Tiago et al. Acta Veterinaria Scandinavica 2012, 54:20 http://www.actavetscand.com/content/54/1/20 Background Horses may harbor Pseudomonas aeruginosa in the gen- italia in percentages that ranged from 4% to 10% in mares and as high as 36% in stallions [1,2]. P. aerugi- nosa can cause venereal disease and infertility in the equine [3,4] and it is generally accepted that stallions contaminated with this bacteria need to be considered potential carriers of a venereal-transmitted disease [5], in particular when used to breed older mares [2]. Pseu- domonas aeruginosa is frequently multi-resistant to anti- biotics and is the most potent biofilm - producing pathogen of the equine reproductive tract [6]. Washing of the stallion penis with disinfectants may lead to unwanted colonization by yeasts and pathogenic bac- teria. Therefore, artificial insemination (AI) associated with minimum contamination techniques is considered to have a distinct advantage over natural service to con- trol the transmission of these bacteria [3]. However, the effect of those techniques on the control of the disease and even the rate of transmission by infected stallions after natural service (NS) is poorly documented and the effect of NS by P. aeruginosa-carrier stallions in embryo quality have not been reported. In the present work we decribe the conception rates obtained after NS with a stallion carrier of P. aeruginosa, the conception rates after AI with fresh extended and frozen/thawed semen of the same stallion, the incidence of uterine disease with either method of breeding, as well as embryo recovery rates and embryo quality after AI and NS. Materials and methods Pseudomonas aeruginosa was repeatedly (6 times) iso- lated from swabbing of the penis, prepuce and distal urethra in a Holstein stallion stationed at the Centre of Animal Reproduction of Vairão, ICBAS, University of Porto. Undiluted samples from eight different ejaculates collected with artificial vagina (Missouri model) after cleaning of the stallion’s penis with warm water were negative for P. aeruginosa. Also negative were 3 samples of semen from the same ejaculates, extended with anti- biotic-free extender (E-Z Mixin®, Animal Reproduction Systems, Chino, CA, USA), and 6 samples of semen fro- zen with Botu-crio®, which contains amikacin. However, at the end of the breeding season one more sample col- lected from the stallion’s genitalia resulted positive to P. aeruginosa. Antibiotic susceptibility tested by the disk diffusion method showed that the bacteria were multi- resistant to 21 of the 25 antibiotics used. No inflamma- tory cells were detected in the ejaculates and ultrasound examination (Aloka Prosound 2 with 5 and 7.5-10 MHz probes, Aloka Holding Europe, Steinhausertrasse, Swit- zerland) of the bulbourethral glands, prostate, seminal vesicles and ampullae did not evidence any sign of Page 2 of 5 pathology. Successful semen collection with artificial vagina (AV) was intermittent, and the stallion’s owner expressed his desire to have his mares bred by NS when a successful collection with AV could not be obtained, despite being aware of the potential risk of P. aeruginosa transmission. Ejaculates (n = 8) had a mean (± SD) volume of 182.5 ± 67.1 mL (gel-free), with a concentra- tion of 103.4 ± 33.0 sperm cells/mL, with a subjective (optical microscopy) progressive motility of 53.1% ± 5.3%. Three ejaculates evaluated by a computer assisted sperm analysis system (ISAS®, Proiser) had a progressive motility of 34.6% ± 16.8%, a total motility of 74.5% ± 14.1%, 21.1% of sperm cells with rapid movements (> 25 μm/s), a linear velocity (VSL) of 25.8 ± 8.8 μm/s, a cur- vilinear velocity (VCL) of 66.7 ± 23.4 μm/s, and a mean velocity (VAP) of 40.5 ± 11.6 μm/s. All mares had an intensive sports background, were 12 to 18 years old and had not been bred for at least sev- eral years. Before being bred or inseminated with the stallion, cli- toral/vestibular samples were collected from all mares at the time of admission in the center, and cultured for isolation of P. aeruginosa. At the first observed estrus, endometrial swabs were also collected. For clitoral/ves- tibular cultures, a sterile swab was utilized and the ven- tral aspect of vestibule, as well as the central clitoral fossa was swabbed. Endometrial swab was performed in mid-to-late estrus, utilizing a guarded swab (Ref. 17214- 2950, Minitüb Ibérica, Barcelona, Spain), passed through the cervix into the uterine lumen, protected in a sterile gloved hand. The swab was then retracted into its sterile sheath. Clitoral/vestibular and endometrial swabs were placed immediately into a sterile centrifuge tube with transport medium and sent refrigerated to the univer- sity’s microbiology lab, in less than two hours. All mares subjected to natural mating with this stallion were re- evaluated for P.aeruginosa by culture of clitoral and endometrial swabs. Mares were teased every other day using a stallion. After signs of behavioral estrus, the mares’ uterus and ovaries where examined daily by ultrasound scanning (Aloka Prosound 2, 5 and 7.5-10 MHz probes) and 1500 UI of human chorionic gonadotropin (hCG - Choru- lon®, Intervet, Portugal) was administered (iv) after detection of a follicle with a diameter ≥ 35 mm. Twenty four hours later semen was collected after cleaning of the penis with warm water and diluted at a concentra- tion of 50 × 106 sperm cells/mL in antibiotic-free exten- der (E-Z Mixin®, Animal Reproduction Systems, Chino, CA, USA), and AI was performed with approximately 450 to 600 × 106 sperm cells with progressive motility. Artificial insemination with frozen semen was per- formed within 6 hours post ovulation with