PARENT SESSION
IVF and Assisted Reproductive Technologies

CYTOGENETIC ANALYSIS OF IN VITRO PRODUCED PORCINE BLASTOCYSTS.

Mazza, M. Rosaria¹, McCauley, Tod², Di Berardino, Dino¹, Didion, Brad³, Day, Bill², ^{1 2 3}

ABSTRACT- Despite improvements in the efficiency of in vitro production of porcine embryos, polyspermic penetration remains a major obstacle to continued advancement in this field. While it is recognized that current porcine in vitro maturation (IVM)/in vitro fertilization (IVF) protocols are not optimized to control polyspermic penetration, the incidence of polyploid blastocysts resulting from IVM/IVF is not known. The objective of this study was to quantify, by chromosomal karyotyping, the incidence of chromosomal abnormalities in day-6 porcine blastocysts produced by IVF. Immature oocytes were matured in vitro for 40-44 h in TCM-199 and inseminated with 3 × 10⁵ sperm/ml in mTBM for 5-6 h. Presumptive zygotes were cultured in NCSU 23 medium with BSA for 144 h. Colcemid (0.2 g/ml) was added to the culture medium and embryos were cultured for 3 h. The zona pellucida was disrupted by pronase treatment and embryos were hypotonized with sodium citrate (0.8%) for 3 min followed by KCl treatment for 2 min. Blastocysts were fixed in methanol/acetic acid (3:1), air-dried and stained with Giemsa (0.4% stock) solution. A total of 318 embryos were examined and 248 (78.0%) were suitable for analysis. Of those, 97 (39.1%) were chromosomally abnormal. The abnormalities observed included haploidy (9.3%), polyploidy (71.1%) and mixoploidy (19.6%); within the polyploidy, triploid and tetraploid showed the highest incidence (56.5 and 27.5%, respectively); within the mixoploidy, the condition 2n/3n was the most represented (36.8%). In addition, abnormal embryos had fewer (p< 0.01) total cells (31.8 ± 1.3 vs. 35.9 ± 1.0) as compared to normal (2n) embryos. These data indicate that a large percentage of porcine embryos produced by IVM/IVF are abnormal as a result of multiple sperm penetration. The development of methods capable of reducing the incidence of polyspermy in porcine IVF systems is required to enhance the efficiency of embryo production for potential commercial application of this technology and is a major focus of current research strategies.

KEY WORDS: chromosome, embryo, polyspermy, porcine