We know more and more about the important role of the microbiome in the human body, how it changes depending on stressful situations. In farm animals, too, stress disrupts the microbiology of the digestive system – says Natalia Szeligowska, a PhD student in the Faculty of Biology and Animal Sciences at the University of Life Sciences, the lead author of the publication, which appeared in the journal “BMC Veterinary Research”.
Sheep under stress
The topic was proposed by MSc. Konrad Wojnarowski, then a doctoral student from the same UPWr institute, currently a researcher at Ludwik and Maximilian University in Munich: – The idea for the study was the result of our interest in the widely understood issue of animal welfare.
– Pregnancy is one of the most physiologically difficult periods in female mammals, hence the idea of comparing the levels of s-glutathione transferase (GST), one of the markers of cell stress, and cortisol, the stress hormone – adds Dr. Paulina Cholewińska of the Faculty of Biology and Animal Breeding, UPWr. Importantly, both factors can influence the microbiology of the digestive system of both humans and animals. We have taken care of sheep because they are considered a model animal for female pregnancy. In the project we combined Konrad Wojnarowski’s experience in cell biology and mine, related to animal microbiology. The project started almost simultaneously with the pandemic, in June 2020. The authors admit that the research was initially complicated: the university was closed, access to laboratories was difficult. Only after the first wave did they receive approval for the study, for which they selected 18 sheep: nine 14-month-olds and nine multiparous women giving birth for the second time. The animals kept at the UPWr research and education station in Swojczyce were kept under the same conditions and fed the same, mainly hay, but also lupine, oats and beet pulp. Faeces were collected several times for examination and also fragments of placenta were collected immediately after delivery.
Sample analyzes showed differences in microbiological composition in both groups. Primiparous women had significantly higher levels of GST and cortisol, indicating higher levels of stress.
– Probably because giving birth was a new, unknown experience for them – says Natalia Szeligowska. – In sheep, as in humans, the adrenal glands secrete more cortisol during pregnancy, which can be toxic to the mother and fetus. In order for the fetus to develop properly, it is important to reduce stressors and maintain maternal homeostasis.
Stress, partly by influencing the microbiome, also increases the susceptibility to infections. Researchers have shown that the composition of the microbiome changed over the course of pregnancy at different gestational periods: In primiparous women during pregnancy, levels of beneficial bacteria were lower than in multiparous women – probably also due to stress.
For the environment, for animals
Scientists relied on placenta and stool tests, although they initially planned a blood analysis.
– However, we have given up on the blood collection, because the procedure for obtaining approval from the Ethics Committee for Animal Experiments of the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław can take a long time and the project can take only a year. Therefore, we developed a plan based on less invasive methods so that the stress caused by our actions does not affect the level of the cortisol tested, but also that these methods are as suitable as possible for our research subject – explains Dr. Paulina Cholewisska
The approval of the ethics committee is one of the elements that must guarantee optimal animal welfare. Anything that is not absolutely necessary is kept to a minimum and that can lead to even more stress. Well-being is also influenced by nutrition adapted to the needs of animals, access to pasturage, good veterinary care and meeting social needs: sheep are herd animals, they should not be isolated.
Konrad Wojnarowski: – Fortunately, the anthropocentric approach to research is slowly disappearing. In 21st century science, increasing emphasis is placed on improving the living conditions of the animals we keep, as well as the entire environment around us. In our small team, we have tried to do all the research in the least invasive way. It is an indispensable practice and at the same time a science and a behavioral model for young scientists. New laboratory methods and techniques allow us to analyze and continuously improve the impact of many factors on animal welfare. We owe this to both the environment and the animals themselves.
On November 17, 2021, the law on the protection of animals used for scientific or educational purposes was amended based on three principles. The principle of substitution is the use, where possible, of a test method or strategy that is scientifically satisfactory and does not involve the use of animals, rather than the procedure. The containment principle is about minimizing the number of animals used in procedures, and the improvement principle recommends that animal husbandry and procedures be improved to eliminate or minimize pain, suffering, distress or permanent damage.
Scientists have conducted research using Olkusz breed sheep, which was established in the Olkusz poviat in the 1930s as a result of mating ewes of the local breed with rams brought from Kashubia. – They are large, medium-muscled, polled sheep with white fur and long ears. They are characterized by very high fecundity and maternal abilities. Since 2005, this breed, like 12 other breeds, has been included in the Genetic Resource Protection Program for its genes responsible for high fertility, says Natalia Szeligowska. In addition to the Olkusz sheep, the fertile breeds also include the Friesian sheep, the Romanian breed and the Finnish breed.
Many sheep breeds are kept in Poland, among others. Polish Merino, Polish mountain sheep, heather or sheep sheep like Cameroon. At the research and education station in Swojczyce, Olkusz sheep adjoin other animal species kept there: alpacas, Holstein-Frisian cows, goats, geese and chickens. Sheep from Swojczyce have already been the subject of many interesting studies at the University of Life Sciences: physicochemical properties of wool, composition and influence of different compounds on sheep milk and cheese. – In previous studies, we initially showed differences in the bacterial composition of the digestive system between different breeds of sheep – adds Paulina Cholewińska.
Many possible paths
– This project was a great scientific adventure for me, which convinced me that I was going in the right direction. It also made it easier for me to start a PhD, Natalia says. The PhD is an extension of the subject of her thesis: while she wrote about the influence of diet on the microbiome of Charolaise beef cows, she will now study the relationship between the microbiology of the digestive system and immunity in dairy cattle. He admits that the microbiome is a fascinating subject.
– The intestines of animals contain archaea, micro-organisms that are involved in the production of methane in the digestive system. It is known to be a gas with a very high greenhouse potential, whose emissions to the atmosphere must be limited to protect the natural environment. That’s why research is being done to reduce the number of archaea in the digestive system – says the PhD student.
Archaea, along with several thousand species of bacteria, protozoa, fungi and viruses, inhabit the digestive system of animals; many of these microorganisms are similar in humans and animals. They create an environment without which we could not exist. They aid in proper digestion, nutrient absorption, production of B vitamins, vitamin K and folic acid. Thanks to competition for the living environment, they prevent colonization by pathogenic bacteria, reducing the risk of inflammatory processes. They act as a specific immune system and influence the homeostasis of the body. There is increasing talk about the relationship between the microbiome and depression, obesity, diabetes and allergy. Once there was talk of microflora, now we use the concept that was introduced in 2021 by the American geneticist and microbiologist Prof. Joshua Lederberg, Nobel Prize winner. Microbiota is a collection of all the microorganisms that inhabit the organism of animals and humans while the microbiome, a term often used interchangeably, is a collection of genomes of these microorganisms.
– It would be worthwhile to get to know the ruminant microbiome better, for example to extend our research to the analysis of other clusters or even sequencing of bacteria inhabiting their digestive system and placenta. In the context of the welfare of pregnant females, it would be good to conduct further research to reduce the effects of cortisol on the mother’s organism and the lambs, says Natalia Szeligowska.
– Recently, animal microbiology research has become increasingly important; the microbiome is a complex ecosystem that influences almost all production parameters important to the grower. There are many possible avenues to continue our research, but it will depend on the results of the current project – adds Paulina Cholewińska. – We chose the sheep for the Wrocław study not because of the breed, but because of the availability of animals. We have now entered into a partnership with the University of Life Sciences in Lublin, where there are more breeds of sheep and, what is very important to us, are kept in the same environment.
Paulina Cholewińska and Natalia Szeligowska are currently working with Prof. Andrzej Junkuszew from the University of Life Sciences in Lublin and Dr inż. Wanda Górniak from Wrocław University of Technology and continued their collaboration with Konrad Wojnarowski from Ludwik and Maximilian University in Munich in determining the variability of bacteria in the digestive system due to the breed of sheep and their physiological state.
Source: UNIVERSITY OF LIFE IN WROCŁAW – Aneta Augustyn