Storage of Chinchilla lanigera semen at 4 degrees C for 24 or 72 h with two different cryoprotectants.

The objectives of this study were to: (a) test the functional activity of Chinchilla lanigera spermatozoa suspended in either glycerol or ethylene glycol, cooled to 4 degrees C, and stored for 24 or 72 h and (b) investigate, after these cooling periods, the effects of incubating sperm at 37 degrees C (for 4 h) upon sperm functional activity. The ejaculate was mixed with the cryoprotectant medium (at 1 M final concentration) and cooled to 4 degrees C. After warming, sperm motility, sperm viability, hypoosmotic swelling test results, and acrosomal integrity were significantly higher for samples containing ethylene glycol than for those in glycerol, stored for 24 or 72 h, and then assayed after 0 or 4 h incubation at 37 degrees C. A significant reduction of sperm motility and viability was detected only when the glycerol cryoprotectant agent was employed, compared to the fresh samples. These results clearly indicate that under our experimental conditions, ethylene glycol is a better protectant for sperm storage than glycerol.

In vitro parthenogenesis of mouse oocytes under several experimental conditions.

Although the in vitro fertilisation index is a parameter commonly employed to investigate sperm functional activity, little attention has been given to the occurrence of parthenogenesis. The purpose of this study was to study at 6 h or 22 h incubation: (a) the cleavage-related events that occur in in vitro incubated mouse oocytes, in the absence (parthenogenesis) or presence of homologous spermatozoa; (b) the effect of mineral oil, commonly used in in vitro fertilisation assays; (c) the effect of piroxicam, a prostaglandin synthesis inhibitor, on the parthenogenetic rate; and (d) the influence on parthenogenesis of spontaneous loss of the cumulus oophorus coat during incubation. Under the experimental conditions employed, there was parthenogenetic activation and activation due to fertilisation. Both increased in a time-dependent manner. The mineral oil enhanced the parthenogenetic rate at 22 h incubation. However, it did not have any effect when the oocytes were inseminated. Since we can not discriminate how much of this activation was due to fertilisation and how much to parthenogenesis we must be very careful with this comparison. Piroxicam 10(-8) M did not show any effect on the mouse oocyte parthenogenetic rate at neither 6 h or 22 h incubation. Our results suggest that oocyte susceptibility to spontaneous parthenogenetic activation may be modified by the presence of the cumulus and corona radiata cells. In conclusion, we consider that further rigorous studies on these influences are necessary in order to confer more reliability on the results.

Functional activity of mouse sperm was not affected by low doses of aspirin-like drugs.

To investigate some possible effects of low doses of nonsteroidal anti-inflammatory drugs upon functional activity of mouse sperm, the authors injected lysine acetyl salicylate (im 14.3 mg/kg day(-1), ibuprofen (ip 5.6 mg/kg day(-1)), or piroxicam (ip 0.28 mg/kg day(-1) to pregnant females (the male cohort was sacrificed at adulthood) (A) or to adult males during 35 (B) or 60 (C) days. Parameters measured were motility, viability, acrosomal integrity, responses to hypoosmotic shock, in vitro fertilization index, and testosterone plasma levels. Salicylate evoked a slight reduction in the percentage of swollen gametes in A, and ibuprofen diminished testosterone plasma levels in B. The other parameters remained unchanged in all groups. Results are well supported by the low doses assayed, which are equivalent to the content of one tablet commercially available for each compound.

Influence of pentoxifylline on sperm membrane functional integrity.

In epididymal mouse spermatozoa, the effects of dibutyryl cyclic adenosine monophosphate 1 mmol/L (dbcAMP), pentoxifylline 5 mmol/L (PX), and/or mastoparan 50 mumol/L (MT) were evaluated for the following parameters: percentage of motile cells and response to hypoosmotic shock (HOS). The gametes were incubated during 80 min (A) or 200 min (B) in Tyrode's medium, and the drugs were added during the last 20 min. In A, dbcAMP + PX (61.5 +/- 5.4%; n = 10) enhanced and MT decreased significantly the population of motile cells (13.4 +/- 5.4%; n = 6) (control: 47.6 +/- 3.9%; n = 11). In B, PX significantly increased this parameter and MT plus PX also exerted a significant detrimental effect. Responses to HOS dropped significantly in the presence of PX + MT in A or in B; in this latter condition a similar decrease was evoked by MT alone. A positive correlation between percentages of swollen and motile spermatozoa was detected in A or in B in samples incubated with PX (r = .58, n = 11 and r = .76, n = 10; p < .05, respectively). These results that support that, in mouse sperm tail, PX would preserve functional membrane integrity, a relevant condition for adequate motility.

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.

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.

Differential effects of pharmacologically generated reactive oxygen species upon functional activity of epididymal mouse spermatozoa.

Several studies indicate that reactive oxygen species (ROS) are involved in defective sperm function pathophysiology. In this study we attempted to determine differentially the effects of xanthine (0.12 mM) plus xanthine oxidase (0.035 U/mL) (X+XO, a ROS promoter system), ROS scavengers (Tiron (TIR, 15 mM); catalase (CAT, 10 micrograms/mL); dimethylsulfoxide (DMSO, 140 mM)), and X+XO plus scavengers on several epididymal mouse spermatozoa functional parameters, incubated in NTPC medium, for 29 min. In the presence of X+XO, progressive gametes significantly diminished. TIR or CAT attenuated this effect, but DMSO did not. Inversely, X+XO increased the bending-forms population; only TIR reversed this phenomenon. The ROS promoter system diminished the viable cell population; all scavengers assayed maintained sperm viability at levels similar to control ones. When exposed to hypoosmotic shock after 29 min incubation with X+XO, the percentage of swollen cells decreased; TIR, CAT, or DMSO did not prevent this effect. Our experiments demonstrate that it is possible to differentiate the deleterious ROS effects upon sperm functional activity. O-2. and H2O2 preferentially seem to modify sperm motility, O-2. exhibiting the greatest ability for generating bending-form gametes, OH-being the most lethal ROS. In addition, sperm membrane clearly appears as the most damaged structure.

Trypanocidal effects of promethazine.

Promethazine is currently used for its antipsychotic and ansiolytic effects. It is a phenothiazine with anticalmodulin action, not toxic for human beings at therapeutic doses. The present results show that promethazine has trypanocidal effect on both epimastigote and trypomastigote stages of T. cruzi; two hundred microM inhibited epimastigote growth in culture medium and 2 microM immobilized and killed bloodstream trypomastigotes. When promethazine (55 mg/Kg/day) was used as treatment of T. cruzi infected mice, it proved effective in reducing parasitemia and it increased the survival of treated animals. Ultrastructural studies suggest that the lethal effect of this phenothiazine is related to a detergent effect that disrupts T. cruzi cell membrane.

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.

[Profile of spermatic morphology in normospermic and dyspermic patients]. / Perfil de la morfologia espermatica en pacientes normospermicos y dispermicos.

The evaluation of spermatic morphology is of importance due to its prognostic value in potential male fertility, both in natural and in assisted reproduction. In the present work, the morphologic profile of samples of semen from normal and dispermic patients was determined, in order to establish a correlation between structure and function. Samples were obtained from male partners of infertile couples who attended andrologic consultation. The smears were stained by the Papanicolaou technique, and the criteria of the OMS and the recent modification of Kruger were followed to evaluate spermatic morphology. Of all the patients studied (n = 46), 35% were normospermics (A), 39% asthenospermics, 7% oligospermics, 2% terastospermics and 17% presented combined alterations. The average percentage of normal spermatozoa was of 23.7 +/- 1.23 in the whole population. When group A patients were compared with asthenospermics, significant differences were found in the percentage of spermatozoa with middle piece and tail alterations, in the last group (p < 0.02). Similarly, when samples were analysed according to the presence of alterations: none (A), one (B) two or more (C), it was observed that the percentage of normal forms decreased in the groups in that order (A < B < C); (A or B vs C p < 0.05). The percentage of tapering spermatozoa was significantly higher in group C (p < 0.01 vs A or B). From the above results, it appears that 1) the larger number of functional alterations is related to a higher percentage of structural anomalies, and 2) the deficient motility in the asthenospermic group, is associated to the middle piece and tail alterations.(ABSTRACT TRUNCATED AT 250 WORDS)

Isometric developed tension and histopathology of myocardium of chagasic mice. II.

In a preceding paper we reported the evolution of chagasic cardiopathy in mice inoculated with low number of T. cruzi from 2 days to 75 days post-infection (p.i.). The present work analyzed the contractility, pharmacological response and histopathology of myocardium isolated from chronic chagasic mice from 90 days until 180 days. p.i. Myocardium contractile force reached values similar to controls until 165 days p.i. From this to the end contractility was significantly lower. At 90 days p.i. NE provoked negative inotropic effect or had no effect in 13% of the cases tested. The others had a reactivity to NE similar to normal ventricles. From 105 days until 180 days p.i. NE induced to a positive inotropic effect significantly lower than in normal. ACh effect was significantly smaller from 165 days to the end. Previously ACh ventricles responses were as control. The effects of dibenamine, propranolol and atropine (10-6M) on chagasic ventricles were similar to those observed in normal tissues. At 90 days p.i. the histopathology showed focalized inflammatory infiltrates. At 180 days p.i. fibers fragmentations and loss of typical striated characteristic of cardiac tissue. The abnormal pharmacological response described could be attributed to alterations in cardiac beta and muscarinic receptors probably due to a lower oxygen support. The present paper shows that during chronic Chagas' disease myocardial function and pharmacological reactivity are seriously and definitively compromised.

Isometric developed tension and histopathology of myocardium of chagasic mice. II.

In a preceding paper we reported the evolution of chagasic cardiopathy in mice inoculated with low number of T. cruzi from 2 days to 75 days post-infection (p.i.). The present work analyzed the contractility, pharmacological response and histopathology of myocardium isolated from chronic chagasic mice from 90 days until 180 days. p.i. Myocardium contractile force reached values similar to controls until 165 days p.i. From this to the end contractility was significantly lower. At 90 days p.i. NE provoked negative inotropic effect or had no effect in 13

of the cases tested. The others had a reactivity to NE similar to normal ventricles. From 105 days until 180 days p.i. NE induced to a positive inotropic effect significantly lower than in normal. ACh effect was significantly smaller from 165 days to the end. Previously ACh ventricles responses were as control. The effects of dibenamine, propranolol and atropine (10-6M) on chagasic ventricles were similar to those observed in normal tissues. At 90 days p.i. the histopathology showed focalized inflammatory infiltrates. At 180 days p.i. fibers fragmentations and loss of typical striated characteristic of cardiac tissue. The abnormal pharmacological response described could be attributed to alterations in cardiac beta and muscarinic receptors probably due to a lower oxygen support. The present paper shows that during chronic Chagas disease myocardial function and pharmacological reactivity are seriously and definitively compromised.

Perfil de la morfologia espermatica en pacientes normospermicos y dispermicos. / [Profile of spermatic morphology in normospermic and dyspermic patients]

The evaluation of spermatic morphology is of importance due to its prognostic value in potential male fertility, both in natural and in assisted reproduction. In the present work, the morphologic profile of samples of semen from normal and dispermic patients was determined, in order to establish a correlation between structure and function. Samples were obtained from male partners of infertile couples who attended andrologic consultation. The smears were stained by the Papanicolaou technique, and the criteria of the OMS and the recent modification of Kruger were followed to evaluate spermatic morphology. Of all the patients studied (n = 46), 35

were normospermics (A), 39

asthenospermics, 7

oligospermics, 2

terastospermics and 17

presented combined alterations. The average percentage of normal spermatozoa was of 23.7 +/- 1.23 in the whole population. When group A patients were compared with asthenospermics, significant differences were found in the percentage of spermatozoa with middle piece and tail alterations, in the last group (p < 0.02). Similarly, when samples were analysed according to the presence of alterations: none (A), one (B) two or more (C), it was observed that the percentage of normal forms decreased in the groups in that order (A < B < C); (A or B vs C p < 0.05). The percentage of tapering spermatozoa was significantly higher in group C (p < 0.01 vs A or B). From the above results, it appears that 1) the larger number of functional alterations is related to a higher percentage of structural anomalies, and 2) the deficient motility in the asthenospermic group, is associated to the middle piece and tail alterations.(ABSTRACT TRUNCATED AT 250 WORDS)

Isometric developed tension and histopathology of myocardium of chagasic mice. II.

In a preceding paper we reported the evolution of chagasic cardiopathy in mice inoculated with low number of T. cruzi from 2 days to 75 days post-infection (p.i.). The present work analyzed the contractility, pharmacological response and histopathology of myocardium isolated from chronic chagasic mice from 90 days until 180 days. p.i. Myocardium contractile force reached values similar to controls until 165 days p.i. From this to the end contractility was significantly lower. At 90 days p.i. NE provoked negative inotropic effect or had no effect in 13

of the cases tested. The others had a reactivity to NE similar to normal ventricles. From 105 days until 180 days p.i. NE induced to a positive inotropic effect significantly lower than in normal. ACh effect was significantly smaller from 165 days to the end. Previously ACh ventricles responses were as control. The effects of dibenamine, propranolol and atropine (10-6M) on chagasic ventricles were similar to those observed in normal tissues. At 90 days p.i. the histopathology showed focalized inflammatory infiltrates. At 180 days p.i. fibers fragmentations and loss of typical striated characteristic of cardiac tissue. The abnormal pharmacological response described could be attributed to alterations in cardiac beta and muscarinic receptors probably due to a lower oxygen support. The present paper shows that during chronic Chagas disease myocardial function and pharmacological reactivity are seriously and definitively compromised.

Perfil de la morfologia espermatica en pacientes normospermicos y dispermicos. / [Profile of spermatic morphology in normospermic and dyspermic patients]

The evaluation of spermatic morphology is of importance due to its prognostic value in potential male fertility, both in natural and in assisted reproduction. In the present work, the morphologic profile of samples of semen from normal and dispermic patients was determined, in order to establish a correlation between structure and function. Samples were obtained from male partners of infertile couples who attended andrologic consultation. The smears were stained by the Papanicolaou technique, and the criteria of the OMS and the recent modification of Kruger were followed to evaluate spermatic morphology. Of all the patients studied (n = 46), 35

were normospermics (A), 39

asthenospermics, 7

oligospermics, 2

terastospermics and 17

presented combined alterations. The average percentage of normal spermatozoa was of 23.7 +/- 1.23 in the whole population. When group A patients were compared with asthenospermics, significant differences were found in the percentage of spermatozoa with middle piece and tail alterations, in the last group (p < 0.02). Similarly, when samples were analysed according to the presence of alterations: none (A), one (B) two or more (C), it was observed that the percentage of normal forms decreased in the groups in that order (A < B < C); (A or B vs C p < 0.05). The percentage of tapering spermatozoa was significantly higher in group C (p < 0.01 vs A or B). From the above results, it appears that 1) the larger number of functional alterations is related to a higher percentage of structural anomalies, and 2) the deficient motility in the asthenospermic group, is associated to the middle piece and tail alterations.(ABSTRACT TRUNCATED AT 250 WORDS)

Disruption of mitochondrial function as the basis of the trypanocidal effect of trifluoperazine on Trypanosoma cruzi.

The tricyclic anti-calmodulin drug trifluoperazine (TFP) inhibited growth and motility of epimastigotes of Trypanosoma cruzi, at concentrations lower than 100 microM, and motility and infectivity of the bloodstream trypomastigote form at 200 microM. Electron microscopy of TFP-treated epimastigotes showed that the major effect was at the mitochondrial level, with gross swelling and disorganization. The oligomycin-sensitive, mitochondrial ATPase was completely inhibited by 20 microM TFP, and the same drug concentration caused a 60% decrease in intracellular ATP content. The results suggest that the trypanocidal effect of TFP may be related more to mitochondrial damage than to the well-known anticalmodulin effect of the drug.

Isometric developed tension and histopathology of myocardium of chagasic mice. I.

Cardiac disease is usually the most serious complication of human infection with Trypanosoma cruzi in our country. This report studies the evolution of the chagasic cardiopathy in mice inoculated with low number of parasites during acute and indeterminate stages in different aspects: contractility, histopathology and pharmacological response. From 2-45 days post infection (p.i.) (acute stage) myocardium contractile force reached higher values than in controls, but norepinephrine (NE) response was significantly lower. Acetylcholine (ACh) caused a negative inotropic effect similar to the observed in control group. In this period cardiac damage evolved to an acute interstitial myocarditis. In the indeterminate phase (45-75 days p.i.) of this parasitosis NE produced either small inotropic effect, negative inotropic effect or had no effect on the ventricles tested. A significantly low ACh effect, sub-endocardiac perivascular fibrosis and necrosis in wall bases were also observed. The abnormal pharmacological response described could be ascribed to modifications in cardiac beta and muscarinic receptors number or affinity. The present results showed that in myocardium isolated from T. cruzi infected mice the histopathology, contractility and pharmacological response were altered from 48 h p.i. reaching a maximum disorder at 60 days p.i.

Isometric developed tension and histopathology of myocardium of chagasic mice. I.

Cardiac disease is usually the most serious complication of human infection with Trypanosoma cruzi in our country. This report studies the evolution of the chagasic cardiopathy in mice inoculated with low number of parasites during acute and indeterminate stages in different aspects: contractility, histopathology and pharmacological response. From 2-45 days post infection (p.i.) (acute stage) myocardium contractile force reached higher values than in controls, but norepinephrine (NE) response was significantly lower. Acetylcholine (ACh) caused a negative inotropic effect similar to the observed in control group. In this period cardiac damage evolved to an acute interstitial myocarditis. In the indeterminate phase (45-75 days p.i.) of this parasitosis NE produced either small inotropic effect, negative inotropic effect or had no effect on the ventricles tested. A significantly low ACh effect, sub-endocardiac perivascular fibrosis and necrosis in wall bases were also observed. The abnormal pharmacological response described could be ascribed to modifications in cardiac beta and muscarinic receptors number or affinity. The present results showed that in myocardium isolated from T. cruzi infected mice the histopathology, contractility and pharmacological response were altered from 48 h p.i. reaching a maximum disorder at 60 days p.i.

Isometric developed tension and histopathology of myocardium of chagasic mice. I.

Cardiac disease is usually the most serious complication of human infection with Trypanosoma cruzi in our country. This report studies the evolution of the chagasic cardiopathy in mice inoculated with low number of parasites during acute and indeterminate stages in different aspects: contractility, histopathology and pharmacological response. From 2-45 days post infection (p.i.) (acute stage) myocardium contractile force reached higher values than in controls, but norepinephrine (NE) response was significantly lower. Acetylcholine (ACh) caused a negative inotropic effect similar to the observed in control group. In this period cardiac damage evolved to an acute interstitial myocarditis. In the indeterminate phase (45-75 days p.i.) of this parasitosis NE produced either small inotropic effect, negative inotropic effect or had no effect on the ventricles tested. A significantly low ACh effect, sub-endocardiac perivascular fibrosis and necrosis in wall bases were also observed. The abnormal pharmacological response described could be ascribed to modifications in cardiac beta and muscarinic receptors number or affinity. The present results showed that in myocardium isolated from T. cruzi infected mice the histopathology, contractility and pharmacological response were altered from 48 h p.i. reaching a maximum disorder at 60 days p.i.

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.