Protein Composition of Seminal Plasma in Fractionated Stallion Ejaculates

Seminal plasma (SP) contains several types of compounds derived from the epididymides and accessory glands. The aim of this study was to examine the protein composition of different ejaculate fractions. Trial I: fractionated ejaculates were collected from two normal and two subfertile stallions. Samples containing pre‐sperm fluid and the first sperm‐rich jets (HIGH‐1), the main sperm‐rich portion (HIGH‐2), the jets with low sperm concentrations (LOW), and a combined whole‐ejaculate (WE) sample was centrifuged, and the SP was filtered and frozen. A part of each SP sample was stored (5°C, 24 h) with spermatozoa from HIGH‐2 and skim milk extender. Sperm motility was evaluated after storage in extender mixed with the stallion’s own SP or SP from one of the other stallions (sperm from a normal stallion stored in SP from a subfertile stallion and vice versa). Protein composition was analysed using reverse‐phase liquid chromatography (RP‐HPLC), N‐terminal sequencing and mass spectrometry. The area‐under‐the‐curve (AUC) was used for quantitative comparison of proteins within fractions. Trial II: semen samples were collected from seven stallions. Fractions with the highest (HIGH) and lowest (LOW) sperm concentrations and WE samples were examined using SDS‐PAGE and densitometry. No significant differences emerged between fractions in the AUC‐values of the Horse Seminal Protein‐1 (HSP‐1) and HSP‐2 peaks, or the peak containing HSP‐3 and HSP‐4 (HSP‐3/4). Levels of HSP‐1, HSP‐2 and HSP‐3/4 were not significantly correlated with total sperm motility, progressive sperm motility or average path velocity after storage. Significant differences between ejaculate fractions in the amount of different protein groups present in SP were not found in Trial I; but in Trial II, the proteins in the 60–70 kDa range were more abundant in LOW than in HIGH and WE, indicating that this band contained proteins derived mainly from the seminal vesicles, which produce most of the SP in LOW.     

Detection of transgenic and endogenous plant DNA in digesta and tissues of sheep and pigs fed Roundup Ready canola meal

The persistence of plant-derived recombinant DNA in sheep and pigs fed genetically modified (Roundup Ready) canola was assessed by PCR and Southern hybridization analysis of DNA extracted from digesta, gastrointestinal (GI) tract tissues, and visceral organs. Sheep (n = 11) and pigs (n = 36) were fed to slaughter on diets containing 6.5 or 15% Roundup Ready canola. Native plant DNA (high- and low-copy-number gene fragments) and the cp4 epsps transgene that encodes 5-enolpyruvyl shikimate-3-phosphate synthase were tracked in ruminal, abomasal, and large intestinal digesta and in tissue from the esophagus, rumen, abomasum, small and large intestine, liver, and kidney of sheep and in cecal content and tissue from the duodenum, cecum, liver, spleen, and kidney of pigs. High-copy chloroplast-specific DNA (a 520-bp fragment) was detected in all digesta samples, the majority (89-100%) of intestinal tissues, and at least one of each visceral organ sample (frequencies of 3-27%) from sheep and swine. Low-copy rubisco fragments (186- and 540-bp sequences from the small subunit) were present at slightly lower, variable frequencies in digesta (18-82%) and intestinal tissues (9-27% of ovine and 17-25% of porcine samples) and infrequently in visceral organs (1 of 88 ovine samples; 3 of 216 porcine samples). Each of the five cp4 epsps transgene fragments (179-527 bp) surveyed was present in at least 27% of ovine large intestinal content samples (maximum = 64%) and at least 33% of porcine cecal content samples (maximum = 75%). In sheep, transgene fragments were more common in intestinal digesta than in ruminal or abomasal content. Transgene fragments were detected in 0 (esophagus) to 3 (large intestine) GI tract tissues from the 11 sheep and in 0-10 of the duodenal and cecal tissues collected from 36 pigs. The feed-ingested recombinant DNA was not detected in visceral tissues (liver, kidney) of lambs or in the spleen from pigs. Of note, however, one liver and one kidney sample from the pigs (different animals) were positive for a 278-bp fragment of the transgenic cp4 epsps (denoted F3). Examination of genomic libraries from these tissues yielded no conclusive information regarding integration of the fragment into porcine DNA. This study confirms that feed-ingested DNA fragments (endogenous and transgenic) do survive to the terminal GI tract and that uptake into gut epithelial tissues does occur. A very low frequency of transmittance to visceral tissue was confirmed in pigs, but not in sheep. It is recognized that the low copy number of transgenes in GM feeds is a challenge to their detection in tissues, but there was no evidence to suggest that recombinant DNA would be processed in the gut in any manner different from endogenous feed-ingested genetic material.     

Impact of Pediococcus pentosaceus and Pichia anomala in combination with chitinase on the preservation of high‐moisture alfalfa hay

This study assessed the effects of applying Pediococcus pentosaceus (PED) or Pichia anomala (PIC) in combination with chitinase (CH) on the conservation characteristics and rumen digestibility of large round‐bale high‐moisture alfalfa hay (HMH). Alfalfa was wilted in the field to 23%–27% moisture and baled without (Control) or with PED + CH, PIC + CH and propionic acid (PA). The study was repeated yearly from 2012 to 2014. The PED + CH‐treated HMH had the numerically lowest high degree day (bale internal temperature >30°C) in all 3 years, which was less (p < .05) than Control HMH and PA in 2012. Treatments had no effect on pH, but mixed effects on chemical and microbial compositions across 3 years. The PED + CH treatment had the highest water‐soluble carbohydrate (WSC) content (p < .05) in 2012 and 2014, whereas PA treatment had the lowest (p < .05) WSC in these years. Total bacteria were decreased (p < .001) by PIC + CH and PED + CH in 2012 only. The PED + CH treatment had greater (p < .01) soluble fraction of dry matter (DM) than Control HMH and PA in both 2012 and 2014, but treatments did not affect the potential degradable fraction of DM. The PED + CH and PIC + CH treatments increased (p < .01) in situ neutral detergent fibre digestibility after 6, 24 and 96 hr of ruminal incubation in 2012 but not in 2014. Combinations of P. pentosaceus or P. anomala with chitinase have potential as high‐moisture hay inoculants to reduce microbial spoilage and to increase fibre digestibility.     

Participation of phosphofructokinase,malate dehydrogenase and isocitrate dehydrogenase in capacitation and acrosome reaction of boar spermatozoa

The aim of this work was to determine the enzymatic activity of phosphofructokinase (PFK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in boar spermatozoa and study their participation in bicarbonate‐induced capacitation and follicular fluid‐induced acrosome reaction. Enzymatic activity of these enzymes was determined spectrophotometrically in extracts of boar spermatozoa. Sperm suspensions were incubated in the presence of bicarbonate (40 mM), a well‐known capacitation inducer, or follicular fluid (30%), as an acrosome reaction inducer, and different concentrations of oxoglutarate, oxalomalate and hydroxymalonate, inhibitors of PFK, IDH and MDH, respectively. Capacitation percentages were determined by the fluorescence technique of chlortetracycline (CTC), and true acrosome reaction was determined by trypan blue and differential–interferential contrast, optical microscopy. The activity of PFK in boar spermatozoa enzymatic extracts was 1.70 ± 0.19 U/10¹⁰ spermatozoa, the activity of NAD‐ and NADP‐dependent IDH was 0.111 ± 0.005 U/10¹⁰ and 2.22 ± 0.14 U/10¹⁰ spermatozoa, respectively, and the activity of MDH was 4.24 ± 0.38 U/10¹⁰ spermatozoa. The addition of the specific inhibitors of these enzymes prevented sperm capacitation and decreased sperm motility during capacitation and inhibited the acrosome reaction (AR), without affecting the sperm motility during this process. Our results demonstrate the participation of PFK, IDH and MDH in bicarbonate‐induced capacitation and follicular fluid‐induced acrosome reaction in boar spermatozoa, contributing to elucidate the mechanisms that produce energy necessary for these processes in porcine spermatozoa.     

The effectiveness of old and new strategies for the long‐term control of Pteridium aquilinum,an 8‐year test

There is a need for management strategies to control dominant perennial weeds and restore seminatural communities. We compared the effects of five weed control treatments on dense Pteridium aquilinum relative to an untreated experimental control over an 8‐year period with the aim of restoring acid grassland. The weed control treatments tested were as follows: cutting and bruising, both twice and thrice annually, and herbicide treatment (asulam in year 1 followed by annual spot retreatment of all emergent fronds). Pteridium aquilinum performance and plant species composition were monitored. Data were analysed using Bayesian mixed‐effect models and multivariate techniques. Cutting twice and thrice yearly and the asulam treatment all reduced frond density to zero; both bruising treatments were ineffective. The plant communities in the cut and asulam‐treated plots showed differences from the untreated and bruised plots; the asulam‐treated plots contained more ruderal species and the cut plots were more typical of acid grassland. Acid grassland recovery was fastest in the asulam‐treated plots, but the cut plots caught up after approximately 5 years. There were two important conclusions. First, an intractable weed like P. aquilinum can be eradicated and a vegetation more suited for grazing can be achieved by the continuous application of some treatments over many years. Here, success was achieved by cutting twice/thrice annually, or by a single asulam application followed by annual spot spraying of all emergent fronds for 8 years. Second, bruising, a treatment favoured by some conservation organisations, did not work and cannot be recommended. The use of long‐term, continuously applied treatments might be considered for all perennial weeds with large underground root/rhizome systems.     

Model uncertainty in the ecosystem approach to fisheries

Fisheries scientists habitually consider uncertainty in parameter values, but often neglect uncertainty about model structure, an issue of increasing importance as ecosystem models are devised to support the move to an ecosystem approach to fisheries (EAF). This paper sets out pragmatic approaches with which to account for uncertainties in model structure and we review current ways of dealing with this issue in fisheries and other disciplines. All involve considering a set of alternative models representing different structural assumptions, but differ in how those models are used. The models can be asked to identify bounds on possible outcomes, find management actions that will perform adequately irrespective of the true model, find management actions that best achieve one or more objectives given weights assigned to each model, or formalize hypotheses for evaluation through experimentation. Data availability is likely to limit the use of approaches that involve weighting alternative models in an ecosystem setting, and the cost of experimentation is likely to limit its use. Practical implementation of an EAF should therefore be based on management approaches that acknowledge the uncertainty inherent in model predictions and are robust to it. Model results must be presented in ways that represent the risks and trade‐offs associated with alternative actions and the degree of uncertainty in predictions. This presentation should not disguise the fact that, in many cases, estimates of model uncertainty may be based on subjective criteria. The problem of model uncertainty is far from unique to fisheries, and a dialogue among fisheries modellers and modellers from other scientific communities will therefore be helpful.     

Effect of exogenous corticosteroids on circulating virus and neutralizing antibodies in striped bass (Morone saxatilis) infected with infectious pancreatic necrosis virus

Corticosteroids have been reported to induce immunosuppression in fish exposed to many types of bacterial antigens. We document a similar phenomenon in fish exposed to infectious pancreatic necrosis virus (IPNV). Fingerling striped bass that were injected with the steroid triamcinolone acetonide (100 mg/kg body weight) 24 hours before receiving intraperitoneal inoculation with IPNV became viremic 3 days post inoculation (dpi) and virus was still detected in the buffy coat cells 14 dpi. In contrast, viremia could not be detected after 7 dpi in fish that received virus but not steroids. Circulating virus neutralizing antibodies were first detected in steroid treated fish at 10 dpi compared to 7 dpi for the virus injected fish and titers were consistently lower in the steroid group. Steroid treatment of chronic IPNV-carriers did not induce detectable viremia nor alter circulating antibody levels in chronic IPNV-carriers. None of the striped bass demonstrated clinical signs of viral disease.     

Results of a new serologic test suggest an association of Waddlia chondrophila with bovine abortion.

Waddlia chondrophila is a little-known intracellular organism belonging to the order Chlamydiales that has twice been isolated from aborted bovine fetuses. To initiate an investigation of the possibility that W. chondrophila may be an abortifacient pathogen, a serologic test was developed and used to screen bovine sera that were submitted to the Washington Animal Disease Diagnostic Laboratory (Pullman, WA). A highly significant statistical association (P < 0.00001) was observed when comparing antibody titers in cows that had aborted with those in other classes of cattle. Although this result is consistent with the possibility that infection with W. chondrophila may be a cause of bovine abortion, it is also possible that seroprevalence simply increases with age or that exposure rates differ between cows and other classes of cattle. Future serologic studies should specifically compare antibody titers between aborting cows and carefully matched nonaborting cohorts.     

Spermatic and oxidative profile of domestic cat (Felis catus) epididymal sperm subjected to different cooling times (24, 48 and 72 hours)

Cooling stored epididymal samples for several days allows facilities to transport and process genetic material post‐mortem. Improvements to this practice allow the preservation of sperm from domestic cats, which are the ideal study model for wild felids. However, the modifications in spermatic features and the oxidative profile are not fully understood in cats. This information is necessary for the development of biotechniques, such as new extenders for cryopreservation. Therefore, the purpose of this study was to evaluate the spermatic and oxidative profile in samples from the epididymal cauda of domestic cats cooled at 5°C for 24, 48 and 72 hr. Spermatozoa were collected from the epididymis cauda. Evaluations consisted of computer‐assisted sperm analysis (CASA), plasma membrane integrity (eosin/nigrosin), acrosome integrity (fast green/rose bengal), sperm morphology, sperm DNA integrity (toluidine blue), mitochondrial activity (3′3 diaminobenzidine), activity of the antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD), measurement of lipid peroxidation (TBARS) and protein oxidation. A decrease in sperm motility parameters was observed after 72 hr of cooling (i.e. total and progressive) with a higher percentage of minor (37.7 ± 6.3%) and total defects (53.4 ± 6.3%). Additionally, a decrease in high mitochondrial activity (Class I: 16.6 ± 2.2%) occurred after 72 hr. The decrease in motility rates after a long cooling time probably was caused by the increase in sperm abnormalities. A long cooling time causes cold shock and mitochondrial exhaustion, but there was no observed change with the oxidative stress condition. Therefore, cat epididymal sperm stored at 5°C appear to maintain a high quality for up to 48 hr of cooling time.