Suitability For Cryopreservation Research Articles

Characteristics of diluted-stored and post-thawed semen of Hutsul stallions.

The use of frozen semen lowers the risk of disease transmission, eliminates geographical limitations and supports the implementation of genetic resource protection programs. However, due to the very rare use of frozen semen from Hutsul stallions, their genetic material is not secured in sperm banks, and very little information is available about their semen, including its suitability for cryopreservation, and sperm survival rates after thawing. The aim of this study was to analyse basic parameters such as sperm motility, vitality and morphology in diluted-stored and post-thawed Hutsul semen, using a CASA system. There were no differences in sperm motility (P = 0.3372) or morphology between the groups, although the progressive motility was higher in thawed semen (P = 0.0151), while the sperm vitality was higher in diluted-stored semen (P = 0.00517). This study demonstrates that semen from Hutsul horses is suitable for cryopreservation, thus supporting the creation of a sperm bank as a genetic reserve for representatives of this breed.

Evaluate the activity of cryopreserved boar ejaculated spermatozoa under different thawing protocols

Aim. To evaluate the activity of cryopreserved boar ejaculated spermatozoa under different thawing protocols to optimize biotechnological approaches in pig reproduction based on the genetic material of the Institute of Animal Breeding and Genetics nd. a. M.V. Zubets of National Academy of Agrarian Science of Ukraine. Methods. Cryopreserved ejaculated boars' sperm was thawed in three different ways. Biotechnological, cryobiological and morphological methods were used to assess the viability of the sperm. Results. We noted the individual feature of semen quality of the studied boars, which affects its suitability for cryopreservation and quality indicators after thawing. It was found that in liquefied boar sperm (sperm activity on average is 86.7%) after its centrifugation and three-hour equilibration at + 4 °C sperm activity decreased by an average of 25.0%. In order to improve the quality of cryopreserved sperm, it was thawed under different conditions. The highest activity of spermatozoa in thawed sperm of boar No. 225 of Myrhorod pig was at the level of 25.0% at the thawing temperature + 70 °C, and the thermal resistance and heat resistance were 70.0% and 60.0%, respectively. Conclusions. During this study was noted that there are peculiarities of boar semen at breed and individual level, which influence its ability to tolerate cryopreservation. Keywords: sperm, in vitro cultivation, cryobank, cryopreservation, boars.

Cryopreservation of sperm in brown-marbled grouper (Epinephelus fuscoguttatus)

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.

Preservation strategies for decellularized pericardial scaffolds for off-the-shelf availability

Decellularized biological scaffolds hold great promise in cardiovascular surgery. In order to ensure off-the-shelf availability, routine use of decellularized scaffolds requires tissue banking. In this study, the suitability of cryopreservation, vitrification and freeze-drying for the preservation of decellularized bovine pericardial (DBP) scaffolds was evaluated. Cryopreservation was conducted using 10% DMSO and slow-rate freezing. Vitrification was performed using vitrification solution (VS83) and rapid cooling. Freeze-drying was done using a programmable freeze-dryer and sucrose as lyoprotectant. The impact of the preservation methods on the DBP extracellular matrix structure, integrity and composition was assessed using histology, biomechanical testing, spectroscopic and thermal analysis, and biochemistry. In addition, the cytocompatibility of the preserved scaffolds was also assessed. All preservation methods were found to be suitable to preserve the extracellular matrix structure and its components, with no apparent signs of collagen deterioration or denaturation, or loss of elastin and glycosaminoglycans. Biomechanical testing, however, showed that the cryopreserved DBP displayed a loss of extensibility compared to vitrified or freeze-dried scaffolds, which both displayed similar biomechanical behavior compared to non-preserved control scaffolds. In conclusion, cryopreservation altered the biomechanical behavior of the DBP scaffolds, which might lead to graft dysfunction in vivo. In contrast to cryopreservation and vitrification, freeze-drying is performed with non-toxic protective agents and does not require storage at ultra-low temperatures, thus allowing for a cost-effective and easy storage and transport. Due to these advantages, freeze-drying is a preferable method for the preservation of decellularized pericardium. Statement of SignificanceClinical use of DBP scaffolds for surgical reconstructions or substitutions requires development of a preservation technology that does not alter scaffold properties during long-term storage. Conclusive investigation on adverse impacts of the preservation methods on DBP matrix integrity is still missing. This work is aiming to close this gap by studying three potential preservation technologies, cryopreservation, vitrification and freeze-drying, in order to achieve the off-the-shelf availability of DBP patches for clinical application. Furthermore, it provides novel insights for dry-preservation of decellularized xenogeneic scaffolds that can be used in the routine clinical cardiovascular practice, allowing the surgeon the opportunity to choose an ideal implant matching with the needs of each patient.

Storage and transportation of Prochilodus lineatus (Characiformes) sperm prior to cryopreservation

In this study we compared post-thaw quality of P. lineatus sperm frozen shortly after collection, with sperm frozen after dilution and transportation, and up to 6h from collection. From each sperm sample (n=10 males) five aliquots were taken. One aliquot was diluted in the freezing medium (1 sperm:8 glucose:1 methyl glycol) and frozen ∼20min after collection in the field (control), while the other four aliquots were transported to the laboratory where freezing took place 3 or 6h after collection. From the transported aliquots, two were diluted 1:4 in glucose solution before transportation (diluted samples), while the other two were kept undiluted until freezing (undiluted samples). Thus the five treatments were: control, undiluted-3h, diluted-3h, undiluted-6h and diluted-6h. Post-thaw sperm was evaluated for membrane integrity, motility rate and velocities (curvilinear=VCL; average path=VAP; straight line=VSL). Post-thaw membrane integrity did not differ among the five treatments (48–60% intact sperm). Sperm motility rate was similar (P>0.05) between control (64%) and undiluted samples (60–62%) and higher (P<0.05) than that in diluted samples (35–45%), regardless the time after collection when freezing took place. Velocities were higher in control and in undiluted-3h samples (VCL of 254–265μm/s, VAP of 219–244μm/s and VSL of 134–147μm/s) than in diluted samples or samples frozen 6h after collection. P. lineatus sperm can be transported/shipped to the laboratory without decreasing its suitability for cryopreservation. Sperm should be kept undiluted during storage and be frozen within 3h.

The activity of N-acetyl-β-hexosaminidase in boar seminal plasma is linked with semen quality and its suitability for cryopreservation

The determination of sperm cryotolerance is an important step in the process of developing optimal techniques for the storage of boar semen. The objective of this study was to determine individual proteome variations in boar seminal plasma and spermatozoa and establish their influence on the cryotolerance of ejaculate. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed the presence of protein with estimated molecular weight of 90 kDa in sperm extracts from ejaculates of selected boars. In all cases, dialysis performed at the initial stage of cryopreservation effectively removed the protein from sperm cells. The protein had an affinity for Zn2+ ions. Mass spectrometry revealed similarities between the discussed protein and the β subunit of N-acetyl-β-hexosaminidase (β-HEX). Seminal plasma β-HEX was purified 252-fold with approximately 27% recovery and specific activity of 1800 U/mg of protein. Enzyme activity in fresh seminal plasma was correlated with superoxide dismutase activity (r = −0.42, P < 0.05), glutathione peroxidase activity (r = −0.42, P < 0.05), mitochondrial function (r = 0.31, P < 0.05), glutathione content (r = 0.34, P < 0.05), total protein content (r = 0.42, P < 0.05), and total oxidant status of seminal plasma (r = 0.37, P < 0.05). After thawing, β-HEX activity in seminal plasma was negatively correlated with the total motile sperm count (r = −0.33, P < 0.05), plasma membrane integrity (r = −0.31, P < 0.05), and lipid peroxidation (r = 0.33, P < 0.05). The observed correlations indicate that lower levels of β-HEX activity in boar seminal plasma are linked with higher quality of sperm after thawing. Based on those observations, the ejaculates were divided into two groups characterized by low (<20,000 U/L) and high (>20,000 U/L) levels of β-HEX activity in seminal plasma. In plasma with high β-HEX activity, spermatozoa were characterized by lower plasma membrane integrity (84.7%, P < 0.05). Higher glutathione levels (1250.3 μM), higher total protein content (50 mg/mL), and higher total oxidant status (6.82-μmol H2O2 Equiv/L) were also observed (P < 0.05). After thawing, lower sperm motility (20.4%), lower plasma membrane integrity (41.7%), and higher lipid peroxidation (30.9-nM malondialdehyde/108 spermatozoa/h) were reported in ejaculates with high seminal plasma β-HEX activity. The results of this study indicate that β-HEX activity in seminal plasma is a useful indicator in preliminary evaluations of boar sperm cryotolerance.

Comparison of seminal parameters in pairs of doses of stallion semen collected at various intervals and their suitability for cryopreservation

Comparison of seminal parameters in pairs of doses of stallion semen collected at various intervals and their suitability for cryopreservation

Recovery and cryopreservation of sika deer ( Cervus nippon) spermatozoa from epididymides stored at 4 °C

The aim of this study is to clarify influence of cold storage of deer epididymides on sperm quality and suitability for cryopreservation. The epididymides were obtained postmortem from sika deer during the breeding season. When epididymides were removed 8–12 h postmortem and stored at 4 °C for 1–4 days, the collected spermatozoa showed low motility (6.4%). When spermatozoa were collected from epididymides removed within 4 h postmortem, sperm motility and viability were 71.8 and 82.4%, respectively. Sperm motility decreased as prolongation of the storage period of the epididymides continued up to 7 days, but sperm viability was not affected. Pyknosis of the epithelial cells and their release into the lumen were observed in the stored epididymides. Epididymal spermatozoa frozen on Day 0 showed 58.1% motility and 83.2% viability. Motility of the frozen–thawed spermatozoa from epididymides stored at 4 °C for 1 day (41.9%) was similar to that of nonfrozen spermatozoa from epididymides stored for 4 days (41.8%). These results suggest that refrigeration of deer epididymides or cryopreservation of spermatozoa from refrigerated epididymides can be used for assisted reproductive techniques when epididymal spermatozoa cannot be collected immediately.

Factors Affecting Cryopreservation Efficiency and Enzyme Activity in Northern Pike, Esox lucius, Sperm

Two freezing techniques (straws and pellets), three cryo-protectants (DMSO, glycerol, and DMA) in four concentrations, and several extenders were tested to determine their suitability for cryopreservation of northern pike, Esox iucius, sperm. Activity of aspar-tate aminotransferase (AspAT) and acid phosphatase (AcP) in cryo-preserved milt was determined. Fertilization ability of cryopreserved milt was affected by the freezing technique, by type and concentration of cryoprotectant, as well as by the kind of extender used. These factors also influenced AspAT and AcP activity assayed in cryopreserved sperm. Extender containing 0.6 M sucrose + 15% DMSO + 10% egg yolk was most suitable for cryopreservation of pike sperm in pellet form (90.5% of eyed eggs, as compared to control group, which was 89.1%).

Biotechnological methods score efficacy the cryopreservation of boar semen

Aim. Estimate boar semen for suitability to cryopreservation using informative biotechnological methods (thermoresistant probe, heat resistance test) to replenish genetic resources of Animal Gene bank Institute of Animal Breeding and Genetics n. M.V. Zubets, NAAS. Methods. In carrying out this work used biotechnology, cryobiological, morphological methods. Results. Mirgorod boars breed investigation we noted feature individual semen quality, which affect its suitability for cryopreservation. All boar genealogy analysis of experiment in Mirgorod breed descended from a line of boar named Lovchyk 1205, which was characterized by negative performance stability to semen cryopreservation. For morphometric and functional parameters of sperm, potentially of better quality sperm for cryopreservation was boar Mirgorod purebred No. 1159, for which thermoresistant probe, heat resistance test indices was respectively 40.0 % and 35.0 %. Most suitable for cryopreservation in terms of heat resistance test boar semen was number 1159 and No. 79, and amounted to 35.0 % and 30 %. Nevertheless, after thawing average sperm activity index fell to 83.7 %. Conclusions. Established a comprehensive method for assessing the suitability of native breeds boar semen cryopreservation involving of morphological and biotechnological methods Keywords: cryobank, spermatozoa, cryopreservation, gene pool.