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-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.

Maternal Selenium and Developmental Programming.

Selenium (Se) is an essential trace element of fundamental importance to health due to its antioxidant, anti-inflammatory, and chemopreventive properties, attributed to its presence within at least 25 selenoproteins (Sel). In this review, we describe some of the recent progress, in our understanding, on the impact of maternal Se intake during the periconceptional period on offspring development and health. Maternal nutrition affects the performance and health of the progeny, and both maternal and offspring Se supplementations are essential for the optimal health and antioxidant protection of the offspring. The case of Se in epigenetic programming and early life nutrition is also discussed.

Epigenetic changes of hepatic glucocorticoid receptor in sheep male offspring undernourished in utero.

The aim of this study was to characterise the effects of maternal undernutrition during gestation on hepatic gluconeogenic enzyme gene expression and to determine whether such effects are mediated through epigenetic changes in the glucocorticoid receptor (GR). Pregnant ewes were fed a 50% nutrient-restricted diet from Day 0 to 30 (R1) or from Day 31 to 100 of gestation (R2) or a 100% diet throughout gestation (Control). After parturition lambs were fed to appetite. At 10 months of age offspring were euthanised and livers were removed. Maternal undernutrition did not affect offspring bodyweight at birth or at 10 months of age. However, liver weight of males of the R2 group was lower (P<0.05) in relation to other groups. A significant (P<0.05) hypomethylation of the hepatic GR promoter was revealed in males of the R2 group and a tendency towards the same in the R1 group, along with increased (P<0.001) GR gene expression in both restricted groups. A significant increase (P<0.05) in hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene expression was found in male lambs of both undernourished groups, accompanied by increased (P<0.01) protein levels, while no differences were detected for glucose-6-phosphatase (G6Pase) mRNA abundance and protein levels. In female lambs, no differences between groups were observed for any parameter studied. These data represent potential mechanisms by which insults in early life may lead to persistent physiological changes in the offspring.

Effects of gestational maternal undernutrition on growth, carcass composition and meat quality of rabbit offspring.

An experiment was conducted in order to evaluate the effects of gestational undernutrition of rabbit does on growth, carcass composition and meat quality of the offsprings. Thirty primiparous non lactating rabbit does were artificially inseminated and randomly divided in three treatment groups: Control (C; fed to 100% of maintenance requirements throughout gestation, n = 10), early undernourished (EU; fed to 50% of maintenance requirements during days 7-19 of gestation, n = 10) and late undernourished (LU; fed to 50% of maintenance requirements during days 20-27 of gestation, n = 10). During the 4th week of the gestation period, LU does significantly lost weight compared to C and EU groups (P<0.05). At kindling, C does produced litters with higher proportions of stillborn kits (P<0.05) while the total litter size (alive and stillborn kits) was not different among groups (10.7, 12.8 and 12.7 kits in C, EU and LU groups, respectively). Kit birth weight tended to be lower in the LU group. During fattening, body weight and feed intake were not different among offsprings of the three experimental groups. Moreover, the maternal undernutrition did not have any impact on carcass composition of the offsprings in terms of carcass parts and internal organs weights as well as meat quality of L. lumborum muscle (pH24, colour, water holding capacity and shear values) at slaughter (70 days of age). Therefore, it can be concluded that the gestational undernutrition of the mother does not have detrimental effects on the productive and quality traits of the offsprings.

Severe maternal undernutrition and post-weaning behavior of rabbits.

The objective of the present experiment was to investigate the implications of severe maternal undernutrition on the post-weaning behavior of rabbits. Thirty two does were randomly assigned to four groups: the control group (C) that was fed 100% of the recommended energy maintenance requirements throughout pregnancy and lactation and the U1, U2 and U3 groups that were fed 50% of the recommended energy maintenance requirements between the 6th and the 19th day of pregnancy, between the 20th and the 27th day of pregnancy and between the 3rd and the 10th day of lactation, respectively. At the age of 50 and 65 days, behavior of rabbits in cages was recorded and rabbits were further subjected to an open-field test; a paradigm used as an indicator of fear and emotional distress. Significant differences were found between the U2 and the other experimental groups for the duration of eating and drinking, and duration of locomotory and investigatory behaviors (P<0.05). At the age of 65 days, duration of comfort behaviors was also lower in the U2 compared to the other groups (P<0.05). On the other hand, duration of resting appeared to be the highest in the group of rabbits born from undernourished does between the 20th and the 27th day of pregnancy (P<0.05). Duration of resting was significantly increased, although frequency and duration of eating and drinking were significantly decreased at the age of 65 days (P<0.05). Male rabbits had greater values for locomotory and investigatory behaviors, and duration of eating and drinking compared to the females (P<0.01). A decreased activity of rabbits during the light period was observed (as expected), whereas an increase in duration of locomotory and investigatory behaviors was observed during the first four and the last 4h of the light and dark periods, respectively (P<0.05). Rabbits born from the U1 and U2 group of does displayed increased rates of latency to leave the start position compared to the other groups during the implementation of the open-field test (P<0.05). No other significant differences in the exhibition of the examined behaviors were found between the treatment groups, with the exception of 'moving forelegs' activity that had the lowest value in the U3 group of rabbits. It is concluded that severe maternal undernutrition (50% of the recommended maintenance requirements) especially between the 20th and the 27th day of pregnancy has significant effects on the exhibition of rabbits' post-weaning behavior, since the offspring from the U2 undernourished does were significantly less active compared to the other experimental groups.

Effects of high selenium and fat supplementation on growth performance and thyroid hormones concentration of broilers.

A total of 400, as hatched, broilers were used to investigate the effect of increase of selenium and energy intake on thyroid hormone metabolism, growth and liver fatty acid profile. There were 5 replicates of 4 dietary treatments namely, TA (0.289mg Se per kg diet and adequate energy content), TB (0.583mg Se per kg diet and adequate energy content), TC (0.267mg Se per kg diet and 9% increase of energy content) and TD (0.576mg Se per kg diet and 9% increase of energy content). Diets were isonitrogenous. Zinc L-selenomethionine complex was used to increase Se content and corn oil was used to increase the energy content. The experiment lasted 42 days. Broiler growth performance was not significantly affected by dietary treatments. Liver glutathione peroxidase (GPx) activity increased (P<0.05) in broilers fed high Se and energy diets compared to other ones. Whole blood GPx activity was higher in Se supplemented groups however, it was reduced by age. Thyroid hormone concentrations were unaffected by dietary treatments. A significant increase of linoleic and arachidonic acid concentration (P<0.001) was observed in the liver of broilers fed diets with moderately increased energy content and supplemented with Se compared to those fed diets with moderately increased energy content alone. In conclusion, zinc L-selenomethionine complex and moderate increase of energy content did not affect growth rate or thyroid hormone metabolism but led to increased liver fatty acid content and hepatic GPx activity.

Human ghrelin decreases pituitary response to GnRH in superovulated ewes.

In addition to its metabolic role, ghrelin has been found to suppress luteinizing hormone secretion in many species acting mainly at the hypothalamic level. The objectives of the present study were to test the hypothesis that besides its effects on the hypothalamic level, ghrelin exerts a direct action on the pituitary. Twelve cycling ewes were synchronized, using progestagen intravaginal sponges and superovulated using eCG. At the time of sponge withdrawal, animals were allocated into two groups, ghrelin-treated (Gh) and control. Two days after the sponge removal, GnRH was given to synchronize ovulations. Simultaneously with GnRH treatment, animals of the Gh group received the first of four treatments of acylated human ghrelin at a dose of 6 µg/kg body weight iv; three additional treatments of ghrelin iv were given every 15 minutes thereafter. Control animals received saline iv. Blood samples were collected before challenge (-30 and 0 minutes) and at 30, 60, 75, 90, 105, 120, 135, 150, and 180 minutes after GnRH treatment, and were analyzed for LH, FSH, estradiol, progesterone, insulin, and insulin-like growth factor-I concentrations. Ghrelin treatment attenuated GnRH-induced a preovulatory surge of both gonadotrophins, with the effect being greater for LH. No difference was detected for insulin, estradiol, and progesterone concentrations, and insulin-like growth factor-I levels were increased in the Gh group. Our results imply that in sheep, ghrelin conducts specific regulatory effects on the GnRH/LH axis, and provide for the first time strong evidence that besides its central action, ghrelin might regulate gonadotrophin release acting at the pituitary level.

The effect of long term under- and over-feeding of sheep on milk and plasma fatty acid profiles and on insulin and leptin concentrations.

Since sheep's milk is mainly used for cheese making and milk chemical composition and fatty acids (FA) profile affect cheese yield and quality, the objective of this study was to determine the effects of different feeding levels on milk chemical composition and FA profile, as well as on plasma FA profile, and on insulin and leptin concentrations. Twenty-four sheep were assigned to three homogeneous sub-groups. Throughout the experimental period each group was fed the same diet but in quantities which met 70% (under-feeding), 100% (control) and 130% (over-feeding) of their respective energy and crude protein requirements. The results showed that the underfed sheep had higher milk fat content compared with overfed. In blood plasma the concentrations of C18:0 and C18:1 in the underfed sheep were significantly higher compared with control and overfed sheep. The concentrations of leptin and insulin were significantly higher in overfed compared with underfed sheep. Underfeeding reduced the concentrations of short chain FA (SCFA) and medium chain FA (MCFA) and increased that of C18:0 and mono unsaturated FA (MUFA) in sheep milk fat compared with controls and overfed. The concentrations of C18:0, long chain FA (LCFA) and monounsaturated FA (MUFA) in milk were significantly higher and those of SCFA, MCFA and saturated FA (SFA) significantly lower in the underfed compared with the overfed sheep. In conclusion, long term under- and over-feeding affected the sheep milk chemical composition and FA profile which consequently has an impact on milk products yield (cheese and yogurt) and quality (human health).

Association between physical properties of cervical mucus and ovulation rate in superovulated cows.

Possible associations between certain physical properties of cervical mucus (CM) and ovulation rate were studied in 21 superovulated Holstein cows. In CM samples collected at the beginning of estrus (0 h) and in 4 h intervals for the following 24 h, the pH, the spinnbarkeit (spinability), and the crystallization value were measured. Blood samples, collected at the same time points with CM samples, were assessed for progesterone and estradiol concentrations. At 48 h the number of ovulated follicles was counted by transrectal ultrasonography and the animals were allotted into 2 groups according to the occurrence of at least one (group A, n = 16) or no (group B, n = 5) ovulations. The pH was lower (P < 0.05) at 8 h (7.00 + 0.24) in group A compared with group B (7.55 + 0.12). In group A, spinnbarkeit was significantly lower at 0 h and 20 h, and higher at 8 h and 16 h compared with group B (0 h: 2.50 + 0.82 versus 6.95 + 0.41; 20 h: 3.00 + 1.89 versus 5.38 + 0.94; 8 h: 7.00 + 0.87 versus 2.75 + 0.43; 16 h: 7.00 + 1.41 versus 4.30 + 0.71, for groups A versus B, respectively). Crystallization was significantly lower at 4 h (2.00 + 0.63) and 20 h (1.50 + 0.82) in group A compared with group B (3.13 + 0.32 at 4 h and 3.00 + 0.41 at 20 h). Progesterone at all time points, and estradiol at 16 h, 20 h, and 24 h were lower (P < 0.05) in group A than in group B. The pH, crystallization, estradiol, and progesterone differed (P < 0.05) within one group, while sbk differed within both groups.Our results imply that during the periovulatory period, steadily low progesterone concentrations trigger alterations of certain CM characteristics, while extremely high estradiol concentration could prevent the occurrence of these alterations.

Phosphodiesterase inhibitor 3-isobutyl-methyl-xanthine affects rabbit ovaries and oviduct.

The aim of our study was to examine the influence of 3-isobutyl-1-methyl-xanthine (IBMX), an inhibitor of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) phosphodiesterases on rabbit reproductive system. The ovarian cycle and ovulation of control rabbits were induced by gonadotropin releasing hormone (GnRH) or equine chorionic gonadotropin (eCG) followed by the human chorionic gonadotropin (hCG) administration. Experimental animals received GnRH together with IBMX (at 50 or 500 microg/animal) or eCG and hCG together with IBMX (at 5 or 257 microg/animal). After mating and insemination, blood samples were collected and analyzed for concentrations of progesterone and estradiol by RIA; some animals from the control and IBMX-treated groups were killed. The presence of follicles at different stages of development was evaluated by a light microscopy. Isolated ovarian fragments were cultured for 2 days. The secretion of progesterone and estradiol was assessed by RIA. The expression of markers of proliferation (PCNA and cyclin B1) and apoptosis (bax) in ovarian fragments was evaluated by Western-blotting. Epithelial cells were isolated from oviducts and cultured for 2 days. The expression of markers of proliferation (PCNA, ERK1,2-related MAP kinase) and apoptosis (bax, bcl-2) in the oviductal cells was evaluated by immunocytochemistry. It was observed, that the ovaries of rabbits treated with IBMX contained more secondary follicles, than control rabbits. Administration of IBMX reduced blood level of progesterone, but did not affect blood estradiol. Fragments of ovaries isolated from rabbits treated with IBMX released more estradiol, but not progesterone, than ovarian cells isolated from the control animals. IBMX injections substantially decreased the expression of the upper (23 kD), but not the bottom (24 kD) fraction of bax. IBMX administration did not affect PCNA, but it caused a decrease in the upper fraction (54 kD) and an increase in the bottom fraction (55 kD) of cyclin B1. Oviductal cells isolated from the IBMX-treated animals, contained less marker of apoptosis - bax (but not bcl-2) and proliferation - ERK1,2-related MAP kinase (but not PCNA) than control animals. These observations demonstrate the involvement of cyclic nucleotide-dependent intracellular mechanisms in control of rabbit reproductive functions: ovarian folliculogenesis, apoptosis, proliferation and steroid hormone release, as well as proliferation and apoptosis of the oviductal cells.

Effect of selenium supplementation on thyroid hormone levels and selenoenzyme activities in growing lambs.

The aim of the present study was to evaluate the effects of selenium supplementation on thyroid hormone metabolism and selenoenzyme activities in lambs. Twelve 20-d-old male lambs were assigned to one of two diets: A (0.11 ppm Se) and B (supplemented with 0.2 ppm selenium as sodium selenite). Blood samples were collected weekly for the determination of T3, T4, and selenium levels. The response to thyrotropin-releasing hormone (TRH) challenge was estimated at the 11th and 20th wk. Animals were slaughtered at wk 20 and tissues were collected for enzyme determination. Plasma selenium concentration was significantly higher in supplemented lambs (p<0.001). Plasma T3 and T4 levels remained similar in both groups. Type I deiodinase activity (ID-I) was decreased in the liver (p<0.05) and increased in the pituitary (p<0.01) of supplemented animals. No ID-I activity was detected in the thyroid. Pituitary type II deiodinase activity (ID-II) remained unchanged. The response to TRH challenge did not differ between the two groups for both challenges, but in group B, the second TRH challenge (20th wk) resulted in a significantly higher T3 response compared to the first one (11th wk) (p<0.05). In conclusion, the lack of effects of Se supplementation on thyroid hormone metabolism demonstrates that enzyme activity is homeostatically controlled and selenium is incorporated in that order to ensure the maintenance of thyroid hormone homeostasis.

Selenoenzyme activities in selenium- and iodine-deficient sheep.

This study was conducted to evaluate the effects of single and combined deficiencies of selenium and iodine on selenoenzyme activities in sheep. Twenty-four male lambs were assigned to one of four semisynthetic diets: combined deficient A (Se-I), Se-deficient B (Se-I+), I-deficient C (Se+I-), and basal diet D (Se+I+). Thyroid hormones (T3, T4), thyroid stimulating hormone (TSH), and inorganic iodine (PII) were determined in plasma. Selenium and glutathione peroxidase activity (GSH-Px) were determined in erythrocytes, and tissue samples, including the thyroid, liver, kidney, and brain, were taken for selenoenzyme analysis. Plasma T3, T4, and TSH concentrations were similar in all groups. Type I deiodinase (ID-I) activity in liver and kidney remained unchanged in Se or I deficiency. In contrast, hepatic ID-I activity was increased by 70% in combined Se-I deficiency. Thyroidal cystolic GSHPx (c-GSH-Px) and phospholipid GSH-Px (ph-GSH-Px) activities remained constant in both Se-deficient groups, whereas thyroidal c-GSH-Px activity increased (57%) in I deficiency. Type II deiodinase (ID-II) activity was not detectable in the cerebrum and cerebellum, whereas cerebellum Type III deiodinase (ID-III) activity was decreased in I deficiency and combined Se-I deficiencies. The results of the present study support a sensitive interaction between Se and I deficiencies in sheep thyroid and brain. Furthermore, the lack of thyroidal ID-I activity, the preservation of the thyroidal antioxidant enzymes, and the increases in hepatic ID-I indicate that a compensatory mechanism(s) works toward retaining plasma T3 levels, mostly by de novo synthesis of T3 and peripheral deiodination of T4 in Se- and I-deficient sheep.