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 %

PARTICIPATION OF APOLIPOPROTEIN E IN TRANSFER AND ABSORPTION OF FATTY ACIDS BY THE CELLS And CAUSING OF HYPERLIPOPROTEINEMIA

ApoE vector protein in association with apoB-100 directly transferring saturated and monounsaturated FA (SFA and MFA) in triglyceride form (composed of very low density lipoproteins (L)) to the cells which are assimilating FA by cooperative receptors of apoE. Only insulin dependent cells have apoe/B-l00 receptors on the cell membrane (skeletal myocytes, cardiac myocytes, periportal hepatocytes, adipocytes of subcutaneous fat and Kupffer's macrophages). Phylogenetically late apoE has a domain for protein-protein interaction unlike the other apos. Apo forms cooperative ligands: apoE/A-l, apoE/B-48 and apoE/B-100 while using this domain. At later stages of phylogenesis while apoE forms cooperative ligands it is also involved in cell transfer and absorption of polyunsatured essential fatty acids in high density L, SFA +MFA +unsatured FA in chylomicrons, SFA+MFA in very low density L. Phenotype E 33 appears to be regular. Phenotypes E2/2 and E4/4 are cause of hypertriglyceridemia of I and V types, which call destructive inflammation of arterial intima with atherothrombosis

MOLECULAR MARKING OF SUNFLOWER LINES WITH DIFFERENT ABILITY TO SUPPRESSION OF THE CYTOPLASMIC MALE STERILITY PHENOTYPE

Ninety five lines of sunflower genetic collection differing by their ability to suppress the CMS phenotype were molecularly marked with the use of 7 primer pairs. Using the STS marker orfH522, a sterile (PET1) cytoplasmon was identified in 79 lines, which confirmed indirectly the presence of fertility restoration genes in their genotypes. The majority of these lines also have a complex of molecular markers linked to the Rf1 gene. The HRG01, HRG02 and STS115 markers showed the best diagnostic value in revealing the Rf1 gene in the examined material. The data on allelic variation of the microsatellite loci ORS224, ORS511 and ORS799 were obtained for the first time

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 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 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 as well as for 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 (one with animals born naturally and another with animals resulting from ART). There were no changes in BP between the groups, but the behavior of ISIAH differed depending on rearing conditions. In particular, grooming time, as well as the number of defecations and the number of urinations during the test were 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 as well as reproductive technologies affect some behavioral characteristics in adult ISIAH rats, although they develop arterial hypertension in all the conditions used in this study

Neuronal density in the brain cortex and hippocampus in Clsnt2-KO mouse strain modeling autistic spectrum disorder

Autistic spectrum disorders (ASD) represent conditions starting in childhood, which are characterized by difficulties with social interaction and communication, as well as non-typical and stereotyping models of behavior. The mechanisms and the origin of these disorders are not yet understood and thus far there is a lack of prophylactic measures for these disorders. The current study aims to estimate neuronal density in the prefrontal cortex and four hippocampal subfields, i. e. СA1, СA2, СA3, and DG in Clstn2-KO mice as a genetic model of ASD. In addition, the level of neurogenesis was measured in the DG area of the hippocampus. This mouse strain was obtained by a knockout of the calsinthenin-2 gene (Clsnt2) in C57BL/6J mice; the latter (wild type) was used as controls. To estimate neuronal density, serial sections were prepared on a cryotome for the above-mentioned brain structures with the subsequent immunohistochemical labeling and confocal microscopy; the neuronal marker (anti-NeuN) was used as the primary antibody. In addition, neurogenesis was estimated in the DG region of the hippocampus; for this purpose, a primary antibody against doublecortin (anti-DCX) was used. In all cases Goat anti-rabbit IgG was used as the secondary antibody. The density of neurons in the CA1 region of the hippocampus was lower in Clstn2-KO mice of both sexes as compared with controls. Moreover, in males of both strains, neuronal density in this region was lower as compared to females. Besides, the differences between males and females were revealed in two other hippocampal regions. In the CA2 region, a lower density of neurons was observed in males of both strains, and in the CA3 region, a lower density of neurons was also observed in males as compared to females but only in C57BL/6J mice. No difference between the studied groups was revealed in neurogenesis, nor was it in neuronal density in the prefrontal cortex or DG hippocampal region. Our new findings indicate that calsyntenin-2 regulates neuronal hippocampal density in subfield-specific manner, suggesting that the CA1 neuronal subpopulation may represent a cellular target for earlylife preventive therapy of ASD

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

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

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