α (77% increase; P < 0.05) and aC (48% increase; P < 0.05). None of the treatments tested affected endogenous production of activin-A or follistatin by cumulus–oocyte complexes. Overall, these results indicate that the inhibin α-subunit (pro-αC) has an inhibitory role in oocyte maturation which is independent of the modulatory effects of activin and follistatin.

Angiogenesis plays an integral role in follicular and luteal development and is positively regulated by several intra-ovarian factors including vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 2 (FGF2). Various transforming growth factor-β (TGF-β) superfamily members function as intra-ovarian regulators of follicle and luteal function, but their potential roles in modulating ovarian angiogenesis have received little attention. In this study, we used a bovine theca interna culture model (exhibiting characteristics of luteinization) to examine the effects of TGF-β1 and bone morphogenetic protein 6 (BMP6) on angiogenesis and steroidogenesis. VEGFA/FGF2 treatment promoted endothelial cell network formation but had little or no effect on progesterone and androstenedione secretion or expression of key steroidogenesis-related genes. TGF-β1 suppressed basal and VEGFA/FGF2-induced endothelial cell network formation and progesterone secretion, effects that were reversed by an activin receptor-like kinase 5 (ALK5) inhibitor (SB-431542). The ALK5 inhibitor alone raised androstenedione secretion and expression of several transcripts including CYP17A1. BMP6 also suppressed endothelial cell network formation under VEGFA/FGF2-stimulated conditions and inhibited progesterone secretion and expression of several steroidogenesis-related genes under basal and VEGFA/FGF2-stimulated conditions. These effects were reversed by an ALK1/2 inhibitor (K02288). Moreover, the ALK1/2 inhibitor alone augmented endothelial network formation, progesterone secretion, androstenedione secretion and expression of several steroidogenesis-related genes. The results indicate dual suppressive actions of both TGF-β1 and BMP6 on follicular angiogenesis and steroidogenesis. Further experiments are needed to unravel the complex interactions between TGF-β superfamily signalling and other regulatory factors controlling ovarian angiogenesis and steroidogenesis.

Summary. An in-vitro superfusion technique was used to study basal and depolarization-induced (32 mmol K⁺/l) release of LHRH from the mediobasal hypothalamus (MBH) of pullets at 8–25 weeks of age. Plasma LH concentrations and the incremental change (ALH) after an i.v. injection of 1 or 15 μg synthetic ovine LHRH/kg body weight were also determined. Between 8 and 25 weeks of age, significant (P < 0·01) increases in basal and depolarization-induced release of LHRH (93 and 330%, respectively) were accompanied by a significant (P < 0·01) rise in the residual LHRH content of MBH tissue (152%), observations which suggest that the ability of the hypothalamus to synthesize and secrete LHRH increases as sexual maturation proceeds. However, plasma LH, which reached a maximum concentration of 2·05 ± 0·43 μg/l at 15 weeks, fell significantly (P < 0·05) to 1·14 ± 0·05 μg/l at 25 weeks. Since ΔLH in response to exogenous LHRH showed a marked and progressive decline between 12 and 20 weeks of age, the low plasma concentration of LH typical of the mature hen is probably attributable to a direct negative-feedback action of ovarian steroids on the anterior pituitary gland rather than to an impaired secretion of LHRH from the median eminence. It is suggested that a dramatic increase in the responsiveness of LHRH nerve terminals in the MBH to depolarization by 32 mmol K⁺/l between 20 and 25 weeks of age (mean age at onset of lay 21 ·9 weeks; range 19–25 weeks) may reflect the development of hypothalamic responsiveness to the positive feedback action of progesterone.

The aim of the present study was to characterize in detail the cytoplasmic and nuclear morphology of cattle oocytes recovered from follicles that are dominant for more than 9 days (with low fertility after ovulation), and to relate morphological changes to intrafollicular markers of follicle health. Beef heifers received prostaglandin F_2α and a synthetic progestagen (3 mg Norgestomet) for 2 or 10 days on the first day of dominance of the second dominant follicle (DF2) of the oestrous cycle, to give a 4 day (n = 19; N2) or 12 day (n = 21; N10) duration of dominance of the dominant follicle at ovariectomy 18 h after implant removal and before the predicted gonadotrophin surge. Ultrasound scanning determined emergence of a new wave of follicles in five N10 heifers the day before (n = 1) or day of ovariectomy (n = 4) (N10-NonDom). Dominant follicles from the remaining N10 heifers (N10-Dom) were larger (P < 0.05) on the day of ovariectomy (17.8 ± 0.6 mm) than those from N2 heifers (13.6 ± 0.4 mm). The oestradiol:progesterone ratio of follicular fluid from N10-Dom heifers was reduced (21.7 ± 3.1 versus 34.1 ± 4.4; P < 0.05), while inhibin A (as measured by immunoradiometric assay) was increased (12.7 ± 1.0 versus 9.0 ± 0.7 μg ml⁻¹; P < 0.05) compared with N2 heifers. Eleven of twelve N2 oocytes demonstrated nuclear activation without germinal vesicle breakdown, while seven of eight N10-Dom oocytes had undergone germinal vesicle breakdown and had progressed to metaphase I (6/8) or II (1/8). In contrast to N2 oocytes, N10-Dom oocytes showed a larger perivitelline space containing more cumulus cell process endings, vacuoles, irregular vesicles, and more mitochrondia and lipid droplets throughout the ooplasm, yet the degree of cumulus cell expansion and atresia was similar. Thus, final oocyte maturation leading to metaphase I is initiated in most dominant follicles with a dominance period of > 9 days before the gonadotrophin surge and is associated with a reduction in dominant follicle health. However, ovulatory ability is maintained and will lead to the ovulation of aged oocytes, markedly reducing subsequent pregnancy rates.

We reported previously that active immunization of heifers using a synthetic peptide-based inhibin vaccine (bIα(1–29)Tyr³⁰) can enhance ovarian follicular development and ovulation rate during spontaneous oestrous cycles. To extend this study, we investigated the effect of inhibin immunization more closely by monitoring plasma hormone profiles and ovarian activity in bIα(1–29)Tyr³⁰-immunized and control (ovalbumin-immunized) heifers (n = 6 per group) over three consecutive oestrous cycles, which were synchronized and shortened by administering a PGF_2α analogue at intervals of 14 days. Blood samples were collected at 2–8 h intervals for 40 days and the ovaries were examined daily using ultrasonography. Repeated-measures anova showed that inhibin immunization significantly increased plasma FSH concentration (by 52% overall; P < 0.01) and ovulation rate (by 58%; P < 0.01). Both immunized and control heifers showed the same cyclic pattern of plasma FSH (treatment × time interaction; not significant), indicating that the increase in plasma FSH was sustained throughout the cycle. Immunization did not affect the concentration or pattern of secretion of LH, oestradiol or progesterone and had no influence on the timing of the LH surge or ovulation after PG injection. While inhibin immunization increased the number of 'large' (i.e. growing to ≥ 10 mm diameter) follicles that developed during both the preovulatory (by 90%, P< 0.02) and postovulatory (by 190%, P< 0.01) period, there was no difference between groups in the temporal pattern of growth or regression of large follicles or of corpora lutea. These observations confirm a physiological role for ovarian inhibin as a component of the ovarian feedback mechanism controlling FSH secretion in heifers, and support the hypothesis that active immunization of heifers against inhibin enhances ovarian follicular development and ovulation rate by promoting a sustained increase in pituitary FSH secretion.

Summary. Passive immunization of male lambs against oestradiol-17β from 2 to 16 weeks of age significantly elevated androgen concentrations in plasma and depressed the median eminence content of dopamine. Removal of endogenous oestrogens had no significant effects on plasma FSH, LH or prolactin concentrations or on testicular growth and hypothalamic content of GnRH. These results suggest that endogenous oestrogens may indirectly suppress testicular androgen secretion by exerting a stimulatory influence on hypothalamic dopaminergic neurones, which in turn may inhibit GnRH secretion by the median eminence.

Summary. A technique is described for the in-vivo determination of mammary gland size and gross composition in goats by using nuclear magnetic resonance imaging (MRI). The volume of test objects determined with MRI had an error of +0·4 ± 1·6% of the actual volume. In lactating goats the in-vivo MRI estimate of mammary parenchymal volume was significantly greater than, but highly significantly correlated with, the weight of parenchyma determined post mortem (for the whole udder, r = 0·88, P < 0·001; for individual glands, r = 0·85, P < 0·001), MRI-determined estimates of the volume of fluid within the mammary gland were within 1·2% of the volume of milk removed from the udders after imaging. The spin-lattice (T₁) relaxation time of the whole udder correlated closely with the volume of fluid within the udder. The T₁ relaxation time of parenchymal tissue measured in vivo did not differ significantly from that determined immediately after post-mortem excision.

Keywords: mammary gland; magnetic resonance imaging; methodology; goat; lactation

Summary. Mammary development and regression were measured in goats in vivo using magnetic resonance imaging (MRI). Measurements were made during the first and second cycles of pregnancy, lactation and involution.

In primiparous goats, an exponential pattern of growth was evident during gestation and for the first 2 weeks of lactation. Parenchyma volume correlated significantly with milk yield across goats during early lactation, and across stage of lactation within goats. Milking was discontinued in Week 26 of the first lactation. Involution was characterized by an initial accumulation of fluid (over 2 days) followed by reabsorption; parenchyma volume did not decrease significantly until the 3rd week of involution, which was also the time at which these goats were mated to start their second gestation. Their udders still contained significant quantities of fluid (40–60% of the gross volume), but parenchyma volume was also greater (by 4·7-fold) than in goats beginning their first gestation. By Week 15 of gestation there was no longer a parity difference in parenchyma; the udders of first-gestation goats had grown significantly, but those of second-gestation goats had not. Conversely, between gestation Week 15 and laction Week 2 mammary growth was significantly more rapid in the second cycle, such that the udder was larger at the start of the second lactation.

Keywords: mammogenesis; mammary gland; lactation; pregnancies; goats; MRI

Summary. Thirty adult Mule (Blue-faced Leicester × Swaledale) ewes were actively immunized against a synthetically produced peptide corresponding to the N-terminus of the α-subunit of bovine inhibin conjugated to tuberculin purified protein derivative (PPD). Primary immunization in the late anoestrous period was followed by two booster injections at 5 week intervals. Control groups were either not immunized (n = 15) or received PPD only (n = 15). Ten days after the second booster, oestrus was synchronized using progestagen sponges and ovulation rate was assessed by laparoscopy on days 9–10 of the cycle. Blood samples were taken at the time of each immunization and immediately before laparoscopy. Ewes were mated with fertile rams in midNovember and the resulting conception, pregnancy and lambing rates monitored. All inhibin-immunized ewes generated antibodies that bound ¹²⁵I-labelled native bovine inhibin (M _r 32 000), and their plasma follicle-stimulating hormone (FSH) concentrations after the second booster were significantly higher than the preimmunization values (30%; P < 0·001) and the corresponding value in the controls (25%; P < 0·025). Inhibin immunization was associated with a 90% increase in ovulation rate (P < 0·005) and had no adverse effect on conception rate (100%), pregnancy rate (100%) or length of gestation (146 days). However, only a 37% increase (P < 0·05) in lambing rate was recorded for inhibin-immunized ewes, indicating a higher incidence of wastage of ova, or embryos, or both, in these ewes. As a consequence of the increased litter size, mean live birthweight of lambs was significantly lower (18%; P < 0·001) in the inhibin-immunized group and a higher proportion of the lambs born to inhibinimmunized ewes (15·4% compared with 0·02% in controls) were stillborn. Stillborn lambs weighed considerably less (38%; P < 0·001) than viable lambs. In terms of the number of viable lambs produced, there was no significant difference between the inhibin-immunized and control group. Although this study confirms the effectiveness of inhibin immunization using a synthetic peptide-based vaccine as a reliable method for increasing ovulation rate in sheep, the results indicate the limitations of this technique for further increasing viable litter size in 'improved' breeds of relatively high inherent prolificacy.

Keywords: inhibin; immunization; sheep; ovulation; litter size; FSH