一种功能标记物在冷冻保护剂对柳珊瑚（Junceella juncea和J. fragilis）精囊影响方面的应用。

Application of a functional marker for the effect of cryoprotectant agents on gorgonian coral (Junceella juncea and J. fragilis) sperm sacs.

作者信息

Tsai S, Kuit V, Lin Z G, Lin C

出版信息

Cryo Letters. 2014 Jan-Feb;35(1):1-7.

Abstract

BACKGROUND

The establishment of coral sperm repositories which retain good post-rewarming viability and fertility play a vital role in species conservation.

OBJECTIVE

This study aimed at obtaining baseline information regarding the effects of cryoprotectant agents (CPAs) on gorgonian coral (Junceella juncea and J. fragilis) sperm sacs.

METHODS

The adenosine triphosphate assay was used to determine the energy level of the gorgonian sperm sacs as an indicator of sperm viability after exposure to cryoprotectants.

RESULTS

The 'no observed effect concentrations' (NOECs) of methanol, dimethyl sulfoxide (DMSO), polypropylene glycol (PG), ethylene glycol (EG) and glycerol for J. juncea sperm sacs were 3 M, 3 M, 1 M, 2 M and 1 M respectively after 20 min exposure; whilst the NOECs for J. fragilis oocytes were 2 M, 3 M, 1 M, 2 M and 2 M, respectively. Methanol and DMSO had the least impact. PG was the most toxic CPA after 10 min exposure. ATP content of J. juncea and J. fragilis sperm sacs did not differ significantly from the control with incubation times of 10-20 min with 2 M EG. However, ATP content dropped significantly after exposing sperm sacs to 2 M EG for 40 min with average values of 2.34 +/- 0.12 and 1.97 +/- 0.48 microg/ml respectively. ATP content for J. juncea and J. fragilis sperm sacs was significantly decreased to 1.79 +/- 0.31 and 2.40 +/- 0.36 microg/ml after 20 min incubation in 2 M PG when compared to the control with 2.98 +/- 0.16 and 4.14 +/- 0.42 microg/ml respectively. Normalized ATP content for sperm sacs of two different gorgonian coral after incubation in methanol, DMSO, PG, EG and glycerol showed that J. juncea sperm sacs were slightly less tolerant to CPAs compared to J. fragilis sperm sacs.

CONCLUSIONS

DMSO or methanol can be considered as efficient CPAs for gorgonian sperm sacs cryopreservation. The ATP luminescence assay provided sensitive and rapid quantification of mitochondrial activity in gorgonian coral sperm sacs. The study on the impact of CPA will contribute to the development of a cryopreservation protocol for coral sperm conservation.

摘要

背景

建立具有良好复温后活力和生育能力的珊瑚精子库对物种保护至关重要。

目的

本研究旨在获取有关冷冻保护剂（CPA）对柳珊瑚（Junceella juncea和J. fragilis）精囊影响的基线信息。

方法

采用三磷酸腺苷测定法来确定柳珊瑚精囊的能量水平，以此作为暴露于冷冻保护剂后精子活力的指标。

结果

在暴露20分钟后，甲醇、二甲基亚砜（DMSO）、聚丙二醇（PG）、乙二醇（EG）和甘油对Junceella juncea精囊的“未观察到效应浓度”（NOECs）分别为3 M、3 M、1 M、2 M和1 M；而对J. fragilis卵母细胞的NOECs分别为2 M、3 M、1 M、2 M和2 M。甲醇和DMSO的影响最小。暴露10分钟后，PG是毒性最大的CPA。在2 M EG中孵育10 - 20分钟时，Junceella juncea和J. fragilis精囊的ATP含量与对照组相比无显著差异。然而，在2 M EG中暴露40分钟后，精囊的ATP含量显著下降，平均值分别为2.34±0.12和1.97±0.48微克/毫升。与对照组（分别为2.98±0.16和4.14±0.42微克/毫升）相比，在2 M PG中孵育20分钟后，Junceella juncea和J. fragilis精囊的ATP含量显著降至1.79±0.31和2.40±0.36微克/毫升。在甲醇、DMSO、PG、EG和甘油中孵育后，两种不同柳珊瑚精囊的归一化ATP含量表明，与J. fragilis精囊相比，Junceella juncea精囊对CPA的耐受性略低。