Effect of nucleocytoplasmic ratio on the in vitro porcine embryo development after in vitro fertilization or parthenogenetic activation.

This study was conducted to examine whether the nuclear to cytoplasmic (N/C) ratio had any influence on the timing of embryo compaction and blastocoel formation, as well as formation rate and quality of blastocyst. First, we produced embryos with increased N/C ratio by removal of approximately one-third of the cytoplasm and with decreased N/C ratio by doubling the oocyte cytoplasm with an enucleated oocyte. The initiation of compaction and cavitation in reduced cytoplasm group was significantly earlier (P < 0.05) compared with the control and doubled cytoplasm groups. The rate of blastocysts in the reduced cytoplasm and doubled cytoplasm groups was significantly lower (P < 0.05) compared with the control group. Blastocyst quality in terms of total cell number in the reduced cytoplasm group was significantly lower (P < 0.05) compared with the doubled cytoplasm group, but not different from the control group. Next, we produced embryos with various N/C ratios by oocyte fusion combined with cytochalasin D treatment. The onset of compaction and cavitation in the 2N/2C group (decreased N/C ratio) was significantly delayed (P < 0.05) or had the tendency to be delayed (P = 0.064), respectively, compared with the control group (2N/1C). A significantly higher rate of blastocyst was observed in the 4N/2C group compared with the 1N/1C group (P < 0.05) but not different from the remaining groups. These results demonstrated that an increase in N/C ratio caused an earlier occurrence of morula compaction and blastocyst formation in both in vitro fertilization (IVF) and parthenogenetically activated pig embryos.

Excess polyspermy reduces the ability of porcine oocytes to promote male pronuclear formation after in vitro fertilization.

Male pronucleus (MPN) formation is a very important physiological event during fertilization, which affects in vitro production of transferrable embryos. The aim of this study was to find out the correlation between the number of penetrated sperm and the occurrence of failure of MPN formation in porcine oocytes. In vitro matured porcine oocytes were fertilized in vitro with frozen epididymal sperm. Two different frozen sperm lots were tested in this study, which were different in terms of polyspermy rates. The numbers and the status of penetrated sperm in oocytes were evaluated 10 h after insemination. Under high polyspermy condition, the polyspermy rate was 83.5% with an average mean of 3.5 sperms per penetrated oocyte, whereas the percentage of polyspermy was 65.5% with an average mean of 2.4 sperms per penetrated oocyte under moderate polyspermic condition. Correlation analysis revealed a negative correlation between the number of penetrated sperm and their MPN formation percentage both in the sperm lot of high polyspermy (R = -0.560, p < 0.05) and in the sperm lot of moderate polyspermy (R = -0.405, p < 0.05) which suggests that penetration of excessive spermatozoa disables the oocyte cytoplasm to promote MPN formation.

Silkworm recombinant bovine zona pellucida protein 4 (bZP4) as a potential female immunocontraceptive antigen; impaired sperm-oocyte interaction and ovarian dysfunction.

Porcine zona pellucida proteins (ZPs) have been utilized as female immunocontraceptive antigens. The purpose of this study was to explore the potential use of silkworm recombinant bovine ZP4 as an alternative. When the protein was injected with monophosphoryl lipid A (MPL) - an immuno-stimulative agent - into two female goats, marked elevation of the anti-ZP4 titer was detected. Application of the purified specific IgG to a porcine in vitro fertilization system reduced the sperm penetration rate. In one goat, the cyclic profile of serum progesterone disappeared as the anti-ZP4 titer increased. Histological examination of the ovaries revealed degeneration of antral follicles with sparse infiltration of inflammatory cells in the theca, indicating that autoimmune oophoritis had been induced. Together, the present results suggest that recombinant ZP4 disturbs fertilization and exerts a pathogenic effect on follicle development in goats, thus indicating its potential as a female immunocontraceptive antigen.

Successful activation of rat T lymphocytes by sperm specific antigens in vitro.

Autoimmune orchitis is a condition related to cellular immunity. A disease model involving transfer of T lymphocytes activated by known antigens would be useful for defining pathogenical molecules. Since no method for activating rat T cells using specific antigens is available, we started the study to develop the method. T cells were collected from draining lymph nodes of immunized rats, then co-cultured with syngeneic splenocytes as antigen-presenting cells (APC) in antigen-supplemented medium (= stimulation). The cells were then incubated in medium without antigens and APC (= resting). Repetitive stimulation and resting increased the number of the T cells more than 100-fold. The antigen-specific activation was demonstrated by cell proliferation assay and ELISA assay for interferon gamma. Flow cytometry revealed that > 95% of the cells expressed tumor necrosis factor alpha, a cytokine responsible for autoimmune orchitis. The present method will provide a new procedure to evaluate antigenicity of sperm molecules.

Doubling of the cytoplasm volume improves the developmental competence of porcine oocytes injected with freeze-dried somatic cells.

In the present study using pig cells, we examined the effect of the cryoprotectant trehalose on the DNA integrity of freeze-dried cells. We then investigated whether donor cell types and storage duration had impact on DNA integrity in freeze-dried cells or developmental competence of oocytes injected with freeze-dried somatic cells. We also examined whether double cytoplasm nuclear transfer (DCNT) would improve developmental competence of such oocytes. Furthermore, using a PCR-based method for sex identification, we determined whether the blastocysts obtained had actually been generated from the freeze-dried cells. It was found that, for a short storage duration at low temperature, trehalose had no beneficial effect on protection from DNA damage, and that donor cell type had no effect on the DNA integrity of freeze-dried somatic cells or the developmental competence of oocytes injected with them. We also confirmed that all of the blastocysts obtained following nuclear transfer were of freeze-dried somatic cell origin. Storage of freeze-dried somatic cells for up to 1 year at low temperature did not degrade DNA integrity in comparison with storage for 1 month, 1 week or 1 day. Following injection of freeze-dried cells, the proportion of oocytes that developed to blastocysts after storage for up to 1 year was similar to that after storage for 1 month, 1 week or 1 day. Moreover, DCNT significantly improved the developmental competence of oocytes treated in this way. In summary, using DCNT, we have demonstrated that freeze-dried porcine somatic cells subjected to long-term storage at 4 °C have nearly the same potential to develop to blastocysts as non-freeze-dried cells.

Pluripotency-associated genes reposition during early embryonic developmental stages in pigs.

We examined the allelic expression and positioning of two pluripotency-associated genes, OCT4 and SOX2, and two housekeeping genes, ACTB and TUBA, in 4- and 8-cell porcine embryos utilizing RNA and DNA fluorescence in situ hybridization (FISH) in single blastomeres. The proportion of blastomeres expressing SOX2 bi-allelically increased from 45% at the 4-cell stage to 60% at the 8-cell stage. Moreover, in 8-cell embryos, SOX2 was expressed bi-allelically in significantly more blastomeres than was the case for OCT4, and this was associated with a tendency for SOX2 alleles to move toward the nuclear interior during 4- to 8-cell transition. However, the radial location of OCT4 alleles did not change significantly during this transition. The locations of active and inactive alleles based on DNA and RNA FISH signals were also calculated. Inactive OCT4 alleles were located in very close proximity to the nuclear membrane, whereas active OCT4 alleles were more centrally disposed in the nucleus. Nevertheless, the nuclear location of active and inactive SOX2 alleles did not change in either 4- or 8-cell blastomeres. Our RNA and DNA FISH data provide novel information on the allelic expression patterns and positioning of pluripotency-associated genes, OCT4 and SOX2, during embryonic genome activation in pigs.

Selection based on morphological features of porcine embryos produced by in vitro fertilization: Timing of early cleavages and the effect of polyspermy.

The aim of this study was to examine whether a morphological approach is efficient for selecting high-quality porcine embryos produced by in vitro fertilization (IVF) under high polyspermy conditions. Frozen-thawed Meishan epididymal spermatozoa showing moderate and high polyspermy were subjected to IVF (1 × 105  sperms/ml). Under conditions of moderate polyspermy, 4-cell embryos selected at 48 hr after IVF (single selection) and 8-cell embryos selected at 79 hr after IVF from the collected 4-cell embryos (double selection) showed high developmental competence. Likewise, 4- and 8-cell embryos produced by IVF under high polyspermy conditions also showed high competence for development to blastocysts. However, blastocysts derived from high polyspermy conditions had significantly fewer cells than those produced under moderate polyspermy conditions. Furthermore, the frequency of nuclear and chromosomal abnormalities in 4- and 8-cell embryos produced under conditions of high polyspermy was significantly (p < .05) higher in comparison to moderate polyspermy conditions. These findings suggest that although high polyspermy affects the frequency of nuclear and chromosomal anomalies in porcine IVF embryos, subsequent selection based on morphological features of 4- and 8-cell embryos even under high polyspermy conditions, could be an alternative option for selecting porcine IVF embryos with high development ability.

Combined refinements to somatic cell nuclear transfer methods improve porcine embryo development.

The discovery of how to utilize CRISPR (clustered, regularly interspaced, short, palindromic repeats)-Cas (CRISPR-associated) systems for genome modification has accelerated development of the field of genome editing, especially in large animals such as pigs. The low efficiency of somatic cell nuclear transfer (SCNT) is now becoming a major obstacle in the production of genome-edited animals via cell-mediated approaches and improving efficacy of this technique is crucial. In this study, we propose a few simple modifications to a zona-free SCNT protocol that are effective to produce numerous high-quality blastocysts. To refine the SCNT protocol we modified the following steps/factors: 1) culture medium for SCNT embryos, 2) chemical treatment to prevent precocious activation of the manipulated/reconstructed oocytes and 3) donor cell serum starvation treatment. Although changes in each of these steps only resulted in small improvements, the combination of all modifications altogether significantly enhanced developmental competence of SCNT embryos. Our modified method yielded approximately three times greater blastocyst formation rates. Moreover, resulting blastocysts had roughly twice as many cells as compared to blastocysts produced by the conventional SCNT method. With these significant in vitro improvements, our refined SCNT method is potentially suited for use in the production of genome edited pigs.

Vitrification of porcine cumulus-oocyte complexes at the germinal vesicle stage does not trigger apoptosis in oocytes and early embryos, but activates anti-apoptotic Bcl-XL gene expression beyond the 4-cell stage.

The aim of the present study was to clarify whether or not our vitrification procedure at the germinal vesicle (GV)-stage triggers the apoptotic cascade in oocytes and subsequent embryos. Immature porcine cumulus-oocyte complexes were either vitrified and warmed (vitrified group) or subjected to cryoprotectant agents (CPA group) or cultured without any treatment (control). Oocytes of all treatment groups were subjected to in vitro maturation (IVM), fertilization, and embryo culture. Apoptosis was assayed in live oocytes at the end of IVM culture and in cleavage-stage embryos after in vitro fertilization (IVF). We detected similar frequencies of DNA fragmentation, levels of caspase activity, phosphatidylserine externalization, and mRNA levels for pro-apoptotic Bax and CASP3 genes in oocytes at the end of IVM and in early embryos among all groups. However, in the vitrified group, the anti-apoptotic Bcl-XL gene was upregulated in 4-8 cell embryos, which caused an 8-fold significant increase in the Bcl-XL/Bax mRNA ratio compared with the control and CPA groups (P < 0.05). In conclusion, vitrification of porcine oocytes at the GV stage by our method did not trigger the apoptotic cascade in oocytes and subsequent embryos but triggered the upregulation of the anti-apoptotic Bcl-XL gene in embryos.

Developmental ability of oocytes retrieved from Meishan neonatal ovarian tissue grafted into nude mice.

Ovarian xenografting makes it possible to obtain oocytes with fertilization ability from immature pigs of Western breeds. In this study, we applied these methods to the Meishan, an indigenous Chinese pig breed, and investigated the developmental competence of oocytes grown in their neonatal tissue after grafting into nude mice. First, mice harboring neonatal ovarian tissue were infused with follicle stimulating hormone (FSH) (62.5 U/ml) for 13 days starting at 10, 30, and 60 days after vaginal opening (D10-, D30-, and D60-FSH groups, respectively). Development of antral follicles and their oocytes was most enhanced in the D60-FSH group. For the next step, we examined the in vitro maturation ability of the oocytes recovered from host mice after infusion with FSH at a dose of 62.5 U/ml or 125 U/ml (FSH-62.5 or -125 group) for 13 days starting at 60 days after vaginal opening. Many more oocytes with maturation ability were obtained from the FSH-125 group. The FSH-125 mature oocytes were fertilized in vitro, as shown by formation of male and female pronuclei, but did not reach the blastocyst stage. These results indicate that Meishan neonatal ovaries are able to produce oocytes with fertilization ability after being grafted into nude mice.

Embryo production by intracytoplasmic injection of sperm retrieved from Meishan neonatal testicular tissue cryopreserved and grafted into nude mice.

Testicular xenografting, combined with cryopreservation can assist conservation of the genetic diversity of indigenous pigs by salvaging germ cells from their neonatal testes. Using Meishan male piglets as an example, we examined whether testicular tissue would acquire the ability to produce sperm after cryopreservation and grafting into nude mice (MS group). For comparison, testicular tissue from neonatal Western crossbreed male piglets was used (WC group). Sixty days after xenografting (day 0 = grafting), MS grafts had already developed seminiferous tubules containing sperm, whereas in the WC grafts, sperm first appeared on day 120. The proportion of tubules containing spermatids and sperm was higher in the MS group than in the WC group between days 90 and 120. Moreover, in vitro-matured porcine oocytes injected with a single sperm obtained from the MS group on day 180 developed to the blastocyst stage. The blastocyst formation rate after injection of the xenogeneic sperm was 14.6%, whereas the ratio in the absence of such injection (attributable to parthenogenesis) was 6.7%. Thus, cryopreserved Meishan testicular tissue acquired spermatogenic activity in host mice 60 days earlier than Western crossbreed tissue. Such xenogeneic sperm are likely capable of generating blastocysts in vitro.

Maturation ability after transfer of freeze-dried germinal vesicles from porcine oocytes.

The purpose of this study was to examine whether freeze-dried germinal vesicles (GV) can be matured in vitro after being injected into enucleated fresh oocytes in pigs as an alternative method for conservation of genetic resources. Although no reduction of the size of GV (p = .094), resveratrol treatment significantly enhanced the survival rates following GV transfer (GVT) (p < .001). Supplementation with 100 or 200 mmol/L trehalose in freeze-drying medium significantly increased the proportions of GVs with intact nuclear membrane and DNA integrity compared with the control group. Following transfer of freeze-dried GVs into enucleated fresh oocytes, the proportion of reconstructed oocytes reached the metaphase-II stage (2.4% ± 1.4%) was significantly lower (p < .05) than that of the in vitro matured control group (83.2% ± 2.5%), it was comparable with the GVT control group (7.4% ± 2.7%). The rates of freeze-dried GVs with intact nuclear membrane and DNA stored at -20°C for 5 days were significantly higher (p < .05) than those at 4°C and room temperature. The rates of intact nuclear membrane and DNA in the freeze-dried GV stored for 15 or 30 days at -20, 4°C and RT were not significantly different. In conclusion, matured oocytes were produced derived from freeze-dried GVs.

Sucrose assists selection of high-quality oocytes in pigs.

We investigated whether high-quality in vitro matured (IVM) oocytes can be distinguished from poor ones based on the morphological changes after treatment with hyperosmotic medium containing 0.2 mol/L sucrose in pigs. We hypothesize that IVM oocytes maintaining round shape have higher quality than mis-shapened oocytes following dehydration. Oocyte quality was verified by determining embryonic developmental competence using in vitro fertilization, nuclear transfer and parthenogenetic activation. In all cases, the round oocytes had greater (p < .05) developmental competence than that of mis-shapened oocytes in terms of blastocyst rate and total cell number in blastocysts obtained after 6 days of in vitro culture. We also confirm that round aged oocytes are higher in quality than mis-shapened aged oocytes. In an attempt to find out why high-quality oocytes maintain a round shape whereas poorer oocytes become mis-shapened following sucrose treatment, we examined the arrangement of actin microfilaments and microtubules. Abnormal organization of these cytoskeletal components was higher (p < .05) in mis-shapened oocytes compared to round oocytes after 52 hr of IVM. In conclusion, sucrose treatment helps selection of high-quality oocytes, including aged oocytes, in pigs. Abnormal cytoskeleton arrangements partly explain for low developmental competence of mis-shapened oocytes.

Ultrasound study of fetal movements in singleton and twin pregnancies at 12-19 weeks.

Objective To evaluate fetal behavioral differences between singleton and twin fetuses before 20 weeks of gestation using four-dimensional (4D) ultrasound. Methods 4D ultrasound was used to examine fetal movements in 58 singleton and 48 twin normal fetuses at 12-19 weeks. The frequencies of eight fetal movements were assessed through 15-min recordings. The fetuses were divided into two gestational age groups (12-13 and 14-19 weeks) to evaluate the changes with advancing gestation in twin versus singleton fetuses. Results Arm and general movements were the most frequent movements in singleton fetuses, whereas only general movement was significantly more frequent than the other seven fetal movements in twin fetuses at 12-13 weeks. At 14-19 weeks, frequencies of arm and leg movements were significantly higher than those of the other six movements in singleton fetuses, while only arm movement was significantly more frequent than the other fetal movements in twin fetuses. Comparisons of fetal movements between singleton and twin fetuses revealed that only arm movement showed a significant difference at 12-13 weeks, while the frequencies of all movements in singleton fetuses were significantly higher than those in twin fetuses at 14-19 weeks. Conclusion Our results suggest that the limitation of available space and crowding of twin fetuses with advancing gestation may have a marked impact on twin fetal movements compared with singleton fetuses, even in the first half of pregnancy. Further studies are needed to assess whether decreased fetal movements in twin pregnancy can affect fetal and neonatal development and maturation before and after birth.

Establishment of a strain of haemophilia-A pigs by xenografting of foetal testicular tissue from neonatally moribund cloned pigs.

Grafting of testicular tissue into immunodeficient mice makes it possible to obtain functional sperm from immature donor animals that cannot be used for reproduction. We have developed a porcine model of human haemophilia A (haemophilia-A pigs) by nuclear transfer cloning from foetal fibroblasts after disruption of the X-linked coagulation factor VIII (F8) gene. Despite having a recessive condition, female F8+/- cloned pigs died of severe bleeding at an early age, as was the case for male F8-/Y cloned pigs, thus making it impossible to obtain progeny. In this study, therefore, we produced sperm from F8-/Y cloned pigs by grafting their foetal testicular tissue into nude mice. Two F8+/- female pigs were generated from oocytes injected with xenogeneic sperm. Unlike the F8+/- cloned pigs, they remained asymptomatic, and delivered five F8-/Y and four F8+/- pigs after being crossed with wild-type boars. The descendant F8-/Y pigs conserved the haemophilia phenotype. Thus, the present F8+/- pigs show resolution of the phenotypic abnormality, and will facilitate production of F8-/Y pigs as founders of a strain of haemophilia-A pigs for the development of new therapeutics for haemophilia A. This strategy will be applicable to other genetically modified pigs.

Production of sperm from porcine fetal testicular tissue after cryopreservation and grafting into nude mice.

A major goal of testicular xenografting is to salvage germ cells from immature animals that cannot be used for reproduction and generate their offspring. In this study, we investigated whether porcine fetal testicular tissue would acquire the ability to produce sperm with full developmental competence after they had been cryopreserved and grafted into nude mice. Testicular fragments from fetuses at 35, 55 and 90 days postartificial insemination (dpi) were vitrified and stored in liquid nitrogen. Immediately after warming, testicular fragments at each fetal stage were transplanted under the back skin of castrated nude mice (Crlj:CD1-Foxn1nu) (35-, 55- and 90-dpi groups, respectively) (day 0 = grafting). Before grafting, the testicular fragments contained seminiferous cords consisting of only gonocytes and Sertoli cells. Histological analyses of the testicular grafts revealed that the differentiation of seminiferous tubules was largely dependent on the time after grafting, and not on donor age. On day 180 in each group, 10-20% of the total number of tubule/cord cross-sections examined had germ cells that had progressed beyond the spermatogonial stage. Fewer than 5% of tubule cross-sections contained elongated spermatids or sperm. Between days 360 and 420, tubule differentiation advanced further, until more than 45% of the tubule cross-sections contained elongated spermatids or sperm. Sperm were recovered for the first time from a single mouse in the 55-dpi group on day 180, although on days 360-420 sperm were recovered from most mice in all of the groups. Serum concentrations of inhibin and testosterone in host mice in all of the groups were higher than those in castrated mice that had received no testicular grafts. Single sperm collected from mice in each group on day 300 or later were injected into individual in vitro-matured oocytes, and these sperm-injected oocytes were transferred to the oviducts of 2 or 3 estrus-synchronized recipient gilts. None of the recipients in any of the groups produced piglets. The present results clearly indicate that porcine fetal testes during the gestational period acquire endocrine and exocrine functions after being cryopreserved and grafted into nude mice. However, the ability of xenogeneic sperm derived from fetal testis to generate piglets was not confirmed in the present study.

Improvement of the developmental competence of porcine oocytes collected from early antral follicles by cytoplast fusion.

In the present study, we propose an alternative technique called cytoplast fusion to improve the maturation rate and developmental competence of growing oocytes collected from early antral follicles in pigs. We examined whether the fusion of a growing oocyte with the cytoplast from a fully-grown oocyte (CFR group) could better promote maturation and developmental competence of the growing oocyte compared to germinal vesicle (GV) transfer (GVTR group). After 44 h of in vitro maturation (IVM), most growing oocytes (GR group) were still arrested at the GV stage (64.0 ± 5.1%); this number was significantly higher (P < 0.01) than that of the other groups. No matured oocyte was observed in the GR group. The maturation rate of GVTR oocytes was significantly improved (18.8 ± 3.5%) compared with that of growing oocytes. The proportion of oocytes that reached the metaphase-II (M-II) stage in the CFR group (37.8 ± 2.0%) was significantly higher (P < 0.05) than that in the GVTR group, although still lower than that in the control group (75.2 ± 4.4%). No blastocyst was derived from growing oocytes. Among in vitro fertilized GVTR oocytes, 3.0 ± 1.9% developed into blastocysts; however, this percentage showed an insignificant increase compared with the GR group. On the other hand, the percentage of CFR embryos that developed into blastocysts (12.0 ± 4.3%) was significantly higher than that of GR embryos (0.0%), although still lower than that of control embryos (27.0 ± 5.5%). Total cell number in blastocysts in the GVTR group (23.3 ± 6.9) was significantly lower (P < 0.05) than that in the control group (50.4 ± 5.0). Meanwhile, the total cell number in blastocysts derived from CFR oocytes (36.3 ± 4.8) was comparable to that of the control group. In summary, cytoplast fusion significantly improves maturation rate and developmental competence of growing oocytes compared with GV transfer.

Overabundance of sika deer and immunocontraception.

The impact of deer overabundance is a worldwide problem. Along with habitat expansion and population increase, damage by sika deer to the forest ecosystem and agriculture has become a serious issue in Japan. Deer also transmit a number of diseases and parasites to humans and livestock. The overabundance of deer is a result of their strong fecundity, and therefore the present situation should, in theory, be tackled by experts in reproductive biology.

Development of a lipopolysaccharide (LPS)-supplemented adjuvant and its effects on cell-mediated and humoral immune responses in male rats immunized against sperm.

Supplementation with lipopolysaccharide (LPS) from non-pathogenic Escherichia coli was found to enhance the adjuvant effects of a veterinary vaccine adjuvant (ISA 71VG®). Sperm immunization using 71VG as an adjuvant in the immature period induced infertility in 25% of male rats, whereas this increased to 62.5% after immunization with 71VG + LPS or Freund's complete adjuvant (FCA). Mean testicular weight of non-sterile males in the 71VG + LPS group was significantly lower than that in the 71VG or FCA group. Histological examination of testicular tissue from sterile males demonstrated severe impairment of spermatogenesis due to experimental autoimmune orchitis, a cell-mediated autoimmune condition. The serum anti-sperm titer was elevated in the three sperm-immunized groups relative to male rats treated with adjuvant alone, but the titer was higher in the 71VG + LPS and FCA groups than in the 71VG group. We consider that this LPS-supplemented adjuvant stimulates both humoral and cell-mediated immune responses to an extent comparable to FCA.

Effects of polyethylene glycol and a synthetic ice blocker during vitrification of immature porcine oocytes on survival and subsequent embryo development.

We evaluated the effects of polyethylene glycol (PEG) and Supercool X-1000 (SC) as supplements during the vitrification of immature cumulus-enclosed porcine oocytes in a solution based on 17.5% ethylene glycol + 17.5% propylene glycol. After warming, the oocytes were subjected to in vitro maturation, fertilization and embryo culture. In Experiment 1, equilibration and vitrification solutions were supplemented with or without 2% (w/v) PEG (PEG+ and PEG-, respectively). The survival rate, cleavage and blastocyst development were similar between PEG+ and PEG- groups; however, all values were lower than those in the non-vitrified control. In Experiment 2, vitrification solution was supplemented with or without 1% (v/v) SC (SC+ and SC-, respectively). The percentages of survival and blastocyst development were similar between SC+ and SC- groups but lower than those in the non-vitrified control. The percentage of cleavage in the SC- group was significantly lower than the control and the SC+ groups, which were in turn similar to one another. In both experiments, the cell numbers in blastocysts were not significantly different among the non-vitrified and vitrified groups. In conclusion, PEG did not improve oocyte survival and embryo development, whereas SC improved the ability of surviving oocytes to cleave but not to develop into blastocysts.