Effect of freezing bull semen in two non-egg yolk extenders on post-thaw sperm quality.

Traditionally, extenders for bull semen included egg yolk or milk, but recently there has been a move to avoid material of animal origin. The aim of this study was to evaluate the effects of two commercial extenders (based on soya lecithin and liposomes) on bull sperm quality after cryopreservation. Post-thaw sperm quality was evaluated by computer-assisted sperm analysis and flow cytometric assessment of membrane integrity, chromatin integrity, mitochondrial membrane potential, production of reactive oxygen species and tyrosine phosphorylation. Furthermore, an artificial insemination (AI) trial was conducted, and 56-day non-return rates were evaluated. Semen frozen in the liposome-based extender showed similar membrane integrity and higher mitochondrial membrane potential compared to those in the soya lecithin-based extender. Chromatin integrity and production of live H2 O2 + reactive oxygen species were similar in both extenders. Less superoxide was produced in the samples extended with liposome-based extender, with or without menadione stimulation. Chromatin integrity and tyrosine phosphorylation were not affected by either type of extender. No differences in 56-day non-return rate between extenders containing soya lecithin and liposomes were observed in the AI trial (66% ± 0.8 and 65% ± 0.8, respectively). In conclusion, the sperm quality of bull semen frozen in the two extenders that do not contain material of animal origin was similar, although the semen frozen in the liposome-based extender had higher mitochondrial membrane potential. Either extender could be used in situations where extenders containing material of animal origin are to be avoided.

Plasma aldosterone concentration and cardiovascular response to low sodium intake in horses in training.

REASON FOR PERFORMING THE STUDY: Horses in training lose large amounts of sodium but little is known about the cardiovascular response to low sodium intake. OBJECTIVES: To investigate the effect of low sodium intake on plasma aldosterone (pAldo) concentrations and the cardiovascular system of athletic horses, and to identify markers of low sodium intake. METHODS: Seven Standardbred geldings in training (trained twice a week) were randomly offered a standardised diet supplemented (NaS, 58 mg Na/kg bwt) and not supplemented (NaN, 3 mg Na/kg bwt) with NaCl for 5 weeks in a changeover design. Blood samples were taken once a week and in Week 5, before and following an exercise test until 22.30 h and analysed for blood sodium (bNa), total plasma protein (TPP), pAldo, troponin I and packed cell volume (PCV). Blood pressure (BP) was measured and pulse wave recorded at rest with high definition oscillometric-technique (HDO). ECG and echocardiography were recorded. Water intake was measured before and on the day of exercise and voluntary saline intake was measured for 2 days after each period. Faecal samples were taken weekly and analysed for sodium and potassium content. RESULTS: The pAldo and the PCV was higher in NaN compared to NaS. There were no differences between diets in BP, ECG, plasma troponin I and echocardiogram but HDO pulse amplitude tended to be smaller on diet NaN. Water intake was lower on diet NaN and saline intake higher. The response to exercise in bNa, pAldo, PCV and TPP was different on the 2 diets. Faecal potassium/sodium ratio was higher on NaN than on NaS. CONCLUSION: This study shows that 5 weeks of low sodium intake increased plasma aldosterone concentration and PCV but no alterations in heart function was observed. Faecal potassium/sodium ratio could be used to assess sodium status in horses.

Cryopreservation of epididymal cat spermatozoa: effects of in vitro antioxidative enzymes supplementation and lipid peroxidation induction.

Reactive oxygen species and lipid peroxidation reaction, causes of sperm damage, can be diminished by action of antioxidative enzymes. This study aimed to investigate effects of (1) the antioxidative enzymes; catalase, glutathione peroxidase and superoxide dismutase, on epipididymal cat sperm quality and (2) the lipid peroxidation reaction induced by a transition metal (ferrous ion (II); Fe(2+)) on sperm quality during the cryopreservation process. Epididymal spermatozoa harvested from 39 male cats were pooled and divided into 13 aliquots (n=13). Each aliquot was resuspended with either a Tris egg yolk extender I (control; EE-I), or the Tris egg yolk extender I supplemented with 200 U/mL catalase (EE-CAT), or 10 U/mL glutathione peroxidase (EE-GPx), or 600 U/mL superoxide dismutase (EE-SOD), and then cryopreserved. After thawing, each sperm sample was subdivided into two groups; with and without lipid peroxidation induction (EE-I plus Fe(2+), EE-CAT plus Fe(2+), EE-GPx plus Fe(2+) and EE-SOD plus Fe(2+)). Subjective sperm motility, membrane, and acrosome integrity were evaluated at the time of collection, after cooling, and at 0, 2, 4, and 6h after thawing. Motility patterns assessed by computer-assisted sperm analysis (CASA), mitochondrial activity, and DNA integrity were evaluated during post-thaw incubation, whereas percentage of lipid peroxidation was detected at 0 and 6h after thawing. The results demonstrate that catalase supplementation reduced linear motility and subjective motility immediately and 2h after thawing (P<0.05). Catalase supplementation, however, improved DNA integrity at 4h (P<0.05). Supplementation with glutathione peroxidase, compared to the control group, had a statistically significant positive effect on subjective motility at 0 and 6h, linear motility at 6h, mitochondrial activity at 6h, membrane integrity at 2 and 6h, and DNA integrity at 4h after thawing. Although superoxide dismutase had a positive effect on sperm membrane integrity at 2h after thawing (P<0.05), it significantly reduced membrane integrity after cooling, linear motility at thawing, and acrosome integrity at 2h after thawing. None of the three selected antioxidative enzymes significantly influenced acrosome integrity and none reduced the level of lipid peroxidation. Furthermore, induction of the lipid peroxidation reaction by Fe(2+) negatively affected most of the sperm quality parameters, i.e., motility and DNA integrity, during post-thaw sperm incubation (P<0.05). After thawing, there were, however, no significant differences between the control plus Fe(2+) and the antioxidative enzymes supplementation plus Fe(2+) groups. We can conclude that (1) glutathione peroxidase exhibits positive effects on post-thaw epididymal cat spermatozoa; but (2) none among the selected antioxidative enzymes could improve all sperm quality parameters; and (3) the lipid peroxidation reaction may be one cause of post-thaw epididymal sperm damage in cats, but the concentrations of antioxidative enzymes used in this study could not protect cat spermatozoa from lipid peroxidation induction.

Antioxidant supplementation of boar spermatozoa from different fractions of the ejaculate improves cryopreservation: changes in sperm membrane lipid architecture.

Previous studies have shown sperm quality after cryopreservation differs depending on the fraction of seminal plasma the boar spermatozoa are contained in. Thus, spermatozoa contained in the first 10 ml of the sperm-rich fraction (portion I) withstand handling procedures (extension, handling and freezing/thawing) better than those contained in the latter part of a fractionated ejaculate (second portion of the sperm-rich fraction and the post-spermatic fraction; portion II). The present study evaluated whether an exogenous antioxidant, the water-soluble vitamin E analogue Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), could, when added to the freezing extender in a split-sample design trial, improve the post-thaw viability and membrane quality of this particular portion of the ejaculate, with particular attention to the status of the plasma membrane. Using a split-sample design, the initial changes in the fluidity status of the sperm plasmalemma after thawing were measured by flow cytometry (FC) after loading with Merocyanine-540 and YO-PRO-1. The FC-derived data revealed a clear ejaculate portion-dependent effect of the antioxidant supplementation. While no beneficial effect of the antioxidant supplementation was visible in spermatozoa from portion I, more spermatozoa with intact membranes were observed in the supplemented samples of portion II, suggesting the protective effect of vitamin E is dependent of the portion of the boar ejaculate considered.

Antioxidant supplementation in vitro improves boar sperm motility and mitochondrial membrane potential after cryopreservation of different fractions of the ejaculate.

Antioxidant supplementation during cooling was assayed to improve the motility of frozen-thawed (FT) boar spermatozoa from two different fractions of the ejaculate, the first component of the sperm-rich fraction (Fraction I) and the rest of the bulk ejaculate (Fraction II). Using a split-sample design, addition of two different concentrations (100 and 200 microMl(-1)) of the water-soluble Vitamin E analogue Trolox (6-hydroxy -2,5,7,8-tetramethylchroman -2-carboxylic acid) was evaluated for an effect on sperm motility (measured both subjectively and by means of a computer assisted motility assessment (CASA)), and on mitochondrial membrane potential using flow cytometry after cell-loading with JC-1. The effect of the Vitamin E analogue was clearly dose-dependent and varied with the fraction of the ejaculate considered. Motility was significantly higher in Trolox-treated spermatozoa (200 microm), from either ejaculate fraction, albeit the effect was more evident in spermatozoa from Fraction II (P<0.05) for any Trolox-concentration. Antioxidant supplementation resulted, also dose-dependent, in a higher number of spermatozoa showing high mitochondrial activity as assessed by the JC-1 staining, in both ejaculate fractions. In the present trial, exogenous Trolox positively affected post-thaw sperm viability (as motility and mitochondrial membrane potential) in both fractions of the ejaculate. The magnitude of the effect appeared, however, to be dependent of the fraction of the ejaculate considered.

Effect of subcutaneous injection of ginseng on cows with subclinical Staphylococcus aureus mastitis.

Cows with subclinical mastitis caused by Staphylococcus aureus were subjected to subcutaneous injections with either an extract from the root of Panax ginseng CA Meyer at a dose of 8 mg/kg body weight per day for 6 days, or with saline as a control. The injection areas were checked for adverse reactions. The daily milk production was measured before and after treatment. Blood was collected for total and differential leucocyte counts, identification of lymphocyte subpopulations using flow cytometry, lymphocyte proliferation test, and neutrophil phagocytosis and oxidative burst assay. Quarter milk samples were collected for bacteriological analysis and somatic cell counts (SCC). After the end of treatment, the numbers of S. aureus-infected quarters and milk SCC tended to decrease in ginseng-treated cows. Phagocytosis and oxidative burst activity of blood neutrophils were significantly increased 1 week after ginseng treatment, but the proliferative response of blood lymphocytes did not change significantly. The number of monocytes in ginseng-injected cows was significantly higher 1 week post-treatment than pre-treatment, and the number of lymphocytes was significantly higher than pre-infusion at 2 and 3 weeks after ginseng treatment. Similar changes were not observed in the control group. The present findings indicate that ginseng treatment can activate the innate immunity of cows and may contribute to the cow's recovery from mastitis. It is therefore suggested that ginseng has a potential as a stimulator of the immune system of dairy cows.

Opsonic capacity of foal serum for the two neonatal pathogens Escherichia coli and Actinobacillus equuli.

Two of the most commonly isolated foal pathogens are Escherichia coli and Actinobacillus equuli. The hypothesis tested in this study was that young foals carry a lower opsonic capacity for these bacteria compared to adult horses. A flow-cytometric method for the phagocytosis of these by equine neutrophils was established. The opsonic capacity of serum from healthy foals from birth to age 6 weeks was evaluated and related to the concentrations of IgGa and IgGb. Phagocytosis of yeast was used as a control. Serum was required for phagocytosis, with higher concentrations for E. coli than for A. equuli. Ingestion of colostrum led to a significantly higher serum opsonic capacity. After that, there was no consistent age-related trend for opsonic capacity for the different microbes. Foal serum showed similar or higher opsonisation of E. coli and A. equuli compared to serum from mature individuals. During the studied period, the predominance among IgG subisotypes switched from IgGb to IgGa. Although the overall correlation between concentrations of IgG subisotypes and serum opsonic capacity was poor, sera with IgGb levels below 1.9 mg/ml induced lower opsonisation of E. coli and yeast, but not of A. equuli. Complement activation was important for opsonisation of all tested microbes. The results of this study are significant to the understanding of a key immunological facet in the pathophysiology of equine neonatal septicaemia in clinical practice.

Dietary zinc oxide in weaned pigs--effects on performance, tissue concentrations, morphology, neutrophil functions and faecal microflora.

The uptake and distribution of zinc in tissues and the effects of 2500 ppm dietary zinc oxide on health, faecal microflora, and the functions of circulating neutrophils were evaluated in weaned pigs. One group was fed a zinc supplement diet and another group was used as a control. All pigs remained healthy throughout the study, but the supplemented animals showed better performance than the controls. The serum zinc values rose rapidly. At autopsy, carried out at the age of 63 days, the zinc concentrations in liver tissue were 4.5 times higher, and in renal tissue two times higher in the supplemented group than in controls (P<0.001). Microscopic examination showed increased lipid accumulation in hepatocytes from supplemented pigs. No effect on the number of excreted Escherichia coli and enterococci per gram faeces or on the functions of circulating neutrophils was observed. Dietary supplementation with 2500 ppm ZnO for up to two weeks after weaning appears to be potentially beneficial in the prevention of postweaning diarrhoea in pigs.

Attempts to use the HPRT-assay as an automated short-term monitor for an acute exposure to mutagens.

Attempts have been made to use the hypoxanthine-guanine-phospho-ribosyl-transferase-assay as a method for automated screening of agent-induced phenotypic variants of human peripheral lymphocytes reflecting 6-thioguanine resistance and assumed to indicate genotoxic action. Different protocols of the hypoxanthine-guanine-phospho-ribosyl-transferase-system were used in this study in order to investigate whether the system can be a candidate for a short-term test for a rapid and reliable identification of biological systems exposed to agents. The current protocols were based on: 1) fluoresceinated monoclonal antibodies against bromodeoxyuridine-DNA for labelling of 6-thioguanine-resistant human lymphocytes and direct flow-cytometric enumeration of bromodeoxyuridine-positive events and: 2) indirect flow-cytometric enrichment of 6-thioguanine-resistant cells labelled with 3H-thymidine followed by autoradiographic enumeration of positive events. Both the direct and the indirect enumeration method yielded similar results down to the range 10(-4) with respect to frequency of variants. For the less time-consuming direct enumeration method the resolution was limited due to non-specific binding of the antibody and false positives. It was, nevertheless, sufficient to score variants induced in vitro with the mutagens EMS, MMC and TT in the same range as e.g. that of cancer patients during and after chemotherapy or radiotherapy, or that of psoriasis patients during the after PUVA (8-methoxypsoralen and long range UV light)-therapy. We conclude that the direct enumeration protocol can be used for a rapid screening of so called outliers, but a more sensitive test, such as the more time-consuming enrichment protocol based on autoradiography, must be used in order to score variants in the range 10(-5)-10(-6).