Leandro Cesar Godoy

Effect of cryoprotectants on the survival of cascudo preto (Rhinelepis aspera) embryos stored at -8 ◦ C

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.

Injuries in pacu embryos (Piaractus mesopotamicus) after freezing and thawing

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.

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.

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.

Efeito do alecrim na defumação da carne de rã (Rana catesbeiana): características sensoriais, composição e rendimento

This work studied the effect of rosemary on the quality of smoked frog meat (Rana catesbeiana) through the analysis of sensory characteristics, centesimal composition, and processing performance. The carcasses were immersed in a solution of brine (20%), in the proportion of 2:1 (volume of brine/weight), soaked in olive oil and smoked at a temperature range from 50 to 90 °C. The experimental delineation was entirely casual with 2 treatments (T1 = frog carcass with rosemary; T2 = frog carcass without rosemary) with 16 repetitions. For the statistical analysis, the SAEG 2004 program, with 5% of significance was used. The smoked carcasses presented medium values of rude protein (28,39%), total lipids (5,13%), and ashes (2,79%) comparatively higher than the values of the in natura carcasses (23,41; 2,29, and 0,85%). The rosemary did not affect the centesimal composition. The frog carcasses smoked with rosemary presented better acceptance concerning aroma. The rosemary did not affect the results of the other sensory characteristics. Frog meat can be smoked with and without rosemary since it does not interfere in the product acceptance.

Cryopreservation of Embryos and Oocytes of South American Fish Species

The importance of animal genetic resources for wildlife maintenance as well as farming production has become more and more evident in recent years. Fish stocks are globally threatened mainly due to overfishing and environmental pollution. Cryopreservation of aquatic germplasm brings the possibility of preserving the genome of endangered species, increasing the representation of genetically valuable animals for farming purposes and avoiding genetic losses through diseases and catastrophes.

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.

Perfis nutricionais e lipídicos dos músculos dorsal e ventral em pirarucus selvagens

The objective of this work was to analyze the proximate and fatty acid composition of the dorsal and ventral muscles of wild pirarucu (Arapaima gigas) captured from a Brazilian Amazonian lake. Dorsal and ventral muscles were dissected out, freeze-dried, vacuum-packed, and had the proximate and fatty acid composition analyzed. Ash, total proteins, and lipids were inversely proportional to moisture and had higher levels in the ventral muscles. Twenty-seven fatty acids were quantified in both muscles without significant differences between them, except for the heneicosylic, palmitoleic, γ-linolenic, and dihomo-γ-linolenic acids. Saturated and monounsaturated fatty acids were predominant in both muscles. The eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids were quantitatively similar: 9.25 (dorsal) to 10.14 (ventral) and 8.50 (dorsal) to 10.63 (ventral) mg g-1 of total lipids, respectively. The EPA+DHA content of the dorsal and ventral muscles were 113.25 and 165.78 mg 100 g-1, respectively. The ratios of polyunsaturated/saturated (0.54 and 0.59 for the dorsal and ventral muscles, respectively), n-3/n-6 (0.20 and 0.21), and hypocholesterolemic/ hypercholesterolemic fatty acids (1.41 and 1.45) ratios, as well as the atherogenicity (0.59 and 0.53) and thrombogenicity (1.02 and 0.94) indices, indicate that pirarucu muscle is a good dietary source of EPA+DHA, and its nutritional lipid quality can be beneficial for human health.

Trypan blue staining is not efficient in determining oocyte viability in Colossoma macropomum and Brycon amazonicus

Currently, there is no effective technique to evaluate the quality of oocytes in fish farming in a practical and affordable way. The cell membrane integrity test with the vital dye trypan blue (TB) could be an option. In this study, Colossoma macropomum and Brycon amazonicus oocytes were exposed to different TB concentrations seeking to verify a possible relationship between the results of staining tests and reproductive rates. Oocytes were exposed to concentrations of 0.05, 0.04, 0.03, 0.02 and 0.01% TB for 1 minute and subsequently were evaluated under a stereomicroscope. The percentage of unstained (viable) oocytes from each sample was correlated with fertilization and hatching rates using a linear regression (P>0.05). We observed a weak correlation between the results of the staining tests and the fertilization and hatching rates in both species. TB integrity tests were not effective in predicting spawning viability in C. macropomum and B. amazonicus.

Benefits of growing tilapia with minimum release of wastewater

Aquaculture has undergone government and the consumer pressure, to reduce the environmental impacts caused by production. Therefore, minimizing the potential problems related to the environment is an important goal. Research has focused on systems that are able to reduce water consumption and effluent release without interfering on production rates. Recirculation systems that enable large water savings are usual in many countries like Israel and Japan, where aquaculture is well developed. Thus, the objective is to compare juveniles tilapias (Oreochromis niloticus) performance in a recirculation system using biological filter without exchange of water with a system of partial exchanges, with renovation of 40% of the tank volume daily. The recirculating system had reduced total ammonia and had a higher alkalinity, and according to these results is the growth performance, which was higher than the Revista Brasileira de Higiene e Sanidade Animal Brazilian Journal of Hygiene and Animal Sanity ISSN: 1981-2965