Genome-wide runs of homozygosity signatures in diverse Indian goat breeds.

The present study analyzed ROH and consensus ROH regions in 102 animals of eleven diverse Indian goat (Capra hircus) breeds using whole genome sequencing. A total of 51,705 ROH and 21,271 consensus regions were identified. The mean number of ROH per animal was highest in the meat breed, Jharkhand Black (2693) and lowest in the pashmina breed, Changthangi (60). The average length of ROH (ALROH) was maximum in Kanniadu (974.11 Kb) and minimum in Tellicherry (146.98 Kb). Long ROH is typically associated with more recent inbreeding, whereas short ROH is connected to more ancient inbreeding. The overall ROH-based genomic inbreeding (FROH) was highest for Jharkhand Black (0.602) followed by Kanniadu (0.120) and Sangamneri (0.108) among all breeds. FROH of Jharkhand Black was higher than Kanniadu  up to 5 Mb ROH length category. However, in > 20 Mb ROH length category, Kanniadu (0.98)  exhibited significantly higher FROH than Jharkhand Black (0.46). This  implies that Kanniadu had higher levels of recent inbreeding than Jharkhand Black. Despite this, due to the presence of both recent and ancient inbreeding, Jharkhand Black  demonstrated higher overall FROH compared to Kanniadu. ROH patterns revealed dual purpose (meat and dairy) and pashmina breeds as less consanguineous while recent inbreeding was apparent in meat breeds. Analysis of ROH consensus regions identified selection sweeps in key genes governing intramuscular fat deposition, meat tenderisation, lean meat production and carcass weight (CDK4, ALOX15, CASP9, PRDM16, DVL1) in meat breeds; milk fat percentage and mammary gland development (POLD1, NOTCH2, ARHGAP35) in dual purpose (meat and dairy) breeds; while cold adaptation and hair follicle development (APOBEC1, DNAJC3, F2RL1, FGF9) in pashmina breed. MAPK, RAS, BMP and Wnt signaling pathways associated with hair follicle morphogenesis in Changthangi were also identified. PCA analysis based on ROH consensus regions revealed that meat breeds are more diverse than other goat breeds/populations. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03921-y.

Canine umbilical cord tissue derived mesenchymal stem cells naturally express mRNAs of some antimicrobial peptides.

Antimicrobial peptides (AMPs) are naturally produced by all living organisms at a constitutive rate. They represent the first line of active defence systems against invading microorganisms, helping in innate immunity. Besides their therapeutic applications, great attention has also been given to the mesenchymal stem cells (MSCs) due to their antimicrobial activities. The study aimed to observe the mRNA expression profile of few antimicrobial peptides (AMPs) in canine MSCs during standard in vitro culture. MSCs were isolated from canine umbilical cord tissue, propagated and characterized by morphology, surface markers and tri-lineage differentiation capability. The mRNA expression of eleven commonly known antimicrobial peptides was checked by Reverse Transcriptase PCR. It has been found for the first time that canine MSCs naturally express the mRNAs of AMPs like C-X-C motif chemokine ligand 8 (CXCL8), Elafin (PI3), Hepcidin (HAMP), Lipocalin 2 (LCN2) and Secretory leukocyte protease inhibitor (SLPI). However, their expressions at protein level and, relation with antimicrobial effect of canine MSCs need to be explored.

Deciphering local adaptation of native Indian cattle (Bos indicus) breeds using landscape genomics and in-silico prediction of deleterious SNP effects on protein structure and function.

India has 50 registered breeds of native cattle (Bos indicus) which are locally adapted to diverse environmental conditions. This study aimed to investigate the genomic basis of adaptation of native Indian cattle and to predict the impact of key SNPs on the amino acid changes that affect protein function. The Illumina 777 K BovineHD BeadChip was used to genotype 178 native cattle belonging to contrasting landscapes and agro-climatic conditions. The genotype-environment association was investigated with R. SamBada, using 5,74,382 QC passed SNPs and 11 predictor variables (10 multi-collinearity controlled environmental variables and 1 variable as "score of PCA" on ancestry coefficients of individuals). In total, 1,12,780 models were selected as significant (q < 0.05) based on G score. The pathway ontology of the annotated genes revealed many important pathways and genes having a direct and indirect role in cold and hot adaptation. Only ten SNP variants had a SIFT score of < 0.05 (deleterious), and only two of them, each lying in the genes CRYBA1 and USP18, were predicted to be deleterious with high confidence. RaptorX predicted the tertiary structures of proteins encoded by wild and mutant variants of these genes. The quality of the models was determined using Ramachandran plots and RaptorX parameters, indicating that they are accurate. RaptorX and I-Mutant 2.0 softwares revealed significant differences among wild and mutant proteins. Adaptive alleles identified in the present investigation might be responsible for the local adaptation of these cattle breeds. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03493-3.

Genome analyses revealed genetic admixture and selection signatures in Bos indicus.

The genomic diversity and relationship among seven diverse cattle breeds viz. Sahiwal, Tharparkar, Gir, Vechur, Ongole, Kangayam and Hariana were investigated in 132 random samples based on high density SNP array comprising > 777 K SNPs. A total of 1993 SNPs (0.25% of the total) having greater power (FST ≥ 0.20) to differentiate these cattle populations were identified, and utilized to partition genome of each animal into a predefined number of clusters. The structure of these cattle indicated shared ancestry of dairy breeds viz. Gir, Tharparkar and Sahiwal. Most of the animals (> 76%) of different populations under study except Vechur clustered into their own group of animals called breed. Vechur population retained highest rate of admixture, consistent with its crossing with other breeds. Ongole, Kangayam and Hariana shared comparatively less of their genome (≤ 15%) with other breeds. The study indicated that all seven breeds evolved from their independent ancestry but there was intermixing of these breeds in the recent past. The selection signatures identified between draft (Kangayam) and dairy breeds included several genes like FAM19A2, RAB31P, BEST3, DGKA, AHCY, PIGU and PFKP which are involved in immune response, metabolic pathway, transportation of glucose and sugars, signaling pathways, cellular processes, cell division and glycolysis regulation, respectively. Moreover, these genomic regions also harbour QTLs affecting milk performance traits. The signatures were also identified even between the dairy breeds. In comparison to large-sized cattle, there were significant differences in the number of QTLs affecting production (body weight, growth rate etc.) and morphological traits (height) in short-statured Vechur breed. The presence of HMGA2 gene in the selection signature on chromosome 5 may explain the variations in stature between these cattle.

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.

Genome-Wide Runs of Homozygosity Revealed Selection Signatures in Bos indicus.

Genome-wide runs of homozygosity (ROH) are suitable for understanding population history, calculating genomic inbreeding, deciphering genetic architecture of complex traits and diseases as well as identifying genes linked with agro-economic traits. Autozygosity and ROH islands, genomic regions with elevated ROH frequencies, were characterized in 112 animals of seven Indian native cattle breeds (B. indicus) using BovineHD BeadChip. In total, 4138 ROH were detected. The average number of ROH per animal was maximum in draft breed, Kangayam (63.62 ± 22.71) and minimum in dairy breed, Sahiwal (24.62 ± 11.03). The mean ROH length was maximum in Vechur (6.97 Mb) and minimum in Hariana (4.04 Mb). Kangayam revealed the highest ROH based inbreeding (FROH > 1Mb = 0.113 ± 0.059), whereas Hariana (FROH > 1Mb = 0.042 ± 0.031) and Sahiwal (FROH > 1Mb = 0.043 ± 0.048) showed the lowest. The high standard deviation observed in each breed highlights a considerable variability in autozygosity. Out of the total autozygous segments observed in each breed except Vechur, > 80% were of short length (< 8 Mb) and contributed almost 50% of the genome proportion under ROH. However, in Vechur cattle, long ROH contributed 75% of the genome proportion under ROH. ROH patterns revealed Hariana and Sahiwal breeds as less consanguineous, while recent inbreeding was apparent in Vechur. Maximum autozygosity observed in Kangayam is attributable to both recent and ancient inbreeding. The ROH islands were harbouring higher proportion of QTLs for production traits (20.68% vs. 14.64%; P≤ 0.05) but lower for reproductive traits (11.49% vs. 15.76%; P≤ 0.05) in dairy breeds compared to draft breed. In draft cattle, genes associated with resistant to diseases/higher immunity (LYZL1, SVIL, and GPX4) and stress tolerant (CCT4) were identified in ROH islands; while in dairy breeds, for milk production (PTGFR, CSN1S1, CSN2, CSN1S2, and CSN3). Significant difference in ROH islands among large and short statured breeds was observed at chromosome 3 and 5 involving genes like PTGFR and HMGA2 responsible for milk production and stature, respectively. PCA analysis on consensus ROH regions revealed distinct clustering of dairy, draft and short stature cattle breeds.

Pentaplex PCR assay for rapid differential detection of Babesia bigemina, Theileria annulata, Anaplasma marginale and Trypanosoma evansi in cattle.

A multiplex PCR (mPCR) assay for simultaneous detection and differentiation of four major haemoparasites in crossbred cattle was established using parasite specific genomic DNA and four sets of primer pairs targeting AMA-1, Tams1, MSP5 and VSG genes of Babesia bigemina, Theileria annulata, Anaplasma marginale and Trypanosoma evansi generating precise amplicons of 448, 156, 382 and 110 bp, respectively. An internal amplification control, 202 bp bovine ß-casein gene fragment, was simultaneously amplified with four target genes to avoid false-negative results. The sensitivity of mPCR was 3.44 × 102, 5.9 × 103, 2.88 × 102 and 3.3 × 103 copies for B. bigemina, T. annulata, A. marginale and T. evansi, respectively. mPCR of cattle clinical samples (n = 516), suspected for haemoparasites, revealed single haemoparasitic infection in 279 (54.06%) cases, whereas mixed infection was recorded in 54 (10.46%) samples. In clinical samples, coinfection with T. annulata and A. marginale was the most common. The findings of mPCR were consistent with uniplex PCR under field conditions except for subtle variations in A. marginale infection. Overall, the mPCR assay represents an economical, reproducible and robust diagnostic tool for concurrent detection of cattle haemoparasites and large scale epidemiological studies.

Hematobiochemical alterations and direct blood polymerase chain reaction detection of Theileria annulata in naturally infected crossbred cows.

AIM: The aim was to determine hemato-biochemical changes and rapid diagnosis of Theileria annulata in naturally infected crossbred cows. MATERIALS AND METHODS: Blood samples from lactating crossbred cows (n=40) between 3 and 7 years of age and showing clinical signs of tropical theileriosis were collected, with or without anticoagulant, and analyzed for tropical theileriosis by direct smear, direct blood polymerase chain reaction (PCR) detection of merozoite-piroplasm surface antigen (Tams1) gene specific amplicon, estimation of hematological and biochemical parameters. Healthy crossbred cows (n=6), examined free from hemoprotozoan infections were included as control. RESULTS: The infected crossbred cows revealed significantly (p<0.001) lower values of total erythrocytic counts (4.46±0.2 × 10(6)/µL), hemoglobin (Hb 6.025±0.39 g%), packed cell volume (17.05±1.1%), mean corpuscular volume (37.94±1.70 fL) and mean corpuscular Hb (13.5±0.48 pg; p<0.002) compared with healthy control. The serum samples of infected cows revealed profound (p<0.05) hyponatremia (Na 133.21±2.36 mEq/l) and hypocalcemia (Ca 8.39±0.34 mg%). Infected crossbred cows showed a significant increase (p<0.05) of mean serum activity of alanine aminotransferase (61.45±13.36 U/L), aspartate aminotransferase (146.1±20.97 U/L), blood urea nitrogen (28.26±3.90 mg%), creatinine (1.55±0.13 mg%), direct bilirubin (0.33±0.04 mg%; p<0.001) and lactate dehydrogenase (3001.32±167.0 U/L; p<001). Blood direct PCR revealed a 721-bp fragment amplified from the target gene encoding 30-kDa major merozoite surface antigen of T. annulata using specific primer pairs. This assay was positive for all the infected animals. CONCLUSION: The assessments of hemato-biochemical parameters in T. annulata infected crossbred cows may be useful in understanding disease pathogenesis, prognosis and corrective measures for supportive therapy. Moreover, blood direct PCR can reliably be used for rapid detection of T. annulata in conjunction with microscopic examination.

Changes in circulatory FSH of Barbari goats following treatment with high molecular weight inhibin isolated from buffalo follicular fluid.

The objective of the present study was to isolate and purify high MW inhibin (≈ 129 kDa) from buffalo ovarian follicular fluid (buFF) and to investigate its biological activity. Throughout the process of purification, the inhibin fractions were evaluated for bioactivity by a specific, sensitive and uniformly reproducible bioassay in mice. The final biological activity of this preparation was tested in normal cycling adult female Barbari goats. Eight animals, randomly divided into two groups, were synchronized for estrus by administering PGF2α twice at an interval of ten days. Following synchronization, the treatment group (n=4) received (i.m.) 0.4 ml (240 µg protein total dose) of purified inhibin (MW ≈ 129 kDa) of buFF in the morning at 08.00 h for the four consecutive days of follicular phase, while the control group (n=4) received only saline (0.4 ml). Blood samples were collected from jugular vein immediately before the first injection and subsequent collections were made daily in the afternoon until day 8 of the experiment including four days (0, 1, 2, & 3 days) of the next cycle. FSH was assayed in all the samples by ELISA. The peripheral FSH concentration sharply declined from 1.854±0.137 to 0.979 ± 0.02 u/l, 8h after the administration of inhibin on the first day. The value in controls was 2.004 ± 0.132 u/l. For the duration of treatment of four consecutive days, the FSH level in experimental group remained significantly low (p<0.05) compared to control group. After cessation of treatment, the FSH level remained low on day 0 and 1 of the next cycle in the experimental and control animals. However, a significant rebound increase in plasma FSH levels occurred on day 2 & 3 (2.73 ± 0.179 & 1.849 ± 0.128 u/l) only in the experimental group compared to control animals (P<0.05). This increment might be caused by the rebound surge of FSH from anterior pituitary which further corroborates the effect of inhibin in treated animals. In conclusion, the present study demonstrates that the high MW form of inhibin (≈ 129 kDa) isolated from buFF has comparable biological activity as revealed by 31-32 kDa inhibin from other species.

Differential expression of protamine 1 and 2 genes in mature spermatozoa of normal and motility impaired semen producing crossbred Frieswal (HF×Sahiwal) bulls.

Mature spermatozoa contain thousands of mRNA transcripts. These untranslated mRNA may perhaps serve as a "footprint" of spermatogenesis since many of them might directly or indirectly be involved in fertilization, early embryo cleavage, poor semen quality and fertility. In this study, we tried to isolate high-quality RNA from mature spermatozoa and to monitor the expression profile of protamine 1 (PRM1) and protamine 2 (PRM2) gene in ejaculated spermatozoa of normal (good, % initial progressive motility: 57.61±1.41, n=9) and motility impaired (poor, % initial progressive motility: 18.45±1.61, n=8) crossbred Frieswal (HF×Sahiwal) bulls semen using real time quantitative PCR. Semen samples were subjected to discontinuous (45:90) Percoll gradient centrifugation, specifically to eliminate damaged spermatozoa and contaminating somatic cells. Total RNA was extracted from sperm pellets and cDNA was synthesized. Furthermore, the absence of contamination of germ cells, epithelial cells and leucocytes in all the RNA extractions was tested by RT-PCR targeting specific molecular markers like KIT, CDH1 and CD4, respectively. The presence of transcripts like PRM1, PRM2, DAZL, and PPIA were demonstrated in ejaculated spermatozoa using appropriate PCR primers without RNA amplification. Expression of PRM1 and PRM2 genes were evaluated by real time quantitative PCR using TaqMan chemistry, where PPIA was used as internal control. The cDNA synthesized from normal buffalo testicular tissue was served as positive control. The good quality semen producing group showed significantly higher level of PRM1 mRNAs expression as compared to the poor quality semen producers (P<0.05) indicating putative role of the gene and semen quality parameters especially initial progressive motility. However, PRM2 transcript levels were not significantly different between the groups (P>0.05).

Multiple molecular forms of inhibin in buffalo (Bubalus bubalis) ovarian follicular fluid.

Inhibin is a heterodimeric glycoprotein hormone involved in the regulation of FSH release from the anterior pituitary gland and it has been characterized from various animals. Although, multiple molecular forms of inhibin have been reported from different species, however, the molecular nature of inhibin has not been studied in buffaloes. In the present study, attempts were made to identify inhibin in buffalo ovarian follicular fluid. Buffalo ovaries were obtained from the local abattoir and follicular fluid was aspirated from surface follicle (with diameter> or =5mm). A combination of techniques (viz., gel filtration, SDS-PAGE, Western blot etc.) was employed for identification and isolation of inhibin(s). Inhibin bands were detected at 129 and 63 kDa by Western blot analysis in non-reducing conditions. In reduced SDS-PAGE, 63 kDa fraction produced a single band while 129 kDa fraction resolved into four components of 63, 43, 29 and 20 kDa. Out of them only 29, 63 and the native 129 kDa fractions produced bands on Western blot analysis. In total five fractions (63, 54, 39, 29, 25 kDa) were obtained by trypsin digestion of 129 kDa form. However, only 63 and 29 kDa fractions showed immunoreactivity. In this study, for the first time, we have identified two major forms of inhibin (129 and 63 kDa) with little proteolytic cleavage/processing of the large precursor in the buffalo follicular fluid.

Association of microsatellite (GT)n polymorphism at 3'UTR of NRAMP1 with the macrophage function following challenge with Brucella LPS in buffalo (Bubalus bubalis).

Brucella abortus is a facultative intracellular pathogen that survives and replicates in host macrophages. Hence, macrophage function plays an important role in influencing natural resistance/susceptibility to intracellular pathogen. The natural resistance associated macrophage protein 1 (NRAMP1; erstwhile referred as Ity/Lsh/Bcg), a transmembrane protein, regulates activity of macrophages against intracellular pathogens. In bovine, natural resistance to brucellosis is significantly associated with (GT)13 allelic variant of microsatellite locus at 3' untranslated region (3'UTR) of the NRAMP1 gene. In the present study we screened 65 Murrah breed of buffalo (Bubalus bubalis) to identify polymorphism at 3'UTR of NRAMP1 gene and evaluate the association of these polymorphisms with the macrophage function. Four allelic variants (viz., GT13, GT14, GT15 and GT16) were identified. Majority of the buffaloes were of either homozygous (GT)14/(GT)14 or heterozygous (GT)14/(GT)15 with (GT)14 allele occurring most frequently (62%). For association study, non-vaccinated and serologically negative animals were divided into three genotypic groups: group 1 (n=2) comprising animals of homozygous (GT)13 genotype, whereas, group 2 (n=4) and group 3 (n=6) consisted animals of heterozygous [(GT)13/(GT)n, where n not equal 13] and non-(GT)13 [(GT)n/(GT)n, where n not equal 13] genotype, respectively. Macrophages, after maturation, were challenged with Brucella LPS to assay the macrophage function in terms of H2O2 and NO production. The (GT)13 allele, either in homozygoous {(GT)13/(GT)13} or heterozygous {(GT)13/(GT)n, where n=14, 15 or 16}, was significantly (p<0.01) associated with increased production of H2O2 and NO. In this manuscript, for the first time, we have identified (GT)13 allelic variant and demonstrated its significant association with the improved macrophage function in buffalo.

Lack of association of brucellosis resistance with (GT)(13) microsatellite allele at 3'UTR of NRAMP1 gene in Indian zebu (Bos indicus) and crossbred (Bos indicus x Bos taurus) cattle.

Natural resistance associated macrophage protein 1 (NRAMP1), an integral transmembrane protein, is reported to influence the intraphagosomal microbial replication and thereby confer resistance to several intracellular pathogens in mice. In bovine, a significant association of (GT)(13) allelic variant of polymorphic microsatellite at 3' untranslated region (UTR) of NRAMP1 gene with natural resistance to brucellosis has been established. The present study was aimed to detect polymorphism at 3'UTR of NRAMP1 gene in Hariana breed of Bos indicus cattle and Holstein Friesian crossbred (B. indicusxBos taurus) cattle, and to determine the association of this polymorphism with resistance/susceptibility to brucellosis. The (GT)(n) polymorphism at 3'UTR in terms of variation in fragment length was determined using denaturing polyacrylamide gel analysis of radioisotope incorporated amplicon of 174 bp. Screening of a total of 100 samples (comprising 50 random samples of each breed) revealed that animals were of same genotype, i.e., homozygous (GT)(13)/(GT)(13). Sequencing of amplicons from representative animals confirmed the presence of (GT)(13) repeat. For association study, the animals that were positive in all three serological tests (viz., RBPT, STAT and ELISA) and had history of abortion were grouped as "affected"; whereas the animals that were negative in all these tests and completed third lactation without any history of abortion were grouped as "non-affected". Since, all animals belonging to either group were homozygous (GT)(13), association could not be established. However, the present study demonstrated that the presence of (GT)(13) allele even in homozygous condition could not provide enough resistance to brucellosis in a naturally infected herd.