Environmental Endocrine Disruptors in Farm Animal Reproduction: Research and Reality
Contents
In this review, possible comparative advantages of studying endocrine disruption in farm animals vs laboratory rodents are discussed. First, using farm animals, the generality of findings in laboratory rodents are challenged. Farm animals may in certain aspects be better models for humans than laboratory rodents, and sometimes there might be methodological advantages in using farm animals. Second, there are several in vitro studies based on cell-culture systems from sows and cows where the effects of chemicals on sex steroid secretion can be measured and maturation and fertilization of oocytes may be assessed. These in vitro systems are powerful tools for dissecting the mechanisms of action for endocrine-disrupting chemicals. Third, in a set of recent in vivo studies using sheep, goats and pigs, in which very different exposure regimens to endocrine-disrupting chemicals have been used, a full panel of reproductive parameters pertinent to farm animals were assessed. Clinically, it is suggested that endocrine disruption in farm animals should be considered when impaired reproduction could be linked to change in source of feed or pasture. Finally, epigenetic and toxicogenomic approaches can be particularly rewarding in elucidating endocrine disruption in future farm animal studies.
Introduction
Over the last two decades, endocrine disruption has gained more public, political and scientific attention. This has been reflected in directed public research funding, new policies and risk assessment regimens, as well as a boom in scientific reports on the subject. A comprehensive report presented in 1992 on chemically induced alterations in sexual development (Clement and Colborn 1992) is often regarded as the starting point for the emergence of the issue. Even so, given the most frequently adopted definition of endocrine disruptors –‘an endocrine disruptor is an exogenous substance or mixture that alter function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub) population’ (Damstra et al. 2002) – it is interesting to note that endocrine disruptors have been around in farm animal reproduction for a long time. The so-called sweet clover disease caused by the phyto-oestrogens formononetin and genistein with prolapsed uterus and embryonic death in sheep is perhaps the most classical example (Cox 1978). Another example is pigs suffering from various signs of hyperoestrogenism such as vaginal prolapse, abortions and stillbirths, owing to phyto-oestrogen produced by the fungi Fusarium in feed stuff (reviewed by Diekman and Green 1992). Furthermore, nowadays reports based on farms animals are just a small proportion of the scientific literature about endocrine disruption.
The rapid expansion of scientific literature in the field has been driven by concern about anthropogenic substances exerting these effects, or specifically chemicals polluting the environment, as recently reviewed (Fowler et al. 2012; Hamlin and Guillette 2010; Hotchkiss et al. 2008; Sharpe 2010). The chemicals mostly discussed are polychlorinated biphenyls (PCBs), dichlorodiphenyldichloroethylenes (DDTs), polybrominated diphenylethers (PBDEs) and phthalates. Therefore, this review focuses on endocrine disruption by chemical pollutants and on the disruption of the reproductive endocrine system, as this system has been of the most concern over the years.
Notably, the debate about endocrine-disrupting chemicals is extensive, with contributors from environmental organizations, chemical industry, regulatory bodies, policymakers and the research community (cf Borgert et al. 2011; Hotchkiss et al. 2008; Sharpe 2011; Shaw et al. 2010). So, is there a role for research on farm animals in this scientific area that is getting so much public attention and scrutiny and is dominated by studies on laboratory rodents? Secondly, is reproduction of farm animals in real-life at risk for endocrine disruption? These are the two main issues that will be elaborated on in this review.
Research on Farm Animals Contributing to Data on Endocrine Disruption
Generally, the data on endocrine disruption are generated from three types of studies: (i) tests performed according to international guidelines (OECD, 2012), (ii) other controlled in vitro or in vivo experiments and (iii) epidemiological or field surveys. A fourth type contributing to our knowledge about endocrine disruption is case reports from the clinical or occupational health literature.
With the aim to refine the risk assessment and regulation of (potentially) endocrine-disrupting chemicals, what is the added value of including experimental studies in farm animals to the bulk of laboratory rodent studies? There are at least three slightly different rationales for performing such studies in farm animals (Magnusson 2005). First, using farm animals, one tests the generality of findings from studies on laboratory rodents – one may find that the laboratory rodent findings are not true for all other mammals and thus possibly not for humans. Second, farm animals may in some aspects be better models for the human than the laboratory species – both when it comes to reproduction or physiology in general. Finally, there might be methodological advantages of using farm animals – for instance sample sizes could be larger and repeated sampling easier.
Examples of studies on endocrine disruption in different farm animal species are listed in Tables 1 and 2. In addition to this, there are recent reviews elaborating on this topic (Pocar et al. 2001; Brevini et al. 2005; Magnusson 2005; Magnusson and Dencker 2010; Veeramachaneni 2008). Here, the two main types of studies, in vitro and experimental in vivo studies, will be discussed in more detail.
| Species | Culture system | Chemical | Main effect | References |
|---|---|---|---|---|
| Pig | Oocytes | PCB | Decreased developmental competence | Brevini et al. (2004) |
| Pig | Theca and granulosa cells | PCB, DDT, Natural mixture of POPs | Decreased (PCB) and increased (DDT) oestradiol secretion. Decresed testosterone secretion (DDT) | Gregoraszczuk et al. (2008a,b) |
| Pig | Theca and granulosa cells | PBDE | Increased testosterone secretion | Karpeta and Gregoraszczuk (2010); Karpeta et al. (2011) |
| Bovine | Oocytes | Octylphenol | Impaired meiotic progression and developmental competence | Pocar et al. (2003) |
| Bovine | Granulosa cells | DDT and Methoxychlor | Loss of viable cells and inhibited progesterone production | Tiemann et al. 1996 |
| Bovine | Oviductal cells | Organochlorine pesticides | Differential reduction in cell viability | Tiemann et al. (1998) |
| Species | Exposure | Main effect | References |
|---|---|---|---|
| Sheep | Gestational and lactational; gestational, prepubertal; bisphenol A, octylphenol | Effects on LH-secretion. Advanced onset of puberty. Increased number of abnormal sperm. | Evans et al. (2004), Wright et al. (2002), Sweeney et al. (2007) |
| Sheep | Gestational, bisphenol A and methoxychlor | Hypergonadotropism, shorter breeding season, differential long-term effect on LH-surge. | Savabieasfahani et al. (2006) |
| Sheep | Gestational and lactational, PCBs | Increased induced LH-levels and increased numbers of follicles. | Kraugerud et al. (2011) |
| Sheep | Gestational; gestational and lactational, sewage sludge | In males: reduction in germ cell number. Lowered GnRH and GnRH receptor mRNA expression; Differentially expressed ovarian proteome and reduced prolactin levels | Bellingham et al. (2010, 2011), Fowler et al. (2008) |
| Pig | Pre-pubertal, DEHP | Lowered induced LH-secretion. Precocious development of bulbourethral glands; reduced percentage of defective sperm | Ljungvall et al. (2006, 2008), Spjuth et al. (2006) |
| Pig | Gestational, octylphenol | Extended gestation length, accelerated onset of puberty and reduced litter size | Bøgh et al. (2001) |
| Goat | Gestational and lactational, PCBs | In females: lower pre-pubertal LH-levels and delayed puberty. Smaller testes, higher proportion of sperm with damaged DNA. | Lyche et al. (2004), Oskam et al. (2005) |
In vitro studies
In farm animal reproduction, we are in the forefront in reproductive biotechnologies, including various in vitro techniques. These techniques can be used as high-throughput screening systems for several chemicals at reasonable costs if the laboratory facilities and techniques are in place. In addition, they may help in establishing more precise understanding of mechanisms of action for endocrine disruption when combined with in vivo studies. Consequently, in vitro systems for parts of the female porcine and bovine reproductive system have been used for investigating effects of various chemicals. For instance, a porcine system using ovarian follicular cells, co-culture of theca and granulosa cells able to produce oestradiol as well as testosterone, has been used to elucidate the effects on mixtures of persistent organic pollutants (Gregoraszczuk et al. 2008a,b). In a neat two-step approach, three natural mixtures were first analysed with respect to effects on oestradiol and testosterone secretion. Somewhat surprisingly, all three mixtures, one from the liver of Atlantic cod and two from livers of burbot from inland lakes in Norway, exerted effects on the steroid secretion. In the second step – when the mixtures had been analysed chemically – specified congeners of PCB at environmentally relevant concentrations were tested alone or in combination. It was thereby possible to pinpoint the most potent congener as well as identify combination effects. Thus, these kinds of studies are very resource efficient for investigating environmentally relevant mixtures and achieving indications of which chemicals are of most concern. Another set of in vitro studies has been investigating the effects of known endocrine-disrupting chemicals on maturation and fertilization of oocytes (Table 1). In a bovine system where oocytes were exposed to octylphenol, there was a dose-related impairment in maturation and decrease in fertilization (Pocar et al. 2003), and these effects did not seem to be caused by oestrogen-mimic activity. This is a good example on how methodological powerful in vitro system can assist in dissecting the mechanism of action for endocrine-disrupting chemicals.
Experimental in vivo studies
The farm animal species most used to study endocrine disruption is the sheep where several exposure regimes have been applied (Table 2). There are also reports from studies in pigs, where there have been gestational or pre-pubertal exposures, and in goats with combined gestational and lactational exposure (Table 2). The chemicals tested have often been known suspected endocrine-disrupting substances like PCBs, phenols and phthalate. Perhaps, one innovative and environmentally relevant exposure regimen applied has been to put grazing ewes on pasture treated with sewage sludge (Fowler et al. 2008; Bellingham et al. 2010, 2011). This regimen, in combination with the fact that the sheep is an outbreed species – compared to the inbred laboratory rodents – gives power to the assessment of the risk for endocrine disruption in domestic animals, wildlife and humans. One weak point, however, in extrapolation to humans, is the difference in the alimentary tract physiology between primates and ruminants. To compare, in kinetic studies of the endocrine-disrupting plastizer Di(2-ethylhexyl)phthalate in pigs, we found that the kinetics after oral intake was more similar to that in primates than in rodents (Ljungvall et al. 2004).
Studies in sheep, goats and pigs show how it is possible to elaborate on different exposure windows during development – it is generally regarded that effects by endocrine disruptors during development are irreversible, whereas those in adulthood are reversible. One interesting observation regarding foetal exposure is that in ewes exposed to sewage sludge during pregnancy, there was a lowered hypothalamic GnRH mRNA expression in the foetus, but not in the dam (Bellingham et al. 2010). Further, sheep is the farm animal in which the most varied exposure periods have been applied: the entire gestational period and a major part of the lactational period (Kraugerud et al. 2011), mid gestation (Savabieasfahani et al. 2006), second part of gestation and/or during lactation (Wright et al. 2002). However, by applying the same chemical exposure and measuring the same end points, more information would be gained about the vulnerability in the different developmental periods. Nevertheless, it may be concluded that gestational exposure as well as post-natal exposure can affect the reproductive endocrine system both in sheep (Evans et al. 2004; Fowler et al. 2008) and pigs (Bøgh et al. 2001; Ljungvall et al. 2005, Ljungvall et al. 2008). Needless to say, different chemicals give different effects as shown in comparisons between, for instance, bisphenol-A and methoxychlor when studying the LH-surge in sheep (Savabieasfahani et al. 2006) or between two PCB congeners when investigating plasma LH-concentrations and sperm quality in goats (Oskam et al. 2005). Taken together, there are endless combinations of exposure regimens, chemicals or mixtures and physiological end points that can be applied in farm animals.
Regarding the physiological end points, besides measuring reproductive hormone concentrations, which were included in most studies cited here, the following end points, to mention some, have been made: assessment of semen quality (Oskam et al. 2005; Spjuth et al. 2006; Sweeney et al. 2007; Bellingham et al. 2011), ovarian follicular dynamics by stereology (Kraugerud et al. 2011) or repeated ultrasonography (Wright et al. 2002), and mating behaviour (Ljungvall et al. 2008). This repertoire can be extended with more end points from the laboratory and, uniquely for farm animals, from the clinic.
Clinical Relevance of Endocrine Disruption in Farm Animals
Is endocrine disruption by anthropogenic compounds in the environment an issue for the reproductive performance of farm animals in real-life? First, one has to realize that wildlife with endocrine-disrupted reproductive systems is mostly on a much higher trophic level in the food web than the herbivorous ruminants. The omnivorous pig, depending on feeding regimen, can, however, be on a higher trophic level, and biomagnification of endocrine disruptors may thus be more prominent. Still, several endocrine-disrupting chemicals has been reported in pigs, cows and sheep from some European countries (Glynn et al. 2009; Petro et al. 2010; Rhind et al. 2011). Encouraging, in Sweden, which is a country with extensive environmental regulations, the burden of organochlorine contaminants in pigs and cows at slaughter declined close to the detection limit for the analytic methods used from 1991 to 2004 (Glynn et al., 2009). Also, in countries where the practice of spreading sewage sludge on pasture occurs, like Belgium and the UK, the concentrations of analysed endocrine-disrupting chemicals in cattle and sheep, respectively, are regarded to be too low to impair the reproductive performance (Petro et al. 2010; Rhind et al. 2010). One may, however, speculate that in regions or countries with a more polluted environment, endocrine disruption might reduce fertility in farm animals.
Obviously, with extreme events like flooding or industrial accidents, endocrine disrupters may contaminate animal feed and may reach harmful concentrations. For instance, following the infamous Seveso disaster in Italy (1976) where several kilograms of dioxin from a factory was released to the environment, several reports disclosed how in utero and/or lactational exposure of children to low dioxin doses can permanently affect the reproductive system, for example reduced sperm quality (Mocarelli et al. 2011). A possible similar example in farm animals is heifers that were drinking water in direct contact with a sewerage overflow and then showed increased age at first calving (Meijer et al. 1999).
All in all, one should consider the possibility of endocrine disruption as a cause to impaired reproductive performance when farm animals are given feed from a new source or put on new pasture or if there has been any suspected exposure to feed by chemically polluted water or solid materials.
Future Perspectives
Given the conceptually and technically well-advanced position of the discipline ‘farm animal reproduction’, this discipline may make significant contributions to research about endocrine disruption. The farm animal models have in some aspects advantages compared with the laboratory rodent models and can be exploited further. For instance, the longer development period in utero and post-natally makes it easier to pinpoint distinct development stages and exposes animals precisely during these stages to explore the mechanisms of action for the disrupting substances. Also, there is a potential to further develop repeated sampling or recording by methods used in the clinic, including recording various kinds of sexually dimorphic behaviour. Finally, epigenetic (Crews and McLachlan 2006) and toxicogenomic (Magnusson and Dencker 2010) approaches in the farm animal research on endocrine disruption are ways forward that may become particularly rewarding.
Acknowledgements
The author acknowledges financial support from the Swedish Environmental Protection Agency and the Swedish International Development Agency.
Conflicts of interest
The author declares no conflicts of interest.
Author contributions
UM scrutinized the literature and wrote the paper.
