Livelli di inquinanti organici persistenti in stenelle (<i>Stenella coeruleoalba</i>) spiaggiate nel nord Sardegna = Concentration of persistent organic pollutants in striped dolphin (<i>Stenella coeruleoalba</i>) stranded in North Sardinia

Long-lived apex predators, such as marine mammals, are particularly at risk from effects of persistent organic pollutants (POPs), e.g. polychlorinated biphenyls (PCBs) and dichlorodiphenylethanes (e.g. DDT), due to bioaccumulation and biomagnification. POPs are lipophilic compounds that tend to accumulate in the lipid-rich blubber. In marine mammals, POPs enter the body almost exclusively through the diet. In the present study, the levels of PCBs and DDTs in the blubber of striped dolphin stranded in North Sardinia were determined. Our results showed that pollutant concentration are related to age and sex of the individuals. Considering the well known harmful consequences of bioaccumulation of POPs in marine mammals, further studies are needed to determine which POPs might be linked to effects on health

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&#60;0.01) and polispermy (14.30% vs 2.21% respectively in prepubertals and adults; P&#60;0.01), lower cleavage rates (60.00% vs 67.08% respectively in prepubertals and adults; P&#60;0.05) and blastocyst output (11.94% vs 34.% respectively in prepubertals and adults; P&#60;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

assessment of cortisol and dhea concentrations in griffon vulture gyps fulvus feathers to evaluate its allostatic load

AbstractThe use of a non-invasive approach to collect biological samples from natural populations represents a great means of gathering information while avoiding handling animals. Even if corticosterone is the main glucocorticoid investigated in birds, there has been observed a proportional direct link between corticosterone and cortisol concentrations. Dehydroepiandrosterone (DHEA) can be produced by the adrenal cortex and should have prominent antiglucocorticoid properties also in birds. The aim of this study was to verify if there is any difference in the cortisol and DHEA feather concentrations between clinically normal and physiologically compromised Griffon vultures (Gyps fulvus) through the non-invasive approach of collecting moulted feathers without having to pluck them from the bird. The study was carried out using 8 physiologically compromised (PC) Griffons and 9 clinically normal Griffons considered as the control (CTRL) group that were necropsied or from the wildlife rehabilitation centre. Primary and secondary covert feathers were either collected directly from the birds' cage floors, or, in the case of dead Griffons, they were plucked off the animals. The results, obtained by RIA, revealed that both cortisol (P<0.01) and DHEA (P<0.05) feather concentrations were higher in the PC than in the CTRL group. No difference was observed by comparing the cortisol/DHEA ratio between the two evaluated groups (P=0.15). Pearson's correlation coefficients showed no correlation between feather hormone concentrations in the PC group (r=0.01, P=0.96) while a positive correlation in the CTRL group (r=0.65, P=0.006) was observed. In conclusion, our study reveals that moulted feathers can be a non-invasive and an interesting tool to evaluate the allostatic load of wild birds and they allowed better understanding the relationship between hormones of the hypothalamic–pituitary–adrenal axis and the physiological status of the birds

Morphofunctional aspects of in vitro matured oocytes from prepubertal and adult sheep

The aim of this study was to analyze the morphofunctional aspects of prepubertal and adult sheep oocytes subjected to in vitro maturation (IVM). The structural and ultrastructural morphology was examined by light and transmission electron microscopy (LM and TEM) while the metabolic competence, as determined by the distribution of active mitochondria, was assessed by confocal microscopy (CM). Cumulus-oocyte complexes underwent IVM in standard condition for 24 h. Half of the mature metaphase II (MII) stage oocytes were processed for LM and TEM observations. The other half was subjected to immunostaining with MitoTracker Red (to stain mitochondria with functionally active membrane potential), SNARF-1 (a pH sensitive fluoroprobe), Hoechst 33342 (to label DNA) and analyzed by CM. Immature germinal vesicle (GV) stage oocytes, retrieved at 0 h IVM, were used as controls. By LM and TEM all the oocytes were regularly rounded, covered by microvilli and surrounded by an intact zona pellucida. Numerous rounded, oval or hooded mitochondria appeared either isolated or grouped in the ooplasm. The GV was usually rounded in prepubertal oocytes. In the adult, the GV often appeared flattened against the oolemma, with a crescent-shaped outline (an early sign of meiotic resumption). Scattered cortical granules (CGs) were rarely found in the ooplasm of both prepubertal and adult GV oocytes. After 24 h of IVM, CGs became abundant and distributed in a single row under the oolemma, particularly in adult oocytes. CM showed a homogeneous fine-to-granular mitochondrial distribution in prepubertal GV and MII oocytes. In adult GV oocytes, the mitochondrial distribution pattern was granular while in MII oocytes mitochondria were arranged in heterogeneous clusters. Thus, both prepubertal and adult oocytes completed maturation after 24 h in culture and showed an overall good preservation after IVM. However, the diverse distribution patterns of mitochondria in prepubertal oocytes reflect their low developmental competence

Use of Propylene-Glycol as a Cosolvent for GnRH in Synchronization of Estrus and Ovulation in Sheep

The foreseen shortage of eCG for estrus synchronization in sheep makes necessary the development of alternative protocols. The aim of the present work was to evaluate the reproductive response of sheep in breeding season to the administration of GnRH using propylene-glycol as a cosolvent and the subcutaneous route for slowing and extending the release of GnRH, as well as the most adequate timing for such administration. In the present study, protocols based on a short-term CIDR treatment and a single subcutaneous dose of GnRH in propylene-glycol at 36 h after CIDR removal induced a similar ovarian response to protocols based on administration of eCG at CIDR removal or intramuscular GnRH in distilled water at 56 h after. In such protocol, 80% of the animals developed estrus in a narrow timing (75% between 36 and 48 h after CIDR removal), and all of them also ovulated in a narrow window (87.5% between 72 and 76 h after CIDR removal, with 62.5% between 72 and 76 h) and showed a similar ovulation rate and plasma progesterone concentrations at the induced estrous cycle. Hence, administration of GnRH in propylene-glycol may constitute an alternative to traditional protocols based on the administration of eCG

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