Understanding the nutritional protein requirements of yak calves is the basis of precise feed formulation. Regulating feed protein can reduce environmental impacts, which is particularly crucial for the rearing and management of yak calves. In this study, we used a combination of comparative slaughter, feeding, and digestibility trials to determine the net protein requirements of suckling yak calves. Thirty-five yak male calves with similar weights at 60 d of age were divided into 5 groups: early slaughter (ES), mid-term slaughter (MS), late slaughter (AL), 70% feeding (R70), and 40% feeding (R40). The ES, MS, and AL groups were used for comparative slaughter trials, while the AL, R70, and R40 groups were used for ad libitum feeding experiments. The results indicated that at different feeding levels, low feeding levels were not conducive to calf growth. For yak calves with body weights of approximately 40-90 kg, the nitrogen digestibility ranged from 49.36% to 59.32%, and the nitrogen retention rate ranged from 36.59% to 48.97%. The net protein requirement for yak calf maintenance is 2.90 g/kgW^0.75·d^-1. The equation for the net protein requirement for yak calf growth is NPg(kg) = 0.0543 × EBW^0.0833(kg). Muscle metabolomics results indicated that the protein content in the muscle tissue did not increase with feeding level or body weight. With an increase in the feeding level, the nutritional protein level provided by the diet increases, which regulates changes in steroid hormone biosynthesis, ovarian steroidogenesis, cortisol synthesis and secretion, and carbon metabolism, promoting an increase in hormone-like metabolites in the muscle tissue. These data we obtained provide guidance for the efficient rearing of yak calves and provide basic data for further research on the nutritional requirements.

To evaluate the impact of different roughages on the intestinal microbiota of yak calves, we fed them oat hay in substitution of alfalfa hay, in addition to milk replacer and starter powder. Twenty-one 45-day-old male yak calves were selected and randomly assigned to three groups: the milk replacer + starter + alfalfa hay group (AH), the milk replacer + starter + oat hay group (OH), and the milk replacer + starter + mixed hay group (AO), in which the alfalfa hay and oat hay were administered in a 1:1 ratio. All calves in the three groups were fed the same milk replacer and an equivalent amount of dry matter. The formal experiment commenced after a 21-day pre-test period and lasted for 120 days. Following the experiment, the contents of the jejunum and colon were collected to investigate the intestinal microbiota and metabolites using 16S rRNA sequencing and LC-MS metabolomics. The result showed that the AO group had greater final body weights overall than the AH group and OH group (p < 0.05). The AH group and OH group had considerably greater feed-to-gain ratios than the AO group (p < 0.05). At the phylum level, the OH group exhibited an increased relative abundance of Bacteroidota and Spirochaetota in the jejunum (p < 0.05). The relative abundance of Actinobacteriota in the colon was increased in the AO group (p < 0.05). At the genus level, the AO group exhibited a decreased abundance of Clostridium sensu_stricto_1 (p < 0.05), and the OH group showed an increased abundance UCG-005 and Alistipes in the jejunum. There were many differential metabolites in the OH group and AO group compared to the AH group, and the different metabolites of the OH group were associated with the metabolic pathways of the nervous system, sensory system, amino acid metabolism, and lipid metabolism in the jejunum and with lipid metabolism, amino acid metabolism, and the nervous system in the colon. In the AO group, these metabolites were associated with the digestive system and the translation and metabolism of cofactors in the jejunum and with the metabolism of cofactors and vitamins in the colon. In summary, it is feasible to replace alfalfa hay with oat hay based on milk replacer and starter. The combination of the two forages enhanced nutrient absorption, improved immune function, maintained the internal homeostasis of yak calves, and was more beneficial to their growth and development.