New indices for canine sperm morphology: the World Health Organization experience of multiple sperm defects

Indices of multiple sperm defects (Multiple Abnormalities Index, MAI; Teratozoospermic Index, TZI; Sperm Deformity Index, SDI) are routinely used in human medicine to evaluate sperm morphology according to the World Health Organization (WHO) guidelines (1). These indices allow a systematic record of all the abnormalities on a single gamete, and they correlate with in vitro (SDI) and in vivo (MAI, TZI) fertility. Our preliminary study applied MAI, TZI and SDI in dogs (2), pointing out that spermatozoa are seldom affected by multiple and associated abnormalities. However, since some sperm defects, often observed in canine species, are not considered in human indices, some adjustments might be needed. Therefore, in this study new indices of multiple sperm defects for canine sperm morphology are proposed, and their potential as fertility parameters based on their correlation with different in vitro function tests was assessed. Eighteen healthy stud dogs were included in the study. Ejaculated spermatozoa were collected by digital manipulation and used fresh (n. 6) or frozen (n. 6) according to the Uppsala method, whereas epididymal spermatozoa (n. 6) were collected from isolated testicles obtained by routine orchiectomies. In each group, subjective motility, membrane integrity (hypo-osmotic swelling test) and acrosome status (FITCPNA/PI) were assessed. Sperm morphology (Bengal Rose and Victoria Blue B staining) was evaluated by the WHO multiple-entry system (1), and hMAI, hTZI and hSDI (i.e. WHO human indices) were obtained. New indices for canine spermatozoa (cMAI, cTZI, cSDI) were calculated including common defects as cytoplasmic droplets (cMAI), detached heads (cMAI, cTZI) and acrosomal abnormalities (cMAI, cSDI). Finally, the Zona Pellucida Binding Assay (ZBA) was performed with homologous oocytes (n. 316) collected from frozen ovaries (3), and ZBA-adhesion indices (ZBA-ai, spermatozoa adherent to the zona divided by spermatozoa total number) were calculated. Spearman non-parametric test and Kruskal-Wallis test followed by multiple comparison test were used to assess correlations and differences among data, respectively. Significance was set at p<0.05. Motility, membrane integrity, acrosome status and ZBA-ai differed significantly between fresh (ejaculated and epididymal) and frozen-thawed samples (p<0.05), whereas indices of multiple sperm defects showed similar values among groups (mean ± SD). Since morphological indices did not differ, their correlation with function tests was analyzed on pooled data (18 dogs). Damaged acrosomes correlated positively (+0.71; p=0.001) with cSDI and negatively (-0.59; p=0.012) with ZBA-ai. A negative correlation (-0.478; p=0.045) between ZBA-ai and cSDI, but not hSDI, was observed, supporting the hypothesis that human indices are not predictive of dog in vitro fertility, and that species-specific indices are necessary. Specific parameters for dog semen evaluation and their correlation with fertility would be of high interest during andrological examination, and cSDI might be the first candidate for a clinical validation.1) World Health Organization, Department of Reproductive Health and Research. WHO laboratory manual for the examination and processing of human semen (fifth edition). 2010.(2) Morselli MG, Vernocchi V, Varesi S et al. Indices of multiple sperm defects applied to dog semen. Proc. 16th Congress EVSSAR, Toulouse (France),5th-6th July 2013, p.158(3) Holst BS, Larsson B, Rodriguez-Martinez H et al. Zona pellucida binding assay- a method for evaluation of canine spermatozoa. J Reprod Fertil 2001;57:137-40.