PhD in Veterinary Sciences for Animal Health and Food Safety

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Curriculum vitae

Phd thesis

Ph.D. thesis title is "Metabolic Status, Immune Function, and Uterine Health in Postpartum Dairy Cows: A Novel Approach to Uterine Pathology Diagnosis and Treatment"

Summary

During the transition period especially after parturition, dairy cows encounter various health issues. These include oxidative stress, immune dysfunction, and alterations in the uterine microbiota, leading to conditions like metritis, Clinical Endometritis (CE), and PVD. Oxidative stress leads to excessive generation of reactive oxygen species (ROS), which intensifies inflammatory responses. Cows experiencing these conditions tend to show elevated OS levels and a decrease in antioxidants. However, there is limited research on the mitochondrial activity and intracellular ROS in the endometrial cells of dairy cows. Postpartum(pp) uterine diseases involve complex signaling processes, cytokine production, and neutrophil mobilization. Transrectal ultrasonography (TUS) is the reliable on-field method for endometritis diagnosis. A thorough understanding of the uterine microbiota is crucial for effective treatments.
The aims of this thesis are centered around several areas: metabolic status, immune function, and uterine health. The first objectives were to assess oxidative stress (OS) and metabolic disease and to evaluate the level of OS in endometrial cells using biomarkers in cows caused by metritis and CE in comparison to healthy cows. In total 121 Holstein cows were enrolled for this study, and blood samples were collected weekly from 7 to 35 days pp to assess OS and metabolic disease in cows diagnosed with metritis (n=17), CE (n=43), and healthy cows (n=61). OS markers that were used in the study were d-ROM, OXY, and OSI. Metabolic disease markers are Glycemia and BHBA. Metritis was diagnosed by abnormal vaginal discharges and fever within 21 days pp. PVD was diagnosed using a mucus score, and the definition of CE is if PVD is combined with >5% endometrial PMNs. A subset of 6 healthy and 5 metritis cows had endometrial samples collected for mitochondrial activity, intracellular ROS levels, and cell nuclear area assessment. Cows with metritis or CE exhibited higher OS levels in comparison to healthy cows (P<0.05). However, mitochondrial activity and intracellular ROS levels did not show a significant difference between metritis and healthy cows (P>0.05), whereas cell nuclear area showed a difference (P<0.05). We found a modest increase in blood glucose in healthy compared to metritis cows at 14 days pp (P<0.05). Significant differences were not observed between blood glucose and BHBA levels in cows with CE compared to healthy (P>0.05).
Second objective was to evaluate the accuracy of Transrectal Ultrasonography (TUS) and OS biomarkers in diagnosing CE and to study the uterine microbiota in cows with PVD before and after local antibiotic treatment. For uterine health analysis, TUS was used for CE diagnosis in 104 cows, including cows diagnosed with CE (n=43) and healthy cows (n=61). To analyze the second objective, we collected uterine microbiota samples from a group of 16 cows, split evenly between 8 healthy cows and 8 with PVD. We found that TUS was the most accurate diagnostic test at 35 d pp, while OS was best for diagnosing CE at 21 and 28 d pp. When analyzing the uterine microbiota, we discovered that Trueperella was more common in cows diagnosed with PVD by bacterial culture method. Furthermore, at 21 and 35 d pp, there were significant differences in both the alpha and beta diversities of the uterine microbiota in PVD cows versus healthy or healed ones. Specifically, at 21 d pp, cows with PVD had higher levels of bacterial genera like Fusobacterium, Porphyromonas, Trueperella, and Parvimonas in comparison to healthy cows. Additionally, at 28 and 35 d pp, cows with PVD had elevated levels of Fusobacterium and Porphyromonas compared to healthy or healed cows.
Third objective was to analyze cytokines in serum samples of healthy cows and CE and to examine the neutrophil's viability and functional capacity in healthy cows. In total 104 Holstein cows were enrolled for evaluation of cytokines, blood samples were collected weekly from 7 to 35 days pp to measure cytokines such as TNF-α, IL-6, and IL-10 using the ELISA kits. The study included cows diagnosed with CE (n=43) and healthy cows (n=61). Levels of IL-10 in CE cows were higher than in healthy cows (P<0.05). Whereas, a significant difference was not found in IL-6 and TNF-α levels in CE compared to healthy cows (P > 0.05). Neutrophil viability and functionality were analyzed using flow cytometry by blood samples collected from 3 cows at multiple time points. For Neutrophil viability, viable PMN showed a difference at 0 hours and > 6 hours of storage (P<0.01). The anticoagulant type did not influence PMN viability parameters at any given time (P>0.05). Phagocytosis and Oxidative burst were not different at any given time point (P >0.05).
In summary, our study highlighted those dairy cows with metritis or CE during the postpartum period showed increased levels of oxidative stress and immune imbalances. This underscores the importance of early detection through uterine health screenings for better management and treatment options.

Research activities

Research products:

1. Paper related to the Ph.D. project
   • Chandrappa, S.M., Pascottini, O.B., Opsomer, G., Meineri, G., Martino, N.A., Banchi, P., Vincenti, L. and Ricci, A., 2023. Circulating and endometrial cell oxidative stress in dairy cows diagnosed with metritis. Theriogenology, 198, pp.217-223. Impact factor - 2.92, Q1 journal.
2. Paper not related to the Ph.D. project
   • Gonzalez Andueza, S., Azari‐Dolatabad, N., Benedetti, C., Fernandez, A., Angel‐Velez, D., Sadeghi, H., Chandrappa, S.M., Opsomer, G., Van Soom, A. and Pascottini, O.B., 2022. Lycopene supplementation to serum‐free embryo culture medium and its effect on development and quality of bovine blastocysts produced in vitro. Reproduction in Domestic Animals, 57(10), pp.1277-1279. Impact factor - 1.85, Q2 journal.
3. Abstracts of National congresses
   • Chandrappa, S. M.; Martino N.A.; Drago S.; Starvaggi Cucuzza A.; Banchi P.; Vincenti L.; Ricci A. Effect of uterine pathologies, treatments, and β-Hydroxybutyrate on recovering time fertility in dairy cows. 74th Annual SISVET conference (Societa Italiana delle Scienze Veterinarie) in Italy from June 23rd to June 26th, 2021, (Oral presentation).
   • Chandrappa S. M.; Donato G.; Alkhawagah A.; Meineri G.; Vincenti L.; Ricci A. 2022. Evaluation of the effect of oxidative stress on uterine pathologies in postpartum dairy cows. 75th Annual SISVET conference in Italy from June 15th to 19th 2022, in presence. Received Best Poster Award 2022. (Poster presentation).
4. Abstracts of International congresses
   • Chandrappa, S.M.; Bogado Pascottini O.; Meineri G.; Martino N.A.; Banchi P.; Starvaggi Cucuzza A.; Vincenti L.; Ricci A. Evaluation of the effect of oxidative stress in blood serum and endometrial cells in cows with metritis. 24th Annual ESDAR (European Society for Domestic Animal Reproduction) Conference 2021 from 12th to 17th October 2021,(Poster presentation).
   • Ricci A.; Chandrappa S. M.; Gaudino T.; Martino N.A.; Starvaggi Cucuzza A.; Vincenti L. 2020. Transrectal ultrasound as a tool for the diagnosis of clinical endometritis in dairy cows. 24th Annual ESDAR Conference 2021 from 12th to 17th October 2021,(Poster presentation).
   • Chandrappa S. M.; Bogado Pascottini O.; Opsomer G.; Meineri G.; Bergagna S.; Vincenti L.; Ricci A. 2022. Evaluation of oxidative stress and peripheral concentrations of inflammatory cytokines in dairy cows with postpartum metritis. 19th International Congress on Animal Reproduction (ICAR) in Bologna (ITALY) from 27th June to 1st July 2022,(Poster presentation).
   • Chandrappa S. M.; Bogado Pascottini O.; Gonzalez Andueza S.; Sadeghi H.; Vincenti L.; Ricci A.; Opsomer G.; Effect of type of anticoagulant, transportation time, and glucose supplementation to the culture medium on circulating neutrophil viability and functionality in dairy cattle. 31st World Buiatrics Congress (WBC) 2022 Madrid, Spain from September 4th to 8th 2022, (Poster presentation).
   • Chandrappa S. M.; Bogado Pascottini O.; Opsomer G.; Grego. E.; Ala U.; Banchi P.; Vincenti L.; Ricci A. 2022. Uterine microbiota in postpartum dairy cows suffering from purulent vaginal discharge and clinical endometritis prior to and following local antibiotic treatment. 25th Annual ESDAR Conference 2022 from 28th September to 2nd October 2022,(Poster presentation).
   • Chandrappa S. M.; Meineri G.; Vincenti L.; Ricci A. 2023. Clinical approach to clinical endometritis based on Oxidative stress markers and Transrectal ultrasound diagnosis. 1st European Symposium on Animal Reproduction (ESAR) in Nantes, France from 21st to 23rd September 2023, (Poster presentation).
5. Third mission activities  

• Game of Research – North edition, 2021- 16^th December 2021 - Evaluation of the effect of oxidative stress in blood serum and endometrial cells in cows with metritis, (Oral presentation).