Post-thaw Storage Research Articles

The administration of blood components: a British Society for Haematology Guideline.

The administration of blood components: a British Society for Haematology Guideline.

The effect of prefreeze rejuvenation on postthaw storage of red blood cells in AS-3 and SAGM.

We investigated whether improving the metabolic status of red blood cell concentrates before freezing could extend the postthaw shelf life beyond 14 days while still meeting the requirements for hemolysis (0.8%) and total adenylate (>82% of original values). At Day 8 after collection, four leukoreduced red blood cell concentrates in saline-adenine-glucose-mannitol (SAGM) were pooled, mixed, and split (n = 4). Of these concentrates, two were rejuvenated in Rejuvesol. In addition, two leukoreduced red blood cell concentrates in phosphate-adenine-glucose-guanosine-gluconate-mannitol (PAGGGM) were pooled, mixed, and split at Day 8 after collection (n = 4). All concentrates were glycerolized, frozen, and stored for at least 2 weeks at -80°C. After thawing and deglycerolization, from each pair, one red blood cell concentrate was resuspended in SAGM, and one was suspended in AS-3. During postthaw storage at 2 to 6°C for 35 days, all concentrates were sampled weekly and analyzed for hematologic, metabolic, and morphologic parameters. Both Rejuvesol and PAGGGM treatment produced increased adenosine triphosphate and total adenylate and 2,3-diphosphoglycerate levels compared with untreated red blood cell concentrates. Regardless of prefreeze Rejuvesol or PAGGGM treatment, postthaw hemolysis remained below 0.8% during 7 days in SAGM and during 35 days in AS-3. At Day 35 of postthaw storage in AS-3, total adenylate in nonrejuvenated red blood cell concentrates had decreased to 72% of the original values; whereas, in prefreeze Rejuvesol-treated and PAGGGM-treated concentrates, adenylate values were still were at 101% and 98%, respectively. Based on maximum allowable hemolysis of 0.8% and total adenylate content greater than 82% of the original value, thawed, prefreeze Rejuvesol-treated or PAGGGM-treated red blood cell concentrates can be stored for 35 days at 2 to 6ºC in AS-3.

Chilled and post‐thaw storage of sperm in different goldfish types

The effective storage time of sperm after stripping (for 48hr in 6-hr intervals) and after thawing (for 6hr in 2-hr intervals) in Black moor, Oranda and Calico goldfish types was investigated. Variations in sperm density were also measured in all lines. The efficiency of a sperm cryopreservation method formerly developed for common carp was recorded in all three goldfish lines. Motility parameters ((pMOT, %), curvilinear velocity (VCL, μm/s) and straightness (STR, %)) of Black moor sperm did not decrease significantly during 48hr of storage. A significant reduction in the Oranda type compared to the fresh control was observed in pMOT after 42 (23±2%) and VCL after 36 (94±12μm/s) hours (pMOT 84±5%, VCL 150±11μm/s). In the Calico type, pMOT decreased significantly already after 18 (42±26%) and VCL after 6 (105±8μm/s) hours (fresh: pMOT 92±5%, VCL 151±6μm/s). A high pMOT immediately following thawing was measured in Oranda (46±12%) and Calico (55±15%) types, whereas a reduced pMOT was recorded in Black moor (24±19%). In Calico, pMOT showed a significant reduction after 6hr (19±11%) in comparison with the initial value, with no changes observed in VCL and STR. None of the parameters changed in the Black moor and Oranda types. Evidence was found that different goldfish lines have different sperm quality and characteristics. Further studies can investigate the possible effects of chilled and post-thaw storage on the fertilizing capacity of sperm in the Black moor, Oranda and Calico goldfish types.

Plasma treated with amotosalen and ultraviolet A light retains activity for hemostasis after 5 days post‐thaw storage at 1 to 6oC

Plasma thawed and stored at 1 to 6° C for up to 5 days (thawed plasma [TP]) provides rapid availability in emergencies and reduces plasma waste, but it carries risks of coagulation factor loss or activation, bacterial outgrowth, and viral contamination. We characterized changes in amotosalen/ultraviolet A (UVA) light pathogen-reduced, fresh-frozen plasma (FFP) and plasma frozen within 24 hours (PF24) with post-thaw storage. Amotosalen/UVA light-treated FFP and PF24 were thawed after approximately 3 to more than 12 months of frozen storage and held at 1 to 6° C for 5 days. Global assessments of coagulation and hemostatic, antithrombotic, and activation markers indicative of function were assessed. Day 5, thawed amotosalen/UVA light-treated FFP and PF24 contained levels of Factors II, V, VIII, IX, X, von Willebrand factor ristocetin cofactor (vWF:RCo), fibrinogen, antithrombin III (ATIII), protein C, and protein S similar to the levels measured in Day 5 TP, as described in the Circular of Information. Thrombin generation was robust on Day 5 (amotosalen/UVA: FFP = 1866 ± 402 nM/minute; PF24 = 1800 ± 277 nM/minute). Most factor activities on Day 5, including von Willebrand factor-cleaving protease (ADAMTS-13), were more than 90% of Day 0 values, except for known labile Factors V and VIII and protein S. All units contained greater than 0.4 IU/mL protein S and α2 plasmin inhibitor on Day 5. Global functional indices, including thrombin-antithrombin complexes, nonactivated thromboplastin time, and thrombin-generation peak height, did not indicate activation of the coagulation cascade, although isolated units showed raised levels of Factor VIIa and Complement 3a. Amotosalen/UVA light-treated FFP and PF24 demonstrated retention of procoagulant and antithrombotic activity after 5 days post-thaw storage at 1 to 6° C.

The effect of supplementation of a trehalose-based extender with KCl on rainbow trout (Oncorhynchus mykiss) sperm freezability and post-thaw motility

The aim of this study was to test if a cryopreservation procedure using methanol as an internal cryoprotectant and trehalose as an external cryoprotectant could be applied to rainbow trout sperm (Oncorhynchus mykiss). Additionally, the effects of potassium ions and Tris-HCl on post-thaw sperm motility, viability, and fertilizing capacity were analyzed. In this study, we demonstrate for the first time that rainbow trout sperm can be cryopreserved using a trehalose and methanol based extender. Trehalose was found to be effective in rainbow trout sperm cryopreservation at concentrations ranging between 175 and 200mM when supplemented with 10% methanol (v/v). The most effective dilution ratio was 1:11.5, which resulted in a final sperm concentration ranging from 0.8 to 1.6×109 spermatozoa mL−1. A 15-min equilibration period of sperm in the extenders did not influence sperm motility parameters. The highest post-thaw sperm motility was found using extenders containing 175mM trehalose (77.4±10.8) and 175mM trehalose with 40mM KCl (79.3±9.2). The supplementation of the extender with Tris-HCl and the adjustment of the pH to 7.5 and 8.5 resulted in a decrease in post-thaw motility. The post-thaw sperm motility in samples cryopreserved in an extender containing 40mM of KCl did not decrease 60min after thawing. The fertilizing capacity of sperm cryopreserved in 175mM trehalose and 40mM KCl was the highest and did not differ from fresh sperm at a sperm-to-egg ratio of 200,000:1. In conclusion, our results indicate that rainbow trout sperm can be successfully cryopreserved with the application of a trehalose based extender. The supplementation of the cryopreservation extender with 40mM KCl resulted in prolonged post-thaw storage of the sperm, allowing prolonged use of the sperm over time for common fishery practices. Statement of relevanceThis work is related to fish production and conservation.

Improvement of common carp (Cyprinus carpio) sperm cryopreservation using a programable freezer

The applicability of a programmable freezer for the increased-scale cryopreservation of common carp sperm was investigated. The effect of different equilibration times, cryopreservation methods, extenders, dilution ratios, activating solutions on the post-thaw motility of common carp sperm was investigated. The suitable post-thaw storage time-interval as well as fertilizing capacity of cryopreserved sperm was also examined. The motility, curvilinear velocity (VCL) and straightness (STR) values did not decrease significantly during 60min of equilibration neither in equilibrated nor thawed groups. Motility parameters of thawed sperm were similar using a conventional cryopreservation technique using a polystyrene box [motility (33%), VCL (47μm/s) and STR (88%)] and a programmable freezer: [motility (32%), VCL (54μm/s) and STR (89%)]. The highest motility and VCL was measured with a sugar based extender (grayling extender) at a ratio 1:9 (motility: 52%, VCL: 76μm/s) and 1:20 (motility: 49%, VCL: 76μm/s). The activating solution for cyprinids (ASC) could prolong sperm movement up for 2min. A storage time of six hours following thawing did not have a significant effect on the motility parameters of thawed carp sperm. Agglutination was observed during cryopreservation of an elevated volume of sperm whereas motility 47%, VCL 62μm/s and STR 91% were measured after thawing. Fertilization rate with thawed sperm (32%) was significantly lower compared to the control group (73%). According to our results, the developed method using a programmable freezer is suitable for the cryopreservation of elevated number of straws. However, carp sperm agglutination during freezing may have a negative effect on the fertilizing capacity.

Utility of different sugar extenders for cryopreservation and post-thaw storage of sperm from Salmonidae species

The effect of replacement of glucose in glucose-methanol extender (GM) with either sucrose and trehalose on the sperm motility characteristics of cryopreserved semen and the post-thaw storage of salmon, brown trout, brook trout, sex-reversed female rainbow trout and whitefish was investigated. Semen samples were diluted 1:5 or 1:9 (for sex-reversed females) in 9% methanol containing 0.18M glucose, sucrose or trehalose. After thawing (40°C, 5s), sperm was stored for 0, 30, 60, 120 and 240min at 4°C and assessed for sperm motility. The type of sugar had no effect on the percentage of sperm motility of brown trout (65–73%), brook trout (51–58%) or sex-reversed female rainbow trout (38–40%). Glucose and trehalose secured the highest post-thaw sperm motility after thawing (0min) in salmon (64±7%) and whitefish (52±8%), respectively. The longest period (determined as the last time point when the percentage of motility did not significantly change) was recorded for cryopreserved semen of brook trout (60–120min). The shortest was recorded for whitefish semen (30min). Our results suggest that glucose, sucrose or trehalose can be used interchangeably for the cryopreservation of brown trout, brook trout and sex-reversed semen. However, the best results in terms of post-thaw sperm motility were achieved for salmon with glucose and sucrose and for whitefish with trehalose and sucrose. Cryopreserved sperm can be stored for prolonged periods of at least 30min without a loss in sperm motility, which can facilitate more organised hatchery work. Statement of relevanceThe results of this study confirmed the high level of utility of cryopreservation employing a 0.18M glucose and 9% methanol extender for freezing, among Salmonidae species. We have demonstrated for the first time the utility of this method for freezing Atlantic salmon semen. Furthermore it was shown that disaccharides are more efficient than glucose for preserving of whitefish post-thaw motility.

Potassium ions in extender differentially influence the post-thaw sperm motility of salmonid fish

Potassium ions are known to have an inhibitory effect on the sperm motility of salmonids. For this reason, the addition of K+ to the extender is frequently applied. However, the effect of the addition of K+ to the extender has not yet been tested. The aim of this study was to test the influence of potassium ion supplementation of the extender on the sperm motility parameters from five Salmonidae species (rainbow trout (Oncorhynchus mykiss), sex-reversed female rainbow trout, whitefish (Coregonus lavaretus), brown trout (Salmo trutta) and brook trout (Salvelinus fontinalis)). Semen samples were diluted in extender containing 0.18 M glucose in 9% methanol (GM) supplemented with 0, 20 or 40 mM potassium chloride. After thawing sperm were stored for 30, 60, 120 and 240 min at 4 °C. Our results demonstrated that the presence of potassium ions in the extender had a negative effect on percentage of motile sperm in four of the salmonid species. In contrast, potassium ions appeared to have a positive effect on percentage of post-thaw motile sperm in whitefish semen. However, this effect could be mimicked by changing the osmolality of the extender (which was achieved by increasing the glucose concentration to 0.22 M). The addition of potassium ions turned out to have no positive effect on post-thaw storage time. Our results suggest that osmolality, rather than potassium ions, seems to be essential for cryopreservation success of salmonids sperm. Further studies should focus on the effects of small changes in osmolality on the post-thaw quality of semen.

Effect of dilution in sperm maturation media and time of storage on sperm motility and fertilizing capacity of cryopreserved semen of sex-reversed female rainbow trout

Masculinized females, also called neomales or sex-reversed females have a male phenotype but retain the female genotype (XX). Therefore, all spermatozoa produced in their functional testes carry an X chromosome, which is desired for the production of all-female rainbow trout populations. Semen of sex-reversed female rainbow trout is of low quality and in vitro maturation is required, which includes dilution of sperm suspensions with specially formulated maturation solutions. The aim of this study was to determine the effect of dilution in different maturation media on sperm quality (sperm motility characteristics and fertilizing capacity) of frozen/thawed sperm of sex-reversed female rainbow trout. The effect of time of post-thaw storage (0, 15, 60 and 120min) on semen quality was also tested. Sperm motility parameters and fertilization rate at the eyed and hatching stages were assessed for post-thaw semen diluted in different media. The cryopreservation procedure resulted in high post-thaw sperm motility of about 57% and did not differ from fresh semen. Unexpectedly, maturation media decreased sperm activation capacity immediately after dilution; however, sperm motility increased over time. Fertilization rates of frozen/thawed semen were high (71–87%) and did not differ significantly between experimental variants at any of tested periods of storage. Our results demonstrated that the effect of the maturation media on frozen/thawed sperm is different from that of fresh sperm. The progressive increase in post-thaw sperm motility in maturation media can potentially be applied to routine hatchery practice.

Microparticles variability in fresh frozen plasma: preparation protocol and storage time effects.

Extracellular vesicles or microparticles exhibiting procoagulant and thrombogenic activity may contribute to the haemostatic potential of fresh frozen plasma. Fresh frozen plasma was prepared from platelet-rich plasma at 20 °C (Group-1 donors) or directly from whole blood at 4 °C (Group-2 donors). Each unit was aseptically divided into three parts, stored frozen for specific periods of time, and analysed by flow cytometry for procoagulant activity immediately after thaw or following post-thaw storage for 24 h at 4 °C. Donors' haematologic, biochemical and life-style profiles as well as circulating microparticles were analysed in parallel. Circulating microparticles exhibited a considerable interdonor but not intergroup variation. Fresh frozen plasma units were enriched in microparticles compared to plasma in vivo. Duration of storage significantly affected platelet- and red cell-derived microparticles. Fresh frozen plasma prepared directly from whole blood contained more residual platelets and more platelet-derived microparticles compared to fresh frozen plasma prepared from platelet-rich plasma. Consequently, there was a statistically significant difference in total, platelet- and red cell-derived microparticles between the two preparation protocols over storage time in the freezer. Preservation of the thawed units for 24 h at 4 °C did not significantly alter microparticle accumulation. Microparticle accumulation and anti-oxidant capacity of fresh frozen plasma was positively or negatively correlated, respectively, with the level of circulating microparticles in individual donors. The preparation protocol and the duration of storage in the freezer, independently and in combination, influenced the accumulation of microparticles in fresh frozen plasma units. In contrast, storage of thawed units for 24 h at 4 °C had no significant effect on the concentration of microparticles.

Quality of Clotting Factor Activity in Fresh Frozen Plasma at Thaw with a Microwave System and after Storage at 4 degrees C for 48 Hours.

Uncontrolled hemorrhage in polytrauma patients usually results in rapid need of blood products. Despite the shorter thawing times of microwave devices for heating fresh frozen plasma (FFP), their use has remained controversial, and just a few laboratory analyses have been published on this topic. The aim of this study was to analyse the quality of clotting factors immediately after thawing FFP with a microwave device and after 48-hour post thaw storage at 4 degrees C. 24 FFP units of all four ABO blood groups (six of each blood group) were thawed with a Transfusio-therm 2000 and later stored at 4 degrees C for 48 hours. Samples were drawn aseptically and investigated on various clotting factors and protein proteases (fibrinogen, antithrombin, FII, FV, FVII, FVIII, FIX, FX, FXI, FXIII, vWF antigen and activity, protein S, and protein C) using standard coagulation and chromogenic assays immediately after thawing and again after a 48-hour storage period at 4 degrees C. All units were tested for both anaerobic and aerobic microbial contamination using standard operating procedures immediately after thawing. After thawing, all coagulation factors and protein protease activities were within normal ranges. Blood group O individuals had approximately 25% lower plasma levels of vWF antigen and activity. After a 48-hour storage period at 4 degrees C, FVIII and FIX activities declined significantly in all blood groups, whereas the remaining clotting factors remained comparably stable. Immediately after rapid thawing using a microwave system, all FFP units contained adequate coagulation factor activities to maintain hemostatic activity at the time of product thaw. The post thaw refrigerated storage caused an anticipated decrease in factor VIII and IX activities, but retained normal coagulation factor levels of many plasma proteins. Therefore we conclude that the Transfusio-therm 2000 has no clinically significant influence on the activity of clotting factors and plasma proteases in FFP units.

Effectiveness of glucose–methanol extender for cryopreservation of Huso huso spermatozoa

The present approach was designed to evaluate the methanol–glucose extender effects on sperm cryopreservation in beluga sturgeon, Huso huso. Sperm quality was examined by measuring post-thaw sperm motility and fertilizing rate at hatching stage. We first tested the effect of glucose concentration (0, 0.10, 0.15, 0.20 and 0.30M) in a methanol extender on post-thaw sperm motility. The optimal cryopreservation conditions were found to be 0.2M glucose in the extender. Then, motility and fertilization rates of sperm cryopreserved with 0.2M glucose and 10% methanol (GM) were compared to Tris–sucrose–KCl in 10% methanol extender (TSKM). Additionally, sperm motility and fertilizing ability in relation to 15 and 30min equilibration in GM extender before and after cryopreservation were measured. Higher post-thaw sperm motility duration and percentage as well as fertilization rate were obtained with the GM extender when compared to TSKM extender. Equilibration of sperm in extender did not affect the motility quality of either fresh-diluted or frozen/thawed sperm, while fertilization rate showed a significant decline alone after 30min of post-thaw storage. Our results indicated that the use of a simple extender consisting of 0.2M glucose in 10% methanol can be an alternative cryopreservation method to those previously described for sturgeons.

Motility and fertilizing ability of cryopreserved Caspian brown trout (Salmo trutta caspius) sperm: Effect of post-thaw storage time and different sperm-to-egg ratios

This study was designed to test the effect of post-thaw storage time on sperm motility parameters of Caspian brown trout (n=7). Furthermore, we investigated the effect of sperm-to-egg ratios of 100,000:1, 300,000:1 and 600,000:1 on fertility of cryopreserved Caspian brown semen. Quality was assessed by measuring sperm motility parameters and fertilization rates at the eyed and hatching stages. The percentage of post-thawed sperm motility, curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) were not affected by 60min of storage, whereas a decrease in straight line velocity (VSL), average path velocity (VAP) and linearity (LIN) were found in cryopreserved semen. Thus, the cryopreserved sperm of Caspian brown trout could be stored up to 60min without loss of the percentage of sperm motility. The fertilization rate was not affected by 60min of post-thaw storage and was over 70% for sperm-to-egg ratios of both 300,000 and 600,000:1. To our knowledge, this study is the first to report the high post-thaw fertilization ability of Caspian brown trout semen at a sperm-to-egg ratio as low as 300,000:1. This procedure after scaling up can be recommended for routine Caspian brown trout sperm cryopreservation.

Fertilizing capacity and motility of tenchTinca tinca(L., 1758) sperm following cryopreservation

Experiments were carried out to develop an optimal cryopreservation protocol for tench sperm by testing the fertilizing capacity and motility parameters including progressive motility, curvilinear velocity (VCL) and linearity (LIN) of cryopreserved sperm. Three experiments were designed to this aim: first experiment where we tested the effects of two extenders (sugar-based Grayling and ion-based Kurokura 180) and two cryoprotectants (DMSO and methanol) on fertilization and hatching success; second where we tested the effect of cryoprotectant type (methanol or DMSO) in different concentrations (5%, 10% and 15%) on fertilization and hatching success; and third where we tested the effect of two cryoprotectants (methanol and DMSO) on sperm motility parameters (progressive motility, VCL and LIN) after 4 h post-thaw storage (4°C). Sperm prepared with the sugar-based Grayling extender displayed better fertilization and hatching rates independently of the applied cryoprotectant most likely due to glucose present which acted as an external cryoprotectant. Concerning cryoprotectant concentrations, the use of 10% methanol yielded the highest fertilization (85 ± 15%) and hatching (80 ± 13%) rates, which were significantly higher than in all other groups. During the post-thaw storage time, 5% methanol, 10% methanol and 5% DMSO groups had significantly higher motility parameters than other groups and we observed no significant decline in any of the parameters during the storage time. Overall, we found that a sugar-based extender in combination with methanol as cryoprotectant is suitable for the cryopreservation of tench sperm and allows storage of cryopreserved sperm for up to 4 h post thaw.

Post-thaw storage of sperm from various salmonid species

The effects of post-thaw storage on the motility and fertilizing capacity of cryopreserved sperm were investigated in four salmonid species, including the Adriatic grayling (Thymallus thymallus; five males), rainbow trout (Oncorhynchus mykiss; six males), marble trout (Salmo marmoratus; five males) and the brown trout (Salmo trutta m. Fario; six males). The sperm of rainbow trout and marble trout was cryopreserved in the presence of either methanol or dimethyl-sulfoxide (DMSO) as a cryoprotectant; for the sperm of the Adriatic grayling and brown trout, only methanol was used. A 1-h storage of sperm did not reduce the fertilizing capacity of the three species, whereas in the marble trout a gradual decrease in the percentage of eyed eggs in parallel with post-thaw storage time was observed. The use of methanol was found to result in higher fertilization than that of DMSO in the rainbow trout and marble trout. Remarkably high post-thaw motility expressed in % motile cells was observed following post-thaw storage of brown trout sperm for 10 (53 ± 12%) or 60 (56 ± 15%) min. The results indicate that immediate use of cryopreserved sperm for fertilization following thawing is not mandatory to attain successful fertilization.

Optimization of conditions for the cryopreservation of Eurasian perch (Perca fluviatilis Linnaeus, 1758) sperm

In this study, a methodical optimization and development of cryopreservation of Eurasian perch (Perca fluviatilis) sperm was carried out. The effects of two extenders (modified Tanaka and Lahnsteiner's immobilizing solution, N = 7 males), 6 dilution ratios (from 1 : 1 to 1 : 50, N = 10 males) and post-thaw storage time (0–6 h, N = 10 males) on post-thaw progressive motility of sperm measured in a CASA system (Computer-assisted Sperm Analysis) were investigated in a wild caught Eurasian perch broodstock collected in the Saska river near Szczytno, Poland. Perch sperm cryopreserved with the modified Tanaka extender showed significantly higher progressive motility after thawing than with the modified Lahnsteiner immobilizing solution. The dilution ratio of 1 : 20 showed higher post-thaw progressive motility than the dilution ratio of 1 : 6 described previously. Post-thaw sperm was sensitive to storage time however the reduction of progressive motility was statistically significant only after 2 h. Thawed sperm resulted in a high fertilization rate (75 ± 5%, N = 9 males) compared to that with fresh sperm (86 ± 5%, N = 9 males). Although the fertilizing capacity has been reduced as a result of cryopreservation, progressive motility did not show a significant reduction in the same experiment (76 ± 24% in fresh sperm and 58 ± 24% in cryopreserved sperm, N = 9 males each). The significant difference in fertilization rates can be attributed to lower individual variation in fertilizing capacity of sperm in comparison to motility.

New extender for cryopreservation of Siberian sturgeon (Acipenser baerii) semen

The goal of this study was to develop a simple glucose–methanol extender for cryopreservation of Siberian sturgeon (Acipenser baerii) semen. Semen quality was assessed by determining post-thaw sperm motility and fertilizing ability at hatching stage. We tested the effect of glucose concentration (0, 0.10, 0.15, 0.20 and 0.30M) in a methanol extender on post-thaw sperm motility. Sperm motility parameters and fertilizing ability of semen cryopreserved in 0.1M glucose in 15% methanol (GM) were compared to previously described Tris–sucrose–KCl in 10% – methanol extender (TSKM). Additionally, sperm motility and fertilizing ability in relation to 30min equilibration in GM extender before cryopreservation and 30min of post-thaw storage were determined. The beneficial effect of the glucose for semen cryopreservation was related to its concentration with a quite narrow optimum of 0.1 to −0.15M. The fertilization rates of frozen/thawed sperm were similar for both (TSKM and GM) tested extenders. The sperm motility and fertilization rate were not affected either by 30min equilibration in GM extender or by 30min of post-thaw storage. Our work indicates that the use a simple extender consisting of 0.1M glucose in 15% methanol can be an alternative cryopreservation method to those previously described for sturgeons. The use of an equilibration period and the possibility of post-thaw semen storage can improve organization of hatchery work and help with logistics of large-scale hatchery operations.

Effect of post-thaw storage time on motility and fertility of cryopreserved beluga sturgeon (Huso huso) sperm.

The aim of this study was to test the influence of post-thaw storage time on the duration of sperm motility, percentage of motile sperm, and fertilization and hatching rates of fresh sperm and sperm stored for 0, 30 and 60 min at 4°C post-thawing. After being frozen in liquid nitrogen and then thawed, the percentage of motile sperm and duration of motility were not affected by 30 min of storage at 4°C, whereas a significant decline in these parameters was observed after 60 min of storage. Similarly, fertilization and hatching rates were significantly affected within 60 min of storage at 4°C, and the fertility of frozen-thawed sperm was significantly lower than that of fresh sperm. We conclude that cryopreserved sperm of beluga sturgeon could be stored for 30 min without the loss of sperm quality. This described procedure for beluga sturgeon cryopreservation is reliable and efficient and therefore can be recommended for hatchery practice after scaling up this technique.

Prolonged post‐thaw shelf life of red cells frozen without prefreeze removal of excess glycerol

The use of a functionally closed system (ACP215, Haemonetics) for (de)glycerolization of RBCs allows for prolonged post-thaw storage. Currently, glycerolization is followed by supernatant glycerol reduction before freezing. The aim of this study was to investigate the influence of supernatant glycerol reduction before freezing on the stability of thawed, deglycerolized RBCs during subsequent cold storage. Leucoreduced RBCs were stored for 6 days at 2-6°C before glycerolization. The RBCs were pooled and split, and glycerol was added using the ACP215 to a final concentration of 40%. Units were either frozen as such (n = 4) or supernatant reduced before freezing (n = 4). After storage at -80°C, the units were thawed, deglycerolized and resuspended in SAGM. An additional sixteen units, frozen without supernatant reduction, were resuspended in either AS-3 (n = 8) or SAGM (n = 8) after deglycerolization. During cold storage (2-6°C), the red cells were analysed for their stability and in vitro quality. The freeze-thaw-wash recovery was comparable for both volume reduced and non-reduced units. During post-thaw storage in SAGM, non-glycerol reduced units showed significantly less potassium leakage and haemolysis and higher ATP levels. AS-3 strongly reduced haemolysis during post-thaw storage of non-glycerol reduced units: haemolysis remained below 0.8% for up to 28 days of storage. Omitting glycerol supernatant reduction before freezing simplifies the cryopreservation procedure and increases the stability and therefore the outdating period of thawed RBCs. This increases the practical applicability of cryopreserved RBCs in both civil (rare blood) and military blood transfusion practice.

Utilization and quality of cryopreserved red blood cells in transfusion medicine.

Cryopreserved (frozen) red blood cells have been used in transfusion medicine since the Vietnam war. The main method to freeze the red blood cells is by usage of glycerol. Although the usage of cryopreserved red blood cells was promising due to the prolonged storage time and the limited cellular deterioration at subzero temperatures, its usage have been hampered due to the more complex and labour intensive procedure and the limited shelf life of thawed products. Since the FDA approval of a closed (de) glycerolization procedure in 2002, allowing a prolonged postthaw storage of red blood cells up to 21 days at 2-6°C, cryopreserved red blood cells have become a more utilized blood product. Currently, cryopreserved red blood cells are mainly used in military operations and to stock red blood cells with rare phenotypes. Yet, cryopreserved red blood cells could also be useful to replenish temporary blood shortages, to prolong storage time before autologous transfusion and for IgA-deficient patients. This review describes the main methods to cryopreserve red blood cells, explores the quality of this blood product and highlights clinical settings in which cryopreserved red blood cells are or could be utilized.