Roland Kozdrowski

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

BackgroundEuropean 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).MethodsEpididymides 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.ResultsMotility 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.ConclusionsThis 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.

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

BackgroundEndometritis 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.MethodsA 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.ResultsPositive growth was observed in 43% of CB samples and in 54% of EB samples (difference not significant). The growth of β-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.ConclusionMicrobiological 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 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.

Type of Inflammation Differentially Affects Expression of Interleukin 1β 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 1β (IL-1β), 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-1β, 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-1β (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.

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.

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.

Inducing ovulation and artificial insemination in the European brown hare (Lepus europaeus Pallas, 1778)

The aim of the study was to show whether it is possible to induce ovulation in the hare by GnRH analogue administration and to carry out an effective artificial insemination (AI). The research was carried out during the breeding and non-breeding season. During the breeding season, plasma progesterone concentrations increased on the 4th day after intramuscular injection of GnRH analogue (buserelin), indicating induced ovulation and corpus luteum development. Prostaglandin F(2)alpha (dinoprost) was an effective luteolytic agent on day 9. During the non-breeding season, the GnRH analogue injection does not cause an increase of progesterone. The 17beta-estradiol concentrations during the breeding and non-breeding season were similar. It was shown that after GnRH analogue administration and artificial insemination with semen diluted in Tris buffer extender 80% females delivered live young (39-43 days after artificial insemination), which proves the effectiveness of inducing ovulation in the hare by means of hormonal stimulation.

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.

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.