High microsatellite and mitochondrial diversity in Anatolian native horse breeds shows Anatolia as a genetic conduit between Europe and Asia.

The horse has been a food source, but more importantly, it has been a means for transport. Its domestication was one of the crucial steps in the history of human civilization. Despite the archaeological and molecular studies carried out on the history of horse domestication, which would contribute to conservation of the breeds, the details of the domestication of horses still remain to be resolved. We employed 21 microsatellite loci and mitochondrial control region partial sequences to analyse genetic variability within and among four Anatolian native horse breeds, Ayvacik Pony, Malakan Horse, Hinis Horse and Canik Horse, as well as samples from indigenous horses of unknown breed ancestry. The aims of the study were twofold: first, to produce data from the prehistorically and historically important land bridge, Anatolia, in order to assess its role in horse domestication and second, to analyse the data from a conservation perspective to help the ministry improve conservation and management strategies regarding native horse breeds. Even though the microsatellite data revealed a high allelic diversity, 98% of the genetic variation partitioned within groups. Genetic structure did not correlate with a breed or geographic origin. High diversity was also detected in mtDNA control region sequence analysis. Frequencies of two haplogroups (HC and HF) revealed a cline between Asia and Europe, suggesting Anatolia as a probable connection route between the two continents. This first detailed genetic study on Anatolian horse breeds revealed high diversity among horse mtDNA haplogroups in Anatolia and suggested Anatolia's role as a conduit between the two continents. The study also provides an important basis for conservation practices in Turkey.

Comparison of different cryopreservation techniques: higher survival and implantation rate of frozen-thawed mouse pronuclear embryos in the presence of beta-mercaptoethanol in post-thaw culture.

The objective of this study was to investigate the effects of beta-mercaptoethanol (ß-ME) on post-thaw embryo developmental competence and implantation rate of mouse pronuclear (PN) embryos that were cryopreserved after slow freezing, solid surface vitrification (SSV) or open-pulled straw (OPS) vitrification methods. Mouse PN embryos were cryopreserved by using slow freezing, SSV and OPS methods. After cryopreservation, freeze-thawed PN embryos were cultured up to blastocyst stage in a defined medium supplemented without or with 50 µM ß-ME. The blastocyst formation rate of embryos that were cryopreserved by slow freezing method (40.0%) or vitrified by OPS method (18.3%) were lower than those vitrified by SSV method (55.6%) and fresh embryos (61.9%) in the absence of 50 ß-ME in the culture media (p < 0.05). The blastocyst formation rate of embryos that were cryopreserved by slow freezing method (53.1%) or by OPS method (41.9%) were lower than those vitrified by SSV method (79.5%) and that of fresh (85.7%) in the presence of ß-ME in the culture media (p < 0.05). The embryos transfer results revealed that the implantation rate of blastocyst derived from mouse PN embryos vitrified by SSV method (31.9% vs 51.2%) was similar to that of the control (39.0% vs 52.5%), but higher than those cryopreserved by slow freezing (28.2% vs 52.0%) and by OPS method (0.0% vs 51.2%) (p < 0.05). In conclusion, supplementation of ß-ME in an in vitro culture medium was shown to increase survival of embryo development and implantation rate of frozen-thawed mouse PN embryos after different cryopreservation protocols.

Effect of chemically defined culture medium supplemented with beta-mercaptoethanol and amino acids on implantation and development of different stage in vivo- or in vitro-derived mouse embryos.

In vitro culture (IVC) systems are required for many biotechnological and assisted reproductive technologies and the researchers have been modifying in vitro embryo culture conditions to reach the comparable efficiencies provided in vivo. In the present study, the effects of beta-mercaptoethanol (Beta-ME) and amino acids (AA) on the development of mouse embryos obtained in vivo or in vitro at different stages were investigated. Chemically defined potassium simplex optimized medium (KSOM) was used as basic culture medium and six experimental groups were established and by supplementation of Beta-ME and AA into KSOM media. The quality of blastocysts was evaluated by counting the cells and determining the ratio of inner cell mass (ICM) to trophoectoderm (TE) cells. In addition, embryo transfer (ET) was performed to investigate the rate of implantation and live fetuses. The results obtained in the present study demonstrated that the combined treatment of Beta-ME and AA to 1-cell stage embryos not only enhanced in vitro development to the blastocyst stage but also improved both the number of blastocysts cells and live fetuses.

Exposure to warmer postoperative temperatures reduces hypothermia caused by anaesthesia and significantly increases the implantation rate of transferred embryos in the mouse.

Embryo transfer (ET) is among the key factors determining the overall efficiency of transgenic technology in the mouse. A successful ET depends among other factors on the quality of the transferred embryos, foster mothers and anaesthetic reagents and on the transfer techniques. Anaesthesia-caused deaths and suboptimal ET procedures are factors which reduce the success of transgenic experiments and mouse colony maintenance. Here we compared the effects of two anaesthetic reagents-a ketamine/xylazine combination, and tribromoethanol (Avertin)-on the rates of implantation and development to term of mouse zygotes transferred into the oviducts of CD-1 foster mothers, and evaluated whether hypothermia caused by anaesthetics after the ET operation could be overcome by postoperative incubation of the foster mothers. We established two experimental groups of fosters, one of which was kept at room temperature (RT, 21 degrees C) with the other in an incubator (33 degrees C) overnight after ET. Rates of implantation, resorption and development to normal fetuses were evaluated by sacrificing the foster mothers on the 15th day of their pregnancy. Our results showed that regardless of the anaesthetic reagents used, the rates of implantation and of development to normal fetuses can be significantly improved by exposing the foster mothers to warmer temperatures (33 degrees C) immediately after the ET operation. These results may have important implications in increasing the success rate of ET with micromanipulated embryos.

Vitrification of pronuclear-stage mouse embryos on solid surface (SSV) versus in cryotube: comparison of the effect of equilibration time and different sugars in the vitrification solution.

The cryopreservation of pronuclear-stage embryos has particular importance in transgenic technology and human assisted reproductive technology (ART). The objective of this study was to improve the efficiency of cryopreservation of pronuclear-stage mouse embryos. Two vitrification methods (solid surface vitrification (SSV) vs. vitrification in cryotube) have been compared with special emphasis on the effect of the exposure of the embryos to the solutions for various times and the sugar content (trehalose, sucrose, or raffinose) of the vitrification solutions. Pronuclear-stage embryos were either exposed to 1 M dimethyl sulfoxide (DMSO) + 1 M propylene-glycol (PG) solution for 2, 5, 10, or 15 min or not exposed to this "equilibration" solution. The vitrification solutions consisted of 2.75 M DMSO and 2.75 M PG in M2 medium supplemented with 1 M trehalose (DPT), 1 M sucrose (DPS), or 1 M raffinose (DPR). In the cryotube method, groups of 15-25 embryos were transferred into a 1.8 ml cryotube containing 30 microl of DPT, DPS, or DPR. After 30 sec, the cryotubes were directly plunged into liquid nitrogen (LN(2)) and stored for 1 day to 1 month. Vitrified samples were warmed by immersing the cryotubes in a 40 degrees C water bath and then immediately diluted with 300 microl of 0.3 M trehalose, sucrose, or raffinose in M2. In the SSV method, after equilibration 15-20 embryos were exposed to DPT, DPS, or DPR solutions for around 20 sec before being dropped in 2-microl drops onto a pre-cooled (-150 to -180 degrees C) metal surface. Vitrified droplets were stored in cryovials in LN(2). Warming was performed by transferring the vitrified droplets into 0.3 M solutions of trehalose, sucrose, or raffinose at 37 degrees C, respectively. Results showed that both SSV and cryotube vitrification methods can result in high rates of in vitro blastocyst development (up to 58.3 and 68.5% with DPR, respectively), not statistically different from that of the controls (58.3 and 64.4%). Even without the equilibration step prior to vitrification, relatively high-survival rates have been achieved, except for the DPS solution. In conclusion, vitrification of pronuclear-stage mouse embryos can result in high rates of in vitro development to blastocyst, and the use of raffinose in the vitrification solution is advantageous to improve cryosurvival.

Production of transgenic mice from vitrified pronuclear-stage embryos.

Cryopreservation of pronuclear-stage embryos would be useful for transgenic technology and genome preservation purposes. We compared a novel vitrification technique (solid surface vitrification, SSV) with another vitrification method in straws for cryosurvival and to generate transgenic progeny from cryopreserved mouse zygotes following microinjection. The SSV solution consisted of 35% ethylene glycol (EG), 5% polyvinyl-pyrrolidone (PVP), and 0.4 M trehalose in M2 supplemented with 4 mg/ml BSA; the in straw vitrification solution was 7 M EG in M2 plus BSA. In experiment I, we compared the effect of the vitrification solutions alone, without cooling. No reduction was detected in survival and cleavage rates. In experiment II, SSV yielded a significantly higher percentage of morphologically normal zygotes (96%) that also cleaved at significantly higher rates (80%) when compared to that following "in straw" vitrification (68 and 66%, respectively). Cleavage rate in the non-vitrified control group (93%) was significantly higher than that of both vitrified groups. Following embryo transfer, there was no difference in the rate of pups obtained from the SSV, "in straw" vitrified, and control groups (97/457, 21%; 15/75, 20% and 56/209, 27%, respectively). In experiment III, SSV vitrified and fresh embryos were used for pronuclear DNA injection. Survival rate of vitrified embryos after microinjection was reduced compared to nonvitrified ones (64 vs. 72%, respectively; P < 0.05); however, development to two-cell stage was not different (76 vs. 72%, respectively). Following embryo transfer of vitrified vs. fresh microinjected embryos, in both cases 10% live pups were generated, including transgenic pups. The results demonstrated that the efficiency of generating transgenic pups from SSV vitrified pronuclear zygotes is comparable to that from fresh embryos.

Accumulation of the proteolytic marker peptide ubiquitin in the trophoblast of mammalian blastocysts.

Ubiquitination is a universal protein degradation pathway in which the molecules of 8.5-kDa proteolytic peptide ubiquitin are covalently attached to the epsilon-amino group of the substrate's lysine residues. Little is known about the importance of this highly conserved mechanism for protein recycling in mammalian gametogenesis and fertilization. The data obtained by the students and faculty of the international training course Window to the Zygote 2000 demonstrate the accumulation of ubiquitin-cross-reactive structures in the trophoblast, but not in the inner cell mass of the expanding bovine and mouse blastocysts. This observation suggests that a major burst of ubiquitin-dependent proteolysis occurs in the trophoblast of mammalian peri-implantation embryos. This event may be important for the success of blastocyst hatching, differentiation of embryonic stem cells into soma and germ line, and/or implantation in both naturally conceived and reconstructed mammalian embryos.