1 ARGININE SUPPLEMENTATION IN VITRO INCREASES PORCINE EMBRYO DEVELOPMENT AND AFFECTS mRNA TRANSCRIPT EXPRESSION

Abstract

In vitro culture systems are suboptimal as compared to in vivo. Previous next-generation sequencing analysis of in vivo fertilized and in vitro cultured (IVC) or in vivo cultured (IVV) porcine blastocyst stage embryos identified an arginine transporter (SLC7A1) expressed 63 fold higher in IVC compared to IVV blastocysts (Bauer et al. 2010 Biol. Reprod. Epub ahead of print). Arginine catabolism may play important roles in placental and conceptus growth and development as it is a substrate for synthesis of nitric oxide synthase and polyamines. The objective of this study was to determine the effects arginine had on both embryo development and mRNA expression in in vitro fertilized embryos. Cumulus–oocyte complexes were matured for 44 h in M199 supplemented with EGF, FSH, and LH. Oocytes with a visible polar body (metaphase II) were selected and fertilized in modified Tris Buffered Medium for 5 h and then placed into one of 5 treatment groups (Porcine Zygote Medium 3 (PZM3) with 0 mM, 0.12 mM (current concentration of arginine in PZM3), 0.36 mM, 0.72 mM, or 1.69 mM arginine). Twenty-eight hours post-fertilization, cleaved embryos were selected and moved into 25 μL drops of respective culture media and cultured to day 6 in 5% CO₂, 5% O₂, 90% N₂ at 38.5°C. To determine the effect arginine had on development the percent of embryos that made it to the blastocyst stage for each treatment group were analysed using PROC GLM in SAS (SAS Institute, Cary, NC). A least significant difference post test comparison was completed to determine if significant differences existed between treatment groups (^a,b,cP < 0.05). The percentage of cleaved embryos on Day 6 that developed to blastocyst was 57.2%^b,c, 50.2%^c, 67.3%^a,b, 67.3%^a,b, 70.4%^a (N = 147, 163, 150, 120, and 134) in 0 mM, 0.12 mM, 0.36 mM, 0.72 mM, and 1.69 mM arginine, respectively. Real-time PCR was then completed to assess the affect arginine supplementation had on SLC7A1 mRNA expression. Three biological replicates, each containing 10 blastocyst pools to ensure enough starting material, were collected for each treatment group. RNA was isolated from each sample and 5 μL was linearly amplified (NuGEN Ovation Pico WTA System) so multiple genes could be compared and then purified using Bio-Rad MicroSpin Columns. Expression levels were calculated relative to the reference sample and the housekeeping gene, YWHAG. The ΔΔCT values were log-transformed and analysed using PROC GLM in SAS. The expression of SLC7A1 mRNA was decreased (P = 0.0006) compared to PZM3 in the 1.69 mM arginine group. These results illustrate the positive effects that additional arginine may be having on porcine embryo development during culture from the 2-cell to the blastocyst stage. Supplementing arginine to a final concentration of 1.69 mM during culture increases development of porcine embryos to blastocyst compared to PZM3 and also decreases the expression of SLC7A1. Evaluation of the transcriptional profile appears to be a good method of letting the embryo tell us what it needs for development, and in this case arginine.