Mediterranean Sea, Egypt is an economically important marine environment. During the last decades there has been extensive increase in the levels of urbanization and industrialization along its coastal area. Therefore, the present work attempts to determine the status of heavy metals distribution in sediment samples, and their ecological risk assessment in the studied area.Twenty surfacial sediment samples were collected from different selected stations along the Egyptian Mediterranean Sea. The samples were homogenized and placed into sealed polyethylene bags, carried to the laboratory in an ice box and stored at −20 °C in the dark until analysis.−1) followed by Mn (17–1086 μgg−1), and a lower concentrations were found for Co (0.43–26.39 μgg−1) and Cd (0.04–0.47 μgg−1). Risk assessment showed that Cd had the highest ecological risk (Er = 21.52), followed by Pb (Er = 3.01), while Zn had the lowest risk (Er = 0.23). Both the ecotoxicological index method and the potential ecological risk index (RI) suggested that the combined ecological risk of the studied metals may be low. Multivariate statistical analysis  suggested that the lithogenic factor dominants the distribution of most part of the considered metals in the study area.The results revealed that Fe had the highest mean value (243–38045 μggMultivariate analysis has been proved to be an effective tool for providing suggestive information regarding heavy metal sources and pathways. The results of this study provide valuable information about metal contamination in sediments along the Mediterranean Sea for over than 1200 km. Heavy metals pollution of aquatic environment has become a great concern in recent years . Heavy mTo date, many methodologies have been developed to assess ecological risks of heavy metals. However, most of them are suitable only for ecological assessment of a single contaminant . In reality, many kinds of heavy metals usually accumulate simultaneously and cause combined pollution. To address this, Hakanson  develope3 per day domestic sewage and 1–2 million cubic meters per day of agricultural wastes [The coastal zone of Egypt on the Mediterranean extends over 1200 km from Rafah to El-Salloum . Unfortul wastes . Rashid,l wastes .The aim of the present study was to: (1) provide the concentration and distribution of some heavy metals in the Egyptian Mediterranean Sea sediments. (2) evaluate the potential ecological risk levels of some heavy metals by applying the Potential Risk Index Method. (3) investigate the biological effects of some heavy metals concentrations using available Sediment Quality Guidelines (SQGs); and (4) identify the sources of the heavy metals with multivariate analyses.The Egyptian Mediterranean coast extends between longitude 25° 30′E and 34° 15′E and extends northward to latitude 33° N  Twenty surfacial sediment samples (0–5 cm depth) were collected during July 2010 from different selected stations along the Egyptian Mediterranean Sea using Peterson grab sampler  according to Oregioni and Aston [For metal analysis, sediment samples were oven dried at 60 °C for almost a week. After drying, sediments were grounded to a size <63 μm in an agate mortar then stored in plastic vials until analysis. Then, the samples were digested in an open system with a mixture of concentrated HNOnd Aston . The detSediment pH was measured according to Yan et al.  with 1: −1, and their measured values are 38334, 460, 252, 47, 78, 31, 14, 77, and 0.70 μgg−1, respectively. The recovery of the selected elements ranged from 90 to 104 % and the measurements of precision was under 10 % RSD. The detection limits of the instrument for each metal were 0.023, 0.033, 0.036, 0.047, 0.039, 0.040, 0.037, 0.044, and 0.025 mg/l for Cd, Cr, Cu, Co, Fe, Mn, Ni, Pb, and Zn, respectively.To remove any contamination, all glassware and plastic vials were washed with 10 % nitric acid solution and rinsed thoroughly with Milli-Q water and dried. All reagents were Merck Chemiclas, Germany analytical grade or super pure quality. In order to check for the quality of the method applied for the analysis of heavy metals, the accuracy of the analytical method was estimated by analyzing sediment Standard Reference Material (IAEA-405): estuarine sediment, International Atomic Energy Agency, Vienna, Austria). Certified values of Fe, Mn, Zn, Cu, Cr, Ni, Co, Pb, and Cd are 37400, 495, 279, 47.7, 84, 32.5, 13.7, 74.8, and 0.73 μggNumerous sediment quality guidelines are used to protect aquatic biota from the harmful and toxic effects related with sediment bound contaminants . These gAnother classification system is the Hong Kong environmental Protection Department (EPD)  ClassifiU.S. National Oceanic Atmospheric Administration has developed Sediment Quality Guidelines (SQGs) for the assessment of sediment quality from the concentrations of contaminants using chemical and biological effects database . The cheAlthough background/reference concentrations do give a base to evaluate SQGs and are important in environmental studies, they provide little insight into the potential ecological impact of contaminants . Based oi is the concentration of element i in sediments, ERMi, PELi the guidelines values for the element i and n is the number of metals. Mean quotients are considered as useful tools for reducing a large amount of contaminants into a single number. By calculating mean quotients it is assumed that adverse effects to marine organisms caused by individual chemicals are additional limitation is that this approach does not consider all the chemicals present in sediments but only those include in the SQG list [Where CSQG list . Mean quSQG list . ERMQ vaSQG list . Consequir of a single element and the potential ecological risk index RI of the multielement can be computed via the following equations:if is the pollution coefficient of a single element of “i”; Cis is the measured level of sedimentary heavy metal; Cin is the background level of sedimentary heavy metal;Tir is the toxic response factor for the given element of “i”, which accounts for the toxic requirement and the sensitivity requirement. The toxic response factors for Pb, Cd, Cr, Cu, Zn and Ni and Mn were 5, 30, 2, 5, 1, 5 and 1, respectively [The assessment of the potential risk of the heavy metal contamination was proposed as a diagnostic tool for water pollution control purposes as a result of the increasing content of heavy metals in sediments and their subsequent release into the water, which could threaten ecological health . Potentiectively , 38. Aveectively  and averectively  were comectively . The aveectively  is selecr and RI values: (1) Er <40, low ecological risk; 40 < Er ≤80, moderate ecological risk; 80 < Er ≤160, appreciable ecological risk; 160 < Er ≤320, high ecological risk; and >320, serious ecological risk; (2) RI <150, low ecological risk; 150 < RI <300, moderate ecological risk; 300 < RI <600, high ecological risk; and RI ≥ 600, significantly high ecological risk.According to Hakanson , the folRI method covers a variety of researching domains, i.e., biological toxicology, environmental chemistry as well as ecology, and can evaluate ecological risks caused by heavy metals comprehensively .Statistical methods were applied to process the analytical data in terms of its distribution and correlation among the studied parameters. MINITAB (version 14) package software was used for statistical analyses of the metal data. Basic statistical parameters such as range, mean, median, standard deviation (SD), and skewness were computed along with correlation analysis, while multivariate statistics in terms of principal component analysis (PCA) and cluster analysis (CA) were also carried out.Figure In general, the results obtained in this study were in the range observed in other Mediterranean countries Table , or evenr = 0.92, p < 0.01), Co , Cr , and Ni . It means that these metals tend to accumulate together. The significantly positive correlation with Fe indicates that the metals were derived from similar sources and also moving together [r = 0.80, p < 0.01) and Pb , which suggested that these metals were redistributed in the sediments by the same physico-chemical processes or had a similar source [r = − 0.81, p < 0.01), Fe , Mn , Co  and Cr  with CaCO3. On the other hand, the negative correlation of TOC with Ni , Fe , Co  and Cr  suggested that TOC doesn’t have important role in the binding of these elements [In order to establish relationships among metals and determine the common source of metals in the Egyptian Mediterranean coast, a correlation matrix was calculated for heavy metals in the sediments. The data showed that strong positive correlation exists between Fe and Mn , respectively which indicate that these metals are not likely to have adverse effects on animals that live in the sediment. Only one station (El-Mex) which had a Pb concentration > ERL, indicated that Pb at El-Mex will likely to has effects on animals that live in this sediment. On the other hand, all the rest of the studied station had a concentration of Pb below the ERL value which indicates that Pb in the study area is not likely to have adverse effects on animals that live in sediments except station 5 in El-Mex. On the other hand, Ni at 30 % of sampling stations  had a value over the ERL value , respectively. This reflects that the adverse effects on animals live at these stations are frequently occurred. Stations 13, 16, and 17 in , respectively had concentration of Ni above the ERM value  which means that Ni probably has adverse effects on animals live in this sediment.Comparing results of the present study with ERL and ERM values, it was observed that Cd, Cu, and Zn at 100 % of sampling stations are below the ERL value  ranged from 0.01 to 0.34    Fig. , indicat24) Fig. , indicatir) and the potential ecological risk index (RI) are summarized in Table ir) of heavy metals in sediments of the Egyptian Mediterranean coast was Cd > Pb > Ni > Cr > Cu > Mn > Zn. The mean potential ecological risk coefficient of Cd, Cr, Cu, Mn, Ni, Pb and Zn were all lower than 40, which belong to low ecological risk. All the sampling sites were at low risk level where the RI values were much lower than 150.To confirm the above evaluation, we further calculated the potential ecological risk index (RI) of surface sediments from the Egyptian Mediterranean coast. The results of evaluation on potential ecological risk factor  has been proved to be an effective tool for providing suggestive information regarding heavy metal sources and pathways .The results of the principal component analysis; PCA on the data matrix obtained from total metal analysis of surface sediments along the study area are shown in Table Cluster analysis is often coupled with PCA to confirm results and provide grouping of variables . In thisThe results of this study provide valuable information about metal contamination in sediments along the Mediterranean Sea from El-Salloum to Rafah for over than 1200 km. The distribution pattern of heavy metals in the sediments followed the sequence: Fe > Mn > Cr > Ni > Zn > Pb > Cu > Co > Cd. Association with adverse biological effects to aquatic biota was also assessed using the classification of sediments and sediment quality Guidelines (SQGs). Ni and Cr exceeded the probable effect level (PEL) in 35 and 15 % of the sampling sites, respectively. The ecotoxicological index expressed as the mean ERM quotients (m-ERM-Q) suggested that the combination of Cd, Cr, Cu, Ni, Pb and Zn may have a 30 % probability of being toxic while, the (m-PEL-Q) showed only a 25 % probability of being toxic due to the combination of these metals. Similar results are also obtained by the potential ecological RI, with the average Er for heavy metals decreased in the order: Cd > Pb > Ni > Cr > Cu > Mn > Zn. Multivariate statistical analysis evidenced significant correlations between Fe, Mn, Co, Cr and Ni, suggesting similar sources and/or similar geochemical processes controlling the occurrence of these metals in the sediments. This study supports metal pollution monitoring and control for the Egyptian Mediterranean Sea. It will be a useful tool to authorities in charge of sustainable marine management.


Furthermore, the genetic differences in autozygosity seen in consanguineous families are currently being delineated at a population level, and support the exploration and comparison of phenotypes seen in this genetic background with other nonconsanguineous genetic backgrounds.IL36RN mutations have been reported to present within 11 years of each other . Here, we present a report of two sisters that carry identical compound heterozygous mutations in IL36RN, yet first presented 34 years apart, highlighting the requirement of further genetic or environmental factors.A 38‐year‐old European woman, with features consistent with severe GPP since 6 months of age, had been attending our department. Widespread small pustules were seen on the trunk and limbs, and numerous hospital admissions with severe pustulation and fever were a recurrent feature of childhood Fig. a. The paGiven the severity of her disease, recombinant soluble tumour necrosis factor‐α receptor treatment was started at the age of 30 years Fig. b, c. FolBy contrast, the proband's sister, who had never previously had skin disease, first developed widespread pustular psoriasis, warranting hospital admission, at the age of 34 years. Her clinical history was negative for antecedent infection, medication use or pregnancy. She was admitted to hospital and managed with topical therapy, prompting resolution of the rash after 12 days. She has subsequently had minimal disease affecting the scalp for a 6‐month period. Notably, she did not have oral pustules.IL36RN was performed given the phenotype, and demonstrated identical mutations (sequencing primers and conditions as previously described).IL36RN: c.338C>T (p.Ser113Leu) and c.368C>T (p.Thr123Met)  protein using the QuikChange® site‐directed mutagenesis kit  with the wild‐type (Wt) IL‐36Ra expression vector as a template.®–IL‐36Ra vectors in HEK293T cells, collected the cell lysate and performed sodium dodecyl sulfate polyacrylamide gel electrophoretic separation of proteins followed by immunoblotting with mouse monoclonal anti‐FLAG® M2 antibody . Similar to the Arg‐mutant of IL‐36Ra protein, expression of the Met‐mutant was markedly reduced compared with the Wt, suggesting instability of the Met‐mutant IL‐36Ra protein , consistent with the mutation resulting in loss of function  .et al. as a useful indicator of IL36RN mutations.We present this case to highlight the strikingly distinct phenotypes seen in two Table S1. Familial homo‐ and heterozygous cases of generalized pustular psoriasis where at least two siblings share familial interleukin 36 receptor antagonist mutations.Table S2. Cases of generalized pustular psoriasis with homozygous interleukin 36 receptor antagonist mutations, illustrating the range in age of onset seen across different populations.Click here for additional data file.


Eighty-four steers  (Charolais or Luing) were used. Temperament was assessed using two standardized tests: restlessness when restrained [crush score (CS)] and flight speed (FS) on release from restraint. Over a 56-day period individual animal dry matter intake (DMI) and weekly body weight was measured. Ultrasound fat depth was measured at the end of 56 days. Average daily gain (ADG), feed conversion ratio (FCR), and residual feed intake (RFI) were calculated. After the 56-day test period, animals were transported in groups of six/week to respiration chamber facilities. Blood samples were taken before and 0, 3, 6, and 9 h after transport. Plasma cortisol, creatine kinase (CK), glucose, and free fatty acids (FFA) were determined to assess physiological stress response. Subsequently, CH4 emissions were measured over a 3-day period in individual respiration chambers. CS (1.7 ± 0.09) and FS (1.6 ± 0.60 m/s) were repeatable  and correlated . Plasma cortisol, CK, and FFA concentrations increased after transport . Temperament (CS) and CK concentration were correlated . The extreme group analysis reveals that excitable animals  and higher stress response  were associated with lower DMI. ADG was lower in more temperamental animals . Fat depth was greater in steers showing calmer CS (P = 0.026) and lower plasma CK (P = 0.058). Temperament did not show any relationship with RFI or CH4 emissions. However, steers with higher cortisol showed improved feed efficiency (lower FCR and RFI) (P < 0.05) and greater CH4 emissions (P = 0.017). In conclusion, agitated temperament and higher stress responsiveness is detrimental to productivity. A greater stress response is associated with a reduction in feed intake that may both increase the efficiency of consumed feed and the ratio of CH4 emissions/unit of feed. Therefore, temperament and stress response should be considered when designing strategies to improve efficiency and mitigate CH4 emissions in beef cattle.The aim of this study was to assess individual differences in temperament and stress response and quantify their impact on feed efficiency, performance, and methane (CH It is widely accepted that breeding for more productive animals would reduce emissions intensity (Ei gram GHG/unit of product), thus mitigating relative GHG emissions (Livestock is a major contributor to greenhouse gas (GHG) emissions, responsible for climate change, which are dominated by cattle, responsible for three-fourths of the total . Methanes oxide NO are the4 emissions (4/unit of product), thus mitigating GHG emissions (4 emissions by reducing the emission intensity (Ei). However, the potential association between stresses with CH4 emissions has never been assessed previously in cattle.Stress responses induce changes in metabolism in order to increase energy availability. For instance, glucocorticoid hormones, with cortisol as the foremost stress mediator in mammals, play a key role in energy metabolism , by regumissions , it is wmissions . Therefo4 emissions derived from divergent stress responses. The objective of the present study was to assess the relationship between individual temperament and physiological response to a commercially relevant stressor (transport) and to quantify their association with feed efficiency, performance, and CH4 emissions in beef cattle.Cattle vary in stress response to handling and physical restraint reflecting differences in temperament . For insThe experiment was approved by the Animal Experiment Committee of SRUC (Home Office License PPL 60/4133) and was conducted in accordance with the requirements of the UK Animals (Scientific Procedures) Act 1986.n = 42; purebred Luing (LU) n = 42] with an average initial body weight (BW) of 547.5 ± 50.49 kg were housed at the SRUC Beef Research Centre in summer 2013. Cattle used in this experiment were part of a larger project to investigate the effect of cattle breed types, diets and dietary CH4 mitigation treatments on performance, feed efficiency, and CH4 emissions that have been published in Ref. , which were filled once a day using a forage wagon. The number of HOKO feeders within each pen was either five feeders (four pens) or six feeders (two pens). The diets consisted of (gram/kilogram DM) forage to concentrate ratios of either 520:480 (mixed) or 84:916 (concentrate). Within each basal diet, the steers were offered one of the following three treatments: (i) control containing rapeseed meal as the main protein source (CON), (ii) rapeseed meal was replaced with nitrate in the form of calcium nitrate , or (iii) rapeseed meal was replaced by higher-oil content rapeseed cake. The ingredient and chemical composition of the diets can be found in Duthie et al. (56), weekly BW, and ultrasound fat depth at the 12/13th rib were recorded  at the end of the test period. Temperament of the steers was assessed three times through the 56-day performance test (referred ahead as 56-day test) by observation of their behavioral response to handling associated with routine weighing.Eighty-four steers  . As no effects of pen, weight, and breed were found to influence temperament, correlation between temperament traits was calculated using Spearman correlation tests (Proc Corr). The effect of transport on the AUC of each stress biomarker  was calculated by variance analysis of the samples through time fitting “sampling time” as a fixed effect and “animal” and “cohort” as random effects. When ANOVA showed significant differences  and to daily CHn” was different for each covariable depending on the number of missing data points ranging from 21 animals in both temperament variables (CS and FS), 17/group in CK and FFA analyses to 10 steers/extreme group in the cortisol analysis. Animals in the two middle quartiles (Q2 and Q3) were not used for this “extreme groups” analysis. This group splitting was made to produce distinct populations of animals based upon the temperamental and physiological stress responses. To evaluate the extreme group effects a Proc Mixed model was used with “breed,” “diet,” and “treatment” as fixed effects, the binomial extreme group as a covariable and “pen” and “methane cohort” were included as random effects. Statistical significance was assumed at P ≤ 0.05 and tendencies at P ≤ 0.1 for all analyses.An “extreme groups” approach was also carried out in which animals were divided into groups that differed with respect to each covariable using quartile splits. In this analysis, animals that scored in the highest quartile (Q1) with respect to each stress biomarker or temperament measure were classified as High extreme and animals that scored in the lowest quartile (Q4) were regarded as Low extreme. The “P < 0.001) and 0.51 (P = 0.028) for CS and FS, respectively. There was a significant correlation between the temperament traits at each sampling point, indicating that an animal which struggled violently in the crush (high CS) exited the crush quickly. The two breeds did not differ (P > 0.1) in either temperament trait (CS and FS).The results of the CS and FS tests are shown in Table P = 0.032) lower levels at 9 h post transport (33.6 ± 6.23 ng/ml) compared to baseline (44.2 ± 6.61 ng/ml) and immediately after transport. The plasma glucose concentration averaged 83.5 ± 1.06 mg/dl and did not differ significantly over time (P > 0.1). The basal plasma CK concentration was 76.4 ± 3.40 U/l which significantly (P = 0.047) increased 3 h after transport (87.6 ± 3.62 U/l) and did not significantly decrease throughout time after transport (85.5 ± 3.47 U/l at 9 h post transport). The plasma FFA concentration increased significantly (P < 0.001) 3 h after transport (5.9 ± 0.67 mg/l) compared to baseline (3.4 ± 0.37 mg/l) and subsequently returned close to the baseline figure 9 h post transport (4.13 ± 0.46 mg/l).The plasma concentration of cortisol, glucose, FFA, and CK is shown in Figure r = 0.41, P = 0.019; FFA, r = 0.43, P = 0.016) and CK was correlated  with FFA. No relationship between glucose and cortisol was found, nor between glucose and either CK or FFA. When comparing the results of physiological stress response with temperament tests, there was a significant positive correlation between serum CK concentration and the average CS ; however, no significant relationship was found between any of the other physiological and temperament traits. Temperament and stress physiology measures did not differ (P > 0.1) according to diet, breed, and treatment.The AUC was then used to examine the relationship between each biomarker  and between biomarkers and temperament traits. The AUC of cortisol correlated with that of other biomarkers , ate less (DMI) (P < 0.001), and had similar ADG. Charolais steers were more efficient , had greater ADG (P < 0.01), lower DMI , and lower fat depth (P < 0.001) than Luing steers. ADG, BW, or DMI did not differ across dietary treatments (P > 0.05).As reported by Duthie et al. , steers 56) was lower in animals with a faster FS (P < 0.05). ADG tended (P < 0.1) to be lower in steers with a high CS. This relationship was confirmed with the FS results where a high escape velocity was associated with a lower ADG (P < 0.05). Fat depth also varied between groups of extreme temperament. The steers that were more excitable during the CS test showed lower fat depth compared to the calm group (P < 0.05). Extreme group analysis revealed no effect of temperament on FCR nor RFI (P > 0.1).No linear association was found between temperament variables (CS and FS) and any of the performance measures. However, extreme group analysis, represented in Table 56)  but also when animals were housed in the respiration chambers (DMICh) . Both the FCR and RFI were significantly higher, indicating poorer efficiency, in the low compared to the high cortisol group (P < 0.05). Fat depth tended to be lower in animals with higher concentrations of CK (P < 0.1).As plasma glucose concentration did not change as a result of transport, its relationship with performance and feed efficiency variables was not investigated. No linear relationship was found between stress biomarkers and performance variables. However, the extreme group analysis showed that high levels of stress biomarkers following transport were associated with a low DMI during the 56-day test (DMI4 than mixed diet (P < 0.001). For the mixed diet, CH4 yield (grams/kilogram DMI) was decreased by 17% when nitrate was added (P < 0.01). However, for the concentrate diet, neither nitrate nor rapeseed cake treatment decreased CH4 yield compared to control. Temperament (CS and FS) showed no relationship with CH4 emissions (gram/day or gram/kilogram DMI) using either linear regression or extreme group analysis  animals categorized with a higher cortisol release following transport emitted significantly (P < 0.05) more CH4 than animals with a lower cortisol response (Table 4/day (P < 0.05). No difference was evident when CH4 was corrected for feed intake. Extreme CK groups showed similar CH4 emissions expressed either as total emissions or corrected for DMI.According to Troy et al.  steers fis Table . None ofse Table . The extTemperament in beef cattle is a measure of the behavioral response to handling that can be assessed using CS and FS tests . AccordiThe potential of transport to cause stress in cattle has been well studied  and, as According to Grandin , animalsTemperament is associated with physiological responses to stress . For insEfficient cattle may have a lower environmental footprint as they consume less feedstuff to produce the same amount of product (meat or milk). The results obtained in this experiment, and published in Duthie et al. , evidenc56 found in these animals. Nkrumah et al. (r = −0.35) between temperament (FS) and DMI. The authors associated different temperaments with changes in feeding behavior that ultimately affected feed intake. Similarly, Café et al.  and lower physiological stress reactivity (higher fat depth) may be partially due to a higher DMIh et al.  also fouSome authors have suggested that differences in energy use may be associated with divergent responsiveness to stressors . Montanh4 emissions can be achieved by a direct antimethanogenesis effect or by improvements in livestock productivity that reduces Ei. In this study, both effects occurred. On the one hand, Troy et al.  increase with DMI  providing evidence that higher stress responsiveness is associated with greater CH4 emissions/feed intake. Again this result may be due to the association between stress and feed intake. Buddle et al. (4 (gram/kilogram DMI) emissions increase proportionally when feed intake is lower, effect of which may be exacerbated by the stress suffered during isolation to respiration chambers. Our results suggest that DMI, while steers were housed in the respiration chambers, was lower in animals categorized as having high expression of each of the stress biomarkers. Indeed, using the same animals and facilities of this experiment, Llonch et al. (4 emissions/unit of feed intake. According to the same authors, an increase of retention time and rumen fermentation of fibrous feed may be the reason of greater CH4 emissions relative to a given quantity of feed.Mitigation of CHy et al.  reportedwith DMI . Conseque et al.  providedh et al.  evidence4 emissions is a novel and interesting discovery. Reducing both individual sensitivity and environmental sources of stress could be important and easy-to-implement strategies to mitigate enteric CH4 emissions in cattle that deserves further investigations. Nevertheless, it is surprising that the association between CH4 and cortisol was not confirmed with the temperament traits. Perhaps transport and isolation in the chambers are stressors that differ physiologically and behaviorally to handling stress in which temperament was measured. Although previous studies have shown that temperament during handling extrapolates to other sources of stress , this m4 emissions. However, lower stress responses, monitored by plasma cortisol, were associated with decreased CH4 expressed as a proportion of DMI. The selection of appropriate breeds and inclusion of concentrate into the diet are strategies with proven efficacy to improve feed efficiency and reduce CH4 emissions. However, the associations of temperament with productivity, on the one hand, and stress responsiveness with CH4 emissions, on the other hand, show also a potential route for breeding and management strategies to improve production efficiency and mitigate GHG emissions. More studies are needed to examine the effects of stress on feed intake, digestibility, and enteric CH4 production.No relationship was found between temperament and feed efficiency. However, calmer cattle ingest higher quantities of feed and are associated with greater growth and fat deposition when comparing animals of extreme temperament. In line with temperament, a higher stress response monitored by cortisol, FFA, and CK serum concentration, is linked to greater feed intake. According to the results of this study, there is no evident relationship between temperament and CHC-AD, JR, ST, and RR contributed to the concept of the work. PL, MS, and SPT initiated, designed the study, and performed the experiment. PL and MS performed statistical analyses. PL, MS, MH, and SPT interpreted data. PL wrote the manuscript. C-AD, JR, ST, MH, and SPT contributed to the manuscript. All authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nycticebus pygmaeus), a small primate inhabiting tropical forests. Here, we show that pygmy slow lorises exposed to natural climatic conditions in northern Vietnam during winter indeed undergo torpor lasting up to 63 h, that is, hibernation. Thus, hibernation has been retained in at least one primate outside of Madagascar.Hibernation and short daily torpor are states of energy conservation with reduced metabolism and body temperature. Both hibernation, also called multiday torpor, and daily torpor are common among mammals and occur in at least 11 orders. Within the primates, there is a peculiar situation, because to date torpor has been almost exclusively reported for Malagasy lemurs. The single exception is the African lesser bushbaby, which is capable of daily torpor, but uses it only under extremely adverse conditions. For true hibernation, the geographical restriction was absolute. No primate outside of Madagascar was previously known to hibernate. Since hibernation is commonly viewed as an ancient, plesiomorphic trait, theoretically this could mean that hibernation as an overwintering strategy was lost in all other primates in mainland Africa, Asia, and the Americas. However, we hypothesized that a good candidate species for the use of hibernation, outside of Madagascar should be the pygmy slow loris ( Galago moholi), which uses daily torpor as an emergency responseOver the past decades, it has become clear that heterothermia and hypometabolism in endotherms, i.e., torpor and hibernation, are not restricted to temperate or arctic conditions. Lemurs, rodents, bats, marsupials, and monotremes use torpor in subtropical or tropical habitats, which can be quite seasonal and energy demanding1234a) reaches 5 °C8b) or metabolism. Moreover, this species is known to exhibit an apparently endogenously controlled body mass gain of up to 50% towards winter, as is typically seen in fattening hibernatorsN. pygmaeus in fact displays any form of torpor we continuously measured core body temperatures using intraperitoneally implanted data loggers over fall, winter, and subsequent spring. The animals were kept in large outdoor enclosures, which allowed for both recapture and veterinary surveillance, but were exposed to natural climatic conditions in northern Vietnam.All three of these criteria apply to pygmy slow lorises, which are small strepsirrhine primates inhabiting forests in Vietnam, Cambodia, Laos and China with mostly quite seasonal environments. In northern Vietnam, for example, the climate is profoundly seasonal with wet warm summers and cool dry winters during which ambient temperature . Minimum Tbs were similar in all animals, i.e., 12.0 °C, 11.0 °C, 11.0 °C and occurred at Tas of 6.5 °C, 6.0 °C, and 8.0 °C, respectively. Times of torpor entrances were not uniformly distributed (Rayleigh’s test P < 0.001) but predominantly occurred in the early morning (mean: 05:19 ± 0.21 h). Times of arousals also significantly clustered around a mean of 14:06 ± 0.19 h (Rayleigh’s test P < 0.001).Mean overall torpor bout duration was 15.0 ± 1.8 h (n = 91). Bouts of multiday torpor lasted on average 43.0 ± 3.0 h . The mean minimum Ta (computed over 24 h prior to noon of the day of torpor entry) decreased . Further, mean daily Tb was positively associated with Ta both on days with torpor (daily minimum Tb < 30 °C) and on euthermic days . During euthermic days, mean Tb obtained from all 5 animals investigated also was a linear function of mean Ta . Interestingly, this was also the case for maximum daily Tb .Lorises were more prone to enter torpor on cold days. The probability to undergo torpor significantly increased as mean Tmic days .. On dayb records from a total of five animals, including three animals recorded during winter. Despite this limited number of individuals, our results leave no doubt that N. pygmaeus has the capability to hibernate, as all animals (of both sexes) monitored during winter did undergo bouts of multiday torpor  occurred typically in the early afternoon, that is, well prior to the onset of nocturnal activitya, which is quite obvious from the temperature traces in a and even use sun-basking to minimise the energetic costs of returning to euthermia28One interesting aspect of hibernation in pygmy slow lorises, and other tropical hibernatorsows e.g.,. This fab, even in the euthermic state, as Ta declined (as this linear decrease of Tb in N. pygmaeus has been observed beforeb (~1.5 °C over a 20 °C Ta range) even during the daily active phase is unusual for primates3132Nyctycebus coucangb, to torpor and hibernation. It would be interesting to see if these physiological responses are even more pronounced in entirely free-living animals that have to rely on natural food resources only.In addition to the use of torpor and hibernation, pygmy slow lorises also reduced energy requirements by significantly lowering both mean and maximum daily Tdeclined . Within In summary, this study shows that hibernation in primates is not restricted to species inhabiting Madagascar. Thus, there are no specific climatic conditions or evolutionary histories that have limited the occurrence of primate hibernation to this island. This finding opens the door for the search for hibernation in further primates living in seasonal habitats.The study was carried out between August 2010 and May 2011 at the Endangered Primate Rescue Center (EPRC), Cúc Phương, Ninh Bình, Vietnam  using 6 adult pygmy slow loris . The animals had been confiscated by Vietnamese authorities from illegal wildlife traders, and were kept at EPRC in outdoor enclosures for at least 3 months prior to the experiments. All experiments were carried out in accordance with the guidelines on “Use if Animals in Research and Education” of the World Organization on Animal Health (OIE), of which Vietnam is a member. All experimental protocols were approved by the Vietnamese Ministry of Agriculture and Rural Development, Decision 59/QD-BNN-KL (dated 09.09.2009). All experimental procedures were also reported to the Ethics Committee of the University of Veterinary Medicine, Vienna, Austria. Animals were housed individually in outdoor enclosures (size 3 m*1.5 m*2 m) with open soil, natural vegetation of a tropical evergreen forest and a furnishing of bamboo and branches. Sleeping boxes were provided as shelters. The animals were fed the same mixture of boiled eggs, fruits and vegetables  throughout the year. In addition, stick insects, grasshoppers and crickets were fed daily (except for periods of rainy weather of up to three days) additionally between May and October, when these insects were abundant, but not during winter, when arthropods are virtually absent in this habitatb was measured at 6 min intervals using intraperitoneally implanted, custom-constructed programmable data loggers based on digitally readable temperature sensors . Loggers were individually calibrated in a water bath prior to implantation and checked for accuracy after explantation. The maximum deviation between measured and actual temperature after explantation was <0.2 °C. Prior to implantation, loggers were coated with a paraffin/beeswax mixture and subsequently EtO-gas-sterilized . However, apparently due to faults in the coating several of the loggers failed after implantation (but retained the stored data). Thus, we obtained records for the full study period (September-May) for 2 out of 6 implanted loggers only , as well as data for September to mid January for one female. For two more females, we retrieved records from autumn (September-October) over 45 and 80 days, respectively. One logger failed after a few days only, and these data were entirely omitted. All together, Tb was measured during 769 days in 5 animals .Core T−1 ketamine  and 50 μg kg−1 medetomidine  and maintained with 0.2–1.5% isoflurane  in an oxygen stream. The animals were placed in dorsal recumbency, the operation field was prepared according to standard surgical procedures and covered with sterile surgical drapes. The abdominal cavity was opened through a ~1.5 cm incision in the central midline. The logger was inserted free-floating. Postimplantation, peritoneum, abdominal muscles were sutured using synthetic absorbable surgical suture material USP 3/0  using a single-button suture technique. The skin was sutured separately using synthetic absorbable surgical suture material USP 3/0  with an intracutaneous suture technique. During the entire procedure, vital parameters  were monitored. Postsurgical analgesia  subcutaneously) was provided.Surgical anaesthesia was induced by intramuscular injection of 5 or 10 mg kgb decreasing below 30 °C for at least 1 h. This slightly conservative definition was used because Tb was highly variable even at night in the absence of clear torpor episodes a had an effect on the probability of animals to enter torpor we computed a variable torpor (yes/no) for each day (n). We then computed mean Ta for the period 12:00 h on day n−1 to 12:00 h on day n, and tested the effect of this mean Ta on the probability of torpor entry on day n using a mixed effects binomial model (R package lme4)Torpor was defined as Tepisodes ,2, and iHow to cite this article: Ruf, T. et al. Hibernation in the pygmy slow loris (Nycticebus pygmaeus): multiday torpor in primates is not restricted to Madagascar. Sci. Rep. 5, 17392; doi: 10.1038/srep17392 (2015).


After surgical repair of traumatically severed peripheral nerves, associated muscles are paralyzed for weeks. Little is known about fascicle length changes in paralyzed muscles during locomotion. The aim of this study was to investigate to what extent, if any, muscle fascicles of denervated feline soleus (SO) change length during stance of walking when intact SO synergists are actively contracting. Hindlimb kinematics, SO fascicle and muscle-tendon unit (MTU) length, and EMG activity of SO, lateral gastrocnemius (LG) and medial gastrocnemius (MG) were measured during level and slope walking in adult cats. Measurements were taken before and 1–2 weeks following SO-LG denervation. Unexpectedly, SO fascicle lengthening and shortening during stance in all walking conditions were evident after denervation. The greatest SO fascicle shortening (17.3 ± 2.2% of a reference length) and least fascicle lengthening (1.5 ± 0.8%) after denervation were found during upslope walking, where MG EMG activity was greatest across slopes (P < 0.05) and greatest discrepancies between post denervation SO fascicle and MTU length changes occurred. These findings suggest that myofascial linkages between denervated SO and its active synergists might affect its fascicle length changes. Further studies are needed to directly test this suggestion. Peripheral nerve injury causes long-lasting and often permanent sensorimotor deficits even after the severed nerve is surgically repaired12357910121314in vivo16in vivo mechanical conditions of muscle fascicles in a denervated muscle surrounded by intact synergists is currently unavailable. The extent to which fascicles of a denervated muscle passively lengthen and shorten while its intact synergists undergo active lengthening and shortening during normal motor behaviors is unknown. Information about muscle fascicle mechanical behavior in the injured muscle, specifically in denervated muscle, would improve our understanding of the mechanical behavior of paralyzed muscles following peripheral nerve injury.Although there are indications of mechanical interactions between passive muscle heads of the human triceps surae o–120o (angles corresponding to the maximum ankle dorsiflexion in the stance phase of cat level and slope ( ± 50% or  ± 27o) walking2023in vivo252723The aim of this study was to investigate to what extent, if any, muscle fascicles of the denervated feline SO change length during the stance phase of walking. In a denervated muscle, there is no active force generation, whereas passive force production by cat SO muscle-tendon unit (MTU) stretched by ankle dorsiflexion up to ~90Preliminary results of this study have been published in abstract formFelis domesticus, mass 3.7 ± 0.8 kg; mean ± SD) were studied. Cats were trained using operant conditioning methods to walk within a custom-made, Plexiglas-enclosed walkway (3.0 m × 0.4 m) with up to three embedded three force platforms  and covered with nonslip rubberized matting. Cats were trained to walk at three walkway slopes – level surface , upslope  and downslope  – for several hours a day, 5 days a week for 3–4 weeks; for more detail see19The surgical and experimental procedures employed corresponded to the “Principles of Laboratory Animal Care”  and were approved by the Institutional Animal Care and Use Committee of the Georgia Institute of Technology. Seven adult female cats , atropine  and isoflurane  and anesthesia was maintained with isoflurane (1–3%). The animal’s vital physiological parameters  were monitored throughout the surgery. After shaving and cleaning with surgical disinfectant the skin overlying the skull, the low back, and dorsal aspects of the right shank, skin incisions were made. Teflon-insulated multi-stranded stainless steel fine wires  and leads with piezoelectric sonomicrometry crystals  were passed subcutaneously along the back from the skull to an incision in the hindlimb. Four of the seven cats were implanted with the sonomicrometry crystals. All wire leads were attached to two multi-pin Amphenol connectors that were secured to the skull with four stainless steel or titanium screws and dental cement. A thin strip of insulation (~1 mm) was removed near the distal end of each pair of the fine wires and secured inside the muscle belly of SO, LG and MG muscles. Mild electrical stimulation of the muscles through the head connectors was used to verify wire placements. The implanted fine wires were used to record electromyographic (EMG) activity. To measure SO fascicle length, one pair of the piezoelectric crystals was implanted near the origin and insertion  of SO muscle fascicles along the muscle belly midline. The superficial surface of the muscle belly at the location of the crystal implantation was reached through a small cut of the fascia surrounding the muscle. A small pocket inside the muscle was created between the muscle fibers. The crystal was inserted in the pocket beneath the muscle surface, and the pocket was closed by a 4–0 silk suture, which also anchored the crystal lead wire.After implantation of EMG electrodes and sonomicrometry crystals, the skin incisions were closed using Vicryl 4–0 suture for the deep fascia and Vicryl 5–0 subcuticular suture for dermal closure. The animal recovered after surgery for 14 days with pain medication administered for at least 3 days and antibiotics for 10 days.After animal recovery, hindlimb mechanics, EMG activity of SO, LG, and MG muscles, and SO muscle fascicle lengths were collected during level, downslope and upslope walking (see below) to obtain baseline values.These surgical procedures were similar to those described previously1314Each cat was tested in two series of locomotion experiments before and after SO-LG nerve transection and repair to measure hindlimb mechanics, EMG of SO and MG, as well as SO fascicle length. Locomotor data collection before nerve injury was performed approximately 2 hours a day, 3–5 days a week for about a month. Post nerve injury locomotor data reported here were collected during the first 3–14 days (1–2 weeks) after SO-LG nerve transection and repair using the same recording schedule as for pre-nerve injury data collection.In order to record hindlimb kinematics, light reflective markers were placed on the following right hindlimb anatomical landmarks using double-sided adhesive tape: iliac spine, greater trochanter, lateral femoral epicondyle, lateral malleolus, 5th metatarsophalangeal (MTP) joint and the distal end of the 5th digit. Marker positions were recorded using a 3D, 6-camera motion capture system  at a sampling rate of 120 Hz. The three components of the ground reaction force vector and coordinates of its application point to the paw were recorded at a sampling rate of 360 Hz by 1 of the 3 small force plates  embedded into the walkway surface and actually contacted during any specific trial. SO fascicle length was recorded by the sonomicrometry system  at a sampling rate of 1059 Hz. EMG signals were collected at a sampling rate of 3000 Hz, band-pass filtered , amplified (100×), and saved on a PC for further analysis. The EMG and sonomicrometry signals were collected via a 16-conductor shielded flexible cable attached to the Amphenol connectors on the animal’s head. The simultaneous data collection onset was triggered by the electronic pulse of the Vicon system to synchronize recordings of the walking mechanics, EMG and SO fascicle length.Hindlimb kinematics. Recorded coordinates of markers on the right hindlimb were low-pass filtered using a Butterworth low-pass, zero-lag filter (cut-off frequency 6 Hz). To reduce knee marker errors due to excessive skin movement, the estimated knee joint center position at each video frame was recalculated using the recorded position of the ankle and hip joints and length of the shank and thigh segmentsref) defined as the mean between the maximum and minimum of the averaged MTU lengths during the swing phase in baseline level walkingEMG. Recorded EMG signals were band-pass filtered (see above) and full-wave rectified. Within each walking cycle an EMG burst of SO, LG or MG was considered on when the EMG signal was above a threshold signal level for at least 50 ms, and off when the signal was below the threshold for at least 50 ms. The threshold level was defined as 2 s.d. above the mean EMG signal during a muscle silent period SO fascicle length. Fascicle length was computed from the measured propagation time of ultrasound between the implanted piezoelectric crystals and the known speed of ultrasound propagation in muscle tissue  was used with the current selected to cause maximum fascicle shortening.To assess the minimum SO fascicle length in relaxed muscle, fascicle length measurements were made in one cat (CO) before SO-LG denervation while the cat was sedated . The ankle joint angle was fully plantar flexed at 180Walking cycles with a steady-state speed and without paw slippage were selected and used for further analysis. For MTU and muscle fascicle length analysis, a total of 186 walking cycles were processed across 4 cats with implanted sonomicrometry crystals, pre and post SO-LG denervation, and three walking slope conditions . For EMG191314P < 0.05.First, a single sample t-test was performed for each cat with implanted sonomicrometry crystals  and slopDuring days 3 to 14 after SO-LG nerve transection and repair, SO and LG muscles were paralyzed, which was evident from the lack of EMG bursts during walking . During ref, F1,47 = 4.45, P ≤ 0.001 for downslope walking; 7.2 ± 0.9 vs 8.8 ± 0.7%Lref, F1,64 = 15.23, P ≤ 0.001 for level; 13.0 ± 1.3 vs 13.9 ± 1.2%Lref, F1,59 = 4.74, P = 0.033 for upslope). Analysis of SO MTU shortening after SO denervation in individual animals revealed a significant increase in 2 out of 4 animals in each of the slope walking conditions (P ≤ 0.01), while one cat showed a decrease during downslope walking . There were no significant differences between level and downslope (P = 0.393), and downslope and upslope (P = 0.595) conditions.SO MTU shortening during the stance phase increased significantly following denervation in all slope conditions , 34% for level walking , and by 200% for upslope walking . SO MTU lengthening following denervation in individual cats increased in most walking conditions and cats . For the change in MTU lengthening in response to denervation, there was a significant difference between downslope and level walking , and downslope and upslope walking . There was no difference between level and upslope walking (P = 0.234).There was a significant increase in SO MTU lengthening following denervation in all walking conditions . It incrref, F1,62 = 7.845, P = 0.007 and from 8.8 ± 2.2 to 14.0 ± 2.9%Lref, F1,46 = 106.330, P ≤ 0.001, respectively); but decreased in upslope condition by 38% , but did not change in one cat . During level walking, SO fascicle lengthening increased after denervation in only one cat from 0.5 ± 0.3 to 3.4 ± 1.3%Lref , whereas the remaining two cats showed no significant changes . We found significant differences in the magnitude of change of SO fascicle lengthening in response to denervation between downslope, level, and upslope conditions , corresponding to its shortest MTU length, was 35.7 mm. When the SO muscle was electrically stimulated via implanted EMG electrodes to cause maximum fascicle shortening at the same joint angle, SO fascicle length decreased to 32.7 mm. Following denervation, SO fascicle lengths of the same cat at the end of stance during downslope, level, and upslope walking were 38.7 ± 0.3 mm (the corresponding ankle joint angle was 103.9 ± 4.3o), 34.8 ± 0.6 mm (130.9 ± 2.6o), and 33.0 ± 0.5 mm (135.4  ±  4.8o), respectively. The fascicle lengths in the denervated SO during level and upslope walking were significantly shorter than the minimum fascicle length of passive SO in the sedated condition . When compared to fascicle length of electrically stimulated SO in the sedated condition, fascicle lengths of the denervated SO at paw lift-off during level and downslope walking were longer , whereas SO fascicle length during upslope walking was not different .SO fascicle length measured in one cat (CO) under sedation at a fully extended ankle  SO MTU in the studied conditions. First, if the muscle fascicles were more compliant than the tendon, as shown to be the case in the cat SO at very small muscle forces, i.e. low EMG levels221333in vivo range of motion at the ankle joint  are very small  lengthening of denervated SO fascicles during the yield phase in stance does not correspond to SO MTU lengthening  of a single muscle in a muscle group may lead to asymmetric loading of the joint and subsequent development of joint tissue degeneration, osteoarthritis, and possible other secondary joint conditions910This study was designed to document fascicle length changes in denervated SO during locomotion. Although the suggested mechanism of myofascial force transmission between denervated SO and its intact synergists (e.g. MG) appears to be the most probable explanation of our unexpected findings (see discussion above), direct testing is required to prove this. One approach could be to sever the myofascial linkages between SO and MG. This, however, will not likely result in the desired condition. Due to extreme plasticity of connective tissues, which tend to quickly (within a few days) reestablish original or grow new connections5253We have presented the first report of increased muscle fascicle length changes in denervated SO fascicles during the stance phase of walking that might potentially affect the extent of muscle atrophy and symmetry of joint loading. Evidence from SO fascicle and MTU length, as well as from MG EMG activity recordings suggests that this behavior might result from the action of epimuscular myofascial linkages between intact and paralyzed ankle extensors. Additional studies are needed to confirm the importance of this mechanism.How to cite this article: Mehta, R. et al. Unexpected Fascicle Length Changes in Denervated Feline Soleus Muscle During Stance Phase Of Walking. Sci. Rep.5, 17619; doi: 10.1038/srep17619 (2015).


Limited studies report on the additional prognostic value of coronary computed tomography angiography (CCTA) and the coronary artery calcium score (CACS).For a median of 637 days, 1551 outpatients with chest pain, without known coronary artery disease (CAD) and low or intermediate pre-test probability of CAD, were followed for major adverse cardiac events (MACE), defined as death, myocardial infarction or late revascularisation. Cox proportional hazard regression was used to evaluate the independent prognostic value of CCTA and CACS.p < 0.001), CACS of 100–400 (p = 0.035) and CACS of > 400 (p = 0.021). CCTA showed obstructive CAD in 3.1 % of patients with CACS = 0. No events occurred in patients with CACS = 0 without obstructive CAD at CCTA, whereas 2/23 patients (9 %) with CACS = 0 with obstructive CAD had a MACE.MACE occurred in 23 patients (1.5 %): death , myocardial infarction  and late revascularisation . Multivariate analysis showed an independent prognostic value of CCTA (Our study shows that both CCTA and higher CACS categories have independent prognostic value in chest pain patients with low to intermediate pre-test probability of obstructive CAD, in which CCTA is appropriate. Furthermore a non-negligible amount of patients with CACS = 0 have obstructive CAD at CCTA. CCTA can be used in these patients to identify those at risk for MACE. Quantification of coronary artery calcium by computed tomography represents a reliable estimate of atherosclerotic plaque burden and the prognostic value of the coronary artery calcium score (CACS) is well established , 2. ReceFrom 13 December 2011 to 26 August 2014 all patients with chest pain with low or intermediate pre-test probability of CAD, referred for CCTA from the outpatient clinic after a diagnostic work up, were prospectively included. None of the patients had a prior history of CAD. The pre-test probability was calculated using the Duke Clinical Score . A pre-tBaseline characteristics including age, gender, and cardiovascular risk factors were prospectively entered in the database. All patients gave written informed consent for usage of their data.Patients were followed for MACE, defined as all-cause mortality, myocardial infarction or revascularisation (either CABG or PCI). A 60-day landmark was used to differentiate between CCTA-driven invasive coronary angiography and late revascularisation, which is considered to be indicative for the prognostic value of CCTA. Patients with referral for invasive coronary angiography in the outpatient setting within 60 days after CCTA and subsequent revascularisation were considered to be CCTA-driven and not MACE. All other revascularisations within follow-up were considered to be MACE. Information on myocardial infarction and revascularisation was obtained from the electronic medical records. Information on mortality was obtained from the municipal personal records database.Patient preparation, image acquisition, and image analysis were performed as previously described  and as bAll scans were performed with a 2 × 64–slice flying focal spot, effectively 2 × 128–slice  and were evaluated by a Certification Board of Cardiovascular Computed Tomography accredited nuclear medicine physician and a cardiologist experienced in the interpretation of CCTA in consensus. In case of disagreement a third opinion was decisive.−1, which is generally used in cardiac CT studies.The radiation dose delivered was generated automatically by the scanner software and represented as dose length product. The effective dose was calculated by multiplying the dose length product with the k-factor of 0.014 mSv × (mGy × cm)Obstructive CAD was defined as a lumen stenosis in any of the large vessels of > 50 %, either the left main artery, left anterior descending artery, circumflex artery or right coronary artery. Normal coronary arteries were defined as CACS = 0 and no coronary plaques. Non-obstructive CAD was defined as CACS > 0 and/or any plaque that did not meet the criteria for obstructive CAD.Statistical analysis was performed using SPSS software, version 22.0.0 . Continuous variables are presented as mean ± SD and categorical variables as frequencies with percentages. Continuous variables were tested for normal distribution.p < 0.10 as threshold for removal of variables. Clinical risk factors included in the univariate analysis were male gender, age, diabetes, smoking, hyperlipidaemia, hypertension, and family history of CAD. Selection of variables for entry in the multivariable Cox proportional hazard regression was based on univariate analysis with a threshold of p < 0.10.Kaplan-Meier analysis was used to assess MACE-free survival stratified by CACS and CCTA results. Univariate and subsequent multivariate Cox proportional hazard regression were used to evaluate the independent prognostic value of CACS and CCTA beyond clinical risk factors. The multivariate Cox regression was done in stepwise fashion according to the backwards approach, with A total of 1560 patients were initially included in the database for follow-up. Follow-up could be obtained in 99.4 % of the patients. Nine patients emigrated to a foreign country and were lost to follow-up. The total number of studied patients was 1551. An overview of the baseline characteristics is shown in Table CACS, CCTA results and radiation dose are summarised in Table The cohort was followed for a median of 637 days. MACE occurred in 23 patients during follow-up. Three (0.2 %) patients died, 4 (0.3 %) patients had a myocardial infarction, and 20 (1.3 %) patients had non-CCTA-driven revascularisations. Four patients (0.3 %) were acute revascularisations in myocardial infarction and 16 (1.0 %) were late revascularisations, referred beyond 60 days after CCTA because of ongoing symptoms.p-value < 0.01).Figure MACE occurred in no patients with normal coronary arteries at CCTA, in 1.0 % of patients with non-obstructive CAD at CCTA and in 7.5 % of patients with obstructive CAD at CCTA. During follow-up, MACE occurred in 0.3 % of patients with CACS = 0, in 1.0 % of patients with CACS 1–100, in 4.2 % with CACS 101–400 and in 7.1 % with CACS > 400.Of all 739 patients with CACS = 0, 23 (3.1 %) had obstructive CAD at CCTA and 62 (8.4 %) had non-obstructive CAD at CCTA. No events occurred in patients with CACS = 0 and no obstructive CAD at CCTA, whereas 2/23 patients (9 %) with CACS = 0 and obstructive CAD at CCTA had a MACE during follow-up: late revascularisation in both cases. MACE-free survival was significantly worse in CACS 0 patients with obstructive CAD, log-rank p-value < 0.001.The evaluation of the predictive value of CACS and CCTA by univariate and multivariate Cox proportional hazard regression is shown in Table p < 0.001). Furthermore, CACS of 100–400 (p = 0.035) and CACS of > 400 (0.021) were independent predictors for the occurrence of MACE, whereas a CACS of 0–100 (p = 0.29) had no independent predictive value for the occurrence of MACE.In the multivariate model, obstructive disease on CCTA was independently associated with increased risk of MACE . Presence of CAD in patients with CACS = 0 was associated with worse prognosis.Furthermore, our study shows that although overall prognosis in this clinical cohort was excellent, presence of obstructive CAD on CCTA had an independent prognostic value. CACS of 0–100 did not show independent prognostic value of CCTA; however CACS 100–400 and > 400 did show independent prognostic value.The prognostic value of CACS has been previously demonstrated in large meta-analyses , 2. A reMultiple studies have previously reported on the prognostic value of CCTA for the occurrence of adverse events –11. Our Our study, in accordance with prior studies, showed an excellent prognosis of symptomatic patients with CACS = 0, with an event rate < 1 % within 2 years of follow-up , 18–20. Several studies have been published on the independent prognostic value of CCTA to CACS. Cho et al. reported that in 7590 studied asymptomatic patients, CCTA did not have prognostic value independent of CACS . Other sData on the independent prognostic value of CACS in the CCTA era are limited –26. ChaiThe recently published PROMISE trial investigated symptomatic patients with suspected CAD who require non-invasive testing and reported no difference in outcome between an initial strategy of CCTA and functional testing . HoweverThe single-centre design of our study allows the evaluation of prognosis in a ‘real world’ clinical setting. However, our results may not be applied to all outpatient chest pain populations, i.e. asymptomatic or high-risk patients.As in previous studies, a 60-day landmark was used to differentiate between CCTA-driven invasive coronary angiography and long-term revascularisation, which is considered to be indicative for the prognostic value of CCTA , 30. In This study shows that both CCTA and CACS of 100–400 and > 400 have an independent prognostic value for the occurrence of MACE in a routine clinical cohort of patients presenting to the outpatient clinic with atypical chest pain with low to intermediate pre-test probability of obstructive CAD, in which CCTA is appropriate. Furthermore our study shows that a non-negligible amount of patients with CACS = 0 have obstructive CAD at CCTA. CCTA can be used in patients with CACS = 0 to identify those at risk for MACE.This work was not supported by any funding.None declared.


Silybum marianum, has been suggested to display neuroprotective properties against various neurodegenerative diseases. In the present study, we observed that neonatal iron (120 μg/g body weight) supplementation resulted in significant abnormality of behavior and depletion of striatal dopamine (DA) in the aging male and female rats while it did not do so in the young male and female rats. No significant change in striatal serotonin content was observed in the aging male and female rats with neonatal supplementation of the same dose of iron. Furthermore, we found that the neonatal iron supplementation resulted in significant increase in malondialdehyde (MDA) and decrease in glutathione (GSH) in the substantia nigra (SN) of the aging male and female rats. No significant change in content of MDA and GSH was observed in the cerebellum of the aging male and female rats with the neonatal iron supplementation. Interestingly, silibinin (25 and 50 mg/kg body weight) treatment significantly and dose-dependently attenuated depletion of striatal DA and improved abnormality of behavior in the aging male and female rats with the neonatal iron supplementation. Moreover, silibinin significantly reduced MDA content and increased GSH content in the SN of the aging male and female rats. Taken together, our results indicate that elevated neonatal iron supplementation may result in neurochemical and behavioral deficits in the male and female rats with aging and silibinin may exert dopaminergic neuroprotection by maintaining redox balance.Aging is a critical risk factor for Parkinson’s disease. Silibinin, a major flavonoid in  Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by cardinal features, including resting tremor, rigidity, slowness of movement, and postural instability. It is due to decreased dopamine (DA) content in the striatum as a result of progressive and selective degeneration of dopaminergic neurons in the substantia nigra (SN). In recent years, more and more studies have demonstrated that the causes of PD are multifactorial, including aging, exposure to environmental toxins, immune/inflammatory factors, genetic predisposition, and innate characteristics of the nigrostriatal dopaminergic system in the brain , has been used clinically for thousands of years in China and Europe as an anti-hepatotoxic agent to treat liver diseases, especially alcoholic liver disease  and the Protocols for Animal Experimentation of Shanghai Jiao Tong University School of Medicine. All efforts were made to minimize the number of animals used and their suffering. Sprague-Dawley rat pups were fed either saline vehicle or carbonyl iron daily by oral gavage from days 10 to 17 post-partum. Based on the previous studies . The latency time to fall was recorded for each test. For locomotor activity, each rat was placed into an open field chamber, which is made of wood covered with impermeable formica. The chamber had a white floor (100 cm × 100 cm) with it divided into 25 squares (20 cm × 20 cm) and 50 cm high walls. Before testing, each animal was placed in the open field center and acclimatized for 10 min. Rat motor behavior was recorded for 30 min. The following parameters were counted: (1) crossing number, defined as entering of another square with all four paws and (2) rearing number, defined as rearing with and without wall contact (standing only on hind legs).High-performance liquid chromatography (HPLC), equipped with electrochemical detector (ECD), was used to determine neurotransmitter content in the rat striata , 2.1 ml of reconstituted Enzyme Mixture (glutathione reductase and glucose-6-phosphate dehydrogenase reconstituted in 2 ml MES buffer), 2.3 ml of water, and 0.45 ml of reconstituted DTNB ). Following incubation of 25 min, the absorbance value was read at 405 nm using a microplate reader.The levels of MDA and GSH were measured according to the manufacturer’s instruction by commercially available kits . For MDA assay, thiobarbituric acid (TBA) reacts with MDA in acidic medium at elevated temperature to form TBA reactive product. SN tissue was homogenized (10% weight/volume) with radioimmunoprecipitation assay (RIPA) homogenizing buffer containing a protease inhibitor. Obtained samples were centrifuged at 1,600 g and 4°C for 10 min. Then, supernatants were used for analysis. Samples or standards (100 μl) were added to 100 μl of sodium dodecyl sulfate solutions followed by the addition of 4 ml of color reagents (530 mg of TBA with 50 ml of diluted TBA acetic acid solution and 50 ml of diluted TBA sodium hydroxide). Obtained solutions were boiled for 1 h followed by the incubation in ice bath for 10 min to stop reaction. Then, solutions were centrifuged at 1,600 g and 4°C for 10 min. The absorbance values of the samples were read at 540 nm using a microplate reader. GSH determination was based on the enzymatic recycling method. In brief, 100 μl of supernatant from SN homogenate was deproteinated by the addition of 100 μl metaphosphoric acid reagent (5 g of metaphosphoric acid in 50 ml water). Then, triethanolamine reagent  was added to the solution and immediately vortexed. Samples or standards (50 μl) were pipetted into the designated wells. This was followed by the addition of 150 μl of freshly prepared Assay Cocktail consisting of 11.25 ml of MES Buffer (0.4 M 2-(N-morpholino) ethanesulphonic acid, 2 mM EDTA, and 0.1 M phosphate), 0.45 ml of reconstituted Cofactor Mixture  and Bonferroni post hoc test for comparison between more than two groups. Normality of sample distribution and homogeneity of variances were tested before each ANOVA. A value of p <0.05 was considered statistically significant.Data were expressed as the mean ± SEM. Differences were determined by two-tailed Student’s p < 0.005 (5 and 10 rpm), p < 0.01 (15 rpm); aging female rats: p < 0.01 ] and the number of crossing and rearing  were observed in the aging male and female rats with neonatal intake of the same dose of iron in comparison with the vehicle-treated rats  was observed in the aging male and female rats with neonatal iron intake compared with the vehicle-treated rats . As shown in Figures 05 5 and 0 rpm, p  Figures . In agre Figures A,B. Howe Figures A,B. Alth Figures C,D.p < 0.005) and decrease in GSH content (p < 0.005) were observed in the SN of the aging male and female rats with neonatal intake of the same dose of iron in comparison with the vehicle-treated rats. Although the neonatal iron intake significantly increased MDA content and decreased GSH content in the SN of the aging male and female rats compared to the rats treated with vehicle, no significant change in the content of MDA and GSH was observed in the cerebellum (CBM) of the aging male and female rats with the neonatal iron intake in comparison with the vehicle-treated rats . As shown in Figure p < 0.005 (5 and 15 rpm), p < 0.01 (10 rpm), p < 0.01 (crossing and rearing number); aging female rats: p < 0.01 (5 and 15 rpm), p < 0.05 (10 rpm), p < 0.01 (crossing and rearing number)] after silibinin administration at the dose of 50 mg/kg body weight. In agreement with behavior tests, neurochemical analysis also demonstrated that silibinin administration at the doses of 25 and 50 mg/kg body weight significantly diminished depletion of striatal DA in the aging male and female rats with increased neonatal iron intake compared with the vehicle-treated rats   treatment on striatal DA depletion and behavioral abnormality in the aging rats  with increased neonatal iron intake. As shown in Figure  Figures A,B.p < 0.05 (25 mg/kg) and p < 0.005 (50 mg/kg)].We finally examined the effect of silibinin treatment on MDA and GSH levels in aging rats with increased neonatal iron intake. As shown in Figures Many evidences have demonstrated that aging is a critical risk factor for idiopathic PD in recent years (Yankner et al., 2O2 production in Aβ1-42-stressed neurons. Tota et al. (+-induced neurotoxicity in the SN in a dose-dependent manner. They also observed that increased levels of inflammatory molecules, such as tumor necrosis factor-alpha, interleukin-1 beta, and inducible nitric oxide synthase by MPP+ treatment, were attenuated by treatment with silibinin. Moreover, Geed et al. (+ injection in rats. In the present study, we found that silibinin treatment significantly improved motor abnormality and diminished striatal DA depletion of the aging male and female rats with increased neonatal iron intake, indicating the potential dopaminergic neuroprotection of silibinin in PD.Silibinin is the major active component of silymarin extracted from milk thistle. By employing a ThT assay and electron microscopic imaging, Yin et al.  identifia et al.  showed ta et al. . Jung eta et al.  observedd et al.  showed tOxidative stress is demonstrated to be involved in aging process and cell injury (Bodhinathan et al., In conclusion, our study suggests that elevated neonatal iron supplementation may induce PD-like neurochemical and behavioral deficits with aging and silibinin may be neuroprotective in PD. Maintaining redox balance may be one of the mechanisms underlying silibinin’s neuroprotection. Further study is required to fully understand the potential role of neonatal iron intake and silibinin in aging process and PD and to develop effective therapeutic strategies to slow aging and PD neurodegeneration.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Concomitant cocaine use is a major problem in clinical practice in methadone maintenance treatment (MMT) and may interfere with successful treatment. Data from European methadone populations is sparse. This register-based study sought to explore the association between prescribed methadone dose and concomitant cocaine and heroin use in the methadone population of Basel City.The study included 613 methadone patients between April 1, 2003 and March 31, 2004. Anonymized data was taken from the methadone register of Basel City. For analysis of the prescribed methadone dose distribution, the patient sample was split into three methadone dosage groups: a low dose group (LDG) , a medium dose group (MDG) , and a high dose group (HDG) . Concomitant drug use was based on self-report.Analysis showed a significant difference in self-reported cocaine use between groups (p < 0.001). Patients in the LDG reported significantly fewer cocaine consumption days compared to the MDG (p < 0.001) and the HDG (p < 0.05). Patients in the HDG reported significantly fewer heroin consumption days than those in the LDG (p < 0.01) and the MDG (p < 0.001). In logistic regression analysis, cocaine use was significantly associated with heroin use (OR 4.9).Cocaine use in methadone patients may be associated with heroin use, which indicates the importance of prescribing appropriate methadone dosages in order to indirectly reduce cocaine use. Opioid dependence is a serious chronic illness with a multitude of somatic and psychosocial risks and which in most cases requires long-term treatment –4. MethaIn Switzerland, the prevalence of opioid dependence was 0.9% in 2012   and cocaThe tendency toward higher methadone doses has been supported by randomised clinical trials –18 and oThis register-based study sought to explore the association between different prescribed methadone dose levels and concomitant cocaine and heroin use in the methadone population of Basel City between April 1, 2003 and March 31, 2004. We were especially interested in the effect of higher methadone doses on concomitant cocaine use, because not enough is known on this topic. We hypothesised that patients with prescribed methadone doses > 100 mg/day would have better treatment outcomes. To answer these research questions, we split the sample into three subsamples. Methadone dosage groups were defined according to a National Institutes of Health (NIH) expert panel that has stated that a daily methadone dose of at least 60 mg is best practice in methadone maintenance . The doshttp://www.ekbb.ch ). As stipulated by legislation, prescribing treatment providers in Basel City are required to submit a registration form to the health authorities each time a heroin-dependent patient begins and ends MMT. The form collects information about methadone patients and their treatment. For monitoring purposes, methadone prescribers were further invited until 2004 to provide anonymized patient and treatment data to the register every 12 months by means of a 2-page questionnaire. Methadone prescribers were instructed to carry out the interviews within one month and send them back to the health authorities. This structured questionnaire contained a core of questions about gender, nationality, marital status, educational level, work situation, age at first heroin and cocaine use, treatment facility , year entering MMT, currently prescribed methadone dose (mg/d), prescribed psychiatric comedication, allowed take-home days per week (0–7 days), number of consultations in the past 6 months, and self-reported substance use  during the previous 30 days . The prAnonymised data from prescription for opioids were collected and analysed by the Addiction Research Center of the Hospital of the University of Basel. A total of 974 patients were surveyed. The response rate was 91% (n = 886). Overall 273 out of 886 patients had to be excluded from the statistical analyses by the study investigators due to missing variables . A further 68 patients were excluded from the analyses, because they had been given a prescription for opioid substitution treatment other than with methadone , and 19 patients were excluded because they were tapering methadone to end MMT. The final sample included 613 patients and was split into three methadone dose groups .2 statistics and non-parametric tests .Dependent variables were concomitant cocaine use (consumption days) and concomitant heroin use (consumption days). Continuous  data were analyzed by one way analysis of variance, and post-hoc tests (Duncan Scheffé’s); categorical data were analyzed by χp < 0.05.To model the determinants of cocaine use in MMT, we used a backward fitting procedure that applied likelihood ratio tests to develop a binary logistic regression model, with cocaine use (coded as a dichotomous variable) as the dependent variable. Initially we included the following predictor variables, that were either associated in bivariate analysis or known from literature: age (years); gender ; employment status (yes = 1); office-based settings (yes = 1); days with take-home (days); prescribed methadone dose group (low methadone dose as the reference category); and concomitant heroin use (no = 0). Because of a large number of missing values, we refrained from including the variables route of administration, age at first heroin and cocaine use. Data analyses were conducted using SPSS . The level of significance was set at Of 613 methadone patients in MMT, 32.6% were in the LDG (n = 200), 44.5% in the MDG (n = 273), and 22.8% in the HDG (n = 140). Patients were aged from 22 to 61 years (38.9 ± 6.6); two thirds were men (67.9%); 10.4% were married; the mean years of education were 10.8 ± 1.6, and 20.6% were employed  , compared to patients in office-based settings.Two thirds of the patients reported no concomitant cocaine use at all in the 30 days prior to the interview , and one third n = 219, 35.7%) reported at least 1 cocaine consumption day. In the LDG, 24.5% (n = 49) reported cocaine use, in the MDG 42.9% (n = 117) and in the HDG 37.9% (n = 53). Patients reported a mean of 2.8 ± 5.9 cocaine consumption days in the past 30 days. As shown in Figure 9, 35.7% Table 2 = 6.138; df = 1; p < 0.05).Two thirds of the patients reported no concomitant heroin use at all in the 30 days , and one third reported at least 1 heroin consumption day n = 223, 36.4%). In the LDG, 37.0% (n = 74) reported heroin use, in the MDG 41.8% (n = 114) and in the HDG 25.0% (n = 35). Patients reported a mean of 2.8 ± 6.1 heroin consumption days in the 30 days prior interview, with significant differences between groups (p < 0.001) , indicating that concomitant heroin and cocaine use is common in a subgroup of methadone patients. Furthermore, the odds for cocaine use were increased in patients with prescriptions of both medium OR 2.3) and high (OR 2.1) methadone doses as compared to the LDG. One explanation could be drawn from the study results that patients in the LDG started cocaine and heroin use later in their lives and seem be more socially integrated, as the LDG is the group with significantly more employed patients compared to the MDG and HDG. Furthermore, the LDG probably has less poly-drug use and less concurrent medication for psychiatric comorbidities. Patients in specialized centers were more likely to have concomitant cocaine use (OR 1.9) than patients in office-based settings. This result corresponds to clinical practice, referring more instable patients to specialized centers. The same accounts for the significant result in less take-home medication days in patients with more concomitant cocaine use , nor about patients who did not attend the interviews; this might have led to underestimation of the amount of illicit cocaine and heroin use. Another limitation is that concomitant drug use in MMT is based on self-reported data and not verified by urine analyses. However, parallel drug use does not usually lead to exclusion from MMT. We are confident that self-reported drug use is quite similar to results from urine analyses. As Decker et al. observed, self-reported cocaine use and urine analyses demonstrate good concordance within treatment . In a prDespite these limitations, the following conclusions can be drawn from the present study. Firstly, two thirds of methadone prescriptions to opioid-dependent patients were within the recommended range. Secondly, prescribed methadone doses > 100 mg/day (HDG) were not associated with significantly fewer cocaine consumption days but with fewer heroin consumption days. Thirdly, concomitant heroin use was the major risk factor for cocaine use in MMT. This means that methadone providers are required to raise awareness for this issue by prescribing appropriate methadone doses, in order to reduce concomitant heroin use directly and cocaine use indirectly.


Enzyme immunoassays were validated for the quantification of reproductive hormones  in the hair of two felid species. The method provides a tool to potentially determine sex and pregnancy states in wild populations, therefore improving the monitoring of felids that often have low reproductive success in captivity. Non-invasive monitoring of wild felid reproductive states is important, given that many species reproduce poorly in captivity. Despite extensive work in faecal hormone analysis in felids, continued development of techniques is necessary, particularly with wild populations. In this study, our aims were as follows: (i) biochemical validation of enzyme immunoassays for estrogen, testosterone and progesterone in Canada lynx and domestic cat hair extracts; (ii) assessment of the use of hair reproductive hormones to differentiate between reproductive states , using domestic cats as a model; and (iii) assessment of the use of hair reproductive hormones to differentiate between age and sex, accounting for potential regional variability in wild lynx populations. Analysis of hair hormone levels showed prospective value in detecting pregnancy states, with pregnant domestic cats having higher levels of progesterone than spayed females. However, intact and pregnant cats did not differ in progesterone levels. Yet, two female domestic cats had higher levels of hair progesterone following a 38-day oral progestin treatment, perhaps providing a preliminary pharmacological validation of the method. Estrogen and testosterone did not differ statistically according to reproductive states of domestic cats, although intact males had higher levels of hair testosterone than neutered males. When we applied these techniques to lynx fur, we determined that hormone levels were not sufficiently precise to differentiate age classes. Hair reproductive hormone ratios differed between sexes, with the estrogen-to-progesterone ratio demonstrating the highest accuracy in differentiating males from females. Hair hormone levels differed regionally for wild lynx, indicating that spatial variability should be a consideration in wildlife hormone studies spanning large spatial scales. We conclude that use of hair hormone analysis by enzyme immunoassay may hold promise for differentiating sex in felids, but the technique will require further refinement and validation before it can be applied broadly and reliably. Lynx are monoestrous, seasonal breeders, with a short breeding season. Canada lynx (Lynx canadensis), which occupy the boreal forest of North America, breed from late February to early April (Lepus americanus), and during periods of high hare density, female lynx can reach sexual maturity at an accelerated rate  reveal that oral progestin increases baseline progesterone levels in faeces  to validate EIAs biochemically for estrogen (E), testosterone (T) and progesterone (P) in Canada lynx and domestic cat hair extracts; (ii) to assess the use of hair reproductive hormones to differentiate between known reproductive states  using domestic cats as a model; and (iii) to assess the use of hair reproductive hormones to differentiate between age and sex, accounting for potential regional variability in wild lynx populations.Assay validation was conducted using methods previously described (see Wild hair samples were collected from lynx pelts at North American Fur Auctions (NAFA) in Toronto and Fur Harvesters Auction (FHA) in North Bay, Canada. All pelts were legally harvested by licensed trappers in the 2008–2009 trapping season and not tanned prior to collection. Peterborough Veterinary Clinic (PVC) in Peterborough, ON, Canada, and Cincinnati Zoo and Botanical Garden, in Cincinnati, OH, USA, provided domestic cat samples. Samples were collected with permission from the cat owners. Animals from PVC were privately owned pets, while those at Cincinnati Zoo were included in studies on felid reproductive biology (see The owners provided the age (6 months to 12 years) and sex of the domestic cats . Individuals that had been spayed or neutered at least 1 year prior to EIA analysis were used to ensure a minimal period of hair growth since castration. Females were classified as in estrus using signs of behavioural estrus , with juveniles (or kits) having a pelt length <90.5 cm in the west and <81 cm in the east, while adults (or yearlings) had a pelt length >90.5 cm in the west and >82 cm in the east . The sexg) 750 or 1500 µl of the extracted solution was transferred to a new glass vial and dried down in a fume hood. Samples were reconstituted in 150 µl EIA buffer  for a 5-fold concentration for lynx hair E, T and P and domestic cat P, and a 10-fold concentration for domestic cat E and T. Reconstituted samples were sonicated for 20 s in an Elmasonic waterbath  before hormone analysis.Hair samples were extracted using methods previously described; see m bicarbonate buffer, pH 9.6) and incubated overnight at 4°C. Unbound antiserum was washed from coated plates with 0.15 m NaCl solution containing 0.05% Tween 20. Immediately, 50 µl of reconstituted hair extracts, hormone standards and controls diluted in EIA buffer were added in duplicate, followed by 50 µl of horseradish peroxidase conjugate diluted in EIA buffer. Plates were incubated for 2 h at room temperature. Following incubation, the plates were washed, and 100 µl of substrate solution  diammonium salt, pH 4.0) was added. Absorbance was measured at 405 nm using a spectrophotometer . Hormone levels are presented as mass per gram dry weight.Estrogen, testosterone and progesterone values were quantified using EIAs based on the protocols from C. Munro, UC Davis, and described in detail by Linear regression analysis was used to determine whether there was a significant relationship between the standard curve and serial dilutions for assay parallelism and hormone added vs. hormone recovered for assay accuracy.post hoc HSD test. Given unequal sample sizes, each model was validated further using residual plots. Post hoc power analyses were completed using the R package ‘stats: anova.power.test’ . While there are limitations to the use of post hoc power analysis  were used to determine whether differences in reproductive states  could be detected, using domestic cats as a model species, given that considerable reproductive hormone data exist for this species . Femalesanalysis , this meet al., 2005; Hair reproductive hormones  were used to determine whether differences in age could be detected, because juveniles generally produce lower levels of reproductive hormones than adults, particularly among males (Rolland P > 0.05). Variances were homogeneous in all cases , except with age and T:P ratio . However, given that this P-value is close to the threshold for determining homogeneity (P = 0.05), a parametric test was used.Hair reproductive hormone ratios  were used to determine whether differences in sex could be detected, given that both sexes can produce testosterone, progesterone and estrogen (The area under the curve (AUC) of a receiver operating characteristic (ROC) curve .r = 0.98; all P > 0.01; see Supplementary Data). Serial dilutions of pooled hair extracts showed parallel displacement with the standard curves . Interassay coefficients of variation were 6.0, 7.4 and 13.0% at 30% binding for E, T and P, respectively, 6.5 and 7.3% at 50% binding for E and P, and 10.4% at 70% binding for T. Intra-assay coefficients of variation were 10.1 at 70% binding for E, and 5.9 and 12.1% at 50% binding for T and P, respectively.Extraction of exogenous E, T and P in domestic cat hair samples resulted in procedural efficiencies averaging 99.7 ± 9.8 (±SEM), 110.7 ± 3.8 and 90.3 ± 6.8%, respectively. The recoveries of known concentrations of E, T and P from domestic cat hair extracts were as follows: 85.8 ± 4.3, 76.4 ± 2.8 and 100.9 ± 3.8%, respectively. The measured hormone concentrations in the spiked samples correlated with the expected concentrations for all hormones . Serial dilutions of pooled hair extracts showed parallel displacement with the standard curves .Extraction of exogenous E, T and P in lynx hair samples resulted in procedural efficiencies averaging 106.6 ± 10.7 (±SEM), 113.1 ± 13.1 and 96.8 ± 14.9%, respectively. The recoveries of known concentrations of E, T and P from lynx hair extracts were 84.1 ± 1.5, 89.9 ± 7.1 and 73.9 ± 4.1%, respectively. The measured hormone concentrations in the spiked samples correlated with the expected concentrations for all hormones  and T levels  did not differ significantly between known reproductive states . Female domestic cat  hair P levels differed according to reproductive states . Pregnant individuals had significantly higher P levels than spayed individuals . However, there was no difference between pregnant and intact or spayed and intact individuals . Incidentally, two female cats were given oral progestin for 38 days ∼6 months prior to analysis, and had discernably higher hair progesterone values (2.7 and 4.13 times the average).Comparisons of hair hormone concentrations for domestic cats of known reproductive states are given in Figs Post hoc power analysis indicated that E and P had sufficient power with a sample size of seven individuals per group , while T had low power (0.47). In order to achieve a power of 0.8 for T, a sample size of ∼9.5 per group (intact and neutered) would be necessary.F1,64 = 7.55, P < 0.01) and age , but main effects of sex and age × sex were not significant . These hair E results were supported by AUC values, which revealed a poor ability to classify according to age .Mean hair hormone values for age (adult vs. juvenile), sex  and region (east vs. west) of lynx are given in Table F1,65 = 8.96, P < 0.01) and age . However, the hair T values were again not accurate at differentiating age . Differences in hair T were not detected between sex, and no age × sex interaction was found .Lynx hair T levels differed between region  and sex  and an age × sex interaction . However, lynx P levels did not differ between age groups (P = 0.14). The significant age × sex interaction term indicates that adult females had higher average hair P values than adult males, while juvenile males and females were not distinguishable ; however, they did differ between sex . The AUC values showed that the T:E ratios were not accurate at differentiating between males and females .Hormone ratios were calculated to determine whether hormone profiles could be used to determine sex using lynx individuals of known sex. Mean hair hormone ratios for age, sex and region are given in Table F1,63 = 23.34, P < 0.01) and age , while there were no differences between region, and no sex × age interaction was found (P > 0.11). The 95% confidence intervals for adult E:P ratios were non-overlapping, indicating they can be used to distinguish sex , while an effect of sex  and a sex × age interaction were detected . Although both E:P and T:P ratios showed differences in sex, further quantifiaction using AUC values showed that E:P ratios  were moderately accurate and superior at differentiating between sexes when compared with T:P ratios . Finally, the level of accuracy was increased for E:P ratios to highly accurate when only adults were used in the ROC analysis .Lynx hormone E:P ratios showed detectable differences between sex  in the hair of a felid species. First, we used domestic cats as a model to determine whether hair reproductive hormones could differentiate between individuals having known reproductive states . Progesterone appeared to be the most discriminating hormone, because pregnant individuals had higher levels than spayed individuals. Elevation in progesterone levels is tied to formation of corpora lutea, and this increase persists through pregnancy . This maEstrogen did not show differences according to domestic cat reproductive states, despite having reasonable statistical power. Although studies have shown that domestic cats are polyestrous and experience brief estrogen surges during estrus , with onTestosterone did not differ between neutered and intact domestic cats, although previous studies have shown that faecal testosterone values differ between intact and neutered male domestic cats .Lynx of known age and sex were used to assess the potential of using hair reproductive hormones to differentiate between groups. Although we detected significant age-specific differences among hair estrogen and testosterone values in lynx pelts, further analysis with AUC showed these differences were not accurate at differentiating between adults and juveniles. Notably, progesterone was effective at differentiating only between the sexes of adult individuals. These findings are similar to faecal reproductive hormone studies that show similarities in estrogen and progesterone values among female lynx aged 2–12 years . The metHair hormone values differed between regions (east and west) for the three hormones of interest. This pattern may be due to differences in the temperature during the time of active hair growth (or moult). For example, the average monthly temperatures from available weather stations were −4.68°C for Ontario and −13.41°C for the Yukon in November 2008 ; such differences in environmental conditions between areas are likely to persist year round. It is possible that this difference leads to differences in fur characteristics that translate to higher reproductive hormone values in the west. However, while the mechanism driving these regional differences is currently unknown, it is an important potential confounding factor that should be investigated further. Depending on the outcome of this investigation, the utility of hair reproductive hormone analysis should be reviewed carefully.Hormone ratios showed differences that may be used to distinguish accurately between sexes, especially for adults. In particular, the E:P ratio holds promise for evaluating sex ratios, which is consistent with previous findings showing that faecal androgen-to-estrogen ratios can distinguish sexes in right whales . UltimatLynx lynx) and bobcat (Lynx rufus)] and biochemically validated an EIA for lynx and domestic cats, which shows that the method holds promise for Lynx spp. , as well as other felids. Our study should serve as a prompt for further investigation into the use of hair analysis for documenting reproductive states. This is particularly important given that felids tend not to reproduce well in captivity (We were able to detect reproductive hormones in the hair of other lynx species [Eurasian lynx (aptivity , making Conservation Physiology online.Supplementary material is available at This work was supported by the Toronto Zoo and by a Natural Sciences and Engineering Council of Canada Strategic Projects Grant to D.L.M.