1. Faculty of Life and Environmental Science, University of Yamanashi, Yamanashi, Japan
2. Advanced Biotechnology Center, University of Yamanashi, Yamanashi, Japan
Abstract Text: Freeze-dried (FD) preservation of sperm allows long-term storage of genetic resources at room temperature without the use of liquid nitrogen. However, compared to fresh sperm, the success rate of offspring from those FD sperm is reduced by one-third. In this study, we focused on the use of monosodium glutamate (MSG) as a new protective agent for mouse FD sperm, which is a type of amino acid and enables yeast to survive after FD. In mammalian sperm, sugars and chelating agents are mainly used as protectants against FD treatment, but no examples have been found using amino acids.
In this study, HTF medium was used as the basic medium. When mouse sperm were preincubated in HTF medium for 1 hour and then replaced with HTF medium containing 0.5-10% MSG, almost all sperm at 5% MSG and all sperm at 10% MSG lost their motility immediately after incubation. To determine the protective effect of MSG during FD treatment, those sperm were freeze-dried just after replacing MSG-HTF, and the appearance and morphology of those sperm after rehydration was observed. Then, those sperm were injected into oocytes and development rate of embryos to blastocyst and its cell number were examined. Some of embryos were transferred into recipient and the birth rate were measured. There was no difference in the appearance of the FD sperm between control and MSG treated sperm after rehydration. However, when embryos were cultured after ICSI, the developmental rate to the blastocysts was significantly increased with increased the concentration of MSG, reaching a maximum of 65% at 3% MSG compared to control (34%; P< 0.05). No significant differences were found in the number of cells in the blastocysts between experiments. When 2-cell embryos were transferred to the recipient, the birth rate peaked at 42% (30 pups) at the 3% MSG, more than twice as high in the control (20%; P< 0.05, 17 pups). Next, to determine whether the addition of MSG in sperm preincubation medium and FD treatment medium had any positive/negative effects, sperm were incubated in 1% MSG-HTF for 1 hour, followed by FD treatment in the same medium. When ICSI were performed using those sperm, both the developmental rate of embryos to the blastocysts (50%) and offspring (37%) were increased compared to controls (38% and 20%, respectively), though the difference was not statistically significant.
This study shows for the first time that MSG, in which usually used for the yeast, also has a protective effect for mammalian sperm against FD treatment. It was also shown that the addition of MSG during preincubation before FD treatment had no adverse effect on FD sperm. Since MSG has been suggested to be involved in membrane protection and antioxidant effects in yeast FD, it is considered to have a similar effect on mouse sperm. Future work is needed to find a new and unique method of freeze-drying mouse sperm to improve the birth rate by referring to freeze-drying methods for more different microorganisms.
