Microbial Flora of Spermatophores from Black Tiger Shrimp (Penaeus monodon) Declines Over Long-Term Cryostorage

Abstract

Microbial contamination in sperm banks is of concern to sperm bank managers to prevent the perceived risk of disease transmission during cryostorage. The objective of this study was to examine the bacterial profile of cryopreserved spermatophores during long-term storage in liquid nitrogen in order to assess the risk of microbial flora during cryostorage. Spermatophores (N = 216) collected from males (N = 108) were equilibrated in calcium-free saline containing 5% dimethyl sulfoxide for 30 min and frozen at a cooling rate of − 2 °C min− 1 prior to storage in liquid nitrogen tanks. Cryopreserved spermatophores were sampled for bacterial profiles after 1 h and 30, 60, 90, 120, 150, 180 and 210 days using Plate count Agar, Thiosulphate Citrate Bile Salts Agar and Pseudomonas Isolation Agar. The incubation temperature was 37 °C for 24–48 h. Long-term storage of spermatophores in liquid nitrogen resulted in the reduction of the bacterial numbers. The number of total heterotrophic bacteria and total Vibrio decreased significantly (P < 0.05) to 2.1 ± 0.4 × 104 CFU/g (95.5% reduction) and 18.7 ± 4.1 × 103 CFU/g (93.8% reduction), respectively, after 30 days in storage, while those of total Pseudomonas declined significantly (P < 0.05) to 1.11 ± 5.9 × 105 CFU/g (62.8% reduction) within 1 h of storage. Average sperm viability of cells from fresh spermatophores (96.4 ± 1.8%) was not significantly different (P > 0.05) from those of cryopreserved spermatophores held in liquid nitrogen for 1 h, 30 or 60 days (94.2 ± 2.1, 89.8 ± 3.5 and 86.7 ± 3.9%, respectively, however it decreased significantly (P < 0.05) after 90 days of storage. There were 23 bacterial species identified from fresh and cryopreserved spermatophores. The bacterial species least able to survive cold storage were Proteus mirabilis, Enterobacter spp., Vibrio spp., Kocuria varians, Bacillus cereus, B. amyloliquefaciens, B. circulans, Pseudomonas aeruginosa and Burkholderia cepacia because they were not detected from cryopreserved spermatophores after 90 or 120 days. The normal flora remaining at the end of the experiment (day 210) were Staphylococcus non aureus, Micrococcus spp., Proteus spp., Ochrobactrum anthropi, Corynebacterium spp., B. licheniformis and Pseudomonas spp. Cryopreservation of P. monodon spermatophores nearly eliminated the occurrence of pathogenic bacteria (Vibrio spp. and Ps. aeruginosa) culturable at 37 °C during long-term storage in liquid nitrogen.