Method for long-term room temperature storage of mouse freeze-dried sperm

Abstract

Permanent preservation of genetic resources may be indispensable for the future of humanity. This requires liquid nitrogen, as is the case for preserving animal sperm. However, this technique is expensive and poses a risk of irrecoverable sample loss on non-replenishment of liquid nitrogen in case of natural disasters. In this study, we demonstrate that lyophilization may be used as a reliable method for long-term preservation of mouse sperm at room temperature. Sperm from four mouse strains were freeze-dried and stored in a non-temperature controlled room for 5-6 years. Although the ability of the stored sperm to activate oocytes had diminished slightly, healthy offspring were obtained by artificially activating the oocytes after sperm injection. Moreover, the birth rate did not decrease even after ≤ 6 years of storage. Furthermore, owing to its low cost, safety, and ease of storage at any location, we believe that this method could be a major mode of preserving mammalian genetic resources in the future.

Keywords: Freeze-drying; Genetic resources; Oocyte activation; Room temperature; Sperm preservation.

Fig. 1

Schematic of storage duration of freeze-dried (FD) sperm at room temperature and various experiments performed (a) Sperm were collected from male ICR, B6, C3H, and BDF1 strain mice to produce FD sperm. The sperm were dispensed into ampoules, frozen in liquid nitrogen, and dried for 6 h. Silica gel was added to certain ampoules after first drying. After being subjected to vacuum once again, the ampoules were sealed by melting the ampoule necks using a gas burner under vacuum. Non-destructive testing was performed using a tesla coil leak detector before the start of the experiment, and a high-vacuum ampoule was used for the experiment. (b) Ampoules containing sperm were placed in paper boxes and stored in drawers in a non-temperature controlled room for short-term (3 months-1 year: ICR and BDF1), medium-term (3 years: ICR), and long-term (5–6 years: all strains). (c) On day 1 of experiment, distilled water was added to the ampoule to determine (1) sperm DNA damage, (2) morphological abnormalities of sperm, (3) fertilization rate after ICSI, (4) DNA damage rate at pronuclear stage, (5) abnormal chromosome segregation (ACS) rate at 2-cell stage, (6) rate of development to blastocyst, (7) Blastocyst quality by cell number, (8) full-term development after embryo transfer, and (9) fertility of offspring.

Fig. 2

Fertilization and in vitro developmental potential of oocyte/embryo with ICR freeze-dried (FD) sperm stored at room temperature for 6 years (a, b) Ampoules prepared in this study were stored in a drawer. (c) FD sperm after rehydration. (d–f) ICSI with FD sperm and fertilization. After sperm injection into the oocyte (d), the embryo is activated and forms a pronucleus (e), and the oocyte is not activated and forms a pseudo-spindle (f). (g) Activation rates of oocytes injected with FD sperm stored for 1, 3, and 6 years. Blue: without artificial activation. Red: with artificial activation; Sr−: without activation treatment; Sr+: with activation treatment. (h) Rate of ACS occurring in two-cell-stage embryos fertilized with FD sperm stored for 6 years. NCS: normal chromosome segregation, ACS: abnormal chromosome segregation. (i) two-cell-stage embryo with micronuclei formation. Image on the right: magnified view. Arrows point to micronuclei. Embryo nuclei and micronuclei were detected using DAPI nuclear staining. (j) Development of embryos fertilized with FD sperm stored for 6 years to blastocysts. 2 C: two-cell stage, 8 C: eight-cell stage, M/Bla: morulae or blastocyst stage, Bla: Blastocyst. (k, l) Immunostaining of blastocysts fertilized with FD sperm with or without artificial activation. Nuclei of embryos detected using DAPI nuclear staining (upper left) are blue. Nanog-positive cells (inner cell mass, ICM) are green (upper right), CDX2-positive cells (trophectoderm, TE) are red (lower left), and merged images (lower right). (m) Cell number for TE and ICM. Photo credits: Yuko Kamada, University of Yamanashi.

Fig. 3

Production of offspring using freeze-dried (FD) sperm stored at room temperature for long periods of time from various mouse strains (a) Development of embryos fertilized with C57BL/6 (B6), C3H/He (C3H), or BDF1 mice FD sperm stored for approximately 5 years to blastocysts. 2 C: 2-cell stage, 8 C: 8-cell stage, M/Bla: morulae or blastocyst stage, Bla: Blastocyst. (b) Birth rate of offspring born with ICR, B6, C3H or BDF1 mice FD sperm stored at room temperature. ICR and BDF1 were compared for birth rate using short- and long-term stored sperm; B6 and C3H were examined only with long-term stored sperm. (c) ICR mice FD sperm were injected into BDF1 fresh oocytes, and embryos were transferred into ICR recipient female. Arrows (colored pup) indicate offspring born from FD sperm stored for 6 years at room temperature. (d) Offspring born from C3H mouse FD sperm stored at room temperature for approximately 5 years. Photo credits: Yuko Kamada, University of Yamanashi.