Relationship between estrus endometrial edema and progesterone production in pregnant mares two weeks after ovulation.

BACKGROUND: Progesterone plays a crucial role in the maintenance of pregnancy from conception to about 100-120 days of gestation when placenta becomes the main source of gestagens. The aim of the study was to test progesterone concentration 14 days after ovulation in pregnant mares and relate it to peak estral endometrial edema and the presence of intrauterine fluid (IUF) after artificial insemination (AI), the number of treatments against IUF, and the time from AI to the day when the uterus was found free of fluid. RESULTS: Mares were divided into two groups: group A (n = 13; age 10.8 ± 4.5 years) in which a normal embryonic vesicle with a diameter ≥ 14 mm and a corpus luteum with a diameter ≥ 15 mm were found 14 days after ovulation, and group B (n = 22; age 9.4 ± 4 .0 years) in which 14 days after ovulation, a small (< 15 mm) corpus luteum and/or a small embryonic vesicle was observed (diameter < 14 mm). Mares from group A had a significantly higher progesterone concentrations at 14 days after ovulation compared with group B mares. The presence of IUF, the number of treatments against IUF, and the time from AI to the day when uterus was found free of fluid did not affect progesterone concentration measured 14 days after ovulation. In group B, a significant correlation was found between progesterone concentration measured 14 days after ovulation and endometrial edema evaluated during estrus. CONCLUSIONS: In some cases poor development of endometrial edema during estrus can be associated with lower progesterone production 14 days after ovulation. Nevertheless, scientific explanation for this finding cannot be given based on our study.

Changes in the Secretion of Anti-Inflammatory Cytokines and Acute-Phase Proteins in the Uterus after Artificial Insemination in the Mare.

The objective of the study was to evaluate the concentrations of interleukin-1 receptor antagonist (IL-1RA), interleukin-10 (IL-10), serum amyloid A (SAA) and haptoglobin (Hp) in uterine lavage fluid before and after artificial insemination (AI). Based on ultrasound examination, mares were divided into: Group 1 (n = 9), no fluid was detected in the uterus during estrus and 7 h after AI; Group 2 (n = 8), no fluid was detected in the uterus during estrus but 7 h after AI fluid was detected in the uterus; Group 3 (n = 8), fluid was detected in the uterus during estrus and also 7 h after AI. In all groups of mares, a significant increase in polymorphonuclear cells (PMN) and a significant increase in IL-1RA and SAA were recorded 7 h after AI. The obtained results show that, regardless of the status of the mare before AI, the endometrial response characterized by PMN influx, and SAA, Hp, IL-1RA and IL-10 production, is similar. The presence of intrauterine fluid during estrus is not connected with PMN influx but can impact uterine IL-1RA production at this time.

The Effectiveness of Pharmacological Synchronization of the Estrous Cycle in Hinds (Cervus elaphus L.): A Pilot Field Trial.

The aim was to estimate the effective pharmacological method of the estrous cycle synchronization by checking the effects of synchronization by measurement of progesterone (P4) and 17-beta estradiol (E2) concentration by RIA and artificial insemination. The experiment was performed at the red deer farm in Rudzie (North-East Poland; 3 year's old). The herd (N = 14) was kept away from bulls and was divided in two groups of seven animals. In the Group I, CIDR insert (0.3 g of P4) was applicated intravaginally for 12 days; a second insert replaced the first one for the next 12 days, and next 200 IU of equine chorionic gonadotropin (eCG) was injected intramuscularly (Folligon). Estrus was expected 48 h after eCG injection. In the Group II, Chronogest sponge (20 mg of flugestone acetate) was applicated intravaginally and after 7 days replaced with second chronogest sponge for 7 days. After removing the sponge, on the same day eCG was injected and estrus was expected after 48 h. Artificial insemination was provided with frozen-thawed semen twice: 12 and 24 h after expected estrus. The peripheral blood from the jugular vein was collected each time when the inserts or sponge were applicated and 40 days after insemination. The concentration of P4 and E2 in plasma was measured by RIA. The effectiveness of insemination was monitored by pregnancy-associated glycoproteins determination and observed by the number of calves born. Two pregnancies were confirmed in Group I and five in Group II based on PAG concentration. One newborn was observed in Group I and five in Group II. Both methods of synchronization are effective in hinds based on the received profile of steroids. Although the sponge shape in case of chronogest is better comparing with CIDR, which was not completely deposited in the vagina of hind, potentially leads to bacteria inflammation, and it disturbs the rightful endocrine regulation. Moreover, pregnancy rate and hormone responsiveness were better in Group II.

Involvement of hemostasis in pathophysiology of RAO in horses.

Recurrent Airway Obstruction (RAO), also called severe asthma or heaves, is a chronic disease in adult horses caused by aeroallergens from straw or hay. Disturbances in hemostasis (intensified coagulation and depressed fibrinolysis) are considered one of the prominent reasons of inflammatory process, injury and dysfunction of the lungs. The aim of the study was to evaluate chosen parameters of hemostasis in horses with active form of RAO. Ten RAO-horses (group R) and ten healthy horses (group C) were exposed to straw and hay allergen challenge. The prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), fibrinogen concentration (Fb), stabilized fibrin degradation product (d-dimer), antithrombin (AT), protein C and coagulation factors II through XII were assessed in plasma obtained from blood of all the horses. Exposure to aeroallergens resulted in prolongation of aPTT in both groups of animals; it was evident in the group R and moderate in the group C. There were no differences in PT and TT. Concentrations of fibrinogen and d-dimer and activity of protein C in both groups were increased but lay within or near to reference values. The activity of AT was depressed in RAO-horses. All exposed horses showed increased activity of coagulation factors II, VIII and X but they had no changes in activity of factor V. Factors VII and XII displayed a reduction in activity. The decrease in factor IX activity was noted in the group C only. Various changes were observed in activity of factor XI; in horses with RAO it was elevated but in healthy horses it was declined. The changes of the parameters tested in RAO-horses indicate the involvement of coagulation and fibrinolysis which apparently remained under control of efficient and active mechanisms of general hemostasis.

Evidence for Increased Content of PGF2α, PGE2, and 6-keto-PGF1α in Endometrial Tissue Cultures From Heavy Draft Mares in Anestrus With Endometritis.

Endometritis is one of the most important causes of infertility in mares. Mares may suffer from endometritis outside the breeding season; however, pathological condition is often undiagnosed in anestrus. The aim of this study was to examine whether the secretion profiles of prostaglandin F2α (PGF2α), prostaglandin E2 (PGE2), and a metabolite of prostacyclin I2 (6-keto-PGF1α) differ in endometrial tissue cultures of heavy draft mares in anestrus with endometritis compared to those without endometritis. The endometrial biopsies were collected from 51 heavy draft mares. Inclusion criteria for the control group were absence of endometritis confirmed by histology and no ovarian activity. Inclusion criteria for the experimental group were presence of endometritis showing polymorphonuclear cells and/or lymphocytes infiltration in endometrium and no ovarian activity. Retrospectively, 22 mares were enrolled in this study. The content of PGF2α (P < .05) and PGE2 (P < .001) in the culture medium was distinctly elevated in mares suffering from endometritis, compared to control mares. The relative mRNA abundance responsible for prostaglandins synthesis, that is, PGF2α synthase (PGFS; P < .01), PGE2 synthase (PGES; P < .01), and prostaglandin-endoperoxide synthase (PTGS2; P < .01), were also increased in endometrial tissue of mares with endometritis compared to control mares. The content of 6-keto-PGF1α (P < .0001) in endometrial tissue cultures from mares with endometritis was strikingly elevated compared to those without endometritis; however, plasma concentration of 6-keto-PGF1α was not significantly different between experimental and control groups. This leads to the conclusion that augmented endometrial secretion of PGF2α, PGE2, and 6-keto-PGF1α is associated with endometritis even in mares in anestrus.

A Survey on West Nile and Usutu Viruses in Horses and Birds in Poland.

West Nile virus (WNV) and Usutu virus (USUV) are members of the family Flaviviridae which, natural life cycles involve mosquito-bird-mosquito transmission. Both represent emerging viruses in Europe with potential to cause neuroinvasive disease in humans. This study investigates the seroprevalence of serum neutralizing antibodies to WNV and to USUV in birds and in horses in Poland. Antibodies against WNV and USUV were detected in 5 (35.7%) and in 1 (7.14%) of 14 birds and in 62 (15.08%) and in 115 (27.98%) of 411 horses, respectively. Twenty-one WNV serologically positive horses (33.87%) and 67 USUV serologically positive horses (58.26%) did not travel outside Polish borders. Given the high abundance of potentially competent mosquito species in Poland, high populations of horses and different bird species, our findings highlight implementation of active control programs, including monitoring of geographic spread and dynamics of WNV and USUV transmission in both primary and accidental hosts. It is also important to improve public health awareness about the disease these viruses may cause.

Reliability of histopathological examination and immunohistochemistry of a single biopsy for evaluation of endometrial health in Icelandic mares.

INTRODUCTION: Endometritis and endometrosis have been and still are the major reasons for infertility in mares. The diagnosis of endometritis can be based on cytology and microbiology, but endometrial biopsy is still the only way to diagnose endometrosis in the mare. Our study attempted to determine if a single biopsy using his-topathology and immunohistochemistry is sufficient to ascertain reasons for infertility in Icelandic mares. The objectives of this study were to examine the relationship between deviations in endometrial biopsies in terms of prostaglandin-endoperoxide synthase-2 (PTGS-2) and fibronectin expression and polymorphonuclear cells (PMNs) infiltration, as well as scoring degeneration in two endometrial biopsies. MATERIAL AND METHODS: Materials were collected from 53 Icelandic breed mares, from whom two endometrial biopsies were collected and they were used for histopathology and for immunohistochemistry for PTGS-2 and fibronectin. RESULTS: In our study, twenty-six of 53 mares (49%) showed differences in the biopsy score between the left and the right uterine horns (p = 0.002). There were statistically significant differences in fibronectin expression (p = 0.001), as well as in PTGS-2 expression in the superficial epithelium (p = 0.017). CONCLUSIONS: Significant differences in the biopsy score, and fibronectin and PTGS-2 expression, between two endometrial biopsies obtained from individual mares demonstrated that a single biopsy could be insufficient for diagnosing uterine health status in Icelandic mares.

Advanced age in mares affects endometrial secretion of arachidonic acid metabolites during equine subclinical endometritis.

Even if mares continue to breed up to an advanced age, in aging mares reproductive failure is quite common. Subclinical endometritis, which occurs more often in aging mares than in younger counterparts, may cause prolongation or shortening of the inter-estrus period or the corpus luteum lifespan. We hypothesized that during subclinical endometritis the secretion of selected arachidonic acid metabolites may differ in aging mares compared to younger females. To verify this thesis, ex vivo organ cultures of endometrium were established with subsequent measurements of concentrations of prostaglandin E2 (PGE2), 6-keto-PGF1α and both leukotrienes (LTs), LTB4 and LTC4 in the culture supernatants. The endometrial biopsies were obtained from 82 mares of known breeding history. This study revealed that the concentrations of the selected arachidonic acid metabolites, which act both as immunological mediators and endocrine modulators in the reproductive organs, depends on the mares' ages. Spontaneous endometrial secretion of PGE2, 6-keto-PGF1α and LTC4 was increased in mares aged 16-23 years that suffered from subclinical endometritis, compared with control counterparts. Moreover, secretion of these metabolites was higher in endometritis-positive mares aged 16-23 years than in younger females. We conclude that advanced age in mares further disturbs the immuno-endocrine balance in endometritis-positive mares.

The usefulness of uterine lavage and acute phase protein levels as a diagnostic tool for subclinical endometritis in Icelandic mares.

BACKGROUND: Endometritis is a common problem in a broodmare practice, often leading to infertility. The diagnosis is based on several methods such as cytology, bacteriology and histopathology; however the outcome of these methods may be inconclusive even when used together. The objectives of this study were: (1) to investigate the usefulness of acute phase proteins as an additional diagnostic tool for diagnosis of subclinical endometritis in mares and (2) to evaluate the association between macroscopic changes in uterine flushes and inflammation of the uterus. Materials were collected from 53 Icelandic mares with subclinical endometritis. Endometrial swabs and uterine lavage for cytological and bacteriological examinations and two endometrial biopsies were taken. Blood samples were collected 12-24 h after ovulation to determine the concentrations of serum amyloid A and haptoglobin in the 53 subfertile mares and, for comparison, from 20 non-pregnant mares that later conceived. RESULTS: Twenty-five mares were classified as positive for endometritis based on endometrial biopsy, which was used as the 'gold standard'. We observed a correlation between cloudy efflux in the lavage and (1) polymorphonuclear cell (PMN) infiltration of the endometrium (P = 0.031), (2) positive cytology in samples obtained by cotton swabs (P = 0.019) and uterine lavage (P = 0.011), and (3) positive microbiology from samples obtained by cotton swabs (P = 0.001) and uterine lavage (P = 0.047). The degree of agreement between PMN infiltration and positive cytology from samples taken by cotton swabs and uterine lavage was fair to moderate. We found no association between the concentration of acute phase proteins and infiltration of the endometrium by PMNs, or with positive results of cytological and microbiological examinations. CONCLUSIONS: Measurement of serum amyloid A and haptoglobin was not proven useful for diagnosis of subclinical endometritis in Icelandic mares. Macroscopic changes in the fluid collected by lavage were not consistently indicative of infection, but when present they indicate inflammation in the uterus with a high probability.

Type of Inflammation Differentially Affects Expression of Interleukin 1ß and 6, Tumor Necrosis Factor-α and Toll-Like Receptors in Subclinical Endometritis in Mares.

Mares that fail to conceive or lose their embryos, without showing typical signs of clinical endometritis, should be suspected of subclinical endometritis (SE). In this study, the question was addressed: does SE fully activate selected mechanisms of innate immunity in mares? For this aim, expression of mRNAs for Toll-like Receptor 2 and 4 (TLR 2/4), interleukin 1ß (IL-1ß), interleukin 6 (IL-6) and tumor necrosis factor α (TNF) was examined in control mares versus either mares suffering from chronic endometritis (ChE) or subacute suppurative endometritis (SSE). The concentrations of IL-1ß, IL-6 and TNF-α in supernatants from endometrial tissue cultures after 4 h incubation were measured using the enzyme immunoassay (EIA) method. Eighty-two warmblood mares, of known breeding history, were enrolled in this study. Based on histopathological assessment, mares were classified as suffering from ChE, SSE or as being healthy. In addition, immuno-localization of both TLR2 and TLR4 as well as TNF-α was investigated in the equine endometria. The mRNA expression of TLR2 (P < 0.01), IL-1ß (P < 0.0001), IL-6 (P < 0.0001) and TLR4 and TNF (P < 0.05) was up-regulated in endometria of mares suffering from SSE compared with unaffected mares. Concentrations of IL-6 and TNF-α were increased only in mares exhibiting SSE, compared with unaffected (P < 0.01 for both) and ChE mares (P < 0.05 for both). Immuno-localization of TNF-α and TLRs was confirmed, both in unaffected and SE-affected endometria, and was present in the luminal and glandular epithelia and stromal cells. The severity of inflammation impacts the immune response and fosters activation of innate immunity mechanisms, as observed in the endometria of mares. The intracellular localization of TLRs and TNF-α in the endometria indicates a key role of endometrial epithelial and stromal cells in the immune response and inflammation.

Relationship between uterine biopsy score, endometrial infection and inflammation in the mare.

OBJECTIVE: Endometrial biopsy score is an accepted marker of uterine health and predicted fertility, and it has been suggested that endometrial alternations are correlated with susceptibility to persistent infectious endometritis. The objective of this study was to investigate associations of endometrial biopsy score with: 1) presence of polymorphonuclear cells (PMNs) in the epithelium and stratum compactum in histopathology; 2) presence of PMNs in cytology and 3) presence of infection in microbiology. MATERIALS AND METHODS: The material for examination was collected from 69 mares suspected for subclinical endometritis (bred three or more times unsuccessfully in the same breeding season) and from 15 maiden mares. Samples were collected by endometrial biopsy and cytobrush technique. RESULTS: Endometrial alterations (biopsy score IIA, IIB, III) were found in 64 of 82 mares (78%). There was an increase in PMN occurrence for grades IIA, IIB and III. When comparing grades and PMNs infiltration, we observed statistically significant differences between grades I and IIA (p  = 0.222) and grades I and IIB (p = 0.042) in samples collected by endometrial biopsy. Statistically significant differences were found in microbiological examination (biopsy p = 0.036; cytobrush p = 0.189), cytological examination (biopsy p = 0.040; cytobrush p = 0.079) and PMN infiltration (p    =    0.042) between mares with biopsy scores I and IIB. Furthermore, the highest percentage of infected mares was in grade IIA and IIB, and we found statistically significant differences between grades I and IIA (p = 0.043), and grades I and IIB (p = 0.036) in biopsy samples. We observed a tendency to higher prevalence of endometrial infection in mares with biopsy score IIA, IIB and III than with biopsy score I in samples collected using cytobrush technique. However, there were no statistical significant differences. CONCLUSION: Degenerative endometrial changes can predispose to uterine infection and inflammation. Our study shows that mares with endometrial score I are less predisposed to infection than mares with category IIA, IIB and III. Endometrial biopsy is a reliable diagnostic tool.

Altered secretion of selected arachidonic acid metabolites during subclinical endometritis relative to estrous cycle stage and grade of fibrosis in mares.

Mares that fail to become pregnant after repeated breeding, without showing typical signs of clinical endometritis, should be suspected of subclinical endometritis (SE). Contact with infectious agents results in altered synthesis and secretion of inflammatory mediators, including cytokines and arachidonic acid metabolites, and disturbs endometrial functional balance. To address the hypothesis that SE affects the immune endocrine status of the equine endometrium, spontaneous secretion of prostaglandin E(2) (PGE(2)), prostaglandin F(2α) (PGF(2α)), 6-keto-PGF(1α )(a metabolite of prostacyclin I(2)), leukotriene B(4) (LTB(4)), and leukotriene C(4) (LTC(4)) was examined. In addition, secretion of these factors was examined relative to the grade of inflammation, fibrosis, and estrous cycle stage. Eighty-two warmblood mares, of known breeding history, were enrolled in this study. On the basis of histopathologic assessment, mares were classified as suffering from first-grade SE, second-grade SE, or being healthy. The grade of fibrosis and the infiltration of endometrial tissue with polymorphonuclear leukocytes were examined by routine hematoxylin-eosin staining. In mares suffering from SE, the secretion profiles of PGE(2), 6-keto-PGF(1α), LTB(4), and LTC(4) were changed compared to mares that did not suffer from endometritis. The secretion of PGE(2) and 6-keto-PGF1α was increased, whereas that of LTB(4) and LTC(4) was decreased. Secretion of 6-keto-PGF(1α) was increased in first- and second-grade SE (P < 0.01). The concentration of PGI(2) metabolite was increased only in inflamed endometrium, independently of the inflammation grade, but was not affected by fibrosis. Prostaglandin E(2) secretion was increased in second-grade SE (P < 0.05). The secretion of LTB(4) decreased in both first- and second-grade SE (P < 0.05), whereas secretion of LTC(4) was decreased only in second-grade SE (P < 0.05). Fibrosis did not change the secretion profile of PGE(2), PGF(2α), and 6-keto-PGF(1α) during the course of SE. However, the secretion profile of LTB(4) was affected during the course of fibrosis. Evident divergences between PGE(2) and PGF(2α) profiles and in PGE2:PGF(2α) ratios in the control versus SE mares observed during the course of diestrus contribute to shortened or prolonged interestrous intervals observed clinically in SE mares.

Effects of cycle stage and sampling procedure on interpretation of endometrial cytology in mares.

The aim of this study was to ascertain if (1) the stage of reproductive cycle influences cytological results, (2) cytology obtained from an endometrial biopsy is more accurate than cytology derived using the cytobrush, and (3) different methods used for evaluation of cytological samples produce similar diagnostic results. Material was collected from 46 mares in estrus, 48 in diestrus and from 33 mares in anestrus. Smears were evaluated using two criteria. In criterion I, a total of 300cells were counted and the percentage of polymorphonuclear cells was recorded. In criterion II, the number of polymorphonuclear cells/high power field was determined. Endometrial biopsy samples were also examined histologically, and comparisons were made between cytological and histological results. Regardless of the method used for sample collection, endometritis was diagnosed significantly more frequently if smears were evaluated according to criterion I. The agreement between the number of polymorphonuclear cells infiltrated into the endometrial luminal epithelium and stratum compactum and the cytology results for smears evaluated according to criterion I was fair for anestrous mares, poor for diestrous mares, and moderate for mares in estrus. When cytological smears were evaluated according to criterion II the agreement between the histological and cytological and results was always poor. In summary, cytological evaluation of the mare endometrium should be based on counting the numbers of polymorphonuclear cells in relation to epithelial cells rather than counting the number of polymorphonuclear cells in relation to high power field.

Comparison of the biopsy and cytobrush techniques for diagnosis of subclinical endometritis in mares.

BACKGROUND: Endometritis is a major cause of infertility in the mare. Therefore, the diagnosis of this disease is very important in veterinary practice. The objective of this study was to compare bacteriological and cytological results obtained from the mare uterus using biopsy (EB) and cytobrush (CB) techniques and relating these findings to the presence of polymorphonuclear cells (PMNs) in endometrial tissue as the gold standard for detection of endometritis. In particular, we tested the hypothesis that endometrial cytology and microbiology data obtained from material collected using the EB and CB techniques are similar, so that the CB technique could preferentially be used to detect subclinical endometritis in clinical practice. METHODS: A total of 69 mares suspected of subclinical endometritis because of previous reproductive history and 15 maiden mares were enrolled in this study. Material collected from both EB and CB was smeared on sterile glass slides for cytological examinations and on culture media for microbiological examinations. Bacteriological cultures and cytological samples were classified as negative (no growth or mixed cultures of more than three microorganisms; <2% PMNs) or positive (pure growth of microorganisms; >2% PMNs) for endometritis. RESULTS: Positive growth was observed in 43% of CB samples and in 54% of EB samples (difference not significant). The growth of ß-hemolytic streptococci was always connected with positive cytology. This relationship was not observed for growth of E. coli or for non-pathogenic flora. The sensitivity of bacterial growth and cytology from EB was 0.63 and 0.73 respectively. The sensitivities of bacterial growth and cytology from CB were 0.50 and 0.71 respectively. CONCLUSION: Microbiological and cytological results obtained from CB are similar to those obtained from EB and based on these findings the CB technique may be recommended for collection of materials from the mare's uterus in clinical practice.

The influence of Sildenafil citrate on uterine tissue perfusion and the cardiovascular system during the luteal phase of the ovarian cycle in cows.

The aim of the study was to evaluate the influence of the Sildenafil citrate on the blood flow in the uterus of cows during dioestrus. Uterine blood flow was examined in five, healthy, adult cows. Between day 6-8 of the ovarian cycle, each cow received 200mg of sildenafil diluted in 10ml of warm saline into the body of the uterus. Analysis of the blood pressure, ECG and the maximum velocity in m/s (V max) in the aorta was performed and selected parameters of the blood flow (PI, pulsatile index; RI, resistance index; SPV, systolic peak velocity; EDV, end diastolic velocity; FVI, flow velocity integral; SV/DV, systolic peak velocity: end-diastolic velocity ratio) were measured in the uterine artery (Arteria uterine) before and after sildenafil infusion. In addition, Color Doppler examination of the uterine wall perfusion was analyzed. A significant decrease of values of PI and SV/DV ratio as well as an increase of end diastolic velocity and time averaged maximum velocity was noted. With the use of color coded sonography, the increased intensity of the blood flow in the uterine wall was observed. It was concluded that intrauterine administration of sildenafil during dioestrus can increase uterine tissue perfusion.

Influence of bitches sex pheromones on the heart rate and other chosen parameters of blood flow in stud dogs (Canis familiaris).

The aim of this study was to verify the usefulness of the measurement of chosen cardiovascular activity parameters for examination of sex pheromones effects in male dogs. We evaluated the influence of the bitches' sexual pheromones (BSP) on heart rate (HR) with the use of a Holter monitor, and chosen parameters of blood flow in vessels of penis, testes and kidney, with color-coded and pulsed Doppler ultrasonography. We found that the BSP increased HR in all examined males even without showing any other signs of arousal. There were no changes in the RI (resistant index) and PI (pulsative index) in any trials during the pheromone presentation. The increase in blood flow intensity was noted in penile vessels but not in the testes and kidney. We concluded that measurement of flow intensity in the penis as well as the changes in heart rate as the male's reaction to the BSP can be useful in research concerning sex pheromones in dogs.

Possibilities of using the European bison (Bison bonasus) epididymal spermatozoa collected post-mortem for cryopreservation and artificial insemination: a pilot study.

BACKGROUND: European bison is the largest mammal in Europe with the population of approximately 4000 individuals. However, there is no report of post-mortem spermatozoa collection and cryopreservation from this species and the aim of this study was to test if the epididymal spermatozoa collected post-mortem from European bison are suitable for cryopreservation and artificial insemination (AI). METHODS: Epididymides were collected post-mortem from two European bison bulls at age of 8 (bull 1) and 11 year (bull 2). Epididymal sperm was harvested by making multiple incisions in caudae epididymidis, which were then rinsed with extender. The left epididymis of bull 1 was rinsed with BioXcell (IMV, France), whereas the right epididymis of bull 1 and the right and left epididymides of bull 2 were rinsed with the extender based on Tris, citric acid, glucose, egg yolk, glycerol, antibiotics and distilled water (extender II). The diluted semen was cooled to 5 degrees C, and frozen in liquid nitrogen vapour. Then, properties of the frozen/thawed semen were examined with the use of computer-assisted semen analysis system, and thirty cows and nine heifers of domestic cattle were artificially inseminated. RESULTS: Motility of fresh spermatozoa collected from the right epididymis of bull 1 was 70% (spermatozoa diluted with extender II), and from the left one was 60% (spermatozoa diluted with BioXcell), whereas motility of fresh spermatozoa collected from bull 2 was 90% (spermatozoa diluted with extender II). Spermatozoa motility just after thawing were 11 and 13% in bull 1, respectively for spermatozoa collected from the left and right epididymis and 48% in bull 2. As a result of AI of domestic cows and heifers with the frozen/thawed European bison spermatozoa, two pregnancies were obtained in heifers. One pregnancy finished with a premature labour after 253 days of pregnancy, and the second one after 264 days of pregnancy. CONCLUSIONS: This is the first report showing pregnancy in the domestic cattle following AI with frozen-thawed European bison spermatozoa collected post-mortem. The protocol of spermatozoa collection, dilution, and cryopreservation presented in this paper may be useful for the creating genetic resource bank in the European bison.

The effect of trehalose on post-thaw viability and fertility of European brown hare (Lepus europaeus Pallas, 1778) spermatozoa.

The aim of the study was to assess the effect of a different trehalose concentration on the post-thaw viability and fertility of European brown hare spermatozoa. The semen was collected under general anaesthesia with electroejaculation method from 4 males. Immediately after collection, the semen was diluted with an extender of the following composition: Tris 250mM, citric acid 80mM, glucose 70mM, DMSO 1.0M, egg yolk (17%, v/v), and kanamycin (80mg/l)-Protocol I. In Protocols II and III, respectively, 50mM and 100mM of trehalose were added to the extender. Immediately after thawing and after 3h incubation at 37 degrees C, motility characteristics of frozen/thawed semen were assessed with computer-assisted semen analysis system, and a percentage of viable, acrosome intact spermatozoa was evaluated using flow cytometry. Immediately after thawing spermatozoa motility (MOT), average path velocity (VAP), straight velocity (VSL) and curvilinear velocity (VCL) were the highest in the semen frozen without addition of trehalose (P<0.01). After 3h of incubation, MOT and spermatozoa with progressive motility (PMOT) were the lowest in the semen frozen with supplementation of 100mM of trehalose (P<0.01) and VAP, VSL, VCL and amplitude of lateral head displacement (ALH) were significantly lower in the semen frozen with supplementation of 50mM and 100mM of trehalose compared to the semen frozen without the addition of trehalose (P<0.01), which indicates an unfavourable effect of trehalose on the motility characteristics of European brown hare spermatozoa. However, the effect of trehalose on a percentage of viable, acrosome intact spermatozoa was not observed. As a result of artificial insemination, 54.55% females became pregnant after insemination with the semen frozen according to Protocol I, 72.73% and 50% females became pregnant after insemination with the semen supplemented with 50mM and 100mM of trehalose, respectively. The number of young born was 1.67+/-0.52, 1.75+/-1.04 and 1.60+/-0.55, in each group, respectively. There were not any significant differences in the results of artificial insemination between groups. Summing up, it should be stated that the addition of trehalose to the extender did not have a favourable effect on post-thaw viability of European brown hare spermatozoa and an influence of trehalose on the results of artificial insemination was not found, either.

Aspergillosis of a dog genital tract-Case report.

The information about aspergillosis locations in the reproductive organ is scarce. This short paper deals with aspergillosis in the dog genital tract with hyphae present in semen. There are two therapy schemes used in visceral mycoses, non-invasive treatment and surgical intervention. Considering the future reproductive career of the dog, we decided on antifungal drugs administration. Based on the microbiological results, we administered amoxycillin with clavulonate (Synulox 500mg, twice daily) orally. Itraconazole was used as an antimycological agent (Orungal, 100mg, twice daily) every other week. In 8th week of therapy no Aspergillus spp. growth was noted, yet slight Penicillium growth was observed. After 12 weeks of treatment, no fungus growth was present. Neither spores or hyphae were seen in the microscopic examination. Three months after the termination of the therapy, the dog mated with two females. In one case, unifetal pregnancy was diagnosed by ultrasound examination on day 42 after mating. Due to purulent discharge on day 45 after mating, the owner decided to terminate the pregnancy. In the other case, severe pyometra appeared 12 days after the second mating and the owner decided to put the female to sleep. The pathogen eradication from the ejaculates may be treated as a serious success, yet the lack of litters after mating calls for an explanation and consequences of Aspergillus spp. infection need to be considered.

Comparison of acetamide and dimethylsulfoxide as cryoprotectants of European brown hare (Lepus europaeus) spermatozoa.

The aim of the present study was to assess the effect of dimethylsulfoxide (DMSO) and acetamide on the post-thaw properties of hare semen and to perform an AI trial with frozen-thawed semen. Semen was collected under general anaesthesia by the electroejaculation method from 6 males. Immediately after collection, the semen was diluted with an extender containing the following components: 250 mM Tris, 80 mM citric acid, 70 mM glucose, 1.0 M DMSO, egg yolk (17% v/v) and kanamycin (80 mg/l); this extender was used for Protocol I (n=17). In Protocol II (n=15), the DMSO was replaced with 1.0 M acetamide. Immediately after thawing and after incubation for 90 and 180 min at 37 C, the properties of semen were evaluated by computer-assisted semen analysis, and the percentage of viable, acrosome intact spermatozoa was evaluated using flow cytometry. During the 3-h incubation, the percentages of motile spermatozoa and spermatozoa with progressive motility were significantly higher in Protocol I (P<0.01). Immediately after thawing, path and straight velocity were significantly higher in Protocol I (P<0.01), as was the curvilinear velocity (P<0.05). The amplitude of lateral head displacement was higher after 3-h incubation in Protocol I (P<0.05), and no differences in beat cross frequency were found between Protocol I and II at any incubation time. The percentage of viable, acrosome intact spermatozoa determined with flow cytometry was higher in Protocol I (P<0.01) at all incubation times. As a result of artificial insemination with the semen frozen with DMSO as a cryoprotectant, two out of three inseminated females delivered two healthy young each. Following artificial insemination with the semen frozen with acetamide as a cryoprotectant, two out of three inseminated females delivered one healthy young each. On the basis of the results, it should be stated that DMSO ensures better post-thaw properties of hare spermatozoa than acetamide.