Neuropeptide FF (NPFF) reduces the expression of morphine- but not of ethanol-induced conditioned place preference in rats

Neuropeptide FF (NPFF) has been described as an anti-opioid peptide. It plays a role in opioid antinociception, dependence and tolerance. Previous study has indicated that 1DMe ([D-Tyr(1), (NMe)Phe(3)]NPFF), a stable analog of NPFF, inhibits acquisition of the rewarding effect of morphine but not of ethanol in mice. The rewarding effects of these drugs were measured in the unbiased paradigm of conditioned place preference (CPP). The present study examines the influence of NPFF on the expression of morphine- and ethanol-induced CPP in the biased procedure in rats. Our experiments showed that NPFF, given intracerebroventricularly (i.c.v.) at the doses of 5, 10 and 20 nmol, inhibited the expression of morphine-induced CPP. NPFF gave itself, neither induced place preference nor aversion, although a tendency to aversive effect was seen at the highest dose of 20 nmol. NPFF did not indicate fear behavior in the elevated plus maze test, and did not disturb locomotor activity of rats. However, NPFF was unable to inhibit the expression of ethanol-induced CPP. Probably this effect is due to the fact that ethanol reward is a more complex process and apart from the role of opioids, there are other neurotransmitters also involved in this mechanism. These results suggest that NPFF is involved in the expression of morphine reward. Moreover, our study supports an anti-opioid character of this peptide.     

Modulation of neuropeptide FF (NPFF) receptors influences the expression of amphetamine-induced conditioned place preference and amphetamine withdrawal anxiety-like behavior in rats

Many data indicate that endogenous opioid system is involved in amphetamine-induced behavior. Neuropeptide FF (NPFF) possesses opioid-modulating properties. The aim of the present study was to determine whether pharmacological modulation of NPFF receptors modify the expression of amphetamine-induced conditioned place preference (CPP) and amphetamine withdrawal anxiety-like behavior, both processes relevant to drug addiction/abuse. Intracerebroventricular (i.c.v.) injection of NPFF (5, 10, and 20 nmol) inhibited the expression of amphetamine CPP at the doses of 10 and 20 nmol. RF9, the NPFF receptors antagonist, reversed inhibitory effect of NPFF (20 nmol, i.c.v.) at the doses of 10 and 20 nmol and did not show any effect in amphetamine- and saline conditioned rats. Anxiety-like effect of amphetamine withdrawal was measured 24h after the last (14 days) amphetamine (2.5mg/kg, i.p.) treatment in the elevated plus-maze test. Amphetamine withdrawal decreased the percent of time spent by rats in the open arms and the percent of open arms entries. RF9 (5, 10, and 20 nmol, i.c.v.) significantly reversed these anxiety-like effects of amphetamine withdrawal and elevated the percent of time spent by rats in open arms at doses of 5 and 10 nmol, and the percent of open arms entries in all doses used. NPFF (20 nmol) pretreatment inhibited the effect of RF9 (10 nmol). Our results indicated that stimulation or inhibition of NPFF receptors decrease the expression of amphetamine CPP and amphetamine withdrawal anxiety, respectively. These findings may have implications for a better understanding of the processes involved in amphetamine dependence.     

Effects of Tasco (a seaweed extract) and heat stress on N metabolism and meat fatty acids in wether lambs fed hays containing endophyte-infected fescue

Wether lambs (n = 27, average BW = 40 kg) were used to test response to forage treated with Tasco-Forage (an extract of the brown kelp Ascophyllum nodosum) prior to conserving, or to direct feeding of the extract (Tasco-EX). Hays made from endophyte (Neoyphodium coenophialum)-infested tall fescue (Festuca arundinacea)-based pasture received 0 or 3 kg of Tasco/ha prior to harvest. Lambs, blocked by weight, were randomly allotted to three diets: (1) control hay, (2) treated hay, and (3) #1 + Tasco-EX fed at 1% of the diet. Hays were low in CP (<7%) so all lambs were fed soybean meal (12% of the diet) in addition to trace mineralized salt. Diets were fed at 1.5% BW to prevent refusals. Total collections (7 d) were made during periods without or with applied heat stress. After each period, rumen contents were obtained to determine pH, NH₃ and VFA. Lambs were sacrificed post-trial. A subset was used to evaluate sensory traits and muscle fatty acids. Lambs were in negative N balance during the study and Tasco treatments did not affect N metabolism. Fecal N tended (P < 0.10) to increase with short duration heat stress causing a concomitant decrease (P < 0.05) in apparent N digestibility (58.6 versus 56.1%; S.E. = 0.7). Urinary N loss decreased (P < 0.001) with heat stress (8.0 versus 5.9 g/d; S.E. = 0.2), resulting in increased (P < 0.001) N retention (−2.1 versus −0.3 g/d; S.E. = 0.2). Apparent OM digestibility was not affected by heat stress but was greater (P < 0.05) for lambs fed Tasco-EX treatment than those fed treated hay. Treatment diets decreased (P < 0.05) ruminal butyrate. Heat stress increased (P < 0.05) acetate and total VFA and decreased (P < 0.01) ruminal pH. A tendency (P < 0.11) of increased 14:1ω5, decreased (P < 0.05) 18:0 and total saturated fatty acids in muscle was observed with Tasco diets. Meat sensory characteristics were not affected by treatment. Tasco may alter some aspects of rumen or lipid metabolism but has no effect on N metabolism or meat sensory characteristics of sheep fed restricted, low-quality diets.     

Surgical castration of pigs affects the behavioural response to a low-dose lipopolysaccharide (LPS) challenge after weaning

The objective of the present study was to evaluate the effect of an acute stressor in the early postnatal life of pigs, surgical castration, on post-weaning behaviour, and on the behavioural, endocrine and immune responses elicited by a low-dose lipopolysaccharide (LPS) challenge after weaning. At 5-days-of-age, 64 male piglets were randomly assigned to undergo surgical castration or were left untreated (treatment). Pigs were weaned at 28 days-of-age. Behaviour post-weaning and mixing was assessed during a 1-h period, during which agonistic interactions were recorded. One day post-weaning, pigs were injected intraperitoneally with a single dose of 0 or 5 μg/kg of BW of LPS from Escherichia coli (challenge). Sickness behaviour was studied by scan sampling every 5 min for 45 min at 0, 1, 2, 3, 4, 6 and 8 h after the challenge. Blood samples were taken at 0, 2, 12 or 24 h after injection and were analysed for plasma concentrations of tumor necrosis factor-alpha (TNF-), interleukin-1beta (IL-1β), C-reactive protein (CRP), serum amyloid A (SAA) and cortisol. Results showed that non-castrated pigs were more aggressive than castrated pigs immediately after weaning (P < 0.05). Administration of LPS provoked behaviours characteristic of sickness including a reduction in general activity, as well as decreased eating and exploratory behaviours (P < 0.05). These altered behaviours occurred predominantly 3-h post injection (P < 0.05). Significant treatment by challenge interactions showed that castration reduced the occurrence of sickness behaviours induced by LPS, such as depressed general activity (P < 0.01), anorexia (P < 0.01) and reduced exploratory behaviours (P < 0.05). LPS administration increased TNF- levels (P < 0.05), with peak concentrations 2 h after injection (P < 0.01). CRP levels of LPS-treated pigs were higher than saline-treated animals at 12 h (P < 0.05). LPS administration tended to increase plasma SAA levels (P < 0.1), but did not increase cortisol levels (P > 0.1). However, castration did not affect the response of pro-inflammatory cytokines, acute phase proteins and cortisol to the challenge. These results show that surgical castration reduces aggressiveness at weaning and affects specific sickness behaviours but not the endocrine and immune responses elicited by low-dose endotoxin challenge in weaned pigs.     

Comparison of independent and combined chronic metabolic effects of GIP and CB1 receptor blockade in high-fat fed mice

GIP receptor antagonism with (Pro³)GIP protects against obesity, insulin resistance, glucose intolerance and associated disturbances in mice fed high-fat diet. Furthermore, cannabinoid CB₁ receptor antagonism with AM251 reduces appetite and body weight gain in mice. The present study has examined and compared the effects of chronic daily administrations of (Pro³)GIP (25 nmol/kg body weight), AM251 (6 mg/kg body weight) and a combination of both drugs in high-fat fed mice. Daily i.p. injection of (Pro³)GIP, AM251 or combined drug administration over 22 days significantly (P < 0.05 to <0.01) decreased body weight compared with saline-treated controls. This was associated with a significant (P < 0.05 to <0.01) reduction of food intake in mice treated with AM251. Plasma glucose levels and glucose tolerance were significantly (P < 0.05) lowered by 22 days (Pro³)GIP, AM251 or combined drug treatment. These changes were accompanied by a significant (P < 0.05) improvement of insulin sensitivity in all treatment groups. In contrast, AM251 lacked effects on glucose tolerance, metabolic response to feeding and insulin sensitivity in high-fat mice when administered acutely. These data indicate that chemical blockade of GIP- or CB₁-receptor signaling using (Pro³)GIP or AM251, respectively provides an effective means of countering obesity and related abnormalities induced by consumption of high-fat energy-rich diet. AM251 lacks acute effects on glucose homeostasis and there was no evidence of a synergistic effect of combined treatment with (Pro³)GIP.     

Lobeline augments and inhibits cocaine-induced hyperactivity in rats

Lobeline has high affinity for nicotinic receptors and alters presynaptic dopamine storage and release in brain. Moreover, lobeline decreases the reinforcing and locomotor-activating properties of methamphetamine, suggesting that lobeline may be a pharmacotherapy for psychostimulant abuse. This study determined if lobeline alters cocaine-induced hyperactivity and if lobeline alters the induction and/or expression of sensitization to cocaine. On Days 1-12, male rats were administered lobeline (0.3 or 1.0 mg/kg) or saline, placed in an automated activity monitor for 20 min, administered cocaine (10, 20 or 30 mg/kg) or saline and returned to the monitor for 60 min. On Day 13, the effect of lobeline on the induction and expression of sensitization to cocaine was determined. Lobeline did not alter the effect of cocaine after acute injection. However, 1.0 mg/kg lobeline attenuated cocaine (10 and 20 mg/kg)-induced hyperactivity after repeated administration and prevented the development of sensitization to these cocaine doses. Interestingly, 0.3 mg/kg lobeline augmented cocaine (10 mg/kg)-induced hyperactivity after repeated administration. Lobeline did not alter the effect of 30 mg/kg cocaine. The present results indicate a complex interaction of lobeline with cocaine and support other research indicating a role for nicotinic receptors in the development of sensitization to psychostimulants.     

Evaluation of sperm functional attributes in relation to in vitro sperm-zona pellucida binding ability and cleavage rate in assessing frozen thawed buffalo (Bubalus bubalis) semen quality

The objective of this study was to evaluate sperm functional attributes in relation to in vitro sperm-zona binding ability and cleavage rate in assessing frozen thawed buffalo (Bubalus bubalis) semen quality. Frozen-thawed forty-eight ejaculates from eight Surti buffalo bulls (six ejaculates/bull) obtained by artificial vagina were used. Frozen semen from each bull was thawed, pooled, and subjected for sperm functional (six replicates) and in vitro fertilization (four replicates) tests. The progressive forward motility, plasmalemma functional integrity assessed by fluorogenic [6-carboxyfluorescein diacetate (CFDA), and propidium iodide (PI)], hypoosmotic swelling (HOS), and hypoosmotic swelling-Giemsa (HOS-G) test, mitochondrial membrane potential, sperm nuclear morphology, the number of sperm bound to zona and cleavage rate differed significantly (P < 0.05) between bulls. When the animals were grouped based on cleavage rate (group I, >40% cleavage rate, n = 5, and group II, <40% cleavage rate, n = 3), in vitro fertility parameters and all the sperm functional attributes except sperm nuclear morphology differed significantly (P < 0.05). The proportions of sperm with functional plasmalemma in the tail and intact acrosome assessed by HOS-G test (25.33, range: 17.48–40.27) were significantly (P < 0.001) lower than the functional plasmalemma in the tail assessed by HOS test (39.80, range: 27.85–54.67). The number of sperm bound to zona had significant correlations with the mitochondrial membrane potential (r = 0.90, P < 0.01) and plasmalemma integrity (fluorogenic, r = 0.74 and HOS, r = 0.79, P < 0.05) and HOS-G, r = 0.87, P < 0.01). The cleavage rate had significant (P < 0.05) correlations with the mitochondrial membrane potential (r = 0.70) and plasmalemma integrity measured by HOS-G test (r = 0.68). The present study indicates that these attributes could represent important determinants of buffalo sperm quality influencing cleavage rate.     

Effect of monensin, fish oil or their combination on in vitro fermentation and conjugated linoleic acid (CLA) production by ruminal bacteria

An in vitro study was conducted to examine the effect of adding monensin, fish oil, or their combination on rumen fermentation and conjugated linoleic acid (CLA) production by mixed ruminal bacteria when incubated with safflower oil. Concentrate (1 g/100 ml) with safflower oil (0.2 g/100 ml) was added to a mixed solution (600 ml) of strained rumen fluid and buffer (control). Monensin (10 ppm), fish oil (0.02 g/100 ml), or monensin plus fish oil was also added into control mixture. All the culture solutions prepared were incubated anaerobically at 39 °C for 12 h. A higher pH and ammonia concentration were observed from the culture solution containing monensin at 12 h of incubation than those from the control or the culture containing fish oil. Monensin increased (P < 0.007) the C₃ content over all the collection times of culture solution while reducing the C₄ content at 6 h (P < 0.018) and 12 h (P < 0.001) of incubations. Supplementation of monensin, fish oil or their combination changed the content of C₁₈-fatty acids of ruminal culture. Monensin alone reduced (P < 0.021) the content of cis-9, trans-11 CLA compared to fish oil at all sampling times, but increased (P < 0.041) the trans-10, cis-12 CLA production compared to fish oil addition and the control which were similar at incubation for 12 h. The combination of monensin and fish oil increased the content of cis-9, trans-11 CLA (P < 0.023) and transvaccenic acid (TVA, P < 0.018) significantly compared to the control or monensin alone at incubation for 12 h.     

Control of postpartum anestrous with an intra-vaginal progesterone device plus eCG or calf removal for 120 h in suckled crossbred cows managed in a pasture-based system

To control postpartum anestrus and reduce calving to conception interval, 167 crossbred non-pregnant cows that were 90–130 days postpartum were allotted randomly to one of the following treatments: PH (n = 59), intra-vaginal sponge with 250 mg of medroxyprogesterone acetate (MAP) for 7 days plus 50 mg of MAP and 5 mg 17-β estradiol (17β-E) in the first day of treatment (day −8), 500 UI eCG (day −3) and 1.5 mg 17β-E in 24 h after sponge removal (day 0); CR (n = 57), temporary calf removal for 120 h; CG (n = 51), control group without treatment. Estrus rate differed among treatments (P < 0.01) being greater in PH (78.2%), followed by CR (52.0%) and CG (22.9%). A greater proportion of cows in the PH (80.0%) and CR (54%) groups had ovulations when compared to CG (35.4%). Intervals to first estrus were 13.5 ± 6.3 days, 26.1 ± 6.4 days and 52.5 ± 7.5 days for the PH, CR and CG groups, respectively. First insemination conception was similar in the three groups. Postpartum intervals to first breeding (PFS) and to conception (PCI) were longer in CG than PH and CR groups (P < 0.05; P < 0.01). The PH and CR groups had a similar PFS but PCI was different (P < 0.02). Accumulated pregnancy rate at 30 and 60 but not at 90 days were different (30 days: P < 0.09; P < 0.01; P < 0.09; 60 days: P < 0.06; P < 0.01; P < 0.03) among treatments. After 90 days post-treatment, 9%, 18% and 33% of cows from the PH, CR and CG groups had not conceived. Similarly, 5.4%, 6.0% and 12.5% of cows from the PH, CR and CG groups, respectively, were culled from the herd because of lack of pregnancy after 180 days post treatment. In the group of cows evaluated by ultrasonography, only those cows having larger ovaries and dominant follicles had ovulations. It was concluded that the hormonal treatment was more efficient in inducing a fertile estrus and reducing calving to conception interval followed by the calf removal for 120 h. Each method can be considered as an important tool to reduce the postpartum anestrous period in dual purpose herds when AI is conduct in the tropics.     

Effect of strategic supplementation with multi-nutrient urea molasses blocks on body weight and body condition score of Lohi sheep owned by tenants of Pakistan

The effect of strategic supplementation with multi-nutrient urea molasses blocks (MNUMB) on BW and body condition score (BCS) in Lohi ewes (treated, n = 514) during late gestation and lactation was compared with those (control, n = 391) grazing on only post-harvest crop residues and road side in the irrigated district of Okara in central Punjab (Pakistan). Analysis of variance revealed highly significant (P < 0.01) differences in body weight (BW) and body condition score (BCS) of ewes of various ages with different reproductive status and seasons under both flocks. Analysis of variance also revealed a significant interaction (P < 0.05) between reproductive status and seasons in favor of BCS. Ewes aged 48 months in average constituted the highest (34.5% and 35.6%), whereas those aged 60 months had the smallest (10% and 4%) proportion in the control as well as in the treated flock. Mean BW and BCS in ewes of control flock was 33.5 and 2.08 kg, and lower (P < 0.05) than 35.0 and 2.31 in ewes in the treated flock, respectively. Ewes aging 12, 24 and 36 months treated with strategic supplementation of MNUMBs were not only heavier (P < 0.01) but also had highest BCS of 2.34. Lactating ewes constituted highest proportion (39%, 51%) followed by pregnant (35%, 32%) ewes in both flocks, respectively. Proportion of dry (16%) and freshly conceived (9.5%) ewes tended to be higher in the control than in the treated flock. BW was 8–11% higher (P < 0.01) in pregnant than in lactating or dry ewes in both flocks with similar BCS. Seasons of autumn and summer were found to affect BW more (P < 0.01) than BCS. Pregnant ewes in treated flocks had gained highest BW, 10–12% higher than ewes under control (P < 0.01) or than non-pregnant ewes (P < 0.05) but lost at a rate of 5–6% at lambing. BW in lactating ewes in treated flock was higher (P < 0.01) than ewes in control. Lambs suckling ewes with strategic supplementation of MNUMBs grew at a faster rate (122 g/day) with 10–15% higher survival rate than those (97 g/day) in the control flock during lactation of 16 weeks but non-significantly. Based on this improvement it can be concluded that supplementation with appropriate sources of energy and N exerts desirable effects on the traits of economic importance in sheep.     

Examination of cyclic changes in bovine luteal echotexture using computer-assisted statistical pattern recognition techniques

B-mode sonography is a well-established diagnostic tool for determination of cycle stage in gynaecology. The aim of this study was to determine whether computer-assisted texture analysis of B- mode sonographic images of bovine luteal glands provides further information about the animal's plasma progesterone concentration and cycle stage. Four Simmenthal cows were examined during two consecutive estrous cycles with an ultrasound device equipped with a 7.5 MHz microconvex probe. During each examination three B-mode images of the corpus luteum (CL) were digitized and analyzed off-line using a computer-assisted texture analysis program. Size, echogeneity, and echotexture of the CL were characterized by the following texture parameters: area of cross-sectional planes of the CL (A), mean gray level (MGL), correlation (CORR), run percentage (RPERC), and long-run emphasis (LREM). Plasma progesterone levels (P4) were also determined. All parameters showed characteristic changes during the estrous cycle (P < 0.05). Variance component estimates for the effect of Day of estrous cycle on A, MGL, CORR, RPERC, and LREM were 56.6%, 64.6%, 77.6%, 89.9%, and 86.0%, respectively, and 20.6%, 24.5%, 7.2%, 0.0%, and 14.0% for the influence of the individual cow. The factor estrous cycle within cows was responsible for 22.8%, 10.9%, 15.2%, 10.1%, and 0.0% of the variability of A, MGL, CORR, RPERC and LREM values, respectively. Cyclic changes were similar in A and P4. In contrast to P4, which decreased already between Days –5 and –3 (Day 0 = ovulation), A stayed at constant high values until Day –3. Mean MGL values were higher (P < 0.05) on Days 7, 9, and 13 compared to Days 3 and –3. Mean CORR values were constantly high (P > 0.05) during the first days after ovulation and decreased continuously (P < 0.05) between Days 5 and 13. Thereafter, mean CORR values remained low (P < 0.05) until the next ovulation, except on Day –3 (P < 0.05). Mean RPERC rose between Days 1 and 9 from low to high values (P < 0.0001) remained at these high values (P > 0.05) between Days 9 and 15, and decreased (P < 0.05) afterwards to baseline values on Day –1. Mean LREM inclined steeply (P < 0.0001) from minimum to maximum between Days 1 and 5. From Days 7 to –3, mean LREM remained (P > 0.05) at a constant level close below the maximum value, and decreased to baseline values on Day –1. The results of this study show that statistical pattern recognition techniques provide new information about the luteal glands, thus facilitating a more accurate differentiation between different cycle stages in cows.     

Different effects of ATP on the contractile activity of mice diaphragmatic and skeletal muscles

Apart from acetyl-choline (Ach), adenosine-5′-trisphosphate (ATP) is thought to play a role in neuromuscular function, however little information is available on its cellular physiology. As such, effects of ATP and adenosine on contractility of mice diaphragmatic and skeletal muscles (m. extensor digitorum longa—MEDL) have been investigated in in vitro experiments. Application of carbacholine (CCh) in vitro in different concentrations led to pronounced muscle contractions, varying from 9.15 ± 4.76 to 513.13 ± 15.4 mg and from 44.65 ± 5.01 to 101.46 ± 9.11 mg for diaphragm and MEDL, respectively. Two hundred micromolars of CCh in both muscles caused the contraction with the 65% (diaphragm) to 75% (MEDL) of maximal contraction force—this concentration was thus used in further experiments. It was found that application of ATP (100 μM) increased the force of diaphragmatic contraction caused by CCh (200 μM) from 335.2 ± 51.4 mg (n = 21) in controls to 426.5 ± 47.8 mg (n = 10; P < 0.05), but decreased the contractions of MEDL of CCh from 76.6 ± 6.5 mg (n = 26) in control to 40.2 ± 9.0 mg (n = 8; P < 0.05). Application of adenosine (100 μM) had no effect on CCh-induced contractions of these muscles.

Resting membrane potential (MP) measurements using sharp electrodes were done at 10, 20 and 30 min after the application of ATP and adenosine. Diaphragm showed depolarization from 75 ± 0.6 down to 63.2 ± 1.05, 57.2 ± 0.96 and 53.6 ± 1.1 mV after 10, 20 and 30 min of exposition, respectively (20 fibers from 4 muscles each, P < 0.05 in all three cases). Adenosine showed no effect on diaphragmatic MP. Both agents were ineffective in case of MEDL.

The effects of ATP in both tissues were abolished by suramin (100 μM), a P2-receptor antagonist, and chelerythrin (50 μM), a specific protein-kinase C (PKC) inhibitor, but were not affected by 1H-[1,2,4]-oxadiazolo-[4,3-]-quinoxalin-1-one (ODQ, 1 μM), a guanylyl-cyclase inhibitor, or by adenosine-3,5-monophosphothioate (Rp-cAMP, 1 μM), a protein-kinase A (PKA) inhibitor.

Besides the action on contractile activity, ATP (100 μM) led to a significant (P < 0.001) depolarization of diaphragm muscle fibers from 74.5 ± 2.3 down to 64 ± 2.1, 58.2 ± 2.2 and 54.3 ± 2.4 mV after 10, 20 and 30 min of incubation, respectively. Incubation of MEDL with the same ATP concentration showed no significant change of MP.

Denervation of the muscles for 28 days led to a decrease of CCh-induced contractions of diaphragm down to 171.1 ± 34.5 mg (n = 11, P < 0.05), but increased the contractile force of MEDL up to 723.9 ± 82.3 mg (n = 9, P < 0.01). Application of ATP elevated the contractility of denervated diaphragm caused by CCh up to normal values (311.1 ± 79.7 mg, n = 6, P > 0.05 versus control), but did not significantly affect of contractility of MEDL, which became 848.1 ± 62.7 mg (n = 6).

These results show that the effects of ATP on both diaphragmatic and skeletal muscles are mediated through P2Y receptors coupled to chelerytrin-sensitive protein-kinase C.     

Dynamics of indirect symptoms of skeletal muscle damage after stretch-shortening exercise

Healthy untrained men (age 20.4 ± 1.7 years, n = 20) volunteered to participate in an experiment in order to establish dynamics of indirect symptoms of skeletal muscle damage (ISMD) (decrease in maximal isometric voluntary contraction torque (MVCT) and torque evoked by electrostimulation at different frequencies and at different quadriceps muscle length, height (H) of drop jump (DJ), muscle soreness and creatine kinase (CK) activity in the blood) after 100 DJs from 0.75 m height performed with maximal intensity with an interval of 20 s between the jumps (stretch-shortening exercise, SSE). All ISMDs remained even 72 h after SSE (P < 0.01–0.001). The muscle experienced greater decrease (P < 0.01) in torque evoked by electrostimulation (at low stimulation frequencies and at short muscle length in particular) after SSE than neuromuscular performance (MVCT and H of DJ) which demonstrated secondary decrease (P < 0.01) in neuromuscular performance during the first 48 h after SSE. Within 24–72 h after the SSE the subjects felt an acute muscle pain (5–7 points approximately) and the CK activity in the blood was significantly increased up to 1200 IU/L (P < 0.001). A significant correlation between decrease in MVCT and H of DJ 24–48 h after SSE on the one hand and muscle soreness registered within 24–48 h after SSE on the other was observed, whereas correlation between the other indirect symptoms of skeletal muscle damage was not significant.     

Effects of anti-oxidant additives on microscopic and oxidative parameters of Angora goat semen following the freeze–thawing process

The anti-oxidant system of reduced glutathione (GSH), glutathione peroxidase (GSH-PX), catalase (CAT), and superoxide dismutase (SOD) has been described as a defense functioning mechanism against lipid peroxidation (LPO) in semen, and is important in maintaining sperm motility and viability. This anti-oxidant capacity of sperm cells may be insufficient in preventing LPO during the freeze–thawing process. The aim of this study was thus to determine the influence of varying doses of anti-oxidant additives on standard semen parameters, lipid peroxidation and anti-oxidant activities after the freeze–thawing of goat semen. Ejaculate samples (artificial vagina) obtained from 4 mature Angora goats were evaluated and pooled at 37 °C. The semen samples diluted with a Tris-based extender, containing taurine (25, 50, 75 mM), trehalose (25, 50, 75 mM), and cysteine (5, 10, 15 mM), and an extender containing no anti-oxidant additives (control) were again evaluated. Diluted semen was cooled down to 5 °C and frozen in 0.25 ml French straws, prior to being stored in liquid nitrogen. Frozen straws were thawed in a water bath (37 °C) for 30 s for microscopic sperm evaluation. Upon evaluation of parameters for semen quality, the use of a Tris-based extender supplemented with anti-oxidant additives was found to cause no significant improvement in sperm mortality, when compared to the controls. Increasing doses of taurine and trehalose decreased (P < 0.05) the sperm motility following the freeze–thawing of the goat semen. In biochemical assays, the application of taurine (75 mM) produced the lowest level of malondialdehyde (MDA) (4.46 ± 0.31 nmol/ml), compared to the controls (P < 0.001). Lower GSH levels were higher in the groups in which cysteine was included at 10 and 15 mM (3.27 ± 0.11 and 3.45 ± 0.28 nmol/ml) – compared to the group which received 5 mM cysteine, as well as the controls (2.27 ± 0.08 and 2.50 ± 0.08 nmol/ml respectively, P < 0.001). Compared to the controls, taurine at a concentration of 25 and 75 mM, and increasing doses (50 and 75 mM) of trehalose, significantly increased the GSH-PX activity (P < 0.01). The maintenance of CAT activity was demonstrated to be higher with the addition of 10 and 15 mM cysteine, compared to the other groups (P < 0.001). Vitamin A (VitA) levels were significantly higher, compared to the controls (267.34 ± 9.68 mg/dl and 267.34 ± 9.68 mg/dl, respectively), when 25 mM taurine (329.61 ± 6.35 mg/dl) and 10 mM (318.64 ± 6.34 mg/dl) cysteine was added to the extender (P < 0.001). The results of this study provide a new approach to the cryopreservation of Angora goat semen and could contribute to the improvement of this technology in the goat industry.     

Prolactin regulation of testicular development and sexual behavior in yearling Suffolk rams

The aim of this study was to determine how the yearly prolactin rhythm might affect the sexual development of Suffolk rams (latitude 50°N). Five rams were injected daily with bromocriptine (35–45 μg kg⁻¹ body weight) for 1 year, beginning in January (early winter) when rams were 11 months of age. Five control rams each received daily injections of the vehicle. In the controls, blood prolactin was <7.5 ng ml⁻¹ in winter, increased (P < 0.01) to a peak of 172.6 ± 11.9 ng ml⁻¹ after the spring equinox, and remained high during summer before declining (P < 0.01) to 29.6 ± 6.6 ng ml⁻¹ at the autumn equinox. Suppression of the seasonal rise in prolactin secretion with bromocriptine slowed testicular growth (50%; P < 0.05) in April and May (spring), thus delaying the time of peak testis size and sperm production by 1 month. Serum testosterone level was lower (50%; P < 0.01) in the treated rams than the controls in June and July (early summer), due mainly to reduced stimulation of the testes by smaller (P < 0.01) LH pulse releases or to smaller (P < 0.01) testosterone responses to LH releases, respectively. Suppression of prolactin also seemed to disrupt the central activation of gonadotropin secretion in that seasonal increases in serum FSH level and LH pulse amplitude and frequency were unusually slow (P < 0.05). These anomalies did not affect testis growth, which was normal from June until development was complete. Rams were sexually inexperienced when libido was first tested in July (non-breeding season). Both groups were equally capable of learning and expressing sexual behavior (i.e. normal mounting and ejaculation frequencies), which was more intense in September (breeding season; P < 0.05). Results support the hypothesis (based on the location of prolactin receptors) that the spring increase in prolactin secretion could target both the testes and the hypothalamic–pituitary system and be involved in the seasonal regulation of sexual function in the young adult Suffolk ram.     

Effects of 1.8 GHz radiofrequency field on DNA damage and expression of heat shock protein 70 in human lens epithelial cells

To investigate the DNA damage, expression of heat shock protein 70 (Hsp70) and cell proliferation of human lens epithelial cells (hLEC) after exposure to the 1.8 GHz radiofrequency field (RF) of a global system for mobile communications (GSM). An Xc-1800 RF exposure system was used to employ a GSM signal at 1.8 GHz (217 Hz amplitude-modulated) with the output power in the specific absorption rate (SAR) of 1, 2 and 3 W/kg. After 2 h exposure to RF, the DNA damage of hLEC was accessed by comet assay at five different incubation times: 0, 30, 60, 120 and 240 min, respectively. Western blot and RT-PCR were used to determine the expression of Hsp70 in hLECs after RF exposure. The proliferation rate of cells was evaluated by bromodeoxyuridine incorporation on days 0, 1 and 4 after exposure. The results show that the difference of DNA-breaks between the exposed and sham-exposed (control) groups induced by 1 and 2 W/kg irradiation were not significant at any incubation time point (P > 0.05). The DNA damage caused by 3 W/kg irradiation was significantly increased at the times of 0 and 30 min after exposure (P < 0.05), a phenomenon that could not be seen at the time points of 60, 120 or 240 min (P > 0.05). Detectable mRNA as well as protein expression of Hsp70 was found in all groups. Exposure at SARs of 2 and 3 W/kg for 2 h exhibited significantly increased Hsp70 protein expression (P < 0.05), while no change in Hsp70 mRNA expression could be found in any of the groups (P > 0.05). No difference of the cell proliferation rate between the sham-exposed and exposed cells was found at any exposure dose tested (P > 0.05). The results indicate that exposure to non-thermal dosages of RF for wireless communications can induce no or repairable DNA damage and the increased Hsp70 protein expression in hLECs occurred without change in the cell proliferation rate. The non-thermal stress response of Hsp70 protein increase to RF exposure might be involved in protecting hLEC from DNA damage and maintaining the cellular capacity for proliferation.     

Biopsied and vitrified bovine embryos viability is improved by trans10, cis12 conjugated linoleic acid supplementation during in vitro embryo culture

Bovine embryos cultured in serum-containing media abnormally accumulate lipids in the cytoplasm. This is well known to contribute to their higher susceptibility to cryopreservation and biopsied embryos are even further susceptible. We aimed to improve in vitro produced (IVP) embryos resistance to micromanipulation and cryopreservation by supplementing serum-containing media with trans-10, cis-12 conjugated linoleic acid (t10, c12 CLA). The effect of t10, c12 CLA on lipid deposition and embryonic development was also tested. After in vitro maturation and fertilization (IVF day = D0), zygotes were cultured on granulosa cells + M199 + 10% serum + 100 μM GSH supplemented with 100 μM of t10, c12 CLA (CLA group, n = 1394) or without supplementation (control group, n = 1431). Samples of D7/D8 embryos were observed under Nomarsky microscopy for lipid droplets evaluation while others were biopsied and vitrified (group B-Control, n = 24; group B-CLA, n = 23). Non-biopsied embryos were also frozen (group NB-Control, n = 49; group NB-CLA, n = 45). Biopsied cells were used for embryo sex determination. Postwarming embryo survival and viability were determined at 0 and 24 h of culture, respectively. Supplementation of t10, c12 CLA did not influence cleavage, embryo sex ratio, D7/D8 embryo rate or morphological quality. CLA embryos had higher number of small lipid droplets (P ≤ 0.003) and a smaller (P < 0.001) fat embryo index being leaner (P = 0.008) than control embryos. Embryo postwarming survival was higher in B-CLA than in B-control group (95.0 ± 7.0% versus 62.5 ± 7.9%; P < 0.001). After 24 h of culture, the viability (expansion rate) of biopsied embryos and nonbiopsied embryos, cultured with t10, c12 CLA was higher than control embryos (B-CLA = 64.6 ± 4.4% and B-control = 27.5 ± 2.5%, P = 0.01; NB-CLA = 86.0 ± 3.5% and NB-Control = 68.6 ± 7.0%, P = 0.05). Results showed that supplying t10, c12 CLA to serum-containing media decreases embryo cytoplasmic lipid deposition during in vitro culture and significantly improves resistance of IVP embryos to micromanipulation and cryopreservation.     

Behaviour of Merinos divergently selected for multiple rearing ability in response to external stimuli

Data of Merino sheep descended from two divergent selection lines for (H line) and against (L line) multiple rearing ability were collected in three different trials to compare response to stressful situations between lines. Trial 1: the behaviour of 138, 20-month-old sheep born in 2001 and 124, 8-month-old sheep born in 2002 were observed in an arena test. Experimental animals amounted to 111 and 101 for the H line in the respective years. Corresponding numbers for the L line were 27 and 23, respectively. Progeny born in 2001 were accustomed to handling, as the tests were performed shortly after routine performance recording. Lambs born in 2002 were unaccustomed to handling when tested. Progeny from the H line born in 2002 allowed a smaller (P < 0.01) minimum distance between them and the human operator seated between them and a group of similar aged sheep than L line contemporaries. Progeny born in the L line during 2002 defecated more (P < 0.05) frequently than H line contemporaries. No line differences were found in progeny born during 2001. Trials 2 and 3 were designed to test whether the putative line difference in stress response would impact on physical meat quality. Trial 2: a total of 103, 18–20-month-old progeny from the two selection lines were slaughtered during 2001, 2002–2003. Mean slaughter weight, dressing percentage and carcass weight of H line animals were between 5 and 13% higher (P < 0.05) than that of L line animals. There were no differences in cooking loss, drip loss and tenderness between the two lines. As pertaining to the meat colour, L line progeny had less vivid red meat, as seen by their lower (P < 0.05) a* colour reading and chroma values. The L line animals had lower (P < 0.01) initial pH values as measured in the Musculus longissimus dorsi. These results are indicative of susceptibility to acute, short-term stress. Trial 3: 20 progeny of the two selection lines were slaughtered and their muscle pH measured at 11 intervals from 45 min to 48 h post slaughter. As in Trial 1, the initial pH of the L line animals were lower (P < 0.05) than that of H line animals. An opposite tendency was observed by 48 h post slaughter. The obtained results support a hypothesis that divergent selection for ewe multiple ability resulted in adaptations to cope with unfamiliar conditions.     

Changes in microRNA expression profile in hippocampus during the acquisition and extinction of cocaine-induced conditioned place preference in rats

Background

MicroRNA (miRNA) emerges as important player in drug abuse. Yet, their expression profile in neurological disorder of cocaine abuse has not been well characterized. Here, we explored the changes of miRNA expression in rat hippocampus following repeated cocaine exposure and subsequent abstinence from cocaine treatment.

Results

Conditioned place preference (CPP) procedure was used to assess the acquisition and extinction of cocaine-seeking behavior in rats. MiRNA microarray was performed to examine miRNAs levels in rat hippocampus. Quantitative RT-PCR was conducted to further confirm results in microarray study. Finally, bioinformatic predictions were made to suggest potential target genes of cocaine-responsive miRNA in this study. MiRNA array found that 34 miRNA levels were changed in rat hippocampus while acquiring cocaine CPP and 42 miRNAs levels were altered after the cocaine-induced CPP were extinguished, as compared to normal controls. The findings from qRT-PCR study support results from microarray analysis.

Conclusions

The current study demonstrated dynamic changes in miRNA expression in rat hippocampus during the acquisition and extinction of cocaine-induced CPP. Some miRNAs which have been previously reported to be involved in brain disorders and drug abuse, including miR-133b, miR-134, miR-181c, miR-191, miR-22, miR-26b, miR-382, miR-409-3p and miR-504, were found to be changed in their expression following repeated cocaine exposure and subsequent abstinence from cocaine treatment. These findings may extend our understanding of the regulatory network underlying cocaine abuse and may provide new targets for the future treatment of drug abuse.     

Effects of ensiled forage legumes on performance of store finishing lambs

Male Beulah speckled face lambs (initial live weight (LW) 28.8 ± 0.31 kg) were allocated to three dietary treatments to evaluate the performance of store lambs of a hill breed when offered ensiled lucerne (Medicago sativa), red clover (Trifolium pratense) or ryegrass. Second-cut silage bales (wilted and inoculated) were prepared from 3-year old lucerne and red clover stands and a 1-year old ryegrass sward. All the lambs were group-housed and offered ad libitum ryegrass silage during a 3-week co-variate period. This was followed by a week of dietary changeover period, after which the lambs were housed individually and offered their treatment diet ad libitum. All the lambs received a flat rate supplement of pelleted molassed sugarbeet (250 g fresh weight/day). Individual intakes were determined daily, and weekly measurements of LW and body condition score (CS) were made. Additional measurements were taken by scanning the lambs for depth of Longissimus dorsi (LD) muscle and subcutaneous fat. Over an experimental period of 7 weeks, the lambs offered red clover silage had a higher voluntary silage dry matter (DM) intake, total DM intake and metabolisable energy (ME) intake (P < 0.001) than lambs offered either lucerne or ryegrass silage. This resulted in a faster (P < 0.001) growth rate and increase (P < 0.001) in CS, with no difference between lucerne and ryegrass silages. The feed conversion efficiency (FCE) was 8.0 ± 0.61 kg feed/kg gain for lambs fed red clover silage, compared with 16.6 ± 2.82 and 10.6 ± 1.94 kg feed/kg gain for lucerne and ryegrass silage, respectively. The CP intake was higher (P < 0.001) for lambs fed the lucerne and red clover silages than for the ryegrass silage treatment. The concentration of plasma total protein (TP) was higher (P < 0.05) for lambs offered ryegrass silage versus lucerne and red clover silage. Urea concentrations were highest for lambs fed lucerne silage and lowest for those fed ryegrass silage (P < 0.05). The glucose concentration was higher (P < 0.05) for lambs offered red clover silage, whereas non-esterified fatty acids (NEFA) concentration was higher (P < 0.05) for lambs offered lucerne silage. Substituting ryegrass silage with red clover silage has the potential to improve the performance of finishing store lambs.