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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.

No abstract

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).

Storage of refrigerated semen is a simple and inexpensive procedure that can facilitate the management and reproduction programmes in aquaculture and allows to store semen for in vitro reproduction, quality analyses, hatchery production support, selective in vitro breeding, disease diagnosis, transportation, cryopreservation and advanced molecular studies. Implementation of semen short‐term storage protocols for threatened or endangered fish represents a useful strategy for preservation of these species. Semen cold storage as technique is a useful strategy for the conservation of fish sperm. Analysing the male broodstock sperm quality during cold storage is a tool to identify the reproductive ability of cultivated fish. Thus, in this review we analysed the most relevant factors that affect sperm quality during cold storage of male broodstock semen as well as its effects on motility, viability, mitochondrial membrane potential, superoxide anion level and DNA fragmentation.