Ultrastructural injury to human spermatozoa after freezing and thawing

The ultrastructure of human spermatozoa at various stages of the freezing and thawing process was studied. In addition to conventional fixations, a freeze-substitution method was used to examine spermatozoa before they were thawed. Dilution in a glycerol-egg yolk-citrate medium caused slight swelling of the acrosome. During slow freezing, when large ice crystals grow in the diluent, the sperm plasmalemma became tighter, the mitochondria had more angular profiles and there was a reduction in electron density of the acrosomal contents. After thawing, the apical segment of the acrosome usually became swollen and the mitochondria appeared rounded. We deduce that these ultrastructural changes occur either during or after the thawing procedure.     

Decrease of fertilizing ability of mouse spermatozoa after freezing and thawing is related to cellular injury

In general, the fertilizing ability of cryopreserved mouse spermatozoa is less than that of fresh spermatozoa. This ability is especially low in C57BL/6, the main strain used for the production of transgenic mice. To solve this problem, the relationship between cell damage and fertilizing ability in cryopreserved mouse spermatozoa was examined in this study. Sperm motility analysis revealed no significant difference among the motilities of cryopreserved C57BL/6J, BALB/cA, and DBA/2N sperm (67.6%, 43.4%, and 60.0%, respectively) after thawing. However, the results of in vitro fertilization (IVF), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) showed a strong correlation between the frequency of aberrant spermatozoa (FAS) and fertilization rates (FR; C57BL/6J: FAS, 83.7%; FR, 17.0%; BALB/cA: FAS, 67.2%; FR, 24.2%; and DBA/2N: FAS, 10.2%; FR, 93.6%), and damage to spermatozoa was localized particularly in the acrosome of the head and mitochondria.     

Ultrastructure of spores ofRhizopus nigricans after repeated freezing and thawing shocks

Disintegration of nuclear envelopes is the only ultrastructural change detectable by freezeetching in dormant spores of the mouldRhizopus nigricans, both dry and swollen, subjected to repeated freezing and thawing. The increase of the number of freeze-inactivated spores corresponds well with the increase of the number of damaged nuclei. This fact led us to formulate a hypothesis that the structure of the nucleus is the primary target of the freezing or thawing damage. As other biomembranes are not damaged it may be assumed that the disintegration of the nuclear membrane is probably secondary. No changes in ultrastructure of metabolically activated spores could be detected, in spite of the fact that the spores lost their germinative ability. Thus, the mechanism of the freeze injury may be different in dormant and growing spores.     

Effect of freezing and thawing rates on the post-thaw viability of boar spermatozoa frozen in FlatPacks and Maxi-straws

The effects of different freezing and thawing rates on the post-thaw motility and membrane integrity of boar spermatozoa, processed as split samples in Maxi-straws or flat PET-plastic packages (FlatPack) were studied. A programmable freezing device was used to obtain freezing rates of either 20, 50 or 80 degrees C/min. Thawing of the samples was performed in a bath of circulating water; for 40s at 50 degrees C or 27s at 70 degrees C for Maxi-straws and 23s at 35 degrees C, 13s at 50 degrees C or 8s at 70 degrees C for the FlatPacks. Sperm motility was assessed both visually and with a computer assisted semen analysis (CASA) apparatus, while plasma membrane integrity was assessed using the fluorescent probes Calcein AM and ethidium homodimer-1. Temperature changes during freezing and thawing were monitored in both forms of packaging. Values for motile spermatozoa, sperm velocity and lateral head displacement variables were significantly (p<0.05) higher for samples frozen in FlatPacks than in Maxi-straws, with superior results at higher thawing rates. Freezing at 50 degrees C/min yielded better motility than 20 or 80 degrees C/min, although the effect was rather small. Neither freezing rate nor thawing rate had any effect on membrane integrity (p>0.05). A significant boar effect was seen for several parameters. The most striking difference in temperature courses between containers was a 4-5-fold lowering of the thawing rate, between -20 and 0 degrees C, in the center of the Maxi-straw, compared with the FlatPack. This is apparently due to the insulating effect of the thawed water in the periphery of the Maxi-straw. The improvement in sperm motility seen when using the FlatPack appears to be related to the rapid thawing throughout the sample, which decreases the risk of cell damage due to recrystallization during thawing. Since sperm motility patterns have been reported to be correlated with fertility both in vitro and in vivo it is speculated that the use of the FlatPack might improve the results when using frozen-thawed boar spermatozoa for artificial insemination.     

DNA integrity of fresh and frozen canine epididymal spermatozoa

The aims of this study were to evaluate: (1) the effect of cryopreservation on DNA fragmentation of canine epididymal spermatozoa, and (2) the potential protective effect of melatonin on post-thaw sperm quality (motility, morphology, acrosomal and DNA integrity). Epididymal spermatozoa were collected after orchiectomy of ten dogs. Sperm samples were frozen in the presence or absence of melatonin (1 mM). DNA fragmentation index (percentage of spermatozoa with fragmented DNA) was similar in fresh samples (3.3 ± 3.6) and samples frozen with (4.2 ± 3.8) or without (3.6 ± 3.7) melatonin. Sperm motility was significantly (p < 0.0001) higher in fresh compared to frozen samples. The presence of melatonin in the freezing extender did not affect the sperm motility. Proportions of spermatozoa with normal morphology were similar in fresh and frozen samples, irrespective of the presence of melatonin in the extender. Acrosome integrity was significantly decreased (p < 0.01) by cryopreservation, and melatonin did not exert any beneficial effects. In conclusion, DNA fragmentation of canine epididymal spermatozoa was not affected by the freezing procedure, and the presence of melatonin did not preserve motility and acrosome integrity which were adversely affected by cryopreservation. The evaluation of DNA status of thawed gametes is particularly relevant for epididymal spermatozoa since these spermatozoa are usually stored and used in assisted reproductive techniques.     

Motility patterns in mouse spermatozoa before and after capacitation

The motility patterns of mouse spermatozoa before and after capacitation are described. The latter is very characteristic, with spermatozoa describing a figure-8 pattern while remaining relatively stationary within a 3-dimensional space.     

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

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.     

Levels of antioxidant defenses are decreased in bovine spermatozoa after a cycle of freezing and thawing

Growing evidence suggests that the generation of reactive oxygen species (ROS) and their detoxification by antioxidants plays a very important role in fertility. However, the relationship between the level of antioxidants in spermatozoa and the decreased fecundity following a freeze/thaw cycle remains poorly understood. We assessed the activities of antioxidant enzymes such as catalase, glutathione peroxidase (GPx), superoxide dismutase (SOD), and levels of reduced/oxidized glutathione (GSH/GSSG) in bovine semen. Sperm cells were isolated using a Percoll gradient to avoid contamination from seminal plasma, cellular debris, and other cell types. We found that bovine spermatozoa are poorly adapted to metabolize the toxic hydrogen peroxide (H(2)O(2)). Indeed, very low levels of GPx and an absence of catalase were observed. We also studied the effect of freezing and thawing bovine spermatozoa in a egg yolk-Tris-glycerol extender (EYTG). Cryopreservation significantly reduced sperm GSH levels by 78% and SOD activity by 50%. We also investigated whether the decrease in GSH level could be linked to oxidative metabolism and found that a greater reduction in intracellular GSH level occurred when fresh sperm cells were incubated in EYTG for 6 hr at 38.5 degrees C under aerobic conditions than when incubated under restricted oxygen availability. Our results strongly suggest the involvement of an oxidative stress during a freeze/thaw cycle and are consistent with the hypothesis that ROS generated during such a cycle are detrimental to sperm function.     

Long-term preservation of mouse spermatozoa after freeze-drying and freezing without cryoprotection

The widespread production of mice with transgenes, disrupted genes and mutant genes, has strained the resources available for maintaining these mouse lines as live populations, and dependable methods for gamete and embryo preservation in these lines are needed. Here we report the results of intracytoplasmic sperm injection (ICSI) with spermatozoa freeze-dried or frozen without a cryoprotectant after storage for periods up to 1.5 years. Freeze-dried samples were stored at 4 degrees C. Samples frozen without cryoprotection were maintained at -196 degrees C. After storage, spermatozoa were injected into the oocytes by ICSI. Zygotic chromosomes and fetal development at Day 15 of gestation were examined after 0, 1, 3, 6, 9, and 12 mo of sperm storage. When fresh spermatozoa were used for ICSI, 96% of resultant zygotes contained normal chromosomes, and 58% of two-cell embryos transferred developed to normal viable fetuses. Similar results were obtained when spermatozoa were frozen without cryoprotection and then used for ICSI (87% and 45%, respectively; P > 0.05) and after 12 mo of sperm storage (mean of six endpoints examined: 87% and 52%, respectively; P > 0.05). Freeze-drying decreased the proportion of zygotes with normal karyoplates (75% vs. 96%; P < 0.001) and the proportion of embryos that developed into fetuses (35% vs. 58%; P < 0.001), but similar to freezing, there was no further deterioration during 12 mo of storage (mean of six endpoints examined: 68% and 34%, respectively; P > 0.05). Live offspring were obtained from both freeze-dried and frozen spermatozoa after storage for 1.5 yr. The results indicate that 1) the freeze-drying procedure itself causes some abnormalities in spermatozoa but freezing without cryoprotection does not and 2) long-term storage of both frozen and freeze-dried spermatozoa is not deleterious to their genetic integrity. Freezing without cryoprotection is highly successful, simple, and efficient but, like all routine sperm storage methods, requires liquid nitrogen. Liquid nitrogen is also required for freeze-drying, but sperm can then be stored at 4 degrees C and shipped at ambient temperatures. Both preservation methods are successful, but rapid freezing without cryoprotection is the preferred method for preservation of spermatozoa from mouse strains carrying unique genes and mutations.     

Preservation of ejaculated and epididymal stallion spermatozoa by cooling and freezing

The suitability of ejaculated and epididymal stallion spermatozoa for cooled storage (5 degrees C) and cryopreservation was examined in 5 ejaculates from each of 6 stallions and in spermatozoa recovered from the cauda epididymidis after castration of these stallions. The percentage of progressively motile spermatozoa, examined by subjective estimation (cooled samples) or by computerized analysis (frozen-thawed samples), was used as parameter. In ejaculated semen samples containing 5 and 25% seminal plasma in a skim milk glucose extender, the lower amount of seminal plasma supported spermatozoal motility significantly better throughout storage at 5 degrees C. Addition of 5 or 25% seminal plasma to perfused epididymal spermatozoa (0% seminal plasma) resulted in a significant stimulation of spermatozoal motility by 25% seminal plasma at 0 h (P<0.05) and to a lesser extent at 24 and 48 h. Post-thaw motility of ejaculated as well as epididymal spermatozoa was not influenced by slow cooling to 15 degrees or 5 degrees C with or without glycerol prior to rapid freezing in liquid nitrogen vapor. During cooled storage, seminal plasma had a stimulatory effect on epididymal spermatozoa and depressed motility in ejaculated spermatozoa. Results on cryopreservation indicate that freezability of equine spermatozoa is already determined when spermatozoa leave the tail of the epididymis.     

Short-term nonfrozen storage of mouse epididymal spermatozoa

Six simple methods for short-term (up to 8 d), nonfrozen (5 to 20 degrees C) storage of mouse epididymides were compared with respect to the motility and fertility of spermatozoa. A high percentage of progressively motile spermatozoa was obtained from epididymis stored for 8 d at 5 degrees C in mineral oil (78.3%), covered with body fat (80.0%), or stored in the intact body of the euthanized donor animal (77.5%). Fertilized eggs (6.4% fertilization rate) were obtained by IVF using spermatozoa that had been stored in mineral oil at 5 degrees C for at least 8 d, and offspring were obtained from 77.5% of transferred eggs that were fertilized by spermatozoa stored for 2 d. These methods inhibited moisture loss from the preserved epididymal spermatozoa, thereby allowing spermatozoa to be stored for a few days without loss of either motility or fertility. These methods make possible such wide-ranging applications as the long-distance transport of epididymis spermatozoa. While in storage at 5 degrees C, the tail of each recovered spermatozoon was bent midway along the tail, possibly owing to damage to the plasma membranes and due to the spermatozoa's hardening in the phospholipid by exposure to the low temperature.     

Preliminary study of water and some element contents in boar spermatozoa, before, during and after freezing

Boar semen was analysed by electron microscopy coupled to image analysis and X-ray energy dispersive spectroscopy, during the usual process for freezing and thawing in field conditions. Freeze-substitution and freeze-quenching permitted recording of real or potential intracellular ice before, during, and after freezing. Heads and flagella displayed two different osmotic properties before freezing. Heads were dehydrated progressively before and during freezing, while flagella were hydrated before freezing and were only dehydrated during freezing. All parts of the thawed cells were rehydrated. Ice crystal damage was mostly present in frozen mitochondria and axonemes and the acrosomes were strongly affected by thawing. The total amounts of Na, Cl, Ca, K, Mg, and Zn per cell were only elevated in frozen and thawed midpieces while the heads were permeable both to water and elements at that time.     

Fertilizing capacity and ultrastructure of fowl and turkey spermatozoa before and after freezing.

The fertilizing capacity, motility and ultrastructure of fowl and turkey spermatozoa were examined at various stages of the freezing process. For both species, fertility and motility were depressed after equilibration with dimethyl-sulphoxide at 5 degrees C. After freezing, motility was maintained at 55% for fowl spermatozoa and 40% for turkey spermatozoa; however, fertility was 55% for the fowl and 0% for the turkey. Qualitatively, the damage to the spermatozoa of both species was nearly identical, as revealed by scanning and transmission electron microscopy. The plasmalemma was the primary site of damage. 'Bent' spermatozoa, coiled tails and swollen mitochondria were also present. Damage to the acrosome was only observed in spermatozoa which had been frozen to -180 degrees or -196 degrees C. These changes were attributed to adverse osmotic conditions. Binding of cationic ferritin to the plasmalemma of spermatozoa from both species remained unaltered.     

Survival of electrical activity of deep frozen fetal mouse hearts after microwave thawing

Hearts removed from 17–19 day fetal mice were frozen in liquid nitrogen and tested for electrical activity after rewarming. After exposure to various cryoprotective agents, hearts were cooled at 0.5–0.7 °C/min. to ?100 °C and then stored in liquid nitrogen for periods between 72 and 216 hr. Exposure to controlled microwaves at 2450 MHz or immersion in a water bath at 25 C was used in thawing. Electrical activity was studied for periods as long as 90 days after subcutaneous implantation into the ear of syngeneic adult mice. Overall, 59% of 54 frozen-thawed fetal hearts showed strong electrical activity after 30 days when the cryoprotective solution that had been used contained 10% ($\text{v}\text{v}$) dimethylsulfoxide (DMSO) and 10% ($\text{v}\text{v}$) fetal calf serum in Hepes buffer. This system consists of a multicellular structure that is nourished by diffusion; it is well suited for the evaluation of different cryoprotective agents and for various thawing techniques.     

Manifestations of cell damage after freezing and thawing

The nature of the primary lesions suffered by cells during freezing and thawing is unclear, although the plasma membrane is often considered the primary site for freezing injury. This study was designed to investigate the nature of damage immediately after thawing, by monitoring several functional tests of the cell and the plasma membrane. Hamster fibroblasts, human lymphocytes, and human granulocytes were subjected to a graded freeze-thaw stress in the absence of cryoprotective compound by cooling at -1 degree C/min to a temperature between -10 and -40 degrees C, and then were either warmed directly in water at 37 degrees C or cooled rapidly to -196 degrees C before rapid warming. Mitochondrial function in the cells was then assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), fluorescein diacetate (FDA), colony growth, and osmometric response in a hypertonic solution. Cells behaved as osmometers after cooling at -1 degree C/min to low temperatures at which there were no responses measured by other assays, indicating that the plasma membrane is not a primary site for injury sustained during slow cooling. These results also indicate that the FDA test does not measure membrane integrity, but reflects the permeability of the channels through which fluorescein leaves the cells. Fewer cells could respond osmotically after cooling under conditions where intracellular freezing was likely, implying that the plasma membrane is directly damaged by the conditions leading to intracellular freezing. A general model of freezing injury to nucleated mammalian cells is proposed in which disruption of the lysosomes constitutes the primary lesion in cells cooled under conditions where the cells are dehydrated at low temperatures.