The present study was conducted to investigate the effect of meiotic stages during in vitro maturation (IVM) on the survival of vitrified-warmed buffalo oocytes, vitrified at different stages of IVM. Cumulus oocyte complexes obtained from slaughterhouse ovaries were randomly divided into 6 groups: control (non-vitrified, matured for 24 h at 38 +/- 1 degrees C, 5% CO2 in humidified air), and those matured for 0 h (vitrified before IVM) or 6, 12, 18 and 24 h before vitrification. Cumulus oocyte complexes were vitrified in solution consisting of 40% w/v propylene glycol and 0.25 mol/L trehalose in phosphate-buffered saline supplemented with 4% w/v bovine serum albumin. Vitrified cumulus oocyte complexes were stored at -196 degrees C (liquid nitrogen) for at least 7 days and then thawed at 37 degrees C; cryoprotectant was removed with 1 mol/L sucrose solution. Cumulus oocyte complexes in the 0, 6, 12, 18 and 24 h groups were then matured for an additional 24, 18, 12, 6 and 0 h, respectively, to complete 24 h of IVM. Among the five vitrification groups, 89-92% of cumulus oocyte complexes were recovered, after warming, of which 84-91% were morphologically normal. Overall survivability of vitrified cumulus oocyte complexes was lower (p < 0.05) than that of non-vitrified cumulus oocyte complexes (94.5%). Survival rates of cumulus oocyte complexes matured 24 h prior to vitrification (61.3%) were higher (p < 0.05) than those matured for 12 h (46.7%), 6 h (40.6%) and 0 h (37.6%). Nuclear status following 24 h IVM was assessed. A higher proportion of non-vitrified (control) oocytes (72.7%) reached metaphase II (M-II) stage in control than oocytes vitrified for 24 h (60.0%), 18 h (54.4), 12 h (42.3%), 6 h (33.3%) and 0 h (31.6%) (p < 0.05). The results suggest that length of time in maturation medium prior to vitrification influences post-thaw survivability of buffalo oocytes; longer intervals resulted in higher survival rates.

Chronic mitral valve insufficiency (CMVI) is the most common acquired heart disease in dogs. In heart failure, the cellular oxygenation and metabolism are affected, which leads to the production of free radicals. Free radicals damage DNA, lipid and protein molecules in cells. In the present experiment, blood samples were collected from CMVI dogs with heart failure and were compared with the results obtained from healthy dogs. A significant increase in the levels of xanthine oxidase, AST, LDH and CK and decrease in the activity of catalase were noticed in CMVI dogs when compared to healthy dogs, which revealed overall cardiac and skeletal muscle damage in CMVI dogs. Results of biochemical parameters revealed an increase in urea level and decrease in sodium, potassium, and calcium levels in CMVI dogs as compared to control dogs, all of which indicate cardiac damage in dogs. Study on hematological parameters revealed a significant decrease in Hb, PCV, RBC and platelet counts and an increase in total WBC counts and percentage of neutrophils, decrease in percentage of the lymphocyte and monocyte in CMVI dogs than control. These results indicate secondary phenomenon to heart failure. The present research data indicates the usefulness of these biomarkers in the diagnosis and prognosis of CMVI with heart failure in dogs.

Antioxidant status determines the susceptibility of tissues to the oxidative stress associated with diabetes and its complications; hence in the present study antioxidant status was explored in streptozotocin-induced diabetic rats treated with vanadium in the form of vanadium pentoxide nanoparticles. Vanadium pentoxide and vanadium pentoxide nanoparticles at the dose rate of 5mg/kg were administered orally in STZ (50mg/Kg) induced diabetic rats for 30 days and glimepiride (reference drug) was administered orally at the dose rate of 800 ìg/kg body weight. Vanadium pentoxide nanoparticles significantly reduced the blood glucose levels than the diabetic control and other treatment group of rats. On exploration of antioxidant status in liver, kidney and pancreas tissues, vanadium in the form of vanadium pentoxide nanoparticles outperformed the vanadium pentoxide by increasing the activities of the enzymes catalase, superoxide dismutase and glutathione peroxidase, and the concentration of reduced glutathione and by decreasing the lipid peroxide levels. The present study also showed that the restoration of antioxidant status by vanadium pentoxide nanoparticles is comparable with that of the reference drug. It can be concluded that vanadium pentoxide nanoparticles, due to its superior control over hyperglycemia and antioxidant properties, outperformed the vanadium pentoxide treatment in enhancing the antioxidant status in diabetic rats.

The present experiment was aimed to detect osteopontin gene transcript from bull spermatozoa. Fresh semen samples from 12 Jersey cross breed bulls were collected using artificial vagina. The volume and concentration of the individual samples were recorded. The spermatozoa were separated from bull semen samples by swim up protocol using sperm TALP. The initial concentration of semen sample was checked immediately before proceeding for RNA isolation and normalization of initial concentration was done. So, initial amount of every sample was made equal. Total RNA from the bull spermatozoa were extracted by the RNeasy® Mini Kit, Qiagen as per manufacturer's protocol. The purity and concentration of the samples were measured at A280/260 by using Nano Drop TM 1000 spectrophotometer. The first strand cDNA was synthesized from 1μg total RNA using M-Mu LV Reverse Transcriptase Revert Aid TM H minus first strand cDNA synthesis kit (Thermo Scientific) as per manufacturer's protocol. PCR product of about 267 bp fragment length of OPN gene was confirmed by conventional PCR. The bulk PCR product of about 100μl was purified by Gen Elute™ PCR Clean-Up Kit Sigma-Aldrich as per manufacturer's protocol. The concentration and purity of PCR product were 64ng/μl and 1.6 respectively. The cloning and expression of OPN gene was carried out by using TA cloning kit. The ligated product was then transformed into E. coli DH5α cells. The plate was incubated for overnight at 37 ˚C for selection of the colonies and stock cultures were maintained in LB media at -80˚ C. The clones which showed the amplification of 400 bp DNA fragment were considered as positive clone carrying desired insert. Plasmid isolation was done as per the protocol followed in Hi Yield TM Plasmid Mini Kit (RBC Cat. NO. YPD100) and the eluted DNA were stored at -20 ˚C. The concentration and purity of PCR purified product were 67ng/μl and 1.6 respectively. The eluted plasmid DNA was sequenced commercially (Amnion, Bangalore) using M13 primers. The cloned partial sequence of OPN gene isolated from bovine spermatozoa was submitted at DDBJ (Accession No. AB983656). On blast analysis using NCBI nucleotide blast (Online tool), 99 per cent identity was found with reported consensus sequences except single nucleotide variation in DNA sequence at 234 th nucleotide. There was substitution of adenine instead of cytosine.

The present study has been undertaken to assess the post thaw recovery of buffalo oocytes vitrified at different stages of in vitro maturation (IVM). Cumulus oocyte complexes (COCs) obtained from slaughterhouse ovaries were randomly divided into 6 different groups: control (non-vitrified oocytes were matured for 24 h in maturation medium (MM) consists of TCM-199 supplemented with 10 per cent w/v fetal calf serum (FCS) at 38±1 0 C and 5 per cent CO 2 in a humidified atmosphere, 0 h (vitrified before the onset of maturation), 6, 12, 18 and 24 h groups (vitrified at 6, 12, 18 and 24 h, respectively, after the onset of maturation). Oocytes were exposed to vitrification solution (VS) consists of 40 per cent w/v propylene glycol and 0.25 M trehalose in phosphate buffered saline (PBS) supplemented with 4 per cent w/v bovine serum albumin (BSA) for 3 min at 20-25 0 C. Oocytes in VS were loaded into 0.25 ml French mini straw with 1M sucrose solution separated by two airspace on either side of VS. The straws were sealed with hot forceps and plunged directly into liquid nitrogen (LN 2 ;-196 0 C). The straws were thawed after storage period of atleast 7 days by transferring them into a water bath at 37°C for 30 sec. The cryoprotectant was removed by exposing the oocytes to 1 M sucrose solution. Oocytes in 0, 6, 12, 18 and 24 h groups were further matured for additional 24, 18, 12, 6 and 0 h, respectively, to complete a total of 24 h maturation period.A sum of 495, 432, 457, 416 and 420 oocytes were vitrified in 0, 6, 12, 18 and 24 h groups, respectively. After thawing, 444 (89.70%), 384 (88.89%), 418(91.47%), 381 (91-59%) and 387 (92.14%) oocytes were recovered in 0, 6, 12, 18 and 24 h groups, respectively. It is evident that no significant difference was observed under different vitrification groups.