Cryobanking of primary somatic cells of elite farm animals - A pilot study in domesticated water buffalo (Bubalus bubalis).

Buffalo is an important farm animal species in South and South-east Asian countries. Cryopreservation allows long-term storage of somatic cells, which can be made available to research communities. This study aimed to 1) establish and cryopreserve somatic cells from elite buffaloes, and 2) share stored somatic cells and their associated data with researchers. To achieve these targets, somatic cells were established successfully from tail-skin biopsies of 17 buffaloes. The informative data such as buffalo details (breed, date of birth, sex, and age at the time of tissue biopsy collection, and production traits), the number of cryovials stored, and freezing dates were recorded in an electronic file and a printed inventory record. The established somatic cells were flat, spindle-shaped morphology, and expressed vimentin (a fibroblast-like cell type marker) and the negative expression of cytokeratin-18 (an epithelial cell type marker). Altogether, we cryopreserved 970 cryovials (0.1 million cells per vial) from two buffalo breeds, namely Murrah and Nili-Ravi (at least 45 cryovials per animal), for cryobanking. Somatic cell nuclear transfer (SCNT) experiments demonstrated the utility of cryopreserved cells to produce cloned buffaloes. Importantly, these cryopreserved somatic cells are made available to scientific communities. This study encourages the cryopreservation of somatic cells of elite farm animals for their utilization in cell-based research.

Semen parameters and fertility potency of a cloned water buffalo (Bubalus bubalis) bull produced from a semen-derived epithelial cell.

Semen contains epithelial cells that can be cultured in vitro. For somatic cell nuclear transfer applications, it is essential to know whether clone(s) produced from semen-derived epithelial cells (SedECs) are healthy and reproductively competent. In this study, the semen and fertility profile of a cloned bull (C1) that was produced from a SedEC were compared with its donor (D1) and with two cloned bulls (C2, C3) that were produced from commonly used skin-derived fibroblast cells (SkdFCs). We observed variations in some fresh semen parameters (ejaculated volume and mass motility), frozen-thawed sperm parameters (plasma membrane integrity, and computer-assisted semen analysis (CASA) indices), but values are within the normal expected range. There was no difference in sperm concentration of ejaculated semen and frozen-thawed semen parameters which include sperm motility, percentage of live and normal morphology sperm, and distance traveled through oestrus mucus. Following in vitro fertilization (IVF) experiments, zygotes from C1 had higher (P < 0.05) cleavage rates (81%) than C2, C3, and D1 (71%, 67%, and 75%, respectively); however, blastocyst development per cleaved embryo and quality of produced blastocysts did not differ. The conception rate of C1 was 46% (7/15) and C2 was 50% (8/15) following artificial insemination with frozen-thawed semen. Established pregnancies resulted in births of 7 and 6 progenies sired by C1 and C2, respectively, and all calves show no signs of phenotypical abnormalities. These results showed that semen from a cloned bull derived from SedECs is equivalent to semen from its donor bull and bulls cloned from SkdFCs.

Successful cloning of a superior buffalo bull.

Somatic cell nuclear transfer (SCNT) technology provides an opportunity to multiply superior animals that could speed up dissemination of favorable genes into the population. In the present study, we attempted to reproduce a superior breeding bull of Murrah buffalo, the best dairy breed of buffalo, using donor cells that were established from tail-skin biopsy and seminal plasma. We studied several parameters such as cell cycle stages, histone modifications (H3K9ac and H3K27me3) and expression of developmental genes in donor cells to determine their SCNT reprogramming potentials. We successfully produced the cloned bull from an embryo that was produced from the skin-derived cell. Growth, blood hematology, plasma biochemistries, and reproductive organs of the produced cloned bull were found normal. Subsequently, the bull was employed for semen production. Semen parameters such as CASA (Computer Assisted Semen Analysis) variables and in vitro fertilizing ability of sperms of the cloned bull were found similar to non-cloned bulls, including the donor bull. At present, we have 12 live healthy progenies that were produced using artificial insemination of frozen semen of the cloned bull, which indicate that the cloned bull is fertile and can be utilized in the buffalo breeding schemes. Taken together, we demonstrate that SCNT can be used to reproduce superior buffalo bulls.

Isolation and culture of epithelial cells from stored buffalo semen and their use for the production of cloned embryos.

The aim of the present study was to isolate somatic cells from semen, a non-invasive source of donor somatic cells, for somatic cell nuclear transfer (SCNT) experiments. The study had two parts: (1) isolation and culture of somatic cells from semen, which was stored at 4°C; and (2) investigating the SCNT competence of semen-derived somatic cells. We successfully cultured somatic cells from freshly ejaculated semen, which was stored for different times (0, 4, 12, 24, 72 and 144h after semen collection) at 4°C, using a Percoll gradient method. Up to 24h storage, 100% cell attachment rates were observed; cell attachment rates of 66% were observed for the 72 and 144h storage groups. The attached cells observed in all groups examined were proliferated (100%). Cultured cells exhibited epithelial cell morphology and culture characteristics, which was further confirmed by positive expression of cytokeratin 18, an epithelial cell-type marker. We compared the SCNT competence of semen-derived epithelial cells and skin-derived fibroblasts. The cleavage rate, blastocyst production rate, total number of cells in blastocysts and the apoptotic index of blastocysts were similar for embryos produced from semen-derived epithelial cells and skin-derived fibroblasts, indicating that semen-derived epithelial cells can serve as donors for SCNT experiments. In conclusion, we demonstrate a method to culture epithelial cells from stored semen, which can be used to produce cloned embryos of breeding bulls, including remote bulls.

A new role for RU486 (mifepristone): it protects sperm from premature capacitation during cryopreservation in buffalo.

The objective of this study was to determine the mechanism by which RU 486 (mifepristone) protects sperm to undergo premature capacitation during cryopreservation. For this, semen ejaculate (n = 20) was divided into four equal fractions and diluted using egg yolk-based extender supplemented with different concentrations of RU 486 (0, 5, 10 and 20 µM) and cryopreserved. We found that RU 486 did not impair the post-thaw sperm kinetics and motility but prevented cholesterol efflux, calcium influx, and protected CatSper channels during cryopreservation. The RU 486 protected sperm from premature capacitation which was confirmed by intracellular calcium level, expression of tyrosine phosphorylated proteins (75 and 80 kDa) and CTC (chlortetracycline) assay. Furthermore, antioxidant ability of RU 486 was reflected by the ferric reducing ability, lower production of sperm malondialdehyde and intracellular reactive oxygen species. Also, we demonstrated that RU 486 treated sperm underwent normal capacitation, zona pellucida binding and zygote cleavage indicating normal fertilizing ability of sperm. In conclusion, we report a new role of RU 486 in protecting buffalo sperm from premature capacitation during cryopreservation.

Transposon mediated reprogramming of buffalo fetal fibroblasts to induced pluripotent stem cells in feeder free culture conditions.

Commonly, induced pluripotent stem (iPS) cells are generated by viral transduction of four core reprogramming genes, but recent evidences suggest that slightly different combination of transcription factors improve the efficiency and quality of generated iPS cells. However, vectors like retro- and lentiviral may cause insertional mutagenesis due to its integrating ability. Hence, alternate methods with safety concerns are needed to be investigated. Therefore, the present study was undertaken to reprogram buffalo fibroblasts using non-viral piggyBac (PB) transposon mediated transfer of six transcription factors. To generate buffalo iPS cells, fibroblasts were isolated from buffalo fetus at passage 2. The cells were co-electroporated with a PB transposon having CAGGS promoter driven cassette of Oct4, Sox2, Klf4, cMyc, Nanog, and Lin28 transcription factors separated by self-cleaving 2A peptide and a helper plasmid pCMV-PB transposase. After 12-14 days post electroporation, fibroblast cells morphology was observed to change to round structures which formed loose aggregates of cells on day 18. Putative iPS cell colonies were propagated in feeder free system and characterized through expression of pluripotency markers such as alkaline phosphatase, SSEA-1, SSEA-4, SSEA-5, TRA-1-81, Oct4, Nanog and Sox2 and endogenous genes supported the stemness property of the generated cells. These cells differentiated in vitro to form embryoid bodies and were found to express three germ layers markers. In conclusion, generation of buffalo iPS cells using transposon system provides insights into viral-free iPS technology which will facilitate genetic modification of the buffalo genome and help in the production of transgenic animals using genetically modified iPS cells.

Generation of Venus fluorochrome expressing transgenic handmade cloned buffalo embryos using Sleeping Beauty transposon.

The objective of this study was to optimise the electroporation conditions for efficient integration of Venus construct in buffalo fetal fibroblasts using Sleeping Beauty (SB) based transposition and to produce Venus expressing transgenic cloned embryos through handmade cloning (HMC) approach. Primary culture of buffalo fetal fibroblast cells was established and subsequently cultured cells were co-transfected with Venus and helper plasmid at different combinations of electroporation condition. In different combinations of voltage, time and plasmid dose, we observed that 300 V, single pulse for 10 ms in 2 mm cuvette and 1.5-2.0 µg transposons with 200-300 ng transposase dose was optimum for expressing Venus fluorescence in cells via electroporation. After electroporation, the cells were cultured for 2-3 days and then Venus expressing cells were picked with the help of a Pasteur pipette under the fluorescence microscope to enrich them through single cell culture method before using as donor cells for HMC. In vitro matured oocytes were reconstructed with either transfected or non-transfected buffalo somatic cells by electric fusion followed by activation. The reconstructed, activated embryos were cultured in 400 µL of Research Vitro Cleave medium supplemented with 1% fatty acid-free BSA in 4-well dish, covered with mineral oil and incubated in an incubator (5% CO2 in air) at 38.5 °C for 8 days and the developmental competence was observed. The percentage of cleaved, 4-8 and 8-16 cells stage embryos generated through Venus expressing cells were comparable with control, whereas, the morula (21.0 vs 53.0%) and blastocysts (10.5 vs 30.6%) produced through Venus expressing cells was found low as compared to control. These results indicate that fetal fibroblasts transfected with Venus could be used as donor cells for buffalo cloning and that Venus gene can be safely used as a marker of foreign gene in buffalo transgenesis.