Developing and validating a practical screening tool for chronic stress in livestock

As the world needs to feed more and more people, the ethical and sustainable production of food becomes of greater importance. In order to produce animal products ethically, animal welfare standards should be kept as high as possible during the production process. Because of this, legislation exists that requires food producers to adhere to minimum standards in terms of animal care. Within these constraints, there are varying methods of rearing to satisfy consumers' demands for high welfare products, for example organic, free range and cage egg production. Objectively assessing which practice benefits the animals' welfare the most, however, is difficult, because while some animals may thrive in one environment, others appear to suffer. But even for the animals that do not show obvious problems, some environments may be more stressful than others. Current methods of animal welfare assessment either depend critically on the animal's immediate state (and would not pick up on problems the previous day, for example), or are difficult and time-consuming to administer, which is not practical in a commercial setting.

Here, we propose to use what we know of the clinical and pre-clinical study of depression and chronic stress to look for markers of long-term poor welfare in chickens. It is well known from rat models of depression and chronic stress that chronic stress leads to a reduction in the survival of newly generated nerve cells in a brain structure called the hippocampus. The exact role of these new cells in the development of depression is still heavily debated, but the reduction in new cells can be used as a marker for whether the animals have undergone chronic stress or not. We propose to develop a rapid and objective method to measure this incorporation of new neurons in the hippocampus by measuring the total amount of a gene or its protein product (called doublecortin) that is produced only by these new neurons.

We will first verify that this method gives the same results as the more laborious method of counting new cells on microscope slides. We will do this in a well-established model of chronic stress, using rats, for which we know chronic stress will lead to a reduction in the number of cells expressing doublecortin. We will then apply this idea to chickens, and compare the amount of doublecortin between chickens housed under laboratory conditions that are stressful or not. Finally, we will compare doublecortin amounts in the hippocampus of chickens collected from different commercial housing environments (e.g. layers in cages vs. free range), and between chickens that are considered to be in good and in poor physical shape, using current criteria of welfare assessment. We will do this for the two most relevant types of chickens: egg-producers (laying hens) and meat-producers (broilers), which are held in different types of facilities. This way we will be able to inform our industrial partners (egg and meat producing companies) about which of their production methods provides the highest welfare for their animals.

Our novel method will provide a fast and reliable method to assess welfare in chickens under different housing conditions, and as such allow producers to optimise animal welfare within their facilities, and for regulators to monitor welfare. If this technique proves useful, it can then later be expanded to other farm animals, such as turkeys, sheep, cattle and pigs.

Chronic stress leads to lower incorporation of new neurons in the hippocampus. We propose to use this as an integrative measure of welfare in animals, starting with chickens. Doublecortin (DCX) is a protein expressed selectively in newly-generated neurons. Counting cells is very time consuming, so we propose to use the expression of the DCX gene and/or the total amount of DCX protein as a proxy measure of new neurons, using real-time PCR and ELISA respectively. We will use Proliferating Cell Nuclear Antigen (PCNA) to control for acute stress effects on cell proliferation.

To validate our method and to ascertain which is the most reliable proxy for counting DCX-positive (DCX+) cells (mRNA or protein), we will treat rats with a chronic mild stress protocol that has been shown to induce robust differences in DCX+ staining in the dentate gyrus (DG). We will compare the stressed rats to control rats using DCX+ and PCNA+ cell counts, as well as quantification of mRNA and protein levels in the DG. These three measures will then be compared between the groups, and correlated with each other to confirm that the molecular methods can be used to quantify adult neurogenesis in the hippocampus.

Once we have validated the faster molecular method in a well-established rat model, we will compare chickens subjected to a chronic stress protocol to a control group, using the same three measures. This experiment will also verify that adult hippocampal neurogenesis can be used as a measure of chronic stress in chickens. Finally, having established a good proxy measure for chronic stress, we will apply this method to laying hens and broilers sampled from commercial establishments, comparing animals from different housing types. As an internal check of our method, we will also compare animals in good and poor physical condition in each housing type. We hope to develop this method as a standard welfare assessment method in farm animals that can be applied at slaughter.

Our project will deliver a new welfare assessment method by measuring impaired neurogenesis in the brain resulting from chronic stress. A welfare assessment method that can be applied to brain tissue collected at slaughter enables comparison between systems or animals within systems.

IMPACTS ON FARMING

1) The proposed technique will benefit farm assurance schemes which include an animal welfare component such as RSPCA Freedom Foods, Red tractor, organic, and supermarkets or poultry industry groups such as BFREPA (the British Free Range Egg Producers Association). Assurance schemes or poultry processors will be able to provide 'benchmarking': farms which rank poorly against their contemporaries can be identified for improvement.

2) Our novel method of measuring welfare will also be of use to expert groups such as FAWC (the Farm Animal Welfare Council) or EFSA (the European Food Safety Authority) which provide the evidence base to policy makers, informing decisions over legislation for systems or husbandry.

3) Citizens and consumers concerned about animal welfare will benefit if sound science is used to inform assurance schemes and legislation.

4) Evidence of chronic stress could be used in welfare enforcement. Defra Animal Health who inspect farms and Meat Hygiene Service who inspect slaughter plants and bodies such as the RSPCA or SSPCA which may prosecute individuals in breach of their responsibility for a 'duty of care' under the Animal Welfare Act (2006), could make use of neurogenesis methodologies.

At the end of the project we expect to have validated a laboratory-based method that will already have informed practice in two commercial chicken companies. It could also be applied to welfare assessment using brain tissue collected at slaughter plants (Red Tractor meat chicken scheme slaughter facilities are already required to inspect and monitor levels of hockburn, pododermatitis, bruising and birds that arrive dead). Before the method could be routinely applied, it will require up-scaling. We will liaise with the Newcastle University Enterprise Team for advice on IP and commercialisation issues, and make an application for BBSRC 'follow-on funding'. The timescale for the realisation of commercial benefits will begin in the final phase of the project but may take a number of years after the project for full realisation of these benefits.

IMPACTS OUTSIDE FARMING

The methodology we are developing can also lead to better animal welfare assessment in other sectors, such as laboratory animal science, wild animal populations, and potentially also zoos and pets. Welfare of laboratory animals can be assessed and different housing conditions or analgesic procedures for chronic pain can be compared to find the one that has the least impact on the animals' welfare. Organisations involved in protecting and improving laboratory animal welfare such as the Home Office, the NC3Rs and the European Union's Animal Legislation are thus likely beneficiaries.

A final and potentially very important beneficiary of our research is the pharmaceutical industry. The incidence of stress-related illnesses is very high and represents a major cost on modern society. There is therefore a strong incentive for biomedical researchers and pharmaceutical companies to develop treatments for such illnesses. These treatments are usually developed in animal models first. Objective assessment of stress-related mental symptoms in laboratory animals is very difficult. Adult neurogenesis has been suggested as an important measure of the impact of chronic stress on an organism, and as an important marker of recovery from such illness. It can therefore be used as a proxy measure to evaluate the effectiveness of novel treatments. The current method of measuring adult neurogenesis is too time-consuming for high-throughput applications. Our method would streamline this procedure.