Transfer Of Mouse Embryos Research Articles

Addition of GM-CSF during in vitro oocyte maturation improves embryo development and implantation and birth rate in mice.

The present study determined whether adding granulocyte macrophage colony stimulating factor (GM-CSF) during in vitro oocyte maturation (IVM) could improve oocyte developmental competence by examining embryo development and implantation and birth rates following embryo transfer in mice. In an initial dose response experiment, we demonstrated that the addition of 2 and 10 ng/mL of GM-CSF during IVM increased cumulus expansion (P<0.05) but did not affect fertilisation rate compared with the control group. The addition of 10 ng/mL increased blastocyst rate (17.0%; P<0.05) and tended to increase the number of good quality blastocysts present at 96 h of culture (+19.4%; P=0.06) and increased blastocyst inner cell mass (+25.2%; P<0.001), trophectoderm (+29.9%; P<0.01), and total cell numbers (+28.6%; P<0.05). GM-CSF also reduced the incidence of DNA damage in blastocysts in the 10 ng/mL group (-16.2%) compared with the control group. These improvements translated into increases in implantation rate (+21.0%; P<0.05) and birth rate (+17.0%; P<0.05) following the transfer of vitrified blastocysts.GM-CSF treatment did not alter any fetal and placental parameters. Together these results suggest that the addition of GM-CSF during IVM may improve livestock in vitro embryo production and human IVM.

#239 : Granulocyte-Macrophage-Colony Stimulating Factor Improve Embryo Development, Mitochondrial Function and Spindle Alignment in a Mouse Model of Advanced Maternal Age

Background and Aims: We have previously shown that GM-CSF can improve embryo development when added during in vitro oocyte maturation (IVM) as well as increase pregnancy and birth rates following embryo transfer in mice. The aim of the present study was to determine whether GM-CSF during IVM can improve embryo development in a mouse model of advanced maternal age (AMA) Method: 12–14-month-old CBAF1 female mice were injected with 5IU of PMSG and cumulus-oocyte complexes (COCs) aspirated 46–48 h post-injection. Ten COCs were cultured per 50 [Formula: see text]L drop in bicarbonate-buffered [Formula: see text]-MEM containing 3 mg/ml BSA, 1 mg/ml Fetuin and 5 mIU/mL FSH plus 0 or 10 ng/ml of GM-CSF. Zygotes were cultured in Cleave Media and cleavage and blastocyst rates were determined. Blastocysts were differentially stained on Day 5 to determine cell numbers. To determine mitochondrial membrane potential (MMP) COCs were incubated for 15 minutes in HEPES buffered alpha-MEM media with JC-1 dye for 15 minutes. To examine spindle formation and chromosomal alignment, MII oocytes were fixed and then incubated with FITC conjugated mouse anti-[Formula: see text]-tubulin for 40 minutes at 25°C and chromosomes stained using Hoechst. Fluorescence was visualised using Cell Voyager CV1000 Confocal Scanner and Z-stack images were analysed using Fiji Image J software. Data were analysed using a univariate general liner model in IBM SPSS. Results: GM-CSF during IVM increased Day 5 blastocyst rate by 21% (P&lt;0.01) and the proportion of expanded blastocysts by 75% (P&lt;0.05). Blastocyst inner cell mass was increased by 23% (P&lt;0.05) and total cell number by 14% (P&lt;0.05). MMP was increased across all fields by three-fold (P&lt;0.05). GM-CSF did not affect spindle formation but tended to reduce chromosomal misalignment by 17% (P&gt;0.05). Conclusion: We have shown that the addition of GM-CSF during IVM can improve embryo development and mitochondrial activity in a mouse of AMA.

Ovarian Stimulation in Mice Resulted in Abnormal Placentation through Its Effects on Proliferation and Cytokine Production of Uterine NK Cells.

Ovarian stimulation is associated with an increased incidence of abnormal placentation. Uterine natural killer (uNK) cells are the major subpopulation of decidual immune cells, which are crucial for placentation. In a previous study, we found that ovarian stimulation impairs uNK cell density on gestation day (GD) 8.5 in mice. However, it was not clear how ovarian stimulation led to a reduction in the density of uNK cells. In this study, we constructed two mouse models, an in vitro mouse embryo transfer model and an estrogen-stimulated mouse model. We used HE and PAS glycogen staining, immunohistochemical techniques, q-PCR, Western blot, and flow cytometry to analyze the mouse decidua and placenta, and the results showed that SO resulted in a fetal weight reduction, abnormal placental morphology, decreased placental vascular density, and abnormal density and function of uNK cells. Our results suggest that ovarian stimulation resulted in aberrant estrogen signaling and may contribute to the disorder of uNK cells caused by ovarian stimulation. Together, these results provide new insights into the mechanisms of aberrant maternal endocrine environments and abnormal placentation.

Comparison of unilateral and bilateral embryo transfer in mice.

Surgical embryo transfer in mice is a key technique in assisted reproduction and applied for different purposes in biomedical research. Due to its frequent application in rodent facilities across the world, further improvement of the procedure can substantially contribute to fulfil the principles of the 3Rs. Here, we investigated the effect of bilateral and unilateral left- or right-sided oviduct transfers on the success of embryo transfers. In total, we performed 223 embryo transfers (56 unilateral left, 56 unilateral right, 111 bilateral), in which we transferred 10-14 two-cell embryos each. We found that the type of transfer significantly influenced both the pregnancy rate of recipients and the survival rate of transferred embryos. Bilateral transfers yielded higher pregnancy and survival rates than left-sided unilateral transfers. Right-sided unilateral transfers yielded higher pregnancy rates than left-sided unilateral transfers and did not differ in embryo survival rates from bilateral transfers. We found no evidence that the number of transferred embryos affected the pregnancy rate. However, the number of born pups increased with the number of transferred embryos. In conclusion, unilateral embryo transfers into the right reproductive tract yield equally high pregnancy and embryo survival rates as bilateral transfers. Given that a second abdominal incision can be prevented and the time of surgery can be reduced, we recommend applying unilateral right-sided transfers, as this would reduce postoperative pain and lower the impact on recipients.

Development of an embryo transfer model to study uterine contributions to pregnancy in vivo in mice.

Graphical abstractA mouse model to study uterine specific contributions to pregnancy.Maternal environmental exposures can exert impacts on the ability of the uterus to sustain healthy pregnancy. To establish an in vivo model to study this, we designed an ovariectomized mouse embryo transfer model. The rationale being future studies could expose recipient female mice to variables such as altered diet, drug, temperature, air, or activity exposure among others to define their impacts on the uterine contribution to pregnancy. Ovariectomy ensures the extent of the variable is limited to exploring outcomes on uterine but not ovarian function. Embryo transfer from healthy, unexposed donor mice guarantees that any impacts of the variable are attributed to the maternal uterine but not the embryonic state. Pregnancy outcomes including pregnancy success (number of implantation sites) and viability (number of viable vs resorbing implantation sites) can be investigated. Numerous functional outcomes can be assessed, including developmental competence encompassing decidual, placental, fetal, and vascular morphology and/or function (e.g. measured using Doppler ultrasound, comparisons of fetal growth, or molecular or histological characterization of the decidua, placenta, and fetal tissues). Lay summaryMany pregnancy complications occur because of problems in the womb (uterus), specifically the womb lining. There is a close relationship between the hormone function of the ovaries and the uterus and distinguishing between the way they both impact pregnancy success is difficult in existing studies using animals. Here, we developed a new animal model to utilize in addressing these gaps in our understanding of pregnancy.

Hormone induced recipients for embryo transfer in mice

Hormone induced recipients for embryo transfer in mice

Effect of different surgical methods in the mouse embryo transfer: Electrosurgery versus cold surgical technique effects on repeated use of surrogate mothers, pregnancy rate and post-surgical behavior.

Using the cold surgical technique (CST) is the most common practice to accomplish embryo transfer (ET). However, it can lead to uncontrolled bleeding and mortality in laboratory animals. Electrosurgery technique (EST) has provided the opportunity to prevent such complications. This study was aimed to evaluate CST versus EST in terms of repeated use of surrogate mothers, litter size, implantation rate and post-surgical behavior. Virgin female NMRI mice were allocated into two different surgical groups (n = 40): 1) CST-ET (control) and 2) EST-ET. Results showed that the first ET in EST-ET and CST-ET groups did not affect litter size, pregnancy rate and survival of surrogate mothers. Following the second and the third ETs, litter size was significantly affected through CST compared to EST, pregnancy rate and survival of surrogate mothers. Litter size, pregnancy rate and surrogate mothers survival rate did not show any significant reduction following the first and the second ETs in EST group. On the other hand, the third ET showed dramatic reduction for all aforementioned parameters regardless of the chosen surgical method for ET. Mice in EST-ET group did not show any significant change in their behavior indicating reduced well-being during the first 24 hr following the first, the second and the third ETs compared to CST-ET group. In conclusion, using EST for ET in mouse made it feasible to reuse surrogate mothers with minimum animal mortality; this could be pivotal with regard to reproductive and animal welfare aspects and research costs. Also, the results indicated that bleeding has severe diverse effects on ET efficiency.

Effects of Myo-inositol on Physiological and Reproductive Traits through Blood Parameters, Oocyte Quality and Embryo Transfer in Mice

Background: The beneficial effects of myo-inositol to mammals on physiological and reproductive functions and treatments of malfunctions have been reported. The aims of present study were to assess the efficacy of myo-inositol supplementation on physiological and reproductive performances through blood parameters, oocyte quality and embryo transfer in mice. Methods: Seventy-five virgin female mice were classified into three equal groups; control group (T1) versus two myo-inositol groups (T2 receive 30 mmol/l and T3 receive 60 mmol/l). Changes of body temperature, blood oxygen and glucose, heart rate, blood parameters (RBCs, WBCs, hematocrit, glucose and total protein), oocyte quality (cumulus enclosed, diameter and brilliant cresyl blue stain) and reproductive performances (litter weight and size) were determined. Result: The results indicated that myo-inositol supplementation resulted in significant increase in values of RBCs, WBCs, hematocrit and total protein in addition to significant hypoglycemia. The quality of oocytes in case of cumulus enclosed, diameter and brilliant cresyl blue staining and reproductive performances were improved due to myo-inositol supplementation. Furthermore, myo-inositol supplementation effectively alleviated hypothermia and hyperglycemia following general anaesthesia.

Modification of Fecal Bacteria Counts and Blood Immune Cells in the Offspring of BALB/c and C57BL/6 Mice Obtained through Interstrain Mouse Embryo Transfer.

The reproducibility of results obtained with rodent models depends on the genetic purity of the strain and the stability of the environment. However, another potential factor is changes in the gut microbiota due to the transmission of mother's bacteria during embryo transfer. In this study, we demonstrate the transmission of the microbiota and immune cell blood phenotype to the offspring of 2 strains, C57BL/6JNskrc and BALB/cJNskrc, from surrogate dams of different genotypes. Interstrain embryo transfer resulted in a change in the number of Enterococcus spp. organisms, as shown by quantitative PCR analysis. The number of blood leukocytes was also affected, as estimated by flow cytometry. The number of blood leukocytes, including B cells and helper T cells, and the number of Enterococcus spp. organisms in male C57BL/6JNskrc offspring born to BALB/cJNskrc surrogate dams became similar to those of male BALB/cJNskrc mice born to BALB/cJNskrc dams. Likewise, the same parameters of male BALB/cJNskrc mice born to C57BL/6JNskrc dams became similar to those of male C57BL/6JNskrc offspring. Researchers should be aware of the possible transmission of the dam's microbiota and immune cell phenotypes to the experimental strains when planning embryo transfer experiments, because these factors could affect the experimental outcomes or the reproducibility of experimental results.

Loss of methylation of H19-imprinted gene derived from assisted reproductive technologies can be mitigated by cleavage-stage embryo transfer in mice.

Studies on rodents have shown that assisted reproductive technologies (ARTs) are associated with perturbation of genomic imprinting in blastocyst-stage embryos. However, the vulnerable developmental window for ART influence on the genomic imprinting of embryos is still undetermined. The purpose of this study was to establish the specific embryonic development stage at which the loss of methylation of H19 imprinting control regions (ICRs) was caused by ART occurrence. Additionally, we explored protocols to safeguard against possible negative impacts of ART on embryo H19 imprinting. Mouse embryos were generated under four different experimental conditions, divided into four groups: control, in vitro culture (IVC), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI). The methylation levels of H19 ICR of the grouped or individual embryos were analyzed by bisulfite-sequencing PCR. Our data showed that the loss of methylation of H19 ICR in mouse blastocysts was inflicted to a similar extent by IVC, IVF, and ICSI. Specifically, we observed a significant loss of methylation of H19 ICR between the mouse 8-cell and morula stages. In addition, we revealed that the transfer of mouse embryos generated by ARTs in the uterus at the 8-cell stage induced the occurrence of methylation patterns in the blastocysts closer to the in vivo ones. Our findings indicate that the loss of methylation of H19 ICR caused by ARTs occurs between the 8-cell and the morula stages, and the transfer of cleavage embryos to the uterus mitigates the loss methylation of H19 derived by mice ARTs.

Transfer of mouse blastocysts exposed to ambient oxygen levels can lead to impaired lung development and redox balance.

In vitro culture under atmospheric oxygen puts embryos under oxidative stress and impairs preimplantation development. However, to what extent this process alters the redox balance in the perinatal period remains largely unknown. The aim of the present study was to examine if the redox balance is altered in the lung tissue of fetuses generated through transfer of mouse embryos exposed to atmospheric oxygen at different stages of development and to determine if this has any effect on lung morphogenesis and gene expression. Two experimental groups (EGs) were generated by transferring in vitro- and in vivo-derived blastocysts to pseudo-pregnant females. In vivo-developed fetuses served as control. Enzymatic/nonenzymatic antioxidants, malondialdehyde (MDA) levels, total antioxidant capacity, stage of lung development and gene expression were evaluated on day 18 of pregnancy. Weight of fetuses was significantly less in both experimental cohorts (ANOVA, P < 0.001 versus control), associated with delayed lung development, higher amounts of MDA (ANOVA, P < 0.001 versus control) and altered expression of several genes in oxidative stress/damage pathways. Evidence gathered in the present study indicates that pre-implantation stress caused by culture under atmospheric oxygen, even for a short period of time, leads to fetal growth restriction, impaired lung development and redox balance along with dysregulation of several genes in oxidative stress response. Absence of an EG in which in vitro embryo culture was performed at 5% oxygen and the use of genetically heterogeneous F2 fetuses are the limitations of the study. In any case, the long-term impact of such dramatic changes in the developmental programming of resulting fetuses warrants further investigations.

Simplified pipelines for genetic engineering of mammalian embryos by CRISPR-Cas9 electroporation†.

Gene editing technologies, such as CRISPR-Cas9, have important applications in mammalian embryos for generating novel animal models in biomedical research and lines of livestock with enhanced production traits. However, the lack of methods for efficient introduction of gene editing reagents into zygotes of various species and the need for surgical embryo transfer in mice have been technical barriers of widespread use. Here, we described methodologies that overcome these limitations for embryos of mice, cattle, and pigs. Using mutation of the Nanos2 gene as a readout, we refined electroporation parameters with preassembled sgRNA-Cas9 RNPs for zygotes of all three species without the need for zona pellucida dissolution that led to high-efficiency INDEL edits. In addition, we optimized culture conditions to support maturation from zygote to the multicellular stage for all three species that generates embryos ready for transfer to produce gene-edited animals. Moreover, for mice, we devised a nonsurgical embryo transfer method that yields offspring at an efficiency comparable to conventional surgical approaches. Collectively, outcomes of these studies provide simplified pipelines for CRISPR-Cas9-based gene editing that are applicable in a variety of mammalian species.

Success of embryo transfer in mice with freshly collected and cryopreserved two-cell embryos with different genetic backgrounds correlated with the number of transferred embryos: A 5-year retrospective analysis.

Embryo transfer of pre-implantation embryos to surrogate dams is a key technique for the hygienic sanitation of strains, cryopreservation, in vitro fertilization, genetic modification and engineering. However, the effects of several parameters, such as the number of transferred embryos, on the success of embryo transfer are not well studied. In this retrospective study, we reanalysed 1320 embryo transfers of two-cell embryos originating from genetically altered donors, which were performed under routine conditions in our facility over a period of 5 years. Of them, 453 embryo transfers were done with freshly collected embryos and 867 transfers were performed with cryopreserved embryos. Despite the fact that the genetic background of the embryo donors was quite heterogeneous, we found that the transfer of ≥ 21 embryos reduced the success of embryo transfers for freshly collected embryos in correlation with the number of pregnancies and born pups, whereas this was not the case for transfer in the cryopreservation group. Most pregnancies were achieved after embryo transfer of 10-20 freshly collected embryos (90.4%), which dropped to 37.5% if more embryos were transferred. The highest pregnancy rates in the cryopreservation group were achieved if 15-17 embryos were transferred (62.9%). Despite the fact that the precise substrains were only rarely defined, we confirmed that beside the number of transferred embryos, the genetic background of the donors had an influence on the success of embryo transfer. Significantly more embryos in a C57BL/6 background developed to term than embryos on a BALB/c background.

Extracellular vesicles derived from donor oviduct fluid improved birth rates after embryo transfer in mice.

Embryo transfer (ET) is an important procedure for assisted reproduction. However, the relatively lower success rate of ET hampers its application potential. In this study we aimed to elucidate the effects of extracellular vesicles derived from donor oviduct fluid (EDOF) on embryo development after ET. Extracellular vesicles from the oviduct were isolated and purified using ultracentrifugation and identified using transmission electron microscopy, NanoSight, bicinchoninic acid (BCA) protein assay and western blotting. The results revealed that extracellular vesicles were present in donor oviduct fluid in higher concentrations (P<0.05) and contained more proteins (P<0.05) than extracellular vesicles derived from recipient oviduct fluid (EROF). EDOF or EROF were supplemented in an ET medium (ETM) and the results showed that EDOF significantly improved birth rate via resisting apoptosis and promoting differentiation. In conclusion, our study indicated that there are differences in EDOF and EROF and that supplementing EDOF to ETM can improve the efficiency of ET; improved ET efficiency promotes the use of gene editing and benefits assisted reproductive technology and animal welfare.

Maternal modulation of paternal effects on offspring development.

The paternal transmission of environmentally induced phenotypes across generations has been reported to occur following a number of qualitatively different exposures and appear to be driven, at least in part, by epigenetic factors that are inherited via the sperm. However, previous studies of paternal germline transmission have not addressed the role of mothers in the propagation of paternal effects to offspring. We hypothesized that paternal exposure to nutritional restriction would impact male mate quality and subsequent maternal reproductive investment with consequences for the transmission of paternal germline effects. In the current report, using embryo transfer in mice, we demonstrate that sperm factors in adult food restricted males can influence growth rate, hypothalamic gene expression and behaviour in female offspring. However, under natural mating conditions females mated with food restricted males show increased pre- and postnatal care, and phenotypic outcomes observed during embryo transfer conditions are absent or reversed. We demonstrate that these compensatory changes in maternal investment are associated with a reduced mate preference for food restricted males and elevated gene expression within the maternal hypothalamus. Therefore, paternal experience can influence offspring development via germline inheritance, but mothers can serve as a modulating factor in determining the impact of paternal influences on offspring development.

Optimization of the balance between effort and yield in unilateral surgical transfer of mouse embryos.

Surgical transfer of embryos is carried out daily in animal facilities worldwide for the rederivation of mouse strains/lines, among other purposes. Current protocols described in laboratory manuals recommend using a high number of embryos during transfer, typically in the range of 15 up to 25. To optimize the use of resources it is necessary to estimate and relate the effort required and the yield obtained. Here, we analyse the balance between the number of embryos transferred (the effort), and the yield as the number of born pups obtained from surgical embryo transfer. To accomplish this, we have analyzed data obtained during rederivation of nearly one hundred lines of mice to a new animal facility. Our results confirm that the use of increasing numbers of embryos per transfer increases the yields of born pups, as has been described previously in the literature, but they also highlight the disproportionate effort required, i.e. in the number of embryos that needed to be transferred. An estimate of the mean expected yields of surgical transfers and their comparison with the actual observed yields indicated that the balance between effort and yield is optimized when using lower numbers of embryos than in currently used protocols, in the range of 8 to 12. Given the heterogeneous nature of the data presented and analyzed here, which is from a population of mice that may be considered as representative of any animal facility, our optimization approach should help save resources in similar facilities and improve the yields of embryo transfer procedures.

Protein-restricted diet during pregnancy after insemination alters behavioral phenotypes of the progeny

BackgroundEpidemiological studies suggest that hyponutrition during the fetal period increases the risk of mental disorders such as attention deficit hyperactivity disorder and autism-spectrum disorder, which has been experimentally supported using animal models. However, previous experimental hyponutrition or protein-restricted (PR) diets affected stages other than the fetal stage, such as formation of the egg before insemination, milk composition during lactation, and maternal nursing behavior.ResultsWe conducted in vitro fertilization and embryo transfer in mice and allowed PR diet and folic acid-supplemented PR diet to affect only fetal environments. Comprehensive phenotyping of PR and control-diet progenies showed moderate differences in fear/anxiety-like, novelty-seeking, and prosocial behaviors, irrespective of folic-acid supplementation. Changes were also detected in gene expression and genomic methylation in the brain.ConclusionsThese results suggest that epigenetic factors in the embryo/fetus influence behavioral and epigenetic phenotypes of progenies. Significant epigenetic alterations in the brains of the progenies induced by the maternal-protein restriction were observed in the present study. To our knowledge, this is first study to evaluate the effect of maternal hyponutrition on behavioral phenotypes using reproductive technology.

Voluntary intake of paracetamol-enriched drinking water and its influence on the success of embryo transfer in mice

Embryo transfer (ET) in mice is a key technique in biomedical research, and is carried out mostly via surgery by transferring founder embryos into pseudo-pregnant recipient females. To cover post-operative analgesic requirements in surrogate mothers, oral self-administration of painkillers has several advantages, but its effectiveness has also been criticized as voluntary ingestion of the drug can be uncertain. Additionally, concerns about potential negative side effects of analgesics on embryo viability and development have been raised. In this regard, we investigated the impact of orally administered analgesia by comparing the outcome of ET with and without paracetamol in the drinking water (3.5mg/ml) of surrogate mothers. Water intake increased significantly when paracetamol, as a sweet-tasting formulation (children's syrup), was added to the drinking water. Measurements of paracetamol concentrations in blood serum confirmed reasonable drug uptake. Success rate of ETs and the body weight of newborn offspring were not different whether paracetamol was administered for two days after surgery or not. In conclusion, paracetamol in drinking water was consumed voluntarily in substantial doses, without detectable side-effects, by freshly operated surrogate mothers, and can therefore be recommended as a feasible method for providing analgesic treatment for surgical ET in mice.

Estrous cycle staging before mating led to increased efficiency in the production of pseudopregnant recipients without negatively affecting embryo transfer in mice

The goal was to increase pseudopregnant mice production by estrous cycle staging by visual examination before pairing and to determine the effect of such pseudopregnant recipients on embryo transfer. To compare methods of estrous cycle staging over 14 days, groups consisted of 10 females in proestrus-estrus and 10 vasectomized males; group 1: only daily visual observation; group 2: daily visual observation and cytological examination on day 1; group 3: daily visual observation and daily cytological examination. The average time to first vaginal plug was 1.8 days in group 1, 2.7 days in group 2, and 3.2 days in group 3, whereas the average time between consecutive vaginal plugs was 9.2 days (group 1), 10 days (group 2), and 9.25 days (group 3). The average time between consecutive estrous cycles was 9.7 days (group 1), 11.8 days (group 2), and 9.4 days (group 3). The congruence between visual and cytological examination in determining proestrus-estrus in group 2 was 100% and that for the four stages in group 3 was 79% with a range of 44% to 100%. From 162 plug-positive females originally selected in proestrus-estrus, 49%, 30%, 19%, and 2% were plug-positive on Day 1, Day 2, Day 3, and Day 4, respectively, showing that pseudopregnant mice production was significantly increased on the first 2 days. From 192 plug-positive females originally selected randomly, these values were 31%, 21%, 35%, 10%, and 3% on d1, d2, d3, d4, and d5, respectively. No significant differences were observed between groups with respect to embryo transfers with fresh or cryopreserved embryos although the number of pups born per litter was higher in group A with fresh (7.57 vs. 6.39) and cryopreserved-thawed embryos (5.0 vs. 4.38). Furthermore, the sex ratio and the genotype of the pups were not significantly affected.

Embryo selection strategy in sibling oocytes: A novel approach to measuring the likelihood of single-embryo transfer using a mouse embryo transfer model

The present study investigated the effect of embryo transfer on post-implantation development using different fertilization approaches in sibling oocyte procedures. C57BL/6, DBA/2, C3H/HeJ and ICR mice were used at 8-10 weeks of age. Mature oocytes were collected, divided into two groups and fertilized using in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Different numbers of the resulting blastocysts were then transferred into recipients and designated as either: The single embryo transfer (SET) I model (one transferred embryo), the SET II model (six transferred embryos) or the multiple embryo transfer (MET) model (24 transferred embryos). The development efficiency, cell number, number of apoptotic cells in blastocysts, pregnancy efficiency, delivery rate and cumulative pregnancy efficiency were analyzed. IVF-fertilized embryos exhibited higher blastocyst development competence and embryo quality compared with ICSI embryos. The pregnancy and delivery efficiency was not identified to be significantly different between the two SET models, but it was lower in these two models than in the MET model. The cumulative pregnancy efficiency in SET models, calculated using a mathematical equation, was not decreased. In conclusion, embryo quality was shown to be the primary factor in selecting embryos prior to embryo transfer using sibling oocytes. In addition, single blastocyst transfer can be performed in sibling oocytes without compromising cumulative pregnancy rates, independent of the fertilization approach.