Cryopreservation of sperm in common carp Cyprinus carpio: sperm motility and hatching success of embryos

In this study, fish sperm cryopreservation methods were elaborated upon for ex situ conservation of nine strains of Bohemian common carp. Common carp sperm were diluted in Kurokura medium and chilled to 4 degrees C and dimethyl sulfoxide was added. Cryotubes of sperm with media were then cooled from +4 to -9 degrees C at a rate of 4 degrees C min(-1) and then from -9 to -80 degrees C at a rate of 11 degrees C min(-1), held for 6 min at -80 degrees C, and finally transferred into liquid N(2). The spermatozoa were thawed in a water bath at 35 degrees C for 110 s and checked for fertilization yield, hatching yield of embryos, and larval malformations. Fresh and frozen/thawed sperm were evaluated for the percentage and for the velocity of motile sperm from video frames using image analysis. The percentage and velocity of sperm motility at 15 s after activation of frozen/thawed sperm was significantly lower than that of fresh sperm (nine males). ANOVA showed a significant influence of fresh vs frozen/thawed sperm on fertilization rate (P < 0.0001), but differences in hatching rate and in larval malformation (0-6.8%) were not significant, and different males had a significant influence on fertilization and hatching rate (P < 0.003 and P < 0.007, respectively). Multiple range analysis (LSD) showed significant differences between fresh and frozen/thawed sperm regarding fertilization rate (68 +/- 11 and 56 +/- 10%, respectively) and insignificant differences between fresh and frozen/thawed sperm on the hatching rate (50 +/- 18 and 52 +/- 9%, respectively). The percentage and velocity of fresh sperm motility were correlated, respectively, with the fertilization yield of frozen/thawed sperm at the levels r = 0.51 and r = 0.54.     

Semen characteristics and their ability to predict sperm cryopreservation potential of Atlantic cod, Gadus morhua L

There is a lack of biomarkers or indices that can be used to predict the quality of fish semen samples following the freezing and thawing cycle. In the present study, a series of semen indices were tested to assess if they could accurately forecast the cryopreservation potential of Atlantic cod (Gadus morhua) semen. Fresh and frozen-thawed sperm activity variables were compared, and relationships between frozen-thawed sperm activity and fertilization success were examined. In comparison with fresh sperm, activity variables of frozen-thawed spermatozoa were reduced. Of the 18 males examined, mean (± SEM) spermatocrit of fresh sperm was 40.72 ± 4.23%, osmolality of the seminal plasma 366.32 ± 4.95 mOsmol/kg, pH 8.32 ± 0.04, protein concentration 1.05 ± 0.08 mg/mL, anti-trypsin activity 153.83 ± 19.25 U/L, and total antioxidant capacity 0.15 ± 0.03 μmol Trolox equivalents/mL. Frozen-thawed fertilization success was highly variable among males with values ranging from 18.5 to 90.2%. Regressions yielded significant positive relationships between frozen-thawed motility, velocity, track crossing frequency, and subsequent fertilization success. Sequential multiple regressions explained up to 95% of the variation in frozen-thawed sperm activity. Spermatocrit and pH of fresh semen were negatively related, whereas osmolality and antioxidant capacity were positively related to frozen-thawed motility and velocity. Each of these indices can be measured within minutes of collecting a fresh sample of semen and are thus early indicators of the capacity of semen samples to withstand cryopreservation. These results have many benefits for conservation of wild stocks, aquaculture production, and for understanding semen biology and cryobiology of fishes.     

Cryopreservation of turbot (Scophthalmus maximus) spermatozoa

The aim of this study was to develop a method for cryopreserving turbot semen and to compare sperm motility characteristics, metabolic status and fertilization capacity of frozenthawed and fresh semen. The best results were obtained when spermatozoa were diluted at a 1:2 ratio with a modified Mounib extender, supplemented with 10% BSA and 10% DMSO. For freezing sperm samples, straws were placed at 6.5 cm above the surface of liquid nitrogen (LN) and plunged in LN. The straws were thawed in water bath at 30 degrees C for 5 sec. Use of this simple method resulted in a 60 to 80% reactivation rate of the thawed spermatozoa. Although the percentage of motile spermatozoa in the frozen-thawed semen samples was significantly lower than in fresh semen, spermatozoa velocity and respiratory rate remained unchanged. The process of cryopreservation significantly decreased intracellular ATP content. The fertilization rate of frozen-thawed spermatozoa was significantly lower than that of fresh spermatozoa, but it increased with sperm concentration.     

Spermatozoa concentration influences cryopreservation success in sea trout (Salmo trutta m. trutta L.)

Variation among individuals is substantial for spermatozoa concentration in fresh milt in sea trout (Salmo trutta m. trutta L.). The objective of the present study was to examine effects of spermatozoa concentration in this species on subsequent cryopreservation success. Milt with high spermatozoa concentration was diluted with seminal plasma to obtain concentrations ranging between 6 and 24 × 10⁹ mL⁻¹ with steps of 2 × 10⁹ mL⁻¹. Diluted milts were cryopreserved in 0.25-mL straws with extender (0.3 M glucose) containing 10% methanol and 10 % (vol/vol) supplement of hen egg yolk. The dilution ratio was 1:3 (milt:cryomedium). Cryopreservation efficacies were assessed according to evaluation of motility of frozen/thawed spermatozoa and quantification of fertilizing ability. Percentage of motility of frozen/thawed spermatozoa was influenced by spermatozoa concentration in the cryomedium (P < 0.05). The highest motility was observed in samples with 3.0 to 4.0 × 10⁹ spermatozoa per mL of cryomedium, which corresponds to 12 to 16 × 10⁹ spermatozoa per mL in fresh milt. Higher sperm concentrations and lower sperm concentrations in cryomedium reduced the effectiveness of cryopreservation when compared with the optimum. Cryopreservation success measured according to fertilization rate was in agreement with results for motility of frozen/thawed spermatozoa, but the optimum could not be determined with statistical precision because of differences in fertilization rate among individual donor males. However, a significant positive correlation was found between postthaw motility and fertilization rate and between cryopreserved spermatozoa velocity and fertilization rate (P < 0.05). In sea trout, cryopreservation efficiency is influenced by spermatozoa concentration in cryomedium. Individual adjustment of the dilution ratio, based on initial spermatozoa density, is recommended in the freezing protocol. Maximum cryoresistance of the cell was obtained when spermatozoa concentration in cryomedium ranged from 3.0 to 4.0 × 10⁹ mL⁻¹.     

Cryopreservation disrupts lipid rafts and heat shock proteins in yellow catfish sperm

Lipid rafts and associated membrane proteins (flotillin, caveolin) play important roles in cell signaling and sperm fertilization while heat shock proteins (Hsp) ensure properly protein folding to fulfill their physiological functions. The markedly reduced fertility in thawed sperm after cryopreservation could result from disrupted membrane lipid rafts and these proteins. To explore the effect of sperm cryopreservation on lipid rafts and heat shock proteins, we compared lipid raft integrity, and the expression levels of lipid raft associated proteins (Flot-1, Flot-2, Cav-1) as well as heat shock proteins (Hsp90, Hsp70) in fresh and thawed sperm cryopreserved under different scenarios in yellow catfish. We found higher lipid raft integrity, higher protein expression levels of Flot-1, Flot-2, Cav-1, Hsp90, and Hsp70 in fresh sperm samples than in thawed sperm samples, in thawed sperm samples cryopreserved with optimal cooling rate than those cryopreserved with sub-optimal cooling rate, and in thawed sperm samples cryopreserved with extenders supplemented with cholesterol than those supplemented with methyl-β-cyclodextrin (for cholesterol removal). Our findings indicate that lipid raft integrity, and expression levels of Flot-1, Flot-2, Cav-1, Hsp90, and Hsp70 are clearly associated with sperm quality, and together they may play a cumulative role in reduced fertility associated with thawed sperm in aquatic species.     

Adenosine triphosphate concentration and beta-D-glucuronidase activity as indicators of sea bass semen quality

The most common parameters used to evaluate sperm quality are motility rate and duration and fertilization ability. In this study, chemical and biochemical parameters of sea bass (Dicentrarchus labrax) sperm were investigated to find an alternative method for evaluating sperm fertilization ability before and after cryopreservation. The biochemical and chemical analyses were performed with fresh and frozen-thawed sperm and seminal plasma. To cryopreserve sperm, 250-microl straws were used. Fertilization ability was evaluated by inseminating eggs (obtained from hormonally stimulated females) with fresh and cryopreserved sperm. The results revealed a linear relationship (P < 0.05) between semen fertilization capacity and some seminal plasma (beta-D-glucuronidase activity, potassium concentration) and sperm (ATP concentration, aspartate aminotransferase activity) parameters. Variations in semen fertilization rate could be best described by two multiple regression models: one including the sperm parameters and another including the seminal plasma parameters. For practical application, the use of simple regression models is of value. Fertilization rate in both fresh and cryopreserved sperm was reliably predicted by determining the ATP concentration or the beta-D-glucuronidase activity or both.     

Cryopreservation of the milt of the northern pike

Seven published extenders, three thawing media and two thawing temperatures were tested in order to determine their suitability for cryopreservation of northern pike ( Esox lucius L.) sperm. Sperm motility during successive steps of cryopreservation was evaluated. Erdahl and Graham's extender with the addition of egg yolk proved to be the most efficient (maximum hatching rate of 74.5%) when semen was thawed in 120 m m NaCl solution warmed up to 30° C. No correlations between motility of sperm (diluted in extenders or diluted in extenders and then activated with thawing solution) and the subsequent hatching success were observed. The relationship between motility of thawed sperm and its fertilization ability was considerable, but correlation was not significant. Spermatozoa frozen in some extenders were frequently motile after thawing but they were not able to fertilize the eggs, this resulted in a poor hatching rate. Depending on the extender, the addition of yolk induced either positive or detrimental effects on fertilization success.     

High-throughput cryopreservation of spermatozoa of blue catfish (Ictalurus furcatus): Establishment of an approach for commercial-scale processing

Hybrid catfish created by crossing of female channel catfish (Ictalurus punctatus) and male blue catfish (Ictalurus furcatus) are being used increasingly in foodfish aquaculture because of their fast growth and efficient food conversion. However, the availability of blue catfish males is limited, and their peak spawning is at a different time than that of the channel catfish. As such, cryopreservation of sperm of blue catfish could improve production of hybrid catfish, and has been studied in the laboratory and tested for feasibility in a commercial dairy bull cryopreservation facility. However, an approach for commercially relevant production of cryopreserved blue catfish sperm is still needed. The goal of this study was to develop practical approaches for commercial-scale sperm cryopreservation of blue catfish by use of an automated high-throughput system (MAPI, CryoBioSystem Co.). The objectives were to: (1) refine cooling rate and cryoprotectant concentration, and evaluate their interactions; (2) evaluate the effect of sperm concentration on cryopreservation; (3) refine cryoprotectant concentration based on the highest effective sperm concentration; (4) compare the effect of thawing samples at 20 or 40 °C; (5) evaluate the fertility of thawed sperm at a research scale by fertilizing with channel catfish eggs; (6) test the post-thaw motility and fertility of sperm from individual males in a commercial setting, and (7) test for correlation of cryopreservation results with biological indices used for male evaluation. The optimal cooling rate was 5 °C/min (Micro Digitcool, IMV) for high-throughput cryopreservation using CBS high-biosecurity 0.5-ml straws with 10% methanol, and a concentration of 1 × 10⁹ sperm/ml. There was no difference in post-thaw motility when samples were thawed at 20 °C for 40 s or 40 °C for 20 s. After fertilization, the percentage of neurulation (Stage V embryos) was 80 ± 21%, and percentage of embryonic mobility (Stage VI embryo) was 51 ± 22%. There was a significant difference among the neurulation values produced by thawed blue catfish sperm, fresh blue catfish sperm (P = 0.010) and channel catfish sperm (P = 0.023), but not for Stage VI embryos (P ? 0.585). Cryopreserved sperm from ten males did not show significant variation in post-thaw motility or fertility at the neurulation stage. This study demonstrates that the protocol established for high-throughput cryopreservation of blue catfish sperm can provide commercially relevant quantities and quality of sperm with stable fertility for hybrid catfish production and provides a model for establishment of commercial-scale approaches for other aquatic species.     

Control of sperm concentration is necessary for standardization of sperm cryopreservation in aquatic species: evidence from sperm agglutination in oysters

A lack of standardization in sperm cryopreservation of aquatic organisms is one of the main reasons for inconsistency observed among various studies. In particular, there have been few attempts to standardize sperm concentration during procedural optimization. This study was intended to call attention to sperm concentration standardization through research of sperm agglutination in Pacific oysters Crassostrea gigas. Sperm agglutination after thawing is a relatively frequent phenomenon observed for various aquatic species, especially when sub-optimal cryopreservation protocols are used; however, no systematic attempts have been made to explain this phenomenon. The present study evaluated various factors affecting sperm agglutination of thawed samples from diploid and tetraploid Pacific oysters, and is the first detailed report addressing the sperm agglutination phenomenon of thawed samples from any aquatic organism. Agglutination of oyster sperm was classified into six levels with a scale ranging from 0 (homogenous suspension) to 5 (well-developed "noodles"). It was found that agglutination in thawed samples was mainly due to the lack of sufficient cryoprotectant for a specific sperm concentration. Interestingly, high levels of agglutination did not necessarily lead to low fertilization. On the contrary, some sperm cells appeared to gain protection from the formation of peripheral agglutination within 0.5-ml French straws. The exact mechanism of sperm agglutination remains unclear. However, morphological examination of cross sections of the noodles (agglutination level 5) indicated at least two forms of agglutination (formed with and without cryoprotectant) which could be used as a tool to understand the cryopreservation process within the micro-environment of the straw. Furthermore, the fact that the level of sperm agglutination was directly determined by sperm concentration, in addition to the type of cryoprotectant, cryoprotectant concentration, and cooling and thawing methods emphasized the importance of procedural standardization and systematic optimization and integration of protocols involving multiple factors.     

Methyl-beta-cyclodextrin improves fertilizing ability of C57BL/6 mouse sperm after freezing and thawing by facilitating cholesterol efflux from the cells

Sperm cryopreservation provides an economical means of storing genetically engineered mouse strains in resource facilities. In general, relatively high fertilization rates are obtained for frozen/thawed sperm of the CBA/JN, DBA/2N, and C3H inbred strains and some F1 hybrid strains. However, the fertilization rate for frozen/thawed sperm of C57BL/6, which is the main strain of genetically engineered mice, remains very low. Therefore, it is necessary to establish an in vitro fertilization (IVF) method for cryopreserved C57BL/6 sperm that can obtain a high rate of fertilization after thawing. In the present study, we focused on the effects of methyl-beta-cyclodextrin (MBCD) on the fertilizing ability of frozen/thawed C57BL/6 sperm. Our results have shown that the highest fertilization rate for frozen/thawed sperm was obtained with MBCD at 1.0 mM for 30 min (63.7% +/- 11.0%), but the effects were attenuated by long-term incubation for 120 min at 1.0 or 2.0 mM. The embryos with frozen/thawed sperm showed good developmental potential, and the offspring had normal fertility. The efflux of cholesterol from frozen/thawed sperm was increased by MBCD in a dose-dependent manner and occurred much earlier and to a greater extent than bovine serum albumin. The localization of cholesterol labeled by filipin in the sperm plasma membrane was drastically decreased by MBCD. In summary, we suggest that MBCD is useful for developing an IVF method for frozen/thawed C57BL/6 mouse sperm achieving a high fertilization rate, being involved in the capacity to sequester cholesterol from sperm membrane.     

Evaluation of gilthead sea bream, Sparus aurata, sperm quality after cryopreservation in 5 ml macrotubes

Cryopreservation produces several types of damage in spermatozoa, leading to fertility impairment. The reduction arises both from a lower viability post-thaw and from sublethal dysfunctions in some of the surviving cells. In the present study, we have analysed the effect of cryopreservation in 5 ml macrotubes on the quality of post-thawed gilthead sea bream sperm. Several standard sperm quality parameters were determined: pH and osmolarity of seminal plasma, sperm concentration, and motility. An exhaustive determination of sperm quality before and after cryopreservation was investigated. Several parameters related with spermatozoal status were determined: ATP content, plasma membrane integrity and functionality, mitochondrial functionality, and sperm fertility. Our results demonstrated that gilthead sea bream spermatozoa suffer several types of damage after freezing/thawing. The percentage of viable cells slightly decreased after cryopreservation, however plasma membrane was affected by cryopreservation, since cells could not resist the hyperosmotic shock. Mitochondrial status was affected by cryopreservation since there was a decrease in the parameters of sperm motility, ATP content (3.17 nmol ATP/10(5) spermatozoa to 1.7 nmol ATP/10(5) spermatozoa in 1:20 frozen samples) and an increase of the percentage of cells with mitochondrial depolarized membranes (11% for fresh and 27% for 1:20 frozen samples). Fertility rate was similar either using fresh or frozen/thawed sperm (77 and 75% hatched larvae, respectively).     

Frozen-thawed rhesus sperm retain normal morphology and highly progressive motility but exhibit sharply reduced efficiency in penetrating cervical mucus and hyaluronic acid gel

The preservation of the genetic diversity of captive populations of rhesus monkeys is critical to the future of biomedical research. Cryopreservation of rhesus macaque sperm is relatively simple to perform, yields high post-thaw motility, and theoretically, provides via artificial insemination (AI) a way to easily transfer genetics among colonies of animals. In the interest of optimizing semen cryopreservation methods for use with vaginal AI, we evaluated the ability of frozen-thawed rhesus sperm to penetrate periovulatory cervical mucus (CM). Motile sperm concentration of pre-freeze (“fresh”) and post-thawed (“thawed”) samples from five different males were normalized for both computer assisted sperm motion analysis and CM penetration experiments. Sperm samples were deposited into slide chambers containing CM or gel composed of hyaluronic acid (HA) as a surrogate for CM and numbers of sperm were recorded as they entered a video field a preset distance from the sperm suspension-CM (or HA) interface. Fresh and thawed sperm were dried on glass slides, “Pap”-stained, and assessed for changes in head dimensions and head and flagellar shape. While retaining better than 80% of fresh sperm progressive motility, thawed sperm from the same ejaculate retained on average only 18.6% of the CM penetration ability. Experiments using HA gel yielded similar results only with reduced experimental error and thus improved detection of treatment differences. Neither the percentage of abnormal forms nor head dimensions differed between fresh and thawed sperm. While findings suggests that sperm-CM interaction is a prominent factor in previous failures of vaginal AI with cryopreserved macaque sperm, neither sperm motility nor morphology appears to account for changes in the ability of cryopreserved sperm to penetrate CM. Our data points to a previously unidentified manifestation of cryodamage which may have implications for assessment of sperm function beyond the cervix and across mammalian species.     

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.     

Motility and fertilizing capability of cryopreserved Acipenser ruthenus L. sperm

Motility before and after cryopreservation of Acipenser ruthenus sperm was determined by computer-assisted sperm motion analysis (CASA). In addition, fertility of fresh and frozen/ thawed sperm was tested by in vitro fertilization. Sperm of A. ruthenus was successfully cryopreserved by 1/1 (v/v) dilution with ethylene glycol in final concentrations from 12.5% to 20%. Stepwise cooling using a programmable freezer and fast thawing in a water bath (40°C) for 3 s and 5 s, respectively, resulted in motility rates of 10% to 28% and fry yields of 44% to 94% compared to controls (fresh sperm). With fresh material, different sperm to egg ratios varying from 10³: 1 to 10⁶: 1 gave similar fertilization rates of more than 90%. Using this cryopreservation method the establishment of sperm banks for endangered sturgeon species and the development of a fertility test for seasonally independent testing seem to be possible.     

Relationship between sperm density, spermatocrit, sperm motility and spawning date in wild and cultured haddock

Semen was collected repeatedly from captive haddock Melanogrammus aeglefinus and the effect of seasonality on various sperm parameters was investigated. No differences in sperm traits were observed for wild and cultured haddock. A highly significant positive relationship existed between spermatocrit and spermatozoa density. A significant increase in mean spermatocrit occurred throughout the spawning season but the amount of variability explained by collection date was low (35·1%) due to variability between males. Each of 10 males sampled repeatedly throughout the spawning season demonstrated an increase in spermatocrit. No relationship existed between spermatocrit and proportion of motile spermatozoa when spermatocrit was ≤70%. Motility was reduced in semen samples with spermatocrits >70%. The proportion of spermatozoa that were motile decreased with time since activation. Some motility was still observed after 60 min in sea water (0·1–15·2%) for sperm collected at all times within the spawning season. Of those spermatozoa that were motile, the proportion that exhibited forward swimming motion decreased and the proportion that had only vibratory movement increased with time post‐activation. The speed of forward swimming spermatozoa showed no significant relationship with spermatocrit at any time between 0 and 60 min after activation. Swimming speed was negatively related to time since activation, decreasing from 174–240 μm s⁻¹ at 0 min to 80–128 μm s⁻¹ at 60 min after activation.     

不同品系小鼠的体外受精、胚胎冷冻及移植的比较研究

目的　探讨不同品系小鼠的体外受精、胚胎和精子的低温保存效果。方法　本实验分别在中国科学院上海实验动物中心 (SLAC)和日本熊本大学动物资源开发中心 (CARD)对 13个品系小鼠 (C57BL 6J、BALB c、C3H HeJ、ICR、KM、FVB、MRL、NOD、CBA、DBA 2、CD 1、BDF1、B6C3F1)的体外受精 (IVF)率、胚胎培养及移植成绩进行了比较研究。结果　各品系小鼠新鲜精子的IVF率 15 1%～ 87 9% ,冻融精子的IVF率 8%～ 80 % ;冷冻胚胎的复苏率4 2 6 %～ 83 9% ;冻融胚胎移植后的产仔率在 17 8%～ 5 1 8%。结论　遗传背景不同的小鼠体外受精率、冷冻胚胎复苏率和胚胎移植的产仔率差异有显著性。但同一品系两个实验室间的新鲜精子的IVF率、冷冻胚胎的复苏率及移植产仔率差异无显著性 (P >0 0 5 ) ;冻融精子的体外受精率CARD明显高于SLAC(P <0 0 1)。     

Proteomic characteristics of human sperm cryopreservation

Human sperm cryopreservation in assisted reproductive technology is the only proven method that enables infertile men to father their own children. However, freezing and thawing reduces spermatozoon motility, viability, and fertilizing ability. An association between dysfunctional spermatozoa due to cryoinjury and protein changes has not been established. We investigated through proteomic analysis the differential protein characteristics between freeze‐thawed and fresh sperm samples obtained from nine normozoospermic donors. Twenty‐seven proteins differed in abundance between the two groups, and results were verified for four proteins via Western blot and immunofluorescent staining. These proteins are putatively involved in sperm motility, viability, acrosomal integrity, ATP and isocitrate content, mitochondrial membrane potential, capacitation, acrosome reaction, and intracellular calcium concentration. These marked differences suggest that dysfunctional spermatozoon after cryopreservation may be due to protein degradation and protein phosphorylation.     

Effect of osmotic immobilization on refrigerated storage and cryopreservation of sperm from a viviparous fish, the green swordtail Xiphophorus helleri

In this study, refrigerated storage and cryopreservation of sperm from the green swordtail Xiphophorus helleri were investigated. Previous cryopreservation research in this species utilized motile sperm because unlike in most fish species, Xiphophorus sperm can remain continuously motile after collection for a week with refrigerated storage. However, this species reproduces by internal fertilization, and given the significant requirements for motility within the female reproductive tract and potential limitations on sperm energetic capacities, immobilization of sperm prior to insemination could be used to improve fertilization success. Thus, the goal in this study was to use osmotic pressure to inhibit the motility of sperm after collection from X. helleri, and to test the effect of immobilization on refrigerated storage and cryopreservation. The objectives were to: (1) estimate the motility of sperm at different osmotic pressures, and determine an osmotic pressure suitable for immobilization; (2) cryopreserve the immobilized sperm, and estimate the motility after thawing with or without dilution, and (3) compare motility of non-immobilized and immobilized sperm after thawing, centrifugation, and washing to remove cryoprotectant. Motility was determined when sperm were suspended in 11 different osmotic pressures (24-500 mOsmol/kg) of Hanks' balanced salt solution (HBSS). Motility was observed between 116 and 425 mOsmol/kg. Sperm were not motile when the osmolality was lower than 116 or higher than 425 mOsmol/kg. Motility of the immobilized (non-motile) sperm could be activated by changing the osmotic pressure to 291-316 mOsmol/kg, and motility of immobilized sperm from hypertonic HBSS (425 mOsmol/kg) was significantly higher than that from hypotonic HBSS (145 mOsmol/kg) after 48 h of storage. At an osmolality of 500 mOsmol/kg, HBSS was used as extender to maintain immobilized sperm during cryopreservation with glycerol as the cryoprotectant. High motility (approximately 55%) was obtained in sperm after thawing when cryopreserved with 10-15% glycerol, and dilution of thawed sperm in fresh HBSS (1:4; V:V) was found to decrease the motility significantly. No difference was found in the motility of thawed sperm cryopreserved with 14% glycerol and extended in 310 and 500 mOsmol/kg HBSS. Washing by centrifugation prolonged the motility of thawed sperm from 24 to 72 h in HBSS at 310 and 500 mOsmol/kg. This study showed that sperm from X. helleri could be immobilized by use of specific osmotic pressures, and that the immobilization did not affect sperm motility after thawing. The immobilization of sperm by osmotic pressure could minimize reduction of the energetic capacities necessary for insemination, traversal, and residence within the female reproductive tract, and fertilization.     

Cryopreservation of European catfish Silurus glanis sperm: sperm motility, viability, and hatching success of embryos

The aim of this study was to elaborate cryopreservation methods for ex situ conservation of European catfish. The success of sperm cryopreservation was evaluated by post-thaw sperm motility and velocity, percentage of live spermatozoa and fertility (hatching rates) using frozen/thawed sperm. The best hatching rates of 82-86% were obtained with sperm stored for 5 h before freezing in immobilizing solution and frozen with Me2SO in concentrations of 8, 10, and 12%, or with a mixture of 5% Me2SO and 5% propandiole. These results did not significantly differ from the fresh sperm control sample. The percentage of live spermatozoa in frozen/thawed sperm did not correlate with hatching rate or motility of spermatozoa, but was negatively correlated with velocity of spermatozoa (r=-0.47, P=0.05). The percentage motility in frozen/thawed sperm ranged from 8 to 62%, when sperm was stored in immobilizing solution 5h before freezing. The average value in the fresh sperm (control) was 96%. The frozen/thawed sperm motility rate significantly correlated with the hatching rate (r=0.76, P=0.0002), but not with the percentage of live spermatozoa (r=0.16, P=0.52) or the sperm velocity (r=0.07, P=0.79). The velocity of frozen/thawed spermatozoa ranged from 37 to 85 microm/s, whereby methanol concentrations of 7.5 and 10% resulted in highest velocities. Freezing sperm volumes of 1-4 ml did not affect the quality of frozen/thawed sperm.     

Laboratory studies of cryopreservation of sperm and trochophore larvae of the eastern oyster

The eastern oyster, Crassostrea virginica, is the most important cultured oyster species of the Atlantic and Gulf coasts of the United States. Cryopreservation of gametes and larvae of aquatic organisms has increased in importance in recent years. However, studies on the cryopreservation of sperm and larvae of mollusks have focused on the Pacific oyster, Crassostrea gigas. The present study was conducted to improve cryopreservation of sperm and trochophore larvae and to assess fertilizing ability and male-to-male variation of thawed sperm of the eastern oyster. Sperm were diluted in 12 cryoprotectant solutions composed of Hanks' balanced salt solution without calcium and 0, 5, 10, 15, 20, and 25% (v/v) propylene glycol with or without 0.25 M sucrose. Trochophore larvae were suspended in artificial seawater and 10 or 15% propylene glycol (v/v). Sperm or trochophore larvae were placed in 5-mL macrotubes and allowed to equilibrate for 15 min. The macrotubes were cooled in a controlled-rate freezer at a rate of 2.5 degrees C per min until reaching a final temperature of -30 degrees C and were plunged into liquid nitrogen. After storage for 2 weeks, the samples were thawed in a water bath at 70 degrees C for 15 s. Overall, for cryopreservation of sperm and larvae, best results were obtained using 10 or 15% propylene glycol. Thawed sperm presented significant male-to-male variation in fertilizing ability. Survival of thawed larvae decreased as the concentration of larvae per macrotube increased. The procedures developed in this study for sperm and larvae are suitable for production of seedstock in commercial oyster hatcheries.