Collection of oocytes:
At abattoir, dogs were separated into two groups based on the growth of tooth (Noden and Delahunta, 1985), one group of younger than 5 month-old dogs, and another group of 5 month-old or older dogs. Concurrently, stages of reproductive cycle were determined for each bitch by considering a combination of clinical signs and ovarian morphology.
The morphology of all ovaries were examined, and then they were transported to laboratory in physiological saline (0.85% [wt./vol.] at temperature of insulated container within 1h. At laboratory, the ovaries were washed carefully with PBS-PVA medium. Cumulus oocyte complexes (COCs) were collected after cutting the ovary into small pieces, and then washed in PBS-PVA added 10% FBS. The number of COCs per bitch in each group was counted, and the total number of oocytes, consisting of oocytes with complete (good oocytes) and incomplete (bad oocytes) layers of cumulus cells was also examined.
Maturation of oocytes (IVM):
The COCs were cultured in groups of 10–30 in 100 μl of 9.5g/l TCM 199, 25mM/ml HEPES, 10% FBS, 50μg/ml gentamycin, 2.2 mg/ml NaHCO3, 22 μg/ ml pyruvic acid, 1 μg/ml estradiol, 0.5 μg/ml FSH and 0.03 UI/ml hCG at 38.50C in a humidified atmosphere of 5% CO2 for 68-72h.
Ocytes were washed 3 times in PBS-PVA in order to remove culture medium properly. They were dipped into a mixture of acetic: ethanol: chloroform (3:6:2), and then into a mixture of ethanol: acetic acid (3:1) for 48h. The oocytes were mixed with a small volume of PVSPVA on a slide and covered gently by a lamella. The slides were immersed in acetone for 15 minutes. Acetone was replaced by purple acetone orcein, and again the slides were immersed for 10 minutes. Finally acetone orcein was used instead of acetone glycerol in the dye. Staining stopped as the purple became white.
One ejaculate from each dog was collected in a calibrated plastic by digital manipulation (referred from Thai Thi My Hanh, 2001). The volume of each collection was determined, and semen was diluted in Trisglucose (3.025 g Tris; 1.25 g glucose; 1.7 g citrate natri; 100 mg penicillin; 100 mg streptomycine; 20ml egg yolk in 100 ml H2O; pH 6.82) at the ratio of 1:1 and preserved at 4 – 80C. Sperms were capacitated and collected by “swim up” protocol in Sperm-TALP-1 (CaCl2 2 mM; KCl 3.1 mM; MgCl2 0.4 mM; Na3PO4 0.3 mM; pyruvate natri 1mM; lactate natri 21.6 mM; BSA 10 g/l; Hepes 10 mM; gentamycin S 0.25 g/l; pH 7,21) in 5% CO2 at 37oC (Sirivaidyapong et al, 2000). The quality of the sperms was evaluated on activity, concentration, abnormal form, integrity of acrosome, intensity of motility before and after capacitation. Six samples of sperm were evaluated after preserving by 0, 24, 48 hours.