In the present study, we provide evidence for the production of reactive oxygen species (ROS) during cryopreservation of bovine spermatozoa. Cooling and thawing of spermatozoa cause an increase in the generation of superoxide radicals. Although nitric oxide production remains unaltered during sperm cooling from 22-4 degrees C, a sudden burst of nitric oxide radicals is observed during thawing. Increase in lipid peroxidation levels have been observed in frozen/thawed spermatozoa and appears to be associated with a reduction in sperm membrane fluidity as detected by spin labeling studies. The data presented provide strong evidence that oxygen free radicals are produced during freezing and thawing of bovine spermatozoa and suggest that these reactive oxygen species may be a cause for the decrease in sperm function following cryopreservation. Mol. Reprod. Dev. 59: 451-458, 2001.

Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing.

BACKGROUND: The effects of cryoinjury were determined simultaneously on the mitochondrial function, motility, morphology and viability of ejaculated human sperm. METHOD: Rhodamine 123 (R123) uptake (% of sperm) and stain intensity were used to determine sperm mitochondrial activity before and after cryopreservation from the semen of 50 men attending for infertility investigation. Morphology was assessed using Tygerberg’s strict criteria and viability was assessed by eosin Y. Sperm motility was measured using computer-assisted semen analysis (CASA). RESULTS: Freeze–thawing caused a 37% (P 0.001) reduction in normal morphological forms of sperm. All CASA sperm motility parameters except amplitude of lateral head displacement were similarly reduced. R123 uptake and intensity within sperm mitochondria decreased by 36 and 47% respectively (both P 0.001). In addition, there was a similar significant decrease (31%, P 0.001) in the viability of the sperm. CONCLUSIONS: Sperm morphology, motility, mitochondrial activities and viability are equally susceptible to cryopreservationinduced damage. R123 intensity is a novel and robust indicator of mitochondrial function before and after such trauma.

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The effects of cryopreservation on sperm morphology, motility and mitochondrial function.

Artificial insemination (AI) was the first great biotechnology applied to improve reproduction and genetics of farm animals. It has had an enormous impact worldwide in many species, particularly in dairy cattle. The acceptance of AI technology worldwide provided the impetus for developing other technologies, such as cryopreservation and sexing of sperm, estrous cycle regulation, and embryo harvesting, freezing, culture and transfer, and cloning. New, highly effective methods of sire evaluation were developed. The history of development of AI is reviewed, particularly in dairy cattle, in which the impact on genetic improvement and control of venereal diseases have been greatest. Other

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The history of artificial insemination: Selected notes and notables

Detection of damage in mammalian sperm cells.

The use of frozen semen in the swine industry is limited by problems with viability and fertility compared with liquid semen. Part of the reduction in sperm motility and fertility associated with cryopreservation may be due to oxidative damage from excessive or inappropriate formation of reactive oxygen species (ROS). Chemiluminescence measurements of ROS are not possible in live cells and are problematic because of poor specificity. An alternative approach, flow cytometry, was developed to identify viable boar sperm containing ROS utilizing the dyes hydroethidine and 2', 7'-dichlorodihydrofluorescein diacetate as oxidizable substrates and impermeant DNA dyes to exclude dead sperm. The percentage of sperm with high mitochondrial transmembrane potential was determined by flow cytometry using the mitochondrial probe 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide with propidium iodide staining to exclude nonviable cells. Sperm were incubated with and without ROS generators and free radical scavengers. Basal ROS formation was low (less than 4%) and did not differ (P = 0.26) between viable fresh and frozen-thawed boar sperm. In addition, fresh and frozen-thawed viable sperm were equally susceptible (P = 0.20) to intracellular formation of ROS produced by xanthine/xanthine oxidase (94.4 and 87.9% of sperm, respectively). Menadione increased (P < 0.05) ROS formation, decreased (P < 0.05) JC-1-aggregate fluorescence intensity, and decreased (P < 0.05) motion variables by 25 to 60%. The mechanism of inhibition of motility by ROS formation may be related to a decrease in mitochondrial charge potential below a critical threshold. Catalase and superoxide dismutase treatment in the presence of xanthine/xanthine oxidase indicated that hydrogen peroxide was the primary intracellular ROS measured. Further, catalase, but not superoxide dismutase, was capable of attenuating ROS-induced inhibition of motility. Whereas basal intracellular hydrogen peroxide formation was low in viable fresh and frozen-thawed boar sperm, both were quite susceptible to external sources of hydrogen peroxide.

Determination of intracellular reactive oxygen species and high mitochondrial membrane potential in Percoll-treated viable boar sperm using fluorescence-activated flow cytometry.

Mitochondrial function and sperm viability were quantified in samples of cryopreserved bovine spermatozoa from 12 bulls using fluorometric techniques. The active mitochondria of the spermatozoa were fluorescently stained using three different fluorophores: rhodamine 123 (R123), 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyan ine iodide (JC-1) or MitoTracker Green FM (MITO). The stained spermatozoa, and companion aliquots that had been stained with SYBR-14 (a living-cell nucleic acid stain) and propidium iodide to assess viability, were quantified using flow cytometry. The resulting fluorescent measurements of mitochondrial function were compared with microscopic assessments of progressive sperm motility immediately after thawing, with motility after 3-h incubation at 37 degrees C, and with the fluorescent assessment of sperm viability. Staining with either R123 or MITO resulted in a single green population. In contrast, the JC-1 staining of mitochondria produced both green and red-orange populations of spermatozoa and sometimes a progressive gradient between the two populations. The ability of JC-1 to discriminate between mitochondria exhibiting high membrane potential from those having low to medium membrane potential provided a more rigorous estimate of metabolic function than the other two fluorescent stains. Overall, the three fluorometric measurements of mitochondrial function were highly correlated with each other, with the SYBR-14 assessment of viability, and with the microscopic estimates of motility.

Fluorometric assessments of mitochondrial function and viability in cryopreserved bovine spermatozoa.

Flow cytometry was used to compare the functional status of fluorescently stained sperm organelles from 12 Holstein bulls after storage for 24 h at 5 degrees C and after cryopreservation. The organelle-specific stains, SYBR-14 and LysoTracker Green DND-26, identified spermatozoa with intact plasmalemma and those with intact acrosomes, respectively. The mitochondria-specific stain, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyan ine iodide (JC-1), identified two populations of spermatozoa. One population stained red-orange because the JC-1 accumulated in the mitochondria as aggregates (characteristic of cells exhibiting a high membrane potential); a second population stained green because of JC-1 monomers within the mitochondria (characteristic of cells exhibiting a lower membrane potential). Analysis of variance revealed that within bulls, the properties of sperm viability, intact acrosomes, and mitochondrial status differed in spermatozoa stored for 24 h (p 0.11). Linear regression analyses resulted in significant models in which the proportions of stained spermatozoa stored for 24 h were indicative of those proportions observed in the cryopreserved fractions. These findings suggest that the plasmalemma, the acrosome, and the mitochondria of unfrozen spermatozoa varied as to their functional status. The cryopreservation process, however, resulted in a more uniform status of sperm organelles.

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Effect of cryopreservation of bovine sperm organelle function and viability as determined by flow cytometry.

The combination of specific fluorometric staining and flow cytometry provides a rapid and precise means of assessing the functional status of cells. We sought to utilize this approach to quantify two important seminal characteristics, acrosomal integrity and sperm viability, and to compare these with classical microscopic measurements of acrosomal integrity and sperm motility. Samples of thawed, cryopreserved sperm packaged in 0.5-ml French straws were obtained from 12 Holstein bulls. Classical acrosomal assessments and sperm motility estimates were made using differential interference contrast microscopy. Fluorescent acrosomal probes included LysoTracker Green DND-26 (LYSO-G), fluorescein-labeled peanut agglutinin, the biotinylated isocoumarin serine protease inhibitor Bi-Aca-Aca-OMe-IC that was secondarily labeled with fluorescein-avidin, and rabbit antibodies to bovine acrosin that were secondarily labeled with fluoresceinated anti-rabbit immunoglobulin. The fluorescent probes for sperm viability were a combination of SYBR-14 and propidium iodide (PI) or of SYTO-17 and PI. Significant differences were found among methods and among bulls, but not among straws (n = 3). All four fluorescent measures of acrosomal integrity showed highly significant correlations with both classical measurements. These data indicated that the quality of cryopreserved bovine sperm samples could be readily quantified using a variety of organelle-specific fluorescent staining techniques.

J M DeJarnette

Papers

Fluorometric assessments of mitochondrial function and viability in cryopreserved bovine spermatozoa.

Effect of cryopreservation of bovine sperm organelle function and viability as determined by flow cytometry.

Fluorometric assessments of acrosomal integrity and viability in cryopreserved bovine spermatozoa

Seminal plasma addition attenuates the dilution effect in bovine sperm.

Seminal plasma addition protects the viability of diluted bovine sperm