Haiying He

Background: Dietary crude protein (CP) acts as a key nutritional factor that affects the growth performance and liver metabolism of fattening Hu sheep, with metabolizable energy (ME) representing a major confounding factor in CP-related responses. To isolate the specific effects of CP on liver metabolism and minimize energy–protein interactions, we standardized dietary ME at 9.4 MJ/kg dry matter. Methods: We then established three isoenergetic CP concentrations: 11.07%, 13.07%, and 15.11%. A total of ninety 4-month-old male Hu sheep (with an initial body weight of 27.09 ± 1.83 kg) were allocated at random to three dietary treatment groups, each containing 30 animals distributed across three replicate pens, and fed pelleted total mixed rations (PTMRs) for 75 days under pen conditions in southern Xinjiang. Exploratory combined transcriptomic and metabolomic profiling of liver tissue was conducted to characterize how graded CP levels modulate growth traits and hepatic metabolic pathways, thereby identifying the appropriate dietary CP level for efficient and sustainable fattening of Hu sheep in this region. Result: Results indicated that animals fed the 15.11% CP diet showed a significantly higher average daily gain (ADG) and cumulative weight gain compared with those fed 11.07% or 13.07% CP (p < 0.05). Exploratory multi-omics enrichment analysis demonstrated significant overrepresentation (p < 0.05) of differentially expressed genes and metabolites in key biological pathways—including bile secretion, AMP-activated protein kinase (AMPK) signaling, steroid biosynthesis, peroxisome proliferator-activated receptor (PPAR) signaling, and oxidative stress-related and oxidative phosphorylation. Correlation analyses characterized two hub genes—ATP6AP1 and LOC101119853—that were significantly and negatively correlated with ADG (p < 0.05), whereas two metabolites—calcidiol and ADP—displayed significant positive relationships with ADG (p < 0.05). Pathway-level comparisons further demonstrated that both the 13.07% vs. 15.11% CP and the 11.07% vs. 15.11% CP contrasts yielded significant enrichment in AMPK signaling and steroid biosynthesis. Notably, calcidiol and ADP both declined numerically in the 13.07% vs. 15.11% CP comparison, whereas only ADP reached statistical significance in the 11.07% vs. 15.11% CP contrast. Conclusions: Collectively, under an ME level of 9.4 MJ/kg, a dietary CP concentration of 15.11% contributes to favorable growth of 4-month-old fattening Hu sheep housed in pens in southern Xinjiang. This level is associated with improved growth performance and coordinated regulation of central hepatic regulatory networks—particularly those involved in energy homeostasis and steroidogenesis—thereby supporting metabolic stability without compromising animal health or production efficiency. These findings provide a preliminary molecular basis for precision protein nutrition in Hu sheep feeding systems and offer translational insights for optimizing ruminant nutrition under arid and semi-arid environmental constraints. All correlations indicate potential associations, not causal relationships.

Natural or artificial selection causes animals to adapt to their environment. The adaptive changes generated by the rumen population and metabolism form the basis of ruminant evolution. In particular, the adaptive drive for environmental adaptation reflects the high-quality traits of sheep that have migrated from other places or have been distant from their origins for a long time. The Hu sheep is the most representative sheep breed in the humid and low-altitude environments (Tai Lake region) in East Asia and has been widely introduced into the arid and high-altitude environments (Tibetan Plateau and Hotan region), resulting in environmental adaptive changes in the Hu sheep. In this study, a joint analysis of the rumen microbial metagenome and metabolome was conducted on Hu sheep from different regions (area of origin and area of introduction) with the objective of investigating the quality traits of Hu sheep and identifying microorganisms that influence the adaptive drive of ruminants. The results demonstrated that the growth performance of Hu sheep was altered due to changes in rumen tissue and metabolism following their introduction to the arid area at relatively high altitude. Metagenomic and metabolomic analyses (five ramsper area) revealed that 3580 different microorganisms and 732 different metabolites were identified in the rumen fluid of arid sheep. Among these, the representative upregulated metabolites were 4,6-isocanedione, methanesulfonic acid and N2-succinyl-L-arginine, while the dominant microorganism was Prevotella ruminicola. The downregulated metabolites were identified as campesterol, teprenone and dihydroclavaminic acid, while the disadvantaged microorganisms were Dialister_succinatiphilus, Prevotella_sp._AGR2160, Prevotella_multisaccharivorax and Selenomonas_bovis. The results of the Pearson analysis indicated that the rumen microbiota and metabolite content of sheep were significantly altered and highly correlated following their relocation from a humid lowland to an arid upland. In particular, the observed changes in rumen microorganisms led to an acceleration of body metabolism, rendering sheep highly adaptable to environmental stress. Prevotella_ruminicola was identified as playing an important role in this process. These findings provide insights into the environmental adaptation mechanisms of sheep.

Objective: The objective of this study was to investigate the effects of prolactin (PRL) on the proliferation and apoptosis of ovine ovarian granulosa cells (GCs) and the secretion of estrogen (E2) and progesterone (P4), as well as to explore the effects of PRL on related genes and proteins.Methods: We isolated ovarian GCs from 1-year-old small-tail Han sheep and identified PRL receptor (PRLR) on ovaries and follicle stimulating hormone receptor (FSHR) on ovarian GCs, respectively, using immunohistochemistry. PRL (0, 0.05, 0.50, 5.00 μg/mL) were added to GCs in vitro along with FSH, cell proliferation was measured by cell counting Kit-8 (CCK-8) and apoptosis by flow cytometry. The measurement of E2 and P4 content by enzyme-linked immunosorbent assays after 48 h and 72 h. The expression of functional genes and proteins was identified by real-time quantitative polymerase chain reaction (RTqPCR) and Western-blot after 48 h.Results: PRLR was expressed in both follicular GCs and corpus luteum, whereas FSHR was expressed specifically. The proliferative activity was lower on day 1 while higher on day 4 and day 5. The apoptosis rate of GCs in the 0.05 μg/mL group was significantly higher than that in the control group after treatment with PRL for 24 h (p<0.05). Compared with the control group, the secretion of E2 in GCs was reduced significantly (p<0.05) in PRL treatment for 48 h and 72 h, while the secretion of P4 was significantly increased (p<0.05). The mRNA expression levels of PRLR, FSHR, LHR, CYP11A1, HSD3B7, and STAR were significantly higher than those in the control group (p<0.01), and the relative abundance of BCL2 in all PRL group were increased after PRL treatment.Conclusion: PRL promoted the proliferation of GCs and supraphysiological concentrations inhibited apoptosis caused by down-regulation of BAX and up-regulation of BCL2. PRL inhibited E2 by down-regulating CYP19A1 and promoted P4 by up-regulating CYP11A1, STAR, and HSD3B7.