Leica rm 2155

Two broilers of from pen were euthanized at 25 days of age to study health parameters. Pancreas, spleen, thymus and bursa of fabricius were weighted and presented as percentage of live weight. Blood haematology and blood chemistry were analyzed at Kasetsart University Veterinary Teaching Hospital, Kasetsart University (Kamphaeng Saen campus). The mid-duodenum of euthanized broilers was immerged in 10% buffered formalin for 30 days and then the tissue samples were processed, embedded in paraffin, sectioned at 5-μm thicknesses by a rotary microtome (Leica RM2155; Leica Instruments GmbH, Nussloch, Germany) and stained with haematoxylin and eosin. Villi height and crypt depth were measured under a microscope using an image analysis programme (Image Pro Plus; Media Cybernetics, Bethesda, MD, USA). All broilers were nasal vaccinated by live attenuated New Castle disease virus (NDV: Bio-Vac LS-H120, Fatro S.p.A., Bologna, Italy) at 5 and 18 days of age. The serum samples were collected at 25 days of age to determine antibody titer against NDV by hemagglutination inhibition assay.

Subcutaneously located ticks were removed with the skin and the surrounding tissue from the complete fur and immediately fixed in 10% buffered formalin, dehydrated in ascending ethanol series (20–100%) and embedded in paraffin blocks. Seven-micron thick sections were cut with a rotary microtome (Leica RM 2155, Leica Microsystems Nussloch GmbH, Nussloch, Germany), placed on glass slides and stained with hematoxylin and eosin (H&E). Microscopy and pictures were performed using a Zeiss Axioplan 2 imaging (Carl Zeiss Microscopy, Jena, Germany) equipped with an objective 25×/0.8 Plan-Neofluar Oil and a color camera ProgRes C14 (Jenoptik, Jena, Germany). Whole section overviews were obtained by tile scans with a 25×/0.8 Plan-Neofluar Oil objective using a tile scan routine under Openlab 5.5 (Improvision, Coventry, UK). During histological examination it was noticed that the inflammatory response was related to the stage of degradation of the tick. Therefore, the ticks were divided into 3 categories: (i) well-preserved ticks with correct position of hypostome and the appendages and intact exoskeleton and body; (ii) deformed ticks with intact exoskeleton; and (iii) ticks with broken, fully deformed exoskeleton.

The internal organ weight and the body weight of the euthanized rabbits were determined. The duodenal part of the small intestine was fixed in 10% buffered formalin for further villus morphometric evaluation. Briefly, small pieces of middle duodenum after fixation were processed, embedded in paraffin, sectioned at 7-µm thicknesses by means of a rotary microtome (Leica RM2155; Leica Instruments GmbH, Nussloch, Germany) and stained by haematoxylin and eosin method. Villi height and crypt depth were evaluated under a microscope using an image analysis programme (Image Pro Plus; Media Cybernetics, Bethesda, MD, USA). Caecal pH was measured directly using a Crison MicropH 2001 pH meter (Crison Instruments, Barcelona, Spain). The caecal content was immediately placed in sterile plastic tubes under ice for enzyme preservation and kept at −20 °C for further analysis of caecal enzyme activity.

Flowers from both groups (for the study of development and viability, as well as fertilization success) were taken at the beginning of flowering, and on the 3rd, 6th, 9th, and 12th days after the beginning of flowering (BOF). The ovaries were fixed in FPA (formalin: propionic acid: 70% ethyl alcohol, 5:5:90) and stored at 4 °C. The material was dehydrated through a series of ethyl alcohol and then molded into paraffin blocks, which were then cut longitudinally at 10 µm by a Leica RM 2155 (Leica Microsystems Nussloch GmbH, D-69226 Nussloch; Germany) rotary microtome. Slices that were mounted on and adhered to glass slides were stained with safranin, crystal violet, and light green SF, as described by Gerlach [60 (link)]. Permanent stained slices were observed and photographed under an Olympus BX61 microscope (Tokyo, Japan).

At 21 and 35 days of age, two birds from each replicate were randomly dissected to collect small intestinal samples. The middle of jejunum samples was sectioned horizontally in 2 cm lengths [14 ]. All samples were fixed in 10% formalin. After fixation, the samples were dehydrated by increasing the concentration of alcohol and embedded in paraffin. Embedded tissue samples were sectioned at a 5 mm thickness using an auto-microtome (Leica RM 2155, Leica Microsystems GmbH, Nussloch, Germany). The slides were stained with hematoxylin and eosin. The villus height and crypt depth were determined in cross-section using Axiolab inverted microscope (Carl Zeiss, Hamburg, Germany) equipped with an HBO 50 camera to capture images and subsequently analyzed using Zen 2012 (blue edition) software (Carl Zeiss Microscopy GmbH, Hamburg, Germany). The measurements were examined on 10 villi and 10 crypts for each segment. The villus height-to-crypt depth ratios were then calculated.