Effects of Cecropia pachystachya and Larrea divaricata aqueous extracts in mice.

Our studies were performed to investigate the effects of the aqueous extracts of Cecropia pachystachya and Larrea divaricata. These plants are used in folkloric medicine in infusion and were administered orally (0.76 g/kg) to male Albino Swiss mice for 16 days, on drink intake, organ weight/body weight (OW/BW x 100) ratio, histology, broqueoalveolar fluid (BALF) and elevated plus-maze (EPM). Feeding as well as body weight were unaffected by the consumption of these extracts. There were no signs of toxicity in BALF, morbidity or mortality during the study. C. pachystachya caused an increase in relative kidney OW/BW (p

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.

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.

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.

A new combined in-vitro test model for the identification of substances affecting essential sperm functions.

Binding of mammalian spermatozoa to the zona pellucida and the induction of the acrosome reaction are prerequisites for successful oocyte fertilization. It has been postulated that xenobiotics that are released in the environment as well as exposure to pharmaceutical medications may be associated with reproductive problems in men and wildlife. Examining physiological and non-physiological effects of particular compounds on sperm functions requires high quality in-vitro test systems. We established a reliable combined in-vitro test system with bovine gametes and evaluated if aliquots of pooled post-thaw spermatozoa are suitable for examining essential sperm functions. Using cryopreserved semen, the PSA-FITC/Hoechst 33258 staining procedure was applicable to evaluate the acrosomal status and cell viability. In the bovine hemizona assay, hemizona indices revealed no differences between cryopreserved and fresh semen. Treatment of post-thaw bovine spermatozoa with progesterone (1 microM or bovine follicular fluid (20%) induced the acrosome reaction from 12% (untreated spermatozoa) to 25% (P < 0.001) and to 22% [corrected] (P < 0.01), respectively. Incubation of both compounds (1 microM progesterone and 20% follicular fluid) raised the percentage of acrosome-reacted spermatozoa to 30% (P < 0001). Our results demonstrate that cryopreserved semen can be integrated into an in-vitro screening model for reproductive toxicology testing. Pooled, cryopreserved bovine spermatozoa will thus permit reproducible experiments for clinical and basic science purposes and may also be applicable for the human system.

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.