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.

Sperm selection of cryopreserved milt of catfish (Rhamdia quelen) by density gradient centrifugation with AllGrad® 90.

Density gradient centrifugation is a technique used to wash or separate samples of cryopreserved milt, mainly in humans and bovines allowing, for example, reducing the concentration of cryoprotectants or choosing the best portion of sperm. The proposed method seeks to reduce the presence of cryoprotectant in the cryopreserved milt of the Rhamdia qhelen and to obtain a fraction of better quality sperm. Gradient centrifugation was formed from 90% AllGrad® and different centrifugation times and forces were compared. The separated sperm presented a low increase in motility and decreased head damage and presence of gout, however, it was better compared to the non-separated samples. The speed of 1000 × g for 10 min, 4 °C, allowed 22.25 ± 4.64% of normal spermatozoa, that is, 9.25% more than the non-centrifuged milt (p = 0.0013).•The centrifugation method allows a fraction of spermatozoa morphologically less affected by cryopreservation.•Density gradient centrifugation with AllGrad® 90% is proposed as a tool of easy adaptation and application for the separation of cryopreserved sperm of R. quelen.•Density gradient centrifugation method at 1000 × g for 10 min allows obtaining a better fraction of normal sperm.

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.

Oxidative Stress and DNA Damage of Zebrafish Sperm at Different Stages of the Cryopreservation Process.

Although gamete cryopreservation has facilitated advancement of reproduction research by allowing the storage of cells over prolonged periods of time, during freezing-thawing cycles, cells inevitably suffer from cryoinjuries. Here, we evaluate oxidative stress and DNA damage of zebrafish sperm at different stages of the cryopreservation process. It was generally observed that the freezing and thawing of the samples led to an increase in the generation of reactive oxygen species and the activity of the catalase enzyme and a reduction in the generation of sulfhydryl groups and superoxide dismutase activity. The alkaline comet assay demonstrated that DNA damage increased after equilibration time, with an even greater increase after freezing and thawing. The comet assay modified with the enzyme formamidopyrimidine glycosylase, and Endonuclease III demonstrated greater DNA damage than the standard comet assay, demonstrating a high degree of oxidation of purines and pyrimidines at all stages of cryopreservation. Our results show that the freeze and thaw processes cause greater oxidative stress and DNA damage than cryoprotectant toxicity during exposure at the equilibrium stage.

Colossoma macropomum females can reproduce more than once in the same reproductive period.

The aim of the present study was to evaluate induced reproduction in Colossoma macropomum females at the beginning of the reproductive period and 75 days after the first spawning in which reproduction was induced. The experiment was conducted in Nova Mutum, MT, Brazil. Eight 4-year-old C. macropomum females with an average body weight of 6.7 ± 2.4 kg were used. Hormonal induction was performed at the beginning of the reproductive period and repeated 75 days after the first spawning. The following variables were then evaluated: weight of released oocytes, production index, absolute fecundity, oocyte diameter, fertilization rate, and hatching rate. Of the eight females that spawned during the first hormonal induction, three (37.5%) spawned again 75 days after the first spawning. Two females died after the first induced spawning. None of the means of the evaluated variables differed between the two induced spawnings, except for fertilization rate, which was greater (P < 0.05) with the first spawning (88.8 ± 6.1%) than in the second (74.1 ± 10.4%). The results of the present study indicate that C. macropomum females can reproduce again 75 days after a first induced spawning.

Ovopel® and carp pituitary extract for induction of reproduction in Colossoma macropomum females.

The aim of this study was to evaluate the efficiency of the hormonal inducers Ovopel® and carp pituitary extract (CPE) for induction of reproduction in Colossoma macropomum females. The treatments were CPE at the dose of 5.5 mg/kg divided into two applications (10%; and 90% after 12 h) and Ovopel® at doses of 0.2 and 0.4 pellet/kg body weight in a single application. Eight replicates were used in each of the three treatments, totaling 24 experimental units. The females spawned when treated with the 0.2 pellet of Ovopel® (100.0%), 0.4 pellet of Ovopel® (62.5%), and CPE (87.5%), but there were no significant differences among the treatment groups in spawning rate. When there was treatment with Ovopel® spawning occurred with greater (P < 0.05) degree-hours (average water temperature × number of hours until spawning; 0.2 pellet: 417.7; 0.4 pellet: 412.3) in relation to the CPE treatment (268.9). The total oocyte weight was similar when there was treatment with Ovopel® (0.2 pellet: 832.3 g; 0.4 pellet: 798.9 g) and CPE (688.3 g). By contrast, the production index was greater (P < 0.05) with the Ovopel® treatments (0.2 pellet: 8.8%; 0.4 pellet: 9.0%) as compared with CPE (6.7%). Fertility and hatching rates were similar among the treatment groups. Ovopel® and CPE are efficient in induction of reproduction in C. macropomum females. Of the two Ovopel® treatments assessed in this study, the dose of 0.2 pellet/kg body weight is sufficient for effective induction of reproductive processes.

Ovopel and carp pituitary extract for the reproductive induction of Colossoma macropomum males.

The objective of this study was to evaluate Ovopel and carp pituitary extract (CPE) in the reproductive induction of Colossoma macropomum males. Nine treatments were tested in triplicate, totaling 27 experimental units. C. macropomum breeders were subjected to the following treatments: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 Ovopel pellet/kg; 2.5 mg CPE/kg (traditional protocol); and a control treatment (no hormone). Breeders under hormone treatment produced a larger (P < 0.05) semen volume (2.4 ± 0.7 to 4.2 ± 0.3 mL) compared with the control (0.9 ± 0.4 mL). Sperm concentration did not differ significantly among treatments (7.2 × 109 ± 1.7 to 10.8 × 109 ± 2.6 spermatozoa/mL). Total sperm count was higher (P < 0.05) after treatment with 0.3, 0.4, and 0.6 Ovopel pellet/kg (41.6 ± 9.3 to 42.3 ± 10.5 × 109 spermatozoa) than the other Ovopel treatments (20.0 ± 2.4 to 26.9 ± 8.2 × 109 spermatozoa) and control (6.6 ± 1.1 × 109 spermatozoa), but did not differ significantly from CPE (33.7 ± 3.2 × 109 spermatozoa). Sperm motility was higher (P < 0.05) in the CPE treated, and 0.2, 0.3, and 0.7 Ovopel pellet/kg (88.3 ± 2.9 to 90.0 ± 5.0) breeders when compared with the other treatments (70.0 ± 10.0 to 78.3 ± 5.8), except for the 0.4 pellet/kg (81.7 ± 2.9) treatment, which did not differ significantly from any of the treatments. The motility period of the spermatozoa did not differ significantly among treatments (93.5 ± 15.7 to 120.0 ± 7.6 s). For the sperm morphological analysis, occurrence of normal spermatozoa was similar across the treatments, with three sperm abnormalities (short tail, bent tail, and detached head) differing (P < 0.05) among the treatments. Ovopel efficiently induced reproduction of C. macropomum breeders, with treatment using 0.3 and 0.4 Ovopel pellet/kg and CPE providing the best semen characteristics.

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.

Identification of TGF-ß, inhibin ßA and follistatin paralogs in the rainbow trout genome.

Since their initial discovery, TGF-ß superfamily members have been considered multifunctional growth and differentiation factors in many cell types. Various studies have clearly demonstrated the key roles of specific TGF-ß members in muscle growth, including myostatin and inhibin as well as genes, such as follistatin. By binding to TGF-ß members, follistatin prevents TGF-ß from binding to its receptors and thus neutralizes its activity. Here, we report the identification of the gene sequences of four TGF-ß isoforms and three paralogs of TGF-ß1, which we called TGF-ß1a, TGF-ß1b and TGF-ß1c, four sequences of inhibin ßA paralogs; and two sequences of follistatin paralogs from rainbow trout. A phylogenetic analysis clearly indicated the existence of four monophyletic clades, corresponding to TGF-ß1, -ß2, -ß3 and -ß6. Based on their sequence identity TGF-ß1a and -ß1c are grouped together, whereas TGF-ß1b appears more divergent even though it is grouped within the TGF-ß1 clade. Alignments and phylogenetic analyses showed that the protein sequences of TGF-ß, inhibin ßA and follistatin are extremely well conserved (>90%) relative to each other; however, their regulation and expression patterns are different. TGF-ß2 and -ß3 showed the most abundant expression in muscle and were the main TGF-ß members expressed in this tissue. Follistatin and inhibin ßA paralogs were expressed in all tissues examined but with different patterns. Our identification of multiple copies of TGF-ß, inhibin ßA and follistatin with different expression patterns suggests non-redundant functions for these paralogs in rainbow trout.

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.

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.

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.