Buffaloes are the second-largest milk-producing animals, and their milk possesses significant nutritional and health benefits. However, the relatively low

The river buffalo is an economically important livestock species supplying milk and meat. However, a multi-tissue transcriptomic atlas for the key dairy river buffalo breeds, Murrah and Nili-Ravi, has not yet been established, and the lack of stable reference genes has hindered in-depth studies of their biological functions and the molecular mechanisms underlying key economic traits such as lactation. We established a multi-tissue gene expression atlas across 20 tissues and identified 717 housekeeping genes (HKGs), and RPL37A and EEF2 were further shown to be stable candidate reference genes under the conditions tested. We found 8368 tissue-specific genes (TSGs), predominantly enriched in the reproductive system. Exploratory analysis of mammary tissue (dry-period vs. public lactating samples, confounded by batch effects) revealed mammary-enriched hub genes including LALBA; these findings are preliminary and require validation. Dynamic analysis across lactation stages (early, peak, mid-, late) identified candidate genes including SEC14L2 and ACSM3. Phenotypic data showed strong negative correlations between milk yield and protein/fat content, and a positive correlation with lactose content. However, causal or regulatory roles were not inferred due to lack of paired individual-level data. Cross-dataset comparisons are descriptive only, and are not key conclusions. In summary, this study lays the foundation for advancing research in lactation trait genetics and functional genomics in river buffalo, with novel reference genes and lactation stage-specific transcriptional dynamics as its main contributions.

Leeches are significant medicinal invertebrates, particularly well-known for their strong bioactive substances and therapeutic uses in analgesia, anticoagulation, and anti-inflammatory conditions. Despite this, insufficient information is available about the molecular processes driving species-specific functional diversity. We conducted a comparative transcriptomic study of three medically valuable leech species, Hirudinaria manillensis, Hirudo nipponia, and Whitmania pigra, and explored the interspecies differences in gene expression and regulatory networks. In total, 18,107 mRNAs and 183 long non-coding RNAs (lncRNAs) were identified from 27 cDNA libraries, and pairwise comparison differential gene expression analysis retrieved 1681, 2156, and 468 DEGs in HM vs HN, HM vs WP, and HN vs WP, respectively, with 81 common genes among all species. These DEGs are mainly enriched in muscle growth, metabolic regulation, and cytoskeletal structure. Muscle-related genes (TPM, TNNT, and TNNI) exhibited higher expressions in H. nipponia, suggesting greater contractile adaptability, whereas H. manillensis displayed high expression of anticoagulant (HIRM2, ANTA, FLNA, PROS), analgesic (AGRIN, MMEL1, NEP), and anti-inflammatory (MMP17, MMP25, TLR4, CYTB) genes, highlighting its robust pharmacological potential. In contrast, W. pigra had comparatively lower expression of these functional genes. Besides, co-expression evaluation presented DElncRNAs, including MSTRG.1617, MSTRG.484, and MSTRG.1673 were shown to be crucial cis-regulators of muscle and bioactive genes (TPM, TNNT, PROS, and MMP25), demonstrating intricate lncRNA–mRNA connections influencing physiological specificity. Together, we provided the first transcriptome comparison of medicinal leech species, signifying their molecular basis of muscle function and therapeutic efficiency, establishing a fundamental resource for future multi-omics integration, functional gene validation, and targeted molecular breeding to advance leech-derived medicinal applications.

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.