Immunisation against inhibin enhances follicular development, oocyte maturation and superovulatory response in water buffaloes.

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.

Study on the inter-subspecies nuclear transfer of river buffalo somatic cell nuclei into swamp buffalo oocyte cytoplasm.

The objective of this study was to explore the feasibility of inter-subspecies somatic cell nuclear transfer (SCNT) of river buffalo (50 chromosomes) somatic cell nuclei into swamp buffalo (48 chromosomes) oocyte cytoplasm. The enucleated swamp buffalo oocytes were fused with four different types of river buffalo cells: freshly thawed ear fibroblasts, serum-starved ear fibroblasts, cumulus cells and ear fibroblasts from a cloned buffalo calf. As a result, the developmental competence of embryos reconstructed with freshly thawed ear fibroblasts was the poorest (P<0.01), while those of the other three types were not different from each other. Furthermore, the efficiency of swamp-swamp buffalo, swamp-river buffalo and bovine-buffalo SCNT were also compared. The results showed that the blastocyst rate of swamp-river reconstructed embryos was not different from swamp-swamp embryos, while significantly higher than that of bovine-buffalo embryos (P<0.01). A total of thirty cloned blastocysts derived from freshly thawed ear fibroblasts were transferred into thirteen recipient buffaloes, four recipients established pregnancy, while three of them aborted on Days 65, 75 and 90 of gestation, respectively. One cross-bred buffalo (Murrah x swamp, 49 chromosomes) receiving three embryos delivered a 39 kg female calf on Day 335 of gestation. These results indicate that the inter-subspecies SCNT is feasible to produce swamp-river buffalo embryos, and these can develop to full term and result in live buffalo calves.

An inter-subspecies cloned buffalo (Bubalus bubalis) obtained by transferring of cryopreserved embryos via somatic cell nuclear transfer.

The aim of this study was to explore the feasibility of cryopreservation of inter-subspecies cloned embryos in buffalo. In our experiment, river buffalo ear fibroblast nucleus was fused into swamp buffalo oocyte cytoplasm. The blastocyst formation rate for nuclear transfer of freshly thawed cells was not different from those of growing cells, confluent or serum-starved cells. A total of 122 cloned blastocysts derived from cryopreserved fibroblasts were cryopreserved and thawed, 37 were survived, the cryosurvival rate was 30.3%. The survived blastocysts were transferred into 15 recipient buffalos. Five of the recipients established pregnancy, but four of them aborted on day 53, 59, 145 and 179 of gestation respectively. One cross-bred buffalo (Murrah × Swamp buffalo (2n = 49) received three embryos delivered a 40.5 kg female calf by natural delivery on day 320 of gestation. Up to now (13-month old), the cloned calf has been growing well with no abnormity observed. These results demonstrated that cryopreservation of inter-subspecies cloned embryos is feasible to produce buffalo offspring.