Magan Singh

Oxygen Concentration and Cysteamine Supplementation During In vitro Production of Buffalo (Bubalus bubalis) Embryos Affect mRNA Expression of BCL-2, BCL-XL, MCL-1, BAX and BID

This study examined the effects of O(2) concentration (5% vs 20%) during in vitro maturation (IVM), fertilization (IVF) and culture (IVC) or supplementation of IVM and IVC media with cysteamine (50 and 100 μm, respectively; IVM, IVF and IVC carried out in 20% O(2)), on blastocyst rate and relative mRNA abundance of some apoptosis-related genes measured by real-time qPCR in immature and in vitro-matured buffalo oocytes and in embryos at 2-, 4-, 8- to 16-cell, morula and blastocyst stages. The blastocyst rate was significantly higher (p < 0.05) while the percentage of TUNEL-positive cells was significantly lower (p < 0.05) under 5% O(2) than that under 20% O(2). The mRNA expression of anti-apoptotic genes BCL-2 and MCL-1 was significantly higher (p < 0.05) and that of pro-apoptotic genes BAX and BID was lower (p < 0.05) under 5% O(2) than that under 20% O(2) concentration at many embryonic stages. Following cysteamine supplementation, the blastocyst rate and the relative mRNA abundance of BCL-XL and MCL-1 was significantly higher (p < 0.05) and that of BAX but not BID was lower (p < 0.05) at many stages of embryonic development, although it did not affect the percentage of TUNEL positive cells in the blastocysts significantly. The mRNA expression pattern of these genes during embryonic development was different in 5% vs 20% O(2) groups and in cysteamine supplemented vs controls. At the 8- to 16-cell stage, where developmental block occurs in buffalo, the relative mRNA abundance of BCL-2 and MCL-1 was highest under 5% O(2) concentration and that of BAX and BID was highest (p < 0.05) under 20% O(2) concentration. These results suggest that one of the mechanisms through which beneficial effects of low O(2) concentration and cysteamine supplementation are mediated during in vitro embryo production is through an increase in the expression of anti-apoptotic and a decrease in the expression of pro-apoptotic genes.

Derivation of buffalo embryonic stem-like cells from in vitro-produced blastocysts on homologous and heterologous feeder cells

PurposeThe aim of the present study is to compare the ability of homologous and heterologous embryonic fibroblast feeder layers to support isolation and proliferation of buffalo ES-like cells generated from hatched and expanded blastocysts produced by in vitro fertilization and characterization of derived cells through expression of pluripotent markers.MethodsEmbryonic stem cells were derived from hatched and expanded blastocysts through intact blastocyst culture and enzymatic method respectively and compared for proliferation rate on homologous (buffalo) and heterologous feeder layers (goat and sheep).ResultsA total of 69 hatched and 83 expanded blastocysts were used for isolation of inner cell masses which were seeded on buffalo, goat and sheep embryonic feeder layers. Following seeding, attachment rate, primary colony formation rate and survival to maximum number of passages were observed to be higher on homologous feeder layers.ConclusionsUpon comparison of different feeder layer cells for derivation and maintenance of buffalo ES-like cells from hatched and expanded blastocysts, buffalo embryonic fibroblast cells were able to provide a better environment for maintaining pluripotency in culture conditions.

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 nitrogen, phosphorus and zinc fertilization on yield and quality of kharif fodder -A review

Nutrient management is one of the most important factors in crop production. The review of knowledge regarding the use of optimum dose of nutrients especially nitrogen, phosphorus and zinc is of prime concern. The literature regarding effect of nitrogen, phosphorus and zinc on growth, yield and quality of kharif fodder is very scanty, therefore, findings of this paper and views of active investigators, theoreticians and practitioners on growth, yield and quality of kharif fodders viz. teosinite, maize, bajra and sorghum will be useful.

Quantitative expression of pluripotency-related genes in parthenogenetically produced buffalo (Bubalus bubalis) embryos and in putative embryonic stem cells derived from them.

Parthenogenetically produced embryos and embryonic stem (ES) cells derived from them offer a unique model for investigating the role of transcription factors in embryonic genome activation (EGA), pluripotent lineage specification and in pluripotency and self-renewal of ES cells because of the unique nature of these embryos. There is little information on the quantitative expression of important genes in parthenogenetically produced embryos and in ES cells derived from them. The present study examined the quantitative expression of some important genes in parthenogenetically produced buffalo embryos and in putative parthenogenetic ES cells (pES) cells. The quantitative expression of OCT-4, SOX-2, NANOG, REX-1, FOXD-3 and NUCLEOSTEMIN, which is very low in immature and mature oocytes, and in embryos at 2-, 4- and 8- to 16-cell stage, increases significantly at morula and blastocyst stage. The expression level of TELOMERASE, c-MYC and STAT-3, which is high in immature oocytes decreases during embryonic development followed by either an increase at the morula stage (TELOMERASE) or a low expression level maintained throughout development till blastocyst stage (c-MYC and STAT-3). There is a progressive decline in the expression level of OCT-4, SOX-2, c-MYC, REX-1, NUCLEOSTEMIN, TELOMERASE and STAT-3 during long term culture of pES cells.

Buffalo (Bubalus bubalis) ES Cell–Like Cells are Capable of In Vitro Skeletal Myogenic Differentiation

When buffalo embryonic stem (ES) cell-like cells that expressed surface markers SSEA-4, TRA-1-60, TRA-1-81, CD9 and CD90 and intracellular markers OCT4, SOX2 and FOXD3, as shown by immunofluorescence, and that expressed REX-1 and NUCLEOSTEMIN as confirmed by RT-PCR, were subjected to suspension culture in hanging drops in absence of LIF and buffalo foetal fibroblast feeder layer support, they differentiated to form three-dimensional embryoid bodies (EBs). Of 231 EBs examined on Day 3 of suspension culture, 141 (61.3 ± 3.09%) were of compact type, whereas 90 (38.4 ± 3.12%) were of cystic type. The cells obtained from EBs were found to express NF-68 and NESTIN (ectodermal lineage), BMP-4 and α-skeletal actin (mesodermal lineage), and α-fetoprotein, GATA-4 and HNF-4 (endodermal lineage). When these EBs were cultured on gelatin-coated dishes, they spontaneously differentiated to several cell types such as epithelial- and neuron-like cells. When EBs were cultured in the presence of 1 or 2% DMSO or 10(-8) M or 10(-7) M retinoic acid for 25 days, ES cells could be directed to form muscle cell-like cells, the identity of which was confirmed by expression of α-actinin by immunofluorescence and of MYF-5, MYOD and MYOGENIN genes by RT-PCR. MYOD was first detected on Day 10 in both treatment groups and on Day 15 in controls, whereas MYOGENIN was first detected on Day 10, Day 15 and Day 25 in the presence of retinoic acid, in the presence of DMSO and in controls, respectively. The present study demonstrates the ability of buffalo ES cell-like cells to undergo directed differentiation to cells of skeletal myogenic lineage.

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.

Yield and Chemical Composition of Fodder Guar (Cyamopsis tetragonoloba L.) as Affected by Harvesting Stage and Zinc Application

An experiment was conducted to study the performance of guar (Cyamopsis tetragonoloba L.) fodder as affected by stage of harvesting and zinc application with four stages of harvesting (50, 60, 70 and 80 DAS) and four levels of ZnSO4 application (Control, 10 kg ZnSO4/ha20 kg ZnSO4/haand 0.5% ZnSO4 spray 30 DAS) with three replications in Split Plot Design. The green fodder yield and DM yield increased at harvest upto 80 DAS and zinc application @ 20 kg/ha while OM content was significantly higher at 50 DAS. The maximum CP level in the fodder was obtained when the crop was harvested at 60 DAS with application of ZnSO4 @ 20 kg/ha. However, the contents of DM, EE, ash contents were higher due to harvesting of crop 80 DAS and application of ZnSO4 @ 20 kg/ha over other treatments. NDF and ADF were recorded significantly higher at 80 DAS while hemicellulose at 70 DAS without any application of Zn (control). However, harvesting at 80 DAS and 20 kg/ha ZnSO4 application was a better combination for yield and quality of guar fodder.

Yield and Quality of Multicut Fodder Sorghum as Affected by Nutrient Levels and Biofertilizer Application

A field experiment was carried out to study the effect of nutrient level and biofertilizer on yield and quality of multicut fodder sorghum (Sorghum bicolor L.). The experiment was laid out in split plot design with three multicut fodder sorghum varieties (CSH 20MF, CSH 24MF and CO(FS) 29) in the main plot and five levels of nutrients (Control, N90P40, N90P40 + BF, N60P40 + BF and N30P20 + biofertilizer (BF)) as subplot treatments with three replications. The higher green fodder yield was obtained with CO(FS)29 variety at N90P40 + BF nutrient level. Among the varieties, maximum DM contents (25.34%) and yield (212.76 q/ha) were obtained for variety CO(FS)29 while in respect to nutrient level, the highest DM content (24.74%) and yield (158.44 q/ha) were recorded with N90P40 + BF level of fertilization. The CP and EE level of three cuts was at par with respect to varieties while the maximum yield was found for variety CO(FS)29. The highest CP and EE content (8.37 and 2.37%, respectively) was obtained at N90P40 + BF. Total ash content was higher by 16.93 and 20.38, 62.68% and 56.15% in variety CO(FS)29 over CSH 24MF and CSH 20MF, respectively. Among nutrient levels, higher total ash (10.76%) was estimated at N90P40 + BF level. The total carbohydrate and cell soluble content was recorded highest in control and N90P40 + BF, respectively, While higher NDF and ADF content was estimated in CSH 20MF and CO(FS)29 variety, respectively over control treatment. CO (FS)29 was the most promising because of higher green fodder, dry matter and nutrient yield than CSH 20MF and CSH 24MF.

Productive performance, body condition score and carcass characteristics of Deccani lambs reared under different farming systems

The present study was carried out to assess the effect of different farming systems on productive performance, body condition score and carcass characteristics of Deccani lambs. Thirty-six weaned lambs having 3 months of age were divided into three groups with twelve lambs in each group in under intensive (T1), semi-intensive (T2) and extensive (T3) farming systems. The comparative mean dry matter intake (kg/day) and (kg/100kg BW) was significantly (p<0.05) higher in T1 group when compared to other groups. T2 group had significantly (p<0.05) higher DM intake (kg/day) than T3 group. The mean values of Average daily gains in Deccani lambs were significantly (p<0.05) higher in T1 group compared to T2 and T3 groups. The feed efficiency was significantly (p < 0.05) higher in T1 compared to T2, T3 groups. Statistically significant difference was found in BCS at six months of age (p<0.05) between T1 and T3 groups. BCS at 9, 12 months and overall BCS, there was a significant (p<0.05) difference between T1 and T3 groups. T1 group had higher pre-slaughter weight, hot carcass weight, meat %, fat% and meat: bone ratio thanT2 and T3. The dressing yields were comparable. The weights of other offals (heart, kidney, spleen, testicles, blood, head and lung and trachea) were comparable. The findings of this study indicated that the intensive and semi-intensive system of rearing could be useful for mutton production. In extensive system, which is predominant in India should change its facet by enriching the grazing land resources as well by the supplementation of required plane of nutrition during growth and special life stages viz., gestation, puberty, post lambing and finisher stages for achieving optimum productivity.