The exact nature of cryo-injury to dog semen and the stage of cryopreservation at which it occurs have not been evaluated fully and were investigated in the present study. Semen samples were examined immediately before and after addition of semen extender, cooling and freeze-thawing in a Tris-based medium. Vital and ultrastructural assessments were made immediately after each treatment and after incubation for 4 h at 39 degrees C after each treatment. Addition of semen extender produced no significant ultrastructural changes in spermatozoa; however, cooling resulted in an immediate increase in the number of acrosomal abnormalities and a subsequent decrease in sperm viability. Freeze-thawing caused both an immediate and a delayed decrease in sperm viability. This effect may have been an exacerbation of the effects of cooling as many acrosomal abnormalities were present or it may have been the result of initially unrecognized damage. Cooling and freezing of dog spermatozoa may have both immediate and delayed effects on the ultrastructure of spermatozoa. The immediate effects of cooling and freezing may either kill spermatozoa or render them incapable of fertilization by damaging the acrosome, whereas delayed effects may reduce sperm longevity by altering plasma membrane structure.

A detergent scrub technique using wash fluid consisting of 0.075 M phosphate-buffered saline, pH 7.9 containing 0.1 per cent Triton X-100 was evaluated for the quantitative culture of Malassezia pachydermatis from canine skin. Preliminary studies showed that the detergents Triton X-100, Tween 40 and Tween 80 were equally able to disperse suspensions of pure cultures of M pachydermatis, but that the yeast counts were significantly reduced (P < 0.001) after suspension in saline, Triton X-100 or Tween 40 for two hours. The counts in skin washings were also reduced (P < 0.001) after suspension for three hours in 0.1 and 0.05 per cent solutions of Triton X-100. Vortexing, or manual or mechanical shaking of the samples yielded comparable counts. The correlation between the counts on diseased skin measured by using detergent scrubs and a contact plate technique was highly significant (P < 0.001). The detergent scrub technique was suitable for the quantitation of M pachydermatis on canine skin provided that the samples were processed without delay.

Boar and ram spermatozoa were incubated in Tyrode's medium in the presence or absence of bicarbonate/CO 2 , a component believed essential for capacitation. At intervals, samples were stained with a range of FITC‐lectins to detect changes in surface glycoconjugates, using a rapid staining technique to avoid problems of lectin toxicity. The samples were then analysed directly by flow cytometry, using propidium iodide to distinguish dead cells. In the presence of bicarbonate, a live subpopulation of spermatozoa developed, which in both animal species showed higher binding affinities towards Phaseolus Vulgaris Agglutinin (PHA‐E), Sophora Japonica Agglutinin (SJA), and Soybean Agglutinin (SBA), and lower binding affinity towards Erythrina Cristagalli Lectin (ECL). In boar samples, the modified subpopulation reached a maximum after 3 hr incubation, whereas in ram samples it maximized after 1.5 hr. No changes were seen when spermatozoa were incubated in bicarbonate‐free medium. The bicarbonate‐induced changes in lectin binding were not due to the onset of acrosome reactions, because spermatozoa induced to undergo acrosome reactions with the ionophore A23187 displayed very different lectin‐binding patterns. Tested on boar spermatozoa, seminal plasma not only inhibited but reversed the bicarbonate‐induced development of the modified subpopulation. EGTA also inhibited development of boar sperm subpopulations; excess Ca 2+ was unable to overcome this inhibition, suggesting that multivalent metal ions might be involved in bicarbonate's action. We conclude that bicarbonate causes a loss of surface coating material with affinity for ECL and an unmasking of binding sites for SBA, SJA and PHA‐E. A modified subpopulation of live spermatozoa is thereby established, which appears to maximize at a rate in accord with reported capacitation times. © 1995 Wiley‐Liss, Inc.

Calcium was identified by a pyroantimonate-osmium fixation technique in ram spermatozoa undergoing a spontaneous acrosome reaction induced by incubation of diluted semen at 39 degrees C. Intracellular calcium was only detected in diluted spermatozoa and increased in amount and distribution over 4 hr At 4 hr, the majority of the spermatozoa displayed ultrastructural evidence of an acrosome reaction. Calcium was initially evident on the outer acrosomal membrane in multiparticulate clusters, which were seen to be located on scalloped crests of acrosomal membrane as fusion developed; it was also located in the region of the acrosomal ridge beneath the outer acrosomal membrane. Vesiculation commenced just anterior to the equatorial segment and proceeded anteriorly. As vesiculation advanced, calcium particles became associated with the periphery of the vesicles attached in the region of the fusion between the two membranes, but were never seen inside the vesicles. The equatorial segment was not labelled until much later in the reaction, at which time calcium particles were also evident on the nuclear membrane; vesiculation of the equatorial segment was also noted at this time.Dense labelling of the postacrosomal dense lamina was seen in all incubated spermatozoa. At the anterior margin of this structure the labelling was seen to be in a ''saw-tooth'' arrangement. The disposition of the calcium both temporally and spatially is discussed in relation to its possible mechanisms in bringing about membrane fusion. (C) 1995 Wiley-Liss, Inc.

Dictyocaulus viviparus were harvested from calves treated daily with albendazole at dose-rates sub-lethal to this parasite and from matched untreated calves. Male and female reproductive tracts from the worms were examined by light and transmission electron microscopy. A first description of the testis of this nematode is provided. Cell division in the germinal zone of both male and female worms was arrested by in vivo exposure to albendazole metabolites and the cytoplasmic threads connecting the germinal cells to the rachis disappeared. In male worms from treated calves, the rachis was absent from the growth zone and severe ultrastructural damage of spermatogonia, spermatocytes and spermatozoa was apparent. In female worms, the rachis remained intact in the growth zone, obvious abnormalities being confined to the germinal zone, where the cytoplasm of the oogonia was disrupted and few nuclei seen, and to the uteri and ovijector which contained only undifferentiated ova. The detrimental effects of albendazole metabolites on male and female worms were quickly reversed after withdrawal of medication.

During incubation of ram spermatozoa at 1 x 10(7) cells mL-1 or less in a simple HEPES-buffered saline medium, high levels of cell death were detected using propidium iodide as a probe of viability (membrane integrity): some 70% of the cells died during 3 h incubation at 37 degrees C. Because the conditions of incubation were similar to those encountered during manipulations for in vitro fertilization, this phenomenon was investigated further. If ram spermatozoa were diluted in an equivalent sucrose-based medium, or if the saline medium was supplemented with 10% seminal plasma, survival was greatly improved (only 5-15% died during a 3-h incubation at 37 degrees C); the protective effect of seminal plasma resided in a 5-10 kDa fraction. Sperm death in the basal saline medium was strongly dependent on cell concentration below 5 x 10(7) spermatozoa mL-1 whereas little effect of concentration was seen in the sucrose medium or in the presence of seminal plasma. The presence of Ca2+ (2 mM), EGTA (1 mM) or mercaptoethanol (1 mM) enhanced sperm survival in saline medium, but no effect was gained by replacing NaCl with KCl, and neither BSA nor fetal calf serum were beneficial. However, when a combination of pyruvate (1 mM), lactate (21.7 mM), Mg2+ (0.4 mM), phosphate (0.3 mM) and Ca2+ (2 mM) was included in the saline medium (to render it similar to Tyrode's medium), cell survival was greatly improved (12% died during the 3-h incubation).(ABSTRACT TRUNCATED AT 250 WORDS)

Dog spermatozoa were evaluated for their ability to swell when exposed to hypo-osmotic conditions. A medium was developed to allow spermatozoal swelling to be identified easily; both the number of swollen spermatozoa and their morphological appearance were influenced by the characteristics of this medium. The number of swollen spermatozoa was inversely proportional to the number of dead spermatozoa in a sample. Although spermatozoal swelling was highly repeatable there was no correlation with spermatozoal motility, morphology or vital staining. Electron microscopic studies demonstrated that swelling of spermatozoa was characterized by ballooning of the tail membrane and internal flexion of the axial filaments. Spermatozoa with damaged membranes were unable to swell. Hypo-osmotic swelling of dog spermatozoa is reliable and repeatable and may be a useful assay for the diagnosis of spermatozoal membrane function.

The ultrastructure of the spontaneous acrosome reaction in ram spermatozoa has been compared with that induced by the ionophore, A23187. The spontaneous event was dependent on incubation for 4 h, on the temperature, and on dilution. Apart from the more rapid occurrence of the ionophore-induced event, the mean diameter and distribution of vesicle size was also different. The ionophore-induced vesicles were larger, more irregular, and heterogeneous in size compared with those occurring in the spontaneous acrosome reaction (average diameter 84 nm vs. 60 nm in the spontaneous acrosome reaction). These observations are interpreted in relation to capacitation.

Twenty-five ununited coronoid processes (UCP) and 24 osteochondritis dissecans (OD) flaps were examined by light and transmission electron microscopy. Chondrocytes showed degenerative changes but remained viable and continued to secrete matrix components, even though the organisation of the matrix was altered. Differences in the histological and ultrastructural appearance of the two lesions tend to suggest that they are two separate disease entities, although they may occur together in the same joint. It is hypothesised that OD results from incorrect cartilage maturation and endochondral ossification. The aetiology of UCP is unclear but there is a possibility of its being a subchondral fracture, with an ineffective fibrous repair in some cases.

The elbows of 13 puppy cadavers were dissected, samples were taken for light and electron microscopy, and the thickness of the articular cartilage of the distal humerus and proximal ulna was measured. Throughout post natal development differences were found in the arrangement of the growth plate and articular chondrocytes. At birth, the articular surface had remnants of a fibrous limiting membrane that was continuous with the perichondrium, a finding not previously recorded in dogs. Orientation of the collagen fibrils within the matrix of the articular cartilage was initially lacking but became established by three weeks. In the humerus cartilage canals were present up to 12 weeks old. The articular cartilage of the humeral condyle varied in thickness across the joint surface, being thicker on the medial than on the lateral side; it was also thicker at the apex of the medial coronoid process. These regions of thick cartilage correspond with the sites where cartilage defects arise in elbow osteochondrosis. No histological evidence was found that the medial coronoid process of the ulna is a separate centre of ossification.

The acrosome reaction has been studied in vitro both by artificial induction using the ionophore A23187, which allows free access of calcium to trigger the reaction, and by observing incubated spermatozoa in which, under certain conditions, a high proportion displayed a spontaneous acrosome reaction. Temperature effects on the acrosome reaction were investigated by incubating spermatozoa in the presence or absence of ionophore at 35, 37, 39, 41 or 43 °C. Maximal numbers of acrosome-reacted spermatozoa were obtained at 39 °C and above after 30 min incubation with A23187, or after 3–8 h incubation in medium without ionophore (spontaneous acrosome reactions). Spermatozoa selected for high-motility characteristics by a swim-up method were found to be more readily induced to undergo an acrosome reaction with ionophore, but selection had no influence on the frequency of the spontaneous event. Variation was found between individual ejaculates in the readiness of the spermatozoa to undergo an acrosome reaction, but this couid not be attributed consistently to any particular ram. However, differences between ejaculates were eliminated by increasing the incubation temperature to 41 °C.

A transmission electron microscope study was undertaken to investigate the details of the synovial tendon sheath and bursal lining in horses. The lining cells appeared to be fibroblasts and were buried in a finely granular ground substance. Generally these cells had poor cytoplasmic organelles, sparse short profiles of rough endoplasmic reticulum (RER), few scattered ribosomes and, occasionally, a poorly developed Golgi complex. However, a few lining cells appeared more active, having pronounced RER with dilated cisternane. The surface of the lining cells, particularly those with dilated RER, had filipodia of different shapes, which in some instances appeared to surround a detached matrix. This feature might indicate that these cells are capable of phagocytosis. In young animals, the lining was more or less akin to that of the adult. However, the lining cells were virtually indistinguishable from the fibroblasts in the subconnective tissue.

The structure of equine synovial tendon sheaths and bursae has been examined by light and scanning electron microscopy. Tissue samples were obtained from horses of various types and ages with no clinical evidence of sheath or bursal disorders. The interior of both structures was lined by a cellular layer superimposed on a vascular zone supported by a fibrous layer. The pattern of cell distribution of the lining varied from site to site within the same structure depending on the nature of the underlying tissue and on the amount of movement to which the structure was subjected. The cellular layer was predominantly fibrous in nature with scanty, widely separated fibroblasts (eg where it lines the palmar ligament, tendons and paratendons). In the mesotendon and bursal extremities, where the lining is subjected to a positive degree of movement, the cellular layer was areolar in type with well established folds populated by abundant cells oval to round in shape. In foals and yearlings, the supportive layer was mainly around the areolar with patches of adipose tissue; which were gradually replaced by fibrous tissue as the animal grew.

Transverse sections of the lateral palmar nerve from a group of horses free from neuromuscular disease were assessed quantitatively, with emphasis on differences in the fibre population related to age. Morphometric analysis of the population of myelinated and unmyelinated fibres was performed. Changes were identified, there being a loss of larger myelinated fibres in older horses. The relationship of myelin thickness to axon diameter and the unmyelinated fibre population did not alter with age. The lateral palmar nerve is suitable for biopsy and electrophysiological study in the horse, but age related changes must be appreciated in the interpretation of results.

Ram spermatozoa treated with the ionophore A23187, to induce the acrosome reaction, were fixed in pyroantimonate-osmium in order to demonstrate calcium at an ultrastructural level. Prior to vesiculation, granules of precipitate occurred at points of contact between plasma and outer acrosomal membranes, suggesting a discrete role for calcium during membrane fusion. The earliest stages of vesiculation always occurred in the region just anterior to the equatorial segment and it was this same site where a marked concentration of precipitate, indicating calcium binding sites, could be demonstrated.