In this study, SweAMI FISH fluorescence in situ hybridization and immunofluorescence were used to analyze the spatiotemporal expression characteristics of the TBXT gene and Brachyury protein in 16-day-old Hulunbuir sheep embryos and 19-day-old Hu sheep embryos and to explore their regulatory effects on the development of sheep tails and related organs. The study showed the following: At 16 days of embryonic age, the TBXT gene was concentratedly expressed in the heart, somites, neural tube, and mesonephros of both sheep breeds; at 19 days of embryonic age, it was concentratedly expressed in the limb ectoderm and tail bud of Hulunbuir sheep, and in the midgut and tail bud of Hu sheep. At 16 days of embryonic age, the BRACHYURY protein was concentratedly expressed in the neural tube, somites, brain vesicles, and mesonephros of both sheep breeds; at 19 days of embryonic age, it was concentratedly expressed in the heart and hindgut of Hulunbuir sheep, and in the tail bud and hindgut of Hu sheep. In summary, this shows that there are differences in the temporal and spatial expressions of the TBXT gene and BRACHYURY protein between the two sheep breeds. There are also site-specific and time-specific differences in the regulation of the above genes and proteins during tail and related organ development between the two breeds, which confirms that the molecular regulation pathways of tail and related organ development are different between the two breeds. This study provides an experimental basis for screening molecular markers related to goat tail development and breed improvement.

In vitro maturation (IVM) of oocytes is a pivotal step in assisted reproductive technologies for livestock. However, oxidative stress (OS) and mitochondrial dysfunction during in vitro culture often lead to oocyte aging, thereby limiting the efficiency of the technologies. To address these challenges, this study investigated the regulatory effects of 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC) on bovine oocyte IVM, aging, and developmental competence to determine the optimal concentration and explore underlying mechanisms. Cumulus–oocyte complexes (COCs) were collected from abattoir-derived bovine ovaries and cultured in IVM medium supplemented with 0 (control), 50, 100, 150, or 200 μmol/mL of POPC (n = 300 per group) at 38.5 °C under 5% CO₂ for 22 h. The optimal concentration was determined based on the first polar body extrusion rate, followed by in vitro fertilization (IVF), fluorescence staining, Smart-seq2 transcriptome sequencing, and quantitative PCR (qPCR) analysis. The results demonstrated that 150 μmol/mL of POPC yielded the highest maturation rate, significantly exceeding the control group (p < 0.05), and enhanced 2-4-cell cleavage rates after IVF. Furthermore, POPC markedly reduced intracellular reactive oxygen species (ROS) levels, increased glutathione (GSH) content, improved mitochondrial function, and restored normal spindle morphology. Transcriptomic analysis identified 350 upregulated and 280 downregulated differentially expressed genes (DEGs), which were enriched in pathways related to OS. qPCR validation confirmed upregulation of SIRT1/2 and BCL-2, along with downregulation of BAX and Caspase-1/3. Collectively, these findings suggest that 150 μmol/mL of POPC alleviates OS and activates the “SIRT–antioxidant–antiapoptotic” signaling axis, thereby providing valuable insights for optimizing assisted reproductive technologies in livestock.

Background: ADAMTS1 participates in ovarian extracellular matrix remodeling and ovulation, but its association with reproductive performance in Mongolian sheep requires further investigation. This study compared ovarian ADAMTS1 and PI3K/Akt pathway-associated gene expression between ewes with documented singleton and twin lambing histories and examined responses of cultured granulosa cells (GCs) to LY294002. Methods: Twenty-four Mongolian ewes were selected, comprising 12 with singleton and 12 with twin births over at least two consecutive lambing seasons. Each category included six ewes in estrus and six in diestrus. Ovarian gene expression was assessed using reverse transcription quantitative real-time PCR (RT-qPCR). Cultured GCs were examined by immunofluorescence and treated with 15 μmol/L LY294002 or dimethyl sulfoxide (DMSO) as the vehicle control. Results: Ovarian ADAMTS1, PI3K, Akt, and PTEN mRNA levels were higher in both twin-history groups than in either singleton-history group (p < 0.05). Bcl-2 expression was highest in the twin-history estrus group, whereas BAX expression was higher in diestrus than in estrus in both lambing-history categories (p < 0.05). Following LY294002 treatment, growth-associated absorbance at 450 nm decreased, and ADAMTS1 and several pathway-associated transcripts were downregulated. BAX mRNA increased significantly in the twin-history groups (p < 0.05), but the increases in the singleton-history groups were not significant (p > 0.05). Conclusions: These findings indicate associations with documented lambing history and cellular responses to LY294002. They do not establish a direct regulatory relationship between ADAMTS1 and PI3K/Akt signaling or a causal role in twinning. Steroidogenic function, gonadotropin responsiveness, and apoptosis require direct functional validation.