Danilo Pedro Streit

Use of amides as cryoprotectants in extenders for frozen sperm of tambaqui, Colossoma macropomum

Amides were tested as cryoprotectants in comparison with glycerol and DMSO (more traditional cryoprotectants) for recovery of Colossoma macropomum (tambaqui fish) sperm. Milt was extended in Beltsville Thawing Solution, then frozen with the addition of 2%, 5%, 8%, or 11% of: (1) dimethylacetamide (DMA), (2) dimethylformamide (DMF), (3) methylformamide (MF), or with 5% glycerol or 10% dimethylsulfoxide. Fertilization rates were greatest (P<0.001) with amides; 8% DMF (91.6±1.3%), 5% DMF (88.9±1.6%), and 8% MF (83.0±1.6%), which did not significantly differ among themselves, when compared with glycerol (51.6±2.4%) and DMSO (61.9±3.1%). The best hatching rates (P<0.001) also occurred for 5% or 8% DMF and 8% MF (79.1±3.1, 87.6±1.5, and 74.8±3.0, respectively) and were also similar (P>0.05). For such treatments, both fertilization and hatching rates were similar (P>0.05) to those with fresh sperm (91.7±1.4 and 87.4±1.4, respectively). The best sperm motility across extenders (at least 55.7%) was with 5%, 8%, and 11% DMF (P<0.001). Those same treatments, along with 11% MF, provided the longest (P<0.001) period of motility (at least 1 min). The greatest sperm integrity (more than 54%) was with 5% and 11% MF and with DMA and DMF at all tested concentrations (P<0.001). The greatest (P<0.001) sperm viability (at least 31%) was for 5%, 8%, and 11% DMA, and with 8% and 11% MF, and also for DMF at all tested concentrations. Sperm DNA integrity was best (more than 50%) for 2%, 5%, and 8% MF and for DMA and DMF at all concentrations (P<0.001), whereas 2% DMA, 11% MF, 11% DMF, and the three amides at both 5% and 8% yielded the highest mitochondrial functionality (at least 44%; P<0.001); thus, 8% MF and both 5% and 8% DMF were the cryoprotectants with the best postthaw quality for C. macropomum sperm.

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.

Methods of cryopreservation of Tambaqui semen, Colossoma macropomum

This study compared three different techniques for sperm cryopreservation of Tambaqui (Colossoma macropomum). Semen was diluted in Beltsville Thawing Solution with the addition of dimethyl sulfoxide (DMSO) at various concentrations (5%, 10%, 15% and 20%). Cryopreservation was performed using three methods: Box Conditioner Method with straws at a 5 cm distance from liquid nitrogen vapor (N2L); Dry Shipper Method placing the straws inside the machine; Vitrification Method placing the straws directly into N2L, amounting to 12 treatments (four DMSO concentrations×three freezing methods). The samples were evaluated for analysis of sperm quality in vivo and in vitro. Use of the Vitrification Method at different concentrations of DMSO provided the least values in the different evaluations. Fertilization, hatching rates and plasma membrane integrity using the Box Conditioner Method with 5% and 10% DMSO did not differ (P>0.05) but use of the concentration of 5% DMSO resulted in greater values than the other treatments (P<0.05) as well as for sperm motility and latency time (P<0.05), although sperm viability was superior using the Dry Shipper Method with 20% of the cryoprotectant. Mitochondrial functionality was impaired by use of the Vitrification Method with all DMSO concentration tested showing the most desirable values when the Box Conditioner Method was used with 5%, 10%, 15% DMSO and the Dry Shipper Method was used with 10% and 15% DMSO. Considering the variables evaluated, the use of the Box Conditioner Method is associated with enhanced Tambaqui semen quality with freeze concentrations of 5% and 10% DMSO.

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.

Microsatellite analysis of the parental contribution of Piaractus mesopotamicus to the production of offspring in the semi-natural system of reproduction

The objective of this study was to evaluate the genetic diversity and the parental contribution of Piaractus mesopotamicus in the production of offspring in the semi-natural system of reproduction. Twenty parental fishes (eleven males and nine females) and the total of 100 larvae were evaluated by microsatellite marker. The parents and offspring had thirty-one alleles and heterozygosity of 0.550 and 0.563, respectively. The females were fertilised by two up to six males while the males fertilised three up to five females. The contribution of the females and males to the offspring were 66.6 and 58%, respectively. Such results indicated no loss in the genetic variability in the offspring, and the parents had multiple paternity and reasonable contribution to the offspring production.

Ovopel and Carp Pituitary Extract as Spawning Inducers in Males of the Amazon Catfish Leiarius marmoratus (Gill, 1970)

The objective of this study was to evaluate Ovopel and carp pituitary extract as spawning inducers in the males of the Amazon catfish L. marmoratus. The following treatments, applied in a single dose, were studied: 0.2, 0.4 and 0.6 Ovopel pellet/kg live weight, and 2.5 mg carp pituitary extract/kg live weight. Each treatment was repeated four times. No significant difference in sperm volume, motility and vigor, time of motility, sperm count, or percentage of normal and abnormal spermatozoa was observed between the treatments. There was also no significant difference in terms of primary or secondary sperm defects, except for the secondary defect of loose heads, which was less frequent in the treatments using 0.4 and 0.6 Ovopel pellet/kg live weight. It was concluded that Ovopel could replace carp pituitary extract for induction spawning in the males of the Amazon catfishL. marmoratus.

Dynamic expression of tgf-β2, tgf-β3 and inhibin βA during muscle growth resumption and satellite cell differentiation in rainbow trout (Oncorhynchus mykiss).

Members of the TGF-β 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-β1, tgf-β2, tgf-β3, inhibin βA (inh) and follistatin (fst) gene expression during muscle growth recovery following a fasting period. We observed that tgf-β1a and tgf-β2 expression were quickly down-regulated after refeeding and that tgf-β3 reached its highest level of expression 7days post-refeeding, mirroring myogenin expression. Inh βA1 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-β1a, tgf-β1b, tgf-β1c and tgf-β6 during refeeding. In vitro, tgf-β2 and inh βA1 expression decreased during the differentiation of satellite cells, whereas tgf-β3 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-β3, inh βA1 and myogenin expression, and that MSTN treatment increased fst b1 and fst b2 expression. In conclusion, we showed that the expression of tgf-β2, tgf-β3 and inh βA1 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.

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.