Growth differentiation factor 9 (GDF9) plays an important role in the early folliculogenesis of sheep. This study investigated the mRNA expression of ovine GDF9 in different tissues by real-time PCR. GDF9 exhibits significantly higher levels of expression (p < 0.01) in the ovary, relative to other tissues, indicating that its expression is tissue specific. To explore the regulatory mechanism of this tissue-specific expression, the methylation level of one CpG island (−1453 to −1854) of GDF9 promoter in ovary and heart was determined. In this region (−1987 to −1750), only the mC-4 site was present in the Sp4 binding site showed differential methylation between the heart and ovary; with increased (p < 0.01) methylation being observed in the heart. Additionally, the methylation level was negatively correlated with GDF9 mRNA expression (R = −0.75, p = 0.012), indicating that the methylation of this site plays an important role in transcriptional regulation of GDF9. The methylation effect of the mC-4 site was confirmed by using dual-luciferase. Site-directed mutation (methylation) of mC-4 site significantly reduced (p < 0.05) basal transcriptional activity of GDF9 promoter in oocytes. These results imply that methylation of GDF9 promoter CpG island mC-4 site may affect the binding of the Sp4 transcription factor to the GDF9 promoter region in sheep, thereby regulating GDF9 expression and resulting in a tissue-specific expression.

Prey animals modify their behaviour and physiology in the presence of predators. Domestic animals differ from wild animals in having less exposure to wild predators, but whether they still retain an antipredator instinct is frequently unknown. In this study, we used domesticated Small Tail Han sheep as a model prey animal to gauge their response to the presence of predators, in the form of odours from the faeces of lion, tiger, and leopard. The faeces of male sheep and male rabbit (as a heterogeneous non-predator) were used as control. We found that the frequency and time of feeding, exploration, moving, watching, and lying down behaviours were significantly affected by predator odour, and that there was an interaction between odour sources and sex. When exposed to predator odour, sheep reduced their frequency and time of feeding, and increased their exploratory, moving, and watching behaviours. Female sheep showed greater motivation towards frequent and lengthy exploration, moving, watching, and lying down behaviours than male sheep, and less motivation towards feeding and drinking behaviours. Serum cortisol levels were lowest in response to tiger stimuli. These results illustrated that Small Tail Han sheep could recognise predator odour and adjust their behaviour to display antipredator strategies, and displayed some physiological responses, although only changing in serum cortisol could be significantly attributed to the odour of predators.

This study investigated the effects of different breeding methods on the cecal microbiota and production traits of Yimeng Black Goats (YBGs). Twenty-seven 3-month-old male YBGs were assigned to three groups (n = 9 each): total mixed ration once daily (A), concentrate in the morning and roughage in the afternoon (B), or grazing with supplementary feeding (C). Cecal bacterial communities were analyzed via 16S rRNA sequencing, and functional potential was predicted using FAPROTAX. Breeding method significantly altered microbial composition (p < 0.05). Beta diversity was highest in Group C, while alpha diversity remained similar across groups. Bacteroidetes, Firmicutes, and Proteobacteria were dominant; Proteobacteria were most abundant in Group A. At the genus level, relative abundances of nine taxa, including Lactobacillus and Fusobacterium, differed significantly (p < 0.05). At the species level, including Lactobacillus mucosae, Bacteroides massiliensis and Alistipes finegoldii, differed significantly (p < 0.05). Chemoheterotrophy and fermentation functions were most enriched, particularly in Group C. Total weight gain was highest in Group A and lowest in Group C (p < 0.05), while carcass rate showed no significant differences (p > 0.05). Correlation analysis revealed that Proteobacteria, Fusobacteria, and Euryarchaeota as the key phylum, and Bacteroides, Tyzzerella, Fusobacterium, unidentified_Prevotellaceae, Methanovrevibacter and Faecalibacterium as the key genera were influencing the production traits of YBGs. These findings highlight the adaptive responses of the cecal microbiota to breeding methods and their potential links to host performance.