O. Merino

Fish (Oncorhynchus mykiss) spermatozoa cryoprotectant-free vitrification: Stability of mitochondrion as criterion of effectiveness

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.

Cryoprotectant‐free vitrification of fish (Oncorhynchus mykiss) spermatozoa: first report

The aims of this investigation were 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 cryo‐injuries. Spermatozoa were isolated and vitrified using three different media: Group 1: standard buffer for fish spermatozoa, Cortland® medium (CM, control); Group 2: CM + 1% BSA + 40% seminal plasma; and Group 3: 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 and cytoplasmic membrane integrity with SYBR‐14/propidium iodide staining technique. Motility (86%, 81% and 82% for groups 1, 2 and 3, respectively) (P > 0.1) was not decreased significantly. At the same time, cytoplasmic membrane integrity of spermatozoa of Groups 1, 2 and 3 was changed significantly (30%, 87% and 76% respectively) (P < 0.05). All tested solutions can be used for vitrification of fish spermatozoa with good post‐warming motility. However, cytoplasmic membrane integrity was maximal in Group 2 (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.

Effect of seminal plasma on Atlantic salmon (Salmo salar) sperm vitrification

This study was designed to test a vitrification method in Atlantic salmon spermatozoa and determine the capacity of seminal plasma (SP) to protect these cells from cryoinjuries. The vitrification medium consisted of a standard buffer for fish spermatozoa (Cortland medium) + 10% DMSO + 2% BSA + 0.13-M sucrose + SP at concentrations of 30% (G30), 40% (G40), or 50% (G50). Fresh sperm was used as a control. To freeze the samples, 30-μL suspensions of spermatozoa from each group were dropped directly into liquid nitrogen. The resulting spheres were placed in cryotubes for storage in liquid nitrogen. The cryotubes with the vitrified spermatozoa were thawed by placing them in a water bath at 37 °C for 45 seconds. After thawing, the following sperm quality parameters were determined by flow cytometry: DNA fragmentation (terminal deoxynucleotidyl transferase dUTP nick end labeling), plasma membrane integrity (SYBR-14/PI, staining technique), and mitochondrial membrane potential (JC-1 staining). An optical microscope was used to assess subjectively sperm motility, whereas fertility was determined by the presence of neurulation using five replicates per treatment in a sample of 30 eggs. Spermatozoa quality variables were preserved best when the highest concentration of SP (50%) was used (DNA fragmentation, 9.2%; plasma membrane integrity, 98.6%; mitochondrial membrane integrity, 47.2%; motility, 44.1%; and fertility, 46.2%).

Cryopreservation of Atlantic salmon Salmo salar sperm: effects on sperm physiology.

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 Cortlands 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).

High temperature is essential for preserved human sperm function during the devitrification process

Sperm vitrification is a cryopreservation method based on high‐speed freezing by direct exposure of cells in liquid nitrogen (N2L), thereby avoiding the traditional cooling curves of freezing. The objective of this work was to determine the optimal warming temperature for vitrified human spermatozoa in order to maintain their fertilisation potential. Spermatozoa were cryopreserved by direct plunging into N2L and warmed at different temperatures for 5 and 10 s at 38, 40 and 42 °C. Sperm motility was evaluated by the CASA system and the sperm membrane function by HOST test. It was detected that progressive motility of sperm warmed at 38, 40 and 42 °C was 26.4 ± 8.4%; 56.6 ± 16.3% and 65.4 ± 15%, respectively, with statistically significant differences between the temperatures of 38 and 40 °C and 38 and 42 °C (P < 0.05). The plasma membrane function evaluated by HOST test was better preserved at 42 °C (76.3 ± 2.0%) compared to 40 °C (43 ± 2%) and 38 °C (65.6 ± 1.5%). The temperature in the thawing process can affect the motility and plasma membrane integrity and function. The warming at 42 °C for thawed vitrified sperm is the optimum temperature to preserve the sperm physiological parameters.

Protective effect of butylated hydroxytoluene on sperm function in human spermatozoa cryopreserved by vitrification technique.

Butylhydroxytoluene (BHT), a synthetic analogue of vitamin E, shows antioxidant and antiviral properties and has been successfully used for mammalian sperm cryopreservation. In this study, BHT was included in a vitrification solution to determine its cryoprotective effect on human spermatozoa. Spermatozoa were selected by swim‐up and vitrified in close sealed straw using either a combination of human tubal fluid (HTF), sucrose and BHT 1 mm (VMBHT), or only HTF and sucrose (VM). The optimal concentration of BHT was determined by the observation of preserved progressive sperm motility (PSM) after warming and detection of plasma membrane (PMI), membrane mitochondrial potential (ΔΨm) and DNA integrity. The presence of reactive oxygen species (ROS) was also detected. The PSM was significantly higher in the VMBHT group (80.86 ± 5.41%) compared with the VM group (68.9 ± 3.67%) (P < 0.05). Butylhydroxytoluene significantly preserved DNA integrity (4.0 ± 0.1% versus 6.1 ± 1.6%; P < 0.05) and reduced ROS production (5.5 ± 2.2 versus 8.6 ± 1.8%; P < 0.05). Plasma membrane and ΔΨm showed no statistical differences. One millimolar BHT effectively maintained cell function and due to its antioxidant and antiviral properties could be used in semen cryopreservation of patients with viral infections transmitted by seminal plasma.

Effect of short‐term semen storage in salmon (Oncorhynchus mykiss) on sperm functional parameters evaluated by flow cytometry

The short‐term storage of salmonid semen is a viable method for in vitro fertilisation. Previous studies have found that short‐term storage affects sperm motility, compromising quality and fertilising capacity. However, the functional characteristics of the spermatozoa of O. mykiss during storage time and its relation to the spawning period are little known. This study was designed to evaluate the effects of in vitro short‐term storage on sperm functional parameters in O. mykiss, determined by flow cytometry. Semen samples of the first spawning – undiluted (SSD) and diluted (SD) (Storfish® 1 : 2v/v; IMV AI solutions, France) – were stored at 4 °C for 14 days. Motility, viability (PMI: plasma membrane integrity) and mitochondrial membrane potential (ΔΨM) were assessed. On the fifth day of storage, spermatozoa showed a motility >70% (SSD: 78.3% versus SD 85.0%), PMI (81.5% SSD/87.2% SD) and ΔΨM (72.5% SSD/SD 80.0%) (P < 0.05). However, a significant decline in the percentage of all functional parameters (P < 0.05) was observed after 5 days of storage for all samples of both undiluted (SSD) and diluted semen. In conclusion, the results here provide new data on O. mykiss sperm quality with respect to in vitro short‐term storage evaluated by flow cytometry.

Short-term storage of salmonids semen in a sodium alginate-based extender.

Short‐term storage of semen is a useful strategy for preservation of fish spermatozoa. However, there is a significantly decrease on sperm function mainly due to oxidative stress. In this way, sodium alginate plays an important role as free radical scavenger compound. Accordingly, the aim of our study was to analyse the effect of a sodium alginate‐based extender on sperm function in the short‐term storage of salmonids semen. Samples of Salmo salar, Oncorhynchus kisutch, and Oncorhynchus mykiss were stored in Storfish® (Ext‐C) and Storfish® supplemented with sodium alginate (Ext‐A) during 10 days at 4°C. After storage, motility, viability, mitochondrial membrane potential (ΔΨmit), superoxide anion (O2−) level and DNA fragmentation (DNA Frag) were assessed. Ext‐A had positive effect in preservation of sperm motility, viability, ΔΨmit, O2− level and DNA integrity in the three species analysed compared to control samples. In Ext‐A, the spermatozoa of S. salar and O. mykiss showed significantly higher motility, viability and ΔΨmit than O. kisutch. However, O. kisutch and O. mykiss had significantly lower O2− level than S. salar, and DNA fragmentation in O. kisutch and S. salar was significantly lower than in samples of O. mykiss (p < 0.05). Dilution of salmonids semen in a sodium alginate‐based extender is effective for protecting sperm quality during 10 days of short‐term storage.

Sperm characteristics of wild and captive lebranche mullet Mugil liza (Valenciennes, 1836), subjected to sperm activation in different pH and salinity conditions

In this article we describe basic aspects of the sperm biology of lebranche mullet (Mugil liza) in the wild and in captivity, in particular assessing the effects of salinity (0, 10, 20, 30, 35, 40, 50 and 60 g L-1) and pH (6, 7, 8, 9 and 10) on sperm motility. Our results indicate that the highest percentage of motility was recorded with salinity 34.6 g L-1 (95 ± 10%) and the longest motility time was obtained with a salinity of 34.8 g L-1 (189 ± 15 s). Variations in the salinity between 30 and 35 g L-1 did not produce any significant alterations in sperm motility; however salinities of 20 and 50 g L-1 produced a significant loss of sperm motility. The highest percentage of motility was obtained at pH 8.5 (93 ± 12%), and the longest motility period at pH 8.7 (218 ± 13 s), while pH lower than or equal to 7 and equal to 10 both produced a significant loss in sperm motility. A positive correlation was found between pH/salinity and the motility percentage (R2 = 0.94 and R2 = 0.97) and motility time (R2 = 0.86 and R2 = 0.98). In seminal and morphometric parameters, statistically significant differences were observed in semen volume, sperm density, plasma membrane integrity and sperm morphometry between the groups studied, showing that the characteristics of the fish have a direct influence on sperm quality. The information generated in this research will be useful for developing biotechnology tools for the effective management of Mugil liza gametes.

Effect of the age of broodstock males on sperm function during cold storage in the trout (Oncorhynchus mykiss)

The knowledge of sperm quality in the broodstock males of different ages is a prerequisite to identify the reproductive ability of cultivated fish for the hatchery management. Thus, in this work, we analysed sperm function of the semen stored of broodstock males of rainbow trout (Oncorhychus mykiss) in different reproductive ages (2, 3 and 4 years old). Sperm samples of each reproductive age were stored in Storfish® during 10 days at 4°C, and then, motility, viability, mitochondrial function (MMP), superoxide anion ( O2− ) level and DNA fragmentation (DNAfrag) were assessed. The results demonstrated that sperm function parameters were affected significantly by the age of the males and the time of storage. Motility, viability and MMP significantly decreased, and DNAfrag and O2− level increased with the age increment and the time of storage. In conclusion, sperm quality of 2 and 3 years old were superior to those of 4 years old, based on higher quality of various sperm functions such as motility, viability, MMP, DNA integrity and level O2− during short‐term storage. This information must be considered for optimum utilization of broodstock males in aquaculture.