{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T15:24:42Z","timestamp":1790954682253,"version":"4.1.0"},"reference-count":44,"publisher":"MDPI AG","issue":"18","license":[{"start":{"date-parts":[[2023,9,5]],"date-time":"2023-09-05T00:00:00Z","timestamp":1693872000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>Blood sampling in rodents is common practice in scientific studies. Some of the refined methods widely used are the puncture of the saphenous vein or tail vein, or even tail docking. The handling needs of these different blood sampling methods are different and can directly affect stress, increasing the variability of the study. Moreover, there is less aversion and stress if the animal is accustomed to the environment, handling and technique. Therefore, our study aimed to assess the influence of these three blood sampling techniques (saphenous puncture, tail vein puncture and tail vein docking) and the use of previous acclimation on different indicators of animal stress, assessing blood glucose concentrations and faecal corticosterone metabolites (FCMs). Twenty-four young adult male and female C57Bl6\/J mice were divided in three groups by sampling method: tail docking (TD), saphenous vein puncture (SV) and caudal vein puncture (CV) groups. All mice were studied with and without acclimation, which was performed during 9 consecutive days. The results showed that both males and females present very similar responses to the different handling and sampling methods without significant differences. Nevertheless, acclimation in all sampling methods decreased glucose and FCM levels significantly. The method that obtained the lowest glucose and FCM levels with significance was saphenous vein puncture. Therefore, we can say that it causes less stress when performing prior acclimation, even when this involves greater handling of the animal. Our results contribute to refinement within the 3R concept and could serve researchers to programme and select a good handling technique and a welfare-friendly blood sampling method for their experiments.<\/jats:p>","DOI":"10.3390\/ani13182816","type":"journal-article","created":{"date-parts":[[2023,9,5]],"date-time":"2023-09-05T09:59:31Z","timestamp":1693907971000},"page":"2816","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Acclimation and Blood Sampling: Effects on Stress Markers in C57Bl\/6J Mice"],"prefix":"10.3390","volume":"13","author":[{"given":"Nerea","family":"Marin","sequence":"first","affiliation":[{"name":"Animal Facility IISLAFE, Hospital La Fe Research Institute, 46026 Valencia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amparo","family":"Moragon","sequence":"additional","affiliation":[{"name":"Animal Facility CIPF, Prince Felipe Research Center, 46012 Valencia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-8034-4845","authenticated-orcid":false,"given":"Domingo","family":"Gil","sequence":"additional","affiliation":[{"name":"Animal Facility CIPF, Prince Felipe Research Center, 46012 Valencia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francisco","family":"Garcia-Garcia","sequence":"additional","affiliation":[{"name":"Bioinformatics & Biostatistics Unit CIPF, Prince Felipe Research Center, 46012 Valencia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Viviana","family":"Bisbal","sequence":"additional","affiliation":[{"name":"Animal Facility IISLAFE, Hospital La Fe Research Institute, 46026 Valencia, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,9,5]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"116","DOI":"10.1258\/002367797780600198","article-title":"Happy animals make good science","volume":"31","author":"Poole","year":"1997","journal-title":"Lab. Anim."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"152","DOI":"10.1038\/laban.1217","article-title":"Improving quality of science through better animal welfare: The NC3Rs strategy","volume":"46","author":"Prescott","year":"2017","journal-title":"Lab Anim."},{"key":"ref_3","unstructured":"National Centre for the Replacement Refinement & Reduction of Animal in Research (NC3Rs) (2013). Blood Sampling Mouse, NC3Rs."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1258\/002367793781082412","article-title":"Removal of blood from laboratory mammals and birds. First report of the BVA\/FRAME\/RSPCA\/UFAW Joint Working Group on Refinement","volume":"27","author":"Morton","year":"1993","journal-title":"Lab. Anim."},{"key":"ref_5","unstructured":"D\u00fclsner, A., Hack, R., Kr\u00fcger, C., Pils, M., Scherer, K., Schmelting, B., Schmidt, M., Weinert, H., and Jourdan, T. (2017). Recommendation for Blood Sampling in Laboratory Animals, Especially Small Laboratory Animals, Gesellschaft f\u00fcr Versuchstierkunde."},{"key":"ref_6","first-page":"47","article-title":"Methods of Blood Collection in the Mouse","volume":"29","author":"Hoff","year":"2000","journal-title":"Lab. Anim."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"236","DOI":"10.1038\/laban0810-236","article-title":"Morbidity and mortality rates associated with serial bleeding from the superficial temporal vein in mice","volume":"39","author":"Forbes","year":"2010","journal-title":"Lab. Anim."},{"key":"ref_8","unstructured":"Russell, W.M.S., and Burch, R.L. (1959). The Principles of Humane Experimental Technique, Universities Federation for Animal Welfare."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"825","DOI":"10.1038\/nmeth.1500","article-title":"Taming anxiety in laboratory mice","volume":"7","author":"Hurst","year":"2010","journal-title":"Nat. Methods"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Meyer, N., Kr\u00f6ger, M., Th\u00fcmmler, J., Tietze, L., Palme, R., and Touma, C. (2020). Impact of three commonly used blood sampling techniques on the welfare of laboratory mice: Taking the animal\u2019s perspective. PLoS ONE, 15.","DOI":"10.1371\/journal.pone.0238895"},{"key":"ref_11","first-page":"42","article-title":"Laboratory routines cause animal stress","volume":"43","author":"Balcombe","year":"2004","journal-title":"J. Am. Assoc. Lab. Anim. Sci."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"142","DOI":"10.30802\/AALAS-JAALAS-18-000057","article-title":"Welfare and Scientific Considerations of Tattooing and Ear Tagging for Mouse Identification","volume":"58","author":"Roughan","year":"2019","journal-title":"J. Am. Assoc. Lab. Anim. Sci."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1258\/002367707780378168","article-title":"Influence of environmental enrichment and handling on the acute stress response in individually housed mice","volume":"41","author":"Meijer","year":"2007","journal-title":"Lab. Anim."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"283","DOI":"10.1177\/026119299802600305","article-title":"Reducing the Use of Laboratory Animals in Biomedical Research: Problems and Possible Solutions","volume":"26","author":"Festing","year":"1998","journal-title":"Altern. Lab. Anim."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"194","DOI":"10.1093\/ilar.43.4.194","article-title":"Control of variability","volume":"43","author":"Howard","year":"2002","journal-title":"ILAR J."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1016\/j.steroids.2018.03.004","article-title":"Comparison of blood sampling methods for plasma corti-costerone measurement in mice with minimal stress-related artefacts","volume":"135","author":"Kim","year":"2018","journal-title":"Steroids"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1258\/la.2008.007075","article-title":"Quality of plasma sampled by different methods for multiple blood sampling in mice","volume":"43","author":"Christensen","year":"2009","journal-title":"Lab. Anim."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1258\/la.2009.009017","article-title":"Quality of blood samples from the saphenous vein compared with the tail vein during multiple blood sampling of mice","volume":"44","author":"Aasland","year":"2010","journal-title":"Lab. Anim."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"414","DOI":"10.1172\/JCI119175","article-title":"Direct glucocorticoid inhibition of insulin secretion. An in vitro study of dexame-thasone effects in mouse islets","volume":"99","author":"Lambillotte","year":"1997","journal-title":"J. Clin. Invest."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"287","DOI":"10.30802\/AALAS-JAALAS-21-000129","article-title":"A Comparison of Blood Collection Techniques in Mice and their Effects on Welfare","volume":"61","author":"Kress","year":"2022","journal-title":"J. Am. Assoc. Lab. Anim. Sci."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"87","DOI":"10.4103\/0976-500X.72350","article-title":"Blood sample collection in small laboratory animals","volume":"1","author":"Parasuraman","year":"2010","journal-title":"J. Pharmacol. Pharmacother."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Gouveia, K., and Hurst, J.L. (2013). Reducing Mouse Anxiety during Handling: Effect of Experience with Handling Tunnels. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0066401"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1159\/000111395","article-title":"Early environmental regulation of forebrain glucocorticoid receptor gene expression: Implications for adrenocortical responses to stress","volume":"18","author":"Meaney","year":"1996","journal-title":"Dev. Neurosci."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"20305","DOI":"10.1038\/s41598-019-56860-7","article-title":"Improving the practicality of using non-aversive handling methods to reduce background stress and anxiety in laboratory mice","volume":"9","author":"Gouveia","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1016\/j.physbeh.2006.12.003","article-title":"Exploration of the hypothalamic\u2013pituitary\u2013adrenal function as a tool to evaluate animal welfare","volume":"92","author":"Andanson","year":"2007","journal-title":"Physiol. Behav."},{"key":"ref_26","first-page":"307","article-title":"Comparing Phlebotomy by Tail Tip Amputation, Facial Vein Puncture, and Tail Vein Incision in C57BL\/6 Mice by Using Physiologic and Behavioral Metrics of Pain and Distress","volume":"56","author":"Moore","year":"2017","journal-title":"J. Am. Assoc. Lab. Anim. Sci."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"219","DOI":"10.33549\/physiolres.931434","article-title":"A non-invasive method for detecting the metabolic stress response in rodents: Characterization and dis-ruption of the circadian corticosterone rhythm","volume":"58","author":"Thanos","year":"2009","journal-title":"Physiol. Res."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1016\/j.physbeh.2018.11.021","article-title":"Non-invasive measurement of glucocorticoids: Advances and problems","volume":"199","author":"Palme","year":"2018","journal-title":"Physiol. Behav."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1016\/j.yhbeh.2003.07.002","article-title":"Analyzing corticosterone metabolites in fecal samples of mice: A noninvasive technique to monitor stress hormones","volume":"45","author":"Touma","year":"2004","journal-title":"Horm. Behav."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1007\/s002130050327","article-title":"Behavioral phenotypes of inbred mouse strains: Implications and recommendations for molecular studies","volume":"132","author":"Crawley","year":"1997","journal-title":"Psychopharmacology"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Ortega-Saez, I., D\u00edez-Solinska, A., Gr\u00edfols, R., Mart\u00ed, C., Zamora, C., Mu\u00f1oz-Culla, M., Vegas, O., and Azkona, G. (2023). Individualized Housing Modifies the Immune\u2013Endocrine System in CD1 Adult Male Mice. Animals, 13.","DOI":"10.3390\/ani13061026"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1016\/0306-4530(93)90001-2","article-title":"Specificity of the effect of repeated handling on sympathetic-adrenomedullary and pitui-tary-adrenocortical activity in rats","volume":"18","year":"1993","journal-title":"Psychoneuroendocrinology"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1016\/j.physbeh.2015.06.021","article-title":"Mouse handling limits the impact of stress on metabolic endpoints","volume":"150","author":"Ghosal","year":"2015","journal-title":"Physiol. Behav."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"390","DOI":"10.2337\/diabetes.55.02.06.db05-0686","article-title":"Considerations in the Design of Hyperinsulinemic-Euglycemic Clamps in the Conscious Mouse","volume":"55","author":"Ayala","year":"2006","journal-title":"Diabetes"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"525","DOI":"10.1242\/dmm.006239","article-title":"Standard operating procedures for describing and performing metabolic tests of glucose homeostasis in mice","volume":"3","author":"Ayala","year":"2010","journal-title":"Dis. Model. Mech."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"190","DOI":"10.1177\/0023677218786982","article-title":"Impact assessment of tail-vein injection in mice using a modified anaesthesia induction chamber versus a common restrainer without anaesthesia","volume":"53","author":"Resch","year":"2019","journal-title":"Lab. Anim."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1177\/0023677217741332","article-title":"A comparison of various methods of blood sampling in mice and rats: Effects on animal welfare","volume":"52","author":"Harikrishnan","year":"2018","journal-title":"Lab. Anim."},{"key":"ref_38","first-page":"215","article-title":"Blood sampling from the tail vein, in comparison with two other techniques, causes less stress to mice","volume":"36","author":"Madetoja","year":"2009","journal-title":"Scand. J. Lab. Anim. Sci."},{"key":"ref_39","first-page":"e62593","article-title":"Handling Techniques to Reduce Stress in Mice","volume":"175","author":"Marcotte","year":"2021","journal-title":"J. Vis. Exp."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"278","DOI":"10.1177\/0023677214541438","article-title":"Manual versus automated blood sampling: Impact of repeated blood sampling on stress parameters and behavior in male NMRI mice","volume":"48","author":"Teilmann","year":"2014","journal-title":"Lab. Anim."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"869","DOI":"10.1007\/s00442-011-1943-y","article-title":"Measuring stress in wildlife: Techniques for quantifying glucocorticoids","volume":"166","author":"Sheriff","year":"2011","journal-title":"Oecologia"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"185","DOI":"10.1016\/0960-0760(91)90061-9","article-title":"The effect of gonadectomy and aromatase inhibition on the excretion of 19-nordeoxycorticosterone in rats","volume":"39","year":"1991","journal-title":"J. Steroid Biochem. Mol. Biol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"343","DOI":"10.1017\/S0962728600030669","article-title":"The effects of individual housing on mice and rats: A review","volume":"15","author":"Krohn","year":"2006","journal-title":"Anim. Welf."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1016\/S0016-6480(02)00620-2","article-title":"Effects of sex and time of day on metabolism and excretion of corticosterone in urine and feces of mice","volume":"130","author":"Touma","year":"2003","journal-title":"Gen. Comp. Endocrinol."}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/13\/18\/2816\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T20:46:47Z","timestamp":1760129207000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/13\/18\/2816"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,9,5]]},"references-count":44,"journal-issue":{"issue":"18","published-online":{"date-parts":[[2023,9]]}},"alternative-id":["ani13182816"],"URL":"https:\/\/doi.org\/10.3390\/ani13182816","relation":{},"ISSN":["2076-2615"],"issn-type":[{"value":"2076-2615","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,9,5]]}}}