Full-Term Development of Gaur–Bovine Interspecies Somatic Cell Nuclear Transfer Embryos: Effect of Trichostatin A Treatment

Abstract

Trichostatin A (TSA) has previously been used in somatic cell nuclear transfer (SCNT) to improve the cloning efficiency in several species, which led our team to investigate the effects of TSA on the full-term development of bovine SCNT and gaur-bovine interspecies SCNT (gaur iSCNT; gaur somatic cells as donors and bovine oocytes as recipients) embryos. Treatment with 50 nM TSA for 10 h after fusion had no positive effects on the rates of fusion, cleavage, or the development to eight-cell or morula stages in both bovine SCNT and gaur iSCNT embryos. However, TSA treatment significantly enhanced the blastocyst formation rate in bovine SCNT embryos (44 vs. 32-34% in the TSA-treated and TSA-untreated groups, respectively), but had no effects on gaur iSCNT embryos. The fresh blastocysts derived from bovine SCNT and gaur iSCNT embryos (fresh groups), as well as vitrified bovine SCNT blastocysts (vitrified group), were transferred to bovine recipients. We found that TSA treatment increased the pregnancy rates only in recipients receiving fresh bovine SCNT embryos. In recipients receiving TSA-treated bovine SCNT embryos, three cloned calves from the fresh group and twin cloned calves from the vitrified group were delivered; however, no calf was born from the TSA-untreated bovine SCNT embryos. In contrast, one gaur iSCNT calf was born from a recipient receiving blastocysts from the TSA-untreated group. In summary, TSA improved the preimplantation development and pregnancy rates of bovine SCNT embryos, but did not have any beneficial effect on gaur iSCNT embryos. However, one gaur iSCNT calf reached full-term development.

Artificial cloning of domestic animals

“…Frequently, these animals do not thrive and die relatively soon after birth. As a case in point, the first gaur calf that was produced by transplantation of a gaur nucleus into a bovine oocyte died shortly after birth (47) although more recent attempts seem to have been more successful (48). Success has also been achieved between endangered cats using oocytes from domestic cat as recipient (46).…”

Segregation of donor cell mitochondrial DNA in gaur–bovine interspecies somatic cell nuclear transfer embryos, fetuses and an offspring

“…iSCNT embryo production 2.1.1. Preparation of donor cells Fibroblast cells were established from the biopsied skin tissue of adult female and male gaur from the Khao Kheow Open Zoo, Chonburi, Thailand according to the protocol of Srirattana et al (2012). Briefly, skin tissues were dissected into small pieces and placed onto glass cover slips.…”

“…Bovine ovaries were collected from a slaughterhouse and processed as previously described (Srirattana et al, 2012). Briefly, cumulus oocyte complexes were aspirated from 2 to 6 mm follicles and cultured in TCM 199 maturation medium supplemented with 10% FBS, 50 IU/ml human chorionic gonadotropin (Intervet, Boxmeer, Netherlands), 0.02 AU/ml follicle-stimulating hormone (Antrin, Kawasaki Seiyaku K.K., Kawasaki, Japan) and 1 μg/ml 17β-estradiol for 21 h at 38.5°C in a humidified atmosphere of 5% CO 2 .…”

“…The samples were incubated at 56°C for 30 min, heated to 95°C for 5 min and stored at −20°C until further use. Total DNA was also extracted from frozen tissue samples from an aborted and mummified female fetuses and a male calf offspring (died at 12 h after birth), including brain, heart, muscle, lung, stomach, liver, kidney and intestine (Srirattana et al, 2012) and donor fibroblasts, using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The DNA concentrations were quantified using Quant-iT PicoGreen (Invitrogen, CA, USA).…”

“…The success of iSCNT embryo production has been achieved in several species, including gaur-bovine (Lanza et al, 2000), panda-rabbit (Chen et al, 2002), macaque-rabbit , human-rabbit , goat-sheep and cat-bovine (Imsoonthornruksa et al, 2011). Furthermore, live gaur (Srirattana et al, 2012;Vogel, 2001), mouflon (Loi et al, 2001), wildcat (Gómez et al, 2004(Gómez et al, , 2008 and wolf (Oh et al, 2008) iSCNT offspring have been obtained. However, the efficiency of iSCNT is low, and the underlying causes for this low efficiency are not well understood.…”