Effects of Donor Fibroblast Cell Type and Transferred Cloned Embryo Number on the Efficiency of Pig Cloning

Abstract

Currently, cloning efficiency in pigs is very low. Donor cell type and number of cloned embryos transferred to an individual surrogate are two major factors that affect the successful rate of somatic cell nuclear transfer (SCNT) in pigs. This study aimed to compare the influence of different donor fibroblast cell types and different transferred embryo numbers on recipients' pregnancy rate and delivery rate, the average number of total clones born, clones born alive and clones born healthy per litter, and the birth rate of healthy clones ( = total number of healthy cloned piglets born / total number of transferred cloned embryos). Three types of donor fibroblasts were tested in large-scale production of cloned pigs, including fetal fibroblasts (FFBs) from four genetically similar Western swine breeds of Pietrain (P), Duroc (D), Landrace (L), and Yorkshire (Y), which are referred to as P,D,LY-FFBs, adult fibroblasts (AFBs) from the same four breeds, which are designated P,D,L,Y-AFBs, and AFBs from a Chinese pig breed of Laiwu (LW), which is referred to as LW-AFBs. Within each donor fibroblast cell type group, five transferred cloned embryo number groups were tested. In each embryo number group, 150-199, 200-249, 250-299, 300-349, or 350-450 cloned embryos were transferred to each individual recipient sow. For the entire experiment, 92,005 cloned embryos were generated from nearly 115,000 matured oocytes and transferred to 328 recipients; in total, 488 cloned piglets were produced. The results showed that the mean clones born healthy per litter resulted from transfer of embryos cloned from LW-AFBs (2.53 -0.34) was similar with that associated with P,D,L,Y-FFBs (2.72 -0.29), but was significantly higher than that resulted from P,D,L,Y-AFBs (1.47 -0.18). Use of LW-AFBs as donor cells for SCNT resulted in a significantly higher pregnancy rate (72.00% vs. 59.30% and 48.11%) and delivery rate (60.00% vs. 45.93% and 35.85%) for cloned embryo recipients, and a significantly higher birth rate of healthy clones (0.5009% vs. 0.3362% and 0.2433%) than that resulting from P,D,L,Y-AFBs and P,D,L,Y-FFBs. This suggests that using LW-AFBs as donor cells results in a higher cloning efficiency in pigs, compared with the other two donor fibroblast cell types. The birth rate of healthy clones was significantly improved when the number of transferred cloned embryos was increased from 150-199 to 200-450 per recipient. However, increase of the number of transferred embryos from 200-249 to 250-450 per surrogate did not change the birth rate of healthy clones. This suggests that transfer of excessive (250-450) cloned embryos to an individual surrogate is not necessary for increasing the cloning efficiency in pigs, and the relatively optimal number of reconstructed embryos transferred to individual recipient is 200-249. Furthermore, our results indicated that the numbers of total born clones, clones born alive, and clones born healthy per litter have a significantly high positive correlation with each other. The present study provi...

Bone Marrow Mesenchymal Stem Cells Are an Attractive Donor Cell Type for Production of Cloned Pigs As Well As Genetically Modified Cloned Pigs by Somatic Cell Nuclear Transfer

“…Our result showed that the cloning efficiency associated with MSCs (0.9%) is significantly higher than that resulting from using Fs as donor cells for cloning (0%). Moreover, our study showed that all the cloned piglets derived from MSCs were born alive and did not exhibit any visible defects, which usually could be seen in piglets cloned from Fs (Li et al, 2013;Schmidt et al, 2010). This strongly suggests that MSCs can serve as suitable donor cells that could be more efficiently reprogrammed to produce cloned pigs through SCNT, as compared with fibroblasts.…”

“…The SCNT experiments for both fibroblasts and MSCs were performed under the same conditions and following the protocol described by us (Li et al, 2013). Briefly, each batch of & 500-600 matured porcine oocytes was randomly separated into two equal portions; one portion was used for SCNT with fibroblasts and the other portion was used for SCNT with the same donor pig's MSCs.…”

“…Transfer of MSCs-embryos and Fs-embryos reconstructed with a same batch of oocytes to recipients were performed under the same conditions and following the protocol described by us (Li et al 2013). Briefly, MSCs-embryos and Fsembryos reconstructed with a same batch of oocytes were transferred to recipient sows (about 250 embryos/recipient) raised in the same swine farm and with similar physiological conditions.…”

“…derived from Yorkshire MSCs were born alive and did not show visible defects, which usually could be found in piglets cloned from Fs (Li et al, 2013;Schmidt et al, 2010). In addition, use of Yorkshire MSCs as donor cells for production of transgenic pigs carrying the Xylanase gene resulted in a transgenic efficiency at 0.42% ( = 2/ 478 = number of born transgenic cloned pigs/number of transferred embryos).…”

Identification of SSEA-1 expressing enhanced reprogramming (SEER) cells in porcine embryonic fibroblasts

“…Furthermore, we also investigated whether differences in these cell populations exist between pEFs derived from different pig breeds, as recent research revealed that pEFs from different breeds affected somatic cell nuclear transfer efficiency. 24 In this case, it was shown that pEFs derived from the Chinese Laiwu pig resulted in significantly improved reprogramming compared with pEFs derived from other pig breeds.…”