Combining dimethyl sulphoxide (DMSO) with different cryoprotectants ensures better cartilage cell cryopreservation.

BACKGROUND: DMSO is a cryoprotective agent (CPA) that is widely used in the cryopreservation of cells. However, evidence suggests that it is more effective when combined with other CPAs. OBJECTIVE: To investigated the effect of combining serum and balanced solutions with DMSO for cryopreservation. MATERIALS AND METHODS: Cartilage cells cultured with Dulbecco's Modified Eagle Medium (DMEM) medium were taken from the ears of adult cattle. Then, these cells were frozen by supplementation with different concentrations of fetal bovine serum (FBS) and different balanced solutions with DMSO. RESULTS: The highest cell viability was obtained using freezing solutions containing 10% DMSO and 40% serum, in dextran 40 or dextrose solution or DMEM. CONCLUSION: Our results indicated that serum concentration is important for cell viability and that supplementing DMSO with dextran or dextrose or DMEM benefits the cryopreservation of bovine cartilage cells.

Treatment with Melatonin Enhances the Embryo Quality and the Development of Vitrified/warmed Eight Cell Mouse Embryos by Solid Surface Vitrification (SSV).

BACKGROUND: Melatonin is an endocrine hormone secreted from the pineal gland located outside the blood-brain barrier. OBJECTIVE: In this study, in vitro propagated eight-cell mouse embryos were vitrified by the Solid Surface Vitrification (SSV) method and after thawing, their in vitro development and embryo qualities in melatonin added media were investigated. METHODS: Pronuclear stage embryos obtained from super ovulated B6CBAF1/J strain mice, were cultured until the eight-cell stage. Then these eight-cell embryos were vitrified by the SSV method and after thawing, cultured in melatonin added media at 37°C and 5 %CO2 conditions until the blastocyst stage. RESULT: In the experimental period, in vitro embryo development rates of the control, SSV and +10-12 M melatonin groups were observed as 97%, 86% and 93%, respectively. CONCLUSION: Our results indicated that melatonin addition slightly increased the development rates and total cell numbers of embryos vitrified by the SSV method.

Are illness perception and coping style associated with the delay between symptom onset and the first general practitioner consultation in early rheumatoid arthritis management? An exploratory study within the CareRA trial.

OBJECTIVES: Persons who are later diagnosed with early rheumatoid arthritis (ERA) often delay their first contact with a health professional after symptom onset. Besides initial symptoms, psychosocial characteristics of individuals may influence their help-seeking behaviour. We explored the role of disease characteristics, illness perception, and coping in patient-related delay before treatment initiation in recently diagnosed patients with ERA. METHOD: This exploratory, cross-sectional study included 112 patients with ERA from the Care for early RA (CareRA) trial for whom complete data on patient-related delay, coping, and illness perception were available. In addition to baseline sociodemographic and clinical data, the patients' psychosocial profiles were assessed with the Utrecht Coping List (UCL) and the revised Illness Perception Questionnaire (IPQ-R). Correlations were measured by Spearman's rho. Using regression analyses, we weighted the association of variables with patient-related delay. RESULTS: Patient-related delay was positively correlated with perceptions of causality including psychological attributions (r = 0.301, p = 0.001), risk factors (r = 0.189, p = 0.045), immunity (r = 0.261, p = 0.005), and passive coping (r = 0.222, p = 0.018). It was negatively correlated with the 28 swollen joint count (SJC28; r = -0.194, p = 0.040), perceptions of treatment control (r = -0.271, p = 0.004), and illness coherence (r = -0.208, p = 0.028). Clinical and psychosocial variables explained 15% and 18%, respectively, of the variability in patient-related delay. CONCLUSIONS: Aside from a lower SJC, a longer patient-related delay was correlated with a passive coping style, a strong conviction of symptom causality, poor expected treatment control, and a feeling of limited illness coherence. Psychosocial aspects influence individuals' help-seeking behaviour and are worth considering when aiming for a reduction in ERA treatment delay.

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.

Development of porcine embryos reconstituted with somatic cells and enucleated metaphase I and II oocytes matured in a protein-free medium.

BACKGROUND: Many cloned animals have been created by transfer of differentiated cells at G0/G1 or M phase of the cell cycle into enucleated M II oocytes having high maturation/meiosis/mitosis-promoting factor activity. Because maturation/meiosis/mitosis-promoting factor activity during oocyte maturation is maximal at both M I and M II, M I oocytes may reprogram differentiated cell nuclei as well. The present study was conducted to examine the developmental ability in vitro of porcine embryos reconstructed by transferring somatic cells (ear fibroblasts) into enucleated M I or M II oocytes. RESULTS: Analysis of the cell cycle stages revealed that 91.2 +/- 0.2% of confluent cells were at the G0/G1 phase and 54.1 +/- 4.4% of nocodazole-treated cells were at the G2/M phase, respectively. At 6 h after activation, nuclear swelling was observed in 50.0-88.9% and 34.4-39.5% of embryos reconstituted with confluent cells and nocodazole-treated cells regardless of the recipient oocytes, respectively. The incidence of both a swollen nucleus and polar body was low (6.3-10.5%) for all nocodazole-treated donor cell regardless of the recipient oocyte. When embryos reconstituted with confluent cells and M I oocytes were cultured, 2 (1.5%) blastocysts were obtained and this was significantly (P < 0.05) lower than that (7.6%) of embryos produced by transferring confluent cells into M II oocytes. No reconstructed embryos developed to the blastocyst stage when nocodazole-treated cells were used as donors. CONCLUSIONS: Porcine M I oocytes have a potential to develop into blastocysts after nuclear transfer of somatic cells.

Gastric tonometer monitoring in infants undergoing repair of coarctation of the aorta.

OBJECTIVE: To evaluate gastric tonometer monitoring for splanchnic hypoperfusion in infants during surgical intervention for aortic coarctation, especially within aortic cross-clamp periods. DESIGN: A prospective study. SETTING: Cardiovascular intensive care unit in a university hospital. PARTICIPANTS: Fourteen infant patients after elective, uncomplicated repair of coarctation of the aorta. INTERVENTIONS: After the anesthesia induction, a 7F tonometry catheter was inserted into the stomach oropharyngeally. Gastric carbon dioxide, arterial blood gases, blood pressure of upper extremities, and hematocrit values were measured in 5 different time intervals. Time periods were as follows: T1 (after the anesthesia induction), T2 (before aortic cross-clamp), T3 (immediately after aortic cross-clamp removal), T4 (40 minutes after aortic cross-clamp removal), and T5 (as the patient reached the intensive care unit). Intramucosal pH was measured by means of the Henderson-Hasselbach equation. The mean values of all parameters were calculated. According to T1 time, T2, T3, T4, and T5 times were compared with Student's t-test. MEASUREMENTS AND MAIN RESULTS: Mean aortic cross-clamp time was 19.4 +/- 6.6 minutes. Intramucosal pH values of T3 (p < 0.001) and T4 (p < 0.01) were found to be lower than values of T1. The gastric carbon dioxide values of T3 were significantly higher than T1 (p < 0.01), and bicarbonate and arterial pH values of T3 were significantly lower (p < 0.01). There were no significant differences in other parameters over time intervals. CONCLUSION: Splanchnic hypoperfusion exists during aortic cross-clamping in infant aortic coarctation surgery, and the tonometric catheter is considered to be a safe method for monitoring this hypoperfusion.

Production of transgenic bovine embryos by transfer of transfected granulosa cells into enucleated oocytes.

Adult granulosa donor cells used in the nuclear transfer (NT) procedure can result in cloned cattle. Subsequently, it may be possible to use the same cell type to produce cloned transgenic cattle. Therefore, this study examined the effect of genetic manipulation and serum levels in culture of donor granulosa cells on developmental rates and cell number of bovine NT embryos. A primary cell line was established from granulosa cells collected by aspirating ovarian follicles. Cells transfected with a plasmid containing the enhanced green fluorescence protein (EGFP) gene, and non-transfected cells were used for cloning between passage 10 and 15 as serum-starved and serum-fed donor cells. There were no significant differences (P > 0.1) in cleavage rates or development to the blastocyst stage for NT embryos from transfected (60.4 and 13.5%, respectively) or non-transfected (61.9 and 14.1%, respectively) and serum-starved (60.6 and 13.4%, respectively) or serum-fed (61.3 and 14%, respectively) cells. Development rates to blastocyst stage of embryos produced using cells at passage 15 (27.1%) were significantly higher than those produced with cells at passage 10,11, and 13 (7, 11.5, and 14%, respectively, P < 0.05). Green fluorescence was observed at different intensity levels in all blastocyst stage embryos resulting from transfected donor cells. The results of the present study indicated that genetically modified granulosa cells can be used to produce transgenic NT embryos and primary transgenic adult cells at late passage may be more effective donor cells than earlier passaged cells.