Effect of cryopreservation and capacitation on expression patterns of ATP synthesis associated genes in bubaline spermatozoa.

This study investigated the expression patterns of certain ATP synthesis associated genes (GAPDHS, PGK2, ENO4 and MDH2) in fresh and frozen-thawed bubaline spermatozoa vis-à-vis capacitation. Eight adult Murrah buffalo bulls, aged 5-7 years, were randomly selected from a bull farm and fresh semen was collected once a week for four weeks (n = 32) from the selected bulls. Simultaneously, frozen straws of the same bulls were collected (n = 32, four straws were pooled per sample). The samples were divided into four groups i.e., fresh non capacitated (control/group 1), fresh capacitated (group 2), frozen non capacitated (group 3) and frozen capacitated (group 4). In all groups mRNA extraction was done. The mRNA expression of GAPDHS was significantly higher in group 3 compared to the other groups, whereas the expression of ENO4 was significantly up-regulated in group 2 and 3 compared to the group 4. Similarly, the mRNA expression of PGK2 was significantly up-regulated in group 3 whereas in case of MDH2, it was significantly higher in group 2, 3 and 4 compared group 1. It can be concluded from our study that freezing and capacitation has a significant effect on the expression dynamics of the ATP synthesis-associated genes warranting necessary interventions in handling procedures to minimise the adverse effect of freezing on ATP synthesis process to ensure persistence of fertilizing ability of sperm.

Implantation associated divergence in neutrophil glucocorticoid and cysteine-X-cysteine receptor genes in cattle.

The differential roles of Interferon-tau (IFNT) and Progesterone (P4) in triggering pro- and anti-inflammatory responses during peri-implantation period were assessed in cows. An ex vivo study was done by isolating neutrophils from the animals post artificial insemination (AI) at defined intervals and analyzing the expression changes of Glucocorticoid receptor-α (GRα) and Interleukin-8 receptor (CXCR1) genes. An in vitro study was also done isolating neutrophils from prepubertal heifers subjecting them to IFNT and Progesterone P4 supplementation separately. We observed that from day 14 post Artificial Insemination, there is a significant up-regulation of neutrophil GRα and CXCR1 in pregnant and non-pregnant cows respectively. The in vitro study showed that IFNT significantly up-regulates GRα whereas P4 significantly up-regulates CXCR1. The total leukocyte count and neutrophil count were also significantly higher in non-pregnant cows. We conclude that IFNT efficiently mediates neutrophil immunosuppression during peri-implantation period in cows by interacting with Interleukin 8 receptor and can also exert ligand independent actions on GRα.

Exploring the concentration-dependent actions of interferon-τ on bovine neutrophils to understand the process of implantation.

Interferon-τ (IFNT) is a major cytokine produced by the ruminant trophoectoderm during the peri-implantation period exerting immunomodulatory actions on various cells including neutrophils. The current in vitro study was undertaken to analyze the concentration-dependent effects of IFNT on neutrophil gene dynamics to understand its possible role in implantation process. The neutrophils were isolated from the blood of heifers and were cultured subjecting them to different IFNT concentrations (1, 5, or 10 ng/mL). The gene-expression patterns of different interferon-stimulated genes, l-selectin, CD31, CD11b, and progesterone-induced blocking factor (PIBF) were analyzed by quantitative real-time PCR. It was observed that at lower concentrations of IFNT, the IFI16, l-selectin, ISG15, and PIBF were upregulated, whereas at higher concentrations the same were down regulated. At all the experimental concentrations, IFI44, OAS1, MX genes were significantly upregulated and CD31, CD11b were significantly downregulated. At lower concentrations of IFNT, the neutrophil activity with respect to chemoattraction is stimulated, whereas at higher concentrations the same is reduced. Hence, it can be concluded that IFNT exerts concentration-dependent actions on neutrophil gene-expression dynamics indicating a fine modulation of its activity depending upon the temporal variation in its destined functions ultimately leading to successful implantation.

HSP70 as a marker of heat and humidity stress in Tarai buffalo.

Heat and humidity stress is a constant challenge to buffalo rearing under tropical climatic conditions. Heat shock proteins (HSPs) constitute a ubiquitous class of highly conserved proteins that contribute to cell survival during different conditions of stress. The present study was carried out in Tarai buffaloes to study the expression of HSP70 in their peripheral blood mononuclear cells during different seasons and establish it as a marker of heat and humidity stress in buffaloes. Blood samples were collected from each healthy, non-lactating and non-pregnant buffalo above 2 years of age, once in the month of January (temperature-humidity index (THI) < 72) and in the month of May (THI > 72). Blood samples were also collected during October (THI = 72) to be used as calibrator/control. Real-time PCR was used to profile the HSP70 gene expression in the peripheral blood mononuclear cells (PBMCs). The relative expression values of HSP70 in Tarai buffalo was found to be significantly higher (P < 0.05) during summer season (2.37 ± 0.12) as compared to winter season (0.29 ± 0.04). The expression positively correlated with changes in physiological parameters like respiration rate (RR), pulse rate (PR) and rectal temperature (RT). In conclusion, it can be said that RR and HSP70 may act as characteristic physiological and cellular markers of heat and humidity stress in buffaloes.

Perspectives of pluripotent stem cells in livestock.

The recent progress in derivation of pluripotent stem cells (PSCs) from farm animals opens new approaches not only for reproduction, genetic engineering, treatment and conservation of these species, but also for screening novel drugs for their efficacy and toxicity, and modelling of human diseases. Initial attempts to derive PSCs from the inner cell mass of blastocyst stages in farm animals were largely unsuccessful as either the cells survived for only a few passages, or lost their cellular potency; indicating that the protocols which allowed the derivation of murine or human embryonic stem (ES) cells were not sufficient to support the maintenance of ES cells from farm animals. This scenario changed by the innovation of induced pluripotency and by the development of the 3 inhibitor culture conditions to support naïve pluripotency in ES cells from livestock species. However, the long-term culture of livestock PSCs while maintaining the full pluripotency is still challenging, and requires further refinements. Here, we review the current achievements in the derivation of PSCs from farm animals, and discuss the potential application areas.

A versatile bulk electrotransfection protocol for murine embryonic fibroblasts and iPS cells.

Although electroporation has been widely accepted as the main gene transfer tool, there is still considerable scope to improve the electroporation efficiency of exogenous DNAs into primary cells. Here, we developed a square-wave pulsing protocol using OptiMEM-GlutaMAX for highly efficient transfection of murine embryonic fibroblasts (MEF) and induced pluripotency stem (iPS) cells using reporter genes as well as gRNA/Cas9-encoding plasmids. An electrotransfection efficiency of > 95% was achieved for both MEF and iPS cells using reporter-encoding plasmids. The protocol was efficient for plasmid sizes ranging from 6.2 to 13.5 kb. Inducing the error prone non-homologous end joining repair by gRNA/Cas9 plasmid transfection, a high rate of targeted gene knockouts of up to 98% was produced in transgenic cells carrying a single-copy of Venus reporter. Targeted deletions in the Venus transgene were efficiently (up to 67% deletion rate) performed by co-electroporation of two gRNA-encoding plasmids. We introduced a plasmid electrotransfection protocol which is straight-forward, cost-effective, and efficient for CRISPRing murine primary cells. This protocol is promising to make targeted genetic engineering using the CRISPR/Cas9 plasmid system.

Systematic optimization of square-wave electroporation conditions for bovine primary fibroblasts.

BACKGROUND: Gene transfer by electroporation is an established method for the non-viral mediated transfection of mammalian cells. Primary cells pose a particular challenge for electroporation-mediated gene transfer, since they are more vulnerable than immortalized cells, and have a limited proliferative capacity. Improving the gene transfer by using square wave electroporation in difficult to transfect cells, like bovine fetal fibroblasts, is a prerequisite for transgenic and further downstream experiments. RESULTS: Here, bovine fetal fibroblasts were used for square-wave electroporation experiments in which the following parameters were systematically tested: electroporation buffer, electroporation temperature, pulse voltage, pulse duration, pulse number, cuvette type and plasmid DNA amount. For the experiments a commercially available square-wave generator was applied. Post electroporation, the bovine fetal fibroblasts were observed after 24 h for viability and reporter expression. The best results were obtained with a single 10 millisecond square-wave pulse of 400 V using 10 µg supercoiled plasmid DNA and 0.3 × 106 cells in 100 µl of Opti-MEM medium in 4 mm cuvettes. Importantly, the electroporation at room temperature was considerably better than with pre-cooled conditions. CONCLUSIONS: The optimized electroporation conditions will be relevant for gene transfer experiments in bovine fetal fibroblasts to obtain genetically engineered donor cells for somatic cell nuclear transfer and for reprogramming experiments in this species.

Applications, challenges, and strategies in the use of nanoparticles as feed additives in equine nutrition.

The rapid expansion of nanotechnology has been transforming the food industry by increasing market share and expenditure. Although nanotechnology offers promising benefits as feed additives, their usage in equines is primarily geared toward immunotherapy, hyper-immunization techniques, drug delivery systems, grooming activities, and therapeutic purposes. Nanoparticles could be engaged as alternatives for antibiotic feed additives to prevent foal diarrhea. Gold nanoparticles are proved to provide beneficial effects for racehorses by healing joint and tendon injuries. Because of the poor bioavailability of micro-sized mineral salts, the usage of nano-minerals is highly encourageable to improve the performance of racehorses. Nano-Vitamin E and enzyme CoQ10 for equines are no longer a simple research topic because of the increased commercial availability. Employing nanotechnology-based preservatives may offer a promising alternative to other conventional preservatives in preserving the quality of equine feed items, even during an extended storage period. While nanoparticles as feed additives may provide multitudinous benefits on equines, they could elicit allergic or toxic responses in case of improper synthesis aids or inappropriate dosages. The safety of nano-feed additives remains uninvestigated and necessitates the additional risk assessment, especially during their usage for a prolonged period. To adopt nano-feed additives in horses, there is an extreme paucity of information regarding the validity of various levels or forms of nanoparticles. Further, the currently available toxicological database on the topic of nano-feed additives is not at all related to equines and even inadequate for other livestock species. This review aims to provide new insights into possible future research pertaining to the usage of nano-feed additives in equines.

Assessment of eco-sustainability vis-à-vis zoo-technical attributes of soybean meal (SBM) replacement with varying levels of coated urea in Nellore sheep (Ovis aries).

The contemporary environmental-stewardship programmes primarily aimed at curbing the global warming potential by adopting a multidisciplinary approach. Manipulating the feeding strategies has great potential in reducing the environmental footprints of livestock production. This study intends to assess the effect of soybean meal (SBM) replacement with varying levels of coated urea (SRU) on both zoo-technical (nutrient digestibility, heat increment, and physio-biochemical parameters) and environmental attributes. The coated urea was used to replace the SBM at 0, 25, 50, and 75 percent levels. Eight adult rams (43.02 ± 0.76) maintained in a conventional shed were used in a replicated 4 x 4 Latin square design. Not all the physiological parameters viz. rectal temperature, pulse rate, and respiratory rate were affected (P>0.05)f by varying levels of SRU incorporation. The SRU fed animals had higher (P<0.05) crude protein digestibility compared to SBM fed animals; however, the replacements did not affect the nutrient digestibility coefficients of DM, OM, NFC, NDFap, ADF, and hemicellulose components. The SRU did not affect various biochemical parameters such as serum glucose, total protein, albumin, globulin, urea, creatinine, ALT, AST, Ca, P and T3, and T4 levels; however, post-prandial serum urea N (SUN) values showed a diurnal quadratic pattern (P<0.05) with a dose-dependent relationship. Further, the SBM replacements had no effect on the calcium excretion, while the SRU incorporation decreased the faecal phosphorous content, thereby abating the eutrophication phenomenon. Although the SBM replacements did not affect in vivo water variables and faecal solid fractions, they managed to decrease the land and virtual water requirement along with global warming potential (GWP) of the entire trial. The GWP-perceptual map unveils the fact that replacement of conventional feed ingredients with NPN compounds aids in eco-friendly livestock production. Further, the conjectural analysis of the carbon footprint methodology revealed that agricultural by-products consideration could cause a huge increase in the GWP share of feed consumed, thus compelling the importance of research pertaining to feed production perspective as equal as ruminal methane amelioration.

Environmental sustainability assessment of tropical dairy buffalo farming vis-a-vis sustainable feed replacement strategy.

Feeding management in dairy animals is crucial for ensuring optimal production apart from making the farming as a whole, a more sustainable activity. In our study we evaluated the production and environmental effects of two different feeding regimens i.e., one dominated by traditional cottonseed meal (CSM) and other with coated urea (slow release urea - SRU) as a replacement for CSM on dairy buffalo production. The SRU at 2% level was evaluated by conducting two different trials using twelve lactating Murrah buffaloes and four adult Murrah buffalo bulls. Neither diet nor dry period management showed any substantial effect on milk components, intakes, nutrients' digestibility coefficients, and nutritive values. The SRU diet revealed increased (P < 0.01) rumen pH, ammonia nitrogen, volatile fatty acids, and microbial nitrogen yield, which were interacted with time of post-prandial sampling. The dynamics of nitrogen metabolism revealed unaltered N-based parameters, except for degradable-N intake and serum urea-N at 3 hr post-feeding. The CSM replacements did not influence (P > 0.05) the residual feed intake, but led to an enhanced milk retention efficiency of nitrogen, calcium, and phosphorus contents, thus reducing their impact on soil pollution and eutrophication of water bodies. Despite an unaltered (P > 0.05) enteric methane emission, SRU diets achieved in decreasing manure methane and nitrous oxide emission. Furthermore, the virtual water flow and lifecycle assessment revealed a water sparing effect and low carbon foot print per unit milk production. In summary, the CSM replacements with SRU could achieve an economical and eco-friendly production system from animal nutrition perspective.

Expression and localization of locally produced growth factors regulating lymphangiogenesis during different stages of the estrous cycle in corpus luteum of buffalo (Bubalus bubalis).

Recent experiments using expression, immunolocalization, and cell culture approaches have provided leading insights into regulation of luteal angiogenesis by different growth factor systems and its role in the function of corpus luteum (CL) in buffalo. On the contrary, lymphangiogenesis and its regulation in the CL are still poorly understood. The aim of this study was to evaluate the expression and localization of lymphangiogenic factors (vascular endothelial growth factor [VEGF]-C and VEGFD), their receptor (VEGFR3), and lymphatic endothelial marker (LYVE1) in bubaline CL during different stages of the estrous cycle and to investigate functional role of VEGFC and VEGFD in luteal lymphangeogenesis. The mRNA and protein expression of VEGFC, VEGFD, and VEGFR3 was significantly greater in mid and late luteal phases, which correlated well with the expression of LYVE1. The lymphangiogenic factors were localized in luteal cells, exclusively in the cytoplasm. Immunoreactivity of VEGFC was greater during midluteal phase and that of VEGFD was greater during the mid and late luteal phases. Luteal cells were cultured in vitro and treated for different time duration (24, 48, and 72 hours) with VEGFC and VEGFD each at 50, 100, and 150 ng/mL concentration and VEGFC with VEGFD at 100 ng/mL concentration. The temporal increase in LYVE1 mRNA expression was significant (P < 0.05) in VEGFC and VEGFC with VEGFD treatment and no significant change was seen in VEGFD treatment. Thus, it seems likely that VEGFD itself has little role in lymphangiogenesis but along with VEGFC it might have a synergistic effect on VEGFR3 receptors for inducing lymphangiogenesis. In summary, the present study provided evidence that VEGFC and VEGFD, and their receptor VEGFR3, are expressed in bubaline CL and are localized exclusively in the cell cytoplasm, suggesting that these factors have a functional role in lymphangiogenesis of CL in buffalo.

Oligomerization and regioselective hydrosilylation of styrenes catalyzed by cationic allyl nickel complexes bearing allylphosphine ligands.

The complexes [Ni(eta(3)-CH(2)CHCH(2))Br(kappa(1)P-PR(2)CH(2)CH=CH(2))] (R = Ph 1, (i)Pr2 ) and [Ni(eta(3)-CH(2)C(R')CH(2))(kappa(1)P-PR(2)CH(2)CH=CH(2))(2)][BAr'(4)] (R' = H, R = Ph 4a, R = (i)Pr 4b; R' = CH(3), R = Ph 5a, R = (i)Pr 5b; Ar' = 3,5-C(6)H(3)(CF(3))(2)) have been prepared and characterized. The X-ray crystal structures of 1, 2 and 5b have been determined. 4a-b and 5a-b are catalyst precursors for the oligomerization of RC(6)H(4)CH=CH(2) to oligostyrene (R = H) or oligo(4-methylstyrene) (R = CH(3)) respectively, without the need of a co-catalyst such as methylalumoxane. The catalytic activities range from moderate to high. The oligomerization reactions are carried out in the temperature interval 25-40 degrees C in 1,2-dichloroethane, using an olefin/catalyst ratio equal to 200, yielding oligostyrenes with a high isotactic fraction content P(m), with M(n) in the range 700-1900 Dalton, and polydispersities between 1.22 and 1.64. The cationic complexes 4a-b and 5a-b are also effective catalyst precursors for the hydrosilylation reactions of styrene or 4-methylstyrene with PhSiH(3) in 1,2-dichloroethane at 40 degrees C using an olefin/catalyst ratio equal to 100, leading selectively to RC(6)H(4)CH(SiH(2)Ph)CH(3) (R = H, CH(3)) in 50-79% yield.