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The aim of the present investigations was to test a novel technology comprising cryoprotectant-free vitrification of the spermatozoa of rainbow trout and to study the ability of sucrose and components of seminal plasma to protect these cells from cryoinjuries. Spermatozoa were isolated and vitrified using five different mediums: Group 1: standard buffer for fish spermatozoa, Cortland(®)-medium (CM, control); Group 2: CM+1% bovine serum albumin (BSA); Group 3: CM+1% BSA+0.125 M sucrose; Group 4: CM+1% BSA+40% seminal plasma; and Group 5: CM+1% BSA+40% seminal plasma+0.125 M sucrose. For cooling, 20 μL suspensions of cells from each group were dropped directly into liquid nitrogen. For warming, the spheres containing the cells were quickly submerged in CM+1% BSA at 37 °C with gentle agitation. The quality of spermatozoa before and after vitrification was analysed by the evaluation of motility, cytoplasmic membrane integrity (SYBR-14/propidium iodide staining technique), and mitochondrial membrane integrity (JC-1 staining). Motility (86%, 71%, 80%, 81%, and 82%, for Groups 1, 2, 3, 4, and 5, respectively) and cytoplasmic membrane integrity (90%, 82%, 83%, 84%, and 87%, respectively) of spermatozoa in all the 5 groups were not decreased significantly. All tested solutions can be used for vitrification of fish spermatozoa with good post-warming motility and cytoplasmic membrane integrity. However, mitochondrial membrane potentials of the spermatozoa in Groups 1, 2, 3, 4, and 5 were changed significantly (6%, 50%, 37%, 55%, and 34%, respectively) (P(1,2,3,4,5)<0.001; P(2,3,4,5) <0.01)(P(3-5)>0.1). This rate was maximal in Group 4 (CM+1% BSA+40% seminal plasma). In conclusion, this is the first report about successful cryoprotectant-free cryopreservation of fish spermatozoa by direct plunging into liquid nitrogen (vitrification). Vitrification of fish spermatozoa without permeable cryoprotectants is a prospective direction for investigations: these cells can be successfully vitrified with 1% BSA+40% seminal plasma without significant loss of important physiological parameters.

Semen conservation methods have been extensively studied for fish species over the last 60 years. Cryopreservation techniques can be divided into two types: one with slow cooling rates like traditional freezing and the other with ultrarapid rates as used in vitrification. These protocols increase the time over which semen samples can be used for reproduction or sperm quality analysis. Marine fish possess greater resistance than freshwater species to variations in osmolality. This favours the application of these methods, which produce better results after thawing and offer high biotechnological potential for aquaculture. In the last 15 years, sperm cryopreservation studies have been carried out in 32 marine species, but there are few analyses of the effects of freezing on the morphology and physiology of sperm cells. The object of this review was to provide recent data on semen cryopreservation in marine fish species and to suggest variables which may be applied in future research.

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The development of sperm cryopreservation has enabled transcendental changes to occur in the reproductive biotechnology of both mammals and fish; it has become a basic tool for animal improvement. Nevertheless, these protocols cause damage to cell structure and physiology, altering sperm functioning due to cryoinjuries during freezing and thawing. However, studies of the effects on the structural, functional and genomic stability of the mitochondria in fish spermatozoa during cryopreservation are still lacking. The object of this review was to analyse the effect of cryopreservation on mitochondrial metabolic pathways in fish spermatozoa. This effect is related with the bioenergy mechanism for flagellar movement during the activation of sperm motility. In teleost fish, the mitochondria may be cylindrical, spherical or irregular in shape and adhere in a helicoidal or conical pattern to the middle piece. The salmonidae have only a single mitochondrion, but this may vary in other species; the mitochondria provide the flagellum with energy during sperm motility, when sperm respiration is essential. The effects of cryopreservation can induce structural damage to the mitochondria, altering the biochemical process involved in ATP production and thus causing a reduction in sperm motility. Fragmentation damage to nuclear DNA and diminution in sperm motility is mainly associated with damage to the structure and metabolic functioning of the mitochondrion. A direct correlation exists between the mitochondrial membrane potential (transmembrane integrity, ΔΨ m ) and the motility and fertilizing capacity of the cryopreserved spermatozoa, confirming that this organelle is the energy nucleus of the spermatozoon and that the cessation or prolongation of motility and successful fertilization depend on the availability of ATP in the spermatozoa. Further works are need to incorporate biotechnology studies, at cell and molecular level, of the possible effects of cryopreservation on mitochondrial DNA, enzymatic or metabolic modifications of the citric acid cycle, and the oxidative phosphorylation process in the inner membrane, as well as studies of the mitochondrial ultrastructure. Thus, mitochondrial dynamics could be established as a potential target for therapeutic strategies.

The objective of this study was to determine the effect of freezing on the function in Atlantic salmon Salmo salar spermatozoa. The semen was frozen in Cortland's medium + 1.3M dimethyl sulphoxide + 0.3M glucose + 2% bovine serum albumin (final concentration) in a ratio of 1:3 (semen:cryoprotectant) as the treatment (T) and fresh semen as the control (F). Straws of 0·5 ml of sperm suspension were frozen in 4 cm of N2 L. They were thawed in a thermoregulated bath (40° C). After thawing, the percentage of spermatozoa with fragmented DNA [transferase dUTP (deoxyuridine triphosphate) nick-end labelling (TUNEL)], plasma membrane integrity (SYBR-14/PI) and mitochondrial membrane potential (ΔΨMMit, JC-1) were evaluated by flow cytometry and motility was evaluated by optical microscope under stroboscopic light. The fertilization rates of the control and treatment semen were tested at a sperm density of 1·5 × 10(7) spermatozoa oocyte(-1) , by observation of the first cleavages after 16 h incubation at 10° C. In the cryopreserved semen (T), the mean ± s.d. DNA fragmentation was 4·8 ± 2·5%; plasma membrane integrity 75·2 ± 6·3%; mitochondrial membrane potential 51·7 ± 3·6%; motility 58·5 ± 5·3%; curved line velocity (VCL ) 61·2 ± 17·4 µm s(-1) ; average-path velocity (VAP ) 50·1 ± 17·3 µm s(-1) ; straight-line velocity (VSL ) 59·1 ± 18·4 µm s(-1) ; fertilization rate 81·6 ± 1·9%. There were significant differences in the plasma membrane integrity, mitochondrial membrane potential, motility, fertilization rate, VCL , VAP and VSL compared with the controls (P < 0·05). Also the mitochondrial membrane potential correlated with motility, fertilization rate, VCL and VSL (r = 0·75; r = 0·59; r = 0·77 and r = 0·79, respectively; P < 0·05); and the fertilization rate correlated with VCL and VSL (r = 0·59 and r = 0·55, respectively).

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