Conservação de sêmen de tilápia-do-Nilo (Oreochromis niloticus) em ultrafreezer (-80 ºC) para fins de melhoramento genético

Abstract

Nile tilapia (Oreochromis niloticus) is a tropical deep-water fish, originally from Africa, and one of the most important species cultivated as a food resource in the world. Reproduction and genetic improvement are essential areas for the increase in tilapiculture productivity. In this context, artificial fertilization, such as the collection and cryopreservation of semen and oocytes from tilapia breeders, can be applied for the purposes of genetic manipulation and transport of genetic materials, assuming a relevant role for aquiculture and for the conservation of its genetic resources. Since sperm cryopreservation has been developed in mammals, different methods have been used to store or freeze fish semen, mainly with the intention of reducing or using liquid nitrogen, since there are still few protocols defined for fish, mainly tilapia. The idea of using deep freezers can, among other advantages, increase storage capacity and facilitate sperm maintenance compared to liquid nitrogen containers. The objective of this study is to develop an protocol for freezing and storing tilapia semen in the ultra-freezer. Were selected 24 specimens of mature male tilapia to proceed with semen extrusion. Prepared pools from every 3 males, resulting in 8 pools of semen that were previously diluted in a solution containing 350 mM glycose, 30 mM Tris and 10% dimethylacetamide (DMA), in a 1:3 ratio (semen: diluent). The diluted semen pool was equilibrated at 4 °C for 10 minutes and placed in 0.5 ml pallets and sealed with polyvinyl alcohol. For each diluted pool, 4 pallets are packaged, totaling 32 samples, which are placed in liquid nitrogen vapor (-40 °C) for 10 minutes. Afterwards, the half of the pallets (n=16) frozen were kept in liquid nitrogen (-196 °C), and the other half (n=16) was kept in ultra-freezer (-80 ºC). After 30 days of storage, the pallets containing the semen are thawed in a double boiler, at 30 ºC for 30 seconds. The following analyzes were carried out: Rate and duration of sperm motility, vigor, plasma membrane integrity, sperm morphology and oxidative stress. It was observed that both semen freezing processes affect the sperm quality of the semen after defrosting. Therefore, semen samples cryopreserved in liquid nitrogen presented higher values for sperm vigor, sperm motility duration and membrane integrity in relation to semen stored in an ultrafreezer (P < 0.05). No differences were observed in the morphological alterations evaluated in frozen sperm in liquid nitrogen and in an ultrafreezer. However, the percentage of normal spermatozoa was higher (P < 0.05) than semen cryopreserved in liquid nitrogen. The most frequent sperm alterations are the isolated head and slightly bent tail, which present a percentage of over 20%. No significant differences were verified (P > 0.05) for the oxidative stress parameters: lipid peroxidation (PL), superoxide dismutase (SOD) and catalase for the treatments used in tilapia semen preparation. It was possible to identify spermatic seminal viability in semen stored in an ultrafreezer for a period of one month.