Proteomics Analysis of Pregnancy in Ewes under Heat Stress Conditions and Melatonin Administration.

Melatonin is an indoleamine with broad spectrum properties that acts as a regulator of antioxidant and immune response in organisms. In our previous studies, melatonin improved redox status and inflammatory response in pregnant ewes under heat stress conditions. In the present study, using proteomics, the proteins regulated by melatonin during different stages of pregnancy and lambing were assessed. Twenty-two ewes equally divided into two groups, the melatonin (M) (n = 11) and control (C) group (n = 11), participated in the study and were exposed to heat stress during the first months of pregnancy. In the M group, melatonin implants were administered throughout pregnancy, every 40 days, until parturition (a total of four implants per ewe). Blood samples were collected at the beginning of the study simultaneously with the administration of the first melatonin implant (blood samples M1, C1), mating (M2, C2), second implant (M3, C3), fourth implant (M4, C4) and parturition (M5, C5), and MALDI-TOF analysis was performed. The results revealed the existence of 42 extra proteins in samples M2, M3 and M4 and 53 in M5 (sample at parturition) that are linked to melatonin. The biological processes of these proteins refer to boosted immune response, the alleviation of oxidative and endoplasmic reticulum stress, energy metabolism, the protection of the maternal organism and embryo development. This proteomics analysis indicates that melatonin regulates protective mechanisms and controls cell proliferation under exogenous or endogenous stressful stimuli during pregnancy and parturition.

Melatonin Administration to Pregnant Ewes for Coccidiosis Control in Their Offspring.

In livestock research, there has been a growing interest in the impact of melatonin on both health and disease conditions. The hypothesis of the present study was that melatonin treatment prenatally could support the immune competence and growth of experimentally infected lambs. This is the first study that aimed to investigate the impact of melatonin administration throughout pregnancy on immunity and oocyst excretion of pre-partum ewes and their offspring after experimental infection with Eimeria species. Thirty pregnant ewes were allocated into five equal groups, ΚΜ, ΚC, CM, CC, and NC, and gave birth to 47 lambs. Ewes of the KM and KC groups were orally challenged with a cocktail of Eimeria-sporulated oocysts (mainly consisting of Eimeria ovinoidalis), on day 120 of pregnancy, as well as all the lambs at the age of 5-9 days apart from those born from the NC group (environmental control). Fecal samples were collected from all ewes before infection and at parturition and from all lambs 14 times (S0-S13), before infection and during the following 8 weeks, for counting oocysts per gram of feces (OPG). Immunoglobulin (IgG) and cytokine (IL-1ß, IL-6, IL-10, IFN-γ) levels were determined in ewes' plasma collected before infection and at parturition, in lambs' plasma at 24 and 72 h after their birth, and in colostrum samples at parturition and 72 h later. Body weight of lambs was recorded five times from birth until the age of 60 days. Accordingly, the leucogram was evaluated in blood samples collected six times within the same period. On average, IgG concentration was higher (p < 0.05) in the blood of KM-ewes compared to KC and CC groups and in colostrum of KM-ewes compared to other groups (p < 0.001). KM-lambs had greater IgG titer and IFN-γ level than the other groups (p < 0.05). The IL-10/ IFN-γ ratio in KM-ewes was lower than the CC group (p = 0.06). Overall, the growth rate of lambs did not differ among groups (p > 0.05). Total oocysts' excretion in KM- and CM-lambs was reduced by 94.9% (p = 0.05) and 92.6% (p = 0.025), respectively, compared to KC-lambs, following the 3-week period after challenge, when E. ovinoidalis predominated in all groups. The dominant type of leucocytes was monocytes in all experimentally infected lambs, but not in NC-lambs, while overall lymphocytes were lower in KC-lambs than in NC-lambs (p < 0.05). Considering that almost all young indoor-reared lambs are exposed to coccidia species during their early life, melatonin treatment prenatally could suggest an alternative management tool in alleviating infection pressure.

Ovarian and Energy Status in Lame Dairy Cows at Puerperium and Their Responsiveness in Protocols for the Synchronization of Ovulation.

The purpose of this study was to assess the ovarian and energy status of multiparous lame dairy cows at the end of puerperium and investigate their responsiveness to estrous synchronization treatment regimens. Initial lameness scoring was performed at 28 ± 5 and 37 ± 5 d post partum, followed by lesion documentation and treatment. Cows were blocked by lameness severity and were randomly allocated to an estrous synchronization treatment regimen with seven days of progesterone supplementation (group LP, n = 26) or with an administration of PGF2α twice, 14 d apart (group LC, n = 26). Non-lame cows served as controls (group C, n = 27) and the same treatment regimen was imposed as that for group LC. Twelve days after estrous presynchronization, an Ovsynch treatment regimen and timed AI were imposed. Ultrasonography of the ovaries and blood sampling for progesterone were used to assess cyclicity status, whereas ß-hydroxybutyrate (BHBA) and non-esterified fatty acids (NEFA) were used to assess energy status. Lame cows were to a greater proportion non-cycling (36.5% vs. 11.1%; p = 0.02), had greater overall NEFA concentrations (0.32 ± 0.02 vs. 0.26 ± 0.02 mEq/L; p = 0.02) and a greater incidence of elevated NEFA concentrations (53.9% vs. 29.6%, p = 0.04) compared to control cows. However, no interaction between energy and lameness status was evident regarding non-cycling cows. The percentage of cows responding to the presynchronization, synchronization and ovulating did not differ between groups LP, LC, and C. The first-service conception rate (FSCR) tended to be greater for group C (37.0%) compared to group LP (16.0%; p = 0.08). Long-term reproductive performance did not differ between lame and control cows, although culling rates did (21.2% vs. 0%, respectivly; p = 0.01). The severity of lameness had an effect on culling rates (30.6% vs. 0% for cows with marked vs. moderate lameness; p = 0.01), whereas the type of lesion largely explained poor reproductive performance (FSCR 13.9% vs. 40.0% for cows with claw horn disruptions vs. infectious lesions; p = 0.04). Conclusively, cows that were lame during puerperium are at a greater risk of not cycling irrespective of energy status. Treatment regimens for the synchronization of ovulation seem to be efficient at resuming ovarian cyclicity. Marked lameness was detrimental to survivability, whereas cows with claw horn lesions had compromised reproductive capacity.

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.

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.

Effect of aflatoxin B1 on blood serum oestradiol-17ß and progesterone concentrations during the luteal phase and the synchronized oestrus of goats.

The effect of prolonged aflatoxin B1 (AFB1) administration on blood serum oestradiol-17ß and progesterone concentrations in goats during the luteal phase and the synchronized oestrus was investigated. Thirty-six Greek indigenous primiparous goats were used, during the oestrus period; 12 goats received, per os, 50 µg (treated group T50) and 12 goats received 100 µg (treated group T100) AFB1/day/head, respectively, for approximately 1.5 month, while 12 goats served as controls (C). On day 36 of the experiment, each goat was injected, i.m, 0.5 ml prostaglandin F2α (PGF2α). Blood samples were collected from each goat twice a week, before PGF2α injection, as well as every 4 hours from the onset to the end of the synchronized oestrus. Oestradiol-17ß and progesterone concentrations in blood serum were determined using radioimmunoassay. During the whole luteal(s) phase(s), linear regression analysis revealed a significant negative dependence (P < 0.05) of oestradiol-17ß and a significant positive dependence (P < 0.05) of progesterone over group (C = 0, T50 = 50, T100 = 100), in a dose dependent manner. During the synchronized oestrus, multiple linear regression analysis revealed a significant negative dependence (P < 0.05) of oestradiol-17ß, as well as a significant positive dependence (P < 0.05) of progesterone over group (C = 0, T50 = 50, T100 = 100) and over time (hours, from the onset to the end of the synchronized oestrus). No significant differences were noticed among the three groups, regarding the body weight of the goats from the onset to the end of AFB1 administration, the occurrence or the duration of the synchronized oestrus presented by the goats (P > 0.05). In conclusion, prolonged AFB1 administration at doses of 100 or even of 50 µg/day/head changes the hormonal pattern in blood during the luteal phase and the synchronized oestrus of goats, being in oestrus period.