PDF《spermatozoa》

in laboratory mice, zygotes generated using ICSI and freeze-dried sperm can develop into healthy, full-term offspring [3]. Moreover, mice derived from the freeze- dried spermatozoa gave rise to ?rst- and second-generation progeny with stable genomes [11]. Many studies have investigated freeze-dried spermatozoa in mammalian spe- cies other than mice and humans. Most of these studies explored whether freeze-dried spermatozoa from cattle [12C15], dog [16], hamster [17], human [17C21], pig [22, 23], Rhesus monkey [24], rabbit [20, 25] or rat [26C29] could develop to the pronuclear stage, the blastocyst stage, and/or to live birth. For assisted reproduction in most species, ICSI must be introduced and improved to ensure that sperm that become non-motile because of harsh isolation, preservation, or storage conditions can support normal development, although storage of freeze-dried mammalian spermatozoa has great potential as an alternative to traditional nitrogen- based cryopreservation. Evaporative drying versus freeze-drying Procedures used to freeze-dry mouse sperm usually include a freezing step (1C10 min) before the sublimation of water in the samples. A lyophilizer is generally used to sublimate the water. Glass ampoules containing the frozen sperm sample are connected to the lyophilizer, and a vacuum is applied at an inner pressure of approximately

1 m Torr for

12 h [3] or 0.04 mbar for

4 h [30];

alternatively, primary drying and subsequent secondary drying pressures (0.37 and 0.001 mbar, respectively) are applied to the ampoules [31]. Evaporative drying is another method used to prepare dried spermatozoa. Reportedly, evaporative drying of mouse spermatozoa is an exceptional method for preparing dried sperm specimens that eliminates the initial freezing step of freeze-drying, which is likely to injure the spermatozoa [32C35]. This evaporative drying method has been used primarily for laboratory mice sperm, and the technique has not been optimized for other mammalian species. Sperm suspension is applied to a glass slide and dried for

5 min at room temperature under a stream of nitrogen gas;

notably, the time required to dry the sample is much shorter than the sublimation time required for freeze- drying, which is at least

4 h. Moreover, the equipment required for the evaporative drying is simpler and cheaper than a lyophilizer [36]. It is unclear whether evaporative drying is superior to freeze-drying for preserving mam- malian spermatozoa, and studies on the long-term main- tenance of the dri........