The Oxidative state of LDL is the major determinant of anti/prooxidant effect of coffee on Cu²⁺ catalysed peroxidation

Antioxidants exert contrasting effect on low density lipoprotein (LDL) oxidation catalysed by metals, acting as pro-oxidants under select in vitro conditions. Through our study on the effect of coffee on LDL oxidation, we identified the parameters governing this phenomenon, contributing to the comprehension of its mechanism and discovering significant implications for correct alimentary recommendations. By measuring conjugated diene formation, we have analysed the quantitative and qualitative effects exerted by an extract of roasted coffee on LDL oxidation triggered by copper sulphate. When the relative effects of different coffee concentrations were plotted against the lag time (LT) of control LDL (C-LDL), the apparently random experimental data arranged in sensible patterns: by increasing the LT the antioxidant activity of coffee decreased progressively to become prooxidant. The critical LT, at which coffee switches from antioxidant to prooxidant, increased by increasing coffee concentration. Also the contrasting results obtained following a delayed addition of coffee to the assay, arranged in a simple pattern when referred to the LT of C-LDL: the prooxidant effect decreased to become antioxidant as the LT of C-LDL increased. The dependence of coffee effect on the LT of C-LDL was influenced by LDL but not by metal catalyst concentration. These novel findings point to the oxidative state of LDL as a major parameter controlling the anti/prooxidant effect of coffee and suggest the LT of C-LDL as a potent analytical tool to express experimental data when studying the action exerted by a compound on LDL oxidation

The Oxidative State of LDL is the Major Determinant of Anti/Prooxidant Effect of Coffee on Cu2+ Catalysed Peroxidation

Antioxidants exert contrasting effect on low density lipoprotein (LDL) oxidation catalysed by metals, acting as pro-oxidants under select in vitro conditions. Through our study on the effect of coffee on LDL oxidation, we identified the parameters governing this phenomenon, contributing to the comprehension of its mechanism and discovering significant implications for correct alimentary recommendations. By measuring conjugated diene formation, we have analysed the quantitative and qualitative effects exerted by an extract of roasted coffee on LDL oxidation triggered by copper sulphate. When the relative effects of different coffee concentrations were plotted against the lag time (LT) of control LDL (C-LDL), the apparently random experimental data arranged in sensible patterns: by increasing the LT the antioxidant activity of coffee decreased progressively to become prooxidant. The critical LT, at which coffee switches from antioxidant to prooxidant, increased by increasing coffee concentration. Also the contrasting results obtained following a delayed addition of coffee to the assay, arranged in a simple pattern when referred to the LT of C-LDL: the prooxidant effect decreased to become antioxidant as the LT of C-LDL increased. The dependence of coffee effect on the LT of C-LDL was influenced by LDL but not by metal catalyst concentration. These novel findings point to the oxidative state of LDL as a major parameter controlling the anti/prooxidant effect of coffee and suggest the LT of C-LDL as a potent analytical tool to express experimental data when studying the action exerted by a compound on LDL oxidation

Differences in the kinetic of the first meiotic division and in active mitochondrial distribution between prepubertal and adult oocytes mirror differences in their developmental competence in a sheep model

Our aim is to verify if oocyte developmental potential is related to the timing of meiotic progression and to mitochondrial distribution and activity using prepubertal and adult oocytes as models of low and high developmental capacity respectively. Prepubertal and adult oocytes were incorporated in an in vitro maturation system to determine meiotic and developmental competence and to assess at different time points kinetic of meiotic maturation, 2D protein electrophoresis patterns, ATP content and mitochondria distribution. Maturation and fertilization rates did not differ between prepubertal and adult oocytes (95.1% vs 96.7% and 66.73% vs 70.62% respectively for prepubertal and adult oocytes). Compared to adults, prepubertal oocytes showed higher parthenogenesis (17.38% vs 2.08% respectively in prepubertals and adults; P<0.01) and polispermy (14.30% vs 2.21% respectively in prepubertals and adults; P<0.01), lower cleavage rates (60.00% vs 67.08% respectively in prepubertals and adults; P<0.05) and blastocyst output (11.94% vs 34.% respectively in prepubertals and adults; P<0.01). Prepubertal oocytes reached MI stage 1 hr later than adults and this delay grows as the first meiotic division proceeds. Simultaneously, the protein pattern was altered since in prepubertal oocytes it fluctuates, dropping and rising to levels similar to adults only at 24 hrs. In prepubertal oocytes ATP rise is delayed and did not reach levels comparable to adult ones. CLSM observations revealed that at MII, in the majority of prepubertal oocytes, the active mitochondria are homogenously distributed, while in adults they are aggregated in big clusters. Our work demonstrates that mitochondria and their functional aggregation during maturation play an active role to provide energy in terms of ATP. The oocyte ATP content determines the timing of the meiotic cycle and the acquisition of developmental competence. Taken together our data suggest that oocytes with low developmental competence have a slowed down energetic metabolism which delays later development

Ejaculate collection efficiency and post-thaw semen quality in wild-caught Griffon vultures from the Sardinian population

This study aimed to test the feasibility of a programme of semen collection and cryopreservation in Griffon vultures. Four wild-caught individuals kept in captivity because of unrecoverable traumas were used. Semen collection attempts were made twice a week during three consecutive reproductive seasons (December – March) using the abdominal massage method. Ejaculation was successfully induced between late January and late February. Semen collection efficiency was rather low (27.9%) and it did not vary among individuals (p > 0.05). No differences were found in ejaculate volumes (12.5 +/- 9.1 μl), spermatozoa concentration (28.4 +/- 30.9 million cells/ml) and viability (61.3 +/- 13.9%) among the 4 vultures. ATP values differed among the four vultures (p < 0.001); B showed higher nucleotide concentration than both C and D, while it did not differ form A, whose values were higher compared with D. After freezing and thawing, semen in vitro viability, DNA integrity and ATP intracellular concentration were determined. Spermatozoa viability after thawing did not differ among the four individuals (52.6 +/- 5.8 in A, 53.4 +/- 4.6 in B, 50.4 +/- 3.2 in C, 42.5 +/- 2.7 in D), but it decreased significantly compared to fresh semen (p < 0.05). During 4 hrs in vitro culture, spermatozoa collected from B maintained over time a higher viability in vitro when compared to A, C and D. As evaluated by the comet assay method, DNA fragmentation after freezing and thawing did not differ in the 4 vultures. ATP concentration in frozen/thawed semen was significantly lower than in fresh semen (p < 0.0001). This study indicates that semen cryopreservation can be considered as a useful tool in the conservation of Griffon vulture genetic resources, but further studies are needed to optimize this technique

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 &lt; 0.05). Moreover, TEAC was higher for the LO group in uterine tissues on Days 14 and 16 (p &lt; 0.05). SOD activity was higher in the LO group in luteal and uterine tissues on Day 14 and Day 16, respectively (p &lt; 0.001). On Day 16, uterine MDA content was lower for the LO group (p &lt; 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

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

15 Pág.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.The experimental work was supported by funds from Regione Autonoma della Sardegna—Progetti ricerca fondamentale o di base—L.R. 7/2007—annualità 2013 (CRP 78167).Peer reviewe

Semen molecular and cellular features: these parameters can reliably predict subsequent ART outcome in a goat model

Currently, the assessment of sperm function in a raw or processed semen sample is not able to reliably predict sperm ability to withstand freezing and thawing procedures and in vivo fertility and/or assisted reproductive biotechnologies (ART) outcome. The aim of the present study was to investigate which parameters among a battery of analyses could predict subsequent spermatozoa in vitro fertilization ability and hence blastocyst output in a goat model. Ejaculates were obtained by artificial vagina from 3 adult goats (Capra hircus) aged 2 years (A, B and C). In order to assess the predictive value of viability, computer assisted sperm analyzer (CASA) motility parameters and ATP intracellular concentration before and after thawing and of DNA integrity after thawing on subsequent embryo output after an in vitro fertility test, a logistic regression analysis was used. Individual differences in semen parameters were evident for semen viability after thawing and DNA integrity. Results of IVF test showed that spermatozoa collected from A and B lead to higher cleavage rates (0 < 0.01) and blastocysts output (p < 0.05) compared with C. Logistic regression analysis model explained a deviance of 72% (p < 0.0001), directly related with the mean percentage of rapid spermatozoa in fresh semen (p < 0.01), semen viability after thawing (p < 0.01), and with two of the three comet parameters considered, i.e tail DNA percentage and comet length (p < 0.0001). DNA integrity alone had a high predictive value on IVF outcome with frozen/thawed semen (deviance explained: 57%). The model proposed here represents one of the many possible ways to explain differences found in embryo output following IVF with different semen donors and may represent a useful tool to select the most suitable donors for semen cryopreservation

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 &lt; 0.001) which increased significantly at glycerol concentrations higher form 200 mg/dL. ROS intracellular level increased at all glycerol concentration tested (p &lt; 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 &lt; 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

Effet des jours longs et de la mélatonine sur la réponse à l’effet mâle chez la chèvre Sarde = Effect of long days and melatonin on the response to male effect in Sarda goats

La chèvre Sarde montre une activité sexuelle saisonnière : les chaleurs apparaissent en été-automne et se terminent en hiver (Casu et al., 1981). La nécessité d’avoir les mises bas en automne oblige l’éleveur à avancer l’activité sexuelle des chèvres à juin. C’est pourquoi il est nécessaire induire des ovulations synchrones en dehors de la saison sexuelle. Normalement, ce but est atteint grâce à un traitement hormonal qui implique l’emploi d’acètate de fluorogestone (FGA), cloprostenol et gonadotrophine sérique de jument gravide (PMSG). Toutefois, ce traitement entraîne la production d’anticorps anti PMSG, avec une réduction progressive de son efficacité, et la présence de résidus hormonaux dans le lait, au-dessus des limites autorisées. Puisque la saison sexuelle est réglée par la photopériode et l’introduction des boucs dans un troupeau de chèvres en anoestrus induit l’activité ovulatoire, la manipulation de la photopériode et l’emploi de l’effet mâle sont deux facteurs non pharmacologiques capables d’induire et de synchroniser l’activité ovarienne des chèvres. Ce travail avait pour but d’étudier l’effet de jours longs (avec ou sans mélatonine) sur la réponse à l’effet mâle chez la chèvre Sarde, afin d’induire et synchroniser l’activité ovarienne en dehors de la saison sexuelle, avec une méthode douce et économiquement acceptable

The Effect of sperm preparation and brief gamete coincubation length on ovine oocytes fertilization, subsequent development and embryo quality

Over the past 70 years, egg yolk (EY) is frequently used as a cryoprotectant in semen diluents and has been confirmed highly effective for the maintenance of sperm fertility. However, the preparation of uniform diluents containing EY is hard. Since Cheng, (1985) reported the beneficial effect of overnight gamete oocytes coincubation length in IVF, this protocol was used a recommended protocol in several species. However, this protocol leading to exposure of oocytes and embryos to toxic effects associated with excessive generation of reactive oxygen species. The aims of this study were to examine (i) whether brief exposure of mature ewe oocytes to frozen-thawed semen prepared with different semi-defined diluents may affect fertilization, enhanced embryonic development, embryo quality before and/or after freezing vs. long coincubation length, and (ii) explore the accompanying effects of different semi-defined diluents and coincubation length on fertilization and embryo development rates