Genetic integrity of black sea bream (Acanthopagrus schlegeli) sperm following cryopreservation

Although semen cryopreservation has been applied successfully in many fish species, extensive variation in post‐thaw semen quality exists between species and individuals. AFLP (amplified restriction fragment length polymorphism) is a powerful method for detecting DNA polymorphisms at the individual, population, and species levels. The method has been successfully applied to boars (Sus domestica, Suidae, Artiodactyla, Mammalia) to detect and evaluate differences in DNA sequences that correspond with semen integretiy after employing various freezing techniques. Freezing and thawing of semen has also an effect of selecting for freezing‐resistant (or intact) and eliminating non‐viable or defective sperm. Only the fully intact and functional sperm, despite potential compromise by adverse freezing and osmotic stresses, retain fertility after thawing. The objective of this study was to use AFLP to assess any genetic changes associated with the effect of employed cryo‐methodology on the genetic integrity of sperm of the black sea bream (Acanthopagrus schlegeli) under different cryopreservation treatments. The cryopreservation protocols had no significant effect on sperm motility or survival rate of fertilized ova regardless of using fresh (% motile sperm 89.6 ± 3.0; % embryonic survival rate 54.4 ± 2.9) and frozen‐thawed semen (% motile sperm 80.2 ± 2.0; % embryonic survival rate 51.8 ± 2.0). The post‐thaw sperm motility and survival rates were not significantly different among the sperm samples of the five black sea bream males examined. In the present study, the remaining black sea bream sperm that survive the cryopreservation limit the power of AFLP to trace the genetic markers which correlate with the differences in the sensitivity of sperm to cryo‐injury. It is also possible that point mutations outside the AFLP priming sites may not have been detected. More thorough investigations are needed to determine whether such DNA fingerprints would be found in fish species.     

Cryopreservation of boar semen: equilibrium freezing in the cryomicroscope and in straws

Supercooling causes very abrupt temperature and osmotic changes and can thus lead to freezing damage. Supercooling can be prevented by seeding, using a sample volume and geometry that allows rapid spreading of the ice throughout the sample. In a split-sample comparison of such samples on the cooling stage of a cryomicroscope and seeded at -5 and -15 degrees C, respectively, the percentages of membrane-intact sperm and sperm with acrosomes with a 'normal apical ridge' (NAR) were 72.5+/-3.8 and 75.8+/-2.0 versus 46.3+/-4.8 and 36.0+/-3.7 (means+/-S.E.M., n=4). In ejaculates of 15 unselected AI boars, after seeding at -5 degrees C, the post-thaw % live and % NAR were 66.3+/-10.4 and 74.8+/-7.5, respectively. Our present research is aimed at translating these findings to freezing in straws and at a high sperm concentration. We have designed a novel type of freezing apparatus for controlled-rate freezing of straws, in which supercooling can be effectively prevented in the entire straw. In a split-sample comparison of semen frozen in straws at a sperm concentration of 1.5 x 10(9) cells/ml with nine ejaculates from eight unselected AI boars, we found 54.8+/-1.9% versus 40.7+/-1.7% (means+/-S.E.M.) membrane-intact sperm for the new apparatus and a conventional freezing apparatus, respectively. With bull semen (eight ejaculates from six bulls), we obtained 67.3+/-3.0% versus 59.3+/-2.9% (means+/-S.E.M.) membrane-intact sperm for the new apparatus and conventional freezing, respectively. Additionally, the temperature curve after ice nucleation is of great importance. We have developed a model that allows us to predict that optimal cryopreservation requires a non-linear cooling curve in which the cooling rate varies as a function of subzero temperature.     

New aspects of boar semen freezing strategies

Although cryopreserved boar semen has been available since 1975, a major breakthrough in commercial application has not yet occurred. There is ongoing research to improve sperm survival after thawing, to limit the damage occurring to spermatozoa during freezing, and to further minimize the number of spermatozoa needed to establish a pregnancy. Boar spermatozoa are exposed to lipid peroxidation during freezing and thawing, which causes damage to the sperm membranes and impairs energy metabolism. The addition of antioxidants or chelating agents (e.g. catalase, vitamin E, glutathione, butylated hydroxytoluene or superoxide dismutase) to the still standard egg-yolk based cooling and freezing media for boar semen, effectively prevented this damage. In general, final glycerol concentrations of 2-3% in the freezing media, cooling rates of -30 to -50 degrees C/min, and thawing rates of 1200-1800 degrees C/min resulted in the best sperm survival. However, cooling and thawing rates individually optimized for sub-standard freezing boars have substantially improved their sperm quality after cryopreservation. With deep intrauterine insemination, the sperm dose has been decreased from 6 to 1x10(9) spermatozoa without compromising farrowing rate or litter size. Minimizing insemination-to-ovulation intervals, based either on estimated or determined ovulation, have also improved the fertility after AI with cryopreserved boar semen. With this combination of different approaches, acceptable fertility with cryopreserved boar semen can be achieved, facilitating the use of cryopreserved boar semen in routine AI programs.     

Cooling rate affects rhesus monkey sperm survival

Background The rate at which lethal intracellular ice formation occurs during cryopreservation is highly dependent on several variables. The objective of this study was to determine the optimal rate at which rhesus sperm can be cooled. Methods Experiments were performed using three rates of cooling. Sperm motility was evaluated by computer‐assisted semen analysis, and post‐thaw viability was determined using propidium iodide labeling and flow cytometry. Semen was frozen at three cooling rates: (i) fast, (ii) slow, and (iii) standard. Straws were thawed for 30 s at 37°C for analysis of motility and viability. Results Post‐thaw motility and viability were comparable between freezing curves. Sperm cryopreserved using the slow freeze curve exhibited lowest motility and viability. Conclusions This study indicates that macaque sperm survive cooling optimally when cooling rates range from ?17 to ?34°C/minute. Conversely, slow cooling was detrimental and resulted in poor quality sperm.     

Systematic factor optimization for cryopreservation of shipped sperm samples of diploid Pacific Oysters, Crassostrea gigas

Despite some 26 published reports addressing oyster sperm cryopreservation, systematic factor optimization is lacking, and sperm cryopreservation has not yet found application in aquaculture on a commercial scale. In this study, the effects of cooling rate, single or combined cryoprotectants at various concentrations, equilibration time (exposure to cryoprotectant), straw size, and cooling method were evaluated for protocol optimization of shipped sperm samples from diploid oysters. Evaluation of cooling rates revealed an optimal rate of 5 degrees C/min to -30 degrees C followed by cooling at 45 degrees C/min to -80 degrees C before plunging into liquid nitrogen. Screening of single or combined cryoprotectants at various concentrations suggested that a low concentration (2%) of polyethylene glycol (FW 200) was effective in retaining post-thaw motility and fertilizing capability when combined with permeating cryoprotetcants such as dimethyl sulfoxide (DMSO), methanol (MeOH), and propylene glycol (P-glycol). However, polyethylene glycol alone was not as effective as MeOH, DMSO, and P-glycol when using the same methods. The highest post-thaw motility (70%) and percent fertilization (98%) were obtained for samples cryopreserved with 6% MeOH. However, this does not exclude other cryoprotectants such as DMSO or P-glycol identified as effective agents in other studies. There was no significant difference in post-thaw motility between straw sizes of 0.25- and 0.5-ml. Equilibration time (exposure to cryoprotectant) of 60 min could be beneficial when the cryoprotectant concentration is low and solution is added in a step-wise fashion at low temperature. Differences in post-thaw sperm quality (e.g., motility or percent fertilization) among individual males were evident in this research. As a consequence, a generalized classification describing males with different tolerances (broad, intermediate, and narrow) to cryopreservation was developed. This classification could be applied to strain or species differences in tolerances to the cryopreservation process. The present study demonstrated that oyster sperm could be collected and shipped chilled to another facility for cryopreservation, and that it could be shipped back to the hatchery for fertilization performed at a production scale yielding live larvae with >90% fertilization. Given the existence of facilities for commercial-scale cryopreservation of dairy bull sperm, the methods developed in the present study for oysters provide a template for the potential commercialization of cryopreserved sperm in aquatic species.     

Cooling rate optimization for zebrafish sperm cryopreservation using a cryomicroscope coupled with SYBR14/PI dual staining

The Zebrafish has gained increased popularity as an aquatic model species in various research fields, and its widespread use has led to numerous mutant strains and transgenic lines. This creates the need to store these important genetic materials as frozen gametes. Sperm cryopreservation in zebrafish has been shown to yield very low post-thaw survival and many protocols suffer from great variability and poor reproducibility. The present study was intended to develop a freezing protocol that can be reliably used to cryopreserve zebrafish sperm with high post-thaw survival. In particular, our study focused on cooling protocol optimization with the aid of cryomicroscopy. Specifically, sperm suspended in 8% DMSO or 4% MeOH were first incubated with live/dead fluorescent dyes (SYBR14/PI) and then cooled at various rates from 4 °C to different intermediate stopping temperatures such as −10, −20, −30 and −80 °C before rewarming to 35 °C at the rate of 100 °C/min. %PI-positive (dead) cells were monitored throughout the cooling process and this screening yielded an optimal rate of 25 °C/min for this initial phase of freezing. We then tested the optimal cooling rate for the second phase of freezing from various intermediate stopping temperatures to −80 °C before plunging into liquid nitrogen. Our finding yielded an optimal intermediate stopping temperature of −30 °C and an optimal rate of 5 °C/min for this second phase of freezing. When we further applied this two-step cooling protocol to the conventional controlled-rate freezer, the average post-thaw motility measured by CASA was 46.8 ± 6.40% across 11 males, indicating high post-thaw survival and consistent results among different individuals. Our study indicates that cryomiscroscopy is a powerful tool to devise the optimal cooling conditions for species with sperm that are very sensitive to cryodamage.     

Membrane status of boar spermatozoa after cooling or cryopreservation

This study tested the hypothesis that sperm membrane changes during cooling contribute substantially to the membrane damage observed after cryopreservation of boar spermatozoa. Flow cytometry was used to assess viability (percentages of live and dead cells) of boar sperm cells after staining with SYBR-14 and propidium iodide (PI) and acrosome status after staining with FITC-pisum sativum agglutenin and PI. Incubation (38 degrees C, 4 h), cooling (to 15 or 5 degrees C) and freezing reduced the proportion of live spermatozoa compared with those in fresh semen. There were more membrane changes in spermatozoa cooled to 5 degrees C than to 15 degrees C. The proportion of live spermatozoa decreased during processing for cryopreservation and cooling to 5 degrees C, but was unaffected by freezing and thawing if held at 15 degrees C for 3.5 h during cooling. Spermatozoa not held during cooling exhibited further loss of viability after freezing and thawing. Holding the spermatozoa also increased the proportion of acrosome-intact spermatozoa at both 15 degrees C and 5 degrees C and at thawing compared with that of the unheld controls. The results of this study suggest that a substantial proportion of the membrane changes associated with cryopreservation of boar spermatozoa may be attributed to the cooling of the cells to 5 degrees C rather than to the freezing and thawing process, and that sperm membrane changes are reduced when semen is held at 15 degrees C during cooling.     

Optimizing conditions for treating goat semen with cholesterol-loaded cyclodextrins prior to freezing to improve cryosurvival

The fertility of goat sperm is highly variable and new methods for improving sperm cryosurvival are needed. Cholesterol plays important roles in membrane fluidity, cold shock sensitivity and cryodamage, and treating sperm from cold-shock sensitive species with cholesterol-loaded cyclodextrins (CLC) prior to cryopreservation enhances sperm cryosurvival. The aim of this study was to develop a CLC-treatment to optimize goat sperm cryopreservation. A total of 45 ejaculates coming from eleven adult Murciano-Granadina bucks were used and three experiments were conducted to determine: (1) the optimal CLC concentration to treat goat sperm; (2) the optimal time to treat the sperm (before or after seminal plasma removal); and (3) optimal freezing diluent (either of two Tris-citrate diluents containing 2% or 20% egg yolk and 4% glycerol or a skim milk diluent with 7% glycerol) to cryopreserve goat sperm. Goat sperm cryosurvival rates were greatest when they were treated with 1 mg CLC/120 × 10⁶ sperm prior to freezing. The benefit was also greatest if the sperm were treated with CLC after seminal plasma removal. Finally, CLC treatment improved sperm cryosurvival rates for sperm frozen in all three diluents, however, CLC treatment was most effective for sperm frozen in egg-yolk diluents. In conclusion, treating goat sperm, with CLC prior to cryopreservation, improved sperm cryosurvival rates. In addition, CLC treatment was effective for all freezing diluents tested, making this technology practical for the industry using current cryopreservation techniques. Nevertheless, additional studies should be conducted to determine how CLC might affect sperm functionality and fertilizing ability.     

Suprazero cooling rate,rather than freezing rate,determines post thaw quality of rhesus macaque sperm

Sperm become most sensitive to cold shock when cooled from 37 °C to 5 °C at rates that are too fast or too slow; cold shock increases the susceptibility to oxidative damage owing to its influence on reactive oxygen species (ROS) production, which are significant stress factors generated during cooling and low temperature storage. In addition, ROS may be a main cause of decreased motility and fertility upon warming. They have been shown to change cellular function through the disruption of the sperm plasma membrane and through damage to proteins and DNA. The objective of this study was to determine which cryopreservation rates result in the lowest degree of oxidative damage and greatest sperm quality. In the rhesus model, it has not been determined whether suprazero cooling or subzero freezing rates causes a significant amount of ROS damage to sperm. Semen samples were collected from male rhesus macaques, washed, and resuspended in TEST-yolk cryopreservation buffer to 100 × 10⁶ sperm/mL. Sperm were frozen in 0.5-mL straws at four different combinations of suprazero and subzero rates. Three different suprazero rates were used between 22 °C and 0 °C: 0.5 °C/min (slow), 45 °C/min (medium), and 93 °C/min (fast). These suprazero rates were used in combination with two different subzero rates for temperatures 0 °C to −110 °C: 42 °C/min (medium) and 87 °C/min (fast). The different freezing groups were as follows: slow-med (SM), slow-fast (SF), med-med (MM), and fast-fast (FF). Flow cytometry was used to detect lipid peroxidation (LPO), a result of ROS generation. Motility was evaluated using a computer assisted sperm motion analyzer. The MM and FF treated sperm had less viable (P < 0.0001) and motile sperm (P < 0.001) than the SM, SF, or fresh sperm. Sperm exposed to MM and FF treatments demonstrated significantly higher oxidative damage than SM, SF, or fresh sperm (P < 0.05). The SM- and SF-treated sperm showed decreased motility, membrane integrity, and LPO compared with fresh semen (P < 0.001). Slow cooling from room temperature promotes higher membrane integrity and motility post thaw, compared with medium or fast cooling rates. Cells exposed to similar cooling rates with differing freezing rates were not different in motility and membrane integrity, whereas comparison of cells exposed to differing cooling rates with similar freezing rates indicated significant differences in motility, membrane integrity, and LPO. These data suggest that sperm quality seems to be more sensitive to the cooling, rather than freezing rate and highlight the role of the suprazero cooling rate in post thaw sperm quality.     

Substantial decrease of heat-shock protein 90 precedes the decline of sperm motility during cooling of boar spermatozoa

The decline in boar semen quality after cryopreservation may be attributed to changes in intracellular proteins. Thus, the aim of the present study was to evaluate the change of protein profiles in boar spermatozoa during the process of cooling and after cryopreservation. A total of 9 sexually mature boars (mean age = 25.5+/-12.3 mo) was used. Samples for protein analysis were collected before chilling, after cooling to 15 degrees C, after cooling to 5 degrees C, following thawing after freezing to -100 degrees C, and following thawing after 1 wk of cryopreservation at -196 degrees C. Semen characteristics evaluated included progressive motility and the percentage of morphologically normal spermatozoa. Total proteins from 5x10(6) spermatozoa were separated and analyzed by SDS-PAGE. The results revealed that there was a substantial decrease of a 90 kDa protein in the frozen-thawed spermatozoa. Western blot analysis demonstrated that this protein was 90 kDa heat-shock protein (HSP90). Time course study showed that the decrease of HSP90 in spermatozoa initially occurred in the first hour during cooling to 5 degrees C. When compared with the fresh spermatozoa before chilling, there was a 64% decrease of HSP90 in spermatozoa after cooling to 5 degrees C. However, the motility and percentage of normal spermatozoa did not significantly decrease during this period of treatment. Both declined substantially as the semen was thawed after freezing from -100 degrees C. The results indicated that the decrease of HSP90 precedes the decline of semen characteristics. The length of time between a decrease of HSP90 and the decline in sperm motility was estimated to be 2 to 3 h. Taken together, the above results suggested that a substantial decrease of HSP90 might be associated with a decline in sperm motility during cooling of boar spermatozoa.     

Cryopreservation of sperm from the coral Acropora humilis

Corals are sensitive to minute changes in their environments, and their continued existence is substantially threatened by the increasing number of destructive anthropogenic activities and unprecedented rates of climate change. Although cryopreservation has been successfully to preserve mammalian gametes for decades, coral cryopreservation was attempted for the first time less than 15 years ago, and freezing protocols exist for only a handful of coral species. The present study developed a cryopreservation protocol for the sperm of the common Indo-Pacific reef-builder Acropora humilis. Colonies of reefs of Sattahip Bay, Chonburi Province, Thailand were collected from 3 m depth with a mesh net during a spawning event. Immediately after collection, the sperm were isolated and subjected to a two-step freezing method featuring DMSO, polyethylene glycol, or methanol as the cryoprotectant. Viability and motility were assessed via a bioluminescence technique and a “computer-assisted semen analysis, and it was found that a 15-min equilibration with 2 M DMSO followed by cooling at 41.7 °C was the optimum cryopreservation protocol for A. humilis sperm. The post-thaw sperm achieved 45% fertilization success, and 35% of the fertilized eggs developed into blastopore larvae. The present optimized protocol can therefore facilitate the preservation of sperm for future propagation efforts of this species and provide an experimental platform for optimizing cryopreservation protocols for gametes of other scleractinian coral species.     

Cryo-scanning electron microscopy (Cryo-SEM) of semen frozen in medium-straws from good and sub-standard freezer AI-boars

A major limiting factor for commercial cryopreservation of boar semen for artificial insemination (AI) is the large individual variation to cooling, where the degree of cell dehydration during ice (re)shaping seems to play a major role. This study investigated, in the frozen state, the degree of dehydration and ice crystal distribution in boar semen doses whose spermatozoa displayed different viability after thawing. Cross-sectioned medium-straws (0.5 mL, n=10) from a total of 10 stud boars classified as "good"(n=5) or sub-standard (e.g., "bad" freezers, n=5) by conventional analyses (computer assisted motility and sperm viability) were examined by Cryo-scanning electron microscopy (Cryo-SEM) to determine whether differences between groups could be already distinguishable prior to thawing. The degree of hydration was monitored in relation to the areas of ice crystal formed extracellularly (lakes), the areas of frozen, concentrated extender (veins) where spermatozoa were located and the degree of compartmentalization (number of lakes) present. Irrespectively of the region studied, the gradient of main dehydration (as lakes) observed along the cross-section area of the straws was very irregular. Most spermatozoa were enclosed in the freezing extender matrix and no obvious signs of external membrane damage were observed. None of the Cryo-SEM variables significantly correlated with post-thaw sperm parameters (p>0.05). However, we identified significant differences (p<0.0001) among boars for all ultrastructure variables studied, including the size of the veins, where differences in solute concentration is expected. We concluded that despite the large variability in ice crystal formation during the conventional freezing process among boars, this is unrelated to inter-boar post-thaw sperm differences.     

Freezing response and optimal cooling rates for cryopreserving sperm cells of striped bass, Morone saxatilis

This study explored the optimization of techniques for sperm cryopreservation of an economically important fish species, the striped bass Morone saxatilis. The volumetric shrinkage or the water transport response during freezing of sperm cells was obtained using a differential scanning calorimeter (DSC) technique. Water transport was obtained in the presence of extracellular ice at a cooling rate of 20 degrees C/min in two different media: (1) without cryoprotective agents (CPAs), and (2) with 5% (v/v) dimethyl sulfoxide (DMSO). The sperm cell was modeled as a cylinder of length of 22.8 microm and diameter 0.288 microm and was assumed to have an osmotically inactive cell volume (V(b)) of 0.6 V(0), where V(0) is the isotonic or initial cell volume. By fitting a model of water transport to the experimentally determined water transport data, the best fit membrane permeability parameters (reference membrane permeability to water, L(pg) or L(pg)[cpa] and the activation energy, E(Lp) or E(Lp)[cpa]) were determined and ranged from L(pg)=0.011-0.001 microm/min-atm, and E(Lp)=40.2-9.2 kcal/mol). The parameters obtained in this study suggested that the optimal rate of cooling for striped bass sperm cells in the presence and absence of DMSO range from 14 to 20 degrees C/min. These theoretically predicted rates of optimally freezing M. saxatilis sperm compared quite closely with independent and experimentally determined optimal rates of cooling striped bass sperm.     

Do different portions of the boar ejaculate vary in their ability to sustain cryopreservation?

Previous studies have shown sperm quality post-cryopreservation differs depending on the fraction of the seminal plasma boar spermatozoa are fortuitously contained in. As such, spermatozoa contained in the first 10 mL of the sperm-rich fraction (portion I) have better sustained handling procedures (extension, handling and freezing/thawing) than those contained in the ulterior part of a fractionated ejaculate (second portion of the sperm-rich fraction and the post-spermatic fraction, portion II). However, those studies were performed using pooled samples. In the present study, individual ejaculates were used. Split ejaculates (portions I and II) from five boars were frozen and thawed using a conventional freezing protocol, followed by computer-assisted motility and morphology analysis (CASA and ASMA, respectively), as well as an Annexin-V assay for spermatozoa from each boar and ejaculate portion. Significant differences between portions were observed in all ASMA-derived variables, except in one boar. Also significant differences were observed between boars and ejaculate portions in sperm quality post-thaw. We identified, however, boars showing best results of motility and sperm membrane integrity post-thaw in portion I, while in other boar the best results was observed in portion II. It is concluded that the identification of the ejaculate portion more suitable to sustain cryopreservation in each individual boar may be a readily applicable and easy technique to diminish variation in sperm freezability among boars.     

Cryopreservation of domestic animal sperm cells

Sperm cells are the endpoint of male spermatogenesis and have particular anatomic and metabolic features. Sperm cryopreservation and storage currently require liquid nitrogen or ultralow refrigeration methods for long or short term storage, which requires routine maintenance and extensive space requirements. Conserving sperms have several purposes such as artificial reproductive technologies (ART), species conservation and clinical medicine. The combinations of storage temperature, cooling rate, chemical composition of the extender, cryoprotectant concentration, reactive oxygen species (ROS), seminal plasma composition and hygienic control are the key factors that affect the life-span of spermatozoa. Sperm preservation protocols vary among animal species owing to their inherent particularities that change extenders used for refrigeration and freezing. Extenders for freezing sperm cells contain buffers, carbohydrates (glucose, lactose, raffinose, saccharose and trehalose), salts (sodium citrate, citric acid), egg yolk and antibiotics. The use of different cryoprotectants, like trehalose or glycerol, as well as different concentrations of egg yolk and other constituents in semen extenders are being studied in our laboratory. Several cooling rates have been tested to freeze sperm cells. The use of faster rates (15–60°C/min) gives rise to best sperm survivals after freezing–thawing, but more studies are needed to find the adequate cooling rates for each animal species. Sheep and goat males of some native breeds are being used in studies performed in EZN. Semen from those males has been frozen and stored as part of the Portuguese Animal Germplasm Bank. In small ruminants, individual variations in the quality of frozen semen have been observed, suggesting specific differences in sperm susceptibility to freezing methods, particularly obvious in goat males. Best quality frozen semen from small ruminants is being used in cervical artificial insemination studies aiming to increase productive parameters in selected flocks. Presented at the International Consensus Meeting “New Horizons in Cell and Tissue Banking” on May 16–20, 2007, Vale de Santarém, Portugal.     

Effect of glycerol addition time on the cryopreserved Korean native brindle cattle (Chikso) sperm quality

Although cryopreservation is an efficient method for maintaining the biological and genetic resources of sperm, the sperm damage during the cryopreservation process cannot be ignored. It should be possible to obtain the most effective cryopreservation performance by accurately grasping the effects of various factors on the cryopreservation of sperm. The previous study demonstrated that a suitable standard protocol for cryopreservation of Korean native brindled cattle (Chikso) does not exist, based on the methods for semen cryopreservation of Chikso differ in each research center. The most obvious difference between most of protocols is the addition of glycerol before and after cooling during the Chikso cryopreserved semen process. Therefore we focused on the effects of glycerol addition time on the quality of cryopreserved Chikso sperm. In the present study, 27 individual Chikso samples were collected by transrectal massage and divided into two parts: the “cryopreservation method A” group (adding glycerol before cooling) and the “cryopreservation method B” group (adding glycerol after cooling). Meanwhile, the values of various sperm parameters were derived from each group, including sperm motility, kinematics, capacitation status, cell viability, and intracellular ATP levels, which we used to compare and evaluate sperm function. The results of this study indicated that during the semen cryopreservation process of the Chikso, the addition of glycerol after cooling yielded superior results in a variety of sperm parameters, such as sperm motility, progressive motility, rapid motility, VCL, VSL, VAP, ALH, capacitation status, viability, and intracellular ATP level after freezing and thawing. Our study is suggested that the glycerol addition time during the cryopreservation process for Chikso should be considered. In addition, our results may be provided reference to develop suitable the cryopreservation procedure of the Chikso sperm.     

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.     

Successful cryopreservation of Pacific oyster (Crassostrea gigas) oocytes

Protocols for cryopreservation of sperm and oocytes would provide the ultimate control over parental crosses in selective breeding programmes. Sperm freezing is routine for many species, but oocyte freezing remains problematic, with virtually zero success in aquatic species to date. This paper describes the development of a successful protocol for cryopreserving high concentrations of Pacific oyster (Crassostrea gigas) oocytes. Ethylene glycol (10%) and dimethyl sulfoxide (15%) were found to be the most effective cryoprotectants resulting in post-thaw fertilization rates of 51.0+/-8.0 and 45.1+/-8.3%, respectively. Propylene glycol was less effective and methanol resulted in zero fertilization post-thaw. The use of Milli-Q water rather than seawater as a base medium significantly improved fertilization (20.4+/-3.0 and 8.7+/-2.2%, respectively) as did the inclusion of a 5 min isothermal hold at -10 or -12 degrees C (35.9+/-5.0 and 31.9+/-4.6%, respectively). The optimal cooling rate post-hold was 0.3 degrees C min(-1), with virtually zero post-thaw fertilization with cooling rates of 3 and 6 degrees C min(-1). Using an optimized protocol, post-thaw fertilization rates for oocytes from eight individual females ranged from 0.8 to 74.5% and D-larval yields from 0.1 to 30.1%. For three individuals, larvae were reared through to spat. Development of D-larvae to eyed larvae and spat was similar for larvae produced from unfrozen (24.8+/-4.1% developed to eyed larvae and 16.5+/-3.2% to spat) and cryopreserved (28.4+/-0.6 and 18.7+/-0.5%, respectively) oocytes. The ability to cryopreserve large quantities of oyster oocytes represents a major advance in cryobiology and selective breeding.     

Seminal plasma damages sperm during cryopreservation, but its presence during thawing improves semen quality and conception rates in boars with poor post-thaw semen quality

To determine the effects of seminal plasma during and after cyopreservation on post-thaw sperm functions in semen from poor freezability boars, seminal plasma was removed immediately after collection, and sperm was subjected to cooling and freezing. Removal of seminal plasma did not significantly affect post-thaw sperm motility in good freezability boars; however, in boars with poor freezability, it increased post-thaw motility relative to control sperm cooled with seminal plasma (64.5+/-3.4% vs. 30.9+/-3.1%, P<0.01). Freezing sperm without seminal plasma increased both loss of the acrosome cap (37.5+/-1.6% vs. 18.4+/-2.8%, P<0.01) and expression of a 15 kDa tyrosine-phosphorylated protein (capacitation marker) in thawed sperm relative to controls; the addition of 10% (v/v) seminal plasma to the thawing solution significantly suppressed both changes and increased conception rate to AI (70% vs. 9% in the control group, P<0.05). In conclusion, our novel cryopreservation and thawing method increased the success of AI with frozen-thawed porcine semen, particularly from boars with poor post-thaw semen quality.     

Subzero water permeability parameters of mouse spermatozoa in the presence of extracellular ice and cryoprotective agents.

Optimization of techniques for cryopreservation of mammalian sperm is limited by a lack of knowledge regarding water permeability characteristics during freezing in the presence of extracellular ice and cryoprotective agents (CPAs). Cryomicroscopy cannot be used to measure dehydration during freezing in mammalian sperm because they are highly nonspherical and their small dimensions are at the limits of light microscopic resolution. Using a new shape-independent differential scanning calorimeter (DSC) technique, volumetric shrinkage during freezing of ICR mouse epididymal sperm cell suspensions was obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and CPAs. Using previously published data, the mouse sperm cell was modeled as a cylinder (122-microm long, radius 0.46 microm) with an osmotically inactive cell volume (V(b)) of 0.61V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best-fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The "combined best-fit" membrane permeability parameters at 5 and 20 degrees C/min for mouse sperm cells in solution are as follows: in D-PBS: L(pg) = 1.7 x 10(-15) m(3)/Ns (0.01 microm/min-atm) and E(Lp) = 94.1 kJ/mole (22.5 kcal/mole) (R(2) = 0.94); in "low" CPA media (consisting of 1% glycerol, 6% raffinose, and 15% egg yolk in D-PBS): L(pg)[cpa] = 1.7 x 10(-15) m(3)/Ns (0.01 microm/min-atm) and E(Lp)[cpa] = 122.2 kJ/mole (29.2 kcal/mole) (R(2) = 0.98); and in "high" CPA media (consisting of 4% glycerol, 16% raffinose, and 15% egg yolk in D-PBS): L(pg)[cpa] = 0.68 x 10(-15) m(3)/Ns (0.004 microm/min-atm) and E(Lp)[cpa] = 63.6 kJ/mole (15.2 kcal/mole) (R(2) = 0.99). These parameters are significantly different than previously published parameters for mammalian sperm obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that damaging intracellular ice formation (IIF) could occur in mouse sperm cells at cooling rates as low as 25-45 degrees C/min, depending on the concentrations of the CPAs. This may help to explain the discrepancy between the empirically determined optimal cryopreservation cooling rates, 10-40 degrees C/min, and the numerically predicted optimal cooling rates, greater than 5000 degrees C/min, obtained using suprazero mouse sperm permeability parameters that do not account for the presence of extracellular ice. As an independent test of this prediction, the percentages of viable and motile sperm cells were obtained after freezing at two different cooling rates ("slow" or 5 degrees C/min; "fast," or 20 degrees C/min) in both the low and high CPA media. The greatest sperm motility and viability was found with the low CPA media under fast (20 degrees C/min) cooling conditions.