Statement of animal ethics
The study was approved by the Institutional Animal Care and Use Committee of Shiraz University (IACUC no: 4687/63). The recommendations of European Council Directive (2010/63/EU) of September 22, 2010, regarding the standards in the protection of animals used for experimental purposes, were also followed.
Semen samples were collected by digital stimulation from 10 healthy, mature, male, mixed-breed dogs with reproductive known history. The quality of the semen was specified based on the sperm concentration (22 × 106 sperm/kg body weight; SpermaCue device, Minitube International, Germany), sperm progressive motility (greater than 70% progressive motility), and sperm morphology (greater than 70% morphologically normal sperm) . The semen samples were pooled together and centrifuged at 1200×g for 10 min at room temperature to separate the sperm cells. The pellet was resuspended in 10 mL sterile phosphate buffered saline (PBS) and the procedure was repeated three more times. Finally, the washed sperm cells were resuspended in sterile PBS at a concentration of 200 × 106 and 100 × 106 cells/mL and stored at − 20 °C until immunization.
Immunization of dogs
A total of 15 healthy, mature, female, mixed-breed dogs with reproductive known history, with an average weight of 15–20 kg and age of 10 months were selected and divided into 3 equal groups (n = 5). All dogs were owned and kept by Shiraz University School of Veterinary Medicine. Anti-parasitic treatment was performed during the first 2 weeks of preparation by using praziquantel (10 mg/kg) and mebendazole (22 mg/kg). All dogs received 300 g/dog/day of commercial dog feed (Nutri® dry dog food; Behintash Co. Iran) and water was provided ad libitum. All dogs were evaluated using ultrasonography to determine stage of cycle and confirm non-pregnancy status.
The sperm vaccine was prepared by emulsifying one mL of whole sperm suspension with an equal volume of Freund’s complete adjuvant (CFA) (Sigma Aldrich, St Louis, MO, USA) in the first immunization and injected subcutaneously at 4 sites across the shoulder area. For the high dose, the dogs received 2 ml of vaccine containing 200 million sperm (which is 100 million sperm/ml) and for the low dose, the dogs received 100 million sperm/ml. Booster immunizations were given at weeks 1, 2, 4, and 6 replacing Freund’s complete adjuvant with Freund’s incomplete adjuvant (IFA; Sigma Aldrich, St. Louis, MO, USA) . The control group (group 3) was administered with 1 mL of sterile PBS emulsified with 1 mL adjuvant in all immunizations.
Blood samples were collected from jugular vein prior to immunizations at days 0, 14, 28, 42, 63, and 84. The serum samples were separated using centrifugation 750×g for 10 min and stored at − 20 °C before anti-sperm antibody analysis. At the end of the 12th week, all the dogs underwent the elective ovariohysterectomy (OHE) surgery and their ovaries and uterus were collected. The status of animals’ reproductive cycle and the presence of follicles and corpora lutea were recorded for all dogs. Finally, the samples of vaginal and uterine secretions were collected by flushing each section with 5 mL sterile PBS. The lavages were centrifuged and the supernatants were stored at − 20 °C to be used later in the analysis of antibodies.
Assessment of anti-sperm antibodies
Anti-sperm antibody production and specificity in serum samples and the reproductive tract secretions were measured using an enzyme-linked immunosorbent assay (ELISA) technique. IgG antibodies were detected using a commercial indirect ELISA kit (Crystal Day Biotech, Shanghai, China. Cat. No. ED0299Ca), according to the manufacturer’s instructions. All the samples were run in duplicate. The inter-assay CV was < 10% and the intra-assay CV was < 12%. Microtiter plates were pre-coated with whole spermatozoa and blocked with bovine serum albumin. 50 μl of diluted serum and reproductive tract secretions (1:5) along with the negative and positive controls were added to the plate and incubated for 30 min at 37 °C. The plates were washed 5 times with PBS-Tween and incubated with 50 μl horseradish peroxidase-conjugated anti-canine IgG for 30 min at 37 °C. The plates were washed five additional times and color reaction was initiated by adding 50 μl tetramethylbenzidine substrate to the plates, incubated at 37 °C for 10 min. At the end of the experiment, the optical density (OD) of each sample was determined at 450 nm with a microtiter plate reader (Immunoskan BDSL, Thermo Lab. Systems, Finland) and used for comparison of anti-sperm antibody titers between groups .
All data were expressed as means ± standard error of mean (SEM). The data were analyzed statistically by two-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test to determine the degree of significance for ASAs titer between the control and experimental groups and also between the two experimental groups at different sampling time points. One-way ANOVA was also used to compare the uterine and vaginal ASAs levels in each group. The data were analyzed using GraphPad Prism software, version 6 for Windows, and the probability of P < 0.05 was considered statistically significant.