Technologies used in the study of sperm function in cryopreserved fish spermatozoa

Sperm cryopreservation has led to transcendental changes in the reproductive biotechnology of both mammals and fish, and is a basic tool for animal improvement. However, these protocols generate damage to cell structure and physiology, altering sperm function as a result of cryoinjuries during freezing and thawing. This review is a compilation of the techniques developed and standardised for assessing sperm function in cryopreserved fish semen. Recent studies have analysed sperm function objectively, applying cellular and molecular techniques to characterise cryodamage. The Computer Assisted Sperm Analysis system has facilitated the assessment of motility, while electron microscopy (SEM and TEM) and cryo-microscopy have made it possible to study sperm morphology and ultra-structure. The effects of cryodamage on nuclear DNA have also been analysed using various methods, including the comet Fluorescence in situ Hybridization test, TUNEL, Sperm Chromatin Structure Assay, specific DNA sequences using RT-PCR and specific genes by qPCR. The latter technique is used to study the mitochondrial genome (mtDNA), together with some candidate genes which are associated with bioenergy activity and sperm motility. Other parameters assessed are mitochondrial membrane potential and ATP content using high performance liquid chromatography, nuclear magnetic resonance spectrometry and cell respiration. All this information makes it possible to establish study and assessment criteria for cryopreserved fish spermatozoa. This work focuses on the use of technologies to study of quality of fish spermatozoa during cryopreservation.