Easy and quick (EQ) sperm freezing method for urgent preservation of mouse strains

Abstract

Cryopreservation of mouse spermatozoa is widely used for the efficient preservation and safe transport of valuable mouse strains. However, the current cryopreservation method requires special containers (plastic straws), undefined chemicals (e.g., skim milk), liquid nitrogen, and expertise when handling sperm suspensions. Here, we report an easy and quick (EQ) sperm freezing method. The main procedure consists of only one step: dissecting a single cauda epididymis in a microtube containing 20% raffinose solution, which is then stored in a -80 °C freezer. The frozen-thawed spermatozoa retain practical fertilization rates after 1 (51%) or even 3 months (25%) with the C57BL/6 J strain, the most sensitive strain for sperm freezing. More than half of the embryos thus obtained developed into offspring after embryo transfer. Importantly, spermatozoa stored at -80 °C can be transferred into liquid nitrogen for indefinite storage. As far as we know, our EQ method is the easiest and quickest method for mouse sperm freezing and should be applicable in all laboratories without expertise in sperm cryopreservation. This technique can help avoid the loss of irreplaceable strains because of closure of animal rooms in emergency situations such as unexpected microbiological contamination or social emergencies such as the COVID-19 threat.

Figure 1

Schematic presentation of sperm freezing protocol by the EQ method.

Figure 2

Schematic presentation of in vitro fertilization (IVF) using sperm frozen-thawed by the EQ method.

Figure 3

The cooling rates of mouse sperm suspensions frozen by various methods. Temperature changes were measured for the following conditions: the standard method using a plastic straw frozen in LN₂ (gray, solid line); using a cryotube frozen in LN₂ (gray, dotted line); the EQ method using a microtube frozen in a −80 °C freezer (black, solid line) or in a −40 °C freezer (black, fine dotted line). The EQ method without an aluminum foil stand was also tested (black, coarse dotted line).

Figure 4

Results of sperm kinetic analysis. The motility rate, progressive motility rate and three kinds of velocity parameters were measured using frozen–thawed spermatozoa from B6N (n = 6) (A,C) and B6J (n = 7) (B,D) mouse strains. The asterisks indicate significant differences between groups (P < 0.05 by Tukey–Kramer test).

Figure 5

Confirmation of developmental abilities after IVF using frozen–thawed spermatozoa cryopreserved using the EQ method. (A) Two-cell stage embryos at 24 h after insemination. (B) Blastocysts after in vitro culture for 120 h. (C) A litter born by Cesarian section from the transfer of 2-cell stage embryos. (D) Pups (black hair color) survived to weaning and the figure shows their foster mother (arrow) and mother’s pups (arrowheads). (E) Newborn pups from the mating of pairs of mice derived from frozen–thawed spermatozoa.

Figure 6

Visualization of plasma membrane integrity and the acrosomal status. (A) Representative images of fresh and frozen–thawed spermatozoa by standard and EQ methods after staining using propidium iodide (PI; red) and peanut agglutinin (PNA; green). (B) The percentages of spermatozoa stained by PI ( +) and PNA ( +). Significant difference indicated by different superscript letters. (^{a, b; c, d; e, f} P < 0.01, ^{A, B} P < 0.05 by Tukey–Kramer test).

Figure 7

Visualization of DNA damage status by TUNEL assay. (A) Representative images of fresh and frozen–thawed spermatozoa cryopreserved by standard or EQ methods after TUNEL staining. (B) The relative fluorescent intensities of TUNEL signals (fresh, n = 93; standard method, n = 96; EQ, n = 83). The asterisks indicate significant differences between groups (**P < 0.01 by Tukey–Kramer test).

Figure 8

Visualization of oxidative stress by CellROX Deep Red staining. (A) Representative images of fresh and frozen–thawed spermatozoa cryopreserved by standard or EQ methods after staining with Hoechst 33,342 and CellROX Deep Red. (B) Relative fluorescent intensities of CellROX Deep Red signals. **P < 0.01 between groups by Tukey–Kramer test.