Biochemical characterization of Siberian sturgeon Acipenser baeri and sterlet Acipenser ruthenus milt plasma and spermatozoa

This paper provides data concerning biochemical composition of milt of two sturgeon species, Siberian sturgeon bred in aquaculture facility of Inland Fisheries Institute in North Poland and sterlet (from two different populations from Danube and Odra). Milt plasma of Siberian sturgeon and sterlet, when compared to teleost fish, is characterized by much lower osmolality (up 70 to 96 mOsm kg⁻¹) and protein concentration (0.24–0.58 g l⁻¹). In spite of the presence of an acrosome and acrosin the antiproteinase activity of seminal plasma was low (12.79–25.40 U l⁻¹). Activities of arylsulfatase and β-N-acetylglucosaminidase were found in spermatozoa. This agrees with the presence of an acrosome in sturgeons sperm. Similarly to mammals, these enzymes are also present in milt plasma. We determined a range of enzymatic activities from the minimal (seminal plasma) to the maximal damage (supernatants obtained after freezing-thawing without cryoprotectant). Activities of arylsulfatase, β-N-acetylglucosaminidase, lactic dehydrogenase and acid phosphatase were released from spermatozoa after freezing-thawing. For this reason they are good potential candidates as a markers of cryoinjury to sperm acrosome and midpiece. This revised version was published online in July 2006 with corrections to the Cover Date.     

Greenlip abalone (Haliotis laevigata Donovan, 1808) sperm cryopreservation using a programmable freezing technique and testing the addition of amino acid and vitamin

This study investigated factors key to the development of sperm cryopreservation in the greenlip abalone Haliotis laevigata using a programmable freezing technique, including (1) permeable cryoprotectant agent (CPA) selection; (2) cooling rate; (3) endpoint temperature; (4) thawing temperature; (5) sperm to egg ratio and (6) sugar, vitamin and amino acid supplementation, using sperm motility, fertilization rate, plasma membrane integrity, mitochondrial membrane potential or acrosome integrity as quality assessment indicators. Results showed that among the permeable CPAs evaluated, 10% dimethyl sulfoxide was the most suitable for greenlip abalone sperm cryopreservation. The highest post‐thaw sperm motility was achieved with the sperm being frozen at a cooling rate of ?5°C min^?1 to ?30°C from 0°C and thawed and recovered in 40°C and 18°C seawater baths respectively. The addition of sugars in 10% dimethyl sulfoxide did not significantly improve the post‐thaw sperm motility and fertilization rate. The addition of 0.6% glycine, 0.2% taurine or 0.02% L‐ascorbic acid, on the other hand, significantly improved the post‐thaw sperm motility. However, only the addition of 0.6% glycine improved the post‐thaw sperm fertilization rate, which was further confirmed by the improvement of the post‐thaw sperm mitochondrial membrane potential and acrosome integrity through flow cytometry analysis.     

低渗溶液浓度对黄颡鱼精子活力和受精率的影响

为提高黄颡鱼(Pelteobagrus fulvidraco)人工授精的稳定性,分别用卵子和精子同步激活法以及预激活精子法,在6个梯度浓度(0%～0.5%)范围的低渗溶液中进行人工授精试验,比较精子活力、受精率和孵化率。结果显示:低渗溶液浓度与精子活力、精子激活率和受精率密切相关,在相同浓度的低渗溶液组中采用预激活精子法的受精率显著高于卵子和精子同步激活法(P<0.05),受精率相对应提高了7.7%～14.5%;低渗溶液的浓度和人工授精方式对孵化率无显著影响。结果表明在浓度0.3%的低渗溶液中采用预激活精子法的效果最好。     

Cryopreservation of Persian sturgeon (Acipenser persicus) sperm: effects of cryoprotectants,antioxidant, membrane stabilizer,equilibration time and dilution ratio on sperm motility and fertility

Three experiments were performed to develop protocols for cryopreservation of Persian sturgeon Acipenser persicus, sperm. In the first experiment, sperm from six males was individually split in three subsamples and cryopreserved using Modified Tsvetkova's extender (mT) supplemented with dimethyl sulfoxide (DMSO), methanol (MeOH), glycerol (Gly) and ethylene glycol (EG) at concentration of 5%, 10%, 15% and 20%. In the second set of experiments, the effects of six equilibration times (0, 5, 10, 20, 40 and 60 min) and dilution ratios (volume sperm: volume extender 1:0.5, 1:1, 1:2, 1:3, 1:5 and 1:10) and the additive advantage of bovine serum albumin (BSA; 0, 2.5, 5 and 10 mg mL^?1) and ascorbic acid (0, 2.5, 5 and 10 U mL^?1), on the post‐thaw survival of sperm (triplicate set of six fish) were evaluated. Then, sperm was diluted in 1:1 mT extender with 10 mg mL^?1 BSA with selected cryoprotectants (15% MeOH and 10% DMSO) for 5 min. After a month of storage in liquid nitrogen, post‐thawed sperm motility; fertilization and hatching rate and viability of derived larvae were measured (Exp.3). Evaluation of cryoprotectants efficiency showed that MeOH 15% and DMSO 10% were suitable for cryopreservation of Persian sturgeon sperm. Gly and EG resulted in very low post‐thaw motility rates even at lowest concentration. No significant difference was observed among the four different equilibration times (0, 5, 10, 20 min) (P > 0.05) although higher equilibration times than 20 min resulted low post‐thaw motility (P < 0.05). The motility of frozen–thawed sperm did not significantly change when dilution ratio was increased from 1:0.5 to 1:3 (P > 0.05). However, higher dilution ratios (1:5 and 1:10) reduced the percentage of motile sperm. Supplementation of the cryoprotectant solution with 10 mg mL^?1 BSA significantly improved post‐thaw motility (P < 0.05), but ascorbic acid did not improve post‐thaw motility (P > 0.05). The results of experiment 3 showed that the highest fertilization (30.2 ± 5.75) and hatching rates (28.2 ± 5.25) were observed when samples were frozen with 15% MeOH (P > 0.05). Our study indicates that the use of mT extender consisting of 10 mg mL^?1 BSA in 15% MeOH diluted with sperm at 1:1 ratio for 5 min can be recommended cryopreservation method for Persian sturgeon sperm.     

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。     

Cryopreservation of strip spawned sperm using non‐programmable freezing technique in the blue mussel Mytilus galloprovincialis

In this study, a non‐programmable freezing technique has been developed with the strip spawned blue mussel (Mytilus galloprovincialis) sperm. The key parameters optimized including (1) cryoprotectant agents (CPAs); (2) cooling temperature; (3) thawing temperature; (4) sugar and amino acid supplementation and (5) sperm to egg ratio. The fertilization rate and/or integrity of plasma membrane and acrosome were used as sperm quality assessment indicators. The highest post‐thaw sperm fertilization rate of 95% was achieved, when sperm were cryopreservated in 8% dimethyl sulfoxide at 7.8 cm above the liquid nitrogen surface and thawed in a 60°C seawater bath. The addition of glucose, sucrose or trehalose in dimethyl sulfoxide did not, whereas 0.8% glycine did significantly improve the post‐thaw sperm fertilization rates. The fluorescent evaluation has demonstrated that the addition of glycine significantly improved the post‐thaw sperm acrosome integrity, revealing a positive role of glycine in the improvement of post‐thaw sperm quality in blue mussels.     

虾夷扇贝精子形态结构和超低温冷冻损伤的电镜观察

应用透射电镜和扫描电镜技术,研究了超低温冷冻保存前后虾夷扇贝精子超微结构和形态的变化。结果显示,虾夷扇贝精子由头部、中段和尾部3部分组成,外被光滑质膜;顶体位于头部最前端,呈倒"v"形,细胞核近似圆柱状,电子密度较高;4个线粒体和两个相互垂直的中心粒构成了精子的中段;鞭毛细长,轴丝为典型的"9 2"结构。经超低温冷冻保存后,冷冻损伤的精子表现为:精子被膜肿胀、被膜与核膜分离、丢失;顶体破裂、内容物流出;线粒体解体、线粒体嵴变形;鞭毛被膜肿胀、部分精子鞭毛脱落。可以推测,超低温冷冻保存对精子膜、顶体、线粒体和鞭毛的损伤可导致冻精活力和受精能力的下降。     

Changes in the flagellum morphology of intact and frozen/thawed Siberian sturgeon Acipenser baerii (Brandt) sperm during motility

Morphological investigations on the changes in flagellar beating was carried out on native (taken from the milt) and thawed sperm of the Siberian sturgeon Acipenser baerii (Brandt). Immediately after activation, the pattern of flagellar wave formation and distribution was the same in native and thawed sperm but, after 27–42 s, depending on the samples, the thawed flagella showed asymmetric and poorly developed waves. The swimming trajectories recorded during 1‐s exposure were much shorter in thawed than in native sperm after 26–28 s motility. In native sperm, the flagella remained in the same axis as the head during the entire motility course, while the head of thawed sperm showed a right angle after 47 s. It is concluded that the freezing/thawing procedure induces some alteration in the dynamics of flagellar beating in many sperm, but these sperm still show progressive displacement. Therefore, the change in morphology of the flagellum during motion is a parameter that should be taken into account in the evaluation of the impact of various treatments on sperm motility.     

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%.     

Role of ion channels and membrane potential in the initiation of carp sperm motility

The exposure of freshly spawned, immotile carp sperm to hypoosmotic media triggers the initiation of calcium-dependent flagellar motility. Intracellular calcium concentration has been thought to be the critical component in motility initiation, possibly acting through a novel signalling pathway. The sensitivity of sperm cells to changes of osmolality of the environment raises the question whether a mechanoregulated osmosensitive calcium pathway is involved in the activation mechanism of carp sperm motility. The sperm cells are in a depolarized state in the seminal plasma (Ψ = –2.6 ± 3 mV) and they hyperpolarize upon hypoosmosis-induced activation of motility (Ψ = –29 ± 4 mV). The intracellular sodium [Na⁺]_i, potassium [K⁺]_i and calcium [Ca²⁺]_i ion concentrations were determined in quiescent cells, and at 20, 60 and 300 s after activation. The [Na⁺]_i and [K⁺]_i of the quiescent cells were similar to the [Na⁺]_e and [K⁺]_e of the seminal plasma. Following hypoosmotic shock-induced motility, both [Na⁺]_i and [K⁺]_i decreased to one-fourth of the initial concentration. The [Ca²⁺]_i doubled at initiation of the motility of the sperm cells and remained unchanged for 5 min. Bepridil (50–250 μM), a blocker of the Na⁺/Ca²⁺ exchanger, blocked carp sperm motility reversibly. Gadolinium, a blocker of stretch-activated channels (10–20 μM), inhibited sperm motility in a dose-dependent manner and its effect was reversible. Hypoosmotic shock fluidized the membrane and gadolinium treatment made it more rigid in both quiescent cells and hypotonic shock treated but immotile sperm cells. Based on these observations, it is suggested that, besides the well-known function of potassium and calcium channels, stretch-induced conformational changes of membrane proteins are also involved in the sperm activation mechanism of common carp.     

Sperm motility and fertilizing ability in the Persian sturgeon Acipenser persicus

The motility and fertilizing ability of the Persian sturgeon, Acipenser persicus, spermatozoa were investigated. Optimum ionic content (Na⁺, K⁺, Ca²⁺ and Mg²⁺) and pH of activation solution as well as the optimum dilution rate were determined. The results show optimum motility characteristics of spermatozoa in buffered solutions containing 25, 0.2, 3 and 10 mM L^?1 Na⁺, K⁺, Ca²⁺ and Mg²⁺, respectively, at dilution rate 1:50 and pH 8.0. To test the fertilizing ability of sperm, two buffered saline solutions were used as activation solution of sperm motility. The present study indicated (1) spermatozoa motility is one of key factors that influence on fertilizing ability of sperm, (2) a high fertilizing ability of sperm is obtained after dilution in saline solutions rather than in freshwater and (3) a maximum fertilization rate occurs in buffered saline solution containing 0.2 mM L^?1 K⁺. There is also a good correlation between biochemical characteristics of seminal plasma and fertilizing ability of sperm.     

Optimization of sperm irradiation protocol for induced gynogenesis in Siberian sturgeon,Acipenser baerii

The great diversity of optimal UV irradiation doses are used for DNA inactivation in fish sperm forcing authors to repeat optimization of irradiation treatment every time. Analysis of sperm UV irradiation protocol for induction of gynogenesis showed the importance of sperm UV light absorption estimations. The UV absorption investigation in Siberian sturgeon sperm showed average extinction coefficient 7.69 × 10^?8 ± 1.04 × 10^?8 cm². It is resulted in high heterogeneity of UV irradiation of undiluted sperm samples. Therefore, it is strongly suggested to specify doses only with defined concentration of spermatozoa; otherwise, the difference in absorbance level between samples can bring a significant error to optimal UV dose estimation. This was confirmed by UV-irradiated sperm motility investigation. Results of motility investigation of UV-irradiated sperm revealed high sensitivity of Siberian sturgeon spermatozoa motion mechanisms to UV irradiation, with complete loss of motility after homogeneous UV irradiation at doses above 2,000 J/m². Partial gynogenesis was conducted using diluted and undiluted sperm. Ploidy level of hatched larvae was estimated by flow cytometry. Percentage of haploid hatched larvae revealed sperm DNA inactivation efficiency. The highest percentage of haploid putative gynogenotes 19.67 ± 4.19 % was obtained at UV irradiation dose 200 J/m² with sperm diluted to 1:4.     

Retention of sperm motility in turbot, Scophthalmus maximus L.: the effects of time from activation, thermal shock and adenosine triphosphate levels

Abstract Four parameters were examined in order to define sperm quality in turbot Scophthalmus maximus L., sperm: (1) sperm motility, measured by direct counts of the number of active spermatozoa, expressed as % of total spermatozoa; (2) retention of motility after activation, measured by direct counts, 0–60min after activation, expressed as a % of the initial level of activity; (3) resistance to thermal stress, measured as change in retention of motility, and (4) adenosine phosphate (ATP) concentration, determined for samples of non-activated sperm. The proportion of motile spermatozoa at activation ranged from 34·8% to 97·6% (mean 76·3%) for the individual males tested. Turbot sperm retained on average 52% (range 27–90%) of its initial activity one hour after activation. Sperm samples which were stressed by cooling to –27°C retained only 8·6% (range 0–25%) of initial activity, compared to control samples which retained 49% (range 38–63%) of initial activity. The retention of motility after activation was not significantly related to the initial motility or the levels of ATP. Concentrations of ATP in turbot sperm (mean 0·46mg ATP/10⁶ spermatozoa, equivalent to 9·2nmol ATP/10⁸ spermatozoa) were comparable to those measured in mammals.     

锯缘青蟹精子入卵过程的扫描电镜观察

利用扫描电镜详细观察了锯缘青蟹精子入卵的过程。精子以其核突起附着在卵膜上,并迅速发生顶体反应。顶体反应时,顶体囊外翻,顶体管前伸,精子核辐射臂收缩,并拖至顶体囊的后部。顶体管迅速穿过卵黄膜,携带核物质一同进入卵子。锯缘青蟹为多精着卵,数精入卵。本文同时探讨了精子顶体反应机制以及受精过程卵子的作用。     

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.     

Chemical composition and osmolality of seminal fluid of Acipenser persicus; their physiological relationship with sperm motility

Determination of semen quality is necessary to understand the basic biochemical processes occurring during motility of sperm and during fertilization to evaluate the reproductive ability of different fish species and to create an optimal environment for storage of spermatozoa; in this regard less information is available for Acipenseridae compared with Cyprinidae and Salmonidae. The aim of the present study is to determine chemical composition and osmolality of seminal fluid and their relationship with sperm motility in Acipenser persicus. The results obtained show that sodium (Na⁺), chloride (Cl^?) and potassium (K⁺) were predominant ions in the seminal plasma and the average of osmolality of seminal plasma was 82.56 mOsm kg^?1. The higher chemical contents and osmolality compared with other sturgeon species reveal species‐specific characteristics and high secretory activity of spermatic duct in A. persicus. Significant positive correlations were observed between osmolality‐Cl^?, Na⁺‐osmolality and Na⁺–Cl^? (P<0.05, P<0.001 and P<0.05 respectively). But statistically significant correlation was not observed between seminal plasma parameters and sperm motility. Probably, the Na⁺ and Cl^? are the main electrolytes playing a major role in maintaining the osmolality of the seminal plasma and the viability of the spermatozoa in vivo.     

Role of sodium bicarbonate on the initiation of sperm motility in the Japanese eel

ABSTRACT:   In order to find out the role of sodium bicarbonate (NaHCO₃) on the initiation of sperm motility in the Japanese eel Anguilla japonica , interactions were investigated between NaHCO₃ and various reagents (K⁺ channel blocker 4-aminopyridine [4-AP], ammonium chloride [NH₄Cl], sodium acetate and calcium chloride [CaCl₂]) that could regulate internal factors (intracellular K⁺, intracellular pH [[pH]_i] and intracellular Ca²⁺) in sperm motility. Contradictory effects of NaHCO₃ were observed (i.e. an inhibitory effect when 4-AP was absent and a promoting effect when 4-AP was present). Sodium bicarbonate inhibited the initiation of sperm motility in the Japanese eel. However, NaHCO₃ restored the motility of immotile sperm that 4-AP inhibited. The inhibitory effect of NaHCO₃ disappeared with the addition of NH₄Cl, which raised [pH]_i, but the promoting effect was not affected by [pH]_i. Although NaHCO₃ recovered motility in the presence of 4-AP, this recovery was also observed with the addition of CaCl₂ instead of NaHCO₃. In the initiation of sperm motility in the Japanese eel, two roles for NaHCO₃ are suggested: an inhibitory role relating to the regulation of [pH]_i and a promoting role relating to the uptake of another initiation factor, which could be Ca²⁺.     

The effects of protein kinase A catalytic subunit on sperm motility regulation in Pacific abalone Haliotis discus hannai

Protein kinase A plays a central role in the regulation of sperm motility from echinoderms to mammals, but the information about its regulatory role in molluscs is very limited. In this study, a protein kinase A catalytic subunit (designated as HdPKA‐C) was identified from Pacific abalone Haliotis discus hannai. The open reading frame of HdPKA‐C was of 1,077 bp, encoding a peptide of 358 amino acids with a typical protein kinase domain. HdPKA‐C shared 82%–87% sequence similarities with other PKA‐Cs, and it was clustered first with gastropod PKA‐Cs in the phylogenetic tree. The mRNA of HdPKA‐C was constitutively expressed in examined tissues, with the highest level detected in hepatopancreas. The phosphorylated form of HdPKA‐C (p‐HdPKA‐C) was localized at the acrosome, connecting piece and flagellum of spermatozoa with variable intensity. Its phosphorylated substrates were also detected in these regions with much lower intensity at the connecting piece. The inhibition of HdPKA‐C activity with H‐89 led to a significant reduction in the percentage of motile sperm and sperm velocities. p‐HdPKA‐C was detected by Western blot in strip‐spawned sperm, naturally spawned sperm and H‐89‐treated sperm with almost the same intensity. The intensity of p‐HdPKA‐C substrates in naturally spawned sperm was higher than that in strip‐spawned sperm, and it was roughly the same as that in H‐89‐treated sperm except for two bands at 50 and 60 kDa. These results collectively indicated that HdPKA‐C played an important role in the regulation of abalone sperm motility by altering its substrates phosphorylation.     

The antioxidant system of sterlet seminal fluid in testes and Wolffian ducts

Oxidative stress is a possible source of spermatozoa function deterioration. Seminal fluid (SF) protects spermatozoa against reactive oxygen species (ROS) attack during development in testes and transit through the reproductive tract. Spermatozoa curvilinear velocity and percent of motile cells as well as changes in thiobarbituric acid-reactive substance (TBARS) content, superoxide dismutase, and catalase activity, and uric acid concentration in SF were evaluated in sterlet sperm collected from testes 24 h after hormone induction of spermiation and from Wolffian ducts at 12, 24, 36, and 60 h after hormone injection (HI). While testicular spermatozoa motility was not initiated in activating medium, Wolffian duct sperm showed low motility at 12 h, significant increase at 24 and 36 h, and decrease at 60 h. Testicular SF was characterized by the highest level of TBARS and activity of studied enzymes compared with SF from Wolffian duct sperm at 24 h post-HI. In fluid from Wolffian duct sperm, a significant increase in TBARS content was shown at 36–60 h post-HI. In contrast to testicular SF, in SF from Wolffian duct sperm, this increase was not counterbalanced by changes in the studied variables of antioxidant system. This may be the source of the observed decrease in spermatozoa motility parameters 60 h post-HI. The results may confirm a dual role of ROS in fish sperm physiology. The data with respect to decrease in sturgeon spermatozoa motility parameters at 60 h post-HI should be taken into account in artificial sturgeon propagation.     

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。