Higher Blastocyst Research Articles

Shading effect on physiological parameters and in vitro embryo production of tropical adapted Nellore heifers in integrated crop-livestock-forest systems

The aim of this study was to evaluate the impact of increased shadow supply in integrated crop-livestock-forest systems on in vitro embryonic development and physiological parameters related to stress response in Nellore heifers (Bos indicus). For the study, animals (n = 16) were randomly divided into two groups and kept in areas with different afforestation systems, the integrated crop-livestock-forest (ICLF) and the integrated crop-livestock (ICL) system. The microclimate of the ICLF system provided better comfort conditions than ICL. No differences of respiratory rate, rectal temperature, cortisol, T3, T4, oocyte quality, and cleavage rate between the systems were verified. A higher blastocyst rate was observed in the ICLF (p < 0.05). The results demonstrate that Nellore heifers managed in ICLF during summer in Midwest of Brazil showed higher production of in vitro embryos, without typical changes in its physiological parameters. The results observed in the present study indicate that zebu females are able to respond satisfactorily to the intense heat conditions; however, we believe that the long period to which these animals are exposed to these conditions interferes in the oocyte competence and embryo development.

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.

Embryo culture in step single and sequential media: influence on morphology and implantation potential of blastocysts

Purpose. To assess the impact of step single and sequential culture media on the morphology and frequency of blastocyst implantation. Materials and methods. In total, 514 embryos obtained from patients aged 30 to 35 years and fertilized by ICSI were studied (2017 to 2018). The pool of embryos from each woman was randomly divided into two groups: the first group was cultured in the sequential media and the second - in the single ones. Evaluation of the effect was carried out by comparing the obtained blastocysts quality cultured in the single and sequential media. Blastocyst morphology was assessed using the modified Gardner D. K. scale. Statistical analysis was performed by Mann–Whitney U-test and Pearson's chi-squared test with Yates' correction. Statistically significant differences between the compared groups were considered at P < 0.05. Results. The studies found that the use of single Origio ® 1-Step medium at embryo culture phase improves the morphological quality of the resulting blastocyst compared to embryos cultured in sequential Origio ® Sequential Cleav™ / Sequential Blast™ media. No statistically significant difference in the pregnancy and implantation rate of blastocysts obtained after embryo cultivation in the step single or sequential media was detected. Conclusions. The priority in terms of obtaining a large number of high quality blastocysts with high implantation potential is to use a step single medium for the embryo cultivation.

13. THE CHROMOSOMAL SET ANALYSIS OF BLASTOCYSTS OBTAINED FROM MODIFIED DONOR OOCYTES BY THE PATIENT'S 1ST POLAR BODY TRANSFER

Introduction Transfer of the patient's 1st polar body (PB) to donor oocyte cytoplasm has proven to be an effective method to help patients with low ovarian reserve and poor response. This technique allows doubling the number of oocytes suitable for fertilization, while maintaining the biological relationship of patient with the future child. The use of transfer in our practice has shown the ability of modified oocytes to form high morphological quality blastocysts and their euploidy have been evaluated in this study. Materials and Methods The study was performed in the Medical Center IGR from March 2017 to December 2018 and involved 330 cells: 168 oocytes (group A) obtained from 39 patients (mean age 40.7±5.3 years) and 162 oocytes (group B) that were received from 28 donors (mean age 28.2±2.4). Donor oocytes have been pre-enucleated and modified by the transfer of patients’ 1stPB with further fertilization. The procedure was made using Nikon Ti Eclipse (Japan) inverted microscope, Saturn 3 laser console (UK). Preimplantation genetic testing for aneuploidy (PGT-A) was performed using trophectoderm (TE) biopsy on the post-fertilization hours 120 or 144. Samples were diagnosed using Ion S5 by Thermo Fisher Scientific(USA). The samples’ ploidy was verified by the FISH. We evaluated the number of euploid and aneuploid blastocysts from maternal and modified oocytes by the 1st PB transfer. Statistical analysis was carried out using Shapiro-Wilk test for normality and Chi-square test. Results In the group A there were 50 blastocysts (29.8%) that formed from 168 original patients’ oocytes and 29 blastocysts (17.9%) developed from 162 modified oocytes (group B). 45 blastocysts from group A and 27 blastocysts from group B were biopsied for PGT-A. The number of euploid embryos was 11 (24.4%) and 6 (22.2%) in the group A and group B, respectively, without statistically significant difference (SSD). Among the aneuploidies, single (only one chromosome was involved in nondisjunction) and complex (several chromosomes involved in nondisjunction) chromosomal anomalies were encountered. In the group A 13 (38.2%) samples carried single chromosomal disorder and 21 (61.8%) samples had complex quantitative abnormalities, in the group B these numbers reached 9 (47.4%).and 10 (52.6%) samples, respectively. Among the aneuploid embryos 79 chromosome abnormalities were found in A group samples and 38 numerical aberration in B group samples.. The following types of chromosomal abnormalities were detected in groups: autosome trisomy (50/63.3% vs. 25/65.8% samples), autosome monosomy (23/29.1% vs.6/15.8% samples), numerical violation of sex chromosomes (6/7.6% vs. 4/10.5% samples), polyploidy (0 vs. 2/5.3% samples), mosaicism (1/1.3% vs. 0 samples) in the group A and the group B, respectively. No SSD was found between the groups (p>0.05). Conclusions The results of our study demonstrated that the 1st PB transfer does not affect the chromosomal set of embryos obtained from modified oocytes in comparison with the original oocytes. This technique is useful to increase the number of female germ cells and avoid the full oocytes donation especially for patients with low ovarian reserve and for poor responders.

131 Fatty acid binding protein inhibition as a strategy to reduce the lipid content of in vitro-produced bovine embryos

Lipid accumulation decreases cryopreservation survival of in vitro-produced embryos, reducing pregnancy rate after embryo transfer. Fatty acid binding protein 3 (FABP3) plays a role in lipid transport from cumulus cells to the oocyte during maturation. Blocking this transport could reduce lipid content in the oocyte and embryo and increase cryopreservation survival. This preliminary study aimed to test the effect of α-truxillic acid (FABP-I), a chemical molecule that inhibits FABP3/5 action by receptor competition, on lipid content of matured oocytes and blastocysts after culture. Slaughterhouse-derived cumulus-oocyte complexes were matured with 0 (control), 10, 50, 100 and 500nM FABP-I for 22h. In Experiment 1, 346 oocytes in 3 replicates were fixed following maturation and stained with 1μg mL−1 Nile Red to evaluate total lipid content; maturation was assessed by nuclear staining with 10μg mL−1 Hoechst 33342[ACD1]. In Experiment 2, 876 cumulus-oocyte complexes in 5 replicates were matured for 22h under the same concentrations of FABP-I, then fertilized for 18h and cultured for 7 days. Cleavage and blastocyst development were evaluated on Day 2 and 7, respectively. Blastocysts were fixed at Day 7 and stained with Nile Red. Fluorescence intensity was measured in arbitrary units using ImageJ (NIH), and data was analysed using GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA). In Experiment 1, maturation rate did not differ among treatments (70.2±8.7; P=0.7). There were significant effects of treatment, replicate and interaction of treatment by replicate on fluorescence intensity. Compared with control (23.6±0.6), intensity was lowest in oocytes matured with 500nM FABP-I (21.2±0.6; P&amp;lt;0.01) and highest in the 10nM group (26.5±0.6; P&amp;lt;0.01). Staining intensity tended to decrease in the 100nM group (22.1±0.6; P=0.09) and was not different in the 50nM group (24.0±0.7; P=0.6). In Experiment 2, cleavage rate (75.8±2.9; P=0.3) did not differ and blastocyst development tended to be different among treatments (P=0.06). Compared with the control (33.3±4.8), the 500nM group had lower development (17.0±4.8; P&amp;lt;0.03); 10 and 50nM groups had numerically lower (24.7 and 24.0±4.8) and the 100nM group had the highest development rate (37.3±4.8), although either was significant. Treatment tended to affect fluorescence intensity of blastocysts (P=0.07; n=209), and there were significant effects of replicate and interaction between replicate and treatment. Compared with the control (11.7±1.3), fluorescence intensity was lower in the 50nM group (6.8±1.3; P&amp;lt;0.01), whereas 10nM had a tendency for lower intensity (8.3±1.2; P=0.06). Groups 100 and 500nM were not significantly different from controls (9.4±0.9 and 10.7±1.5, respectively). In conclusion, addition of FABP-I up to 500nM did not affect maturation or embryo cleavage but altered blastocyst development. Exposure to 50nM reduced staining intensity in blastocysts without significant decrease in development, whereas 100nM resulted in numerically lower oocyte staining intensity and higher blastocyst development. Future experiments will evaluate cryopreservation survival of embryos treated with FABP-I, and embryo transfer.

Increased Elasticity of the Zona Pellucida Affects Micromanipulation Procedures for Preimplantation Genetic Screening at the Cleavage Stage: Possible Role of Blastomere Alterations on Inconclusive or “No Results” Interpretations

Objective Occasionally, submission of blastomere(s) or trophectoderm cells for preimplantation genetic screening (PGS) may be interpreted as inconclusive (“no results”). The main reasons for those results include failure of DNA amplification, degraded DNA, or failure to load cell(s) into the biopsy tube. In a particular case at our clinic, blastomere biopsies were submitted for PGS and all embryos were labeled as “no results”. Interestingly, the zona pellucida (ZP) of all embryos was extremely elastic and blastomere displacement was difficult. We theorize that the high elasticity and extended micromanipulation procedures may have affected the DNA in such a way as to interfere with the accurate assessment/interpretation of results. Design Case Report. Materials and Methods A total of 43 oocytes were harvested at retrieval. Oocytes were inseminated via standard IVF or ICSI. Ten cleavage stage embryos with a minimum of 6 blastomeres were selected for biopsy/PGS screening. Assisted hatching (AH) via partial zona dissection (PZD; tridimensional) was performed prior to blastomere biopsy. Embryo biopsy was performed via blastomere displacement. Results The ZP of all biopsied embryos was highly elastic. In addition, the inner layer of the ZP appeared to be detached in certain areas for some embryos. Those embryos acquired an oval-elliptical shape. The lack of rigidity prevented generation of internal pressure inside the embryo, which is necessary to displace the blastomere(s). The ZP of each embryo had to be probed at different angles in order to displace the selected blastomere(s). The time required to harvest blastomeres was extended to approximately ≥5 min per embryo. Selected blastomeres were eventually isolated without evidence of damage to the remaining blastomeres. All embryos were labeled as “no results” after assessment by the genetic laboratory. The report and additional options were discussed with the patients, and it was decided to proceed with embryo transfer (D5) based on blastocyst morphology. Two high quality blastocysts were transferred, resulting in a singleton term pregnancy. The remaining viable embryos were considered for vitrification. Conclusions Zona pellucida elasticity and extended manipulation of embryos/blastomeres may result in a form DNA degradation that interferes with the accuracy or interpretation of results during genetic assessment. Further analysis of similar cases may shed light into the relationship between difficult biopsies and inconclusive PGS results. Disclosures Nothing to disclose.

Acrosome reaction and chromatin integrity as additional parameters of semen analysis to predict fertilization and blastocyst rates

BackgroundTraditional semen parameters have shown little to none predictive value for fertilization and blastocyst viability for a successful pregnancy. Therefore, the purpose of this study was to explore the usefulness of incorporating the acrosome reaction (AR) and chromatin integrity to conventional semen analysis to individually predict the fertile potential of sperm samples.MethodsA cross-sectional study was conducted in 69 participants undergoing IVF using oocyte donation. Semen samples were collected and evaluated for: AR [spontaneous (sAR) and induced (iAR)] by flow cytometry using anti-CD46-FITC, Acrosome Response to an Ionophore Challenge (ARIC), chromatin integrity by Sperm Chromatin Structure Assay (DNA Fragmentation Index-%DFI and High DNA Stainability-%HDS), WHO semen analysis, fertilization and blastocyst rates.ResultsThe participant age was 40.0 ± 6.1 years (66% were normozoospermic). Sperm morphology, sAR, iAR, and ARIC were associated with the fertilization (β = 3.56, R2 = 0.054; β = − 5.92, R2 = 0.276; β = 1.83, R2 = 0.150; and β = 2.10, R2 = 0.270, respectively, p < 0.05). A logit model was developed to calculate the probability of fertilization (≥ 60%) for each participant, using the sperm morphology and ARIC as independent variables, followed by ROC analysis to determine a cutoff probability of 0.65 (specificity = 80.6%, sensitivity = 63.2%). %DFI was inversely associated with the viable blastocyst rate (β = − 1.77, R2 = 0.057, p = 0.003), by the logit model and ROC analysis, a cutoff probability of 0.70 (specificity = 80.6%, sensitivity = 72.3%) was obtained to predict blastocyst viability (≥ 40%). There was no difference in the results with normozoospermic samples (n = 46).ConclusionsThe incorporation of ARIC and %DFI allowed to obtain predictive models for high fertilization and blastocyst rates in an individualized way, being promising tools to improve the diagnosis of male fertility potential for research or assisted reproduction, even in men with unknown infertility.

79 Effect of Polydatin on Development and Cryotolerance of In Vitro-Produced Bovine Embryos

In vitro-produced (IVP) cattle embryos have high reactive oxygen species levels resulting in poor development and cryotolerance. Polydatin, a naturally occurring antioxidant, improves embryonic metabolism when added to maturation media; however, it has not been evaluated at other stages of embryo production. We hypothesised that embryos cultured with polydatin during maturation and fertilization would have increased development and cryotolerance. Therefore, IVP embryos were produced in 8 treatment groups supplemented with 1 µM polydatin during in vitro maturation, fertilization, and culture, or a combination of the different production stages, and each assigned a letter (Table 1). Embryos were produced in 7 replicates by oocytes (n = 3320) aspirated from abattoir ovaries, matured for 23 h in TCM-199 plus 10% fetal bovine serum and gonadotropins, fertilized with semen from 1 of 3 bulls, and cultured in SCF1 (SOF for Conventional Freezing 1; Owen et al. 2017 Reprod. Fertil. Dev. 29, 129-130) in 38.5°C in 5% O2, 5% CO2, and 90% N2. Stage 7 blastocysts were stained with 1 µg mL−1 Nile Red for lipid content or 300 nM Mitotracker Red CMX-Rosamine (Thermo Fisher Scientific, Waltham, MA, USA) for mitochondrial activity. Ten images per embryo were acquired using confocal microscopy at 5 µM step size at 40× magnification, and fluorescence was measured by Image Pro software (Media Cybernetics, Rockville, MD, USA). Remaining blastocysts were slow frozen following a 20-min equilibration in conventional freezing medium (1.5 M ethylene glycol and 0.5 M sucrose in holding medium) with 1 mm l-ascorbic acid. Embryos were thawed and assessed for re-expansion at 48 h. Blastocyst rate, Nile Red, Mitotracker, and re-expansion data were analysed by one-way ANOVA and means separated by least significant difference. Results indicate that treatment B had a higher blastocyst rate than treatment H (P &lt; 0.01), lower lipid content than all other treatment groups (P &lt; 0.01 or 0.05), and higher level of mitochondrial polarity than treatments A, D, E, and G (P &lt; 0.01 or 0.05), suggesting enhanced metabolic activity. Additionally, this treatment enhanced cryotolerance compared with treatment H (P &lt; 0.01). These results suggest that adding polydatin to maturation media has the most effect on embryo developmental competence and cryotolerance. Table 1.Effect of polydatin addition during in vitro maturation (IVM), fertilization (IVF), and culture (IVC) on blastocyst rate, lipid content, Mitotracker, and cryotolerance (± SEM)

208 Cryotolerance of In Vitro-Produced Cattle Embryos Produced with Different Maturation and Culture Media

In vitro-produced (IVP) bovine embryos suffer from damage due to suboptimal culture conditions involving altered metabolism, reactive oxygen species generation and high lipid accumulation, which could be impacted by oocyte maturation × embryo culture interaction. We hypothesised that optimizing oocyte and embryo culture conditions would lead to a higher post-thaw embryo survival rate. The experiment was replicated 9 times in a factorial design with 3 oocyte maturation media: TCM-199 plus 10% serum and gonadotropins (control), BO-HEPES-IVM (BO, IVF Bioscience UK), and HMM (HEPES-buffered maturation medium) and 3 embryo culture media: BO-IVC (BOC), BBH7 (Cooley Biotech, Gainesville, FL, USA), and SCF1 (SOF for Conventional Freezing 1; Owen et al. 2017 Reprod Fertil Dev. 29, 129-130). Bovine oocytes (n = 2907) were aspirated from 2- to 8-mm follicles of abattoir ovaries, matured at 38.5°C for 22 to 24 h in 5% CO2 (control) or in BO and HMM in air, fertilized with semen from 1 of 3 bulls, and cultured in BOC, BBH7, and SCF1 at 38.5°C in 5.5% CO2, 5% O2, and 90% N2. Cleavage and blastocyst rates were evaluated at Day 3 and 7, respectively, after IVF. Selected stage 7 blastocysts were slow frozen using 1.5 M ethylene glycol supplemented with 1 mm l-ascorbic acid for 20 min (equilibration). The embryos were cooled to and seeded at –6°C, and then cooled to −32°C at 0.5°C/min. Embryos were thawed and re-expansion was assessed at 24 and 48 h. Data (Table 1) was arcsin transformed and analysed by ANOVA, and means separated by l.s.d. Results indicate that oocytes matured in BO and embryos cultured in SCF1 had a higher blastocyst rate than all oocytes matured in control medium and all embryos cultured in BBH7 (P &lt; 0.05). However, oocytes matured in control medium and embryos cultured in BOC had lower post-thaw re-expansion rates than other treatment groups (P &lt; 0.05). These results suggest that a combination of oocyte maturation and embryo culture media plays an important role in post-thaw embryo survival, al though interactions of present treatments need to be further evaluated. Table 1.Effect of maturation and culture media on blastocyst rate and cryotolerance ± SEM)

Ефективність лікування трубно-перитонеальної форми безплідності у програмах допоміжних репродуктивних технологій залежно від протоколу стимуляції суперовуляції

The objective: of the investigation was to study the effectiveness of treatment of women with tubal infertility, depending on the protocol of ovarian stimulation. Materials and methods. To achieve tht goal, the medical documentation of patients who contacted the «Mother and Child» Medical Center in 2012-2013 was analyzed. To compare the efficacy of different ovarian stimulation protocols were selected 387 patients under the age of 35 years with tubal infertility, who were stimulated by standard protocols with further separation into two groups: S (short stimulation protocol, n=246) and L (long stimulation protocol, n=141). In both groups, an intracytoplasmic sperm injection (ICSI) was performed. Results.The mean duration of stimulation in a long protocol was on 2 days longer. The total amount of spent rFSH was also significantly (by 300 IU) more. There was no significant difference between the number of blastocysts and high quality blastocysts. The cumulative efficacy of treatment after 1 year was 82.33% and 77.30% (p&gt;0.05). Conclusion. The retrospective study showed that women with tubal infertility in a long protocol require a longer stimulation, using a larger total dose of recombinant FSH, with the same end-point effectiveness. Key words: tubal peritoneal infertility, in vitro fertilization, stimulation of superovulation, treatment effectiveness.

In vitro production of bovine embryos derived from individual donors in the Corral\xae dish

BackgroundSince the identity of the embryo is of outmost importance during commercial in vitro embryo production, bovine oocytes and embryos have to be cultured strictly per donor. Due to the rather low yield of oocytes collected after ovum pick-up (OPU) per individual cow, oocyte maturation and embryo culture take place in small groups, which is often associated with inferior embryo development. The objective of this study was to improve embryonic development in small donor groups by using the Corral® dish. This commercial dish is designed for human embryo production. It contains two central wells that are divided into quadrants by a semi-permeable wall. In human embryo culture, one embryo is placed per quadrant, allowing individual follow-up while embryos are exposed to a common medium. In our study, small groups of oocytes and subsequently embryos of different bovine donors were placed in the Corral® dish, each donor group in a separate quadrant.ResultsIn two experiments, the Corral® dish was evaluated during in vitro maturation (IVM) and/or in vitro culture (IVC) by grouping oocytes and embryos of individual bovine donors per quadrant. At day 7, a significantly higher blastocyst rate was noted in the Corral® dish used during IVM and IVC than when only used during IVM (12.9% ± 2.10 versus 22.8% ± 2.67) (P < 0.05). However, no significant differences in blastocyst yield were observed anymore between treatment groups at day 8 post insemination.ConclusionsIn the present study, the Corral® dish was used for in vitro embryo production (IVP) in cattle; allowing to allocate oocytes and/or embryos per donor. As fresh embryo transfers on day 7 have higher pregnancy outcomes, the Corral® dish offers an added value for commercial OPU/IVP, since a higher blastocyst development at day 7 is obtained when the Corral® dish is used during IVM and IVC.

Molecular characterization and expression analysis during embryo development of CD4-1 homologue in large yellow croaker Larimichthys crocea

CD4+ helper T (Th) cells are a master component of the adaptive immune response. CD4 is one of the most effective surface markers for identifying Th cells. In the present study, we cloned and characterized a CD4-1 homologue, LycCD4-1, from large yellow croaker Larimichthys crocea. The full-length cDNA of LycCD4-1 is 1695 bp long, encoding a protein of 462 amino acids. The deduced LycCD4-1 protein has a typical domain architecture as found in mammalian CD4 molecules, including a signal peptide, four extracellular immunoglobulin-like (Ig-like) domains, a transmembrane region, and a CXC signaling motif in the cytoplasmic tail. Four N-glycosylation sites and 10 cysteine residues were also found in LycCD4-1, which may be essential for its tertiary structure and succeeding function. Homology comparison showed that LycCD4-1 has 27.9–58.4% identity to other teleost fish CD4-1 molecules, and 16.4–20% identity to those of higher vertebrates. Genomic analysis revealed that the LycCD4-1 gene consisted of nine exons and eight introns and exhibited a similar exon-intron organization to other species CD4 genes except for a different intron length. Phylogenetic analysis showed that LycCD4-1 form a cluster with CD4-1 molecules in other fish species. The LycCD4-1 was constitutively expressed in all tissues tested, with a higher expression in gills and spleen. LycCD4-1 mRNA expression in the spleen and head kidney tissue was increased by poly (I:C) at 48 h, whereas its expression levels were somewhat down-regulated at 6 h and 72 h after bacterial vaccine induction in spleen. Unexpectedly, LycCD4-1 mRNA could be detected in each stage of early embryo development since fertilized eggs, with a higher level before mid-gastrula and the highest level in high blastocysts. These results will be helpful for better understanding molecular characteristics of CD4-1 and tracing origin of CD4-1+ cell precursors in fish.

Study of TALP and TRIS citrate medium on caprine sperm capacitation and subsequent in vitro embryo production.

The aim of the present investigation was to compare Tyrode's albumin lactate pyruvate (TALP) medium and TRIS citrate medium for capacitation of caprine sperm. In experiment 1 capacitation was assessed by chlortetracycline assay and in experiment 2 with in vitro fertilization and embryo development. In experiment 2, cumulus oocyte complexes (COCs) recovered by slicing the caprine ovaries were matured in maturation medium for 27 h in humidified atmosphere at 38.5°C with 5% CO2. After 27 h of culture a total of 2480 in vitro matured oocytes were selected and randomly divided into two groups. Group 1 (n=1124) matured oocytes were fertilized by the spermatozoa capacitated in TALP medium and in group 2 (n=1356) matured oocytes were fertilized by the spermatozoa capacitated in TRIS citrate medium. The results of experiment 1 indicated a comparatively more number of sperms with Chlortetracycline (CTC) Pattern B in TRIS citrate than TALP medium (55.32 ± 0.91% vs 47.96 ± 0.20%). In experiment 2, the cleavage rate and blastocyst production were higher following capacitation of spermatozoa in TRIS citrate than TALP medium. In conclusion, TRIS citrate can be used as an alternative and effective media for sperm capacitation to get higher cleavage rate and blastocyst production in goat.

17 SPERM FERTILITY RATE ASSESSED BY EMBRYO PRODUCTION IN VIVO AND IN VITRO IN SOUTH AFRICAN BULLS

The aim of this study was to determine the sperm fertility rate by embryo production in vivo and in vitro in South African bulls and further compare the embryo quality developed from different oocyte recovery methods. A total of 15 frozen semen straws (5 Bonsmara; 5 Nguni; 5 Boran) were thawed and evaluated for sperm motility characteristics using sperm class analyzer. The fertilizing ability of frozen–thawed semen was assessed by performing AI and in vitro fertilization. For AI, 6 cows were superovulated and inseminated with frozen–thawed semen followed by flushing on Day 7 post-insemination and then evaluated for embryo developmental stages. For IVF, oocytes were retrieved using two recovery methods namely ovum pick-up (OPU) and ovary aspiration. A total of 383 (106, OPU; 277, ovary aspiration) oocytes were matured in M199 + 10% fetal bovine serum (FBS) maturation medium at 38.5°C for 24h. Oocytes were washed in Bracket and Oliphant’s fertilization medium, co-incubated with frozen–thawed (Boran) semen at 38.5°C for 6 h, and then cultured in SOF-BSA medium, incubated at 38.5°C, 5% CO2 for 7 days, and further evaluated for embryo development. Data were analysed by ANOVA. Total sperm motility was &gt;70% in all breeds. Boran had a significantly (P &lt; 0.05) higher total post-thaw sperm motility (93.2 ± 3.6) compared with Nguni (75.1 ± 4.2) and Bonsmara (80.7 ± 6.9). Furthermore, Boran had higher (P &lt; 0.05) progressive motility (39.7 ± 3.4) and rapid motility (36.1 ± 5.9) compared with other breeds. Interestingly, Boran produced significantly (P &lt; 0.05) higher blastocyst rate (56.34%) compared with Bonsmara (38.03%) Nguni (31.08%). Superovulation and OPU resulted in a significantly higher (P &lt; 0.05) number of blastocysts (10.5 ± 3.3 and 10.5 ± 3.3) respectively, compared with aspiration (1.3 ± 3.3). Moreover, the OPU method yielded a significantly higher (P &lt; 0.05) number of grade 2 blastocyst (3.0 ± 0.1) compared with aspiration (0.50 ± 0.1). However, there was no significant (P &gt; 0.05) difference in the number of grade 1 and grade 3 blastocysts obtained when the 3 recovery methods were used. In conclusion, the Boran breed showed better a sperm fertility rate following in vivo and in vitro embryo production. The superovulation and OPU methods resulted in higher numbers and better quality blastocysts compared with aspiration.

178 TRANSCRIPTIONAL DIFFERENCES IN GENES RELATED TO GLUCOSE METABOLISM BETWEEN COMPETENT AND INCOMPETENT BOVINE IMMATURE CUMULUS-OOCYTE COMPLEXES SELECTED BY BCB

In vitro maturation is a key step of in vitro embryo production, being the main factor responsible for the low blastocyst yield. In vitro maturation requires the selection of competent immature cumulus-oocytes complexes (COC), which is usually accomplished based on morphological criteria and follicle size. Competent immature COC have finished their growth phase and show decreased G6PDH activity. Brilliant cresol blue (BCB) is a dye that is degraded by G6PDH and, therefore, can be used to distinguish COC that have finished their growth phase (BCB+) from those that are still growing and are less competent (BCB-). The objective of this study was to determine the metabolic differences between BCB- and BCB+ COC by performing a transcriptional analysis of genes related to glucose metabolism. The COC obtained from slaughterhouse ovaries were selected based on BCB staining. The BCB+ and BCB- COC were fertilized and cultured in vitro to determine the differences in developmental ability. For gene expression analysis, BCB+ and BCB- COC were denuded by vortexing and 5 groups of 10 oocytes; their corresponding cumulus cells per group were snap-frozen until analysis. Messenger RNA was extracted by Dynabeads (Dynal Biotech, Lake Success, NY, USA) and relative mRNA abundance was analysed by quantitative PCR using PPIA1 as housekeeping. Statistical differences were determined based on ANOVA (P &lt; 0.05). The genes analysed were G6PDH, its positive regulator SIRT2, 2 glucose transporters SLC2A1 and SLC2A5, 2 genes involved in anaerobic glycolysis GAPDH and LDHA, 2 genes related with Krebs cycle CS and ATP5A1, and 1 gene related to glutathione metabolism GPX1. As expected, the BCB+ group showed a higher cleavage rate (85.6 ± 1.8 v. 74.2 ± 1.3%, BCB+ v. BCB-; P &lt; 0.05) and blastocyst yield (Day 9: 33.3 ± 3.8 v. 16.1 ± 1.4%, BCB+ v. BCB-; P &lt; 0.05) compared with BCB-. Genes SIRT2, GAPDH, and LDHA were significantly up-regulated in BCB- cumulus cells (SIRT2: 1 ± 0.04 v. 1.45 ± 0.21; GAPDH: 1 ± 0.17 v. 1.46 ± 0.15; LDHA: 1 ± 0.22 v. 1.65 ± 0.12; BCB+ v. BCB-; P &lt; 0.05), whereas no significant differences were found in the other genes and in oocytes. In conclusion, the differences in G6PDH activity between BCB+ and BCB- COC are not controlled by G6PDH transcript abundance, but seem to be mediated by SIRT2 regulation of G6PDH activity. The BCB- cumulus cells showed an up-regulation of GAPDH and LDHA, suggesting a higher activity of anaerobic glycolysis in BCB- COC compared with BCB+.

181 RESVERATROL DURING IN VITRO MATURATION IMPROVES THE QUALITY OF BOVINE OOCYTE AND ENHANCES EMBRYONIC DEVELOPMENT IN VITRO

It is known that reactive oxygen species (ROS) are accumulated within the oocyte during in vitro maturation (IVM) and have been related to poor quality and decreased embryo development in vitro. The use of antioxidants in culture media is an alternative to overcome oxidative stress damage in the oocyte. Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a phenol produced naturally by several plants and has shown protection against oxidative damage in numerous cell types. Two different experiments were performed to evaluate the effect of resveratrol on the quality of bovine oocytes matured in vitro assessed by levels of ROS and intracellular glutathione (GSH) as well as in vitro embryo development rates. Experiment 1 used different concentrations of resveratrol [0 (Control), 1 (R1), 10 (R10), 20 (R20), and 40 (R40) μM] were used to supplement IVM media. Ovaries were collected from Bos indicus cows at a local abattoir and cumulus-oocyte complexes were matured in vitro for 24 h in TCM199 with 6 mg mL−1 of fatty acid-free BSA, 5% fetal bovine serum, 0.2 mM Na-pyruvate, 50 μg mL−1 of gentamicin, 0.5 μg mL−1 of FSH, and 0.5 µg mL−1 of LH at 38°C in 5% CO2 and 90% humidity. The ROS were evaluated by 2′,7′-dichlorodihydrofluorescein diacetate staining (n = 301) and intracellular GSH levels were determined by Cell Tracker Blue fluorescent stain (n = 310). Denuded oocytes were observed under an epifluorescence microscope. Fluorescence intensities of oocytes were analysed by ImageJ software (Version 1.49v, National Institutes of Health, Bethesda, MD, USA) and normalized to control oocytes. Experiment 2 used cumulus-oocyte complexes (n = 674) collected and matured in vitro under the same conditions described for Exp. 1. In vitro fertilization was performed for 18 h at 38°C in 5% CO2 in Tyrode’s medium with 25 mM bicarbonate, 22 mM Na-lactate, 1 mM Na-pyruvate, and 6 mg mL−1 of fatty acid-free BSA. Additionally 10 µg mL−1 of heparin and 20 μM d-penicillamine, 10 μM hypotaurine, and 1 μM epinephrine were added. The presumptive zygotes were cultured in vitro in SOFaa medium with 5% fetal bovine serum, at 38°C, in 5% CO2, 5% O2, and 90% humidity until Day 7, when embryonic development was assessed. Data were analysed by ANOVA followed by Fisher´s multiple range test using Statgraphics Centurion XVI (Version 16.2.04, Statpoint Technologies Inc., Warrentown, VA). Data are presented as percentage mean ± standard error of the mean (P &lt; 0.05). All concentrations of resveratrol in treated oocytes showed reduced intracellular levels of ROS compared to control (R1: 0.66 ± 0.04, R10: 0.55 ± 0.04, R20: 0.62 ± 0.04, R40: 0.64 ± 0.04, and Control: 1 ± 0.04 pixel/oocyte; P &lt; 0.01). Intracellular levels of GSH were significantly higher for R1 (1.4 ± 0.06; P &lt; 0.01) and R10 (1.3 ± 0.06; P &lt; 0.01) compared with the control. On the other hand, R10 showed a significantly higher blastocyst rate (51% ± 3) compared with R1 (39% ± 4), R20 (39% ± 3), R40 (33% ± 3), and control (38% ± 4). Treatments R1, R20, and R40 showed no significant differences compared to control. These results indicate that resveratrol at 10 μM during IVM improves maturation conditions by decreasing ROS level, increasing intracellular GSH, and improving embryonic developmental competence.

The effects of blastocyst morphological score and blastocoele re-expansion speed after warming on pregnancy outcomes

ObjectiveThe aim of this study was to investigate associations between the morphology score of blastocysts and blastocoele re-expansion speed after warming with clinical outcomes, which could assist in making correct and cost-effective decisions regarding the appropriate time to vitrify blastocysts and to transfer vitrified-warmed blastocysts.MethodsA total of 327 vitrified-warmed two-blastocyst transfer cycles in women 38 years old and younger were included in this retrospective study.ResultsThe clinical pregnancy rate (CPR) and implantation rate (IR) of transfers of two good-morphology grade 4 blastocysts vitrified on day 5 (64.1% and 46.8%, respectively) were significantly higher than the CPR and IR associated with the transfers of two good-morphology grade 3 blastocysts vitrified on day 5 (46.7% and 32.2%, respectively). No significant differences were found in the CPR and IR among the transfers of two good-morphology grade 4 blastocysts regardless of the day of cryopreservation. Logistic regression analysis showed that blastocoele re-expansion speed after warming was associated with the CPR.ConclusionThe selection of a good-morphology grade 4 blastocyst to be vitrified could be superior to the choice of a grade 3 blastocyst. Extending the culture of grade 3 blastocysts and freezing grade 4 or higher blastocysts on day 6 could lead to a greater likelihood of pregnancy. Since re-expansion was shown to be a morphological marker of superior blastocyst viability, blastocysts that quickly re-expand after warming should be prioritized for transfer.

103 OVARIAN STIMULATION OF ANESTROUS CATS WITH FOLLICLE-STIMULATING HORMONE IMPROVES OOCYTE QUALITY AND DEVELOPMENTAL CAPABILITY AFTER PARTHENOGENETIC ACTIVATION

The domestic cat is a valuable model for developing assisted reproductive technologies in endangered felids. However, the in vitro production of cat embryos is negatively affected during the anestrous season. It seems that a low FSH level or the few FSH receptors in the granulosa cells might affect oocyte quality. The objective of this research was to evaluate the effect of ovarian stimulation of anestrous cats with FSH on oocyte quality and developmental capability after parthenogenetic activation. For this, two experimental groups were made. The first (FSH group) consisted of 9 anestrous cats treated with a subcutaneous dose of 5 mg of porcine FSH, every 24 h, for 4 days. The second (control group) consisted of 10 untreated anestrous cats. The ovaries were recovered by ovariohysterectomy. Cumulus-oocyte complexes (COCs) were collected by slicing of the ovaries and classified morphologically as grade I (excellent), grade II (good), grade III (fair), and grade IV (poor) quality. Grade I and II COCs were matured in vitro during 30 h and matured oocytes were used for parthenogenetic activation. Matured oocytes were activated using 7% ethanol for 5 min, followed by incubation in 10 μg mL–1 cycloheximide and 5 μg mL–1 cytochalasin B for 5 h. Presumed zygotes were in vitro cultured in SOF for 8 days, and blastocyst and hatching blastocyst rates were assessed. The relative expression of pluripotency (OCT4, SOX2, and NANOG) and differentiation markers (CDX2 and GATA6) were evaluated by RT-qPCR in the blastocysts. The results were normalized using the geometric mean of GAPDH and SDHA. Statistical analysis was performed using the Wilcoxon non-parametric test. A total (mean ± s.d.) of 741 COCs (82.33 ± 36.61 per cat) were recovered from the FSH group and 660 COCs (66 ± 20.67 per cat) from the control group (P &gt; 0.05). The FSH group had a higher rate of grade I COCs and a lower rate of grade III and IV COCs (22.28% and 50.62%, respectively) than the control group (13.2% and 60.08%, respectively; P &lt; 0.05). However, no significant differences were found in grade II COCs between FSH (26.21%) and control groups (27.01%; P &gt; 0.05). Thereafter, 190 matured oocytes were activated in the FSH group and 198 in the control group. The FSH group had higher blastocyst and hatching blastocyst rates (30.52% and 13.15%, respectively) than the control group (13.13% and 1.01% respectively; P &lt; 0.05). The blastocysts from the FSH group had a higher cell number (± s.d.) than their counterparts from the control group (199.9 ± 53.6 and 121 ± 34.91, respectively; P &lt; 0.05). Furthermore, the blastocysts from FSH group had an increased relative expression of OCT4 and GATA6 (P &lt; 0.05). In conclusion, ovarian stimulation of anestrous cats with FSH improves oocyte quality and embryo developmental capability, which agrees with an increase in relative expression of OCT4 and GATA6.

248 SUPERSTIMULATION STRATEGIES FOR OVUM PICKUP IN HOLSTEIN DONORS

In vitro embryo production (IVP) is an important tool to enhance genetic gain in cattle. However, oocyte quality is a limiting factor for the success of IVP programs in high-producing donors. A series of studies using protocols for follicular wave synchronization and superstimulation before ovum pickup were performed to improve the efficiency of ovum pickup and in vitro production in dairy cattle. The first study evaluated superstimulation with FSH (Folltropin-V®) before ovum pickup in lactating (n = 15) and non-lactating (n = 15) Holstein donors in a crossover design. Cows underwent synchronization of follicle wave emergence (FWE) and at the expected time of FWE, the FSH group received a total dosage of 200 mg of FSH in 4 decreasing doses 12 h apart; controls received no FSH, and ovum pickup was conducted 72 h after the expected FWE in all cows. The FSH-treated cows had a higher (P &lt; 0.01) percentage of medium-sized follicles (6 to 10 mm) at the time of ovum pickup (55.1%) than control cows (20.8%) as well as lower cumulus‐oocyte complexes (COC) recovery rates (60.0 v. 69.8%, respectively; P = 0.002). However, FSH-treated cows had a higher blastocyst production rate (34.5 v. 19.8%; P &lt; 0.01) and more transferable embryos per ovum pickup session (3.0 ± 0.5 v. 1.8 ± 0.4; P = 0.02). Subsequent trials evaluated plasma FSH profiles in 23 heifers and in vitro production following ovum pickup in 90 non-lactating Holstein donors superstimulated with a single IM injection of FSH in 0.5% hyaluronan (HA; MAP-5®, 50 mg). Controls received no treatment, while the F200 group received 200 mg of FSH in 4 decreasing doses 12 h apart. The F200HA and F300HA groups received 200 or 300 mg of FSH in 5 or 7.5 mL, respectively, of 0.5% HA by a single IM injection. Circulating FSH area under curve (AUC) in FSH-treated animals was greater than in the control group (P = 0.02). Although the AUC for F200 group did not differ from HA groups (P = 0.56), the total period of time plasma FSH levels were elevated was greater than in the HA groups (P &lt; 0.01). In the IVP trial, FSH-treated cows had a greater proportion of medium-sized (6–10 mm) follicles than controls (P &lt; 0.001). Also, numbers of follicles (P = 0.01) retrieved (control: 13.1 ± 1.0; F200: 16.5 ± 1.2; F200HA: 19.5 ± 2.1; F300HA: 15.4 ± 1.4; P = 0.01) and blastocysts produced per ovum pickup session (control: 2.4 ± 0.5; F200: 3.7 ± 0.7; F200HA: 4.7 ± 0.7; F300HA: 3.1 ± 0.6; P = 0.06) were greater in cows receiving FSH, regardless of treatment. Cows in the F200HA group had a greater recovery rate (P = 0.009), number of COC cultured (P = 0.04), and blastocysts per ovum pickup session (P = 0.06) than cows in the F300HA group. In conclusion, superstimulation of Holstein donors before ovum pickup increased the efficiency of in vitro production. Additionally, a single IM dose of FSH in 0.5% HA resulted in similar plasma FSH profiles to twice-daily FSH treatment. Non-lactating donors treated with FSH produced more embryos per ovum pickup session regardless of FSH treatment. Lastly, all in vitro-produced endpoints were greater following a single dose of 200 mg of FSH in 0.5% HA than 300 mg of FSH in 0.5% HA.

101 TOWARDS OPTIMAL IN VITRO CULTURE CONDITIONS FOR PIG-MONKEY AGGREGATION CHIMERAS

Induced pluripotent stem cells (iPSCs) are promising for developing novel cell-based medical treatments. One potential use of iPSCs is for xenotransplantation via production of chimeric organisms. The generation of organs originated from the iPSCs of one individual in a foreign organism could allow the production of immune compatible organ transplants. One of the major problems down this road is to define the different nutrient needs of chimeric embryos (i.e. iPSCs and the host embryos). Here, we evaluated different media for supporting development of chimeric embryos consisting of parthenogenetic porcine embryos and iPS cells, either from pig or nonhuman primate, to identify the optimal medium conditions for pig-monkey aggregation chimeras. First, we cultured 3-day-old porcine parthenogenetic embryos in porcine zygote medium (PZM), iPSC-medium, and mixtures of the two media to identify the most suitable culture conditions. Three-day-old parthenogenetic embryos developed poorly in pure iPSC-medium (6.3% blastocyst rate), but grew in a mixture of PZM and iPSC medium. The best results were achieved with PZM and PZM with 10% or 25% iPSC medium (38.8% and 30% blastocyst rates, respectively). Next, we checked aggregation results of chimeric embryos produced with two different iPSC lines in the respective media (PZM, PZM+10% iPSC medium, PZM+25% iPSC medium). The porcine iPSC line (piPSC) and cynomolgus monkey iPSC line (ciPSC) carry fluorescent markers (piPSC: GFP, ciPSC: venus), thus facilitating detection of integration into the host embryos. After aggregation of iPS cells between two Day-3 parthenogenetic embryos (sandwich technique), blastocyst rates at Day 6 were determined (see Table 1). While culture in PZM allowed for the highest blastocyst rate (&gt;40%), the iPS cell participation was very low (0–11.1%). Cultivation in PZM+25% iPS medium was compatible with a high rate of embryo-iPSC chimeras (50–100%), but with a low blastocyst rate (7.7–16.7%). Possibly, the iPS cells proliferate more rapidly than blastomeres and the embryo is overgrown. The best results were obtained in medium mixture PZM+10% iPSC medium (25–40% blastocyst rate with 66.7–100% iPS cell participation). These results show that supplementation of the basic culture medium with 10% iPSC medium yields high blastocyst rates for chimeric embryos, and also ensures much higher iPS cell participation. This improves the use of 3-day-old embryos in aggregation chimeras. Table 1.Development of parthenogenetic porcine embryo aggregates with porcine (p) or cynomolgus monkey (c) iPS cells