218 ANTIBIOTIC-RESISTANT MICROBIAL CONTAMINATION (ENTEROBACTER CLOACAE) DERIVED FROM EGG YOLK, FROZEN SEMEN EXTENDER, IN PORCINE IN VITRO-FERTILIZED EMBRYOS

Abstract

The semen storage medium and the embryo culture environment are important for the further development of pre-implantation embryos, and these environments provide an ideal habitat for the propagation of a variety of microorganisms. Even though all precautions are taken to prevent contamination, this happens frequently during IVF and in vitro culture (IVC). Therefore, the aim of the present study was to investigate the source of contamination during semen processing for in vitro uses. In the present study, frozen semen was prepared from liquid semen (liquid semen was prepared according the method of (Weitze 1990 Reprod. Dom. Anim., 231–253 suppl. 1), and purchased from the Veterinary Service Laboratory, Department of Livestock Research (Yongin city, Gyeonggi-do, Korea) in our laboratory for IVF experiments because of a lack of fresh semen. Antibiotics were added to the frozen semen extender (kanamycin and gentamicin) and IVC medium (gentamicin) to further inhibit the growth of microorganisms. Nevertheless, microorganisms were observed to proliferate in the IVC drop when culturing IVF embryos using frozen semen. Three samples were randomly taken from the liquid semen, frozen semen, and egg yolk. Contaminated IVC medium, frozen–thawed semen, liquid semen, and egg yolk were cultured in de Man, Rogosa, and Sharpe agar medium. Identical whitish colonies were detected in the contaminated IVC drop, frozen–thawed semen samples, and egg yolk, but no colonies were formed in liquid semen samples. Identical gram-negative and rod-shaped bacteria were found in both the frozen–thawed semen sample and the contaminated IVC drop and egg yolk samples. Enterobacter cloacae were confirmed by API 20E kit according to the manufacturer’s instructions, with an identification value of 94.3% and a T index of 0.88. Antibiotic susceptibility tests were done according to CLSI (Wayne, PA) by using an ampicillin, amikacin, cephalothin, gentamicin, kanamycin, tetracycline, oxytetracycline, sulfamethoxazole, trimethoprim, norfloxacin, and ciprofloxacin test. Among them, E. cloacae were resistant to ampicillin, amikacin, cephalothin, gentamicin, and kanamycin but were susceptible to tetracycline, oxytetracycline, sulfamethoxazole and trimethoprim, norfloxacin, and ciprofloxacin. We suggest, based on these findings, that the sources of contamination might be from egg yolk. Therefore, synthetic semen extenders, which are free of egg yolk, might be considered during semen preparation.