Tushar Kumar Mohanty

Effects of pedigree and exotic genetic inheritance on semen production traits of dairy bulls

Abstract Objective To study the effects of different levels of exotic inheritance on ejaculate quality in bulls and its passage through different generations. Methods Data on semen production traits and ejaculate quality were obtained for 38 crossbred bulls and grandsire-sire-progeny relationship in relation to semen quality was studied. The bulls were classified into three groups based on the level of exotic inheritance viz. F1, 50.0%-62.5% exotic germplasm and >75% exotic germplasm. Results Results of the present study indicated that about 40% of the ejaculates obtained from the crossbred bulls were rejected from further processing due to poor ejaculate quality. The F1 bulls produced significantly higher proportions (57.00±10.00) of poor quality ejaculates compared to the interse mated bulls. The age at first semen collection in crossbred bulls ranged from 567 to 1 010 days with an average of 738.89±18.18 days while the mean age at first semen freezing was 865.72±34.60 days. Conclusions It may be inferred that the “acceptable quality semen producing ability” decreased from grandsire through sire to male progeny and among the increasing exotic genetic levels of CB cattle, F1 bulls produced significantly higher “low grade ejaculates” that were unfit for cryopreservation.

Profiling of urinary proteins in Karan Fries cows reveals more than 1550 proteins

Urine is a non-invasive source of biological fluid, which reflects the physiological status of the mammals. We have profiled the cow urinary proteome and analyzed its functional significance. The urine collected from three healthy cows was concentrated by diafiltration (DF) followed by protein extraction using three methods, namely methanol, acetone, and ammonium sulphate (AS) precipitation and Proteo Spin urine concentration kit (PS). The quality of the protein was assessed by two-dimensional gel electrophoresis (2DE). In-gel digestion method revealed more proteins (1191) in comparison to in-solution digestion method (541). Collectively, 938, 606 and 444 proteins were identified in LC-MS/MS after in-gel and in-solution tryptic digestion of proteins prepared by AS, PS and DF methods, respectively resulting in identification of a total of 1564 proteins. Gene ontology (GO) using Panther7.0 grouped the majority of the proteins into cytoplasmic (location), catalytic activity (function), and metabolism (biological processes), while Cytoscape grouped proteins into complement and coagulation cascades; protease inhibitor activity and wound healing. Functional significance of few selected proteins seems to play important role in their physiology. Comparative analysis with human urine revealed 315 overlapping proteins. This study reports for the first time evidence of more than 1550 proteins in urine of healthy cow donors. This article is part of a Special Issue entitled: Proteomics in India.

Effect of lameness (hoof disorders) on productivity of Karan Fries crossbred cows

In present study production performance of 96 lame cows was compared with 67 healthy cows. No significant effect of parity and year of calving on milk yield were observed but the effect of season of calving was significant (P < 0.01). Effect of lameness on milk yield at the second, third and fourth months and 305 days was highly significant (P < 0.01), and was also significant (P < 0.05) on lactation yield of the fifth and tenth months. The effect of lameness on monthly and 305-day milk yield was significant (P < 0.01) only for those cows diagnosed lame before calving and during the first month of lactation. The differences in mean monthly yield were highly significant (P < 0.01) at the second, third and fourth months; and significant (P < 0.05) in the first and fifth months. The loss in the first lactation month of cows which were diagnosed as lame in the second month, was found to be significant (P < 0.05). Thus the yield of the month previous to the diagnosis (sub-clinical stage) was also affected. A significant (P < 0.01) total loss of 498.95 kg of milk yield was observed during a period of 305 days.

Investigations on the influence of 100 MeV O7+ ion irradiation on the structural, surface morphology and optical studies of gallium nitride epilayers

Swift heavy ion irradiations were carried out on metal organic chemical vapour deposition- grown GaN epilayers on sapphire (0 0 0 1) substrates at room temperature, with 100 MeV O7+ ions and fluences varying from 1×1012, 1×1013 and 5×1013 ions cm−2. The pristine and irradiated GaN samples were characterized by X-ray diffraction (XRD), atomic force microscopy (AFM), photoluminescence (PL) and UV–visible optical transmittance studies. XRD results indicate the presence of gallium oxide phases after O7+ ion irradiation and the increase in the Ga2O3 peak intensity and full-width half-maximum of irradiated GaN with increasing ion fluences. Surface morphology was observed from AFM analysis, which indicates a decrease in the root-mean square of roughness from 3.9 to 0.5 nm. PL measurements show a red shift as compared to the as-grown GaN. The UV–visible optical transmittance studies show a decrease in band gap after ion irradiation.

Spermatozoa with high mitochondrial membrane potential and low tyrosine phosphorylation preferentially bind to oviduct explants in the water buffalo (Bubalus bubalis)

Although it is understood that spermatozoa are subjected to selection processes to form a functional sperm reservoir in the oviduct, the mechanism remains obscure. With the aim to understand the sperm selection process in the oviduct, in the present in vitro study, we analyzed mitochondrial membrane potential and tyrosine phosphorylation status in oviduct-explants bound and unbound spermatozoa. Frozen semen from Murrah buffalo bulls (n=10) used under progeny testing programme were utilized for the study. Oviduct explants were prepared by overnight culture of epithelial cells in TCM- 199 and washed spermatozoa were added to the oviduct explants and incubated for 4h. Mitochondrial membrane potential (MMP) and tyrosine phosphorylation status of bound and unbound spermatozoa were assessed at 1h and 4h of incubation. The proportion of spermatozoa with high MMP was significantly higher (P<0.001) among the bound spermatozoa (range 84.67-96.56%) compared to unbound (range 8.70-21.03%) spermatozoa. The proportion of tyrosine phosphorylated spermatozoa was significantly higher (P<0.001) among unbound population as compared to bound population. The proportion of spermatozoa displaying tyrosine phosphorylation at acrosomal area was significantly (P<0.05) lower in bound sperm population compared to unbound population. It was inferred that spermatozoa with high MMP and low tyrosine phosphorylation were preferred for oviduct-explants binding in the buffalo.

Functional characterization of Mammary Gland Protein-40, a chitinase-like glycoprotein expressed during mammary gland apoptosis

MGP-40 is a chitinase-like protein which is over expressed during mammary gland involution. However, its physiological function in the mammary gland is poorly understood. In the present investigation, we have reported the functional significance of buffalo specific MGP-40 in the mammary gland by using an in vitro model of the buffalo mammary epithelial cell (BuMEC) line. MGP-40 was highly up regulated in BuMECs in serum starved condition as well as after treatment with prolactin suggesting its role in the stress response. Subsequently, to study the effect of MGP-40 on BuMECs, the cells were transfected with a mammalian expression construct of pCI neo harboring MGP-40 gene. It was observed that over expression of MGP-40 enhanced proliferation of BuMECs and protected the cells from apoptosis under serum free condition. In contrast, MGP-40 attenuated the mitogenic effect of insulin in BuMECs. Besides, over expression of the MGP-40 reduced dome formation, acinar polarization and casein synthesis in BuMECs in the presence of lactogenic hormones, it also induced Stat3 phosphorylation and epithelial to mesenchymal transition (EMT) -like features. Together, our data suggest that MGP-40 is involved in protection of BuMECs under stress conditions, inhibits cellular differentiation and induces EMT-like features. A schematic diagram depicting possible association of MGP-40 in various molecular pathways has been presented.

Development of a wireless sensor network for animal management: Experiences with Moosense

A wireless sensor network based precision animal management system developed by us called Moosense is presented. Moosense is deployed in the Livestock Research Centre (LRC) of National Dairy Research Institute (NDRI) Karnal. It is spread across a cluster of sensor networks where each cluster has a set of specialized systems, installed on the ground or mounted on the animals, which are responsible for live-monitoring of a variety of cattle-health parameters. Each cluster has a gateway to which the devices are connected either directly or through a set of relay nodes. The gateways are connected to a central station where all sensor data of LRC is archived and analysed. Through Moosense, we share our experiences with the development of structurally and functionally complex sensor networks for challenging environments such as in animal husbandry. Moosense is one of the first operational multi-objective wireless sensor networks in India being actively used for precision dairy research.

Anandamide exerts a suppressive effect on sperm binding to oviduct explants through CB1 receptors in the water buffalo (Bubalus bubalis)

An endocannabinoid system comprising of Anandamide (AEA) and its receptor has been shown to play a role in sperm acquisition of fertilizing potential and sperm-oviduct interaction. In the present study, we assessed the effect of sperm pre-treatment with AEA or co-incubation of sperm-oviduct explants with AEA in the presence or absence of CB1 receptor antagonist (SR141716A) on sperm-oviduct binding in the water buffalo. Cryopreserved spermatozoa from 3 Murrah buffalo bulls (3 ejaculates from each bull) were utilized for the study. Oviduct explants were prepared by overnight culture of epithelial cells in TCM- 199 and washed spermatozoa were added to the oviduct explants and incubated for 1h. Then, sperm-oviduct explants were stained with a fluorescent stain (JC-1) and sperm binding index (BI - No. of bound spermatozoa/unit area of oviduct explants) was assessed. The results indicate that BI decreased significantly (P<0.05) when spermatozoa were either pre-treated with AEA (14.16±0.87) or sperm-oviduct explants were co-incubated with AEA (16.27±0.86) at 1nM concentration compared to the control group (29.12±2.17), however such effect was not observed when AEA was used at 1μM concentration. Incorporation of SR141716A in the incubation medium inhibited the suppressive effect of AEA on BI. It was concluded that AEA, at 1nM concentration, decreased the number of spermatozoa bound to the oviduct explants and the suppressive effect of AEA on sperm-oviduct binding was inhibited by CB1 receptor antagonist suggesting that the effect of AEA was mediated through CB1 receptor in the water buffalo.

Incubation of spermatozoa with Anandamide prior to cryopreservation reduces cryocapacitation and improves post-thaw sperm quality in the water buffalo ( Bubalus bubalis )

Anandamide (AEA), an endocannabinoid, has been shown to reduce capacitation and acrosomal exocytosis in human spermatozoa. Because buffalo spermatozoa are highly susceptible to cryopreservation induced damage, AEA was assessed as to whether it could protect spermatozoa from cryo-damage. Six ejaculates from six Murrah buffalo bulls (total 36 ejaculates) were utilized for the study. Each ejaculate was divided into four aliquots; spermatozoa in Aliquot 1 were extended in Tris-Citrate-Egg Yolk and frozen as per the standard protocol. Spermatozoa in Aliquots 2, 3 and 4 were incubated with AEA at 1 nM, 1 μM and 10 μM, respectively in Tris-Citrate extender for 15 min at 37 °C before cryopreservation. Cryopreserved spermatozoa were thawed at 37 °C for 30 s before assessment of sperm motility, membrane integrity, capacitation, acrosome reaction, mitochondrial membrane potential (MMP) and lipid peroxidation status. The proportion of motile and membrane intact spermatozoa were greater (P < 0.05) with use of 1 μM AEA incorporated group compared with other groups. The proportion of un-capacitated and acrosome intact spermatozoa was greater (P < 0.05) with use of 1 or 10 μM of AEA compared with the other groups. When compared to the control group, use of 1 μM AEA resulted in a greater proportion of spermatozoa with high MMP (P < 0.05). There was no significant difference in the lipid peroxidation status of spermatozoa among any of the four groups. It was inferred that the protective role of AEA during cryopreservation of buffalo spermatozoa was dose dependent and incubation of spermatozoa with AEA at 1 μM concentration prior to cryopreservation reduced cryo-capacitation and improved post-thaw sperm quality in buffalo.

Transcriptional abundance of type-1 endocannabinoid receptor (CB1) and fatty acid amide hydrolase (FAAH) in bull spermatozoa: Relationship with field fertility

A highly sophisticated endogenous cannabinoid system (ECS) has been shown to play a crucial role in controlling sperm functions and fertility in men. In the present study, we report the differences in the expression level of components of ECS [type-1 endocannabinoid receptor (CB1) and fatty acid amide hydrolase (FAAH)] in spermatozoa from bulls with different field fertility ratings. Cryopreserved spermatozoa from crossbred cattle bulls (n = 40) were utilized for the study. The bulls were classified into high-, medium- and low-fertile bulls based on field conception rates. Sperm viability, capacitation status and protamine deficiency were assessed. Spermatozoa RNA was isolated from all the bulls, cDNA was synthesized and quantitative real time PCR was carried out to study the transcriptional abundance of CB1 and FAAH genes. Sperm viability was lower and capacitation was higher (p < 0.05) in low fertile bulls compared to medium and high fertile bulls. The expression level of CB1 gene was significantly (p < 0.05) lower in spermatozoa from low and medium fertile bulls compared to high fertile bulls. The expression of CB1 gene was 21.07 and 4.23 times greater in high and medium fertile bulls, respectively compared to low fertile bulls. The correlation between CB1 gene expression and field conception rate of bulls was positive and significant (r = 0.57; p < 0.001). Unlike CB1 receptors, FAAH gene expression was similar among high, medium and low fertile bulls. The correlation of FAAH expression with bull conception rate was positive but not significant. It was concluded that the transcriptional abundance of type-1 endocannabinoid receptor (CB1) was positively and significantly related to bull fertility.