<br><br />Conclusions<br /><br><br />Conflict of interest statement<br /><br><br />Acknowledgment<br />We are grateful to Alireza Fazeli (University of Sheffield, UK) for his help and critical comments regarding the manuscript. This research was supported by the National Natural Science Foundation of China (grant number 31172282) and Priority Academic Program for Development of Jiangsu Higher Education Institutions, China.<br /><br><br />Equine pituitary pars intermedia dysfunction (PPID) is a disease <img src="http://farm5.static.flickr.com/4398/36363832443_f7ce914c8a.jpg
" align="right" width="209" style="padding:10px;"/> frequently seen in the aged equine population () and endogenous adrenocorticotrophic hormone (ACTH) has become the accepted test for the majority of cases (). Equine ACTH has been shown to have increased stability in samples stored up to 48 h in normal horses and between 4 and 8 h in PPID positive horses ().<br />The objectives of the study were to determine the stability of ACTH in whole blood and plasma over 72 h at either 4 or 21 °C and to determine the stability of ACTH when plasma was frozen at −20 or −80 °C for up to 30 days. The stability of various <a href='http://www.lipopeptide.net/viewtopic.php?f=2&t=4539
'>selective androgen receptor modulators</a> in whole blood has been shown to be low () and the stability of equine ACTH after sample collection and varying storage conditions has not been fully reported previously.<br />Blood samples were collected from nine clinically healthy horses >16 years of age in January and February 2013. The samples were collected into plastic KEDTA (ethylene diamine tetracetic acid) Vacutainers and were immediately placed on ice (4 °C) or stored at 21 °C. Half of the samples stored at both 4 and 21 °C were centrifuged immediately following collection and divided into 500 µL aliquots. Aliquots where stored for 1, 4, 8, 24, 48 and 72 h at their respective temperatures prior to analysis. Plasma samples were stored at −20 °C for 24, 48, 72, 168 and 720 h and at −80 °C for 168 and 720 h prior to analysis. Finally, samples were stored at 4 or 21 °C for 4, 8, 24, 48 and 72 h as 1 mL whole blood aliquots prior to centrifugation and analysis.<br /><br><br />Contagious agalactia in goats is most frequently associated with infection by (). The syndrome is characterised by clinical signs of acute mastitis that usually progress to chronic disease (). The host–pathogen interaction during infection with this particular mycoplasma organism is, however, unclear; in particular, the biological basis for its persistence in milk and host tissues, despite a prominent host inflammatory response.<br />Cyclooxygenase (COX) is the enzyme that converts arachidonic <a href='http://en.wikipedia.org/wiki/ACID
'>acid</a> to prostanoids, including prostaglandins (PGs). Expression of the COX-2 isoform is induced in host cells (particularly leukocytes) by bacterial products and inflammatory cytokines, as part of the innate immune response to infection (). COX-2-derived mediators can participate in both pro- and anti-inflammatory processes, but it remains to be established how PGs specifically contribute to initiation, progression and resolution of the inflammatory process during mastitis. COX-2 seems to have a pro-inflammatory role during the early phase of inflammation, but might also participate in the recovery and resolution stages (). The aim of the present study was to investigate the expression and localisation of COX-2 in mammary gland lesions from goats infected with , and to investigate its relationship with the development of such inflammatory lesions.<br />Mammary gland tissues were obtained from five goats affected with mastitis during an outbreak of contagious agalactia in a dairy goat herd in Gran Canaria, Spain. The animals were culled due to progression of clinical signs and the presence of in the milk. Archived tissues from five goats without mastitis and five goats that had been experimentally infected with as part of a previous study () were also included. For the experimental infection, 2 mL inoculum, containing ~10 colony-forming units (cfu) of the same strain of , cultured in 10 mL of mycoplasma broth for 5 days at 37 °C, was intra-cisternally inoculated into the right halves of the mammary glands. Left mammary gland halves were inoculated in the same way with 2 mL of sterile mycoplasma broth. Goats were clinically examined each day and euthanased 2 weeks after inoculation. The experiment had been carried out in accordance with the Code of Practice for Housing and Care of Animals used in Scientific Procedures (EU Directive 86/609/EEC).