1. Impact of aspartate and serine on mouse embryo development in vitro BACKGROUND Attempts to improve in vitro culture conditions of early embryos must determine the nutritional requirement of individual nutrient components that impact embryo development in vitro. The findings of the present research effort is to assist elucidate the effect of two amino acids on embryo development. Amino acids are crucial for protein synthesis, energy metabolism and for its role as osmolytes. A 15-20 YEARS RESEARCH EFFORT: This study is part of a 15-20 years of research effort to culture embryos in synthetic and chemically defined conditions (Ali, 1997; Ali et al., 2000; Ali, 2007) devoid of added donor serum proteins. OBJECTIVE The objective is to determine the optimal concentrations, tolerance levels and effects of aspartate and serine on mouse embryo development in vitro. MATERIALS AND METHODS Earle’s Basal salt solution was supplemented with 5mg/ml of bovine serum albumin, antibiotics penicillin/ streptomycin sulfate, and pyruvate/ lactate. Treatments consisted of culture media supplemented with graded amounts (0, 250,500,750 and 1000mM) of aspartate and serine. Quakenbush sibling mice zygotes were randomly apportioned to individual treatments. The numbers of replicates were 5 and 4 respectively. The end point was the development of day 6 (expanded, hatching, hatched) embryos. RESULTS The proportion of day 6 embryos developed decreased when supplemented with aspartate with the portion of dead increased 29.1, 34.2, 41.8, 40.3, 32.9% with the addition of graded amounts (0, 250,500,750 and 1000mM respectively) of aspartate with maximum effect seen with treatment supplemented with 500mM but this was not significant. Likewise all treatments supplemented with serine resulted in insignificant increases in embryonic death and was harmful at all concentrations tested (%dead: c =51.6, 250mM=58.3%, 500mM=53.3%, 750mM=75% and 1000mM=61.7% respectively; r = 0.6271; p=0.2575). Image 1: Human embryo generated in synthetic protein- free ECM; now a youngster aged about 17 yrs. REFERENCE Ali,1997. FSA Annual Conference, Adelaide, Australia Ali et al., 2000, Human Reprod 15:145-156 Ali, 2004, MEFS J. 9(2):118-127 Ali, J. United States Patent US2009/0226879 A1;Dec 2007; PCT 2008 (No. US2009/0226879 A1); (PCT US No. PCT/US2008087818) Jaffar Ali 1* and Nafisa Ali 2 CONCLUSION Supplementation of both aspartate and serine in embryo culture medium appear detrimental but these effects were insignificant. These responses were obtained under the confounding influences of bovine serum albumin that is present in conventional embryo culture media (ECM). The present findings showed the addition of these two amino acids in conventional ECM containing serum proteins may not be useful and may even be harmful. This suggests the practice of supplementing ECM with aspartate and serine be re-examined. Table 1: Effect of Aspartate on embryo development in vitro Table 2: Effect of Serine on embryo development in vitro 1* Department of Obstet. & Gynaecol, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; Email: jaffarali@um.edu.my; Tel: +603 7949 6898; Fax: +603 7949 4193jaffarali@um.edu.my 2 School of Archaeology & Anthropology, ANU College of Arts and Social Sciences, AD Hope Building, The Australian National University, Canberra, ACT 2601, Australia 1* Department of Obstet. & Gynaecol, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; Email: jaffarali@um.edu.my; Tel: +603 7949 6898; Fax: +603 7949 4193jaffarali@um.edu.my 2 School of Archaeology & Anthropology, ANU College of Arts and Social Sciences, AD Hope Building, The Australian National University, Canberra, ACT 2601, Australia Concentration of Aspartate (mM) 0250500750 1000 Day 1 Embryos 7976797776 Day 2 717375 73 Day-3 4037484237 Day-4 4036484236 Day-5 2726343327 Day-6 2326333125 %Dead r=0.4093 (p=1) 29.134.241.840.332.9 p-value Chi-square for trend + 0.99010.86720.93180.9944 Replicates=5; n= 387 embryos + Treatments compared individually with control Concentration of Serine (mM) 02505007501000 Day 1 Embryos 6260 Day 2 57565755 Day 3 40364031 Day 4 4033 2831 Day 5 3932282425 Day 6 3025281523 % dead embryos r = 0.6271 (p=0.2575) 51.658.353.37561.7 P-value; Chi-square for trend+ 0.98040.89630.33820.7604 Abstract No. Aspir-0176 Replicates = 4; n= 302 embryos + Treatments compared individually with control www.um.edu.my