Effect of sperm cryopreservation on sperm DNA stability and progeny development in rainbow trout

This study was carried out to test how sperm cryopreservation affected nuclear DNA stability and whether progeny development was modified when eggs were fertilized with cryopreserved spermatozoa. The "comet assay" (alkaline single-cell gel electrophoresis assay) was adapted to trout spermatozoa to estimate DNA stability as measured by alkali-induced DNA strand break formation. Because trout eggs develop in water after fertilization (oviparous species) and that eggshell is easy to clear up after fixative treatment, progeny development was assessed from the blastodisc flattening stage of the embryos to the first feeding stage of the hatched fries by direct observation. All parameters under study were analyzed on each sperm and comparisons between parameters were made using paired data. Freeze-thawing of sperm slightly but significantly increased the percentage of nuclei showing altered DNA after comet assay. This increase was correlated to the decrease in fertilization rates of sperm, but the absolute percentage of altered nuclei was not predictive of the absolute fertilization ability of sperm. Assessment of progeny development showed that survival rate and abnormality rate obtained after fertilization with cryopreserved sperm were not different from those obtained with fresh sperm. It is concluded that trout sperm cryopreservation only slightly affected sperm DNA stability and that the use of cryopreserved spermatozoa did not impair offspring survival and quality.     

Evaluation of DNA damage as a quality marker for rainbow trout sperm cryopreservation and use of LDL as cryoprotectant

Defining reliable and objective biomarkers of sperm quality is a complex matter, because it does not rely on a particular characteristic of the milt. Susceptibility to cryopreservation varies between ejaculations and throughout the year, and the evaluation of fresh sperm does not always provide accurate information about their fertilization ability after freezing and thawing. DNA is one of the cell components prone to suffering cryodamage and several studies have pointed out the importance of the maintenance of its integrity during sperm cryostorage. The authors analysed sperm from rainbow trout for four weeks during the natural reproductive season. Viability, DNA integrity, and fertilization ability were evaluated. Furthermore, in order to increase membrane and DNA protection during sperm cryopreservation, the authors optimized the use of LDL fraction from egg yolk as a cryoprotectant during the analysed period. Results revealed that the evaluation of DNA damage in fresh sperm reveals subtle cell damage, not evidenced in fresh sperm by the other parameters. DNA fragmentation increased from 8 to 31% during the reproductive season, indicating pre-freezing differences that render the cells more susceptible to cryodamage. Also, the use of 12% LDL (low density lipoprotein) fraction, instead of the commonly used pure egg yolk, improved sperm quality after freezing. When LDL was used, post-thaw quality remained constant throughout the analysed period, providing around 60% of eyed embryos. In contrast, when egg yolk was used, post-thaw quality decreased significantly at the end of the season and the percentage of eyed embryos dropped from 60% to 27%. Results demonstrated that reduction in DNA integrity takes place during the reproductive season affecting susceptibility to cryodamage and that the protective effect of egg yolk is very much improved when only their LDL fraction is added to the cryopreservation extender.     

Characterization of sperm plasma membrane properties after cholesterol modification: consequences for cryopreservation of rainbow trout spermatozoa

During cryopreservation, the cell plasma membrane faces severe perils, including lipid phase separation, solute effects, and osmotic stresses associated with ice crystallization. How the initial biophysical properties of the plasma membrane can be modulated before cryopreservation in order to influence cellular resistance to the freeze-thaw stress is addressed in this study. Rainbow trout (Oncorhynchus mykiss) spermatozoa were chosen because the lack of an acrosome in this species suppresses potential interactions of cryopreservation with capacitation. Methyl-beta cyclodextrin-induced modulation of membrane cholesterol revealed the presence of a significant cholesterol exchangeable pool in the trout sperm plasma membrane, as membrane cholesterol content could be halved or doubled with respect to the basic composition of the cell without impairing fresh sperm motility and fertilizing ability. Biophysical properties of the sperm plasma membrane were affected by cholesterol changes: membrane resistance to a hypo-osmotic stress increased linearly with membrane cholesterol whereas membrane fluidity, assessed with DPH (1,6-diphenyl-1,3,5-hexatriene) and with several spin-labeled analogues of membrane lipids, decreased. Phosphatidyl serine translocation between the bilayers was slowed at high cholesterol content. The increased cohesion of fresh trout sperm plasma membrane as cholesterol increased did not improve the fertilizing ability of frozen-thawed sperm whereas the lowest cholesterol contents impaired this parameter of sperm quality. Our study demonstrated that cholesterol induced a stabilization of the plasma membrane in rainbow trout spermatozoa, but this stabilization before cryopreservation brought no improvement to the poor freezability of this cell.     

Evaluation of oxidative DNA damage promoted by storage in sperm from sex-reversed rainbow trout

Short-term storage and cryopreservation of sperm are two common procedures in aquaculture, used for routine practices in artificial insemination reproduction and gene banking, respectively. Nevertheless, both procedures cause injuries affecting sperm motility, viability, cell structure and DNA stability, which diminish reproductive success. DNA modification is considered extremely important, especially when sperm storage is carried out with gene banking purposes. DNA damage caused by sperm storage is not well characterized and previous studies have reported simple and double strand breaks that have been attributed to oxidative events promoted by the generation of free radicals during storage.The objective of this study was to reveal DNA fragmentation and to explore the presence of oxidized bases that could be produced by oxidative events during short-term storage and cryopreservation in sex-reversed rainbow trout (Oncorhynchus mykiss) spermatozoa. Sperm from six males was analyzed separately. Different aliquots of the samples were stored 2 h (fresh) or 5 days at 4 °C or were cryopreserved. Then spermatozoa were analyzed using the Comet assay, as well as combining this method with digestion with two endonucleases from Escherichia coli (Endonuclease III, that cut in oxidized cytosines, and FPG, cutting in oxidized guanosines). Both storage procedures yielded DNA fragmentation, but only short-term storage oxidative events were clearly detected, showing that oxidative processes affect guanosines rather than cytosines. Cryopreservation increases DNA fragmentation but the presence of oxidized bases was not noticed, suggesting that mechanisms other than oxidative stress could be involved in DNA fragmentation promoted by freezing.     

Changes in sperm parameters of sex-reversed female rainbow trout during spawning season in relation to sperm parameters of normal males

The production of all-female populations has important economic benefits in commercial rainbow trout aquaculture. The procedure commonly implemented to produce all-female stocks centers on the sex reversal of rainbow trout females via the administration of androgens in the early developmental stages, followed by the egg fertilization of normal females with semen from sex-reversed females (srf). However, there is no information regarding the quality of semen from srf rainbow trout throughout the spawning season. This information is critical because the quality of srf semen is highly variable. The aim of the study was to determine the changes in the semen parameters of srf rainbow trout throughout the duration of the spawning season. Sperm concentration, sperm motility parameters, and the biochemical parameters of seminal plasma (protein concentration, antitrypsin activity, osmolality, and lactate dehydrogenase activity) from srf were monitored during the spawning season and compared with normal male rainbow trout. The observed values of sperm, protein concentration, antitrypsin activity, osmolality, and lactate dehydrogenase activity of seminal plasma were all higher in comparison with normal males. Semen from srf was therefore characterized by a lower sperm motility during each period of the spawning season, in comparison with normal males, approximately 1.8, 1.5, and 1.7 times, respectively for the beginning, middle, and end of the spawning season. The percentage of sperm motility from srf and normal males were affected by the spawning season in the same way, as the highest values in the middle of the spawning season demonstrate (60% and 91% for srf and normal males, respectively). Spermatozoa of srf are characterized by a lower speed and a more curvilinear trajectory of movement as compared with that of normal males. The patterns of changes during the spawning season in sperm concentration, sperm motility parameters, as well as osmolality, and lactate dehydrogenase activity of the seminal plasma of srf were different in comparison with normal males. Our results could be important for fish breeders in regard to the spawning control of srf rainbow trout, as well as for the development of short- and long-term sperm storage procedures.     

Trehalose improves rabbit sperm quality during cryopreservation

High levels of reactive oxygen species are associated with spermatozoa cryopreservation, which bring damage to functional spermatozoa. The aim of the present study was to investigate whether and how the freezing extenders supplemented with trehalose was beneficial for the survival of rabbit spermatozoa. semen was diluted with Tris-citrate-glucose extender addition of different concentrations of trehalose. Addition of 100 mM trehaose significantly improved post-thaw rabbit sperm parameters, such as motility, acrosome integriy, membrane integrity and mitochondrial membrane potential. Moreover, when freezing extenders supplemented with trehalose, activities of catalase (CAT), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) of post-thaw spermatozoa were enhanced, meanwhile, reactive oxygen species (ROS) level and Malondialdehyde (MDA) content were decreased. The results suggest that freezing extenders supplemented with 100 mM trehalose resulted in less ROS level and MDA content, higher motility and mitochondrial membrane potential as well as the integrity of acrosome and plasma membrane. Supplementation of trehalose with freezing extenders is beneficial to the rabbit breeding industry.     

Evaluation of DNA damage in Dicentrarchus labrax sperm following cryopreservation

In this paper, DNA laddering analysis and single-cell gel electrophoresis (SCGE) or Comet assay, were used to detect DNA damage in response to a cryopreservation process in sea bass spermatozoa. The results obtained demonstrate that the cryopreservation protocol used to cryopreserve the sea bass sperm cause significantly damage at DNA level. In fact, the degree of DNA damage in frozen-thawed sperm (%DNAT=38.2+/-11.2, MT=498.9+/-166.4, n=3) was different (P<0.01) from that measured in fresh sperm (%DNAT=32.7+/-11.1, MT=375.2+/-190.7, n=3). Data here reported also demonstrated the fundamental role played by cryoprotectants (BSA and Me2SO) in reducing fish sperm DNA fragmentation. Finally, from our results, the ability of SCGE to reveal DNA fragmentation in fish sperm is also confirmed.     

Semen from rainbow trout produced using cryopreserved spermatozoa is more suitable for cryopreservation

Oocytes from three female rainbow trout Oncorhynchus mykiss were inseminated separately with untreated or cryopreserved semen, which had been produced using either untreated (three males) or cryopreserved (three males) spermatozoa. In half of variants, the cryopreservation did not significantly affect fertilization efficiency. Regardless of whether the sperm donors were produced from cryopreserved or intact semen, insemination of oocytes with their intact sperm resulted in the same percentage of eyed embryos (94.4 and 94.3%, respectively). When eggs were inseminated with cryopreserved semen, the use of sperm from males produced with cryopreserved spermatozoa resulted in a significantly higher percentage of eyed eggs than in case of donors produced with intact sperm (89.6 and 81.7%, respectively). The production of rainbow trout using cryopreserved sperm does not appear to negatively affect reproductive abilities of male progeny and semen from donors, which were produced using cryopreserved sperm, is more suitable for cryopreservation than the semen from donors produced with intactspermatozoa.     

Protective effects of iodixanol during bovine sperm cryopreservation

The aim of cryopreservation is to maintain cellular integrity, thereby enabling resumption of proper biological functioning after thawing. Here we propose OptiPrep™ (60% iodixanol in water) as a protectant during sperm cryopreservation using pooled bull semen as the model. We evaluated OptiPrep concentration effect and its relation to cryopreservation by comparing frozen-thawed and chilled samples. Semen, extended in Andromed^® with 0 (control), 1.25%, 2.5%, and 5% OptiPrep™, was compared after either chilling or freezing in large volume by directional freezing. Sample evaluation included sperm motility upon thawing and after 3 h incubation at 37 °C for frozen-thawed samples and after 3 h and 6 h of chilling for chilled samples; viability, acrosomal integrity, and hypoosmotic swelling were also tested for frozen-thawed and chilled samples. Chilled samples with 5% OptiPrep™ showed inferior viability (P = 0.047) and 3 h motility (P = 0.017) relative to that for chilled samples with 2.5% OptiPrep and inferior viability (P = 0.042), acrosomal integrity (P = 0.045), and 0 h motility (P = 0.024) relative to that for chilled samples with 1.25% OptiPrep. The 1.25%, 2.5%, and control samples did not differ. In frozen-thawed samples, 2.5% OptiPrep was superior to all other concentrations for 3 h motility (control, P = 0.007; 5% OptiPrep, P = 0.005; 1.25% OptiPrep, P = 0.004) and to 1.25% OptiPrep for acrosomal integrity (P = 0.001). In a search for a protection mechanism, we measured glass transition temperature (T_g) of Andromed^® and of Andromed^® with 1.25%, 2.5%, and 5% OptiPrep™. Andromed^® (-58.78 °C) and 1.25% OptiPrep™ (-58.75 °C) groups had lower mean T_g than that of the 2.5% (-57.67 °C) and the 5% (-57.10 °C) groups. Directional cryomicroscopy revealed that the presence of iodixanol alters ice crystal formation into an intricate net of dendrites. Thus, iodixanol appears to possess cryoprotective properties by helping spermatozoa maintain motility and membrane integrity, possibly through altering ice crystals formation into a more hospitable environment and increasing the glass transition temperature.     

Effect of external cryoprotectants as membrane stabilizers on cryopreserved rainbow trout sperm

The process of freezing and thawing induces certain cellular damage in rainbow trout (Oncorhynchus mykiss) spermatozoa. We have previously demonstrated that after freezing and thawing decreased fertility in rainbow trout (Oncorhynchus mykiss) spermatozoa, is related to sublethal damage to the plasma membrane. External cryoprotectants are known to stabilize the sperm cell membrane against such damage. In the current study, we used a basic freezing extender containing #6 Erdahl and Graham and 7% DMSO and added egg yolk, BSA, and a soybean-protein complex (DanPro S760) singly and in various combinations. To assess the effect of these cryoprotectants we evaluated the percentage of cells with progressive motility, permeability of cells to propidium iodide (viability) after exposure for 30 sec, 2, 5, 10 and 15 min. to hypo- and isoosmotic solutions of 10 and 300 mOsm, and the in vitro fertility rate. Fertility trials were performed using 1.87 x 10(7) spermatozoa/egg. Some of the tested stabilizers increased motility, increased viability, or reduced cell fragility after freezing and thawing. Nevertheless these quality improvements demonstrated by the "in vitro" tests do not always correlate with high fertility. The best membrane protection in terms of resistance to hypoosmotic shock was achieved when BSA and egg yolk were added to the extender. The highest fertility rates were obtained with DanPro S760 alone or in combination with BSA; the use of BSA with egg yolk did not improve this parameter. Our results demonstrated that some external cryoprotectants effectively increased membrane resistance during freezing and thawing, but some of the tested mixtures interfered with fertilization. Soybean protein concentrate provided good protection and increased fertility rates in cryopreserved trout spermatozoa.     

UV-irradiation of rainbow trout sperm as a practical method for induced gynogenesis

The goal of the experiment was to refine a simple and practical method for inducing gynogenesis in rainbow trout. To eliminate the male genome, UV-irradiation was used in combination with seminal fluid dilution and continuous stirring. Sperm motility was controlled. A visible recessive marker (yellow colour) as well as the Hertwig Effect were applied to assess the outcome of induced gynogenesis. Sperm irradiation by UV (energy output 2075 μW/cm²) for more than 5 minutes and 10 minutes in dilutions of seminal fluid of 1:40 and 1:20, respectively, resulted in yellow coloured feeding fry in the diploidized groups, whereas non-viable larvae were produced in the haploid groups. The highest survival rate for gynogenomes (from insemination until first-feeding fry) was 19 %.     

The effect of two cryopreservation methods on human sperm DNA damage

Several methods are currently available for selection when conducting sperm cryopreservation, however, these methods might cause different degrees of damage on sperm DNA. The aim of the this study is to compare the effects of storage at −80 °C (in ultra-low temperature refrigerator) and at −196 °C (in liquid nitrogen) on sperm DNA damage, thus to provide a reference for choosing the right method according to different aims. We randomly collected 28 semen samples from college students of Chongqing city. The samples stored at −80 °C were neat semen samples and the samples stored at −196 °C were mixed with additional cryoprotectants. Each sample was subjected to two freezing-thawing cycles, and the sperm DNA damage levels of fresh and thawed samples were measured by single cell gel electrophoresis (SCGE) and sperm chromatin structure assay (SCSA). Both SCGE and SCSA assays showed cryopreservation induced significant damage to sperm DNA. However, storage at −196 °C lead to more severe damage to sperm DNA than storage at −80 °C measured by SCSA. Sperm DNA damage increased simultaneously with the higher frequency of freezing-thawing cycles. We concluded that storage of neat semen samples at −80 °C had milder damage to sperm DNA than storage at −196 °C mixed with cryoprotectants. To avoid additional sperm DNA damage, repeated freezing and thawing should be prevented.     

No transgenic rainbow trout produced with sperm incubated with linear DNA.

We attempted to produce transgenic rainbow trout embryos by fertilizing eggs with sperm incubated with linearized plasmids. One experiment was conducted with the construct pBGH7 in the medium MMSF, with or without DMSO, at 2 concentrations of sperm cells and a relatively low concentration of DNA. The DNA was also in contact with the eggs during insemination and during the first minutes of egg activation. The second experiment was conducted with the construct CMVCAT in the medium MMSF, at 2 concentrations of sperm cells and a much higher concentration of DNA. The DNA was also present during the insemination. DNA analyses and dosages of CAT activity did not permit detection of any transgenic fry. However, one result suggests that sperm cells can capture part of the linear DNA in teh conditions tested.     

Sublethal copper exposure induces respiratory stress in common and gibel carp but not in rainbow trout

Rainbow trout, common carp, and gibel carp were exposed to sublethal Cu levels (1.0 or 1.7 microM) for 1 week. In rainbow trout, arterial oxygen tension (P(aO(2))) remained normal and there was no indication of anaerobic metabolism. P(aO(2)) was considerably lower in common and gibel carp and Cu exposure decreased this further. The decrease was transient for common carp but persistent in gibel carp and coincided with an elevation in arterial carbon dioxide tension (P(aCO(2))) indicating that all gas exchange was compromised in both cyprinid species. The disturbed gas exchange resulted in acidosis, which was respiratory and metabolic for common carp but mainly respiratory for gibel carp. Gibel carp produced ethanol as end product of their alternative anaerobic pathway. The hypothesis that hypertrophy and hyperplasia, resulting in increased diffusion distances, are reducing P(aO(2)) appeared invalid. Hypoventilation seems a more likely cause. Ionoregulatory parameters responded more uniform among species. Fast and pronounced decreases in plasma sodium and chloride developed for all three species, independent of the observed gill damage. Rainbow trout lost 20% of their plasma Na in the first 3 days, while common and gibel carp had only lost 13 and 16% respectively at that time. This difference might be crucial when challenged with Cu exposure and allow a fish to survive the first shock phase and supports it the hypothesis that sodium turnover is a key factor in predicting Cu toxicity.     

Evaluation of DNA damage in rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata) cryopreserved sperm

Cryopreservation causes several types of damage to spermatozoa, such as loss of plasma membrane integrity and functionality, loss of motility, and ATP content, resulting in decrease of fertility rates. This spermatozoal damage has been widely investigated for several marine and freshwater fish species. However, not much attention has been paid to the nuclear DNA. The objective of this study was to determine the degree to which cryopreservation induces spermatozoal DNA damage in two commercially cultured species, rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), both of which could benefit from the development of cryopreservation strategies on a large scale. We have used the single-cell gel electrophoresis, commonly known as Comet assay to detect strand breaks in DNA. This technique was performed on fresh and cryopreserved sperm from both species. In rainbow trout there was a significant increase in the averages of fragmented DNA and Olive tail moment after cryopreservation (11.19-30.29% tail DNA and 13.4-53.48% Olive tail moment in fresh and cryopreserved sperm, respectively), as well as in the proportion of cells with a high percentage of DNA fragmentation. For gilthead sea bream there were no significant differences in the percentage of tail DNA between the control samples and sperm diluted 1:6 and cryopreserved (28.23 and 31.3% DNA(t), respectively). However, an increase in the sperm dilution rate produced an increase in the percentage of DNA fragmentation (41.4%). Our study demonstrates that cryopreservation can induce DNA damage in these species, and that this fact should be taken into account in the evaluation of freezing/thawing protocols, especially when sperm cryopreservation will be used for gene bank purposes.     

Sublethal ammonia and urea concentrations inhibit rainbow trout (Oncorhynchus mykiss) erythrocyte glucose-6-phosphate dehydrogenase

In vitro and in vivo effects of sublethal ammonia and urea concentrations were assayed on glucose-6-phosphate dehydrogenase (G6PD) of rainbow trout (Oncorhynchus mykiss) erythrocyte. G6PD was purified from erythrocytes with a specific activity of 16.7 EU (mmol NADP+/min)/mg protein and approximately 1600-fold in a yield of approximately 60% by ammonium sulphate precipitation and 2',5'-ADP Sepharose 4B affinity chromatography. The purity of the enzyme was confirmed using SDS polyacrylamide gel electrophoresis. Experiments with ammonia (2.2-5.5 microM) and urea (20-50 microM) showed the inhibitory effects on the enzyme, in vitro. Inhibition effects were determined in vitro by Lineweaver-Burk and regression graphs. The dissociation constant of the enzyme inhibitor complex (Ki) and 50% inhibitory values were 2.26+/-1.21 and 2.86+/-3.51 microM for ammonia and 18.69+/-6.75 and 23.77+/-4.58 microM for urea, respectively. In vivo studies in rainbow trout erythrocytes showed significant (p < 0.01) inhibition of G6PD by ammonia and urea. However, ammonia inhibited more than urea since there were significant differences between the final values of erythrocyte G6PD activities.     

Deep - freezing of rainbow trout Salmo gairdneri sperm at varying intervals after collection

Rainbow trout semen cryopreserved 0.33, 5, 60 and 360 min after collection gave postthaw fertilization rates of 71.9, 68.4, 65.5 and 49.1% of eyed eggs, respectively. The corresponding result obtained with chilled semen stored for 2 wk under oxygen was 96.9%. This suggests that the freezing of rainbow trout semen should be done immediately after collection.