*1.1.1. History of ovariohysterectomy*

If taken from another person, the bitch may have been sterilized, which a new owner may overlook. Presence of a tattoo in the inguinal region and palpation or inspection of a scar from operation in the ventral wall of the abdomen may be indicators of ovariohysterectomy. However, it should be kept in mind that such a scar may be present if any intra-abdominal operation has been performed, so it may be premature to conclude that the scar resulted from ovariohysterectomy. Serum LH level measurement may be used to identify bitches that have undergone ovariohysterectomy, as serum LH is continuously high in such bitches as a result of the absence of negative feedback on LH (because the ovaries have been removed). Although elevation in serum LH provides information about ovariohysterectomy, note that this indicator may also be seen in ovarian dysfunction or during the preovulatory period (i.e., the preovulatory LH peak). Therefore, repeated measurements are required to confirm and experimental laparotomy may also be performed for a definitive diagnosis [8]. Whether ovariohysterectomy has been performed may also be detected by measuring the serum estrogen level before and 60–90 min after intravenous administration of 0.02–0.03 μg/kg buserelin. If ovariohysterectomy has been performed, estrogen levels will be found above 15–20 pg/mL.

Anti-müllerian hormone (AMH) measurement may also show whether a bitch has undergone ovariohysterectomy. AMH levels are found to be significantly lower in bitches that have undergone ovariohysterectomy compared to nonsterilized bitches [9, 10].

## *1.1.2. Silent heat (subestrus)*

Silent heat is defined as the maintenance of ovarian functions without the presence of vulvar edema, serosanguinous vaginal discharge, and charm for male dogs. Silent heat may be observed for several cycles before first estrus in younger bitches of smaller breeds. These animals may be evaluated as "anestrus" because the pet owner may not find the external signs of estrus, or they may be identified as healthy male dogs although ovarian functions continue normally.

If silent heat is suspected, serum progesterone level should be measured once monthly in order to verify that the ovaries are functioning. A serum progesterone level above 2 ng/mL indicates functional luteal tissue. Observation of increasing superficial epithelial cells in regular vaginal cytological examinations is an indicator of functional ovaries [5].
