IGF-1 stabilizes goat sperm mitochondrial transmembrane potential and reduces dna fragmentation.

BACKGROUND: Antioxidant present in sperm cells protects them from oxidative damage. However, sperm are more susceptible to peroxidative damages due to the loss of these enzymes during cryopreservation and their survival and fertility may be compromised. Insulin like growth factor-1 (IGF-1) has an antioxidant effect and could maintain sperm motility. OBJECTIVE: To improve seminal parameters, mitochondrial membrane potential (MMP), oxidative status and DNA integrity of buck semen after freeze-thawing by fortification of goat semen diluent with various concentrations of IGF-1. MATERIALS AND METHODS: Fifty ejaculates were collected and were extended with tris- citric acid- fructose diluent with 10% egg yolk and 6% glycerol with sperm concentrations of 1×108 mL-1. Post-cryopreserved sperm were assessed for motility and a range of other functional parameters. RESULTS: In post-thaw semen sperm motility, live sperm count, acrosome integrity, hypo-osmotic swelling positive spermatozoa, malondialdehyde (MDA), protein carbonyl content (PCC), TUNEL positive sperm differed significantly (P<0.05) with the various concentrations of IGF-1 used. Sperm functional parameters post-thawing were significantly (P<0.05) better in 250 ng/mL IGF-1. IGF-1 protects against lipid peroxidation by lowering MDA and PCC production, thus reducing the harmful effect of reactive oxygen species. The kidding percentage using the artificial insemination technique was significantly higher ( i.e., 40%) in the group supplemented with 250 ng/mL of IGF-1 than in the non-supplemented group (i.e., 30%). CONCLUSION: IGF-1 may be used to improve post-thaw semen quality and fertility as measured by actual kidding rate. Doi.org/10.54680/fr23610110312.

Establishment of effective and safe recipient preparation for germ-cell transplantation with intra-testicular busulfan treatment in pre-pubertal Barbari goats.

The busulfan, an alkylating agent, suppresses endogenous spermatogenesis in recipient testes. However, considering a wide variation in the effects of busulfan among animal species, its dosage and route of infusion need optimization to prepare effective and safe recipients. Thus, the current study aimed to create a suitable recipient goat model for germ cell (Gc) transplantation through a single intra-testicular (i.t.) busulfan infusion under ultrasonographic (USG) guidance. As observed through the infusion of trypan blue under USG guidance into mediastinum testis (MT) of pre-pubertal Barbari bucks, 3-5 mL of trypan blue solution could fill almost 80% of seminiferous tubules. Thereafter, in Experiment-1, the effect of different busulfan doses (mg/kg) i.e. 0 [negative control, Group (Gr) 1; 0 mg/kg-MT], 1 (Gr 2; 1 mg/kg-MT), 2 (Gr 3; 2 mg/kg-MT), and 3 (Gr 4; 3 mg/kg-MT) were studied. Further, in Experiment-2, sterilizing effects of busulfan infusion through two different routes [MT or cavum vaginale (CV)] were compared. Following i.t. busulfan treatment, no adverse physiological effects or body weight loss were detected. The histological analyses demonstrate a dose-dependent depletion of Gc with almost complete loss of Gc and spermatogenic activities in Gr 3 and 4, and extensive fibrosis in Gr 4. A considerable suppression of spermatogenesis marked with devoid of endogenous spermatogonial population and absence of significant (P > 0.05) effect on key hematological variables were observed in 2 mg/kg-MT Gr. These findings coupled with the results of significant (P < 0.05) down-regulation of marker genes of undifferentiated spermatogonia (THY-1 and PLZF), Gc pluripotency (UCHL-1, OCT-4, and DDX-4), and adhesion (E-cadherin and ß-integrin); up-regulation of apoptotic genes (ID - 4 and BCL-6), and unchanged expression of Sertoli cell marker (vimentin), confirmed the safe and efficient depletion of endogenous Gc in 2 mg/kg-MT Gr. Furthermore, the effect of busulfan infusion on scrotal-testicular biometry, endocrine variables (plasma cortisol and testosterone), and Gc removal was more evident when busulfan was infused into MT than into CV. Overall, the results demonstrated that 2.0 mg/kg is an optimal single dose of busulfan when infused into the MT under USG guidance for the preparation of pre-pubertal recipient bucks. Overall, this study provides a basis to prepare suitable recipients through providing an available niche for efficient Gc transplantation in goats.

Expression of the developmental important candidate genes in oocytes, embryos, embryonic stem cells, cumulus cells, and fibroblast cells of buffalo (Bubalus bubalis).

The present study was undertaken to study the expression of the developmental important gene transcripts in immature oocytes, mature oocytes, different stages of IVF produced embryos, embryonic stem (ES), cumulus (BCC), fetal fibroblast (BFF), newborn fibroblast (NBF) and adult fibroblast (BAF) cells of buffalo by semi-quantitative RT-PCR. The expression of GLUT1, HSP70.1, POL A Polymerase, GDF9, BMP15, and SURVIVIN transcripts was found in immature oocytes, mature oocytes, 2-cell, 4-cell, 8-16 cell, morula, and the blastocyst. Interestingly, the CX43 expression was found in oocytes, embryos, and other cell types, but it was not detected in the blastocyst. However, the IFNT expression was found in the blastocyst only, but not in other cells. The buffalo ES cells showed the expression of intracellular and cell surface markers (NANOG, OCT4, SOX2, FOXD3, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) and alkaline phosphatase activity. Two ES cell lines (S-line and M-line-II) were continued to survive up to 98th passages (~630 days) and 97th passages (~624 days), respectively. It was interesting to note that GLUT1, CX43, HSP70.1, POL A Polymerase, GDF9, BMP15, and SURVIVIN transcripts (except the IFNT) were expressed in buffalo ES, BCC, BFF, NBF and BAF cells. This is the first preliminary report that the buffalo ES, BCC, BFF, NBF, and BAF cells expressed the several developmental important candidate genes. It is concluded that the expression of the major developmental important genes was not only expressed in the oocytes and embryos but also expressed in the ES, BCC, BFF, NBF, and BAF cells of buffalo.

Parthenogenetic activation of buffalo (Bubalus bubalis) oocytes: comparison of different activation reagents and different media on their developmental competence and quantitative expression of developmentally regulated genes.

This study was carried out to compare the efficacy of different methods to activate buffalo A + B and C + D quality oocytes parthenogenetically and to study the in vitro developmental competence of oocytes and expression of some important genes at the different developmental stages of parthenotes. The percentage of A + B oocytes (62.16 ± 5.06%, range 53.8-71.3%) was significantly higher (P < 0.001) compared with that of C + D oocytes (37.8 ± 5.00%, range 28.6-46.1%) retrieved from slaughterhouse buffalo ovaries. Among all combinations, ethanol activation followed by culture in research vitro cleave medium gave the highest cleavage and blastocyst yields for both A + B and C + D grade oocytes. Total cell numbers, inner cell mass/trophectoderm ratio and apoptotic index of A + B group blastocysts were significantly different (P < 0.05) from their C + D counterpart. To determine the status of expression patterns of developmentally regulated genes, the expression of cumulus-oocyte complexes, fertilization, developmental competence and apoptotic-related genes were also studied in parthenogenetically produced buffalo embryos at different stages, and indicated that the differential expression patterns of the above genes had a role in early embryonic development.

Effects of treatment with a microRNA mimic or inhibitor on the developmental competence, quality, epigenetic status and gene expression of buffalo (Bubalus bubalis) somatic cell nuclear transfer embryos.

Expression levels of 13 microRNAs (miRNAs) were compared between buffalo blastocysts produced by somatic cell nuclear transfer through hand-made cloning and IVF to improve cloning efficiency. Expression of miR-22, miR-145, miR-374a and miR-30c was higher, whereas that of miR-29b, miR-101, miR-302b, miR-34a, miR-21 and miR-25 was lower, in nuclear transferred (NT) than IVF embryos; the expression of miR-200b, miR-26a and miR-128 was similar between the two groups. Based on these, miR-145, which is involved in the regulation of pluripotency, was selected for further investigation of NT embryos. miR-145 expression was lowest at the 2-cell stage, increased through the 4-cell stage and was highest at the 8-cell or morula stage in a pattern that was similar between NT and IVF embryos. miR-145 expression was higher in NT than IVF embryos at all stages examined. Treatment of reconstructed embryos 1h after electrofusion with an inhibitor of miR-145 for 1h decreased the apoptotic index and increased the blastocyst rate, total cell number, ratio of cells in the inner cell mass to trophectoderm, global levels of acetylation of histone 3 at lysine 18 and expression of Krueppel-like factor 4 (KLF4), octamer-binding transcription factor 4 (OCT4) and SRY (sex determining region Y)-box 2 (SOX2) in blastocysts. Treatment with an miR-145 mimic had the opposite effects. In conclusion, treatment of NT embryos with an miR-145 inhibitor improves the developmental competence and quality, and increases histone acetylation and expression of pluripotency-related genes.

Successful transplantation of transfected enriched buffalo (Bubalus bubalis) spermatogonial stem cells to homologous recipients.

Genetic modification of spermatogonial stem cells (SSCs) is an alternative method to pronuclear microinjection and somatic cell nuclear transfer for transgenesis in large animals. In the present study, we optimized the process of homologous SSC transplantation in the water buffalo (Bubalus bubalis) using transfected enriched SSCs generated by a non-viral transfection approach. Firstly, the SSC enrichment efficiencies of extracellular matrix components viz. collagen, gelatin, and Datura stramonium agglutinin (DSA) lectin were determined either individually or in combination with Percoll density gradient centrifugation. The highest enrichment was achieved after differential plating with DSA lectin followed by Percoll density gradient centrifugation. Nucleofection showed greater transfection efficiency (68.55 ±â€¯4.56%, P < 0.05) for enriched SSCs in comparison to fugene HD (6.7 ±â€¯0.25%) and lipofectamine 3000 (15.57 ±â€¯0.74%). The transfected enriched SSCs were transplanted into buffalo males under the ultrasound guidance and testis was removed by castration after 7-8 weeks of transplantation. Persistence and localization of donor cells within recipient seminiferous tubules was confirmed using fluorescent microscopy. Further confirmation was done by flow cytometric evaluation of GFP expressing cells among those isolated from two-step enzymatic digestion of recipient testicular parenchyma. In conclusion, we demonstrated for the first time, generation of buffalo transfected enriched SSCs and their successful homologous transplantation in buffaloes. This study represents the first step towards genetic modifications in buffaloes using SSC transplantation technique.

Utility of 18 F-FDG PET/CT in pre-surgical risk stratification of patients with breast cancer.

OBJECTIVE: To determine the correlation between fluorine-18-fluorodeoxyglucose (18F-FDG) uptake values and clinicopathological prognostic markers using preoperative 18F-FDG positron emission tomography/computed tomography (PET/CT) in primary breast cancer (BC). SUBJECTS AND METHODS: One hundred and twelve patients with primary BC were studied prospectively. Pretreatment 18F-FDG PET/CT was performed. Maximum standardized uptake values (SUVmax) were compared with various clinicopathological variables. RESULTS: In a univariate analysis, SUVmax correlated well with the following prognostic variables: T stage, absence of progesterone receptor (PR), absence of estrogen receptor (ER), triple negative lesions (ER/PR and Her 2 negative) and high histologic grade. Metastatic lesions and ductal lesions had higher SUVmax than lobular carcinoma. No significant correlation was found between SUVmax,and human epidermal growth factor receptor 2 (Her-2) statusor perineural and lymphovascular invasion. Multivariate analyses showed that breast density, tumor size and PR negativity were significantly correlated with SUVmax (P=0.046 and 0.009, respectively). CONCLUSION: The pre-treatment tumor SUVmax could be utilized as an independent imaging biomarker of the tumor aggressiveness and poor prognosis. Risk stratification based on this index could play a pivotal role in alteration of treatment planning, such as neoadjuvant chemotherapy (precision oncology).

Reducing the cytoplasmic volume during hand-made cloning adversely affects the developmental competence and quality, and alters relative abundance of mRNA transcripts and epigenetic status of buffalo (Bubalus bubalis) embryos.

Hand-made cloning (HMC) is a method of choice for somatic cell nuclear transfer (SCNT). There is 20% to 50% of cytoplasm lost during manual enucleation of oocytes with HMC. To compensate, two enucleated demicytoplasts, instead of one, are fused with each donor cell, which leads to cytoplasm pooling from two different demicytoplasts. In this study, effects of using one, instead of two demicytoplasts (controls) was examined, for production of embryos using HMC. Use of one demicytoplast decreased blastocyst development (12.7 ±â€¯1.98% compared with 47.6 ±â€¯3.49%, P < 0.001), total cell number (TCN, 167.6 ± 14.66 compared with 335.9 ± 58.96, P < 0.01), apoptotic index (2.11 ± 0.38 compared with 3.43±0.38, P < 0.05) but did not significantly alter inner cell mass:trophectoderm cell number ratio (0.17 ± 0.01 compared with 0.19 ± 0.02) and the global content of H3K9ac and H3K27me3 of blastocysts, compared to controls. There were gene expression alterations in pluripotency- (SOX2 and NANOG but not OCT4), epigenetic- (DNMT1 but not DNMT3a and HDAC1), apoptosis- (CASPASE3 but not BCL-2 and BAX), trophectoderm- (CDX2), development- (G6PD but not GLUT1) and cell cycle check point control-related related genes (P53) compared with controls. Transfer of cloned blastocysts from one demicytoplast (n = 8) to recipients resulted in a live calf birth that after 12 days died whereas, with transfer of control blastocysts (n = 14) there was birth of a healthy calf. In conclusion, use of one, instead of two demicytoplasts for HMC, compromises in vitro developmental competence, and alters expression of several important genes affecting embryo development.

Comparative analysis of buffalo (Bubalus bubalis) non-transgenic and transgenic embryos containing human insulin gene, produced by SCNT.

Somatic cell nuclear transfer (SCNT), using transgenic donor cells, is a highly efficient method for producing transgenic embryos. We compared the developmental competence, quality and gene expression of transgenic embryos produced by Hand-made cloning from buffalo fetal fibroblasts (BFFs) containing human insulin gene, with non-transgenic embryos produced from BFFs (Controls). The expression vector (pAcISUBC), constructed by inserting human insulin gene between DNA fragments containing mammary gland-specific buffalo ß-lactoglobulin (buBLG) promoter and terminator buBLG 3'UTR regions into pAcGFP-N1 vector, was used for obtaining the 11 kb insert for transfection of BFFs by nucleofection. Presence of the transgene in embryos was confirmed by examining GFP expression by RT-PCR and immunofluorescence. The blastocyst rate was lower (P < 0.05) for transgenic embryos than for controls (35.7 ±â€¯1.8% vs 48.7 ±â€¯2.4%). The apoptotic index was higher (P < 0.05) for transgenic than for control blastocysts which, in turn, was higher (P < 0.05) than for IVF counterparts (6.9 ±â€¯0.9, 3.8 ±â€¯0.5 and 1.8 ±â€¯0.3, respectively). The total cell number was similar for transgenic and non-transgenic blastocysts (143.2 ±â€¯17.0 and 137.2 ±â€¯7.6, respectively). The expression level of pro-apoptotic genes BAX and BID but not that of CASP3 and CASP9, and cell cycle check point control-related gene P53 was higher (P < 0.05), and that of development- (IGF-1R and G6PD) and pluripotency-related gene NANOG was lower (P < 0.05) in transgenic than in control embryos. The expression level of epigenetic-related genes DNMT1, DNMT3a and HDAC1 and pluripotency-related gene OCT4 was similar in the two groups. The expression level of BAX, BID, CASP9, P53, DNMT1 and DNMT3a was higher (P < 0.05) and that of OCT4, NANOG IGF-1R and G6PD was lower (P < 0.05) in cloned transgenic than in IVF blastocysts whereas, that of CASP3 and HDAC1 was similar between the two groups. In conclusion, these results suggest that transgenic embryos produced by SCNT have lower developmental competence and quality, and altered gene expression compared to non-transgenic embryos.

Calcium ionophore enhanced developmental competence and apoptotic dynamics of goat parthenogenetic embryos produced in vitro.

Parthenogenetically developed embryos are efficient sources of in vitro embryo production, having less ethical issue and being useful for investigating culture conditions/treatments, early developmental, genomic studies, and homonymous source of stem cells. Keeping its advantages in mind, we aimed to study the effects of different activating agents on embryo production and its quality and gene expression. In the present study, 1348 immature oocytes recovered were parthenogenetically developed to embryos. Usable-quality immature oocytes were collected by puncturing the surface follicles and matured in in vitro maturation (IVM) medium for 27 h in a humidified 5% CO2 incubator at 38.5°C. The matured oocytes were parthenogenetically activated by exposure to 5 µM calcium ionophore for 5 min or 7% ethanol for 7 min sequentially followed by 4 h incubation in 2 mM 6-DMAP and then in vitro cultured (IVC) in RVCL/G-2 medium for 8 days. Matured oocytes were activated by calcium ionophore, the cleavage rate observed was 76.67 ± 3.47%, and further they developed into 4-cell, 8-16-cell, morula, blastocyst, and hatched blastocyst with 85.30 ± 1.57%, 70.60 ± 2.00%, 45.05 ± 2.66%, 22.89 ± 2.40%, and 5.70 ± 1.97%, respectively. Whereas ethanol-activated oocytes showed cleavage rate of 87.60 ± 1.70% and further culture developed into 4-cell, 8-16 cell, morula, blastocyst, and hatched blastocyst with 86.14 ± 1.03%, 71.56 ± 2.21%, 40.90 ± 2.45%, 19.02 ± 1.26%, and 2.22 ± 0.38%, respectively. Blastocyst developed from calcium ionophore-activated oocytes showed significantly (P < 0.05) higher total cell number (282.25 ± 27.02 vs 206.00 ± 40.46) and a lower apoptotic index (2.42 ± 0.46 vs 4.07 ± 1.44) than blastocyst developed from ethanol-activated oocytes. The relative expression of anti-apoptotic genes (BCL2, BCL2A1, MCL) at different stages of embryos produced by either calcium ionophore or ethanol activation was found to be increased in earlier stages and decreased in later stages of embryonic development. Similarly, when these embryos were subjected to pro-apoptotic genes (BAX, BAD, BAK), expression was found to be slightly higher in blastocysts than other stages. This study shows that calcium ionophore-activated blastocysts were developmentally more competent than the ethanol-activated blastocysts.

m-carboxycinnamic acid bishydroxamide improves developmental competence, reduces apoptosis and alters epigenetic status and gene expression pattern in cloned buffalo (Bubalus bubalis) embryos.

Incomplete or aberrant reprogramming of nuclear genome is one of the major problems in somatic cell nuclear transfer. In this study, we studied the effect of histone deacetylase inhibitor m-carboxycinnamic acid bishydroxamide (CBHA) on in vitro development of buffalo embryos produced by Hand-made cloning. Cloned embryos were treated with CBHA (0, 5, 10, 20 or 50 µM) for 10 hr from the start of reconstruction till activation. At 10 µM, but not at other concentrations examined, CBHA increased (p < .05) the blastocyst rate (63.77 ± 3.97% vs 48.63 ± 3.55%) and reduced (p < .05) the apoptotic index of the cloned blastocysts (8.91 ± 1.94 vs 4.36 ± 1.08) compared to untreated controls, to levels similar to those in IVF blastocysts (4.78 ± 0.74). CBHA treatment, at all the concentrations examined, increased (p < .05) the global level of H3K9ac in cloned blastocysts than in untreated controls to that observed in IVF blastocysts. Treatment with CBHA (10 µM) decreased (p < .05) the global level of H3K27me3 in cloned blastocysts than in untreated controls but it was still higher (p < .05) than in IVF blastocysts. CBHA (10 µM) treatment increased (p < .05) the relative expression level of pluripotency-related genes OCT-4 and NANOG, and anti-apoptotic gene BCL-XL, and decreased (p < .05) that of pro-apoptotic gene BAX than in untreated controls but did not affect the relative expression level of apoptosis-related genes p53 and CASPASE3 and epigenetics-related genes DNMT1, DNMT3a and HDAC1. These results suggest that treatment of cloned embryos with 10 µM CBHA improves the blastocyst rate, reduces the level of apoptosis and alters the epigenetic status and gene expression pattern.

Tumor characteristics and metabolic quantification in carcinoma breast: An institutional experience.

BACKGROUND: In India, carcinoma breast is the most common cancer among urban women population and second most common cancer after carcinoma cervix in rural areas. One in 22 women in India develops carcinoma of the breast in their lifetime. Fluorine-18-fluoro-2-deoxy-D-glucose (18F-FDG) uptake in breast cancer usually indicates the degree of tumor metabolism and hence can predict its behavior and prognosis. On the other hand, the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2) or neu state of breast cancer is a biomarker that provides important prognostic information in addition to predicting response to therapy. AIMS: The main objective of this study is to assess whether a correlation exists between 18F-FDG uptake in untreated cases of breast cancer, their receptor status (ER, PR, and HER-2 or neu), tumor histology, and tumor size. SUBJECTS AND METHODS: Sixty consecutive female patients, with biopsy-proven primary breast cancer, were enrolled in this prospective study for whom 18F-FDG positron emission tomography-computed tomography scan was done in the Department of Nuclear Medicine. Results obtained were analyzed using appropriate statistical tests (t-test and Pearson Chi-square tests), and interpretation was made with 95% confidence level. RESULTS: In our series, a positive correlation between tumor size, high tumor grade, and standardized uptake value (SUV) was found. Tumors with positive receptor status for estrogen, progesterone, and HER-2/neu receptors had statistically insignificant lower maximum SUV (SUVmax) values than their negative counterparts. Triple-negative breast tumors (ER-, PR-, and no overexpression of HER-2/neu) are currently a subject of major interest because of their aggressiveness, poor prognosis, and lack of targeted therapy. Based on receptor status when the SUVmaxof the group with triple-negative receptor status (ER-/PR-/HER-2/neu-) was compared to rest of the patient group, it was seen that patients with negative receptor status had significantly higher mean SUVmaxvalues. CONCLUSIONS: We have inferred that in patients with breast cancer, various biological parameters such as tumor size, grade, histology, and hormonal receptor status have different impact on tumor metabolic activity.

Analysis of the contaminants released from municipal solid waste landfill site: A case study.

Release and transport of leachate from municipal solid waste landfills pose a potential hazard to both surrounding ecosystems and human populations. In the present study, soil, groundwater, and surface water samples were collected from the periphery of a municipal solid waste landfill (located at Ranital of Jabalpur, Madhya Pradesh, India) for laboratory analysis to understand the release of contaminants. The landfill does not receive any solid wastes for dumping now as the same is under a landfill closure plan. Groundwater and soil samples were collected from the bore holes of 15m deep drilled along the periphery of the landfill and the surface water samples were collected from the existing surface water courses near the landfill. The landfill had neither any bottom liner nor any leachate collection and treatment system. Thus the leachate generated from the landfills finds paths into the groundwater and surrounding surface water courses. Concentrations of various physico-chemical parameters including some toxic metals (in collected groundwater, soil, and surface water samples) and microbiological parameters (in surface water samples) were determined. The analyzed data were integrated into ArcGIS environment and the spatial distribution of the metals and other physic- chemical parameter across the landfill was extrapolated to observe the distribution. The statistical analysis and spatial variations indicated the leaching of metals from the landfill to the groundwater aquifer system. The study will help the readers and the municipal engineers to understand the release of contaminants from landfills for better management of municipal solid wastes.

Production of hand-made cloned buffalo (Bubalus bubalis) embryos from non-viable somatic cells.

Use of non-viable somatic cells for hand-made cloning (HMC) can enable production of cloned animals from tissues obtained from elite or endangered dead animals. Buffalo skin fibroblast cells were rendered non-viable by heat treatment and used for HMC. Although fusion (93.6 ± 1.72 vs 67.1 ± 2.83%) and cleavage (90.3 ± 1.79 vs 65.8 ± 1.56%) rate was lower (P < 0.001) than that for controls, blastocysts could be successfully produced. However, blastocyst rate (34.1 ± 2.43 vs 6.9 ± 2.18%, P < 0.001) and total cell number of blastocysts (TCN, 221.3 ± 25.14 vs 151.1 ± 21.69, P < 0.05) were lower and apoptotic index (4.8 ± 1.06 vs 10.9 ± 1.21) was higher (P < 0.001) than that of controls. In another experiment, ear tissue of slaughterhouse buffaloes was preserved in mustard oil at room temperature for 48 h following which somatic cells were harvested by enzymatic digestion and used for HMC. Although fusion (96.8 ± 1.48 vs 84.2 ± 3.19%), cleavage (89.6 ± 3.59 vs 77.2 ± 3.99%), and blastocyst rate (36.9 ± 7.45 vs 13.1 ± 6.87%) were lower (P < 0.01), TCN (223.0 ± 27.89 vs 213.3 ± 28.21) and apoptotic index (3.97 ± 0.67 vs 5.22 ± 0.51) of blastocysts were similar to those of controls. In conclusion, HMC can be successfully used for production of blastocysts from non-viable cells and from cells obtained from freshly slaughtered buffaloes. This can pave the way for the restoration of farm or wild animals by HMC if somatic cells could be obtained within a few hours after their death.

Use of peripheral blood for production of buffalo (Bubalus bubalis) embryos by handmade cloning.

Buffalo embryos were produced by handmade cloning using peripheral blood-derived lymphocytes as donor cells. Although the blastocyst rate was lower (P < 0.01) for lymphocyte- than control skin fibroblast-derived embryos (6.6 ± 0.84% vs. 31.15 ± 2.97%), the total cell number (152.6 ± 23.06 vs. 160.1 ± 13.25) and apoptotic index (6.54 ± 0.95 vs. 8.45 ± 1.32) were similar. The global level of H3K9ac was higher (P < 0.05) in lymphocyte- than that in skin-derived blastocysts; whereas in IVF blastocysts, the level was not significantly different from the two cloned groups. The level of H3K27me3 was similar among the three groups. The expression level of DNMT1, DNMT3a, HDAC1, and IGF-1R was higher (P < 0.01) in lymphocytes than that in skin fibroblasts. The expression level of CDX2 was higher (P < 0.05) than that of DNMT3a, IGF-1R, OCT4, and NANOG was lower (P < 0.05) in lymphocyte-derived than in IVF blastocysts; that of DNMT1 and HDAC1 was similar in the two groups. The expression level of all these genes, except that of NANOG, was lower (P < 0.05) in lymphocyte- than in skin fibroblast-derived blastocysts. It is concluded that, peripheral blood-derived lymphocytes can be used for producing handmade cloning embryos in bubaline buffaloes.

Caspase-9 inhibitor Z-LEHD-FMK enhances the yield of in vitro produced buffalo (Bubalus bubalis) pre-implantation embryos and alters cellular stress response.

The present investigation was done to study the effect of caspase-9 inhibitor Z-LEHD-FMK, on in vitro produced buffalo embryos. Z-LEHD-FMK is a cell-permeable, competitive and irreversible inhibitor of enzyme caspase-9, which helps in cell survival. Buffalo ovaries were collected from slaughterhouse and the oocytes were subjected to in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC). The culture medium was supplemented with Z-LEHD-FMK at different concentrations i.e. 0 µM (control), 10 µM, 20 µM, 30 µM and 50 µM during IVM and IVC respectively. After day-2 post-insemination, the cleavage rate was significantly higher (74.20 ± 5.87% at P<0.05) in the group treated with 20 µM of Z-LEHD-FMK than at any other concentration. Same trend was observed in the blastocyst production rate which was higher at 20 µM (27.42 ± 2.94% at P<0.05). The blastocysts obtained at day-8 of the culture at different concentrations were subjected to TUNEL assay, to determine the level of apoptosis during the culture medium supplied with 20 µM Z-LEHD-FMK which showed apoptotic index significantly lower (1.88 ± 0.87 at P<0.05). There was a non-significant increase in total cell number in all Z-LEHD-FMK treated blastocysts. The quantitative gene expression of CHOP and HSP10 genes showed significant increase (P<0.05) in the group treated with 50 µM Z-LEHD-FMK, while, HSP40 showed significant increase (P<0.05) at 30 µM and 50 µM Z-LEHD-FMK concentrations. From the afore mentioned results we conclude that, Z-LEHD-FMK at 20 µM increased the cleavage and blastocyst rate of buffalo pre-implantation embryos also affecting the rate of apoptosis and cellular stress at various concentrations.

Heat stress and antioxidant enzyme activity in bubaline (Bubalus bubalis) oocytes during in vitro maturation.

In vitro environments like heat stress usually increase the production of reactive oxygen species in bubaline oocytes which have been implicated as one of the major causes for reduced developmental competence. Oocytes during meiotic maturation are sensitive to oxidative stress, and heat stress accelerates cellular metabolism, resulting in the higher production of free radicals. Therefore, the aim of present work was to assess the impact of heat stress during meiotic maturation on bubaline cumulus-oocyte complexes (COC), denuded oocytes (DO), and cumulus cell mass in terms of their oxidative status. Accordingly, for control group, COC were matured at 38.5 °C for complete 24 h of meiotic maturation and heat stress of 40.5 and 41.5 °C was applied to COC during the first 12 h of maturation and then moved to 38.5 °C for rest of the 12 h. In another group, COC after maturation were denuded from the surrounding cumulus cells by manual pipetting. Results indicated that the production of reactive oxygen species (ROS), lipid peroxides, and nitric oxide (NO) was significantly (P < 0.05) higher in the oocytes subjected to heat stress (40.5 and 41.5 °C) during meiotic maturation compared to the oocytes matured under standard in vitro culture conditions (38.5 °C). Also, the antioxidant enzymatic activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase were significantly (P < 0.05) increased in all the treatment groups compared to the control group. Therefore, the present study clearly establishes that heat stress ensues oxidative stress in bubaline oocytes which triggers the induction of antioxidant enzymatic defense system for scavenging the ROS.

Treatment of buffalo (Bubalus bubalis) donor cells with trichostatin A and 5-aza-2'-deoxycytidine alters their growth characteristics, gene expression and epigenetic status and improves the in vitro developmental competence, quality and epigenetic status of cloned embryos.

We examined the effects of treating buffalo skin fibroblast donor cells with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, and 5-aza-2'-deoxycytidine (5azadC), a DNA methyltransferase (DNMT) inhibitor, on the cells and embryos produced by hand-made cloning. Treatment of donor cells with TSA or 5azadC resulted in altered expression levels of the HDAC1, DNMT1, DNMT3a, P53, CASPASE3 and CASPASE9 genes and global levels of acetylation of lysine at position 9 or 14 in histone 3 (H3K9/14ac), acetylation of lysine at position 5 in histone 4 (H4K5ac), acetylation of lysine at position 18 in histone 3 (H3K18ac) and tri-methylation of lysine at position 27 in histone 3 (H3K27me3). Moreover, global levels of DNA methylation and activity of DNMT1 and HDAC1 were decreased, while global acetylation of H3 and H3K9 was significantly increased in comparison to untreated cells. Simultaneous treatment of donor cells with TSA (50nM) and 5azadC (7.5nM) resulted in higher in vitro development to the blastocyst stage, reduction of the apoptotic index and the global level of H3K27 me3 and altered expression levels of HDAC1, P53, CASPASE3, CASPASE9 and DNMT3a in cloned blastocysts. Transfer of cloned embryos produced with donor cells treated with TSA led to the birth of a calf that survived for 21 days. These results show that treatment of buffalo donor cells with TSA and 5azadC improved developmental competence and quality of cloned embryos and altered their epigenetic status and gene expression, and that these beneficial effects were mediated by a reduction in DNA and histone methylation and an increase in histone acetylation in donor cells.

Developmental competence of different quality bovine oocytes retrieved through ovum pick-up following in vitro maturation and fertilization.

In the present study, oocytes retrieved from cross bred Karan Fries cows by ovum pick-up technique were graded into Group 1 and Group 2, based on the morphological appearance of the individual cumulus-oocyte complexes (COCs). To analyze whether the developmental potential of the COCs bears a relation to morphological appearance, relative expression of a panel of genes associated with; (a) cumulus-oocyte interaction (Cx43, Cx37, GDF9 and BMP15), (b) fertilization (ZP2 and ZP3), (c) embryonic development (HSF1, ZAR1 and bFGF) and (d) apoptosis and survival (BAX, BID and BCL-XL, MCL-1, respectively) was studied at two stages: germinal vesicle (GV) stage and after in vitro maturation. The competence was further corroborated by evaluating the embryonic progression of the presumed zygotes obtained from fertilization of the graded COCs. The gene expression profile and development rate in pooled A and B grade (Group 1) COCs and pooled C and D grade (Group 2) COCs were determined and compared according to the original grades. The results of the study demonstrated that the morphologically characterized Group 2 COCs showed significantly (P<0.05) lower expression for most of the genes related to cumulus-oocyte interplay, fertilization and embryonic development, both at GV stage as well as after maturation. Group 1 COCs also showed greater expression of anti-apoptotic genes (BCL-XL and MCL1) both at GV stage and after maturation, while pro-apoptotic genes (BAX and BID) showed significantly (P<0.05) elevated expression in poor quality COCs at both the stages. The cleavage rate in Group 1 COCs was significantly higher than that of Group 2 (74.46±7.06 v. 31.57±5.32%). The development of the presumed zygotes in Group 2 oocytes proceeded up to 8- to 16-cell stages only, while in Group 1 it progressed up to morulae (35.38±7.11%) and blastocyst stages (9.70±3.15%), indicating their better developmental potential.

Effect of donor cell type on developmental competence, quality, gene expression, and epigenetic status of interspecies cloned embryos produced using cells from wild buffalo and oocytes from domestic buffalo.

This study compared the cloning efficiency of donor cells of fibroblast and epithelial origin isolated from ear skin of a wild buffalo (Bubalus arnee) and used with cytoplasts from domestic buffalo (Bubalus bubalis) in interspecies SCNT by hand-made cloning. The cleavage (93.0 ± 2.8% vs. 85.6 ± 2.4%) and blastocyst rates (50.6 ± 4.0% vs. 20.5 ± 2.6%) were higher (P < 0.05) for fibroblasts than those for epithelial cells, whereas the total cell number (490 ± 42 and 492 ± 95, respectively) and apoptotic index (2.3 ± 0.3 and 2.5 ± 0.6, respectively) of blastocysts were similar. The global level of H3K18ac and H3K27me3 was lower (P < 0.05) in fibroblasts than that in epithelial cells. The global level of H3K18ac was higher (P < 0.05) in fibroblast than that in epithelial cell-derived blastocysts, whereas that of H3K27me3 was similar between the two groups. The expression level of HDAC1, DNMT1, DNMT3a, and P53 was higher (P < 0.05) in fibroblasts than that in epithelial cells; that of CASPASE3 showed an opposite pattern (P < 0.001), whereas CASPASE7 expression level was similar in the two groups. In the embryos, the expression level of HDAC1, DNMT3a, and CDX2 was lower (P < 0.05) in fibroblast than that in epithelial cell-derived blastocysts; that of NANOG showed an opposite pattern (P < 0.05), whereas that of OCT4 was similar between the two groups. In conclusion, donor cells of fibroblast origin are easier to reprogram than those of epithelial origin in interspecies SCNT, and cloning efficiency, epigenetic status, and gene expression pattern vary among cells having different origin although they may be from the same tissue.