Motility and oxidative stress of common carp Cyprinus carpio sperm during short-term storage.

Short-term storage of semen is a simple and inexpensive procedure to deal with logistics of large-scale hatchery operations but can lead to oxidative stress and a significant decrease in sperm motility and velocity. To better understand the mechanisms responsible for the association of poor sperm quality with oxidative stress, in the present study we investigated the effect of refrigerated storage for 0, 24, 48, 72, and 144 h on sperm motility and curvilinear velocity and oxidant/antioxidant balance of common carp Cyprinus carpio. Percentage of motile sperm was significantly (p < 0.05) reduced stored for 72 h storage compared to that of fresh sperm. Sperm stored 144 h showed < 40% motility with an average velocity of 91.12 ± 10.4 µm s-1. A time-dependent increase in the level of the oxidative stress indices lipid peroxidation and carbonyl derivatives of proteins was observed. Increase (p > 0.05) in total superoxide dismutase was detected after 48 h, and glutathione reductase and glutathione peroxidase activity demonstrated a significant increase after 72 h. These results provide an additional tool for the development and improvement of short-term sperm preservation procedures commonly applied in aquaculture.

Seminal plasma fractions can protect common carp (Cyprinus carpio) sperm during cryopreservation.

This study aimed to investigate the effect of fractionated seminal plasma on characteristics of common carp Cyprinus carpio cryopreserved sperm. Nanosep® centrifugal devices yielded four seminal plasma fractions with different total protein content ranging in molecular weight from less than 17 to almost 74 kDa. Each protein fraction was added to semen extender medium prior to freezing. Spermatozoon motility characteristics and DNA integrity were analyzed in supplemented and non-supplemented cryopreserved samples. The cryopreservation process strongly affected the swim-up sperm quality. Treatment with fractions 1, 2, 3, and 4 was associated with significantly higher spermatozoon motility rate and curvilinear velocity than seen in extender only, with highest values obtained with fraction 4 (78.21 ± 2.41% and 168.05 ± 4.46 µm/s, respectively). Significantly less DNA damage, expressed as percent tail DNA (12.23 ± 1.27) and olive tail moment (0.68 ± 0.12), was recorded in fraction 4. The findings indicated that addition of fractionated seminal plasma to cryopreservation medium can preserve the quality of common carp sperm. The protective effect of each fraction varied, suggesting the presence of distinct components exerting different effects on cryopreserved sperm function.

Transferrin Identification in Sterlet (Acipenser ruthenus) Reproductive System.

Transferrins are a superfamily of iron-binding proteins and are recognized as multifunctional proteins. In the present study, transcriptomic and proteomic methods were used to identify transferrins in the reproductive organs and sperm of out-of-spawning and spermiating sterlet (Acipenser ruthenus) males. The results showed that seven transferrin transcripts were identified in the transcriptome of sterlet, and these transcripts were qualified as two different transferrin genes, serotransferrin and melanotransferrin, with several isoforms present for serotransferrin. The relative abundance of serotransferrin isoforms was higher in the kidneys and Wolffian ducts in the spermiating males compared to out-of-spawning males. In addition, transferrin was immunodetected in sterlet seminal plasma, but not in sterlet spermatozoa extract. Mass spectrometry identification of transferrin in seminal plasma but not in spermatozoa corroborates immunodetection. The identification of transferrin in the reproductive organs and seminal plasma of sterlet in this study provides the potential function of transferrin during sturgeon male reproduction.

Seminal plasma transferrin effects on cryopreserved common carp Cyprinus carpio sperm and comparison with bovine serum albumin and antifreeze proteins.

In the current study, there was evaluation of the cryopreservation effectiveness of common carp Cyprinus carpio sperm when cryopreservation medium was supplemented with proteins. Semen was diluted with Kurokura's extender composing 180 mM NaCl, 2.68 mM KCl, 1.36 mM CaCl2, 2.38 mM NaHCO3, and 10% dimethylsulfoxide (DMSO). Cryopreservation medium was supplemented with purified seminal plasma transferrin (Tf), bovine serum albumin (BSA) or antifreeze protein (AFP) Types I and III. Concentration of proteins evaluated was 0.1 µg/ml, 1 µg/ml, and 10 µg/ml. Motility and curvilinear velocity of spermatozoa was evaluated by the Computer Assisted Semen Analyzer (CASA). The percent of motile cells and spermatozoa curvilinear velocity of frozen-thawed sperm with supplementation of Tf and AFP III at all concentrations were greater compared to samples with no added proteins. The protective effect of BSA and AFP I was less and dose-dependent. Thus, it is concluded that incorporation of Tf in the extender before freezing improves crypreservation of common carp spermatozoa whereas supplementation with AFP III in greater concentrations was more effective.

Effects of antifreeze proteins on cryopreserved sterlet (Acipenser ruthenus) sperm motility variables and fertilization capacity.

The effect of antifreeze proteins on sterlet, Acipenser ruthenus sperm motility variables and fertilization rate were investigated after cryopreservation. Two types of antifreeze proteins (AFPI or AFPIII) were used at concentrations of 0.1, 1, 10 and 100 µg/mL. The motility variables of fresh and cryopreserved sperm with and without addition of antifreeze proteins were evaluated by the Computer Assisted Semen Analyzer (CASA). The fertilization rate using about 200,000 spermatozoa per egg was evaluated after 54 h incubation at 17 °C during the early stage of organogenesis. The motility, curvilinear velocity and straight-line velocity of fresh sperm was 93 ± 5%, 128 ± 13 µm/s and 89 ± 9 µm/s, respectively. There was a significant decrease of sperm motility rate between fresh sperm and cryopreserved sperm with/without addition of antifreeze proteins. The greatest motility among thawed samples was in the sperm cryopreserved with 10 µg/mL of AFPI (56 ± 20%), however, these data were not different compared to the sperm without antifreeze proteins (49 ± 14%). No statistical variations were detected in curvilinear velocity nor straight-line velocity. The fertilization rate with fresh sperm was 67 ± 7%. No significant differences were detected in fertilization rate between fresh and cryopreserved spermatozoa with/without addition of antifreeze proteins, except the sperm cryopreserved with 100 µg/mL of AFPIII (39 ± 14%). Thus, it is concluded that addition of antifreeze proteins to cryopreservation medium do not improve nor have toxicity effects on the quality and fertilization capacity of sterlet sperm after thawing.

Protective role of antifreeze proteins on sterlet (Acipenser ruthenus) sperm during cryopreservation.

The loss of sperm quality in sterlet (Acipenser ruthenus) due to freeze-thaw process in cryopreservation was investigated in the present study. Two antifreeze proteins (AFPI or AFPIII) were used at different concentrations of 0.1, 1, 10, and 100 µg/mL. We compared motility, curvilinear velocity, and plasma membrane integrity of fresh, cryopreserved sperm, and sperm cryopreserved in the presence of antifreeze proteins. Fresh sperm (control) had 85 ± 4% motility and 160 ± 2 µm/s curvilinear velocity, respectively. After cryopreservation, the motility of frozen-thawed sperm without addition of antifreeze proteins significantly decreased (44 ± 9%), compared to the control. The highest motility of frozen-thawed sperm was obtained in cryopreserved sperm with addition of 1 µg/mL of AFPIII (58 ± 14%). No significant differences were observed in curvilinear velocity between fresh sperm and cryopreserved sperm with/without addition of AFPI or AFPIII. The flow cytometry analysis revealed that fresh sperm contained 94.5 ± 6% live cells, while the cryopreserved sperm only contained 26.6 ± 14% live cells. Supplementation of antifreeze proteins has significantly improved the percentage of live cells in frozen-thawed sperm, except 0.1 µg/ml of AFPI group. No significant difference in percentage of live cells was detected in the sperm cryopreserved with 10 µg/mL of AFPI or AFPIII, compared to fresh sperm. Thus, addition of antifreeze proteins to cryopreservation medium could be considered to improve the post-thawed sperm quality of sterlet.

Impact of cryopreservation on sterlet, Acipenser ruthenus sperm motility and proteome.

Fish sperm cryopreservation is a well-established technique allowing for artificial insemination on a commercial scale. The extent of proteome alterations in seminal plasma and sperm due to cryopreservation, however, is not known. This study was conducted to evaluate the effect of cryopreservation on motility variables of sterlet Acipenser ruthenus sperm and to detect the differences in protein profiles of fresh and cryopreserved sterlet sperm and seminal plasma. Fresh sperm had 89 ±â€¯3% motility and 160 ±â€¯14 µm/s curvilinear velocity at 15 s post-activation. The motility rate of cryopreserved sperm (37 ±â€¯5%) was less at 15 s post-activation. No difference (ANOVA; P > 0.05) in mean curvilinear velocity of fresh and cryopreserved sperm was detected. The protein profiles of seminal plasma and sperm were characterized using comparative proteomics to determine the influence of cryopreservation. Six altered protein spots in seminal plasma and thirteen altered spots in sperm were detected in fresh and thawed sperm. Subsequent protein characterization suggested that the proteins identified were involved in sperm metabolism, cytoskeleton, and stress response. The results broaden the understanding of the effects of cryopreservation and identify the proteins associated with cryo-injury. These data may help to determine the function of altered proteins and provide new insights into improving sperm cryopreservation.

Protein phosphorylation in spermatozoa motility of Acipenser ruthenus and Cyprinus carpio.

Spermatozoa of externally fertilizing freshwater fish possess several different modes of motility activation. Spermatozoa of common carp (Cyprinus carpio L.) are activated by hypoosmolality, whereas spermatozoa of sterlet (Acipenser ruthenus) require Ca2+ and low concentration of K+ for motility activation. Intracellular signaling differs between these two species as well, particularly in terms of utilization of secondary messengers (cAMP and Ca2+), and kinase activities. The current study was performed in order to determine the importance of protein phosphorylation and protein kinases for activation of sperm motility in carp and sterlet. Treatment with kinase inhibitors indicates that protein kinases A and C (PKA and PKC) participate in spermatozoa motility of both species. Immunodetection of phospho-(Ser/Thr) PKA substrates shows that phosphorylated proteins are localized differently in spermatozoa of carp and sterlet. Strong phosphorylation of PKC substrate was observed in flagella of sterlet spermatozoa, whereas in carp sperm, PKC substrates were lightly phosphorylated in the midpiece and flagella. Motility activation induced either phosphorylation or dephosphorylation on serine, threonine and tyrosine residues of numerous proteins in carp and sterlet spermatozoa. Proteomic methods were used to identify proteins whose phosphorylation state changes upon the initiation of sperm motility. Numerous mitochondrial and glycolytic enzymes were identified in spermatozoa of both species, as well as axonemal proteins, heat shock proteins, septins and calcium-binding proteins. Our results contribute to an understanding of the roles of signaling molecules, protein kinases and protein phosphorylation in motility activation and regulation of two valuable fish species, C. carpio and A. ruthenus.

Progress and challenges of fish sperm vitrification: A mini review.

To survive low temperature is required for a long-term storage (cryopreservation), cells should be vitrified to a state in which intracellular water is solidified without ice crystal formation. Two different approaches are described for fish sperm cryopreservation: 1) sperm conventional cryopreservation, in which extracellular water is partially crystallized and 2) sperm vitrification, in which both intra- and extra-cellular liquids are vitrified. Sperm vitrification has been applied to some fish species with limited success. Traditional vitrification requires rapid cooling/warming rates, small sample carriers, and using high permeable cryoprotectant concentrations. The latter cause cytotoxic effects which must be well managed and will require continuous effort to match an appropriate cryoprotectant with suitable apparatus and warming methods. Novel cryoprotectant-free sperm vitrification approach has been applied to several fishes. This review summarizes development of basic procedures and discusses advantages and disadvantages of vitrification when applied it to fish sperm.

The in vitro effect of nonylphenol, propranolol, and diethylstilbestrol on quality parameters and oxidative stress in sterlet (Acipenser ruthenus) spermatozoa.

The sturgeon is a highly endangered fish mostly due to over-fishing, habitat destruction, and water pollution. Nonylphenol (NP), propranolol (PN), and diethylstilbestrol (DES) are multifunctional xenobiotic compounds used in a variety of commercial and industrial products. The mechanism by which these xenobiotic compounds interfere with fish reproduction is not fully elucidated. This study assessed the effect of NP, PN, and DES on motility parameters, membrane integrity, and oxidative/antioxidant status in sterlet Acispenser ruthenus spermatozoa. Spermatozoa were incubated with several concentrations of target substances for 1h. Motility rate and velocity of spermatozoa decreased in the presence of xenobiotics in a dose-dependent manner compared with controls. A significant decrease in membrane integrity was recorded with exposure to 5µM of NP, 25µM of PN, and 50µM of DES. After 1h exposure at higher tested concentrations NP (5-25µM), PN (25-100µM), and DES (50-200µM), oxidative stress was apparent, as reflected by significantly higher levels of protein and lipid oxidation and significantly greater superoxide dismutase activity. The results demonstrated that NP, PN, and DES can induce reactive oxygen species stress in fish spermatozoa, which could impair sperm quality and the antioxidant defence system and decrease the percentage of intact sperm cells.

Protein profile of seminal plasma and functionality of spermatozoa during the reproductive season in the common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss).

The purpose of the study was to analyze seminal plasma composition, sperm production, and sperm motility over the course of the spawning season in the common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss). The highest mean percent of motile sperm (carp, 98.3 ± 1.6%; rainbow trout, 92.8 ± 5.6%) and highest spermatozoon velocity (carp, 286.3 ± 7.8 µm sec-1 ; rainbow trout, 245.3 ± 8.3 µm sec-1 ) were observed in the middle phase of the spawning period, at 5 sec post-activation. Sperm volume and concentration increased in the early spawning period, then decreased significantly toward the end of the season in both species. Seminal plasma osmolality was 262-270 mOsmol kg-1 from carp and 232-248 mOsmol kg-1 from rainbow trout, with little variation over the spawning period. Protein concentration in carp seminal plasma was stable throughout the reproductive season, whereas the highest protein level (2.1 ± 0.3 mg mL-1 ) in rainbow trout was observed in the middle phase of the spawning period. Seminal plasma composition in both species was analyzed using two-dimensional polyacrylamide gel electrophoresis combined with mass spectrometry. Thirteen differentially expressed protein spots in carp seminal plasma and sixteen spots in rainbow trout seminal plasma varied over the course of the reproductive season. The unique proteins identified are involved in the regulation of spermatogenesis, sperm motility, maintenance of sperm membrane lipid stability, and antioxidant protection. These results provide a deeper understanding of the role of fish seminal plasma proteins as well as a list of novel markers of sperm quality. Mol. Reprod. Dev. 83: 968-982, 2016 © 2016 Wiley Periodicals, Inc.

The influence of cryoprotectants on sturgeon (Acipenser ruthenus) sperm quality, DNA integrity, antioxidant responses, and resistance to oxidative stress.

This study examined the effect of cryoprotectants on DNA integrity, antioxidant defense, and resistance to oxidative stress in cryopreserved sterlet Acipenser ruthenus sperm. The freeze-thaw process significantly influenced sperm motility, with significant differences among cryoprotectants. In vitro exposure of cryopreserved sperm to the xanthine-xanthine oxidase (X-XO) system as a model reactive oxygen species inducer resulted in a lesser motility rate and velocity compared to the control, and there was a decrease in these variables in a time- and dose-dependent manner. The greatest X (0.6mM)-XO (0.05U/mL) concentration and incubation period (30min) was associated with 62% DNA fragmentation in sperm cryopreserved with 10% ethylene glycol (EG). The maximum lipid peroxidation (LPO) and carbonyl derivatives of proteins (CP) was also observed in sperm cryopreserved with 10% EG and exposed to the X-XO system at a concentration of 0.6mM X-0.05U/mL XO. The frozen/thawed sperm containing 10% EG and that with 10% dimethyl sulfoxide (DMSO) had a significant enhancement of superoxide dismutase (SOD) and glutathione reductase (GR) activity. The current study confirms that EG is not effective for cryopreservation, and sterlet sperm were highly sensitive to free radicals after cryopreservation with EG.

The effect of reactive oxygen species on motility parameters, DNA integrity, tyrosine phosphorylation and phosphatase activity of common carp (Cyprinus carpio L.) spermatozoa.

The effect of reactive oxygen species production on the motility parameters, DNA integrity, acid phosphatase activity, and protein tyrosine phosphorylation in spermatozoa of the common carp (Cyprinus carpio) was investigated. Spermatozoa were exposed to different concentrations of xanthine and xanthine oxidase (X-XO) either in the presence or absence of antioxidants for 15 and 60 min. A dose- and time-dependent reduction in spermatozoa motility and velocity was observed. Comet assays showed a dramatic increase in DNA fragmentation after 15 min. Changes in tyrosine phosphorylation of spermatozoa proteins were observed by Western blotting with anti-phosphotyrosine antibodies, and proteins of interest were identified by mass spectrometry. After a 60 min exposure to X-XO, O-linked N-acetylglucosamine transferase, isoform 4 was phosphorylated and septin-8-A was dephosphorylated. Acid phosphatase activity also decreased in a dose-dependent manner after a 60 min exposure to oxidative stress. The results demonstrate that oxidative stress impaired functional variables (sperm motility, velocity, DNA integrity) of carp spermatozoa, and altered intracellular signalling pathways through changes in tyrosine phosphorylation and acid phosphatase activity.

Effects of tetrabrombisphenol A on DNA integrity, oxidative stress, and sterlet (Acipenser ruthenus) spermatozoa quality variables.

The sperm of sterlet (Acispenser ruthenus) was used to investigate the effect of the xenobiotic tetrabrombisphenol A (TBBPA) on sperm quality variables (ATP content, spermatozoa motility, and velocity), DNA integrity, and oxidative stress indices. Sperm was diluted to obtain a spermatozoa density of 5 × 10(8) cells/mL and exposed for 2 h to final concentrations of TBBPA (0.5, 1.75, 2.5, 5, and 10 µg/L). The oxidative stress indices, including lipid peroxidation, carbonyl derivatives of proteins, and antioxidant activity were significantly higher with increased concentrations of TBBPA. There was significantly less intracellular ATP in sperm samples at TBBPA concentrations of 2.5 µg/L and above. Spermatozoa velocity and percent motile sperm were significantly lower at each sampling time post-activation compared to controls. DNA damage expressed as percent DNA in Tail and Olive Tail moment was significantly higher with exposures ≥2.5 µg/L TBBPA. The results demonstrated that TBBPA and other xenobiotics can induce reactive oxygen species stress in fish spermatozoa, which could impair the sperm quality, DNA integrity, ATP content, and the antioxidant defense system. This study confirmed that fish spermatozoa can be used in in vitro assays for monitoring residual pollution in aquatic environments.