Progesterone and Androstenedione Are Important Follicular Fluid Factors Regulating Porcine Oocyte Maturation Quality.

Oocytes matured in vitro are useful for assisted human and farm animal reproduction. However, the quality of in vitro matured oocytes is usually lower than that of in vivo matured oocytes, possibly due to the absence of some important signal regulators in vitro. In this study, untargeted metabolomics was used to detect the changes in the metabolites in the follicular fluid (FF) during in vivo pig oocyte maturation and in the culture medium during in vitro maturation. Our results showed that the total metabolite changing profile of the in vivo FF was different from that of the in vitro maturation medium, but the levels of 23 differentially expressed metabolites (DEMs) changed by following the same trend during both in vivo and in vitro pig oocyte maturation. These 23 metabolites may be important regulators of porcine oocyte maturation. We found that progesterone and androstenedione, two factors in the ovarian steroidogenesis pathway enriched from the DEMs, were upregulated in the FF during in vivo pig oocyte maturation. The levels of these two factors were 31 and 20 fold, respectively, and they were higher in the FF than in the culture medium at the oocyte mature stage. The supplementation of progesterone and androstenedione during in vitro maturation significantly improved the pig oocyte maturation rate and subsequent embryo developmental competence. Our finding suggests that a metabolic abnormality during in vitro pig oocyte maturation affects the quality of the matured oocytes. This study identified some important metabolites that regulate oocyte maturation and their developmental potential, which will be helpful to improve assisted animal and human reproduction.

Source and Follicular Fluid Treatment During the In Vitro Maturation of Recipient Oocytes Affects the Development of Cloned Pig Embryo.

Pig cloning technique is valuable in agriculture, biomedicine, and life sciences. However, the full-term developmental efficiency of cloned pig embryos is only about 1%, which limits pig cloning application. The quality of recipient oocytes greatly affects the developmental competence of cloned pig embryos. Thus, this study investigated the effects of a recipient oocyte source (in vivo matured [IVVM] oocytes vs. slaughter house-derived in vitro matured [IVTM] oocytes), and follicular liquid treatment (slaughter house-derived immature follicle-derived fluid [IFF] vs. in vivo-matured follicle-derived fluid [MFF]) during the in vitro maturation (IVM) of oocytes on the development of the cloned pig embryos. Our results showed that using IVVM oocytes to replace IVTM oocytes as recipient oocytes, and using 10% MFF IVM medium to replace 10% IFF IVM medium could enhance the development of the cloned pig embryos. IFF and MFF contained different levels of oocyte quality-related proteins, resulting in different oocyte quality-related gene expression levels and reactive oxygen species levels between the 10% MFF medium-cultured oocytes and 10% IFF medium-cultured oocytes. This study provided useful information for enhancing the pig cloning efficiency by improving the quality of recipient oocytes.

Influence of embryo handling and transfer method on pig cloning efficiency.

The somatic cell nuclear transfer (SCNT) technique could be used to produce genetically superior or genetically engineered cloned pigs that have wide application in agriculture and bioscience research. However, the efficiency of porcine SCNT currently is very low. Embryo transfer (ET) is a key step for the success of SCNT. In this study, the effects of several ET-related factors, including cloned embryo culture time, recipient's ovulation status, co-transferred helper embryos and ET position, on the success rate of pig cloning were investigated. The results indicated that transfer of cloned embryos cultured for a longer time (22-24h vs. 4-6h) into pre-ovulatory sows decreased recipient's pregnancy rate and farrowing rate, and use of pre-ovulatory and post-ovulatory sows as recipients for SCNT embryos cultured for 22-24h resulted in a similar porcine SCNT efficiency. Use of insemination-produced in vivo fertilized, parthenogenetically activated and in vitro fertilized embryos as helper embryos to establish and/or maintain pregnancy of SCNT embryos recipients could not improve the success rate of porcine SCNT. Transfer of cloned embryos into double oviducts of surrogates significantly increased pregnancy rate as well as farrowing rate of recipients, and the developmental rate of transferred cloned embryos, as compared to unilateral oviduct transfer. This study provided useful information for optimization of the embryo handling and transfer protocol, which will help to improve the ability to generate cloned pigs.

Effects of donor fibroblast cell type and transferred cloned embryo number on the efficiency of pig cloning.

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 provides useful information for improving SCNT efficiency in pigs.