T H Nguyen’s scientific contributions

Cell co-culture during in vitro maturation or embryo culture has been reported as a method to improve the efficiency of maturation or embryo development (Kidson et al. 2003 Theriogenology 59, 1889; Romar et al. 2005 Anim. Reprod. Sci. 85, 287). Here, we present the impact of different methods of co-culture with mouse embryonic fibroblasts or oviduct epithelial cells on in vitro embryo production in pigs. Cumulus-oocyte complexes (COC) were collected from follicles with diameter larger than 3mm and used for in vitro embryo production based on the method of Kikuchi et al. 2002 (Biol. Reprod. 66, 1033) with minor modifications. There were 8 groups; group 1: maturation and embryo culture without cell co-culture (control group); group 2: maturation in the presence of fibroblasts; group 3: embryo culture in the presence of fibroblasts; group 4: both maturation and embryo culture in the presence of fibroblasts; group 5: maturation in the presence of oviduct cells; group 6: embryo culture in the presence of oviduct cells; group 7: both maturation and embryo culture in the presence of oviduct cells; group 8: both maturation and embryo culture in the presence of both fibroblast and oviduct cells. In vitro maturation (IVM) was carried out at 39(o)C under 5% CO(2) in air for 44h using NCSU-37 as basic medium. Matured oocytes were inseminated using epididymal frozen semen in IVF medium modified Pig-FM supplemented with 2mM caffeine and 5mgmL(-1) bovine serum albumin (Kikuchi et al. 2002). The percentage of cleaved embryos and percentage of cleaved embryos which developed to the compact morula and early blastocyst stage were recorded. Results were analysed by one-way ANOVA followed by Dunnett's test. To investigate the effects of co-culture with mouse embryonic fibroblasts and oviduct epithelial cells on oocyte maturation, some COCs cultured in groups 1, 2, 5, and 8 were fixed to assess their nuclear maturation to the metaphase II stage. The rate of matured oocytes in the groups 2, 5, and 8 was 76.85±3.39% (n=102), 79.11±3.75% (n=64), and 81.84±3.93% (n=66), respectively; these rates were increased significantly compared to the group 1 (55.87±1.88%, n=94; P<0.05). The effect of co-culture on the fertilization and embryo development is shown in Table 1. Our results indicate that co-culture increases the rates of embryonic cleavage in all groups by comparison with the control group. However, a significant increase in the rate of morula-blastocyst was only observed when embryos were co-cultured with fibroblasts or when both maturation and culture were performed in co-culture with either fibroblasts or oviduct cells (groups 3, 4, 7, and 8). The most important increase in morula-blastocyst rate was recorded for the group of embryos co-cultured with fibroblasts (group 3). In conclusion, the co-culture with fibroblast or oviduct cells during maturation can improve oocyte maturation and cleavage rate, while co-culturing the embryos with fibroblasts seems sufficient to improve both the cleavage and the morula-blastocyst rates.

Embryo cryopreservation is an important need for industrial catfish production, as well as for biodiversity conservation. However, due to the limit of membrane permeability and dehydration in fish embryos, no successful cryopreservation method has been reported. We present herein results of the effect of dehydration and low temperature on the survival of catfish embryos, to understand their cryobiological behaviour and develop an efficient freeze method for this species. Embryos at blastocyst stage were collected at 10h after insemination and kept in a water tank with an air pump. Embryos with somites and optic cups clearly distinguished were selected and treated according to 4 treatment groups: group 1, control group: embryos were kept in water at room temperature for 2 h; group 2, treated for dehydration: embryos were kept in 1M or 2M sucrose solution for 2h at room temperature; group 3, treated for low temperature: embryos were kept in water for 2h at 0°C; and group 4, treated for dehydration and low temperature: embryos were kept in 1M or 2M sucrose solution for 2h at 0°C. After treatments, all embryos were washed twice in water and incubated in separated water tanks for 2 days. The survival of embryo was evaluated by the rate of intact and hatched embryos during the first day after treatment. Statistical analysis was performed using one-way ANOVA followed by Dunnett's test. Our results showed that in the control group, all of the embryos were intact and hatched. In the group 2, the survival rate of embryos treated with 1M sucrose was not different compared with the control group. However, in this group, no hatched embryos were obtained when treated with 2M sucrose (Table 1). Also, no hatched embryos were observed when they were kept at 0°C without dehydration (group 3). A significant improving hatching rate was obtained in group 4 for embryos treated at 0°C and dehydrated in 2M sucrose (90 v. 0% in group 3; P<0.05). In conclusion, the combined treatment of dehydration (in 2M sucrose concentration) and low temperature (at 0°C) can result in synergistic effect that are able to protect embryos from damages caused by treatment with either low temperature or dehydration conditions.