Activation of Zona‐Free Buffalo (Bubalus bubalis) Oocytes by Chemical or Electrical stimulation,and Subsequent Parthenogenetic Embryo Development

Parthenogenetic activation using zona‐free oocytes offers an alternative model that could be applied to develop protocols for the activation of reconstructed embryos for cloning. The aim of this study was to compare the efficacy of different methods for the activation of zona‐free buffalo oocytes in terms of their effects on the developmental competence of parthenogenetic embryos. The effects of zona removal on parthenogenetic activation and in vitro developmental competence of metaphase II oocytes were also examined. All activation methods were followed by incubation of 2 mm 6‐dimethylaminopurine (6‐DMAP) for 4 h. Out of three different pulse strengths (1.2, 2.1 or 3.3 kV/cm) used, 2.1 kV/cm resulted in the highest blastocyst rate (25.3%). On comparing different chemical agents and electric pulse, highest blastocyst rate was observed for calcium ionophore (CaI) (28.6%) followed by ethanol (25.0%), electric pulse (22.5%) and combined CaI and ethanol treatment (16.7%) although differences among them were not significant. Furthermore, a significantly reduced developmental potential was observed in zona‐free oocytes when compared to zona‐intact ones up to the blastocyst stage (44.3% vs 27.1%). In conclusion, zona‐free buffalo oocytes can be successfully activated for parthenogenetic development using chemical or electrical stimulation. Out of different agents examined, CaI followed by 6‐DMAP resulted in the highest blastocyst rate.     

Production of nuclear transfer embryos by using somatic cells isolated from milk in buffalo (Bubalus bubalis)

Somatic cells in milk are a potential source of nuclei for nuclear transfer to produce genetically identical animals; this is especially important in animals that are susceptible to risks of bacterial infection on biopsy collection. In this study, a minimum of 10 milk samples were collected from each of the three buffaloes representing Murrah breed. All the samples were processed immediately and cell colonies were obtained. Cell colonies from one buffalo (MU-442) survived beyond 10 passages and were evaluated by fluorescence microscopy and used in nuclear transfer experiments. In culture, these cells expressed vimentin, indicating they were of fibroblast origin similar to ear cells. We compared the effectiveness of cloning using those milk-derived fibroblast (MDF) cells and fibroblast cells derived from the ear derived fibroblast (EDF). Fusion and cleavage rates of MDF-NT and EDF-NT embryos were found to be similar (92.43 ± 1.28% vs 94.98 ± 1.24%, and 80.27 ± 1.75% vs 84.56 ± 3.73%, respectively; p > 0.01); however, development to blastocyst stage and total cell number was higher for EDF-NT embryos (50.24 ± 2.54%, 227.14 ± 13.04, respectively, p < 0.01), than for MDF-NT embryos (16.44 ± 0.75%, 170.57 ± 4.50 respectively). We conclude that somatic cells from milk can be cultured effectively and used as nucleus donor to produce cloned blastocyst-stage embryos.     

Presence of nitric oxide synthase immunoreactivity and mRNA in buffalo (Bubalus bubalis) oocytes and embryos

This study examined the presence of immunoreactivity and mRNA for different nitric oxide synthase (NOS) isoforms in immature and in vitro matured oocytes and in embryos at two‐, four‐ and eight‐cell, and morula and blastocyst stages in buffalo. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to in vitro maturation in TCM‐199 + 10% FBS + 5 μg/ml pFSH + 1 μg/ml estradiol‐17β + 0.81 mm sodium pyruvate + 10% buffalo follicular fluid + 50 μg/ml gentamycin sulphate for 24 h in a CO₂ incubator (5% CO₂ in air) at 38.5°C. Following in vitro fertilization carried out by incubating them with 2–4 million spermatozoa/ml for 18 h, the presumed zygotes were cultured in mCR2aa medium containing 0.6% BSA and 10% FBS for up to 8 days post insemination. Immunofluorescence staining of NOS using antibodies that cross‐reacted either with all the NOS isoforms i.e., universal (uNOS) or specifically with inducible (iNOS) or endothelial (eNOS) isoforms revealed that NOS was present in oocytes and embryos at all the stages examined. Examination of the semi‐quantitative expression of NOS genes by RT‐PCR revealed that the iNOS, eNOS and nNOS mRNA was present in the immature and mature oocytes and in all the embryonic stages examined. In conclusion, it was demonstrated in the present study that immunoreactivity and mRNA for different NOS isoforms was present in buffalo oocytes and pre‐implantation stage embryos.     

Optimizing embryo age and media for enhancing hybrid seedling recovery in sour orange (Citrus aurantium) × Sacaton citrumelo (C. paradisi × Poncirus trifoliata) crosses through embryo rescue

In citrus, development of new hybrids is difficult due to failure of the germination of zygotic embryos. Hence, this study was conducted to standardize embryo age and media for maximizing the germination and subsequent seedling growth in sour orange × Sacaton citrumelo crosses followed by the identification of simple sequence repeats (SSR) markers for distinguishing the hybrids. A factorial experiment in complete randomized design was conducted with two each embryo age (110–120 and 130–140 days after pollination) and culture media (G‐B5 supplemented with 1.5 mg/L GA₃ plus malt extract and G‐B5 plus malt extract) for in vitro embryo culture of sour orange × Sacaton citrumelo progenies. Older embryos germinated well in both media, though highest on M1 medium (100%). In fact, M1 medium gave higher plantlet survival in both age groups. The seedling growth of older embryos had highest root length (37.80 mm) and shoot length (24.20 mm) in M1 medium at 60 days after inoculation (DAI). Three SSR markers (TAA45, CAC15 and CAC39) showed polymorphism between female and male parents and were able to identify their hybrids.     

Evaluation of candidate genotype of leptin gene associated with fertility and production traits in Hardhenu (Bos taurus × Bos indicus) cattle

The present study was conducted on Hardhenu cattle to screen genomic region of leptin gene with an objective to find the association of genotypes with fertility and production traits. The association analysis with traits under study was analysed by least squares analysis of variance by taking SNPs genotype as fixed effects in the statistical model. The genotypic frequencies with respect to targeted loci g.92450765 G > A indicated that AG (0.54) genotype was highest in Hardhenu cattle. Chi-squared tests showed that g.92450765G > A SNP meet with the Hardy–Weinberg equilibrium (p > .05).The association analysis revealed significant association of genotypes with total milk yield (TMY) and 305 days milk yield (MY) (p < .05). Service period (SP) and calving interval (CI) were also found significantly associated with genotypes (p < .05). Whereas, lactation length (LL), dry days (DD) and age at first calving (AFC) did not divulge any significant association with genotype. The AG and GG genotypes were associated with higher milk yields as compared to AA genotype, indicating that allele G was associated with superior milk performance. However, AA genotyped cattle found to be favourable with SP, CI and artificial insemination (AI) per conception compared to AG and GG genotyped cows. Chi-square analysis revealed that genetic variants of g.92450765 G > A SNP of leptin gene differ significantly with regard to reproductive disorders incidence (p < .05). The frequency of GG genotype (88.89%) in the affected animal group was very high followed by AG. The animals with GG genotype were found to be more susceptible to reproductive disorders as suggested by the higher odd ratio value (16.00) in logistic model. These observations and their differential association with the fertility and production traits can be utilized as an aid to selection for genetic improvement of antagonistic traits in dairy cows.     

Detection and assessment of risk factors associated with natural concurrent infection of Trypanosoma evansi and Anaplasma marginale in dairy animals by duplex PCR in eastern Punjab

Tropical Animal Health and Production - Duplex PCR consisting of two primer sets within a single mixture for the simultaneous detection of Anaplasma marginale and Trypanosoma evansi was...     

Effect of biogas digested slurry based-biochar and digested liquid on N2O,CO2 flux and crop yield for three continuous cropping cycles of komatsuna (Brassica rapa var. perviridis)

Biogas production generates digested slurry, as a byproduct, which can be used as fertilizer after its conversion into digested liquid and biochar. A microcosm-based study was conducted to evaluate the effects of chemical fertilizer (CF), digested liquid (DL) and varying concentrations of biogas digested slurry based-biochar along with DL on N₂O flux, CO₂ flux, soil chemical properties and crop yield for three continuous cropping cycles of komatsuna (Brassica rapa var. perviridis) from April to July 2013. Analyses revealed that DL-treated soils released almost equal cumulative amounts of N₂O and CO₂ as soils treated with CF. The soil mineral-N contents were also similar for the DL- and CF-treated soils while DL application increased the soluble organic carbon (SOC) content of the soil compared to CF treatment. The application of slurry-based biochar increased N₂O and CO₂ flux, which, in turn, appeared to depend upon biochar concentration. The application of biochar probably increased the nitrification rate as biochar-treated soils had higher values of NO₃ ^?-N and lower values of NH₄ ⁺-N compared to soils not treated with biochar at most of the observations. The SOC content was also the highest in biochar-treated soils. The overall crop yield for three cropping cycles was the highest in DL and biochar at low application rate (BL), and it was lower in CF, biochar at medium (BM) and high (BH) application rate. This study indicates that the application of DL could be an effective strategy to minimize the use of CF, without affecting N₂O flux, CO₂ flux, soil mineral N, and increasing crop productivity. The effects of slurry-based biochar on greenhouse gases flux and crop yield depends on the application rate of biochar.