Frequency of sex chromosomal mosaicism in bovine embryos and its effects on sexing using a single blastomere by PCR

Assessment of nuclear status is important when a biopsied single blastomere is used for embryo sexing. In this study we investigated the nuclear status of blastomeres derived from 8- to 16-cell stage in vitro fertilised bovine embryos to determine the representativeness of a single blastomere for embryo sexing. In 24 embryos analysed, the agreement in sex determination between a biopsied single blastomere and a matched blastocyst by polymerase chain reaction (PCR) was 83.3%. To clarify the discrepancies, karyotypes of blastomeres in 8- to 16-cell stage bovine embryos were analysed. We applied vinblastine sulfate at various concentrations and for different exposure times for metaphase plate induction in 8- to 16-cell stage bovine embryos. The 1.0 mg/ml vinblastine sulfate treatment for 15 h was selected as the most effective condition for induction of a metaphase plate (> 45%). Among 22 embryos under these conditions, only 8 of 10 that had a normal diploid chromosome complement showed a sex chromosomal composition of XX or XY (36.4%) and 2 diploid embryos showed mosaicism of the opposite sex of XX and XY in blastomeres of the embryo (9.1%). One haploid embryo contained only one X-chromosome (4.5%). Four of another 11 embryos with a mixoploid chromosomal complement contained a haploid blastomere with a wrong sex chromosome (18.2%). In conclusion, assessment of nuclear status of 8- to 16-cell stage bovine embryos revealed that morphologically normal embryos had a considerable proportion of mixoploid blastomeres and sex chromosomal mosaicism; these could be the cause of discrepancies in the sex between biopsied single blastomeres and matched blastocysts by PCR.     

Rapid sexing of preimplantation bovine embryo using consecutive and multiplex polymerase chain reaction (PCR) with biopsied single blastomere.

The objective of this study was to establish a rapid and reliable PCR method for the sexing of 8- to 16-cell stage bovine embryos. The BOV97M and bovine 1.715 satellite DNA sequences were selected for amplification of male- and bovine-specific DNA, respectively. But the unequal number of copies of these two repetitive sequences required some modification of the multiplex PCR method. In consecutive and multiplex PCR, the first 10 PCR cycles were done with male-specific primer followed by an additional 23 cycles with bovine-specific primer. In this PCR method, the appearance of male- and bovine-specific bands was independent of the DNA concentration. This PCR method was applied successfully using groups of 8, 4, 2, and 1 blastomeres dissociated from the embryos, and the sexing efficiency was 100.0, 96.3, 94.3 and 92.1%, respectively. The coincident rate of sex determination between biopsied single blastomere and matched blastocyst was 90.0%. Therefore the developmental potential from 8- to 16-cell stage embryos to the blastocyst stage was not significantly different (P>0.2) for intact embryo (42.3%) than for demi-embryos (53.8%), suggesting that trauma to the demi-embryo caused by single-blastomere aspiration using a bevelled micropipette was very small. In conclusion, we developed a rapid (within 2 hours) and effective PCR method for the sexing of 8- to 16-cell stage bovine embryos using a single blastomere.     

Sexing of mouse preimplantation embryos by detection of Y chromosome-specific sequences using polymerase chain reaction.

Detection of genes known to be present on the mammalian Y chromosome was adapted for sexing mouse early embryos using the polymerase chain reaction (PCR) method. Sry and Zfy genes located in the sex-determining region of the Y chromosome were chosen for Y-specific target sequences, and DXNds3 sequence on the X chromosome was chosen for control. The two-step PCR method using two pairs of primers for each of the target sequences was employed for detecting the sequences. When DNAs of male and female mice were amplified with these primers, male-specific fragments were detected even in DNAs that were equivalent in amount to two cells. Mouse embryos at the two-cell stage were separated into two individual blastomeres, and one blastomere was karyotyped at the second cleavage. The remaining blastomere was subjected to PCR amplification immediately or after having been cultured for 48 h up to the morula stage. The Sry and Zfy sequences were detected in about half the embryos; detection of the Sry and Zfy sequences corresponded exactly to the presence of the Y chromosome, except in one sample of male morula in which embryos may have been lost before the PCR amplification. It is concluded that the sex of mouse preimplantation embryos can be accurately determined through detection of the Y-specific sequences using the two-step PCR method, even with the single blastomeres separated at the two-cell stage.     

Gender determination in single bovine blastomeres by polymerase chain reaction amplification of sex-specific polymorphic fragments in the amelogenin gene.

A sensitive technique for the sexing of bovine embryos was developed using polymerase chain reaction (PCR) amplification of the bovine amelogenin (bAML) gene on the X- and Y-chromosomes of Holstein dairy cattle. Cloning and DNA sequencing showed a 45.1% homology between the fifth intron of the bAML-X and bAML-Y gene with multiple deletions. A pair of sex-specific primers was designed to allow amplification of a single fragment of 467-bp from the X-chromosome of female cattle and two fragments of 467-bp and 341-bp from the X- and Y-chromosomes of male cattle. The primers were successfully applied to bovine sexing from single blastomeres isolated from day-6 to day-7 cow embryos by direct cell lysis and PCR. Our protocol of embryo sexing should be applicable to the diagnosis of defective genes in vitro in human embryos and in other domestic or recreational animals.     

Sexing single bovine blastomeres using TSPY gene amplification

The testis-specific protein Y-encoded gene (TSPY) is a Y-specific gene present in variable copy number in many mammalian species, including cattle. We tested the applicability of the TSPY gene as a Y-specific marker to predict preimplantation embryo sex in Nelore (Bos indicus) cattle. Two blastomeres were removed from each embryo. A total of 36 single blastomeres and the remaining cells of their 18 matched in vitro conceived embryos were screened for TSPY amplification by nested-PCR. The results obtained from a single blastomere and the remaining cells of the same embryo were concordant in all cases. All blastomeres (16/16) from eight embryos produced with sexed sperm (specific for production of male embryos) were TSPY-positive. We conclude that TSPY is a good male-specific marker, the usefulness of which is probably enhanced by the high copy number. Other methods that are less time-consuming, such as real-time PCR, could be improved with the use of the TSPY gene sequences to generate primers and/or probes. This is the first report to demonstrate the applicability of the TSPY gene for sexing single cells in cattle.     

Development of single mouse blastomeres enlarged to zygote size in conditions of nucleo-cytoplasmic synchrony

The following blastomeres were enlarged to the size of the zygote by one, two or three rounds of blastomere enucleation and electrofusion: (1) from the 2-cell stage (referred to as 2/1 embryos), (2) from the 4-cell stage (referred to as 4/1 embryos), (3) from the 8-cell stage (referred to as 8/1 embryos). Such single enlarged blastomeres developed into blastocysts in vivo in 55.5% (2/1), 28% (4/1) and 6.6% (8/1) of cases. Their mean cell numbers were 45.3, 24.5 and 13.0 in 2/1, 4/1 and 8/1 embryos, respectively. When a blastomere nucleus from another mouse strain (heterologous nucleus) was substituted for a blastomere's own (homologous) one, then fewer blastocysts were formed from 2/1 embryos (34.6%), but not from 4/1 and 8/1 embryos. Five young (10.4%) were born from 2/1 embryos with a homologous nucleus, and nine (8.3%) from 2/1 embryos with heterologous nuclei. Four young (7.1%) were born from 4/1 embryos with heterologous nuclei. No young were obtained from 8/1 embryos. Incorrect cavitation resulting in trophoblastic vesicles and false blastocyst formation was common in 4/1 embryos (18.7% of those with homologous nuclei and 41.3% with heterologous nuclei) and in 8/1 embryos (53.3% and 43.7%, respectively). The results show that neither enlargement to zygote size nor nucleo-cytoplasmic synchrony improve postimplantation development of 4- and 8-cell stage blastomeres when compared with less enlarged non-synchronous ones; therefore, it appears that an insufficient number of inner cell mass cells in blastocysts and not too small a size of isolated blastomeres precludes their postimplantation development.     

Sexing using single blastomere derived from IVF bovine embryos by fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) is a sensitive technique for molecular diagnosis of chromosomes on single cells and can be applied to sex determination of embryos. The objective has been to develop an accurate and reliable bovine Y chromosome-specific DNA probe in order to sex biopsed blastomeres derived from IVF bovine embryos by FISH. Bovine Y chromosome-specific PCR product derived from BtY2 sequences was labeled with biotin-16-dUTP (BtY2-L1 probe), and FISH was performed on karyoplasts of biopsed blastomeres and matched demi-embryos. Our FISH signal was clearly detected in nuclei of blastomeres of male embryos. FISH analysis of bovine embryos gave high reliability (96%) between biopsied blastomeres and matched demi-embryos. These results indicated that the BtY2-L1 bovine Y chromosome-specific FISH probe was an effective probe for bovine embryo sexing, and the FISH technique of probe detection could improve the efficiency and reliability.     

Nuclear transplantation of mouse fetal germ cells into enucleated two-cell embryos

Mouse fetal germ cells were fused with enucleated blastomeres of two-cell embryos. Donor germ cells were obtained from fetuses of albino CD-1 strain or pigmented F(1) (C57BL x CBA) female mice mated with the same strain males at 11.5 to 16.5 days post coitum. Recipient two-cell embryos, which were of a different strain from the donors, were obtained at 37 to 42 hours (Group 1), 42 to 47 hours (Group 2), and 47 to 52 hours (Group 3) after treatment with human chorionic gonadotropin (hCG). After removing the nucleus from one two-cell blastomere, a single germ cell was fused with the enucleated blastomere using the Sendai virus; the second blastomere was left intact. The reconstituted embryos were cultured for 3 days in vitro, to examine their developmental capacity. The fused blastomeres in Groups 1 and 2 did not divide, but a few transplanted blastomeres in Group 3 divided several times, and some of them developed into normal blastocysts. Most embryos developed into blastocysts from one blastomere, with an undivided blastomere remaining. Embryos developing into normal blastocysts or blastocysts with small blastomeres were transferred to the oviducts of Day-1 or the uteri of Day-3 pregnant albino CD-1 mice. None of the young showed any contribution of the germ cells, judging by the eye and coat colors and by the germ cells in the germ line following mating with albino mice. Possible reasons for failure of pluripotency of the germ cells are discussed here.     

Laser microbeam-induced DNA damage inhibits cell division in fertilized eggs and early embryos

DNA double-strand breaks are caused by both intracellular physiological processes and environmental stress. In this study, we used laser microbeam cut (abbreviated microcut or cut), which allows specific DNA damage in the pronucleus of a fertilized egg and in individual blastomere(s) of an early embryo, to investigate the response of early embryos to DNA double-strand breaks. Line type γH2AX foci were detected in the cut region, while Chk2 phosphorylation staining was observed in the whole nuclear region of the cut pronuclei or blastomeres. Zygotes with cut male or female pronucleus showed poor developmental capability: the percentage of cleavage embryos was significantly decreased, and the embryos failed to complete further development to blastocysts. The cut blastomeres in 2-cell, 4-cell, and 8-cell embryos ceased cleavage, and they failed to incorporate into compacted morulae, but instead underwent apoptosis and cell death at the blastocyst stage; the uncut part of embryos could develop to blastocysts, with a reduced percentage or decreased cell number. When both blastomeres of the 2-cell embryos were cut by laser microbeam, cell death occurred 24 h earlier, suggesting important functions of the uncut blastomere in delaying cell death of the cut blastomere. Taken together, we conclude that microbeam-induced DNA damage in early embryos causes compromised development, and that embryos may have their own mechanisms to exclude DNA-damaged blastomeres from participating in further development.     

Preimplantation diagnosis of the β1 integrin knockout mutation as a model for aneuploid gene testing

The autosomal beta1 integrin knockout mouse mutation was selected as a model to experimentally determine preimplantation diagnosis test reliability for autosomal gene deletions and duplications. In experiment 1, which analyzed 198 individually disaggregated single blastomeres, the observed test frequencies matched the mathematically predicted frequencies calculated from the independently derived values of 90% normal allele amplification, 92% mutant allele amplification, 4% alternate allele contamination, and 4% failure to transfer amplifiable target DNA into the PCR reaction mix. This experiment correctly predicted a normal embryonic phenotype in 143 (99.3%) of the 144 phenotypically normal autosomal recessive results. Experiment 2 compared single biopsied blastomere test results to test results on the remaining embryonic cells cultured 1 week until trophoblast outgrowth. Single biopsied blastomere analysis correctly predicted a normal autosomal recessive phenotype in 87 (98%) of the 89 embryos that would have been selected for implantation. Experiment 3 compared the PCR results of two biopsied blastomeres tested independently to the PCR result from the remaining cultured blastomeres to improve test reliability. Given that embryos would have been implanted only when two normal results were obtained, 17 of 17 phenotypically normal embryos would have been implanted from among the 44 embryos tested. These experiment 3 results are consistent with the mathematical prediction that about 99.9% of embryos implanted with two unaffected biopsied blastomere results would have had a phenotypically normal genotype.     

Cloning and sequencing of buffalo male-specific repetitive DNA: sexing of in-vitro developed buffalo embryos using multiplex and nested polymerase chain reaction

Buffalo Y-chromosome specific repetitive DNA (BuRY.I) was cloned and sequenced in order to develop a sensitive method for sexing of buffalo preimplantation stage embryos using polymerase chain reaction (PCR). A highly sensitive and reliable sex determination assay using a primary (BRY.I), nested (BuRYN.I) and multiplex (BuRYN.I, ZFX/ZFY) PCR was developed. The BRY.I and BuRYN.I primers are targeted to amplify Y-specific sequences, while the ZFX/ZFY loci was amplified to serve as a positive control for both male and female samples. Accuracy of the sex determination assay was initially verified with genomic DNA obtained from blood of known gender. Further sensitivity and reproducibility of the assay was examined using DNA obtained from 1 or 2 blastomeres to demi embryos. Altogether, 80 IVF-derived embryos ranging from the 2 to 4 cell to the blastocyst stage were used for sex determination. Definite and clear signals following PCR amplification were obtained from all embryo samples. Accuracy of assays was determined by comparing results from a single cell with those of blastocyst stage embryos, thereby indicating that 1 or 2 blastomeres from a preimplantation buffalo embryo is sufficient for sex determination by PCR. No misidentification was observed within the embryo samples using nested (BuRY.I), primary (BRY.I) and multiplex (BuRYN.I; ZFX/ZFY) PCR, suggesting that this technique is a highly reliable method for sexing buffalo embryos.     

Rapid sexing of bovine preimplantation embryos using loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method that amplifies a target sequence specifically under isothermal conditions. The product of LAMP is detected by the turbidity of the reaction mixture without electrophoresis. The objective of this study was to develop a rapid sexing method for bovine preimplantation embryos using LAMP. The first experiment was conducted to optimize the DNA extraction method for LAMP-based embryo sexing. The DNA of single blastomeres was extracted using three methods: heat, NaOH, and proteinase K-Tween 20 (PK-TW) treatments. Sexing was performed with two LAMP reactions, male-specific and male-female common reaction, after DNA extraction. The rates of correct determination of sex were 88.9-94.4%, with no difference among methods. The sensitivity and accuracy of LAMP-based embryo sexing were evaluated in the next experiment. The proportion of samples in which the sex was correctly determined was 75-100% for one to five biopsied cells. Lastly, in vivo-derived embryos were examined to verify the usefulness of LAMP-based embryo sexing, and some of these fresh, sexed embryos were transferred into recipient animals. The time needed for sexing was <1 h. The pregnancy rate was 57.4% and all calves born were of the predicted sex (12 male and 21 female). Therefore, LAMP-based embryo sexing accurately determined gender and is suitable for field application.     

Intravital selection of early mouse embryos by sex and karyotypic characteristics

The possibility of live karyotyping by a single blastomere isolated at the 2-, 4-, and 8-cell stage has been investigated. It is expedient to use to this end a single blastomere isolated from a 4-cell embryo. Three rest blastomeres formed normal morulae or blastocysts upon cultivation during 44 or 68 hrs. When the isolated blastomeres were cultivated for 14-16 hrs in a medium 0.5 micrograms/ml colcemide, 97% of blastomeres were at the metaphase stage and 72% of chromosome plates were suitable for karyotyping. The prospects of the method proposed in experimental embryology and for selection of the early embryos of farm animals by sex are discussed.     

Production of monozygotic twin calves using the blastomere separation technique and Well of the Well culture system

The present study was conducted to establish a simple and efficient method of producing monozygotic twin calves using the blastomere separation technique. To produce monozygotic twin embryos from zona-free two- and eight-cell embryos, blastomeres were separated mechanically by pipetting to form two demi-embryos; each single blastomere from the two-cell embryo and tetra-blastomeres from the eight-cell embryo were cultured in vitro using the Well of the Well culture system (WOW). This culture system supported the successful arrangement of blastomeres, resulting in their subsequent aggregation to form a demi-embryo developing to the blastocyst stage without a zona pellucida. There was no significant difference in the development to the blastocyst stage between blastomeres separated from eight-cell (72.0%) and two-cell (62.0%) embryos. The production rates of the monozygotic pair blastocysts and transferable paired blastocysts for demi-embryos obtained from eight-cell embryos (64.0 and 45.0%, respectively) were higher than those for demi-embryos obtained from two-cell embryos (49.0 and 31.0%, P<0.05). The separated demi-embryos obtained from eight-cell embryos produced by IVM/IVF of oocytes collected by ovum pick-up (OPU) from elite cows and cultured in wells tended to have a higher pregnancy rate (78.9% vs. 57.1%) and similar monozygotic twinning rate (40.0% vs. 33.3%) compared with monozygotic twin blastocysts obtained by the conventional bisection of in vivo derived blastocysts. In conclusion, producing twins by separation of blastomeres in OPU-IVF embryos, followed by the WOW culture system, yielded viable monozygotic demi-embryos, resulting in high rates of pregnancy and twinning rates after embryo transfer.     

Developmental potential of isolated blastomeres from early murine embryos

Experiments were designed to evaluate the effect of blastomere separation on blastocoele formation and development of viable fetuses. Two-cell and four-cell murine embryos were dissociated into individual blastomeres and cultured to the blastocyst stage. For embryos of both stages, zona removal and blastomere separation reduced (P<0.05) the number of viable embryos at the onset of culture and reduced (P<0.01) the frequency of continuation of development of blastomeres to the blastocyst stage. Attempts to repeatedly split two-cell stage embryos decreased in vitro development to blastocysts. The number of cells in two-cell embryos that were cultured to blastocyst was not different for control (64.8 +/- 11.5) or for two-cell embryos cultured without the zona pellucida (60.9 +/- 10.1) but was reduced (P<0.01) for one-half embryos that were cultured to blastocysts (35.6 +/- 10.6). The cell number of blastocysts obtained from dissociated four-cell (1/4) embryos (17.4 +/- 1.4) was similarly reduced (P<0.01). In vivo development was assessed after cultured embryos were transferred to the uteri of day 3 pseudopregnant females. Zona free intact embryos (2/36, 6%) and zona free half embryos (7/36; 19%) developed less frequently (P<0.05) than intact controls (45/100). Noncultured morula briefly exposed to pronase to thin the zona had similar impaired development. Embryos with thinned zona or no zona developed less frequently (21/82, 2/72 respectively, P<0.05) than nonpronase-treated controls (50/83).     

Generation of transgenic rabbits by the novel technique of chimeric somatic cell cloning

A novel technique of chimeric somatic cell cloning was applied to produce a transgenic rabbit (NT20). Karyoplasts of transgenic adult skin fibroblasts with Tg(Wap-GH1) gene construct as a marker were microsurgically transferred into one, previously enucleated, blastomere of 2-cell non-transgenic embryos, while the second one remained intact. The reconstructed embryos either were cultured in vitro up to the blastocyst stage (Experiment I) or were transferred into recipient-females immediately after the cloning procedure (Experiment II). In Experiment I, 25/102 (24.5%) embryos formed blastocysts from whole embryos and 46/102 (44.12%) embryos developed to the blastocyst stage from single non-operated blastomeres, while the reconstructed blastomeres were damaged and degenerated. Thirteen (12.7%) embryos did not exceed 3- to 4-cell stages and 18 (17.7%) embryos were inhibited at the initial 2-cell stage. Out of 14 blastocysts which were subjected to molecular analysis, the transgene was detected in the cells of 4 blastocysts. In Experiment II, 163/217 (75.0%) embryos were transferred into 9 pseudopregnant recipient-rabbits (an average of 18 embryos per recipient). Four recipient-females (44.4%) became pregnant and delivered a total of 24 (14.7%) pups. Molecular analysis confirmed that two pups (1.2%), one live and one stillborn, showed a positive transgene signal. Live transgenic rabbit NT20 appeared healthy and anatomically as well as physiologically normal. The results of our experiments showed that transgenic adult skin fibroblast cell nuclei, which have been introduced into the cytoplasmic microenvironment of single enucleated blastomeres from 2-cell stage rabbit embryos, are able to direct the development of chimeric embryos not only to the blastocyst stage but also up to term.     

运用PCR对小鼠植入前胚胎进行性别诊断

根据C57BL6小鼠Y染色体重复序列145C5的碱基顺序,设计并合成一对引物,运用PCR扩增昆明白小鼠入前胚胎卵裂球DNA,以确定其性别,共对108枚活检胚胎的相应卵裂球进行了性别诊断,获雄性胚46枚,雌性胚62枚,移植后分别获雄性仔鼠4只,准确率100％（4／4）,雌性仔鼠9只,准确率70％（9／13）,本研究结果表明小鼠Y染色体重复序列145C5的碱基顺序在C57BL6小鼠和昆明白小鼠中基本一致,为农牧业动物进行性别选择和运用PCR进行单基因病植入前遗传学诊断提供了方法学基础。     

Blastomeres from somatic cell nuclear transfer embryos are not allocated randomly in chimeric blastocysts

A marker has been developed to allow detection of blastomeres that originate from embryos produced by nuclear transfer (NT) of genetically engineered fetal fibroblasts. A plasmid (phEFnGFP) was constructed with a G418 resistance cassette for selection in fibroblasts and a nuclear localized green fluorescent protein (nGFP) expression cassette that expresses in every cell of day-6, -7, and -8 bovine embryos. This construct was utilized to follow the blastomere distribution in aggregation chimeras produced from fertilized embryos (in vitro produced, IVP) or parthenotes and NT embryos. Fluorescent and nonfluorescent NT embryos were aggregated early on day 4 and evaluated on day 8. Nuclei of blastomeres that carried the transgene were fluorescent under both UV epifluorescence (Hoechst 33342) and blue epifluorescence (nGFP). There was no bias in the distribution of green fluorescent blastomeres in the inner cell mass (ICM) or trophectoderm in NT<>NT chimeras. However, there was a strong bias for NT blastomeres to populate the ICM when aggregated with IVP embryos or parthenotes. There was also a strong bias against NT blastomeres in the trophectoderm when aggregated to IVP embryos. However, the bias against NT blastomeres in the trophectoderm was significantly less (p < 0.05) when aggregated with parthenotes as compared to aggregation with IVP embryos. In NT<>NT aggregates, no chimeric embryos were produced that had an ICM composed of blastomeres from a single origin. However, in NT<>Parthenote aggregates, 67% of the blastocysts had an ICM composed exclusively of NT origin. The remaining blastocysts ranged from 0% to 83% of the ICM that expressed nGFP. Similarly, in NT<>IVP aggregates 50% of the blastocysts had an ICM composed exclusively of NT origin. The remaining blastocysts ranged from 19% to 71% of the ICM being of NT origin. We conclude that production of divaricated chimeras from NT origin is feasible. Other applications of this technology are discussed.     

Efficient production of sex-identified and cryosurvived bovine in-vitro produced blastocysts

To establish a protocol for production of bovine in-vitro produced (IVP) blastocysts that were sex-identified and cryopreserved, we examined the sexing efficiency and accuracy of Day-3 and Day-4 embryos by polymerase chain reaction (PCR), the development of the biopsied embryos into Day-7 blastocysts and the freezability of these blastocysts by vitrification in gel-loading tips. One or two blastomeres were isolated from IVP embryos at either the 8-cell or 16-cell stage (Days 3 and 4, respectively) by a pressing-out method, and were then subjected to primer extension preamplification (PEP)-PCR. The successful sex-identification rate of biopsied samples amplified, purified and analyzed for sex by a second PCR (88.9%) was higher than that of those amplified and analyzed without purification (32.0%). Developmental rates into Day-7 blastocysts of biopsied embryos (Day-3, 65.5%; Day-4, 70.8%) were similar to those of non-biopsied control embryos (Day-3, 74.5%; Day-4, 65.1%). Total cell numbers and the inner cell mass (ICM) ratio of blastocysts derived from biopsied embryos were also comparable with those of control embryos. Blastocysts were vitrified-warmed in the presence of 20% DMSO, 20% ethylene glycol and 0.6M sucrose using gel-loading tips as containers. The proportions of biopsied blastocysts that were hatched or hatching rates after warming were high, regardless of the biopsy time (Day-3, 94.1%; Day-4, 91.9%), similar to the rates for control blastocysts (Day-3, 97.5%; Day-4, 96.9%). In conclusion, a protocol that allows sexing of Day-3 and Day-4 bovine embryos without compromising either the developmental ability to the blastocyst stage or freezability of Day-7 blastocysts was developed.     

小鼠2—细胞胚胎单个卵裂球体外培养及其发育形成囊胚的玻璃化冷冻保存技术的研究（简报）

The present experiments were designed to study the effects of glucose, EDTA, glutamine on the in vitro development of single blastomeres from 2-cell embryos in mouse, and the efficiency of cryopreservation of blastocysts from single blastomers with different vitrification. Single blastomeres derived from female ICR x male BDF1 2-cell embryos were cultured in mKRB with or without glucose, EDTA and glutamine, respectively. The expanded blastocyst rates were significantly different between in mKRB with glucose and without glucose (34% vs 65%); The blastomeres were cultured in mKRB with EDTA and glutamine but glucose, the expanded blastocyst rate (90%) was significantly higher than other groups. The blastocysts derived from single blastomeres were vitrified in liquid nitrogen after equilibration in GFS40 for 0.5-2 min, the survival rate 24%-51%. The blastocysts were pretreated in mPBS with 10% glycerol for 5 min, followed by exposure to GFS40 at 25 degrees C for 0.5 min, then vitrified in liquid nitrogen(two-step method), the survival rate was 61%. However, the survival rates increased to 64% and 70% when the blastocysts were vitrified(one-step method) ater equilibration in EFS40 at 25 degrees C for 0.5-1 min.