Biodegradable capsules as a sustainable and accessible container for vitrification of gonadal tissue using the zebrafish animal model.

Cryopreservation of fish gonadal tissue is an important technique for preserving genetic variability. However, this technique involves the use of cryotubes, plastic containers with low degradability that are expensive and difficult to obtain in certain parts of the world. Therefore, this study aimed to evaluate the efficiency of gelatin and hypromellose hard capsules as a sustainable and accessible alternative container to the cryotube for vitrification of zebrafish (Danio rerio) gonadal tissue. The gonadal tissues (testicular or ovarian) were vitrified in cryotubes, hard-gelatin, and hard-hypromellose capsules. Gelatin capsules exhibited comparable efficacy to cryotubes in preserving spermatogonia viability (33.03 ± 10.03 % and 37.96 ± 8.35 %, respectively), whereas hypromellose capsules showed decreased viability (18.38 ± 2.09 %). Immature oocyte viability remained unaffected by the capsule materials, with no difference compared to cryotubes at all oocyte stages (Primary Growth: p < 0.0001; Cortical Alveolar: p < 0.0001; Vitellogenic: p < 0.0001). Mitochondrial activity and lipid peroxidation demonstrated no difference among cryotubes and capsules for both gonadal tissues. However, antioxidant activity was notably higher in gelatin capsules (Testes: 147.2 ± 32.32 µg; Ovary: 87.98 ± 10.91 µg) than in cryotubes (Testes: 81.04 ± 26.05 µg; Ovary: 54.35 ± 11.23 µg) and hypromellose capsules (Testes: 62.36 ± 17.10 µg; Ovary: 63.96 ± 7.51 µg), likely due to the inherent antioxidant properties of gelatin. The results obtained in this study demonstrate that the cryotube can be replaced by gelatin capsules for vitrification of both gonadal tissues of zebrafish, being a sustainable and accessible alternative as it is a low-cost and environmentally friendly container.

Electronarcosis of Nile tilapia (Oreochromis niloticus): Effects on Dynamic Physiological Balance and Meat Quality.

INTRODUCTION: Electronarcosis is the most commonly used stunning method for large animals, but its consequences in tilapia still need to be evaluated. The aim of the study was to evaluate the application of electronarcosis in the pre-slaughter stunning of Nile tilapia (Oreochromis ni-loticus) and verify its effects on dynamic physiological balance and meat quality. METHODS: Nile tilapia specimens, totaling 184, with an average weight of 247.08 37.04 g, were randomly distributed. Each fish was individually placed in a rectangular tank constituted by a voltage regulator and aluminum electrode. The behavior of the fish subjected to different expo-sure times (5, 10, 20, and 30 seconds) and electric currents (1.50, 3.00, 4.50, and 6.00 amperes) with alternating and continuous currents was evaluated. Subsequently, the quality of the chilled fillets was checked after slaughter over a period of 35 days. The longest stun time was achieved using an alternating current of 3.00, 4.50, and 6.00A for 30 seconds. RESULTS: The fillet quality index (FQI) showed a high correlation with the storage time. In the first 15 days of storage, the fish stunned with different alternating currents maintained a higher MQI, meeting the meat quality standard when compared to fish slaughtered by ice stunning. The fish fillets obtained using different electrical currents showed a pH similar to the fish fillets stunned with ice. CONCLUSION: Therefore, electronarcosis can be applied in the slaughter of tilapia using al-ternating current between three and six amps for 30 seconds, with euthanasia time of 37 and 46 seconds, ensuring safety in the slaughter procedures in the industry, the quality of the meat, and the well-being of the animal.

Use of Powdered Milk in Semen Cryopreservation Protocols for Fish: A Systematic Review.

This systematic review provides an overview of the history and current status of cryopreservation of fish sperm and a detailed evaluation of cryoprotocols using powdered milk. A literature search was performed in PubMed, Scopus, Web of Science, and SciELO databases. Twenty-nine articles were selected after excluding duplicate articles or articles that did not meet the eligibility criteria. Rhamdia quelen and Danio rerio were the most studied species. Slow freezing method, dry-shipper, freezing rate of -35.6°C/min, thawing in water bath (35.93°C ± 10°C), and 0.25 and 0.5 mL plastic straws were the main approaches evaluated. Methanol was the most used permeable cryoprotectant in combination with powdered milk, yielding the best results at 10% concentration. Motility rate was the main analysis performed after cryopreservation in virtually all studies, being subjectively evaluated by most authors. Powdered milk at 15% promoted the best results in the analyzed studies. For motility rate, the gains with the addition of powdered milk were observed in the orders Perciformes (Oreochromis mossambicus), Siluriformes (Pangasius pangasius, Pseudoplatystoma corruscans, and Pseudoplatystoma mataense), and Cypriniformes (Tor soro and Barbonymus gonionotus). For fertilization, gains were observed in the order Siluriformes (P. mataense) and Cypriniformes (T. soro). Sperm viability gains were observed in the orders Siluriformes (P. pangasius), Characiformes (Piaractus brachypomus), and Cypriniformes (B. gonionotus). The scientific evidence we present in this study may contribute and serve as a starting point for new and more refined studies to be developed in the field.

Pogonias courbina sperm characteristcs in its first reproductive season.

Southern black drum (Pogonias courbina) is a species distributed along the western Atlantic Ocean, and it is the largest Sciaenidae observed in the coast of Rio Grande do Sul state, Brazil. However, it is listed as a vulnerable species at The IUCN Red List of Threatened Species™, and their fishing is prohibited. The objective of this study was to determine the sperm characteristics of P. courbina. Sperm samples of five young males (two-year-old fish) were collected through abdominal pressure. The sperm kinetics parameters were sperm motility (MOT) 10.7 ± 5.6%, curvilinear velocity (VCL) 120.07 ± 16.16 mm s ± 1, average path velocity (VAP) 75.64 ± 23.78 mm s ± 1, straight-line velocity (VSL) 62.49 ± 15.83 mm s ± 1, straightness (STR) 83.9 ± 5.3%, wobble (WOB) 61.9 ± 12.7%, beat cross frequency (BCF) 42.981 ± 4.627 Hz and progression (PRG) 1,805.4 ± 564.5 µm. The proportion of normal spermatozoa was 35.6 ± 6.1%. About the abnormalities observed, 22.7% occurred in the tail (short tail = 0.6 ± 0.5%, distally curled tail = 2.4 ± 1.6%, strongly curled tail = 1.9 ± 1.3%, broken tail = 7.9 ± 5.1%, folded tail = 5.5 ± 0.8%, loose tail = 4.4 ± 1.9%); 14.2% occurred in the head (degenerate head = 4.2 ± 1.6%, microcephaly = 1.8 ± 2.5%, loose head = 8.2 ± 2.1%) and 27.5% of the spermatozoa showed cytoplasmatic gouts (proximal gout = 20.0 ± 8.4%, distal gout = 7.5 ± 2.8%). Besides that, a correlation analysis was performed between sperm morphology and kinetics parameters, and the spermatozoa were measured for the morphometric parameters. There was a positive correlation between BCF and normal spermatozoa (r =  0.9269). A negative correlation occurred between BCF and loose head (r =  -0.9047); WOB and strongly curled tail (r =  -0.8911); and PROG and strongly curled tail (r =  -0.9191). The morphometric measures found for the head were length of 2.50 ± 0.21 µm and width of 2.12 ± 0.22 µm, and for the tail it was length of 37.97 ± 2.01 µm. It was possible to verify that the animals have sperm characteristics that indicate reproductive aptitude, but an abnormal behavior on sperm activation and high presence of the cytoplasmic gout abnormality indicates that the animals are not fully mature in their first reproductive season. This work contributes to a better understanding of the P. courbina spermatic parameters, what can be allies to recovery this species population in nature and promote its production in fish farms.

Post-thaw dilution of Rhamdia quelen sperm improves the reproductive success.

The aim was to evaluate the effect of a post-thaw dilution of Rhamdia quelen sperm in 1.1% NaCl (325 mOsm kg-1; pH 7.6; 24 °C) solution on the quality and reproductive capacity. Sperm from eight males were cryopreservation in nitrogen vapor at - 170 °C for 18 h in 0.25 mL straws in a freezing medium containing 5% fructose, 5% Powdered milk, and 10% methanol. The samples were thawed and post-thaw diluted (1:20) in NaCl solution or not (control). The higher spermatozoa velocities were observed in the post-thaw diluted samples (curvilinear (VCL) - 69 ± 11 µm s-1; average path (VAP) - 45 ± 8 µm s-1; straight-line (VSL) - 43 ± 8 µm s-1) compared to the control (VCL - 47 ± 10 µm s-1; VAP - 31 ± 6 µm s-1; VSL - 30 ± 6 µm s-1). Greater straightness (STR), progression (PROG), and beat cross frequency (BCF) were observed in the post-thaw diluted samples (STR - 96 ± 7%; PROG - 666 ± 128 µm; BCF - 42 ± 2 Hz) than in control (STR - 95 ± 5%; PROG - 463 ± 92 µm; BCF - 40 ± 2 Hz). The strongly curled tail was the only morphology change that differ between the post-thaw diluted (5 ± 2%) and control (2 ± 1%). Membrane integrity, mitochondrial activity, and normal larvae rate were not different between treatments. Fertilization and hatching were higher in the post-thaw diluted sperm (93 ± 3%; 82 ± 9%) when compared to control samples (65 ± 13%; 55 ± 17%). Were used oocytes from one female, limiting these results. The post-thaw dilution improved the sperm kinetics and reproductive parameters. Thus, this methodology can be included in the sperm cryopreservation protocol for R. quelen.

Successive seminal collections from Leiarius marmoratus during the reproductive period.

This study aim was to verify whether milt quality of male Leiarius marmoratus is maintained among successive samples collected during the same reproductive period. Ten reproductively mature males were used to evaluate four successive sperm samples collected at 10-day intervals. For these collections, seven males were injected with carp pituitary homogenate (CPH) at a dosage of 3.0 mg/kg body weight, in two applications (30% and 70%), at an interval of 10 h. The other three males were administered only saline (control). Injection with CPH or saline occurred prior to each of the four collections. Only one male from the control group released a small volume of milt (0.33 mL), and only during the first collection period. Of the seven males treated with CPH, five released milt during all four collections. Milt volume of the first sample collected (0.63 mL) did not differ from that of other samples (0.59-1.38 mL; P > 0.05). Sperm concentration was greater in the first samples collected (1.98 × 109 spermatozoa/mL) compared to the other samples (0.35 × 109 at 0.92 × 109 spermatozoa/mL; P < 0.05). Sperm motility, curvilinear velocity, straightness, and morphological normality did not differ among the consecutive samples (P > 0.05). Average path velocity, straight-line velocity, oscillation, linearity, progression, and membrane integrity decreased slightly in the samples collected subsequent to the first sample (P > 0.05). In conclusion, milt quality decreased among successive collections; however, quality of all samples from all collections was sufficient for use for fertilization of oocytes.

Sperm selection of cryopreserved milt of catfish (Rhamdia quelen) by density gradient centrifugation with AllGrad® 90.

Density gradient centrifugation is a technique used to wash or separate samples of cryopreserved milt, mainly in humans and bovines allowing, for example, reducing the concentration of cryoprotectants or choosing the best portion of sperm. The proposed method seeks to reduce the presence of cryoprotectant in the cryopreserved milt of the Rhamdia qhelen and to obtain a fraction of better quality sperm. Gradient centrifugation was formed from 90% AllGrad® and different centrifugation times and forces were compared. The separated sperm presented a low increase in motility and decreased head damage and presence of gout, however, it was better compared to the non-separated samples. The speed of 1000 × g for 10 min, 4 °C, allowed 22.25 ± 4.64% of normal spermatozoa, that is, 9.25% more than the non-centrifuged milt (p = 0.0013).•The centrifugation method allows a fraction of spermatozoa morphologically less affected by cryopreservation.•Density gradient centrifugation with AllGrad® 90% is proposed as a tool of easy adaptation and application for the separation of cryopreserved sperm of R. quelen.•Density gradient centrifugation method at 1000 × g for 10 min allows obtaining a better fraction of normal sperm.

Urine, feces, and blood contamination of frozen and fresh tambaqui (Colossoma macropomum Cuvier, 1818) sperm.

Contamination of fish milt during collection can have an important effect on the quality of fresh and frozen samples. The aim of this study was to evaluate the effects of biological contaminants (urine, feces, and blood) on the sperm of Colossoma macropomum. After hormonal induction, contaminated and contaminant-free milt samples from thirteen males (6.48 ± 2.82 kg) were collected and frozen. The sperm motility was evaluated in fresh and frozen-thawed sperm. Membrane and DNA integrity and mitochondrial functionality were evaluated only in frozen samples. The results revealed lower motility for contaminated sperm in both fresh and frozen-thawed samples [urine (76.15 ± 19.38% and 8.08 ± 6.63%), feces (78.85 ± 26.07% and 1.67 ± 3.26%), and blood (79.62 ± 20.96% and 2.69 ± 4.39%), respectively] than for contaminant-free sperm (95.77 ± 6.07% and 40.00 ± 12.25%, respectively). Motility was different between contaminant-free (118.50 ± 52.08 s) and feces-contaminated (77.00 ± 42.54 s) fresh samples. However, in frozen samples, there was no difference in motility among the groups. The membrane integrity was lower in the contaminated (urine: 72.38 ± 15.55%, blood: 77.00 ± 11.50%, and feces: 68.00 ± 13.64%) than in the contaminant-free (91.46 ± 5.12%) sperm. DNA integrity and mitochondrial functionality were greater in the contaminant-free (82.85 ± 12.19% and 87.15 ± 9.01%, respectively) than in the feces-contaminated (93.38 ± 5.49% and 94.92 ± 6.73%, respectively) samples. C. macropomum sperm contaminated with urine, blood, or feces should not be used for cryopreservation, as these contaminants have detrimental effects on sperm quality.

Oxidative Stress and DNA Damage of Zebrafish Sperm at Different Stages of the Cryopreservation Process.

Although gamete cryopreservation has facilitated advancement of reproduction research by allowing the storage of cells over prolonged periods of time, during freezing-thawing cycles, cells inevitably suffer from cryoinjuries. Here, we evaluate oxidative stress and DNA damage of zebrafish sperm at different stages of the cryopreservation process. It was generally observed that the freezing and thawing of the samples led to an increase in the generation of reactive oxygen species and the activity of the catalase enzyme and a reduction in the generation of sulfhydryl groups and superoxide dismutase activity. The alkaline comet assay demonstrated that DNA damage increased after equilibration time, with an even greater increase after freezing and thawing. The comet assay modified with the enzyme formamidopyrimidine glycosylase, and Endonuclease III demonstrated greater DNA damage than the standard comet assay, demonstrating a high degree of oxidation of purines and pyrimidines at all stages of cryopreservation. Our results show that the freeze and thaw processes cause greater oxidative stress and DNA damage than cryoprotectant toxicity during exposure at the equilibrium stage.

Effects of anesthetic MS-222 on stress and reproduction of South American silver catfish (Rhamdia quelen) males.

Anesthesia is a common practice used in fish research and aquaculture. It is important to understand anesthetic effects on the animal and tissues of interest to ensure validity of data and to improve animal welfare in research and fish production endeavors. The production of some captive fish species is only possible by imposing artificial reproduction procedures, and manipulation of fish for these purposes is a stressor. The purpose of this study, therefore, was to evaluate effects of different concentrations (100, 200, and 300 mg/L) of the anesthetic MS-222 (tricaine methanesulfonate) on cortisol concentrations and effects on sperm quality in Rhamdia quelen. After hormonal induction of gamete production, 28 sexually mature males were randomly assigned to treatments, and milt and blood samples were collected. Anesthesia induction time, motility rate, sperm concentration and morphology, plasma cortisol concentrations, and reproductive hormone concentrations (testosterone, 17-α-hydroxyprogesterone, and estradiol) were evaluated. Sperm motility was greater in the control than 300 mg/L treatment group but did not differ among the control, 100, and 200 mg/L groups. The estradiol concentration was greater in non- anesthetized than anesthetized Rhamdia quelen, but plasma cortisol concentrations did not differ among treatment groups (182.50 ±â€¯42.03 ng/mL). The anesthetic MS-222 at concentrations of 100, 200, and 300 mg/L did not inhibit the stress response due to handling of Rhamdia quelen males. In addition, treatment with MS-222 was not effective in inhibiting detrimental effects on sperm quality because this treatment was associated with impaired sperm motility and lesser concentrations of plasma estradiol.

Skim milk powder used as a non-permeable cryoprotectant reduces oxidative and DNA damage in cryopreserved zebrafish sperm.

Cryoprotectants play a vital role in the cryopreservation process, protecting biological samples from freezing damage. Here, we evaluate the effects of the combination and interaction of different extenders with permeable and non-permeable cryoprotectants, on the cryopreservation of Danio rerio sperm, analyzing the effects of cryopreservation through a broad approach to variables. Two extenders were used, Hank's balanced salt solution (HBSS) and Ginsburg's solution. Eight cryoprotective solutions (CS) were used: CS1 (HBSS + Me2SO 8%), CS2 (HBSS + Methanol 8%), CS3 (HBSS + Me2SO 8% + Skim milk powder 15%), CS4 (HBSS + Methanol 8% + Skim milk powder 15%), CS5 (Ginsburg + Me2SO 8%), CS6 (Ginsburg + Methanol 8%), CS7 (Ginsburg + Me2SO 8% + Skim milk powder 15%) and CS8 (Ginsburg + Methanol 8% + Skim milk powder 15%). The samples were cryopreserved in cryovials for 20 min on dry ice, stored in liquid nitrogen, thawed at 38 °C for 10 s, and analyzed. In addition to increasing viability, we show that powdered milk also allows for better preservation of the membrane and normal cell morphology, and protects the sperm cells from DNA damage and oxidative stress caused by cryopreservation.

Morphological abnormalities in zebrafish cryopreserved sperm.

The aim of the present study was to evaluate the effect of cryopreservation on the morphology of zebrafish sperm (Danio rerio). Sperm from 30 males were collected and divided in two treatments: fresh and cryopreserved semen. The following were measured sperm morphology, motility and membrane integrity. Cryopreservation reduced motility, the number of normal cells and the membrane integrity, as well as increased the percentage of sperm abnormalities. The most frequent types of morphological changes found in cryopreserved semen were macrocephaly, loose head, degenerated head, proximal gout, curled tail and short tail. This study opens the way for further investigations on morphological changes and for a new classification of these changes in fish semen due to cryopreservation.