Title: COLLAGEN AND GLYCOSAMINOGLYCAN PROFILES IN THE CANINE 4 CERVIX DURING DIFFERENT STAGES OF THE ESTROUS CYCLE AND IN OPEN-

26 The extracellular matrix of the cervix that comprises collagen, elastin, 27 proteoglycans and glycosaminoglycans (GAGs) is thought to have an essential role in 28 cervical relaxation. This study investigated the proportion of collagen and smooth muscle 29 as well as the GAGs in cervices obtained from healthy bitches at different stages of the 30 estrous cycle and bitches with openand closed-cervix pyometra. Cervices were collected 31 after ovariohysterectomy. The proportion of collagen to smooth muscle was determined 32 using Masson’s trichrome staining. Alcian blue staining was used to evaluate the relative 33 distribution of cervical GAGs. The proportion of cervical collagen relative to smooth 34 muscle was higher at estrus compared to anestrus (P≤0.05). It was also higher (P≤0.05) in 35 bitches with opencompared to those with closed-cervix pyometra. Overall, hyaluronan 36 (HA) was the predominant GAG in the canine cervix. In the luminal epithelium, the 37 staining intensity for HA was stronger in estrus than in anestrus (P≤0.05), but not in 38 diestrus (P>0.05). On the contrary, the intensity for the combined keratan sulfate (KS) and 39 heparan sulfate (HS) was stronger in anestrus than in estrus and diestrus (P≤0.05). In 40 bitches with pyometra, the staining intensity of the stroma for KS and HS was weaker in 41 opencompared to closed-cervix pyometra (P≤0.05). Collectively, the different profiles of 42 collagen and GAG suggest that the metabolism of both collagen and GAGs in the canine 43 cervix is associated with hormonal statuses during the estrous cycle and cervical patency 44 of bitches with pathological uterine conditions, such as pyometra. 45 46