Developmental potential of pig embryos reconstructed by use of sow versus pre-pubertal gilt oocytes after somatic cell nuclear transfer

Abstract

In this study, the developmental ability of cloned embryos using gilt versus sow oocytes was evaluated under the hypothesis that the efficiency of nuclear transfer using gilt oocytes was lower than that of sow oocytes, but that it could be optimized. Five experiments were performed with routine production of cloned embryos with sow oocytes serving as the control. Results showed that: Experiment 1: Blastocyst rates of cloned embryos with gilt oocytes was about half compared with control. Experiment 2: An extended maturation time of 48 h used for gilt oocytes resulted in lower blastocyst rates after cloning. Experiment 3: Development of cloned embryos with gilt oocytes was improved by co-culture with sow oocytes. Experiment 4: After maturation of gilt oocytes using follicular fluid from gilt instead of sow, the oocytes were sorted into large and small oocytes, and after cloning, blastocyst rates were higher using large gilt oocytes compared with small oocytes; however, the rate remained lower compared with control. Experiment 5: Six sow recipients received a total of 503 morulae and blastocysts cloned from gilt oocytes (four recipients) and 190 cloned from sow oocytes (two recipients). All recipients became pregnant and went to term, resulting in 26 (gilt oocytes) and six (sow oocytes) piglets. In conclusion, results confirmed that nuclear transfer efficiency was higher using sow versus gilt oocytes, but the use of gilt oocytes can be optimized by sorting after ooplasm size following maturation and by maturing gilt and sow oocytes together.

Porcine oocyte mtDNA copy number is high or low depending on the donor

“…The same principle was used by Iwata et al (2011), demonstrating a positive correlation between mtDNA copy number and abnormal fertilization in older cows. In our study, there were no indications of mtDNA copy number being related to decreased developmental competence of small oocytes/follicles (Marchal et al , 2002; Bagg et al , 2007) and prepubertal oocytes (Ikeda & Takahashi 2003; Li et al , 2013; Pedersen et al , 2014). These examples illustrate the need for further research of the importance of mtDNA copy number and the complex mechanisms regulating the mitochondrial complement of an oocyte.…”

Generation and characterization of a novel knockin minipig model of Hutchinson-Gilford progeria syndrome

“…HGPS knockin Yucatan minipigs carrying a heterozygous LMNA c.1824C > T mutation were generated using CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) gene editing 25,26 in male Yucatan minipig skin fibroblasts, followed by somatic cell nuclear transfer (SCNT) by handmade cloning to enucleated oocytes from Large White sows 27 (Supplementary Fig. 2a).…”

“…Moreover, this HGPS cell clone showed no evidence of off-target Cas9 effects or random integration of the vectors used (sgRNA, Cas9, EGFP-N3, and the donor molecule backbone). The clone was therefore used as the nuclear donor for SCNT into enucleated oocytes from Large White sows 27 . Viable reconstructed embryos were transferred to three surrogate Large White sows 29 .…”

A genetically modified minipig model for Alzheimer’s disease with SORL1 haploinsufficiency

“…Two of the validated SORL1 3′ KO/ SORL1 5′ KO gene edited cell clones were used as nuclear donor cells for somatic cell nuclear transfer (SCNT) by handmade cloning as previously described. 75 Cumulus-oocyte complexes harvested from slaughterhouse-derived sow ovaries were in-vitro matured and treated to remove cumulus cells and partially zonae pellucidae. The oocytes were bisected manually, and the cytoplasts without chromatin were collected.…”