Cluster of cases of malignant schwannoma in cattle.

Between 1998 and 2001, several cases of ataxia and paresis followed by recumbency and death were reported in cows from different farms in a restricted area of the Argentinian Patagonia. Five cases of this cluster were studied and a diagnosis of malignant schwannoma was established. Electron microscopy (em) of tumour samples from three of the animals revealed intracytoplasmic or interstitial structures resembling retroviral particles. Attempts to isolate a viral agent from the tumours were unsuccessful but the epidemiological data and the em findings suggest a viral aetiology.

Randomised study of single dose (8 Gy vs. 6 Gy) of analgesic radiotherapy plus zoledronic acid in patients with bone metastases.

OBJECTIVE: To assess the effectiveness of a single dose of radio therapy (8 Gy vs. 6 Gy) plus zoledronic acid in cancer patients with bone metastases in treating pain; quality of life, time to onset of skeletal events and functional status. MATERIAL AND METHODS: A total of 139 patients from 22 Spanish hospitals were randomly assigned to: Group A, administered a single dose of 8 Gy+zoledronic acid (4 mg iv, in 15-min infusions), and Group B, administered a single dose of 6 Gy+zoledronic acid (4 mg iv, in 15-min infusions). The main variable was pain, which was assessed with the Visual Analogue Pain Scale (VAS) in supine, seated and standing positions. RESULTS: There was a total of 118 patients for intention to treat (n=67 in Group A and n=51 in Group B). The most frequent primary neoplasms were the lung (29.66%), prostate (22.03%) and breast (21.19%). Sixty patients were analysed per protocol, n=34 in group A and n=26 in group B. Improvements were observed in the VAS scores for pain in all three positions. The mean time to onset of the event was greater (p=0.0211) in Group A than in Group B (122 vs. 81.62 days). Functional status improved in Group A, and quality of life improved in both groups. CONCLUSION: The two groups achieved similar levels of pain control in supine, seated and standing positions. Quality of life also improved in both groups. However, the higher dose (8 Gy dose) in combination with zoledronic acid is associated with a longer period without skeletal events.

Chronic administration of propylthiouracil ameliorates hyperdynamic circulation in portal hypertensive rats.

OBJECTIVE: To evaluate the effect of a hypothyroid state, induced by chronic propylthiouracil administration, on splanchnic and systemic hemodynamic parameters in rats with portal hypertension due to portal vein ligation. METHODS: Portal hypertension was induced by surgical stenosis of the portal vein. Cardiac index and portal blood flow were measured using radioactive microspheres. Measurements were performed after treatment with propylthiouracil (1 mg/ml in drinking water) for 5 days. RESULTS: Propylthiouracil-treated portal hypertensive rats had a lower portal pressure (12.4 +/- 1.9 versus 16.3 +/- 0.7 mmHg; p < 0.05) and portal blood flow (11.6 +/- 0.7 versus 13.2 +/- 1.3 ml/min/100 g; p < 0.05) than non-treated animals. Splanchnic vasoconstriction in treated animals was associated with a higher peripheral vascular resistance (2.3 +/- 0.4 versus 1.8 +/- 0.3 mmHg/ml/min/100 g; p < 0.05) than controls. CONCLUSION: These results suggest that portal pressure can be lowered by inducing a hypothyroid state by chronic administration of propylthiouracil.

Ovine pulmonary adenomatosis in Patagonia, Argentina.

An outbreak of pulmonary adenomatosis (OPA) occurred in sheep in Patagonia, Argentina's southernmost region. On the affected farm, nine animals died over a 6-month period with pulmonary lesions of OPA. In all cases, the histology of the lungs was characterized by proliferation of cuboideal and prismatic cells lining the alveoli. Inflammatory exudates and accumulation of alveolar macrophages were marked in most cases, but in six of the cases there was no excess fluid in the airways. The presence of the Jaagsiekte retrovirus was demonstrated in the lungs by immunocytochemistry and PCR. To the best of our knowledge, this is the first report of OPA in Patagonia.

Vitrification of in vitro produced bovine embryos: in vitro and in vivo evaluations.

The efficacy of different vitrification solutions to cryopreserve in vitro produced bovine blastocysts was evaluated based upon in vitro development of embryos in culture and on in vivo development of embryos transferred into recipients. In the first experiment, ethylene glycol + glycerol (Eg + Gly) + different sucrose concentrations were evaluated. There were no significant differences in development rates among solutions. As for hatching, the Eg + Gly + 0.1 M sucrose group had a greater rate as compared with Eg + Gly + 0 M sucrose and Eg + Gly + 0.5 M sucrose groups in the evaluations of Day 6, Day 7 and Day 6 + Day 7 embryos; and, Eg + Gly + 0.3 M sucrose group had a greater rate as compared with the Eg + Gly + 0 M sucrose and Eg + Gly + 0.5 M sucrose groups in evaluations of Day 6 and Day 6 + Day 7 embryos. There were no significant differences in development and hatching rates between Day 6 and 7 in in vitro produced bovine embryos within each treatment group. There were significant differences in nuclei number after vitrification between Eg + Gly + 0.1 M and Eg + Gly + 0 M sucrose groups and the Eg + Gly + 0.5 M sucrose group. Pregnancy after 60 days of transfer and calving rates showed a difference between in vivo produced embryos freshly transferred and in vitro produced embryos vitrified with Eg + Gly + 0.3 M. There were no significant differences in gestation length and sex ratio between treatments. As for birth weight, there were significant differences between fresh in vivo produced embryos and all treatments of in vitro produced embryos. There were significant differences in dystocial parturition between in vivo produced embryos and all treatments with in vitro produced embryos. These results demonstrate that vitrification can be used successfully in the cryopreservation of in vitro produced bovine embryos, and that it might be considered for use in commercial programs.

[Dexamethasone, an inhibitor of the expression of inducible nitric oxide synthase, does not modify the hyperdynamic state in cirrhotic rats]. / La dexametasona, un inhibidor de la expresion de la óxido nítrico sintasa inducible, no modifica el estado hiperdinámico en ratas cirróticas.

Increased nitric oxide formation has been shown to be involved in the hyperdynamic circulation of portal hypertension. It has been proposed that it could be related to stimulation of the inducible nitric oxide synthase by endotoxin. Therefore, the aim of the present study was to evaluate whether dexamethasone treatment, an inhibitor of the expression of the inducible enzyme, ameliorates the hyperdynamic circulation observed in cirrhotic rats due to chronic bile duct ligation. Systemic and splanchnic hemodynamic parameters were measured after administration of dexamethasone (3 mg/kg/day during 3 days, i.p.) or its vehicle. In cirrhotic rats dexamethasone treatment caused a mild but not significantly higher mean arterial pressure in comparison with vehicle while similar values of cardiac output, peripheral vascular resistance, portal blood flow and portal pressure were observed in both group of animals. A significant body weight loss over the three days of treatment was observed in rats receiving dexamethasone. In sham-operated rats, dexamethasone administration caused similar changes as observed in cirrhotic animals. Endotoxemia was observed in five of six cirrhotic rats while it was not detected in the control group. Our results show that dexamethasone administration does not modify systemic and splanchnic hemodynamic parameters in endotoxemic cirrhotic rats. This finding suggests that stimulation of the inducible nitric oxide synthase may not play a role in the increased nitric oxide production in portal hypertension.

La dexametasona, un inhibidor de la expresion de la óxido nítrico sintasa inducible, no modifica el estado hiperdinámico en ratas cirróticas. / [Dexamethasone, an inhibitor of the expression of inducible nitric oxide synthase, does not modify the hyperdynamic state in cirrhotic rats]

Increased nitric oxide formation has been shown to be involved in the hyperdynamic circulation of portal hypertension. It has been proposed that it could be related to stimulation of the inducible nitric oxide synthase by endotoxin. Therefore, the aim of the present study was to evaluate whether dexamethasone treatment, an inhibitor of the expression of the inducible enzyme, ameliorates the hyperdynamic circulation observed in cirrhotic rats due to chronic bile duct ligation. Systemic and splanchnic hemodynamic parameters were measured after administration of dexamethasone (3 mg/kg/day during 3 days, i.p.) or its vehicle. In cirrhotic rats dexamethasone treatment caused a mild but not significantly higher mean arterial pressure in comparison with vehicle while similar values of cardiac output, peripheral vascular resistance, portal blood flow and portal pressure were observed in both group of animals. A significant body weight loss over the three days of treatment was observed in rats receiving dexamethasone. In sham-operated rats, dexamethasone administration caused similar changes as observed in cirrhotic animals. Endotoxemia was observed in five of six cirrhotic rats while it was not detected in the control group. Our results show that dexamethasone administration does not modify systemic and splanchnic hemodynamic parameters in endotoxemic cirrhotic rats. This finding suggests that stimulation of the inducible nitric oxide synthase may not play a role in the increased nitric oxide production in portal hypertension.

GABA release mechanism in the golden hamster retina.

High K+ medium and glutamate elicited a significant [3H]-GABA release in the golden hamster retina. High K+ -induced GABA release was largely calcium-dependent, while the effect of glutamate was Ca2+ -independent. After replacing Na+ by Li+, glutamate-evoked [3H]-GABA release was abolished, while high K+ -evoked release remained unchanged. The effect of glutamate was completely blocked by DNQX but not by APV. Furthermore, kainate induced [3H]-GABA release, whereas NMDA was ineffective. Assessment of endogenous GABA efflux further confirmed results obtained for [3H]-GABA. GABA-like immunoreactivity was observed in amacrine cells, in neurons localized in ganglion cell layer, as well as in fibers and terminals at the inner plexiform layer. In addition a few horizontal cells showed GABA-like immunolabeling. The present results suggest the existence of at least two pools of GABA in the hamster retina, compatible with both vesicular and carrier-mediated mechanisms of transmitter release, being the amacrine cells the main gabaergic source in this tissue.

A lineal equation for the classification of progressive and hyperactive spermatozoa.

Capacitation of spermatozoa, a complex sequence of events that render them able to fertilize the egg, is generally associated with a switch from lineal, progressive movement to a vigorous, non-progressive pattern characterized by starlike tracks, a process known as hyperactivation. Development of a method for the analysis of progressive and hyperactive tracks is thus important for the assessment of capacitation in biochemical, physiological and clinical studies. In this study, we have applied a two-step heuristic model to deduce a lineal equation that discriminates hyperactive from progressive spermatozoa. The kinetic parameters (curvilinear velocity (VCL), linearity (LIN), amplitude of lateral head displacement (ALH), straightness (STR), wobble (WOB), mean 'dance' (DAN) and velocity of the average path (VAP)) of ram spermatozoa were evaluated with a computerized motility analyzer, and classified one by one as progressive or hyperactive by the appearance of their tracks. In a first step, a discriminating plane was defined by minimizing the number of misclassified spermatozoa ('conflicting points'); then, the plane was adjusted by an iterative process to minimize the distance from conflicting points to it. The resulting plane showed a discriminating capacity of over 95% for both classes, higher than that achieved by setting a threshold value for the parameters taken separately or in group. When included in a standard semen analysis, application of the equation allowed a rapid assessment of the percentage of hyperactive spermatozoa. The method described, developed in ram spermatozoa, can be applied to different species for a variety of purposes.

Pentoxifylline reduces nephrotoxicity associated with cyclosporine in the rat by its rheological properties.

BACKGROUND: The goals of this study were to evaluate whether administration of pentoxifylline (POF) reduces the nephrotoxicity associated with cyclosporine (CsA) in the rat, and whether the effect of POF is related to its rheological properties. METHODS: Mean arterial pressure was measured by an intraarterial catheter. Glomerular filtration rate and renal plasma flow were determined by measuring inulin and para-aminohippurate clearances, after double-blind coadministration for 10 days of CsA (25 mg/kg/day) with either vehicle or POF (45 mg/kg every 12 hr). These results were compared with those obtained in control rats. Blood viscosity and erythrocyte deformability were also evaluated after treatment using a cone plate viscometer and a filtration method, respectively. RESULTS: No changes were observed in mean arterial pressure in both groups compared with controls. Glomerular filtration rate was significantly lower in CsA-treated rats (0.3+/-0.1 ml/min/100 g) than in control animals (0.6+/-0.1 ml/min/100 g, P<0.02). The coadministration of CsA with POF normalized the glomerular filtration rate (0.6+/-0.1 ml/min/100 g). A parallel decrease in renal plasma flow was observed in CsA-treated rats compared with controls (CsA+vehicle: 1.5+/-0.2 vs. control: 2.2+/-0.1 ml/min/100 g, P<0.02), this effect completely reversed by cotreatment with POF (3.1+/-0.2 ml/min/100 g). Blood viscosity was significantly higher in CsA-treated rats than in the control group (CsA+vehicle: 5.6+/-0.7 vs. control: 5.0+/-0.4 m x Pa x s, P<0.05). This effect was associated with a lower erythrocyte deformability (CsA+vehicle: 1.2+/-0.2 vs. control: 1.5+/-0.3 ml/min, P<0.05). These rheological abnormalities were normalized by coadministration with POF (blood viscosity: 4.9+/-0.7 m x Pa x s and erythrocyte deformability: 1.9+/-0.4 ml/min, P<0.05). CONCLUSIONS: Our results show that administration of POF prevents the nephrotoxicity associated with CsA. This beneficial effect could be related to its rheological properties.

In vitro capacitating effect of gamma-aminobutyric acid in ram spermatozoa.

We have evaluated the capacitating effect of gamma-aminobutyric acid (GABA) in ram spermatozoa in vitro, in a chemically defined medium, by means of the chlortetracycline (CTC) binding assay. Semen from adult Australian Merino rams was collected in an artificial vagina; spermatozoa were washed once in modified Biggers, Whitten, and Wittingham medium (m-BWW), without BSA or serum, and incubated in m-BWW alone or in m-BWW containing GABA, GABA agonists, or antagonists for 2 h at 38.5 degrees C under 5% CO2 in air. Samples were taken for assessment of CTC binding pattern or were further incubated for 15 min in the presence of 5 microM calcium ionophore A23187. Acrosomal exocytosis was evaluated by Pisum sativum agglutinin binding. Addition of GABA to the incubation medium resulted in a concentration-dependent increase in the percentage of CTC forms II and III, corresponding to mid-capacitated and capacitated spermatozoa, respectively. The effect was marginally significant at 1 microM and maximal at 20 microM. The action of 20 microM GABA was mimicked by the GABAB-receptor agonist, muscimol, but not by the GABAA-receptor agonist, baclofen, and completely blocked by the GABAA-receptor antagonists, bicuculline and picrotoxin, which lacked effect per se. In a separate set of experiments, incubation of spermatozoa with GABA at a concentration of 1 microM, which was insufficient to stimulate sperm capacitation, together with the neuroactive steroid allopregnanolone (1 microM) provoked a capacitating effect similar to that achieved by 20 microM GABA alone. These results show that GABA has a capacitating action on ram spermatozoa through a GABAA receptor-mediated mechanism.

Daily variation in portal blood flow and the effect of propranolol administration in a randomized study of patients with cirrhosis.

A nocturnal increase in portal pressure and blood flow was demonstrated in patients with cirrhosis, suggesting that these hemodynamic changes may contribute to the triggering of the hemorrhagic episodes observed during the night in these patients. It is known that propranolol reduces portal flow, thus reducing the risk of variceal bleeding. In a double-blind, placebo-controlled study, we evaluated the effect of long-term propranolol administration on the daily fluctuation of systemic and splanchnic hemodynamic parameters in 14 patients with cirrhosis. Cardiac output and portal blood flow were measured by the Doppler technique. A daily fluctuation of both cardiac output and portal blood flow was observed, peaking at midnight. beta-Adrenergic blockade was manifested by a significant reduction in heart rate (-21% +/- 4%, P < .01) and cardiac output (-12% +/- 2%, P < .05). A significant decrease in portal blood flow (-20% +/- 4%, P < .01) was also observed in these patients. Propranolol administration blunted the time-related changes in cardiac output and portal blood flow. In contrast, patients receiving placebo had a nocturnal peak of both parameters similar to that observed under basal conditions. Our study shows that chronic propranolol administration abolishes the nocturnal peak of portal blood flow in patients with cirrhosis and indicates a preventive effect of propranolol in these patients.

Actin localization in ram spermatozoa: effect of freezing/thawing, capacitation and calcium ionophore-induced acrosomal exocytosis.

We have analyzed, by immunofluorescence, the localization of actin in ram spermatozoa, its colocalization with the actin-binding protein, gelsolin, and the effect of freeze/thawing, in vitro capacitation, and induced acrosomal exocytosis on its distribution. The monoclonal anti-actin and anti-gelsolin antibodies used recognized single bands at 43,000 and 90,000 kDa, respectively. In all spermatozoa, intense actin staining was observed in the whole length of the flagellum and, depending on the protocol used, in the neck and postacrosomal region of the head. Comparison of three staining methods, together with the use of NBD-phallacidin, allowed us to characterize ram sperm actin as a monomeric, intracellular, membrane-associated protein. Gelsolin was also present in ram spermatozoa and precisely colocalized with actin. Processes involving alterations in membrane structure such as freezing/thawing, in vitro capacitation, and calcium ionophore-induced acrosomal exocytosis provoked changes in the exposure of actin to the antibody. This strongly suggests a physical association of this protein to the plasma membrane, most likely by its intracellular side. The possible role of actin in sperm function is discussed.

Assessment of the acrosomal status of membrane-intact ram spermatozoa after freezing and thawing, by simultaneous lectin/Hoechst 33258 staining.

We have evaluated the effect of freezing and thawing on the acrosomal status of ram spermatozoa, especially those that withstood cryopreservation as assessed by membrane integrity. To this end, we performed simultaneous lectin/Hoechst 33258 staining, and compared the ability of three fluoresceinated lectins. Ram spermatozoa were treated with fluorescein isothiocyanate-labelled Pisum sativum lectin (PSA), fluorescein isothiocyanate-labelled Arachis hypogea lectin (PNA) and fluorescein isothiocyanate-labelled Triticum vulgaris lectin (WGA) and simultaneously with Hoechst 33258 for determination of membrane integrity and acrosomal status. In all cases, three forms were readily distinguished by their distribution pattern. For both PSA and PNA, the most abundant form found in fresh semen consisted of fluorescence on the acrosomal area. This form corresponds to acrosome-intact spermatozoa, as assessed by Differential Interference Contrast (DIC) microscopy. Two minor forms showed weak fluorescence on the equatorial segment or no fluorescence on the head. DIC microscopy revealed that both forms were associated with acrosome-lost spermatozoa. WGA labelling showed two forms, one of which consisted of fluorescence on the entire head, albeit more intensely on its anterior segment. Spermatozoa in this form were acrosome-intact by DIC. The other form lacked fluorescence on the acrosomal region, but still showed faint fluorescence in the posterior region. This form was acrosome-lost by DIC. Incubation of fresh spermatozoa with calcium ionophore A23187 for up to 1 h significantly increased the percentage of those forms identified as acrosome-reacted as described above. This was confirmed by the time-dependent accumulation of these forms, as well as by DIC microscopy. At all times, differences among values obtained using these three lectins were not significant. Freezing and thawing led to a decrease of both membrane integrity and acrosomal integrity, irrespective of the lectin used. However, almost all spermatozoa that withstood cryopreservation, as evaluated by Hoechst exclusion, showed intact acrosomes. In this case, no differences between fresh and frozen/thawed samples were observed. These results suggest that the structural integrity of ram spermatozoa is mostly unaffected after cryopreservation, suggesting that it is damage to the plasma membrane that is primarily responsible for the low fertility of cryopreserved samples.

The storage of pure ram semen at room temperature results in capacitation of a subpopulation of spermatozoa.

We investigated whether storage of pure ram semen at room temperature would facilitate the sperm capacitation process, as assessed by means of the chlortetracycline method. Objective motility, membrane integrity and ability of spermatozoa to undergo acrosome reaction induced by A23187 for 15 min were simultaneously evaluated to gain further insight into this process. Storage for 4 h at room temperature had a clear capacitating effect in approximately 50% of spermatozoa and increased their ability to respond to A23187. Beyond that time, the percentage of motility and membrane integrity remained unchanged. Moreover, storage did not alter the ability of those spermatozoa that remained noncapacitated under these conditions to become capacitated in SOF-m medium. Storage for 4 h increased the percentage of spermatozoa showing swelling of the apical ridge from 3 to 13%. In conclusion, storage of ram semen at room temperature for 4 h in the dark has a marked capacitating effect on a subpopulation of spermatozoa, without changes in motility or membrane integrity, and a low effect on the appearance of the acrosome. Since semen storage is generally included in different IVF protocols, the results presented here contribute toward a clearer understanding of its role in these procedures.

A large-scale program in laparoscopic intrauterine insemination with frozen-thawed semen in Australian Merino sheep in Argentine Patagonia.

This report shows the results of a large-scale laparoscopic intrauterine insemination program on a flock of Australian Merino sheep in Argentine Patagonia. The study was carried out on a total of 1824 ewes (3-to-7-yr-old) and 480 ewe hoggets (19-20 months old) on 2 farms in the southeastern region of Santa Cruz Province, in April and May 1996. The animals, divided into 15 groups, were synchronized with vaginal sponges containing 60 mg medroxyprogesterone acetate for 14 d and injected with 200 IU PMSG upon sponge removal. Estrus was screened every 12 h by means of vasectomized marker rams. The animals were inseminated laparoscopically by the intrauterine route using 2 schemes: 1) at a fixed time (12 h) after estrus detection, or 2) at a fixed time (60 h) after sponge removal irrespective of estrus. Pregnancy was determined at 30 d by transrectal ultrasound imaging. The results showed that 1) the onset of estrus occurs most often between 24 and 48 h after sponge removal, 2) ewe hoggets undergo estrus significantly earlier than sexually mature ewes, 3) in those animals showing estrus, there appears to be no relationship between fertility (as assessed by pregnancy outcome) and time of estrus, 4) there is a significant association between the percentage of estrus occurrence and pregnancy rate, 5) fertility is significantly higher in ewes than in hoggets, 6) for practical purposes insemination at a fixed time after the onset of estrus has no advantage over that of to insemination at a fixed time after sponge removal. It is concluded that large-scale laparoscopic intrauterine insemination can be successfully applied in Australian Merino ewes and ewe hoggets in low-productivity areas such as that of Argentine Patagonia and that estrus detection is unnecessary when insemination is performed at 60 h after sponge removal.

Changes in sperm-bound amidase activity suggest subtle damage to ram sperm acrosomes by freezing/thawing, not detected by light microscopy.

We have measured sperm-bound amidase activity in fresh, cooled and frozen/thawed ram spermatozoa, in order to study if freezing and thawing led to some degree of acrosome damage of motile/viable spermatozoa not detected by optical methods. This assay was based on the fact that membrane damage would result in an increased access of the enzyme substrate to the sperm acrosome. Semen was collected from adult Australian Merino rams, and spermatozoa were washed by centrifugation through a Ficoll solution. Sperm-bound amidase activity was measured in whole spermatozoa using the protease substrate benzoyl-arginyl-p-nitroanilide (BAPNA). Acrosomal status was also assessed by light microscopy after Giemsa staining. Most amidase activity was shown to be sperm-bound, as only a minor fraction of the enzyme activity was release into the medium after induced damage. Simultaneous assessment of sperm-bound amidase activity and the percentage of spermatozoa with microscopically evident acrosomal damage, after mild sonication for different times, showed a high correlation between both parameters (r = 0.97, p < 0.001). In separate experiments, fresh, cooled and frozen/thawed semen samples were filtered through Sephadex G-10 to obtain a subpopulation of motile, mostly acrosome-intact spermatozoa. As controls, spermatozoa from the same samples to which extensive acrosome damage was induced were evaluated. Slow cooling to 4 degrees C had no effect on amidase activity or percent acrosomal damage with respect to fresh samples. Freezing and thawing resulted in a sperm population that, after filtration through Sephadex, had a low percentage of acrosome damage (9.4%, vs. 2.1% for fresh filtered controls), which was 11% of that obtained after extensive acrosome damage (83%). However, amidase activity in these samples was markedly increased, showing values of activity that were 56% of those obtained in extensively damaged spermatozoa. This effect was not due to an alteration in the enzyme kinetics. We conclude that sperm-bound amidase activity is useful to detect subtle changes, provoked by a standard freezing/thawing procedure, in the permeability of acrosomes from ram spermatozoa which are not detected by direct observation of the acrosomes after Giemsa staining.

In vitro capacitation and induction of acrosomal exocytosis in ram spermatozoa as assessed by the chlortetracycline assay.

We have described the different patterns of chlortetracycline (CTC) binding to ram spermatozoa, immediately after ejaculation and upon in vitro capacitation and calcium ionophore-induced acrosomal exocytosis. Four different forms of CTC distribution were found. Form I showed an even distribution of fluorescence over the entire head, with a brighter band in the equatorial region. In Form II, uniform fluorescence was observed without equatorial band. Form III consisted of fluorescence in the anterior portion of the head. Form IV showed no fluorescence over the head. In all cases, fluorescence in the middle piece of the flagellum was observed as well. Immediately after ejaculation, Form I was the most abundant one (78%) in fresh semen with Forms II and III being relatively scarce (less than 15%). Form IV was virtually absent or appeared only occasionally. Incubation under in vitro capacitating conditions led to a significant decrease in Form I and to a significant increase in Forms II and III. Form II was mainly associated to intact acrosomes, while most spermatozoa in Form III showed intermediate forms of acrosomal status. Incubation of spermatozoa with the calcium ionophore A23187 resulted in 55% of spermatozoa showing Form IV, suggesting that it represents the acrosome-reacted stage. Form I was abruptly decreased at 30 min of incubation and was neglectible after 60 min. In contrast, Forms II and III increased at 30 min but decreased later on, suggesting that both forms represent intermediate stages before the acrosomal exocytosis. Analysis of acrosomal status in spermatozoa from individual CTC forms revealed that all spermatozoa that remained in Form II after incubation had intact acrosomes. Intermediate stages were predominant in Form III-spermatozoa, while most Form IV-spermatozoa underwent full acrosomal exocytosis. These results show that CTC binding can be used to monitor changes in ram spermatozoa during capacitation and acrosome-reaction.

Daily variations in 2-[125I]melatonin specific binding in the golden hamster retina.

Daily variations in melatonin content and 2-[125I]melatonin specific binding in retinas of golden hamsters were studied. Both parameters showed significant variations throughout the 24 h period. Maximal specific binding was observed at 24.00 h, while melatonin content peaked at 04.00 h. Saturation studies performed at 12.00 and 24.00 h indicated that the maximal concentration of 2-[125I]melatonin binding sites (Bmax) was significantly higher at 24.00 h than at 12.00 h, whereas the dissociation constant (Kd) remained unchanged. As 2-[125I]melatonin specific binding decreased at times when retinal melatonin content was high, these findings suggest that daily variations in retinal melatonin levels may be implicated in the regulation of the density of melatonin binding sites, probably through mechanisms of up- and down-regulation induced by melatonin on its own binding sites.

Use of computerized motility analyzer for the evaluation of frozen-thawed ram spermatozoa.

A computerized motility analyzer (CellTrak/STM) was calibrated for its use with ram semen. Adjustment of the several setup variables allowed an accurate measurement of kinetic parameters such as percentage of motile cells, straight velocity, curvilinear velocity, linearity and amplitude of lateral displacement of the head. All kinetic parameters, except the lateral displacement of the head, showed significant changes after freezing and thawing. The curvilinear velocity exhibited the least significative post-thaw decrease. The alterations in kinetic parameters provoked by freezing and thawing could account for the low success obtained with frozen semen by cervical insemination, as it is accepted that during the initial steps of fertilization high motility and linearity are required.