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

Effect of exogenous human chorionic gonadotropin on ovulation in mice

The implementation of assisted reproductive technologies (ART), hormonal stimulation in particular, may change the quality of ovulated oocytes. The purpose of our work was to study ovulation in CD1 mice after their stimulation with human chorionic gonadotropin (hCG) and to investigate the effects of such hormonal stimulation on the pregnancy duration, fetal losses and the weight of the offspring. No significant differences were found in the total number of ovulated oocytes or in the number of immature (without a polar body) ovulated oocytes; nor were there differences between the groups in the number of oocytes with a developing polar body. However, the number of matured oocytes with a distinct polar body was significantly higher (p < 0.05) in mice stimulated with hCG (experimental group) as compared with the controls (6.2 ± 0.86 and 2.2 ± 0.97, respectively). No significant differences were observed between the experimental and control mice in the duration of pregnancy or in the numbers of term offspring, including the percentage of live and stillborn pups. However, the body weight of the offspring in the experimental group was significantly lower (p < 0.001) as compared with the controls on the fifth day after birth (3.16 ± 0.09 and 3.76 ± 0.07, respectively). Thus, exogenous hCG facilitates the development of mouse oocytes in vivo, which leads to the larger number of their mature forms at ovulation, however, the offspring born after hCG-stimulated pregnancy was characterized by a lower body weight on the fifth day after birth

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

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

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