Biodegradable capsules as a sustainable and accessible container for vitrification of gonadal tissue using the zebrafish animal model.

Cryopreservation of fish gonadal tissue is an important technique for preserving genetic variability. However, this technique involves the use of cryotubes, plastic containers with low degradability that are expensive and difficult to obtain in certain parts of the world. Therefore, this study aimed to evaluate the efficiency of gelatin and hypromellose hard capsules as a sustainable and accessible alternative container to the cryotube for vitrification of zebrafish (Danio rerio) gonadal tissue. The gonadal tissues (testicular or ovarian) were vitrified in cryotubes, hard-gelatin, and hard-hypromellose capsules. Gelatin capsules exhibited comparable efficacy to cryotubes in preserving spermatogonia viability (33.03 ± 10.03 % and 37.96 ± 8.35 %, respectively), whereas hypromellose capsules showed decreased viability (18.38 ± 2.09 %). Immature oocyte viability remained unaffected by the capsule materials, with no difference compared to cryotubes at all oocyte stages (Primary Growth: p < 0.0001; Cortical Alveolar: p < 0.0001; Vitellogenic: p < 0.0001). Mitochondrial activity and lipid peroxidation demonstrated no difference among cryotubes and capsules for both gonadal tissues. However, antioxidant activity was notably higher in gelatin capsules (Testes: 147.2 ± 32.32 µg; Ovary: 87.98 ± 10.91 µg) than in cryotubes (Testes: 81.04 ± 26.05 µg; Ovary: 54.35 ± 11.23 µg) and hypromellose capsules (Testes: 62.36 ± 17.10 µg; Ovary: 63.96 ± 7.51 µg), likely due to the inherent antioxidant properties of gelatin. The results obtained in this study demonstrate that the cryotube can be replaced by gelatin capsules for vitrification of both gonadal tissues of zebrafish, being a sustainable and accessible alternative as it is a low-cost and environmentally friendly container.

Fish sperm cryopreservation using biodegradable containers: New low-cost and environment-friendly methodology.

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.

Urine, feces, and blood contamination of frozen and fresh tambaqui (Colossoma macropomum Cuvier, 1818) sperm.

Contamination of fish milt during collection can have an important effect on the quality of fresh and frozen samples. The aim of this study was to evaluate the effects of biological contaminants (urine, feces, and blood) on the sperm of Colossoma macropomum. After hormonal induction, contaminated and contaminant-free milt samples from thirteen males (6.48 ± 2.82 kg) were collected and frozen. The sperm motility was evaluated in fresh and frozen-thawed sperm. Membrane and DNA integrity and mitochondrial functionality were evaluated only in frozen samples. The results revealed lower motility for contaminated sperm in both fresh and frozen-thawed samples [urine (76.15 ± 19.38% and 8.08 ± 6.63%), feces (78.85 ± 26.07% and 1.67 ± 3.26%), and blood (79.62 ± 20.96% and 2.69 ± 4.39%), respectively] than for contaminant-free sperm (95.77 ± 6.07% and 40.00 ± 12.25%, respectively). Motility was different between contaminant-free (118.50 ± 52.08 s) and feces-contaminated (77.00 ± 42.54 s) fresh samples. However, in frozen samples, there was no difference in motility among the groups. The membrane integrity was lower in the contaminated (urine: 72.38 ± 15.55%, blood: 77.00 ± 11.50%, and feces: 68.00 ± 13.64%) than in the contaminant-free (91.46 ± 5.12%) sperm. DNA integrity and mitochondrial functionality were greater in the contaminant-free (82.85 ± 12.19% and 87.15 ± 9.01%, respectively) than in the feces-contaminated (93.38 ± 5.49% and 94.92 ± 6.73%, respectively) samples. C. macropomum sperm contaminated with urine, blood, or feces should not be used for cryopreservation, as these contaminants have detrimental effects on sperm quality.

Vitrification protocol for immature Brycon orbignyanus ovarian tissue as an extinction escape strategy.

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.

Morphological abnormalities in zebrafish cryopreserved sperm.

The aim of the present study was to evaluate the effect of cryopreservation on the morphology of zebrafish sperm (Danio rerio). Sperm from 30 males were collected and divided in two treatments: fresh and cryopreserved semen. The following were measured sperm morphology, motility and membrane integrity. Cryopreservation reduced motility, the number of normal cells and the membrane integrity, as well as increased the percentage of sperm abnormalities. The most frequent types of morphological changes found in cryopreserved semen were macrocephaly, loose head, degenerated head, proximal gout, curled tail and short tail. This study opens the way for further investigations on morphological changes and for a new classification of these changes in fish semen due to cryopreservation.

Skim milk powder used as a non-permeable cryoprotectant reduces oxidative and DNA damage in cryopreserved zebrafish sperm.

Cryoprotectants play a vital role in the cryopreservation process, protecting biological samples from freezing damage. Here, we evaluate the effects of the combination and interaction of different extenders with permeable and non-permeable cryoprotectants, on the cryopreservation of Danio rerio sperm, analyzing the effects of cryopreservation through a broad approach to variables. Two extenders were used, Hank's balanced salt solution (HBSS) and Ginsburg's solution. Eight cryoprotective solutions (CS) were used: CS1 (HBSS + Me2SO 8%), CS2 (HBSS + Methanol 8%), CS3 (HBSS + Me2SO 8% + Skim milk powder 15%), CS4 (HBSS + Methanol 8% + Skim milk powder 15%), CS5 (Ginsburg + Me2SO 8%), CS6 (Ginsburg + Methanol 8%), CS7 (Ginsburg + Me2SO 8% + Skim milk powder 15%) and CS8 (Ginsburg + Methanol 8% + Skim milk powder 15%). The samples were cryopreserved in cryovials for 20 min on dry ice, stored in liquid nitrogen, thawed at 38 °C for 10 s, and analyzed. In addition to increasing viability, we show that powdered milk also allows for better preservation of the membrane and normal cell morphology, and protects the sperm cells from DNA damage and oxidative stress caused by cryopreservation.

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.

Dynamic expression of tgf-ß2, tgf-ß3 and inhibin ßA during muscle growth resumption and satellite cell differentiation in rainbow trout (Oncorhynchus mykiss).

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.

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.

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).

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.

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.

Cryopreservation-Induced Morphological Changes in Freshwater Fish Sperm: A Systematic Review.

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.

Use of Powdered Milk in Semen Cryopreservation Protocols for Fish: A Systematic Review.

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.

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.

Chilling curves for Piaractus mesopotamicus (Holmberg, 1887) embryos stored at -8°C.

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.

Hydrogel encapsulation as a handling and vitrification tool for zebrafish ovarian tissue.

Zebrafish is an important animal model, thousands lines have been developed, thus having a great need for their preservation. However, the cryopreservation of fish oocytes is still limited and needs improvement. The sodium alginate hydrogel, in addition to providing support for the cells, has been shown to be a potential cryoprotectant. Therefore, the aim of this study was to evaluate the sodium alginate hydrogel encapsulation technique efficiency during zebrafish ovarian tissue vitrification. The encapsulation methodology was standardized in the first experiment. In Experiment 2, we evaluated four vitrified groups: standard protocol without encapsulation (VS); encapsulated with cryoprotectants (VS1-A); encapsulated with half the cryoprotectants concentration (VS2-A); encapsulated without cryoprotectants (VA). VS treatment (54.6 ± 12.3%; 23.7 ± 9.9%; 12.6 ± 5.0%) did not differ from the VS1-A and VA showed a lower membrane integrity percentage (1.2 ± 1.4%; 0.3 ± 0.6%; 0.5 ± 1.5%). Mitochondrial activity was significantly greater in non-encapsulated treatment (VS) when compared to the encapsulated treatments. VS1-A and VS obtained the lowest lipid peroxidation (39.4 ± 4.4 and 40.5 ± 3.3 nmol MDA/mg respectively) in which VS was not significantly different from the VS2-A treatment (63.6 ± 3.1 nmol MDA/mg), unlike, VA obtained the highest lipid peroxidation level (124.7 ± 7.9 nmol MDA/mg). The results obtained in this study demonstrate that the sodium alginate hydrogel encapsulation technique did not have a cryoprotective action, but maintained the membrane integrity when used the standard concentration of cryoprotectants. However, halving the cryoprotectant concentration of fragments encapsulated in alginate hydrogel did not cause an increase in lipid peroxidation. In addition, it provided support and prevented the oocytes from loosening from the tissue during the vitrification process, being an interesting alternative for later in vitro maturation.

Pogonias courbina sperm characteristcs in its first reproductive season.

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.