Cell culture media dependent in vitro dynamics and culture characteristics of adult caprine dermal fibroblast cells.

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 < 0.05) lower in FGM compared with other media tested. Overall, our results illustrate the media-dependent deviation in in-vitro dynamics and culture characteristics of cadFibroblasts that may be useful to develop strategies to cultivate these cells efficiently for research and downstream applications.

Different Effects of Sugars and Methods to Preserve Post-Thaw Functional Properties of Cryopreserved Caprine Spermatogonial Stem Cells.

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 spermatogonial stem cells (cSSCs) and the cells obtained from cryopreserved testis tissue of prepubertal Barbari bucks. For this, in experiment 1, cSSCs were isolated and cryopreserved by either SF or FF method with different non-permeable (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 dimethyl sulfoxide) cryoprotectants. After 1 week 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 prepubertal Barbari bucks using the SF or FF method was evaluated. After 1 week of cryopreservation, the tissues were thawed and cSSCs were isolated and cultured for 3 weeks. 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-fold higher in the 140T group compared with EG, 140S, and 400S groups, respectively. Similarly, the expression of stemness markers (protein gene product 9.5 and octamer-binding transcription factor-4) was relatively higher in 140T group compared with the other groups. In experiment 2, the recovery rate of cells per unit tissue weight was significantly (p < 0.05) higher when cryopreserved using 140 mm trehalose compared with other groups. The results of immunocytochemical analyses imply the expression of pluripotent stem cell markers in cSSCs following cryopreservation. Overall, the outcome of the study demonstrates different effects of sugars and methods on post-thaw functional properties of cSSCs with superiority of 140 mm trehalose using SF method over other treatment groups. These results are important for ex vivo expansion and differentiation of cSSCs for fertility preservation and their other downstream applications.

Reproductive stage- and season-dependent culture characteristics of enriched caprine male germline stem cells.

The present study aims to evaluate season- and reproductive-stage dependent variation in culture characteristics and expression of pluripotency and adhesion markers in caprine-male germline stem cells (cmGSCs). For this, testes from pre-pubertal (4-6 months) and adult (~ 2 years) bucks during non-breeding (July-August; n = 4 each) and breeding (October-November; n = 4 each) seasons were used to isolated testicular cells by two-step enzymatic digestion. After cmGSCs enrichment by multiple methods (differential platting, Percoll density gradient centrifugation, and MACS), cell viability of CD90+ cells was assessed before co-cultured onto the Sertoli cell feeder layer up to 3rd-passage (P-3). The culture characteristics of cmGSCs were compared during primary culture (P-0) and P-3 with different assays [BrdU-assay (proliferation), MTT-assay (senescence), and Cluster-forming activity-assay] and transcript expression analyses by qRT-PCR. Moreover, the co-localization of UCHL-1, CD90, and DBA was examined by a double-immunofluorescence method. In adult bucks, significantly (p < 0.05) higher cell numbers with the ability to proliferate faster and form a greater number of cell clusters, besides up-regulation of pluripotency and adhesion markers expression were observed during the breeding season than the non-breeding season. In contrast, such season-dependent variation was lacking in pre-pubertal bucks. The expression of transcripts during non-breeding seasons was significantly (p < 0.05) higher in pre-pubertal cmGSCs than in adult cells (UCHL-1 = 2.38-folds; CD-90 = 6.66-folds; PLZF = 20.87-folds; ID-4 = 4.75-folds; E-cadherin = 3.89-folds and ß1-integrin = 5.70-folds). Overall, the reproductive stage and season affect the population, culture characteristics, and expression of pluripotency and adhesion specific markers in buck testis. These results provide an insight to develop an efficient system for successful cell culture processes targeting cmGSCs. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00515-x.

Low oxygen tension potentiates proliferation and stemness but not multilineage differentiation of caprine male germline stem cells.

The milieu of male germline stem cells (mGSCs) is characterized as a low-oxygen (O2) environment, whereas, their in-vitro expansion is typically performed under normoxia (20-21% O2). The comparative information about the effects of low and normal O2 levels on the growth and differentiation of caprine mGSCs (cmGSCs) is lacking. Thus, we aimed to investigate the functional and multilineage differentiation characteristics of enriched cmGSCs, when grown under hypoxia and normoxia. After enrichment of cmGSCs through multiple methods (differential platting and Percoll-density gradient centrifugation), the growth characteristics of cells [population-doubling time (PDT), viability, proliferation, and senescence], and expression of key-markers of adhesion (ß-integrin and E-Cadherin) and stemness (OCT-4, THY-1 and UCHL-1) were evaluated under hypoxia (5% O2) and normoxia (21% O2). Furthermore, the extent of multilineage differentiation (neurogenic, adipogenic, and chondrogenic differentiation) under different culture conditions was assessed. The survival, viability, and proliferation were significantly (p < 0.05) improved, thus, yielding a significantly (p < 0.05) higher number of viable cells with larger colonies under hypoxia. Furthermore, the expression of stemness and adhesion markers were distinctly upregulated under lowered O2 conditions. Conversely, the differentiated regions and expression of differentiation-specific genes [C/EBPα (adipogenic), nestin and ß-tubulin (neurogenic), and COL2A1 (chondrogenic)] were significantly (p < 0.05) reduced under hypoxia. Overall, the results demonstrate that culturing cmGSCs under hypoxia augments the growth characteristics and stemness but not the multilineage differentiation of cmGSCs, as compared with normoxia. These data are important to develop robust methodologies for ex-vivo expansion and lineage-committed differentiation of cmGSCs for clinical applications.

Assessment of pregnancy-associated glycoprotein profile in milk for early pregnancy diagnosis in goats.

OBJECTIVE: This study was conducted to assess the level of pregnancy-associated glycoprotein (PAG) in whole and skim milk samples, and its suitability for early pregnancy diagnosis in goats. METHODS: A two-step sandwich enzyme-linked immunosorbent assay (ELISA) system for estimation of milk PAG was developed and validated, which employed caprine-PAG specific polyclonal antisera. Whole and skim milk samples (n = 210 each) from fifteen multiparous goats were collected on alternate days from d 10 to d 30, and thereafter weekly till d 51 postmating. PAG levels in milk samples were estimated by ELISA and the pregnancies were confirmed at d40 post-mating by transrectal ultrasonography (TRUS). RESULTS: The level of PAG in whole and skim milk samples of both pregnant and nonpregnant goats remained below the threshold values until d 24 after mating. Thereafter, PAG concentration in whole and skim milk increased steadily in pregnant goats, whereas it continued below the threshold in non-pregnant does. The PAG profiles in whole and skim milk of pregnant goats were almost similar and exhibited strong positive relationship (r = 0.891; p<0.001). Day 26 post-mating was identified as the first time-point for significantly (p<0.05) higher milk PAG concentration in pregnant goats than to non-pregnant goats. When compared to TRUS examination for pregnancy diagnosis, the accuracy and specificity of PAG ELISA using whole and skim milk samples were 94.5% and 95.4%; and 95.3% and 100%, respectively. The high values of area-under-curve (0.904 [whole milk] and 0.922 [skim milk]), demonstrate outstanding discrimination ability of the milk assays. Among the sampling dates chosen, d 37 post-mating was identified as the best suitable time point for collection of milk samples to detect pregnancy in goats. CONCLUSION: The PAG concentration in whole and skim milk of goats collected between days 26 and 51 post-breeding can be used for the accurate prediction of pregnancy and may be useful for assisting management decisions in goat flocks.

Growth and proliferation of caprine bone marrow mesenchymal stem cells on different culture media.

The growth and proliferation of mesenchymal stem cells are very sensitive in in vitro and a number of factors like media play a significant role in that context. In this study we assessed effect of different media on growth and proliferation of bone marrow derived mesenchymal stem cells (BMMSCs). The BMMSCs were isolated from caprine bone marrow and were subjected to magnetic activated cell sorting against CD90+, CD105+, CD271+and CD34- along with FC blocker. After characterisation, 2 × 104 cells were seeded in 12 well culture plates in four different media viz. MesenCult, MesenPRO, StemPro and complete DMEM (15 % FBS) to study their growth kinetic for 6 days from passage 0 (P0) to passage 3 (P3). The population doubling time (PDT) was derived from growth curve using logarithmic formula. The results showed that the BMMSCs growth and proliferation was highest in MesenCult media in P0 which varied significantly (p < 0.05) from rest of media and from P1 to P3, it was MesenPRO which yielded maximum cells (p < 0.05). The PDT was also in line with growth curve findings. In conclusion, the MesenPRO media had higher growth and proliferation rate from P1 to P3 although MesenCult had higher cell numbers in P0. In conclusion, the use of MesenPRO media could be a better option than conventional media when mesenchymal stem cells are used in clinical applications and other therapeutic purposes taking consideration to its higher growth and proliferation rate. And MesenCult would be a great option to harvest MSCs from P0.

Temporal changes in plasma profile of pregnancy-associated glycoprotein, progesterone and estrone sulfate associated with fetal number during early- and mid-pregnancy in goats.

This study was designed to investigate plasma profile of pregnancy-associated glycoprotein (PAG), progesterone (P4) and estrone sulfate (E1S) during early- and mid-pregnancy. The goal was to explore the relationships with values for reproductive variables, to detect the most reliable predictor variable, and to identify the most desirable time point for blood collection for determining fetal number in goats. After ultrasonographic examination at d35-40 post-mating, blood sampling of 15 pregnant goats (total 18) was continued until d114. The PAG profile was characterized by gradual increase during early pregnancy from d26 to d51 and thereafter concentrations were relatively constant until d114 of gestation. The effect of fetal number on plasma PAG, P4 and E1S was first evident on d28, d51 and d26, respectively. During mid-pregnancy, does with twins had a greater (P < 0.05) PAG (S-N = 2.54 ± 0.12 compared with 1.59 ± 0.11), P4 (18.91 ± 0.67 compared with 14.51 ± 0.47 ng/mL) and E1S (16.34 ± 0.76 compared with 11.32 ± 0.44 ng/dL) as compared with does with a singleton fetus. Plasma PAG but not P4 and E1S was positively correlated with fetal number and birth weight of kids during early pregnancy. Multivariate linear regression and discriminant function analyzes allowed for identification of plasma PAG as the most reliable predictor for fetal number and birth weight of kids. Furthermore, d58 was the most suitable single time point for prediction of fetal number using PAG as a biomarker. In conclusion, plasma profile of PAG, P4 and E1S was affected by fetal count. Plasma PAG was identified as the most reliable predictor variable of fetal number and birth weight of kids as compared to plasma P4 and E1S.