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National Dairy Research Institute, Karnal
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ThesisItem Open Access ELUCIDATING THE EFFICACY OF SODIUM ALGINATE AS AN ANTIBACTERIAL ALTERNATIVE IN CRYOPRESERVATION OF SAHIWAL BULL SEMEN(ICAR-NDRI, KARNAL, 2022) MOHIT KUMAR; NISHANT KUMARThe present study was conducted to investigate the efficacy of sodium alginate (S.A.) as an antibacterial alternative in cryopreservation of Sahiwal bull semen. In 1st objective, each of the pre-dominant Gram –ve (Proteus spp.) & Gram +ve (Bacillus spp.) bacterial isolates from Sahiwal bulls were collected and the antibacterial potency of sodium alginate was determined. Minimum inhibitory concentration (MIC) was determined by the broth microdilution method, in which two-fold serial dilutions of S.A. were done from 10 to 0.078 mg/ml concentration. It was found that the MIC of S.A. against Proteus spp. & Bacillus spp. was 0.625 and 1.25 mg/ml respectively. Similarly, the minimum bactericidal concentration (MBC) of S.A. against Proteus spp. & Bacillus spp. was found to be 0.625 and 2.5 mg/ml respectively. In 2nd objective, effect of S.A. as an antibacterial alternative on microbial load and freezability of Sahiwal bull semen was studied. Dose tolerance test in semen, as a pilot study was done by using six different S.A. concentrations (without streptopenicillin) based on MIC doses keeping one group as negative control (without streptopenicillin). Among these treated groups, three best doses (i.e. 1.25, 0.625 & 0.313 mg/ml) were selected on the basis of progressive motility & CFU/ml count for the standardization experiment. In standardization experiment, extended ejaculates were split into 3 treatment groups (1.25 mg/ml T1; 0.625 mg/ml T2; and 0.353 mg/ml T3- no antibiotics) and one control (Penicillin-1000IU/ml; Streptomycin-1mg/ml) group. Post-dilution, pre-freeze, post-thaw seminal parameters & bacterial load (CFU/ml) were evaluated, and T2 (0.625 mg/ml S.A.) was selected as the best dose for the final experiment. In the final experiment, ejaculates were split into 3 groups; one was kept as control (Penicillin-1000IU/ml; Streptomycin-1mg/ml), T1 (0.625 mg/ml S.A., no antibiotics) and T2 (0.625 mg/ml S.A. + antibiotics) groups. It was found that progressive motility, viability, HOST and acrosomal integrity of spermatozoa in the post-thaw stages was found to be significantly higher (p<0.05) in the T2 group as compared to the control & T1 group. Bacterial load (CFU/ml) at all the cryopreservation stages was found to be significantly lower (p<0.05) in the T2 group as compared to the control. Cryo-capacitation were significantly (p<0.05) reduced in the T2 group as compared to the control and T1 group at the post-thaw stages. No significant difference (p<0.05) was recorded in the protamine deficiency of the spermatozoa between the control, T1 & T2 groups at post-thaw stages. In the CASA study, total motility (TM), and progressive motility (PM) was found to be significantly higher (p<0.05) in T2 as compared to the control and T1 group. While there was no significant difference recorded in curvilinear velocity (VCL), straight linear velocity (VSL), average path velocity (VAP), and beat cross frequency (BCF) in the control and T2 groups. It can be concluded that S.A. has limited anti-bacterial activity as compared to strepto-penicillin, however it potentiates the antibacterial activity of strepto-penicillin if used in the supplementation. Also, S.A. in supplementation with antibiotics, enhances progressive motility, viability, plasma membrane integrity and acrosomal integrity of the sperm, as it protects the sperm from the adverse effects of cryopreservation.ThesisItem Open Access Early pregnancy diagnosis using color doppler ultrasonographic monitoring of luteal blood flow and growth dynamics in cattle(ICAR-SRS-NDRI, KARNAL, 2022) Majumder, Kaushik; S. JEYAKUMAREarly pregnancy diagnosis is a fundamental aspect of reducing the calving interval by identifying open animals early and rebreeding them as soon as possible. The Corpus luteum (CL) is a highly vascularized transitory endocrine gland that produces progesterone to support pregnancy. Luteal blood flow is a more accurate predictor of the CL function and serum P4 concentration than the CL size, particularly during the CL's regression phase. In this context, the evaluation of luteal blood flow by color Doppler ultrasonography can be a reliable parameter for diagnosing pregnancy. The aim of this study was to evaluate the CL growth dynamics and luteal blood flow changes in relation to early pregnancy. Multiparous (n=18) Deoni cows between 3-5 parity were selected for this study. Ultrasonographic examination and serum progesterone estimation was performed on days 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 33, 40, and 47 after estrus or until exhibition of next estrus, with or without insemination. Grayscale (B-mode) wasused for examining CL morphometry like diameter, tissue area, volume, and color-flow Doppler was used for assessing luteal blow area. Deoni cows were retrospectively allocated as being pregnant (n=6) or non-pregnant (interoestrus interval 18-21 days; n=6) by per rectal pregnancy diagnosis method on day 47, and were compared with nonbred cyclic control Deoni cows (n=6). The results revealed that the CL morphometry and blood flow area were progressively increasing from day 5 to 47 of the gestation period in pregnant cows, whereas it decreased from day 19 in inseminated non-pregnant and cyclic control cows. The CL diameter, volume and tissue area of pregnant cows was significantly higher than non-pregnant, and cyclic control (P<0.05) on day 19. Similarly, corpus luteal blood flow area was significantly higher (P<0.05) on day 19 in pregnant than in non-pregnant, or cyclic control cows. Though the CL vascularity index showed a similar trend, there was no significant difference between the groups. The serum P4 concentration declined from day 17 in non-pregnant and cyclic control cows and on day 19 it was significantly higher (P<0.05) in pregnant cows. In the present study, the CL morphometry and luteal blood flow on day 19 was significantly higher in pregnant than non-pregnant cows and are reflective of serum P4 concentration. Therefore, it was concluded that CL morphometry and CL blood flow area could be potential non-invasive predictors for early pregnancy diagnosis on day 19 in Deoni cows.ThesisItem Open Access EFFECT OF Murraya koenigii AND MELATONIN ON MODULATION OF BUFFALO GRANULOSA CELL FUNCTION IN HEAT STRESS CONDITION(ICAR-NDRI, KARNAL, 2022) CHIRAG PRUTHI; RUBINA K. BAITHALUThis study aimed to establish the buffalo granulosa cells (GCs) culture model mimicking the intra-follicular environment in order to access the effect of heat stress on cell viability and functional characteristics of buffalo GCs and its amelioration by treatment of melatonin and Murraya koengii plant extracts. Buffalo GCs isolated from small antral follicles were cultured for 6 days in 3D-like by using gelatine pre-coated plates and a 3D culture model by using the hanging drop (HD) method. Three different cells seeding densities (2, 3, and 4X105/mL) were compared and found that seeding density of 4X105/mL was optimum to develop GCs of preovulatory phenotype after 96 hrs of culture based on morphology and functional attributes (CYP19, FSHR, RUNX, and PCNA) in both the culture models. Based on structural and functional characteristics, a 3D culture model using the hanging drop method was selected to access the effect of heat stress by exposing GCs to higher temperatures (40.5˚C & 41.5˚C) and the functional attributes of cells were studied. Exposure to acute heat stress for 2-24 hrs after attaining preovulatory phenotype showed a significant difference in morphology, growth characteristics, and functional attributes. Chronic heat stress exposure for 24-120 hrs after 24 hrs of culture affected the GCs growth and cells didn’t form compact spheroids and started to lose their phenotype earlier (after 72 hrs) than in the control group (120 hrs). Further, heat stress exposure affected the function of GCs by reducing the expression of PCNA, CYP19, FSHR, and RUNX and increasing the expression of HSP70 after 72 hrs. Subsequently, the GCs in the 3D culture model were treated with melatonin and Murraya koenigii plant extracts to study their ameliorating effect against heat stress on GCs functional attributes. Total phenolics (TP), Total flavonoid content (TFC), and reducing power of Murraya koenigii leaves were 459.67±9.1 μg GAE/mg, 788.83±5.49 μg QE/mg, and 16556.81±1445.2 μmol TE/mg in methanolic extract respectively and 417±69.9 μg GAE/mg, 52.21±14. μg QE/mg and 20042.95±1866.32 μmol TE/mg in aqueous extract respectively. Cytotoxicity assay revealed maximum cell proliferation at concentrations of 0.390 mg/mL, 3.125 mg/mL, and 300 ng/mL for methanolic extract, aqueous extract, and melatonin, respectively. Melatonin & plant extracts (aqueous and methanolic) treatment to GCs has shown an ameliorative response against heat stress by the maintenance of their structural & functional phenotype evidenced by significantly higher expression (p<0.05) of PCNA, CYP19, FSHR, antioxidant genes (CAT and SOD2) & reduced expression (p<0.05) of HSP70 & BAX transcript under acute & chronic heat stress condition. The estradiol-17-β (E2) analysis in the spent culture medium revealed reduced basal production of estradiol in heat stress conditions (acute- 6.48 pg/μg of cellular protein; chronic- 6.76 pg/μg of cellular protein) compared to the control (acute-7.52 pg/μg of cellular protein and chronic-8.26 pg/μg of cellular protein) whereas, melatonin treatment improved basal production of estradiol under control (acute-9.06 pg/μg of cellular protein and chronic-8.07 pg/μg of cellular protein) and heat stress condition (acute-8.48 pg/μg of cellular protein and chronic-7.27 pg/μg of cellular protein). It could be concluded that the hanging drop method with a seeding density of 4X105 cells/ mL is one of the best strategies to culture buffalo GCs mimicking the inta-follicular environment. Heat stress exposure to GCs hampers the growth, differentiation, and steroidogenesis which can be ameliorated by the use of Murraya koenigii leaves extracts and melatonin.ThesisItem Open Access PROTEOMIC PROFILING OF OVARIAN FOLLICULAR FLUID FROM NORMAL AND PROLONGED FOLLICULAR DOMINANCE IN DEONI COWS(ICAR-SRS-NDRI, KARNAL, 2022) NIRIBILI RAJBANGSHI; S. JEYAKUMARDairying is an integral part of animal husbandry, and efficient reproduction in dairy cattle makes a distinct contribution to the socio-economic growth of the country. Prolongation of follicular dominance is one of the conditions associated with disconcerted follicular dynamics that result in substantial economic losses through low reproductive efficiency. Despite the fact that there is a lot of research available on the mechanisms of prolonged follicular dominance and oocyte viability, the proteomic alterations in the microenvironment of the prolonged follicle remain unknown. In this regard, we investigated the ultrasonographic characteristics of follicles as well as the proteomic alterations in follicular fluid from normal (NDFF) and prolonged dominant follicles (PDFF). Prolonged dominance was induced in Deoni cows (n = 6) by placing CIDR (previously used for 7 days) intravaginally from day 4 to 8 of estrus, with a luteolytic dose of PGF2 on day-6 and day-7resulting in subluteal progesterone concentration. Ultrasonographic examination revealed no significant difference in the size and growth pattern of normal and prolonged dominant follicles. The concentration of serum progesterone in the prolonged follicular dominance group was significantly higher (P<0.05) than in the normal follicular dominance group on the day of transvaginal follicular aspiration (day-8). Furthermore, global proteomic analysis of follicular fluid detected 217 proteins in the Deoni cow, with the majority of the identified proteins involved in 21 pathways, 42 molecular functions, and 106 biological processes. Complement and coagulation cascades (22.8%) and cholesterol metabolism (4.68%) were the major pathways in which identified proteins were involved. In comparison to NDFF, 26 proteins were dysregulated in PDFF. Among them, 15 proteins showed upregulation, and 11 proteins showed downregulation. Proteins involved in complement and coagulation cascades, and vitamin digestion and absorption were found to be dysregulated. In this study, the ovarian follicular characteristics were not different in cows experiencing prolonged follicular dominance from those of normal follicular dominance. However, the expression of proteins involved in inflammation, oocyte metabolism, and ovulation of follicles was dysregulated in the follicular fluid of cows with prolonged follicular dominance, which might be responsible for delayed ovulation.ThesisItem Open Access SEASONAL VARIATIONS IN TESTICULAR BIOMETRY, SEMINAL ATTRIBUTES AND FREEZABILITY OF BLACK BENGAL BUCKS(ICAR-NDRI, KARNAL, 2022) PRINCE CLINTON RAVA; M. KARUNAKARANThe present research work entitled “Seasonal variations in testicular biometry, seminal attributes and freezability of Black Bengal bucks” was undertaken at goat farm, ICAR National Dairy Research Institute, Eastern Regional Station, Kalyani, West Bengal during the months of November, 2021 to May, 2022. The investigation was undertaken to study the testicular biometry and seminal parameters of Black Bengal bucks during pre- and postpubertal age and to study the changes in semen quality parameters during different seasons like autumn, winter, spring and summer. Black Bengal males of different ages were selected and categorized into different groups viz. aged between 4-12 months, between 13-24 months, above 15 months. Further male kids aged between 2-8 months were used to study the testicular parameters vis-avis semen quality obtained from cauda epididymis of bucks after castration at different age intervals. The body weight (kg), scrotal circumference (cm), testicular length(cm), testicular width(cm), testicular thickness(cm), scrotal skinfold thickness was recorded every 15 days from 120±10 days along with serum testosterone (ng/ml) estimated by Goat Testosterone ELISA Kit at monthly interval. Collection of semen was attempted from 4 months onward during pre- and post- pubertal age at weekly interval period to confirm the age at puberty. The semen was evaluated for macroscopic and microscopic tests. It was found that body weight differed significantly across the age groups. The mean scrotal circumference increased with the advancement of age and attained maximum circumference of 22.53±0.44 (cm). Testicular length, width and thickness gain was maximum at 5 to 6 months of age. Body weight had highest correlation with age followed by scrotal circumference, testicular length, testicular width, testicular thickness, scrotal skinfold thickness. All these biometrical parameters were highly correlated with each other, and with age and body weight. The semen collected at 6 months of age revealed the presence of structurally and functionally matured sperm cells. The mean testosterone concentration increased from 2.07 ±0.74 to 6.086 ± 0.27 ng/ml between 2 and 6 months of age, respectively. Significant effect of the seasons (autumn, winter, spring and summer) was observed on the semen parameters with significantly higher sperm concentration, mass motility, post thaw motility and significantly lower abnormal sperm cell during summer season. It can be concluded that Black Bengal bucks attain puberty by six months of age and semen collected during summer season had significantly better pre and post freeze thaw seminal parameters.ThesisItem Open Access IMPACT OF OXIDATIVE STRESS ON BULL SPERM PROTEOME AND FUNCTIONAL ATTRIBUTES(ICAR-SRS-NDRI, KARNAL, 2022) APOORVA VERMA; KUMARESAN A.Reactive oxygen species (ROS) are involved in several sperm functions, but when their levels exceed beyond physiological concentrations, leads to oxidative stress and associated alterations in sperm fertilizing potential. Recent studies suggested that ROS is the major cause of infertility in case of human being, however the consequence of oxidative stress on bovine sperm are unclear, and the alterations in the spermatozoa at the protein level due to oxidative stress is not known. In this regard, the current study aimed to explore sperm functional attributes and proteome of the oxidative stress induced spermatozoa. Freshly ejaculated spermatozoa from Deoni (Bos indicus) bulls were subjected to oxidative stress (50μM H2O2) either in the presence or absence of seminal plasma. Flow cytometric analysis revealed that oxidative stress resulted in higher (p<0.05) proportion of ROS positive spermatozoa, dead spermatozoa, altered intra-cellular calcium concentrations and lipid peroxidation status but did not have any significant effect on sperm acrosome reaction status and mitochondrial membrane potential. Global proteomic analysis detected a total of 1441 proteins in Deoni bull spermatozoa, which are involved in 70 pathways, 118 molecular functions and 141 biological processes. The major pathway in which the identified proteins were involved was metabolic process (26%) and oxidative phosphorylation (7%). In the absence of seminal plasma, a total of 1434 proteins were identified in oxidative stress induced bull spermatozoa; among dysregulated proteins, 260 were upregulated while 191 were downregulated. A total of 78 proteins were identified to be specific to oxidative stressed spermatozoa. Among the differentially expressed proteins, 27% were involved in metabolic process, 8% were involved in reactive oxygen species and 7% were involved in oxidative phosphorylation. Significant number of proteins were involved in biological process like response to oxidative stress, spermatogenesis, protein stabilization and folding, and sperm motility. In the presence of seminal plasma, a total of 1325 proteins were identified in oxidative stress induced bull spermatozoa; among dysregulated proteins, 32 were upregulated while 21 were downregulated. It is concluded that oxidative stress significantly altered the proteomic profile of bull spermatozoa and presence of seminal plasma did not have any beneficial effect on sperm oxidative stress, functional attributes and protein profile in bulls. The findings provide valuable information regarding functional and subcellular changes in sperm due to oxidative stress.ThesisItem Open Access DEVELOPMENT OF PHOTOBIOMODULATION SYSTEM FOR ASSESSING BULL SPERM QUALITY(ICAR-NDRI, KARNAL, 2022) HITESH KUMAR BAGRI; T.K. MOHANTYThe goal of the current study was to determine the impact of various light spectra with different timings of exposure over the critical characters of sperm. An in-housing light chamber was constructed from the modifications of a spectrophotometer. This apparatus uses a halogen LED to produce non-chromatic light, which is then transformed into a monochromatic light by the filter. Light from the 400-700nm wavelength range was used in the pilot study with a time range of 1-4 min. 28 combinations of wavelength and time were used to evaluate the sperm motility immediately and two hours after the exposure. Six ejaculates were used in the PBS diluent in the pilot trial and kept at 37 °C. Among the 28 combinations, we selected the top 5 combinations based on motility for the objective-I, which were 600 nm for 2 min (T1), 600 nm for 3 min (T2), 650 nm for 2 min (T3), 650 nm for 3 min (T4), and 700 nm for 3 min (T5). In objective 1, extended ejaculates were split into 6 aliquots and exposed to all different treatments, with one split kept as a control. Motility, viability, HOST and acrosome integrity were basic semen parameters that were measured with time gaps ranging from 30 minutes and analysed up to 2 hours. It was found that motility was significantly higher (p<0.05) in all treatment groups after light exposure. The T1 and T4 groups had significantly higher (p<0.05) than control 60 min after light exposure, although no significant difference was found in the motility of all groups after 60 min. Viability was found to be non-significant between groups, although the T4 group exhibited greater values. There were no appreciable variations in the acrosome and membrane integrity between the control group and the other groups. Based on the values observed, T1 and T4 were selected for further experiments in Objective II. In objective-II, T1 (600 nm for 2 min) and T2 (650 nm for 3 min) were used for exposing semen. Under this objective, semen was evaluated at different cryopreservation stages: post-dilution, pre-freeze, and post-thaw. Progressive motility was significantly higher (p<0.05) in both treatment groups at all stages. CFDA-PI analysis revealed that the post-thaw viability of sperm in the T1 and T2 groups were significantly higher (p<0.05) than that of the control group. At the post-thaw stage, membrane intact spermatozoa were discovered to be significantly higher (p<0.05) in T1 and T2. The average percentage of intact acrosome sperm at the pre-freeze and post-thaw stages was higher in both treatment groups. The proportion of spermatozoa with higher MMP was significantly elevated (p<0.05) in T1 and T2 compared to control. In contrast, cryocapacitation and lipid peroxidation of spermatozoa were substantially reduced (p<0.05) in T1, compared to control group. In summary, polarised light selected wavelength and time exposure can improve seminal characteristics by reducing lipid peroxidation and enhancing the mitochondrial respiratory chain activity.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.
