J. Risopatrón

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.

Effect of Escherichia coli and its soluble factors on mitochondrial membrane potential, phosphatidylserine translocation, viability, and motility of human spermatozoa

OBJECTIVE To evaluate the effect of Escherichia coli and its soluble factors on the viability and function of human spermatozoa. DESIGN In this prospective study, after removal of seminal plasma, the sperm suspension was incubated in vitro with E. coli or with supernatant from E. coli culture. SETTING Andrology laboratory in a medical research institution. PATIENT(S) Semen was obtained from normozoospermic men. INTERVENTION(S) Semen samples were evaluated to determine the effect of E. coli and its soluble factors on sperm viability, motility, mitochondrial membrane potential (DeltaPsim), phosphatidylserine translocation, and reactive oxygen species generation. MAIN OUTCOME MEASURE(S) To verify the effect of E. coli and its soluble factors on sperm function. RESULT(S) After incubation with E. coli, the percentage of sperm with intact DeltaPsim decreased significantly, as did sperm viability and motility. Reactive oxygen species levels and phosphatidylserine translocation did not increase significantly. After sperm incubation with E. coli supernatant, a significant reduction in DeltaPsim, viability, and motility were also observed. CONCLUSION(S) Escherichia coli and its soluble factors affect sperm function, suggesting that the harmful effects of bacterial infection do not require that the spermatozoon come into direct contact with bacteria.

Canine sperm vitrification with sucrose: effect on sperm function.

The ability of sucrose to protect spermatozoa against mitochondrial damage, artificial acrosome reaction and DNA fragmentation during ultra‐rapid cryopreservation in canine sperm was investigated. Swim‐up selected spermatozoa of second‐fraction semen were vitrified with different concentrations of sucrose (0.1, 0.25 and 0.4 m) in proportion 1 : 1 v/v with HTF–BSA 1%. From each group, 30‐μl suspensions of cells were dropped directly into liquid nitrogen and stored for at least 24 h. Cells were thawed by submerging the spheres in HTF with 1% BSA at 37 °C. The number of progressively motile spermatozoa was significantly higher in the sucrose 0.25 m + HTF–BSA 1% (42.5 ± 2.3%, P < 0.01) than in HTF only (1.66 ± 0.3%). The same combination of sucrose 0.25 m + HTF–BSA 1% (42.7 ± 1.5%) had a stronger cryoprotective effect on the integrity of mitochondrial membrane potential (P < 0.05) and decreased the DNA fragmentation (2.8 ± 0.5%) as compared with HTF only (1.93 ± 0.6% and 5.6 ± 0.6% respectively). With respect to acrosome‐reacted spermatozoa, no significant difference was found between the groups investigated (P > 0.05). It is concluded that sucrose, a nonpermeable cryoprotectant, can effectively preserve important physiological parameters such as mitochondrial membrane potential and DNA integrity during ultra‐rapid cryopreservation.

Vitrification of Human ICSI/IVF Spermatozoa Without Cryoprotectants: New Capillary Technology

The aim of this study was to develop and to test the standardized aseptic technology of permeable cryoprotectant-free vitrification of human spermatozoa in capillaries (for intracytoplasmic sperm injection [ICSI] or in vitro fertilization [IVF]). To test the effect of vitrification on basic sperm parameters, each of 68 swim-up-prepared ejaculates from oligo-astheno-terato-zoospermic patients were aliquoted and distributed into 3 groups: 1) nontreated control, 2) 10 μL of spermatozoa cryopreserved by slow conventional freezing with glycerol-contented medium, and 3) 10 μL of spermatozoa vitrified in 50-μL plastic capillaries in culture medium with 0.25 M sucrose. Spermatozoa motility (1, 24, and 48 hours after warming), plasma membrane integrity, acrosomal integrity, and spontaneous capacitation-like changes were determined after warming. Aseptic cryoprotectant-free vitrification showed a significantly stronger cryoprotective effect compared with conventional freezing. One hour after warming, motility, plasma membrane integrity, and acrosomal integrity were significantly higher than is observed for conventionally frozen spermatozoa (28% vs 18%, 56% vs 22%, and 55% vs 21%, respectively; P < .05), although lower than in fresh spermatozoa (35%, 96%, and 84%, respectively; P < .05). Capacitation-like changes did not differ significantly between vitrified and conventionally frozen samples (8% vs 9%, respectively; P > .1) (2% in fresh spermatozoa). The newly developed technology of aseptic vitrification of human spermatozoa in capillaries can effectively preserve these cells from cryo-injures. Spermatozoa, vitrified by this technology, are free from seminal plasma owing to swim-up preceding vitrification and are free from permeable cryoprotectants. They are ready for further use immediately after warming without any additional treatment. Therefore, the reported technology has a great potential for use in ICSI/IVF.

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.

Live Birth After Intrauterine Insemination With Spermatozoa From an Oligoasthenozoospermic Patient Vitrified Without Permeable Cryoprotectants

We report the first case of a healthy baby born after intrauterine insemination with vitrified swim-up spermatozoa from an oligoasthenozoospermic patient. A 39-year-old patient was subjected to intrauterine insemination with spermatozoa from her 35-year-old husband, diagnosed with oligoasthenozoospermia. The swim-up spermatozoa from 2 ejaculates were suspended in a culture medium supplemented with 1% human serum albumin and 0.25 M sucrose. Three hermetically packaged 100-μL sperm portions (each containing 1.0 × 10(6) spermatozoa/mL) were vitrified by direct plunging into liquid nitrogen. The presence of leukocytes in the ejaculates was determined by indirect immunofluorescence assay. Production of reactive oxygen species was measured by chemiluminescent assay. Before intrauterine insemination, all portions were warmed in culture medium at 37°C, and the spermatozoa were concentrated by centrifugation; they were then resuspended in 500 μL of culture medium and inseminated. The cell suspension used for insemination displayed 60% progressive motility. Clinical pregnancy was confirmed at 7 weeks of gestation, and a healthy baby was born at term. Our results show that a successful intrauterine insemination can be achieved with aseptic vitrification of spermatozoa.

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%).

Alpha-glucosidase in the human epididymis: topographic distribution and clinical application

Summary.  α‐Glucosidase activity (EC.3.2.1.20) is present in human seminal plasma, and the neutral form of the enzyme originates almost exclusively from the epididymis. In this study, the specific immunocytochemical location of α‐glucosidase in the human epididymis was evaluated using a polyclonal antibody. Furthermore, a spectrophotometric assay was employed to assess epididymal obstruction in infertile patients. The enzymatic activity of α‐glucosidase free of prostate isoform (AGFPI) was determined spectrophotometrically at 405 nm. According to AGFPI activity, patients with leucocytospermia, oligozoospermia and azoospermia were recorded as having normal values or low values indicating epididymal obstruction. Specific immunochemistry staining was demonstrated in the cytoplasmic cells at the epithelial level, in the transition area and in the efferent ducts. The values of the three groups and the control were as follows (mean ± SEM): normozoospermia (control): 20.2 ± 1.4 mU ml−1; azoospermia: normal value: 17.6 ± 2.2 mU ml−1, low value: 7.4 ± 1.8 mU ml−1; oligozoospermia: normal value: 22.3 ± 2.5 mU ml−1, low value: 7.3 ± 0.7 mU ml−1; leucocytospermia: increase value: 38.9 ± 3.7 mU ml−1, low value: 11.1 ±1.3 mU ml−1. This study suggests that determination of α‐glucosidase might be helpful to evaluate functions of the epididymis and particularly to exclude epididymal obstruction.

Migration/sedimentation sperm selection method used in bovine in vitro fertilization: Comparison with washing/centrifugation

Sperm selection methods are usually considered for in vitro fertilization (IVF) programs. To achieve a population of viable, motile and morphologically normal spermatozoa, seminal plasma and contaminants must be removed. In this study 2 sperm selection methods were compared with regard to conventional parameters: 1) repeated washing/centrifugation, and 2) migration/sedimentation. Their effect on the fertilization of oocytes aspired from ovaries of slaughtered cows was determined. Frozen-thawed semen was used for all assays (n = 8). The sperm concentration was adjusted to 1.0 × 106 cells/ml for in vitro insemination. Selected oocytecumulus complexes were matured in vitro for 24 h and were co-incubated with spermatozoa prepared by the 2 different methods. Presumptive zygotes were co-cultered in TCM-199. The percentage of motile, viable and morphologically normal spermatozoa was markedly higher (p < 0.05) with the migration-sedimentation method. Triple stain showed that the integrity of the acrosome was significantly better maintained after migration/sedimentation (68.3%) than after washing/centrifugation (53.2%; p < 0.05). In accordance with these results, a high fertilisation rate was achieved after application of the migration/sedimentation method (92.5 vs 69.1%;p < 0.05). It is concluded, that this method is very promising for obtaining spermatozoa with optimal fertilization capacity.

Addition of superoxide dismutase mimics during cooling process prevents oxidative stress and improves semen quality parameters in frozen/thawed ram spermatozoa.

High levels of reactive oxygen species (ROS), which may be related to reduced semen quality, are detected during semen cryopreservation in some species. The objectives of this study were to measure the oxidative stress during ram semen cryopreservation and to evaluate the effect of adding 2 antioxidant mimics of superoxide dismutase (Tempo and Tempol) during the cooling process on sperm motility, viability, acrosomal integrity, capacitation status, ROS levels, and lipid peroxidation in frozen and/or thawed ram spermatozoa. Measuring of ROS levels during the cooling process at 35, 25, 15, and 5 °C and after freezing and/or thawing showed a directly proportional increase (P < 0.05) when temperatures were lowering. Adding antioxidants at 10 °C confered a higher motility and sperm viability after cryopreservation in comparison with adding at 35 °C or at 35 °C/5 °C. After freezing and/or thawing, sperm motility was significantly higher (P < 0.05) in Tempo and Tempol 1 mM than that in control group. Percentage of capacitated spermatozoa was lower (P < 0.05) in Tempo and Tempol 1 mM in comparison with that in control group. In addition, ROS levels and lipid peroxidation in group Tempo 1 mM were lower (P < 0.05) than those in control group. These results demonstrate that ram spermatozoa are exposed to oxidative stress during the cooling process, specifically when maintained at 5 °C and that lipid peroxidation induced by high levels of ROS decreases sperm motility and induces premature sperm capacitation. In contrast, the addition of Tempo or Tempol at 0.5 to 1 mM during the cooling process (10 °C) protects ram spermatozoa from oxidative stress.