ADSL gene. The disease is characterized by wide clinical variability. Here we provide an updated clinical profiling of the disorder and discuss genotype–phenotype correlations.Adenylosuccinate lyase deficiency (ADSLD) is an ultrarare neurometabolic recessive disorder caused by loss-of-function mutations in the Data were collected through "Our Journey with ADSL deficiency Association" by using a dedicated web survey filled-in by parents.ADSL coding sequence and were predicted to have a variable structural impact by impairing proper homotetramerization or catalytic activity of the enzyme. Six variants had not previously been reported. All but two variants were missense.Clinical and molecular data were collected from 18 patients , from 13 unrelated families. The age at onset ranged from birth to the first three years (median age 0.63 years ± 0.84 SD), and age at diagnosis varied from 2 months to 17 years, (median age 6.4 years ± 6.1 SD). The first sign was a psychomotor delay in 8/18 patients, epilepsy in 3/18, psychomotor delay and epilepsy in 3/18, and apneas, hypotonia, nystagmus in single cases. One patient (sibling of a previously diagnosed child) had a presymptomatic diagnosis. The diagnosis was made by exome sequencing in 7/18 patients. All patients were definitively diagnosed with ADSL deficiency based on pathogenic variants and/or biochemical assessment. One patient had a fatal neonatal form of ADSL deficiency, seven showed features fitting type I, and nine were characterized by a milder condition (type II), with two showing a very mild phenotype. Eighteen different variants were distributed along the entire The study adds more details on the spectrum of ADSLD patients’ phenotypes and molecular data. ADSL gene (MIM *608222), spanning over 23 kb on chromosome 22q13.1–13.2 [Adenylosuccinate lyase deficiency (ADSLD) MIM #103050) is a rare autosomal recessive disease first described by Jaeken and van den Berghe in 1984 [03050 is  MIM *60822, spannADSL mutation described [https://www.ncbi.nlm.nih.gov/clinvar) and ADSLD (http://www1.lf1.cuni.cz/udmp/adsl) databases, the majority being missense changes. Among the pathogenic variants, c.1277G > A (R426H) represents the most common event [Since the first escribed , approxion event , 6. BaseThe ADSL enzyme is a homotetramer with a subunit size of ~ 50 kDa consisting of four active sites, each of which is contributed by three distinct subunits (PDB 2VD6) . ADSL caAlthough a wide clinical variability has been described, three different major phenotypes have emerged over the years. The fatal neonatal form is characterized by encephalopathy, intractable seizures, and respiratory failure, which lead to early death –10. The The non-specific clinical spectrum and lack of awareness of the condition may prevent the correct and prompt diagnosis, which is essential for improving outcomes and quantifying reproductive risk.This study aims to delineate clinical features of ADSL from a cohort of affected patients and investigate genotype–phenotype correlation.Over one year, 18 patients , belonging to 13 unrelated families (including three pairs of siblings and a family with three affected siblings) from different geographical areas  were enrolled. There was one case of consanguinity (patient 4) with first-degree cousin parents.The age at onset was between birth and the first three years of life  but the age at diagnosis varied between two months and 17 years . The diagnostic delay was about six years . In two cases having a very mild and non-specific phenotype, patients had to wait for 13 years to reach a diagnosis. The first noted sign/symptom was psychomotor delay in 8/18 patients (44.4%), epilepsy in 3/18 (16.7%), psychomotor delay and epilepsy in 3/18 (16.7%). One patient was asymptomatic at the time of enrollment and 3/18 patients (16.7%) had other symptoms .Cardiotocographic abnormalities were detected in only one patient (patient 9). Some patients showed minor craniofacial features .The diagnosis was made by whole-exome sequencing (WES) in 7/18 patients (38.9%), by biochemical analysis in 8/18 (44.4%), and by the combination of the two approaches in 2/18 (11.1%). In one patient, the diagnosis was attained by segregation analysis.ADSL VoUS in compound heterozygosity, c.1343_1345delCTT (Ser448del), and c.502G > A . Urine samples showed the presence of S-Ado , SAICAr  and S-Ado/SAICAr less than 1.One patient presented fatal neonatal form, seven type I (38.9%), and nine type II (50%), two of which with a new very mild phenotype. The newborn with fatal form (patient #14) was a Qatar child, born at 37 weeks of gestational age from unrelated parents; he presented hypotonia, severe apneas, respiratory failure, microcephaly, drug-resistant epilepsy characterized by myoclonic seizures (more than 20 seizures/week), and severe psychomotor delay. He needed mechanical ventilation. He died at the age of 15 months. WES analysis documented the presence of two The seven patients with type I, including two siblings, had an early onset of disease (birth-nine months). In most cases, they showed an onset of epilepsy within the first six months of life. Severe cognitive and language delay and seizures were present in all patients. The epileptic phenotype included generalized tonic–clonic seizures, partial seizure, absence epilepsy, myoclonus and atonic seizure. The frequency of seizures was variable, ranging between 1 and 5 episodes/week and 11–15/week. Treatment, generally multidrug therapy, resulted in a complete response in one patient and partial response in four cases. Two patients had drug-refractory epilepsy. Neurologically, symptoms included hypotonia associated with spasticity in more than half of cases. The eye phenotype included strabismus and nystagmus. Cranial magnetic resonance imaging (MRI) showed cerebral atrophy in six patients. Microcephaly was reported in only one patient. Three patients had ASD, and behavioral abnormalities  were reported in some patients, while poor eye contact was reported in all patients. Sleep apneas were observed in two patients, with the need for mechanical ventilation for one of the two. Three needed oxygen therapy in the post-critical period.Table The nine patients with type II, including two pairs of siblings, presented mild to moderate neurodevelopmental and speech delay. Among them, two siblings from Germany showed a very mild isolated psychomotor delay without other manifestations; they also achieved numerous personal autonomies of daily life and showed an improvement in language and motor skills . Six chiBrain MRI showed cerebral atrophy and hypomyelination in only one patient, as well as microcephaly. Four patients presented ASD and four children stereotypies. No respiratory problems were reported.ADSL gene sequencing, which documented the presence of the homozygous c.1277G > A missense change (p.Arg426His).The apparently asymptomatic patient (#13), sibling of patient #12, was a child of 4 months at the time of enrollment. She was born at 42 weeks of gestational age from unrelated parents. The diagnosis was made at two months by Table Urine samples of nine patients showed high levels of SAICAr and S-Ado. Analysis of cerebrospinal fluid was performed in four patients and evaluation of enzyme catalytic activity in erythrocytes in two patients Table .Table 3BBased on the American College of Medical Genetics and Genomics criteria , one patThe recurrent R426H substitution was identified in seven patients (38.8%), either as homozygous change (five cases) or in compound heterozygosity with a second variant in two children. The six novel variants affected different regions and functional domains of the protein. Positive biochemical test in children carrying novel variants  confirmed their causative role in disease presentation and their bona fide pathogenic impact.The location of the newly reported variants in the structural model of homotetrameric ADSL is illustrated in Fig. The six novel variants were predicted to have a similar structural/functional impact on the ADSL holoenzyme by affecting the proper oligomerization of the ADSL monomer. V168I is invariant from humans to insects and replaced by a similar residue (leucine) in lower organisms. Val168 interacts with Arg149, which is the site of the previously reported disease-causing Arg149Gly substitution. Nevertheless, it was predicted to cause conformational changes at the nearby region of interaction with another ADSL monomer. Similarly, the highly conserved Trp175 is also close to the region of interaction with another ADSL monomer; W175C change is non-conservative and is expected to induce significant conformational changes impairing the homotetramerization. D332H substitution should be detrimental to ADSL functions since it represents a non-conservative change involving the invariant Asp332 at the region of tetramerization and in the proximity of the binding region of the enzymatic products adenosine monophosphate and fumaric acid. The E343K change implies electrostatic charge inversion inside the region of tetramerization and is predicted to cause defects in this process. Similarly, the invariant Gly418 residue contributes to the stabilization of the ADSL tetramer. The methyl side chain introduced by the G418A mutation decreases the flexibility of the affected protein site in an ADSL monomer and also introduces novel interactions with residues of another bound ADSL monomer; thus, it could have an essential effect on tetramerization.Finally, Ser448 lies near the region of interaction with another ADSL monomer. Although Ser448 is conserved only in higher organisms , the effect of its deletion is to perturb the ADSL structure locally and impair ADSL tetramerization considerably.In this study, we describe parent-reported clinical, biochemical, and molecular features of 18 patients affected by ADSLD.The majority of patients described so far presented a severe clinical phenotype, with only about 15–20% of cases with a milder form . While oDespite an early onset of the disease, diagnosis is often delayed, leading to a "diagnostic odyssey" for young patients and their families. Indeed, albeit an early manifestation of the isolated psychomotor delay was already observed before the age of two in more than half of the cohort, the average age at diagnosis was much later, at about six. DNA sequencing techniques have partially solved this problem. In recent years, the development of modern technologies for sequencing the human genome, together with the growth of knowledge on genetics, has made available genetic tests for molecular diagnosis of Mendelian conditions characterized by marked clinical heterogeneity and partial overlapping of phenotypes , 20. TheIn our cohort, 7 out of 18 patients were diagnosed by WES, and by the two combined methods  in two other patients, almost all had a type II form, suggesting that more patients with mild/nonspecific presentations could be identified "genotype first" approaches that are less affected by selection bias.The collected data indicate that type II cases have a mild neurological involvement with absence or a lower incidence of epilepsy, in line with results reported by Jurecka et al. In this cohort, the frequency of ASD is similar in the two phenotypic classes, while there is a higher percentage of brain imaging anomalies in the severe group. In literature, various anomalies in brain tissue, including cerebral cortex atrophy, corpus callosum agenesis, delayed or absent myelination, white matter anomalies and lissencephaly, were reported in all three phenotypic classes , 22. In ASD was initially reported as a typical sign of ADSLD and found in about a third of patients , 23. In Similar to previous reports , craniofTherefore, our data confirmed the wide clinical heterogeneity. As previously reported, two German siblings showed a very mild isolated psychomotor retardation without other manifestations defining a new phenotype .ADSL gene have been described, and most of them are missense changes (ClinVar database). In our cohort, we identified 34 missense variants, confirming an evident prevalence of these variants compared to other types. Nonsense variants represent only a small fraction (< 5%) of all the pathogenic ADSL variants reported, probably because complete loss of ADSL function is incompatible with development. The most common disease-associated variant, c.1277G > A (R426H), leading to structural instability of the enzyme [More than 150 variants of e enzyme , has beeADSL variants reported are classified as pathogenic according to the ACMG criteria, while more than 50% are classified as VoUS. Ten VoUS (10/36) have been identified in the cohort; eight of them were in compound heterozygosity defining a not previously reported genotype. In all subjects carrying these variants, a biochemical assessment confirmed ADSLD diagnosis, validating the clinical relevance of the identified variant.Only slightly more than 50 of all Structural inspection of the disease-associated variants documented that their impact on proper oligomerization of the ADSL monomer is a general feature. Structural considerations, however, do not allow to appreciate the specific impact of a subset of identified variants. This is exemplified by the predicted structural consequences of the E343K substitution, which was identified as a homozygous change in a patient with a mild phenotype. The side chain of Glu343 of each ADSL monomer is in contact with the side chain of Glu343 located in another monomer, producing potential electrostatic intermolecular repulsions. Although the E343K mutation implies an anionic to cationic electrostatic charge inversion of the affected site, homozygosity, by inverting all Glu343 anions to Lys343 cations, results in the "conservation" of repulsive intermolecular interactions among ADSL monomers. Since Glu343 side chains are located at the monomer–monomer interfaces but do not present further interactions with other residues, we are not able to predict significant detrimental effects for the E343K mutation. The "conservation" of repulsive-type forces in ADSL dimers and tetramers might explain the mild phenotype associated with E343K in homozygosis.The two novel variants, Ser448del and V168I, were associated with a fatal neonatal form of ADSLD. In particular, Ser448del is detrimental to ADSL tetramerization; it could, therefore, determine more severe functional consequences and explain the phenotype severity.The variant W175C is expected to destroy the protein fold for its location and type of amino acid replacement, as well as the R149G mutation, has an important structural role. This might explains the severe phenotype presented by the patient carrying the two mutations on distinct alleles.Intriguingly, the novel variant D332H should be detrimental to ADSL function; however, in one patient, D332H is in compound heterozygosity with T450S mutation, and it is associated with a mild phenotype. This allows hypothesizing the presence of factors that may mitigate the final phenotype or, most likely, the T450S mutation may preserve a residual enzymatic activity.There is no effective treatment for ADSLD; only a few reports propose therapeutic strategies to prevent intractable epilepsy. Of these, two showed some beneficial effects , 27, notIn conclusion, our study adds more details on the spectrum of ADSLD phenotypes. Our experience also underlines the crucial role of patient/parent association as a critical resource to connect families who share common experiences, collect patient information, and support pediatricians in diagnostic tools. Furthermore, genotype characterization may help to define the biological effects of novel variants better. A considerable effort is still needed to understand the molecular mechanism on cellular metabolism, thus fostering the development of therapeutic strategies to limit the adverse effects on the nervous system and muscles.Patients were enrolled through the Patient's Association "Our Journey with ADSL deficiency." Families were advised through the association's Facebook Page of the possibility of participating in the study. Parents who expressed their interest were contacted by clinicians, who collected the informed consent and administered the information sheet. Data collection was carried out through a survey filled directly by patients' parents. The survey, built on a literature review, is aimed to investigate clinical, biochemical, and molecular features of affected patients .Our genotype–phenotype correlation is based on a structural analysis that consisted of the visual inspection of the amino acids affected by mutations within the crystal structure of the ADSL protein, tetramerized and complexed with its substrate and catalytic products . This allowed us to observe that the various mutations directly affect  functional regions of ADSL  and consequently we envisaged the effects as explained in detail in the text.æwww.pymol.org).Molecular graphs were made with PyMOL (Additional file 1. ADSL variants in silico evaluation.Additional file 2. ADSL survey aimed to investigate clinical, biochemical, and molecular features of affected patients.


A weakly positive correlation exists between cfcDNA concentration and total PSA  but not with age or Gleason score in PCa patients. Conclusion: cfcDNA concentration was relatively nonsignificantly higher in PCa patients in comparison to persons with BPH, whereas cfcDNA integrity was similar in both groups. Though limited in sample size, this study shows that cfcDNA concentration may be a potentially valuable noninvasive biomarker for the diagnosis of PCa. Background: Cell free circulating DNA (cfcDNA) is a promising diagnostic tool for prostate cancer (PCa). This study aimed to measure the cfcDNA concentration and integrity in PCa patients using quantitative polymerase chain reaction (qPCR) analysis. This study also assessed the correlation between these molecular biomarkers with total prostate-specific antigen (PSA), Gleason score, prostate volume, and age. Methods: Eleven PCa patients and 9 persons with benign prostatic hyperplasia (BPH) were recruited. Blood samples were collected before prostate biopsy and plasma quantified by qPCR amplification of the ALU 115 DNA sequence, with the ratio of ALU 247 to ALU 115 reflecting cfcDNA integrity. Results: There were no significant differences in median, interquartile range (IQR) cfcDNA concentration or cfcDNA integrity between the patients with PCa (47.9 (214.93) ng/mL; 0.61 (0.49)) and persons with BPH (41.5 (55.13) ng/mL,  In the diagnosis and monitoring of prostate cancer (PCa), the most notable biomarker is prostate-specific antigen (PSA). PSA is detectable in the blood because some of it escapes across the basement membrane, enters the interstitium of the prostate gland, and is absorbed by the lymphatic system and blood circulation . While tThe PSA test is complemented by the digital rectal exam (DRE) to increase diagnostic accuracy. However, the findings on DRE are also not specific for PCa. The primary means of diagnosing PCa is through histopathologic confirmation of a prostate biopsy. Biopsies also have their own limitations such as being an invasive procedure and increasing the risk of serious infection . ConsequThe chief source of cfcDNA in the plasma of healthy persons is apoptotic cells which cleave their own DNA into shorter fragments and secrete homogenous truncated DNA fragments with length of approximately 185–200 base pairs . ConversCirculating cell free DNA is a noninvasive and sensitive biomarker, and a number of methods have been employed for its measurement in plasma and serum including quantitative polymerase chain reaction (PCR) using PicoGreen assay , real-tiThe clinical utility of cfcDNA concentrations and cfcDNA integrity has been investigated in many studies including prostate cancer ,16,17. CUmetani et al. (2006) conceptualized a method using real-time quantitative PCR to directly measure cfcDNA integrity . CirculaThis study aimed to measure cfcDNA concentration and integrity in patients with PCa and to compare them to those with BPH. This study aimed at also assessing whether there is a relationship between cfcDNA concentration with baseline PSA, Gleason score, total prostate volume, age, and number of preexisting comorbidities.The study was reviewed and approved by the ethics committee of The University of the West Indies. The study was a pilot, cross-sectional study that recruited male participants from 1 August 2016 to 31 April 2017 through opportunity (convenience) sampling. Participants were recruited from the urology clinic prior to histological confirmation of prostate cancer with their attending physician. Blood samples were collected from consenting participants who were suspected of having PCa for the measurement of cfcDNA concentrations, cfcDNA integrity, and total prostate-specific antigen (tPSA). Patients with a negative or positive biopsy for prostate cancer were included in this study. Data collection involved chart review to determine patients’ demographic, clinicopathologic, and biochemical information. A correlation of the number of comorbidities present associated with cfcDNA concentration was also investigated.A blood sample of 8 mL was collected from each consented participant after recruitment to determine cfcDNA concentration, cfcDNA integrity, and tPSA before the initiation of therapeutic intervention.The measurement of serum tPSA was performed using a cobas 6000 analyzer  in the Chemical Pathology Laboratory, The University of the West Indies. The Roche Elecsys tPSA assay is centered on a sandwich electrochemiluminescent immunoassay test principle that involves the reaction of a monoclonal PSA-specific antibody labeled with ruthenium complex and a biotinylated monoclonal PSA-specific antibody to form a complex. There is a resulting generation of chemiluminescent emission measured by a photomultiplier and the determination of tPSA concentration by the use of a calibration curve .Four milliliters of blood were collected in an ethylenediaminetetraacetic acid (EDTA) tube and processed within 2 h. The blood collected from patients was centrifuged at 1200 rpm for 10 min in a Thermo Scientific Cl40 centrifuge  to obtain the plasma. The plasma was separated, and a second centrifugation at 2400 rpm for 10 min was performed. Plasma samples were aliquoted into tubes and stored at −20 °C before DNA extraction.v:v:v) , and centrifuged for 10 min at 14,000× g. The aqueous phase was precipitated overnight with 1/10 volume of 3M sodium acetate (NaOAc) and 2.5 volume of 100% ethanol at −20 °C. The DNA pellet was washed with ethanol, air-dried, and resuspended in 50 μL of ddH2O.DNA extraction was performed using the organic DNA extraction method adapted by Xue et al. 2009 . Five huThe amount of DNA was determined by a quantitative real-time PCR (qPCR) technique developed by Umetani et al. (2006) . This me® 480 Real-Time PCR System  and involves pre-cycling heat activation of DNA polymerase at 95 °C for 10 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 64 °C for 30 s, and extension at 72 °C for 30 s.The standard reaction volume was 20 μL and consisted of 5 μL of isolated templates of DNA sample, 0.2 μL of forward and reverse primer, and 10 μL of SYRB Green Master Mix . The real-time PCR (qPCR) was carried out using The LightCyclerThe DNA levels were calculated from a standard curve with serial dilutions (10 ng–0.01 pg) and were prepared with standardized genomic DNA (standardized genomic from a qPCR kit). For each plate, a negative control was used for quality control, and mean values were calculated from triplicate quantification reactions. Participants were interviewed and their demographic, clinical, and pathological data (collected from dockets); results from qPCR analysis; and tPSA results were entered on a data collection form.p values less than 0.05 considered statistically significant. The statistical analysis was done using Statistical Package for Social Sciences, version 20 .Demographic data were evaluated using descriptive statistics. Continuous variables were expressed as medians, interquartile range (IQR), and categorical variables were expressed as counts and percentages. The relationship between cfcDNA concentration and integrity with continuous variables, such as age, was investigated using Mann–Whitney U test. The relationship between cfcDNA concentration and integrity with categorical variables was investigated using the Chi-square test or, where appropriate, Fisher’s exact test. The association between cfcDNA concentration and cfcDNA integrity with baseline tPSA, Gleason score, total prostate volume, age, and the presence and number of comorbidities was assessed using Spearman’s correlation, with two-sided Thirty-five (35) eligible patients were approached, and 29 patients consented to participating in the study. Consenting patients were recruited at a rate of approximately one patient per week. Twenty  of these patients completed their biopsy for histological confirmation for prostate cancer (PCa); the remaining patients’ results  were not available (NA). Eleven  patients were confirmed to have PCa, and 9 (45%) patients had BPH. p = 0.382). The median (IQR) cfcDNA integrity for PCa and BPH patients were 0.6159 (0.49) and 0.6734 (0.45), respectively (p = 0.342).The median (IQR) cfcDNA concentrations for PCa and BPH patients were 47.9 (214.93) ng/mL and 41.5 (55.13) ng/mL, respectively . There wectively . There wp = 0.555) but not with other clinicopathological parameters such as age and Gleason score in PCa patients  previously developed by Umetani et al. (2006) .To the best of our understanding, this is the first investigation of the importance of cfcDNA concentration and cfcDNA integrity using ALU 115 and ALU 247 sequences in plasma from Caribbean men of African Ancestry with prostate cancer and the control group composed of BPH patients. In this study, the median cfcDNA concentration for PCa patients was higher in comparison to BPH patients; however, this trend was not statistically significant. Feng et al. (2013) conducted a similar study using ALU markers involving 96 PCa and 112 BPH patients and found significantly higher levels of cfcDNA concentration among PCa patients . FurtherThere are other studies that have examined the importance of cfcDNA concentration and cfcDNA integrity using the ALU 115-qPCR marker in plasma ,32,33. FRegarding cfcDNA integrity, this parameter was lower in PCa patients in comparison to BPH persons, but there was no statistical difference between groups. This trend was not consistent with previous studies ,34. In pThe cfcDNA integrity was mildly elevated in BPH persons compared to PCa patients, and these observations diverge from the pattern reported by other investigators for both cfcDNA ALU 115 and ALU 247 sequences ,32,33. IProstate cancer is believed to progress via a stepwise development that involves the conversion of benign prostatic epithelial cells to high-grade prostatic intraepithelial neoplasia and eventually to invasive adenocarcinoma . The traHypermethylation of the GSTP1 promoter leads to its expression in PCa patients compared with those with BPH, signifying its critical role in prostate cancer progression ,44. Hypep = 0.160). There are disparities in a number of studies examining the relationship between cfcDNA concentration, cfcDNA integrity, and total PSA. Khani et al. demonstrated a significant direct association between cfcDNA concentration and low total PSA in prostate cancer patients, as approximately 20% with normal PSA levels had higher cfcDNA concentrations compared to those persons with higher total PSA values [In this study, there was a weakly positive correlation between cfcDNA concentration and total PSA but not with other clinicopathological parameters such as age and Gleason score in PCa patients. Feng et al. showed a significant association between cfcDNA concentration and total PSA. Circulating cell free DNA concentration and cfcDNA integrity could distinguish prostate cancer from BPH in patients with serum total PSA ≥ 4 ng/mL . FurtherA values . In anotA values , which wA values ,46,47.In this study, the lack of statistically significant difference with cfcDNA concentration as well as cfcDNA integrity in the two groups of patients may be associated with the small sample size. Furthermore, cfcDNA concentration and cfcDNA integrity may vary due to sample preparation. Serum typically has higher cfcDNA concentrations in comparison to plasma, and this is primarily due to the coagulation of serum and subsequent leukocyte lysis, which releases additional DNA . ExtractThe determination of cfcDNA concentrations present some limitations such as variations in methodology as well as the absence of standardization in existing methodologies which impede the application of these biomarkers in clinical practice ,51. MoreThe current study found that cfcDNA (ALU 115 sequence) levels were relatively nonsignificantly higher in PCa patients in comparison to BPH persons, whereas cfcDNA integrity was somewhat similar in both groups. There was a weakly positive correlation between cfcDNA and tPSA but not with other clinicopathological parameters such as age and Gleason score in PCa patients.The most significant limitation of this study is the sample size, which may have impacted the concentration of cfcDNA, the cfcDNA integrity in PCa patients, and the significance level. Therefore, although the cfcDNA concentration in plasma is nonsignificantly higher in PCa patients, it may be a potentially valuable noninvasive biomarker for screening and diagnosis of the disease. Our study is preliminary and requires a larger sample size for further investigations to confirm this assumption.


Principal component analysis performed simultaneously on FTIR and Raman spectra enabled a clear-cut, time-dependent discrimination between intra-lag phase bacteria probed every 30 min. This demonstrates the unique capability of multimodal vibrational spectroscopy to probe the chemistry of bacterial growth even at the intra-phase level, which is particularly important for the lag phase, where low bacterial numbers limit conventional analytical approaches.Bacterial growth in batch cultures occurs in four phases  that differ distinctly in number of different bacteria, biochemistry and physiology. Knowledge regarding the growth phase and its kinetics is essential for bacterial research, especially in taxonomic identification and monitoring drug interactions. However, the conventional methods by which to assess microbial growth are based only on cell counting or optical density, without any insight into the biochemistry of cells or processes. Both Raman and Fourier transform infrared (FTIR) spectroscopy have shown potential to determine the chemical changes occurring between different bacterial growth phases. Here, we extend the application of spectroscopy and for the first time combine both Raman and FTIR microscopy in a multimodal approach to detect changes in the chemical compositions of bacteria within the same phase (intra-phase). We found a number of spectral markers associated with nucleic acids (IR: 964, 1082, 1215 cm Bacterial growth in batch cultures typically occurs in four distinct phases that are clearly visible through the growth curve: 1) lag, 2) exponential (log), 3) stationary and 4) death phase [ lag, (2) exponent stationa death phKnowledge of the bacterial growth phase, bacterial numbers and growth kinetics is essential for research and commercial applications. Conventional methods of determining the growth phase are based on cell numbers, which are established either through standard plate counting or through optical density . These mOver the past decade, both Raman and FTIR spectroscopy have become widely used in microbiology research on fungi ,7,8,9 anBoth IR and Raman were also applied to study various aspects of growth ,25,26,27Staphylococcus aureus over a timeframe of 6 h of growth in liquid medium, with particular emphasis on intra-lag differences. We probed at regular intervals of 30 min for the first 2 h of experimentation, then subsequently every 2 h . We collected both IR (population level) and Raman (single-cell level) spectra and complemented them by monitoring the growth using standard optical density measurements. Using principal component analysis (PCA) together with an analysis of spectral patterns and integral intensities of selected bands for single and combined modalities, we identified a range of spectra markers, reflective of inter- and intra-phase biochemical changes.Here, we present a multimodal spectroscopic approach in order to study alterations in the biochemical composition of S. aureus (strain AP308) was used. An overnight culture was used to inoculate 600 mL of heart infusion (HI) medium to achieve a bacterial density of 0.2 . The culture was incubated at 37 °C with aeration for 6 h. Samples (30 mL) were collected at time points of 0, 30, 60, 90, 120, 240 and 360 min. We used 1-mL samples for OD625 measurements, to monitor growth. The remaining volume of each sample was centrifuged at 10,000× g for 2 min and subsequently washed three time, using ultrapure, sterile water. After the final wash, the pallets were resuspended in water. Next, 20 µL of suspension was placed on Raman grade CaF2 window and air-dried. The remaining volume of samples were centrifuged  and used directly for ATR measurements. The experiments were conducted in triplicate.For bacterial growth experiments, −1). For each sample, nine technical replicates were recorded, with each spectrum from a different cell. Spectra were recorded in the spectral range of −73 to 3800 cm−1, with a spectral resolution of 3 cm−1. The integration time was set to 1 s, with 50 accumulations per spectrum.Data were collected using a WITec confocal CRM alpha 300 Raman microscope  equipped with a CCD detector cooled to −60 °C, 600 grooves/mm grating and an air-cooled solid-state laser operating at 532 nm. A dry Olympus MPLAN (100x/0.90NA) objective was used, with the laser coupled to the microscope by an optical fiber 50 µm in diameter. Prior to measurements, the instrument was calibrated using a silicon plate .For infrared data collection, we used attenuated total reflectance—Fourier transform infrared spectroscopy (ATR-FTIR). ATR spectra were collected using a Bruker Alpha FTIR  spectrometer equipped with an attenuated total reflection (ATR) sampling device that contained a single bounce diamond internal reflection element and a KBr beam splitter with a globar source and a deuterated triglycine sulfate detector. We transferred 0.5 µL of bacterial pallets directly onto the crystal surface and dried until no change in spectra could be seen (monitored via a live view). Spectra were collected in the range of 4000–800 cm−1 (ATR) and 1800–600 cm−1 (Raman).Data analysis was conducted with MATLAB 8.6 2015b , PLS toolbox v8.2 , WITec Project Plus  and Origin Pro 9.1 . Raman spectra were preprocessed using cosmic spike removal. Second derivatives of Raman spectra were calculated using the Savitzky–Golay (SG) algorithm with 17 smoothing points. The whole dataset was then normalized using a standard normal variate (SNV). Preprocessing of ATR spectra included the calculation of second derivatives  and SNV normalization. Average spectra for each time point were obtained by averaging firstly the technical replicates and subsequently the biological replicates. The average spectra for lag and log phases were calculated as averages of the corresponding time-points . The integral intensities of bands were calculated for second derivatives, with statistical significance calculates using a T-test and ANOVA. The patterns of spectral behaviors were calculated in MATLAB using second derivatives (in-house script) and represent the change of spectra for each point relative to the previous time point or previous phase. Data were analyzed for Raman and ATR modalities independently, then in combination . Multimodal PCA was conducted after mean centering using spectral ranges of 1800–900 cmS. aureus strain based on optical density measurements. It is evident that within the timeframe of the experiment (360 min) bacteria went through the lag phase (0–120 min) then entered an exponential (log) phase (>120 min) of growth. This is consistent with literature reports [We first determined the growth phases of bacteria observed over the 6 h course of the experiment.  reports ,29.We first focused on a comparison between average spectra of bacteria in lag (30–120 min) and log (>120 min) phases. The average ATR and RS spectra, together with an analysis of spectral patterns and relative intensities, are shown in −1) and expressed through bands at 1215, 1117, 1082, 1057, 1032, 994 and 965 cm−1. A detail assignment of these is given in −1) and carbohydrates (1032 cm−1). These clearly increased during the lag phase compared to the log phase (3)) together with a decrease of Amide II intensity (relative to Amide I), indicating a change in protein composition , proteins) decreased in the log phase relative to lag phase (−1) in the log phase compared to lag phase was observed [The Raman spectra B also shic acids D,F. Simiag phase D,F. Thesobserved . This weobserved .−1 (−1. In Raman spectra (−1 (−1 (−1 together with varying relative intensities of bands at 1480 and 1453 cm−1.We further focused on differences observed between spectra of bacteria within the lag phase (30–120 min.). The second derivatives of the average IR and Raman spectra from each time point are given in −1 B, predom spectra C, the mactra (−1 D and 151 (−1 (−1 E throughSpectra recorded at the beginning of the experiment (time point 0) were not included in the general comparison of average spectra from intra-lag bacteria for clarity of presentation. The experiments were initiated using an overnight culture. In such culture, cells were no longer in the lag phase, but rather in the log or even stationary phase. Indeed, spectra recorded for bacteria at the beginning of the experiment resembled bacteria in the log phase to a much greater extent than bacteria in the lag phase . Thus, tS. aureus , along with the time point of the most prominent changes. As expected, both IR and Raman spectra indicated that by far the largest extent of differences existed between the 0 and 30 min time points. This clearly reflects the multitude of biochemical processes occurring upon the adaptation of bacteria from the overnight culture to ‘new’ conditions. At the same time, it indicates that the vast majority of these adaptive processes occur in fact within the first 30 min.To further elucidate the observed intra-lag spectral variability, we performed a detailed analysis of the patterns of changes for IR and Raman spectra recorded over the growth of the . aureus . Figure −1) progressed significantly from the beginning, reaching its maximum at 60 (964 cm−1) or 90 min . Of note is the fact that not all nucleic acid-associated bands reached their maximum relative intensity at the same time point. This may have been a result of the contribution of vibrational modes from other compounds to individual bands, or it could reflect ongoing structural changes in nucleic acids. In addition, a decrease in the relative intensity of the band at 1394 cm−1 and amide II is visible, with the major changes occurring within the first 30 min and further progressing slightly until 60 min. Finally, a significant change can be observed in the relative intensity of the band at 1035 cm−1, with a very prominent increase after 30 min followed by a further increase after 60 min and a rapid drop after 90 min. The pattern of intensity changes in this band differs from the one for nucleic acids and proteins, indicating the likelihood of a different origin—most probably carbohydrate.Several bands showing large variability in the analysis of patterns of spectral changes were further selected and their relative integral intensities were calculated together with their statistical significance C,D. From−1) or 90 min (1483 cm−1). A rapid decrease in the relative intensity of the band at 1453 cm−1 is visible after the first 30 min. This relative decrease may have resulted from a decrease in protein and lipid content, or it might reflect an increase in nucleic acid content. A prominent difference is also visible in the band at 1045 cm−1, which is typically associated with carbohydrates. This band rapidly increases within the first 30 min of the lag phase and then steadily decreases over the next 2 h.The outcomes of the analysis of Raman data D remaine−1 and in Raman at 1483 cm−1) are more intense in the spectra of bacterial pellets collected between 60 and 120 min. The pellets collected at the beginning (0 min) and after 30 min of the culture were associated with higher relative content of proteins, visible particularly in the IR through bands at 1540, 1515 and 1394 cm−1. The decrease of the band at 1459 cm−1 could potentially reflect reduced/changed protein content, or it could be a secondary-effect of increased nucleic acid content. Nevertheless, the Raman part of loading 1 clearly indicates that out of the plethora of nucleic acid-associated bands in the Raman spectra, the ratio of 1483/1459 cm−1 appears to have been the most significant probe of relative nucleic acid content. Loading 2 clearly separates the spectra of intra-lag bacterial collected after 30 and 60 min from the others. Interestingly, this separation is driven in the Raman part of the loading by one band located at 1047 cm−1 and assigned to carbohydrates. This band is positively correlated with the IR band at 1035 cm−1 in loading 2, also attributed to carbohydrates. The IR part of loading 2 further reveals the contribution of one nucleic acid band (1082 cm−1) and one protein band (1550 cm−1) towards the discrimination. However, unlike in loading 1, the correlation between nucleic acid and protein bands for loading 2 is positive. Both are negatively correlated with carbohydrate-related bands.Multimodal PCA  performeThe presented results clearly demonstrate the utility of both IR and RS in probing the chemical changes of bacteria, even within the same growth phase. The changes observed in this study reflect the cellular activity that may be associated with adaptation to new environments and in preparation for division. However, it should also be highlighted that these changes may have been affected by continuous probing of the culture, which resulted in a steady decrease of the culture volume. Such decreases affect the nutrient availability and metabolic activity of cells.−1 in ATR and at 1483 cm−1 in Raman. Further to that, we observed an alteration in the protein composition (Amide II/Amide I) and substantial changes in relative carbohydrate content, visible via bands at 1035 cm−1 (ATR) and 1045 cm−1 (Raman). These changes may reflect the cellular activity aimed at adaptation to a new environment or in preparation for division. Altogether, our results demonstrate the possibilities offered by multimodal vibrational spectroscopies towards providing a biochemical characterization, enabling the study of microbial physiology even given low bacterial numbers. Such biochemical probing opens a new door towards studying lag-phase related events.We demonstrated that multimodal vibrational spectroscopy is a powerful tool to probe the biochemical composition of bacteria and is sensitive to even minor alterations in composition. Both FTIR and Raman spectroscopic methods detected significant changes in the chemical composition of bacteria in different growth phases. For the lag and log phases these changes were related predominantly to different relative nucleic acid contents, and were accompanied by alterations in protein composition. Moreover, for the first time we demonstrated changes in the biochemical compositions of bacteria within the lag phase probed at regular 30 min intervals. We conducted an analysis of spectral patterns and relative intensities together with multimodal PCA, performed simultaneously on ATR and Raman data. This enabled us to achieve a clear-cut, time-dependent discrimination between intra-lag bacteria. The dominant spectral differences were associated with relative nucleic acid content, which reached its highest level after 60 and 90 min. This was expressed by bands at 1215, 1085 and 965 cm


The spaying of female cattle is a routine husbandry procedure conducted in some extensive beef systems, including in northern Australia. Female cattle may be spayed to control stocking rates, reduce mortalities associated with breeding, or to enable surplus females to be sold in compliance with live export requirements. The more widely practiced Willis dropped ovary technique involves severing the ovarian attachments via use of an ovariotome, which is inserted trans-vaginally to enter the abdominal cavity. While the procedure has been shown to cause pain, stress, morbidity, and mortality, it is mostly conducted without the use of veterinary pharmaceuticals. This study evaluates the efficacy of a topical anaesthetic, haemostatic wound dressing, and a non-steroidal anti-inflammatory drug for minimising pain and haemorrhage in the acute period post-spaying via the Willis dropped ovary technique. Adverse behavioural responses observed in spayed heifers were reduced in those cattle that received the non-steroidal anti-inflammatory drug, suggesting an improvement in animal welfare.n = 75) were randomly allocated to the following treatment groups: (1) rectal palpation/control (CON); (2) WDOT spay (S); (3) WDOT spay with meloxicam (SM); (4) WDOT spay with TA (STA); and (5) WDOT spay with TA and meloxicam (STAM). Individual behavioural responses, body weight, packed cell volume (PCV), and total plasma protein (TPP) were monitored for up to 24 h following treatment. Head tucking behaviour and tail stiffness was increased in all spay groups compared to the CON group (p < 0.001), with the lowest proportional increase in the SM group. Rumination was initially reduced in S, SM, and STA heifers compared to CON heifers (p < 0.001), though SM heifers ruminated more than S heifers (p < 0.001). CON and SM heifers stood with an arched back the least, spent the most time eating, and spent less time lying down and more time standing compared to other treatment groups (p < 0.001). There was no significant effect of treatment on weight change (p = 0.519), PCV (p = 0.125) or TPP (p = 0.799). The administration of meloxicam is suggested as an effective, currently available method for improving the welfare of cattle undergoing WDOT spaying.Multiple physiological and neuroendocrine changes consistent with stress and pain have been demonstrated in cattle spayed via the Willis dropped ovary technique (WDOT). The procedure is routinely conducted without the use of anaesthetics or analgesics and has major implications for animal welfare. This study aimed to evaluate the effect of a topical anaesthetic (TA), haemostatic wound dressing, and meloxicam on pain behaviour and haemorrhage in the acute period following spaying. Yearling Brahman heifers ( In the rangeland beef cattle production systems of northern Australia, female cattle that are not required for breeding are spayed to control stocking rates and to enable surplus females, particularly heifers and aged animals, to be sold ,2,3. In The three methods used for the routine spaying of cattle are flank laparotomy (FL), the Willis dropped ovary technique (WDOT), and passage spaying/webbing ,2,6. In ®; Bayer Animal Health, Australia) is registered for use in lambs and calves undergoing routine husbandry procedures. It contains the anaesthetic agents lignocaine (40.6 g/L) and bupivacaine (4.5 g/L), as well as cetrimide (5.0 g/L) and adrenaline (24.8 mg/L). The wound dressing is applied post-operatively to exposed wounds and effects rapid and prolonged pain alleviation, as observed through a reduction in pain-related behaviours [® and all its components will now be referred to as topical anaesthetic (TA). The NSAID meloxicam is another effective analgesic option available for use in cattle, which may be administered as an injection or orally following routine painful procedures for a prolonged duration of effect [Pain management is a progressively important consideration in livestock production given its role in improving animal welfare outcomes . Multimohaviours ,16. Thisf effect . The useThe current study aimed to evaluate the effect of a topical anaesthetic, haemostatic wound dressing, and meloxicam, alone and in combination, on pain and haemorrhage in the acute period following spaying of heifers via the WDOT. The primary objectives were to assess pain via behavioural observation, and to evaluate post-operative haemorrhage through an assessment of packed cell volume (PCV) and total plasma protein (TPP). It was hypothesised that both the TA and meloxicam would reduce pain following spaying, with a combination of the two being most effective, and that the adrenaline included in the TA would reduce haemorrhage following spaying.The experimental protocol was approved by the University of Sydney Animal Ethics Committee . The study was conducted in compliance with national standards for the care and welfare of animals.The experiment was conducted on a commercial beef cattle property located approximately 39 km north-west of Kununurra in the East Kimberley region of Western Australia . The experiment occurred over a 3-day period in the early wet season (late November 2017). The daily minimum and maximum temperature ranges during the experimental period were between 23.5 and 26.2 °C and 36.8 and 39.8 °C, respectively. The average relative humidity ranged from 39% to 69%, and there was a total of 7.4 mm rainfall.n = 75, body weight 260.8 ± 16.5 kg) selected for spaying were representative of cattle routinely spayed in the region. Animals were mustered to the yards two days prior to the experimental period. One of the heifers included had been recently dehorned (within the two weeks preceding the study) and none were found to be pregnant using rectal palpation during the experiment.The yearling Brahman heifers  was modified by a local engineering company prior to commencement of the experiment. The modified ovariotome featured a thin, cylindrical metal tube attached laterally parallel to the length of the tool, and opening at the distal end near the cutting slot . On the The experiment was conducted in a set of steel commercial cattle yards and adjacent holding yards. Three holding yards were used, each approximately 20 by 25 m . The procedures started at 0630 h, with three replicates of 14 heifers (2–3 of each treatment), and 2 replicates of 14 and 19 heifers (2–4 of each treatment) processed respectively on two successive days . This prOn day 1, the cattle within each replicate group were moved from the holding yard into a crush via a set of scales where animals were weighed immediately prior to being restrained for the procedure. A blood sample was taken from the tail vein, and the treatment, as previously assigned to each individual animal, was performed. After the procedure, heifers were head-bailed, ear-tagged (left ear), and a corresponding number was spray-painted on the flank bilaterally for individual identification. The animal was then released into an adjacent holding yard.Once all cattle within the replicate had been sampled and treated, the group was moved to another yard to allow for the next replicate group. This was repeated until the 3 replicates were completed on day 1. In each yard, feed and water were provided ad libitum with good quality pasture hay and 1–2 water troughs per yard. The following morning (day 2), the cattle from day 1 were observed for an hour at the start of the day, approximately 24 h post-treatment. All replicate groups were then moved through the crush and individually restrained for repeat weighing and blood sampling. Cattle were then released into a larger holding yard where they were kept for a week following spaying for observation by staff to detect any post-operative mortalities. All animals were then moved to a paddock to graze as a single group with ad libitum pasture, hay, and water. These procedures were repeated for the 2 replicate groups on day 2.n = 14); (2) a WDOT spay ; (3) a WDOT spay with intra-operative delivery of meloxicam ; (4) a WDOT spay with intra-operative delivery of TA ; and (5) a WDOT spay with intra-operative delivery of TA and meloxicam . The WDOT involves rectal palpation and manipulation of the reproductive tract prior to the insertion of a device into the vagina. Hence, we examined the responses of cattle to this initial, less invasive component of cattle spaying procedures in the control group. The procedures performed are detailed below.Each animal was randomly allocated to 1 of 5 treatment groups, with 14-16 heifers assigned to each of the treatments. The treatments were: (1) rectal palpation  Rectal palpation (CON)Each heifer was physically restrained in a commercial cattle crush, with the kick-gate closed behind the back legs. Rectal palpation was performed with brief manipulation of the reproductive tract (10–15 s) . The ent(2) WDOT spay using modified ovariotome (S)The heifers were physically restrained as described above. Cattle were spayed according to procedures described by de Witte, Jubb, and Letchford , using t(3) WDOT spay using modified ovariotome with intra-operative delivery of meloxicam (SM)® 20 mg/mL; Boehringer Ingelheim Vetmedica, Germany) at a dose of 0.5 mg/kg body weight was administered subcutaneously immediately after the procedure. Doses were pre-calculated for body weight groups at 50 kg intervals from 150–400 kg to facilitate ease of drug administration and to minimise the overall handling time of animals. On average, the entire procedure was performed within 3 min.The heifers were physically restrained and the spay was performed as described above. An injection of meloxicam (Metacamvia modified ovariotome (STA)(4) WDOT spay with delivery of TA ® TA administered at two key points during the procedure by an assistant: prior to piercing through the vaginal wall, and immediately prior to ovary excision. These were identified to be the most invasive and likely more painful parts of the procedure. Approximately 6–8 mL of TA were administered per heifer. On average, the entire procedure was performed within 5 min.The heifers were physically restrained and the spay was performed as above, with the addition of Tri-solfenvia modified ovariotome and the intra-operative delivery of meloxicam (STAM)(5) WDOT spay with the delivery of TA As described above for the SM and STA procedures, cattle received a combination of TA during the procedure and meloxicam intra-operatively.2 EDTA 18 mg Plus Vacutainer tubes . A repeat sample was obtained the following morning at approximately 24 h post-treatment. The blood samples were placed in a portable refrigerator maintaining an ambient temperature below 10 °C until all replicates for the day were completed. The samples were then centrifuged using a 24 place microhaematocrit centrifuge at RCF 13,000× g for 7 min at room temperature . The PCV and TPP for each animal sampled that morning was recorded [A blood sample was taken from each heifer immediately following restraint in the crush, prior to treatment. Approximately 10 mL of blood was collected from the tail vein into labelled lithium heparin or Krecorded .® XT 13 observational data software program .Video recordings of heifers in the holding yards were taken in order to observe and comprehensively quantify the behavioural responses following spaying with minimal disturbance by human activity. On day 1, four HD 1080p Sports Action Cam video cameras  were mounted on Yards 1 and 2 in each corner, and 3 cameras were mounted on Yard 3 . As therSeven observations were recorded in total for each replicate group, corresponding to time after procedures had been conducted. Observation 1 was recorded one hour after the procedure was performed, and, similarly, Observations 2–6 were documented their respective hours after the procedure (between 0730 and 1700). Observations were analysed by a single observer to standardize the recordings, and the observer was blinded to the treatments. For each observation (O1-7), each animal was observed over a 10 min period within the hour. At one minute intervals, the behaviours exhibited by the animal were recorded, giving a total of 10 instantaneous samples reflective of the frequency of each behaviour. The key behaviours analysed  were derThe general health status of the heifers was visually assessed and monitored daily for a week following the procedures by property staff. All mortalities were systematically examined by necropsy by the attending veterinarian.For the behavioural analyses, data was exported from The Observer XT13 programme and all analyses were conducted using Genstat statistical software . Data from the first 6 h on frequency of behaviours were combined to provide an improved overall assessment of post-procedure behaviour. Data from the 24 h timepoint was assessed separately. Regression analysis was performed with generalised linear modelling, using binomial proportions. A macro in Microsoft Excel 2013 was then used to generate the odds and probabilities for each behaviour. Heifer 47 died on day 3 of the experiment and was hence removed from all statistical analysis to avoid any bias. As not all cattle were located at every sample, for data analysis, results were expressed as proportions of the number of observations.p values ≤ 0.05 were considered statistically significant.As animals were not divided into treatment groups according to weight, an ANOVA was also performed to test for significant differences in the effect of treatments in relation to initial body weights. Data on changes in weight, PCV, and TPP at 0 h and 24 h for each animal was entered into Excel 2013. Data was subjected to linear mixed modelling (REML) to account for individual animal variation and to determine if time or treatment had a significant effect on these factors. Outliers were identified using boxplots. Outliers were removed from the data regarding changes in weight and the data was re-analysed, as this was likely due to inaccurate scale readings rather than a true dramatic change in weight. For all statistical calculations, One mortality was documented during the experiment, where a STAM heifer treated on day 1 was found dead on arrival on the morning of day 3. A post-mortem was performed, and findings included free blood in the abdomen, a larger left ovarian pedicle stump and corresponding blood clot indicating increased haemorrhage from the area previously. The puncture point in the vaginal wall was skewed to the left into the broad ligament—a structure with increased vascularity. The mortality occurred as a result of internal haemorrhage, though it was unclear whether haemorrhage from the ovarian pedicle stump or the broad ligament was the primary cause.p = 0.392). There was no significant effect of treatment on PCV (p = 0.125), TPP (p = 0.799), or weight (p = 0.519) from 0 to 24 h. Outliers were identified in each data set. Heifers 87 (STAM), 88 (SM), and 67 (SM) had marked increases in post-procedure PCV, while heifer 65 (STA) had a marked decrease in PCV. Heifer 86 (STA) had a marked increase in TPP and a marked decrease in weight. Heifer 56 (STAM) had a marked increase in weight.No significant differences were found between treatments in terms of initial body weight , as well as on walking (p = 0.047) and self lick behaviours (p = 0.002). There were insufficient recordings of drinking and licking other for statistical analysis.Between 1 and 6 h, there was a significant effect of treatment on lying, standing, repetitive head movement, head tuck, head down, head up, back arch, repetitive tail movement, relaxed tail, and stiff tail behaviours (p < 0.001), as well as on walking (p = 0.036). There was no significant effect at this timepoint of treatment on repetitive head movement, head tuck, self lick, or back arching behaviours.At the 24 h timepoint, there was a significant effect of treatment on lying, standing, head down, repetitive tail movement, relaxed tail, and stiff tail behaviours (p < 0.001). The SM and CON heifers spent the least amount of time lying down. The STA heifers spent the greatest proportion of time lying down. The reciprocal for this pattern was shown for standing, with S, STA, and STAM heifers spending significantly less time standing compared to CON and SM heifers (p < 0.001). CON and SM heifers stood for the longest duration, and STA heifers spent the least proportion of time standing. SM and STAM heifers spent a significantly greater proportion of time standing compared to S heifers (p < 0.001). STA heifers spent significantly less time walking than S and STAM heifers (p = 0.047).The results for this are summarised in p < 0.001). The S and CON heifers spent the least amount of time lying down, and STAM heifers spent the most time lying down. SM, STA, and STAM heifers spent significantly less time standing than CON and S heifers (p < 0.001). Significantly more STA heifers were observed walking compared to CON and STAM groups (p = 0.036).At 24 h post-procedure, the SM and STAM groups spent a significantly greater amount of time lying down compared to the CON and S groups (p < 0.001). SM heifers stood with their head down significantly more than S heifers, while STA heifers stood with their head down for significantly less time compared to S heifers (p < 0.001). Conversely, STA heifers stood with their head up for a greater proportion of time compared to both the CON and S groups (p < 0.001). S and SM heifers exhibited significantly more repetitive head movements compared to CON heifers, while STA heifers were observed performing this behaviour significantly less frequently (p < 0.001). The frequency of head tucking behaviour was significantly increased in all groups compared to the CON group (p < 0.001), with the highest frequency in the STA and STAM groups. SM, STA, and STAM heifers exhibited self-licking behaviour significantly less frequently than S heifers (p = 0.002).The results for this are summarised in p < 0.001). SM heifers stood with their head down the least. Reciprocally, SM, STA, and STAM heifers stood with their head up for a greater proportion of time compared to CON heifers (p < 0.001).At the 24 h timepoint, SM, STA, and STAM heifers spent significantly less time standing with their head down compared to CON heifers (p < 0.001). CON and SM heifers stood least frequently with an arched back, and STA heifers stood with an arched back most frequently. In all treatment groups , heifers stood with a stiff tail for significantly longer than heifers in the CON group (p < 0.001). STA heifers stood the greatest proportion of time with a stiff tail, while SM heifers stood for the least amount of time with a stiff tail out of the spayed groups. Conversely, CON heifers spent the greatest proportion of time with a relaxed tail. There was a significant reduction in the proportion of heifers with a relaxed tail in all treatment groups compared to the CON group (p < 0.001). SM and STAM heifers had significantly increased repetitive tail movements post-procedure compared to CON heifers (p < 0.001). STA heifers spent the least proportion of time with repetitive tail movements, followed by CON and S heifers.The results for this are summarised in p < 0.001). CON heifers stood with a stiff tail for the least amount of time, and spent the greatest proportion of time with a relaxed tail. There was a significant reduction in the proportion of heifers with a relaxed tail in all treatment groups compared to the CON group (p < 0.001). Significantly more repetitive tail movements were observed in SM, STA, and STAM groups compared to the CON group (p < 0.001). SM heifers showed the most repetitive tail movements proportionally, while CON heifers showed the least.At 24 h post-procedure, S heifers spent a significantly greater proportion of time with a stiff tail compared to all other treatment groups (p < 0.001). The CON heifers spent the most amount of time eating, followed by SM heifers. The STA heifers spent the least amount of time eating. In addition, CON heifers spent the greatest proportion of time ruminating. Rumination was significantly reduced in S, SM, and STA heifers compared to CON heifers (p < 0.001). Compared to S heifers, SM and STAM heifers ruminated significantly more, while STA heifers ruminated less (p < 0.001).The results for this are summarised in p < 0.001). Significantly less rumination was observed in S heifers (p < 0.001), while rumination was significantly increased in SM and STAM heifers compared to CON heifers (p < 0.001).At the 24 h timepoint, all treatment groups spent a significantly reduced amount of time eating compared to the CON group , which often had similar results to the CON group, indicating that meloxicam is effective at mitigating the pain and discomfort associated with spaying for at least 6 h post-procedure. Non-steroidal anti-inflammatory drugs function by inhibiting cyclooxygenase (COX) enzymes, resulting in a decreased production of prostaglandins and a reduced inflammatory response . Meloxic® is licensed for use on external wounds, it is possible that cetrimide, an antiseptic contained in the product, is irritant to the abdominal viscera with resultant chemical peritonitis. The TA gel was applied intra-vaginally and intra-abdominally prior to ovariectomy, and may have caused local inflammatory reactions resulting in increased pain behaviours. Interestingly, STA heifers demonstrated the least repetitive head and tail movements from all groups, and the lack of these behaviours could potentially be reflective of a strong pain response. Behavioural responses of the STAM group were often intermediate between STA and CON/S groups, which may be attributed to the analgesic effects of meloxicam countering any local irritation from Tri-solfen®.The use of TA, both alone and in combination with meloxicam, increased the incidence of pain-related behaviours after spaying. TA has been shown to achieve rapid wound anaesthesia within 1 min and is effective in the amelioration of pain for up to 24 h after surgical castration in calves . In this® combination in this experiment did not yield better results than when meloxicam was administered solely. The effectiveness of a local anaesthetic and NSAID combination on pain mitigation has been well documented [While multimodal analgesia is ideal for optimising pain management, the use of the meloxicam and Tri-Solfencumented ,24, and cumented . For exacumented ,13,14. Ncumented . For exacumented . Further® could assist in local vasoconstriction and potentially minimise haemorrhage from severing the ovarian pedicle. However, no significant difference was found between the PCV and TPP readings of CON and spayed animals at 0 and 24 h; therefore, spaying via the WDOT may only cause severe haemorrhage in few cases.It was also hypothesised that the adrenaline contained in Tri-solfenIn order for the development and approval of effective analgesic drug protocols for use in livestock, validated, repeatable methods of measuring pain must first be established . HoweverThis study demonstrates the animal welfare implications of spaying cattle without the use of pain relief, as it occurs according to current industry practice and state regulations. There were significant behavioural changes in WDOT-spayed heifers consistent with discomfort and pain in the initial 6 h and up until 24 h post-procedure. It appears that meloxicam is effective at mitigating the pain and discomfort associated with spaying, and could be practically incorporated into routine operations for the improvement of animal welfare. Further research into an effective multimodal analgesic protocol and haemorrhage prevention for spaying are warranted, with alternate methods for administering a local anaesthetic agent solely being suggested for future investigations.


Recently, there has been a worldwide concern regarding soil contamination by heavy metals and organic compounds, especially in the developing countries including Lebanon that has suffered from solid waste mismanagement for decades. Deir Kanoun Ras El Ain is a village in southern Lebanon that possesses one of the country's worst dumps, and its leachates influx into a running canal that irrigates surrounding agricultural lands. The aim of this study was to determine the levels of some toxic heavy metals and organic compounds in different soil samples collected from the dump and along the canal during winter and summer seasons. Six research sites  were selected, and the soil samples for analysis were collected from a depth of around 10 cm. Heavy metals  and organic compounds  content were determined using atomic absorption and high pressure liquid chromatography, respectively. The conducted research confirmed high levels of contamination in the collected soil samples by both heavy metals and organic compounds. The present study provided evidence that different sampling sites accumulated heavy metals at concentrations that exceeded the average maximum permissible levels for sewage sludge and agricultural land. These findings suggest the need for mitigation measures by the Lebanese authorities and new waste management programs to resolve the problems associated with uncontrolled dumping of solid wastes in Lebanon. Recently, environmental contamination by high levels of heavy metals and organic compounds has raised the global public health concerns especially in the developing countries . The majIn addition, soil contamination by heavy metals and organic compounds may affect water resources and agricultural areas where the soil-crop system plays a chief role in the exposure of humans to contaminants , 10. HumLebanon has suffered from solid waste mismanagement for decades . It lackSamples were collected in triplicate during winter and summer seasons of 2017. Sampling sites included two along the canal and four surrounding the dump as presented in The concentrations of heavy metals such as lead, cadmium, arsenic, and mercury  were assessed in soil samples using atomic absorption spectrophotometer . Analysis was performed at the Lebanese Agricultural Research Institute (LARI) in Lebanon. Details of the analysis procedure are available in Supplemental Materials.The methods for phthalates and bisphenol A extraction and fractionation were carried out according to those described by Fromme et al. . MethodsThe concentrations of heavy metals assessed in soil samples collected during summer season are presented in Pb, Cd, As, and Hg were abundant in all of the collected samples from dump and canal, where their concentrations ranged 504.3–1365 mg/kg, 77–131.1 mg/kg, 51–603.3 mg/kg, and 0.16–6.48 mg/kg, respectively. Pb, As, and Hg concentrations were the highest in soil samples collected from C1 site. Concerning samples collected around the dump, D4 site contained the highest concentrations of Pb (1306.07 mg/kg), while Cd and Hg were at the highest levels at D1 site . As was highly abundant in soil samples collected from D2 (471.97 mg/kg) and D3 (521.7 mg/kg) sites.While absolute values of heavy metal contamination are important, especially during remediation efforts, these values do not take into account the relative toxicity of the individual metals present at each site. These relative toxicities are considered in the determination of the maximum permission limits (MPL) set by various governmental regulatory agencies. The maximal permissible levels for Pb, Cd, As, and Hg in sewage sludge are shown in Comparison of the levels of heavy metals to the sewage sludge MPLs promulgated by several countries showed that Pb levels at D3, D4, and C1 sites surpassed the quality standards of EEC, France, US EPA, and CCME. In addition, the levels of Cd at all sites except D4 and C1 surpassed the sewage sludge MPLs of EEC, France, US EPA, and CCME. The D4 and C1 sites had Cd levels that fell below the MPLs promulgated by the US EPA; however, these Cd levels exceeded the Cd MPLS set by the European Union, Canada, and France. The levels of As at all sites except D1 highly exceeded the US EPA and CCME sewage sludge standards. Only three EU member states  currently regulate As in sewage sludge, with an MPL of 25–30 mg/kg ; the levAn average MPL value for each metal was calculated  were also assessed in soil samples collected from the dump and canal sites during two seasons. These data are presented in α-pyrene (BAP), a carcinogenic polycyclic hydrocarbon produced during incomplete combustion, was detected only in samples collected from the D3 (0.022 mg/kg) and D4 (0.012 mg/kg) sites. Samples collected from C2 site showed detectable concentrations of BAP (0.075 mg/kg). Summer collections (S) revealed a similar abundance of residues from plastic waste among different sites. DBH, DEHP, and BPA concentrations showed decreasing patterns where the highest concentrations were in samples collected from D1 site  while the lowest concentrations were in samples collected from D4 site . Regarding soil samples collected near the canal, DBH concentrations in samples collected from C2 site (1.79 mg/kg) were higher than the ones collected from C1 site (1.23 mg/kg). Likewise, DEHP and BPA concentrations in C2 site  were higher than those from C1 site (3.16 and 0.064 mg/kg). Benzo α-pyrene was undetectable in all samples collected during the summer season.As seen in The lack of waste management policies in Lebanon has led to uncontrolled dumping of both household and hazardous solid wastes –19. DeirRegarding heavy metals, our findings indicate that Pb, Cd, and As were the most serious metal pollutants in soil samples collected from Deir Kanoun dump and canal. Upon comparing our data to previously published research studies, it was noted that our findings are similar to those obtained by Tang et al.  where thAdditionally, the levels of Pb, Cd, and As exceeded the sewage sludge maximum permissible limits (MPL) of several quality standards. Fold above average MPL for each metal at each site showed that As levels had the highest fold above average MPL followed by Cd and Pb. Comparing our results to the MPL of heavy metals in agricultural soil set by different guidelines showed that the concentrations of Pb, Cd, and As in all soil samples greatly surpassed the quality standards of several agencies. It should be noted that the dump area in Deir Kanoun Ras El Ain is surrounded by agricultural lands, and the canal from which samples were collected irrigates these lands. The fact that Cd and As greatly exceed the MPLs for both sewage sludge and agricultural soil indicates that these two metals may pose a risk to residents who live near the dumpsite and consume crops grown in contaminated agricultural lands. In fact, high levels of Pb, Cd, and As have been shown to exert damaging effects and serious disorders in living organisms , 38. ForMoreover, the findings reported herein are of serious concern since Cd has been widely associated with decreased bone mineral density which subsequently leads to increased risk of bone fracture in both experimental models and populations-based studies , 45. Thiα)pyrene in most of the collected soil samples. Regarding phthalates, a study by Zorníková & Hřivna in Czech Republic showed that agricultural soil samples contained 0.28–1.59 mg/kg di-n-butyl phthalate (DEHP) and 0.03–0.73 mg/kg bis(2-ethylhexyl) phthalate (DBP) which are lower than the findings of the present study [μg/kg and 1110–1900 μg/kg, respectively [μg/kg and 12–40 μg/kg, respectively. According to the Danish guidelines, the levels of DBP and DEHP should be limited to 0.01–0.98 μg/kg in fertilized soils and 1450–2430 μg/kg in sewage sludge [In addition to heavy metals, we were also able to detect the presence of phthalates (DBP and DEHP), bisphenol A, and benzo is an important organic compound that acts as an endocrine disruptor, mainly as an estrogen, and it is widely used in the industrial manufacture of plastics . Due to α)pyrene which is one of the highly toxic PAHs in the soil-crop system [α)pyrene in three sites only at concentrations of 0.012–0.075 mg/kg, which are similar to those reported in soil samples from regions in Poland , Spain 9 mg/kg; ), South 9 mg/kg; ), and Ca9 mg/kg; ). Also, 9 mg/kg; ).Finally, it is crucial to provide a brief comparison of pollution between the different sites of soil collection. Among all the studied sites, sites C1, D4, and D3 were the most heavily contaminated with heavy metals, while site D1 had the highest levels of organic compounds. There are several factors that lead to the different levels and distribution of heavy metals and organic compounds, such as the type of dumped waste at each site, soil properties, soil organic matter (SOM), distance to the sources of contamination, use of lands, type of grown crops, and dry or wet depositions which may explain why our summer collections had higher levels of organic compounds than winter collections , 3. MoreIt should be taken into consideration that the toxicological effects of such compounds depend on their bioavailability, metabolism, and excretion into organisms and humans. Thus, the current results suggest further investigation of the levels of heavy metals and organic compounds in crops grown in agricultural lands near the dump and irrigated from the canal. Furthermore, serious actions and mitigation measures must be taken by the Lebanese authorities to solve the current environmental disaster as well as other similar dumps. Additionally, it is hoped that these findings lead to a greater awareness of the hazardous consequences of unregulated waste dumping.The results of the current study show that soil samples collected from Deir Kanoun Ras El Ein dump and canal were heavily contaminated with heavy metals. The presence of organic compounds including phthalates, bisphenol, and PAHs was also noted. The concentrations of heavy metals highly exceeded the permissible limits recommended by different agencies. The presence of heavy metals and organic compounds in the irrigation canal may also affect agricultural lands that are irrigated by canal's water and, ultimately, the health of inhabitants who consume the crops grown in these contaminated soils. More studies are clearly needed to determine the extent of heavy metal contamination at various distances from the dump's site and in the crops grown in fields both adjacent to and more distant from this dump. Since both the heavy metals and organic pollutants are a result of the uncontrolled dumping of solid waste at this site, stricter environmental policies regarding waste management should be implemented by the Lebanese authorities and guidance should be given to local inhabitants about healthy management practices regarding information pertinent to their health status.