Effect of Culture Medium Supplementation with b-mercaptoethanol and Amino Acid on Canine Intergeneric Embryo Development with Porcine Oocyte Cytoplasm Recipient

The present study investigated the effect of culture medium supplementation with mercaptoethanol ( ME) and amino acid (AA) on canine intergeneric embryo development with porcine oocyte cytoplasm. Porcine cumulus oocyte complexes (COCs) were collected from slaughterhouse and matured in TCM-199 supplemented with 26.2 mM NaHCO , 3.05 mM glucose, 0.91 mM sodium pyruvate, 0.57 mM L-cysteine, 75 mg/l kanamycin, 10 ng/ml 3 epidermal growth factor, equine chorionic gonadotropin (eCG), 10 IU/ml human chorionic gonadotropin (hCG), and 10% (v/v) porcine follicular fluid (pFF) at 39 °C in a humidified atmosphere of 5% CO for 42-44 h and donor cell 2 collected from ear skin afghanhound male dog. After somatic cell nuclear transfer (SCNT), embryo development were examined for cleavage rate and 144 hr for final development after cultured in media. The result shows that, amino acid and mercapoethanol addition in culture medium (NCSU-23) have no effect on embryo development. The development rate of embryo until 16 cell stage in NCSU and NCSU supplement are 4.67% and morula stage are

Interspecies nuclear transfer also provides a possible approach to clone animal species whose oocytes were difficult to obtain (Jumnian et al., 2002;Wen et al., 2003).Several studies have shown that oocyte cytoplasm from bovine, rabbits and sheep can support early development of embryos produced by nuclear transfer of s o m a t i c c e l l s n u c l e i f r o m v a r i o u s mammalian species (Dominko et al., 1999;White et al., 1999;Cohen et al., 1999;Lanza et al., 1999;Chen et al., 1999;2002;Wen et al., 2003).Recently, the successes of cloning gaur (Lanza et al., 2000) and mouflon (Loi et al., 2001) have demonstrated that the technique of interspecies cloning can be practically applied to save highly endangered species, such as the giant panda, Ovis orientalis musilmon, buffalo, bos gaurus (White et al.,

Introduction
Interspecies somatic cell nuclear transfer method was firstly applied for conservation of endangered animals.The highly publicized that an adult sheep had been cloned from the nucleus of a frozen somatic cell (Wilmut et al., 1997) speculated that cloning technologies might be applied to increase population sizes of endangered species, or even restore them following extinction (Cohen, 1997;Wen et al., 2005).1999; Lanza et al., 2000;Vogel et al., 2001;Chen et al., 2002;Jumnian et al., 2002;Sansisena et al., 2005).
The establishment of optimum culture medium for embryos of domestic animals derived from in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) is very critical for applied as well as basic research.Many factors are known to influence the in vitro culture (IVC) of mammalian embryos.Among them, media composition, culture atmosphere, temperature, oxygen tension, osmotic pressure, free radical scavengers, volume of culture drops, and embryo manipulation such as microinjection and cryopreservation are reported to be influence embryo development and quality (Quinn and Harlow, 1978;Harlow and Quinn, 1982;Umaoka et al., 1992;Kooyman and Pinkert, 1994;Bagis et al., 2002;Bagis and Odaman, 2004).Factors that have a negative impact on the in vitro development of the embryo include oxidative stress and composition of culture medium (Umaoka et al., 1992).
Amino acids serve a variety of physiological functions, including: the synthesis of proteins and nucleotides (Epstein and Smith 1973;Alexiou and Leese 1992;Katchadourian et al., 1994), nutrition and energy provision (Lane and Gardner, 1997;1998;Houghton et al., 2002), o s m o r e g u l a t i o n ( V a n W i n k l e a n d Campione, 1996;Dumoulin et al., 1997;Dawson et al., 1998), protection against oxidative stress (Lindenbaum 1973;Nasr-Esfahani et al., 1992),pH regulation (Bavister and McKiernan, 1993;Edward et al., 1998), signalling molecule biosynthesis (Wu and Morris, 1998), trophectoderm differentiation (Martin and Sutherland, 2001)and basement membrane formation between primitive endoderm and ectoderm (Biggers et al., 2000).Although achievements have made early stages of feasible embryonic development in vitro, the adequate culture conditions for the preimplantation porcine embryo have yet to be determined.Differential developmental competence in response to various culture media has been demonstrated between IVF and SCNT embryos (Chung et al., 2002).Four-cell developmental block was overcome by using NCSU-23 in vivo porcine oocytes (Machaty et al., 1998).
Glutathione is the major non-protein sulfidryl compound present in mammalian cells.Multiple actions have been described for GSH, including increasing amino acid transport, stimulating DNA and protein synthesis, reduction of disulfides and protection against toxic effects of oxidative damage (Meister, 1983;Lafleur et al., 1994).It has been demonstrated that adding beta m e r c a p t o e t h a n o l ( M E ) i n c r e a s e d intracellular GSH in the mo use lymphocytes (Zmuda and Friedenson, 1983).In mouse embryo, it was reported that glutathione content decreases about tenfold during preimplantation development and later stage embryos would be more sensitive to oxidative stress because of their lower GSH content (Nasr-Esfahani and Johnson, 1992).As for embryotrophic effect of ME in e m b r y o s , T a k a h a s h i e t a l .( 1 9 9 3 ) demonstrated that the adding low molecular weight thiols such â-ME and cysteamine into culture medium enhanced cysteine mediated GSH synthesis and improved the production of 6-to 8-cell bovine embryos in vitro.In addition to this, increased intracellular GSH content in oocytes and embryos of varied developmental stages improves embryonic development and embryo quality, resulting in higher blastocyst yield (Takahashi et al., 1993).The results obtained by Bagis and Odaman (2004a) demonstrated that the combined treatment of ME and amino acid to 1-cell stage embryos not only enhanced in vitro development to the blastocyst stage but also improved both the number of blastocyst Although studies have reported using mouse embryos, in intergeneric canine cellnuclear transfer embryo culture, the effect of b-ME and AA supplement in culture medium on embryo development is largely not known.Therefore, the present study was conducted to examine the efficacy of NCSU-23 medium supplemented with ME and AA on the developmental competence of intergeneric canine embryo using porcine oocyte in order to improve in vitro culture conditions.

Collection of porcine oocytes and invitro maturation (IVM)
Ovaries were obtained from a local abattoir and transported to the laboratory in o physiological saline at 30 to 35 C. Antral follicles 3 to 6 mm in diameter were aspirated using an 18-gauge needle attached to a 5-ml disposable syringe.Cumulusoocyte complexes (COCs) with compact cumulus cells were collected from the aspirate and washed several times in HEPES-buffered tissue culture medium (TCM)-199 (Life Technologies, Rockville, MD, USA).The COCs were then placed in IVM medium (Earle's salts-and Lg l u t a m i n e -c o n t a i n i n g T C M -1 9 9 supplemented with 26.2 mM NaHCO , 3.05 3 mM glucose, 0.91 mM sodium pyruvate, 0.57 mM L-cysteine, 75 mg/l kanamycin, 10 ng/ml epidermal growth factor (Sigma-Aldrich), equine chorionic gonadotropin (eCG, Intervet, Boxmeer, Netherland), 10 IU/ml human chorionic gonadotropin (hCG, Intervet Boxmeer Netherland), and 10% (v/v) porcine follicular fluid (pFF).The pFF was aspirated from superficial antral follicles 8 to 10 mm in diameter from prepubertal gilts.After centrifugation at 1,600 x g for 30 min, supernatant was collected and filtered sequentially through 1.2 mm and 0.45 mm syringe filters (Gelman b-Sciences, Ann Arbor, MI, USA).Prepared o pFF was then stored at -20 C until use.
A group of 50 COCs was cultured in 500ml IVM medium at 39°C in a humidified atmosphere of 5% CO and 95% air.After 2 culturing for 22 hr, COCs were transferred to eCG-and hCG-free IVM medium and cultured further for 20-22 h.At the end of the culture, oocytes were freed from cumulus cells by repeated pipetting in IVM medium containing 0.1 % hyaluronidase.Oocytes with a first polar body, intact zona pellucida, evenly granulated cytoplasm, expanded cumulus cells and distinct ooplasmic membrane were provided for SCNT of this study.

Preparation of recipient oocytes for somatic cell nuclear transfer
After 42-44 h of maturation, the oocytes were freed from cumulus cell by pipetting in H EPES-b uf f er ed NCSU-23 medium supplemented with 0.1% hyaluronidase.Oocytes were cultured in NCSU-23 containing 5 mg/ml bisbenzimide (Hoechst 33342; Sigma-aldrich Co.) and 7.5 mg/ml cytochalasin B for 30 min.Oocytes were placed in a 4 ml drop of HEPES-buffered NCSU-23 medium on working dishes.Each recipient oocyte was held with a holding micropipette (110 µm in outer and 24 µm in inner diameter) and zona pellucida was partially dissected with a fine glass needle to create a slit near the polar body.Then, the first polar body and adjacent cytoplasm containing metaphase plate were removed by squeezing.Enucleated oocyte were visually verified by ultraviolet fluorescence, keeping exposure to a minimum.The enucleated oocytes were then placed in NCSU23-D (Table 7) and used for SCNT.

Injection, Electrofusion and activation
Injection was performed in 4 ml drop of HEPES-buffered NCSU23-W medium and covered with light mineral oil.A single cell

Preparation of donor canine cells
Canine fibroblast cells were isolated from ear skin.The external surface of canine ear skin was shaved and cleaned aseptically.

3
A piece of ear skin tissue about 100 mm wide and 2 mm thick was biobsied and immediately immersed in D-PBS (Life technologies).After washing, the tissues were minced by a surgical blade on a 100 mm culture dish, followed by dissociation by 0.25% (w/v) trypsin containing 1 mM EDTA for 1 to 2 h at 38°C.Trypsinized cells were washed once by centrifugation (300xg, 2 min) and subsequently seeded into 100 mm culture dishes and cultured for 6-8 day in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 1 mM sodium pyruvate, 1 % (v/v) non-essential amino acid and 10 ml/ml penicillin/streptomycin solution in a humidified atmosphere of 95% air, 5% CO at 2 38°C before removal of unattached clumps of cells or explants.The attached cells were passage by trypsinization when confluent.
with smooth membrane was transferred into the perivitelline space of an enucleated oocyte.Before nuclear transfer, transfected donor cell were identified emission of greenfluorescence under an epifluorescent microscope using a standard fluorescein isothiocyanate (FITC) filter set.Roundshaped and green-colored small cell were individually injected into perivitelline space of enucleated oocytes.
Reconstructed oocytes were fused and activated simultaneously.The reconstructed oocytes were equilibrated for 10 sec in fusion medium (0.26 M mannitol, 0.1 mM MgCl , 0.1 2 mM CaCl , 0.5 mM HEPES and 0.05% BSA, 2 Table 3) and transfer to a fusion chamber with two electrodes (3.2 mm gap, BTX Inc., San Diego, CA) overlaid with the mannitol medium.Reconstructed oocytes (5-7 oocytes) were aligned with a fine mouthcontrolled Pasteur pipette in parallel with a fusion chamber.
Fusion was induced with a single DC pulse of 1.86 kV/cm for 30, by on a Electrocell Manipulator 2001 (BTX Inc.).All treated oocytes were washed three times with NCSU23-W supplemented with 4 mg/ml BSA, placed in 25 ml microdrops (10-15 oocytes per drop) of NCSU23-D under 0 mineral oil and cultured at 39 C, 5% CO , 5% 2 O and 90% N .Fused oocytes were 2 2 determined one hour after the electrical pulse under microscope.

Subsequent culture
The reconstructed embryos were cultured in 25 ml drops of NCSU23-D overlaid with mineral oil at 39ºC in humidified 5% CO , 5% O and 90% N

Discussion
The aim of this study was wanted to learn about good supporting media culture for optimal development of intergeneric cloned dog embryo using porcine oocyte as cytoplasm donor.In this study we used NCSU-23 or NCSU-23 plus ßME and amino acid.
The supplementation of amino acids to a culture medium significantly improved the development of hamster (Schini and Bavister, 1988), mouse (Lane and Gardner, 1994;Lamb and Leese, 1994; Lane and G a r d n e r , 1 9 9 7 ) , a n d c o w e m b r y o s (Rosenkrans and First;1994;Bavister and Arlotto, 1990;Bavister and Mckiernan, 1993).It has also been suggested that in vitro produced embryos should be exposed to amino acids as early as the oocyte stage, as this increases oocyte maternal mRNA levels a n d p r o m o t e s p r e i m p l a n t a t i o n development (Watson et al., 2000).In support of this opinion, it is notable that a brief exposure of zygotes to amino acid-free conditions depresses their developmental capacity and blastocyst cell numbers (Gardner and Lane,1996).
In bovine, a major step toward ameliorating media for cultureembryos was discovery that the addition of Eagle's amino

Statistical analysis
Data from all experiment in this experiment were analyzed using the statistical Analysis System (SAS) program.Data were subjected to analysis of variance (ANOVA) and protected least significant different (LSD) test to determine differences among experimental groups.When a significant model effect was found in each experimental parameter, data were compared by the least squares method.Statistical significance was determined where P value was less than 0.05.

Experimental studies Effect of amino acid and ß-mercaptoetanol supplement on NCSU-23 on intergeneric canine embryo development with porcine oocyte cytoplasm recipient
Canine intergeneric embryos with porcine oocyte cytoplasm recipient cultured in different media with randomly distribution.Embryo development were examined for cleavage rate and 144 h for final development after cultured in media.

Results
In this experiment, amino acid and ßmercapoethanol addition in medium NCSU-acids improved embryo development (Takahashi and First, 1992;Kim et al., 1993;Gardner 1994;Rosenkrans and First;1994).Much of understanding of the way that amino acids affect mammalian embryo development and subsequent viability has come from studies on the hamster (Carney and Bavister, 1987;Bavister and Arlotto, 1990;Bavister and Mckiernan, 1993;Mckiernan et al., 1995), mouse (Mehta and Kiessling, 1990;Gardner and Lane, 1993;1996;Lane and Gardner, 1994;1997;1997a), and rat ( Zhang and Armstrong, 1990).These studies have determined that amino acids can be either stimulatory or inhibitory to embryo development in vitro and that the presence of amino acids in culture media has a significant effect on the viability of e m b r y o s a n d p o s t i m p l a n t a t i o n development.
On the other hand, low molecular weight thiol compounds, such as b-ME, have been reported to reduce cystine to cysteine and also to promote the uptake of cysteineenhancing glutathione synthesis (Takahashi et al., 2002).Moreover, it has been reported that b-ME transports cystine, forming a mixed disulphide that is taken up to facilitate the uptake of cysteine into the cells (Ishii et al., 1981).Data on the effect of the concentration of b-ME are conflicting.In a study of Abeydeera et al. (1998) reported that adding 0 to 50 µM b-ME during in vitro maturation of pig oocytes increased intracellular glutathione concentration and subsequent embryo development and blastocyst cell numbers.The concentration (10 µM) of b-ME used in the present study was different from that (50 µM) reported by Takahashi et al., (1993) but it was the same as that reported by Hamano et al., (1997) (10 to 50 mM).Glutathione has many important functions in cells or embryos for the mechanism of cell defense during oxidative stress and toxicants (Gardner et al., 2000).It is possible that GSH synthesis in embryos was increased by addition both of b-ME and AA into NCSU-23 medium and will be resulted in higher blastocysts and total cell numbers than that of control group.
Amino acids and b-ME both can play a vital role for preventing the oxidative stress of embryo (Ishii et al., 1981;Issels et al., 1988;Hamano et al., 1994;Reed, 1994;Gardner and Reed, 1995;1995a;Lane and Gardner, 1997;Takahashi et al., 2002).There is a positive correlation between glutathione (GSH) and oxidative stress of the embryo.Glutathione is a tripeptide thiol synthesized by glutamic acid, cysteine, and glycine in pathway of the glutamyl cycle (Abeydeera et al., 1998;Gardner et al., 2000).Most cells do not take GSH intact from outside the cell; instead, GSH is broken down at the cell membrane, the constituent AA are taken up, and GSH is resynthesized inside the cell or the AA are used for other pathways (Reed, 1994).It has been reported that during IVC, GSH level drops approximately ten fold beginning from the unfertilized oocyte to blastocyst stage in mouse (Gardner and Reed, 1994;1995).The availability of precursor AA is a regulatory factor in GSH synthesis, and it is likely that AA supplied from outside the cell provide a control point in mammalian cells (Issels et al., 1988).It has been reported that nonessential AA and glutamine accelerate the time of the first three cleavage divisions and increase the time of the first three cleavage divisions and increase the time of compaction in the mouse when added to culture medium (Lane and Gardner, 1997).In another findings observed that NEAA improves development during cleavage, whereas EAA supports development after 8 cell stage (Van Winkle and Dickinson, 1995) Unfortunately, the in vitro embryo culture media used in this study does not which matched with macaca embryo.Another research with Canine cell with bovine oocyte recipient in SOF medium got 0.4% blastocyst although the number of blastocyst cell only 50 (Murakami et al., 2005).
These indication told that, for in vitro culture canine embryo, NCSU-23 are suboptimal for embryo development although for several mammalian these media supported the development (Rosenkrans, 1998;Biggers, 2000) and improvement culture system for the development of canine embryo still necessary.

Acknowledgment
This study was done in Laboratory of Theriogenology and biotechnology, College of Veterinary Medicine Seoul National University.The authors acknowledge to my professor, Hwang Woo-Suk PhD, Lee Byeong-Chun, PhD and Kang Song-Keun PhD for their supporting my study in this laboratory.(Wen et al., 2003).In this study we used media which usually we used as donor cytoplasm (NCSU-23) and also subsequent development is dependent on successful transfer of control to the embryonic genome, which may be deficient in embryos produced by somatic nuclear transfer.Establishment embryos from different mammalian species require species-specific embryo culture conditions (Leibfried-Rudledge et al., 1997).The ability of NCSU to support embryonicdevelopment of interspecies units may be attributable to the fact that this initial development of embryo is driven by recipient cytoplasm, and also thus any media supporting bovine and porcine embryo development might also support the initial development of interspecies NT embryos.Another morphological features that characterize the time of the transition is a developmental block in non permissive in vitro culture conditions (Crosby et al., 1988), embryos reconstructed by SCNT are more susceptible to suboptimal culture condition than IVP embryos, indicating a need to improve c u l t u r e f o r m u l a t i o n s t o e n h a n c e development of these compromised embryos which may have abnormal metabolic activities (Chung et al., 2002).It means that embryo from different mammalian species require species-specific c u l t u r e m e d i a .Y o n g e t a l .( 2 0 0 3 ) demonstrated that the interspecies nuclear transfer embryo (macaca fibroblast cell and rabbit oocyte) have been block at 4 cell development stage when used media for rabbit embryo culture and reached the blastocyst stage when culture in media

References
to fifteen embryos were cultured together.Cleavage rate was recorded after 48 h post fusion.On 7 days p o s t f u s i o n , t h e d e v e l o p m e n t o f reconstructed embryos was recorded, and GFP expression rate in embryo was examined under FITC filter.

Figure 1 .
Figure 1. Adult canine ear skin fibroblast cell line

Table 1 .
Composition of North Carolina State University (NCSU)-23 medium

Table 2 .
Effect of amino acid and â-mercaptoetanol supplementation in NCSU-23 medium on canine embryo development with porcine oocyte cytoplasm recipient