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Abstract: The objectives of this study were to determine the effect of cryoprotectants on sperm viability and develop a freezing protocol for long-term storage of P. monodon spermatophores. Spermatophores suspended for 30min in calcium-free saline (Ca-F saline) containing the cryoprotectants dimethyl sulfoxide (DMSO), ethylene glycol (EG), 1,2-propylene glycol (PG), formamide, and methanol at concentrations of 5, 10, 15, or 20% were studied using a modified eosin–nigrosin staining technique. The smallest reductions in apparent sperm viability occurred with DMSO; therefore, a freezing protocol was developed using Ca-F saline containing 5% DMSO. Spermatophores were cryopreserved using three protocols; cooling to a final temperature of −30, −80 or −80°C and immediately stored in liquid nitrogen (cooling rates of −2, −4, −6, −8, −10, −12, −14 or −16°C/min). Frozen spermatophores were thawed (2min) at 30, 60, 70, or 90°C. Successful cryopreservation of spermatophores in liquid nitrogen was achieved by a one-step cooling rate of −2°C/min between 25 and −80°C before storing in liquid nitrogen. Optimal thawing was in a 30°C water bath for 2min; this yielded live sperm after storage in liquid nitrogen for 210 days. Average sperm viability for fresh (97.8±2.9%) and cryopreserved spermatophores held for less than 60 days (87.3±4.1%) did not differ (P >0.05); however, that for spermatophores stored in liquid nitrogen between 90 and 210 days were lower (P <0.05) and varied from 27.3±3.4 to 53.3±4.3%. Thawed spermatophores previously held in liquid nitrogen for less than 62 days fertilized eggs (fertilization and hatching rates of 71.6–72.2% and 63.6–64.1%, respectively) at rates comparable to fresh spermatophores (70.8–78.2% and 66.3–67.8%, respectively). In conclusion, sperm within cryopreserved spermatophores stored in liquid nitrogen retained their viability for up to 210 days. [Copyright &y& Elsevier]

Abstract: Sperm cryopreservation of red snapper (Lutjanus argentimaculatus) is essentially unexplored, although many species of the Lutjanidae family are considered to be high-value commercial species. The objective of this study was to develop a species-specific cryopreservation protocol for red snapper (L. argentimaculatus) sperm by optimizing cryoprotectants and cooling rates in the cryopreservation procedure. Ten cryoprotectants at four concentrations and two freezing protocols were examined in two separate experiments. In the first experiment, toxicity studies of dimethyl sulfoxide (DMSO), glycerol, propylene glycol (PG), ethylene glycol (EG), formamide, methanol, ethanol, sucrose, trehalose, and dimethylacetamide (DMA) on sperm motility were performed. Semen diluted 1:1 in Ringer solution were exposed to cryoprotectants at four final concentrations of 5%, 10%, 15%, or 20% for periods of 10, 20, 30, 40, 50, 60, 90, and 120min at room temperature (25°C). The cryoprotectants and concentrations that showed the least toxic effect on sperm motility were selected for cryopreservation trials. In the second experiment, selected cryoprotectants were then assessed for freezing capacity of sperm as follows: DMSO 5% and 10%, PG 5% and 10%, EG 5% and 10%, ethanol 5%, and methanol 5%. Semen was diluted 1:1 in Ringer solution and equilibrated with selected cryoprotectants for 10min at room temperature. Sperm were frozen in a controlled-rate programmable freezer at four cooling rates of 3, 5, 10, and 12°C/min from an initial temperature of 25°C to final temperatures of −40 or −80°C before plunging into liquid nitrogen. Sperm equilibrated in 10% DMSO and cooled at a rate of 10°C/min to a final temperature of −80°C had the highest motility (91.1±2.2%) and viability (92.7±2.3%) after thawing. The fertilization rate of frozen-thawed sperm (72.4±2.4%) was not different (P>0.05) from that of fresh sperm (75.5±2.4%). This study apparently represents the first reported attempt for cryopreservation of L. argentimaculatus sperm. [Copyright &y& Elsevier]