Effects of growth factors during in vitro culture of mouse and rat embryos

In vitro culture of preimplantation embryos of ICR, HT1AN/Icgn, HT1AC/Icgn and C57BL/6J-Ay mouse strains as well as in OXYS/Icgn rat strain in media containing granulocyte-macrophage colony stimulating factor (GM-CSF) or epidermal growth factor (EGF) has been studied. Both mouse and rat embryos were first frozen in a programmable freezer after a standard protocol using a mixture of glycerol and sucrose as cryoprotectants, thawed and cultured in vitro in R1ECM (rat one-cell embryo culture medium) for 24 hours (mice) and 72 hours (rats). For the in vitro culture experiments with these growth factors, 8-cell frozen-thawed mouse embryos and 2–4-cell frozen-thawed rat embryos were used. Supplementation of the culture medium with GM-CSF improved the rate of embryonic development in HT1AC/Icgn and C57BL/6J-Ay strain mice, while EGF had no effect. The reverse was true of the rats. Supplementation of the culture medium with EGF increased the percentage of deve­loping blastocysts in OXYS/Icgn rat strain, while GM-CSF had no effect. Co-culture of four-cell embryos of HT1AN/ Icgn strain mice with more advanced embryonic stages (morulas) of a different strain ICR led to the facilitation preimplantation embryo development. Experimental results presented here reveal the species-specific effects of growth factors on mouse and rat embryos and indicate that co-culture of different stages of embryo development have stimulatory effects on earlier stages

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 %

Psycho-emotional stress, folliculogenesis, and reproductive technologies: clinical and experimental data

Modern life, especially in large cities, exposes people to a high level of noise, high density of population, disrupted sleeping, large amount of excessive and controversial information as well as to other negative factors; all this may cause chronic psycho-emotional stress. The latest publications often use the term “Syndrome of megalopolis”, which means disruption of sleeping, high anxiety, and altered reproductive function. Medical treatment of infertility may also be considered as a stress factor, especially when infertility lasts for years and is aggravated with emotional frustration. Long-lasting distress may worsen health in general and suppress reproductive function, in particular. The review presents the data on the effects of maternal stress on folliculogenesis, especially when assisted reproductive technologies (ARTs) are used. Clinical data are presented alongside data from laboratory animal experiments. Different maternal stress models are taken into account in respect of their inf luence on oocyte maturation and embryo development. The interfering of psycho-emotional stress and reproductive function is the focus of the review. In these situations, exogenous hormones compensate for the stress-related disruption of the hypothalamic-pituitary-gonadal axis. When ARTs are implemented, stress-induced disruption of oogenesis is realized not via a decrease in hypothalamic and pituitary hormones, but by other ways, which involve paracrine mechanisms described in this review. Based on the literature analysis, one may conclude that stress negatively affects oocyte maturation in the ovary and suppresses subsequent embryo development. The role of some ovarian paracrine factors, such as BDNF, GDF-9, HB-EGF, TNF-α, and some others has been elucidated

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

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

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

Sensitivity of the skin test with tuberculous allergen in TB/HIV patients

The sensitivity of skin test with tuberculous allergen in TB/HIV patients has need studied (n = 36) depending on the immune deficiency and the character of tuberculous inflammation. The comparison group included patients with HIV infection and no tuberculosis (n = 54). The test results in HIV patients but without tuberculosis and in the patients with the concurrent condition are directly connected to the degree of immune deficiency. In the patients with HIV infection and progressing tuberculosis the negative results of the test are due to secondary anergy. The patients with severe and expressed immune deficiency the negative results do not exclude the activeness of the infection, thus patients with CD4 level up to 350 + cl/mcl and negative results can be referred as a group of high risk of developing tuberculosis