Curva de acumulación de especies para el estudio de escarabajos

The collected materials were routinely fixed for light, fluorescent, and electron microscope.

3.2.1.1. Tissue fixation and processing for light microscope (LM)

For light microscopic examination, 3 different fixatives were used. These include Bouin’s, methanol-glacial acetic acids (2:1), and 3.7 % formalin solutions. Embryos of small size (2.5- 6 cm/CRL) and small samples of the embryonic (10-90 cm/CRL) and adult testicular tissue (0.5-1 cm) were fixed in Bouin’s fluid and methanol-glacial acetic acid solutions for 12-24 h

according to the age. Thereafter, fixed samples were extensively washed in 70% ethanol (3 x 24 hr) to get rid of the fixative before the subsequent step of tissue processing. Exceptionally, formalin-fixed material was washed for 2 hr under the running tape water before ethanol immersion. Small embryos were transversely cut directly behind the level of the forelimb. Using automatic tissue processor (Shandon Duplex Processor, Shandon, Frankfurt, Germany), the tissue samples were dehydrated in graded series of ethanol (80%, 95% and absolute), cleared in xylene and impregnated with Paraplast® (Monoject Scientific Inc., Kildare, Irland). Subsequently, samples were embedded in paraplast blocks using Histostat Tissue Embedding Centre (Reichert-Jung, Wien). Sections (5µm) were cut on Leitz rotatory microtome (type 1521)and mounted on both 3-aminopropyltriethoxysilane-coated and uncoated glass slides. Paraffin sections were kept in incubator at 40°C until used for conventional staining, glyco (lectin)-and immuno-histochemical analysis.

3.2.1.2. Conventional histological staining.

As a general scheme for the ordinary stains, sections of paraffin-embedded testicular tissue were dewaxed (2 x 30 min), rehydrated in descending series of ethanol (100%, 95%, 70%) and distilled water. The subsequent stained sections were dehydrated again in ascending grades of ethanol (70%, 95%, 100%), cleared in xylene (2 x 10 min) and mounted with Eukitt® (Riedel de Haen AG, Seelze).

In this study, several conventionalstains were carried out to investigate the general histological structure of fetal and adult testis in bovine. All of the staining techniques employed were performed according to Romeis (1989). These could be briefly described as following:

Haematoxylin and Eosin (H&E)

A selection of slides was routinely stained with H&E to differentiate the gonadal sex of the small-sized bovine embryos (2.5-6 cm/CRL) and for general histological studies. Generally, the cellular nuclei stained with H&E appear blue while the remaining elements of the testicular tissue show rosy red color.

Trichrome stain after Masson and Goldner

This stain is mainly used to differentiate between the different constituent of the tissues. Indeed, cellular nuclei are constantly dark blue (stained by Weigertُ s

Haematoxylin), collagen fibers exhibit green stain whereas the other cellular and tissue components (cytoplasm, muscle fibers,..) appear red.

Alcian blue 8 GX (pH 1.0 and pH 2.5)

Alcian blue stains the acidic mucosubstances (acidic mucopolysaccharides) with light blue color while the nuclei and background appear red and light pink respectively. At different pH values, Alcian blue is able to distinguish between sulfated and non-sulfated acidic

mucosubstances. It is frequently clear that the acidic sulfated mucosubstances are easily demonstrable at highly acid pH (1.0) while the acidic non-sulfated ones, containing carboxyl groups, are detectable at comparatively lower pH value (2.5).

Periodic acid-Schiff reaction after McManus (PAS-Reaction) with and without Amylase digestion

PAS is essentially used to identify the aldehyde groups, which are formed through the oxidation of glycol with periodic acid. Therefore, PAS detect the carbohydrates or carbohydrate-rich macromolecules such as glycoproteins and glycogen. Basal lamina,

reticular and collagen fibrils are known to contain glycoprotein and react positively with PAS. The PAS positive structures stain mainly with rose to purple red color while the background appears light pink. Importantly, since Amylase has the ability to digest glycogen, Amylase digestion was done to discriminate between glycoproteins and glycogen.

Elastic stain after Weigert (Resorcin-fuchsin)

With Weigertُ s elastic stain, the reactive elastic fibers appear predominantly blue black. Toluidine blue stain

This stain is employed for identification of mast cells metachromasia. The metachromatic granules have constantly blue-violet appearance.

3.2.1.3. Light microscopic examination of the stained sections

Stained sections were evaluated by Leitz Dialux 20 Microscope and photos were taken by a Cannon digital camera (Cannon Powershot A95).

3.2.1.4. Morphometric studies

Measurements were taken with an eyepiece micrometer scale, which was calibrated with a stage micrometer at x 40. The diameter of tunica albuginea was measured from the basal lamina of the surface epithelium to the end of the inner vascular layer. The measurements of the diameter of the seminiferous cords represent in every case the average measurements of 20 seminiferous cords, 10 taken from the region near the tunica albuginea and 10 from the

rete testis region. The diameter of the seminiferous cords was measured from the basal lamina to the basal lamina and only tubules of a perfectly clear transversal section were measured. The Sertoli and germ cells found in these transversal sections were counted as well. Leydig cell count was performed using image tool program (UTHSCSA, v 3.00). First, 10 photos/ age/embryo were taken by Cannon digital camera (Cannon Powershot A95). Importantly, all photos were taken at the same zoom and with the same objective (x 40). Then, the surface area of the photo was measured in photoshop program (Adobe® Photoshop® 7.0) and adjusted to 0.03 mm² of the testicular area. Finally the Leydig cells per photo was counted using image tool program and the average number for the 10 photos/age/embryo was calculated.

3.2.2. Tissue fixation and processing for electron microscope (EM)

Ultrastructure of the adult bovine testis was studied using transmission electron microscope. To accomplish this aim, small samples (about 1 mm) from different areas of the testes of 3 sexually mature bulls were directly collected within 30 min after slaughtering and fixed by immersion method. These samples were immediately fixed in formaldehyde-glutaraldehyde mixture (Karnovsky, 1965) at 4°C overnight. Subsequently, the specimens were washed (4 x 15 min) in 0.1 M cacodylate buffer (pH 7.2). All samples were then contrasted in 1.5% potassium ferrocyanide and 1% osmium tetroxide at 4°C for 2 hr in the dark. Later, they were again washed (3 x 20 min) in 0.1 M cacodylate buffer (pH 7.2). After dehydration in a graded series of ethanol (50%, 70%, 90%, 100%) and propylene oxide (Merck, Darmstadt, Germany) the specimens were gradually embedded in Epon (Polysciences, Eppelheim, Germany). Briefly, samples were firstly dipped (2 x 15 min) in propylene oxide (Merck, Darmstadt, Germany) then in propylene-Epon mixture (2:1) for 1 hr, in propylene-Epon mixture (1:1) overnight and finally in pure Epon for 30 min. Thereafter, testicular specimens were embedded in gelatin capsules (Plannet, Wetzlar) containing Epon and polymerized at 60°C for 24 hr. For general morphology, semithin sections (1µm) were cut using an ultramicrotome Ultracut E (Reichert-Jung, Wien) and stained with methylene blue (Sigma-Aldrich Chemicals GmbH, Deisenhofen, Germany) to be examined by light microscope. Ultra thin sections (60 nm) were cut from selected blocks, mounted on uncoated copper grids (SSI, Science Services, Munich, Germany) and routinely contrasted with uranyl-acetate and lead citrate (Reynolds, 1963) prior to examination with a Zeiss EM 902 (Carl Zeiss, Oberkochen) electron

microscope. Photos of selected structures were taken on Maco ort 25c-films (Maco Photo Products, Hamburg, Germany).