Male Pronuclear Formation Rates Research Articles

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 < 0.05) and male pronuclear formation were significantly higher (P < 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.

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.

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 < 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 < 0.05) polyspermic penetration rates and significantly increased (P < 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 < 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.

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.

Pretreatment of bovine sperm with dithiobutylamine (DTBA) significantly improves embryo development after ICSI

We assessed the effect of pretreating sperm with dithiobutylamine (DTBA) to improve embryo development by intracytoplasmic sperm injection (ICSI) in cows. Acridine Orange staining revealed that when applied at different concentrations (2.5, 5, and 10 mM) and exposure times (5 min, 20 min, 1 h, and 2 h), DTBA reduced disulfide bonds in spermatozoa with the highest efficacy at 5 mM for 5 min. DTBA enhanced the percentage of spermatozoa with free protamine thiol groups compared with untreated spermatozoa (control) (P < 0.05); however, this result did not differ from that of dithiothreitol (DTT) treatment. The percentage of live spermatozoa after DTBA treatment was identical to that in the control, but significantly higher than that after DTT treatment (P < 0.05). After ICSI, DTBA treatment tended to improve male pronuclear formation rate (P = 0.071) compared with non-treated sperm injection. Blastocyst formation rate was significantly improved by DTBA treatment compared with that in DTT, control, and sham injection groups (P < 0.05). Blastocyst quality in terms of cell numbers and ploidy was not different among these groups. In conclusion, DTBA increases the efficacy of blastocyst production by ICSI even if DTT treatment does not work.

Fertilization Ability of Porcine Oocytes Reconstructed from Ooplasmic Fragments Produced and Characterized after Serial Centrifugations

Mitochondria are reported to be critical in in vitro maturation of oocytes and subsequent embryo development after fertilization, but their contribution for fertilization has not been investigated in detail. In the present study, we investigate the contribution of mitochondria to fertilization using reconstructed porcine oocytes by fusion of ooplasmic fragments produced by serial centrifugations (centri-fusion). Firstly, we evaluated the characteristics of ooplasmic fragments. Three types of fragments were obtained by centrifugation of porcine oocytes matured in vitro for 46 h: brownish (B), transparent (T) and large (L) fragments containing both B and T parts in a fragment. The production efficiencies of these types of fragments were 71.7, 91.0 and 17.8 fragments/100 oocytes, respectively. In experiments, L fragments were excluded because they contained both brownish and transparent components that were apparently intermediate between B and T fragments. Observations by confocal microscopy after staining with MitoTracker Red CMXRos® and transmission electron microscopy revealed highly condensed active mitochondria in B fragments in contrast to T fragments that contained only sparse organelles. We reconstructed oocytes by fusion of a karyoplast and two cytoplasts from B and T fragments (B and T oocytes, respectively). The B oocytes showed higher sperm penetration (95.8%) and male pronuclear formation rates (94.2%) by in vitro fertilization than T oocytes (66.7% and 50.0%, respectively). These results suggest that the active mitochondria in oocytes may be related to their ability for fertilization.

Effects of porcine granulocyte-macrophage colony-stimulating factor on porcine in vitro-fertilized embryos

This study investigated the effects of porcine granulocyte-macrophage colony-stimulating factor (pGM-CSF) on the developmental potential of porcine in vitro-fertilized (IVF) embryos in chemically and semidefined (with BSA) medium. In experiment 1, zygotes were treated with different concentrations of pGM-CSF (0, 2, 10, 100 ng/mL). The results indicated that 10 ng/mL pGM-CSF significantly (P < 0.05) increased blastocyst development and total cell number (15.1% and 53.5, respectively) compared with the control (6.1%, and 38.8, respectively). Comparing blastocyst formation, early and expanded blastocyst formation was significantly higher in the 10 ng/mL-pGM-CSF group than in the control on Days 6 and 7 of the culture period. However, there was no significant difference in cleavage rate. Experiment 2 demonstrated that pGM-CSF influenced the percentage of blastocyst formation and total cell number when pGM-CSF was added during Days 4 to 7 (14.6% and 53.9, respectively) or Days 0 to 7 (15.2% and 54.0, respectively) compared with the control (7.8% and 43.1, respectively) and compared with Days 0 to 3 (8.7% and 42.5, respectively). Similarly, early blastocyst formation rates were significantly higher at Days 4 to 7 than in the control, and expanded blastocyst formation was significantly higher at Days 4 to 7 or Days 0 to 7. No significant difference in cleavage rates appeared among the groups. In experiment 3, in the presence of BSA, pGM-CSF also increased the percentage of embryos that developed to the blastocyst stage and the total cell number (20.3% and 59.8, respectively) compared with the control (14.9% and 51.4, respectively), whereas there was no significant difference in cleavage rate. Experiment 4 found that the total cell number and the number of cells in the inner cell mass (ICM) were significantly increased compared with the control when zygotes were cultured in either porcine zygotic medium (PZM)-3 or PZM-4 supplemented with 10 ng/mL pGM-CSF. The number of trophectoderm (TE) cells was significantly higher in PZM-3 medium supplemented with pGM-CSF than in the control, and the number tended to increase (P = 0.058) in PZM-4 medium supplemented with pGM-CSF. The ratio of inner cell mass to trophectoderm cells was significantly higher in PZM-4 supplemented with 10 ng/mL pGM-CSF, but not in PZM-3. In experiment 5, it was found that the male pronuclear formation rate, monospermic penetration and sperm/oocyte were 95.4%, 37.2%, and 2.4, respectively. Together, these results suggest that pGM-CSF may have a physiological role in promoting the development of porcine preimplantation embryos and regulating cell viability and that addition of pGM-CSF to IVC medium at Days 4 to 7 or 0 to 7 improves the developmental potential of porcine IVF embryos.

Viability of ICSI oocytes after caffeine treatment and sperm membrane removal with Triton X-100 in pigs

Non-adequate decondensation of injected sperm nucleus is one the main problems of intracytoplasmic sperm injection (ICSI) in porcine. With the aim of improving pronuclear formation, the effects on activation and embryo development rates of 0.1% Triton X-100 (TX) sperm pre-treatment for membrane removal and/or 5 mM Caffeine (CAF) addition in oocyte manipulating and culture medium for 2 h after ICSI or artificial activation were studied. The effects of 4 different Ca2+ concentrations contained in the injection medium on embryo development after sham injection were also analysed. In Experiment 1, no significant effect on cleavage or blastocyst rate was detected independently of Ca2+ concentration contained in the injection medium. In Experiment 2, oocytes injected with TX pre-treated sperm showed a significant higher rate of male pronuclear formation in comparison with oocytes from control group (2PN; 54.1 vs 36.6%). However, no differences on in vitro embryo development, cleavage or blastocyst rates were observed. In Experiment 3, oocytes treated with CAF during and after micromanipulation and injected with sperm pre-treated with TX had a significantly lower oocyte activation rate than any other experimental groups (25.7 vs 56.3–66.3%). No differences were observed in cleavage rates among different experimental groups. However, the CAF group showed a higher blastocyst rate significantly different from TX+CAF group (12.0 vs 1.9%, respectively). In a second approach, the effect of electric field strengths and CAF treatments on oocyte activation was studied. In Experiment 4, oocytes submitted to 0.6 kV/cm showed significant higher activation rates than 1.2 kV/cm ones regardless of the caffeine treatment (83.7 vs 55.9% and 75.7 vs 44.3%; in control and caffeine groups, respectively). No effect of caffeine treatment was observed in any experimental group. In conclusion, TX sperm treatment before ICSI without an additional activation procedure improved male pronuclear formation, but did not improve embryo development until blastocyst stage. No significant effect of caffeine was found when sperm was not treated with TX, although in membrane absence caffeine avoided oocyte activation and embryo development. Finally, caffeine had no effect on female pronuclear formation regardless of electric field strengths applied to the parthenogenetic activation.

Supplementation with vascular endothelial growth factor during in vitro maturation of porcine cumulus oocyte complexes and subsequent developmental competence after in vitro fertilization

The aim of the present study was to investigate whether the effects of vascular endothelial growth factor (VEGF) on porcine cumulus oocyte complexes (COCs) and subsequent blastocyst formation following in vitro fertilization are attributable to improved fertilization and cytoplasmic maturation. Porcine COCs were cultured for 42 h in TCM199 medium with 5 ng/mL human recombinant VEGF, and the resultant metaphase II oocytes were fertilized in vitro. COCs without VEGF supplementation served controls. Supplementation with VEGF during in vitro maturation (IVM) significantly (P < 0.05) improved the blastocyst formation rate and total cell number (46.7 ± 3.1% and 82.8 ± 6.7, respectively) compared with controls (32.5 ± 3.4% and 64.1 ± 5.6, respectively). On day 2, the percentage of four-cell stage embryos was significantly higher in the VEGF-matured group (49.1 ± 2.7%) than in the control (33.1 ± 5.8%), and the percentage of two-cell stage embryos was significantly higher in the control group (10.4 ± 1.4%) than in the VEGF-matured group (6.6 ± 0.9%). At 10 h after the onset of in vitro fertilization (IVF), oocytes with two pronuclei were considered as monospermically or normally fertilized, and oocytes with more than two pronuclei were considered as polyspermically fertilized. Monospermy was significantly higher in VEGF-matured oocytes (47.2 ± 4.3%) than in the control (20.0 ± 2.4%), and polyspermy and sperm penetration per oocyte were significantly higher in the control group (54.4 ± 3.8% and 2.3 ± 0.1, respectively) than in the VEGF-matured oocytes (43.9 ± 3.6% and 1.8 ± 0.1, respectively). Supplementation with VEGF during IVM significantly (P < 0.05) improved male pronuclear formation as compared with the control (91.1 ± 1.9 vs 74.4 ± 3.8%). Type III cortical granule distribution in oocytes was more common in VEGF-matured oocytes (78.0%) than in the control (52.1%). These results suggest that VEGF supplementation during IVM enhanced the developmental potential of porcine IVF embryos through higher male pronuclear formation and higher monospermic fertilization rates as a consequence of improved cytoplasmic maturation.

Effects of oxygen tension and follicle cells on maturation and fertilization of porcine oocytes during in vitro culture in follicular fluid

The objective was to investigate the effects of oxygen tension and follicle cells (FCs) during in vitro maturation of porcine oocytes in only porcine ( Sus scrofa domesticus) follicular fluid (pFF), using static and non-static (rotating) culture systems, on the nuclear maturation and subsequent in vitro fertilization of the oocytes. In the first experiment, cumulus-oocyte complexes (COCs) were matured for 48 h in pFF supplemented with (+) or without (−) FCs (5.2 × 10 6 cells/mL), using the static (S) and rotating (R) culture systems (+FC/S, −FC/S, +FC/R, and −FC/R) under 5% or 20% O 2. Co-culture with FCs in the static culture system (+FC/S) had a detrimental effect on the meiotic competence of oocytes, whereas co-culture with FCs in the rotating culture system (+FC/R) increased maturation rates. In both culture systems, oxygen tension had no apparent effects on meiotic competence of oocytes, irrespective of culture system and FC addition. In the second experiment, COCs were matured under 5% or 20% O 2 using the −FC/S or +FC/R culture systems and then fertilized. Oxygen tension had no significant effects on fertilization parameters, irrespective of the culture system. The rotating culture system increased rates of sperm penetration and male pronuclear formation and decreased polyspermic fertilization compared with the static culture system (P < 0.05). In conclusion, both −FC/S and +FC/R culture systems supported meiotic competence, irrespective of oxygen tension. However, the +FC/R culture system may be superior to the −FC/S culture system for promoting fertilization.

Effects of liquid preservation of sperm on their ability to activate oocytes and initiate preimplantational development after intracytoplasmic sperm injection in the pig

The objective of this study was to clarify the effects of liquid preservation conditions on the ability of pig sperm to activate oocytes, form a male pronucleus, and initiate preimplantational development of embryos after intracytoplasmic sperm injection (ICSI). Porcine ejaculates were preserved at 4, 14, and 24 °C for up to 48 h, and then damage to the plasma membrane, morphologic changes of the acrosome, and the amount of phospholipase Cζ (PLCζ) in the sperm were assessed by SYBR-14/propidium iodide staining, fluorescein isothiocyanate–conjugated peanut agglutinin staining, indirect immunofluorescence, and Western blots, respectively. The proportion of sperm with a disintegrated plasma membrane or damaged acrosome increased in all samples as the duration of preservation increased, although the time courses of the increases varied among preservation temperatures. The immunolocalization and immunoreactivity of PLCζ in the sperm showed its reduction concurrent with disintegration of the plasma membrane and acrosome. Rates of oocyte activation, male-pronuclear formation, and blastocyst formation after ICSI using sperm preserved for 18 h at 24 °C (78%, 62%, and 35%, respectively) and for 48 h at 14 °C (63%, 53%, and 28%, respectively) were significantly higher than those of any other sperm sample. We concluded that the damage to the plasma membrane and acrosome, and a sufficient amount of PLCζ in the sperm head, enhanced successful oocyte activation, fertilization, and early development of the oocytes after ICSI. Moreover, we inferred that appropriate liquid preservation of sperm improved the efficiency of blastocyst production in vitro after ICSI in pigs.

Nuclear Replacement ofIn Vitro-Matured Porcine Oocytes by a Serial Centrifugation and Fusion Method

The objective of the present study was to establish a method for nuclear replacement in metaphase-II (M-II) stage porcine oocytes. Karyoplasts containing M-II chromosomes (K) and cytoplasts without chromosomes (C) were produced from in vitro-matured oocytes by a serial centrifugation method. The oocytes were then reconstructed by fusion of one karyoplast with 1, 2, 3 or 4 cytoplasts (K + 1C, K + 2C, K + 3C and K + 4C, respectively). Reconstructed oocytes, karyoplasts without fusion of any cytoplast (K) and zona-free M-II oocytes (control) were used for experiments. The rates of female pronucleus formation after parthenogenetic activation in all groups of reconstructed oocytes (58.2-77.4%) were not different from those of the K and control groups (58.2% and 66.0%, respectively). In vitro fertilization was carried out to assay the fertilization ability and subsequent embryonic development of the reconstructed oocytes. The cytoplast : karyoplast ratio did not affect the fertilization status (penetration and male pronuclear formation rates) of the oocytes. A significantly high monospermy rate was found in K oocytes (p < 0.05, 61.6%) compared with the other groups (18.2-32.8%). Blastocyst formation rates increased significantly as the number of the cytoplasts fused with karyoplasts increased (p < 0.05, 0.0-15.3%). The blastocyst rate in the K + 4C group (15.3%) was comparable with that of the control (17.8%). Total cell numbers in both the K + 3C and K + 4C groups (16.0 and 15.3 cells, respectively) were comparable with that of the control (26.2 cells). Our results demonstrate that a serial centrifugation and fusion (Centri-Fusion) is an effective method for producing M-II chromosome transferred oocytes with normal fertilization ability and in vitro development. It is suggested that the number of cytoplasts fused with a karyoplast plays a critical role in embryonic development.

Cytoplasmic glutathione regulated by cumulus cells during porcine oocyte maturation affects fertilization and embryonic development in vitro

It is generally accepted that cumulus cells support the nuclear maturation of mammalian oocytes. In the present study, we examined relationships between the cytoplasmic glutathione (GSH) content of porcine oocytes, and oocyte nuclear maturation, fertilization or subsequent embryonic development. Cumulus-oocyte complexes (COCs; control group) and oocytes denuded of cumulus cells after collection (DO 0 h group) were cultured for 24 h with dibutyryl cAMP, eCG and hCG (first culture step) and then for a further 20 h without supplements (second culture step; 44 h total culture). After the first culture step, some of the COCs were denuded, either completely (DO 24 h group) or partly (H-DO 24 h group), and then matured by the second culture step. Also, in the second culture step, some DOs were co-cultured with cumulus cells that had been pre-cultured for 24 h (DO 24 h + CC group). The maturation rates of all the cumulus-removed groups (DO 0 h, DO 24 h, H-DO 24 h and DO 24 h + CC groups) were lower (34.3–45.0%) than that of the control group (64.5%; P < 0.05). The GSH contents of matured oocytes in the completely denuded groups (DO 0 h, DO 24 h and DO 24 h + CC groups) were lower (4.03–5.26 pmol/oocyte) than that of the control group (9.60 pmol/oocyte; P < 0.05); however, the H-DO 24 h group had an intermediate value (7.0 pmol/oocyte). The male pronuclear formation rates of completely denuded oocytes were lower (41.4–59.3%) than that of the control group (89.4%; P < 0.05), whereas the H-DO 24 h group had an intermediate rate (80.0%). The blastocyst formation rates of the completely denuded oocytes were lower (3.0–4.5%) than that of the control group (19.9%; P < 0.05), and the H-DO 24 h group again had an intermediate rate (11.6%). The GSH content was correlated with the rates of male pronuclear formation ( P < 0.01) and blastocyst formation ( P < 0.01), and also with the number of cells per blastocyst ( P < 0.01). In conclusion, we inferred that GSH synthesized by intact cumulus cells during maturation culture improved oocyte maturation and played an important role in fertilization and embryonic development.

225 DOES THE SUPPLEMENTATION OF THE BOAR SEMEN EXTENDER WITH CARNOSINE, L-HISTIDINE, AND TAURINE PRESERVE SPERM FUNCTION DURING STORAGE?

High levels of reactive oxygen species (ROS: superoxide, hydroxyl, hydrogen peroxide, nitric oxide, peroxynitrile) endanger sperm motility, viability, and function by interaction with membrane lipids, proteins, and nuclear and mitochondrial DNA (Sikka 2004 J. Androl. 25, 5–18). ROS generation has a significant negative effect on the fertilization rate after IVF, and so measurement of ROS levels in semen specimens before IVF may be useful in predicting the IVF outcome (Agarwal et al. 2005 Fertil. Steril. 84, 228–231). Several compounds of the antioxidant systems have been identified in the epididymal environment, spermatozoa, and seminal plasma. The antioxidants carnosine, L-histidine (Ducci et al. 2006 Pol. J. Vet. Sci. 9, 159–163), and taurine (Van der Horst and Grooten 1966 Biochim. Biophys. Acta. 117, 495–497) have been detected in boar semen and added to the extender in freezing procedures in several species. The main objective of this study was to evaluate the effect of carnosine, L-histidine, and taurine supplementation of the extender on boar sperm functionality as measured by sperm motility during computer-assisted semen analysis (CASA) and by IVF ability using mature oocytes, as previously described (Selles et al. 2003 Reprod. Domest. Anim. 38, 66–72). The sperm-rich fraction from mature fertile boars was diluted with isothermal Beltsville thawing solution (BTS) extender. Diluted semen was placed at 15�C and centrifuged at 800g for 10 min. The semen pellet was resuspended with BTS supplemented by 5 mm of carnosine, L-histidine, or taurine or not supplemented (control) to provide 75 � 106 spermatozoa mL–1 and stored at 15�C for 24 h (IVF assay), or 48 or 120 h (for CASA assay). We observed that the motility parameters were affected by storage time and that the addition of taurine increased the motility at 48 h of storage. Alternately, the addition of L-histidine to the extender reduced significantly the motility parameters after 120 h. The results showed that the addition of L-histidine induced a significant (P ≤ 0.01) decrease of the penetration rate (L-histidine 75.8% v. control 89.9%) and the number of sperm per oocyte penetrated (L-histidine 3.1 v. control 4.1). The rate of male pronuclear formation was not affected by the addition of antioxidants to the extender (over 85% in all cases). The addition of carnosine and taurine had no effect on the IVF parameters. In conclusion the antioxidants carnosine, taurine, and L-histidine affect sperm functionality differently, and further studies are necessary to elucidate what changes in sperm function take place during storage and the mechanisms by which these antioxidants exert their effects. This work was supported by Italian-Spanish research project HI2005-0165 and AGL2006-03495.

44 EFFECT OF CYTOPLAST VOLUME ON FERTILIZATION AND EMBRYO DEVELOPMENT IN PORCINE M-II OOCYTES RECONSTRUCTED WITH KARYOPLASTS AND CYTOPLASTS OBTAINED BY THE 'CENTRI-FUSION' METHOD

Metaphase-II chromosome transfer (M-II transfer) of oocytes is considered to be one of the advanced procedures to improve fertilization and developmental abilities of oocytes with poor cytoplasmic maturation. The aim of this study was to investigate the developmental capacity after IVF and IVC of porcine oocytes reconstructed from karyoplasts and cytoplasts produced by centri-fusion (Fahrudin et al. 2007 Cloning Stem Cells 9, 216–228). In brief, IVM oocytes (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041) with a visible first polar body were centrifuged at 13 000g for 9 min to stratify the cytoplasm. Then the zonae pellucidae were removed with pronase treatment. Zona-free oocytes were layered on a 300-µL discontinuous gradient of Percoll in TCM-HEPES with 5 µg mL–1 of cytochalasin B. After centrifugation at 6000g for 4 s, fragmented cytoplasms with approximately equal volumes were obtained, stained with Hoechst-33342, and classified into cytoplasm with (K; karyoplast) or without (C; cytoplast) chromosomes. One karyoplast was fused with 0, 1, 2, 3, and 4 cytoplasts (K, K + 1C, K + 2C, K + 3C, and K + 4C, respectively) by an electric stimulation with a single DC pulse (1.5 kV cm–1 for 20 µs) and cultured for 1 h. Zona-free oocytes without any reconstruction served as control oocytes. The diameters of the reconstructed and control oocytes were measured. All specimens were fertilized in vitro with frozen–thawed boar sperm, and cultured using the well of the well (WOW) system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264). Their fertilization status and developmental competence were examined. Data were analyzed by ANOVA followed by Duncan's multiple range tests. The diameter differed significantly among K to K + 4C oocytes (75.0–127.1 µm; P &lt; 0.05), whereas the diameter of K + 2C oocytes was similar to that of the control oocytes (110.5 µm). Regardless of the cytoplast volume, sperm penetration rates (73.1–93.8%) for K to K + 4C oocytes were not significantly different compared to control oocytes (78.0%). Male pronuclear formation rates of K to K + 4C oocytes (92.3–97.1%) were also not different significantly different compared to control oocytes (96.6%). However, monospermy rates of K oocytes was significantly higher (61.6%; P &lt; 0.05) than those of the reconstructed (K + 1C to K + 4C; 18.2–34.9%) and control oocytes (32.9%). The blastocyst formation rates in K, K + 1C, K + 2C, and K + 3C groups (0.0–9.8%; P &lt; 0.05) were significantly lower than those in the control and K + 4C groups (17.8% and 15.3%, respectively; P &lt; 0.05). The total cell numbers per blastocyst in K + 1C and K + 2C groups (7.5 and 8.3 cells, respectively) were significantly lower than in the control, K + 3C, and K + 4C groups (15.3–26.2 cells; P &lt; 0.05). These results suggest that the cytoplast volume of porcine M-II transferred oocytes, produced by reconstruction from a karyoplast and cytoplast(s) and centri-fusion, is important for their ability to develop to the blastocyst stage and influences cell number.

The predictive value of routine semen evaluation and IVF technology for determining relative boar fertility

Practical techniques for assessing semen quality in order to predict male fertility are still needed. The principal objective of this experiment was to evaluate routine laboratory evaluation and in vitro fertilization (IVF) techniques as predictors of relative boar fertility using a low-dose AI protocol. Nine boars were evaluated during a 6.5±1mo period, beginning at 29–32wk of age. Ejaculates were evaluated for motility, morphology and concentration, diluted to 1.5 billion sperm in 50mL extender, and used to breed 50±5 gilts over the same period. On nine occasions, a specific aliquot of the ejaculate's first sperm-rich fraction was evaluated using IVF procedures. Boars differed (P<0.001) consistently for pregnancy rate (from 73 to 98%), farrowing rate (71–98%) and total born (8.8–12.0). Routine semen evaluation and IVF parameters that presented significant differences between boars, but no differences in time and no boar by time interaction, were used to correlate in vivo fertility. A multiple regression model based on routine semen evaluation parameters accounted for up to 27 and 22% of the variation of fertility index and total piglets born, respectively, whereas male pronuclear formation rate was the IVF variable that accounted for 17 and 12% of the variation in farrowing rate and fertility index, respectively. Collectively, we inferred that the use of low sperm numbers for AI, determination of pregnancy rate at Day 30, motility of extended semen after 7 and 10d, and specific IVF parameters may be useful for identifying relatively infertile boars that are not currently excluded from use in existing commercial boar studs.

In vitro maturation and fertilization of porcine oocytes after a 48 h culture in roscovitine, an inhibitor of p34 cdc2/cyclin B kinase

Maintaining oocytes at the germinal vesicle (GV) stage in vitro may permit enhanced acquisition of the developmental competence. The objective of the current study was to evaluate the nuclear and cytoplasmic maturation in vitro of porcine oocytes after pretreatment with S-roscovitine (ROS). Cumulus oocyte complexes (COC) were treated with 50 μM ROS for 48 h and then matured for various lengths of time in a conventional step-wise in vitro maturation (IVM) system by using dibutyryl cyclic AMP. The COC that were matured in the same system for 44 h without pretreatment with ROS were used as the control group. At various periods after the start of IVM, oocytes were assessed for the meiotic stages and subjected to in vitro fertilization (IVF) with fresh spermatozoa. The ROS treatment inhibited GV breakdown of 94.4% oocytes, with the majority arrested at the GV-I stage (67.4%). Maximum maturation rate to the metaphase-II stage after ROS treatment was achieved by 44 h of IVM (92.1%) and no differences were observed with control oocytes (95.0%). Penetration rate was correlated to the maturation rate. The duration of IVM had no effects on polyspermy and male pronuclear (MPN) formation rates at 8 h post insemination (hpi), whereas both rates increased at 22 hpi. Direct comparison with controls assessed at 22 hpi confirmed a lesser MPN formation in ROS-treated oocytes (73.7% compared with 53.6%). Glutathione (GSH) concentrations were less in oocytes treated with ROS than in control oocytes (5 compared with 7.7 pmol/oocyte) as well as blastocyst rate (22.0% compared with 38.1%, respectively). These results demonstrate that cytoplasmic maturation in porcine oocytes pretreated with ROS for 48 h did not equal that of control oocytes in the current IVM system.

357 FERTILIZATION AND EMBRYONIC DEVELOPMENT OF PORCINE OOCYTES FOLLOWING INTRACYTOPLASMIC SPERM HEAD INJECTION ENHANCED BY ELECTRIC STIMULATION TO OOCYTES BUT NOT BY PRE-TREATMENT OF SPERMATOZOA WITH DITHIOTHREITOL

Failure of sperm nuclear decondensation has been reported after injection into oocytes in pigs (Kren et al. 2003 J. Reprod. Dev. 49, 271-273). We examined the effects of pretreatment of spermatozoa with Triton X-100 (TX-100) and dithiothreitol (DTT) and electric stimulation of oocytes after injection on sperm decondensation, male pronuclear formation, and in vitro development to the blastocyst stage. We performed three replicates in each experimental group, with a total of about 70 oocytes per group. In Experiment 1, spermatozoa were pretreated with 1% TX-100 and 5 mM DTT (T+D), and injected into IVM oocytes that were collected from crossbred gilts. Electric stimulation (1.5 kV/cm, 20 �s; Nakai et al. 2003 Biol. Reprod. 68, 1003-1008) was applied 1 h to the oocytes after the injection (the stimulated group) or was not applied (the nonstimulated group). Some of the oocytes in each group were evaluated for morphological changes of sperm nuclei at hourly intervals until 10 h post-injection. Of nonstimulated oocytes, those injected with untreated spermatozoa showed a delayed peak in nuclear decondensation (39.4 to 44.1%, 3-6 h after the injection) compared to that of oocytes injected with T+D treated spermatozoa (57.0 to 52.6%, 1-1 h). The rate of male pronuclear formation increased after 4 h post-stimulation (by 40 to 60%) when the injected oocytes were stimulated, whether or not spermatozoa were pretreated. In nonstimulated oocytes, the rate of male pronuclear formation stayed at the basal level (less than 20%) throughout the culture period regardless of sperm treatments. Thus, the T+D treatment of spermatozoa did not affect decondensation and pronuclear formation. In Experiment 2, the effects of electric stimulation and sperm treatments with T+D on the rate of blastocyst formation and the mean numbers of cells per blastocyst were evaluated. Oocytes that were stimulated after injection of either T+D-treated or untreated spermatozoa showed significantly higher percentages of blastocyst formation (24.8% and 27.1%, respectively) than did nonstimulated oocytes (1.1% and 4.1% for T+D-treated and untreated, respectively; P &lt; 0.01). The rate of blastocyst formation was not different between the T+D-treated and the untreated groups. The mean number of cells per blastocyst was not different among all groups (14.0-29.4). In conclusion, the pretreatment of sperm with TX-100 and DTT shifted the timing of sperm nuclear decondensation forward. However, pronuclear formation and development to the blastocyst stage in vitro were not improved by the sperm treatment. Electric stimulation to the injected oocytes enhances in vitro development to the blastocyst stage in pigs.

283 EFFECTS OF CUMULUS CELL REMOVAL ON IN VITRO OOCYTE MATURATION, FERTILIZATION, AND EMBRYONIC DEVELOPMENT IN PIGS

It is generally accepted that cumulus cells (CCs) support the nuclear maturation of immature oocytes in mammals. However, the precise mechanism of interaction between cumulus cells and oocytes has not been clarified. Furthermore, the role of cumulus cells in embryonic development has not been reported. In the present study, the effect of denuding cumulus cells from porcine oocytes on oocyte maturation, ertilization, and their subsequent development to the blastocyst stage was examined in vitro. In vitro maturation, fertilization, and culture were carried out as previously reported (Kikuchi et al. 2002 Biol. Reprod. 66, 1033-1041). Porcine cumulus-oocyte complexes (COCs) were collected; some of them were completely denuded of cumulus cells immediately after the collection (DO-0 group). The remaining intact COCs and the DO-0 oocytes were cultured for 24 h in the presence of dbcAMP and hormones. After the initial culture, some of the intact COCs were denuded either completely (DO-24 group) or partially (H-DO-24 group). Additionally, some of DO-24 oocytes were co-cultured with the cumulus cells removed at 0 h and pre-cultured for 24 h (DO-24 + CCs group). The denuded oocytes in each experimental group and intact COCs (control) were further cultured for total 46 h. The remaining oocytes with a first polar body were either examined for the levels of intracellular glutathione (GSH) or fertilized in vitro with frozen-thawed boar spermatozoa. The inseminated oocytes were cultured and examined for their fertilization status after 10 h and for their developmental competence after 6 days. Data were analyzed by ANOVA, followed by the Duncan's multiple range tests. The maturation rates of all denuded groups were significantly lower (P &lt; 0.05; 34.3 to 45.0%) than that of the control group (64.5%). Intracellular GSH concentrations of all denuded groups were also significantly lower (P &lt; 0.05; 4.03 to 7.00 pmol/oocyte) than that of the control group (9.60 pmol/oocyte); however, the GSH level of H-DO-24 oocytes was significantly higher (P &lt; 0.05) than the GSH levels in the other denuded groups. Male pronuclear formation rates of completely denuded oocytes (DO-0, DO-24, and DO-24 + CCs groups) were significantly lower (P &lt; 0.05; 41.4 to 59.3%) than those of the control (89.4%) and the H-DO-24 (80.0%) groups. The blastocyst rate of the control group was significantly higher (P &lt; 0.05; 19.9%) than that of H-DO-24 group (11.6%), and these rates were significantly higher (P &lt; 0.05) than those of the completely denuded groups (3.0 to 4.5%). The results suggest that the presence of cumulus cells during maturation culture improves nuclear maturation of oocytes and plays an important role in embryonic development to the blastocyst stage in vitro.

262IN VITRO FERTILIZATION IN MICROFLUIDIC CHANNELS ENHANCES MONOSPERMIC PENETRATION OF SWINE OOCYTES

In traditional porcine IVF systems, there is a high incidence of polyspermy, a pathological condition which results in aberrant embryonic development. Efforts to improve the in vitro embryo production process in pigs have included modifying the culture medium, the number of spermatozoa inseminated, and the quantity of medium used. Recently, the development of microscale embryo culture devices (microchannels) has opened new avenues for manipulation of the IVF system to improve the efficiency and overall production of porcine embryos by more closely mimicking the function of the oviduct. The volume of medium in the local vicinity of the embryo is smaller (0.125μL) compared to the typical 5–50μL microdrops. Additionally, it is believed that the delivery of sperm cells in the microchannel simulates the flow pattern of spermatozoa past the oocytes similar to that in the oviduct. This study was designed to compare the incidence of polyspermy of pig oocytes fertilized in PDMS-glass microchannels (MC) to conventional microdrop methods (controls). Oocytes were obtained by aspiration of ovarian antral follicles. Fifty oocytes were placed into 500μL of TCM199 medium supplemented with LH, FSH, EGF, cysteine, PVA, and antibiotics. Extended semen was washed with mTBM and re-suspended to 6×105 sperm/mL. The sperm suspension was placed in humidified 5% CO2 in air atmosphere at 39°C for 1h to allow for capacitation. Concurrently, pre-warmed microchannels were filled with 200μL of mTBM and allowed to equilibrate for 1h at 39°C. Cumulus cells were removed from the oocytes using 0.1mgmL−1 hyaluronidase in mTBM. At 44h of maturation, 15 oocytes were placed into a pre-equilibrated 50μL drop of mTBM covered with warm paraffin oil in a petri dish, and placed into the microchannel. Capacitated sperm cells were then added to the oocytes to give a final concentration of 3×105 sperm/mL; the mixture was incubated for 6h. Presumptive zygotes were cultured in 100μL drops of NCSU-23 covered with oil at 39°C in a humidified 5% CO2 in air for 12h. At this time, the zygotes were fixed in 1:3 (v/v) glacial acetic acid in absolute ethanol for 48–72h. Aceto-orcein staining data revealed a higher incidence of monospermic penetration and a lower number of spermatozoa per oocyte in the microchannels as compared to the controls (Table 1). Data from six replicates were arranged in a randomized block design and analyzed by the generalized linear model in SAS. These data support the idea that the microchannel environment reduces the incidence of polyspermy during IVF of porcine oocytes (P&amp;lt;0.05) while maintaining comparable penetration and male pronuclear formation rates. Furthermore, it is possible that the number of sperm present near the oocytes during fertilization is decreased using the microchannel. In conclusion, microfluidic technology has shown the potential to improve in vitro fertilization in swine by an increasing monospermic penetration of oocytes. Table 1 Effect of IVF system on fertilization parameters