257 THE PRESENCE OFAMMONIUM IN CHEMICALLY DEFINED MATURATION MEDIUM INHIBITS PORCINE OOCYTE NUCLEAR MATURATIONAND SUBSEQUENT EMBRYONIC DEVELOPMENT IN VITRO

Abstract

Exogenous supplementation with amino acids is important to the success of oocyte IVM and embryonic development in vitro. However, free ammonium ions will be generated by amino acid degradation or as a by-product of amino acid metabolism. Ammonium in embryo culture medium has detrimental effects on embryo development and viability. Nevertheless, the effects of ammonium in maturation medium on oocyte nuclear maturation and resulting developmental potential are still unknown. The objective of this study was to examine the effect of ammonium in maturation medium on porcine oocyte nuclear maturation and subsequent embryonic development after parthenogenetic activation (PA). Cumulus–oocyte complexes were matured in a chemically defined medium, Purdue Porcine Medium (PPM; experimental treatments), or TCM199 with 10% porcine follicle fluid (TCM + pFF; positive control) for 44 h, in 7% CO₂ in air at 38.7°C. For experimental treatments, ammonium chloride was added to PPM: 0 mm, 0.02 mm. 0.2 mm, 2 mm, and 20 mm. To examine nuclear maturation, oocytes were fixed and stained after IVM (20–40/treatment/replicate; 6 replicates). Each oocyte was scored from 1 to 7 based on meiotic stage. Oocytes were activated by exposure to a DC electrical pulse of 120 V mm^–1, 80 μs. The PA embryos were cultured in porcine zygote medium (PZM) containing 2 mm 6-DMAP for 6 h, then in PZM supplemented with 4 mg mL^–1 BSA for 6 days, at which time cleavage, blastocyst development, and blastocyst cell number were determined (40–60/treatment/replicate; 4 replicates). Data were analyzed by ANOVA with Bonferroni multiple comparison test; percentage data were arcsin transformed (significance, P < 0.05). There was no significant difference in maturation between TCM + pFF (6.17 ± 0.11), 0 mm (5.76 ± 0.14), 0.02 mm (5.70 ± 0.13), or 0.2 mm (5.61 ± 0.14), but maturation was significantly decreased in 2 mm (4.57 ± 0.15) and 20 mm (3.76 ± 0.14) ammonium. Embryonic development is shown in Table 1. Blastocyst development (of total activated oocytes) was significantly reduced in 0.2 mm, 2 mm, and 20 mm groups compared to the positive control. In conclusion, the presence of 0.2 mm ammonium in maturation medium compromised oocyte developmental competence, although nuclear maturation was not affected until ammonium concentrations reached 2 mm. These results demonstrate the deleterious effects of ammonium on both nuclear and cytoplasmic maturation in vitro, and point to the importance of minimization of ammonium build-up during this time period.

Table 1. Development of parthenogenetically activated oocytes after exposure to ammonium during IVM