<div style="text-align:justify">Fetal Bovine Serum (FBS) is an expensive source of macronutrients which are required for proper nutrition of Leishmania parasite in the culture medium. An alternative, cost effective source of macronutrients which can replace the use of FBS in tissue culture medium is required. The potential of Soy Protein Isolate (SPI) to replace FBS in RPMI-1640 medium for the in vitro cultivation of Leishmania donovani was evaluated. Commercially available SPI powder was used in RPMI-1640 medium as a substitute of FBS to cultivate L. donovani promastigotes. The growth, multiplication and morphology of cultivated parasites was observed in conventional RPMI-1640 with 10% FBS (v/v) and RPMI-1640 containing 10% SPI (v/v) by using light microscopy, measurement of absorbance and cell counting. The growth of Leishmania promastigotes in the medium containing 10% SPI was slower in initial phase; however, the parasites were morphologically larger as compared to those in RPMI-1640 medium containing 10% FBS. Cell count in the SPI-containing RPMI-1640 medium was 2.3 &times; 108 cells/ml whereas it was 1.9 &times; 107 cells/ml in RPMI-1640 with 10% FBS. This study concludes that RPMI-1640 may be supplemented with SPI instead of FBS for the in vitro cultivation of Leishmania donovani promastigotes to decrease the culture maintenance cost in developing countries.&nbsp;</div>

Soy protein isolate: A substitute of fetal bovine serum for the in vitro cultivation of Leishmania donovani

<div>Background: Intestine of pigs (chitterling) is one of the important edible offal with substantial yield and accounts for 1.8% of the slaughter weight. At present its utilization as direct consumption is limited only to some economically weaker section of population. Therefore the present study was undertaken to develop a shelf stable chitterling pickle recipe incorporated with fermented bamboo shoot (FBS) and lemon juice as natural acidulant.&nbsp; 
 
Methods: Freshly collected intestines of pig were washed, pressure cooked, cut into 3 to 4 cm3 and deep fried. Spices and condiments were fried separately then mixed with the fried chitterlings along with FBS and lemon juice to prepare the shelf stable chitterling pickle. The pickles were studied for various parameters such as physico-chemical, microbial and sensory attributes.&nbsp; 
 
Result: Our study showed significantly (p&lt;0.05) higher pH, product yield and moisture content in the chitterling pickle added with FBS and lemon juice. Protein and fat content did not differ significantly. No adverse effects were noted on hedonic score for appearance, flavor, juiciness, texture, saltiness, sourness and overall acceptability up to 12 days of storage at ambient temperature. The overall acceptability of chitterling pickle with FBS and lemon juice was significantly (p&lt;0.05) higher as compared to control. Therefore, it could be concluded that highly acceptable shelf stable pickle can be prepared from intestine of pig with substantial value addition to the by-products.</div>

Development of Shelf Stable Chitterlings Pickle Incorporated with Natural Acidulant

<div>Background: Sperm cells are susceptible to oxidative stress during cryopreservation. Therefore, an antioxidant is necessary to protect them from damages. Fetal bovine serum (FBS) is one of potent antioxidants for fish sperm cryopreservation. Hence, the aims of this study are to examine the effect of FBS on sperm quality after a short period and to determine its optimum concentration on depik (Rasbora tawarensis). 
 
Methods: Depik fish were obtained from the Fish hatchery of Lukup Badak, Aceh Tengah District, Indonesia. Sperms collected from the fish were diluted in Ringer extenders containing FBS concentration of 10% (P1), 20% (P2), 30% (P3), 40% (P4), 50% (P5) and 60% (P6), filled into 2 ml cryotubes and equilibrated prior immersed into liquid nitrogen for 15 days. The parameters observed were sperm motility, consistency, pH, fertilization and hatching rates and DNA fragmentation post-thawing.&nbsp; 
 
Result: The ANOVA test indicates that the application of FBS in Ringer had a significant effect on sperm motility, fertilization and hatching rates (P&lt;0.05). The highest motility (58.33%) was recorded at FBS 60% and significantly different from those at other concentrations. The laddering analysis showed that applying FBS protected the integrity of depik sperms DNA. It is concluded that the optimum concentration of FBS on depik sperm led to a short-term cryopreservation of 60%.</div>

Effect of Fetal Bovine Serum on the Sperm Quality of Depik (Rasbora tawarensis) after Short-term Cryopreservation

<div>Induced Pluripotent stem cells (iPSC) have a high ability to renew and differentiate themselves into various lineages and as vehicles of cell based therapy. Stem cell can differentiate under appropriate in vitro and in vivo conditions into different cell types. This study described the establishment of condition for in vitro expression of alpha MHC gene in cardiac differentiated canine iPSC (ciPSC). In vitro differentiation of canine iPSCs via embryoid bodies (EBs) were produced by &lsquo;Hanging Drop&rsquo; method. EB&rsquo;s were differentiated by using IMDM differentiation media: FBS &ndash; 10%, NEAA (100X) &ndash; 0.5%, &Acirc;-Mercaptoethanol- 100mM, Gentamycin- 5&micro;g/ml supplemented with Azacytidine- 0.5&micro;M. During differentiation, EBs were collected on day 4, 6, 8, 12, 16, 20 and 24 for characterization of cardiomyocytes specific marker expression. Total RNA from EBs were extracted by using Trizol method and subsequently cDNA were synthesized. The differentiated cells expressed cardiac specific gene (Alpha MHC) which started from day 6 of differentiation upto day 24 Immunocytochemistry and relative expression of cardiac specific genes revealed that ciPSC have the potential to differentiate into cardiomyocytes which can be used for cardiac tissue regeneration and as disease models for pharmaceutical testing.</div>

<div>Expression of Cardiac Specific Cell Marker in Ex Vivo Differentiated Canine iPSC</div>

Background: Mesenchymal stem cells are a potential candidate for regenerative medicine. Many factors may influence the physiological characteristics of stem cells that may lead to the alteration in their potential.&nbsp; 
 
Methods: The impact of age on mesenchymal stem cell characteristics was investigated in older buffaloes (aged over 10 years) and compared to younger counterparts (aged less than 2 years). Following isolation through enzymatic digestion, the mesenchymal stem cells were cultured in DMEM containing 12.5% FBS and antibiotics. Different variables indicating stem cell characteristics were evaluated at subsequent cultures.&nbsp; 
 
Result: The results revealed both groups displayed positive staining for alkaline phosphatase (AP) and expressed stem cell markers, including CD73, CD105 and OCT4, while remain negative for CD34. Furthermore, both sets of stem cells demonstrated the ability to differentiate into adipogenic, osteogenicand chondrogenic lineages. There were no notable variations observed between the two groups in terms of the cell yield, percentages of senescent cells, necrotic cells, dead cells and the apoptotic index. Expression of the P53 gene in RT-PCR amplicon in both groups confirms that stem cells are dividing normally as healthy cells and not tumor cells. Consequently, it was concluded from the present investigation that the age of the animal does not influence the characteristics of mesenchymal stem cells derived from adipose tissue. Thus, the stem cells derived from older and younger buffaloes are suited equally as a cell source for regenerative therapeutics.

A Comparative Analysis of the Age-dependent Features of Mesenchymal Stem Cells from Buffalo Adipose Tissue

Isolation of goat spermatogonial stem cells (gSSC) from pre-pubertal testes (3 to 6 months of age) by double enzymatic digestion is reported. The isolated cells were further enriched for spermatogonial stem cell population by filtration through 80- and 60-&micro;m nylon mesh filters, followed by differential plating on DSA-Lectin coated dishes. After overnight incubation, the unattached cells (putative gSSCs) were cultured on sertoli cell feeder layers in SSC medium, composed of DMEM supplemented with 10% FBS, GDNF (Glial cell line derived neurotrophic factor, 40 ng mL-1), bFGF (Basic fibroblast growth factor, 10 ng mL-1) and EGF (Epidermal growth factor, 10 ng mL-1). After 10-15 days, the putative SSCs started to develop and the cultures were maintained till the colonies stopped growing. The colonies were characterized for their affinity to Dolichos biflorus Agglutinin (DBA), alkaline phosphatase (AP) activity and various spermatogonial stem cell markers (PLZF, THY1, UCHL1, BCL6B and ID4). The developed colonies were positive for DBA, AP and all other markers, which is an indication of their being spermatogonial stem cells. In the present study, goat SSCs have been successfully isolated and characterized and the culture conditions will be improved further for developing more efficient and long term in vitro spermatogonial stem cell culture system in goat.

Derivation, enrichment and characterization of goat (Capra hircus) spermatogonial stem cells from pre-pubertal testes

Background: The mammary gland is a crucial organ in dairy animals, responsible for milk production, regulated by intricate hormonal and cellular interactions. Traditional two-dimensional (2D) cell culture systems used in mammary gland research fail to replicate the complex in vivo environment, limiting the understanding of mammary gland biology. The study explores the advantages of three-dimensional (3D) culture systems for Buffalo Mammary Epithelial Cells (BuMECs), aiming to enhance morphological and functional differentiation compared to 2D cultures. 
 
Methods: BuMECs were cultured in Dulbecco&rsquo;s Modified Eagle&rsquo;s Medium (DMEM/F12) supplemented with 10% FBS and specific growth factors. For 2D culture, BuMECs were grown on a plastic substratum. For 3D culture, cells were cultured on top of and embedded in growth factor-reduced (GFR) Matrigel. Morphological differentiation was observed under phase-contrast microscopy. Total RNA was extracted and cDNA was synthesized to measure &beta;-casein gene expression using quantitative real-time PCR (qPCR). Protein expression was analyzed by Western blotting, targeting total casein. 
 
Result: BuMECs grown in 3D cultures exhibited enhanced morphological differentiation, forming alveolar and ductal structures, unlike the monolayer formation observed in 2D cultures. Rapid formation of duct-like structures was noted within 24 hours in 3D cultures. Functional differentiation was significantly improved, evidenced by a five-fold increase in &beta;-casein mRNA expression and higher protein levels of &alpha;, &beta; and &kappa;-casein in 3D cultured cells compared to 2D cultures. Western blot analysis confirmed the elevated casein protein expression in 3D cultures.

Three-dimensional Culture of Buffalo (Bubalus bubalis) Mammary Epithelial Cell Line

Background: The study was conducted to compare the development of goat embryos in simple media with BSA and complex culture media supplemented with serum on the development of goat embryos. Also, studied the effect of oviductal cell co-culture on the developmental competence of goat oocytes. 
 
Methods: The A and B quality goat oocytes subjected to in vitro maturation in TCM 199 + 10% FCS + FSH + Estradiol for 24 hours in a CO2 incubator (6% CO2) at 38.5&deg;C. The oocytes that exhibited cumulus expansion subjected to in vitro fertilization in 1) BO - IVF and 2) TALP-fertilization media supplemented with heparin. The cleavage percent found significantly higher in BO-IVF (66.66%) compared to TALP- fertilization media (42.07%). After 18 hours of sperm-oocyte incubation, the presumptive zygotes from BO-IVF media divided randomly into 3 groups and cultured in one of the following 3 media, group 1) CR1aa media; group 2) BO-IVC media and group 3) TCM-199 + 10% FBS. 
 
Result: The cleavage percentage was significantly higher (P&gt;0.05) in BO-IVC media than CR1aa media and TCM-199 media (72.32, 64.35 and 35.23 percent respectively). The blastocyst development observed significantly higher (P&gt;0.05) in CR1aa compared to commercial BO-IVC media and TCM-199 media (9.23, 5.55, and 4.05 percent, respectively). Among the different media compared, CR1aa and BO-IVC media were effective for supporting the development of goat zygotes to the morula and blastocyst stages than those obtained when a complex TCM-199 medium used with serum supplementation. The effect of oviductal cell co-culture on the developmental competence of goat oocytes studied. The blastocyst development rate found higher in CR1aa + oviductal and TCM- 199 + oviductal cells as compared to CR1aa and TCM-199 alone.

Comparative Efficiency of Different Culture Media on&nbsp;in vitro Embryonic Development of Goat Oocytes

Background: The overall objective of this study is to confirm that normal expression of yeast NADH- dehydrogenase (NDI1) can occur in neural stem cell lines of minipig with resistance to rotenone exposure, an environmental factor responsible for dysfunction of mitochondrial enzyme complex I. In modern society, there are many diseases that cannot be treated. Diseases including LHON, Parkinson disease and dystonia, have been associated with defects in mitochondrial complexes. This experiment was performed to demonstrate that it was not sensitive. The overall objective of this study is to confirm that normal expression of yeast NADH-dehydrogenase (NDI1) can occur in neural stem cell lines of minipig with resistance to rotenone exposure, an environmental factor responsible for dysfunction of mitochondrial enzyme complex I. 
 
Methods: A Mini Pig Neural stem cell line (MPV) was used for transfection of the NDI gene, DMEM/F-12 culture medium and MPV was inoculated in a six-well plate (Corning, USA) at a concentration of 1&times;105 cells/3ml/well, with inoculation of MPV at 37&deg;C for 24 hours, 5% CO2, followed by incubation in an incubator with 95% humidity and attached. The plate containing MPV cells was treated with recombinant adeno-associated virus ndi1 (rAAV-ndi1) for transfection, with periodic replacement with a cell selection culture solution containing 10% FBS, 1% P/S and 0.2 mM rotenone. The experiment was performed for restoration of mitochondrial activity of thawed cells. RNA was extracted from MPV cells and transfection of MPV cells with the NDI1 gene was performed using Trizol (Invitrogen, USA). Reverse transcription PCR (RT-PCR) and Western blot were performed to confirm normal expression of the NDI1 gene in MPV cells transfected with the NDI1 gene. Immunofluorescence was performed to determine the presence of the NDI1 protein in the cell and the cell count was used for LUNA and the rate of cell death for rotenone was determined using the MTS [3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay. 
 
Result: rAAV-NDI1 was successfully introduced into MPV cells and the proliferation rates for the cells were compared with those of the transformed cells; after three days, the non-infected cells were killed and the infected cells proliferated. The results after differentiating the cell lines were similar to those of previously reported studies. Toxicity analysis on rotenone was also performed using the MTS assay and the rates of cell death over three days were compared; the results showed significantly lower levels of NDI1-transformed minipig neural stem cells compared with those of minipig. The present work will be a complementary contribution to the comprehensive study of the scorpion sting syndrome. These results were similar to those of previously reported studies. Previous studies have reported that oxidoreductive stress can be a cause of apoptosis. Therefore, conduct of additional studies for measurement of ROS and oxygen utilization to determine oxidative stress in cells will be necessary.

Soy protein isolate: A substitute of fetal bovine serum for the in vitro cultivation of Leishmania donovani

Development of Shelf Stable Chitterlings Pickle Incorporated with Natural Acidulant

Effect of Fetal Bovine Serum on the Sperm Quality of Depik (Rasbora tawarensis) after Short-term Cryopreservation

Expression of Cardiac Specific Cell Marker in Ex Vivo Differentiated Canine iPSC

A Comparative Analysis of the Age-dependent Features of Mesenchymal Stem Cells from Buffalo Adipose Tissue

Derivation, enrichment and characterization of goat (Capra hircus) spermatogonial stem cells from pre-pubertal testes

Three-dimensional Culture of Buffalo (Bubalus bubalis) Mammary Epithelial Cell Line

Comparative Efficiency of Different Culture Media on in vitro Embryonic Development of Goat Oocytes

Effects of Rotenone Exposure on Apoptosis in rAAV-NDI1-infected Neural Stem Cell Line of Minipig