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.     

Sperm cryopreservation of the noble Scallop Chlamys nobilis by a programmable freezing method: Effect of cryoprotectant

A study on Chlamys nobilis sperm cryopreservation by a programmable freezing method was conducted under laboratory condition. Four cryoprotectant agents (dimethyl sulfoxide [DMSO], methanol [MET], propanediol[PG] and ethylene glycol [EG]) and four concentrations (5%, 10%, 20% and 30%) were evaluated for their ability to retain sperm motility, movement characteristics and fertility. Results showed that cryopreserved sperm total motility produced by DMSO and MET at 5%, 10% and 20% were higher than other cryoprotectant treatment groups (CPA groups), as well as rapid sperm percentage. The curvilinear (VCL) and straight line (VSL) velocity produced by DMSO at 5% significantly higher than other CPA groups (p < 0.05), while no significant differences were found for average path (VAP) velocity. The lateral head displacement (ALH) in all CPA groups was similar and without significant difference (p > 0.05), as well as the beat‐cross frequency (BCF). A significant higher fertilization rate was produced in DMSO than that in MET at same concentration (p < 0.05), and no significant differences were found for differing concentrations of the same cryoprotectant (p > 0.05). Overall, 5%‐20% DMSO was more suitable for Chlamys nobilis sperm programmable cryopreservation when the calcium‐free Hanks’ balanced salt solution was used as the extender, and 10°C/min from 0°C to ?80°C was used as freezing rate. The findings presented in this study will benefit conservation programs for Chlamys nobilis.     

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.     

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.     

The Effects of an Oxygen Scavenger and Coconut Water on Equine Sperm Cryopreservation

Alternative sources of lipoproteins in semen extenders could replace animal by-products. We hypothesized that: (1) post-thaw semen parameters and fertility would not be different in coconut water (CW)–treated samples compared with egg yolk (EY)–treated samples and (2) the use of an oxygen scavenger (Oxyrase) would improve post-thaw sperm motility and membrane integrity and decrease lipid peroxidation. Experiment 1: three ejaculates each from five stallions were split into four treatments: EY, CW, egg yolk with Oxyrase, and coconut water with Oxyrase. Computer-assisted sperm analysis measured progressive and total motility, velocity, and linearity. Membrane integrity, apoptosis, and lipid peroxidation were evaluated using propidium iodide, annexin, and BODIPY fluorescent probes, respectively. Samples were cryopreserved, stored in liquid nitrogen, and then thawed to 37°C and analyzed again. Experiment 2: one ejaculate was divided into two aliquots and cryopreserved using either CW or EY. In a crossover design, 12 mares were bred on two consecutive cycles with either EY or CW. Pregnancy evaluations were at 14-day gestation. No differences were detected in sperm parameters between CW and EY (P > .05). Oxyrase did not improve sperm motility parameters in post-thaw samples, nor did it show protective effects for viability or against membrane damage (P > .05). More mares became pregnant using CW than EY (11/12 vs. 6/12, respectively; P = .013). Use of CW is a viable alternative to animal-based products in the cryopreservation of stallion semen.     

Effect of vitamin E on spermatophore regeneration and quality of pond‐reared,black tiger shrimp (Penaeus monodon)

The objective of the present work was to evaluate the effects of different levels of vitamin E in feed on the spermatophore regeneration and quality of male Penaeus monodon. The experiment was carried out with the four following treatments: the basal diet no added vitamin E, the diet added 200, 600 and 1,000 mg/kg respectively. Spermatophore regeneration and quality were evaluated by spermatophore weight, sperm count and spermatophore absence rates, which male P. monodon were extruded spermatophore for feeding 20 and 40 days. In the experiment, the weight of the twice regenerated spermatophore of the males added to the vitamin E group was higher than that of the untreated control group. The weight of the first regenerated spermatophore with the addition of 1,000 mg/kg group was the highest and significantly higher than the control group (p < .05), but there was no significant difference among the three groups with different levels of vitamin E. The weight of the second regenerated spermatophore with the addition of 600 mg/kg group was the highest, followed by 1,000 mg/kg group, both of which were higher than the control group and the addition of 200 mg/kg group. Within the same group, the regeneration spermatophore weight showed overall upward trend as the feeding time, twice regenerate experiment spermatophore weight with added to the vitamin E groups were significantly higher than the initial value (p < .05), but three spermatophore weight of male shrimp at the control group had no significant difference. The sperm quantity and the percentage of normal sperm of the twice regenerated spermatophore of the males with added to the vitamin E group was higher than that of the untreated control group, and those of the addition of 200 mg/kg group was significantly higher than that of control group (p < .05). The total number of sperm and the percentage of living sperm of male shrimp in the experimental group decreased with the increase of vitamin E in the feed. Within the same group, the total number of sperm and the percentage of living sperm of male shrimp with added to the vitamin E groups showed overall upward trend as the feeding time and were significantly higher than the initial value (p < .05), but the control group was slightly down and had no significant difference. Comprehensive sperm weight, sperm quantity and living sperm percentage of three indicators, that adding 200 mg/kg of vitamin E in feed could effectively promote the spermatophore regeneration in the male P. monodon and improve the sperm quantity. The experimental results provide a scientific basis for the breeding of P. monodon.     

Evidence of LAT1 expression in canine caput epididymis

L-type amino acid transporter 1 (LAT1), the first isotype of amino acid transport system L, transports aromatic and branched amino acids pivotal for fundamental cellular activities such cellular growth and proliferation. LAT1 expression was high only in the brain in contrast to its limited distribution and low level of expression in normal tissues. We found potent LAT1 expression in canine caput epididymis by quantitative RT-PCR and Western blotting analysis. Immnuno-histochemical examination revealed observable LAT1 in microvillous epithelial cells.     

Evaluation of Commercial‐scale Approaches for Cryopreservation of White Crappie,Pomoxis annularis,Sperm

Crappie, Pomoxis spp., are popular game fish throughout North America and are produced by public and private hatcheries. However, production is limited by a lack of information on tank culture and induced spawning methods. Development of techniques for storage of sperm and in vitro fertilization would increase flexibility in spawning. Therefore, techniques for sperm cryopreservation were examined in white crappie, Pomoxis annularis. Sperm from adult wild white crappie were used to evaluate sperm extender, cryoprotectant agent and concentration, and cooling technique based on post‐thaw sperm motility. Percent egg fertilization was also compared between sperm stored in the two best cryopreservation protocols and two different osmotic activator solutions. Sperm were cryopreserved using treatment combinations of two extenders (350 mOsmol/kg Hanks' balanced salt solution [HBSS] and 350 mOsmol/kg Ca²⁺free HBSS) and two cryoprotectants (dimethyl sulfoxide [DMSO] and methanol) at concentrations of 5, 10, and 15% that were cooled at four different rates: 5, 10, 20, and 40 C/min. Post‐thaw sperm motility and fertilization rates indicated white crappie sperm can be cryopreserved using either extender, cryoprotectants of either 5% DMSO or 10% methanol, and cooling at 40 C/min. A follow‐up experiment demonstrated sperm in suspensions on ice retained viability after overnight transport.     

硫酸铜对泥鳅精子活力的影响

以泥鳅精子为受试材料,应用计算机辅助精子分析系统,研究硫酸铜(0.1、1、10 mg/L)对泥鳅的精子活力的影响.结果表明,在0 h试验组,0.1 mg/L的硫酸铜明显降低泥鳅精子运动速度,1 mg/L的硫酸铜对运动时间和速度产生显著抑制作用,10 mg/L的硫酸铜时运动百分数、运动时间和速度都产生明显影响(P<0.0...