RESUMO

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.

Assuntos

Criopreservação , Peixes , Água Doce , Preservação do Sêmen , Espermatozoides , Criopreservação/métodos , Animais , Masculino , Espermatozoides/citologia , Peixes/fisiologia , Análise do Sêmen

RESUMO

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.

Assuntos

Preservação do Sêmen , Sêmen , Animais , Masculino , Leite , Motilidade dos Espermatozoides , Preservação do Sêmen/veterinária , Preservação do Sêmen/métodos , Criopreservação/métodos , Peixes , Revisões Sistemáticas como Assunto

RESUMO

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.

Assuntos

Sêmen , Motilidade dos Espermatozoides , Animais , Masculino , Motilidade dos Espermatozoides/fisiologia , Estações do Ano , Espermatozoides , Reprodução

RESUMO

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.

Assuntos

Gelatina , Preservação do Sêmen , Animais , Masculino , Cápsulas , Motilidade dos Espermatozoides , Sêmen , Preservação do Sêmen/veterinária , Preservação do Sêmen/métodos , Criopreservação/veterinária , Criopreservação/métodos , Espermatozoides

RESUMO

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.

Assuntos

Caraciformes , Vitrificação , Animais , Criopreservação/métodos , Feminino , Oócitos , Ovário

RESUMO

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.

RESUMO

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.

Assuntos

Criopreservação , Peixe-Zebra , Animais , Criopreservação/métodos , Crioprotetores/toxicidade , Dano ao DNA , Masculino , Estresse Oxidativo , Espermatozoides

RESUMO

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.

Assuntos

Caraciformes , Animais , Cruzamento , Caraciformes/crescimento & desenvolvimento

RESUMO

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.

Assuntos

Criopreservação , Congelamento , Ovário/fisiologia , Vitrificação , Peixe-Zebra/fisiologia , Animais , Antioxidantes/metabolismo , Membrana Celular/efeitos dos fármacos , Crioprotetores/farmacologia , Dano ao DNA , Feminino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/ultraestrutura , Ovário/efeitos dos fármacos , Ovário/ultraestrutura , Espécies Reativas de Oxigênio/metabolismo

RESUMO

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.

Assuntos

Peixes-Gato/fisiologia , Criopreservação/veterinária , Eugenol/farmacologia , Hidrocortisona/sangue , Preservação do Sêmen/veterinária , Espermatozoides/fisiologia , Anestésicos , Animais , Peixes-Gato/sangue , Relação Dose-Resposta a Droga , Eugenol/administração & dosagem , Fertilidade , Congelamento , Masculino , Distribuição Aleatória , Análise do Sêmen

RESUMO

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.

Assuntos

Caraciformes/fisiologia , Reprodução/fisiologia , Animais , Brasil , Feminino , Fertilidade , Oócitos

RESUMO

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.

Assuntos

Caraciformes/fisiologia , Hipófise/química , Extratos de Tecidos/farmacologia , Animais , Relação Dose-Resposta a Droga , Feminino , Masculino , Distribuição Aleatória , Reprodução/efeitos dos fármacos , Extratos de Tecidos/administração & dosagem

RESUMO

This study aimed to assess the effects of carp pituitary extract (CPE), follicle stimulating hormone (FSH) and luteinizing hormone (LH) on zebrafish oocyte maturation and the ability of these mature oocytes to be fertilized and developed until hatching. Stage III follicles were matured in eight treatments: five concentrations of CPE (16, 32, 48, 64 and 80 µg/mL), one of FSH (0.5 µg/mL), one of LH (0.5 µg/mL), or one combination of FSH (0.5 µg/mL) and LH (0.5 µg/mL). Maturation rates in CPE treatments were 12.8% (16 µg/mL), 24.8% (32 µg/mL), 27.0% (48 µg/mL), 22.7% (64 µg/mL) and 9.6% (80 µg/mL); in FSH was 15.7% (0.5 µg/mL), in LH was 31.8% (0.5 µg/mL) and in FSH (0.5 µg/mL) combined with LH (0.5 µg/mL) it was 50.4%. In vitro fertilization was performed in all treatments; however, only the treatment combining FSH and LH resulted in fertilized oocytes. After maturation using FSH combined with LH, the cleavage rate was 33.3% and hatching rate of live larvae was 20.0%. These results showed that FSH combined with LH was effective in IVM of zebrafish oocyte.

Assuntos

Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/crescimento & desenvolvimento , Peixe-Zebra/fisiologia , Animais , Relação Dose-Resposta a Droga , Larva/fisiologia , Oócitos/efeitos dos fármacos , Hipófise/química , Extratos de Tecidos/administração & dosagem , Extratos de Tecidos/química , Extratos de Tecidos/farmacologia

RESUMO

The objective of this study was to evaluate Ovopel and carp pituitary extract (CPE) in the reproductive induction of Colossoma macropomum males. Nine treatments were tested in triplicate, totaling 27 experimental units. C. macropomum breeders were subjected to the following treatments: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 Ovopel pellet/kg; 2.5 mg CPE/kg (traditional protocol); and a control treatment (no hormone). Breeders under hormone treatment produced a larger (P < 0.05) semen volume (2.4 ± 0.7 to 4.2 ± 0.3 mL) compared with the control (0.9 ± 0.4 mL). Sperm concentration did not differ significantly among treatments (7.2 × 109 ± 1.7 to 10.8 × 109 ± 2.6 spermatozoa/mL). Total sperm count was higher (P < 0.05) after treatment with 0.3, 0.4, and 0.6 Ovopel pellet/kg (41.6 ± 9.3 to 42.3 ± 10.5 × 109 spermatozoa) than the other Ovopel treatments (20.0 ± 2.4 to 26.9 ± 8.2 × 109 spermatozoa) and control (6.6 ± 1.1 × 109 spermatozoa), but did not differ significantly from CPE (33.7 ± 3.2 × 109 spermatozoa). Sperm motility was higher (P < 0.05) in the CPE treated, and 0.2, 0.3, and 0.7 Ovopel pellet/kg (88.3 ± 2.9 to 90.0 ± 5.0) breeders when compared with the other treatments (70.0 ± 10.0 to 78.3 ± 5.8), except for the 0.4 pellet/kg (81.7 ± 2.9) treatment, which did not differ significantly from any of the treatments. The motility period of the spermatozoa did not differ significantly among treatments (93.5 ± 15.7 to 120.0 ± 7.6 s). For the sperm morphological analysis, occurrence of normal spermatozoa was similar across the treatments, with three sperm abnormalities (short tail, bent tail, and detached head) differing (P < 0.05) among the treatments. Ovopel efficiently induced reproduction of C. macropomum breeders, with treatment using 0.3 and 0.4 Ovopel pellet/kg and CPE providing the best semen characteristics.

Assuntos

Caraciformes/fisiologia , Hormônio Liberador de Gonadotropina/análogos & derivados , Hipófise/química , Reprodução/efeitos dos fármacos , Extratos de Tecidos/farmacologia , Animais , Aquicultura , Relação Dose-Resposta a Droga , Hormônio Liberador de Gonadotropina/administração & dosagem , Hormônio Liberador de Gonadotropina/farmacologia , Masculino , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Extratos de Tecidos/administração & dosagem

RESUMO

Was evaluated the pattern of growth among females and males of tambaqui by Gompertz nonlinear regression model. Five traits of economic importance were measured on 145 animals during the three years, totaling 981 morphometric data analyzed. Different curves were adjusted between males and females for body weight, height and head length and only one curve was adjusted to the width and body length. The asymptotic weight (a) and relative growth rate to maturity (k) were different between sexes in animals with ± 5 kg; slaughter weight practiced by a specific niche market, very profitable. However, there was no difference between males and females up to ± 2 kg; slaughter weight established to supply the bigger consumer market. Females showed weight greater than males (± 280 g), which are more suitable for fish farming purposes defined for the niche market to larger animals. In general, males had lower maximum growth rate (8.66 g / day) than females (9.34 g / day), however, reached faster than females, 476 and 486 days growth rate, respectively. The height and length body are the traits that contributed most to the weight at 516 days (P <0.001).

Assuntos

Caraciformes/crescimento & desenvolvimento , Animais , Brasil , Caraciformes/classificação , Feminino , Masculino , Modelos Biológicos , Fatores Sexuais

RESUMO

Members of the TGF-ß superfamily are involved in numerous cell functions; however, except for myostatin, their roles in the regulation of muscle growth in fish are completely unknown. We measured tgf-ß1, tgf-ß2, tgf-ß3, inhibin ßA (inh) and follistatin (fst) gene expression during muscle growth recovery following a fasting period. We observed that tgf-ß1a and tgf-ß2 expression were quickly down-regulated after refeeding and that tgf-ß3 reached its highest level of expression 7days post-refeeding, mirroring myogenin expression. Inh ßA1 mRNA levels decreased sharply after refeeding, in contrast to fst b2 expression, which peaked at day 2. No significant modification of expression was observed for tgf-ß1a, tgf-ß1b, tgf-ß1c and tgf-ß6 during refeeding. In vitro, tgf-ß2 and inh ßA1 expression decreased during the differentiation of satellite cells, whereas tgf-ß3 expression increased following the same pattern as myogenin. Surprisingly, fst b1 and fst b2 expression decreased during differentiation, whereas no variation was observed in fst a1 and fst a2 expression levels. In vitro analyses also indicated that IGF1 treatment up-regulated tgf-ß3, inh ßA1 and myogenin expression, and that MSTN treatment increased fst b1 and fst b2 expression. In conclusion, we showed that the expression of tgf-ß2, tgf-ß3 and inh ßA1 is dynamically regulated during muscle growth resumption and satellite cell differentiation, strongly suggesting that these genes have a role in the regulation of muscle growth.

Assuntos

Diferenciação Celular/genética , Subunidades beta de Inibinas/genética , Desenvolvimento Muscular/genética , Oncorhynchus mykiss , Células Satélites de Músculo Esquelético/fisiologia , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta3/genética , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hormônio do Crescimento/farmacologia , Subunidades beta de Inibinas/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Músculos/efeitos dos fármacos , Músculos/fisiologia , Miostatina/farmacologia , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/crescimento & desenvolvimento , Oncorhynchus mykiss/metabolismo , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Fator de Crescimento Transformador beta2/metabolismo , Fator de Crescimento Transformador beta3/metabolismo

RESUMO

Since their initial discovery, TGF-ß superfamily members have been considered multifunctional growth and differentiation factors in many cell types. Various studies have clearly demonstrated the key roles of specific TGF-ß members in muscle growth, including myostatin and inhibin as well as genes, such as follistatin. By binding to TGF-ß members, follistatin prevents TGF-ß from binding to its receptors and thus neutralizes its activity. Here, we report the identification of the gene sequences of four TGF-ß isoforms and three paralogs of TGF-ß1, which we called TGF-ß1a, TGF-ß1b and TGF-ß1c, four sequences of inhibin ßA paralogs; and two sequences of follistatin paralogs from rainbow trout. A phylogenetic analysis clearly indicated the existence of four monophyletic clades, corresponding to TGF-ß1, -ß2, -ß3 and -ß6. Based on their sequence identity TGF-ß1a and -ß1c are grouped together, whereas TGF-ß1b appears more divergent even though it is grouped within the TGF-ß1 clade. Alignments and phylogenetic analyses showed that the protein sequences of TGF-ß, inhibin ßA and follistatin are extremely well conserved (>90%) relative to each other; however, their regulation and expression patterns are different. TGF-ß2 and -ß3 showed the most abundant expression in muscle and were the main TGF-ß members expressed in this tissue. Follistatin and inhibin ßA paralogs were expressed in all tissues examined but with different patterns. Our identification of multiple copies of TGF-ß, inhibin ßA and follistatin with different expression patterns suggests non-redundant functions for these paralogs in rainbow trout.

Assuntos

Folistatina/metabolismo , Genoma , Subunidades beta de Inibinas/metabolismo , Oncorhynchus mykiss/genética , Fator de Crescimento Transformador beta/metabolismo , Sequência de Aminoácidos , Animais , Folistatina/genética , Subunidades beta de Inibinas/genética , Dados de Sequência Molecular , Oncorhynchus mykiss/metabolismo , Especificidade de Órgãos , Filogenia , RNA Mensageiro/metabolismo , Transcriptoma , Fator de Crescimento Transformador beta/genética

RESUMO

Cryopreservation of germplasm provides a promising method to preserve fish genetic material, which is of great importance in preservation of species diversity, aquaculture, and management of fish models used in biomedical research. In the present study, cryopreservation of Rhinelepis aspera embryos, a Brazilian endangered species, was studied for the first time using a short-term cooling protocol. Embryos at blastoporous closing stage were selected, placed in 6-ml glass vials and stored at -8 °C for 6 h in 10 different cryoprotectant solutions: S1 (17.1% sucrose + 9% methanol); S2 (17.1% sucrose + 9% DMSO); S3 (8.5% sucrose + 8.5% glucose + 9% methanol); S4 (8.5% sucrose + 8.5% glucose + 9% DMSO); S5 (17.1% sucrose + 9% ethylene glycol); S6 (8.5% sucrose + 8.5% glucose + 9% ethylene glycol); S7 (17.1% sucrose + 4.5% methanol + 4.5% DMSO); S8 (17.1% sucrose + 4.5% methanol + 4.5% ethylene glycol); S9 (17.1% sucrose + 4.5% DMSO + 4.5% ethylene glycol); and S10 (100% water). Embryo viability was assessed by hatching rate, counting live larvae and number of failed eggs under a stereomicroscope. The results showed that only the cryoprotectant solutions that contained methanol associated to sucrose (S1, S7 and S8) provided partial protection of Rhinelepis aspera embryos from cold damage (over 50% hatching rate in S1), while the use of DMSO and ethylene glycol, isolated or in combination, resulted in no hatching rate. Further studies are needed in order to extend the storage time and to improve the hatching rate for the species.

Assuntos

Peixes-Gato/embriologia , Criopreservação/métodos , Crioprotetores/farmacologia , Embrião não Mamífero/citologia , Desenvolvimento Embrionário/efeitos dos fármacos , Animais , Aquicultura , Brasil , Embrião não Mamífero/efeitos dos fármacos , Etilenoglicol/farmacologia , Metanol/farmacologia , Sacarose/farmacologia

RESUMO

Although the sperm cryopreservation of freshwater and marine teleosts has been feasible for years, the cryopreservation of some fish embryos still remains elusive. Thus, the objective of this experiment was to analyze the embryo morphology after freezing and thawing 40 embryos of Piaractus mesopotamicus immersed into methanol and ethylene glycol, both at 7, 10 and 13% plus 0.1 M sucrose for 10 min. Soon after thawing, three embryos were treated with historesin, stained with hematoxylin-eosin and analyzed under an optical microscope. From every treatment, one palette containing embryos was thawed and incubated, but none of the eggs hatched. Samples containing two embryos were immersed into 10% methanol or 10% ethylene glycol both in association with sucrose, and embryos immersed into only water or sucrose solution were frozen, processed and analyzed using scanning electron microscopy (SEM). In both cases, the control group was immersed into only water. Although the embryos had the chorion, vitello, yolk syncytial layer and blastoderm, all of them were found altered under the optical microscope and by SEM. The chorion was irregular and injured; there was no individuality in the yolk granules; the yolk syncytial layer had an irregular shape, thickness and size; the blastoderm showed injuries in the nucleus shape and sometimes was absent; the blastoderm was located in atypical areas and absent in some embryos. In conclusion, no treatment was effective in preserving the embryos, and none of the embryos avoided injury from intracellular ice formation. These morphological injuries during the freezing process made the P. mesopotamicus embryos unfeasible for hatching.

Assuntos

Characidae , Criopreservação/métodos , Embrião não Mamífero/patologia , Embrião não Mamífero/ultraestrutura , Congelamento , Animais , Crioprotetores/farmacologia , Microscopia Eletrônica de Varredura

RESUMO

The present study investigates the effect of different slow chilling curves on the storage of pacu (Piaractus mesopotamicus) embryos submitted to chilling at -8°C. Embryos at the blastopore closure stage were divided into two groups: G1 - embryos exposed to cryoprotectant solution containing methanol (10%) and sucrose (0.5 M), treated as follows: (T1) taken directly from room temperature to the refrigerator without being submitted to the curve; (T2) chilling curve of 0.5°C/min; and (T3) chilling curve of 1°C/min; and G2 - the cryoprotectant solution alone was submitted to these same temperatures, receiving the embryos only after temperature had decreased, corresponding to treatments T4, T5 and T6, respectively. Treatments were kept at -8°C for a period of 6 h. Embryo development was evaluated for each treatment, with six replicates in an entirely randomized design. Survival among embryos not submitted to refrigeration was 94.3 ± 8.05%. Percentage of total larvae (TL) and addled eggs (AE) did not differ statistically between the groups, although percentage of swimming larvae (SL) exhibited higher values in G1 for the 1°C/min curve. Furthermore, when comparing the three chilling curves, a decrease of 1°C/min resulted in the highest TL percentage (90.85%), followed by the 0.5°C/min curve (78.52%). Thus, the use of 1°C/min chilling curves is recommended for P. mesopotamicus embryos stored for 6 h at -8°C.

Assuntos

Characidae/embriologia , Temperatura Baixa , Criopreservação , Embrião não Mamífero/fisiologia , Desenvolvimento Embrionário , Animais , Sobrevivência Celular , Embrião não Mamífero/citologia , Larva/crescimento & desenvolvimento , Fatores de Tempo