E. Yu. Brusentsev

Rederivation by embryo transfer in strains of laboratory mice and rats

Rederivation enables one to decontaminate colonies of laboratory mice and rats from specific pathogens and to convert them to the SPF (specified pathogen free) state. In this study the results of the rederivation of two unique rat strains that were selected at the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences, viz., tame Norway rats, rats with inherited stress-induced arterial hypertension (ISIAH), and the ICR mouse strain, are presented. The SPF state of the rederivated rats was confirmed by indicator animals, which are also called sentinel animals. An optimized rederivation model of laboratory animals is suggested in the article, which includes a series of embryotechnological methods, viz., freezing and cryopreservation of embryos, their decontamination by washing in sterile media, cultivation for 48 h, and, finally, transfer to recipients (with the SPF state). As a result of the application of this model to the ICR strain mice, it was possible to obtain 39 offspring that were born in the conditions of an SPF animal facility. It is worth mentioning that the efficacy of the procedure is in agreement with international standards, with all three lines representing a specific phenotype after undergoing all the procedures of rederivation.

Coats of preimplantation mammalian embryos as a target of reproductive technologies

The structure and function of the mammalian oocyte and preimplantation embryo coverings are described in this review. The integrity of embryonic coverings is the main prerequisite for the success of such technology as preimplantation embryo freezing and, especially, for successful rederivation. On the other hand, results of in vitro fertilization and, sometimes, the results of embryo freezing are improved after perforation of the oocyte/embryonic coverings. Modern reproductive technologies focusing on oocyte/embryonic coverings, such as preimplantation embryo freezing/cryopreservation, in vitro fertilization, intracytoplasmic sperm injection, assisted hatching, immunocontraception, and rederivation, are reviewed. Application of these technologies to different mammalian species is discussed with a special emphasis on the oocytes/preimplantation embryos coverings.

Embryo and gamete cryopreservation for genetic resources conservation of laboratory animals

The article reviews the use of embryo and gamete cryopreservation for cryobanking the laboratory animal species. Special emphasis is given to the mechanisms of cryoinjury and cryoprotection during program freezing and vitrification. The species specific cryobanking problems are discussed and the prospects to overcome these problems are outlined.

Traditional and modern approaches to culture of preimplantation mammalian embryos in vitro

This review covers the basic principles and methods of in vitro culture of preimplantation mammalian embryos. The features of in vitro development of embryos of various species of animals with allowance for the composition of nutrient media are described, with special attention paid to those species that have traditionally been considered as laboratory (i.e., mice, rats, and hamsters). The effects of suboptimal culturing conditions of preimplantation embryos on the formation of the phenotype of individuals developed from these embryos are discussed. New approaches to optimize the conditions of the development of preimplantation mammalian embryos in vitro are analyzed.

Developmental aspects of senescence

Different types of senescence and major theories of aging are reviewed, and mechanisms of this complex biological phenomenon are discussed. Emphasis is placed on changes in the nervous systems of mammals and humans with age. Experimental animal models for studying aging and modern approaches to the correction of age-related deterioration are considered. Chemicals and other factors that may alleviate agerelated disorders and slow down senescence are critically reviewed.

Cryoprotectant redistribution along the frozen straw probed by Raman spectroscopy.

The distribution of cryoprotectant (10% glycerol) and ice along the frozen plastic straw (the most useful container for freezing mammalian semen, oocytes and embryos) was studied by Raman scattering technique. Raman spectroscopy being a contactless, non-invasive tool was applied for the straws filled with the cryoprotectant solution and frozen by controlled rate programs commonly used for mammalian embryos freezing. Analysis of Raman spectra measured at different points along the straw reveals a non-uniform distribution of the cryoprotectant. The ratio between non-crystalline solution and ice was found to be increased by several times at the bottom side of the solution column frozen by the standard freezing program. The increase of the cryoprotectant fraction occurs in the area where embryos or oocytes are normally placed during their freezing. Possible effects of the cooling rate and the ice nucleation temperature on the cryoprotectant fraction at the bottom side of the solution column were considered. Our findings highlight that the ice fraction around cryopreserved embryos or oocytes can differ significantly from the averaged one in the frozen plastic straws.

A comparison of different cryoprotectant solutions and thawing methods for the cryopreservation of embryos of mice and rats

The proper choice of the cryoprotectant and thawing method affects the efficiency of cryopreservation. A freezing-thawing method aimed at the preservation of blastomere cells was evaluated in experiments with ICR mice. The cleavage-stage embryos of ICR mice, GC rats, and OXYS rats were collected on Day 3 of pregnancy and frozen in plastic straws according to the standard procedure. We compared the effect of permeating (ethylene glycol and glycerol) and nonpermeating (sucrose) cryoprotectants and their combinations on the survival rate of embryos after thawing. We also compared the effect of rapid (water bath, 10 s, 37°С) and slow (40 s, room temperature; then 40 s, 30°С) thawing methods. The viability of the embryos of mice and rats after cryopreservation was evaluated by their in vitro culturing after thawing. Our data prove that slow thawing is more suitable for mice embryos and provides a higher survival rate; the addition of sucrose to the basic cryoprotectant (ethylene glycol or glycerol) improves the parameters of the in vitro cultures of embryos after thawing, especially if glycerol is used as the basic cryoprotectant. This freezing-thawing method (glycerole and sucrose as the cryoprotectant solution and slow thawing) was used for cryopreservation of GC and OXYS rats. As a result, the survival rate of embryos after freezing was 68–83.3% and the rate of in vitro development after thawing was 64.7–66.6%.

Applying reproductive technologies and genome resource banking to laboratory animals

The Genome Resource Bank (GRB) is a repository of frozen biological material, including semen and embryos. Cryobanking is often used in combination with modern reproductive technologies, such as rederivation, in vitro culture, and embryo transfer. Thirteen mouse and rat strains have been rederived, and 32 are kept in cryostorage at the Institute of Cytology and Genetics, Novosibirsk. Other laboratory animal species have been cryopreserved as well. Embryos of two hamster species (Djungarian and Campbell’s) of the Phodopus genus were cryopreserved, and the viability of thawed embryos was confirmed by their successful development in vitro and in vivo (by transfer to a recipient). The positive effect of the Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) was demonstrated for both of these Phodopus species. Furthermore, the sperm of Djungarian (Phodopus sungorus) and Campbell’s (Phodopus campbelli) hamsters, domestic cat (Felis catusf),leopard cat (Prionailurus bengalensis euptilurus), and bobcat (Lynx rufus) was frozen and cryopreserved. Double staining by SYBR Green/PI, followed by confocal microscopy, demonstrated that more than 40% of amur cat semen retained their viability after cryopreservation. This is the world’s first reported successful freezing of semen of these wild feline species (Prionailurus bengalensis euptilurus). The article reviews the results and discusses the prospects of using reproductive technologies for the conservation of laboratory animal species.

Effects of reproductive technologies and SPF status on some physiological and behavioral characteristics in rats with arterial hypertension (ISIAH Strain)

Modern standards of Laboratory Animal Science include working with laboratory animals of a high quality, in particular, with specific pathogen-free (SPF) mice and rats. On the other hand, assisted reproductive technologies (ART) are widely used in modern medicine for human infertility treatment and genome resource banking. In the present study, a comparison of body weight, blood pressure (BP), and behavior in the elevated plus maze (EPM) test was made between three groups of ISIAH (inherited stress induced arterial hypertension) rats: a group of animals that were born and raised in a conventional animal facility and two groups from an SPF animal facility (animals born naturally and animals resulting from ART). There were no changes in BP between the groups, but the behavior of ISIAH rats differed depending on rearing conditions. In particular, the grooming time, as well as the number of defecations and the number of urinations during the test decreased in both groups of ISIAH rats born in the SPF animal facility as compared to ISIAH rats born in the conventional animal facility. The behavior of the ISIAH rat offspring resulting from ART was different from that of the naturally born group: the EPM test revealed reduced anxiety in the former. The results of the present study indicate that the rearing conditions and the reproductive technologies affect some behavioral characteristics in adult ISIAH rats, although they posessed arterial hypertension in all the conditions used in this study.

Assisted reproductive technologies and arterial hypertension

The effects of assisted reproductive technologies on the development of hypertensive phenotype were reviewed. Special attention was paid to the effects of cultivation and transplantation of preimplantation embryos on arterial pressure in individuals developed from these embryos. The analysis of studies performed on the laboratory models (mostly on hypertensive strains of rats) was performed. These data were discussed in the context of application of assisted reproductive technologies in medicine.