REPLACING GLUTAMINE WITH ALANYL-GLUTAMINE IN CHEMICALLY DEFINED MATURATION MEDIUM CAN INCREASE PORCINE OOCYTE NUCLEAR MATURATION IN VITRO

Abstract

Glutamine is an important energy substrate that can be utilized by oocytes during nuclear maturation. Glutamine easily breaks down and thus generates free ammonium ions, which are very toxic to the cell. The objective of this study was to compare in vitro nuclear maturation of porcine oocytes using alanyl-glutamine (ala-gln), a more stable dipeptide consisting of glutamine and alanine, to replace glutamine (gln) in the maturation medium. Cumulus-oocyte complexes (COCs) (10–40/treatment/replicate; 5 replicates) were matured in a chemically defined medium, Purdue Porcine Medium (PPM), or TCM199 with 10% porcine follicle fluid (pFF) for 44 h, in 7% CO2 in air at 38°C. Maturation treatments included: PPM without gln or ala-gln as a negative control (none); PPM with 1.0 mM gln (gln); PPM with 1.0 mM ala-gln (ala-gln); PPM with 1.0 mM ala-gln and 0.2 mM gln (both); and TCM 199 with 10% pFF as a positive control (TCM + pFF). After maturation, oocytes were fixed and stained for determination of meiotic stage. The total number of COCs used for each treatment was: none, n=107; gln, n=96; ala-gln, n=106; both, n=118; TCM + pFF, n=116. Each oocyte was scored from 1 to 7 based on meiotic stage: Germinal vesicle, GV=1; Germinal vesicle breakdown, GVBD=2; Condensed Chromatin, CC=3; Metaphase I, MI=4; Anaphase, A=5; Telophase, T=6; Metaphase II, MII=7. Data were analyzed by ANOVA with Fishers LSD multiple comparison test. The P value used to determine significance was 0.05. The mean maturation score ± SEM for each treatment was: none, 4.37 ± 0.21; gln, 5.04 ± 0.21; alagln, 5.47 ± 0.19; both, 5.25 ± 0.18; TCM + pFF, 5.78 ± 0.17). Overall oocyte maturation percentage was determined by calculating the proportion of oocytes scored 5, 6 and 7 (A, T and MII) of the total number of oocytes in each treatment (none, 38.32 ± 4.72; gln, 52.08 ± 5.13; alagln, 61.32 ± 4.75; both, 55.93 ± 4.59; TCM + pFF, 70.69 ± 4.24). Oocytes without any gln supplementation (none) had a significantly lower maturation score than oocytes in all other treatment groups, and also had a significantly lower overall maturation percentage. There was no significant difference between gln, ala-gln or both in maturation score or overall maturation percentage. The maturation score and percentage of oocytes in TCM + pFF were significantly higher than those of oocytes matured with gln or both. Oocytes matured in vitro in defined maturation medium containing ala-gln underwent nuclear maturation equally as well as those matured in TCM + pFF. In conclusion, using ala-gln instead of gln was beneficial to oocyte IVM and increased oocyte maturation percentage. Including ala-gln instead of gln may result in increased developmental competence after fertilization by reducing ammonium buildup during the maturation period. (poster)