Improved approach for the cryopreservation of mouse sperm by combining monothioglycerol and l-glutamine

Abstract

The CryoPreservation Media (CPM) for mouse sperm using raffinose and skim milk have been improved by adding either monothioglycerol (MTG) or l-glutamine to reduce the oxidative damage during sperm freezing and thawing. The CARD-CPM utilizing l-glutamine, but not MTG, has been widely used to meet the rising demand for cryopreservation of genetically modified mice, as the CARD method also improved sperm capacitation and in vitro fertilization (IVF). However, the viability of sperm frozen in the CARD-CPM is highly variable, indicating a room for improvement. To develop a more dependable technique for mouse sperm cryopreservation, we investigate whether combining MTG and l-glutamine in the CPM (MG-CPM) can produce a synergistic impact on sperm thawing and IVF rate. We found that MG-CPM reduced the incidence of infertility and increased the IVF success rate. Therefore, cryopreservation of mouse sperm in MG-CPM is a reliable method to ensure embryo generation from frozen sperm.

Keywords: CARD method; Cryopreservation; IVF; In vitro fertilization; MTG; Monothioglycerol; Sperm; l-glutamine.

Fig. 1.

The main question and experimental design. The three media are used for the cryopreservation of mouse sperm and the subsequent production of embryos with thawed sperm using CARD method: Cryopreservation medium for sperm (CPM), sperm capacitation medium, and in vitro fertilization (IVF) medium. Here, we examined whether altering the composition of CPM improves the efficiency of embryo generation by thawed sperm (CARD-CPM vs. MG-CPM). The main question was presented in red text and a red box. The composition of the sperm capacitation medium and IVF medium remained unchanged from the CARD method. Figure was created by BioRender.com. MTG: monothioglycerol; TYH medium: Toyoda, Yokoyama, Hoshi medium; MBCD: methyl-β-cyclodextrin; HTF: human tubal fluid.

Fig. 2.

MG-CPM is a more reliable medium for sperm cryopreservation than CARD-CPM. Sperm that had been frozen in either CARD-CPM or MG-CPM was thawed and used for in vitro fertilization according to the CARD IVF protocol. Sperm and eggs from various mouse lines in a C57BL/6 background were used. At day 2, embryos reaching the 2-cell stage were counted from the total eggs utilized to calculate fertilization rate. Medians were labelled as solid line, and quartiles were labelled as dotted line. Average: 57.01% (CARD-CPM); 72.8% (MG-CPM). Sample size: n = 34 (CARD-CPM); n = 50 (MG-CPM); **: P < 0.0023 with student’s t-test.