ABSTRACT. We evaluated transcervical insemination (TCI) using frozen semen by flexible endoscopy in dogs. Eight female and eight male beagles were used in this study. A flexible endoscope and a washing tube were used for TCI. A tracheal tube was used as an alternative to the penis and was an auxiliary for inserting the flexible endoscope. The mean time required to insert the washing tube into the external os of the uterus after inserting the endoscope into the tracheal tube was 7.5 min. Slight or mild pain was observed in all bitches during TCI. However, TCI could be easily performed with retention in all bitches and without sedation anesthesia. The tracheal tube was useful to ensure the visual field using air sufflation. Clinical signs suspicious of infection were not observed in any bitches from the TCI to the pregnancy diagnosis. The conception rate was 87.5%, and the mean number of fetuses was 6.3. TCI using a flexible endoscope in bitches was performed quickly with minimal invasiveness. We present a new method of TCI in dogs. This method should be studied in small and large breeds to obtain more detailed results. Artificial insemination (AI) using frozen semen in dogs has been increasing in recent years. However, AI using cryopreserved semen generally yields lower pregnancy rates with vaginal deposition than with uterine deposition because of the short life span of frozen-thawed spermatozoa [5,19]. Therefore, AI methods usually use uterine deposition with frozen semen. Surgical insemination is highly invasive and requires general anesthesia, whereas transcervical insemination (TCI) using metal catheterization is minimally invasive but requires a skilled technique [24]. In contrast, TCI using a rigid endoscope is easy and less invasive [13,30]. TCI with a flexible endoscope is also less invasive because the scope is soft, and it has an air supply and instrument channel, which endows it with good operability [21,29]. Therefore, a flexible endoscope might be useful as a new tool for TCI.The purpose of this study was to evaluate TCI using frozen semen by flexible endoscope in dogs. MATERIALS AND METHODSEight female beagles aged 1-8 years (mean ± standard deviation: SD, 4.1 ± 2.5 years) and weighing 10.7-15.1 kg (mean ± SD, 12.8 ± 1.3 kg) were used in this study; 4 were parous, and the others were nulliparous. Eight male beagles aged 1-7 years (mean ± SD, 3.6 ± 1.8 years) and weighing 11.4-15.8 kg (mean ± SD, 13.1 ± 1.4 kg) were used as sperm donors. Unrelated dogs were used to prevent inbreeding. This study was performed in accordance with the Guide for the Experimentation of Animals of the College of Bioresource Sciences, Nihon University (AP11B018, 2010).The sperm-rich fraction of a single ejaculate was collected from each dog by digital manipulation [11]. The collected semen was centrifuged at 400 × g for 5 min. The supernatant was discarded, and the pellets were gently suspended in 1 ml with egg-yolk Tris-fructose citrate extender supplemented with 1% Orvus ES paste (OEP) [26][27][28]. Sperm concentration w...