Spotted wolffish (Anarhichas minor) sperm cryopreservation in 5-mL cryovials.

In spotted wolffish Anarhichas minor aquaculture, cryopreservation is used to secure sperm availability throughout the entire spawning season. Under current protocols, sperm is cryopreserved in 0.5-mL straws. This implies thawing a considerable number of straws for insemination with cryopreserved sperm. In this work, we scale up the spotted wolffish sperm cryopreservation procedure through the development of a protocol for sperm cryopreservation in 5-mL cryovials. Different freezing (distances from the liquid nitrogen surface) and thawing rates were tested. The best results were obtained with cryovials frozen at a distance of 1.5 cm from the liquid nitrogen surface and thawed either at 15 or 10 °C for 4 and 6 min, respectively. Under these conditions, similar percentage of motile cells, sperm velocity and percentage of viable cells were obtained in comparison with the sperm cryopreserved in the traditional 0.5-mL straws. This protocol will facilitate the process of insemination with cryopreserved sperm in the spotted wolffish hatcheries.

Is it possible to store spotted wolffish (Anarhichas minor) sperm by refrigeration?

Spotted wolffish Anarhichas minor reproduction in captivity is dependent on in vitro fertilization. However, it is often challenging to acquire sufficient fresh sperm to fertilize the eggs that are obtained. In this study, we evaluate the possibility to store spotted wolffish sperm by refrigeration. Spotted wolffish sperm has the particularity that is already motile on stripping, and currently it is not possible to immobilize and reactivate. Thus, sperm refrigeration protocols should focus in extending this motility period that usually lasts up to 2 days. In a first experiment, we evaluated the possibility that the motility period of the sperm was limited by contamination with urine. The urea concentration in the sperm obtained both by stripping (17.10 ± 1.98 mg/dL) and directly from the testis (12.59 ± 2.37 mg/dL) was similar (p > 0.05), which indicate that the sperm collection method used avoid contamination with urine. Afterwards, we tested the possibility that the sperm motility period was limited by energy stores. The ATP concentration (initial value 5.65 ± 0.86 nmol/109 cells) remained stable (p = 0.099) during 30 h after sperm collection, and similar values (p = 0.329) were recorded at end of sperm storage in both diluted (3.88 ± 1.35 nmol/109 cells) and undiluted samples (4.76 ± 1.08 nmol/109). This indicates that the low intracellular ATP consumption, derived from the slow sperm motility, can probably be compensated rapidly enough by mitochondrial synthesis of ATP in the spotted wolffish sperm. In both experiments, diluted sperm kept higher percentage of motile cells during the storage time.

Inter-population ovarian fluid variation differentially modulates sperm motility in Atlantic cod Gadus morhua.

This study tested the hypothesis that the effects of Atlantic cod Gadus morhua ovarian fluid on sperm motility variables are population specific. Sperm from a northern G. morhua population were activated in the presence of ovarian fluid from either northern or southern G. morhua at different concentrations. Ovarian fluid acted as a filter, in some cases reducing sperm swimming performance compared with seawater. Fluid from females foreign in population (southern) to the males (northern) had a greater inhibiting effect than those from the native population. Follow-up analysis indicated that the ovarian fluids had lower Ca(2+) concentration in northern than southern G. morhua, which could be the causative mechanism. If widespread, such cryptic female choice could reduce the incidence of intraspecific hybridization among diverged populations and contribute to reproductive isolation.

Effect of cryopreservation on fish sperm subpopulations.

The evaluation of the motility data obtained with a CASA system, applying a Two-Step Cluster analysis, identified in seabream sperm 3 different sperm subpopulations that correlated differently with embryo hatching rates. Hence, we designed an experiment to understand the effect of the application of different cryopreservation protocols in these sperm motility-based subpopulations. We analyzed Sparus aurata frozen/thawed semen motility 15, 30, 45 and 60s after activation, using CASA software. Different protocols were applied for cryopreservation: three different cryoprotectants (Dimethyl Sulfoxide (Me(2)SO), Ethylene Glycol (EG) and Propylene Glycol (PG)) each at two different concentrations and two packaging volumes (0.5ml straws, and 1.8ml cryovials) were tested. Different freezing rates were evaluated corresponding to 1, 2, 3, 4 and 8cm above the liquid nitrogen surface for the straws and 1, 2 and 4cm for the cryovials. Motility parameters rendered by CASA were treated with a Two-Step Cluster analysis. Three different subpopulations were obtained: SP1 - slow non-linear spermatozoa, SP2 - slow linear spermatozoa and SP3 - fast linear spermatozoa. We considered SP3 as the subpopulation of interest and focused further analyses on it. Generally, SP3 was the best represented subpopulation 15s after activation and was also the one showing a greater decrease in time, being the least represented after 60s. According to the applied univariate general linear model, samples frozen in straws with 5% Me(2)SO and in cryovials with 10% Me(2)SO at 2 and 1cm from the LN(2,) respectively, produced the best results (closer to the control). Clustering analysis allowed the detection of fish sperm subpopulations according to their motility pattern and showed that sperm composition in terms of subpopulations was differentially affected by the cryopreservation protocol depending on the cryoprotectant used, freezing rates and packaging systems.

Fertilization capacity with rainbow trout DNA-damaged sperm and embryo developmental success.

Mammalian spermatozoa undergo a strong selection process along the female tract to guarantee fertilization by good quality cells, but risks of fertilization with DNA-damaged spermatozoa have been reported. In contrast, most external fertilizers such as fish seem to have weaker selection procedures. This fact, together with their high prolificacy and external embryo development, indicates that fish could be useful for the study of the effects of sperm DNA damage on embryo development. We cryopreserved sperm from rainbow trout using egg yolk and low-density lipoprotein as additives to promote different rates of DNA damage. DNA fragmentation and oxidization were analyzed using comet assay with and without digestion with restriction enzymes, and fertilization trials were performed. Some embryo batches were treated with 3-aminobenzamide (3AB) to inhibit DNA repair by the poly (ADP-ribose) polymerase, which is an enzyme of the base excision repair pathway. Results showed that all the spermatozoa cryopreserved with egg yolk carried more than 10% fragmented DNA, maintaining fertilization rates of 61.1+/-2.3 but a high rate of abortions, especially during gastrulation, and only 14.5+/-4.4 hatching success. Furthermore, after 3AB treatment, hatching dropped to 3.2+/-2.2, showing that at least 10% DNA fragmentation was repaired. We conclude that trout sperm maintains its ability to fertilize in spite of having DNA damage, but that embryo survival is affected. Damage is partially repaired by the oocyte during the first cleavage. Important advantages of using rainbow trout for the study of processes related to DNA damage and repair during development have been reported.

Step by step optimization of a sperm cryopreservation protocol for spotted wolffish (Anarhichas minor Olafsen, 1772).

Spotted wolffish Anarhichas minor reproduction in captivity is dependent on in vitro fertilization. However, low sperm volume with relatively low cell concentration and the lack of gametes synchronization (simultaneous availability of mature eggs and sperm) represent a challenge for the industry. Thus, the development of protocols for sperm storage are crucial. Four sequential experiments were conducted to optimize a sperm cryopreservation protocol for this species. First, three different cryoprotectants (DMSO; 1, 2-propanediol; and methanol) at different concentrations (5, 10, and 20%) were tested for their toxicity. No significant differences (p > 0.05) were detected between the control samples and cryoprotectants at concentration up to 10% DMSO, 10% propanediol, and 20% methanol in terms of motility parameters. Second, using the highest non-toxic concentrations of cryoprotectants, sperm was cryopreserved in 0.5 mL straws, at different distances from the liquid nitrogen (1.5, 2.5, 4.5, and 7.5 cm) that correspond to different freezing rates. Motility parameters after freezing/thawing decreased for all the cryoprotectants (p < 0.001), however, methanol had the lowest protective capacity while DMSO the highest. Afterwards, two different thawing rates (1 min at 5 °C; and 25 s at 10 °C) were tested using only 10% DMSO and 10% propanediol. Both for the DMSO and propanediol, there were no significant differences (p > 0.05) between the two thawing rates. The best results were obtained using 10% DMSO. Finally, the fertilization capacity of cryopreserved sperm (10% DMSO and thawed at 5 °C for 1 min) was tested against fresh sperm using two spermatozoa:egg ratios and 4 h gametes contact time. The ratio of eggs with normal cell cleavage, abnormal cleavage or undeveloped were counted at the 2-4 cell stage. Cryopreserved sperm showed lower fertilization capacity at a concentration of 5 × 104 spermatozoa:egg compared with fresh sperm (p < 0.001). At a concentration of 5 × 105 spermatozoa:egg, similar fertilizations rates to the fresh sperm were obtained. The presence of the cryoprotectant DMSO during the 4 h contact time did not affect the fertilization rate or the percentage of embryos with abnormal cleavage (p > 0.05). To cryopreserve spotted wolffish sperm it is recommended to use 10% DMSO, loaded in 0.5 mL straws, freeze at a height between 4.5 (-14.05 °C/min) and 7.5 cm (-5.9 °C/min) from liquid nitrogen for 10 min and thaw for 1 min at 5 °C (177.9 °C/min). In vitro fertilization with cryopreserved sperm should be performed with a concentration of at least 5 × 105 spermatozoa per egg.

[Cerebrovascular disorders in young subjects and deficit of protein S]. / Acidente vascular cerebral no jovem e déficit de proteína S.

The authors describe a right hemiparesis and global aphasia, suddenly developed in a 33-Year-old woman, with a previous aphasia, 7 years ago, from which she recovered without sequels. They discuss the relationship between protein inhibitor of blood coagulation and arterial thrombosis, and make references to what has been published about it.

[The semiology and classification of myoclonias]. / Semiologia e classificação das mioclonias.

Myoclonus can present itself in various distinct clinical contexts. The authors review the possible different types of myoclonus, as a single manifestation or included in a syndrome, based on a semiological and aetiological classification. Special emphasis is given to the group of progressive myoclonus epilepsies.

[Prefrontal syndrome as a manifestation of intracranial dural arterio-venous malformation]. / Síndrome pré-frontal como manifestação de malformação artério-venosa dural intracraniana.

The authors present a clinical case of an intracranial dural arteriovenous malformation with clinical manifestations of a prefrontal syndrome, documented with its respective diagnostic tests, including imaging studies. A number of considerations are being enlarged upon, concerning possible pathophysiological mechanisms.

[Familial peripheral neuropathy caused by susceptibility to entrapment (tomaculous neuropathy)]. / Neuropatia periférica familiar por susceptibilidade ao entrapment (neuropatia tomaculosa).

The authors describe a case of hereditary neuropathy with liability to pressure palsies (entrapment), and compare it to reports from literature. The main characteristics are: autosomal dominant inheritance, recurrent mononeuropathies (ulnar, median, peroneal, brachial plexus), and specific features at nerve biopsy. The sensory nerve reveals predominantly demyelinating alterations, having the remaining myelin fibres focal thickenings, the so called tomaculous, and shows numerous subperineural structures named Renaut Bodies. The EMG findings show a slowing of the nerve conduction velocities and an increase of distal latencies.

Changes in Solea senegalensis sperm quality throughout the year.

Some of the Senegalese sole (Solea senegalensis) broodstock reproductive constraints are related to sperm quality. Although they present two defined spawning season (spring and autumn), males gave semen during all the year thus an exhaustive annual sperm analysis is important to determine the seasonal changes in semen quality. Sampling was performed monthly during one year, analyzing different cellular parameters to better understand sperm quality limitations obstructing sole mass production. The percentage of progressive motile cells and their linear velocity showed a decrease from March (beginning of the first spawning season) to July (when the highest temperatures were observed), followed by a slight increase in August and October (second spawning season). DNA fragmentation values showed highest values between the two spawning seasons and decreased to the end of the year. The percentage of apoptotic cells was lowest in March (beginning of the first spawning season) and the highest in November. The percentage of cells resistant to seawater exposure presented two peaks related with both spawning seasons. There was a tendency for the semen to attain a quality peak between the beginning and the middle of the first spawning season (March-May), followed by a pronounced decrease, achieving the lowest values during the months with the highest temperature. Also, the different males present in the broodstocks reach their sperm quality peak at different times, which will result in an unequal contribution for the next generation.

Endocrine and milt response of Senegalese sole, Solea senegalensis, males maintained in captivity.

Improving fertilization success in captive Senegalese sole broodstocks has been a challenge in the last years. Recent reports suggest that low sperm volume and quality could be one of the reasons leading to poor fertilization rates, although further studies are needed to reach a conclusive explanation. Here, we report on several experiments focused on this issue. Seasonal profiles of plasma androgen levels (testosterone and 11-ketotestosterone) and sperm production and quality parameters were assessed, although no statistical correlations among them were identified. The response of males to female presence/absence was also analyzed. Long-term isolation from females decreased male androgen levels at the peak of the reproductive period, suggesting some kind of disrupting effects on the endocrine system. On the other hand, short-term exposure of previously isolated males to ripe females decreased androgen levels, possibly reflecting a rapid steroidogenic shift promoting final maturation of spermatozoa, and increased sperm viability, motility and velocity, thus, supporting the concept of positive effects of female contact on male sole performance. Further evidence sustaining the relevant female-to-male communication in sole reproduction was obtained after treating the females with progestagen 17α,20ß-dihydroxy-4-pregnen-3-one (regarded as pre-ovulatory pheromone in fish) and registering a significant increase in sperm viability, velocity and motility in surrounding males. Finally, we found that a single administration of a 20 µg/kg GnRH analogue in males was effective in stimulating androgen release and sperm quality, although the effects were transient and thus, the use of sustained hormone delivery methods were suggested for improving efficiency. Our results point to velocity, viability, and motility as the most sensitive parameters in sole sperm, although further studies will have to evaluate whether these parameters have any relation with fertilization success in captive broodstocks of this important aquaculture species.

Altered gene transcription and telomere length in trout embryo and larvae obtained with DNA cryodamaged sperm.

Sperm cryopreservation could entail DNA damage, promoting base oxidization and strand breaks. In a previous work we showed that trout DNA damaged sperm is able to fertilize leading to embryo loss when the repair system of the oocyte is inhibited. Here we have analysed the later effects on embryo and larvae of fertilizing trout oocytes with cryopreserved DNA-damaged spermatozoa. Fish have weak sperm selection mechanisms, are very prolific and have external embryo development, being convenient models for this type of study. We cryopreserved rainbow trout semen using extenders containing egg yolk or their low density lipoprotein fraction to obtain samples with different degrees of DNA damage. DNA fragmentation was evaluated using the Comet assay and telomere length using quantitative-PCR. Fertilization trials were performed and the transcription at different developmental stages of telomerase reverse transcriptase (Tert) and eight genes related with embryo growth and development (Igf1, Igf2, Igfr1a, Igfr1b, Gh1, Gh2, Ins1 and Ins2) were analyzed using quantitative-PCR in surviving embryos and larvae. Results showed an increase in sperm DNA fragmentation after both cryopreservation procedures as well as a decrease in sperm telomere length. Larvae obtained with damaged sperm showed longer telomeres and Tert overexpression. The transcription of the analyzed genes in these embryos and larvae was also modified with respect to the control, most of them as an increase at hatch. We conclude that fertilization with cryopreserved DNA-damaged spermatozoa significantly affects offspring performance, detectable as an increase in telomere length as well as some alterations in gene expression in surviving embryo and larvae.

Evaluation of DNA damage as a quality marker for rainbow trout sperm cryopreservation and use of LDL as cryoprotectant.

Defining reliable and objective biomarkers of sperm quality is a complex matter, because it does not rely on a particular characteristic of the milt. Susceptibility to cryopreservation varies between ejaculations and throughout the year, and the evaluation of fresh sperm does not always provide accurate information about their fertilization ability after freezing and thawing. DNA is one of the cell components prone to suffering cryodamage and several studies have pointed out the importance of the maintenance of its integrity during sperm cryostorage. The authors analysed sperm from rainbow trout for four weeks during the natural reproductive season. Viability, DNA integrity, and fertilization ability were evaluated. Furthermore, in order to increase membrane and DNA protection during sperm cryopreservation, the authors optimized the use of LDL fraction from egg yolk as a cryoprotectant during the analysed period. Results revealed that the evaluation of DNA damage in fresh sperm reveals subtle cell damage, not evidenced in fresh sperm by the other parameters. DNA fragmentation increased from 8 to 31% during the reproductive season, indicating pre-freezing differences that render the cells more susceptible to cryodamage. Also, the use of 12% LDL (low density lipoprotein) fraction, instead of the commonly used pure egg yolk, improved sperm quality after freezing. When LDL was used, post-thaw quality remained constant throughout the analysed period, providing around 60% of eyed embryos. In contrast, when egg yolk was used, post-thaw quality decreased significantly at the end of the season and the percentage of eyed embryos dropped from 60% to 27%. Results demonstrated that reduction in DNA integrity takes place during the reproductive season affecting susceptibility to cryodamage and that the protective effect of egg yolk is very much improved when only their LDL fraction is added to the cryopreservation extender.

Sperm quality evaluation in Solea senegalensis during the reproductive season at cellular level.

Sperm quality seems to be one of the reasons for the reproduction constraints faced by Senegalese sole (Solea senegalensis) aquaculturists. Previous studies in this species indicated that the sperm quality of individuals kept in culture varies throughout the year and that different sperm subpopulations can be identified in ejaculates according to the motility pattern of spermatozoa. Aiming to better understand factors affecting sole sperm quality in captivity, sperm of 11 males was assessed during the reproductive season using different parameters: motility characteristics using CASA analysis; cell plasma membrane resistance to seawater hyperosmolarity; DNA fragmentation with single-cell gel electrophoresis; and early apoptosis, labeled with Annexin-V FITC. Computer-assisted sperm analyses motility data were treated using multivariate analysis to identify the presence of different spermatozoa subpopulations according to their motility pattern. Four distinct sperm subpopulations were obtained: Subpop1, which includes fast linear spermatozoa; Subpop2, made up of fast nonlinear spermatozoa; Subpop3, which includes slow linear spermatozoa; and Subpop4, which contains slow nonlinear spermatozoa. The sperm subpopulation structure varied with time after activation and with male. Low cell resistance to the seawater hyperosmotic conditions was noticed. The Annexin-V assay allowed the identification of an apoptotic population ranging from 6% to 20%. A high percentage of cells (64.1%) showed a DNA fragmentation level below 30%, but these values varied significantly between males. DNA fragmentation appears to be related to cell membrane resistance to hyperosmotic conditions faced by the cells when in contact with seawater. This condition seems to modulate the composition of the motile sperm population and performance after activation. This phenomenon could be related to the spermatozoa maturation process.