Quality parameters and fertility of ram semen cryopreserved in egg yolk and soybean lecithin supplemented extenders.

The aim of the study was to investigate the effect of soybean lecithin as a substitute for egg yolk in milk and tris based extenders in ram semen cryopreservation. Twenty ejaculates were col- lected from four healthy, mature Wrzosówka rams (2-3 years of age). Each ejaculate was divided into four equal aliquots and diluted with four different extenders: 1) milk extender containing 5% egg yolk, 2) milk extender containing 1.5% soybean lecithin, 3) tris extender containing 20% egg yolk, 4) tris extender containing 1.5% soybean lecithin. Extended semen was loaded into 0.25 ml French straws, cooled and frozen in liquid nitrogen vapor. Total motility, curvilinear velocity, plasma membrane integrity and fertilizing ability of sperm were assessed after thawing. Total mo- tility was lower (p⟨0.05) in tris-soybean lecithin extender when compared to other extenders. Curvilinear velocity was higher (p⟨0.05) for spermatozoa cryopreserved in milk-soybean lecithin extender compared to other extenders tested. For the percentage of live sperm no significant difference was observed between extenders. The lambing rate were higher (not statistically signifi- cant) in ewes inseminated with semen doses frozen in milk-soybean lecithin extender (42.9%) than in the tris-egg yolk extender (16.7%). In conclusion, replacing the egg yolk with soybean lecithin was effective in milk but not in tris extender.

Apoptotic-like changes of boar spermatozoa in freezing media supplemented with different antioxidants.

This study evaluated the effect of supplementing the freezing extender with exogenous anti-oxidants on apoptotic-like changes in post-thaw boar spermatozoa. A total of 36 ejaculates were resuspended in standard lactose-egg yolk-glycerol extender supplemented with antioxidant to final concentrations of 0 (as control), 2.5mM GSH (group I), 5.0 mM GSH (group II), 150 IU/mL SOD (group III), 300 IU/mL SOD (group IV), 200 IU/mL CAT (group V), 400 IU/mL CAT (group VI), 150 IU/mL SOD+200 IU/mL CAT (group VII), 300 IU/mL SOD+400 IU/mL CAT (group VIII). Sperm motility and apoptotic-like changes were determined before and after freeze-thawing. The various markers of apoptotic-like changes were measured: plasma membrane permeability by YO-PRO-1/PI assay, phosphatidylserine (PS) translocation across the plasma membrane using fluorescein-labeled Annexin-V, mitochondrial transmembrane potential detected by JC-1, and DNA fragmentation evaluated by TUNEL assay. The highest percentage of progressive motile sperm was noticed in group II (PM% 64.2±15.4) compared with control (PM% 36.8±5.5). The supplementation of 400 IU/mL CAT (group VI) revealed significant (P<0.01) reduction of apoptotic-like changes (YO-PRO-1+/PI-: 13.1±7.5%, AnV+/PI-: 9.9±4.1%) in frozen-thawed spermatozoa compared with extender supplemented with 200 IU/mL CAT (group V). Irrespective of the concentration used, SOD and CAT in combination (group VII and group VIII) significantly (P<0.01) improved post-thaw sperm survival compared with the control. Evaluation by TUNEL assay revealed that cryopreservation and thawing did not induce DNA fragmentation in boar spermatozoa.

Motility, mitochondrial membrane potential and ATP content of rabbit spermatozoa stored in extender supplemented with GnRH analogue [des-Gly10, D-Ala6]-LH-RH ethylamide.

The present study was aimed to determine the effect of GnRH analogue [des-Gly10, D-Ala6]-LH-RH ethylamide on the quality of rabbit spermatozoa stored at 17°C for 3 days. Semen from 5 bucks (13 ejaculates) was used in the experiment. Ejaculates were divided and diluted at a 1:10 ratio with rabbit semen extender Galap (IMV, France) (Control) or with Galap extender supplemented with GnRH analogue [des-Gly10, D-Ala6]-LH-RH ethylamide (50 µg/ml) and stored for 3 days. Sperm motility parameters, mitochondrial membrane potential (MMP) and ATP content were as- sessed on each day of the experiment. Motility analysis was performed using a computer-assisted sperm analysis (CASA) system. The following sperm motility parameters were recorded: total motile spermatozoa, progressively motile spermatozoa, curvilinear velocity, straight-line velocity, average path velocity, linearity, straightness and amplitude of lateral head displacement. MMP was evaluated using JC-1 fluorescent dye. ATP content was assessed using a bioluminescence method. The addition of GnRH analogue [des-Gly10, D-Ala6]-LH-RH ethylamide to Galap extender did not affect any of the quality parameters studied. However, in both groups (Control and GnRH), significant changes in motility parameters (except straight-line velocity) and proportion of spermatozoa showing high MMP and ATP content were observed throughout 3 days of storage.

Apoptotic-like changes in the spermatozoa of fresh and stored boar semen and the quality of embryos produced in vivo.

The aim of this study was to determine the apoptotic-like changes in the spermatozoa of fresh and stored boar semen and to investigate the relationship between this phenomenon and the quality of embryos produced in vivo. The experiments were divided into two series. In the first series, ten ejaculates were collected from five boars, which were crossbreeds of the Polish Landrace and Large White breeds. The semen was stored as a liquid until Day A (the day on which sperm motility decreased to 30%). Three fluorescence methods were used to evaluate semen quality: an assay to assess the early changes in sperm membrane integrity using the fluorophore YO-PRO-1, an assay for phosphatidylserine (PS) translocation across the plasma membrane using fluorescein-labeled annexin-V and the mitochondrial-specific probe JC-1 (5,5',6,6'-tetrachloro-1,1',3,3' tetraethylbenzimidazolylcarbocyanine iodide) for measuring changes in mitochondrial membrane potential. Our results showed that liquid preservation of boar semen causes apoptotic-like changes in the sperm, and a significant increase in both: apoptotic sperm (YO-PRO-1(+)/PI(-)) and early apoptotic sperm (annexin-V(+)/PI(-)) were observed between Day 0 (fresh semen) and Day A only in semen from three of the five boars. In the second series of experiments, the semen from boar nos. 1, 2, and 3 was selected for insemination of superovulated gilts. The fertilizing capacity of fresh and stored semen with different levels of apoptotic spermatozoa was measured based on the morphology and the number of cells of embryos that were obtained after insemination with this semen. Our studies indicated no significant differences in the fertilization rate of gilts after insemination with fresh and stored semen with increased levels of apoptotic spermatozoa. After insemination with stored semen, a significantly greater number of degenerated embryos were observed, but the morphologically normal blastocysts obtained after insemination with either fresh or stored semen had a similar number of nuclei.

Effect of semen quality in transgenic boars on the developmental competence of preimplantation embryos.

The aim of this study was to compare the fertilising capacity of sperm from 6 transgenic (TG) and 6 non-transgenic (NTG) boars based on analyses of embryos resulting from insemination with sperm from these particular boars. Expanded blastocysts were collected from five groups of synchronised gilts (six gilts per group) inseminated by TG boars bearing a gene construct containing the human alpha1,2-fucosyltransferase gene and by NTG boars. The ejaculates used for insemination were analysed to detect apoptotic changes using two fluorescence methods: an assay to assess early changes in the membrane integrity of the sperm using the YO-PRO-1 fluorophore and an assay for phosphatidylserine (PS) translocation across the plasma membranes using fluorescein-labelled Annexin-V. Our results, using a combination of YO-PRO-1 and PI fluorophores, revealed no significant differences in the percentage of sperm subpopulations between non-transgenic and transgenic boars (P<0.01). Moreover, the second fluorescent probe also revealed no significant differences between the average values of live (Ann-V(-)/PI(-)), early apoptotic (Ann-V(+)/PI(-)), and late apoptotic/early necrotic sperm (Ann-V(+)/PI(+)) as calculated for TG and NTG boars. Only the percentage of necrotic sperm (Ann-V(-)/PI(+)) was significantly different (P<0.05) between transgenic and non-transgenic boars (3.4%+/-2.7; 7.2%+/-2.1, respectively). The quality of the preimplantation embryos at the blastocyst stage was determined by counting the number of cells, observing a TUNEL-positive reaction and by caspase-3 labelling. We found that expanded blastocysts that were derived from gilts inseminated with TG and NTG boar semen showed almost no DNA fragmentation (80%) and 70% caspase-3 activity. The expanded blastocysts that were derived from gilts inseminated with TG and NTG boar semen did not differ significantly in their DNA fragmentation, and there were no differences in caspase-3 activity. These results revealed a positive correlation between the percentage of blastocysts with TUNEL-positive nuclei and the percentage of blastocysts with caspase-3 activity (r=0.9787; P<0.0001).

Assessment of plasma membrane and chromatin structure of sperm from transgenic and non-transgenic boars.

The aim of this study was to determine the apoptotic changes and chromatin damage in non-transgenic and transgenic boars carrying the human alpha1,2-fucosyltransferase gene. Five ejaculates were collected from six transgenic (TG) and six non-transgenic (NTG) boars. Five ejaculates were collected from six transgenic (TG) and six non-transgenic (NTG) boars both crossbreds of Polish Landrace and Large White. Two fluorescence methods were employed to measure apoptosis: an assay to assess the early changes in sperm membrane integrity using fluorophore YO-PRO-1 and an assay for phosphatidylserine (PS) translocation across the plasma membrane using fluorescein-labeled Annexin-V. The chromatin damage was assessed based on the sperm chromatin structure assay method. No significant differences in the proportion of all detected subpopulations of spermatozoa were found between TG and NTG boars. Similarly, the analysis of the chromatin structure revealed no statistical differences in the sperm chromatin damage between TG and NTG boars. In conclusion, the presence of the human alpha1,2-fucosyltransferase gene in the genome of TG boars did not cause any spermatogenesis process disturbances leading to the increased production of apoptotic spermatozoa. Moreover, the low level of sperm with damaged chromatin in TG boars confirms the high stability of the spermatogenesis process in the TG boars analyzed.

CGP 44532, a GABAB receptor agonist, is hedonically neutral and reduces cocaine-induced enhancement of reward.

Drugs that alter the function of gamma-aminobutyric acid (GABA) neurotransmission seem to reduce cocaine reinforcement, and as such may be useful in pharmacologically treating cocaine addiction. In the present experiment, the anti-cocaine effects of CGP 44532, a phosphinic acid analogue of GABA, and a highly selective GABA(B) receptor agonist were examined in male Sprague-Dawley rats using brain stimulation reward (BSR) paradigm. In this method, the relationship between the rate of bar pressing and the frequency of stimulation pulses was analyzed in two measures: the maximum rate of responding (MAX) and the frequency necessary to sustain half maximal rate of responding known as the locus of rise (LOR). CGP 44532 was found to be hedonically neutral without producing any measurable effects on performance (MAX). It also dose-dependently reduced cocaine-induced BSR enhancement, in the order of 15-31%, as shown by progressive shifts in LOR towards baseline. Thus, in theory, administration of CGP 44532 might reduce cocaine's hedonic effects, while also maintaining patient compliance. Whether this agent would also be effective at curbing craving, a long-term consequence of drug abuse, remains to be determined.

Effects of repeated GBR 12909 administration on brain stimulation reward.

Male rats were trained at three separate currents to bar press for intracranial self-stimulation. On days 1 and 15, all subjects were given 1-(2-bis(4-fluorophenyl)-methoxy)-ethyl-4-(3-phenylpropyl) piperazine, also known as GBR 12909 (10 mg/kg, i.p.), prior to test session. Between these days, the paired Chronic-before group was injected (every other day) with GBR 12909 prior to intracranial self-stimulation, while unpaired, Chronic-after group was given the drug just after the end of the session. A third group (Control) received saline injections (i.p.) 20 min following the session. Although GBR 12909 was found to be reward enhancing, neither sensitization nor tolerance developed to the rewarding and performance/motor effects regardless of the injection regimen. In addition, the rewarding effects of intracranial self-stimulation were found to be independent of both current and environment-specific pairing. The present data obtained for GBR 12909 agree with previous observations of the effects of repeated administration of drugs of abuse on intracranial self-stimulation.

Repeated exposure to rewarding brain stimulation downregulates GluR1 expression in the ventral tegmental area.

There is considerable evidence that drug reward and brain stimulation reward (BSR) share common neural substrates. Although it is known that exposure to drugs of abuse causes a variety of molecular changes in brain reward systems, little is known about the molecular consequences of BSR. We report that repeated exposure to rewarding stimulation of the medial forebrain bundle (MFB) selectively decreases expression of GluR1 (an AMPA receptor subunit) in the VTA, without effect on expression of several other proteins (GluR2, NMDAR1, tyrosine hydroxylase). This effect of BSR on GluR1 expression is opposite of that caused by intermittent exposure to cocaine and morphine, which are known to elevate GluR1 expression in the VTA. Considering that elevated GluR1 expression in the VTA has been associated with increased sensitivity to drug reward, the finding that BSR and drugs of abuse have opposite effects on GluR1 expression in this region may provide an explanation for why the reward-related effects of many drugs (cocaine, morphine, amphetamine, PCP, nicotine) do not sensitize with repeated testing in BSR procedures that quantify reward strength.

Novel monoamine transporter ligands reduce cocaine-induced enhancement of brain stimulation reward.

Six novel monoamine reuptake inhibitors were screened for their intrinsic effects on brain stimulation reward (BSR), as well as for their potential to reduce cocaine-induced reward-enhancement in that paradigm. Two of the compounds, nocaine-3B and 5-ara-74A (disubstituted piperidines) significantly reduced locus of rise (LOR), threshold measure of reward, at some doses. One compound, 1-RV-96A (a hybrid of the GBR and WIN-like agents) significantly reduced reward (increased LOR), but only at the highest dose tested. No effect of dose was found for MC9-20 (a GBR-like acyclic analogue of the N-bisarylmethoxyethyl-N'-phenylpropyl piperazine), nocaine-250B or 4-ara-42C (disubstituted piperidines). When cocaine (10 mg/kg, ip) and selected, hedonically neutral doses of novel compounds were combined, the following findings were obtained: MC9-20 (2.5 mg/kg, ip) showed a significant increase in cocaine-induced reward enhancement (0.2 log units or 53%). In contrast, nocaine-250B and 1-RV-96A (both at 10 mg/kg, ip) demonstrated a significant reduction (0.13 log units or 41%) in cocaine-induced reward enhancement (P<.01 and P<.05, respectively), as measured by changes in LOR. There were no differences in the maximum behavioral output (MAX) at either dose of each of the six drugs, or when selected doses were combined with cocaine. These results indicate that nocaine-250B and 1-RV-96A constitute two potential anticocaine compounds worthy of further behavioral and biochemical evaluation.

Prenatally protein-malnourished rats are less sensitive to the amnestic effects of medial septal infusions of chlordiazepoxide.

Evidence is mounting that prenatal protein malnutrition affects the physiological properties of the GABAergic neurotransmitter system in rats. To investigate the functional behavioral consequences of these changes, chlordiazepoxide (CDP, a positive modulator of the GABA(A) receptor) was applied directly to the medial septum and the amnestic response appraised. In adulthood, male offspring of rats provided with a protein-deficient diet (6% casein) for 5 weeks prior to mating and throughout pregnancy underwent stereotaxic surgery to implant steel cannulae aimed at the medial septum. After recovery, spatial learning performance in the submerged platform version of the Morris water maze task was assessed immediately following a 1 microl infusion of either artificial cerebrospinal fluid (aCSF), or one of three doses of CDP (15, 30 and 60 nmol). Well-nourished control rats demonstrated a robust amnestic response to intraseptal CDP. During task acquisition, well-nourished rats administered each of the doses exhibited significantly longer escape latencies than those given aCSF. On the probe trial (platform removed) a lower proportion of time was spent in the target quadrant (all three doses) at a greater average distance from the former platform location (30 and 60 nmol doses). In contrast, prenatally malnourished rats exhibited a muted sensitivity to CDP, most notable at the 30 nmol dose. These findings provide further support for functional changes within the GABAergic system consequent to malnutrition.

An enantioselective synthesis and biobehavioral evaluation of 7-fluoro-3-(p-fluorophenyl)-2-propyltropanes.

Optically pure 7-fluorotropanes 3a-c, were synthesized as structural probes of the dopamine transporter. The synthesis of these compounds was accomplished through the asymmetric 1,3-dipolar cycloaddition reaction of the oxidopyridinium betaine 4 with the chiral dipolarophile (R)-p-tolyl vinyl sulfoxide. In the preliminary analysis, tropane 3a was found to reduce the rewarding effects of cocaine in the brain stimulation reward paradigm.

Dopaminergic agents for the treatment of cocaine abuse.

Cocaine is a major drug of abuse whose devastating effects have captured the attention of health officials and policy makers. Based upon the alarming health and crime-related costs associated with the use of this powerful reinforcing drug, immediate therapies are needed for the treatment of cocaine addiction. In this review, some of the small-molecule-based approaches that have been pursued in the search for such medications are highlighted. Because the pharmacological actions of cocaine stem laargely from its ability to block the dopamine transporter, many intervention strategies have focused on the dopaminergic pathway.

Activation of reward-relevant neurons in the caudate-putamen influences the development of medial prefrontal cortex self-stimulation: a moveable electrode mapping study.

Two hundred fifty five medial prefrontal cortical (MPFC) and 187 caudate-putamen (CPu) sites were evaluated for intracranial self-stimulation in 67 animals using moveable electrodes and collecting trade-off functions between current and frequency. Eleven percent of the examined areas, located predominantly in the ventromedial aspects of MPFC and CPu, showed reliable self-stimulation and the average charge of 1.12 and 1.11 microC respectively, values that are in line with those reported for the Medial forebrain bundle. The distribution of charge, however, was greater than reported for the latter region, and ranged between 0.68 to 1.63 microC across sites. Some subjects were implanted with two electrodes, one aimed at the MPFC, and the other at the CPu, ventral tegmental area, or lateral hypothalamus. Only animals with CPu placements showed transference of self-stimulation to the MPFC, suggesting that these two regions might form part of the same reward substrate, a view that has anatomical, electrophysiological and recently behavioral support.

Functional connections between medial prefrontal cortex and caudate-putamen in brain-stimulation reward of rats.

Rats were trained to self-stimulate for trains of cathodal pulses delivered via electrodes placed in the medial prefrontal cortex (MPFC) and caudate-putamen (CPu). When the pulses were delivered via 9 ipsilateral MPFC and CPu sites alternately, summation varied from 13% to 40%. However, the overall summation for 2 contralateral MPFC-CPu pairs was 5%, thus indicating a greater integration of ipsilateral than contralateral reward signals. When the interval between alternate pulses decreased in 4 of the 9 ipsilateral pairs, the summation also decreased, an outcome consistent with collision of action potentials passing between the MPFC and CPu sites. The size of the collision effect ranged from 15% to 33%. Estimates of conduction velocity varied between 0.4 to 5.4 m/s, with a 1.7 m/s average. According to these values, the neurons connecting the MPFC and CPu self-stimulation sites appear to be slower than the ones that have been shown to link reward fibers that course between posterior brain regions.

Prenatal malnutrition-induced changes in blood pressure: dissociation of stress and nonstress responses using radiotelemetry.

A link between prenatal malnutrition and hypertension in human populations has recently been proposed. Rat models of prenatal malnutrition have provided major support for this theory on the basis of tail-cuff measurements. However, this technique requires restraint and elevated temperature, both potential sources of stress. To determine the effect of prenatal protein malnutrition on blood pressure under nonstress conditions, 24-hour radiotelemetric measurements were taken in the home cage. Male rats born to dams fed a 6% casein diet for 5 weeks before mating and throughout pregnancy were studied in early adulthood (from 96 days of age). During the waking phase of their cycle but not the sleep phase, prenatal malnutrition gave rise to small but significant elevations of diastolic blood pressure and heart rate compared with well-nourished controls. Direct effects of stress on blood pressure responses were determined in a second experiment using an olfactory stressor. Prenatally malnourished rats showed a greater increase in both systolic and diastolic pressures compared with well-nourished controls during the first exposure to ammonia. A different pattern of change of cardiovascular responses was also observed during subsequent presentations of the stressor. These findings of a small baseline increase in diastolic pressure consequent to prenatal malnutrition, but an augmented elevation of both systolic and diastolic pressures after first exposure to stress, suggest the need to reevaluate interpretation of the large elevations in blood pressure previously observed in malnourished animals using the stressful tail-cuff procedure.

Characteristics of stimulation-induced feeding sites in the sulcal prefrontal cortex.

Double pulse tests were used to infer the refractory periods of the substrate underlying stimulation-induced feeding in the sulcal prefrontal cortex of the rat. Eleven sites were examined, of which five supported the behaviour at currents of 250 to 400 microA; pulse duration was 100 microseconds. The average profile indicates a recovery function that begins at 0.5 ms and ends at 3.0 ms, with no apparent contribution from local potential summations. The mean effectiveness value corresponding to the asymptotic portion of the curves was 91%. These results suggest that there is substantial overlap in the excitability of neurons underlying stimulation-induced feeding in the sulcal prefrontal cortex and that reported for the medial forebrain bundle, but unlike the latter structure, there is no evidence of self-stimulation from the same sulcal prefrontal cortex placements. Response rates were collected for a maximum of 21 days from each of the cortical sites at which feeding was recorded and at the same currents used to evaluate the refractory periods underlying stimulation-induced feeding. The rates averaged between 0 and 3 responses per minute whether the current was available or not. These data represent the first demonstration of a site that supports stimulation-induced feeding in the absence of brain stimulation reward, at least at these specific placements and stimulation parameters.

Behaviourally derived estimates of excitability in striatal and medial prefrontal cortical self-stimulation sites.

The refractory periods of the substrate underlying brain-stimulation reward were investigated in three rats with moveable electrodes implanted in the rostral caudate-putamen and the medial prefrontal cortex. Acquisition of caudate-putamen self-stimulation occurred within the first session, while self-stimulation for medial prefrontal cortex was observed only after three sessions of caudate-putamen stimulation. The currents required for self-stimulation ranged from 300 to 800 microA (0.1 ms pulse duration) across animals; the maximum response rates averaged roughly 40 bar presses per minute for both structures. Refractory period estimates were obtained from ten caudate-putamen and four medial prefrontal cortex sites. The time course of recovery had the following profile: the curves began to rise at 0.65 ms and 0.95 ms for caudate-putamen and medial prefrontal cortex stimulation, respectively, thereafter increasing to approach an asymptote at 6.00 ms for the caudate-putamen and 6.25 ms for the medial prefrontal cortex. The mean effectiveness value corresponding to the asymptotic portion of the curves was 73% for the caudate-putamen and 69% for the medial prefrontal cortex. Like other forebrain structures, the behaviourally derived refractory periods underlying caudate-putamen and medial prefrontal cortex stimulation, at least at these particular sites, are significantly longer than those observed in most medial forebrain bundle areas, both beginning and ending later. One interpretation for the similarity in their refractory period profiles and the apparent facilitating effect of caudate-putamen stimulation on acquisition of medial prefrontal cortex self-stimulation is that these two regions form part of the same reward substrate.

Total thyroidectomy in the treatment of Basedow's ophthalmopathy.

Four patients in the age 42-63 (3 females and 1 male) with Basedow's disease and hyperthyroidism were investigated. Visual changes were classified to the IV group of American Thyreologic Society's classification. A total thyroidectomy was performed when status of euthyreosis has been obtained. Check examinations were carried out 6, 12 and 18 months after surgery. It has been confirmed that total thyroidectomy performed in patients with thyroid ophthalmopathy leads to reduction of infiltrative changes in the orbit. However, this can be stated not earlier than 12 months after surgery.