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BOR - Papers in Press, published online ahead of print August 20, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.020081
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BIOLOGY OF REPRODUCTION 69, 2092–2099 (2003)
DOI: 10.1095/biolreprod.103.020081
© 2003 by the Society for the Study of Reproduction, Inc.

Reproductive Technology

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 Fertilization1

Koji Yoshioka2,3,4, Chie Suzuki3, Seigo Itoh5, Kazuhiro Kikuchi6, Shokichi Iwamura3, and Heriberto Rodriguez-Martinez4

National Institute of Animal Health,3 Tsukuba, Ibaraki 305-0856, Japan Department of Obstetrics and Gynaecology,4 Swedish University of Agricultural Sciences, Uppsala 750 07, Sweden Azabu University,5 Sagamihara, Kanagawa 229-8501, Japan National Institute of Agrobiological Sciences,6 Tsukuba, Ibaraki 305-8602, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To further develop defined conditions for in vitro fertilization (IVF) and in vitro culture (IVC) of in vitro-matured porcine oocytes, we evaluated the effects of theophylline, adenosine, and cysteine in a chemically defined medium during IVF. Viability to full term of in vitro-produced blastocysts after IVF and IVC in chemically defined medium was also investigated by embryo transfer to recipients. A chemically defined medium, porcine gamate medium (PGM), was modified from porcine zygote medium (PZM-4), which was previously established. PGM was used as a basal medium for IVF and PZM-4 was for the culture of presumptive zygotes. Addition of 2.5 mM theophylline to PGM significantly increased the percentage of male pronuclear formation compared with controls (no addition). Addition of 1 µM adenosine to PGM supplemented either with or without 2.5 mM theophylline significantly reduced the number of penetrated spermatozoa compared with controls (no addition of adenosine). Supplementation with 0.2 µM cysteine in PGM containing both 2.5 mM theophylline and 1 µM adenosine further increased the percentage of development to the blastocyst stage, compared with no supplementation of cysteine, but there was no difference in fertilization parameters, such as monospermy and pronuclear formation, regardless of presence or absence of theophylline and adenosine. When Day 5 blastocysts were transferred into four recipients (20–25 blastocysts per recipient), all recipients became pregnant and farrowed a total of 21 live piglets. The present results clearly demonstrate that porcine blastocysts can be produced by IVF and IVC in chemically defined media and that they can develop to full term after embryo transfer.

assisted reproductive technology, early development, embryo, in vitro fertilization


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
An in vitro production (IVP) system for preimplantation embryos that can develop to full-term after transfer would contribute to a better understanding of the physiology of embryonic development in early pregnancy and the control of animal reproduction, including embryo transfer, transgenesis, and cloning [1]. Recently, piglets have been obtained from in vitro-produced porcine blastocysts [2, 3], but their developmental efficiency to term is still low.

An abnormally high incidence of polyspermic penetration in porcine in vitro fertilization (IVF) systems is one of the persistent problems for IVP of porcine embryos. Low developmental rates of in vitro-produced porcine embryos may, therefore, be caused not only by inadequate culture conditions but by a high incidence of polyspermy, a lethal condition in mammals [4]. Although polyspermic porcine embryos can develop to blastocysts, they have fewer numbers of inner cell mass cells compared with monospermic embryos [5]. Polyspermic penetration in vitro is not due to delayed or incomplete cortical granule exocytosis [6] but more likely is caused by a delayed zona reaction and/or the simultaneous penetration by a number of spermatozoa with a reacted acrosome [7, 8].

In a previous study, we demonstrated that in vivo-derived porcine zygotes could develop into blastocysts in the chemically defined porcine zygote medium (PZM-4) and to full term after embryo transfer [9]. Because a chemically defined medium eliminates undefined factors present in biological materials such as serum or serum albumin, application of a chemically defined medium to IVP of embryos has certain advantages. For instance, the use of defined media facilitates the analysis of the physical action of substances on the development of preimplantation embryos, improves reliability of formulations, yields a higher reproducibility of results, and ensures biosafety of culture media by elimination of protein preparations, which may be contaminated with pathogens [10]. BSA in fertilization medium accelerated the ability of bovine [11] and porcine [12] spermatozoa to penetrate in vitro-matured oocytes. Use of a BSA-free chemically defined medium for IVF may thus decrease the incidence of polyspermic penetration of porcine oocytes by suppressing simultaneous sperm penetration under a delayed fertilization process.

In porcine IVF, caffeine and adenosine stimulate and/or modulate sperm penetration of in vitro-matured oocytes by different modes of action [8]. Methylxanthines, such as caffeine and theophylline, can enhance the ability of sperm to penetrate in vitro-matured bovine oocytes [11], stimulating and maintaining sperm motility by acting as phosphodiesterase (PDE) inhibitors, presumably by elevating cAMP levels [13, 14]. However, information is not yet available on the effect of theophylline on porcine IVF, despite the fact that theophylline is more effective than caffeine as an inhibitor of PDE [15]. Cysteine has been reported to improve male pronuclear formation of porcine oocytes when added to in vitro maturation (IVM) medium [16]. The effect of cysteine in the IVF medium is, however, yet unclear.

In the present study, we attempted to establish a chemically defined system for IVP of porcine in vitro-matured oocytes, evaluating the effects of three reagents, theophylline, adenosine, and cysteine. These reagents were added during IVF, and we evaluated fertilization and subsequent in vitro development to the blastocyst stage. Viability to term of blastocysts produced in the chemically defined IVF and in vitro culture (IVC) media was also determined after embryo transfer to recipient gilts.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
IVM of Porcine Oocytes

Ovaries from prepubertal gilts were obtained at a local abattoir and transported to the laboratory at 30–35°C. Cumulus-oocyte complexes (COCs) were aspirated from antral follicles (3–6 mm in diameter) using a 20-ga needle and a regulated vacuum pump (KMAR-5100; Cook, Eight Mile Plains, Australia) at -32 mm Hg of pressure and were collected into a 50-ml conical tube (Sumilon; Sumitomo Baklite, Tokyo, Japan). After washing the COCs twice with Hepes-buffered Tyrode medium (TALP-Hepes [9]), only COCs with uniform ooplasm and a compact cumulus cell mass were selected for IVM, which was carried out by a previously described method [3]. The COCs were washed twice with modified North Carolina State University-37 medium supplemented with 10% (v/v) porcine follicular fluid, 10 IU/ml hCG (Puberogen; Sankyo, Tokyo, Japan) and eCG (Peamex; Sankyo), 1 mM dibutyryl cAMP (Sigma Chemical Co., St. Louis, MO), 0.6 mM cysteine (Sigma), and 50 µg/ml gentamicin sulfate (Sigma). COCs were then cultured for 20–22 h in the same medium and subsequently for 24 h in the same medium but without hormone and dibutyryl cAMP at 39°C in a humidified atmosphere containing 5% CO2:5% O2:90% N2.

Preparation of Frozen Boar Semen

The method of semen cryopreservation was essentially the same as that described by Niwa [17] and Kikuchi et al. [18] with minor modifications. The sperm-rich fraction of the ejaculation was collected from Landrace boars by the glove-hand method and filtered through double gauze to remove gel particles. After checking immediately for sperm motility, the semen samples were extended 1:2 with collecting solution [17, 18], kept at room temperature (25°C) for 1 h, and then cooled down to 15°C during about 2 h. Subsequently, the extended semen was centrifuged at 800 x g for 10 min and the supernatant was removed. The sperm pellet was resuspended in Niwa and Sasaki freezing (NSF) I extender [17, 18] to give 1.6 x 109 cells/ml and then cooled to 5°C, taking about 1.5 h. Spermatozoa resuspended in NSF-I were then mixed with an equal volume of NSF-II [17, 18], which determined the final 3% (v/v) concentration of glycerol as cryoprotectant. The sperm suspension was transferred to 0.5-ml plastic straws, which were frozen in liquid nitrogen vapor for 10 min and finally stored in liquid nitrogen until use.

In Vitro Fertilization

IVF was performed using the same batch of frozen semen treated with a two-step Percoll gradient, except for experiment 4, in which the semen from three different boars were used for IVF. A 90% Percoll was prepared with a 9:1 mixture of Percoll (Amersham-Pharmacia Biotech, Uppsala, Sweden) and 10x modified Modena solution [19] supplemented with 50 µg/ml gentamicin sulfate. The Percoll gradient was prepared in a 15-ml conical tube (Sumilon) with 2 ml of 90% Percoll added to the tube, which was layered with 2 ml of 45% Percoll. Frozen-thawed semen was layered on the Percoll gradient and centrifuged at 700 x g for 20 min. Porcine gamate medium (PGM) was used to modify PZM-4 [9] and as basal fertilization medium. PGM consisted of 108.00 mM NaCl, 10.00 mM KCl, 0.40 mM MgSO4·7H2O, 0.35 mM KH2PO4, 25.07 mM NaHCO3, 1.00 mM glucose, 2.50 mM Ca-(lactate)2·5H2O, 0.20 mM sodium pyruvate, 50 µg/ml gentamicin sulfate, and 3 mg/ml polyvinyl alcohol (P-8136; Sigma). The sperm pellet was resuspended with PGM supplemented with some reagents as described in the experimental protocol and then washed by centrifugation at 500 x g for 5 min. The COCs were coincubated for 4–20 h, depending on each experiment, with spermatozoa at a concentration of 5 x 106 spermatozoa/ml in 100-µl droplets of PGM. The conditions for incubation were 39°C in a humidified atmosphere containing 5% CO2:5% O2:90% N2. Each droplet contained 15–20 COCs.

Evaluation of Fertilization

After coincubation with spermatozoa, the COCs were transferred to a 15-ml conical tube and vortexed for 4 min in TALP-Hepes to remove cumulus cells from the oocytes. Some of the denuded oocytes were randomly selected, mounted on slides, fixed for 24–48 h in 25% (v/v) acetic acid in ethanol, stained with 1% (w/v) orcein in 45% (v/v) acetic acid, and examined under a phase-contrast microscope. Oocytes were considered fertilized when they had one or more swollen sperm head(s) and/or male pronuclei with corresponding sperm tails. The frequency of normal fertilization was determined as the proportion of oocytes with a second polar body, a pair of pronuclei, and corresponding sperm tail out of the total number of oocytes evaluated.

IVC of Embryos

Following IVF, presumptive zygotes were washed twice with TALP-Hepes and twice with PZM-4 and then cultured in 40-µl droplets of PZM-4 covered with paraffin oil (Nacalai Tesque, Kyoto, Japan) at 39°C in a humidified atmosphere containing 5% CO2:5% O2:90% N2 until Day 5 (Day 0 is the day of IVF). Each droplet contained approximately 25 presumptive zygotes. The percentage of presumptive zygotes that cleaved (at or beyond the two-cell stage) and developed to blastocysts was assessed under a stereomicroscope at 48 and 125–130 h postinsemination, respectively. After the end of culture, blastocysts that developed in each treatment were collected and the total number of cells in each blastocyst was counted.

Determination of Cell Number

Total cell numbers in blastocysts were determined by an air-drying method as described previously [20]. Embryos were put in a hypotonic solution of 0.9% (w/v) sodium citrate and 1% (v/v) calf serum for 15 min. They were then treated with fixative I (10:3:7 methanol:acetic acid:distilled water) and fixative II (3:1 methanol:acetic acid). After staining with 2% (v/v) Giemsa solution, the total number of cells, including metaphase plates but excluding pyknotic nuclei, was counted under a bright-field microscope.

Experimental Design

Experiment 1: Effects of various dosages of theophylline on IVF Following IVM, COCs were coincubated with spermatozoa in PGM containing theophylline (Sigma) at concentrations of 0, 1.25, 2.5, or 5 mM for 20 h. The dosages of theophylline investigated were determined by preliminary experiments and other reagents. After IVF, some of the oocytes were fixed for evaluation of fertilization, and the other oocytes were cultured in PZM-4.

Experiment 2: Effects of various dosages of adenosine in the presence of theophylline on IVF COCs were coincubated with spermatozoa in PGM containing 2.5 mM theophylline and 0, 0.1, 1, or 10 µM adenosine (Sigma) for 20 h. After IVF, some of the oocytes were fixed for evaluation of fertilization, and the remaining oocytes were cultured in PZM-4.

Experiment 3: Effects of various dosages of cysteine in the presence of theophylline and adenosine on IVF COCs were coincubated with spermatozoa in PGM containing 2.5 mM theophylline, 1 µM adenosine, and 0, 0.2, 1, or 5 µM cysteine for 20 h. After IVF, some of the oocytes were fixed for evaluation of fertilization, and the remaining oocytes were cultured in PZM-4.

Experiment 4: Effects of theophylline, adenosine, and cysteine alone and the combination of the three reagents on IVF using semen from different boars COCs were coincubated with spermatozoa from three boars in PGM alone, PGM containing the one of 2.5 mM theophylline, 1 µM adenosine, or 0.2 µM cysteine, or all of three reagents together (TAC) for 20 h. After IVF, the oocytes were fixed for evaluation of fertilization.

Experiment 5: Time course of fertilization in PGM supplemented with theophylline, adenosine, and cysteine COCs were coincubated with spermatozoa in PGM containing 2.5 mM theophylline, 1 µM adenosine, and 0.2 µM cysteine for 4–20 h. After IVF for various periods, oocytes were fixed and examined for total fertilization, pronucleus formation, and percentage of polyspermic oocytes. Some of the oocytes that were coincubated for 8–20 h with sperm were cultured in PZM-4 following IVF.

Experiment 6: Developmental competence of blastocysts produced by IVF and IVC in the chemically defined media COCs were coincubated with spermatozoa in PGM containing 2.5 mM theophylline, 1 µM adenosine, and 0.2 µM cysteine for 12 h. Following IVF, presumptive zygotes were cultured in PZM-4 until Day 5. Crossbred prepubertal gilts (5 mo old, 75–85 kg body weight) were treated with a combination of 400 IU eCG and 200 IU hCG (PG600; Suigonan, Intervet International B.V., Boxmeer, The Netherlands) to induce puberty. Twice daily i.m. injections of prostaglandin F2{alpha}, as 15 mg dinoprost (Panacelan Hi; Daiichi Pharmaceutical, Tokyo, Japan), were given for 3 days from 14 to 16 days after PG600 treatment. Ovulation was induced by i.m. injections of 1500 IU hCG 5 days after the start of prostaglandin F2{alpha} treatments. Day 5 embryos that developed to blastocysts were surgically transferred into four recipients (20 or 25 blastocysts/recipient) 5 days after hCG injection, as previously described [9].

Statistical Analysis

Data analyses were carried out by one-way (experiments 1, 2, 3, and 5) or two-way (boars and treatments; experiment 4) ANOVA and the Fisher protected least significant difference test using the StatView 5 software package (SAS Institute, Cary, NC). All percentage data were subjected to arcsine transformation before statistical analysis. The total numbers of cells in the blastocysts were subjected to logarithmic transformation and then analyzed by one-way ANOVA. A P value of <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1: Effects of Various Dosages of Theophylline During IVF

A significant reduction in fertilization rate (P < 0.05) was observed after treatment with 5 mM theophylline compared with 2.5 mM theophylline (Fig. 1). Normal fertilization rate was significantly higher (P < 0.05) in the presence of theophylline at a concentration of 25 mM than at 0, 1.25, or 5 mM. The proportion of oocytes that formed male pronuclei was significantly higher (P < 0.05) with 2.5 mM theophylline compared with control or addition of 5 mM theophylline.



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  		FIG. 1. Effects of theophylline on sperm penetration. Porcine oocytes were coincubated with frozen-thawed boar spermatozoa for 20 h in PGM containing 0, 1.25, 2.5, or 5 mM theophylline. Results are reported as penetration rates (A), normal fertilization rates (B), and rates of male pronuclear formation (C) per oocytes examined and the number of spermatozoa per penetrated oocyte (D). Data are presented as mean and SEM. Different letters above the bars denote significant differences (P < 0.05). Each group had six replicates and 113–119 COCs

Cleavage rates of presumptive zygotes were significantly lower (P < 0.05) in the presence of 5 mM theophylline during IVF compared with 2.5 mM theophylline (Table 1). However, the percentage of zygotes that developed to the blastocyst stage and the total number of cells in blastocysts did not differ among treatments.


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  		TABLE 1. Effects of theophylline in IVF medium on in vitro development of porcine presumptive zygotes.*

Experiment 2: Effects of Various Dosages of Adenosine in the Presence of Theophylline During IVF

Normal fertilization rate was significantly higher (P < 0.05) when oocytes were coincubated with spermatozoa in the medium containing adenosine at a concentration of 1 µM than at 0, 0.1, or 10 µM (Fig. 2). The addition of 1 µM adenosine significantly reduced sperm numbers per penetrated oocyte (P < 0.05) compared with 0 or 0.1 µM adenosine.



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  		FIG. 2. Effects of adenosine on sperm penetration. Porcine oocytes were coincubated with frozen-thawed boar spermatozoa for 20 h in PGM containing 2.5 mM theophylline and 0, 0.1, 1, or 10 µM adenosine. Results are reported as penetration rates (A), normal fertilization rates (B), and rates of male pronuclear formation (C) per oocytes examined and the number of spermatozoa per penetrated oocyte (D). Data are presented as mean and SEM. Different letters above the bars denote significant differences (P < 0.05). Each group had six replicates and 114–118 COCs

The percentage of zygotes that developed to the blastocyst stage was significantly higher in the presence of adenosine at a concentration of 1 µM than no addition of adenedosine (P < 0.05), but cleavage rates of presumptive zygotes did not differ among treatments (Table 2).


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  		TABLE 2. Effects of adenosine in IVF medium supplemented with 2.5 mM theophylline on in vitro development of porcine presumptive zygotes.*

Experiment 3: Effects of Various Dosages of Cysteine in the Presence of Theophylline and Adenosine During IVF

There was no significant difference among treatments in the proportion of oocytes penetrated, fertilized normally, and containing male pronuclei and the numbers of spermatozoa per penetrated oocyte (Fig. 3).



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  		FIG. 3. Effects of cysteine on sperm penetration. Porcine oocytes were coincubated with frozen-thawed boar spermatozoa for 20 h in PGM containing 2.5 mM theophylline, 1 µM adenosine, and 0, 0.2, 1, or 5 µM cysteine. Results are reported as penetration rates (A), normal fertilization rates (B), and rates of male pronuclear formation (C) per oocytes examined and the number of spermatozoa per penetrated oocyte (D). Data are presented as mean and SEM. Each group had eight replicates and 147–158 COCs

The percentage of zygotes that developed to blastocysts was significantly higher in the presence of cysteine at a concentration of 0.2 or 1 µM than at 0 or 5 µM (P < 0.05), but no differences were found among treatments for cleavage rates of presumptive zygotes and for total cell number in blastocysts (Table 3).


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  		TABLE 3. Effects of cysteine in IVF medium supplemented with 2.5 mM theophylline and 1 µM adenosine on in vitro development of porcine presumptive zygotes.*

Experiment 4: Effects of Theophylline, Adenosine, and Cysteine Alone and the Combination of the Three Reagents on IVF Using Semen from Different Boars

The proportions of oocytes penetrated and formed male pronuclei were significantly higher in boar B (P < 0.01) and normal fertilization rate was significantly lower in boar C (P < 0.01) compared with other boars. Overall percentages of fertilized oocytes after addition to PGM of theophylline alone or in combination with TAC were significantly higher (P < 0.05) than after no addition of any reagents (control) (Fig. 4). Normal fertilization rates after addition to PGM of adenosine alone or of TAC were significantly higher (P < 0.05) than those of controls. The proportion of oocytes that formed male pronuclei was significantly higher (P < 0.05) with theophylline alone than in controls, adenosine alone, or cysteine alone groups, and further increases were observed (P < 0.05) with the addition of TAC compared with the addition of theophylline alone. Moreover, the addition of adenosine alone significantly reduced sperm numbers per penetrated oocyte (P < 0.05) compared with controls, theophylline alone, or TAC groups.



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  		FIG. 4. Effects of theophylline alone, adenosine alone, cysteine alone, and the combination of the three reagents on sperm penetration using semen from different boars. Porcine oocytes were coincubated with frozen-thawed spermatozoa from three boars for 20 h in PGM alone (None), PGM containing 2.5 mM theophylline (Theo), 1 µM adenosine (Ado), 0.2 µM cysteine (Cys), or all three reagents (TAC). Results are reported as penetration rates (A), normal fertilization rates (B), and rates of male pronuclear formation (C) per oocytes examined, the number of spermatozoa per penetrated oocyte (D) for each boar (bars), and mean and SEM of all data (circles). Different letters (a–c) denote significant differences (P < 0.05). Each group had three replicates and 45–48 COCs per batch of semen

Experiment 5: Time Course of Fertilization in the Presence of Theophylline, Adenosine, and Cysteine During IVF

Evidence of sperm penetration, oocyte activation, and enlarged sperm heads was observed in 2% of oocytes examined after 4 h of coincubation with spermatozoa (Fig. 5). The fertilization rate then increased rapidly and reached a maximum level within another 8 h of coincubation. Oocytes with male and female pronuclei were first recognized 6 h after IVF, and the percentage of oocytes possessing male and female pronuclei (pronucleus formation rate) increased in parallel with an increase in total fertilization rate. Rates of normal fertilization and polyspermia gradually increased to reach a plateau by 12 h post-IVF. There was no significant difference in the results of in vitro development between the insemination periods of 8–20 h (Table 4).



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  		FIG. 5. Time course of porcine sperm penetration in PGM containing 2.5 mM theophylline, 1 µM adenosine, and 0.2 µM cysteine. The incidences of penetration, pronucleus (PN) formation, normal fertilization, and polyspermy were monitored from 4 h to 20 h after IVF. Data are presented as mean and SEM. Each group had five replicates and 83–90 COCs


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  		TABLE 4. In vitro development of porcine presumptive zygotes coincubated with sperm for various periods.*

Experiment 6: Embryo Transfer

A total of 434 presumptive zygotes were cultured in PZM-4. Three hundred forty-two embryos (78.8%) were cleaved at 48 h post-IVF, and 97 embryos (22.4%) developed to the blastocyst stage on Day 5. Of a total of 97 blastocysts, 90 embryos were transferred into four recipients. All of the recipients became pregnant and farrowed a total of 21 live piglets (Table 5). The mean (±SD) litter size was 5.3 ± 2.2. Both mean gestation period and mean body weight of piglets born were within expected normal ranges.


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  		TABLE 5. Pregnancy results after transfer of embryos fertilized and cultured in the chemically defined media


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results clearly demonstrated that in vitro-matured porcine oocytes can be fertilized and can develop to the blastocyst stage in chemically defined, protein-free media and that the blastocysts produced are developmentally competent to full term after embryo transfer. Furthermore, presence of specific doses of theophylline, adenosine, and cysteine during IVF improved the fertilization rate for porcine oocytes and frozen-thawed spermatozoa or the subsequent embryo development under the chemically defined conditions of culture.

Methylxanthines, such as caffeine and theophylline, are considered inhibitors of the cyclic nucleotide PDE, resulting in an increase in intracellular cAMP [13, 14]. In bovine IVF systems, both caffeine and theophylline have been used to induce sperm capacitation [11, 20–23]. Although theophylline has a greater ability to inhibit PDE than does caffeine [15], only caffeine has been applied for a majority of current porcine IVF systems [2, 3, 6–8, 12, 24, 25]. However, caffeine stimulates both capacitation and spontaneous acrosome reaction of freshly ejaculated boar spermatozoa [26, 27], resulting in the induction of polyspermic penetration in porcine oocytes [8, 27]. In the present study, we demonstrated that theophylline at a concentration of 2.5 mM could stimulate the ability of spermatozoa to penetrate in vitro-matured porcine oocytes without being accompanied by polyspermy. The presence of cumulus cells surrounding the oocytes was reported not to be essential for IVF, and boar spermatozoa could penetrate into cumulus-denuded oocytes in the presence of caffeine [8, 12, 14, 24]. However, it has been suggested that theophylline facilitates cumulus-induced sperm capacitation and the zona pellucida-induced acrosome reaction by increasing cytoplasmic cAMP in cattle, because theophylline has not shown any beneficial effect on bovine sperm fertilizing ability in the absence of cumulus cells [11]. Therefore, theophylline may enhance sperm fertilizing ability by mechanisms different from those of caffeine. The effects of theophylline on sperm fertilizing ability in the absence of cumulus cells surrounding porcine oocytes and on alterations of sperm membrane should be investigated to clarify differences between the effects of theophylline and those of caffeine.

It has been reported that the replacement of caffeine with adenosine in porcine IVF systems increases the incidence of monospermic penetration while decreasing total penetration rates [8, 27]. In the present study, 1 µM adenosine either with or without 2.5 mM theophylline increased rates of morphologically normal IVF and decreased the number of spermatozoa per penetrated oocyte without decreasing the total fertilization rate. The percentage of presumptive zygotes that developed to blastocysts was also increased when IVF occurred in the presence of a combination of 1 µM adenosine and 2.5 mM theophylline. The mode of action of adenosine in modulating sperm penetration of pig oocytes may differ from that of methylxanthines. A recent study using a chlortetracycline fluorescence assay has shown that caffeine stimulates both capacitation and spontaneous acrosome reaction of ejaculated boar spermatozoa, whereas adenosine promotes capacitation and inhibits spontaneous acrosome reaction, regardless of the presence or absence of caffeine [27, 28]. In mouse spermatozoa, adenosine modulates the adenylyl cyclase/cAMP pathway [29, 30] and induces sperm response via adenosine A2 receptors in uncapacitated cells and through adenosine A1 receptors in capacitated cells [31, 32]. In boar spermatozoa, adenosine appears to act via mechanisms similar to those observed in mice [8, 26].

Cysteine is the precursor of glutathione, which plays an important role as an antioxidant in protecting mammalian cells from oxidative damage [33]. Addition of cysteine to the maturation medium can increase intracellular glutathione levels in porcine mature oocytes, resulting in an enhancement of male pronucleus formation [16]. Addition of cysteine during IVM enhanced the subsequent blastocyst development in pigs, but the rates of fertilization and cleavage were not affected [34]. In the hamster, addition of cysteine during IVF stimulated sperm penetration in a chemically defined, protein-free medium [35]. Although high concentrations of reactive oxygen species (ROS), such as the superoxide anion, hydrogen peroxide, and nitric oxide, cause sperm damage [36–39], small amounts of ROS are necessary for optimal fusion between the oocyte and spermatozoon [40] and even for sperm capacitation [41]. In the present study, cysteine during IVF did not affect the fertilization parameters or cleavage rates, regardless of the presence or absence of theophylline and adenosine. However, the percentage of presumptive zygotes that developed to blastocysts was increased by addition of 0.2 or 1 µM cysteine in the presence of theophylline and adenosine. Thus, cysteine during pig IVF may affect the oocyte (and spermatozoa) rather than the spermatozoa alone. The mechanism by which cysteine during IVF enhances subsequent blastocyst development is not clear. However, the effective dose of cysteine during IVF in this study (0.2–1 µM) was clearly low compared with that during IVM (0.04–2.28 mM) [16, 34, 42]. Levels of intracellular glutathione are reduced after fertilization [16]. In cattle, high doses of exogenous cysteine (0.6 mM) during IVF had detrimental effects on embryo development, but the same dose of cysteine during IVM appears to enhance morula and blastocyst yields [43]. Thus, a fine balance between production and scavenging of ROS is an important factor for fertilization [44]. The low dose (0.2–1 µM) of cysteine assayed during IVF may maintain levels of intracellular glutathione and counterbalance ROS generation.

The fertilization ability of the frozen-thawed spermatozoa varies among boars [45]. In the present study, addition of TAC during IVF was effective with three batches of semen from different boars. Moreover, porcine blastocysts could be produced in our IVP system using 12 frozen-thawed and 5 "liquid"-stored spermatozoa from different boars [46]. 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 any boar for porcine IVF.

Serum albumin has numerous properties that could play a role in capacitation, including removal or alteration of sperm membrane surface components [47], alteration of cholesterol:phospholipid ratios in sperm membranes [48], and removal of zinc [49]. Fraser [50] suggested that under albumin-free conditions, acrosome loss in mouse spermatozoa occurred more slowly than when albumin was present, resulting in a low rate of sperm penetration. Moreover, addition of serum albumin to the fertilization medium accelerates sperm fertilizing ability in cattle [11] and pig [12]. In the present study, 2% of the oocytes inseminated had an enlarged sperm head 4 h after IVF, and sperm penetration of oocytes reached a plateau at 12 h after IVF. Wang et al. [51] also reported that frozen-thawed ejaculated boar spermatozoa in a chemically defined, protein-free medium started to penetrate cumulus-enclosed oocytes 4 h after IVF and reached maximum penetration at 10 h postinsemination. In contrast to porcine IVF in chemically defined media, IVF with BSA-containing media has been generally performed 3–6 h after insemination to obtain a satisfactory proportion of fertilized oocytes [3, 8, 25]. Porcine IVF in the chemically defined, protein-free medium may slow down capacitation of boar spermatozoa, which may prevent polyspermy because the incidence and degree of polyspermy is an indication of the number of capacitated spermatozoa present in the immediate vicinity of the oocyte [4]. According to Suzuki et al. [12], replacement of BSA with polyvinyl alcohol decreases the number of polyspermic oocytes and the number of spermatozoa per penetrated oocyte.

Mean cell numbers in the present blastocysts ranged from 37 to 46 by 5 days after IVF in the medium containing 2.5 mM theophylline, 0.1 µM adenosine, and 0.2 µM cysteine. This value is almost the same as those for in vitro-produced blastocysts at 5 days after IVF [3] and for in vivo-fertilized and then in vitro-cultured blastocysts at 6 days after hCG injection [9], whose developmental potential was confirmed by embryo transfer. In the present study, we clearly demonstrated that porcine zygotes fertilized in the chemically defined medium PGM developed into blastocysts in the chemically defined medium PZM-4 and then developed to full term after their transfer to recipients. Production of offspring is the only unequivocal test of viability of in vitro-produced embryos [10]. The present results prove that for successful IVP of porcine blastocysts, protein supplementation during IVF and IVC is not essential.

It is important to increase the incidence of monospermic penetration (or normal fertilization) to produce piglets efficiently from in vitro-produced blastocysts. In the present study, transfer of 20 or 25 blastocysts per recipient resulted in 100% pregnancy rates and 24% developmental rates for transferred blastocysts to piglets. Marchal et al. [2] reported that the transfer of a total of 80 in vitro-produced blastocysts to four recipients (14–26 blastocysts per recipient) resulted in one pregnancy (25% pregnancy rate) and the birth of only two piglets (2.5% developmental rate). Kikuchi et al. [3] obtained 13% developmental rate by transferring a total of 150 in vitro-produced blastocysts into three recipients (50 blastocysts per recipient), and all recipients became pregnant and farrowed. Our results of embryo transfer are excellent compared with these reports, and the gestation period and body weights of newborn piglets seem to be within normal limits. Although we could use only four recipients, we demonstrated that porcine blastocysts produced in our IVP system had full-term developmental potential. Our IVP system for porcine embryos should be useful for embryo manipulation techniques in transgenesis and cloning.

We have established an IVP system for porcine blastocysts that can successfully produce piglets after embryo transfer. Porcine in vitro-matured oocytes were fertilized and developed to the blastocyst stage in the chemically defined, protein-free medium. To verify the widespread beneficial effect of TAC during IVF, a larger number of embryos derived from different boars and from respective controls without TAC should be transferred and evaluated. Only 20%–30% of presumptive zygotes matured and fertilized in vitro developed to the blastocyst stage in the present study, whereas >70% of single-cell zygotes derived in vivo developed to blastocysts in PZM-4 [9]. Therefore, further improvements of this porcine IVP system are required to optimize embryonic growth and to maximize the number of IVP embryos that can survive after embryo transfer.


   ACKNOWLEDGMENTS
 
The authors thank the staff of Nagano Animal Industry Experiment Station for providing frozen boar semen.


   FOOTNOTES
 
1 This work was supported by grants from the Ministry of Agriculture, Forestry and Fisheries of Japan and from the Swedish Foundation for International Cooperation in Research and Higher Education (STINT; SLU-Japan Programme on Reproductive Biotechnology), Sweden. Back

2 Correspondence: Koji Yoshioka, Theriogenology Section, Department of Production Diseases, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan. FAX: 81 29 838 7880; kojiyos{at}affrc.go.jp Back

Received: 6 June 2003.

First decision: 24 June 2003.

Accepted: 18 August 2003.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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