Canine spermatozoa may be stored for several days within the genital tract of the bitch since natural matings 8 to 9 days before ovulation may result in litters. Several studies have suggested that the sperm reservoir in the dog is located in the uterine crypts and the uterotubal junction (UTJ). In the present study, we investigated the effect of the timing of artificial insemination (AI) in relation to ovulation on the sperm distribution in the genital tract of the bitch. Ten beagle dogs were inseminated intravaginally with 500 × 10⁶ spermatozoa. Based on progesterone concentration, three dogs were inseminated 1–2 days before ovulation, four dogs during ovulation, and three dogs 2–3 days after ovulation. Ovariohysterectomy was performed 24 h after AI. The genital tract was divided into eight segments (i.e. corpus uteri; caudal, middle, and cranial parts of the uterine horn; UTJ; isthmus; ampulla; and infundibulum) which were processed for histology. From each segment, 30 histological sections were evaluated. For the UTJ and the different segments of the oviduct, the total number of spermatozoa was determined. For the different parts of the uterus, on each of these 30 sections, 100 uterine crypts of comparable size were evaluated for the presence of spermatozoa. The crypts were divided into crypts without spermatozoa, crypts with 1 sperm cell, crypts with 2 to 5 spermatozoa, and crypts with either more than 5 spermatozoa or in which the spermatozoa were clustered. The data were analyzed using univariate analysis of variance. Histology revealed that the spermatozoa were located mainly in the uterine crypts and at the UTJ, while very few spermatozoa were detected in the different parts of the oviduct. Insemination during ovulation resulted in higher percentages of crypts with spermatozoa in the different parts of the uterus (P < 0.05). Moreover, for the ovulatory group, 54.7% of the uterine crypts with spermatozoa contained more than 5 spermatozoa (or clusters) compared to 19.9% and 28.2% for the pre- and post-ovulatory groups, respectively (P < 0.05). In the pre-ovulatory group, 59.6% of the uterine crypts with spermatozoa contained only 1 sperm cell whereas in the post-ovulatory group, frequently 1 (34.0%) or 2 to 5 spermatozoa (37.9%) were found per crypt. In conclusion, sperm transport in the genital tract of the bitch is affected by the time of AI in relation to ovulation. Insemination during the ovulation period resulted in higher percentages of uterine crypts with spermatozoa, and most of these crypts contained 5 or more spermatozoa. Further research should determine whether the number of sperm binding sites expressed on the epithelium of the canine uterine crypts is influenced by the ovulation event.

This research was supported by the UGent Special Research Fund, Grant numbers 011 B8698 and 011 B8301.