The role of negative substrate bias voltage in influencing the microstructural evolution, along with the mechanical and scratch behavior of magnetron sputtered Ni–Zr alloyed thin films, has been investigated. The films have been deposited on a Si(100) substrate by direct current (DC) magnetron co-sputtering of high-purity elemental Ni and Zr targets, using an optimized target power in an argon atmosphere at room temperature by altering the negative substrate bias voltage (0 to −80 V). The increase in negative substrate bias voltages leads to an increase in Zr content of the investigated films. The characterization techniques such as grazing incidence X-ray diffraction and high-resolution transmission electron microscopy studies confirm that an increase in the negative substrate bias voltage leads to an increase in the volume fractions of amorphous phase and Ni3Zr, but a decrease in the deposition rate, surface roughness, and average grain sizes. Hardness and Young's modulus obtained by nanoindentation, along with the coefficient of friction obtained from nano-scratch experiments, appear to be related to the relative volume fractions of both nanocrystalline and the amorphous phase. Furthermore, increase in Ni3Zr volume fraction with decrease in grain size within the crystalline part of the film, with increase in substrate bias used during deposition may have contributed to both increase in both hardness and scratch resistance.

We evaluated the usefulness of lissamine green B (LB) staining of cumulus–oocyte complexes (COC) as a non-invasive method of predicting maturational and developmental competence of slaughterhouse-derived porcine oocytes cultured in vitro. Cumulus cells of freshly aspirated COCs were evaluated either morphologically on the basis of thickness of cumulus cell layers, or stained with LB, which penetrates only non-viable cells. The extent of cumulus cell staining was taken as an inverse indicator of membrane integrity. The two methods of COC grading were then examined as predictors of nuclear maturation and development after parthenogenetic activation. In both cases LB staining proved a more reliable indicator than morphological assessment (P < 0.05). The relationship between LB staining and cumulus cell apoptosis was also examined. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for DNA fragmentation revealed that oocytes within COCs graded as low quality by either LB staining or visual morphology showed significantly greater DNA fragmentation (P < 0.05) than higher grades, and that LB and visual grading were of similar predictive value. Expression of the stress response gene TP53 showed significantly higher expression in COCs graded as low quality by LB staining. However expression of the apoptosis-associated genes BAK and CASP3 was not significantly different between high or low grade COCs, suggesting that mRNA expression of BAK and CASP3 is not a reliable method of detecting apoptosis in porcine COCs. Evaluation of cumulus cell membrane integrity by lissamine green B staining thus provides a useful new tool to gain information about the maturational and developmental competence of porcine oocytes.