Vuthiphandchai V, Chomphuthawach S, Nimrat S
Department of Aquatic Science, Faculty of Science, Burapha University, Chonburi 20131, Thailand.
Theriogenology. 2009 Jul 1;72(1):129-38. doi: 10.1016/j.theriogenology.2009.02.013. Epub 2009 Apr 5.
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 120 min at room temperature (25 degrees 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 10 min at room temperature. Sperm were frozen in a controlled-rate programmable freezer at four cooling rates of 3, 5, 10, and 12 degrees C/min from an initial temperature of 25 degrees C to final temperatures of -40 or -80 degrees C before plunging into liquid nitrogen. Sperm equilibrated in 10% DMSO and cooled at a rate of 10 degrees C/min to a final temperature of -80 degrees 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.
尽管笛鲷科的许多物种被认为是高价值的商业物种，但紫红笛鲷（Lutjanus argentimaculatus）精子的冷冻保存基本上尚未得到探索。本研究的目的是通过优化冷冻保存过程中的冷冻保护剂和降温速率，为紫红笛鲷（L. argentimaculatus）精子制定一种物种特异性的冷冻保存方案。在两个独立的实验中，研究了四种浓度的十种冷冻保护剂和两种冷冻方案。在第一个实验中，进行了二甲基亚砜（DMSO）、甘油、丙二醇（PG）、乙二醇（EG）、甲酰胺、甲醇、乙醇、蔗糖、海藻糖和二甲基乙酰胺（DMA）对精子活力的毒性研究。将在林格氏溶液中按1:1稀释的精液在室温（25摄氏度）下暴露于四种终浓度分别为5%、10%、15%或20%的冷冻保护剂中10、20、30、40、50、60、90和120分钟。选择对精子活力毒性最小的冷冻保护剂和浓度进行冷冻保存试验。在第二个实验中，对选定的冷冻保护剂的精子冷冻能力进行如下评估：DMSO 5%和10%、PG 5%和10%、EG 5%和10%、乙醇5%和甲醇5%。精液在林格氏溶液中按1:1稀释，并在室温下与选定的冷冻保护剂平衡10分钟。精子在程序控制的速率冷冻机中以3、5、10和12摄氏度/分钟的四种降温速率从初始温度25摄氏度冷冻至最终温度-40或-80摄氏度，然后投入液氮中。在10% DMSO中平衡并以10摄氏度/分钟的速率冷却至最终温度-80摄氏度的精子在解冻后具有最高的活力（91.1±2.2%）和存活率（92.7±2.3%）。冻融精子的受精率（72.4±2.4%）与新鲜精子的受精率（75.5±2.4%）没有差异（P>0.05）。本研究显然是首次报道的对紫红笛鲷精子进行冷冻保存的尝试。
