Antonia Zarrilli

Disuse of rat muscle in vivo reduces protein kinase C activity controlling the sarcolemma chloride conductance

Muscle disuse produced by hindlimb unloading (HU) induces severe atrophy and slow‐to‐fast fibre type transition of the slow‐twitch soleus muscle (Sol). After 2 weeks HU, the resting ClC‐1 chloride conductance (gCl) of sarcolemma, which controls muscle excitability, increases in Sol toward a value typical of the fast‐twitch EDL muscle. After 3 days of HU, the gCl increases as well before initiation of fibre type transition. Since ClC‐1 channels are acutely silenced by PKC‐dependent phosphorylation, we studied the modulation of gCl by PKC and serine–threonine phosphatase in Sol during HU, using a number of pharmacological tools. We show that a fraction of ClC‐1 channels of control Sol are maintained in an inactive state by PKC basal activity, which contributes to the lower gCl in control Sol compared to EDL. After 14 days of HU, PKC/phosphatase manipulation produces effects on Sol gCl that corroborate the partial slow‐to‐fast transition. After 3 days of HU, the early increase of gCl in Sol is entirely attributable to a reduction of PKC activity and/or activation of phosphatase, maintaining ClC‐1 channels in a fully active state. Accordingly, we found that HU reduces expression of PKCα, ɛ, and θ isoenzymes in Sol and EDL muscles and reduces total PKC activity. Moreover, we show that the rheobase current is increased in Sol muscle fibres as soon as after 3 days of HU, most probably in relation to the increased gCl. In conclusion, Sol muscle disuse is characterized by a rapid reduction of PKC activity, which reduces muscle excitability and is likely to contribute to disuse‐induced muscle impairment.

Stress-related hormones in horses before and after stunning by captive bolt gun.

In this work the slaughter-linked plasma modifications of some stress-related hormones in horses subject to standardized butchering procedures were investigated in order to highlight the compromised animal welfare during pre-slaughter handling. During pre-slaughter, animals show strong hardship behavioural patterns, probably due to being under life-threatening conditions. Blood samples from 12 male horses, ageing from 3 to 5 years, were collected before slaughtering in lairage, and during exsanguination after stunning. Catecholamines, cortisol and beta-endorphin concentrations were assessed in plasma samples by EIA. Results show that plasma beta-endorphin concentration did not increase significantly after stunning, while cortisol (P<0.05) and catecholamines (P<0.001) increased significantly. The ratio between the plasma level of norepinephrine and epinephrine decreased significantly (P<0.001) during the time considered for observation underlining a greater involvement of adrenal medulla in the stress response. Moreover these results suggest that, under stress, the release of beta-endorphin could be different from that of ACTH.

Delta opioid receptor on equine sperm cells: subcellular localization and involvement in sperm motility analyzed by computer assisted sperm analyzer (CASA)

BackgroundOpioid receptors and endogenous opioid peptides act not only in the control of nociceptive pathways, indeed several reports demonstrate the effects of opiates on sperm cell motility and morphology suggesting the importance of these receptors in the modulation of reproduction in mammals. In this study we investigated the expression of delta opioid receptors on equine spermatozoa by western blot/indirect immunofluorescence and its relationship with sperm cell physiology.MethodsWe analyzed viability, motility, capacitation, acrosome reaction and mitochondrial activity in the presence of naltrindole and DPDPE by means of a computer assisted sperm analyzer and a fluorescent confocal microscope. The evaluation of viability, capacitation and acrosome reaction was carried out by the double CTC/Hoechst staining, whereas mitochondrial activity was assessed by means of MitoTracker Orange dye.ResultsWe showed that in equine sperm cells, delta opioid receptor is expressed as a doublet of 65 and 50 kDa molecular mass and is localized in the mid piece of tail; we also demonstrated that naltrindole, a delta opioid receptor antagonist, could be utilized in modulating several physiological parameters of the equine spermatozoon in a dose-dependent way. We also found that low concentrations of the antagonist increase sperm motility whereas high concentrations show the opposite effect. Moreover low concentrations hamper capacitation, acrosome reaction and viability even if the percentage of cells with active mitochondria seems to be increased; the opposite effect is exerted at high concentrations. We have also observed that the delta opioid receptor agonist DPDPE is scarcely involved in affecting the same parameters at the employed concentrations.ConclusionsThe results described in this paper add new important details in the comprehension of the mammalian sperm physiology and suggest new insights for improving reproduction and for optimizing equine breeding.

Changes in the expression of the μ-opioid receptor in the mare oviduct during oestrus and anoestrus

The presence of the mu-opioid receptor (MOR) was investigated in the mare oviduct during oestrus and anoestrus, by means of immunoblotting and immunohistochemistry. Immunoblotting analysis showed that the MOR protein is expressed as 65, 50 and 30 kDa forms in the infundibulum and ampulla both in oestrus and anoestrus, while the 30 kDa form is absent in the isthmus. Moreover, different levels of expression were observed along the ampulla in the two periods examined. Immunohistochemistry revealed MOR in the mucosal epithelium, stromal cells, myocytes and blood vessels. Ciliated cells expressed MOR in the apical cytoplasm and, except for the isthmus of oestrous mares, also in the nucleus. Non-ciliated cells showed MOR only in the isthmus segment during oestrus. Stromal cells showed different immunoreactivity along the oviduct segments and during the oestrous and anoestrous phases. The myosalpinx displayed immunostained myocytes in the intrinsic musculature of the ampulla and in the extrinsic and intrinsic musculature of the isthmus without significant differences between anoestrus and oestrus. Blood vessels expressed MOR in endothelial cells and smooth muscle cells in the isthmus myosalpinx of oestrous mares only. In conclusion, these findings show diverse MOR expression in the three segments constituting the oviduct, as well as changes in MOR expression linked to the mares physiological condition.

Reduction of the olfactory cognitive ability in horses during preslaughter: Stress-related hormones evaluation

As horses may perceive several odour signals of danger at slaughter, application of mentholated ointment to their nostrils may limit their perception of danger. To assess the effect of the application of a mentholated ointment to horse nostrils on the stress response during pre-slaughter handling, plasma levels were evaluated for cortisol, beta-endorphin, epinephrine and norepinephrine prior to and after stunning. Twenty draught-type horses were divided into control (n=10) and treated (n=10) groups and a mentholated ointment applied to the nostrils of the treated horses following blood sampling in lairage 45 min prior to slaughter. Treatment did not affect plasma concentrations of beta-endorphin or cortisol but significantly reduced the concentrations of epinephrine and norepinephrine observed in post-stun plasma. These results indicated that mentholated ointment applied to the nostrils of horses pre-slaughter reduced their adrenergic response to the slaughter environment, implying that the stress response may be reduced with this technology.

Evaluation of animal welfare and milk production of goat fed on diet containing hydroponically germinating seeds

Abstract Hydroponic fodder is a particularly nutritious feed, rich in protein and vitamins such as ß-carotene, trace elements and enzymes. It may also offer the advantage of a continuous availability. A pilot plant for hydroponically production of germinating seeds was built in an area of the same farm where the trial took place. Three homogeneous groups of 30 Jonica breed goats in lactation (4th-5th parity) were used to evaluate the effects of two different levels of partial dietary substitution with hydroponically germinating (h.g.) oat on plasma levels of cortisol and milk production. Germinated oat was used after 7 days of hydroponic growth. Control group (T) received only feed (fodder and oat integrated with complement feed). The other 2 groups were fed on diet containing different levels (1,5Kg - group A; 3Kg - group B) of hydroponically germinating oat. Goats showed a small interest in fresh feed during the trial period. The integration with hydroponically germinating oat in partial substitution of the traditional feed in the diet of goat did not significantly affect biochemical and haematological parameters.

Stress indicators in steers at slaughtering

Abstract This work aimed to assess the blood modifications of some slaughtering-linked stress hormones in cattle subject to butcher standardized procedures. The blood samples of 20 Limousine 12-13 months old steers have been collected before slaughtering, during lairage, and after stunning by captive bolt gun, during exsanguination. The plasma level of epinephrine, norepinephrine, cortisol and beta-endorphin have been assayed by EIA. The data indicate that catecholamines, cortisol and beta-endorphin did not significantly increase after stunning in these animals.

Improvement of sheep welfare and milk production fed on diet containing hydroponically germinating seeds.

Abstract Plasma cortisol and milk production responses of 45 lactating Comisana sheeps (4th5th parity), divided into three homogeneous groups of 15 subject each, were used to evaluate the effects of two different levels of partial substitution of a complete feed with hydroponically germinating seeds. Germinated oat was employed after 7 days of hydroponic growth. The three groups received the following diets: Control group (T) received only complete feed. The other 2 groups were fed on diet containing different levels of hydroponically germinating oat (1,5 kg – group A; 3 kg – group B). All the subjects have shown to accept the diets because the per capita ration was always completely consumed. In the second month, the A and B groups showed lower average values of cortisol (P<0.01) and a statistically significant increase in milk production as compared to T (P<0.05 and P<0.001). The obtained data induced to conclude that integration with hydroponically germinating oat in partial substitution of the complete feed does not modify biochemical and hematological parameters and seems to produce an improvement in animal welfare and production of milk.

A functional study on L-type calcium channels in granulosa cells of small follicles in laying and forced molt hens

To investigate Ca(2+) dynamics in earlier phases of follicular development we compared the resting [Ca(2+)](i) and tested the functional responses to agonist/antagonist of L-type voltage-operated calcium channels (VOCCs) in small follicles GCs from hens during oviposition (O-GCs) and forced molt (M-GCs), using the microspectrofluorimetric [Ca(2+)](i) imaging. O-GCs were obtained from prehierarchical follicles (F(6)-F(5)-F(4)<8mm). In basal and agonist/antagonist stimulated M-GCs we did not observe a change in the [Ca(2+)](i) under any of condition in all cells analyzed. Based on basal measurements we can distinguish three different patterns reflecting cells variability within O-GCs group: (a) 39% cells showed small oscillations and [Ca(2+)](i) was 108±11nM; (b) 36% cells displayed yet small oscillations and [Ca(2+)](i) was 167±14nM; (c) 25% were cells with repetitive irregular oscillations that peaked until 2 fold basal value and [Ca(2+)](i) very variable, was 248±41nM. In O-GCs L-type VOCCs stimuli displayed different effects on [Ca(2+)](i) for both treatment in three basal patterns. In our study we demonstrated: (1) at resting the [Ca(2+)](i) is low (111±5nM) in M-GCs and tend to increasing in prehierarchical O-GCs; (2) L-type Ca(2+) channels are functionally expressed in the major part of O-GCs whereas they are not activated nor inhibited in M-GCs and in a percentage of O-GCs; (3) there are three different cellular types in prehierarchical O-GCs that may be associated with increasing stages of follicular development, based on their Ca(2+) pathway. Therefore, the functional response of L-type Ca(2+) channels in cultured laying hen prehierarchical GCs may be correlated with the functional maturation phase of laying hens ovarian. We hypothesize that the L-type Ca(2+)-dependent signaling could have a critical role in the regulatory mechanisms hormone mediated in hen ovarian cycle.

Cashmere production from Scottish Cashmere kids and crossbreed Scottish Cashmere x Jonica kids

Abstract This study is part of a much wider research programme to evaluate the possibility of producing valuable textile fibres, such as cashmere, from goat breeds reared in Italy. In order to achieve this, we have used crossbreeding. The first stage of the programme consisted of evaluating cashmere production in F1 kids obtained by crossing white-haired Jonica does, which have no secondary fibres, with Scottish Cashmere bucks. The trial lasted one year starting in March 2007, and took place in the Department of Animal Production of the University of Bari (Italy). We used 14 male kids: 7 Scottish Cashmere (SC group), and 7 F1 (SC x J group) derived from crossing Scottish Cashmere bucks with does of the Jonica breed, commonly reared in southern Italy. All the parameters considered (live weight, number and active percentage of primary and secondary follicles, S/P ratio, patch weight, growth and length of guard hair and down, yield, down production and diameter, blood protein and T3 and T4) were significantly influenced (P<0.01) by age. Genotype also had a significant effect (P<0.01) on all parameters except for the active percentage of primary follicles and the blood protein level. The factors which influence down production showed the heterosis effect to a varying extent in F1, but they still produced significantly less than the SC group kids (38.5±4.04 vs 68.5±9.16 g; P<0.01). These results are largely due to both their low number of secondary follicles (30.0±1.46 vs 39.3±1.02; P<0.01), which also have a lower percentage of activity (64.7±2.47 vs 90.0±1.53; P<0.01), and also to the down length which was 28% shorter than in SC group. This genetic combination is clearly unsatisfactory so others must be sought, probably by using more rustic local breeds, as well as more productive breeds for crossbreeding.