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The enhanced availability of functional fibroblasts from precious tissue samples requires an ideal cell-culture system. Therefore, this study was designed to investigate the performance of caprine adult fibroblast cells (cadFibroblast) when cultivated in different culture media. The cadFibroblast cell lines from adult Barbari bucks were established and the effect of different media viz. DMEM/F-12 [with low glucose (5.5 mM; DL) and high glucose (30 mM; DH)], α-MEM [with low glucose (5.5 mM; ML) and with high glucose (30 mM; MH)], and fibroblast growth medium (FGM) were evaluated and compared for growth characteristics and in-vitro dynamics through cellular morphology, proliferation, population doubling time, double-immunocytochemistry, colony forming units, wound healing, transwell migration and differential expression of fibroblast-specific markers [FSP-1 and vimentin]. The results of immunocytochemistry, transwell migration/invasion, and wound healing assays showed the superiority of DH over DL and other media tested. Whereas, similar effects of glucose supplementation and expression of FSP-1 were not observed in α-MEM. Transwell migration was significantly (p

The present study aimed to identify the effects of sugar and methods [slow freezing (SF) vs fast freezing (FF)] on post-thaw in-vitro functional characteristics of cryopreserved caprine SSCs (cSSCs) and the cells obtained from cryopreserved testis tissue of pre-pubertal Barbari bucks. For this, in experiment-1, cSSCs were isolated and cryopreserved by either SF or FF method with different nonpermeable [sugars; trehalose (140 mM; 140T or 400 mM; 400T), and sucrose (140 mM; 140S or 400 mM; 400S)] or/and permeable [5% ethylene glycol (EG) and DMSO] cryoprotectants. After one-wk of cryopreservation, the cSSCs were thawed and cultured for evaluation of their characteristics. Further, in experiment-2, the effectiveness of sugars [trehalose (140 mM) or sucrose (140 mM)] for cryopreservation of testicular tissues of pre-pubertal Barbari bucks using the SF or FF method was evaluated. After one-wk of cryopreservation, the tissues were thawed and cSSCs were isolated and cultured for 3-wk. In both experiments, cSSCs were evaluated for recovery rate, proliferation, metabolic viability, senescence, and stemness markers expression. The recovery rate was 1.3, 1.3, and 1.1-folds higher in the 140T group compared with EG, 140S, and 400S groups, respectively. Similarly, the expression of stemness markers (PGP9.5 and OCT-4) was relatively higher in 140T group compared with the other groups. In experiment 2, the recovery rate of cells per unit tissue wt. was significantly (P

This study was conducted to evaluate the effect of 5% and 21% oxygen concentrations on chimeric embryo development. Inner cell mass (ICM) from parthenogenetic activated blastocyst were used to produce ES cell-like cells. A pair of zona-free tetraploid embryos and one clump of pESCs at passage 2 and 3 were aggregated on granulosa monolayer in such a manner that the clump of pESCs was sandwiched between the tetraploid embryos. The aggregates prepared were randomly divided into two groups viz. Group 1 (21% O2) (n=54) and Group 2 (5% O2) (n=50) followed by culture in humidified atmosphere of 5% CO2 at 38.5°C in a CO2 incubator. Aggregation rate and embryo quality was similar in both the groups. The percentage of aggregation, 8-16 cell, morula and blastocyst Group 1 (21% O2) was 91.66±4.04%, 49.07±5.08%, 25.92±6.05% and 16.66±4.95%, respectively while the percentage of aggregation, 8-16 cell, morula and blastocyst Group 2 (5% O2) was 96.59±2.83%, 51.89±9.14%, 16.28±5.52% and 28.40±8.10%, respectively. The aggregates and 8-16 cell stage embryos formed in Group 2 (5% O2) were comparatively higher than in Group 1 (21% O2) while morula formation in Group 2 (5% O2) was comparatively lower than in Group 1 (21% O2). Also, Group 2 (5% O2) had significantly more blastocysts as compared to Group 1 (21% O2). To conclude, culture under 5% O2 leads to improved aggregation and also enhances the production of blastocysts rather than with atmospheric oxygen concentrations (21%) during embryo incubation.

The present study aimed to investigate whether follicular fluid, gonadotropins (FSH, LH and estradiol-17β), and combination of both types of supplementation (follicular fluid + gonadotropins) in maturation medium influence the transcript abundance of germ cell marker genes ( MATER , ZAR1 , GDF9 and BMP15 ) in goat oocytes. Grade I and II oocytes were collected from the goat ovaries and matured further under in-vitro conditions using four different maturation regimens viz , group B containing basal media (TCM 199 + BSA + serum), group C (basal media + 10% follicular fluid), group D (basal media + FSH + LH + estradiol-17β) and group E (basal media + 10% follicular fluid + FSH + LH + estradiol-17β). Group A consisted of immature oocytes. Expression profile of MATER, ZAR1, GDF9, and BMP15 was analyzed in matured oocytes of different groups as well as immature oocytes using real-time PCR. The relative abundance of MATER gene in immature oocytes (group A) was found to be significantly higher (p ZAR1 was found to be significant different (P GDF9 and BMP15 was significantly higher (P MATER and ZAR1 transcript were down regulated after maturation; however BMP15 and GDF9 transcripts were upregulated after maturation. For better in vitro embryo production (IVEP) out comes goat oocytes may be matured in vitro in TCM-199 supplemented with follicular fluid and gonadotropins.

A study was aimed to optimize the electrofusion parameters for generation of caprine tetraploid in vitro fertilized embryos. Freshly recovered 2-cell goat in vitro fertilized embryos were submitted to electrofusion with varying voltages (1.0, 1.2 and 1.4 kV/cm) and time duration (2, 4 and 6 μ sec) combinations to produce tetraploid goat embryos. Fused embryos were cultured in RVCL (Research vitro cleave) media further for 8 to 12 days for subsequent embryo development. Fusion, cleavage and blastocyst rate at voltage 1.2kV/cm for 4 μ sec duration was significantly higher (P

The objective of this study was to compare the effectiveness of different activation treatments of in vitro matured oocytes and their developmental potency in potassium simplex optimization medium. Ovaries were collected from the local abattoir and transported within 4 h to the laboratory in warm saline (37 °C) containing 100 IU penicillin-G and 100 μg streptomycin sulphate per ml. A total of 1004 cumulus oocyte complexes (COC's) were collected from 454 ovaries. Oocytes were matured in TCM-199 medium containing FSH (5 μg/ml), LH (10 μg/ml), oestradiol-17β (1 μg/ml) supplemented with 10% fetal bovine serum and 3 mg/ml BSA at 38.5 °C and 5% CO2 in an incubator under humidified air for 27 h. After 27 h of IVM, oocytes were denuded, washed and selected 933 in vitro matured oocytes were randomly divided into four groups. Group 1 in vitro matured oocytes (n = 579), were exposed to 7% ethanol for 5 min followed by treatment with 2.0 mM DMAP for 4 h in KSOM. Group 2 in vitro matured oocytes (n = 145) were exposed to 7% ethanol for 5 min followed by treatment with 10 μg/ml CHX for 4 h in KSOM. Group 3 in vitro matured oocytes (n = 100) were exposed to 7% ethanol for 5 min followed by treatment with 2.0 mM DMAP and 10 μg/ml CHX for 4 h in KSOM. Group 4 in vitro matured oocytes (n = 109) were cultured for 4 h without any chemical activation treatment in KSOM medium (control). After 4 h of culture in different chemicals, the oocytes were washed five to ten times in the culture medium (KSOM) and cultured in 50 μl drops of KSOM. Development of activated oocytes was observed at every 48 h till day 10 post activation under an inverted phase contrast microscope (200x, Nikon, Japan). The cleavage rate in groups 1, 2, 3 and 4 were 42.83%, 58.62%, 74.0% and 0.00%, respectively and morula production in groups 1, 2 and 3 were 24.59%, 30.58% and 31.08%, respectively. These results indicated that the activation of in vitro matured oocytes by 7% ethanol for 5 min followed by treatment with 2.0 mM DMAP and 10 μg/ml CHX for 4 h in KSOM is most favorable for parthenogenetic caprine embryos production.

The aim of the present investigation was to study the effect of calcium ionophore activation on blastocyst production following intracytoplasmic sperm injection (ICSI) in in vitro-matured Caprine oocytes. A total of 470 in vitro-matured oocytes were selected and randomly divided in to three groups. Cumulus oocyte complexes (COCs) recovered by slicing the Caprine ovaries were matured in TCM199 supplemented with 10% foetal bovine serum (FBS) + 10% follicular fluid + FSH (5 μg/ml) + LH (10 μg/ml) + estradiol (1 μg/ml) + EGF (10 ng/ml) + BSA (3 mg/ml) for 27 h in humidified atmosphere at 38.5°C with 5% CO2 in CO2 incubator. After 27 h of culture, selected COCs (n = 470) were separated from cumulus cells by treating with 0.1% hyaluronidase enzyme and passing repeatedly through a fine pipette and randomly divided into three groups. In group 1, (n = 168) matured oocytes were injected with injection micropipette without sperm as control. In group 2, (n = 152) capacitated spermatozoa were injected into cytoplasm of in vitro-matured oocytes through injection micropipette. In group 3, (n = 150) capacitated spermatozoa were injected into cytoplasm of in vitro-matured oocytes through injection micropipette and then activated with 5 μm Ca ionophore for 5 min. The oocytes of all groups were then culture in RVCL media for embryo development. The cleavage rate was observed after 48-72 h of injection. The cleavage rate and blastocyst production in group 1, 2 and 3 were 0.00 and 0.00, 18.42 and 3.57 and 61.33% and 16.30%, respectively. The result indicated that mechanical activation failed to induce cleavage in in vitro-matured Caprine oocytes, whereas chemical activation of intracytoplasmic sperm-injected in vitro-matured Caprine oocytes showed significantly higher cleavage rate and blastocyst production as compare to non-activated oocytes.