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In vitro fertilization and subsequent development of porcine oocytes using cryopreserved and liquid-stored spermatozoa from various boars
Theriogenology 64 (2005) 1287–1296 www.journals.elsevierhealth.com/periodicals/the
In vitro fertilization and subsequent development of porcine oocytes using cryopreserved and liquid-stored spermatozoa from various boars Chie Suzuki a,*, Koji Yoshioka a, Seigo Itoh b, Tatsuo Kawarasaki c, Kazuhiro Kikuchi d a Department of Production Diseases, National Institute of Animal Health, Theriogenology Section, Kannondai 3-1-5, Tsukuba, Ibaraki 305-0856, Japan b School of Veterinary Medicine, Azabu University, Fuchinobe 1-17-71, Sagamihara, Kanagawa 229-8501, Japan c Shizuoka Swine and Poultry Experiment Station, Nishikata 2780, Ogasa, Shizuoka 439-0037, Japan d Genetic Diversity Department, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan
Received 2 August 2004
Abstract We had previously developed a porcine IVF system using a chemically defined medium, i.e., porcine gamete medium supplemented with theophylline, adenosine, and cysteine (PGMtac). In the present study, we investigated the utility of this IVF system using different types of semen: (1) cryopreserved ejaculated (n = 8); (2) cryopreserved epididymal (n = 4); and (3) liquid-stored ejaculated (n = 5). Cryopreserved spermatozoa were prepared by three methods. In vitro-matured porcine oocytes were fertilized for 20 h in PGMtac using each type of semen, and the presumptive zygotes were cultured in porcine zygote medium (PZM)-4 for 5 days. In the case of frozen–thawed spermatozoa, the number of spermatozoa per penetrated oocyte (1.1–1.7), rate of blastocyst formation (26–56%), and total number of cells per blastocyst (34–49) differed (P < 0.05) among freezing methods. However, blastocysts were produced using all types of cryopreserved spermatozoa (14–75%). When spermatozoa were liquid-stored for 1–14 days after semen collection, the rate of sperm penetration (P < 0.05) decreased as storage time increased, although there was no significant reduction in sperm motility during storage. In all groups, semen that had been stored within 10 days * Corresponding author. Tel.: +81 29 838 7784; fax: +81 29 838 7880. E-mail address: [email protected] (C. Suzuki). 0093-691X/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2005.03.009
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after collection enabled blastocyst production in vitro (20–48%). In conclusion, this IVF system, which uses a chemically defined medium, had widespread utility with both frozen–thawed and liquidstored spermatozoa. # 2005 Elsevier Inc. All rights reserved. Keywords: Pig; IVF; IVC; Frozen–thawed Spermatozoa; Liquid-stored Spermatozoa
1. Introduction It is important to produce large numbers of preimplantation embryos from abbatoirderived ovaries to reduce the time and cost of biomedical and basic research [1]. Despite recent improvements in porcine IVM and IVF techniques, rates of embryonic development to the blastocyst stage have generally been extremely low. Polyspermic penetration of porcine IVM oocytes remains the major obstacle to successful production of large numbers of IVP embryos with sufficient developmental competence to reach full-term [2], together with unsuitability of the in vitro culture (IVC) system [3]. The low developmental rate and poor quality of in vitro-produced (IVP) blastocysts are caused by a high incidence of polyspermy [1]. The incidence of polyspermy appears to be influenced by variation among boars, batches, and sources, among different fractions within the same ejaculate, and among the storage and IVF protocols used in different laboratories [1,4–6]. Therefore, specific batches of semen are selected to obtain superior results for IVF. However, IVF using spermatozoa derived from specific boars may be required to produce transgenic pigs or to use valuable genetic sources. To improve IVP techniques for porcine embryos, a universal porcine IVF system that can use any batch of available semen needs to be developed. Various types of fertilizing medium, such as tissue culture medium (TCM)-199 [7], Brackett and Oliphant medium (BO) [8], modified Tris-buffered medium (mTBS) [9], and Tyrode’s medium (TALP) [10], have been used for in vitro penetration of pig oocytes. Most of these media are supplemented with fetal calf serum or BSA as a protein source [1]. Suzuki et al. [11] showed that the type of macromolecule added to IVF medium has a significant influence on sperm penetration. In that regard, the addition of BSA to IVF medium accelerated the fertilization rate in pigs [11]. Application of a chemically defined medium to the IVF system is useful not only for analyzing the physical action of substances, but also for improving the reproducibility of results, by eliminating unknown factor(s). Recently, Yoshioka et al. [12] established a novel IVP system for porcine embryos, using a chemically defined medium for IVF. This IVF medium, a porcine gamete medium containing theophylline, adenosine, and cysteine (PGMtac), improved the fertilizing ability of frozen–thawed ejaculated spermatozoa. The combination of multiple exogenous reagents such as theophylline, adenosine, and cysteine to modulate the porcine IVF environment may make it possible to use semen from unselected boars for porcine IVF by minimizing the differences between batches of semen in response to each reagent. The objective of the present study was to determine the utility of our established IVF system for porcine oocytes. We evaluated fertilization and subsequent in vitro development
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to the blastocyst stage using both frozen–thawed (ejaculated and epididymal) and liquidstored spermatozoa, derived from a total of 17 boars.
2. Materials and methods 2.1. Preparation of boar semen Individual batches of semen collected from a total of 17 boars were used. Three (derived individually from three Landrace boars) and five (derived individually from three Duroc and two Large White boars) batches of ejaculated semen were cryopreserved by previously described methods A [12] and B [13], respectively. Briefly, the sperm-rich fraction of the ejaculate was cooled to 15 8C. After centrifugation of the extended semen, the sperm pellet was resuspended in Niwa and Sasaki freezing (NSF)-I extender (method A) or Beltsville F5 (BF5) extender (method B), and then cooled to 5 8C. Spermatozoa resuspended in each extender were then mixed with an equal volume of NSF-II containing 6% (v/v) glycerol (method A) or BF5 containing 5% (v/v) glycerol (method B). The sperm suspension was transferred to 0.5-mL straws, which were frozen in liquid nitrogen vapor and finally stored in liquid nitrogen until use. Four batches of cryopreserved epididymal spermatozoa derived individually from one Hampshire, one Meishan, and two Landrace boars were prepared by a previously described freezing method (method C [14]). Briefly, epididymal spermatozoa was centrifuged at room temperature, and then cooled to 15 8C. Spermatozoa were resuspended in a mixture of NSF-I and NSF-II by the protocol of method A, and then transferred to 0.25-mL straws. The straws including the sperm suspension were frozen in liquid nitrogen vapor and finally stored in liquid nitrogen until use. Five batches of liquid-stored semen (derived individually from one Landrace, two Large White, and two Duroc boars), which were commercially prepared and diluted for artificial insemination (Cimco, Tokyo, Japan), were delivered to the laboratory on the day after semen collection. The liquid-stored semen batches were divided into 14-mL round tubes and stored at 15 8C until use. 2.2. Evaluation of sperm motility in liquid-stored semen Sperm motility in liquid-stored semen was evaluated at 1, 4, 7, 10, and 14 days after ejaculation. The spermatozoa were transferred to a modified Modena extender (mME) [12] and subsequently incubated for 10 min. The percentage of motile spermatozoa was evaluated by a computer-assisted semen analysis (CASA) system (Hamilton Thorn Research, Danvers, MA, USA). The motility analyzer settings were: image type phase contrast; frame acquired: 30; minimum contrast: 80; minimum size: 7; low size gate: 0.72; high size gate: 8.82; low intensity gate: 0.14; high intensity gate: 1.84; medium average path velocity (VAP): 50 mm/sec; and low VAP: 20 mm/s. 2.3. IVM and IVF of porcine oocytes Oocytes were collected from the ovaries of slaughtered prepubertal gilts and matured in vitro by a previously described method [12]. Briefly, intact cumulus–oocyte complexes
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(COCs) were cultured for 20–22 h in a modified North Carolina State University-37 medium containing 10% (v/v) porcine follicular fluid; 0.6 mM cysteine (Sigma Chemical Co., St. Louis, MO, USA); 1 mM dibutyryl cAMP (Sigma); 10 IU/mL eCG (Peamex, Sankyo, Tokyo, Japan); 10 IU/mL hCG (Puberogen, Sankyo); and 50 mg/mL gentamicin sulfate (Sigma), and were subsequently cultured in the same medium without dbcAMP or hormones for 24 h. Maturation cultures as well as all other cultures were maintained at 39 8C in a humidified atmosphere containing 5% CO2, 5% O2, and 90% N2. The IVF procedure was essentially the same as that described by Yoshioka et al. [12]. Liquid-stored semen at 1, 4, 7, 10, and 14 days after collection was centrifuged at 400 g for 5 min and resuspended in mME. After being washed once, the spermatozoa pellet was resuspended in 500 mL of mME by centrifugation at 500 g for 5 min. Frozen–thawed or resuspended liquid-stored spermatozoa were layered on a 45% or 90% Percoll (Amersham Biosciences, Piscataway, NJ, USA) gradient and centrifuged at 700 g for 20 min. The spermatozoa pellet was resuspended with PGM containing 2.5 mM theophylline, 1 mM adenosine, and 0.2 mM cysteine [12] and then washed by centrifugation at 500 g for 5 min. The COCs were co-incubated for 20 h with spermatozoa at a concentration of 2 106 spermatozoa/mL in 100-mL droplets of PGMtac. Each droplet contained 15–20 COCs. 2.4. Evaluation of fertilization After co-incubation with spermatozoa, the COCs were stripped of cumulus cells by vortexing for 4 min in TALP-HEPES. Some of the denuded oocytes were randomly selected, mounted on slides, fixed with a mixture of glacial acetic acid and methanol (1:3), stained with 1% (w/v) aceto-orcein, and examined under a phase-contrast microscope [12]. Oocytes were considered as penetrated by sperm when they had one or more swollen sperm head(s) and or male pronuclei. The frequency of normal fertilization was determined as the ratio of oocytes with a second polar body and a pair of pronuclei among the total number of penetrated oocytes. 2.5. IVC of embryos and evaluation of embryonic development After IVF, presumptive zygotes were washed twice with TALP-HEPES and twice with PZM-4 [15], and then cultured in 40-mL droplets of PZM-4 for 5 days. Each droplet contained approximately 25 presumptive zygotes. The rate of cleavage ( 2-cell stage) was evaluated under a stereomicroscope at 48 h after IVF, and the rate of blastocyst formation was evaluated at 144 h after IVF. After the end of culture, the blastocysts were collected and the total number of cells per blastocyst was counted by an air-drying method, as described previously [12]. 2.6. Statistical analysis Data analyses were carried out by the general linear models procedures of the Statistical Analysis System and by using the Scheffe post hoc comparison [16]. Percentage data and the total numbers of cells per blastocyst were subjected to arcsine and logarithmic transformation, respectively, before analysis. A P value of <0.05 was considered significant.
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3. Results 3.1. Frozen–thawed spermatozoa Fertilization parameters for 12 batches of semen cryopreserved by using three different protocols are shown in Table 1. Effect of ejaculates on fertilization parameters were irrespective of freezing methods. Following IVF and IVC, blastocysts derived from all batches of semen were produced (14–75%). The results of IVF and IVC of oocytes with frozen–thawed spermatozoa are shown grouped according to freezing protocol (Fig. 1). There were no significant differences among the freezing protocols in the proportions of oocytes penetrated, fertilized normally, and polyspermic (Fig. 1(I–III)). However, the number of spermatozoa per penetrated oocyte was lower (P < 0.05) with method A than with method C (Fig. 1(IV)). The percentages of presumptive zygotes that cleaved did not differ among the freezing protocols (Fig. 1(V)). However, the rate of blastocyst formation (Fig. 1(VI)) with method C was less (P < 0.05) than with method B, and the total number of cells per blastocyst (Fig. 1(VII)) with method C was less (P < 0.05) than with either of the other two methods.
Fig. 1. Effects of freezing protocols for boar spermatozoa on penetration into porcine oocytes (I–IV) and subsequent embryonic development (V–VII). In total, 17 batches of frozen–thawed spermatozoa cryopreserved using different three protocols (method A: n = 3; method B: n = 5; and method C: n = 4) were used in porcine IVF. Rates of normal fertilization (II) and polyspermic penetration (III) were calculated as percentages of penetrated oocytes. The blastocyst formation rate (VI) was calculated as a percentage of the number of cleaved embryos. Each group had 3–5 replicates, and 46–79 oocytes and 64–108 presumptive zygotes were examined for each batch of boar semen. Data are presented as mean S.D. Different letters above the bars denote differences (P < 0.05).
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Table 1 In vitro fertilization of porcine oocytes matured in vitro, using various batches of boar semen cryopreserved by different methods Freezing methoda
Sourceb
Boar (breed)
Percentage of oocytesc Penetrated
Normally fertilizedd
Polyspermicd
No. of spermatozoa per penetrated oocytec
A
Ej
#1 (Landrace) #2 (Landrace) #3 (Landrace)
75 68 66
79 6 13 13 85 9
81 15 67 58 75 18
13 11 00 16 12
1.1 0.4 1.0 0.0 1.2 0.4
B
Ej
#4 #5 #6 #7 #8
(Large White) (Large White) (Duroc) (Duroc) (Duroc)
75 79 79 75 75
73 18 92 8 34 19 89 10 49 15
26 9 44 19 56 11 37 21 61 11
70 7 44 18 29 21 45 20 13 12
2.0 0.8 1.6 0.7 1.3 0.6 1.6 0.7 1.1 0.4
C
Ep
#9 (Landrace) #10 (Landrace) #11 (Hampshire) #12 (Meishan)
46 64 62 60
91 11 95 6 47 28 75 18
33 19 33 14 64 32 42 34
55 30 56 17 18 37 33 40
2.0 1.0 1.8 0.9 1.5 0.7 1.6 0.8
a b c d
See text for details; A [12], B [13], C [14]. Ej: ejaculated; Ep: epididymal. Data are expressed as the mean S.D. of 3–5 replicates. Percentages of normally fertilized or polyspermic oocytes among the penetrated oocytes.
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No. of oocytes examined
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3.2. Liquid-stored spermatozoa Results for IVF and IVC of oocytes with ejaculated semen stored for 1–14 days are shown in Fig. 2. There was no significant difference in sperm motility among the storage periods (Fig. 2(I)). The rate of sperm penetration was reduced (P < 0.05) in semen stored for 14 days (Fig. 2(II)) compared to 1-, 4- and 7-days storage, although the normal fertilization rate as a percentage of oocytes was higher (P < 0.05) than that using semen stored for 1, 4, or 7 days (Fig. 2(III)). Blastocysts were produced in all IVF experiments using all batches of semen stored for 1–10 days after collection (20–48%). Furthermore, when semen stored for 14 days was inseminated, blastocysts were still produced from two of five batches (14 and 18%; Fig. 2(VII)). With the use of 14-day stored semen, the rate of cleavage was reduced (P < 0.05) compared with semen stored for 1, 4, or 10 days (Fig. 2(VI)), and the rate of blastocyst formation was also reduced (P < 0.05) compared with storage for 4 or 7 days (Fig. 2(VII)). There were no significant differences among the storage periods in terms of total cell numbers per blastocyst (Fig. 2(VIII)).
4. Discussion We demonstrated that both frozen–thawed (ejaculated and epididymal) and liquidstored spermatozoa derived from various boars could fertilize IVM porcine oocytes in our IVF system. Moreover, blastocysts were produced by IVC of presumptive zygotes after IVF using all batches of semen tested. In a previous study, we demonstrated that the fertilizing ability of porcine oocytes and frozen–thawed ejaculated spermatozoa, and subsequent embryonic development, were improved by the addition of specific doses of theophylline, adenosine, and cysteine to PGM [12] during IVF. In porcine IVF, the fertilizing ability of semen varies among boars, batches, and sources, in different fractions within the same ejaculation, and with storage [4–6]. Furthermore, the high incidence of polyspermic penetration remains a major unsolved problem with IVF of pig oocytes [1]. In the present study, we were able to produce blastocysts derived from IVM oocytes that were fertilized with 17 batches of semen from different boars, although these batches had a wide range of fertilizing ability. It may therefore be possible to use almost any batch of available semen in our porcine IVF system. However, optimal IVF conditions, such as the addition of optimal concentrations of theophylline, adenosine, and cysteine (to PGM), an optimal concentration of spermatozoa at insemination, and an optimal co-incubation period of oocytes with spermatozoa, should be determined for each batch of semen. Cryopreservation of spermatozoa provides an effective solution for long-term storage of valuable genetic material and permits the production of embryos with a desirable genetic composition through IVF. In heneral, both frozen–thawed ejaculated and epididymal spermatozoa seem to have the ability to penetrate oocytes [17–19]. Due to damage in the acrosome and flagellar region [20], however, the quality of frozen–thawed spermatozoa (e.g., viability and motility), degrades rapidly, resulting in poor fertilization [21,22]. The freezing protocol directly influences the success of frozen storage. The determinants of successful freezing may differ among boars and ejaculates; furthermore, there are
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Fig. 2. Effects of storage of boar spermatozoa at 15 8C on sperm motility (I); penetration of spermatozoa into oocytes (II–V) and subsequent embryonic development (VI-VIII). A total of five batches of spermatozoa diluted and stored for 1–14 days were used for porcine IVF. The rates of normal fertilization (III) and polyspermic penetration (IV) were calculated as percentages of penetrated oocytes. The blastocyst formation rate (VII) was calculated as a percentage of the number of cleaved embryos. Each group had 76–86 oocytes and 125–135 presumptive zygotes were examined per day of storage. Data are presented as means S.D. Different letters above the bars denote differences (P < 0.05).
differences due to composition of diluents, cooling, equilibration, and/or methods for freezing and thawing [23]. In our study, the number of spermatozoa per penetrated oocyte, the rate of blastocyst formation, and the total number of cells per blastocyst were statistically different among two or three freezing protocols. Therefore, further improvements are required, not only in the porcine IVF system, but also in the freezing protocols for boar spermatozoa to maximize the number of IVP embryos. Under in vivo conditions, sperm motility is considered to be an important parameter in the fertility of porcine-ejaculated spermatozoa [24]. In our study, sperm motility was still as high as 40%, 14 days after collection. Percoll treatment of diluted boar spermatozoa can very efficiently separate motile spermatozoa [25]. However, the penetration rate of oocytes decreased as the storage period increased, even though we used Percoll-separated spermatozoa. Furthermore, rates of cleavage and blastocyst formation were lowest when we used semen stored for 14 days. The relationship between sperm motility and the other parameters examined is unclear. According to Suzuki et al. [26], there is no correlation between sperm motility and the IVF ability of pre-incubated unfrozen ejaculated spermatozoa. Ikeda et al. [18] suggested that acrosomal integrity rather than sperm motility was more important for penetration in vitro when using frozen–thawed epididymal spermatozoa. Thus, acrosomal damage of liquid-stored spermatozoa is also an important subject that needs to be investigated.
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In conclusion, our established porcine IVF system has widespread utility, including the use of both frozen–thawed (ejaculated and epididymal) and liquid-stored spermatozoa. We produced porcine IVP blastocysts from all batches of semen examined. However, the fertilizing ability and subsequent embryonic development varied with the origin of the spermatozoa. Therefore, further improvements to this porcine IVF system are required if it is to be used with almost any batch of available semen and if we are to optimize normal fertilization. Acknowledgment This study was supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan.
References [1] Abeydeera LR. In vitro fertilization and embryo development in pigs. Reproduction 2001;58:159–73. [2] Day BN. Reproductive biotechnologies: current status in porcine reproduction. Anim Reprod Sci 2000;60– 61:161–72. [3] Kikuchi K, Onishi A, Kashiwazaki N, Iwamoto M, Noguchi J, Kaneko H, et al. Successful piglet production after transfer of blastocysts produced by a modified in vitro system. Biol Reprod 2002;66:1033–41. [4] Sirard MA, Dubuc A, Bolamba D, Zheng Y, Coenen K. Follicle-oocyte-sperm interactions in vivo and in vitro in pigs. J Reprod Fertil Suppl 1993;48:3–16. [5] Xu X, Ding J, Seth PC, Harbison DS, Foxcroft GR. In vitro fertilization of in vitro matured pig oocytes: effects of boar and ejaculated fraction. Theriogenology 1996;45:745–55. [6] Xu X, Seth PC, Harbison DS, Cheung AP, Foxcroft GR. Semen dilution for assessment of boar ejaculate quality in pig IVM and IVF systems. Theriogenology 1996;46:1325–37. [7] Nagai T, Takahashi T, Masuda H, Shioya Y, Kuwayama M, Fukushima M, et al. In vitro fertilization of pig oocytes by frozen boar spermatozoa. J Reprod Fertil 1988;84:585–91. [8] Yoshida M. In vitro fertilization of pig oocytes matured in vivo. Jpn J Vet Sci 1987;49:711–8. [9] 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. [10] Cordova A, Ducolomb Y, Jimenez I, Casas E, Bonilla E, Betancourt M. In vitro fertilizing capacity of frozen– thawed boar semen. Theriogenology 1997;47:1309–17. [11] Suzuki K, Mori T, Shimizu H. In vitro fertilization of porcine oocytes in chemically defined medium. Theriogenology 1994;42:1357–68. [12] Yoshioka K, Suzuki C, Itoh S, Kikuchi K, Iwamura S, Rodriguez-Martinez H. Production of piglets derived from in vitro produced blastocysts fertilized and cultured in chemically defined media: effects of theophylline, adenosine, and cysteine during in vitro fertilization. Biol Reprod 2003;69:2092–9. [13] Kawarasaki T, Akamatsu H, Chikyu M, Horiuchi A, Banba K. Simple method for freezing boar spermatozoa packaged in 0.5-ml plastic straws: effects of freezing conditions and glycerol equilibration times. Jpn J Swine Sci 2002;39:183–90. [14] Kikuchi K, Nagai T, Kashiwazaki N, Ikeda H, Noguchi J, Shimada A, et al. Cryopreservation and ensuing in vitro fertilization ability of boar spermatozoa from epididymides stored at 4 8C. Theriogenology 1998;50:615–23. [15] Yoshioka K, Suzuki C, Tanaka A, Anas IMK, Iwamura S. Birth of piglets derived from porcine zygotes cultured in a chemically defined medium. Biol Reprod 2002;66:112–9. [16] SAS/STAT User’s Guide. 6.11 ed. Cary, NC: SAS Institute Inc., 1988. [17] Watson PF. Recent developments and concepts in the cryopreservation of spermatozoa and assessment of their post-thawing function. Reprod Fertil Dev 1995;7:871–91.
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[18] Ikeda H, Kikuchi K, Noguchi J, Takeda H, Shimada A, Mizokami T, et al. Effect of preincubation of cryopreserved porcine epididymal sperm. Theriogenology 2002;57:1309–18. [19] Nagai T, Niwa K, Iritani A. Effect of sperm concentration during preincubation in a defined medium on fertilization in vitro of pig follicular oocytes. J Reprod Fertil 1984;70:271–5. [20] Courtens JL, Paquignon M. Ultrastructure of fresh, frozen, and frozen–thawed spermatozoa of the boar. In: Courtens JL, Paquignon M, Johnson LA, Larsson KS, editors. Proceedings of the First International Conference on Deep Freezing of Boar Semen. Uppsala, Sweden: Swedish University of Agricultural Sciences; 1985. p. 61–87. [21] Beck WW, Silversten I. Variable motility recovery of spermatozoa following freeze preservation. Fertil Steril 1975;26:863–7. [22] Keel BA, Webster BW, Roberts DK. Effects of cryopreservation on the motility characteristics of human spermatozoa. J Reprod Fertil 1987;81:213–20. [23] Johnson LA, Weitze KF, Fiser P, Maxwell WMC. Storage of boar semen. Anim Reprod Sci 2000;62:143–72. [24] World Health Organization. WHO Laboratory Manual for the Examination of Human Semen and SpermCervical Mucus Interaction. New York Cambridge University Press, 1992. [25] Grant SA, Long SE, Parkinson TJ. Fertilizability and structural properties of boar spermatozoa prepared by Percoll gradient centrifugation. J Reprod Fertil 1994;100:477–83. [26] Suzuki K, Mori T, Shimizu H. Effect of the duration of preincubation on the ability of pig spermatozoa to penetrate oocyte in vitro. Anim Sci Tech Jpn 1996;67:24–7.
In vitro fertilization and subsequent development of porcine oocytes using cryopreserved and liquid-stored spermatozoa from various boars Chie Suzuki a,*, Koji Yoshioka a, Seigo Itoh b, Tatsuo Kawarasaki c, Kazuhiro Kikuchi d a Department of Production Diseases, National Institute of Animal Health, Theriogenology Section, Kannondai 3-1-5, Tsukuba, Ibaraki 305-0856, Japan b School of Veterinary Medicine, Azabu University, Fuchinobe 1-17-71, Sagamihara, Kanagawa 229-8501, Japan c Shizuoka Swine and Poultry Experiment Station, Nishikata 2780, Ogasa, Shizuoka 439-0037, Japan d Genetic Diversity Department, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan
Received 2 August 2004
Abstract We had previously developed a porcine IVF system using a chemically defined medium, i.e., porcine gamete medium supplemented with theophylline, adenosine, and cysteine (PGMtac). In the present study, we investigated the utility of this IVF system using different types of semen: (1) cryopreserved ejaculated (n = 8); (2) cryopreserved epididymal (n = 4); and (3) liquid-stored ejaculated (n = 5). Cryopreserved spermatozoa were prepared by three methods. In vitro-matured porcine oocytes were fertilized for 20 h in PGMtac using each type of semen, and the presumptive zygotes were cultured in porcine zygote medium (PZM)-4 for 5 days. In the case of frozen–thawed spermatozoa, the number of spermatozoa per penetrated oocyte (1.1–1.7), rate of blastocyst formation (26–56%), and total number of cells per blastocyst (34–49) differed (P < 0.05) among freezing methods. However, blastocysts were produced using all types of cryopreserved spermatozoa (14–75%). When spermatozoa were liquid-stored for 1–14 days after semen collection, the rate of sperm penetration (P < 0.05) decreased as storage time increased, although there was no significant reduction in sperm motility during storage. In all groups, semen that had been stored within 10 days * Corresponding author. Tel.: +81 29 838 7784; fax: +81 29 838 7880. E-mail address: [email protected] (C. Suzuki). 0093-691X/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2005.03.009
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after collection enabled blastocyst production in vitro (20–48%). In conclusion, this IVF system, which uses a chemically defined medium, had widespread utility with both frozen–thawed and liquidstored spermatozoa. # 2005 Elsevier Inc. All rights reserved. Keywords: Pig; IVF; IVC; Frozen–thawed Spermatozoa; Liquid-stored Spermatozoa
1. Introduction It is important to produce large numbers of preimplantation embryos from abbatoirderived ovaries to reduce the time and cost of biomedical and basic research [1]. Despite recent improvements in porcine IVM and IVF techniques, rates of embryonic development to the blastocyst stage have generally been extremely low. Polyspermic penetration of porcine IVM oocytes remains the major obstacle to successful production of large numbers of IVP embryos with sufficient developmental competence to reach full-term [2], together with unsuitability of the in vitro culture (IVC) system [3]. The low developmental rate and poor quality of in vitro-produced (IVP) blastocysts are caused by a high incidence of polyspermy [1]. The incidence of polyspermy appears to be influenced by variation among boars, batches, and sources, among different fractions within the same ejaculate, and among the storage and IVF protocols used in different laboratories [1,4–6]. Therefore, specific batches of semen are selected to obtain superior results for IVF. However, IVF using spermatozoa derived from specific boars may be required to produce transgenic pigs or to use valuable genetic sources. To improve IVP techniques for porcine embryos, a universal porcine IVF system that can use any batch of available semen needs to be developed. Various types of fertilizing medium, such as tissue culture medium (TCM)-199 [7], Brackett and Oliphant medium (BO) [8], modified Tris-buffered medium (mTBS) [9], and Tyrode’s medium (TALP) [10], have been used for in vitro penetration of pig oocytes. Most of these media are supplemented with fetal calf serum or BSA as a protein source [1]. Suzuki et al. [11] showed that the type of macromolecule added to IVF medium has a significant influence on sperm penetration. In that regard, the addition of BSA to IVF medium accelerated the fertilization rate in pigs [11]. Application of a chemically defined medium to the IVF system is useful not only for analyzing the physical action of substances, but also for improving the reproducibility of results, by eliminating unknown factor(s). Recently, Yoshioka et al. [12] established a novel IVP system for porcine embryos, using a chemically defined medium for IVF. This IVF medium, a porcine gamete medium containing theophylline, adenosine, and cysteine (PGMtac), improved the fertilizing ability of frozen–thawed ejaculated spermatozoa. The combination of multiple exogenous reagents such as theophylline, adenosine, and cysteine to modulate the porcine IVF environment may make it possible to use semen from unselected boars for porcine IVF by minimizing the differences between batches of semen in response to each reagent. The objective of the present study was to determine the utility of our established IVF system for porcine oocytes. We evaluated fertilization and subsequent in vitro development
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to the blastocyst stage using both frozen–thawed (ejaculated and epididymal) and liquidstored spermatozoa, derived from a total of 17 boars.
2. Materials and methods 2.1. Preparation of boar semen Individual batches of semen collected from a total of 17 boars were used. Three (derived individually from three Landrace boars) and five (derived individually from three Duroc and two Large White boars) batches of ejaculated semen were cryopreserved by previously described methods A [12] and B [13], respectively. Briefly, the sperm-rich fraction of the ejaculate was cooled to 15 8C. After centrifugation of the extended semen, the sperm pellet was resuspended in Niwa and Sasaki freezing (NSF)-I extender (method A) or Beltsville F5 (BF5) extender (method B), and then cooled to 5 8C. Spermatozoa resuspended in each extender were then mixed with an equal volume of NSF-II containing 6% (v/v) glycerol (method A) or BF5 containing 5% (v/v) glycerol (method B). The sperm suspension was transferred to 0.5-mL straws, which were frozen in liquid nitrogen vapor and finally stored in liquid nitrogen until use. Four batches of cryopreserved epididymal spermatozoa derived individually from one Hampshire, one Meishan, and two Landrace boars were prepared by a previously described freezing method (method C [14]). Briefly, epididymal spermatozoa was centrifuged at room temperature, and then cooled to 15 8C. Spermatozoa were resuspended in a mixture of NSF-I and NSF-II by the protocol of method A, and then transferred to 0.25-mL straws. The straws including the sperm suspension were frozen in liquid nitrogen vapor and finally stored in liquid nitrogen until use. Five batches of liquid-stored semen (derived individually from one Landrace, two Large White, and two Duroc boars), which were commercially prepared and diluted for artificial insemination (Cimco, Tokyo, Japan), were delivered to the laboratory on the day after semen collection. The liquid-stored semen batches were divided into 14-mL round tubes and stored at 15 8C until use. 2.2. Evaluation of sperm motility in liquid-stored semen Sperm motility in liquid-stored semen was evaluated at 1, 4, 7, 10, and 14 days after ejaculation. The spermatozoa were transferred to a modified Modena extender (mME) [12] and subsequently incubated for 10 min. The percentage of motile spermatozoa was evaluated by a computer-assisted semen analysis (CASA) system (Hamilton Thorn Research, Danvers, MA, USA). The motility analyzer settings were: image type phase contrast; frame acquired: 30; minimum contrast: 80; minimum size: 7; low size gate: 0.72; high size gate: 8.82; low intensity gate: 0.14; high intensity gate: 1.84; medium average path velocity (VAP): 50 mm/sec; and low VAP: 20 mm/s. 2.3. IVM and IVF of porcine oocytes Oocytes were collected from the ovaries of slaughtered prepubertal gilts and matured in vitro by a previously described method [12]. Briefly, intact cumulus–oocyte complexes
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(COCs) were cultured for 20–22 h in a modified North Carolina State University-37 medium containing 10% (v/v) porcine follicular fluid; 0.6 mM cysteine (Sigma Chemical Co., St. Louis, MO, USA); 1 mM dibutyryl cAMP (Sigma); 10 IU/mL eCG (Peamex, Sankyo, Tokyo, Japan); 10 IU/mL hCG (Puberogen, Sankyo); and 50 mg/mL gentamicin sulfate (Sigma), and were subsequently cultured in the same medium without dbcAMP or hormones for 24 h. Maturation cultures as well as all other cultures were maintained at 39 8C in a humidified atmosphere containing 5% CO2, 5% O2, and 90% N2. The IVF procedure was essentially the same as that described by Yoshioka et al. [12]. Liquid-stored semen at 1, 4, 7, 10, and 14 days after collection was centrifuged at 400 g for 5 min and resuspended in mME. After being washed once, the spermatozoa pellet was resuspended in 500 mL of mME by centrifugation at 500 g for 5 min. Frozen–thawed or resuspended liquid-stored spermatozoa were layered on a 45% or 90% Percoll (Amersham Biosciences, Piscataway, NJ, USA) gradient and centrifuged at 700 g for 20 min. The spermatozoa pellet was resuspended with PGM containing 2.5 mM theophylline, 1 mM adenosine, and 0.2 mM cysteine [12] and then washed by centrifugation at 500 g for 5 min. The COCs were co-incubated for 20 h with spermatozoa at a concentration of 2 106 spermatozoa/mL in 100-mL droplets of PGMtac. Each droplet contained 15–20 COCs. 2.4. Evaluation of fertilization After co-incubation with spermatozoa, the COCs were stripped of cumulus cells by vortexing for 4 min in TALP-HEPES. Some of the denuded oocytes were randomly selected, mounted on slides, fixed with a mixture of glacial acetic acid and methanol (1:3), stained with 1% (w/v) aceto-orcein, and examined under a phase-contrast microscope [12]. Oocytes were considered as penetrated by sperm when they had one or more swollen sperm head(s) and or male pronuclei. The frequency of normal fertilization was determined as the ratio of oocytes with a second polar body and a pair of pronuclei among the total number of penetrated oocytes. 2.5. IVC of embryos and evaluation of embryonic development After IVF, presumptive zygotes were washed twice with TALP-HEPES and twice with PZM-4 [15], and then cultured in 40-mL droplets of PZM-4 for 5 days. Each droplet contained approximately 25 presumptive zygotes. The rate of cleavage ( 2-cell stage) was evaluated under a stereomicroscope at 48 h after IVF, and the rate of blastocyst formation was evaluated at 144 h after IVF. After the end of culture, the blastocysts were collected and the total number of cells per blastocyst was counted by an air-drying method, as described previously [12]. 2.6. Statistical analysis Data analyses were carried out by the general linear models procedures of the Statistical Analysis System and by using the Scheffe post hoc comparison [16]. Percentage data and the total numbers of cells per blastocyst were subjected to arcsine and logarithmic transformation, respectively, before analysis. A P value of <0.05 was considered significant.
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3. Results 3.1. Frozen–thawed spermatozoa Fertilization parameters for 12 batches of semen cryopreserved by using three different protocols are shown in Table 1. Effect of ejaculates on fertilization parameters were irrespective of freezing methods. Following IVF and IVC, blastocysts derived from all batches of semen were produced (14–75%). The results of IVF and IVC of oocytes with frozen–thawed spermatozoa are shown grouped according to freezing protocol (Fig. 1). There were no significant differences among the freezing protocols in the proportions of oocytes penetrated, fertilized normally, and polyspermic (Fig. 1(I–III)). However, the number of spermatozoa per penetrated oocyte was lower (P < 0.05) with method A than with method C (Fig. 1(IV)). The percentages of presumptive zygotes that cleaved did not differ among the freezing protocols (Fig. 1(V)). However, the rate of blastocyst formation (Fig. 1(VI)) with method C was less (P < 0.05) than with method B, and the total number of cells per blastocyst (Fig. 1(VII)) with method C was less (P < 0.05) than with either of the other two methods.
Fig. 1. Effects of freezing protocols for boar spermatozoa on penetration into porcine oocytes (I–IV) and subsequent embryonic development (V–VII). In total, 17 batches of frozen–thawed spermatozoa cryopreserved using different three protocols (method A: n = 3; method B: n = 5; and method C: n = 4) were used in porcine IVF. Rates of normal fertilization (II) and polyspermic penetration (III) were calculated as percentages of penetrated oocytes. The blastocyst formation rate (VI) was calculated as a percentage of the number of cleaved embryos. Each group had 3–5 replicates, and 46–79 oocytes and 64–108 presumptive zygotes were examined for each batch of boar semen. Data are presented as mean S.D. Different letters above the bars denote differences (P < 0.05).
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Table 1 In vitro fertilization of porcine oocytes matured in vitro, using various batches of boar semen cryopreserved by different methods Freezing methoda
Sourceb
Boar (breed)
Percentage of oocytesc Penetrated
Normally fertilizedd
Polyspermicd
No. of spermatozoa per penetrated oocytec
A
Ej
#1 (Landrace) #2 (Landrace) #3 (Landrace)
75 68 66
79 6 13 13 85 9
81 15 67 58 75 18
13 11 00 16 12
1.1 0.4 1.0 0.0 1.2 0.4
B
Ej
#4 #5 #6 #7 #8
(Large White) (Large White) (Duroc) (Duroc) (Duroc)
75 79 79 75 75
73 18 92 8 34 19 89 10 49 15
26 9 44 19 56 11 37 21 61 11
70 7 44 18 29 21 45 20 13 12
2.0 0.8 1.6 0.7 1.3 0.6 1.6 0.7 1.1 0.4
C
Ep
#9 (Landrace) #10 (Landrace) #11 (Hampshire) #12 (Meishan)
46 64 62 60
91 11 95 6 47 28 75 18
33 19 33 14 64 32 42 34
55 30 56 17 18 37 33 40
2.0 1.0 1.8 0.9 1.5 0.7 1.6 0.8
a b c d
See text for details; A [12], B [13], C [14]. Ej: ejaculated; Ep: epididymal. Data are expressed as the mean S.D. of 3–5 replicates. Percentages of normally fertilized or polyspermic oocytes among the penetrated oocytes.
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No. of oocytes examined
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3.2. Liquid-stored spermatozoa Results for IVF and IVC of oocytes with ejaculated semen stored for 1–14 days are shown in Fig. 2. There was no significant difference in sperm motility among the storage periods (Fig. 2(I)). The rate of sperm penetration was reduced (P < 0.05) in semen stored for 14 days (Fig. 2(II)) compared to 1-, 4- and 7-days storage, although the normal fertilization rate as a percentage of oocytes was higher (P < 0.05) than that using semen stored for 1, 4, or 7 days (Fig. 2(III)). Blastocysts were produced in all IVF experiments using all batches of semen stored for 1–10 days after collection (20–48%). Furthermore, when semen stored for 14 days was inseminated, blastocysts were still produced from two of five batches (14 and 18%; Fig. 2(VII)). With the use of 14-day stored semen, the rate of cleavage was reduced (P < 0.05) compared with semen stored for 1, 4, or 10 days (Fig. 2(VI)), and the rate of blastocyst formation was also reduced (P < 0.05) compared with storage for 4 or 7 days (Fig. 2(VII)). There were no significant differences among the storage periods in terms of total cell numbers per blastocyst (Fig. 2(VIII)).
4. Discussion We demonstrated that both frozen–thawed (ejaculated and epididymal) and liquidstored spermatozoa derived from various boars could fertilize IVM porcine oocytes in our IVF system. Moreover, blastocysts were produced by IVC of presumptive zygotes after IVF using all batches of semen tested. In a previous study, we demonstrated that the fertilizing ability of porcine oocytes and frozen–thawed ejaculated spermatozoa, and subsequent embryonic development, were improved by the addition of specific doses of theophylline, adenosine, and cysteine to PGM [12] during IVF. In porcine IVF, the fertilizing ability of semen varies among boars, batches, and sources, in different fractions within the same ejaculation, and with storage [4–6]. Furthermore, the high incidence of polyspermic penetration remains a major unsolved problem with IVF of pig oocytes [1]. In the present study, we were able to produce blastocysts derived from IVM oocytes that were fertilized with 17 batches of semen from different boars, although these batches had a wide range of fertilizing ability. It may therefore be possible to use almost any batch of available semen in our porcine IVF system. However, optimal IVF conditions, such as the addition of optimal concentrations of theophylline, adenosine, and cysteine (to PGM), an optimal concentration of spermatozoa at insemination, and an optimal co-incubation period of oocytes with spermatozoa, should be determined for each batch of semen. Cryopreservation of spermatozoa provides an effective solution for long-term storage of valuable genetic material and permits the production of embryos with a desirable genetic composition through IVF. In heneral, both frozen–thawed ejaculated and epididymal spermatozoa seem to have the ability to penetrate oocytes [17–19]. Due to damage in the acrosome and flagellar region [20], however, the quality of frozen–thawed spermatozoa (e.g., viability and motility), degrades rapidly, resulting in poor fertilization [21,22]. The freezing protocol directly influences the success of frozen storage. The determinants of successful freezing may differ among boars and ejaculates; furthermore, there are
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Fig. 2. Effects of storage of boar spermatozoa at 15 8C on sperm motility (I); penetration of spermatozoa into oocytes (II–V) and subsequent embryonic development (VI-VIII). A total of five batches of spermatozoa diluted and stored for 1–14 days were used for porcine IVF. The rates of normal fertilization (III) and polyspermic penetration (IV) were calculated as percentages of penetrated oocytes. The blastocyst formation rate (VII) was calculated as a percentage of the number of cleaved embryos. Each group had 76–86 oocytes and 125–135 presumptive zygotes were examined per day of storage. Data are presented as means S.D. Different letters above the bars denote differences (P < 0.05).
differences due to composition of diluents, cooling, equilibration, and/or methods for freezing and thawing [23]. In our study, the number of spermatozoa per penetrated oocyte, the rate of blastocyst formation, and the total number of cells per blastocyst were statistically different among two or three freezing protocols. Therefore, further improvements are required, not only in the porcine IVF system, but also in the freezing protocols for boar spermatozoa to maximize the number of IVP embryos. Under in vivo conditions, sperm motility is considered to be an important parameter in the fertility of porcine-ejaculated spermatozoa [24]. In our study, sperm motility was still as high as 40%, 14 days after collection. Percoll treatment of diluted boar spermatozoa can very efficiently separate motile spermatozoa [25]. However, the penetration rate of oocytes decreased as the storage period increased, even though we used Percoll-separated spermatozoa. Furthermore, rates of cleavage and blastocyst formation were lowest when we used semen stored for 14 days. The relationship between sperm motility and the other parameters examined is unclear. According to Suzuki et al. [26], there is no correlation between sperm motility and the IVF ability of pre-incubated unfrozen ejaculated spermatozoa. Ikeda et al. [18] suggested that acrosomal integrity rather than sperm motility was more important for penetration in vitro when using frozen–thawed epididymal spermatozoa. Thus, acrosomal damage of liquid-stored spermatozoa is also an important subject that needs to be investigated.
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In conclusion, our established porcine IVF system has widespread utility, including the use of both frozen–thawed (ejaculated and epididymal) and liquid-stored spermatozoa. We produced porcine IVP blastocysts from all batches of semen examined. However, the fertilizing ability and subsequent embryonic development varied with the origin of the spermatozoa. Therefore, further improvements to this porcine IVF system are required if it is to be used with almost any batch of available semen and if we are to optimize normal fertilization. Acknowledgment This study was supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan.
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