Nili-Ravi buffalo is a core dairy breed for China’s genetic improvement, yet its assisted reproductive technologies are restricted by low ovum pick-up (OPU) efficiency and unstable embryo transfer (ET) pregnant rates. This study established statistically optimized key parameters for OPU procedures in Nili-Ravi buffalo, and evaluated the effects of multiple ET-related protocols. For OPU, 50–55 mmHg vacuum pressure balanced oocyte recovery and usable rate best (p < 0.05). Young and adult buffalo performed significantly better than prepubertal heifers (p < 0.05). Four consecutive weekly consecutive OPU sessions did not significantly reduce oocyte yield (p > 0.05). FSH treatment markedly enhanced oocyte quantity, quality and recovery efficiency (p < 0.05), with no significant difference between two FSH protocols (p > 0.05). For ET, three estrus synchronization protocols showed no significant effects on estrus, ovulation, transferable recipient and pregnant rates in breeding or non-breeding seasons (p > 0.05), though optimized protocols showed a numerical improvement. Combinations of CL grade and embryo quality, as well as post-transfer treatments with vitamin ADE, hCG and progesterone, did not significantly affect pregnancy or early abortion rates (p > 0.05). This study built a practical reference framework for OPU-ET procedures in Nili-Ravi buffalo, with key OPU parameters statistically optimized and ET protocols showing numerical improvements that require further validation with larger sample sizes.

Melatonin (MT) is an important bioactive functional component of milk, where its content and biosynthetic mechanism are of considerable relevance to the quality regulation of dairy buffalo milk. This study aimed to investigate the variation patterns and biosynthetic mechanisms of melatonin in milk from dairy buffaloes. A total of 125 buffalo milk samples were collected, and MT content was determined using liquid chromatography–tandem mass spectrometry (LC–MS/MS). MT secretion patterns were evaluated according to breed, lactation, parity, and milking time (light exposure). In addition, whole-genome sequencing (WGS) data were used to screen for single-nucleotide polymorphisms (SNPs), which are significantly associated with melatonin biosynthetic enzyme genes. The results showed that breed, milking time, and lactation were the major factors affecting MT content in buffalo milk. Based on the WGS data, two SNPs significantly associated with MT secretion and synthesis were identified within the aralkylamine N-acetyltransferase (AANAT) region, among which the GG genotype at 3-8019407 and the CC genotype at 3-8019446 were associated with higher MT content. The MT content for both wild-type homozygotes was 59.54 pg/mL, which was higher than that of heterozygotes (39.12 pg/mL) (p < 0.05). With the exception of SNP2 and SNP8, all other SNPs were in Hardy–Weinberg equilibrium. These findings will serve as a reference for the subsequent selection and identification of breeding materials for high MT dairy buffalo, as well as for the development of high MT buffalo milk products.