Exploring molecular dynamic indicators associated with reproductive performance of Bos indicus cattle in blood plasma samples through data-independent acquisition mass spectrometry.

Improving reproductive performance of cattle is of paramount importance for sustainable dairy farming. Poor reproduction performance (RP) hinders the genetic improvement of important Bos indicus cattle breeds. It is well known that incorporation of molecular information along with conventional breeding method is far better than use of conventional method alone for the genetic improvement of reproductive performance traits in cattle. Therefore, the present study sought to investigate the plasma proteome of the Deoni cows in cyclical (n = 6) and pregnant (n = 6) reproductive phases with varying reproductive performance (high and low). High-throughput data independent acquisition (DIA) based proteomics was performed to understand corresponding proteome. We identified a total of 430 plasma proteins. Among cyclic cows, twenty proteins were differentially regulated in low RP as compared to high RP. BARD1 and AFP proteins were observed upregulated in cyclical cows whose upregulation reported to affect reproductive performance in cattle. Among the pregnant cows, thirty-five proteins were differentially regulated, including the downregulation of FGL2 and ZNFX1 that modulates the maternal immune response mechanism which is required for successful implantation of the embryo. Also, proteins such as AHSG, CLU and SERPINA6 were upregulated in the pregnant cows whose upregulation reported to reduced reproductive performance. The results of this study will be helpful in establishing a framework for future research on the aspect of improving reproductive performance in Bos indicus cattle breeds. SIGNIFICANCE: The Indian subcontinent is the center of domestication for Bos indicus cattle breeds and they are known for their disease resistance, heat tolerance, ability to survive in low input regime and harsh climatic conditions. In recent times, population of many important Bos indicus breeds including Deoni cattle is declining due to various factors, especially due to reproductive performance. Traditional breeding methods are not sufficient enough to understand and improve the reproductive performance traits in important Bos indicus cattle breeds. Proteomics approach is a promising technology to understand the complex biological factors which leads to poor reproductive performance in cattle. The present study utilized DIA based LC- MS/MS analysis to identify the plasma proteins associated with reproductive performance in cyclical and pregnant cows. This study if improved further, can be used to develop potential protein markers associated with reproductive performance which is useful for the selection and genetic improvement of important Bos indicus breeds.

Quantitative proteomics profiling of spermatozoa and seminal plasma reveals proteins associated with semen quality in Bos indicus bulls.

Cattle breeding approaches are an evolving field of research in veterinary science. Certain factors such as Ejaculate Rejection Rate (ERR) pose a limitation to such approaches. In this regard, we sought to investigate the spermatozoa and seminal plasma proteome of Hallikar bulls with low (n = 3) and high (n = 3) ERR. Through the Tandem mass spectrometry approach, we identified a total of 2409 proteins, in which 828 proteins were common in both the semen components, whereas 375 and 378 proteins were unique to spermatozoa and seminal plasma respectively. Tandem mass tags (TMT) based protein quantification resulted in 75 spermatozoal, and 42 seminal plasma proteins being differentially regulated between high and low ERR bulls. Proteins such as SPADH2, TIMP-2, and PLA2G7 which are negative regulators of motility were upregulated in the seminal plasma of high ERR bulls. Proteins such as OAZ3, GPx4, and GSTM3 whose upregulation leads to reduced motility were upregulated in the spermatozoa of high ERR bulls. Caltrin and ADM proteins that enhance sperm motility were downregulated in the seminal plasma of high ERR bulls. The regulation of ACE, a negative regulator of sperm motility was upregulated in both the spermatozoa and seminal plasma of high ERR bulls. SIGNIFICANCE: The saying "Bull is more than half of the herd" signifies the importance of bull in the genetic improvement of the herd. Traditionally used semen quality tests will provide limited information about the potential fertility of bulls. The proteomics approach is a promising omics technology to understand the factors involved in male fertility. The present study identified the spermatozoal and seminal plasma proteins that are differentially regulated between high and low ERR bulls. Sperm motility-associated proteins are differentially regulated. This study if improved further, can be used to develop markers associated with semen quality which is useful for the selection of bulls.

Expression analysis of pro-apoptotic BAX and anti-apoptotic BCL-2 genes in relation to lactation performance in Deoni and Holstein Friesian crossbred cows.

The study was designed with the objective of expression analysis of pro-apoptotic BAX and anti-apoptotic BCL-2 genes on lactation performance in Bos indicus and HF crossbred cows during early lactation. BAX/BCL-2 mRNA expression ratio in HF crossbreds showed a steady increase from 30th day to 90th day, but in Deoni cows the ratio exhibited a different pattern, which increased from day 30 to day 60, decreased on day 75, and then increased on day 90. BAX/BCL-2 expression ratio in Deoni and HF crossbreds were lowest on day 30 and highest on day 90. On contrary, the milk yield was highest on day 30 and lowest on day 90 suggesting BCL-2 gene favors milk production and BAX gene oppose milk production. In comparison to HF crossbreds, Deoni cows exhibited highest BAX/BCL-2 ratio at the end of early lactation, indicating Bos indicus cows were more sensitive to apoptosis than HF crossbreds. Comparison of daily milk yield with BAX/BCL-2 mRNA expression ratio revealed significant negative correlation with a correlation coefficient of -0.98 (P < 0.01) and -0.95 (P < 0.05) in Deoni and HF crossbred cows, respectively. Our study provides new insights into understanding the genetic control of mammary apoptosis between Bos indicus and HF crossbreds.

Anti-Müllerian hormone as an endocrine biomarker of reproductive longevity and assessment of single nucleotide polymorphisms in AMH gene of Bos indicus breeds of cattle.

Anti-Müllerian hormone (AMH) is a member of the TGF-ß superfamily produced by follicular granulosa cells in women and cattle and is considered an endocrine biomarker of ovarian follicular reserve. The study examined how age and parity influence serum AMH concentration and investigated the presence of single nucleotide polymorphisms in AMH gene in Bos indicus breeds viz Malnad Gidda Amritmahal and Hallikar. All five exons of AMH gene amplified by polymerase chain reaction were subjected to sanger sequencing and identified important SNP and its effects. We observed a highly significant relationship between parity and AMH concentration in Amritmahal cattle, whereas Malnad Gidda and Hallikar breeds did not show a significant difference. We identified one SNP located in exon 5 (rs21402788) with base change A>G, a non-synonymous mutation resulting in a change in amino acid Q>R and the protein product. It is concluded that AMH level could be considered as an indicator of the ovarian reserve and productive herd life (longevity) irrespective of age/parity, especially in B. indicus breeds of cattle.

Sperm Transcripts Associated With Odorant Binding and Olfactory Transduction Pathways Are Altered in Breeding Bulls Producing Poor-Quality Semen.

Spermatozoa carries a reservoir of mRNAs regulating sperm functions and fertilizing potential. Although it is well recognized that a considerable proportion of high genetic merit breeding bulls produce poor-quality semen, the transcriptomic alterations in spermatozoa from such bulls are not understood. In the present study, comparative high-throughput transcriptomic profiling of spermatozoa from good and poor-quality semen-producing bulls was carried out to identify the transcripts associated with semen quality. Using next-generation sequencing (NGS), we identified 11,632 transcripts in Holstein Friesian bull spermatozoa; after total hit normalization, a total of 544 transcripts were detected, of which 185 transcripts were common to both good and poor-quality semen, while 181 sperm transcripts were unique to good quality semen, and 178 transcripts were unique to poor-quality semen. Among the co-expressed transcripts, 31 were upregulated, while 108 were downregulated, and 46 were neutrally expressed in poor-quality semen. Bioinformatics analysis revealed that the dysregulated transcripts were predominantly involved in molecular function, such as olfactory receptor activity and odor binding, and in biological process, such as detection of chemical stimulus involved in sensory perception, sensory perception of smell, signal transduction, and signal synaptic transmission. Since a majority of the dysregulated transcripts were involved in the olfactory pathway (85% of enriched dysregulated genes were involved in this pathway), the expression of selected five transcripts associated with this pathway (OR2T11, OR10S1, ORIL3, OR5M11, and PRRX1) were validated using real-time qPCR, and it was found that their transcriptional abundance followed the same trend as observed in NGS; the sperm transcriptional abundance of OR2T11 and OR10S1 differed significantly (p < 0.05) between good and poor-quality semen. It is concluded that poor-quality semen showed altered expression of transcripts associated with olfactory receptors and pathways indicating the relationship between olfactory pathway and semen quality in bulls.

Deciphering metabolomic alterations in seminal plasma of crossbred (Bos taurus X Bos indicus) bulls through comparative deep metabolomic analysis.

The incidence of sub-fertility is higher in crossbred bulls compared to zebu bulls. In the present study, we analysed the metabolomic profile of seminal plasma from crossbred and zebu bulls and uncovered differentially expressed metabolites between these two breeds. Using a high-throughput LC-MS/MS-based approach, we identified 990 and 1,002 metabolites in crossbred and zebu bull seminal plasma respectively. After excluding the exogenous metabolites, we found that 50 and 68 putative metabolites were unique to crossbred and zebu bull seminal plasma, respectively, whilst 87 metabolites were common to both. After data normalisation, 63 metabolites were found to be dysregulated between crossbred and zebu bull seminal plasma. Observed pathways included Linoleic acid metabolism (observed metabolite was phosphatidylcholine) in crossbred bull seminal plasma whereas inositol phosphate metabolism (observed metabolites were phosphatidylinositol-3,4,5-trisphosphate/inositol 1,3,4,5,6-pentakisphosphate/myo-inositol hexakisphosphate) was observed in zebu bull seminal plasma. Abundance of Tetradecanoyl-CoA was significantly higher, whilst abundance of Taurine was significantly lower in crossbred bull seminal plasma. In conclusion, the present study established the seminal plasma metabolomic profile in crossbred and zebu bulls and suggest that increased lipid peroxidation coupled with low concentrations of antioxidants in seminal plasma might be associated with high incidence of sub-fertility in crossbred bulls.

Sperm phenotypic characteristics and oviduct binding ability are altered in breeding bulls with high sperm DNA fragmentation index.

In the present study, we standardized an in vitro oviduct explants model for cattle and assessed the oviduct explants binding ability and phenotypic characteristics of spermatozoa obtained from breeding bulls with high- and low-sperm DNA fragmentation index (DFI%). Cryopreserved spermatozoa from Holstein Friesian crossbred breeding bulls (n = 45) with known field fertility were assessed for DFI% and were classified into either high DFI% or low DFI% category. Flow cytometry was used to assess sperm membrane integrity, acrosome reaction status, mitochondrial membrane potential and intracellular calcium concentrations. It was found that spermatozoa from bulls with low DFI% had significantly higher (P < 0.05) membrane integrity, acrosome intactness, and mitochondrial membrane potential. To assess the sperm oviduct binding ability, oviduct explants were prepared by incubating the oviduct cells overnight in TCM-199 medium at 38.5 °C under 5% CO2. Different sperm concentrations and times of incubation were evaluated and found that 2 million spermatozoa and 1-h incubation yielded high binding index (BI). The BI was also significantly (P < 0.01) higher (>2 times) in the bulls with low-DFI% as compared to high DFI% bulls. The correlation between binding index and DFI% was negative and significant (r = -0.528; P < 0.05). Further, the binding index was positively correlated with conception rate (r = 0.703), intact sperm membrane (r = 0.631) and mitochondrial membrane potential (r = 0.609). It is inferred that sperm phenotypic characteristics and oviduct binding ability are impaired in breeding bulls with high sperm DFI%, which might be associated with low conception rates in these bulls.

Preliminary comparative deep metabolomic analysis of spermatozoa from zebu and crossbred cattle suggests associations between metabolites, sperm quality and fertility.

Poor semen quality and infertility/subfertility are more frequent in crossbred than zebu bulls. Using a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach, we established the preliminary metabolomic profile of crossbred and zebu bull spermatozoa (n=3 bulls each) and identified changes in sperm metabolomics between the two groups. In all, 1732 and 1240 metabolites were detected in zebu and crossbred bull spermatozoa respectively. After excluding exogenous metabolites, 115 and 87 metabolites were found to be unique to zebu and crossbred bull spermatozoa respectively whereas 71 metabolites were common to both. In the normalised data, 49 metabolites were found to be differentially expressed between zebu and crossbred bull spermatozoa. The significantly enriched (P<0.05) pathways in spermatozoa were taurine and hypotaurine metabolism (observed metabolites taurine and hypotaurine) in zebu and glycerophospholipid metabolism (observed metabolites phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine) in crossbred bulls. The abundance of nitroprusside (variable importance in projection (VIP) score >1.5) was downregulated, whereas that of l-cysteine, acetyl coenzyme A and 2'-deoxyribonucleoside 5'-diphosphate (VIP scores >1.0) was upregulated in crossbred bull spermatozoa. In conclusion, this study established the metabolomic profile of zebu and crossbred bull spermatozoa and suggests that aberrations in taurine, hypotaurine and glycerophospholipid metabolism may be associated with the higher incidence of infertility/subfertility in crossbred bulls.

Differences in milk metabolites in Malnad Gidda (Bos indicus) cows reared under pasture-based feeding system.

The milk and milk products from cows reared under grazing system are believed to be healthier and hence have high demand compared to milk from cows reared in the non-grazing system. However, the effect of grazing on milk metabolites, specifically lipids has not been fully understood. In this study, we used acetonitrile precipitation and methanol:chloroform methods for extracting the milk metabolites followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) run to identify the different metabolites between the milk of grazing and non-grazing early lactating Malnad Gidda cows. Various carbohydrates, amino acids, nucleosides and vitamin derivatives were found to be differentially abundant in grazing cows. A total of 35 metabolites were differentially regulated (fold change above 1.5) between the two groups. Tyrosyl-threonine, histidinyl-cysteine, 1-methyladenine, L-cysteine and selenocysteine showed fold change above 3 in grazing cows. The lipid profile of milk showed a lesser difference between grazing and non-grazing cows as compared to polar metabolites. To the best of our knowledge, this is the largest inventory of milk metabolomics data of an Indian cattle (Bos indicus) breed. We believe that our study would help to emerge a field of Nutri-metabolomics and veterinary omics research.

Deep Metabolomic Profiling Reveals Alterations in Fatty Acid Synthesis and Ketone Body Degradations in Spermatozoa and Seminal Plasma of Astheno-Oligozoospermic Bulls.

Male fertility is extremely important in dairy animals because semen from a single bull is used to inseminate several thousand females. Asthenozoospermia (reduced sperm motility) and oligozoospermia (reduced sperm concentration) are the two important reasons cited for idiopathic infertility in crossbred bulls; however, the etiology remains elusive. In this study, using a non-targeted liquid chromatography with tandem mass spectrometry-based approach, we carried out a deep metabolomic analysis of spermatozoa and seminal plasma derived from normozoospermic and astheno-oligozoospermic bulls. Using bioinformatics tools, alterations in metabolites and metabolic pathways between normozoospermia and astheno-oligozoospermia were elucidated. A total of 299 and 167 metabolites in spermatozoa and 183 and 147 metabolites in seminal plasma were detected in astheno-oligozoospermic and normozoospermic bulls, respectively. Among the mapped metabolites, 75 sperm metabolites were common to both the groups, whereas 166 and 50 sperm metabolites were unique to astheno-oligozoospermic and normozoospermic bulls, respectively. Similarly, 86 metabolites were common to both the groups, whereas 45 and 37 seminal plasma metabolites were unique to astheno-oligozoospermic and normozoospermic bulls, respectively. Among the differentially expressed metabolites, 62 sperm metabolites and 56 seminal plasma metabolites were significantly dysregulated in astheno-oligozoospermic bulls. In spermatozoa, selenocysteine, deoxyuridine triphosphate, and nitroprusside showed significant enrichment in astheno-oligozoospermic bulls. In seminal plasma, malonic acid, 5-diphosphoinositol pentakisphosphate, D-cysteine, and nicotinamide adenine dinucleotide phosphate were significantly upregulated, whereas tetradecanoyl-CoA was significantly downregulated in the astheno-oligozoospermia. Spermatozoa from astheno-oligozoospermic bulls showed alterations in the metabolism of fatty acid and fatty acid elongation in mitochondria pathways, whereas seminal plasma from astheno-oligozoospermic bulls showed alterations in synthesis and degradation of ketone bodies, pyruvate metabolism, and inositol phosphate metabolism pathways. The present study revealed vital information related to semen metabolomic differences between astheno-oligozoospermic and normospermic crossbred breeding bulls. It is inferred that fatty acid synthesis and ketone body degradations are altered in the spermatozoa and seminal plasma of astheno-oligozoospermic crossbred bulls. These results open up new avenues for further research, and current findings can be applied for the modulation of identified pathways to restore sperm motility and concentration in astheno-oligozoospermic bulls.

Sub-fertility in crossbred bulls: deciphering testicular level transcriptomic alterations between zebu (Bos indicus) and crossbred (Bos taurus x Bos indicus) bulls.

BACKGROUND: The incidence of poor semen quality and sub-fertility/infertility is higher in crossbred as compared to Zebu males. Several attempts have been made to understand the possible reasons for higher incidence of fertility problems in crossbred males, at sperm phenotype, proteome and genome level but with variable results. Since the quality of the ejaculated spermatozoa is determined by the testicular environment, assessing the testicular transcriptome between these breeds would help in identifying the possible mechanisms associated with infertility in crossbred bulls. However, such information is not available. We performed global transcriptomic profiling of testicular tissue from crossbred and Zebu bulls using Agilent Bos taurus GXP 8X60k AMADID: 29411 array. To the best of our knowledge, this is the first study comparing the testicular mRNAs between crossbred and Zebu bulls. RESULTS: Out of the 14,419 transcripts detected in bovine testis, 1466 were differentially expressed between crossbred and Zebu bulls, in which 1038 were upregulated and 428 were downregulated in crossbred bulls. PI4KB and DPY19L2 genes, reported to be involved in sperm capacitation and acrosome formation respectively, were among the top 10 downregulated transcripts in crossbred testis. Genes involved in ubiquitination and proteolysis were upregulated, while genes involved in cell proliferation, stem cell differentiation, stem cell population maintenance, steroidogenesis, WNT signalling, protein localization to plasma membrane, endocannabinoid signalling, heparin binding, cAMP metabolism and GABA receptor activity were downregulated in crossbred testis. Among the 10 genes validated using qPCR, expression of CCNYL, SOX2, MSMB, SPATA7, TNP1, TNP2 and CRISP2 followed the same trend as observed in microarray analysis with SPATA7 being significantly downregulated and transition proteins (TNP1, TNP2) being significantly upregulated in crossbred bulls. CONCLUSIONS: Abundant proteolysis by ubiquitination and downregulation of WNT signaling, cell proliferation, differentiation and steroidogenesis might be associated with higher incidence of poor semen quality and/or sub-fertility/infertility in crossbred bulls as compared to Zebu bulls. Downregulation of SPATA7 (Spermatogenesis Associated 7) and upregulation of transition proteins (TNP1 and TNP2) in crossbred bull testis might be associated with impaired spermatogenesis processes including improper chromatin compaction in crossbred bulls.

Deep Proteome Profiling of Semen of Indian Indigenous Malnad Gidda (Bos indicus) Cattle.

Malnad Gidda is a dwarf indigenous cattle breed of India, which is known for its uniqueness of calving every year under a low input grazing system of rearing. Bulls of Malnad Gidda are known to be highly fertile even in stress conditions. However, the proteomic profiling of semen of this breed has not been investigated so far, which might provide a platform for a better understanding of its semen quality and male fertility. Therefore, we made an effort to characterize and quantify the proteome of seminal plasma and spermatozoa components of Malnad Gidda semen using a high-resolution mass spectrometry platform. We identified 2814 proteins from spermatozoa and 1974 proteins from the seminal plasma of this breed. Furthermore, >90% of proteins from each fraction were quantified using the intensity-based absolute quantification. We observed signal peptides in 33% of seminal plasma proteins, indicating their secretory nature. Gene Ontology analysis revealed their involvement in cytoskeletal assembly associated with sperm head, sperm motility, acrosome reaction, seminal plasma binding, and spermatogenesis-associated protein. An in-depth proteome profiling of semen of a unique indigenous cattle breed of India was carried out. Our findings could provide a reference for further studies on sperm functions, semen quality, and reproductive health of Bos indicus cattle. Mass spectrometry data generated in this study is deposited and publicly made available through ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD014172.

Metabolomic fingerprinting of bull spermatozoa for identification of fertility signature metabolites.

The objective of the study was to identify the fertility-associated metabolites in bovine spermatozoa using liquid chromatography-mass spectrometry (LC-MS). Six Holstein Friesian crossbred bulls (three high-fertile and three low-fertile bulls) were the experimental animals. Sperm proteins were isolated and protein-normalized samples were processed for metabolite extraction and subjected to LC-MS/MS analysis. Mass spectrometry data were processed using iMETQ software and metabolites were identified using Human Metabolome DataBase while, Metaboanalyst 4.0 tool was used for statistical and pathway analysis. A total of 3,704 metabolites belonging to various chemical classes were identified in bull spermatozoa. After sorting out exogenous metabolites, 56 metabolites were observed common to both the groups while 44 and 35 metabolites were found unique to high- and low-fertile spermatozoa, respectively. Among the common metabolites, concentrations of 19 metabolites were higher in high-fertile compared to low-fertile spermatozoa (fold change > 1.00). Spermatozoa metabolites with variable importance in projections score of more than 1.5 included hypotaurine, d-cysteine, selenocystine. In addition, metabolites such as spermine and l-cysteine were identified exclusively in high-fertile spermatozoa. Collectively, the present study established the metabolic profile of bovine spermatozoa and identified the metabolomic differences between spermatozoa from high- and low-fertile bulls. Among the sperm metabolites, hypotaurine, selenocysteine, l-malic acid, d-cysteine, and chondroitin 4-sulfate hold the potential to be recognized as fertility-associated metabolites.

Scrotal infrared digital thermography predicts effects of thermal stress on buffalo (Bubalus bubalis) semen.

The objective was to use scrotal infrared digital thermography to evaluate effects of thermal stress on semen quality of Murrah buffalo (Bubalus bubalis) breeding bulls. Ejaculates from 109 Murrah bulls maintained at three semen stations were evaluated for: ejaculate volume, sperm concentration (SCON), initial motility (IM), percent live sperm (LIVE), acrosome integrity of fresh semen (AIFS), plasma membrane integrity of fresh semen (PMIFS), head abnormalities of fresh semen (HAFS), midpiece abnormalities of fresh semen (MPAFS), tail abnormalities of fresh semen (TAFS), post-thaw motility (PTM), acrosome integrity of post-thawed semen (AIPT) and plasma membrane integrity of post-thawed semen (PMIPT). Scrotal and ocular surface temperatures were acquired during rainy, winter and summer seasons, using an FLIR i5 infrared camera. Thermographic images were analyzed with Quick Report 1.2 SP2 software and temperature data acquired. Daily mean temperature and mean relative humidity were used to determine the temperature-humidity index (THI). Environmental factors were analyzed using CORR to determine collinearity among independent variables. There was a high correlation among THI, proximal, mid and distal scrotal temperatures (r ≥ 0.73). Therefore, distal pole temperature (DPT), THI, temperature gradient (TG) and ocular temperature (OcT) were used in the analysis of variance (ANOVA) and in regression analysis of dependent variables. The THI, DPT, TG and OcT had significant influences on sperm abnormalities, acrosome and plasma membrane integrity of fresh as well as post-thawed semen. The TG had significant effects on LIVE and SCON. All semen quality parameters were predicted (regression analysis) as a function of the three independent factors. We concluded that scrotal infrared thermography was useful for assessing influences of thermal stress and environmental factors on characteristics of buffalo semen.

Bovine Milk Comparative Proteome Analysis from Early, Mid, and Late Lactation in the Cattle Breed, Malnad Gidda (Bos indicus).

Bovine milk is important for both veterinary medicine and human nutrition. Understanding the bovine milk proteome at different stages of lactation has therefore broad significance for integrative biology and clinical medicine as well. Indeed, different lactation stages have marked influence on the milk yield, milk constituents, and nourishment of the neonates. We performed a comparative proteome analysis of the bovine milk obtained at different stages of lactation from the Indian indigenous cattle Malnad Gidda (Bos indicus), a widely available breed. The milk differential proteome during the lactation stages in B. indicus has not been investigated to date. Using high-resolution mass spectrometry-based quantitative proteomics of the bovine whey proteins at early, mid, and late lactation stages, we identified a total of 564 proteins, out of which 403 proteins were found to be differentially abundant at different lactation stages. As is expected of any body fluid proteome, 51% of the proteins identified in the milk were found to have signal peptides. Gene ontology analyses were carried out to categorize proteins altered across different lactation stages based on biological process and molecular function, which enabled us to correlate their significance in each lactation stage. We also investigated the potential pathways enriched in different lactation stages using bioinformatics pathway analysis tools. To the best of our knowledge, this study represents the first and largest inventory of milk proteins identified to date for an Indian cattle breed. We believe that the current study broadly informs both veterinary omics research and the emerging field of nutriproteomics during lactation stages.

Influence of season, age and management on scrotal thermal profile in Murrah bulls using scrotal infrared digital thermography.

The aim of the present study was to examine the effects of non-genetic factors on scrotal thermographic profile viz., proximal pole temperature (PPT °C), mid pole temperature (MPT °C), distal pole temperature (DPT °C) and ocular temperature (OcT) of Murrah (Bubalus bubalis) breeding bulls. A total of 109 buffalo bulls, maintained at three semen stations (SS), were monitored for scrotal surface and ocular temperatures using infrared thermography twice daily during rainy, winter and summer seasons using an FLIR i5 infrared camera and temperatures were measured. Thermograms were analysed by FLIR QuickReport v.1.2 SP2 software. Statistical analysis revealed that semen station, season, temperature humidity index (THI), housing system and timing of observations had significant (P < 0.05) effect on scrotal surface temperature (SST) and OcT. In SS-I, the PPT and MPT were significantly (P < 0.05) higher as compared to SS-II and SS-III. THI had significant (P < 0.05) effect on SST and OcT, whereas PPT (°C), MPT (°C), DPT (°C) and OcT (°C) values during high THI (>80.88; <0.05) period were higher as compared to medium THI period (70.06-80.88) and during low THI period (<70.06). Temperature gradient (TG) of the testes was significantly (P < 0.05) higher during low THI period (4.50 ± 0.06 °C) as compared to medium THI (2.38 ± 0.03 °C) and high THI (1.61 ± 0.05 °C). Season of the year had a significant effect (P < 0.05) on the SST and OcT. During the rainy season, PPT (34.50 ± 0.09 °C), MPT (33.44 ± 0.12 °C) and DPT (32.11 ± 0.15 °C) were significantly (P < 0.05) higher as compared to winter and summer seasons. Age of the bulls had non-significant effect on SST and OcT but had a marked influence on thermal profile of scrotum. It could be concluded semen station, season, temperature humidity index, housing system and timing of observations had a significant influence on scrotal surface temperature. The monitoring of scrotal surface temperature by infrared thermography was found to be useful in evaluating the effects of thermal stress on physiology and health of buffalo bulls.

Controlled breeding and reproductive management in water buffaloes (Bubalus bubalis) using Eazi Breed controlled internal drug release.

Buffalo reproduction is considerably affected by late maturity, poor oestrus symptoms and long postpartum periods. This study was undertaken to evaluate the efficiency of Eazi Breed controlled internal drug release (CIDR), an intravaginal progesterone-releasing device, in relation to oestrus and fertility. Five hundred true anoestrus buffalo cows, in the age group 4-6 years in 10 villages of Dharwad district in Karnataka state in India, were randomly selected and treated with CIDR for 9 days. Two mL of Cidirol (1 mg oestradiol benzoate) was administered intramuscularly to all animals on day 10. Forty-two buffaloes (8.4%) that failed to show oestrus signs (1.6%) or showed weak signs of oestrus (6.8%) after the first treatment were treated again 72 h after the Cidriol injection with a new device, and inseminated after the expression of oestrus. After the second treatment all the animals showed oestrus signs. The percentage of buffaloes showing intense oestrus was 67.40%, intermediate oestrus was shown by 25.80%, whilst 6.80% buffaloes showed weak oestrus even after the second treatment. The buffaloes showing oestrus signs were inseminated twice with an interval of 12 h, starting 12 h after the start of the oestrus signs. In 86 buffaloes showing prolonged oestrus signs a third insemination was done. The conception rates were 85.16%, 60.47% and 44.11% respectively in buffaloes showing intense, intermediate and weak oestrus. Transrectal palpation of the genital tract was performed 45-60 days post-insemination to diagnose pregnancy status, and in doubtful cases pregnancy was reconfirmed at 90 days after insemination. Out of 500 buffaloes treated in this way 380 animals became pregnant and the pregnancy rate was 76%. This study revealed the usefulness of Eazi Breed CIDR along with Cidirol treatment in buffaloes to improve their reproductive performance.

Calves born from anestrus yaks (Poephagus grunniens L.) subjected to Ovsynch and superovulation treatment.

An attempt was made to induce estrus and ovulation in eight anestrus yaks by use of the Ovsynch protocol. Six out of eight yaks were successfully induced into estrus, and ovulation occurred in all the responding yaks 1-2 days after the second GnRH administration. Out of the six animals that responded to the treatment, two mated naturally with yak bulls, and calves were obtained from them. The other four animals were further administered a superovulatory regimen of Folltropin (FSH-P). Following Folltropin and Ilerin (a PGF(2alpha) analog) treatment, the animals were subjected to natural insemination. Only one animal in which natural mating occurred was flushed non-surgically for embryo recovery 7 days post-insemination. Thereafter, all the donor animals were administered with Ilerin. After 48-72 h, they came into heat and mated naturally with yak bulls, and calves were obtained from them after expiration of the normal gestation period. Following superovulation, the average numbers of palpable corpora lutea in the right and left ovaries were 2.25+/-0.6 and 1.75 +/-0.3, respectively. Three embryos were recovered by non-surgical flushing from a single animal. One embryo was transferred to a recipient yak, who produced one female calf after 258 days. This is the first report of production of a yak calf through embryo transfer-technology.