Genome resource banking in the family Felidae

Many of the extant Felidae species are endangered or vulnerable. Others being not endangered as a whole species contain endangered subspecies. Only a very few cat species, besides domestic cats, are not in the risk group. Cryopreservation of embryos and gametes is a modern approach for ex situ mammalian genetic resources conservation. Freezing of semen has been successfully applied to the domestic cat and to more than 25 wild members of this family. However, embryos/oocytes cryopreservation was successful for only a small number of felids. Domestic cat and four wild Felidae species produced offspring after cryopreservation and subsequent embryo transfer. Regarding freezing of oocytes, so far different cryopreservation methods are still being experimentally tried exclusively for domestic cat. Genome Resource Bank (GRB) containing frozen semen of Amur leopard cat, bobcat and Eurasian lynx was established at the Institute of Cytology and Genetics, Novosibirsk. As a result of this project, original methods of feline semen freezing have been developed; embryos of domestic cat have been successfully frozen as well. Approaches to freeze domestic cat’s oocytes have also been tried. During this work, we combined biological and physical methods. In particular, the process of freezing embryos and oocytes was monitored with Raman spectroscopy. Different methods of frozen-thawed spermatozoa and embryonic viability testing were used in this study, including vital staining and subsequent fluorescent and light microscopy, and heterologous in vitro fertilization

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

The proper choice of cryoprotectant and thawing method affects cryopreservation efficiency. A freezing-thawing method for sparing embryonic cells was evaluated in experiments with ICR mice. 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 a standard protocol. Permeating (ethylene glycol and glycerol) and nonpermeating (sucrose) cryoprotectants and their combinations were compared during the freezing of ICR mouse embryos. With these mice, two thawing methods were compared: rapid (water bath, 10 s, 37 °С) and slow (40 s, room temperature; 40 s, 30 °С). Embryo viability in mice and rats was evaluated by their in vitro culturing after thawing. Our data on mice indicate that slow thawing is more suitable for sparing the integrity of embryonic cells; moreover, supplementation of the main cryoprotectant (either ethylene glycol or glycerol) with sucrose is beneficial for subsequent in vitro culture, especially in the case of glycerol. This freezing-thawing protocol (with glycerol and sucrose as cryoprotectant agents and slow thawing) was applied to rats of the GC and OXYS strains; the survival rate after cryopreservation was 68–83.3 %, and the rate of in vitro development was 64.7–66.6 %

Cryopreservation of epididymal semen of domestic cat

Domestic cat (Felis silvestris catus) is used as a model species for developing effective methods of wild felids’ semen cryopreservation. The present study represents a comparison of domestic cat semen cryopreservation with two commercially available cryoprotectant agents (CPAs): CaniPlus Freeze (CPF) and SpermFreeze (SF). Semen was collected from the caudal epididymises of adult males and frozen with CPF and SF, correspondingly. The viability of frozen-thawed spermatozoa was evaluated by VitalScreen kit, staining with hematoxylin and eosin was performed for analysis of the spermatozoa morphology; both analyses were combined with the light microscopy. The viability rate of the frozenthawed semen cryopreserved with CPF and SF did not differ: 32.3±4.4 % for CPF and 43.3±4.0 % for SF. Total percentage of morphologically normal spermatozoa after freezing and thawing domestic cat semen was 26.0±2.3 % for CPF and 23.9±1.9 % for SF. In both cases, there were no differences from non-frozen semen, in the latter group the total percentage of spermatozoa with normal morphology was 29.0±4.1 %. The most frequent anomalies were the anomalies of tail, and the rarest anomalies were head defects. The percentages of spermatozoa with anomalies of the head, mid piece, tail and combined did not differ in these three groups. Taken together these results suggest that both CPAs are suitable for the purpose of domestic cat semen freezing and cryopreservation, although CPF was designed for Canidae semen cryopreservation and SF was developed for human semen freezing and so far was used exclusively in reproductive medicine. It might be concluded that these two commercially available cryoprotectant media are applicable for the purposes of domestic cat breeds’ semen cryopreservation

Comparison of in vivo and in vitro preimplantation embryo development in OXYS and WAG rats

OXYS rats are the model of precocious senescence. Numerous studies addressed physiology and behavior in rats of this strain during a postnatal period of their life, however, preimplantation development in OXYS rats has not yet been investigated. This study is addressing preimplantation embryonic development in OXYS rats both in vivo and in vitro. Rats of the WAG strain were used as controls. For studying the in vivo development, the embryos were collected from OXYS and WAG rats on day 5 post coitum, the stages of embryo development were estimated, the percentage of embryos at blastocyst stage and the cell numbers in these blastocysts were counted. In a special experiment, for studying in vitro development, the embryos were collected from both rat strains on day 4 post coitum and were cultured in vitro in P1 medium for 48 hours with or without supplementation with IGF-1 (200 ng/mL). Thereafter the percentage of embryos at blastocyst stage and the cell numbers in these blastocysts were counted in the same manner as for the in vivo experiment. This study reports that in vivo derived blastocysts of OXYS rats contain fewer cells on day 5 of their development than in vivo derived blastocysts of WAG rats. In vitro culture of the early preimplantation embryos in P1 medium mitigated the difference in the rate of embryo development between these two strains, the addition of IGF-1 into culture medium exerts neither negative nor positive effect on the rate of in vitro embryo development in rats of both strains

Effects of a high-fat diet on the lipid profile of oocytes in mice

There are evidences that obese women exhibit a detrimental oocyte quality. However, it remains unclear how this change is associated with obesity, indirectly – or directly through a change in the content and/or composition of lipids in oocytes. The aim of this work was to study effects of a high-fat diet applied to female donor mice on the amount and qualitative composition of lipids of immature and in vivo matured oocytes. A high-fat diet caused larger body weight in female mice compared with the control (p < 0.001; 44.77±1.46 and 35.22±1.57, respectively), and increased the blood levels of cholesterol (p < 0.05; 2.06±0.10 and 1.78±0.10, respectively) and triglycerides (p < 0.05; 2.13±0.23 and 1.49±0.21, respectively). At the same time, this diet does not affect the level of unsaturation of lipids in immature (0.207±0.004 in the experiment and 0.206±0.002 in the control) and matured oocytes (0.212±0.005 in the experiment and 0.211±0.003 in the control). Total lipid content increased during in vivo maturation of mouse oocytes. The amount of lipids was greater in mature oocytes in the experimental group compared to the control (p < 0.01; 8.15±0.37 and 5.83±0.14, respectively). An increase in intracellular lipid amount during oocyte maturation was revealed both after a standard diet (p < 0.05; 4.72±0.48 and 5.83±0.14, respectively) and after a fat-rich diet (p < 0.001; 3.45±0.62 and 8.15±0.37, respectively). Thus, during in vivo oocyte maturation in mice the content of intracellular lipids enhanced, the high-fat diet aggravated this dynamics of lipid increase during in vivo maturation of oocytes

Alterations in the social-conditioned place preference and density of dopaminergic neurons in the ventral tegmental area in Clsnt2-KO mice

The incidence of autistic spectrum disorders (ASD) constantly increases in the world. Studying the mechanisms underlying ASD as well as searching for new therapeutic targets are crucial tasks. Many researchers agree that autism is a neurodevelopmental disorder. Clstn2-KO mouse strain with a knockout of calsyntenin 2 gene (Clstn2) is model for investigating ASD. This study aims to evaluate the social-conditioned place preference as well as density of dopaminergic (DA) neurons in the ventral tegmental area (VTA), which belongs to the brain reward system, in the males of the Clstn2-KO strain using wild type C57BL/6J males as controls. Social-conditioned place preference test evaluates a reward-dependent component of social behavior. The results of this test revealed differences between the Clstn2-KO and the control males, as the former did not value socializing with the familiar partner, spending equal time in the isolationand socializing-associated compartments. The Clstn2-KO group entered both compartments more frequently, but spent less time in the socializingassociated compartment compared to the controls. By contrast, the control males of the C57BL/6J strain spent more time in socializing-associated compartment and less time in the compartment that was associated with loneness. At the same time, an increased number of DA and possibly GABA neurons labeled with antibodies against the type 2 dopamine receptor as well as against tyrosine hydroxylase were detected in the VTA of the Clstn2-KO mice. Thus, a change in social-conditioned place preference in Clstn2-KO mice as well as a higher number of neurons expressing type 2 dopamine receptors and tyrosine hydroxylase in the VTA, the key structure of the mesolimbic dopaminergic pathway, were observed

Long-term effects of maternal exposure to surgical stress at the earliest stage of pregnancy on blood pressure and behavior in offspring of OXYS rats

The use of some assisted reproductive technologies, in particular, embryo transfer, may cause various physiological and behavioral changes in the offspring. The purpose of our study was to study the effects of surgery (which is used for embryo transfer) done with pregnant dams on the weight, blood pressure and behavior in the open field and elevated plus­maze tests in adult offspring. Thus, long­term effects on the offspring after maternal exposure to surgical stress given to dams at the 4th day of pregnancy were studied in OXYS rats. OXYS females were mated in estrus with fertile males of the same strain. 96 hours after spermatozoa were found in vaginal smears the surgery (sham operation, imitating embryo transfer) was performed. Body weight (BW), systolic (SAP) and diastolic (DAP) arterial pressure as well as behavior in open field (OF) and elevated plus maze (EPM) tests were studied in the offspring of females exposed to surgical treatment during pregnancy (OXYS­PS) at the age of 3 mo. Untreated offspring of OXYS rats were used as controls. BW in naturally born OXYS rats did not differ from those of the OXYS­PS group. OXYS and OXYS­PS rats exhibited higher SAP (more than 150 mm Hg) and DAP; it is noteworthy that both SAP and DAP were higher in the OXYS­PS group than in the control group. The time spent in the center of arena, the area studied, the time and number of rearing were decreased in OXYS­PS rats in the OF test as compared to the OXYS controls. Moreover, OXYS­PS rats were characterized by the absence of grooming in the OF test. As was demonstrated by the EPM test, the duration and numbers of peeking out from closed arms were decreased in the OXYS­PS rats as compared to the OXYS controls. Thus, OXYS dams’ exposure to surgical stress at their early pregnancy led to such effects in the offspring as elevated SAP and DAP, decreased overall activity and increased anxiety

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 used in combination with modern reproductive technologies such as rederivation, in vitro culture and embryo transfer. Thirteen mouse and rat strains have been re-derived and 32 are kept frozen in the cryostorage at the Institute of Cytology and Genetics, Novosibirsk. Some other laboratory animal species have been cryopreserved as well. Embryos of two hamster species (Djungarian and Campbell’s) in the genus Phodopus were cryopreserved and the viability of thawed embryos was proved by their successful development in vitro and in vivo (by transfer to a recipient). A positive effect of the granulocyte-macrophage colony-stimulating factor (GM-CSF) was demonstrated with both these Phodopus species. Furthermore, semen of Djungarian (Phodopus sungorus) and Campbell’s (Phodopus campbelli) hamsters, domestic cat (Felis catus), amur cat (Prionailurus bengalensis euptilurus) and bobcat (Lynx rufus) was frozen and cryopreserved. Double staining by SYBR Green/PI and subsequent confocal microscopy demonstrated that more than 40 % of amur cat semen retained viability after cryopreservation. This is the world’s first reported successful freezing of semen of this wild felid (Prionailurus bengalensis euptilurus). This article reviews the results and discusses prospects of using reproductive technologies for conservation of laboratory species

REDERIVATION BY EMBRYO TRANSFER IN STRAINS OF LABORATORY MICE AND RATS

Rederivation allows laboratory animal populations to be purged from specified pathogens and thus turns these animals to the SPF (specified pathogens free) status. Results of the rederivation of two unique rat strains selected at the Institute of Cytology and Genetics and one mouse strain are presented. The two rat strains are: tame Norway rats and rats with Inherited Stress Induced Arterial Hypertension (ISIAH strain). The ICR mouse strain has been named as abbreviation of the Institute of Cancer Research wherefrom these mice were distributed to laboratories all over the world. The SPF status of the rats after rederivation was confirmed by the method of indicator animals (sentinel animals). The optimized model of rederivation offered here involves a combination of such embryotechnological methods as freezing/cryopreservation of embryos, their washing through the number of fresh volumes of sterile media, growing in vitro for 48 hours, and subsequent transfer into either one or both uterine horns of recipient females. Application of this model to rederivation of ICR mice yielded 39 pups born in an SPF vivarium. It should be noticed that the effectiveness of the procedure met international standards, and characteristic features of phenotype were retained in all the three strains after rederivation