Hypoxia related genes modulate in similar fashion in skin fibroblast cells of yak (Bos grunniens) adapted to high altitude and native cows (Bos indicus) adapted to tropical climate during hypoxia stress.

The present study was conducted to understand transcriptional response of skin fibroblast of yak (Bos grunniens) and cows of Bos indicus origin to hypoxia stress. Six primary fibroblast cell lines derived from three individuals each of Ladakhi yak (Bos grunniens) and Sahiwal cows (Bos indicus) were exposed to low oxygen concentration for a period of 24 h, 48 h and 72 h. The expression of 10 important genes known to regulate hypoxia response such as HIF1A, VEGFA, EPAS1, ATP1A1, GLUT1, HMOX1, ECE1, TNF-A, GPx and SOD were evaluated in fibroblast cells of Ladakhi yak (LAY-Fb) and Sahiwal cows (SAC-Fb) during pre- and post-hypoxia stress. A panel of 10 reference genes (GAPDH, RPL4, EEF1A1, RPS9, HPRT1, UXT, RPS23, B2M, RPS15, ACTB) were also evaluated for their expression stability to perform accurate normalization. The expression of HIF1A was significantly (p < 0.05) induced in both LAY-Fb (2.29-fold) and SAC-Fb (2.07-fold) after 24 h of hypoxia stress. The angiogenic (VEGFA), metabolic (GLUT1) and antioxidant genes (SOD and GPx) were also induced after 24 h of hypoxia stress. However, EPAS1 and ATP1A1 induced significantly (p < 0.05) after 48 h whereas, ECE1 expression induced significantly (p < 0.05) at 72 h after exposure to hypoxia. The TNF-alpha which is a pro-inflammatory gene induced significantly (p < 0.05) at 24 h in SAC-Fb and at 72 h in LAY-Fb. The induction of hypoxia associated genes indicated the utility of skin derived fibroblast as cellular model to evaluate transcriptome signatures post hypoxia stress in populations adapted to diverse altitudes.

Selection of reference genes for normalizing gene expression data across seasons in spermatozoa of water buffalo (Bubalus bubalis).

Selection of the most stably expressed reference genes is key to monitoring accurate target gene expression across any tissue or cell type. The mRNA in spermatozoa stores valuable information related to changes in spermatogenesis due to variations in environmental conditions, especially during heat stress, which affects various sperm functions. Semen quality in buffalo bulls is significantly influenced by the seasons. In the study, a panel of nine genes was evaluated to identify the most stably expressed internal control gene (ICG) for the normalization of real-time gene expression data generated across various seasons for Murrah buffalo bulls' spermatozoa. Sperm cells were purified from the semen samples collected during different seasons, with temperature-humidity index (THI) ranging from 80.80 ± 1.47 (hot summer) to 55.88 ± 1.98 (winter), using the BoviPure™ gradient purification method. The RNA isolated from the purified spermatozoa fraction was quality checked prior to reverse transcription and subjected to qPCR (quantitative real-time PCR) based expression analysis. An automated 'endoGene' pipeline was employed to apply the geNorm, NormFinder, and BestKeeper algorithms for data analysis. The result indicated that GAPDH and PP1A were the most stably expressed among the gene panel, whereas ATPSF1 and ACTB were the two least stable expressed reference genes. Further, the most suitable ICGs identified were validated by normalization of real time expression data of heat stress and sperm quality genes, HSFY2 and AKAP4, respectively. The genes identified would help in generating the most reliable results for the expression profiling of the genes dictating sperm quality and heat stress cope-up mechanism in buffalo spermatozoa, collected during different seasons.

Genome-wide 5'-C-phosphate-G-3' methylation patterns reveal the effect of heat stress on the altered semen quality in Bubalus bubalis.

Semen production and quality are closely correlated with different environmental factors in bovines, particularly for the buffalo (Bubalus bubalis) bulls reared under tropical and sub-tropical conditions. Factors including DNA methylation patterns, an intricate process in sperm cells, have an impact on the production of quality semen in buffalo bulls under abiotic stress conditions. The present study was conducted to identify DNA methylome signatures for semen quality in Murrah buffalo bulls, acclaimed as a major dairy breed globally, under summer heat stress. Based on semen quality parameters that significantly varied between the two groups over the seasons, the breeding bulls were classified into seasonally affected (SA = 6) and seasonally non-affected (SNA = 6) categories. DNA was isolated from purified sperm cells and sequenced using the RRBS (Reduced Representation Bisulfite Sequencing) technique for genome-wide methylome data generation. During the hot summer months, the physiological parameters such as scrotal surface temperature, rectal temperature, and respiration rate for both the SA and SNA bulls were significantly higher in the afternoon than in the morning. Whereas, the global CpG% of SA bulls was positively correlated with the afternoon's scrotal surface and rectal temperature. The RRBS results conveyed differentially methylated cytosines in the promoter region of the genes encoding the channels responsible for Ca2+ exchange, NPTN, Ca2+ activated chloride channels, ANO1, and a few structure-related units such as septins (SEPT4 and SEPT6), SPATA, etc. Additionally, the hypermethylated set of genes in SA was significantly enriched for pathways such as the FOXO signaling pathway and oocyte meiosis. The methylation patterns suggest promoter methylation in the genes regulating the sperm structure as well as surface transporters, which could contribute to the reduced semen quality in the Murrah buffalo bulls during the season-related heat stress.

Development and validation of most efficient RNA isolation method from buffalo bull spermatozoa.

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.

ddRAD sequencing based genotyping of six indigenous dairy cattle breeds of India to infer existing genetic diversity and population structure.

The present investigation aimed to identify genome wide SNPs and to carry out diversity and population structure study using ddRAD-seq based genotyping of 58 individuals of six indigenous milch cattle breeds (Bos indicus) such as Sahiwal, Gir, Rathi, Tharparkar, Red Sindhi and Kankrej of India. A high percentage of reads (94.53%) were mapped to the Bos taurus (ARS-UCD1.2) reference genome assembly. Following filtration criteria, a total of 84,027 high quality SNPs were identified across the genome of 6 cattle breeds with the highest number of SNPs observed in Gir (34,743), followed by Red Sindhi (13,092), Kankrej (12,812), Sahiwal (8956), Tharparkar (7356) and Rathi (7068). Most of these SNPs were distributed in the intronic regions (53.87%) followed by intergenic regions (34.94%) while only 1.23% were located in the exonic regions. Together with analysis of nucleotide diversity (π = 0.373), Tajima's D (D value ranging from - 0.295 to 0.214), observed heterozygosity (HO ranging from 0.464 to 0.551), inbreeding coefficient (FIS ranging from - 0.253 to 0.0513) suggested for the presence of sufficient within breed diversity in the 6 major milch breeds of India. The phylogenetic based structuring, principal component and admixture analysis revealed genetic distinctness as well as purity of almost all of the 6 cattle breeds. Overall, our strategy has successfully identified thousands of high-quality genome wide SNPs that will further enrich the Bos indicus representation basic information about genetic diversity and structure of 6 major Indian milch cattle breeds which should have implications for better management and conservation of valuable indicine cattle diversity.

Identification of stably expressed Internal Control Genes (ICGs) for normalization of expression data in liver of C57BL/6 mice injected with beta casomorphins.

In recent years, beta-casomorphin peptides (BCM7/BCM9) derived from the digestion of cow milk have drawn a lot of attention world over because of their proposed impact on human health. In order to evaluate the transcriptional modulation of target genes through RT-qPCR in response to these peptides, availability of appropriate reference or internal control genes (ICGs) will be the key. The present study was planned to identify a panel of stable ICGs in the liver tissue of C57BL/6 mice injected with BCM7/BCM9 cow milk peptides for 3 weeks. A total of ten candidate genes were evaluated as potential ICGs by assessing their expression stability using software suites; geNorm, NormFinder and BestKeeper. The suitability of the identified ICGs was validated by assessing the relative expression levels of target genes, HP and Cu/Zn SOD. Based on geNorm, PPIA and SDHA gene pair was identified to be most stably expressed in liver tissue during the animal trials. Similarly, NormFinder analysis also identified PPIA as the most stable gene. BestKeeper analysis showed crossing point SD value for all the genes in the acceptable range that is closer to 1. Overall, the study identified a panel of stable ICGs for reliable normalization of target genes expression data in mice liver tissues during BCM7/9 peptides trial.

ASIP gene polymorphism associated with black coat and skin color in Murrah buffalo.

The melanogenesis pathway regulates pigmentation through the synergic action of various genes. We are interested in analyzing the genetic variations in the ASIP which determine eumelanin production in the dermis layer. In the present study, the ASIP gene was characterized in buffalo and 268 genetically unrelated buffaloes belonging to 10 different populations were genotyped for the non-synonymous SNP (c.292C>T) identified in the exon 3 region of the gene using Tetra-ARMS-PCR. The TT genotype occurred at a higher rate in Murrah, followed by Nili Ravi, Tripura, and Paralakhemundi (42.63%, 19.30%, 3.45%, and 3.33%). These results convey the association of the black coat color of Murrah with the ASIP gene TT genotype and the lighter shades of black coat (brown and grayish-black) color phenotype in other breeds with the CC genotype.

Editing of HSF-1 and Na/K-ATPase α1 subunit by CRISPR/Cas9 reduces thermal tolerance of bovine skin fibroblasts to heat shock in vitro.

A follow-up to our previous findings, the present study was planned to evaluate the role of Na/K-ATPase alpha1-subunit (ATP1A1) gene in heat shock tolerance. The primary fibroblast culture was established using ear pinna tissue samples of Sahiwal cattle (Bos indicus). The knockout cell lines of Na/K-ATP1A1 and HSF-1 (heat shock factor-1, as a positive control) genes were developed by CRISPR/Cas9 method and the gene-editing was confirmed by the genomic cleavage detection assay. The two knockout cell lines (ATP1A1 and HSF-1) and wild-type fibroblasts were exposed to heat shock at 42 °C in vitro and different cellular parameters viz., apoptosis, proliferation, mitochondrial membrane potential (ΔΨm), oxidative stress, along with expression pattern of heat-responsive genes were studied. The results showed that in vitro heat shock given to knockout fibroblast cells of both ATP1A1 and HSF-1 genes resulted in decreased cell viability, while increasing the apoptosis rate, membrane depolarization, and ROS levels. However, the overall impact was more in HSF-1 knockout cells as compared to ATP1A1 knockout cells. Taken together, these results indicated that the ATP1A1 gene plays a critical role as HSF-1 under heat stress and helps cells to cope with heat shock.

Expression profile of different classes of proteases in milk derived somatic cells across different lactation stages of indigenous cows (Bos indicus) and riverine buffaloes (Bubalus bubalis).

Proteases play a significant role in milk and its products by affecting flavor, texture and longevity. The expression of endogenous proteases varies across different stages of lactation. The study was conducted to understand the transcriptional pattern of different classes of protease-pathways associated genes (CTSB, CTSD, CTSH, CTSL, CTSK, CTSS, CTSZ, PLAU, PLAT) and potential protease inhibitors (SERPIN E2 and SERPIN F2) in 40 milk somatic cells (MSC) samples isolated during early, peak, mid and late lactation stages of Sahiwal cows and Murrah buffaloes - the two most important dairy breeds of India. In Sahiwal cows, except CTSK and PLAU, the expression of other proteases class was not affected significantly (p > 0.05) across lactation stages. However, in Murrah buffaloes, the expression of different proteases increased as the lactation progressed. Most of the proteases showed lower expression during early and peak lactation stages while their expression tends to increase during mid to late lactation stages. The overall trend was somewhat similar in both the dairy species albeit the level of expression was higher in buffalo MSC as compared to cow MSC. The study has provided valuable information on expression kinetics of different proteases in milk somatic cells of two major dairy breeds of India.

Selection of species specific panel of reference genes in peripheral blood mononuclear cells of native livestock species adapted to trans-Himalayan region of Leh-Ladakh.

The identification of appropriate references genes is an integral component of any gene expression-based study for getting accuracy and reliability in data interpretation. In this study, we evaluated the expression stability of 10 candidate reference genes (GAPDH, RPL4, EEF1A1, RPS9, HPRT1, UXT, RPS23, B2M, RPS15, ACTB) in peripheral blood mononuclear cells of livestock species that are adapted to high altitude hypoxia conditions of Leh-Ladakh. A total of 37 PBMCs samples from six native livestock species of Leh-Ladakh region such as Ladakhi cattle, Ladakhi yak, Ladakhi donkey, Chanthangi goat, Double hump cattle and Zanskar ponies were included in this study. The commonly used statistical algorithms such as geNorm, Normfinder, BestKeeper and RefFinder were employed to assess the stability of these RGs in all the livestock species. Our study has identified different panel of reference genes in each species; for example, EEF1A1, RPL4 in Ladakhi cattle; GAPDH, RPS9, ACTB in Ladakhi yak; HPRT1, B2M, ACTB in Ladakhi donkey; HPRT1, B2M, ACTB in Double hump camel, RPS9, HPRT1 in Changthangi goat, HPRT1 and ACTB in Zanskar ponies. To the best of our knowledge, this is the first systematic attempt to identify panel of RGs across different livestock species types adapted to high altitude hypoxia conditions. In future, the findings of the present study would be quite helpful in conducting any transcriptional studies to understand the molecular basis of high altitude adaptation of native livestock population of Leh-Ladakh.

Identification of differential methylome signatures of white pigmented skin patches in Nili Ravi buffalo of India.

The DNA methylation events mark a major epigenetic change in the genome, reflecting non-genetic disease developments and varied phenotypes. The water buffalo is a dairy production animal with wide agro-climatic distribution in India. Breed-wise the coat color of water buffalo varies from ash-gray to jet black. A typical pigmentation pattern is found in one of the breeds of North India, Nili Ravi, with variedly distributed white patches. The DNA methylation pattern could potentially reveal the epigenetic factors responsible for the pigmentation patterns. To address this question, the DNA isolated from the skin tissues of Nili Ravi with varied white pigmentation and black Murrah buffaloes was subjected to reduced representation bisulfite sequencing. DNA methylation analysis revealed, 68.44%, 63.39%, and 47.94% of the promoter regions were hypermethylated in Nili Ravi over-white versus Murrah, Nili Ravi under-white versus Murrah, and Nili Ravi under-white versus Nili Ravi over-white, respectively. Major genes identified to be differentially methylated among over-white and under-white skin tissues in Nili Ravi included TBX2, SNAI2, HERC2, and CITED1. Overall the results have indicated differential methylation patterns to be potentially involved in hyper or hypopigmentation in Nili Ravi and Murrah buffaloes.

Demographic pattern of A1/A2 beta casein variants indicates conservation of A2 type haplotype across native cattle breeds (Bos indicus) of India.

Genetic variations of the beta casein gene hold importance because of their probable association with human health. Comparative sequence analysis of ß-casein gene across Indian native, crossbred and exotic breeds in India revealed 15 SNPs and 4 INDELs corresponding to 14 haplotypes. The frequency of A2 type haplotype was maximum (0.941) across all Indian native breeds. Among the 15 variants reported for taurine breeds, only three (A1, A2 and B) were observed in analysed populations. Allelic profiling of A1/A2 ß-casein variants in ~ 4000 animals belonging to three cattle types and breeding bulls also revealed the predominance of A2 allele (0.95) in Indian cattle. The high proportion of A2 allele/haplotype indicates that Indian native cattle are the best suited to meet the demands for A2 milk globally. However, a higher percentage of heterozygous genotype (A1A2) in breeding bulls warrants the need to screen sire lines so as to drift the herd towards A2. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03232-0.

Characterization of thermo-physiological, hematological, and molecular changes in response to seasonal variations in two tropically adapted native cattle breeds of Bos indicus lineage in hot arid ambience of Thar Desert.

The selection of climate resilient animal is necessary to secure the future of sustainable animal production. The present investigation therefore was an effort to unravel answers to the adaptation at physiological, hematological, and molecular levels in cows of hot arid region that helps them to survive harsh environment, to continue production and reproduction. This investigation was carried out in indicine cows over a period of one year, encompassing four seasons, wherein physiological data of 50 animals, hematological data of 15 animals, and gene expression profile of 5 animals from each of Sahiwal and Kankrej breeds per season was generated. In total, 5600 physiological observations, 1344 hematological observations, and 480 molecular samples were processed. The meteorological data revealed a high diurnal variation of temperature across seasons, with THI exceeding 80 during the months of summer and hot-humid seasons, indicating significant heat stress (HS). The physiological parameters showed an increasing trend with the incremental THI, with significantly (p < 0.05) higher values of rectal temperature (RT), respiration rate (RR), pulse rate (PR), and body surface temperature (BST) at ventral (VT), lateral (LT), dorsal (DT), and frontal (FT), in both breeds recorded during HS. The hematological pictures also revealed significant (p < 0.05) seasonal perturbations in erythrocytic and leucocytic parameters. Moreover, the molecular response was driven by a significant (p < 0.05) upregulation of all the key HSPs, HSP70, HSP90, HSP60, and HSP40, except HSP27 during the hotter months of summer and hot-humid seasons. The expression of HSF1, an important transcriptional regulator of  HSP70 was also significantly (p < 0.05) upregulated during summer season in both breeds. All the molecular chaperones revealed a significant upregulation during the summer season, followed by a decreasing trend by hot-humid season. The study indicated a well-developed thermotolerance mechanism in animals of both breeds, with Kankrej cows exhibiting better thermotolerance compared to Sahiwal cows.

Genetic admixture and population structure analysis of Indian water buffaloes (Bubalus bubalis) using STR markers.

BACKGROUND: India has a vast riverine and swamp buffalo diversity adapted to various agro-ecological conditions. In the present study, genetic diversity data for 10 different buffalo populations of India, using 20 highly polymorphic microsatellite markers has been generated for the genetic diversity analysis. The buffalo populations of Eastern Odisha state, were the primary focus. METHODS AND RESULTS: The minimal spanning network based on Bruvo's distance, PCA (Principal Component Analysis) based on the Fst (Fixation Index) values, and genetic admixture analysis using both the STRUCTURE and 'snapclust' were performed. The analysis could identify the Manda population as distinct from other Odisha buffalo breeds as well as adjoining Chhattisgarhi buffalo breeds. The total observed number of alleles ranged between 143 (Manda) and 301 (Paralakhemundi) with an average of 204 alleles per breed. The Sambhalpuri buffalo population also clustered into two separate subpopulations, half of the unique sub-population located geographically south-wards, displayed no admixture with any of the adjacent buffalo populations. The Manda buffalo population has shown sufficient allelic richness and heterozygosity under random mating being practiced in the field conditions. CONCLUSIONS: The study has led to the identification of the Manda as a distinct buffalo population, and the germplasm has been registered as a new Indian buffalo breed. Whereas, the Sambhalpuri population requires elaborate analysis to confirm the existence of two distinct sub-populations.

Current status and unique attributes of Indian Chilika buffalo for adaptation to brackish water ecology.

Chilika buffalo is native to the Eastern coast of India and well adapted to the largest coastal brackish water lagoon of Asia, Chilika Lake. We present here a report on the Chilika buffalo breed emphasizing the conservational urgency based on unique biochemical and molecular evidence related to liver and kidney functions while comparing it with tropically adapted other water buffalo breeds (Bubalus bubalis) of India. It is found that the Chilika buffalo breed has a better ability to withstand a long dehydration period as evident from its better glomerular filtration and higher expression of the ion transport channel. Mitochondrial D-loop sequencing results have shown these buffaloes being closer to swamp-type buffaloes of Bangladesh and northeast India and represent a unique "hybrid zone" on the eastern coast of India. Conservation of such uniquely adapted germplasm is crucial owing to the current global trend, where the introduction of exotic breeds has negatively impact "sui-generis" germplasm and they require higher managerial resource consumption for maintaining higher productivity. Further, the introduction of unconventional fisheries activities has proved detrimental to the lagoon ecosystem, potentially causing more threat to the buffalo's population.

In Silico Analysis of HSP70 Gene Family in Bovine Genome.

Heat shock proteins (HSPs), members of molecular chaperones families fulfill essential roles under normal conditions and provide protection and adaptation during and after stress. Among different HSPs, HSP70 kDa family of proteins is most abundant and well-studied in human and mouse but has not yet been characterized in bovines. In silico analysis was performed to characterize members of HSP70 gene family in bovine genome and a total of 17 genes of bovine HSP70 gene family were identified. The members of HSP70 family were distributed over 12 chromosomes with gene size ranging from 1911 (HSPA2) to 54,017 bp (HSPA4). Five genes were intronless, while rest of 12 genes were multiexonic. Phylogenetic analysis of HSP70 gene family distinguished them into eight major evolutionary groups wherein members of group 1 were most divergent and quite dissimilar than from rest of the HSP70 sequences. Domain structure of all bovine HSP70 genes was conserved and three signature patterns HSP70_1, HSP70_2, and HSP70_3 were identified. HSPA8, HSP9, and HSPA1A showed comparatively higher expression in majority of tissues. Like humans, bovine HSP70 family was characterized by remarkable evolutionary diversity. The analysis also suggested resemblance of bovine HSP70 family to that of human compared to mouse. Overall, the study indicates the presence of diversity for structure, function, localization, and expression in the bovine HSP70 family chaperons which could form the basis to understand thermotolerance/adaptive changes in the bovines.

Identification of Internal Reference Genes in Peripheral Blood Mononuclear Cells of Cattle Populations Adapted to Hot Arid Normoxia and Cold Arid Hypoxia Environments.

To estimate gene expression in a reliable manner, quantitative real-time polymerase chain reaction data require normalisation using a panel of stably expressed reference genes (RGs). To date, information on an appropriate panel of RGs in cattle populations reared at cold arid high-altitude hypoxia and hot arid tropical normoxia environments is not available. Therefore, the present study was carried out to identify a panel of stably expressed RGs from 10 candidate genes (GAPDH, RPL4, EEF1A1, RPS9, HPRT1, UXT, HMBS, B2M, RPS15, and ACTB) in peripheral blood mononuclear cells (PBMCs) of cattle populations reared at cold arid high-altitude hypoxia and hot arid normoxia environments. Four different statistical algorithms: geNorm, NormFinder, BestKeeper, and RefFinder were used to assess the stability of these genes. A total of 30 blood samples were collected: six adult heifers each of Ladakhi (LAC) and Holstein Frisian crosses (HFX) and 4 Jersey (JYC) cows from cold arid high-altitude hypoxia environments (group I) and five adult heifers each of Sahiwal (SAC), Karan Fries (KFC), and Holstein Friesian (HFC) cows from hot arid normoxia environments (group II). Combined analysis of group I and group II resulted in identification of a panel of RGs like RPS9, RPS15, and GAPDH that could act as a useful resource to unravel the accurate transcriptional profile of PBMCs from diverse cattle populations adapted to distinct altitudes.

Genetic characterization and population structure of different coat colour variants of Badri cattle.

The present study aimed to genetically characterize the Badri cattle and its three colour variants and assess their population structure using 24 microsatellite markers. Out of 96 animals analyzed, 32 each were collected from grey (GVBC), brown (BrVBC) and black (BVBC) colour variants of Badri cattle (BC). The genetic diversity parameters including allele frequencies, observed and effective number of alleles, observed and expected heterozygosity, PIC, Shannon's indices and F-statistics were estimated using POPGENE software. Bottleneck analysis was performed using both qualitative and quantitative approaches. A total of 274 alleles (50 private and 224 shared) were scored for BC, GVBC, BrVBC and BVBC with mean number of 11.417, 9.083, 9.125 and 9.083 alleles, respectively. All populations exhibited average heterozygosity estimate > 0.5 indicating existence of substantial genetic variability, concurrent with revelations from Shannon's indices. Observed mean PIC estimates (> 0.74) were indicative of optimum informativeness of used microsatellite markers. The mean inbreeding estimates (F) in GVBC, BrVBC and BVBC were 0.041, - 0.024 and 0.016, respectively. The pair wise genetic (> 0.91) pointed towards similarity between different colour variant populations. STRUCTURE analysis also revealed clear admixture for the three Badri colour variants indicating absence of genetic differentiation. The present study revealed first-hand information that populations of Badri cattle with different phenotypes with respect to coat colour are genetically related and can be considered as a single breed. The comprehensive knowledge generated for Badri cattle will help in designing breeding plan for its genetic improvement and deciding the conservation priorities.

Heat stress modulates differential response in skin fibroblast cells of native cattle (Bos indicus) and riverine buffaloes (Bubalus bubalis).

Heat stress in hot climates is a major cause that negatively affects dairy animals, leading to substantial economic loss. The present study was aimed to analyze the effect of heat stress on cellular and molecular levels in dermal fibroblast of cattle and buffaloes. Primary fibroblast culture was established using ear pinna tissue samples of cattle (Bos indicus) and riverine buffaloes (Bubalus Bubalis). The cells were exposed to thermal stress at 42°C for 1 h and subsequently allowed to recover and harvest at 37°C at different time points (0, 2, 4, 8, 16, and 24 h) along with control samples. Different cellular parameters viz., apoptosis, proliferation, mitochondrial membrane potential (ΔΨm), oxidative stress, along with expression pattern of heat responsive genes and miRNAs were determined. Cell viability and proliferation rate of heat-stressed fibroblasts decreased significantly (P < 0.05) albeit to a different extent in both species. The cell cytotoxicity, apoptosis, production of reactive oxygen species, and ΔΨm increased more significantly (P < 0.01) in heat stressed fibroblasts of buffalo than cattle. The pattern of heat shock proteins, inflammation/immune genes, and heat responsive miRNA showed differences in induction of their expression level in buffalo and native cattle fibroblasts. Conclusively, finding indicates that heat stress induces more profound impact on buffalo fibroblasts than native cattle fibroblasts. The differential response of cellular parameters, HSP genes, and miRNA expression could be due to better adaptive capacity of skin fibroblast of Bos indicus cattle in comparison with riverine buffaloes.

Evaluation of Milk Colostrum Derived Lactoferrin of Sahiwal (Bos indicus) and Karan Fries (Cross-Bred) Cows for Its Anti-Cancerous Potential.

Lactoferrin (Lf) is an iron-binding glycoprotein protein known to have immune-modulatory role and recently, its anticancerous effect against different cancer cell types was emphasized. In the present investigation, a comparative evaluation of anticancer potential of colostrum-derived lactoferrin from Indian native zebu cow (Sahiwal, SAC), crossbred (Karan Fries, KFC) and commercially available (C-Lf) lactoferrin from exotic cow using cellular models was made. A protocol was standardized successfully to purify Lf protein from colostrum of both breeds using HPLC and purity was confirmed by LC-MS. A standardized dose of 750 µg/mL Lf was used to treat two cell types MDA-MB-231 and MCF-7 with Lf from three different sources; SAC-Lf, KFC-Lf and C-Lf for 48 h and 72 h. Different cellular parameters including cytotoxicity, viability, apoptosis and cell proliferation were determined. Comparatively, Lf from commercial source (C-Lf) had maximum effect in both cell types followed by SAC-Lf and KFC-Lf. Further, transcriptional changes in genes associated with apoptosis (Bax and Bcl-2), tumor progression (p53, p21, CD44 and NF-κß) and survival (survivin) were evaluated in Lf treatment. The overall results strongly emphasized to the fact that Lf purified from cow colostrum has the capacity to inhibit the in vitro growth of cancerous cell lines albeit to a varied extent.