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Pig oocytes have been used increasingly for in vitro production techniques in recent years. The slaughterhouse-derived oocytes that are often used are mostly of prepubertal origin. The aims of the present study were to compare the developmental competence between pre- and postpubertal pig oocytes, and to develop a simple and practical method for the selection of prepubertal pig oocytes for parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) based on oocyte morphology after IVM and oocyte inside zona pellucida (ZP) diameter ('small' ≤110 µm; 'medium' >110 µm; 'large' ≥120 µm). Meiotic competence and blastocyst rates after PA and SCNT of prepubertal oocytes increased with oocyte size, with the large prepubertal oocytes reaching a level similar to postpubertal oocytes after SCNT. Blastocyst cell number was not related to oocyte inside ZP diameter and oocyte donor to the same extent as blastocyst rate. Very low blastocyst rates were obtained after PA of morphologically bad pre- and postpubertal oocytes. In conclusion, measurement of inside ZP diameter combined with morphological selection is useful to remove incompetent oocytes. Further studies are needed to clarify the relative importance of cytoplasmic volume and stage in oocyte growth phase. Oocytes from sexually immature pigs are used for in vitro production techniques studying, for example, human diseases. The objective of the present study was met by developing a method for the selection of prepubertal oocytes with high developmental competence by measuring the oocyte's diameter in combination with morphological evaluation. The study is important for understanding oocyte growth and development and for increasing the efficiency of in vitro work. [ABSTRACT FROM AUTHOR]

Extracts from eggs of Xenopus laevisfrogs can induce nuclear remodelling or increase transcriptional reprogramming in somatic cells. However, it is not known if this effect is passed on from one cell generation to another, or how it affects somatic cell nuclear transfer in porcine cells. This study aimed to investigate the effect of extract-treated cells over several generations on porcine cloning. Extracts were prepared from 2 frogs (B1 and B2) by the same protocol (Higa et al.2006 Methods 39, 284–290). Fetal fibroblasts grown on poly-L-lysine coated coverslips were permeabilized by digitonin (7μgmL-1, 2min, 4°C) and incubated with 1 extract batch at 37°C for 30min. After resealing the membrane in DMEM supplemented with 2mM CaCl2at 37°C for 2h, the remaining cells were cultured in ES medium (Vejlsted et al.2005 Mol. Reprod. Dev. 70, 445–454) for 7 to 8 days when they formed colonies. The colonies were trypsinized and divided onto 2 coverslips for subculture, defined as Experimental Passage 1 (XP1). New subcultures were made every 7 to 8 days when 70 to 80% clusters become colonies until XP15. Colonies from XP3, 8 and 15 were isolated and trypsinized before being used in handmade cloning. Nontreated cells grown in DMEM were used as controls (no colony formation was observed). On each cloning day, cells from different XP number and controls were used. Rates of cleavage (Day 2) and blastocyst development (Day 6) were analysed with chi-square test (SAS version 9.2, SAS Institute Inc., Cary, NC, USA). Results are summarised in Table 1. No difference was observed in cleavage rate between groups. Blastocyst rates of all XP colony cells were significantly higher than their controls. For the same XP number and their controls, blastocyst rates were similar between the colony cells from the 2 extract batches, and there was no difference between their controls, either. In conclusion, the cloning efficiency in porcine cells could be increased with extract-treated cells used for several generations, and this effect was present at XP3, 8, and 15. Table 1.Developmental competence of cloned porcine embryos with extract-treated cells from different batches of extract (B1 and B2) and Experimental Passage (XP) numbers [ABSTRACT FROM AUTHOR]

Extract from Xenopus eggs can induce reprogramming in somatic cells. In our previous study, cell colony formation was induced during culture of porcine fetal fibroblasts after a single treatment with Xenopus egg extract and culture for several passages and using these long-term cultured cells for cloning increased the resulting blastocyst rate (Liu et al. 2011 Reprod. Fertil. Dev. 23, 130). However, both colony number and cloned blastocyst rate decreased after Passage 15 and no colonies formed after Passage 18. Therefore, in this study we investigated the effect of a second extract treatment on colony formation and cloned blastocyst formation. Extract-treated (ExT) porcine fetal fibroblasts at Passage 13 (P13) grown on poly-L-lysine-coated coverslips were permeabilized by digitonin (7 μg mL-1, 2 min, 4°C) and incubated in extract at 37°C for 30 min. After resealing the membrane in DMEM supplemented with 2 mM CaCl2, the remaining cells were cultured in ES medium (Vejlsted et al. 2005 Mol. Reprod. Dev. 70, 445). The treated cells were split onto 2 coverslips on Day 7 after the second extract treatment (2ExT), defined as Passage 1 (2ExT P1, comparable with ExT P14). New subcultures were made every 7 to 8 days when 70 to 80% clusters became colonies (i.e. 2ExT P8). Colony cells from both ExT (P14 and P16) and 2ExT (P1, P3 and P6) were used for handmade cloning and nontreated cells were used as control (Day 0). Blastocyst rates were analysed by chi-square test and colony numbers were analysed by 1-way ANOVA (SAS version 9.2). Colony numbers and cloned blastocyst rates on Day 6 are summarised in Table 1. Colonies continued to form in treated cells from 2ExT P1 to P8. The colony number maintained at a high level (60 to 80) from 2ExT P4 to P8 and it was significantly higher than that of ExT cells at the comparable passage numbers. No colonies formed in control cells. When using 2ExT colony cells at P3 and P6 for cloning, the blastocyst rates increased compared with controls and they were also higher than in the ExT group. Cloned blastocyst rates were not different between 2ExT P1 and ExT P14 groups. In conclusion, a second extract treatment can induce colony formation and increase cloned blastocyst rates, indicating that this repeated extract treatment again could activate the extract-treated cells to an activity level similar to that achieved after the first treatment. Table 1. Summary of colony number and cloned blastocyst rate with ExT and 2ExT colony cells [ABSTRACT FROM AUTHOR]

One of the primary factors influencing the developmental ability of cloned embryos is the oocyte′s diameter (Hirao et al.1994 J. Reprod. Fertil. 100, 333–339). However, the oocyte donor''s age (i.e. its sexual maturity) is also important to consider, because a high proportion of immature oocytes can be expected (Ikeda and Takahashi 2003 Reprod. Fertil. Dev. 15, 215–221). The present study was to investigate the effect of diameter of oocytes collected from prepubertal gilts weighing 100 to 120kg on the developmental ability of cloned and parthenogenetically activated (PA) embryos. Cumulus–oocyte complexes collected from ovaries of prepubertal gilts were in vitromatured for 42 to 44h as described for sow oocytes (Li et al.2008 Theriog 70, 800–808). After removal of the cumulus cells, the matured oocytes were sorted into 2 groups based on visual inspection: large (L) and small (S) oocytes, whereas non-sorted oocytes were used as control (C). In addition, 1 batch from each of the 3 groups of oocytes had their mean size measured. Subsequently, all 3 groups were used for handmade cloning (HMC; Li et al.2009 Reprod. Domest. Anim. 44, 122–127) or parthenogenetic activation (PA; Kragh et al.2005 Theriogenology 64, 1536–1545). Then a chemical activation with 5μgmL-1cytochalasin B and 10μgmL-1cycloheximide in PZM-3 medium was applied for 4h on both HMC and PA embryos. Finally, the activated embryos were washed and cultured in PZM-3 medium using the WOW system. The embryo development was evaluated by cleavage rate (Day 2), blastocyst rate (Day 6), and total cell number in blastocysts. Data were analysed by ANOVA with single factor in Excel (Microsoft Excel 2007, Redmond, WA, USA). The results showed (Table 1) that by simple visual observation, oocytes could be easily sorted into the following groups: L group (mean diameter 110μm, from 105 to 116μm), S group (mean diameter 101μm, from 93 to 106μm) and C group (mean diameter 107μm, from 93 to 116μm). Cleavage rates and total cell number were similar in the 3 groups. However, the blastocyst rate in L group either for HMC or PA was higher than S group. The data confirm that prepubertal gilt oocytes are useful for cloning and PA, but developmental rates can be increased by selection of large oocytes by simple visual observation. Table 1.Data analysis results [ABSTRACT FROM AUTHOR]

Pretreatment of somatic cells to promote subsequent reprogramming during somatic cell nuclear transfer (SCNT) may significantly improve efficiency of the technique. The aim of this study was to evaluate the effect of Xenopus laevisegg extract pretreatment of porcine fetal fibroblast cells using different permeabilization agents prior to SCNT. Fibroblasts were permeabilized using streptolysin O (SLO; 300 ng mL-1, 30 min, 37°C) or digitonin (7 μg mL-1, 2 min, 4°C), and exposed to egg extract for 1 h or 0.5 h, respectively. Cell membranes were resealed in DMEM supplemented with 2 mMCaCl2for 2 h. After culture for 1, 3, and 5 days (for SLO) or 3 and 5 days (for digitonin), the SLO extract-treated cells (SETC) and digitonin extract-treated cells (DETC) were used as donor karyoplasts for handmade cloning. Controls were SCNT with nontreated cells. Embryos were evaluated for cleavage rate (Day 2), blastocyst rate (Day 6), and total cell numbers of blastocysts. Statistical differences were analyzed by ANOVA. Results are summarized in Table 1. When SETC were used as donors, blastocyst rates were significantly lower compared with the controls, except when the donor cells were cultured for 3 days after treatment. Blastocysts of the latter group also had higher total cell number. With DETC as donors, blastocyst rates and total cell number of embryos at Day 6 reconstructed with cells cultured for 5 days were higher than those in other groups. Results indicate that extract treatment of the donor cells after SLO-permeabilization can give higher number of cells in cloned blastocysts but not improve overall embryo development. However, digitonin treatment for donor cell permeabilization improved both embryo development and cell number of blastocyst. The latter effect was detected only 5 days after the treatment. In conclusion, qualitative efficiency of porcine SCNT could be improved with a combined donor cell permeabilization and extract treatment. Table 1.Effect of different permeabilization agents prior to SCNT [ABSTRACT FROM AUTHOR]