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Abstract 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 (Capra hircus) 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. Cells were then 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

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 aim of this study was to investigate the effect of increasing volume of bone marrow (BM) aspirate of goats on yield of mesenchymal stem cells (MSCs). For this, yield of MSCs in seven groups (Gr; 1-7 Grs, including a control Gr) were compared. After isolation and enrichment of BM MSCs with density gradient centrifugation using increasing volume of Histopaque-1077, the properties of MSCs to adhere the plastic surfaces and expression of alkaline phosphatase (ALP) were used for the identification of MSCs. The results indicated that 18 ml of BM aspirate (Gr 5) provided maximum number of MSCs using 3 ml of histopaque compared to the control and other Gr. The yield in Gr 5 was about 4.4 folds higher compared to the control Gr. In conclusion, these results demonstrated that in a single procedure the modified protocol yield significantly higher number of caprine MSCs from goat BM aspirate compared to the standard protocol. Thus, it can be used for isolation of more number of MSCs from a large quantity of bone marrow with least volume of consumables required for density-gradient centrifugation.

Parthenogenetic activation of oocytes has gained new interest in recent years as an alternative approach to create embryos with no reproductive purpose for research in areas such as assisted reproduction technologies itself and for derivation of clinical grade pluripotent embryonic stem cells for regenerative medicine. In this study, we described the production of goat parthenogenetic ES like cells from different stages of parthenogenetic embryos. We compared the source material on the pESCs outgrowth and culture. 8-16 cell stage embryos, morula, blastocysts and ICM (inner cell mass) from hatched blastocysts produced from chemical activation were cultured on goat fetal fibroblast monolayer in stem cell culture media. Colonies were passaged when signs of differentiation were visible. ESC like colonies were cultured on feeder cells in the presence of hLIF however, some ESC like colonies were also cultured in absence of hLIF for random differentiation. In our study, ESC like colony formation with ICM of hatched blastocyst was comparatively higher as compared to blastocysts while ESC like colony formation with blastocysts and ICM of hatched blastocyst was significantly higher as compared 8-16 cells and morula. It is observed that inner cell mass source is an important criterion for the ES like cells derivation in goats. Also, in comparison to the expanded blastocysts (80.10%), hatched blastocysts showed higher (86.06%) attachment rate and primary colony formation rate. ESC like colonies in absence of hLIF differentiated in vitro into epithelial like and neuronal like cells. Undifferentiated ESC like colonies stained positive for alkaline phosphatase, SSEA-3 and OCT-4.

Enrichment of cell suspension with germ cells prior to injection into recipient seminiferous tubules is of importance in spermatogonial stem cells (SSCs) transplantation. Fetal bovine serum (FBS) is the most widely used growth supplement for cell cultures, primarily because of its high levels of growth stimulatory factors and low levels of growth inhibitory factors. This study was undertaken to investigate the effect of serum concentration on colony formation and development of different types of SSC colonies with respect to passage number. Cells were isolated from pre-pubertal buck testes by two step enzymatic digestion method. The filtered cells were enriched by differential adherence selection method. Cells were then randomly divided into 8 groups, depending on concentration of FBS in culture medium ranging from 0% to 35%. In experiment 1, effect of different concentrations of FBS on total number pSSCs with reference to differential plating was observed while in experiment 2, effect of different concentrations of FBS on types of pSSC colonies with respect to passage number was observed. No colony formation was observed in control group (0% FBS) while significantly higher number of single, paired, cluster and rosette colonies observed were with 20% FBS group in differential 2 (D2) as compared to other groups. Alkaline phosphatase staining and immunocytochemistry staining (PGP9.5 and OCT4) were positive in SSCs colonies. The growth rate of the culture was significantly and consistently higher with 20% FBS.

The aim of the present study was to evaluate the development of caprine chimeric embryos in different culture media, viz. RVCL and mCR2aa + 10% FBS. Inner cell mass (ICM) from hatched blastocysts of parthenogenetic activated embryos were used to produce ES cell-like cells (103) while 4-cell embryos obtained from IVF were utilized to produce tetraploid embryos (231). The aggregates prepared were randomly divided into 2 groups, viz. Gr 1 (RVCL) (53) and Gr 2 (mCR2aa +10% FBS) (50) followed by culture in humidified atmosphere of 5% CO2 at 38.5°C in a CO2 incubator. Outcome measures were aggregation, 8–16 cell, morula and blastocyst formation. The percentage of aggregation, 8–16 cell, morula and blastocyst in Gr 1 (RVCL) was 83.33±6.00%, 42.24±7.71%,29.59±7.64% and 11.49±5.36%, respectively while the percentage of aggregation, 8–16 cell, morula and blastocyst in Gr 2 (mCR2aa +10% FBS) was 91.66±4.32%, 54.76±7.75%, 22.32±5.75% and 14.58±5.18%, respectively. In conclusion, both the media supported the development of chimeric embryos up to blastocyst.