The Effect of By-Pass Linseed Oil Supplementation on the Maternal Antioxidant System during the Embryo-Maternal Recognition Period in Ewes.

This study analyzed the effects of dietary supplementation with by-pass linseed oil (LO; rich in α-linolenic acid) on maternal antioxidant systems at Days 14 and 16 of pregnancy in Sarda ewes. This trial used sixteen dry ewes. Eight ewes (CT group) were fed with a control diet without LO, and eight ewes (LO group) were fed with a diet supplemented with LO (10.8 g of α-linolenic acid/ewe/day). Both diets had similar crude protein and energy levels. The experiment included 10 days of an adaptation period and 31 days of a supplementation period. This supplementation period was divided into Period -2 (from Day -15 to -8), Period -1 (from Day -7 to -1; before synchronized mating period/Day 0), Period +1 (from Day +1 to + 7 after mating), and Period +2 (from Day +8 to +15 after mating). Estrous synchronization was induced in all the ewes using an intravaginal sponge (45 mg fluorgestone acetate) for 14 days and equine chorionic gonadotropin (350 UI/ewe) at the end of the treatment. On Days 14 (CT, N = 4; LO, N = 4) and 16 (CT, N = 4; LO, N = 4) after mating, the ewes were slaughtered. Samples of plasma, uterine, and luteal tissues were collected. Thiols, total antioxidant activity (TEAC), superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content were measured. On Day 16, thiol and TEAC in luteal tissues were higher in the LO group when compared with the control one (p < 0.05). Moreover, TEAC was higher for the LO group in uterine tissues on Days 14 and 16 (p < 0.05). SOD activity was higher in the LO group in luteal and uterine tissues on Day 14 and Day 16, respectively (p < 0.001). On Day 16, uterine MDA content was lower for the LO group (p < 0.001). No differences were found between groups at the plasmatic level. However, the by-pass LO supplementation enhanced the analyzed antioxidant parameters in luteal and uterine tissues. In conclusion, these results demonstrate that by-pass LO supplementation exerted a positive effect on antioxidative defenses on maternal structures during the embryo-maternal recognition period in ewes. Thus, this could contribute to improving the maternal environment during the embryo-maternal recognition period in mammals.

Application of the "echo-synch protocol" to advance pregnancy onset in ewe lambs at the first reproductive season.

Introduction: This study assessed the efficacy and economic impact of a reproductive protocol based on repeated ultrasound scanning (US) associated with the use of GnRH to advance pregnancy onset in ewe lambs. Methods: Prepubertal ewe lambs (n = 133) were divided into three weight groups (High: HW n = 35; Medium: MW n = 65; Low: LW n = 33). Thereafter, animals were randomly allocated into two subgroups: GnRH, ewe lambs treated with GnRH analog and then exposed to rams; CTR, ewe lambs exposed to rams only. CTR groups were joined with rams as a single flock. GnRH groups were kept separate from rams receiving a single dose of gonadorelin (40 µg/head) and then were evaluated after a week of US. Animals showing corpora lutea received an injection of PGF2α analog (100 µg/head) and then were joined with rams. The remaining ewe lambs received a second dose of gonadorelin and were kept separate from the rams. After another week, animals were checked again and the ones showing corpora lutea were injected with the PGF2α analog, while the others received a third injection of gonadorelin. On the same day, all the animals were joined with rams. Pregnancies were confirmed within 30 days by US. The efficacy of the protocol was determined by assessing differences in the number of days required to achieve pregnancy rates of 25, 50, and 75% and in the total costs and incomes from birth to the end of first lactation within the groups. Results: The GnRH-MW group showed the best performances in reaching the threshold pregnancy rates of 25, 50, and 75%, but the effect of treatment was significant only at the 25% threshold (p < 0.01). Both low groups displayed an overall poorer performance at 50 and 75% thresholds than medium and high-weight groups (p = 0.01 and p < 0.01, respectively). The GnRH administration did not advance pregnancy onset in GnRH-HW compared with CTR-HW. In the balance between costs and income, the HW-CTR and MW-GnRH groups showed higher gross margins than the other groups. Conclusion: Using the US/GnRH protocol in ewe lambs appears technically and economically effective in animals that have not reached the optimal weight at the first breeding season, advancing ewe lambs' pregnncies and increasing farm profitability.

Effect of Strategic Supplementation of Dietary By-Pass Linseed Oil on Fertility and Milk Quality in Sarda Ewes.

The aim of the present study was to assess whether the strategic supplementation of bypass LO can enhance reproductive indexes­fertility, lambing rate, and prolificacy­in dairy Sarda ewes at the end of lactation. To assess whether LO supplementation leads to the adsorptions of PUFAs and their subsequent utilization by the body tissues, milk composition and fatty acid content were analyzed. Forty-eight ewes were assigned to the following groups: the control group (CT; N = 24), fed with a control diet without LO; and the treatment group (LO; N = 24), fed with a diet supplemented with LO (10.8 g/ewe/day). Both diets had similar crude protein and energy levels and were offered for 38 days (−21 to +17 days after artificial insemination). The trial included an adaptation period (7 days) followed by a regular supplementation (31 days) period. Estrus synchronization was induced in all the ewes using an intravaginal sponge and equine chorionic gonadotropin. Fifty-five hours after pessaries withdrawal, all ewes were inseminated using the cervical route and fresh semen. Cholesterol (p < 0.01), high-density lipoprotein (p < 0.001), and triglyceride (p < 0.05) levels in plasma were higher in the LO group. Plasmatic levels of non-esterified fatty acids were lower in the LO group after the end of the supplementation period (p < 0.05). Milk unsaturated fatty acids (UFAs), monounsaturated fatty acids (MUFAs), total polyunsaturated fatty acids (PUFAs), PUFAs omega 3 (PUFAs-ω3) and 6 (PUFAs-ω6), and trans fatty acids were higher in the LO group (p < 0.001), while saturated fatty acids (SFAs) were higher in the CT group during the supplementation period (p < 0.001). Three days after the end of the supplementation period, the content of milk UFAs (p < 0.05), PUFAs (p < 0.001), MUFAs, and PUFAs-ω6 (p < 0.01) were still higher in the LO group. whereas SFA was higher in the CT group (p < 0.01). There was no difference between groups in terms of ovulation rate, progesterone levels in plasma, fertility rate, prolificacy, and total reproductive wastage. However, the total area of luteal tissue was higher in the LO group (p < 0.01). Results obtained demonstrated that LO supplementation exerts a positive role in corpus luteum size at the onset of the peri-implantation period in Sarda dairy ewes. Additionally, the results obtained in the present study showed that the use of dietary bypass LO affects lipid metabolites in plasma and milk fatty acid profiles, demonstrating the ALA uptake by body tissues.

Measurement of fecal T3 metabolite levels in sheep: Analytical and biological validation of the method.

Introduction: Biological sample collection from wild and farms animals is often associated with difficulties related to the handling and restraint procedures, and most of the time it could induce stress, altering the welfare and physiological homeostasis. The analysis of fecal T3 metabolites (FTMs) allows to test samples collected in a non-invasive manner, providing several information about the animal's physiological conditions and the effects related to environmental and nutritional variations. This procedure has found wide application in wild species, but less in domestic ones. Methods: The aim of this work was to validate the use of an immuno-enzymatic competitive ELISA kit, designed for T3 quantification in human blood serum samples, for the assessment of FTMs in the sheep. For the analytical validation, precision, recovery and parallelism were evaluated; for biological validation the variations of FTMs in relation to age, sex and the physiological status of the animal were determined. Results: After a verification of the precision (RSD % < 15%), mean recovery (75%) and parallelism (CV% < 10%), the kit was used to measure FTMs in cyclic, pregnant, and early lactating ewes as well as in rams and ewe lambs. The results showed that FTMs concentrations in pregnant ewes were significantly lower (p < 0.05) than in cyclic and early lactation ones. Furthermore, there were no significant differences in FTMs levels between ewes and rams, while in lambs FTMs levels were higher than in adults (p < 0.001). Conclusion: In conclusion the present study demonstrates that FTMs can be reliably and accurately determined in sheep feces, using an ELISA kit formulated for human serum T3 assay. The application of this method in the livestock sector could allow to improve our knowledge about the response of animals to different physiological and environmental conditions, and thus assess their welfare.

Does the timing of pasture allocation affect rumen and plasma metabolites and ghrelin, insulin and cortisol profile in dairy ewes?

A study was undertaken to assess the impact of the timing of grazing on rumen and plasma metabolites and some metabolic hormones in lactating dairy sheep allocated to an Italian ryegrass (Lolium multiflorum Lam) pasture in spring for 4 h/d. Twenty-four mid lactation Sarda ewes stratified for milk yield, body weight, and body condition score, were divided into four homogeneous groups randomly allocated to the treatments (2 replicate groups per treatment). Treatments were morning (AM, from 08:00 to 12:00) and afternoon pasture allocation (PM, from 15:30 to 19:30). Samples of rumen liquor (day 39) and blood plasma (days 17 and 34 of the experimental period) were collected before and after the grazing sessions. Moreover, on days 11 and 35, grazing time was assessed by direct observation and herbage intake measured by the double weighing procedure. Grazing time was longer in PM than AM ewes (P < 0.001) but herbage intake was undifferentiated between groups. The intake of water-soluble carbohydrates at pasture was higher in PM than AM ewes (P < 0.05). The post-grazing propionic and butyric acid concentration, as measured on day 39, were higher in PM than AM ewes (P < 0.05). The basal level of glucose on day 34 and insulin (on both sampling days) were higher in PM than AM (P < 0.05). The opposite trend was detected for non-esterified fatty acids (P < 0.05, day 34) and urea (both days). Pasture allocation in the afternoon rather than in the morning decreased plasma concentration of ghrelin (P < 0.001) and cortisol (P < 0.001), with a smoothed trend on day 34 in the latter variable. To conclude, postponing the pasture allocation to afternoon increased the intake of WSC, favoring a glucogenic pattern of rumen fermentation and a rise of glucose and insulin levels in blood, although these effects were not consistent across the whole experimental period. Moreover, the afternoon grazing decreased the level of cortisol and ghrelin, suggesting a higher satiation-relaxing effect.

Non-invasive Assay for Measurement of Fecal Triiodothyronine (T3) Metabolite Levels in European Mouflon (Ovis aries musimon).

Thyroid hormones (THs) are important indicators of metabolism and animal health. Traditionally, they have been determined from blood or urine samples. However, as their collection may be stressful and requires ethical approval, alternative non-invasive matrices are preferred when dealing with wild animals. Triiodothyronine (T3) is the active form of THs in blood and the major metabolite excreted in feces. This creates the ideal conditions for its assay in fecal samples. Fecal sampling eliminates the stress of the animals and the need to physically capture them. However, in wild species it is rare to find species-specific kits for the hormone assay. So, the objective of this work was to validate a method for the quantification of T3 metabolite (FTM) levels in feces of European mouflon by using an economic and easily available ELISA kit designed to quantify T3 in human plasma. Analytical and biological validations were performed in feces collected from 10 mouflons (5 ewes and 5 rams). An efficient liquid-extraction method was optimized. Precision, dilution linearity, parallelism, recovery and stability of T3 in fecal samples were calculated. Obtained data were considered acceptable according to international guidelines. The reliability of the results was verified comparing human plasma and mouflon fecal samples fortified with the same T3 standard solutions. The biological validation showed higher FTM levels in March compared to June, and no differences between mouflon ewes and rams. The validation of the present method provides a non-invasive and affordable tool for the quantification of FTM in European mouflon.

Measurement of progesterone in sheep using a commercial ELISA kit for human plasma.

Determination of serum or plasma progesterone (P4) concentrations is important to recognize pregnant and non-pregnant ewes, and also to predict the number of carried lambs. The 2 most common methodologies for the detection of plasma P4 are radioimmunoassay (RIA) and enzyme immunoassay (EIA). RIA is very expensive, and not all laboratories are equipped to perform this test; EIA is commercially available for human use, but only a few companies produce species-specific kits, which are expensive. We verified for ovine plasma a less expensive and easily available ELISA kit (DiaMetra) designed to quantify P4 in humans. Pools of ovine and human plasma were used to compare repeatability, accuracy, sensitivity, and stability of P4 measured by the DiaMetra kit. Repeatability data were within 15%, and accuracy values were ~90% for both plasma matrices. Stability data showed a loss of <20% for freeze-thaw and <30% for 30-d storage. All parameters were acceptable under international guidelines for method validation. The human ELISA kit was used successfully to quantify plasma P4 in 26 ewes during pregnancy until delivery. P4 concentrations were also correlated with the number of carried lambs.

Effect of Media with Different Glycerol Concentrations on Sheep Red Blood Cells' Viability In Vitro.

The use of high doses of glycerol as a livestock feed supplement is followed by a rapid increase in plasma concentrations and consequently in plasma osmolality. Moreover, glycerol is a highly diffusible molecule that can readily permeate the red blood cell (RBC) membrane following a concentration gradient. A rise in glycerol plasma concentrations can thus alter RBC homeostasis. The present study aimed at investigating both glycerol osmotic effects on sheep RBCs and their oxidative response under in vitro conditions. Sheep blood samples were suspended in media supplemented with increasing glycerol concentrations (0, 25, 50, 100, 150, 200, 250, 300, 350, 400 mg/dL), which reflected those found in vivo in previous studies, and incubated at 37 °C for 4h. Thereafter, osmolality and hemolysis were determined in spent media, while cell extracts were used to assay intracellular concentration of glycerol, ATP, Ca2+ ions, oxidative stress markers and reactive oxygen species (ROS).The study confirmed that glycerol intracellular concentrations are directly related with its concentration in the incubation media, as well as hemolysis (p < 0.001) which increased significantly at glycerol concentrations higher form 200 mg/dL. ROS intracellular level increased at all glycerol concentration tested (p < 0.01) and total thiols decreased at the highest concentrations. However, RBCs proved to be able to cope by activating their antioxidant defense system. Superoxide dismutase activity indeed increased at the highest glycerol concentrations (p < 0.001), while total antioxidant capacity and malonyldialdehyde, a typical product of lipid peroxidation by ROS, did not show significant changes. Moreover, no alterations in intracellular Ca2+ ions and ATP concentrations were found. In conclusion, glycerol-induced hemolysis can be related to the induced osmotic stress. In sheep, nutritional treatments should be designed to avoid reaching glycerol circulating concentrations higher than 200 mg/dL.

Towards a Sustainable Reproduction Management of Dairy Sheep: Glycerol-Based Formulations as Alternative to eCG in Milked Ewes Mated at the End of Anoestrus Period.

This study investigated whether the administration of equine chorionic gonadotrophin (eCG) in a protocol to induce and synchronize ovulations before mating could be replaced by the administration of glycerol-based formulations in milked ewes at the end of their seasonal anoestrus. Forty-eight late-lactation dairy ewes of the Sarda breed were synchronized using sponges impregnated with progestogen and then joined with fertile rams (day (D) 0, ram introduction). From D-4 to D-1, the ewes received by gavage either 100 mL of a glucogenic mixture (70% glycerol, 20% propylene glycol and 10% water; GLU group; n = 24) or 100 mL of water (GON group; n = 24) twice daily. Moreover, on the day of sponge withdrawal (D-1), GON ewes received 200 IU of eCG. There were no differences in reproductive performances between groups. GLU ewes showed higher glycemia (p < 0.001), insulinemia (p < 0.05), plasma glycerol (p < 0.001), triglycerides (p < 0.001) and lower cholesterol (p < 0.001), non-esterified fatty acids (NEFA; p < 0.05) and urea (p < 0.001). Plasma osmolality was higher in GLU but only 4 h after dosing (p < 0.001). Milk yield and milk composition were not affected by the treatments with exception of milk glycerol (p < 0.001) and milk urea (p < 0.001), which were higher and lower in GLU than GON ewes, respectively. In conclusion, the administration of the glucogenic mixture to late lactation dairy ewes at the end of anoestrus period resulted in reproductive responses as good as the ones obtained by the eCG treatment, suggesting that the objective of a sustainable reproductive management of dairy sheep can be successfully pursued.

Confocal investigation on colocalization between tubulin posttranslational modifications and associated proteins in rat C6 glioma cells.

Glioblastoma multiforme is the most lethal brain tumor. In the study of mechanisms underlying its development attention has been paid to the microtubular network of its cells, mainly on ßIII tubulin, considered as a marker of malignancy. In the present work, we chose to investigate the tubulin code in glioblastoma cells, analyzing the degree of interaction between tubulin post-translational modifications and different proteins associated with them. The pattern of diverse associated proteins such as EB-1, CLIP-170 and kinesin-1 and their degree of co-distribution with the most abundant post-translational tubulin modifications (tyrosination, acetylation and polyglutamylation) were evaluated. Through immunofluorescence we have shown that EB-1, CLIP-170 and kinesin-1 were well detectable in glioblastoma cells. The double fluorescence and colocalization index between the post-translational modifications of tubulin and associated proteins showed that tyrosinated α-tubulin has significantly high affinity with EB-1, CLIP-170 and kinesin-1, while for acetylated and polyglutamylated tubulin, the degree of interaction with the three associated proteins evaluated was less apparent. Data presented in this paper underline the importance of a thorough analysis of the microtubular mechanics in glioblastoma cells. This may suggest new experimental therapeutic approaches able to act more selectively on the microtubular network of cells in this type of cancer.

Administration of glycerol-based formulations in sheep results in similar ovulation rate to eCG but red blood cell indices may be affected.

BACKGROUND: The objective of this study was to investigate the metabolic and osmotic effects of different doses of glycerol or a glycerol - propylene glycol mixture in Sarda sheep with the aim to identify those able to beneficially modify ewe's metabolic status without harmful changes in red blood cell (RBC) indices. Thereafter, the selected doses were tested for their effects on ewe's ovarian activity during an induced follicular phase and compared to the effects of a hormonal treatment with equine chorionic gonadotrophin (eCG). RESULTS: Glycerol was administered alone (G groups: 90% glycerol and 10% water; % v/v) or in combination with propylene glycol (M groups: 70% glycerol, 20% propylene glycol, 10% water; % v/v). Treatments were formulated to provide 100, 75, 50 and 25% of the amount of energy supplied in previous experiments. Obtained results showed that the formulations G75 and M75 (22.5 and 18.2% on DM basis, respectively) induce metabolic changes comparable to those induced by M100. The latter dose has been already evaluated for its effects on sheep metabolism and reproductive performance. However, with these high doses, plasma osmolality increased significantly, and RBC indices showed significant alterations. The low dose groups (G25 and M25, 8.6 and 6.9% on DM basis, respectively) did not show any alterations in plasma osmolality and RBC indices, but the metabolic milieu differed markedly from that of M100. Between the medium dose groups, M50 (12.9% on DM basis) showed a more comparable milieu to M100 than G50 (15.9% on DM basis) and no RBC alterations. Therefore, M75, G75 and M50 doses were tested for their effect on ovarian functions and proved to be equally effective as eCG. CONCLUSION: The results of the present study evidenced an alteration of RBC indices, and possibly of their functions, as a side effect of glycerol administration at high doses in the diet of ewes. Therefore, protocols foreseeing the administration of glycerol should be tested for their effects on RBC indices and functions. In general terms, the medium dose of the glucogenic mixture (12.9% of dietary DM on offer) should be preferred.

Increased number of large non-atretic follicles and co-dominance effects account for high litter sizes in Bonga sheep.

To understand the ovarian basis for prolificacy of Bonga sheep, a total of 31 ewes were selected based on litter size (LS) records and divided into two groups: High Prolificacy (HP) (n = 20) with LS ≥ 2 and Low Prolificacy (LP) (n = 11) with LS = 1. At a synchronized estrus, follicular dynamics were determined using transrectal ultrasonography. Plasma estradiol concentrations were also monitored. In total 27 ewes were observed in estrus being 9/11 LP (82%) and 18/20 HP (90%). On the day of estrus (day 0), the mean number of large follicles was higher (p < .05) in HP (1.78 ± 0.19) than in LP (1.0 ± 0.28) ewes. Prior to estrus, more (p < .05) medium follicles were visible for HP compared to LP ewes. Plasma estradiol concentrations were higher in HP compared to LP ewes (18.91 ± 0.41 vs. 14.51 ± 0.65 pg/ml; p < .05) and similarly was ovulation number (2.3 ± 0.15 vs. 1.28 ± 0. 14; p < .05). Higher ovulation rates and litter size in Bonga sheep are evidenced by the previous presence of more large follicles and the existence of co-dominance effects as most likely medium follicles are selected to ovulate.

Circulating Concentrations of Key Regulators of Nitric Oxide Production in Undernourished Sheep Carrying Single and Multiple Fetuses.

The aim of this study was to investigate the blood concentrations of L-arginine, asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and L-homoarginine, which are regulators of nitric oxide (NO) synthesis, in single, twin, and triplet pregnancies in ewes undergoing either a dietary energy restriction or receiving 100% of their energy requirements. From day 24 to 100 of pregnancy, the ewes were fed ryegrass hay and two different iso-proteic concentrates fulfilling either 100% of ewes' energy requirements (control group; n = 30, 14 singleton pregnancies, 12 twin pregnancies, and 4 triplet pregnancies) or only 45% (feed-restricted group; n = 29; 11 singleton pregnancies, 15 twin pregnancies, and 3 triplet pregnancies). Blood samples were collected monthly to measure, by capillary electrophoresis, the circulating concentrations of arginine, ADMA, homoarginine, SDMA, and of other amino acids not involved in NO synthesis to rule out possible direct effects of diet restriction on their concentrations. No differences between groups were observed in the circulating concentrations of most of the amino acids investigated. L-homoarginine increased markedly in both groups during pregnancy (p < 0.001). SDMA (p < 0.01), L-arginine, and ADMA concentrations were higher in feed-restricted ewes than in controls. The L-arginine/ADMA ratio, an indicator of NO production by NOS, decreased towards term without differences between groups. The ADMA/SDMA ratio, an index of the ADMA degrading enzyme activity, was higher in controls than in feed-restricted ewes (p < 0.001). Obtained results show that circulating concentrations of L-arginine, of its metabolites, and the ratio between NO synthesis boosters and inhibitors are altered in energy-restricted ewes, and that these alterations are more marked in ewes carrying multiple fetuses.

Commercial human kits' applicability for the determination of biochemical parameters in sheep plasma.

Currently, several commercially available biochemical kits are validated for their use in human but not in animals. The purpose of this work is to demonstrate the applicability of human kits for alanine-aminotransferase, aspartato-aminotransferase, albumin, total protein, total cholesterol, and triglycerides in ovine plasma. Assays were validated according to international guidelines and stability was explored. Accuracy values were between 67 and 100%, and intra and interday precisions (%RSD) were <15% for all studied parameters. These results confirm the suitability of the studied human kits for their use in ovine plasma and they were used in plasma collected from pregnant ewes.

A recovery time after warming restores mitochondrial function and improves developmental competence of vitrified ovine oocytes.

The aim of the present study was to assess the ability of vitrified/warmed oocyte to recover from vitrification-induced damages after warming. In vitro matured, vitrified/warmed ovine oocytes were assessed for developmental competence, mitochondrial activity and distribution, ATP, ROS and catalase levels during 6 h of in vitro culture using fresh oocytes as control. ATP content in vitrified oocytes was lower than control during 4 h of post warming culture (p < .01). Vitrified oocytes were able to fill this gap only after 6 h of post-warming incubation. Moreover, mitochondrial activity was significantly lower (p < 0.01) in vitrified oocytes compared to controls, and this difference was maintained up to 2 h of incubation. Then the activity increased and at 4 h it was higher compared to controls (p < 0.01). These oocytes showed an increasing rate of clustered distribution of mitochondria which was lower than controls during the first 4 h of post warming culture (p < 0.01). ROS level was significantly higher at 0 h in vitrified compared to control oocytes and this difference was maintained also at 2 h and 6 h of incubation (p < 0.01). Catalase level was higher in vitrified oocytes than controls (p < 0.01) during the entire culture period. Cleavage and blastocyst rates were lower in vitrified oocytes compared to control ones during the two first time point of incubation period (p < .01), indeed they increased significantly from 0 to 4 h of incubation post warming (p < 0.01). The study demonstrated that vitrified/warmed oocytes need an extra time to restore damage due to cryopreservation procedures and to increase their developmental potential. Thus, time of damage recovery after vitrification could be used to standardize the vitrification protocols and to improve the developmental competence of vitrified/warmed oocytes.