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The present study describes the structural components of the bovine vaginal fluid at estrus by scanning electron microscopy (SEM) following critical point- and freeze-drying preparation procedures. Confocal scanning laser microscopy (CLSM) was also used to evaluate the structural integrity of samples, and a control sample was assessed by adding sperm to the vaginal fluid. Samples were collected from 10 cows at the time of artificial insemination, prepared for SEM by using critical point- and freeze-drying procedures, gold coated, and observed by SEM. Mesh size and filament thickness were measured with an image analyzer. Of the 10 samples processed, 4 were considered altered following critical point drying. Compaction and lack of filaments were observed in these samples. A small area of one sample showed a honey comb-like structure when freeze drying was used. Nonoriented filaments with different thicknesses and with a network-like structure were observed throughout the remainder of the samples. Filaments throughout all samples were also observed by CSLM. After critical point drying, the mesh area ranged from 0.8 to 101.4 microns 2; the minor axis from 0.7 to 10.8 microns; and filament thickness from 40 to 442 nm. Using freeze drying, the mesh area ranged from 0.9 to 493.8 microns 2; the minor axis from 0.7 to 27.5 microns; and filament thickness from 40 to 800 nm. When samples were freeze dried, mesh values were similar to the interstrand channels observed by CSLM. In sperm-vaginal fluid samples, following critical point- or freeze-drying procedures, spermatozoa were oriented randomly in the vaginal fluid and did not seem to alter filamentous structure. Our data suggest that the freeze-drying procedure better preserves the true structural dimensions of the vaginal fluid. Furthermore, the filamentous structure of the vaginal fluid does not appear to impede sperm transport.

SummaryThe aspect of calcified tissues involved in fracture healing was studied by means of backscattered electron imaging. Bilateral transverse midshaft osteotomies were performed in the tibiae of 16 dogs. The osteotomies were reduced by means of a type II external skeletal fixator, and the clinical and radiographic course was assessed weekly until the moment of euthanasia, one, two, four and eight weeks after the operations. The osteotomized areas were removed and their structure examined in the scanning electron microscope, using backscattered electron images, to determine the general aspect of the extracellular matrix of the calcified tissues present. Four different tissues were observed: lamellar bone, woven bone, calcified cartilage and chondroid tissue. The backscattered electron contrast and fibre arrangement of the matrix, as well as the size and shape of the cellular lacunae, allow identification of the tissue. Chondroid tissue, which seems to have a leading role in the early phases of fracture healing, shows a characteristic pattern of a highly calcified and fibrous matrix with a large number of irregular and confluent cell lacunae.The morphological characteristics of the calcified tissues involved in fracture healing were studied by means of backscattered electron imaging. Lamellar bone, woven bone, calcified cartilage and chondroid tissue were the four calcified tissues observed during the healing process of canine midshaft tibial experimental fractures.

This article reports the study of the structural elements of bovine vaginal fluid at oestrus using a confocal scanning laser microscope (CSLM) to examine samples collected from 10 lactating cows at the time of insemination. The filamentous glycoproteins, which form the structural component of vaginal fluid, were stained with fluorophore acridine orange for 10 min. This microscopy technique permits visualization of a network of non-oriented filaments of different thickness in a three-dimensional array. This arrangement was repeated throughout the sample and was common to all samples. Interfilament spaces varied in width between cows and ranged from 1 to 20 microns. The present results provide a new, non-aggressive technique for the study of biological samples that show hydrogel-like behaviour. Due to its simplicity, the CSLM technique proposed offers the advantages of (1) providing realistic images of vaginal fluid structure, free from artifacts arising from sample preparation, and (2) the possibility of observing specimens shortly after collection. CSLM proved to be a useful tool for the examination of vaginal fluid and may also serve as a good control for artifacts associated with scanning electron microscopy procedures.