Cryopreservation of banana shrimp (Fenneropenaeus merguiensis) spermatophores with supplementation of medicinal plant extracts: Development of a programmable controlled-rate method and a practical method

Abstract

The objectives of this study were to generate the optimal cryopreservation protocols (programmable controlled-rate and practical methods) for banana shrimp (Fenneropenaeus merguiensis) spermatophores and investigate the efficacy of plant extracts (moringa, Moringa oleifera Lam and ginger, Zingiber officinale Roscoe) on sperm survival and bacterial abundance in cryostored spermatophores. Spermatophores were exposed to various cryoprotectants at different concentrations and equilibrium times prior to examining sperm viability. A minimal toxicity to banana shrimp sperm was observed in spermatophores suspended in 5% DMSO. The optimal freezing protocol using a programmable controlled-rate freezer included exposure of spermatophores in calcium-free saline (Ca–F saline) containing 5% DMSO with 20 min equilibration time, and then utilization of a one-step freezing at the rate of −2 °C min−1 from 25 °C to −80 °C and holding for 30 s before storage in a liquid nitrogen tank. Highest sperm viability of post-thawed spermatophores was obtained after thawing at 30 °C for 2 min in a water bath. Cryopreservation of F. merguiensis spermatophores using a practical method was achieved by suspending spermatophores in Ca–F saline containing 5% DMSO with equilibration time of 20 min at 25 °C and placing at 4 cm over liquid nitrogen surface (a freezing rate of about −2.5 °C min−1) prior to plunging into liquid nitrogen. In order to evaluate the efficacy of plant extracts on sperm survival and bacterial abundance of cryostored spermatophores, two freezing protocols (programmable and practical methods) with ethanolic moringa and ginger extracts (0.1 mg mL−1 each) were developed for long-term cryostorage. Moringa and ginger extracts had no detrimental effect on F. merguiensis sperm due to retaining high viable sperm without significant differences (P > 0.05) compared to the control after a 12-month cryostorage. Supplementation of moringa or ginger extract into sperm extender resulted in a reduction of bacterial abundance in cryostored spermatophores. This report, for the first time, indicates the promising potential of plant extracts as an alternative method for controlling bacterial contaminants in cryopreserved spermatophores of penaeid shrimp.