Post-rewarming Developmental Competence of in vitro Produced Buffalo (Bubalis Bubalis) Embryos Vitrified Using the Solid Surface Technique.

BACKGROUND: Vitrification is an ultra-rapid freezing technique for germplasm preservation under high salt concentration with very short exposure time. OBJECTIVE: To assess the post-thawed developmental potential of in vitro-produced buffalo embryos vitrified by solid surface technique using different concentrations of cryoprotectants. MATERIALS AND METHODS: The slaughterhouse derived oocytes were in vitro matured and fertilized with epididymal sperm. IVF embryos at the morula stage were vitrified under two protocols; (i) Protocol-1: ethylene glycol (35%) (ii) Protocol-2: ethylene glycol (15%) and dimethyl sulfoxide (15%). The vitrified-thawed embryos were in vitro cultured up to the blastocyst stage. RESULTS: Post-thawed development of embryos vitrified under Protocol-1 was significantly higher in terms of compact morula formation as compared to Protocol-2. However, blastocyst developmental rates were not significantly different between the two protocols. The developmental rates of the non-vitrified control were significantly higher than embryos vitrified by either protocols. CONCLUSION: The process of cryopreservation, under both protocols, significantly affected the developmental potential of pre-implant embryos as compared to fresh embryos. Hence the nature and concentrations of cryoprotectants needs to be optimized for efficient, viable embryonic development.

Molecular characterization of the complete mitochondrial genome sequence of Indian wild pig (Sus scrofa cristatus).

The Indian wild pig is a sub-species (Sus scrofa cristatus) which is different from the other pig breeds and is protected under Schedule-III of the Indian Wildlife (Protection) Act, 1972. In this study, complete mitogenome of two Indian wild pigs was sequenced and characterized by shotgun sequencing and de novo assembly, which revealed sequence size of 16,738 and 16,251 bp, respectively, (Accession no. MG725630 and MG725631). The mitogenome sequence in this study displayed 98% homology with previously reported mitogenome of pigs from different parts of the world. Mitogenome analysis by MITOS Web server revealed similarity of gene organization with the other vertebrates (13 protein-coding, 22 tRNAs, 2 rRNAs genes, and a control region). The mitogenomic sequences of Indian wild pig maintained a separate clade in the phylogenetic tree constructed by using 62 whole mitogenome sequences across the world. The phylogeny derived from mitogenomic sequences revealed distinct separate European-American and Asiatic pig clades. It was concluded that whole mitogenome sequencing using NGS without designing mitogenome-specific primer for amplification, is possible thereby reducing the cost and labor. This study is the first report of complete sequence of mitogenome of Indian wild pig.

Assessment of developmental potential of caprine cloned embryos with ooplasm replenishment under two culture media.

The present study was designed to assess the developmental potential of somatic cell nuclear transfer (SCNT) embryos, with and without replenishment of ooplasm into enucleated oocytes, by culturing separately in two culture media. The enucleated oocytes were replenished with exogenous matured ooplasm under replenished nuclear transfer (RNT) method and compared with conventional nuclear transfer (CNT) method without replenishment. The fusion efficiency in RNT group was found to be significantly higher (P < 0.05) than CNT group (59.39 ± 7.36 vs 45.57 ± 3.68%). The completely fused reconstructed oocytes from both groups were cultured separately in research vitro cleave medium (RVCL) and RVCL-Blast medium. The embryonic development of 2 cell, 4 cell, 8-16 cell and 16-32 cell stages in the RNT group was superior to the CNT group regardless of the culture media used (P < 0.05). The embryonic development of the 8-16 cell, 16-32 cell, morula, and blastocyst stages in the RVCL-Blast medium was significantly higher (P < 0.05) than the RVCL alone for both RNT as well as CNT groups. RNT method with RVCL-Blast produced highly significant (P < 0.01) embryonic development for 8-16 cell and 16-32 cell stage when compared to CNT with RVCL. Conclusively, the combination of RNT with RVCL-Blast culture media enabled an overall increase in the embryonic developmental potential.

Molecular and biochemical evaluation of Indian draft breeds of cattle (Bos indicus).

Glutathione peroxidase-1 (GPX-1) enzyme detoxifies peroxides by reacting with the GSH (reduced glutathione) responsible for the maintenance of the integrity of essential biomolecules. This study was conducted on 100 animals of two Indian draft breeds of cattle (Bos indicus), Nimari, and Malvi. Genomic DNA was isolated from blood samples, and four fragments (80, 71, 78, and 442 bp) of the GPX-1 gene were amplified by polymerase chain reaction. PCR-SSCP analysis in 12% PAGE with silver staining revealed polymorphism in three of the fragments (80, 71, and 442 bp) in these two cattle breeds. Breed differences for the blood biochemical parameters (serum creatine kinase and lactic acid level) and overall draft ability were studied. The genetic polymorphism identified for the GPX-1 gene in this investigation would help in the identification of alleles related to draft capacity in these animals for future genetic improvement.

Knockdown of the myostatin gene by RNA interference in caprine fibroblast cells.

Myostatin (MSTN), a member of transforming growth factor-beta superfamily is a negative regulator of the skeletal muscle growth. It suppresses the proliferation and differentiation of myoblast cells. Dysfunction of MSTN gene either by natural mutation or induced through genetic manipulation (knockout or knockdown) has been reported to increase the muscle mass in mammalian species. RNA interference (RNAi) is the most promising method for inhibition of gene expression that can be utilized for MSTN gene knockdown by developing short hairpin RNA (shRNA) construct against it. In the present investigation silencing of MSTN gene in caprine fibroblast cell line was evaluated using four different shRNA expressing constructs. Variation in the efficiency of silencing (22-92%) was obtained among different constructs. It was observed that sh1 and sh4 constructs downregulated the MSTN gene expression by reducing 92.4 and 80.5% (P<0.05) level of downregulation MSTN mRNA, respectively. On the contrary, the sh3 construct significantly upregulated the MSTN mRNA level (P<0.05). These two promising constructs (sh1 and sh4) need to be further tested for interferon (IFN) response before their use in long term stable expression of anti-MSTN shRNA in muscle cells to improve chevon production.

Nucleic acids in the spermatozoa of pure and strain crosses of White Leghorn cockerels.

Nucleic acids (DNA and RNA) were estimated in the spermatozoa of three pure strains (M, N and P) and three strain crosses (N X P, M X N and P X M) of White Leghorn cockerels. The average DNA content was 2.31 +/- 0.06 X 10(-9)mg/sperm. Most was found in the P X M strain cross and least in the P strain. The DNA content of the P strain was significantly lower than that in the other strains and it also had the lowest fertility and hatchability of all the strains. The average RNA content in poultry spermatozoa was 0.0522 +/- 0.0038 X 10(-9)mg/sperm. Again, most RNA was found in the P X M strain cross and least in the P strain. The P X M strain cross had a significantly higher RNA content than the rest of the strains. Thus, the P X M strain contained the most DNA and RNA whereas the P strain had least.

Studies on dimensional characteristics of spermatozoa in pure and strain crosses of White Leghorn cockerels.

Sperm head dimensional characteristics were evaluated in three pure strains (M, N, and P) and three strain crosses (N x P, M x N, and P x M) of White Leghorn cockerels. A total of 120 collections from 24 cockerels were taken. The average sperm head length recorded was 14.58 +/- .09 mu, with an average maximum value of 15.29 +/- .19 mu for spermatozoa from the P x M strain cockerels. The average sperm breadth was found to be .722 +/- .005 mu. Maximum values were found for spermatozoa from the M x N strain cockerels that averaged .735 +/- .004 mu. There was a significant difference (P less than .01) between strains for sperm head length. The P x M strain had a significantly longer sperm head length than the other strains. This may be associated with heterosis. There were no significant differences among strains for sperm head breadth. The mean sperm head area was recorded to be 10.61 +/- .08 mu 2. Maximum sperm head area was observed in semen from P x M strain cockerels (11.03 +/- .15 mu 2). The average sperm head shape was found to be 20.14 +/- .22. There were significant differences (P less than .01) among strains for sperm head area and sperm head shape. The repeatability estimates for all the dimensional characteristics showed that they are highly repeatable traits, indicating that additive gene action is playing an important role in the expression of these traits.