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<0,05). Houve uma redução significativa na porcentagem de espermatozoides móveis e velocidade curvilinear após a adição de GO independente da concentração. Logo, a adição de GO ao Ringer + DMSO teve efeito negativo sobre a motilidade do sêmen congelado de tambaqui.
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.
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