In vitro fertilization (IVF) in straws and a short gamete coincubation time improves the efficiency of porcine IVF.
ABSTRACT 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.
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ABSTRACT: 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 x 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 x 10(4) to 575.0 +/- 56.3 x 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.Theriogenology 09/2010; 74(5):863-70. · 2.08 Impact Factor
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ABSTRACT: The in vitro production of porcine embryos has presented numerous challenges to researchers over the past four decades. Some of the problems encountered were specific to porcine gametes and embryos and needed the concerted efforts of many to overcome. Gradually, porcine embryo in vitro production systems became more reliable and acceptable rates of blastocyst formation were achieved. Despite the significant improvements, the problem of polyspermic fertilization has still not been adequately resolved and the embryo in vitro culture conditions are still considered to be suboptimal. Whereas early studies focused on increasing our understanding of the reproductive processes involved, the technology evolved to the point where in vitro-matured oocytes and in vitro-produced embryos could be used as research material for developing associated reproductive technologies, such as SCNT and embryo cryopreservation. Today, the in vitro procedures used to mature oocytes and culture embryos are integral to the production of transgenic pigs by SCNT. This review discusses the major achievements, advances, and knowledge gained from porcine embryo in vitro production studies and highlights the future research perspectives of this important technology.Theriogenology 01/2014; 81(1):24–37. · 2.08 Impact Factor