Advances in canine semen evaluation techniques

Abstract

 Veterinarians are frequently asked to evaluate dog semen for a variety of reasons, 6 including but not limited to: breeding soundness examination, shipment of fresh, cooled semen, 7 cryopreservation; after a conception failure; after an illness suspect to affect fertility; or after 8 testicular neoplasia or prostatic disease is diagnosed. Ultimately, the goal of any semen 9 evaluation is to predict how likely it is for a male to be successful when used in a breeding 10 situation. Standard semen evaluation in the dog includes determination of semen volume, 11 spermatozoal motility (both total and progressive), velocity, concentration, total 12 spermatozoa/ejaculate, and morphology. Semen morphology during a typical breeding 13 soundness examination is typically performed using one of two stains: Wright-Giemsa or eosin14 nigrosin. In the dog, standards for normal semen parameters include a semen concentration of 15 > 10 million spermatozoa/kg bodyweight; >70% progressively motile sperm, and >70% 16 morphologically normal spermatozoa. Many times, semen quality will exceed these parameters 17 yet fertility of the dog may still be suboptimal. If infection and prostatic disease can be ruled out 18 as causes of the subfertility, the clinician is left with attempting to further evaluate the ejaculate 19 to determine the cause of the infertility. Evaluation of the sperm’s function is the next logical 20 step. 21 Male factor infertility is said to account for up to 50% of the failed pregnancy attempts in humans.1 22 No estimates have been made for the canine, but clinical experience would tell us 23 that male factor infertility accounts for a significant portion of either non-pregnancy, early 24 embryonic death, or small litter size. When a standard semen evaluation fails to elicit a clear 25 diagnosis of infertility, additional testing of the ejaculate is desirable. This testing may include 26 additional bright field spermatozoal staining techniques which differentiate different parts of the 169 27 sperm cell; alternate microscopic evaluation of sperm morphology using differential interference 28 contrast or phase contrast morphology; electron microscopy (EM), both scanning and 29 transmission; acrosomal testing; hypo-osmotic swelling testing (HOST); sperm chromatin 30 structure analysis (SCSA); computer assisted spermatozoal analysis (CASA/ASMA); fluorescent 31 antibody staining techniques using flow cytometric analysis; anti-sperm antibody (ASA) testing; 32 assays for reactive oxygen species (ROS); chromosomal studies; and sperm function testing 33 including zona binding assays, sperm penetration assays. 34 While there is some information on the canine in this regard, much of the information 35 presented will be from human, bovine, and other domestic species research, where this topic 36 has been far more extensively evaluated. The data from other species can be extrapolated to 37 the canine although more research is needed to determine if its use is appropriate in the dog as 38 a predictor of fertility. This paper describes the use of these additional diagnostic methods of 39 spermatozoal analysis for infertility assessment. CASA will only be covered briefly as another 40 paper in this symposium is dedicated to its use.