PVP contrasted with dextran and a multifactor theory of cryoprotection

Washed human erythrocytes were suspended in 0, 5, 10, 15, and 20% PVP in phosphate-buffered saline (PBS). Fifty lambda samples were frozen in alcohol baths at temperatures ranging from ?10 ° to ?80 °C. The specimens were frozen either for 1 or 16 min, rapidly thawed, and resuspended in PBS or PBS plus PVP. Percent hemolysis was determined colorimetrically. Results indicate that there is a high degree of latent damage when red cells are frozen in the presence of PVP. This damage is evident from the large increase in hemolysis when freeze-thawed, intact red cells are resuspended in the PBS. Under some circumstances 16 min freezing is significantly less damaging than 1 min freezing. This indicates a partial recovery from the freezing stress during subzero storage of the red cells.The general cryoprotective properties of PVP were described in terms of: (1) latent damage; (2) storage damage; (3) optimal cooling and rewarming rates (as a function of freezing bath temperature); (4) optimum PVP concentration; and (5) post-thaw cryoprotection. The data were compared with that from a similar study using dextran-40. This comparison indicated six similarities and ten differences in the cryoprotective properties of dextran and PVP. The remarkable differences between dextran and PVP was counted as an important common characteristics of macromolecular cryoprotective agents. That is, their cryoproteetive properties cannot be reduced to one or a few physical characteristics held in common. Nine other common characteristics were listed. Several of these, which include latent damage and recovery from latent damage, cannot be explained by current theories of cryoprotection. A multifactor theory was proposed to account for these ten common features of macromolecular cryoprotective agents.     

Effect of two cooling protocols on the post-thaw characteristics of Iberian ibex sperms

The rate at which lethal intracellular ice forms during sperm cryopreservation is highly dependent on the cooling protocol. The present work compares two cooling protocols for use with Iberian ibex (Capra pyrenaica) sperm by assessing the effects on the motility, viability, and size of frozen-thawed sperm cells. Ejaculates, obtained from six adult ibex males via transrectal, ultrasound-guided massage of the accessory sex glands plus electroejaculation if necessary, were cooled via either 1) Protocol 1 (decelerating cooling), involving cooling in liquid nitrogen vapor from 5 °C to −35 °C (40 °C/min), from −35 °C to −65 °C (17 °C/min), and then from −65 °C to −85 °C (3 °C/min); or 2) Protocol 2 (accelerating cooling) involving cooling in a biological freezer from 5 °C to −5 °C (4 °C/min), from −5 °C to −110 °C (25 °C/min), and then from −110 °C to −140 °C (35 °C/min). Compared to fresh ejaculates, sperm quality at thawing was found to be reduced by both protocols (p < .05), but especially by Protocol 1. Sperm head size was also significantly reduced by both protocols, although the Protocol 1 sperm heads were also significantly smaller than those of Protocol 2 sperms heads (p < .05). In fresh sperm samples, clustering analyses revealed two subpopulations of sperms with different morphometric characteristics, SP1 with larger cells, and SP2 with smaller cells. Both cooling protocols caused reduction in the proportion of SP1 cells, and an increase in the proportion of SP2 cells. In conclusion, the decelerating cooling protocol (Protocol 1) caused greater cryodamage to the sperm cells than the accelerating protocol (Protocol 2).     

The effect of warming velocity on motility and acrosomal integrity of boar sperm as influenced by the rate of freezing and glycerol level

The effect of thawing velocities ranging from 10°C/min to 1.800°C/min on the motility and acrosomal integrity of boar spermatozoa frozen at 1°C/min (suboptimal), 5°C/min, and 30°C/min (optimal) rate was studied with the sperm suspended for freezing in diluent containing 2, 4, or 6% of glycerol (v/v). The influence of thawing on sperm survival depends on the rate at which the sperm had been frozen. In semen frozen at a suboptimal rate of 1°C/min, the percentage of motile sperm (FMP) initially fell to 3.5–4.0% when the thawing rose to 200°C/ min, but, with further increases in thawing rate, increased and reached peak values (10.3–11.0% FMP) after thawing at 1,800°C/min. The percentage of sperm with normal apical ridge (NAR) also increased moderately with thawing rate, but the degree of improvement decreased as the glycerol level was increased. In semen frozen at 1°C/min, acrosomal integrity (NAR) was best maintained in 2% glycerol, reaching 22.9% NAR after thawing at 1,800°C/min. In semen frozen at the optimal rate of 30°C/min, the increases in thawing rates above 200°C/min substantially improved motility. Motility was generally higher in semen protected by 4 or 6% glycerol, with the peak values of 44 or 46% FMP, respectively, after thawing at 1,200°C/min. The proportion of sperm with NAR also increased with thawing rate, but as in the case of suboptimally frozen sperm it was influenced negatively by the glycerol concentration. The peak value 53% NAR was recorded in semen protected by 2% glycerol, frozen at 30°C/min, and thawed at 1,200°C/min. In view of the inverse relationship between FMP and NAR, selection of optimal conditions from among the interacting variables, freezing rate, glycerol concentration, and thawing rate requires compromising between maximal FMP and maximal NAR. Accordingly, we have adopted as optimal a protocol with a thawing rate of 1,200°C/min, a freezing rate of 30°C/min and concentrations of 3% glycerol. © 1993 Wiley-Liss, Inc.     

Role of serum on cryopreservation and subsequent viability of mouse bone marrow hemopoietic stem cells

Normal mouse marrow cells were frozen in an automatically controlled freezer at a cooling rate of 1 °C/min to ?40 °C and 7 °C/ min to ?100 °C using dimethylsulfoxide as a cryoprotective agent. The freezing solution contained in addition either 10% homologous serum or 10% fetal calf serum. Control samples were frozen with serum-free medium. After thawing, stepwise dilution, and washing, the cells were counted, checked for CFU-s content, and cultured in Millipore diffusion chambers for 2 and 7 days.HS resulted in a recovery of 59.7% nucleated cells and 100.5% CFU-s whereas FCS and serum-free medium resulted in 59.8 and 34.7% nucleated cells and 24.5 and 18.2% CFU-s, respectively. After 2 days of culture, D.C. data showed a correlation with the CFU-s results. After 7 days of culture, no significant difference was observed between the three groups. The results of these experiments indicate that HS is required for an optimal stem cell cryopreservation and that a 2-day D.C. culture is a reliable assay system for transplantable hemopoietic tissue.     

Optimal temperature ranges for control of cooling rate.

Survival of hamster fibroblasts following cooling at 1 °C/min to various subzero temperatures in the presence of penetrating or nonpenetrating cryoprotective agents was examined. In the presence of nonpenetrating agents maximum recovery was obtained when the cooling rate was controlled between ?5 and ?20 °C followed by rapid cooling to ?196 °C. For penetrating agents recovery was maximal in samples cooled at 1 °C/min to ?30 °C or lower. These different temperature ranges for maximum recovery indicate different modes of actions of penetrating and nonpenetrating cryoprotective agents. The action of penetrating agents appear to be based on their colligative properties. Nonpenetrating agents may promote electrolyte leaks out of the cell and a corresponding osmotic efflux of cell water during slow cooling, thereby reducing the amount of intracellular ice present at ?196 °C.     

The effect of cooling and warming rates on the survival of cryopreserved L-cells

A tissue culture assay has been used to measure the survival of murine lymphoma cells (L-cells) after freezing and thawing in the presence of 2 M glycerol or 1.6 M dimethyl sulfoxide. The effect of variations in cooling rate (0.1 to 10.0 °C/min) and warming rate (0.3 to 200 °C/min) were studied. It was found that survival exhibited a peak at the “conventional” combination of slow cooling and rapid warming (~1 and 200 °C/ min, respectively). It was also shown, however, that a second peak of similar magnitude occurred when the cells were cooled and rewarmed at 0.2-0.3 °C/min. These results are interpreted on the basis of current theories of freezing injury, stressing the importance of damage produced by the recrystallization of intracellular ice and by solute loading. The ultraslow rates of cooling and rewarming which produced the second survival peak are practicable for whole organs, and their potential importance for organ cryopreservation is apparent.     

Ultrastructure before freezing,while frozen,and after thawing in assessing cryoinjury of mouse epididymal spermatozoa

Tails of mouse epididymides were treated as follows: control, unfrozen with and without cryoprotective agents (CPA); frozen (to below ?80 °C), slowly (8 °C/min), and rapidly (18 °C/sec), with and without CPA. Intracellular and/or extracellular location of CPA, at least glycerol, was influenced, respectively, by high (22 °C) or low (0 °C) exposure temperature. Standard procedures in electron microscopy were employed and the frozen state preserved by freeze-substitution. Motility before freezing and after thawing was the criterion of cryosurvival.Results showed no evidence of deleterious ultrastructural effects of freezing at rates compared, or of benefits of CPA, regardless of their cellular location. Differences were noted, however, in the appearance of spermatozoa in the frozen state, as a function of the rate of freezing but not as a function of the presence, absence, or location of either glycerol of DMSO. Rapidly frozen cells showed intracellular ice formation in the acrosome, neck, midpiece, and tail regions; there was no intranuclear ice, and extracellular ice artifacts were small. Slowly frozen cells showed large extracellular ice artifacts with evidence of shrinkage distortion due to the dehydration induced by extracellular ice. No spermatozoa survived any of the freezing treatments, showing the lethal effect of both extracellular ice during slow freezing and of intracellular and/or extracellular ice during rapid freezing.     

Regeneration of Plants from Cell Suspensions Frozen at −20, −70 and −196°C

Cell suspensions of carrot, Datura, tobacco and soybean subjected to ?20°C, ?70°C and ?196°C in the presence of a suitable cryoprotective agent, and stored for various lengths of time have been revived. After revival these cells divided to form callus masses. Direct immersion in liquid nitrogen invariably killed the cells, whereas cooling at the rate of 1 or 2°C/min, or pre-freezing briefly at ?20 and ?70°C, followed by freezing at ?196°C retained the viability. Depending on the plant species up to 70% of the cell clumps could withstand ultra-cooling. Tobacco and Datura cell suspensions were more sensitive to cold treatment than were those of carrot. Actively growing cell suspensions containing small cell-clumps revived rapidly, while filtered cell-suspensions of free cells only occasionally survived. Calli of tobacco and carrot obtained from frozen suspensions have been regenerated into plants.     

Methodology for reliable and reproducible cryopreservation of human cervical tissue

BackgroundIn order to conduct laboratory studies on donated cervical tissue at suitable times an effective and reliable cryopreservation protocol for cervical tissue is required.MethodsAn active freezing approach was devised utilising 10% dimethyl sulfoxide in foetal bovine serum as a cryoprotective agent with a cooling rate of 1 °C/min to −50 °C then 10 °C/min to −120 °C; a related thawing protocol was also optimised which would allow for the bio-banking of cervical tissue. Viability of freshly harvested cervical tissue was compared to frozen-thawed samples utilising colorimetric MTT assay. In parallel, fresh and freeze-thawed samples were cultured and tested on days 1, 7 and 14 to determine whether bio-banking had detrimental effects on tissue viability over time.ResultsRepeat testing revealed that tissue viability between fresh and freeze-thawed samples was comparable at all four time points (days 0, 1, 7 and 14) with no apparent reductions of viability, thus demonstrating this method of cryopreserving cervical tissue is reliable and reproducible, without detrimental effects on live tissue culture. We believe this methodology creates the opportunity for bio-banking donated cervical tissues, which aids improved experimental design and reduces time pressures and wastage.     

Cryopreservation of granulocytes for transfusion: Studies on human granulocyte isolation,the effect of glycerol on lysosomes,kinetics of glycerol uptake and cryopreservation with dimethyl sulfoxide and glycerol

Existing methods for the cryopreservation of granulocytes employ primarily dimethyl sulfoxide (Me₂SO) rather than glycerol as the cryoprotective additive of choice. Although Me₂SO has been demonstrated to be an effective cryoprotective additive for granulocyte preservation to yield viable cells (dye exclusion, phagocytosis, etc.), the inherent toxicity and clinical objections of Me₂SO as a cryoprotective additive for granulocyte preservation preclude its extensive and routine use in patients. Therefore, glycerol, with its important advantage of nontoxicity, has been investigated for its potential usefulness as a cryoprotective additive for preserving human granulocytes for transfusion.Granulocyte preparations were isolated from impure leukocyte concentrates obtained from the buffy coats of human whole blood. Studies on the isolation and purification of the granulocytes involved separation by sedimentation with dextran, removal of red cells by hypotonic shock with water, resuspension with Plasmatein and further purification by centrifugation. Intact viable granulocytes were obtained with a purity in excess of 90%.Lysosomes were studied as indicators of cryoinjury in granulocytes using β-glucuronidase as the key marker enzyme. This enzyme has been characterized as a sensitive indicator of damage to lysosomes and a direct linear relationship has been established between damage to granulocytes by freezing and amount of lysosomal enzyme released. Addition or presence of the cryoprotectant, glycerol, did not appear to have any adverse effect on lysosomes of intact granulocytes.Studies on the permeation kinetics of glycerol in granulocytes indicated that the additive was freely permeable and did not cause any potentially damaging osmotic changes in cell volume. Granulocytes in various concentrations of glycerol were then frozen at slow, moderate, and rapid cooling rates. Based on the small amount of β-glucuronidase released, good preservation of granulocyte lysosomes has been obtained with a slow cooling rate of 5 °C/min and a concentration of 15% glycerol. Further studies now are necessary to define those conditions of cooling rate and glycerol concentration required to develop a simple method for optimal preservation of granulocytes based on additional functional criteria of viability.     

Studies with dextran 40 in cryopreservation of blood

Whole blood from healthy donors was washed twice with phosphate-buffered saline (PBS) and then resuspended in sufficient PBS to give a final concentration of 2 × 10⁹/cells/ml. Aliquots were combined with equal volumes of the required diluents to give final dextran 40 concentrations of 0, 5, 10, 15, and 20% in PBS. Fifty-lambda samples in 50-lambda Micropets (Clay Adams) were frozen in alcohol baths at temperatures ranging from ?10 to ?80 °C. The specimens were frozen either for 1 min or 16 min, rapidly thawed, and resuspended in PBS or PBS plus dextran. Percentage of hemolysis was determined colorimetrically. Results indicate that concentraitons as low as 5% dextran exert a cryoprotective effect. Increased dextran concentration increases cryoprotection at high subzero bath temperatures (?10 ° and ?20 °C). Dextran concentrations beyond 12% have a damaging effect at low subzero bath temperatures (below ?30 °C). Based on this a two-factor hypothesis for cryopreservation is proposed. Apparent partial recovery of red blood cells without dextran or with 5% dextran during subzero storage was demonstrated.     

The effect of freeze rate,duration of phase transition,and warming rate on survival of frozen canine kidneys

The effect of cooling rate, warming rate, and duration of phase transition upon survival of frozen canine kidneys was investigated. In the present study, 11 kidneys out of 14 rapidly cooled (2–4 °C/min) to ?22 °C and thawed (70–110 °C/min) were viable following contralateral nephrectomy. The serum creatinine and BUN levels rose to a maximum of 8.4 and 30 mg%, respectively, on the eighth day post-contralateral nephrectomy. Average survival time was 10 days; however, two of the dogs in this group were allowed to survive, one for 3 months and one for over 2 years. Eight kidneys out of 16 slowly cooled (0.25–1.0 °C/min) and either rapidly or slowly warmed (20–30 °C/min) had function to produce small amounts of urine; however, they did not survive more than 5 days after contralateral nephrectomy.Cooling rates of 0.1 and 10 °C/min were too harmful to the kidney to have renal function after reimplantation.The minimum renal cell damage as assessed by LDH and GOT in the post-freeze perfusate was found in the 2–4 °C/min cooling rate following rapid warming (70 °–110 °C/min).Correlation of the duration of phase transition time to renal cell damage was linear for LDH and GOT (r = 0.93). This result suggests that the duration of phase transition time also is an important factor during the freezing process, affecting postthaw survival of canine kidneys.     

Comparisons of glucose,sucrose, and dimethyl sulfoxide as cryoprotective agents for Babesia rodhaini, with estimates of survival rates

Comparisons were made between glucose, sucrose, and dimethyl sulfoxide (DMSO) as cryoprotective agents for the hemoprotozoan parasite, Babesia rodhaini, using infectivity for mice as the criterion of survival. Concentrations of the cryoprotectants tested were from 0.1 to 0.5 M for the sugars, and 1.5 to 2.5 M for DMSO. Glucose and sucrose were comparable as cryoprotectants, although glucose reduced infectivity of the parasites slightly more than did sucrose at above-freezing temperatures. When sucrose and DMSO were compared for cryoprotection during cooling to ?196 °C at nominal rates of 5, 100, and 500 °C/min, parasite survival varied with the type and concentration of cryoprotectant, but was higher in blood containing DMSO at all three cooling rates. The percentages of parasites that survived cooling at 100 °C/min and frozen storage in the presence of DMSO ranged from 20 to 36%.     

A comparative study of the effects of freezing and frozen storage on intact and demembranated bull spermatozoa

The damage caused to bull sperm by freezing and thawing them without cryoprotectants was assessed in both intact and membrane-extracted cells. Preparations of membrane-extracted cells were produced by treating the sperm with 0.1% Triton X-100 and motility was restored with exogenously applied ATP and Mg²⁺. Motile demembranated sperm showed no detectable reduction in motility after freezing and thawing. In contrast, when intact cells where subjected to freezing and thawing they lost all motility. These damaged cells were also restored to motility when exogenous ATP and Mg²⁺ were added to the sperm mixture. Apparently freezing and thawing sperm cells causes damage to the plasma membrane which permits ATP and Mg²⁺ to freely enter or leave the cells, but does not damage the components of the sperm cell which generate motility.The effects of storage temperature on frozen demembranated sperm were also explored. Sperm held at ?20 °C showed marked structural changes and progressively decreased motility after prolonged storage. When sperm were frozen at ?20 °C the mitochondrial structures were completely lost after 48 to 72 hr and ATP caused the disintegration of the flagellum rather than initiating motility. Sperm which were frozen at ?76 °C retained motility after short periods of storage, but showed a significant decline in motility when thawed after 8 days. Demembranated sperm which were kept frozen at ?196 °C showed no significant loss of motility when thawed after 1 year of storage.     

Cryopreservation of Plagiognathops microlepis sperm

Sperm was collected from cultured male fish and cryopreserved in 0.25 ml straws for the study of sperm cryopreservation. Different parameters were evaluated, including extender, dilution ratio, cryoprotectant type and concentration, equilibrium time, cooling height (in a two-step cooling protocol), and thawing temperature. The optimum result was obtained when the sperm was diluted at a 1:7 ratio in D-16 with 5% DMSO as a cryoprotectant, equilibrated for 20 min, held at 3 cm above liquid nitrogen for 10 min, and then stored in liquid nitrogen. After thawing in a water bath at 40 °C, the percentage of motile cells and fertilization rates of frozen-thawed sperm were 35.33 ± 2.52% and 39.00 ± 4.58%, respectively, while the corresponding rates for fresh sperm were 87.67 ± 3.06% and 88.67 ± 4.62%. We also used a programmed cooling protocol in which temperature was decreased from 4 °C to −80 °C by a rate of 30 °C/min, and then straws (0.25 ml) were placed above the surface of liquid nitrogen for 2 min before being stored in liquid nitrogen. This protocol provided a post-thaw activation rate of 36.67 ± 4.77%. Further parametric optimization is required to improve the quality of frozen-thawed sperm.     

Some factors affecting the viability of mouse and cattle embryos frozen to −40°C before transfer to liquid nitrogen

A total of 1161 8- to 16-cell mouse embryos and 31 cattle early morulae and late blastocysts were frozen to ?40°C before transfer to liquid nitrogen. After thawing, mouse embryo viability was determined by in vitro development to the blastocyst stage and cattle embryo viability by both in vivo and in vitro development.Using glycerol as the cryoprotective agent, 88% of the mouse embryos developed to the blastocyst stage: thawing at 45 and 360° C/min gave the best results (88.8 and 84.8%, respectively). In another test with holding times at ?40°C of up to 60 min, about 70% of embryos developed to blastocysts with holding time 30–60 min.In cattle, 11 embryos frozen in DMSO and thawed at 360°C/min were transplanted to eight recipients. Four pregnancies (six fetuses) resulted. Thawing rates of 200 and 360°C/min resulted in the best in vitro development of cattle embryos.     

The status within,an Autobiography by Francois Jacob; Basic Book Inc., New York, 1988, 326 pp. $22.95

The interactions of ram spermatozoa with exogenous liposomes of varying composition were studied, with the aim of examining the mechanisms by which some lipids protect against cold-induced damage during cryostorage. Liposomes containing various preparations of phosphatidylcholine and cholesterol enhanced sperm survival during storage at 5°C, both in terms of motility and acrosomal integrity. A membrane-fluidizing agent, A₂C, was slightly deleterious in this respect. Cholesterol-containing liposomes were not superior in their effects to those prepared without cholesterol. Thus stabilization of the plasma membrane by cholesterol loading may be unimportant. When sodium vanadate was used as a functional probe of membrane integrity, the cryoprotective effects of lipids were apparent despite increased plasma membrane permeability. Incubation of spermatozoa with positively charged liposomes, containing stearyl-amine, caused considerable loss of motility and acrosomal damage, coupled with cellular aggregation. There was also some evidence that the presence of calcium lessened the effectiveness of liposomes in protecting spermatozoa against damage during cooling.     

Two-step accelerating freezing protocol yields a better motility,membranes and DNA integrities of thawed ram sperm than three-steps freezing protocols

The present study compares a protocol that mimics freezing of ram semen in static nitrogen vapor with two protocols with an initial low cooling rate in the first step, followed by higher cooling rates where ice nucleation occurs. Semen ejaculates, obtained from twelve adults rams, were diluted with TEST-based extender and frozen with either Protocol 1 (three-step decelerating cooling): from +5 °C to −35 °C (40 °C/min), from −35 °C to −65 °C (17 °C/min), and then from −65 °C to −85 °C (3 °C/min); or Protocol 2 (three-step accelerating cooling): from +5 °C to −5 °C (4 °C/min), from −5 °C to −110 °C (25 °C/min), and then from −110 °C to −140 °C (35 °C/min); or Protocol 3 (two-step accelerating cooling), from +5 °C to −10 °C (5 °C/min), and then from −10 °C to −130 °C (60 °C/min). Post-thaw sperm quality was reduced for all protocols (p < .05) compared with fresh semen. Post-thaw percentages of sperm motility characteristics and sperm with intact plasma membrane, intact acrosome, and intact mitochondrial membrane were greater using Protocol 3 than Protocol 2 (p < .05) and Protocol 1 (p < .01). In addition, the post-thaw percentage of sperm with fragmented DNA was lower (p < .05) using Protocol 3 compared with Protocol 1. The present results indicate that a cooling rate of 60 °C/min around and after the time point of ice nucleation provided better post thaw survival and function of ram sperm than lower (and/or decelerating) cooling rates.     

Temperatures in Pigs During 3 T MRI Temperatures,Heart Rates,and Breathing Rates of Pigs During RF Power Deposition in a 3 T (128 MHz) Body Coil

Exposure to radiofrequency (RF) power deposition during magnetic resonance imaging (MRI) induces elevated body‐tissue temperatures and may cause changes in heart and breathing rates, disturbing thermoregulation. Eleven temperature sensors were placed in muscle tissue and one sensor in the rectum (measured in 10 cm depth) of 20 free‐breathing anesthetized pigs to verify temperature curves during RF exposure. Tissue temperatures and heart and breathing rates were measured before, during, and after RF exposure. Pigs were placed into a 60‐cm diameter whole‐body resonator of a 3 T MRI system. Nineteen anesthetized pigs were divided into four RF exposure groups: sham (0 W/kg), low‐exposure (2.7 W/kg, mean exposure time 56 min), moderate‐exposure (4.8 W/kg, mean exposure time 31 min), and high‐exposure (4.4 W/kg, mean exposure time 61 min). One pig was exposed to a whole‐body specific absorption rate (wbSAR) of 11.4 W/kg (extreme‐exposure). Hotspot temperatures, measured by sensor 2, increased by mean 5.0 ± 0.9°C, min 3.9; max 6.3 (low), 7.0 ± 2.3°C, min 4.6; max 9.9 (moderate), and 9.2 ± 4.4°C, min 6.1, max 17.9 (high) compared with 0.3 ± 0.3°C in the sham‐exposure group (min 0.1, max 0.6). Four time‐temperature curves were identified: sinusoidal, parabolic, plateau, and linear. These curve shapes did not correlate with RF intensity, rectal temperature, breathing rate, or heart rate. In all pigs, rectal temperatures increased (2.1 ± 0.9°C) during and even after RF exposure, while hotspot temperatures decreased after exposure. When rectal temperature increased by 1°C, hotspot temperature increased up to 42.8°C within 37 min (low‐exposure) or up to 43.8°C within 24 min (high‐exposure). Global wbSAR did not correlate with maximum hotspot. Bioelectromagnetics. 2021;42:37–50. © 2020 The Authors. Bioelectromagnetics published by Wiley Periodicals LLC on behalf of Bioelectromagnetics Society     

Effect of different procedures of ejaculate collection, extenders and packages on DNA integrity of boar spermatozoa following freezing-thawing

Whole ejaculate or sperm-rich fraction, collected from four sexually mature boars, was frozen in an extender containing lactose-hen egg yolk with glycerol (lactose-HEY-G) or extender containing lactose, lyophilized lipoprotein fractions isolated from ostrich egg yolk and glycerol (lactose-LPFo-G), and Orvus Es Paste, respectively. The sperm samples were also frozen in a standard boar semen extender (Kortowo-3), without the addition of cryoprotective substances. Sperm DNA integrity was assessed using a modified neutral comet assay. Sperm characteristics such as motility, plasma membrane integrity (SYBR-14/PI), mitochondrial function (rhodamine 123) and acrosome integrity were monitored. Freezing-thawing caused a significant increase (P<0.05) in sperm DNA fragmentation, irrespective of the procedures of ejaculate collection and extender type. Sperm DNA fragmentation was significantly lower (P<0.05) in the whole ejaculate compared with the sperm-rich fraction, indicating that spermatozoa maintained in the whole seminal plasma prior to its removal for freezing-thawing procedure were less vulnerable to cryo-induced DNA fragmentation. Furthermore, spermatozoa frozen in lactose-HEY-G or lactose-LPFo-G extender exhibited lower (P<0.05) DNA fragmentation than those frozen in the absence of cryoprotective substances. The levels of sperm DNA damage, as expressed by comet tail length and tail moment values, were significantly higher (P<0.05) in sperm samples frozen in the absence of cryoprotective substances. The deterioration in post-thaw sperm DNA integrity was concurrent with reduced sperm characteristics. It can be suggested that evaluation of DNA integrity, coupled with different sperm characteristics such as motility, plasma membrane integrity and mitochondrial function, may aid in determining the quality of frozen-thawed boar semen.