Over the last four decades, there has been increasing concern over declining human male reproductive health. Reduced sperm counts have been widely used as an index of male subfertility and meta-analytical studies indicate a 50% global reduction in quality from 1938 to 20111,2,3. Sperm morphology has also been reported to decrease over a period of 17 years in France with some geographical regional variation; Aquitaine and Midi-Pyrenees having the lowest morphology combined with the lowest concentrations4,5. These data are indicative of an environmental aetiology and, in support of this, epidemiological studies showing increased incidences of testicular cancer and malformations at birth have been linked to regions with reduced sperm counts6,7.

Temporal trends in human semen quality are paralleled by a similar trend in dogs that live in the human household, where sperm motility declined by 30% over a 26 year period8. In this latter study, all data was generated from a single laboratory using consistent techniques and thus did not suffer from changes in methodology and quality assurance over the time span encompassed in human meta-analytical studies9. These observations support the hypothesis that temporal trends in semen quality, both in the human and dog, are due to shared environmental factors and that the dog may be a sentinel for human exposure to such factors. Access to a controlled breeding population of assistance dogs that are routinely sampled for sperm quality provides a cost-effective means of sperm analyses without the stigma and social complications that accompany analogous human studies. Furthermore, there is considerable potential to extend these analyses to any individual or population of dogs. For example, although not investigated in the current study, this could be achieved by semen collection from the tail of the epididymis10 immediately after removal of dog testes at routine surgical neutering; a procedure that is performed on hundreds of thousands of dogs worldwide each year. In addition, semen collections from live dogs is a procedure that is tolerated by a majority of breeds11 unaccustomed to routine fertility monitoring and can therefore easily be carried out by a trained technician.

Declining sperm quality has been linked with the exposure to persistent anthropogenic chemicals, many of which exhibit endocrine disrupting activity6. Although the mechanisms underlying these putative effects are uncertain, historically, the period of fetal development has been highlighted as being particularly sensitive to chemicals with endocrine disrupting activity12. However, a number of studies have shown that environmental chemicals (ECs) are present in semen in a range of species, including the human, raising the possibility of a direct acute effect of chemicals on sperm13,14,15. In support of this theory, an elevated concentration of seminal bisphenol A (BPA) has been associated with infertility in men15,16 and elevated human seminal phthalate metabolites have been associated with reduced sperm counts17. In a separate study, the phthalates DEHP and di-n-butyl-phthalate (DBP) in human semen were reported to be inversely associated with motility and this was confirmed by the direct application of the same phthalates, at seminal concentrations, to sperm in vitro18. By contrast, PCB congeners 118, 126 and 153 were reported to have no negative effect on human sperm motility in vitro, both individually and in combination19. Similar findings have been reported in the dog where DEHP and PCB153, at concentrations detected in dog semen and testis, exhibited inhibitory and stimulatory effects respectively when tested on sperm motility in vitro8. In the same study, both DEHP and PCB153 were detected in a range of dry and wet dog foods indicative of a dietary source. Both DEHP and PCB153 are widely present in the environment and have been detected in tissues/fluids ranging from human breast milk to ovine liver. DEHP is a widely used plasticizer that leaches out into food and liquids and PCBs are lipophilic, and are therefore present in fatty foods20,21,22. Consequently, exposure occurs largely through the diet and these chemicals are deemed as risk factors for reproductive function23,24,25. In support of this, our own published study has shown that DEHP, PCB153 and other PCB congeners are present within both dry and wet dog food sources8 [DEHP: wet and dry food, 0.37 ± 0.10 and 0.20 ± 0.03 μg/g respectively; ∑PCBs: wet and dry food, 1.35 ± 0.5792 and 0.78 ± 0.223 μg/kg respectively; PCB153: wet and dry food, 0.39 ± 0.193 and 0.22 ± 0.11 μg/kg respectively].

Another parameter of ejaculate quality is the proportion of sperm that exhibit DNA fragmentation26. Environmental chemicals have been shown to induce both human and dog sperm DNA fragmentation8,27 and a commercial mixture of PCBs (Arochlor), administered to rats in vivo and added to sperm in vitro, also increased sperm DNA fragmentation28.

In total, these data suggest that environmental chemicals induce similar acute effects on human and dog sperm in vitro: measurement of sperm motility and DNA fragmentation are tried and tested measures of such chemical effects. Since sperm concentration would not alter during the period of in vitro culture and morphology is confounded by abnormality classification, partly due to swelling that may occur during culture and the generation of artefacts during processing, these parameters were not selected for testing acute chemical effects in vitro29,30. Notwithstanding, testing the effects of individual chemicals on sperm functional parameters does not represent “real-life” exposure to a mixture of chemicals, many of which exhibit synergistic, antagonistic or additive effects. Two chemicals known to be present in dog seminal plasma and testis were therefore selected and their effects tested both independently and in combination, on sperm motility and DNA fragmentation in both the human and dog.