Tiago et al. Acta Veterinaria Scandinavica 2012, 54:20 http://www.actavetscand.com/content/54/1/20 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 includ- ing 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 exami- nations 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 uter- ine 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 pregnan- cies 12 to 16 days post ovulation. For embryo collection, 2 mares were inseminated with fresh-extended semen (1 AI/mare), and 2 addi- tional 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 collec- tion. A total of 9 natural services were performed in these 2 mares. Embryo recoveries were attempted pas- sing 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 (Labor- atorio sorologico, Amadora, Portugal) for embryo col- lections 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 trans- ferred to a Petri dish and embryos searched using a stereoscope. The embryos were graded from 1 (excel- lent) to 4 (degenerated/dead) as reviewed by McCue et al. [8]. Page 3 of 5 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 fre- quencies of Grade 1 embryos. Differences were consid- ered significant when P < 0.05. Authorization was granted, from the owner of the ani- mals, to use the data for a scientific publication. Results Pregnancy rates; metritis/delayed uterine clearance Pregnancy rates per cycle were not significantly different (P > 0.3). Data on pregnancy rates obtained after AI and NS are detailed in Table 1. All 7 mares inseminated with fresh-extended semen became pregnant. Only one of these mares required post-AI treatment for delayed uterine clearance. No delayed uterine clearance was detected in the 2 mares inseminated with fresh-extended semen for embryo collection, and one embryo was collected. Four of the 7 mares inseminated with frozen semen got pregnant (57%). Embryos were recovered in 3 out of 4 flushings (75% recovery rate). Two of the 3 mares bred by NS became pregnant. P. aeruginosa was isolated from one pregnant mare (cli- toral swab) and from one of the mares that did not get pregnant (clitoral and uterine swab). Embryos were col- lected in 6 out of 9 flushings (66% recovery rate). Differences in uterine pathology between mares inse- minated or subjected to NS approached significance (P = 0.07). In total, 6 cases (22%) of delayed uterine clear- ance/endometritis were noted after the 27 AIs per- formed with fresh or frozen/thawed semen, either for pregnancy or for embryo collection, while 7 cases (54%) of endometritis were diagnosed after the 13 natural ser- vices performed by the stallion for the establishment of pregnancies or for embryo collection. Embryo quality The difference in frequency of Grade 1 embryos pro- duced by AI or NS approached significance (P < 0.08). The embryo recovered after AI with fresh semen was classified as a day 8, Grade 1 blastocyst. Two day 8 Grade 1 blastocysts and a day 10 Grade 1 expanded blastocyst were obtained after insemination with frozen semen. In relation to embryo quality after natural breed- ing, the results were as follow: one Grade 4 morula after a day 8 flushing, a Grade 2 morula and a grade 1 blasto- cyst after a day 8 flushing of a mare with two ovulations within a 3 day interval, a Grade 2 and a Grade 4 blasto- cysts in two nine-day flushings, respectively, and a Grade 1 expanded blastocyst after a day 10 flushing.