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.

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.

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.

Allelic diversity at BoLA DRB3 locus and association with predisposition to clinical mastitis in indicus and crossbred cattle.

Bovine lymphocyte antigen (BoLA) DRB3 locus in healthy and mastitis affected cattle has been genotyped by a polymerase chain reaction and restriction fragment length polymorphisms (PCR-RLFP) using RsaI restriction enzyme, followed by sequencing. In 130 farm animals, 25 BoLA DRB3 alleles have been detected by PCR-RFLP. Three distinct allelic patterns significantly associated with mastitis in Karan Fries crossbred and Sahiwal indicus cattle have been identified, whereas, four other allelic patterns were significantly high in frequency among healthy animals. Sequencing of RFLP genotypes revealed 25 and 47 alleles among healthy Sahiwal and Karan Fries, respectively, while 17 and 38 patterns observed in mastitis affected Sahiwal and Karan Fries animals, respectively. From Tajima's D-test of neutrality, it was concluded that alleles associated with mastitis were expanding in the population, whereas those of healthy were under contraction. Phylogenetic analysis carried out to delineate the evolutionary relationship of the farm and field animals at DRB3 locus, differentiating allelic patterns into six different clusters. Among the phylogenetic lineages, five patterns DRB3*028:01, DRB3*011:03, DRB3*031:01, DRB3*001:01 and DRB3*043:01, were previously reported, whereas one novel allelic variant was observed in indicus and crossbred cattle. This information will help in further exploring the association between BoLA-DRB3 genetic diversity and disease resistance in distinct cattle breeds, important in designing breeding strategies for increasing the distribution of favorable alleles.

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.

Genetic characterization and diversity assessment in 'Bhangor' indigenous swamp buffalo population using heterologous microsatellite markers.

'Bhangor' newly identified swamp buffalo population from North East Indian, was characterized using microsatellite markers. Genomic DNA was isolated from blood samples of 76 unrelated animals, 15 microsatellite markers (CSSM33, BM1818, CSRM60, HEL13, ILSTS019, ILSTS025, ILSTS028, ILSTS029, ILSTS033, ILSTS036, ILSTS056, ILSTS058, ILSTS061, ILSTS089 and ETH003) were found to be highly polymorphic in the population of the selected markers. A total of 114 alleles were observed, which ranged from 3 in CSRM60 and ILSTS025 locus to 12 in ILSTS056 and ILSTS061. The mean effective number of alleles across all polymorphic loci was found to be 3.76. The overall mean expected heterozygosity and unbiased expected heterozygosity values were 0.67 and 0.68, ranging from 0.067 (ILSTS025) to 0.85 (ILSTS058) and 0.068 (ILSTS025) to 0.86 (ILSTS058), respectively. Within the population, the inbreeding estimates (FIS) ranged between -0.4352 and 0.804, with an average FIS of 0.114 ± 0.033. The outcome for infinite allele model (IAM), two-phase model (TPM) and test for mode shift revealed the absence of any recent bottleneck in the investigated buffalo population. The population was found to be in optimum diversity based on polymorphic microsatellite markers. With fast changing agro-climatic conditions; there is an urgent need to characterize the nondescript livestock populations.

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.

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.

Identification of novel allelic patterns and evolutionary lineage of BoLA MHC class II DQA locus in indicine and taurine cattle.

Among different cattle types, Bos indicus are known for their ability to better resist the tropical microbial infections comparatively, wherein MHC molecules play a significant role. In this study allelic diversity at MHC locus, DQA of Bos indicus, Bos taurus and crossbred of taurine-indicus has been explored to understand the possible role of MHC region in differential immune response. Thirty nine different DQA alleles were identified, out of which 14 were novel, along with documentation of duplication of DQA alleles. Indicus cattle population presented diverse types of DQA alleles compared to crossbred and exotic. Translated amino acid sequence analysis indicated, codon 64 and 50 of peptide binding sites being highly polymorphic and most of the indicus cattle presented alanine and arginine amino acid at position 64 and 50. Within breed genetic variation found to be higher than between breeds. Because of their ability to bind and subsequently respond to a wide array of antigens, the newly identified DQA alleles with high diversity present in the form of duplicated haplotypes in different combinations in cattle populations provided significant insights into probable role of this MHC locus in better tropical disease combating ability and genetic fitness of indicus cattle.

Casein (CSN) gene variants and parity affect the milk protein traits in crossbred (Bos taurus x Bos indicus) cows in sub-tropical climate.

Genotypes at four casein (CSN) loci-A26181G of CSN1S1, C6227T of CSN1S2, A8101C of CSN2, and A13104C of CSN3-along with non-genetic factors were studied for their effects on various milk protein traits in 100 crossbred cows with major inheritance of Holstein Frisian (Bos taurus) and Tharparkar (Bos indicus). Results demonstrated the presence of all CSN genotypes with a predominance of heterozygotes. At CSN2 (A8101C; His67Pro) locus, the A2 allele, desirable for human health, was present in 62% as heterozygous and 29% in homozygous condition. Among non-genetic factors, parity of the cows had a significant influence on the milk protein traits in these crossbreds. The genotypes at CSN1S1, CSN2, and CSN3 loci were found to influence (p<0.05 to 0.01) the casein and whey protein yields and composition traits. The casein index and total milk yield were most influenced by the CSN1S2 locus. The AA (A1 milk) genotype of CSN2 had significantly higher yields and percentages of casein and whey proteins. Positive influence of CC genotype of CSNS3 on milk proteins of was observed similar to Bos taurus cows; however, such influence of AA genotype of CSN2 locus may be distinctive to the crossbred cows maintained in subtropical condition. Overall, the results revealed the diverse effects of CSN genotypes on milk proteins in crossbred cattle.

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.

Y-chromosome genetic diversity of Bos indicus cattle in close proximity to the centre of domestication.

Y-chromosome genetic diversity in and around its domestication origin and a better understanding of indicine-specific microsatellite alleles are imperative concerns but less -targeted. We analysed Y-chromosome markers in 301 bulls representing 19 native Indian cattle (Bos indicus) and identified new alleles and haplotypes. Compared to other indicine studies, the high Y-haplotype diversity found in Indian cattle supports the hypothesis of greater genetic variability across the centre of origin decreasing along migratory routes with increasing distance. Hence, a considerable paternal genetic diversity of Indian cattle appears to have been lost in transboundary commercial indicine breeds. The Khillar and Gir are the most diversified populations where the first tends to be the well-differentiated traditional breed carrying strikingly distinct Y-lineages with typical BM861-158 bp allele, characteristics of taurine cattle, while retaining standard indicine lineages for all other markers. Geographical distribution found to be an unreliable predictor of parental variation, and Y-lineages seemed closely related to Indian breed function/utility. The comprehensive Y-chromosome information will be useful to examine the demographic expansion/spread of Bos indicus lineages from close proximity to the domestication centre across different countries worldwide and such diversity should be preserved through effective management and conservation programs.

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.

Characterizing binding sites of heat responsive microRNAs and their expression pattern in heat stressed PBMCs of native cattle, exotic cattle and riverine buffaloes.

It is generally believed that due to evolutionary differences and adaptation to tropical conditions, Indian native cattle has superior heat tolerant ability than Bos taurus cattle. In the present study, 3'-UTR of two most important heat responsive genes i.e., heat shock protein 70.1 (HSP70.1) and heat shock factor- 1 (HSF-1) were sequence characterized in different breeds of Indian native cattle to identify the variations and miRNA binding sites. In addition, the impact of heat stress was assessed in a total of 57 PBMCs samples of native Sahiwal cows (Bos indicus), exotic Holstein cows (Bos taurus) and Murrah buffaloes (Bubalus bubalis) using various cellular parameters like cell viability, cytotoxicity and apoptosis. Further, expression profile of 12 heat responsive miRNAs were also evaluated in unstressed and stressed PBMCs to understand post transcriptional changes in native cows, exotic cows and Murrah buffaloes. The sequence data showed 3'-UTR of HSP70.1 gene of Indian cattle to be exactly similar to Bos taurus with no miRNA binding site. Whereas, sequencing of 3'-UTR of HSF-1 gene revealed 3 SNPs at positions G1762T; C1811T and C1983T with 7 well conserved miRNA binding sites. The impact of heat stress on various cellular parameters in terms of cell viability, cytotoxicity and apoptosis was highest in PBMCs of Holstein cows followed by Murrah buffaloes and Sahiwal cows. Further, in contrast to Holstein Frisian cows and Murrah buffaloes, the expression pattern of 12 heat responsive miRNAs, in heat stressed PBMCs of Sahiwal cows were quite distinct. There was a significant (p < 0.05) induction in expression of most of the miRNAs after heat stress in PBMCs of Sahiwal cows followed by a rapid decline. The distinct cellular response and pattern of miRNA expression across cattle types and buffaloes might be influencing their PBMCs tolerance level to heat stress.