Sperm chromatin condensation as an in vivo fertility biomarker in bulls:a flow cytometry approach

Background:Genetic selection in cattle has been directed to increase milk production.This,coupled to the fact that the vast majority of bovine artificial inseminations(AI)are performed using cryopreserved sperm,have led to a reduction of fertility rates over the years.Thus,seeking sensitive and specific sperm biomarkers able to predict fertility rates is of vital importance to improve cattle reproductive efficiency.In humans,sperm chromatin condensation evaluated through chromomycin A3(CMA3)has recently been purported to be a powerful biomarker for sperm functional status and male infertility.The objectives of the present study were:a)to set up a flow cytometry method for simultaneously evaluating chromatin condensation and sperm viability,and b)to test whether this parameter could be used as a predictor of in vivo fertility in bulls.The study included pools of three independent cryopreserved ejaculates per bull from 25 Holstein males.Reproductive outcomes of each sire were determined by non-return rates,which were used to classify bulls into two groups(highly fertile and subfertile).Results:Chromatin condensation status of bovine sperm was evaluated through the combination of CMA3 and Yo-Pro-1 staining and flow cytometry.Sperm quality parameters(morphology,viability,total and progressive motility)were also assessed.Pearson correlation coefficients and ROC curves were calculated to assess their capacity to predict in vivo fertility.Sperm morphology,viability and total motility presented an area under the ROC curve(AUC)of 0.54,0.64 and 0.68,respectively(P>0.05),and thus were not able to discriminate between fertile and subfertile individuals.Alternatively,while the percentage of progressively motile sperm showed a significant predictive value,with an AUC of 0.73(P=0.05),CMA3/Yo-Pro-1 staining even depicted superior results for the prediction of in vivo fertility in bulls.Specifically,the percentage of viable sperm with poor chromatin condensation showed better accuracy and precision to predict in vivo fertility,with an AUC of 0.78(P=0.02).Conclusions:Chromatin condensation evaluated through CMA3/Yo-Pro-1 and flow cytometry is defined here as a more powerful tool than conventional sperm parameters to predict bull in vivo fertility,with a potential ability to maximising the efficiency of dairy breeding industry.     

Characterization of platelet and soluble-porcine P-selectin (CD62P)

P-selectin (CD62P), an adhesion molecule expressed on activated endothelial cells and platelets, mediates the initial attachment of leukocytes to the stimulated endothelium upon inflammation and the interaction between leukocytes and platelets. A soluble form of P-selectin is present in the serum of healthy individuals as a circulating protein and high levels have been described in various pathological situations. The aim of this study was to characterize P-selectin on porcine platelets and investigate the soluble form of this protein, which are uncharacterized in several animal species including pigs. A new monoclonal antibody (mAb) (SwPsel.1.9) against porcine P-selectin was produced using a mouse cell line transfected with pig P-selectin cDNA. This mAb together with a previously described mAb (P-sel.KO.2.5), produced in our laboratory, was used to develop an ELISA to quantify porcine P-selectin. No significant levels of soluble-porcine P-selectin were observed in healthy animals. However, the total amount of P-selectin measured in porcine platelets was similar to that found in humans. Increased levels of this circulating protein were detected in the plasma from pigs after allograft implantation. In vitro, P-selectin expression on platelet membrane was rapidly induced by PMA and thrombin, as assessed by flow cytometry. However, these activators did not stimulate the release of soluble P-selectin. Analysis of the proteolytic cleavage of this protein from COS-transfected cells revealed that PMA treatment failed to cause the shedding of membrane-bound P-selectin. These data suggest that porcine P-selectin is a suitable marker for inflammation and that the mechanism involved in the generation of circulating P-selectin is not proteolytic release.     

Resistance of potato cultivars to Synchytrium endobioticum in field and laboratory tests, risk of secondary infection, and implications for phytosanitary regulations

Laboratory (Spieckermann) tests, pot tests and field tests provided concordant evidence for the partial nature of resistance of potatoes to pathotypes 1 (D1) and 6 (O1) of Synchytrium endobioticum . Susceptible potato cultivars produced large warts (> 16 mm in diameter) in Spieckermann tests and had low field resistance levels (1–6). Field-resistant cultivars (levels 7–9) produced small warts or no warts at all in Spieckermann and field tests. In pot tests, at low inoculum levels (1 sporangium per 25 g soil) susceptible cultivars still developed warts, whereas field-resistant ones did not develop any warts below 25 sporangia per g soil. Above 35 sporangia per g soil, 100% disease incidence was observed in susceptible cultivars but only minimal wart development in field-resistant ones. Tests with continuous cultivation of potato cultivars in infected soil during three consecutive years showed that field-resistant cultivars will not support build-up of inoculum in soil. It is concluded that field-resistant cultivars do not create a risk of secondary infection, the criterion given for resistance in EU Directive 69/464/EC.     

Manipulation of growth in pigs through treatment of the neonate with clenbuterol and somatotropin

Neonatal pigs were treated with lipolytic agents to determine whether this would cause a long-term decrease in their ability to deposit fat, with a consequent increase in muscle growth and feed efficiency. Groups of 25 female piglets were given clenbuterol (100 microg/kg BW), porcine somatotropin (pST; 100 microg/kg BW), pST plus clenbuterol, or saline injections from 3 d to 40 d of age. Five piglets from each group were then slaughtered to determine body composition. Clenbuterol and pST both increased ADG up to weaning when given separately (24%, P < 0.05; 20%, P < 0.1 respectively) but did not reduce fat deposition. In contrast, pigs given clenbuterol plus pST showed no increase in ADG and a 41% reduction in carcass fat (P < 0.05). Clenbuterol caused a marked decrease in beta2-adrenoceptor density in porcine adipose tissue (P < 0.001) and skeletal muscle (P < 0.01). This effect was attenuated by concurrent pST treatment, which helps to explain the synergistic effect of these drugs on fat deposition. Once the drugs were withdrawn at 40 d, the anabolic effect of pST gradually disappeared, so that the live weight of pST-treated and control pigs was identical at 168 d. Clenbuterol withdrawal caused the rapid loss of extra weight gained, plus an additional 4 to 5 kg live weight that was never recovered. During the 4-wk finishing period there was an increase in feed intake in pigs that had previously undergone treatment with pST (23%, P < 0.1), with no increase in ADG, and so feed efficiency was impaired (P < 0.05). Pigs that were treated with pST plus clenbuterol showed no marked increase in feed intake during this period. Carcasses from clenbuterol-treated pigs tended to be leaner at 168 d, but there was no long-term effect of pST or the combined treatment on carcass composition. Overall, the treatment of neonatal pigs with repartitioning agents was counter-productive, due to the withdrawal effects of the beta-adrenefgic agonist and the delayed long-term effect of pST on feed intake.     

Mokola virus infection: description of recent South African cases and a review of the virus epidemiology

Five cases of Mokola virus, a lyssavirus related to rabies, are described. The cases occurred in cats from the East London, Pinetown and Pietermaritzburg areas of South Africa from February 1996 to February 1998. Each of the cats was suspected of being rabid and their brains were submitted for laboratory confirmation. Four of the cases were positive, but with atypical fluorescence, and 1 was negative. Mokola virus infection was identified by anti-lyssavirus nucleocapsid monoclonal antibody typing. As in rabies cases, the predominant clinical signs were of unusual behaviour. Aggression was present, but only during handling. Four of the 5 cats had been vaccinated for rabies, which is consistent with other studies that show that rabies vaccination does not appear to protect against Mokola virus. Since Mokola may be confused with rabies, the incidence of Mokola virus may be more common in Africa than is currently reported. As human infections may be fatal, the emergence of this virus is a potential threat to public health.     

Changes in populations of the eyespot fungi Tapesia yallundae and T. acuformis under different fungicide regimes in successive crops of winter wheat, 1984–2000

Effects of regular treatments with the fungicides carbendazim and prochloraz applied to whole plots divided into subplots with different initial population mixtures of carbendazim-sensitive or carbendazim-resistant Tapesia yallundae or T. acuformis were studied in successive crops of winter wheat from 1984/85 to 1999/2000. In unsprayed and carbendazim-sprayed whole plots, a stable coexistence of about 50% each of T. yallundae and T. acuformis developed within five seasons, but in whole plots sprayed with prochloraz or prochloraz plus carbendazim, the proportion of T. acuformis increased to > 80%. A discrete time difference equation model was derived from knowledge of the biology of eyespot and competition theory to describe the population changes. The model was fitted to the data from treatments where coexistence occurred [subplots in unsprayed (1985–92) and carbendazim-sprayed (1985–89) whole plots], using nonlinear least squares regression. The optimized value of the resource overlap coefficient was small, suggesting niche differences between the two species. Populations were nearly 100% carbendazim-resistant in carbendazim-sprayed whole plots by July 1985 (one season) and in whole plots sprayed with prochloraz plus carbendazim by July 1986 (two seasons). In prochloraz-sprayed whole plots, the proportion of carbendazim-resistant isolates decreased more rapidly than in unsprayed whole plots in the 1980s, but by July 1992 a shift in populations in unsprayed and prochloraz-sprayed whole plots towards predominantly carbendazim-resistant strains had occurred.     

Comparative in vitro fermentation activity in the canine distal gastrointestinal tract and fermentation kinetics of fiber sources

The current study aimed to evaluate the variation in fermentation activity along the distal canine gastrointestinal tract (GIT, Exp. 1). It also aimed to assess fermentation kinetics and end product profiles of 16 dietary fibers for dog foods using canine fecal inoculum (Exp. 2). For Exp. 1, digesta were collected from the distal ileum, proximal colon, transverse colon, and rectum of 3 adult dogs. Digesta per part of the GIT were pooled for 3 dogs, diluted (1:25, wt/vol), mixed, and filtered for the preparation of inoculum. A fructan, ground soy hulls, and native potato starch were used as substrates and incubated for cumulative gas production measurement as an indicator of the kinetics of fermentation. In addition, fermentation bottles with similar contents were incubated but were allowed to release their gas throughout incubation. Fermentation fluid was sampled at 4, 8, 12, 24, 48, and 72 h after initiation of incubation, and short-chain fatty acids and ammonia were measured. Results showed comparable maximal fermentation rates for rectal and proximal colonic inocula (P > 0.05). Production of short-chain fatty acids was least for the ileal and greatest for the rectal inoculum (P < 0.05). Therefore, for in vitro studies, fecal microbiota can be used as an inoculum source but may slightly overestimate in vivo fermentation. Experiment 2 evaluated the gas production, fermentation kinetics, and end product profiles at 8 and 72 h of incubation for citrus pectin, 3 fructans, gum arabic, 3 guar gums, pea fiber, peanut hulls, soy fiber, sugar beet fiber, sugar beet pectin, sugar beet pulp, wheat fiber, and wheat middlings. Feces of 4 adult dogs were used as an inoculum source. Similar techniques were used as in Exp. 1 except for the dilution factor used (1:10, wt/vol). Among substrates, large variations in fermentation kinetics and end product profiles were noted. Sugar beet pectin, the fructans, and the gums were rapidly fermentable, indicated by a greater maximal rate of gas production (R(max)) compared with all other substrates (P < 0.05), whereas peanut hulls and wheat fiber were poorly fermentable, indicated by the least amount of gas produced (P < 0.05). Sugar beet fiber, sugar beet pulp, soy fiber, and wheat middlings were moderately fermentable with a low R(max). Citrus pectin and pea fiber showed a similar low R(max), but time at which this occurred was later compared with sugar beet fiber, sugar beet pulp, soy fiber, and wheat middlings (P < 0.05). Results of this study can be used to formulate canine diets that stimulate dietary fiber fermentation along the distal GIT that may optimize GIT health and stimulate the level of satiety in dogs.     

Are protected areas preserving ecosystem services and biodiversity? Insights from Mediterranean forests and shrublands

Landscape Ecology - Protected areas (PAs) are essential for biodiversity conservation and the provision of ecosystem services (ES), representing 15% of the earth’s surface and targeted to...     

Spatiotemporal patterns of urbanization in three Swiss urban agglomerations: insights from landscape metrics,growth modes and fractal analysis

Landscape Ecology - Urbanization is the most important form of landscape change and is increasingly affecting biodiversity and ecosystem functions. Understanding how landscape patterns change in...     

猪喘气病免疫应考虑的因素——如何发挥疫苗的最大效力

猪喘气病是由猪肺炎支原体(Mycoplasma hy-opneumoniae,MH)感染引起的一种慢性消耗性疾病。临床表现咳嗽和气喘,患病动物体温、食欲变化