30 EFFECT OF THE CYTOPLAST SOURCE AND KARYOPLAST TYPE ON THE DEVELOPMENT OF HANDMADE CLONED EMBRYOS IN GOATS

Abstract

The aim of this study was to compare the effect of 2 donor cell types (bone marrow-derived mesenchymal stem cells, BM-MSC and skin fibroblast cells, SFC) and the source of oocytes (in vivo- and in vitro-matured goat oocytes) on the developmental capacity of handmade cloned goat embryos, following our procedures adapted from cattle (Ribeiro et al., 2009, Cloning Stem Cells 11, 377–386). In vivo- and in vitro-matured oocytes obtained postmortem from 36 superovulated and 90 nonstimulated goats were used as cytoplasts for cloning, after 26 h from the induction of ovulation or 20 h from the onset of IVM, respectively. Subsequent to cumulus cell removal and polar body selection, a total of 242 in vivo- and 580 in vitro-matured oocytes were subjected to zona removal, bisection in cytochalasin B and screening under ultraviolet light. Enucleated hemi-cytoplasts were exposed to phytohemoagglutinin and adhered to a single somatic cell (BM-MSC or SF) and electrofused by two 1.2 kV cm^–1 DC pulses for 20 μs. Cell primary cultures were established from lysozyme transgenic goats. Prior to cloning, cells between the 3rd and 8th passage and at 50 to 60% (BM-MSC) or >95% (SFC) confluence were evaluated for size and viability using the Countess^TM Automated Cell Counter (Invitrogen, Carlsbad, CA, USA). Fused structures were activated in ionomycin/6-DMAP and in vitro-cultured in the well of the well system in SOFaa + 5% FCS + 0.2% BSA, at 38.5°C, in 5% CO₂, 5% O₂ and 90% N₂, for 6 days. After 8 replications, fusion, cleavage (Day 2) and embryo developmental (Day 6) rates were compared by the χ² test. Data obtained on cell size and viability were analysed by ANOVA (P < 0.05). Cell viability was similar between SFC (86.7 ± 2.2%) and BM-MSC (89.0 ± 2.2%). However, mean cell size was significantly smaller in SFC (14.4 ± 0.4 μm) than in BM-MSC (20.1 ± 0.4 μm). Cell size appeared to be associated with fusion efficiency because fusion rates were also significantly lower with SFC than with BM-MSC (Table 1). However, cell type or oocyte source did not affect any other parameter for embryo production by cloning between groups. A total of 63 compact morulas and blastocysts from both cell and oocyte types were transferred, in groups of 4 to 5 embryos, to 15 synchronous recipients. Pregnancy diagnosis is performed by ultrasonography on Days 28 to 32. Thus far, one pregnancy derived from an embryo reconstructed with in vivo-matured oocytes and BM-MSC was obtained out of 9 recipients that received 37 embryos from all treatment groups. Six recipients with 26 embryos transferred are still pending diagnosis. In conclusion, the handmade cloning procedure using in vivo- and in vitro-matured oocytes and BM-MSC and SFC appears to be an effective alternative for the production of transgenic goats.

Table 1.  In vitro development of goat embryos produced by handmade cloning using human lysozyme (hLZ) transgenic cell lines

Funded by the RECODISA Project, FINEP/MCT/Brazil.