Proteomics Analysis in Dairy Products: Cheese, a Review

Cheese is a worldwide produced and consumed commodity. There are many varieties of cheese from soft to hard, white to yellow, and fresh to aged after ripening. Especially, each category has its own producing technology. Many countries have labeled their most traditional cheese as Protective Designation of Origin (PDO). Moreover, several studies using advanced technologies, such as proteomics, have been performed to enhance labeling. In this review, broadly diffused and marketed, as well as Mediterranean countries’ special interest in Mediterranean diet-related PDO cheeses have been chosen as a reference. The aim of this work was to highlight the use of proteomics methods to examine how cheese proteins and peptides rearrange after ripening and use of starters. Further, we aimed to examine what kind of proteins are produced. Finally, we focused on bioactive molecules in cheeses and distinction of the original product from its counterfeit

Melatonin administration during pregnancy in heat-stressed ewes: study of reproductive traits, redox status, and proteome of ewes as well as of growth, oxidative balance, and immune response of their offspring

The present doctoral thesis focuses on the harmful effects of heat stress on the reproductive process in ewes and how they can be mitigated through the administration of melatonin implants. Specifically, the aim of this thesis is to clarify the effects of melatonin on i) the reproductive performance of ewes under heat stress conditions, ii) the antioxidant and immune competence of ewes during pregnancy and parturition, iii) the antioxidant and immune competence of offspring during the first days of their life, and iv) the molecular mechanisms of proteins expression during pregnancy and parturition. The thesis is divided into five chapters, followed by the conclusions. In the first chapter, the literature is reviewed in six thematic sections: i) stress, ii) oxidative stress, iii) heat stress, iv) melatonin, v) coccidiosis, and vi) proteomics. In the second chapter, the published research paper entitled "Effects of Melatonin Administration to Pregnant Ewes under Heat-Stress Conditions, in Redox Status and Reproductive Outcome" is presented verbatim. The experiment investigated the impact of melatonin on the reproductive performance, redox balance, and milk yield of ewes subjected to heat stress during the first three months of pregnancy. A total of sixty-eight ewes participated in the study, which were randomly allocated into two equal groups, the melatonin group (Μ), and the control (C). At the beginning of summer, synchronization of estrus was applied to all animals by inserting progestogen sponges (day 0) and administering equine chorionic gonadotropin hormone (eCG, 300IU, i.m.) at sponges’ removal (day 14), followed by natural mating. On day 0, melatonin implants were placed subcutaneously at the base of the ear in ewes of the M group, one in each ewe. This procedure was repeated three more times during pregnancy, at intervals of 40 days (days 40, 80, and 120). In total, ewes in the M group received four implants. Pregnancy diagnosis was performed on days 50 and 84, by means of ultrasonography, and ewes diagnosed as non-pregnant were removed from the study. Throughout the summer, environmental conditions (temperature, humidity) were recorded to assess heat stress using the Temperature Humidity Index (THI), as well as the body temperature and respiratory rate of the animals to estimate the degree of their stress. Blood samples were collected from all animals on days 0, 23, 40, 80, and 120, and then on days 0, 1, 2, 5, 10, and 40 post-partum, to assay antioxidant markers [total antioxidant capacity (TAC), glutathione (GSH), and thiobarbituric acid reactive substances (TBARS)] in blood plasma. The milk yield of ewes was assessed on days 5, 10, and 40 post-partum, the day of the weaning of the lambs. The results showed that melatonin administration improved the redox status of ewes in the M group throughout pregnancy, despite the heat stress that all animals experienced. This led to improved reproductive performance compared to the control ones. This result was amplified by the strong correlation found between the number of fetuses estimated on D50 and the levels of GSH assayed at the stage of blastocyst (D23), as well as the increased number of offspring in the M group. Additionally, the birth weight of lambs born per ewe in the M group improved. Finally, a significant increase in milk yield was recorded in the ewes of the M group compared to those of the C group in all three measurements. From the above results, it can be concluded that the administration of melatonin implants before and during pregnancy in ewes subjected to heat stress exerts a beneficial effect on improving their reproductive performance and milk yield, as well as the antioxidant capacity of the maternal organism. The third chapter is the sequel to the second chapter and focuses on the offspring of the first experiment. This chapter is presented as an independent published work entitled "Association of Melatonin Administration in Pregnant Ewes with Growth, Redox Status, and Immunity of Their Offspring." Out of the lambs born during the experiment described in the second chapter, thirty-seven lambs participated in this experiment. These lambs were distributed into the same groups as their mothers, namely the melatonin group (MEL) and the control group (CON), based on whether their mothers received melatonin implants or not. No interventions were made on these lambs during the study period, except for their mothers' exposure to heat stress before and during pregnancy and the insertion of melatonin implants in ewes of the MEL group. The body weight of lambs was recorded on days 0 (birth), 15, and 40, the last of which coincided with their weaning. Blood samples were collected from the lambs on days 0, 1, 2, 5, 10, and 40 to measure antioxidant markers (TAC, GSH, TBARS) in blood plasma. At the same time points, samples of colostrum/milk were, also, collected for chemical analysis and determination of their antioxidant capacity. Additionally, blood samples were collected from ewes at lambing (day 0) and one day later. Immunological parameters, such as interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), and interferon-γ (IFN-γ), as well as immunoglobulins IgG, were assayed in blood samples and colostrum samples collected from ewes at time points 0 and 1, together with blood samples collected from lambs at time points 0, 1 and 2. The results indicated that the lambs’ growth rate did not significantly differ between the two groups. However, the colostrum/milk of the ewes in the MEL group had a better antioxidant profile and higher protein content on the day of lambing (day 0), as well as higher fat content on day 1, compared to the control group. Regarding the immunological profile, the MEL ewes showed an increase in interleukin-6 on day 1, with a simultaneous increase in interleukin-10 in the plasma of their lambs on day 2, compared to the control group. The correlations conducted demonstrated that the MEL lambs exhibited better absorption of immunoglobulins, despite the significant increase in immunoglobulins in the blood of the CON lambs on day 2. These results suggest that the administration of melatonin during embryonic development has a positive impact on offspring during the critical hours following birth by improving their redox status. Additionally, based on the immunological parameters, antioxidant capacity and chemical analysis of colostrum/milk, it can be concluded that melatonin administration to ewes throughout their pregnancy enhances the quality of the milk produced. The fourth chapter follows the same pattern as the previous two and includes a previously published study entitled "Melatonin Administration to Pregnant Ewes for Coccidiosis Control in Their Offspring." In this experiment, thirty ewes were exposed to heat stress during the first months of pregnancy and gave birth to total forty-seven lambs. The ewes were divided into five equal groups as follows: the KM group, which was subjected to melatonin treatment and experimental infection; the KC group, which underwent experimental infection only; the CM group, which received melatonin implants only; the CC group, serving as control for the previous three groups; and the NC groups, which was the control group for natural environmental infection. The number of lambs born per group of ewes was 9, 11, 10, 8, and 9 for the KM, KC, CM, CC, and NC group, respectively. Hormonal control of the estrous cycle was applied to all ewes, while melatonin implants were administered to the ewes of the KM and CM groups, as described in the second chapter. Additionally, experimental infection with a mixture of sporulated oocysts of Eimeria spp. took place on the one hundred and twentieth day of pregnancy in the KM- and CM- ewes, as well as in the KM-, CM-, KC-, and CC-lambs at the age of five to nine days. Fecal samples were collected from all ewes on the day of infection and at parturition, as well as from all lambs on the day of infection and thirteen more times within eight weeks (a total of fourteen samples) to determine the number of oocysts per gram of feces. Blood samples were collected from ewes on the 120th day of pregnancy and at parturition, as well as from lambs on days 1 and 2 after birth, while colostrum/milk samples were collected at parturition and seventy-two hours later for the determination of cytokines (IL-1β, IL-6, IL-10, and IFN-γ) and IgG immunoglobulins. The growth rate of lambs was recorded from birth until the age of sixty days, every fifteen days, for a total of five measurements. Meanwhile, the leukogram in blood smears of lambs was assessed in blood samples at six-time points during the same period as fecal sampling. Results showed significantly increased levels of immunoglobulins in both the blood plasma of lambs and ewes, as well as in the colostrum of the KM group compared to the controls. KM-lambs also exhibited a higher concentration of IFN-γ compared to lambs from other groups. Furthermore, the ratio IL-10/IFN-γ was significantly reduced in ewes belonging to the KM group, which is related to melatonin's ability to upregulate the production of IFN-γ and downregulate IL-10. The growth rate of lambs did not differ significantly, suggesting that melatonin contributes to increased and sustained body weight despite the severity of clinical symptoms. Moreover, the oocyst excretion per gram of feces was significantly reduced by 94.9% and 92.6% in KM and CM lambs, respectively, compared to the KC group during the first three weeks, when the pathogen E. ovinoidalis was predominant. Finally, the leukogram showed that the predominant type of leukocytes in all experimentally infected lambs was mononuclear cells, but not in NC lambs. In conclusion, the administration of melatonin during embryonic development appears to have a positive impact on the immune system of offspring when exposed to a high parasitic load. Additionally, the reduction in oocyst excretion in feces contributes to a decrease in the environmental parasitic load, and thus in the farm. These findings suggest that melatonin could be applied as a management practice for coccidiosis control in lambs. In the fifth and final chapter, a self-contained study entitled "Proteomics analysis of pregnancy in ewes under heat stress conditions and melatonin administration" is presented. In this study, proteomic techniques were employed to elucidate the biochemical pathways in which melatonin is involved, both during the early months of pregnancy in ewes under thermal stress conditions and at parturition. The reason behind this experiment stemmed from the previous experiments that demonstrated the positive effects of melatonin on oxidative stress, immune response, and reproductive performance in ewes who received it. To achieve this goal, the exact protocol from the first experiment was maintained, regarding the control of estrous cycles and melatonin implant administration. A total of twenty-two ewes participated in the study, which were divided into two equal groups: the M group, which received melatonin implants, and the C group, which served as the control. Blood samples were collected from all animals on days 0 (start of estrus synchronization and the insertion of the first melatonin implant, samples M1-C1), 16 (before natural mating, M2-C2), 40 (second implant, M3-C3), 80 (fourth implant, M4-C4), and at parturition (M5-C5). The blood plasma samples were prepared for proteomic analysis by separating proteins using two-dimensional polyacrylamide gel electrophoresis, and differentially expressed proteins were detected in the gels. These proteins were identified using mass spectrometry, specifically matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The results revealed the presence of a total of forty-two additional proteins in samples M2, M3, and M4, as well as fifty-three additional proteins in M5, at parturition. These proteins can be categorized into five groups related to immune response, embryo and placental development during pregnancy, antioxidant defense, cell cycle regulation, and ion exchange and transport within the cell. Melatonin was found to be a regulatory factor in cell division and ion transport, as it regulates cellular respiration and ATP production in mitochondria. Furthermore, it appears to influence the increase or not of cytokines production related to the immune response, and it promotes the formation and development of embryonic organs. Finally, it serves as a regulatory factor in the antioxidant defense system, as it enhances the expression of enzymes that regulate endogenous antioxidants. The most important conclusions drawn from the findings of this thesis are summarized below. The exogenous melatonin, when administered to ewes throughout the entire reproductive process under heat stress conditions to maintain consistently elevated levels i) improves the reproductive performance of ewes, increases the number of fetuses, and enhances the body weight of lambs at birth. It also protects both the maternal organism and the fetus when pregnancy proceeds under heat stress and, consequently, increased oxidative stress; ii) increases the activity of endogenous antioxidant enzymes and reduces lipid peroxidation, which can be detrimental to both the cell and the organism, leading to apoptotic phenomena; iii) positively impacts the milk yield of ewes by improving both the quantity and quality of milk produced; iv) protects to neonates during the critical early hours of their life by significantly reducing the oxidative stress they experience; v) exerts immunomodulatory effects on both the maternal organism and neonates during the first days of life. It increases the levels of cytokines in the blood of ewes and their lambs, as well as in their colostrum under normal conditions; vi) improves the immune response of both ewes and their offspring after experimental infection with a mixture of coccidia. This includes increasing antibody and cytokine titers while presenting a milder clinical picture, maintaining a steady rate of lamb’s growth, and decreasing the oocysts’ excretion to the environment; vii) enhances the expression of proteins related to the immune response, regulation of endogenous antioxidants, the cell cycle, cellular respiration, and the formation and development of embryonic organs and placenta. This protective effect extends to both the maternal organism and the fetus under stressful conditions.Η παρούσα διδακτορική διατριβή εστιάζει στη μελέτη των επιπτώσεων της θερμικής καταπόνησης κατά τη διάρκεια της αναπαραγωγικής διαδικασίας στις προβατίνες και πώς αυτές μπορούν να μετριαστούν μετά από χορήγηση εμφυτευμάτων μελατονίνης. Ειδικότερα, η διατριβή αποσκοπεί στο να διαλευκανθούν οι επιδράσεις της μελατονίνης: α) στην αναπαραγωγική απόδοση των προβατίνων σε καταστάσεις θερμικής καταπόνησης, β) στην αντιοξειδωτική ικανότητα και ανοσολογική απόκριση των προβατίνων κατά τη διάρκεια της εγκυμοσύνης και στον τοκετό, γ) στην αντιοξειδωτική και ανοσολογική ικανότητα των απογόνων τις πρώτες ημέρες της ζωής τους και δ) στους μηχανισμούς έκφρασης των πρωτεϊνών κατά τη διάρκεια της εγκυμοσύνης και στον τοκετό. Η διατριβή χωρίζεται σε πέντε κεφάλαια και έπονται τα συμπεράσματα. Στο πρώτο κεφάλαιο γίνεται ανασκόπηση της σχετικής βιβλιογραφίας γύρω από έξι θεματικές ενότητες: α) στρες, β) οξειδωτικό στρες, γ) θερμική καταπόνηση, δ) μελατονίνη, ε) κοκκιδιακή παρασίτωση και στ) πρωτεωμική τεχνολογία. Στο δεύτερο κεφάλαιο παρατίθεται αυτούσια η δημοσιευμένη ερευνητική εργασία, με τίτλο «Effects of Melatonin Administration to Pregnant Ewes under Heat-Stress Conditions, in Redox Status and Reproductive Outcome». Στον πειραματισμό διερευνήθηκε η επίδραση της μελατονίνης στην αναπαραγωγική απόδοση, την οξειδοαναγωγική ισορροπία και την γαλακτοπαραγωγική ικανότητα των προβατίνων όταν αυτές βρίσκονται σε θερμική καταπόνηση κατά τους τρεις πρώτους μήνες της κυοφορίας τους. Στον πειραματισμό συμμετείχαν συνολικά εξήντα οχτώ προβατίνες, οι οποίες χωρίστηκαν τυχαία σε δύο ισάριθμες ομάδες, την ομάδα της μελατονίνης (Μ) και την ομάδα των μαρτύρων (C). Στις αρχές του καλοκαιριού εφαρμόσθηκε σε όλα τα ζώα συγχρονισμός των οιστρικών κύκλων με εισαγωγή σπόγγων προγεσταγόνων (ημέρα 0) και χορήγησης 300IU ίππειας χοριακής γοναδοτροπίνης (eCG) ενδομυικά κατά την αφαίρεση των σπόγγων (ημέρα 14). Ακολούθησε γονιμοποίηση των ζώων με φυσική οχεία. Την ημέρα 0 στην ομάδα Μ τοποθετήθηκαν υποδόρια στη βάση του ωτός εμφυτεύματα μελατονίνης, ένα σε κάθε προβατίνα. Η διαδικασία επαναλήφθηκε άλλες τρεις φορές, κατά τη διάρκεια της εγκυμοσύνης, ανά 40 ημέρες (ημέρες 40, 80 και 120). Συνολικά, οι προβατίνες της ομάδας Μ έλαβαν τέσσερα εμφυτεύματα. Τις ημέρες 50 και 84 πραγματοποιήθηκε διάγνωση της εγκυμοσύνης με υπερηχοτομογραφική εξέταση και οι προβατίνες στις οποίες δε διαγνώσθηκε εγκυμοσύνη απομακρύνθηκαν από τη μελέτη. Καθόλη τη διάρκεια του καλοκαιριού πραγματοποιήθηκε καταγραφή των περιβαλλοντολογικών συνθηκών (θερμοκρασία, υγρασία) για την εξακρίβωση της θερμικής καταπόνησης μέσω του δείκτη ΤΗΙ (Temperature Humidity Index), καθώς και καταγραφή της θερμοκρασίας του σώματος και της συχνότητας των αναπνοών των ζώων, για να εκτιμηθεί ο βαθμός καταπόνησής τους. Δείγματα αίματος ελήφθησαν από όλα τα ζώα τις ημέρες 0, 23, 40, 80, 120, καθώς και τις ημέρες 0, 1, 2, 5, 10 και 40 μετά τον τοκετό, για τη μελέτη αντιοξειδωτικών δεικτών [ολική αντιοξειδωτική ικανότητα (TAC), γλουταθειόνη (GSH), δείκτης λιπιδικής υπεροξείδωσης (TBARS)] στο πλάσμα του αίματος. Η γαλακτοπαραγωγική ικανότητα των προβατίνων εκτιμήθηκε τις ημέρες 5, 10 και 40 μετά τον τοκετό, ημέρα που εφαρμόσθηκε και ο απογαλακτισμός των αρνιών. Από τη στατιστική επεξεργασία των αποτελεσμάτων διαπιστώθηκε ότι η χορήγηση μελατονίνης βελτίωσε σημαντικά την οξειδοαναγωγική κατάσταση των προβατίνων της ομάδας Μ καθόλη τη διάρκεια της εγκυμοσύνης παρά τη θερμική καταπόνηση που υπέστησαν όλα τα ζώα, με αποτέλεσμα τη βελτίωση της αναπαραγωγικής τους απόδοσης συγκριτικά με την ομάδα των μαρτύρων. Το αποτέλεσμα αυτό ενισχύθηκε από την ισχυρή συσχέτιση που βρέθηκε μεταξύ του αριθμού των εμβρύων που καταμετρήθηκαν την ημέρα 50 και της γλουταθειόνης που προσδιορίστηκε στο στάδιο της βλαστοκύστης (ημέρα 23), αλλά και από τον αυξημένο απογόνων στην ομάδα Μ. Παράλληλα, βελτιώθηκε σημαντικά το βάρος των αρνιών που γεννήθηκαν ανά προβατίνα στην ομάδα Μ. Τέλος, καταγράφηκε σημαντική αύξηση στην παραγωγή γάλακτος στις προβατίνες της ομάδας Μ σε σύγκριση με εκείνες της ομάδας C και στις τρεις μετρήσεις που πραγματοποιήθηκαν. Από τα παραπάνω αποτελέσματα συμπεραίνεται ότι η χορήγηση εμφυτευμάτων μελατονίνης πριν και κατά τη διάρκεια της εγκυμοσύνης σε προβατίνες που υποβάλλονται σε θερμική καταπόνηση δρα ευεργετικά ως προς τη βελτίωση της αναπαραγωγικής και γαλακτοπαραγωγικής τους απόδοσης, καθώς και της αντιοξειδωτικής ικανότητας του μητρικού οργανισμού κατά τη διάρκεια της εγκυμοσύνης.Το τρίτο κεφάλαιο αποτελεί τη συνέχεια του δεύτερου, καθώς αφορά τους απογόνους του πρώτου πειραματισμού. Το κεφάλαιο αυτό παρουσιάζεται ως αυτοτελής δημοσιευμένη εργασία, με τίτλο «Association of Melatonin Administration in Pregnant Ewes with Growth, Redox Status and Immunity of Their Offspring». Από τα αρνιά που γεννήθηκαν κατά τον πειραματισμό που περιγράφεται στο κεφάλαιο δύο, τριάντα επτά έλαβαν μέρος στον συγκεκριμένο πειραματισμό. Τα αρνιά αυτά κατανεμήθηκαν στις ίδιες ομάδες που ανήκαν οι μητέρες τους, δηλαδή στην ομάδα της μελατονίνης (MEL) και στην ομάδα των μαρτύρων (CON), με βάση το εάν οι μητέρες τους είχαν λάβει ή όχι εμφυτεύματα μελατονίνης, αντίστοιχα, όπως περιγράφεται αναλυτικά στο δεύτερο κεφάλαιο. Στα αρνιά αυτά δεν έγινε καμία παρέμβαση κατά το χρονικό διάστημα της μελέτης, πλην της έκθεσης των μητέρων τους σε θερμική καταπόνηση πριν και κατά τη διάρκεια της εγκυμοσύνης και της τοποθέτησης των εμφυτευμάτων μελατονίνης στις μητέρες των αρνιών της ομάδας ΜEL. Το βάρος των αρνιών καταγράφηκε τις ημέρες 0 (γέννηση), 15 και 40, όταν έλαβε χώρα ο απογαλακτισμός τους. Δείγματα αίματος συλλέχθηκαν από τα αρνιά τις ημέρες 0, 1, 2, 5, 10 και 40 για τη μέτρηση αντιοξειδωτικών δεικτών στο πλάσμα του αίματος. Τις ίδιες χρονικές στιγμές συλλέχθηκαν και δείγματα πρωτογάλακτος/γάλακτος, με σκοπό τη χημική τους ανάλυση και τον προσδιορισμό της αντιοξειδωτικής ικανότητας (TAC, GSH, TBARS). Επίσης, δείγματα αίματος συλλέχθηκαν από τις προβατίνες στον τοκετό (ημέρα 0) και μία ημέρα αργότερα. Τις χρονικές στιγμές 0 και 1 αναλύθηκαν δείγματα του πλάσματος αίματος από τις προβατίνες και δείγματα πρωτογάλακτος για τον προσδιορισμό κυτταροκινών [ιντερλευκίνη-1β (IL-1β), ιντερλευκίνη-6 (IL-6), ιντερλευκίνη-10 (IL-10), ιντερφερόνη-γ (IFN-γ)] και ανοσοσφαιρινών γ (IgG). Οι ίδιοι ανοσολογικοί δείκτες μελετήθηκαν στα δείγματα του πλάσματος του αίματος των αρνιών τις χρονικές στιγμές 0, 1 και 2. Από τη στατιστική επεξεργασία των αποτελεσμάτων διαπιστώθηκε ότι ο ρυθμός ανάπτυξης των αρνιών δε διέφερε σημαντικά μεταξύ των δύο ομάδων. Παρόλα αυτά το πρωτόγαλα/γάλα των προβατίνων της ομάδας Μ είχε καλύτερο αντιοξειδωτικό προφίλ και μεγαλύτερη περιεκτικότητα σε πρωτεΐνες την ημέρα του τοκετού (ημέρα 0), καθώς και μεγαλύτερη περιεκτικότητα σε λιπαρά την ημέρα 1, σε σύγκριση με τα αντίστοιχα δείγματα της ομάδας των μαρτύρων. Επίσης, τα αρνιά της ομάδας MEL εμφάνισαν βελτιωμένη οξειδοαναγωγική κατάσταση από τη γέννησή τους μέχρι και την ημέρα 2 σε σύγκριση με τους μάρτυρες. Όσον αφορά το ανοσολογικό προφίλ, οι προβατίνες της ομάδας Μ εμφάνισαν αύξηση της ιντερλευκίνης-6 την ημέρα 1, με ταυτόχρονη αύξηση της ιντερλευκίνης-10 στο πλάσμα των αρνιών τους συγκριτικά με τα αρνιά της ομάδας CON την ημέρα 2. Οι συσχετίσεις που πραγματοποιήθηκαν έδειξαν ότι τα αρνιά της ομάδας MEL παρουσίασαν καλύτερη

Redox Status of Pregnant Ewes after Vaccination against Clostridial Diseases

The redox status shortly after the vaccination of pregnant ewes is rather unexploited. Thus, the present study was designed to evaluate the fluctuation of redox status after the administration of the annual booster dose of a polyvalent clostridial vaccine in pregnant ewes, 3 to 4 weeks before lambing, with or without a simultaneous injection of Vit E/Se. In total, 24 pregnant Lacaune ewes 3–4 weeks before lambing were randomly allocated into four equal groups: the V (vaccinated with a polyvalent clostridial vaccine), VE (vaccinated and injected IM with Vit E/Se), E (injected IM with Vit E and Se), and C (neither vaccinated nor injected with Vit E/Se). The study period lasted for 21 days, starting on the day of administration. Four redox biomarkers, the antioxidant capacity (TAC), the thiobarbituric acid reactive substances (TBARS), the reduced glutathione (GSH), and the catalase (CAT) were evaluated in blood samples collected from all ewes before the injections (0 h) and then at 12 (12 h), 24 (D1), and 48 h (D2), and thereafter on days 4 (D4), 6 (D6), 10 (D10), 14 (D14), and 21 (D21). The results reveal that the TAC was the only biomarker evaluated that was significantly affected by group and significantly lower in vaccinated animals (V and VE groups) compared to non-vaccinated (E and C groups). The absence of an increase in the TBARS values after vaccination in groups V and VE indicates the absence of significant oxidative stress. Overall, it can be assumed that annual booster immunizations against clostridial diseases do not impose acute oxidative stress on pregnant ewes in the last month of pregnancy

Redox Status and Hematological Variables in Melatonin-Treated Ewes during Early Pregnancy under Heat Stress

The preovulatory follicles and preimplantation stage embryos are found to be rather sensitive to heat stress due to their low potential for scavenging reactive oxygen species (ROS). The aim of the present study was to assess the impact of melatonin administration on redox status and hematological variables during the preovulatory period and early stages of embryogenesis in heat-stressed ewes in vivo. Forty Karagouniko-breed ewes were divided in two groups, the melatonin (M, n = 20) group and control (C, n = 20) one. All animals were subjected to heat stress throughout the study, which lasted forty days (D0 to D40). In M group, melatonin implants were administered on D0. Then, oestrous synchronization was applied (D19-D33). On D34, six rams were introduced into the ewe flock for mating. Ultrasonographic examination was conducted on D73 for pregnancy diagnosis. The temperature humidity index (THI), the rectal temperature (RT), and the number of breaths per minute (BR) were evaluated twice daily. Redox biomarkers, namely total antioxidant capacity (TAC), reduced glutathione (GSH), and thiobarbituric acid reactive substances (TBARS), were assayed in blood samples collected on D0, D33, and D40. In addition, packed cell volume (PCV), white blood cells (WBCs), leukocyte differential count, and cortisol assessment were conducted in blood samples on D33 and D40. The results indicated improved fertility rate and mean number of lambs born per ewe due to improved redox status (p < 0.05) in ewes that received melatonin implants 34 days approximately before the onset of oestrus. The PCV decreased in both groups between the two time-points (p < 0.05). However, the NEU/LYMPH ratio decreased (p < 0.05) only in group M. The low cortisol levels and the decreased NEU/LYMPH ratio in both groups support the hypothesis that ewes of the indigenous Karagouniko breed may exhibit adaptation to environmental thermal stress. The administration of melatonin as an antioxidant regime may improve the reproductive competence of heat stressed ewes and may also enhance their ability to adapt at high ambient temperatures

Association of Melatonin Administration in Pregnant Ewes with Growth, Redox Status and Immunity of Their Offspring

In this study, the effects of melatonin treatment on growth, redox status and immunity in prenatally stressed newborn lambs were evaluated. Thirty-seven newborn lambs were allocated into two groups (melatonin-MEL and control-CON), based on whether their mothers were treated with melatonin implants or not, respectively. All pregnant ewes were exposed to heat stress. The body weight of lambs was recorded at birth (L0), and then on days 15 (L15) and 40 (L40). Redox biomarkers [total antioxidant capacity (TAC), glutathione (GSH), thiobarbituric acid reactive substances (TBARS)] were assayed in blood samples collected from lambs on days L0, L1, L2, L5, L10 and L40. Chemical analysis and antioxidant capacity were evaluated in colostrum and milk samples collected at the same time points with blood samples. Cytokines (IL-1β, IL-6, IL-10, IFN-γ) and immunoglobulin (IgG) were assayed in blood and colostrum samples collected from ewes on days L0 and L1, and in lambs’ blood on days L0, L1 and L2. The results revealed that body weight gain of newborn lambs did not differ between the two groups (p > 0.05). Better redox status was found in MEL lambs until L2, as well as higher antioxidant capacity in the colostrum of MEL ewes compared to CON ones on day L0 (p < 0.05). In MEL ewes’ colostrum, higher protein content was measured on day L0 and higher fat content on L1 compared to CON group (p < 0.05). The highest level of IL-6 was found in MEL ewes on L1, with a concomitant increase of IL-10 level in MEL lambs in comparison to CON lambs on L2. Moreover, CON colostrum resulted in a higher level of IL-10 within time, coupled with an increased level of IgG found in lambs’ plasma on L2 (p = 0.04). This study indicated that melatonin could be administered as antioxidant and immune-modulatory regime in prenatally stressed offspring in order to cope with the crucial first days of their life. This effect of melatonin was also amplified by crosstalk between IL-6, IL-10 and IgG production, resulting in an improved quality of produced milk

Association of melatonin administration in pregnant ewes with growth, redox status and immunity of their offspring.

SIMPLE SUMMARY: Melatonin is a known antioxidant and anti-inflammatory regime, while in sheep it is broadly used to accelerate the onset of the breeding season. Our recent study showed that melatonin administration during pregnancy in heat-stressed ewes improved fertility rate and number of lambs born per ewe, the redox status of the maternal organism and the produced milk quantity until weaning. In this study, we present the impact of melatonin administration in stressed ewes during pregnancy considering: (a) humoral response of both maternal organism and offspring during the first two days after parturition, (b) chemical composition and antioxidant parameters of colostrum and milk until weaning and (c) redox status of the offspring until weaning. The results indicated that melatonin improved the redox status of the offspring and the quality of colostrum. Moreover, melatonin could be administered as immune-modulatory regime, apart from antioxidant, in prenatally stressed offspring in order to cope with the crucial first days of their life, as the humoral response results suggested. ABSTRACT: In this study, the effects of melatonin treatment on growth, redox status and immunity in prenatally stressed newborn lambs were evaluated. Thirty-seven newborn lambs were allocated into two groups (melatonin-MEL and control-CON), based on whether their mothers were treated with melatonin implants or not, respectively. All pregnant ewes were exposed to heat stress. The body weight of lambs was recorded at birth (L0), and then on days 15 (L15) and 40 (L40). Redox biomarkers [total antioxidant capacity (TAC), glutathione (GSH), thiobarbituric acid reactive substances (TBARS)] were assayed in blood samples collected from lambs on days L0, L1, L2, L5, L10 and L40. Chemical analysis and antioxidant capacity were evaluated in colostrum and milk samples collected at the same time points with blood samples. Cytokines (IL-1β, IL-6, IL-10, IFN-γ) and immunoglobulin (IgG) were assayed in blood and colostrum samples collected from ewes on days L0 and L1, and in lambs’ blood on days L0, L1 and L2. The results revealed that body weight gain of newborn lambs did not differ between the two groups (p > 0.05). Better redox status was found in MEL lambs until L2, as well as higher antioxidant capacity in the colostrum of MEL ewes compared to CON ones on day L0 (p < 0.05). In MEL ewes’ colostrum, higher protein content was measured on day L0 and higher fat content on L1 compared to CON group (p < 0.05). The highest level of IL-6 was found in MEL ewes on L1, with a concomitant increase of IL-10 level in MEL lambs in comparison to CON lambs on L2. Moreover, CON colostrum resulted in a higher level of IL-10 within time, coupled with an increased level of IgG found in lambs’ plasma on L2 (p = 0.04). This study indicated that melatonin could be administered as antioxidant and immune-modulatory regime in prenatally stressed offspring in order to cope with the crucial first days of their life. This effect of melatonin was also amplified by crosstalk between IL-6, IL-10 and IgG production, resulting in an improved quality of produced milk

Redox biomarker baseline levels in cattle tissues and their relationships with meat quality

Cattle breeds or crossbreds with high productivity traits have been developed to meet a growing demand for food. When intensive farming practices are followed, animals face several challenges which can result in poor performance, compromised welfare and the reduced quality of their products. Our study aims to highlight the resting values of the physiological oxidative stress that three cattle breeds exhibit, and their potential relationship with meat quality. For this purpose, we determined the levels of five common redox biomarkers (glutathione (GSH), catalase (CAT), total antioxidant capacity (TAC), thiobarbituric reactive substances (TBARS) and protein carbonyls (CARBS)) in the tissues of three commonly used beef cattle breeds (Charolais (CHA), Limousin (LIM) and Simmental (SIM)) and their association with specific meat quality traits that depend on color, pH and texture. The results revealed that LIM cattle breed animals have elevated intrinsic antioxidant defense systems in comparison to CHA and SIM cattle breed animals. In addition, the meat quality parameters were associated with the redox biomarkers. We propose that the determination of specific antioxidant parameters in the blood might be used as potential biomarkers to predict meat quality. This would allow farmers to nutritionally intervene to improve the quality of their products