Browsing by Author "KUMARESAN, A."
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ThesisItem Open Access ANTISPERM ANTIBODIES IN BOVINE: PREVALENCE AND RELATIONSHIP WITH FIELD FERTILITY(ICAR-NATIONAL DAIRY RESEARCH INSTITUTE, BENGALURU, 2019) KUNTAREDDI, CHANNAREDDI; KUMARESAN, A.ThesisItem Restricted CONTINUOUS RATE INFUSION OF BUTORPHANOL - LIDOCAINE - KETAMINE FOR ABDOMINAL SURGERIES IN CALVES(TANUVAS, CHENNAI, 2022) MERIL RAJ C.; TANUVAS; SENTHIL KUMAR S.; KUMARESAN, A.; SARAVANAN M.The study was conducted in 21 clinical cases of calves presented to Department of Veterinary Surgery and Radiology, Veterinary College and Research Institute, Orathanadu for various major abdominal surgical interventions. The study was conducted during March 2021 to October 2021. The animals were randomly divided into two groups viz., group I and group 11 consisting of fifteen animals and six animals, respectively. General clinical examination of all calves selected for the study was carried out and physiological parameters were recorded before anaesthesia in all the animals. Age, sex and bodyweight in kilograms of the animals were recorded. In all the animals, xylazine at the dose rate of 0.03 mg per kg body weight, butorphanol at the dose rate of 0.003 mg per kg body weight and ketamine at the dose rate of 0.25 mg per kg body weight were administered intramuscularly as premedicant.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 Restricted EVALUATION OF BALANCED ANAESTHETIC PROTOCOLS IN GERIATRIC DOGS WITH VARIOUS SURGICAL AILMENTS(TANUVAS, CHENNAI, 2021) VIGNESHWARAN S.; TANUVAS; KATHIRVEL S.; KUMARESAN, A.; VIJAYAKUMAR, G.; SRINIVASAN, P.The study was conducted on thirty six medium sized geriatric dogs. A total of 1610 dogs were screened for the selection and out of it 36 animals were selected for this study. The animals were divided into two categories viz., category IC (Cardiac) and category IIR (Renal) comprising of 18 animals each based on the cardiac and renal status. The category IC and IIR animals were further divided into three groups viz., ICK, ICP, ICE, IIRK, IIRP and IIRE and accordingly ketamine (K), propofol (P) and etomidate (E) were used for induction of anaesthesia. The demographic data of the selected animals revealed non-descript breed contributed major portion than the other breeds. The incidence of geriatric affections were high in males having cardiac aliments in group I animals and in females having renal affections in group II animals. The body weight of the selected animals was found between 20 to 27 kg regardless of cardiac and renal category. Higher incidence of surgical ailments was found in male dogs. The age of the selected animals in this study was above 11 years in both the cardiac and renal category.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 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.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 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 SALIVARY MUCIN PROFILING IN RELATION TO OESTRUS AND EARLY PREGNANCY IN DAIRY CATTLE(ICAR-SRS-NDRI, KARNAL, 2021) SHAJI, ARSHA; KUMARESAN, A.Accurate detection of oestrus and diagnosis of early pregnancy in dairy cattle still remains as a challenge, especially in field conditions. With the aim to identify potential method/tool for identification of oestrus and early pregnancy, the present study assessed salivary crystallization pattern in cows during the oestrus cycle and early pregnancy and also carried out global proteomic analysis of salivary proteins. Saliva was collected from a total of 15 Holstein Friesian crossbred cows during oestrous cycle and during early pregnancy. Salivary crystallization patterns were assessed using microscopy and proteomic profiling was done using a highthroughput LC-MS/MS-based method. At least 6 types of salivary crystallization patterns were discerned that include Fern-like, Branch-like, Fir-like, Branch like + Fir like, Fir like + Fern like, and Branch like + Fern like. Typical fern pattern is observed on the day of oestrus only, therefore it could be used as a tool for oestrus detection. The absence of a typical fern pattern in saliva on days 20-22 after breeding could be considered as an indication of pregnancy. Regarding proteomic analysis, a total of 3707 proteins were detected in the saliva of crossbred cows. A total of 2463, 1456, 2453 and 1574 non-redundant proteins were identified on the day of oestrus, day 9 of dioestrus, day 16 of pregnancy, and day 18 of pregnancy. A total of 549, 161, 486, and 197 proteins were specific to oestrus, dioestrus, day 16 of pregnancy, and day 18 of pregnancy. The presence of oestrus specific salivary proteins like 17-β hydroxysteroid dehydrogenase, Inhibin beta A chain, Heat shock 70kDa protein 1A, and, Alpha enolase have the potential to be used to identify oestrus in cattle. Most of the proteins specific to oestrus were involved in the metabolic process and the rest of them were involved in estrogen signaling, steroid biosynthesis pathways. Pregnancy-related proteins like Pregnancy Associated Glycoprotein, Gametocytespecific factor 1 like protein, Placental protein-9, Chorionic somatomammotropins hormone 2, and Interferon-tau were detected in the saliva of crossbred cows during early pregnancy, indicating that saliva could be used as a non-invasive method for early pregnancy diagnosis. Major pathways and protein-protein interaction found to be represented by the pregnant animal saliva proteins was the notch signaling pathway, hippo signaling pathway, p53 signaling pathway, phagosome, JAK-STAT cascade, etc. The study also identified few salivary mucins specific to oestrus that need to be validated on large number of cows before arriving at a conclusion on their usage for oestrus detection in cows.ThesisItem Open Access TESTICULAR TRANSCRIPTOMIC ANALYSIS OF CROSSBRED AND INDIGENOUS CATTLE(ICAR-NATIONAL DAIRY RESEARCH INSTITUTE, BENGALURU, 2019) K, ELANGO.; KUMARESAN, A.ThesisItem Open Access TRANSCRIPTOMIC PROFILING OF CROSSBRED BULL SPERMATOZOA IN RELATION TO FERTILITY AND CAPACITATION(ICAR-SRS-NDRI, KARNAL, 2020) M. ARUL PRAKASH; KUMARESAN, A.Although the significance of bull infertility has been documented well, the underlying reasons for high incidence of infertility in crossbred bulls still remains elusive. The present investigation was carried out to identify fertility related sperm transcripts in HF crossbred bulls and to find out the ability of spermatozoa to undergo capacitation on exposure to oestrous oviductal fluid and its relationship with bull fertility. The present study identified a total of 13,563 transcripts in crossbred bull spermatozoa; after total hit normalization, a total of 776 transcripts were detected, of which 524 transcripts were common to both high- and low-fertile bulls, while 84 sperm transcripts were unique to high-fertile bulls and 168 transcripts were unique to low-fertile bulls. Among the co-expressed genes, 176 genes were up-regulated while 209 genes were down regulated in low-fertile bulls. Using bioinformatics analysis, we identified that the biological processes down-regulated in low fertile bull spermatozoa included multicellular organism development and spermatogenesis. Further, ribosomal pathway was upregulated whereas oxidative phosphorylation was down-regulated in low fertile bull spermatozoa. Moreover, sperm transcripts unique to high fertile bulls were involved in oxidative phosphorylation pathway and transcripts unique to low fertile bulls were involved in ribosomal pathway. Select transcripts (TPT1, PFN1, ZNF706, CRISP2, MDB4, TNP2, ADIPOR1 and TNP1) were validated using qPCR and found that the transcriptional abundance of ZNF706, CRISP2, MDB4, TNP2 and TNP1 genes were significantly (p<0.05) higher in high-fertile bulls compared to low-fertile bulls and were positively correlated with conception rate (p<0.01). During incubation of spermatozoa from above- and below-average bulls with NL-ODF, it was observed that the extent of reduction in sperm viability and acrosome reaction between 0 h and 4 h of incubation was lower in above average bulls compared to below average bulls. Further, the hallmarks of sperm capacitation were significantly higher in above- compared to below-average bulls at 4 h of incubation indicating the dual role of oviductal secretions on spermatozoa. Transcriptional abundance of genes related to sperm capacitation (CATSPER1, PEBP1, CRISP2, ADIPOR1, ADIPOR2 and IQCF1) were upregulated in below average bulls. Conception rate was positively correlated (p<0.01) with sperm viability, intracellular calcium level and acrosome intactness at 4 h incubation with NL-ODF. It was therefore concluded that oxidative phosphorylation was the most affected pathway in low-fertile bull spermatozoa and the sperm transcriptional abundance of ZNF706, CRISP2, MDB4, TNP2 and TNP1 genes was significantly related to bull fertility and thus these genes could serve as potential candidate biomarkers for fertility in crossbred bulls. Further, the ability to undergo capacitation upon exposure to NL-ODF varied with individual bulls and also significantly related to bull fertility.