Seasonal assessment of mastitis using thermogram analysis in murrah buffaloes.

Mastitis is a global threat that challenges dairy farmers' economies worldwide. Sub-clinical mastitis (SCM) beholds the lion's share in it, as its visible clinical signs are not evident and are challenging to diagnose. The treatment of intramammary infection (IMI) demands antimicrobial therapy and subsequent milk withdrawal for a week or two. This context requires a non-invasive diagnostic tool like infrared thermography (IRT) to identify mastitis. It can form the basis of precision dairy farming. Therefore, the present study focuses on thermal imaging of the udder and teat quarters of Murrah buffaloes during different seasons to identify SCM and clinical mastitis (CM) cases using the Darvi DTL007 camera. A total of 30-45 lactating Murrah buffalo cows were screened out using IRT regularly throughout the year 2021-22. The IMI was further screened using the California mastitis test. The thermogram analysis revealed a significant difference (p < 0.01) in the mean values of the udder and teat skin surface temperature of Murrah buffaloes between healthy, SCM, and CM during different seasons. The mean values of udder skin surface temperature (USST) during different seasons ranged between 30.28 and 36.81 °C, 32.54 to 38.61 °C, and 34.32 to 40.02 °C among healthy, SCM, and CM-affected quarters. Correspondingly, the mean values of teat skin surface temperature (TSST) were 30.52 to 35.96 °C, 32.92 to 37.55 °C, and 34.51 to 39.05 °C, respectively. Further results revealed an increase (p < 0.01) in the mean values of USST during winter, summer, rainy, and autumn as 2.26, 4.04; 2.19, 3.35; 1.80, 3.21; and 1.45, 2.64 °C and TSST as 2.40, 3.99; 2.28, 3.26; 1.59, 3.09; and 1.68, 2.92 °C of SCM, CM-affected quarters to healthy quarters, respectively. The highest incidence of SCM was observed during autumn and CM during winter. Henceforth, irrespective of the seasons studied in the present study, IRT is an efficient, supportive tool for the early identification of SCM.

Reduced sperm number of murrah (Bubalus bubalis) bull semen in french mini-straw affects kinetic and functional competence after cryopreservation.

BACKGROUND: Extensive dilution of cattle semen with tris-based extender compromises certain sperm kinetic and functional traits following cryopreservation. OBJECTIVE: To study sperm functions of buffalo bulls under high dilution rates. MATERIALS AND METHODS: Twenty-four ejaculates were harvested twice a week from four buffalo bulls, and diluted to sperm concentrations of 80, 60, 40 and 20 million/mL. Diluted samples were filled in straws, equilibrated at refrigeration temperature for 4 h, and frozen in liquid nitrogen. Frozen sperm samples were thawed for evaluation of kinetic and functional attributes. RESULTS: Compared to 20 million/mL (million/mL) sperm sample, the total motility, progressive motility and rapid motility were reduced (P < 0.05) in 5 million/mL sample. The proportion of live sperm were significantly (P < 0.05) higher in 10, 15 and 20 million/mL samples than in 5 million/mL sample. The percentage of moribund sperm, dead sperm, and sperm with lipid per oxidation increased significantly (P < 0.05) in 5 million/mL sample. CONCLUSION: The reduction of sperm concentrations to < 10 million/mL affects post-thaw Buffalo sperm kinetic and functional attributes.. https://doi.org/10.54680/fr24110110712.

Seasonal assessment of mastitis using thermogram analysis in Sahiwal cows.

"India is the world's leading producer of milk" and demands a non-invasive diagnostic tool like infrared thermography (IRT) to identify the costliest production disease, mastitis. It can form the basis of precision dairy farming. Therefore, the present study focuses on thermal imaging of the udder and teat quarters of Sahiwal cows during different seasons to identify subclinical (SCM) and clinical mastitis (CM) cases using the Darvi DTL007 camera. A total of 24-69 lactating Sahiwal cows were screened out using IRT regularly throughout the year. The intramammary infection status was further assessed using the CMT. The receiver operating characteristic analysis was carried out to develop the current study's cut-off for various thermographic parameters. The incidence for SCM and CM ranged from 26.47 to 38.75% and 17.83-22.79%, respectively during different seasons in Sahiwal udder quarters. The thermogram analysis revealed a significant difference (p < 0.01) in the mean values of the udder and teat surface temperature of Sahiwal cows between healthy, SCM, and CM during different seasons. The mean values of udder skin surface temperature (USST) during different seasons ranged between 29.07 and 36.91 °C, 31.51 to 37.88 °C and 32.42 to 38.79 °C among healthy, SCM, and CM-affected quarters, and correspondingly, the mean values of teat skin surface temperature (TSST) were 28.28 to 36.77 °C, 30.68 to 37.88 °C and 31.70 to 38.73 °C, respectively. Further results revealed an increase (p < 0.01) in the mean values of USST during winter, summer, rainy, and autumn as 2.44, 3.35; 0.97, 1.88; 1.06, 1.83; 1.29, 2.39 °C and TSST as 2.4, 3.42; 1.11, 1.96; 1.21, 2.19, 1.3, 2.4 °C of SCM, CM-affected quarters to healthy quarters, respectively, in Sahiwal cows. Thermograms showed a strong positive correlation with the CMT scores of SCM, CM cases, and healthy samples. Henceforth, irrespective of the seasons studied in the present work, IRT is an efficient, supportive tool for the early identification of subclinical mastitis.

Short milking tube thermogram analysis: an indicator of mastitis in Murrah buffaloes.

In the present study, thermal images of the short milking tube of the milking machine representing four udder quarters independently attached to a milking animal, along with pre-milking and post-milking udder and teat thermograms, were taken using a hand-held digital infrared thermal camera (DarviDTL007) during morning milking of lactating Murrah buffaloes (n = 132) to assess the mastitis status. California mastitis test (CMT) and somatic cell count (SCC) of milk samples were carried out to screen the udder quarters as healthy, subclinical (SCM), and clinical mastitis (CM). The thermograms revealed an increase (p < 0.05) of 2.19 and 3.72ºC in the mean values of short milking tube (SMT) surface temperature among SCM and CM quarters compared to healthy quarters, respectively. The mean values of udder skin surface temperature (USST) for pre-milking, milking, and post-milking of SCM and CM compared to healthy quarters showed an increase (p < 0.05) of 2.17, 1.96, and 1.61ºC and 3.11, 2.88, and 2.73ºC, respectively. Similarly, compared to healthy quarters, the mean values of teat skin surface temperature (TSST) for pre-milking and post-milking of SCM and CM showed an increase (p < 0.05) of 2.12 and 1.66ºC and 3.07 and 2.45ºC, respectively. Also, CMT and SCC results showed a strong positive correlation (r = 0.68-0.91, p < 0.01) with all the thermographic parameters. Thus, thermograms of SMT alone can be used as an efficient detection tool in assessing SCM among Murrah buffaloes.

Short-milking-tube thermograms: An alternative to udder thermograms for mastitis detection in Sahiwal cows.

Mastitis is a multi-etiological production disease that causes substantial financial loss to dairy farmers. In this context, early detection of mastitis using thermograms can aid the dairy sector in managing mastitis efficiently, and this technology could be a supportive tool in precision dairy farming. Infrared cameras can detect minor temperature changes on the udder surface by taking multiple images of the udder and teat. In the current study, a thermogram of the short milking tube (SMT) of the milking machine, as well as the udder and teat of lactating Sahiwal cow (n = 100 quarters of 25 Sahiwal cows), was captured using a hand-held digital infrared thermal camera (DarviDTL007) during morning milking to assess the mastitis status. CMT and SCC of milk samples were carried out for further confirmatory diagnosis of healthy, sub-clinical (SCM), and clinical mastitis (CM). Cut-offs for short milking tube temperature were developed using the receiver operating characteristics analysis. Results of thermal image analysis revealed that the pre-milking, milking, and post-milking parameters of the udder and the teat skin surface temperatures showed a significant difference in the healthy, SCM, and CM-affected quarters. The thermogram analysis showed a significant increase (p < 0.05) of 1.11 and 2.04°C in the mean values of SMT surface temperature among SCM and CM quarters compared to healthy quarters, respectively. In addition, the values of CMT and SCC revealed a significant increase (p < 0.05) in SCM and CM samples and a positive correlation to SMT surface temperatures. Short milking tube thermograms can be a useful assessment tool for detecting sub-clinical mastitis in dairy animals.

Optimized addition of nitric oxide compounds in semen extender improves post-thaw seminal attributes of Murrah buffaloes.

Semen dilution and cryopreservation alter the homogeneity of seminal plasma, resulting in a non-physiological redox milieu and consequently poor sperm functionality. Considering the concentration-specific bimodal action of nitric oxide (NO) in the regulation of sperm functions, cryopreservation media supplemented with optimized concentrations can improve the semen attributes. The present study aimed to evaluate the effect of adding an optimized concentration of sodium nitroprusside (SNP) and N-nitro-L-arginine methyl ester (L-NAME) in an extender on in vitro semen quality. An aliquot of semen samples (n = 32) from Murrah buffalo bulls (n = 8) was divided into control (C) and treatment (T-I: SNP in extender at 1 µmol/L; T-II: L-NAME in extender at 10 µmol/L). Fresh semen quality parameters showed no significant difference at 0 h except for the structural integrity in the T-II group. Post-thaw semen quality parameters and sperm kinematics using computer-aided sperm analysis (CASA) revealed significantly higher (p < 0.05) cryoresistance in the treatment groups. Viability, acrosome integrity, and membrane integrity were significantly higher (p < 0.05) in both treatment groups; however, the results were pervasive in T-II. Lower abnormal spermatozoa were observed in both T-I and T-II. SNP supplementation led to a significant rise (p < 0.05) in NO, whereas L-NAME reduced the NO concentration in post-thawed samples, which was directly correlated with different sperm functionality and associated biomarkers viz. total antioxidant capacity (TAC) and thiobarbituric acid reactive substance (TBARS). It was concluded that the cryopreservation media supplemented with SNP and L-NAME at 1 µmol/L and 10 µmol/L, respectively, lower the cryo-damage and improve post-thaw seminal attributes.

Time and dose-dependent effect of preconditioning with sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1) on post-thaw semen quality of Karan-Fries (KF) bulls.

A novel strategy, focused on the induction of sub-lethal oxidative stress to optimize sperm cryosurvival, has been used before cryopreservation. The present study compared the effect of preconditioning with various concentrations of nitric oxide-donor (sodium nitroprusside, SNP) and peroxynitrite-generator (3-morpholinosydnonimine, SIN-1) on in vitro sperm functions and lipid peroxidation status (LPO) of cryopreserved Karan-Fries (KF) crossbred bull semen. To optimize the concentration of additives, spermatozoa obtained from 36 ejaculates were supplemented with different concentrations of SNP (0.01, 0.05, 0.1 µM) and SIN-1 (80, 160, 200 µM) versus control in the extender. The post-freezing sperm motility and viability were greater (p < 0.05) in 0.1 µM SNP and 80 µM SIN-1 in comparison to other concentrations used. Furthermore, the spermatozoa obtained from 48 ejaculates were supplemented with 0.1 µM SNP and 80 µM SIN-1 in the extender. A significant increase (p < 0.05) was observed in progressive motility, viability and membrane integrity in SNP and SIN-1 treated extender at 24 h, 15 days, and 2-month post-cryopreservation (PC) periods. There was no significant difference in sperm abnormality in the extended groups and the control group. The seminal plasma of SNP-treated extender had less (p < 0.05) lipid peroxidation as compared to SIN-1 treated and control groups. In post-thaw semen, both SNP and SIN-1 showed a higher (p < 0.05) proportion of acrosome intact (FITC-PNA) sperm with a greater decrease (p < 0.05) in membrane scrambling and lipid peroxidation. SNP and SIN-1 improved (p < 0.05) the proportion of sperm with higher mitochondrial membrane potential (Δψm) as compared to the control. In conclusion, it seems that the preconditioning of SNP and SIN-1 at lower doses may have beneficial effects on post-thawed crossbred bull sperm quality.

Iodine Supplementation Improved Antioxidant Status, Hormonal Status, Sexual Behavior, and Semen Production Performance of Bos indicus Bulls Under Tropical Climatic Condition.

Iodine is anessential micronutrient that plays a crucial role in male reproduction (sexual behavior and semen production performance) by modulating thyroid function and the antioxidant status of the animal. Nonetheless, in Bos indicus bulls, a thorough evaluation of the effects of dietary iodine supplementation on antioxidant status, seminal quality parameters, and its interaction with other minerals is not documented. Twelve Bos indicus (Sahiwal) bulls were distributed into three groups (n = 4 in each group) viz. T1 (control), T2, and T3 and fed diets containing 0.250, 0.375, and 0.500 ppm iodine/ kg dry matter intake, corresponding to 0%, 50%, and 100% higher than ICAR (2013) recommendations, respectively. The experimental feeding was carried out for 60 days and the effects on nutrient utilization, hormonal and antioxidant status, and sperm function tests were investigated. Results revealed that body weight, dry matter intake, and nutrient digestibility remained unaffected by dietary supplementation of iodine. Testosterone and thyroxine hormone concentrations were improved (p<0.05) in T2 and T3 groups. Blood and seminal iodine content were also higher (p<0.05) in both the supplemented groups (T2 and T3). Sperm functions viz. viability, physical membrane integrity, acrosomal integrity, motility, and mitochondrial membrane potential were improved (p<0.05) due to iodine supplementation. Furthermore, lipid peroxidation and membrane scrambling in spermatozoa were reduced (p<0.05) in T2 and T3 groups. Blood antioxidant status (total antioxidant activity and GPx levels) was improved (p<0.05) in T2 and T3. Sexual behavior was also improved (p<0.05) in iodine-supplemented groups. Hence, it can be concluded that iodine supplementation at the dose rate of 0.500 ppm in the Bos indicus bull diet is beneficial in improving hormonal status, antioxidant status, and semen quality.

Butylated hydroxytoluene (bht) improves the post-thaw semen quality in low-dose sperm cryopreservation in murrah buffalo bull.

BACKGROUND: Cryopreservation is an important technique for the long-term storage of semen for artificial insemination (AI). Buffalo spermatozoa are sensitive to cryopreservation procedures because of the presence of a high amount of polyunsaturated fatty acids in the plasma membrane. OBJECTIVE: To study the effect of different concentrations of BHT on the quality of Murrah buffalo bull semen for low-dose cryopreservation. MATERIAL AND METHODS: Semen was collected from four high fertile Murrah buffalo bulls (6 ejaculates each) using an artificial vagina. A total of 24 ejaculates were collected from each bull twice a week using an artificial vagina. Every sample was split into four parts: Control without additives; and three treatments with BHT at 0.5 mM, 1 mM or 2 mM. Semen was cryopreserved at low-dose sperm cryopreservation of 20, 15, 10 and 5 million sperm per aliquot after supplementation of BHT. Semen samples were evaluated for fresh, pre-freeze and post-thaw stages. RESULTS: There was a significant increase (p<0.05) in sperm quality parameters, such as progressive motility (%), viability (%), HOST response (%), acrosome integrity (%) and post-thaw motility, with the addition of 0.5-1 mM BHT. CONCLUSION: The addition of BHT in Murrah buffalo semen improves the low dose cryopreservation quality in a dose-dependent manner. doi.org/10.54680/fr23110110612.

Targeted antioxidant delivery modulates mitochondrial functions, ameliorates oxidative stress and preserve sperm quality during cryopreservation.

Mitochondria are vital organelles with a multifaceted role in cellular bioenergetics, biosynthesis, signaling and calcium homeostasis. During oxidative phosphorylation, sperm mitochondria generate reactive oxygen species (ROS) at physiological levels mediating signaling pathways essential for sperm fertilizing competence. Moreover, sperm subpopulation with active mitochondria is positively associated with sperm motility, chromatin and plasma membrane integrity, and normal morphology. However, the osmotic and thermal stress, and intracellular ice crystal formation generate excess ROS to cause mitochondrial dysfunction, potentiating cryoprotectant-induced calcium overload in the mitochondrial matrix. It further stimulates the opening of mitochondrial permeability transition pores (mPTP) to release pro-apoptotic factors from mitochondria and initiate apoptotic cascade, with a decrease in Mitochondrial Membrane Potential (MMP) and altered sperm functions. To improve the male reproductive potential, it is essential to address challenges in semen cryopreservation, precisely the deleterious effects of oxidative stress on sperm quality. During semen cryopreservation, the supplementation of extended semen with conventional antioxidants is extensively reported. However, the outcomes of supplementation to improve semen quality are inconclusive across different species, which is chiefly attributed to the unknown bioavailability of antioxidants at the primary site of ROS generation, i.e., mitochondria. Increasing evidence suggests that the targeted delivery of antioxidants to sperm mitochondria is superior in mitigating oxidative stress and improving semen freezability than conventional antioxidants. Therefore, the present review comprehensively describes mitochondrial-targeted antioxidants, their mechanism of action and effects of supplementation on improving semen cryopreservation efficiency in different species. Moreover, it also discusses the significance of active mitochondria in determining sperm fertilizing competence, cryopreservation-induced oxidative stress and mitochondrial dysfunction, and its implications on sperm fertility. The potential of mitochondrial-targeted antioxidants to modulate mitochondrial functions and improve semen quality has been reviewed extensively.

Comparative evidence support better antioxidant efficacy of mitochondrial-targeted (Mitoquinone) than cytosolic (Resveratrol) antioxidant in improving in-vitro sperm functions of cryopreserved buffalo (Bubalus bubalis) semen.

The present study compared the effect of mitochondria-targeted (Mitoquinone, MitoQ) and untargeted cytosolic antioxidant (Resveratrol, RESV) supplementation on lipid peroxidation (LPO) and in-vitro sperm functions of cryopreserved buffalo bull semen. To optimize additive's concentration, sperm pellet obtained from twenty-four ejaculates was supplemented with different concentrations of MitoQ (20 nM, 100 nM, 200 nM); and RESV (10 µM, 25 µM, 50 µM) against control in the extender. The post-thaw sperm motility, livability, and membrane integrity were higher (P < 0.05) in 200 nM MitoQ and 50 µM RESV than other concentrations used. In another experiment, sperm pellet from thirty-two ejaculates was supplemented with 200 nM MitoQ and 50 µM RESV in the extender. Pre-freeze and post-thaw progressive motility and livability were higher (P < 0.05) in MitoQ (200 nM) than RESV (50 µM) treatment. MitoQ supplementation improved post-thaw membrane integrity (CFDA-PI) higher (P < 0.05) than RESV, however, hypo-osmotic swelling response observed no improvement with RESV treatment. Post-thaw LPO rate was lower (P < 0.05) and Bovine cervical mucus penetration was higher (P < 0.05) in MitoQ than RESV treatment. In post-thaw semen, MitoQ showed higher (P < 0.05) proportion of acrosome intact (FITC-PNA), live non-apoptotic (P < 0.01) sperm with a higher reduction (P < 0.05) in membrane scrambling. MitoQ improved (P < 0.01) proportion of sperm with high Mitochondrial Membrane Potential and low LPO (P < 0.01) than RESV treatment. In conclusion, improvement in post-thaw in-vitro sperm functions and cryo-tolerance was more evident in MitoQ than RESV supplemented buffalo bull semen. Our study provides a better strategy to mitigate oxidative stress by enhancing mitochondrial antioxidant system with targeted antioxidants than cytosolic antioxidant supplementation.

Targeted transcript analysis revealed association of suboptimal expression of certain endometrial immunity-related genes with disparate uterine diseases in zebu cows.

The onset of uterine infection during postpartum period compromises uterine health, fertility, and productivity of dairy cattle. Endometrial innate immunity plays a key role in eliminating uterine infection and keeping the uterus healthy. Hence, the present study has been designed with the hypothesis that altered endometrial immune response around calving may compromise uterine health during postpartum period. Expression of interleukins (IL-1ß, IL-6, IL-8, IL-10, and TNF-α), prostaglandin synthase (PGFS, PGES), and antimicrobial peptides (beta-defensins (BDEF-4, BDEF-5), lingual antimicrobial peptide (LAP), and calcium-binding proteins (S100A8, S100A9, and S100A12) in endometrial tissues on the day of calving was studied using qRT-PCR, and circulating concentrations of prostaglandin E and F metabolites (PGEM and PGFM) during peripartum period (on days - 7, - 4, - 1 (before calving), 0 (on the day of calving), + 1, + 4, and + 7 (post calving)) of normal (healthy) cows (n = 11) that did not develop postpartum uterine infection and cows that developed puerperal metritis (n = 7) and clinical endometritis (n = 6) were studied. Endometrial expression of IL-1ß, TNF-α, BDEF-4, BDEF-5, S100A8, S100A12, and PGFS was higher (P < 0.05), and expression of IL-6, IL-8, IL-10, and PGES was lower (P < 0.05) in normal (healthy) cows than puerperal metritic and clinical endometritic cows. The PGFM concentration in serum was high (P < 0.05) on days 0, + 1, and + 4 of calving in puerperal metritic cows followed by normal and clinical endometritic cows. However, PGEM concentration in serum was high (P < 0.05) during peripartum period in uterine-infected (puerperal metritic and clinical endometritic) cows compared with normal cows. From the above findings, it is concluded that higher constitutive expression of IL-1ß, TNF-α, PGFS, BDEF-4, BDEF-5, S100A8, and S100A12 genes in the endometrium and lower concentration of PGEM during the period immediate to calving might be beneficial for uterine health of cows.

Effect of cattle-specific estrus molecules on libido and semen production of zebu bulls under tropical climate.

Zebu bulls are a shy breeder and they exhibit optimum libido in the presence of females with estrus phase. Continuous semen collection with the use of male dummy leads to lack of adequate sexual stimulation. Therefore, the present study was designed to test the effect of estrus-specific molecule(s) for effective sexual preparation of donor bulls. The bulls were divided into normal and poor libido group, five bulls in each group by taking 1-month control study data after collecting the information of individual bull's sexual behaviour during semen collection by regular semen collector. The bulls were never being exposed to female animals and semen was collected by an artificial vagina. The ten animals were exposed to a glycerol-water solution (50/50 v:v) as control and then exposed to estrus-specific molecules one by one. The estrus-specific molecules like squalene, 1-iodoundecane, acetic acid, coumarin, propionic acid, oleic acid, and 2-butanone were purchased from Sigma-Aldrich Company, USA, and the molecules were solubilised individually in a non-pressurised aerosol dispenser as 1.0% concentration in glycerol-water solution (50/50, v:v). Identical bulls were used as the control and exposed to each molecule one by one by giving a refractory period of 14 days. A nasal spray of acetic acid or 2-butanone significantly (p < 0.05) reduced reaction time (RT) and total time taken to ejaculate (TTTE) in normal libido bull group. Semen volume, sperm concentration, and the total number of sperm per ejaculation obtained did not show significant improvement in the normal libido group of bulls after the application of estrus-specific molecules as compared to the control. In poor libido group, acetic acid, oleic acid, and 2-butanone application showed significant (p < 0.01) improvement in RT and TTTE as compared to the control group, whereas semen production variables like sperm concentration and total sperm output per ejaculation increased significantly (p < 0.05) except semen volume. There was significant (p < 0.01) reduction in RT (%) and TTTE (%) after the application of acetic acid followed by 2-butanone and oleic acid. The sperm concentration and total sperm output per ejaculation were more after the application of each molecule but significant increase (p < 0.05) in sperm concentration was observed with 2-butanone (11.42%), acetic acid (11.42%), and oleic acid (10.13%), whereas total sperm output per ejaculation increased significantly (p < 0.05) only after the application of acetic acid and 2-butanone (24.75% and 26.84%). Hence, it can be concluded that acetic acid, 2-butanone, and oleic acid are effective for better sexual preparation of Sahiwal bulls and total sperm output per ejaculation.

Identification of biomarker candidates for fertility in spermatozoa of crossbred bulls through comparative proteomics.

Associations between expression of some proteins in spermatozoa and fertility have been sought in recent years to identify the male fertility markers. Since the incidence of sub-fertility is high in crossbred bulls, the present investigation was carried out on high- and low-fertile crossbred bulls to identify fertility markers in spermatozoa through proteomics approach. Sperm proteome of high-fertile bulls were compared with low-fertile bulls using 2D-DIGE and MALDI-TOF-MS techniques and the results were validated with immuno-blotting. The proteins MDH2, ENO1, RIBC1, CAPN7, ATP5D, LacA like protein-2 like, NCAPD3, DECR1, GCNT2, GDI2, TOP and USP12 were over expressed in high-fertile spermatozoa, whereas DST like isoform 1, TMEM43 and BSP1 were over expressed in low-fertile spermatozoa (P < 0.05). The differential expression ranged from 1.57 (GDI2) to 5.1 (BSP1) fold between the two groups. Based on the GO annotation, majority of them were involved in cellular and metabolic processes, with catalytic and binding activities, and localized in cell and organelles. Among these proteins, ENO1 and BSP1 were selected based on the degree of differential expression and reliability in identification, for further validation. Immuno-blotting studies indicated that ENO1expression was positively correlated (P < 0.05) while the expression of BSP1 was negatively (P < 0.01) correlated with bull fertility. The proportion of capacitated spermatozoa in frozen thawed spermatozoa of low-fertile bulls was higher (P < 0.05) as compared to high-fertile bulls. Collectively, the study identified some potential molecules in spermatozoa of bulls, which may act as a panel of biomarkers for fertility.

Improvement in sperm functional competence through modified low-dose packaging in French mini straws of bull semen.

To achieve the targeted artificial insemination coverage with the current rate of semen production, without affecting the conception rate, it needs to reduce the number of spermatozoa per insemination dose in India as per international practice. Therefore, this study was planned to perform different levels of semen dilution, compare in vitro post-thaw semen quality and develop a modified low-dose semen packaging method in French mini straw to minimise semen dilution effect. Sixteen ejaculates were collected from Karan Fries bulls (n = 4). The mean percentage post-thaw motility, viability, membrane integrity, acrosome integrity, lipid peroxidation and capacitation status were estimated as post-thaw sperm function assays in semen sample diluted to 20, 15, 10 and 5 million spermatozoa per 0.25 ml and filled in the French mini straw by conventional packaging. No significant (p > .05) difference in post-thaw sperm quality was observed between 15 and 20 million doses; however, below 15 million sperm quality get reduced. There was no significant difference in post-thaw semen quality traits between 20 million conventional packaging and 5 million spermatozoa/dose in modified packaging. In conclusions, the modified packaging is a very effective method for low-dose cryopreservation with acceptable post-thaw semen quality.

Global proteomic analysis of water buffalo (Bubalus bubalis) saliva at different stages of estrous cycle using high throughput mass spectrometry.

Accurate and efficient detection of estrus is one of the major constraints for exploitation of the production potential of buffalo owing to its poor manifestation of estrus signs, seasonal differences in expression and higher incidences of silent estrus (29%). The current study focused on identification of estrus specific candidate proteins in saliva of buffaloes. Estrus was detected based on behavioral signs in response to the teaser and changes in reproductive organs and confirmed by per-rectal examination, trans-rectal USG of reproductive organs, cervico-vaginal mucus characteristics and blood serum progesterone estimation. Day of onset of estrus was considered as day 0 and day -3, +3, +10 were considered as proestrus, metestrus and diestrus stage of the estrous cycle respectively. A total of 19 animals and their 38 estrous cycles (two from each) were included in this study. Saliva was collected from these animals during different stages of estrous cycle. Out of these, 08 animals were selected for global proteome analysis of saliva using in-solution digestion and nano-LC-MS/MS. A total of 275, 371, 304 and 565 proteins were identified with ≥2 peptides during proestrus, estrus, metestrus and diestrus stages of estrous cycle. Among the identified proteins 31, 62, 32 and 104 proteins were found specific to proestrus, estrus, metestrus and diestrus stage of the estrous cycle. Few salivary proteins such as Cullin-associated NEDD8-dissociated protein 1, Heat shock 70 kDa protein 1A, 17-beta-hydroxysteroid dehydrogenase type 1, Inhibin beta A chain, testin were identified as estrus specific and are important for estrus physiology. Taken together, these estrus specific proteins could be considered as the candidate biomarker for detection and confirmation of estrus in buffalo after thorough validation.

Biochemical analysis of uterine fluid for identification of indicators for subclinical endometritis in the water buffalo (Bubalus bubalis).

Alterations in biochemical constituents of uterine fluid have been suggested for diagnosis of subclinical uterine infection in the bovine. This study was undertaken to investigate whether uterine fluid biomolecules could act as tool for diagnosis of subclinical endometritis in the buffalo. Uterine fluid samples from normal (n = 22) and subclinical endometritis (n = 18; diagnosed based on uterine cytology)-affected buffaloes were subjected to biochemical analysis. Among the different biochemical constituents estimated, urea, urea N, cholesterol, total bilirubin and alkaline phosphatase (ALP) concentrations were significantly (p < 0.05) higher in uterine fluid obtained from subclinical endometritis-affected buffaloes. The extent of difference between normal and subclinical endometritis-affected buffaloes was highest in ALP (69%) followed by cholesterol (55%), bilirubin (48%), urea (30%) and urea N (30%) concentrations. Receiver operating characteristic (ROC) analysis indicated that the likelihood ratio (LR) was 3.63 for urea, indicating that buffaloes having less than the threshold concentration (47.5 mg/dl) of urea in their uterine fluid were at 3.6 times more risk to be affected with SE. The LRs for urea N, cholesterol, ALP and bilirubin were 2.33, 2.54, 2.12 and 1.65, respectively. It was concluded that ALP, urea, urea N and cholesterol concentrations in uterine fluid may serve as an aid for diagnosing subclinical endometritis in the buffalo.

Development of an in vitro oviduct epithelial explants model for studying sperm-oviduct binding in the buffalo.

In this study, we developed an in vitro model for studying sperm-oviduct binding in the buffalo. Oviduct explants were prepared by overnight culture of epithelial cells in TCM-199 medium under 5% CO2 at 38.5 °C. Cryopreserved spermatozoa from buffalo bulls (n = 4) were incubated with the oviduct explants, and the sperm-oviduct explants complex was stained with JC-1. The effect of sperm concentration (2, 3 and 4 million), size of the oviduct explants (<0.2, 0.2-0.3, 0.3-0.4 and >0.4 mm2 ) and time of incubation (1 hr and 4 hr) on binding index (BI-number of sperm bound to unit area of explants) was studied. No significant difference was observed in the BI among <0.2, 0.2-0.3 and 0.3-0.4 mm2 size of explants; however, the BI decreased significantly (p < .05) when the size of explants exceeded 0.4 mm2 . The BI decreased significantly (p < .05) when the sperm concentration was increased to 4 million, while the duration of incubation did not have any significant effect on the BI. The interaction of bulls with explants size, sperm concentration and incubation time was not significant. The developed assay has the potential to be used as an in vitro model for studying sperm-oviduct binding in the buffalo.

Age-related changes in transcriptional abundance and circulating levels of anti-Mullerian hormone and Sertoli cell count in crossbred and Zebu bovine males.

Age-related changes in peripheral anti-Mullerian hormone (AMH) concentrations and transcriptional abundance of AMH gene in testicular tissue were studied in crossbred (Holstein Friesian × Tharparkar) and Zebu (Tharparkar) males. In both the breeds, basal AMH concentrations were estimated using ELISA method in blood plasma obtained from six males each at 1, 6, 12, 18, and 24 months age. After blood collection at respective ages, all the males were castrated and expression and immunolocalization of AMH was performed in the testicular tissue. The concentration of AMH in blood plasma was found to be highest at 1 month of age in both crossbred and Zebu males, which subsequently decreased with advancing age. Significantly (P < 0.05) lower concentration of AMH was observed in crossbred as compared with Zebu males at 24 months of age. In line with peripheral AMH concentrations, the expression of AMH gene was also higher (P < 0.05) at 1 month of age, which thereafter declined significantly with advancement of age in crossbred males. Furthermore, the expression of AMH gene differed significantly between Zebu and crossbred males at all the age groups studied. Immunolocalization of AMH in testicular tissue also revealed a stronger expression at 1 month age, which gradually decreased till 24 months of age. The true Sertoli cell count was significantly higher in Zebu compared with crossbred males at all age groups studied except at 6 months age. The relationship between Sertoli cell count and circulating AMH concentrations was negative and significant (r = -0.81; P = 0.004). In conclusion, expression of AMH gene in testicular tissue and peripheral blood concentrations of AMH were higher in young compared with adults in both crossbred and Zebu males; however, the transcriptional abundance and circulating levels of AMH were higher in Zebu compared with crossbred males.

Efficiency of uterine fluid cytology in the diagnosis of subclinical endometritis in the water buffalo (Bubalus bubalis).

This study compared endometrial cytology vis-a-vis uterine fluid cytology for assessment of uterine health in clinically normal and subclinical endometritis (SE)-affected buffaloes. Uterine fluid samples and endometrial samples were collected from the buffaloes (n = 38) at oestrus using blue sheath and cytobrush, respectively. The smears were stained with Field stain for 3 minutes, and a minimum of 400 cells were counted in each smear for determination of the percentage of polymorphonuclear (PMN) leucocyte. The incidence of subclinical endometritis, based on the cytobrush cytology, was 23.08%. The correlation between cytobrush cytology with uterine fluid cytology was positive and significant (r = .37; p = .02). The ratio of PMN leucocyte in cytobrush cytology to uterine fluid cytology was 1:2.4. ROC analysis revealed that the threshold value of 6.16% PMN leucocyte in uterine fluid cytology showed a diagnostic sensitivity and specificity of 100% in differentiating normal from SE-affected buffaloes. In conclusion, collection of uterine fluid was easier compared to collection of endometrial samples using cytobrush and the percentage of PMN leucocyte in uterine fluid cytology can be used as a tool for diagnosis of subclinical endometritis in buffaloes.