Polyspermic Penetration Research Articles

Melatonin decreases excessive polyspermy for single oocyte in pigs through the MT2 receptor

Melatonin supplementation during in vitro maturation (IVM) improves porcine oocyte maturation and embryonic development by exerting antioxidative effects. Nevertheless, the mechanism by which melatonin prevents polyspermy after in vitro fertilization (IVF) remains unclear. Here, we examined the effects of melatonin on cytoplasmic maturation and the incidence of polyspermic penetration in porcine oocytes. No statistically significant difference was observed in the rate of first polar body formation between the groups (Control, Melatonin, Melatonin + Luzindole, and Melatonin + 4-P-PDOT). Interestingly, melatonin supplementation significantly improved the cytoplasmic maturation of porcine oocytes by enhancing the normal distribution of organelles (Golgi apparatus, endoplasmic reticulum and mitochondria) and upregulating organelle-related gene expressions (P < 0.05). However, these promotional effects were counteracted by melatonin antagonists, suggesting that melatonin enhances cytoplasmic maturation through its receptors in porcine oocytes. Melatonin supplementation also significantly improved the rate of diploid and blastocyst formation after IVF by promoting the normal distribution of cortical granules (P < 0.05). In conclusion, melatonin supplementation during in vitro maturation of porcine oocyte improves fertilization efficiency and embryonic developmental competence by enhancing cytoplasmic maturation.

PSIV-11 The Effects of Vanillic Acid Supplementation During in Vitro Oocyte Maturation in Pigs

Abstract In vitro fertilization (IVF) typically has low success rates in pigs partially due to the negative effects of oxidative stress on both the sperm and egg. Antioxidants, such as vanillic acid, are known to have protective effects on the oocyte in terms of maturation. The purpose of this study was to determine the effects of vanillic acid supplementation during porcine oocyte maturation on reactive oxygen species (ROS) generation, IVF, early embryonic development success rates, membrane lipid peroxidation, and catalase activity. Oocytes were matured for 40 to 44 h supplemented with vanillic acid (0, 3, 6, 12 mg/mL; Sigma-Aldrich Co.). After maturation, oocytes were denuded and either evaluated for ROS production (n = 183, r = 3) by measuring the fluorescent intensity from the oxidation of 2’,7’-dichlorodihydrofluorescein diacetate, membrane lipid peroxidation (n = 360), catalase activity (n = 724), or fertilized using frozen-thawed boar semen (1.0 x 105 sperm cells/mL) and co-incubated for 6 to 8 h followed by embryo culture for 144 h. After IVF, presumptive zygotes were fixed, permeabilized, and stained with bisBenzimide H 33342 trihydrochloride and evaluated for penetration, polyspermy, and pronucleus formation rates (n = 390) and embryos were evaluated for cleavage and blastocyst formation (n = 529) at 48 h and 144 h post-IVF, respectively. Fluorescent intensities indicating ROS of individual oocytes, lipid peroxidation, catalase activity, and were analyzed using GLM, and means were compared using LSD. The IVF and embryo development data were reported as the percent observed/drop, and mean percentages, and means were analyzed using a GLM with differences being compared using Tukey’s test. Supplementation of 6 g/mL vanillic acid or 12 mg/mL vanillic acid reduced (P &amp;lt; 0.05) the amount of ROS (0.68 ± 0.06, 0.63 ± 0.06; respectively) compared with no vanillic acid supplementation (1 ± 0.06). Vanillic acid supplementation did not have any effect on oocyte penetration rates; however both 6 mg/mL and 12 mg/mL vanillic acid decreased (P &amp;lt; 0.05) polyspermic penetration rates (23.79 ± 0.02, 24.03 ± 0.02; respectively), and increased (P &amp;lt; 0.05) pronuclear formation (83.76 ± 0.02, 84.80 ± 0.02; respectively). Supplementation of vanillic acid had no effect on cleavage rates (78.69 ± 0.02) by 48 h post-IVF or blastocyst formation (31.18 ± 0.03) by 144 h post-IVF. Supplementation of 6 mg/mL vanillic acid decreased (P &amp;lt; 0.05) membrane lipid peroxidation (0.46 ± 0.06) and catalase activity (0.01 ± 0.01) compared with no vanillic acid supplementation (0.79 ± 0.06, 0.03 ± 0.01; respectively). Based on these results, supplementation of 6 mg/mL vanillic acid or 12 mg/mL vanillic acid during oocyte maturation reduces the amount of ROS formed in vitro and improves IVF success rates. Supplementation of 6 mg/mL decreases catalase activity and membrane lipid oxidation following oocyte maturation.

Tannin Reduces the Incidence of Polyspermic Penetration in Porcine Oocytes.

Tannin (TA) improves porcine oocyte cytoplasmic maturation and subsequent embryonic development after in vitro fertilization (IVF). However, the mechanism through which TA blocks polyspermy after IVF remains unclear. Hence, the biological function of organelles (cortical granule [CG], Golgi apparatus, endoplasmic reticulum [ER], and mitochondria) and the incidence of polyspermic penetration were examined. We found no significant difference in oocyte nuclear maturation among the 1 µg/mL, 10 µg/mL TA, and control groups. Moreover, 100 μg/mL TA significantly reduced 1st polar body formation rate compared to the other groups. Additionally, 1 and 10 μg/mL TA significantly increased the protein levels of GDF9, BMP15, and CDK1 compared to the control and 100 μg/mL TA groups. Interestingly, 1 and 10 μg/mL TA improved the normal distribution of CGs, Golgi, ER, and mitochondria by upregulating organelle-related gene expression and downregulating ER stress (CHOP) gene expression. Simultaneously, 1 and 10 μg/mL TA significantly increased the proportion of normal fertilized oocytes (2 pronuclei; 2 PN) and blastocyst formation rate compared to the control, as well as that of 100 μg/mL TA after IVF by upregulating polyspermy-related genes. In conclusion, TA during IVM enhances 2PN and blastocyst formation rates by regulating organelles’ functions and activities.

Zinc Chloride Supplemented during Ovarian Tissue Vitrification Improves In Vitro Follicle Development and Fertilization in Pigs

Ovarian tissue vitrification is a promising method to preserve follicles and gametes, but can be improved with mineral supplementation to the vitrification medium. The objective of this study was to determine the effects of supplementing 0.5 mg/mL zinc chloride to the media during cryopreservation of pig ovarian tissue. After thawing, the following criteria were evaluated: (1) follicular development and damage, (2) in vitro fertilization (IVF) characteristics, and (3) embryonic development. The number of damaged antral follicles (72.0 ± 3.8%) was less (p &lt; 0.05) in ovarian tissue vitrified in media supplemented with zinc chloride compared to those not supplemented with zinc chloride (86.7 ± 4.1%). Oocytes obtained from the antral follicles on ovarian tissue vitrified in media supplemented with zinc chloride had less (p &lt; 0.05) polyspermic penetration and higher (p &lt; 0.05) male pronuclear formation during IVF than oocytes obtained from ovarian tissue not supplemented with zinc chloride. There were no statistical differences in embryonic development rates. Based on these results, supplementing zinc chloride during the vitrification protocol improves follicular development and subsequent IVF in pigs.

PSV-11 Efficacy of Quisqualic Acid as an Amino Acid Transporter Inhibitor in Pig Oocytes

Abstract Quisqualic acid is a known inhibitor of sodium-dependent amino acid transporters. However, it is unknown if quisqualic acid has similar effects in in vitro mature oocytes. Therefore, the objective of this study was to determine the optimal dose and effects of quisqualic acid supplemented during maturation. Oocytes (n=362) were supplemented during maturation with quisqualic acid (0, 0.5, 0.75, 1.0, 2.5 mM) to determine the minimum concentration of quisqualic acid that had no effect on oocyte maturation but significantly decreased the intracellular glutathione concentration. The addition of 1.0 mM quisqualic acid was the lowest concentration observed to cause intracellular glutathione levels to be significantly less (P &amp;lt; 0.05) without affecting maturation compared to no quisqualic acid. Based on those results, oocytes were supplemented with or without 1.0 mM quisqualic acid then evaluated for cumulus cell expansion (n=410) and stage of meiosis (n=380) at the end of maturation. Additional oocytes were fertilized and assessed for cortical granule exocytosis (n=400) and kinetics at 12 h after IVF (n=420). Supplementing quisqualic acid to the media did not have an effect on stage of meiosis, fertilization, polyspermic penetration, or cortical granule exocytosis. Supplementing 1.0 mM quisqualic acid significantly decreased (P &amp;lt; 0.05) cumulus cell expansion by the end of maturation and male pronuclear formation by 12 h after IVF. These results suggest that quisqualic acid supplementation during maturation in pigs inhibits sodium-dependent amino acid transporters.

PSV-10 Effects of l-α-amino Butyrate Supplementation on Oocyte Maturation

Abstract L-α-amino butyrate is a low-molecular weight thiol compound that acts to increase the levels of glutathione in the oocyte. Glutathione acts as an antioxidant during oocyte maturation and promotes male pronuclear formation during fertilization. Supplementing the L-α-amino butyrate helps to decrease polyspermic penetration rates and improve early embryonic development in swine. However, it is unknown if L-α-amino butyrate supplementation affects the environment of the oocyte or the oocyte directly. Therefore, the objective of this study was to determine if L-α-amino butyrate supplementation to the maturation media acted on the oocyte or had alternative beneficial effects in the surrounding environment. Oocytes were randomly assigned to a maturation media containing an amino acid transport inhibitor, quisqualic acid (QA) (0 or 1 mM) and then supplemented with L-α-amino butyrate (0 or 3.3 mM). Oocytes were evaluated for stage of meiosis (n=380) and cumulus cell expansion (n=411) at the end of maturation. The remaining oocytes were fertilized and evaluated for cortical granule exocytosis (n=400) and IVF kinetics (n=456). Supplementation of L-α-amino butyrate with or without QA significantly increased (P &amp;lt; 0.05) cumulus cell expansion, cortical granule exocytosis and male pronuclear formation compared to no supplementation or QA supplementation. There was no difference in meiotic progression, fertilization or polyspermic penetration rates between the treatment groups. Results suggest that when L-α-amino butyrate is supplemented during maturation, it improves the maturation of the oocyte by acting directly on the oocyte and not through the surrounding environment of the oocyte.

PSVII-17 Effects of zinc chloride supplementation during ovarian cortex vitrification prior to in vitro fertilization on embryo development in pigs

Abstract Ovarian tissue has an increased risk of damage during vitrification due to the presence of different cell types and water permeability levels within the tissue. Supplementation of antioxidant-like compounds such as zinc chloride improves follicular integrity and increases antral follicle development post-thawing. However, the effects of zinc chloride supplementation on the oocytes within the follicles are unknown. Therefore, the objective of this study is was to determine the effects of 5 μg/mL zinc chloride supplementation during the vitrification process on post-thawing fertilization characteristics and embryonic development. Ovarian cortex samples (5 x 5 mm; n=27) were isolated from ovaries extracted from cycling gilts. Cortical pieces were loaded onto a 25-gauge needle and incubated for five min in equilibrium solution followed by five min in vitrification solution, both supplemented with 5 μg/mL zinc chloride. Following incubation, the cortexes were place in liquid nitrogen for 7 d. Cortexes were then thawed and oocytes were aspirated from antral follicles. Oocytes (n=162) were incubated in maturation media for 40-44 h and then subjected to IVF and embryo culture. Frozen-thawed semen from three boars was used (30 oocytes/well, 200 sperm/oocyte). Post-IVF, a portion of the potential embryos (n=25) were evaluated for penetration, polyspermy, and male pronuclear formation rates. The remaining embryos (n=137) were evaluated 48 h after IVF for cleavage and 144 h for blastocyst formation. Data were analyzed using ANOVA and Tukey’s test. There were no differences between treatment groups when comparing penetration, 2-cell and blastocyst formation rates. However, polyspermic penetration rates were significantly lower (P &amp;lt; 0.05) and male pronuclear formation were significantly higher (P &amp;lt; 0.05) in the zinc chloride supplemented treatment group compared to no supplementation. These results indicate that supplementing zinc chloride during ovarian cortex freezing improves fertilization characteristics of their oocytes post-thawing and maturation.

In vitro fertilization and embryo development in pigs

Considerable progress has been made in the in vitro production of pig embryos using improved methods for in vitro maturation (IVM) and fertilization (IVF). Despite the progress, polyspermic penetration remains a problem for in vitro-matured oocytes. Variation among boars, ejaculates and IVF protocols used in different laboratories appears to influence the incidence of polyspermy. Recent studies indicate that oviduct cells and their secretions play a role in reducing polyspermy. Very early attempts to culture in vivo-derived pig embryos met with little success and most were arrested at the four-cell stage. At present, many culture media are available that can overcome the four-cell block and support development to the blastocyst stage. In contrast, blastocyst development of in vitro-produced (IVP) embryos in these culture media varies significantly. Significant differences in morphology and numbers of cells have been observed in in vitro-produced blastocysts compared with in vivo-derived blastocysts. Surgical transfer of in vitro-produced embryos to recipient animals has resulted in acceptable pregnancy rates with moderate litter sizes. Although several systems are available for the generation of in vitro-produced embryos, the problems of polyspermy and poor embryo survival prevent large-scale production of embryos. Further research should be directed to improve oocyte and embryo quality, and to develop methods to minimize polyspermy through development of better IVM, IVF and embryo culture techniques.

Advances in in vitro production of pig embryos

A series of integrated, effective techniques is required to produce pig embryos from follicular oocytes in vitro. The failure to form a male pronucleus and polyspermy have been serious problems in efforts to produce embryos efficiently in vitro from pig oocytes. The former problem is now considered to be due to oxidative stress and the latter has been partially solved by reducing the number of capacitated spermatozoa reaching the oocytes. By the use of new technology for in vitro production of embryos, an acceptable rate of blastocyst formation and the birth of live piglets has been achieved. However, even with the use of these improved in vitro maturation (IVM) and fertilization (IVF) conditions, the efficiency of production of in vitro blastocysts and offspring still remains relatively low. More recently the developmental competence of embryos matured and fertilized in vitro has been investigated through modification of culture conditions of oocytes during the germinal vesicle stage. Oocyte competence for early embryonic development appears to be achieved by active communication between the oocyte and follicular cells. Since the ovarian oocytes available for IVM are primarily those present in mid-size antral follicles of prepubertal gilts, more research is needed to gain an improved understanding of the culture conditions required to induce developmental competence in oocytes from both preantral and antral follicles as well as additional modifications in IVF systems to overcome the problem of polyspermic penetration.

Cyanidin improves oocyte maturation and the in vitro production of pig embryos.

The objective of this study was to reduce the negative effects of oxidative stress by decreasing the levels of reactive oxygen species (ROS) through supplementation of the major antioxidants present in elderberries: kuromanin and cyanidin. Oocytes (n = 1150) were supplemented with 100 or 200μM of kuromanin or cyanidin during maturation, and then evaluated for ROS levels or fertilized and evaluated for penetration, polyspermic penetration, male pronucleus formation, and embryonic development. The ROS levels and incidence of polyspermic penetration were lower (P < 0.05) in oocytes supplemented with 100μM cyanidin when compared with other treatments. Supplementation of 100μM cyanidin increased (P < 0.05) MPN and blastocyst formation compared with other treatments. However, supplementation of 100μM kuromanin did not have significant effects on the criteria evaluated, and supplementation of 200μM kuromanin had significant (P < 0.05) detrimental effects for each criterion. Additional oocytes (n = 1438) were supplemented with 100μM cyanidin during maturation and evaluated for glutathione, glutathione peroxidase, catalase, and superoxide dismutase activity. Supplementation of 100μM cyanidin increased (P < 0.05) catalase activity and intracellular GSH levels compared with no supplementation of cyanidin. These results indicate that supplementing cyanidin during maturation reduces oxidative stress by reducing ROS levels and increasing GSH concentrations within the oocyte.

Effects of glucuronic acid and N-acetyl-D-glucosamine supplementation on the perivitelline space during the IVM of pig oocytes.

The objective of this study was to minimise polyspermic penetration by increasing the perivitelline space (PVS) thickness through supplementation of the hyaluronic acid components glucuronic acid and N-acetyl-d-glucosamine (GlcNAc). Oocytes (n=4690) were supplemented during the first 24h and/or the remainder of maturation (final 16-18h) with 0.01mM glucuronic acid and 0.01mM GlcNAc and then evaluated for PVS thickness, hyaluronic acid, glutathione and glutathione peroxidase concentrations. Fertilised oocytes were evaluated for polyspermic penetration and embryo development. The PVS thickness and amount of hyaluronic acid was significantly (P<0.05) greater in oocytes supplemented with 0.01mM glucuronic acid and 0.01mM GlcNAc during the second part or all of maturation compared with the other treatments. In addition, polyspermic penetration was significantly (P<0.05) less in oocytes supplemented with 0.01mM glucuronic acid and 0.01mM GlcNAc during the second part or all of maturation compared with the other treatments. Supplementing 0.01mM glucuronic acid and GlcNAc during maturation significantly (P<0.05) increased the percentage of cleaved embryos by 48h after IVF and blastocysts formed by 144h after IVF compared those not supplemented. These results indicate that supplementing PVS components during maturation decreases polyspermic penetration by increasing PVS thickness.

Coculture of porcine cumulus-oocyte complexes with porcine luteal cells during IVM: effect on oocyte maturation and embryo development.

Coculture with somatic cells is an alternative to improve suboptimal invitro culture conditions. In pigs, IVF is related to poor male pronuclear formation and high rates of polyspermy. The aim of this study was to assess the effect of a coculture system with porcine luteal cells (PLCs) on the IVM of porcine cumulus-oocyte complexes (COCs). Abattoir-derived ovaries were used to obtain PLCs and COCs. COCs were matured invitro in TCM-199 with or without the addition of human menopausal gonadotrophin (hMG; C+hMG and C-hMG respectively), in coculture with PLCs from passage 1 (PLC-1) and in PLC-1 conditioned medium (CM). In the coculture system, nuclear maturation rates were significantly higher than in the C-hMG and CM groups, but similar to rates in the C+hMG group. In cumulus cells, PLC-1 coculture decreased viability, early apoptosis and necrosis, and increased late apoptosis compared with C+hMG. PLC-1 coculture also decreased reactive oxygen species levels in cumulus cells. After IVF, monospermic penetration and IVF efficiency increased in the PLC-1 group compared with the C+hMG group. After invitro culture, higher blastocysts rates were observed in the PLC-1 group. This is the first report of a coculture system of COCs with PLCs. Our model could be an alternative for the conventional maturation medium plus gonadotrophins because of its lower rates of polyspermic penetration and higher blastocysts rates, key issues in porcine invitro embryo production.

11. TRANSCRIPTOME ANALYSIS AND TIME-LAPSE OBSERVATION OF A CASE WITH REPEATED MULTIPLE PRONUCLEI AFTER ICSI

Introduction Pronucleus (PN) formation is a process by which the parental chromosome complements unite and engender the zygotic genome. During fertilization, the maternal PN is pulled toward the male counterpart, and the 2PN are juxtaposed and positioned in the central or paracentral region of the cytoplasm (Terada et al., 2010). More than 2PN can sometimes be observed, which is generally assumed to be due to either a polyspermic penetration or to the failure of a second polar body extrusion, especially in the case of 3PN (Rosenbusch et al., 2010). However, for oocytes with >3PN, the reason of their incidence remains unclear. The aim of the study was to identify the cause of repeated multipronuclei (MPN) formation in zygotes of a patient after intracytoplasmic sperm injection (ICSI). Material and Methods This is a case study of a patient with repeated MPN formation after five ICSI cycles (four in other clinics and one in Istanbul Memorial Hospital). Time-lapse monitoring of PN formation was carried out. Embryos developed from seven MPN zygotes of the sixth cycle were analyzed by FISH with the informed consent of the patient. Six MPN day-3 embryos and two surplus day-3 embryos for which the couples did not accept freezing were collected with informed consent from the patient and two couples, respectively. They were analyzed by whole genome expression analysis (Clariom™ D Assay, ThermoFisher Scientific). Results The time-lapse evaluation of the patient's 17 oocytes showed 9 MPN zygotes, one 3PN zygote, two unfertilized oocytes and three normally fertilized zygotes. The paternal pronuclei was of normal size and 7-9 micropronuclei appeared beneath the second polar body and migrated to the center of the cytoplasm. The ensuing cleavages were unequal and gave rise to a high rate of fragmentation. All MPN and the 3PN embryos arrested their development on day-3. Seven MPN embryos were analyzed by FISH and 1-20 micronuclei were observed in each blastomere. Six embryos were diagnosed as being chaotic aneuploid and one as being monosomy18 and nullisomy13. Transcriptomes of six MPN arrested day-3 embryos and two surplus 2PN good quality day-3 embryos were compared. Three pathways involving the AXIN1 gene were found to be significantly affected: Wnt signaling pathway and pluripotency, embryonic stem cell pluripotency pathways and the apoptosis-related network due to altered Notch3 in ovarian cancer. The chemokine signaling pathway (CCL19, FGR, PREX1 genes) known to be involved in the regulation of the actin cytoskeleton, which has a role in cell division, vesicle and organelle movement and interestingly documented to be disrupted in patients with premature ovarian failure was also found to be involved. Conclusions Several candidate coding genes involved in cell cycle, pluripotency of stem cells and actin cytoskeleton pathways have been identified as a new cause of MPN formation and infertility. However, transcriptional data from six MPN embryos can only provide clues for further investigation. Future studies, including whole exon sequencing of candidate genes of the patient, her family and other patients with MPN zygotes might help in identifying the exact mechanism of this phenomenon.

PSI-21 The effects of kuromanin supplementation during oocyte maturation on the in vitro production of pig embryos

Abstract The imbalance of reactive oxygen species levels and antioxidants impact oocyte matruation. Elderberries (Sambucus sp.) have been identified as possessing the ability to alleviate oxidative stress. An antioxidant class found in elderberry is anthocyanidin, which includes kuromanin. The objective of this study was to determine the effects of kuromanin supplementation to the media during oocyte maturation. Oocytes (n = 692, r=3) were supplemented with 100 or 200 μM kuromanin during 40-44 h of maturation and then evaluated at the end of maturation for the formation of reactive oxygen species, fertilization characteristics, and rates of embryonic cleavage and blastocyst development at 48 h and 144 h after IVF, respectively. There were no significant differences between no kuromanin supplementation and 100 μM kuromanin supplementation when comparing reactive oxygen species generation at the end of oocyte maturation. Supplementation of 200 μM kuromanin significantly increased (P &lt; 0.05) reactive oxygen species generation in oocytes compared to the other groups. There were no significant differences between no kuromanin supplementation and 100 μM kuromanin supplementation when comparing penetration and polyspermic penetration rates and male pronuclear formation. Supplementation of 200 μM kuromanin significantly decreased (P &lt; 0.05) penetration and polyspermic penetration rates and male pronuclear formation compared to the other groups. There were no significant differences between no kuromanin supplementation and 100 μM kuromanin supplementation when comparing the percentage of cleaved embryos by 48 h after IVF and the percentage of those reaching the blastocyst stage by 144 h after IVF. Supplementation of 200 μM kuromanin significantly decreased (P &lt; 0.05) the cleavage rates by 48 h after IVF and the blastocyst formation rates by 144 h after IVF compared to all other treatment groups. These results indicate that supplementing 200 μM kuromanin is detrimental to oocyte maturation and lower levels (100 μM) do not have a significant effect compared to not supplementing the oocytes when evaluating in vitro fertilization and early embryonic development characteristics.

PSI-22 The effects of cyanidin supplementation during oocyte maturation on the in vitro production of pig embryos

Abstract Oxidative stress has a negative effect on embryonic development during the in vitro production of pig embryos. The imbalance of reactive oxygen species and antioxidants impact oocyte maturation. Berries from the elder plant (Sambucus sp.) have been identified as containing high levels of a broad spectrum of antioxidants. One of the predominant antioxidant classes of compounds found in elderberry extract is anthocyanin, which includes the antioxidant cyanidin. Therefore, the objective of this study was to determine the effects of cyanidin supplementation to the media during oocyte maturation. Oocytes (n = 815, r=3) were supplemented with 100 or 200 μM cyanidin during 40–44 h of maturation and then evaluated at the end of maturation for the formation of reactive oxygen species, fertilization characteristics, and rates of embryonic cleavage and blastocyst development at 48 h and 144 h after IVF, respectively. There were no significant differences between no cyanidin supplementation and 200 μM cyanidin supplementation when comparing reactive oxygen species generation at the end of oocyte maturation. Supplementation of 100 μM cyanidin significantly decreased (P &lt; 0.05) reactive oxygen species generation in oocytes compared to the other groups. There were no significant differences between no cyanidin supplementation and 200 μM cyanidin supplementation when comparing penetration and polyspermic penetration rates and male pronuclear formation. Supplementation of 100 μM cyanidin significantly decreased (P &lt; 0.05) polyspermic penetration rates and significantly increased (P &lt; 0.05) male pronuclear formation rates compared to the other groups. There were no significant differences between the treatment groups when comparing the percentage of cleaved embryos by 48 h after IVF. Supplementation of 100 μM cyanidin significantly increased (P &lt; 0.05) the blastocyst formation rates by 144 h after IVF compared to all other treatment groups. These results indicate that supplementing 100 μM cyanidin to the media during oocyte maturation reduces reactive oxygen species formation and improves in vitro fertilization and early embryonic development in pigs.

Coenzyme Q10 Supplementation Effects on In Vitro Maturation, Fertilization, and Early Embryonic Development in Pigs

The objective of this study was to determine the reduction of polyspermic penetration, and increase of mitochondrial activity, in early pig embryonic development by supplementing different concentrations of coenzyme Q10 during oocyte maturation. Oocytes (n = 1,100) were supplemented during the last 24 h of maturation with 0 (control), 10, 50, or 100 μM of coenzyme Q10. After in vitro fertilization (IVF), embryos were evaluated for fertilization kinetics (penetration, polyspermic penetration, male pronuclear formation), and subsequent embryonic development and mitochondrial activity. Supplementation of 100 μM coenzyme Q10 was detrimental to the oocytes, as they had significantly lower (p &lt; 0.05) fertilization kinetic and early embryonic development rates to the other treatment groups. There were no differences in fertilization kinetic and early embryonic development rates between the 0, 10 and 50 μM coenzyme Q10 treatment groups. Oocytes, matured in medium supplemented with 50 μM coenzyme Q10, ultimately developed into embryos with a significantly greater (p &lt; 0.05) presence of intact mitochondrial membranes (observed at both 48 and 144 h post-IVF) compared to oocytes not supplemented with coenzyme Q10. In summary, supplementation of 100 μM coenzyme Q10 during oocyte maturation is detrimental, yet supplementation of 50 μM coenzyme Q10 leads to a higher occurrence of intact mitochondrial membranes in the in vitro produced pig embryos.

146 Quercetin supplementation during boar semen thawing and incubation improves the in vitro production of pig embryos

Elevated levels of reactive oxygen species in the in vitro environment cause oxidative stress, which leads to membrane damage, decreased fertility, and morphological deformities of spermatozoa. Antioxidants, such as quercetin (a polyphenol flavonoid), are often supplemented to reduce the effects of oxidative stress on spermatozoa. Supplementing frozen-thawed boar semen with quercetin improves sperm forward progressive motility, viability and lipid peroxidation up to 10h after thawing. However, the effects of fertilizing with quercetin-supplemented sperm are unknown. Therefore, the objective of this study was to determine the effects of supplementing quercetin (0.25, 0.50, 0.75mM) during the thawing and incubation of frozen-thawed boar semen on oocyte fertilization characteristics (n=400) and subsequent embryonic development (n=1340) at 48 and 144h for cleavage and blastocyst formation, respectively. Oocytes from aspired aspirated mature follicles (3-6mm diameter) were obtained from a local abattoir and matured in medium 199 for 40 to 44h at 38.5°C in an atmosphere of 5% CO2. Fertilization was performed using pooled frozen-thawed semen from 3 different boars, and co-incubation of the sperm (2×105 sperm mL−1) and oocytes (30 oocytes/well) lasted for 6 to 8h at 38.5°C in an atmosphere of 5% CO2. Data were analysed using ANOVA with the main effects including treatment, well and replicate. Chi-squared analysis was used to determine percentages of embryos reaching the different developmental stages for each treatment. There were no differences in penetration rates and male pronuclear formation between treatment groups; however, supplementation of 0.25 (18.18±10.63%), 0.50 (20.93±9.89%) and 0.75mM (18.07±12.02%) quercetin significantly decreased (P&amp;lt;0.05) polyspermic penetration rates compared with no supplementation (40.00±11.34%). Embryos produced from frozen-thawed boar sperm supplemented with 0.25 and 0.50mM quercetin had a significantly higher percentage (P&amp;lt;0.05) of embryos reaching the 2-cell stage of development by 48h after IVF (75.00±7.89%, 68.75±2.23%, respectively) compared with 0.75mM quercetin supplementation (64.62±3.88%) and no supplementation (62.97±4.11%). Supplementation of 0.25 (44.12±6.23%), 0.50 (43.75±7.02%) and 0.75mM (43.08±2.98%) quercetin to the sperm significantly increased (P&amp;lt;0.05) the percentage of embryos reaching the blastocyst stage of development by 144h after IVF compared with no supplementation (28.27±8.07%). These results indicate that supplementing frozen-thawed boar semen with quercetin decreases the incidence of polyspermic penetration and improves early embryonic development in pigs.

Removal of sialic acid from bull sperm decreases motility and mucus penetration ability but increases zona pellucida binding and polyspermic penetration in vitro.

This study tested the hypothesis that sperm sialic acid (Sia) is required to reach the site of fertilization, and that successful fertilization requires recognition of Sia from both the sperm and oocyte to occur. In addition, it has recently been reported that Siglecs (Sia-binding-immunoglobulin-like lectins) are present on the sperm surface. Thus, the possibility that the recognition of oocyte Sia was sperm-Siglec-mediated was also addressed. Sperm exposed to neuraminidase (NMase) exhibited lower overall and progressive motility, which translated to a decreased ability to swim through cervical mucus from cows in oestrus. In addition, when either sperm or cumulus-oocyte complexes (COCs) were treated with NMase, a decrease in cleavage and blastocyst rate was observed. However, incubation of sperm with increasing concentrations of anti-Siglec-2, -5, -6 and -10 antibodies prior to fertilization had no effect on their fertilizing ability. Interestingly, treatment with NMase increased the number of sperm bound to the ZP but also the rate of polyspermic fertilization. Flow cytometry analysis revealed no differences in the percentage of capacitated or acrosome-reacted sperm. These results suggest that Sia are required to reach the site of fertilization but need to be removed for sperm-oocyte interaction. However, fine regulation is needed to avoid abnormal fertilization which can lead to impaired embryo development.

Melatonin and tannic acid supplementation in vitro improve fertilization and embryonic development in pigs.

The objective of this study was to determine the effects of melatonin supplementation during maturation and tannic acid supplementation during IVF on fertilization kinetics and early embryonic development. Experiment 1 determined the optimum concentration of melatonin supplemented to the oocytes for subsequent embryonic development. Oocytes (n = 400) were supplemented at 22 h of maturation with 0, 75, 100, or 150 nm melatonin and then subjected to IVF and embryo culture. After IVF, a portion of the embryos were evaluated for penetration, polyspermy, and male pronuclear (MPN) formation rates. Embryos were evaluated 48 h after IVF for cleavage and 144 h for blastocyst formation. There were no significant differences between treatment groups with respect to penetration and polyspermy. Supplementation of 150 nm melatonin produced a significantly greater (P < 0.05) percent of embryos with MPN compared to those supplemented with 75 nm or 100 nm. Supplementation of 150 nm melatonin produced significantly less (P < 0.05) embryos cleaved by 48 h after IVF while 75 nm melatonin supplementation had a significantly higher (P < 0.05) percentage of blastocyst formation by 144 h after IVF. Based on the optimal concentration of melatonin observed in experiment 1, experiment 2 determined the effects of supplementing 75 nm melatonin to the maturation media and 5.0 μg/ml tannic acid supplementation during IVF on oxidative stress, fertilization kinetics, and embryonic development. Oocytes (n = 720) were supplemented at 22 h of maturation with or without 75 nm melatonin and then fertilized with frozen-thawed sperm supplemented with or without 5 μg/ml tannic acid. Reactive oxygen species levels were measured in matured oocytes using 2’,7’-dichlorodihydrofluorescein diacetate. Oocytes supplemented with 75 nm melatonin had significantly less (P < 0.05) reactive oxygen species generation and oocytes fertilized with sperm incubated with tannic acid had a significantly less (P < 0.05) incidence of polyspermic penetration compared to no supplementation. All treatment groups had significantly greater (P < 0.05) incidence of male pronuclear formation compared to oocytes not supplemented with melatonin and fertilized without tannic acid. Oocytes that were supplemented with melatonin and fertilized with sperm incubated with tannic acid had a significantly higher (P < 0.05) percentage of blastocyst formation by 144 h post-IVF compared all other treatment groups. Results indicate that supplementation of 75 nm melatonin during oocyte maturation and 5 μg/ml tannic acid during IVF leads to a decrease in oxidative stress, increase in IVF success and subsequent embryo development in pigs.

175 EFFECTS OF GLUCURONIC ACID AND N-ACETYL-D-GLUCOSAMINE SUPPLEMENTATION ON THE PERIVITELLINE SPACE DURING THE IN VITRO MATURATION OF PORCINE OOCYTES

Pig oocytes fertilized in vitro experience high polyspermic penetration rates due to inadequate cortical granule exocytosis into reduced perivitelline space (PVS) thickness. The objective of this study was to minimize polyspermic penetration by increasing the PVS thickness through supplementation of its hyaluronic acid components, glucuronic acid (GA), and N-acetyl-d-glucosamine (GlcNAc) during maturation. Oocytes were supplemented during the first 24 h or second 24 h of maturation with 0.01 mM GA and 0.01 mM GlcNAc and then evaluated for nuclear maturation (n = 200), PVS thickness (n = 245), and the amount of hyaluronic acid (n = 245) present. The PVS thickness was determined at the equatorial plane of the oocyte using a micrometer. Hyaluronic acid concentrations were determined using an enzyme-linked immunosorbent assay method. Oocytes (n = 800) were fertilized using frozen-thawed semen and evaluated for fertilization characteristics and subsequent embryonic development at 48 and 144 h for cleavage and blastocyst formation, respectively. The PVS thickness was significantly thicker (P &lt; 0.05) with oocytes supplemented with 0.01 mM GA and 0.01 mM GlcNAc during the first half of maturation (3.20 ± 0.29) and all of maturation (2.78 ± 0.21) compared with no supplementation (2.22 ± 0.13) and supplementation during only the second half of maturation (2.02 ± 0.16). The amount of hyaluronic acid present at 24 h of maturation was significantly greater (P &lt; 0.05) in oocytes supplemented with the PVS components (2.03 ± 0.07 pg/oocyte) compared with no supplementation (0.21 ± 0.02 pg/oocyte). At the end of maturation, oocytes supplemented with 0.01 mM GA and 0.01 mM GlcNAc during the entire maturation had significantly greater (P &lt; 0.05) amounts of hyaluronic acid present (4.16 ± 0.19 pg/oocyte) compared with all other groups. There was no significant difference in penetration rates between the groups. Polyspermic penetration was significantly less (P &lt; 0.05) in oocytes supplemented with 0.01 mM GA and 0.01 mM GlcNAc during the first half of maturation compared with no supplementation or supplementation during only the second half of maturation. Oocytes supplemented with 0.01 mM GA and 0.01 mM GlcNAc during the first half of maturation (87.36 ± 4.01) compared with no supplementation (76.47 ± 5.67) or supplementation during only the second half of maturation (80.23 ± 3.21) had significantly higher percentages (P &lt; 0.05) of male pronuclear formation by 12 h after IVF. Supplementing 0.01 mM GA and 0.01 mM GlcNAc during maturation significantly increased (P &lt; 0.05) the percentage of cleaved embryos by 48 h after IVF and the percentage of those reaching the blastocyst stage by 144 h after IVF compared with those that were not supplemented during maturation (55.00 ± 6.43, 20.00 ± 6.16). There were no significant differences between the supplementation treatment groups at 48 or 144 h after IVF. These results indicate that supplementing GA and GlcNAc to the media during maturation, specifically during the first 24 h, decreases polyspermic penetration by increasing PVS thickness, hyaluronic acid amount, and male pronuclear formation, which improves subsequent embryonic development.