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Effect of short periods of sperm–oocyte coincubation during in vitro fertilization on embryo development in pigs
Theriogenology 62 (2004) 544–552
Effect of short periods of sperm–oocyte coincubation during in vitro fertilization on embryo development in pigs Maria A. Gila,*, Maria Ruiza, Juan M. Vazqueza, Jordi Rocaa, Billy N. Dayb, Emilio A. Martineza a
Departamento Medicina y Cirugı´a Animal (Reproduccio´n y Obstetricia), Hospital Clı´nico Veterinario, Universidad de Murcia, Murcia E-30071, Spain b Department of Animal Sciences, University of Missouri, Columbia, MO, USA Received 6 September 2003; received in revised form 4 November 2003; accepted 6 November 2003
Abstract The present study was conducted to determine if the efficiency of in vitro pig embryo production could be improved by a reduction in the period of time that oocytes are exposed to sperm during in vitro fertilization. A total of 1596 immature cumulus–oocyte complexes from five replicates were matured in vitro and inseminated with frozen–thawed spermatozoa (2000 spermatozoa/oocyte) for 10, 30, 60 min or 6 h (control group). The oocytes from short coincubation times were washed three times in fertilization medium to remove spermatozoa not bound to the zona and transferred to another droplet of the same medium (containing no sperm) for 6 h. After 6 h, the oocytes from each group were cultured in embryo culture medium for another 6 h to assess fertilization parameters and for 7 days to assess embryo development. After each period of coincubation, some oocytes were stained with Hoechst-33342 to count zona-bound sperm. Although the number of zona-bound sperm increased with the coincubation time (34:1 1:7, 46:8 2:8, 62:8 3:8, and 139:5 6:1 for 10, 30, 60 min and 6 h, respectively, P < 0:02), the penetration rate was not significantly different among groups (61.3–68.2%). However, the efficiency of fertilization (number of monospermic oocytes/total number of inseminated oocytes) increased (P < 0:04) as the coincubation time was increased (26:6 2:9%, 29:0 4:4%, 39:5 6:2%, and 49:3 3:0% for 10, 30, 60 min and 6 h, respectively). Nevertheless, there were no significant differences among groups in blastocyst formation rates (17.5– 25.5%). These results demonstrate that although a sperm–oocyte coincubation time of as little as 10 min results in fertilization rates similar to a 6-h coincubation, the reduction in the period of time of sperm–oocyte coincubation does not improve the efficiency of in vitro pig embryo production. # 2003 Elsevier Inc. All rights reserved. Keywords: In vitro fertilization; Oocyte; Porcine; Coincubation time * Corresponding author. Tel.: þ34-968364812; fax: þ34-968367069. E-mail address: [email protected] (M.A. Gil).
0093-691X/$ – see front matter # 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2003.11.001
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1. Introduction Although techniques for in vitro maturation (IVM) and in vitro fertilization (IVF) of pig oocytes have been improved in recent years, the high incidence of polyspermy penetration remains the main obstacle to successful production of a large number of in vitro porcine embryos [1–3]. Many studies have been conducted in an attempt to decrease the incidence of polyspermy in vitro, simulating the in vivo conditions. In vivo, the incidence of polyspermic penetration is less than 5% when the number of spermatozoa reaching the site of fertilization is very low as occurs after natural breeding or cervical insemination. However, polyspermy increases significantly [4,5] if a high number of spermatozoa are deposited directly into the oviduct. Similarly, in vitro, a reduction of the absolute number of spermatozoa during coincubation with the oocytes increases the monospermic penetration rates, however, this is usually accompanied by a reduction in penetration rates [6]. A reduction of the period of sperm–oocyte exposure has also been suggested to increase the incidence of monospermy [7,8]. In fact, most of the current IVF systems use a 5- to 6-h sperm–oocyte coincubation time [6,9–12], compared to 12- to 18-h coincubation times used in the original porcine IVF systems [13–15]. Recently, both penetration rate and blastocyst development were improved with a simple modification of the IVF procedure by reducing the coincubation time from 5 h to 10 min [16]. Thus, the aim of this study was to evaluate the effect of short periods of sperm–oocyte coincubation during in vitro fertilization on embryo development in pigs.
2. Materials and methods 2.1. Culture media All chemicals used in this study were purchased from Sigma-Aldrich Co. (Alcobendas, Madrid, Spain) unless otherwise stated. The medium used for oocyte maturation was the protein-free tissue culture medium (PF-TCM) 199 (Gibco, Life Technologies S.A. Barcelona, Spain) supplemented with 0.57 mM cysteine, 0.1% (w/v) polyvinylalcohol (PVA), 10 ng/ml epidermal growth factor (EGF), 75 mg/ml potassium penicillin G, 50 mg/ ml streptomycin sulfate, 0.5 mg/ml LH, and 0.5 mg/ml FSH. The basic medium used for IVF was essentially the same as that used by Abeydeera and Day [17]. This medium, designated as modified Tris-buffered medium (mTBM), consists of 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl2H2O, 20 mM Tris, 11 mM glucose, 5 mM sodium pyruvate, and no antibiotics. The embryo culture (EC) medium was North Carolina State University (NCSU) 23 [18] containing 0.4% (w/v) BSA (fraction V; A 8022, initial fractionation by cold alcohol precipitation). 2.2. Recovery and culture of oocytes Ovaries were obtained from prepubertal hybrid gilts at a local slaughterhouse and transported to the laboratory in 0.9% (w/v) NaCl containing 75 mg/ml potassium penicillin
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G and 50 mg/ml streptomycin sulfate maintained at 25–30 8C. Oocytes were aspirated from medium-sized follicles (3–6 mm in diameter) with an 18-gauge needle fixed to a 10 ml disposable syringe. Oocytes surrounded by a compact cumulus mass and having evenly granulated cytoplasm were washed three times in maturation medium and 45–50 oocytes were transferred into each well of a Nunc 4-well multidish containing 500 ml of preequilibrated maturation medium, previously covered with warm mineral oil, and cultured for 44 h in 5% CO2 in air at 39 8C. 2.3. In vitro fertilization After the completion of IVM culture, cumulus cells were removed with 0.1% (w/v) hyaluronidase in maturation medium and the oocytes were washed three times with preequilibrated IVF medium containing 2 mM caffeine and 0.2% (w/v) BSA (fraction V; A 7888, initial fractionation by cold alcohol precipitation). After washing, batches of 30 oocytes were placed in 50 ml drops of the same medium that had been covered with warm mineral oil in a 35 mm 10 mm petri dish. The dishes were kept in the incubator for about 30 min until spermatozoa were added for fertilization. A semen medium-straw from the same mature Pietrain boar, cryopreserved as described by Roca et al. [19], was thawed in a circulating water-bath at 37 8C for 20 s and washed three times by centrifugation at 1900 g for 3 min in Dulbecco’s PBS (Gibco) supplemented with 0.1% (w/v) BSA, 75 mg/ml potassium penicillin G, and 50 mg/ml streptomycin sulfate (pH 7.2). At the end of the washing procedure, the sperm pellet was resuspended in IVF medium (same as above), and then 50 ml of this sperm suspension was added to the medium that contained oocytes so that each oocyte was exposed to 2000 spermatozoa. Just before fertilization, motility was assessed by placing a drop of sperm suspension on a warm glass slide and examining it subjectively at 100 magnification. Oocytes were coincubated with spermatozoa at 39 8C in an atmosphere of 5% CO2 in air for 10, 30, 60 min or 6 h (control group). The oocytes from short coincubation times were washed by mechanical pipetting three times in fresh IVF medium to remove spermatozoa not bound to the zona, transferred to another droplet of mTBM (containing no sperm), and incubated until the 6 h were completed. Following incubation, some oocytes from each group were stained with Hoechst-33342 (6 mM) and the number of sperm bound to the zona were counted by using a fluorescence microscope at 400 magnification. 2.4. Embryo culture After the 6-h incubation, oocytes from each group were washed three times in preequilibrated EC medium and transferred (30 oocytes per well) to a Nunc 4-well multidish containing 500 ml of the same medium per well covered with oil. Oocytes were cultured at 39 8C, 5% CO2 in air for 6 h to assess fertilization parameters or 7 days to assess embryo development. 2.5. Assessment of fertilization parameters Twelve hours after insemination, oocytes were mounted on slides, fixed for 48–72 h in 25% (v/v) acetic acid in ethanol at room temperature, stained with 1% lacmoid in 45%
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(v/v) acetic acid, and examined under a phase-contrast microscope at magnifications of 200 and 400. Oocytes were considered penetrated when they had one or more swollen sperm heads and/or male pronuclei and their corresponding sperm tails present. The fertilization parameters evaluated were: penetration (percentage of the number of oocytes penetrated/total inseminated), monospermy (percentage of the number of monospermic oocytes/total penetrated), number of spermatozoa/oocyte (mean number of spermatozoa in penetrated oocytes), and efficiency of fertilization (percentage of the number of monospermic oocytes/total inseminated). Degenerated oocytes or oocytes with a broken oolemma or abnormal appearance of the cytoplasm were not counted. 2.6. Evaluation of embryo development At 48 and 168 h after IVF, cleavage rate (percentage of the number of oocytes divided to 2–4 cells/total cultivated) and blastocyst formation (percentage of the number of blastocyst/total cultivated), respectively, were evaluated under a stereomicroscope. 2.7. Statistical analysis The experiment was conducted in five replicates, each on a different day. Data from each replicate were pooled. The results are expressed as mean S:E:M. Data were analyzed by ANOVA using the GLM of SPSS 10.0/PC Statistics package (SPSS Inc., Chicago, IL, USA). Data in percentages were modeled according to the binomial model of parameters, as described by Fisz [20]. When ANOVA revealed a significant effect, values were compared by Tukey’s test for multiple comparisons. A probability of P < 0:05 was considered to be statistically significant.
3. Results To determine if the efficiency of in vitro pig embryos could be improved by a reduction in the period of coincubation time, a total of 1596 oocytes from five replicates were inseminated, from which 142 were stained with Hoechst to determine the number of zonaTable 1 Effect of short periods of sperm–oocyte coincubation during in vitro fertilization on fertilization parameters Time
Total examined (number)
Oocytes penetrated (%)
10 min 30 min 60 min 6 h (control)
179 196 204 172
64.8 61.6 61.3 68.2
5.1 5.5 7.2 3.2
Oocytes monospermic1 (%) 40.8 47.2 63.9 72.3
2.1a 5.6a 4.4b 2.6b
Spermatozoa in penetrated oocytes (number) 1.7 1.6 1.5 1.3
0.03a 0.1a 0.03ab 0.05b
Efficiency of fertilization2 (%) 26.6 29.0 39.5 49.3
Different superscripts within a column differ significantly (a, b: P < 0:04; c, d: P < 0:03). 1 Percentage of the number of monospermic oocytes/total of penetrated oocytes. 2 Percentage of the number of monospermic oocytes/total number of inseminated oocytes.
2.9c 4.5c 6.2cd 3.0d
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Table 2 Mean S:E:M: number of sperm bound to the zona pellucida of oocytes coincubated for different periods of time Time
Zona-bound sperm (number)
10 min 30 min 60 min 6 h (control)
34.1 46.8 62.8 139.5
1.7a 2.8a 3.8b 6.1c
Values differ (a, b: P < 0:001; b, c: P < 0:01; a, c: P < 0:001).
bound sperm, 751 were fixed and stained to assess fertilization parameters, and 703 were cultured for 7 days to assess embryo development. As shown in Table 1, the penetration rates were similar among groups, with values between 61.3 and 68.2%. However, the monospermy rate increased significantly (P < 0:04) with increasing coincubation time. When the oocytes were coincubated with spermatozoa for 10 or 30 min, the monospermic rates were indeed reduced to 48%, increasing to approximately 65 and 70% when the coincubation time increased to 60 min and 6 h, respectively. Therefore, the efficiency of fertilization increased as the coincubation time was increased (P < 0:03). The mean number of spermatozoa present per
Fig. 1. Effect of short periods of sperm–oocyte coincubation times during in vitro fertilization on the development of porcine embryos in vitro. Oocytes from five replicates were coincubated with frozen–thawed spermatozoa for 10, 30, 60 min and 6 h. The oocytes from short-time coincubations were washed by mechanical pipetting three times in fresh IVF medium to remove spermatozoa not bound to the zona and transferred to another droplet of mTBM (containing no sperm) until the 6 h were completed. The oocytes were washed and transferred to embryo culture medium and cultured for 7 days. Data represent the mean rate S:E:M: of ( ) cleaved and ( ) blastocyst from the total inseminated. No effect of coincubation time was observed on cleavage or blastocyst formation.
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penetrated oocyte followed a similar effect to monospermy, reaching the minimum value (1:34 0:05) in the control group (6 h). To the contrary, the number of zona-bound sperm (Table 2) increased significantly (P < 0:02) with 60 min of coincubation, and was especially pronounced after 6 h of coincubation (P < 0:001). Nevertheless, there were no significant differences among groups in cleavage or blastocyst formation rates (Fig. 1), with values between 40–50% and 17–25%, respectively.
4. Discussion The modification of gamete coincubation time during porcine IVF has been investigated in the literature to decrease the rate of polyspermy in vitro [6,7,21,22]. It has been reported that penetration of in vitro matured porcine oocytes as well as polyspermic fertilization occur as early as 2-h postinsemination with frozen–thawed sperm [23] and/or with fresh semen [24]. Moreover, it is also known that the incidence of acrosome-reacted living spermatozoa, under capacitating conditions, remains constant after 2, 4, and 6 h of coincubation with oocytes [25]. This suggests that extending sperm– oocyte coincubation times tends to increase sperm–oocyte interactions resulting in a high incidence of polyspermic penetration. Furthermore, a reduction of the time that oocytes are exposed to spermatozoa from 12–18 to 8 h [21] as well as the removal of adherent spermatozoa from the zona pellucida after 6–8 h of coincubation [22], have been suggested to decrease the incidence of polyspermy. On the other hand, when the coincubation time is less than 4 h, the penetration rate decreases significantly without affecting the rate of monospermic fertilization, at least when in vivo matured oocytes are used [7]. At present, most laboratories discontinue the sperm–oocyte coculture after 5–6 h of coincubation time, transferring the oocytes to embryo culture medium [6,9–12]. However, it is necessary to develop new strategies to minimize the number of capacitated spermatozoa present at the fertilization site to obtain high penetration and monospermy rates. In that regard, Grupen and Nottle [16] proposed to decrease the gametes exposition from 5 h to 10 min and maintain the oocytes with the zona-bound sperm in a fresh IVF medium drop without sperm for an additional 5-h incubation. This modification of the IVF conditions resulted in a higher penetration rate (57% versus 80%) and blastocyst development (8% versus 30%) as compared to the conventional procedure of gametes coincubated together for 5 h. In the present experiment, we reduced the coincubation time from 6 h (control group) to 60, 30, and 10 min, washing and transferring the short-time coincubation groups to another droplet of IVF medium (containing no sperm) until the 6 h of the control group were completed. Our results demonstrated that the penetration rates were not significantly different among groups (with values between 60 and 70%). Nevertheless, and despite that the number of zona-bound sperm increased significantly as the coincubation time increased, the oocytes coincubated with spermatozoa for only 10 or 30 min displayed lower monospermy rates (40.8 and 47.2%, respectively), significantly different (P < 0:04) from the monospermy rate obtained after 60 min and or 6-h coincubation (63.9 and 72.3%, respectively). From these results, it is evident that the sperm bound to the zona within the
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first minutes of insemination were able to fertilize a similar number of oocytes as the control group. Thus, when the oocytes were washed and transferred to a medium without spermatozoa, it prevented new interactions but did not prevent the zona-bound spermatozoa from penetrating oocytes. Surprisingly, the monospermy rate increased with longer coincubation times. Grupen and Nottle [16] also observed an increase in polyspermic penetration when the oocytes were exposed to sperm for 10 min compared to the control group of 5 h, although it was not significantly different. Furthermore, they observed a significantly higher penetration rate with the short coincubation time than the control whereas our penetration rates did not vary. The reasons for this discrepancy could be due, at least in part, to the different source and concentration of spermatozoa used in these two studies, fresh semen was used for Grupen and Nottle [16] and frozen–thawed sperm was used in the present work. The high incidence of polyspermy obtained with short time of coincubation is difficult to explain. It is known that frozen–thawed sperm cells generate increased amounts of reactive oxygen species (ROS) as by-products of metabolism and cell death [26,27]. ROS plays an important role in mediating sperm–zona interactions because it increases DNA fragmentation [28], modifies the cytoskeleton [29], and produces a loss of fluidity, integrity and competence of the sperm membrane to participate in the membrane events associated with fertilization [30–32]. In our experiment, because oocytes were washed and transferred to a fresh IVF medium drop (containing no sperm), the short-time coincubation groups may have suffered less oxidative damage than the control group. This could explain the high incidence of polyspermic penetration observed after short-term coincubation. More studies will be needed to explain the high incidence of polyspermic penetration when the gamete coincubation time is shorter than 30 min. The differences encountered in monospermy rates in the present study, did not affect embryo development since there were no differences among groups in cleavage or blastocyst formation rates. On the contrary, in the two-step IVF method proposed by Grupen and Nottle, the blastocyst formation was largely improved compared to the conventional procedure, which allowed a rate of blastocyst development of only 8%, percentage excessively low compared to the 25% obtained with our control group of 6 h. The disparity between the two studies could possibly be accounted for by the different fertilization media used. Indeed, we used mTBM as fertilization medium, whereas Grupen and Nottle used TALP-PVA. It is assumed that the selection of IVF medium is a critical factor on the fertilization parameters and developmental competence of IVM-IVF embryos [33]. Furthermore, the components added during IVF, the concentration of BSA, caffeine and calcium also modulate fertilization and embryo parameters [17]. In any case, it might be expected that the group with highest monospermy showed highest blastocyst development, but this was not the case under our experimental conditions. A recent work from Herrick et al. [34], also showed an increased efficiency of fertilization without affecting the development to the blastocyst stage. Thus, the hypothesis currently admitted that polyspermy remains the main obstacle in porcine IVP is questionable. Recent studies have indicated that polypronuclear oocytes produced from IVF develop to the blastocyst stage in vitro or in vivo at a similar rate to two-pronuclear oocytes [35–37]. Thus, we may assume that some blastocyst from our short periods of coincubation come from polyspermic penetrated oocytes. However, there is always the chance that the monospermic oocytes
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from the short coincubation times have more competence to develop to blastocyst stage since the detrimental effects caused by ROS have been avoided. Further studies have to be performed before conclusions regarding the relationship between gamete coincubation times, ROS production, and embryo development can be reached. The results presented here indicate that, under the in vitro conditions studied, short periods of sperm–oocyte coincubation during in vitro fertilization do not improve the efficiency of in vitro pig embryo production. Further research is required to re-examine this IVF strategy taking into consideration parameters, such as sperm concentration and boar effect.
Acknowledgements The authors wish to thank Dr. Tod McCauley for helpful discussion and critical reading of the manuscript. This study was supported by CDTI (01/0317) project.
References [1] Niwa K. Effectiveness of in vitro maturation and in vitro fertilization techniques in pigs. J Reprod Fertil 1993;48:49–59. [2] Funahashi H, Day BN. Advances in in vitro production of pig embryos. J Reprod Fertil 1997;52:271–83. [3] Day BN, Abeydeera LR, Prather RS. Recent progress in pig embryo production through in vitro maturation and fertilization techniques. In: Johnson LA, Guthrie HD, editors. Boar semen preservation IV. Kansas: Allen Press Inc.; 2000. p. 81–92. [4] Hunter RHF. Polyspermic fertilization in pig after tubal deposition of excessive numbers of spermatozoa. J Exp Zool 1973;183:57–64. [5] Hunter RHF. Oviduct function in pigs, with particular reference to the pathological condition of polyspermy. Mol Reprod Dev 1991;29:385–91. [6] Abeydeera LR, Day BN. Fertilization and subsequent development in vitro of pig oocytes inseminated in a modified Tris-buffered medium with frozen–thawed ejaculated spermatozoa. Biol Reprod 1997;57: 729–34. [7] Coy P, Martinez E, Ruiz S, Vazquez JM, Roca J, Matas C, et al. In vitro fertilization of pig oocytes after different coincubation intervals. Theriogenology 1993;39:1201–8. [8] Ocampo MB, Ocampo LC, Mori T, Ueda J, Kanagawa H. Timing of sequential changes in chromosome configurations during the second meiotic division and cytoplasmic events of pig oocytes matured and fertilized in vitro. Anim Reprod Sci 1994;34:281–8. [9] Abeydeera LR, Wang WH, Cantley TC, Rieke A, Murphy CN, Prather RS, et al. Development and viability of pig oocytes matured in a protein-free medium containing epidermal growth factor. Theriogenology 2000;54:787–97. [10] Wang WH, Abeydeera LR, Han Y, Prather RS, Day BN. Morphologic evaluation and actin filament distribution in porcine embryos produced in vitro and in vivo. Biol Reprod 1999;60:1020–8. [11] Funahashi H, Mcintush EW, Smith MF, Day BN. The presence of Tissue Inhibitor of Matrix Metalloproteinase-1 (TIMP-1) during meiosis improves porcine ‘‘oocyte competence’’ as determined by early embryonic development after in vitro fertilization. J Reprod Dev 1999;45:265–71. [12] Gil MA, Abeydeera LR, Day BN, Vazquez JM, Roca J, Martinez EA. Effect of the volume of medium and number of oocytes during in vitro fertilization in embryo development in pigs. Theriogenology 2003;60:767–76. [13] Iritani A, Niwa K, Imai H. Sperm penetration in vivo of pig follicular oocytes matured in culture. J Reprod Fertil 1978;54:379–83.
552
M.A. Gil et al. / Theriogenology 62 (2004) 544–552
[14] Nagai T, Niwa K, Iritani A, Leidl W. Improved rates of sperm penetration in vitro of pig follicular oocytes matured in culture. Jpn J Fertil Steril 1983;28:313–8. [15] Toyoda Y, Itagaki Y, Minato Y, Fukuda Y. Fertilization in vitro of pig eggs matured in vivo and in vitro. Proc 10th Int Congr Anim Reprod AI 1984;1:395. [16] Grupen CG, Nottle MB. A simple modification of the in vitro fertilization procedure. Theriogenology 2000;53:422. [17] Abeydeera LR, Day BN. In vitro penetration of pig oocytes in a modified Tris-buffered medium: effect of BSA, caffeine and calcium. Theriogenology 1997;48:537–44. [18] Petters RM, Wells KD. Culture of pig embryos. J Reprod Fertil 1993;48:61–73. [19] Roca J, Carvajal G, Lucas X, Vazquez JM, Martinez EA. Fertility of weaned sows after deep intrauterine insemination with a reduced number of frozen–thawed spermatozoa. Theriogenology 2003;60:77–87. [20] Fisz M. Some probability distributions. In: Krieger RE, editor. Probability theory and mathematical statistics. Malabar, FL: Wiley; 1980. p. 129–74. [21] Cheng WTK, Polge C, Moor RM. In vitro fertilization of pig and sheep oocytes. Theriogenology 1986;25:146. [22] Mattioli M, Bacci ML, Galeati G, Seren E. Developmental competence of pig oocytes matured and fertilized in vitro. Theriogenology 1989;31:1201–7. [23] Marchal R, Pelaez J, Terqui M, Mermillod P. Effect of sperm survival and CTC staining pattern on in vitro fertilization of porcine oocytes. Theriogenology 2002;57:1917–27. [24] Matas C, Coy P, Romar R, Marco M, Gadea J, Ruiz S. Effect of sperm preparation method on in vitro fertilization in pigs. Reproduction 2003;125:133–41. [25] Vazquez JM, Martinez E, Roca J, Coy P, Pastor LM. Acrosome reaction of boar spermatozoa in homologous in vitro fertilization. Mol Reprod Dev 1993;36:84–8. [26] Kessopolou E, Tomlinson JJ, Barrat CLR, Bolton AE, Cooke ID. Origin of reactive oxygen species in human semen: spermatozoa or leucocytes? J Reprod Fertil 1992;94:463–70. [27] Jones R, Mann T, Sherins RJ. Peroxidative breakdown of phospholipids in human spermatozoa: spermicidal effects of fatty acid peroxides and protective action of seminal plasma. Fertil Steril 1979; 31:531–7. [28] Lopes S, Jurisicova A, Sun JG, Casper RF. Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa. Hum Reprod 1998;13:896–900. [29] Hindshaw DB, Sklar LA, Bohl B. Cytoskeletal and morphologic impact of cellular oxidant injury. Am J Pathol 1986;123:454–64. [30] Aitken RJ. A free radical theory of male infertility. Reprod Fertil Dev 1994;6:19–24. [31] Alvarez JG, Storey BT. Differential incorporation of fatty acids into and peroxidative loss of fatty acids from phospholipids of human spermatozoa. Mol Reprod Dev 1995;42:334–6. [32] Mammoto A, Masumoto N, Tahara M, Ikebuchi Y, Ohmichi M, Tasaka K, et al. Reactive oxygen species block sperm–egg fusion via oxidation of sperm sulfhydryl proteins in mice. Biol Reprod 1996;55:1063–8. [33] Abeydeera LR. In vitro production of embryos in swine. Theriogenology 2002;57:257–73. [34] Herrick JR, Conover-Saprman ML, Krisher RL. Reduced polyspermic fertilization of porcine oocytes utilizing elevated bicarbonate and reduced calcium concentrations in a single-medium system. Reprod Fertil Dev 2003;15:249–54. [35] Han YM, Abeydeera LR, Kim JH, Moon HB, Cabot RA, Day BN, et al. Growth retardation of inner cell mass cells in polyspermic porcine embryos produced in vitro. Biol Reprod 1999;60:1110–3. [36] Han YM, Wang WH, Abeydeera LR, Petersen AL, Kim JH, Murphy C, et al. Pronuclear location before the first cell division determines ploidy of polyspermic pig embryos. Biol Reprod 1999;61:1340–6. [37] McCauley TC, Mazza MR, Didion BA, Mao J, Wu G, Coppola G, et al. Chromosomal abnormalities in day-6, in vitro-produced embryos. Theriogenology 2003;60:1569–80.
Effect of short periods of sperm–oocyte coincubation during in vitro fertilization on embryo development in pigs Maria A. Gila,*, Maria Ruiza, Juan M. Vazqueza, Jordi Rocaa, Billy N. Dayb, Emilio A. Martineza a
Departamento Medicina y Cirugı´a Animal (Reproduccio´n y Obstetricia), Hospital Clı´nico Veterinario, Universidad de Murcia, Murcia E-30071, Spain b Department of Animal Sciences, University of Missouri, Columbia, MO, USA Received 6 September 2003; received in revised form 4 November 2003; accepted 6 November 2003
Abstract The present study was conducted to determine if the efficiency of in vitro pig embryo production could be improved by a reduction in the period of time that oocytes are exposed to sperm during in vitro fertilization. A total of 1596 immature cumulus–oocyte complexes from five replicates were matured in vitro and inseminated with frozen–thawed spermatozoa (2000 spermatozoa/oocyte) for 10, 30, 60 min or 6 h (control group). The oocytes from short coincubation times were washed three times in fertilization medium to remove spermatozoa not bound to the zona and transferred to another droplet of the same medium (containing no sperm) for 6 h. After 6 h, the oocytes from each group were cultured in embryo culture medium for another 6 h to assess fertilization parameters and for 7 days to assess embryo development. After each period of coincubation, some oocytes were stained with Hoechst-33342 to count zona-bound sperm. Although the number of zona-bound sperm increased with the coincubation time (34:1 1:7, 46:8 2:8, 62:8 3:8, and 139:5 6:1 for 10, 30, 60 min and 6 h, respectively, P < 0:02), the penetration rate was not significantly different among groups (61.3–68.2%). However, the efficiency of fertilization (number of monospermic oocytes/total number of inseminated oocytes) increased (P < 0:04) as the coincubation time was increased (26:6 2:9%, 29:0 4:4%, 39:5 6:2%, and 49:3 3:0% for 10, 30, 60 min and 6 h, respectively). Nevertheless, there were no significant differences among groups in blastocyst formation rates (17.5– 25.5%). These results demonstrate that although a sperm–oocyte coincubation time of as little as 10 min results in fertilization rates similar to a 6-h coincubation, the reduction in the period of time of sperm–oocyte coincubation does not improve the efficiency of in vitro pig embryo production. # 2003 Elsevier Inc. All rights reserved. Keywords: In vitro fertilization; Oocyte; Porcine; Coincubation time * Corresponding author. Tel.: þ34-968364812; fax: þ34-968367069. E-mail address: [email protected] (M.A. Gil).
0093-691X/$ – see front matter # 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2003.11.001
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1. Introduction Although techniques for in vitro maturation (IVM) and in vitro fertilization (IVF) of pig oocytes have been improved in recent years, the high incidence of polyspermy penetration remains the main obstacle to successful production of a large number of in vitro porcine embryos [1–3]. Many studies have been conducted in an attempt to decrease the incidence of polyspermy in vitro, simulating the in vivo conditions. In vivo, the incidence of polyspermic penetration is less than 5% when the number of spermatozoa reaching the site of fertilization is very low as occurs after natural breeding or cervical insemination. However, polyspermy increases significantly [4,5] if a high number of spermatozoa are deposited directly into the oviduct. Similarly, in vitro, a reduction of the absolute number of spermatozoa during coincubation with the oocytes increases the monospermic penetration rates, however, this is usually accompanied by a reduction in penetration rates [6]. A reduction of the period of sperm–oocyte exposure has also been suggested to increase the incidence of monospermy [7,8]. In fact, most of the current IVF systems use a 5- to 6-h sperm–oocyte coincubation time [6,9–12], compared to 12- to 18-h coincubation times used in the original porcine IVF systems [13–15]. Recently, both penetration rate and blastocyst development were improved with a simple modification of the IVF procedure by reducing the coincubation time from 5 h to 10 min [16]. Thus, the aim of this study was to evaluate the effect of short periods of sperm–oocyte coincubation during in vitro fertilization on embryo development in pigs.
2. Materials and methods 2.1. Culture media All chemicals used in this study were purchased from Sigma-Aldrich Co. (Alcobendas, Madrid, Spain) unless otherwise stated. The medium used for oocyte maturation was the protein-free tissue culture medium (PF-TCM) 199 (Gibco, Life Technologies S.A. Barcelona, Spain) supplemented with 0.57 mM cysteine, 0.1% (w/v) polyvinylalcohol (PVA), 10 ng/ml epidermal growth factor (EGF), 75 mg/ml potassium penicillin G, 50 mg/ ml streptomycin sulfate, 0.5 mg/ml LH, and 0.5 mg/ml FSH. The basic medium used for IVF was essentially the same as that used by Abeydeera and Day [17]. This medium, designated as modified Tris-buffered medium (mTBM), consists of 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl2H2O, 20 mM Tris, 11 mM glucose, 5 mM sodium pyruvate, and no antibiotics. The embryo culture (EC) medium was North Carolina State University (NCSU) 23 [18] containing 0.4% (w/v) BSA (fraction V; A 8022, initial fractionation by cold alcohol precipitation). 2.2. Recovery and culture of oocytes Ovaries were obtained from prepubertal hybrid gilts at a local slaughterhouse and transported to the laboratory in 0.9% (w/v) NaCl containing 75 mg/ml potassium penicillin
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G and 50 mg/ml streptomycin sulfate maintained at 25–30 8C. Oocytes were aspirated from medium-sized follicles (3–6 mm in diameter) with an 18-gauge needle fixed to a 10 ml disposable syringe. Oocytes surrounded by a compact cumulus mass and having evenly granulated cytoplasm were washed three times in maturation medium and 45–50 oocytes were transferred into each well of a Nunc 4-well multidish containing 500 ml of preequilibrated maturation medium, previously covered with warm mineral oil, and cultured for 44 h in 5% CO2 in air at 39 8C. 2.3. In vitro fertilization After the completion of IVM culture, cumulus cells were removed with 0.1% (w/v) hyaluronidase in maturation medium and the oocytes were washed three times with preequilibrated IVF medium containing 2 mM caffeine and 0.2% (w/v) BSA (fraction V; A 7888, initial fractionation by cold alcohol precipitation). After washing, batches of 30 oocytes were placed in 50 ml drops of the same medium that had been covered with warm mineral oil in a 35 mm 10 mm petri dish. The dishes were kept in the incubator for about 30 min until spermatozoa were added for fertilization. A semen medium-straw from the same mature Pietrain boar, cryopreserved as described by Roca et al. [19], was thawed in a circulating water-bath at 37 8C for 20 s and washed three times by centrifugation at 1900 g for 3 min in Dulbecco’s PBS (Gibco) supplemented with 0.1% (w/v) BSA, 75 mg/ml potassium penicillin G, and 50 mg/ml streptomycin sulfate (pH 7.2). At the end of the washing procedure, the sperm pellet was resuspended in IVF medium (same as above), and then 50 ml of this sperm suspension was added to the medium that contained oocytes so that each oocyte was exposed to 2000 spermatozoa. Just before fertilization, motility was assessed by placing a drop of sperm suspension on a warm glass slide and examining it subjectively at 100 magnification. Oocytes were coincubated with spermatozoa at 39 8C in an atmosphere of 5% CO2 in air for 10, 30, 60 min or 6 h (control group). The oocytes from short coincubation times were washed by mechanical pipetting three times in fresh IVF medium to remove spermatozoa not bound to the zona, transferred to another droplet of mTBM (containing no sperm), and incubated until the 6 h were completed. Following incubation, some oocytes from each group were stained with Hoechst-33342 (6 mM) and the number of sperm bound to the zona were counted by using a fluorescence microscope at 400 magnification. 2.4. Embryo culture After the 6-h incubation, oocytes from each group were washed three times in preequilibrated EC medium and transferred (30 oocytes per well) to a Nunc 4-well multidish containing 500 ml of the same medium per well covered with oil. Oocytes were cultured at 39 8C, 5% CO2 in air for 6 h to assess fertilization parameters or 7 days to assess embryo development. 2.5. Assessment of fertilization parameters Twelve hours after insemination, oocytes were mounted on slides, fixed for 48–72 h in 25% (v/v) acetic acid in ethanol at room temperature, stained with 1% lacmoid in 45%
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(v/v) acetic acid, and examined under a phase-contrast microscope at magnifications of 200 and 400. Oocytes were considered penetrated when they had one or more swollen sperm heads and/or male pronuclei and their corresponding sperm tails present. The fertilization parameters evaluated were: penetration (percentage of the number of oocytes penetrated/total inseminated), monospermy (percentage of the number of monospermic oocytes/total penetrated), number of spermatozoa/oocyte (mean number of spermatozoa in penetrated oocytes), and efficiency of fertilization (percentage of the number of monospermic oocytes/total inseminated). Degenerated oocytes or oocytes with a broken oolemma or abnormal appearance of the cytoplasm were not counted. 2.6. Evaluation of embryo development At 48 and 168 h after IVF, cleavage rate (percentage of the number of oocytes divided to 2–4 cells/total cultivated) and blastocyst formation (percentage of the number of blastocyst/total cultivated), respectively, were evaluated under a stereomicroscope. 2.7. Statistical analysis The experiment was conducted in five replicates, each on a different day. Data from each replicate were pooled. The results are expressed as mean S:E:M. Data were analyzed by ANOVA using the GLM of SPSS 10.0/PC Statistics package (SPSS Inc., Chicago, IL, USA). Data in percentages were modeled according to the binomial model of parameters, as described by Fisz [20]. When ANOVA revealed a significant effect, values were compared by Tukey’s test for multiple comparisons. A probability of P < 0:05 was considered to be statistically significant.
3. Results To determine if the efficiency of in vitro pig embryos could be improved by a reduction in the period of coincubation time, a total of 1596 oocytes from five replicates were inseminated, from which 142 were stained with Hoechst to determine the number of zonaTable 1 Effect of short periods of sperm–oocyte coincubation during in vitro fertilization on fertilization parameters Time
Total examined (number)
Oocytes penetrated (%)
10 min 30 min 60 min 6 h (control)
179 196 204 172
64.8 61.6 61.3 68.2
5.1 5.5 7.2 3.2
Oocytes monospermic1 (%) 40.8 47.2 63.9 72.3
2.1a 5.6a 4.4b 2.6b
Spermatozoa in penetrated oocytes (number) 1.7 1.6 1.5 1.3
0.03a 0.1a 0.03ab 0.05b
Efficiency of fertilization2 (%) 26.6 29.0 39.5 49.3
Different superscripts within a column differ significantly (a, b: P < 0:04; c, d: P < 0:03). 1 Percentage of the number of monospermic oocytes/total of penetrated oocytes. 2 Percentage of the number of monospermic oocytes/total number of inseminated oocytes.
2.9c 4.5c 6.2cd 3.0d
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Table 2 Mean S:E:M: number of sperm bound to the zona pellucida of oocytes coincubated for different periods of time Time
Zona-bound sperm (number)
10 min 30 min 60 min 6 h (control)
34.1 46.8 62.8 139.5
1.7a 2.8a 3.8b 6.1c
Values differ (a, b: P < 0:001; b, c: P < 0:01; a, c: P < 0:001).
bound sperm, 751 were fixed and stained to assess fertilization parameters, and 703 were cultured for 7 days to assess embryo development. As shown in Table 1, the penetration rates were similar among groups, with values between 61.3 and 68.2%. However, the monospermy rate increased significantly (P < 0:04) with increasing coincubation time. When the oocytes were coincubated with spermatozoa for 10 or 30 min, the monospermic rates were indeed reduced to 48%, increasing to approximately 65 and 70% when the coincubation time increased to 60 min and 6 h, respectively. Therefore, the efficiency of fertilization increased as the coincubation time was increased (P < 0:03). The mean number of spermatozoa present per
Fig. 1. Effect of short periods of sperm–oocyte coincubation times during in vitro fertilization on the development of porcine embryos in vitro. Oocytes from five replicates were coincubated with frozen–thawed spermatozoa for 10, 30, 60 min and 6 h. The oocytes from short-time coincubations were washed by mechanical pipetting three times in fresh IVF medium to remove spermatozoa not bound to the zona and transferred to another droplet of mTBM (containing no sperm) until the 6 h were completed. The oocytes were washed and transferred to embryo culture medium and cultured for 7 days. Data represent the mean rate S:E:M: of ( ) cleaved and ( ) blastocyst from the total inseminated. No effect of coincubation time was observed on cleavage or blastocyst formation.
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penetrated oocyte followed a similar effect to monospermy, reaching the minimum value (1:34 0:05) in the control group (6 h). To the contrary, the number of zona-bound sperm (Table 2) increased significantly (P < 0:02) with 60 min of coincubation, and was especially pronounced after 6 h of coincubation (P < 0:001). Nevertheless, there were no significant differences among groups in cleavage or blastocyst formation rates (Fig. 1), with values between 40–50% and 17–25%, respectively.
4. Discussion The modification of gamete coincubation time during porcine IVF has been investigated in the literature to decrease the rate of polyspermy in vitro [6,7,21,22]. It has been reported that penetration of in vitro matured porcine oocytes as well as polyspermic fertilization occur as early as 2-h postinsemination with frozen–thawed sperm [23] and/or with fresh semen [24]. Moreover, it is also known that the incidence of acrosome-reacted living spermatozoa, under capacitating conditions, remains constant after 2, 4, and 6 h of coincubation with oocytes [25]. This suggests that extending sperm– oocyte coincubation times tends to increase sperm–oocyte interactions resulting in a high incidence of polyspermic penetration. Furthermore, a reduction of the time that oocytes are exposed to spermatozoa from 12–18 to 8 h [21] as well as the removal of adherent spermatozoa from the zona pellucida after 6–8 h of coincubation [22], have been suggested to decrease the incidence of polyspermy. On the other hand, when the coincubation time is less than 4 h, the penetration rate decreases significantly without affecting the rate of monospermic fertilization, at least when in vivo matured oocytes are used [7]. At present, most laboratories discontinue the sperm–oocyte coculture after 5–6 h of coincubation time, transferring the oocytes to embryo culture medium [6,9–12]. However, it is necessary to develop new strategies to minimize the number of capacitated spermatozoa present at the fertilization site to obtain high penetration and monospermy rates. In that regard, Grupen and Nottle [16] proposed to decrease the gametes exposition from 5 h to 10 min and maintain the oocytes with the zona-bound sperm in a fresh IVF medium drop without sperm for an additional 5-h incubation. This modification of the IVF conditions resulted in a higher penetration rate (57% versus 80%) and blastocyst development (8% versus 30%) as compared to the conventional procedure of gametes coincubated together for 5 h. In the present experiment, we reduced the coincubation time from 6 h (control group) to 60, 30, and 10 min, washing and transferring the short-time coincubation groups to another droplet of IVF medium (containing no sperm) until the 6 h of the control group were completed. Our results demonstrated that the penetration rates were not significantly different among groups (with values between 60 and 70%). Nevertheless, and despite that the number of zona-bound sperm increased significantly as the coincubation time increased, the oocytes coincubated with spermatozoa for only 10 or 30 min displayed lower monospermy rates (40.8 and 47.2%, respectively), significantly different (P < 0:04) from the monospermy rate obtained after 60 min and or 6-h coincubation (63.9 and 72.3%, respectively). From these results, it is evident that the sperm bound to the zona within the
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first minutes of insemination were able to fertilize a similar number of oocytes as the control group. Thus, when the oocytes were washed and transferred to a medium without spermatozoa, it prevented new interactions but did not prevent the zona-bound spermatozoa from penetrating oocytes. Surprisingly, the monospermy rate increased with longer coincubation times. Grupen and Nottle [16] also observed an increase in polyspermic penetration when the oocytes were exposed to sperm for 10 min compared to the control group of 5 h, although it was not significantly different. Furthermore, they observed a significantly higher penetration rate with the short coincubation time than the control whereas our penetration rates did not vary. The reasons for this discrepancy could be due, at least in part, to the different source and concentration of spermatozoa used in these two studies, fresh semen was used for Grupen and Nottle [16] and frozen–thawed sperm was used in the present work. The high incidence of polyspermy obtained with short time of coincubation is difficult to explain. It is known that frozen–thawed sperm cells generate increased amounts of reactive oxygen species (ROS) as by-products of metabolism and cell death [26,27]. ROS plays an important role in mediating sperm–zona interactions because it increases DNA fragmentation [28], modifies the cytoskeleton [29], and produces a loss of fluidity, integrity and competence of the sperm membrane to participate in the membrane events associated with fertilization [30–32]. In our experiment, because oocytes were washed and transferred to a fresh IVF medium drop (containing no sperm), the short-time coincubation groups may have suffered less oxidative damage than the control group. This could explain the high incidence of polyspermic penetration observed after short-term coincubation. More studies will be needed to explain the high incidence of polyspermic penetration when the gamete coincubation time is shorter than 30 min. The differences encountered in monospermy rates in the present study, did not affect embryo development since there were no differences among groups in cleavage or blastocyst formation rates. On the contrary, in the two-step IVF method proposed by Grupen and Nottle, the blastocyst formation was largely improved compared to the conventional procedure, which allowed a rate of blastocyst development of only 8%, percentage excessively low compared to the 25% obtained with our control group of 6 h. The disparity between the two studies could possibly be accounted for by the different fertilization media used. Indeed, we used mTBM as fertilization medium, whereas Grupen and Nottle used TALP-PVA. It is assumed that the selection of IVF medium is a critical factor on the fertilization parameters and developmental competence of IVM-IVF embryos [33]. Furthermore, the components added during IVF, the concentration of BSA, caffeine and calcium also modulate fertilization and embryo parameters [17]. In any case, it might be expected that the group with highest monospermy showed highest blastocyst development, but this was not the case under our experimental conditions. A recent work from Herrick et al. [34], also showed an increased efficiency of fertilization without affecting the development to the blastocyst stage. Thus, the hypothesis currently admitted that polyspermy remains the main obstacle in porcine IVP is questionable. Recent studies have indicated that polypronuclear oocytes produced from IVF develop to the blastocyst stage in vitro or in vivo at a similar rate to two-pronuclear oocytes [35–37]. Thus, we may assume that some blastocyst from our short periods of coincubation come from polyspermic penetrated oocytes. However, there is always the chance that the monospermic oocytes
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from the short coincubation times have more competence to develop to blastocyst stage since the detrimental effects caused by ROS have been avoided. Further studies have to be performed before conclusions regarding the relationship between gamete coincubation times, ROS production, and embryo development can be reached. The results presented here indicate that, under the in vitro conditions studied, short periods of sperm–oocyte coincubation during in vitro fertilization do not improve the efficiency of in vitro pig embryo production. Further research is required to re-examine this IVF strategy taking into consideration parameters, such as sperm concentration and boar effect.
Acknowledgements The authors wish to thank Dr. Tod McCauley for helpful discussion and critical reading of the manuscript. This study was supported by CDTI (01/0317) project.
References [1] Niwa K. Effectiveness of in vitro maturation and in vitro fertilization techniques in pigs. J Reprod Fertil 1993;48:49–59. [2] Funahashi H, Day BN. Advances in in vitro production of pig embryos. J Reprod Fertil 1997;52:271–83. [3] Day BN, Abeydeera LR, Prather RS. Recent progress in pig embryo production through in vitro maturation and fertilization techniques. In: Johnson LA, Guthrie HD, editors. Boar semen preservation IV. Kansas: Allen Press Inc.; 2000. p. 81–92. [4] Hunter RHF. Polyspermic fertilization in pig after tubal deposition of excessive numbers of spermatozoa. J Exp Zool 1973;183:57–64. [5] Hunter RHF. Oviduct function in pigs, with particular reference to the pathological condition of polyspermy. Mol Reprod Dev 1991;29:385–91. [6] Abeydeera LR, Day BN. Fertilization and subsequent development in vitro of pig oocytes inseminated in a modified Tris-buffered medium with frozen–thawed ejaculated spermatozoa. Biol Reprod 1997;57: 729–34. [7] Coy P, Martinez E, Ruiz S, Vazquez JM, Roca J, Matas C, et al. In vitro fertilization of pig oocytes after different coincubation intervals. Theriogenology 1993;39:1201–8. [8] Ocampo MB, Ocampo LC, Mori T, Ueda J, Kanagawa H. Timing of sequential changes in chromosome configurations during the second meiotic division and cytoplasmic events of pig oocytes matured and fertilized in vitro. Anim Reprod Sci 1994;34:281–8. [9] Abeydeera LR, Wang WH, Cantley TC, Rieke A, Murphy CN, Prather RS, et al. Development and viability of pig oocytes matured in a protein-free medium containing epidermal growth factor. Theriogenology 2000;54:787–97. [10] Wang WH, Abeydeera LR, Han Y, Prather RS, Day BN. Morphologic evaluation and actin filament distribution in porcine embryos produced in vitro and in vivo. Biol Reprod 1999;60:1020–8. [11] Funahashi H, Mcintush EW, Smith MF, Day BN. The presence of Tissue Inhibitor of Matrix Metalloproteinase-1 (TIMP-1) during meiosis improves porcine ‘‘oocyte competence’’ as determined by early embryonic development after in vitro fertilization. J Reprod Dev 1999;45:265–71. [12] Gil MA, Abeydeera LR, Day BN, Vazquez JM, Roca J, Martinez EA. Effect of the volume of medium and number of oocytes during in vitro fertilization in embryo development in pigs. Theriogenology 2003;60:767–76. [13] Iritani A, Niwa K, Imai H. Sperm penetration in vivo of pig follicular oocytes matured in culture. J Reprod Fertil 1978;54:379–83.
552
M.A. Gil et al. / Theriogenology 62 (2004) 544–552
[14] Nagai T, Niwa K, Iritani A, Leidl W. Improved rates of sperm penetration in vitro of pig follicular oocytes matured in culture. Jpn J Fertil Steril 1983;28:313–8. [15] Toyoda Y, Itagaki Y, Minato Y, Fukuda Y. Fertilization in vitro of pig eggs matured in vivo and in vitro. Proc 10th Int Congr Anim Reprod AI 1984;1:395. [16] Grupen CG, Nottle MB. A simple modification of the in vitro fertilization procedure. Theriogenology 2000;53:422. [17] Abeydeera LR, Day BN. In vitro penetration of pig oocytes in a modified Tris-buffered medium: effect of BSA, caffeine and calcium. Theriogenology 1997;48:537–44. [18] Petters RM, Wells KD. Culture of pig embryos. J Reprod Fertil 1993;48:61–73. [19] Roca J, Carvajal G, Lucas X, Vazquez JM, Martinez EA. Fertility of weaned sows after deep intrauterine insemination with a reduced number of frozen–thawed spermatozoa. Theriogenology 2003;60:77–87. [20] Fisz M. Some probability distributions. In: Krieger RE, editor. Probability theory and mathematical statistics. Malabar, FL: Wiley; 1980. p. 129–74. [21] Cheng WTK, Polge C, Moor RM. In vitro fertilization of pig and sheep oocytes. Theriogenology 1986;25:146. [22] Mattioli M, Bacci ML, Galeati G, Seren E. Developmental competence of pig oocytes matured and fertilized in vitro. Theriogenology 1989;31:1201–7. [23] Marchal R, Pelaez J, Terqui M, Mermillod P. Effect of sperm survival and CTC staining pattern on in vitro fertilization of porcine oocytes. Theriogenology 2002;57:1917–27. [24] Matas C, Coy P, Romar R, Marco M, Gadea J, Ruiz S. Effect of sperm preparation method on in vitro fertilization in pigs. Reproduction 2003;125:133–41. [25] Vazquez JM, Martinez E, Roca J, Coy P, Pastor LM. Acrosome reaction of boar spermatozoa in homologous in vitro fertilization. Mol Reprod Dev 1993;36:84–8. [26] Kessopolou E, Tomlinson JJ, Barrat CLR, Bolton AE, Cooke ID. Origin of reactive oxygen species in human semen: spermatozoa or leucocytes? J Reprod Fertil 1992;94:463–70. [27] Jones R, Mann T, Sherins RJ. Peroxidative breakdown of phospholipids in human spermatozoa: spermicidal effects of fatty acid peroxides and protective action of seminal plasma. Fertil Steril 1979; 31:531–7. [28] Lopes S, Jurisicova A, Sun JG, Casper RF. Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa. Hum Reprod 1998;13:896–900. [29] Hindshaw DB, Sklar LA, Bohl B. Cytoskeletal and morphologic impact of cellular oxidant injury. Am J Pathol 1986;123:454–64. [30] Aitken RJ. A free radical theory of male infertility. Reprod Fertil Dev 1994;6:19–24. [31] Alvarez JG, Storey BT. Differential incorporation of fatty acids into and peroxidative loss of fatty acids from phospholipids of human spermatozoa. Mol Reprod Dev 1995;42:334–6. [32] Mammoto A, Masumoto N, Tahara M, Ikebuchi Y, Ohmichi M, Tasaka K, et al. Reactive oxygen species block sperm–egg fusion via oxidation of sperm sulfhydryl proteins in mice. Biol Reprod 1996;55:1063–8. [33] Abeydeera LR. In vitro production of embryos in swine. Theriogenology 2002;57:257–73. [34] Herrick JR, Conover-Saprman ML, Krisher RL. Reduced polyspermic fertilization of porcine oocytes utilizing elevated bicarbonate and reduced calcium concentrations in a single-medium system. Reprod Fertil Dev 2003;15:249–54. [35] Han YM, Abeydeera LR, Kim JH, Moon HB, Cabot RA, Day BN, et al. Growth retardation of inner cell mass cells in polyspermic porcine embryos produced in vitro. Biol Reprod 1999;60:1110–3. [36] Han YM, Wang WH, Abeydeera LR, Petersen AL, Kim JH, Murphy C, et al. Pronuclear location before the first cell division determines ploidy of polyspermic pig embryos. Biol Reprod 1999;61:1340–6. [37] McCauley TC, Mazza MR, Didion BA, Mao J, Wu G, Coppola G, et al. Chromosomal abnormalities in day-6, in vitro-produced embryos. Theriogenology 2003;60:1569–80.