Bloodmeal Identification in Field-Collected Sand Flies From Casa Branca, Brazil, Using the Cytochrome b PCR Method.

PCR-based identification of vertebrate host bloodmeals has been performed on several vectors species with success. In the present study, we used a previously published PCR protocol followed by DNA sequencing based on primers designed from multiple alignments of the mitochondrial cytochrome b gene used to identify avian and mammalian hosts of various hematophagous vectors. The amplification of a fragment encoding a 359 bp sequence of the Cyt b gene yielded recognized amplification products in 192 female sand flies (53%), from a total of 362 females analyzed. In the study area of Casa Branca, Brazil, blood-engorged female sand flies such as Lutzomyia longipalpis (Lutz & Neiva, 1912), Migonemyia migonei (França, 1924), and Nyssomyia whitmani (Antunes & Coutinho, 1939) were analyzed for bloodmeal sources. The PCR-based method identified human, dog, chicken, and domestic rat blood sources.

Associação entre sorologia para Neospora caninum e taxa de prenhez em vacas receptoras de embriões / Association between seropositivity for Neospora caninum and pregnancy rate in bovine receipts submitted to embryo transfer technology

The association between seropositivity for Neospora caninum and pregnancy rate in cows belonging to a surrogate herd submitted to embryo transfer technology was determined. The serological status was evaluated in 275 heifers, aging from 14 to 20-month-old. For N. caninum serology analysis of a monoclonal competitive ELISA test Kit was used, and 81 animals (29.5 percent) showed seropositive. Thus, two groups were randomly formed selecting 33 seropositive heifers and other 33 seronegative animals out of the remaining 194 animals. Seronegative animals were followed up by serological analysis until the end of the trial in order to identify persistently infected individuals. The pregnancy rate was 72.7 percent in the group of N. caninum-positive sera, and, 81.8 percent in the seronegative group.No significant difference was observed between groups according to Chi-square test. No association between N. caninum seropositivity and pregnancy rates in surrogate heifers was found

Separation of sperm cells by sedimentation technique is not suitable for in vitro fertilization purposes.

Two methods of sperm preparation for in vitro fertilization were compared: the swim-up technique vs. the migration-sedimentation technique. The study comprised fresh semen samples obtained from 25 couples treated in the In Vitro Fertilization Unit. Oocytes aspirated in a single cycle were divided into two groups, each inseminated by sperm prepared by one of these techniques. Motility, degree of motility, and normal morphology were improved by both methods. The improvement was greater when the migration-sedimentation technique was applied. However, fertilization rate was significantly higher after the swim-up technique. In order to clarify this contradiction, an additional group of 26 semen samples was divided and then prepared by the swim-up or migration-sedimentation techniques. Sperm quality was examined up to 72 h after separation. Compared with the swim-up technique, sperm characteristics were better after separation by the migration-sedimentation technique. However, this difference abated after 24 h. The better results of the swim-up technique in the "survival experiment' may explain its improved performance in in vitro fertilization, despite lower separation capacity. Thus, the migration-sedimentation technique is not recommended for sperm preparation in in vitro fertilization.

Management of bilateral nonpalpable testes: laparoscopic diagnosis and orchidectomy.

Bilateral nonpalpable testes in the adult human are associated with testicular malignancy, infertility and other abnormalities. Investigation for localization of the testes is mandatory, and either orchidopexy or orchidectomy is indicated. Laparoscopy was performed in an azoospermic male with bilateral unpalpable testes. A diagnosis of intra-abdominal testes was made and bilateral orchidectomy was performed. Laparoscopy is recommended for diagnosis and precise localization, as well as for orchidectomy, thus avoiding open abdominal operation in cases of intra-abdominal testes.

Migration sedimentation technique as a predictive test for the fertilizing capacity of spermatozoa in an in-vitro fertilization programme.

The migration-sedimentation technique (MST) has been proposed as a means of separating high quality motile spermatozoa. The present study was conducted in order to evaluate whether sperm performance following separation by MST predicts their fertilizing capacity in an in-vitro fertilization (IVF) programme. Ninety semen specimens were analysed for use in an IVF-embryo transfer (ET) programme. Each specimens was divided into two parts: one was processed in the IVF programme and was used after sperm swim-up separation for insemination of human ova. The other aliquot (0.2 ml) was separated by MST, and the sperm then characterized by their concentration, motility, degree of motility and morphology. Sperm characteristics after separation by MST were then correlated with the results of the IVF-fertilization rates. In 79 of 90 IVF-ET cycles, at least one oocyte was fertilized. All post-MST sperm characteristics were significantly higher in cycles with fertilizations compared to IVF cycles without fertilization. A larger percentage of the total motile spermatozoa were recovered after MST in semen specimens with fertilization, compared to semen specimens without fertilization (39.9 +/- 3.6 and 20.6 +/- 6.6%, respectively; P < 0.05). This value was correlated with the percentage of fertilized oocytes (r = 0.24; P < 0.02). More IVF cycles with fertilizations were recorded in cases in which the recovery of motile sperm was > 25% (P < 0.005), or when more than 1.5 x 10(6) motile spermatozoa were recovered after MST (P < 0.0001). As sperm characteristics after MST correlated significantly with their fertilizing capacity, the MST test could be used in evaluation of the fertilizing capacity of spermatozoa.

Ca(2+)-independent induction of acrosome reaction by protein kinase C in human sperm.

We report that activated protein kinase C (PKC) can induce acrosome reaction independently of elevated Ca2+. Addition of 12-O-tetradecanoyl phorbol-13-acetate or the membrane-permeable diacylglycerol analog 1-oleoyl-2-acetylglycerol to ejaculated human sperm resulted in stimulation of acrosomal reaction (2- to 3-fold), provided the sperm underwent capacitation. Induction of acrosome reaction by 12-O-tetradecanoyl phorbol-13-acetate was blocked by the PKC inhibitor staurosporine or by down-regulation of endogenous PKC, but not by removal of extracellular Ca2+. Acrosome reaction was also enhanced by the Ca2+ ionophore ionomycin in a Ca(2+)-dependent, PKC-independent fashion. Immunohistochemical analysis with type-specific PKC antibodies revealed the presence of PKC alpha and PKC beta II in the equatorial segment, whereas PKC beta I and PKC epsilon staining was found in the principal piece of the tail. Acrosome reaction, thus far believed to be induced only by elevated Ca2+, can therefore be triggered by activated PKC in a Ca(2+)-independent fashion. The PKC subtypes potentially involved in acrosome reaction are most likely alpha and beta II, whereas the beta I- and epsilon-subspecies might be involved in regulation of flagellar motility of human sperm.

The predictive fertilization value of the hypoosmotic swelling test (HOST) for fresh and cryopreserved sperm.

BACKGROUND: The hypoosmotic swelling test (HOST) was recommended as a predictive test for in vitro fertilization (IVF) outcome. These results, however, were controversial and the results for thawed semen were insufficient. The present study was conducted in order to clarify the predictive value of the HOST for IVF in fresh and thawed sperm. METHODS: The hypoosmotic swelling test was performed in three groups: husband's fresh semen "subfertile" group, donor's thawed semen group, and donor's fresh semen "fertile" group. RESULTS: No correlation was found between HOST values and sperm characteristics in fresh or thawed sperm. Fresh sperm HOST values correlated with IVF fertilizations. No such correlations were found when thawed sperm was used. HOST values were significantly higher in the fresh fertile donor group than in the fresh subfertile group (P less than 0.001). Following the freezing and thawing process, HOST values decreased dramatically. Nevertheless, the fertilization rate was still higher compared to that of the fresh subfertile group (P less than 0.001). There were significantly more IVF cycles with no fertilizations when HOST values were below 45%. CONCLUSION: The HOST has a predictive value for fertilization of oocytes in IVF cycles when fresh semen, but not thawed sperm, is used. The freezing-thawing process affects the outer membrane of the spermatozoon and changes its characteristics, leading to a decrease in HOST values. Sperm characteristics that play a role in the fertilization process are not expressed directly by HOST values.

Further studies on the involvement of protein kinase C in human sperm flagellar motility.

Addition of the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) or the membrane-permeable diacylglycerol analog, 1-oleoyl-2-acetylglycerol to human sperm resulted in increased motility. The biologically inactive 4 alpha-phorbol 12,13 didecanoate had no effect on flagellar motility. Basal motility was markedly reduced in the absence of Ca2+ in the incubation medium, but TPA-induced sperm motility persisted even in the absence of Ca2+. Sperm motility was also enhanced by the Ca2+ ionophore ionomycin in a Ca2(+)-dependent, protein kinase c (PKC)-independent fashion. Although all stimulants examined here reached maximal response at about 15 min of incubation, nevertheless whereas the effect of TPA and 1-oleoyl-2-acetylglycerol declined at 60 min of incubation, that of ionomycin still persisted. Human sperm PKC activity is extremely low and represents only about 20% and 25% of the specific activity recovered from PC-12 and rat pituitary cells, respectively. Immunohistochemical analysis using various type-specific PKC antibodies revealed staining only in the equatorial segment and the principal piece of the tail. Thus, PKC is present in human ejaculated sperm and is involved in flagellar motility.

Luteinizing hormone secretion as a response to a second naltrexone administration.

Previous studies with naltrexone (Nalt), a "long-lasting" opioid antagonist, demonstrated a rapid increase in luteinizing hormone (LH) secretion which gradually declined, reaching baseline values after 1 hr. A second Nalt challenge, 120 min later, caused only a blunted response. This poor reaction has been shown in this study not to be due to lack of pituitary responsiveness, because LH-releasing hormone treatment revealed a normal response. A time-response study was carried out in order to establish the refractory period length, by administering a second Nalt injection at 0 hr (immediately after the first injection) and at 2, 4, 8, 16, and 24 hr after the first bolus. Partial responsiveness could be achieved 2 and 4 hr after the first challenge. However, only after 8 hr was a full response recorded. The diurnal changes in serum LH (nadir at 18.00 hr) did not affect the response to Nalt challenge. It is suggested that in the presence of a Nalt blockade, nonopioid systems are able to "normalize" LH blood levels. However, when Nalt blood levels have fallen sufficiently to allow the endogenous opioid system to take primary control again, then a second Nalt injection will provoke a renewed response.

Protein kinase C is present in human sperm: possible role in flagellar motility.

We report the presence of protein kinase C (PKC) in ejaculated human sperm as revealed by enzymatic activity assay and indirect immunohistochemistry. PKC is localized in the equatorial segment and in the principal piece of the tail. Addition of phorbol 12-myristate 13-acetate resulted in increased flagellar motility that was blocked by known PKC inhibitors such as sphingosine, staurosporine, and 1-(5-isoquinoylinylsulfonyl)-2-methylpiperazine. A very good correlation (r = 0.9, P less than 0.001) was found between the percentage of PKC-stained sperm cells and motility. We propose that PKC is involved in the regulation of flagellar motility in human sperm.

Transferrin in seminal plasma and in serum of men: its correlation with sperm quality and hormonal status.

Transferrin concentrations in the seminal plasma and in serum were measured and correlated with sperm quality (concentration, motility and morphology) and hormonal status (FSH, LH and testosterone) of 75 men aged from 21 to 46 years. A significant positive correlation was found between the seminal plasma concentration of transferrin and the sperm concentration (r = 0.69, P less than 0.0001) and motility (r = 0.39, P less than 0.0001). No other correlations were found. These data confirm that seminal plasma levels of transferrin can be used as a reliable index of sperm quality, and possibly as a Sertoli cell marker.

Possible dopaminergic system involvement in LH and PRL responses to naltrexone treatment.

Naltrexone (Nalt) causes a rapid increase in luteinizing hormone (LH) level. This short term increase of LH concentration declines to baseline levels in less than 1 hour. Addition of pimozide (0.1 mg) caused a blunted response to Nalt challenge, with significantly reduced LH peak values compared with Nalt treatment alone. Pimozide alone caused a delayed decrease compared with baseline LH values. By following plasma prolactin (PRL) levels it was shown that pimozide administration increased PRL levels rapidly for more than 2 hours. Addition of Nalt to pimozide-treated rats significantly decreased plasma PRL values compared with pimozide alone. Nalt injected by itself attenuated PRL baseline levels. Thus, the mechanism by which pimozide caused PRL elevated level is via the dopaminergic as well as the opioid system. It is suggested that the opioid system controls plasma PRL and LH levels through other hypothalamic neurotransmitters in addition to dopamine.

Clomiphene citrate treatment in oligozoospermia: comparison between two regimens of low-dose treatment.

Clomiphene citrate (CC) is a well-known drug in fertility clinics that is used for increasing gonadotropin secretion. The present study was planned in order to evaluate the efficiency of a new regimen of treatment by 25 mg on alternate days (group A), compared to a daily dose of 25 mg (25 days on, 5 days off, group B), for 4 months. Semen quality was assessed in two matched groups, which consisted of 45 and 44 normogonadotropic oligoterato-asthenozoospermic (OTA) men, respectively. Nine men in group A and 22 in group B did not respond to therapy by improvement in semen quality. The statistical evaluation of the results revealed group A to yield the highest improvement in sperm concentration (P less than 0.0008) and total sperm count (P less than 0.004). Sperm motility was improved only in group A. No changes were recorded in the morphology of the sperm cells or in semen volume. Pregnancy rate after 6 months of follow-up was 26.7% and 20.5%, in couples of groups A and B, respectively. This study implicates the use of CC (25 mg on alternate days) in andrologic clinics as one of the recommended drugs for normogonadotropic OTA subfertile men in order to achieve a significant increase in sperm concentration and total sperm count.

Opiate system involvement in the control of LH secretion in diabetic and normoglycemic male rats.

The effect of Naltrexone (Nalt), a specific opiate receptor blocker, on LH secretion was studied at frequent intervals during the first hour following treatment. Nalt was injected i.v. by one bolus (1 mg/rat) to diabetic and normoglycemic rats. Blood samples (0.8 ml) were withdrawn at short intervals after injection, through an indwelling cannula. The diabetic rats responded by secretion of LH, which was lower, but not significantly, than that of normal rats, (peak levels 0.74 +/- 0.17 and 0.97 +/- 0.21 ng/ml respectively). After 45 min., LH levels were in the same range as baseline level in the diabetic group; but were still significantly elevated in the control rats. Thus, it can be concluded that in normal rats, as well as in diabetics, LH secretion as a response to Nalt was episodic in spite of Nalt's long half life time. In order to explain the rapid fall in LH levels after Nalt administration, normal rats were injected with a second bolus of Nalt, 2 hours after the first. The second bolus caused only a blunted response of LH secretion. In another experiment, administration of morphine (1 mg/rat) 2 hours after pretreatment with Nalt did not stimulate the prolactin secretion which normally follows morphine treatment. These results indicate that the rapid decrease of LH levels after Nalt treatment in normal rats is not due to absence of the drug in the system. It is suggested that other neural mechanisms, such as the dopaminergic system, are activated during Nalt influence.

Changes in LH and prolactin levels in diabetic male rats and the role of the opiate system in the control of their secretion.

Diabetic male rat has low serum levels of luteinizing hormone (LH) and testosterone (T), which are accompanied by atrophy of the testes and accessory glands. The present study investigated changes in the serum levels of LH, prolactin (PRL) and glucose, following diabetes induction by streptozotocin. In addition, involvement of the opiate system in the control of LH and PRL secretion was evaluated. There was no difference in PRL levels between diabetic and control animals, except at 8 hours after streptozotocin injection. In contrast, the diabetic animals had consistently lower levels of LH, starting on the second day of diabetes. Blockade of the opiate system by naltrexone caused a sharp increase of LH levels in normoglycemic rats, while only a gradual decrease was observed in hyperglycemic animals. PRL secretion was inhibited by naltrexone, both in diabetic and control groups. It is concluded that, unlike normoglycemic rats, inhibition of LH secretion in diabetes is not under the control of the opiate system, probably as a result of T deficiency. In contrast, PRL secretion in diabetic rats, as in the control group, is under the influence of endogenous opiates.

Treatment of cervical ectropion by cryosurgery: effect on cervical mucus characteristics.

Eighteen women with cervical ectropion and 12 women with ectropion and vaginal discharge were treated by cryosurgery. Evaluation of the cervical mucus characteristics by cervical score and in vitro penetration test was performed before treatment and 2 months later. In the group with ectropion only (group A) the total cervical score was 5.7 +/- 0.4 and 11.9 +/- 0.06 (P less than 0.001) (mean +/- standard error) before treatment and 2 months later, respectively. In the group with ectropion and vaginal discharge (group B) the total cervical score before and after cryosurgery was 3.8 +/- 0.4 and 11.8 +/- 0.1 (P less than 0.001), respectively. In vitro penetration tests in group A before and after treatment were 0.72 +/- 0.1 and 2.9 +/- 0.08 (P less than 0.001), respectively. In group B, in vitro penetration tests before and after cryosurgery were 0.25 +/- 0.1 and 2.8 +/- 0.1 (P less than 0.001), respectively. It appears that cryosurgery improves the cervical mucus characteristics. It is recommended that infertile patients with hostile cervical mucus and ectropion will be treated by cryosurgery.

The use of technetium in evaluation of the male accessory glands activity.

This study was performed with the purpose to elucidate the possibility of using Technetium 99m (Tc) as a marker for the evaluation of male sex glandular activity of the rat. Tc was injected into the femoral vein of the rat and its cpm was counted. The studies have shown that 22 hours after injection of Tc the material was accumulated in the glands in much higher values than its activity in the blood (which was only 1% of its zero time activity). In the rat, the dorsolateral coagulating gland has the highest capacity of accumulating Tc. Studies on castrated rats, or rats treated with testosterone enanthate, revealed that the activity of the cells in the glands and the volume of the excretion play an important role in the evaluation of the results. Thus, the use of Tc 99m in evaluating male accessory glands activity is a new interesting approach in the study of the physiology of the male sexual glands.

Effect of the antifertility agent, gossypol acetic acid, on the metabolism and testosterone secretion of isolated rat interstitial cells in vitro.

Gossypol acetic acid is a polyphenolic compound present in the seed of cotton plants. Its antifertility activity by inhibition of spermatogenesis was proven in a large group of animals, including man. In the present study, the direct effect of gossypol acetic acid on collagenase isolated rat I-cells (interstitial cells) was investigated. It was shown that gossypol acetic acid depressed significantly the metabolic rate of the cells. Glucose utilization was abolished by a starting dose of 100 micrograms/ml. Oxygen consumption of I-cells was reduced even at a smaller dose of gossypol (50 micrograms/ml). At these doses, the vitality of the cells remained (proven by trypan blue exclusion test). 3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) histochemical stain was slightly decreased. Increasing doses of gossypol caused a marked decrease in the vitality of I-cells and a dramatic drop in histochemical stain for 3 beta-HSD. The pH of the medium was not changed at any dose of treatment. In cultures of I-cells not stimulated by hCG, gossypol did not affect the tonic slow release of testosterone. Thus, gossypol acetic acid has a direct inhibitory effect on isolated rat I-cells, depressing cell metabolism. The failure of some of the other groups to show such an effect, especially in vivo, can be attributed to differences in the dose of treatment and strain of animals.