Polyspermic Penetration Research Articles

156 OVIDUCTAL FLUID SUPPLEMENTATION DURING MATURATION AND FERTILIZATION IMPROVES IN VITRO EMBRYONIC DEVELOPMENT IN PIGS

Oviducal fluid has a major role in the maturation of gametes and the process of fertilization. The objective of this study was to determine the effects of oviducal fluid supplementation in vitro, during oocyte maturation and IVF on fertilization characteristics and early embryonic development rates. Oocytes from aspired aspirated mature follicles (3–6 mm diameter) were obtained from a local abattoir. During the last 24 h of maturation, oocytes (n = 1303) were placed into maturation media supplemented either 1% (vol/vol) or 5% (vol/vol) thawed snap-frozen oviducal fluid. Fertilization was performed using pooled frozen-thawed semen from 3 different boars. During IVF, the fertilization medium was supplemented with 1% (vol/vol) or 5% (vol/vol) oviducal fluid. Fertilization characteristics were evaluated 12 h after IVF and rates of embryonic cleavage and blastocyst development were observed at 48 and 144 h after IVF, respectively. Data were analysed using ANOVA with the main effects including treatment, well, and replicate. Chi-square analysis was used to determine percentages of embryos reaching the different developmental stages for each treatment. There were no significant differences in the percentages of oocytes that reached metaphase II by the end of maturation or in sperm penetration rates after IVF. However, oocytes treated with 1% (vol/vol) oviducal fluid during the end of maturation and IVF (33.33 ± 2.61) and 5% (vol/vol) oviducal fluid during maturation (33.33 ± 2.66) or IVF (39.53 ± 3.78) had significantly less (P < 0.05) incidence of polyspermic penetrations and a significantly higher (P < 0.05) incidence of male pronuclear formation (87.50 ± 4.01; 86.67 ± 4.83; 86.05 ± 3.19, respectively) compared with no oviducal fluid supplementation. Oocytes supplemented with 5% (vol/vol) oviducal fluid during maturation and IVF had significantly lower (P < 0.05) incidences of polyspermic penetration (27.91 ± 2.50) and significantly higher (P < 0.05) percentages of embryos reaching the 2-cell stage (81.76 ± 3.72) and blastocyst stage of development (37.74 ± 1.09) by 48 and 144 h, respectively, compared with all other groups. The results of this study suggest that supplementing 5% (vol/vol) oviducal fluid during maturation and IVF improves the success rates of in vitro embryo development in pigs.

Pre-incubation of porcine semen reduces the incidence of polyspermy on embryos derived from low quality oocytes

ABSTRACT: The main cause of low efficiency of in vitro produced porcine embryos is the high polyspermic penetration rates at fertilization, which is aggravated in low quality oocytes. Experiment 1 evaluated the embryo development in high and low quality oocytes. Experiment 2 evaluated the embryo development and quality of low quality oocytes fertilized with sperm pre-incubated during 0h (control), 0.5h, 1h and 1.5h. Experiment 3 investigated fertilization and monospermic rates of the same groups of Experiment 2. Experiment 4 evaluated embryo development, cell density, fertilization and monospermic rates of high quality oocytes using semen pre incubated during the best time observed in the previous experiments. Cleavage and blastocyst rates were analyzed by chi-square test, and remaining data by ANOVA and Tukey test (P≤0.05). The cleavage (74.8 vs 51.7%) and blastocyst (33.7 vs 9.8%) rates were greater in oocytes of high versus low quality, with no differences in cell density. Fertilization rates (65.6 to 79.5%) were not influenced by pre-incubation time. However, semen pre-incubation during 1.5h increased monospermic penetration (53.3%) and cleavage rates (92.5%) in low quality oocytes. Blastocyst rate was improved with 1.5h of semen pre incubation; however they were still lower than that observed with high quality control oocytes. Ultimately, pre-incubation did not influence fertilization, monospermic penetration, embryo development rates, nor cell density in oocytes of high quality. Low-quality porcine oocytes resulted in better rates of embryo development if in vitro fertilized with sperm pre-incubated for 1.5 hour.

Methods for Improving In Vitro and In Vivo Boar Sperm Fertility.

Fertility of boar spermatozoa is changed after ejaculation invivo and invitro. During processing for invitro fertilization (IVF), although spermatozoa are induced capacitation, resulting in a high penetration rate, persistent obstacle of polyspermic penetration is still observed with a high incidence. For artificial insemination (AI), we still need a large number of spermatozoa and lose a majority of those in the female reproductive tract. Fertility of cryopreserved boar spermatozoa is still injured through freezing and thawing process. In the present brief review, factors affecting fertility of boar sperm during IVF, AI and cryopreservation are discussed in the context of discovering methodologies to improve it.

The effects of glucuronic acid and N-acetyl-D-glucosamine on in vitro fertilisation of porcine oocytes.

High incidences of polyspermic penetration continue to challenge researchers during porcine in vitro fertilisation (IVF). The aim of this study was to reduce the incidence of polyspermy by increasing the perivitelline space thickness with glucuronic acid and N-acetyl-D-glucosamine (GlcNAc) supplementation during oocyte maturation. After maturation, zona pellucida and perivitelline space thicknesses, intracellular glutathione concentrations and fertilisation kinetics were measured, in addition to embryonic cleavage and blastocyst formation at 48h and 144h after IVF, respectively. There were no significant differences between the treatments for zona pellucida thickness, penetration rates, male pronuclear formation or cortical granule exocytosis. Glucuronic acid supplementation significantly increased (PPPP<0.05) of cleavage and blastocyst formation by 48 and 144h after IVF compared with all other groups. These results indicate that supplementing with 0.005mM glucuronic acid and 0.005mM GlcNAc during oocyte maturation decreases the incidence of polyspermic penetration by increasing perivitelline space thickness and improving embryo development in pigs.

A new rolling culture-based in vitro fertilization system capable of reducing polyspermy in porcine oocytes.

The high incidence of polyspermy is one of the major obstacles during in vitro fertilization (IVF) in pigs. To overcome this, we developed a novel IVF method, which involves constant rotation. Oocytes matured in vitro were mixed with spermatozoa (0.2 × 10(5) sperm/mL) in an IVF medium (200 μL) using a 200 μL PCR tube. This tube was then rotated at 1 rpm for 6 h at 38.5°C in a rotation mixer (experimental group). A second PCR tube was simultaneously cultured without rotation (control group). The rate of polyspermy was evaluated 12 h after insemination and was significantly (P < 0.05; 21.0% vs. 48.3%) lower in the experimental group than in the control group. Sperm penetration rate was similar in oocytes from the experimental and control groups (75.2% vs. 83.1%). However, monospermic fertilization rate of the oocytes was significantly (P < 0.05; 44.8% vs. 21.2%) higher in the experimental group than in the control group. Furthermore, the rate of blastocyst formation (30.1% vs. 20.8%) increased in the experimental group, as compared to the control group. This present system will contribute to increase the efficacy of blastocyst production through reduction of polyspermic penetration.

Evaluation of Zona Pellucida Function for Sperm Penetration During &lt;i&gt;In Vitro&lt;/i&gt; Fertilization in Pigs

In porcine oocytes, the function of the zona pellucida (ZP) with regard to sperm penetration or prevention of polyspermy is not well understood. In the present study, we investigated the effects of the ZP on sperm penetration during in vitro fertilization (IVF). We collected in vitro-matured oocytes with a first polar body (ZP+ oocytes). Some of them were freed from the ZP (ZP− oocytes) by two treatments (pronase and mechanical pipetting), and the effects of these treatments on sperm penetration parameters (sperm penetration rate and numbers of penetrated sperm per oocyte) were evaluated. There was no evident difference in the parameters between the two groups. Secondly, we compared the sperm penetration parameters of ZP+ and ZP− oocytes using frozen-thawed epididymal spermatozoa from four boars. Sperm penetration into ZP+ oocytes was found to be accelerated relative to ZP− oocytes. Thirdly, we evaluated the sperm penetration of ZP+ and ZP− oocytes at 1−10 h after IVF (3 h gamete co-incubation). The proportions of oocytes penetrated by sperm increased significantly with time in both groups; however, the number of penetrated sperm per oocyte did not increase in ZP− oocytes. Finally, we performed IVF using ZP− oocytes divided into control (3 h) and prolonged gamete co-incubation (5 h) groups. Greater numbers of sperm penetrated in the 5 h group than in the control group. These results suggest that the ZP and oolemma are not competent factors for prevention of polyspermy in our present porcine IVF system. However, it appears that ZP removal is one of the possibilities for reducing polyspermic penetration in vitro in pigs.

Sperm head binding to epithelium of the oviduct isthmus is not an essential preliminary to mammalian fertilization - review

In endeavouring to understand the nature of sperm-oviduct interactions in mammals, attention was focused on experimental models in which fertilization can occur without a preliminary phase of sperm head binding to the isthmus epithelium. The ovarian endocrine milieu imposed on the oviduct tissues plays an important role in the binding phenomenon, although less so after the time of ovulation. Nonetheless, a sperm suspension introduced into the peritoneal cavity or surgical insemination directly into the oviduct ampulla before ovulation can result in fertilization, as can a surgical model in which the isthmus has been resected and the remaining portions of the duct reanastomosed. Mating or artificial insemination after ovulation in pigs permits rapid sperm transport to the site of fertilization, and the frequency of polyspermic penetration increases with the post-ovulatory age of eggs.Strategies underlying sperm binding were considered, especially in terms of preovulatory sperm storage and suppression of full membranous maturation. These, in turn, raised the problem of how sperm binding in vitro to oviduct cells from prepuberal animals or to cells harvested during the luteal phase of the estrous cycle, or to cells from the ampulla or even the tracheal epithelium, can act to regulate sperm storage and maturation with precision. In an evolutionary perspective, preovulatory binding of diverse populations of cells to the endosalpinx may have developed as a form of fine tuning to assist in sperm selection, to synchronize completion of capacitation with the events of ovulation, and to promote monospermic fertilization by a controlled release of competent gametes.

Application of a microfluidic sperm sorter to the in-vitro fertilization of porcine oocytes reduced the incidence of polyspermic penetration

The objective of this study was to use a microfluidic sperm sorter (MFSS), designed to isolate motile human spermatozoa with laminar flows (no centrifugation), for porcine IVF. Boar spermatozoa were diluted at 1 × 10 8 cells/mL with a diluent containing 20% seminal fluid and flowed with modified TCM-199 (mM199, with 5 mM caffeine) to introduce motile sperm into the exit chamber for IVF. In Experiment 1, after flowing for 5 min, sperm concentration varied significantly among specific sites within the MFSS collecting chamber (range, 0.8 ± 0.5 × 10 4 to 575.0 ± 56.3 × 10 4 cells/mL; mean ± SEM). In Experiment 2, when porcine IVM oocytes were placed at three locations in the MFSS exit chamber (where only motile spermatozoa accumulated) and subsequently cultured in caffeine-free mM199 for 8 h, sperm penetration rate was not significantly different among places (86.1 ± 10.5 to 100%), but the monospermic penetration rate was lower (P < 0.05) in oocytes 3.5 mm from the exit position (12.5 ± 4.8%) than those at 7.5 mm (53.1 ± 6.0%) or further (41.9 ± 2.8%) from the exit. In Experiment 3, the normal fertilization index (ratio of monospermic oocytes to number of oocytes examined) 8 h after insemination was higher (P < 0.05) in the MFSS-IVF system (0.375 ± 0.040) than both standard IVF and transient IVF (0.222 ± 0.028 and 0.189 ± 0.027, respectively, with co-culture for 8 h and for 5 min). Developmental competence of fertilized oocytes (blastocyst formation) was higher (P < 0.05) in the MFSS-IVF system (40.9 ± 2.3%) than in either standard or transient IVF (22.6 ± 1.4 and 33.7 ± 3.5%). In conclusion, brief co-culture of porcine oocytes with spermatozoa gradually accumulated in the MFSS chamber improved the efficiency of producing monospermic fertilized embryos and blastocysts. Furthermore, efficiencies were significantly affected by oocyte location within the chamber.

Advances in Swine In Vitro Embryo Production Technologies

Recent advances in new technologies to produce cloned and genetically modified pigs involve manipulating oocytes and/or embryos in vitro. Although a great deal of progress has been made, the current IVM-IVF systems still result in major problems: a high rate of polyspermy; and a low development rate and low quality of blastocysts for in vitro compared with the in vivo-produced embryos. This study summarizes recent advancements in IVM-IVF-IVC porcine systems. Recent methods to select monospermic embryos are also discussed. Finally, achievements in vitrification and in somatic cell nuclear transfer are discussed.

A modified swim-up method reduces polyspermy during in vitro fertilization of porcine oocytes

The general method of porcine in vitro fertilization (IVF), involving the co-culture of both gametes in a medium drop, is thought to be the main reason for the high incidence of polyspermy. The aim of this study was to reduce the polyspermic fertilization of porcine embryos during IVF by the modified swim-up method, based on general sperm swim-up technique. Within this design, a 70 μm pore sized cell strainer was used to separate the sperm pellet placed at the bottom of a tube from the mature oocytes placed within the upper region. The separation of gametes using this permeable barrier was to ensure that only motile sperm gained access to the oocytes. It was found that the rate of polyspermy was significantly lowered for the sperm preparations from three boar breeds in modified swim-up method when compared with that of the general microdrop method ( p < 0.05). However, the penetration rates were found to be similar in both methods for two boar breeds. The average occurrence of blastocysts with more total cell number was higher in the modified swim-up method, while no significant difference in blastocyst rates between the two IVF methods was observed. The frequency of normal diploid embryos was also significantly higher in the modified swim-up method and polyploidy was more frequently observed in microdrop method ( p < 0.05). Our results demonstrated that the modified swim-up IVF method could reduce polyspermic penetration, and consequently produce better quality and karyotypically normal embryos in porcine IVF.

Bovine Oocyte Quality in Relation to Ultrastructural Characteristics of Zona Pellucida, Polyspermic Penetration and Developmental Competence

In the present study, the effect of bovine oocyte quality related to ultrastructural characteristics of zona pellucida (ZP), polyspermic penetration and embryo developmental competence was evaluated. Cumulus-oocyte complexes were punctioned from 453 ovaries, classified as 1, 2, 3 and 4 according to their morphological aspect, matured for 24 h and then divided into two groups. In group A, oocytes were fixed in 2.5% glutaraldehyde and 0.1 m sodium cacodylate and examined under a scanning electron microscope. Photomicrographs were taken and ZP's pores were evaluated in squares of 6.4-microm width. In group B, oocytes were fertilized in vitro. After 48 h, non-cleaved oocytes were fixed for polyspermy evaluation. On days 7, 9 and 10, embryos were classified as developed (blastocysts and hatched blastocysts). Results showed that quality 1 oocytes revealed a ZP pore diameter of 0.50 +/- 0.07 microm, which was smaller than the observed on oocytes of quality 2 (0.83 +/- 0.10 microm), quality 3 (1.02 +/- 0.22 microm) and quality 4 (1.38 +/- 0.59 microm) (p < or = 0.05). For In Vitro Fertilization (IVF), results showed that embryos originating from oocytes classed as 3 and 4 had lower cleavage rate (68.4% and 43.8%) than those belonging to class 1 and 2 (79.5% and 69.3%) (p < or = 0.05). None oocyte classified as 3 and 4 developed to hatch blastocysts, while for oocytes belonging to quality 1 and 2, these values were, respectively, 15.2% and 12.5%. Concerning polyspermy, oocytes class 1 and 2 had lower polyspermic penetration than those belonging to class 3 and 4 (respectively 4.1%, 4.5%, 11.1% and 9.8%, for class 1, 2, 3 and 4). In conclusion, the present study demonstrated that oocytes with low qualities result in lower developmental competence and with high percentage of polyspermy after IVF, which can be the result of the ZP structure such as the number and the pore's diameter.

In Vitro Fertilization (IVF) in Straws and a Short Gamete Coincubation Time Improves the Efficiency of Porcine IVF

The present study was designed to evaluate three different in vitro fertilization (IVF) systems: a straw-IVF system with 10 min of coincubation, a straw-IVF system with 6-h coincubation and the microdrop-IVF system with 6-h coincubation (the traditional IVF system used routinely in most of IVF laboratories) in an attempt to reduce polyspermic penetration (Experiment 1). When the straw-IVF system was tested in combination with two coincubation times, the use of 10 min of coincubation significantly increased (p < 0.001) the penetration rate and the efficiency of fertilization (67.7 +/- 6.4% vs 31.9 +/- 6.5% and 41.5 +/- 2.5% vs 17.6 +/- 2.5% for 10 min and 6 h, respectively), while there were no significant differences in the incidence of monospermy between both systems (64.3 +/- 5.1% and 67.7 +/- 3.4%, for 10 min and 6 h, respectively). The penetration rate in the 6-h microdrop-IVF system was higher (93.8 +/- 3.6%; p < 0.001) compared with the 10-min straw-IVF system (67.7 +/- 6.4%), however, monospermy was severely reduced (25.0 +/- 4.3% vs 67.7 +/- 3.4%, for the 6-h microdrop-IVF system and 10-min straw-IVF system, respectively). The efficiency of the IVF showed similar values between microdrop and 6-h straw-IVF systems, but efficiency was significantly improved (p < 0.05) when the 10-min straw-IVF system was used. Experiment 2 was designed to compare porcine in vitro embryo production in two IVF systems, the 6-h microdrop-IVF system (1000 sperm per oocyte) and 10-min straw-IVF system (30 000 sperm per oocyte). The blastocyst formation rates tended (p = 0.06) to be higher when the 10-min straw-IVF system was used compared with the 6-h microdrop-IVF system. In addition, the number of total cells per blastocyst increased significantly (p < 0.05) in the 10-min straw-IVF system. These results showed that the 10-min straw-IVF system is an effective way to decrease polyspermic penetration, and improve the efficiency of fertilization and the quality of blastocysts in terms of cell number per embryo.

A MAPK pathway is involved in the control of cortical granule reaction and mitosis during bovine fertilization

In order to understand the mechanism by which mitogen-activated protein kinase (MAPK) regulates fertilization, we examined the effect of the MAPK pathway inhibitor U0126 on polyspermy, cortical granule reaction and mitosis in bovine oocytes during and after fertilization. Oocytes were treated with 30 microM U0126 for 30 min prior to insemination, or from 15 to 27 hr following insemination. Western blotting with antibodies that detect active, phosphorylated MAPK revealed that MAPK activity was decreased in U0126 treated oocytes. Oocytes that were treated with U0126 before insemination displayed a significantly higher incidence of polyspermic penetration and incomplete cortical granule reaction than that observed in untreated oocytes (P < 0.05). Exposure of oocytes to 30microM U0126 15-27 hr after insemination induced aberrant microtubule assembly and cell division, often resulting in the formation of two or three daughter cells with altered shapes and sizes. These results suggest that an ERK-like cascade is part of a mechanism that controls cortical granule reaction and the formation of the mitotic spindle following sperm penetration in the bovine.

244 AMPHIREGULIN CAN ASSIST IMMATURE PORCINE OOCYTES TO DEVELOP IN AN IN VITRO SYSTEM

Porcine IVM-IVF technique has been used and improved. However, polyspermic penetration, low rate of blastocyst (BL) formation, and poor quality of BLs are induced by imperfect nuclear and cytoplasmic maturation. It has been reported that epidermal growth factor (EGF) is beneficial for oocyte maturation to improve the IVM system, and amphiregulin (AR) is a growth factor containing an EGF-like domain. Consequently, the present study was performed to investigate whether TCM199 supplemented with EGF and/or AR is profitable for improving oocyte maturation and embryo development. The experimental groups were control (A), EGF 15 ng mL–1 (B), AR 15 ng mL–1 (C), and EGF 15 ng mL + AR 15 ng mL–1 (D) in TCM199. In Experiment 1, oocytes were selected and maturated in each group. After 44 h, oocytes were stained with Hoechst, and metaphase II and development rate were evaluated. In Experiment 2, maturated oocytes were fertilized with proven sperm and stained with Hoechst; the penetration rate after 10 h of post-insemination was evaluated. In Experiment 3, after 168 h of in vitro culture, cleavage rate and BL formation rate were recorded. BLs were stained with differential stain and the cell numbers calculated. In Experiment 4, porcine oocytes were maturated for 44 h and denuded by hyaluronidase. Thereafter, oocytes were stained with fluorescein isothiocyanate-labeled peanut agglutinin and the distribution of cortical granules (CGs) was evaluated by laser confocal microscopy. All data was analyzed by a Duncan test using SPSS (SPSS, Inc., Chicago, IL, USA). In Experiment 1, the maturation rate into metaphase II stage in groups B, C, and D (71.0 � 15.5%, 75.8 � 9.8%, and 71.5 � 10.8%, respectively) was significantly different (P &lt; 0.05) from that of group A (51.4 � 18.3%). In Experiment 2, there was no difference among all groups in relation to penetration rate. But polyspermy was decreased (P &lt; 0.05) in group D (26.2 � 11.3%) compared with group A (42.3 � 13.0%). In Experiment 3, there was no significant difference among all groups regarding the cleavage rate. However, BL developmental rates in group D (17.3 � 11.5%) were increased (P &lt; 0.05) compared with groups A and B (9.6 � 3.2% and 13.3 � 4.6%, respectively). In groups B, C, and D, the cell number in BL increased significantly (P &lt; 0.05) compared with that of group A(81.4 � 32.0%, 79.7 � 27.3%, and 80.3 � 28.5% v. 56.3 � 25.5%). In Experiment 4, group D had the largest CG area among the groups. The results of this study indicate that the addition of EGF or AR or both to TCM199 promotes oocyte maturation and increases the total cell number. EGF plus AR increases development to the BL stage and decreases polyspermy. It is suggested that AR can assist immature porcine oocytes to the metaphase II stage, that AR may enhance developmental ability in the in vitro system, and that there is a synergistic effect between EGF and AR during the maturation period.

215 APPLICATION OFA MICROFLUIDIC SPERM SORTER TO THE IN VITRO FERTILIZATION OF PORCINE OOCYTES

In porcine IVF, polyspermy is a persistent obstacle to the efficient production of normal embryos in vitro. A microfluidic sperm sorter (MFSS; Strex Inc., Osaka, Japan) has been developed to isolate motile human spermatozoa (from diluted semen by 2 laminar flows in the microchannel). The motile spermatozoa can gradually accumulate in a chamber of the MFSS. In addition, we have succeeded in reducing the incidence of polyspermic penetration by a transient coincubation of oocytes with spermatozoa in the presence of caffeine, followed by culture of the oocytes in the absence of caffeine (2004 Reproduction 128, 789–800). If porcine oocytes could be cocultured with motile spermatozoa in the chamber of the MFSS, the gradual accumulation of motile spermatozoa might further improve the efficiency of production of monospermic fertilized porcine embryos. The current study was undertaken to apply the MFSS technology to porcine IVF. The sperm-rich fraction from 3 Berkshire boars was diluted at 1 � 108 cells mL–1 with modified Modena containing 20% seminal fluid, cooled to 15�C for 4 h, and kept at the same temperature until use within 2 days. Stored, diluted semen with greater than 80% viability was flowed with Tyrode lactate-HEPES-polyvinyl alcohol medium for 5 or 10 min at room temperature. The concentration and viability of spermatozoa mixed in the flowed medium were determined. In the next experiment, a diluted semen sample was flowed with modified TCM-199 containing 5 mm caffeine for 5 min at room temperature. Before flowing, porcine IVM oocytes were put into the chamber of the MFSS, where motile spermatozoa will accumulate with modified TCM-199 containing 5 mm caffeine. After flowing for 5 min, those oocytes were transferred into a 500-µL droplet of caffeine-free modified TCM-199, and culture was continued for 8 h. Statistical analyses were carried out by ANOVA and Fisher's PLSD post hoc test. After flowing for 5 min, the concentration of spermatozoa recollected from the chamber was 5.7 � 105 cells mL–1. The viability of recollected spermatozoa was significantly higher than the original flowed semen. However, when spermatozoa were flowed for 10 min, the viability of recollected spermatozoa was not different from the original flowed semen, whereas the concentration of recollected spermatozoa was 9.7 � 105 cells mL–1. When IVM oocytes were cocultured with spermatozoa gradually accumulated in the chamber of MFSS for 5 min, 35% (22 to 48%) of oocytes were penetrated, and 95% (90 to 100%) of the penetrated oocytes were monospermic. These observations demonstrate that the MFSS can separate penetrable boar spermatozoa with a high viability and sufficient concentration for IVF. Furthermore, the current data suggest the possibility of improving the efficiency of monospermic fertilization of porcine IVM oocytes by a transient coculture with MFSS.

305 INCREASED MONOSPERMIC PENETRATION OF PORCINE OOCYTES USING A VISCOUS IN VITRO FERTILIZATION SYSTEM

The high incidence of polyspermy remains a major problem in porcine IVF systems. The high number of sperm bound to the oocyte is one of the major causes for pathological polyploidy (Funahashi 2003 Reprod. Fertil. Dev. 15, 167–177). In this experiment, we tested the hypothesis that limiting the number of competent sperm able to participate in fertilization during IVF will result in a reduction in polyspermic penetration. Combinations of various sperm (3 levels) and Percoll concentrations (3 levels) were used in a modified Tris-buffered medium (mTBM) with 3% polyvinylpyrrolidone supplementation to find the optimal combination that could result in a significant reduction in polyspermy rate compared with that resulting from the current porcine IVF system (Abeydeera and Day 1997 Theriogenology 48, 537–544). In vitro-matured gilt oocytes (n = 844) were randomly allocated into 1 of 10 treatment groups in groups of 30. Motile sperm were selected from frozen–thawed samples by density gradient centrifugation using a discontinuous Percoll gradient consisting of 3 mL of 54% Percoll in PBS on top of 1 mL of 72% Percoll in a 15-mL conical tube at 600g for 15 min. After centrifugation, the supernatant was removed as much as possible without disturbing the sperm pellet. Sperm stock solutions were made in a manner that resulted in the desired final concentration of sperm when 5 µL of the sperm solution were added to 95 µL of fertilization medium. During IVF, the sperm stock solutions were placed at one side of an IVF droplet so that most sperm were trapped inside the viscous Percoll solution and only a limited number of sperm, with high progressive motility, were capable of fertilizing the oocytes. A group of oocytes fertilized using our standard IVF protocol with a sperm concentration of 5 × 105 sperm mL−1 in mTBM served as a control. Pronuclear formation after 12 h of fertilization was used to assess sperm penetration. The data from 3 replicates were analyzed by standard ANOVA procedures using the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). The treatment group of 5 × 105 sperm mL−1 + 10% Percoll resulted in a significantly higher monospermy rate than the control or the treatment group of 1 × 106 sperm mL−1 + 30% Percoll (Table 1; P &amp;lt; 0.05). These data support the hypothesis that the polyspermy rate in a porcine IVF system can be reduced by limiting the number of competent sperm fertilizing the oocytes. Table 1.Monospermy rates in a viscous IVF system (mean ± SEM%) This project was supported by NIH U42 RR 018877.

OC12 Combination of IVF Strategies to Reduce Porcine Polyspermic Fertilization: Straw IVF System and Short Gamete Coincubation Time

This study was conducted to evaluate the combined effect of a short period of gamete coincubation and a new in vitro fertilization (IVF) system in straw (Li et al., 2003; Biol. Reprod. 69: 1580) to attempt to reduce polyspermic penetration. A total of 723 in vitro matured oocytes were inseminated with thawed spermatozoa. Various sperm concentrations (20 000, 30 000, 40 000, and 50 000 sperm/oocyte) and two coincubation times (6 h and 10 min) were used for straw‐IVF and compared to control group (1000 sperm/ovocito, in microdrop). The oocytes from 10 min group were washed in mTBM medium to remove spermatozoa not bound to the zona and transferred to the same medium (containing no sperm) for 6 h. After 6 h, oocytes from each group were cultured in NCSU‐23 medium for 12–15 h to assess fertilization parameters. The sperm concentration did not affect the efficiency of the IVF in both periods of coincubation. The short coincubation time significantly (p &lt; 0.001) increased penetration rate and efficiency of the IVF (67.7 ± 6.4% vs 31.19 ± 6.5% and 41.5 ± 2.5% vs 17.6 ± 2.5% for 10 min and 6 h, respectively), while there were no significant differences in the incidence of monospermy between 10 min (64.3 ± 5.1) and 6 h (67.7 ± 3.4%) coincubation times. The penetration rate in the control group was higher (95.5 ± 5.6%; p &lt; 0.001) than in the straw groups, but monospermy was severely reduced (25.0 ± 4.3%). These results show that short coincubation time together with the straw IVF system improves the efficiency of the porcine IVF. (Supported by SENECA).

The effect of FF-MAS on porcine cumulus–oocyte complex maturation, fertilization and pronucleus formation in vitro

Follicular fluid meiosis-activating sterol (FF-MAS) has been isolated from the follicular fluid (FF) of several species including man. FF-MAS increases the quality of in vitro oocyte maturation, and thus the developmental potential of oocytes exposed to FF-MAS during in vitro maturation is improved. The aim of the present study was to investigate the effects of FF-MAS on porcine oocyte maturation and pronucleus formation in vitro. Porcine cumulus-oocyte complexes (COCs) were isolated from abattoir ovaries and in vitro matured for 48 h in NCSU 37 medium supplemented with 1 mg/l cysteine, 10 ng/ml epidermal growth factor and 50 microM 2-mercaptoethanol with or without 10% porcine follicular fluid (pFF). For the first 22 h, 1 mM db-cAMP and 10 I.E PMSG/hCG was added. The medium was supplemented with 1 microM, 3 microM, 10 microM, 30 microM or 100 microM FF-MAS dissolved in ethanol. After maturation the COCs were denuded mechanically using a fine glass pipette under constant pH and in vitro fertilized with fresh semen (5 x 10(5) spermatozoa/ml). The presumptive zygotes were evaluated 18 h after fertilization. The addition of pFF increased the monospermic as well as the polyspermic penetration of oocytes. In the absence of pFF, the addition of FF-MAS decreased the polyspermic penetration rate, whereas FF-MAS in combination with pFF decreased monospermic and increased polyspermic penetration. The degeneration rate of ova decreased in the presence of FF-MAS irrespective of the presence or absence of pFF. In the absence of pFF, FF-MAS at 3-10 microM increased the number of zygotes with advanced maternal pronuclear stages. In supraphysiological doses, i.e. 30-100 microM, FF-MAS dose-dependently and reversibly inhibited nuclear maturation in the absence of pFF.

Mechanism and Control of Mammalian Cortical Granule Exocytosis

ABSTRACT Polyspermy is penetration of the egg cytoplasm by more than a single spermatozoon, and in humans, polyspermy usually results in spontaneous abortion. Following sperm penetration in mammals, cortical granules (CGs), special organelles in eggs, release their contents into the perivitelline space. The CG exudates act on the zona pellucida, causing biochemical and structural changes that result in zona sperm receptor modification and zona hardening, and thus block polyspermic penetration. Significant advances have been made in elucidating signal molecules and signal transduction cascades that play important roles in the CG exocytosis and subsequent polyspermy block. Ca2+ oscillation is necessary and sufficient for CG exocytosis as well as for other events of egg activation. The Ca2+-dependent pathways and the proteins involved in membrane fusion may play pivotal roles in the regulation of CG exocytosis. Mammalian oocytes develop their ability to undergo CG exocytosis during maturation. This article r...

306 IN VITRO PENETRATION OF SWINE OOCYTES MATURED IN TCM-199 WITH ADDED DIBUTYRYL CYCLIC ADENOSINE MONOPHOSPHATE AND FOLLICULAR FLUID

During the in vitro maturation (IVM) of pig oocytes, a large variation in the nuclear morphology of the germinal vesicle stage is observed. Thus, some oocytes can start meiosis earlier than others. A reversible alternative to inhibit meiotic resumption is the use of dibutyryl cyclic adenosine monophosphate (dbcAMP) in the early period of IVM, which may synchronize the oocytes to a specific germinal vesicle stage and improve early embryonic development. This study investigated the effects of additional dbcAMP and porcine follicular fluid (PFF) on monospermic and polyspermic penetration rates after IVF. Oocytes from prepuberal females were selected under a stereomicroscope, and those with uniform ooplasm and surrounded by several layers of compact cumulus cells were divided into 2 groups: T1 (control) group: TCM-199 supplemented with polyvinyl alcohol (0.1%), FSH (0.5 �g mL-1), LH (0.5 �g mL-1), epidermal growth factor (10 ng mL-1), pyruvate (0.9 mM), d-glucose (3.05 mM), cysteine (0.1 mg mL-1), and gentamycin (50 �g mL-1); and T2 group: T1 with the addition of 25% PFF and 1 mM dbcAMP. Both the T1 and T2 groups were IVM for 42 to 46 h, with FSH, LH, and dbcAMP used only in the first 22 h. At the end of the maturation period, cumulus cells were chemically removed; the oocytes were washed 3 times in IVF medium (modified Tyrode&amp;apos;s buffered medium, mTBM) and placed in petri dishes containing 50 &amp;micro;L of the same medium. The sperm-rich fraction was collected from 2 boars by digital pressure with a gloved hand, extended in Beltsville thawing solution, and incubated for 24 h at 17&amp;deg;C. It was then centrifuged at 1200g for 3 min and standardized for 1 &amp;times; 105 spermatozoa mL&amp;minus;1. Oocytes were co-incubated with the sperm for 6 h in mTBM at 38.5&amp;deg;C and 5&amp;percnt; CO2. After insemination, oocytes were cultured in porcine zygote medium-3 (80 &amp;micro;L), covered with paraffin oil, for 18 h. The presumptive zygotes were fixed and stained with 1&amp;percnt; orcein in 45&amp;percnt; acetic acid and evaluated under phase-contrast microscopy at a 400&amp;times; magnification. Differences among groups were determined by one-way ANOVA. In the T1 group, the penetration rate was 39.3 &amp;plusmn; 9.6&amp;percnt; (162/412), and no difference was observed in comparison with the T2 group, 29.5 &amp;plusmn; 4.9&amp;percnt; (113/383). The monospermic penetration rate was 31.5 &amp;plusmn; 6&amp;percnt; (51/162) in the T1 group and differed from that in the T2 group, 71.7 &amp;plusmn; 3.3&amp;percnt; (81/113). Moreover, the polyspermic penetration was significantly higher in the T1 group, 68.5 &amp;plusmn; 6&amp;percnt; (111/162), compared with the T2 group, 28.3 &amp;plusmn; 3.3&amp;percnt; (32/113). These data suggest that the IVM with TCM-199 with added dbcAMP &amp;plus; PFF can improve in vitro production of swine embryos and decrease polyspermic penetration.