Measurement of 16-Androstenes (5α-Androst-16-en-3-One, 5α-Androst-16-en-3α-ol, 5α-Androst-16-en-3β-ol) in Saliva of German Landrace and Gottingen Minipig Boars

Two C16-unsaturated steroids (16-androstenes), 5a-androst-16-en-3-one with a urine-like odour and 5a-androst-16-en-3a-ol with a musk-like odour are present in salivary glands and saliva of mature male pigs. Both 5a-androstenol and 5a-androstenone have a pheromonal function in the pig. They are exhaled by sexually excited boars and stimulate oestrous behaviour in females. Concentrations of 2 ng/ml 5a-androst-16-en-3-one occur in saliva. In European wild and domestic boars there is seasonal variation in gonadal function with plasma testosterone increasing from similar to 0.5 to similar to 10.0 ng/ml during the mating season under short-day photoperiod. Anecdotal evidence suggests that Gottingen minipig boars (GMB) are more potent in stimulating females during oestrus than boars commonly used in swine production, which might be due in part to differences in salivary 16-androstene content. We analysed 16-androstene in saliva of GMB and German landrace boars (GLB). A total of 10 GMB and 16 GLB were sampled either in December (GMB) or February (GLB), and saliva (0.5 to 2.0 ml) was obtained during semen collection using a collection systems designed for use in humans. Salivary 5a-androst-16-en-3-one, 5a-androst-16-en-3a-ol and -3b-ol were determined using GC-MS analyses with external standard curves. The MS acquisition was performed in SIM by monitoring the ions m/z 272 for 5a-androst-16-en-3-one, m/z 274 for 5a-androst-16-en-3a-ol/-3b-ol and m/z 202 for 5a-androst-3-one (internal standard). A calibration line prepared by adding various amounts of the analytes and a constant amount of 5a-androst-3-one was used for quantification. In addition, salivary testosterone was determined using an in-house enzyme immunoassay (EIA). Mean concentrations of 5a-androst-16-en-3a-ol were 0.18 +/- 0.13 and 9.20 +/- 8.57 mu g/ml, respectively, in GLB and GMB. 5a-androst-16-en-3b-ol concentrations were 0.02 +/- 0.01 and 0.42 +/- 0.37 mu g/ml, respectively. The urine-like compound 5a-androst-16-en-3-one was 0.04 +/- 0.03 and 1.70 +/- 1.42 mu g/ml, respectively. Our results show that both odorous substances were about 50 times higher in GMB than in GLB which indicates a difference in salivary 16-androstene in these two pig breeds. The fact that salivary testosterone was 3.05 +/- 3.32 ng/ml in GLB versus 223.7 +/- 266.3 ng/ml in GMB supports an effect of breed. However, a seasonal effect cannot be ruled out since samples from GMB were obtained in the middle of the breeding season when sexual activity was highest, while GLB were collected at the end of the breeding season.