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Topic:Biomarkers

Biomarkers in horses are measurable indicators of biological processes, states, or conditions that can be used to assess health and disease. These biomarkers can be identified in various biological samples such as blood, urine, or tissue. They provide valuable information for diagnosing diseases, monitoring health status, and evaluating treatment responses in equine medicine. Common biomarkers in horses include enzymes, hormones, and metabolites, which may change in response to physiological or pathological conditions. This page compiles peer-reviewed research studies and scholarly articles that explore the identification, quantification, and application of biomarkers in equine health and disease management.
Transrectal ultrasonographic assessment of the fetal proximal phalanx: A new tool to assess fetal age and bone development in horses.
Theriogenology    June 13, 2024   Volume 226 167-172 doi: 10.1016/j.theriogenology.2024.06.010
Renaudin C, Wensley F, Morgan J, Cassano J, Spriet M.Fetal age in Quarter Horses can be predicted within 2 weeks from 100- to 200- days of gestation using femur length, biparietal diameter (cranium diameter) and eye approximated volume. However, as pregnancy advances, the femur and cranium become too large to be imaged in their entirety using ultrasound and the corresponding biometric parameters can no longer be measured. In this longitudinal study, the proximal phalanx (P1) was evaluated as a novel biometric parameter for late gestation to predict fetal age and bone maturation. Transrectal ultrasound was performed in ten pregnant mares with kno...
Non-Synonymous Substitutions in Cadherin 13, Solute Carrier Family 6 Member 4, and Monoamine Oxidase A Genes are Associated with Personality Traits in Thoroughbred Horses.
Behavior genetics    June 10, 2024   doi: 10.1007/s10519-024-10186-x
Yokomori T, Tozaki T, Ohnuma A, Ishimaru M, Sato F, Hori Y, Segawa T, Itou T.Retraining retired racehorses for various purposes can help correct behavioral issues. However, ensuring efficiency and preventing accidents present global challenges. Based on the hypothesis that a simple personality assessment could help address these challenges, the present study aimed to identify genetic markers associated with personality. Eight genes were selected from 18 personality-related candidate genes that are orthologs of human personality genes, and their association with personality was verified based on actual behavior. A total of 169 Thoroughbred horses were assessed for their...
Comparative Analysis of mRNA and lncRNA Expression Profiles in Testicular Tissue of Sexually Immature and Sexually Mature Mongolian Horses.
Animals : an open access journal from MDPI    June 7, 2024   Volume 14, Issue 12 doi: 10.3390/ani14121717
Liu Y, Du M, Zhang L, Wang N, He Q, Cao J, Zhao B, Li X, Li B, Bou G, Zhao Y, Dugarjaviin M.Testicular development and spermatogenesis are tightly regulated by both coding and non-coding genes, with mRNA and lncRNA playing crucial roles in post-transcriptional gene expression regulation. However, there are significant differences in regulatory mechanisms before and after sexual maturity. Nevertheless, the mRNAs and lncRNAs in the testes of Mongolian horses have not been systematically identified. In this study, we first identified the testicular tissues of sexually immature and sexually mature Mongolian horses at the tissue and protein levels, and comprehensively analyzed the express...
Measurement of Trace Elements (Zinc, Copper, Magnesium, and Iron) in the Saliva of Horses: Validation Data and Changes in Equine Gastric Ulcer Syndrome (EGUS).
Animals : an open access journal from MDPI    June 7, 2024   Volume 14, Issue 12 doi: 10.3390/ani14121724
Muñoz-Prieto A, Cerón JJ, Tecles F, Cuervo MM, Contreras-Aguilar MD, Ayala I, Oí«š-Guillén A, Pardo-Marín L, Hansen S.The objective of this study was to evaluate the possible use of spectrophotometric assays for the measurement of trace elements, including Zinc (Zn), Copper (Cu), Magnesium (Mg), and iron (Fe) in the saliva of horses and study their possible changes in equine gastric ulcer syndrome (EGUS). EGUS is a highly prevalent disease, with a current high incidence due to the increase in intensive management conditions. There are two EGUS diseases: equine squamous gastric disease (ESGD) and equine glandular gastric disease (EGGD), which can appear individually or together. For this purpose, automated spe...
Ultrasound biomicroscopic imaging parameters associated with outcome in equine infectious ulcerative keratitis and stromal abscesses.
Journal of the American Veterinary Medical Association    June 6, 2024   Volume 262, Issue S2 S57-S64 doi: 10.2460/javma.24.02.0097
Collins EN, Barr EM, Westermeyer H, Gilger BC, Oh A.To determine the predictive value of corneal ultrasound biomicroscopy (UBM) findings for the outcome of equine corneal disease. Methods: 46 horses with a clinical diagnosis of either infectious ulcerative keratitis or stromal abscess. Methods: Corneal UBM (VevoMD; UHF70; VisualSonics) of horses with infectious corneal disease presenting to the North Carolina State University Equine Ophthalmology Service from 2019 to 2023 were evaluated. Size and depth of lesion, presence of Descemet membrane disruption (DMD), corneal thickness, and aqueous humor cell counts (AHCC) were assessed. Comparisons of...
Heart rate and heart rate variability in horses undergoing hot and cold shoeing.
PloS one    June 6, 2024   Volume 19, Issue 6 e0305031 doi: 10.1371/journal.pone.0305031
Huangsaksri O, Wonghanchao T, Sanigavatee K, Poochipakorn C, Chanda M.Heart rate variability (HRV) is a frequently used indicator of autonomic responses to various stimuli in horses. This study aimed to investigate HRV variables in horses undergoing cold (n = 25) or hot (n = 26) shoeing. Multiple HRV variables were measured and compared between horses undergoing cold and hot shoeing, including the time domain, frequency domain, and nonlinear variables pre-shoeing, during shoeing, and at 30-minute intervals for 120 minutes post-shoeing. The shoeing method interacted with time to change the HRV variables standard deviation of RR intervals (SDNN), root mean square ...
Effect of an endothelial regulatory module on plasma proteomics in exercising horses.
Comparative biochemistry and physiology. Part D, Genomics & proteomics    June 6, 2024   Volume 52 101265 doi: 10.1016/j.cbd.2024.101265
Amiri Roudbar M, Rosengren MK, Mousavi SF, Fegraeus K, Naboulsi R, Meadows JRS, Lindgren G.Elite performing exercise requires an intricate modulation of the blood pressure to support the working muscles with oxygen. We have previously identified a genomic regulatory module that associates with differences in blood pressures of importance for elite performance in racehorses. This study aimed to determine the effect of the regulatory module on the protein repertoire. We sampled plasma from 12 Coldblooded trotters divided into two endothelial regulatory module haplotype groups, a sub-elite performing haplotype (SPH) and an elite performing haplotype (EPH), each at rest and exercise. Th...
Changes in plasma metabolite concentrations and enzyme activities in aging riding horses.
Frontiers in veterinary science    May 31, 2024   Volume 11 1345548 doi: 10.3389/fvets.2024.1345548
Asahi Y, Arai T, Tanaka Y.In older horses, basal metabolic rate decreases, and plasma metabolite and hormone concentrations related to energy metabolism change. The occurrence of age-related diseases, which increases in old animals, may enhance inflammatory reactivity (inflammaging). Finding the appropriate treatment for inflammaging at an early stage may prevent various age-related diseases. Changes in metabolite and hormone concentrations and enzyme activities involved in energy metabolism in the plasma of clinically healthy riding horses of various ages were measured to identify biomarkers of inflammaging (persisten...
Proteomics is advancing the understanding of stallion sperm biology.
Proteomics    May 29, 2024   e2300522 doi: 10.1002/pmic.202300522
Peña FJ, Martín-Cano FE, Becerro-Rey L, Ortega-Ferrusola C, Gaitskell-Phillips G, da Silva-Álvarez E, Gil MC.The mammalian ejaculate is very well suited to proteomics studies. As such, research concerning sperm proteomics is offering a huge amount of new information on the biology of spermatozoa. Among domestic animals, horses represent a species of special interest, in which reproductive technologies and a sizeable market of genetic material have grown exponentially in the last decade. Studies using proteomic approaches have been conducted in recent years, showing that proteomics is a potent tool to dig into the biology of the stallion spermatozoa. The aim of this review is to present an overview of...
Obesity and gestation in Criollo mares: endocrine and metabolic profiles.
Domestic animal endocrinology    May 28, 2024   Volume 89 106857 doi: 10.1016/j.domaniend.2024.106857
Pardié M, Kalpokas I, Freccero F, Castagnetti C, Meikle A.The aim of this study was to determine the effect of body condition score (BCS) on metabolic and endocrine parameters in pregnant Criollo mares (n=41), which were categorized according to their BCS as obese (7 to 9 BCS, n=26) or normal (5 to 7, n=15). Blood samples were taken during gestation in 3 periods: between 3.5 and 5 months (I), 8 and 9 months (II) and in the last month of gestation (III). The data was analyzed in the statistical model by mixed procedures, including BCS, gestational period and their interaction as fixed effects. BCS was only different in period I, as normal mares increa...
ECEIM consensus statement on equine kidney disease.
Journal of veterinary internal medicine    May 27, 2024   doi: 10.1111/jvim.17101
van Galen G, Divers TJ, Savage V, Schott HC, Siwinska N.The aim of this consensus statement is to summarize and appraise scientific evidence and combine this with the clinical experience of a panel of experts to optimize recommendations on how to recognize and manage kidney disease in horses.
Monitoring of inflammatory blood biomarkers in foals with Rhodococcus Equi pneumonia during antimicrobial treatment.
Journal of equine veterinary science    May 24, 2024   Volume 138 105103 doi: 10.1016/j.jevs.2024.105103
Deniz Ö, Ekinci G, Onmaz AC, Derelli FM, Fazio F, Aragona F, Hoven RVD.Rhodococcus equi (R. equi), a gram-positive facultative intracellular pathogen, is a common cause of pneumonia in foals and represents a major cause of disease and death. The aim of the present study was to investigate the time-depended changes in White Blood Cells (WBC), basophils (Baso), neutrophils (Neu), lymphocytes (Lymf), monocytes (Mon), eosinophils (Eos), platelet (PLT) counts, fibrinogen (Fbg) concentration, interferon (IFN-α, IFN-γ) and interleukins (IL-2 and IL-10) in foals with clinical R. equi pneumonia. The main treatment was with azithromycin-rifampicin for 14 days. Blood was ...
Evaluation of gastrointestinal tract lesions and serum malondialdehyde levels after repeated oral administration of phenylbutazone in horses.
Veterinary research communications    May 21, 2024   Volume 48, Issue 4 2343-2355 doi: 10.1007/s11259-024-10415-y
Tesena P, Vinijkumthorn R, Preuksathaporn T, Piyakul P, Chotikaprakal T, Sirireugwipas R, Wong-Aree K, Prapaiwan N.Phenylbutazone (PBZ) is a widely used nonsteroidal anti-inflammatory drug for horses. However, because of its gastrointestinal side effects, its administration requires careful attention in veterinary practice. Malondialdehyde (MDA) is a serum biomarker associated with increased damage to the equine gastrointestinal system. This study investigated the hematological effects and alterations in the gastrointestinal tract and assessed serum MDA concentrations following repeated oral PBZ administration at clinical doses. Fourteen horses were randomly divided into control and treatment groups. All h...
Biomarkers of in vivo platelet activation in thoroughbreds during their first long-term training.
Frontiers in veterinary science    May 20, 2024   Volume 11 1395423 doi: 10.3389/fvets.2024.1395423
Miglio A, Falcinelli E, Mezzasoma AM, Busechian S, Rueca F, Gresele P, Antognoni MT.Physical exercise has an activating effect on platelet function that differs between trained and untrained subjects, depending on the type of exercise and training status. In humans, soluble P-selectin (sP-sel) and platelet-derived extracellular vesicles (PEVs) are considered reliable markers of platelet activation during exercise. In untrained humans, they increase after transient physical exercise, whereas long-term training induces a decrease in their resting levels due to an improved ability to adapt to hemodynamic changes. The aim of this study was to assess whether circulating levels of...
Biomolecular evidence reveals mares and long-distance imported horses sacrificed by the last pagans in temperate Europe.
Science advances    May 17, 2024   Volume 10, Issue 20 eado3529 doi: 10.1126/sciadv.ado3529
French KM, Musiał AD, Karczewski M, Daugnora L, Shiroukhov R, Ropka-Molik K, Baranowski T, Bertašius M, Skvortsov K, Szymański P....Horse sacrifice and deposition are enigmatic features of funerary rituals identified across prehistoric Europe that persisted in the eastern Baltic. Genetic and isotopic analysis of horses in Balt cemeteries [1st to 13th centuries CE (Common Era)] dismantle prevailing narratives that locally procured stallions were exclusively selected. Strontium isotope analysis provides direct evidence for long-distance (~300 to 1500 kilometers) maritime transport of Fennoscandian horses to the eastern Baltic in the Late Viking Age (11th to 13th centuries CE). Genetic analysis proves that horses of both sexe...
Salivary analysis to unveil the paradigma of stress of domestic horses reared in the wild.
Scientific reports    May 17, 2024   Volume 14, Issue 1 11266 doi: 10.1038/s41598-024-62172-2
Bazzano M, Marchegiani A, La Gualana F, Petriti B, Spaterna A, Laus F.Horse welfare is the product of multiple factors, including behavioral and physiological adjustments to cope with stressful situation regarding environment and housing condition. Collectively, it is supposed that a horse kept in the wild has a lower level of stress than other housing system, and the aim of the present study was to investigate the level of stress in domestic horses reared in the wild and then moved to human controlled housing, through saliva analysis. Twelve clinically healthy Catria (Italian local breed) mares, usually reared in the wild, were moved into collective paddocks fo...
The use of genomic assisted selection could allow early selection among Thoroughbreds and Polo Argentino horses for practicing polo.
Journal of equine veterinary science    May 17, 2024   105098 doi: 10.1016/j.jevs.2024.105098
Azcona F, Karlau A, Trigo P, Molina A, Demyda-Peyrás S.The Polo Argentino (PA) horse is a recognized breed, developed originally by mixing crossbred and Thoroughbred (TB) horses to play polo. Early PA selection is difficult due to unreliable performance estimations. This study investigated the usefulness of genomic markers previously linked to morphological and functional traits as a tool for the early selection of PA. To this, we genotyped 520 PA and 30 TB horses using the Equine GGPArray (Illumina, n=71,778 SNPs). Analyses included a genetic characterization of six genetic markers associated with behavioral (DRD4), muscular development (MSTN), a...
Current insights into equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy.
Veterinary journal (London, England : 1997)    May 10, 2024   Volume 305 106129 doi: 10.1016/j.tvjl.2024.106129
Young AE, Finno CJ.Equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM) is an inherited neurodegenerative disease associated with vitamin E deficiency in the first year of life. It is the second most common cause of spinal ataxia in horses euthanized for neurologic disease. Equine NAD/EDM is characterized by neurologic signs including a symmetric proprioceptive ataxia (> grade 2/5) and a wide-base stance at rest. There are currently no antemortem tests for eNAD/EDM in any breed. Conclusive diagnosis requires postmortem histologic evaluation of the brainstem and spinal cord at necropsy....
Immunology of Physical Exercise: Is Equus caballus an Appropriate Animal Model for Human Athletes?
International journal of molecular sciences    May 10, 2024   Volume 25, Issue 10 5210 doi: 10.3390/ijms25105210
Witkowska-Piłaszewicz O, Malin K, Dąbrowska I, Grzędzicka J, Ostaszewski P, Carter C.Domestic horses routinely participate in vigorous and various athletic activities. This enables the horse to serve as a model for studying athletic physiology and immunology in other species, including humans. For instance, as a model of physical efforts, such as endurance rides (long-distance running/aerobic exercise) and races (anaerobic exercise), the horse can be useful in evaluating post-exercise response. Currently, there has been significant interest in finding biomarkers, which characterize the advancement of training and adaptation to physical exercise in the horse. The parallels in c...
Nuclear Magnetic Resonance (NMR) Metabolomics: Current Applications in Equine Health Assessment.
Metabolites    May 7, 2024   Volume 14, Issue 5 269 doi: 10.3390/metabo14050269
Laus F, Bazzano M, Spaterna A, Laghi L, Marchegiani A.Metabolomics can allow for the comprehensive identification of metabolites within biological systems, at given time points, in physiological and pathological conditions. In the last few years, metabolomic analysis has gained popularity both in human and in veterinary medicine, showing great potential for novel applications in clinical activity. The aim of applying metabolomics in clinical practice is understanding the mechanisms underlying pathological conditions and the influence of certain stimuli (i.e., drugs, nutrition, exercise) on body systems, in the attempt of identifying biomarkers th...
Single-Cell Transcriptome Sequencing Reveals Molecular Expression Differences and Marker Genes in Testes during the Sexual Maturation of Mongolian Horses.
Animals : an open access journal from MDPI    April 23, 2024   Volume 14, Issue 9 doi: 10.3390/ani14091258
Liu Y, Du M, Li X, Zhang L, Zhao B, Wang N, Dugarjaviin M.This study aimed to investigate differences in testicular tissue morphology, gene expression, and marker genes between sexually immature (1-year-old) and sexually mature (10-year-old) Mongolian horses. The purposes of our research were to provide insights into the reproductive physiology of male Mongolian horses and to identify potential markers for sexual maturity. The methods we applied included the transcriptomic profiling of testicular cells using single-cell sequencing techniques. Our results revealed significant differences in tissue morphology and gene expression patterns between the tw...
Selecting potential biomarkers of plasma proteins in mares with endometritis.
Equine veterinary journal    April 14, 2024   doi: 10.1111/evj.14092
Zhang X, Mai Z, Gao Y, Zhao X, Zhang Y.Endometritis is a common condition in mares that causes significant economic loss. Lacking obvious clinical signs, the clinical diagnosis of endometritis in mares relies on case-by-case clinical examinations, which can be particularly inefficient in large-scale farms. Therefore, the identification of potential biomarkers can serve as a non-invasive and efficient screening technique for endometritis in mares. Objective: To compare the blood proteome between fertile mares and mares with endometritis to identify biomarkers potentially associated with the development of endometritis and validate t...
Plasma and synovial fluid extracellular vesicles display altered microRNA profiles in horses with naturally occurring post-traumatic osteoarthritis: an exploratory study.
Journal of the American Veterinary Medical Association    April 13, 2024   1-12 doi: 10.2460/javma.24.02.0102
Connard SS, Gaesser AM, Clarke EJ, Linardi RL, Even KM, Engiles JB, Koch DW, Peffers MJ, Ortved KF.The objective of this study was to characterize extracellular vesicles (EVs) in plasma and synovial fluid obtained from horses with and without naturally occurring post-traumatic osteoarthritis (PTOA). Methods: EVs were isolated from plasma and synovial fluid from horses with (n = 6) and without (n = 6) PTOA. Methods: Plasma and synovial fluid EVs were characterized with respect to quantity, size, and surface markers. Small RNA sequencing was performed, and differentially expressed microRNAs (miRNAs) underwent bioinformatic analysis to identify putative targets and to explore potential associa...
Serial injections of 4% polyacrylamide hydrogel have no detrimental effects in equine joints following clinical, histologic, and synovial biomarker evaluation.
American journal of veterinary research    March 23, 2024   1-7 doi: 10.2460/ajvr.24.01.0016
McClure SR, Peitzmeier MD, Jackman BR, Evans RB, Ziegler CL, Ganta CK.Polyacrylamide hydrogel (4% PAHG) is an inert viscoelastic supplement used to manage osteoarthritis in horses. Even with a prolonged clinical effect, horses may be administered multiple doses during their performance career. The effect of the serial 4% PAHG treatments is not known. The objectives of this study were to evaluate the clinical, histologic, and synovial fluid biomarker effects following serial administration of 4% PAHG in normal equine fetlock joints. Methods: 8 healthy horses. Methods: In a blinded, controlled in vivo study, horses received serial intra-articular injections of 4% ...
Analysis of the season-dependent component in the evaluation of morphological and biochemical blood parameters in Shetland ponies of both sexes during exercise.
Journal of veterinary research    March 23, 2024   Volume 68, Issue 1 155-166 doi: 10.2478/jvetres-2024-0017
Determination of morphological and biochemical blood indices facilitates assessment of the health and welfare of horses, their nutrient demand, the effects of training already undertaken, and the horses' suitability for exercise. Identification of the season-dependent components and the effects of sex and exercise on changes in frequently referenced haematological and biochemical parameters was the main goal of the current study. Unassigned: The blood morphology of 21 healthy adult Shetland ponies (11 mares and 10 stallions) aged 6.5 ± 1.4 years from the central Pomeranian region in Poland wa...
Changes in the blood redox status of horses subjected to combat training.
Research in veterinary science    March 7, 2024   Volume 171 105219 doi: 10.1016/j.rvsc.2024.105219
Bujok J, Pavľak A, Walski T, Zigo F, Trochanowska-Pauk N, Bohara R, Miśta D.Combat training of police horses, involving physical activity in the presence of environmental stressors, poses a risk of oxidative stress. This study compared the oxidative imbalance after combat training in horses in the regular police service and in horses that had just been schooled. Blood collection was performed immediately after training and after 16 h rest. The activity of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), and total antioxidant status (TAS) were determined as the markers of enzymatic antioxidant defence. At the same time, lipid peroxidation (TBAR...
Equine Models of Temporomandibular Joint Osteoarthritis: A Review of Feasibility, Biomarkers, and Molecular Signaling.
Biomedicines    February 28, 2024   Volume 12, Issue 3 542 doi: 10.3390/biomedicines12030542
Jasiński T, Turek B, Kaczorowski M, Brehm W, Skierbiszewska K, Bonecka J, Domino M.Osteoarthritis (OA) of the temporomandibular joint (TMJ) occurs spontaneously in humans and various animal species, including horses. In humans, obtaining tissue samples is challenging and clinical symptoms appear late in the disease progression. Therefore, genetically modified, induced, and naturally occurring animal models play a crucial role in understanding the pathogenesis and evaluating potential therapeutic interventions for TMJ OA. Among the naturally occurring models, the equine TMJ OA model is characterized by slow, age-related progression, a wide range of clinical examinations, and ...
microRNAs are differentially expressed in equine plasma of horses with osteoarthritis and osteochondritis dissecans versus control horses.
PloS one    February 23, 2024   Volume 19, Issue 2 e0297303 doi: 10.1371/journal.pone.0297303
Antunes J, Salcedo-Jiménez R, Lively S, Potla P, Coté N, Dubois MS, Koenig J, Kapoor M, LaMarre J, Koch TG.Osteoarthritis (OA) is a leading cause of lameness in horses with no effective disease-modifying treatment and challenging early diagnosis. OA is considered a disease of the joint involving the articular cartilage, subchondral bone, synovial membrane, and ligaments. Osteochondritis dissecans (OCD) is a joint disease consisting of focal defects in the osteochondral unit which may progress to OA later in life. MicroRNAs (miRNAs) have been recognized as small non-coding RNAs that regulate a variety of biological processes and have been detected in biological fluids. MiRNAs are currently investiga...
The Welfare of Horses Competing in Three-Barrel Race Events Is Shown to Be Not Inhibited by Short Intervals between Starts.
Animals : an open access journal from MDPI    February 9, 2024   Volume 14, Issue 4 583 doi: 10.3390/ani14040583
Filho HCM, Trindade KLG, Silva CJFL, Cruz RKS, Vilela CF, Coelho CS, Filho JDR, Manso HECCC.Equestrian sports require precise animal welfare and health evaluations. To test the hypothesis that horses maintain their welfare when subjected to two three-barrel (3TB) races with 2 min intervals, an experiment was designed to evaluate their surface temperature using infrared thermography (IRT) in regions of interest (barrel, flank, neck, jaw, corner of the mouth, and ocular caruncle) and also measure blood biomarkers (hemogram, total plasma protein, fibrinogen, urea, creatinine, GGT, CK, cortisol, IL-6, and IL-1β). Ten Quarter Horses were monitored through thermography (pre-race, +1, +4, ...
Freezing Stallion Semen-What Do We Need to Focus on for the Future?
Veterinary sciences    February 2, 2024   Volume 11, Issue 2 doi: 10.3390/vetsci11020065
Al-Kass Z, Morrell JM.Artificial insemination (AI) is used frequently in the breeding of sport horses, apart from Thoroughbreds. Most AIs are carried out with cooled semen rather than frozen semen because of the difficulties in identifying a protocol that is suitable for freezing most ejaculates and the necessity to inseminate close to ovulation because of the short life of the thawed spermatozoa. More widespread use of frozen semen would improve biosecurity, allow greater choice of stallions, and offer more flexibility when managing deliveries of semen to the stud. It would even decrease the amount of antibiotics ...
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