- SCIENZE VETERINARIE PER LA SALUTE ANIMALE E LA SICUREZZA ALIMENTARE
- Phd: 37th cycle
- Dottorato in Scienze Veterinarie per la Salute Animale e la Sicurezza Alimentare
- Matriculation number: 866605
- ORCID: orcid.org/ 0000-0002-7110-0960
Curriculum vitaeCurriculum Vitae
State of the art
MicroRNAs (miRNAs) are a class of small non-coding RNA molecules, highly conserved among different species, with an average length of 22 nucleotides. Their post-transcriptional regulation is crucial for a wide range of cellular processes, including development, immune responses and homeostasis. miRNAs are expressed in tissues and released in extracellular fluids; in this case, they are known as circulating-miRNAs (c-miRNAs). In the last few years, interest in c-miRNAs has grown since they are readily available (from the blood) and are highly stable in body fluids1. C-miRNAs could be useful biomarkers, changing profiles under conditions such as viral and bacterial infections2, physiological states (eg. pregnancy)3 and environmental stressful stimuli4. Therefore, combination between c-miRNAs expression profiles and other hormonal profiles, like female reproductive hormones or cortisol, can give useful information about oestrous cycle, often affected by stressful situations, and be relevant tools to assess animal welfare5.
The studies related to miRNAs in farm animals are mostly focused on tissue miRNAs and on the most commonly bred species and breeds. In literature there are really few information about c-miRNAs in sheep6. In addition, Frabosana-Roaschina sheep is an autochthonous, rustic breed that is inserted in the extinction-threatened breeds list of the Piemonte Region. For this reason, reproduction and welfare issues become crucial.
This project aims to identify which c-miRNAs are expressed in bloodstream in Frabosana-Roaschina sheep during oestrus cycle and to understand their possible regulatory role in it.
Therefore, the first task of the project is to detect differentially expressed c-miRNAs (DE-miRNAs) during different phases of the oestrus cycle and to associate the potential candidates, related to the regulation of reproductive aspect, to the hormonal trend of progesterone and 17β-oestradiol in plasma. Moreover, c-miRNAs differential expression will be analysed in two different physiological conditions, ewes at the first oestrus and after at least one pregnancy, in an attempt to find variations linked with reproductive competence.
A second goal would be to locate possible correlations between c-miRNAs and animal welfare during the reproductive cycle. Indeed, behavioural data analyses and average trends of cortisol from saliva and faeces could be used to assess the correlation between animal welfare of sheep and stressful conditions during oestrus cycle. Then, through these analyses, a possible relation between c-miRNAs expression and stress may be examined.
Materials and methods
The experimental design is articulated in two trials, one held in January 2020 and the other in February 2022. Samples from the first trial were collected before the start of the PhD project.
Each trial involved 20 ewes (Frabosana-Roaschina breed) from a farm in Ormea (CN). The sheep of the first trial were nulliparous in their first oestrous cycle, those of the second were pluriparous. Upon their arrival in the Department sheepfold all ewes were synchronized with a standard protocol7.
Blood, saliva and faeces samples were taken every other day, along with ultrasound images, on ewes during an oestrus cycle. Video recordings were made throughout the sampling period by 3 cameras located inside and outside the sheepfold.
ELISA immunoassays will provide quantification of progesterone and oestradiol throughout the oestrus cycle and their trends in plasma will be used to identify the moment of oestrus (eg. spike in oestrogen) in which to evaluate the expression of c-miRNAs.
Then, c-miRNAs of a subset of animal for each trial will be sequenced to identify a panel of detectable and/or DE-miRNAs during oestrous cycle and under the two different reproductive conditions.
Using quantitative real-time PCR (qRT-PCR), candidates miRNAs from sequencing will then be validated and gene expression variations of these miRNAs will be analysed.
ELISA immunoassays to determine cortisol levels in faeces and saliva, along with behavioural scan sampling analyses, will provide a stress assessment of ewes.
The final purpose of this study is to identify differentially expressed circulating plasma miRNAs related to the reproductive cycle in sheep.
Along with the identification of potential c-miRNAs targets and their regulation pathways such results would allow to define a c-miRNA panel associated with the different reproductive phases of the Frabosana-Roaschina breed.
In this regard, deepening the reproductive aspects of an endangered breed of sheep becomes an extremely important factor. Indeed, thanks to this knowledge and especially using non-invasive assessment tools, not only reproductive performance could be predicted and evaluated, but also the stress condition of the animals, which can often affect the reproductive sphere too.
In this perspective, circulating miRNAs that would emerge from this study can be investigated as potential biomarkers in sheep for the evaluation of animal welfare during reproductive period.
 J. O’brien et al., “Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation” Front. Endocrinol. | www.frontiersin.org, vol. 1, p. 402, 2018, doi: 10.3389/fendo.2018.00402.
 D. N. Do et al., “miRNA regulatory functions in farm animal diseases, and biomarker potentials for effective therapies,” International Journal of Molecular Sciences, vol. 22, no. 6. MDPI AG, pp. 1–28, 2021, doi: 10.3390/ijms22063080.
 C. Zhou et al., “Deep-Sequencing Identification of MicroRNA Biomarkers in Serum Exosomes for Early Pig Pregnancy,” Front. Genet., vol. 11, p. 536, 2020, doi: 10.3389/fgene.2020.00536.
 J. Lee et al., “Analysis of circulating-microRNA expression in lactating Holstein cows under summer heat stress,” PLoS One, vol. 15, no. 8 August, p. e0231125, 2020, doi: 10.1371/journal.pone.0231125.
 S. Miretti et al., “MicroRNAs as Biomarkers for Animal Health and Welfare in Livestock.” Front. Vet. Sci. 7:578193, 2020, doi: 10.3389/fvets.2020.578193
 D.N. Do et al., “miRNA Regulatory Functions in Farm Animal Diseases, and Biomarker Potentials for Effective Therapies”, Int. J. Mol. Sci. vol. 22, p. 3080, 2021, doi: 10.3390/ijms22063080
 N. Hameed et al., “Approaches of estrous synchronization in sheep: developments during the last two decades: a review.” Trop Anim Health Prod 53, 485, 2021, doi: 10.1007/s11250-021-02932-8