Somatic cell nuclear transfer in horses: effect of oocyte morphology, embryo reconstruction method and donor cell type

Abstract

The objective of the present work was to investigate and clarify the factors affecting the efficiency of somatic cell nuclear transfer (NT) in the horse, including embryo reconstruction, in vitro culture to the blastocyst stage, embryo transfer, pregnancy monitoring and production of offspring. Matured oocytes, with zona pellucida or after zona removal, were fused to cumulus cells, granulosa cells, and fetal and adult fibroblasts, and fused couplets were cultured in vitro. Blastocyst development to Day 8 varied significantly among donor cells (from 1.3% to 16%, P < 0.05). In total, 137 nuclear transfer-embryos were transferred nonsurgically to 58 recipient mares. Pregnancy rate after transfer of NT-embryos derived from adult fibroblasts from three donor animals was 24.3% (9/37 mares transferred corresponding to 9/101 blastocysts transferred), while only 1/18 (5.6%) of NT-blastocysts derived from one fetal cell line gave rise to a pregnancy (corresponding to 1/33 blastocysts transferred). Overall, seven pregnancies were confirmed at 35 days, and two went to term delivering two live foals. One foal died 40 h after birth of acute septicemia while the other foal was healthy and is currently 2 months old. These results indicate that (a) the zona-free method allows high fusion rate and optimal use of equine oocytes, (b) different donor cell cultures have different abilities to support blastocyst development, (c) blastocyst formation rate does not correlate with pregnancy fate and (d) healthy offspring can be obtained by somatic cell nuclear transfer in the horse.

High developmental potential in vitro and in vivo of cattle embryos cloned without micromanipulators

“…Concerns about removing zona from embryos include the possibility of viral infection of them (however the risk is the same for conventional cloning, due to the rupture of the zona), the potential failures in DNA methylation in these embryos, as shown for mouse zona free embryos [18] and the impossibility for group culture of embryos due to aggregation of adjacent embryos; however this problem has been overcame by the use of WOW culture [3]. In the last years this method has been successfully used for production of bovine [2,[19][20][21], horse [22][23], mouse [24] and pig [25] offspring. Finally, and probable one of the most important features of HMC is its simplicity, speed of work and low cost when compared to traditional cloning using micromanipulators.…”

Production of horse foals via direct injection of roscovitine-treated donor cells and activation by injection of sperm extract

“…There are two reports of horse foals produced by NT from adult somatic cells (Galli et al 2003, Lagutina et al 2005; in addition, three mule foals have been produced by NT using fibroblasts obtained from a mule fetus (Woods et al 2003, Vanderwall et al 2004b.…”

“…To make maximum use of the available oocytes, alternative methods yielding higher recombination rates have been developed in the horse. These have included addition of sendai virus during fusion (Li et al 2002), fusion after zona removal (Galli et al 2003, Lagutina et al 2005 and direct injection of the donor cell (Choi et al 2002a). Of the recombination methods used, only fusion after zona removal has been shown to produce live young after nuclear transfer using adult somatic cells.…”

“…The reported rates of blastocyst production after equine NT have been low (0-16%; Galli et al 2003, Li et al 2004, Lagutina et al 2005. Because rates of blastocyst production after intracytoplasmic sperm injection of in vitro-matured oocytes are acceptable (up to 38%; Hinrichs et al 2005), the low blastocyst development after NT in the horse may be related to inefficiency in activation of equine oocytes.…”

Nuclear reprogramming: what has been done and potential avenues for improvements

“…Such allele-specific gene expression is programmed by chromatin features established in the germ line which are maintained throughout development. However, despite the progress achieved in our knowledge of nuclear reprogramming, witnessed by 11 mammalian species successfully cloned [3], and the simplification of micromanipulation procedures [12,13], the frequency of offspring production in SCNT remains low, between 1-5% of transferred embryos.…”