314 OPTIMIZING TREATMENT FOR IVF WHEN USING HEAVILY BACTERIAL CONTAMINATED SEMEN

Abstract

Following semen collection, the nonspecific bacterial count is usually kept low by addition of antibiotics to the extender. Ordinary protocols for semen processing in preparation for IVF are characterized by a washing procedure, followed by spermatozoa selection by either swim-up or Percoll gradient. In this study, commercially available bovine semen to be used for IVF was found to be heavily contaminated with Escherichia coli and Leclercia adecarboxilata despite the addition of tylosin (5 mg/100 mL), gentamicin (25 mg/100 mL), spectomycin (30 mg/100 mL), and lincomycin (15 mg/100 mL) to the extender. Bacterial concentrations varied among straws examined (n = 7), ranging from 10 × 4 UFC mL^-1 to 10 × 8 UFC mL^-1. Noncontaminated semen from the same valuable bull, although derived from a different semen collection, was known to be used with great efficiency for IVF in a different laboratory at a very low concentration (i.e. 350 000 sperm mL^-1). Therefore, to rescue the fertilizing capacity of such contaminated semen, an improved treatment was applied and compared with the ordinary semen treatment. From the previous use of semen from the same bull, a concentration of 2 million sperm/mL^-1 and 20 µg mL^-1 of heparin was chosen for IVF. During ordinary treatment, the semen was washed using 6 mL of fertilization medium into a 15-mL tube and subjected to centrifugation at 800 rpm for 5 min. After removal of the supernatant, the resulting pellet was layered under 1 mL of the same medium and left for 45 min in an incubator for swim-up. The supernatant was moved into a new 15-mL tube for additional centrifugation. The newly formed pellet was then resuspended for the final count and co-incubation with oocytes. With such a procedure, the bacterial contamination was found for 3 consecutive replicates in culture droplets starting at Days 3 to 4 post-IVF. In the course of 5 additional replications, the semen treatment was improved by doubling the volume for initial washing to 12 mL of fertilization medium. The resuspended pellet following the first centrifugation was equally partitioned into 6 round-bottomed tubes and left for 60 to 80 min in an incubator for swim-up. The supernatant was then moved into a new 15-mL tube for additional centrifugation and a final count. The IVF medium was supplemented with 100 IU mL^-1 of penicillin G in both treatments. Following the ordinary and improved semen treatment, a large number of sperm cells were found agglutinated head-to-head. A subsequent embryo culture was performed for both treatments in SOF containing 0.025 mg mL^-1 of kanamycin sulfate. When using sperm and heparin concentrations at 2 million and 20 µg mL^-1, respectively, under the improved semen treatment, cleavage was 84.8%. The rates of blastocyst production at Day 7 from used cumulus-oocyte complexes and from cleavage were 29.5 and 35.9%, respectively. In conclusion, the improved washing procedure rescued valuable but heavily contaminated semen for use in IVF procedures.