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Animal Science Journal (2015) 86, 494–498
doi: 10.1111/asj.12327
ORIGINAL ARTICLE A new rolling culture-based in vitro fertilization system capable of reducing polyspermy in porcine oocytes Hideki KITAJI,1 Shoji OOKUTSU,1 Masahiro SATO2 and Kazuchika MIYOSHI1 1
Laboratory of Animal Reproduction, Faculty of Agriculture and 2Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima, Japan
ABSTRACT 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 × 105 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.
Key words: in vitro fertilization, oocytes, pig, polyspermy, rolling culture.
INTRODUCTION Porcine embryos produced by in vitro maturation (IVM)/ in vitro fertilization (IVF) techniques can successfully develop to the blastocyst stage (Abeydeera & Day 1997a; Abeydeera et al. 1998a,b, 2000). On the other hand, a high incidence (> 50%) of polyspermy remains a major obstacle to the improvement of developmental rates in IVF-derived porcine eggs (Wang et al. 1994, 1998; Abeydeera & Day 1997a). However, such a high incidence of polyspermy is rarely observed in fertilized eggs obtained in vivo (Hunter 1990; Wang et al. 1998). It is believed that the microenvironment of the oviduct prevents polyspermy during in vivo fertilization of ovulated oocytes (Hunter 1990). To date, a few attempts to mimic in vivo conditions to reduce polyspermy during porcine IVF have been reported, but without satisfactory results (Li et al. 2003). The causes for a high incidence of polyspermy in IVF-derived porcine eggs include the use of excessive spermatozoa, and sub-optimal IVM and/or IVF conditions (Hunter 1990; Niwa & Wang 2001; Wang et al. 2003). Reduction in the concentration of spermatozoa used for IVF indeed decreased polyspermy, leading to a decrease in IVF rates (Abeydeera & Day 1997a). Therefore, an optimal number of fully capacitated sperma© 2014 Japanese Society of Animal Science
tozoa at the site of fertilization is extremely important for reducing the incidence of polyspermy in pigs (Li et al. 2003). During in vivo fertilization, motile spermatozoa that are deposited within the cervix canal move toward the site of fertilization, namely the oviduct, where they must pass through the ampullary-isthmic junction, which is the site where active spermatozoa capable of fertilizing the oocyte are selected. Therefore, the oviductal isthmus is considered as the anatomical base of the sperm reservoir that regulates the number of sperm that reach the site of fertilization (Hunter 1991). In this context, the number of spermatozoa required for fertilization is strictly regulated in vivo. In contrast, high concentrations of spermatozoa are used for the insemination of isolated oocytes during IVF. Consequently, the frequency of polyspermy increases, even if oocytes have the ability to block polyspermy.
Correspondence: Masahiro Sato, Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan. (Email: [email protected]) Received 30 March 2014; accepted for publication 7 August 2014.
ROLLING CULTURE IVF IN PIGS
A method capable of mimicking the oviductal microenvironment would be ideal to prevent polyspermy without reducing the overall fertilization rate. The in vivo oviduct is dynamic due to peristaltic movement, like the digestive organs (Croxatto & Ortiz 1975; Rodriguez-Martinez et al. 1982a,b; RodriguezMartinez 1984). Therefore, we proposed that polyspermy can be reduced in a condition where the microtube containing fertilization medium, oocytes and spermatozoa is rotated gently using a rotation mixer. In this study, we examined whether this rolling culture-based method is effective in improving IVF and subsequent in vitro development of porcine eggs.
MATERIALS AND METHODS Oocyte collection and IVM Ovaries were collected from pre-pubertal gilts at a local slaughterhouse and transported to the laboratory within 2 h in 0.9% (w/v) NaCl containing 100 mg/L kanamycin sulfate (Meiji Seika, Tokyo, Japan) at 33–37°C. Cumulus-oocyte complexes (COCs) were aspirated from antral follicles, 2-5 mm in diameter with an 18-gauge needle fixed to a 5 mL disposable syringe (Terumo, Tokyo, Japan). They were then washed twice with Hepes-buffered Tyrode-lactate-pyruvate (TLP) medium supplemented with polyvinyl alcohol (PVA) (Hepes-TLP-PVA) (Bavister & Yanagimachi 1977). The COCs consisting of more than three layers of compact cumulus cells encasing oocytes with evenly granulated ooplasm were selected for IVM. The selected COCs were then washed twice in maturation medium that had been equilibrated for a minimum of 3 h at 38.5°C in an atmosphere of 5% CO2 in air. A total of 40 COCs were transferred into 200 μL maturation medium covered with paraffin oil (Nacalai Tesque, Kyoto, Japan) in a 30 mm dish (#1008; Becton Dickinson & Co., Franklin Lakes, NJ, USA), which was previously equilibrated at 38.5°C in an atmosphere of 5% CO2 in air, and subsequently cultured for 42 h to induce IVM. The maturation medium comprised of TCM-199 with Earle’s salts (Gibco BRL, Grand Island, NY, USA), supplemented with 3.05 mmol/L D-glucose, 0.91 mmol/L sodium pyruvate, 0.57 mmol/L cysteine, 24.71 mmol/L NaHCO3, 10 ng/mL epidermal growth factor (EGF; Sigma-Aldrich Chemical, St. Louis, MO, USA), 10 IU/mL equine chorionic gonadotropin (eCG; Teikoku-Zoki, Tokyo, Japan), 10 IU/mL human chorionic gonadotropin (hCG; Teikoku-Zoki), 0.1 mg/mL amikacin sulfate (Meiji Seika), 0.1% (w/v) PVA (Abeydeera et al. 1998b) and 10% (v/v) pig follicular fluid (pFF). The pFF was prepared by aspirating follicular fluid from antral follicles that were 3-7 mm in diameter from prepubertal gilt ovaries and collecting the supernatant after centrifugation (1900 g, 20 min). The supernatant was then filtered and stored at −20°C until further use.
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evaluated in comparison to the quality standard (i.e. total sperm motility > 80%, progressive sperm motility > 60%, morphologically normal spermatozoa > 85% and sperm viability > 85%) according to the criteria proposed by Yeste et al. (2013). Semen samples were then washed thrice with 0.9% (w/v) NaCl containing 10 mg/L bovine serum albumin (BSA) (Nacalai Tesque) and 100 mg/L kanamycin sulfate. These washed spermatozoa were subsequently diluted to 2 × 107 sperm/mL in the IVF medium without caffeine and were used for IVF. The IVF medium comprised of modified TLP (mTLP) medium (Parrish et al. 1988) supplemented with 0.27 mmol/L CaCl2.2H2O, 5.07 mmol/L NaHCO3, 0.1 mmol/L sodium pyruvate, 2.0 mmol/L caffeine (Sigma-Aldrich Chemical) and 0.3% (w/v) BSA (Sigma-Aldrich Chemical).
IVF of oocytes After induction of IVM, cumulus cells were partially removed from oocytes by treatment with 0.1% (w/v) hyaluronidase (Sigma-Aldrich Chemical) and pipetting. These isolated oocytes (50 to 55) were washed four times in IVF medium pre-equilibrated with 5% CO2 in air at 38.5°C and transferred to the pre-equilibrated IVF medium (180 μL) in a 0.2 mL thin-wall PCR tube (Watson, Kobe, Japan). The spermatozoa (20 μL) were then added into the IVF medium containing oocytes to a final concentration of 2 × 105 cells/ mL. The PCR tube was then tightly sealed with parafilm to prevent evaporation and loss of CO2. The PCR tube containing the oocytes/spermatozoa mixture was set in a rotation mixer (IWAKI, Chiba, Japan; Fig. 1) placed in an incubator (ICV-300; AS ONE, Osaka, Japan) and rotated at 1 rpm at 38.5°C for 6 h. Simultaneously, a second PCR tube was cultured for 6 h at 38.5°C without rotation as a control. During 6 h of IVF, CO2 was not provided within the incubator chamber.
Embryo culture After culturing the 0.2 mL thin-wall PCR tubes for 6 h postinsemination, oocytes were transferred to a drop of IVF medium (50 μL) on a 30 mm dish (#1008; Becton Dickinson
Preparation of spermatozoa The sperm-rich fractions of ejaculates were obtained from Clawn miniature pigs (Japan Farm, Kagoshima, Japan) using the gloved-hand method. Immediately after collection, the total volume of the sperm-rich fraction was diluted 1:1 (v:v) in Mulberry III (VMD, Hoge Mauw, Belgium). These diluted sperm-rich fractions were then stored at 15°C overnight, prior to use for IVF. The quality of the semen samples was Animal Science Journal (2015) 86, 494–498
Figure 1 A new IVF system based on rotation of a small tube containing IVF medium, porcine spermatozoa and porcine oocytes. The rotation mixer was placed within an incubator at 38.5°C and rotated at 1 rpm.
© 2014 Japanese Society of Animal Science
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& Co.). The residual spermatozoa attached to the zona pellucida (ZP) of oocytes were then removed by pipetting in pre-equilibrated IVF medium, and the denuded oocytes were washed three times with mPZM-3 (Yoshioka et al. 2002). They were then transferred to a drop (50 μL) of mPZM-3 covered with paraffin oil (Nacalai Tesque) on a 30 mm dish (#1008; Becton Dickinson & Co.) and cultured at 38.5°C in an atmosphere of 5% CO2, 5% O2 and 90% N2 in air for 7 days to assess the fertilization rate and embryonic development.
Assessment for sperm penetration and embryonic development After 12 h post-insemination, an aliquot of putative zygotes was mounted on slides, fixed for 48-72 h in 25% (v/v) acetic acid in ethanol at room temperature, stained with 1% (w/v) orcein in 45% (v/v) acetic acid, and examined for polar body extrusion and pronuclear formation under a Nomarski differential interference microscope (Olympus, Tokyo, Japan). Embryos can only be considered polyspermic when they have more than one male pronucleus or decondensed sperm head, with corresponding sperm tails. If an embryo had both male and female pronuclei (one each) and both the first and second polar bodies, it was considered an oocyte showing monospermic fertilization. The rest of the putative zygotes were cultivated in a dish, as mentioned previously, and the cleavage rate of fertilized eggs to develop to two-cell stage and blastocyst formation rate at 2 and 7 days of culture, respectively, were assessed. Fragmented embryos were not considered in the present study. At the end of the culture period, nuclei within blastocyststage embryos were counted after staining with Hoechst 33342 (Sigma-Aldrich Chemicals), a nuclear staining dye. The blastocysts were then briefly placed on slides with a drop of mounting medium consisting of glycerol and PBS (9:1) containing 0.1 mg/mL Hoechst 33342. A cover slip was
placed over the drop and the edge was sealed with nail polish. The number of fluorescent nuclei was counted under ultraviolet light. Embryos having more than 32 nuclei were defined as normal blastocysts.
Statistical analysis An arcsine transformation was applied to all percentage data in each replicate. The transformed values and numbers of cells in blastocysts were analyzed by one-way analysis of variance (ANOVA) followed by Fisher’s Protected Least Significant Difference test. A probability of P < 0.05 was considered statistically significant.
RESULTS AND DISCUSSION We first evaluated sperm penetration rate, 12 h postinsemination by staining oocytes with 1% (w/v) orcein. As shown in Table 1, sperm penetration rate was similar in oocytes from the experimental and control groups (75.2% vs. 83.1%). However, the incidence of polyspermy was significantly reduced in the experimental group compared with the control group (P < 0.05; 21.0% and 48.3%, respectively). The number of oocytes showing monospermic fertilization, identified by the presence of two pronuclei in the cytoplasm, was significantly higher in the experimental group than in the control group (P < 0.05; 44.8% vs. 21.2%). We next evaluated the ability of fertilized eggs to develop to the two-cell stage (cleavage rate) after 2 days in culture. As shown in Table 2, there were no significant differences between the two groups (50.5% vs. 52.7%). The blastocyst formation rate, assessed
Table 1 Results of IVF in static (Control) and rotating (Experiment) systems
IVF
No. of oocytes Treatment†
% (no.) of oocytes Tested penetrated‡
% (no.) of oocytes§ Polyspermy
% (no.) of oocytes¶ Monospermic
Experiment Control
105 118
75.2 (79) 83.1 (98)
21.0 (22)a 48.3 (57)b
44.8 (47)a 21.2 (25)b
†IVF was performed using a rolling culture method with a PCR tube and rotation mixer (Experimental group), as described in Materials and Methods. Simultaneously, a second PCR tube containing oocytes/spermatozoa was cultured at 38.5°C (Control group). Experiments were performed a total of eight times. ‡Oocytes having two or more nuclei/swollen heads/sperm tails in their cytoplasm and plasma membrane were designated as penetrated oocytes. §Oocytes with more than one male pronuclei or sperm heads in their cytoplasm and plasma membrane were designated as polyspermic oocytes. ¶Oocytes with both male and female pronuclei (one each) were designated as monospermic oocytes. ab Values with different superscripts within the same column are significantly different (P < 0.05).
Table 2 In vitro development of porcine oocytes after IVF in a static (Control) and rotating (Experiment) IVF systems
IVF
No. of oocytes
% (no.) of oocytes
% (no.) of
Mean no. of
Treatment† Experiment Control
cultured‡ 283 283
cleaved§ 50.5 (143) 52.7 (149)
blastocysts¶ 30.1 (43) 20.8 (31)
cells in blastocysts 54.5 ± 5.4 60.3 ± 5.6
†Treatment is the same as described in Table 1. Experiments were performed a total of eight times. ‡Oocytes penetrated with spermatozoa, as described in Table 1, were cultured up to the blastocyst stage. §Percentage of oocytes developed to the two-cell stage. ¶Percentage of oocytes developed to the blastocyst stage.
© 2014 Japanese Society of Animal Science
Animal Science Journal (2015) 86, 494–498
ROLLING CULTURE IVF IN PIGS
after 7 days in culture, appeared to be higher in the experimental group than in the control group (30.1% vs. 20.8%), although the difference was not statistically significant (P < 0.1). The present study indicated that several obstacles (including a high incidence of polyspermy and low fertilization rate) associated with IVF can be overcome if porcine oocytes are inseminated under constant rotation using a rotation mixer with microtubes used for PCR analysis. Previously, several attempts have been made to improve success rates of IVF in pigs by using a co-culture of spermatozoa with oviductal cells (Nagai & Moor 1990) and follicle cells (Wang et al. 1992). Addition of oviductal fluid (Kim et al. 1996) or follicular fluid (Funahashi & Day 1993) to medium (0.1 ∼10% and 10%, respectively) has also been used as IVF medium. Furthermore, addition of adenosine, fertilization promoting peptide (FPP; pGlu-GluProNH2), and other substances to the IVF medium can reduce the incidence of polyspermic penetration (Funahashi et al. 2000). All of these experiments were designed to mimic in vivo conditions of the ovary and oviducts. As a result, in some trials, the rate of polyspermy was reduced to some extent. However, these attempts were often laborious due to the need to prepare the oviductal or follicular fluids/ cells. Furthermore, the resulting data was sometimes variable, probably due to the differences in the preparation procedures or characteristics of individual animals used. In contrast, our rolling culturebased IVF system does not include such cells/fluids, and only requires a PCR tube, rotation mixer and synthetic IVF medium, which is very simple and convenient. Interestingly, Gil et al. (2003) demonstrated that IVF-medium volume and oocyte numbers are important factors affecting the efficiency of IVF and subsequent development in pigs. This group reported that a low volume (100 μL) of IVF medium, 30-50 oocytes and 2000 spermatozoa/oocyte was optimal. These results led us to believe that as IVF procedures are generally performed in a static cultural condition, the resultant proportion of dead spermatozoa could generate metabolites that may affect the IVF success rate. Probably, such materials may be localized, that is in an area near the ZP into which spermatozoa penetrate. If the IVF medium containing oocytes/ spermatozoa are mixed by rotation, not only would the number of spermatozoa attached to oocytes be reduced (thus preventing polyspermy), but the concentration of toxic material released by dead spermatozoa that accumulate near the ZP would also be reduced. In this study, we observed the effective prevention of polyspermy and increased monospermy rate using a rolling system. However, the cleavage rate of the IVFAnimal Science Journal (2015) 86, 494–498
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derived eggs did not differ between the ‘rolling’ and static groups. We defined normally fertilized eggs as those with two pronuclei in their cytoplasm. However, the number of fertilized eggs derived from rolling culture, which developed to the two-cell stage, was not as high as expected. The reason for this discrepancy is unclear. It is possible that a few of the fertilized eggs derived from the rolling culture system had abnormal embryos which were unable to develop further. Further research is required to overcome this discrepancy in the future. Notably, as shown in Table 2, the cleavage rate of fertilized eggs and blastocyst formation rate were not significantly affected. Thus, our rolling-based IVF system appears to be effective at the level of fertilization, through prevention of polyspermy. In the present IVF system, the number of spermatozoa used for IVF was physically limited by the size of the tube used, namely a 0.2 mL PCR tube. An increase in the number of spermatozoa employed increases the chances of motile spermatozoa binding to the ZP, although there is a risk for polyspermy. Fortunately, our rolling-based system can prevent polyspermy. Thus, a larger tube (e.g. a 0.6 or 1.5 mL microtube) could improve IVF rate in porcine oocytes, as this would accommodate a greater amount of spermatozoa than the 0.2 mL tube used in this study. In fact, in a preliminary test, fertilization in a 1.5 mL microcentrifuge tube resulted in increased blastocyst formation rate (> 30%) (unpublished data). Interestingly, there are several reports describing that polyspermic porcine oocytes (especially dispermic ones) can cleave and develop to the blastocysts in vivo and in vitro (Han et al. 1999a, b; Funahashi 2003; Somfai et al. 2008). The morphology of blastocysts derived from polyspermic oocytes did not differ from that of those derived from monospermic oocytes. Han et al. (1999a) demonstrated that the inner cell mass : trophectoderm ((ICM:TE) ratio in the blastocysts derived from polyspermic oocytes was smaller than that in blastocysts derived from monospermic oocytes when blastocysts were subjected to an immunosurgery assay after nuclear staining. Unfortunately, we did not explore the ICM:TE ratio using blastocysts obtained by the present rolling-based IVF system. This is thus one of our major subjects to be tested in the future. In conclusion, we developed a new IVF system based on rotation of a small tube that contained IVF medium, spermatozoa and oocytes. Using this system, we achieved an increased monospermic fertilization rate and a reduced polyspermic fertilization rate. Our present rolling-based IVF system is based on IVF experiments using fresh ejaculated sperm. It would be of interest to test whether IVF can be performed with frozen-thawed sperm or epididymal sperm using this rolling-based IVF system. © 2014 Japanese Society of Animal Science
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ACKNOWLEDGMENTS The authors express their gratitude to the staff at the Kagoshima Meat Inspection Office and Meat Center Kagoshima (Kagoshima, Japan) for supplying pig ovaries.
REFERENCES Abeydeera LR, Day BN. 1997a. Fertilization and subsequent development in vitro of pig oocytes inseminated in a modified Tris-buffered medium with frozen-thawed ejaculated spermatozoa. Biology of Reproduction 57, 729–734. Abeydeera LR, Day BN. 1997b. In vitro penetration of pig oocytes in a modified Tris-buffered medium: effect of BSA, caffeine and calcium. Theriogenology 48, 537–544. Abeydeera LR, Wang WH, Cantley TC, Rieke A, Murphy CA, Prather RS, Day BN. 2000. Development and viability of pig oocytes matured in a protein-free medium containing epidermal growth factor. Theriogenology 54, 787– 797. Abeydeera LR, Wang WH, Cantley TC, Rieke A, Prather RS, Day BN. 1998a. Presence of epidermal growth factor during in vitro maturation of pig oocytes and embryo culture can modulate blastocyst development after in vitro fertilization. Molecular Reproduction and Development 51, 395–401. Abeydeera LR, Wang WH, Prather RS, Day BN. 1998b. Maturation in vitro of pig oocytes in protein-free culture media: fertilization and subsequent embryo development in vitro. Biology of Reproduction 58, 1316–1320. Bavister BD, Yanagimachi R. 1977. The effects of sperm extracts and energy sources on the motility and acrosome reaction of hamster spermatozoa in vitro. Biology of Reproduction 16, 228–237. Croxatto HB, Ortiz ME. 1975. Egg transport in the fallopian tube. Gynecologic Investigation 6, 215–225. Funahashi H. 2003. Polyspermic penetration in porcine IVMIVF systems. Reproduction, Fertility and Development 15, 167–177. Funahashi H, Day BN. 1993. Effects of follicular fluid at fertilization in vitro on sperm penetration in pig oocytes. Journal of Reproduction and Fertility 99, 97–103. Funahashi H, Fujiwara T, Nagai T. 2000. Modulation of the function of boar spermatozoa via adenosine and fertilization promoting peptide receptors reduces the incidence of polyspermic penetration into porcine oocytes. Biology of Reproduction 63, 1157–1163. Gil MA, Abeydeera LR, Day BN, Vazquez JM, Roca J, Martinez EA. 2003. Effect of the volume of the medium and number of oocytes during in vitro fertilization on embryo development in pigs. Theriogenology 60, 767– 776. Han YM, Abeydeera LR, Kim JH, Moon HB, Cabot RA, Day BN, Prather RS. 1999a. Growth retardation of inner cell mass cells in polyspermic porcine embryos produced in vitro. Biology of Reproduction 60, 1110–1113. Han YM, Wang WH, Abeydeera LR, Petersen AL, Kim JH, Murphy C, et al. 1999b. Pronuclear location before the first cell division determines ploidy of polyspermic pig embryos. Biology of Reproduction 61, 1340–1346.
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Hunter RH. 1991. Oviduct function in pigs with particular reference to the pathological condition of polyspermy. Molecular Reproduction and Development 29, 385–391. Hunter RHF. 1990. Fertilization of pig eggs in vitro and in vivo. Journal of Reproduction and Fertility 40 (Suppl), 211–226. Kim NH, Funahashi H, Abeydeera LR, Moon SJ, Prather RS, Day BN. 1996. Effects of oviductal fluid on sperm penetration and cortical granule exocytosis during fertilization of pig oocytes in vitro. Journal of Reproduction and Fertility 107, 79–86. Li YH, Ma W, Li M, Hou Y, Jiao LH, Wang WH. 2003. Reduced polyspermic penetration in porcine oocytes inseminated in a new in vitro fertilization (IVF) system: straw IVF. Biology of Reproduction 69, 1580–1585. Nagai T, Moor RM. 1990. Effect of oviduct cells on the incidence of polyspermy in pig eggs fertilized in vitro. Molecular Reproduction and Development 26, 377–381. Niwa K, Wang WH. 2001. Cortical granules and cortical reaction in pig oocytes. In: Miyamoto H, Manabe N (eds), Reproductive Biotechnology, pp. 27–34. Hokuto Shobo, Kyoto, Japan. Parrish JJ, Parrish JS, Winer MA, First NL. 1988. Capacitation of bovine sperm by heparin. Biology of Reproduction 38, 1171–1180. Rodriguez-Martinez H. 1984. Effects of adrenergic agents on the in vitro motility of porcine oviducts. Zentralblatt für Veterinärmedizin. Reihe A 31, 91–104. Rodriguez-Martinez H, Einarsson S, Larsson B. 1982a. Spontaneous motility of the oviduct in the anaesthetized pig. Journal of Reproduction and Fertility 66, 615–624. Rodriguez-Martinez H, Einarsson S, Larsson B, Akusu M, Settergren I. 1982b. Spontaneous motility of the pig oviduct in vitro. Biology of Reproduction 26, 98–104. Somfai T, Ozawa M, Noguchi J, Kaneko H, Karja NWK, Fahrudin M, et al. 2008. In vitro development of polyspermic porcine oocytes: relationship between early fragmentation and excessive number of penetrating spermatozoa. Animal Reproduction Science 107, 131–147. Wang WH, Abeydeera LR, Okuda K, Niwa K. 1994. Penetration of porcine oocytes during maturation in vitro by cryopreserved, ejaculated spermatozoa. Biology of Reproduction 50, 510–515. Wang WH, Abeydeera LR, Prather RS, Day BN. 1998. Morphologic comparison of ovulated and in vitro-matured porcine oocytes, with particular reference to polyspermy after in vitro fertilization. Molecular Reproduction and Development 49, 308–316. Wang WH, Day BN, Wu GM. 2003. How does polyspermy happen in mammalian oocytes. Microscopy Research and Technique 61, 335–341. Wang WH, Uchida M, Niwa K. 1992. Effects of follicle cells on in vitro penetration of pig oocytes by cryopreserved, ejaculated spermatozoa. Journal of Reproduction and Development 38, 125–131. Yeste M, Estrada E, Casas I, Bonet S, Gil JER. 2013. Good and bad freezability boar ejaculates differ in the integrity of nucleoprotein structure after freeze-thawing but not in ROS levels. Theriogenology 79, 929–939. Yoshioka K, Suzuki C, Tanaka A, Anas IMK, Iwamura S. 2002. Birth of piglets derived from porcine zygotes cultured in a chemically defined medium. Biology of Reproduction 66, 112–119.
Animal Science Journal (2015) 86, 494–498
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Animal Science Journal (2015) 86, 494–498
doi: 10.1111/asj.12327
ORIGINAL ARTICLE A new rolling culture-based in vitro fertilization system capable of reducing polyspermy in porcine oocytes Hideki KITAJI,1 Shoji OOKUTSU,1 Masahiro SATO2 and Kazuchika MIYOSHI1 1
Laboratory of Animal Reproduction, Faculty of Agriculture and 2Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima, Japan
ABSTRACT 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 × 105 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.
Key words: in vitro fertilization, oocytes, pig, polyspermy, rolling culture.
INTRODUCTION Porcine embryos produced by in vitro maturation (IVM)/ in vitro fertilization (IVF) techniques can successfully develop to the blastocyst stage (Abeydeera & Day 1997a; Abeydeera et al. 1998a,b, 2000). On the other hand, a high incidence (> 50%) of polyspermy remains a major obstacle to the improvement of developmental rates in IVF-derived porcine eggs (Wang et al. 1994, 1998; Abeydeera & Day 1997a). However, such a high incidence of polyspermy is rarely observed in fertilized eggs obtained in vivo (Hunter 1990; Wang et al. 1998). It is believed that the microenvironment of the oviduct prevents polyspermy during in vivo fertilization of ovulated oocytes (Hunter 1990). To date, a few attempts to mimic in vivo conditions to reduce polyspermy during porcine IVF have been reported, but without satisfactory results (Li et al. 2003). The causes for a high incidence of polyspermy in IVF-derived porcine eggs include the use of excessive spermatozoa, and sub-optimal IVM and/or IVF conditions (Hunter 1990; Niwa & Wang 2001; Wang et al. 2003). Reduction in the concentration of spermatozoa used for IVF indeed decreased polyspermy, leading to a decrease in IVF rates (Abeydeera & Day 1997a). Therefore, an optimal number of fully capacitated sperma© 2014 Japanese Society of Animal Science
tozoa at the site of fertilization is extremely important for reducing the incidence of polyspermy in pigs (Li et al. 2003). During in vivo fertilization, motile spermatozoa that are deposited within the cervix canal move toward the site of fertilization, namely the oviduct, where they must pass through the ampullary-isthmic junction, which is the site where active spermatozoa capable of fertilizing the oocyte are selected. Therefore, the oviductal isthmus is considered as the anatomical base of the sperm reservoir that regulates the number of sperm that reach the site of fertilization (Hunter 1991). In this context, the number of spermatozoa required for fertilization is strictly regulated in vivo. In contrast, high concentrations of spermatozoa are used for the insemination of isolated oocytes during IVF. Consequently, the frequency of polyspermy increases, even if oocytes have the ability to block polyspermy.
Correspondence: Masahiro Sato, Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan. (Email: [email protected]) Received 30 March 2014; accepted for publication 7 August 2014.
ROLLING CULTURE IVF IN PIGS
A method capable of mimicking the oviductal microenvironment would be ideal to prevent polyspermy without reducing the overall fertilization rate. The in vivo oviduct is dynamic due to peristaltic movement, like the digestive organs (Croxatto & Ortiz 1975; Rodriguez-Martinez et al. 1982a,b; RodriguezMartinez 1984). Therefore, we proposed that polyspermy can be reduced in a condition where the microtube containing fertilization medium, oocytes and spermatozoa is rotated gently using a rotation mixer. In this study, we examined whether this rolling culture-based method is effective in improving IVF and subsequent in vitro development of porcine eggs.
MATERIALS AND METHODS Oocyte collection and IVM Ovaries were collected from pre-pubertal gilts at a local slaughterhouse and transported to the laboratory within 2 h in 0.9% (w/v) NaCl containing 100 mg/L kanamycin sulfate (Meiji Seika, Tokyo, Japan) at 33–37°C. Cumulus-oocyte complexes (COCs) were aspirated from antral follicles, 2-5 mm in diameter with an 18-gauge needle fixed to a 5 mL disposable syringe (Terumo, Tokyo, Japan). They were then washed twice with Hepes-buffered Tyrode-lactate-pyruvate (TLP) medium supplemented with polyvinyl alcohol (PVA) (Hepes-TLP-PVA) (Bavister & Yanagimachi 1977). The COCs consisting of more than three layers of compact cumulus cells encasing oocytes with evenly granulated ooplasm were selected for IVM. The selected COCs were then washed twice in maturation medium that had been equilibrated for a minimum of 3 h at 38.5°C in an atmosphere of 5% CO2 in air. A total of 40 COCs were transferred into 200 μL maturation medium covered with paraffin oil (Nacalai Tesque, Kyoto, Japan) in a 30 mm dish (#1008; Becton Dickinson & Co., Franklin Lakes, NJ, USA), which was previously equilibrated at 38.5°C in an atmosphere of 5% CO2 in air, and subsequently cultured for 42 h to induce IVM. The maturation medium comprised of TCM-199 with Earle’s salts (Gibco BRL, Grand Island, NY, USA), supplemented with 3.05 mmol/L D-glucose, 0.91 mmol/L sodium pyruvate, 0.57 mmol/L cysteine, 24.71 mmol/L NaHCO3, 10 ng/mL epidermal growth factor (EGF; Sigma-Aldrich Chemical, St. Louis, MO, USA), 10 IU/mL equine chorionic gonadotropin (eCG; Teikoku-Zoki, Tokyo, Japan), 10 IU/mL human chorionic gonadotropin (hCG; Teikoku-Zoki), 0.1 mg/mL amikacin sulfate (Meiji Seika), 0.1% (w/v) PVA (Abeydeera et al. 1998b) and 10% (v/v) pig follicular fluid (pFF). The pFF was prepared by aspirating follicular fluid from antral follicles that were 3-7 mm in diameter from prepubertal gilt ovaries and collecting the supernatant after centrifugation (1900 g, 20 min). The supernatant was then filtered and stored at −20°C until further use.
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evaluated in comparison to the quality standard (i.e. total sperm motility > 80%, progressive sperm motility > 60%, morphologically normal spermatozoa > 85% and sperm viability > 85%) according to the criteria proposed by Yeste et al. (2013). Semen samples were then washed thrice with 0.9% (w/v) NaCl containing 10 mg/L bovine serum albumin (BSA) (Nacalai Tesque) and 100 mg/L kanamycin sulfate. These washed spermatozoa were subsequently diluted to 2 × 107 sperm/mL in the IVF medium without caffeine and were used for IVF. The IVF medium comprised of modified TLP (mTLP) medium (Parrish et al. 1988) supplemented with 0.27 mmol/L CaCl2.2H2O, 5.07 mmol/L NaHCO3, 0.1 mmol/L sodium pyruvate, 2.0 mmol/L caffeine (Sigma-Aldrich Chemical) and 0.3% (w/v) BSA (Sigma-Aldrich Chemical).
IVF of oocytes After induction of IVM, cumulus cells were partially removed from oocytes by treatment with 0.1% (w/v) hyaluronidase (Sigma-Aldrich Chemical) and pipetting. These isolated oocytes (50 to 55) were washed four times in IVF medium pre-equilibrated with 5% CO2 in air at 38.5°C and transferred to the pre-equilibrated IVF medium (180 μL) in a 0.2 mL thin-wall PCR tube (Watson, Kobe, Japan). The spermatozoa (20 μL) were then added into the IVF medium containing oocytes to a final concentration of 2 × 105 cells/ mL. The PCR tube was then tightly sealed with parafilm to prevent evaporation and loss of CO2. The PCR tube containing the oocytes/spermatozoa mixture was set in a rotation mixer (IWAKI, Chiba, Japan; Fig. 1) placed in an incubator (ICV-300; AS ONE, Osaka, Japan) and rotated at 1 rpm at 38.5°C for 6 h. Simultaneously, a second PCR tube was cultured for 6 h at 38.5°C without rotation as a control. During 6 h of IVF, CO2 was not provided within the incubator chamber.
Embryo culture After culturing the 0.2 mL thin-wall PCR tubes for 6 h postinsemination, oocytes were transferred to a drop of IVF medium (50 μL) on a 30 mm dish (#1008; Becton Dickinson
Preparation of spermatozoa The sperm-rich fractions of ejaculates were obtained from Clawn miniature pigs (Japan Farm, Kagoshima, Japan) using the gloved-hand method. Immediately after collection, the total volume of the sperm-rich fraction was diluted 1:1 (v:v) in Mulberry III (VMD, Hoge Mauw, Belgium). These diluted sperm-rich fractions were then stored at 15°C overnight, prior to use for IVF. The quality of the semen samples was Animal Science Journal (2015) 86, 494–498
Figure 1 A new IVF system based on rotation of a small tube containing IVF medium, porcine spermatozoa and porcine oocytes. The rotation mixer was placed within an incubator at 38.5°C and rotated at 1 rpm.
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& Co.). The residual spermatozoa attached to the zona pellucida (ZP) of oocytes were then removed by pipetting in pre-equilibrated IVF medium, and the denuded oocytes were washed three times with mPZM-3 (Yoshioka et al. 2002). They were then transferred to a drop (50 μL) of mPZM-3 covered with paraffin oil (Nacalai Tesque) on a 30 mm dish (#1008; Becton Dickinson & Co.) and cultured at 38.5°C in an atmosphere of 5% CO2, 5% O2 and 90% N2 in air for 7 days to assess the fertilization rate and embryonic development.
Assessment for sperm penetration and embryonic development After 12 h post-insemination, an aliquot of putative zygotes was mounted on slides, fixed for 48-72 h in 25% (v/v) acetic acid in ethanol at room temperature, stained with 1% (w/v) orcein in 45% (v/v) acetic acid, and examined for polar body extrusion and pronuclear formation under a Nomarski differential interference microscope (Olympus, Tokyo, Japan). Embryos can only be considered polyspermic when they have more than one male pronucleus or decondensed sperm head, with corresponding sperm tails. If an embryo had both male and female pronuclei (one each) and both the first and second polar bodies, it was considered an oocyte showing monospermic fertilization. The rest of the putative zygotes were cultivated in a dish, as mentioned previously, and the cleavage rate of fertilized eggs to develop to two-cell stage and blastocyst formation rate at 2 and 7 days of culture, respectively, were assessed. Fragmented embryos were not considered in the present study. At the end of the culture period, nuclei within blastocyststage embryos were counted after staining with Hoechst 33342 (Sigma-Aldrich Chemicals), a nuclear staining dye. The blastocysts were then briefly placed on slides with a drop of mounting medium consisting of glycerol and PBS (9:1) containing 0.1 mg/mL Hoechst 33342. A cover slip was
placed over the drop and the edge was sealed with nail polish. The number of fluorescent nuclei was counted under ultraviolet light. Embryos having more than 32 nuclei were defined as normal blastocysts.
Statistical analysis An arcsine transformation was applied to all percentage data in each replicate. The transformed values and numbers of cells in blastocysts were analyzed by one-way analysis of variance (ANOVA) followed by Fisher’s Protected Least Significant Difference test. A probability of P < 0.05 was considered statistically significant.
RESULTS AND DISCUSSION We first evaluated sperm penetration rate, 12 h postinsemination by staining oocytes with 1% (w/v) orcein. As shown in Table 1, sperm penetration rate was similar in oocytes from the experimental and control groups (75.2% vs. 83.1%). However, the incidence of polyspermy was significantly reduced in the experimental group compared with the control group (P < 0.05; 21.0% and 48.3%, respectively). The number of oocytes showing monospermic fertilization, identified by the presence of two pronuclei in the cytoplasm, was significantly higher in the experimental group than in the control group (P < 0.05; 44.8% vs. 21.2%). We next evaluated the ability of fertilized eggs to develop to the two-cell stage (cleavage rate) after 2 days in culture. As shown in Table 2, there were no significant differences between the two groups (50.5% vs. 52.7%). The blastocyst formation rate, assessed
Table 1 Results of IVF in static (Control) and rotating (Experiment) systems
IVF
No. of oocytes Treatment†
% (no.) of oocytes Tested penetrated‡
% (no.) of oocytes§ Polyspermy
% (no.) of oocytes¶ Monospermic
Experiment Control
105 118
75.2 (79) 83.1 (98)
21.0 (22)a 48.3 (57)b
44.8 (47)a 21.2 (25)b
†IVF was performed using a rolling culture method with a PCR tube and rotation mixer (Experimental group), as described in Materials and Methods. Simultaneously, a second PCR tube containing oocytes/spermatozoa was cultured at 38.5°C (Control group). Experiments were performed a total of eight times. ‡Oocytes having two or more nuclei/swollen heads/sperm tails in their cytoplasm and plasma membrane were designated as penetrated oocytes. §Oocytes with more than one male pronuclei or sperm heads in their cytoplasm and plasma membrane were designated as polyspermic oocytes. ¶Oocytes with both male and female pronuclei (one each) were designated as monospermic oocytes. ab Values with different superscripts within the same column are significantly different (P < 0.05).
Table 2 In vitro development of porcine oocytes after IVF in a static (Control) and rotating (Experiment) IVF systems
IVF
No. of oocytes
% (no.) of oocytes
% (no.) of
Mean no. of
Treatment† Experiment Control
cultured‡ 283 283
cleaved§ 50.5 (143) 52.7 (149)
blastocysts¶ 30.1 (43) 20.8 (31)
cells in blastocysts 54.5 ± 5.4 60.3 ± 5.6
†Treatment is the same as described in Table 1. Experiments were performed a total of eight times. ‡Oocytes penetrated with spermatozoa, as described in Table 1, were cultured up to the blastocyst stage. §Percentage of oocytes developed to the two-cell stage. ¶Percentage of oocytes developed to the blastocyst stage.
© 2014 Japanese Society of Animal Science
Animal Science Journal (2015) 86, 494–498
ROLLING CULTURE IVF IN PIGS
after 7 days in culture, appeared to be higher in the experimental group than in the control group (30.1% vs. 20.8%), although the difference was not statistically significant (P < 0.1). The present study indicated that several obstacles (including a high incidence of polyspermy and low fertilization rate) associated with IVF can be overcome if porcine oocytes are inseminated under constant rotation using a rotation mixer with microtubes used for PCR analysis. Previously, several attempts have been made to improve success rates of IVF in pigs by using a co-culture of spermatozoa with oviductal cells (Nagai & Moor 1990) and follicle cells (Wang et al. 1992). Addition of oviductal fluid (Kim et al. 1996) or follicular fluid (Funahashi & Day 1993) to medium (0.1 ∼10% and 10%, respectively) has also been used as IVF medium. Furthermore, addition of adenosine, fertilization promoting peptide (FPP; pGlu-GluProNH2), and other substances to the IVF medium can reduce the incidence of polyspermic penetration (Funahashi et al. 2000). All of these experiments were designed to mimic in vivo conditions of the ovary and oviducts. As a result, in some trials, the rate of polyspermy was reduced to some extent. However, these attempts were often laborious due to the need to prepare the oviductal or follicular fluids/ cells. Furthermore, the resulting data was sometimes variable, probably due to the differences in the preparation procedures or characteristics of individual animals used. In contrast, our rolling culturebased IVF system does not include such cells/fluids, and only requires a PCR tube, rotation mixer and synthetic IVF medium, which is very simple and convenient. Interestingly, Gil et al. (2003) demonstrated that IVF-medium volume and oocyte numbers are important factors affecting the efficiency of IVF and subsequent development in pigs. This group reported that a low volume (100 μL) of IVF medium, 30-50 oocytes and 2000 spermatozoa/oocyte was optimal. These results led us to believe that as IVF procedures are generally performed in a static cultural condition, the resultant proportion of dead spermatozoa could generate metabolites that may affect the IVF success rate. Probably, such materials may be localized, that is in an area near the ZP into which spermatozoa penetrate. If the IVF medium containing oocytes/ spermatozoa are mixed by rotation, not only would the number of spermatozoa attached to oocytes be reduced (thus preventing polyspermy), but the concentration of toxic material released by dead spermatozoa that accumulate near the ZP would also be reduced. In this study, we observed the effective prevention of polyspermy and increased monospermy rate using a rolling system. However, the cleavage rate of the IVFAnimal Science Journal (2015) 86, 494–498
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derived eggs did not differ between the ‘rolling’ and static groups. We defined normally fertilized eggs as those with two pronuclei in their cytoplasm. However, the number of fertilized eggs derived from rolling culture, which developed to the two-cell stage, was not as high as expected. The reason for this discrepancy is unclear. It is possible that a few of the fertilized eggs derived from the rolling culture system had abnormal embryos which were unable to develop further. Further research is required to overcome this discrepancy in the future. Notably, as shown in Table 2, the cleavage rate of fertilized eggs and blastocyst formation rate were not significantly affected. Thus, our rolling-based IVF system appears to be effective at the level of fertilization, through prevention of polyspermy. In the present IVF system, the number of spermatozoa used for IVF was physically limited by the size of the tube used, namely a 0.2 mL PCR tube. An increase in the number of spermatozoa employed increases the chances of motile spermatozoa binding to the ZP, although there is a risk for polyspermy. Fortunately, our rolling-based system can prevent polyspermy. Thus, a larger tube (e.g. a 0.6 or 1.5 mL microtube) could improve IVF rate in porcine oocytes, as this would accommodate a greater amount of spermatozoa than the 0.2 mL tube used in this study. In fact, in a preliminary test, fertilization in a 1.5 mL microcentrifuge tube resulted in increased blastocyst formation rate (> 30%) (unpublished data). Interestingly, there are several reports describing that polyspermic porcine oocytes (especially dispermic ones) can cleave and develop to the blastocysts in vivo and in vitro (Han et al. 1999a, b; Funahashi 2003; Somfai et al. 2008). The morphology of blastocysts derived from polyspermic oocytes did not differ from that of those derived from monospermic oocytes. Han et al. (1999a) demonstrated that the inner cell mass : trophectoderm ((ICM:TE) ratio in the blastocysts derived from polyspermic oocytes was smaller than that in blastocysts derived from monospermic oocytes when blastocysts were subjected to an immunosurgery assay after nuclear staining. Unfortunately, we did not explore the ICM:TE ratio using blastocysts obtained by the present rolling-based IVF system. This is thus one of our major subjects to be tested in the future. In conclusion, we developed a new IVF system based on rotation of a small tube that contained IVF medium, spermatozoa and oocytes. Using this system, we achieved an increased monospermic fertilization rate and a reduced polyspermic fertilization rate. Our present rolling-based IVF system is based on IVF experiments using fresh ejaculated sperm. It would be of interest to test whether IVF can be performed with frozen-thawed sperm or epididymal sperm using this rolling-based IVF system. © 2014 Japanese Society of Animal Science
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ACKNOWLEDGMENTS The authors express their gratitude to the staff at the Kagoshima Meat Inspection Office and Meat Center Kagoshima (Kagoshima, Japan) for supplying pig ovaries.
REFERENCES Abeydeera LR, Day BN. 1997a. Fertilization and subsequent development in vitro of pig oocytes inseminated in a modified Tris-buffered medium with frozen-thawed ejaculated spermatozoa. Biology of Reproduction 57, 729–734. Abeydeera LR, Day BN. 1997b. In vitro penetration of pig oocytes in a modified Tris-buffered medium: effect of BSA, caffeine and calcium. Theriogenology 48, 537–544. Abeydeera LR, Wang WH, Cantley TC, Rieke A, Murphy CA, Prather RS, Day BN. 2000. Development and viability of pig oocytes matured in a protein-free medium containing epidermal growth factor. Theriogenology 54, 787– 797. Abeydeera LR, Wang WH, Cantley TC, Rieke A, Prather RS, Day BN. 1998a. Presence of epidermal growth factor during in vitro maturation of pig oocytes and embryo culture can modulate blastocyst development after in vitro fertilization. Molecular Reproduction and Development 51, 395–401. Abeydeera LR, Wang WH, Prather RS, Day BN. 1998b. Maturation in vitro of pig oocytes in protein-free culture media: fertilization and subsequent embryo development in vitro. Biology of Reproduction 58, 1316–1320. Bavister BD, Yanagimachi R. 1977. The effects of sperm extracts and energy sources on the motility and acrosome reaction of hamster spermatozoa in vitro. Biology of Reproduction 16, 228–237. Croxatto HB, Ortiz ME. 1975. Egg transport in the fallopian tube. Gynecologic Investigation 6, 215–225. Funahashi H. 2003. Polyspermic penetration in porcine IVMIVF systems. Reproduction, Fertility and Development 15, 167–177. Funahashi H, Day BN. 1993. Effects of follicular fluid at fertilization in vitro on sperm penetration in pig oocytes. Journal of Reproduction and Fertility 99, 97–103. Funahashi H, Fujiwara T, Nagai T. 2000. Modulation of the function of boar spermatozoa via adenosine and fertilization promoting peptide receptors reduces the incidence of polyspermic penetration into porcine oocytes. Biology of Reproduction 63, 1157–1163. Gil MA, Abeydeera LR, Day BN, Vazquez JM, Roca J, Martinez EA. 2003. Effect of the volume of the medium and number of oocytes during in vitro fertilization on embryo development in pigs. Theriogenology 60, 767– 776. Han YM, Abeydeera LR, Kim JH, Moon HB, Cabot RA, Day BN, Prather RS. 1999a. Growth retardation of inner cell mass cells in polyspermic porcine embryos produced in vitro. Biology of Reproduction 60, 1110–1113. Han YM, Wang WH, Abeydeera LR, Petersen AL, Kim JH, Murphy C, et al. 1999b. Pronuclear location before the first cell division determines ploidy of polyspermic pig embryos. Biology of Reproduction 61, 1340–1346.
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Hunter RH. 1991. Oviduct function in pigs with particular reference to the pathological condition of polyspermy. Molecular Reproduction and Development 29, 385–391. Hunter RHF. 1990. Fertilization of pig eggs in vitro and in vivo. Journal of Reproduction and Fertility 40 (Suppl), 211–226. Kim NH, Funahashi H, Abeydeera LR, Moon SJ, Prather RS, Day BN. 1996. Effects of oviductal fluid on sperm penetration and cortical granule exocytosis during fertilization of pig oocytes in vitro. Journal of Reproduction and Fertility 107, 79–86. Li YH, Ma W, Li M, Hou Y, Jiao LH, Wang WH. 2003. Reduced polyspermic penetration in porcine oocytes inseminated in a new in vitro fertilization (IVF) system: straw IVF. Biology of Reproduction 69, 1580–1585. Nagai T, Moor RM. 1990. Effect of oviduct cells on the incidence of polyspermy in pig eggs fertilized in vitro. Molecular Reproduction and Development 26, 377–381. Niwa K, Wang WH. 2001. Cortical granules and cortical reaction in pig oocytes. In: Miyamoto H, Manabe N (eds), Reproductive Biotechnology, pp. 27–34. Hokuto Shobo, Kyoto, Japan. Parrish JJ, Parrish JS, Winer MA, First NL. 1988. Capacitation of bovine sperm by heparin. Biology of Reproduction 38, 1171–1180. Rodriguez-Martinez H. 1984. Effects of adrenergic agents on the in vitro motility of porcine oviducts. Zentralblatt für Veterinärmedizin. Reihe A 31, 91–104. Rodriguez-Martinez H, Einarsson S, Larsson B. 1982a. Spontaneous motility of the oviduct in the anaesthetized pig. Journal of Reproduction and Fertility 66, 615–624. Rodriguez-Martinez H, Einarsson S, Larsson B, Akusu M, Settergren I. 1982b. Spontaneous motility of the pig oviduct in vitro. Biology of Reproduction 26, 98–104. Somfai T, Ozawa M, Noguchi J, Kaneko H, Karja NWK, Fahrudin M, et al. 2008. In vitro development of polyspermic porcine oocytes: relationship between early fragmentation and excessive number of penetrating spermatozoa. Animal Reproduction Science 107, 131–147. Wang WH, Abeydeera LR, Okuda K, Niwa K. 1994. Penetration of porcine oocytes during maturation in vitro by cryopreserved, ejaculated spermatozoa. Biology of Reproduction 50, 510–515. Wang WH, Abeydeera LR, Prather RS, Day BN. 1998. Morphologic comparison of ovulated and in vitro-matured porcine oocytes, with particular reference to polyspermy after in vitro fertilization. Molecular Reproduction and Development 49, 308–316. Wang WH, Day BN, Wu GM. 2003. How does polyspermy happen in mammalian oocytes. Microscopy Research and Technique 61, 335–341. Wang WH, Uchida M, Niwa K. 1992. Effects of follicle cells on in vitro penetration of pig oocytes by cryopreserved, ejaculated spermatozoa. Journal of Reproduction and Development 38, 125–131. Yeste M, Estrada E, Casas I, Bonet S, Gil JER. 2013. Good and bad freezability boar ejaculates differ in the integrity of nucleoprotein structure after freeze-thawing but not in ROS levels. Theriogenology 79, 929–939. Yoshioka K, Suzuki C, Tanaka A, Anas IMK, Iwamura S. 2002. Birth of piglets derived from porcine zygotes cultured in a chemically defined medium. Biology of Reproduction 66, 112–119.
Animal Science Journal (2015) 86, 494–498
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