Effect of cryopreservant combinations on the motility and morphology of curimba (Prochilodus lineatus) sperm

This study investigated the application of intra- and extra-cellular cryoprotectant combinations on the quality of curimba Prochilodus lineatus semen subjected to cryopreservation. Semen treatments were tested with 8% DMSO or methanol as intracellular cryoprotectant, 5% egg yolk or lactose as extracellular cryoprotectant and 5% BTS. These cryoprotectant combinations are suitable for curimba but have not been tested at the lesser concentrations proposed or in combination with BTS. Semen samples collected from 19 curimbas were diluted into one of four cryoprotectant combinations: DMSO+yolk; DMSO+lactose; methanol+yolk; and methanol+lactose. After dilution, semen samples were cryopreserved in 0.5 mL straws for 10 days in a liquid nitrogen tank. Semen was thawed in a water bath at 60°C for 8s. We evaluated the quality of fresh, diluted (pre-freezing) and post-freezing semen according to sperm motility rate (%) and duration (s). Sperm morphology was also analyzed in thawed semen. Sperm motility rate decreased progressively after dilution and thawing. The motility rate in post-freezing semen was higher in the treatments using DMSO+lactose and methanol+yolk. Sperm motility duration in post-freezing sperm was greater in the treatments using methanol rather than DMSO as intracellular cryoprotectant, irrespective of the extracellular cryoprotectant used. Abnormality frequency in thawed sperm was less in semen treated with egg yolk than with lactose. Thus the use of methanol intracellular cryoprotectant is recommended along with yolk extracellular cryoprotectant in the cryopreservation process for curimba semen.     

Cryopreservation of European catfish Silurus glanis sperm: sperm motility, viability, and hatching success of embryos

The aim of this study was to elaborate cryopreservation methods for ex situ conservation of European catfish. The success of sperm cryopreservation was evaluated by post-thaw sperm motility and velocity, percentage of live spermatozoa and fertility (hatching rates) using frozen/thawed sperm. The best hatching rates of 82-86% were obtained with sperm stored for 5 h before freezing in immobilizing solution and frozen with Me2SO in concentrations of 8, 10, and 12%, or with a mixture of 5% Me2SO and 5% propandiole. These results did not significantly differ from the fresh sperm control sample. The percentage of live spermatozoa in frozen/thawed sperm did not correlate with hatching rate or motility of spermatozoa, but was negatively correlated with velocity of spermatozoa (r=-0.47, P=0.05). The percentage motility in frozen/thawed sperm ranged from 8 to 62%, when sperm was stored in immobilizing solution 5h before freezing. The average value in the fresh sperm (control) was 96%. The frozen/thawed sperm motility rate significantly correlated with the hatching rate (r=0.76, P=0.0002), but not with the percentage of live spermatozoa (r=0.16, P=0.52) or the sperm velocity (r=0.07, P=0.79). The velocity of frozen/thawed spermatozoa ranged from 37 to 85 microm/s, whereby methanol concentrations of 7.5 and 10% resulted in highest velocities. Freezing sperm volumes of 1-4 ml did not affect the quality of frozen/thawed sperm.     

Influence of osmolality and ions on the activation and characteristics of zebrafish sperm motility

Despite the prevalence of zebrafish as a model scientific organism, understanding sperm function in this species is essentially limited to observations that osmotic shock initiates motility. During natural spawning, sperm encounter a range of environmental salinities as well as freshwater mixed with egg-associated ovarian fluid (OF), thus sperm are likely to be exposed to saline prior to egg contact. Effects of saline on sperm function in this model species are unknown, but likely to be important. Using computer assisted sperm analysis, this study addressed the effects of osmolality of spawning media and ionic composition and pH on the proportion of sperm becoming motile at activation (motility), as well as sperm velocity and path. When activated with tap water, motility was maximal (80%) at 10 s (earliest time measured), declining to 5% by 87 s postactivation. With activation at moderate osmolalities (∼160-200 mmol/kg) initial motility was decreased relative to low osmolality, increased from 10 to 30 s, and subsequently declined less rapidly (motility in 80 mM NaCl was 35%, 80%, and 60% at 10, 30 and 147 s, respectively). Thus, moderate osmolality increased duration, but introduced a temporal lag in motility onset. With moderate osmolalities, the rate of velocity decay was less than that with tap water activation. Sodium chloride and sucrose similarly impacted both motility and velocity. Replacement of NaCl with KCl, pH values ranging from 6.8 to 8.4, or the presence of gadolinium were without effect. Motility, but not velocity, was slightly supressed by Ca²⁺. Therefore, whereas pH and concentrations of Ca²⁺ or K⁺ of OF are unlikely to impact fertility via sperm motility, the OF contribution to spawning media osmolality may have pronounced effects on motility and velocity of sperm, factors previously correlated with fertility in other species.     

Relationships between reproductive characteristics in male Vimba vimba L. and the effects of osmolality on sperm motility

In Vimba vimba, GSI, sperm volume, and spermatozoa concentration range from 3.4-7.4 %, 0.1-1.1 ml, and 13.3-34.8 × 10⁹ spz ml⁻¹, respectively. Gonad mass (r = 0.90) and sperm volume (r = 0.35) significantly correlated with weight of males. Significant correlation was also found between gonad mass and length of males (r = 0.85). Sperm motility (r = 0.99) and velocity (r = 098) significantly decreased after activation in Tris-HCl 20 mM, pH 8.5. Osmolality of the seminal plasma was 273.2 mOsmol kg⁻¹. Sperm motility and velocity were significantly affected by the osmolality of the activation medium (P < 0.01). Hyper-osmolality compared to seminal plasma osmolality totally suppressed the sperm activation. At 15 s post-activation, the sperm motility significantly decreased at 240 mOsmol kg⁻¹ in KCl or NaCl media. The highest sperm motility and velocity (at 60 s post-activation) were observed at 200 mOsmol kg⁻¹ in NaCl, KCl, or sucrose media. In all treatments, the tip of the flagellum of spermatozoa became curled into a loop shape after activation of sperm in distilled water containing 20 mM Tris-HCl, pH 8.5 that shortened the flagellum.     

Cryopreservation of red snapper (Lutjanus argentimaculatus) sperm: Effect of cryoprotectants and cooling rates on sperm motility, sperm viability, and fertilization capacity

Sperm cryopreservation of red snapper (Lutjanus argentimaculatus) is essentially unexplored, although many species of the Lutjanidae family are considered to be high-value commercial species. The objective of this study was to develop a species-specific cryopreservation protocol for red snapper (L. argentimaculatus) sperm by optimizing cryoprotectants and cooling rates in the cryopreservation procedure. Ten cryoprotectants at four concentrations and two freezing protocols were examined in two separate experiments. In the first experiment, toxicity studies of dimethyl sulfoxide (DMSO), glycerol, propylene glycol (PG), ethylene glycol (EG), formamide, methanol, ethanol, sucrose, trehalose, and dimethylacetamide (DMA) on sperm motility were performed. Semen diluted 1:1 in Ringer solution were exposed to cryoprotectants at four final concentrations of 5%, 10%, 15%, or 20% for periods of 10, 20, 30, 40, 50, 60, 90, and 120 min at room temperature (25 °C). The cryoprotectants and concentrations that showed the least toxic effect on sperm motility were selected for cryopreservation trials. In the second experiment, selected cryoprotectants were then assessed for freezing capacity of sperm as follows: DMSO 5% and 10%, PG 5% and 10%, EG 5% and 10%, ethanol 5%, and methanol 5%. Semen was diluted 1:1 in Ringer solution and equilibrated with selected cryoprotectants for 10 min at room temperature. Sperm were frozen in a controlled-rate programmable freezer at four cooling rates of 3, 5, 10, and 12 °C/min from an initial temperature of 25 °C to final temperatures of −40 or −80 °C before plunging into liquid nitrogen. Sperm equilibrated in 10% DMSO and cooled at a rate of 10 °C/min to a final temperature of −80 °C had the highest motility (91.1 ± 2.2%) and viability (92.7 ± 2.3%) after thawing. The fertilization rate of frozen-thawed sperm (72.4 ± 2.4%) was not different (P > 0.05) from that of fresh sperm (75.5 ± 2.4%). This study apparently represents the first reported attempt for cryopreservation of L. argentimaculatus sperm.     

Effects of osmolality on sperm morphology, motility and flagellar wave parameters in Northern pike (Esox lucius L.)

Northern pike (Esox lucius L.) spermatozoa are uniflagellated cells differentiated into a head without acrosome, a midpiece and a flagellar tail region flanked by a fin structure. Total, flagellar, head and midpiece lengths of spermatozoa were measured and show mean values of 34.5, 32.0, 1.32, 1.17 μm, respectively, with anterior and posterior widths of the midpiece measuring 0.8 and 0.6 μm, respectively. The osmolality of seminal plasma ranged from 228 to 350 mOsmol kg⁻¹ (average: 283.88 ± 33.05). After triggering of sperm motility in very low osmolality medium (distilled water), blebs appeared along the flagellum. At later periods in the motility phase, the tip of the flagellum became curled into a loop shape which resulted in a shortening of the flagellum and a restriction of wave development to the proximal part (close to head). Spermatozoa velocity and percentage of motile spermatozoa decreased rapidly as a function of time postactivation and depended on the osmolality of activation media (P < 0.05). In general, the greatest percentage of motile spermatozoa and highest spermatozoa velocity were observed between 125 and 235 mOsmol kg⁻¹. Osmolality above 375 mOsmol kg⁻¹ inhibited the motility of spermatozoa. After triggering of sperm motility in activation media, beating waves propagated along the full length of flagella, while waves appeared dampened during later periods in the motility phase, and were absent at the end of the motility phase. By increasing osmolality, the velocity of spermatozoa reached the highest value while wave length, amplitude, number of waves and curvatures also were at their highest values. This study showed that sperm morphology can be used for fish classification. Sperm morphology, in particular, the flagellar part showed several changes during activation in distilled water. Sperm motility of pike is inhibited due to high osmolality in the seminal plasma. Osmolality of activation medium affects the percentage of motile sperm and spermatozoa velocity due to changes in flagellar wave parameters.     

Cryopreservation of turkey semen: Effect of breeding line and freezing method on post-thaw sperm quality,fertilization, and hatching

Cryopreservation methods for poultry semen are not reliable for germplasm preservation, especially for turkeys, where fertility rates from frozen/thawed semen are particularly low. The objective was to evaluate cryopreservation methods for effectiveness in promoting cryosurvival and post-thaw function of sperm from five turkey lines: one commercial line and four research (RBC1; E; RBC2; F) lines from Ohio State University (OSU). The model for cryopreservation was set up as a 2 × 2 × 2 × 5 design for cryoprotectant (glycerol or dimethylacetamide (DMA)), cryopreservation medium (Lake or ASG), method of dilution (fixed dilution volume versus fixed sperm concentration) and turkey line, respectively. The final cryoprotectant concentrations were 11% glycerol or 6% DMA. Thawed sperm were evaluated for plasma membrane integrity and quality, motility, acrosome integrity and, after artificial insemination, for egg fertility and hatchability. Commercial turkey hens were used for all fertility trials, regardless of semen source. Turkey sperm frozen with glycerol exhibited higher membrane integrity and membrane quality upon thawing than turkey sperm frozen with DMA although no differences in total motility, and only minimal differences in progressive motility, were detected among the eight cryopreservation treatments. Within line, fertility was affected by cryoprotectant, medium and dilution method, where the overall highest percentages of fertile, viable embryos (Day 7) occurred for the DMA/ASG/fixed sperm concentration method, while high percentages (15.8–31.5%) of fertile, non-viable embryos (Day 1–6) were observed for multiple cryopreservation methods, including two glycerol treatments. From a single insemination, the duration of true and viable fertility in all lines was 10–13 weeks and 9–10 weeks, respectively. The duration of hatchability was 4–6 weeks after insemination for four of the turkey lines. The highest percentage of viable embryos was observed for the commercial line (9.5 ± 2.4%), followed by the E line (5.3 ± 1.3%), F line (3.7 ± 2.0%) and RBC2 line (2.6 ± 0.8%). For the RBC1 line, there was 100% embryonic death by Day 6 of incubation. Overall, better fertility results were obtained with the cryoprotectant DMA, the ASG diluent and fixed sperm concentration. However, the applicability of this method for preserving semen from research populations may be line dependent.     

In vitro effect of zearalenone on sperm parameters,oocyte maturation and embryonic development in buffalo

The negative impact of zearalenone (ZEN; potent estrogenic mycotoxin) exposure on buffalo embryo production has not yet been determined. In the current study, buffalo sperm and oocytes were exposed to ZEN at different concentrations during maturation. Sperms (with and without ZEN exposure) were incubated for 2 h and evaluated for motility, viability, acrosome integrity, normality, and ultrastructure. Matured oocytes exposed to ZEN were stained to determine their nuclear maturation. Further, their developmental ability was evaluated after in vitro fertilization. Our results showed the toxic effects of ZEN at high concentrations (2000 ng/mL) on different buffalo sperm parameters. The number of acrosome-intact sperm was reduced at 0 h after exposure to a concentration of ≥ 100 ng/mL. Furthermore, the maturation rate of buffalo oocytes (telophase I + metaphase II) was significantly decreased in ZEN-treated oocytes with a higher degeneration rate. Oocytes matured in 1000 ng/mL ZEN and subsequently exhibited considerable reduction in cleavage rate and blastocyst formation compared with control oocytes (2.6% vs. 13.1%). Moreover, the morula rate was decreased (p < 0.001) in ZEN-treated oocytes at concentrations of ≥ 10 ng/mL. Overall, the adverse effects of in vitro ZEN exposure on buffalo sperm parameters and oocyte meiotic progression with a notable reduction in cleavage, morula, and blastocyst rates were defined by these results. Altogether, buffaloes should be considered sensitive to ZEN exposure with respect to their reproductive function.     

Effect of cryopreservation on phosphorylation state of proteins involved in sperm motility initiation in sea bream

We have previously demonstrated that in sea bream Sparus aurata motility initiation determined changes in the phosphorylation state of some proteins. This paper describes an investigation of the effect of a freezing-thawing procedure on the protein phosphorylation/dephosphorylation pattern. Proteins extracted from fresh and cryopreserved spermatozoa (before and after motility activation) were separated on SDS-PAGE, blotted on nitrocellulose membrane and treated with anti-phosphotyrosine, anti-phosphothreonine, or anti-phosphoserine antibodies. The results obtained demonstrate that the cryopreservation protocol has a strong effect on the phosphorylation state of proteins. In general, compared to fresh sperm, phosphorylated proteins are most numerous in both activated and non-activated cryopreserved sperm, and in particular we observed a dramatic increase in threonine phosphorylation. However, frozen-thawed sperm showed a minor number of proteins that changed their phosphorylation state after motility activation. Among these, we identified the acetyl-coenzyme A synthetase that plays a role in sperm motility initiation in both fresh and cryopreserved sperm.     

Role and regulation of sperm gelsolin prior to fertilization

To acquire fertilization competence, spermatozoa should undergo several biochemical changes in the female reproductive tract, known as capacitation. The capacitated spermatozoon can interact with the egg zona pellucida resulting in the occurrence of the acrosome reaction, a process that allowed its penetration into the egg and fertilization. Sperm capacitation requires actin polymerization, whereas F-actin must disperse prior to the acrosome reaction. Here, we suggest that the actin-severing protein, gelsolin, is inactive during capacitation and is activated prior to the acrosome reaction. The release of bound gelsolin from phosphatidylinositol 4,5-bisphosphate (PIP(2)) by PBP10, a peptide containing the PIP(2)-binding domain of gelsolin, or by activation of phospholipase C, which hydrolyzes PIP(2), caused rapid Ca(2+)-dependent F-actin depolymerization as well as enhanced acrosome reaction. Using immunoprecipitation assays, we showed that the tyrosine kinase SRC and gelsolin coimmunoprecipitate, and activating SRC by adding 8-bromo-cAMP (8-Br-cAMP) enhanced the amount of gelsolin in this precipitate. Moreover, 8-Br-cAMP enhanced tyrosine phosphorylation of gelsolin and its binding to PIP(2(4,5)), both of which inactivated gelsolin, allowing actin polymerization during capacitation. This actin polymerization was blocked by inhibiting the Src family kinases, suggesting that gelsolin is activated under these conditions. These results are further supported by our finding that PBP10 was unable to cause complete F-actin breakdown in the presence of 8-Br-cAMP or vanadate. In conclusion, inactivation of gelsolin during capacitation occurs by its binding to PIP(2) and tyrosine phosphorylation by SRC. The release of gelsolin from PIP(2) together with its dephosphorylation enables gelsolin activation, resulting in the acrosome reaction.     

The developments between gametogenesis and fertilization: ovulation and female sperm storage in Drosophila melanogaster

In animals with internal fertilization, ovulation and female sperm storage are essential steps in reproduction. While these events are often required for successful fertilization, they remain poorly understood at the developmental and molecular levels in many species. Ovulation involves the regulated release of oocytes from the ovary. Female sperm storage consists of the movement of sperm into, maintenance within, and release from specific regions of the female reproductive tract. Both ovulation and sperm storage elicit important changes in gametes: in oocytes, ovulation can trigger changes in the egg envelopes and the resumption of meiosis; for sperm, storage is a step in their transition from being "movers" to "fertilizers." Ovulation and sperm storage both consist of timed and directed cell movements within a morphologically and chemically complex environment (the female reproductive tract), culminating with gamete fusion. We review the processes of ovulation and sperm storage for Drosophila melanogaster, whose requirements for gamete maturation and sperm storage as well as powerful molecular genetics make it an excellent model organism for study of these processes. Within the female D. melanogaster, both processes are triggered by male factors during and after mating, including sperm and seminal fluid proteins. Therefore, an interplay of male and female factors coordinates the gametes for fertilization.     

Investigations of motility and fertilization potential in thawed cryopreserved mouse sperm from cold-stored epididymides

Cold transport of epididymides from genetically modified mice is an efficient alternative to the shipment of live animals between research facilities. Mouse sperm from epididymides cold-stored for short periods can maintain viability. We previously reported that cold storage of mouse epididymides in Lifor® perfusion medium prolonged sperm motility and fertilization potential and that the sperm efficiently fertilized oocytes when reduced glutathione was added to the fertilization medium. Cryopreservation usually results in decreased sperm viability; an optimized protocol for cold storage of epididymides plus sperm cryopreservation has yet to be established. Here, we examined the motility and fertilization potential of cryopreserved, thawed (frozen-thawed) sperm from previously cold-stored mouse epididymides. We also examined the protective effect of sphingosine-1-phosphate (S1P) on sperm viability when S1P was added to the preservation medium during cold storage. We assessed viability of frozen-thawed sperm from mouse epididymides that had been cold-transported domestically or internationally and investigated whether embryos fertilized in vitro with these sperm developed normally when implanted in pseudo-pregnant mice. Our results indicate that frozen-thawed sperm from epididymides cold-stored for up to 48 h maintained high fertilization potential. Fertilization potential was reduced after cold storage for 72 h, but not if S1P was included in the cold storage medium. Live pups were born normally to recipients after in vitro fertilization using frozen-thawed sperm from cold-transported epididymides. In summary, we demonstrate an improved protocol for cold-storage of epididymides that can facilitate transport of genetically engineered-mice and preserve sperm viability after cryopreservation.     

Human sperm proteins from testicular and epididymal origin that participate in fertilization: modulation of sperm binding to zona-free hamster oocytes, using monoclonal antibodies.

In order to identify human sperm surface proteins involved in the gamete recognition process, mouse monoclonal antibodies were directed against human spermatozoa and screened with live spermatozoa by enzyme-linked immunosorbent assay (ELISA). Immunoperoxidase staining of human testis showed the early presence of four corresponding proteins on germinal cells, while six were detected primarily in testis fluid. The presence of 17 proteins was evidenced in the epididymis. Eight were detected with a decreasing gradient from the beginning to the end of the organ, including vasa efferentia for three of them. The other nine were observed in only one defined segment, usually the caput epididymis, which was found to be the most active region. Comparison of spermatozoa patterns from testis, vasa efferentia, and the three regions of epididymis pointed out a progressive coating. By contrast, three antibodies displayed a migration of spermatozoa surface domains in the course of epididymal transit. Six antibodies were found to inhibit human spermatozoa adherence to zona-free hamster oocytes, while nine promoted it. Molecular weights of antigens corresponding to nine of the antibodies ranged from 11 to 215 kDa. No correlation could be established with previously described human proteins. These observations emphasize the role of epididymis in human sperm maturation.     

Relief effect of vitamin A on the decreased motility of sperm and the increased incidence of malformed sperm in mice exposed neonatally to bisphenol A

Administration of 50 g of bisphenol A (BPA) for the first 5 days after birth resulted in a decrease in the percentage of moving sperm, and an increase in the incidence of malformed sperm, in the epididymides of mice at 10 weeks of age, although no marked changes were found in the testicular histology between BPA-treated and vehicle-treated control mice. The deteriorating effects of 50 g of BPA were ameliorated by the concurrent administration of 100 IU of retinol acetate (RA). Neonatal treatment with 0.5 g of BPA for 5 days resulted in an increase in the incidence of malformed sperm, whereas the BPA effect became more severe in mice nursed by mothers fed a vitamin A-deficient (VAD) diet only a few days before and after parturition. On the other hand, neonatal treatment with 20 g of estrogen for the first 5 days after birth resulted in an increase in the number of estrogen receptor (ER)-positive cells in the epithelium of the vas deferens, whereas only a few epithelial cells showed weak ER-positive signals in the vehicle-treated control mice at 18 days after birth. This increase, however, was suppressed by the concurrent administration of RA. Although five daily treatments with 50 g BPA led to no significant increase in the number of ER-positive cells, it may have been due to the weak estrogenic activity of BPA, as discussed. These findings clearly showed that in mice, neonatal exposure to a relatively large dose of BPA causes damage to the motility and morphology of sperm, but the BPA effect is, to some extent, inhibited by a supplement of VA, and enhanced under a VAD condition.This work was supported by Grants-in-Aid for Encouragement of Young Scientists, Scientific Research (C) and Scientific Research on Priority Areas (A) from the Ministry of Education, Science, Sports, and Culture, Japan     

Use of amides as cryoprotectants in extenders for frozen sperm of tambaqui, Colossoma macropomum

Amides were tested as cryoprotectants in comparison with glycerol and DMSO (more traditional cryoprotectants) for recovery of Colossoma macropomum (tambaqui fish) sperm. Milt was extended in Beltsville Thawing Solution, then frozen with the addition of 2%, 5%, 8%, or 11% of: (1) dimethylacetamide (DMA), (2) dimethylformamide (DMF), (3) methylformamide (MF), or with 5% glycerol or 10% dimethylsulfoxide. Fertilization rates were greatest (P < 0.001) with amides; 8% DMF (91.6 ± 1.3%), 5% DMF (88.9 ± 1.6%), and 8% MF (83.0 ± 1.6%), which did not significantly differ among themselves, when compared with glycerol (51.6 ± 2.4%) and DMSO (61.9 ± 3.1%). The best hatching rates (P < 0.001) also occurred for 5% or 8% DMF and 8% MF (79.1 ± 3.1, 87.6 ± 1.5, and 74.8 ± 3.0, respectively) and were also similar (P > 0.05). For such treatments, both fertilization and hatching rates were similar (P > 0.05) to those with fresh sperm (91.7 ± 1.4 and 87.4 ± 1.4, respectively). The best sperm motility across extenders (at least 55.7%) was with 5%, 8%, and 11% DMF (P < 0.001). Those same treatments, along with 11% MF, provided the longest (P < 0.001) period of motility (at least 1 min). The greatest sperm integrity (more than 54%) was with 5% and 11% MF and with DMA and DMF at all tested concentrations (P < 0.001). The greatest (P < 0.001) sperm viability (at least 31%) was for 5%, 8%, and 11% DMA, and with 8% and 11% MF, and also for DMF at all tested concentrations. Sperm DNA integrity was best (more than 50%) for 2%, 5%, and 8% MF and for DMA and DMF at all concentrations (P < 0.001), whereas 2% DMA, 11% MF, 11% DMF, and the three amides at both 5% and 8% yielded the highest mitochondrial functionality (at least 44%; P < 0.001); thus, 8% MF and both 5% and 8% DMF were the cryoprotectants with the best postthaw quality for C. macropomum sperm.     

Relationship between sperm motility, morphology and the fertility of stallions

Sperm quality has an important role in determining fertility. Although there have been numerous studies to document the relationship between sperm quality and fertility, the methods of determining this association and conclusions vary. In the present study, computer-assisted sperm analysis (CASA) was used for evaluation of sperm motility, and differential interference contrast (DIC) microscopy was used for evaluating sperm morphologic features of breeding stallions. Fertility was measured using three endpoints: seasonal pregnancy rate (PR), percent pregnant/cycle (PC), and percent pregnant/first cycle (FCP). Increased total sperm motility (P = 0.08) and progressive path velocity (P = 0.06) tended to be associated with higher PR, whereas percent coiled tails (P = 0.02) was associated with a lower PR. Sperm motility variables associated with an increase in PC and FCP included total, progressive, and rapid sperm motility, and increased path and progressive velocity. Percent pregnant/first cycle was the only fertility measure able to discriminate among high, average, and low fertility groups, based on total and progressive sperm motility. Percent normal sperm was the only morphology variable associated with an increased PC and FCP, whereas increased levels of most sperm morphologic abnormalities (including abnormal and detached heads, proximal and distal droplets, general midpiece abnormality, and coiled tails) were associated with a decline in PC and FCP. Sperm quality variables most highly correlated with fertility included percent total sperm motility (PR, r = 0.37, P < 0.05; PC, r = 0.59, P < 0.05; and FCP, r = 0.64, P < 0.05), and percent morphologically normal sperm (PC, r = 0.42, P < 0.05; and FCP, r = 0.39, P < 0.05).     

Subpopulation distribution of motile sperm relative to activation medium in steelhead (Oncorhynchus mykiss)

In the present study, steelhead sperm were activated in artificial tap water, ovarian fluid, activating saline, or in combinations of these media, and motility characteristics were determined using computer-assisted sperm analysis. Motility characteristics of individual sperm were then assessed to test the hypothesis that motile sperm are distributed among discrete subpopulations and that their distribution is influenced by the activation medium. Analysis with k-means clustering detected three discrete motile sperm subpopulations in steelhead semen, regardless of the activation medium. Based on multivariate analysis of variance, proportions of these subpopulations did not differ between sperm activated with ovarian fluid and activating saline, or any combination of these two media. However, subpopulation distributions for sperm activated with either ovarian fluid or activating saline were influenced by the level of dilution of these media in artificial tap water. There was an increase in the number of sperm in high velocity (curvilinear), high straightness, and high wobble subpopulation with increased levels of ovarian fluid or activating saline. The change in sperm motility characteristics with a change in activation medium may play a role in normal fertilization, as discharged sperm pass from seminal plasma and water through ovarian fluid en route to the egg.     

Ionic factors affecting motility,respiration and fertilization rate of the sperm of the bivalvePecten maximus (L.)

Fertilization of the scallopPecten maximus occurs after gametes were naturally released in sea water by the bivalve which has undergone stimulation. The motility of the spermatozoa requires their dilution in sea water (1/40). Dilution triggers an immediate increase of oxygen consumption by sperm, reflecting an activation of a cyanide-sensitive respiration of a cellular origin. When scallops were stimulated by thermal shocks or by serotonin injection, sperm sampled at the urogenital pore output duct shows a respiration-motility activation after sea water dilution which is not seen in sperm scarified from the gonad. Dilution of kidney-sampled sperm into acidic (pH 5) or Na⁺-free artificial sea water reversibly inhibits both respiration and motility. In all cases fertilization rate of sperm is correlated to the increase of respiratory rate and motility measured after dilution in different media. Whether the scallop was stimulated or not, the pH of haemolymph and pericardic fluids were one pH unit below the value of sea water, the pH of the gonad and of the kidney tissues being more acidic (6.5 in average). Our results suggest that the acidic pH of the genital tract maintains the spermatozoa in a quiescent state and that capacitation occurs when male gametes move from the gonad to the kidney from where it is naturally released.Abbreviations ASW artificial sea water - SW sea water - TRIS trishydroxymethyl-aminomethane     

Effects of gonadotropin-releasing hormone agonist administered in microparticles on sperm quality and quantity,and plasma sex steroid levels in northern pike

Artificial reproduction of northern pike Esox lucius is impeded by the likelihood of obtaining only a small volume of sperm of inconsistent quality. A controlled-release hormone delivery system has the potential to enhance sperm production while avoiding multiple injections The objective of this study was to investigate the effects of mammalian gonadotropin-releasing hormone agonist (mGnRHa) incorporated into poly(lactic-co-glycolic acid) (PLGA) microparticles on milt production, spermatozoon characteristics, and secretion of 17β-estradiol (E2), 11-keto testosterone (11-KT), and testosterone in northern pike. Fish were divided into four groups and injected with 2 mg/kg BW carp pituitary extract (CPE), 20 µg/kg BW mGnRHa in PLGA microparticles, or 20 µg/kg BW mGnRHa plus 20 mg/kg BW metoclopramide (MET) in PLGA microparticles (PLGA + MET), along with a control group injected with 1 ml/kg 0.9% NaCl. At 48 h postinjection, the volume of milt produced was significantly greater in groups treated with CPE and PLGA + MET than in other groups. At 96 h postinjection, all hormone-treated groups exhibited significantly higher spermatozoon average velocity than recorded in the control group. Spermatozoon motility was significantly increased (P < 0.05) in the CPE and PLGA groups compared to baseline values. All treated groups showed significantly lower levels of 11-KT after the hormone injection compared to baseline values and to controls. Plasma testosterone levels increased in all hormone-treated groups. The use of PLGA microparticles, with or without metoclopramide, is suitable for use as a carrier of hormone treatments to regulate spermiation in mature northern pike.     

Survivorship, development, and DNA damage in echinoderm embryos and larvae exposed to ultraviolet radiation (290-400 nm)

Laboratory experiments utilizing ecologically relevant irradiances of ultraviolet radiation (UVR) known to occur in shallow Gulf of Maine waters were conducted on the planktonic embryos and larvae of two common benthic echinoids; the green sea urchin Strongylocentrotus droebachiensis and the sand dollar Echinarachnius parma. Significant decreases in survivorship were observed in freshly fertilized embryos of both species with greater mortality in E. parma that was associated with the absence of UVR-absorbing compounds, the mycosporine-like amino acids. Experiments on blastula, gastrula, and prism larval stages of S. droebachiensis also showed significant decreases in survivorship, delays in development, and abnormal embryos and larvae associated with exposure to UVR. Additionally, all developmental stages of S. droebachiensis experimentally exposed to UVR resulted in significant increases in DNA damage, measured as cyclobutane pyrimidine dimer photoproducts. The observed delays in early cleavage and subsequent developmental stages for S. droebachiensis are correlated with DNA damage. It is postulated that cell cycle arrest at critical checkpoints after DNA damage, mediated by a suite of cell cycle genes, is a component of the observed UVR induced developmental delays.