Single layer colloid centrifugation technique improves motility, viability and chromatin integrity of ram spermatozoa after thawing.

The cell membrane of ram spermatozoa is more sensitive to the freezing process than in other species due to its composition. As a result, the quality and viability of frozen thawed ram spermatozoa are often poor, which together with the specific structure of the ewe's cervix are the main reasons for lower fertility in ewes after intracervical insemination. In the present study we investigated the effects of semen centrifugation through a single layer of a species-specific colloid (Androcoll-O) on post-thaw quality of ram spermatozoa. Motility, viability and morphology were analysed 0, 6, 12 and 24 h after thawing. DNA fragmentation index (%DFI) of the samples was assessed 0 h after thawing, by SCSA™. Membrane and acrosome integrity of spermatozoa were analysed by Sybr-14/PI/PNA test 0 h after thawing. The proportion of motile spermatozoa was significantly higher in SLC - selected samples in comparison to control (not SLC - selected) samples at 0, 6, 12 (P < 0.001) and 24 h (P < 0.05). The proportion of viable spermatozoa was also significantly higher in SLC - selected samples in comparison to control samples at all times (P < 0.001). The proportion of abnormal acrosomes and morphologically abnormal spermatozoa (MAS) were significantly lower in SLC - selected samples compared to control samples at all times (P < 0.001). Analysis of chromatin stability revealed significantly lower %DFI values in SLC - selected samples compared to control samples (P < 0.001). The SYBR-14/PI/PNA test also revealed significantly better values in SLC - selected compared to control samples (P < 0.05). In conclusion, single layer colloid centrifugation significantly improved post-thaw quality and longevity of ram spermatozoa, making it suitable for artificial insemination initiatives.

Long-term leptin fluctuations in female donkeys.

REASONS FOR PERFORMING STUDY: The interest in donkeys is growing due to their integration in the systems of ecological farming, among other reasons. OBJECTIVES: Due to limited reports on leptin concentrations in donkeys, the aim of the present study was to examine age-dependent and seasonal changes in the circulating leptin concentration in female donkeys (jennies) and thus contribute to knowledge about the physiological characteristics of this species. STUDY DESIGN: Prospective longitudinal study. METHODS: The study was performed over a year (September 2008 to September 2009) on 20 yearling and young adult (pregnant, lactating or barren) jennies aged 1-5 years at the onset of the study; the animals were kept on pasture from May to September and stabled for the rest of the year. Blood samples were taken monthly and analysed for serum leptin concentrations by a commercial radioimmunoassay kit. RESULTS: Circulating leptin concentrations in studied jennies were lower than those reported for donkeys and horses. Despite the tendency for lower values in yearling vs. young adult jennies, the age range of the examined animals was insufficient to confirm any age-related leptin variations. Significant seasonal leptin fluctuations with peak levels in late spring and the lowest levels in autumn months, correlated with photoperiod, were detected in yearling, barren as well as pregnant jennies. Therefore, it was impossible to identify any effects of gestation or lactation on leptin concentrations of jennies. CONCLUSIONS: The results of this study cannot be used as evidence of a causal relationship between the photoperiod and seasonal circulating leptin fluctuations in donkeys, but could reflect changes induced by various external or internal factors enabling adaptations of grazing animals in variable submediterranean environments.

Lipid mobilisation and oxidative stress as metabolic adaptation processes in dairy heifers during transition period.

The objective of this study was to evaluate metabolic disorders and oxidative stress in dairy heifers during the transition period. Possible relationships between lipid mobilisation indicators and oxidative stress markers were investigated as well. Nineteen dairy heifers were included in the study. Blood samples were collected at the time of estrus synchronisation in heifers, at insemination, three weeks after insemination, one week before calving, at calving and 1, 2, 4 and 8 weeks postpartum. Common metabolic parameters, beta-hydroxybutyrate (BHB), free fatty acids (FFA), paraoxonase-1 (PON1) activity and total antioxidative status (TAS) were analysed. Around insemination, no significant difference was observed in the majority of tested parameters (P>0.05). However, the transition period markedly affected the concentration of triglycerides, total cholesterol, HDL-C, BHB, FFA, TAS and PON1activity. Positive correlations between PON1 activity and total cholesterol, HDL-C and triglycerides were noted but inverse correlations with FFA, BHB and bilirubin were found indicating that PON1 activity changed with lipid metabolism and was influenced by negative energy balance. These findings suggest that lipid mobilisation and oxidative stress are part of a complex metabolic adaptation to low energy balance which reaches equilibrium later in advanced lactation.

Lift-off PMN-PT Thick Film for High Frequency Ultrasonic Biomicroscopy.

Piezoelectric 0.65Pb(Mg(1/3)Nb(2/3))O(3)-0.35PbTiO(3) (PMN-35PT) thick film with a thickness of approximately 12 µm has been deposited on the platinum buffered Si substrate via a sol-gel composite method. The separation of the film from the substrate was achieved using a wet chemical method. The lifted-off PMN-35PT thick film exhibited good dielectric and ferroelectric properties. At 1 kHz, the dielectric constant and the dielectric loss were 3,326 and 0.037, respectively, while the remnant polarization was 30.0 µC/cm(2). A high frequency single element acoustic transducer fabricated with this film showed a bandwidth at -6 dB of 63.6% at 110 MHz.

Serum leptin concentrations in Lipizzan fillies.

To determine seasonal changes in circulating leptin concentrations, as well as leptin fluctuations in various patterns of seasonal ovarian activity, blood samples were taken in 107 Lipizzan fillies aged 1-4 years over the whole year from January to December. Serum concentrations were measured in samples obtained once a month (leptin) or twice a month (progesterone), and evaluated with respect to the age of animals (yearlings, 2-, 3- and 4-year olds) and the pattern of ovarian activity (anoestrous, seasonal cycle, prolonged cyclicity, continuous cyclicity). The mean and monthly leptin concentrations in 3- and 4-year olds were significantly higher than in yearlings and 2-year olds (p < 0.001). Circulating leptin levels in 3- and 4-year olds increased significantly in summer, to peak in August and then decline significantly (p < 0.001) in autumn. The fluctuations in younger fillies were less evident. The highest mean serum leptin concentrations were observed in fillies exhibiting continuous cyclicity and the lowest in those, exhibiting seasonal cyclicity (p < 0.001). In anoestrous fillies, leptin concentrations were lower than in fillies with prolonged or continuous cyclicity but higher than in fillies with seasonal cyclic activity (p > 0.05).

Comparison of different molecular methods for assessment of equine arteritis virus (EAV) infection: a novel one-step MGB real-time RT-PCR assay, PCR-ELISA and classical RT-PCR for detection of highly diverse sequences of Slovenian EAV variants.

In the present study, a new one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) strategy with minor-groove-binder (MGB) technology for the detection of EAV from 40 semen samples of Slovenian carrier stallions was tested. A novel MGB probe (EAVMGBpr) and a reverse primer (EAV-R) based on the multiple sequence alignment of 49 different EAV strain sequences of the highly conserved ORF7 (nucleocapsid gene) were designed. The performance of the assay was compared with different molecular detection methods. Three different primer pairs targeting the ORF1b and ORF7 were used, respectively. The real-time RT-PCR assay was at least 2 log(10) more sensitive than the classical RT-PCR and at least 1 log(10) more sensitive than the primer set used in the semi-nested PCR. The specificities of the amplification reactions were confirmed with biotinylated probes in the PCR-enzyme-linked immunosorbent assay (PCR-ELISA). Under the conditions described in our study, the sensitivity of the real-time RT-PCR was found to be superior to the PCR-ELISA assay. Thus, while the PCR-ELISA method was found to be both relatively demanding and time consuming, better sensitivity coupled with high specificity and speed of the assay makes the real-time RT-PCR a valuable tool for diagnosis of EAV infection.

Onset of puberty and duration of seasonal cyclicity in Lipizzan fillies.

REASONS FOR PERFORMING STUDY: The onset of puberty and duration of seasonal cyclicity in Lipizzan fillies and young mares have been determined mostly by empirical data, but very little is known about the hormonal changes at puberty and during the seasonal cyclicity in this breed. OBJECTIVES: To determine the onset of puberty and the course of the cyclicity in young Lipizzan mares from age 1-4 years. METHODS: Blood samples were collected twice a month for 2 years from 46 Lipizzan fillies age 1-4 years. Progesterone concentrations, indicating ovarian activity, were measured using an EIA kit. Values above 6.4 nmol/l were taken to indicate luteal phase and those below 3.4 nmol/l, oestrus. RESULTS: Fifty percent of Lipizzan fillies entered puberty during the first spring after their birth at mean +/- s.e. age 15.25 +/- 0.31 months and 50% during the second spring, at age 25.2 +/- 0.57 months. For several fillies of all ages, manifesting anoestrus during the winter months, the oestrus started in March and reached a peak in June to September. The duration of the oestrus activity was age-dependent, at 2.37 +/- 0.62 months in yearlings and 8.56 +/- 0.46 months in fillies age 4 years. In December, >50% of fillies age 2-4 years still exhibited oestrus activity and some entered anoestrus in January the following year. A small proportion of fillies age 3-4 years remained cyclic during the whole year, indicating that factors other than photoperiod can be involved in regulating seasonal cyclicity. CONCLUSIONS AND POTENTIAL RELEVANCE: Our results demonstrate the influence of seasonal factors on the onset of puberty and on the course of oestrus activity in the majority of Lipizzan fillies. However, certain exceptions were also demonstrated but reasons for the prolonged periods of oestrus activity in some Lipizzan fillies were not identified, and the role of the hormone leptin should be studied in future investigations.

Establishment of a pregnancy following intravaginal insemination with epididymal semen from a dog castrated due to benign prostatic hyperplasia.

Benign prostatic hyperplasia was diagnosed in an American Staffordshire Terrier of high breeding value presenting concurrent haematuria. Castration as a treatment was synchronized with the oestrus cycle of a bitch selected for insemination. After castration the cauda epididymis was flushed with Gent semen extender and collected spermatozoa were filtered and analysed by Hamilton Thorn computer assisted sperm analysis. A total of 7 ml semen containing 742 x 10(6) spermatozoa with 76.5% mean motility was used for insemination. Intravaginal insemination of the bitch was performed with an insemination catheter for dogs (Kruuse, Marslev, Denmark) on the day when plasma progesterone levels reached 9.9 ng/ml. Normal pregnancy without complications resulted in eight live-born puppies 63 days after insemination. This is the first report of a normal pregnancy and birth of puppies from a bitch inseminated with epididymal semen obtained from a dog affected by benign prostate hyperplasia.

Freezability of equine semen after glass beads column separation.

REASONS FOR PERFORMING STUDY: The success rate of artificial insemination following the freezing of stallion semen is limited; therefore, improving the stallion semen quality after the freezing and thawing process is a necessary objective. OBJECTIVES: To investigate the influence of glass bead column separation on the freezability of stallion semen. HYPOTHESIS: Glass beads in a column separator remove damaged and dead spermatozoa in the ejaculate during centrifugation. METHODS: In total, 50 ejaculates from 6 Lipizzaner stallions were studied. Each ejaculate was divided into 2 parts, one half processed following standard procedure and the second half used for the column separation procedure. After freezing, semen quality was evaluated using standard tests for motility, morphology and viability of semen. RESULTS: Motility and progressive motility of the column-separated (CS) semen were significantly higher (P < 0.001) before freezing and immediately, 24 and 48 h after thawing. A significant increase (P < 0.001) in the percentage of hypoosmotic positive spermatozoa was observed in CS samples. The percentage of total morphological changes in the separated samples before and after freezing was significantly lower (P < 0.001) compared with samples prepared using the standard procedure. A substantial decrease (P < 0.001) was found in the percentage of spermatozoa with damaged acrosomes. However, the percentage of spermatozoa with coiled tails was increased in the separated samples (P < 0.001). CONCLUSIONS: Column separation before freezing has a positive effect on the quality of thawed equine semen. POTENTIAL RELEVANCE: The quality of CS frozen/thawed samples indicates their potential use for increasing insemination success in mares.

Spectrophotometric application of resazurin reduction assay to evaluate boar semen quality.

The resazurin reduction assay depends on the ability of metabolically active cells to reduce the resazurin redox dye to resorufin. In the present study we applied and made a diagnostic evaluation of a spectrophotometric application of the resazurin reduction assay to assess the colour change of resazurin reduction in butanol extracted colour to evaluate boar semen quality. Forty-one samples of boar semen from various breeds were included in the study. The absorption peaks for resazurin and resorufin were found to be 610 and 575 nm, respectively. Absorbance at 610 nm, where the minimum overlap of the two peaks was observed, was used in further analysis. Spearman rank correlation analysis was used to determine the correlation between the resazurin reduction assay and various semen parameters. The highest correlations were observed with the concentration of motile spermatozoa (r = -0.841; p < 0.001), sperm concentration (r = -0.833; p < 0.001), sperm index (-0.826; p < 0.001) and concentration of viable spermatozoa (r = -0.763; p < 0.001). Sensitivity and specificity, at 94.1 and 91.7%, respectively, indicate that the present test is highly accurate in discriminating between the samples according to the sperm index. When motile sperm concentration was used to distinguish between good and poor samples, high sensitivity (93.6%) was also found, whereas the test was only moderately, 80%, specific. The stability of butanol extracts in terms of A610 at different times of measurement confirmed that the resazurin reduction could be spectrophotometrically measured within 7 days from the time of assay performance, making the assay much more useful. Based on these results, the assay could be used as an additional tool for evaluating the quality of boar semen.

The variations of white blood cell count in Lipizzan horses.

Total and differential leucocyte counts (lymphocytes, neutrophils, monocytes, eosinophils and basophils) were measured in 140 stallions, 101 mares and 25 foals of Lipizzan breed. The values fell in the normal ranges for warm-blooded horses. Differences between mares and stallions were not significant with the exception of foals, having higher white blood cell, neutrophil, lymphocyte, monocyte and basophil values in females than in males. Foals exhibited an age-related increase of total leucocyte count during the first 4 months of life, accompanied by a decrease in neutrophil and increase in lymphocyte and eosinophil counts. In mares and stallions, the total number of leucocytes, lymphocytes, monocytes and basophils significantly decreased but the number of neutrophils and eosinophils remained almost unchanged with age gain.

Microcystin-LR affects cytoskeleton and morphology of rabbit primary whole embryo cultured cells in vitro.

Microcystin-LR is the most frequently studied cyclic heptapeptide produced by different genera of cyanobacteria and is hepatotoxic to livestock and human populations. The adverse effects of microcystin-LR on morphology and cytoskeletal elements in different stages of early embryonal development have been studied in vitro. Embryos and whole embryo cultures have been exposed to microcystin-LR (10-100 microM). Actin filaments were visualized by fluorescence staining and the microtubular network labelled by immunostaining. Growth, development and cytoskeleton organization of the embryos embedded in zona pellucida are not affected by microcystin-LR in concentrations up to 100 microM, while whole embryo cell cultures are affected by the presence of microcystin-LR in the culture medium. High microcystin-LR concentrations (100 microM) cause cells to be detached and destroyed, while lower concentrations (10-20 microM) profoundly affect actin and microtubule organization. These effects are confirmed also by the presence of transformed microcystin-LR in all the media at the lowest concentrations. It seems that the changes to the cells are far more serious than that expressed in cell morphology. From our experiments we conclude that the presence of zona pellucida is an effective way of embryo protection against xenobiotics like microcystin-LR.

Postnatal hypothyroidism does not affect prepubertal testis development in boars.

Young boars were treated with propiothiouracil to induce hypothyroidism to examine its effects on postnatal testicular development. Treatments with 0.1% 4-propyl-2-thiouracil (PTU) in drinking water started after weaning, at 3 weeks of age and all boars were severely hypothyroid at 6 weeks of age as determined by measuring T3 and T4 in blood plasma. Boars were castrated at different ages up to 20 weeks and their testes used for histological and immunohistochemical analyses. Although small but significant reduction in testis weight was observed from 8 to 12 weeks of age, this was not accompanied by significant difference in testicular volume. By 20 weeks of age, at the beginning of puberty, the differences in testis weights between control and treated groups of boars disappeared suggesting there is no lasting effect of hypothyroidism on postnatal development of boar testis. Immunohistochemical staining was used to determine the presence of molecular markers in both Sertoli and Leydig cells. Again, there were no differences between testes from control and treated boars in the pattern or intensity of immunostaining using antibodies against 3beta-hydroxysteroid dehydrogenase, antimullerian hormone or proliferating cell nuclear antigen (PCNA). Immunostaining with antibodies against PCNA showed interesting results as it was observed that Sertoli cells still express this marker of proliferating cells at 14 weeks of age, later than previously suggested cessation of Sertoli cell proliferation. This study suggests that hypothyroidism in boars does not have similar effects on postnatal testis development as reported in some other species.

Chemical solution-deposited PbZr 0.53 Ti 0.47 O3 on La 0.5 Sr 0.5 Co O3. SIMS investigation of the effect of different precursor additives on the layer structure.

Chemical solution-deposited thin films of PbZr(0.53)Ti(0.47)O(3)/La(0.5)Sr(0.5)CoO(3) on Pt/TiO(2)/SiO(2)/Si substrates have been investigated by dynamic SIMS. The PbZr(0.53)Ti(0.47)O(3) (PZT) is intended to serve as a ferroelectric layer for microelectronic or microelectromechanical applications; conducting La(0.5)Sr(0.5)CoO(3) (LSCO) is a buffer layer intended to eliminate fatigue effects which usually occur at the Pt/PZT interface. Depth profiles of the main components were obtained and revealed that significant diffusion occurred during the deposition and crystallisation processes. Two types of sample, with different thickness of PZT and different types of poly(vinyl alcohol) (PVA) added to the LSCO precursor, were investigated.

The influence of age and gender on haematological parameters in Lipizzan horses.

Haematological parameters [red blood cell count (RBC), white blood cell count (WBC), packed cell volume (PCV), haemoglobin concentration, mean cell volume (MCV), mean cell haemoglobin (MCH) and mean cell haemoglobin concentration (MCHC)] in resting Lipizzan horses were determined for 143 stallions, 104 mares and 25 foals. The mean RBC and WBC values in Lipizzans were in the lower part of the normal range for warm-blooded horses. The mean PCV, MCV and MCH values were higher, but the mean haemoglobin concentration and MCHC values were lower than reported for other warm-blooded horses. In foals, the mean RBC, WBC, PCV, haemoglobin concentration and MCHC values were higher, whereas MCV and MCH were lower than in older animals. Results indicating a significant decrease in WBC (P < 0.01) and an increase in MCV, MCH and MCHC (P < 0.05) with increasing age are consistent with some other reports on warm-blooded horses. The age-related variations in RBC and PCV were less marked. Contrary to some reports, RBC (P < 0.01), WBC and haemoglobin concentration (P < 0.001) were higher in Lipizzan stallions than in mares, but differences in MCHC, MCH and MCV were insignificant. The specific haematological values determined in Lipizzans are presumably a result of selection and should be taken into consideration when dealing with this race of horses.

Subchronic liver injuries caused by microcystins.

The subchronic effects of cyanobacterial lyophilizate (CL) containing microcystins on liver were investigated in female New Zealand rabbits. Sterilised CL containing microcystins was injected i.p. Liver toxicity was assessed by histological examination of liver samples. Non-invasive magnetic resonance imaging (MRI) of liver was also performed in order to assess changes in the homogeneity of liver tissue. Subchronical intoxication with microcystins caused morphological changes of liver tissue that were also detected by use of MRI. Histological analysis showed that changes seen on MRI represent liver injury characterised with fatty infiltration and periportal fibrosis. This demonstrates that subchronic exposure to microcystins can lead to liver degeneration, which can easily be detected in vivo by use of MRI.

Decreased cortisol secretion by adrenal glands perfused with the P-glycoprotein inhibitor valspodar and mitotane or doxorubicin.

The aim of this study was to investigate the role of P-glycoprotein (P-gp) in the adrenal gland. It has been presumed that P-gp, rather than being involved in physiological cortisol secretion, plays a role in protecting the adrenacortical cells from xenobiotics. To explore this a study was performed on perfused bovine adrenal glands. Individual experimental groups were perfused with either a selective P-gp blocker (valspodar) alone, with a xenobiotic (mitotane or doxorubicin) alone or with both valspodar and a xenobiotic. The cumulative amounts of cortisol secreted in each individual group were calculated and the two-sample t-test was used to compare the mean values of cumulative amounts. The mean value of cortisol secreted from the group of adrenals perfused with the P-gp blocker was not significantly different from that of the control group. Treatment with either mitotane or doxorubicin decreased the amount of cortisol secreted but not significantly when compared to the amount of cortisol secreted in basal conditions. However, treatment with the P-gp blocker valspodar in addition to either mitotane or doxorubicin significantly decreased cortisol secreted compared to the amount of cortisol secreted by the glands treated with either mitotane (p=0.009) or doxorubicin (p=0.017) alone. The regressive changes discovered in all experimental groups were most prominent when valspodar was used with either mitotane or doxorubicin. We found that P-gp blockade increases by xenobiotic (mitotane and doxorubicin)-induced damage of adrenocortical cells, which points to a role of P-gp in the protection of adrenal gland from xenobiotics.

[Ultrasound guided surgical reduction of twin pregnancies in the horse up to the 46th day of pregnancy]. / Ultraschallgestützte chirurgische Reduktion von Zwillingsanlagen beim Pferd bis zum 46. Graviditätstag.

Methods for elimination of one of two embryos in twin pregnancies of mares were studied. Transvaginal ultrasound guided punction of yolk sac or allantoic cavity with fluid aspiration was used. Punction was done in 8 mares, pregnant between 22 and 46 days. In six cases, pregnancy did continue normally with one embryo only while in two cases, both embryos died following the procedure.

Higher thyroid hormone levels in neonatal life result in reduced testis volume in postpubertal bulls.

Thyroid hormones appear to determine adult testis size in rodents by regulating the period of Sertoli cell proliferation in the neonatal period. In the present study, the correlation between neonatal thyroid hormone levels (T3 and thyroxin, T4) and postpubertal testis size in Simental bulls was examined. T3 and T4 levels were measured in blood plasma from 35 calves immediately after their arrival at the AI centre at age 3-6 months. Testis size (height and width) was measured at 12 months of age in the same live animals. A significant negative correlation (r = -0.55; p < or = 0.001) was found between T4 and calculated testicular volume using either the Pearson correlation test or linear regression analysis, while the levels of T3 and testis volume showed a negative correlation, although this did not reach statistical significance (r = -0.20, p < or = 0.05). The results of this study suggest, therefore, that neonatal thyroid hormone levels might have the same effect on testicular size in cattle as they do in rodents.