In-straw cryoprotectant dilution for bovine embryos vitrified using Cryotop

The aim of this study was to develop an in-straw dilution method suitable for 1-step bovine embryo transfer of vitrified embryos using the Cryotop vitrification-straw dilution (CVSD) method. The development of embryos vitrified using the CVSD method was compared with those of embryos cryopreserved using in-straw vitrification-dilution (ISVD) and conventional slow freezing, outside dilution of straw (SFODS) methods. In Experiment 1, in vitro-produced (IVP) embryos cryopreserved using the CVSD method were diluted, warmed and exposed to the dilution solution at various times. When vitrified IVP embryos were exposed to the dilution solution for 30 min after warming, the rates of embryos developing to the hatched blastocyst stage after 72 h of culture (62.0-72.5%) were significantly lower (P<0.05) than those of embryos exposed to the solution for 5 and 10 min (82.4-94.3%), irrespective of supplementation with 0.3 M sucrose in the dilution solution. In Experiment 2, the rate of embryos developing to the hatching blastocyst stage after 48 h of culture in IVP embryos cryopreserved using the SFODS method (75.0%) was significantly (P<0.05) lower than those of embryos cryopreserved using the CVSD and ISVD methods (93.2 and 97.3%, respectively). In Experiment 3, when in vivo-produced embryos that had been cryopreserved using the CVSD, ISVD and SFODS methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception and delivery rates among groups. In Experiment 4, when IVP embryos derived from oocytes collected by ovum pick-up that had been cryopreserved using the CVSD and ISVD methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception rates among groups. Our results indicate that this simplified regimen of warming and diluting Cryotop-vitrified embryos may enable 1-step bovine embryo transfer without the requirement of a microscope or other laboratory equipment.     

Cryopreservation and sexing of in vivo- and in vitro-produced bovine embryos for their practical use

My research awarded includes contributions to cryopreservation and sexing of bovine embryos produced in vitro and in vivo, as follows; (1) In vivo-derived morulae and blastocysts were cryopreserved in the presence of 10% glycerol, and the embryos were transferred into recipients after two-step dilution of glycerol in straw, with a practically acceptable pregnancy rate. (2) The survival rate of 16-cell stage embryos frozen in the medium with ethylene glycol was higher than that with DMSO or 1,2-propanediol. Addition of linoleic acid-albumin to culture medium enhanced the survival rate of post-thaw bovine 16-cell stage in vitro-produced (IVP) embryos. (3) Polarization of cytoplasmic lipid droplets by centrifugation of 2-cell stage embryos was found effective to increase freezing tolerance in 16-cell stage embryos developed from the centrifuged embryos, because blastomeres of 16-cell stage embryos were mostly lipid-free. (4) The usefulness of gel-loading tip (GL-Tip) as a container for ultra-rapid vitrification was demonstrated in IVP embryos from 2-cell to blastocyst stages, with a higher in vitro survival than the conventional two-step freezing. (5) PCR analysis for sexing of in vivo-derived Day-7 embryos indicated that male embryos developed faster and graded higher than female embryos. But such correlation between genetic sex and embryonic development was not found in IVP embryos obtained from individual cows. (6) Addition of 0.1-1.0% deproteinized hemodialysate product from calf blood to culture medium increased the producing efficiency of demi-embryos with good quality. Female embryos rather than male embryos required a longer time to repair after bisection. (7) In vivo-derived bovine embryos after biopsy for sexing by PCR analysis and subsequent vitrification using GL-Tips are available to practical use in the field. (8) Introduction of primer extension preamplification-PCR and purification of DNA product before standard sexing PCR of biopsy samples from Day 3-4 in vitro-derived embryos allowed accurate sex determination, and Day-7 blastocysts developed from Day 3-4 embryos were cryopreserved by GL-Tip vitrification without a loss of their viability. Thus the field application of bovine embryo transfer is in part supported by improvements of technologies in embryo cryopreservation and sex pre-determination.     

Growth and metabolism of murine and bovine embryos in bovine uterine flushing-supplemented culture media.

The aim of this study was to compare the development and metabolic activity of cultured murine and bovine embryos in 2 standard media (HAM F-10 and RPMI) in the presence or absence of bovine uterine flushings. Murine morulae (n = 653) and day 7 bovine embryos (n = 273) were cultured for 18 h or 36 h in either HAM F-10 or RPMI in the presence or absence of bovine uterine flushings. After culture, the development, quality, and metabolic activity (glucose utilization or methionine uptake and incorporation) of embryos was assessed. It was found that HAM F-10 (without uterine flushings) was a more suitable medium than RPMI for optimal development and metabolism of murine and bovine embryos. Poor quality and development, as well as decreased metabolism, were evident after culture of murine embryos in RPMI; in contrast, this medium had no adverse effects on bovine embryos in culture. Supplementation of HAM F-10 with bovine uterine flushings improved the growth of murine embryos and the protein synthesis (as measured by an increased methionine incorporation) for both murine and bovine embryos. However, supplementation with bovine uterine flushings could not overcome deficiencies of an inappropriate medium (RPMI) for murine embryos. Supplementation of a well-defined culture medium with uterine flushings increased metabolism of embryos in culture, and thus might help to increase pregnancy rates after transfer of such embryos to recipient cows.     

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.     

牛IVP胚胎玻璃化冷冻技术的研究

作者比较了乙二醇传统冷冻和OPS玻璃化冷冻牛IVP胚胎的效果,目的是改善其冷冻效果,提高IVP胚胎的移植妊娠率。结果表明,玻璃化冷冻后的存活率(D7-71)明显高于D7-71L(P<0.01),D7-72组与D7-71L组(传统冷冻方法)的存活率基本一致(P>0.05)。     

Propagation of elite rescue dogs by somatic cell nuclear transfer

The objective of the present study was to compare the efficiency of two oocyte activation culture media to produce cloned dogs from an elite rescue dog and to analyze their behavioral tendencies. In somatic cell nuclear transfer procedure, fused couplets were activated by calcium ionophore treatment for 4 min, cultured in two media: modified synthetic oviduct fluid (mSOF) with 1.9 mmol/L 6‐dimethylaminopyridine (DMAP) (SOF‐DMAP) or porcine zygote medium (PZM‐5) with 1.9 mmol/L DMAP (PZM‐DMAP) for 4 h, and then were transferred into recipients. After embryo transfer, pregnancy was detected in one out of three surrogate mothers that received cloned embryos from the PZM‐DMAP group (33.3%), and one pregnancy (25%) was detected in four surrogate mothers receiving cloned embryos from the SOF‐DMAP group. Each pregnant dog gave birth to one healthy cloned puppy by cesarean section. We conducted the puppy aptitude test with two cloned puppies; the two cloned puppies were classified as the same type, accepting humans and leaders easily. The present study indicated that the type of medium used in 6‐DMAP culture did not increase in cloning efficiency and dogs cloned using donor cells derived from one elite dog have similar behavioral tendencies.     

Development and application of a chemically defined medium for the in vitro production of porcine embryos

Recent advances in systems for in vitro production (IVP) of porcine embryos, including in vitro oocyte maturation, fertilization and embryo culture, have enabled us to generate viable embryos that can develop to full term after transfer into recipients. This technology is being applied now to developments in gamete/embryo biology and agriculture, as well as in producing cloned and genetically modified pigs. Chemically defined media for IVP of embryos are useful for a precise analysis of the physical action of substances on gametogenesis and early embryogenesis, because they eliminate undefined factors present in biological materials, such as serum or serum albumin. Use of a chemically defined medium also improves the reliability of media formulations, yields a higher reproducibility of results and ensures biosafety of culture media by eliminating protein preparations, which may be contaminated with pathogens. Therefore, it has certain advantages for research and for commercial purposes. We have recently developed a defined IVP system for porcine embryos using a single basic medium based on the composition of porcine oviductal fluid. This paper discusses the developmental ability and normality of porcine IVP embryos, and limitations and advancements in this system.     

The efficiency of in vitro ovine embryo production using an undefined or a defined maturation medium is determined by the source of the oocyte

In vitro oocyte maturation can be influenced by oocyte source and maturation media composition. The aim of the present study was to compare the efficiency of a defined in vitro maturation medium (TCM199 supplemented with cysteamine and epidermal growth factor; Cys + EGF) with an undefined medium (TCM199 supplemented with follicle-stimulating hormone and follicular fluid; FSH + FF) for in vitro production (IVP) of ovine embryos, using oocytes obtained by laparoscopic ovum pick-up from FSH-stimulated [n=11; 158 cumulus-oocyte complexes (COCs)] and non-stimulated (n=16; 120 COCs) live ewes, as well as abattoir-derived oocytes (170 COCs). The produced blastocysts were vitrified and some of them were transferred to synchronized recipients. The best and the worst final yields of embryo IVP observed in this study were obtained using oocytes from FSH-stimulated ewes matured in FSH + FF (41.3%; 33/80) and in Cys + EGF (19.2%; 15/78) medium, respectively (p<0.01). No significant differences between both media were attained in the blastocyst development rate or in the final yield of embryo IVP using oocytes from non-stimulated ewes or abattoir-derived oocytes. The overall in vivo survival rate of the transferred vitrified blastocysts was 13.1% (8/61), without significant differences between oocyte sources or maturation media. In conclusion, under the experimental conditions of the present study, TCM199 supplemented with cysteamine and EGF is a convenient defined maturation medium for IVP of embryos from oocytes of live non-stimulated ewes or from oocytes of abattoir-derived ovaries. However, the best final yield of embryo IVP observed in this study was attained when oocytes came from FSH-stimulated donors and TCM199 was supplemented with FSH and follicular fluid.     

Assessment of Actin Cytoskeleton and Nuclei in Bovine Blastocysts Developed Under Different Culture Conditions Using a Novel Computer Program

This study was performed to investigate the effects, in terms of nuclear material and actin cytoskeleton quantities (fluorescent pixel counts), of four different bovine blastocyst culturing techniques (in vitro, stepwise in vitro‐to‐in vivo, or purely in vivo). Cumulus oocyte complexes from abattoir‐sourced ovaries were matured in vitro and allocated to four groups: IVP‐group embryos developed up to blastocyst stage in vitro. Gamete intra‐fallopian transfer (GIFT)‐group oocytes were co‐incubated with semen for 4 h before transfer to oviducts of heifers. Following in vitro fertilization, cleaved embryos (day 2 of embryo development, day 2–7 group) were transferred into oviducts on day 2. Multiple ovulation embryo transfer (MOET)‐group embryos were obtained by superovulating and inseminating heifers; the heifers’ genital tracts were flushed at day 7 of blastocyst development. Within each group, ten blastocysts were selected to be differentially dyed (for nuclei and actin cytoskeleton) with fluorescent stains. A novel computer program (ColorAnalyzer) provided differential pixel counts representing organelle quantities. Blastocysts developed only in vivo (MOET group) showed significantly more nuclear material than did blastocysts produced by any other technique. In terms of actin cytoskeleton quantity, blastocysts produced by IVP and by day 2–7 transfer did not differ significantly from each other. Gamete intra‐fallopian transfer‐ and MOET‐group embryos showed significantly larger quantities of actin cytoskeleton when compared with any other group and differed significantly from each other. The results of this study indicate that culturing under in vitro conditions, even with part time in vivo techniques, may adversely affect the quantity of blastocyst nuclear material and actin cytoskeleton. The software employed may be useful for culture environment evaluation/developmental competence assessment.     

Programming of postnatal phenotype caused by exposure of cultured embryos from Brahman cattle to colony-stimulating factor 2 and serum

Alterations in the environment of the preimplantation embryo can affect competence to establish pregnancy and phenotype of resultant calves. In this study, the bovine embryo produced in vitro was used to evaluate postnatal programming actions of the embryokine colony-stimulating factor 2 (CSF2) and serum, which is a common additive of culture media. Oocytes were collected by ovum pick up from Brahman donors and fertilized with semen from Brahman bulls. Embryos were randomly assigned to one of the three treatments: vehicle, CSF2 10 ng/mL, or 1% (v/v) serum. Treatments were added to the culture medium from day 5 to 7 after fertilization. Blastocysts were harvested on day 7 and transferred into crossbred recipients. Postnatal body growth and Longissimus dorsi muscle characteristics of the resultant calves were measured. The percent of cleaved embryos becoming blastocysts was increased by serum and, to a lesser extent, CSF2. Treatment did not affect survival after embryo transfer but gestation length was shortest for pregnancies established with serum-treated embryos. Treatment did not significantly affect postnatal body weight or growth. At 3 mo of age, CSF2 calves had lower fat content in the Longissimus dorsi muscle and less subcutaneous fat over the muscle than vehicle calves. There was a tendency for cross-sectional area of the muscle to be smaller for serum calves than vehicle calves. Results confirm the importance of the preimplantation period as a window to modulate postnatal phenotype of resultant calves. In particular, CSF2 exerted actions during the preimplantation period to program characteristics of accumulation of intramuscular and subcutaneous fat of resultant calves. The use of a low serum concentration in culture medium from day 5 to 7 of development can increase the yield of transferrable embryos without causing serious negative consequences for the offspring.     

Transition of the pregnancy rate of bisected bovine embryos after co-transfer with trophoblastic vesicles prepared from in vivo-cultured in vitro-fertilized embryos

Bisected bovine embryos were co-transferred with trophoblastic vesicles (TVs). These TVs were prepared by dissection of conceptuses that were collected by uterine flushing after culture for seven days in the uterus following transfer of embryos derived by in vitro fertilization (IVF). Pregnancy diagnoses were performed twice, between Day 26 and Day 43 and between Day 38 and Day 73 post-estrus by ultrasonography. The pregnancy rate was significantly increased at first pregnancy diagnosis when demi-embryos were transferred with TVs (66.7%, 16/24) compared with the control group (34.5%, 10/29) (P < 0.05). Three losses occurred in the co-transfer group between the first and second pregnancy diagnosis. The final pregnancy rates according to delivered offspring were 41.7% (10/24) and 27.6% (8/29), respectively. There were no statistically significant differences between the pregnant and non-pregnant groups with regard to the average diameter of the TVs measured before transfer at three points during the gestation period. The birth weight and gestation lengths of the offspring were almost the same for the co-transfer and control groups. In the co-transfer group, the genetic identities of calves from the separated embryos were not affected by the TVs, as confirmed by parental blood type testing. Delivered offspring in co-transferred groups showed normal morphology. In conclusion, the present study indicates that co-transfer of TVs prepared from conceptuses cultured in vivo following transfer of IVF embryos enhances the fertility of demi-embryos during the early stages of pregnancy, as has similarly been shown in previous research for those prepared from in vivo embryos.     

Three-dimensional live imaging of bovine embryos by optical coherence tomography

While embryo transfer (ET) is widely practiced, many of the transferred embryos fail to develop in cattle. To establish a more effective method for selecting bovine embryos for ET, here we quantified morphological parameters of living embryos using three-dimensional (3D) images non-invasively captured by optical coherence tomography (OCT). Seven Japanese Black embryos produced by in vitro fertilization that had reached the expanded blastocyst stage after 7 days of culture were transferred after imaged by OCT. Twenty-two parameters, including thickness and volumes of the inner cell mass, trophectoderm, and zona pellucida, and volumes of blastocoel and whole embryo, were quantified from 3D images. Four of the seven recipients became pregnant. We suggest that these 22 parameters can be potentially employed to evaluate the quality of bovine embryos before ET.     

Birth weight and gestation length of Japanese black calves following transfer of embryos produced in vitro with or without co-culture.

Birth weight and gestation length of calves following the transfer of in vitro produced (IVP) embryos with or without co-culture of cumulus cells, were compared to those produced in vivo (IVD). Spermatozoa from one Japanese Black bull were used for both IVP and IVD. IVP embryos were produced using two types of culture method: 1) co-culturing with cumulus cells in TCM 199 supplemented with calf serum (IVP-Co), and 2) non-co-culturing without cumulus cells in CR1aa supplemented with BSA / calf serum (IVP-NON-Co). Both IVP and IVD embryos were transferred non-surgically to Holstein recipients on day 7+/-1 of the estrous cycle. Birth weight and gestation length of half-sib single calves were analyzed. No differences were observed in birth weight and gestation length between IVP-Co and IVP-NON-Co calves (31.0 kg and 31.8 kg, and 291.9 days and 291.0 days, respectively). However, the birth weight of the IVP-Co and IVP-NON-Co calves was significantly higher than that of the IVD calves (P<0.01). Gestation length of the IVP-Co and IVP-NON-Co calves was also significantly longer than that of the IVD calves (P<0.01).     

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.     

Susceptibility of In Vivo- and In Vitro-produced Porcine Embryos to Classical Swine Fever Virus

The objective of this study was to investigate the susceptibility of in vivo- and in vitro-produced (IVP) porcine embryos to classical swine fever virus (CSFV). IVP zona pellucida (ZP)-intact porcine embryos (n = 721) were co-cultured with CSFV for 120 h. After washing according to the International Embryo Transfer Society guidelines (without trypsin) and transferring embryos to CSFV-susceptible porcine kidney cells (PK15 cell line), no virus was isolated. However, when 88 IVP ZP-intact porcine embryos were co-cultured with CSFV for only 48 h before being transferred to PK15 cells, virus was isolated in three of six replicates. Similarly, 603 in vivo-produced porcine embryos were co-cultured with CSFV for 120 h. Subsequently, CSFV was isolated in eight of 50 groups (16%) and the ability of these to form a blastocyst was significantly reduced when compared with the control group (68.2 +/- 19.9% vs 81.9 +/- 9.7%; p < or = 0.001). In contrast, the development of CSFV-exposed IVP porcine embryos was not affected when compared with control embryos (19.1 +/- 10.8% vs 18.9 +/- 10.6%; p > or = 0.05). After removal of the ZP of IVP embryos and subsequent co-culture with CSFV, the virus was isolated from all groups of embryos. These data suggest that virus replication had occurred in the embryonic cells. In conclusion, data indicate that in vivo- and in vitro-produced ZP-intact porcine embryos differ in their susceptibility to CSFV. Hatched or micro-manipulated embryos may increase the risk of transmission of CSFV by embryo transfer, which has to be confirmed by in vivo tests under isolation conditions.     

基因表达差异对牛胚胎发育潜能的影响

随着牛胚胎体外生产(IVP)技术的不断成熟,体外胚胎生产在一些国家已进入商业化运营阶段.但与体内胚胎相比其移植受体妊娠率、产犊率及胎儿的后天发育能力均较差.因此,开发一种既可靠又实用的检测胚胎发育潜能的技术显得尤为重要.应用实时荧光定量PCR技术检测早期胚胎基因表达,通过分析mRNA的表达丰度来评价卵母细胞成熟情况和胚...     

The Effect of Long‐term In Vivo Culture in Bovine Oviduct and Uterus on the Development and Cryo‐tolerance of In Vitro Produced Bovine Embryos

The objective of this study was to compare the embryo production and quality carried out entirely in vitro or partly in vitro combined with short‐ vs long‐term in vivo culture using the homologous cattle oviduct. The IVM oocytes were in vitro fertilized and cultured for 7 and 8 days (IVP‐Group), or after IVF and 2–3 days of IVC, 4–8 cell stage embryos were endoscopically transferred into oviducts of synchronized heifers (In Vivo‐Group) or IVM oocytes were co‐incubated with spermatozoa for 3–4 h and transferred into the oviducts of synchronized heifers (GIFT‐Group). Embryos of the In Vivo‐Group and the GIFT‐Group were recovered on day 7 from the oviducts and uterine horns. Embryos of all groups were either cryopreserved at day 7 (day 7 blastocysts) or cultured in vitro in CR1aa‐medium supplemented with 5% ECS for further 24 h and cryopreserved (day 8 blastocysts). The total blastocyst yield found in the in vivo cultured groups was similar to the results of the IVP‐Group. But the appearance of blastocysts was dependent on the duration of in vivo culture. The more time the embryos spent in the in vivo environment, the more blastocysts appeared at day 8. The quality of produced blastocysts assessed by cryo‐survival was also correlated to the culture conditions; the in vivo cultured embryos showed higher cryo‐tolerance. However, the duration of in vivo culture crucially influenced the cryo‐tolerance of produced blastocysts. It is concluded that tubal access is a promising tool to provide a further basis for studying embryo sensitivity to environmental changes.     

Different thermotolerances in in vitro‐produced embryos derived from different maternal and paternal genetic backgrounds

The present study evaluated the effects of genetic backgrounds on the developmental competence and thermotolerance of bovine in vitro‐produced (IVP) embryos. First, Holstein (Hol) and Japanese Black (JB) oocytes were fertilized with sperm from Hol, JB and a thermotolerant breed (Brahman), and in vitro development was evaluated when the embryos were exposed to heat shock on Day 2 (Day 0 = day of fertilization). Sperm genetic backgrounds affected the developmental competence in controls (P < 0.05). Second, the effect of sperm pre‐incubation for 4 h on subsequent in vitro fertilization was assessed using different sperm genetic backgrounds. The pre‐incubation of sperm did not decrease the embryonic development regardless of the breed of the sperm. A milder heat shock (40.0°C) effect on parthenotes (Hol and JB) and IVP embryos were evaluated. JB parthenotes showed developmental arrest after Day 4, and the rate of development to the blastocyst stage decreased by heat shock, but not in Hol parthenotes. Heat shock decreased developmental competence after cleavage of IVP embryos regardless of genetic background. The thermotolerance of IVP embryos would be controlled by both maternal and paternal factors but genetic involvement was still unclear. Further evaluation is needed to reveal the genetic contribution to thermotolerance.