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Biology of Reproduction 66, 112-119 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Birth of Piglets Derived from Porcine Zygotes Cultured in a Chemically Defined Medium1

Koji Yoshioka2,a, Chie Suzukia, Atsushi Tanakaa, Idris M.-K. Anasa, and Shokichi Iwamuraa

a Department of Production Diseases, National Institute of Animal Health, Tsukuba, Ibaraki 305-0856, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We evaluated the in vitro development of porcine zygotes that were cultured in a novel culture medium, porcine zygote medium (PZM), under different conditions and compared to in vivo development. The viability of these zygotes to full term after culture was also evaluated by embryo transfer to recipients. Porcine single-cell zygotes were collected from gilts on Day 2 after hCG injection. Culture of zygotes in PZM containing 3 mg/ml of BSA (PZM-3) produced better results in terms of proportion of Day 6 blastocysts, Day 8 hatching rate, and numbers of inner cell mass (ICM) cells and total cells in Day 8 embryos than that in North Carolina State University (NCSU)-23 medium. In culture with PZM-3, embryo development was optimized in an atmosphere of 5% CO2:5% O2:90% N2 compared to 5% CO2 in air. The ICM and total cell numbers in Day 6 embryos cultured in PZM-3 or in PZM-3 in which BSA was replaced with 3 mg/ml of polyvinyl alcohol (PZM-4) were also greater than those of NCSU-23 but less than those developed in vivo. However, no difference was found in the ratio of ICM to total cells among embryos developed in PZM-3, PZM-4, or in vivo. When the Day 6 embryos that developed in PZM-4 (99 embryos) or in vivo (100 embryos) were each transferred into six recipients, no difference was found in the farrowing rate (83.3% for both treatments) and in the number of piglets born (33 and 42 piglets, respectively). Our results indicate that porcine zygotes can develop into blastocysts in a chemically defined medium and to full term by transfer to recipients after culture.

early development, embryo, oviduct, reproductive technology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In vitro culture systems for preimplantation embryos in domestic species are important for studying the physiology of embryos during early pregnancy and for controlling animal reproduction, including embryo transfer, transgenesis, and cloning [1]. Numerous studies have been conducted on culture conditions for mammalian preimplantation embryos, but to validate the normality of cultured embryos and the physiological relevance of data on regulation of development, evaluation of embryo viability is necessary in vivo after transfer [2].

Pig embryos can develop from the zygote to the blastocyst stage in vitro. Several media, such as modified Whitten medium [3], North Carolina State University (NCSU)-23 medium [4], modified Chatot, Ziomek, Bavister medium [5], and Beltsville embryo culture medium (BECM)-3 [6], are available for the successful culture of embryos to the blastocyst stage. In particular, NCSU-23, which was tailored to the metabolic and nutrient needs of pig embryos, is the most successful, compared to other culture media, for the culture of pig embryos derived in vivo and in vitro to the blastocyst stage [4, 7, 8]. Moreover, transfer of blastocysts that are cultured for 120 h from 2- to 4-cell stage embryos following in vivo fertilization [9] and of the 8-cell to morula-stage embryos that are cultured for 96 h after in vitro fertilization [10, 11] has resulted in the birth of piglets. Recently, two live offspring from a recipient were obtained by transfer of in vitro-produced blastocysts [12]. However, the pregnancy rate and average litter size after transfer of cultured pig embryos is apparently low [1, 7, 9, 13]. In vitro development of pig embryos is delayed and results in fewer cell numbers in blastocysts compared to that with in vivo development [5, 7, 14, 15]. In particular, both of the number of inner cell mass (ICM) cells and the ratio of ICM to trophectoderm (TE) cell number for embryos cultured in vitro were less than for in vivo embryos [7]. Thus, culture conditions for pig zygotes have yet to be substantially improved.

A chemically defined medium is useful for analyzing the physical action of substances, such as inorganic compounds, energy substrates, hormones, cytokines, and vitamins, on the development of preimplantation embryos, because it eliminates undefined factors present in serum or serum albumin. Such medium could also be used as a powerful tool for optimizing embryonic growth and for maximizing the number of embryos that survive after transfer [2]. However, to our knowledge, no piglet has yet been obtained from embryos cultured in a chemically defined medium after transfer into recipients.

For in vitro culture of preimplantation embryos, synthetic oviductal fluid [16] and human tubal fluid [17] media have been developed, which are based on the composition of sheep and human oviductal fluid, respectively. These media have been useful for in vitro culture of preimplantation sheep and cattle [16] and for mouse and human [17] embryos, respectively. However, the composition of the inorganic elements and energy substrates in the mammalian oviducts differs slightly between species. For example, in porcine oviductal fluid, the concentration of potassium (12.4 mM [18]) is high compared to that in sheep (8.12 mM [19]) and cow (4.53 mM [20]) oviducts, whereas concentrations of energy substrates (pyruvate, 0.21 mM; lactate, 5.71 mM; and glucose, 0.59 mM [21]) are low in some respects compared to those in the mouse (0.14, 4.26, and 5.19 mM, respectively [22]), rabbit (0.30, 3.67, and 1.46 mM, respectively [22]), and human (0.32, 10.50, and 0.50 mM, respectively [23]) oviducts. Moreover, addition of glutamine and/or (hypo)taurine, or of up to 20 amino acids, to the culture medium stimulates development of embryos [4, 24–28].

In the present study, we developed a medium for in vitro culture of porcine zygotes based on the composition of pig oviductal fluid, which was supplemented with amino acids. The aim of our study was to investigate the suitability of this medium for in vitro development of porcine zygotes. The quality of embryos was assessed in vitro by the number of ICM and TE cells in embryos that developed to the morula, blastocyst, and hatching blastocyst stages. Moreover, in vivo viability of embryos that were cultured in a chemically defined medium was also determined after transfer to recipient gilts.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collection of Pig Embryos

A total of 56 cross-bred prepuberal gilts (Landrace x Large White x Duroc; age, 135–170 days; body weight, 70–117 kg) were used in the experiments. All animal-related procedures followed in this study were approved by the Institutional Care and Use Committee for Laboratory Animals of the National Institute of Animal Health (protocol no. 165) and the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching. Gilts were treated with a combination of 400 IU of eCG and 200 IU of hCG (PG600; Suigonan; Intervet International B.V., Boxmeer, The Netherlands) to induce puberty [29]. Luteolysis was induced by i.m. injections of prostaglandin F2{alpha} as 15 mg of dinoprost (Panacelan Hi; Daiichi Pharmaceutical, Tokyo, Japan) twice daily for 3 days from 14 to 16 days after PG600 treatment [30]. Sixteen days after PG600 treatment, gilts were superovulated with i.m. injections of 1500 IU of eCG (Peamex; Sankyo, Tokyo, Japan), followed 72 h later by 1500 IU of hCG (Puberogen; Sankyo). Artificial insemination was carried out with commercial Duroc semen (Cimco, Tokyo, Japan) 27 h after hCG injection. At 52–54 h after hCG injection, reproductive tracts were recovered at slaughter, and embryos were flushed from the oviducts with 20 ml of glucose-free, Hepes-buffered Tyrode medium (TALP-Hepes [31]) as described previously [32]. As a control, in vivo-developed embryos were also collected from uteri on Day 6 (Day 0 = the day of hCG injection) by flushing with 50 ml of TALP-Hepes.

Embryo Culture

After collection on Day 2, embryos were washed three times with TALP-Hepes and then evaluated under a stereomicroscope for morphology of the cumulus investment, presence of polar bodies, and cleavage. Only zygotes with a single cell, visible polar bodies, and spermatozoa embedded in the zona pellucida, but without expanded cumulus, were used. Zygotes were randomly divided into treatment groups after washing twice with each culture medium. Newly developed porcine zygote medium (PZM) was used as the basal culture medium (Table 1). The inorganic and energy substrate composition of this medium was calculated according to the concentrations of pig oviductal fluid reported previously [18, 21]. In all experiments, either 3 mg/ml of fatty acid-free BSA (A6003; Sigma Chemical Co., St. Louis, MO) or 3 mg/ml of polyvinyl alcohol (PVA; P8136; Sigma) were added to the basal cultured medium as a macromolecular component (designated as PZM-3 and PZM-4, respectively). The NCSU-23 medium [4], in which antibiotics and fraction-V BSA were replaced with 50 µg/ml of gentamicin and fatty acid-free BSA, respectively, was also used for comparison. Zygotes were cultured in 30-µl droplets covered with mineral oil (Nakarai, Kyoto, Japan) at 38.7°C in a humidified atmosphere containing 5% CO2:5% O2:90% N2 or 5% CO2 in air. Each droplet contained 10–14 zygotes.


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  		TABLE 1. Composition of culture media

Differential Staining

Differential cell count was performed as described previously [7, 15] with some modification. Briefly, the zona pellucida was removed by treatment with 0.5% (w/v) pronase (Protease; P8811; Sigma) at 37°C. When the zona became wrinkled, the embryo was transferred to acid Tyrode (pH 2.1) for 1.0–1.5 min and then plunged into TALP-Hepes. The zona-free embryos were cultured individually in drops of PZM-3 for 1–2 h to recover and then subjected to differential staining. Zona-free embryos were incubated in rabbit anti-pig whole serum (P3164; Sigma) diluted 1:9 (v/v) in PZM-4 for 40 min at 38.7°C in a humidified atmosphere containing 5% CO2:5% O2:90% N2. After washing in PZM-4, the embryos were finally incubated in a 1:9 (v/v) dilution of guinea pig complement (S1639; Sigma) in PZM-4 supplemented with 10 µg/ml of propium iodide (Sigma) for 1 h at 38.7°C in a humidified atmosphere containing 5% CO2:5% O2:90% N2. After complement-mediated cell lysis and staining of lysed cells by propium iodide, the embryos were fixed in ice-cold ethanol for 5 min and then stained with 10 µg/ml of Hoechst 33342 (Sigma) in ethanol for 5 min at room temperature. The stained embryos were transferred to 100% glycerol on a microscopic slide and carefully covered with a coverslip. Embryos were examined in whole mount under a fluorescence microscope (DMRE; Leica Microsystems Wetzlar GmbH, Wetzler, Germany). Thus, ICM nuclei labeled with Hoechst 33342 appeared blue, and TE nuclei labeled with both Hoechst 33342 and propium iodide appeared pink to red. Numbers of ICM and TE nuclei were counted directly under the microscope.

Experiment 1

Experiment 1 represented preliminary studies designed for development in different culture media and different gas atmospheres. The single-cell zygotes recovered on Day 2 were cultured to Day 8 in NCSU-23 or PZM-3 under a gas atmosphere of either 5% CO2:5% O2:90% N2 or 5% CO2 in air (~20% O2). The initial cleavage (>=2-cell stage) was determined under a stereomicroscope at 18–20 h after onset of culture. The percentages of cleaved zygotes that developed to morulae, blastocysts, and hatching blastocysts were assessed under a stereomicroscope at 24-h intervals from Days 6 to 8. Blastocysts and hatching blastocysts in each treatment were collected on Day 8, and their ICM and TE nuclei were counted after differential staining.

Experiment 2

Experiment 2 was conducted to determine the effect of replacement of BSA by PVA on in vitro development of porcine zygotes and to compare with in vivo-developed embryos. The single-cell zygotes harvested on Day 2 were cultured to Day 6 in NCSU-23 under a gas atmosphere of 5% CO2 in air or in PZM-3 or PZM-4 under a gas atmosphere of 5% CO2:5% O2:90% N2. The initial cleavage was determined under a stereomicroscope at 18–20 h after onset of culture. The percentages of cleaved zygotes that developed to morulae and blastocysts were assessed under a stereomicroscope on Day 6. As a control, in vivo-developed embryos were also collected on Day 6 from three gilts. The ICM and TE nuclei in Day 6 morulae and blastocysts were counted after differential staining.

Experiment 3

To examine the developmental competence of embryos cultured in a chemically defined medium for 4 days, embryos were surgically transferred into recipients as described previously [31]. The single-cell zygotes recovered on Day 2 were cultured to Day 6 in PZM-4 under a gas atmosphere of 5% CO2:5% O2:90% N2. After culture for 4 days, embryos that developed to the morula and blastocyst stages were transferred into six recipients (16–18 embryos, including 8–13 blastocysts/recipient) who received hormonal treatments similar to the donors but without eCG injection. The estrous cycle of the recipients was asynchronous to that of the donors, with a 1-day delay. Morulae and blastocysts developed in vivo were also collected on Day 6 and then transferred into six recipients at Day 5 (16–18 embryos, including 5–17 blastocysts/recipient) as a control.

Statistical Analysis

Data were analyzed with the StatView 5 (SAS Institute, Inc., Cary, NC) software package. Differences in the mean percentages of cleavage, morulae, blastocysts, and hatching blastocysts among the treatments were analyzed by factorial ANOVA. The ICM and total number of cells and the ratio of ICM to total cells in embryos were also subjected to ANOVA. All percentage and ratio data were subjected to arcsine transformation before statistical analysis. When ANOVA revealed a significant effect of the treatments, treatments were compared by the Fisher protected least-significant difference test. Linear relationships and correlation coefficients between ICM and total number of cells in embryos were determined by simple regression analysis and Pearson correlation coefficient analysis, respectively. Data from embryo transfer were analyzed by the test for equal variance, followed by Student t-test. A P value less than 0.05 denoted a statistically significant difference.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1

A total of 187 single-cell zygotes collected from 12 gilts were cultured in each treatment. One hundred and thirty-eight zygotes were cleaved at 18–20 h after onset of culture. Cleavage rates of zygotes (68%–77%) were not significantly different among the treatments. The percentages of zygotes that developed to the morula and blastocyst stages were not different among the treatments (Table 2). However, the percentages of zygotes that developed to the blastocyst stage on Day 6 and the hatching blastocyst stage on Day 8 in PZM-3 at both oxygen tensions (5% and 20%) were significantly higher (P < 0.05) than those in NCSU-23. In culture with PZM-3, the percentages of embryos that developed to blastocysts on Day 6 and hatching blastocysts on Day 8 in 5% CO2:5% O2:90% N2 were significantly higher (P < 0.05) than in 5% CO2 in air, whereas the percentages of embryos that developed to blastocysts or hatching blastocysts in culture with NSCU-23 were not significantly different between the gas atmosphere of 5% CO2:5% O2:90% N2 and 5% CO2 in air. In Day 8 blastocysts/hatching blastocysts, the mean total and ICM cell numbers in PZM-3 at both 5% and 20% O2 were significantly greater (P < 0.05) than those in NCSU-23. The maximum mean total and ICM cell numbers were obtained in culture with PZM-3 at 5% O2 (P < 0.05). Moreover, the ratio of ICM to total cell number in Day 8 embryos cultured in PZM-3 at 5% O2 was significantly greater (P < 0.05) than that in NCSU-23. The correlations between ICM and total cell numbers of Day 8 embryos were significant (P < 0.01) in all treatments (Fig. 1).


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  		TABLE 2. Effects of culture system on the development of porcine zygotes to the morula, blastocyst, and hatching blastocyst stages and the numbers of total and ICM cells in Day 8a embryos



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  		FIG. 1. Correlation between the number of ICM cells and total cells of Day 8 porcine blastocysts (open circles) and hatching blastocysts (solid circles). Day 2 zygotes were cultured in NCSU-23 under a gas atmosphere of either 5% CO2 in air (A) or 5% CO2:5% O2:90% N2 (B) and PZM-3 under a gas atmosphere of either 5% CO2 in air (C) or 5% CO2:5% O2:90% N2 (D). Regression lines are drawn to show the relationship between ICM cell number and total cell number

Experiment 2

Of a total of 139 single-cell zygotes collected from 10 gilts and cultured in each treatment, 104 zygotes were cleaved at 18–20 h after the onset of culture. Cleavage rates of zygotes (73%–77%) were not significantly different among the treatments. Thirty-six embryos were also collected from three gilts on Day 6 and used as in vivo controls. No significant difference was found in the percentages of zygotes that developed to the morula and blastocyst stages on Day 6 among the treatments (Table 3). However, the percentages of zygotes that developed to the blastocyst stage in PZM-3 and PZM-4 were significantly higher (P < 0.05) than in NCSU-23 but did not differ significantly from those of in vivo embryos. In Day 6 morulae/blastocysts, the mean total and ICM cell numbers in PZM-3 and PZM-4 were significantly greater (P < 0.05) than those in NCSU-23 but significantly less (P < 0.05) than in vivo controls. Although the ratio of ICM to total cell number in Day 6 embryos cultured in NCSU-23 was significantly lower (P < 0.05) than those in other treatments, no significant difference was found in this ratio between PZM-3, PZM-4, or in vivo controls. The correlations between ICM and total cell numbers of Day 6 embryos were significant (P < 0.0001) in all treatments (Fig. 2). Typical photographs of embryos stained with the differential staining method are shown in Figure 3.


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  		TABLE 3. Effects of culture system on the development of porcine zygotes to the morula and blastocysts stages, and the numbers of total and ICM cells in Day 6a embryos



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  		FIG. 2. Correlation between the number of ICM cells and total cells of Day 6 porcine morulae (solid circles) and blastocysts (open circles). Embryos were obtained from the culture of zygotes with NCSU-23 (A), PZM-3 (B), or PZM-4 (C) and from in vivo development (D). Regression lines are drawn to show the relationship between ICM cell number and total cell number



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  		FIG. 3. Differentially stained, Day 6 porcine blastocysts with blue nuclei representing inner cells (ICM) and pink-to-red nuclei representing outer cells (TE). Embryos were obtained from the culture of zygotes with NCSU-23 (A) or PZM-3 (B) and from in vivo development (C)

Experiment 3

A total of 115 single-cell zygotes collected from 10 gilts were cultured in PZM-4. One hundred zygotes were cleaved at 18–20 h after the onset of culture, and 99 embryos developed to the morula and blastocyst stages on Day 6. Moreover, 100 morulae/blastocysts of a total of 104 embryos that were collected from nine gilts on Day 6 were used as in vivo controls. Of six recipients, five gilts were pregnant and farrowed in both treatments (Table 4). No significant difference was found in the numbers (total, alive, and litter size) and body weight of piglets born between in vitro- and in vivo-developed embryos. Sixteen embryos cultured in PZM-4 and piglets born after transfer of the identical cultured embryos into a recipient are shown in Figure 4.


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  		TABLE 4. Pregnancy results after transfer of embryos cultured in the chemically defined medium (PZM-4) for 4 days



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  		FIG. 4. Porcine embryos after culture in a chemically defined medium (A) and live piglets born after transfer of the identical cultured embryos into a recipient (B). Sixteen zygotes were cultured in PZM-4 for 4 days. Six morulae and 10 blastocysts were then transferred into a recipient, resulting in birth of seven live and two stillborn piglets


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results clearly demonstrated that our novel PZM is efficient for in vitro development of porcine zygotes. We also showed that porcine zygotes could successfully develop to the blastocyst stage in a chemically defined medium (PZM-4), and that the cultured Day 6 embryos have developmental competence to full term. To our knowledge, this is the first report of the birth of piglets after transfer of pig zygotes cultured in a chemically defined medium.

In the present study, we developed a culture medium (PZM) that was devised on the basis of the concentrations of inorganic elements and energy substrates in the porcine oviduct [18, 21]. In vitro viability of porcine zygotes in the PZM was high (a maximum of 97% of cleaved zygotes developed to blastocysts) and in no way inferior to that in other media described previously [3–8]. Thus, a medium designed to closely mimic in vivo environmental conditions in the porcine oviduct may support the normal development of porcine zygotes in vitro.

The NCSU-23 medium has been the most successful for the culture of pig embryos compared to other culture media, such as modified Whitten medium [4] and potassium simplex optimized medium [7]. In the present study, the proportion of Day 6 blastocysts and Day 8 hatching frequency were greater in embryos that were cultured with PZM-3 than in those cultured with NCSU-23 (Tables 2 and 3). Furthermore, embryos that were cultured with PZM-3 up to Day 6 or Day 8 had more ICM and total cells than those cultured with NCSU-23. The ICM and total cell numbers in Day 6 embryos cultured with NCSU-23 in the present study, however, were similar to those of a previous report in which embryos were cultured in NCSU-23 [7]. These results indicate that our PZM provides a better environment for the development of cultured pig zygotes compared to NCSU-23.

Culture of pig embryos in NCSU-23 resulted in an increased number of TE and total cells in Day 6 blastocysts that developed in 5% CO2 in air (20% O2) compared with those developed in 5% O2 [7]. In our study, no significant effect of oxygen tension on embryo development in the culture with NCSU-23 was observed. However, culture of porcine zygotes at 5% O2 produced better results in terms of the proportion of Day 6 blastocysts, Day 8 hatching rate, and ICM and total cell numbers of Day 8 embryos than did culture in 20% O2 when PZM-3 was used (Table 2). Thus, PZM appears to provide appropriate conditions for culture of porcine zygotes under low-oxygen conditions rather than under a gas atmosphere of 5% CO2 in air. Embryo metabolism in the pig, which is reflected by glucose and pyruvate uptake as well as by lactate production, differs depending on the culture medium used [33]. The pyruvate:lactate ratio is important for maintenance of the intracellular NAD+:NADH ratio, thus balancing the oxidation-reduction potential of embryos [34, 35]. It has been suggested that the oxygen environment could influence metabolism [36] and the oxidation-reduction potential [34, 37] in the embryo. The oxygen concentrations for maintenance of the NAD+:NADH ratio and redox equilibrium, resulting in viable embryonic growth and development [34, 38], may vary with the culture medium; hence, PZM may provide porcine embryos with more suitable conditions for cellular oxidation-reduction equilibrium compared to NCSU-23.

It has been reported that omission of BSA (protein-free, defined BECM-3) from BECM-3 supplemented with fatty acid-free BSA does not affect in vitro development of porcine zygotes [6]. Our results also demonstrated that replacement of fatty acid-free BSA with PVA in PZM had no detrimental effect on development of porcine zygotes up to Day 6. Although the mechanism through which BSA affects embryo development is not yet fully understood, due in part to inclusion of numerous undefined impurities, such as citrate and lipid transfer protein [2], BSA may be beneficial for embryo culture, because it can be taken up by embryos and broken down to provide amino acids for metabolic and anabolic processes as well as chelation of heavy metals or other toxic substances in the medium [2, 39]. In the absence of protein, chelation of heavy metal ions likely is one reason that amino acids are required by mouse embryos [40]. Both essential and nonessential amino acids, which have been supplemented in both BECM-3 and PZM-4 defined media, may compensate for the needs of the porcine embryo in the absence of BSA. Because it has been reported that specific amino acids, such as glutamine and (hypo)taurine, stimulate porcine embryo development in vitro [4, 27], further studies are required to elucidate the influence of individual amino acids on porcine embryo development in culture with PZM.

The relationship between the ICM and total cell numbers appears to depend on the developmental environment, such as the in vivo and in vitro conditions of the medium and gas atmosphere (Figs. 1 and 2). It has been suggested that bovine embryos cultured in vitro are retarded by approximately one cell cycle compared to embryos cultured in vivo, resulting in a lower ratio of ICM to total cells in embryos [41, 42]. It is thought that the ratio of ICM to total cells in embryos is more important for subsequent normal development after transfer than is the total cell number, because pig demiembryos that had only half the cell numbers of intact embryos, but were bisected at the correct ratio of ICM to total cells, could develop to term [43, 44]. Although the ICM and total cell numbers in Day 6 embryos cultured with PZM did not reach the level of in vivo embryos (Table 3), we observed no difference in the ratio of ICM to total cells between embryos cultured in PZM and in vivo embryos. Inner cell allocation of porcine zygotes cultured in PZM may be temporally correct for the subsequent birth of piglets, whereas progress of their cell cycle is delayed compared to that of in vivo controls.

Production of offspring is the only unequivocal test of normality of cultured embryos [2]. In the present study, transfer of 16–18 embryos cultured in PZM-4 for 4 days per recipient led to high pregnancy rates (83.3%), equivalent to those obtained after transfer of in vivo embryos. Given the numbers of transferred morulae and blastocysts, and offspring obtained after transfer, we consider that some piglets were derived from blastocysts (Fig. 4). Moreover, none of parameters investigated from pregnancy to parturition, such as farrowing rate, gestation period, litter size, and birth weight, was significantly different between cultured embryos and in vivo controls. Our results of pregnancy rate and litter size in cultured embryos did not deviate from the ranges of pregnancy rate (53%–80%) and litter size (5.7–8.1 piglets) that have been reported previously for pig embryo transfer without embryo culture [13]. Whereas we demonstrated that porcine zygotes cultured for 4 days in a chemically defined medium showed full-term developmental potential without any severe reduction in embryo viability, recent studies have shown that the preimplantation environment affects subsequent fetal and postnatal growth [45, 46]. Therefore, further studies will be also required to elucidate the normality of porcine embryos cultured with PZM on fetal-postnatal growth.

In conclusion, we have developed a new medium, PZM, for porcine embryo culture and could obtain piglets from porcine zygotes that were cultured in our chemically defined medium, PZM-4, for 4 days after transfer to recipients. Our culture medium should be useful as a basal medium for in vitro production systems in the pig and for embryonic manipulation techniques in transgenesis and cloning. The use of chemically defined media can lead to improvements in the reliability of media formulations, reproducibility of results, and biosafety aspects of protocols by elimination of protein preparations [2]. However, detailed comparisons, such as optimal concentrations of medium components, could not be performed in this study due to the limitation of the source of embryos. Further studies are required to optimize embryonic growth and to maximize the number of in vitro-produced pig embryos that can survive after transfer to recipients.


   ACKNOWLEDGMENTS
 
The authors thank Dr. A. Onishi (National Institute of Agrobiological Sciences) for his useful advice regarding embryo transfer and Dr. K. Kikuchi (National Institute of Agrobiological Sciences) for critical reading of the manuscript.


   FOOTNOTES
 
First decision: 26 June 2001.

1 Supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan. Back

2 Correspondence: Koji Yoshioka, Theriogenology Section, Department of Production Diseases, National Institute of Animal Health, National Agricultural Research Organization, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan. FAX: 81 298 38 7880; kojiyos{at}affrc.go.jp Back

Accepted: August 20, 2001.

Received: May 29, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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