IntroductionSperm preservation plays an important role in the lineage maintenance of genetically modified mice, conservation of wildlife or rare animal species, and assisted reproductive technology. Typically, long-term sperm preservation is achieved using liquid nitrogen1,2. Sperm preserved by this method can be stored semi-permanently, and since they maintain motility after thawing, they can fertilize and develop normal embryos after in vitro fertilization. However, regular replenishment of liquid nitrogen is necessary, and if not properly controlled, there is a risk of frostbite and asphyxiation. Furthermore, if the liquid nitrogen cannot be replenished after natural disasters, valuable samples can get lost3. Further, it is a costly preservation method because it requires special containers such as dry sippers and large amounts of dry ice to transport small amounts of samples4.Freeze-dried sperm (FD sperm) is a room-temperature preservation method that addresses the issues of cryopreservation. Mouse FD sperm lose their motility after rehydration, but can produce normal offspring by intracytoplasmic sperm injection (ICSI), if the nuclei are undamaged5,6. This technique has been successfully used to produce offspring from the FD sperm of rabbits7rats8horses9and hamsters10and it has been used in domestic animals such as cattle11pigs12sheep13dogs14and cats15 as well. Interestingly, the chromosomal integrity of mouse FD sperm has been demonstrated after storage at high temperatures16,17 and exposure to radiation18,19. In fact, offspring can be produced even from mouse FD sperm exposed to space radiation for extended periods at the International Space Station19,20. Recently we have successfully produced offspring from mouse FD sperm stored at room temperature for > 6 years without decreased birth rate21 which suggests the feasibility of longer storage at room temperature. Further, simplified preservation methods are being investigated; now, it is possible to obtain normal offspring from mouse FD sperm stored simply in plastic microtubes22 or thin plastic film, which allows mailing sperm attached to a postcard4. Similarly, sheep FD sperm can be preserved in stainless steel mini capsules23. Although ICSI is necessary to produce offspring because FD sperm do not move after rehydration, this technique is used worldwide, including for humans24,25. However, the birth rate obtained with FD sperm remains low compared with that of fresh sperm3,5,21,26,27. Given the common occurrence of DNA damage in FD sperm28for this technology to become practical, more effective desiccant protectants are needed to reduce DNA damage in FD sperm and increase birth rates.There are four steps involved in the preparation and use of FD sperm: freezing, drying, storage at room temperature, and rehydration. The damage caused during room-temperature storage and rehydration can be improved by adding trehalose29 or rehydration with high-temperature water (submitted). However, the major cause of the decreased birth rate of embryos derived from FD sperm is drying rather than freezing30. Thus, to prevent the DNA damage caused by FD, sperm can be treated with protective agents such as EDTA, a chelating agent with a retention effect on endonuclease activity during storage31; silica gel as a dehydrating agent32; and rosmarinic acid as an antioxidant33. However, these agents only achieved minor improvements and did not substantially increase birth rates. Therefore, we focused on freeze-drying and vacuum-drying methods for microorganism preservation34,35. Numerous drying media have been optimized for different microbial species, typically incorporating monosodium glutamate (MSG), an amino acid. MSG not only acts as a cryoprotectant36,37,38 but also protects cell membranes and acts as an antioxidant39. Moreover, it stabilizes the protein structure through reaction of amino groups with the carboxyl groups of proteins in microorganisms and can retain a large amount of residual moisture40making it useful for drying and storage of microorganisms. Since microorganisms such as dried yeast are viable after dry storage, MSG may be an effective desiccation protectant for mammalian FD sperm.In mammals, it has been reported that sperm produced from rats that consumed large amounts of MSG exhibited functional disorders41. However, to the best of our knowledge, there are no examples of MSG being added to sperm culture medium or cryoprotectant agents. Thus, this study aimed to determine whether MSG addition acts as a protective agent in mouse FD sperm. Accordingly, 1–10% (w/v) MSG was added to the drying medium during FD sperm production to determine its protective effect on sperm and its influence on embryonic development after ICSI. Additionally, the effects of MSG on several mouse strains, including C57BL/6 N (B6N), were examined (Fig. 1).Fig. 1Schematic representation of the preparation of freeze-dried spermatozoa and other experiments.Full size imageResultsEffect of MSG on the separation rate of sperm heads and TailsThe ampoules of FD sperm preserved in the desk drawer (Fig. 2a) were tested with a Tesla coil leak detector, and only those ampoules that showed a positive reaction were used (Fig. 2b).To determine the protective effect of MSG on FD sperm, we first evaluated the physical damage caused by FD by quantifying the sperm head and tail separation rate. The percentage of tailless sperm in FD sperm was 23%, much higher than the fresh control (3%). Conversely, MSG addition significantly decreased the percentage of tailless sperm for FD sperm in a concentration-dependent manner, up to 6% (Fig. 2c). Similar results were obtained for freeze-thaw sperm (Table S1).Fig. 2Analysis of FD sperm produced with MSG. (a) A glass ampoule containing FD sperm (left) stored in a desk blower (right). (b) Vacuum level determination using a Tesla coil leak detector. A glowing ampoule (right ampoule) with high vacuum was used. (c) Ratio of tailless FD sperm produced by different MSG concentrations. Error bars indicate SE. (d) Detection of acrosomes in FD sperm produced with 5% MSG. Acrosomes were stained with lectin PNA. Upper left; DAPI. Upper right; lectin PNA. Bottom left; merge. Bottom right; merge + bright field (BF). Arrows indicate PNA negatives. Arrowheads indicate PNA positives. (e) Ratio of PNA-positive sperm with and without Triton X-100 (Tx) treatment. Yellow; with Tx. Red; without Tx. Error bars indicate SE. (f–h) Comet assay with 0%, 3%, and 10% MSG-added FD sperm, respectively. (i) Relative values of the comet tail of FD spermatozoa at different MSG concentrations. Different characters indicate significant differences (P 5% MSG applied to fresh sperm made them lose their motility, this motility was not fully recovered even after washing. As high concentrations of MSG may substantially increase the osmolarity, it cannot be ruled out that high osmotic pressure had a negative effect rather than MSG itself. The addition of 3% MSG to the drying medium may have yielded the highest birth rate because it balanced the reduction of morphological and minor DNA damage caused by MSG and the increase in the cell membrane and severe DNA damage caused by high concentrations of MSG.Herein, we demonstrated that adding MSG to the drying medium successfully improved the birth rate from FD sperm, to levels comparable to fresh sperm (46–60%) or freeze-thaw sperm (23–48%) in previous studies30,45,46. However, in this study, the birth rate from FD sperm stored for 3 months was lower than that stored for 1 month. If the addition of MSG caused the birth rate to decrease, this method would not be suitable for long-term storage at RT. However, as we already reported that storing FD sperm in a desk drawer at room temperature for up to 6 years does not decrease the birth rate21. Furthermore, it has been found that even when using sperm from the same male to prepare FD sperm ampoules simultaneously, there are significant differences in the birth rate between ampoules32. Thus, further detailed verification of the protective effect of MSG is necessary, but it is likely that the decrease in birth rate is due to differences between ampoules rather than the storage period. Whether permanent preservation is possible remains unclear, but it appears possible to reliably preserve FD sperm with a high birth rate for a sufficient time. Although FD of oocytes is not yet possible, healthy offspring have been produced from embryos fertilized with FD round spermatid47 or cloned embryos with FD somatic cells48indicating that FD can be applied not only to sperm but also to other cells. If MSG had a protective effect on oocytes as well, it may allow producing offspring from FD oocytes.Currently, researchers are paying high costs to maintain a growing number of genetically modified mice strains being produced around the world. However, using this method not only eliminates the cost of maintaining mouse strains and makes it easy to store them anywhere, it also eliminates the fear of losing all strains due to the inability to replenish liquid nitrogen in the event of a disaster27. As FD sperm is feasible in other species26it could be used to safely and inexpensively preserve the genetic resources of endangered species and original livestock species in developing countries3. When humans expand into space in the future, they will take their livestock and pets with them to other planets. FD sperm can be transported in large numbers in a spacecraft, thus avoiding the inbreeding of livestock and pets at the destination20. In addition, if the Earth’s genetic resources were stored under the moon in FD, it would be possible to revive all species in the event of any major disaster on Earth49. The preservation of genetic resources by FD technology could contribute greatly to the future of humankind.Materials and methodsAnimalsICR and B6D2F1 (BDF1) female and male mice and C57BL/6 N (B6N) male mice (8–10 weeks of age) were obtained from SLC Inc (Hamamatsu, Japan). Surrogate pseudopregnant ICR females, used as embryo recipients, were mated with vasectomized ICR males with proven sterility. The GFP transgenic mice carrying the acrosin/eGFP (Acr3-EGFP) transgenes50 was kindly provided by Dr. M. Okabe. On the day of the experiment or after all experiments were completed, the mice were euthanized by CO2 inhalation or cervical dislocation and used for further experiments. All animal experiments were conducted according to the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Committee of Laboratory Animal Experimentation of the University of Yamanashi (reference number: A4-10) following ARRIVE guidelines. All mice were maintained under SPF conditions, with controlled temperature (25 °C), relative humidity (50%), and photoperiod (14 L-10D). They were fed a commercial diet and provided distilled water ad libitum.MediaHTF medium51 was used for capacitation and FD of spermatozoa. HEPES-CZB medium52 and CZB medium53 were used for oocyte/embryo manipulation and for incubation in 5% CO2 at 37 °C, respectively.Preparation of FD spermatozoaBoth epididymides were collected from male mice (ICR, BDF1, and B6N) and the ducts severed with sharp scissors. A few drops of dense spermatozoa mass were then placed into a centrifuge tube containing 1 mL HTF medium and incubated for 60 min at 37 °C in 5% CO2. Spermatozoa concentration and motility were determined, and 800 µL supernatant was collected. Then, the sperm suspension was centrifuged at 2300 g for 10 min before removing the supernatant and replacing it with HTF medium containing 1% (w/v), 3% (w/v), 5% (w/v), and 10% (w/v) MSG. The 50 µL aliquots of the spermatozoa suspension were then dispensed into glass ampoules and connected to an FDU-2200 freeze dryer (EYELA, Tokyo, Japan) for vacuum drying. After freezing in liquid nitrogen for 1 min, the cork of the freeze dryer was open for ≥ 6 h until all samples were completely dry. After drying, the ampoules were sealed by melting the ampoule necks using a gas burner under vacuum, as previously described29. All ampoules were then stored at room temperature until further use (Fig. 2a).Detection of trapped air in ampoules using the Tesla coil leak detectorAmpoules containing air were identified using a Tesla coil leak detector (Sanko Electronic Laboratory, Kanagawa, Japan) according to manufacturer’s instructions. When the tip of the Tesla coil comes near the ampoule, the tip will spark around the glass. If a lot of air is trapped inside the ampoule, the air cannot be ionized. However, if the ampoule contains only a small amount of residual air, its ionization produces a spark inside the ampoule (Fig. 2b). Only Tesla-positive ampoules indicating highly vacuumed ampoules were used for all experiments32.Measurement of sperm head and tail separation rateThe sperm suspension was replaced with MSG-HTF as for the FD sperm preparation described above, and 50 µL was dispensed into plastic tubes. The plastic tubes were then frozen in liquid nitrogen for 1 min and incubated at room temperature until completely thawed. The freeze-thawed sperm suspension was diluted 3–5 times with distilled water, and 150–300 sperm were counted in the frame using a blood cell calculator. The sperm head-tail separation rate corresponded to the amount of sperm heads divided by the total sperm count. For FD sperm, water was added within 3 days after FD to avoid the effects of the storage period and measured as described above.Observation of the acrosomeWe optimized the methods based on previous study54. After water addition, ICR FD sperm were centrifuged at 800 g for 5 min. For Tx (+) treatment, the sperm supernatant was replaced by 0.1% Triton X-100 and incubated for 15 min at room temperature. For Tx (−) treatment, this treatment was skipped. After centrifugation, they were then replaced with lectin PNA and Alexa Flur 568 conjugate (1:500, 2 µg/mL, Invitrogen, MA, USA) and incubated for 30 min at room temperature. After centrifugation, the fluid was replaced by a 4′,6-diamidino-2-phenylindole (DAPI) solution (1:1000), the samples were mounted on a slide and observed under a confocal microscope (FV1200, Olympus, Tokyo, Japan). When fresh sperm were used, they were fixed in paraformaldehyde (PFA) for 15 min on ice and washed with chilled PBS. They were then treated in the same way as FD sperm.Analysis and scoring of comet slidesSpermatozoa DNA damage, potentially caused by single- and double-stranded breaks55was measured using the CometAssay® Kit (Trevigen, MD, USA), according to manufacturer’s instructions. In brief, spermatozoa specimens were collected from ampoules immediately after opening and rehydrated in water. Both the specimen and its counterpart were mounted on the same slide, and 100–300 spermatozoa heads on each slide were analyzed by electrophoresis. To standardize the results across conditions under, the length of each DNA comet tail was divided by the mean length of the one-side results in each experiment. In this comet assay, fresh and freeze-thaw spermatozoa were unsuitable as controls because their preparation techniques differ. This discrepancy would have hindered an accurate comparison between specimens on the same slide.Oocyte preparationFemale ICR or BDF1 mice were superovulated by injection of 7.5 IU equine chorionic gonadotropin, followed by7.5 IU human chorionic gonadotropin after 48 h. Cumulus-oocyte complexes (COCs) were collected from the oviducts of females after 14–16 h and moved to a Falcon dish containing HEPES-CZB media. To disperse the cumulus, COCs were transferred into a 100 µL droplet of HEPES-CZB medium containing 0.1% bovine testicular hyaluronidase for 3 min. Cumulus-free oocytes were then washed twice and transferred to a 20 µL droplet of CZB for culture.ICSI and embryo transferICSI was performed as previously described52. Just before starting the ICSI, the ampoule neck was broken and 50 µL of sterile distilled water was added and mixed with a pipette. For ICSI, 1–2 µL of the sperm suspension was transferred directly to the injection chamber. The sperm suspension was replaced every 30 min during ICSI. The application of several piezo pulses separated the sperm head from the tail, and the head was then injected into ICR or BDF1 oocytes. The oocytes that survived ICSI were incubated in CZB medium at 37 °C with 5% CO2. Pronuclear formation was verified 6 h after ICSI. Next, embryos at the 2-cell stage were transferred into day 0.5 pseudopregnant mice mated with a vasectomized male the night before transfer. At that point, 5–12 embryos were transferred into each oviduct. On day 18.5 of gestation, the offspring were delivered by cesarean section and allowed to mature. The remaining unused embryos were cultured for up to 4 days to evaluate their potential for developing into blastocysts.Toxicity assessment of MSGThe effect of MSG on oocytes was observed by its injection into oocytes before activation for parthenogenesis. As previously reported, 3–5 pL of 0%, 3%, and 10% MSG-HTF were injected into fresh oocytes using piezo-driven micropipettes (Prime Tech, Ibaraki, Japan)56. Briefly, microinjection was performed in HEPES-buffered CZB on an inverted microscope (Olympus) with a micromanipulator (Narishige, Tokyo, Japan). The zona pellucida and cell membrane were penetrated with a piezo drive. After MSG-HTF was injected into the oocytes, they were diploidized by incubation for 5–6.5 h in activated medium with 5 µg/mL cytochalasin B57. After washing three times in CZB medium, embryos were incubated in CZB medium for 4 days to evaluate the developmental potential of the blastocyst.The impact of MSG on sperm was assessed by examining motility and embryo development following ICSI. Sperm was precultured for 1 h in HTF medium before replacing it with 0%, 3%, or 10% MSG-HTF incubated for 1 h. For the sperm motility assay, sperm were washed three times in HTF medium and motility visually evaluated under an inverted microscope. For the developmental potential assay, ICSI and embryo culture were performed using the method described above.ACS detectionThe day after ICSI, two-cell stage embryos were fixed and permeabilized with 4% PFA and 0.5% Tx for 15 min. These embryos were observed using fluorescence microscopy (BZ-X710, KEYENCE, Osaka, Japan) in DAPI and 1% BSA containing PBS. ACS was categorized into four groups: “light,” “moderate,” “heavy,” and “lethal,” as described previously19. Light ACS was considered when a single micronucleus was detected. Moderate ACS was considered when two small, 1–2 medium, or one large micronucleus was detected. Heavy ACS was considered when three small, medium, or 2–3 large micronuclei were detected. Lethal ACS was considered when embryos had multiple micronuclei. Importantly, when both conditions co-occurred, the evaluation was more severe. For example, a moderate micronucleus and two small micronuclei were detected in the embryo and classified as “heavy.”ImmunostainingWe modified previous methods58and performed immunofluorescence staining on blastocysts. After imaging with BZ-X710 (KEYENCE, Osaka, Japan), we counted the total number of nuclei, as well as the number of TE cells and ICM blastomeres. At 96 h after ICSI, blastocysts were fixed in 4% PFA with 0.2% Tx for 15 min at room temperature. Fixed embryos were washed three times in PBS containing 1% (w/v) BSA (BSA-PBS) for 15 min. The primary antibodies used were an anti-CDX2 mouse monoclonal antibody (1:500, BioGenex, CA, USA) to detect TE cells and an anti-Nanog rabbit polyclonal antibody (1:500, abcam, Cambridge, UK) to detect ICM cells. The secondary antibodies used were an Alexa Fluor® 488-labeled goat anti-mouse IgG (1:500, invitrogen, MA, USA) and an Alexa Fluor® 568-labeled goat anti-rabbit IgG (1:500 invitrogen, MA, USA). DNA was stained with DAPI. The total cell number was counted as DAPI-positive cells.Statistical analysis and reproducibilityAll experiments were repeated at least thrice, and similar results were obtained irrespective of the experimentalists. Results of the comet assay were analyzed using the Bonferroni-Dunn test. Blastocyst cell counts were analyzed using the Tukey-Kramer’s HSD test. Sperm head-tail separation rate, lectin-positive rate, blastocyst rate, and birth rates were evaluated using the Tukey’s WSD test or chi-square test. Statistical significance of differences between variables was determined at P of