Search

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.

The present study was designed to determine whether different calcium concentrations in the vitrification solutions could improve the developmental competence of in vitro matured ovine oocytes after cryopreservation. In vitro matured oocytes were vitrified with 16.5% ethylene glycol (EG) + 16.5% dimethylsulfoxide (DMSO) vitrification media. The base media contain different calcium concentrations, so that five experimental groups were obtained: TCM/FCS (TCM 199 + 20% fetal calf serum (FCS), [Ca(2+)] 9.9 mg/dl); PBS/FCS (Dulbecco Phosphate Buffered Saline (PBS) + 20% FCS, [Ca(2+)] 4.4 mg/dl); PBS(CaMg free)/FCS (PBS without Ca(2+) and Mg(2+) + 20% FCS [Ca(2+)] 2.2 mg/dl); PBS/BSA (PBS + 0.4% bovine serum albumin (BSA), [Ca(2+)] 3.2 mg/dl) and PBS(CaMg free)/BSA (PBS without Ca(2+) and Mg(2+) +0.4% BSA, [Ca(2+)] 0.4 mg/dl). After warming, the oocytes from the five experimental groups were assessed for survival, spontaneous parthenogenetic activation and developmental capacity via in vitro fertilization. Oocyte survival after vitrification procedures was better preserved in group PBS(CaMg free)/FCS compared to the others (P < 0.05). In addition, a positive correlation was found between calcium concentration in vitrification solutions and spontaneous parthenogenetic activation (correlation index 0,82; P < 0.001). Development of vitrified oocytes was significantly affected by vitrification media composition (P < 0.01). In particular, oocytes from group PBS(CaMg free)/FCS led to higher cleavage rates and blastocyst rate compared to the others. Our data showed that lowering calcium concentration in the vitrification medium improves the blastocyst rate of vitrified ovine oocytes, probably reducing the effect of EG + DMSO during vitrification. On the contrary, the replacement of FCS with BSA dramatically reduces the developmental potential of these oocytes., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Simple Summary: The placenta serves as the vital interface between maternal and fetal circulation, playing a pivotal role in ensuring the fetus's nutrition and oxygenation. It is, in fact, a highly sophisticated and multifaceted organ, capable of integrating signals from both the mother and the fetus with remarkable efficiency. PAG levels are indicative of the number of fetal cotyledons in the placenta and, consequently, the size of the placenta. This provides insight into placental development and fetal–placental well-being. However, to the best of our knowledge, the effect of maternal undernutrition on PAG secretion has not been extensively investigated in ruminant species. Starting from this premise, this study aimed to determine whether undernutrition might exert an influence on the production of this glycoprotein, with subsequent implications for embryonic and fetal well-being. To the best of our knowledge, no studies in the literature have reported on the relationship between the pregnancy-associated glycoprotein (PAG) concentration and undernutrition during pregnancy in sheep. Therefore, the aim of this study was to investigate the effects of undernutrition on the blood concentration of PAGs in pregnant ewes carrying single and multiple fetuses, undergoing either dietary energy restriction or receiving 100% of their energy requirements during the period of maximal placenta growth. From d 24 to 100 of pregnancy, the ewes were fed ryegrass hay and two different iso-proteic concentrates, fulfilling either 100% of the ewes' energy requirements (control group; n = 30, 14 singleton pregnancies, 16 multiple pregnancies) or only 50% (feed-restricted group; n = 29; 11 singleton pregnancies, 18 multiple pregnancies). Blood samples were collected from all the ewes the day before intravaginal sponge insertion (T0) and, thereafter, every 30 days starting from d 24 of gestation (T1) until d 30 after parturition (T6), to determine the PAG, progesterone, NEFA, total protein, and glucose concentrations. Our results showed that PAG concentrations are not only influenced by the number of fetuses, but also by the ewe's diet. In particular, the PAG marginal means were higher in the feed-restricted group than in the control group (7.8 ± 0.6 and 10.8 ± 0.9 ng/mL for the control and feed-restricted groups, respectively; p = 0.040). This finding confirms that a dietary restriction between d 24 and d 100 of gestation leads to a growth in placental functions as inferred from the increase in the concentration of the PAG levels. Therefore, the measurement of PAG concentrations, in addition to serving as a marker of pregnancy, can also serve as a marker of placental functionality. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The importance of postnatal pituitary activation as regards female reproductive development is not yet understood. By taking advantage of the experimental model developed in a previous study, i.e. ewe lambs expressing markedly different ovarian phenotypes at 50 days of age, we designed this study to determine whether differences found in ovarian status during the early prepubertal period are due to different patterns of postnatal pituitary activation, and to assess whether these differences have long lasting effects on subsequent reproductive performance. Results showed that ewe lambs with high antral follicle count (AFC) at 50 days of age had significantly lower plasma FSH concentrations and higher anti-Mullerian hormone (AMH) concentrations during the first 9 weeks of age compared with low AFC ewe lambs (P!0.0001).With a longitudinal experiment we showed that a high AFC in the early prepubertal period is associated with consistently higher AMH concentrations and numbers of antral follicles up to the postpubertal period, and with higher pregnancy rates in the first breeding season. In addition, the effect of age in decreasing AMH concentrations was more marked in the low AFC group. Results of the present study demonstrate that ewe lambs undergo different patterns of postnatal pituitary activation. A high AFC at 50 days of age indicates an advanced phase of ovarian maturation, which was accompanied by constantly higher AMH concentrations up to the postpubertal period, a greater ovarian response to FSH stimulation and by higher pregnancy rates at first mating, as compared with the low AFC group. [ABSTRACT FROM AUTHOR]

Abstract: After cryopreservation, embryos become sensitive to the oxidative stress, resulting in lipid peroxidation, membrane injury, and structural destruction. The present study aimed to assess the effect of increasing concentration of melatonin during postwarming culture on embryo's ability to restore its functions after cryopreservation. In vitro–produced blastocysts were vitrified, warmed, and cultured in vitro in TCM 199 with 5 different supplementations: control (CTR): 10% fetal calf serum; bovine serum albumin (BSA): 0.04% (wt/vol) BSA; and MEL−3, MEL−6, MEL−9: BSA plus melatonin 10−3, 10−6, and 10−9 M. The medium with the highest melatonin concentration had the highest trolox equivalent antioxidant capacity, whose values were comparable with those determined in plasma sampled from adult ewes (8.7 ± 2.4 mM). The other media had lower trolox equivalent antioxidant capacity values (P < 0.01), below the range of the plasma. At the same time, embryos cultured with the highest melatonin concentration reported a lower in vitro viability, as evaluated by lower re-expansion and hatching rates, and lower total cell number compared with the other groups (P < 0.05). Their metabolic status was also affected, as evidenced by higher oxidative and apoptotic index and lower ATP concentration. The beneficial effects of melatonin on embryo development during postwarming culture were observed only at low concentration (10−9 M). These results suggest that melatonin at high concentration may exert some degree of toxic activity on pre-implantation embryos. Thus, the dose at which the embryos are exposed is pivotal to obtain the desiderate effect. [Copyright &y& Elsevier]

Simple Summary: Glycerol is widely used as a feed supplement in ruminant nutrition. However, its administration at moderate and high doses results in an increase in plasma osmolality and in an alteration of red blood cell (RBC) indices. The present study aimed at further elucidating the effect of glycerol on RBCs' functionality by evaluating the effect of different glycerol concentrations on RBC homeostasis under in vitro conditions. Obtained results showed that glycerol permeates the RBC membrane and leads to hemolysis when glycerol concentration exceeds 200 mg/dL. However, RBCs' antioxidant defenses appear to protect cell membranes without causing an increase of oxidative stress markers. Moreover, no alteration in RBCs' intracellular Ca2+ ion concentrations and metabolic activity were found. In conclusion, glycerol-based nutritional treatment should be designed in sheep to avoid exceeding 200 mg/dL glycerol circulating concentration to prevent RBC osmotic stress. 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. [ABSTRACT FROM AUTHOR]

Simple Summary: Reproductive management of sheep for autumnal lambing often require induction and synchronization of oestrus and ovulation, either for natural mating or artificial insemination, by the use of pharmacological treatments. Such treatments are mostly based on the administration of progesterone followed by a single intramuscular dose of equine chorionic gonadotrophin (eCG) at progesterone withdrawal. However, repeated eCG treatments in consecutive mating seasons can result in the outbreak of resistance with a rise of anti-eCG antibodies. Furthermore, the future use and availability of eCG appears to be strongly challenged by the highly active animal-rights movement because the hormone is obtained from pregnant mares. The present study demonstrated that the administration of glycerol-based formulations to milked ewes is a valid alternative to eCG treatment in reproductive management protocols based on the induction of ovulation with progesterone-releasing devices at the end of anoestrus period. The glucogenic treatment administration to late lactation dairy ewes at the end of the anoestrus period improved their metabolism without harming animal production or animal welfare, thus promoting a sustainable reproductive management of dairy sheep. 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. [ABSTRACT FROM AUTHOR]

The potential of using long in vitro culture (LIVC) of cumulus-oocyte complexes (COCs) from early antral follicles (EAFs) as an assisted reproductive technology in cattle has shown promising results. This study explored the feasibility of applying this technology to sheep as seasonal breeding animals. Ovaries from sheep were collected during both the breeding and non-breeding seasons. COCs were isolated from EAFs (350–450 μm) and cultured in TCM199 medium supplemented with 0.15 μg/mL Zn sulfate, 10−4IU/mL FSH, 10 ng/mL estradiol, 50 ng/mL testosterone, 50 ng/mL progesterone, and 5 μM Cilostamide. After five days of LIVC, the COCs were submitted to an in vitro maturation procedure. The results indicate successful in vitro development of COCs, evidenced by a significant increase in oocyte diameter (p < 0.000) and the preservation of gap junction communication between oocyte and cumulus cells. The gradual uncoupling was accompanied by a progressive chromatin transition from the non-surrounded nucleolus (NSN) to the surrounded nucleolus (SN) (p < 0.000), coupled with a gradual decrease in global transcriptional activity and an increase in oocyte meiotic competence (p < 0.000). Maintenance of oocyte-cumulus investment architecture, viability, and metaphase II capability was significantly higher in COCs collected during the breeding season (p < 0.000), suggesting higher quality than those obtained during the non-breeding season. In conclusion, our study confirms LIVC feasibility in sheep, emphasizing increased effectiveness during the breeding season in isolating higher-quality COCs from EAFs. These findings can influence improving the LIVC system in mammals with seasonal reproduction. • LIVC protocol promotes efficient oocyte growth from sheep EAFs in the breeding season. • LIVC supports chromatin transition, reduces transcriptional activity, and enhances meiotic competence. • Cultured COCs showed higher viability, morphological integrity, and open gap junctions during the breeding season. [ABSTRACT FROM AUTHOR]

Simple Summary: The present study aimed to determine the blood concentrations of L-arginine, asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and L-homoarginine, modulating nitric oxide (NO) synthesis, in single, twin. and triplet pregnancies in ewes undergoing either dietary energy restriction or receiving 100% of their energy requirements. Blood concentrations of L-arginine, of its metabolites. and the ratio between NO synthesis boosters and inhibitors are altered in energy-restricted ewes, these alterations being higher in ewes carrying 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. [ABSTRACT FROM AUTHOR]

The effects of the intra-ruminal dosing of a glucogenic mixture including 70% glycerol, 20% propylene glycol and 10% water was studied on thirty late lactation dairy ewes of Sarda breed. The animals were divided in two homogeneous groups receiving by gavage either 200 mL of water (CTR group; body weight 40.9 ± 1.5 kg) or 200 mL of the above mixture (GLY group; body weight 39.4 ± 1.3 kg) twice daily from d 16 to d 19 of the oestrus cycle, synchronised by “ram effect”. The ewes were then mated and their reproductive responses to the synchronised mating evaluated by scanning on d 50 and at lambing. During the treatment, the ewes were housed in an open hut, machine milked twice daily and fed concentrate and hay to meet their nutrient requirements. During the treatment, concentrate intake was markedly reduced in GLY when compared with CTR (P < 0.001), without any effect on ewe body weight or body condition. The administration of the glucogenic mixture increased plasma osmolarity and blood volume as estimated by serum total protein concentration. Moreover, it increased plasma content of glycerol, glucose (P < 0.001) and insulin (P < 0.01) while decreasing plasma level of NEFA (P < 0.001) and urea (P < 0.05). Milk yield (P < 0.01) and milk lactose content (P < 0.001) were decreased by the glucogenic treatment, whereas milk protein and casein contents were increased (P < 0.001). As for reproductive performance, the glucogenic treatment numerically increased ewe’s conception rate, but the difference was not statistically significant. Prolificacy did not change between groups. In conclusion, the administration of a glucogenic mixture to late lactation dairy ewes caused significant changes both in plasma and in milk composition during the treatment. However, reproductive performances were unaffected by the treatment. [ABSTRACT FROM AUTHOR]