Osteopontin and calbindin gene expression in the eggshell gland as related to eggshell abnormalities.

Eggshell quality is a major concern to the poultry industry: eggs with poor-quality shells hatch poorly and are rejected in the processing plant. The eggshell gland (ESG) proteins and the matrix proteins, which participate in crystallization, fulfill important functions during formation of the calcified tissues and contribute to the biomechanical properties of the mature product. We selected layers that consistently produced eggshells with specific abnormalities, and continued to do so after molting, and evaluated the expression of 2 genes-osteopontin (OPN) and calbindin-as related to particular eggshell abnormalities. These genes are synthesized by the ESG and appear to participate in the calcification process. When the ESG produces normal eggshells, OPN was expressed uniformly by all of the epithelial cells facing the lumen, and calbindin was expressed by the glandular epithelium. In contrast, in the layers producing pimpled eggs, OPN was expressed only in sections of the pseudostratified epithelium, separated by areas of cells devoid of OPN gene expression, whereas calbindin was expressed at much greater levels throughout the glandular epithelium. Almost no OPN gene expression was observed in the ESG of layers producing corrugated shells, but their pattern of calbindin expression was similar to but somewhat greater than that in ESG that produced normal eggshells. In cases in which eggs had cracks at the sharp or blunt poles, OPN was expressed only at the side opposite to the cracks, whereas calbindin was expressed at both sides equally independent of the cracks. The results suggest that synthesis of the proteins associated with the formation of eggshells with the various abnormalities is controlled by different mechanisms. This may imply that more than 1 strategy will be required to improve eggshell quality.

Co-localization analysis of complex formation among membrane proteins by computerized fluorescence microscopy: application to immunofluorescence co-patching studies.

Fluorescence imaging of two independently labelled proteins is commonly used to determine their co-localization in cells. Antibody-mediated crosslinking can mediate the patching of such proteins at the cell surface, and their co-localization can serve to determine complex formation among them. However, manual analysis of such studies is both tedious and subjective. Here we present a digital co-localization analysis that is independent of the fluorescence intensity, is highly consistent and reproducible between observers, and dramatically reduces the analysis time. The approach presented is based on a segmentation procedure that creates binary objects, and then determines whether objects belonging to two different groups (e.g. green- and red-labelled) are co-localized. Two methods are used to determine co-localization. The 'overlap' analysis defines two objects as co-localized if the centre of mass of one falls within the area of the other. The 'nearest-neighbour distance' analysis considers two objects as co-localized if their centres are within a threshold distance determined by the imaging modality. To test the significance of the results, the analysis of the actual images is tested against randomized images generated by a method that creates images with uncorrelated distributions of objects from the two groups. The applicability of the algorithms presented to study protein interactions in live cells is demonstrated by co-patching studies on influenza haemagglutinin mutants that do or do not associate into mutual oligomers at the cell surface via binding to AP-2 adaptor complexes. The approach presented is potentially applicable to studies of co-localization by other methods (e.g. electron microscopy), and the nearest-neighbour distance method can also be adapted to study phenomena of correlated placement.

Effect of age at molting on postmolting performance.

Molt was induced at the 431, 501, or 571 d, in Lohmann (L) and Hy-Line W-77 (H) hens, by 8 or 14 d, respectively, of feed withdrawal followed by a rest period of 16 d. Induced molt resulted in increases in egg production, numbers of intact eggs, egg mass per housed or surviving hen, and shell quality and in decreases in egg breakage (not significant), mortality, and culling. Egg weight was only slightly affected by molt, and the EW of hens induced to molt at 431 or 501 d of age were slightly lower than those of the unmolted hens or of those induced to molt at 571 d. Both strains reacted similarly to molt, although the L hens responded better, and expressed their responses more intensively when induced to molt earlier (431 d). This finding suggests that although different breeds have some effects of molt in common, molt protocols should be finely tuned for each breed. Total intact egg production and egg mass of the molted hens became higher than those of the unmolted hens at 650 to 728 d, which suggests that no benefit would be achieved by rearing molted hens for less than 700 to 730 d.