PhD in Veterinary Sciences for Animal Health and Food Safety

Contacts

Supervisor

Eugenio Martignani

Curriculum vitae

Curriculum Vitae

Phd thesis

^{PhD Project}

^{Application of circulating-miRNAs expression profiles in an endangered Piedmontese sheep breed during the oestrous cycle}

^Background

^{MicroRNAs (miRNAs) are small non-coding RNAs (~22 nucleotides), that play a role in post-transcriptional regulation of several cellular processes. Expressed by tissue, they are released in body fluids and therefore called circulating miRNAs (c-miRNAs) [1]. Changing their profile under different conditions like physiological, pathological and psychological states, they can be used as minimally invasive biomarkers [2].}

^{Oestrus cycle is a complex physiological status, regulated by multiple factors. C-miRNAs could be useful tools to assess it and to identify any causes of abnormal conditions that undermine the reproduction efficiency [3].}

^{The combination of c-miRNAs profiles, hormonal trends and behaviour analyses can provide us information about animal welfare. Given the little information about c-miRNAs in sheep, investigating them in the Frabosana-Roaschina, a threatened local breed, can also be useful for its conservation.}

 

^{Specific aims of the project, Methods, Results}

^{The main aims of the project are to define a pattern of differentially expressed c-miRNAs (DE-miRNAs) in plasma during the different phases of oestrus cycle in Frabosana-Roaschina sheep and to investigate if the potential target genes of these c-miRNAs are involved in animal welfare during the reproductive activity.}

^{The experimental design is structured in two trials of 20 ewes each, with different reproductive competence: nulliparous ewes at first oestrus (10-12 months old) and pluriparous ewes (4-6 years old). All ewes were synchronised with a standard protocol and, after that, the ram was introduced in the flock. Blood and saliva or faeces samples were collected on alternate days (I personally contributed to the sampling of the second trial), along with ultrasound images. The animals were also recorded by 3 cameras throughout the sampling period.}

^{The first goal is to identify DE-miRNAs in bloodstream related to oestrus cycle phases. Sexual hormones (progesterone and 17β-oestradiol) were extracted from plasma and then ELISA immunoassays were performed to detect trends during oestrus cycle. Based on hormonal levels and ultrasound images, representative animals in specific phases from each trial will be selected for miRNAs isolation and gene expression analysis, at first by sequencing to identify DE-miRNAs and subsequently by validating candidate miRNAs by qRT-PCR.}

^{The second task of the project is to detect sheep stress markers during oestrus cycle. Cortisol will be extracted from different matrices. ELISA immunoassays will be performed on extracts to identify hormonal trends. In addition, scan sampling of videorecording for behavioural analyses will be carried out.}

^{The final goal is to investigate possible relations among circulating DE-miRNAs, cortisol levels or behaviour patterns in order to identify critical points during the reproductive activity of this breed and define c-miRNAs specific signatures.}

^{Currently, preliminary results on hormonal trends and behaviour of first trial are available. No sexual behaviours were evident, but different play behaviours were detected among the ewes, although the oestrous cycle was highlighted by hormonal trends.}

 

^{Future developments}

^{In the coming years, small-RNA sequencing will be performed by an external service on pilot samples, based on hormonal trends. The expression analysis and subsequently a panel of detectable and/or DE-miRNAs will be validated on all the animals to identify the differences in expression during oestrus cycle and under two different reproductive conditions.}

^{By combining cortisol trends and/or behavioural analyses I will assess stress condition of animals during the oestrus cycle period. After this, I will look for possible correlation between DE-miRNAs and stressful conditions related to reproductive sphere of ewes.}

^{Moreover, c-miRNAs extracted from saliva will be compared with those from plasma. The presence of matching miRNAs reinforces the idea of ​​miRNAs as biomarkers and the collection of saliva is a less invasive procedure than blood collection.}

 

_References

_{[1] 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.}

_{[2] 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.}

_{[3] 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}

Research activities

PhD Project

Application of circulating-miRNAs expression profiles in an endangered Piedmontese sheep breed during the oestrous cycle

 

Background

Circulating-microRNAs (c-miRNAs) are small non-coding RNAs, released in body fluids, involved in post-transcriptional regulation of several cellular processes [1]. Changing their profile under different physiological, pathological and psychological states, they can be used as minimally invasive biomarkers [2].

Oestrus cycle is a complex physiological status, regulated by multiple factors. c-miRNAs could be useful tools to assess it and to identify any stress conditions that thwart the reproduction efficiency [3].

 

Specific aims of the project, Methods, Results

The main aims of the project are to define a pattern of differentially expressed c-miRNAs (DE-miRNAs) during the different phases of oestrus cycle in Frabosana-Roaschina sheep, a threatened local breed, and to investigate if the potential target genes of these c-miRNAs are involved in animal welfare during the reproductive activity.

The experimental design is structured in two trials of 20 ewes each, with different reproductive competence: nulliparous at first oestrus (10-12 months old) and pluriparous (4-6 years old). All ewes were synchronised with a standard protocol and, after that, the ram was introduced in the flock. Blood and saliva or faeces samples were collected on alternate days (I personally contributed to the second trial), plus ultrasound images. The animals were recorded by 3 cameras during the sampling period for behavioural evaluation.

For the first task, I determined plasma progesterone and 17β-oestradiol levels to detect oestrus phases. Two key points, immediately after synchronization and at approximate ovulation time, were selected in six representative animals for plasma miRNA isolation and sequencing by an external service. Sequencing data identified the most expressed plasma miRNAs in the subset of animals at each time point. The identification of DE-miRNAs is currently underway. Validation of the sequencing data by qPCR is underway.

Meanwhile, I detected cortisol levels from various matrices with ELISA immunoassays to investigate the individual stress response during oestrus cycle. Cortisol trends did not highlight stress situations between animals.

I applied a similar protocol to saliva samples as a less invasive matrix and thus better suited for assessing welfare. I extracted c-miRNAs from saliva and with qPCR I highlighted different expression of 2 miRNAs (miR-16b and miR-21) in sheep clustered according to different cortisol trends. In literature, these miRNAs are involved in the stress response in other species. In such instance, the objective was to evaluate DE-miRNAs linked to cortisol trends. Moreover, behavioural analyses will be carried out to potentially associate with cortisol or c-miRNAs quantification.

The ultimate objective is to look at potential relationships between circulating DE-miRNAs in plasma and saliva, cortisol levels, or behavioural patterns in order to pinpoint crucial moments in this breed's reproductive activity and define c-miRNAs specific signals.

 

Future developments

Over the next six months, I will validate the panel of plasma detectable and/or DE-miRNAs collected by sequencing to determine the variations in expression during the oestrus cycle and under two different reproductive proficiencies. Once a list of DE miRNA is ready, putative targets will be identified through TargetScan database and a KEGG pathway analysis will be carried out (in collaboration) to detect changes in gene expression that could be caused by the different c-miRNA profiles.

After assessing stress condition during oestrus cycle with cortisol trends and/or behavioural analyses, I will assess possible correlations between DE-miRNAs and stressful conditions related to reproductive sphere of ewes.

Finally, c-miRNAs extracted from saliva will be compared with those from plasma. The presence of matching miRNAs might reinforce the idea of miRNAs as biomarkers while providing a less invasive collection than blood samples.

 

References

[1] 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.

[2] 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.

[3] 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

 

National Congress attendance (as the first author)

• Manenti I., Macchi E., Baratta M., Miretti S., Viola I., Toschi P., Cornale P. Does hormonal profile influence behaviors in pubescent ewes?, 75° Convegno SISVET, 15-17 giugno 2022, Lodi, Italia
• Manenti I., Viola I., Ala U., Macchi E., Baratta M., Martignani E., Miretti S. The extreme phenotypes of cortisol levels reveal different expression patterns of miRNAs in sheep saliva, 76° Convegno SISVET, 21-23 giugno 2023, Bari, Italia

International Congress attendance (as the first author)

• Manenti I., Viola I., Ala U., Macchi E., Baratta M., Martignani E., Miretti S., Adaptation response in sheep: ewes in different cortisol clusters reveal changes in the expression of salivary miRNAs, 5° Annual Meeting of the European Veterinary Congress of Behavioural Medicine and Animal Welfare, 19-20 ottobre 2023, Pisa, Italia

 

Period Abroad & Courses

Facultad de Veterinaria, Universidad de Zaragoza (UNIZAR) with supervisor Dr. Genaro C. Miranda de la Lama (4 months)

 

Publications

• Ogun S.; Viola I.; Obertino M.; Manenti I.; Ala U.; Brugiapaglia A.; Battaglini L.; Perona G.; Baratta M. (2022). Using sensors to detect individual responses of lambs during transport and preslaughter handling and their relationship with meat quality, Animal Welfare, 31(4): 505-516, DOI: 10.7120/09627286.31.4.010
• Viola I.; Toschi P.; Manenti I.; Accornero P.; Baratta M. (2023). Modulatory role of mTOR in trophoblast adaptive response in the early stage of placentation in sheep, Reproduction, 165(3): 313-324, DOI: 10.1530/REP-22-0356
• Manenti, I.; Viola, I.; Ala, U.; Cornale, P.; Macchi, E.; Toschi, P.; Martignani, E.; Baratta, M.; Miretti, S. (2023) Adaptation Response in Sheep: Ewes in Different Cortisol Clusters Reveal Changes in the Expression of Salivary miRNAs. Animals, 13(20): 3273, DOI: 10.3390/ani13203273

 

Academic bodies

• Teaching Committee