Vaginal isolation of beta-haemolytic Streptococcus from bitches with and without neonatal deaths in the litters.

The aim of the study was to identify beta-haemolytic streptococci in the vagina of bitches who had delivered healthy litters and bitches who had delivered litters in which neonatal deaths occurred. Fifty-one bitches divided into two groups were used. Group 1 (G1) included 28 bitches that had delivered healthy litters and group 2 (G2) included 23 bitches that had delivered puppies who died in the neonatal period. Two vaginal samples were taken, one in proestrus and the other at the end of gestation (EG). Beta-haemolytic Streptococcus (BS) was isolated from 16 bitches (57%) in G1 and from 21 bitches (91%) in G2. The bacteriological cultures, serological tests (Streptex® ) and PCR assay allowed identification of Streptococcus canis and Streptococcus dysgalactiae in G1 and G2. Ultramicroscopic studies allowed the observation of M Protein and capsules in strains of S. dysgalactiae and S. canis in G1 and G2. The S. canis strains isolated from G2 showed thicker capsules than S. canis strains isolated from G1 (234 ± 24.2 vs 151.23 ± 28.93 nm; p < .001.). No differences were observed in capsule thickness between strains of S. dysgalactiae isolated from G1 and G2 (210 ± 13.54 vs 211.66 ± 19.67 nm; p > .70). All strains of beta-haemolytic Streptococcus isolated were penicillin sensitive. Penicillin was administered from EG to 5 days post-partum in 10 G2 females with isolation of BS (G2A). Saline solution was administered in eleven G2 females with isolation of BS (G2B). Ninety per cent of the puppies survived in G2A and 25% survived in G2B. Our results suggest BS is involved in canine neonatal deaths.

A quantitative ultramorphological approach for systematic assessment of sperm head regions: an example in rams.

Examination of the type and frequency of damage to the head of spermatozoa using electron microscopy can be used to evaluate the quality of differently treated sperm. This report describes a systematic approach based on 29 morphological categories of sperm heads assessed from discrete regions in raw, chilled and frozen-thawed spermatozoa. Injury occurred principally at the plasma membrane and could be present or absent in all regions. In the anterior segment, when the plasma membrane is present, it can be intact, dilated, very dilated, disrupted, or contain vesicles characteristic of acrosomal reaction-like capacitation changes. When the plasma membrane is absent, the acrosome may be intact, exhibit a complete loss of contents, or retain some contents of the apical ridge and present a very dilated outer acrosomal membrane. The plasma membrane in the equatorial segment and the boundary between regions can be intact, dilated, very dilated or disrupted. The post-acrosomal plasma membrane is classified as intact, dilated or very dilated, whereas the dense lamina is intact, dilated or fragmented. The morphology of the heads most frequently observed in chilled spermatozoa consists of anterior and equatorial segments with a dilated, or dilated and disrupted plasma membrane; a boundary between regions with an intact and dilated plasma membrane; and a post-acrosomal region with an intact plasma membrane and dense lamina, both dilated. In frozen-thawed spermatozoa, the morphology of the heads is more frequently characterised by no plasma membrane and an acrosome showing complete or some loss of contents in the apical ridge and very dilated outer acrosomal membrane, presenting mostly dilated and fragmented dense lamina in the post-acrosomal region. These findings are consistent with the conclusion that the freezing process produces an increase in the degree of damage to the cells when they are subjected to increasing degrees of cold shock. There are still difficulties in developing a good diluent and process for preserving the plasma membrane in ram spermatozoa. This systematisation, using different categories, allows characterisation of multiple transmission electron microscopy images. Thus, the different changes observed due to cryopreservation may be correlated.

Effect of storage media and storage time on histological and ultrastructural changes in cat epididymal cells.

The aim of this study was to assess the histological and ultrastructural changes in cat epididymides (n = 22) stored at 4 °C in two different media [saline solution (SAL) or tris-egg yolk (TEY)]. Our hypothesis was that epididymides stored in TEY would have delayed epithelial cell autolysis. Four epididymides were fixed and processed immediately, and the remaining 18 epididymides were stored at 4 °C in SAL or TEY for 24, 48 or 72 h. In histological sections, the nuclear features and stereocilia morphology were scored from 0 to 3. Ultrastructurally, nuclear chromatin and stereocilia morphology were scored from 0 to 3. In addition, using transmission electron microscopy nuclear number, nuclear area, mitochondrial number and mitochondrial area were recorded. In the histological study, parameters changed with time and media (p < 0.01). A significant effect of time was observed (p < 0.01), and the morphological changes were greatest when the storage time increased. Morphological changes were higher in SAL compared with TEY (p < 0.01). In the ultrastructural study, nuclear chromatin and stereocilia morphology decreased with time and media as in the histological study (p < 0.01). In addition, nuclear number and nuclear area changed with time (p < 0.004; p < 0.001) but not with media. Conversely, mitochondrial number and mitochondrial area did not change with media or time (p > 0.05). In conclusion, these results show that TEY preserved epididymal epithelial cells better than SAL; this finding could help improve sperm quality of stored epididymides.

Ultrastructural and quantitative immunohistochemical changes induced by nonsteroid antiandrogens on pituitary gonadotroph population of prepubertal male rats.

Specific blockade of the androgen receptor by the nonsteroid antiandrogens flutamide and Casodex has proven to be a useful tool for studying androgens in vivo. The aim of the present study was to investigate the effect of antiandrogen administration at the pituitary level by evaluating the ultrastructural changes in gonadotrophs, in correlation with the quantitative immunohistochemical findings, and by comparing these alterations with the effect of androgen deprivation by castration either with or without subsequent androgen replacement. Male Sprague-Dawley rats (23 days old) were grouped as follows: (1) controls, (2) flutamide-injected (10 mg/rat/day), (3) Casodex-injected (10 mg/rat/day), (4) castrated, and (5) castrated plus androgen-replaced (dihydrotestosterone propionate; 40 microg/rat/day). Groups were sacrificed after 10 days of maintenance under each condition. Pituitaries were processed for both light and electron microscopy. Serial sections (4 microm) were obtained at different levels and immunostained by means of the primary murine monoclonal antibodies anti-FSH and anti-LH and a peroxidase-mediated EnVision System (Dako). Volume density, cell density and mean cell area were measured with an image analysis system (Imaging Technology, Software Optimas 5.2). The mean cell area (p < 0.001) and the volume density (p < 0.05) increased significantly in the flutamide- and Casodex-treated groups as well as the castrated group of FSH and LH cells. On the other hand, androgen replacement in the castrated rats, however, reduced in both parameters related to control animals. The cell density of FSH-secreting cells was increased (p < 0.05) in the Casodex and flutamide treatment as well as castrated group. The cell density of LH-secreting cells was augmented (p < 0.05) in the Casodex-treated group, while there was no increase in such parameter with flutamide and castration. The ultrastructure of all groups showed two types of gonadotrophs. Type I cells contained large (300-500 nm) and small (150-200 nm) secretory granules, while type II cells were smaller, and exhibited only small granules (100-200 nm). Flutamide-treated, Casodex-treated and castrated groups presented a decreased number of secretory granules with some exocytotic profiles, well-developed rough endoplasmic reticulum and an expanded Golgi complex of both types of cells. The gonadotrophs from the castrated group exhibited numerous mitochondria with electron-dense ring-shaped laminar figures, while in the castrated plus androgen-replaced rats only a few mitochondria had similar changes to those observed in castrated animals, as a possible residual alteration. Finally, the gonadotrophs from flutamide-treated rats showed mitochondrial alterations with clear areas and isolated electron-dense laminar figures. In summary, we conclude that lack of androgen reaction through the effects of nonsteroid antiandrogens and castration on prepubertal rats produced a hypertrophia-hyperplasia of the FSH cells, and hypertrophia of LH-secreting cells, with marked alterations at the ultrastructural level suggestive of a hyperstimulation stage.

Effect of undernutrition on cranial components and somatotroph-lactotroph pituitary populations in the squirrel monkey (Saimiri sciureus boliviensis).

The aim of the present study was to investigate in monkeys the effects of undernutrition on neurocranial and facial components, correlated with a histometric and ultrastructural analysis of somatotroph (growth hormone, GH) and lactotroph (prolactin, PRL) pituitary populations. Twenty Saimiri sciureus boliviensis (Cebidae) of both sexes were employed. The monkeys were born in captivity and when they reached 1 year of age, they were separated into two groups: control and undernourished animals. They were fed ad libitum a 20% and 10% protein diet, respectively. The monkeys were radiographed when they were 3 years old in order to measure the length, width and height of the anterior, middle and posterior components of the neurocranium, as well as those of the masticatory, respiratory and optic components of the face. The volumetric and morphometric indices were then calculated. After the sacrifice, pituitary glands were processed for light and electron microscopy. The quantitative immunohistochemistry revealed a decrease in the volume density and cell density of both GH and PRL cells from malnourished animals when compared to control ones. The ultrastructural study showed changes suggestive of cellular hyperfunction for both types of cells in the former experimental group. Under nutrition also affected the size of the cranial components, with males being more affected than females; brain weight was, however, nonmodified by stress, with the brain/body ratio difference being the same for both sexes. We conclude that in monkeys, experimental undernutrition produces a decrease in the pituitary GH and PRL cell populations, in some way related to changes in the cranio-facial morphometric patterns.

Morphometric and ultrastructural analysis of different pituitary cell populations in undernourished monkeys.

Undernutrition elicited by a low-protein diet determines a marked reduction of hypophyseal activity and affects the function of the respective target organs. The objective of the present investigation was to study the ultrastructural and quantitative immunohistochemical changes of the different pituitary cell populations in undernourished monkeys that had been previously shown to have significant changes in craniofacial growth. Twenty Saimiri sciureus boliviensis monkeys of both sexes were used. The animals were born in captivity and were separated into two groups at one year of age, i.e., control and undernourished animals. The monkeys were fed ad libitum a 20% (control group) and a 10% (experimental group) protein diet for two years. Pituitaries were processed for light and electron microscopy. The former was immunolabeled with anti-GH, -PRL, -LH, -FSH, -ACTH, and -TSH sera. Volume density and cell density were measured using an image analyzer. Quantitative immunohistochemistry revealed a decrease in these parameters with regard to somatotrophs, lactotrophs, gonadotrophs and thyrotrophs from undernourished animals compared to control ones. In these populations, the ultrastructural study showed changes suggesting compensatory hyperfunction. On the contrary, no significant changes were found in the morphometric parameters or the ultrastructure of the corticotroph population. We conclude that in undernourished monkeys the somatotroph, lactotroph, gonadotroph, and thyrotroph cell populations showed quantitative immunohistochemical changes that can be correlated with ultrastructural findings.

Morphometric and ultrastructural analysis of different pituitary cell populations in undernourished monkeys

Undernutrition elicited by a low-protein diet determines a marked reduction of hypophyseal activity and affects the function of the respective target organs. The objective of the present investigation was to study the ultrastructural and quantitative immunohistochemical changes of the different pituitary cell populations in undernourished monkeys that had been previously shown to have significant changes in craniofacial growth. Twenty Saimiri sciureus boliviensis monkeys of both sexes were used. The animals were born in captivity and were separated into two groups at one year of age, i.e., control and undernourished animals. The monkeys were fed ad libitum a 20 percent (control group) and a 10 percent (experimental group) protein diet for two years. Pituitaries were processed for light and electron microscopy. The former was immunolabeled with anti-GH, -PRL, -LH, -FSH, -ACTH, and -TSH sera. Volume density and cell density were measured using an image analyzer. Quantitative immunohistochemistry revealed a decrease in these parameters with regard to somatotrophs, lactotrophs, gonadotrophs and thyrotrophs from undernourished animals compared to control ones. In these populations, the ultrastructural study showed changes suggesting compensatory hyperfunction. On the contrary, no significant changes were found in the morphometric parameters or the ultrastructure of the corticotroph population. We conclude that in undernourished monkeys the somatotroph, lactotroph, gonadotroph, and thyrotroph cell populations showed quantitative immunohistochemical changes that can be correlated with ultrastructural findings

Immunohistochemical and ultrastructural study of pituitary folliculostellate cells during aging in rats.

The impact of aging on pituitary folliculostellate (FS) cells is not well known. The aim of the work reported here was to carry out a quantitative immunohistochemical assessment of the FS population in male and female rats during aging and to correlate the findings with possible changes at the ultrastructural level. Young (4 months), old (20 months) and senescent (29 months) Sprague-Dawley rats of both sexes were sacrificed by rapid decapitation, their pituitaries dissected and processed by both light immunohistochemistry and electron microscopy. Serial sections (4 microm) were obtained at different levels and immunostained by means of rabbit anti-S100 serum as the primary antibody and a peroxidase-mediated EnVision System (Dako). Measurement of volume density (VD) and cell density (CD) was made in S100-reacting elements by means of an image analysis system (Imaging Technology, Optimas). These parameters were found to be significantly (p < 0.05) decreased in old and senescent rats as compared to young animals. In senescent females, which presented a high incidence of microprolactinomas, a significant (p < 0.01) increment of VD and CD was observed in FS cells in the area surrounding the adenomas, together with a marked decrease in those parameters within the tumors. Sexual dimorphism was not found except for the prolactinoma-bearing female group. The ultrastructure of FS cells showed the typical characteristics previously described in the pituitary gland. Only moderate changes in the endoplasmic reticulum were observed in old and senescent animals. We conclude that aging has a clear effect on the morphology of the pituitary FS cell population.

Immunohistochemical and ultrastructural age-related changes in rat lactotroph cells

Ageing produces alterations in some functions of the hypothalamo-pituitary axis leading to sexually dimorphic changes in the prolactin (PRL)-secreting cells. Since quantitative morphological data of these age-associated alterations are scarce, we carried out a morphometric immunohistochemical assessment as well as an ultrastructural study of the PRL cell population in male and female rats of different ages. Young (3-month-old), old (20-month-old), and senescent (31-month-old) Sprague-Dawley rats of both sexes were sacrificed by rapid decapitation, their pituitaries immediately dissected out and processed for both immunohistochemistry and electron microscopy. Analysis of different morphometric parameters revealed that the cell density (CD) and volume density (VD) significantly decreased with age in male rats. In females, while CD showed a significant age-related diminution when young rats were compared to old ones, this parameter increased in senescent animals. The VD presented higher values in senescent rats. When the data were compared between sexes, VD was found to be higher in females if old and senescent rats were considered. Finally, CD increased significantly in females when compared to males. The ultrastructure of the PRL cells from old and senescent animals of both sexes exhibited changes suggestive of an hyperstimulation state, with some prolactotrophs having the appearance of cells undergoing an involutive process. We conclude that ageing has a differential impact on the PRL cells of male and female rats with respect to the immunohistochemical and ultrastructural features of that cell population

Immunohistochemical and ultrastructural age-related changes in rat lactotroph cells

Ageing produces alterations in some functions of the hypothalamo-pituitary axis leading to sexually dimorphic changes in the prolactin (PRL)-secreting cells. Since quantitative morphological data of these age-associated alterations are scarce, we carried out a morphometric immunohistochemical assessment as well as an ultrastructural study of the PRL cell population in male and female rats of different ages. Young (3-month-old), old (20-month-old), and senescent (31-month-old) Sprague-Dawley rats of both sexes were sacrificed by rapid decapitation, their pituitaries immediately dissected out and processed for both immunohistochemistry and electron microscopy. Analysis of different morphometric parameters revealed that the cell density (CD) and volume density (VD) significantly decreased with age in male rats. In females, while CD showed a significant age-related diminution when young rats were compared to old ones, this parameter increased in senescent animals. The VD presented higher values in senescent rats. When the data were compared between sexes, VD was found to be higher in females if old and senescent rats were considered. Finally, CD increased significantly in females when compared to males. The ultrastructure of the PRL cells from old and senescent animals of both sexes exhibited changes suggestive of an hyperstimulation state, with some prolactotrophs having the appearance of cells undergoing an involutive process. We conclude that ageing has a differential impact on the PRL cells of male and female rats with respect to the immunohistochemical and ultrastructural features of that cell population

Immunohistochemical and ultrastructural age-related changes in rat lactotroph cells.

Ageing produces alterations in some functions of the hypothalamo-pituitary axis leading to sexually dimorphic changes in the prolactin (PRL)-secreting cells. Since quantitative morphological data of these age-associated alterations are scarce, we carried out a morphometric immunohistochemical assessment as well as an ultrastructural study of the PRL cell population in male and female rats of different ages. Young (3-month-old), old (20-month-old), and senescent (31-month-old) Sprague-Dawley rats of both sexes were sacrificed by rapid decapitation, their pituitaries immediately dissected out and processed for both immunohistochemistry and electron microscopy. Analysis of different morphometric parameters revealed that the cell density (CD) and volume density (VD) significantly decreased with age in male rats. In females, while CD showed a significant age-related diminution when young rats were compared to old ones, this parameter increased in senescent animals. The VD presented higher values in senescent rats. When the data were compared between sexes, VD was found to be higher in females if old and senescent rats were considered. Finally, CD increased significantly in females when compared to males. The ultrastructure of the PRL cells from old and senescent animals of both sexes exhibited changes suggestive of an hyperstimulation state, with some prolactotrophs having the appearance of cells undergoing an involutive process. We conclude that ageing has a differential impact on the PRL cells of male and female rats with respect to the immunohistochemical and ultrastructural features of that cell population.

An ultrastructural study of the Sertoli cell in the Shiba goat.

The ultrastructure of the Shiba goat Sertoli cell was observed using light and transmission electron microscopy. In the basal portion of the Sertoli cell, the nucleus was located near the basement membrane and was irregular in outline. A number of filaments with intermediate width were observed around the nucleus. A multivesicular nuclear body, composed of numerous vesicles of different sizes and ribosome-like structures, was detected within the nucleus. The majority of cell organelles was situated in this basal portion of the cytoplasm. Oval mitochondria were randomly distributed. A well-developed laminated smooth endoplasmic reticulum (sER) was frequently found within the cytoplasm of Sertoli cell. Lysosomes and large lipid droplets were also recognized. While, the outline of the basal lamina was almost straight. No nodular processes were observed in the lamina. The Golgi complex appeared only in the supranuclear region. In the middle and apical portions of the Sertoli cell, elongated mitochondria and microtubules were oriented parallel to the long axis of the cell. The laminated sER was also detected in these portions. The apical head of slightly-elongated spermatids was occasionally surrounded by the laminated sER. The head of maturing spermatids without laminated sER revealed a bleb-like shape. The microfilament bundles at the ectoplasmic specialization site were regularly arranged. Judging from the present observations, we have suggested that only the multivesicular nuclear body and the laminated sER may be the peculiar structures which are commonly found in the Sertoli cell of ruminants.