Volume 58 (2014): Issue 1 (March 2014)

The review describes the role of cells of extracellular matrix (ECM) as a source of neoplastic outgrowths additional to the original tumour. The cells undergo a spontaneous transformation or stimulation by the original tumour through intercellular signals, e.g. through Shh protein (sonic hedgehog). Additionally, cells of an inflammatory infiltrate, which frequently accompany malignant tumours and particularly carcinomas, may regulate tumour cell behaviour. This is either by restricting tumour proliferation or, inversely, by induction and stimulation of the proliferation of another tumour cell type, e.g. mesenchymal cells. The latter type of tumour may involve formation of histologically differentiated stromal tumours (GIST), which probably originate from interstitial cells of Cajal in the alimentary tract. Occasionally, e.g. in gastric carcinoma, proliferation involves lymphoid follicles and lymphocytes of GALT (gut-associated lymphoid tissue), which gives rise to lymphoma. The process is preceded by the earlier stage of intestinal metaplasia, or is induced by gastritis alone. This is an example of primary involvement of inflammatory infiltrate cells in neoplastic progression. Despite the numerous histogenetic classifications of tumours (zygotoma benignum et zygotoma malignum, or mesenchymomata maligna et mesenchymomata benigna), currently in oncological diagnosis the view prevails that the direction of tumour differentiation and its degree of histologic malignancy (grading) are more important factors than the histogenesis of the tumour.

In order to evaluate the prevalence of the Mycoplasma mycoides cluster in goats in Chongqing, China, an epidemiological survey in this area was carried out. A total of 68 samples were subjected to bacteria isolation on Hartley’s medium. Four isolates (three from lung tissue and one from nasal discharges) were recovered from the samples and identified as the Mycoplasma species by their morphological and biochemical characteristics. They were further confirmed by PCR using 16S rRNA specific primer pairs and by restriction enzyme analysis. In vitro antimicrobial susceptibility of the isolates indicated that some strains had developed resistance to the antibiotics tested. This is the first report on the isolation, identification, and molecular characterisation of Mycoplasma species isolated from goats in Chongqing. This study also revealed a prevalence of Mycoplasma species infection in goats in this area.

Expression of CD4, CD8, and CD25 surface markers on T lymphocytes and levels of IFNγ, IL-10, and TNF-α in colostrum and milk were determined in sows vaccinated against Trueperella pyogenes in the final stage of pregnancy. The autovaccine, prepared from Trueperella pyogenes, administered twice to pregnant sows six and three weeks before the anticipated delivery significantly increased the percentages of TCD4+, TCD8+, and TCD25+ as well as levels of IFNγ, TNF-α, and IL-10 in colostrum and milk. The enhanced immune potential of colostrum effectively protected the piglets against T. pyogenes infections during weaning and thus reduced the economic losses on the particular farm concerned, where T. pyogenes infections occur endemically. Knowledge of the profile of cellular and humoral immune response in colostrum and milk of vaccinated sows will enable the design of a T. pyogenes infection prophylactic programme for suckling pigs and weaners, which are most susceptible to infections.

The expression of galanin (GAL) and its three receptors (GalR1, GalR2, and GalR3) were studied with real-time PCR in the colonic wall of pigs suffering from experimental colitis caused by the infection with Brachyspira hyodysenteriae. The expression was studied in the muscular membrane, mucosa/submucosa layer, and in lymphocytes isolated from mucosa/submucosa. The expression levels were normalized to glyceraldehyde-6-phosphate dehydrogenase (GAPDH) expression and compared to expression levels in control animals. GAL expression was found in all three studied compartments of the colonic wall. A significant decrease in GAL expression level was found in the mucosa/submucosa and in isolated lymphocytes, whereas the decrease was much less profound in the muscular membrane. In the case of galanin receptors their expression was found in all studied compartments of the colonic wall, however at different levels, as compared to GAPDH expression. The decrease of galanin receptors expression was found in all studied compartments of the colonic wall of the sick animals.

The aim of the study was to assess the distribution of Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, and Babesia canis in adult females and males of Ixodes ricinus and Dermacentor reticulatus ticks, inhabiting meadows near large forest complexes throughout the Lubelskie Voivodship (eastern region of Poland). Ticks were collected using the flagging method. Among 720 ticks collected, 506 were identified as D. reticulatus, and 214 as I. ricinus. DNA of B. canis and B. burgdorferi s.l. was detected in 21.3% and 0.6% of D. reticulatus ticks, respectively. In I. ricinus ticks, DNA specific to B. burgdorferi s.l. and A. phagocytophilum was detected in 5.6% and 10.3%, respectively. Co-infections of B. burgdorferi s.l. and A. phagocytophilum were found in two I. ricinus ticks. These results indicate that the Lublin region is an area at risk of tick-borne diseases of humans and animals, which must be considered in clinical practice.

The aim of the study was to determine the prevalence of canine dirofilariosis in four provinces of central-eastern Poland and to describe pathological changes occurring during the course of this parasitosis. A total of 188 dogs aged from one and a half up to 16 years were examined for dirofilariosis. Blood samples were analysed for the presence of microfilariae using Knott method, as well as the method of Kingston and Morton. Pathomorphological examination of two dogs was also performed. Histological sections were stained with H&E, AB-PAS, van Gieson, and von Kossa methods. Microfilariae of Dirofilaria repens were found in blood samples of dogs that originated from the Mazowieckie, Lubelskie, and Podlaskie provinces. The mean prevalence of that species was 12.7%. Autopsy revealed the adult nematodes in the subcutaneous connective tissue. Microscopic examinations indicated the presence of microfilariae in the liver, kidneys, lungs, heart muscle, and intestines. Necrotic foci, thrombosis, and infiltrations of different intensity with eosinophils in internal organs were also observed. Significant lesions in different internal organs together with the presence of numerous microfilariae suggest their important role in the disease process.

The aim of this study was to investigate pathomorphological changes in the duodenum, jejunum, ileum, and caecum of Japanese quails naturally infected with Eimeria tsunodai. Samples of the intestines were collected from 6-week-old cockerels and hens before laying and from laying quails aged 12, 24, and 48 weeks. The tissue sections were stained with haematoxylin and eosin, PAS method according to McManus, and Ziehl-Neelsen method. Morphometric and morphological analyses of coccidia revealed that Eimeria tsunodai was responsible for the infection. Different developmental stages of the coccidia were observed almost exclusively in the caecum, and they led to the complete damage of caecal mucosa. The main changes observed in the caecum involved mucosal damage, atrophy of the folds and crypts, and mucosal desquamation. No significant inflammation changes were detected. The degree of damage to caecal mucosa increased with age and the greatest damage was noted at the age of 48 weeks. Eimeria tsunodai infections occur in Japanese quails during the entire laying period. Different developmental stages of coccidia are responsible for total damage to caecal mucosa.

The aim of the study was to determine a growth rate of Salmonella Enteritidis in cooked ham stored under different temperatures and to compare usefulness of the mathematical models for describing the microbiological data. The samples of cooked pork ham were inoculated with the mixture of three Salmonella Enteritidis strains and stored at 5°C, 10°C, 15°C for 21 d, and at 20°C and 25°C for 5 d. The number of salmonellae was determined at 10 periods of storage at each temperature. From each sample a series of decimal dilutions were prepared and plated onto Brilliant Green Agar. The plates were incubated at 37°C for 24-48 h under aerobic conditions. The colonies grown on culture media were counted, bacterial counts were multiplied by the appropriate dilutions, and number of bacteria (colony-forming units) was calculated. The bacterial counts were transformed into logarithms and analysed using IBM SPSS Statistics 20. The experiment was performed in five replicates. The obtained growth curves of bacteria were fitted to primary growth models, namely Gompertz, logistic, and Baranyi models. The goodness-of-fit test was evaluated by calculating mean square error and Akaike’s criterion. Growth kinetics values from the modified Gompertz and logistic equations were calculated. It was found that in samples of ham stored at 5°C and 10°C for 21 d, the number of bacteria remained almost at the same level during storage. In samples stored at 15°C, 20°C, and 25°C growth of salmonellae was observed. It was found that logistic model gave in most cases the best fit to obtained microbiological data describing the behaviour of S. Enteritidis in cooked ham. The growth kinetics values calculated in this study from logistic equations can be used to predict potential S. Enteritidis growth in cooked ham stored at 15°C, 20°C, and 25°C.

The aim of the study was to provide a detailed phenotypic and genotypic characterisation of Staphylococcus aureus strains and the group of microorganisms with unusual biochemical patterns (called Staphylococcus aureus-like) isolated from table chicken eggs. All of the strains tested exhibited resistance to at least one of the 17 antibiotics tested, and 55.55% of isolates were found to be resistant to five or more of them. PCR used for detection of the methicillin resistance gene (mecA) confirmed the presence of a specific product of 533 bp in the case of two of the isolated S. aureus-like strains. Analysis of the phylogenetic relationship between eight of S. aureus and ten S. aureus-like strains distinguished 18 macrorestriction profiles following digestion with SmaI endonuclease, indicating that there were no identical strains with the same macrorestriction profile. However, the presence of methicillin-resistant strains indicates a serious risk to consumer health.

A chromatographic procedure for determination of oxytetracycline (OXT), tetracycline (TC), chlorotetracycline (CTC), and doxycycline (DC) in water samples was developed and was applied for the analysis of water samples collected from poultry and pig farms and environmental water samples. Samples were acidified with trifluoroacetetic acid to pH 3 and further purified by solid phase extraction using Oasis HLB cartridges. The samples were dried up and redissolved in the mixture of oxalic acid and methanol. Separation was performed on reserved phase column (Phenomenex column C18 , 250 mm × 4.6 mm, 5 μm) by multistep gradient elution, and detection was carried out at 360 nm for OTC and TC, 370 nm for CTC, and 350 nm for DC. The tetracyclines were eluted with the mobile phase of 0.05 M oxalic acid (pH 2.5), acetonitrile, and methanol. This method provided average recoveries of 83.53% to 108.59%, with coefficient of variations (CVs) of 2.41% to 8.64% in the range of 10 to 1000 μg/L OTC, TC, CTC, and DC in water. The linearity for the tetracyclines was determined by HPLC-DAD in the range 10 to 1000 μg/L, with the correlation coefficient (R) > 0.99. The LOD and LOQ for the tetracyclines in water samples ranged from 1.51 to 4.00 and 2.51 to 5.93 μg/L, respectively.

The aim of the study was to determine and compare the concentrations of chlorinated hydrocarbons residues (DDT, DDE, DDD, γ-HCH) in the milk fat of selected species of farm animals. The experiment was carried out on cow’s, sheep’s, goat’s, and mare’s milk samples originating from different parts of north-eastern Poland. The samples were prepared using Röse-Gottlieb’s and Ludwicki’s methods. The determination of the compounds was conducted with gas chromatography. All tested samples contained the residues of chlorinated hydrocarbons. The results varied depending on the animal species as well as the places of sample collections. The highest content of γ-HCH and ΣDDT was determined in cow’s milk (22.75; 53.12 μg/kg of fat, respectively). The lowest level of γ-HCH and ΣDDT was observed in sheep’s milk (0.25; 5.94 μg/kg of fat, respectively). The content of chlorinated hydrocarbons did not exceed the maximum acceptable levels of these compounds.

The aim of the study was to determine the concentrations of lead (Pb), cadmium (Cd), and mercury (Hg) in the liver, kidneys, and muscles of European beavers (Castor fiber) and thus to evaluate the degree of heavy metals contamination in Warmia and Mazury region in Poland. The study was conducted on free-living beavers captured in region of Warmia and Mazury during autumn 2011. Concentrations of the elements were determined by atomic absorption spectrometry. The presence of the metals was detected in all individual tissue samples. Mean Pb and Hg concentrations were relatively low. However, the high mean Cd level, especially in the kidneys (7.933 mg/kg) and liver (0.880 mg/kg) was demonstrated. Despite the fact that region of Warmia and Mazury is considered to be “ecologically clean”, the conducted studies indicate that systematic monitoring for the presence of heavy metals is necessary not only in industrialised but also in agricultural regions, as well as in natural ecosystems.

The undertaken study aimed at analysing the effect of linseed oil used in diets for turkey hens on contents of pro- and antioxidants in their tissues. Additionally, correlations were analysed between the contents of these compounds in blood, liver, and muscles of the birds. The experiment was conducted on 240 one-week-old turkey hens reared until 16 weeks of age, randomly allocated into two feeding groups. Hens from the first group received soybean oil in their complete feed mixture, whereas linseed oil was the source of fat for turkey hens from the second group. The oil content of the diet was on the level appropriate for the period of feeding: Starter - 0.5%; Grower I - 1.0%, Grower II - 2.5%; Finisher I - 3%. The redox parameters were assayed in breast and thigh muscles, blood, and liver. Introduction of PUFA n-3 high linseed oil to a feed mixture contributed to the enhancement of lipid peroxidation processes, which was indicated by a significant increase in concentrations of H₂O₂ and malondialdehyde in muscles, and by an increase of superoxide dismutase activity and concentrations of vitamin C and minerals (Cu⁺², Zn⁺², Fe⁺², Se⁺²). This points out the need of strengthening the antioxidative defence by the use of suitable antioxidants. The use of linseed oil as a feed material for turkey hens additionally contributed to changes in parameters determining the ferric reducing ability of plasma (FRAP). The increased FRAP values resulted from a significant increase in vitamin E level.

The study aimed at evaluation of pathological lesions on flexor surface of navicular bone and deep digital flexor tendon in horses graded in standard X-ray examination as 2 (fair). The evaluation was performed on fifteen horses (6-9 years of age). Analysis procedure involved examining navicular bones on X-ray pictures, post-slaughter preparation of navicular bones from the hoof capsule, macroscopic evaluation of fibrocartilage on flexor surface, and analysis of histologic preparations. In horses with navicular bones graded as 2, early pathological changes have already developed, even if such horses were not lame. The pathological changes included fibrillation and disruption of deep digital flexor tendon surface, loss of fibrocartillage in sagittal ridge area of navicular bone, thinning of subchondral bone on its flexor surface, and fibromyxoid changes in chondroid matrix. In terms of clinical relevance, more studies are needed to understand the sequence of changes in a better way.

The aim of this study was to investigate the causes of chronic respiratory disorders in Polish Konik horses maintained in a stable and pasture, under condition typical for pleasure horses in Poland. The study was conducted on 14 adult horses, consistent with regard to environment and living conditions. The horses were divided into two groups: seven horses not affected by any respiratory problem (control group) and seven horses with history of lower airway disease (study group). Clinical and laboratory evaluation, endoscopic examination, bronchoalveolar lavage, tracheal wash, and lung ultrasound were performed in all horses. Median (25^th and 75^th percentiles) for bronchoalveolar lavage fluid differential cell count in control horses was 55.8 (49.8 and 59.1) of macrophages, 41 (38.5 and 45.9) of lymphocytes, and 5.1 (4.1 and 5.3) of neutrophils, whereas in the study group they were 32.8 (25.9 and 35.7) of macrophages, 59.8 (51.3 and 64.8) of lymphocytes, and 38.1 (34.8 and 41.1) of neutrophils. It was concluded that chronic respiratory disorders in Polish Konik horses were probably caused by recurrent airway obstruction.

Cytometric analysis was performed in order to detect the expression of surface molecules CD11b and CD11/18 on lymphocytes, granulocytes, and monocytes of peripheral blood of dogs with atopic dermatitis (AD) complicated by purulent dermatitis. The percentage of peripheral blood lymphocytes with CD11b and CD11/18 was found to be significantly higher (P < 0.05) in dogs with AD than in healthy dogs. The percentage of granulocytes expressing CD11b molecule was significantly lower in dogs with complicated AD compared to healthy dogs and dogs with uncomplicated AD. The expression of CD11b and CD11/18 integrin on monocytes of dogs with complicated AD depended on the severity of symptoms and was higher in dogs with deep purulent dermatitis. It was concluded that the expression of CD11b and CD11/18 integrin on leukocytes of dogs with AD depends on the stage of the disease.

The aim of the study was to investigate S100β protein in astrocytes of CA1 and CA3 areas of the hippocampus proper and the dentate gyrus with the hilus yet undefined in mature males of chinchilla. The presence of S100β was determined using indirect immunohistochemical peroxidase-antiperoxidase method with specific monoclonal antibody against this protein. Most of the S100β-positive cells were detected in the subgranular zone of the dentate gyrus and in the middle part of the hilus. In CA3 area, it was found that the most numerous cells with S100β are in stratum radiatum. In CA1 area, there were single astrocytes expressing this protein. This data demonstrates species differences and a large quantity of S100β immunoreactive cells in the subgranular zone of the dentate gyrus of chinchilla, which may be associated with structural reorganisation of the hippocampus and with neurogenesis, learning, and memorising process dependent on the hippocampus.

Immune and oxidative parameters were evaluated as indicators of the influence of stress on the occurrence of respiratory syndrome in feedlot calves. The study was carried out on 60 2 and a half-month-old Simmentaler calves transported at feedlot. Mean daily feed consumption, daily weight gain, and behavioural activity were evaluated. Blood was collected on days 1, 3, 7, 14, 21, and 28 of feedlot. Serum NO ions, lipid peroxidation, acute phase proteins, IgG, and IgM were determined. The calves showed a decrease in feed consumption during the first 7 d of the feedlot. Sporadic stereotypies were noted. NO concentration showed a significant (P < 0.05) increase on days 7 and 14. Concentration of thiobarbituric acid reactive substances (TBARS) increased (P < 0.05) on days 1 and 3. IgM concentration was found to be considerably lower on days 14 and 28. Serum haptoglobin level showed a significant increase in stressed calves on days 1, 3, 7, and 14 of the feedlot. Based on these results, it can be suggested that stressors associated with transport and adaptation to the feedlot induce a stress reaction in calves, resulting in behavioural disorders, reduced weight gain, suppression of the humoral immunity and increased morbidity during the first weeks. These parameters seem to be crucial in evaluating the animals' health and welfare.

The study aimed to demonstrate the expression of hypoxia-inducible factor (HIF-1α) in soft tissue mesenchymal tumours (fibroma and fibrosarcoma) in dogs. An attempt was made to correlate the obtained results with density of blood vessels (expression of von Willebrand Factor, vWF), expression of Ki-67 proliferation antigen, and with intensity of apoptosis in studied tumours. The study was performed on paraffin sections of 15 fibromas and 40 fibrosarcomas sampled from 55 female dogs aged 6 to 16 years. Immunohistochemical staining against HIF-1α, vWF, and Ki-67 was performed. Apoptosis was detected with the use of TUNEL reaction. A significantly higher HIF-1α expression was noted in fibrosarcomas in comparison to fibromas (P < 0.0001). HIF-1α expression in fibromas manifested strong positive correlation with tumour vascularity (r = 0.67, P = 0.007). Moreover, HIF-1α expression in fibrosarcomas manifested a moderate positive correlation with tumour malignancy grade (r = 0.44, P = 0.004), tumour vascularity (r = 0.52, P < 0.001), Ki-67 antigen expression (r = 0.42; P = 0.007), and TUNELpositive cells (r = 0.37, P = 0.017). Expression of HIF-1α was detected in 86.7% of fibroma type tumours and in 100% of fibrosarcomas. In all studied tumours expression of HIF-1α manifested positive correlation with the density of blood vessels, and in fibrosarcomas it correlated also with malignancy grade, intensity of Ki-67 expression, and with intensity of apoptosis in tumour cells.

The aim of this study was to develop and examine a model of apoptosis and necrosis of hepatocytes induced by a damaging factor - adriamycin, correlating time after its administration with cell death type, and to investigate the localisation within the liver acinus of hepatocytes dying in these two ways. The results obtained in the present and previous studies were compared in order to make a map of cell death localisation in the liver acinus, showing the effect of time in action and dose of adriamycin. The experiment was performed on 32 female Wistar rats, divided into four groups: I and II - experimental, and III and IV - control. Adriamycin (3 mg/kg b.w.) was administered intraperitoneally to rats in groups I and II, and the rats were decapitated after four (group I) and eight (group II) weeks. Animals in control groups III and IV were given 0.5 mL of 0.9% NaCl solution, and decapitated after four and eight weeks respectively. Sections of the liver were examined with a three-stage immunohistochemical method. This method allowed to examine hepatocytes qualitatively and quantitatively for the presence of proteins involved in three types of apoptosis: induced by the mitochondrial pathway (caspase 3, 9), the intrinsic pathway related to endoplasmic reticulum stress (caspase 3, 12), and the extrinsic pathway (caspase 3, 8). One of the inflammatory markers, caspase 1, was also examined. The zonal localisation of all three types of apoptosis was assessed in the liver tissue. More oxidated hepatocytes indicated only signs of the internal mitochondrial pathway, whereas less oxidated hepatocytes induced the internal reticular pathway and the external apoptotic pathway. The period between adriamycin administration and hepatic cell investigation was a main factor of the process. A longer period post insult resulted in a more pronounced effect of the activation of apoptosis. Sections explored eight weeks after treatment with different doses of the drug (3 and 5 mg/kg in the previous study) showed a similar intensity of apoptosis.

The aim of the study was to demonstrate differences in the gene expression of signalling pathways between healthy dogs and dogs with chronic mitral valve disease in different heart failure groups. Blood samples were collected from 49 dogs of various breeds between 1.4 and 15.2 years of age. Isolated RNA samples were analysed for quality and integrity and the gene expression profile was determined. The study demonstrated that nucleated cells from peripheral blood can be used to assess the status of heart failure in dogs. Furthermore, significant differences in the expression of the genes were noticed between healthy dogs and dogs with clinical signs of chronic mitral valve disease. This is a preliminary non-invasive study showing the feasibility of genetic testing from peripheral blood nucleated cells, which at the same time has made it possible to set the future directions of genetic studies in clinical cases of canine chronic mitral valve disease.

The experiment was conducted on 30 Holstein-Friesian cows: 10 cows in the follicular phase of the cycle and in the luteal phase 10 d later, 10 cows with follicular cysts, and 10 with luteal cysts. The presence of the ovarian structures was confirmed by ultrasonography. Serum levels of progesterone and 17β-oestradiol were tested with ELISA. Samples for cytological examination were collected from the uterus of all cows using a cytological brush. Following staining, the smears were evaluated in terms of quality and percentages of endometrial cells. In the follicular phase of the oestrous cycle, cells of type A - superficial cells (64.6 ± 4.48) were proportionally the largest group of cells. Cells of type C - basal cells (19.8 ± 2.75) were also present. In the luteal phase, the highest percentage of cells was of type B - intermediate cells (76.9 ± 4.26). When follicular cysts were present on the ovaries, the cytology resembled the follicular phase of the cycle, but with many younger type C cells (33.1 ± 4.11). In the case of luteal cysts on the ovaries, the cytology was similar to that of the luteal phase of the cycle, however with a lower percentage of type B cells (58.1 ± 5.71), and a slightly higher percentage of the other types. The differences in the cytological image of the uterus when different ovarian structures are present, depend on the hormonal activity of those structures. Due to the lack of literature data, the results of the study are important as a model, and may substantially facilitate identification of phases of the oestrus cycle, or the pathologies described, as well as indicate the current status of the endometrium

The aim of this study was to investigate the usefulness of new software Pixel Flux (PXFX) for clinical evaluation of tissue perfusion in the field of reproduction in dogs. The experiment was performed on six adult Beagle dogs. Different organs and tissues of the animals were examined with the MyLab25 Gold ultrasound system. Blood flow in the ovary, testicle, prostate, the ramification of the penile artery, and the network of blood vessels of the pampiniform plexus were examined with the use of colour coded Doppler technique, and obtained data was evaluated with the PXFX software. The more objective digital evaluation of data obtained with colour Doppler sonography through the application of dynamic tissue perfusion measurements provides new opportunities for diagnosis, as well as continuous monitoring of the function of the examined tissues and organs. The use of PXFX software is strongly indicated as a tool in small animal practice as an additional method for evaluation of tissue perfusion, especially in the cases when other methods like pulsed wave Doppler techniques are difficult to be performed

The aim of the study was to investigate the presence of α (Erα) and β (Erβ) oestrogen receptors in rabbit male’s claustrum, and thus to examine whether oestrogens may influence nerve cells’ functions of this structure. The material for the study was obtained from the brains of seven rabbits. To detect the presence of Erα and Erβ, specific monoclonal antibodies were used. The expression of Erα and Erβ was demonstrated in proper claustrum, as well as in the nucleus endopiriformis. For Erβ, the reaction occurred mainly in the cell nucleus, whereas Erα was localised predominantly in the neuron cytoplasm. For both Erα and Erβ, the nuclear and cytoplasmic localisation was observed in a small percentage of the examined cells. The obtained results may indicate oestrogen influence on the brain area and also suggest a further research on oestrogen-dependent pathways in males, which still remain greatly unexplored.

Sperm surface binding sites for non-zinc-binding proteins (nZnBPs) and zinc-binding proteins (ZnBPs) were studied by the fluorescence technique with biotin-labelled proteins. The nZnBPs binding pattern was unspecific, no characteristic sites on plasmalemma were found. ZnBPs were attached mainly to the acrosomal region of sperm head and to the sperm flagellum. ZnBPs added to the incubation mixture of the canine spermatozoa allowed the preservation of higher values for total motility, progressive motility, curvilinear line velocity, straight line velocity, and beat cross frequency (P < 0.05), both at time 0 and after 1 h incubation at 5ºC. The addition of nZnBPs to the incubation mixture caused only weak positive effects when compared with control sample (PBS). A higher percentage of canine-ejaculated spermatozoa with intact membranes were observed when ejaculate was incubated with ZnBPs in comparison to control sample stored with PBS (P < 0.05) or nZnBPs (P < 0.05). Spermatozoa diluted with ZnBPs and nZnBPs exhibited a higher percentage of cells with active mitochondria when compared with control, both at time 0 and after 1 h; however, no statistical differences were observed. Our results emphasise the role of seminal plasma protein in securing the correct quantity and availability of zinc ions as a component regulating the motility of canine spermatozoa. The protective effect of ZnBPs against the cooling effect may be due to their ability of preventing sperm membrane damage.