Impact of probiotic supplementation on the growth, health, and fecal microbiota of Argentine Holstein calves

Impact of probiotic supplementation on the growth, health, and fecal microbiota of Argentine Holstein calves | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Impact of probiotic supplementation on the growth, health, and fecal microbiota of Argentine Holstein calves María Laura Mon, María Jaureguiberry, Marcelo A. Soria, Fiorella Alvarado Pinedo, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6864752/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study aimed to evaluate the impact of probiotic administration on the growth, health status, and fecal microbiota of Argentine Holstein calves. Thirty calves from a commercial dairy farm were assigned to two groups. The treated group received a daily oral capsule containing four Lactobacillus strains (1x10 9 CFU), from 2 to 30 days of age, while the control group received a placebo capsule following the same schedule. After colostrum feeding, all animals were provided with a milk replacer and formulated compound feed. Health status was monitored using the Wisconsin test to assess diarrhea, and body weight was measured at 2 and 60 days of age. Fecal microbiota was evaluated at 30 ± 3 days using 16S rRNA gene sequencing targeting the V3-V4 regions. No significant differences were observed between groups in terms of diarrhea and weight gain. Similarly, 16S rRNA gene sequencing revealed no notable differences in bacterial community structure between groups. The most abundant family identified was Muribaculaceae; while dominant genera included Bacteroides , Prevotella , Collinsella , Alloprevotella , and Lactobacillus. The probiotic treatment proposed had no effect on diarrhea incidence or growth performance but caused no harmful effects either. Animal Science Probiotics Lactobacillus Holstein calves 16S rRNA sequencing Figures Figure 1 Introduction The gut microbiota is crucial in the health and development of calves (Du et al., 2023 ). Previous studies have associated the incorrect use of antimicrobials, including antibiotics, with increased antimicrobial resistance and disruption of the gut microbiota, negatively affecting animal health and growth (Kim et al., 2021 ). In response, different regulatory measures, such as the Veterinary Medicinal Products, have been implemented (Schmerold et al., 2023 ). As an alternative to antimicrobials, researchers have proposed the use of probiotics to help reduce antimicrobial resistance (Jessop et al., 2024 ). When administered in sufficient quantities, probiotics confer health benefits, particularly in preweaning animals (Du et al., 2023 ). Lactic acid bacteria (LAB), known for their probiotic properties, stimulate the immune system, support intestinal microbiota, and reduce diarrhea in production animals (Fan et al., 2021 ; Wu et al., 2021 ). Our research group has previously shown that a probiotic formulation containing Lactobacillus johnsonii CRL 1693, L. murinus CRL 1695, L. mucosae CRL 1696, and L. salivarius CRL 1702, administered as fermented milk, improved calf performance (Maldonado et al., 2018 ). To simplify the treatment protocol, this study aimed to evaluate the impact of the same probiotic formulation, but administered as capsules and during a shorter time, on the health, growth, and fecal microbiota of preweaning dairy calves. Materials and methods Preparation of microcapsules with multispecies The probiotic strains used to prepare the capsules at CERELA were previously isolated from calf feces (Maldonado et al., 2018 ). The strains were freeze-dried and encapsulated with 1x109 CFU, as described previously (Marchesi et al., 2020 ). Animals and experimental design Thirty Holstein female newborn calves from a commercial dairy farm (Buenos Aires, Argentina) were randomly assigned to treated and control groups (n = 15 each). Within 8 h of birth, calves ingested 3 L of colostrum. They were kept individually outdoors with 2-meter-long neck chains which restricted movement. A milk replacer (Teknal S.A., 2 L) was provided twice daily in individual buckets. Additionally, 200 g of calf starter (Teknal S.A.) was provided once daily along with fresh water. The amount was increased weekly until 2 kg per day on the weaning date. Blood samples were collected from the jugular vein into sterile tubes at 1–4 days of age and stored in a cooling container until analysis within 6 h (Centro de Diagnóstico e Investigaciones Veterinarias, Universidad Nacional de la Plata (UNLP), Argentina). Total serum protein concentration was determined by manual refractometry (REF_CLI_8107; Alla France, Chemillé-en-Anjou, France). The cutoff point used as indicative of failure of passive transfer (FPT) was < 5.5 g/dL (Elsohaby et al., 2015 ). Treated calves received one probiotic capsule per day orally from 1 to 30 days of age, while controls received placebo capsules in a double-blind randomized trial. Calves were monitored daily for diarrhea using a 4-point fecal scale (0 = normal, 1 = semi-formed, 2 = loose, and 3 = watery), considering a fecal score ≥ 2 as positive. Weight was estimated at 2 (SD ± 1.7) and 60 (SD ± 2.1) days of age, using a heart-girth measuring tape around the thorax, caudal to the forelimb, with the calf standing in a neutral position. Fecal samples from all animals were collected at 30 ± 3 days of age directly from the rectum into sterile tubes. Samples were transported on ice to the laboratory and stored at -20ºC until processing. The experimental protocol was approved by the Institutional Animal Care and Use of Experimentation Animals Committee of the UNLP (Nº 230831-2). DNA extraction Total DNA from fecal samples was extracted using the QIAamp PowerFecal Pro DNA Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions with some modifications: two cycles at 70ºC for 5 min prior a bead-beating step of 2 cycles of 5 min at 26 hertz (TissueLyser II, Qiagen, Chadstone, Australia). The extracted DNA was checked on 1% agarose gel, and DNA concentration and purity were determined using a NanoDrop 2000 UV − vis spectrophotometer (Thermo Scientific, Wilmington, USA) and stored (-80°C) until further processing. The V3-V4 hypervariable regions of the 16S rRNA gene were amplified using primers 341F (CCTAYGGGRBGCASCAG) and 806R (GGACTACNNGGGTATCTAAT) (Caporaso et al., 2011 ; Klindworth et al., 2013 ). Sequencing was performed on an Illumina NovaSeq PE250 at Novogene (Sacramento, CA, USA). Sequence analysis Sequence quality was checked using FastQC software ( https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ). The processing and analysis of the data continued with QIIME2 (Caporaso et al., 2010 ). The paired-end sequences were assembled, denoised and consolidated into amplicon sequence variants (ASVs) using the DADA2 algorithm (Callahan et al., 2016 ). ASVs were then taxonomic assigned using the "Naive Bayes" method with the SILVA 132 reference database. ASV-based abundance tables and taxonomic entity tables were constructed and used to perform beta diversity analyses by calculating Unifrac and Bray-Curtis distance matrices. Sequences were deposited in the NCBI Sequence Read Archive (BioProject accession number PRJNA1244342). Statistical Analysis Descriptive and statistical analyses were performed with R version 4.2.0 (R Core Team, 2024 ). Data normality was verified with the Shapiro-Wilk test. The average daily gain (ADG) was calculated to evaluate growth performance. The effect of the probiotic treatment on diarrhea occurrence and growth performance was evaluated with Chi-squared and t-Student tests. Also, each daily fecal score was summarized to calculate the number of days having a specific score. The median of days having a specific score was compared between groups (i.e., number of days having score 0 in control calves vs. number of days having score 0 in treated calves) using the Wilcoxon test. Results Clinical parameters The total FPT of calves was 46.67% (7/15) in both groups. Diarrhea occurrence was 60% (9/15) in treated calves and 40% (6/15) in control ones. The ADG was 0.65 kg in treated calves and 0.68 kg in control ones. The probiotic treatment administered did not affect the occurrence of diarrhea, the number of days of a specific fecal score or the growth performance (Table 1 ). Table 1 Effect of probiotic treatment on body weight, average daily gain (ADG), and fecal score, in newborn calves (n = 30) Items Treatment P-value Control a Treatment b Initial BW, kg 37.21 36.64 0.61 Final BW, kg 76.36 74.36 0.52 ADG, kg/day 0.68 0.65 0.57 Total days of fecal scores Fecal score = 0 53 52 0.13 Fecal score = 1 5.5 5.5 0.96 Fecal score = 2 0 1 0.57 Fecal score = 3 0 0 1 Medians with different superscript letters in the same row differ significantly (P < 0.05), medians no superscript letter in the same row are not significantly different (P ≥ 0.05). a control group. b treatment group. Fecal microbiota Microbiota analysis revealed no significant differences in community structure between groups, as determined by distance matrix calculations using UniFrac and Bray-Curtis analysis and PCoA visualizations (Fig. S1). In both groups, the dominant phylum was Bacteroidota (̴50%), followed by Firmicutes (̴30%), Actinobacteriota (̴10%) and Proteobacteria (̴4%). These phyla accounted for approximately 94% of the total microbiota (Fig. 1 a). At the family level, the dominant taxa in both groups were: Muribaculaceae, Prevotellaceae, Bacteoroidaceae, and Lachnospiraceae followed by Coriobacteriaceae, Erysipelatoclostridiaceae, Lactobacillaceae, Oscillospiraceae, Rikenellaceae, Bifidobacteriaceae, Succinivibrionaceae, Selenomonadaceae, Veillonellaceae and Tannerellaceae (Fig. 1 b). However, treated calves showed lower abundance of Prevotellaceae and higher abundance of Coriobacteriaceae compared to controls (Fig. 1 b). At the genus level, Bacteroides , Prevotella , Colinsella , Aloprevotella and Lactobacillus were the most abundant, with higher quantities, although not significant, of Lactobacillus in treated calves, which was expected since they were administered with four Lactobacillus strains (Fig. 1 c). However, no major structural changes in microbiota composition were observed between groups. Discussion Prevotellaceae, typically associated with carbohydrate degradation and normal digestive function, was reduced in treated calves, whereas Coriobacteriaceae and Collinsella, involved in carbohydrate metabolism and bile acid modification, were more abundant (Wang et al., 2019 ; Wu et al., 2021 ). Different studies have proposed probiotic treatment to improve health and growth performance in newborn calves and as an alternative to reduce antimicrobial usage in animal production systems (Antanaitis et al., 2024 ; Wang et al., 2022 ; Wu et al., 2021 ). Also, researchers have evaluated the use of probiotics to reduce the prevalence of diarrhea, one of the most prevalent diseases during the calf-rearing period, and antimicrobial usage, with encouraging results. Wang et al. ( 2022 ) reported that dietary supplementation with compound probiotics improved calf health, improving fecal score and reducing medical treatments. Similarly, Wu et al. ( 2021 ) showed that the supplementation of probiotic strains, including L. acidophilus , Bacillus subtilis , and Saccharomyces cerevisiae in milk for eight weeks also reduced diarrhea incidence, increased the ADG, and enhanced immune indexes. However, Signorini et al. ( 2012 ) observed that the positive effects of LAB supplementation are more pronounced in farms with high morbidity and mortality rates, particularly when these are primarily associated with enteric pathogens. Antanaitis et al. ( 2024 ) evaluated the use of a B. subtilis probiotic solution in a milk replacer over 30 days in calves and found no differences in fecal scores but found a beneficial effect on growth performance and metabolic status. In our study, probiotic administration via capsules for 30 days did not significantly improve health or growth, despite some differences reported in earlier studies (Maldonado et al., 2018 ). This aligns with findings by Karamzadeh-Dehaghani et al. ( 2021 ), who supplemented calves with a commercial probiotic blend for 28 days without benefits on diarrhea or growth. These inconsistencies could be explained by differences in probiotic strains, doses, or delivery methods. It is important to emphasize the need for in vivo trials using specific hosts, to provide substantiation of in vitro effects and determination of probiotic mechanism for microorganisms selected based on their in vitro properties under laboratory conditions (FAO/WHO, 2002 ). The great variability in treatment protocols makes it difficult to compare and interpret results. A previous study using the same probiotic strains but administered as fermented milk instead of capsules for 60 days showed positive effects on health and growth (Maldonado et al., 2018 ). This suggests that factors such as treatment duration or mode of administration may play a crucial role in probiotic efficacy. Moreover, FPT and diarrhea prevalence in this study suggest suboptimal calf-rearing practices, potentially overshadowing probiotic effects. It is also worth noting that the milk replacer used contained two Bacillus strains, which could explain the lack of observed differences, as a probiotic was already being administered. Although the composition of the capsules was different, this may have masked the effect of our probiotic. In conclusion, while probiotic administration influenced certain bacterial groups, it did not significantly affect calf health or growth under the conditions assayed. Future studies should refine treatment parameters including dosage, duration, and delivery method to optimize probiotic efficacy in preweaning calves. Declarations Acknowledgments MLM, MJ, NM, FNM and PMT are members of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) Argentina. Funding source This study was supported by grants from the Instituto Nacional de Tecnología Agropecuaria (INTA) (PI 089 and PI 102), the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) Proyectos de Investigación Científica y Tecnológica (PICT) 2019#3156. Competing Interest The authors have no relevant financial or non-financial interests to disclose. Author contributions MLM and MJ performed the experiments, analyzed the data, prepared figures and tables, contributed to manuscript writing, authored or reviewed drafts of the paper, and approved the final draft. MAS analyzed the data, prepared figures, contributed to manuscript writing, and approved the final draft. FAP and GT contributed to the experiments at the dairy farm, authored or reviewed drafts of the paper, and approved the final draft. NM and FNM contributed to the probiotic’s capsules and the design of the experiment at the dairy farm, contributed to manuscript writing, authored or reviewed drafts of the paper, and approved the final draft. PMT conceived, designed, and performed the experiments, analyzed the data, prepared figures and/or tables, was responsible for writing the manuscript, authored and reviewed drafts of the paper, and approved the final draft. Ethics approval Ethical approval was obtained by the Institutional Animal Care and Use of Experimentation Animals Committee of the Universidad Nacional de la Plata (Nº 230831-2). References Antanaitis R, Džermeikaitė K, Krištolaitytė J, Armonavičiūtė E, Arlauskaitė S, Girdauskaitė A, Rutkauskas A, Baumgartner W (2024) Effects of Bacillus subtilis on growth performance, metabolic profile, and health status in dairy calves. 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Universidad Nacional de la Plata, Chascomús, Buenos Aires, Argentina","correspondingAuthor":false,"prefix":"","firstName":"Fiorella","middleName":"Alvarado","lastName":"Pinedo","suffix":""},{"id":469340434,"identity":"99905fd8-a9ce-4dc3-8e5b-7ab7ca0c10ac","order_by":4,"name":"Gabriel Travería","email":"","orcid":"https://orcid.org/0000-0001-7840-074X","institution":"Centro de Diagnóstico e Investigaciones Veterinarias (CEDIVE). Universidad Nacional de la Plata, Chascomús, Buenos Aires, Argentina","correspondingAuthor":false,"prefix":"","firstName":"Gabriel","middleName":"","lastName":"Travería","suffix":""},{"id":469340435,"identity":"25bb199e-e694-48cb-966c-fcc04a988845","order_by":5,"name":"Natalia Maldonado","email":"","orcid":"https://orcid.org/0009-0002-4405-4986","institution":"Centro de Referencia para Lactobacilos (CERELA-CONICET), San Miguel de Tucumán, Tucumán, Argentina","correspondingAuthor":false,"prefix":"","firstName":"Natalia","middleName":"","lastName":"Maldonado","suffix":""},{"id":469340436,"identity":"29721303-94d7-495c-8c14-0c7491b88d99","order_by":6,"name":"Fátima Nader Macías","email":"","orcid":"https://orcid.org/0000-0001-7526-1860","institution":"Centro de Referencia para Lactobacilos (CERELA-CONICET), San Miguel de Tucumán, Tucumán, Argentina","correspondingAuthor":false,"prefix":"","firstName":"Fátima","middleName":"Nader","lastName":"Macías","suffix":""},{"id":469340437,"identity":"0d6ccc00-eb94-423e-bd1b-00e4f934f61f","order_by":7,"name":"Paola M. Talia","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIie3OoWoDMRjA8S8EEhOIjRjrK6QMzpV7lQsnagc1FRMZhcyUna7uC1zdZI7AqT5ARRl35tTEasoGZSzsCpvJrXKw/E1C+H58AYjF/mRY26+TAmqy/okMAgboTDBgmYG8jJy3ARFwCUlpdW/fYD/iGHfz9uk0Ar6uG7h7Dm9hSldL6MarBUl2aivHWnRTCfVs4GNKWwYOlQ48MRJpsU0EkCxMeKurE7i0dPR460nak48BIpR2fosqHUvAE6X5MhHIDJBdq92V7PLVgs2EMje5EWQq1WOY0CJ3h5f5flLQh83h3VxPCu7q5vUYJn3Sft+J8NO/Ad8PAtwGhmKxWOy/9gkma1T0aI4xvAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-2877-8271","institution":"Instituto de Agrobiotecnología y Biología Molecular (IABIMO), Instituto Nacional de Tecnología Agropecuaria (INTA), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Hurlingham, Buenos Aires, Argentina","correspondingAuthor":true,"prefix":"","firstName":"Paola","middleName":"M.","lastName":"Talia","suffix":""}],"badges":[],"createdAt":"2025-06-10 15:45:39","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6864752/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6864752/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84388759,"identity":"5627540e-6b60-4d11-9dfe-c673a859ac4b","added_by":"auto","created_at":"2025-06-11 10:53:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":346935,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAmplicon sequence variants\u003c/strong\u003e \u003cstrong\u003erelative abundance at phylum level (a), family level (b) and genus level (c) in control and treated calves.\u003c/strong\u003e The percentages of relative abundance of the most representative phyla, families and genera in both groups are plotted.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6864752/v1/09a6fed1e831435d54dd0f7a.png"},{"id":84390022,"identity":"61a0c005-df4e-4cef-bba6-b00e4f40c347","added_by":"auto","created_at":"2025-06-11 11:09:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":956750,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6864752/v1/64a548b1-d84f-41bf-bfc4-a29caf4a34a9.pdf"},{"id":84388766,"identity":"b9742f4e-88e2-43f8-a1e2-63edc52ac279","added_by":"auto","created_at":"2025-06-11 10:53:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":68330,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6864752/v1/aeaaaa8fcddc8eeffcb07cab.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eImpact of probiotic supplementation on the growth, health, and fecal microbiota of Argentine Holstein calves\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe gut microbiota is crucial in the health and development of calves (Du et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Previous studies have associated the incorrect use of antimicrobials, including antibiotics, with increased antimicrobial resistance and disruption of the gut microbiota, negatively affecting animal health and growth (Kim et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In response, different regulatory measures, such as the Veterinary Medicinal Products, have been implemented (Schmerold et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs an alternative to antimicrobials, researchers have proposed the use of probiotics to help reduce antimicrobial resistance (Jessop et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). When administered in sufficient quantities, probiotics confer health benefits, particularly in preweaning animals (Du et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Lactic acid bacteria (LAB), known for their probiotic properties, stimulate the immune system, support intestinal microbiota, and reduce diarrhea in production animals (Fan et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur research group has previously shown that a probiotic formulation containing \u003cem\u003eLactobacillus johnsonii\u003c/em\u003e CRL 1693, \u003cem\u003eL. murinus\u003c/em\u003e CRL 1695, \u003cem\u003eL. mucosae\u003c/em\u003e CRL 1696, and \u003cem\u003eL. salivarius\u003c/em\u003e CRL 1702, administered as fermented milk, improved calf performance (Maldonado et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). To simplify the treatment protocol, this study aimed to evaluate the impact of the same probiotic formulation, but administered as capsules and during a shorter time, on the health, growth, and fecal microbiota of preweaning dairy calves.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of microcapsules with multispecies\u003c/h2\u003e \u003cp\u003eThe probiotic strains used to prepare the capsules at CERELA were previously isolated from calf feces (Maldonado et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The strains were freeze-dried and encapsulated with 1x109 CFU, as described previously (Marchesi et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimals and experimental design\u003c/h3\u003e\n\u003cp\u003eThirty Holstein female newborn calves from a commercial dairy farm (Buenos Aires, Argentina) were randomly assigned to treated and control groups (n\u0026thinsp;=\u0026thinsp;15 each). Within 8 h of birth, calves ingested 3 L of colostrum. They were kept individually outdoors with 2-meter-long neck chains which restricted movement. A milk replacer (Teknal S.A., 2 L) was provided twice daily in individual buckets. Additionally, 200 g of calf starter (Teknal S.A.) was provided once daily along with fresh water. The amount was increased weekly until 2 kg per day on the weaning date.\u003c/p\u003e \u003cp\u003eBlood samples were collected from the jugular vein into sterile tubes at 1\u0026ndash;4 days of age and stored in a cooling container until analysis within 6 h (Centro de Diagn\u0026oacute;stico e Investigaciones Veterinarias, Universidad Nacional de la Plata (UNLP), Argentina). Total serum protein concentration was determined by manual refractometry (REF_CLI_8107; Alla France, Chemill\u0026eacute;-en-Anjou, France). The cutoff point used as indicative of failure of passive transfer (FPT) was \u0026lt;\u0026thinsp;5.5 g/dL (Elsohaby et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTreated calves received one probiotic capsule per day orally from 1 to 30 days of age, while controls received placebo capsules in a double-blind randomized trial. Calves were monitored daily for diarrhea using a 4-point fecal scale (0\u0026thinsp;=\u0026thinsp;normal, 1\u0026thinsp;=\u0026thinsp;semi-formed, 2\u0026thinsp;=\u0026thinsp;loose, and 3\u0026thinsp;=\u0026thinsp;watery), considering a fecal score\u0026thinsp;\u0026ge;\u0026thinsp;2 as positive. Weight was estimated at 2 (SD\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7) and 60 (SD\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1) days of age, using a heart-girth measuring tape around the thorax, caudal to the forelimb, with the calf standing in a neutral position. Fecal samples from all animals were collected at 30\u0026thinsp;\u0026plusmn;\u0026thinsp;3 days of age directly from the rectum into sterile tubes. Samples were transported on ice to the laboratory and stored at -20\u0026ordm;C until processing.\u003c/p\u003e \u003cp\u003e The experimental protocol was approved by the Institutional Animal Care and Use of Experimentation Animals Committee of the UNLP (N\u0026ordm; 230831-2).\u003c/p\u003e\n\u003ch3\u003eDNA extraction\u003c/h3\u003e\n\u003cp\u003eTotal DNA from fecal samples was extracted using the QIAamp PowerFecal Pro DNA Kit (Qiagen, Hilden, Germany) according to the manufacturer\u0026rsquo;s instructions with some modifications: two cycles at 70\u0026ordm;C for 5 min prior a bead-beating step of 2 cycles of 5 min at 26 hertz (TissueLyser II, Qiagen, Chadstone, Australia). The extracted DNA was checked on 1% agarose gel, and DNA concentration and purity were determined using a NanoDrop 2000 UV\u0026thinsp;\u0026minus;\u0026thinsp;vis spectrophotometer (Thermo Scientific, Wilmington, USA) and stored (-80\u0026deg;C) until further processing. The V3-V4 hypervariable regions of the 16S rRNA gene were amplified using primers 341F (CCTAYGGGRBGCASCAG) and 806R (GGACTACNNGGGTATCTAAT) (Caporaso et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Klindworth et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Sequencing was performed on an Illumina NovaSeq PE250 at Novogene (Sacramento, CA, USA).\u003c/p\u003e\n\u003ch3\u003eSequence analysis\u003c/h3\u003e\n\u003cp\u003eSequence quality was checked using FastQC software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.babraham.ac.uk/projects/fastqc/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The processing and analysis of the data continued with QIIME2 (Caporaso et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The paired-end sequences were assembled, denoised and consolidated into amplicon sequence variants (ASVs) using the DADA2 algorithm (Callahan et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). ASVs were then taxonomic assigned using the \"Naive Bayes\" method with the SILVA 132 reference database. ASV-based abundance tables and taxonomic entity tables were constructed and used to perform beta diversity analyses by calculating Unifrac and Bray-Curtis distance matrices. Sequences were deposited in the NCBI Sequence Read Archive (BioProject accession number PRJNA1244342).\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eDescriptive and statistical analyses were performed with R version 4.2.0 (R Core Team, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Data normality was verified with the Shapiro-Wilk test. The average daily gain (ADG) was calculated to evaluate growth performance. The effect of the probiotic treatment on diarrhea occurrence and growth performance was evaluated with Chi-squared and t-Student tests. Also, each daily fecal score was summarized to calculate the number of days having a specific score. The median of days having a specific score was compared between groups (i.e., number of days having score 0 in control calves vs. number of days having score 0 in treated calves) using the Wilcoxon test.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eClinical parameters\u003c/h2\u003e \u003cp\u003eThe total FPT of calves was 46.67% (7/15) in both groups. Diarrhea occurrence was 60% (9/15) in treated calves and 40% (6/15) in control ones. The ADG was 0.65 kg in treated calves and 0.68 kg in control ones. The probiotic treatment administered did not affect the occurrence of diarrhea, the number of days of a specific fecal score or the growth performance (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of probiotic treatment on body weight, average daily gain (ADG), and fecal score, in newborn calves (n\u0026thinsp;=\u0026thinsp;30)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eItems\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTreatment\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInitial BW, kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e37.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFinal BW, kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e76.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e74.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eADG, kg/day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal days of fecal scores\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFecal score\u0026thinsp;=\u0026thinsp;0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFecal score\u0026thinsp;=\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFecal score\u0026thinsp;=\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFecal score\u0026thinsp;=\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMedians with different superscript letters in the same row differ significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), medians no superscript letter in the same row are not significantly different (P\u0026thinsp;\u0026ge;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003econtrol group. \u003csup\u003eb\u003c/sup\u003etreatment group.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFecal microbiota\u003c/h3\u003e\n\u003cp\u003eMicrobiota analysis revealed no significant differences in community structure between groups, as determined by distance matrix calculations using UniFrac and Bray-Curtis analysis and PCoA visualizations (Fig. S1). In both groups, the dominant phylum was Bacteroidota (̴50%), followed by Firmicutes (̴30%), Actinobacteriota (̴10%) and Proteobacteria (̴4%). These phyla accounted for approximately 94% of the total microbiota (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eAt the family level, the dominant taxa in both groups were: Muribaculaceae, Prevotellaceae, Bacteoroidaceae, and Lachnospiraceae followed by Coriobacteriaceae, Erysipelatoclostridiaceae, Lactobacillaceae, Oscillospiraceae, Rikenellaceae, Bifidobacteriaceae, Succinivibrionaceae, Selenomonadaceae, Veillonellaceae and Tannerellaceae (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). However, treated calves showed lower abundance of Prevotellaceae and higher abundance of Coriobacteriaceae compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). At the genus level, \u003cem\u003eBacteroides\u003c/em\u003e, \u003cem\u003ePrevotella\u003c/em\u003e, \u003cem\u003eColinsella\u003c/em\u003e, \u003cem\u003eAloprevotella\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e were the most abundant, with higher quantities, although not significant, of \u003cem\u003eLactobacillus\u003c/em\u003e in treated calves, which was expected since they were administered with four \u003cem\u003eLactobacillus\u003c/em\u003e strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). However, no major structural changes in microbiota composition were observed between groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrevotellaceae, typically associated with carbohydrate degradation and normal digestive function, was reduced in treated calves, whereas Coriobacteriaceae and Collinsella, involved in carbohydrate metabolism and bile acid modification, were more abundant (Wang et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDifferent studies have proposed probiotic treatment to improve health and growth performance in newborn calves and as an alternative to reduce antimicrobial usage in animal production systems (Antanaitis et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Also, researchers have evaluated the use of probiotics to reduce the prevalence of diarrhea, one of the most prevalent diseases during the calf-rearing period, and antimicrobial usage, with encouraging results. Wang et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported that dietary supplementation with compound probiotics improved calf health, improving fecal score and reducing medical treatments. Similarly, Wu et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) showed that the supplementation of probiotic strains, including \u003cem\u003eL. acidophilus\u003c/em\u003e, \u003cem\u003eBacillus subtilis\u003c/em\u003e, and \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e in milk for eight weeks also reduced diarrhea incidence, increased the ADG, and enhanced immune indexes. However, Signorini et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) observed that the positive effects of LAB supplementation are more pronounced in farms with high morbidity and mortality rates, particularly when these are primarily associated with enteric pathogens.\u003c/p\u003e \u003cp\u003eAntanaitis et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) evaluated the use of a \u003cem\u003eB. subtilis\u003c/em\u003e probiotic solution in a milk replacer over 30 days in calves and found no differences in fecal scores but found a beneficial effect on growth performance and metabolic status.\u003c/p\u003e \u003cp\u003eIn our study, probiotic administration via capsules for 30 days did not significantly improve health or growth, despite some differences reported in earlier studies (Maldonado et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This aligns with findings by Karamzadeh-Dehaghani et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), who supplemented calves with a commercial probiotic blend for 28 days without benefits on diarrhea or growth. These inconsistencies could be explained by differences in probiotic strains, doses, or delivery methods. It is important to emphasize the need for in vivo trials using specific hosts, to provide substantiation of in vitro effects and determination of probiotic mechanism for microorganisms selected based on their in vitro properties under laboratory conditions (FAO/WHO, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The great variability in treatment protocols makes it difficult to compare and interpret results. A previous study using the same probiotic strains but administered as fermented milk instead of capsules for 60 days showed positive effects on health and growth (Maldonado et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This suggests that factors such as treatment duration or mode of administration may play a crucial role in probiotic efficacy. Moreover, FPT and diarrhea prevalence in this study suggest suboptimal calf-rearing practices, potentially overshadowing probiotic effects.\u003c/p\u003e \u003cp\u003eIt is also worth noting that the milk replacer used contained two \u003cem\u003eBacillus\u003c/em\u003e strains, which could explain the lack of observed differences, as a probiotic was already being administered. Although the composition of the capsules was different, this may have masked the effect of our probiotic.\u003c/p\u003e \u003cp\u003eIn conclusion, while probiotic administration influenced certain bacterial groups, it did not significantly affect calf health or growth under the conditions assayed. Future studies should refine treatment parameters including dosage, duration, and delivery method to optimize probiotic efficacy in preweaning calves.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMLM, MJ, NM, FNM and PMT are members of the Consejo Nacional de Investigaciones Cient\u0026iacute;ficas y T\u0026eacute;cnicas (CONICET) Argentina.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFunding source\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from the Instituto Nacional de Tecnolog\u0026iacute;a Agropecuaria (INTA) (PI 089 and PI 102), the Agencia Nacional de Promoci\u0026oacute;n Cient\u0026iacute;fica y Tecnol\u0026oacute;gica (ANPCyT) Proyectos de Investigaci\u0026oacute;n Cient\u0026iacute;fica y Tecnol\u0026oacute;gica (PICT) 2019#3156.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMLM and MJ performed the experiments, analyzed the data, prepared figures and tables, contributed to manuscript writing, authored or reviewed drafts of the paper, and approved the final draft. MAS analyzed the data, prepared figures, contributed to manuscript writing, and approved the final draft. FAP and GT contributed to the experiments at the dairy farm, authored or reviewed drafts of the paper, and approved the final draft. NM and FNM contributed to the probiotic\u0026rsquo;s capsules and the design of the experiment at the dairy farm, contributed to manuscript writing, authored or reviewed drafts of the paper, and approved the final draft. PMT conceived, designed, and performed the experiments, analyzed the data, prepared figures and/or tables, was responsible for writing the manuscript, authored and reviewed drafts of the paper, and approved the final draft.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval was obtained by the Institutional Animal Care and Use of Experimentation Animals Committee of the Universidad Nacional de la Plata (N\u0026ordm; 230831-2).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAntanaitis R, Džermeikaitė K, Kri\u0026scaron;tolaitytė J, Armonavičiūtė E, Arlauskaitė S, Girdauskaitė A, Rutkauskas A, Baumgartner W (2024) Effects of \u003cem\u003eBacillus subtilis\u003c/em\u003e on growth performance, metabolic profile, and health status in dairy calves. Animals 14, 2489. https://doi.org/10.3390/ani14172489\u003c/li\u003e\n\u003cli\u003eCallahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJ, Holmes SP (2016) DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods 13, 581-583. https://doi.org/10.1038/nmeth.3869\u003c/li\u003e\n\u003cli\u003eCaporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Pe\u0026ntilde;a AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld J, Knight R (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7, 35-336. https://doi.org/10.1038/nmeth.f.303\u003c/li\u003e\n\u003cli\u003eCaporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Lozupone CA, Turnbaugh PJ, Fierer N, Knight R (2011) Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Natl Acad Sci U S A 108 Suppl 1, 4516-4522. https://doi: 10.1073/pnas.1000080107\u003c/li\u003e\n\u003cli\u003eCore R, Team (2024) R: A language and environment for statistical computing. R Foundation for Statistical Computing,Vienna, Austria.\u003c/li\u003e\n\u003cli\u003eDu W, Wang X, Hu M, Hou J, Du Y, Si W, Yang L, Xu L, Xu Q (2023) Modulating gastrointestinal microbiota to alleviate diarrhea in calves. Front Microbiol 14, 1181545. https://doi: 10.3389/fmicb.2023.1181545\u003c/li\u003e\n\u003cli\u003eElsohaby I, Hou S, McClure JT, Riley C, Shaw RA, Keefe GP (2015) A rapid field test for the measurement of bovine serum immunoglobulin G using attenuated total reflectance infrared spectroscopy. BMC Vet Res 11, 218. https://doi.org/10.1186/s12917-015-0539-x\u003c/li\u003e\n\u003cli\u003eFan P, Kim M, Liu G, Zhai Y, Liu T, Driver JD, Jeong KC (2021) The Gut Microbiota of Newborn Calves and Influence of Potential Probiotics on Reducing Diarrheic Disease by Inhibition of Pathogen Colonization. Front Microbiol 12, 772863. https://doi: 10.3389/fmicb.2021.772863\u003c/li\u003e\n\u003cli\u003eFAO/WHO (2002) Guidelines for the Evaluation of Probiotics in Food, Canada.\u003c/li\u003e\n\u003cli\u003eJessop E, Li L, Renaud DL, Verbrugghe A, Macnicol J, Gamsj\u0026auml;ger L, Gomez DE (2024) Neonatal Calf Diarrhea and Gastrointestinal Microbiota: Etiologic Agents and Microbiota Manipulation for Treatment and Prevention of Diarrhea. Vet Sci 11, 108. https://doi.org/10.3390/vetsci11030108\u003c/li\u003e\n\u003cli\u003eKaramzadeh-Dehaghani A, Towhidi A, Zhandi M, Mojgani N, Fouladi-Nashta A (2021) Combined effect of probiotics and specific immunoglobulin Y directed against \u003cem\u003eEscherichia coli\u003c/em\u003e on growth performance, diarrhea incidence, and immune system in calves. Animal 15, 100124. https://doi.org/10.1016/j.animal.2020.100124\u003c/li\u003e\n\u003cli\u003eKim ET, Lee SJ, Kim TY, Lee HG, Atikur RM, Gu BH, Kim DH, Park BY, Son JK, Kim MH (2021) Dynamic Changes in Fecal Microbial Communities of Neonatal Dairy Calves by Aging and Diarrhea. Animals (Basel) 11,1113. https://doi.org/10.3390/ani11041113\u003c/li\u003e\n\u003cli\u003eKlindworth A, Pruesse E, Schweer T, Peplies J, Quast C, Horn M, Gl\u0026ouml;ckner FO (2013) Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res 41, e1. https://doi.org/10.1093/nar/gks808\u003c/li\u003e\n\u003cli\u003eMaldonado NC, Chiaraviglio J, Bru E, De Chazal L, Santos V, Nader-Macias MEF (2018) Effect of Milk Fermented with Lactic Acid Bacteria on Diarrheal Incidence, Growth Performance and Microbiological and Blood Profiles of Newborn Dairy Calves. Probiotics Antimicrob Proteins 10, 668-676. https://doi.org/10.1007/s12602-017-9308-4\u003c/li\u003e\n\u003cli\u003eMarchesi A, Silva JA, Wiese B, Nader-Mac\u0026iacute;as MEF (2020) Survival of Beneficial Vaginal Lactobacilli (BVL) to Different Gastrointestinal Tract Conditions. Curr Pharm Des 26, 3608-3618. https://doi.org/10.2174/1381612826666200218093607\u003c/li\u003e\n\u003cli\u003eSchmerold I, van Geijlswijk I, Gehring R (2023) European regulations on the use of antibiotics in veterinary medicine. Eur J Pharm Sci 189, 106473. https://doi.org/10.1016/j.ejps.2023.106473.\u003c/li\u003e\n\u003cli\u003eSignorini ML, Soto LP, Zbrun MV, Sequeira GJ, Rosmini MR, Frizzo LS (2012) Impact of probiotic administration on the health and fecal microbiota of young calves: a meta-analysis of randomized controlled trials of lactic acid bacteria. Res Vet Sci 93, 250-258. https://doi.org/10.1016/j.rvsc.2011.05.001\u003c/li\u003e\n\u003cli\u003eWang H, Yu Z, Gao Z, Li Q, Qiu X, Wu F, Guan T, Cao B, Su H (2022) Effects of compound probiotics on growth performance, rumen fermentation, blood parameters, and health status of neonatal Holstein calves. J Dairy Sci 105, 2190-2200. https://doi.org/0.3168/jds.2021-20721\u003c/li\u003e\n\u003cli\u003eWang L, Zhang G, Xu H, Xin H, Zhang Y (2019) Metagenomic Analyses of Microbial and Carbohydrate-Active Enzymes in the Rumen of Holstein Cows Fed Different Forage-to-Concentrate Ratios. Front Microbiol 10, 649. https://doi.org/10.3389/fmicb.2019.00649\u003c/li\u003e\n\u003cli\u003eWu Y, Wang L, Luo R, Chen H, Nie C, Niu J, Chen C, Xu Y, Li X, Zhang W (2021) Effect of a Multispecies Probiotic Mixture on the Growth and Incidence of Diarrhea, Immune Function, and Fecal Microbiota of Pre-weaning Dairy Calves. Front Microbiol 12, 681014-681014. https://doi.org/10.3389/fmicb.2021.681014\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Probiotics, Lactobacillus, Holstein calves, 16S rRNA sequencing","lastPublishedDoi":"10.21203/rs.3.rs-6864752/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6864752/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study aimed to evaluate the impact of probiotic administration on the growth, health status, and fecal microbiota of Argentine Holstein calves. Thirty calves from a commercial dairy farm were assigned to two groups. The treated group received a daily oral capsule containing four \u003cem\u003eLactobacillus\u003c/em\u003e strains (1x10\u003csup\u003e9\u003c/sup\u003e CFU), from 2 to 30 days of age, while the control group received a placebo capsule following the same schedule. After colostrum feeding, all animals were provided with a milk replacer and formulated compound feed. Health status was monitored using the Wisconsin test to assess diarrhea, and body weight was measured at 2 and 60 days of age. Fecal microbiota was evaluated at 30\u0026thinsp;\u0026plusmn;\u0026thinsp;3 days using 16S rRNA gene sequencing targeting the V3-V4 regions. No significant differences were observed between groups in terms of diarrhea and weight gain. Similarly, 16S rRNA gene sequencing revealed no notable differences in bacterial community structure between groups. The most abundant family identified was Muribaculaceae; while dominant genera included \u003cem\u003eBacteroides\u003c/em\u003e, \u003cem\u003ePrevotella\u003c/em\u003e, \u003cem\u003eCollinsella\u003c/em\u003e, \u003cem\u003eAlloprevotella\u003c/em\u003e, and \u003cem\u003eLactobacillus.\u003c/em\u003e The probiotic treatment proposed had no effect on diarrhea incidence or growth performance but caused no harmful effects either.\u003c/p\u003e","manuscriptTitle":"Impact of probiotic supplementation on the growth, health, and fecal microbiota of Argentine Holstein calves","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-11 10:53:00","doi":"10.21203/rs.3.rs-6864752/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9aa9574d-0414-4bf1-8360-fa8ea39fb8a9","owner":[],"postedDate":"June 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49831689,"name":"Animal Science"}],"tags":[],"updatedAt":"2025-06-11T10:53:00+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-11 10:53:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6864752","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6864752","identity":"rs-6864752","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}