史氏鲟精子超低温冷冻保存研究

比较了史氏鲟精子在3种不同配比浓度稀释液的保存效果。试验结果表明,配方Ⅲ作为稀释液,8%甲醇作为抗冻剂,二步法超低温(-196℃)冷冻保存,5h后取出,38℃水浴解冻取得最好的冻后激活率,解冻后激活率为(52.3±3.5)%。解冻精子分别采用井水和激活液D(10mmol/L Tris+10mmol/L NaCl+25mmol/L Glu,pH 8.0)激活,进行人工授精。结果显示配方Ⅲ冻精采用激活液D激活授精获得最高受精率为68.56%,最高孵化率为52.91%。本次试验表明,1～2mmol/L范围内,低浓度K+比高浓度K+对史氏鲟精子保存有利;52～82mosmol/kg范围内,高渗稀释液有利于史氏鲟精子的保存;且激活授精方法是影响冻精受精率和孵化率的关键因素之一。     

对虾输精管用于人工授精的研究

越冬后期雄虾输精管中的精子已达成熟程度,可以作为人工授精用精子来源。采取越冬后期雄虾的输精管用于人工授精,除一批卵子未受精外,其余两批的平均受精率为73.6%,孵化率为86.6%。保存72小时后的输精管用于人工授精,平均受精率为54.2%,孵化率为86.7%。类似于用精荚进行人工授精的实验结果。     

瓦氏黄颡鱼精子的生理特性及其超低温冷冻保存的初步研究

对瓦氏黄颡鱼(Pelteobagrus vachelli)精子在不同盐度和pH下的精子活力进行观察,同时研究了精子在4种不同稀释液与2种不同浓度抗冻剂组成的保存液中的超低温冷冻保存,并开展了冻精的授精实验。结果表明,瓦氏黄颡鱼精子浓度为(2.035±0.179)×1012cell·mL-1,在盐度为5.8、pH为7.17时,精子的活力都高达95%。以A液作为稀释液、10%甲醇作为抗冻剂时,冷冻保存精子效果最好,解冻后精子活力为(81.7±0.9)%。用解冻后的精子进行人工授精,获得的受精率为(88.4±2.1)%,孵化率为(74.0±0.8)%;而鲜精受精率为(91.0±0.8)%,孵化率(82±1.6)%,冻精与鲜精均无显著性差异。人工授精实验证明了解冻后的精子能正常用于该鱼的人工繁殖。     

舟山渔场银鲳人工授精及孵化

2005-2008年,在舟山海域银鲳(Pampus argenteus)繁殖季节(4-5月),随流刺网或张网捕鱼船出海,捕捞性腺成熟的银鲳,进行人工授精和孵化.人工授精试验采用干法、湿法和半干法,比较受精率和孵化率.通过4年连续测量银鲳成熟卵的卵径、油球径等形态指标,对卵子成熟度进行判别,在水温16.0～18.5 ℃、盐度28.0±0.5的条件下进行孵化.结果表明,以干法受精后间隔3～5 min水洗和半干法受精的方法受精率最高,受精率达18.50%～33.50%,最高达40%;孵化率达到43.83%～51.00%,最高达66%.银鲳成熟卵子的卵径为(1.368±0.082)mm,油球径为(0.550±0.031)mm,油球1个.对影响海捕银鲳亲体受精率和孵化率的环境条件也进行了讨论.     

杂色鲍与盘鲍种间杂交受精率的影响因素

研究卵子排放后的时间、精子排放后的时间、授精精子浓度、胰蛋白酶、钙离子、pH和水温等因子对杂色鲍(Haiotisdiversicolor,以下简称S)与盘鲍(H.discus discu,以下简称J)种间杂交受精率的影响。结果表明,卵子排放后的时间和授精精子浓度是影响杂交受精率两个最重要的因素。与自繁组合相比,正反杂交受精率均较低且下降速度快得多。水温21.5℃时,两亲本自繁对照组中,卵子排放1 h对受精率均无显著影响。而SJ杂交,卵子排放时间2 min时,受精率为55.9%±8.1%,4.7 min时受精率已下降了50%;JS杂交,卵子排放时间为4 min时,受精率为31.5%±9.7%,10 min时受精率下降至4 min时的29.1%。相比之下,精子排放时间对杂交受精率的影响小得多,3 h内对正反交杂交受精率没有显著影响。杂交适宜精子浓度约为母本自繁的100倍,其中SJ杂交适宜授精精子浓度为6.0×106~2.4×107mL-1,JS杂交适宜授精精子浓度为3.8×107~7.5×107mL-1。水温对于杂交受精率也有影响,短时间内将水温控制在26~29℃可以得到较高受精率。其他实验因素,如添加胰蛋白酶、Ca2+或改变授精体系pH对杂交受精率均无正向作用。     

精子密度对杂交黄颡鱼“黄优1号”受精率、孵化率及出苗率的影响

为研究瓦氏黄颡鱼(Pelteobagrus vachelli)不同精子密度对杂交黄颡鱼"黄优1号"(hybrid yellow catfish "Huangyou-1")受精率、孵化率和出苗率的影响,设置8组不同精子密度组(1.0×10~3～1.0×10~6个·mL~(-1))开展人工授精实验。结果表明:当精子密度从1.0×10~3个·mL~(-1)提高到2.5×10~4个·mL~(-1)时,受精率、孵化率和出苗率均显著上升(P0.05);当精子密度为5.0×10~4个·mL~(-1)时,受精率达到最大值为(88.8±2.0)%;当精子密度为2.5×10~4个·mL~(-1)时,孵化率及出苗率达到最大值,分别为(83.4±2.8)%和(79.4±4.6)%。最后,通过建立回归方程确定了精子密度与受精率、孵化率和出苗率之间的关系。研究筛选出了最适精子密度,研究结果可为杂交黄颡鱼规模化高效繁育提供理论参考。     

家鱼冷冻精液激活、授精方法的研究

激活—授精溶液渗透压对家鱼冻精的能育性有明显影响。当用1毫升冻精与4 毫升卵子授精时,冻精与鲜卵在水中与在 171和 342 mOsm/l NaCl 溶液中的受精率均很高(91％左右),无明显差别;当精卵量比为 1:16时,在水中的受精率明显低于在上述 NaCl 溶液中的受精率;当精卵量比升高到 1:32以上时,冻精在 174mOsm/l 溶液中的受精率明显高于其它二组。鲢卵在171和342 mOsm/l 溶液中维持受精能力的有效时间(6 分钟)明显长于在淡水中(2 分钟)。当精子稀释难的 pH 值偏碱(8.7)和冻精激活—授精溶液偏酸(pH6.3)时,冻精的受精率明显下降。解冻后精子在 4℃存放1.5小时活力和受精率下降不多,少数冻精可存活长达 16小时。冻精活力与受精率之间存在着明显正相关,相关系数为0.97,回归方程为 Y=3.68 1.23X。     

氯化钠浓度对鲈鲤精子活力的影响

为了解鲈鲤精子的生理特性,研究了不同氯化钠浓度试验条件下鲈鲤精子的活力情况,结果表明:在0%～0.65%氯化钠溶液中鲈鲤精子均能被激活;当氯化钠溶液浓度为0.35%时,鲈鲤精子激烈运动时间和寿命最长,分别为(66.33±7.99)s、(635.66±95.78)s,而且该组的鲈鲤精子的寿命极显著高于其他处理组(P0.01),激烈运动时间除显著高于0.60%组和0.65%组(P0.05)外,与其他处理组的差异不显著(P0.05);当氯化钠浓度超过0.35%时,鲈鲤精子活动强度减弱,寿命缩短;当氯化钠溶液浓度超过0.70%后,精子无法被激活。本研究可为鲈鲤精子的保存提供参考依据。     

中华鲟子一代配子质量及其子二代生长特征分析

通过测定卵的直径、卵粒质量、受精率和孵化率系列指标以及精子的激活率、快速运动时间和寿命系列指标对人工驯养子一代中华鲟配子进行质量评价,并通过该批受精卵孵出的子二代系列生长指标进行其发育状况分析。结果显示,子一代所产卵子形态饱满,卵径平均3.71 mm,小于自然繁殖群体。与历年在本基地养殖的自然繁殖群体统计资料相比,其受精率(60.1%)和孵化率(36.8%)处在中等偏下的水平。2012年和2013年的精子活力监测结果发现,子一代精子有效运动时间(漩涡运动+快速运动)均较野生群体高。表明子一代卵子质量较野生群体有所降低,而精子活力则较野生群体高。与历年该基地集约化养殖系统培育出的子一代中华鲟相比,子二代的生长表现出显著的阶段差异性,其中仔鱼阶段生长较慢,稚鱼和幼鱼阶段生长优势则较明显。研究表明,促进人工驯养条件下的子一代后备亲鱼性腺发育完善,以获得较高质量的配子和子二代,对防止中华鲟种质退化至关重要。     

黄鳝精子活力检测和精子入卵早期过程观察

采用Olympus3×51相差系统显微镜和SQIAS—1000彩色精液质量图文分析系统检测黄鳝精子活力。结果表明,在NaCl溶液浓度为0～0.3%时,黄鳝精子激活比例随溶液浓度升高而极显著增加(P<0.01);当NaCl浓度达到0.7%时,精子激活比例、直线运动速度和鞭毛摆动频率均显著(P<0.05)或极显著(P<0.01)降低。扫描电镜观察显示:黄鳝成熟卵卵壳膜上的精孔区呈漏斗状凹陷,其底部中央可见一精孔管外孔,口径约4.22±0.66μm;黄鳝精子入卵速度缓慢,受精过程较长,从精子附着于卵球表面到精孔管完全堵塞,约30s～5min。     

Fertilization by refrigerator stored sperm of the Indian major carp, Labeo calbasu (Hamilton, 1822)

This study reports on the spermatological properties, and on the development of a protocol for refrigerator storage (4°C) of Labeo calbasu (Hamilton, 1822) sperm for artificial breeding. Volume, motility, concentration and pH of the freshly collected sperm were 2.21 ± 0.53 (μL g^?1 of fish weight) (mean ± SD), 95 ± 1 (%), 1.93 ± 0.44 × 10⁹ (cells mL^?1) and 7.56 ± 0.17 respectively. Sperm activation was evaluated at different osmolalities of NaCl solution, and motility ceased completely when osmolality of the extender was ≥287 mOsmol kg^?1. Sperm retained motility for 24, 72 and 108 h, after refrigerator storage when sperm were undiluted, suspended in Alsever's solution and suspended in Alsever's solution containing 5% methanol respectively. Fertilization rate of fresh eggs with sperm stored at 4°C in Alsever's solution and Alsever's solution containing 5% methanol was 77% and 60% with a hatching rate of 60% and 43% respectively. The fertilization and hatching success of the stored sperm suggests potential to use refrigeration for transporting genetic material to hatcheries for artificial breeding of L. calbasu in Bangladesh.     

Kurokura Solution as Immobilizing Medium for Spermatozoa of Tench (Tinca tinca L.)

This study tested KUROKURA solution (Kurokura et al., 1984, Aquaculture 37, 267–274) and its modifications (by increasing NaCl content to 160, 180 and 200 mM) on immobilizing properties for sampling and short-term preservation of potential motility of tench spermatozoa. The immobilizing solution is used because, when collected, the sperm of most samples is contaminated by urine, causing spermatozoa to be of poor quality, with low motilities and velocities (almost 0), thus resulting in a worsened fertilization and hatching rate. Sperm was sampled with a syringe containing an immobilizing solution (IS), allowing an IS:sperm ratio of 2:1, under aerobic conditions at 0–4°C. This sperm solution was stored for 10 h and untreated sperm was collected prior to fertilization as a control. Spermatozoa quality was evaluated for the cell motility and velocity parameters and also for fertilization ability and hatching rate. Results obtained for tench sperm motility, velocity, fertilization and hatching rate showed that only sperm collected in the various immobilizing solutions can be successfully used for artificial insemination and preservation after 10 h at 0–4°C. The best immobilizing solution was found to be KUROKURA 180 (180 mM NaCl, 2.68 mM KCl, 1.36 mM CaCl₂· 2H₂O and 2.38 mM NaHCO₃), giving a fertility and hatching rate of 41%, with no change in rates after 10 h storage of sperm. Control sperm without immobilizing solution showed a fertilization and hatching rate of only 6–7%.     

紫外辐射灭活大黄鱼和黄姑鱼精子及其激活大黄鱼卵子发育为胚胎的效果

为探讨大黄鱼(Larimichthys crocea)和黄姑鱼(Nibea albiflora)精子的紫外辐射灭活适宜剂量及其激活大黄鱼卵子发育为胚胎的效果,以Ringer氏液为稀释液,按1:30稀释大黄鱼和黄姑鱼精子,采用自制紫外灭活装置[紫外辐射强度2200μW/(cm~2·s),紫外波长254 nm]对这2种鱼的精子进行紫外照射处理及活力测定,然后与正常大黄鱼卵子进行人工授精,授精后一部分卵未作冷休克处理,另一部分卵进行了冷休克处理(受精2 min 30 s,3℃海水,冷休克10 min),并进行了早期胚胎成活率和仔鱼孵化率的测定与比较。结果显示:1)大黄鱼和黄姑鱼精子的激活率与紫外照射处理时间呈负相关,精子的快速运动时间变化呈典型的Hertwig效应。2)未冷休克组中大黄鱼和黄姑鱼诱导的早期胚胎成活率与精子的紫外照射时间总体呈负相关,而仔鱼孵化率呈Hertwig效应。3)冷休克组中大黄鱼和黄姑鱼精子诱导的早期胚胎成活率和仔鱼孵化率随紫外照射时间的增加呈Hertwig效应,分别于2 min 20 s和1min 30 s时达相对峰值,此时,大黄鱼早期胚胎成活率和仔鱼孵化率分别为(38.3±4.3)%和(66.5±5.1)%,黄姑鱼早期胚胎成活率和仔鱼孵化率分别为(43.3±3.3)%和(67.7±6.3)%。分析认为,大黄鱼和黄姑鱼精子遗传失活的紫外辐射剂量分别以308 m J/cm~2和198 m J/cm~2为佳。本研究旨在为大黄鱼雌核发育技术的改进提供依据。     

Artificial insemination in tench, Tinca tinca L.

Abstract. The sperm of tench, Tinca tinca L., is characterized by a milky colour and consistency, and is of very low density. After collecting the sperm, motion of spermatozoa was recorded even without water activation. A better motility value (value 4·36 on average) was observed in spermatozoa collected in immobilizing solution (collecting medium) and stored for 3h, when compared with spermatozoa without collecting medium. Average total and relative numbers of spermatozoa were 12·16 × 10⁹ per male and 18·50 × 10⁹ per kg of body weight, respectively. When testing the effect of activating solution in artificial propagation of tench, the highest fertilization rates (81·3 and 85% in two cases) were found for NaCl solution with an osmotic concentration of 34 or 69 mOsmol and for fresh water, respectively. The fertility rate was reduced significantly ( P < 0·01) by any increase above 105 mOsmol in NaCl concentration in the activating solution. In the tests of optimal method of artificial fertilization, the highest hatching rate of sac fry (71·35%) was found in sperm collected into immobilizing solution. The application of immobilizing solution significantly increased the number of sac fry at the levels P < 0·1 and P < 0·01, if compared with intact sperm stored for 3 h and fresh sperm, respectively.     

Effects of ultra-violet irradiation on sperm motility and diploid gynogenesis induction in large yellow croaker (<Emphasis Type="Italic">Pseudosciaena crocea</Emphasis>) undergoing cold shock

Large yellow croaker, Pseudosciaena crocea, exhibit sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. We have established a protocol to produce all-female croaker P. crocea through induction of meiotic gynogenesis with homologous sperm. The first set of experiments investigated the ultra-violet (UV) irradiation on sperm motility and duration of sperm activity to determine the optimal UV dosage for genetic inactivation of sperm, yet retaining adequate motility for activation of eggs. Milt from several males was diluted 1:100 with Ringer’s solution and UV irradiated with doses ranging from 0–150 J cm⁻². The results indicated that motility and duration of activity generally decreased with increased UV doses. At UV doses greater than 105 J cm⁻², after fertilization, motility was <10% and fertilization rates were significantly lower. Highest hatching rate was obtained at 75 J cm⁻². A second set of experiments was carried out to determine appropriate conditions of cold shock for retention of the 2nd polar body in P. crocea eggs after fertilization with UV-inactivated sperm by altering the timing, temperature and duration of shock. At 20°C, shock applied at 3 min after fertilization resulted in higher survival rate of larvae at 6 h after hatching. Results of different combinations of three shock temperatures (2°C, 3°C or 4°C) and five shock durations (4 min, 8 min, 12 min, 16 min or 20 min) at 3 min after fertilization demonstrated that shocks of 12 min gave highest production of diploid gynogens. Statistical analysis revealed that maximum production of diploid gynogens (44.55 ± 2.99%) were obtained at 3°C. The results of this study indicate that the use of UV-irradiated homologous sperm for activation of P. crocea eggs and cold shock for polar body retention is an effective method for producing gynogenetic offspring.     

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%.     

Production of androgenetic amago salmon<Emphasis Type="Italic"> Oncorhynchus masou ishikawae</Emphasis> with dispermy fertilization

With the aim of improving artificial androgenesis in teleost fishes, we tested two methods for producing androgenetic diploids of amago salmon (Oncorhynchus masou ishikawae), namely, fertilization of gamma-ray irradiated eggs with fused spermatozoa (sperm-fusion method) and the fertilization of irradiated eggs with untreated sperm followed by the blocking of cell division (mitosis-inhibition method). Our results showed that the optimal condition for sperm fusion was to treat the sperm with 50% polyethylene glycol (molecular weight 7500) for 100 s. The efficiency of the two methods of androgenesis was compared in terms of fertilization rate, hatching rate, and larval survival after hatching. The rate of fertilization was lower with the sperm-fusion method than with the mitosis-inhibition method, but the reverse was true for the hatching rate. The survival rate of hatched larvae was the same with the two methods. Androgenesis was confirmed with a recessive albino color marker, and all viable offspring were found to be heterozygous based on analysis of the microsatellite markers. Our results suggest that androgenesis with the sperm-fusion method is a promising approach with potential applications in both aquaculture breeding programs and the preservation of endangered freshwater fishes.     

Improved Cryopreservation of Sperm of Paddlefish (Polyodon spathula)

Experiments were performed to improve protocols for sperm cryopreservation of paddlefish (Polyodon spathula), a species for which there has been limited study. The first experiment was conducted to investigate the effects of two extenders (modified Tsvetkova’s extender: mT and modified Hanks’ balanced salt solution: mHBSS) in combination with methanol (MeOH) and dimethyl sulfoxide in two concentrations (5 and 10%) on the postthaw motility and fertilization rates of cryopreserved sperm. The highest postthaw motility (85 ± 5%) was observed when sperm were frozen using mT extender with 10% MeOH as cryoprotectant. Extenders (P = 0.0018) and cryoprotectants (P = 0.0040) each had a significant effect on the postthaw motility of paddlefish sperm. The highest fertilization (80 ± 3%) was found when eggs were fertilized with sperm frozen with mT extender in combination with 10% MeOH. However, there was no significant difference among fertilization rates when MeOH was used as a cryoprotectant in either concentration or in combination with either mT or mHBSS extenders. In the second experiment, 4000 eggs were fertilized with the pooled contents of five straws of thawed sperm (total volume of 1.25 mL) using mT extender in combination with 5% MeOH, and hatch rates as high as 79 ± 5% were observed. A third experiment was also conducted to clarify the role of MeOH concentration; however, no significant difference was found among fertilization and hatch rates when either 5 or 10% MeOH was used as a cryoprotectant. These results suggest that MeOH is a safe and reliable cryoprotectant for freezing of paddlefish sperm and obtaining viable postthaw sperm for consistent fertilization and hatch rates. Further, this experimental protocol is relatively simple and applicable for commercial hatchery production of paddlefish.     

Cryopreservation of common carp sperm

Experiments were carried out to investigate the effect of five extenders (sucrose, glucose, fructose, KCl and a saline carp sperm extender) and two cryoprotectants (dimethyl-sulfoxide (DMSO) and methanol) on the cryopreservation of common carp sperm. Freezing of sperm using glucose extender and methanol as cryoprotectant resulted in the highest post-thaw motility, fertilization as well as hatching rates (63 ± 9%, 74 ± 15% and 67 ± 17% vs. 87 ± 5%, 84 ± 14% and 69 ± 14% using fresh sperm, respectively). In general, sugar-based extenders combined with methanol as cryoprotectant yielded higher motility, fertilization and hatching rates than ionic extenders in combination with DMSO. The jelly-like agglutination observed after thawing in samples frozen with sugar-based extenders did not reduce fertilization and hatching rates. Frozen–thawed sperm samples were able to successfully fertilize 10 g (8000) eggs.