Closed-loop optimal and automatic tuning of pulse amplitude and width in EMG-guided controllable transcranial magnetic stimulation.

This paper proposes an efficient algorithm for automatic and optimal tuning of pulse amplitude and width for sequential parameter estimation (SPE) of the neural membrane time constant and input-output (IO) curve parameters in closed-loop electromyography-guided (EMG-guided) controllable transcranial magnetic stimulation (cTMS). The proposed SPE is performed by administering a train of optimally tuned TMS pulses and updating the estimations until a stopping rule is satisfied or the maximum number of pulses is reached. The pulse amplitude is computed by the Fisher information maximization. The pulse width is chosen by maximizing a normalized depolarization factor, which is defined to separate the optimization and tuning of the pulse amplitude and width. The normalized depolarization factor maximization identifies the critical pulse width, which is an important parameter in the identifiability analysis, without any prior neurophysiological or anatomical knowledge of the neural membrane. The effectiveness of the proposed algorithm is evaluated through simulation. The results confirm satisfactory estimation of the membrane time constant and IO curve parameters for the simulation case. By defining the stopping rule based on the satisfaction of the convergence criterion with tolerance of 0.01 for 5 consecutive times for all parameters, the IO curve parameters are estimated with 52 TMS pulses, with absolute relative estimation errors (AREs) of less than 7%. The membrane time constant is estimated with 0.67% ARE, and the pulse width value tends to the critical pulse width with 0.16% ARE with 52 TMS pulses. The results confirm that the pulse width and amplitude can be tuned optimally and automatically to estimate the membrane time constant and IO curve parameters in real-time with closed-loop EMG-guided cTMS.

A formalism for sequential estimation of neural membrane time constant and input-output curve towards selective and closed-loop transcranial magnetic stimulation.

Objective.To obtain a formalism for real-time concurrent sequential estimation of neural membrane time constant and input-output (IO) curve with transcranial magnetic stimulation (TMS).Approach.First, the neural membrane response and depolarization factor, which leads to motor evoked potentials with TMS are analytically computed and discussed. Then, an integrated model is developed which combines the neural membrane time constant and IO curve. Identifiability of the proposed integrated model is discussed. A condition is derived, which assures estimation of the proposed integrated model. Finally, sequential parameter estimation (SPE) of the neural membrane time constant and IO curve is described through closed-loop optimal sampling and open-loop uniform sampling TMS. Without loss of generality, this paper focuses on a specific case of commercialized TMS pulse shapes. The proposed formalism and SPE method are directly applicable to other pulse shapes.Main results.The results confirm satisfactory estimation of the membrane time constant and IO curve parameters. By defining a stopping rule based on five times consecutive convergence of the estimation parameters with a tolerances of 0.01, the membrane time constant and IO curve parameters are estimated with 82 TMS pulses with absolute relative estimation errors (AREs) of less than 4% with the optimal sampling SPE method. At this point, the uniform sampling SPE method leads to AREs up to 16%. The uniform sampling method does not satisfy the stopping rule due to the large estimation variations.Significance.This paper provides a tool for real-time closed-loop SPE of the neural time constant and IO curve, which can contribute novel insights in TMS studies. SPE of the membrane time constant enables selective stimulation, which can be used for advanced brain research, precision medicine and personalized medicine.

Statistical Model of Motor-Evoked Potentials.

Motor-evoked potentials (MEPs) are widely used for biomarkers and dose individualization in transcranial stimulation. The large variability of MEPs requires sophisticated methods of analysis to extract information fast and correctly. Development and testing of such methods relies on the availability for realistic models of MEP generation, which are presently lacking. This paper presents a statistical model that can simulate long sequences of individualized MEP amplitude data with properties matching experimental observations. The MEP model includes three sources of trial-to-trial variability: excitability fluctuations, variability in the neural and muscular pathways, and physiological and measurement noise. It also generates virtual human subject data from statistics of population variability. All parameters are extracted as statistical distributions from experimental data from the literature. The model exhibits previously described features, such as stimulus-intensity-dependent MEP amplitude distributions, including bimodal ones. The model can generate long sequences of test data for individual subjects with specified parameters or for subjects from a virtual population. The presented MEP model is the most detailed to date and can be used for the development and implementation of dosing and biomarker estimation algorithms for transcranial stimulation.

Comparison of the effects of gelatin, Ringer's solution and a modern hydroxyl ethyl starch solution after coronary artery bypass graft surgery.

OBJECTIVE: The aim of this study was to compare the effect of 6% hydroxyl ethyl starch solution with 4% gelatin and Ringer's solutions on the haemodynamic stability of patients after coronary artery bypass graft (CABG) surgery and immediately after discontinuation of cardiopulmonary bypass (CPB). METHODS: This was a randomised, double-blind clinical trial of 92 patients who were candidates for on-pump CABG. After discontinuation of CPB, all patients were transferred to the intensive care unit (ICU) and divided randomly into three groups. The first group received Ringer's solution, the second group 4% gelatin, and the third 6% hydroxyl ethyl starch (HES) solution (Voluven). Haemodynamic parameters such as heart rate, mean arterial pressure, systolic blood pressure, diastolic blood pressure, central venous pressure, cardiac output and the presence of arrhythmias were documented. RESULTS: The volume needed for maintaining normal blood pressure and central venous pressure in the range of 10-14 mmHg was less in the HES group than in the other groups. The volume was similar however in the gelatin and Ringer's groups in the first 24 hours after surgery. Urinary output in the first four and 24 hours after surgery were significantly higher in the HES group than in the other two groups. Mean creatinine levels were significantly lower in the HES group. CONCLUSION: HES (6%) had a better volume-expanding effect than gelatin (4%) and Ringer's solutions, and its short-term effects on renal function were also better than gelatin and Ringer's solutions.

Identification and cloning of the Cu/Zn superoxide dismutase gene from halophyte plant Aeluropus littoralis.

Reactive oxygen species (ROS) derived from molecular oxygen under biotic and abiotic stress such as salinity which have deleterious effects on cell metabolism. The toxic effect of ROS counteract by enzymatic as well as non-enzymatic antioxidant system. Superoxide dismutase (SOD) has a potential role for elimination of ROS. Halophytes respond to salt stress at different levels and can be a model for increasing salt tolerance in crop plants. Thus salt tolerance gene isolation and cloning of gene as well as subsequent transformation are first step for sensitive crop improvement. Aeluropus littoralis is a halophyte plant from poaceae family can be as a beneficial plant with high potential for creal breeding. There was no report on isolation of SOD gene from A. littoralis and little genomic study of this plant carried out. In this study a novel gene from A. littoralis isolated. This gene amplified by reverse transcription-PCR and cloned in E. coli pTZ57R/T cloning vector. The AlSOD gene sequence contained 456 bp and the deduced transcripts encoding 152 amino acids shared a high homology with those putative CuZnSOD of higher plants like Zea mays and Oryza sativa.

The relative frequency of Mycobacterium tuberculosis and Mycobacterium avium infections in HIV positive patients, Ahvaz, Iran.

OBJECTIVE: To estimate the prevalence of Mycobacterium tuberculosis (M. tuberculosis) and Mycobacterium avium (M. avium) infections in HIV-positive patients suspected to have pulmonary and extrapulmonary mycobacterial co-infection using PCR technique. METHODS: Totally 50 samples comprising sputum, pleural fluid and CSF taken from HIV positive patients suspected to have mycobacterial infection, were processed. The demographic information and results of acid fast staining and culture were recorded for each patient. The PCR for detecting of M. tuberculosis comprised of specific primers targeting IS6110 gene sequence. For detecting of M. avium, PCR with primers that amplifies the mig gene were used. RESULTS: From 50 samples processed, 45 were sputum (90%), 3 pleural fluid (6%) and 2 CSF (4%). In total, 8 (16%) were culture positive, 7 had positive acid fast staining (14 %) and 13 samples (26%) were positive using PCR technique. All the positive samples were sputum and belonged to patients with pulmonary infection. Of these, 9 were positive for M. tuberculosis (69.2%) and 4 were identified as M. avium (30.8%), which 2 out of 13 positive samples showed mixed infections by both mycobacteria. CONCLUSIONS: The PCR shows the highest detection rate (26%) of mycobacteria compared with culture and acid fast staining. The majority of infections were with M. tuberculosis (18%) and this shows the importance of this mycobacterial co-infection in HIV positive patients in the region of study.

Automated sperm head morphology analyzer for open-source software.

Sperm head morphology has been identified as a characteristic that can be used to predict a male's semen quality. In the present study, we have developed an automated sperm head morphology analysis (ASMA) plug-in for open-source ImageJ software (http://rsbweb.nih.gov/ij/). We describe the plug-in's functionality, and confirm its validity for sperm head morphology analysis using fish sperm. Sperm head morphological measurements (length and width) made with the ASMA plug-in did not differ from manual measurements. Using the plug-in to measure sperm head-shaped objects of known size, the associated plug-in error rate was < 0.5%. Brightness and contrast ratios influenced sperm head measurements, suggesting the need for standardized protocols. This plug-in was effective at measuring elliptical (i.e., Atlantic cod) as well as slightly irregular (i.e., Chinook salmon) shaped sperm heads. In conclusion, our ASMA plug-in represents a versatile alternative to costly sperm morphology software.

Roles of osmolality, calcium - Potassium antagonist and calcium in activation and flagellar beating pattern of sturgeon sperm.

The present study shows the roles of osmolality, calcium (Ca(2+))-potassium (K(+)) antagonist and Ca(2+) in sperm activation and flagellar beating of a sturgeon species, sterlet (Acipenser ruthenus). Sperm motility was activated at hypoosmolality relative to seminal plasma and suppressed at 175 mOsmol kg(-1). Sperm activation was totally suppressed by 0.35mM K(+), but Ca(2+) could fully reverse K(+) inhibitory effect at Ca(2+): K(+) ratio of 0.25. Neither EGTA (a chelator of Ca(2+) ions) nor nifedipine (a Ca(2+) channel blocker) prevented sperm activation. But, sperm motility and velocity were significantly decreased by EGTA, nifedipine and an inhibitor for Ca(2+)/calmodulin activated phosphodiesterase (w-7) that suggest role of Ca(2+) signaling after triggering sperm activation through hypoosmolality. Symmetric flagellar beating was also turned to asymmetric after activation in w-7, which is an evidence for modulation of Ca(2+)-binding proteins activity. Sturgeon sperm, similar to salmonids, is immotile in seminal plasma due to high K(+) concentrations, but the mechanism of sperm activation seems to be closer to other fish species where osmolality prohibits sperm activation in seminal plasma. In these species, hypoosmolality is the primary signal for sperm Ca(2+)-dependent signaling of axonemal beating.

The potential inhibitory effect of cuminum cyminum, ziziphora clinopodioides and nigella sativa essential oils on the growth of Aspergillus fumigatus and Aspergillus flavus

The goals of this study were to evaluate the effectiveness of Cuminum cyminum, Ziziphora clinopodioides and Nigella sativa essential oils to inhibit the growth of Aspergillus fumigatus and A. flavus and to evoke ultrastructural changes. The fungi were cultured into RPMI 1640 media in the presence of oils at concentrations of 8, 6, 5, 4, 3, 2, 1.5, 1.25, 1, 0.75 and 0.5 mg/ml in broth microdilution and 2, 1.5, 1 and 0.5 mg/ml in broth macrodilution methods with shaking for 48 h at 28ºC. Conidial and mycelial samples exposed to 0.25, 0.5, 1, 1.5 and 2 mg essential oils/ml for 5 days in 2 percent yeast extract granulated plus 15 percent Saccharose media were processed for transmission electron microscopy (TEM). Based on broth dilution methods, C. cyminum and to a lesser extent Z. clinopodioides oils exhibited the strongest activity against A. fumigatus and A. flavus with MIC90 ranging from 0.25 to 1.5 mg/ml, while the oil from N. sativa exhibited relatively moderate activity against two above fungi with MIC90 ranging from 1.5 to 2 mg/ml. The main changes observed by TEM were in the cell wall, plasma membrane and membranous organelles; in particular, in the nuclei and mitochondria. These modifications in fungal structure were associated with the interference of the essential oils with the enzymes responsible for cell wall synthesis, which disturbed normal growth. Moreover, the essential oils caused high vacuolation of the cytoplasm, detachment of fibrillar layer of cell wall, plasma membrane disruption and disorganization of the nuclear and mitochondrial structures. Aspergillus fumigatus and A. flavus growth inhibition induced by these oils were found to be well-correlated with subsequent morphological changes of the fungi exposed to different fungistatic concentrations of the oils. Our results show the anti-Aspergillus activities of C. cyminum, Z. clinopodioides and N. sativa essential oils, which strengthens the potential use of these substances as anti-mould in the future.

Tuberculous spondylitis: risk factors and clinical/paraclinical aspects in the south west of Iran.

BACKGROUND AND OBJECTIVES: Tuberculous spondylitis (TS) is both the most common and the most dangerous form of TB infection. Delay in diagnosis and management causes spinal cord compression and spinal deformity. The aim of this study was to identify the clinical and paraclinical aspects and also to describe its risk factors in Khuzestan, a province located in the south west of Iran. METHOD: In this medical record-based retrospective study 69 cases of TS registered in Khuzestan Health Center from 1999 to 2008, were reviewed. For each TS case two extra pulmonary TB cases (without spinal involvement) were randomly selected as control. Related data in patients were analyzed in SPSS software (version 16, USA) using chi square and Fishers exact test. Differences with P<0.05 were considered significant. RESULTS: The mean age of patients was 43.7±18.3 years, and 60.8% were males. The mean time of delay: for patients' delay, doctors' delays and from diagnosis to initiation of treatment was 1.8±1.1, 6.8±4.3 and 1.3±1.2 months, respectively. In 56 cases (81.1%), TS was diagnosed on lumbosacral radiograph, in remaining cases by lumbosacral MRI. Twenty patients (30.4%) had a previous TB history, 30.4% had underlying medical disorders such as diabetes mellitus (30%), steroid use (45%), chronic renal failure (50%). The most common clinical findings were: backache 98.5%, fever 26.1%, spinal tenderness 84.1%, paraparesis 26.1%, and kyphosis 28.9%. Laboratory results were: elevated sedimentation rate (ESR) 92.8% and positive CRP 86.9%. There were statistically significant differences in age, gender, CRF, imprisonment and previous TB infection between the two groups. CONCLUSION: TS is therefore a diagnosis that attending doctors need to consider early. Patients in endemic area of TB, presenting with back pain in association with constitutional symptoms and elevated ESR and or positive CRP should be investigated to exclude TS. Older age, male gender, chronic peritoneal dialysis, imprisonment and previous TB infection may be the main risk factors of TS.

Relationships between reproductive characteristics in male Vimba vimba L. and the effects of osmolality on sperm motility.

In Vimba vimba, GSI, sperm volume, and spermatozoa concentration range from 3.4-7.4 %, 0.1-1.1 ml, and 13.3-34.8 x 10(9) spz ml(-1), respectively. Gonad mass (r = 0.90) and sperm volume (r = 0.35) significantly correlated with weight of males. Significant correlation was also found between gonad mass and length of males (r = 0.85). Sperm motility (r = 0.99) and velocity (r = 098) significantly decreased after activation in Tris-HCl 20 mM, pH 8.5. Osmolality of the seminal plasma was 273.2 mOsmol kg(-1). Sperm motility and velocity were significantly affected by the osmolality of the activation medium (P < 0.01). Hyper-osmolality compared to seminal plasma osmolality totally suppressed the sperm activation. At 15 s post-activation, the sperm motility significantly decreased at 240 mOsmol kg(-1) in KCl or NaCl media. The highest sperm motility and velocity (at 60 s post-activation) were observed at 200 mOsmol kg(-1) in NaCl, KCl, or sucrose media. In all treatments, the tip of the flagellum of spermatozoa became curled into a loop shape after activation of sperm in distilled water containing 20 mM Tris-HCl, pH 8.5 that shortened the flagellum.

Relationship between semen characteristics and body size in Barbus barbus L. (Teleostei: Cyprinidae) and effects of ions and osmolality on sperm motility.

The objectives of the present study were to determine the relationships among length and weight of males, sperm volume, spermatozoa concentration, total number of spermatozoa, ionic contents and osmolality of seminal plasma in Barbus barbus. The effect of osmolality on sperm motility parameters after activation in NaCl, KCl, or sucrose solutions was also examined. There were significant correlations between spermatozoa concentration - length (R=+0.7) and - weight (R=+0.8) of males. No significant correlations were observed between the total number of spermatozoa, sperm volume, and length and weight of males. Seminal plasma osmolality was higher when the total number of spermatozoa (R=+0.6) and sperm volume (R=+0.6) were higher. Sperm motility and velocity was positively correlated with osmolality (R=+0.5). The correlation between sperm motility and K(+) was negative (R=0.5), but positively correlated with Ca(2+) (R=0.8), Na(+) (R=0.8), and Cl(-) (R=0.8). There was a rapid decrease (P<0.05) in sperm motility parameters after sperm activation. Just after sperm activation, beating waves propagated along the full length of flagella. At latter stages post sperm activation, the waves appeared only in proximal part of the flagellum. The highest spermatozoa velocity and percentage of motility were observed at 215-235 mOsmol kg(-1) in NaCl, KCl or sucrose. The tip of the flagellum became curled into a loop shape which shortened the flagellum after activation of sperm in distilled water. B. barbus sperm is very similar to that of other cyprinids in terms of ionic contents and osmolality of the seminal plasma, mechanism of sperm activation and behavior and motility of sperm during swimming period.

Effects of osmolality on sperm morphology, motility and flagellar wave parameters in Northern pike (Esox lucius L.).

Northern pike (Esox lucius L.) spermatozoa are uniflagellated cells differentiated into a head without acrosome, a midpiece and a flagellar tail region flanked by a fin structure. Total, flagellar, head and midpiece lengths of spermatozoa were measured and show mean values of 34.5, 32.0, 1.32, 1.17 microm, respectively, with anterior and posterior widths of the midpiece measuring 0.8 and 0.6 microm, respectively. The osmolality of seminal plasma ranged from 228 to 350 mOsmol kg(-1) (average: 283.88+/-33.05). After triggering of sperm motility in very low osmolality medium (distilled water), blebs appeared along the flagellum. At later periods in the motility phase, the tip of the flagellum became curled into a loop shape which resulted in a shortening of the flagellum and a restriction of wave development to the proximal part (close to head). Spermatozoa velocity and percentage of motile spermatozoa decreased rapidly as a function of time postactivation and depended on the osmolality of activation media (P<0.05). In general, the greatest percentage of motile spermatozoa and highest spermatozoa velocity were observed between 125 and 235 mOsmol kg(-1). Osmolality above 375 mOsmol kg(-1) inhibited the motility of spermatozoa. After triggering of sperm motility in activation media, beating waves propagated along the full length of flagella, while waves appeared dampened during later periods in the motility phase, and were absent at the end of the motility phase. By increasing osmolality, the velocity of spermatozoa reached the highest value while wave length, amplitude, number of waves and curvatures also were at their highest values. This study showed that sperm morphology can be used for fish classification. Sperm morphology, in particular, the flagellar part showed several changes during activation in distilled water. Sperm motility of pike is inhibited due to high osmolality in the seminal plasma. Osmolality of activation medium affects the percentage of motile sperm and spermatozoa velocity due to changes in flagellar wave parameters.

Semen of Perca fluviatilis L.: sperm volume and density, seminal plasma indices and effects of dilution ratio, ions and osmolality on sperm motility.

The objectives of the present study were to characterize sperm volume and density, seminal plasma indices (ionic contents and osmolality) and to study the effects of dilution ratio, ions and osmolality on sperm motility parameters (percentage of motile sperm and sperm velocity) in farmed European perch (Perca fluviatilis L.). The means of sperm volume (ml), sperm density (x10(9)spermml(-1)) and total number of sperm (volumexdensity) per fish were 2.75+/-0.51, 29.19+/-3.15 and 82.19+/-15.26. The seminal plasma osmolality (mOsmkg(-1)), sodium, chloride, potassium and calcium ions concentrations (mM) were measured to be 298.07+/-5.09, 130.97+/-2.19, 106.75+/-2.37, 10.70+/-0.61 and 2.41+/-0.09, respectively. At 15s post-activation of stripped sperm, the percentage of motile sperm (%) and sperm velocity (mums(-1)) were 91.90+/-1.27 and 115.54+/-1.25, respectively, and decreased significantly following sperm activation (P<0.05). The optimal sperm motility was observed when the sperm was prediluted in immobilizing solution (IS) at a ratio 1:50. Prediluted sperm showed the maximum velocity when activated in 2.5mM Ca(2+), 50mM K(+) and sucrose with osmolality 100mOsmkg(-1). Neither Ca(2+) nor K(+) showed a significant effect on the percentage of motile sperm at 15s post-activation. Osmolality higher than 200mOsmkg(-1) significantly decreased the percentage of motile sperm, while osmolality of 300mOsmkg(-1) or above totally suppressed sperm motility.

Ultrastructure and morphology of spermatozoa in Chinese sturgeon (Acipenser sinensis Gray 1835) using scanning and transmission electron microscopy.

The Chinese sturgeon (Acipenser sinensis Gray 1835) is an endangered anadromous sturgeon inhabiting the Yangtze River in China. In this study, the ultrastructure and morphology of spermatozoa was studied using transmission and scanning electron microscopy with a cryo-holder. The spermatozoon consisted of an elongated head with a distinct acrosome and nucleus region, a midpiece and a flagellum. The mean length of the head and midpiece, the flagellum and total length of spermatozoon were 4.48, 33.3 and 37.8 microm, respectively. The nucleus was an elongated trapezoid shape with anterior (acrosome) end narrower than the posterior. Granular material and an actin filament were observed within the anterior acrosome. Three to five endonuclear canals were present. The midpiece was eudipleural along its longitudinal axis. Compared to other sturgeon species, the data from the present study suggest a more recent evolutionary linkage between Chinese sturgeon and white sturgeon (Acipenser transmontanus Richardson 1836).

Morphology and ultrastructure of Siberian sturgeon (Acipenser baerii) spermatozoa using scanning and transmission electron microscopy.

BACKGROUND INFORMATION: Available data concerning the sperm morphology of teleost fishes demonstrate wide variation. In the present study, the spermatozoa of Siberian sturgeon (Acipenser baerii Brandt, 1869), a chondrostean fish, was investigated. In contrast with teleost fish, chondrostean spermatozoa have a head with a distinct acrosome, whereas other structures, such as a midpiece and a single flagellum, are present in spermatozoa of most species. RESULTS: The average length of the head including the acrosome and the midpiece was 7.01+/-0.83 microm. Ten posterolateral projections derived from the acrosome were present on a subacrosomal region, with mean lengths of 0.94+/-0.15 microm and widths of 0.93+/-0.11 microm. The nucleus consisted of electrodense homogeneous nuclear chromatin. Three intertwining endonuclear canals, bound by membranes, traversed the nucleus longitudinally from the acrosomal end to the basal nuclear fossa region. There were between three and six mitochondria, two types of centrioles (proximal and distal) in the midpiece and two vacuoles composed of lipid droplets. The flagellum (44.75+/-4.93 microm in length), originating from the centriolar apparatus, had a typical 9+2 eukaryotic flagellar organization. In addition, there was an extracellular cytoplasm canal between the cytoplasmic sheath and the flagellum. CONCLUSIONS: A principal components analysis explained the individual morphological variation fairly well. Of the total accumulated variance, 41.45% was accounted for by parameters related to the head and midpiece of the sperm and the length of the flagellum. Comparing the present study with previous studies of morphology of sturgeon spermatozoa, there were large inter- or intra-specific differences that could be valuable taxonomically.

Cryopreservation of tench, Tinca tinca, sperm: Sperm motility and hatching success of embryos.

The aim of the present study was to elaborate cryopreservation methods for ex situ conservation of tench. Success of cryopreservation was tested during two series of experiments. The first set of experiments studied the effects of two types of cryoprotectants (DMSO and a combination of DMSO with propanediol at ratio 1:1) at concentrations of 8 and 10% and three different equilibration times in two different immobilization solutions (IS) (Kurokura 180 and Kurokura) before freezing (0.0, 2.0 and 4.0h after T(0)). The K4 cooling programme was used to freeze 1ml of cryoextended sperm using 1.8ml cryotubes. Main monitored parameter was hatching rate after using of cryopreserved sperm. The second set of experiments studied the volume effect of 0.5, 1 and 5ml straws and compared these with 1.8ml cryotubes as well as the effect of the cooling programme (K4 and L1). Following the results of the first study, a combination of DMSO and propanediol (ratio 1:1) at concentration of 10% was added to extended sperm in Kurokura 180 IS. Main monitored parameter was hatching rate after using cryopreserved sperm, supplementary parameters were sperm velocity and motility percentage assessed at 10s post-activation. Sperm was collected directly into IS and stored at 4 degrees C for 2.5h. Thereafter were sperm samples pooled, equlibred in IS (first set of experiments) or directly mixed with cryoprotectants (DMSO or a mixture of DMSO with propanediol at ratio 1:1) and transferred to 1.8ml cryotubes or straws (0.5, 1 and 5ml). Then the cryotubes/straws were directly transferred to pre-programmed PLANER Kryo 10 series III and cooled using two different cooling programmes including a slow cooling programme (a) named K4 (from +4 to -9 degrees C at a rate of 4 degrees Cmin(-1) and then from -9 to -80 degrees C at a rate of 11 degrees Cmin(-1)) and a rapid cooling programme (b) named L1 (directly from +4 to -80 degrees C at a rate of 20 degrees Cmin(-1)). Both slow (K4) and rapid (L1) cooled samples were held 6min at -80 degrees C. Finally, samples were transferred into liquid N(2). The frozen spermatozoa were thawed in a water bath (40 degrees C) according to the frozen volume and checked for fertilization and hatching rates. Percentage of sperm motility and sperm velocity were measured using video recorded frames. ANOVA showed a significant influence of frozen and fresh sperm in all treatments. The hatching rates of 33.8% were obtained when sperm was equilibrated for 0h before freezing in IS of Kurokura 180 and frozen with a 10% of mixture 1:1 of DMSO and propanediol into straws of 5ml and cooled using program L1. The velocity of frozen-thawed spermatozoa ranged from 31 to 46microms(-1) and in post-thawed sperm was not significantly different according to frozen sperm volume, but a higher velocity was obtained when sperm was fast frozen using programme L1. A large volume of frozen sperm could reveal the best procedure for freezing, but also for simulating methods of artificial propagation for future practical use of frozen tench sperm at a large scale.

Cyclic Amp and the release of antral gastrin.

The isolated, vagally innervated antral pouches in anesthetized dogs were irrigated with 095% acetylcholine chloride (Ach) or a 0.4 M mixture of non-essential amino acids (AA), or distended at 30 cm H2O pressure. In addition,2-deoxy-D-glucose (2-DG) was administered intravenously. Significant increases in antral venous gastrin, caused by gastrin releasers, were associated with a significant rise in cAMP content in the antral mucosa. Acidification of the antrum with simultaneous administration of 2-DG, and acidified AA abolished gastrin release, and significantly reduced mucosal cAMP. Acidified Ach and distension of the antrum with 0.1 N HC1 supressed gastrin levels without significantly changing the mucosal cAMP content. Since the augmented gastrin release was accompanied by an increased mucosal cAMP, the results suggest that cAMP participates in the action of gastrin secretagogues.