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ThesisItem Open Access INFRARED THERMOGRAPHY AND BEHAVIOURAL BIOMETRICS ASSOCIATED WITH CALVING IN BUFFALOES (Bubalus bubalis)(ICAR-SRS-NDRI, BENGALURU, 2022) TEJA ALLU; JEYAKUMAR, S.Calving is the process of delivery of the fully grown fetus on the completion of the normal pregnancy period. Parturition is an interesting biological process in the sense that the uterus that was quiescent during the entire pregnancy starts contracting and the cervix that was tightly contracted relaxes sufficiently to allow the passage of the young one to the world outside the mother’s womb, passing through the birth canal. This is one of the most important events for the farmers as by this act of his animal he would derive gain in terms of milk or sale of animal and its progeny. Prediction of calving time is a key element in livestock farming; it is used to decide when to move a buffalo to the calving pen and to decide whether human supervision is required, especially at night. Approximately 50% death of the dam/calf loss which occurs due to dystocia could be prevented by giving timely correct obstetrical assistance. As the calving approaches there are distinguish behavioural changes and temperature variation of the dairy animals. These changes can be used to predict approximate time of calving. The behavioural biometrics and the infrared thermographic profile of body temperature around calving are lacking in buffaloes. Therefore, aim of the present study was to investigate behavioural changes and thermal profile before and after calving in buffaloes to identify the imminent calving signs. The current study was conducted between August 2021 to February 2022 in buffaloes maintained at Buffalo Research Station of SVVU, Venkataramannagudem. Twenty-eight (28) multiparous pregnant Murrah buffaloes with parity ranged between 3rd and 7th were included for the study. Behavioural studies were monitored by digital video recording system (DVRS) and Infrared thermal imaging of five region of interest (ROI) viz., eye, muzzle, vulval region, tail base surface and udder skin surface temperature (USST) were recorded using FLUKE IR camera. Observations were made for all 28 buffaloes at six hourly intervals for a period of 96 hours before the expected date of calving, at the time of calving and 96 hours post calving continuously. Data were compiled and analysed statistically. The results (Mean±SE) of the present study are as follows. Behavioural activities of buffaloes revealed that both lying and standing time differed significantly (P<0.05) and transition occurred during last 12 h prior to calving. Frequencies of lying bouts and tail raising activity were found to increase significantly (P<0.000) 9.50±0.5 and 10.35±0.8 times during last 6 h, respectively. Rumination time decreased significantly (P<0.000) 36 h prior to calving from 193.23 ± 5.47 to 126.92 ± 2.56 min/ 6 h and observed marked reduction in 6 h before calving. Infrared thermographic (IRT) profile of eye and muzzle in buffaloes revealed that there was a significant (P<0.005) decrease in temperature from 48 h prior to onset of calving with a ΔT of 0.56 oC and 0.45 oC, respectively. Vulval region and tail base surface temperature showed significant (P<0.05) reduction from 48 h prior to onset of calving, with a ΔT of 0.45 oC and 0.48 oC, respectively. However, the vulval skin temperature increased from six hours prior to calving. Interestingly, in the present study, USST was significantly (P<0.0001) declined from 48 h prior to calving with a ΔT of 0.85 oC. Plasma progesterone concentration decreased from 72 h prior to calving and differed significantly (P<0.0001) between days to calving and reached 0.69 ng/mL at the time of calving. A wearable wireless thermal sensor prototype has been developed for continuous measurement of ventral tail base surface temperature. It is concluded that behavioural activities (lying bouts, tail raising and rumination time) and thermal biometrics (eye, muzzle, vulval region, tail base) and most importantly udder skin surface temperature could be used as potential non-invasive indicators for calving prediction within 6-12 h in buffaloes. On-farm tail base wearable thermal sensor device prototype can be explored for prediction of calving in dairy cattle and buffaloes in future.ThesisItem Open Access GENOME-WIDE ANALYSIS OF SEMEN MICRORNAS AS POTENTIAL BIOMARKERS FOR BULL FERTILITY(ICAR-SRS-NDRI, BENGALURU, 2022) ELANGO K; KUMARESAN, A.The bull fertility has been choreographed by the physical, endocrinological, genetic and epigenetic factors, among which the last two factors are quite intriguing as well as intricate to comprehend. Due to the failure of semen evaluation assays in fertility prediction, the omics technologies emerged to evaluate molecules i.e., DNA, RNA and protein in semen. Though miRNAs were studied extensively as a biomarker for various diseases, their ability in fertility prediction is barely addressed. Therefore, the present study analyzed the genome-wide miRNAs using small RNA next gen seq in semen of bulls with contrasting fertility, with the aim to find out fertility associated miRNAs. We detected 2628 miRNAs (Known-534, Novel-2094) in bull semen. A total of 1002 miRNAs were detected in sperm (Known-349, Novel-653) while 2005 miRNAs were detected in seminal plasma (Known-511, Novel-1494). The 250 known miRNAs were detected common between high- and low-fertile bull sperm, in which 77 were upregulated and 54 were downregulated. The 237 known miRNAs were detected common between high- and low-fertile bull seminal plasma, in which 100 were upregulated and 94 were downregulated in low-fertile bulls. The miRNAs involved in spermatogenesis (miR-135a, -197), sperm motility (miR-151-5p, -874), embryonic development (miR-34c, -451) and epigenetic inheritance (miR-1) were among the top 20 differentially expressed miRNAs in sperm and seminal plasma. The target genes of 10 abundantly expressed known miRNAs in sperm and seminal plasma of low-fertile bulls were predicted to be involved in MAPK signaling pathway and PI3K-Akt signaling pathway which are related to male fertility. Among the 2094 miRNAs novel to Bos taurus genome, 2071 miRNAs were novel to whole miRbase 22.1 database. Validation of ten select miRNAs in sperm and seminal plasma samples revealed that miR-2285bu and bull-miR-291 were significantly upregulated in low-fertile than high-fertile bulls. Bull-miR-291 and bull-miR-772 were not reported earlier in any organism but our study confirmed their expression in both bull sperm and seminal plasma using sequencing and qPCR. Moreover, we found bull-miR-291 to be related to bull fertility. The altered MAPK and PI3K-Akt signaling pathway were reported earlier as the reason for infertility, but this study points out miRNAs might be the major player in the background for bull infertility by targeted repression of mRNAs involved in these pathways.ThesisItem Open Access STUDIES ON SPERM FUNCTION TESTS AND ABUNDANCE OF FERTILITY-RELATED TRANSCRIPTS (CRISP2, PRM1, CCT5 AND CCT8) IN SPERMATOZOA OF SAHIWAL AND MURRAH BULLS(ICAR-NDRI, KARNAL, 2022) RAJU KUMAR DEWRY; MOHANTY, T.K.Insemination of females with low-fertile frozen-semen accounts for a significant economic loss to the dairy industry. Stressful factors like seasonal variation and vaccination effects deteriorate the quality of semen in breeding bulls. Assessment of the frozen semen through advanced sperm functions concerning fertility is of prime importance in the dairy animal breeding industry. There is an ardent need for developing in -vitro tests or a battery of tests that can rapidly and inexpensively determine the quality of semen to fertility. The study of sperm RNA transcripts may prove to be an essential tool in understanding the molecular mechanism behind motility, capacitation status, and fertilization regarding conception rate. The objectives of the present study were 1. Developing prediction model for sperm fertility using conception rate data and suitable combinations of in vitro sperm function tests for Indigenous dairy bulls and 2. To study the abundance of fertility-related candidate transcripts (CRISP2, PRM1, CCT5 and CCT8) in spermatozoa of dairy bulls in relation to fertility, season and vaccination effect. Sahiwal and Murrah breeding bulls were classified into high (52 and 45 %) and low (26 and 43%) fertile based on conception rate. As per the standard protocols, routine semen analysis was done on fresh semen ejaculates and cryo-preserved semen samples. Advanced sperm functions test for sperm acrosomal integrity, liveability, membrane integrity, CTC assay, lipid peroxidation and CASA parameters in a cryopreserved semen sample. Fresh semen sample was standardized by percoll gradient for RNA isolation, quantification, and identification of transcripts followed by bioinformatics analysis for differential gene expression in relation to fertility such as CRISP2, PRM1, CCT5 and CCT8. The results revealed that the most favorable period for all the seminal parameters was significantly (p≤0.05) higher in the winter season and the most affected season on seminal parameters was observed in the summer season in both high and low fertile Sahiwal and Murrah bulls. Most affected period for all the parameters was found within 15 days postvaccination and restoration were observed at 60 days post-vaccination in all the semen quality parameters in both high and low fertile bulls. The expression of CRISP2 and PRM1 gene was found to be up-regulated and significantly (p≤0.05) increased during rainy (2, 3, 2 fold) and winter (7, 10, 5 fold) season whereas, all the three genes were down-regulated in summer in low fertile Sahiwal and Murrah bulls. The expression of CCT5 and CCT8 gene was found to be significantly (p≤0.05) up-regulated during summer (9, 8 fold) as compared to winter in low fertile bulls. The expression of CRISP2 and PRM1 gene was found to be increasing gradually post-vaccination up to 60th day in both high and low fertile Sahiwal and Murrah bulls whereas, the expression of CCT5 and CCT8 gene was found to be significantly (p≤0.05) up-regulated during the post-vaccination period on 15th day and gradually decreased in both high and low fertile bulls. It can be concluded that the winter season is favorable for seminal attributes compared to the summer and rainy season between high and low-fertile bulls and the quality can be restored at 60 days post-vaccination in both breeds. The expression of positive fertility-related genes (CRISP2 and PRM1) was higher in the winter than in the summer and rainy seasons. The expression of negative fertility genes (CCT5 and CCT8) was up-regulated during summer compared to winter in low fertile Sahiwal and Murrah bulls. The expression of positive fertility-related genes was found to be decreased in the post-vaccination period (15th to 60th days) in both high and low fertile bulls and the expression of negative fertility genes was found to be increased in the postvaccination period (15th to 60th days) than pre-vaccination in low fertile as compared to high fertile Sahiwal and Murrah bulls.ThesisItem Open Access DEVELOPMENT OF A METHOD FOR NANOPURIFICATION OF FUNCTIONALLY COMPETENT BULL SPERMATOZOA(ICAR-SRS-NDRI, BENGALURU, 2022) Nilendu Paul; KUMARESAN, A.Poor conception rate following artificial insemination with cryopreserved semen is a matter of great concern as it results in slow genetic progress, increased generation interval and incurs a substantial economic loss to the farmers. Inferior sperm quality is often cited as the major reason for reduced conception rates (CR) in the bovine. Bull ejaculate contains different sperm subpopulations with different fertilizing potential. Therefore, enriching sperm subpopulations with superior functions are expected to yield high CR. In this regard, the present study was undertaken to develop a rapid nanopurification method to remove inferior spermatozoa so as to enrich spermatozoa with high fertility. For this purpose, dextran coated iron magnetic nanoparticles were produced using the coprecipitation method and characterized using dynamic light scattering, scanning electron microscopy-energy dispersive, X-Ray spectroscopy and Fourier-transform infrared spectroscopy. The present study found that dextran coated iron oxide nanoparticles did not alter sperm functional attributes in a time-dependent manner, thus nullifying any detrimental effects of nanoparticles on spermatozoa. FITC-PNA (0.6mg), Ubi-Ab (0.4mg) as well as Annexin V (0.6mg) coated MNPs were found to be effective in enriching the frozen thawed semen with more proportion of viable, non-apoptotic and acrosome intact spermatozoa with low intracellular calcium level (p<0.05). Further, we have used a combination of above mentioned three types of conjugated MNPs, and the resultant nanopurified semen showed significant enrichment of samples with spermatozoa population having higher viability (29.25%), acrosome intactness (26.69%), low intracellular calcium status (49.89%), and live nonapoptotic status (34.68%) as compared to control semen. It is concluded that nanopurification of spermatozoa successfully removed inferior quality spermatozoa in a given semen sample and the developed “Combo” nanopurification method could successfully enrich superior quality spermatozoa, which can be beneficial in artificial insemination as well as assisted reproductive techniques.ThesisItem Open Access MODULATION OF OVIDUCT MICROENVIRONMENT FOR PREFERENTIAL BINDING OF X-BEARING SPERMATOZOA IN DAIRY(ICAR-SRS-NDRI, KARNAL, 2021) PRADEEP NAG B S; KUMARESAN, A.The present study was undertaken to find out if modulation of micromilieu of oviductal cells alters the proportion of X- and Y- bearing spermatozoa binding to oviduct in cattle. As a prerequisite we standardized sperm oviduct explant binding model for cattle and also standardized a method for denudation of oviductal monolayer bound spermatozoa. oviductal cells were extruded from slaughterhouse derived non-luteal oviducts cultured in TCM-199 medium for 12 hours at 5% CO2 and 38.5°C to prepare oviduct explants. Sperm concentration (2 million), incubation timings (1 hr) and staining method Hoechst 33342 were standardized for obtaining optimal binding index. Using the standardized sperm oviduct model, the phenotypic and functional characteristics of oviductal cells bound and unbound sperm were assessed and found that membrane integrity, acrosome intactness and mitochondrial membrane potential were significantly (P<0.05) higher in bound sperm population in comparison with unbound sperm population. Sperm phenotype characteristics assessment indicated that highfertile bulls had significantly (P<0.05) higher oviduct explant binding index, membrane integrity, acrosomal intactness and mitochondrial membrane potential, and significantly (P<0.05) lower % DNA fragmentation index when compared to low-fertile bulls. In vivo experiment was conducted on cows (n=20) to study the effect of oral administration of calcium preparation on sex of the offspring; out of 6 calves, 5 calves were females. Based on the biochemical profile assessed in cows (n=6) three doses of calcium (0, 1 and 3 mM), magnesium (0, 1 and 3 mM) and glucose (0, 1 and 2 mM) were selected for in vitro modulation, Modulation of oviduct monolayer revealed that calcium at 1mM and 3mM facilitate 15.6 and 5.2 times, respectively more X chromosome bearing spermatozoa. In contrast, magnesium at 1mM and 3mM facilitated 22 and 37.4 times, respectively more Y chromosome bearing spermatozoa. However, we did not observe any clear deviation in sex ratio of bound spermatozoa. it was concluded that sperm phenotypes viz. sperm viability, acrosome intactness and high mitochondrial membrane potential are prerequisite characters to bind to oviduct. Further, and incorporation of calcium in incubation medium skewed sex ratio of oviductal cells bound spermatozoa towards X-bearing spermatozoa, while incorporation of magnesium skewed the sex ratio towards Y-bearing spermatozoa.ThesisItem Open Access IDENTIFICATION AND VALIDATION OF ESTRUS ASSOCIATED CANDIDATE PROTEINS IN BUFFALOES(ICAR-NDRI, KARNAL, 2020) SINGH, LAISHRAM KIPJEN; MOHANTY, TUSHAR KUMAREstrus detection is a major problem in buffaloes due to its poor manifestation of estrus signs and higher incidences of silent estrus (29%) especially during summer. Therefore, identification of potential estrus biomarkers in easily accessible bio-fluid like saliva and cervico-vaginal mucus fluid (CVF) will be of immense use. The present study was designed to identify and validate estrus associated proteins in saliva and CVF using label free (LFQ) and labeled (TMT) method coupled with LC-MS/MS analysis. Pluriparous Murrah buffaloes (n=14) maintained at the Livestock research centre, NDRI, Karnal and a total of 18 estrous cycles were included for this study. Global proteome profiling of saliva resulted in the identification of 936, 844, 809 and 870 proteins during the proestrus, estrus, metestrus and diestrus stage of the estrous cycle. Out of these, 70, 27, 25 and 43 proteins were found specific to proestrus, estrus, metestrus and diestrus stages of estrous cycle. Differential proteome analysis of saliva using LFQ revealed the identification of 742 differentially expressed proteins (DEPs) and out of these, 133, 162 and 210 proteins were up-regulated (fold change ≥1.5) in estrus vs. proestrus, metestrus and diestrus stage. Similarly, using TMT-LC-MS/MS analysis revealed the identification of 689 DEPs in buffalo saliva and out of these, 201, 213 and 252 proteins were up regulated (fold change ≥1.5) during estrus compared to proestrus, metestrus and diestrus stage. Few vital proteins in saliva found important for estrous physiology viz., HSPA1A, VMO1, SERPINB1, SDF4, NUCB1, LCN1, OBP, and ENO3. Differential proteome analysis in CVF using LFQ-LC-MS/MS analysis identified 2463 DEPs and out of which 455, 476 proteins were up regulated during estrus compared to pre-estrus and post-estrus stage. Few important CVF proteins found upregulated during estrus phase include NUCB2, LCN1, SDF4, B2M and OBP. This study highlights the identification and validation of candidate estrus biomarker in saliva and CVF, which can be used to develop on-spot diagnostic for estrus identification, staging of the estrous cycle and deciding the exact time of insemination in buffaloes.ThesisItem Open Access STUDIES IN BULL SEMINAL PLASMA PROTEOME AND REDUCTION OF SPERM DOSES ON CRYO-SURVIVABILITY AND IN VITRO SPERM FUNCTIONS(ICAR-NDRI, KARNAL, 2020) YADAV, HANUMAN PRASAD; MOHANTY, TUSHAR KUMARThe present study was conducted for the assessment of in vitro sperm functions and seminal plasma proteome of bulls with differential fertility, method(s) for immobilization and revival of bull sperm and cryo-survivability of low sperm number per dose in Murrah bulls. A total no. of Murrah (n=8) and Sahiwal bulls (n=8) were divided into high-fertile (HF) and lowfertile (LF) groups based on conception rate of 43-58 % and 23-34%, respectively. In post-thaw, PM, live and AI in HF bulls was found to be highly significant (p≤0.05) as compared to LF Murrah bulls, however: VAP, STR, TM, PM, moribund and dead sperm, B-pattern, AR-pattern were differed significantly (p≤0.05) in HF and LF bulls. For sperm kinetics and sperm function, VCL, ALH, LIN, RM, live sperm, F-pattern, LAI and MMP were significantly (p≤0.05) higher in HF group as compared to LF Murrah bulls. Furthermore, a significant (p≤0.05) higher difference was also found in LAR, DAR, CMA3 deficiency and lipid peroxidation in LF as compared to HF Murrah bulls. For Sahiwal bulls, sperm kinetics and sperm function, which includes post-thaw, PM, HOST, VCL and ALH differ significantly (p≤0.05) in HF as compared to LF. Further, postthaw sperm live, AI, TM, PM and RM, live sperm, LAI and JC-1 was highly significant (p≤0.05) in HF as compared to LF. However, significant (p≤0.05) differences in STR, LIN, and SM, moribund and dead sperm, LAR, DAR and CMA3 were found in LF as compared to HF. In the case of sperm immobilization, the PM of sperm was highly significant (p≤0.05) at zero, 30, 60, 90,120, 150 and 180 mins in different groups of Ouabain viz. I (Control) II, III, IV, V and VI respectively and above time interval different groups of Amiloride viz. Groups; I (Control), II, III, IV and V respectively in HF Murrah bulls. In revival with Nigericin, PM, live, HOST and AI of sperm were found to be highly significant (p≤0.05) at 185 mins in different groups of Ouabain viz. I (control), II, III, IV, V and VI respectively, however PM, live, HOST and AI of sperm was highly significant (p≤0.05) in different groups of Amiloride viz. Groups; I (Control), II, III, IV and V respectively. In case of low dose sperm Murrah bulls, the post-thaw PM, live, HOST, AI, VAP and VSL were significantly (P≤0.05) high in 10, 15 and 20 million (M) sperm doses as compared to 5M sperm doses in HF Murrah bulls. Further, TM differed significantly (P≤0.05) 5, 10, 15 and 20M sperm doses in HF bulls. The RM was significantly (P≤0.05) higher in 15 and 20 M sperm doses as compared to 5 and 10 M sperm doses in HF bulls. The post-thaw live sperm, Fpattern, LAI and MMP was significantly (P≤0.05) high in 10, 15 and 20M sperm doses as compared to 5 M sperm doses in HF bulls. The post-thaw AR, LAR, DAI and LPO of sperm were significantly (P≤0.05) high in 5, 10 and 15M sperm doses as compared to 20 M sperm doses in HF bulls. For seminal plasma proteome studies: 8 Murrah bulls (4HF + 4LF) and 8 Sahiwal bulls (4HF + 4LF) were included and undergone with Tandem Mass Tag. The total no. of proteins in Murrah bulls was found to be 1545. In conclusion, both HF Murrah and Sahiwal bulls exhibited better post-thaw in vitro sperm functional attributes in comparison to their respective LF bulls. Differential sperm motility inhibition potential of Ouabain and Amiloride with the revival of sperm functionality with Nigericin treatment was found to be significant. In low dose sperm /straw (20, 15, 10 and 5 M); high sperm doses (20, 15 and 10 M) performed better during in vitro sperm function tests than the low sperm dose (<10 M). The total no. of SP protein group and peptides in Murrah bulls identified was 934 and 9868. Up and down-regulated proteins were 30 and 6.ThesisItem Open Access IDENTIFICATION OF NOVEL TRANSCRIPTS AND METABOLITES IN SPERMATOZOA OF MALNAD GIDDA(ICAR-SRS-NDRI, KARNAL, 2019) GUPTA, MOHUA DAS; KUMARESAN, A.Breed variations in fertility have been reported in literature with comparatively lesser infertility in zebu breeds as compared to other breeds. While the problem of infertility is comparatively higher in crossbred males as compared to pure bred males, the reason for the same has not been elucidated yet. We hypothesized that comparative analysis of molecular profile of spermatozoa from dwarf zebu and crossbred bull might help in identification of possible alterations in spermatozoa of crossbred bull. On the other hand, the possible molecular pathways associated with high fertility in dwarf could also be identified. With this back drop, global transcriptomic and metabolomic profile of a dwarf zebu (Malnad Gidda) and crossbred (HF crossbred) bull spermatozoa was carried out. A total of 15814 and 17324 transcripts were identified in dwarf zebu bull spermatozoa and crossbred bull spermatozoa respectively, of which 521 transcripts were differentially expressed between dwarf zebu bull and crossbred bull spermatozoa. Among the differentially expressed transcripts 156 were upregulated while 365 were down regulated in dwarf zebu bull spermatozoa as compared to crossbred bull spermatozoa. Olfactory transduction pathway was upregulated whereas Ribosomal pathways, Oxidative phosphorylation and metabolic pathways were downregulated in dwarf zebu bull spermatozoa. Metabolomics analysis of sperm and seminal plasma revealed 1372 and 1240 metabolites in dwarf zebu bull and crossbred bull spermatozoa, respectively of which 71 metabolites were differentially expressed between dwarf zebu bull and crossbred bull spermatozoa respectively. A total of 1002 and 990 metabolites were identified in dwarf zebu bull and crossbred bull seminal plasma, respectively; of which 6 metabolites were downregulated in dwarf zebu bull spermatozoa as compared to crossbred bull spermatozoa. A metabolite Nitroprusside was 2.6 fold higher whereas LCysteine and Acetyl-CoA were 2.4 fold lower in dwarf zebu bull as compared to crossbred bull spermatozoa whereas, Tetradecanoyl-CoA and Phosphotidylethanolamine were 3.5 and 3 fold lower in dwarf zebu bull seminal plasma as compared to crossbred bull seminal plasma. It is concluded that transcriptional abundance of genes associated with Olfactory transduction were significantly higher in dwarf zebu bull spermatozoa as compared to crossbred bull spermatozoa. Expression of transcripts involved in ribosome pathway and oxidative phosphorylation were significantly upregulated in crossbred bull spermatozoa as compared to dwarf zebu bull spermatozoa. A total of 2 novel transcripts and 3 noncoding RNAs were identified in dwarf zebu bull spermatozoa. Taurine and hypotaurine metabolism pathway were highly enriched and concentration of nitroprusside was significantly higher in dwarf zebu bull spermatozoa as compared to crossbred bull spermatozoa. These findings might help in understanding the reasons for high incidence of infertility/ subfertility in crossbred bulls and to improve the quality of cryopreserved spermatozoa.ThesisItem Open Access IDENTIFICATION OF CANDIDATE BIOMARKERS FOR FERTILITY IN CROSSBRED BULL SPERMATOZOA THROUGH FUNCTIONAL GENOMICS APPROACH(ICAR-SRS-NDRI, KARNAL, 2020) SARAF KAUSTUBH KISHOR; KUMARESAN, A.The incidence of infertility/ sub-fertility is higher in crossbred males, however the aetiology remains obscure. Altered sperm functional attributes have been reported to be associated with infertility in this breed of cattle. To understand the molecular mechanisms involved in spermatozoa functionality, the first step is to characterize the molecular signatures present in the spermatozoa. Here, our aim was to profile the transcripts, proteins and metabolites in crossbred bull spermatozoa and to compare the differences between high- and low-fertile crossbred bulls. Frozen thawed spermatozoa (from three high- and low-fertile bulls each) were analysed using advanced molecular techniques like DNA microarray and Liquid chromatography & mass spectrometry (LC-MS) for transcriptomic, proteomic and metabolomic profiling coupled with Bioinformatics tools. Microarray experiment detected a total of 561 gene transcripts in crossbred bull spermatozoa. Among these, 321 and 240 transcripts were up- and down-regulated, respectively in low-fertile compared to high-fertile bulls. The most significantly affected pathway in low fertile spermatozoa includes GnRH signaling pathway, MAPK signaling pathway, oocyte meiosis and Wntsignaling pathway. Proteomic analysis of sperm membrane protein identified 456 proteins. Among all proteins, 399 proteins were commonly present in both the study groups; 108 proteins had higher abundance while 26 proteins had lower abundance in low fertile bull spermatozoa. The significantly enriched pathways were Oxidative phosphorylation and Citrate cycle (TCA cycle). We observed the presence of 3704 metabolites in bull spermatozoa.The results showed that, 33 metabolites were differentially regulated between high and low fertile bulls. Spermatozoa metabolites with variable importance in projections of more than 1.5 included hypotaurine, D-cysteine, selenocystine. In addition, metabolites such as spermine, L-cysteine were identified exclusively in high-fertile spermatozoa.Multi-omic data integration revealed that, 133 proteins were found to have corresponding transcripts in microarray data. The translated proteins play a significant (p < 0.05) role in oxidation-reduction process, ATP synthesis coupled proton transport, protein stabilization and binding of sperm to zona pellucida. In conclusion, the most affected pathways in low-fertile crossbred bull spermatozoa are oxidative phosphorylation, GnRH signaling, MAPK signalling, oocyte meiosis and Wntsignaling pathway. Hypotaurine, selenocysteine, L-malic acid, D- cysteine and chondroitin 4-sulfate holds potential to be recognized as a potential fertility associated metabolites.
