Effect of potassium simplex optimization medium (KSOM) and embryo screening on the production of human lactoferrin transgenic cloned dairy goats

Abstract

In this study, we produced cloned transgenic dairy goat based on dairy goat ear skin fibroblast as donor cells for nuclear transfer (NT), which were modified by human lactoferrin (hLF) gene. The developmental competence of NT embryos was compared with either between different embryo culture medium, potassium simplex optimization medium (KSOM) and tissue culture medium (TCM 199), or different classification of NT embryos (48 h after fusion). First we cultured NT embryos to cleavage stage (48 h after fusion) by TCM 199 supplemented with 1 mg/ml bovine serum albumin BSA and KSOM, then used TCM 199 supplemented with 10% FBS to culture them to blastula stage. The results show that the NT embryos in KSOM (19.5%) were superior to TCM 199 (10.6%) in blastulation. In the second experiment, we found that the growth rate of NT embryos (48 h after fusion) was different, then we divided them into four groups: 2-cell, 3- to 4-cell, 5- to 8-cell and >8-cell in stereo microscope and cultured them in vitro respectively. The results show day-2 embryos at 3-4cell and 5-8cell stage (31.9 and 28.2%, P < 0.05) had higher blastocyst formation rates than those at both 2-cell (9.1%) and >8-cell (8.3%) stage, and finally three healthy cloned transgenic goat were successfully produced using 3-8 cell embryos at Day-2 (82%). Using Hoechst 33342 staining, we also found that the >8 cells embryos at Day- 2 demonstrated higher frequency of fragmentation, which may be the one cause of the low blastocyst formation rate. This study therefore demonstrates that KSOM medium could be selected as the early embryo culture medium, and 3-8 cell embryos at day-2 (48 h after fusion) may be the suitable embryos for transplantation, which could reduce the nuclei fragmentation and result in good quality blastocysts that may also enhance the efficiency of transgenic cloned dairy goats production, as well as decrease the economic loss due to embryonic mortality when embryos are transferred to synchronized recipients.

Key words: Nuclear transfer, KSOM, transgenic, human lactoferrin, dairy goat.