Flaxseed oil modulates testicular biometrics, hormone, libido, antioxidant and semen profiles in endangered Teressa goat of Andaman and Nicobar Islands.

Teressa goat is a unique goat breed in Andaman and Nicobar Islands (ANI) of India. Effects of Flaxseed oil (FSO) supplementation in body weight (BW), scrotal circumference (SC), testicular volume (TV) and testicular weight (TW), endocrinological profiles, sex behavioural profiles (SBPs), oxidative stress markers and semen production and its quality profiles in rainy and dry summer season were studied in Teressa goat. Male goats (n = 12) of 3-4 years old were equally divided into control and treated groups. Treated animals received 25 mL FSO per day. Oral drenching of FSO was done in the morning before feeding the concentrate ration. Body weight, scrotal circumference, TV and TW were measured in bucks of FSO treated and untreated during rainy and dry summer seasons. Blood follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone, thyroid stimulating hormone (TSH), triiodothyronine (T3), thyroxine (T4), cortisol and prolactin, total antioxidant capacity (TAC), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and malondialdehyde (MDA) were measured in bucks of FSO treated and untreated during rainy and dry summer seasons. Libido score (LS), mating ability score (MAS) and sex behavioural score (SBS) were estimated at time of semen collection in bucks of FSO treated and untreated during rainy and dry summer seasons. Semen samples (n = 100; 50 semen samples from each season; each 25 semen samples from control and treatment groups per season) were collected and analysed for semen quality profiles. One-way ANOVA (control rainy, control dry, treated rainy and treated dry) revealed that BW, SC, TV and TW, FSH, LH, testosterone, TSH, T3 and T4 were higher (P < 0.05) and cortisol and prolactin were lower (P < 0.05) in FSO treated bucks of rainy season followed by untreated bucks of rainy season, FSO treated bucks of dry summer season and were lower (P < 0.05) in untreated bucks of dry summer season. Similarly, TAC, CAT, SOD and GSH, LS, MAS and SBS, and volume, pH, sperm concentration, mass activity, total motility (TM), viability, acrosomal integrity (AcI), plasma membrane integrity (PMI) and nuclear integrity (NI) were higher (P < 0.05) and MDA and TSA were lower (P < 0.05) in FSO treated bucks of rainy season followed by FSO treated bucks of dry summer season, untreated bucks of rainy season and were lower (P < 0.05) in untreated bucks of dry summer season. The results of the present study indicated that the breeding bucks suffered physiological stress (higher cortisol), oxidative stress (higher MDA and deficiency of antioxidants), hormonal imbalance (higher prolactin and cortisol and deficiency of gonadotropins, gonadal hormone and thyroid hormones) and infertility due to poor libido and poor semen production and its quality profiles during dry summer season. Thus, dry summer was more stressful season compared to rainy season for the goat bucks. FSO supplementation mitigated these stresses and improved the scrotal and testicular biometrics, libido, antioxidants, hormones and semen quality profiles in Teressa goat bucks. The current study concluded that FSO effectively improved the hormones, libido, antioxidant profiles, and scrotal and testicular biometrics with cascading beneficial effects on semen quality profiles in Teressa goat bucks under humid tropical island ecosystem of Andaman and Nicobar Islands.

Maternal lineage of Nicobari pig (Sus scrofa nicobaricus) correlated with migration of Nicobarese, a native tribal population of Andaman and Nicobar Islands, India.

Nicobari pig is reared by Nicobarese, a native tribal population of Andaman and Nicobar Islands. Nicobari pig has maintained its genetic identity due to geographical isolation. This communication is the first report on maternal inheritance of Nicobari pigs. DNA polymorphism data showed seven haplotypes. D-loop sequence information and mitogenome analysis were able to earmark Nicobari pigs to Asian clade. The domestication process of pigs and its expansion pattern help to understand human migration pattern. Based on this hypothesis, this communication elucidates the probable origin of Nicobarese. Earlier studies indicated that Nicobarese had genetic affinities to races distributed in China, Malaysia and Thailand. Our data on maternal inheritance of Nicobari pig correlates with the data on migration of Nicobarese. Moreover, we could establish a novel connection of Nicobarese with people of Northeastern parts of India, Philippines and Vietnam through phylogenetic signal and geographical provenance of Nicobari pig. We further concluded that migration of Nicobarese happened during Western route of migration (WRM) ∼4000 years before present. Therefore, we propose one wave hypothesis of peopling of Nicobar based on our study and existence of Ausrtroasiatic language, Mon-Khmer in these islands.

Designing, optimization, and validation of whole blood direct T-ARMS PCR for precise and rapid genotyping of complex vertebral malformation in cattle.

BACKGROUND: DNA testing in the cattle industry undergoes multiple hurdles. Successful genotyping involves the transportation of samples from the field to the laboratory in a chilled environment followed by DNA extraction, and finally, a specific genotyping protocol is followed. Various researches are focused on overcoming these issues. Microcards offer blood transportation at ambient temperature. Direct PCR methods can save the time of DNA extraction but available only for simplex PCR. Tetra Primer-Amplification Refractory Mutation System based Polymerase Chain Reaction (T-ARMS PCR) can make DNA testing faster in a low-cost setting. The present study was aimed to design, optimize, and validate a T-ARMS PCR for faster DNA testing of SNP responsible for Complex Vertebral Malformation (CVM)-an important genetic disease of the cattle industry. Further, a direct T-ARMS PCR from whole blood was developed to avoid the DNA extraction steps. Lastly, using the optimized protocol, genotyping of blood spotted on Microcard eliminates the need for cold chain maintenance in the transportation of samples. RESULTS: The present study demonstrated a novel T-ARMS PCR-based genotyping of the SNP rs438228855, which is responsible for CVM. Here, wild genotypes were recognized by 389 bp and 199 bp bands in agarose gel, while the carrier genotype showed an additional 241 bp band. The developed protocol was validated using PCR-Primer Introduced Restriction Analysis (PCR-PIRA) and sequencing. The present study further established a direct T-ARMS PCR for this SNP from whole blood. Different conditions such as heparin and EDTA treated blood, the need for pre-treatment, and two different DNA Polymerases for the direct PCR were optimized. Finally, our optimized protocol successfully genotyped the whole blood samples dried on Insta™DNA cards. CONCLUSIONS: The present study reported the usefulness of primer modified T-ARMS PCR for detecting CVM for the first time. To the best of our knowledge, direct PCR in T-ARMS PCR has never been reported. Lastly, the use of microcards in the developed protocol can make the assay useful in the DNA testing of field samples.

Daily and seasonal rhythmic secretary pattern of endocrinological profiles in mithun bull.

Mithun (Bos frontalis) is a unique domestic free-range bovine species available in North Eastern hilly (NEH) regions of India and is reared for its protein rich meat. Mithun suffers severe non-cyclical population fluctuations; however, it is not yet declared as endangered species. Mithun follows some sort of seasonality based on the calving trend and semen production, although it is a perennial breeder. However, there is need to study the rhythmic changes of endocrine profiles to understand the hormone flow pattern in mithun to select the suitable time for blood collection to assess the exact endocrine profiles and to select the suitable time for natural breeding or semen collection by artificial methods for further research, conservation and propagation of its germplasm. Therefore, the present study was designed to evaluate the reproductive as well as metabolic endocrinological profiles in 24:00 h in intact adult mithun bulls during different seasons (winter, spring, summer and autumn) to know the rhythmic changes and flow pattern of the endocrinological profiles to improve its reproductive efficiency. Experimental mithun bulls (n = 6; age: 5-6 years; body condition score: 5-6 out of 10, classified as good) were selected for the study. Endocrinological profiles, follicle stimulating hormone (FSH), luteinizing hormone/interstitial cell stimulating hormone (LH/ICSH), testosterone, cortisol, thyroxine (T4), insulin like growth factor-1 (IGF-1), prolactin and melatonin (MT) were estimated at 04:00 h interval for one whole day in four seasons. The analysis was completed in two different ways as different times of collection (08:00, 12:00, 16:00, 20:00, 24:00 and 04:00 h) and day (08:00 to 16:00 h) & night time (20:00 to 04:00 h) collections. Repeated measures ANOVA analysis revealed that the bulls in winter and spring had significantly (p < 0.05) higher FSH, LH, testosterone, T4, IGF-1 and MT than those in summer whereas the bulls in summer had significantly higher cortisol and prolactin than those in winter and spring seasons. Similarly FSH, LH, testosterone, T4, IGF-1 and MT were significantly (p < 0.05) higher in night than in day time collections whereas cortisol and prolactin were significantly (p < 0.05) higher in day than in night time collections in different seasons. Correlation analysis revealed that FSH, LH, testosterone, T4, IGF-1 and MT had significant (p < 0.05) positive correlation with each other whereas these had significant (p < 0.05) negative correlation with cortisol and prolactin. The study concludes that season and time of blood collection had significant effect on the endocrinological profiles in mithun bulls. Estimation of FSH, LH, testosterone, T4, IGF-1 and MT during night time and cortisol and prolactin during day time was more appropriate to get correct value of the endocrinological profiles. Spring and winter have significantly greater beneficial effects than summer on reproduction and artificial breeding programs in mithun species in the semi-intensive management in the present location.

Assessment of Protein Profiles of RNAlater Stored and Fresh PBMC Cells Using Different Protein Extraction Buffers.

For proteome analyses, the tissue samples are mostly preserved either snap frozen or formalin-fixed, paraffin-embedded form. Use of RNAlater-a non-toxic solution primarily used to stabilize the RNA content of samples-in tissue preservation for proteome analysis recently described equally reliable with snap-frozen preservation in human tissues. Even though RNALater storage has great potential in the preservation of Peripheral Blood Mononuclear Cells (PBMC), its impact on the results of proteome analysis is poorly described at qualitative and quantitative measures. The present study investigated protein profiles of RNAlater preserved and fresh PBMCs using three extraction buffers viz. Triton X-100, RIPA and SDS. Proteins are separated in SDS-PAGE and quantified using densitometry. On an average 19.3 bands from fresh and 15.6 bands from RNAlater storage cells were obtained with a molecular weight ranging from 25 to > 250 kDa. RNAlater storage generated a fewer number and lesser quantity of low molecular weight proteins while yielded a similar or high quantity of high molecular weight protein fractions. The principal component analysis showed that Triton X-100 is inferior as compared to SDS and RIPA with respect to their protein bands and quantity yielded. While RNAlater is effective in preserving PBMC for proteome analysis, our findings warrant caution in its use in proteomics experiments especially if the target is low molecular weight proteins.

Identification of differentially expressed microRNAs in Sahiwal (Bos indicus) breed of cattle during thermal stress.

microRNAs (miRNAs) are a class of small non-coding RNAs that play key roles in post transcriptional gene regulation that influence various fundamental cellular processes, including the cellular responses during environmental stresses. However, perusal of literatures revealed few reports on the differential expression of miRNA during thermal stress in Indian native (Bos indicus) cattle breeds. The present investigation aimed to identify differentially expressed miRNAs during thermal stress in Sahiwal (Bos indicus) dairy cattle breed of India, adapted with tropical climate over a long period of time. Stress responses of the animals were characterized by determining various physiological as well as biochemical parameters and differential expression profile of major heat shock protein genes. Ion Torrent deep sequencing and CLC-genomic analysis identified a set of differentially expressed miRNAs during summer and winter seasons. Most of the identified differentially expressed miRNAs were found to target heat shock responsive genes especially members of heat shock protein (HSP) family. Real-time quantification-based analysis of selected miRNAs revealed that bta-mir-1248, bta-mir-2332, bta-mir-2478, and bta-mir-1839 were significantly (p < 0.01) over expressed while bta-mir-16a, bta-let-7b, bta-mir-142, and bta-mir-425 were significantly (p < 0.01) under expressed during summer in comparison to winter. The present study enlists differentially expressed miRNAs at different environmental temperatures in Sahiwal (Bos indicus) that may be importance for further understanding the role of miRNAs on thermo-regulatory mechanisms.

Using PCR-PIRA based genotyping for identifying complex vertebral malformation allele in Frieswal young bulls in India.

Complex vertebral malformation (CVM) has considerable economic impact on dairy cattle breeding due to extensive use of artificial insemination (AI). Identifying the carrier is an important factor to reduce the incidence of the genetic disorder. The study was conducted to identify the carriers of CVM in Frieswal cattle by polymerase chain reaction-primer-introduced restriction analysis (PCR-PIRA) method, which was further confirmed by sequencing. Carrier prevalence of 1% was observed in the Frieswal cattle. The results of the study clearly demonstrated the existence of carriers of CVM among Frieswal bull calves. Due to the widespread use of AI it is recommended to screen young bulls at early stages for this defective allele in order to avoid its rapid spread within the population.

A Synonymous Mutation at Bovine Alpha Vitronectin Domain of Integrin Host Receptor (ITGAV) Gene Effect the Susceptibility of Foot-and-Mouth Disease in Crossbred Cattle.

Integrins are one of the major biologically active proteins responsible for Foot-and-mouth disease virus (FMDV)- host interaction. Out of various heterodimeric integrins discovered, αVß6 heterodimer serves as the chief receptor for FMDV host tropism. Earlier studies reported that, SNPs at beta 6 subunit (ITGB6) were associated with the occurrence of the diseases in cattle. In this study we report the association between a synonymous SNP (rs719257875) at bovine alpha vitronectin domain of integrin receptor (ITGAV) gene and FMD susceptibility in cattle. A strong significant association (P < 0.0001) of the genotypes with FMD susceptibility were obtained, where the CC genotypes play a major role in occurrence of FMD in crossbred cattle.

Differential expression of microRNAs associated with thermal stress in Frieswal (Bos taurus x Bos indicus) crossbred dairy cattle.

Environmental temperature is one of the important abiotic factors that influence the normal physiological function and productive performance of dairy cattle. Temperature stress evokes complex responses that are essential for safeguarding of cellular integrity and animal health. Post-transcriptional regulation of gene expression by miRNA plays a key role cellular stress responses. The present study investigated the differential expression of miRNA in Frieswal (Holstein Friesian × Sahiwal) crossbred dairy cattle that are distinctly adapted to environmental temperature stress as they were evolved by using the temperate dairy breed Holstein Friesian. The results indicated that there was a significant variation in the physiological and biochemical indicators estimated under summer stress. The differential expression of miRNA was observed under heat stress when compared to the normal winter season. Out of the total 420 miRNAs, 65 were differentially expressed during peak summer temperatures. Most of these miRNAs were found to target heat shock responsive genes especially members of heat shock protein (HSP) family, and network analysis revealed most of them having stress-mediated effects on signaling mechanisms. Being greater in their expression profile during peak summer, bta-miR-2898 was chosen for reporter assay to identify its effect on the target HSPB8 (heat shock protein 22) gene in stressed bovine PBMC cell cultured model. Comprehensive understanding of the biological regulation of stress responsive mechanism is critical for developing approaches to reduce the production losses due to environmental heat stress in dairy cattle.

Association analysis of novel polymorphisms in 2', 5'-oligoadenylate synthetase gene with reproductive traits in indigenous and cross-bred cattle of Indian Origin.

2', 5'-Oligoadenylate synthetases (OAS) are important components of an interferon-mediated antiviral pathway. No polymorphisms in exonic regions of bovine OAS1 gene have been identified and associated with reproduction traits. The objective of the study was to detect and evaluate the effects of mutations in exonic region of bovine OAS1 gene with reproduction traits in cattle. DNA samples collected from 250 individual cows of two Indian dairy breeds (Sahiwal and Frieswal) of cattle were used in the study. The genetic variants of the OAS1 gene were identified with polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and sequence analysis using seven set of primer pairs. The PCR-SSCP analysis revealed polymorphism in the fragments comprising of exon 2, exon 5 and first fragment of exon 6 while the fragments of exons 1, 3, 4 and second fragment of exon 6 were monomorphic in Sahiwal and Frieswal cattle. The mutations in the amplified region comprising of exon 2 were found to have significant association with age at first breeding and calving, service period, dry period and pregnancy rate. Significant associations were found between SNPs in the exon 5 and service and dry periods of the animal, whereas the genetic variants in the first fragment of the exon 6 showed significant association with age at first breeding and calving. To our knowledge, this study demonstrated for the first time that the polymorphisms in OAS1 gene were associated with reproductive traits and it can be chosen as a candidate gene for improvement of reproductive performance of cattle.

LAMP assay for rapid diagnosis of cow DNA in goat milk and meat samples.

Animal species detection is one of the crucial steps for consumer's food analysis. In the present study we developed an in-house built loop-mediated isothermal amplification (LAMP) assay for rapid detection of adulterated cow DNA in goat milk/meat samples. The cow milk/tissue DNA in goat milk/meat samples were identified in the developed LAMP assay by either naked eye visualizing with SYBR Green I dyes or by detecting the typical ladder pattern on gel electrophoresis. This test can detect up to minimum 5% level of cow components admixed in goat milk/meat samples and can be completed within 1 h 40 min starting from DNA extraction from milk/meat samples and can be performed in a water bath. Developed LAMP methodology is simple; rapid and sensitive techniques that can detect adulterant like cow components in goat milk/meat are more accurate than other existing DNA based technologies.

RT-LAMP assay: an alternative approach for profiling of bovine heat shock protein 70 gene in PBMC cultured model.

The purpose of this study is to develop a novel Reverse Transcriptase Loop-mediated isothermal amplification (RT-LAMP) based assay for in vitro profiling of heat shock protein 70 (Hsp70) in bovine peripheral blood mononuclear cell (PBMC) culture model utilizing the absorbance level of magnesium pyrophosphate-a by-product of LAMP reaction. A set of bovine Hsp70 specific RT-LAMP primers were designed to detect the differential absorbance level of magnesium pyrophosphate by-product which signifies the degree of Hsp70 amplification from cDNA of thermally induced cultured cells at different recovery periods. The study revealed significant (P < 0.05) correlation between absorbance level and the fold change of Hsp70 transcripts at different kinetic intervals of heat stress recovery in bovine PBMC cell culture models. RT-LAMP based absorbance assay can be used as an indicator to measure the degree of bovine Hsp70 transcripts produced during thermal stress and can be used as an alternative to the traditional Real time PCR assay. Developed RT-LAMP assay can be used as a cost-effective method for profiling of bovine HSP70 gene.

Comparative expression profile of NOD1/2 and certain acute inflammatory cytokines in thermal-stressed cell culture model of native and crossbred cattle.

Thermotolerance depends mainly on the health and immune status of the animals. The variation in the immune status of the animals may alter the level of tolerance of animals exposed to heat or cold stress. The present study was conducted to investigate the expression profile of two important nucleotide binding and oligomerization domain receptors (NLRs) (NOD1 and NOD2) and their central signalling molecule RIP2 gene during in vitro thermal-stressed bovine peripheral blood mononuclear cells (PBMCs) of native (Sahiwal) and crossbred (Sahiwal X HF) cattle. We also examined the differential expression profile of certain acute inflammatory cytokines in in vitro thermal-stressed PBMC culture among native and its crossbred counterparts. Results revealed that the expression profile of NOD1/2 positively correlates with the thermal stress, signalling molecule and cytokines. Present findings also highlighted that the expression patterns during thermal stress were comparatively superior among indigenous compared to crossbred cattle which may add references regarding the better immune adaptability of Zebu cattle.

Application of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cow Components Adulterated in Buffalo Milk/Meat.

Loop-mediated isothermal amplification (LAMP) is a diagnostic method for amplification of DNA with rapid and minimal equipment requirement. In the present study, we applied the LAMP assay for rapid detection of cow components adulteration in buffalo milk/meat samples. The test can be completed within around 1 h 40 min starting from DNA extraction and can be performed in water bath without requirement of thermocycler. The cow DNA in buffalo samples were identified in the developed LAMP assay by either visualizing with SYBR Green I/HNB dyes or observing the typical ladder pattern on gel electrophoresis. The test can detect up to 5 % level of cow milk/meat mixed in buffalo counterparts. Due to the simplicity and specificity, the developed LAMP test can be easily adapted in any laboratory for rapid detection of cow species identification in livestock by products.

Differential effect of thermal stress on HSP70 expression, nitric oxide production and cell proliferation among native and crossbred dairy cattle.

In a tropical country like India, thermal stress is one of the major factors which significantly affects the productivity of dairy cattle. The present study was aimed to identify the effect of heat and cold stress on cell viability, mitogen stimulation indices, nitric oxide production and HSP70 expression in Sahiwal and Holstein crossbred (Frieswal) population in India. The results indicated that the Sahiwal breed can better withstand the effect of heat and cold stress significantly (P<0.05) when compared to the crossbred cattle due to the higher survivability of the Peripheral Blood Mononuclear Cells (PBMCs) and Phytohemagglutinin (PHA-P) mitogen based stimulation indices. The study also revealed the significant differences (P<0.05) in the level of nitric oxide (µM) production amongst the pre and post thermal stressed samples of Sahiwal and Frieswal crossbred samples. Further, the expression of HSP70 was significantly (P<0.05) higher in Sahiwal compared to Frieswal immediately after heat/cold shock to 6h of recovery as indirect ELISA analysis showed gradual rise in the Hsp70 protein concentration (ng/ml) immediately after heat and cold stress (0h) and reached the peak at 6h of recovery. Western blot and immune fluorescent assay results were also corroborated with the findings of indirect ELISA. In Sahiwal cattle the mRNA expression of HSP70 and its protein concentration were higher (P<0.05) during peak summer (44°C) and winter (10°C) as compared to Frieswal cattle. This investigation supports the earlier information on the higher adaptability of indigenous cattle breeds to hot and humid conditions compared to the crossbreds of temperate cattle breeds.