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.

Cryopreservation-Induced Morphological Changes in Freshwater Fish Sperm: A Systematic Review.

A systematic review was performed to summarize the scientific evidence and critically evaluate the effects of cryopreservation on sperm morphology in freshwater fish, and to assess the methodologies for sperm morphology classification. The search strategy was applied to four electronic databases (CAB Direct, Pub Med, Scopus, and ISI Web of Science). The main inclusion criteria involved studies on semen from freshwater fish subjected to the cryopreservation process and evaluation of sperm quality through morphology. The risk of bias was assessed with respect to randomization, allocation concealment, blinding, incomplete outcome data, and selective reporting. A total of 6 publications reporting sperm cryopreservation from 4 species with a total 74 fish individuals were included in this review. A high methodological variability among the results of the studies was observed due to the species-specific protocols and diversity of freshwater fish species studied. All included studies reported negative effects of cryopreservation on sperm quality, especially morphology, highlighting the increase in incidence of sperm abnormalities. However, only five studies statistically compared abnormalities between groups (fresh and cryopreserved sperm). Our results suggest the need to elaborate on a new morphological classification of fish spermatozoa, by considering the structure and physiology of fish sperm. This classification should be developed based on the sperm characterization and observing damage caused by different cryopreservation protocols.

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.

Fish sperm cryopreservation using biodegradable containers: New low-cost and environment-friendly methodology.

In brief: The containers used in cell cryopreservation are essential to maintain cell integrity and viability after thawing. This paper reveals the methodology of using biodegradable containers for fish sperm cryopreservation. Cryopreserved sperm in biodegradable containers showed high fertility capability. Biodegradable capsules could be alternative containers to plastic straws for sperm cryopreservation. Abstract: Containers used to cryopreserve sperm are made with non-biodegradable plastic compounds, having a high monetary and environmental cost. Therefore, the development of biodegradable alternative containers for cell cryopreservation is necessary. Thus, this study aimed to evaluate the efficiency of hard-gelatin and hard-hydroxypropyl methylcellulose (HPMC) capsules as low-cost and biodegradable alternative containers for sperm cryopreservation. Sperm from 12South American silver catfish Rhamdia quelen were individually cryopreserved in plastic straws 0.25 mL (as control), hard-gelatin, and hard-HPMC capsules. The quality of post-thaw sperm cryopreserved in the different containers was checked by measuring spermatozoa membrane integrity, kinetic parameters, mitochondrial activity, fertilization, hatching, and normal larvae rates. The samples cryopreserved in straws showed a higher percentage of membrane integrity (68%) than those frozen in hard-gelatin (40%) and hard-HPMC capsules (40%). However, we did not observe differences between the samples stored in straws and hard capsules for the rest of the tested sperm parameters. Thus, based on the high sperm fertility capability, both capsules were efficient as cryopreservation containers for maintaining sperm functionality.

Hydrogel encapsulation as a handling and vitrification tool for zebrafish ovarian tissue.

Zebrafish is an important animal model, thousands lines have been developed, thus having a great need for their preservation. However, the cryopreservation of fish oocytes is still limited and needs improvement. The sodium alginate hydrogel, in addition to providing support for the cells, has been shown to be a potential cryoprotectant. Therefore, the aim of this study was to evaluate the sodium alginate hydrogel encapsulation technique efficiency during zebrafish ovarian tissue vitrification. The encapsulation methodology was standardized in the first experiment. In Experiment 2, we evaluated four vitrified groups: standard protocol without encapsulation (VS); encapsulated with cryoprotectants (VS1-A); encapsulated with half the cryoprotectants concentration (VS2-A); encapsulated without cryoprotectants (VA). VS treatment (54.6 ± 12.3%; 23.7 ± 9.9%; 12.6 ± 5.0%) did not differ from the VS1-A and VA showed a lower membrane integrity percentage (1.2 ± 1.4%; 0.3 ± 0.6%; 0.5 ± 1.5%). Mitochondrial activity was significantly greater in non-encapsulated treatment (VS) when compared to the encapsulated treatments. VS1-A and VS obtained the lowest lipid peroxidation (39.4 ± 4.4 and 40.5 ± 3.3 nmol MDA/mg respectively) in which VS was not significantly different from the VS2-A treatment (63.6 ± 3.1 nmol MDA/mg), unlike, VA obtained the highest lipid peroxidation level (124.7 ± 7.9 nmol MDA/mg). The results obtained in this study demonstrate that the sodium alginate hydrogel encapsulation technique did not have a cryoprotective action, but maintained the membrane integrity when used the standard concentration of cryoprotectants. However, halving the cryoprotectant concentration of fragments encapsulated in alginate hydrogel did not cause an increase in lipid peroxidation. In addition, it provided support and prevented the oocytes from loosening from the tissue during the vitrification process, being an interesting alternative for later in vitro maturation.

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.

Vitrification protocol for immature Brycon orbignyanus ovarian tissue as an extinction escape strategy.

Piracanjunba (Brycon orbignyanus) is an endangered South American fish, and ovarian tissue cryopreservation is an alternative method for preserving maternal germplasm and genetic diversity. Therefore, our aim was to test a vitrification protocol for ovarian tissue containing primary growth (PG) oocytes of B. orbignyanus as a strategy to avoid the threat of extinction. Two vitrification solutions were evaluated (VS1: 1.5 M methanol + 4.5 M propylene glycol and VS2: 1.5 M methanol + 5.5 M Me2SO) and compared using control/fresh ovarian tissue. After vitrification, the following factors were analyzed: membrane integrity using trypan blue, morphology using a histological assessment, oxidative stress (total reactive antioxidant potential (TRAP) and reduced thiol [-SH]), mitochondrial activity using MTT, and DNA damage using a comet assay. The vitrified oocytes (VS1 = 24.3 ± 0.49% and VS2 = 24.8 ± 0.69%) showed higher DNA damage than the control group (control = 20.7 ± 1.03%) (P = 0.004). In contrast, in most evaluations (membrane integrity, membrane damage, oxidative stress, and mitochondrial activity), there were no discernible differences between the control group and the vitrified samples. In addition, oocyte (P = 0.883) and nuclear diameter (P = 0.118) did not change after vitrification. VS2 treatment resulted in higher nuclear damage (15.7 ± 1.45%) than in the control treatment (3.5 ± 1.19%); however, VS1 treatment did not result in significantly more damage (9.5 ± 3.01%) than in the control (P = 0.015). Therefore, the protocol for ovarian tissue vitrification tested in this study resulted in high maintenance of PG oocyte cell integrity, making it a promising alternative for B. orbignyanus maternal genome preservation.

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.

Growth curve of selectively bred and non-selectively bred tambaqui (Colossoma macropomum).

The aim of this study was to evaluate the growth curve of selectively bred and non-selectively bred tambaqui (Colossoma macropomum). The experiment involved 388 fish (weight: 65.38 ± 20.00 g; age: 217 days), consisting of 252 fish from seven selectively bred families (18 fish per family) and 18 non-selectively bred fish (control group). Groups were placed in two 800-m² tanks. Biometric measurements were taken on nine occasions at 30-day intervals, for a period of 254 days. Weight and morphometric traits were evaluated. To describe the tambaqui growth behavior, we adopted the Gompertz nonlinear regression model. Greater growth (p < 0.05) was observed in selectively bred families compared with control group. Four families stood out with higher (p < 0.05) asymptotic values for weight (F1: 2448.7 g; F7: 2284.7 g; F5 2180.1 g; F4: 2080.5 g; and control: 1808.4 g) and other morphometric traits. None of the selectively bred families (except F5) had a higher growth rate and age at inflection point than the fish from control group. In conclusion, selectively bred and non-selectively bred fish present distinct growth curves, but some families have greatly superior growth.

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.

Slow freezing versus vitrification for the cryopreservation of zebrafish (Danio rerio) ovarian tissue.

The aim of the present study was to compare the efficiency of vitrification and slow freezing techniques for the cryopreservation of zebrafish ovarian tissue containing immature follicles. In Experiment 1, assessment of cell membrane integrity by trypan blue exclusion staining was used to select the best cryoprotectant solution for each cryopreservation method. Primary growth (PG) oocytes showed the best percentage of membrane integrity (63.5 ± 2.99%) when SF4 solution (2 M methanol + 0.1 M trehalose + 10% egg yolk solution) was employed. The vitrification solution, which presented the highest membrane integrity (V2; 1.5 M methanol + 5.5 M Me2SO + 0.5 M sucrose + 10% egg yolk solution) was selected for Experiment 2. Experiment 2 aimed to compare the vitrification and slow freezing techniques in the following parameters: morphology, oxidative stress, mitochondrial activity, and DNA damage. Frozen ovarian tissue showed higher ROS levels and lower mitochondrial activity than vitrified ovarian tissue. Ultrastructural observations of frozen PG oocytes showed rupture of the plasma membrane, loss of intracellular contents and a large number of damaged mitochondria, while vitrified PG oocytes had intact mitochondria and cell plasma membranes. We conclude that vitrification may be more effective than slow freezing for the cryopreservation of zebrafish ovarian tissue.

The germ cell marker dead end reveals alternatively spliced transcripts with dissimilar expression.

Since the late 19th century, the Amazon species Colossoma macropomum (tambaqui) has been exploited commercially and the climate change has contributed to decline in tambaqui numbers. Although germ cell cryopreservation and transplantation can help preserve the species' genetic resources semipermanently, its germ cell behavior has not been analyzed to date. In this study, we isolated the tambaqui's dead end gene (dnd) homolog (tdnd) and used it as a molecular marker for germ cells to obtain basic information essential for transplantation. The amino acid sequence showed 98% similarity and 53% identity with the zebrafish dnd. Phylogenetic analysis and the presence of consensus motifs known for dnd revealed that tdnd encodes the dnd ortholog and its transcript is detectable only in the testes and ovaries, showing a strong positive signal in oocytes and spermatogonia. The tambaqui possesses, at least, three different transcripts of tdnd which show dissimilar expression profile in undifferentiated and sexually mature animals, suggesting that they play distinct roles in germline development and they may influence the choice of donors for the cell transplantation study.

Effects of different doses of eugenol on plasma cortisol levels and the quality of fresh and frozen-thawed sperm in South American catfish (Rhamdia quelen).

The production of captive fish is only possible through artificial reproduction, but manipulation is a known stressor stimulus. Thus, the objective of the present study was to evaluate the effects of different eugenol concentrations (0, 30, 40, 50 and 60 mg/L) during reproductive management of Rhamdia quelen. Seventy-five mature male R. quelen were randomly distributed among the five treatments, and blood samples were collected at the time of semen collection to measure plasma cortisol. The following parameters were evaluated in the fresh semen samples: motility, motility duration, concentration and fertilization rate. The following parameters were evaluated in the frozen semen samples: motility, motility duration, morphology, membrane integrity, DNA integrity and mitochondrial functionality. The animals anesthetized with eugenol at concentrations of 40 and 50 mg/L had lower levels of plasma cortisol (88.4 and 83.3 ng/mL, respectively) than the control (147.1 ng/mL). For fresh semen, the control treatment presented the highest rate and time of motility but differed (P < 0.05) only from the animals treated with 60 mg/L eugenol. For the cryopreserved semen the highest rates and motility time were observed in the control treatment and in the animals anesthetized with 40 mg/L eugenol, differing (P < 0.05) from anesthetized animals with 50 and 60 mg/L. Mitochondrial functionality was higher in fish anesthetized with 30 mg/L eugenol differing only for animals anesthetized with 60 mg/L. There was no difference between treatments for sperm concentration and fertilization rate of fresh semen. There were no differences (P > 0.05) between treatments in the parameters of membrane integrity, DNA integrity and% of normal spermatozoa after thawing of the cryopreserved semen samples. The use of 30, 40 and 50 mg/L eugenol maintained the seminal quality of the fresh semen, and the quality of the thawed semen was maintained with 30 and 40 mg/L eugenol. These results show that stress reduction can be reconciled with reproductive management without compromising reproductive performance.

Isolation and characterization of a germ cell marker in teleost fish Colossoma macropomum.

The native Amazonian fish tambaqui (Colossoma macropomum) is the second-largest scaled fish in South America and the most common native species in Brazil. To preserve genetic resources with sufficient genetic diversity through germ cell cryopreservation and transplantation techniques, a molecular marker for identifying the cells is required to trace them during the manipulation processes. The vasa gene is a promising candidate, as its specific expression in germ cell lineage has been well-conserved throughout animal evolution. In this study, the full sequence of the vasa cDNA homolog from tambaqui was isolated and characterized, showing an open reading frame of 2010 bp encoding 669 amino acids. The putative protein was shown to contain eight conserved motifs of the DEAD-box protein family and high similarity to vasa homologs of other species. Tambaqui vasa (tvasa) mRNA expression was specific to the gonads, and in situ hybridization showed signals only in oocytes and spermatogonia. The results suggested that tvasa could be a useful germ cell marker in this species.

Colossoma macropomum females can reproduce more than once in the same reproductive period.

The aim of the present study was to evaluate induced reproduction in Colossoma macropomum females at the beginning of the reproductive period and 75 days after the first spawning in which reproduction was induced. The experiment was conducted in Nova Mutum, MT, Brazil. Eight 4-year-old C. macropomum females with an average body weight of 6.7 ± 2.4 kg were used. Hormonal induction was performed at the beginning of the reproductive period and repeated 75 days after the first spawning. The following variables were then evaluated: weight of released oocytes, production index, absolute fecundity, oocyte diameter, fertilization rate, and hatching rate. Of the eight females that spawned during the first hormonal induction, three (37.5%) spawned again 75 days after the first spawning. Two females died after the first induced spawning. None of the means of the evaluated variables differed between the two induced spawnings, except for fertilization rate, which was greater (P < 0.05) with the first spawning (88.8 ± 6.1%) than in the second (74.1 ± 10.4%). The results of the present study indicate that C. macropomum females can reproduce again 75 days after a first induced spawning.

Ovopel® and carp pituitary extract for induction of reproduction in Colossoma macropomum females.

The aim of this study was to evaluate the efficiency of the hormonal inducers Ovopel® and carp pituitary extract (CPE) for induction of reproduction in Colossoma macropomum females. The treatments were CPE at the dose of 5.5 mg/kg divided into two applications (10%; and 90% after 12 h) and Ovopel® at doses of 0.2 and 0.4 pellet/kg body weight in a single application. Eight replicates were used in each of the three treatments, totaling 24 experimental units. The females spawned when treated with the 0.2 pellet of Ovopel® (100.0%), 0.4 pellet of Ovopel® (62.5%), and CPE (87.5%), but there were no significant differences among the treatment groups in spawning rate. When there was treatment with Ovopel® spawning occurred with greater (P < 0.05) degree-hours (average water temperature × number of hours until spawning; 0.2 pellet: 417.7; 0.4 pellet: 412.3) in relation to the CPE treatment (268.9). The total oocyte weight was similar when there was treatment with Ovopel® (0.2 pellet: 832.3 g; 0.4 pellet: 798.9 g) and CPE (688.3 g). By contrast, the production index was greater (P < 0.05) with the Ovopel® treatments (0.2 pellet: 8.8%; 0.4 pellet: 9.0%) as compared with CPE (6.7%). Fertility and hatching rates were similar among the treatment groups. Ovopel® and CPE are efficient in induction of reproduction in C. macropomum females. Of the two Ovopel® treatments assessed in this study, the dose of 0.2 pellet/kg body weight is sufficient for effective induction of reproductive processes.