2011: Neutrophil prevalence in dismount semen samples of Thoroughbred stallions   Neutrophil prevalence in dismount semen samples of Thoroughbred stallions* Allan J Gunn, Victoria J. Brookes Barn Veterinary Services, Muswellbrook, NSW, Australia Introduction Semen dismount samples from Thoroughbred stallions are routinely monitored after natural mating.1,2 Due to the relative ease of obtaining the sample, it is tempting to use features such as neutrophil presence to predict fertility.3-6 There are reports that neutrophils in a dismount sample from a stallion may indicate an inflammatory or infectious process in the reproductive tract of that stallion, or that they may be from the mare with an inflamed, and possibly infected reproductive tract with which the stallion has mated.2 The presence of neutrophils in dismount samples has been reported to decrease the odds of pregnancy.7,8 However, neutrophil prevalence in dismount samples of stallions with no reported fertility problems is unknown. The aim of this study was to investigate the prevalence of neutrophils in stallion dismount samples from stallions with no perceived fertility problems. The study group consisted of commercial Thoroughbred stallions on a breeding farm in Eastern Australia. Keywords: Stallion, dismount semen sample, neutrophils Materials and methods Study design The seven Thoroughbred stallions chosen for the study were all used commercially for the full length of the 2009 southern hemisphere breeding season. No abnormalities of the reproductive tracts of any of the stallions were detected. The stallions all resided on the same farm. The sampling strategy was convenience sampling. Dismount semen samples from the distal penis and urethra were collected after natural mating from as many matings as possible, Monday through Friday from 10 September to 7 December 2009 (Figure 1). The date, the time of collection and the stallion were recorded for each sample. Sample processing and assessment The samples were examined within 24 hours of collection. After agitating the sample, a 25 microliter drop was smeared across a micrscope slide, air dried and stained with modified Wright Giemsa stain according to the procedure described by Schumacher and Moll9 (Figures 2 and 3). The length of the slide was scanned initially at 100x magnification, then more critically evaluated at 1000x magnification with an oil immersion objective. Numbers of neutrophils were counted in each field and the sample was categorized according to Table 1. The number of slides falling into each category was counted. Category Number of neutrophils per high power field (HPF) 0 0 1 <1 per 5 HPF 2 1 per 5 HPF - <1 per 3 HPF 3 >1 per 3 HPF- <1 per HPF 4 1-2 per HPF 5 >2 per HPF Table 1. Sample category according to number of neutrophils in stallion semen dismount sample.                                                              *Data presented at Australian College of Veterinary Scientists Science Week, 1-3 July 2010 and Association of Applied Animal Andrology 7th Biennial Meeting, Sydney, Australia, 28-29 August 2010.   Clinical Theriogenology • Volume 3 Number 4 • December 2011477   Figure 1. Collection of a dismount sample from the penis of a stallion. Analysis The prevalence of neutrophils in each category was calculated, with a 95% confidence interval. The number of samples collected from each stallion was compared to expected numbers (chi-square test), to determine that a particular stallion was not under- or over-represented by the sampling method. The number of samples containing neutrophils for each stallion was also compared to expected numbers (chi-square test), to investigate whether samples with neutrophils were more likely to come from one stallion than another. Data on the number of services for each stallion per week were not available. However, the number of samples collected each week and the number of samples with neutrophils each week was examined, to ascertain that the samples had been taken over the entire season, and to investigate whether neutrophil presence could be associated with a particular time of season. Clinical Theriogenology • Volume 3 Number 4 • December 2011 478     Figure 2. Neutrophils, stratified squamous cells and a spermatozoan.   Figure 3. Spermatozoa and ciliated epithelial cell. Anucleate Stratified  Squamous Epithelial  Cells.  Active Neutrophils  Aged/Degenerating  Neutrophils Spermatozoan Ciliated Epithelial  Cell Spermatozoa Clinical Theriogenology • Volume 3 Number 4 • December 2011479   Results Four hundred eighteen samples were collected (Table 2). Stallion Total services (n) Samples (n) Samples with neutrophils (n) 1 257 91 27 2 167 63 8 3 96 38 9 4 210 93 20 5 115 42 8 6 115 40 6 7 148 51 6 Total 1108 418 84 P value of x2 statistic P=0.71 P=0.20 Table 2. Total number of services, number of samples collected and number of samples containing neutrophils, by stallion. The samples were categorized according to neutrophil presence, and the prevalence of neutrophils calculated for categories 1 to 5 (Table 3). Category Number of Samples Percent of Total Prevalence of Neutrophils (95% confidence interval) 0 334 80.0 1 42 10.0 0.10 (0.16, 0.24) 2 11 2.6 0.03 (0.01, 0.04) 3 9 2.1 0.02 (0.01, 0.04) 4 12 2.9 0.03 (0.01, 0.04) 5 10 2.4 0.02 (0.01, 0.04) Total 418 100 0.20 (0.16, 0.24) Table 3. Number and percent of stallion semen dismount samples in each neutrophil score category. A graph was plotted of the number of samples collected weekly and the number of samples with neutrophils (categories 1-5) over time (Figure 4). Figure 4. Graph showing the weekly number of stallion semen dismount samples collected and the number of samples with neutrophils. 0 10 20 30 40 50 60 1 2 3 4 5 6 7 8 9 10 11 12 13 Week (10.09.09-07.12.09) N um be r o f S am pl es Samples Collected Samples with neutrophils Clinical Theriogenology • Volume 3 Number 4 • December 2011 480   Discussion The results of this study show that neutrophils are present in 20% (95% confidence interval, 16%-24%) of dismount semen samples from this population of Thoroughbred stallions with no detected fertility problems from a commercial breeding operation in Australia. The sampling strategy (convenience) did not significantly over- or under-represent any of the stallions (p>0.05). The number of samples containing neutrophils was not significantly different between each stallion (p>0.05). The sample collection appears to be spread over the entire breeding season. Samples with neutrophils are also evenly distributed over time, and appear to be of similar proportion to number of samples collected each week (Figure 1), indicating that neutrophil presence in stallion dismount samples is a consistent feature. The origin of the neutrophils was not determined in this study, and further work is required to establish whether they are from the mare or the stallion, or both. Further work is also required to determine the outcome of individual matings where neutrophils are present compared to those without neutrophils. Conclusion In this study, approximately 20% of dismount samples from Thoroughbred stallions contained neutrophils. In the authors’ opinion, neutrophil presence in a dismount semen sample should not be used as an indicator of stallion fertility. Acknowledgements The authors thank John Chopin for advice in preparing the manuscript. References 1. Umphenour NW, Sprinkle TA, Murphy HQ: Natural service. In: McKinnon AO, Voss JL, editors. Equine reproduction. Philadelphia: Lea and Febiger; 1992. p. 805-807. 2. Conboy HS: Significance of bacteria affecting the stallion’s reproductive system. In: Samper JC, Pycock JF, McKinnon AO, editors. Current therapy in equine reproduction. St. Louis: W.B. Saunders; 2007. p. 234. 3. Haag FM: Evaluation of ‘dismount’ semen in Thoroughbred horse breeding. J Am Vet Med Assoc 1959;134:312- 314. 4. Gravance CG, Champion Z, Liu IKM, et al: Sperm head morphometry analysis of ejaculate and dismount semen samples. Anim Reprod Sci 1997;47:149-155. 5. Love CC, Varner DD, Thompson JA: Intra- and inter-stallion variation in sperm morphology and their relationship with fertility. J Reprod Fertil Suppl 2000;56:93-100. 6. Koyago M, Nakada K, Tsunoda N, et al: Change in morphology of spermatozoa from dismount semen during the breeding season in Thoroughbred stallions in Japan. J Equine Sci 2009;20:1-5. 7. Blanchard TL: The subfertile stallion. In: Samper JC, Pycock JF, McKinnon AO, editors. Current therapy in equine reproduction. St. Louis: W.B. Saunders; 2007. p. 240-241. 8. Blanchard TL, Thompson JA, Brinsko SP, et al: Sources of variation in fertility of Thoroughbred stallions. Anim Reprod Sci 2010;121 Suppl:28-29. 9. Schumacher J, Moll H.D: Handling of fluid specimens. In: Schumacher J, Moll HD, editors. A manual of equine diagnostic procedures. Jackson (WY): Teton NewMedia. Internet Publisher: International Veterinary Information Service, Last updated: 31-May-2010. Available at: http://sss.ivis.ort/advances/schumacher/toc.asp Clinical Theriogenology • Volume 3 Number 4 • December 2011481 Clinical Theriogenology • Volume 3 Number 4 • December 2011 482 OMNIBLANK: