Utilising male stimulus to improve the reproductive efficiency of 8-month-old nulliparous ewes and adult parous ewes.

We tested whether utilising the male effect to stimulate ewes before the mating period can reduce the time to conception following the introduction of entire rams, and increase fertility, prolificacy, and reproductive rate (number of fetuses per 100 ewes exposed to fertile rams). A retrospective analysis was used to analyse records from 59,716 ewes collected over 34 years (1986-2020) from seven genotypes: Border Leicester, Composite (crossbred), Dorset, Merino, Dorset x Polypay, Rambouillet, White Suffolk. The dataset also included nulliparous young ewes (mated at age 8 months) and adult parous ewes. Vasectomized rams were used to stimulate 20,632 ewes before a mating period that lasted 2 or 3 estrous cycles, and the outcomes were compared with those from 39,084 ewes that had not been stimulated. Independently of genotype, utilising the male stimulus advanced the average conception date by 8 days for young ewes (P < 0.0001) and by 1 day for adult ewes (P < 0.0001). The male stimulus also increased the proportion of ewes that conceived in their first cycle by 33 % for young ewes and by 6 % for adult ewes (P < 0.0001). For the cycle of conception, there were significant (P < 0.0001) effects of two interactions: male stimulus x age at mating and male stimulus x live weight at mating. The male stimulus improved fertility in both adult ewes (99.8 % vs 89 %; P < 0.001) and young ewes (77.7 % vs 81.3 %; P < 0.001). The male stimulus increased the number of young ewes (41.9 % vs 11.1 %; P < 0.001) and adult ewes (16.6 % vs 2.7 %; P < 0.001) that conceived multiple fetuses in the first 17 days of the mating period. The reproductive rate was improved by the male stimulus in young ewes (129 % vs 135 %; P < 0.001) but not in adult ewes (120 % vs 122 %; P = 0.12). When all animals for all breeds were included in the analyses, there were improvements in fertility, prolificacy, and reproductive rate as age and live weight increased at mating (P < 0.0001). We conclude that, independently of genotype, utilising the male stimulus before the mating period reduces the time to conception and improves reproductive performance in both young and adult ewes.

Non-invasive assessment of fecal glucocorticoid and androgen metabolites in the pygmy hippopotamus (Choeropsis liberiensis).

The pygmy hippopotamus (Choeropsis liberiensis) is an endangered species endemic to the Upper Guinea Forest ecosystem in West Africa. We have limited information concerning the species' reproduction and well-being under managed care. We therefore developed non-invasive methods for characterizing gonadal androgen and adrenal hormone profiles in pygmy hippos using fecal samples collected from 12 males and 12 females housed in North American zoological institutions. We aimed to: 1) identify and validate enzyme immunoassays (EIAs) for measuring metabolites of corticosteroids and testosterone in feces; and 2) test whether gonadal activity is correlated with previous breeding history, season or type of housing. For glucocorticoids, several EIAs for measuring metabolites were investigated. A group-specific EIA exhibiting cross-reactivity with 11,17-dioxoandrostane (DOA) metabolites of cortisol most clearly reflected adrenocortical activity in response to pharmocological challenge with adrenocorticotropic hormone (ACTH) in both males and females. However, day-to-day concentrations of this metabolite in the feces of pygmy hippos that did not undergo ACTH challenge were near the detection limits of the assay, making this EIA impractical for assessing glucocorticoid activity in this species. Another group-specific EIA, exhibiting cross-reactivity with 5α-pregnane-3ß,11ß,21-triol-20-one, produced biologically relevant data and evidence of an appropriate response to pharmacological challenge with exogenous ACTH. The testosterone metabolite assay C196 (Arbor Assays, Ann Arbor, Michigan, USA) also produced biologically coherent data: adult males exhibited the highest mean androgen metabolite concentrations (477 ng/g), followed by adult females (259 ng/g) and juvenile males (160 ng/g). Proven breeding males had higher, but not significantly different, mean concentrations (472 ng/g) to unproven males (352 ng/g; P = 0.400). Similarly, adult males housed outdoors year-round in subtropical climates exhibited higher, but not statistically different mean concentrations (554 ng/g) to males in temperate climates that were housed indoors at least part of the year (412 ng/g; P = 0.208). There were, however, significant differences in mean concentrations among seasons for adult males, with higher values in spring (546 ng/g) and summer (542 ng/g) than in autumn (426 ng/g) and winter (388 ng/g, P = 0.003). In conclusion, we identified EIAs for the measurement of fecal metabolites of androgens and glucocorticoids that can be used for further studies to monitor gonadal activity in male pygmy hippos and adrenocortical activity in both sexes. We also identified a seasonal trend in male gonadal activity in this species under managed care in North America. Finally, our findings highlight an important consideration when using non-invasive methods for evaluating fecal cortisol metabolites: ACTH used for pharmacological validation of an EIA does not necessarily equate to biological relevance.

Heat shock protein HSP90 immunoexpression in equine endometrium during oestrus, dioestrus and anoestrus.

Heat shock proteins play a crucial role in cellular development, proliferation, differentiation and apoptosis. Heat shock protein 90 (HSP90) has been localised in the human endometrium, where its immunoexpression changes during the menstrual cycle. Similar studies have not been done for the equid species, so the present study aimed to describe endometrial HSP90 immunoexpression in mare endometrium. Endometrial biopsies were formalin-fixed and paraffin-embedded, and sections were stained with haematoxylin-eosin in preparation for HSP90 immunohistochemistry. Immunostaining and morphometric analyses were performed on the epithelial lining, endometrial glands and connective stroma during oestrus, dioestrus phase and anoestrus period (n = 7 per phase or period). Immunoexpression was localised in the basal region of the epithelial cells lining the lumen. Immunoexpression was greater during oestrus than during either dioestrus or anoestrus. During anoestrus, there was little immunostaining in the endometrium, suggesting that HSP90 is involved in the functional modulation of sex steroid receptors in cyclic mares. Indeed, the function of HSP90 as a chaperone in the folding of proteins, such as steroid receptors, might explain the greater intensity of immunostaining during the oestrus and dioestrus phases, compared the anoestrus period. We conclude that, in the mare, HSP90 plays a role in endometrial function and that further studies are needed to test whether it is important in pathological conditions as endometritis.

Palm oil protects α-linolenic acid from rumen biohydrogenation and muscle oxidation in cashmere goat kids.

BACKGROUND: In ruminants, dietary C18:3n-3 can be lost through biohydrogenation in the rumen; and C18:3n-3 that by-passes the rumen still can be lost through oxidation in muscle, theoretically reducing the deposition of C18:3n-3, the substrate for synthesis of poly-unsaturated fatty acids (n-3 LCPUFA) in muscle. In vitro studies have shown that rumen hydrogenation of C18:3n-3 is reduced by supplementation with palm oil (rich in cis-9 C18:1). In addition, in hepatocytes, studies with neonatal rats have shown that cis-9 C18:1 inhibits the oxidation of C18:3n-3. It therefore seems likely that palm oil could reduce both rumen biohydrogenation of C18:3n-3 and muscle oxidation of C18:3n-3. The present experiment tested whether the addition of palm oil to a linseed oil supplement for goat kids would prevent the losses of C18:3n-3 and thus improve the FA composition in two muscles, Longissimus dorsi and Biceps femoris. To investigate the processes involved, we studied the rumen bacterial communities and measured the mRNA expression of genes related to lipid metabolism in Longissimus dorsi. Sixty 4-month-old castrated male Albas white cashmere kids were randomly allocated among three dietary treatments. All three diets contained the same ingredients in the same proportions, but differed in their fat additives: palm oil (PMO), linseed oil (LSO) or mixed oil (MIX; 2 parts linseed oil plus 1 part palm oil on a weight basis). RESULTS: Compared with the LSO diet, the MIX diet decreased the relative abuandance of Pseudobutyrivibrio, a bacterial species that is positively related to the proportional loss rate of dietary C18:3n-3 and that has been reported to generate the ATP required for biohydrogenation (reflecting a decrease in the abundance of rumen bacteria that hydrogenate C18:3n-3 in MIX kids). In muscle, the MIX diet increased concentrations of C18:3n-3, C20:5n-3, C22:6n-3, and n-3 LCPUFA, and thus decreased the n-6/n-3 ratio; decreased the mRNA expression of CPT1ß (a gene associated with fatty acid oxidation) and increased the mRNA expression of FADS1 and FADS2 (genes associated with n-3 LCPUFA synthesis), compared with the LSO diet. Interestingly, compared to Longissimus dorsi, Biceps femoris had greater concentrations of PUFA, greater ratios of unsaturated fatty acids/saturated fatty acids (U/S), and poly-unsaturated fatty acids/saturated fatty acids (P/S), but a lesser concentration of saturated fatty acids (SFA). CONCLUSIONS: In cashmere goat kids, a combination of linseed and palm oils in the diet increases the muscle concentration of n-3 LCPUFA, apparently by decreasing the relative abundance of rumen bacteria that are positively related to the proportional loss rate of dietary C18:3n-3, by inhibiting mRNA expression of genes related to C18:3n-3 oxidation in muscle, and by up-regulating mRNA expression of genes related to n-3 LCPUFA synthesis in muscle, especially in Longissimus dorsi.

Follicle-stimulating hormone (FSHß) gene polymorphisms and associations with reproductive traits in Rex rabbits.

Follicle-stimulating hormone (FSH) stimulates granulosa cell proliferation and controls the development and maturation of oocytes. In this study with Rex rabbit, there was exploration of the relationships between reproductive traits and SNPs and haplotypes of FSHß, and tested whether FSHß SNPs would segregate between two lines, using 70 females from the White line and 100 females from the Beaver line. Three SNPs (FSH2, FSH30, FSH31) in exon1 and exon3 of FSHß were strongly associated with reproductive traits in the combined population. For FSH2, the GG variant was associated with greater (P <  0.05) values for total number born (TNB) and number born alive (NBA), compared to those with the TT variant. For FSH30, for the AA variant there was greater (P <  0.05) values for TNB and NBA than with the GG and AG variants. For FSH31, the GG variant was associated with greater (P <  0.05) values for TNB, litter weight at birth (LWB) and litter size at 21 days of age (LS21), than the AG variant, and with greater (P < 0.01) values for NBA than the AG variant. When analyzed separately, FSH2 SNP was associated with LW21 in the Beaver line (P <  0.05), whereas FSH30 and FSH31 SNPs were associated with TNB and NBA in the White line (P <  0.05). It is concluded that genetic variation in FSHß gene is associated with reproductive traits in the Rex rabbit, therefore, the FSH2, FSH30 and FSH31 SNPs can be used as molecular markers in genetic selection of rabbits.

Linseed oil and heated linseed grain supplements have different effects on rumen bacterial community structures and fatty acid profiles in cashmere kids1.

This study investigated the effects of dietary supplementation with alternative sources of α-linolenic acid on growth, the composition of rumen microbiota, and the interactions between rumen microbiota and long-chain fatty acid (FA) concentrations, in goat kids. Sixty 4-month-old castrated male Albas white cashmere kids (average BW 18.6 ± 0.1 kg) were randomly allocated among three dietary treatments: (i) basal diet without supplementation (Control), (ii) basal diet supplemented with linseed oil (LSO), (iii) basal diet supplemented with heated linseed grain (HLS). The concentrate:forage ratio was 5:5 and the LSO and HLS treatments provided the kids with similar dietary FA profiles. The diets were fed for 104 d, consisting of 14 d for adaptation followed by 90 d of experimental observation. Treatment did not significantly influence BW, DMI, or bacterial richness or diversity. On the other hand, the relative abundance of bacteria participating in hydrogenation differed significantly among the three groups: the Veillonellaceae and Christensenellaceae were more abundant in LSO kids, Prevotellaceae were more abundant in HLS kids, and the Fibrobacteriaceae were more abundant in Control kids (P < 0.05). Spearman correlation analysis indicated that Ruminobacter, Selenomonas_1, Fretibacterium, Prevotellaceae_UCG-001, Succinimonas, and Ruminococcaceae_NK4A214_group were the genera that participated in hydrogenation of long-chain FAs. HLS-fed kids had a lower relative abundance of Ruminobacter, but a higher abundance of Prevotellaceae_UCG-001 and Fretibacterium than LSO-fed kids. These changes were associated with greater rumen concentrations of C18:3n3 and n-3 PUFA, but lower concentrations of n-6 PUFA and lower n-6/n-3 ratios, in HLS than in LSO-fed kids. In conclusion, feeding kids with HLS increased rumen concentrations of C18:3n3 and n-3 PUFA, but decreased the n-6/n-3 ratio by decreasing the abundance of bacteria that hydrogenate C18:3n3 and increasing the abundance of bacteria that hydrogenate C18:2n6.

Modeling the Male Reproductive Endocrine Axis: Potential Role for a Delay Mechanism in the Inhibitory Action of Gonadal Steroids on GnRH Pulse Frequency.

We developed a compartmental model so we could test mechanistic concepts in the control of the male reproductive endocrine axis. Using SAAM II computer software and a bank of experimental data from male sheep, we began by modeling GnRH-LH feed-forward and LH-T feedback. A key assumption was that the primary control signal comes from a hypothetical neural network (the PULSAR) that emits a digital (pulsatile) signal of variable frequency that drives GnRH secretion in square wave-like pulses. This model produced endocrine profiles that matched experimental observations for the testis-intact animal and for changes in GnRH pulse frequency after castration and T replacement. In the second stage of the model development, we introduced a delay in the negative feedback caused by the aromatization of T to estradiol at the brain level, a concept supported by empirical observations. The simulations showed how changes in the process of aromatization could affect the response of the pulsatile signal to inhibition by steroid feedback. The sensitivity of the PULSAR to estradiol was a critical factor, but the most striking observation was the effect of time delays. With longer delays, there was a reduction in the rate of aromatization and therefore a decrease in local estradiol concentrations, and the outcome was multiple-pulse events in the secretion of GnRH/LH, reflecting experimental observations. In conclusion, our model successfully emulates the GnRH-LH-T-GnRH loop, accommodates a pivotal role for central aromatization in negative feedback, and suggests that time delays in negative feedback are an important aspect of the control of GnRH pulse frequency.

The 'male effect' in sheep and goats--revisiting the dogmas.

Male-induced ovulation in sheep and goats (the 'male effect'), documented during the period 1940-1960, has long been shrouded in preconceptions concerning how, when and why it worked. These preconceptions became dogmas but recent research is challenging them so, in this review, we have re-visited some major physiological (breed seasonality; characteristics of the response; the nature of the male stimuli) and physical factors (duration of male presence; isolation from male stimuli) that affect the phenomenon. We reject the dogma that ewes must be isolated from males and conclude that male 'novelty' is more important than isolation per se. Similarly, we reject the perception that the neuroendocrine component of the male effect is restricted to anovulatory females. Finally, we re-assess the relative importance of olfactory and non-olfactory signals, and develop a perspective on the way male-induced ovulation fits with preconceptions about pheromonal processes in mammals. Overall, our understanding of the male effect has evolved significantly and it is time to modify or reject our dogmas so this field of research can advance. We can now ask new questions regarding the application of the male effect in industry and develop research so we can fully understand this biological phenomenon.

Rapid induction of cell proliferation in the adult female ungulate brain (Ovis aries) associated with activation of the reproductive axis by exposure to unfamiliar males.

In many species, the reproductive centers of the brain are profoundly affected by sociosexual stimuli. This is particularly evident in female ungulates such as sheep, in which exposure to males switches them from reproductively quiescent to fertile. In two experiments with female sheep, we tested whether the brain centers that control gonadotropin-releasing hormone (GnRH) neuronal activity respond differentially to "novel" vs. familiar males and whether the neuroendocrine response is associated with increased cell proliferation in the hippocampus, a site associated with memory formation. In experiment 1, groups of 10 female sheep that had previously been habituated to males for 3 mo were re-exposed to familiar males or were exposed to novel males. Only the novel males increased luteinizing hormone (LH) pulse frequency, indicating stimulation of GnRH neuronal activity. In experiment 2, groups of six female sheep were injected with bromodeoxyuridine (BrdU) and then maintained in isolation from males or exposed to novel males. Two days later, the hippocampus and hypothalamus were removed and processed for fluorescence immunohistochemistry. Again, exposure to males increased LH pulse frequency. Most important, male exposure also doubled the number of BrdU-positive cells in the dentate gyrus of the hippocampus. No BrdU-positive cells were detected in the hypothalamus. We conclude that the stimulus from novel males switches on the reproductive centers of the brain of female sheep and rapidly doubles the rate of cell proliferation in the hippocampus. The rapidity of this response contrasts with rodents, in which several days of exposure to male pheromones seem necessary for an effect on neurogenesis.

Effects of artificial social stimuli on the reproductive schedule and hormone levels of yellow-eyed penguins (Megadyptes antipodes).

The effects of social stimuli on avian reproductive behaviors such as breeding schedules and courtship behaviors are well known due to numerous field studies. However, studies that have simultaneously examined the effects of social stimuli on reproductive behavior and the mediating endocrine mechanisms have been largely restricted to captive populations, which may not be representative of free-living populations. This study, conducted over two breeding seasons, aimed to simultaneously measure the effects of experimentally increasing auditory stimuli on the breeding schedule and endocrinology (levels of total androgen, estradiol, progesterone and prolactin) on free-living yellow-eyed penguins (Megadyptes antipodes). The yellow-eyed penguin is the least colonial of all penguins, nesting far apart from each other under dense vegetation, and, therefore, is presumed to experience much lower levels of social stimuli than other penguins. Egg laying was significantly more synchronous and tended to be earlier when birds were exposed to playbacks of the calls of conspecifics in 1 year of the study. We also found that levels of total androgen and estradiol of males in 1 year, and prolactin in another year, were proportionally higher among treated birds compared control birds that received no artificial auditory stimuli. These results show that even among supposedly solitary nesters, social stimuli could still play a role in influencing reproductive behavior and physiology. For the first time in free-living seabirds, we have demonstrated that behavioral responses to increased social stimuli are associated with hormonal changes.

Dynamics of the responses in secretion of luteinising hormone, leptin and insulin following an acute increase in nutrition in mature male sheep.

In Merino rams, an increase in the plane of nutrition increases the frequency of luteinising hormone (LH) pulses within a few days and this response is correlated with changes in the circulating concentrations of metabolic hormones. To analyse early dynamic aspects of these responses, we studied mature rams fed with diets that contained either low or high amounts of energy and protein. Jugular blood was sampled every 20 min for 96 h, including a control period of 24 h before the change of diet in the high-diet group. In the high-diet group, a significant increase in LH pulse frequency was first detected 6 h after the increase in nutrition on Day 1 and the frequency remained significantly elevated throughout the 72-h treatment period, except for a 12-h period on Day 2. Following the change of diet, insulin concentrations increased within 3 h and leptin concentrations increased within 7 h, after which time the concentrations of both hormones remained high. Dietary treatment did not affect the concentrations of thyroxine or insulin-like growth factor-I, but the high diet increased the concentrations of tri-iodothyronine. These observations are consistent with insulin and leptin playing a role in the early activation of the gonadotrophin-releasing hormone-LH axis by nutritional inputs.