2.1. Experimental Animals and Sample Collection

Animal care and experimental protocols were approved by the Animal Ethics Committee of Inner Mongolia University (No. IMU-CATTLE-2022-073) and were carried out in accordance with the committee’s guidelines for animal research. As mentioned in our previous report, we used CRISPR/Cas9 technology and somatic cell nuclear transplantation to produce MSTN knockout Luxi cattle (MSTN^−/− Luxi cattle ♂, Bos taurus), and used lineage progenitor hybridization with wild-type female Luxi cattle to successfully produce a population of MSTN^+/− Luxi cattle [9,26]. In this study, a total of twelve 2-year-old steers were used, with six MSTN^+/− Luxi cattle (MT) as the experimental group and six wild-type Luxi cattle (WT) as the control group. The wild-type cattle were not genetically edited and all are purebred Luxi cattle.

All experimental animals were housed in Hohhot, Inner Mongolia, China (111°85′ E, 40°55′ N, 1040 m above sea level). Each barn was equipped with a temperature-controlled (15 °C) automatic watering system, and all cattle were allowed to drink freely. The total mixed ration (TMR) consists of 68% silage, 12% hay and 20% grain feed. Each barn was a separate compartment containing approximately 30 m² of indoor area and approximately 300 m² of outdoor area shared by each group of cattle (n = 6), with all cattle being allowed unrestricted access to both indoor and outdoor areas. Cattle were fed until 24 months of age for slaughter, and fasted for 24 h before slaughter and allowed to drink freely. Slaughter was carried out in the morning, and all cattle were slaughtered by bloodletting. The slaughtering process followed national standard operating procedures (GB/T 19477-2018 [27], Cattle slaughter, Beijing, China). Liver tissue was collected within 30 min following slaughter, along with semitendinosus tissue from the rump of the cattle. These samples were subsequently divided into smaller portions and rapidly submerged in liquid nitrogen for freezing. Afterward, the frozen specimens were transferred to a −80 °C freezer for storage until needed for further analysis. The ileal contents were also collected within 30 min after slaughter and these contents were taken from the middle segment of the cattle ilea. The contents were packed into clean collection tubes, tightly capped, and then first immersed in liquid nitrogen and frozen, then transferred to a −80 °C refrigerator until further analysis.