Body mass index and human sperm quality: neither one extreme nor the other.

The aim of the present study was to investigate the still contentious association between body mass index (BMI) and seminal quality. To this end, 4860 male patients (aged 18-65 years; non-smokers and non-drinkers), were classified according to BMI as either underweight (UW; BMI <20kgm-2; n=45), normal weight (NW; BMI 20-24.9kgm-2; n=1330), overweight (OW; BMI 25-29.9kgm-2; n=2493), obese (OB; BMI 30-39.9kgm-2; n=926) or morbidly obese (MOB; BMI ≥40kgm-2; n=57). Conventional semen parameters and seminal concentrations of fructose, citric acid and neutral α-glucosidase (NAG) were evaluated. The four parameters that reflect epididymal maturation were significantly lower in the UW and MOB groups compared with NW, OW and OB groups: sperm concentration, total sperm count (103.3±11.4 and 121.5±20.6 and vs 157.9±3.6, 152.4±2.7 or 142.1±4.3 spermatozoa ejaculate-1 respectively, P<0.05), motility (41.8±2.5 and 42.6±2.6 vs 47.8±0.5, 48.0±0.4 or 46.3±0.6 % of motile spermatozoa respectively, P<0.05) and NAG (45.2±6.6 and 60.1±7.9 vs 71.5±1.9, 64.7±1.3 or 63.1±2.1 mU ejaculate-1 respectively, P<0.05). Moreover, the percentage of morphologically normal spermatozoa was decreased in the MOB group compared with the UW, NW, OW and OB groups (4.8±0.6% vs 6.0±0.8%, 6.9±0.1%, 6.8±0.1 and 6.4±0.2%, respectively; P<0.05). In addition, men in the MOB group had an increased risk (2.3- to 4.9-fold greater) of suffering oligospermia and teratospermia (P<0.05). Both morbid obesity and being underweight have a negative effect on sperm quality, particularly epididymal maturation. These results show the importance of an adequate or normal bodyweight as the natural best option for fertility, with both extremes of the BMI scale as negative prognostic factors.

Reproductive performance and fertility in male and female adult mice chronically treated with hexarelin.

Hexarelin (HEXr), a synthetic ghrelin analogue, has been associated with modifications of reproductive physiology. In previous studies of adult mice, we detected that HEXr induced significantly reduced ovulation rate and significant correlation coefficients between sexual maturation and corporal weight in offspring. In this study, we investigated the effects of chronic HEXr administration on sperm concentration and functional activity, oestrous cyclicity and pregnancy index, in addition to the number of fetuses and its correlation with the number of corpora lutea. Adult Albino swiss mice were injected (sc) daily with HEXr: 100 µgkg(-1) day(-1) (HEXr D1) or 200 µgkg(-1) day(-1) (HEXr D2) for 53 days in males and 30 days in females. We detected a significantly decreased ratio in the number of fetuses per corpora lutea in females treated with HEXr D2 for 30 days before mating and during the first 6 days of pregnancy, in addition to a downward trend in the pregnancy index and percentage of females impregnated by each male treated with both doses of the analogue. Although we did not find any significant effect on additional parameters evaluated in both genders, we propose certain effects of HEXr on the implantation process and/or early development of embryos and over the in vivo reproductive capability of males.

Inhibitory effects of ghrelin on sexual behavior: role of the peptide in the receptivity reduction induced by food restriction in mice.

Ghrelin (Ghr) is a gut/hypothalamus peptide with inhibitory actions on reproductive physiology; however, there are no previous reports of its role on estrous behavior. Under the hypothesis that the increase of plasma Ghr during food restriction (FR) is responsible for receptivity reduction, we intended to evaluate the receptivity percentage of female mice subjected to: exp. 1) acute and chronic FR and Ghr administration (3 nmol/animal/day, s. c.) and exp. 2) the co-administration of a ghrelin antagonist [ant=(d-Lys3)-GHRP-6; 6 nmol/animal/day s. c.]. All females were ovariectomized, primed with steroids, trained, and randomly subjected every week to each one of several protocols, followed by a behavioral test. Experiment 1 (n=8): basal, no treatment; acute FR (aFR), 24-h fasting; chronic FR (cFR), 50% FR for 5 days; acute ghrelin (aGhr), Ghr 30 min before test and chronic ghrelin (cGhr), Ghr for 5 days. Except for cGhr, all treatments significantly decreased the percentage of receptivity (mean±SEM): basal 61.9±6.0, aFR 33.1±8.1, cFR 18.8±7.7, aGhr 45.6±10.6, p<0.05 vs. basal. In exp. 2 (n=11), except for cFR+ant (55.0±6.4) the co-administration of the antagonist reversed the deleterious effects detected in exp. 1: basal 70.9±5.4; aFR+ant 72.3±7.6; aGhr+ant 73.6±4.7. As expected, the administration of vehicle or antagonist alone did not modify receptivity. Besides, we found a significant correlation between percentage of body weight loss and percentage of receptivity reduction (r=0.62, p=0.0004). This is the first study demonstrating that ghrelin is able to inhibit female mice sexual behavior and that is involved, at least in part, in receptivity reduction after food scarcity.

Effects of hexarelin (a ghrelin analogue) on fertilisation and the pre- and postnatal development of mice.

Ghrelin (Ghr) has been associated with reproductive physiology and pre- and postnatal development. The objectives of the present study were to evaluate the effects of hexarelin (HEX; 100 or 200 microg kg(-1) day(-1)), a therapeutic Ghr analogue, on: (1) embryo development 60 h post ovulation, induced pharmacologically, in pregnant mice; (2) the physical, neurobiological and sexual development of offspring of female mice injected with HEX during the first, second or third week of pregnancy or throughout the entire pregnancy; and (3) adult memory acquisition in these offspring. We also evaluated the effects of chronic HEX administration on memory acquisition in adult mice. Treatment of non-pregnant female mice with HEX decreased ovulation rate. However, treatment of pregnant mice with HEX at any time during pregnancy tended to accelerate offspring maturation, regardless of bodyweight. This effect was only significant on neurobiological parameters following treatment during the first week. HEX treatment during the first week and/or throughout the entire pregnancy resulted in impaired memory acquisition in the offspring, with female mice being more susceptible to these effects. Similar results were observed for the effects of chronic HEX treatment on memory acquisition in adult mice. In conclusion, HEX seems to exert differential effects depending on when it is administered. Because HEX has started to be used therapeutically, its deleterious effects on ovulation and memory acquisition must be further evaluated.

Decreased memory for novel object recognition in chronically food-restricted mice is reversed by acute ghrelin administration.

It has been demonstrated, in normal and aged rats and mice, that acute i.c.v. ghrelin (Ghr) administration increases memory retention. In order to evaluate if this treatment, restores memory retention in animals exhibiting impaired memory, in the present work we selected a chronic food restriction mouse model (since undernutrition prejudices higher nervous functions). We employed adult female mice with 28 days of 50% food restriction and evaluated: a) behavioral performance using novel object recognition test for memory, and plus maze for anxiety-like behavior, b) some morphometric parameters as body and hepatic weights and c) plasma Ghr levels. The animals with 50% food restriction showed an increase in plasma Ghr levels and a decrease in morphometric parameters and in the percentage of novel object recognition time. When the peptide was i.c.v. injected in food-restricted animals (0.03, 0.3 or 3.0 nmol/microl), memory increases in relation to food-restricted mice injected with vehicle, reaching a performance similar to controls.

Functional activity of frozen thawed Chinchilla lanigera spermatozoa cryopreserved with glycerol or ethylene glycol.

The cryopreservation of spermatozoa constitutes a valuable tool for the captive breeding management of valuable and/or threatened species. Chinchilla lanigera is a species almost extinct in the wild, and the domestic counterpart has one of the most valuable pelts in the world. The objectives of this study were to: (i) compare the functional activity of post-thawed chinchilla spermatozoa cryopreserved at -196 degrees C either with glycerol (G) or ethylene glycol (EG) as cryoprotectants (1 m final concentration) and (ii) investigate the effects of incubating the gametes for 4 h in the presence or in the absence of the cryoprotectants; evaluations were performed taking into account motility, viability, response to hypo-osmotic shock and acrosome integrity of the cells. Parameters reflecting post-thaw (0 h) sperm functional activity were significantly lower than those of freshly ejaculated gametes. When comparing the cryoprotectant efficiency of G vs EG, neither cryoprotectant agent offered appreciable advantages. After 4 h of incubation, in the presence or absence of the cryoprotectant agent, a rapid and significant decrease was found in all functional parameters and remained at approximately 20-30% motile, viable and viable acrosome intact cells. Viability was significantly lower when the cryoprotectant was removed from the media (possibly due to the centrifugation process). With respect to the maintenance of sperm membrane integrity, only approximately 10% of cells showed membrane resistance to hypo-osmotic conditions after the 4 h incubation period. These results constitute new insights for cryopreservation protocols and the development of assisted reproductive techniques in this species.

Electroejaculation in the Chinchilla (Chinchilla lanigera): effects of anesthesia on seminal characteristics.

Repetitive electroejaculation is probably extremely stressful in conscious animals and could adversely affect fertility. The present study was designed to (a) evaluate the effects of anesthesia (40 mg ketamine/kg body weight, i.m.) on a method of electroejaculation used previously in conscious chinchillas (Chinchilla lanigera), and (b) determine the quality of the electroejaculated semen obtained under anesthesia. In Experiment 1 (8 animals), a 4 x 4 Latin square design was used to study the effects of anesthesia and ejaculatory voltage on semen collection, ejaculate volume, sperm concentration, motility, viability, response to the hypoosmotic swelling test (HOST), and acrosomal status. In Experiment 2 (12 animals), the effects of differing voltages and the number of stimuli on ejaculation by conscious or anesthetized males was determined. In both experiments, all the conscious animals ejaculated, but only 60% ejaculated under anesthesia and they required more stimuli and higher voltages to achieve ejaculation. Ejaculate volume was significantly lower in anesthetized (<5 microl) than in conscious animals (>40 microl), but sperm concentration was unaffected. None of the indices of sperm quality were affected by anesthesia. The techniques we have developed in anesthetized domestic chinchillas should be applicable to endangered chinchillas in the wild, and though the number of sperm available is reduced, there are still sufficient for assisted reproduction.

Effects of vasoactive agents on oxygen uptake in portal venous smooth muscle.

The studies concerned the effects of epinephrine, norepinephrine, phenylephrine, acetylcholine and K+ ions, in doses inducing maximal contraction on oxygen consumption of isolated rat portal veins. All the agonists tested stimulated the rate of O2 uptake, whereas high K+ did not. After specific blockade of alpha-adrenoceptors, the effects of epinephrine and norepinephrine on O2 consumption were not affected, whilst those of phenylephrine were abolished, and beta-adrenoceptor blockade suppressed the metabolic effect of epinephrine and norepinephrine. Acetylcholine was the agonist that most elevated O2 consumption; atropine blocked both contractile and metabolic effects of this drug. It appears that there is a clear dissociation between contractile and metabolic actions of some pharmacological agents, and a question is posed concerning phenylephrine and acetylcholine effects. Results with K+ suggest that different metabolic pathways might be involved in the activation and maintenance of drug-induced contraction in smooth muscle.

Activity of Chinchilla laniger spermatozoa collected by electroejaculation and cryopreserved.

Because reproductive studies and the application of assisted reproductive techniques are relevant issues for an endangered species such as Chinchila laniger, the availability of a source of viable spermatozoa becomes of utmost importance. In this paper, we evaluate several functional parameters (motility, viability, response to hypoosmotic swelling test and acrosomal integrity) of fresh or frozen-thawed spermatozoa. Electro-ejaculation trials (50-cyc/sec sinusoidal wave was applied for 5 of every 10 sec) were successful in all unanesthetized animals. After volume (108.3 +/- 12.0 microL, n = 15) and concentration (421.8 +/- 34.4 x 10(6) cells/mL, n = 15) measurements, the above mentioned parameters were determined. In frozen-thawed semen samples sperm motility, viability, hypoosmotic swelling test and acrosomal integrity were significantly lower than in fresh semen samples. The results clearly indicated that electro-ejaculation is a useful method for evaluating spermatozoa for genetic analysis or for used in Al in this species. In addition, the cryopreservation procedure in this study preserved adequate levels of functional sperm activity.

Functional activity of epididymal Chinchilla laniger spermatozoa cryopreserved in different extenders.

Chinchilla laniger is an endangered species and improved cryopreservation of spermatozoa would constitute a significant advance in the development of assisted reproductive techniques in this species. The functional activity of epididymal spermatozoa from adult males was studied immediately after extrusion and after 24 hours incubation, and the ability of five extenders to protect these gametes during cryopreservation was determined. A decrease in sperm motility, viability, acrosome intact cells and response to hypo-osmotic swelling test was detected 24 hours later. The extenders here assayed showed well-defined differences in their cryoprotective ability; however such differences could not be attributed to any one of their individual components. The presence of TES plus Tris, the proportion of the individual constituents and/or the differences in metabolic substrate content could explain the above-mentioned finding. The results indicate that cryo-buffer II (TES-Tris-egg yolk-fructose-glycerol) is the most powerful protector of sperm functional activity in this species.

Changes in corticosterone levels and sperm functional activity by chronic stress in rats.

Corticosterone levels and epididymal sperm functional activity have been studied in rats subjected to immobilization stress (A: 12 days) or variable chronic stress (B: 24 days; C: 32 days and D: 32 days plus 28 days without stress). Plasma corticosterone levels were significantly enhanced in B and C groups. Body and testicular weight significantly decreased in C and D groups. A decrease in viability or percentage of progressive epididymal spermatozoa was detected in C and D groups. Responses of spermatozoa to hypoosmotic shock remained unaltered in all groups. Results suggest that sustained increase in activity of the hypothalamo-adenohypophyseal-adrenal cortical system alters the activity of the hypothalamo-adenohypophyseal-testicular axis.

Time-related changes in functional activity of mouse spermatozoa during in vitro or in vivo incubation.

The kinetics of functional modifications during in vivo or in vitro incubation was studied in spermatozoa from Albino Swiss mice, obtained from cauda epididymis (E) or from uterus (U) or oviduct (O) post copulation (p.c.). Results clearly suggest that functional activity as reflected by measurements of motility, hypoosmotic swelling test and acrosome reaction differs according to the segments of the reproductive tract from which they were collected. The comparison of results obtained after equivalent periods of in vitro or in vivo incubation, shows that the time course of changes in sperm motility and percentage of swollen sperm is similar in both conditions. Since the percentage of acrosome-reacted gametes was higher in O, at all times scored, oviductal environment appeared to be capable of inducing this phenomenon. Our experimental conditions seemed to be adequate for completion of spermatozoa maturation and successful interaction between male and female gametes as reflected by the results of fertilization rate and embryo cleavage.

Seasonal evaluations of urinary androgen metabolites and semen quality in domestic long-tailed chinchilla (Chinchilla lanigera) under natural photoperiod.

Chinchilla spp. is a South American hystricomorph rodent genus currently considered almost extinct in the wild. The high quality of chinchilla fur motivated the harvesting of chinchillas for the fur market. Reproductive biology advances come from studies on commercially exploited animals, especially Chinchilla lanigera. We studied seasonal variation of urinary androgen metabolites, sperm concentration and sperm functional activity in males of domestic Chinchilla lanigera under natural photoperiod. In Córdoba city (31° S-64° W; Argentina), within the same latitudes as those of the historic Andean distribution (tropical deserts; 15°-34° S), domestic males (n=7) were studied in May (autumn), August (winter), November (spring), and February (summer). Urine was seasonally collected (over 24h; once for season, 4 in total) to measure urinary androgen metabolites (RIA), before semen collection by electroejaculation. The results indicated that although testicular volume (relative to body weight) and values of sperm functional activity did not show seasonal changes, a seasonal variation in androgen excretion was detected, with the highest values occurring during "short" light/dark cycles (autumn-winter). In addition, viable spermatozoa with intact acrosome mean values during winter-spring were higher than in autumn or summer. This study provides information that might contribute to the assessment of testicular activity in male chinchilla subjected to genetic selection in the fur industry. In addition, since domestic chinchilla still share some genomic characteristics with their counterparts in the wild, results presented may alsocontribute to ex situ breeding program of endangered chinchilla. In conclusion, natural photoperiod cycle affects testicular activity in domestic chinchilla.

Reproduction in chinchilla (Chinchilla lanigera): current status of environmental control of gonadal activity and advances in reproductive techniques.

A review of the biology of reproduction of chinchilla, focusing on environmental control of the gonadal activity, is presented. Chinchilla is a South American hystricomorph rodent genus currently considered almost extinct in the wild. However, a domestic form is still widespread in breeding farms around the world. Information regarding their reproductive biology has been obtained from studies on captive animals. In the case of Chinchilla lanigera, a seasonal reproductive pattern has been frequently reported in breeding facilities, but factors that might trigger gonadal activity have not been identified. The available information on reproductive productivity in farms worldwide shows a range of 1.2 to 2.4 deliveries per female per yr (with up to 2.1 weaned young per female per yr). Indeed, as found in all rodents, chinchillas can multiply at high fecundity and fertility rates (4 to 6 follicles mature during estrous cycles). Some new research avenues are postulated to improve the control of gonadal activity by means of environmental and/or pharmacologic factors. Furthermore, reproductive techniques that have been validated in chinchilla are reviewed (noninvasive hormone monitoring, semen collection, sperm cryopreservation, estrus induction), and several technical steps are proposed to be able to achieve AI. Because domesticated chinchilla still share some genomic characteristics with their counterparts in the wild, validated reproductive techniques in chinchilla males and females might contribute to the success of breeding programs.

Seminal quality and neutral alpha-glucosidase activity after sequential electroejaculation of chinchilla (Ch. lanigera).

The objectives of this study of seminal quality of chinchilla (Chinchilla lanigera) males were to evaluate (1) the use of neutral alpha-glucosidase (NAG) as a functional epididymal marker in this species, and (2) the effects of repetitive electroejaculation on sperm function and seminal NAG concentration. Semen was obtained by electroejaculation from sexually mature domestic Ch. lanigera males (n=6) once a week for six consecutive weeks, and evaluated to assess seminal volume and NAG activity, and sperm concentration and functional activity (motility, viability, membrane and acrosome integrity). Body weight was determined in all animals before each electroejaculation procedure. NAG activity was detected in all semen samples obtained, reaching a concentration of 55.4±6.8µU/mL. No significant variations were observed throughout the experimental period in any of the semen quality parameters evaluated. A reduction (P=0.001) in body weight was detected from the third week of experimentation and by the end of the experiments the animals lost a 7.2±0.9% of body weight. Overall, results in the present study confirm that NAG activity is detectable in chinchilla seminal plasma and that repetitive electroejaculation is not deleterious, on a short-term basis, to the semen characteristics.

Hippocampal effects of neuronostatin on memory, anxiety-like behavior and food intake in rats.

A 13-amino acid peptide named neuronostatin (NST) encoded in the somatostatin pro-hormone has been recently reported. It is produced throughout the body, particularly in brain areas that have significant actions over the metabolic and autonomic regulation. The present study was performed in order to elucidate the functional role of NST on memory, anxiety-like behavior and food intake and the hippocampal participation in these effects. When the peptide was intra-hippocampally administered at 3.0 nmol/µl, it impaired memory retention in both, object recognition and step-down test. Also, this dose blocked the hippocampal long-term potentiation (LTP) generation. When NST was intra-hippocampally administered at 0.3 nmol/µl and 3.0 nmol/µl, anxiolytic effects were observed. Also, the administration in the third ventricle at the higher dose (3.0 nmol/µl) induced similar effects, and both doses reduced food intake. The main result of the present study is the relevance of the hippocampal formation in the behavioral effects induced by NST, and these effects could be associated to a reduced hippocampal synaptic plasticity.

Contractility and pharmacological reactivity of isolated vascular smooth muscle from diabetic rats.

Conflicting reports exist about the effects of diabetes on vascular function. In the present study we investigated (1) the influences of diverse stages of diabetes on mechanical activity and pharmacological reactivity of portal vein and tail ventral artery isolated from male rats 7, 21 or 30 days after alloxan injection (150-180 mg/kg) and (2) the effects of in vitro or in vivo insulin treatment. Various parameters were used to assess the diabetic state (serum glucose levels, body weight, percentage of glycosylated hemoglobin and glucosuria). Isometric developed tension of portal vein from control rats was 10.86 +/- 0.41 mN (n = 54), and was enhanced significantly in diabetics (+56% at 21 days and +45% at 30 days; p less than 0.001 vs. controls). When challenged with noradrenaline, portal veins from diabetics exhibited a greater contractility and lower reactivity (as reflected by EC50 values). The magnitude of responses to KCl remained similar to those obtained in controls, but nonetheless the reactivity seems to be higher. Tail ventral artery from diabetics also exhibits a greater contractility in response to noradrenaline with no significant changes in EC50 values. The results demonstrate that diabetes affects mechanical performance of the vascular smooth muscle in a differential manner depending on the stage of the endocrinopathy and on the types of vessel studied. These modifications were not avoided by insulin treatment.

Modifications evoked by piretanide in the mechanical activity of cardiovascular tissues.

The effects of piretanide upon mechanical activity and pharmacological reactivity of vascular and myocardial tissues from normotensive rats were investigated. Magnitude of phasic contractions of isolated rat portal vein was diminished by the drug in a dose-related manner; contractile depression induced by piretanide (10(-4)M) was less in the presence of insulin (0.1 U/mL), glucose (22 mM) or pyruvate (5 mM). Responses to KCl (90 mM), or norepinephrine (2.5 X 10(-5)M) were also reduced. Contractile activity of atria and ventricle strips was diminished only when piretanide reached 10(-4)M. Results support direct actions of piretanide upon cardiac and vascular tissues. Possible mechanisms of action are discussed.

Effects of helio-neon laser radiation upon cellular cycle in a plant model.

The scope of this study was to investigate possible relationships between He-Neon laser radiation and mitotic and phase indices in meristematic cells of Allium cepa L. bulbs. Our results indicate that mitotic index increased after irradiation depending this modification on the time exposure and the potency of the He-Neon beam. Phase indices were also modified: frequency of prophase increased, while inter- meta- and anaphase decreased: telophases remain unchanged. These variations were significative only when the preparations were irradiated a) with 5 mW for 10 min. or more, b) with 10 mW or c) when the preparations were processed 60 min. after irradiation. These findings could not be attributed to thermal changes. Modifications in RNA or protein synthesis could be responsible.

Influence of the metabolic environment on oxygen uptake and isometric developed tension of isolated rat portal vein.

Oxygen uptake and isometric developed tension (IDT) of isolated rat portal vein were simultaneously measured after glucose removal, following the readmission of metabolic substrates and addition of 2-deoxy-D-glucose (2-DG) or insulin. In control conditions, basal O2 uptake was 0.128 +/- 0.004 microliter O2/mg dry weight/min (n = 50). After 40 min of incubation in a glucose-free medium, IDT and O2 uptake decayed to a similar degree. Addition of 11 mM glucose, 5 mM pyruvate, acetate, lactate or butyrate to hypodynamic veins, elicited differential recoveries in the mechanical and metabolic parameters, pyruvate and acetate being the most effective. Insulin (0.1 U/ml) added 5 min prior to readmission of 11 mM glucose, increased the restoration of IDT without any effect on oxygen uptake. 2-DG 5.5 mM, when added in glucose-free medium, abolished IDT but only depressed the cellular respiration by 38% at 20 min; these effects were decreased in presence of glucose (11 mM). These results suggest that the oxygen uptake of this vessel depends, among other factors, on the exogenous substrate offered. They show also the relationship between peak of developed isometric force and metabolic activity as well as the relevance of glycolytic and oxidative pathways in this tissue.