C-reactive protein, tumor necrosis factor α, and interleukin-6 in dogs with pyometra and SIRS

Objective: To determine the frequency of the systemic inflammatory response syndrome (SIRS) in canine pyometra and to evaluate the relationship between C‐reactive protein (CRP), tumor necrosis factor α (TNFα), interleukin‐6 (IL‐6), and SIRS. Design: Prospective clinical study. Setting: Veterinary teaching hospital. Animals: Fifty‐three clinical cases of canine pyometra and 19 healthy control bitches. Interventions: Upon admission to the veterinary hospital, history and physical examination findings, including previously defined clinical SIRS parameters, were documented. Blood samples were obtained for hematology and biochemical tests and for CRP, TNFα, and IL‐6 analysis. The diagnosis of pyometra was confirmed by histopathology of the uterus after ovariohysterectomy. After surgery, clinical SIRS parameters, length of hospitalization, and mortality were recorded. Measurements and main results: Pyometra dogs were grouped as SIRS positive (30/53; 57%) or SIRS negative (23/53; 43%). Logistic regression showed that CRP was the only parameter that significantly related to SIRS apart from the clinical criteria that define this syndrome. The mortality rate was low (2/53; 3.8%), and conclusions regarding association with SIRS could not be drawn. A positive SIRS status, high plasma CRP concentration, and high body temperature were variables that related to increased morbidity reflected by the length of hospitalization. Conclusions: SIRS was seen in 57% of canine pyometra cases and a positive SIRS status showed a positive association with prolonged hospitalization. The mortality rate was low (3.3%) among SIRS positive dogs, indicating that progression to multiple organ dysfunction syndrome (MODS) rarely occurs in surgically treated cases of pyometra. CRP was associated with SIRS and with prolonged hospitalization. Further studies of plasma CRP may be warranted in canine intensive care cases susceptible to development of SIRS and MODS.     

Quantity of IL‐2, IL‐4, IL‐10, INF‐γ, TNF‐α and KC‐Like Cytokines in Serum of Bitches With Pyometra in Different Stages of Oestrous Cycle and Pregnancy

The occurrence of the pyometra is most common in the first half of the dioestrus when there is decreased cellular immunity associated with increased serum concentration of progesterone in females. The aim of this study was to determine the immunological profile of bitches with pyometra, studying serum levels of IL‐2, IL‐4, IL‐10, IFN‐γ, KC‐like and TNF‐α and comparing them with those of healthy bitches in anoestrus, dioestrus and pregnant. Forty females were divided into four experimental groups: group 1 (G1): with pyometra (n = 10); group 2 (G2): bitches in the second week of gestation (n = 10); group 3 (G3): in anoestrus (n = 10); and group 4 (G4): in dioestrus (n = 10). The serum levels for IL‐2, KC‐like, INF‐γ and TNF‐α were similar for all experimental groups. The values obtained for IL‐10 were found increased (p < 0.001) in animals in dioestrus and pyometra compared with females in anoestrus and pregnant, and the levels of IL‐4 observed were significantly greater (p < 0.001) in bitches with pyometra when compared with others. The cytokine profile in animals with pyometra is similar to bitches in dioestrus for IL‐10 and had increase in IL‐4 for bitches with pyometra, which represents an anti‐inflammatory these cases. This suggests the presence of an immunosuppressive state in both cases, which may explain the propensity of bitches in dioestrus to be affected by pyometra and the severity of the disease on these animals.     

Lactoferrin in Canine Sera: A Pyometra Study

The concentration of lactoferrin was measured in canine sera from groups of healthy male dogs as well as pregnant and non-pregnant female dogs and was compared with that of bitches with pyometra. Lactoferrin concentrations were higher in bitches with pyometra. The role of elevated lactoferrin concentrations in the suppression of lymphocyte activity was examined in sera from bitches with pyometra in a series of investigations. Although the sera from bitches with pyometra were capable of suppressing lymphocyte activity, lactoferrin was not found to be involved in this action.     

Anti‐Muellerian hormone concentration in bitches with histopathologically diagnosed ovarian tumours and cysts

Increased concentrations of Anti‐Muellerian hormone (AMH) can indicate a granulosa cell tumour as shown in women, mares and cows. To investigate AMH to differentiate canine granulosa cell tumour from other ovarian pathologies, we evaluated the ovaries of 63 bitches. Blood serum samples were collected before surgery for AMH analysis. Ovaries were submitted for histopathological examination. Fourteen bitches showed normal ovaries. These bitches had AMH values between 0.12 and 0.99 ng/ml. In 20 bitches ovarian cysts i.e., follicular cysts (n = 8), corpora lutea cysts (n = 7), subsurface cysts (n = 5) were diagnosed. These dogs had AMH values of 0.11–2.09 ng/ml. Bitches with small luteinized follicular cysts had slightly higher AMH values than those without ovarian alteration. In 29 cases ovarian neoplasms i.e., granulosa cell tumour (n = 9), epithelial tumours (n = 16), dysgerminomas (n = 3) and one sarcoma were identified. Anti‐Muellerian hormone values of bitches with an ovarian neoplasm except granulosa cell tumour ranged from 0.18 to 1.18 ng/ml. The AMH values of bitches with granulosa cell tumour ranged from 1.12 to ≤23 ng/ml and were significantly higher (p < .05) than in all of the other bitches. The cut‐off of 0.99 ng/ml gave a sensitivity of 100% and a specificity of 94.44% to diagnose granulosa cell tumour. In conclusion, markedly elevated AMH concentrations in bitches are indicative for a granulosa cell tumour. However, negative testing does not rule out the existence of small one. Differentiation of GCT from luteinized follicular cysts may especially be difficult.     

Comparison of clinical and inflammatory parameters in dogs with pyometra before and after ovariohysterectomy

This study aimed to identify potential biomarkers of canine pyometra and their correlations with clinical parameters. First, 90 dogs with pyometra and 26 healthy female dogs were compared. Then, paired samples (before and after ovariohysterectomy) from 22 dogs with pyometra and 9 healthy controls from the initial cohort were compared.Concentrations of acute inflammatory proteins, C-reactive protein (CRP) and serum amyloid A (SAA), and cell-free DNA (cfDNA), were significantly higher in dogs with pyometra than in clinically healthy dogs. Cell-free DNA was the most sensitive biomarker for systemic inflammation, based on the receiver operating characteristic curve analysis (area under the curve = 0.959). In addition, cfDNA and CRP were significantly associated with inflammation and organ injury-related clinical parameters.Following the surgical removal of the inflamed uterus, interleukin-6 (IL-6), high-mobility group box 1 (HMGB1), and procalcitonin (PCT) significantly decreased, whereas changes in CRP, SAA, and cfDNA were not significant. These findings indicate that cfDNA, CRP, and SAA are potential clinical biomarkers of systemic inflammation in dogs with pyometra and PCT, IL-6, and HMGB1 are potential biomarkers of clinical recovery.     

Immunohistochemical and qPCR determination of the expression and serum level of anti‐Müllerian hormone in pre‐pubertal,intact and ovarian remnant syndrome detected bitches

The aim of this study was to determine the serum concentrations, ovarian presence and expression of anti‐Müllerian hormone (AMH) in pre‐pubertal, bitches with signs of ovarian remnant syndrome (ORS) and intact bitches. In addition, we aimed to verify the suitability of serum AMH concentrations for diagnostic purposes in sterilized bitches and/or in suspected cases of ORS in the field of veterinary medicine. For this purpose, 36 healthy female dogs divided into six groups: proestrus, oestrus, dioestrus, anoestrus, pre‐pubertal and ORS. Serum AMH concentrations were determined by electrochemiluminescence immunoassay, and ovarian presence and distribution of AMH was confirmed by immunohistochemical and qPCR techniques. According to the results of qPCR, while the expression values of AMH were at the highest concentrations in the proestrus and oestrus, there was a statistically significant decrease in these values at the later stages of the cycle (p < 0.05). According to hormone analysis, the serum AMH values of the ORS group had decreased significantly compared with the proestrus and oestrus (p < 0.05). Although serum AMH levels of ORS group were increased compared with anestrus and pre‐pubertal groups, this increase was statistically non‐significant (p > 0.05). Immunohistochemically, AMH expression was first observed in the granulosa cells of primordial follicles in folliculogenesis. Expression values were the highest in the proestrous and oestrus groups, but values from bitches in later stages of the cycle were statistically significant decrease in comparison with these groups (p < 0.05). As a result, AMH concentration and expression were found to be higher in proestrus and oestrus than in other periods (p < 0.05). In addition, the measurable level of AMH concentration in bitches with ORS is an indication that it can be used in the diagnosis of ORS.     

Blood lactate levels in 31 female dogs with pyometra

Background

Canine pyometra is a life-threatening disease common in countries where spaying of dogs is not routinely performed. The disease is associated with endotoxemia, sepsis, systemic inflammatory response syndrome (SIRS) and a 3–4% mortality rate. Blood lactate analysis is clinically valuable in predicting prognosis and survival, evaluating tissue perfusion and treatment response in human and veterinary critical care settings. The aims of the present study were to investigate 1) the blood lactate levels of female dogs with pyometra by a hand-held analyser and 2) if these levels are related with the clinical status or other biochemical or hematological disorders.

Methods

In total 31 female dogs with pyometra admitted for surgical ovariohysterectomy and 16 healthy female control dogs were included in the present study. A complete physical examination including SIRS-status determination was performed. Blood samples for lactate concentrations, hematological and biochemical parameters, acid-base and blood gas analysis and other laboratory parameters were collected and subsequently analysed. The diagnosis pyometra was verified with histopathological examination of the uterus and ovaries. Increased hospitalisation length and presence of SIRS were used as indicators of outcome.

Results

In the pyometra group the median blood lactate level was 1,6 mmol l^-1 (range <0.8–2.7 mmol l^-1). In the control group the median lactate level was 1,2 mmol l^-1 (range <0.8–2.1 mmol l^-1). Of the 31 bitches 19 (61%) fulfilled 2 or more criteria for SIRS at inclusion, 10 bitches (32%) fulfilled 3 of the SIRS criteria whereas none accomplished more than 3 criteria. Lactate levels did not differ significantly between the pyometra and control group, or between the SIRS positive and SIRS negative dogs with pyometra. Increased lactate concentration (>2.5 mmol l^-1) was demonstrated in one female dog with pyometra (3%), and was not associated with longer hospitalisation or presence of SIRS. Lactate measurement was not indicative of peritonitis. None of the bitches died during or within two months of the hospital stay. The measurements of temperature, heart rate, respiratory rate, percentage bandforms of neutrophilic granulocytes, α₂-globulins, creatinin, pvCO₂, TCO₂ and base excess showed significant differences between the SIRS positive and the SIRS negative pyometra cases.

Conclusion

Increased blood lactate concentrations were demonstrated in 3% (1/31), and SIRS was present in 61% (19/31) of the female dogs with pyometra. Preoperative lactate levels were not related with presence of SIRS or prolonged hospitalisation. Lactate measurement was not indicative of peritonitis. The value of a single and repeated lactate analysis in more severely affected cases remains to be determined.     

The Concentrations of Inflammatory Cytokines and Acute‐Phase Proteins in the Peripheral Blood and Uterine Washings in Cows with Pyometra

The development of pyometra in cows depends largely on the state of local immunity of the uterus. The objective of the study was to evaluate the concentration of the following proinflammatory cytokines: tumour necrosis factor (TNF‐α) and interleukin‐6 (IL‐6); anti‐inflammatory cytokine: interleukin‐10 (IL‐10); and acute‐phase proteins (APPs): haptoglobin (Hp) and serum amyloid A (SAA), in serum and uterine washings in cows with pyometra and healthy animals. The study was performed on 20 cows divided into two groups based on the results of cytological and ultrasonographic tests: a pyometra and a healthy group (10 cows per group). Experimental material consisted of blood serum and uterine washings. The levels of the following cytokines, TNF‐α, IL‐6, IL‐10 and APPs – Hp and SAA, in the study material were determined by ELISA. The results showed that the values of TNF‐α, IL‐6, IL‐10 as well as SAA and Hp were significantly higher in serum of cows with pyometra compared to controls (p < 0.001). The uterine washings had significantly higher levels of IL‐6, IL‐10, and Hp in pyometra cows compared to the control (p < 0.001). Our results indicate that it is possible to monitor the course of pyometra in cows based on the evaluation of the concentration of cytokines and Hp in the serum and uterine washings. Simultaneous evaluation of selected indicators of antagonistic interaction can be helpful in determining the current status of local immunity of the uterus. On this basis, it could be possible to properly select an adjunctive therapy in the form of immunomodulating preparations.     

Evaluation of kidney injury in dogs with pyometra based on proteinuria, renal histomorphology, and urinary biomarkers

Background: Proteinuria is a feature of pyometra‐associated renal dysfunction, but its prevalence and clinical relevance are not well characterized. Objectives: To define which subset of dogs with pyometra has clinically relevant kidney injury by quantification of proteinuria; light, immunofluorescence, and electron microscopic examination of kidney biopsy specimens; and measurement of urinary biomarkers. Animals: Forty‐seven dogs with pyometra. Ten clinically healthy intact bitches of comparable age. Methods: Prospective study. Routine clinicopathological variables including urinary protein to creatinine ratio (UPC) were analyzed. Validated assays were used to quantify urinary biomarkers for glomerular (urinary albumin, urinary immunoglobulin G, urinary C‐reactive protein, urinary thromboxane B₂) and tubular function (urinary retinol‐binding protein, urinary N‐acetyl‐β‐d ‐glucosaminidase). Kidney biopsy specimens from 10 dogs with pyometra and dipstick urine protein concentrations of 2+ or 3+ were collected during ovariohysterectomy. Urinalysis was repeated within 3 weeks after surgery in 9 of the 10 dogs. Results: UPC (median, range) was significantly higher in dogs with pyometra (0.48, 0.05–8.69) compared with healthy bitches (0.08, 0.02–0.16) (P < .01). Twenty‐two of 47 dogs with pyometra had UPC>0.5, 12 had UPC>1.0, and 7 had UPC>2.0. Glomerulosclerosis and tubulointerstitial nephritis were common kidney biopsy findings in proteinuric dogs with pyometra. Dogs with glomerulosclerosis (5/10), either global or focal and segmental, had UPC>1.0 at ovariohysterectomy and afterward. Dogs with structural glomerular and tubular changes mostly had urinary biomarker to creatinine ratios above the 75th percentile. Conclusion: Dogs with pyometra and UPC>1.0 or high ratios of urinary biomarkers appear likely to have clinically relevant renal histologic lesions and require monitoring after ovariohysterectomy. Future studies should evaluate the role of pyometra‐associated pathogenic mechanisms in causing or exacerbating focal and segmental glomerulosclerosis in dogs.     

Assessment of three automated assays for C-reactive protein determination in dogs

OBJECTIVE: To determine the characteristics of an automated canine C-reactive protein (CRP) assay and evaluate 2 human CRP assays for use in dogs. Animals-56 client-owned dogs with pyometra and 11 healthy control dogs. PROCEDURES: Samples from 11 dogs with high (> 100 mg/L) or low (< 10 mg/L) CRP concentrations (determined by use of a canine ELISA) were evaluated by use of the automated canine CRP assay. Intra- and interassay imprecision was determined (by use of those 2 plasma pools), and assay inaccuracy was assessed by use of logistic regression analysis of results obtained via ELISA and the automated canine CRP assay. Two automated human CRP assays were used to measure plasma CRP concentration in 10 dogs. RESULTS: By use of the ELISA, mean +/- SD plasma CRP concentration was 96.1 +/- 38.5 mg/L and 10.1 +/- 23.2 mg/L in dogs with pyometra and control dogs, respectively. The automated canine assay had intra-assay coefficients of variation (CVs) of 7.8% and 7.9%, respectively, and interassay CVs of 11.1% and 13.1%, respectively. Results from the automated assay were highly correlated with results obtained via ELISA. The human assay results did not exceed 0.4 mg/L in any dog. CONCLUSIONS AND CLINICAL RELEVANCE: The automated canine CRP assay had less interassay imprecision, compared with the ELISA. The 2 human CRP assays were not suitable for analysis of canine plasma samples. The automated canine CRP assay was more precise than the ELISA for serial evaluations of plasma CRP concentration in dogs.     

Does serum anti‐Müllerian hormone levels always discriminate presence of the ovaries in adult bitches? Comparison of two ELISA kits

Anti‐Müllerian hormone (AMH) is produced in the ovary, and thus, it is an excellent marker of follicle pool in females. Current interest is the clinical use of this parameter as a biomarker to assess presence or absence of an intact ovary and to diagnose ovarian remnant syndrome (ORS) following incomplete ovariohysterectomy (OHE) in bitches. The aim of this study was to evaluate serum AMH concentrations in bitches (n = 34) before and after OHE using two different commercial ELISA kits, one of which is based on detecting human AMH and the other is based on detecting human AMH and the other specified for canine AMH. Furthermore, serum AMH levels were also measured in six ORS cases to compare the diagnostic utility of the two different ELISA kits. Serum AMH concentrations measured using the human and canine kit prior to and after OHE were 0.32 ± 0.24, 0.006 ± 0.22 ng/ml (p < .001) and 12.08 ± 22.81, 9.55 ± 15.42 ng/ml (p = .868), respectively. Thus, the canine‐based kit was not able to reveal the significant drop in serum AMH levels. In conclusion, the human‐based ELISA kits successfully detected the drop in serum AMH concentrations. Reliable results can only be achieved from well‐designed ELISA kits, and AMH levels might be a useful diagnostic tool for the evaluation of presence or absence of ovaries as well as for the detection of ORS cases in bitches.     

Is Mechanically Induced Cystic Endometrial Hyperplasia (CEH) a Suitable Model for Study of Spontaneously Occurring CEH in the Uterus of the Bitch?

Cystic endometrial hyperplasia (CEH) was experimentally reproduced by the intraluminal insertion of a silk wire in the uterus of metestral bitches to obtain a model for the study of spontaneously occurring CEH in the bitch. This mechanically reproduced CEH corresponded histologically very well to spontaneously occurring CEH. With respect to sex hormone receptor expressions, however, there was no similarity. In the mechanically induced CEH, progesterone receptor expression was reduced in the epithelial cells (surface epithelium and endometrial glands) and slightly increased in the stromal fibroblasts and myometrium as compared with the normal metestrus uterus. The oestrogen receptor expression in mechanically induced CEH was reduced in the epithelial cells (surface epithelium and endometrial glands) and more or less unchanged in the stromal fibroblast and myometrium as compared with the normal metestrus uterus. This is in contrast to the increased sex hormone receptor expressions in all uterine cell types, observed in spontaneously occurring CEH. The mechanically induced CEH model corresponds immunohistochemically more to early placentation and to pyometra in the bitch. Thus this experimental CEH model is not suitable for pathogenic studies of spontaneously occurring CEH in the bitch.     

Hysterectomy leads to fast improvement of haematological and immunological parameters in bitches with pyometra

OBJECTIVES: To investigate whether a combination of hysterectomy and antibiotic treatment leads to an improvement of altered haematological and immunological parameters in bitches affected by pyometra. METHODS: Blood samples obtained from 13 bitches affected by pyometra were investigated before hysterectomy and seven days later for a total and differential leucocyte count, activity of neutrophils and lymphocytes, and quantification of total serum immunoglobulins, lysozyme and circulating immune complexes. RESULTS: The parameters most affected included changes in blood profile (leucocytosis because of neutrophilia or leucopenia) and inhibition of lymphocyte activity. Seven days after hysterectomy, all affected parameters returned to normal levels comparable to clinically healthy dogs. CLINICAL SIGNIFICANCE: Application of immunostimulating therapy is not necessary to manage changes in blood cell counts and suppression of lymphocyte activity in bitches with pyometra. Removal of the infected uterus, being the source of infection, leads to improvement.     

Molecular studies on oestrogen α and progesterone receptors and histomorphometric analysis of canine uteri following aglepristone treatment

Aglepristone, a competitive progesterone antagonist, is successfully used in various progesterone-dependent conditions. This study investigated uterine histomorphometric analysis, and expressions of the oestrogen α receptor (ERα) and progesterone receptor (PR) in uteri of bitches following the single dose of aglepristone treatment. Twelve client-owned healthy diestrous bitches were used in the study. The single dose of aglepristone (Alizine^®, 10 mg/kg) was injected subcutaneously 5 days before ovariohysterectomy in the treatment group (n = 6); bitches without treatment served as a control group (n = 6). Uteri were collected for histomorphometric analysis, ERα and PR gene, and protein expressions studies. The mRNA expressions of ERα and PR were determined by RT-qPCR. Immunohistochemical analysis was used to evaluate the ERα and PR protein expressions using an H-score in five parts of the uterus. The results demonstrated glandular epithelium height significantly decreased (p < .05) and ERα mRNA increased (p < .01) in treated dogs. Of the treated bitches, lower expression levels of ERα were observed in the luminal epithelium, crypt and glandular epithelium, with higher expression in the endometrial stroma and myometrium (p < .05); however, PR expression decreased in the luminal epithelium, crypt and glandular epithelium (p < .01). In conclusion, reduction of the uterine glandular epithelium and ERα mRNA upregulation together with changes in ERα and PR expressions were observed in the treated bitches. However, changes in uterine ERα and PR expressions in the treated bitches depended on tissue layers. The treatment had no effect on serum oestradiol and progesterone levels.     

Prostaglandin F2 alpha treatment of canine pyometra

Immediate and long-term outcomes of prostaglandin F2 alpha treatment for canine pyometra were studied in 10 bitches. Examination of pretreatment uterine biopsy specimens, taken for histopathologic diagnosis and classification of disease severity, revealed either type III or IV pyometra. Dinoprost tromethamine (0.25 or 0.5 mg/kg of body weight, SC) was given once daily for 3 days. Bitches were bred at the first posttreatment estrus and monitored for a minimum of one year. When pure cultures of Escherichia coli (n = 3) or Staphylococcus aureus (n = 1) were obtained from the vagina, these bacteria also were found in the uterus. Pretreatment WBC counts often did not reflect the severity of histopathologic findings in the uterus, but posttreatment WBC counts were useful in monitoring response to treatment. Four bitches produced a litter within one year of treatment. Four bitches (40%) had recurrence of pyometra within one year of treatment, and these same bitches had another recurrence after an additional prostaglandin treatment. Three additional bitches had a recurrence by 27 months after therapy, establishing a total recurrence rate of 77% (7/9). Results suggested that subclinical disease may persist after treatment, with clinical recurrence during diestrus. Despite the high recurrence rate, it was concluded that this treatment is a practical treatment for canine pyometra when reproduction is desired.     

Ultrasonography and Cystic Hyperplasia–Pyometra Complex in the Bitch

Cystic endometrial hyperplasia-pyometra complex is the most frequent and important endometrial disorder encountered in bitches. The pathogenesis of the disease is related to the activity of progesterone [Feldman and Nelson, Canine and Feline Endocrinology and Reproduction (1996) W.B. Saunders, Philadelphia]. Cystic endometrial hyperplasia (CEH) is an abnormal response of the bitch's uterus to ovarian hormones [De Bosschere et al. Theriogenology (2001) 55, 1509]. CEH is considered by many authors to be an exaggerated response of the uterus to chronic progestational stimulation during the luteal phase of the oestrous cycle, causing an abnormal accumulation of fluid within the endometrial glands and uterine lumen (De Bosschere et al. 2001). The resulting lesions of pyometra are due to the interaction between bacteria and hormones. The aim of this study was to evaluate if transabdominal uterine ultrasonography can be a useful and reliable diagnostic method to confirm Dow's [Veterinary Record (1958) 70, 1102] and De Bosschere's histopathological classification of CEH-pyometra complex. The study was carried out on 45 bitches with pyometra, 10 purebreds and 35 crossbreeds, 1-15 years old, 20% of which had whelped at least once. None of these animals had received exogenous oestrogen or progesterone treatment. On admission the 45 animals were in the luteal phase of the oestrus cycle. Clinical signs, blood parameters, uterine ultrasonography, bacterial swabs and uterine histopathological results were recorded. Results suggest that ultrasonographic examination is a useful and reliable tool for the diagnosis of cystic endometrial hyperplasia.     

Measurement of serum interleukin-10 in the dog

The objective was to evaluate independently the reliability of a commercially available canine serum interleukin-10 (IL-10) enzyme-linked immunoassay (ELISA) and to investigate canine serum IL-10 concentrations in healthy dogs, in dogs with a naturally-occurring acute phase reaction and in dogs following surgical stimulus by assessing intra- and interassay imprecision, inaccuracy and detection limits. Median (and range) serum IL-10 concentrations (ng/L) in the various groups were as follows: healthy dogs (n=15), 18.9 (11.2-71.5); dogs with pyometra (n=9), 37.9 (12.4-201.8); dogs with angiostrongylosis (n=8), 20.29 (14.3-108.7) and values in dogs following surgical stimulus (n=15), 14.8 (10.7-65.8). The assay measured canine serum IL-10 reliably (intra- and interassay imprecision 4.9-8.3% and 9.9-10.9%, respectively; detection limit 10.7 ng/L with no significant inaccuracy). No significant increases in IL-10 were observed following surgical stimulus and no difference in IL-10 was observed between the diagnostic groups. IL-10 values showed a higher degree of variation in dogs with an inflammatory response, i.e. those with elevated serum C-reactive protein (CRP) concentrations, compared to healthy dogs. As anticipated, healthy dogs had low levels of both analytes, whereas dogs with an acute phase response had IL-10 levels with no clear relationship to CRP concentrations, with observed low IL-10 values even when there was a marked inflammatory response.     

Investigation of the usability of kisspeptin and oxidative stress parameters in the early diagnosis of asymptomatic cystic endometrial hyperplasia in dogs

This study aimed to investigate the differences in oxidative stress index (OSI) and kisspeptin levels in clinically asymptomatic dogs with cystic endometrial hyperplasia (CEH) compared to healthy and pregnant dogs, and to determine the usability of the obtained results in the diagnosis of asymptomatic CEH. The study comprised three groups; a healthy (n = 8), a pregnant (n = 10) and a CEH (n = 10). All dogs in the three groups were included in the study at the 30 ± 3th day after estrus, and blood samples were collected for analysis of kisspeptin, total antioxidant status (TAS), total oxidant status (TOS), progesterone (P4), estradiol (E2) and some biochemical parameters (TSH; thyroid stimulating hormone, ALT; alanine aminotransferase, AST; aspartate aminotransferase, ALP; alkaline phosphatase, LDH; lactate dehydrogenase, CRE; creatine and BUN; blood urea nitrogen). In addition, OSI value was calculated. P4 and ALT and BUN levels were significantly lower and higher in CEH group than the pregnant group, respectively (p < .05). While kisspeptin and TAS levels were significantly lower in CEH group compared to the healthy and pregnant groups (p < .01), OSI level increased dramatically. In conclusion, it was confirmed that CEH clearly affected kisspeptin and OSI levels, and it is thought that these parameters may be an alternative diagnostic tool for the detection of CEH after further studies.     

Plasma concentrations of folic acid, vitamin B12 and progesterone of cyclic bitches, bitches during pregnancy and induced abortion and bitches with pyometra

The aim of this study was to investigate the plasma concentrations of folic acid, vitamin B12 and progesterone at different stages of the sexual cycle and pregnancy, during induced abortion and in bitches with pyometra. Bitches (n = 97) were assigned to groups as follows: a) oestrous cycle (n = 42) b) pregnancy (n = 25) c) induction of abortion (n = 10 and d) pyometra (n = 20). Oestrous cycle stages were determined by vaginal inspection and cytology. Pregnancies were estimated by ultrasound (5.0 Mhz; linear transducer; Schimadzu) at days 15-25, 35-45 and 46-63 of pregnancy. Treatments for the induction of abortion were started between days 25 and 35 after mating (5 microg/kg cabergoline daily, Galastop; 5-10 microg/kg Alfaprostol every other day, Gabbrostim). Diagnosis of pyometra was confirmed by ultrasound and vaginoscopy. Folic acid and vitamin B12 concentrations did not differ among different stages of the oestrous cycle. The mean concentration of folic acid during early pregnancy (days 15-25) exceeded levels of later stages (days 46-63): 9.4 +/- 3.7 microg/ml and 4.7 +/- 1.8 microg/ml, respectively (p < 0.01). A positive correlation between folic acid and vitamin B12 was determined in pregnant dogs ( r = 0.925; p < 0.02). Before the induction of abortion, the concentration of folic acid was 9.6 +/- 5.2 microg/ml; during abortion it decreased to 5.0 +/- 3.2 microg/ml (p < 0.01). A significant correlation (r = 0.925; p < 0.02) between progesterone and folic acid was obtained in bitches with abortion. The mean concentration of folic acid in bitches with pyometra significantly differed from that of bitches at different stages of the oestrous cycle (p < 0.05). The mean concentration of folic acid was significantly lower in metoestrous bitches when compared to bitches with pyometra (p < 0.05). The decrease of serum concentrations of folic acid during pregnancy and induced abortion show that fetal growth and abortion caused higher consumption of folic acid. Concerning bitches did not show any deficiency symptoms, which is why it can be concluded that this decrease is physiological.