Influence of Cooling Time and Diluents on the Freezability of Prochilodus brevis semen

Abstract

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.

Determination of the ideal volume of activating solution and the optimal ratio of spermatozoa per oocyte for Prochilodus brevis fertilization

“…The mean volume of semen obtained from the breeders used in the present study was higher than that reported by Nunes et al (2016) and Nascimento et al (2017), who presented a mean of 0.56 ± 0.24 and 0.54 ± 0.23 ml, respectively. A similar result was observed with sperm concentration, with the values obtained in the present study being higher than those reported by these authors.…”

“…These data can also be used to work with cryopreserved semen. As cryopreserved semen of this species is generally diluted 1:9 (semen:diluent, Nunes et al, 2016;Nascimento et al, 2017), it is recommended to use 0.25 ml straws of cryopreserved semen (motile > 80%) to 0.76 g ova or 37.4 straws per female (spawn weight ∼28.40 g). In the case of cryopreserved semen with motility close to 50%, it is recommended to use twice the dose mentioned above.…”

Use of glycosaminoglycans from Oreochromis niloticus skin as an antioxidant supplement for milt cryopreservation of Brazilian bocachico

“…They belong to the Laboratory of Biotechnology of Fish Reproduction (LBRP), located at the State University of Ceará, Fortaleza, Ceará, Brazil. Hormonal induction to reproduction and management to obtain milt from P. brevis were carried out according to standard laboratory procedures (Nunes et al, 2016;Nascimento et al, 2017). The doses of carp pituitary extract administered were 0.4 and 4.0 mg kg -1 of body weight.…”

“…Studies related to the seminal cryopreservation of P. brevis have already been carried out, in which were tested different dilution rates (Lopes et al, 2014), forms of milt storage (Nascimento et al, 2017), forms of dilution (Lopes et al, 2014;Nunes et al, 2016) and freezing methods (Pinheiro et al, 2016). There is a good interaction of P. brevis sperm with the freezing medium composed of 5% glucose and 10% dimethyl sulfoxide (DMSO), both for sperm kinetics parameters (Nunes et al, 2016) and the fertilization capacity (Nunes et al, 2019).…”

“…There is a good interaction of P. brevis sperm with the freezing medium composed of 5% glucose and 10% dimethyl sulfoxide (DMSO), both for sperm kinetics parameters (Nunes et al, 2016) and the fertilization capacity (Nunes et al, 2019). Besides, the use of this freezing medium with an addition of antioxidant agents, such as vitamin C, shows good results for thawed sperm (Almeida-Monteiro et al, 2017). This confirms the importance of supplementing the freezing medium with compounds that improve its protective action, such as antioxidant agents.…”