四大家鱼人工繁殖技术与生产实践(三)

4.催产亲鱼的选择(1)催产用雄亲鱼的选择标准:从头向尾方向轻挤腹部即有精液流出,若精液浓稠,呈乳白色,入水后能很快散开,为性成熟的优质亲鱼;若精液量少,入水后     

人类绒毛膜促性腺激素（HCG）、鲢鳙鱼垂体及下丘脑促黄体素释放激素（LRH）类似物对家鱼免疫反应的初步研究

前言 自1958年我国先后用鲤鱼垂体和人类绒毛膜促性腺激素(HCG)催产四大家鱼以来，对淡水养殖事业的迅速发展起了重要的作用。但随着生产的发展，发现催产剂量有逐年提高的趋势，常见一些催产的亲鱼不产及难产。这除了与亲鱼的培育、生态条件有关外，可能生理上由于亲鱼多年连续注射HCG或垂体后，产生体液性抗体——抗激素。     

亲鱼催产严把三关

立夏至小满．是鱼类催产的最好时期．此时水温适宜，亲鱼成熟好，产孵率、孵化率高。鱼苗下塘和成活率也高。若抓住这一有利时机，严把亲鱼催产选择关、药物使用关、催产管理关，是取得催产成功的关键。     

减少催产亲鱼死亡的技术措施

减少催产亲鱼的死亡，是鱼苗生产者一项重要的技术工作。在生产实践中笔者发现，引起催产亲鱼死亡的主要因素有两个方面，一是亲鱼在产卵生殖过程中体能消耗很大，加上催产药物的副作用，使亲鱼肌体抵抗能力下降，如果亲鱼受伤严重，各种病原体会趁机侵染鱼体，引发疾病，导致亲鱼死亡；二是亲鱼的体质差，或     

对白鲢亲鱼催产效果差原因的初步探讨

对白鲢亲鱼催产效果差原因的初步探讨包振学（辽宁省朝阳县水利局）关键词：白鲢，亲鱼，催产在鲢鱼的人工繁殖过程中，往往遇到这种情况：被选中的亲鱼在年龄和体重等方面都达到了催产标准，催产后或不发情，或半产，且受精率、孵化率很低；或腹部膨胀，甚至造成死亡。经...     

采用挖卵观察 准确选择催产成熟亲鱼

选择成熟度良好的亲鱼，是催产成败的重要因素。但目前生产中选择催产亲鱼普遍采用体外观察法(看、比、摸)。我们在多年生产实践中深刻体会到用这种方法来选择催产的雌性亲鱼，很难掌握，特别是在催产的早期。笔者近四年来在九观桥水库鱼苗场亲鱼催产过程中，将选择雌性亲鱼成熟度，采用先体外观察初选，再进行挖卵观察鉴别的方法。本文谈谈肤浅体会，以期对提高亲鱼催产率有所裨益。     

家鱼亲鱼产后精心培育两点措施

家鱼亲鱼产后精心培育两点措施一、要采取防病措施产后亲鱼体质虚弱，鱼体在拉网过程中常会有些受伤，较易感染疾病．因此，亲鱼产后要及时拉上检查产卵情况和受伤情况．检查亲鱼，发现如果没有顺产或半产．要把亲鱼体内的精液和卵子挤出，进行人工授精，提高催产率．同时...     

LRH—A对草、鲢、鳙亲鱼的催产效果

今年我场使用LRH—A(宁波产品)催产草、鲢、鳙亲鱼共49组，获产41组，催产率84%(其中草鱼33组，获产29组，催产率88%；鲢鱼8组，获产7组，催产率87%；鳙鱼8组，获产5组，催产率63%)，获得鱼卵4060万粒，获苗1245万尾。     

鲢亲鱼的手术催产法

鲢亲鱼的手术催产法鲢鱼人工繁殖选择亲鱼时，会发现有部分雌亲鱼的性腺发育良好，腹部膨大，卵巢轮廊明显，但其生殖孔发炎肿胀，呈深红色而突出，开口很小，有的甚至溃烂。这样的雌亲鱼在注射催产药物后，会因输卵管（生殖孔）被堵塞而产不出卵来，造成难产，并因此引起...     

亲鱼产后保健剂应用初报

近七年来，农村经济体制改革推动了淡水养殖业的发展，全国使用各类鱼用催产剂(如绒膜激素，促黄体素释放激素类似物，鱼垂体等)对经济鱼类进行催产，据初步调查，各地在催产亲鱼时，因操作不当和机械损伤引起的炎症、产后体弱、未产雌亲鱼卵子膨胀、雄亲鱼流精过多、激素用量不当等原因，使亲鱼产后死亡率不断增高，一般为10-30％，有的竟高达80％以上，     

Progestin is important for testicular development of male turbot (<Emphasis Type="Italic">Scophthalmus maximus</Emphasis>) during the annual reproductive cycle through functionally distinct progestin receptors

In teleost, sex steroid hormones are critical for reproduction. Progestin is known to promote spermiation. To further understand the functions of progestin via its receptors during the annual reproductive cycle in male turbot (Scophthalmus maximus), we observed testicular development, quantified several sex steroid hormones, detected the expression of progestin receptors, and measured various sperm parameters. Results showed that the turbot testicular structure was of the lobular type. During breeding season, a number of spermatocytes (stage III) developed into spermatids (stage IV), then differentiated into sperm during spermiogenesis (stage V), and finally regressed to spermatocytes (stage VI). Concomitant with testicular development, serum progesterone (P4) and 17α,20β-dihydroxy-4-pregnen-3-one (DHP) exhibited higher levels from stage IV to V than other stages. Furthermore, males with higher motility sperm showed higher levels of P4 and DHP compared with fish with lower motility sperm. These results indicated that P4 and DHP might induce spermatogenesis due to seasonal changes. Concurrently, in testes, the nuclear progesterone receptor (pgr) was expressed throughout the reproductive cycle and its level peaked during spermiogenesis while expression of membrane progestin receptor alpha (mPRα) did not change significantly. However, in sperm, mPRα expression was higher than in testes and had a significant positive correlation with curvilinear velocities (VCL), sperm motility, and motility duration. In conclusion, progestin appears to exert a direct pgr-mediated effect on spermiogenesis and improve sperm motility characteristics depending on the abundance of mPRα protein in sperm during spermiation.     

长牡蛎精子膜蛋白的提取及其部分生化性质的研究

从提取时间和TritonX-100浓度两个方面对长牡蛎(Crassosstrea gigas)精子膜蛋白提取条件进行优化,确定最佳提取条件:将精液150 g离心去除精原细胞,经PBS缓冲液和Tris-HCl缓冲液清洗后,3 000 g离心10 min(4 ℃)得精子悬液;然后加入3倍体积含1% TritonX-100的膜蛋白提取液,冰浴振摇1 h;50 000 g离心15 min(4 ℃),收集上清液即为精子膜蛋白溶液.SDS-PAGE电泳后,考马斯亮蓝R-250染色检测到21种膜蛋白,分子量在26～156 kD之间,PAS染色检测到糖蛋白有14种,苏丹黑B染色检测到脂蛋白有17种,其中有13种膜蛋白既是糖蛋白又是脂蛋白.实验结果还发现1种分子量为38 kD的r16膜蛋白既为糖蛋白又为脂蛋白,且高碘酸-Shiff试剂和苏丹黑B染色最深,条带最清晰,蛋白丰度最高,与此相对的分子量为55 kD的r12膜蛋白,考马斯亮蓝R-250染色也较深,条带清晰,但其糖蛋白和脂蛋白染色相对r16膜蛋白较浅,说明r16和r12两种膜蛋白其蛋白丰度相差不大,其糖基化程度及脂化程度可能相差较大,此两种膜蛋白有待于进一步分离纯化.此研究结果可为长牡蛎精子膜蛋白在精子发生和受精过程中作用的研究提供基础资料.     

A Preliminary Bacteriological Study of Refrigerated Channel Catfish Sperm

Abstract.— This study was designed to simulate conditions encountered routinely during refrigerated storage of channel catfish sperm. Sperm samples were stored at 4 C in non-sterile and sterile Hanks' balanced salt solution (HBSS). Non-sterile HBSS was prepared with distilled water stored for 2 wk in a plastic carboy prior to use. Observations were made on the frequency and abundance of bacteria in samples, and on changes in sperm motility and quality. Sperm samples stored in non-sterile HBSS had a complete loss of motility within 72 h. Samples maintained in sterile HBSS showed an initial decrease in motility between 48 and 72 h, and a complete loss of motility within 10 d. Quality of the sperm in each buffer decreased as motility decreased; morphologic changes and reduced motility of sperm were coincident with increased bacterial numbers. Bacteria were cultured on tryptic soy agar and Pseudomonas F agar (PFA) by spread-plating 10–μL aliquots from each sample onto bacteriologic media and incubating for 5 d. The dominant bacteria observed were members of the genus Pseudomonas , representing 67% of the total bacteria identified. The dominant pseudomonad ( Pseudomonas sp.) cultured from sperm samples stored in sterile buffer produced caseinase, lecithinase, and was β-hemolytic, whereas the dominant bacteria ( P. putida ) cultured from samples stored in the non-sterile buffers did not. Highly motile pseudomonads, present in two samples stored in sterile buffer, colonized below the surface of the PFA media at 4 C. The attributes of the bacterial contaminants that likely contributed to the decrease in sperm quality were production of extracellular enzymes, consumption of oxygen, and a high level of motility. Potential sources of degradative bacteria were commensal flora of channel catfish and the water used in preparing the storage buffer.     

Structure of the intratesticular duct system for sperm emission in the starspotted smooth-hound Mustelus manazo

We conducted histological observation of male germ cells and reproductive organs of the starspotted smooth-hound Mustelus manazo in Tokyo Bay to reveal any abnormality in male reproductive traits, as part of a study to elucidate the factors causing recent fluctuation in abundance of the population. Spermatogenesis proceeded in spermatocysts from the germinal zone in the ventral part of the testis to the degenerative zone in the dorsal part, where the spermatozoa were conveyed into the ciliated lumina of the attached terminal branches of the intratesticular ducts. The intratesticular ducts were classified from their terminal ends into branch, stem, and collecting tubules. The ducts formed in the germinal zone and grew as the spermatocysts developed. An opening formed through the wall of each of the most mature spermatocysts into a branch tubule; bundles of spermatozoa were evacuated through this opening into the branch and then the stem tubule and subsequently into the collecting tubules in the rete testis and the efferent duct connected to the epididymis. Spermatocysts that were unable to emit sperm because of failure of adhesion to the branch tubules were disorganized in situ, as were their spermatozoa. The collapsed spermatocysts seem to be cleared by hemophagocytosis with lymphocytes and leukocytes, which may have been recruited from the epigonal organ. There were no specific abnormalities in the spermatogenesis or the morphological structure of testes, which suggested that an abnormality of male reproductive traits was not the major cause of the recent fluctuation in the population abundance of this species. Details of the intratesticular duct system for sperm emission to the epididymis are the first findings in elasmobranchs worldwide.     

Evaluation of fertilizing capacity of cryopreserved rainbow trout sperm

ABSTRACT:   Experimental insemination was performed using artificially produced low-motility sperm. A mathematical model was applied to the results of the insemination in order to clarify the relationship between sperm motility, the density of sperm and the fertilization rate of eggs. In the model, the probability of fertilization by individual spermatozoa was a function of sperm density in the insemination solution. The results showed that the probability of fertilization clearly decreased with increased sperm density, and the maximum possible fertilizing rate by increasing the sperm density was constrained by the proportion of motile sperm (% motility). The model was also applied to the results of insemination tests of cryopreserved sperm in order to evaluate the fertilizing capacity of cryopreserved sperm. It was proven that cryopreserved sperm needed a higher density to obtain the maximum fertilization rate compared with fresh sperm, and it was anticipated that the ratio of the motile inseminated cryopreserved sperm should be more than 5.0% to achieve an egg fertilization rate greater than 90%.     

Relationships between body size and secondary sexual characters,and sperm characters in male Dolly Varden char (Salvelinus malma)

In Salmonidae, subordinate males are exposed to higher risks of sperm competition than dominant males and thus are expected to improve the sperm characteristics (sperm concentrations, sperm velocity and sperm longevity). In this study, we investigated the relationships between body size and secondary sexual characters (breeding colour, hump height and snout length), and sperm characteristics of one‐year‐old (newly matured) Dolly Varden char. Small males displayed higher sperm concentrations than large males. Moreover, males with dull breeding colours, but not with lesser snout length and hump height, displayed an increased sperm velocity compared to males with bright colours, suggesting a trade‐off between sperm quantity and the investment in breeding colour. In addition, sperm longevity decreased as sperm swimming velocity increased. These findings indicate that small males with dull breeding colours improve the quantity and quality of their sperm to a great extent to enhance their chances of reproductive success.     

鱼类精子质量评价研究进展

在鱼类人工繁育中,研究者主要关心的是卵子质量,长期以来对精子质量未引起足够重视.而精子质量同样会影响繁育效果的重要因素.鱼类精子质量的评价指标有多种,如精子活力、运动时间、密度、形态、受精率和生理功能等.其中最传统的评价指标是精子活力,其测定方便,能较准确地预测受精率.将精子运动时间和活力综合考虑可更好地反映精子的运动能力.而精子受精率则是精子质量的直接反映,但会受到卵质等因素的影响.质膜完整性、线粒体功能、染色质结构完整性等可体现精子的质量,但测定方法较繁琐.近年来,鱼类精子质量检测技术迅速发展,计算机辅助精子分析(CASA)、流式细胞术(FCM)分析、低渗肿胀(HOS)、单细胞凝胶单泳(SCGE)等技术的建立,使得测定指标更多样、客观、准确.本文逐一介绍了评价精子质量的各种指标,并对各指标的测定方法、测定原理、国内外研究情况进行详细叙述,旨为我国鱼类精子质量评价研究提供背景资料.     

Motility Parameters of Rainbow Trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) Spermatozoa in Relation to Sequential Collection of Milt,Time of <Emphasis Type="Italic">Post-mortem</Emphasis> Storage and Anesthesia

The present study investigated the effects of sequential collection of milt, time of post-mortem storage and anesthesia on rainbow trout (Oncorhynchus mykiss) sperm motility parameters (using computer-assisted sperm analysis – CASA) as well as seminal plasma osmolality and sperm concentration. The post-mortem storage and time of anesthesia altered motility characteristics of rainbow trout sperm to different extents. The moderate impact of time of anesthesia was manifested in a shortened duration of sperm motility after 10 min exposure of fish to anesthetic. The prolonged post-mortem storage (≥40–60 min), in addition to lowering sperm motility duration, also significantly influenced sperm motility parameters, such as sperm velocities, percentage of motile sperm and sperm trajectory parameters. These results clearly demonstrate that when milt from sacrificed fish is used for sperm motility studies, the time of post-mortem storage significantly alters sperm motility characteristics. Since sperm motility rate and swimming velocity could predict fertilizing ability, detrimental effects of prolonged post-mortem storage may lead to reduced fertilization success. Sperm concentration and seminal plasma osmolality were lower in the first fractions and increased with successive collections of milt. It suggests the presence of urine contamination of the first milt fractions which were collected by stripping. Therefore, testing of sperm concentration and/or seminal plasma osmolality should be mandatory while handling stored milt.     

Protective role of antifreeze proteins on sterlet (<Emphasis Type="Italic">Acipenser ruthenus</Emphasis>) sperm during cryopreservation

The loss of sperm quality in sterlet (Acipenser ruthenus) due to freeze-thaw process in cryopreservation was investigated in the present study. Two antifreeze proteins (AFPI or AFPIII) were used at different concentrations of 0.1, 1, 10, and 100 μg/mL. We compared motility, curvilinear velocity, and plasma membrane integrity of fresh, cryopreserved sperm, and sperm cryopreserved in the presence of antifreeze proteins. Fresh sperm (control) had 85?±?4% motility and 160?±?2 μm/s curvilinear velocity, respectively. After cryopreservation, the motility of frozen-thawed sperm without addition of antifreeze proteins significantly decreased (44?±?9%), compared to the control. The highest motility of frozen-thawed sperm was obtained in cryopreserved sperm with addition of 1 μg/mL of AFPIII (58?±?14%). No significant differences were observed in curvilinear velocity between fresh sperm and cryopreserved sperm with/without addition of AFPI or AFPIII. The flow cytometry analysis revealed that fresh sperm contained 94.5?±?6% live cells, while the cryopreserved sperm only contained 26.6?±?14% live cells. Supplementation of antifreeze proteins has significantly improved the percentage of live cells in frozen-thawed sperm, except 0.1 μg/ml of AFPI group. No significant difference in percentage of live cells was detected in the sperm cryopreserved with 10 μg/mL of AFPI or AFPIII, compared to fresh sperm. Thus, addition of antifreeze proteins to cryopreservation medium could be considered to improve the post-thawed sperm quality of sterlet.     

Cryopreservation of sea cucumber Apostichopus japonicus (Selenka) sperm

A simple and convenient method for the cryopreservation of sea cucumber Apostichopus japonicus (Selenka) sperm was tested in the present study. The highest motility (76.7±2.9%) of post‐thawing sperm was obtained in 15% dimethyl sulphoxide (DMSO) with a 1:9 dilution (semen volume to DMSO volume) when 0.5 mL semen–DMSO mixture was frozen at 6 cm above liquid N₂ in a closed styrofoam box. After thawing, sperm cryopreserved in glycerol almost lost motility entirely. Although there was no significant difference in percentage of motile sperm between 15% and 20% DMSO, the duration of sperm motility of 15% DMSO group was longer than that of 20% DMSO group. The motility of post‐thawing sperm enhanced when the dilution ratio of semen increased from 1:1 to 1:9. Morphological changes such as the loss of mitochondria, swollen plasma membrane and broken or rolled‐up tails were observed in post‐thawing sperm using an eosin–nigrosin staining. The fertility of cryopreserved sperm was significantly lower than that of unfrozen sperm. The 10‐fold increase in sperm to egg ratio resulted in double fertility for cryopreserved sperm, and about 70% fertility relative to the control.