Ekaterini K. Theodosiadou

The proteins most frequently associated with pregnancy are the pregnancy-associated glycoproteins (PAGs), which are abundantly expressed placental products in species belonging to the order Cetartiodactyla. Multiple PAG isoforms are expressed across different species and stages of gestation, with their expression influenced by factors, such as breed and bodyweight. These proteins can be detected in both blood and milk samples using ELISA or RIA assays, serving as early indicators of pregnancy. The present study aimed to detect PAGs in caprine milk through a non-invasive, high-throughput, mass-spectrometry-based proteomic approach. Milk samples were collected from 20 Skopelos breed goats at 20 and 45 days post-mating. Following appropriate processing for whey protein extraction, the samples underwent tryptic digestion to generate peptides for LC-MS/MS analysis. The proteomic investigation identified five distinct PAGs (caPAG₂ on day 20 and caPAG₃, caPAG₅, caPAG₆, and caPAG₁₂,on day 45 post-mating) annotated to Capra hircus, along with 22 additional proteins associated with the fetal–maternal interface, pregnancy progression, and immune-related pathways. These findings demonstrate that LC-MS/MS provides a non-invasive, sensitive, and reliable method for detecting PAGs in caprine milk during the early stages of pregnancy (day 20, as the presence of caPAG₂ revealed), applicable to both individual animals and flock-level monitoring.

The effect of four extenders on buck semen quality parameters was examined during a 48 h liquid storage. Semen was collected from six Skopelos bucks and diluted in the following extenders, containing: soy lecithin (SL, OviXcell^®), plant phospholipids (PP, AndroMed^®), egg yolk lecithin (EY, Steridyl^®), or no phospholipids (basic extender). Samples were stored at 5 °C for 48 h and assessed at 0, 24 and 48 h for viability (eosin-nigrosin), acrosome integrity (SpermBlue^®), membrane functional integrity (HOST), mitochondrial function (Rhodamine 123/SYBR-14/PI) and motility parameters (CASA). No significant reduction in total or progressive spermatozoa motility and mitochondrial function was observed at 24 h, whereas they all dropped significantly at 48 h, in all extenders. Spermatozoa viability, cell membrane functionality and acrosome integrity dropped progressively (0 h > 24 h > 48 h) in all groups. No significant difference among extenders was observed concerning spermatozoa mitochondrial function. Overall, spermatozoa viability, cell membrane functionality and acrosome integrity were higher in the three commercial extenders, compared to the basic extender. SL and EY extenders (OviXcell^® and Steridyl^®, respectively) preserved viability more effectively than the PP extender (AndroMed^®). Total motility was higher in the PP extender, compared to the SL extender. Spermatozoa acrosome integrity tended to be higher in the EY extender compared to all the other extenders. Further investigation of the protective potential of different types of cryoprotectants on liquid buck semen storage is important.

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.

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-1β, 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.

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 PGF_2α 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 β-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.

Milk is newborn’s food and an emulsion full of all necessary components for neonatal growth. Its consumption is worldwide and is the base for all dairy products. Because of the latter, many new technologies are growing, among them proteomics; in order to give new insights in milk’s compounds and to maximize the beneficial potential for consumers’ health. In this review, we aimed to gather data of proteomics studies for the majority of dairy animals and elucidate the role of milk bioactive compounds. Furthermore, special reference was made to milk fat globule membrane (MFGM) peptides and the result of thermal treatment in milk proteins. Finally, the proteomic approach regarding adulterations was included in the review.

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.

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.

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.

Heat stress is a known promoter of reactive oxygen species generation, which may compromise pregnancy and foetal development. Melatonin is a pleiotropic molecule that regulates various processes including pregnancy. Thus, it could be used to ameliorate the redox status of pregnant heat-stressed ewes and the outcome of their pregnancy. Sixty-eight ewes participated in the study, which were allocated into two equal groups, i.e., Melatonin (M) and Control (C) group. All ewes were exposed to heat stress from D0 to D120. In both groups, after oestrus synchronization of ewes, rams were introduced to them for mating (D16). In M group, starting with sponges’ insertion (D0), melatonin implants were administered four-fold every 40 days. Pregnancy diagnosis was performed by means of ultrasonography. Daily evaluation of temperature humidity index (THI), rectal temperature, and breathing rate were performed throughout the study. Blood samples were collected repeatedly from D0 until weaning for assaying redox biomarkers. Milk yield was measured thrice during puerperium. The results showed that melatonin administration throughout pregnancy improved the redox status of heat-stressed ewes and increased the mean number and bodyweight of lambs born per ewe, as well as the milk production. Therefore, melatonin may be used as antioxidant regimen in heat-stressed ewes for improving their reproductive traits.