Tambaqui (Colossoma macropomum) is a native freshwater fish that is of great importance for Brazilian aquaculture. Because of this importance, several techniques have been developed to improve the reproduction of this species in captivity. One of these techniques is the cryopreservation of sperm. In an effort to increase the efficiency of cryopreservation protocols, researchers have tried to determine suitable diluting solutions and freezing methods, which will provide a better post-thaw sperm quality. Thus, this study aimed to evaluate the efficiency of different diluents and freezing methods for the cryopreservation of tambaqui (C. macropomum) sperm. Samples of fresh semen were diluted in different treatments (Glucose 5% + 10% Dimethyl sulfoxide -DMSO, Glucose 5% + 10% Methyl glycol -MG, BTS + 10% DMSO and BTS + 10% MG) at a 1:9 dilution rate and frozen in a programmed freezing machine and a dry shipper. The semen samples were thawed and evaluated for vitality, sperm morphology and kinetics. Cryopreserved semen with DMSO and using the programmed freezing machine provided a greater percentage of motile sperm (15.44 ± 1.04%) after thawing compared to the dry shipper (3.99 ± 0.55%), regardless of the diluent. Additionally, DMSO showed better sperm velocities than MG regardless of the freezing method and the extender employed. A higher percentage of living spermatozoa was observed when glucose (37.28 ± 1.32%) (regardless of the freezing method and cryoprotectant) and DMSO (37.98 ± 1.25%) was used in the programmed freezing machine. For morphology, a greater amount of normal spermatozoa (46.10 ± 1.82%) was observed when the semen was cryopreserved using a freezing machine programmed with DMSO as the cryoprotectant and Glucose or BTS (38.16 ± 1.9% and 39.26 ± 1.87%, respectively) as extenders. Therefore, we suggest the use of the DMSO (10%) cryoprotectant in association with the Glucose (5%) extended in the programmed freezing machine for cryopreservation of C. macropomum semen. ResumoO tambaqui (Colossoma macropomum) é uma espécie nativa de peixe de água doce de grande importância para aquicultura brasileira. Devido a isso, diversas técnicas têm sido desenvolvidas para aperfeiçoar a reprodução desta espécie em cativeiro, dentre elas a criopreservação de sêmen de peixe. Como uma forma de melhorar os protocolos de criopreservação, tem-se buscado utilizar soluções diluidoras e métodos de congelação adequados, proporcionando uma boa qualidade seminal pós-descongelação. Dessa forma, este estudo objetivou avaliar a eficiência de diferentes diluidores e métodos de congelação na criopreservação do sêmen de tambaqui (C. macropomum). As amostras de sêmen fresco foram diluídas em diferentes tratamentos (Glicose 5% + 10% Dimetilsufóxido -DMSO; Glicose 5% + 10% Metil glicol -MG; Beltsville Thawing Solution -BTS + 10% DMSO e BTS + 10% MG) na proporção 1:9 e congeladas em máquina de congelação programada e em Dry shipper. As amostras seminais foram descongeladas e avaliadas para vitalidade, morfologia e cinética espermát...

This study evaluated the effect of glucose or Beltsville Thawing Solution (BTS™) combined with dimethyl sulfoxide (DMSO) or methylglycol (MG) under two different freezing protocols on the kinetics and morphology of cryopreserved Prochilodus brevis sperm. The semen samples were diluted using one of four different treatments (glucose+DMSO, glucose+MG, BTS™+DMSO, and BTS™+MG), loaded into 0.25-ml straws and subjected to two different freezing processes (programmed freezing machine and dry shipper). After 10 days, the semen samples were thawed, and the sperm morphology and kinetics were evaluated. The physicochemical parameters of the semen in natura were similar to those observed in other studies of Characiformes, indicating the feasibility of semen cryopreservation. Glucose, when used as a diluent with the cryoprotectant MG (glucose+MG), yielded higher percentages of mobile spermatozoa after freezing in a dry shipper (76.88 ± 4.84%) and in a programmed freezing machine (70.95 ± 1.76%) compared with the combination of glucose and DMSO. Moreover, the glucose+MG treatment yielded a higher sperm velocity (curvilinear velocity: 79.52 ± 2.88 µm s -1 ; straight-line velocity: 45.46 ± 3.01 µm s -1 ; average path velocity: 67.92 ± 3.08 µm s -1 ) than the other studied treatments, and a higher amount of normal sperm (74.56 ± 0.77%) was observed in the semen samples cryopreserved using a programmed freezing machine. The sperm abnormalities observed included a bent tail morphology. Therefore, the use of glucose+MG in combination with either a dry shipper or a programmed freezing machine is recommended for the cryopreservation of P. brevis sperm because these methods yielded high numbers of motile and morphologically normal spermatozoa.

Seminal cryopreservation allows the long-term conservation of gametes of various species, including endangered species, such as Prochilodus brevis. However, the application of this biotechnology can cause damage to sperm cells, reducing seminal quality. Thus, we have sought substances that minimize the damage caused by this process, such as antioxidants. Thus, this study aimed to evaluate the association between two cryoprotectants and two vitamins, in different concentrations, on the quality of cryopreserved semen of P. brevis. For cryopreservation, the experiment was performed in two stages. In the first stage, the semen of 10 animals was submitted to six different freezing means, coming from the combination of 5% glucose, two cryoprotectants (Dimethyl sulfoxide [DMSO] or Methyl glycol) and two vitamins (C or E to 0.0001 mg) for cryopreservation. In the second stage, semen samples of eight animals were diluted in 5% glucose and the best cryoprotectant found in the first stage, associated with three different concentrations of vitamins C or E (0.01, 0.001, and 0.0001 mg). In both steps, the in natura and post-thawed samples were submitted to kinetic analysis, morphology, and sperm membrane integrity. The cryopreserved semen with DMSO presented significantly higher results (p < 0.05) than that frozen with Methyl glycol, regardless of the vitamin used. The morphologically normal spermatozoa rate was higher (p < 0.05) in the vitamin-containing samples, however, vitamin E reduced sperm motility rates, independent of the cryoprotectant used. As for vitamin concentrations, higher motility rates were obtained when cryopreserved semen with 0.01 and 0.0001 mg of any of the vitamins. However, the higher concentration had a deleterious effect on the spermatic morphology of P. brevis. Therefore, the glucose associated with DMSO and the lower concentration of vitamin C provides good quality for the post-thawed semen of P. brevis.

Background: Prochilodus brevis is a rheophilic fish of economic and ecological importance. However, anthropic action has made its population vulnerable. Thus, the development of reproductive biotechnologies, such as seminal conservation, is necessary to subsidize their fish farming. However, seminal collections are often performed in places with few laboratory resources, demanding studies to determine the maximum time for which sperm can be cooled, as well as its process until frozen. Thus, the present study aimed to evaluate the influence of cooling time and the presence of dilution solutions on cryopreservation of P. brevis semen.Material, Methods & Results: After seminal collection, nine pools were formed and analyzed for seminal pH, concentration, membrane integrity, morphology and spermatic kinetics - motility, curvilinear velocity (VCL), average path velocity (VAP) and straight line velocity (VSL). After the analysis of the pools in natura (control 1), they were processed as follows: 1)- immediate freezing (control 2); 2)- cooling: undiluted, diluted in coconut water powder (ACP-104) or diluted in 5% glucose, followed by cooling at different times (6, 12, 24 or 48 h); 3)- Post-refrigeration freezing: the pools were diluted in their respective diluents and 10% dimethyl sulfoxide. After 15 days, the samples were thawed and analyzed for the aforementioned parameters. For the cooled and post-thawed semen, a completely randomized design with 2 (diluent × cooling time) and 3 (storage form × cooling time and storage form × diluent) factors, respectively, was utilized. ANOVA and Dunnett tests were applied to compare the means. In case of seminal cooling, there was no difference (P > 0.05) in sperm motility between control 1 and the undiluted and diluted treatments in ACP-104 for up to 24 h. After 48 h, only the VCL of the sample diluted in ACP-104 was similar (P > 0.05) to that of control 1. When comparing forms of storage (undiluted, diluted in ACP-104 or diluted in glucose) and cooling times, the undiluted samples and the samples diluted in ACP-104 were better (P < 0.05) for all the kinetics parameters analyzed, than those diluted in glucose after 24 h. After 48 h, the cooled semen diluted in ACP-104 presented greater (P < 0.05) motility than the other treated semen samples. The samples diluted in glucose for 48 h presented lower spermatic velocity (P < 0.05) than those subjected to other treatments. Regardless of the diluent used, the post-thawed semen and the cooled semen diluted for 6 h, presented higher sperm kinetic values (P < 0.05) than those of control 2 and other treated samples. Overall, the samples diluted in ACP-104 showed satisfactory results when cooled for up to 48 h or cooled for up to 6 h and frozen.Discussion: This is the first study that froze semen from P. brevis after cooling. Although glucose is a commonly used diluent during seminal freezing and has good post-thawing stability for this species, it is not recommended for cooling before seminal freezing, as prolonged exposure of spermatozoa to glucose may cause osmotic stress to sperm cells. Conversely, good results with ACP-104 might be because of its rich composition, mainly the presence of indole-3-acetic acid (IAA), an auxin with proven potential for seminal conservation of other species. Therefore, for fertilization trials, it is recommended to use ACP-104 as diluent for seminal cooling of P. brevis for up to 48 h or semen that has been frozen after cooling in ACP-104 for a maximum of 6 h.

Seminal cryopreservation allows the long-term conservation of gametes of various species, including endangered species, such as Prochilodus brevis. However, the application of this biotechnology can cause damage to sperm cells, reducing seminal quality. Thus, we have sought substances that minimize the damage caused by this process, such as antioxidants. Thus, this study aimed to evaluate the association between two cryoprotectants and two vitamins, in different concentrations, on the quality of cryopreserved semen of P. brevis. For cryopreservation, the experiment was performed in two stages. In the first stage, the semen of 10 animals was submitted to six different freezing means, coming from the combination of 5% glucose, two cryoprotectants (Dimethyl sulfoxide [DMSO] or Methyl glycol) and two vitamins (C or E to 0.0001 mg) for cryopreservation. In the second stage, semen samples of eight animals were diluted in 5% glucose and the best cryoprotectant found in the first stage, associated with three different concentrations of vitamins C or E (0.01, 0.001, and 0.0001 mg). In both steps, the in natura and post-thawed samples were submitted to kinetic analysis, morphology, and sperm membrane integrity. The cryopreserved semen with DMSO presented significantly higher results (p < 0.05) than that frozen with Methyl glycol, regardless of the vitamin used. The morphologically normal spermatozoa rate was higher (p < 0.05) in the vitamin-containing samples, however, vitamin E reduced sperm motility rates, independent of the cryoprotectant used. As for vitamin concentrations, higher motility rates were obtained when cryopreserved semen with 0.01 and 0.0001 mg of any of the vitamins. However, the higher concentration had a deleterious effect on the spermatic morphology of P. brevis. Therefore, the glucose associated with DMSO and the lower concentration of vitamin C provides good quality for the post-thawed semen of P. brevis. Key words: Antioxidants. Cryopreservation of semen. ResumoA criopreservação seminal permite a conservação em longo prazo dos gametas de diversas espécies, inclusive as ameaçadas, como a Prochilodus brevis. Contudo, a aplicação dessa biotecnologia pode causar danos às células espermáticas, reduzindo a qualidade seminal. Assim, tem-se buscado substâncias que minimizem os danos causados por esse processo, como os antioxidantes. Deste modo, este estudo objetivou avaliar a associação entre dois crioprotetores e duas vitaminas, em diferentes concentrações, sobre a qualidade do sêmen criopreservado de P. brevis. Para a criopreservação, o experimento foi realizado em duas etapas. Na primeira, o sêmen de 10 animais foi submetido a seis diferentes meios de congelação, oriundos da combinação de glicose 5%, dois crioprotetores (Dimetilsulfóxido [DMSO] ou Metilglicol) e duas vitaminas (C ou E a 0,0001 mg), para a criopreservação. Na segunda etapa, amostras de sêmen de oito animais foram diluídas em Glicose 5% e DMSO (melhor crioprotetor encontrado na primeira etapa), associados a três diferentes conce...

The Brazilian bocachico, Prochilodus brevis, is a rheophilic fish. Although there is evidence that this species shows reproductive seasonality in the wild, in captivity hormonal induction techniques allow semen sampling in different seasons. This study aimed to compare the kinetics, morphology and biochemical composition of the semen of Brazilian bocachico in captivity when hormonally induced to breed in the reproductive and non-reproductive seasons. During sampling spermiation was hormonally induced in breeders. The concentrations of total protein, glucose, fructose, triglyceride, calcium and chloride were evaluated with biochemical kits. The pH data (6.5 to 8.5) suggest semen requires alkaline conditions, as expected for freshwater fish. Seminal plasma contained more protein (1.51 ± 0.06 dL g-1), glucose (79.44 ± 1.88 mg dL-1) and triglycerides (61.59 ± 8.10 mg dL-1) in the non-reproductive than the reproductive season, but calcium ions (15.98 ± 1.02 mg dL-1) showed the opposite pattern. There was a significant seasonal difference in sperm morphology, with a higher percentage of normal sperm in the reproductive season. From these data it can be concluded that the physical, kinetic, morphological and biochemical characteristics of semen of captive Prochilodus brevis are influenced by reproductive season.

Currently, aquaculture has been growing due to factors such as increased fish consumption and production, population growth, higher family income and urbanization (Food and Agriculture Organization of the United Nations (FAO), 2019). Therefore, studies that focus on investigating the reproductive biology of fish have been intensified to develop species conservation and genetic improvement programmes (Denniston, Michelet, & Godke, 2000), as well as to enable an increase in aquaculture production and reduce pressure on natural stocks. Thus, species belonging to genus Prochilodus, like Prochilodus brevis, a rheophilic species native to Northeast Brazil, have shown increasing potential for use in Brazilian aquaculture (Peixe Br, 2019) to meet the great demand for fish, as they are among the most important species in both commercial and subsistence continental

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