Microbiome – a fascinating ecosystem

• Pregnancy affects the microbiology of the digestive system of both humans and animals. Sheep are considered to be a model animal for female pregnancy.
• UPWr scientists conducted research on sheep in a minimally invasive manner and with animal welfare in mind.
• The microbiome is a complex ecosystem. The microorganisms in the digestive system create an environment that we could not exist without.

The article is a result of the year-long 'Young Minds Project', in which Natalia Szeligowska participated while still a Master's student. – I applied for the best student research paper project, in which the criteria included the average grade, a research plan and cost estimate, and above all an innovative and as yet unexplored topic – says the PhD student from the Institute of Animal Breeding. The research, supervised by Dr. Katarzyna Czyż, was conducted with the involvement of other academics, students and doctoral students of the UPWr: Dr. Paulina Cholewińska, Dr. Krystyna Pogoda-Sewerniak, Dr. Przemysław Pokorny, Jakub Smoliński, Konrad Wojnarowski. They are all co-authors of a publication that describes the extent to which the stress of giving birth changes the microflora in sheep.

Sheep in distress

The topic was suggested by Konrad Wojnarowski M.Sc., then a doctoral student from the same institute at the UPWr, now a researcher at the Ludwig Maximilian University of Munich: – The idea for the research stemmed from our interest in the broadly understood issue of animal welfare.

– Pregnancy is one of the most physiologically difficult periods for female mammals, hence the idea of comparing the levels of s-glutathione transferase (GST), i.e. one of the markers of cellular stress and cortisol, the stress hormone – adds Dr. Paulina Cholewińska from the Department of Biology and Animal Breeding at the UPWr. – Importantly, both of these factors can affect the microbiology of the digestive system in both humans and animals. We looked into sheep because they are considered to be model animals for female pregnancy. For this project, we combined Konrad Wojnarowski's experience in cell biology and my experience in animal microbiology. The project started more or less at the same time as the pandemic, in June 2020. The authors admit that this initially complicated the research, as the university was closed and access to laboratories was difficult. It was only after the first wave that they got approval for the study, for which they selected 18 sheep: nine 14-month-old primiparous sheep and nine two-year-old multiparous sheep that were giving birth for the second time. Animals kept at the UPWr Research and Training Station in Swojczyce were kept under the same conditions and fed the same, mainly hay, as well as lupine, oats and beet pulp. Their faeces were collected several times for examination, and placental fragments were also collected shortly after they gave birth.

Analyses of samples showed differences in the microbiological composition in both groups. The primiparas had significantly higher GST and cortisol levels, indicating higher levels of stress.

– This was probably because giving birth was a new, unknown experience for them – says Natalia Szeligowska. – With sheep, as with humans, the adrenal glands secrete more cortisol during pregnancy, which can have toxic effects on the mother and the foetus. In order for the foetus to develop properly, it is important to reduce stressors and maintain maternal homeostasis.

Stress also increases susceptibility to infections through, among other things, its impact on the microbiome. The scientists showed that the composition of the microbiome changed during different periods of pregnancy: first time mothers had lower levels of beneficial bacteria throughout their pregnancies than multiparous sheep – presumably also a result of stress.

For the environment and for animals

The scientists relied on placenta and faecal tests, although they had initially planned to analyse blood.

– However, we gave up blood sampling because the procedure for obtaining permission from the Ethics Committee for Experiments on Animals at the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław can be lengthy and the project could only last a year. We therefore developed a plan involving less invasive methods so that the stress caused by our activities did not affect the studied cortisol levels, but also so that the methods were as relevant to our research topic as possible – explains Dr. Paulina Cholewińska

The ethics committee's approval is one of the elements to ensure optimal animal welfare. Everything that is not absolutely necessary and that could lead to even greater stress is kept to a minimum. Animal welfare is also affected by nutrition, access to pastures, proper veterinary care and social needs: sheep are herd animals, so they should not be isolated.

Konrad Wojnarowski: – Fortunately, the anthropocentric approach to research is slowly becoming less popular. In 21st century science there is more and more emphasis on improving the living conditions of livestock, as well as the environment around us. In our small team we tried to conduct all our research in the least invasive way possible. This is an essential practice, as well as a learning and behavioural model for young scientists. Thanks to new laboratory methods and techniques, we can analyse the impact of many factors on animal welfare, continuously improving it. We owe this to both the environment and the animals themselves.

On 17 November 2021 the Act on the Protection of Animals used for Scientific or Educational Purposes was amended, based on three principles. The principle of replacement is to use, wherever possible, a scientifically satisfactory method or testing strategy that does not involve the use of animals, but instead of a particular procedure. The principle of reduction states that the number of animals used in procedures should be reduced to a minimum, and the principle of refinement recommends refinement of animal husbandry and methods used in procedures so as to eliminate or minimise pain, suffering, distress or permanent impairment.

The scientists conducted their research using the Olkuska sheep breed, which originated in the 1930s in the Olkusz district as a result of mating ewes of the local breed with rams imported from Kashubia. – These are large, moderately muscled, hornless sheep with a white coat and long ears. They have a very high fertility rate and maternal ability. Since 2005, together with 12 other breeds, this breed has been included in the Programme for the Protection of Genetic Resources because of the genes responsible for high fertility - says Natalia Szeligowska. In addition to Olkuska, fertile breeds include the Friesian, Romanov and Finnish breeds.

Many breeds of sheep are kept in Poland, including Polish merino, Polish mountain sheep, wrzosowka (heath) and broadleaf sheep such as Cameroonian. At the Swojczyce Research and Training Station Olkusz sheep live side by side with other animal species kept there: alpacas, Holstein-Friesian cows, goats, geese and hens. Sheep from Swojczyce have already been the subject of many interesting studies at the Wrocław University of Environmental and Life Sciences: such as physicochemicalical properties of wool and the composition and influence of various compounds on sheep milk and cheese have. – In earlier studies, we initially demonstrated differences in the bacterial composition of the digestive system between different breeds of sheep – adds Paulina Cholewińska.

Many possible routes

– This project was a great scientific adventure for me, which confirmed my conviction that I had chosen the right route. It also made it easier for me to get accepted for my doctoral studies – says Natalia. The doctoral thesis is an extension of the subject of her master's thesis: at the time she wrote about the impact of diet on the microbiome of Charolais cows, while now she will be studying the relationship between the microbiology of the digestive system and immunity in dairy cattle. She admits that microbiology is a fascinating topic.

– The intestines of animals are home to archaeons, which are microorganisms involved in the production of methane in the digestive system. We know that it has a very high global warming potential and its emission into the atmosphere should be limited in order to protect the environment. Research is therefore being conducted to reduce the number of archaeons in the digestive system – says the doctoral student. 

Archaeons, 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, and help us properly digest, absorp nutrients, produce B vitamins, vitamin K and folic acid. By competing for a habitat, they prevent colonisation by pathogenic bacteria, which reduces the risk of inflammatory processes, while acting as a specific immune system they influence the body's homeostasis. The relationship between the microbiome and depression, obesity, diabetes and allergies is becoming an increasingly important issue, and while we used to refer to microflora, we now use a term that was introduced in 2021 by the American geneticist, microbiologist and Nobel Prize Winner Prof. Joshua Lederberg. The microbiota is the collection of all microorganisms that inhabit the animal and human body, while the microbiome, a term often used interchangeably, is the collection of the genomes of these microorganisms.

– It would be useful to gain a better understanding of the ruminant microbiome, for example to extend our research to analyse other clusters or even to sequence the bacteria inhabiting their digestive system and placenta. In the context of the welfare of pregnant females, it would be good to carry out further research to reduce the effects of cortisol on the mother and lambs – says Natalia Szeligowska.

– Recently, research related to animal microbiology has become increasingly important; the microbiome is a complex ecosystem that affects almost all production parameters relevant for farmers. There are many potential routes for continuing our research, but it will depend on the results of the current project – adds Paulina Cholewińska. – We chose sheep for the Wrocław study not based on their breed, but availability. We have now started cooperation with the University of Life Sciences in Lublin, where there are more sheep breeds and, what is very important for us, they are kept in the same environment.

Paulina Cholewinska and Natalia Szeligowska are currently cooperating as part of a new project called 'Innovative Scientist', together with Prof. Andrzej Junkuszew from the University of Life Sciences in Lublin and Dr. Wanda Górniak of the Wrocław University of Technology, and they are continuing to work with Konrad Wojnarowski of the Ludwig Maximilian University of Munich to determine the variability of bacteria in the digestive system in relation to the breed of sheep and their physiological state.