Darci Carlos Fornari

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.

Freezing injuries in the embryos of Piaractus mesopotamicus

Cryopreservation of mammal embryos has been technically feasible for many years, but morphological injuries still persist in fish embryos during cryopreservation. Thus, the objective of the present study was to describe these freezing injuries in Piaractus mesopotamicus embryos. Two hundred and twenty-five embryos were collected at the post-gastrula stage and assigned into four treatments of sucrose at 8.5, 17.0, 25.0 or 34.0% plus 9.0% methanol. The control was prepared with distilled water only. The gradual decrease in the temperature was 0.5°C/min. After the seeding stage, the fish embryos were stored in liquid nitrogen at -33°C. Thereafter, they were thawed for evaluating per cent hatching, and the samples collected from every treatment were submitted to scanning electron microscopy for morphological analysis. The micrographic images showed that there was substantial alterations in embryo morphology under the highest concentrations of sucrose. These solutions did not prevent the formation of ice crystals, which lead to deformities and killed the embryos, but the observed reduced level of morphological structure in these embryos when treated with 17.0% sucrose plus 9.0% methanol is a compelling argument for additional studies.

Oocyte quality of tambaqui (Colossoma macropomum) during the reproductive season.

The study aimed to analyze the Colossoma macropomum reproductive behavior and quality of the female gametes throughout the reproductive season. The experiment was carried out in Pimenta Bueno - Rondônia State (Northern Brazil) during the reproductive season (2010-2011) using 36 females. Each sampling was performed on a 15 ± 5 days interval. Female gametes were collected by stripping and the following analyses were performed: weight of oocytes released (g); productivity index, fertilization and hatching rate. During the sampling period was verified effect (p < 0.05) of collecting time into the season for oocytes weight, productivity index and fertilization rate. Although the period 3 (December) did not differ significantly from other periods, it showed better parameters for the quality of C. macropomum oocytes.

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.

Growth curve by Gompertz nonlinear regression model in female and males in tambaqui (Colossoma macropomum)

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).

Sperm quality of the Amazon catfish Leiarius marmoratus (Gill, 1870) after cold storage

This study aimed at assessing the sperm quality of the Amazon catfish, Leiarius marmoratus ¸ after refrigeration without extenders. After capturing the animals and stripping of semen, the following parameters were analyzed: progressive motility, motility quality score, duration of motility and sperm morphology. An aliquot of fresh semen from each male was kept at room temperature (28 ± 2°C) as a control, for further comparison with cooled semen. The semen from each animal was stored in extenders-free individual syringes. The syringes were kept in ice within polystyrene boxes at 13 ± 2°C. For both fresh and cooled semen, seminal parameters were evaluated every one-hour interval, reaching seven hours of analysis. Fresh semen showed a significant decrease in motility, motility quality score and duration of motility remaining viable only for three hours. Progressive motility of the cooled semen displayed a negative linear pattern (P<0.05). The duration of motility increased (P<0.05), reaching its peak after three hours of storage. The motility quality score showed a quadratic pattern. No statistical differences were observed when sperm morphology was assessed (P>0.05), even though the mean values of total abnormalities have increased over the storage time. Further studies focusing on the application of this technique should be performed, including the addition of extenders and cryoprotectants for preservation of the sperm over longer periods.

Quality of fresh and cryopreserved semen and their influence on the rates of fertilization, hatching and quality of the larvae of Piaractus mesopotamicus

In this work, the seminal parameters of P. mesopotamicus were evaluated fresh and after cryopreservation, focusing on the sperm variables that affect the rates of fertilization, hatching and post-hatching parameters such as larval survival and morphology. The semen and oocytes from the animals were collected after extrusion, and seminal quality and oocyte fertilization were analyzed. Subsequently, a portion of each semen sample was cryopreserved and, after two days, the oocytes from three new females were fertilized with cryopreserved semen from the males. The analyzes showed that progressive motility, spermatic vigor, motility duration, number of normal sperm and secondary abnormalities were higher in fresh semen than in semen after thawing (P <0.0001). Similarly, fertilization and hatching rates and the percentage of normal and abnormal larvae in fertilized oocytes were higher when fresh semen was used (P <0.0001). The cryopreservation process affected the qualitative parameters of the semen of Piaractus mesopotamicus. The primary abnormality of spermatozoa was the main variable that influenced both fertilization and hatching rates, both in fresh and thawed semen. The second most important variable that influenced, particularly, thawed semen, was the spermatic vigor.