Validation of an enzyme-linked immunosorbent assay developed for measuring cortisol concentration in human saliva and serum for its applicability to analyze cortisol in pig saliva

Acta Veterinaria Scandinavica

Ola Thomsson²  Bodil Ström-Holst¹  Ylva Sjunnesson²  Ann-Sofi Bergqvist² 

[1] Division of Diagnostics and Large Animal Clinical Sciences, Department of Clinical Sciences, Swedish University of Agricultural Sciences, SLU, Uppsala, SE-750 07, Sweden

[2] Division of Reproduction, Department of Clinical Sciences, Swedish University of Agricultural Sciences, SLU, Uppsala, SE-750 07, Sweden

关键词: Primiparous;    Multiparous;    Group housing;    Sow;    Porcine;    Stress;   

Others  :  1082560
DOI  :  10.1186/s13028-014-0055-1

 received in 2013-11-01, accepted in 2014-08-27,  发布年份 2014

【 摘 要 】

Background

The purpose of this study was to validate a commercially available enzyme-linked immunosorbent assay (ELISA) developed for measuring free cortisol in human saliva and total cortisol concentration in diluted human serum, for its applicability in measuring cortisol concentration in pig saliva. Collection of saliva is less stressful than e.g. blood sampling, and is a non-invasive method.

Findings

Saliva was collected by allowing sows to chew on cotton swabs held by forceps. Thereafter, the swabs were centrifuged to retrieve the saliva. The ELISA was performed according to instructions provided by the manufacturer. To validate the ELISA, determination of the intra-assay coefficient of variation (CV), inter-assay CV, recovery, linearity and parallelism was performed. The intra-assay CV was below 10% and inter-assay CV below 15% for samples of high, medium and low cortisol concentrations. The mean recovery was 117% and the linearity and parallelism showed an r²-value of 0.994 and 0.993, respectively. For biological assessment of induced social stress, two saliva samples were collected in the morning from 6 primiparous and 21 multiparous sows. One sample was collected when the sows were individually housed in a farrowing pen and a second sample was collected when the sows were group housed. The primiparous sows had a significant higher cortisol concentration compared to the multiparous sows when group housed.

Conclusion

The results obtained in this validation study indicate that the ELISA is suitable for measuring cortisol concentration in porcine saliva.

【 授权许可】

   
2014 Thomsson et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

【 参考文献 】

• [1]Colson V, Martin E, Orgeur P, Prunier A: Influence of housing and social changes on growth, behaviour and cortisol in piglets at weaning. Physiol Behav 2012, 107:59-64.
• [2]Brandt Y, Lang A, Rodriguez-Martinez H, Madej A, Einarsson S: Impact of ACTH during oestrus on the ultrastructure of the spermatozoa and their environment in the tubal reservoir of the postovulatory sow. Anim Reprod Sci 2006, 93:231-245.
• [3]Cook NJ, Schaefer AL, Lepage P, Jones SDM: Radioimmunoassay for cortisol in pig saliva and serum. J Agric Food Chem 1997, 45:395-399.
• [4]Pol F, Courboulay V, Cotte J-P, Martrenchar A, Hay M, Mormède P: Urinary cortisol as an additional tool to assess the welfare of pregnant sows kept in two types of housing. Vet Res 2002, 33:13-22.
• [5]Möstl E, Palme R: Hormones as indicators of stress. Domest Anim Endocrinol 2002, 23:67-74.
• [6]Brandt Y, Einarsson S, Ljung A, Lundeheim N, Rodríguez-Martínez H, Madej A: Effects of continuous elevated cortisol concentrations during oestrus on concentrations and patterns of progesterone, oestradiol and LH in the sow. Anim Reprod Sci 2009, 110:172-185.
• [7]Kerlik J, Penesova A, Vlcek M, Imrich R, Vogeser M, Radikova Z: Comparison of salivary cortisol and calculated free plasma cortisol during low-dose ACTH test in healthy subjects. Clin Biochem 2010, 43:764-767.
• [8]Coutellier L, Arnould C, Boissy A, Orgeur P, Prunier A, Veissier I, Meunier-Salaün M-C: Pig’s responses to repeated social regrouping and relocation during the growing-finishing period. Appl Anim Behav Sci 2007, 105:102-114.
• [9]Parrott RF, Misson BH: Changes in pig salivary cortisol in response to transport simulation, food and water deprivation, and mixing. Br Vet J 1989, 145:501-505.
• [10]Soede NM, van Sleuwen MJW, Molenaar R, Rietveld FW, Schouten WPG, Hazeleger W, Kemp B: Influence of repeated regrouping on reproduction in gilts. Anim Reprod Sci 2006, 96:133-145.
• [11]Ekkel ED, van Doorn CE, Hessing MJ, Tielen MJ: The Specific-Stress-Free housing system has positive effects on productivity, health, and welfare of pigs. J Anim Sci 1995, 73:1544-1551.
• [12]Li YZ, Wang LH, Johnston LJ: Sorting by parity to reduce aggression toward first-parity sows in group-gestation housing systems. J Anim Sci 2012, 90:4514-4522.
• [13]Smulders D, Verbeke G, Mormede P, Geers R: Validation of a behavioral observation tool to assess pig welfare. Physiol Behav 2006, 89:438-447.
• [14]Escribano D, Fuentes-Rubio M, Ceron JJ: Validation of an automated chemiluminescent immunoassay for salivary cortisol measurements in pigs. J Vet Diagn Invest 2012, 24:918-923.
• [15]Fuentes M, Tecles F, Gutiérrez A, Otal J, Martínez-Subiela S, Cerón JJ: Validation of an automated method for Salivary Alpha-Amylase measurements in Pigs (Sus Scrofa Domesticus) and its application as a stress biomarker. J Vet Diagn Invest 2011, 23:282-287.
• [16]Jaedicke KM, Taylor JJ, Preshaw PM: Validation and quality control of ELISAs for the use with human saliva samples. J Immunol Methods 2012, 377:62-65.
• [17][http:/ / www.fda.gov/ downloads/ Drugs/ GuidanceComplianceRegulatoryInforma tion/ Guidances/ ucm070107.pdf] webcite Guidance for industry- bioanalytical method validation. .
• [18]Ekkel ED, Dieleman SJ, Schouten WGP, Portela A, Cornélissen G, Tielen MJM, Halberg F: The circadian rhythm of cortisol in the saliva of young pigs. Physiol Behav 1996, 60:985-989.
• [19]Strawford ML, Li YZ, Gonyou HW: The effect of management strategies and parity on the behaviour and physiology of gestating sows housed in an electronic sow feeding system. Can J Anim Sci 2008, 88:559-567.
• [20]De Leeuw J, Ekkel E: Effects of feeding level and the presence of a foraging substrate on the behaviour and stress physiological response of individually housed gilts. Appl Anim Behav Sci 2004, 86:15-25.
• [21]Merlot E, Vincent A, Thomas F, Meunier-Salaün M-C, Damon M, Robert F, Dourmad J-Y, Lebret B, Prunier A: Health and immune traits of Basque and Large White pigs housed in a conventional or enriched environment. Animal Int J Anim Biosci 2012, 6:1290-1299.