The findings from the current study are significant for the following reasons. They document, for the first time, a decline in the semen quality of a population of stud dogs over a period of 26 years. Although in some years semen quality parameters temporarily increased as noted in other studies22, there was a repeated decline in motility even after removal of animals with the poorest semen quality. Contemporaneous with the decline in semen quality, offspring from the same population of dogs showed an increased incidence of cryptorchidism from 1995 (first available records) to 2014, a decline in the proportion of male pups at birth, and a temporal increase in female mortality. The decline in number of males relative to the number of females was lost when stillborn pups/early post-natal deaths were excluded. Significantly, we report that the endocrine disrupting chemicals PCB153 and DEHP are detectable in canine testes collected from dogs from the same location as the temporal study. Furthermore, the same chemicals were detected in a range of commercially available dog foods. Since testicular concentrations of these chemicals directly perturbed sperm motility and viability, this may be a mechanism by which environmental chemicals directly affect male fertility.

In the current study, it was not our intension to demonstrate temporal changes in semen or testicular chemical content and to correlate with changes in semen quality. Indeed, such screening is confounded by the need to pool individual semen samples, the lack of historical samples and access to the technology at the start of the study. Rather, we have focussed on temporal changes in semen quality and have demonstrated that ECs are able to influence motility and other parameters of semen quality. Similarly, correlating the decline in semen quality in individual stud dogs with cryptorchidism in their offspring would be difficult since an individual stud dog may have a breeding career of 8 years and may produce several hundred male offspring. Thus although the relationship between semen quality, cryptorchidism and ECs is not demonstrated through a longitudinal or cross-sectional study design, we postulate that the rapidity of the temporal changes observed are indicative of an environmental aetiology and that our in vitro data support this contention.

The original meta-analysis reporting a decline in human semen quality spanned a period of 53 years (1938–1991)1. Although this time frame is extensive, the study outcomes have recently been criticised on the basis of changes in laboratory methods, training of laboratory personnel and improved quality assurance over the years4. Since additional studies have either supported or refuted the original claims, declining human semen quality remains controversial. In contrast, data reported here on canine sperm quality have been generated using sperm analysis methods that have remained consistent throughout the 26 years of the study with uniform input from three experienced technical staff and one of the senior authors (GE). Indeed, although there was some variability between technical staff, this was in line with, or better than, other studies23. Given that the current study is devoid of the confounding factors inherent in human meta-analyses, we present a unique dataset showing a reliable temporal decline in semen quality which, in this case, was observed in a controlled population of stud dogs. This raises the tantalising prospect that the decline in canine semen quality has an environmental cause. Indeed it is unlikely that the effect is heritable since in this population of dogs heritability measures were low for all parameters except for the heritability of high sperm motility and low total sperm output24.

The decline in percent normal morphology sperm from 1988 to 1994 followed by an increase and apparent subsequent plateau is difficult to interpret. Although a temporal decline in human sperm morphology has been reported and attributed to environmental factors3, the degree to which this accounts for the temporal trends in canine sperm morphology is uncertain. Furthermore, the increase in total sperm output contrasts with the decline in motility and morphology and with human studies and meta-analyses showing a decline in sperm counts. One factor that may have influenced the increased sperm output from 2002 to 2005 is that between 1999 and 2001 dogs with poor semen quality were removed. Since this included dogs with less than 150 million sperm, their removal may have constituted a bias towards increased sperm output. Indeed a similar temporal increase in sperm output has been reported in bull semen between 1985 and 1995, and a comparable selection bias was suggested22.

In the human, cryptorchidism affects 2 to 9% of new born infants25 and published data generally supports a temporal increased incidence and clear geographic differences in the prevalence25,26. As a key manifestation of “testicular dysgenesis syndrome” in the human, data presented here illustrates a similar increased incidence of cryptorchidism in a population of stud dogs comprising 5 breeds. While inbreeding in dogs has been linked to cryptorchidism, it is generally accepted that the pathology is underpinned by an interaction between genetic, epigenetic and environmental factors27. It is however difficult to tease out the relative contributions of these drivers. Whilst we were unable to undertake chemical analysis on the cryptorchid testes, it is an interesting observation that all of these cryptorchid pups were produced from the stud dogs donating the semen and described in Fig. 1. Clearly, study of the effect of exposure of the pregnant dam on male fetuses are warranted. In a study of 1,339 litters of 4 different canine breeds, litters with 1 or 2 cryptorchids were reported to exhibit an imbalanced sex ratio in favour of males28. In a separate canine study in which cryptorchidism carriers were intentionally selected for further breeding, a similar increased male to female ratio (in favour of males) was observed at weaning along with reduced numbers of females per litter29. Both of these reports contrast with the current study in which cryptorchidism carriers were purposely excluded from the breeding stock and there was no effect on the male:female ratio of live pups at birth. However, if stillbirths and early post-natal deaths were included, there was a decrease in male:female ratio, at the expense of males, accompanied by an increase in female mortality and decline in male mortality. Although previous studies of temporal trends in canine stillbirth rates have shown a significant fluctuation with time (1978–2005), gender specific changes have not previously been described28.

In the current study, although the contemporaneous increase in cryptorchidism and female mortality likely occurs through a separate mechanism, a lethal effect on females induced by genes that cause cryptorchidism has previously been proposed30. However, this contention was put forward to account for a ‘high sex ratio’ in favour of males in litters with cryptorchids, which contrasts with the reduced proportion of males per litter reported in the current study. Notwithstanding, the rise in cryptorchidism, fall in male:female ratio and increased female mortality all alter with time and the former is also related to declining sperm quality for which there is substantial evidence of an environmental trigger.

The dog is probably man’s closest companion and by sharing the same habitat, is likely to be exposed to similar environmental conditions including environmental chemicals. This was especially true in the current study since the dogs lived in homes with their handlers. Twelve chemicals detected in adult dog testes (DEHP, 7 PCB congeners, 4 PBDE congeners) were also detected in the 15 commercially available dog foods analysed. Since this indicated a possible food source of exposure, two chemicals (PCB153 and DEHP) were selected for further in vitro study on the basis of (1) high abundance in both testis and some pet foods and (2) both chemicals, or their metabolites, are reported to be present in seminal plasma of farm animals and in men at similar concentrations to those reported in the current study31,32. In addition, dietary/oral exposure to DEHP or PCB153 alters semen quality in animal models33,34 and human male infertility has been associated with elevated levels of a mix of PCB congeners in seminal plasma and DEHP35. In the current study, 6 PCB congeners and 4 PBDE congeners were detected in entire ejaculate pools at concentrations greater than that observed in the testes. Since testicular concentrations of PCB 153 and DEHP directly impacted on sperm function and viability, it is axiomatic that the higher concentrations found in seminal plasma will similarly alter the parameters measured.

In the current study, we recognise that the lowest chemical concentration used did not equate with a “no adverse effect level (NOAEL)”. Although a wider range would be preferable, we have shown effects of PCB153 and DEHP at low environmentally relevant concentrations. The mechanism by which PCB153 and DEHP alter sperm motility and viability is uncertain. Indeed, previous studies of PCB and DEHP effects on sperm quality have yielded mixed results. For example, in the human, a number of studies report an inverse relationship between serum PCBs and sperm quality36. In separate studies of seminal ECs across human populations selected on the basis of variable organochlorine exposure, increased seminal PCB153 was consistently associated with reduced sperm motility37,38. In contrast, the sum of 4 PCB congeners in human blood plasma, including PCB153, has been reported to be lower in samples from men with infertility and, although negatively associated with testosterone, was positively associated with sperm quality39. Although this latter study is consistent with the positive effect we report of PCB153 on sperm motility, establishing a clear effect of single or multiple PCB congeners is complex, likely due to effects of confounding blood and seminal ECs. Similar mixed results have been observed in relation to DEHP and sperm quality. For example, one study from China reported that human urinary phthalate metabolites, measured as an index of DEHP exposure, were associated with increased sperm DNA damage and apoptosis40. In contrast, a separate study from the USA found very little association between DEHP metabolites and a range of sperm parameters including motility41. A further study from Sweden however, reported a negative correlation between urinary DEHP metabolites and sperm motility42. Phthalate esters have also been measured directly in human semen and reported to be inversely associated with sperm motility43. Notably, the authors report that the same inverse relationship was recapitulated in vitro when semen concentrations of phthalates were added directly to sperm. These findings support our in vitro findings of an inhibitory effect of DEHP on parameters of sperm motility.

In the current study, although the chemical effect on any independent parameter of sperm motility was subtle, the consistent differing effects of PCB153 and DEHP on all four motility parameters may have greater physiological relevance. Both chemicals are known endocrine disruptors with PBDE and PCB considered to have pro-estrogenic or anti-androgenic activity44 whereas DEHP and its primary metabolite mono-2-ethylhexyl phthalate (MEHP) are reported to exhibit pro- and anti-androgenic activity respectively45,46. The differing effects that PCB153 and DEHP have on sperm motility may reflect their respective actions on estrogen or androgen receptors, both of which are expressed by sperm47,48. However, it is recognised that many environmental chemicals operate through other endocrine and non-endocrine mechanisms, one or more of which may account for the direct effects on reproductive function. In support of this, neither chemical blunted LH stimulated testosterone secretion however acute direct effects were observed on sperm motility. In the human, environmental concentrations of DEHP and PCB153 have previously been reported to reduce sperm motility49,50 and interactions have been reported for some PCBs and phthalates mediated by PCB metabolites and enzymes important for phthalate metabolism51. With respect to DEHP exposure in vivo, the main anti-androgenic effects reported are attributed to its primary metabolite MEHP52. For example the exposure of canine testis interstitial cell suspension cultures to MEHP both inhibits hCG induced testosterone secretion and increases basal testosterone secretion53. In the current study, although DEHP adversely affected motility, vitality and DNA fragmentation, it had no anti- or pro-androgenic effect on testosterone secretion. This likely reflects the use of DEHP rather than MEHP and/or the lack of metabolic breakdown in culture, as reported for cultured rat fetal testis cells46 or relatively low levels of activity as reported in human testis extract54. The lack of a pro-androgenic response may reflect the use of canine adult testis explants rather than fetal or adult dispersed cells as described above46,53. Although we recognise that expanding our study to encompass the effects of PCB and DEHP metabolites on sperm would help consolidate the mechanism, our data are indicative of an interaction between PCB153 and DEHP as reported by others. Furthermore, our observation of an increase in sperm DNA fragmentation and reduced viability, as measured by the hypo-osmotic swelling test, supports previous studies on human sperm showing similar effects and also, increased urinary phthalate concentrations are reported to be associated with lower sperm counts and altered morphology55,56. Notwithstanding, it is recognised that the response of sperm to chemicals in vivo will inevitably reflect exposure to a more complex mixture of pollutants, many of which may interact. Thus establishing clinical relevance and ‘cause and effect’ would require the generation of a mixture of chemicals representative of what is present in semen and/or testis.

In conclusion, this study demonstrates that in a population of stud dogs, sperm motility has declined over a 26 year period. Although the mechanism remains to be determined, we have shown that chemicals present in testis and ejaculate directly affect sperm function and viability. Since the increased incidence of cryptorchidism coupled with declining sperm quality in males is indicative of canine “testicular dysgenesis syndrome”, the domestic dog may be a useful sentinel for the study of environmental influences on human male fertility.