Chloride channels (ClCs) have received global interest due to their significant role in the regulation of ion homeostasis, fluid transport, and electrical excitability of tissues and organs in different mammals and contributing to various functions, such as neuronal signaling, muscle contraction, and regulating the electrolytes’ balance in kidneys and other organs. In order to define the chloride voltage-gated channel (CLCN) gene family in buffalo, this study used in silico analyses to examine physicochemical properties, evolutionary patterns, and genome-wide identification. We identified eight CLCN genes in buffalo. The ProtParam tool analysis identified a number of important physicochemical properties of these proteins, including hydrophilicity, thermostability, in vitro instability, and basic nature. Based on their evolutionary relationships, a phylogenetic analysis divided the eight discovered genes into three subfamilies. Furthermore, a gene structure analysis, motif patterns, and conserved domains using TBtool demonstrated the significant conservation of this gene family among selected species over the course of evolution. A comparative amino acid analysis using ClustalW revealed similarities and differences between buffalo and cattle CLCN proteins. Three duplicated gene pairs were identified, all of which were segmental duplications except for CLCN4-CLCN5, which was a tandem duplication in buffalo. For each gene pair, the Ka/Ks test ratio findings showed that none of the ratios was more than one, indicating that these proteins were likely subject to positive selection. A synteny analysis confirmed a conserved pattern of genomic blocks between buffalo and cattle. Transcriptional control in cells relies on the binding of transcription factors to specific sites in the genome. The number of transcription factor binding sites (TFBSs) was higher in cattle compared to buffalo. Five main recombination breakpoints were identified at various places in the recombination analysis. The outcomes of our study provide new knowledge about the CLCN gene family in buffalo and open the door for further research on candidate genes in vertebrates through genome-wide studies.

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.