Post-thaw sperm morphology of Prochilodus lineatus throughout the spawning season

Abstract

Cryopreservation of fish sperm is a valuable tool used for the conservation of genetic resources. However, this technique causes cryoinjuries to the cell and induces damage at structural level, which is a key factor for post-thaw sperm fertilization capacity. Thus, Prochilodus lineatus post-thaw sperm morphology was assessed throughout the spawning season with the aim to evaluate the best period for the collection and cryopreservation of this fish sperm. Sperm was collected throughout the spawning season (2017/2018), on November (n=7), December (n=8), January (n=9), February (n=11), and March (n=8). After collection, sperm was diluted in a 325 mOsm/kg glucose (pH=7.6) and methyl glycol [CH3O(CH2)2OH] solution (1:8:1, sperm: glucose: methyl glycol), drawn in 0.5 mL straws, frozen in a nitrogen vapour vessel (freezing rate of -36.5 °C/min) and transferred to a cryogenic tank within 24 h for storage. After thawed at 60 °C for 8 s, a sperm aliquot from each straw was diluted (1:1000) in a citrate formaldehyde solution for spermatozoa morphologic analysis. The fixed sample was stained with Rose Bengal (3:20; stain: sperm) and two slides per sample (a duplicate) were prepared with 10µL of stained sperm each. The slides were observed using a light microscope (×1000), and the morphology of 200 sperm cells was evaluated on each slide. For analysis, normal cells, primary (head degeneration, midpiece degeneration, tail stump, fractured tail, strongly coiled tail, macrocephaly, and microcephaly) and secondary (free normal head, simple bent tail, proximal and distal droplet) damages were considered. Data were compared by ANOVA, followed by Student– Newman–Keuls test (p<0.05). Significantly less (p<0.05) normal spermatozoa were observed in samples cryopreserved in March (72.06 ± 2.49 %). The lowest (p<0.05) primary damages values were observed in December to February (11.21 – 13.78 %), while secondary damages were higher (p<0.05) in March (8.94 ± 2.04 %) than in February (5.07 ± 1.56 %). Prochilodus lineatus post-thaw sperm presented better morphologic characteristics from December to February. Therefore, this species sperm cryopreservation should take place during these months.