Research Article: Conception rate, uterine infection and embryo quality after artificial insemination and natural breeding with a stallion carrier of Pseudomonas aeruginosa: a case report

Date Published: March 29, 2012

Publisher: BioMed Central

Author(s): Guimarães Tiago, Carvalheira Júlio, Rocha António.


Pseudomonas aeruginosa may cause venereal disease and infertility in horses. A Pseudomonas aeruginosa – carrier stallion, often unresponsive to artificial vagina collection, was used to naturally breed mares. Semen collected from the same stallion was also used to perform artificial inseminations. Pregnancy rates, embryo quality and incidence of uterine infection were compared between inseminated or naturally-bred mares.

P. aeruginosa was isolated from swabbing of the penis, prepuce and distal urethra of the stallion. Before being bred or inseminated, clitoral/vestibular samples were collected from all mares, and cultured for isolation of P. aeruginosa. At the first observed estrus, endometrial swabs were also collected. All mares subjected to natural mating (NS) were re-evaluated for P.aeruginosa by culture of clitoral and endometrial swabs. Artificial inseminations (AI) were performed either with fresh-extended semen (11 AI/7 mares) or frozen semen (10 AI/7 mares). The stallion was also used to breed 3 mares (4 services). For embryo collection, 2 mares were inseminated with fresh-extended semen (1 AI/mare), and 2 additional mares were inseminated with frozen semen (2 AI/mare). Two mares were naturally-bred with a total of 9 services, for embryo collection. All mares were examined after AI or natural service (NS), for uterine pathologies. Embryo recoveries were attempted passing a catheter with inflatable cuff connected to a sterile flexible 2-way flushing catheter, through the cervix. Flushed media was recovered into an Em-Con filter, and embryos searched using a stereoscope. Embryos were graded from 1 (excellent) to 4 (degenerated/dead).

Pregnancy rates obtained after NS was 50% per cycle. However, more than half of the NS resulted in uterine disease, while uterine pathology was seen only in 22% of the time following AI. Half of the mares bred by NS got positive to P. aeruginosa. Percentage of embryo recovery rates was identical after AI or NS (66.7%). The 4 embryos recovered after AI were classified as Grade 1, while after NS only 2 out of the 6 recovered embryos were Grade 1.

a) there was no evidence of reduced fertilization after AI or NS, b) a numerically higher incidence of uterine disease was noticed after NS, c) venereal transmission of P. aeruginosa after NS was confirmed, d) a lower percentage of G1 embryos may be obtained after NS. Overall, the data supports the indication for P. aeruginosa-carrier stallions to be bred by AI rather than by NS, and raises the possibility that P. aeruginosa may affect embryo quality.

Partial Text

Horses may harbor Pseudomonas aeruginosa in the genitalia in percentages that ranged from 4% to 10% in mares and as high as 36% in stallions [1,2]. P. aeruginosa 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]. Pseudomonas aeruginosa is frequently multi-resistant to antibiotics 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 bacteria. Therefore, artificial insemination (AI) associated with minimum contamination techniques is considered to have a distinct advantage over natural service to control 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.

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.

The results of this study were based on a limited number of observations and thus no solid inferences can be made about the fertilizing ability of inseminated semen vs. NS for this stallion. Notwithstanding that, conception rates and embryo recovery rates after AI with fresh and frozen semen were in the normal range obtained in our centre. Not all ejaculates were tested for the presence of P. aeruginosa, but the fact that the bacteria was not isolated from 8 fresh, undiluted ejaculates raises the possibility that it was not present in the semen or had a very low concentration. As no isolations were obtained in undiluted semen, none should be expected in extended semen even if without antibiotics (E-Z Mixin®) or even less when containing antimicrobials (Botu-crio®). The somewhat lower conception rate achieved with frozen semen may be attributed, at least in part [10], to degenerative conditions of the endometrium of two older mares (18 years-old) which had a grade III Kenny’s endometrial classification. Pregnancy rates as well as embryo recovery rates obtained after NS with the infected stallion were acceptable despite having more than half of the NS resulting in uterine disease compared to only in 22% following insemination. These positive results may have been due, at least in part, to the intensive uterine treatments performed right after every natural service, up till day 4 post ovulation. Confirmation of venereal transmission of P. aeruginosa and was obtained with 2 out of the 4 mares bred by natural service getting positive to P. aeruginosa and thus acquiring the potential to transmit the bacteria.

With this carrier stallion, there was no evidence of low fertilization rates after AI or NS, but uterine disease was noted more often after NS than after AI. Venereal transmission of P. aeruginosa after NS did occur, and was associated with lower embryo quality after NS compared to AI. Overall, the data supports the indication for P. aeruginosa – carrier stallions to be bred by AI rather than by NS, both when used to impregnate mares or to breed embryo donors.

The authors declare that they have no competing interests.

AR and TG executed the field work and collected the data, TG wrote the first draft of the manuscript, AR and JC produced the final version of the manuscript. All authors read and approved the final manuscript.