Higher Blastocyst Formation Rate Research Articles

Does Mitochondrial Transfer An Alternative to Egg Donation?

Objective
 To evaluate the utility of pronuclear transfer using healthy oocytes from donors in poor prognosis patients.
 Design
 Retrospective study.
 Subjects
 A total of 29 patients with no blastocyst development or total fertilization failure were included.
 Main Outcome Measures
 Our primary outcome was the blastocyst rate. Secondary outcomes measures included euplody rate, clinical pregnacy rate and live birth rate.
 Results
 On day 5/6 of blastocyst development, an average blastocyst rate of 49.2% was obtained. The average rate of euploid blastocysts obtained after PGT-A was 69.5%. All patients underwent frozen embryo transfer. Single blastocyst transfer was performed in all patients, and the clinical pregnancy rate was 48.3%. Of these patients, 14 had a live birth, and the live birth rate was 48.3%. No genetic or morphologic abnormalities have been detected in the babies born. The 29 patients were grouped based on their age, as <35 (n=2), 35-27 (n=6), 38-40 (n=9), 41-42 (n=6) and >42 (n=6). Based on these age groups, blastocyst formation rates were 47.8%, 50.0%, 45.0%, 50.0% and 66.7%, respectively; euploidy rates were 63.6%, 61.1%, 66.7%, 100.0% and 83.3%, respectively; clinical pregnancy and live birth rates were 100.0%, 50.0%, 44.4%, 50.0% and 33.3%, respectively.
 Conclusions
 The results of this study show that pronuclear transfer can result in a high blastocyst formation rate, euploidy rate and live birth rate. This was shown among patients across all age groups, particularly in older patients who often show diminished IVF success rates.

Effects of niacin supplementation during in vitro culture on the developmental competence of porcine embryos.

Niacin is a water-soluble vitamin belonging to the vitamin B complex. It has been found to possess various biological activities, including antioxidant and lipid modification capacities. This study aimed to elucidate the effects of niacin treatment in porcine in vitro culture (IVC) medium on embryo developmental competence after parthenogenetic activation. IVC medium was supplemented with different concentrations of niacin (0 [control], 300, 600 and 900 μM). The results showed that embryos cultured in an IVC medium supplemented with 300 and 600 μM niacin had an increased cleavage rate (p < .05). In addition, 300 μM niacin treatment resulted in a higher blastocyst formation rate than the control and other niacin-treated groups. However, the total cell number did not differ significantly among the experimental groups. Niacin supplementation at 600 μM decreased reactive oxygen species, whereas treatment with 300, 600 and 900 μM increased glutathione levels in day two embryos. On day seven, 300 μM niacin exhibited improved fatty acid levels and fewer lipid droplets than the control group. Furthermore, gene expression at the mRNA level was performed on day two and day seven embryos, treated with or without 300 μM niacin. The expression of anti-apoptotic BCL2 and lipid metabolism PLIN2-related genes were upregulated, whereas the pro-apoptotic BAX and CASPASE3 were downregulated with niacin supplementation compared with the control group. However, SIRT1, a gene related to energy and the oxidative state, was up-regulated in niacin-treated day two embryos (p < .05). Overall, the results indicate that niacin has a beneficial effect on pre-implantation embryo development by modulating lipid metabolism and reducing oxidative stress and apoptosis. The expression patterns of PLIN2 and SIRT1 reported here suggest that these transcripts may be involved in the mechanism by which niacin affects the developmental capacity of IVC embryos.

Effects of crocin supplementation during in vitro culture of intact and half-destroyed four-cell mouse embryos.

We evaluated the effects of crocin supplementation during culture of intact and half-destroyed four-cell mouse embryos. Outcomes measured included rate of cleavage arrest, blastocyst formation, and blastocyst cell number. We used laser to create two zonal holes without blastomere destruction in Groups 1 (n=100) and 2 (n=100), and to destroy two of the four blastomeres in Groups 3 (n=150) and 4 (n=150). Embryos were cultured in groups of ten in drops of medium without (Groups 1 and 3) or with 20 μg/ml of crocin supplementation (Groups 2 and 4). Embryos in Groups 1 and 2 had no difference in the rate of cleavage arrest (6.0% vs. 7.0%, respectively; p=0.774) or blastocyst formation (89.0% vs. 86.0%, respectively; p=0.521). Neither was there a difference in the number of cells in the blastocysts (99.6±23.5 vs. 95.6± 8.2, respectively, p=0.83). Half-destroyed embryos cultured in crocin-supplemented medium (Group 4) had a lower rate of cleavage arrest (14.7% vs. 30.0%, p=0.001), and a higher rate of blastocyst formation (51.3% vs. 37.3%, p=0.015), than those in non-supplemented medium (Group 3). In blastocysts derived from half-destroyed embryos, there was no difference in the number of cells in ICM (14.5±3.9 vs. 13.7±2.9, p=0.285), TE (45.2±12.3 vs. 46.0±13.3, p=0.764), or total cells (59.7±12.2 vs. 59.7±14.8, respectively, p=0.990) among the two groups. Crocin supplementation during in vitro development of impaired embryos improved their development, but had no effect on intact embryos.

Effect of method and media type for in vitro fertilization on equine (Equus ferus caballus) embryo development

Demonstrating the role of in vitro fertilization in the equine family, the current study aimed to monitor the rates of equine in vitro-fertilized and cleaved oocytes based on microdroplet and intracytoplasmic sperm injection (ICSI) methods. The fertilization process was carried out in four main media consisting of Tyrod?s albumin lactate pyruvate and TCM-199 (1:1): F (TCM-199); assigned for the ICSI method, FI (contained 0.01 mg/ml heparin + 0.01 ng/ml hypotaurine), FII (contained 0.3 mg/ml calcium chloride + 0.1 mg/ml magnesium chloride) and FIII (contained 0.05 mg/ml hypotaurine-epinephrine). The results indicated an increase in the fertilization rate by the ICSI method (65.71%) compared to the microdroplet method (39.25%) (p 0.04). The rates of unfertilized and degenerated oocytes in the ICSI method decreased, compared to the microdroplet method, to 8.57% and 25.72%, respectively; p&lt; 0.001. The rate of fertilized oocytes in the FI medium increased to 47.61% (p&lt; 0.05) compared to oocytes of FII and FIII treatments (37.64% and 30.00%, respectively). On in vitro culture, the rate of cleavage increased to 74.62% in the CI medium (SOF) compared to those in the CII medium (DMEM-F12) (61.29%); p &lt;0.001. Zygotes cultured in the CII medium achieved a higher rate of blastocyst formation (30%) compared to those in the CI medium (26.31%); p&lt; 0.001. Applying the ICSI method and SOF culture media led to high yields of equine embryos.

O-099 The use of cumulus cells (CCs) to select spermatozoa for intracytoplasmic sperm injection improves blastocyst development and quality

Abstract Study question Is there a role for sperm selection based on the capacity to pass through cumulus oophorous column in improving the laboratory outcomes in ICSI cycles? Summary answer A higher rate of blastocyst formation and quality are obtained with the use of CCs to select spermatozoa for ICSI compared to conventional density gradients. What is known already The sperm selection for ICSI is an operator-dependent procedure based on sperm morphology and motility. ICSI bypasses all natural barriers of sperm selection, which may impact embryo development. During natural fertilization, only the spermatozoa that pass-through cumulus cells will have the chance to fertilize the egg. Spermatozoa capable of traverse COCs have better morphology, higher chromatin integrity and greater acrosomal reaction. Hyaluronic acid is an important component of these cells and plays a key role in the selection of spermatozoa with intact DNA. However, there is scarce data regarding this selection method for ICSI on the development of the embryo. Study design, size, duration This is a prospective randomized sibling oocyte pilot study (February-December 2021). A total of 996 metaphase II (MII) oocytes were randomized into two groups. In the control group (Group 0, n = 489 MII), ICSI was performed with sperm selected by conventional density gradients. In the study group (Group 1, n = 507 MII) the oocytes were microinjected with sperm selected through CCs. The main outcome was blastocyst formation rate and quality. Reproductive results were analyzed as secondary outcomes. Participants/materials, setting, methods Ninety-eight couples undergoing ICSI (private center) with ≥6 fresh own MII obtained after ovarian stimulation, embryo culture until blastocyst stage and ejaculated sperm samples were included in the study. All seminal samples were processed using density gradients and those in the study group were additionally passed through CCs (the patient's own CCs were used in all cases). Fertilization and day 5 blastocyst formation rates were assessed. Embryo quality was scored according to Gardner's criteria. Main results and the role of chance Data was similar with regards to cause of infertility, maternal age (36.47 vs 36.63 y.o.), sperm count (60.53 vs 58.25 mill/ml) and progressive motility (57.45 vs 57.50%) between group 0 vs 1, respectively. Fertilization rate was similar between groups (74.0% vs 78.7%; p = 0.419. Group 0 vs 1, respectively). However, the blastocyst formation rate was significantly higher in the study group compared to control (70.9% vs 60.1%; p = 0.002). In addition, high-quality blastocyst rate was significantly higher in the CCs compared to the control group (49.6% vs 38.5%; p = 0.003). After the analysis of 145 treatments (131 frozen vs 14 fresh transfers) the cumulative clinical pregnancy rate in the study group was 47.2%, higher than in the control group (32.1%), but without reaching statistical difference (p = 0.07). Limitations, reasons for caution This is an exploratory study, aimed to evaluate the effectiveness of CCs as a more “physiologic” selection of spermatozoa for ICSI treatments in terms of laboratory outcomes (blastocyst formation rate and quality). Albeit not reaching statistical significance, the higher cumulative pregnancy rate in the study group seems clinically relevant. Wider implications of the findings Our results suggest that CCs can select more competent sperm for ICSI. This “physiologic-resembling”, operator-independent selection method has the potential of increasing pregnancy outcomes in ICSI cycles, pending of confirmation in larger studies including cumulative embryo-transfer cycles. Trial registration number NA

Effects of Trichostatin A on the Timing of the First Cleavage and In Vitro Developmental Potential of Bovine Somatic Cell Nuclear Transfer Embryos.

This study examined the relationship between the timing of the first cleavage and in vitro development of bovine somatic cell nuclear transfer (SCNT) embryos treated with trichostatin A (TSA). SCNT embryos were visually assessed at 22, 26, and 48 hours after activation. Each embryo with two or more distinct blastomeres was transferred into a microwell and cultured until day 7. Irrespective of TSA treatment, approximately half of the cleaved embryos were observed at 22 hours, and a significantly higher blastocyst formation rate was shown in the SCNT embryos cleaved at 22 hours than those cleaved at ≥26 hours. The blastocyst formation rate of TSA-treated embryos cleaved at 22 hours (80%) was slightly higher than that of the control embryos (70%). In addition, interferon-τ (IFN-τ) expression was significantly lower in control SCNT embryos and late-cleaving (>26 hours) TSA-treated embryos than in in vitro fertilized (IVF) embryos. However, a significant difference was not observed between TSA-treated SCNT embryos cleaved at 22 and 26 hours, and IVF embryos. These results suggest that TSA treatment has no influence on the timing of the first cleavage of SCNT embryos; however, it slightly improves the blastocyst formation rate and the expression level of IFN-τ in early-cleaving embryos.

The Resurrection of Mabrokan: Production of Multiple Cloned Offspring from Decade-Old Vitrified Tissue Collected from a Deceased Champion Show Camel.

Simple SummarySomatic cell nuclear transfer (SCNT) is a technique used to reproduce individuals from their somatic cell nucleus, which is commonly known as cloning. In SCNT, viable cell lines are usually established from living animals and preserved for future use. In the present study, tissues were collected and cryopreserved (rather than cells) from a suddenly deceased champion show camel. We established fibroblast cell lines from this decade-old vitrified tissue and used them as nuclear donors. Both the in vitro and in vivo matured oocytes were used to produce cloned embryos. Blastocysts were transferred to synchronized recipients to establish pregnancies. A total of 18 pregnancies (5 from in vitro matured oocytes and 13 from in vivo matured oocytes) were established, and 11 live offspring were born (2 from in vitro matured oocytes and 9 from in vivo matured oocytes). We concluded that fibroblast cell lines could be established from long cryopreserved tissues, and these cells could be used as nucleus donors to clone animals.Somatic cell nuclear transfer (SCNT) provides a unique opportunity to reproduce animals with superior genetics. Viable cell lines are usually established from tissues collected by biopsy from living animals in the SCNT program. In the present study, tissues were collected and preserved from a suddenly deceased champion camel. We established cell lines from these decade-old tissues and used them as nuclear donors. After 42 h of in vitro maturation, 68.00 ± 2.40% of oocytes reached the metaphase II (M II) stage while 87.31 ± 2.57% in vivo collected oocytes were matured at collection (p < 0.05). We observed a higher blastocyst formation rate when in vivo matured oocytes (43.45 ± 2.07%) were used compared to in vitro matured oocytes (21.52 ± 1.74%). The live birth rate was 6.45% vs. 16.67% for in vitro and in vivo matured oocytes, respectively. Microsatellite analysis of 13 camel loci revealed that all the SCNT-derived offspring were identical to each other and with their somatic cell donor. The present study succeeded in the resurrection of 11 healthy offspring from the decade-old vitrified tissues of a single somatic cell donor individual using both in vitro and in vivo matured oocytes.

P–218 Analysis of the occurrence of microbial contamination in IVF culture system and the effect of microorganisms on embryo development and clinical outcomes

Abstract Study question what is the source, prevalence, and influence of microbial contamination on in vitro fertilization (IVF) and embryo transfer (ET) cycles? Summary answer Microbial contamination mainly occurs on Day 2, most caused by Escherichia coli carried with semen. ICSI could prevent contamination effectively and get good clinical outcomes. What is known already Microbial contamination occurs in IVF-ET system occasionally, which is hard to stop happening. The IVF culture system and laboratory environment, the patients’ follicular fluid and semen are not absolutely sterile, while the antibiotics in culture medium isn’t effective for all microbe types, and the artificial operations may bring in microbes. Generally, microbial contamination leads to degradation of embryos, reduction the number of embryos available, and infection of female reproductive tract, which would increase the cost of patients’ time, money, and bring psychological damages. A better understanding of embryo contamination in IVF culture system is of added value. Study design, size, duration A total of 29583 IVF-ET cycles were enrolled in this prospective observational study, from January 2010 to December 2020, included 70 microbial contamination cycles discovered in Day1-Day3 (D1-D3) of in vitro culture. Follicular fluid and semen saved on oocyte retrieval day, and culture medium contaminated were examined and identified for microorganisms at each contamination cycle. Participants/materials, setting, methods Compared the contamination rate of different insemination methods (IVF/ICSI/IVF+ICSI), different in vitro culture days (D1-D3), and different samples examination (follicular fluid, semen, culture medium) respectively, identified the source of microorganism types, compared the IVF culture outcomes and clinical outcomes between total contamination group (TC group, 42 cases) and partial contamination group (PC group, 28 cases). Main results and the role of chance A total of 70 microbial contamination cases occurred in 29583 oocyte retrieving cycles (0.24%), and it was observed only in IVF embryos but never in ICSI (Intracytoplasmic sperm injection) embryos. 38 contamination cases occurred on D2 with a highest ratio (54.3%) compared to D1 (32.9%) and D3(12.9%); Compared with follicular fluid, semen was the main cause inducing contamination from D1 to D3, and Escherichia coli in semen and culture medium, Enterococcus faecalis in follicular fluid proved to be the most common sources. Compared with TC group, the PC group showed a lower rate of No-available embryos (21.4% vs 81.0%) and a higher rate of blastocyst formation (41.2% vs 28.6%), In addition, the clinical pregnancy rate of PC group was higher than that of TC group in both fresh and frozen-thawed embryo transfer cycles (31.3% vs 16.7%, 38.5% vs 0.0%). Limitations, reasons for caution Further study is still necessary to better understand the sources that induce microbial contamination embryos, and more efficient methods are required to remove the microbes on these contaminated embryos so as better develop and manage a sterile micro-environment for successful embryo growth. Wider implications of the findings: The differential embryonic microbe types associated to different IVF culture and clinical outcomes in patients undergoing IVF-ET might have profound implications for understanding the microbial sources and making a better management of IVF culture system. Trial registration number Not applicable

P–291 Searching for a suitable serum progesterone range at triggering day to achieve optimal cumulative live birth rate in high responders – Which range is better?

Abstract Study question Is there a suitable range of serum progestereone level at triggering day to optimize the cumulative live birth rate (LBR) in high responders? Summary answer Fom the point of view of cLBR, the optimal P4 range for triggering is between 1.5 to 2.5 ng/ml generally and in the high responders. What is known already It is well established that premature progesterone rise (PPR) affect adversely the pregnancy outcome in fresh embryo transfer cycle. It is inferred that PPR alters synchrony between endometrium and the embryos. However, detailed study of the effect of PPR on efficiency of oocyte retrieval , embryo quality and the subsequent cumulative pregnancy outcome is still lacking. Hence we sort to analyze the effect of PPR on the final cumulative LBR in our program especially focused on high responders. Study design, size, duration ART Database in our center was retrospectively reviewed. Total 1523 cycles between 20160101 and 20191231 were recruited under the condition of GnRH antagonist cycle with duration of ovulation induction for more than 5 days and available serum P4 level data on triggering day for data analysis for the relationship between serum P4 value and final cumulative LBRs. Participants/materials, setting, methods Cycles with serum P4 level &amp;lt; 1.5 ng/ml were defined as without PPR (Group A: n = 1383). Cycles with serum P4 level &amp;gt;1.5 were defined as with PPR: P4 between 1.5 and 2.5 as Group B (n = 113), P4 &amp;gt; 2.5 as Group C (n = 27). Those high responding cycles (n = 404) were analyzed similarly and separately as Group A’ (n = 304), B’ (n = 81) and C’(n = 19). The statistics were carried out by SPSS-PC ver. 22.0 with p &amp;lt; 0.05 as statistical significance. Main results and the role of chance Group A had significantly lower number of oocytes (9.8 + 8.0) retrieved as compared to Group B (19.3 + 11.2) and Group C (18.2 + 9.9). However there were no differences in fertilization rate, good embryo rates and BC formation rates between groups. The cumulative LBR (cLBR) were significantly higher in Group B (65.1%) as compared to Group A (40.9%, p &amp;lt; 0.001) and Group C (37.0%, p = 0.008). For the high responding cycles, Group B’ also had marginally significant higher cLBR (75.3%) as compared to group A’(63.8% ; p=0.051) and Group C’ (52.6%; p = 0.050). Comparisons between Group A’ and C revealed significantly less oocytes retrieved but significantly higher blastocyst formation rates in Group A’ and the resultant cLBR were comparable between these two groups. Comparisons between Groups B’ and C’ revealed comparable oocytes retrieved but significant lower blastocyst formation rates and cLBRs in Group C’. The baseline of the first part analysis revealed higher age and lower AMH in Group A, but comparable age and AMH between groups B and C.The lower cLBR in group A could be due to selection bias.The second part (high responders) showed comparable baselines between three groups. However, the case numbers are too few in group C’ which might also result in uncertainty. Limitations, reasons for caution Although the data revealed interesting, significantly different results between groups, this is only a retrospective analysis from our ART patient series. Selection bias could not be precluded. Analysis restricted to high responders could have a more balanced population for comparisons. However, more cases are needed to affirm the findings. Wider implications of the findings: We still do not know the tolerable ceiling of serum P4 at the triggering day in high responders if future FET already planned. Pushing P4 value too high not only could not increase mature oocyte yields and possibly may decrease the number of available good blastocysts for optimizing final cLBRs. Trial registration number Not applicable

P–168 RCT comparing the effect of Continuous ( Single Step ) embryo culture system versus a Sequential embryo culture system on the outcome of IVF/ICSI cycles

Abstract Study question Is the outcome of IVF/ICSI cycles done with continuous (single step ) embryo culture system different from that with sequential embryo culture system ? Summary answer Yes the outcome of IVF / ICSI cycles done with continuous (single step ) embryo-culture system is better than that with sequential embryo-culture system . What is known already Embryo culture media are important factors in IVF, which can significantly influence the clinical outcome of IVF/ICSI cycles. However it is not clear which formulation is most optimal and whether sequential or continuous media (single step) should be favored. Sequential media complies with embryo demands based on developmental stage , taking into account metabolic changes embryos undergo in-vivo, while moving from the oviduct to the uterus. The embryos in the early cleavage stage prefer to use pyruvate to produce energy, whereas once development nears the blastocyst stage , the embryos start using glucose in the process of glycolysis . Study design, size, duration A prospective RCT was carried out at our centre between 2018–2019 and IVF-ICSI patients meeting inclusion criteria (at least six normal MII - Oocytes) were included in this study. The aim of study was to compare blastocyst formation rates after embryo-culture in two different culture media systems. 436 metaphase II Oocytes from 62 women were randomly and equally divided to be fertilized and cultured to the blastocyst stage in either sequential media or single-step media. Participants/materials, setting, methods In this prospective trial with sibling oocytes, 436 metaphase II oocytes from 62 women were randomly and equally divided to be fertilized and cultured to the blastocyst stage in either sequential media ( n = 218 MII oocytes) or a single medium ( n = 218 MII oocytes). In both groups, embryos were cultured in an interrupted fashion with media changes on day 3. Embryo transfer was performed on day 5. Main results and the role of chance Blastocyst formation rates on day 5 were significantly higher following culture in single step media 60.55% (132 / 218 ) as compared to sequential media 34.86% ( 76 / 218) . The percentage of good quality blastocysts was also significantly higher in single step media. In conclusion, culture in single step media was associated with higher blastocyst formation rates compared to sequential media , suggesting that the single medium may provide better support to the developing embryo. The proportion of poor quality embryos was significantly higher in the sequential media group. Results indicate that embryo culture in continuous media could be as efficient as embryo culture in sequential media. A significant difference observed was the proportion of poor quality embryos on day 5 , which was significantly higher when the embryos were cultured in sequential media. Our results suggest that the type of embryo culture media can influence the quality of embryos both at the cleavage stage and blastocyst stage. The use of continuous embryo culture media does not seem to cause an adverse effect; in fact, their use can lower the workload in busy IVF labs and lower the stress that embryos are exposed to during handling. Limitations, reasons for caution Although single-step-medium for extended culture has practical advantages and blastocyst formation rates appear to be higher, there is insufficient evidence to recommend either sequential or single-step media as being superior for the embryo-culture to days 5/6. Further studies comparing these two media systems in well-designed trials should be performed. Wider implications of the findings: When employing sequential media for embryo culture , it is necessary to transfer the embryos from one medium to another ( cleavage stage medium to blastocyst stage medium) which increases stress related embryo damage . Therefore, single-step media is beneficial as the embryos can develop undisturbed till blastocyst stage. Trial registration number Not applicable

INDIVIDUAL CULTURE LEADS TO DECREASED BLASTULATION BUT DOES NOT AFFECT PREGNANCY OUTCOMES IN THE SETTING OF A SINGLE, VITRIFIED-WARMED EUPLOID BLASTOCYST TRANSFER

While there is ongoing debate regarding ideal conditions in the embryology laboratory, it has been suggested that group culture protocols lead to higher quality embryos than protocols utilizing individual culture. This may be due to higher levels of beneficial embryotrophic factors in the group setting [1]. Group culture appears to result in higher rates of blastocyst formation and has been associated with improved live birth rates following fresh embryo transfers [2]. However, an evaluation of embryology and pregnancy outcomes comparing individual culture to group culture has not been performed in the setting of modern laboratory practices and freeze-all cycles.

Curcumin supplementation in the maturation medium improves the maturation, fertilisation and developmental competence of porcine oocytes.

This study was conducted to determine the effects of supplementing the maturation medium with the antioxidant curcumin on the in vitro maturation (IVM), fertilisation and development of porcine oocytes. Curcumin supplementation was performed at concentrations of 0, 5, 10, 20, and 40 µM. At concentrations of 5-20 µM, curcumin had significant positive effects (P < 0.05) on maturation and fertilisation rates compared to the non-treated group. Of the groups cultured with 5-20 µM curcumin, the number of oocytes with DNA-fragmented nuclei after IVM was significantly lower than in groups matured without curcumin. Moreover, curcumin supplementation at 10 µM also gave a significantly higher rate of blastocyst formation compared with oocytes matured without curcumin. Increasing the curcumin concentration to 40 µM yielded negative effects on fertilisation and embryonic development compared with the groups treated with lower concentrations of curcumin. Supplementation with 10 µM curcumin had beneficial effects on the oocyte maturation rate and DNA fragmentation index compared to the non-treated group both in the presence and absence of hydrogen peroxide. These results indicate that curcumin supplementation at a suitable concentration (10 µM) is potentially useful for porcine oocyte culture systems, in terms of protecting oocytes from various forms of oxidative stress.

114 Omega-3-enriched diet improves fertilization competence of cryopreserved sperm

Intensive reproductive management in dairy herds is mostly based on AI using high-merit bulls. Therefore, semen quality of bulls is of high importance. An association between semen quality and fatty acid content in feed has been suggested. Accordingly, the aim of this study was to examine the effect of omega-3 supplementation on sperm traits and fertilization competence. Fifteen Israeli Holstein bulls were assigned to three experimental groups. Bulls were fed over 13 weeks with a standard ration top-dressed with encapsulated-fat supplementation-fish oil (FO) or flaxseed oil (FLX; i.e. omega-3 sources), or saturated fatty acids (SFA, control). Ejaculates were collected before initiation of the study, during the feeding trial, and 1 month after feeding trial. Ejaculates were treated according to the routine procedure of the Israeli AI centre (Sion Ltd.), frozen, and stored in straws. Frozen-thawed samples were subjected to “swim-up” procedure, and spermatozoon viability, mitochondrial membrane potential, reactive oxygen species (ROS) level, acrosome membrane integrity, and DNA fragmentation were evaluated via flow cytometry, using sperm-specific kits (EasyCyte, IMV Technologies). Feeding with FO, FLX, or SFA did not affect the viability or mitochondrial membrane potential of sperm collected before, during, or after the feeding trial. On the other hand, a reduced proportion of sperm with ROS expression was recorded in the FLX samples compared to the SFA sample at the end of the feeding trial (42.2±1.2 vs. 47.3±4.3%, respectively; P&amp;lt;0.05) and one month later (36.3±2.2 vs. 41.6±4.6%, respectively; P&amp;lt;0.05). A low proportion of sperm with damaged acrosomal membrane was observed in both FLX and FO samples compared with SFA at the end of the feeding trial (48.8±3.4 and 41.7±2.7 vs. 59.8±3.4%, respectively; P&amp;lt;0.05). The proportion of sperm with fragmented DNA was lower in the FLX group than in the SFA group, collected one month after the end of the feeding trial (2.3±0.6 vs. 5.4±1.2%, respectively; P&amp;lt;0.05). To examine fertilization competence, oocytes were aspirated from ovaries collected from a local abattoir, then matured (n=216; 3 replicates) and fertilized invitro with a pool of samples from each group, collected one month after the end of the feeding trial (n=5 samples per group). The proportions of 2- to 4-cell-stage embryos and of blastocysts were determined 42h and 8 days after fertilization, respectively. Although the proportion of cleaved embryos did not differ between groups, a higher blastocyst formation rate was recorded in the FLX group (P&amp;lt;0.05), and a moderate increase was noted in the FO group, relative to the SFA group (28.1±4.4, 19.1±2.6, and 11.9±3.4%, respectively). Results imply that feeding bulls with omega-3 originating from FLX improves sperm quality, most likely due to improved redox status and decreased DNA fragmentation. This nutritional approach seems to be an effective tool for improving bull fertility competence. Nevertheless, invivo examination is required.

The use of pituitary adenylate cyclase-activating polypeptide in the pre-maturation system improves in vitro developmental competence from small follicles of porcine oocytes

ObjectiveWe investigated how pituitary adenylate cyclase-activating polypeptide (PACAP) affects embryonic development during pre-in vitro maturation (pre-IVM) using porcine oocytes isolated from small follicles.MethodsWe divided the follicles into the experimental groups by size (SF, small follicles; MF, medium follicles) and treated with and without PACAP and cultured for 18 hours (Pre-SF[−]PACAP; without PACAP, Pre-SF[+]PACAP; with PACAP) before undergoing IVM. The gene expression related to extracellular matrix formation (amphiregulin, epiregulin, and hyaluronan synthase 2 [HAS2]) and apoptosis (Bcl-2-associated X [BAX], B-cell lymphoma 2, and cysteine-aspartic acid protease 3) was investigated after maturation. The impact on developmental competence was assessed by the cleavage and blastocyst rate and total cell number of blastocysts in embryos generated from parthenogenesis (PA) and in vitro fertilization (IVF).ResultsCleavage rates in the Pre-SF(+)PACAP after PA were significantly higher than SF and Pre-SF(−)PACAP (p<0.05). The cleavage rates between MF and Pre- SF(+)PACAP groups yielded no notable differences after IVF. Pre-SF(+)PACAP displayed the higher rate of blastocyst formation and greater total cell number than SF and Pre-SF(−)PACAP (p<0.05). Cumulus cells showed significant upregulation of HAS2 mRNA in the Pre-SF(+)PACAP compared to the SF (p<0.05). In comparison to other groups, the Pre-SF(+)PACAP group displayed a downregulation in mRNA expression of BAX in matured oocytes (p<0.05).ConclusionThe PACAP treatment during pre-IVM improved the developmental potential of porcine oocytes derived from SF by regulating cumulus expansion and apoptosis of oocytes.

Effect of Alpha Lipoic Acid on in vitro Maturation of Porcine Oocytes and Subsequent Embryonic Development after Parthenogenetic Activation

Alpha lipoic acid (ALA) is a biological membranes compound. As the antioxidant, it decreases the oxidized forms of other antioxidant substances such as vitamin C, vitamin E, and glutathione (GSH). To examine the effect of ALA on the in vitro maturation (IVM) of porcine oocytes, we investigated intracellular GSH and reactive oxygen species (ROS) levels, and subsequent embryonic development after parthenogenetic activation (PA). Intracellular GSH levels in oocytes treated with 50uM ALA increased significantly (P < 0.05) and exhibited a significant (P < 0.05) decrease in intracellular ROS levels compared with the control group. Oocytes matured with 50 uM of ALA during IVM displayed significantly higher cleavage rates (67.8% vs. 83.4%, respectively), and higher blastocyst formation rates and total cell number of blastocysts after PA (31.6%, 58.49 vs. 46.8%, 68.58, respectively) than the control group. In conclusion, these results suggest that treatment with ALA during IVM improves the cytoplasmic maturation of porcine oocytes by increasing the intracellular GSH levels, thereby decreasing the intracellular ROS levels and subsequent embryonic developmental potential of PA.

166 Paracrine Regulation of Epidermal Growth Factor-Like Factors in Oviduct Cells and its Effect on Oocyte Maturation and Subsequent Embryo Development

Progesterone (P4) and progesterone receptor signalling appears essential for maintenance of a proper cumulus cell expansion during the oocyte maturation by regulating the epidermal growth factor-like factors (EGF-F) related pathway during the ovulatory process. It is known that expression of EGF-F including amphiregulin (AREG), epiregulin (EREG), and betacellulin (BTC) is critical for cumulus–oocyte complex (COC) expansion and resumption of meiosis. Therefore, we hypothesised that oviduct cells might be involved in nonexclusive mechanisms of actions of P4 that in turn modulate oocyte meiosis resumption by regulating the levels of EGF-F. First, we added different concentrations of P4 (0, 0.5, 1, and 2 μg mL−1) to oviduct cell culture medium and assessed the effect of P4 on expression of AREG, EREG, and BTC in oviduct cells by immunocytochemical analysis. Then, the oviduct cells were used for co-culturing under the proper concentration of P4 with porcine oocytes. The COC were randomly cultured in 3 groups: (1) culturing without oviduct cells, (2) co-culturing with oviduct cells, and (3) co-culturing with oviduct cells treated with P4. After IVM, extrusion of the 1st polar body was observed under the microscope. To evaluate embryo development competence, the matured oocytes were activated with electrical stimulus and parthenotes were cultured in porcine zygote medium-5 for 7 days at 39°C, 5% CO2 and O2 in a humidified atmosphere. The cleavage and blastocyst formation rates were observed under the microscope to evaluate developmental competence. To count the total cell number of blastocysts, they were stained with 5 μg mL−1 of Hoechst 33342 for 10 min. The data were analysed by one-way ANOVA using GraphPad Prism 5.0 (GraphPad Inc., San Diego, CA, USA). Values are means ± standard error of mean (P &lt; 0.05). Significantly higher levels of EGF-F were observed in oviduct cells treated with 1 μg mL−1 progesterone. The oocyte maturation rate of co-culture group treated with P4 (80.7 ± 1.6%) was significantly higher than that of the control (69.7 ± 2.1%). There was a significant difference between co-culture treated with P4 and the control in cleavage rate (67.2 ± 2.4% and 82.0 ± 1.6%). However, no significant difference was observed between the co-culture groups. The co-culture treated with P4 group showed significantly higher rate of blastocyst formation (37.7 ± 0.8%) and total cell number of blastocyst (72.8 ± 1.0) than control and co-culture groups. In conclusion, co-culturing with oviduct cell treated with P4 improved oocyte maturation and subsequent embryo development. Thus, we suggested that oviduct cells induce the expression of EGF-F under the treatment of P4, which has a beneficial effect on porcine oocyte development. This research was supported by NRF-20142A1021187, Korea IPET (#316002-05-2-SB010), RDA (#PJ010928032017) and Research Institute for Veterinary Science, the BK21 plus program.

Effects of gel-embedded embryos on developmental competence of separated bovine blastomeres

Abstract This study aimed to examine how gel embedding compared with the Well-of-Well (WOW) system when culturing bovine separated blastomeres, in terms of developmental competence rates and blastocyst quality. We first optimized the gel-embedding method via culturing intact zygotes in either 1% agarose or 1% calcium alginate gel. Gel-embedded groups and control did not differ in development rates, but the 1% calcium alginate group was selected for subsequent experiments due to higher blastocyst recovery. The separated embryo group had higher potential for blastocyst production than the intact embryo group. Among separated blastomeres (2- and 8-cell), WOW and 1% calcium alginate did not differ in blastocyst formation rate, except between the 2-cell alginate and WOW groups, with the latter exhibiting the highest rate of blastocyst formation. Within the WOW system, the separated 2-cell embryo resulted in significantly higher rates of OPU (ovum pick-up)-derived blastocysts than the intact 2-cell embryo; however, the latter had more total cells than the former. In conclusion, the 1% calcium alginate gel can support cell growth of separated 2- and 8-cell bovine embryos during blastomere aggregation. Moreover, the gel-embedded technique is a suitable replacement for WOW in producing blastocysts from bovine separated blastomeres.

Effect of Alpha Lipoic Acid as an Antioxidant Supplement during In Vitro Maturation Medium on Bovine Embryonic Development

This study was conducted to investigate the effects of alpha-lipoic acid (aLA) as an antioxidant that decrease the reactive oxygen species (ROS) in bovine embryonic development. Slaughterhouse derived bovine immature oocytes were collected and 4 different concentrations (0, 5, 10 and 20 mM) of aLA was supplemented in bovine in Vitro maturation (IVM) medium. After 20 hrs of IVM, maturation rates, levels of ROS and glutathione (GSH), and further embryonic development after parthenogenetic activation (PA) and in Vitro fertilization (IVF) was investigated according to aLA concentrations. Maturation rate was significantly higher in 10 mM group than other groups (80.5% vs. 62.9, 73.9, 64.2%; P<0.05). In the levels of ROS and GSH in matured oocytes as an indicator of oocyte quality, significantly better results were shown in 5 and 10 mM groups compared with other 2 groups. After IVM, significantly higher rates of blastocyst formation were shown in 10 mM groups in both of PA (27.9% vs. 18.8, 22.3, 14.2%; P<0.05) and IVF (32.6% vs. 23.9, 27.3, 16.2%; P<0.05) embryos. In addition, significantly more cell total cell number and higher inner cell mass ratio in 10 mM PA and IVP blastocysts showed developmental competence in 10 uM groups. Therefore, based on the entire data from this study, using 10 μM of aLA confirmed to be the optimal concentration for bovine oocyte maturation and embryonic development.

Parthenogenetic activation and somatic cell nuclear transfer of porcine oocytes activated by an electric pulse and AZD5438 treatment.

We examined the in vitro developmental competence of parthenogenetic activation (PA) oocytes activated by an electric pulse (EP) and treated with various concentrations of AZD5438 for 4 h. Treatment with 10 µM AZD5438 for 4 h significantly improved the blastocyst formation rate of PA oocytes in comparison with 0, 20, or 50 µM AZD5438 treatment (46.4% vs. 34.5%, 32.3%, and 24.0%, respectively; P 0.05). Furthermore, 66.67% of blastocysts derived from these AZD5438-treated PA oocytes had a diploid karyotype. The blastocyst formation rate of PA and somatic cell nuclear transfer (SCNT) embryos was similar between oocytes activated by an EP and treated with 2 mM 6-dimethylaminopurine for 4 h and those activated by an EP and treated with 10 µM AZD5438 for 4 h (11.11% vs. 13.40%, P > 0.05). In addition, the level of maturation-promoting factor (MPF) was significantly decreased in oocytes activated by an EP and treated with 10 µM AZD5438 for 4 h. Finally, the mRNA expression levels of apoptosis-related genes (Bax and Bcl-2) and pluripotency-related genes (Oct4, Nanog, and Sox2) were checked by RT-PCR; however, there were no differences between the AZD5438-treated and non-treated control groups. Our results demonstrate that porcine oocyte activation via an EP in combination with AZD5438 treatment can lead to a high blastocyst formation rate in PA and SCNT experiments.

H2O2-induced mild stress in relation with in vitro ovine oocyte developmental competence: implications for blastocyst apoptosis and related genes expression.

In this study, in vitro maturation was performed in presence of various concentrations (0, 10, 100, or 1000 µM) of H2O2. The intracellular glutathione (GSH) level, fertilization, cleavage, and blastocyst rates, total cell number, and apoptotic cell number and expression of Bax, Bcl-2, and p53 genes in blastocyst-stage embryos were studied. At 10 μM H2O2 concentration, a higher GSH level was detected in comparison to the other groups while oocytes exposed to 1000 μM H2O2 had the lowest GSH level. Treatment of oocytes with 1000 μM H2O2 decreased the rate of two pronuclei formation as compared with other groups. A higher rate of blastocyst formation was seen in 100 μM H2O2 group as compared with the control group. However, exogenous H2O2 in maturation medium did not affect total cell numbers and apoptotic cell ratio at the blastocyst stage. Moreover, mRNA transcript abundance of Bax, Bcl-2, and p53 genes was similar between blastocysts derived from H2O2-induced oocytes and control blastocysts. Treatment of oocytes with H2O2 at mild level during in vitro maturation had a positive effect on GSH level and this, in turn, may lead to improvement in preimplantation embryonic development.