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.

Viability assessment of primary growth oocytes following ovarian tissue vitrification of neotropical teleost pacu (Piaractus mesopotamicus).

Vitrification of ovarian tissue containing immature oocytes provides an important tool for protecting the endangered species and genetic diversity in aquatic species. Therefore, the main objective was to assess primary growth (PG) oocytes viability following ovarian tissue vitrification using histological analysis, two staining protocols (trypan blue or fluorescein diacetate combined with propidium iodide) and mitochondrial activity assay (MTT assay). In addition, oocyte histomorphometry was performed to evaluate the morphometric parameters after vitrification and the relationship with the occurrence of damage (nucleus and/or membrane) in PG oocytes. There was no significant difference among the vitrified oocytes using trypan blue dye or FDA + IP staining. Oocyte viability assessed using histological analysis showed that vitrification solution 2.0 M Me2SO + 2.5 M etilenoglycol +0.5 M sucrose (VS3; 66.43 ± 4.68%) and 1.5 M methanol + 5.5 M Me2SO + 0.5 M sucrose (VS5; 74.14 ± 3.71%) had the lowest viability rate. Similar results were observed in MTT assay where VS3 (1.63 ± 0.12) and VS5 (1.58 ± 0.09) had the lowest averages when compare with VS1 (2.39 ± 0.14), VS2 (1.78 ± 0.06) and VS4 (2.34 ± 0.19) (P = 0.0002). In membrane damage evaluation by histology, there was no difference among vitrified oocytes and control. However, the highest percentages of nucleus damage were observed in treatments VS3 (26.00 ± 5.55) and VS5 (26.00 ± 5.55). Oocyte diameter did not change after vitrification; however, nucleus diameter was significantly higher in control group (49.03 ± 1.07). Oocyte viability by histological analysis was positive-correlated to the occurrence of nucleus (r2 = 0.78) and membrane (r2 = 0.45) damage after vitrification/warming. The high viability of PG oocytes obtained after ovarian tissue vitrification of Piaractus mesopotamicus suggests that the protocol applied here might be used successfully in other teleost species for food production.

The effect of cryoprotectant agents on DNA methylation patterns and progeny development in the spermatozoa of Colossoma macropomum.

DNA methylation patterns are inherited from parents and are imperative for proper embryonic development; however, alterations in these patterns can compromise fertilization and development into a fully functioning adult animal because DNA methylation is part of a complex program of gene transcription. In this study, we investigated the impact of cryoprotectant agents (CPAs) on DNA methylation patterns in spermatozoa and the consequences on embryonic development and the survival rate of progeny. Global methylation was assessed by enzymatic reactions in Colossoma macropomum spermatozoa that were cryopreserved using dimethylsulfoxide, dimethylformamide, methanol, ethyl glycol and glycerol as CPAs. Fertilization was carried out to evaluate survival rates and abnormalities in embryonic development upon treatment with each of the CPAs. Fresh semen served as the control. Our results indicated that, compared to the control group, spermatozoa cryopreservation decreased the fertilization rate and delayed embryonic development from the midblastula stage. Furthermore, spermatozoa cryopreserved in all CPAs had lower methylation levels and exhibited more delays and abnormalities during embryonic development than did fresh semen. Methanol resulted in fertilization, hatching rates and embryonic development that were closer to the control but had lower methylation levels. In conclusion, ours results show significant alterations on spermatozoa DNA methylation patterns caused by CPAs that are used in the semen cryopreservation process. DNA methylation pattern alterations affected the viability of progeny (r=0.48); however, these effects can be minimized by choosing the CPA that will compose the freezing solution.

Viability of zebrafish (Danio rerio) ovarian follicles after vitrification in a metal container.

Cryopreservation of ovarian tissue has been studied for female germline preservation of farm animals and endangered mammalian species. However, there are relatively few reports on cryopreservation of fish ovarian tissue and especially using vitrification approach. Previous studies of our group has shown that the use of a metal container for the cryopreservation of bovine ovarian fragments results in good primordial and primary follicle morphological integrity after vitrification. The aim of this study was to assess the viability and in vitro development of zebrafish follicles after vitrification of fragmented or whole ovaries using the same metal container. In Experiment 1, we tested the follicular viability of five developmental stages following vitrification in four vitrification solutions using fluorescein diacetate and propidium iodide fluorescent probes. These results showed that the highest viability rates were obtained with immature follicles (Stage I) and VS1 (1.5 M methanol + 4.5 M propylene glycol). In Experiment 2, we used VS1 to vitrify different types of ovarian tissue (fragments or whole ovaries) in two different carriers (plastic cryotube or metal container). In this experiment, Stage I follicle survival was assessed following vitrification by vital staining after 24 h in vitro culture. Follicular morphology was analyzed by light microscopy after vitrification. Data showed that the immature follicles morphology was well preserved after cryopreservation. Follicular survival rate was higher (P < 0.05) in vitrified fragments, when compared to whole ovaries. There were no significant differences in follicular survival and growth when the two vitrification devices were compared.

A study on the vitrification of stage III zebrafish (Danio rerio) ovarian follicles.

Attempts to cryopreserve fish embryos have been conducted over the past three decades, nevertheless successful cryopreservation protocol for long-term storage still remains elusive. Fish oocytes offer some advantages when compared to embryos, which may help in improving the chances of cryopreservation. In the present study, a series of cryo-solutions were designed and tested for their vitrifying ability using different devices (0.25ml plastic straw, vitrification block and fibreplug™). Toxicity of vitrification solutions was evaluated by assessing follicle membrane integrity with trypan blue staining. In addition, the effect of vitrification protocol on stage III zebrafish ovarian follicles was investigated by measuring the cytoplasmic ATP content and the mitochondrial distribution and activity using JC-1 probe and confocal microscopy. After vitrification, follicles showed membrane integrity of 59.9±18.4% when fibreplug and V16 (1.5M methanol+4.5M propylene glycol) solution were employed. When vitrified in V2 (1.5M methanol+5.5M Me2SO) the membrane integrity decreased to 42.0±21.0%. It was observed that follicles located in the middle of the fragments were more protected from injuries and some of them showed good morphological appearance 2h post-warming. Mitochondria integrity of granulosa cells layer was clearly damaged by the vitrification protocol and ATP level in the follicles declined significantly after warming. Vitrification of zebrafish follicles in ovarian tissue fragments and its effect at sub-cellular level is reported here for the first time. Information gained from this study will help in guiding development of optimal protocol for cryopreservation of fish oocytes.

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.

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.

Production of live larvae following in vitro maturation of zebrafish oocytes.

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.

Cooling of pacu (Piaractus mesopotamicus) embryos at various stages of development for 6 or 10 hours.

The objective of this research was to verify the effects of cooling embryos of pacu, Piaractus mesopotamicus, in four stages of development during two stocking periods. The stages of embryo development were at: blastoderm, ∼ 64 cells-1.4 h after fertilization (haf); 25% of the epiboly movement--5.2 haf; blastoporous closing--8.0 haf; and optical vesicle appearing--13.3 haf. Embryos were exposed to a cryoprotectant solution containing methanol (10%) and sucrose (0.5 M). Thereafter, embryos were submitted to a cooling curve until they reached -8 °C, and then kept cooled for 6 or 10 h. In addition, for each stage of embryonic development, a control group with uncooled embryos was used to compare hatching rates. The total number of larvae from the first two stages of ontogenetic development (1.4 and 5.2 haf) was lower compared to the other stages (0.0 and 8.0 haf). There was no significant difference between stages 8.0 and 13.3 haf for the total number of larvae (49.9 ± 6.7% and 55.2 ± 6.7%, respectively). Embryo diameter varied according to embryonic stage, providing evidence of differences in membrane permeability. There was a negative correlation between embryo diameter and the total number of larvae (r = -0.372). In conclusion, use of embryonic stages 8.0 and 13.3 haf were recommended for maintaining cooled pacu embryos at -8 °C for 6 or 10 h.

Avaliação de locos ssr (single sequence repeats) desenvolvidos para oreochromis niloticus e tropheus moorii em alguns ciclidios neotropicais / Avaliation of SSR (single sequence repeats) locus developed from Oreochromis niloticus and Tropheus morrii in some neotropical ciclhids

ôPrimersö de locos SSR (Single Sequence Repeats) desenvolvidos a partir de tilápia (Oreochromis niloticus) (UNH104, UNH108 e UNH160) e de Tropheus moorii (TmoM27) foram testados em alguns ciclídios brasileiros. Destes microssatélites avaliados, somente o TmoM27 apresentou amplificação para os gêneros Geophagus e Crenicichla (Cichlidae). Esse loco foi monomórfico em Crenicichla iguassuensis e nos morfotipos Crenicichla sp1 e sp2, com um tamanho estimado da ordem de 346 pb. Pelo menos nesse segmento de DNA não há diferenciação entre C. iguassuensis e as supostas sp1 e sp2. A espécie Geophagus brasiliensis apresentou, contudo, dois alelos com tamanhos de 346 e 358 pb. O menor alelo de G. brasiliensis correspondia ao alelo encontrado nas populações de Crenicichla, contudo sua identidade de bases não foi estabelecida. Observou-se equilíbrio de Hardy-Weinberg para o loco TmoM27, tanto em G. brasiliensis como em Oreochromis niloticus. O loco TmoM27 apresentou o mesmo nível de polimorfismo obtido por outros pesquisadores que analisaram este microssatélite nas espécies de Crenicichla saxatilis e Oreochromis niloticus. O locus TmoM27 pode ser usado para análise da heterozigosidade em Geophagus brasiliensis. Os locos UNH 104, UNH108 e UNH160 não tem aplicação em estudos de estrutura de populações para Crenicichla e Geophagus, pois nenhum produto de amplificação foi obtido nestas espécies.