Search

Background: Buffalo is a globally important livestock species, but its reproductive performance is relatively low than cattles. At present, dominant follicle development specific process and mechanistic role of follicular growth related genes in water buffaloes are not well understood. Therefore, we comprehensively performed transcriptomics of granulosa cells and oocytes from different-sized follicles in water buffalo to identify key candidate genes that influence follicle development and diameter, and further explored the potential regulatory mechanisms of granulosa cells and oocytes in the process of water buffalo follicle development., Results: In this study, we found918 granulosa cell transcripts and 1401 oocyte transcripts were correlated in follicles of different diameters, and the expression differences were significant. Subsequent enrichment analysis of the co-expressed differentially expressed transcripts identified several genes targeted by long non-coding RNAs (lncRNAs) and associated with follicular development. Notably, the upregulation of BUB1 regulated by MSTRG.41325.4 and interactive action of SMAD2 and SMAD7 might have key regulatory role in follicular development. Additionally, we also detected key differentially expressed genes that potentially influence follicular hormone metabolism and growth, like ID2, CHRD, TGIF2 and MAD2L1, and constructed an interaction network between lncRNA transcripts and mRNAs., Conclusions: In summary, this study preliminarily revealed the differences in gene expression patterns among buffalo follicles of different sizes and their potential molecular regulatory mechanisms. It provides a new perspective for exploring the mechanism of buffalo follicular dominance and improving buffalo reproductive performance., (© 2024. The Author(s).)

Background: Buffaloes are crucial to agriculture, yet mitochondrial biology in these animals is less studied compared to humans and laboratory animals. This research examines tissue-specific variations in mitochondrial succinate dehydrogenase (SDH) gene expression across buffalo kidneys, hearts, brains, and ovaries. Understanding these variations sheds light on mitochondrial energy metabolism and its impact on buffalo health and productivity, revealing insights into enzyme regulation and potential improvements in livestock management., Materials and Methods: RNA-seq data from buffalo kidney, heart, brain, and ovary tissues were reanalyzed to explore mitochondrial SDH gene expression. The expression of SDH subunits (SDHA, SDHB, SDHC, SDHD) and assembly factors (SDHAF1, SDHAF2, SDHAF3, SDHAF4) was assessed using a log2 fold-change threshold of + 1 for up-regulated and - 1 for down-regulated transcripts, with significance set at p < 0.05. Hierarchical clustering and differential expression analyses were performed to identify tissue-specific expression patterns and regulatory mechanisms, while Gene Ontology and KEGG pathway analyses were conducted to uncover functional attributes and pathway enrichments across different tissues., Results: Reanalysis of RNA-seq data from different tissues of healthy female buffaloes revealed distinct expression patterns for SDH subunits and assembly factors. While SDHA, SDHB, and SDHC showed variable expression across tissues, SDHAF2, SDHAF3, and SDHAF4 exhibited tissue-specific profiles. Significant up-regulation of SDHA, SDHB, and several assembly factors was observed in specific tissue comparisons, with fewer down-regulated transcripts. Gene ontology and KEGG pathway analyses linked the up-regulated transcripts to mitochondrial ATP synthesis and the respiratory electron transport chain. Notably, tissue-specific variations in mitochondrial function were particularly evident in the ovary., Conclusion: This study identifies distinct SDH gene expression patterns in buffalo tissues, highlighting significant down-regulation of SDHA, SDHB, SDHC, and assembly factors in the ovary. These findings underscore the critical role of mitochondria in tissue-specific energy production and metabolic regulation, suggest potential metabolic adaptations, and emphasize the importance of mitochondrial complex II. The insights gained offer valuable implications for improving feed efficiency and guiding future research and therapies for energy metabolism disorders., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Milk phospholipids have multiple health benefits, but the deficiency of detailed phospholipid profiles in dairy products brings obstacles to intake calculation and function evaluation of dairy phospholipids. In present study, 306 phospholipid molecular species were identified and quantified among 207 milk, yogurt and cream products using a HILIC-ESI-Q-TOF MS and a HILIC-ESI-QQQ MS. The phospholipid profiles of five mammals' milk show that camel milk contains the most abundant phosphatidylethanolamine, phosphatidylserine and sphingomyelin; cow, yak and goat milk have similar phospholipidomes, while buffalo milk contains abundant phosphatidylinositol. Fewer plasmalogens but more lyso-glycerolphospholipids were found in ultra-high-temperature (UHT) sterilized milk than in pasteurized milk, and higher proportions of lyso-glycerolphospholipid/total phospholipid were observed in both cream and skimmed/semi-skimmed milk than whole milk, indicating that UHT and skimming processes improve glycerolphospholipid degradation and phospholipid nutrition loss. Meanwhile, more diacyl-glycerolphospholipids and less of their degradation products make yogurt a better phospholipid resource than whole milk., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Colostrum from yaks and buffaloes possesses substantial nutritional value, yet the complete array of metabolites within remains insufficiently elucidated. This study scrutinizes the metabolite profiles of yak, buffalo, and cow colostrum utilizing targeted metabolomics paired with ultra-performance liquid chromatography-tandem triple quadrupole linear ion trap mass spectrometry (UPLC-QTRAP-MS). The analysis detected 362 metabolites across all samples. Furthermore, 63, 77, and 46 differential metabolites were selected between yak and buffalo colostrum, yak and cow colostrum, and buffalo and cow colostrum, respectively. Yak colostrum notably contained higher concentrations of inositol, glycine, and carnitine, whereas buffalo colostrum was distinguished by a substantial presence of primary bile acids, which facilitate fat digestion. These findings offer profound insights into yak and buffalo colostrum, providing critical data to propel advancements in the dairy industry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Recently, much interest has been raised for the characterization of signaling molecules carried by extracellular vesicles (EVs), which are particularly enriched in milk (mEVs). Such interest is linked to the capability of EVs to cross biological barriers, resist acidification in the gastric environment, and exert modulation of the immune system, mainly through their microRNA (miRNA) content. We characterized the small-RNA cargo of colostrum EVs (colosEVs) and mEVs from Italian Mediterranean buffalo through next generation sequencing. Colostrum (first milking after birth) and milk (day 50 of lactation) were sampled from seven subjects from five farms. ColosEVs and mEVs were subjected to morphological characterization, followed by high-depth sequencing of small RNA libraries produced from total RNA. The main difference was the amount of EV in the two samples, with colostrum showing 10 to 100-fold higher content than milk. For both matrices, miRNA was the most abundant RNA species (95% for colosEVs and 96% for mEVs) and three lists were identified: colosEV-specific, mEV-specific and shared most expressed. Gene ontology (GO) enrichment analysis on miRNA targets highlighted many terms related to the epigenetic, transcriptional and translational regulations across the three lists, with a higher number of enriched terms for colosEV-specific miRNAs. Terms specific to colosEVs were related to "cell differentiation" and "microvillus assembly", while for mEV "cardiac and blood vessel development" and "mitochondria" emergerd. Immune modulation terms were found for both sample-specific miRNAs. Overall, both matrices carry a similar molecular message in terms of biological processes potentially modulated into receiving cells, but there is significant difference in the abundance, with colostrum containing much more EVs than milk. Moreover, colosEVs carry molecules involved in signal transduction, cell cycle and immune response, as for mEVs and EVs of other previously characterized species, but with a special enrichment for miRNAs with epigenetic regulation capacities. These beneficial characteristics of colosEVs and mEVs are essential for the calf and could also be exploited for the therapeutic purposes in humans, although further studies are necessary to measure the sanitization treatment impact on EV conservation, especially in buffalo where milk is consumed almost exclusively after processing., (© 2024. The Author(s).)

The reproductive efficiency in buffalo is highly influenced by seasonal variability. Angiogenesis in the reproductive cycle is important for optimal physiological functioning of uterus. Estrogen receptor-α (ERα), vascular endothelial growth factor (VEGF) and reduced nicotinamide adenine dinucleotide phosphatase diaphorase (NADPH-d) are vital indicators for the uterine angiogenic process. This study was conducted to see the effect of season on the expression of different uterine angiogenic factors. Season wise (winter and summer) and phase wise (follicular and luteal), immune staining intensity of buffalo uterus was measured by calculating the optical density value (OD) for ERα and VEGF. Percentage of immuno-positive cell count for ERα was done. Histoenzymic NADPH-d expression was analysed. Expression of all these factors increased during follicular phase of oestrous cycle in order to support the angiogenesis; however, the expression was significantly lower (p ≤ 0.05) in term of OD value as well as percentage count of immuno-positive cells during summer season indicating lower angiogenic activity that subsequently affected reproduction in buffalo., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Background: Mitochondria, essential for cellular energy production through oxidative phosphorylation (OXPHOS), integrate mt-DNA and nuclear-encoded genes. This cooperation extends to the mitochondrial translation machinery, involving crucial mtDNA-encoded RNAs: 22 tRNAs (mt-tRNAs) as adapters and two rRNAs (mt-rRNAs) for ribosomal assembly, enabling mitochondrial-encoded mRNA translation. Disruptions in mitochondrial gene expression can strongly impact energy generation and overall animal health. Our study investigates the tissue-specific expression patterns of mt-tRNAs and mt-rRNAs in buffalo., Material and Methods: To investigate the expression patterns of mt-tRNAs and mt-rRNAs in different tissues and gain a better understanding of tissue-specific variations, RNA-seq was performed on various tissues, such as the kidney, heart, brain, and ovary, from post-pubertal female buffaloes. Subsequently, we identified transcripts that were differentially expressed in various tissue comparisons., Results: The findings reveal distinct expression patterns among specific mt-tRNA and mt-rRNA genes across various tissues, with some exhibiting significant upregulation and others demonstrating marked downregulation in specific tissue contexts. These identified variations reflect tissue-specific physiological roles, underscoring their significance in meeting the unique energy demands of each tissue. Notably, the brain exhibits the highest mtDNA copy numbers and an abundance of mitochondrial mRNAs of our earlier findings, potentially linked to the significant upregulation of mt-tRNAs in brain. This suggests a plausible association between mtDNA replication and the regulation of mtDNA gene expression., Conclusion: Overall, our study unveils the tissue-specific expression of mitochondrial-encoded non-coding RNAs in buffalo. As we proceed, our further investigations into tissue-specific mitochondrial proteomics and microRNA studies aim to elucidate the intricate mechanisms within mitochondria, contributing to tissue-specific mitochondrial attributes. This research holds promise to elucidate the critical role of mitochondria in animal health and disease., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: The milk's nutritional value is determined by its constituents, including fat, protein, carbohydrates, and minerals. The mammary gland's ability to produce milk is controlled by a complex network of genes. Thereby, the fat, protein, and lactose synthesis must be boost in milk to increase milk production efficiency. This can be accomplished by fusing genetic advancements with proper management practices. Therefore, this study aimed to investigate the association between the Lipoprotein lipase (LPL), kappa casein CSN3, and Glucose transporter 1 (GLUT1) genes expression levels and such milk components as fat, protein, and lactose in different dairy breeds during different stages of lactation., Methods: To achieve such a purpose, 94 milk samples were collected (72 samples from 36 multiparous black-white and red-white Holstein-Friesian (HF) cows and 22 milk samples from 11 Egyptian buffaloes) during the early and peak lactation stages. The milk samples were utilized for milk analysis and genes expressions analyses using non- invasive approach in obtaining milk fat globules (MFGs) as a source of Ribonucleic acid (RNA)., Results: LPL and CSN3 genes expressions levels were found to be significantly higher in Egyptian buffalo than Holstein-Friesian (HF) cows as well as fat and protein percentages. On the other hand, GLUT1 gene expression level was shown to be significantly higher during peak lactation than early lactation. Moreover, lactose % showed a significant difference in peak lactation phase compared to early lactation phase. Also, fat and protein percentages were significantly higher in early lactation period than peak lactation period but lactose% showed the opposite pattern of Egyptian buffalo., Conclusion: Total RNA can be successfully obtained from MFGs. The results suggest that these genes play a role in glucose absorption and lactose synthesis in bovine mammary epithelial cells during lactation. Also, these results provide light on the differential expression of these genes among distinct Holstein-Friesian cow breeds and Egyptian buffalo subspecies throughout various lactation phases., (© 2024. The Author(s).)

Peroxisome proliferator-activated receptor γ (PPARG) has various splicing variants and plays essential roles in the regulation of adipocyte differentiation and lipogenesis. However, little is known about the expression pattern and effect of the PPARG on milk fat synthesis in the buffalo mammary gland. In this study, we found that only PPARG-X17 and PPARG-X21 of the splicing variant were expressed in the buffalo mammary gland. Amino acid sequence characterization showed that the proteins encoded by PPARG-X17 and PPARG-X21 are endonuclear non-secreted hydrophilic proteins. Protein domain prediction found that only the PPARG-X21 -encoded protein had PPAR ligand-binding domains (NR&#95;LBD&#95;PPAR), which may lead to functional differences between the two splices. RNA interference (RNAi) and the overexpression of PPARG-X17 and PPARG-X21 in buffalo mammary epithelial cells (BMECs) were performed. Results showed that the expression of fatty acid synthesis-related genes ( ACACA , CD36 , ACSL1 , GPAT , AGPAT6 , DGAT1 ) was significantly modified ( p < 0.05) by the RNAi and overexpression of PPARG-X17 and PPARG-X21 . All kinds of FAs detected in this study were significantly decreased ( p < 0.05) after RNAi of PPARG-X17 or PPARG-X21 . Overexpression of PPARG-X17 or PPARG-X21 significantly decreased ( p < 0.05) the SFA content, while significantly increased ( p < 0.05) the UFA, especially the MUFA in the BMECs. In conclusion, there are two PPARG splicing variants expressed in the BMECs that can regulate FA synthesis by altering the expression of diverse fatty acid synthesis-related genes. This study revealed the expression characteristics and functions of the PPARG gene in buffalo mammary glands and provided a reference for further understanding of fat synthesis in buffalo milk.

Colostrum/Milk is a chief repertoire of antioxidant peptides. Nuclear factor-erythroid 2 related factor 2 (Nrf2) is a viable target for Parkinson's Disease (PD), as this pathway deduced to be impaired in PD. Cullin-3 is one of the crucial E3 ligase responsible for its regulation. The present study screened peptide libraries of buffalo colostrum & milk peptides for Cullin-3 inhibition, thus ensuing activation of Nrf2 to alleviate the molecular etiopathology in PD using the C. elegans as a model. The structure was modelled, binding sites analyzed and peptide-interactions analyzed by docking. Among the 55 sequences (≤1 kDa), the peptide SFVSEVPEL having the highest dock score (-16.919) was synthesized and evaluated for its effects on oxidative stress markers, antioxidant enzymes, neurochemical marker and Nrf2/Skn-1 levels. The lead peptide alleviated the oxidative pathophysiology and behavioural deficits associated with PD in C. elegans., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Background: Early embryonic mortality is one of the major intriguing factors of reproductive failure that causes considerable challenge to the mammalian cell biologists. Heat stress is the major factor responsible for reduced fertility in farm animals. The present study aimed to investigate the influence of heat stress on prostaglandin production and the expression of key genes, including COX-2, PGES, PGFS, ITGAV and LGALS15, in buffalo endometrial epithelial cells., Methods and Results: Buffalo genitalia containing ovaries with corpus luteum (CL) were collected immediately post-slaughter. The stages of the estrous cycle were determined based on macroscopic observations of the ovaries. Uterine lumens of the mid-luteal phase (days 6-10 of the estrous cycle) were washed and treated with trypsin to isolate epithelial cells, which were then cultured at control temperature (38.5 °C for 24 h) or exposed to elevated temperatures [38.5 °C for 6 h, 40.5 °C for 18 h; Heat Stressed (HS)]. The supernatant and endometrial epithelial cells were collected at various time points (0, 3, 6, 12, and 24 h) from both the control and treatment groups. Although heat stress (40.5 °C) significantly (P < 0.05) increased COX-2, PGES, and PGFS transcripts in epithelial cells but it did not affect the in vitro production of PGF 2α and PGE 2 . The expression of ITGAV and LGALS15 mRNAs in endometrial epithelial cells remained unaltered under elevated temperature conditions., Conclusion: It can be concluded that elevated temperature did not directly modulate prostaglandin production but, it promoted the expression of COX-2, PGES and PGFS mRNA in buffalo endometrial epithelial cells., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Genetic and genomic analyses of longitudinal traits related to milk production efficiency are paramount for optimizing water buffaloes breeding schemes. Therefore, this study aimed to (1) compare single-trait random regression models under a single-step genomic BLUP setting based on alternative covariance functions (i.e., Wood, Wilmink, and Ali and Schaeffer) to describe milk (MY), fat (FY), protein (PY), and mozzarella (MZY) yields, fat-to-protein ratio (FPR), somatic cell score (SCS), lactation length (LL), and lactation persistency (LP) in Murrah dairy buffaloes (Bubalus bubalis); (2) combine the best functions for each trait under a multiple-trait framework; (3) estimate time-dependent SNP effects for all the studied longitudinal traits; and (4) identify the most likely candidate genes associated with the traits. A total of 323,140 test-day records from the first lactation of 4,588 Murrah buffaloes were made available for the study. The model included the average curve of the population nested within herd-year-season of calving, systematic effects of number of milkings per day, and age at first calving as linear and quadratic covariates, and additive genetic, permanent environment, and residual as random effects. The Wood model had the best goodness of fit based on the deviance information criterion and posterior model probabilities for all traits. Moderate heritabilities were estimated over time for most traits (0.30 ± 0.02 for MY; 0.26 ± 0.03 for FY; 0.45 ± 0.04 for PY; 0.28 ± 0.05 for MZY; 0.13 ± 0.02 for FPR; and 0.15 ± 0.03 for SCS). The heritability estimates for LP ranged from 0.38 ± 0.02 to 0.65 ± 0.03 depending on the trait definition used. Similarly, heritabilities estimated for LL ranged from 0.10 ± 0.01 to 0.14 ± 0.03. The genetic correlation estimates across days in milk (DIM) for all traits ranged from -0.06 (186-215 DIM for MY-SCS) to 0.78 (66-95 DIM for PY-MZY). The SNP effects calculated for the random regression model coefficients were used to estimate the SNP effects throughout the lactation curve (from 5 to 305 d). Numerous relevant genomic regions and candidate genes were identified for all traits, confirming their polygenic nature. The candidate genes identified contribute to a better understanding of the genetic background of milk-related traits in Murrah buffaloes and reinforce the value of incorporating genomic information in their breeding programs., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The aim was to compare the UF proteomics of pregnant and non-pregnant buffalo during early pregnancy. Forty-four females were submitted to hormonal estrus synchronization and randomly divided into two groups: pregnant (n = 30) and non-pregnant (n = 14). The pregnant group was artificially inseminated and divided into a further two groups: P12 (n = 15) and P18 (n = 15). Conceptus and uterine fluid samples were collected during slaughter at, respectively, 12 and 18 days after insemination. Of all the inseminated females, only eight animals in each group were pregnant, which reduced the sample of the groups to P12 (n = 8) and P18 (n = 8). The non-pregnant group was also re-divided into two groups at the end of synchronization: NP12 (n = 7) and NP18 (n = 7). The UF samples were processed for proteomic analysis. The results were submitted to multivariate and univariate analysis. A total of 1068 proteins were found in the uterine fluid in both groups. Our results describe proteins involved in the conceptus elongation and maternal recognition of pregnancy, and their action was associated with cell growth, endometrial remodeling, and modulation of immune and antioxidant protection, mechanisms necessary for embryonic maintenance in the uterine environment. SIGNIFICANCE: Uterine fluid is a substance synthesized and secreted by the endometrium that plays essential roles during pregnancy in ruminants, contributing significantly to embryonic development. Understanding the functions that the proteins present in the UF perform during early pregnancy, a period marked by embryonic implantation, and maternal recognition of pregnancy is of fundamental importance to understanding the mechanisms necessary for the maintenance of pregnancy. The present study characterized and compared the UF proteome at the beginning of pregnancy in pregnant and non-pregnant buffaloes to correlate the functions of the proteins and the stage of development of the conceptus and unravel their processes in maternal recognition of pregnancy. The proteins found were involved in cell growth and endometrial remodeling, in addition to acting in the immunological protection of the conceptus and performing antioxidant actions necessary for establishing a pregnancy., Competing Interests: Declaration of Competing Interest The authors declare that no conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

Intramuscular fat (IMF) is a crucial determinant of meat quality and is influenced by various regulatory factors. Despite the growing recognition of the important role of long noncoding RNAs (lncRNAs) in IMF deposition, the mechanisms underlying buffalo IMF deposition remain poorly understood. In this study, we identified and characterized a lncRNA, lncFABP4, which is transcribed from the antisense strand of fatty acid-binding protein 4 (FABP4). lncFABP4 inhibited cell proliferation in buffalo intramuscular preadipocytes. Moreover, lncFABP4 significantly increased intramuscular preadipocyte differentiation, as indicated by an increase in the expression of the adipogenic markers peroxisome proliferator-activated receptor gamma (PPARG), CCAAT enhancer binding protein alpha (C/EBPα), and FABP4. Mechanistically, lncFABP4 was found to have the potential to regulate downstream gene expression by participating in protein-protein interaction pathways. These findings contribute to further understanding of the intricate mechanisms through which lncRNAs modulate intramuscular adipogenesis in buffaloes., (© 2024 Japanese Society of Animal Science.)

In this study, the whey and milk fat globule membrane (MFGM) proteomes of buffalo, cow, goat, and yak milk were analyzed using label-free proteomic technology. Totally, 1,292 MFGM proteins and 686 whey proteins were identified from these four species, and GO analysis revealed there were specific proteins with different functions in both whey (376) and MFGM (982) proteomes. The principal component analysis showed that ALB, TF, CSN1S1, and GLYCAM1 are characteristic markers of the milk for each of the four species. Furthermore, the conserved and differential in the expression of whey and MFGM proteins across the four species were identified by limma, and subsequent KEGG pathway analysis showed that immune-related proteins are both conserved and species-specific in the four species. These results provide a deepening of the understanding of the characteristics of proteins in whey and MFGMs from these four common dairy animals and new insight into developing dairy production., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Han Juan reports financial support was provided by National Natural Science Foundation of China., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Whey protein hydrolysate from Binglangjiang buffalo, a unique genetic resource, has anti-inflammatory activity, but its anti-inflammatory composition and effects are unknown. The aim of this study was to investigate the anti-inflammatory peptides from Binglangjiang buffalo whey protein hydrolysate. A total of 1483 peptides were identified using LC-MS/MS, and 12 peptides were chosen for chemical synthesis using peptidomics, and then two novel anti-inflammatory peptides (DQPFFHYN (DN8) and YSPFSSFPR (YR9)) were screened out using LPS-stimulated RAW264.7 cells. The molecular weights of DN8 and YR9 with β-turn conformations were 1067.458 Da and 1087.52 Da, respectively, and showed a high in-vitro safety profile and thermal stability, but were intolerant to pepsin. Furthermore, ELISA and Western blot analysis indicated that peptides DN8 and YR9 significantly suppressed the secretion of pro-inflammatory cytokines NO, TNF-α, and IL-6 and the expression of mediators iNOS, TNF-α, and IL-6 in LPS-stimulated RAW264.7 cells. The study provides insights into the development of novel food-based anti-inflammatory nutritional supplements., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

AGPAT6 plays a crucial role in the triglyceride (TG) synthesis pathway in mammals. However, its roles in buffalo lactation remain unknown. Therefore, we investigated the functional roles of AGPAT6 in milk fat synthesis by transfecting overexpression and lentivirus interference vectors in buffalo mammary epithelial cells (BuMECs) in vitro. AGPAT6 overexpression in BuMECs significantly enhanced the mRNA expression of FABP4 , SLC27A6, ACSL1 , DGAT1 , DGAT2 , LPIN1 , INSIG1 , CEBPA and SREBF1 genes, and significantly reduced that of XDH , CPT1A , LIPE , INSIG2 and PPARGC1A , but has no significant influence to the mRNA abundance of FABP3 , GPAM, PPARG and SREBF2 . However, the interference with AGPAT6 , the mRNA expression of FABP4 , SLC27A6 , ACSL1 , DGAT1 , DGAT2 , INSIG1 , CEBPA, SREBF1 , XDH , CPT1A , LIPE , INSIG2 and PPARGC1A genes in BuMECs changed contrary to the overexpression experiment, and that of GPAM , PPARG and SREBF2 also did not change significantly, but the expression of FABP3 was significantly decreased. In addition, the overexpression/interference of AGPAT6 gene significantly increased/decreased TG content in BuMECs. The results here indicate that AGPAT6 gene is involved in TG synthesis in BuMECs, and affects the expression of major genes associated with FA transport and activation, TG synthesis and transcription regulation, FA oxidation and TG degradation during the lipogenesis of milk.

In this study, Wingless-type MMTV (mouse mammary tumor virus) integration site family member ( WNT10B ) gene was sequence characterized in the Indian water buffalo. Sequence analysis revealed an open reading frame of 1176 nucleotides in buffalo, encoding 391 amino acids long protein. Nineteen nucleotide variations were observed between cattle and buffalo resulting in six amino acid changes. Phylogenetic analysis showed the clustering of ruminant species together. Real-time expression analysis of WNT10B in tissues collected from different organs of fetal and adult buffalo, revealed, the gene being abundantly expressed in the rumen and liver of the fetus. The fetal ovary, heart, kidney, lung, testis and mammary gland showed moderate expression, while in adult tissues, expression was high in the ovary, testis, brain, kidney, small intestine and liver, whereas lower expression was observed in the adult rumen. Significant differences in WNT10B expression levels were found for the brain, small intestine, testes, kidney, heart, rumen, and ovary when adult and fetal tissues were compared. A moderate level of genetic variation was found between cattle and buffalo WNT10B and expression patterns in a variety of tissues in adult buffalo implies that in addition to possible roles in adipogenesis and hematopoiesis, the WNT10B gene might be playing a significant role in other regulatory pathways as well.

Follistatin (FST), a member of the transforming growth factor-β (TGF-β) superfamily, has been identified as an inhibitor of follicle-stimulating hormone. Previous studies showed that it plays an important role in animal reproduction. Therefore, this study aims to investigate its effect on the maturation of buffalo oocytes in vitro, and the underlying mechanism of FST affecting oocyte maturation was also explored in buffalo cumulus cells. Results showed that FST was enriched in the ovary and expressed at different stages of buffalo ovarian follicles as well as during oocyte maturation and early embryo development. The FST expression level was up-regulated in MII buffalo oocytes compared with the GV stage (p < .05). To study the effects of FST on buffalo oocytes' maturation and early embryonic development, we added the pcD3.1 skeleton vector and PCD3.1-EGFP-FST vector into the maturation fluid of buffalo oocytes, respectively. It was demonstrated that FST promoted the in vitro maturation rate of buffalo oocytes and the blastocyst rate of embryos cultured in vitro (p < .05). By interfering with FST expression, we discovered that FST in cumulus cells plays a crucial role in oocyte maturation. Interference with the FST expression during the buffalo oocyte maturation did not affect the first polar body rate of buffalo oocyte (p > .05). In contrast, the location of mitochondria in oocytes was abnormal, and the cumulus expansion area was reduced (p < .05). After parthenogenetic activation, the cleavage and blastocyst rates of the FST-interfered group were reduced (p < .05). Furthermore, RT-qPCR was performed to investigate further the underlying mechanism by which FST enhances oocyte maturation. We found that overexpression of FST could up-regulate the expression level of apoptosis suppressor gene Bcl-2 and TGF-β/SMAD pathway-related genes TGF-β, SMAD2, and SMAD3 (p < .05). In contrast, the expression levels of SMAD4 and pro-apoptotic gene BAX were significantly decreased (p < .05). The FST gene could affect buffalo oocyte maturation by regulating the oocyte mitochondria integrity, the cumulus expansion, cumulus cell apoptosis, and the expression levels of TGF-β/SMAD pathway-related genes., (© 2023 Wiley-VCH GmbH. Published by John Wiley & Sons Ltd.)

The current study investigated the effect of adding different doses of galangal rhizome powder ( Alpinia galanga ) to the diets of lactating buffaloes on milk production, composition and in vitro ruminal fermentation kinetics. Twenty buffaloes were divided into four groups: (T1) a control diet comprising of concentrates, corn silage and rice straw at 50: 35: 15 on a DM basis, (T2) a control diet plus 15 g of galangal/animal/day and (T3) a control diet plus 30 g of galangal/animal/day and (T4) a control diet plus 45 g of galangal/animal/day. Results showed that inclusion of galangal in diets decreased ( p  < 0.05) the concentrations of butyrate, ammonia-nitrogen, CO 2 , and total protozoa count as compared with T1. Acetate concentration was lower in T4 as compared with other groups ( p  < 0.05). While propionate concentration was lower ( p  < 0.05) in T2 as compared with T1. T3 and T4 lowered ( p  < 0.05) methane, CO 2 productions, and protozoa count. T3 increased ( p  < 0.05) milk yield, energy corrected milk (ECM), yield of total solids, total protein, and solids not fat (SNF) than recorded for T1. While T4 recorded the lowest values ( p  < 0.05) of milk yield, ECM, total solids, fat, and total protein. The current results support the hypothesis that the inclusion of galangal in lactating buffaloes' diets at 30 g per animal daily improved feed intake and milk yield.

Buffalo plays a compelling role in reducing malnutrition and ensuring food to the people of Asian countries by its major contribution to milk and meat pool of the livestock agriculture farming system in the region. As Asia is the home for more than 90% of world buffalo population, they are also one of the largest emitters of greenhouse gasses. Eucalyptus (Eucalyptus sp.) leaves are rich sources of naturally occurring essential oils and phenolic compounds, which could modulate rumen fermentation through mitigation of methanogenesis and nitrogen excretion along with stimulation of immune system and production performances of animals. Therefore, the present study investigated the impact of dietary inclusion of eucalyptus (Eucalyptus citriodora) leaf meal (ELM) on voluntary feed intake, rumen functions, methane emission, nutrient utilization, milk yield and fatty acids profile, and immune response in lactating buffalo (Bubalus bubalis). An in vitro experiment conducted with graded dose (10-40 g/kg) inclusion of ELM into the total mixed ration to select ideal level for feeding to lactating buffaloes, an improvement (P < 0.05) in feed degradability (IVDMD), microbial biomass and ruminal volatile fatty acids concentration with reduced (P < 0.05) methane and ammonia-N production were evidenced when ELM was added at 10-20 g/kg DM, beyond which negative effects on rumen fermentation were pronounced. An in vivo experimentation was conducted with sixteen Murrah (Bubalus bubalis) buffaloes of mean live weight, 544.23 ± 10.02 kg; parity, 2-4 at initial stage (~60 days) of lactation with average milk yield of 11.43 ± 1.32 kg and were divided into two groups (CON, ELM) of eight each in a completely randomized design. All the animals were kept individually on wheat straw-based diet with required quantity of concentrate mixture and green fodder. The control group buffaloes were fed a total mixed ration; however, the treatment group (ELM) was supplemented with 10 g/kg DM diet of dry grounded eucalyptus (Eucalyptus citriodora) leaves by mixing with the concentrate mixture. The feeding experiment was conducted for 120 days, including 15 days for adaptation to the experimental diets and 105 days for data recording. The nutrient digestibility (DM, OM, CP, and EE) was improved (P < 0.05) without affecting feed intake (P > 0.05) and fiber digestibility (NDF and ADF) in ELM supplemented buffaloes. Increased (P < 0.05) milk production and rumenic acid concentration (cis 9 trans 11 C18:2 CLA) were demonstrated with comparable (P > 0.05) milk composition and major fatty acids profile of milk in the supplemented buffaloes. Dietary inclusion of ELM reduced (P < 0.05) enteric methane production and fecal excretion of nitrogen. The health status of buffaloes fed ELM improved throughout the experimental period was improved by enhancing cell mediated (P = 0.09) and humoral (P < 0.01) immune responses without affecting (P > 0.05) major blood metabolites. The study described feeding ELM at 10 g/kg diet to lactating Murrah buffaloes as a natural source of phenols and essential oils to increase milk production and CLA content, reduce methane and nitrogen emissions, and improve health status. Thus, feeding of ELM could be beneficial for climate smart buffalo production system for enhancing milk production with lesser impact on environment., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

During triacylglycerol synthesis, the acylglycerol-3-phosphate acyltransferase (AGPAT) family catalyzes the conversion of lysophosphatidic acid to phosphatidic acid and the acylation of sn-2 fatty acids. However, the catalytic activity of different AGPAT members is different. Therefore, this study aimed to investigate the mechanism through which different AGPAT s affect the efficiency of TAG synthesis and fatty acid composition. The conservation of amino acid sequences and protein domains of the AGPAT family was analyzed, and the functions of AGPAT1 , AGPAT3 , and AGPAT4 genes in buffalo mammary epithelial cells (BMECs) were studied using RNA interference and gene overexpression. Prediction of the protein tertiary structure of the AGPAT family demonstrated that four conservative motifs (motif1, motif2, motif3, and motif6) formed a hydrophobic pocket in AGPAT proteins, except AGPAT6. According to cytological studies, AGPAT1 , AGPAT3 , and AGPAT4 were found to promote the synthesis and fatty acid compositions of triacylglycerol, especially UFA compositions of triacylglycerol, by regulating ACSL1 , FASN , GPAM , DGAT2 , and PPARG gene expression. This study provides new insights into the role of different AGPAT gene family members involved in TAG synthesis, and a reference for improving the fatty acid composition of milk.

The purpose of this study was to explore the effects of Rice straw and King grass on apparent digestibility, ruminal bacterial, and fungus composition in buffaloes. Three ruminal fistulated buffaloes were used in a 3 × 2 Latin square design. The dietary treatments were king grass and straw hay. Experimental animals were kept in individual pens and concentrate was offered at 1 kg/d while roughage was fed ad libitum . Each period lasted for 15d, with the first 12d for an adaptation period, followed by a 3-day formal trial period. King grass has higher digestibility of protein. Rice straw has higher digestibility to cellulose. The results showed that when buffaloes were fed king grass and straw, Bacteroidetes were dominant in the rumen normal flora, but firmicutes were not. In addition, the results of this experiment suggest that increasing protein content in diets may be beneficial to increase the relative abundance of Proteobacteria . Similarly, higher dietary fiber content may be beneficial for increasing relative abundance of Prevotella and Staphylococcus . The dominant fungi in ruminal fluid 2 h after ingestion were aerobic fungi. These aerobic fungi most likely entered the rumen with food. Whether and how long aerobic fungi can survive in the rumen needs more research.

Dietary mix and host species have both been shown to have a significant impact on rumen microbial diversity, enteric methane emission and animal performance. The goal of this study was to see how the roughage concentrate ratio 70:30 (Low concentrate; LC) vs 40:60 (High concentrate; HC) and the host species crossbred cattle vs buffalo affected rumen microbial diversity, enteric methane emissions and nutrient utilization. Dry matter intake (kg/d) and dry matter percent digestibility were considerably ( p  < 0.05) higher in the HC ration and buffalo compared to LC ration and crossbred cattle, respectively. Both dietary mix and host species had a substantial ( p  < 0.05) impact on intake of various nutrients, including organic matter (OM), crude protein (CP), ether extract (EE), neutral detergent fiber (NDF), and acid detergent fiber (ADF). Increased concentrate proportion in the ration improved nitrogen balance, resulting in increased average daily gain and considerably reduced methane (g/d) output ( p  < 0.05). Furthermore, 16S rRNA genes were sequenced using Oxford Nanopore Technology (ONT) and subsequently annotated using the Centrifuge workflow to uncover ruminal bacterial diversity. Firmicutes was considerably ( p  < 0.01) greater in the LC diet, whereas, Bacteroidetes was higher in the HC ration. Genus Prevotella dominated all rumen samples, and buffalo fed LC ration had significantly ( p  < 0.01) higher Oscillospira abundance. At the species level, simple sugar-utilizing bacteria such as Prevotella spp. and Selenomonas ruminantium predominated in the crossbred cattle, but fibrolytic bacteria such as Oscillospira guilliermondii were statistically ( p  < 0.01) more abundant in the buffalo. Overall, dietary mix and host species have both been shown to have a significant impact on rumen microbial diversity, enteric methane emission and animal performance, however, host species remained a major driving force to change ruminal community composition as compared to roughage concentrate ratio under similar environmental conditions.

Granulosa cells (GCs) synthesize estrogens needed for follicular growth. However, the effects of hypoxia on steroidogenesis in buffalo GCs remain unclear. In this study, the impacts of hypoxic conditions (5% oxygen) on estrogen synthesis in buffalo GCs were examined. The results showed that hypoxia improved both the expression levels of estrogen synthesis-related genes (CYP11A1, CYP19A1, and 3β-HSD) and the secretion levels of estradiol in buffalo GCs. Hypoxic conditions promoted the sensitivity of buffalo GCs to FSH. Furthermore, inhibition of cAMP/PKA signaling pathway (H89, a cAMP/PKA signaling pathway inhibitor) reduced both the expression levels of estrogen synthesis-related genes (CYP11A1, CYP19A1, and 3β-HSD) and the secretion levels of estradiol in hypoxia-cultured buffalo GCs. Besides, inhibition of cAMP/PKA signaling pathway lowered the responsiveness of buffalo GCs to FSH under hypoxic conditions. The present study indicated that hypoxia enhanced the steroidogenic competence of buffalo GCs principal by affecting cAMP/PKA signaling pathway and subsequent sensitivity of GCs to FSH., Competing Interests: Declaration of competing interest None of the authors have any conflict of interest to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

In pregnant animals, communication between the mother and conceptus occurs via extracellular vesicles (EVs) that carry several biomolecules such as nucleic acids (miRNAs, mRNAs), proteins, and lipids. At the time of implantation, the endometrium undergoes several morphological and physiological changes, such as angiogenesis, apoptosis, and cell proliferation regulation at the implantation site, to attain a receptive state. This study was conducted to detect pregnancy-specific miRNAs derived from extracellular vesicles in the systemic circulation of Bubalus bubalis (water buffalo) and to assess their functional significance in the modulation of endometrial primary cells. The extracellular vesicles were isolated from the blood plasma using a precipitation-based method and further characterized by various methods such as Differential light scattering, Nanoparticle tracking assay, Western blot, and transmission electron microscopy. The relative expression of the selected extracellular vesicles associated miRNAs (EV-miRNA) at different intervals (days 15, 19, 25, and 30) post artificial insemination (AI) was analyzed using RT-qPCR, and expression of miR-195-5p was found to be significantly higher (P < 0.01) in pregnant animals on day 19 post AI (implantation window) as compared to day 15 post AI. The elevated expression might indicate the involvement of this miRNA in the maternal-conceptus cross-talk occurring during the implantation period. The KEGG pathway enrichment and Gene Ontology analyses of the miR-195-5p target genes revealed that these were mostly involved in the PI3-Akt, MAPK, cell cycle, ubiquitin-mediated proteolysis, and mTOR signaling pathways, which are related to the regulation of cell proliferation. Transfecting the in vitro cultured cells with miR-195-5p mimic significantly suppressed (P < 0.05) the expression of its target genes such as YWHAQ, CDC27, AKT-3, FGF-7, MAPK8, SGK1, VEGFA, CACAND1, CUL2, MKNK1, and CACAN2D1. Furthermore, the downregulation of the miR-195-5p target genes was positively correlated with a significant increase in the apoptotic rate and a decrease in the proliferation. In conclusion, the current findings provide vital information on the presence of EV miR-195-5p in maternal circulation during the implantation window indicating its important role in the modulation of buffalo endometrium epithelial cells via promoting cell death. Altogether, the milieu of miR-195-5p may serve as a novel and potential molecular factor facilitating the implantation of the early embryo during the establishment of pregnancy in buffaloes. Thus, miR-195-5p may be identified as a unique circulatory EV biomarker related to establishing pregnancy in buffaloes as early as day 19 post-AI., (© 2023. Springer Nature Limited.)

Glycosylation of milk whey proteins, specifically the presence of sialic acid-containing glycan residues, causes functional changes in these proteins. This study aimed to analyze the N-glycome of milk whey glycoproteins from various milk sources using a linkage-specific ethyl esterification approach with MALDI-MS (matrix-assisted laser desorption/ionization-mass spectrometry). The results showed that the N-glycan profiles of bovine and buffalo whey mostly overlapped. Acetylated N-glycans were only detected in donkey milk whey at a rate of 16.06%. a2,6-linked N-Acetylneuraminic acid (a2,6-linked NeuAc, E) was found to be the predominant sialylation type in human milk whey (65.16%). The amount of a2,6-linked NeuAc in bovine, buffalo, goat, and donkey whey glycoproteomes was 42.33%, 44.16%, 39.00%, and 34.86%, respectively. The relative abundances of a2,6-linked N-Glycolylneuraminic acid (a2,6-linked NeuGc, Ge) in bovine, buffalo, goat, and donkey whey were 7.52%, 5.41%, 28.24%, and 17.31%, respectively. Goat whey exhibited the highest amount of a2,3-linked N-Glycolylneuraminic acid (a2,3-linked NeuGc, Gl, 8.62%), while bovine and donkey whey contained only 2.14% and 1.11%, respectively., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Sperm membrane glycan-binding proteins (lectins) interact with the counterpart glycans in the oviduct, oocytes, and vice-versa. It has already been well known that specific glycans are present on oviductal epithelium and zona pellucida (ZP) in different mammalian species. Some of these glycans are necessary for oviductal sperm reservoir formation and gamete recognition. The specific binding phenomenon of lectin-glycans is one of the vital factors for successful fertilization in mammals. We hypothesized that buffalo sperm membrane glycan-binding proteins have specific glycan targets in the oviduct and ZP supporting the fertilization event. In the present investigation, sperm membrane proteins were extracted and assessed for their binding capacity with glycans using a high-throughput glycan microarray. The most promising glycan binding signals were evaluated to confirm the sperm putative receptors for glycan targets in the oviductal epithelial cells (OEC) and on ZP using an in-vitro competitive binding inhibition assay. Based on an array of 100 glycans, we found that N-acetyllactosamine (LacNAc), Lewis-a trisaccharide, 3'-sialyllactosamine and LacdiNAc were the most promising glycans and selected for further in-vitro validation. We established an inhibitory concentration of 12 mM Lewis-a trisaccharide and 10 μg/ml Lotus tetragonolobus (LTL) lectin for the sperm-OEC binding interaction, indicating its specificity and sensitivity. We observed that 3 mM 3'-sialyllactosamine, and LacdiNAc were the most competitive inhibitory concentration in sperm-ZP binding, suggesting a specific and abundance-dependent binding affinity. The competitive binding affinity of Maackia amurensis (MAA) lectin with Neu5Ac(α2-3)Gal(β1-4)GlcNAc further supports the abundance of 3'-sialyllactosamine on ZP responsible for sperm binding. Our findings develop the strong evidence on buffalo sperm putative receptors underlying their locking specificities with Lewis-a trisaccharide in oviduct and 3'-sialyllactosamine on ZP. The functional interaction of buffalo sperm lectins with the target glycans in OEC and ZP appears to be accomplished in an abundance-dependent manner, facilitating the fertilization event in buffaloes., Competing Interests: Declaration of competing interest All authors declare no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Being highly fragmented and low in concentration, isolation of good quality RNA from sperm cells is a big challenge. Attempts have been made to evaluate various sperm RNA isolation methods from purified buffalo bull sperm cells., Methods: Both, non-membrane and membrane-based methods have been evaluated for isolating RNA from Murrah buffalo sperms and compared for their respective efficacies. The traditional TRIzol, TRIzol-heat lysed (H-TRIzol) and cocktail of TCEP-RLT lysis buffer (Qiagen RNeasy mini kit)-TRIzol (C-TRIzol) based isopropanol isolation methods have been evaluated., Results: H-TRIzol yielded best results among conventional methods. The combined T-RLT RNA isolation protocol yielded best quality and quantity compared to other membrane-based methods, due to high lytic property of cocktail of lysis reagents, necessary for complete breakdown of sperm membrane and RNA binding membrane for RNA isolation. Combined lysis performed by treatment with RLT-T and T-RLT differing in order of reagents used were also evaluated. T-RLT combination giving better results compared to RLT-T due to high gDNA contamination and membrane clogging in later protocol steps., Conclusion: Overall, in terms of total RNA quantity and quality per million spermatozoa, the heat-lysed TRIzol method (H-TRIzol) performs best among RNA separation techniques employed and is also quite easy to perform. This comparative evaluation of sperm RNA isolation protocols can be useful in deciding the best protocol for isolation of good quality and high concentration sperm RNA from buffalo semen, for transcriptome and other downstream studies., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

The current study focuses on molecular cloning, expression and structural characterization of growth hormone-receptor (GHR) and its extracellular domain as growth hormone binding protein (GHBP) from the liver of Nili-Ravi buffalo (Bubalus bubalis; Bb). RNA was isolated, genes were amplified by reverse transcriptase-polymerase chain reaction and sequence was characterized. The BbGHR sequence showed three amino acid variations in the extracellular domain when compared with Indian BbGHR. For the production of full length BbGHR and BbGHBP in Escherichia coli (E. coli) BL21 (RIPL) Codon Plus, expression plasmids were constructed under the control of T7lac promoter and isopropyl β-D thiogalactopyranoside was used as an inducer. BbGHR and BbGHBP were expressed as inclusion bodies at ~ 40% and > 30% of the total E. coli proteins, respectively. The BbGHBP was solubilized and refolded by dilution method using cysteine-cystine redox potential. The recombinant BbGHBP was purified and biological activity was checked on HeLa cell lines showing increase cell proliferation in the presence of ovine GH (oGH), hence justifying the increase in the half-life of GH in the presence of BbGHBP. For the molecular interactions of oGH-BbGHBP multiple docking programs were employed to explore the subsequent interactions which showed high binding affinity and presence of large number of hydrogen bonds. Molecular Dynamics studies performed to examine the stability of proteins and exhibited stable structures along with favorable molecular interactions. This study has described the sequence characterization of BbGHR in Nili-Ravi buffaloes and hence provided the basis for the assessment of GH-GHR binding in other Bovidae species., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Cryopreservation commonly decreases the cellular functionality and post-thaw viability of cells. Reactive oxygen species (ROS) generated during cryopreservation degrade mitochondrial activity and promote the release of cytochrome C which activates caspases required for apoptosis. Antioxidants have the potential to improve the recovery efficiency of cells by reducing ROS production and maintaining mitochondrial membrane potential (MMP). The present study was conducted to explore the role of MitoQ, a derivative of coenzyme Q10 on cryopreserved fibroblasts derived from buffalo skin. To achieve our goal, buffalo skin fibroblasts were treated with varying concentrations of MitoQ (0, 0.1, 0.5, 1, 2, and 10 μM) for 24, 48, and 72 h. The MMP, ROS generation, cell viability was measured by flow cytometry. Furthermore, expression of genes related to mitochondrial oxidative stress (NRF2, GPX, and SOD), apoptosis (BAK and caspase 3) and cell proliferation (AKT) were also assessed. The results showed that over a period of 72 h lower concentrations of MitoQ (0.1-0.5 μM) decrease the ROS production, improves MMP and cell viability whilst the high concentration of MitoQ (2-10 μM) increased the oxidative damage to the cells. Taken together, our study provide important insights into the novel role of MitoQ in cryopreserved buffalo skin fibroblasts. In conclusion, we demonstrated the dose-dependent functional role of MitoQ on cryopreserved fibroblasts for improving post-thaw cell viability and cellular function., Competing Interests: Competing interests The authors declare that they have no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Cellular metabolism is most invariant process, occurring in all living organisms, which involves mitochondrial proteins from both nuclear and mitochondrial genomes. The mitochondrial DNA (mtDNA) copy number, protein-coding genes (mtPCGs) expression, and activity vary between various tissues to fulfill specific energy demands across the tissues., Methods and Results: In present study, we investigated the OXPHOS complexes and citrate synthase activity in isolated mitochondria from various tissues of freshly slaughtered buffaloes (n = 3). Further, the evaluation of tissue-specific diversity based on the quantification of mtDNA copy numbers was performed and also comprised an expression study of 13 mtPCGs. We found that the functional activity of individual OXPHOS complex I was significantly higher in the liver compared to muscle and brain. Additionally, OXPHOS complex III and V activities was observed significantly higher levels in liver compared to heart, ovary, and brain. Similarly, CS-specific activity differs between tissues, with the ovary, kidney, and liver having significantly greater. Furthermore, we revealed the mtDNA copy number was strictly tissue-specific, with muscle and brain tissues exhibiting the highest levels. Among 13 PCGs expression analyses, mRNA abundances in all genes were differentially expressed among the different tissue., Conclusions: Overall, our results indicate the existence of a tissue-specific variation in mitochondrial activity, bioenergetics, and mtPCGs expression among various types of buffalo tissues. This study serves as a critical first stage in gathering vital comparable data about the physiological function of mitochondria in energy metabolism in distinct tissues, laying the groundwork for future mitochondrial based diagnosis and research., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

This study is a comparative analysis of the biochemical, hormonal, and mineral compositions of follicular fluid in preovulatory and cystic follicles of water buffalo ( Bubalus bubalis ). In total, reproductive tracts from 215 buffalo along with intact ovaries were collected randomly from an abattoir. The incidence of cystic conditions found in this study was 3.72% (8/215), involving the right ovary in 62.5% of instances and the left ovary in 37.5% of instances during the non-breeding season. Follicular fluid was aspirated from preovulatory follicles (12-15 mm diameter, oestrogen-active, follicular phase or stage IV corpus luteum on one of the two ovaries, n = 10) and cystic follicles (at least 20 mm diameter, no corpus luteum on any one of the two ovaries, n = 8). The follicular fluid samples were assayed for biochemical components (uric acid, creatinine, blood urea nitrogen, cholesterol, total protein, glucose, ascorbic acid, and alkaline phosphatase), hormones (progesterone, estradiol, and insulin), and minerals (calcium, magnesium, phosphorus, copper, zinc, and cobalt). Cystic follicles had greater ( P < 0.05) concentrations of creatinine, blood urea nitrogen, cholesterol, progesterone, copper, zinc, and cobalt, and lesser ( P < 0.05) concentrations of uric acid, glucose, ascorbic acid, estradiol, insulin, calcium, magnesium, and phosphorus compared with preovulatory follicles. These results indicated the marked differences in follicular fluid composition between preovulatory and cystic follicles in buffalo. Some of the changes were indicative of oxidative stress and disturbed steroidogenesis, two important mechanisms shown to be associated with cystic ovarian disease in various species. Further studies are warranted to investigate whether these differences are directly or indirectly involved in the formation of cystic follicles or are mere manifestations of the condition.

Metabolic disorders as ketosis are manifestations of the animal's inability to manage the increase in energy requirement during early lactation. Generally, buffaloes show a different response to higher metabolic demands than other ruminants with a lower incidence of metabolic problems, although ketosis is one of the major diseases that may decrease the productivity in buffaloes. The aim of this study was to characterize the metabolic profile of Mediterranean buffaloes (MB) associated with 2 different levels of β-hydroxybutyrate (BHB). Sixty-two MB within 50 days in milk (DIM) were enrolled and divided into 2 groups according to serum BHB concentration: healthy group (37 MB; BHB <0.70 mmol/L; body condition score: 5.00; parity: 3.78; and DIM: 30.70) and group at risk of hyperketonemia (25 MB; BHB ≥0.70 mmol/L; body condition score: 4.50; parity: 3.76; and DIM: 33.20). The statistical analysis was conducted by one-way ANOVA and unpaired 2-sample Wilcoxon tests. Fifty-seven metabolites were identified and among them, 12 were significant or tended to be significant. These metabolites were related to different metabolic changes such as mobilization of body resources, ruminal fermentations, urea cycle, thyroid hormone synthesis, inflammation, and oxidative stress status. These findings are suggestive of metabolic changes related to subclinical ketosis status that should be further investigated to better characterize this disease in the MB., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Background: The orbital glands, viz . lacrimal gland, superficial and deep gland of third eyelid (LG, SGT and HG), are important for normal eye functions. These glands have different functions in various animals. The information about the enzyme histochemical nature of prenatal orbital glands in Indian buffalo seems to be unavailable. Therefore, the study was planned on orbital glands of six full term recently died fetuses from animals with dystocia., Methods: The frozen sections of all these glands were subjected to standard localization protocols for Alkaline Phosphatase (AKPase), Glucose 6 phosphatase (G-6-Pase), Lactate dehydrogenase (LDH), Succinate dehydrogenase (SDH), Glucose 6 phosphate dehydrogenase (G-6-PD), Nicotinamide Adenine Dinucleotide Hydrogen Diaphorase (NADHD), Nicotinamide Adenine Dinucleotide Phosphate Hydrogen diaphorase (NADPHD), Dihydroxy phenylalanine oxidase (DOPA-O), Tyrosinase, non-specific esterase (NSE) and Carbonic anhydrase (CAse)., Results: The results revealed a mixed spectrum of reaction for the above enzymes in LG, SGT and HG which ranged from moderate (for LDH in SGT) to intense (for most of the enzymes in all three glands). However, DOPA-O, Tyrosinase and CAse did not show any reaction. From the present study, it can be postulated that the orbital glands of fetus have a high activity of metabolism as it has many developmental and functional activities which were mediated with the higher activity of the enzymes involved., Competing Interests: Mahendra Pratap Singh Tomar is an Academic Editor for PeerJ., (© 2023 Tomar and Bansal.)

Adult male and female Murrah buffalo fibroblast cells were used as donors for the production of embryos using handmade cloning. Both donor cells and reconstructed embryos were treated with 50 nM trichostatin-A (TSA) and 7.5 nM 5-aza-2'-deoxycytidine (5-aza-dC). The blastocyst rate of both treated male (40.1% ± 2.05) and female (37.0% ± 0.83) embryos was significantly lower than in untreated control males (49.7% ± 3.80) and females (47.2% ± 2.44) but their apoptotic index was lower (male, control: 5.90 ± 0.48; treated: 4.96 ± 0.31): (female, control: 8.11 ± 0.67; treated: 6.65 ± 0.43) and epigenetic status in terms of global acetylation and methylation of histone was significantly improved. The expression level of hypoxanthine-guanine phosphoribosyltransferase ( HPRT) was higher ( P < 0.05) and that of PGK , G6PD , OCT 4 , IFN-tau and CASPASE3 was significantly lower ( P < 0.05) in treated male blastocyst than control and the expression levels of DNMT1 , IGF1R and BCL-XL were not significantly different between the two groups. In the female embryos, the relative mRNA abundance of OCT4 was significantly higher ( P < 0.05), and that of XIST and CASPASE3 was significantly lower ( P < 0.05) in the epigenetic modifier-treated group compared with that of the control group, whereas the expression levels of HPRT , PGK , G6PD , DNMT1 , IFN-tau , IGF1R and BCL-XL were not significantly different between the two groups. In both embryos, a similar effect of treatment was observed on genes related to growth and development, but the effect on the expression of X-linked genes varied. These results indicate that not all X-linked genes respond to TSA and 5-aza-dC treatment in the same manner.

The capacity of buffalo milk proteins to release bioactive peptides was evaluated and novel bioactive peptides were identified. The sequential similarity between buffalo milk proteins and their cow counterparts was analysed. Buffalo milk proteins were simulated to yield theoretical peptides via in silico proteolysis. The potential of selected proteins to release specific bioactive peptides was evaluated by the A value obtained from the BIOPEP-UWM database (Minkiewicz et al. in Int J Mol Sci 20(23):5978, 2019). Buffalo milk protein is a suitable precursor to produce bioactive peptides, particularly dipeptidyl peptidase IV (DPP-IV) and angiotensin I-converting enzyme (ACE) inhibitory peptides. Two novel ACE inhibitory peptides (KPW and RGP) and four potential DPP-IV inhibitory peptides (RGP, KPW, FPK and KFTW) derived from in silico proteolysis of buffalo milk proteins were screened using different integrated bioinformatic approaches (PeptideRanker, Innovagen, peptide-cutter and molecular docking). The Lineweaver-Burk plots showed that KPW (IC 50  = 136.28 ± 10.77 μM) and RGP (104.72 ± 8.37 μM) acted as a competitive inhibitor against ACE. Similarly, KFTW (IC 50  = 873.92 ± 32.89 μM) was also a competitive inhibitor of DPP-IV, while KPW and FPK (82.52 ± 10.37 and 126.57 ± 8.45 μM, respectively) were mixed-type inhibitors. It should be emphasized that this study does not involve any clinical trial., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Buffalo holds an excellent potential for beef production, and circRNA plays an important role in regulating myogenesis. However, the regulatory mechanism of circRNAs during buffalo skeletal muscle development has not been fully explored. In this study, circRNA expression profiles during the proliferation and differentiation stages of buffalo myoblasts were analysed by RNA-seq. Here, a total of 3,142 circRNAs candidates were identified, and 110 of them were found to be differentially expressed in the proliferation and differentiation stages of buffalo myoblast libraries. We focused on a 347 nt circRNA subsequently named circCLTH. It consists of three exons and is expressed specifically in muscle tissues. It is a highly conserved non-coding RNA with about 95% homology to both the human and the mouse circRNAs. The results of cell experiments and RNA pull-down assays indicated that circCLTH may capture PLEC protein, promote the proliferation and differentiation of myoblasts as well as inhibit apoptosis. Overexpression of circCLTH in vivo suggests that circCLTH is involved in the stimulation of skeletal muscle regeneration. In conclusion, we identified a novel noncoding regulator, circCLTH, that promotes proliferation and differentiation of myoblasts and skeletal muscles.

Semen cryopreservation is used for the propagation of variety among species and domestic breeding. Mitochondria are implicated in sperm freezability, and their proteins are prone to succinylation, but the relationship between sperm freezability and mitochondrial protein succinylation is unclear. In this study, six bulls were classified as having good or poor freezability ejaculates (GFE or PFE, each 3 bulls). The fresh sperm mitochondrial membrane potential (MMP) and pan succinylation level of the two groups were first detected. Then the lysine succinylome and fatty acid content of the two groups were analyzed using label-free LC-MS/MS and GC-MS/MS in multiple reaction monitoring (MRM) modes, respectively. The results indicated that the GFE sperm had significantly higher MMPs than the PFE group (p < 0.05). A total of 1393 succinylation sites corresponding to 426 proteins were assessed and 5 succinylated peptides of the GFE group were markedly upregulated, while 3 were significantly downregulated (FC > 2.0 - < 0.5 and p-value < 0.05) when compared to the PFE group. Forty-six succinylated proteins were identified to have consistent presence/absence expression. The upregulated succinylated proteins in the GFE sperm were enriched in lipid metabolic processes. A total of 31 fatty acids were further subjected to quantitative analysis of which 23 including arachidic (C20:0), linolenic (C18:3n3), and docosahexaenoic acids (C22:6n3) were decreased in GFE sperm when compared with PFE (p < 0.05). These results suggest that lysine succinylation can potentially influence the sperm freezability of Mediterranean buffaloes through mitochondrial lipid metabolism. This novel study provides our understanding of sperm succinylation and the molecular basis for the mechanism of sperm freezability., (© 2022 Federation of American Societies for Experimental Biology.)

DNA synthesis and methylations are crucial during pre-implantation embryonic development, and are mediated by one-carbon metabolism of folates. Folates, transported into the cells via folate receptors (FOLR1 and FOLR2) and carriers (SLC19A1), are metabolized by various enzymes involved in folate-methionine cycle. However, the variations in temporal expression of folate transporters and folate-methionine cycle enzymes during pre-implantation embryo development is obscure. Thus, the present study aimed to investigate the differential expression of the genes for folate transporters and folate-methionine cycle enzymes. We also examined the expression of folate transport proteins in different pre-implantation development stages. Immature buffalo oocytes were matured in maturation medium followed by in vitro fertilization and culture at standard culture conditions. The temporal pattern of gene expression in buffalo, when compared to previous studies, indicated an inter-specific variation. The transcripts of some enzymes and folate transporters were significantly upregulated after zygotic genome activation. The transcripts as well as proteins for FOLR1, FOLR2 and SLC19A1 were present in oocytes and all the pre-implantation embryo stages. FOLR1 was present in the nuclei of different stages of developing embryos but not in the metaphase (MII) oocytes. As a result, the present study advocates the existence of active folate transport in buffalo oocytes and pre-implantation embryos. The data provided by the analysis of differential gene expression of folate transporters and metabolic enzymes would likely contribute to a better understanding of the role of folates in embryo development as well as advancements in assisted reproductive technologies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The relationship between dietary constituents and the onset and prevention of colorectal cancer (CRC) is constantly growing. Recently, the antineoplastic profiles of milk and whey from Mediterranean buffalo (Bubalus bubalis) have been brought to attention. However, to date, compared to cow milk, the potential health benefits of buffalo milk exosome-miRNA are still little explored. In the present study, we profiled the exosomal miRNA from buffalo milk and investigated the possible anticancer effects in CRC cells, HCT116, and HT-29. Results indicated that buffalo milk exosomes contained higher levels of miR-27b, miR-15b, and miR-148a compared to cow milk. Mimic miR-27b transfection in CRC cells induced higher cytotoxic effects (p < 0.01) compared to miR-15b and miR-148a. Moreover, miR-27b overexpression in HCT116 and HT-29 cells (miR-27b+) induced apoptosis, mitochondrial reactive oxygen species (ROS), and lysosome accumulation. Exposure of miR-27b+ cells to the bioactive 3kDa milk extract aggravated the apoptosis rate (p < 0.01), mitochondrial stress (p < 0.01), and advanced endoplasmic reticulum (ER) stress (p < 0.01), via PERK/IRE1/XBP1 and CHOP protein modulation (p < 0.01). Moreover, GSK2606414, the ER-inhibitor (ER-i), decreased the apoptosis phenomenon and XBP1 and CHOP modulation in miR-27b+ cells treated with milk (p < 0.01 vs. miR-27b++Milk), suggesting the ER stress as a cell-death-aggravating mechanism. These results support the in vitro anticancer activity of 3kDa milk extract and unveil the contribution of miR-27b in the promising beneficial effect of buffalo milk in CRC prevention.

Vulture declines in South Asia were caused by accidental poisoning by the veterinary non-steroidal anti-inflammatory drug (NSAID) diclofenac. Although veterinary use of diclofenac has been banned, other vulture-toxic NSAIDs are legally available, including aceclofenac, which has been shown to metabolise into diclofenac in domestic cattle. We gave nine domestic water buffalo the recommended dose of aceclofenac (2 mg kg -1 body weight), collected blood at intervals up to 48 h, and carried out a pharmacokinetic analysis of aceclofenac and its metabolite diclofenac in plasma. Aceclofenac was rapidly converted to diclofenac, and was barely detectable in plasma at any sampling time. Diclofenac was present within 20 min, and peaked 4-8 h after dosing. Aceclofenac is a prodrug of diclofenac, and behaves similarly in domestic water buffalo as it did in domestic cattle, posing the same risk to vultures. We recommend an immediate ban on the veterinary use of aceclofenac across vulture-range countries., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Milk-derived protein hydrolysates have generated a great deal of interest recently due to their numerous beneficial health effects. However, there are few comparative studies on protein hydrolysates from different dairy species, their production, characterization, and bioactivity. In the present study, skimmed milk from both major and minor dairy species was hydrolyzed with alcalase, and its protein profiles were studied using tricine polyacrylamide gel electrophoresis and reverse phase-high protein liquid chromatography. The antioxidant and in vitro immunostimulatory properties were determined., Results: Iron chelation activity was highest in hydrolysates of whey (25.00 ± 0.32 mmol L -1 ), casein (25.14 ± 0.34 mmol L -1 ), colostrum (24.52 ± 0.28 mmol L -1 ), and skimmed cattle milk (24.21 ± 0.26 mmol L -1 ). α,α-Diphenyl-β-picrylhydrazyl scavenging and 2,2'-azobis(2-amidino-propane) dihydrochloride activity was lowest in skimmed donkey milk protein hydrolysates (MPHs) (IC 50 : 5.37 ± 0.05 mg mL -1 and 151.59 ± 2.1 mg mL -1 ). Production of nitric oxide and phagocytosis activity in RAW 264.7 (murine macrophage cell line) was significantly higher among whey and buffalo skimmed milk protein hydrolysate-treated groups as compared with the untreated group. The incorporation of whey protein hydrolysate and skimmed buffalo milk protein hydrolysate were sensorially acceptable at 10% level in beverage mix., Conclusion: This study comparatively evaluates the antioxidative and immunomodulatory properties of different skimmed MPHs and their potential applications as ingredients in pediatric, geriatric, and other health-promoting foods. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Relaxin family peptides significantly regulate reproduction, nutrient metabolism, and immune response in mammals. The present study aimed to identify and characterize the relaxin family peptides in cattle and buffalo at the genome level. The genomic and proteomic sequences of cattle, buffalo, goat, sheep, horse, and camel were accessed through the NCBI database, and BLAST was performed. We identified four relaxin peptides genes ( RLN3 , INSL3 , INSL5 , and INSL6 ) in Bos taurus , whereas three relaxin genes ( RLN3 , INSL3 , and INSL6 ) in Bubalus bubalis . Evolutionary analysis revealed the conserved nature of relaxin family peptides in buffalo and cattle. Physicochemical properties revealed that relaxin proteins were thermostable, hydrophilic, and basic peptides except for INSL5 which was an acidic peptide. Three nonsynonymous mutations (two in RLN3 at positions A16 > T and P29 > A, and one in INSL6 at position R32 > Q) in Bos taurus , whereas two nonsynonymous mutations (one in RLN3 at positions G105 > w and one in INSL3 at position G22 > R) in Bubalus bubalis , were identified. INSL3 had one indel (insertion) at position 55 in Bos taurus . Gene duplication analysis revealed predominantly segmental duplications ( INSL5/RLN3 and INSL6/INSL3 gene pairs) that helped expand this gene family, whereas Bubalus bubalis showed primarily tandem duplication ( INSL3/RLN3 ). Our study concluded that relaxin family peptides remained conserved during the evolution, and gene duplications might help to adapt and enrich specific functions like reproduction, nutrient metabolism, and immune response. Further, the nonsynonymous mutations identified potentially affect these functions in buffalo., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2022 Muhammad Saif-ur Rehman et al.)

Dairy proteins in the diet are beneficial for the growth of probiotics; however, what is unknown is the gut-mediated immune responses under protein energy malnutrition (PEM) and if dairy protein hydrolysates can be effective as dietary interventions. This study compares the composition of the gut microbiota of rats with moderate protein deficiency (M.PEM) and severe protein deficiency (S.PEM) induced by feeding 5% and 1% hypoprotein diets, followed by replenishment with buffalo and whey protein hydrolysates. Fecal samples were collected, and the composition of the gut bacteria was analyzed by whole genome sequencing using long-read sequencing. Gene expression studies of the immunomodulatory cytokines involved and quantification of sIgA were carried out. IL-6 and IFN-γ were downregulated by about 0.17 ± 0.06 and 0.12 ± 0.10 fold when supplemented with whey protein hydrolysate in SP-RWC rats and by about 0.02 ± 0.06 and 0.35 ± 0.12 fold when using buffalo milk hydrolysate. The percentage of Firmicutes decreased in M.PEM and S.PEM rats (33.57%, 28.83 versus 47.73% of control at 3 weeks) but increased upon protein replenishment for all three protein sources at the end of nine weeks. The percentage of Bacteroidetes increased to 31.03% in S.PEM-induced rats as against 28.17% in control rats. The relative abundance of Lactobacillus sp. decreased in M.PEM and S.PEM rats while it showed the opposite effect upon protein replenishment. Gut microbiota modulated the pathogenesis of PEM differentially based on protein intervention along with a significant increase in the relative abundance of the keystone Lactobacillus genus.

High-quality semen with high viability is critical to improving the in-vitro fertilization efficiency. This study aimed to understand the effect of ambient temperature and humidity on semen quality and seminal plasma biochemical parameters of Mediterranean buffalo in March and July. The metabolites of seminal plasma in two seasons were detected using the UPLC-MS/MS method. The results showed that temperature and humidity index (THI) in March were 66.86 ± 2.98, and 82.94 ± 3.52 in July. Compared with in March, breath frequency, rectal temperature, and heat shock protein 70 expressions of seminal plasma were significantly increased in July ( p <  0.05), motility of sperm was dramatically reduced, and sperm deformity rate was significantly increased ( p <  0.05). Fructose, acid phosphatase and α-glucosidase in seminal plasma were significantly increased ( p <  0.05) in July, while testosterone level was significantly reduced ( p <  0.05). Six different metabolites were found in the two groups, which involved in three metabolic pathways, the tricarboxylic acid cycle, glycerophospholipid, glyoxylic acid and dicarboxylic acid. The above results indicate that the increased ambient temperature has obvious side effects on the semen quality of Mediterranean buffalo, and the compromised quality is associated with the change of metabolites related to male hormone secretion, energy metabolism and fatty acid oxidation.

Mammalian individuals differ in their somatic cell cloning efficiency, but the mechanisms leading to this variation is poorly understood. Here we found that high cloning efficiency buffalo fetal fibroblasts (BFFs) displayed robust energy metabolism, looser chromatin structure, high H3K9 acetylation and low heterochromatin protein 1α (HP1α) expression. High cloning efficiency BFFs had more H3K9ac regions near to the upstream of glycolysis genes by ChIP-seq, and involved more openness loci related to glycolysis genes through ATAC-seq. The expression of these glycolysis genes was also found to be higher in high cloning efficiency BFFs by qRT-PCR. Two key enzymes of glycolysis, PDKs and LDH, were confirmed to be associated with histone acetylation and chromatin openness of BFFs. Treatment of low cloning efficiency BFFs with PS48 (activator of PDK1) resulted in an increase in the intracellular lactate production and H3K9 acetylation, decrease in histone deacetylase activity and HP1α expression, less condensed chromatin structure and more cloning embryos developing to blastocysts. These results indicate that the cloning efficiency of buffalo somatic cells is associated with their glycolytic metabolism and chromatin structure, and can be improved by increasing glycolytic metabolism., (© 2022. Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature.)

Around 60% dairy animals developed moderate to severe hepatic lipidosis at the time of parturition or during early lactation stage. Most of clinician suspect the hepatic lipidosis during above time window only. However, negative energy balance or feeding of high concentrate diet can lead to hepatic lipidosis at any phase of life. The aim of the present study was to evaluate the potential for diagnosis of hepatic lipidosis by means of hemato-biochemical parameters and ultrasonography of the liver at any stage of life. Here, ultrasonographic back fat thickness measurement was correlated with ultrasonographic features of hepatic lipidosis. A total 60 buffaloes were included under the study and sampled for hematological and biochemical parameters. Hematological parameters did not exhibit any significant difference between healthy and hepatic lipidosis-affected buffaloes. Biochemical parameters like beta hydroxy butyric acid, non esterified fatty acid, aspartate amino transferase, gamma glutamyl transferase and alkaline phosphatase revealed a significant increase, while triglyceride, cholesterol, and glucose declined significantly in hepatic lipidosis-affected buffaloes. Total protein, albumin, and total bilirubin levels did not exhibit any significant difference. Based on ultrasonographic findings, the hepatic lipidosis-affected buffaloes were further sub divided into mild, moderate, and severe groups. Portal vein diameter and depth of portal vein were also estimated in current study. Ultrasonographic examination could diagnose 53.33% hepatic lipidosis cases in buffaloes. Among it, 37.50% buffalo had mild hepatic lipidosis, 33.33% had moderate hepatic lipidosis, and 29.16% had severe hepatic lipidosis. Depth of portal vein significantly increased in hepatic lipidosis cases. However, portal vein diameter exhibited a non-significant difference in mild, moderate, and severe groups of hepatic lipidosis. Back fat thickness also revealed a non-significant difference in mild, moderate, and severe hepatic lipidosis. Above study indicate that B mode ultrasonography of the liver can be employed to differentiate various grades of hepatic lipidosis in buffaloes. Biochemical parameters like NEFA, BHBA, AST, GGT, ALP, TG, cholesterol, and glucose can be helpful to screen the hepatic lipidosis at farm level., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

In buffalo (Bubalus bubalis) reproductive seasonality, causing cycles of milk production, is one of the major factors affecting farming profitability. Follicular fluid (FF) contains extracellular vesicles (EVs) playing an important role in modulating oocyte developmental competence and carrying microRNAs (miRNAs) essential for in vitro fertilization outcomes. The aim of this work was to characterize the FF-EVs-miRNA cargo of antral (An) and preovulatory (pO) follicles collected in the breeding (BS) and non-breeding (NBS) seasons, to unravel the molecular causes of the reduced oocyte competence recorded in buffalo during the NBS. In total, 1335 miRNAs (538 known Bos taurus miRNAs, 324 homologous to known miRNAs from other species and 473 new candidate miRNAs) were found. We identified 413 differentially expressed miRNAs (DE-miRNAs) (FDR < 0.05) between An and pO groups. A subset of the most significant DE-miRNAs between An and pO groups targets genes which function is related to the lipid and steroid metabolism, response to glucocorticoid and oestradiol stimulus. Comparison between BS and NBS showed 14 and 12 DE-miRNAs in An-FF-EVs and pO-FF-EVs, which regulate IL6 release and cellular adhesion, respectively. In conclusion, these results demonstrated that the miRNA cargo of buffalo FF-EVs varies in relation to both follicular development and season., (© 2022. The Author(s).)

The epithelial cell is the main basic unit of the udder in which milk synthesis takes place. Curcumin is well known for its antioxidant, anti-apoptotic, and anti- inflammatory properties. The present study was performed to test whether in vitro curcumin supplementation can alleviate the unfavorable impact of hyperthermia on buffalo mammary epithelial cells (BuMECs). The spontaneously immortalized BuMECs were divided into 7 groups (n = 9); 1) unstressed BuMECs (negative control, 37 °C); 2) BuMECs exposed to hyperthermia without curcumin treatment (positive control); 3-7) BuMECs cultured with different concentrations of curcumin (5, 10, 20, 40 and 60 μM), respectively, followed by hyperthermic exposure (42ºC) for 1 h and then returned to 37ºC. Changes in viability (MTT assay), proliferation (BrdU colorimetric immunoassay) and concentrations of antioxidant enzymes, CAT, and SOD (ELISA) of BuMECs were recorded. The gene expression study was performed using qRT-PCR. Lower concentrations of curcumin (5, 10 μM) maintained viability, enhanced proliferation, and content of antioxidant enzymes of heat stressed BuMECs. Curcumin induced thermotolerance and antioxidant status by upregulating the expression of antioxidants genes, anti-apoptotic genes and heat shock proteins in heat stressed BuMECs compared to the positive control group. Besides, curcumin reduced apoptosis and inflammation in BuMECs exposed to hyperthermia by downregulating the expression of genes and transcriptional factors associated with apoptosis and inflammatory immune response. The results reveal the potential roles of curcumin in eliminating the negative impact of hyperthermia on BuMECs by regulating the pathways of apoptosis, inflammation, and oxidative stress., (Copyright © 2022 Elsevier B.V. All rights reserved.)