CAJ | 학술논문

实验所用翘嘴鳜(♀, Siniperca chuatsi)2~3龄,体质量1000~1500 g；斑鳜(♂, Siniperca scherzeri Stein-dachner)1~2龄,体质量300~500 g。于繁殖季节选取成熟度好的亲鱼以促黄体素释放激素类似物(LHRH-A2)、绒毛膜促性腺激素(HCG)和马来酸地欧酮(DOM)进行催产。通过筛选D-15、D-17、Ringer液和 M-Hank’s液4种稀释液和二甲基亚砜(DMSO)、甘油(Gly)、乙二醇(EG)、1,2-丙二醇(PG)和二甲基甲酰胺(DMF)5种抗冻剂,发现DMSO和D-17分别是优选的抗冻剂和稀释液。以D-17作为斑鳜精子的稀释液,按照1︰3(精液︰稀释液)体积比稀释,添加10%体积的DMSO作为抗冻剂,按照三步冷冻法超低温冷冻保存,37℃水浴解冻的斑鳜精子,经CASA分析精子活率为(83.26±18.20)%, SCGE检测表明70%以上的精子核DNA不会发生损伤； FCM分析表明26.74%的解冻精子有完整的细胞膜且线粒体功能正常；用冷冻复苏斑鳜精子与翘嘴鳜精卵进行人工授精,最高受精率(39.6±6.5)%,温度24~28℃孵化时间38 h,孵化后的鱼苗发育正常,开口1周以后的鱼苗全长(1.346±0.255) cm,体高(0.438±0.103) cm,体质量(0.045±0.020) g。鲜精受精鱼苗和冻精受精鱼苗在开口后1周的生长没有显著差异。因此认为D-17+10% DMSO可用于斑鳜精液的超低温冷冻保存。本研究将有助于斑鳜种质资源的收集保存和冷冻保存的斑鳜精液在翘嘴鳜(♀)×斑鳜(♂)杂交中的应用。
실험소용교취궐(♀, Siniperca chuatsi)2~3령,체질량1000~1500 g；반궐(♂, Siniperca scherzeri Stein-dachner)1~2령,체질량300~500 g。우번식계절선취성숙도호적친어이촉황체소석방격소유사물(LHRH-A2)、융모막촉성선격소(HCG)화마래산지구동(DOM)진행최산。통과사선D-15、D-17、Ringer액화 M-Hank’s액4충희석액화이갑기아풍(DMSO)、감유(Gly)、을이순(EG)、1,2-병이순(PG)화이갑기갑선알(DMF)5충항동제,발현DMSO화D-17분별시우선적항동제화희석액。이D-17작위반궐정자적희석액,안조1︰3(정액︰희석액)체적비희석,첨가10%체적적DMSO작위항동제,안조삼보냉동법초저온냉동보존,37℃수욕해동적반궐정자,경CASA분석정자활솔위(83.26±18.20)%, SCGE검측표명70%이상적정자핵DNA불회발생손상； FCM분석표명26.74%적해동정자유완정적세포막차선립체공능정상；용냉동복소반궐정자여교취궐정란진행인공수정,최고수정솔(39.6±6.5)%,온도24~28℃부화시간38 h,부화후적어묘발육정상,개구1주이후적어묘전장(1.346±0.255) cm,체고(0.438±0.103) cm,체질량(0.045±0.020) g。선정수정어묘화동정수정어묘재개구후1주적생장몰유현저차이。인차인위D-17+10% DMSO가용우반궐정액적초저온냉동보존。본연구장유조우반궐충질자원적수집보존화냉동보존적반궐정액재교취궐(♀)×반궐(♂)잡교중적응용。
The golden mandarin or Aucha perch, Sinniperca scherzeri, is an important genetic resource. To preserve the species, experimental trials were carried out to find suitable sperm cryopreservation methods by comparing the effects of four extenders, D-15, D-17, Ringer’s and M-Hank’s, and five cryoprotectants, dimethyl sulfoxide (DMSO), glycerol (Gly), ethylene glycol (EG), propylene glycol (PG) and dimethylformamide (DMF), on sperm motility after thawing. The present study demonstrates that DMSO and D-17 are a suitable choice as cryoprotectant and extender, respectively. Using a protocol of diluting a sperm sample with D-17 at a sperm/extender ratio of 1︰3, adding DMSO to a final con-centration of 10% in volume, loading to 2-mL cryotubes as containers, following the three-step method to cool the mixture and thawing the frozen semen in a 37℃ water bath for 80 s, a post-thaw motility of>80%could be obtained using computer-assisted sperm analysis (CASA). Single cell gel electrophoresis showed that>70%of sperm DNA was intact and practically no sperm nuclear DNA was severely damaged. Flow cytometric analysis showed that 26.74%of the frozen-thawed sperm had intact membrane and functional mitochondria. The highest fertilization rate was (39.6±6.5)%in the hybridization of Siniperca chautsi (♀) ×Siniperca scherzeri (♂). The hatching time was 38 h after fertilization at 24-28℃ and body parameters after being fed for 7 day were (0.045±0.020) g in weight, (1.346±0.255) cm in length and (0.438±0.103) cm in depth. Thus, it is recommended that D-17+10%DMSO be used for cryopreserva-tion of Siniperca scherzeri semen. This will assist the preservation of S. scherzeri germplasm resources and promote crossbreeding with S. chautsi (♀) through the successful application of sperm cryopreservation skills.