Laboratory and Clinical Evaluation of a Feia Method for Canine Serum Progesterone Assay A Rota 1 , I Vannozzi 1 , S Marianelli 2 , A Gavazza 1 and G Lubas 1 1 Department of Veterinary Sciences, University of Pisa, Pisa, Italy; 2 Practitioner, Perignano (PI), Italy Contents The evaluation of progesterone (P4) concentration is a valuable tool in assessing physiological reproductive events and repro- ductive disorders in bitches. A reliable and rapid (preferable, point of care) determination of P4 is advisable in most cases. Aims of this study were to evaluate a fluorescence enzyme immunoassay (FEIA) for canine serum P4 concentration by (i) the agreement with liquid chromatography–tandem mass spec- trometry (LC/MS/MS), (ii) the association with vaginal cytol- ogy and (iii) the accuracy in the prediction of the parturition date calculated from the estimated day of ovulation. Serum samples were collected from client-owned bitches presented between 2011 and 2014 for the evaluation of their oestrous cycle, pregnancy or reproductive disorders. The agreement between FEIA and LC/MS/MS, evaluated on 19 samples, was statisti- cally significant (R 2 = 95.7%, p < 0.001), although FEIA showed significantly higher values than LC/MS/MS (p < 0.05). In the different phases of oestrous cycle, as determined by vaginal cytology, P4 concentrations (by FEIA) were statistically different (p < 0.05): anoestrus (n = 7) 0.38 Æ 0.14 ng/ml, proestrus (n = 14) 1.04 Æ 0.67 ng/ml and oestrus (n = 72) 6.8 Æ 7.26 ng/ml. Mean pregnancy length from the estimated day of ovulation was 62.9 Æ 1.8 days. In 13 of 22 (59.1%), 19 of 22 (86.3%) and 21 of 22 (95.5%) bitches pregnancy lasted 63 Æ 1, 63 Æ 2 and 63 Æ 3 days, respectively. Three pregnancies were outside the 61–65 days range (60, 60 and 67 days). In conclusion, the FEIA method employed can be considered reliable and, in association with vaginal cytology, effective in evaluating the canine oestrous cycle. Introduction In Italy, more than 130 000 puppies are registered at the kennel club (Ente Nazionale della Cinofilia Italiana, the national organization responsible to issue dogs’ pedi- grees) every year, and hundreds of working competition (hunting, utility, agility, sheepdog, coursing, etc.) and dog shows are organized. Dog breeding is an econom- ically important activity, as it drives an economic engine including the sale of puppies, pet food and accessories, animal health and veterinary services, pet insurances, and dog activities (FECAVA 2007). It has been estimated that companion animals in the United States move nearly forty billion dollars per year for food, accessories, veterinary care and other support services and products (Zawistowski 2008). Canine events are today organized in most world’s countries and success- ful show or working dogs together with their owners travel the whole year around, adding journey and housing expenses. The value of these dogs can be very high and owners require increasingly sophisticated veterinary evaluations and treatments, including spe- cialists in reproduction, for the examinations of both males and females. The evaluation of progesterone (P4) concentration is a valuable tool in assessing reproductive events in bitches. Stage of oestrous cycle and ovulation time can be determined by the combination of vaginal cytology and serum P4 concentration. Ovulation occurs when a bitch in cytological oestrus reaches 4–8 ng/ml of P4, and best breeding days for natural mating are considered those in the range from 1 to 4 days post-ovulation, while for frozen semen, the window is narrower and closer to day 4 (Badinand et al. 1993; Steckler et al. 2013). Pregnancy length from the day of ovulation has been described to range 62–64 days (Concannon et al. 2001; Tsutsui et al. 2006; Fontbonne 2008), 61–65 days (Okkens et al. 2001; Elits et al. 2005) or even 58–67 (Mir et al. 2011). Progesterone levels decrease significantly (<1–2 ng/ml) 24 h before parturition (Concannon et al. 1978; Chak- raborty 1987; Hoffmann et al. 1994; England and Verstegen 1996; Veronesi et al. 2002). Progesterone can thus be employed both to predict with enough accuracy the date of parturition and to evaluate whether natural parturition is impending or a planned Caesarean section (C-S) should be performed (Smith 2007), which is particularly important due to the late pulmonary maturation of canine foetuses (Sipriani et al. 2009). Moreover, knowledge of circulating progesterone con- centration is useful for the diagnosis and treatment of reproductive disorders such as pyometra, abnormalities of oestrous cycle, diseases of pregnancy and ovarian remnant syndrome (e.g. Meyers-Wallen 2007; Sontas et al. 2014; Krauss and Krauss 2015). A reliable and rapid (preferable, point of care) determination of P4 is often advisable, that is when the time of ovulation needs to be determined for a mating or an artificial insemination, or when a decision has to be made whether a C-S should be performed or a treatment initiated. Several laboratory methods have been employed for the quantification of P4. Radioim- munoassay (RIA) is today still considered the reference method; however, its use is limited due to practical problems, such as those linked to the use of gamma- radioactive isotopes. For this reason, methods more often used today in canine practice and research are immunoassays, both by chemiluminescence (CLIA, e.g. Forsberg et al. 1993; Kutzler et al. 2003; Chapwanya et al. 2008; Mir et al. 2011) and by fluorescence enzyme Reprod Dom Anim 51, 69–74 (2016); doi: 10.1111/rda.12647 ISSN 0936–6768 © 2015 Blackwell Verlag GmbH