Growth-related micromorphological characteristics of the porcine common carotid artery.

The purpose of this study was to gain knowledge about the micromorphology of the porcine common carotid artery (CCA) during the period of growth over the bodyweight range of 10-40 kg. CCA samples from German landrace pigs (DL) aged either 2 or 3 months (DL-2 and DL-3) were compared with samples from Göttingen minipigs (GM) aged either 18 or 40 months (GM-18 and GM-40) using transmitted light (phase-contrast mode) and transmission electron microscopy. The GM-18, GM-40 and the DL-3 groups had typical muscular artery histological characteristics. Contrasting to this, the 2-month-old DL pigs had a transitional artery type being characterized by a significantly higher proportion of elastic fibres and a significantly lower number of smooth muscle cells than did the 1 month older DL-3. During the period of maturation, the tunica media of the CCA in GM animals thickened by 1.3× and in DL animals by 2.5× resulting in an overall increased vessel wall thickness. The cumulated thickness of the tunica interna (endothelium, stratum subendotheliale and internal elastic lamina) and the tunica media (including the external elastic lamina) of DL-3 and GM-40 pigs were similar to each other and comparable to that of humans. With an increasing vessel wall thickness, the luminal diameter decreased in GM by 19% and in DL by 11%. Additionally, in the older age groups, GM-40 and DL-3, the internal elastic lamina principally was continuous, but there were also interrupted large segments of elastic lamina separated by gaps. In addition, the principal internal elastic lamina was duplicated in several places.

Expression of phospholipase A2 receptor in primary cultured podocytes derived from dog kidneys.

Phospholipase A2 receptor (PLA2R) expressed in human podocytes has been highlighted as a causative autoantigen of human idiopathic membranous nephropathy. However, its expression was found to be minimal or absent in murine and rat podocytes. In this study, immunofluorescence revealed the expression of PLA2R in the glomerular podocytes in the kidney tissue sections of dogs. We then attempted to culture canine podocytes and investigate the expression of PLA2R in these cells. Glomeruli were isolated from dog kidneys and cultured to obtain podocytes using nylon mesh-based isolation method as followed for isolating rat podocytes. The cultured cells expressed PLA2R mRNA and protein in addition to other podocyte markers (synaptopodin, podocin and nephrin). These results indicate that the canine podocytes express PLA2R.

Geotrichum candidum as a possible cause of bovine abortion.

Geotrichum spp. are ubiquitous, saprotrophic fungi found in soil, organic matter, and silage, as a contaminant in food products and in the digestive tracts of mammals. The current study reports a case of Geotrichum candidum infection with dermatitis in an aborted bovine fetus with skin and lung lesions. A 6-month-old aborted male Holstein Friesian fetus displayed unusual lesions on the skin of the abdomen, thorax, and head, which was excessively thickened and wrinkled. These changes corresponded to orthokeratotic hyperkeratosis, neutrophil accumulation in the stratum corneum, a pyogranulomatous inflammatory infiltrate, and superficial dermal necrosis. Moderate suppurative multifocal pneumonia was observed. Large numbers of mononuclear cells and occasional fibrin thrombi within blood vessels were found in the lungs, brain, and cerebellum. Gridley staining revealed fungal structures within the skin lesions. The mycological exam demonstrated the growth of G. candidum, and phase contrast microscopy conducted on the abomasal fluid revealed hyphae compatible with this agent. The skin lesions observed, in association with the fungus isolated, indicated that the abortion was due to G. candidum infection of the bovine fetus.

Suitability and effectiveness of single layer centrifugation using Androcoll-P in the cryopreservation protocol for boar spermatozoa.

The goal of the present experiment was to evaluate the suitability and effectiveness of single layer centrifugation (SLC), using the pig-specific colloid Androcoll-P, as a routine procedure for selecting boar spermatozoa for cryopreservation. The study focuses special attention on the effectiveness of SLC for processing a whole sperm rich ejaculate fraction and the fertilizing ability of frozen-thawed (FT) sperm selected using SLC prior to freezing. Thirteen sperm rich ejaculate fractions (one per boar) were split into three aliquots. Two aliquots of 15 and 150mL were SLC-processed (500×g for 20min) using 15 and 150mL (v/v) of Androcoll-P-Large and Androcoll-P-XL, respectively. The third aliquot remained un-processed as a control. The percentages of spermatozoa that were morphologically normal and showed rapid and progressive motility (assessed by CASA) spermatozoa were higher (P<0.01) and those with fragmented nuclear DNA (sperm chromatin dispersion test) were lower (P<0.01) after SLC than control semen samples, regardless of the Androcoll-P used. The recovery rates of total, motile, viable (flow cytometric evaluated after staining with H-42, PI and FITC-PNA) and morphologically normal spermatozoa ranged between 20 and 100% and those with intact nuclear DNA ranged between 60 and 100%, irrespective of the Androcoll-P used. Thereafter, the semen samples were cryopreserved using a standard 0.5-mL straw freezing protocol. Post-thaw percentages of sperm motility (both total motility and rapid progressive motility), viability and intact nuclear DNA were higher (P<0.05) in SLC-processed than in control semen samples, irrespective of the Androcoll-P used. SLC-processing also improved the in vitro fertilizing ability of FT-sperm (679 in vitro matured oocytes inseminated with a viable sperm:oocyte ratio of 300:1 and coincubated for 6h), measured as the percentage of penetrated oocytes and the mean number of swollen sperm heads and/or male pronuclei in penetrated oocytes. However, there was no effect of SLC-processing on the in vitro ability of putative zygotes to develop to blastocysts. Overall these results indicate that SLC-processing of boar ejaculates using Androcoll-P improves the quality and fertilizing ability of cryosurvival boar sperm. However, efforts should be made to ensure continued high recovery yields before considering the inclusion of SLC as a routine procedure in the cryopreservation protocol of boar ejaculates.

Identification of sperm head subpopulations with defined pleiomorphic characteristics in ejaculates of captive Goeldi's monkeys (Callimico goeldii).

The aim of this study was to evaluate the incidence of pleiomorphisms and its influence on the distribution of sperm morphometric subpopulations in ejaculates from the vulnerable Goeldi's monkey (Callimico goeldii) by using a combination of computerized analysis system and Principal Component Analysis (PCA) methods. Each sperm head was measured for four primary spermatozoal head dimensional parameters (area [A (µm(2))], perimeter [P (µm)], length [L (µm)] and width [W (µm)]) and three head shape derived parameters (ellipticity [(L/W)], elongation [(L-W)/(L+W)] and rugosity [(4πA/P(2))]). Six separate subpopulations (SPs) were identified: SP1, constituted by very large, narrow and very elliptical spermatozoa (A=16.85±1.56µm(2), W=2.75±0.42µm and ellipticity=2.16±0.24); SP2, characterized by average sized, short, wide and round spermatozoa (A=15.00±1.92µm(2), L=5.06±0.49µm, W=3.51±0.31µm and ellipticity=1.44±0.15); SP3, represented by small, wide and slightly round spermatozoa (A=14.95±1.75µm(2), W=3.47±0.29µm and ellipticity=1.48±0.14); SP4 included very small, short and very round spermatozoa (A=14.15±2.38µm(2), L=4.90±0.57µm and elongation=0.18±0.05); SP5 consisted of average sized and slightly elliptical spermatozoa (A=15.14±1.72µm(2) and ellipticity=1.49±0.14); and SP6 included large and round spermatozoa (A=16.30±1.62µm(2) and elongation=0.19±0.04). There were differences in the sperm subpopulation distribution (P<0.001) among the five donors analyzed. In conclusion, the results of the current study confirmed that the use of computer sperm analysis methods combined with PCA cluster analyses are useful methods to identify, classify, and characterize different sperm head morphometric subpopulations in neotropical primates. Broadening our knowledge of C. goeldii sperm morphometric abnormalities as well as developing reliable techniques for sperm evaluation may be essential for ex situ conservation of this threatened species.

Pyrethroids cypermethrin, deltamethrin and fenvalerate have different effects on in vitro maturation of pig oocytes at different stages of growth.

Pesticides can significantly harm reproduction in animals and people. Pyrethroids are often used as insecticides, and their toxicity for mammals is considered to be low. However, cypermethrin, deltamethrin and fenvalerate - as potent specific inhibitors of protein phosphatase calcineurin - can influence the meiosis of mammalian oocytes. The objective of this study was to evaluate the effects of these pyrethroids on the in vitro maturation of pig oocytes at different levels of meiotic competence. Under the tested concentrations, cypermethrin, deltamethrin and fenvalerate neither had a significant effect on the viability of oocytes nor did they induce significant degeneration of oocytes. However, these pyrethroids significantly affected meiotic maturation. The effects depended on the stage of meiotic competence of the oocytes. Maturation of growing pig oocytes with partial meiotic competence was induced. On the other hand, in fully grown pig oocytes with full meiotic competence, maturation in vitro was delayed. The specificity of these effects was further supported by the same effect of non-pyrethroidal inhibitors of calcineurin - cyclosporin A or hymenistatin I - on the maturation of oocytes with different levels of meiotic competence. However, pyrethroids, which do not inhibit calcineurin - allethrin or permethrin - had no effect on pig oocyte maturation. We demonstrated a significant effect of pyrethroids on the maturation of mammalian oocytes under in vitro conditions. This indicates that exposure to these substances could affect the fertility of people or animals.

Ureaplasma diversum in bull semen in Australia: its detection and potential effects.

BACKGROUND: The primary objective of this study was to confirm the infection status of Ureaplasma diversum in Australian bulls and to identify morphological changes of sperm from U. diversum-positive bulls. METHODS: Fresh semen samples were taken from 29 sexually active beef bulls from suspect herds in the Riverina/Upper Murray region. U. diversum was identified using PCR analyses and culture of the organism. RESULTS: Nine of the bulls were PCR-positive for U. diversum but none of these had genital lesions. Sperm from infected bulls showed increased incidence of abnormal tails (bent and coiled), as well as surface abnormalities (i.e. small protuberances or lumps). CONCLUSIONS: The results suggest impairment of sperm function and possibly fertility. Further investigations into the potential role of U. diversum as a pathogen for Australian cattle are warranted.

Artificial insemination in pigs: predicting male fertility.

Efficient artificial insemination (AI) is essential for future challenges in the pig industry. Knowledge on the exact relation between semen quality characteristics and fertility can have a major impact on both the genetic merit of future animals and the efficiency of AI. Variation in fertility is caused not only by farm- or sow-related parameters but also by boar- and semen-related parameters. In pig AI there is no gold standard concerning semen quality assessment. Assessing semen quality characteristics objectively and relating them to large field fertility datasets leads to an efficient production of insemination doses, which results in an efficient dissemination/descent of the breeding program required genes. Overall, this contributes to the development of semen quality assessments, which improves the prediction of porcine male fertility. Knowing which semen characteristics, and to what extent, contribute to male fertility and makes the field fertility more predictable.

Growth curves and morphology of three Treponema subtypes isolated from digital dermatitis in cattle.

Digital dermatitis (DD) is an infectious claw disease of cattle that causes painful lesions, principally along the coronary band of the claws. In the US alone, the estimated economic impact of DD is estimated to be $190 million. The etiology of DD remains unclear and there is no reliable laboratory test, so DD is most often diagnosed clinically. Spirochetal bacteria of the genera Treponema have been implicated in DD infections following their isolation using culture techniques, serological detection of bovine antibodies against treponemes, and amplification of treponemal 16s DNA sequences by PCR. During in vitro growth of spirochetes and treponemes isolated from DD, morphological changes have been observed indicating the presence of a spiral form and an encysted form. It is not known why encysted forms appear or what role they have in the progression of DD. The current study established growth curves for three subtypes of treponemes, Treponema denticola-like, Treponema phagedenis-like, and Treponema medium-like, while photographically monitoring changes in morphology. In addition to observing spiral and encysted forms, two intermediate forms were also observed. These appeared as either spiral forms with spherical bodies or as enveloped clusters of granules. The observation of encysted forms adds further support to the theory that treponemes causing recurrent infections deep in bovine skin have mechanisms to facilitate persistence and the chronic character of DD.

Effect of active immunization against GnRH on testosterone concentration, libido and sperm quality in mature AI boars.

BACKGROUND: The aim of the present study was to investigate the efficacy of the Improvac on testosterone concentration in blood serum, sexual behavior and sperm quality in matured AI boars. A total of nine Danish Landrace AI boars were included in the analysis. METHODS: The trial period lasted for 15 weeks and was divided into four periods: Control period: three weeks before vaccination; Period I - four weeks after first vaccination; Period II - four weeks after second vaccination, Period III - four weeks after third vaccination. Blood and sperm samples were collected at weekly intervals. Freshly collected sperm samples were analyzed. RESULTS: Testosterone concentration correlated with libido (r = 0.531; p < 0.001), volume of ejaculate (r = 0.324; p < 0.001) and the percentage of morphologically normal spermatozoa (r = 0.207; p < 0.05). Testosterone concentration rised significantly (p < 0.05) in 5-6 week of trial, e. i. after the first dose of Improvac and after this peak the level of testosterone further progressively decreased (p < 0.05). CONCLUSIONS: Results from this study indicate that active immunization of sexually matured boars against GnRH has negative impact on testosterone concentration, sexual behavior, volume of ejaculate and total number of normal spermatozoa in ejaculate.

Evaluation of duck egg yolk for the cryopreservation of Nili-Ravi buffalo bull semen.

This study was carried out to investigate if the substitution of chicken egg yolk (CEY) with duck egg yolk (DEY) in extenders can improve the quality of frozen-thawed semen of Nili-Ravi buffalo bulls and to study if reducing DEY level in extender affects the freezability results. Thirty semen samples collected from three buffalo bulls were diluted in extenders A, B, C, D and E containing tris, citric acid, fructose, egg yolk, glycerol and antibiotics. Extender A contained 20% CEY (control), while extenders B, C, D and E contained 5, 10, 15 and 20% DEY, respectively. After freezing and storage for 24h in liquid nitrogen, samples were evaluated for post-thaw quality. The post extension sperm motility did not differ between extenders A (control) and E (20% DEY). The same was true for post-thaw percentage of sperm with functional plasma membrane and percentage of sperm with abnormal heads or mid pieces. However, extender E showed higher (P<0.05) values for post-thaw sperm motility, livability and absolute index of livability of spermatozoa at 37 °C compared to extender A. Spermatozoa with abnormal tail were lower (P<0.05) in extender E compared to extender A. Values of these parameters of post-thaw semen quality were highest for extender E containing 20% DEY and decreased significantly with decrease in the concentration of DEY, except sperm abnormalities (head, mid-piece and tail) which increased with decrease in DEY level. These results showed that replacement of 20% CEY with 20% DEY in extenders significantly improved post-thaw sperm motility, livability and absolute index of livability of spermatozoa and reduced tail abnormalities. Reduction in the level of DEY in extenders from 20% adversely affected post-thaw semen quality of Nili-Ravi buffalo bulls.

Mycoplasma synoviae induces upregulation of apoptotic genes, secretion of nitric oxide and appearance of an apoptotic phenotype in infected chicken chondrocytes.

The role of chondrocytes in the development of infectious arthritis is not well understood. Several examples of mycoplasma-induced arthritis in animals indicate that chondrocytes come into direct contact with bacteria. The objective of this study was to analyze the interaction of an arthrogenic Mycoplasma synoviae strain WVU 1853 with chicken chondrocytes. We found that M. synoviae significantly reduces chondrocyte respiration. This was accompanied by alterations in chondrocyte morphology, namely cell shrinkage and cytoplasm condensation, as well as nuclear condensation and formation of plasma membrane invaginations containing nuclear material, which appeared to cleave off the cell surface. In concordance with these apoptosis-like events in chondrocytes, transcription was increased in several pro-apoptotic genes. Twenty-four hours after infection, strong upregulation was assayed in NOS2, Mapk11, CASP8 and Casp3 genes. Twenty-four and 72 h incubation of chondrocytes with M. synoviae induced upregulation of AIFM1, NFκB1, htrA3 and BCL2. Casp3 and NOS2 remained upregulated, but upregulation ceased for Mapk11 and CASP8 genes. Increased production of nitric oxide was also confirmed in cell supernates. The data suggests that chicken chondrocytes infected with M. synoviae die by apoptosis involving production of nitric oxide, caspase 3 activation and mitochondrial inactivation. The results of this study show for the first time that mycoplasmas could cause chondrocyte apoptosis. This could contribute to tissue destruction and influence the development of arthritic conditions. Hence, the study gives new insights into the role of mycoplasma infection on chondrocyte biology and development of infectious arthritis in chickens and potentially in humans.

Isolation, expansion, and differentiation of goat adipose-derived stem cells.

A goat adipose-derived stem cell (ADSC) line was established and compared to a rat line. Goat ADSC cells had normal diploidy after subculture. Proliferation of goat ADSCs was faster than rat cells in the same conditions. Both rat and goat ADSCs stained positively for vimentin, CD49d, CD44 and CD13, but stained negatively for CD34 and CD106. Bone nodules were apparent, and alizarin staining was positive after osteogenic induction. Cells expressing osteocalcin were positive by alkaline phosphatase (ALP) staining. After osteogenic induction, ossification nodules of goat ADSCs were larger than in rats, with dense ALP staining. Adipogenic induction resulting in lipid droplets and peroxisome proliferator-activated receptor (PPARγ2) expression were observed. Cartilage lacunae were formed and COL2A1 was expressed. More cartilage lacunae with better morphology were seen following differentiation of goat ADSC's using the hang-drop method. For goat ADSCs, results with both adherent-induced and hanging-drop induced cultures were better than for three-dimensional cultures.

Effect of butylated hydroxytoluene on cryopreservation of Boer goat semen in Tris egg yolk extender.

The aim of this study was to determine the effect of butylated hydroxytoluene (BHT), a lipid-soluble anti-oxidant added in different concentrations to the Tris egg yolk extenders on semen cytological parameters pre freezing and post thawing (motility, morphology, viability, acrosome integrity and membrane integrity) of Boer goat spermatozoa. A total of 40 ejaculates from four Boer goat bucks were collected using an artificial vagina. Ten replicates of the ejaculates were diluted with a Tris egg yolk based extender which contained various concentrations (0.5mM, 1.0mM, 2.0mM and 3.0mM) of butylated hydroxytoluene while one sample was processed without supplementation of antioxidant and served as control. The diluted semen was cooled at 4°C and loaded into the straw and then stored in liquid nitrogen. It was evident that supplementation of BHT produces positive effect in terms of motility, membrane integrity and acrosome integrity in comparison with the control group in cooled and frozen Boer goat semen. Results showed significant differences in motility, membrane integrity, acrosome integrity and viability of cooled and frozen Boer goat spermatozoa at different concentrations. Motility, membrane integrity, acrosome integrity and viability was significantly higher in all treated groups than the control group (P<0.05) while there was no significant differences (P>0.05) in morphology trait between all group in cooled semen. However, improvement (P<0.05) was observed only in terms of the membrane integrity and acrosome integrity compared to the control and other treated groups in frozen semen. In conclusion, BHT can be used in cryopreservation of Boer goat semen in order to reduce the oxidative stress on spermatozoa.

Isolation and characterization of multipotent mesenchymal stromal cells from the gingiva and the periodontal ligament of the horse.

BACKGROUND: The equine periodontium provides tooth support and lifelong tooth eruption on a remarkable scale. These functions require continuous tissue remodeling. It is assumed that multipotent mesenchymal stromal cells (MSC) reside in the periodontal ligament (PDL) and play a crucial role in regulating physiological periodontal tissue regeneration. The aim of this study was to isolate and characterize equine periodontal MSC. Tissue samples were obtained from four healthy horses. Primary cell populations were harvested and cultured from the gingiva, from three horizontal levels of the PDL (apical, midtooth and subgingival) and for comparison purposes from the subcutis (masseteric region). Colony-forming cells were grown on uncoated culture dishes and typical in vitro characteristics of non-human MSC, i.e. self-renewal capacity, population doubling time, expression of stemness markers and trilineage differentiation were analyzed. RESULTS: Colony-forming cell populations from all locations showed expression of the stemness markers CD90 and CD105. In vitro self-renewal capacity was demonstrated by colony-forming unit fibroblast (CFU-F) assays. CFU-efficiency was highest in cell populations from the apical and from the mid-tooth PDL. Population doubling time was highest in subcutaneous cells. All investigated cell populations possessed trilineage differentiation potential into osteogenic, adipogenic and chondrogenic lineages. CONCLUSIONS: Due to the demonstrated in vitro characteristics cells were referred to as equine subcutaneous MSC (eSc-MSC), equine gingival MSC (eG-MSC) and equine periodontal MSC (eP-MSC). According to different PDL levels, eP-MSC were further specified as eP-MSC from the apical PDL (eP-MSCap), eP-MSC from the mid-tooth PDL (eP-MSCm) and eP-MSC from the subgingival PDL (eP-MSCsg). Considering current concepts of cell-based regenerative therapies in horses, eP-MSC might be promising candidates for future clinical applications in equine orthopedic and periodontal diseases.

Freezing stallion semen with the new Cáceres extender improves post thaw sperm quality and diminishes stallion-to-stallion variability.

Ejaculates from 7 stallions were split and simultaneously frozen in three different extenders, INRA 96 egg yolk glycerol, Ghent and the newly developed extender Caceres. After thawing, samples were evaluated for motility (CASA system) sperm membrane integrity and early membrane changes (YoPro-1/Eth staining), acrosome integrity (FICT-PNA), and mitochondrial membrane potential (JC-1) (flow cytometry). Samples frozen in Caceres extender consistently showed the best results in post-thaw motility (increases ranging from 11 to 17%, p<0.05) and velocity (p<0.05), membrane integrity (increases ranging from 11 to 14%, p<0.05) and mitochondrial membrane potential (p<0.05). It is concluded that this new extender should be included in a freezeability test to determine the best extender for each individual.

Validating the assessment of bull sperm morphology by veterinary practitioners.

The objective of this study was to validate the assessment of bull sperm morphology done by veterinary practitioners. Out of 1606 bulls, 1400 (87.2%) and 1344 (83.7%) were designated by practitioners and an experienced andrologist, respectively, as having > 70% morphologically normal sperm. In 92% of the evaluations, there was agreement between the designations chosen.

Effects of centrifugation through three different discontinuous Percoll gradients on boar sperm function.

In this study, different combinations of 2-step, discontinuous gradient centrifugation were used, consisting of three different combinations of isotonic Percoll (45/60, 60/75 and 45/90%) that allowed us to select different sperm subpopulations from fertile and normozoospermic boars. Our objective in this study is to evaluate the effects of centrifugation through three different discontinuous Percoll gradients on sperm function parameters (motility, viability, morphology, acrosome status, chromatin condensation, DNA fragmentation, ROS generation, tyrosine phosphorylation and intracellular calcium concentration) and the sperm penetrating capacity in an IVF system. All the Percoll treatments evaluated increased the percentage of spermatozoa with normal morphology, the proportion of un-damaged DNA, normal chromatin condensation, motion parameters measured by CASA and the percentage of capacitated spermatozoa with tyrosine phosphorylated proteins compared to control group. Finally, the in vitro oocyte penetrating capacity of boar spermatozoa was significantly affected by Percoll centrifugation. All the Percoll treatments increased the penetration rates and mean number of sperm per penetrated oocyte. Despite the efficiency of all three of the sperm treatments tested in selecting spermatozoa with improved sperm parameters and capacity to penetrate oocytes in vitro, the optimum performance of this system was demonstrated after preselecting spermatozoa by centrifugation on a discontinuous 45/90 Percoll gradient. The P45/90 treatment leads to obtain a higher percentage of spermatozoa which develop properly the capacitation process as it was shown measuring tyrosine phosphorylation and intracellular calcium concentration.

Effect of method and clinician on stallion sperm morphology evaluation.

The objective of this study was to determine the effects of method and clinician on stallion sperm morphology evaluation. Five clinicians evaluated 60 semen samples using wet-mount preparations with phase-contrast, eosin/nigrosin-stained semen smears, and Papanicolaou-stained semen smears. There were significant differences among methods for all sperm morphology categories and most intra-class correlation coefficients were only fair to moderate. The use of wet-mount preparations facilitated detection of acrosome defects, nuclear vacuoles, and cytoplasmic droplets when compared to stained smears. Smearing stallion semen samples onto slides increased the proportion of detached sperm heads. In addition, acrosome defects, nuclear vacuoles, rough/swollen midpieces, and cytoplasmic droplets were difficult to observe with Papanicolaou stain; this method resulted in overestimation of normal sperm when compared to other methods. There were significant differences among clinicians for all sperm morphology classification categories. In conclusion, this study demonstrated that sperm morphology evaluation results varied, depending on the evaluation method and clinician. Wet-mount preparation with phase-contrast microscopy appeared to be more sensitive for identification of abnormal stallion sperm when compared to stained smears. Veterinary andrology laboratories should invest in training, continuing education, proficiency testing, and other quality control measures to minimize the variation of sperm morphology evaluation results among clinicians.

Quantitative comparison of different purification and detection methods for Cryptosporidium parvum oocysts.

UNLABELLED: Cryptosporidium parvum (C. parvum) is the causal agent of cryptosporidiosis in many animals, mainly cattle, and possesses a high zoonotic potential. It occurs worldwide and ubiquitously. Detection of C. parvum is mainly performed directly but purification of the oocysts is useful to increase sensitivity and to obtain oocyst material for further use. The study was designed to compare (a) three different direct diagnostic methods, namely modified Ziehl-Neelsen staining, carbol fuchsin staining and conventional PCR, and (b) three routine oocyst purification methods, in particular flotation with saturated sodium chloride solution, Sheather's sucrose solution and a Percoll(®) gradient. During comparison of purification methods, special regard was paid to the ability to separate morphologically intact oocysts from the morphologically degenerated fraction or viable from non-viable oocysts, respectively. RESULTS: (a) DIAGNOSTIC METHODS: Most effective in C. parvum oocysts detection in calf faeces was PCR; carbol fuchsin and modified Ziehl-Neelsen stainings achieved comparable results. (b) Purification methods: Oocyst flotation using sodium chloride solution showed to be superior to Percoll(®) gradient centrifugation and sugar flotation in terms of purification quality, recovery efficacy (yield) and reduction of the proportion of degenerated or non-viable oocysts.