Vpliv biotopa na genetsko variabilnost, fitnes osebkov in embrionalno diapauzo pri Evropski srni

Predstavitev projekta

Naslov projekta: Vpliv biotopa na genetsko variabilnost, fitnes osebkov in embrionalno diapauzo pri Evropski srni

Šifra projekta: N1-0281

Vodja projekta: prof. dr. Elena Bužan

Vodilna institucija: UP FAMNIT

Partnerske institucije: Visoka šola za varstvo okolja, Research Institute of the Polish Academy of Sciences (Poljska)

Financer projekta: Javna agencija za znanstvenoraziskovalno in inovacijsko dejavnost Republike Slovenije (ARIS)

Vrsta projekta: Mednarodni temeljni projekt (WEAVE)

Raziskovalno področje (ARIS): 1.03 Biologija

Trajanje projekta: 1. 1. 2023–31. 12. 2026

Letni obseg projekta: 0,96 FTE (1640 raziskovalnih ur)

Člani raziskovalnega projekta:

Ime in priimek	Vloga na projektu	Organizacija	Evidenčna št.:
prof. dr. Elena Bužan	vodja projekta / PI	UP FAMNIT	24375
dr. Tilen Komel	raziskovalec / researcher	UP FAMNIT	53504
dr. Felicita Urzi	raziskovalec / researcher	UP FAMNIT	34584
Aja Bončina	tehnični sodelavec / technical staff	UP FAMNIT	56934
prof. dr. Boštjan Pokorny	raziskovalec / researcher	FVO	18112

Opis projekta:

Evropska srna je ena najpogostejših vrst parkljarjev in pomembna lovna vrsta v Evropi. Zaradi svoje vseevropske razširjenosti se sooča z raznolikimi okoljskimi in podnebnimi dejavniki, ki skupaj z intrinzičnimi dejavniki oblikujejo ključne značilnosti njenih življenjskih strategij, kot so fitnes, imunski odziv oziroma odpornost ter mikrobiom prebavil. Prav zato lahko srnjad služi kot modelna vrsta za preučevanje dolgoročnega vpliva okoljskih dejavnikov na biološke procese in genetsko variabilnost.

Dosedanje ekološke raziskave so prepoznale dva ekotipa srnjadi: poljski ekotip, ki se bistveno razlikuje od srnjadi, ki živi pretežno v gozdovih ali gozdnih krajinskih območjih (t. i. gozdni ekotip). Različne krajinske značilnosti vplivajo na razpoložljivost prehranskih virov med ekotipi, kar povzroča razlike v telesni velikosti in masi ter v ekologiji in fiziologiji pripadnikov obeh ekotipov. Pomembno je poudariti, da je prekrivanje med ekotipi zaradi teritorialnega vedenja in navezanosti na življenjski okoliš matere zelo redko.

Populacije srnjadi, ki živijo v odprtih pokrajinah s spremenljivimi podnebnimi razmerami in omejenim zatočiščem, a z večjo razpoložljivostjo hranilno bogatih prehranskih virov, so v povprečju večje in težje od populacij, ki prebivajo v gozdovih. Na Poljskem prevladuje gozdni ekotip srnjadi, saj več kot 30 % države prekrivajo gozdovi, medtem ko prostrana kmetijska območja osrednje Poljske naseljuje poljski ekotip. Na primerljivem območju osrednje Slovenije, ki je vključeno v projekt, prevladuje mozaična pokrajina (gozdovi z odprtimi kmetijskimi površinami; gozdnatost 50–60 %) in mešani ekotip.

Splošni cilj projekta:

Cilj projekta je oceniti povezavo med genomsko variabilnostjo in epigenetskimi mehanizmi okoljsko izbranih parametrov fitnesa prežvekovalcev, pri čemer je srnjad uporabljena kot modelna vrsta.

Hipoteze projekta:

Project presentationna vrh

Title: Biotope influence on genetic diversity, fitness traits and embryonic diapause in the European roe deer

Project acronym: N1-0281

Leading institution: UP FAMNIT

Partner institutions: Faculty of Environmental Protection, Research Institute of the Polish Academy of Sciences (Poland)

Principal investigator: prof. dr. Elena Bužan

Funding organization: Slovenian Research and Innovation Agency (ARIS)

Research field (ARIS): 1.03 - Biology

Duration: 1. 1. 2023–31. 12. 2026

European roe deer is one of the most common ungulates and an important game species, which is spread throughout Europe. Across its pan-European distribution, it faces a wide diversity of environmental and climatic conditions, therefore several factors, including intrinsic ones, shape its life-history traits, welfare as well as immune and microbiome status, causing significant variability among fitness traits. Therefore, roe deer can serve as a model species for studying the long-term effect of environmental factors on biological processes and genetic background. In previous studies on roe deer ecology, a distinct field ecotype of European roe deer was recognised, which significantly differs from roe deer living mainly in forests or forest landscape (i.e., forest ecotype). Different landscape features affecting availability of resources between the ecotypes lead to inter-ecotype differences in body size and body mass, but also in their distinct ecology and physiology; importantly, because of the territorial behaviour and high site fidelity of roe deer compared to other cervids, admixing between each of two ecotypes is not common. Roe deer which live in open landscapes with more severe and variable climatic conditions and limited shelter but with higher availability of nutrient-rich food are in average larger and heavier compared to their counterparts living mainly in forests. Almost 30% of Poland is covered by forests, and forest ecotype of roe deer is prevalent there. On the contrary, Central Poland mainly contains agriculture areas, where field ecotype developed. In a comparative area of central Slovenia, mosaic landscape (forests with open agriculture land; forest cover 50–60%) is prevalent.

General aim: to assess the association between genomic diversity and epigenetic mechanisms of environmental selected parameters of ruminant fitness by using the European roe deer as a model species.

We hypothesized that:

1. Roe deer genetic heterogeneity differs between habitats, and the type of biotope affects the fitness of the individual.
2. Susceptibility to stress varies with the environment.
3. Body condition and immune status of roe deer differ between biotopes.
4. Rumen microbiota depends on the available feeding base in the biotope and correlates with immune status and stress susceptibility.
5. The duration of embryonic diapause depends on the biotope of the mother and is regulated by environmental factors, such as food availability/quality, social relations, and/or exposure to stress.

The following tasks (T) will be realized:

• T1 – Heterogeneity of roe deer across biotopes
• T2 – Susceptibility of roe deer to different stressors according to the biotope
• T3 – Connections between diet, rumen microbiota and rumen content in roe deer across biotopes
• T4 – Correlation between biotope and predisposition to inflammatory processes in roe deer
• T5 – Involvement of maternal factors in embryonic diapause regulation in the roe deer ecotypes.

Applicability of the project results:

Considering how climatic changes and human alterations of the landscape might change habitat availability, it is important to understand the effect of different biotopes on the life history, stress level and welfare of roe deer. Genetic differentiation between roe deer living in the same climatic conditions, but in different biotopes, has not been determined yet. Furthermore, the colonization of different biotopes entails the exposure of individuals to different stressors, which will be measured through cortisol and copeptin concentrations in faeces, blood, hair, and urine. It will allow us to profile and correlate stress parameters immediately prior to death of the individual (blood), over the last 24 h (urine, faeces), and over the course of several months (retrospective profile; hair).

Food availability varies across habitats and seasons, and nutrition may affect the rumen microflora. Indeed, particular food items might trigger changes in biome of rumen and consequently assimilation of nutrition and body condition. Comparison of forage base composition, rumen microbiota, nutrient content and immune parameters in different areas (and ecotypes) will indicate which habitat is beneficial for roe deer.

The approach of oocyte vitrification, maturation, in vitro fertilization and culture of blastocyst, which will be developed in the project, is innovative, and may be used as reproductive technique in cervid species.

Project phases and description of their implementation

Genetic Structure of the European roe deer

In addition to the analysis of mitochondrial DNA gene segments, whole-genome sequencing will be conducted to provide a more detailed insight into the genetic diversity of European roe deer across Europe and to clarify its phylogeography and population structure. Both mitochondrial and nuclear DNA will be analyzed, allowing for a broader understanding of the species evolutionary history and differences between habitats. It is expected that postglacial migrations will have significantly shaped roe deer population dynamics, and that introgression of the Siberian lineage will be present in Eastern Europe. The highest genetic diversity will likely be observed in Central and Eastern Europe, while peripheral populations will appear to be more homogeneous. Differences between mtDNA and nuclear markers will indicate varying levels of genetic introgression and the influence of geographical barriers on gene flow. The combined results, including data obtained from whole-genome sequencing, will contribute to understanding the effects of climate change and human activities on the genetic structure of the European roe deer. Whole-genome analyses will be continued.

Variability of Major Histocompatibility Complex (MHC) genes

The variability of MHC genes in roe deer in Slovenia will be analyzed to determine its role in adaptation to environmental factors. Ten MHC alleles, including three new ones, will be identified, and evidence of historical positive selection pressure on certain codons will be found. The analysis is expected to show that MHC polymorphism will not fully align with the neutral genetic structure of the population, indicating the influence of balancing selection. Further immunogenomic analyses will be conducted using whole-genome sequencing.

Susceptibility of European roe deer to different stressors

Urine, hair, plasma, and feces will be collected from the same individuals analyzed previously. Stress susceptibility will be assessed by determining copeptin and cortisol levels using ELISA tests. The correlation between the type of sampled material, habitat, and the health condition of the animals will be analyzed.

Relationship between diet and  European roe deer microbiota

The relationship between nutrient content (proteins, fiber, fat, and nitrogen-free extracts) and microbiota will be examined based on dietary composition analysis. Plant species and microbiota will be identified using next-generation sequencing (NGS).

Susceptibility of European roe deer to inflammatory processes

Red and white blood cell counts, hemoglobin concentration, albumin, creatinine, and urea levels, as well as white blood cell distribution and circulating antibody levels, will be determined in blood samples using biochemical and hematological veterinary analyzers. The level of IgG will be measured with an EIA assay. In various tissues, mRNA and protein expression will be analyzed using real-time PCR and Western blot for TLRs (1-10), MHC-I, MHC-II, TNFα, IL-1β, and IL-6.

The role of maternal factors in the regulation of embryonic diapause

Preliminary experiment: evaluation of epididymal sperm quality and cryopreservation A preliminary study will be conducted to assess epididymal sperm quality and cryopreservation. Epididymal sperm will be collected post-mortem from roe deer males during the rutting season (late July – early August; N=10 per habitat). Sperm concentration, motility (CASA system), plasma membrane integrity (PMI, %; SYBR-14/PI), mitochondrial membrane potential (MMP, %; JC-1/PI), and DNA fragmentation (DFI, %; acridine orange) will be determined.

Evaluation of In vitro fertilization and embryo culture to the blastocyst stage

Oocytes will be isolated from ovarian reserves and vitrified. After thawing, cumulus-oocyte complexes (COCs) will be aspirated from follicular fluid, selected, and classified as either mature or immature. Mature oocytes will be fertilized immediately, while immature ones will be cultured to reach maturity in vitro. Fertilized zygotes will be cultured to the blastocyst stage, and mRNA expression of quality markers (CTS, BAX/BCL2, AREG, EREG), cumulus expansion levels, and glucose concentration in the culture medium will be analyzed. Blastocyst quality will be assessed by measuring mRNA expression of OCT4, SOX2, and PLAC8 using real-time PCR. The final outcome will be the percentage of blastocysts obtained relative to matured follicles. Steroid hormones, PRL, and LIF levels will be determined in plasma and culture medium using an LC-MS-based approach.

Influence of maternal regulators on diapause regulation

Morulas (5th day post-insemination) from all three habitats will be stimulated with P4, E4, PRL, and LIF, and their effects will be assessed by measuring mRNA expression of key implantation factors (HB-EGF, ODC-1, c-Myc) using real-time PCR. Culture will be continued until the hatched blastocyst stage (maximum 9th day of culture). The doses of stimulatory factors will be determined based on preliminary experiments and existing literature. The effects of stimulation will be compared with expression levels in control blastocysts from the previous experiment. Culture media will be collected for glucose level analysis.

Znanstvene objave / Scientific publications: