Unlicensed and off label drug use is an issue recognized worldwide in pediatric pharmacotherapy. The study was designed to assess the prevalence and predictors of unlicensed and off label drug use in neonatal population of Pakistan.A prospective, observation study was conducted in nursery units at pediatric department of four tertiary care hospitals during the 1 year. Micromedex DRUGDEX was used to evaluate the case notes of 1300 patients. Logistic regression was employed to calculate the odds ratio for the predictors of unlicensed and off label drug use.A total of 1300 patients were included in this study who were treated with 52 different drugs. The prevalence of off label drug use was higher (52.14%) as compared to unlicensed drug use (33.35%). Dose (61.29%) and indication (13.68%) were the most frequent reasons for off label prescribing. In comparison to the corresponding reference categories, females and preterm infants were less likely to receive unlicensed prescriptions. While patients staying at hospital more than 5 days and infants receiving 3 or more medications were significantly more likely to receive unlicensed prescriptions. Moreover, in comparison to the corresponding reference categories, females were less likely to receive off label prescriptions while infants receiving 3 or more medications were 7 times more likely to receive off label prescriptions.The significant prevalence of unlicensed and off label drug prescriptions was found in neonatal population of Pakistan. The findings imply that more data on prevalence of unlicensed and off label prescriptions are required to provide a better picture of pediatric therapy in developing countries. Furthermore, advance formulations with new dosing in pediatrics is also necessary to minimize the risk of adverse drug events.The online version of this article (10.1186/s12887-018-1211-y) contains supplementary material, which is available to authorized users. Challenges faced by healthcare professionals are unique in pediatric population as compared to that of adults, as varied drug response can occur in the absorption, distribution, metabolism, and elimination, affecting the efficacy or safety of the drug. . To combUnlicensed drug refers to employment of a drug in lack of product license, while off label drug refers to drug prescribed outside the specifications labelled on the product license . Lack ofTo date, no national or regional surveys have been conducted in Pakistan to evaluate unlicensed and off label drug use in neonates. The study was designed to assess the prevalence and predictors of unlicensed and off label prescriptions.Data for this study was collected from each nursery at pediatric department of four tertiary care hospitals in Peshawar, Pakistan. Nursery was the second most populous ward after medical unit in our region. The nursery of private hospital (NWGH) was better facilitated as compared to government hospitals . However, number of beds were more in government hospitals than in private hospital. No neonatal intensive care unit was present in any of the hospitals and all neonates who required critical care were treated in the nurseries of the respective hospitals.The prospective observational study was conducted from 15th May, 2014 to 15 April, 2015. Sample size was determined using an appropriate formula for known populations. A total of 1345 case notes were collected, in order to complete sample size (1300 patients were included which fulfil our inclusion criteria). Cluster sampling technique was employed to collect the data. Primary data was collected on predesigned proforma. Data source was the patient medication charts which provided information of identification number, name, age, gender, duration of stay, diagnosis and prescribed medications. Patients admitted to nursey unit with a minimum duration of stay of 24 h in the hospital were included in the study. Topical medications, oxygen therapy and IV’s stock solutions were not considered for evaluation.Age groups of neonates were categorized on the basis of recommended World Health Organization (WHO) guidelines. Patients of age less than 38 weeks were considered as preterm, while those of age above 38 weeks were categorized as infants . AnatomiY = 1, N = 0) in model. Multivariate binary logistic regression was employed to determine the odds ratio with 95% confidence interval (CI) for the predictors of dependent variables. Age, gender, duration of stay and number of prescribed drugs were independent variables in model and considered as predictors [Age, gender, number of prescribed drugs, duration of stay, unlicensed and off label drugs use were summarized using frequencies. Unlicensed and off label prescriptions were dependent variables and presented as  were from HMC, 240 (18.46%) were from NWGH, while 355 (27.31%) were from KTH and LRH each. Out of total neonates, 271 (20.76%) were born with premature status in nursery unit of four tertiary care hospitals. Median gestational age for preterm babies was 33 weeks (range 26 to 35 week). The male to female ratio was 17:8. The mean age of the population was 8 (±18.45) days and age of the patients ranged from 1 day to 1 month. Of the total patients, 74.92% patients were between the ages of 1 to 7 days, 11.00% were between the age of 8 to 14 days, 3.54% were between the ages of 15 to 21 days and 10.54% were 22 to 30 days. Mean number of prescribed drugs were 2.85 (±1.358). While Mean duration of stay in the nurseries was 3.15 (±2.8) days.as shown in Table The primary diagnosis of the neonates was neonatal sepsis (18.23%), followed by hypoxic ischemic encephalopathy (17.92%), neonatal jaundice (15.00%), preterm observation (9.08%), respiratory distress syndrome (7.15%), birth asphyxia (5.46%), meconium aspiration syndrome (3.23%), pneumonia (2.92%), meningitis (2.77%) and kernicterus (2.31%).Anti-infective for systemic use (72.97%) and central nervous system (12.3%) were prevalent therapeutic drug categories in nursery units of pediatric department of four tertiary care hospitals. Commonly prescribed medications in all the four hospitals were ampicillin (29.23%), ceftriaxone (28.57%), amikacin (5.83%), phenobarbitone (5.28%), ceftazidime (5.16%), phenytoin (2.32%), gentamicin (2.26%), captopril (2.03%), Paracetamol (1.94%), and aminophylline (1.80%).A total of 52 drugs were prescribed 3448 times, of which 1150 (33.35%) were unlicensed and 1798 (52.14%) were off label. Out of the total patients, 1001 (77.00%) received at least one unlicensed drug and 1016 (78.15%) received at least one off label drug. The detail of patients received at least one unlicensed and one off label drug is given in Table Dose 61.29%) and indication (13.68%) were the most frequent off label categories in all the therapeutics classes. Other observed off label categories were; age (10.79%), indication-dose (8.12%), age-dose (2.45%), indication-dosage form (1.67%), age-dosage form (0.67%), dosage form (0.50%), dosage form-dose (0.50%), age-indication (0.11%), age-indication-dose (0.11%) and age-dosage from-dose (0.11%). %). The commonly prescribed off label drugs along with their categories are shown in Table 9% and inp < 0.0001). Further indicating that prevalence of unlicensed drug prescribing was high in the private setup  as compared to government setups . A significant association was also found between unlicensed drugs prescribing with age groups (p < 0.0001). Unlicensed drugs were prescribed in 72.94% of 7 days old, in 88.81% of 8 to 14 days old, 95.96% 15 to 21 days old and in 86.86%% in 22 days and above category.Chi square value determine that unlicensed drug use was significantly associated with the type of hospital (p < 0.001) when analyzed using chi square. Off label drug prescribing was lesser in private hospital as compared to government hospitals i.e., government hospitals; in HMC-91.42%, in KTH-82.81%, in LRH-70.98%, and private hospital; in NWGH-62.5%. Medications prescribed off label was insignificantly associated with age groups (p < 0.332). Drugs prescribed off-label were more frequently found in children  and infants .There was significant association between off label drug use and type of hospitals  were significantly less likely to receive unlicensed prescriptions as compare to males. Similarly, preterm infants  were significantly less likely to receive unlicensed drugs as compared to term infants. While patient staying at hospital more than 5 days  were 1.7 times significantly more likely to receive unlicensed medications as compared to patients staying at hospital less than 5 days. Infants receiving 3 or more medications  were also 3.3 times significantly more likely to receive unlicensed prescriptions as compared to reference category.While evaluating off label prescriptions, females  were significantly less likely to receive off label prescriptions as compare to males. Infants receiving 3 or more medications  were 7 times more likely to receive off label drugs as compare to patients receiving 1 or 2 drugs as shown in Table In Neonates, proportion of unlicensed prescriptions (33.35%) were comparatively less as compared to off label prescriptions 52.14%). A Turkish study conducted in neonatal intensive care revealed a high prevalence (62.3%) of unlicensed or off label drug use in neonates . In cont%. A TurkAn Irish and Australian studies reported 39 and 47% of off label prescriptions which is low as compared to our results , 18. TheDose (61.29%) was observed to the most common reason for off label drug use. A similar reason for off label drug use was reported by many other studies –20. Off-ly prescribed therapeutic drug category [Anti-infective for systemic use (51.37%) and drugs used in respiratory system were the frequently prescribed therapeutic drug categories. A multicenter study performed at NICU in Italy reported similar findings . Anothercategory . A Turkicategory . A prospcategory . The freTwo antibiotics ampicillin and cefotaxime were the most frequently prescribed in neonatology units. Warrier et al., conducted a study at Detroit, United states reported concomitant findings . HoweverPreterm neonates, patients receiving 3 or more medications, infants staying at hospital more than 5 days and female gender were significant risk factors for unlicensed drug use while preterm infants and infants receiving 3 or more medications were significant risk factors for off label prescriptions in current study. Gender is not taken into consideration during the drug development . HoweverPakistan follows the both the Food and Drug Dosage guidelines and European Union Regulation On Medicinal Products for Pediatric Use (Regulation n° 1901/2006) implemented in 2007. European Medicinal Agency (EMA) published a report on clinical experience obtain with the application of this module which introduced and implemented a system of obligations, incentives and rewards to the development of medicines for pediatric age in European Member States . The repThe number of pediatric clinical drug trial remained constant between 2006 and 2012, but there was a sophisticated upsurge in the number of pediatric studies, in particular for the infant age group, who were normally not considered in the clinical trials before 2008. More than 18,000 reports were submitted to European medicine agency since 2008 regarding the use of medicine in pediatrics, but it originated only 65 changes in approved products . These oNeonates with stay less than 24 h were not included in this study. Further studies are also required in other pediatric units at tertiary care hospitals in our country. So the results should be generalized with caution in population.The prevalence of unlicensed and off label drug use was high in neonates and not reported in Pakistan till date. Physicians must be reminded of the unlicensed and off label drug use when prescribing medications for newborns. Suitable medical interventions must also be established by drug manufactures and government agencies for betterment of neonatal pharmacotherapy.Additional file 1:Nursery all cases. File contains all the data which has been analyzed for this manuscript. (XLSX 584 kb)


Salmonella enterica serovar Enteritidis, which was previously isolated from conveyor belts during chicken slaughter and has the potential to form biofilms on several surfaces. The genome is predicted to contain 110 noncoding small RNAs on the chromosome.Here, we report the complete genome sequence of strain NMC 61 of  Salmonella enterica serovar Enteritidis, which was previously isolated from conveyor belts during chicken slaughter and has the potential to form biofilms on several surfaces. The genome is predicted to contain 110 noncoding small RNAs on the chromosome.Here, we report the complete genome sequence of strain NMC 61 of  Salmonella belonging to Enterobacteriaceae is a globally widespread foodborne pathogen. All are at risk for Salmonella infection but especially those with weakened immune systems. Salmonella enterica serovar Enteritidis is the most frequently detected causative agent in foodborne outbreaks and recalls  and incubated at 37°C for 18 h with shaking. The DNA was extracted using a QIAamp DNA microbiome kit and used to prepare a library with a fragment size of >10 Kb selected with the BluePippin system. The genome of S. Enteritidis was sequenced on a Pacific Biosciences instrument  using P4-C2 chemistry. Hierarchical Genome Assembly Process (HGAP) software (version 2.3.0) was used to assemble a total of 86,659 reads with an average length of 11,156 bp. The de novo assembled chromosome genome of S. Enteritidis NCM 61 is 4,679,739 bp with GC content of 52.17%. The 4,660 coding sequences, 106 structural RNAs (22 rRNAs and 84 tRNAs), and 14 gene islands (GIs) were annotated using Glimmer version 1.1, rRNAmmer 1.2 server, tRNAscan-SE 2.0, Rfam 14.0, and IslandViewer 4 , S. Heidelberg SL476 (CP001120), and S. Enteritidis Durban (CP007507) were 95.04%/91.54%, 94.18%/90.14%, and 99.12%/99.14%, respectively, which were determined using NCBI BLAST between two genomes with an E value less than or equal to 10−5 and alignments exceeding 80% identity within 1,000 bp of the query sequence. In addition, using the Rfam database, a total of 110 noncoding small RNAs were identified on the chromosome. The availability of the whole-genome sequence will provide future insight into the adhesion and pathogenic profiles of the S. Enteritidis NCM 61 strain.A single colony of Viewer 4 . One 59,Viewer 4 , includiSalmonella Enteritidis strain NCM 61 has been deposited in NCBI under the BioProject accession number PRJNA492709. The GenBank accession number is CP032851 for the chromosome and CP032850 for the plasmid. The SRA accession number is SRS3887548.The whole-genome sequence for


In contrast, application of the gap junction blocker carbenoxolone prevents the appearance of SLEs in the low-[Mg2+] epilepsy model, but aggravates seizures in non-convulsive absence epilepsy, in vivo. Pharmacological dissection of neuronal vs. astrocytic connexins shows that astrocytic Cx43 contribute to seizure formation to a significantly higher extent than neuronal Cx36. We conclude that astrocytic gap junctions are key players in the formation of epileptiform activity and we provide a scheme for the different mode of action in the convulsive and non-convulsive epilepsy types.The influence of astrocytic cell networks on neuronal network activity is an emerging issue in epilepsy. Among the various mechanisms by which astrocytes modulate neuronal function, synchronization of astrocytes via gap junction channels is widely considered to be a crucial mechanism in epileptic conditions, contributing to the synchronization of the neuronal cell networks, possibly inducing recurrent epileptiform activity. Here, we explored whether modulation of astrocytic gap junctions could alter epileptic seizures in different types of epilepsy. Opening of gap junctions by trimethylamine intensifies seizure-like events (SLEs) in the low-[Mg Epilepsy is one of the most common neurological disorder affecting 1–2% of the population worldwide. It is considered to be an enhanced neuronal activity characterized by spontaneous recurrent seizures, which can be prevented by appropriate medication only in 70% of patients, but one-third remains pharmacoresistant . The epi+ buffering through Na+/K+ pumps, Na+/K+/Cl– cotransporters and Kir channels  in response to intracellular Ca2+ concentration changes triggered by ionotropic and metabotropic receptor activation  are also significant contributors of epileptic activity . In addichannels , water rchannels  as well nd EAAT2 , astrocytivation . Moreovetivation , 2012.2+, IP3, cyclic AMP, and oligonucleotides   . Increass (SLEs) , becauses (SLEs) . These fExcessive neuronal synchronization and excitability have a role in the generation of mechanistically different epilepsy types, like the temporal lobe epilepsy (TLE) and absence epilepsy . As astrin vitro low-[Mg2+] model (in vitro model of TLE) and in vivo in the animal model of human absence epilepsy Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats  rats . In contij) rats . Convuls, the Hungarian Act of Animal Care and Experimentation , European Communities Council Directive 24 November, 1986 (86/609/EEC) and EU Directive 2010/63/EU on the use and treatment of animals in experimental laboratories. The experiments on WAG/Rij rats were approved by the Animal Care and Experimentation Committee of the Eötvös Loránd University (Savaria University Centre) and National Scientific Ethical Committee on Animal Experimentation (Hungary) under License No. VA/ÉBNTF02/85-8/2016. All efforts were made to reduce animal suffering and the number of animals used.Animals were housed in groups of 3–4 under standard laboratory conditions .2; 1.8 MgSO4; 1.25 NaH2PO4; 21 NaHCO3; 10 glucose. To induce epilepsy, MgSO4 was eliminated and 2 mM KCl was added (low-[Mg2+] ACSF). In the in vitro experiments, CBX (200 μM), TMA (5 mM), quinine (100 μM) and La(NO3)3 were diluted in ACSF or low-[Mg2+] ACSF. The pH value of 7.4 was not affected by the applied TMA concentration. All solutions were continuously oxygenated . In the in vivo experiments on WAG/Rij rats, both CBX (100 mg/kg) and quinine (40 mg/kg) was dissolved in saline, whereas TMA (200 mM) in aseptic ACSF . Unless otherwise stated, all drugs were purchased from Sigma-Aldrich, Budapest, Hungary.Artificial cerebrospinal fluid (ACSF) contained in mM: 129 NaCl; 3 KCl; 1.6 CaCl2; 7 mM MgSO4; 1.25 mM NaH2PO4; 25 mM NaHCO3; 25 mM glucose) at 4°C. Slices were incubated in an interface-type chamber in continuously oxygenated ACSF for 1 h at 37°C followed by incubation in room temperature before performing the experiments.Transverse, 400 μm thick, hippocampal-entorhinal slices from 11 to 14 day old Wistar rats  were prepared in modified ACSF , low-pass filtered at 2 kHz and digitized at 10 or 20 kHz . Recordings were analyzed after high pass filtering at 1 or 2 Hz.The electrophysiological field potential (FP) recordings were performed at 31°C. For FP recordings glass microelectrodes (1 to 4 MΩ) were filled with ACSF solution and were inserted in the CA3 2+] ACSF (ACSF with no added MgSO4 and KCl elevated to 5 mM). Drugs were applied according to two different protocols. To test the effect of the drugs on the appearance of SLEs, they were continuously present in the ACSF and low-[Mg2+] ACSF solutions. First, normal ACSF solution was applied for 20 min as control condition. Then normal ACSF solution with 5 mM TMA or 200 μM CBX was applied for further 20 min. Finally, low-[Mg2+] ACSF, containing the same concentration of drugs was applied. Another protocol was used to identify the connexin form involved in the modulation of epileptiform activity. In these experiments, drugs were applied after the detection of two fully developed SLEs. The drugs were present either during the next two SLEs or at least for 10 min . To avoid excessive consumption of the Cx43 antibody  it was applied according to a special protocol: perfusion of ACSF was stopped after the second SLE and Cx43 antibody was added to the chamber at a relatively high concentration . After 10 min, the perfusion was restarted, and the antibody was washed out. In control measurements, the same protocol was applied without adding Cx43 antibody after perfusion was stopped. In these control measurements, SLE occurrence was not affected by the interruption of perfusion.Epileptiform activity was induced by switching the perfusing solution (ACSF) to low-[Mgin vivo experiments . Isoflurane-air mixture (2.0–2.5%) anesthesia was used for electrode implantation. Screw electrodes were implanted into the bone above two cortical areas of all rats for EEG recording   . A groun 2.1 mm) .One stainless steel guide cannula (27G) was inserted into the lateral ventricle  to i.c.v. injection of TMA by infusion pump . The location of the cannula tip in the third ventricle was verified by the free outflow of cerebrospinal fluid (CSF) through the guide cannula. Patency of guide cannula was maintained with a sterile stainless steel dummy stylet .After implantations, all rats were allowed to recover for 2 weeks.Electroencephalogram (EEG) was recorded by a differential biological amplifier . The amplifier was attached to a CED 1401 mkII  data capture and analysis device. The bandwidth of the EEG recording was 0.16 to 150 Hz and the sampling rate was 1 kHz . As handTo adapt the WAG/Rij rats to the experimental procedures, all animals were handled daily for 4 days, which adaptation period was started 2 weeks after surgery .n = 8) was intraperitoneally (i.p.) injected by 0.5 ml/100 g body weight saline for 3 days (3-day control period) to establish average control SWD number and SWD time. On the 4th day, animals of group 1 were injected i.p. by 0.5 ml/100 g body weight saline containing CBX (100 mg/kg). The second group of animals (n = 5) received the i.c.v. TMA injection after the 3-day control period. The TMA solution (200 mM TMA in 5 μl aseptic ACSF/rat) was injected into the lateral ventricle through the guide cannula. At the beginning of the i.c.v. injection the animals were gently restrained by a towel. The dummy stylet was removed from the guide cannula and a stainless steel needle (which was connected to an infusion pump by a polyethylene tube) was inserted. The injection flow rate was 0.5 μl/min. The i.c.v. administration of 5 μl ACSF alone had no effect on SWD number (n = 7) were injected similar to animals of the first group by saline for 3 days (3-day control period) whereas they were injected i.p. by 0.5 ml/100 g body weight saline containing quinine (40 mg/kg) on the 4th day.Animals were separated into three groups. The first group of WAG/Rij rats (D number . The thip < 0.05.The Clampfit (Axon Instruments) program was used to evaluate electrophysiological data. Recordings were analyzed after high pass filtering at 1 or 2 Hz. SLE onset was determined by the negative FP deflection and a high-frequency oscillation at the start of discharges. This is the paroxysmal initiation period, which is followed by the tonic and clonic periods of paroxysmal spike discharges . The intIn vivo EEG recordings were split into 30 min sections and were evaluated separately  recurrent SLEs appeared in 27 of 37 slices (73%). The average onset of the first SLE was 17.0 ± 1.0 min. Under these circumstances the average duration of SLEs were decreasing . The interictal intervals during these recurrent SLEs were gradually declined  indicating a growing incidence of seizure propagation . The onset of SLEs also diminished from 17.0 ± 1.0 to 10.6 ± 1.0 min compared to control condition  was not affected by the gap junction opener TMA (5 mM) . Howeverondition , increasp = 0.01 for SLE1, p > 0.05 for ongoing SLEs compared to the control period; n = 15), showing that TMA also has a slight antiepileptic effect.However, the duration of SLEs also decreased to 83.6 ± 7.7 s (SLE1); 87.4 ± 8.1 s (SLE2); 100.3 ± 17.5 s (SLE3); 67.2 ± 9.2 s (SLE4); 73.4 ± 9.8 s (SLE5) . In accordance with our previous reports , we obseA receptor-mediated excitatory and inhibitory post-synaptic currents  compared to the control periods where it was only 72.97% (n = 37). In the remaining two treatments SLE formation was significantly declined. In the presence of CBX only 16.6% of slices generated SLEs (n = 12), while simultaneously applying TMA and CBX induced SLEs only in 8.3% of slices (n = 12)  .Although we confirmed that TMA and CBX share a common target in regard to their modulatory activity on SLEs, the molecular identity of this target was not fully resolved. First, to differentiate between the neuronal and astrocytic connexins, we compared the effects of a Cx43- and a Cx36-specific inhibitor. To specifically block the astrocytic Cx43 subtype, we used an antibody that was raised against the gating peptide segment of Cx43 , which wTo further confirm the connexin subtype-specificity of the gap junction-related anti-epileptic effect, we also investigated the contribution of the neuronal Cx36 subtype, which is known to be involved in neuronal synchronization . Cx36 wa3+), which is supposed to selectively block hemichannel function  significantly decreased the SWD number in all 30-min segments compared to controls . ParalleIn contrast to TMA, i.p. administration of 100 mg/kg CBX significantly increased the SWD number and total time of SWDs. The length of interictal intervals significantly decreased .in vivo absence epilepsy model as well. Similar to CBX, i.p. administration of the Cx36-specific inhibitor quinine (40 mg/kg) also increased the number and total time of SWDs and decreased the length of the interictal intervals  SWDs. These results reflect the dual role of gap junctions in epilepsy. Intercellular gap junctions play a pivotal role in K+ buffering and Glu clearance, since they contribute to the spatial distribution of these excitatory agents , the Hungarian Act of Animal Care and Experimentation , European Communities Council Directive 24 November, 1986 (86/609/EEC) and EU Directive 2010/63/EU on the use and treatment of animals in experimental laboratories. The experiments on WAG/Rij rats were approved by the Animal Care and Experimentation Committee of the Eötvös Loránd University (Savaria University Centre) and National Scientific Ethical Committee on Animal Experimentation (Hungary) under License No. VA/ÉBNTF02/85-8/2016. All efforts were made to reduce animal suffering and the number of animals used.in vitro experiments and wrote the manuscript. MP performed the in vitro experiments. ZS analyzed the data. JK designed the experiments and wrote the manuscript. LH designed the experiments, analyzed the data, and wrote the manuscript. ZK performed the in vivo experiments and wrote the manuscript.RV performed the 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.


To the Editor: Childhood undernutrition continues to be a significant health problem in resource-limited settings, contributing to morbidity, mortality and a frequent cause of hospitalization . More than half (n = 10) of the deaths occurred in age group 13–16 mo. Risk factors of mortality were analyzed on binary logistic regression for independent association, and it was found that children with fatal outcome were 11.29 times more likely to have shock (p = 0.001), 10.2 times more likely to have dehydration (p < 0.001), 17.2 times more likely to have acute kidney injury (p < 0.001) and 7.1 times more likely to have hyponatremia (p = 0.01). The presence of acute diarrhea had six times the odds of mortality (p < 0.001).We conducted an observational study from September 2016 through May 2018 to identify risk factors of mortality in hospitalized children, aged 6 to 60 mo, fulfilling the WHO criteria of SAM. Children with preterm birth or intrauterine growth retardation (IUGR) at birth, inborn error of metabolism, congenital anomalies, chronic renal failure, cerebral palsy, chronic liver disease, and chromosomal abnormalities were excluded. One hundred and twenty two children with SAM were assessed for eligibility. One hundred and ten children met the inclusion criteria and 12 were excluded. Of these 110 children, 57 (51.8%) had edematous malnutrition and 53 (48.1%) had non edematous malnutrition. Out of total children, 90 (81.8%) were discharged from the hospital, 18 (16.6%) died and 2 (1.8%) left against medical advice . Males constituted 72 (65.5%) of the children; 51 (46.6%) of the children were in age group of 13 to 36 mo. Nine (50%) children died within 3 d and 16 (88.9%) within 5 d of hospitalization. More children died in edematous group than in non edematous group [12 (21%) In this study we found that features of shock, severe dehydration, oliguria and hyponatremia were independent predictors of mortality. The presence of one or more of these risk factors in children with SAM should alert the physician of increased mortality risk in these children and requires early stabilization, close monitoring, and appropriate therapy.


The cow-calf industry in North America is in a period of rapid consolidation withcorresponding increases in herd sizes and changes in management. The objectivesof this study were to examine longitudinal data on reproductive performance incow-calf herds and identify benchmarks for the most critical measures andimportant sources of differences among herds. To address these questions, asurveillance network was established in western Canada to collect data between2013 to 2017 privately owned cow-calf herds during calving (n = 105 herds) andat pregnancy testing (n = 94 herds). Data were summarized for a number ofindices of herd performance. However, the values considered to be most reliableand accurate were the percentage of females not pregnant when tested by aveterinarian, the percentage of calves dead within 24 hours of birth, and thepercentage of calves dead from 24 hours to weaning. The mean and variationbetween herds for heifers, measured using standard deviation, was greater thanfor cows for: non-pregnancy (cows 6.8% (mean)±3.4%(SD), heifers 9.7%±8.2%), calfdeath from birth to 24 hours , and calf deathfrom 24 hours to weaning . Benchmarks orperformance targets derived from the 25th percentiles of these data for bothcows and heifers were <5% for non-pregnancy risk and <1% for calf losswithin 24 hours of birth. The suggested benchmark for calf loss from 24 hours toweaning was <2% for cows and <1% for heifers. All outcomes consistentlydisplayed greater variation between herds as compared to year to yeardifferences within herds with the exception of calf loss before 24 hours incows. The timing of the start of breeding season was a consistent source ofvariation in risks of non-pregnancy and calf losses. Cows bred in April orearlier to start calving in late December or January were at increased risk oflow pregnancy percentages (p<0.001) and calf losses at birth (p<0.04), aswell as increased calf loss before weaning in both cows and heifers (p<0.02).There was also an increase in the risk of non-pregnancy for cows and heifers(p<0.001) where first exposure to breeding was not until July or August. Incontrast, the risks of calf loss within 24 hrs of birth (p<0.001) and from 24hrs to weaning in cows (p<0.02) first exposed to breeding in July and Augustwere significantly lower than for herds that had earlier breeding seasons. The Canadian beef industry is undergoing important changes characterized bydecreasing herd numbers and increasing herd sizes . The conTo assess the potential impacts from ongoing industry changes to reproductiveperformance, surveillance data are needed. These data must be collected on anappropriate geographic scale to consider regional variation in climate, nutritionand management decisions on herd performance. While other one time surveys have beensuccessful in providing data to inform performance expectations –9, thereMore than 80% of Canadian cow-calf production is located in the western provinces ofAlberta, Saskatchewan and Manitoba . In respEnrollment targets for cow-calf herds within Alberta, Saskatchewan, and Manitobawere established using data from 2011 Census of Agriculture  as were herds with some purebred sales  after accounting for province(P = 0.06) and year of data collection (P= 0.06).Herds that bred < 300 cows were more likely than larger herds to startbreeding season in April or earlier as compared to later [OR 2.2, 95%CI 1.0–4.5,th percentile for the risk of non-pregnancy whentested was 4.3% for cows and 4.2% for heifers. That is, 25% of herds pregnancytested achieved less than 4.3% non-pregnant cows and 4.2% heifers,respectively.The mean percentage of cows that were not pregnant when tested were slightlylower than the percentage of heifers that were not pregnant . The perThe percentage of total variation in non-pregnancy risk of cows attributed todifferences between herds was 3.6% and the percentage of total variationattributed to year to year differences within herds for cows was calculated tobe 2.7%. The percentage of total variation in non-pregnancy risk of heifersattributed to differences between herds was 7.9% and the percentage of totalvariation attributed to year to year differences within herds for heifers wascalculated to be 5.8%.The model examining risk factors for non-pregnancy in cows included 276 herdobservations with complete cow herd outcome and risk factor data from 94different herds.P = 0.26) and between cows bred in May and June (P= 0.31).Cows first exposed to breeding in April or earlier were less likely to bepregnant  than cows from herds where thebreeding season started between May and June. In addition, cows from herds thatstarted the breeding season in July or August were also less likely to bepregnant (OR 1.3 95%CI 1.2–1.4 p<0.001) than cows from herds that started thebreeding season in May or June. There was no difference in the risk ofnon-pregnancy between cows bred in April or earlier and July and August(Cows from herds in Manitoba were less likely to be pregnant compared to herdsfrom Saskatchewan (OR 1.3 95%CI 1.1–1.4 p<0.001) and Alberta (OR 1.3 95%CI1.2–1.4 p<0.001).P =0.02) (Cows were less likely to be pregnant in 2017 than in 2015 ( =0.02) .P = 0.002) wereless likely to be pregnant than cows from herds that reported selling at leastsome purebred cattle. The total number of females pregnancy tested was notassociated with the risk of non-pregnancy in cows (P =0.10).Cows from commercial herds (OR 1.2 95%CI 1.1–1.3 The model examining risk factors for non-pregnancy in heifers included 265observations with complete heifer herd data from 93 herds.P =0.25).Similar to what was observed with the cows, heifers from herds that started thebreeding season in July or August were less likely to be pregnant (OR 1.4 95%CI1.2 to 1.5 p<0.001) than heifers from herds that started breeding season inJune or earlier. However, in contrast to what was seen with the cows, herds thatstarted breeding their heifers early were not at greater risk. Heifers firstexposed to breeding in April or earlier were more likely to be pregnant  than heifers from herds where the breeding seasonstarted between May and August. There was no difference in the risk ofnon-pregnancy between heifers bred in May and June (Heifers from herds in Manitoba were less likely to be pregnant compared to herdsfrom Saskatchewan (OR 1.4 95%CI 1.2–1.7 p<0.001) and Alberta (OR 1.4 95%CI1.2–1.7 p<0.001).P = 0.27) (The percent of heifers that were not pregnant did not differ significantly byyear ( = 0.27) .P= 0.84) compared to those that reported selling at least somepurebred cattle. Heifers were, however, less likely to be pregnant from largerherds that pregnancy tested >300 females (OR 1.2 95%CI 1.0–1.4 P= 0.016).There was no difference between heifers from commercial herds , May (22%), June (31%), July or August(22%) . Thelatth percentile 8.0%, 95th percentile 42.2%).The average percentage of heifers across all annual observations for herdsproviding breeding data was 22.6% (SD 10.7%); the median was 20.3% or heifers (P= 0.86)There was no association between the start of breeding season and the risk ofabortion for either cows (P = 0.02), but there was no difference across provincesfor heifers (P = 0.27).The risk of abortion from Alberta herds was higher than from Saskatchewan forcows (P= 0.01) and heifers (P = 0.03) and it was alsohigher in 2017 than in 2015 (p<0.001) for cows and for heifers (P= 0.01) (P = 0.01), but just for heifers.The risk of abortion was higher in 2014 than in 2015 for cows (= 0.01) . FinallyP = 0.34) or heifers (P= 0.70).There was no association between the risk of abortion based on whether herd sizewas >300 cows for cows (P = 0.01) from herds thatsold at least some purebred cattle than those that did not.The risk of abortion was also higher for cows (OR 1.4 95%CI 1.1–1.6 p<0.001)and for heifers  Tables  and 4. TThe models for calves dead from birth to 24 hours for cows included 330observations with complete data from 102 herds and the model for heifersincluded 318 observations with complete data from 102 herds.P= 0.13) or between those bred in May or June (P =0.41). The risk of calf death from birth to 24 hours was also higher for cowsbred in April or earlier than for cows bred in May (OR 1.2 95%CI 1.0–1.5P = 0.04) or June (OR 1.3 95%CI 1.1–1.6 P= 0.01). There was no association between the month that breedingbegan and the risk of calf death from birth to 24 hours in heifers (P= 0.66).Risk of calf death from birth to 24 hours was lower for cows first exposed tobreeding in July or August than those bred in May or earlier (OR 1.4 95%CI1.2–1.6 p<0.001). There was no difference in risk of calf death from birth to24 hours for cows bred in June and those bred in July and August (P = 0.87) orheifers (P = 0.51).There was also no significant difference in the risk of calf death from birth to24 hours among provinces for calves from cows (P = 0.001) or 2015 (P = 0.03) for cows(P = 0.03) or 2017 (P = 0.02) forheifers.Risk of calf death from birth to 24 hours was significantly lower in 2016 than in2014 (for cows. Risk ofP= 0.12) or for heifers (P = 0.07) and whether ornot the herd sold any purebred cattle for cows (P = 0.95) orfor heifers (P = 0.61).There was no significant difference in risk of calf death from birth to 24 hoursbased on whether the herd size at calving was >300 for cows . Therisk significantly lowered again from May to June (OR 1.2 95%CI 1.0–1.5P = 0.03), and from June to July and August (OR 1.2 95%CI1.0–1.4 P = 0.02).Calf death loss between 24 hours and weaning was highest for cows exposed tobreeding in April or earlier with the risk lowering significantly for cows firstexposed to breeding in May (OR 1.2 95%CI 1.0–1.5 P= 0.02), June (OR 2.0 95%CI 1.4–2.8 p<0.001) or July or August(OR 1.4 95%CI 1.1–1.9 P = 0.02). However, calves were also morelikely to die before weaning if born to heifers exposed to breeding in July orAugust as compared to June (OR 1.4 95%CI 1.1–1.9 P = 0.01).Calves were more likely to die before weaning if born to heifers exposed tobreeding in April or earlier as compared to May (OR 1.5 95%CI 1.1–2.2 P = 0.004) andManitoba (p<0.001) than in Saskatchewan.Calf death loss from 24 hours to weaning was significantly higher for calves fromcows in Alberta than in either Saskatchewan (p<0.001) or Manitoba(p<0.001). Calf death loss from 24 hours to weaning was also significantlyhigher for calves born to heifers in Alberta (P = 0.047), 2015 (p<0.001), or 2016 (P= 0.008) than in 2017 for calves born to cows (P= 0.001), 2016 (p<0.001), and 2017 (P = 0.04).It was also lower in 2017 than 2016 (P = 0.003).Calf death loss between 24 hours and weaning was significantly lower in 2014( to cows . The deaP = 0.30) or for heifers (P = 0.18) orwhether the herd size at calving was >300 for cows (P =0.83). The risk of death between 24 hours and weaning was higher for calves fromheifers from herds with >300 cows at calving (OR 1.8 95%CI 1.4–2.4p<0.001).There was no significant difference in the risk of death between 24 hours andweaning based on whether or not the herd sold any purebred cattle for cows 0.8% to 2.9%). These numbers are lower than that reportedfrom a large 2002 study of 203 cow-calf herds in western Canada where the median forcalves dead within 1 hour of birth was 2.4% (IQR 1.3% to 3.9%) for cows and heiferscombined . The difCalf deaths from 24 hours to weaning were not broken down into smaller risk periodsin this report as questions on the details of when calves died were not asked everyyear of the study. Calf loss data were expected to be reasonably complete up to thetime cow-calf pairs are moved to summer pasture. However, calf losses on extensiveand heavily treed summer pasture could potentially be missed and infrequent lossesduring the summer season that are identified could miss being recorded in busyoperations. Half of the herd reports included losses from birth to weaning that were<2% for both heifers and cows. A quarter of herd reports were just over 1% forcows and were 0% for heifers. Five percent of reported calf losses before weaningwere >6.9% for cows and 10.4% for heifers.A large survey from the USA in 2007 reported overall calf mortality of 6.4%; with 28%of these mortalities occurring after 24 hours of age. This would equate to a 2.4%mortality percentage of calves from 24 hours to weaning . A studyYear-to-year variation was significant for all outcomes of interest with theexception of pregnancy rates in heifers. Year-to-year variation was more apparentfor calf losses both at and after birth than for pregnancy rates in this sample.Environmental conditions such as temperature and precipitation have been shown tohave a significant effect on calf mortality and these factors could be responsiblefor some of this year to year variation in productivity outcomes , 22. TheAll outcomes of interest consistently displayed greater variation between herds ascompared to year to year differences within herds with the exception of abortion andcalf death from birth to 24 hours in cows. This would suggest that for theseproduction traits, management differences between herds plays a greater role inaffecting the variation of these traits then do year to year changes within herdsthat are seen due to climatic conditions. However, the abortion risk and risk ofcalf death from birth to 24 hours in cows did not follow this pattern and variationbetween herds was very similar to variation within herds.There were no consistent patterns of differences among provinces, but what we didobserve suggests the potential for significant geographic variability in performanceacross the study region that needs to be considered in designing surveillanceprograms and in developing management recommendations. Previous herd productivitystudies across large regions of western Canada identified geographic variationthrough the inclusion of precipitation . VariabiThe timing of the start of breeding season was a consistent source of variabilityamong the outcome measures of interest. Cows bred in April or earlier to startcalving in late December or January were at increased risk of low pregnancypercentages and higher calf losses at birth, as well as higher calf losses beforeweaning in both cows and heifers. This is not surprising given the extreme cold andresulting confinement and increased infectious disease risk necessary to manage coldweather calving and the challenges of getting cows pregnant on stored feed. Smallerherds and herds having at least some purebred cattle were more likely to startbreeding April or earlier.However, perhaps the more interesting finding was the significant increase in therisk of non-pregnancy for cows and heifers where first exposure to breeding was notuntil July or August. In contrast, the risks of calf loss at and after birth in cowsfirst exposed to breeding in July and August were significantly lower than for herdsthat had earlier breeding seasons. One of the most plausible reasons proposed forlower pregnancy percentages in cows first exposed to breeding later in the summer isthe challenge of matching the cow’s nutritional needs early in the breeding seasonduring peak lactation, with the stage of the growing season and pasture quality.Larger herds were more likely than smaller herds to start breeding in July orAugust. It is important to recognize that although there were differences innon-pregnancy risk for different timing of breeding seasons, the decision onselecting the timing of breeding and calving season is based on a wide variety ofeconomic, marketing and labour issues that may offset any disadvantages of lowpregnancy percentages.Increasing herd size was only a significant risk factor only for pregnancy percentageand calf loss before weaning in heifers. Larger herds could be at an increased riskof biosecurity challenges if there have been recent increases in herd size frompurchased animals. One previous report did link increased risk of infectious diseasein calves to larger herd sizes . One impNo consistent patterns of differences were observed between herds with onlycommercial cattle and those that sold at least some purebred animals. However, therewere a relatively small number of herds that reported being exclusively purebred inthe present study limiting the power to examine the impact of potential managementdifferences in seed stock operations.In most previous studies we have relied on intensive contact with herd owners andindividual animal records to ensure data quality –6. This Others have described asking producers to report the number of cows calving in thefirst, second and third cycle of the calving season . We askeFor future studies of reproductive performance in client-owned herds, we suggestfocussing effort on the accurate collection of pregnancy testing data, stillbirthand calf loss information. Investigators should work with producers to encouragemore complete reporting of abortion losses and consistently reporting calf deathlosses within meaningful categories for age at death rather than just reporting alllosses between 24 hours of age and weaning. While the present study was not based ona random sample of all cow-calf producers, the results are most applicable tocow-calf clients of veterinary practices and the data were comparable to previousreports from western Canada. Future studies should also focus on accurate andspecific production information for defined periods of risk for calf loss  as compared to summarizing all lossesfrom birth to weaning. Reporting bias due to sales and purchases of cattle are lesslikely to be an issue in defined periods during the calving season as compared tolater during and after the breeding season.The percentiles reported when summarizing individual production metrics can be usedby veterinarians and producers to benchmark herd performance, to set realistic goalsfor improvement and to define thresholds requiring diagnostic investigation. Thefinding of greater variation among herds in heifer performance than among cowssuggests opportunities for improvement in heifer management. The finding that laterbreeding and calving herds were more at risk for poor pregnancy percentages providesdocumentation of a problem suspected by producers and veterinarians and will requirefurther investigation to determine the best way to minimize the nutritionalchallenges of getting cows pregnant later in the growing season.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.


Under phenotypic selection design, the accuracies of prediction by ssGBLUP method were 11.8% and 15.4% higher than those obtained by GBLUP for heritability of 0.10 and 0.20, respectively. However, the efficiency of ssGBLUP and GBLUP was not influenced by selection design at higher level of heritability (h2 = 0.50). Furthermore, higher selection intensity increased the bias of predictions in both pedigree-based and genomic evaluations. Regardless of selection design, TS sizes for GBLUP and ssGBLUP methods should be at least 3000 to achieve more accuracy than using BLUP for heritability of 0.50 and marker density of 10k and 50k. Overall, more accurate predictions were obtained using ssGBLUP method particularly for lowly heritable traits and low density of markers. Our results indicated that TS sizes should be optimized in accordance with heritability level, marker density, selection design and prediction method for genomic selection in American mink. The results provided an initial framework for designing genomic selection in mink breeding programs.Genomic selection can be considered as an effective tool for developing breeding programs in American mink. However, the genetic gains for economically important traits can be influenced by the accuracy of genomic predictions. The objective of this study was to investigate the prediction accuracies of traditional best linear unbiased prediction (BLUP), multi-step genomic BLUP (GBLUP) and single-step GBLUP (ssGBLUP) methods in American mink using simulated data with different levels of heritability, marker density, training set (TS) sizes and selection designs based on either phenotypic performance or estimated breeding values (EBVs). Under EBV selection design, the accuracy of BLUP predictions was increased by 38% and 44% for h GEBVs) is an important factor influencing the efficiency of genomic selection in livestock species [QTL) effects [Availability of cost-effective and high-throughput genotyping technologies has facilitated the use of genomic selection as a useful tool to select animals for breeding in livestock species. Genomic evaluations improve the genetic gains for economically important traits through reducing generation intervals and enhancing the accuracy of selection. Furthermore, selection based on genome-wide information can be particularly effective for traits that are expensive or hard to measure and have low heritability . Predict species . Accurac species , heritab species , marker  species , effecti species , distrib effects , 8, numb effects  and stat effects –11.BLUP) method is based on pedigree information and phenotypic performance of animals [DGVs) are predicted based on the effects of single nucleotide polymorphism (SNP) markers across the whole genome under the assumption of linkage disequilibrium (LD) between QTL alleles and SNP markers at some loci [GBLUP) methods, pseudo-phenotypes are obtained for genotyped animals using BLUP procedures at the first step, and subsequently these pseudo-phenotypes are used to solve prediction equations [ssGBLUP) blends marker-based and pedigree-based relationship matrices and makes it possible to include all genotyped and non-genotyped animals in the genetic evaluations simultaneously [The traditional best linear unbiased prediction  for genomic selection of American mink. The results provide useful information for designing genomic selection programs in the mink industry.Mink has been considered as one of the most desirable sources of pelt in the fur industry. Improvement of pelt size, fur quality, disease resistance and reproductive performance are defined as the main breeding objectives in mink production . The useNe) in livestock populations due to domestication [Ne in American mink was reported to be in the range of 17.5–70.8 [2 statistic. Pair-wise r2 was computed for all SNPs located in inter-marker distances from 0 up to 1 Mb. In addition, pedigree-based inbreeding, inbreeding based on excess homozygosity, heterozygosity levels and effective population size were calculated for each scenario using SNP1101 software [Populations were simulated using QMSim software  with 10ktication . The Ne 7.5–70.8 , which w7.5–70.8 . The bre7.5–70.8 . Considesoftware .−5 for both QTL and markers loci. Recurrent mutations are usually very rare between SNPs and do not contribute significantly to the reduction of LD between markers [The genome was consisted of 14 autosomal chromosomes with total length of 1192 cM mimicking the structure of mink genome reported by Anistoroaei et al. . Phenoty markers . It was y is the vector of phenotypic performances, μ is the overall mean, Z is the incidence matrix relating phenotypes to additive genetic effects, a is the vector of additive genetic effects and e is the vector of random residuals. It was assumed that random effects are independent and normally distributed:A is the numerator relationship matrix, I is the identity matrix, The following basic animal model was used to estimate breeding values using pedigree and phenotype information:y is the vector of phenotypes, μ is the overall mean, Z is the incidence matrix relating phenotypes to DGVs, g is the vector of DGV e is the vector of random residuals G is the genomic relationship matrix, I is the identity matrix and G matrix was computed according to VanRaden method [M is the matrix of marker genotypes for each individual and P is a matrix of 2pj where pj is the frequency of the second allele p at locus j. The GBLUP analyses were performed using GS3 software [The DGVs were estimated for all genotyped individuals using the following equation:n method  as follosoftware .H-1) blending the pedigree-based additive relationship matrix (A) with genomic relationship matrix (G) as follow [A for genotyped individuals. Genomic relationship matrix (G) was built using the same approach described above for GBLUP analyses [A-1 matrix with H-1 matrix. The ssGBLUP predictions were computed based on default options in BLUPF90 software [The ssGBLUP method was implemented to combine information from genotyped and non-genotyped animals using the inversed of H matrix  and estimated breeding values (EBVs) obtained from different genetic evaluation methods. The regression coefficient of TBV on EBV was used to evaluate the bias (inflation or deflation) of predictions [Effects of all segregating markers were estimated for training sets composed of 1000, 2000, 3000, 4000 and 5000 individuals. The prediction set included 500 individuals randomly selected from the 10dictions . The meaNe in the recent four generations of different scenarios. Compared to phenotypic selection scenarios, reduction in heterozygosity and Ne were greater under EBV selection scenarios. Genomic inbreeding rates were in the range of -0.045 (±0.001) to -0.048 (±0.001) for different populations. These results were in accordance with genomic inbreeding rates (-0.150 to 0.005) reported by Thirstrup et al. [p et al.  using 19p et al.  in feral2 decayed with increase in physical distances between markers and reached to 0.118–0.130 for 0.9–1 Mb distance. To our knowledge, genome-wide pattern of LD decay has not been reported for farmed American mink and it would be worth further investigation. However, Zalewski et al. [Ne range of 17.5–70.8 using LD method for feral American mink on the Swedish coast which was in accordance with our simulated Ne (47 to 72). On the other hand, lower effective sizes (7.2–34.8) were estimated for American mink populations in Spain based on LD method [Ne in American mink might be due to line selection in farmed mink and founder effects of farm mink escaping into feral populations [i et al.  estimateD method . This diD method  obtainedulations . Furtherizes 7.2–.8 were e2 = 0.10, 0.417 to 0.441 for h2 = 0.20 and 0.620 to 0.662 for h2 = 0.50. Compared to BLUP, the prediction accuracy of GBLUP and ssGBLUP was increased by 38% and 44% for h2 = 0.10, 27% and 29% for h2 = 0.20, and 5.8% and 6% for h2 = 0.50, respectively. In accordance with our results, higher average accuracies were reported for ssGBLUP and GBLUP methods compared to traditional pedigree-based method in genomic evaluations of dairy cattle [Accuracy of prediction was evaluated based on the correlation between TBV and EBV. y cattle , beef cay cattle , 37, broy cattle . Additioy cattle , 41 and y cattle .2 = 0.10 and marker density of 700k was higher than that was obtained from BLUP (0.322±0.036) in 1000 TS scenario (2 = 0.10 and h2 = 0.20) to obtain more accurate predictions than BLUP. Furthermore, number of animals in TS should be at least 3000 for scenarios with high heritability of 0.50 and marker densities of 10k and 50k to achieve more accuracy than BLUP evaluation. These results indicated that TS sizes should be optimized in accordance with heritability, marker density and prediction method of genomic evaluations in American mink.The prediction accuracy (±SD) of GBLUP (0.357±0.067) and ssGBLUP (0.368±0.059) for hscenario . However2 = 0.50 and marker density of 700k. Increasing effects of heritability, marker density and number of genotyped animals on the prediction accuracy has been previously confirmed in other studies on domestic animals using simulated [2 = 0.532, accuracy = 0.490), pelt length , pelt density  and under wool density  were comparable with those obtained in the present study for TS size of 1000 using 10k  and 50k marker density . However, the predictive abilities of silky appearance of pelt  and overall general impression  were higher than the predicted accuracies in the present study. Furthermore, the prediction accuracies of pelt silkiness  and pelt quality  were lower than those obtained in our simulated data. This inconsistency can be due to the differences in the genetic architecture of these traits, number of markers, reliability of phenotypic measurements, statistical models and TS sizes in these studies.Accuracy of genomic methods was increased with enhancement in heritability levels, marker densities and TS sizes. The lowest prediction accuracy (±SD) of GBLUP 0.314±0.079) and ssGBLUP (0.298±0.084) was observed in 1000 TS scenario with 10k marker density and 0.10 heritability. On the other hand, the highest accuracy (±SD) of GBLUP (0.787±0.016) and ssGBLUP (0.780±0.038) was obtained in TS size of 5000 with h14±0.079 imulated –47. Villimulated  used 28,Compared to marker density of 10k, the prediction accuracies of GBLUP and ssGBLUP were increased by 1.5% and 3.2% using 50k marker density and 6.4% and 5.7% using 700k marker density, respectively. Enhancement of marker density could improve the accuracy of genomic predictions due to increased extent of LD between markers and QTLs across the genome . In addiOverall, the prediction accuracies of ssGBLUP were 4.4%, 1.6% and 0.28% higher than those obtained using GBLUP method for heritability of 0.10, 0.20 and 0.50, respectively. These results indicated that ssGBLUP method can be more efficient particularly for lowly heritable traits and low marker density. The average reliabilities of predictions obtained from ssGBLUP were 1.8% higher than GBLUP for 16 various traits in Nordic Holstein cattle , which i2 = 0.10, 0.463 to 0.503 for h2 = 0.20 and 0.657 to 0.700 for h2 = 0.50. On average, the prediction accuracies of BLUP under phenotypic selection were 11.6%, 11.3% and 5.6% higher than those predicted under EBV selection design for h2 = 0.10, h2 = 0.2 and h2 = 0.50, respectively. For heritability of 0.50, the average accuracy of GBLUP method under phenotypic selection ranged from 0.570 to 0.792, which was 2.65% higher than those estimated under EBV selection. However, predictions of GBLUP method under phenotypic selection were 13.24% and 11.82% less accurate than those obtained under EBV selection for heritability of 0.10 and 0.20, respectively. Gowane et al. [The prediction accuracies obtained from BLUP, GBLUP and ssGBLUP methods for different scenarios under phenotypic selection are presented in e et al.  reportede et al. , 61. Howe et al. , 62.2 = 0.10 with 10k and 50k marker densities should be >2000 to obtain more accuracy than BLUP method, the TS size of 2000 in ssGBLUP method provided higher accuracies than BLUP in the same scenarios . These results revealed that ssGBLUP approach can be more efficient for improvement of lowly heritable traits such as reproduction and health traits in mink breeding programs. Therefore, considering the fact that the current selection of animals in the mink industry is mainly based on their phenotypic performances, the use of ssGBLUP method can have more advantages for mink breeding programs.Overall, the increased accuracy of predictions using ssGBLUP in comparison with GBLUP were 13.5%, 7.9% and 5.4% for marker densities of 10k, 50k and 700k, respectively. In addition, the accuracies of prediction by ssGBLUP method were 11.8% and 15.4% higher than those obtained by GBLUP method for heritability of 0.10 and 0.20, respectively. However, this difference was reduced to -0.47% for hThe bias of predictions (inflation or deflation) was measured by the regression coefficient of TBV on EBV. 2 = 0.10, 4.04% and 6.26% for h2 = 0.20, and 1.67% and 3.24% for h2 = 0.50, respectively. In accordance with our results, Vitezica et al. [-1 and G-1 and incorporating inbreeding coefficients in the genomic models [The regression coefficients of prediction methods were higher in phenotypic selection designs compared to EBV selection designs . In compa et al.  obtaineda et al.  reporteda et al. . Under ta et al. . Howeverc models , 65.In this study, we investigated the potential advantages of genomic selection in mink breeding programs using simulated data with different levels of marker density, heritability, selection designs and TS sizes. Our results indicated that use of lower marker density (10k) can also be useful to improve genetic merit in mink farming. However, TS sizes should be optimized based on the selection designs, levels of heritability, population structure and genetic architecture of traits. In general, ssGBLUP method could provide more accurate predictions particularly for lowly heritable traits with a low density of markers. Our results indicated that higher selection intensity can increase the bias of predictions in both pedigree-based and genome-based evaluations. Overall, less biased and more accurate predictions were obtained using ssGBLUP method due to incorporating information of both genotyped and non-genotyped animals in the genetic evaluations. Genomic selection can be used as an effective strategy for improving the genetic merits of animals in mink breeding. Identification of genomic variations using sequencing technologies, development of commercial SNP panels and adaptation of statistical approaches to apply in mink breeding are the main steps to develop genomic selection programs in the mink industry. The results of this research can be helpful in designing initial frameworks of genomic selection in American mink.


The two cohorts did not demonstrate statistical differences in the PFS (10.5 months (95%CI 6.4-14.6) vs 8.0 months (95%CI 3.2-12.8), P=0.686) and the OS (23.3 months (95%CI 12.1-34.5) vs 20.0 months (95%CI 10.1-29.9), P=0.776). In conclusion, our study suggests that patients with extra gained HER2 positivity may not show compromised efficacy to trastuzumab treatment.Dual block HER2 assessment can effectively increase the HER2 positive rate in resected specimens of gastric cancer (GC). The aim of this study is to explore whether GC patients with extra gained HER2 positivity by dual block assessment can benefit from trastuzumab therapy. Twenty-eight GC patients receiving gastrectomy prior to trastuzumab treatment were retrospectively analyzed. All the cases routinely accepted dual block HER2 assessment. The cases were divided into 2 cohorts based on HER2 status: cohort A with concordant HER2 results and cohort B with discordant HER2 results between the two blocks (cases with extra gained HER2 positivity). Response rate (RR), progress free survival (PFS) and overall survival (OS) were compared between the two cohorts. The results showed that no significant differences were found between the two cohorts in main clinicopathologic characteristics. No statistical difference was found in response rate (47.6%  Gastric cancer is the fifth most common malignancy and the third common cause of cancer related death worldwide . In ChinEarly detection and treatment of GC is vital to acquire satisfying prognosis, however, many patients present with inoperable tumors during their first visit to hospitals in China, and for them, the prognosis is insufficient. Five-year survival rate of advanced GC patients is approximately 5% to 20%, and overall survival (OS) is approximately 10 months for those who receive conventional chemotherapy . Recent HER2 characteristics of GC have been extensively studied since the ToGA trial. Compared with breast cancer (BC), heterogeneity is an important feature in GC. Studies have shown that 30% to 79.3% of HER2 positive GCs show heterogeneity –8. For tTo cope with the adverse impact of HER2 heterogeneity on HER2 evaluation, we are the first to propose that dual block HER2 assessment (conducting HER2 assessment on two tumor-containing blocks) in resected specimens of GC is a practical way to increase HER2 positive rate , 12. AndTherefore, in the current cohort study, patients who accepted gastrectomy prior to trastuzumab treatment were retrospectively collected. Trastuzumab efficacy was compared between the two cohorts with concordant HER2 results or discordant HER2 results (extra gained HER2 positivity) between the two blocks. The purpose of this study is to explore if the patients with extra gained HER2 positivity are eligible candidates for the molecular targeted therapy.Patient characteristics were shown in Trastuzumab was used as the first line treatment in 20 patients. Eighteen patients were given further trastuzumab administration beyond first line treatment progression. The total response rate was 50.0%. The mPFS of total patients was 10.5 months (range 5.5–15.5 months). And the mOS in total patients was 24.6 months (range 17.3–31.9 months).Among the 28 patients, 21 patients demonstrated concordant HER2 results between both blocks. These patients were enrolled into cohort A. Among them, 15 patients showed HER2 IHC 3+ in both blocks, and 6 patients showed HER2 IHC 2+/FISH+ in both blocks.In the remaining 7 patients, the two blocks exhibited discordant HER2 results, and these patients were enrolled in cohort B . In 5 of the 7 patients, the first block was HER2 3+ and the second block was HER2 negative (HER2 2+/FISH- or HER2 0/1+). In the remaining 2 patients, the first block was HER2 2+/FISH+, and the second block was HER2 negative.HER2 status was shown in P=1.0), age (P=0.418), tumor location (P=0.574), Lauren classification (P=0.393), tumor differentiation (P=0.398), pTNM stage  guidelines) (P=0.173), and number of metastasis (P=0.721)  .P=0.131), palliative chemotherapy (P=1.0) and the trastuzumab administration (P=1.0)  .P=1.0) (P=0.689) (P=0.690) (The response rate of cohort A (47.6%) was slightly lower than cohort B (57.1%) with no statistical difference (P=1.0) . With reP=0.689) . SimilarP=0.690) .HER2 evaluation of GC can be challenging for the protein`s affinity of heterogeneous expression , 8, 10. vs 52.4%), and more patients in cohort B started trastuzumab as the second line treatment. We mainly attribute such bias to small sample size. These result indicated that patients with extra gained HER2 positivity by dual block assessment may not show inferior treatment outcome to trastuzumab treatment. And these patients may also be eligible for the targeted treatment.The current study showed that the RR, mPFS, mOS of both cohorts did not demonstrate statistical difference, although visually, mPFS and mOS were slightly shorter in the cohort B with extra gained HER2 positivity. This visual difference in the mPFS and mOS may be caused by the bias of the clinicopathologic features between the two cohorts. Although without statistical significance, more cases in cohort B were with later stage tumors than those in cohort A , homogeneously HER2 over expression showed more benefit from trastuzumab treatment . NeverthThere are some limitations in this research. First of all, it is a retrospective study from a single institution. Secondly, sample size is relatively small. This is mainly because most GC patients who accepted trastuzumab therapy are inoperable late stage patients. The direct impact of small sample size is the limited statistical power. Therefore, independent studies with larger sample size are needed to confirm the finding of this research.In conclusion, GC patients with extra gained HER2 positivity by dual block assessment may not show compromised efficacy of trastuzumab treatment. Considering the retrospective nature of this study and limited statistical power, this result would need to be confirmed in independent studies. The findings provided the evidence of the clinical value of dual block HER2 assessment. Oncologists should be aware of the clinical significance of identifying GC patients with heterogeneous HER2 positivity. We recommended that dual block assessment routinely performed in resected specimens in GC to obtain more eligible patients for the molecular targeted therapy. With more oncoming data, conclusions of the current study can be further verified.One hundred and seventy five patients with advanced or metastatic gastric cancer of histologically confirmed HER2 positivity were given trastuzumab based chemotherapy in Zhongshan hospital, Fudan University from February 2010 to March 2018. Among them, 28 patients accepted gastrectomy prior to trastuzumab treatment and their primary tumor samples were available. All these patients accepted palliative trastuzumab based chemotherapy after tumor recurrence or metastasis. The HER2 status of all the 28 patients was routinely evaluated by dual block assessment. The research protocols were approved by the ethics board of Zhongshan Hospital. Prior written informed consents were received from all patients.All the patients received standard palliative treatment including trastuzumab with a dose of 6 mg/kg every 3 weeks after a first infusion of 8 mg/kg. There were no protocol-specified chemotherapy regimens.The specimens were fixed in 10% buffered formalin within 30 minutes after excision. Samples were processed following routine procedures after fixation for 24 hours. Haematoxylin and eosin staining was routinely performed.HER2 IHC staining was conducted on a BenchMark XT automated stainer  with an iView DAB Detection Kit  according to the procedures previously described .HER2 FISH was performed with a Pathvysion HER2 DNA Probe Kit . All the procedures followed the manufactures` instructions.After pathological evaluation, two primary tumor blocks were chosen for HER2 IHC assessment. Blocks were given priority in the selection with the following conditions: containing an intestinal-type component, demonstrating the lowest grade and rich in tumor cells.HER2 IHC status was evaluated by 2 independent observers who were blinded with the aim of the study. A discussion panel involving the third observer was introduced for cases with discrepant HER2 scores. HER2 was scored following the established criteria for resected specimens of GC –26. BrieFor HER2 2+ blocks, HER2 FISH was performed to confirm the HER2 status. A case was considered to be with HER2 amplification when a minimum of 20 cancer cell nuclei exhibited a ratio of HER2: CEP17 (centromeric probe 17) of ≥2, or when HER2 signal clusters were observed.A case was considered HER2 positive if either of the 2 blocks showed HER2 3+ or 2+/FISH+. Cases in which both 2 blocks were HER2 positive were considered to be those with originally gained HER2 positivity (cohort A). On the other hand, cases in which only 1 block showed HER2 positivity were considered to be those with extra gained HER2 positivity by dual block assessment (cohort B).th, 2018.Medical records were reviewed to get the follow up data. Response was evaluated by CT/MRI scanning every 8 weeks after the trasutzumab administration following the Response Evaluation Criteria in Solid Tumors (RECIST) (version1.1) or earlier when indications of treatment failure were present. Patients were considered on study until death or loss to follow-up. The last date of follow up was December 20Progression free survival (PFS) was defined as the time from the starting date of trastuzumab administration to the date of tumor progression. Overall survival (OS) was defined as the time from the start of trastuzumab treatment to the death of the patient. Response rate (RR) was defined as the ratio of the number of the patients with complete response (CR) plus partial response (PR) to the number of total patients.P-value of <0.05 was considered statistically significant. No adjustments were made. All the analyses were conducted with the SPSS version 22.0 .Clinicopathological parameters were evaluated with Chi-squared tests, Fisher's exact tests, and Mann-Whitney U test. Kaplan–Meier curves were constructed for survival analysis. Variables associated with prognostic value were selected to evaluate by univariate and multivariate analysis using Cox proportional hazard regression models. Data were presented as an HR and a 95% CI. For each analysis, a two-sided


The objective of the research is to measure prevalence of hypertension and associated factors adult outpatient clients in Addis Ababa, Ethiopia.A total of 487 participants were included in the study. The prevalence of hypertension was (34.7%), 95% CI (30.6–38.8), of them 53.8 were Male and 46.2% were female. Among 169 participants identified as having high blood pressure, 131 (66.5%) were aware of their blood pressure, from those 96 (48.7%) of them were receiving antihypertensive medication. Mean elevated systolic blood pressure was 124.7 ± 20 (SD) and diastolic blood pressure was 81.5 ± 9.8 (SD). The multivariable regression analysis showed that having ever been told hypertension ; using animal product butter ; physical inactivity  and BMI 25.0 to 29.9 and greater than 30  were statistically significant predictors of hypertension. The prevalence of hypertension was considerably high. The health system needs to develop strategies to increase the reach of relevant screening and diagnostic services.The online version of this article (10.1186/s13104-019-4127-1) contains supplementary material, which is available to authorized users. Hypertension accounts for approximately 50% of coronary heart disease and 67% for the cerebra vascular disease burden worldwide . It is oHypertension is not only an important public health problem; rather, it will also have a big economic impact . Many faWorld Health Organization (WHO) urges countries to develop measures and strengthen health care services to reduce the level of exposure risk factors for non communicable diseases (NCDs) . In wellIn sub-Saharan Africa (SSA), hypertension and other cardiovascular diseases were not given due attention . An incrThe study was conducted at Yekatit 12 hospital in Addis Ababa, Ethiopia. A hospital based cross sectional study conducted from Nov to Dec 2015 in Addis Ababa, Ethiopia.Simple random sampling procedure was conducted in outpatient departments. A total of 487 outpatient department clients participated in this research whose age ≥ 18 years. Patients with serious illnesses, acute life-threatening conditions, and severe injury were excluded from the study.Data was collected using a structured interview questionnaire and physical measurements which was adapted from “WHO STEPS’’ tool that contain Socio-demographic, behavioural, and other variables . A digitBlood pressure (BP) was measured twice in a sitting position (using a standard sphygmomanometer BP cuff with an appropriate size to cover two-thirds of the upper arm) after the participant rested for at least 5 min, the second measurement was taken 5–10 min after the first measurement. Participants were inquired whether they had consumed any hot beverage such as tea or coffee, smoked cigarette or undertaken any physical activity 30 min before measurement. Hypertension defined as systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg or treatment with antihypertensive agents .To ensure the data quality, there was 2-day training for the data collectors and supervisors. Pre-test to see the effectiveness of the questionnaire and if there were additional needs, continuous and strict supervision was conducted on the spot.® SPSS Statistics software version 20. Descriptive statistics such as frequency and cross tabulation were calculated for selected variables. Multivariable logistic regression model was used to identify factors associated with hypertension. Adjusted odds ratio (AOR) with 95% confidence interval (CI) and P-value ≤ 0.05 in the final model were used to determine significant predictors.Data was entered to Epidemiological Information (Epi Info) version 3.5 and analysed by IBMA total of 487 clients participated in the study with 100% response rate. The mean age of the participants was 46.71 with the standard derivation of 15.67. The majority, 380 (78%) were Orthodox and 294 (60.4%) were married. About 94 (13.5%) had no formal education (Table From 487 participants 146 (30%) had family history of hypertension. Of all 430 (88.3%) which were measured their blood level by the health professional they know their hypertension status. From these 96 (48.7%) take antihypertensive medicine currently. Among all 487 respondents 96 (19.7%) were take antihypertensive medicine currently. Only 1/4th of the participants, 121 (24.8%) had family history of diabetes. Ninety-two 18.9% had past history of tobacco smoking. The habit of eating fruits of the respondents was satisfactory, which 228 (46.8%) of them ate fruit 1–2 days per week, 166 (34.1%) less than 1 day per week. Only 155 (31.8%) of the respondents were participate daily on vigorous working activity.The overall prevalence of hypertension was 169 (34.7%) with 95% CI 30.6–38.8. Among 169 participants identified as having high blood pressure, 131 (66.5%) were aware of their blood pressure and 96 (48.7%) of them were receiving antihypertensive medication, of which 16 (16.7%) had normal (controlled) blood pressure at the time of the study.In the multivariable logistic regression analysis, having ever been told hypertension; using animal product butter; physical inactivity and BMI were statistically significant predictors of hypertension. The odds of developing hypertension among respondents who had ever been told hypertension was 15.47-times more likely compared to the counterparts who had not .Using animal product butter type was the other significant predictor of hypertension, so that respondents who ate animal product butter type were 2.66-times more likely to develop hypertension as compared to those who did not use any type of butter . Also, respondents who did not physical exercise were 2.83-times more likely to have hypertension compared to those who did physical exercise .Moreover, the BMI of the respondents was statistically associated with developing hypertension, the odds of hypertension being more than four-, five- and three-times higher among respondents whose BMI ranged from 18.5 to 24.9, 25.0 to 29.9 and greater than 30.0 compared to whose BMI was less than 18.5, respectively , respectively  and Northern Africa (33.3%) . Also, sMore than one-third of adults was overweight/obese (35.9%) in this study; and 47.4% of them were hypertensive which was consistent with findings reported in community-based studies in Sub-Saharan Africa countries. Moreover, BMI was statistically associated with hypertension, with the odds of hypertension being more than five- and four-times higher among respondents who were overweight and obese as compared to respondents whose BMI was less than 18.5. This finding is in accordance with studies conducted in our country and other countries in the world, where higher BMI was associated with hypertension , 23–25.Respondents who didn’t do exercise/sports were more than 2.83-times more likely to have hypertension compared to those who did physical exercise. The prevalence of physical activity in this study is (17.2%) which is higher than the study conducted in Bahirdar (4.9%)  but muchThe prevalence of hypertension was found to be high among Outpatient clients. Awareness, treatment, and control rates in those with the disease were low this supports the notion that hypertension is a silent killer. Factors like physical inactivity, family history of hypertension, eating animal product butter type and being overweight and obesity were found to be associated with high BP whereas, sex, vegetable and fruit consuming not associated with high blood pressure. The findings in this study and other recent studies conducted in the country have clearly shown that hypertension is becoming a serious public health concern. Therefore, mass screening of hypertension and life style modifications and motivating the community for adequate BP control are recommended. It is also better to give special emphasis for health education regarding the daily live events like healthy dietary habit and regular exercise and preventing alcohol consumption and cigarette smoking. Policy makers need to focus on community level intervention through integration with the open-door health extension program. Furthermore, there must be national strategy for hypertension screening at least for adults visiting health institutions for various reasons.This finding is not free from limitations; most of our patients don’t knew their exact birth date, as a result there was no reliable account of their age. Secondly, which was based on only self-reports of a previous diagnosis and history of personal history of diabetes, alcohol drinking, cigarette smoking. This study may lack generalizability for the community at large.Additional file 1. Prevalence of hypertension data. The prevalence of hypertension and factors associated to it.


P = 0.032 (ambient temperature), r = –0.39 vs. r = –0.45, P = 0.012 P = 0.015 (relative humidity), r = 0.41 vs. r = 0.46, P = 0.022 (THI)]. No differences were found between groups during the control day for any of the physiological parameters. During days 2 and 3, average and maximal values of respiratory and heart rates were higher in unshaded calves than in shaded ones. Maximal respiratory rates were in average by 25.9, 17.8 and 10.1 breaths/min lower in shaded calves than in unshaded calves for days 2, 3 and 4, respectively . Maximal heart rate was 127.4 ± 8.5 vs. 99.2 ± 6.3 beats/min on the heat stress day (P < 0.001), and 121.0 ± 6.9 vs. 103.4 ± 7.7 beats/min on day 3 (P = 0.006) in unshaded and shaded calves, respectively. Maximal body temperatures were higher measured either in the rectum or on the ear skin in unshaded calves than in shaded ones , but only on the heat stress day. Based on our results, shading of young calves may be adequate for alleviating acute heat stress in continental regions. Ambient temperature is appropriate to estimate acute heat stress in dairy calves.We estimated thermal stress in 7-week old Holstein bull calves during a warm episode in summer to study acute physiological responses of calves to heat stress. Data were collected over a 5-day period: day 1 (control), day 2 (heat stress), and a 3-day post-stress period in shaded (n = 8) and unshaded (n = 8) thermal environments. On the control day, both groups were shaded. Thermal environment was characterized by relative humidity, ambient temperature, and the temperature–humidity index (THI). Physiological variables included respiratory rate, rectal temperature, ear skin temperature and heart rate. Correlations between animal-based and meteorological indices were calculated, and ambient temperature correlated slightly better with physiological measures than THI. Rectal temperature was the only animal-based parameter that showed stronger correlations with the thermal indices when calculated for the shaded than for the unshaded environment [r = 0.42 vs. r = 0.47,  A persistent rise in global mean temperature has severe impacts on natural and societal systems . It has Based on ambient temperature and relative humidity, thermos-hygrometric indices are used for the assessment of the thermal environment in cattle. The temperature-humidity index (THI) represents the combined effects of air temperature and relative humidity to estimate the level of thermal stress in adult cows .In dairy , 6 and bAll sampling procedures and experimental manipulations with animals were specifically approved by the Pest County Government Office, Hungary, Department of Animal Health (Permit Number: PE/EA/1973-6/2016).The experiment was carried out at a large-scale dairy farm in Hungary (N47°18'191'' E18°48'336''), which has a herd of 1000 lactating Holstein cows. The farm was visited between August 15 and 20 in 2016. As a field study, our experiment was timed based on the expected meteorological patterns of the regional weather forecast. The 6-day study started with a 24-h habituation to the study environment when preparation of the shading structure over the hutches of both shaded (n = 8) and unshaded calves (n = 8) were done. On day 1  all calves were shaded for 24-h from 0:00 o’clock until 24:00 o’clock and shade was removed from the unshaded group at 24:00 o’clock. Day 2 was considered as the heat stress day (maximum of 37.7°C for the shaded hutch environment), and days 3–5 covered a post-stress period .2 exercise pen that were arranged in rows based on their sex (heifer row and bull row) and birth date. To eliminate the possible effect of individual-related  and environmental factors (i.e. row orientation) on our results, bull calves with similar age and body weight  one week before weaning from the same row were selected for the study and assigned to the shaded or unshaded groups. The shading structure measured 32.5 × 3.4 m and covered the experimental hutches and pens as well. After removing the shading structure above unshaded calves, their hutches were exposed to direct sunlight 5 m from the shaded environment. A green raschel net , located 1.9 m above the ground was used as shading material that provided 80% shading, according to the manufacturer. Calves received one feeding of 3.8 liters of milk replacer in the morning (5:00 o’clock) and had ad libitum access to chopped alfalfa hay and the starter grain diet , which met the requirements for preweaned Holstein calves . We tested whether a difference between correlation coefficients exists according to the environment (shaded or unshaded). For this purpose, the Fisher-type t-test at the significance level of 0.05.To evaluate the effect of shading on the thermal environment of the animals, maximal and average values of selected thermal indices were calculated for all experimental days and compared between unshaded and shaded groups in the hutch environment, using all recorded meteorological data with 30-min sampling frequency. Statistical comparisons between environments were made with the t-test. Significance was set at the level of 0.05.The effect of shading was tested by comparing physiological responses of shaded and unshaded groups by using the individuals’ maximum and average values of respiratory rate, heart rate, ear skin and rectal temperatures calculated for each experimental day. All recorded animal-based data were used in this analysis, irrespective of the location of the calves (measured either in the hutch or the pen). Statistical comparisons between groups were made with the Figs 1 and 2). The THI formula used in our study was found to be appropriate for continental weather characteristics (low daytime relative humidity) in dairy cows . Any other correlations showed no significant differences between shaded and unshaded environments . An earlier study reported an r = 0.52 correlation between THI and rectal temperature in dairy cows both recorded between 15:00 and 17:00 o’clock, under high ambient temperatures . Reduced heat load in the shaded hutch environment compared to the unshaded one was reflected by daily averages of temperature as well on the heat stress day  and THI . Earlier studies have shown that shading reduces hutch temperature by around 1 to 2°C [P < 0.001 and P = 0.005) and 4 (P = 0.008 and P = 0.012), respectively. Maximal and average THI showed similar differences between the shaded and unshaded environments for the first two days of the post-stress period . On day 5 no differences were found in maximal and daily average values either for ambient temperature (P = 0.395 and P = 0.420) or THI (P = 0.526 and P = 0.545).There were no statistical differences either for maximal or average values of relative humidity between shaded and unshaded environments during the experimental period. On day 1 (control) no differences were found in maximal and daily average values either for ambient temperature , in unshaded calves compared to shaded ones (P < 0.001 for both days). Daily averages of respiratory rates differed also between groups either on day 2 (P = 0.008) and day 3 (P = 0.010) . Perhaps, calves of the present study were less adapted to extreme temperatures or to other features of their microenvironment. Another explanation for the substantial differences from previous results could be that the range of mean air temperature for individual sample days was 16.5 to 38.2°C in the cited paper, whereas animals were exposed to extremely high ambient temperatures during days 2 and 3 that ranged from 17.1 to 43.7°C for the unshaded hutch, and from 17.8 to 46.5°C for the unshaded pen environment in our study, respectively.Lower respiratory rates in calves in shaded hutches are consistent with earlier observations that have shown in average 10.4 breaths/min lower respiratory rates (47.3 vs. 57.7 breaths/min) for shaded calves than for calves in hutches without shade when measured at 15:00 o’clock . In the P = 0.865 and P = 0.760; P = 0.046) or on the ear skin , respectively , suggesting their prolonged acclimatization to the challenging environment; however, based on this difference (10.1 breaths/min in average) a seriously impaired well-being of unshaded animals could not be assumed. Higher maximal and average heart rates of unshaded calves during day 2 (P < 0.001 and P = 0.010) and day 3 (P = 0.005 and P = 0.012) are possibly due to the increased physical activity associated with the high respiratory effort .Following the heat stress day, differences in respiratory and heart rates were also meaningful with higher maximal and daily average values in unshaded animals for both parameters. Unshaded calves exhibited significantly higher maximal respiratory rates even on day 4 than calves with shading , even the maximal values of all animal-based parameters were in the physiological range. The available literature on critical cut-off points of different THIs of cattle focused only on adult cattle. Mader et al. [Although unshaded calves were exposed to significant daytime heat load during the afternoon hours on day 3  that also limited the study population. Furthermore, we used only pre-weaned calves (up to 50 days of age) because weaning might have caused additional stress to the animals.Thermal variables correlated well with animal-based heat stress indicators and, in case of rectal temperature all correlations were stronger in the unshaded than in the shaded thermal environment. Ambient temperature correlated better with physiological measures than THI therefore should be used as measure of acute heat stress in continental regions. Shading of preweaned dairy calves may be adequate to alleviate heat stress in continental regions. Respiratory rate and heart rate support that the well-being of unshaded dairy calves can be impaired even during decreasing thermal load following a short-term heat stress event. However, to provide practical recommendations on shading of dairy calves, further studies might be necessary using larger number of animals.S1 Table(XLSX)Click here for additional data file.