Effects of a Curcumin/Silymarin/Yeast-Based Mycotoxin Detoxifier on Redox Status and Growth Performance of Weaned Piglets under Field Conditions.

The aim of this in vivo study was to investigate the effects of a novel mycotoxin detoxifier whose formulation includes clay (bentonite and sepiolite), phytogenic feed additives (curcumin and silymarin) and postbiotics (yeast products) on the health, performance and redox status of weaned piglets under the dietary challenge of fumonisins (FUMs). The study was conducted in duplicate in the course of two independent trials on two different farms. One hundred and fifty (150) weaned piglets per trial farm were allocated into two separate groups: (a) T1 (control group): 75 weaned piglets received FUM-contaminated feed and (b) T2 (experimental group): 75 weaned piglets received FUM-contaminated feed with the mycotoxin-detoxifying agent from the day of weaning (28 days) until 70 days of age. Thiobarbituric acid reactive substances (TBARSs), protein carbonyls (CARBs) and the overall antioxidant capacity (TAC) were assessed in plasma as indicators of redox status at 45 and 70 days of age. Furthermore, mortality and performance parameters were recorded at 28, 45 and 70 days of age, while histopathological examination was performed at the end of the trial period (day 70). The results of the present study reveal the beneficial effects of supplementing a novel mycotoxin detoxifier in the diets of weaners, including improved redox status, potential hepatoprotective properties and enhanced growth performance.

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.

Patterns of Reproductive Management in Sheep and Goat Farms in Greece.

This paper reports findings regarding patterns of reproductive management in 325 sheep flocks and 119 goat herds throughout Greece. The objectives were to describe the patterns of reproductive management in sheep and goat farms, to highlight factors that were associated with these management patterns and the clinical application of the various reproductive management approaches in the farms and to establish potential associations with production outcomes. The median months of the start of the mating period for adult sheep and goats were May and June, respectively and for ewe lambs and replacement goats these were August and September, respectively. The median duration of the mating period was 2 months for adults and 1 month for replacements. The median ratio of female to male animals was 22:1 and 25:1 in sheep and goat farms, respectively. Control of reproduction was applied in 33.2% and 16.8% of sheep and goat farms, respectively, mostly by intravaginal progestagen sponges and less often by melatonin implants; multivariable analysis indicated five (availability of milking parlour, number of ewes in a farm, number of daily milkings, daily period spent by farmers at the farm, farming tradition in the family) and two (number of daily milkings, availability of milking parlour) predictors in sheep and goat farms, respectively. Pregnancy diagnosis ultrasonographically was performed in 36.6% and 16.8% of sheep and goat farms, respectively; multivariable analysis indicated as significant three (management system applied in farms, age of farmer, farming tradition in the family) and two (management system applied in farms, availability of milking parlour) predictors in sheep and goat farms, respectively. The control of reproduction and pregnancy diagnosis were associated with a higher number of vaccinations during pregnancy: 2.6 and 2.7, respectively, versus 2.2 and 2.1 in farms where they were not performed. The average per farm number of lambs/kids born per female animal was 1.33 ± 0.01 and 1.30 ± 0.02 for sheep and goat farms, respectively; five (control of reproduction, location of the farm, presence of working staff, collaboration with veterinary practice, breed of ewes) and two (control of reproduction, breed of female goats) predictors were identified in sheep and goat farms, respectively, for high numbers of lambs/kids. Lambs/kids were taken away from their dams at the age of 50 and 65 days, respectively. The average culling age (females, males) was 5.9 and 4.4 years and 6.9 and 4.9 years for sheep and goats, respectively. Farmers sourced animals to be used as replacements for those removed from the farm, mostly from the animals in their own farms, considering criteria related to milk production. Finally, the application of the control of reproduction was associated with increased milk production and the number of newborns, whilst an inverse correlation between the culling age of animals and milk production was seen.

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.

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-1ß, 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.

Identification of Polymorphisms of the CSN2 Gene Encoding ß-Casein in Greek Local Breeds of Cattle.

This e research focused on the detection and identification of genetic polymorphisms in exon 7 of the ß-casein CSN2 gene in blood samples from Greek Holstein cows and from local breeds of cattle, such as Vrachykeratiki, Katerinis, and Sykias. For this purpose, DNA was isolated from 780 blood samples obtained from Greek Holstein cows, 86 from three local breeds of cattle, namely Brachyceros, Katerinis, and Sykias, and 14 from Greek buffalo. The desired region of exon 7 was amplified by PCR, resulting in 121 and 251 bp products in bovine and buffalo samples. The PCR product was digested with restriction fragment length polymorphism (RFLP) on agarose gels. The restriction enzymes DdeI and TaqI were used. All of the blood samples had the amplified size. The results showed that 74.4% of the Greek Holstein cows had the A2A2 ß-casein genotype, the three native breads Vrachykeratiki had 57.7%, and the other two had 100% of the A2A2 ß-casein. From the 14 Greek buffalo, 100% had the A2A2 ß-casein.

Mortality of Dairy Sheep during the Peri-Parturient Period: Results of a Field Investigation in Greece.

Objectives of the study were (a) to investigate the incidence risk of peri-parturient mortality in dairy flocks in Greece, (b) to study when such mortality occurs in relation to lambing, (c) to identify clinical problems associated with the deaths, and (d) to evaluate potentially associated factors. The deaths of ewes during the peri-parturient period (10 days before to 7 days after lambing) were monitored in 60 flocks over two years. In the ewes that died, detailed post-mortems were performed. The incidence risk of peri-parturient deaths was 0.7%. The daily incidence rate of deaths before, at, and after lambing was 0.05, 0.04, and 0.03%, respectively. Clinical problems most frequently associated with peri-parturient deaths were pregnancy toxaemia, ruminal acidosis and post-partum genital problems. For the occurrence of peri-parturient deaths in flocks, the average lambing rate in the flocks was the only significant factor. Regarding deaths before or after lambing, the season during which lambing took place and the number of animals in the flock, respectively, were found to be significant. Most ewes (54.5%) that died at lambing (dystocia) were primigravidae.

Effects of Melatonin Administration to Pregnant Ewes under Heat-Stress Conditions, in Redox Status and Reproductive Outcome.

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.

Ultrasonographic findings in the ovine udder during lactogenesis in healthy ewes or ewes with pregnancy toxaemia.

Objective of the study was to record, by means of ultrasonographic examination, changes occurring during lactogenesis in the udder of healthy ewes and of ewes with pregnancy toxaemia. The work was carried out in 28 ewes, 16 with pregnancy toxaemia (group A) and 12 healthy controls (group B). B-mode and Doppler ultrasonographic examination of the udder of ewes was performed. During the last month of pregnancy, grey-scale intensity values of mammary parenchyma in group A were significantly greater than in group B (P = 0.007), as was also the progressive increase in grey-scale intensity values in both groups (P < 0.001). Blood mammary input was significantly greater in ewes of group B than in ewes of group A (P < 0.05), as was also the progressive increase in blood input in both groups (P < 0.001). Further, differences between the two groups were identified in pulsatility index (P = 0.007) and in mean blood velocity (P = 0.036), but only during the last fortnight of pregnancy. After lambing, grey-scale values decreased sharply compared to those in pregnancy (P < 0.01), whilst blood input, pulsatility index and mean blood velocity continued the same trend as at the last stage of pregnancy, with differences between the two groups still prevalent (P < 0.05). There was a reverse correlation between grey-scale intensity values and milk quantities (P < 0.035) and a correlation between blood input and milk quantities (P < 0.07). The progressive increase in the diameter of the external pudendal artery was significant (P < 0.001), but no significant differences were evident between the two groups (P > 0.35). Differences between group A and group B in all other haemodynamic parameters studied were not significant, neither throughout the last month of pregnancy (P > 0.25), nor during the first week of lactation (P > 0.06). However, their progressive changes during the last month of pregnancy were significant (P < 0.02).

Ultrasonographic findings in the ovine udder during involution.

The objective of the study was to record, by means of ultrasonographic examination, changes occurring in the udder of ewes during involution and to compare differences between progressive or abrupt udder drying-off. In group A ewes, udder drying-off took place progressively during a period of 22 d; ewes were milked twice daily for the last time on day(D) 0; then, they were hand-milked once daily for a week (D1-D7), which was followed by another week during which ewes were hand-milked once every 2 d (D9, D11, D13), followed by a third week during which ewes were hand-milked once every 3 d (D16, D19, D22). In group B ewes, the procedure took place abruptly; ewes were milked twice daily for the last time on D0 and no milking was carried out after that. B-mode and Doppler ultrasonographic examination of the udder of all ewes was performed throughout the drying-off procedure. Appropriate data management and analysis were performed. Progressive changes of the various parameters evaluated throughout the study period were significant in both groups (P<0.005). Gray-scale results of mammary parenchyma progressively decreased during the study and differed significantly between group A and group B (P=0.049). A temporary increase in cistern volume was evident after cessation of lactation, but differences were not significant between the two groups (P>0.3). Diameter of the external pudendal artery progressively decreased during the study and differed significantly between the two groups (P=0.037). Both resistance index and pulsatility index progressively increased throughout the study period in both groups; for both parameters, differences between the two groups were significant (P<0.0005). B-mode ultrasonographic examination indicated differences in remodelling of the extracellular matrix in relation to the procedure for udder drying-off. Volume of the gland cistern did not appear to be affected by the procedure for udder drying-off. Doppler ultrasonographic examination confirmed that blood flow during initiated involution was lower than during a progressive procedure for drying-off.

Effects of melatonin treatment on follicular development and oocyte quality in Chios ewes - short communication.

Follicular development and oocyte quality were assessed by laparoscopic observation and in vitro fertilisation, respectively, in melatonin-treated (Group M) and control (Group C) anoestrous Chios ewes (n = 10 in each group). Fourteen days after melatonin insertion, all ewes had laparoscopic evaluation of the follicular population followed by oocyte pick-up (OPU); on day 22 intravaginal progestagen sponges were inserted for 14 days. Two days after sponge removal the follicular population was re-evaluated and a second follicular aspiration was performed. Collected oocytes from the second OPU underwent in vitro maturation, fertilisation and culture. The number of large follicles was higher in Group M than in the control ewes during the first OPU and tended to be so (P = 0.06) at the second. Morphologically, oocytes collected from controls were of better quality than those from Group M; however, more oocytes collected from melatonintreated animals fertilised and developed in vitro . These results indicate that melatonin is a potent regulator of follicular development and oocyte competence during the anoestrous period of the ewe.

Oestradiol concentration as a predictor of ovarian response in FSH stimulated ewe-lambs.

We investigated the prediction of ovarian response using oestradiol determination, in 37 gonadotrophin stimulated Karagouniko ewe-lambs. Ovarian stimulation was induced by serial FSH administrations, and laparoscopic follicular aspiration (OPU) was conducted 12h after the last FSH injection. Oestradiol concentration was assessed in six blood samples collected prior to each FSH injection and in one sample collected prior to follicular aspiration. According to ovarian response, ewe-lambs were allotted in three groups: good, L1 (n=17); moderate, L2 (n=10); and poor, L3 (n=10). Based on the data obtained from 28 (75%) randomly selected animals, a statistical model was designed and tested on the remaining nine lambs for its ability to predict the probability of good ovarian response. From the 2nd sample, oestradiol concentration was constantly higher in group L1 in comparison with L3 lambs (all p-values for the contrasts were

Oocyte pick-up in juvenile lambs affects neither onset of puberty nor their future fertility.

A study was designed to evaluate whether the time of onset of puberty and fertility of young ewe lambs would be affected by oocyte pick-up conducted in single or repeated sessions during the first months of lambs' live. Five groups of lambs from the Karagouniko breed were used (A-E each n=12). In group A no treatments were applied (control group) while, laparoscopical follicular aspiration (OPU) was performed early in the third, fourth and fifth month of lambs age (groups C-E, respectively). From the second to fifth month of their age, group B lambs were aspirated four times in monthly intervals. All lambs were weighed at birth, weaning, at second month and monthly thereafter until the eighth month of age. Progesterone priming and ovarian stimulation by serial FSH administrations proceeded each OPU session. To determine onset of puberty blood progesterone concentration was assayed in samples collected initially every week and after the seventh month of age twice weekly. From the seventh month a fertile ram was introduced in each group and oestrous behavior/mating was daily monitored and recorded. Pregnancy diagnosis was carried out by transabdominal ultrasound scanning 55 days after rams' removal. At the fourth and fifth month of age group B lambs were lighter (p<0.05) than controls, but this difference was later equalized. The time of onset of puberty did not differ between groups (p=0.069) and ranged between 224 and 270 days. Some animals (n=15) entered puberty with a full-length luteal phase having progesterone concentration greater than 1ng/ml, while others (n=32) exhibited one or two short luteal phases before luteal length restoration. During the first breeding season 41 animals were fertilized and maintained pregnancy to term, without noticeable differences between groups (p=0.555). During the second breeding season, all ewes were naturally served and lambed at the expected time. It is concluded that OPU in young dairy lambs does not affect the time of onset of puberty, the endocrine profile of the lambs and it does not compromise their future fertility even if it is applied at four successive months.