序贯培养液G1/G2对山羊核移植胚胎发育和质量的影响

为了优化山羊核移植胚胎体外培养体系,提高核移植效率,本研究检测了山羊体细胞核移植(SCNT)胚胎在序贯培养液G1/G2中的发育率和囊胚细胞凋亡,以及核移植胚胎移植后的妊娠率,以传统mSOF-FBS培养液作为对照组,评估序贯培养液G1/G2支持山羊核移植胚胎的发育能力。结果显示,与对照组相比,G1/G2组的囊胚发育率差异不显著((27.7±3.1)%vs(25.3±1.0)%,P>0.05),囊胚细胞数和囊胚细胞凋亡率显著降低(分别为(93.2±4.5)vs(109.1±6.2)和(4.9±0.2)%vs(11.3±0.1)%,P<0.05),但移植后的妊娠率显著增高(21.4%vs 8.0%,P<0.05)。结果表明,与传统的培养液mSOF-FBS相比,序贯培养液G1/G2能更好地支持山羊核移植胚胎的发育。     

Effect of mSOF and G1.1/G2.2 Media on the Developmental Competence of SCNT‐Derived Bovine Embryos

The objective of this study was to compare the effect of two culture media: modified synthetic oviductal fluid (mSOF) and G1.2/G2.2, on the developmental competence of bovine somatic cell–cloned embryos. Cloned embryos were produced by transferring adult skin fibroblasts into enucleated MII oocytes. After activation, the reconstructed embryos were randomly allotted to either mSOF or G1.2/G2.2 for culture (the embryos were transferred from G1.2 to G2.2 on days 3 of culture). The development competence of cloned embryos in these two culture systems was compared in terms of cleavage rate, blastocyst formation rate and apoptosis cell number in day 7 blastocyts. To investigate the in vivo developmental competence of cloned embryos in the two culture systems, a total of 87 and 104 blastocysts derived from mSOF and G1.2/G2.2 medium groups were transferred individually to recipient Angus cows, respectively. No differences were observed in terms of cleavage rate, day 7 blastocyst rate and blastocyst cell number between these two culture systems. However, the day 6 blastocyst formation rate was significantly higher in G1.2/G2.2 than that in mSOF. In addition, blastocysts cultured in mSOF have a higher percentage of apoptotic blastomeres compared to those in G1.2/G2.2 (8.5 ± 1.2 vs 16.8 ± 1.5, p < 0.05). Although difference in pregnancy rate was not observed 40 days after embryo transfer, significantly higher pregnancy rate was observed in G1.2/G2.2 group after 90 days of embryo transfer (12.4% vs 37.5%, p < 0.05). Moreover, calving rate was significantly improved in G1.2/G2.2 group compared to mSOF group (27.9% vs 6.7%, p < 0.05). In conclusion, our results indicate that G1.2/G2.2 can improve developmental competence of bovine SCNT embryos both in vitro and in vivo, which is more suitable for culture of bovine SCNT embryos than mSOF medium.     

Cloned calves derived from somatic cell nuclear transfer embryos cultured in chemically defined medium or modified synthetic oviduct fluid

Somatic cell nuclear transfer (SCNT) is considered to be a critical tool for propagating valuable animals. To determine the productivity calves resulting from embryos derived with different culture media, enucleated oocytes matured in vitro were reconstructed with fetal fibroblasts, fused, and activated. The cloned embryos were cultured in modified synthetic oviduct fluid (mSOF) or a chemically defined medium (CDM) and developmental competence was monitored. After 7 days of culturing, the blastocysts were transferred into the uterine horn of estrus-synchronized recipients. SCNT embryos that were cultured in mSOF or CDM developed to the blastocysts stages at similar rates (26.6% vs. 22.5%, respectively). A total of 67 preimplantational stage embryos were transferred into 34 recipients and six cloned calves were born by caesarean section, or assisted or natural delivery. Survival of transferred blastocysts to live cloned calves in the mSOF and the CDM was 18.5% (to recipients), 9.6% (to blastocysts) and 42.9% (to recipients), 20.0% (to blastocysts), respectively. DNA analysis showed that all cloned calves were genetically identical to the donor cells. These results demonstrate that SCNT embryos cultured in CDM showed higher viability as judged by survival of the calves that came to term compared to blastocysts derived from mSOF cultures.     

Effects of synchronization of donor cell cycle on embryonic development and DNA synthesis in porcine nuclear transfer embryos

The relationship between donor cell cycle and the developmental ability of somatic cell nuclear transfer (SCNT) embryos has not fully been elucidated. Donor cells that are usually prepared by serum starvation or confluent-cell culture for SCNT represent a heterogeneous population that includes mainly G0 phase cells, other cells in different phases of the cell cycle and apoptotic cells. In this study, we compared the developmental ability of porcine SCNT embryos reconstructed from G0 phase cells (G0-SCNT embryos) and strictly synchronized-G1 phase cells (G1-SCNT embryos), and examined the developmental rates and timing of first DNA synthesis. The G0 phase cells were synchronized by confluent culture, and the G1 phase cells were prepared from actively dividing M phase cells. The G1-SCNT embryos showed a significantly higher (P<0.05) developmental rate to the blastocyst stage per cleaved embryo (59%) than the G0-SCNT embryos (43%). Moreover, initiation of first DNA synthesis and cleavage occurred significantly earlier in the G1-SCNT embryos than in the G0-SCNT embryos. Delay of initiation of first DNA synthesis in the SCNT embryos by aphidicolin resulted in decreased developmental rates to the blastocyst stage without any effect on cleavage rates. Our data demonstrates that synchronized-G1 phase cells can be used as donor cells for SCNT embryos and that earlier initiation of first DNA synthesis may be important for subsequent development of SCNT embryos. The SCNT system using G1-synchronized cells, in terms of their highly uniform and viable cell states, can be useful for studying the reprogramming processes and embryonic development of SCNT embryos.     

Production of blastocysts after intergeneric nuclear transfer of goral (Naemorhedus goral) somatic cells into bovine oocytes

Interspecies cloning may be a useful method to help conserve endangered species and to study nuclear-cytoplasm interaction. The present study investigated in vitro development of goral (Naemorhedus goral) intergeneric nuclear transfer embryos produced by fusing goral fibroblasts with enucleated metaphase II (MII) bovine oocytes. After two to five passages, serum-starved or non-starved goral skin fibroblast cells were transferred into enucleated MII bovine oocytes. Couplets were electrically fused and chemically activated, and then cultured in either modified synthetic oviduct fluid (mSOF) or tissue culture medium-199 (TCM-199) supplemented with 10% FBS. Serum starvation of donor cells did not affect the fusion rate and or development to of cells to the two-cell stage, to more than 9-cells, or to morulae, regardless of culture medium. Three blastocysts from 202 fused embryos were obtained when embryos reconstructed with non- serum- starved donor cells were cultured in mSOF. However, no blastocysts were obtained when the embryos reconstructed with serum-starved donor cells were cultured in mSOF. The total cell number of goral intergeneric embryos averaged 130.3 (range 105-180). In conclusion, this study demonstrated that bovine oocytes can support blastocyst development after intergeneric SCNT with goral fibroblasts.     

Treatment of porcine oocytes with MEM vitamins during in vitro maturation improves subsequent blastocyst development following nuclear transfer

This study was carried out to investigate the effects of minimum essential medium (MEM) vitamins during in vitro maturation (IVM)/in vitro culture (IVC) of porcine nuclear transfer (NT) embryos on subsequent developmental capacity in vitro. Porcine cumulus-oocyte complexes (COCs) were divided into five groups, matured for 44 h in maturation medium with various concentrations of MEM vitamins (0, 0.05, 0.1, 0.2 and 0.4%), and observed for maturation rate. Also, COCs were matured in NUSU-23 media without MEM vitamins for 44 h and cultured in PZM-3 media with various concentrations of MEM vitamins (0, 0.05, 0.4 and 1.0%) for 6 days following nuclear transfer. Factorial (IVM/IVC) experiments were also performed in NCSU-23 medium with or without 0.05% MEM vitamins and PZM-3 medium with or without 0.4% MEM vitamins. They were then tested by examining in vitro development of the porcine reconstructed embryos. The maturation rates of the COCs treated with the MEM vitamins did not differ significantly among the MEM vitamin-treated groups. Addition of vitamins to culture medium did not affect development of porcine reconstructed embryos in vitro. However, addition of low concentrations of MEM vitamins only to maturation medium increased (P<0.05) the proportion of NT embryos developing into blastocysts compared with the control group. Addition of MEM vitamins to IVC medium did not enhance the developmental rate compared with the control group. Thus, addition of MEM vitamins to IVM medium could improve subsequent blastocyst development of porcine NT embryos.     

猪植入前胚胎体外培养条件的优化

探讨了更换胚胎培养液及添加FBS、高渗透压和不同浓度VE对猪卵母细胞体外受精(IVF)和孤雌激活(PA)胚胎体外发育的影响,进一步优化了猪植入前胚胎体外培养体系。试验一：在第2天、第4天更换新的培养液(换液组),在换液基础上第4天更换为添加10%FBS的培养液(FBS组)。试验二：胚胎分别在0.05 mol/L蔗糖(蔗糖组)和138 mmol/L氯化钠(氯化钠组)的PZM-3(300～320 mOsmol)中培养2 d后移至PZM-3(288 mOsmol)中培养5 d。试验三：在培养液中分别添加50、100和200 μmol/L VE。对照组均在PZM-3(288 mOsmol)中培养7 d。结果表明：试验一,IVF和PA胚胎FBS组囊胚率显著高于对照组和换液组(P<0.05);试验二,IVF胚胎氯化钠组卵裂率、囊胚率均显著高于对照组与蔗糖组(P<0.05);试验三,IVF胚胎添加100 μmol/L VE组囊胚率显著高于对照组(P<0.05)。结果提示,在换液的基础上添加FBS有利于猪IVF和PA胚胎的体外发育;氯化钠调节的高渗透压可以促进猪IVF胚胎的早期发育;添加100 μmol/L VE可以改善猪IVF胚胎的体外发育体系。     

Effect of synchronization of donor cells in early G1‐phase using shake‐off method on developmental potential of somatic cell nuclear transfer embryos in cattle

In this study, we compared the developmental ability of somatic cell nuclear transfer (SCNT) embryos reconstructed with three bovine somatic cells that had been synchronized in G0‐phase (G0‐SCNT group) or early G1‐phase (eG1‐SCNT group). Furthermore, we investigated the production efficiency of cloned offspring for NT embryos derived from these donor cells. The G0‐phase and eG1‐phase cells were synchronized, respectively, using serum starvation and antimitotic reagent treatment combined with shaking of the plate containing the cells (shake‐off method). The fusion rate in the G0‐SCNT groups (64.2 ± 1.8%) was significantly higher than that of eG1‐SCNT groups (39.2 ± 1.9%) (P < 0.05), but the developmental rates to the blastocyst stage of SCNT embryos per fused oocytes were similar for all groups. The overall production efficiency of the clone offspring in eG1‐SCNT groups (12.7%) per recipient cow was higher than that in G0‐SCNT groups (3%) (P < 0.05). The mean birth weight of cloned calves and the average calving score in the G0‐SCNT groups (48.1 ± 3.4 kg and 3.3 ± 0.3, respectively) was significantly higher (P < 0.05) than those of eG1‐SCNT groups (37.2 ± 2.1 kg and 2.3 ± 0.2, respectively). Results of this study indicate that synchronization of donor cells in eG1‐phase using the shake‐off method improved the overall production efficiency of the clone offspring per transferred embryo.     

Efficiency of Two Enucleation Methods Connected to Handmade Cloning to Produce Transgenic Porcine Embryos

The purpose of our work was to establish an efficient-oriented enucleation method to produce transgenic embryos with handmade cloning (HMC). After 41–42 h oocytes maturation, the oocytes were further cultured with or without 0.4 μg/ml demecolcine for 45 min [chemically assisted handmade enucleation (CAHE) group vs polar body (PB) oriented handmade enucleation (OHE) group respectively]. After removal of the cumulus cells and partial digestion of the zona pellucida, oocytes with visible extrusion cones and/or polar bodies attached to the surface were subjected to oriented bisection. Putative cytoplasts without extrusion cones or PB were selected as recipients. Two cytoplasts were electrofused with one transgenic fibroblasts expressing green fluorescent protein (GFP), while non-transgenic fibroblasts were used as controls. Reconstructed embryos were cultured in Well of Wells (WOWs) with porcine zygote medium 3 (PZM-3) after activation. Cleavage and blastocyst rates were registered on day 2 and day 7 of in vitro culture respectively. Meanwhile, the total blastocyst cell number was counted on day 7. We found that the difference was only observed between blastocyst rates (38.6 ± 2% vs 48.1 ± 3%) of cloned embryos with GFP transgenic fibroblast cells after CAHE vs OHE. With adjusted time-lapse for zonae-free cloned embryos cultured in WOWs with PZM-3, it was obvious that in vitro developmental competence after CAHE was compromised when compared with the OHE method. OHE enucleation method seems to be a potential superior alternative method used for somatic cell nuclear transfer (SCNT) with transgenic fibroblast cells.     

In vitro development and postvitrification survival of cloned feline embryos derived from preadipocytes

The aim of the present study was to optimize the conditions for in vitro development and postvitrification survival of somatic cell cloned feline embryos. To determine the effects of cell cycle synchronization of the nuclear donor cells, we cultured preadipocytes under serum starvation or conventional conditions. After two days in serum starvation culture, the proportion of synchronized donor cells at the G0/G1 phase was 91.6%. This was significantly higher than the proportion of non-synchronized cells in the proliferative phase (72.6%, P<0.05). The in vitro development of somatic cell nuclear transfer (SCNT) embryos reconstructed using donor cells treated under serum starvation conditions (normal cleavage rate of 65.7%, 46/70, and blastocyst formation rate of 20.0%, 14/70) was comparable to that of the serum supplemented group (52.5%, 31/59, and 20.3%, 12/59). Use of in vitro or in vivo matured oocytes as recipient cytoplasts equally supported development of the SCNT embryos to the blastocyst stage (11.9%, 5/42, vs. 9.5%, 2/21). SCNT-derived blastocysts were vitrified using the original minimum volume cooling (MVC) or the modified (stepwise) MVC method. Although none (n=10) of the SCNT blastocysts survived following vitrification by the original MVC method, the stepwise MVC method resulted in 100% survival after rewarming (n=11). In conclusion, we demonstrated that feline somatic cell cloned embryos with a high developmental ability can be produced irrespective of cell cycle synchronization of donor cells using either in vivo or in vitro matured oocytes. Furthermore, by utilizing a stepwise vitrification method, we showed that it is possible to cryopreserve cloned feline blastocysts.     

Optimization of parthenogenetic activation of rabbit oocytes and development of rabbit embryo by somatic cell nuclear transfer

The present study explored a suitable parthenogenetic activation (PA) procedure for rabbit oocytes and investigated the developmental potential of somatic cell nuclear transfer (SCNT) embryos using rabbit foetal fibroblasts (RFFs). The electrical activation had the optimal rate of blastocyst (14.06%) when oocytes were activated by three direct current (DC) pulses (40 V/mm, 20 μs each) followed by 6‐dimethylaminopurine (6‐DMAP) and cycloheximide (CHX) treatment; the blastocyst rate of ionomycin (ION) + 6‐DMAP + CHX (12.07%) activation was higher than that of ION + 6‐DMAP (8.6%) activation or ION + CHX (1.24%) activation; there was no significant difference in blastocyst rate between ION + 6‐DMAP + CHX and DC + 6‐DMAP + CHX groups. The blastocyst rate of ION + 6‐DMAP + CHX‐activated oocytes in the basic rabbit culture medium (M‐199) + 10% foetal bovine serum (FBS; 14.28%) was higher than that in buffalo conditioned medium (5.75%) or G1/G2 medium (0), and the blastocyst rate was increased when M‐199 + 10% FBS was supplemented with amino acids. Refreshing culture medium every day or every other day significantly increased the blastocyst rate. Treatment of donor cells with 0.5% FBS for 3–5 days increased blastocyst rate of SCNT embryos (33.33%) than no serum starvation (22.47%) or 0.5% FBS treatment for 6–9 days (23.61%); the blastocyst rate of SCNT embryos derived from nontransgenic RFFs was higher than that derived from transgenic RFFs by electroporation. The blastocyst development ability of SCNT embryos derived from RFFs by electroporation (32.22%) was higher than that of liposome (19.11%) or calcium phosphate (20.00%) transfection, and only the embryos from electroporation group have the EGFP expression (24.44%). In conclusion, this study for the first time systematically optimized the conditions for yield of rabbit embryo by SCNT.     

Extended exposure to trichostatin A after activation alters the expression of genes important for early development in nuclear transfer murine embryos

The low viability of embryos reconstructed by somatic cell nuclear transfer (SCNT) is believed to be associated with epigenetic modification errors, and reduction of those errors may improve the viability of SCNT embryos. The present study shows the effect of trichostatin A (TSA), a strong inhibitor of histone deacetylase, on the development of murine SCNT embryos. After enucleation and nuclear injection, reconstructed murine oocytes were activated with or without TSA for 6 hr (TSA-6 hr). After activation, TSA treatment was extended to 3 hr (TSA-9 hr), 5 hr (TSA-11 hr) and 18 hr (TSA-24 hr) during culture. As a result, the SCNT embryos in the TSA-11 hr group showed a remarkably higher blastocyst rate (21.1%) when compared with the nontreated embryos (3.4%), while the concentration of TSA did not significantly affect embryonic development. The expressions of histone deacetylase (HDAC1 and HDAC2) and DNA methylation (DNMT3a and DNMT3b) genes decreased in the TSA-11 hr and TSA-24 hr groups, while there was an increase in the expression of histone acetyltransferase (P300 and CBP), pluripotency (OCT4 and NANOG) and embryonic growth/trophectoderm formation (FGF4)-related genes in the same groups. The expression of CDX2, a critical gene for trophectoderm formation was upregulated only in the TSA-24 hr group. Our results show that TSA treatment during the peri- and postactivation period improves the development of reconstructed murine embryos, and this observation may be explained by enhanced epigenetic modification of somatic cells caused by TSA-induced hyperacetylation, demethylation and upregulation of pluripotency and embryonic growth after SCNT.     

Zona-free and with-zona parthenogenetic embryo production in goat (Capra hircus) — effect of activation methods, culture systems and culture media

Studies on parthenogenetic activation of oocytes are important to improve the efficiency of nuclear transfer as artificial activation of oocytes is an essential component of nuclear transfer protocol. The present study was carried out to investigate the effects of different activation methods, culture systems and culture media on in vitro development of zona-free and with-zona parthenogenetic embryos in goat. In case of zona-free parthenogenesis, there was a significant (p < 0.05) increase in cleavage rate and blastocyst yield when oocytes were activated by electrical pulse (76.29 ± 0.52% and 19.07 ± 0.39% respectively) than when Ca-ionophore was used for activation (63.45 ± 0.73% and 14.09 ± 0.65% respectively). The quality of blastocysts was evaluated by determination of cell number and by expression profile of pluripotent related gene Oct-4. No significant (p < 0.05) difference was found in quality of blastocysts produced by different activation methods. In culturing of zona-free parthenogenetic embryos, flat surface (FS) was proved to be the best system. A significant (p < 0.05) decrease in cleavage rate and blastocyst yield was found in Microdrop culture of zona-free embryos (43.67 ± 2.08% and 0.72 ± 0.72% respectively) in comparison to WOW of zona-free embryos (73.88 ± 1.70% and 15.51 ± 1.34% respectively) and FS of zona-free (75.14 ± 0.81% and 23.93 ± 2.71% respectively) as well as with-zona (72.16 ± 1.55% and 18.16 ± 0.68%) embryos. Zona-free flat culture system yielded significantly (p < 0.05) higher blastocyst rate than zona-free WOW system as well as with-zona flat culture system. The zona-free and with-zona parthenogenetic embryos were cultured in three different media — Research Vitro Cleave media from Cook® Australia (RVCL), Embryo Development Medium (EDM) and Modified Synthetic Oviductal Fluid (mSOF). In case of zona-free parthenogenesis, significant (p < 0.05) increase was found in blastocyst development rate in RVCL medium (18.61 ± 1.52%) than EDM (11.29 ± 0.77%) or mSOF (11.53 ± 1.86%). In case of with-zona parthenogenesis, RVCL medium and EDM were found superior to mSOF. The results of the study will be helpful to improve the efficiency of nuclear transfer in goat.     

Osmolarity- and Stage-Dependent Effects of Glycine on Parthenogenetic Development of Pig Oocytes

The osmolarities of media that are most effective for in vitro culture of mammalian oocytes and embryos are lower than that of oviductal fluid. Oocytes and embryos can survive the high physiological osmolarity in vivo perhaps owing to the presence of amino acids such as glycine, which serve as organic osmolytes in the female reproductive tract. In the present study, the effects of glycine on the parthenogenetic development of pig oocytes were examined in hypotonic or isotonic media. The results showed that culturing oocytes in isotonic media improved the cleavage rates (P<0.01) at 2 days in culture but inhibited any further development beyond cleavage when compared with the hypotonic media. However, addition of 4 mM glycine to the isotonic media resulted in improved blastocyst formation rates compared with that observed in the hypotonic media (P<0.01), and there was no inhibition of development beyond the cleavage stages in oocytes. The beneficial effects of glycine were observed only when oocytes were cultured in isotonic media and glycine was added at day 2 or 3 in culture. The results from the present study indicate that an isotonic medium with glycine is useful for in vitro culture of pig oocytes and that glycine may protect pig oocytes against the detrimental effects of increased osmolarity.     

Development of interspecies cloned monkey embryos reconstructed with bovine enucleated oocytes

This study was carried out to determine whether culture media reconstructed with bovine enucleated oocytes and the expression pattern of Oct-4 could support dedifferentiaton of monkey fibroblasts in interspecies cloned monkey embryos. In this study, monkey and bovine skin fibroblasts were used as donor cells for reconstruction with bovine enucleated oocytes. The reconstructed monkey interspecies somatic cell nuclear transfer (iSCNT) embryos were then cultured under six different culture conditions with modifications of the embryo culture media and normal bovine and monkey specifications. The Oct-4 expression patterns of the embryos were examined at the two-cell to blastocyst stages using immunocytochemistry. The monkey iSCNT embryos showed similar cleavage rates to those of bovine SCNT and bovine parthenogenetic activation (PA). However, the monkey iSCNT embryos were not able to develop beyond the 16-cell stage under any of the culture conditions. In monkey and bovine SCNT embryos, Oct-4 could be detected from the two-cell to blastocyst stage, and in bovine PA embryos, Oct-4 was detectable from the morula to blastocyst stage. These results suggested that bovine ooplasm could support dedifferentiation of monkey somatic cell nuclei but could not support embryo development to either the compact morula or blastocyst stage. In conclusion, we found that the culture conditions that tend to enhance monkey iSCNT embryo development and the expression pattern of Oct-4 in cloned embryos (monkey iSCNT and bovine SCNT) are different than in bovine PA embryos.     

Autologous somatic cell nuclear transfer in pigs using recipient oocytes and donor cells from the same animal

The objective of the present study was to examine the feasibility of the production of autologous porcine somatic cell nuclear transfer (SCNT) blastocysts using oocytes and donor cells from slaughtered ovaries. Therefore, we attempted to optimize autologous SCNT by examining the effects of electrical fusion conditions and donor cell type on cell fusion and the development of SCNT embryos. Four types of donor cells were used: 1) denuded cumulus cells (DCCs) collected from in vitro-matured (IVM) oocytes; 2) cumulus cells collected from oocytes after 22 h of IVM and cultured for 18 h (CCCs); 3) follicular cells obtained from follicular contents and cultured for 40 h (CFCs); and 4) adult skin fibroblasts. The DCCs showed a significantly (p < 0.01) lower rate of fusion than the CCCs when two pulses of 170 V/mm DC were applied for 50 µsec (19 ± 2% vs. 77 ± 3%). The rate of DCC fusion with oocytes was increased by the application of two DC pulses of 190 V/mm for 30 µsec, although this was still lower than the rate of fusion in the CCCs (33 ± 1% vs. 80 ± 2%). The rates of cleavage (57 ± 5%) and blastocyst formation (1 ± 1%) in the DCC-derived embryos did not differ from those (55 ± 6% and 3 ± 1%, respectively) in the CCC-derived SCNT embryos. Autologous SCNT embryos derived from CFCs (5 ± 2%) showed higher levels of blastocyst formation (p < 0.01) than CCC-derived autologous SCNT embryos (1 ± 0%). In conclusion, the results of the present study show that culturing cumulus and follicular cells before SCNT enhances cell fusion with oocytes and that CFCs are superior to CCCs in the production of higher numbers of autologous SCNT blastocysts.     

Effect of postactivation treatment with latrunculin a on in vitro and in vivo development of cloned embryos derived from kidney fibroblasts of an aged clawn miniature boar

The objective of this study was to examine the effect of postactivation treatment with latrunculin A (LatA), an actin polymerization inhibitor, on in vitro and in vivo development of somatic cell nuclear transfer (SCNT) embryos derived from kidney fibroblasts of an aged Clawn miniature boar (12 years old). After electric activation, SCNT embryos were treated with 0, 0.5 or 1 μM LatA and cultured in vitro. The rate of blastocyst formation was significantly higher (P<0.05) in SCNT embryos treated with 0.5 μM LatA (38%) than those in control (14%). When cloned embryos treated with 0.5 μM LatA were transferred into the oviducts of two recipient miniature gilts to assess their development in vivo, both recipients became pregnant; one maintained pregnancy to term, and a live piglet (weighing 220 g) was delivered by Caesarean section. The results of this study indicated that the postactivation treatment with LatA was effective in improving in vitro developmental capacity of SCNT miniature pig embryos derived from kidney fibroblasts of an aged animal and that miniature pig cloned embryos treated with LatA had the ability to develop to term.     

Effects of cumulus cells on in vitro maturation of oocytes and development of cloned embryos in the pig

The purpose of this study was to investigate the role of porcine cumulus cells (CC) in oocyte maturation and somatic cell nuclear transfer (SCNT) embryo development in vitro. Denuded pig oocytes were co-cultured with CC or routinely cultured in maturation medium without a feeder layer. Porcine CC inactivated with mitomycin C or non-inactivated were used for the feeder layer in co-culture with porcine SCNT embryos to investigate comparatively the developmental competence of cloned embryos. The DNA damage aspects of apoptosis and expression pattern of genes implicated in apoptosis (Fas/FasL) as well as the mRNA expression of DNA methyltransferase (Dnmt1, Dnmt3a) of porcine SCNT embryos were also evaluated by comet assay or real-time RT-PCR, respectively. The results showed that co-culture with CC improved the extrusion rate of pbI (49.3% vs 31.5%, p<0.05) and survival rate (75.7% vs 53.3%, p<0.05) of denuded oocytes, but had no effects on blastocyst developmental rate or 2-cell-stage survival rate of in vitro fertilization embryos. Co-culture with CC inactivated by mitomycin C improved the blastocyst developmental rate (26.6% vs 13.0%, p<0.05) and decreased the apoptotic incidence (27.6% vs 46.2%, p<0.05) of porcine cloned embryos. Co-culture with inactivated CC reduced Fas and FasL mRNA expression of cloned embryos at the blastocyst stage compared with NT controls (p<0.05), but there were no differences in Dnmt1 and Dnmt3a mRNA expression among groups. Co-culture with inactivated cumulus cell monolayer significantly increased blastocyst formation and decreased the apoptotic incidence in porcine cloned embryos during in vitro development.     

Successful Production of Piglets Derived from Expanded Blastocysts Vitrified Using a Micro Volume Air Cooling Method without Direct Exposure to Liquid Nitrogen

This study was conducted to clarify the feasibility of newly developed vitrification techniques for porcine embryos using the micro volume air cooling (MVAC) method without direct contact with liquid nitrogen (LN₂). Expanded blastocysts were vitrified in a solution containing 6 M ethylene glycol, 0.6 M trehalose and 2% (wt/vol) polyethylene glycol in 10% HEPES-buffered PZM-5. The blastocysts were collected from gilts and vitrified using the new device (MVAC) or a Cryotop (CT). Blastocysts were stored in LN₂ for at least 1 month. After warming, cryoprotective agents were removed using a single step. Survival of the embryos was assessed by in vitro culture (Experiment 1) and by embryo transfer to recipients (Experiment 2). In Experiment 1, the embryos vitrified by the MVAC or CT and fresh embryos without vitrification (Control) were used. The survival rates of embryos in the MVAC, CT and Control groups were 88.9% (32/36), 91.7% (33/36) and 100% (34/34), respectively, after 48 h culture, and the hatching rates of embryos after 48 h incubation were 69.4% (25/36), 63.9% (23/36) and 94.1% (32/34), respectively. In Experiment 2, 64 vitrified embryos were transferred to 5 recipient gilts, and 8 healthy piglets were produced from 3 recipients in the MVAC group. Similarly, 66 vitrified embryos were transferred to 5 recipient gilts, and 9 healthy piglets were produced from 2 recipients in the CT group. These results indicated that porcine expanded blastocysts can be cryopreserved using the MVAC method without potential pathogen contamination from LN₂.