The initiation of spermatogenesis at puberty leads to expression of new proteins by developing germ cells. These proteins are recognized as foreign by the immune system since immune self-tolerance is established in utero. However, these new antigens are normally tolerated without inducing immune responses due to the maintenance of the testicular immune privileged status by tissue physical structure, local immunosuppressive milieu, and systemic immune tolerance working cooperatively. Dysregulation of the tolerogenic environment leads to the development of autoimmune orchitis, which can be primary, or secondary to inflammation, infection, trauma, or toxins, among others [1]. Autoimmune orchitis is as an autoimmune reaction to testicular antigens characterized clinically by infertility and presence of antisperm antibodies (ASAs) [1]. Canine autoimmune orchitis associated with progressive infertility was first reported in 32% of beagle dogs in an inbred colony, the majority of which had concurrent lymphocytic thyroiditis, presumably of genetic origin [2]. More recently, autoimmune orchitis was recognized as the main cause of non-obstructive azoospermia in dogs characterized by the presence of lymphoplasmacytic infiltration of the testicular parenchyma and tubular atrophy [[3], [4], [5], [6]]. Dogs with non-obstructive azoospermia due to autoimmune orchitis had a common history of progressive decline in total sperm count over 4 to 5 months, ending in irreversible azoospermia and infertility [[3], [4], [5]].

In humans, autoimmune orchitis is diagnosed clinically by the presence of ASAs [1]. Canine ASAs of the IgA isotype were reported in the seminal plasma and serum of dogs with chronic Brucella canis infection and sperm agglutination [7]. The presence of canine sperm-directed IgG was reported after testicular biopsy, hemicastration and epididymal aspirates [8], and presumably in a dog with asthenospermia associated with sperm granuloma [9]. Each isotype may have a different effect on sperm function. Immunoglobulin A is typically found in semen and may interfere with sperm motility, sperm capacitation, and binding to the oocyte [10,11]. On the other hand, IgG reduced sperm binding to oviductal epithelial cells in bulls [12]. Together, ASAs can significantly impair fertilization and contribute to infertility [11]. Unfortunately, the lack of standardized tests to detect canine ASAs precludes an early diagnosis of autoimmune orchitis that could prevent the progression of the condition by early intervention. Given that invasive testicular biopsies are not routinely done in clinical practice, the prevalence of subclinical or early autoimmune orchitis in dogs is not known. Studies aimed at standardizing tests and evaluating the prevalence of ASAs and concurrent andrological findings in dogs would benefit canine infertility investigation and would add another clinical diagnostic tool. Flow cytometry was standardized in veterinary medicine for the diagnosis of ASAs in bulls and stallions, but not in dogs [13,14].

The objectives of the study were to determine the prevalence of ASAs and their association with semen quality and cooling ability in dogs, and to determine the origin of ASA binding and the diagnostic ability of indirect immunofluorescence tests. It was hypothesized that: 1. ASA binding is higher in semen samples with low sperm motility, morphology or output, and more prevalent among dogs failing the breeding soundness exam, 2. ASA binding impairs motility of cooled shipped sperm, 3. ASAs are present in prostatic fluid of ASA-positive dogs.