Derivation and transcriptional profiling analysis of pluripotent stem cell lines from rat blastocysts

Embryonic stem （ES） cells are derived from blastocyst-stage embryos. Their unique properties of self-renewal and pluripotency make them an attractive tool for basic research and a potential cell resource for therapy. ES cells of mouse and human have been successfully generated and applied in a wide range of research. However, no genuine ES cell lines have been obtained from rat to date. In this study, we identified pluripotent cells in early rat embryos using specific antibodies against markers of pluripotent stem ceils. Subsequently, by modifying the culture medium for rat blastocysts, we derived pluripotent rat ES-like cell lines, which expressed pluripotency markers and formed embryoid bodies （EBs） in vitro. Importantly, these rat ES-like cells were able to produce teratomas. Both EBs and teratomas contained tissues from all three embryonic germ layers. In addition, from the rat ES-like cells, we derived a rat primitive endoderm （PrE） cell line. Furthermore, we conducted transcriptional profiling of the rat ES-like cells and identified the unique molecular signature of the rat pluripotent stem cells. Our analysis demonstrates that multiple signaling pathways, including the BMP, Activin and mTOR pathways, may be involved in keeping the rat ES-like cells in an undifferentiated state. The cell lines and information obtained in this study will accelerate our understanding of the molecular regulation underlying pluripotency and guide us in the appropriate manipulation of ES cells from a particular species.

High in vitro survival rate of sheep in vitro produced blastocysts vitrified with a new method and device

Background:To advance the use of embryo vitrification in veterinary practice,we developed a system in which embryo vitrification,warming and dilution can be performed within a straw.Ovine in vitro produced embryos(IVEP)were vitrified at either early(EBs:n=74)or fully expanded blastocyst stage(FEBs:n=195),using a new device named"E.Vit",composed by a 0.25-m L straw with a 50-μm pore polycarbonate grid at one end.Embryos at each stage(EBs and FEBs)were vitrified by either Two-step(TS)or Multi-step(MS;6 different concentrations of vitrification solutions)protocol.Non-vitrified embryos(n=102)were maintained in in vitro culture as a control.Warming consisted of placing the straws directly into 1.5 m L tubes containing a TCM-199 solution with three decreasing concentrations of sucrose.Blastocyst re-expansion,embryo survival and hatching rate were evaluated at2,24 and 48 h post warming.The number of apoptotic cells was determined by TUNEL assay.Results:Blastocyst re-expansion(2 h)after warming was higher(P<0.05)in FEBs group,vitrified with the MS and TS methods(77.90%and 71.25%,respectively)compared with the EBs group(MS:59.38%and TS:48.50%,respectively).Survival rates of vitrified FEBs after 24 h IVC were higher(P<0.001)in both methods(MS and TS)than vitrified EBs(MS:56.25%;TS:42.42%)and was higher(P<0.05)in the MS method(94.19%)compared with those in TS(83.75%).After 48 h of culture the hatching rate for FEBs vitrified in MS system(91.86%)was similar to control(91.89%),but higher than FEB TS(77.5%)and EBs vitrified in MS(37.5%)and TS(33.33%).Number of apoptotic cells were higher in EBs,irrespective of the system used,compared to FEBs.The number of apoptotic cells in FEBs vitrified with MS was comparable to the control.Conclusions:A high survival rate of IVP embryos can be achieved by the new"E.Vit"device with hatching rates in vitro comparable with control fresh embryos.This method has the potential for use in direct embryo transfer in field conditions.

Controlled Freezing and Open-Pulled Straw (OPS) Vitrification of In vitro Produced Bovine Blastocysts FollowingAnalysis of ATP Content and Reactive Oxygen Species (ROS) Level

To our knowledge,no single study has systemically compared cryopreservation efficiencies of bovine blastocysts derived from in vitro fertilization （IVF）,intracytoplasmic sperm injection （ICSI） and somatic cell nuclear transfer （SCNT） by controlled freezing and vitrification.This experiment,therefore,was designed to compare the cryopreservation of these blastocysts with controlled freezing and OPS vitrification.Adenosine-5’-triphosphate （ATP） content and reactive oxygen species （ROS） level in blastocysts were also analyzed.Firstly,for each type of blastocyst （IVF,ICSI or SCNT）,significant differences were observed between the survival rates of the controlled freezing （（81.56±2.33）,（68.18±4.72） or （47.89±5.83）%） and OPS vitrification groups （（92.24±4.54）,（82.40±3.76） or （78.71±5.91）%;P〈0.05）.Secondly,for each type of blastocyst （IVF,ICSI or SCNT）,ATP content was significantly decreased after controlled freezing or vitrification,and the ATP content in the controlled freezing group （0.43±0.06）,（0.35±0.05） or （0.21±0.02） pmol） was significantly lower than that found in the OPS vitrification group （0.62±0.04）,（0.46±0.03） or （0.30±0.01） pmol;P〈0.05）.Thirdly,ROS level in fresh IVF （（47.33±3.56） c.p.s （counted photons per second）,ICSI （（36.51±2.58） c.p.s） or SCNT blastocysts （（26.44±1.49） c.p.s） was significantly lower than that found in the OPS vitrification group （（72.14±4.31）,（58.89±3.89） or （40.11±5.73） c.p.s;P〈0.05）,but higher than that of the controlled freezing group （34.41±3.32）,（23.13±1.26） or （15.46±2.45） c.p.s;P〈0.05）.The present study indicated that vitrification is more efficient in the cryopreservation of bovine blastocysts derived from IVF,ICSI or SCNT than controlled freezing.Furthermore,both vitrification and controlled freezing significantly altered the ATP content and ROS level in those blastocysts.

Developmental competence and ultrastructural changes of heat-stressed mouse early blastocysts produced in vitro

Mouse early blastocysts were exposed to temperatures of 39℃ and 41℃ for 2 h, respectively, to determine their developmental competence and ultrastructural changes. The results showed that heat stress at 41 ℃ for 2 h, significantly reduced the percentages of expanded and hatched blastocysts, but not at 39℃ for 2 h. The average cell numbers in expanded blastocysts, which developed from early blastoeysts heat-stressed at temperatures of 39℃ and 41 ℃, were significantly reduced. The average cell numbers in hatched blastocysts subjected to heat stress were no different from those in the control group cultured at 37 ℃ . The mitochondria of the early blastocysts heat-stressed at 39℃ for 2 h, were slightly swollen, but they had recovered after culturing at 37 ℃ for 2 h. However, the mitochondria in the blastocysts heat stressed at 41 ℃ for 2 h were severely swollen, and their number increased. The ribosomes shed from the rough endoplasmic reticulum, and the number of secondary lysosomes in the plasma increased. The integrity of desmosomes was disrupted. The space between the nuclear envelope and the perivitelline membrane enlarged. The fibre fraction and the particulate fraction of nueleoli were separated. The heterochromatin in nueleoli was also increased in its quantity. There were some lamellar-shape structures and heterogeneous dense materials exhibiting in the cytoplasm. The ultrastructural changes induced by heat shock at 41 ℃ for 2 h were not reversible. In conclusion, the damage of heat stress to mitoehondria, lysosomes, ribosomes and cell nucleus, may be one of the most important factors that inhibit the normal development of mouse early blastoeysts .

Beneficial effect of reduced oxygen concentration with transfer of blastocysts in IVF patients older than 40 years old

The aim of the present study was to determine the impact of oxygen concentration on implantation, pregnancy and delivery rates in IVF patients older than 40 year old with transfer of blastocysts. Included were 558 women aged 23-45 years old undergoing IVF/ICSI procedures whose embryos were cultured at blastocyst stage under two different oxygen environments (a bi-gas system: 5.6% CO2 in air and a tri-gas system: 5.6% CO2, 5% de O2 and 89.4% N2). The main outcome measures of this study are implantation, pregnancy and delivery rates. Implantation, pregnancy and delivery rates are found to be reduced in women older than 40 years old. The implantation and pregnancy rates are significantly higher in women older than 40 years old from the 5% of O2 group, in comparison to the 20% group (25.00% versus 2.70% and 41.38% versus 5.56%;P < 0.05). The deliveries rates were 13.79% and 5.56% in the 5% and 20% oxygen groups respectively (P: NS). The birthweight was similar in both study groups (P: NS). Gestational age was significantly longer in wo- men from the 5% of O2 group, in comparison to the 20% (36.87 versus 35.87 weeks, P < 0.05). Results indicated that the embryonic culture with 5% of oxygen and transfer of blastocysts in women older than 40 years old improve the results in the in Vitro fertilization/intracytoplasmic injection procedures (IVF/ICSI).

Effect of Retinoic Acid on Apoptosis and Expression of Fas Proteins in Mouse Blastocysts Cultured In Vitro

Mouse blastocysts were exposed to doses of 0, 1 and 10 μmol/L retinoic acid （RA） for 24 h and the cytotoxic effect of RA on the mouse blastocysts in vitro was observed. FITC-labeled terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling （TUNEL-FITC） assay was employed to stain apoptotic cells and immunohistochemical S-P staining method was used to detect the expression of Fas protein in mouse blastocysts in vitro. The results showed that RA could induce apoptosis and increase the expression of Fas proteins of trophectoderm （TE） and inner cell mass （ICM） cells in blastocysts. Compared with the findings for the control blastocysts, exposure to RA （10 μmol/L） resulted in a more significant apoptosis and higher expression level of Fas proteins （P〈0.01）. It was concluded that RA could induce apoptosis, which may result in a significant reduction in the average number of total cells and the trophectoderm/inner cell mass in blastocysts and an increased expression of Fas protein, suggesting that RA had a cytotoxic effect on the growth and development of early embryos in mice.

Effects of Mifepristone (RU486) on Development and Ultrastructure of Mouse Blastocysts

Objective To investigate effects of mifepristone （MP） on the development and ultrastructure of mouse blastocysts in vivo Methods Sixty female mice were equally divided into 4 groups： control group （group A）, 1.9 mg/kg MP group （group B）, 5.6 mg/kg MP group （group C）, and 16.8 mg/kg MP group （group D）. The female mice of 4 groups undertook a superovulation method. The development of obtained blastocysts was evaluated, and ultrastructural changes of the blastocysts were observed by transmission electron microscope. Results In comparison with group A, the development rate of blastocysts in group B showed no difference （P〉0.05）, while the development rate of blastocysts in both group C and group D was significantly lower （P〈0.05）. With doses of 5.6 mg/kg and 16.8 mg/kg, the blastocysts showed granular appearance of the cytoplasm, irregular cell borders, enlarged perivitelline space and degeneration. Ultrastructure of the blastocysts in group B was similar to group A, except a little number of fat droplets in the cytoplasm. In group C, the microvilli on apical surface was decreased in number or even disappeared, mitochondria were under developed, a lot of filamentous crystals were found in the cytoplasm. Cellular junctions were defected. In group D, the blastocyst cells were irregular in shape, mitochondria were frequently vacuolated, the nucleolus was enlarged, nuclear membrane was ruptured, and chromatin was slack. Conclusion MP could damage to the ultrastructure of mouse blastocyst, and was responsible for the inhibition of blastocyst development. The inhibitory effect of MP would be in a dose-dependent fashion.

Factors Analysis of Spontaneous Abortion after Thawed-vitrified Blastocysts Transfer

Objective To investigate the factors resulting in spontaneous abortion after transferring frozen-thawing blastocysts.Methods A total of 108 cases transferring vitrified blastocysts were divided into two groups. abortion group （n=20） and ongoing group （n=88）. Cytogenetic analysis of apoblemas was performed in 12 cases of the abortion.Results The overall spontaneous abortion rate was 18.50%（20/108） and the early spontaneous rate was 16.67%（18/108）. A significant difference in maternal age was observed （abortion group： 33.3±4.0years, ongoing group： 31.0 ±3.6years, P=0.02）. No difference in other parameters was found. Cytogenetic analysis of apoblemas was obtained for 12 cases, and 2 specimens were contaminated. Seven often patients had abnormal karyotypes.Conclusion The underlying cause of spontaneous abortion after transferring frozenthawing blastoeysts appears to be abnormal karyotypes. Advancing maternal age seems to increase the risk of spontaneous abortion.

Clinical scenarios of quadruplet pregnancy by transfer of two blastocysts—A case report

Blastocyst transfer is advocated to reduce the risk of multiple gestations in pregnancies by assisted reproduction. Nevertheless, there remains the rare inherit possibility of embryo splitting that can result in monozygotic twins leading to high-order multiples. Also, when a patient is found to have a higher gestation than the number of embryos transferred it calls into question the IVF facility’s competency and credibility. The case report presented such a rare phenomenon of embryo splitting and the clinical consequences precipitated from it. In the patient, two blastocysts were transferred 5 days after transvaginal oocyte retrieval. The pregnancy confirming hCG test did not predict high-order multiples in this case. Early ultrasonography documented trichorionic-quadramnionic gestation. Selective reduction of the monochorionic twins was done at 11 weeks. Dichorionic twin gestation continued uneventful until 22 weeks at which point the patient experienced preterm premature rupture of membranes with subsequent delivery. The pathology report confirmed trichorionic-quadramnionic gestation. The mode of splitting was different for the two embryos one leading to monozygotic dichorionic and the other to monozygotic monochorionic. Furthermore, the implantation was also apparently asynchronous with one implanted considerable later than the other. The first β-hCG value seemingly did not represent the entire initial implementation events otherwise the value would be higher carrying the early signal of high-order multiple.

Generation of rat-mouse chimeras by introducing single cells of rat inner cell masses into mouse blastocysts

In the field of developmental biology and regenerative medicine,mammalian interspecific chimeras have been proved very useful for investigating early embryonic development and the immune system establishment,and extended to a promising potential for human organ generation（Rossant et al.,1982）.

Identification of a suitable endogenous control gene in porcine blastocysts for use in quantitative PCR analysis of microRNAs

To obtain reliable results in quantitative PCR （qPCR） reactions, an endogenous control （EC） gene is needed to correct for systematic variations. In this study, a TaqMan low density array was used to quantify the expression levels of microRNA （rniRNA） genes in in vivo fertilized, in vitro fertilized, parthenogenetic and somatic cell nuclear transfer blastocysts. The aim was to identify suitable EC genes for the qPCR analysis of rniRNAs in porcine blastocysts. The results showed that thirty-six miRNAs were commonly expressed in the four kinds of embryos and the expression levels of eleven miRNAs were similar in the different embryo types （P-value〉0.05）. These 11 miRNAs were selected as candidate EC genes for further analysis and, of these, miR-16 was identified as the most stable EC gene by the GeNorm （a tool based on a pair-wise comparison model that calculates the internal control genes stability measure and determines the most reliable pair of EC genes） and NorrnFinder （an excel plug-in that uses an ANOVA-based model to estimate intra- and inter- group variation to indicate the single most stable EC gene） programs. In addition, a cell number normalization method validated miR-16 as a suitable EC gene for use in future qPCR analysis of miRNAs in porcine blastocysts.

Pregnancy Outcomes for Day 5 Versus Day 6 Single Frozen-thawed Blastocyst Transfer with Different Qualities of Embryos: A Large Matched-cohort Study

Objective This study aimed to determine whether the day of blastocyst expansion affects pregnancy outcomes in frozen-thawed blastocyst transfer(FBT)cycles.Methods A retrospective match-cohort study was conducted.Patients who underwent blastocyst transfer in frozen-thawed cycles at day 5 or 6 were matched for potential confounding factors.A total of 2207 matched pairs of FBT cycles were included from January 2016 to December 2019 in our Reproductive Medicine Center.Results The clinical pregnancy rate(CPR)and live birth rate(LBR)were significantly increased in day 5 blastocyst transfers when compared to day 6 blastocyst transfers,in terms of the same embryo quality.For FBT cycles with good-quality embryo,the CPR at day 5 and 6 was 61.30%and 57.56%,respectively(P=0.045),and the LBR was 44.79%and 36.16%,respectively(P<0.001).For FBT cycles with poor-quality embryo,the CPR at day 5 and 6 was 48.61%and 40.89%,respectively(P=0.006),and the LBR was 31.71%and 25.74%,respectively(P=0.019).The CPR for FBT cycles with good-quality embryo was statistically higher at day 6 than that at day 5 with poor-quality embryo transferred(57.56%vs.48.61%,P=0.001).Maternal age,anti-Müllerian hormone(AMH),endometrial thickness,embryo quality,and the day of blastocyst expansion were independently correlated with the CPR and LBR.The FBT cycles at day 5 had significantly higher CPR(adjusted odds ratio[OR]=1.246,95%confidence intervals[CI]:1.097–1.415,P=0.001)and LBR(adjusted OR=1.435,95%CI:1.258–1.637,P<0.001)than those at day 6.Conclusion The embryo quality is the primary indicator for FBT cycles.Day 5 blastocysts should be preferred when the quality of embryo at day 5 is the same as that at day 6.

Embryo quality and chromosomal abnormality in embryos from couples undergoing assisted reproductive technology using preimplantation genetic screening

Objective:To detect common chromosomal aneuploidy variations in embryos from couples undergoing assisted reproductive technology and preimplantation genetic screening and their possible associations with embryo quality.Methods:In this study,359 embryos from 62 couples were screened for chromosomes 13,21,18,X,and Y by fluorescence insitu hybridization.For biopsy of blastomere,a laser was used to remove a significantly smaller portion of the zona pellucida.One blastomere was gently biopsied by an aspiration pipette through the hole.After biopsy,the embryo was immediately returned to the embryo scope until transfer.Embryo integrity and blastocyst formation were assessed on day 5.Results:Totally,282 embryos from 62 couples were evaluated.The chromosomes were normal in 199(70.57%)embryos and abnormal in 83(29.43%)embryos.There was no significant association between the quality of embryos and numerical chromosomal abnormality(P=0.67).Conclusions:Embryo quality is not significantly correlated with its genetic status.Hence,the quality of embryos determined by morphological parameters is not an appropriate method for choosing embryos without these abnormalities.

Effects of Different Bovine Sperm Capacitation Methods on Fertilization in vitro

In order to improve the in vitro fertilization rate of bovine, the effects of different sperm capacitation methods on fertilization were investigated. Total two treatments were designed： two-time centrifugation （C）, and one-time centrifugation and swim-up method （CS）. The results showed that the cleavage rate in the C treatment group was （75.6±4.5）%, which showed no difference compared with that （（76.4±1.9）%） in the CS treatment group （P〉0.05）; there was also no significant dif- ference in blastocyst rate between the two treatment groups （（35.7±4.1）% vs. （36.3± 2.7）%, P〉0.05）. However, the CS treatment significantly saved the capacitation time in vitro.

Effects of Cumulus Cells on in vitro Fertilization of Bovine

[Objective] The aim was to explore the effect of cumulus cells on the in vitro fertilization of in vitro matured bovine oocytes. [Method] The in vitro matured oocytes were divided into three groups of cumulus cells removal, partial removal and no removal. [Result] In the co-culture with cumulus cells, the oocytes of the removal group had higher cleavage rate and blastocyst rate （74.4%±4.1, 53.7%±5.1） than those of the no removal group （72.7%±5.1, 52.4%±3.5）, but the difference was not significant （P〉0.05）, while both groups had better performances than the re- moval group （39.6%±4.5, 18.8%±4.6） with the difference reaching the significant level （P〈0.05）. All the three groups showed significant difference with the control. The combination of cumulus cells and melatonin achieved the best effects as the cleavage rate and blastocyst rate of the partial removal group （79.8%±3.7, 56.5%±5.1） were better than those of the no removal group （78.2%±2.6, 55.8%±4.6）, and the difference was not significant, while both group had better performances than the removal group （48.3%±5.5, 22.7%±4.3） and the control group with the differences reaching the significant level （P〈0.05）. [Conclusion] The study provided technical support for the production of dairy cows and beef cattle.

Relationships Between Icariin and Anti-Apoptotic miRNA-21 in Mouse Blastocyst Development In vitro

In this study, the effect of icariin, a flavonoid from the Chinese traditional medicine epimedium, on miRNA-21 of mouse developmental blastocysts in vitro and the development of preimplantation embryos were studied. The possible effective targets of icariin promoting preimplantation embryo development in vitro and anti-apoptosis were determined. The embryos were cultured in modified CZB medium （mCZB） as control group. The experimental group （Ica group） was supplemented with 0.6 μg mL-1 icariin. Mouse pronuclear embryos were cultured in vitro until blastocysts. The development rates of preimplantation embryos were observed. The total cell number, apoptotic cell number and the rate of apoptotic cells in blastocysts were analysed by the staining of Hoechst33342 and labeling of TUNEL and detected under a laser confocal scanning microscope. The miRNA-21 expression, the mRNA levels of pro-apoptotic Caspase3, and the target genes of miRNA-21： pro-apoptotic PTEN, anti-apoptotic Bcl-2 were detected by real-time RT-PCR. The results showed that percentages of morulaes and blastocysts in Ica group were both extremely higher than control group （（85.14±6.57）% vs. （72.04±11.58）%; （82.50±7.11）% vs. （66.80±11.70）%, respectively, P〈0.01）. The total cell number ofblastocysts had extreme difference between Ica group and control group （（61.40±9.64） vs. （46.23±4.50）, P〈0.01）. The apoptotic cell number and rate of apoptotic cells of blastocysts were both reduced in Ica group （（1.47±0.51） vs. （2.94±0.66）; （2.40±0.27）% vs. （6.25±0.62）%, respectively, P〈0.01）. Compared to control group, addition of icariin into mCZB extremely increased the expression of anti-apoptotic miRNA-21 （P〈0.01）, down-regulated pro-apoptotic Caspase3 （P〈0.05） and PTEN （P〈0.01）, up-regulated anti-apoptotic Bcl-2 （P〈0.01）. In conclusion, icariin could reduce the apoptosis, promote the embryo development in vitro by enhancing miRNA-21 expression to up-regulated anti-apoptotic genes and down-regulated pro- apoptotic genes. These apoptosis-related genes were regulated by miRNA-21.

Effect of PMSG/hCG Superovulation on Mouse Embryonic Development

Kunming mouse strain is widely used in China, and the superovulation was administrated with 10 IU PMSG combined with 10 IU hCG. In this study, the effects of the exogenous gonadotropins on superovulation of Kunming mice and embryo quality derived from the superovulated mice were assessed. Female mice at 6-8-wk old were superovulated with 0, 5, 7.5 and 10 IU PMSG/hCG and mated with male mice. The embryos were retrieved at 2.5 d post coitum. No statistic difference was observed for the number of 2-cell embryos collected per mouse between control and 5 IU PMSG/hCG treatment group, but the number significantly increased for 7.5 and 10 IU PMSG/hCG treatment group （P〈0.05）. The average number of 4- cell and 8-cell embryos collected from each mouse significantly differed between control and 5, 7.5, 10 IU PMSG/hCG treatment groups （P〈0.05）. When 8-cell embryos derived from mice administrated with 0, 5, 7.5 and 10 IU PMSG/hCG were cultured in KSOM, the blastocyst development rates were 88.1, 94.7, 96.1 and 94.3%, respectively, which were similar to control （P〉0.05）. This indicated that exogenous gonadotropins have no effects on development of Kunming mouse embryos. The quality of blastocyst was assessed by labelling with Hoechst and propidium iodide for inner cell mass and trophectoderm cells, the result showed that ICM/TE ratio significantly decreased for 10 IU PMSG/hCG treatment group compared with control, 5 and 7.5 IU PMSG/hCG treatment group （P〈0.05）. This suggested that the embryo quality of Kunming mouse has been affected by high dose of gonadotropins.

High-quality Cleavage Embryo versus Low-quality Blastocyst in Frozen-thawed Cycles:Comparison of Clinical Outcomes

This study compared the clinical outcomes of the frozen-thawed cycles of high-quality cleavage embryos with low-quality blastocysts to provide a reference for the choice of frozen-thawed embryo transfer schemes and to improve clinical pregnancy rates.A retrospective analysis was performed on the clinical data of patients undergoing frozen-thawed embryo transfer at the Reproductive Medicine Center of Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology from 2016 to 2017.In total,845 cases were divided into a high-quality cleavage embryo group(group A)and a low-quality blastocyst group(group B).Each group was further divided into subgroups based on the number of transplants.Group A was categorized into two subgroups comprising of 94 cases in subgroup Al(1 high-quality 8-cell group)and 201 cases in subgroup A2(2 high-quality 8-cell group).Group B was divided into four subgroups consisting of 73 cases in subgroup B I(D53BC group),65 cases in subgroup B2(D54BC group),110 cases in subgroup B3(D63BC group),and 282 cases in subgroup B4(D64BC group).The pregnancy outcomes and neonatal outcomes between the groups were compared.The clinical pregnancy rates(56.72%and 60.00%)and live birth rates(47.76%and 46.15%)in subgroups A2 and B2 showed no significant differences,but these rates were significantly higher in subgroups A2 and B2 than in the rest subgroups(P<0.05).The multiple birth rate(26.32%)in the subgroup A2 was significantly higher than that in the rest subgroups(P<0.05).There were no statistically significant differences in the abortion rates among all groups(P>0.05).In terms of neonatal outcomes,there were no statistically significant differences in the proportion of premature births,sex ratios,and birth defects among the low-weight and gigantic infants(P>0.05).Transplanting two high-quality cleavage embryos during the frozen-thawed embryo transfer cycles could significantly increase clinical pregnancy rates and live birth rates,but at the same time,it also increased the risks of multiple births and complications to mothers and infants.The D54BC subgroup had the most significant advantages among all groups(P<0.05).The rest low-quality blastocysts had clinical outcomes similar to the single high-quality cleavage embryo group.

Trans-10,cis-12 conjugated linoleic acid reduces neutral lipid content and may affect cryotolerance of in vitro-produced crossbred bovine embryos

Background：Due to high neutral lipids accumulation in the cytoplasm,in vitro-produced embryos from Bos primigenius indicus and their crosses are more sensitive to chilling and cryopreservation than those from Bos primigenius taurus.The objective of the present study was to evaluate the effects of trans-10,cis-12 conjugated linoleic acid（CLA） on the development and cryotolerance of crossbred Bos primigenius taurus x Bos primigenius indicus embryos produced in vitro,and cultured in the presence of fetal calf serum.Bovine zygotes（n = 1,692）were randomly assigned to one of the following treatment groups：1） Control,zygotes cultured in Charles Rosenkrans 2 amino acid（CR2aa） medium（n = 815） or 2） CLA,zygotes cultured in CR2 aa medium supplemented with 100 μmol/L of trans-10,cis-12 CLA（n =877）.Embryo development（cleavage and blastocyst rates evaluated at days 3 and 8 of culture,respectively）,lipid content at morula stage（day 5） and blastocyst cryotolerance（re-expansion and hatching rates,evaluated 24 and 72 h post-thawing,respectively） were compared between groups.Additionally,selected mRNA transcripts were measured by Real-Time PCR in blastocyst stage.Results：The CLA treatment had no effect on cleavage and blastocyst rates,or on mRNA levels for genes related to cellular stress and apoptosis.On the other hand,abundance of mRNA for the 1-acylglycerol-3-phosphate0-acyltransferase-encoding gene（AGPAT）,which is involved in triglycerides synthesis,and consequently neutral lipid content,were reduced by CLA treatment.A significant increase was observed in the re-expansion rate of embryos cultured with trans-10,cis-12 CLA when compared to control（56.3 vs.34.4%,respectively,P = 0.002）.However,this difference was not observed in the hatching rate（16.5 vs.14.0%,respectively,P=0.62）.Conclusions：The supplementation with trans-10,ds-12 CLA isomer in culture medium reduced the lipid content of in vitro produced bovine embryos by reducing the gene expression of 1-acylglycerol 3-phosphate 0-acyltransferase（AGPAT） enzyme.However,a possible improvement in embryo cryotolerance in response to CLA,as suggested by increased blastocyst re-expansion rate,was not confirmed by hatching rates.

Gene Expression and Regulation of Blastocyst Formation

Blastocyst formation is a crucial stage of early embryo development.Cell junction proteins and cell adhesion associated proteins are involved in the establishment of cell junction,and subsequently induce cell compaction,blastocyst formation,differentiation of trophectoderm and maintenance of blastocyst expansion.Genes regulating development and differentiation participate in embryo development and differentiation of inner cell mass and trophectoderm,which controls the transition from the undifferentiation to differentiation state.Furthermore,cytokine and growth factor have influence on the proliferation of cells of inner cell mass.In a word,many proteins and factors are involved in the gene expression and regulation of blastocyst formation.