Pseudomonas aeruginosa was repeatedly (6 times) isolated 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 antibiotic-free extender (E-Z Mixin®, Animal Reproduction Systems, Chino, CA, USA), and 6 samples of semen frozen with Botu-crio®, which contains amikacin. However, at the end of the breeding season one more sample collected 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 inflammatory cells were detected in the ejaculates and ultrasound examination (Aloka Prosound 2 with 5 and 7.5-10 MHz probes, Aloka Holding Europe, Steinhausertrasse, Switzerland) of the bulbourethral glands, prostate, seminal vesicles and ampullae did not evidence any sign of 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 concentration 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 curvilinear 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 several years.
Before being bred or inseminated with the stallion, clitoral/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/vestibular cultures, a sterile swab was utilized and the ventral 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 university'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 - Chorulon®
, 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 concentration of 50 × 106
sperm cells/mL in antibiotic-free extender (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 performed within 6 hours post ovulation with approximately 300 × 106
sperm cells with progressive motility. The stallion was also used to breed mares by NS. For that, the same management used for AI including the cleaning of the stallion's penis was used, with the AI replaced by NS. No filling of the uterus with extender immediately prior to NS as described originally for the minimum contamination technique [7
] was done, to allow for the confirmation of ejaculated semen in the uterus by ultrasound scanning. Ultrasound examinations were repeated daily after AI or natural service to confirm ovulation and to assess uterine clearance until day 4 post-ovulation.
When uterine fluid was present 24 hrs post AI (delayed uterine clearance/endometritis) mares were treated with 30 IU of oxytocin i.v. (Placentol®, CEVA, Portugal) 2 to 3 times daily and the uterus was flushed with saline. One mg/day of Cetiofur (Excenel®, Pfizer Manufacturing, Puurs, Belgium) was infused in the uterus up till day 4 post ovulation when purulent uterine exudates were recovered after uterine flushing, or when a neutrophile-positive uterine swabbing was obtained 24-hrs after AI, and in all mares bred by NS.
Eleven inseminations were performed with fresh-extended semen in 7 mares. Ten inseminations were performed with frozen semen in 7 mares. Three mares were hand mated by NS to get impregnated, two of which in a single estrous cycle and other in two estrous periods. One of these mares had turned positive to P. aeruginosa after previously being bred by NS for embryo collection. Ultrasound examination confirmed pregnancies 12 to 16 days post ovulation.
For embryo collection, 2 mares were inseminated with fresh-extended semen (1 AI/mare), and 2 additional mares were also inseminated with frozen semen (2 AI/mare), in two consecutive cycles. Two mares were allocated to be bred by NS for embryo collection. A total of 9 natural services were performed in these 2 mares. Embryo recoveries were attempted passing a catheter with inflatable cuff through the cervix, connected to a sterile flexible 2-way flushing catheter, after careful washing of the vulva. Once in the body of the uterus, the cuff was inflated with 50 to 70 mL of air and the catheter pulled backwards onto the internal os of the cervix to seal off the uterus. Between 2 and 3 Lts of warmed Ringer lactate (Laboratorio sorologico, Amadora, Portugal) for embryo collections were infused into the uterus. The media was recovered by gravity flow into an Em-Con filter, after which the cuff was deflated to allow the withdrawal of the catheter. The medium in the filter was than transferred to a Petri dish and embryos searched using a stereoscope. The embryos were graded from 1 (excellent) to 4 (degenerated/dead) as reviewed by McCue et al.
All statistical procedures for analysis of the data were computed using SAS software [9
]. Fisher's exact test was dimmed the appropriate statistic to assess the univariate associations between the outcome of interest (positive vs negative to AI with fresh and frozen semen and NS) with pregnancy per cycle, uterine pathology and frequencies of Grade 1 embryos. Differences were considered significant when P < 0.05.
Authorization was granted, from the owner of the animals, to use the data for a scientific publication.