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SummaryProchilodus brevis is a rheophilic species with a threatened natural population that promotes studies aimed at optimizing reproduction in captivity. The correct quantity of inseminating dose and activating solution volume significantly improves fertilization rates, thereby increasing productivity in captivity. The objective of this study was to determine the proportion of sperm per oocyte and the ideal volume of activating solution to be used in the assisted fertilization of P. brevis. Gametes were collected and fertilization performed in two steps. In step 1, the ideal proportion of spermatozoa was determined based on the fertilization rate:oocyte by testing six doses of semen: D1 = 30 × 103, D2 = 150 × 103, D3 = 300 × 103, D4 = 3 × 106, D5 = 5 × 106, and D6 = 10 × 106. In step 2, the fertilization and hatching rates were evaluated using different volumes of activating solution (V1 – 25 ml, V2 – 50 ml, V3 – 75 ml,V4 – 100 ml, V5 – 125 ml, and V6 – 150 ml). A linear regression equation was estimated from steps 1 and 2. The Student–Newman–Keuls test was used to compare the means. In step 1, the percentage of fertilization increased linearly, reaching a plateau of 51.69%. In step 2, the best fertilization rates were obtained with an estimated ideal volume of 75.64 ml per 2 ml of oocytes. Therefore, the proportion of 928,410.29 sperm:oocyte, associated with the volume of 75.64 ml of water per 2 ml of oocytes, provided the maximum reproductive performance for P. brevis.

Background: Seminal cryopreservation is a technique that optimizes aquacultural production, as it requires less breeding and enables reproduction outside of the breeding season. This technique also helps to preserve species, thus reducing the pressure on the natural stocks. Several studies have sought to develop freezing protocols that result in semen of a good quality. However, some studies do not evaluate the ability of frozen semen to produce viable larvae. Therefore, the aim of this study was to verify the fertilizing capacity of the frozen semen of Prochilodus brevis.Materials, Methods & Results: Semen from twenty adult males of the Brazilian bocachico was collected and evaluated to establish the total motility, curvilinear velocity, straight linear velocity, average path velocity, membrane integrity, pH, and concentration. Six pools were formed, each of which was diluted in a freezing medium containing 5% glucose with 10% dimethyl sulfoxide (DMSO) or 5% glucose with 10% methyl glycol (MG). The samples were loaded into 0.25 mL French straws, frozen in a dry shipper, and stored in a liquid nitrogen canister. The semen was then thawed and evaluated to establish the total motility, curvilinear velocity, straight linear velocity, average path velocity, and membrane integrity. For the fertilization test, four females were used. The oocytes from each female were divided into three batches and fertilized with either fresh or cryopreserved semen. The rates of fertilization, hatching, and larval survival were then measured. Data were expressed as the mean ± standard deviation and analyzed using SAS (2002). The frozen semen with glucose + DMSO was significantly higher (P < 0.001) than the frozen semen with glucose + MG, in all seminal quality parameters evaluated (63.95 ± 15.88% and 25.36 ± 3.53% for the motility, 36.38 ± 7.02 μm.s-1 and 20.45 ± 2.84 μm.s-1 for the curvilinear velocity, 19.26 ± 2.74 μm.s-1 and 3.03 ± 1.40 μm.s-1 for the straight linear velocity, 25.70 ± 6.51 μm.s-1 and 6.90 ± 1.12 μm.s-1 for the average path velocity, and 63.58 ± 6.95% and 35.58 ± 11.26% for the membrane integrity, respectively). MG were very close to zero and not statistically significant. Regarding these same parameters, there were no significant differences (P > 0.05) when the fresh semen was compared to the cryopreserved semen with glucose + DMSO (36.25 ± 2.5% and 29.16 ± 5.64% for the fertilization rate, 38.56 ± 11.23% and 29.33 ± 11.75% for the hatching rate, and 11.59 ± 5.16% and 7.63 ± 5.46% for the larval survival rate, respectively).Discussion: This is the first study of the artificial fertilization of Prochilodus brevis using cryopreserved semen. Seminal quality parameters are important for predicting the success of the cryopreservation technique, however, in vivo tests are essential to confirm such success. Thus, obtaining larvae is a major step towards the standardization of a cryopreservation protocol for a particular species. It is known that cryopreservation reduces the seminal quality but is a necessary process for the conservation of male gametes in the long term and, as shown in this study, good results can be obtained. In this study, the best results were obtained with the inclusion of DMSO in the freezing medium. This effect can be attributed to DMSO having a very low molecular weight, which decreases the formation of ice crystals. Considering the results obtained, we concluded that it is feasible to obtain larvae of the Brazilian bocachico using frozen semen in a 5% glucose solution with 10% DMSO.

Tambaqui, Colossoma macropomum, is an ideal fish for cultivation in captivity and it is valued in the consumer market. Studies on various aspects of aquaculture, including semen freezing, have been conducted. Despite its many benefits, cryopreservation damages the samples. Thus, to refine the existing protocols, products with antioxidant properties can be added, such as sulfated polysaccharides. This study evaluated the effect of adding sulfated polysaccharides from seaweed (green: Caulerpa cupressoides; red: Acanthophora muscoides and Solieria filiformis) and Nile tilapia skin into the tambaqui semen freezing medium. The resulting polysaccharidic samples were diluted and added to the cryopreservation medium. The semen was then collected, pooled, and subsequently frozen. The pools were diluted and frozen in solutions containing powdered coconut water (ACP-104) and 10% dimethyl sulfoxide (DMSO), and were supplemented with different polysaccharide concentrations (100, 250, 500, or 1000 μg/mL) from seaweed or Nile tilapia skin. Semen cryopreserved in an un-supplemented solution was used as control. After 45 days, the samples were thawed by immersion in a water bath (45 °C/8 s) and the membrane integrity, the morphology and, the sperm motility kinetics were evaluated. There were no differences in sperm kinetics for any of the analyzed parameters that could be linked to the polysaccharide source that was used. For membrane integrity, semen supplemented with sulfated polysaccharides from Nile tilapia skin showed better results (76.3 ± 1.9%) compared to treatments supplemented with A. muscoides (65.0 ± 1.8%), C. cupressoides (66.1 ± 1.7%), or S. filiformis (68.8 ± 1.0%), but there was no difference compared to the control (73.6 ± 2.9%). For morphology, the best results were observed in semen supplemented with C. cupressoides (86.7 ± 0.8%) and S. filiformis (87.7 ± 1.0%) polysaccharides at 100 μg/mL. For the mean path velocity (VAP), semen treated with 250 μg/mL of polysaccharides was significantly faster for A. muscoides (21.1 ± 2.4 μm/s) compared to S. filiformis (15.8 ± 1.7 μm/s). However, at 1000 μg/mL, these two red algae shown to be more efficient than the green algae. In C. cupressoides, 1000 μg/mL of polysaccharides showed lower velocity (13.8 ± 1.5 μm/s) than the other concentrations. In S. filiformis, 100 μg/mL and 1000 μg/mL of polysaccharides did not differ and presented higher VAP than 250 μg/mL and 500 μg/mL. Therefore, the addition of sulfated polysaccharides extracted from aquatic sources to ACP-104 and 10% DMSO solution is safe and has a potential of applicability on C. macropomum semen cryopreservation.

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.

The objective of the current study was to observe the performance kinetics (motilities and velocities) of the spermatozoa from Prochilodus brevis (curimatã), Colossoma macropomum (tambaqui) and Piaractus brachypomus (pirapitinga) species in different times post-activation. The sperm of P. brevis, C. macropomum and P. brachypomus species were collected after hormonal induction with carp pituitary extract. The samples with not contamination with water, urine or feces had motility subjective, morphology, osmolality and concentration analyzed. The samples selected were analyzed with Sperm Class Analyzer. Spermatozoa motility and velocities were captured at 10, 30, 60 and 120 s post-activation. No significant differences in total motility of P. brevis spermatozoa were observed between 10 s and 30 s post-activation. However, significant reduction was observed in 60 s. This reduction was more accentuated after 120 s. The same pattern of spermatozoa motility decline happened for C. macropomum and P. brachypomus. Velocities also followed the same pattern for the three species. There was significant reduction in velocities after 30 s; this reduction was more significant after 60 s. There was no significance difference between 60 s and 120 s post-activation. Sperm of C. macropomum and P. brachypomus show satisfactory sperm quality up to 60 s after activation. On the other hand, sperm of P. brevis up to 120 s after activation. These findings show that the rate of sperm motility in different times post activation is change for each species tested.

Background: The addition of antioxidant substances to a cryoprotective solution can increase its protective capacity, shielding spermatozoa from the oxidative stress caused by the seminal cryopreservation process. However, there is no record of a seminal cryopreservation protocol of tambaqui (Colossoma macropomum) using antioxidants as a supplement to the cryoprotective solution. Thus, the objective of this study was to evaluate the effects of adding vitamin C, vitamin E, cysteine, and/or taurine to the seminal cryopreservation of tambaqui.Materials, Methods & Results: Pools of semen (n = 10) were diluted in cryoprotective solutions supplemented with: vitamin C (T1), vitamin E (T2), vitamin C + vitamin E (T3), cysteine (T4), taurine (T5), and taurine + cysteine (T6). The control treatment (T7) was not supplemented. Diluted semen was loaded in 0.5 mL straws, frozen in a dry-shipper, stored in a cryogenic cylinder, and then thawed in a water bath (45ºC for eight seconds). The quality of fresh and cryopreserved semen was evaluated by measuring total motility, progressive motility, curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity, and straightness using a computerized system of sperm analysis. Sperm membrane integrity parameters were analyzed using eosin-nigrosin staining, sperm morphology was assessed using pink bengal staining, and motility duration was measured by a digital timer. Data were analyzed using the statistical program SAS (2002) and the results were expressed as means ± standard error of the mean. The results showed that, in general, there was no significant increase in seminal quality when antioxidants were added to the cryoprotective solution. The T5 treatment promoted an increase (P < 0.05) in progressive motility when compared to T1 (6.33 ± 1.14% and 2.98 ± 0.88%, respectively). However, it did not differ significantly (P > 0.05) from the other treatments. Treatments T2 and T5 presented the highest values of VCL (34.74 ± 2.58 and 33.60 ± 1.81 μm.s-1, respectively). These were higher (P < 0.05) than T1 (26.31 ± 1.64 μm.s-1) but not different (P > 0.05) from the T7 control (30.87 ± 1.49 μm.s-1). The VSL and VAP results showed that T1 presented the lowest velocity (9.89 ± 1.75 and 15.06 ± 1.92 μm.s-1, respectively) compared to the other treatments (P < 0.05) that did not differ from each other. Combining the two vitamins (T3) or the two amino acids (T6) was not advantageous in relation to the use of only one of these antioxidants.Discussion: The present study reports, for the first time, results of the addition of antioxidants to the tambaqui seminal freezing medium. The addition of taurine and vitamin E, although not significantly different from the control treatment, resulted in a tendency to increase sperm kinetics. This effect may be due to the action of taurine as a regulator of Ca2+ transporters, which is necessary to trigger sperm activation, and to the ability of vitamin E to scavenge reactive oxygen species produced during lipid peroxidation. On the other hand, the reduced sperm quality observed when vitamin C was used may have been related to the toxicity caused by a high concentration of this vitamin. In addition, once the safe dose of antioxidants has been exceeded, the normal physiological functions of reactive oxygen species can be inhibited. Thus, it is concluded that the use of vitamin E and taurine promotes promising results of curvilinear velocity after thawing of sperm. Therefore, these treatments are recommended, as well as more tests to determine their optimal concentrations.

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.

Resumo O objetivo deste trabalho foi avaliar o efeito da adição de gema de ovo (GO) sobre a cinética dos espermatozoides de tambaquis após a criopreservação. Utilizaram-se vinte machos de tambaquis (n= 4 pools), que foram induzidos hormonalmente com extrato hipofisário de carpa, para espermiação. Quatorze horas após a indução, realizou-se a coleta seminal. O sêmen de cada pool foi diluído em Ringer adicionado de 10% de dimetilsulfóxido (DMSO) acrescido ou não de GO (T1: sem acréscimo de GO; T2: com 5% de GO e T3: com 10% de GO). O sêmen tratado foi envasado em palhetas de 0,5 mL, congelado em vapor de nitrogênio líquido (dry shipper-30 min/-153 °C) e posteriormente transferidas para nitrogênio líquido. As palhetas foram descongeladas em banho-maria a 37 °C/30 segundos. A taxa de motilidade (%) e a velocidade curvilinear espermática (µm/s) foram analisadas em sistema computadorizado (CASA). Os dados foram expressos em média ± desvio padrão e foi aplicado o teste de Tukey (P

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.