Motility characteristics and membrane integrity of cryopreserved human spermatozoa.

The motility characteristics of washed spermatozoa from 50 normal ejaculates were measured by time-lapse photography, before and after cryopreservation. Plasma membrane integrity was assessed by the hypo-osmotic swelling test and with the supravital fluorescent dye bisbenzimide (H33258). There was a marked decline in the percentage of progressively motile spermatozoa after cryopreservation, the extent varying widely among donors. Results were, however, consistent between different ejaculates from the same individual. The ability of spermatozoa to survive cryopreservation could not be predicted from the properties of the semen beforehand. The mean velocity of the spermatozoa was significantly reduced after freezing, but the lateral head displacement was unaltered. There was a significant reduction in the proportion of spermatozoa with intact plasma membranes after cryopreservation and the results of the hypo-osmotic swelling test and H33258 tests correlated closely. There was no correlation between the declines in the percentage of motile spermatozoa, or intact spermatozoa and the sperm velocity. We conclude that membrane rupture is not the sole cause of loss of motile spermatozoa during freezing and that the decrease in the proportion of motile spermatozoa is caused, at least in part, by a separate process from that responsible for the decrease in the average swimming speed of spermatozoa.     

Nonbeneficial effects of glycerol on the oocyte penetrating capacity of cryopreserved and incubated human spermatozoa

The effect of cryopreservation on human spermatozoa in the presence or absence of glycerol was assessed by using sperm motility, functional integrity of sperm membrane, and denuded hamster oocyte penetration tests. Glycerol treated cryopreserved spermatozoa yielded a significantly higher (P less than 0.01) percentage of motile sperm and percentage of sperm with functionally intact membrane immediately after thawing than the spermatozoa not treated with glycerol but cryopreserved. However, no significant difference was observed between these cryopreserved spermatozoa (either treated or untreated with glycerol) on the percentage of motile sperm and the rate of oocyte penetration when the sperm were washed and incubated for 2 hr in a medium containing no glycerol. Thus, it appears glycerol may not be beneficial, since cryopreservation of spermatozoa either treated or untreated with glycerol essentially yields similar oocyte-penetrating capacity of sperm.     

Positive effect of butylated hydroxytoluene (BHT) on the quality of cryopreserved cat spermatozoa

The semen cryopreservation processes are associated with state of oxidative stress induced by high levels of reactive oxygen species (ROS), causing damage to functional spermatozoa. Whereby, antioxidants have been utilized to scavenge or neutralize the elevated levels of ROS. The aim of at the present study was to evaluate the effect of adding BHT to the freezing extenders on post-thaw characteristics of domestic cat spermatozoa. Semen samples were frozen in Tris-fructose-citric acid-based extender, supplemented with different concentrations of BHT (0.5 mM, 1.0 mM and 2.0 mM) and a control sample without antioxidant. After thawing, sperm samples were assessed for motility by computer‐assisted sperm analysis and viability, acrosome integrity, superoxide anion production and membrane lipid peroxidation status by flow cytometry. In the study, the parameters of sperm motility and acrosome integrity were significantly higher in 2.0 mM BHT compared to sperm frozen in the extender with other concentrations and control (P < 0.05), in addition, this concentration reduced significantly the superoxide anion production and lipid peroxidation of the sperm. The results demonstrated that the supplementation of BHT to the freezing extender could protect the function and cellular structure of domestic cat sperm from cryoinjuries.     

Comet assay of fresh and cryopreserved bull spermatozoa

In this study, we describe DNA fragmentation of fresh and cryopreserved bull spermatozoa using the comet assay. Cryopreservation caused a significant but low (3.8%) decrease in the percentage of DNA in the comet head and an increase (5.3%) in the tail length. Our results suggest that in addition to motility and viability, low levels of DNA fragmentation after cryopreservation is a characteristic of bull spermatozoa and can be a part of remarkable cryoresistance of bull spermatozoa.     

Androgenesis in rainbow trout using cryopreserved spermatozoa: the effect of processing and biological factors

In addition to producing homozygous lines for biomedical and genomic research and monosex stocks for commercial purposes, androgenesis is the biotechnology considered most promising and reliable for recovering complete nuclear genome information from cryopreserved fish cells. That is because procedures of cryopreserving spermatozoa, contrary to procedures for oocytes or entire eggs, are being well developed. Application of androgenesis in genome banking programs addresses the needs of both the aquaculture industry (safeguard for valuable strains and lines) and natural resource conservation (in vitro protection of endangered species or populations). The present study was focused on successful production of an androgenetic rainbow trout stock using cryopreserved spermatozoa. Our report constitutes the first time a full factorial analysis of processing and biological factors affecting androgenesis efficacy has been presented. Also for the first time, the survival of androgenetic individuals during 2 years of life was recorded. Remarkably high survival rates were observed in one of the two experiments-up to 42.5 +/- 2.8% of hatched larvae, 22.5 +/- 0.1% of swim-up larvae and 10.5% of androgenetic alevins 0.4 g. Mortality rates in androgenetic groups were high especially during the first 6 months. In all, 114 androgenetic individuals (0.9%) survived 2 years. Cryopreservation of spermatozoa generally did not affect androgenesis efficiency significantly, however, this effect was significantly dependent on the method of ploidization shock and on the duration of treatment. Significant interactions were revealed between the irradiation dose and the magnitude of pressure applied, and between the treatment of sperm and duration of pressure shock. Individual variability of spermatozoa donors significantly affected androgenesis efficiency regardless of their genetic (outbred or inbred) origin. Genetic source of the oocytes, contrary to spermatozoa, proved to be an important factor. Following findings of other researchers that androgenesis using cryopreserved spermatozoa is possible, we demonstrated that viable stock could be successfully established from cryopreserved nuclear genome information. Complex statistical analysis of previously developed procedures resulted in information-rich data regarding factorial interactions helpful for developing protocols in genome-restoration programs.     

Effect of quercetin on the motility of cryopreserved canine spermatozoa

The addition of an antioxidant to cryopreservation solutions for preventing oxidative stress to sperm from several species, including that from humans, has been studied previously. Quercetin is a flavonoid contained in subarctic trees with freeze resistance and is known to be a strong antioxidant. Therefore, the effect of quercetin on the cryopreservation of dog spermatozoa was examined in this study. The proportions of total motile spermatozoa were significantly higher at 30, 60, 90, 120, and 150 min and at 60, 120, and 150 min after thawing in groups treated with 5 μg/ml and 10 μg/ml of quercetin dissolved in 0.1% DMSO added to the second extender based on skim milk compared to that in the control group, respectively. There were no differences between the experimental groups in the proportion of total motile spermatozoa during the observation periods. The proportion of total motile spermatozoa among those treated with 5 μg/ml of quercetin in 0.1% DMSO was improved by approximately 10–20% at 30–180 min after thawing compared to that in the control group. To evaluate the fertility of cryopreserved spermatozoa treated with quercetin, 2 × 10⁸ spermatozoa were transcervically inseminated into bitches, and a total of 18 puppies were delivered in three bitches. These results indicated that supplementation of quercetin as a cryoprotectant to the skim milk-based extender improved the motility of cryopreserved spermatozoa from dogs compared to those of the control group. And fertility of cryopreserved spermatozoa with quercetin supplementation was proven with higher efficiency.     

Effects of cooling and warming conditions on post-thawed motility and fertility of cryopreserved buffalo spermatozoa.

The principal objective of this study was to derive an improved procedure for cryopreservation of swamp buffalo (Bubalus bubalis) spermatozoa. Experiments were conducted to determine effects of cooling rate, intermediate plunge temperature and warming rate on motility and acrosome integrity of spermatozoa. Spermatozoa were obtained from three bulls (three ejaculates/bull) and were subjected to nine cooling conditions before being frozen in liquid nitrogen: cooling at 10, 20, or 30 degrees C/min each to -40, -80, or -120 degrees C before being plunged into liquid nitrogen. The spermatozoa frozen under a given condition were then thawed either at 1000 or 200 degrees C/min. Cooling rate, intermediate temperature and warming rate significantly affected survival of spermatozoa obtained from the three bulls. Cooling spermatozoa from 4 to -120 degrees C either at 20 or 30 degrees C/min yielded better progressive motility compared to other cooling conditions (50 versus 30%). Rapid warming was superior to slow warming. In an additional study, motility and fertility of spermatozoa frozen after being cooled to -120 degrees C at 20 degrees C and 30 degrees C/min and those frozen by a standard protocol used routinely for semen processing were assessed. Progressive motility of cryopreserved spermatozoa cooled at 20 degrees C and 30 degrees C/min was 40%, while that of spermatozoa cryopreserved using a standard protocol was 25%. A total of 178 buffalo cows were inseminated with cryopreserved spermatozoa obtained from one bull, and their pregnancy status was assessed 60 days later by rectal palpation. Out of the 60, 26 (43%) and 23 of 58 (40%) cows inseminated with sperm cooled at 20 and 30 degrees C/min, respectively, became pregnant, whereas 17 of 60 (28%) cows inseminated with sperm frozen by a standard protocol became pregnant. This study demonstrates that an effective cryopreservation procedure for buffalo spermatozoa can be derived by systematic examination of various cryobiological factors.     

Positive effect of partial zona pellucida digestion on in vitro fertilization of mouse oocytes with cryopreserved spermatozoa

Cryopreservation of mouse spermatozoa has been widely used; however, fertility of frozen spermatozoa in some strains, especially when inseminating cryopreserved oocytes, is low and may be improved by assisted fertilization techniques. The present study was performed to investigate the effect of partial zona pellucida (ZP) digestion on the in vitro fertilization (IVF) capacity of frozen mouse spermatozoa. Mouse oocytes were subjected to partial ZP digestion using acidic Tyrode's solution (pH 3.1). Fertilization rates in digestion groups (30 or 45 s) were higher (P < 0.05) than that of zona-intact control (78.3% or 86.3% vs. 52.5%). The recovery rate at 45 s was lower (P < 0.05) than that at 30 s (84.2% vs. 97.3%). Among vitrified oocytes, the fertilization rate in treatment group (digested for 30 s) was higher (P < 0.05) than that of zona-intact group (50.8% vs. 22.1%). After embryo transfer at the two-cell stage, 17.7% and 11.8% of transferred embryos derived from fresh and vitrified digested oocytes developed to term and showed no significant difference as compared with that from zona-intact oocytes (24.1%, P > 0.05). These results indicate that partial ZP digestion improves IVF efficiency of fresh and vitrified oocytes with frozen mouse spermatozoa, which can provide valuable information for in vitro assisted fertilization using cryopreserved gametes in the re-establishment of mouse colonies.     

Improved recovery of post-thaw motility and vitality of human spermatozoa cryopreserved in the presence of dithiothreitol

Semen was collected in the laboratory from nine healthy donors. The concentrations and the percentages of live and motile spermatozoa in all semen samples were within the normal range. Each sample was diluted with citrate-egg yolk-glycerol medium with and without 5 mM dithiothreitol (DTT). Samples were frozen in liquid nitrogen vapor (?70 °C) for 7 min and subsequently stored in liquid nitrogen. The effect of DTT in cryopreservation of sperm was determined by comparing percentage of motile and live spermatozoa between controls and DTT-treated post-thaw samples. Percentage of motile spermatozoa was determined by two techniques, laser Doppler velocimetry (LDV) and light microscopy. The percentage of live spermatozoa was measured by microscopic evaluation after staining with eosin-nigrosin. It was shown that the addition of DTT to the freezing medium significantly improved the recovery of motile and live spermatozoa in the post-thaw samples. The mean motility recovery, as measured by LDV, was 44.9% in the controls as compared to 73.9% in the DTT-treated samples. Similarly the mean recovery of live spermatozoa in the controls and DTT-treated samples was 66.5 and 86.6%, respectively. Based on these results, a new hypothesis implicating lipid peroxidation in cryoinjury is proposed. It is also suggested that the use of DTT in the freezing medium may offer an advantage over the commonly used techniques of human sperm cryopreservation.     

The effect of liquid nitrogen submersion on cryopreserved human heart valves

Cryopreservation and storage of human heart valves have become an accepted means of maintaining a usable supply of heart valves for outflow track reconstructive surgery. Valves are typically stored at the vapor phase temperature of liquid nitrogen, -130 degrees C and below, to reduce the chance of recrystallization within the tissues. Concern over the effects of submersion of the valves in liquid nitrogen, i.e., plunging to -196 degrees C, prompted this study. Cryopreserved valves were plunged into liquid nitrogen, held for 5 min, and then processed (thawed) by standardized protocols. The thawed valves were then assessed using scanning electron microscopy and the more traditional histology at the light microscope level. Cuspal tissues plunged into liquid nitrogen appear to have numerous microfractures over both surfaces of the tissue, penetrating into the collagen/proteoglycan matrix. Control cryopreserved valves do not exhibit these microfractures. Histologically, the submerged valves appear normal. The clinical use of valves which have been submerged in liquid nitrogen is discussed.     

Activity of Chinchilla laniger spermatozoa collected by electroejaculation and cryopreserved

Because reproductive studies and the application of assisted reproductive techniques are relevant issues for an endangered species such as Chinchila laniger, the availability of a source of viable spermatozoa becomes of utmost importance. In this paper, we evaluate several functional parameters (motility, viability, response to hypoosmotic swelling test and acrosomal integrity) of fresh or frozen-thawed spermatozoa. Electro-ejaculation trials (50-cyc/sec sinusoidal wave was applied for 5 of every 10 sec) were successful in all unanesthetized animals. After volume (108.3 +/- 12.0 microL, n = 15) and concentration (421.8 +/- 34.4 x 10(6) cells/mL, n = 15) measurements, the above mentioned parameters were determined. In frozen-thawed semen samples sperm motility, viability, hypoosmotic swelling test and acrosomal integrity were significantly lower than in fresh semen samples. The results clearly indicated that electro-ejaculation is a useful method for evaluating spermatozoa for genetic analysis or for used in Al in this species. In addition, the cryopreservation procedure in this study preserved adequate levels of functional sperm activity.     

Localization of various ATPases in fresh and cryopreserved bovine spermatozoa

Different ATPases may control the various functional changes that spermatozoa undergo just prior to fertilization, with the enzyme's specific location within the cell reflecting its function. The activities of Mg²⁺-ATPase, Ca²⁺Mg²⁺-ATPase, Na⁺K⁺-ATPase and Ca²⁺-ATPase were determined for head plasma membranes (HPM) and sperm body membrane (SBM) from both fresh (n = 4) and cryopreserved bovine spermatozoa (n = 4) and fresh homogenized whole spermatozoa (HWS) (n = 6). No activity of Ca²⁺Mg²⁺-ATPase was found in any preparation from spermatozoa. Ca²⁺-ATPase was detected in fresh SBM and HWS but not in HPM. Activity of Mg²⁺-ATPase and Na⁺K⁺-ATPase was higher in HPM than HWS or SBM (P < 0.01). Cryopeserving the whole sperm reduced the activities of all three enzymes, but Na⁺K⁺-ATPase was more sensitive to cryopreservation than Mg²⁺-ATPase (P ≤ 0.05). Enzyme location suggests that Ca²⁺-ATPase may be associated with events in the flagellum, while Mg²⁺-ATPase and Na⁺K⁺-ATPase may affect functions in the sperm head. Cryopreservation-induced damage to ATPases might be involved in reducing the fertilizing ability of cryopreserved spermatozoa.     

Reliable recovery of inbred mouse lines using cryopreserved spermatozoa

Since the mouse has become the most detailed model system to investigate the genetics and pathogenesis of human diseases, large numbers of new mouse strains have and continue to be produced. In nearly all animal facilities, the maintenance of breeding colonies is limited and mouse strains have to be archived in an efficient way. This study was undertaken to test the reliability of recovering mouse lines by use of cryopreserved spermatozoa from individual male mice. In contrast to many studies, spermatozoa and oocytes were derived from the same genetic background. 30 C3HeB/FeJ males belonging to three different categories (wild-type, F1-generation of ENU-treated males, and defined mutants) were recovered by producing at least 20 offspring from each donor. Independent of the experimental group, every single male was successfully recovered. Archiving mouse strains by cryopreservation of spermatozoa may, therefore, offer a reliable way to preserve genetically valuable mouse strains and provides an efficient management strategy for animal facilities. Received: 15 February 1999 / Accepted: 19 April 1999     

Comparative effects of rifabutin and rifampicin on cytochromes P450 and UDP-glucuronosyl-transferases expression in fresh and cryopreserved human hepatocytes.

The aim of this study was to evaluate rifabutin (RBT) and rifampicin (RIF) capabilities in inducing various xenobiotic metabolizing enzymes such as cytochromes P450 (CYPs) and UDP-glucuronosyl-transferases (UGTs) in cultured fresh and cryopreserved human hepatocytes. Enzyme induction was assessed through the use of several diagnostic markers, i.e. testosterone, midazolam (MDZ), diazepam (DZP) and 7-ethoxyresorufin for CYP-dependent enzyme reactions; and AZT for UGT-dependent enzyme reactions. RBT concentrations (0.118, 0.708 microM) were selected according to previously published pharmacokinetic data in patients. The known CYP3A4 inducer in humans, RIF, was used as a positive control. At the concentrations used, no sign of cytotoxicity was evidenced. Both compounds were able to dose-dependently induce the overall metabolism of testosterone (approximately 2-fold for RBT, 4-fold for RIF) and the formation of the 6beta-hydroxylated-derivative (up to approximately 4-fold over control for RBT and approximately 10-fold for RIF), which is CYP3A4 dependent. The other hydroxylated metabolites (16alpha-OH and 2alpha-OH) were also enhanced. The metabolism of MDZ, which is specifically metabolized by CYP3A4 in humans, was also investigated following drug's exposure to hepatocytes. DZP one, which is governed by various CYPs, including CYP3A, was also investigated. RBT was shown to increase the biotransformation of both benzodiazepines (approximately 1.9-fold over control). Moreover, the effects of both drugs on ethoxyresorufin O-deethylase activity (EROD), which is representative of CYPIA1/2 isoforms, were tested. Results showed only a moderate induction of this marker (approximately 2-fold over control) when compared to the high effect observed after hepatocyte exposure to 3-methylcholantene (approximately 14-fold over control). Finally, the action of RBT and RIF on UGTs expression was investigated by using AZT as diagnostic substrate: glucuronides formation was not significantly affected by the two rifamycin derivatives. On the whole, exposure of fresh or cryopreserved human hepatocytes to RBT dose-dependently affected the levels of drug metabolizing enzymes in a dose-dependent manner. However, as already demonstrated by in vivo pharmacokinetic studies, its inducing properties towards CYPs, CYP3A in particular, are less pronounced than RIF.     

L-arginine promotes capacitation and acrosome reaction in cryopreserved bovine spermatozoa

Sperm capacitation and acrosome reaction are essential for fertilization and they are considered as part of an oxidative process involving superoxide and hydrogen peroxide. In human spermatozoa, the amino acid L-arginine is a substrate for the nitric oxide synthase (NOS) producing nitric oxide (NO*), a reactive molecule that participates in capacitation as well as in acrosome reaction. L-arginine plays an important role in the physiology of spermatozoa and has been shown to enhance their metabolism and maintain their motility. Moreover, L-arginine has a protective effect on spermatozoa against the sperm plasma membrane lipid peroxidation. In this paper, we have presented, for the first time, the effect of L-arginine on cryopreserved bovine sperm capacitation and acrosome reaction and the possible participation of NOS in both processes. Frozen-thawed bovine spermatozoa have been incubated in TALP medium with different concentrations of L-arginine and the percentages of capacitated and acrosome reacted spermatozoa have been determined. L-arginine induced both capacitation and acrosome reaction. NO* produced by L-arginine has been inhibited or inactivated using NOS inhibitors or NO* scavengers in the incubation medium, respectively. Thus, the effect of NOS inhibitors and NO* scavengers in capacitated and non-capacitated spermatozoa treated with L-arginine has also been monitored. The data presented suggest the participation of NO*, produced by a sperm NOS, in cryopreseved bovine sperm capacitation and acrosome reaction.     

The effect of oviductal fluid on protein tyrosine phosphorylation in cryopreserved boar spermatozoa differs with the freezing method

Sperm capacitation takes place in the oviduct and protein tyrosine phosphorylation of sperm proteins is a crucial step in capacitation and acquisition of fertilizing potential. Cryopreserved spermatozoa show altered expression of protein tyrosine phosphorylation in the oviduct. The present study compared two freezing methods (conventional-conventional freezing (CF) and simplified-simplified freezing (SF) methods) for their effect on the ability of boar spermatozoa to undergo protein tyrosine phosphorylation in response to oviductal fluid (ODF). Cryopreserved boar-spermatozoa were incubated with pre- and post-ovulatory ODF for 6 h at 38 °C under 5% CO₂. Aliquots of sperm samples were taken at hourly intervals and analyzed for kinematics and protein tyrosine phosphorylation. Global protein tyrosine phosphorylation in spermatozoa was measured using flow cytometry and different patterns of phosphorylation were assessed using confocal microscopy. Immediately after thawing, no significant difference was observed in post-thaw sperm motility, velocity and global tyrosine phosphorylation between the two methods of freezing although the freezing method significantly (P < 0.05) influenced the effect of oviductal fluid on these parameters during incubation. While spermatozoa frozen by the CF method showed a significantly higher (P < 0.001) proportion of phosphorylation in response to preovulatory ODF during incubation, spermatozoa frozen by the SF method did not elicit such significant response as there was no significant difference in the proportion of tyrosine phosphorylated spermatozoa between treatments at any given time during incubation. If the CF method was used, the proportion of spermatozoa displaying either tail or full sperm phosphorylation increased in response to both preovulatory (EODF) and postovulatory oviductal fluid. However, if the SF method was used, a significant increase in these patterns was noticed only in the EODF treated group. The present study demonstrates that preovulatory isthmic ODF induce tyrosine phosphorylation in a higher proportion of boar spermatozoa compared to the post-ovulatory fluid and that the method of freezing significantly influences the response of post-thaw spermatozoa to porcine ODF.     

Defining the salt effect on human RAD51 activities

Previous work by Sung and colleagues identified unusual salt requirements for hRAD51 strand exchange compared to RecA [S. Sigurdsson, K. Trujillo, B. Song, S. Stratton, P. Sung, Basis for avid homologous DNA strand exchange by human Rad51 and RPA, J. Biol. Chem. 276 (2001) 8798-8806]. Later studies showed that this salt [(NH4)2SO4] appeared to enhance the ability of hRAD51 to distinguish ssDNA from dsDNA [Y. Liu, A.Z. Stasiak, J.Y. Masson, M.J. McIlwraith, A. Stasiak, S.C. West, Conformational changes modulate the activity of human RAD51 protein, J. Mol. Biol. 337 (2004) 817-827]. The mechanism of this salt effect remains enigmatic. Here, we detail the properties of several neutral salts on hRAD51 activities. We found that the cation identity correlated with the stimulatory effect of these neutral salts on hRAD51 ATPase and strand exchange activities. The salt effect appears to be related to the size of the cation, which may be largely mimicked with the cesium ion. These results are consistent with the hypothesis that stimulating cations induce an important conformation and/or transition state in hRAD51. In the presence of an optimal ammonium-based salt (NaNH4HPO4), hRAD51 mediated strand exchange was successfully performed using a simplified protocol. We confirmed and extend the observation that efficient strand exchange correlated with preferential binding of ssDNA over dsDNA. In addition we observed an induced stability of the hRAD51-DNA complex in the presence of ATP that becomes unstable following ATP hydrolysis (the ADP form or nucleotide free form). These salt-induced characteristics of hRAD51 increasingly resemble RecA-mediated recombinase activities, which should help in dissecting the mechanism of these proteins in homologous recombination.