Higher Blastocyst Development Rate Research Articles

Ovarian stimulation with luteinizing hormone supplementation: the impact of timing on ovarian response and ICSI outcomes.

To investigate whether the timing of rLH addition to rFSH during controlled ovarian stimulation (COS) impacts ovarian response and the outcomes of intracytoplasmic sperm injection (ICSI) cycles. Data of 1278 patients undergoing ICSI between 2015 and 2018, in a private university-affiliated IVF center were analyzed. Patients were divided into groups according to the timing of LH addition to the COS protocol: Group LH-start (n=323), in which LH was administered since day 1 of ovarian stimulation; and Group LH-mid (n=955), in which LH was administered concomitantly with gonadotropin releasing hormone (GnRH) antagonist. Data were also stratified according to female age and response to COS. The outcomes of COS and ICSI were compared between the groups. For the general group and in patients aged ≥ 35 years, higher blastocyst development rates were in Group LH-mid compared to Group LH-start. In patients with poor response to COS (POR), higher fertilization rate, blastocyst development rate and implantation rate were observed in Group LH-mid. rLH supplementation in POR patients may improve laboratorial and clinical outcomes when started in the mid-follicular phase, in GnRH antagonist ICSI cycles.

P–215 The degree of perivitelline space (PS) at the pronuclear stage affects subsequent embryonic development in human zygotes

Abstract Study question Was embryonic development affected by the degree of perivitelline space (PS) at the pronuclear stage in human zygotes? Summary answer Zygotes with a fully surrounding PS showed less cytoplasmic fragmentation and a higher blastocyst development rate (BDR) than zygotes with a partially surrounding PS. What is known already We previously used abnormally-fertilized oocytes (zygotes with three pronuclei; 3PN), donated by ART patients in our clinic who gave written consent for the research. The zona pellucida (ZP) was artificially removed from these oocytes at the pronuclear stage, termed ZP-free culture. The resultant ZP-free 3PN embryos showed less cytoplasmic fragmentation and a higher rate of good-quality embryos (GQE) compared with ZP-intact embryos. Furthermore, in our clinical setting, the rate of GQE and BDR of normally-fertilized embryos were clearly improved by ZP-free culture in patients with recurrent failure of ART treatments due to severe cytoplasmic fragmentation at the early cleavage stage. Study design, size, duration This study included 49 patients who gave written informed consent for our study and were treated with ART in our clinic between March and December 2020. Embryonic development was compared between zygotes with a fully surrounding PS [PS(+)] with those with a partially surrounding PS [PS(-)] at the pronuclear stage. Furthermore, the ZP of PS(-) embryos were artificially removed at the pronuclear stage, and the rate of GQE and BDR were compared with ZP-intact embryos. Participants/materials, setting, methods The degree of PS in 128 zygotes was confirmed by hypertonic preparation using 0.125M sucrose-containing HEPES medium. PS(+) and PS(-) embryos were both cultured as ZP-intact, and the rate of GQE was compared. Furthermore, 223 zygotes were divided into three groups: 1) PS(-)/ZP-intact, 2) PS(-)/ZP-free, and 3) PS(+)/ZP-intact, and cultured in an incubator equipped with time-lapse monitoring up to Day 7, and the rate of GQE, BDR and useable embryos were compared between each groups. Main results and the role of chance The degree of PS was confirmed by a hypertonic preparation (shrinkage of the ooplasm) in 128 normally-fertilized zygotes obtained from 44 cases. There were 86 PS(-) (67.2%) and 42 PS(+) (32.8%) zygotes. The mean maternal age was 35.9 in PS(-) and 40.5 in PS(+) (P < 0.01), and the rate of GQE was significantly higher in PS(+) [64.3% (27/42)] than in PS(-)[38.4% (33/86)] (P < 0.01). In addition, of 223 normally-fertilized zygotes obtained from 41 cases, there were 51 PS(-)/ZP-intact (Group 1), 132 PS(-)/ZP-free (Group 2) and 40 PS(+)/ZP-intact (Group 3) zygotes. The rate of GQE was significantly lower in Group 1 [29.4% (15/51)] compared with Group 2 [59.8% (79/132)] and Group 3 [62.5% (25/40)] (P < 0.01). BDR was also significantly lower in Group 1 [51.3% (10/39)] compared with Group 2 [75.0% (99/132)] and Group 3 [65.0% (13/20) (P < 0.01). Limitations, reasons for caution Although the artificial removal of ZP at the pronuclear stage (ZP-free culture) clearly increased the rate of GQE, embryonic development was not improved in all cases. It seems that this procedure is only effective in embryos with a viable ooplasm. Wider implications of the findings: The degree of PS at the pronuclear stage affects subsequent embryonic development in human zygotes. The artificial removal of ZP at the pronuclear stage (ZP-free culture) helps to suppress fragmentation and leads to an increase in GQE and BDR, and eventually, improves pregnancy rate in cases with severe fragmentation. Trial registration number non

P–594 Ovarian stimulation with luteinizing hormone supplementation: the impact of timing on ovarian response and ICSI outcomes

Abstract Study question Is there an impact of recombinant luteinizing-hormone (rLH) administration timing during controlled ovarian stimulation (COS) on ovarian response and intracytoplasmic sperm injection (ICSI) cycles outcomes? Summary answer rLH supplementation in patients with poor ovarian response (POR) improves laboratorial and clinical outcomes when started in the mid-follicular phase, in GnRH antagonist ICSI cycles. What is known already Meta-analyses demonstrated that the use of rLH combined with rFSH for COS may lead to more ongoing pregnancies than rFSH alone. However, there is limited evidence that the timing of rLH addition to rFSH may impact the ovarian response or the outcomes of ICSI, based on a limited casuistic, which demonstrated improved ovarian response, embryo quality and pregnancy rate with LH supplementation from GnRH antagonist administration day, in estimated POR patients. The objective of the present study was to further investigate this hypothesis in a larger population, and in subpopulations of patients stratified by age and response to COS. Study design, size, duration This historical cohort study included data obtained via chart review of 1278 ICSI cycles performed in 1278 patients between 2015 and 2018, in a private university-affiliated in vitro fertilization center. Post hoc power analysis was calculated, given α of 5%, sample size of 1278, and effect size for implantation rate. The achieved power was superior to 99%. Participants/materials, setting, methods Two groups were formed according to timing of LH administration: Group LH-start (n = 323), in which LH was started on day–1; and Group LH-mid (n = 955), in which LH was started with GnRH antagonist. Then, data were stratified according to female age (<35 years-old, n = 283, and ≥35 years-old, n = 995) and response to COS (poor response (POR): ≤4 retrieved oocytes, n = 423, and normal response: >5 retrieved oocytes, n = 855). Ovarian response and ICSI outcomes were compared among the groups. Main results and the role of chance In POR patients, significantly higher fertilization rate (68.3% ± 2.5 vs. 78.6% ± 3.7, p = 0.023), blastocyst development rate (22.5% ± 7.2 vs. 44.7% ± 6.2, p = 0.022) and implantation rate (17.6% ± 59.1 vs. 20.2% ± 43.2, p < 0.001) were observed in Group LH-mid, even though the amount of LH used in these patients was not significant different from that used in Group LH-mid from patients with normal response to COS (1062.35 IU ± 54.33 vs. 925.81 IU ± 414.41, p: 0.431, respectively). For the general group and in patients aged ≥ 35 years, higher blastocyst development rates were observed in Group LH-mid compared to Group LH-start (33.0% ± 31.9 vs. 40.8% ± 32.6, p = 0.012, and 28.8% ± 30.4 vs 38.5% ± 32.3, p = 0.006, respectively). In patients aged < 35 years and in those with normal response to COS, similar outcomes were obtained irrespective of timing of LH administration. Limitations, reasons for caution The limitations included the retrospective design and limited sample size in subpopulations. In addition, the reduced clinical outcomes related to POR patients may hamper the true estimation of the differences between the stimulation groups in terms of pregnancy and miscarriage rates. Wider implications of the findings: In POR patients, mid-follicular phase LH supplementation starting with 150 IU daily doses, may rescue the ongoing cycle by compensating an initial slow response, and balancing the deprivation of endogenous LH in GnRH antagonist cycles, with no need of expending more gonadotropin compared to patients with normal response to COS. Trial registration number Not applicable

Dual trigger improves response to ovarian stimulation and ICSI outcomes in patients with a previous r-hCG triggered ICSI cycle.

ObjectiveTo evaluate if ovarian response to controlled ovarian stimulation (COS) and intracytoplasmic sperm injection (ICSI) outcomes are improved by the use of dual trigger (gonadotropin-releasing hormone (GnRH) agonists plus recombinant human chorionic gonadotropin (r-hCG)) in patients with previous cycles triggered with r-hCG.MethodsThis case-control study included 88 matched cycles performed in 88 patients, which had the first ICSI cycle triggered with r-hCG (n=44), and the following ICSI cycle with dual trigger (n=44). We compared the cycle outcomes between the groups. In a second case-control within-subject analyses, we compared the ICSI outcomes between patients which had the first ICSI cycle triggered with r-hCG only (n=18), and the following ICSI cycle with dual trigger (n=18) or r-hCG only (n=18).ResultsUpon investigating repeated cycles (r-hCG only vs. dual trigger), we found higher oocyte yield and mature oocyte rates, lower immature oocyte rates, higher fertilization rates, and higher blastocyst development rates; and higher rates of cycles with embryos transferred and implantation in the dual trigger cycle.ConclusionsThe dual trigger regimen is a more effective approach than r-hCG trigger in patients with a previous r-hCG triggered ICSI cycle, yielding improved response to COS, and better laboratorial and clinical outcomes.

Fertilisation and early embryonic development of immature and rescue in vitro-matured sibling oocytes

The objective of this study was to assess the effect of rescue in vitro maturation and immediate intracytoplasmic sperm injection (ICSI) application on fertilisation success and early embryonic development of metaphase I (MI) oocytes. This was a retrospective cohort study including 2425 sibling oocytes in 259 ICSI cycles. ICSI was performed on 104 GV (germinal vesicle) oocytes which had reached the metaphase II (MII) stage (Group 1) and 231 MI oocytes which had reached the MII stage (Group 2) following IVM (in vitro maturation). Immediate ICSI was applied following oocyte aspiration on 292 MI stage (Group 3) and 1798 MII stage oocytes (Group 4). Normal fertilisation rates in Groups 1, 2, 3 and 4 were 51.9%, 39%, 30.1% and 59.5%, respectively. The rates of blastocyst development per oocyte and per zygote were calculated as 3.8%, 3.0%, 6.8%, 14.1% and 7.4%, 7.7%, 22.7%, 23.6% for Groups 1, 2, 3 and 4, respectively. The blastocyst development rate was significantly higher in the MI-ICSI group compared with other immature oocytes. Even though performing ICSI on the oocytes at the MI stage on the day of oocyte aspiration resulted in lower fertilisation rates, it was associated with significantly higher rates of blastocyst development.

The IMSI Procedure Improves Laboratory and Clinical Outcomes Without Compromising the Aneuploidy Rate When Compared to the Classical ICSI Procedure.

PURPOSEThe intracytoplasmic morphologically selected sperm injection (IMSI) procedure has been associated with better laboratory and clinical outcomes in assisted reproduction technologies. Less information is available regarding the relationship between embryo aneuploidy rate and the IMSI procedure. The aim of this study is to compare the clinical outcomes and chromosomal status of IMSI-derived embryos with those obtained from intracytoplasmic sperm injection (ICSI) in order to establish a clearer view of the benefits of IMSI in infertile patients.METHODSWe retrospectively analyzed a total of 11 cycles of IMSI and 20 cycles of ICSI with preimplantation genetic diagnosis. The fertilization rate, cleavage rate, embryo quality, blastocyst development, aneuploidy rate, pregnancy rate, implantation rate, and miscarriage rate were compared between the groups.RESULTSSimilar rates of fertilization (70% and 73%), cleavage (98% and 100%), and aneuploidy (76.9% and 70.9%) were observed in the IMSI and ICSI groups, respectively. The IMSI group had significantly more good quality embryos at day 3 (95% vs 73%), higher blastocyst development rates (33% vs 19%), and greater number of hatching blastocysts (43% vs 28%), cycles with at least one blastocyst at day 5 (55% vs 35%), and blastocysts with good trophoectoderm morphology (21% vs 6%) compared with the ICSI group (P < 0.001). Significantly higher implantation rates were observed in the IMSI group compared with the ICSI group (57% vs 27%; P < 0.05). Pregnancy and miscarriage rates were similar in both groups (80% vs 50% and 0% vs 33%, respectively).CONCLUSIONThe IMSI procedure significantly improves the embryo quality/development by increasing the implantation rates without affecting the chromosomal status of embryos. There is a tendency for the IMSI procedure to enhance the pregnancy rates and lower the miscarriage rates when compared with ICSI.

Maturation rate and subsequent blastocyst development of human oocytes derived from various follicle size at the time of oocyte pick up

Mature oocytes were often gotten from various follicle diameter by ultrasound diagnosis. We have estimated follicle size based on amount of follicle fluid at the time of oocyte pick up (opu) in conventional IVF and ICSI since 2013. The objective of this study was to make it appear that the relationship between follicle size at the time of opu ,maturation rate of oocyte and subsequent development. This study was a retrospective analysis of maturation rate of oocytes derived from large (≥18 mm; LF), medium (16-17 mm; MF) and small (≤15 mm; SF) follicle at the time of opu, and subsequent cleavage of the oocytes and blastocyst development were assessed on day 2 and day 5 after ICSI between 2013 and 2014. From August 2013 to March 2014, we studied maturation rate of oocytes and developmental rate of embryos to the blastocyst stage derived from large (≥18 mm; LF), medium (16-17 mm; MF) and small (≤15 mm; SF) sized follicles for 306 cycles induced by clomiphene or letrozole-rFSH-GnRHa administration. Follicle fluid was measured individually follicles after oocyte picked up. Then, the follicle size was calculated with volume of the fluid. The oocytes were examined for maturation and fertiization. Cleavage of the oocytes and blastocyst development were assessed on day 2 and day 5. All blastocysts were vitrified and stored for a single blastocyst transfer in the next cycle. Maturation rate of oocytes derived from large follicle 87.5% (239/273) were significantly higher than medium and small follicle (69.5% (41/59) and 36.8% (53/144) P<0.05, respectively).The percentages of normal fertilization did not differ between large 62.8% (150/239), medium 78.0% (32/41) and small 64.2% (34/53) follicle size. Although there was not significantly different in the blastocyst development rates, the oocytes derived from medium 51.2% (20/41) follicle were higher blastocyst development rate than large 39.3% (145/239) and small 30.2% (37/53) follicle. These results suggest that follicle size affected maturation rate at the time of opu. Although follicle size did not affected fertilization, the blastocyst development could be higher with oocytes derived from medium follicle. Follicle size based on amount of follicle fluid at the time of opu could be one of criteria for selecting to transfer embryo.

Selection of &lt;i&gt;In Vitro&lt;/i&gt;-Matured Porcine Oocytes Based on Localization Patterns of Lipid Droplets to Evaluate Developmental Competence

Localization patterns of lipid droplets in the cytoplasm of porcine oocytes were evaluated as a novel marker for in vitro maturation (IVM) of oocytes with high developmental competence. Porcine oocytes were cultured in TCM-199, which is a complete synthetic medium, for 44 h at 38.5 C. Localization patterns were divided into 2 classes: lipid droplets localized uniformly in the whole cytoplasm (class I) and those that were centrally located (class II). After IVM in TCM-199, 60% of matured oocytes exhibited the class II pattern. To investigate the relation between the distribution of lipid droplets and the developmental rate of the oocyte, the developmental rates of class I and class II oocytes were compared after in vitro fertilization (IVF). Class II oocytes showed a significantly higher rate of blastocyst development than class I oocytes. These results suggest that porcine oocytes with high developmental competence can be selected based on the localization patterns of lipid droplets.

Enucleation of Demecolcine-Treated Bovine Oocytes in Cytochalasin-Free Medium: Mechanism Investigation and Practical Improvement

Demecolcine-assisted/induced enucleation has been used in nuclear transfer cloning procedures for many species, yet its mechanism of action remains unclear. Primarily because oocytoplasm protrusion induced by demecolcine is inhibited by the presence of cytochalasin, its use has had limited application. In this experiment, we investigated the microtubule and microfilament alterations in bovine oocytes after demecolcine and/or cytochalasin B (CB) treatments by immunocytochemical staining. We also examined mechanical enucleation of demecolcine-treated oocytes in cytochalasin-free medium. The results showed that demecolcine-treatment disrupts the balance between microtubule/microfilament interactions primarily by deleting microtubules and with little effect on the microfilaments that we believe accounts for the membrane protrusion. The CB treatment reduced the amount of microfilaments but had little effect on the microtubules. Most demecolcine-induced membrane protrusions disappeared when exposed to CB. Western blotting showed that CB treatment increases G-actin, which indicates a decrease in the microfilaments. High oocyte enucleation, survival, and developmental rates occurred when demecolcine-assisted enucleation was carried out in a CB-free solution. Higher blastocyst development rates and blastocyst cell numbers were achieved compared to control, indicating that CB is not necessary in the enucleation procedure of bovine oocytes. This study provides a clearer understanding of the mechanism for the demecolcine-induced oocyte membrane protrusion, and substantiates the practical use of demecolcine-assisted enucleation in a CB-free environment.

Oocyte Source and Hormonal Stimulation forIn VitroFertilization Using Sexed Spermatozoa in Cattle

The aim of this study was to investigate the efficiency of in vitro embryo production in cattle utilizing sexed sperm from two bulls and oocytes recovered by OPU. Twenty donor animals were employed in eight OPU replicates: the first four OPU trials were conducted on animals without hormone treatment, and the last four were run on the same animals, following FSH subcutaneous and intramuscular administration. A higher rate of blastocyst development was recorded in stimulated, as compared to nonstimulated animals, (25.2% versus 12.8%, P = .001). Ocytes derived from slaughterhouse (SH) ovaries were also fertilized with sperm from the same bulls. Overall, non-sexed sperm used with oocytes derived from SH ovaries was significantly more efficient for blastocyst development than was sexed sperm with these same SH derived oocytes and sexed sperm with stimulated donor oocytes (39.8% versus 25.0% and 25.2%, P = .001). In conclusion, the use of sexed sperm with OPU-derived oocytes resulted in a significantly higher blastocyst development when donors were hormonally stimulated; furthermore, the level of efficiency achieved was comparable to that attained when the same sexed sperm was tested on oocytes derived from SH ovaries.

46 RECLONING USING TRANSGENIC FETAL FIBROBLASTS AS NUCLEI DONORS INCREASES DEVELOPMENTAL POTENTIAL OF RECONSTRUCTED EMBRYOS IN CATTLE

Genetically modified animals have numerous applications ranging from basic research to agriculture production. Cloning by nuclear transfer (NT) has made possible the production of transgenic animals using previously genetically modified cell lineages. Gene expression studies and adequate selection of the nuclei donor cell for NT guarantees the presence of the gene construction in the offspring and the absence of deleterious mutations caused by the random insertion of transgenes in functional areas of the genome. Embryonic development after NT requires a change in the transcriptome of the donor cell from a somatic to an embryonic pattern, causing cloning efficiencies to be low because of incomplete or defective nuclear reprogramming. Therefore, the establishment of methodologies able to increase cloning success is highly desirable. The experiment was designed to test if recloning of transgenic fetal fibroblasts increases cloned blastocyst production and the pregnancy rates of transgenic cloned embryos produced by NT. This study compared the developmental potential of cloned embryos reconstructed with fetal fibroblasts genetically modified by lentivirus random integration (control group) expressing the green fluorescent protein gene (eGFP), with a transgenic fetal fibroblast cell line established from a 30-day transgenic pregnancy (recloning group). Fusion, cleavage (72 h post-activation, hpa), blastocyst production (168 hpa), and 30-day pregnancy rates were analyzed. A total of 1213 embryos were reconstructed; 884 (10 replicates) with random transgene insertion fibroblasts and 329 (4 replicates) with cells derived from the cloned fetus. Results were analyzed by chi-square test at 5% significance. No difference was observed (P &gt; 0.05) between control and recloned groups regarding fusion rate (n = 550, 62.22% and n = 189, 57.25%; respectively) or cleavage rate (n = 383, 69.45% and n = 132, 69.84%, respectively). The recloned group, however, showed a higher blastocyst development rate (P &lt; 0.01) compared with the control group (n = 51, 26.98%, and n = 79, 14.36%, respectively) and a higher 30-day pregnancy rate (n = 6, 15.38% and n = 3, 5.56%, respectively). In conclusion, recloning of transgenic fibroblasts from a successfully established pregnancy augments the efficiency in the production of embryos and pregnancy establishment compared with the control group. We speculate that a second round of NT enhances the nuclear reprogramming of donor cells, and moreover, the use of a transgenic cell lineage already proven to be successfully reprogrammed may indicate that transgene integration is not deleterious in that specific cell lineage, resulting in a good source of donor cells to be used in order to produce a homogeneous bioreactor herd. Financial support: FAPESP, Brazil.

Influence of oocyte recovery method, in vitro fertilization method and serum source on embryonic development of in vitro matured bovine oocytes

Abstract. The objective of this study was to investigate the influence of various factors on blastocyst development in a bovine in vitro embryo production system. Two production systems were originally compared: Protocol 1, which utilized oocyte recovery by slicing, 20 % fetal bovine serum with FSH (FBS+) for oocyte maturation, and sperm preparation system 1 (F1), and Protocol 2, which utilized oocyte recovery by aspiration, 5 % estrous cow serum with FSH, HCG and estradiol (ECS+) for oocyte maturation, and sperm preparation system 2 (F2). Because a significantly higher blastocyst development rate was found for Protocol 2, the different factors of oocyte recovery technique (slicing vs. aspiration), sperm preparation technique (F1 vs. F2) and serum and hormone supplementation during maturation (FBS+ vs. ECS+) were evaluated. Recovering oocytes using the alternative technique (slicing vs. aspiration) did not significantly alter blastocyst rate on Day 8 within either production system 1 (FBS+/F1) or production system 2 (ECS+/F2). Although investigations of influence of the type of fertilization method revealed no effect, a significant effect of the type of serum was observed on the blastocyst rates with ECS+ proving better as compared to the use of FBS+ (P&lt;0.03). When embryos produced from these investigations were evaluated by differential staining, the number of ICM cells did not differ among treatment, but the number of TE cells, and thus the total cell number, and the ICM : TCN ratio, was significantly increased when oocytes were matured in ECS+ supplemented medium, regardless of sperm preparation method. In conclusion, serum supplementation, but not oocyte recovery method or sperm preparation method, was responsible for the difference in blastocyst rate between the two original IVP protocols.

103 BIRTH OF CLONED PIGLETS DERIVED FROM AN OPTIMIZED IN VITRO BLASTOCYST PRODUCTION SYSTEM BY TRICHOSTATIN A TREATMENT

The present study was designed to examine the effect of trichostatin A (TSA, a histone deacetylase inhibitor) treatment on in vitro developmental ability of pig cloned embryos and to evaluate the feasibility of producing piglets from these embryos. Cell lines were established from 40-day-old fetuses, and adult ear skin was used as nuclear donor. In vitro-matured oocytes from abattoir-derived sow ovaries were used as cytoplast recipients for micromanipulator-assisted somatic cell nuclear transfer (SCNT). Data were analyzed by using SPSS (11.0) with one-way ANOVA, and each experiment was replicated at least 3 times. In Experiment 1, immediately after simultaneous fusion and activation, the reconstructed couplets were randomly cultured in porcine zygote medium 3 (PZM3; Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) with 10 �g mL-1 cytochalasin B (CB), 10 �g mL-1 cycloheximide (CHX), and 0 nM, 5 nM, or 50 nM TSA for the first 4 h. Cloned embryos (fused reconstructed couplets) were moved to the same culture media but without CB and CHX and further cultured at 38.5�C, under 5% CO2, 5% O2, 90% N2 and 100% humidity. After incubation for a total of 8–14 h in 50 nM, 19–24 h in 50 nM or 5 nM, and 31–36 h in 50 nM TSA in PZM3 (0 nM TSA serves as control for each group), the embryos were further cultured in vitro without TSA in PZM3 for up to 168 h. Cleavage and blastocyst development rates, based on embryos cultured, were recorded at 48 and 168 h of IVC, respectively. Results showed that 50 nM TSA treatment for 19-24 h supported a higher blastocyst development rate than the control group [No. blastocysts/No. embryos cultured (mean � SEM): 107/258, 47.4 � 5.9% vs. 65/324, 20.0 � 2.3%, respectively; P &amp;lt; 0.05], whereas similar pre-implantation development was obtained between the other 3 test groups and the control. In Experiment 2, TSA-treated cloned embryos at the one-cell stage or blastocyst stage were transferred to recipients to examine the possibility of producing piglets. Ten cloned piglets (2 are healthy and 8 died shortly after birth) and one ongoing pregnancy were obtained from 3 recipients who received an average of 110 one-cell stage embryos, whereas 4 piglets originating from traditional cloning were produced from one recipient which received 28 traditional cloned blastocysts (produced from the effective group in Experiment 1) and 30 handmade but non-TSA-treated ones. Our data demonstrate that TSA treatment after SCNT in porcine can significantly improve the in vitro blastocyst production, and embryos treated with TSA could support full-term development and result in healthy offspring.

Chromatin Configuration Within the Germinal Vesicle of Horse Oocytes: Changes Post Mortem and Relationship to Meiotic and Developmental Competence1

We evaluated the relationship of initial chromatin configuration to time of oocyte recovery and to nuclear maturation after culture in horse oocytes having compact (Cp) and expanded (Ex) cumuli. In addition, we evaluated the effect of oocyte type, time of recovery, and duration of culture on blastocyst development after intracytoplasmic sperm injection. In oocytes collected within 1 h of slaughter, fibrillar and intermediate chromatin configurations were more prevalent in Cp than in Ex oocytes (68% and 12%, respectively). In Cp oocytes collected after a 5- to 9-h delay, the proportions in the fibrillar and intermediate configurations decreased significantly, and the proportions of degenerating and homogeneously fluorescent configurations increased. When cultured, 20% of oocytes classified as having fibrillar chromatin resumed meiosis, whereas 82% of intermediate and 81% to 86% of condensed chromatin oocytes did so. Meiotic resumption was higher in oocytes recovered immediately after slaughter, but these oocytes took longer to mature. Duration of maturation significantly affected blastocyst development rates in Cp oocytes recovered after a delay (13% and 38% for oocytes matured 24 and 36 h, respectively). Oocytes recovered after a delay had higher blastocyst development rates than did those collected immediately after slaughter. We conclude that the fibrillar and intermediate chromatin configurations may degenerate during ovary storage, resulting in decreased maturation rates, especially of Cp oocytes. Time of oocyte recovery and duration of maturation significantly affect the rate of blastocyst development. Oocytes with Cp and Ex cumuli have similar developmental competence to the blastocyst stage.

Parthenogenetic and nuclear transfer rabbit embryo development and apoptosis after activation treatments

Previous studies mainly evaluated the effect of culture conditions on preimplantation embryo apoptosis. In order to inhibit apoptosis of nuclear transfer (NT) embryos, putative apoptosis inhibitors were used to treat donor cells. However, little is known about the effect of activation treatments on embryo apoptosis. We firstly investigated the effect of various parthenogenetic activation (PA) treatments on embryo development, blastocyst cell number, and apoptosis, and then one of these activation treatments proved to be most efficient was selected for activation rabbit NT embryos. The activation by electrical pulses and 30 min later, electroporation with 25 muM D-myoinositol 1,4,5-trisphosphate (IP3) in Ca(2+)- and Mg(2+)-free PBS, then exposure to 2.0 mM 6-dimethylaminopurine (6-DMAP) for 3 hr effectively activated rabbit oocytes, and resulted in significantly a higher blastocyst development rate (72.7%) and total cell number (175 +/- 14.1), and markedly lower apoptosis level of blastocyst (4.3 +/- 0.5) than all the other groups. When the same activation protocol was applied in NT embryo activation, we found that exposure of the embryos to 6-DMAP for 3 hr could decrease the apoptosis level of blastocyst and increase blastocyst rate and cell number. The results demonstrate that oocyte activation affects not only embryo development and quality but also embryo apoptosis.

66EFFECTS OF CONTACT INHIBITION INTERVALS V. SERUM DEPRIVATION ON DEVELOPMENT OF PORCINE NUCLEAR TRANSFER DERIVED EMBRYOS

Contact inhibition and serum deprivation are commonly used to synchronize donor cells at the G0/G1 state of the cell cycle prior to use in nuclear transfer. Here we compared the effects of serum deprivation (SD) and different intervals of contact inhibition (CI) of the donor cells on the blastocyst rate. One batch from pooled porcine fetal fibroblasts (passage 3) was used in this study. The cells were thawed, seeded to a six-well plate and cultured in Dulbecco‘s Modified Eagles Medium (DMEM) supplemented with 2mM glutamine, 1% non-essential amino acids, 0.1mM mercaptoethanol, 100UmL−1 penicillin, 100mgmL−1 streptomycin containing 10% fetal calf serum (FCS). Serum deprivation was achieved by culturing cells in DMEM containing 0.5% FCS for 48h. Cells of the CI groups were grown to 100% confluency and kept in that state for 24h, 48h and 72h. Immediately after cell cycle synchronization, cells were used in nuclear transfer. Cell cycle state of the cells was evaluated by FACS analysis at 24h after beginning of CI and prior to nuclear transfer. Blastocyst rate was determined 7 days after nuclear transfer. An average of 38-42h in vitro matured oocytes were used in nuclear transfer (NT). NT was performed as described previously (Betthauser J et al., 2000 Nat. Biotechnol. 17, 456–461). There were no differences in the proportion of cells in G0/G1 of the cell cycle in any of the treatment groups (85.0%, 85.8%, 85.5% and 86.3% for SD and CI at either 24h, 48h and 72h, respectively). After nuclear transfer (for each CI group n=336–384 reconstr. embryos; SD n=215) there was a statistically significant difference in the fusion rate between 48h CI and SD cells (74.8% v. 87.5%, t-test P&amp;lt;0.050). Blastocyst rate (blastocysts/fused) differed significantly between SD, 24h CI and 48h CI (17.4%; 9.1%, 9.6%, t-test P&amp;lt;0.050), there was no difference between SD and 72h CI and within the CI groups (72h CI 10.6%). Four transfers of reconstructed embryos (72h CI, n=138–163 embryos/gilt, 1-cell embryos) to prepuberal Landrace gilts led to 2 initial pregnancies determined at Day 25 by ultrasound. One pregnancy was lost at Day 35; the other recipient remained pregnant and farrowed 4 piglets. One piglet was stillborn and one died 7h after birth; the remaining two piglets are healthy and now 4 months old. Four transfers of embryos (n=96–110) reconstructed with SD cells revealed two initial pregnancies determined on Day 25 by ultrasound. Again, one was lost on Day 35, and the other one is now at Day 100. Our results show that, despite similar proportions of cells being in G0/G1 of the cell cycle, cells either contact-inhibited for 72h or serum-deprived both show higher rates of blastocyst development compared to cells contact-inhibited for shorter time periods. Both donor cell preparations can lead to full term development of nuclear transfer-derived embryos. This work was funded by the Deutsche Forschungsgemeinschaft (DFG, SFB 265).

Differential cytoplast requirement for embryonic and somatic cell nuclear transfer in cattle.

Effective activation of a recipient oocyte and its compatibility with the nuclear donor are critical to the successful nuclear reprogramming during nuclear transfer. We designed a series of experiments using various activation methods to determine the optimum activation efficiency of bovine oocytes. We then performed nuclear transfer (NT) of embryonic and somatic cells into cytoplasts presumably at G1/S phase (with prior activation) or at metaphase II (MII, without prior activation). Oocytes at 24 hr of maturation in vitro were activated with various combinations of calcium ionophore A23187 (A187) (5 microM, 5 min), electric pulse (EP), ethanol (7%, 7 min), cycloheximide (CHX) (10 micro g/ml, 6 hr), and then cultured in cytochalasin D (CD) for a total of 18 hr. Through a series of experiments (Exp. 1-4), an improved activation protocol (A187/EP/CHX/CD) was identified and used for comparison of NT efficiency of embryonic versus somatic donor cells (Exp. 5). When embryonic cells from morula and blastocysts (BL) were used as nuclear donors, a significantly higher rate of blastocyst development from cloned embryos was obtained with G1/S phase cytoplasts than with MII-phase cytoplasts (36 vs. 11%, P < 0.05). In contrast, when skin fibroblasts were used as donor cells, the use of an MII cytoplast (vs. G1/S phase) was imperative for blastocyst development (30 vs. 6%, P < 0.05). Differential staining showed that parthenogenetic, embryonic, and somatic cloned BL contained 26, 29, and 33% presumptive inner cell mass (ICM) cells, respectively, which is similar to that of frozen-thawed in vivo embryos at a comparable developmental stage (23%). These data indicate that embryonic and somatic nuclei require different recipient cytoplast environment for remodeling/ reprogramming, and this is likely due to the different cell cycle stage and profiles of molecular differentiation of the transferred donor nuclei.

Sperm and oocyte treatments to improve the formation of male and female pronuclei and subsequent development following intracytoplasmic sperm injection into bovine oocytes.

This study assessed pronuclear formation, the chromosomal constitution, and the developmental capacity of bovine zygotes formed by intracytoplasmic injection of oocytes with sperm, treated or not with dithiothreitol (DTT). Oocytes were matured in vitro for 22-24 h and then centrifuged so that sperm, prepared by swim-up in the presence or absence of 5 mM DTT, could be injected into the cleared area of the ooplasm. Injected oocytes were activated by treatment with 5 microM ionomycin (5 min) and, after a 3-h interval, with 1.9 mM 6-dimethylaminopurine (DMAP) for 3 h. They were then cocultured with bovine oviductal epithelial cells in M199. Sperm treatment resulted in a significantly higher proportion of male pronucleus formation 16 h after injection (40% vs. 11%; p < 0.0001) and a significantly higher rate of blastocyst development (24% vs. 10%; p < 0.005). Sixty-one percent of blastocysts produced with treated sperm were diploid. Of 12 blastocysts produced with treated sperm and sexed by a polymerase chain reaction, 4 were male and 7 female, and in one a definite diagnosis could not be made. Embryo transfer (2 embryos per heifer) resulted in pregnancies in 6 of 16 recipients at Day 49, but none was carried to term. These results show that the efficiency of bovine intracytoplasmic sperm injection can be improved by sperm pretreatment with DTT and by oocyte activation with ionomycin plus DMAP, although the developmental capacity of the resulting embryos remains limited.

Effects of Oxygen Concentration and Free-Radical Scavengers on the In Vitro Development of Bovine Oocytes Matured and Fertilized &lt;I&gt;In Vitro&lt;/I&gt;

The present study was conducted to evaluate effects of oxygen concentration and free-radical scavengers on in vitro development of bovine oocytes matured and fertilized in vitro. Oocytes were matured in TCM-199+10% FCS supplemented with hormones and granulosa cells, and were fertilized in vitro with frozen-thawed, swim-up separated, and heparin-treated spermatozoa. After insemination, oocytes were cultured for 8 days in SOFM supplemented with 10% HS and with 5 different SOD (super-oxide dismutase) levels (0 to 750 μg/ml) [Experiment 1] and 5 different catalase levels (0 to 100 μg/ml) [Experiment 2]. In Experiment 1, high levels of SOD (500 and 750 μg/ml) significantly (P<0.01) reduced the rates of blastocyst development. In Experiment 2, there was no significant difference on the develop-ment to the blastocysts stage among the catalase levels. In Experiments 3 and 4, effects of the addition of SOD+ catalase under two gas atmospheres (5% CO2, 5% O2, 90% N2 vs 5% CO2 in Air) were examined. The addition of the free-radical scavengers in the medium under 5% CO2, 5% O2, 90% N2 resulted in sig-nificantly (P<0.05) higher rate of blastocyst development than embryos cultured under 5% CO2 in Air. The free-radical scavengers did not improve the blastocyst development under 5% CO2 in Air. It appeared that the cause of the stress of high oxygen tension (20% O2) was not only due to the free radicals but also other undetermined inhibiting factors.