Developmental Competence of Early Stage Porcine Embryos Cultured in Medium with Different Energy Substrate in vitro

To elucidate the effect of energy requirement during the early embryonic development on their developmental ability to develop to blastocyst stage, in vitro fertilized (IVF) porcine one-cell embryos were cultured in modified North Carolina State University (NCSU)-37 supplemented with different energy substrate. Result indicated that the cleavage rate of embryos in Pyr-Lac and Gluc-Pyr-Lact groups was significantly higher than in those in Gluc group and Gluc-Rib group (P < 0.05). At Day 6 of culture, the highest proportion of embryos develop to the blastocyst stage was obtained in the presence of pyruvate-lactate only. In the medium with glucose, the addition of pyruvate-lactate or ribose slightly increased the proportion of embryos develop to the blastocyst stage, however the value were not significantly different form those obtained in the presence of glucose only. The mean cell number in blastocysts derived from Pyr-Lac and Gluc-Pyr-Lact groups were significantly higher than those in the Gluc group (P < 0.05). These results indicated that the presence of glucose only, as energy substrate, during the first 2 days of in vitro culture (IVC) caused a decrease in development of in vitro produced (IVP) porcine embryos to the blastocyst stage and mean cell number in blastocysts .


Introduction
The development of culture system to support early embryonic development is important for study of the factor that are involved as well as for practical use in such biotechnologies as in vitro fertilization (IVF), nuclear transplantation and gene transfer by DNA microinjection.Embryos from most s p e c i e s e x h i b i t l e s s t h a n o p t i m a l development in vitro and may 'block' cleavages with elevated glucose and oxygen consumption as they approach cavitation (Houghton et al., 1996).Most mammalian embryos need pyruvate as a substrate of energy in the early stages of development (Rieger et al., 1992;Gardner et al., 1993).Pyruvate metabolism was relatively high at the two-and four-cell stages, declined to a minimum at the compacted morula stage, and then increased at blastulation.Glucose metabolism via glycolysis, on the other hand, increased between the eight-cell and 16-cell stage, and continued to increase up to the blastocyst stage (Rieger et al., 1992).Similarly, glucose uptake by sheep embryos is low before the 16-cell stage and increases steadily thereafter (Gardner et al., 1993).Unlike cattle embryos, pyruvate uptake by sheep embryos is relatively constant up to the morula stage and then increases significantly through the blastocyst stage (Thompson et al., 1992;Gardner et al., 1993).In the mouse, although the zygote and early cleavage stage embryo cannot use glucose as the sole substrate of energy, the blastocysts exhibit a large capacity to utilize glucose both oxidatively and through aerobic glycolysis (Chatot et al., 1989).In pigs, it has been reported that the embryos consumed glucose and produced lactate at all stages of development, utilization of glucose was low during the early cleavage stages, increased slightly at the morula stage and reached a peak at the early blastocyst stage (Sturmey and Leese, 2003).Therefore, the uptake and metabolism of energy substrate such as, glucose and pyruvate are appears to be developmentally related.
Although IVF and embryo production using in vitro matured oocytes have been successful and many laboratories have tried to overcome inadequacies of their IVC system by supplementing culture medium with various energy substrate or using stepwise culture system with components that may be advantageous for different stages of embryo development (Petters and Wells 1993;Gandhi et al., 2001;Kikuchi et al., 2002;Karja et al., 2004aKarja et al., , 2004b)), their developmental ability and the quality of blastocyst yielded are also still low compared to in vivo-derived blastocyst.Therefore, the purpose of this study was to observe developmental ability of early stage porcine embryos after culturing in medium with different energy substrate.We have focused specifically the experiment on the first 2 days of culture, early stage of embryo development, since the first 2 days of culture in porcine embryos seem to have a critical effect on further development (Kikuchi et al., 2002), at period when the activation of embryonic genome occurs.In the suboptimal in vitro culture conditions, embryos are blocked in developmental or slowing down in cleavages at the stage when the activation of embryonic genome occurs (Telford et al., 1990)

In vitro fertilization (IVF)
Spermatozoa were thawed and preincubated for 15 min at 38.5°C in a tissue culture medium (TCM) 199 with Earle's salts (Gibco) adjusted to pH 7. lactate, 3 mg/ml bovine serum albumin (BSA; Fatty acid free, Sigma), 5 mM caffeine (Sigma) and 50 µg/ml gentamicin.The final sperm concentration was adjusted to 1 x 6 10 /ml.The oocytes were co-incubated with spermatozoa for 3 h.And then, the inseminated oocytes were denuded from the cumulus cells and attached spermatozoa by mechanical pipetting and transferred to culture medium to which they had been designed, as bellow.

Embryo culture
The basic medium used for embryo culture was glucose-free NCSU-37 containing 4 mg/ml BSA and 50µM bmercaptoethanol (IVC medium).To observe developmental ability o developmental ability of early stage porcine embryos after culturing in medium with different energy substrate for the first 2 days of culture, resumptive zygotes were cultured from p Days 0 (the day of insemination was defined as day 0) to 2 in IVC medium supplemented with (1) 5.5 mM glucose (Gluc group), (2) 0.17 mM sodium pyruvate and 2.73 mM lactate (Pyr-Lact group), (3) 5.5 mM glucose, 0.17 mM pyruvate and 2.73 mM lactate (Gluc-Pyr-Lact group), or (4) 5.5 mM glucose and 5.5 mM Ribose-5 phosphate (Gluc-Rib group).Subsequently, embryos in all groups were cultured until Day 6 in IVC medium supplemented with 5.5 mM glucose only.
Culture was performed at 38.5°C and 5% CO 2 under 5% O and 90% N .

Embryos evaluation
For examination of the embryo development, on Day 6 all embryos in all groups were fixed and permeabilized for 15 min at room temperature with 3.7% paraformaldehyde in PBS containing 1% (v/v) Triton X-100 (Sigma), and then placed in PBS supplemented with 0.3ÿ (w/v) polyvinylpyrrolidone (PVP; Sigma) for 15 min at room temperature.The embryos were placed on a microslide in a drop of mounting medium consisting of 90% (v/v) glycerol containing 1.9 µM bis-benzimide (Hoechst 33342; Calbiochem; EMD Biosciences, Inc. ).A coverslip was placed on top of the embryos and the staining of the nuclei was examined with a fluorescence microscope (BX 51; Olympus).The numbers of cleaved embryos including the stage of embryo development, blastocysts formation rate, and the cell number in blastocyst were recorded.

Statistical Analysis
All percentage data were subjected to arc-sine transformation before statistical analysis.Then all data, including the arcsine-transformed percentages of blastocyst formation and the data on mean number of cells in the blastocyst were subjected to ANOVA by a post hoc, Fisher's protected least significant difference test (PLSD test) using the STATVIEW (Abacus Concepts Inc., Berkeley, CA, USA) program Data were expressed as means ± SEM.Differences at P < 0.05 were considered significant.

Results and Discussion
The results of in vitro cleavage and development of porcine zygotes to the blastocyst stage and total cell number in blastocyst are shown in Table 1.When the embryos were cultured in the medium without any energy substrate, no cleavage and development to blastocyst stage were observed.In the medium supplemented with pyruvate-lactate only (Pyr-Lac group) or together with glucose (Gluc-Pyr-Lact groups), the proportion of embryos that cleaved at 48 hours after IVC was significantly higher than in medium with glucose only (Gluc group) or together with ribose (Gluc-Rib group) (P < 0.05).At Day 6 of IVC, the highest proportion of embryos develop to the blastocyst stage was obtained in the presence of pyruvate-lactate only.In the medium with glucose, the addition of pyruvate and lactate or ribose slightly increased the proportion of embryos develop to blastocyst stage, but the values were not significantly different from those obtained in gluc-group.
. As shown in Table 1, the mean cell number in blastocysts derived from Pyr-Lac and Gluc-Pyr-Lact groups were significantly higher than those in the Gluc group (P < 0.05).Developed porcine embryos at blastocyst stage and blastocyst with Hoechst-labeled nuclei at Day 6 of culture were represented in Figure 1.
The types and concentrations of energy substrates added to a culture medium alter metabolic profiles, development and quality of embryos.The results from this study has demonstrated that when porcine IVM-IVF oocytes were cultured in IVC medium supplemented with pyruvate and lactate for the first 2 days and then cultured in the medium containing glucose for subsequent 4 days, the rate of blastocyst formation the cell number in blastocysts were higher as compared with that of glucose supplement for the first 2 days.Although porcine embryos has been reported are able to utilize glucose throughout their development, results from the present study indicate that glucose alone as energy substrate during early stage of porcine embryos development could not support development of the embryos to the blastocyst stage.The lack of response to the glucose in this study could be explained as the limited ability of early stage  porcine embryos to utilize glucose (Sturmey and Leese, 2003) or the embryos use only a fraction of the glucose that they take up (Swain et al., 2002).Porcine embryos appear to behave similar fashion metabolically as do other domestic livestock species embryos, with limited glucose utilization before the morula stage and a heavy reliance on glycolysis during blastocoele formation (Flood and Wiebold, 1988;Rieger et al., 1992;Gandhi et al., 2001).On the other hand, culturing porcine embryos in a combined medium that provided 5.5 mM glucose, pyruvate and lactate, or glucose combining with 5.5 mM ribose 5-phosphate, the end product of glucose metabolism through pentose phosphate pathway (PPP), as energy substrates for the first 2 days and followed by culture in medium with glucose (5.5 mM) the next 4 days slightly improved blastocyst development and cell number in blastocysts compared with those cultured for 144 h in medium with glucose.Taken together, these results clearly indicate that glucose in IVC medium for the first 2 days of culture is detrimental to the development of embryos, while pyruvate and lactate appear to be important energy substrates for both early embryonic development in vitro and for improving the number of cells in blatocysts.Our observation confirms the previous reports, which reported that pyruvate and lactate sseem to be predominant energy substrates for the first cleavage division for porcine embryos (Kikuchi, 2002, Kim et al., 2004).Pyruvate and lactate may also provide the embryos with more suitable conditions for cellular oxidation reduction equilibrium, resulting in viable embryo growth and development (Thompson et al. 1993).Butcher et al. (1998) recently suggested that pyruvate might be converted to alanine and may play a role in removing ammonia from embryos.Another possible function for pyruvate is to protect embryos against oxidative stress as suggested by Leese (1991).
Glucose is usually included in standard tissue culture media at a concentration of 5.5 mM, on the basis of its concentration in serum.This is much higher compared than in the oviduct, in which in vivo porcine -1 embryos are exposed (0.17 mmol glucose l ) (Nichol et al., 1992).The deleterious effects of glucose on embryo development in vitro have been reported.The most likely reason that glucose affects the development of early stage embryo, therefore, is that one or more metabolites generated by the PPP leads to a failure in development of the embryos.One possibility is that NADPH, although generally regarded as protective against free radical damage (Salvemini et al., 1999), is produced in such quantities that its reoxidation actually generates oxygen free radicals, an occurrence that is well documented in some cell types (Babior, 2000).The use of glucose as an energy substrate results in the production of reactive oxygen species (ROS), particularly the superoxide anion and the hydroxyl radical.Production of superoxide anion and H O via glucose metabolism through PPP 2 2 has been described on rabbit blastocyst (Manes and Lai, 1995) extremely reactive OHÉ.The overgeneration of intracellular ROS during culture of mammalian embryos in vitro is generally thought to be detrimental to embryo development (Johnson and Nasr-Esfahani, 1994;Guerin et al., 2001).
8. A portion (10 µl) o f p r e i n c u b a t e d s p e r m a t o z o a w a s introduced into 90 µl of fertilization medium c o n t a i n i n g 1 0 -2 0 m a t u r e d C O C s .Fertilization medium consists of 90 mM NaCl, 12 mM KCl, 25 mM NaHCO , 0.5 mM 3 NaH PO , 0.5 mM MgSO , 10 mM sodium 2 4 4

Figure 1 .
Figure 1.Developed porcine embryos at blastocyst stage (A) and blastocyst with Hoechst-labeled nuclei (B) at Day 6 of culture (original magnification 100x).
. Reactive oxygen species are highly active electron acceptors, able to strip electrons from other molecules that, in turn, become free radicals.Hydrogen peroxide (H O ) is not a radical per se, but is a