Noradrenergic substrates sensing light within brainstem reticular formation as targets for light-induced behavioral and cardiovascular plasticity.

The occurrence of pure light exerts a variety of effects in the human body, which span from behavioral alterations, such as light-driven automatic motor activity, cognition and mood to more archaic vegetative functions, which encompass most organs of the body with remarkable effects on the cardiovascular system. Although empirical evidence clearly indicates occurrence of these widespread effects, the anatomical correlates and long-lasting changes within putatively specific neuronal circuitries remain largely unexplored. A specific role is supposed to take place for catecholamine containing neurons in the core of the brainstem reticular formation, which produces a widespread release of noradrenaline in the forebrain while controlling the vegetative nervous system. An indirect as well as a direct (mono-synaptic) retino-brainstem pathway is hypothesized to rise from a subtype of intrinsically photosensitive retinal ganglion cells (iPRGCs), subtype M1, which do stain for Brn3b, and project to the pre-tectal region (including the olivary pre-tectal nucleus). This pathway provides profuse axon collaterals, which spread to the periacqueductal gray and dorsal raphe nuclei. According to this evidence, a retino-reticular monosynaptic system occurs, which powerfully modulate the noradrenergic hub of reticular nuclei in the lateral column of the brainstem reticular formation. These nuclei, which are evidenced in the present study, provide the anatomical basis to induce behavioral and cardiovascular modulation. The occurrence of a highly interconnected network within these nuclei is responsible for light driven plastic effects, which may alter persistently behavior and vegetative functions as the consequence of long-lasting alterations in the environmental light stimulation of the retina. These changes, which occur within the core of an archaic circuitry such as the noradrenaline-containing neurons of the reticular formation, recapitulate, within the CNS, ancestral effects of light-driven changes, which can be detected already within the retina itself at the level of multipotent photic cells.

The inhibition of spermatic cystine/glutamate antiporter xCT (SLC7A11) influences the ability of cryopreserved stallion sperm to bind to heterologous zonae pellucidae.

Sperm are redox-regulated cells, and deregulation of their redox status is considered to affect male fertility and to reduce their fertilizing ability following biotechnological procedures, such as cryopreservation. Cystine (CysS), after incorporation in sperm via SLC7A11 antiporter, has been demonstrated to increase intracellular GSH content, the most important non enzymatic antioxidant. This study was aimed at investigating the role of SLC7A11 antiporter on frozen-thawed stallion sperm ability to respond to in vitro capacitating environment after post-thaw incubation with CysS and/or Sulfasalazine (SS), a specific inhibitor of SLC7A11 antiporter. Viability, motility, immunolocalization of tyrosine phosphorylated proteins and the ability to bind to heterologous zonae pellucidae were evaluated. Thawed sperm from seven stallions (2 ejaculates/stallion) was washed and resuspended in Tyrodes media; each thawed ejaculate was divided in Control (CTR) and 3 samples supplemented with: 0.5 mM Cystine (CysS), 500 µM Sulfasalazine (SS) and 0.5 mM CysS + 500 µM SS (CysS + SS). After 1 h of incubation at 37 °C, samples were washed twice, resuspended in capacitating BWW medium and incubated at 38 °C under 5% CO2. After 30 and 60 min, sperm motility, viability and tyrosine phosphorylated protein immunolocalization, used as capacitation status index, were evaluated. After 30 min of capacitation, 4 × 105 sperm were co-incubated with denuded pig oocytes in capacitation medium for 30 min for the heterologous binding assay. None of the sperm parameters studied (motility, viability and tyrosine phosphorylation) showed any difference respective to control. The number of sperm bound per oocyte (mean ± SEM) tended to increase in CysS group (44.0 ± 12.3) respect CTR (40.8 ± 10.8) while decreased in SS group (32.4 ± 7.8) (p < 0.01). Moreover, CysS + SS group showed a lower binding rate (32.0 ± 10.0) compared to CysS (p < 0.001). Our results suggest that CysS supplementation of thawed stallion sperm can influence their ability to bind to heterologous zona pellucidae as the inhibition of CysS incorporation by SLC7A11 reduced the number of sperm bound per oocyte. This effect does not seem to be ascribed to a modification of sperm motility, membrane integrity and tyrosine phosphorylation.

Step by step procedure for stereological counts of catecholamine neurons in the mouse brainstem.

This work represents a detailed methodological description of automated stereology dedicated to all brainstem catecholamine nuclei. Each tyrosine-hydroxylase-containing nucleus was analyzed to count the following features: i) nuclear volume; ii) neuron number per nucleus; iii) neuron area per each nucleus.A number of reports described catecholamine-containing neurons within brainstem of a variety of animal species. In a recently published work, we reported a simultaneous quantitative analysis of tyrosine hydroxylase-positive neurons in the whole brainstem. Here we report the detailed step by step stereological procedure which allowed to perform a morphometric assessment of each catecholamine nucleus. This protocol provides the method chance to analyze simultaneously various morphological features in the same experimental setting to avoid variability when single nuclei are analyzed in different experiments. This improves the reliability of comparisons between brainstem catecholamine nuclei within the reticular formation to increase our insight about the key functional roles played by these cells in the mammalian brain. In fact, despite being a discrete number of neurons scattered in a small brain area, these cells provide remarkable axonal collateralization which allows the modulation of neuronal activity in the entire CNS. The step by step description of brainstem stereology provided here is reported in order to share these methods and enhance quantitative studies about these fascinating nuclei. At the same time we aim to provide a tool to be used routinely when analyzing the morphology and physiology of brainstem catecholamine cells.

Long-term efficacy of imepitoin in the treatment of naive dogs affected by idiopathic epilepsy.

The purpose of this study was to evaluate the long-term (12 months) efficacy and tolerability of imepitoin as first-choice treatment in 56 dogs suffering from idiopathic epilepsy and identify possible factors affecting the outcome. Primary treatment success (PTS) was defined as the achievement of a seizure-free interval three times longer than the pretreatment interictal interval (at least three months). Secondary treatment success (STS) was achieved by a decrease in seizure frequency ≥50 per cent compared with the pretreatment frequency. In the long-term follow-up, PTS was recorded in 14 (25 per cent) dogs and responder-dogs (PTS+STS) were 30 (54 per cent) showing significant reduction in the monthly average number of seizures (P<0.001). Median seizure frequency per month was 1.69 pretreatment and 0.3 at 12-month follow-up. Dogs with cluster seizures were significantly reduced (P=0.02). PTS at three and six months was associated with PTS (P=0.006 and <0.001, respectively) and with the status of responder dogs (P=0.002) at 12-month follow-up. Dogs aged >36 months at the start of imepitoin treatment had a positive association to become responder dogs (P<0.001) and achieve PTS (P=0.004). 16 dogs (29 per cent) discontinued imepitoin due to its inefficacy. The receiver operator curve highlighted ≥19 mg/kg twice a day as the most effective minimal dosage. Mild and transient side effects were observed in 16 dogs (29 per cent).

Pig sperm preincubation and gamete coincubation with glutamate enhance sperm-oocyte binding and in vitro fertilization.

As the taste receptor for monosodium glutamate (umami) is expressed in both murine and human spermatozoa and the presence of α-gustducin and α-transducin, G proteins involved in the umami taste signaling, has been described in boar germ cells, the aim of this study was to evaluate if monosodium glutamate (MSG) would exert any effect on sperm-oocyte binding, in vitro fertilization (IVF) and sperm parameters during in vitro induced capacitation. For sperm-zona pellucida binding assay, boar spermatozoa were preincubated for 1 h and then coincubated for 1 h with denuded in vitro matured oocytes in presence of different concentrations of MSG (0, 0.1, 1, 10 mM). MSG 1 and 10 mM significantly (P < 0.05) increased the mean number of sperm bound to ZP compared with control (12.3 ± 9.0, 17.8 ± 11.3, 17.6 ± 10.8, MSG 0, 1 and 10 mM respectively). For in vitro fertilization trials, both sperm preicubation (1 h) and gamete coincubation (1 h) were performed in presence of different concentrations of MSG (0, 0.1, 1, 10 mM). After 19 h of culture in fresh IVF medium, oocytes were fixed. MSG 1 mM significantly (P < 0.05) increased the penetration rate compared with control (53.7 ± 20.4 vs. 36.8 ± 16.2). The addition of MSG during in vitro induced capacitation of boar spermatozoa did not cause any significant difference, compared with control, on the percentage of viable cells, spermatozoa with intact acrosome and the percentage of spermatozoa displaying tyrosine-phosphorylation of sperm tail proteins. In order to evaluate whether the effect elicited by MSG could be due to glutamate uptake in boar spermatozoa, fertilization trials were performed in presence of either 1 mM MSG or 1 mM MSG + 100 µM DL-threo-beta-hydroxyaspartic acid (THA), a non selective inhibitor of glutamate uptake. A significant increase (P < 0.05) in the penetration rate in both MSG and MSG + THA groups compared to control was recorded (39.8 ± 15.7, 53.7 ± 22.1, 52.2 ± 23.7, Control, MSG and MSG + THA respectively) while no difference in penetration rate between MSG and MSG + THA treatment was observed suggesting that sperm glutamate transporters are not involved in the pathway mediating this effect. Our study demonstrates for the first time that glutamate exerts a positive effect on sperm-oocyte binding and fertilization. Further studies are needed to clarify the mechanism by which glutamate exert his effect.

Resveratrol and Epigallocatechin-3-gallate addition to thawed boar sperm improves in vitro fertilization.

Thawing is one of the most delicate process after semen cryopreservation as spermatozoa pass from a dormant metabolic stage to a sudden awakening in cellular metabolism. The rapid oxygen utilization leads to an overproduction of reactive oxygen species that can damage sperm cells, thus causing a significant decrease of fertilizing potential of frozen-thawed spermatozoa. Resveratrol (Res) is a natural grape-derived phytoalexin and Epigallocatechin-3-gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis); both molecules are known to possess high levels of antioxidant activity. The objective of the present study was to assess the effect of different concentrations of Res (0.5, 1 or 2 mM; Experiment 1) or EGCG (25, 50 or 100 µM; Experiment 2) supplementation to thawing boar semen extender on sperm quality parameters (viability and acrosome integrity) and in vitro fertilization (IVF). Semen after thawing and dilution with three volumes of Beltsville Thawing Solution (BTS), was immediately divided in control group without antioxidants addition (CTR) and either Res or EGCG groups. Sperm viability and acrosome integrity were evaluated in CTR, Res or EGCG groups after 1 h of incubation at 37 °C. The addition of different doses of Res or EGCG to thawing extender for 1 h did not induce any effect on boar sperm viability and acrosome integrity. However, both Res and EGCG treated samples exhibited a significantly higher penetration rate compared with CTR when used for IVF. In particular the treatment with all the EGCG concentrations increased the penetration rate (P < 0.01) while only Res 2 mM induced a significant increase of this parameter (P < 0.01). In addition, EGCG 25 and 50 µM supplementation significantly increased total fertilization efficiency as compared to control (EGCG 25 µM: 40.3 ± 8.2 vs 26.8 ± 9.5, P < 0.05; EGCG 50 µM: 40.4 ± 7.8 vs 26.8 ± 9.5, P < 0.01). The same effect was observed with Res 2 mM (51.0 ± 7.6 vs 29.6 ± 11.3, P < 0.01). In conclusion, our results indicate that the addition of different doses of the two antioxidants to thawed spermatozoa for one hour, even if does not exert any effect on sperm viability and acrosome integrity, efficiently improves in vitro penetration rate. Moreover, both molecules (EGCG 25 and 50 µM and Res 2 mM) significantly increases the total efficiency of fertilization.

Epigallocatechin-3-gallate (EGCG) and green tea polyphenols do not improve stallion semen parameters during cooling at 4°C.

Stallion semen storage for artificial insemination is mainly based on liquid cooled storage. In many stallions this technique maintains sperm quality for an extended period of time (24-72 hr) at 7°C. While this technique is commonly used in the horse industry, there can be a decline in fertility in some stallions, due to an inability of their sperm to tolerate the cool storage process. The aim of the present work was to evaluate the effect of two natural antioxidants (epigallocatechin-3-gallate (EGCG) at 20, 60 and 120 µm and green tea polyphenols, and p at .001, .01 and .1 mg/ml) on some sperm parameters (sperm motility, viability/acrosome integrity and DNA quality) in extended semen immediately after its collection (T0) and after 2, 6, 24 and 48 hr of cool storage. Two ejaculates from three trotter stallions were analysed after 48 hr of storage at 4°C. No beneficial effect on the analysed parameters was observed: the two antioxidants were not able to improve sperm quality after 48 hr of storage. These results are in agreement with previous findings on the effect of different antioxidants reported by other researches, who have demonstrated that stallion semen keeps good antioxidant capacity after dilution for 24 hr. In conclusion, the positive effect exerted by antioxidant molecules in other species is not confirmed in the equine one.

Quantitative 3D investigation of Neuronal network in mouse spinal cord model.

The investigation of the neuronal network in mouse spinal cord models represents the basis for the research on neurodegenerative diseases. In this framework, the quantitative analysis of the single elements in different districts is a crucial task. However, conventional 3D imaging techniques do not have enough spatial resolution and contrast to allow for a quantitative investigation of the neuronal network. Exploiting the high coherence and the high flux of synchrotron sources, X-ray Phase-Contrast multiscale-Tomography allows for the 3D investigation of the neuronal microanatomy without any aggressive sample preparation or sectioning. We investigated healthy-mouse neuronal architecture by imaging the 3D distribution of the neuronal-network with a spatial resolution of 640 nm. The high quality of the obtained images enables a quantitative study of the neuronal structure on a subject-by-subject basis. We developed and applied a spatial statistical analysis on the motor neurons to obtain quantitative information on their 3D arrangement in the healthy-mice spinal cord. Then, we compared the obtained results with a mouse model of multiple sclerosis. Our approach paves the way to the creation of a "database" for the characterization of the neuronal network main features for a comparative investigation of neurodegenerative diseases and therapies.

Overstocking dairy cows during the dry period affects dehydroepiandrosterone and cortisol secretion.

Stressful situations trigger several changes such as the secretion of cortisol and dehydroepiandrosterone (DHEA) from the adrenal cortex, in response to ACTH. The aim of this study was to verify whether overstocking during the dry period (from 21±3 d to the expected calving until calving) affects DHEA and cortisol secretion and behavior in Holstein Friesian cows. Twenty-eight cows were randomly divided into 2 groups (14 animals each), balanced for the number of lactations, body condition score, and expected date of calving. Cows in the far-off phase of the dry period (from 60 to 21 d before the expected calving date) were housed together in a bedded pack. Then, animals from 21±3 d before the expected calving until calving were housed in pens with the same size but under different crowding conditions due to the introduction of heifers (interference animals) into the pen. The control condition (CTR) had 2 animals per pen with 12.0m2 each, whereas the overstocked condition (OS) had 3 interference animals in the same pen with 4.8m2 for each animal. On d -30±3, -21±3, -15±3, -10±3, and -5±3 before and 10, 20, and 30 after calving, blood samples were collected from each cow for the determination of plasma DHEA and cortisol concentrations by RIA. Rumination time (min/d), activity (steps/h), lying time (min/d), and lying bouts (bouts/d) were individually recorded daily. In both groups, DHEA increased before calving and the concentration declined rapidly after parturition. Overstocking significantly increased DHEA concentration compared with the CTR group at d -10 (1.79±0.09 vs. 1.24±0.14 pmol/mL), whereas an increase of cortisol was observed at d -15 (3.64±0.52 vs. 1.64±0.46ng/mL). The OS group showed significantly higher activity (steps/h) compared with the CTR group. Daily lying bouts tended to be higher for the OS group compared with CTR group in the first week of treatment. The overall results of this study documented that overstocking during the dry period was associated with a short-term changes in DHEA and cortisol but these hormonal modifications did not influence cow behavior.

Embelin supplementation of in vitro maturation medium does not influence nuclear and cytoplasmic maturation of pig oocytes.

Oxidative stress caused from in vitro culture contributes to inadequate oocyte maturation which leads to a poor embryo development. Therefore, it is important to protect oocytes and embryos against oxidative stress. This study was aimed at evaluating the effect of Embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone), an antioxidant with various pharmacologic activities, on nuclear and cytoplasmic maturation of pig oocytes as well as on steroidogenesis of cumulus cells (CCs). Another objective was to determine the influence of Embelin on developmental competence of pig oocytes as well as the expression levels of three key genes (Nanog, Sox2 and Oct4) involved in the control of pluripotency in parthenogenetically activated embryos. Embelin (0, 10, 20 and 40 µM) was added during in vitro maturation of cumulus oocyte complexes; media of both the first and the second day of culture were collected and assayed for progesterone and estradiol-17ß. At the end of the maturation period, the oocytes were fixed (to determine nuclear maturation) or partenogenically activated to evaluate cytoplasmic maturation and genes expression. Embelin did not exert any effect on the proportion of MII oocytes, steroidogenesis of CCs, percentage of embryos that developed to blastocyst stage and the number of blastomeres/blastocyst. Moreover, no significant differences of Oct4, Nanog and Sox2 transcripts were detected in blastocyst stage embryos. In conclusion, Embelin did not influence the reproductive parameters assessed, confirming that it is not possible to predict whether the beneficial effect exerted by an antioxidant in a particular tissue could be present also in another one.

Beta-mercaptoethanol supplementation of in vitro maturation medium does not influence nuclear and cytoplasmic maturation of equine oocytes.

In vitro embryo production in the horse is still not as efficient as in other species. Oxidative stress negatively affects oocyte and embryo culture. To attenuate/minimize the oxidative stress, antioxidants such as low-molecular thiol compounds can be added to culture media. Beta-mercaptoethanol (BME) has been shown to improve maturation and embryo development in different species. The aim of this study was to investigate whether the addition to maturation medium of BME at common (0.1 mM) and high (0.7 mM) concentration could improve oocyte maturation also in the horse. Equine oocytes recovered from slaughterhouse ovaries were used. Meiotic configuration after in vitro maturation (IVM) and early embryo production after intracytoplasmic sperm injection (ICSI) were considered as criteria for assessing nuclear and cytoplasmic maturation, respectively. A total of 1,076 oocytes were analysed over two experiments: 848 (control n = 293, BME 0.1 n = 270, BME 0.7 n = 285) were stained with Hoechst 33342 and examined for nuclear stage after 26 hr of IVM, and 228 MII oocytes were fertilized by ICSI (control n = 83, BME 0.1 n = 65, BME 0.7 n = 80). Cleavage rates were determined after 60 hr of culture. Unlike results obtained in other species, the addition of BME did not influence maturation rates (51.9% control vs 55.6% BME 0.1 mM and 55.1% BME 0.7 mM), nor cleavage rates after ICSI (38.6% vs 38.5% and 41.3%, respectively). In conclusion, the addition of BME at 0.1 and 0.7 mM to the maturation medium, in our culture conditions, has no effect on nuclear and cytoplasmic maturation of equine oocytes.

Storage of sexed boar spermatozoa: Limits and perspectives.

Despite the great potential application of sex-sorted spermatozoa in swine, the technology is not practiced in the pig industry because of technical factors and species-specific issues. The susceptibility of boar spermatozoa to stresses induced by the sorting procedure, the relative slowness of the sex-sorting process together with the high sperm numbers required for routine artificial insemination in pig are some of the main factors limiting the commercial application of this technology in pigs. This review briefly describes the damage to spermatozoa during sex sorting, focusing on an additional limiting factor: increased susceptibility of sexed boar spermatozoa to injuries induced by liquid storage and cryopreservation that, in turn, impairs sperm quality leading to unsatisfactory results in vivo. Strategies to extend the lifespan of sex-sorted boar spermatozoa and to improve their fertilizing ability after liquid storage or cryopreservation need to be implemented before this technology can be used in pig farms. In this regard, encapsulation in barium alginate membranes could be a promising technique to optimize the in vivo use of sexed boar spermatozoa, by protecting, targeting, and controlling the release of sperm into the female genital tract.

Epigallocatechin-3-Gallate (EGCG) Reduces Rotenone Effect on Stallion Sperm-Zona Pellucida Heterologous Binding.

Stallion spermatozoa are highly dependent on oxidative phosphorylation for ATP production to achieve normal sperm function and to fuel the motility. The aim of this study was to evaluate the response of equine sperm under capacitating conditions to the inhibition of mitochondrial complex I by rotenone and to test whether epigallocatechin-3-gallate (EGCG), a natural polyphenol component of green tea, could counteract this effect. After 2-h incubation of stallion spermatozoa in modified Tyrode's medium, rotenone (100 nm, 500 nm and 5 µm) and EGCG (10, 20 and 60 µm), alone or in combination, did not induce any significant difference on the percentage of viable cells, live sperm with active mitochondria and spermatozoa with intact acrosome. The inhibition of complex I of mitochondrial respiratory chain of stallion sperm with rotenone exerted a negative effect on heterologous ZP binding ability. EGCG at the concentrations of 10 and 20 µm (but not of 60 µm) induced a significant increase in the number of sperm bound to the ZP compared with that for control. Moreover, when stallion sperm were treated with rotenone 100 nm, the presence of EGCG at all the concentrations tested (10, 20 and 60 µm) significantly increased the number of sperm bound to the ZP up to control levels, suggesting that this green tea polyphenol is able to reduce the toxicity of rotenone.

Sperm Encapsulation from 1985 to Date: Technology Evolution and New Challenges in Swine Reproduction.

In the last 30 years, encapsulation technology has been applied to different species to minimize the loss of spermatozoa after artificial insemination. In particular, the vehiculation of boar sperm cells in barium alginate membrane has proved a valid strategy to reduce the risk of polyspermy and optimize in vivo fertilizing yields. Controlled release of male gametes into the female genital tract has reduced the minimum fertilizing dose of spermatozoa. Notwithstanding these results, encapsulation has not yet reached commercial application, largely due to the additional costs of production. However, encapsulation could be useful in advanced reproductive technology, such as sex sorting, to store sorted boar semen. The controlled release of flow cytometrically sorted spermatozoa could be a promising strategy to reduce the number of cells necessary for each insemination and hence allow the widescale use of sex sorting in this species.

Effect of cushioned or single layer semen centrifugation before sex sorting on frozen stallion semen quality.

The aim of this study was to compare the effect of presorting centrifugation (cushioned [CC] or single-layer colloid [SLC]), with simple dilution (SD), on the quality of sex-sorted stallion semen before and after sorting and after freezing and thawing. Four ejaculates from each of two fertile stallions were collected 1 week apart and evaluated for percent total sperm motility (TM), percent viable acrosome-intact sperm (VAI), and DNA quality (percentage of DNA fragmentation index). Freezing caused, independently from CC and SLC treatments, a significant decrease of TM (P < 0.05) and VAI (P < 0.05) in both unsorted and sorted semen. On the other hand, sorting did not impair TM and VAI and, interestingly, improved DNA quality in all treatments only before freezing (28 vs 13, 28 vs 10, 22 vs 7 in SD, CC, and SLC for unsorted vs sorted groups, respectively; P < 0.05); this positive effect was lost in the same samples after freezing and thawing, suggesting that the freezing process reduces the DNA quality of sex-sorted sperm. Our results suggest that CC and SLC are not able to select those spermatozoa that possess a better ability to withstand sperm processing associated with sperm sorting and freezing.

Expression of α-gustducin and α-transducin, G proteins coupled with taste receptors, in boar sperm.

During the transit in the female genital tract, spermatozoa are exposed to an environment that varies in composition from the vagina to the oviduct. Because G proteins, α-gustducin and α-transducin, are accepted as markers of chemosensitive cells, this study was aimed at assessing whether these proteins are expressed in boar germ cells. Ejaculated sperm extracts were analyzed by Western blot, and indirect immunofluorescence was performed on testis sections, smears of epididymal and ejaculated spermatozoa, sperm cells after in vitro induction of capacitation and acrosome reaction (IVAR), and in sperm cells bound to zona pellucida during IVF. Based on immunoblot results, both G proteins are present in boar sperm. In the testicular tissue sections, α-gustducin and α-transducin positivity was recorded in the germinal cells near the tubular lumen, whereas no positive signal was evident in spermatogonia located in the outer region of the seminiferous tubules. α-Gustducin expression in epididymal and ejaculated spermatozoa was mainly detectable in both the acrosome and the principal piece of the tail, whereas α-transducin was confined to the acrosome and the midpiece. No changes after in vitro induction of capacitation and IVAR were observed, except for the disappearance of acrosomal positivity in reacted spermatozoa. In sperm bound to zona pellucida, the G protein signal was congruent with that observed in IVAR cells. To the best of our knowledge, this is the first description of α-transducin in mammalian sperm and the first description of α-gustducin in boar sperm. Further studies are needed to clarify the possible role of these G proteins in sperm physiology.

Alkaline phosphatase in boar sperm function.

Alkaline phosphatase (AP) catalyses the detachment of phosphate residues from different substrates. Its activity has been demonstrated in seminal plasma and spermatozoa from porcine and other mammalian species; anyway, the role of AP in male reproduction has not been clarified yet and the aim of this study was to determine AP function in boar sperm capacitation and in vitro fertilization (IVF). AP activity was assayed in seminal plasma and in uncapacitated and in vitro capacitated (IVC) spermatozoa; in addition, capacitation was studied in presence of different doses of AP (1.2 and 2.5 IU/mL). The effect of different doses of AP (1.2 and 2.5 IU/mL) on several sperm parameters after IVC (viability, acrosome integrity with FITC-PSA, capacitation status with CTC staining, tyrosine phosphorylation) and on fertilizing ability during IVF were also evaluated. High AP activity was detected in seminal plasma, in particular in sperm-rich fraction; a lower activity was detected in uncapacitated spermatozoa while a significant decrease was evidenced after IVC. Viability was not changed by AP supplementation of the capacitating medium, whereas acrosome integrity and capacitation status were significantly affected by 1.2 and 2.5 doses, with a dose-dependent decrease in acrosome-reacted cells as well as in CTC B pattern displaying cells. As for sperm head protein phosphorylation, a decrease in relative fluorescence was detected in AP 2.5 group, if compared with capacitated one. After IVF, a dose-dependent decrease in penetrated oocytes was recorded, with an increase in monospermic zygote rate. In conclusion, we demonstrated that AP activity decreases under capacitating condition and that addition of AP to spermatozoa during capacitation results in a depression of the capacitating process and IVF. We can infer that AP plays a role in keeping spermatozoa quiescent until they are ejaculated and in modulating the acquisition of the fertilizing ability.

Physical exercise during pregnancy improves object recognition memory in adult offspring.

Exercising during pregnancy has been shown to improve spatial learning and short-term memory, as well as increase brain-derived neurotrophic factor mRNA levels and hippocampal cell survival in juvenile offspring. However, it remains unknown if these effects endure into adulthood. In addition, few studies have considered how maternal exercise can impact cognitive functions that do not rely on the hippocampus. To address these issues, the present study tested the effects of maternal exercise during pregnancy on object recognition memory, which relies on the perirhinal cortex (PER), in adult offspring. Pregnant rats were given access to a running wheel throughout gestation and the adult male offspring were subsequently tested in an object recognition memory task at three different time points, each spaced 2-weeks apart, beginning at 60 days of age. At each time point, offspring from exercising mothers were able to successfully discriminate between novel and familiar objects in that they spent more time exploring the novel object than the familiar object. The offspring of non-exercising mothers were not able to successfully discriminate between objects and spent an equal amount of time with both objects. A subset of rats was euthanized 1h after the final object recognition test to assess c-FOS expression in the PER. The offspring of exercising mothers had more c-FOS expression in the PER than the offspring of non-exercising mothers. By comparison, c-FOS levels in the adjacent auditory cortex did not differ between groups. These results indicate that maternal exercise during pregnancy can improve object recognition memory in adult male offspring and increase c-FOS expression in the PER; suggesting that exercise during the gestational period may enhance brain function of the offspring.