Cloning of buffalos (Bubalus bubalis) through nuclear transfer is a potential alternative approach in genetic improvement of buffalos. However, to our knowledge, cloned offspring of buffalos derived from embryonic, fetal, or somatic cells have not yet been reported. Thus, factors affecting the nuclear transfer of buffalo somatic cells were examined, and the possibility of cloning buffalos was explored in the present study. Treatment of buffalo fibroblasts and granulosa cells with aphidicolin plus serum starvation resulted in more cells being arrested at the G0/G1 phase, the proportion of cells with DNA fragmentation being less, and the number of embryos derived from these cells that developed to blastocysts being greater. In addition, a difference was found in the development of embryos reconstructed with fetal fibroblasts from different individuals (P < 0.001). Forty-two blastocysts derived from granulosa cells and fetal fibroblasts were transferred into 21 recipient swamp buffalos, and 4 recipients were confirmed to be pregnant by rectal palpation on Day 60 of gestation. One recipient received two embryos from fetal fibroblasts aborted on Day 300 of gestation and delivered two female premature calves. Three recipients maintained pregnancy to term and delivered three female cloned calves after Days 338-349 of gestation. These results indicate that buffalo embryos derived from either fetal fibroblasts or granulosa cells can develop to the term of gestation and result in newborn calves.

Domesticated buffaloes have been integral to rice-paddy agro-ecosystems for millennia, yet relatively little is known about the buffalo genomics. Here, we sequenced and assembled reference genomes for both swamp and river buffaloes and we re-sequenced 230 individuals (132 swamp buffaloes and 98 river buffaloes) sampled from across Asia and Europe. Beyond the many actionable insights that our study revealed about the domestication, basic physiology and breeding of buffalo, we made the striking discovery that the divergent domestication traits between swamp and river buffaloes can be explained with recent selections of genes on social behavior, digestion metabolism, strengths and milk production.

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The solubility of veratric acid (3,4-dimethoxybenzoic acid, with measured melting point of 453.12 K by differential scanning calorimetry, DSC) in eight monosolvents, including ethanol, 1-propanol, 2-propanol, 1-butanol, 2-methyl-1-propanol, methyl acetate, ethyl acetate, and 2-butanone, and binary mixtures of ethanol + 1-butanol was determined at (278 to 323) K and atmospheric pressure using a dynamic method. The modified Apelblat equation and two local composition models (NRTL and UNIQUAC) were used to correlate the solubility of veratric acid in pure solvents. The modified Apelblat and the Jouyban–Acree model were used to correlate the solute solubility in binary mixtures. A combination of the Jouyban–Acree model and van’t Hoff equation was used to predict solubility data in the mixed solvents at different temperatures and gives a reasonable prediction. Each of the correlation equations selected gives a good description of the relationship of solubility and the temperature, and correlated data of the modified Apelblat equation show the best agreement with the experimental data, with the overall relative average deviations values of 0.61 % and 0.62 % in pure solvents and binary mixtures, respectively.

Ectopically, expression of defined factors could reprogram mammalian somatic cells into induced pluripotent stem cells (iPSCs), which initiates a new strategy to obtain pluripotent stem cell lines. Attempts have been made to generate buffalo pluripotent stem cells by culturing primary germ cells or inner cell mass, but the efficiency is extremely low. Here, we report a successful method to reprogram buffalo fetal fibroblasts (BFFs) into pluripotent stem cells [buffalo induced pluripotent stem cell (biPSCs)] by transduction of buffalo defined factors (Oct4, Sox2, Klf4, and c-Myc) using retroviral vectors. The established biPSCs displayed typical morphological characteristics of pluripotent stem cells, normal karyotype, positive staining of alkaline phosphatase, and expressed pluripotent markers including Oct4, Sox2, Nanog, Lin28, E-Cadherin, SSEA-1, SSEA-4, TRA-1-81, STAT3, and FOXD3. They could form embryoid bodies (EBs) in vitro and teratomas after injecting into the nude BALB/C mice, and 3 germ layers were identified in the EBs and teratomas. Methylation assay revealed that the promoters of Oct4 and Nanog were hypomethylated in biPSCs compared with BFFs and pre-biPSCs, while the promoters of Sox2 and E-Cadherin were hypomethylated in both BFFs and biPSCs. Further, inhibiting p53 expression by coexpression of SV40 large T antigen and buffalo defined factors in BFFs or treating BFFs with p53 inhibitor pifithrin-a (PFT) could increase the efficiency of biPSCs generation up to 3-fold, and nuclear transfer embryos reconstructed with biPSCs could develop to blastocysts. These results indicate that BFFs can be reprogrammed into biPSCs by buffalo defined factors, and the generation efficiency of biPSCs can be increased by inhibition of p53 expression. These efforts will provide a feasible approach for investigating buffalo stem cell signal pathways, establishing buffalo stem cell lines, and producing genetic modification buffaloes in the future.

Cryopreservation of human gonadal tissue and oocytes has brought about new and exciting research in reproductive medicine, as well as new cryopreservation techniques that are dramatically improving post-thaw viability and freezing damage. The work done on gonadal tissues is aimed at improving the quality of life for infertile patients and for prepubertal patients undergoing gonadotoxic chemotherapy, patients for whom hormonal stimulation /IVF is not an option, and women without partners. Cryopreservation of mature oocytes is the best model for timing IVF. Vitrification is likely to benefit the field, and since 2005, implantation and pregnancy rates from vitrified oocytes have matched or eclipsed results from conventional methods, due to new cell-specific methods and formulas. Cryopreservation of immature oocytes leads to a new direction of egg banking in future. Preserving ovarian tissue for autografting is still promising and has resulted in folliculogenesis, resumed hormone production and live births in limited cases. The use of small cortical blocks, or mechanical/chemical digestion of ovarian tissue for isolation of follicles is a new direction for ovary preservation for reasons of cryoprotectant permeation and graft revascularization. Maturation of follicles in vitro has become more feasible with the use of alginate microencapsulation. Testicular tissue preservation has taken a sharp turn towards preservation of gonadal stem cells. Research into the mechanism for spermatogenesis points to the ability for male germ cells to resume spermatogenesis. The cryopreservation of minced testicular tissue for isolation of germ cells via chemical digestion has produced viable cells, however structural damage that may be avoided by vitrification has been noted to the surrounding cell junctions and supporting cells.

This study was carried out to test the feasibility of enhancing embryo production in vivo and in vitro by immunoneutralisation against inhibin or follistatin. In Experiment 1, multi-parity buffaloes were assigned into three groups: High group (n=8), which received one primary (2mg) and two booster (1mg) vaccinations (28-day intervals) with a recombinant inhibin α subunit in 1 mL of white oil adjuvant; Low group (n=8), which received half that dose; and Control group (n=7), which received only adjuvant. Immunisation against inhibin stimulated development of ovarian follicles. Following superovulation and artificial insemination, inhibin-immunised buffaloes had more developing follicles than the Control buffaloes. The average number of embryos and unfertilised ova (4.5±0.6, n=6) in the High group was higher (P<0.05) than in the Control group (2.8±0.6, n=5) and was intermediate (4.1±0.7, n=7) in the Low group. The pooled number of transferable embryos of the High and Low groups (3.2±0.5, n=13) was also higher (P<0.05) than that (1.6±0.7, n=5) of the controls. The immunised groups also had higher plasma concentrations of activin, oestradiol and progesterone. In Experiment 2, the addition of anti-inhibin or anti-follistatin antibodies into buffalo oocyte IVM maturation medium significantly improved oocyte maturation and cleavage rates following parthenogenic activation. Treatment with anti-follistatin antibody also doubled the blastocyst yield from activated embryos. These results demonstrated that immunisation against inhibin stimulated follicular development, enhanced oocyte quality and maturation competence, yielded more and better embryos both in vivo and in vitro.

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