Improved assessment of frozen/thawed mouse spermatozoa using fluorescence microscopy
نویسندگان
چکیده
Genetically modified (GM) animals are unique mutants with an enormous scientific potential. Cryopreservation of pre-implantation embryos or spermatozoa is a common approach for protecting these lines from being lost or to store them in a repository. A mutant line can be taken out of a breeding nucleus only if sufficient numbers of samples with an appropriate level of quality are cryopreserved. The quality of different donors within the same mouse line might be heterogeneous and the cryopreservation procedure might also be error-prone. However, only limited amounts of material are available for analysis. To improve the monitoring of frozen/thawed spermatozoa, commonly used in vitro fertilization (IVF) followed by embryo transfer were replaced with animal-free techniques. Major factors for assessing spermatozoa quality (i.e., density, viability, motility, and morphology) were evaluated by fluorescence microscopy. For this, a live/dead cell staining protocol requiring only small amounts of material was created. Membrane integrity was then examined as major parameter closely correlated with successful IVF. These complex analyses allow us to monitor frozen/thawed spermatozoa from GM mice using a relatively simple staining procedure. This approach leads to a reduction of animal experiments and contributes to the 3R principles (replacement, reduction and refinement of animal experiments).
منابع مشابه
P-134: In Vitro Assessment of Cysteine Effect on DNA Integrity of Frozen-Thawed Buffalo Spermatozoa
Background: Cysteine has been used as additive in cryopreservation extenders. It contains of thiol group which plays an antioxidant role to eliminate reactive oxygen species (ROS) during freeze -thaw procedure. Excessive ROS production cause DNA damage and subsequently reduce sperm fertility. On the other hand, protamine protein have high levels of cysteine which is critical in packaging and co...
متن کاملEffects of storage duration at 5 ºC and type of cryoprotectant on cooled and frozen-thawed ram epididymal spermatozoa
Freezing of ejaculated and epididymal spermatozoa is currently a subject of interest with the purpose of establishing an efficient gene banking model for valuable animals or endangered species. Therefore, the present study evaluated the influence of different storage duration )0, 1.5 and 5 h) at 5 ºC and type of cryoprotectant on freezability of ram epididymal spermatozoa. With increasing the s...
متن کاملP-133: Effect of Thawing Rate on Kinetic Parameters of Frozen - Thawed Buffalo Spermatozoa Extended in Tris - Egg Yolk
Background: Optimal thawing rate is critical for quality and fertilizability of frozen -thawed spermatozoa. Kinetic parameters are the most important factors which are affected by thawing rate so, that have close relation between kinetic parameters and fertility of the spermatozoa. Tris - egg yolk have been widely used for cryopreservation of the buffalo and other species semen. According to ou...
متن کاملLaser-Assisted In Vitro Fertilization Facilitates Fertilization of Vitrified-Warmed C57BL/6 Mouse Oocytes with Fresh and Frozen-Thawed Spermatozoa, Producing Live Pups
The utility of cryopreserved mouse gametes for reproduction of transgenic mice depends on development of assisted reproductive technologies, including vitrification of unfertilized mouse oocytes. Due to hardening of the zona pellucida, spermatozoa are often unable to penetrate vitrified-warmed (V-W) oocytes. Laser-assisted in vitro fertilization (LAIVF) facilitates fertilization by allowing eas...
متن کاملDevelopmental competence of Dromedary camel oocytes fertilized in vitro by frozen-thawed ejaculated and epididymal spermatozoa
The present study aimed to compare the in vitro fertilizing capacity of frozen-thawed ejaculated and epididymal spermatozoa in order to standardize the semen preparation protocol for camel in vitro fertilization (IVF). Semen samples were collected from 7 Dromedary camels by means of artificial vagina (AV). Ten cauda epididymes were obtained from slaughtered adult camels, isolated, incised and r...
متن کامل