Cryopreservation of Prochilodus brevis semen: freezing media and thawing rates

Abstract

Prochilodus brevis is a migratory fish, an important component of its river ecosystem and an appreciated animal in northeastern cuisine. However, human activities have threatened its survival. Thus, researchers have become interested in developing genetic material storage protocols, such as seminal cryopreservation. Therefore, determination of the appropriate freezing media and thawing rate is a fundamental step toward the use of this biotechnology in the production of common curimatã and for reducing risks to the species' survival. As such, the aim of the current study was to evaluate the effect of different freezing media and thawing rates on the quality of cryopreserved semen from P. brevis. For this study, males received a single dose of pituitary extract of carp 18 hours before semen collection. The semen samples were diluted in 5% glucose + 10% methyl glycol (MG), 5% glucose + 10% dimethyl sulfoxide (DMSO), 0.9% NaCl + 10% methyl glycol, and 0.9% NaCl + 10% dimethyl sulfoxide, loaded into 0.25-mL straws and frozen in liquid nitrogen vapor. The semen from each treatment was thawed at three different thawing rates: 25 °C for 30 sec, 30 °C for 16 sec and 40 °C for 12 sec. Motility, vitality and morphology analyses were performed by computer-assisted sperm analysis (CASA). The characteristics of the fresh sperm mostly resembled those found in the literature. For the parameters analyzed, fresh sperm presented higher sperm quality in comparison to all treatments with cryopreserved sperm (p<0.05), except for the characteristic of normal morphology, for which the sperm cryopreserved in glucose and MG did not differ statistically from the fresh sperm. For the cryopreserved semen, the greatest results of total motility and curvilinear velocity (VCL) were obtained using glucose and DMSO, regardless of the thawing rate employed. For the straight-line velocity (VSL) and average path velocity (VAP), DMSO showed the best results, regardless of the diluent and thawing rate. With regard to vitality, the highest values were achieved when DMSO and thawing rates of 30 °C for 16 sec or 40 °C for 12 sec were used. In the morphological analysis, the greatest percentage of normal sperm cells was obtained using thawing rates of 25 °C for 30 sec and 40 °C for 12 sec, regardless of the freezing media. Sperm quality was found to suffer interference from the freezing media, as well as from interaction between its components (diluent and cryoprotectant) and the thawing rate used. Under the methodological conditions employed, the use of 5% glucose + 10% DMSO and a thawing rate of 30 °C for 16 seconds or 40 °C for 12 seconds is recommended for P. brevis semen cryopreservation. ResumoO Prochilodus brevis é um peixe reofílico, importante componente do ecossistema fluvial e apreciado na culinária nordestina. No entanto, ações antrópicas têm ameaçado sua sobrevivência. Desta forma, surge, nos pesquisadores, o interesse no desenvolvimento de protocolos de conservação do material genético, como a criopreservação seminal. Logo, a deter...

Influence of vitamins C and E on the quality of cryopreserved semen Prochilodus brevis (Prochilodontidae, Teleostei)

“…In relation to the cryoprotectants, a significant difference was found among them, so that for all analyzed parameters (spermatic kinetics, membrane integrity, and morphologically normal spermatozoa), the cryopreserved samples with DMSO were superior (p <0.05) to those with Methyl glycol, regardless of whether or not the vitamins are used (Table 1). DMSO, as well as most cryoprotectants, is a substance toxic to cells; however, it is considered the best cryoprotectant for semen of freshwater fish (SALMITO-VANDERLEY et al, 2012), being in agreement with the results found for several species of freshwater fish, such as Colossoma macropomum , P. lineatus (NASCIMENTO et al, 2012), and P. magdalenae , including P. brevis as reported by Nunes et al (2016). Despite the toxicity, the use of cryoprotectants for the freezing of semen is indispensable, since the reduction of temperature during the process leads to the formation of ice crystals, which can damage the plasma membrane of sperm cells (SALMITO-VANDERLEY et al, 2012).…”

“…According to Solis-Murgas et al (2011), these characteristics can be influenced by several factors, such as nutritional status, age, and weight of animals, health status, type, and dosage of the hormone used for induction, climate, time of year, and frequency of collection, among other aspects. In relation to pH and sperm kinetics data (total motility, VCL, VSL and VAP), the values obtained in the present study were similar to those found by Lopes et al (2014) and Nunes et al (2016).…”

“…The seminal volume and sperm concentration and morphology data obtained in this study were similar to those found for the same species by Nunes et al (2016); (0.56 ± 0.24 mL, 27.89 ± 6.13 sptz mL -1 and 74.18 ± 10.63%, respectively), but differed from the values reported by Lopes et al (2014); (1.31 ± 0.64 mL, 22.26 ± 9.34 sptz mL -1 and 97.21 ± 1.46%, respectively). According to Solis-Murgas et al (2011), these characteristics can be influenced by several factors, such as nutritional status, age, and weight of animals, health status, type, and dosage of the hormone used for induction, climate, time of year, and frequency of collection, among other aspects.…”

“…As for membrane integrity, a greater than 58% rate of intact membrane spermatozoa was found in all samples, whereas Nunes et al (2016) observed 41.61 ± 3.05% intact cells (Tables 1 and 2). However, no significant differences (p> 0.05) were found between the cryopreserved samples with and without vitamins.…”

Influence of vitamins C and E on the quality of cryopreserved semen Prochilodus brevis (Prochilodontidae, Teleostei)

“…Characiformes, native to the northeast region and endemic in the states of Piauí, Ceará, and Rio Grande do Norte, and can also be found in the southeast of the country (DOURADO, 1981;ARAÚJO;GURGEL, 2002). Besides the economic importance to the region, mainly by the appreciation of its ova, the curimatã has a strong social appeal because its consumption is linked to subsistence; and ecological, due to their iliophage habit when adult (NUNES et al, 2016;DOURADO, 1981), since they recycle the organic matter of the rivers, supporting the upper trophic levels (MOORE et al, 2004).…”