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IPVS 2022
The following technical article is related to the event::
IPVS 2022

Towards the development of epigenetic marks of stress and welfare in farm animals

Published on: 12/20/2021
Author/s : Carlos Guerrero-Bosagna / Linkoping University. Department of Physics, Chemistry and Biology. B-Huset, Entrance 23, Room 3E.637. Campus Valla. Sweden.

Epigenetic mechanisms have been extensively studied in many organisms in relation to diverse biological questions. In production animals, however, despite the enormous implications that epigenetics could have for welfare and production aspects, its application is incipient. One of the concerns regarding production practices is the stress that animals experience while in captivity. Despite regulations around the globe, stressful practices are very common in some regions. In animals undergoing stress, a plethora of hormonal responses are produced. Because of this, short-term stress in animals is usually determined by cortisol and epinephrine levels. However, stress hormones can show an acute but not a sustained exposure to stressful conditions. An important challenge is to determine the history of the exposure of an organism to stress in live specimens. We postulate that epigenetic marks in peripheral cells (e.g., blood cells, buccal swabs) can serve as epigenetic biomarkers of a history of stress with enormous potential to be converted in an application in real situations within the production environment, particularly in hens and pigs. Epigenetic marks of stress would greatly improve the ability of authorities, welfare inspectors and veterinarians to objectively diagnose welfare in farming systems.

While meat production generates food and a livelihood for billions of people around the planet, it is also associated with environmental and health consequences (OECD 2016). Among production animals, chickens and pigs are species of enormous economic importance for humans, with chickens being the most consumed meet source in the world (13.8 kg/capita in 2016) followed by pigs (12.3 kg/per capita in 2016) (OECD 2016). Given the cultural and economic importance of these meet sources and the growing concern about climate change and animal welfare, it is extremely important to create tools that foment sustainable production practices in the meat industry. One of the concerns regarding production practices is the stress that animals experience while in captivity. Although different levels of regulation around the globe exist, some detrimental practices are common in some regions, such as beak trimming to avoid pecking, exposure to random illumination patterns to promote accretion, or exposure to arousing odors, loud noises, too low or too high temperatures, among others (MORGAN AND TROMBORG 2006). 
In animals undergoing stress, hormonal responses are produced such as changes in testosterone, norepinephrine, epinephrine, prolactin, adrenocorticotropic hormone, and cortisol (HENRY 1992). Because of this, short-term stress in animals is usually determined by cortisol and epinephrine levels (ISHIBASHI et al. 2013; MULLER et al. 2013). However, the release of stress hormones can show an acute but not a sustained exposure to stressful conditions (HENRY 1992). Thus, an important challenge is to determine the history of the exposure of an organism to stress in live specimens. Epigenetic marks in peripheral cells (e.g., blood cells, buccal swabs) could serve as epigenetic biomarkers of a history of stress (PROVENCAL et al. 2012; WANG et al. 2012) with enormous potential to be converted in an application in real situations within the production environment. 
Epigenetic mechanisms involve chemical modifications of the DNA that can regulate gene expression and be maintained after cell divisions (SKINNER et al. 2010). Epigenetic mechanisms are fundamental players in the development of phenotypes, and are sensitive to environmental influences (JIRTLE AND SKINNER 2007). Thus, external influences that affect early life stages (pre- and post-birth) can have dramatic consequences on epigenetic processes that ultimately shape the adult phenotype (GUERRERO-BOSAGNA AND SKINNER 2012). Several factors are among the environmental influences reported to interfere with epigenetic mechanisms during early development, among them endocrine disrupting (SUSIARJO et al. 2013) or inorganic (KILE et al. 2014) chemicals, nutritional compounds (DOLINOY et al. 2007; GUERRERO-BOSAGNA et al. 2008) or stressful conditions (FAGIOLINI et al. 2009). 
Although research on epigenetics has permeated many fields of biological research, from molecular biology to evolution (STEIN AND DAVIS 2012), and has employed a variety of organism models (e.g., laboratory rodents (DOLINOY et al. 2007; GUERRERO-BOSAGNA et al. 2008; SUSIARJO et al. 2013), invertebrates (LYKO et al. 2010), plants (MANNING et al. 2006) or yeast (ZHANG et al. 2013)), the understanding of epigenetic mechanisms in farm animals is minimal. The consideration of epigenetics is essential to fully understand molecular mechanisms related to the phenotypes of production animals, and thus can lead to uncover the molecular basis of behavioral traits not well predicted by genotype, such as those related with detrimental behaviors in farm animals (e.g., tail biting in pigs, feather pecking in chickens).
Epigenetics can also provide mechanistic cues in processes of transgenerational epigenetic inheritance (TEI), which involves effects of environmental stimuli on exposed individuals and on their unexposed descendants (GUERRERO-BOSAGNA AND SKINNER 2012). TEI is thought to be mediated by environmentally-altered epigenetic marks (epigenome) in the gametes that are transmitted across generations. Transgenerational effects have been reported in a variety of organisms, including lab rodents (ANWAY et al. 2005; GUERRERO-BOSAGNA et al. 2010; GUERREROBOSAGNA et al. 2012), fish (BAKER et al. 2014a; BAKER et al. 2014b; BHANDARI et al. 2015), quails(LEROUX et al. 2017), ducks (BRUN et al. 2015) and chickens (GOERLICH et al. 2012). Although initial evidence of TEI was generated by environmental exposures acting in early developmental stages, recent reports in rodents have shown that juvenile or adult exposures could also affect the germ line epigenome with consequences in future generations (CARONE et al. 2010; RODGERS et al. 2013; DIAS AND RESSLER 2014). 
In chickens, early social isolation has been shown to affect the HPA axis and gene expression in the thalamus/hypothalamus of the offspring (GOERLICH et al. 2012). Transcriptomic changes in the hypothalamus of chickens raised with unpredictable light patterns are also observed in their offspring (NATT et al. 2009). These effects observed in parents and their offspring suggest transmission of stress-induced germ line epigenetic alterations to future generations.
Overview of ongoing research
We previously showed that epigenetic marks in red blood cells (RBCs)of chickens reflect their previous rearing condition, in cage or open aviaries (PERTILLE et al. 2017). These conditions associate with different stress levels and cognitive abilities. Two questions originated from this experiment that were studied in the present investigation: i) are epigenetic marks in RBCs associated with specific stressors? and ii) how long in life these epigenetic alterations persist? In follow up experiments (unpublished data) we exposed 4-day old male chickens to social isolation stress incrementally for three weeks. This stress has been previously shown to have long-term and transgenerational effects. We then collected RBCs immediately after the stress treatment ended and six months after, in a completely renewed RBC population. We performed DNA methylation analysis in a reduced fraction of the genome and interrogated whether correlations exist between the DNA methylation changes observed in these two time points. Our analyses revealed that even in the control group old individuals tend have hypermethylated sites in relation to young individuals. Interestingly, DNA methylation is incrementally disrupted in response to the stress as animals age, predominantly through hypermethylation, while the enriched environment produces predominantly hypomethylated sites in old individuals in comparison to young ones. 
This abovementioned experiment was performed in parallel at Linköping University in Sweden (SW population) and the Brazilian Agricultural Research Corporation (Embrapa Swine and Poultry National Research Center; BR population). The aim was to identify stress associated with DNA methylation profiles in RBC across these populations, in spite of the variable conditions to which birds are exposed in each facility, and of chickens coming from independent lineages. Interestingly, we found three significant (P < 0.05) differentially methylated regions overlapping between the BR and SW lineages. This is of high relevance because these are putative epigenetic biomarkers of stress in production animals from different lineages, breeding programs and biomes. 
These results in chickens prompted us to expand our research towards pigs. In collaboration with the group of Dr. Adroaldo Zanella at the University of Sao Paulo, we are investigating the relationship between stereotypies observed during pregnancy in saws and the brain methylome of the offspring. In collaboration with Dr. Linda Keeling from the Swedish Agricultural University we are investigating how the genome and the brain methylome varies among performers, victims and non-involved individuals in relation to tail biting 
Our current studies are giving epigenetics a central role as a tool to evaluate detrimental exposures in the production environment. We expect the results obtained in the near future will represent a tipping point in relation to the use of epigenetic tools within the production context. The results emerging from our ongoing experiments should pave the way for the development of toolboxes based on epigenetic marks that will result in improved animal welfare. The aim is that epigenetic toolboxes will identify if animals have been exposed to detrimental conditions (e.g., stress, exposure to chemicals) in the production environment. This will allow for rapid evaluation of previous exposures or health/welfare status using peripheral cells from live animals. The potential incorporation of these tools in the production environment would help to transition animal production practices towards the new demands in terms of animal welfare and sustainability.
Published in the proceedings of the International Pig Veterinary Society Congress – IPVS2020. For information on the event, past and future editions, check out https://ipvs2022.com/en.

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