Improved Birth Rates via Rehydration of Mouse Freeze-Dried Spermatozoa using High-Temperature Ultrapure Water

Improved Birth Rates via Rehydration of Mouse Freeze-Dried Spermatozoa using High-Temperature Ultrapure Water | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Improved Birth Rates via Rehydration of Mouse Freeze-Dried Spermatozoa using High-Temperature Ultrapure Water Kango Yamaji, Sayaka Wakayama, Natsuki Ushigome, Daiyu Ito, Teruhiko Wakayama This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5994995/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Freeze-drying (FD) is a promising method for achieving the long-term, low-cost, and safe preservation of mammalian sperm at room temperature (RT). However, the birth rate of embryos fertilized with FD sperm is reduced to less than half compared to those fertilized with fresh sperm. Moreover, the underlying causes and potential solutions remain unclear. In this study, we investigated a previously unexamined rehydration process using FD sperm to determine its effects on sperm DNA damage. We also attempted to optimize this rehydration method to improve birth rates. Initially, we examined the effects of slowing water infiltration into FD sperm using a high osmolarity or viscosity solution, but this increased DNA damage and decreased birth rates. Next, to accelerate infiltration speed, we performed rehydration of FD sperm using ultrapure water heated up to as hot as 90℃. However, we found that the DNA damage of the FD sperm decreased as the temperature increased. The level of DNA damage in the male pronucleus at the zygote stage and of abnormal chromosome segregation (ACS) at the two-cell stage were also decreased at 37℃ or 50℃. Finally, the birth rates of embryos derived from FD sperm also significantly improved when rehydration was performed using 50℃ ultrapure water (37%) compared with the RT control (21%). Taken together, the results of this study demonstrate that the DNA of FD sperm can be damaged during the rehydration process and that rapid rehydration significantly improves the birth rate. Biological sciences/Developmental biology/Embryology Biological sciences/Biotechnology/Animal biotechnology Rehydration Freeze-dry Intracytoplasmic sperm injection Infiltration speed Birth rate Figures Figure 1 Figure 2 Figure 3 Introduction Mammalian gametes are preserved for a variety of purposes, including infertility treatments, the conservation of endangered species, the cost-effective storage of genetically modified organisms, and the efficient transportation of mouse strains 1 – 3 . The standard method for preserving mammalian spermatozoa is cryopreservation using liquid nitrogen 4 . This technique maintains sperm motility post-thaw, which makes it suitable for in vitro fertilization (IVF) 5 . However, handling liquid nitrogen is challenging and poses risks of frostbite, asphyxiation, and rapid gas expansion. Moreover, it is also necessary to frequently replenish liquid nitrogen to keep temperatures low, and specialized containers are required for storage and transport, leading to high maintenance costs 1 , 2 . Furthermore, during disruptions of the liquid nitrogen supply (e.g., during natural disasters) there is a risk of losing all stored samples 6 . Freeze-drying technologies offer a promising solution to simultaneously address the multiple challenges associated with cryopreservation. Since 1998, when the first successful Freeze-drying of mouse sperm was demonstrated 7 , viable offspring have been produced from embryos fertilized with freeze-dried (FD) sperm from rats 8 , hamsters 9 , rabbits 10 , and horses 11 . Initially, the room temperature (RT) storage period of FD sperm was limited to only one month 7 . However, by sealing FD sperm in ampoules under a high vacuum, sperm storage at RT for longer periods became possible, with some studies reporting successful storage for periods of up to six years 12 , 13 . This technology eliminates the need for liquid nitrogen, and permits storage in compact spaces such as desk drawers 14 . Overall, this technique enables space-efficient and cost-effective storage while reducing the risk of sample loss during disasters. In addition, the development of safer and more user-friendly storage containers, such as plastic sheets 15 , microtubes 16 , and mini stainless steel tubes 17 , is currently underway. Furthermore, sperm subjected to FD pretreatment show improved resistance to extreme temperatures, including from − 196°C to 150°C 18 . FD pretreatment has also been found to improve survival following exposure to space radiation aboard the International Space Station for over 200 years 19 , 20 . Taken together, these findings highlight the advantages of FD preservation over cryopreservation for the long-term storage of sperm. However, the birth rates of embryos derived from FD sperm are less than one-third of those of embryos derived from fresh or cryopreserved sperm 13 , 21 , 22 . Thus, for FD methods to become more widely used for preserving genetic resources, it is essential to improve the rate at which they reliably produce offspring from preserved sperm. Recently, our lab has produced mouse offspring from FD sperm stored at RT for six years. Although the reliability of this method for long-term storage at RT has increased, the low birth rate remains an unsolved problem 13 . In animals such as cattle, which produce only one offspring per pregnancy, low birth rates necessitate multiple attempts, which can result in high overall costs despite improvements in sperm storage efficiency and safety. Overall, despite the significant advantages of FD technology, the low birth rate of embryos derived from FD sperm is a major barrier to its widespread use. Embryos fertilized with FD sperm frequently exhibit both DNA damage in the paternal pronucleus and chromosomal segregation abnormalities (ACS) 23 . These findings suggest that sperm DNA damage occurs at some stage of FD sperm preparation or use. Preparing and using FD sperm is a process that consists of four steps: freezing, drying, storage at RT, and rehydration of FD sperm. During the first sperm freezing, cryoprotectants such as DMSO or glycerol cannot be used because they cannot be dried, therefore all the sperm die after freezing. However, intracytoplasmic sperm injection (ICSI) allows injection of all sperm—including dead sperm—into oocytes 24 , thereby achieving normal fertilization and full-term development. This indicates that decreases in the birth rate of FD sperm are not due to the freezing step 22 , 25 , 26 . During the drying step, experiments using vacuum-dried sperm, in which sperm were dried without freezing, demonstrated that viable offspring could be obtained, although they showed lower birth rates than FD sperm 27 . Taken together, these findings suggest that damage to FD sperm primarily occurs during drying, whereas introducing a freezing step mitigates damage caused by drying. During RT storage, damage to FD sperm is primarily caused by air contamination. Therefore, storing ampoules under a high vacuum reduces damage to FD sperm and enables long-term RT preservation 12 . Interestingly, the addition of exogenous trehalose to the storage medium did not mitigate damage to sperm during freezing and drying, and was found to significantly improved birth rates when FD sperm were preserved for longer periods at RT 14 . This finding indicates that reduced birth rates of embryos derived from FD sperm following preservation at RT results not only from damage to FD sperm during freezing and drying but also from additional damage sustained during RT storage. In this study, we focused on the rehydration of FD sperm, a step that has not yet been thoroughly investigated. To do so, we prepared three types of solutions with varying osmotic pressures, viscosities, and temperatures to alter the speed of water infiltration during rehydration. We therefore used these infiltration treatments to investigate the effect of different rehydration regimes on DNA damage in FD sperm, fertilization rates following ICSI using FD sperm, embryo development rates, and offspring rates. Results Effects of the Osmotic Pressure of Rehydration Solutions on FD Sperm To investigate whether water infiltration speed during rehydration of FD sperm caused DNA damage, we rehydrated FD sperm with either ultrapure water or HTF medium, i.e., the basal medium used for FD sperm production (Fig. 1a, b, 2a, b). First, to measure solution infiltration speed, we dropped ultrapure water and HTF medium at RT onto filter paper and measured the diffusion distance per unit time (Fig. 2c). The infiltration speed of the HTF medium was approximately 0.85 times slower than ultrapure water (Fig. 2d; Supplementary Table 1). Next, to assess DNA damage in FD sperm, we performed a comet assay immediately after rehydration with either ultrapure water or HTF medium (Fig. 1c). When rehydration was performed with HTF medium, the comet tail, representing DNA damage, showed a slight but significant increase compared to ultrapure water (ultrapure water: 1.00 vs. HTF medium: 1.02, p < 0.05) (Fig. 2e, f; Supplementary Table 2). Next, we injected rehydrated sperm into oocytes via ICSI, and evaluated the fertilization rate, the development rate to the two-cell stage, and the birth rate. We found no significant differences at any of these stages between embryos derived from FD sperm rehydrated with ultrapure water and those rehydrated with HTF medium (Fig. 2g; Supplementary Table 3). Therefore, our results indicate that rehydration with HTF medium, in which the infiltration speed was slightly slower than in ultrapure water, did not result in reduced sperm DNA damage. Effect of Rehydration Solution Viscosity on FD Sperm Next, to investigate whether changes in the infiltration speed caused by the viscosity of the rehydration solution affected the degree of DNA damage, we added PVP to ultrapure water to achieve concentrations ranging from 1.5% to 12%, then used these higher-viscosity solutions for rehydration (Fig. 1b). First, we assessed the infiltration speed of PVP solutions by dropping each solution onto filter paper and measuring the diffusion distance per unit time. These results showed that the infiltration speed of PVP solutions decreased in a concentration-dependent manner relative to that of ultrapure water (Fig. 2d; Supplementary Table 1). Next, we performed a comet assay to evaluate the degree of DNA damage in FD sperm rehydrated with different PVP solutions. Except for the 1.5% and 3% concentrations, we found significant increases in DNA damage as the PVP concentration increased (Fig. 2e, f; Supplementary Table 4). Subsequently, FD sperm rehydrated with 6% and 12% PVP solutions were used for ICSI, and we examined ACS at the two-cell stage. ACS can detect large-scale chromosomal damage that cannot be assessed by comet assays. These results showed that ACS rates increased when rehydration was performed using 6% PVP, and the incidence of severe or lethal ACS was 3.5 times higher relative to the ultrapure water control (Supplementary Fig. 1a, b; Supplementary Table 5). Furthermore, in 12% PVP samples, we also observed elevated ACS rates, although not as markedly as for 6% PVP samples (Supplementary Fig. 1a, b; Supplementary Table 5). When ICSI was performed using FD sperm rehydrated with 0–12% PVP solutions, we did not observe significant differences in fertilization rates or developmental rates to the two-cell stage (Supplementary Fig. 1d; Supplementary Table 6). However, we did observe that in vitro development rates decreased as the PVP concentration increased, and the blastocyst formation rate was significantly lower for samples containing the highest viscosity solution, 12% PVP (Supplementary Fig. 1c, d; Supplementary Table 6). Furthermore, when some of the two-cell stage embryos were transferred to recipient mice to assess birth rates, we also observed a significant decrease in birth rates as the PVP concentration increased (Fig. 2g, Supplementary Table 7). Based on these results, we concluded that ultrapure water is the most suitable medium for rehydrating FD sperm. In addition, since slower infiltration speeds cause more damage to FD sperm and reduce the birth rate to a greater degree, we next performed experiments using heated ultrapure water to increase infiltration speed during the rehydration of FD sperm (Fig. 1b). Effect of Rehydration Solution Temperature on FD Sperm Next, to examine the infiltration speed of the rehydration solution, ultrapure water cooled or heated between 0.5°C and 90°C was applied to filter paper, and the diffusion distance per unit time was measured (Fig. 2c). As a result, the infiltration speed increased proportionally with increasing temperature (Fig. 2d, Supplementary Table 1). To investigate the effect of the water temperature on DNA damage in sperm, we then performed a comet assay on FD sperm rehydrated using medium at each temperature. When rehydration was performed at 0.5°C, we observed significantly higher DNA damage in sperm relative to the RT (23°C–25°C) (Fig. 3a; Supplementary Table 8). Furthermore, DNA damage significantly decreased as the temperature of the rehydration solution increased above RT, with the lowest DNA damage observed at 70°C (relative value: 1.00 vs. 0.85, p < 0.05) (Fig. 3a; Supplementary Table 8). Next, we performed ICSI using FD sperm rehydrated at each temperature. In preliminary experiments, the activation rate after ICSI tended to be lower when FD sperm were rehydrated with water at 70°C. To account for this, in this experiment some oocytes were artificially activated with SrCl₂ after ICSI (Table 1). When male pronuclei of one-cell embryos were examined using gamma-H2Ax antibodies, we found that rehydration at 0.5°C resulted in higher gamma-H2Ax fluorescence intensity relative to RT rehydration, which indicates that increased DNA damage was present (Fig. 3b, c; Supplementary Table 9). In contrast, rehydration at 37°C resulted in the lowest gamma-H2Ax fluorescence intensity compared to the RT control, which is suggestive of reduced DNA damage. However, rehydration at temperatures above 50°C showed DNA damage levels that were comparable to or slightly higher than those observed in samples dehydrated at RT. Table 1. Full-term development rates of ICSI embryos using FD sperm rehydrated with ultrapure water at different temperatures. Temperature (℃) Oocyte activation No. of oocytes surviving after ICSI No. (%) of oocytes surviving after oocyte activation No. (%) of fertilized zygotes No. (%) of embryos developed to 2 cell No. of transferred embryos [no. of recipients] No. (%) [min-max] of offspring 0.5 － 186 － 146 (78) 122 (66) 116 [8] 28 (24) ab [8-37] RT － 261 － 231 (89) 197 (75) 197 [16] 41 (21) a [7-43] 37 － 202 － 160 (79) 132 (65) 117 [9] 34 (29) ab [0-56] 50 － 333 － 259 (78) 237 (92) 226 [14] 83 (37) b [8-83] 70 － 54 － 33 (61) 28 (52) 28 [2] 4 (14) ab [0-21] 70 ＋ 282 251 (89) 231 (92) 184 (80) 175 [9] 55 (31) ab [18-79] 90 － 59 － 48 (81) 42 (71) 23 [3] 8 (35) ab [17-60] 90 ＋ 172 141 (82) 126 (89) 85 (67) 83 [6] 29 (35) ab [13-43] Next, we examined ACS in two-cell embryos, and found that rehydration at 0.5°C increased the incidence of severe ACS (i.e., classified as ”heavy” or ”lethal”) relative to the RT control (Fig. 3d, e; Supplementary Table 10). In contrast, increasing the rehydration temperature above RT tended to reduce the incidence of severe ACS, with the least damage observed at 50°C. Moreover, embryos showing light ACS can sometimes result in viable offspring 28 ; if we combine both those embryos scored as normal chromosome segregation (NCS) and Light as “normal” embryos, and the remaining embryos to be “abnormal” embryos, we find that the lowest proportion of abnormal embryos (29%) was observed at 50°C. Finally, we transferred two-cell embryos to recipients, and evaluated the resulting birth rate. We found that rehydration at 0.5°C resulted in a birth rate of 24%, which was similar to the rate obtained with RT (21%) (Table 1). However, rehydration at temperatures above RT improved the birth rate to 29% (at 37°C) and 37% (at 50°C) (Fig. 3f, g). In contrast, rehydration at 70°C or higher resulted in a slight decrease in birth rate compared to 50°C (70°C: 31%; 90°C: 35%). Discussion Previous studies have reported that several kinds of damage affect sperm during the drying process. However, this study revealed—for the first time—that sperm DNA can also be damaged during rehydration. In addition, here we show that rehydrating with high-temperature ultrapure water—which has previously been considered to be harmful to sperm—reduces DNA damage in FD sperm. Furthermore, using conventional methods, the birth rate for mouse FD sperm-derived embryos is approximately 20%, but when following a rehydration protocol using ultrapure water at 50°C, we observed birth rates as high as 37%. Here, we first hypothesized that—just as riverbanks erode when exposed to rapid currents 29 —faster infiltration speeds may damage the DNA of FD sperm. Therefore, we predicted that the current method used to rehydrate FD sperm may cause DNA damage. Based on this hypothesis, we proposed that employing a slower infiltration method during rehydration of FD sperm can mitigate DNA damage and improve the birth rate. However, despite attempts to slow down the infiltration speed by increasing osmotic pressure and viscosity, we found—contrary to our expectations—that lower infiltration speed was associated with increased DNA damage in FD sperm, as well as significant decreases in blastocyst formation and birth rates. In contrast to our initial hypothesis, when pure water was heated to high temperatures to speed up infiltration into the nucleus, we observed reduced DNA damage in FD sperm, as well as birth rates that were up to 1.8 times higher than those in FD sperm rehydrated with pure water at RT. Taken together, these results suggest that during FD sperm rehydration, rapid water infiltration into the nucleus is more effective in preventing DNA damage than slower water infiltration. It has been reported that when cellular DNA experiences drying, it adopts a compact A-DNA structure 30 . In sperm, the nucleus is composed of protamine instead of histones 31 , making it unclear whether drying can induce an A-DNA structure in sperm DNA as it does in other cell types. However, since FD alters the sperm plasma membrane and increases DNA damage 7 , it is likely that structural changes also occur within the sperm nucleus. When the structural changes are returned from the dried state to the original during rehydration, a structural inconsistency may occur between the surface region, where the structure recovers first, and the central region, which can remain dry due to delayed water infiltration. Such a structural inconsistency can cause a high level of damage to chromosomes and/or can exacerbate minor sources of DNA damage already caused by other FD processes. Therefore, when rehydration is performed with hot water, which infiltrates the nucleus quickly, the structure of DNA may be restored uniformly and immediately, thereby minimizing structural inconsistencies and consequently improving the birth rate. Indeed, when we evaluated the DNA damage on each FD sperm treatment, we found that rehydration at low temperatures increased DNA damage relative to the RT control. Conversely, rehydration at higher temperatures resulted in reduced DNA damage. However, while a comet assay showed the lowest level of DNA damage in FD sperm samples rehydrated in water at 70℃, the incidence of ACS in two-cell stage embryos was lowest when the rehydration water was 50℃, and this value increased at temperatures above 70℃ compared to the RT control. We note that the comet assay is most suitable for visualizing minor DNA damage and for evaluating the total amount of DNA damage, whereas the ACS assay is better suited for detecting the presence of severe DNA damage 32 . Taken together, these results indicate that increasing the temperature of the ultrapure water used for rehydration can accelerate infiltration speed into the nucleus, thereby reducing minor DNA damage as the temperature increases. However, although we observed decreases in the level of severe DNA damage at temperatures between 37℃ and 50℃, we also note that the level of damage can increase when temperatures exceed this range. In the past, heat treatments of sperm were considered undesirable since they were known to impair the oocyte activation potential of spermatozoa 33 , 34 . However, subsequent studies revealed that offspring can be obtained via the artificial activation of oocytes following ICSI 34 . In addition, we also found that FD sperm could produce offspring even after brief exposures to temperatures as high as 150℃ 18 . In this study, the maximum temperature of the hot ultrapure water was 90°C, and only 50 µL was added to the FD sperm sample. Therefore, the FD sperm sample was exposed to a temperature of 90°C for only a short time, and it was thought that this would not affect its ability to activate oocytes or facilitate embryo development due to high temperatures. However, the results of the gamma-H2Ax and ACS assays confirmed that DNA damage increased at temperatures 70℃ or higher. It is therefore likely that while FD sperm can tolerate heat exposure within ampoules, they cannot withstand the direct infiltration of hot water into the nucleus. It is then possible that high temperatures not only destroy oocyte activation factors present on the sperm surface but can also denature proteins such as protamine and cause double-stranded breaks in DNA during the process of replacing protamine with histone following fertilization 35 , 36 . Next, we observed that birth rates improved as the temperature of the ultrapure water increased, with the highest birth rates being recorded at 50℃. Interestingly, we observed higher levels of severe DNA damage in sperm when water was added at over 50℃, but the birth rate did not decrease. Perhaps when hot water at temperatures over 50°C is added to FD sperm, the number of embryos with chromosomal abnormalities increases, thereby reducing the proportion of embryos that can develop into live offspring. However, as the water temperature increased, minor DNA damage continued to decrease, leading to an improvement in the overall quality of the embryos. As a result, the rate of live offspring remained unchanged. In addition, if it were possible to exclude ACS embryos before embryo transfer, or to reduce the incidence of ACS 23 , we speculate that the birth rate might be further improved. Overall, this study revealed that DNA damage occurs not only during FD but also during rehydration, and that rapid infiltration can suppress this damage. However, the birth rate of embryos derived from FD sperm remains lower than that of fresh sperm. To further improve birth rate, it is necessary to elucidate which mechanisms of DNA damage are induced by all processes involved in FD sperm production and to develop new methods for minimizing this damage. If DNA damage can be prevented throughout the process, it should be possible to achieve birth rates comparable to those of fresh sperm. In recent years, global warming and the spread of emerging infectious diseases has highlighted the importance of genetic resources 1 – 3 . While sperm is the most suitable genetic resource, for individuals from whom sperm cannot be collected, a round spermatid (i.e., a sperm progenitor cell) can be harvested from infertile males 37 , 38 and somatic cells 39 , which are present in any individual regardless of age or sex, are also a valuable genetic resources 40 . Although research on FD these cells remains ongoing, the success rate remains very low, and a practical use has not yet been established 41 , 42 . If rapid infiltration using hot water can reduce DNA damage and improve the birth rate of FD cells, their utility as genetic resources would increase significantly. Although oocytes have not yet been successfully preserved using FD techniques, the chances of success may increase if they are rehydrated using hot water. The findings of this study are expected to contribute significantly to future enhancements of the reliability and practical application of FD technology for genetic resource preservation. Materials and Methods Animals Female and male ICR mice (8–10 weeks old) were first obtained from SLC Inc. (Hamamatsu, Japan). Surrogate pseudo pregnant ICR females were then prepared for embryo implantation by mating with vasectomized ICR males that had been previously confirmed as sterile. All mice were euthanized (see Embryo transfer of this Method section) either on the day on which the experiment was performed or following the completion of experiments via CO 2 inhalation or cervical dislocation. All animal experiments followed the ARRIVE animal care guidelines. Moreover, all experiments were also performed in accordance with the guidelines for the Care and Use of Laboratory Animals and the specific experimental protocols employed in this study were approved by the Institutional Committee of Laboratory Animal Experimentation of the University of Yamanashi (Ref. No.: A29-24). Media HTF medium was used for capacitation and FD of spermatozoa as well as for spermatozoa rehydration 43 . HEPES-CZB 44 and CZB 45 media were used for oocyte/embryo manipulation and embryos were incubated in 5% CO 2 at 37°C. Preparation of FD spermatozoa To prepare FD sperm, both epididymides were first collected from male ICR mice, and the ducts were severed using sharp scissors. A few drops of the dense spermatozoa mass was then added into a centrifuge tube containing 850 μL of HTF medium. This mixture was then incubated for 30 min at 37°C and 5% CO 2 . Next, the concentration and motility of spermatozoa were determined under a microscope, and 400 µl of supernatant was collected. An aliquot of 50 µl spermatozoal suspension was then dispensed into each glass ampoule. Next, the ampoules were flash frozen in LN 2 then freeze-dried using an FDU-2200 freeze dryer (EYELA, Tokyo, Japan) 12,15 . The cork of the freeze-dryer was opened for at least 6 h until all samples had completely dried. After drying, ampoules were sealed by melting their glass necks under vacuum using a gas burner. All ampoules were then stored in a freezer at −30°C until further use to avoid damage during storage. Ampoules were preserved for different periods, ranging from three days to one month before use 18 . Detection of air trapped in ampoules using a Tesla coil leak detector Air within the ampoules were detected using a Tesla coil leak detector (Sanko Electronic Laboratory, Kanagawa, Japan) with all procedures performed according to the manufacturer’s instructions. When the tip of the Tesla coil is brought near the ampoule, the tip forms sparks near the glass. However, if a large amount of air is trapped within the ampoule, this air cannot be ionized. However, if the ampoule contains only a small amount of residual air, its ionization produces a spark within the ampoule. Only Tesla-positive ampoules, i.e., those containing almost no air if not no air whatsoever, were used for all experiments 12,13 . Rehydration of FD Spermatozoa For rehydration, we first opened FD sperm ampoules immediately prior to use, and to each ampoule 50 μL of rehydration solution was added. The rehydration solution used was specific to each experimental condition. After this addition, ampoule contents were immediately mixed thoroughly by pipetting multiple times. Ultrapure water at room temperature was used as the control solution for all experiments. For osmotic pressure experiments, we used HTF medium (Fig. 1b, left), while for high-viscosity experiments, we used PVP solutions with concentrations of 1.5%, 3%, 6%, and 12% (Fig. 1b, center). Finally, for temperature experiments, we used ultrapure water heated to 0.5°C, 37°C, 50°C, 70°C, or 90°C (Fig. 1b, right). The temperature of ultrapure water was adjusted using an ice-water bath or a water bath. Measurement of Infiltration Speed Next, to verify whether the liquid infiltration speed varied in response to different experimental conditions, we performed a simplified measurement of infiltration speed using filter paper. Here, we used ultrapure water at RT as a control condition for all experiments. The experimental conditions included HTF medium, PVP solutions at concentrations of 1.5%, 3%, 6%, and 12%, as well as ultrapure water adjusted to 0.5°C, 37°C, 50°C, 70°C, and 90°C. For each experimental condition, a 50 μL aliquot of rehydration solution was dropped onto a filter paper cut to a width of 5 mm, and measured the distance permeated on the filter paper after 5–10 s. By recording the distance of medium infiltration over time, we were able to calculate the infiltration speed. Oocyte preparation To superovulate female mice, we injected each with 5 IU of equine chorionic gonadotropin, followed by 5 IU of human chorionic gonadotropin 48 h later. After 14–16 hours, we collected cumulus-oocyte complexes (COCs) from the oviducts of all female mice and transferred the COCs to a Falcon dish containing HEPES-CZB medium. Next, we dispersed the cumulus by transferring COCs to a 50 µl droplet of HEPES-CZB medium containing 0.1% bovine testicular hyaluronidase and incubating at RT for 3 minutes. The resulting cumulus-free oocytes were then washed twice before being transferred to a 20 μL droplet of CZB for further culturing. ICSI and artificial oocyte activation ICSI was performed as per a previously described protocol 44 , and the sperm involved were prepared as mentioned above. For each sperm microinjection, 1–2 μL of sperm suspension was transferred directly to the injection chamber. The sperm suspension was then replaced every 30 min during ICSI. For injection, the sperm head was first separated from the tail by applying piezoelectric pulses, and the tailless head was then microinjected into the oocyte. Next, all oocytes that survived ICSI were incubated in CZB medium at 37°C with 5% CO 2 . After 15 min, some oocytes were artificially activated by immersion in 5 mM strontium chloride for 1 h. After incubation, these were cultured again at 37°C in 5% CO 2 CZB medium. Finally, pronucleus formation was assessed 6 h after ICSI. Embryo transfer For embryo transfer, embryos at the two-cell stage implanted into a day 0.5 pseudo pregnant ICR female mouse that had mated with a vasectomized male the night before the transfer. On the day of embryo transfer, recipients were first anesthetized via intraperitoneal injection of medetomidine, midazolam, and butorphanol. After completion of embryo transfer, atipamezole was administered, and mice were kept warm until they regained consciousness. A total of 6–10 embryos were transferred into each oviduct. On day 18.5 of gestation, offspring were delivered via cesarean section and allowed to mature normally. Remaining unused embryos were cultured for up to four days to evaluate their potential for development into blastocysts. Analysis and scoring of comet slides DNA damage to spermatozoa, including single- and double-strand breaks 46 was measured using a CometAssay™ Kit (Trevigen, MD, USA) with all procedures performed as per the manufacturer’s protocol. Briefly, spermatozoa specimens were first collected from ampoules immediately after opening and were then rehydrated in water. Specimens were then mounted on slides, and 100–300 spermatozoa heads on each slide were subsequently analyzed via electrophoresis. To standardize the results obtained from the different conditions under which spermatozoa were produced, the length of each DNA comet tail was divided by the mean length of the one-sided results of each experiment. Gamma-H2Ax assays Histone H2Ax is an important variant of H2A, as it contains a serine residue at position 139 that is rapidly phosphorylated within seconds of DNA damage. The resulting phosphorylated H2Ax, known as gamma-H2Ax, then forms foci at the sites of DNA damage, which leads to the recruitment of various repair and cell-cycle checkpoint proteins. Given this role, we used gamma-H2Ax foci formation as a marker of DNA double-strand breaks observed in male and female pronuclei. For this experiment, all specimens were first fixed with 4% paraformaldehyde (PFA; Wako Pure Chemical, Osaka, Japan) containing 0.2% Triton X at RT for 20 min before being stored in a refrigerator until staining. The primary antibody used for zygote immunostaining was the anti-phospho-H2Ax (Ser139) rabbit polyclonal antibody (1:500; Millipore-Merck, Darmstadt, Germany), while the secondary antibody used was the Alexa Fluor 568-labeled goat antirabbit IgG (1:500; Molecular Probes). Moreover, DNA was stained using 4′6-diamidino-2-phenylindole (2 µg/ml; Molecular Probes). We detected several DNA repair sites in male pronuclei. However, we found that counting the number of repair sites within a pronucleus was challenging. Therefore, the brightness of each male pronucleus was measured using NIH ImageJ and was then subtracted from the brightness of the zygote cytoplasm. Detection of abnormal chromosome segregation (ACS) One day after ICSI, embryos at the two-cell stage were fixed and permeabilized with 4% PFA and 0.5% Triton X-100 for 15 min. These embryos were then immersed in PBS containing DAPI and 1% BSA before being analyzed using a fluorescence microscope (Olympus IX-73, Tokyo, Japan). As previously described, we classified ACS as belonging to on of four groups: light, moderate, heavy, and lethal. Specifically, light ACS was defined as the presence of only one micronucleus 19 , moderate ACS by the presence of two small micronuclei, one to two medium micronuclei, or a single large micronucleus. Heavy ACS involved detection of three small or medium micronuclei or two to three large micronuclei, while lethal ACS involved embryos that contained multiple micronuclei. In some instances, two conditions occurred simultaneously, leading to a more severe evaluation. For example, if an embryo contained one medium and two small micronuclei, we classified it as “heavy.” However, since embryos with low-level ACS can develop normally—i.e., to full term 28 —we instead focused on embryos with severe (i.e., moderate or higher) ACS in this study. Statistical analysis Comet assay results were analyzed using Wilcoxon–Mann–Whitney nonparametric tests or Dunn’s tests. Gamma-H2Ax assay results were analyzed using Kruskal–Wallis tests. Normal fertilization rates, ACS assays, in vitro development rates, and birth rates were evaluated using chi-square tests or Tukey’s WSD tests. For all statistical tests, statistical significance was determined as p < 0.05. Declarations Acknowledgements We thank Y. Kanda for assistance in preparing this manuscript. This work was partially funded by JST SPRING, Grant Number JPMJSP2133 to N. U.; the Research Fellowships of Japan Society for the Promotion of Science for Young Scientists to D.I. (23K19330), S. W. (23K08843) to T.W. (23K18124 and 24K01779); the Naito Foundation and Takahashi Industrial and Economic Research Foundation (189) to S.W.; Asada Science Foundation and the Canon Foundation (M20-0008) to T.W. Author contributions K.Y., D.I., S.W., and T.W. conceived and designed the study. K.Y., S.W., N.U., D.I., and T.W. performed experiments, analyzed the data, and interpreted the results. K.Y. and T.W. wrote the manuscript. All authors read and edited the manuscript. Declaration of competing interests The authors declare that they have no competing interests. Data availability All data generated or analyzed during this study are included in this published article and are available from the corresponding author (Teruhiko Wakayama) on reasonable request.. References Comizzoli, P., Amelkina, O. & Lee, P. C. Damages and stress responses in sperm cells and other germplasms during dehydration and storage at nonfreezing temperatures for fertility preservation. Mol Reprod Dev . 89 , 565-578 (2022). Loi, P., Iuso, D., Czernik, M., Zacchini, F. & Ptak, G. Towards storage of cells and gametes in dry form. Trends Biotechnol . 31 , 688-695 (2013). Saragusty, J. et al. Dry biobanking as a conservation tool in the Anthropocene. Theriogenology . 150 , 130-138 (2020). Benson, J. D., Woods, E. J., Walters, E. M. & Critser, J. K. The cryobiology of spermatozoa. Theriogenology . 78 , 1682-1699 (2012). Sztein, J. M., Takeo, T. & Nakagata, N. History of cryobiology, with special emphasis in evolution of mouse sperm cryopreservation. Cryobiology . 82 , 57-63 (2018). Nollet, K. E., Komazawa, T. & Ohto, H. Transfusion under triple threat: Lessons from Japan's 2011 earthquake, tsunami, and nuclear crisis. Transfus Apher Sci . 55 , 177-183 (2016). Wakayama, T. & Yanagimachi, R. Development of normal mice from oocytes injected with freeze-dried spermatozoa. Nat Biotechnol , 57-63 (1998). Hirabayashi, M., Kato, M., Ito, J. & Hochi, S. Viable rat offspring derived from oocytes intracytoplasmically injected with freeze-dried sperm heads. Zygote . 13 , 79-85 (2005). Muneto, T. & Horiuchi, T. Full-term Development of Hamster Embryos Produced by Injecting Freeze-dried Spermatozoa into Oocytes , Vol. 28 (2011). Liu, J. L. et al. Freeze-dried sperm fertilization leads to full-term development in rabbits. Biology of Reproduction . 70 , 1776-1781 (2004). Choi, Y. H., Varner, D. D., Love, C. C., Hartman, D. L. & Hinrichs, K. Production of live foals via intracytoplasmic injection of lyophilized sperm and sperm extract in the horse. Reproduction . 142 , 529-538 (2011). Kamada, Y. et al. Assessing the tolerance to room temperature and viability of freeze-dried mice spermatozoa over long-Term storage at room temperature under vacuum. Scientific Reports . 8 (2018). Kamada, Y. et al. Method for long-term room temperature storage of mouse freeze-dried sperm. Sci Rep . 15 , 303 (2025). Ito, D. et al. Effect of trehalose on the preservation of freeze-dried mice spermatozoa at room temperature. J Reprod Dev . 65 , 353-359 (2019). Ito, D. et al. T. Mailing viable mouse freeze-dried spermatozoa on postcards. iScience . 24 (2021). Yang, L. L. et al. A novel, simplified method to prepare and preserve freeze-dried mouse sperm in plastic microtubes. J Reprod Dev . 69 , 198-205 (2023). Palazzese, L. et al. Reviving vacuum-dried encapsulated ram spermatozoa via ICSI after 2 years of storage. Front Vet Sci. 10 (2023). Wakayama, S. et al. Tolerance of the freeze-dried mouse sperm nucleus to temperatures ranging from −196 °C to 150 °C. Scientific Reports . 9 , 1270266 (2019). Wakayama, S. et al. Evaluating the long-term effect of space radiation on the reproductive normality of mammalian sperm preserved on the International Space Station. Sci Adv . 7 (2021). Wakayama, S. et al. Healthy offspring from freeze-dried mouse spermatozoa held on the International Space Station for 9 months. Proceedings of the National Academy of Sciences of the United States of America . 114 , 5988-5993 (2017). Kusakabe, H., Szczygiel, M. A., Whittingham, D. G. & Yanagimachi, R. Maintenance of genetic integrity in frozen and freeze-dried mouse spermatozoa (2001). Ward, M. A. et al. Long-Term Preservation of Mouse Spermatozoa after Freeze-Drying and Freezing Without Cryoprotection. Biology of Reproduction . 69 , 2100-2108 (2003). Shibasaki, I. et al. Extracting and analyzing micronuclei from mouse two-cell embryos fertilized with freeze-dried spermatozoa. Commun Biol . 8 , 6 (2025). Wakayama, T. & Ogura, A. In memory of Dr. Ryuzo Yanagimachi (Yana) (1928-2023). J Reprod Dev . 70 , i-iv (2024). Kuretake, S., Kimura, Y., Hoshi, K. & Yanagimachi, R. Fertilization and Development of Mouse Oocytes Injected with Isolated Sperm Heads' , Vol. 55 (1996). Wakayama, T., Whittingham, D. G. & Yanagimachi, R. Production of normal offspring from mouse oocytes injected with spermatozoa cryopreserved with or without cryoprotection (1998). Ushigome, N. et al. Production of offspring from vacuum-dried mouse spermatozoa and assessing the effect of drying conditions on sperm DNA and embryo development. J Reprod Dev . 68 , 262-270 (2022). Mashiko, D. et al. Chromosome segregation error during early cleavage in mouse pre-implantation embryo does not necessarily cause developmental failure after blastocyst stage. Sci Rep . 10 , 854 (2020). Saadon, A., Abdullah, J., Muhammad, N. S., Ariffin, J. & Julien, P. Y. Predictive models for the estimation of riverbank erosion rates. Catena . 196 (2021). Franklin, R. E. & Goslino, X. d. R. G. The Structure of Sodium Thymonucleate Fibres. I. The Influence of Water Content , Vol. 6 (1953). Bao, J. & Bedford, M. T. Epigenetic regulation of the histone-to-protamine transition during spermiogenesis , Vol. 151 (BioScientifica Ltd., 2016). Yamagata, K., Suetsugu, R. & Wakayama, T. Assessment of chromosomal integrity using a novel live-cell imaging technique in mouse embryos produced by intracytoplasmic sperm injection. Hum Reprod . 24 , 2490-2499 (2009). Chao, S. B. et al. Heated spermatozoa: Effects on embryonic development and epigenetics. Human Reproduction . 27 , 1016-1024 (2012). Cozzi, J., Monier-Gavelle, F., Liè, N., Bomsel, M. & Wolf, J. P. Mouse Offspring after Microinjection of Heated Spermatozoa , Vol. 65 (2001). Aitken, R. J. & De Iuliis, G. N. On the possible origins of DNA damage in human spermatozoa. Mol Hum Reprod . 16 , 3-13 (2010). Yanagida, K., Yanagimachi, R., Perreault, S. D. & Kleinfeld, R. G. Thermostability of sperm nuclei assessed by microinjection into hamster oocytes. Biol Reprod . 44 , 440-447 (1991). Kimura, Y. & Yanagimachi, R. Mouse oocytes injected with testicular spermatozoa or round spermatids can develop into normal offspring. Development . 121 , 2397-2405 (1995). Ogura, A., Matsuda, J. & Yanagimachi, R. Birth of normal young after electrofusion of mouse oocytes with round spermatids. Proc Natl Acad Sci U S A . 91 , 7460-7462 (1994). Wakayama, T., Perry, A. C., Zuccotti, M., Johnson, K. R. & Yanagimachi, R. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature . 394 , 369-374 (1998). Wakayama, S. & Wakayama, T. Can humanity thrive beyond the galaxy? J Reprod Dev (2024). Wakayama, S., Ito, D., Hayashi, E., Ishiuchi, T. & Wakayama, T. Healthy cloned offspring derived from freeze-dried somatic cells. Nat Commun . 13 , 3666 (2022). Wakayama, S., Ito, D., Ooga, M. & Wakayama, T. Production of mouse offspring from zygotes fertilized with freeze-dried spermatids. Sci Rep . 12 , 18430 (2022). Quinn, P., Moinipanah, R., Steinberg, J. M. & Weathersbee, P. S. Successful human in vitro fertilization using a modified human tubal fluid medium lacking glucose and phosphate ions. Fertil Steril . 63 , 922-924 (1995). Kimura, Y. & Yanagimachi, R. Intracytoplasmic Sperm Injection in the Mouse' , Vol. 52 (1995). Chatot, C. L., Ziomek, C. A., Bavister, B. D., Lewis, J. L. & Torres, I. An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. J Reprod Fertil . 86 , 679-688 (1989). Haines, G., Marples, B., Daniel, P. & Morris, I. DNA damage in human and mouse spermatozoa after in vitro-irradiation assessed by the comet assay. Adv Exp Med Biol . 444 , 79-91; discussion 92-73 (1998). Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5994995","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":414457484,"identity":"666be23b-daba-4164-bad1-1eb22961d034","order_by":0,"name":"Kango Yamaji","email":"","orcid":"","institution":"University of Yamanashi","correspondingAuthor":false,"prefix":"","firstName":"Kango","middleName":"","lastName":"Yamaji","suffix":""},{"id":414457485,"identity":"fe737185-7d20-4723-a410-0b837c11c5df","order_by":1,"name":"Sayaka Wakayama","email":"","orcid":"","institution":"University of Yamanashi","correspondingAuthor":false,"prefix":"","firstName":"Sayaka","middleName":"","lastName":"Wakayama","suffix":""},{"id":414457486,"identity":"da2d2217-d0a7-4476-b29e-0e865add5603","order_by":2,"name":"Natsuki Ushigome","email":"","orcid":"","institution":"University of Yamanashi","correspondingAuthor":false,"prefix":"","firstName":"Natsuki","middleName":"","lastName":"Ushigome","suffix":""},{"id":414457487,"identity":"5d6ed8fa-a1e8-49f0-b119-fdf9a7d42ab5","order_by":3,"name":"Daiyu Ito","email":"","orcid":"","institution":"University of Yamanashi","correspondingAuthor":false,"prefix":"","firstName":"Daiyu","middleName":"","lastName":"Ito","suffix":""},{"id":414457488,"identity":"649a4831-040d-46fa-a445-4216fedde569","order_by":4,"name":"Teruhiko Wakayama","email":"data:image/png;base64,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","orcid":"","institution":"University of Yamanashi","correspondingAuthor":true,"prefix":"","firstName":"Teruhiko","middleName":"","lastName":"Wakayama","suffix":""}],"badges":[],"createdAt":"2025-02-10 02:38:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5994995/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5994995/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":76089477,"identity":"58d98ba1-48cb-47d6-a62a-973ced7301bb","added_by":"auto","created_at":"2025-02-12 08:20:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":740444,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of preparation of freeze-dried spermatozoa, rehydration conditions, and subsequent experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Spermatozoa were first collected from male mice and were capacitated before being aliquoted into glass ampoules in aliquots of 50 μL. Ampoules were frozen in liquid nitrogen and freeze-dried using a vacuum freeze-dryer. Glass ampoules were then opened prior to subsequent experiments and rehydration was then performed.\u003c/p\u003e\n\u003cp\u003e(b) Rehydration was conducted using media subjected to various conditions. To decrease infiltration speed, we used an HTF medium with a higher osmotic pressure than ultrapure water. To further reduce infiltration speed, we also used PVP solutions with higher viscosity. In addition, low-temperature ultrapure water was used to decrease infiltration speed, while high-temperature ultrapure water was used to increase it. To evaluate the changes in the infiltration speed under these conditions, ewe performed a simplified measurement of the infiltration distance per unit time using filter paper.\u003c/p\u003e\n\u003cp\u003e(c) After adding different rehydration solutions to glass ampoules, we conducted the following experiments: (1) a comet assay to evaluate the degree of DNA damage in spermatozoa, (2) an analysis of gamma-H2Ax foci to assess damage at the pronuclear stage after fertilization, (3) measurements of ACS rates in two-cell embryos, (4) an evaluation of the rate of development to the blastocyst stage, and (5) an assessment of offspring rates after transferring two-cell embryos.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5994995/v1/6b862cac57fc7668b9262148.png"},{"id":76090806,"identity":"b3d07b7e-79e8-4083-b86a-0a29f9963696","added_by":"auto","created_at":"2025-02-12 08:28:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":728239,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in infiltration speed, DNA damage, and offspring rates under rehydration conditions under varying osmotic pressure or viscosity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Glass ampoules containing freeze-dried (FD) spermatozoa.\u003c/p\u003e\n\u003cp\u003e(b) Freeze-dried spermatozoa showing separation of the sperm head during freeze-drying.\u003c/p\u003e\n\u003cp\u003e(c, d) Simplified measurement of infiltration speed using filter paper. Briefly, rehydration solutions reflecting different experimental conditions were dropped onto filter paper, and we measured the infiltration distance per unit time. Background colors on the graph represent different conditions; shown are ultrapure water (the control for all experimental conditions) in white, HTF medium in blue, PVP solution in yellow, and ultrapure water (various temperatures) in red. RT indicates room temperature. Each data point represents an independent measurement of infiltration speed, with bars indicating the mean and error bars representing the standard error of the mean (SEM).\u003c/p\u003e\n\u003cp\u003e(e) DNA damage in FD sperm rehydrated with HTF medium or PVP solution evaluated using comet assays. Background colors on the graph represent different conditions; blue indicates experimental conditions where osmotic pressure was altered using HTF medium, yellow represents conditions where viscosity was modified using PVP solution. Comet tail lengths were normalized to those of FD sperm rehydrated with ultrapure water (control). The vertical axis represents relative DNA damage, with higher values indicating greater damage. Each data point represents avalue recorded for an individual spermatozoa, witherror bars showingstandard deviation. Anasterisk denotes statistically significant differences between pairs of samples (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Different letters indicate statistically significantly different group means (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e(f) Images show the ultrapure water control group (top left), the HTF medium experimental group(top right), the 0% PVP solution control group (bottom left), and the 12% PVP solution experimental group (bottom right).\u003c/p\u003e\n\u003cp\u003e(g) Offspring rates were evaluated in intracytoplasmic sperm injection (ICSI) embryos using FD sperm rehydrated with HTF medium or PVP solution. Background colors on the graph represent different conditions; blue indicates experimental conditions where osmotic pressure was altered using HTF medium, yellow represents conditions where viscosity was modified using PVP solution. Asterisks denote statistically significant differences between pairs of samples (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5994995/v1/cc570b282de6a1256e173eea.png"},{"id":76089474,"identity":"0a409cd3-cd22-424c-83d3-26b46568896b","added_by":"auto","created_at":"2025-02-12 08:20:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":999755,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in DNA damage and developmental potential following rehydration using ultrapure water at different temperatures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Observed DNA damage in FD sperm rehydrated with ultrapure water at different temperatures as evaluated using comet assays. Different letters indicate statistically significantly different group means (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e(b) gamma-H2Ax assay of fertilized embryos derived from FD sperm rehydrated with ultrapure water at different temperatures. Images show male and female pronuclei stained with 4′6-diamidino-2-phenylindole (DAPI, blue, top left), gamma-H2Ax signals indicating double-stranded DNA breaks (red, top right), bright-field images (bottom left), and merged images (bottom right).\u003c/p\u003e\n\u003cp\u003e(c) Relative brightness of male pronuclei sourced from fertilized embryos derived from FD sperm rehydrated at different temperatures. Higher brightness values indicate more DNA damage.\u003c/p\u003e\n\u003cp\u003e(d) Two-cell embryos derived from FD sperm rehydrated with ultrapure water at 50°C after staining with DAPI. The upper left image shows a two-cell embryo with normal chromosome segregation (NCS), while the lower left image shows a two-cell embryo with abnormal chromosome segregation (ACS). The enlarged image on the right shows a highlighted portion of the ACS embryo, and the arrowhead indicates the micronucleus.\u003c/p\u003e\n\u003cp\u003e(e) Proportion of embryos with moderate or higher ACS among all embryos derived from FD sperm rehydrated with ultrapure water at different temperatures.\u003c/p\u003e\n\u003cp\u003e(f) Offspring derived from ICSI embryos using FD sperm rehydrated with ultrapure water at 50°C.\u003c/p\u003e\n\u003cp\u003e(g) Normal fertilization rates and offspring rates of ICSI embryos using FD sperm rehydrated with ultrapure water at different temperatures. Asterisks denote statistically significant differences in sample means (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5994995/v1/e39981aa3caece5d611220fe.png"},{"id":83773532,"identity":"eb7cdadc-12b8-4725-a3fb-6920cdb4a6b3","added_by":"auto","created_at":"2025-06-02 13:16:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3644192,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5994995/v1/d14523b4-2d95-4580-bef7-5806cfdc60b7.pdf"},{"id":76089480,"identity":"233b4de9-c131-4a63-bb41-3c6ef60fa51e","added_by":"auto","created_at":"2025-02-12 08:20:48","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":162193,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5994995/v1/44b8456431904b12fd4c8d51.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eImproved Birth Rates via Rehydration of Mouse Freeze-Dried Spermatozoa using High-Temperature Ultrapure Water\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMammalian gametes are preserved for a variety of purposes, including infertility treatments, the conservation of endangered species, the cost-effective storage of genetically modified organisms, and the efficient transportation of mouse strains \u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The standard method for preserving mammalian spermatozoa is cryopreservation using liquid nitrogen\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. This technique maintains sperm motility post-thaw, which makes it suitable for \u003cem\u003ein vitro\u003c/em\u003e fertilization (IVF)\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. However, handling liquid nitrogen is challenging and poses risks of frostbite, asphyxiation, and rapid gas expansion. Moreover, it is also necessary to frequently replenish liquid nitrogen to keep temperatures low, and specialized containers are required for storage and transport, leading to high maintenance costs\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Furthermore, during disruptions of the liquid nitrogen supply (e.g., during natural disasters) there is a risk of losing all stored samples\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFreeze-drying technologies offer a promising solution to simultaneously address the multiple challenges associated with cryopreservation. Since 1998, when the first successful Freeze-drying of mouse sperm was demonstrated\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, viable offspring have been produced from embryos fertilized with freeze-dried (FD) sperm from rats\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, hamsters\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, rabbits\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, and horses\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Initially, the room temperature (RT) storage period of FD sperm was limited to only one month\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. However, by sealing FD sperm in ampoules under a high vacuum, sperm storage at RT for longer periods became possible, with some studies reporting successful storage for periods of up to six years \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. This technology eliminates the need for liquid nitrogen, and permits storage in compact spaces such as desk drawers\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Overall, this technique enables space-efficient and cost-effective storage while reducing the risk of sample loss during disasters. In addition, the development of safer and more user-friendly storage containers, such as plastic sheets\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, microtubes\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, and mini stainless steel tubes\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, is currently underway. Furthermore, sperm subjected to FD pretreatment show improved resistance to extreme temperatures, including from \u0026minus;\u0026thinsp;196\u0026deg;C to 150\u0026deg;C\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. FD pretreatment has also been found to improve survival following exposure to space radiation aboard the International Space Station for over 200 years\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Taken together, these findings highlight the advantages of FD preservation over cryopreservation for the long-term storage of sperm.\u003c/p\u003e \u003cp\u003eHowever, the birth rates of embryos derived from FD sperm are less than one-third of those of embryos derived from fresh or cryopreserved sperm\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Thus, for FD methods to become more widely used for preserving genetic resources, it is essential to improve the rate at which they reliably produce offspring from preserved sperm. Recently, our lab has produced mouse offspring from FD sperm stored at RT for six years. Although the reliability of this method for long-term storage at RT has increased, the low birth rate remains an unsolved problem\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In animals such as cattle, which produce only one offspring per pregnancy, low birth rates necessitate multiple attempts, which can result in high overall costs despite improvements in sperm storage efficiency and safety. Overall, despite the significant advantages of FD technology, the low birth rate of embryos derived from FD sperm is a major barrier to its widespread use.\u003c/p\u003e \u003cp\u003eEmbryos fertilized with FD sperm frequently exhibit both DNA damage in the paternal pronucleus and chromosomal segregation abnormalities (ACS)\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. These findings suggest that sperm DNA damage occurs at some stage of FD sperm preparation or use. Preparing and using FD sperm is a process that consists of four steps: freezing, drying, storage at RT, and rehydration of FD sperm. During the first sperm freezing, cryoprotectants such as DMSO or glycerol cannot be used because they cannot be dried, therefore all the sperm die after freezing. However, intracytoplasmic sperm injection (ICSI) allows injection of all sperm\u0026mdash;including dead sperm\u0026mdash;into oocytes \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, thereby achieving normal fertilization and full-term development. This indicates that decreases in the birth rate of FD sperm are not due to the freezing step\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. During the drying step, experiments using vacuum-dried sperm, in which sperm were dried without freezing, demonstrated that viable offspring could be obtained, although they showed lower birth rates than FD sperm\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Taken together, these findings suggest that damage to FD sperm primarily occurs during drying, whereas introducing a freezing step mitigates damage caused by drying.\u003c/p\u003e \u003cp\u003eDuring RT storage, damage to FD sperm is primarily caused by air contamination. Therefore, storing ampoules under a high vacuum reduces damage to FD sperm and enables long-term RT preservation\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Interestingly, the addition of exogenous trehalose to the storage medium did not mitigate damage to sperm during freezing and drying, and was found to significantly improved birth rates when FD sperm were preserved for longer periods at RT\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. This finding indicates that reduced birth rates of embryos derived from FD sperm following preservation at RT results not only from damage to FD sperm during freezing and drying but also from additional damage sustained during RT storage.\u003c/p\u003e \u003cp\u003eIn this study, we focused on the rehydration of FD sperm, a step that has not yet been thoroughly investigated. To do so, we prepared three types of solutions with varying osmotic pressures, viscosities, and temperatures to alter the speed of water infiltration during rehydration. We therefore used these infiltration treatments to investigate the effect of different rehydration regimes on DNA damage in FD sperm, fertilization rates following ICSI using FD sperm, embryo development rates, and offspring rates.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEffects of the Osmotic Pressure of Rehydration Solutions on FD Sperm\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate whether water infiltration speed during rehydration of FD sperm caused DNA damage, we rehydrated FD sperm with either ultrapure water or HTF medium, i.e., the basal medium used for FD sperm production (Fig. 1a, b, 2a, b).\u0026nbsp;First, to measure solution infiltration speed, we dropped ultrapure water and HTF medium at RT onto filter paper and measured the diffusion distance per unit time (Fig. 2c). The infiltration speed of the HTF medium was approximately 0.85 times slower than ultrapure water (Fig. 2d; Supplementary Table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, to assess DNA damage in FD sperm, we performed a comet assay immediately after rehydration with either ultrapure water or HTF medium (Fig. 1c). When rehydration was performed with HTF medium, the comet tail, representing DNA damage, showed a slight but significant increase compared to ultrapure water (ultrapure water: 1.00 vs. HTF medium: 1.02, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) (Fig. 2e, f; Supplementary Table 2). Next, we injected rehydrated sperm into oocytes via ICSI, and evaluated the fertilization rate, the development rate to the two-cell stage, and the birth rate. We found no significant differences at any of these stages between embryos derived from FD sperm rehydrated with ultrapure water and those rehydrated with HTF medium (Fig. 2g; Supplementary Table 3). Therefore, our results indicate that rehydration with HTF medium, in which the infiltration speed was slightly slower than in ultrapure water, did not result in reduced sperm DNA damage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of Rehydration Solution Viscosity on FD Sperm\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, to investigate whether changes in the infiltration speed caused by the viscosity of the rehydration solution affected the degree of DNA damage, we added PVP to ultrapure water to achieve concentrations ranging from 1.5% to 12%, then used these higher-viscosity solutions for rehydration (Fig. 1b). First, we assessed the infiltration speed of PVP solutions by dropping each solution onto filter paper and measuring the diffusion distance per unit time. These results showed that the infiltration speed of PVP solutions decreased in a concentration-dependent manner relative to that of ultrapure water (Fig. 2d; Supplementary Table 1).\u003c/p\u003e\n\u003cp\u003eNext, we performed a comet assay to evaluate the degree of DNA damage in FD sperm rehydrated with different PVP solutions. Except for the 1.5% and 3% concentrations, we found significant increases in DNA damage as the PVP concentration increased (Fig. 2e, f; Supplementary Table 4). Subsequently, FD sperm rehydrated with 6% and 12% PVP solutions were used for ICSI, and we examined ACS at the two-cell stage. ACS can detect large-scale chromosomal damage that cannot be assessed by comet assays. These results showed that ACS rates increased when rehydration was performed using 6% PVP, and the incidence of severe or lethal ACS was 3.5 times higher relative to the ultrapure water control (Supplementary Fig. 1a, b; Supplementary Table 5). Furthermore, in 12% PVP samples, we also observed elevated ACS rates, although not as markedly as for 6% PVP samples (Supplementary Fig. 1a, b; Supplementary Table 5). When ICSI was performed using FD sperm rehydrated with 0\u0026ndash;12% PVP solutions, we did not observe significant differences in fertilization rates or developmental rates to the two-cell stage (Supplementary Fig. 1d; Supplementary Table 6). However, we did observe that\u0026nbsp;\u003cem\u003ein vitro\u003c/em\u003e development rates decreased as the PVP concentration increased, and the blastocyst formation rate was significantly lower for samples containing the highest viscosity solution, 12% PVP (Supplementary Fig. 1c, d; Supplementary Table 6). Furthermore, when some of the two-cell stage embryos were transferred to recipient mice to assess birth rates, we also observed a significant decrease in birth rates as the PVP concentration increased (Fig. 2g, Supplementary Table 7).\u003c/p\u003e\n\u003cp\u003eBased on these results, we concluded that ultrapure water is the most suitable medium for rehydrating FD sperm. In addition, since slower infiltration speeds cause more damage to FD sperm and reduce the birth rate to a greater degree, we next performed experiments using heated ultrapure water to increase infiltration speed during the rehydration of FD sperm (Fig. 1b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of Rehydration Solution Temperature on FD Sperm\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, to examine the infiltration speed of the rehydration solution, ultrapure water cooled or heated between 0.5\u0026deg;C and 90\u0026deg;C was applied to filter paper, and the diffusion distance per unit time was measured (Fig. 2c). As a result, the infiltration speed increased proportionally with increasing temperature (Fig. 2d, Supplementary Table 1).\u003c/p\u003e\n\u003cp\u003eTo investigate the effect of the water temperature on DNA damage in sperm, we then performed a comet assay on FD sperm rehydrated using medium at each temperature. When rehydration was performed at 0.5\u0026deg;C, we observed significantly higher DNA damage in sperm relative to the RT (23\u0026deg;C\u0026ndash;25\u0026deg;C) (Fig. 3a; Supplementary Table 8). Furthermore, DNA damage significantly decreased as the temperature of the rehydration solution increased above RT, with the lowest DNA damage observed at 70\u0026deg;C (relative value: 1.00 vs. 0.85, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) (Fig. 3a; Supplementary Table 8).\u003c/p\u003e\n\u003cp\u003eNext, we performed ICSI using FD sperm rehydrated at each temperature. In preliminary experiments, the activation rate after ICSI tended to be lower when FD sperm were rehydrated with water at 70\u0026deg;C. To account for this, in this experiment some oocytes were artificially activated with SrCl₂ after ICSI (Table 1). When male pronuclei of one-cell embryos were examined using gamma-H2Ax antibodies, we found that rehydration at 0.5\u0026deg;C resulted in higher gamma-H2Ax fluorescence intensity relative to RT rehydration, which indicates that increased DNA damage was present (Fig. 3b, c; Supplementary Table 9). In contrast, rehydration at 37\u0026deg;C resulted in the lowest gamma-H2Ax fluorescence intensity compared to the RT control, which is suggestive of reduced DNA damage. However, rehydration at temperatures above 50\u0026deg;C showed DNA damage levels that were comparable to or slightly higher than those observed in samples dehydrated at RT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Full-term development rates of ICSI embryos using FD sperm rehydrated with ultrapure water at different temperatures.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"742\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTemperature\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(℃)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOocyte\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eactivation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. of\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eoocytes\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003esurviving\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eafter ICSI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. (%) of oocytes\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003esurviving after\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eoocyte activation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. (%) of\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003efertilized\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ezygotes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. (%) of\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eembryos\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003edeveloped\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eto 2 cell\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. of\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003etransferred\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eembryos\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e[no. of\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003erecipients]\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. (%)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e[min-max]\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eof offspring\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e－\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e186\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e－\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e146 (78)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e122 (66)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e116 [8]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e28 (24)\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e[8-37]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003eRT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e－\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e261\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e－\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e231 (89)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e197 (75)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e197 [16]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e41 (21)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e[7-43]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e－\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e202\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e－\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e160 (79)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e132 (65)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e117 [9]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e34 (29)\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e[0-56]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e－\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e333\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e－\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e259 (78)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e237 (92)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e226 [14]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e83 (37)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e[8-83]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e－\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e－\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e33 (61)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e28 (52)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e28 [2]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e4 (14)\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e[0-21]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e＋\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e282\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e251 (89)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e231 (92)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e184 (80)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e175 [9]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e55 (31)\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e[18-79]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e－\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e－\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e48 (81)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e42 (71)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e23 [3]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e8 (35)\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e[17-60]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e＋\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e172\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e141 (82)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e126 (89)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e85 (67)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e83 [6]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 98px;\"\u003e\n \u003cp\u003e29 (35)\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e[13-43]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNext, we examined ACS in two-cell embryos, and found that rehydration at 0.5\u0026deg;C increased the incidence of severe ACS (i.e., classified as \u0026rdquo;heavy\u0026rdquo; or \u0026rdquo;lethal\u0026rdquo;) relative to the RT control (Fig. 3d, e; Supplementary Table 10). In contrast, increasing the rehydration temperature above RT tended to reduce the incidence of severe ACS, with the least damage observed at 50\u0026deg;C. Moreover, embryos showing light ACS can sometimes result in viable offspring\u003csup\u003e28\u003c/sup\u003e; if we combine both those embryos scored as normal chromosome segregation (NCS) and Light as \u0026ldquo;normal\u0026rdquo; embryos, and the remaining embryos to be \u0026ldquo;abnormal\u0026rdquo; embryos, we find that the lowest proportion of abnormal embryos (29%) was observed at 50\u0026deg;C. Finally, we transferred two-cell embryos to recipients, and evaluated the resulting birth rate. We found that rehydration at 0.5\u0026deg;C resulted in a birth rate of 24%, which was similar to the rate obtained with RT (21%) (Table 1). However, rehydration at temperatures above RT improved the birth rate to 29% (at 37\u0026deg;C) and 37% (at 50\u0026deg;C) (Fig. 3f, g). In contrast, rehydration at 70\u0026deg;C or higher resulted in a slight decrease in birth rate compared to 50\u0026deg;C (70\u0026deg;C: 31%; 90\u0026deg;C: 35%).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrevious studies have reported that several kinds of damage affect sperm during the drying process. However, this study revealed\u0026mdash;for the first time\u0026mdash;that sperm DNA can also be damaged during rehydration. In addition, here we show that rehydrating with high-temperature ultrapure water\u0026mdash;which has previously been considered to be harmful to sperm\u0026mdash;reduces DNA damage in FD sperm. Furthermore, using conventional methods, the birth rate for mouse FD sperm-derived embryos is approximately 20%, but when following a rehydration protocol using ultrapure water at 50\u0026deg;C, we observed birth rates as high as 37%.\u003c/p\u003e \u003cp\u003eHere, we first hypothesized that\u0026mdash;just as riverbanks erode when exposed to rapid currents\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e\u0026mdash;faster infiltration speeds may damage the DNA of FD sperm. Therefore, we predicted that the current method used to rehydrate FD sperm may cause DNA damage. Based on this hypothesis, we proposed that employing a slower infiltration method during rehydration of FD sperm can mitigate DNA damage and improve the birth rate. However, despite attempts to slow down the infiltration speed by increasing osmotic pressure and viscosity, we found\u0026mdash;contrary to our expectations\u0026mdash;that lower infiltration speed was associated with increased DNA damage in FD sperm, as well as significant decreases in blastocyst formation and birth rates. In contrast to our initial hypothesis, when pure water was heated to high temperatures to speed up infiltration into the nucleus, we observed reduced DNA damage in FD sperm, as well as birth rates that were up to 1.8 times higher than those in FD sperm rehydrated with pure water at RT. Taken together, these results suggest that during FD sperm rehydration, rapid water infiltration into the nucleus is more effective in preventing DNA damage than slower water infiltration.\u003c/p\u003e \u003cp\u003eIt has been reported that when cellular DNA experiences drying, it adopts a compact A-DNA structure\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In sperm, the nucleus is composed of protamine instead of histones\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, making it unclear whether drying can induce an A-DNA structure in sperm DNA as it does in other cell types. However, since FD alters the sperm plasma membrane and increases DNA damage\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, it is likely that structural changes also occur within the sperm nucleus. When the structural changes are returned from the dried state to the original during rehydration, a structural inconsistency may occur between the surface region, where the structure recovers first, and the central region, which can remain dry due to delayed water infiltration. Such a structural inconsistency can cause a high level of damage to chromosomes and/or can exacerbate minor sources of DNA damage already caused by other FD processes. Therefore, when rehydration is performed with hot water, which infiltrates the nucleus quickly, the structure of DNA may be restored uniformly and immediately, thereby minimizing structural inconsistencies and consequently improving the birth rate. Indeed, when we evaluated the DNA damage on each FD sperm treatment, we found that rehydration at low temperatures increased DNA damage relative to the RT control. Conversely, rehydration at higher temperatures resulted in reduced DNA damage.\u003c/p\u003e \u003cp\u003eHowever, while a comet assay showed the lowest level of DNA damage in FD sperm samples rehydrated in water at 70℃, the incidence of ACS in two-cell stage embryos was lowest when the rehydration water was 50℃, and this value increased at temperatures above 70℃ compared to the RT control. We note that the comet assay is most suitable for visualizing minor DNA damage and for evaluating the total amount of DNA damage, whereas the ACS assay is better suited for detecting the presence of severe DNA damage\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Taken together, these results indicate that increasing the temperature of the ultrapure water used for rehydration can accelerate infiltration speed into the nucleus, thereby reducing minor DNA damage as the temperature increases. However, although we observed decreases in the level of severe DNA damage at temperatures between 37℃ and 50℃, we also note that the level of damage can increase when temperatures exceed this range.\u003c/p\u003e \u003cp\u003eIn the past, heat treatments of sperm were considered undesirable since they were known to impair the oocyte activation potential of spermatozoa\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, subsequent studies revealed that offspring can be obtained via the artificial activation of oocytes following ICSI\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. In addition, we also found that FD sperm could produce offspring even after brief exposures to temperatures as high as 150℃\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In this study, the maximum temperature of the hot ultrapure water was 90\u0026deg;C, and only 50 \u0026micro;L was added to the FD sperm sample. Therefore, the FD sperm sample was exposed to a temperature of 90\u0026deg;C for only a short time, and it was thought that this would not affect its ability to activate oocytes or facilitate embryo development due to high temperatures. However, the results of the gamma-H2Ax and ACS assays confirmed that DNA damage increased at temperatures 70℃ or higher. It is therefore likely that while FD sperm can tolerate heat exposure within ampoules, they cannot withstand the direct infiltration of hot water into the nucleus. It is then possible that high temperatures not only destroy oocyte activation factors present on the sperm surface but can also denature proteins such as protamine and cause double-stranded breaks in DNA during the process of replacing protamine with histone following fertilization\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNext, we observed that birth rates improved as the temperature of the ultrapure water increased, with the highest birth rates being recorded at 50℃. Interestingly, we observed higher levels of severe DNA damage in sperm when water was added at over 50℃, but the birth rate did not decrease. Perhaps when hot water at temperatures over 50\u0026deg;C is added to FD sperm, the number of embryos with chromosomal abnormalities increases, thereby reducing the proportion of embryos that can develop into live offspring. However, as the water temperature increased, minor DNA damage continued to decrease, leading to an improvement in the overall quality of the embryos. As a result, the rate of live offspring remained unchanged. In addition, if it were possible to exclude ACS embryos before embryo transfer, or to reduce the incidence of ACS\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, we speculate that the birth rate might be further improved.\u003c/p\u003e \u003cp\u003eOverall, this study revealed that DNA damage occurs not only during FD but also during rehydration, and that rapid infiltration can suppress this damage. However, the birth rate of embryos derived from FD sperm remains lower than that of fresh sperm. To further improve birth rate, it is necessary to elucidate which mechanisms of DNA damage are induced by all processes involved in FD sperm production and to develop new methods for minimizing this damage. If DNA damage can be prevented throughout the process, it should be possible to achieve birth rates comparable to those of fresh sperm. In recent years, global warming and the spread of emerging infectious diseases has highlighted the importance of genetic resources\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. While sperm is the most suitable genetic resource, for individuals from whom sperm cannot be collected, a round spermatid (i.e., a sperm progenitor cell) can be harvested from infertile males\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e and somatic cells\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, which are present in any individual regardless of age or sex, are also a valuable genetic resources \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Although research on FD these cells remains ongoing, the success rate remains very low, and a practical use has not yet been established \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. If rapid infiltration using hot water can reduce DNA damage and improve the birth rate of FD cells, their utility as genetic resources would increase significantly. Although oocytes have not yet been successfully preserved using FD techniques, the chances of success may increase if they are rehydrated using hot water. The findings of this study are expected to contribute significantly to future enhancements of the reliability and practical application of FD technology for genetic resource preservation.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFemale and male ICR mice (8\u0026ndash;10 weeks old) were first obtained from SLC Inc. (Hamamatsu, Japan). Surrogate pseudo pregnant ICR females were then prepared for embryo implantation by mating with vasectomized ICR males that had been previously confirmed as sterile. All mice were euthanized (see Embryo transfer of this Method section) either on the day on which the experiment was performed or following the completion of experiments via CO\u003csub\u003e2\u003c/sub\u003e inhalation or cervical dislocation. All animal experiments followed the ARRIVE animal care guidelines. Moreover, all experiments were also performed in accordance with the guidelines for the Care and Use of Laboratory Animals and the specific experimental protocols employed in this study were approved by the Institutional Committee of Laboratory Animal Experimentation of the University of Yamanashi (Ref. No.: A29-24).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMedia\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHTF medium was used for capacitation and FD of spermatozoa as well as for spermatozoa rehydration \u003csup\u003e43\u003c/sup\u003e. HEPES-CZB\u003csup\u003e44\u003c/sup\u003e and CZB\u003csup\u003e45\u003c/sup\u003e media were used for oocyte/embryo manipulation and embryos were incubated in 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of FD spermatozoa\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo prepare FD sperm, both epididymides were first collected from male ICR mice, and the ducts were severed using sharp scissors. A few drops of the dense spermatozoa mass was then added into a centrifuge tube containing 850 \u0026mu;L of HTF medium. This mixture was then incubated for 30 min at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. Next, the concentration and motility of spermatozoa were determined under a microscope, and 400 \u0026micro;l of supernatant was collected. An aliquot of 50 \u0026micro;l spermatozoal suspension was then dispensed into each glass ampoule. Next, the ampoules were flash frozen in LN\u003csub\u003e2\u003c/sub\u003e then freeze-dried using an FDU-2200 freeze dryer (EYELA, Tokyo, Japan)\u003csup\u003e12,15\u003c/sup\u003e. The cork of the freeze-dryer was opened for at least 6 h until all samples had completely dried. After drying, ampoules were sealed by melting their glass necks under vacuum using a gas burner. All ampoules were then stored in a freezer at \u0026minus;30\u0026deg;C until further use to avoid damage during storage. Ampoules were preserved for different periods, ranging from three days to one month before use\u003csup\u003e18\u003c/sup\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of air trapped in ampoules using a Tesla coil leak detector\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAir within\u0026nbsp;the ampoules were detected using a Tesla coil leak detector (Sanko Electronic Laboratory, Kanagawa, Japan) with all procedures performed according to the manufacturer\u0026rsquo;s instructions. When the tip of the Tesla coil is brought near the ampoule, the tip forms sparks near the glass. However, if a large amount of air is trapped within the ampoule, this air cannot be ionized. However, if the ampoule contains only a small amount of residual air, its ionization produces a spark within the ampoule. Only Tesla-positive ampoules, i.e., those containing almost no air if not no air whatsoever, were used for all experiments\u003csup\u003e12,13\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRehydration of FD Spermatozoa\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor rehydration, we first opened FD sperm ampoules immediately prior to use, and to each ampoule 50 \u0026mu;L of rehydration solution was added. The rehydration solution used was specific to each experimental condition. After this addition, ampoule contents were immediately mixed thoroughly by pipetting multiple times. Ultrapure water at room temperature was used as the control solution for all experiments. For osmotic pressure experiments, we used HTF medium (Fig. 1b, left), while for high-viscosity experiments, we used PVP solutions with concentrations of 1.5%, 3%, 6%, and 12% (Fig. 1b, center). Finally, for temperature experiments, we used ultrapure water heated to 0.5\u0026deg;C, 37\u0026deg;C, 50\u0026deg;C, 70\u0026deg;C, or 90\u0026deg;C (Fig. 1b, right). The temperature of ultrapure water was adjusted using an ice-water bath or a water bath.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of Infiltration Speed\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, to verify whether the liquid infiltration speed varied in response to different experimental conditions, we performed a simplified measurement of infiltration speed using filter paper. Here, we used ultrapure water at RT as a control condition for all experiments. The experimental conditions included HTF medium, PVP solutions at concentrations of 1.5%, 3%, 6%, and 12%, as well as ultrapure water adjusted to 0.5\u0026deg;C, 37\u0026deg;C, 50\u0026deg;C, 70\u0026deg;C, and 90\u0026deg;C. For each experimental condition, a 50 \u0026mu;L aliquot of rehydration solution was dropped onto a filter paper cut to a width of 5 mm, and measured the distance permeated on the filter paper after 5\u0026ndash;10 s. By recording the distance of medium infiltration over time, we were able to calculate the infiltration speed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOocyte preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo superovulate female mice, we injected each with 5 IU of equine chorionic gonadotropin, followed by 5 IU of human chorionic gonadotropin 48 h later. After 14\u0026ndash;16 hours, we collected cumulus-oocyte complexes (COCs) from the oviducts of all female mice and transferred the COCs to a Falcon dish containing HEPES-CZB medium. Next, we dispersed the cumulus by transferring COCs to a 50 \u0026micro;l droplet of HEPES-CZB medium containing 0.1% bovine testicular hyaluronidase and incubating at RT for 3 minutes. The resulting cumulus-free oocytes were then washed twice before being transferred to a 20 \u0026mu;L droplet of CZB for further culturing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eICSI and artificial oocyte activation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eICSI was performed as per a previously described protocol\u003csup\u003e44\u003c/sup\u003e, and the sperm involved were prepared as mentioned above. For each sperm microinjection, 1\u0026ndash;2 \u0026mu;L of sperm suspension was transferred directly to the injection chamber. The sperm suspension was then replaced every 30 min during ICSI. For injection, the sperm head was first separated from the tail by applying piezoelectric pulses, and the tailless head was then microinjected into the oocyte. Next, all oocytes that survived ICSI were incubated in CZB medium at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. After 15 min, some oocytes were artificially activated by immersion in 5 mM strontium chloride for 1 h. After incubation, these were cultured again at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e CZB medium. Finally, pronucleus formation was assessed 6 h after ICSI.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEmbryo transfer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor embryo transfer, embryos at the two-cell stage implanted into a day 0.5 pseudo pregnant ICR female mouse that had mated with a vasectomized male the night before the transfer. On the day of embryo transfer, recipients were first anesthetized via intraperitoneal injection of medetomidine, midazolam, and butorphanol. After completion of embryo transfer, atipamezole was administered, and mice were kept warm until they regained consciousness. A total of 6\u0026ndash;10 embryos were transferred into each oviduct. On day 18.5 of gestation, offspring were delivered via cesarean section and allowed to mature normally. Remaining unused embryos were cultured for up to four days to evaluate their potential for development into blastocysts.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis and scoring of comet slides\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA damage to spermatozoa, including single- and double-strand breaks\u003csup\u003e46\u003c/sup\u003e was measured using a CometAssay\u0026trade; Kit (Trevigen, MD, USA) with all procedures performed as per the manufacturer\u0026rsquo;s protocol. Briefly, spermatozoa specimens were first collected from ampoules immediately after opening and were then rehydrated in water. Specimens were then mounted on slides, and 100\u0026ndash;300 spermatozoa heads on each slide were subsequently analyzed via electrophoresis. To standardize the results obtained from the different conditions under which spermatozoa were produced, the length of each DNA comet tail was divided by the mean length of the one-sided results of each experiment. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGamma-H2Ax assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHistone H2Ax is an important variant of H2A, as it contains a serine residue at position 139 that is rapidly phosphorylated within seconds of DNA damage. The resulting phosphorylated H2Ax, known as gamma-H2Ax, then forms foci at the sites of DNA damage, which leads to the recruitment of various repair and cell-cycle checkpoint proteins. Given this role, we used gamma-H2Ax foci formation as a marker of DNA double-strand breaks observed in male and female pronuclei.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor this experiment, all specimens were first fixed with 4% paraformaldehyde (PFA; Wako Pure Chemical, Osaka, Japan) containing 0.2% Triton X at RT for 20 min before being stored in a refrigerator until staining. The primary antibody used for zygote immunostaining was the anti-phospho-H2Ax (Ser139) rabbit polyclonal antibody (1:500; Millipore-Merck, Darmstadt, Germany), while the secondary antibody used was the Alexa Fluor 568-labeled goat antirabbit IgG (1:500; Molecular Probes). Moreover, DNA was stained using 4\u0026prime;6-diamidino-2-phenylindole (2 \u0026micro;g/ml; Molecular Probes). We detected several DNA repair sites in male pronuclei. However, we found that counting the number of repair sites within a pronucleus was challenging. Therefore, the brightness of each male pronucleus was measured using NIH ImageJ and was then subtracted from the brightness of the zygote cytoplasm.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of abnormal chromosome segregation (ACS)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne day after ICSI, embryos at the two-cell stage were fixed and permeabilized with 4% PFA and 0.5% Triton X-100 for 15 min. These embryos were then immersed in PBS containing DAPI and 1% BSA before being analyzed using a fluorescence microscope (Olympus IX-73, Tokyo, Japan). As previously described, we classified ACS as belonging to on of four groups: light, moderate, heavy, and lethal. Specifically, light ACS was defined as the presence of only one micronucleus\u003csup\u003e19\u003c/sup\u003e, moderate ACS by the presence of two small micronuclei, one to two medium micronuclei, or a single large micronucleus. Heavy ACS involved detection of three small or medium micronuclei or two to three large micronuclei, while lethal ACS involved embryos that contained multiple micronuclei. In some instances, two conditions occurred simultaneously, leading to a more severe evaluation. For example, if an embryo contained one medium and two small micronuclei, we classified it as \u0026ldquo;heavy.\u0026rdquo; However, since embryos with low-level ACS can develop normally\u0026mdash;i.e., to full term \u003csup\u003e28\u003c/sup\u003e\u0026mdash;we instead focused on embryos with severe (i.e., moderate or higher) ACS in this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComet assay results were analyzed using Wilcoxon\u0026ndash;Mann\u0026ndash;Whitney nonparametric tests or Dunn\u0026rsquo;s tests. Gamma-H2Ax assay results were analyzed using Kruskal\u0026ndash;Wallis tests. Normal fertilization rates, ACS assays,\u0026nbsp;\u003cem\u003ein vitro\u003c/em\u003e development rates, and birth rates were evaluated using chi-square tests or Tukey\u0026rsquo;s WSD tests. For all statistical tests, statistical significance was determined as \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Y. Kanda for assistance in preparing this manuscript. This work was partially funded by JST SPRING, Grant Number JPMJSP2133 to N. U.; the Research Fellowships of Japan Society for the Promotion of Science for Young Scientists to D.I. (23K19330), S. W. (23K08843) to T.W. (23K18124 and 24K01779); the Naito Foundation and Takahashi Industrial and Economic Research Foundation (189) to S.W.; Asada Science Foundation and the Canon Foundation (M20-0008) to T.W. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK.Y., D.I., S.W., and T.W. conceived and designed the study. K.Y., S.W., N.U., D.I., and T.W. performed experiments, analyzed the data, and interpreted the results. K.Y. and T.W. wrote the manuscript. All authors read and edited the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and are available from the corresponding author (Teruhiko Wakayama) on reasonable request..\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eComizzoli, P., Amelkina, O. \u0026amp; Lee, P. C. 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DNA damage in human and mouse spermatozoa after in vitro-irradiation assessed by the comet assay. \u003cem\u003eAdv Exp Med Biol\u003c/em\u003e. \u003cstrong\u003e444\u003c/strong\u003e, 79-91; discussion 92-73 (1998).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Rehydration, Freeze-dry, Intracytoplasmic sperm injection, Infiltration speed, Birth rate","lastPublishedDoi":"10.21203/rs.3.rs-5994995/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5994995/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFreeze-drying (FD) is a promising method for achieving the long-term, low-cost, and safe preservation of mammalian sperm at room temperature (RT). However, the birth rate of embryos fertilized with FD sperm is reduced to less than half compared to those fertilized with fresh sperm. Moreover, the underlying causes and potential solutions remain unclear. In this study, we investigated a previously unexamined rehydration process using FD sperm to determine its effects on sperm DNA damage. We also attempted to optimize this rehydration method to improve birth rates. Initially, we examined the effects of slowing water infiltration into FD sperm using a high osmolarity or viscosity solution, but this increased DNA damage and decreased birth rates. Next, to accelerate infiltration speed, we performed rehydration of FD sperm using ultrapure water heated up to as hot as 90℃. However, we found that the DNA damage of the FD sperm decreased as the temperature increased. The level of DNA damage in the male pronucleus at the zygote stage and of abnormal chromosome segregation (ACS) at the two-cell stage were also decreased at 37℃ or 50℃. Finally, the birth rates of embryos derived from FD sperm also significantly improved when rehydration was performed using 50℃ ultrapure water (37%) compared with the RT control (21%). Taken together, the results of this study demonstrate that the DNA of FD sperm can be damaged during the rehydration process and that rapid rehydration significantly improves the birth rate.\u003c/p\u003e","manuscriptTitle":"Improved Birth Rates via Rehydration of Mouse Freeze-Dried Spermatozoa using High-Temperature Ultrapure Water","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-12 08:20:38","doi":"10.21203/rs.3.rs-5994995/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"65f46c82-8dc7-45e4-a8ca-0f6ffaef0853","owner":[],"postedDate":"February 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":44213889,"name":"Biological sciences/Developmental biology/Embryology"},{"id":44213890,"name":"Biological sciences/Biotechnology/Animal biotechnology"}],"tags":[],"updatedAt":"2025-06-02T13:08:48+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-12 08:20:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5994995","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5994995","identity":"rs-5994995","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}