Preservation of ejaculated mouse spermatozoa

本应用程序的主要目标是开发一种简单有效的方法，通过使用ICSI保存有价值的小鼠基因组。这种应用的假设是，射精精子可以反复从男性身上取出，保存下来，然后用于ICSI，高效率地产生活的后代。从老鼠身上收集精子仅限于涉及安乐死的技术，并导致雄性死亡，使其无法进一步繁殖。一种反复获得射精的方法可以保持雄性存活并能够繁殖。在具体目标1中，我们将测试通过ICSI将保存的射精精子注射到卵母细胞中是否能够高效地获得活的后代。我们将从几个C57BL/6雄性精子中反复获得精子，并通过常规的红细胞保存和不冷冻的简单冷冻保存。我们将使用ICSI将这些精子注射到C57BL/6雌性的卵母细胞中进行生育

The major goal of this application is to develop a simple and efficient method for preservation of valuable mouse genomes by using ICSI with preserved ejaculated spermatozoa. The hypothesis of this application is that ejaculated spermatozoa can be repeatedly retrieved from a male, preserved, and then used for ICSI yielding live offspring with high efficiency. The collection of spermatozoa from mice is limited to techniques that involve euthanasia and result in a death of the male, precluding him from further breeding. A method for repeatedly obtaining ejaculates allows for keeping the male alive and able to breed. In the Specific Aim 1 we will test if ejaculated preserved spermatozoa injected into the oocytes by ICSI allow obtaining live offspring with high efficiency. We will repeatedly obtain spermatozoa from several C57BL/6 males and preserve them by conventional eryopreservation and by simple freezing without cryoprotection. We will use ICSI to inject these spermatozoa into the oocytes from C57BL/6 females to produce embryos. We will evaluate the effects of these methods on sperm DNA integrity by analysis of patemal chromosomes in the zygotes and perform embryo transfer and produce live offspring. Finally, we will apply the proposed technology to one mutant mouse strain (azh - abnormal spermatozoon head-shape) with fertility problems to demonstrate its usefulness in preservation of a medically important mouse strain. In the Specific Aim 2 we will evaluate the mechanism of chromosome degradation in ejaculated preserved mouse spermatozoa. We will explore the biological significance of observed phenomenon that spermatozoa exposed to uterine content are more susceptible to DNA damage. We will establish if the unknown factor present in the uterus after mating affects live spermatozoa or rather interferes with the conditions of unprotected sperm freezing. We will also analyze independently the effects of two compounds of the uterine content: uterine fluid and seminal vesicle fluid. The significance of this proposal is that it will vigorously test the novel approach of using ICSI with preserved ejaculated spermatozoa to maintain valuable mouse genomes. This method will allow increasing the overall efficiency of reconstituting mouse strains and will accelerate sharing of given genetic material (i.e. a novel mutation) within the scientific community. Our expectations are that the results of this application will allow us to recommend ICSI with preserved ejaculated spermatozoa as a simple and efficient method for preservation of valuable mouse genomes.