Effect of MEM Vitamins Supplementation of In vitro Maturation Medium and In vitro Culture Medium on the Development of Porcine Embryos

This study was carried out to examine the influence of minimum essential medium (MEM) vitamins supplementation to in vitro maturation medium and in vitro culture medium on the development of porcine embryos. Porcine embryo development was investigated following cultivation in both in vitro maturation and culture medium with the supplementation of MEM vitamins (0, 0.1, 0.2 and 0.4%) using immature oocytes collected from the ovary of prepubertal gilts. Embryo development was observed and the total cell number in each blastocyst generated under the culture conditions was quantified following supplementation of the medium. The embryonic development rate of the blastocyst and hatched blastocyst was higher, but not significantly so, when 0.4% MEM vitamins were supplemented to the in vitro maturation medium of the porcine oocyte. Interestingly, the total number of cells in the blastocyst was significantly higher in the in vitro maturation MEM vitamins supplemented group compared to either the untreated group or the group which had MEM vitamins supplemented to both in vitro maturation and in vitro culture medium simultaneously (p<0.05). Therefore, the supplementation of 0.4% MEM vitamins to the in vitro mature medium has a beneficial effect on the embryonic development of in vitro produced blastocysts derived from the immature porcine oocytes. (


INTRODUCTION
In vitro production (IVP) of porcine embryos is used not only to study the generation process of mammalian embryogenesis but also widely used for both veterinary animal husbandry and biotechnology such as in the production of individuals containing superior genetic characters by embryo transplantation, the development of micro manipulation techniques, and stem cell studies.
Over the past several years, researchers have achieved in vitro fertilization of porcine oocytes (Iritani et al., 1978) and have succeeded in birthing piglets by in vitro embryo transplantation (Mattioli et al., 1989).Three main factors which determine the success of in vitro produced mammalian embryos are the in vitro maturity level of immature oocytes, the in vitro fertilization process and the method of embryos in vitro culture.Although multiple babies can be produced using in vitro produced blastocysts in other mammalian animals (Kikuchi et al., 2002), embryo transplantation technology is not as well established in the case of porcine.For example, cytoplasm maturation along with nucleus maturation is not well described in porcine, compared with other animals.Therefore, there are still many difficulties in securing high quality embryos due to the developmental retardation and stoppage (Camous et al., 1984;Heyman et al., 1987) in the 4-cell stage during in vitro culture because of unstable in vitro maturation (Wang et al., 1997), unstable male pronucleus generation (Motlik et al., 1984) and polyspermy during in vitro fertilization.
Several new media have been developed such as mWM, beltsville embryo culture medium-3 (BECM-3) consisting of simple minerals, an energy source, amino acids, a pH buffer, trace elements and antibiotics (Dobrinsky et al., 1996), low concentrated sodium lactate and NCSU23 with glucose (Petters and wells, 1993;Abeydeera et al., 2000;Abeydeera et al., 2001) and free-glucose and porcine zygote medium-3 (PZM-3) containing various amino acids (Yoshioka et al., 2002).In addition, improvements have been made to the in vitro production methods of generating porcine oocytes by co-culture with somatic cells (Xu et al., 1992), a static/perifusion culture system (Lim et al., 1997), the supplementation of growth factors (May et al., 1988;Chang et al., 2000), and the supplementation of antioxidants (Ali et al., 2003) since active oxygen in the medium is harmful for embryo development (Legge et al., 1991).In spite of these developments, less than 25% of total oocytes produce blastocysts following the in vitro culture of porcine oocytes (Stoke et al., 2005) with very low reproducibility and this leads to a remarkably low pregnancy rate following embryo transplantation (Prather and Day, 1998).
Among the alterations of in vitro culture conditions, it was reported that the supplementation of water-soluble minimum essential medium (MEM) vitamins to the in vitro culture medium is effective for the blastocole expansion of the blastocysts of rabbits (Kane, 1988).Furthermore, in the case of the hamster, it was also reported that MEM vitamins supplementation increased the embryo development rate of blastocysts and the hatched blastocysts from the 8-cell stage (Mishra and Seshagiri, 1998).However, MEM vitamins supplementation was not effective for porcine blastocyst development (Yoshioka et al., 2002).Likewise, the effect of MEM vitamins supplementation to in vitro embryo production is different for each animal species and the effect of MEM vitamins has not been clearly determined.Therefore, in this study, we researched the effects of MEM vitamins supplementation to in vitro maturation and culture medium on the development of in vitro porcine embryos in order to establish enhanced culture conditions.

Chemicals and media
Unless otherwise stated, all chemicals used in this study were purchased from Sigma Chemical Co.(St, Louis, MO, USA).Solutions are expressed as percent dilutions (v:v) and all media used for in vitro maturation, in vitro fertilization, and in vitro culture were pre-warmed to 39°C in a 5% CO 2 incubator with maximum humidity for 4 h before use.

Oocyte collection and in vitro maturation
Ovaries were collected from prepubertal gilts at a local slaughterhouse and transported to the laboratory within 2 to 3 h in saline supplemented with 25 μg/ml gentamicin at 25-30°C.Cumulus oocyte complexes (COCs) were obtained by aspiration from follicles 2 to 6 mm in diameter using an 18gauge needle connected to a 10-ml disposable syringe.Only COCs with compact cumulus cell layers and evenly granulated ooplasm were selected.The COCs were washed three times in HEPES-Tyrode-albumin-lactate-pyruvate medium (TALP medium) supplemented with 25 mM HEPES and 3 mg/ml BSA.Groups of 50 COCs were placed into 500 μl of BSA-free NCSU-23 solution with 0.57 mM cysteine, 10% porcine follicular fluid (pFF), 2.5 mM β-mercaptoethanol, 10 ng/ml epidermal growth factor (EGF), 10 IU/ml human chorionic gonadotropin (hCG) and 10 IU/ml pregnant mare serum gonadotropin (PMSG) in each well of a 4-well multidish (Nunc, Roskilde, Denmark).After 22 h for maturation, oocytes were washed twice in the same maturation medium without PMSG and hCG and cultured in this medium for 22 h at 39°C in an atmosphere of 5% CO 2 and maximum humidity.

Supplementation of MEM vitamins on in vitro maturation medium
0.1, 0.2 and 0.4% of MEM vitamins (minimum essential medium vitamins, 11120-052, Life Technologies Inc., Grand Island, NY, USA) were added to in vitro maturation medium and in vitro culture medium as indicated.The blastocyte and hatched blastocyst development rate was then monitored following in vitro fertilization.

Sperm preparation and in vitro fertilization
Diluted porcine semen was produced by Dabby A.I. Center and stored at 17°C for 5 d.The semen was layered on top of a discontinuous Percoll density gradient (2 ml of 45% Percoll over 2 ml of 90% Percoll) in a 15-ml centrifuge tube.The sample was centrifuged for 20 min at 500×g at room temperature.The spermatozoa collected in the bottom fraction were washed three times: twice in D-PBS containing 1 mg/ml BSA, 100 μg/ml penicillin and 75 μg/ml streptomycin at 500×g for 5 min, and once in modified Tris-buffered medium (mTBM).The spermatozoa were diluted with mTBM to give a final concentration of 3×10 6 spermatozoa/ml.
After the IVM period, oocytes were briefly treated with 0.1% hyaluronidase in Dulbecco's phosphate-buffered saline (D-PBS, Gibco, USA) supplemented with 1 mg/ml BSA to remove cumulus cells, and were washed 2 to 3 times with mTBM containing 1 mg/ml BSA and 2.5 mM caffeine sodium benzoate (Abeydeera et al., 1997).After washing, groups of 25 to 30 oocytes were placed in 48-μl droplets of mTBM in 60-mm petri dishes that had been covered with warm mineral oil.Two microliters of spermatozoa suspension was added to each fertilization drop, resulting in a final concentration of 2.5×10 5 spermatozoa/ml.Oocytes and spermatozoa were coincubated for 6 h at 39°C and 5% CO 2 with maximum humidity.

In vitro culture
After sperm-oocyte co-incubation, presumptive zygotes (d 0) were washed 2 to 3 times in PZM3 supplemented with 3 mg/ml BSA.Thirty oocytes were transferred to 50 μl of PZM3 in a 60-mm petri dish, covered with warm mineral oil, and cultured for 168 h at 39°C in a humidified atmosphere of 5% CO 2.

MEM vitamin supplementation on in vitro culture medium
The presumptive zygotes were cultured in medium containing 0.1, 0.2 and 0.4% of MEM vitamins as indicated.0.4% MEM vitamins was supplemented on d 0, d 2 and d 4.After 48 h of in vitro culture, the fertilization rate was examined and the blastocyst and hatched blastocyst development rate was examined after 168 h.

Blastocyst differential staining
The zona pellucida of the blastocysts was removed with a 0.5% protease solution and washed 4 to 5 times in TL-HEPES solution with 0.1% PVA (TL-PVA).Zona-free blastocysts were incubated in a 1:5 dilution of rabbit antibovine whole serum in TL-PVA medium for 1 h.After being washed five more times in TL-PVA medium, blastocysts were re-incubated in a 1:10 dilution of a guinea pig complement in TL-PVA medium supplemented with 4 μg/ml propidium iodide (PI) and 4 μg/ml bisbenzimide for 1 h.The presumptive stained blastocysts were mounted on a slide and the cells were counted under a fluorescence microscope (Olympus, Tokyo, Japan).The bisbenzimidestained inner cell mass (ICM) nuclei fluoresced blue, and the trophectoderm (TE) nuclei, which were stained with both bisbenzimide and PI, fluoresced red or pink.

Statistical analysis
Data on the embryo development were analyzed by the x 2 -test.All data for cell number were arcsine-transformed and analyzed by the General Linear Models Procedure with the Statistical Analysis System (SAS; Cary, USA).Treatment means were compared with Duncan's multiple range test; p-values less than 0.05 were considered statistically significant.

Altering the concentration of MEM vitamins on in vitro maturation medium has no effect on embryonic development rate
The concentration of supplemented MEM vitamins for in vitro maturation medium ranged from 0.1% to 0.4% in treated groups (Table 1).The in vitro fertilization rate was similar among all groups, ranging from 61.1% to 67.8%.Similarly, there was no significant difference among the treated groups on the embryonic development rate of the blastocyst which ranged from 11.4% to 14.3%.In addition the embryonic development rate of the hatched blastocyst ranged from 3.4% to 18.2%, which was also not significantly different among the treated groups.

Altering the concentration of MEM vitamins on in vitro culture medium has no effect on embryonic development rate
The concentration of supplemented MEM vitamins for in vitro culture medium ranged from 0.1% to 0.4% in treated groups (Table 2).The in vitro fertilization rate was not significantly different among the treated groups, ranging from 58.3% to 63.5%.Similarly, there was no significant difference among the treated groups on the embryonic development rate of the blastocyst which ranged from 4.3% to 14.4%.In addition, the embryonic development rate of the hatched blastocyst ranged from 7.7% to 31.3%, which was also not significantly different among the treated groups.

Altering the timing of addition of MEM vitamins supplementation to in vitro culture medium has no effect on embryonic development
The time to supplement of the MEM vitamins was experimentally altered from d 0 to d 4 in the treated groups (Table 3).The in vitro fertilization rate was not significantly different among the treated groups, ranging from 62.8% to 65.2%.Similarly, there was no significant difference among the treated groups on the embryonic development of the blastocyst which ranged from 11.5% to 13.6%.In addition, the embryonic development rate of the hatched blastocyst ranged from 7.1% to 26.7%, which was also not significantly different among the groups.

Altering the order of cultivation medium which contain MEM vitamins has no effect on embryonic development
To determine if addition of MEM vitamins to either or both in vitro media has an effect on embryonic development rate, four groups were grown under the following conditions: the double treated group in which 0.4% MEM vitamins supplementation was applied to both the in vitro maturation medium and the in vitro culture medium (+, +), one in which 0.4% MEM vitamins was supplemented only to the in vitro maturation medium (+, -), one in which 0.4% MEM vitamins was supplemented only to the in vitro culture medium (-, +), and the untreated group which had no MEM vitamins in any medium (-, -) (Table 4).
Within these various conditions, the in vitro fertilization rate ranged from 60.8% to 65.2%, which was not significantly different among the treated groups.The embryonic development of the blastocyst ranged from 12.0% to 15.1%, which was also not significantly different among the treated groups.Finally, the embryonic development rate of the breeding blastocyst ranged from 5.9% to 18.9%, which was also not significantly different among the treated groups.

MEM vitamins supplementation of in vitro maturation medium alone increases the total number of cells in the blastocyst
The total number of cells was 39.0±1.7 in the treated group (+, -) where MEM vitamins were supplemented to the in vitro maturation medium but not the in vitro culture medium (Table 5).This was significantly higher than 22.0±3.0 cells of the untreated group (-, -) and 29.5±8.6 cells of the double treated group (+, +) where both the in vitro maturation medium as well as the in vitro culture medium were supplemented with MEM vitamins (p<0.05).In addition, the number of trophectoderm (TE) cells was 34.7±1.5 in the treated group (+, -) with supplemented in vitro maturation medium and untreated in vitro culture medium, which was significantly higher than 18.3±2.1 of the untreated group (-, -) and 20.0±2.0 of the double treated group (+, +) (p<0.05)In contrast, the number of inner cell mass (ICM) cells among the total number of cells ranged from 3.7±1.5 to 4.3±1.1,which was not significantly different among the groups.

DISCUSSION
The embryonic development rate of the blastocyst and hatched blastocyst in the group where 0.4% MEM vitamins were supplemented to the in vitro maturation medium but not the in vitro culture medium (+, -) was slightly higher than that of other treated groups, however it was not effective for significantly increasing embryo development.In contrast, supplementation of MEM vitamins to the in vitro maturation medium but not the in vitro culture medium (+, -) resulted in a significant increase in the total numbers of cells compared to the untreated group (-, -) or the double treated group (+, +) (p<0.05).
There have been many attempts to create an in vitro culture condition for oocytes which mimic the natural environment of the internal mammalian condition.As results of these studies, several effective improvements have been made such as a method to decrease the oxygen concentration in the in vitro culture medium (Fukui et al., 1992), supplementation of an alternative energy source (Choi et al., 2002;Rizos et al., 2003) growth factor addition (Saito et al., 1984;Ogawa et al., 1987), supplementation of antioxidant that protects in vitro oocyte from free radicals (Murray et al., 1990;Ali et al., 2003), and supplementation of serum (Nagai et al., 1988;Mattioli et al., 1989).Additionally, in the case of bovine, embryo development was improved when it was co-cultured with mouse fetus fibroblast, by producing either an unknown growthpromoting factor or by removing a risk factor for oocytes that exist in the medium from the coculture cell (Eyston and First, 1989).However, currently developed in vitro environment was not yet appropriate for oocyte culture.
Although Yoshioka et al. (2002) reported that MEM vitamins was not effective in porcine embryo development, Rabbit (Kand and Bavister, 1988) and hamster (Mishra and Seshagiri, 1998) were effective in improving the embryo development rate.Likewise, there is still controversy regarding the effect of supplementation of water-soluble vitamins on the in vitro development of embryos of mammalian animals.
The kinds of vitamins that are supplemented with MEM vitamins generally consist of myo-inositol (0.02%), nicotinamide (0.01%), pantothenic acid (0.01%) which is the complex of vitamin B, pyridoxine hydrochloride (B6, 0.01%), riboflavin (0.001%), thamine HCl (0.01%), choline chloride (0.01%) and folic acid (0.01%) (Hong et al., 2004).It was reported that these vitamins are imported actively or passively into the cell and prevent the loss of oocyte viability that occurs in suboptimal culture conditions.Although the overall transfer system as well as the mutual cooperation of these vitamins are still unknown, the roles of individual vitamins have been identified.For example, choline plays an important role in maintaining the metabolism of phospholipids and cell structure (McDowell, 2000), folic acid is important for proteolysis, DNA synthesis and in energy production (McDowell, 2000), nicotinamide is important for glycolysis and the redox reactions of cells including most of the citric acid cycle during cell respiration (Stryer, 1988), riboflavin plays a role as a precursor for two important subsidiary enzymes which are important for energy production and flavin mononucleotide as well as flavin adenine dinucleotide act as hydrogen carriers to produce energy in the form of ATP through the metabolism of lipids and carbohydrates (McDowell, 2000).
Also, regarding the embryo, pantothenate promotes the development of the blastocyst and is important for embryo vitality, at least in the hamster (McKiernen and Bavister, 2000).Inositol, which is known to play an important role in the in vitro maturation of oocyte, is involved in phospholipid synthesis for new cell membranes (Hawthorne, 1982;Vance, 1986), pronucleus formation during in vitro fertilization (Kane and Bavister, 1988), and it acts as the phosphatidylinositol second messenger (Kane and Bavister, 1988).
These processes are responsible for mediating numerous hormone and growth factor reactions.When these vitamins are supplemented to in vitro maturation medium, they have the potential to improve the effect of PMSG, HCG and EGF on the in vitro maturation medium.Also, it is known that vitamin B6 displays antioxidant activity that inhibits free radicals (Olson and Seidel, 2000) during the in vitro culture of embryo (Kamat and Devasagayam, 1996;Mcdowell, 2000;Kitagawa et al., 2004).In addition, Lane and Gardner (1998) reported that vitamins play roles in inhibiting the vitality loss and unstable metabolism due to inappropriate culture conditions.
In conclusion, the results of this study demonstrate that MEM vitamins supplementation to in vitro maturation medium alone does have an effect on embryo development as seen by an increase in cell numbers.Therefore, the effect of MEM vitamins can be offset by hormones, albumin or other components that are supplemented to in vitro maturation medium.However, the supplementation of MEM vitamins to in vitro culture medium did not affect development of the porcine embryo in vitro.Further studies are required to understand the reason for the suppression of porcine embryos produced in vitro with supplementation of MEM vitamins to the culture medium.

Table 1 .
Effect of increasing concentration of MEM vitamins during in vitro maturation on the development of porcine oocytes

Table 2 .
Effect of increasing concentration of MEM vitamins during in vitro culture on the development of porcine in vitro

Table 3 .
Effect of altering the addition time of MEM vitamins during in vitro culture the on development of porcine in vitro fertilized embryos

Table 4 .
Effect of addition of MEM vitamins during in vitro 1 -: without, +: with.

Table 5 .
Effect of MEM vitamins supplements on cell number of porcine blastocysts developed from in vitro produced embryos