No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Effect of cryopreservation on motility, DNA integrity and gene expression in grey mullet, Mugil cephalus sperm
Abstract
This study documents the effect of cryopreservation on motility, DNA integrity, and gene expression in Mugil cephalus sperm. Fresh sperm were cryopreserved using V2 extender (V2E) or 0.3 M glucose, each in combination with one of three cryoprotective agents (CPAs), i.e., 10 % of dimethylsulfoxide, ethylene glycol, or glycerol, all at once. After two different storage (7- vs 60- day) periods in liquid nitrogen, sperm samples were thawed. Single-cell gel electrophoresis was used to detect the DNA integrity. Heat shock proteins (HSPs), HSP70, HSP90 and glutathione peroxidase (GPx2) genes mRNA expression levels was documented using qRT-PCR. The results demonstrated that among 0.3 M glucose + CPAs combinations, EG recorded higher frozen-thawed motility 69 % (7- day) and 59 % (60- day). Similarly, in V2E + CPAs combinations, EG recorded higher frozen-thawed motility 31 % (7- day) and 26 % (60- day). The DNA integrity of all thawed sperm (both periods) did not differ from that of fresh sperm. The qRT-PCR results revealed that in the combination of 0.3 M glucose + CPAs, the level of HSP90 and GPx2 gene expression was found to be upregulated in frozen-thawed sperm on both periods. Whereas, the expression level of the HSP70 gene was down-regulated. On the contrary, in the combination of V2E + CPAs, the expression levels of HSP70, HSP90 and GPx2 genes could not be detected on both periods. Overall, the findings of this study demonstrate that the cryomedium (extender + cryoprotectant) has a more influential role in the motility and levels of gene expression in the frozen-thawed sperm of M. cephalus.
... FBS also provides nutritional support and promotes cell adhesion [48]. Consequently, MET and FBS are more suitable for preserving high bioactivity and cell integrity in sensitive biological samples at high concentrations, whereas other cryoprotectants may be less effective in providing added protection at elevated concentrations [49]. Studies show that 10% methanol as a cryoprotectant resulted in the highest post-thaw motility and longest swimming duration for zebrafish (Danio rerio) sperm [50]. ...
In cryopreservation technology, the choice of cryoprotectant plays a crucial role in cell survival and function. Different types of cryoprotectants, each with unique protective mechanisms, mitigate cellular damage from ice crystal formation during freezing. This study investigated the effects of different types and concentrations of cryoprotectants on the cryopreservation efficacy of noble scallop Mimachlamys nobilis sperm. Six cryoprotectants were tested, including four permeable cryoprotectants (dimethyl sulfoxide (DMSO), ethylene glycerol (EG), propylene glycerol (PG), methanol (MET)) and two non-permeable cryoprotectants (trehalose (TRE), fetal bovine serum (FBS)). The results showed that permeable cryoprotectants, which penetrate the cell membrane, regulate the osmotic pressure inside and outside cells to reduce dehydration damage. Among them, 10% DMSO provided the best protection, significantly preserving sperm motility, velocity, and morphology. Non-permeable cryoprotectants, although unable to penetrate cells, stabilized the extracellular environment at higher concentrations (such as FBS). Additionally, MET and FBS exhibited enhanced protective effects with increasing concentration, indicating their potential in reducing sperm structural damage at higher concentrations. Morphological observations indicated that freezing caused varying degrees of structural damage to sperm, with flagellar integrity being crucial for motility. Overall, selecting an appropriate cryoprotectant and concentration is essential for the efficient cryopreservation of M. nobilis sperm, providing a valuable reference for conserving germplasm resources of marine species.
This study aimed to produce market-sized Pacific abalone, Haliotis discus hannai progeny from cryopreserved sperm (PCS) and assess their mRNA stability. Embryonic development in PCS was moderately delayed until the veliger larval stage compared to control progeny (CP), although both groups exhibited similar morphological structures. Shell width (SW) and shell height (SH) in PCS and CP remained consistent throughout the experimental periods. However, the shell length (SL) and body weight (BW) of PCS were significantly lower than those of CP during the early developmental stages (up to 9 months). After this period, PCS and CP exhibited comparable body traits at 18 months and 3 years. At 9 months, the mRNA expression patterns of growth-associated genes (Hdh-HGAP, Hdh-IGFBP7, Hdh-MSTN), shell-forming genes (Hdh-PRLSTN, and Hdh-SMP5), immune regulatory genes (Hdh-CAT, Hdh-GR, Hdh-GPx, Hdh-Caspase 3, Hdh-BAX, and Hdh-BCL2) were similar between PCS and CP. However, Hdh-MGF, Hdh-SOD, Hdh-IL17, and Hdh-NF-kB exhibited significantly lower mRNA expression, whereas Hdh-MIRP3 showed significantly higher expression in PCS compared to CP at 9 months. Furthermore, the expression patterns of all examined genes were similar in both PCS and CP at 3 years. In summary, PCS initially demonstrated lower growth traits (SL, BW) and impaired mRNA stability until 9 months; however, these traits normalized over time, resulting in comparable growth traits and gene expression to CP by 3 years. These findings suggest that cryopreserved sperm can effectively produce viable offspring with normal growth traits and mRNA stability for vital growth (Hdh-MGF, Hdh-HGAP, Hdh-IGFBP7, Hdh-MIRP3 and Hdh-MSTN), shell formation (Hdh-SMP5 and Hdh-PRLSTN), reproduction (Hdh-GnRH, Hdh-GnRH-R, Hdh-APGWamide and Hdh-5HT), and immune (Hdh-SOD, Hdh-CAT, Hdh-GR, Hdh-GPx, Hdh-BCL2, Hdh-BAX, Hdh-Caspase 3, Hdh-IL17 and Hdh-NF-kB) associated genes.
To date, little attention has been paid to identifying the effects of long-term cryopreservation on sperm quality for Piaractus orinoquensis. The object of this study was therefore to evaluate the effect of long-term cryopreser-vation (24 h, 1, 6 and 12 months) on sperm motility, viability, DNA integrity, ATP content, total antioxidant capacity (TAC), morphology and sperm ultrastructure in this species. Milt samples from six males were cry-opreserved in a medium containing final concentrations of 7.5% Me 2 SO, 4.1% glucose and 9.0% egg yolk. The samples were frozen in liquid nitrogen (LN) vapor and stored in LN for periods of 24 h and 1, 6 and 12 months. After thawing, both the motility rate and the viability decreased significantly compared with fresh sperm; however, these parameters did not differ among the four cryopreservation times. The DNA integrity and ATP content decreased significantly after 6 months of cryopreservation. There were no significant differences in TAC values between fresh and cryopreserved sperm. The total sperm abnormalities in cryopreserved samples were about 5-fold higher than in fresh sperm; short tail was the most common defect occurring after cryostorage. The ultrastructural analysis reveals that P. orinoquensis spermatozoa consist of an ovoid head without acrosome, a cylindrical mid-piece, and a single flagellum. The nuclear fossa is located at the base of the nucleus and contains the centriolar complex. There are 1-2 ring-shaped mitochondria located in the mid-piece. The flagellum shows a 9 + 2 organization of microtubules in the axoneme. Post-thaw spermatozoa presented damage such as swelling and rupture of the plasma membrane, mitochondrial damage, loss of the electron-dense chromatin of the nucleus , and degeneration in the middle region.
Despite the relatively long history of captive breeding, the Arctic charr still exhibits a generally low, but highly variable reproductive performance in aquaculture. A recent publication exposed potential paternal factors influencing the reproductive outcome of Arctic charr broodstock from the Swedish breeding program. Interestingly, the paternal factor appeared to be more closely connected to embryo survival than to fertilisation rates. This lead to speculations on whether e.g. chromatin related issues, potentially related to oxidative stress could be involved. In order to investigate this hypothesis the present study assessed the levels of DNA fragmentation, using the SCD-method, and membrane integrity, using flow cytometry, in sperm of farmed Arctic charr. Moreover, the existence of associations was tested between DNA fragmentation and membrane integrity in individual semen samples and viability of their resulting progeny. We found high levels of DNA fragmentation in sperm from the Arctic charr sires, ranging from 24% to 86% with a median of 67%. Membrane integrity values were high, with individual levels of 93.1% to 99.6% viable sperm cells, median 98.8%. DNA fragmentation and membrane integrity values were moderately correlated (r = 0.304, p < 0.05). Fertilisation rates and proportions of eyed eggs showed substantial individual variation and were correlated (r = 0.497, p < 0.05). However, large differences between proportion of eyed eggs and fertilisation rate, median 52% and 81.6% respectively, highlight that the main loss occurred due to embryo mortality rather than failed fertilisation. No correlation was found between either DNA fragmentation or membrane integrity and the resulting reproductive outcome (fertilisation rate and eyed eggs) of the individual Arctic charr sires. Overall, our study identified very high levels of DNA fragmentation, which could influence the fertility of the broodstock in question and thereby be a mitigating mechanism involved in the low reproductive success most often observed in farmed Arctic charr. Further exploration of this relationship would be needed, though.
Mugil cephalus has been identified as a suitable species for feeding populations in developing countries, and it represents a traditionally harvested and consumed species in various European countries. A decline in wild populations and a growing demand have promoted new interest in mullet production in hatcheries, considered imperative for the production of a steady supply of fry and the expansion of M. cephalus aquaculture. In the present study, wild adults collected in Sardinian lagoons were induced to spawn during their natural reproductive period by treating them with a single dose of a slow release GnRHa preparation (200 µg kg bw-1). In 2016, a protocol for reducing stress in capturing, transporting and selecting wild adults was set up, and 64% of the females with an oocyte diameter larger than 550 µm were successfully induced to spawn. Five spawning induction trials were carried out in the period 2016-2019. The mean fertilization rate and the mean hatching rate obtained were 83 ± 14% and 76 ± 13%, respectively. The individuals obtained were reared indoor in a recirculating aquaculture system (RAS) at a density of 40 individuals L-1 for 60 days, and their survival ranged from 10 ± 2 to 19 ± 1%. During the whole larval rearing phase, from 0 to 34 days post hatching (dph), the mean standard growth rates recorded were 5.0 ± 0.7% in total length and 14.4 ± 3.9% in body weight. The rearing density used resulted in an average survival rate comparable or even better than those obtained by other authors, thus indicating the possibility of producing M. cephalus fries in captivity from sexually mature adults captured in Sardinian lagoons, reducing the costs and optimizing the production.
Pacific abalone, Haliotis discus hannai, is a high commercial seafood in South-East Asia. The aim of the present study was to determine effects of cryopreservation on gene expression and post thaw sperm quality of Pacific abalone. Two ions, Na⁺ (459.1 ± 3.1 mM) and Cl– (515.9 ± 1.1 mM), were predominant in the seminal plasma (pH: 6.8 ± 0.1; osmolarity: 1,126 ± 3 mOsmL–1). Cryopreservation reduced mRNA expression levels of protein kinase A (PKA-C) and heat shock proteins (HSP70 and HSP90) genes in sperm. Fluorescent technique was used to compare morphological defects, acrosome integrity (AI), plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), and DNA integrity of sperm cryopreserved with five different cryopreservation solutions (8% Me2SO, 8% EG, 6% PG, 2% GLY, and 2% MeOH). Droplet in tail and coiled tail defects was not observed for sperm cryopreserved with 8% Me2SO or 2% GLY. Sperm cryopreserved with 8% Me2SO showed improved DNA integrity and lower cryodamage than sperm cryopreserved with other cryoprotectants. Sperm to egg ratio of 10,000:1 was found to be the most suitable ratio for in vitro fertilization among different ratios tested. The fertilization rate of sperm cryopreserved with 8% Me2SO was not significantly (p > 0.05) different from that of sperm cryopreserved with 2% GLY. DNA fragmentation showed strongly negative relationships with sperm quality parameters. Sperm cryopreserved with 8% Me2SO showed higher post thaw quality and mRNA expression of sperm motility associated gene than those cryopreserved with other cryoprotectants. The present research suggests to use 8% Me2SO for cryopreservation of Pacific abalone sperm as well as for hatchery production.
Maintenance of cellular redox control is pivotal for normal cellular functions and cell fate decisions including cell death. Among the key cellular redox systems in mammals, the glutathione peroxidase (GPX) family of proteins is the largest conferring multifaceted functions and affecting virtually all cellular processes. The endoplasmic reticulum (ER)-resident GPXs, designated as GPX7 and GPX8, are the most recently added members of this family of enzymes. Recent studies have provided exciting insights how both enzymes support critical processes of the ER including oxidative protein folding, maintenance of ER redox control by eliminating H 2 O 2 , and preventing palmitic acid-induced lipotoxicity. Consequently, numerous pathological conditions, such as neurodegeneration, cancer and metabolic diseases have been linked with altered GPX7 and GPX8 expression. Studies in mice have demonstrated that loss of GPX7 leads to increased differentiation of preadipocytes, increased tumorigenesis and shortened lifespan. By contrast, GPX8 deficiency in mice results in enhanced caspase-4/11 activation and increased endotoxic shock in colitis model. With the increasing recognition that both types of enzymes are dysregulated in various tumor entities in man, we deem a review of the emerging roles played by GPX7 and GPX8 in health and disease development timely and appropriate.
For several years Tunisia has opted to breed freshwater fish in reservoirs and artificial lakes, created for irrigation, as a strategy of providing high quality aquatic protein to the interior regions and providing work opportunities for local communities. The aim of this study was to summarize the main results accumulated by the last 20 year efforts made by the members of the National Institute for Marine Science and Technology (INSTM)-Aquaculture Laboratory to develop thicklip (Chelon labrosus) and flathead (Mugil cephalus) grey mullets fry production from captive broodstocks intended for stocking inland reservoirs (artificial lakes) and grow-out purposes Administration of human chorionic gonadotropin (hCG) at a priming dose of 10.000 IU kg⁻¹ female bw, followed by 10.000 IU hCG and (100 or 200 μg kg⁻¹ female bw of thicklip or flathead grey mullet respectively) as resolving dose resulted at the highest egg production for both species. Mean fecundity was 494.655 eggs kg⁻¹ bw for thicklip grey mullet and 418.945 eggs kg⁻¹ bw for flathead grey mullet. Fertilization rate of eggs produced was high (mean rate 83 % and 63 % for thicklip and flathead grey mullet respectively) and larvae hatched from those eggs at high rates (77 % and 88 %, respectively). The green method was more efficient than clear method to produce thicklip and flathead grey mullet fry, (i.e larvae had higher growth rate and better survival rates). The results of this study demonstrate the possibility to control reproduction and produce grey mullet fry in captivity. However, further research is needed to optimize the production protocol of eggs and fry of both grey mullet species.
The brown-marbled grouper, Epinephelus fuscoguttatus, is a highly commercial fishery and mariculture species listed as Near Threatened by IUCN. Adequate supply of good quality seed, at present, has become a bottleneck for grouper farming due to the rapid expansion of aquaculture practices. As sperm cryopreservation is of great potential not only for fish farming but also for species conservation, a series of sequential experiments were carried out to determine optimum cryoprotectant, freezing condition, dilution ratio, equilibration time, and thawing temperature for freezing brown-marbled grouper sperm. Based on post-thaw motility, 10% DMSO combined with 0.3 M glucose shown to be an effective cryomedium among the cryoprotectants evaluated here, and sperm was better cryopreserved by freezing 1–5 cm above the liquid nitrogen (LN) surface and cooled below − 80 °C before plunging into LN. Significantly higher post-thaw motility was observed for dilution ratios ranging from 4:1 to 1:3 than at higher dilution ratios (1:5, 1:9, and 1:19). Besides, equilibrated and post-thaw sperm motility was not affected by a 2-h equilibration period, indicating a high level of toxicity tolerance for 10% DMSO. Further evaluation of thawing temperatures showed that 30 °C and 40 °C yielded significantly higher post-thaw motility than at 25 °C, 50 °C, or 60 °C. In addition, brown-marbled grouper sperm could be refrigerated up to 48 h without decreasing its suitability for cryopreservation. Compared with fresh sperm, a decrease in motility and curvilinear velocity (VCL) was detected in thawed sperm. However, the thawed sperm could be stored up to 2 h at 4 °C without losing its fertilizing capacity, which was equal to that of fresh sperm (fertilization rate, 92.7 ± 2.5%; hatching rate, 87.7 ± 2.1%). The cryopreservation protocol developed in this study is efficient and will facilitate seed production for both aquaculture and conservation purposes.
This study was designed to optimize the semen freezing protocol of the native Mediterranean brown trout inhabiting the Molise rivers through two experiments: an in vitro analysis of the effects of two basic extenders combined with three cryoprotectants on post-thaw semen quality; and an in vivo test to assess the fertilization and hatching rate. Semen was diluted at a ratio of 1:3 in a freezing medium composed of a glucose extender (A) or mineral extender (B). Each basic component contained 10% dimethylsulfoxide, dimethylacetamide or methanol. The post-semen quality was evaluated considering motility, duration of motility, viability and DNA integrity. The basic extender and cryoprotectant were shown to have significant effects on these variables, and the best results were obtained using extender A or B combined with dimethylsulfoxide (P < 0.05). These freezing protocols were selected for fertilization trials in vivo. Fertilization and hatching rates were significantly higher in fresh semen. No significant differences were observed in frozen semen using extender A or B, although higher percentages of eyed eggs and hatching rates were recorded using extender A. According to our in vitro and in vivo results, the glucose-based extender and dimethylsulfoxide emerged as the best combination for an effective cryopreservation protocol for semen of this trout.
Temperature is an abiotic factor that affects various biological and physiological processes in fish. Temperature stress is known to increase the production of reactive oxygen species (ROS) that subsequently cause oxidative stress. Fish is known to evolve a system of antioxidant enzymes to reduce ROS toxicology. Glutathione peroxidase (GPx) family consists of key enzymes that protect fish from oxidative stress. In this study, full-length GPx1 cDNA (GenBank accession no. KY984468) of Tor tambroides was cloned and characterized by rapid amplification of cDNA ends (RACE). The 899-base-pair (bp) GPx1 cDNA includes a 576-bp open reading frame encoding for 191 amino acids, plus 28 bp of 5′-untranslated region (UTR) and 295 bp of 3′-UTR. Homology analysis revealed that GPx1 of T tambroides (Tor-GPx1) shared high similarity with GPx1 sequences of other fish species. The phylogenetic construction based on the amino acid sequence showed that Tor-GPx1 formed a clade with GPx1 sequences of various fish species. Real-time polymerase chain reaction (PCR) was performed to assess the levels of GPx1 gene expression in the liver and muscle of T tambroides under thermal stress. The results indicated that GPx1 gene expression was down-regulated under decreased temperature. However, there was no significant difference between GPx1 gene expression in fish exposed to high temperature and control. Our study provides the first data regarding GPx gene expression in T tambroides under thermal stress.
The effects of osmolality and pH of the seawater and non-ionic media (glucose) on sperm activation in Mugil cephalus was evaluated. The effect of cryopreservation was documented by cryopreserving the sperm diluted with a cryomedium (V2 extender + 10% dimethylsulfoxide) in a programmable freezer. The highest motility grade (4) or (3) was recorded when sperm were activated with seawater with osmolality above 600 (mOsmol/ kg) and pH 6 to 9. Significant difference (P < 0.05) was found in the duration of sperm motility with pH 7 (316 ± 8 s) followed by pH 6 and pH 8.2. In non-ionic media, the highest motility grade (4) and maximum duration of sperm motility (152 ± 23 s) was recorded when sperm activation carried out with 800 mM of glucose. The frozen-thawed sperm registered a motility grade of 2.44 ± 0.72. Frozen-thawed spermatozoa revealed changes in ultrastructure like damage of plasma membrane around the sperm head, shrinkage and swelling of the mid-piece region, partial fragmentation, and complete loss of flagellum when observed under electron microscope. However, comet assay indicated a non-significant (P > 0.05) DNA damage in frozen-thawed spermatozoa (3.68 ± 2.69% of tail DNA) compared to fresh spermatozoa (3.01 ± 2.13% of tail DNA). Overall , the sperm activation experiments indicated that sperm of M. cephalus can be activated by a media having osmolality above 600 (mOsmol/kg) and pH ranging from 6 to 9. Although DNA damage is minimal in frozen-thawed spermatozoa, the ultrastructural changes are prominent. Therefore, further experiments are required for the modifications of the composition of cryomedium to minimize the cryodamage.
We investigated various factors, including cryoprotective agents (CPAs), diluents, and freezing rates, to develop an optimal cryopreservation protocol for Epinephelus akaara sperm. In experiments using 10% dimethyl sulfoxide (DMSO), glycerol, and methanol with various diluents, 10% DMSO and 300 mM glucose yielded the highest post-thaw sperm motility. The combination of 10% glycerol and 300 mM sucrose yielded significantly higher post-thaw sperm motility than did combinations using other diluents. Glycerol and DMSO at a concentration of 10% as CPAs with 300 mM glucose as the diluent resulted in the highest MSR and sperm activity index (SAI). An investigation to determine the effects of glycerol and DMSO concentrations on post-thaw sperm survival rate revealed no significant differences among 5, 10, 15, and 20% concentrations of either CPA. In assessing the effects of CPA concentration on the fertilization rate, the 10% concentration yielded the highest fertilization rate (81.4 ± 4.3%) in DMSO, whereas 15% was the optimal concentration for glycerol (fertilization rate = 66.7 ± 6.1%). The hatching rate was also highest in 10% DMSO (40.1 ± 0.4%) and in 15% glycerol (27.8 ± 2.3%). In conclusion, the optimal rates of post-thaw sperm motility, fertilization, and hatching were achieved when E. akaara sperm were cryopreserved in a diluent of 300 mM glucose with 10% DMSO as the CPA at a freezing rate of -5 °C/min. We therefore recommend this protocol for the cryopreservation of E. akaara sperm.
Cryopreservation of Senegalese sole sperm can represent an alternative to overcome some reproductive problems of this species. However, it is important to guarantee the safe use of cryopreserved sperm by selecting an appropriate protocol according to a high demand quality need to be ensured. It has been demonstrated that traditional assays such as motility and viability do not provide enough information to identify specific damage caused by cryopreservation process (freezing and thawing). Specific tests, including lipid peroxidation and DNA damage, should be performed. In the present study, motility and lipid peroxidation were performed as specific tests allowing us to discard cryopreservation conditions such as methanol as internal cryoprotectant and bovine serum albumin as external cryoprotectant. In addition, a caspase 3/7 detection by flow cytometry was performed to analyze apoptosis activity in the best selected conditions. Moreover, new highly sensitive tests based on transcript number detection have recently been described in fish sperm cryopreservation. For this reason, a transcript level detection assay was performed on certain oxidative and chaperone genes related to fertilization ability and embryo development (hsp70, hsp90BB, hsp90AA, gpx) to select the best cryopreservation conditions. DMSO+ egg yolk proved to be the best cryoprotectant combination in terms of transcript level. This study describes an optimized cryopreservation protocol for Solea senegalensis sperm demonstrating for the first time that transcript degradation is the most sensitive predictor of cell status in this species after cryopreservation.
Cryopreservation techniques allow the long-term storage of a wide variety of biological materials without significant deterioration in quality. Immediate post-thaw survival is most often used to assess the effect of the freeze-thaw process on cells. However, this parameter provides no information on possible subtle effects of cryopreservation, including DNA damage, and alteration of mRNA levels and protein function that may not be evident immediately post thaw. These potential adverse effects do not necessarily result in cell death. While there are many comprehensive reviews of gamete and embryo cryopreservation in vertebrate species, we review here the publications relating to the impact of cryopreservation on the genome of sperm, embryos and oocytes.
Sperm chromatin in mammals is packaged in different blocks associated to protamines (PDNA), histones (HDNA), or nuclear matrix proteins. Differential packaging has been related to early or late transcription and also to differential susceptibility to genotoxic damage. Genes located in the more accessible HDNA could be more susceptible to injuries than those located in PDNA, being potential biomarkers of paternal DNA damage. Fish sperm chromatin organization is much diversified, some species lacking protamines and some others totally depleted of histones. Analyzing genotoxic damage in a species homogeneously compacted with some sperm nuclear basic protein type, could help in deciphering the clues of differential susceptibility to damage. In the present study we analyzed in rainbow trout the differential susceptibility of nine genes to UV irradiation and H2O2 treatment. The absence of histones in the sperm nuclei was confirmed by Western blot. The chromatin fractionation in sensitive and resistant regions to PvuII (presumably HDNA-like and PDNA-like, respectively) revealed that the nine genes locate in the same resistant region. The number of lesions promoted was quantified using a qPCR approach. Location of 8-hydroxyguanosine (8-OHdG) was analyzed by immunocytochemistry and confocal microscopy. UV irradiation promoted similar number of lesions in all the analyzed genes and a homogenous distribution of 8-OHdG within the nuclei. 8-OHdG was located in the peripheral area of the nucleus after H2O2 treatment, which promoted a significantly higher number of lesions in developmental-related genes (8.76-10.95 lesions/10 kb) than in rDNA genes (1.05-1.67 lesions/10 kb). We showed for the first time, that differential susceptibility to damage is dependent on the genotoxic mechanism and relies on positional differences between genes. Sensitive genes were also analyzed in cryopreserved sperm showing a lower number of lesions than the previous treatments and a predominant peripheral distribution of oxidative damage (8-OHdG).
Glutathione peroxidase (GPx; EC 1.11.1.9) is an important family of enzymes that protects organisms from oxidative damage. Four full-length GPx cDNAs were cloned and characterized by rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) from the liver of gilthead sea bream (Sparus aurata), an economically important species for Mediterranean aquaculture. Structural and functional annotations were performed for all paralogs, which suggested possible differences in function and subcellular localization. The phylogenetic analysis, based on the amino acid sequences, revealed four groups corresponding to teleostean GPx1a, GPx1b, GPx4a, GPx4b and three groups for mammalian GPx1, GPx2 and GPx4. The tree topology indicated past duplication events for fish genes, unlike their mammalian homologs. Transcriptional analysis in ten tissues by reverse transcription quantitative polymerase chain reaction (RT-qPCR) evidenced a tissue-specific pattern for each GPx homolog. Fish experimental groups were exposed to stress factors such as fasting and confinement. Relative expression analysis in fish liver demonstrated that GPx1 genes were not regulated by dietary restriction; GPx4b was differentially expressed opposed to regularly fed fish. On the other hand, both GPx1 and GPx4 genes were up-regulated in fish post exposed to confinement, considered as a response to acute stress. The results underline the role of GPx genes as indicators of stress and welfare status in gilthead sea bream aquaculture.
Striped mullet Mugil cephalus is an economically important species to commercial and recreational fishermen, as well as an ecologically significant detritivore linking lower trophic levels with a wide variety of estuarine and marine fish and birds. Despite this importance, striped mullet migration patterns and mortality rates are poorly understood. Approximately 15,000 striped mullet were tagged in North Carolina between 1997 and 2001, and monthly movement information was collected on recovered individuals (n = 384) from commercial and recreational fishermen and state agency personnel. A tag return model was used to estimate an instantaneous total mortality rate, and this rate was partitioned into natural and fishing components by means of life history methods. Nearly all (98.2%) striped mullet were recovered in North Carolina, the remaining few being recovered in nearby states. Striped mullet moved southward of their tagging locations and had the highest daily movement rate between the months of August and November. Movements corresponded to the months when spawning migrations are thought to occur in North Carolina. A smaller but substantial proportion of fish were recovered north of their tagging locations in late fall. Instantaneous total mortality rates of 2.12 and 1.71 were estimated using the two most parsimonious models for individuals larger than 300 mm fork length, comparing well with preliminary estimates from an independent statistical catch-at-age model. Concomitant holding tank experiments suggested that tag retention and posttagging survival, two central assumptions of the tag return model, were extremely high for adult striped mullet. These results will be incorporated into the North Carolina striped mullet fishery management plan currently in development.
In this study, we optimized a simple method of cryopreservation for Mugil cephalus sperm based on post-thaw motility and viability. A series of experiments were conducted by changing the extender, cryoprotectant and freezing height above the liquid nitrogen (LN) surface. First, we carried out the cryopreservation using the extender V2E and cryoprotective agents (CPAs) namely, propylene glycol (PG), methanol (MeOH), glycerol (GLY), ethylene glycol (EG), dimethylsulfoxide (Me2SO) and dimethylacetamide (DMA) at a final concentration of 5% and 10%. We found that 10% of GLY, EG and Me2SO were more suitable compared to other CPAs. Then, different freezing heights (6, 8, 10 and 12 cm) above the LN surface were experimented with extender V2E and optimized CPAs. Then, 0.3 M of glucose, sucrose and trehalose were tested as extender along with optimized CPAs and freezing height. Additionally, the effect of fast-rate freezing and storage days (7, 30 and 180) on post-thaw sperm quality was documented using the factors optimized in earlier experiments. For all experiments, the fresh sperm was diluted at a ratio of 1:1 with cryomedium (CPA + extender), loaded into cryovials (2.0 mL) and frozen. The cryopreserved sperm was thawed at 30 °C for 90-120 s and their quality was evaluated. Among the experimented factors, sperm diluted in cryomedium (0.3 M glucose + 10% EG) and frozen at 4 cm above the LN surface registered significantly (P < 0.05) highest post-thaw motility (73 ± 2%) and (71 ± 1%) viability. Fast-rate freezing has resulted in lower (about 30%) post-thaw motility and viability of sperm. The storage days (7, 30 and 180) did not have a significant effect on post-thaw sperm quality. Overall results show that using the factors optimized through this study, high-quality sperm can be obtained after cryopreservation.
The climbing perch, Anabas testudineus is a freshwater fish that has economic value in Indonesia. It is cultured in the country, but the breeding technology, specifically sperm storage, is not well developed. Sperm cryopreservation is one of the preservation methods that need to be developed to support fish breeding technology. The type of cryoprotectants and its concentration are species-dependent and determines the success of this approach. Therefore, this study is aimed at determining the optimal type and concentration of cryoprotectant for sperm cryopreservation of A. testudineus. Four separate study series were performed, each of which evaluated one type of cryoprotectant at five concentration levels. The cryoprotectants used were DMSO, methanol, glycerol, and ethanol, and the tested concentrations were 0%, 5%, 10%, 15%, and 20%, which were combined with 5% egg yolks. Each treatment was conducted with three replications. The results showed that the type of cryoprotectant and its concentration significantly affected sperm motility, viability, and fertility of climbing perch (P < 0.05). The best outcome was obtained in DMSO, and methanol at a concentration of 10%, glycerol at 5%, and ethanol at 15%. However, the highest motility, viability, and fertility values were observed at 10% DMSO, indicating it is the best type and concentration for sperm cryopreservation of climbing perch A. testudineus.
The feeding habits and effect of the diameter of pelleted feeds on the feeding responses of wild juvenile and adult flathead grey mullet (Mugil cephalus) in captivity were examined. Optimal pellet size for feeding was defined according to the behavioural responses and ingestion of pellets with different diameters (2, 4, 6, 8 mm) that were dropped into the tank in a random sequence. Larger pellets (6 and 8 mm) were more attractive (lower reaction time, high percentage of capture), but the small to medium-sized pellets (2 and 4 mm) were consumed the most. The optimal size was the 2- and 4-mm pellet diameter for juvenile individuals (365.50 ± 36.90 g; 28.8 ± 0.84 cm) and the 4-mm diameter pellet for adults (937.49 ± 146.54 g; 40 ± 1.12 cm). The preferred feeding area of adult mullet was also studied to estimate preference in relation to pellet characteristics such as floating or sink. Two pellet types, floating or sinking, were offered simultaneously in the water column: at the surface, mid-water column and bottom of the tank. The flathead grey mullet had a preference to feed in the mid-water column and the bottom of the tanks indicating that sinking or slow-sinking pellets would be the optimal feed type in relation to mullet feeding behaviour.
In fish hatcheries, sperm cryopreservation is a helpful tool for managing broodstock and artificial fertilization. However, cold shock can cause sperm injury resulting in decreasing sperm motility, low cell viability, structural damage to the plasma membrane and DNA fragmentation. This parameters are critical factor for the assessment of cryopreserved sperm for commercial scale application. In recent years, antioxidants have been increasingly useful for treating physiological issues in fish, including in reproduction and cryopreservation procedures. The purpose of this study was to examine the effects of supplementation of various antioxidants, including trehalose, reduced glutathione (GSH), Mito-TEMPO (MT), and rosmarinic acid (RA), during fish sperm cryopreservation on sperm motility, cell survival rate, and DNA damage. One-way ANOVA and Duncan's multiple range tests (p > 0.05) were used to compare the parameters. Sperm from adult fish were cryopreserved in 10% DMSO with 300 mM sucrose (1:1) supplemented with different concentrations of trehalose (0, 50, 100, 150, 200, and 250 mM), GSH (0, 2, 4, 6, 8, and 10 mM), MT (0, 25, 50, 75, 100, 125, 150, 175, and 200 μM), or RA (0, 25, 50, 75, and 100 μM). Compared with the control groups, antioxidant supplementation in sperm was more efficient in increasing post-thaw motility, maintaining cell survival rates, and inhibiting the increase in DNA damage that occurs during non-activated sperm during storage. The results demonstrated that the post-thaw motility of sperm was higher in 8 mM GSH (85.71 ± 0.04%), followed by 6 mM GSH (82.97 ± 1.30%), 75 and 25 μM RA (82.93 ± 0.93% and 82.76 ± 1.42%, respectively) and 100 mM trehalose (80.39 ± 0.61%) with not statistically different. The cell survival rate, VCL, and VSL of frozen–thawed sperm were higher in 100 mM trehalose resulted 84.22 ± 2.24%, 114.72 ± 2.00 μm/s, and 95.49 ± 0.70 μm/s, respectively. Mito-TEMPO at concentrations 100 μM resulted lower percentages of tail DNA (1.60 ± 0.08%) with not statistically different with 100 mM trehalose. In conclusion, the present study indicated that antioxidant 100 mM trehalose provided the most pronounced protective effect in improving frozen-thawed quality of frozen–thawed spotted halibut sperm.
Characterized by the extremely fast growth performance, giant grouper (Epinephelus lanceolatus) is one of the most promising marine culture species in South East Asia. Motivated by the rapid expansion in grouper aquaculture, giant grouper semen remains in increasing demand for massive seed production, but which is often constrained by the shortage of broodstock and asynchronous availability of gametes for the bulk of farmers. Sperm cryopreservation has a great potential for increased flexibility and efficiency for artificial seed production in aquaculture. To develop an efficient and standardized cryopreservation protocol for giant grouper semen, some key factors were investigated in this study, including the effects of different extenders (MPRS, E2, ELRS3), freezing heights (3, 5, 7 and 9 cm) above the liquid nitrogen surface corresponding to different freezing rates, final sperm concentration in the straw (1–10 × 10⁹ spermatozoa/ml), equilibration (0–180 min) and pre-freezing storage time (0–60 h). Moreover, the fertilizing ability of cryopreserved semen was tested at sperm:egg ratios ranging from 100:1 to 1× 10⁶:1. With 10% DMSO as cryoprotectant, the glucose-based extender ELRS3 at freezing height 5–7 cm produced higher post-thaw sperm motility. The optimal sperm concentrations in straws ranged from 2 to 8 × 10⁹ spermatozoa/ml. Besides, sperm motility after thawing was not affected by a 120-min equilibration, but a significant decrease was detected after 180 min of equilibration. Concerning pre-freezing storage, semen stored within 24 h after collection could be successfully cryopreserved, while extended storage time would compromise the cryopreserved sperm motility. Similar fertilization results were obtained at the range of 1000 to 1 × 10⁶ spermatozoa per egg for cryopreserved semen. Compared with fresh semen, the fertilization capacity of thawed sperm was not affected by 120 min of post-thaw storage, although with reduced motility parameters. This standardized procedure will be useful for the implementation of cryopreserved giant grouper semen in hatchery practice.
The giant grouper, Epinephelus lanceolatus, is one of the most valuable tropical commercial fishes. The asynchronous maturity periods between males and females hampers breeding programs. Sperm cryopreservation for gamete conservation is important for fish reproduction. We evaluated the effects of storage for 1, 3, 4, and 5 years on cryopreserved sperm of the giant grouper, E. lanceolatus, in 10% DMSO as a cryoprotectant with artificial seminal plasma and 300 mM sucrose as a diluent. Sperm motility, cell survival rate, DNA damage, fertilization rate, and hatching rate were investigated in this study. Post-thaw sperm motility peaked after 1 year of storage (63.27 ± 2.87%; p < 0.05), followed by 3 and 4 years (54.50 ± 4.33% and 54.40 ± 0.30%, respectively). Post-thaw sperm motility decreased after 5 years of storage. The cell survival rate after 1 year of storage (91.54 ± 3.02%) was similar to that of fresh sperm (98.12 ± 0.44%). The rate of DNA damage was unaffected by storage but significantly higher than that of fresh sperm. The fertility rate was significantly higher after storage for 1 and 3 years (98.06 ± 0.57% and 93.26 ± 1.73%, respectively). Samples stored for 1 and 3 years also resulted in high hatching rates (95.90 ± 1.53% and 88.73 ± 1.15%, respectively), which tended to decrease with increasing storage duration. Sperm cryopreserved for 4 years were of good quality in terms of motility and cell survival rate. However, storage for longer than 3 years decreased the fertility and hatching rates.
This study uses the CASA system and enzyme activity kit to detect movement parameters and enzyme activities of fresh and cryopreserved yellowfin seabream (Acanthopagrus latus) sperm, which could provide theoretical support for the protection of the germplasm resources of yellowfin seabream and the sustainable and healthy development of its aquacultural industry. The results showed that MOT, ALH, BCF and other indices were significantly reduced after cryopreservation (P < 0.05). VCL, VSL and WOB and others indicators were not significantly different (P > 0.05). CAT, SOD, CK and other enzyme activities of fresh sperm decreased significantly after cryopreservation (P < 0.05), and there was no significant difference in the enzyme activity of ATP and SDH (P > 0.05). After the second freezing treatment, the enzyme activities of SOD, T-GSH, ATP, CK and SDH of sperm were significantly reduced (P < 0.05), and there was no significant difference in the enzyme activities of CAT, GR, GSH-Px and LDH (P > 0.05). When the cryopreservation time was extended from 5 days to 20 days, the enzyme activities of CAT, SOD, ATP and CK in sperm tended to increase first and then decrease, while the enzyme activities of GSH-Px and LDH tended to remain stable first and then decreased. This study indicates that cryopreservation has some effect on sperm motility parameters and that the activity compared with fresh sperm is slightly reduced. Cryopreservation causes damage to the antioxidant system of sperm to some extent. It also reduces the antioxidant capacity of antioxidant enzymes and energy metabolizing enzymes to generate energy.
The effects of herbicide Roundup (based on glyphosate) on the embryonic development, survival and hatching of common carp (Cyprinus carpio L.) larvae and alteration in foxr1 and hsp70 gene expression were determined. The eggs (obtained from 6 females) were fertilised and incubated in water containing 0; 1 or 10 μl L⁻¹ of Roundup formulation. During early embryonic development (24 and 48 h post-fertilisation – hpf), Roundup caused a statistically important decrease in the embryonic survival rate of common carp. Moreover, retardation of the hatching rate was observed in the group treated with the higher concentration of Roundup at 81 to 99 hpf. At the end of the experiment (99 hpf), an important increase in number of deformed larvae was observed in both groups treated with Roundup in comparison to the control group (52.06; 16.02 and 5.08%, respectively). Significant differences in transcript of the gene foxr1 were found in Roundup-intoxicated groups in comparison to the controls. In the case of hsp70 transcripts, no important changes in exposed groups were observed.
These results showed that even small, environmentally relevant amount of Roundup present in the aquatic environment is able to affect the early life stages of common carp and change the transcripts of foxr1, which may have an adverse effect on the later proper development of the reproductive system.
Under intensive captive conditions, wild-caught flathead grey mullet (Mugil cephalus) females remained arrested in previtellogenic stages of gonadal development and no sperm could be obtained from males. With the aim to induce oogenesis from previtellogenesis to oocyte maturation, induce the release of sperm and obtain fertilized eggs, female and male flathead grey mullet were treated with Mugil cephalus single-chain recombinant gonadotropins (rGths), follicle-stimulating (rFsh) and luteinizing (rLh) hormones. In Experiment 1, fish were treated with a weekly dose of rFsh (15 μg kg⁻¹), which in females significantly (P < 0.001) increased plasma concentration of 17β-estradiol and induced vitellogenic oocyte growth up to a maximum mean diameter of 425 ± 19 μm after 9 weeks of treatment. In Experiment 2, fish were treated with weekly injections of both rFsh and rLh at different doses (from 2.5 to 12 μg kg⁻¹). Oocyte diameter reached 609 ± 5 μm, from which final oocyte maturation and ovulation was induced with 30 μg kg⁻¹ of rLh and 40 mg kg⁻¹ of progesterone. Good quality sperm (> 75% motile spermatozoa) was obtained from males in both experiments, and in Exp. 2 the addition of rLh induced the production of higher quantities of sperm that were used to fertilise the eggs. Although fertilisation was low (0.4%), these fertilized eggs with embryo development produced viable larvae (71% hatching). In comparison, control females remained arrested at previtellogenesis and control males did not produce sperm. The study demonstrated that both rGths are effective to induce the process of oogenesis in female flathead grey mullet and to obtain flowing sperm from males, adding more data to confirm the roles of the Gths in teleost gametogenesis. This is the first report, in a teleost species, of the use of rGths (rFsh and rLh) to induce oogenesis from previtellogenesis through to maturation to obtain eggs and larvae. This advance provides the bases for the development of therapies for the use in the aquaculture of teleost of commercial interest or the conservation of endangered species.
Success in captive reproduction and hatchery-based seed production is fundamental for the development of sustainable aquaculture of grey mullet in India. In the present study, the induced breeding and reproductive performance of two geographically different groups of grey mullet, Mugil cephalus sourced from east and west coasts of India were studied under captivity. Grey mullets sourced from east coast, Chennai, India (EP-E: eastern mullet population in situ; 782.4 ± 58.26 g) and west coast, Ernakulam, India (WP-E: western mullet population transferred to eastern location; 726.1 ± 31.09 g) were reared under uniform captive conditions at Muttukadu Experimental Station, ICAR-CIBA, Chennai. On the east coast, captive maturation and reproductive performance were studied for two years from October 2015 – January 2018 (EP-E and WP-E; N = 140). To accelerate reproductive maturation, female mullets were treated with intramuscular cholesterol pellets of gonadotropin-releasing hormone (GnRHa, 200 μg/fish) and males with silastic implants of 17-α-methyl testosterone (5 mg/fish). On the west coast, grey mullets (WP-W: western mullet population in situ; N = 130) were reared in earthen ponds under similar experimental conditions as EP-E and WP-E. Fourteen induced breeding trials were carried out in eastern population whereas 24 induced breeding trials were carried out in the western population. The percentages of mature fish initially and at 12 and 24 months for EP-E were 48.7, 57.5, and 50% whereas for WP-E, they were 0, 2.4, and 14.7% respectively. The maximum average oocyte diameter recorded for EP-E was 538 ± 6 μm and that for WP-E was 564 ± 21 μm. The peak reproductive periods of captive grey mullet on the east coast were November (EP-E) and December – January (WP-E). Induced breeding trials using EP-E were successful using females with an average oocyte size in the range of 520–535 μm from 6th – 20th November and the fertilisation rates ranged from 1 to 35%. The peak reproductive period of WP-W extended from June to August and successful induced breeding trials were conducted using females with average oocyte size of 540–570 μm, from 15th June – 20th July. The fertilisation percentage varied from 45 to 85%. Western mullet population exhibited reproductive dysfunctions when maintained under captivity on the east coast. The reproductive periods of captive grey mullets on the east and west coasts of India are asynchronous and provide the opportunity for two nonoverlapping breeding periods for captive reproduction of grey mullets in the region.
This study was performed as part of the effort to develop artificial seed production techniques for stone flounder Kareius bicoloratus, which has a great potential for farming in northeast Asian countries. To develop a sperm cryopreservation protocol that can be applied for artificial fertilization, we carried out experiments on the key factors for successful fish sperm cryopreservation, including cryoprotective agents (CPAs), diluents, equilibration times, and dilution ratios. The CPAs tested in this study were dimethyl sulfoxide (DMSO), glycerol, methanol, and ethylene glycol, and the diluents were glucose and sucrose. The equilibration times tested in the experiments ranged from 0 to 210 s and the dilution ratios ranged from 1:1 to 1:1000. According to the results of our experiments, the optimal CPA for stone flounder sperm cryopreservation was DMSO in concentrations ranging from 7.5% to 10% when 300 mM glucose was used as diluent and DMSO in concentrations ranging from 15% to 17.5% when 300 mM sucrose was used as diluent. Post-thaw sperm motility was high, with equilibration times up to 150 s, and dilution ratios up to 1:10 (semen:CPA + diluent) showed no significant differences in the effect on post-thaw sperm motility. The cryopreserved sperm showed more significant DNA damage than the fresh sperm with the severity of DNA damage being in inverse proportions to post-thaw sperm motility and survival rate. It is expected that these results will be useful for applications of artificial fertilization at stone flounder hatchery farms.
Immunity occupies the top priority in aquaculture. Great efforts have been made to study fish immunity, but little is known about the immunity of grey mullet, Mugil cephalus despite of its importance. We evaluated the effect of different doses (0, 1, 10 and 100 µg/kg body weight) of lipopolysaccharide (LPS) on the expression of non‐specific immune response genes (interleukin (il) 1β, il8, il10 and tumour necrosis factor alpha (tnfα)) in the liver, as well as its effect on the fractionation of serum protein in juvenile grey mullet along different times and concentrations. Analysis revealed that LPS initially caused an upregulation in the studied cytokines. After that, transcripts levels were switched sequentially between downregulation and upregulation, ending with an upregulation at 1 week post injection in il8, il10 and tnfα. Regarding il1β, its initial upregulation was followed by gradual decreases, which reached a marked downregulation at 1 week post injection. Protein fractionation exhibited many changes among doses and time. Nine fractions were discernable in protein electrophoresis of serum in all doses. The most pronounced changes were detected in fractions 7–9 (generally refer to γ globulins or immunoglobulins) using densitometric analysis. In addition, LPS suggested to modulate the levels of serum transferrin. It is known that LPS is a component of the cell wall of Gram negative bacteria, and this study sheds light on the molecular fitness mechanisms against bacterial infection, which could help in developing a strategy to improve resistance to bacterial diseases, or possibly to enhance immune response in grey mullet.
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.
We analyzed the effect of cryopreservation on the expression of glutathione peroxidase (GPX1) and glutathione reductase (GSR) genes in human sperm cells (15 sperm samples from fertile donors and 10 samples from infertile patients). The relative expression of GPX1 and GSR genes was determined by real-time PCR. The rate of post-thaw recovery was 2.1 times higher in the group of fertile donors. A significant increase in the expression of GPX1, but not GSR, was observed in sperm samples from infertile patients, while in patients with infertility, GPX1 expression significantly decreased after cryopreservation/thawing, in samples from fertile donors after the same procedure it increased to the level observed in the sperm samples from infertile patients. A positive correlation was revealed between GPX1 expression and sperm cryotolerance.
Sperm cryobanking is a common procedure in animal production, including aquaculture, and animal conservation. The accuracy of the method relies in the faithful preservation of the sperm quality in order to provide a healthy progeny. Cryopreservation is prone to promote DNA damage that could compromise this objective. As we demonstrated in a previous study, trout embryos obtained with sperm damaged with UV irradiation, can generate tolerant conditions to genotoxic damage that allow the progression of development. This tolerant scenario would increase the chances of affecting gene expression in the progeny. In order to check whether the DNA damage promoted by cryopreservation provides a similar progeny to those from fresh sperm, we have developed an in silico analysis of the transcriptomic profile of trout larvae, with apparently normal phenotype, obtained from fresh sperm or from sperm frozen using two procedures that rendered two levels of DNA damage. Data, deposited in the Gene Expression Omnibus database (GEO bank_Accession number GSE52217), were processed and the Panther online database was used for the functional and ontological study. We found differential expressed genes (DEGs) in those progenies from cryo-damaged sperm, which were mainly implied in metabolic processes and in other cellular processes such as differentiation, signalization, trafficking, etc. We have demonstrated that the effects of sperm DNA cryodamage go beyond from its fertilization ability and, particularly in fish, affect the transcriptomic profiling of the obtained larvae. Our results encourage to strictly evaluating the DNA integrity prior to the adoption of any cryopreservation sperm protocol, mainly in the aquaculture field.
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 ± 3% motility and 160 ± 14 μm/s curvilinear velocity at 15 s post-activation. The motility rate of cryopreserved sperm (37 ± 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.
This study developed the cryopreservation of brown-marbled grouper spermatozoa for practical application. We examined 32 cryodiluents, developed from four types of cryoprotectants [propylene glycol (PG), dimethyl-sulphoxide (Me2SO), dimethyl-acetamide (DMA) and ethylene glycol (EG)] at four concentrations of 5, 10, 15 and 20% in combination with two extenders [Fetal bovine serum (FBS) and artificial seminal plasma (ASP). Cooling rates were examined by adjusting the height of straws (2.5-12.5 cm) from the liquid nitrogen (LN) vapour and cooled for 5 min before immersion into LN. DNA laddering was used to detect DNA damage in cryopreserved sperm. In fertilization trials, 0.5 g of eggs was mixed with cryopreserved sperm stored for 30 days in LN. The best motility of post-thaw sperm was achieved using 15% PG + 85% FBS (76.7 ± 8.8%); 10% PG + 90% FBS was also effective as cryodiluent. Generally, FBS gave better post-thaw motility compared to ASP. The optimum cooling rate was at 17.6 °C min-1 obtained by freezing at the height of 7.5 cm surface of LN. The results obtained showed that cryopreserved sperm of brown-marbled grouper suffered slight DNA fragmentation, which resulted in significantly lower motility. However, the fertilization (90.9 ± 0.5%), hatching (64.5 ± 4.1%) and deformity rates (3.8 ± 0.2%) obtained from cryopreserved sperm showed no significant difference with fresh sperm. These finding show that the developed protocol for cryopreservation of brown-marbled grouper sperm was viable and will be useful for successful breeding and seed production of brown-marbled grouper.
The aim of this study was to document the effects of cryopreservation on sperm motility and viability in Grey mullet Mugil cephalus. Cryopreservation of sperm was attempted by using two extenders ringer solution for marine fish (RSMF) and V2 extender (V2E) and cryoprotectants dimethylacetamide (DMA), dimethylsulfoxide (DMSO), ethylene glycol (EG), glycerol (GLY), propylene glycol (PG) and methanol (MeOH). Cryoprotectants were assessed at different concentrations individually as well as in combination with varying equilibration times (10 and 30. min). For optimization of freezing rate, four freezing protocols (-5, -10, -20 and -30. °C/min) were evaluated. After achieving final temperature, samples were plunged in liquid nitrogen (-196. °C) and stored for a week. Samples were subsequently thawed in a water bath at 30. °C for assessment of sperm motility and viability. Results indicated that cryomedium constituting of V2E extender + 10% glycerol with a dilution ratio of 1:1 (sperm: cryomedium) at an equilibration time of 5 to- 10. min and freezing rate of -20. °C/min was more desirable compared with other factors that were assessed. Use of this protocol resulted in retaining the greatest sperm motility grade 3.0. ±. 0.0 (50%-80% sperm movement, fast swimming) and 48.19. ±. 3.12% of sperm viability. The results of the present study, therefore, provide base-line data for establishing a protocol for sperm cryopreservation in M.cephalus. Further studies are, however, required for optimization of most suitable sperm cryopreservation protocol.
Cryopreservation has been extensively used in various mega-industries and has recently been applied in genetic banking for conservation purposes. Compared with conventional cell preservation methods, cryopreservation can maintain the viability of cryopreserved cells for an indefinite time at a relatively lower cost and lesser manpower, with lower probabilities of contamination and genetic changes. This study presents the crucial role of sugar, a cryoprotectant supplement in cryopreservation. Sugar molecules typically interact with the lipid bilayer during the freezing phase to maintain plasma membrane integrity when cells undergo dehydration. When combined with other permeable cryoprotectants such as glucose with methanol and trehalose with dimethyl sulphoxide, sugar prolongs and enhances cellular post-thaw viability. Moreover, terrestrial and marine organisms have benefited from the inclusion of sugar in the cryopreservation protocol. For wide range of cells such as the sperm, oocytes, embryos and larvae of marine vertebrates and invertebrates, as well as marine algae, cryopreservation with sugar produced positive results compared with cryopreservation without sugar. Not all sugar is beneficial, and the type and concentration of sugar should be applied according to the species. Moreover, the freezing method may also affect the function of sugar. Nevertheless, understanding the role of sugar in cryobiology and conducting a preliminary trial of sugar for cryopreserving cells would benefit future research on cryopreservation.
... Thomas D Schmittgen 1 & Kenneth J Livak 2 . ABSTRACT. ... N. Engl. J . Med. ... 32, e178 (2004). | Article | PubMed | ChemPort |; Livak , KJ & Schmittgen , TD Analysis of relative gene expression data using real - time quantitative PCR and the 2 (- Delta Delta C(T)) Method . ...
... Thomas D Schmittgen 1 & Kenneth J Livak 2 . ABSTRACT. ... N. Engl. J . Med. ... 32, e178 (2004). | Article | PubMed | ChemPort |; Livak , KJ & Schmittgen , TD Analysis of relative gene expression data using real - time quantitative PCR and the 2 (- Delta Delta C(T)) Method . ...
... Thomas D Schmittgen 1 & Kenneth J Livak 2 . ABSTRACT. ... N. Engl. J . Med. ... 32, e178 (2004). | Article | PubMed | ChemPort |; Livak , KJ & Schmittgen , TD Analysis of relative gene expression data using real - time quantitative PCR and the 2 (- Delta Delta C(T)) Method . ...
Heat shock protein 70 (HSP70) is considered as a gene which affects semen quality traits. The present study attempted to investigate the relationship between the HSP70 expression level and motility of bull sperm during the process of freezing-thawing. Semen samples were collected from 5 QinChuan bulls by artificial vagina. Sperm motility and plasma membrane integrity of the semen samples at three stages (fresh, after equilibration, and frozen-thawed) were evaluated. The HSP70 expression level at the three stages was detected using real-time PCR. The results indicated that HSP70 expression level, membrane integrity, and sperm motility in the fresh semen were higher than those of the sperm after equilibration and freezing-thawing (P < 0.05), the HSP70 expression level, plasma membrane integrity, and sperm motility in sperm after equilibration were higher than those of the frozen-thawed sperm (P < 0.05). The correlation between HSP70 expression level and sperm motility was positive (ranging from 0.327 to 0.785). The results suggest that HSP70 expression level in bull spermatozoa was gradually decreased following the process of freezing-thawing, and might be associated with bull sperm motility. © 2015, Institute of Agricultural and Food Information. All rights reserved.
The correlation between the 90 kDa heat-shock protein (HSP90) and sperm quality following the process of freezing-thawing in bulls has not been studied clearly. Therefore, the objective of the present was to clarify the relationship between HSP90 level and semen parameters during the process of cryopreservation in bulls. Semen samples from 5 Holstein bulls were obtained by artificial vagina. Characteristics of these semen at three stages (fresh, after equilibration and frozen-thawed), including motility, plasma membrane integrity and acrosome integrity were evaluated. The mRNA expression level of HSP90 at the three stages was evaluated by using quantitative Real-Time PCR. Meanwhile, the protein level of HSP90 expression at the three stages was detected according to western blot. The results showed that sperm parameters evaluated in fresh semen was the highest in the three groups. Sperm parameters in semen after equilibration were lower than those in fresh semen (P > 0.05) and higher than those in post-thawed semen (P < 0.05). Sperm parameters in frozen-thawed semen were the lowest among the three groups (P < 0.05). This study indicated that HSP90 expression is proportional to sperm quality. HSP90 expression level in fresh semen was significantly higher than that in frozen-thawed semen (P < 0.05). Although no significant differences in HSP90 expression were observed between fresh semen and semen after equilibration (P > 0.05). Results in this sutdy suggest that HSP90 level in bull spermatozoa was gradually declined following the process of freezing-thawing, and might be associated with sperm motility, plasma membrane integrity and acrosome integrity.
The efficacy of spermatocrit (the ratio of packed sperm cell in semen after centrifugation) as an indicator of sperm density was tested in snowtrout (Schizothorax richardsonii). Semen samples were collected from 98 live ripe male brooders over two consecutive breeding seasons for the sperm density analysis. A significant, positive, linear relationship between spermatocrit and sperm density (measured with a haemocytometer) was established during regression analysis (r=0.817, Pb0.001), thus supporting the use of spermatocrit as a rapid and reliable estimator of sperm concentration in this species. A decrease in sperm density and spermatocrit was noticed with the advancement of breeding season.
Growth, survival, biomass production and body composition of striped grey mullet (Mugil cephalus L.) fingerlings were evaluated in two pond experimental trials as a function of two stocking densities and three management systems in brackishwater pond rearing. The three management systems tested were feeding, fertilization and combined fertilization-feeding. The Experiment 1 had a 2×2 factorial design with two levels of stocking density and two types of pond management system (feeding and fertilization) resulting in four treatments in triplicates: stocking density 1+feeding (S1F1), stocking density 1+fertilization (S1F2), stocking density 2+feeding (S2F1) and stocking density 2+fertilization (S2F2). Twelve ponds (600m2 each) were stocked with grey mullet fry (0.17±0.02 g/23.8±0.6 mm) at 7500 number ha−1 in treatments S1F1, and S1F2 and at 15,000 number ha−1 in treatments S2F1, and S2F2. In Experiment 2, three different pond management systems evaluated as three treatments (in triplicate ponds) were FR: fingerling rearing with fertilization; FD: fingerling rearing with feeding and FF: fingerling rearing with combination of fertilization+feeding. Nine ponds (600m2 each) under the three treatments were stocked with grey mullet fry (0.55±0.08 g/36.0±2.1 mm) at 15,000 number ha−1. In S1F1, S2F1, FD and FF, formulated feed containing 27.5% crude protein was provided at 3.5–20% body weight daily, whereas, in S1F2, S2F2, FR and FF systems, ponds were fertilized fortnightly with cattle manure, urea and single super phosphate at 500, 30 and 30 kg ha−1, respectively. In Experiment 1, although stocking density and application of feed or fertilization did not affect the growth parameters, survival and apparent feed conversion ratio (P>0.05) of grey mullet, coefficient of variation at harvest weight (CVhw) and total harvested biomass were influenced by feeding or fertilization (P<0.05). In Experiment 2, significantly better growth, lower CVhw and higher total harvested fish biomass were obtained in fishes of FF compared with that of FR and FD (P<0.05). From the pond trials it can be concluded that combined fertilization–feeding system with a stocking density of 15,000 fry ha−1 would be appropriate for production of striped grey mullet fingerlings in brackishwater ponds. The present findings indicate feasibility of establishing M. cephalus seed rearing practice using a cost-effective and resource-efficient approach.
Cryopreservation is a common procedure for long-term storage of sperm in animal reproduction and is also employed in the aquaculture industry. The freezing–thawing process originates reactive oxygen species (ROS) which cause serious damage at chromatin level. Analyzing and characterizing this type of damage, especially in specific DNA regions, could be particularly interesting for germplasm banking purposes. This type of study could provide information about damage in some regions of interest or specific genes with important roles in fertilization and embryo development. This is the first report on quantification of DNA lesions in specific genes after cryopreservation in any species. We performed a specific DNA assay based on the quantitative PCR (qPCR) approach to quantify lesions generated after sperm cryopreservation in two Sparus aurata nuclear genes with important roles in embryo development (Igf1 and Gh) and two mitochondrial genes (Cytb and CoI). We tested different cryopreservation protocols using DMSO as permeable cryoprotectant, with or without BSA supplementation. The maximum number of lesions per 10 kb observed after cryopreservation was 2.28 (for Igf1) and 0.8 (Gh), significantly lower than that detected for mitochondrial genes, 8.43 (Cytb) and 5.34 (CoI). A complementary analysis of DNA fragmentation (Comet assay) and telomere length measurement (qPCR) was done. The results demonstrated that the protocol used did not induce telomere shortening and that DNA integrity was very well preserved (%DNAt lower than 2.47 ± 0.19%). The results suggest that this protocol offers a high degree of DNA protection; however the qPCR assay demonstrated different vulnerability of the DNA regions to undergoing damage during freezing/thawing processes. This study demonstrates the usefulness of using qPCR approaches to study gene-specific damage after cryopreservation, which can be an excellent complement for traditional techniques.
During recent years, several studies have pointed out the importance of key paternal transcripts in early embryo development. Sperm cryopreservation is commonly applied in assisted reproductive technologies (ARTs) and it is important to know if it produces any relevant effect at this level. In this study, using normozoospermic donors, we present a candidate transcript approach in which we quantify the presence of sperm mRNAs considered as markers for male fertility and pregnancy success. Analyses were done using quantitative PCR. Our results demonstrated that the used cryopreservation protocol, which is routinely employed in clinical practice, alter transcripts considered as spermatozoa quality markers and markers for pregnancy success. Most of the studied transcripts considered as male quality markers (PRM1, PRM2, and PEG1/MEST) and one of studied mRNAs reported as markers of pregnancy success (ADD1) were reduced after cryopreservation. In order to check if vitrification protocols could reduce this alteration, another assay was carried out analyzing those transcripts with higher differences in the first study (PRM1 and PRM2). The results showed the same tendency. Although maternal mRNAs can compensate these deficiencies, these results could make advisable the optimization of freezing/thawing procedures.
Summary To date, there has been little improvement in cryopreservation of bull sperm due to lack of understanding of the freezing mechanisms. Therefore, this study set out to investigate expression levels of fertility-associated proteins in bull sperm, and in particular the relationship between the 90 kDa heat-shock protein (HSP90) and the sperm characteristics after freezing-thawing. Semen was collected from eight Holstein bulls by artificial vagina. Characteristics of these fresh semen, including sperm motility, morphology, viability and concentration, were evaluated. Sperm quality was also assessed after freezing-thawing. Eight ejaculates were divided into two groups based on freezing resistance and sperm motility. Sperm proteins were extracted and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and western blotting were performed. SDS-PAGE results showed that there was substantial diversity in 90 kDa proteins in the frozen-thawed sperm and HSP90 was confirmed as one of the 90 kDa proteins by western blot. This study indicated that HSP90 expression correlated positively with sperm quality. The amount of expressed 90 kDa proteins in the high freezing resistance (HFR) group was significantly higher than that in the low freezing resistance (LFR) group (P < 0.05). Thus, higher expression of HSP90 could probably lead to the higher motility and freezing resistance of sperm found after freezing-thawing. Therefore, we concluded that level of HSP90 expression could be used to predict reliably and simply the freezing resistance of bull sperm.
The Cantabrian brown bear survives as a small remnant population in northern Spain and semen cryopreservation for future artificial insemination is one of the measures being implemented for conservation of this species. As part of this program we investigated the value of adding heat shock protein A8 (HSPA8) to media (N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid-TRIS-fructose with 20% egg yolk) used for chilling and cryopreserving the spermatozoa. Semen samples from eight brown bears were obtained by electroejaculation during the breeding season. In experiment 1, we tested three concentrations of HSPA8 (0.5, 1, and 5 μg/mL) to determine whether sperm motility (computer assisted sperm analysis system) and sperm survival could be improved during refrigeration (5 °C) up to 48 hours. Results showed that sperm viability (test with propidium iodide) was improved by the addition of 0.5 and 5 μg/mL HSPA8. In experiment 2, HSPA8 was added to the cryopreservation media (6% final glycerol concentration) before the freezing process. Though there were no differences in sperm viability immediately after thawing (analyses to 0 hours), plasma membrane permeability (test with YO-PRO-1) was significantly lower by the presence of HSPA8 (1 μg/mL) and acrosomal damage (test with peanut agglutinin-fluorescein isothiocyanate conjugate) was reduced by higher concentrations of HSPA8 (1 and 5 μg/mL) (analyses after thermal stress test incubating over 2 hours to 37 °C). In experiment 3, results of a simple progression test carried out through artificial mucus (hyaluronic acid 4 mg/mL) showed a significant decrease in the total number of sperm able to swim a distance of 0.5 to 2 cm through a capillary tube for all HSPA8-based extenders. Nevertheless, the distance traveled by the vanguard spermatozoa, which represent a highly motile subpopulation, was restored by the inclusion of 1 and 5 μg/mL HSPA8 in the cryopreservation media. Thus, the HSPA8 addition to extender improves the quality of brown bear (Ursus arctos) sperm during chilling (viability) and after cryopreservation (number of sperm with damaged acrosomes and "apoptotic-like" changes).
Sperm cryopreservation could entail DNA damage, promoting base oxidization and strand breaks. In a previous work we showed that trout DNA damaged sperm is able to fertilize leading to embryo loss when the repair system of the oocyte is inhibited. Here we have analysed the later effects on embryo and larvae of fertilizing trout oocytes with cryopreserved DNA-damaged spermatozoa. Fish have weak sperm selection mechanisms, are very prolific and have external embryo development, being convenient models for this type of study. We cryopreserved rainbow trout semen using extenders containing egg yolk or their low density lipoprotein fraction to obtain samples with different degrees of DNA damage. DNA fragmentation was evaluated using the Comet assay and telomere length using quantitative-PCR. Fertilization trials were performed and the transcription at different developmental stages of telomerase reverse transcriptase (Tert) and eight genes related with embryo growth and development (Igf1, Igf2, Igfr1a, Igfr1b, Gh1, Gh2, Ins1 and Ins2) were analyzed using quantitative-PCR in surviving embryos and larvae. Results showed an increase in sperm DNA fragmentation after both cryopreservation procedures as well as a decrease in sperm telomere length. Larvae obtained with damaged sperm showed longer telomeres and Tert overexpression. The transcription of the analyzed genes in these embryos and larvae was also modified with respect to the control, most of them as an increase at hatch. We conclude that fertilization with cryopreserved DNA-damaged spermatozoa significantly affects offspring performance, detectable as an increase in telomere length as well as some alterations in gene expression in surviving embryo and larvae.
Heat shock proteins (HSPs), also known as stress proteins and extrinsic chaperones, are a suite of highly conserved proteins of varying molecular weight (c. 16-100 kDa) produced in all cellular organisms when they are exposed to stress. They develop following up-regulation of specific genes, whose transcription is mediated by the interaction of heat shock factors with heat shock elements in gene promoter regions. HSPs function as helper molecules or chaperones for all protein and lipid metabolic activities of the cell, and it is now recognized that the up-regulation in response to stress is universal to all cells and not restricted to heat stress. Thus, other stressors such as anoxia, ischaemia, toxins, protein degradation, hypoxia, acidosis and microbial damage will also lead to their up-regulation. They play a fundamental role in the regulation of normal protein synthesis within the cell. HSP families, such as HSP90 and HSP70, are critical to the folding and assembly of other cellular proteins and are also involved in regulation of kinetic partitioning between folding, translocation and aggregation within the cell. HSPs also have a wider role in relation to the function of the immune system, apoptosis and various facets of the inflammatory process. In aquatic animals, they have been shown to play an important role in health, in relation to the host response to environmental pollutants, to food toxins and in particular in the development of inflammation and the specific and non-specific immune responses to bacterial and viral infections in both finfish and shrimp. With the recent development of non-traumatic methods for enhancing HSP levels in fish and shrimp populations via heat, via provision of exogenous HSPs or by oral or water administration of HSP stimulants, they have also, in addition to the health effects, been demonstrated to be valuable in contributing to reducing trauma and physical stress in relation to husbandry events such as transportation and vaccination.