Tubule detection in testis images using boundary weighting and circular shortest path.

In studies of germ cell transplantation, measureing tubule diameters and counting cells from different populations using antibodies as markers are very important. Manual measurement of tubule sizes and cell counts is a tedious and sanity grinding work. In this paper, we propose a new boundary weighting based tubule detection method. We first enhance the linear features of the input image and detect the approximate centers of tubules. Next, a boundary weighting transform is applied to the polar transformed image of each tubule region and a circular shortest path is used for the boundary detection. Then, ellipse fitting is carried out for tubule selection and measurement. The algorithm has been tested on a dataset consisting of 20 images, each having about 20 tubules. Experiments show that the detection results of our algorithm are very close to the results obtained manually.

Nurse:patient ratio and achievement of oxygen saturation goals in premature infants.

BACKGROUND: Premature newborns often experience oxygen saturations outside policy-specified targets, which may be associated with increased morbidity. Nurse workload may affect oxygen management. OBJECTIVE: To examine the relationship between number of patients assigned to neonatal intensive care unit (NICU) nurses and achievement of oxygen saturation goals in premature newborns. DESIGN: The authors linked nurse-patient assignment data with continuous oxygen saturation data for infants <29 weeks' gestation in a single NICU between January and June 2008. The proportion of time oxygen saturation was in policy-specified target range (85-92%) and proportion of time hyperoxaemic (98-100%) were determined for multiple 6 h monitoring periods. Each period was characterised by a single nurse, respiratory support mode and fraction of inspired oxygen (Fio(2)) level (0.22-0.49 or ≥0.5). The nurse:patient ratio for the infant's nurse for each monitoring period was determined. Factors associated with Spo(2) target achievement and hyperoxaemia were identified. RESULTS: The authors analysed 1019 monitoring periods from 14 infants with a mean (SD) birth weight of 860 (270) g and gestational age of 26.6 (1.6) weeks. The mean (range) postmenstrual age for all monitoring periods was 31.6 (24.1-40.7) weeks. Eighty-seven nurses provided care. In a multivariate cross-classified hierarchical regression, the nurse:patient ratio, postmenstrual age, respiratory support mode and Fio(2) were significantly associated with oxygen saturation outcomes. Fewer patients per nurse was significantly associated with a higher saturation target achievement among patients on high-frequency ventilation, and with reduced hyperoxaemia among patients on nasal cannula. CONCLUSIONS: Fewer patients per nurse may be associated with improved achievement of oxygen saturation goals and may be an important modifiable factor influencing oxygen-related outcomes in premature newborns. This effect may vary with mode of respiratory support.

Cell microarrays for the screening of factors that allow the enrichment of bovine testicular cells.

Cell microarrays can serve as high-throughput platforms for the screening of a diverse range of biologically active factors and biomaterials that can induce desired cellular responses such as attachment, proliferation, or differentiation. Here, we demonstrate that surface-engineered microarrays can be used for the screening and identification of factors that allow the enrichment and isolation of rare cells from tissue-derived heterogeneous cell populations. In particular, we have focused on the enrichment of bovine testicular cells including type A spermatogonia and Sertoli cells. Microarray slides were coated with a copolymer synthesized from poly(ethylene glycol) methacrylate and glycidyl methacrylate to enable both the prevention of cell attachment between printed spots and the covalent anchoring of various factors such as antibodies, lectins, growth factors, extracellular matrix proteins, and synthetic macromolecules on printed spots. Microarrays were incubated with mixed cell populations from freshly isolated bovine testicular tissue. Overall, cell attachment was evaluated using CellTracker staining, whereas differential attachment of testicular cells was determined by immunohistochemistry staining with Plzf and vimentin antibodies as markers for type A spermatogonia and Sertoli cells, respectively. The results indicate that various surface immobilized factors, but in particular Dolichos biflorus lectin, allowed the enrichment of Plzf positive cells. Furthermore, Pisum sativum lectin, concanavalin A, collagen type IV, and vitronectin were identified as suitable negative selection factors. To our best knowledge, this work is the first to demonstrate the utility of surface engineered cell-based microarrays for the identification of factors that allow the selective capture of rare cells from tissue isolated heterogeneous mixtures.

Irradiation enhances the efficiency of testicular germ cell transplantation in sheep.

Testis germ cell transplantation in livestock has the potential for production of transgenic genotypes and for use as an alternative to artificial insemination in animal breeding systems. In a pilot experiment, we investigated a workable protocol for testis germ cell transplantation in sheep, including donor cell isolation, rete testis injection, and microsatellite detection of donor spermatozoa in recipient semen. In a second experiment, the effect of depletion of endogenous stem cells with a single irradiation dose of 9 Gy (n = 5) or 15 Gy (n = 5) on the outcome of germ cell transplantation was investigated. Irradiation of recipient testes with a single dose of 15 Gy, followed by transplantation 6 wk after depletion, may be most advantageous because it resulted in all recipients (five of five) producing donor-derived spermatozoa, while the 9-Gy and control groups had limited success rates (two of five and one of three, respectively). Using microsatellite markers to detect the presence of donor DNA, 10 rams were identified that produced spermatozoa of donor origin. The proportion of donor DNA was between 1% and 30% of total ejaculate DNA. When three of these positive rams were used in breeding experiments, four donor-derived offspring (four of 50 [8% of progeny])resulted from a recipient in Merino to Merino transplantation. Six lambs (six of 41 [15% of progeny]) were sired by donor-derived Border Leicester sperm produced in a Merino recipient ram; however, no donor-derived offspring were detected among 34 progeny from a second Border Leicester to Merino combination. These results confirm that preparation of recipient animals with a correct dose of irradiation not only enhances the success rate of the transplantation procedure but also increases the proportion of donor spermatozoa in recipient semen. This study represents the first report of the production of live progeny following testis germ cell transplantation using irradiated recipients in a livestock species.

Large quantity cryopreservation of bovine testicular cells and its effect on enrichment of type A spermatogonia.

Cryopreservation has become an integral component of any cell transplantation technique helping to overcome the issues associated with known spatial and temporal barriers between donor and recipient. The aim of this study was to develop a protocol for large quantity cryopreservation of bovine testicular germ cells. The impact of 3 different packaging methods (5 ml semen straw, 20 ml freezing bag and 1.5 ml cryovial) and varying cell densities (3 x 10(6), 9 x 10(6), or 18 x 10(6)cells/ml) on the survival of testis germ cells was examined. Cells processed in 5 ml semen straws had a significantly higher viability (70.7+/-1.2%, P<0.05) compared to those cells in 20 ml freezing bags (46.7+/-0.1%) or 1.5 ml cryovials (46.3+/-2.2%). For 5 ml straws, a 20 min cooling prior to cryopreservation resulted in a higher post thaw viability (73.2+/-0.6%) than a 10 min cooling (56.0+/-2.2%), while the density of the cell suspension did not impact on post thaw viability. Thus cryopreservation of testicular germ cells in 5 ml straws at a density between 3 x 10(6) and 18 x 10(6)cells/ml in liquid nitrogen vapour for 20 min cooling appears to be a simple and practical way to preserve cells. Subsequent testing of frozen/thawed cells exhibited viable cultures and retained the ability to proliferate. The freezing protocol does not preferentially preserve type A spermatogonia. However, the cell surface properties of somatic cells appear to be affected by the freezing procedure and therefore the frozen/thawed cells are less suitable for enriching type A spermatogonia by differential plating.