In Vitro Development of Marbled Cat Embryos Derived from Interspecies Somatic Cell Nuclear Transfer

The objective of the study was to investigate interspecies Somatic Cell Nuclear Transfer (iSCNT) techniques in marbled cats (Pardofelis marmorata), using domestic cat and rabbit oocytes as the recipient cytoplasm. The recipient oocytes were obtained from ovariohysterectomized cats and superovulated rabbits. The donor cells were collected from a male marbled cat that had died in captivity. Experiment 1 was conducted to observe the development of cloned marbled cat embryos (marbled cat donor cells-domestic cat oocytes; MC-DC), derived from oocytes matured for 24, 36 and 42 h. The result showed that the developmental rates of MC-DC cloned embryos at the 4-8 cell and the morula stages derived from oocytes cultured for 24 h were significantly greater than those cultured for 36 and 42 h (p < 0.05). Experiment 2 was conducted to compare the fusion rate of MC-DC couplets, fused by inducing different fusion voltages, 2.1 or 2.4 kV/cm. The result showed that there was no difference in fusion efficiency between the 2.1 and 2.4 kV/cm fusion protocols. Experiment 3 was conducted to compare the developmental rate of MC-DC and domestic cat (DC-DC) cloned embryos. In vitro fertilized cat embryos served as a control. The development of MC-DC and DC-DC cloned embryos to the 4- to 8-cell, morula and blastocyst stages was not significantly different. However, the development rates at morula and blastocyst stages of control were significantly greater than those of cloned embryos (p < 0.05). Experiment 4 rabbit (RB) oocytes were used as a recipient cytoplasm for marbled cat and domestic cat cloned embryos (MC- RB and DC-RB). RB-RB cloned embryos served as a control. There were no differences in the developmental rates between MC-RB, DC-RB and RB-RB embryos. In conclusion, marbled cat fibroblast cells can be reprogrammed in domestic cat and rabbit oocytes, and by using iSCNT it might be possible to produce marbled cat offspring in the future.     

Effects of Serum-Free Culture Media on in vitro Development of Domestic Cat Embryos Following In Vitro Maturation and Fertilization

This study was conducted to determine the adequate medium for a serum‐free culture system of domestic cat embryos produced by in vitro maturation (IVM) and fertilization (IVF). Cumulusoocyte complexes recovered from cat ovaries were matured in vitro for 24 h, and then inseminated in vitro for 12 h. After insemination, the oocytes were cultured in five media [Ham's F10, Waymouth 752/1 (Waymouth), TCM199, modified Earle's balanced salt solution (MK‐1) and CR1aa], each of which contained 0.4% bovine serum albumin. There were no significant differences among the rates of fertilization of oocytes cultured in five media following IVF. The rate of oocytes/embryos developed to at least the morula stage was significantly lower (p < 0.05) in Waymouth than in MK‐1, TCM199 and CR1aa. Moreover, none of the embryos cultured in Ham's F10 and Waymouth developed to the blastocyst stage. There were no differences among the rates of development to the blastocyst stage of oocytes/embryos cultured in MK‐1, TCM199 and CR1aa. These results indicate that the type of serum‐free medium has a major impact on in vitro development of domestic cat embryos derived from IVM/IVF, and MK‐1, TCM199 and CR1aa media are suitable for in vitro culture of cat embryos in a serum‐free culture system.     

Influence of intergeneric/interspecies mitochondrial injection; parthenogenetic development of bovine oocytes after injection of mitochondria derived from somatic cells

Interspecies/intergeneric mitochondrial heteroplasmy can occur in interspecies/intergeneric hybrid embryos or following nuclear transfer. In the present study, intergeneric buffalo (Bubalus bubalis) mitochondria (WB-mt) or interspecies murine (Mus spretus) mitochondria (M-mt) were injected into bovine (Bos taurus) oocytes, and the subsequent embryonic development was characterized. Fibroblast mitochondria (WB-mt or M-mt) were microinjected into in vitro matured bovine oocytes followed by oocyte activation by a combination of electrical stimulation and 6-dimethylaminopurine treatment. After seven days of culture, embryo development was evaluated. The copy number of specific mtDNA populations (introduced and native mtDNA) from heteroplasmic oocytes was estimated using real-time PCR. The results illustrated that oocytes injected with either WB-mt or M-mt can develop to the blastocyst stage (20.6% and 19.6%). Cleavage division rates and development to the morula stage in oocytes injected with WB-mt were lower (76.2% and 45.9%, respectively) in comparison with uninjected oocytes (89.2% and 59.1%, respectively) (P<0.05). However, no differences were found in comparing M-mt injected oocytes and controls (P>0.05). An increase in bovine mtDNA copy number was observed at the expanded blastocyst stage of injected embryos (P<0.01), while the number of injected mtDNA was stable throughout development. This study demonstrates that interspecies/intergeneric mitochondrial injected bovine oocytes have the ability to develop to the blastocyst stage after parthenogenetic activation and that injected mtDNA was neither selectively destroyed nor enhanced through development. Moreover, injected intergeneric mitochondria had a demonstrated influence on bovine parthenogenetic development and mtDNA replication.     

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.     

Interspecies nuclear transfer embryos reconstructed from cat somatic cells and bovine ooplasm

This study was conducted to investigate the developmental capacity of domestic cat-bovine reconstructed embryos via interspecies somatic cell nuclear transfer (iSCNT) and to observe the mitochondrial DNA (mtDNA) content of the iSCNT embryos. The iSCNT embryos were generated using mixed-breed domestic cat fibroblasts as donor cells and enucleated bovine oocytes as the recipient cytoplasm. When the developmental capacities of iSCNT embryos and parthenogenic bovine embryos were compared, there was no difference (P>0.05) in the rates of cleavage and development to the 8-cell stage (86.6 vs. 84.0% and 32.2 vs. 36.2%, respectively). However, in contrast to development of parthenogenic embryos to the morula and blastocyst stages, no iSCNT embryos (0/202) developed beyond the 8-cell stage. For mtDNA analysis, iSCNT embryos at the 1-cell, 2-cell, 4-cell and 8-cell stages were randomly selected. Both cat and bovine mtDNA quantification analysis were performed using quantitative PCR. The levels of both cat and bovine mtDNA in cat-bovine iSCNT embryos varied at each stage of development. The cat mtDNA concentration in the iSCNT embryos was stable from the 1-cell to 8-cell stages. The bovine mtDNA in the iSCNT embryos at the 8-cell stage was significantly lower than that at the 4-cell stage (P<0.05). No difference in the proportions of cat mtDNA in the iSCNT embryos was found in any of the observed developmental stages (1- through 8-cell stages). In conclusion, bovine cytoplasm supports domestic cat nucleus development through the 8-cell stage. The mtDNA genotype of domestic cat-bovine iSCNT embryos illustrates persistence of heteroplasmy, and the reduction in mtDNA content might reflect a developmental block at the 8-cell stage.     

Developmental Competence of Frozen-thawed Blastocysts from Fair-quality Bovine Embryos Cultured with β-Mercaptoethanol

Two-hundred-and-thirty-one fair-quality embryos at the compacted morula stage collected from 89 superovulated cows were cultured in TCM199 or Brinster's BMOC-3 medium with or without 100 microM beta-mercaptoethanol (beta-ME). After 24 h culture, a total of 142 fair-quality embryos developed to the blastocyst stage, of which 106 were subsequently frozen with 1.8 M ethylene glycol. The mean cell number and development rates of frozen-thawed blastocysts from the fair-quality embryos cultured in TCM199 containing beta-ME were higher than those of the fair-quality embryos directly frozen without culture. The pregnancy rates obtained with frozen blastocysts from fair-quality embryos tended to be lower than those of non-cultured fresh fair-quality embryos and cultured fresh blastocysts. These results indicate that the inclusion of beta-ME in pre-freezing culture media improve the development of frozen-thawed blastocysts from fair-quality embryos, but not the pregnancy rate.     

Development of one-cell porcine embryos to the blastocyst stage in simple media

Porcine embryos were flushed from mated donors and examined for cleavage stage. One- and two-cell embryos were randomly allotted to one of the five following in vitro treatments: M199 with Earle's salts, a modified Tyrode's medium (TL), TL supplemented with 10 mM N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES) (TLH), TLH supplemented with 5.5 mM glucose (TLHG), or TLH supplemented with 5 mM glutamine (TLHGL). The bicarbonate concentration of TLH, TLHG, and TLHGL was 2 mM, compared with the 25 mM concentration in M199 and TL. Embryos in M199 and TL were incubated in 95% air:5% CO2 at 39 degrees C. Those in the remaining three treatments were incubated in air at 39 degrees C. Embryos incubated in TL and M199 did not develop past the four- to eight-cell stage, whereas the proportions of embryos developing to the compact morula or blastocyst stage by d 7 of culture in the other treatments were as follows: TLHG, 49.1%; TLHGL, 59.4%; TLH, 63.5% (P less than .005). These results indicate that porcine embryos can be cultured from the one-cell stage to blastocyst in a simple HEPES-buffered medium in air. The ability of porcine embryos to develop without supplemental CO2 may be an important finding for use in situations in which embryos must be transported for long periods before embryo transfer.     

Effects of slow freezing and vitrification on embryo development in domestic cat

Cryopreservation of gametes and embryos is used to maintain genetic diversity of domestic and wild felids. However, felid oocytes and preimplantation embryos contain large amount of intracellular lipids, which affect their cryosensitivity. The objective was to compare the effects of slow freezing and vitrification and to study lipid phase transition (LPT) during cooling in cat embryos. In vitro-derived embryos were cultured 48 hr up to 4–8 cell stage, thereafter were either slow frozen or vitrified. Propylene glycol (PG) alone was used as a cryoprotective agent (CPA) for slow freezing, and a mixture of PG and dimethyl sulfoxide (DMSO) were used as CPAs for vitrification. After thawing/warming, embryos were in vitro cultured additionally for 72 hr. The total time of in vitro culture was 120 hr for all the groups including non-frozen controls. Effects of both cryopreservation procedures on the subsequent embryo development and nuclear fragmentation rate in embryonic cells were compared. There was no significant differences among the percentages of embryos achieved morula and early blastocyst stage in frozen-thawed group (36.4% and 20.0%), in vitrified-warmed group (34.3% and 28.6%) and in controls (55.6% and 25.9%). Cell numbers as well as nuclear fragmentation rate did not differ in these three groups. Average lipid phase transition (LPT) temperature (T*) was found to be relatively low (–2.2 ± 1.3°C) for the domestic cat embryos. It is supposed that the low LPT of LDs may provide a good background for successful application of slow freezing to domestic cat embryos. Generally, our study indicates that slow freezing and vitrification are both applicable for domestic cat embryo cryopreservation.     

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.     

Development In Vitro and Mitochondrial Fate of Interspecies Cloned Embryos

Although the technique of interspecies somatic cell nuclear transfer can be used to increase the population size of endangered mammals, the mitochondrial heteroplasmy in cloned embryos and animals makes this idea doubtful. In present study, goat–sheep cloned embryos were constructed by fusing goat foetal fibroblasts (GFFs) into sheep oocytes and then cultured in vitro to investigate the capability of sheep oocyte dedifferentiating GFF nucleus. Moreover, at each stage of 1‐ (immediately after fused), 2‐, 4‐, 8‐, 16‐cell, morula and blastocyst, the copy number of mtDNA from GFF and sheep oocyte was examined using real‐time PCR. The results showed that: 7.4% of the fused cloned embryos can develop to the blastocyst stage; in the process of one cell to the morula stage, the copy number of two kinds of mtDNA was stable relatively; however, in the process of morula to the blastocyst stage, the decreasing in the copy number of GFF‐derived mtDNA, while the increasing in sheep oocyte‐derived, resulted in their ratio of decreasing sharply from 2.0 ± 1.0% to 0.012 ± 0.004%. This study demonstrates that: (i) the goat–sheep cloned embryos have the ability to develop to blastocyst in vitro; (ii) from the morula stage to the blastocyst stage of goat–sheep cloned embryos, goat derived mitochondria can be gradually replaced with those from sheep oocyte.     

The use of human and bovine commercial media for oocyte maturation and embryo development in the domestic cat (Felis catus)

The aim of this study was to examine the suitability of commercial media designed for humans and cattle for oocyte maturation and embryo culture in the domestic cat. In Exp. I, feline oocytes collected ex vivo were subjected to in vitro maturation in a laboratory‐made culture medium (based on M199) or a commercial medium designed for cattle cells (BO‐IVM^®). In Exp. II, ICSI‐derived feline embryos were cultured for 7 days in a commercial human (Continuous Single Culture^®) or bovine (BO‐EC^®) cell medium. The rates of cleavage, morula and blastocyst formation were evaluated at 24 hr, 6 days and 7 days after ICSI, respectively, and compared between experimental groups. At the end of culture, embryos were assessed for viability and apoptotic changes. In Exp. I, no statistically significant difference in oocyte maturation outcome between laboratory‐made (52.7%) and commercial media (58.9%) was observed. However, the use of a commercial medium prepared for use with bovine cells resulted in a significantly lower variance of the maturation rate. In Exp. II, no statistically significant differences between two commercial media were observed for cleavage (67.5% and 64.5%), morula (39.3% and 47.1%) and blastocyst rates (25.0% and 19.6%), as well as for the percentage of late apoptotic blastomeres. Morulae cultured in medium marketed for humans exhibited significantly more early apoptotic (43.2 ± 31.2% vs. 23.4 ± 23.2%) and necrotic (60.6 ± 47.6% vs. 29.4 ± 22.6%) blastomeres. In conclusion, both commercial media tested are suitable for in vitro oocyte maturation and embryo culture procedures in cats. It is remarkable that a culture medium designed for use in cattle for in vitro maturation of cat oocytes provides more reproducible results.     

胚胎质量和发育阶段对奶牛胚胎移植妊娠率的影响

[目的]为了评估胚胎质量和发育阶段对奶牛胚胎移植妊娠率的影响。[方法]使用63头青年奶牛作为供体进行超数排卵,评估回收胚胎质量和发育阶段。选择334头青年奶牛作为受体鲜胚移植不同质量和发育阶段胚胎。对胚胎质量分布、发育阶段分布、不同质量胚胎和不同发育阶段胚胎移植30 d妊娠率进行统计分析。[结果]可用胚胎中A级胚胎比例(60.78%)显著高于B级和C级胚胎比例(36.70%和2.52%)(P<0.05);致密桑椹胚比例(54.36%)显著高于早期囊胚,囊胚和扩张囊胚比例(18.35%,25.0%和2.29%)(P<0.05)。A级和B级胚胎移植30 d妊娠率(63.55%和64.35%)显著高于C级胚胎移植30 d妊娠率(44.44%)(P<0.05);致密桑椹胚、早期囊胚、囊胚和扩张囊胚移植30 d妊娠率差异不显著(P<0.05),早期囊胚、囊胚移植30 d妊娠率高于致密桑椹胚、扩张囊胚移植30 d妊娠率(P<0.05)。[结论]选择不同发育阶段的A级和B级胚胎能够获得较高胚胎移植妊娠率,增加早期囊胚和囊胚阶段胚胎移植数量能够提高胚胎移植妊娠率。     

Developmental competence of frozen-thawed blastocysts from fair-quality bovine embryos cultured with beta-mercaptoethanol

Two-hundred-and-thirty-one fair-quality embryos at the compacted morula stage collected from 89 superovulated cows were cultured in TCM199 or Brinster's BMOC-3 medium with or without 100 microM beta-mercaptoethanol (beta-ME). After 24 h culture, a total of 142 fair-quality embryos developed to the blastocyst stage, of which 106 were subsequently frozen with 1.8 M ethylene glycol. The mean cell number and development rates of frozen-thawed blastocysts from the fair-quality embryos cultured in TCM199 containing beta-ME were higher than those of the fair-quality embryos directly frozen without culture. The pregnancy rates obtained with frozen blastocysts from fair-quality embryos tended to be lower than those of non-cultured fresh fair-quality embryos and cultured fresh blastocysts. These results indicate that the inclusion of beta-ME in pre-freezing culture media improve the development of frozen-thawed blastocysts from fair-quality embryos, but not the pregnancy rate.     

不同培养液及血清浓度对绵羊孤雌生殖胚胎发育的影响

体外成熟的绵羊卵母细胞，经5 μmol/L A23187 5 min和2 mmol/L 6-DMAP 4 h激活后，分别在SOFaa、M-199两种培养液中进行培养，试验比较了SOFaa、M-199液分别添加BSA和不同浓度（10%、20%）胎牛血清对绵羊孤雌生殖胚体外发育的影响。M-199+FCS组的卵裂率显著低于SOFaa+BSA和SOFaa+FCS组（P<0.05），SOFaa+BSA组的囊胚率显著低于M-199+BSA、SOFaa+FCS、M-199+FCS三组（P<0.05）；SOFaa+10% FCS与SOFaa+20% FCS组的卵裂率及囊胚率均无显著差异，M-199+10% FCS和M-199+20% FCS组的卵裂率及囊胚率间均无显著差异。结果表明：SOFaa比M-199更适合绵羊孤雌生殖胚体外发育，此外添加10%的FCS即可达到较好的培养效果。     

Mitochondrial activity and morphology in developing porcine oocytes and pre-implantation non-cultured and cultured embryos

Mitochondria are important determinants of developmental competence for oocytes and embryos owing to their central role in cellular metabolism, yet mitochondrial activity and morphometry during early porcine development have not been quantified. In this study, we examined the membrane potential Δψ(m) and the surface density Sv(in,m) of the inner mitochondrial membrane in pig oocytes and pre-implantation embryos using fluorescent probes and confocal microscopy. Mitochondria and their cristae were also examined by transmission electron microscope. Δψ(m) was consistently low from immature oocytes up to morulae and increased significantly in the early blastocyst before decreasing at the expanded blastocyst stage. This stage-dependent pattern of Δψ(m) changes differs from that reported for other mammals. We also determined that Δψ(m) is lower in cultured when compared to non-cultured porcine early blastocysts. Sv(in,m) was higher in immature oocytes than mature oocytes and remained constant up to the 4- to 8-cell embryo stage. It increased significantly at morula and early blastocyst stages. No differences in Sv(in,m) were found between developmentally matched non-cultured and cultured embryos. These results indicate that the inner mitochondrial membrane potential and surface density change significantly during pre-implantation porcine development in relation to metabolic alterations of the embryo. It is possible that modification of Δψ(m) by manipulating culture conditions may improve the performance of embryos that develop in vitro.     

Preimplantation development of embryos in labrador retrievers

Preimplantation development of canine embryos is not well understood. To understand the timing of preattachment embryogenesis relative to the luteinizing hormone (LH) surge, early embryonic development was examined in Labrador Retrievers after artificial insemination. The embryos migrated from the oviduct to the uterus beginning on day 11 after the LH surge. This transport must be completed within 24 h. By day 13 after the LH surge, all of the embryos had moved and were localized in the uterus. The embryos developed to the morula stage within 11-13 days and to the blastocyst stage within 14 days after the LH surge, respectively. These findings add to the current understanding concerning the physiology of preimplantation development and should help further develop assisted reproductive techniques in canine species, such as cryopreservation and subsequent embryo transfer.     

Influence of cysteine in conjunction with growth factors on the development of in vitro-produced bovine embryos

Cysteine supplementation to in vitro maturation (IVM) media of bovine oocytes increases cellular glutathione production. Beneficial effects of growth factors for improving the rate of blastocyst development have been reported, but combined effects are unknown. This study was conducted to determine the additive effect of cysteine with epidermal growth factor (EGF) and/or insulin-like growth factor-I (IGF-I) on embryo development. Bovine oocytes from slaughterhouse ovaries were matured in TCM-199 (control), with or without the addition of 0.6 mm cysteine (C) at 0 or 12 h of maturation. After in vitro fertilization, embryos were allocated to culture treatments containing synthetic oviductal fluid medium. Culture treatments included fetal calf serum (FCS, 4%) alone; IGF-I (100 ng/ml); EGF (10 ng/ml); and IGF-I + EGF (100 + 10 ng/ml). Although rates for blastocysts development were not different among treatments, an increased proportion of embryos attaining morula formation was achieved when cysteine was added to the maturation media (12 h C IGF-I + EGF, 41.4%; 0 h C EGF, 40.0%) as compared to control (FCS: 34.6%). When cysteine treatments were combined, percent cleavage was greater for IGF-I + EGF (70.8%) compared to FCS (61.2%). The abundance of mRNA from the apoptotic genes, Bax and Bcl-2, and the oxidative stress genes, copper (Cu)-zinc (Zn) superoxide dismutase (SOD) and manganese (Mn) SOD in embryos was assessed. No treatment effect was observed on the expression of these genes. In conclusion, supplementation of cysteine during IVM of oocytes, in conjunction with growth factors could effectively be used as a replacement for FCS.     

猪胚胎的OPS玻璃化冷冻

以广西巴马小型猪为供体,采用超数排卵技术,采集5~6日龄具有完整透明带的胚胎(囊胚/桑葚胚),采用二步法OPS(Open pulled straw)玻璃化冷冻技术进行保存,即胚胎首先在冷冻液1(TCM199 20?S 10%EG 10%DMSO)中平衡3 min,然后立即转入冷冻液2(TCM199 20?S 20%EG 20%DMSO 0.4 mol/LSUC)中并在1 min内装管(每管含2~6枚胚胎),直接投入液氮保存;3个月后解冻移植给8头受体母猪(每头移入25~26枚胚胎),其中1头怀孕产仔(8头活仔),获得猪胚胎超低温(-196℃)冷冻后代。