[Is there a difference in the postoperative follow up of ureteropelvic junction obstruction according to age?] / ¿Existe diferencia en la evolución postoperatoria de la estenosis pieloureteral según la edad?

OBJECTIVE: To compare postoperative follow up in patients older and younger than 12 months who underwent surgical treatment of ureteropelvic junction obstruction (UJO). MATERIAL AND METHODS: Retrospective study of 77 patients, 78 kidney units, intervened from UJO (2007-2014). We analyzed epidemiological, clinical, echographic, and pre and postoperative renogram variables, outcomes and complications. We divided the patients into 2 groups according to age: group A ≤ 12 months and group B > 12 months, comparing the results by statistical analysis, considering p < 0.05 statistically significant. RESULTS: Group A: 38 patients, 26 males (68.4%), one bilateral UJO and 22 rights (57.9%), 36 prenatal diagnoses (92.3%) and mean age of intervention 5.28 months [range 0.24 -11,28]. We performed 9 minilumbotomies, 29 assisted by retroperitoneoscopy (ARP) and 1 pneumatic dilation (PD). Group B: 39 patients, 26 males (66.7%), 10 rights (25.64%), 19 prenatal diagnoses (48.7%) and mean age 6.13 years [range 1.13-14.52]. 15 minilumbotomies, 20 ARP, 3 laparoscopic and 1 PD. Preoperative mean renal function (MRF) of group A: 35.9 ± 13.4 [range 8-57] vs. 39.74 ± 13.91 [range 9-57] in group B (p = 0.347). Postoperative MRF 43.29 ± 18.2 [range 12-100] group A and 39.41 ± 12.89 [range 11-54] group B (p = 0.464). Group A and B: 11 and 8 complications, respectively (p = 0.429). We did not find statistically significant differences in the mean preoperative anteroposterior diameter (DAP) between both groups (p = 0.313). We compared DAP at 3, 6, 12, 24 and 48 postoperative months, observing a greater reduction of DAP from group A compared to B; however, we found only statistically significant differences in DAP at 3 months postoperatively (p = 0.047). CONCLUSION: Renal DAP is reduced postoperatively more in patients younger than 1 year. Moreover, an improvement of the DRF after pieloplasty can be observed despite not being statistically significant.

OBJETIVO: Comparar la evolución postquirúrgica en pacientes mayores y menores de 12 meses intervenidos de estenosis pieloureteral (EPU). MATERIAL Y METODOS: Estudio retrospectivo de 77 pacientes, 78 unidades renales, intervenidos por EPU (2007-2014). Analizamos variables epidemiológicas, clínicas, ecográficas y de renogramas pre y postoperatorios, resultados y complicaciones. Dividimos a los pacientes en 2 grupos según la edad: grupo A ≤ 12 meses y grupo B > 12 meses, comparando los resultados mediante análisis estadísticos (p < 0,05 estadísticamente significativo). RESULTADOS: Grupo A: 38 pacientes, 26 varones (68,4%), una EPU bilateral y 22 derechas (57,9%), 36 diagnósticos prenatales (92,3%) y edad media de intervención 5,28 meses [rango 0,24-11,28]. Realizamos 9 minilumbotomías, 29 asistidas por retroperitoneoscopia (ARP) y una dilatación neumática (DN). Grupo B: 39 pacientes, 26 varones (66,7%), 10 derechas (25,64%), 19 diagnósticos prenatales (48,7%) y edad media 6,13 años [rango 1,13-14,52]. Realizamos 15 minilumbotomías, 20 ARP, 3 laparoscópicas y 1 DN. Función renal diferencial media (FRDM) preoperatoria del grupo A: 35,9 ± 13,4 [rango 8-57] vs. 39,74 ± 13,91 [rango 9-57] grupo B (p = 0,347). FRDM postoperatoria 43,29 ± 18,2 [rango 12-100] grupo A y 39,41 ± 12,89 [rango 11-54] grupo B (p = 0,464). Grupos A y B: 11 y 8 complicaciones, respectivamente (p = 0,429). No encontramos diferencias estadísticamente significativas en la media del diámetro anteroposterior (DAP) de la pelvis preoperatoria entre ambos grupos (p = 0,313). Comparamos los DAP a los 3, 6, 12, 24 y 48 meses postoperatorios, observando una reducción mayor del DAP del grupo A frente al B, sin embargo, solo encontramos diferencias estadísticamente significativas en el DAP a los 3 meses postoperatorios (p = 0,047). CONCLUSION: El DAP de la pelvis renal se reduce más en los pacientes menores de 1 año a los 3 meses postoperatorios. Además, podemos observar una evidente mejoría de la FRDM tras la pieloplastia a pesar de no encontrar diferencias estadísticamente significativas.

[The diagnosis of pilomatrixoma in children is not as easy as it may seem. A review of 126 cases]. / El diagnóstico del pilomatrixoma infantil no es tan fácil como parece: revisión de 126 casos.

OBJECTIVE: To describe our experience with pilomatrixomas, clinical presentation, clinical accuracy, associated diseases, and surgical treatment. METHODS: A retrospective review of patients with a pathology report of an excised pilomatrixoma between 2011 and 2014. Data regarding gender, age of intervention, number of masses, size, location, and preoperative clinical diagnosis, pathology report, recurrences, and associated diseases were collected. Statistical analysis was performed using EPIinfo 7.1.5. RESULTS: A total of 151 tumors in 138 patients were removed, 25 were excluded for having a clinical diagnosis of pilomatrixoma without confirmation of the pathology report. The remaining 126 cases have an average age of 8.26 years (range 1-14 years) and 50.72% are males. The tumors were located above the umbilicus in 97% of the cases, with a higher concentration of 50.72% in the head and neck. The clinical diagnosis corresponds with the pathology report in 69.05% of cases. Out of these confirmed cases the most frequent differential diagnosis is cystic lesion (11.90%) and dermoid cyst (10.22%). There were no cases of malignant degeneration. The average diameter is 1.13 cm, there were no recurrences, but there were 11 cases of a new lesion in a different location. There is no statistical association with other pathologies. The most frequent diseases found were obesity (3.62%) and attention deficit syndrome (3.62%). CONCLUSION: The pilomatrixoma is a tumor that appears predominantly in the head and neck, and has a difficult clinical diagnosis. Complete removal is curative, although it does not prevent the appearance of new lesions.

OBJETIVO: Describir nuestra experiencia con pilomatrixomas, examinando la presentación clínica, acierto clínico, enfermedades asociadas y tratamiento quirúrgico. MATERIAL Y METODOS: Estudio retrospectivo de los pacientes con diagnóstico anatomo-patológico (AP) de pilomatrixoma, en el periodo de 2011 a 2014. Se recopilaron datos sobre genero, edad de intervención, número de tumoraciones, tamaño, localización, diagnóstico preoperatorio, anatomía patológica, reintervenciones y patología asociada. Se realizó el análisis estadístico con EPIinfo 7.1.5. RESULTADOS: Se extirparon un total de 151 tumores en 138 pacientes, excluyendo 25 tumoraciones por tener diagnóstico clínico de pilomatrixoma sin confirmación patológica. Los 126 casos que tienen confirmación AP de pilomatrixoma tienen una edad media de exéresis de 8,26 años de edad (1-14 años) y el 50,72% son del género masculino. El 97% de los tumores se localizan por encima del ombligo, concentrándose en la cabeza y cuello el 52,9%. La sospecha clínica corresponde con AP en el 69,05%. De los casos confirmados, los diagnósticos clínicos diferenciales más frecuentes son lesión quística (11,90%) y quiste dermoide (10,22%); no hubo pilomatrixomas con degeneración maligna. La media de diámetro es de 1,13 cm, no hubo recidivas locales, pero sí 11 nuevos casos de pilomatrixomas en otra localización. No hay asociación estadísticamente significativa a otras patologías. Las enfermedades más frecuentes encontradas son la obesidad (3,62%) y el síndrome de déficit de atención (3,62%). CONCLUSION: El pilomatrixoma es una tumoración predominantemente de aparición en cara y cuello, de difícil diagnóstico clínico. La exéresis completa es curativa, aunque no previene la aparición de nuevas lesiones.

[Efficacy of the retroperitoneal-assisted laparoscopic pyeloplasty versus the mini posterior lumbotomy approach]. / Eficacia de la pieloplastia asistida por retroperitoneoscopia versus a la realizada por minilumbotomía posterior.

OBJECTIVE: To evaluate the results of the retroperitoneal-assisted laparoscopic pyeloplasty (RALP) versus the mini posterior lumbotomy pyeloplasty (MPLP). MATERIAL AND METHODS: A retrospective study of 77 patients diagnosed with ureteropelvic junction obstruction between 2007 and 2013 was made, analyzing the surgical technique, complications and results. The anteroposterior pelvic diameter of the kidney, the thickness of the renal parenchyma, the renal function and the morphology of the renogram curve were also evaluated and compared. RESULTS: We performed 50 RALP and 21 MPLP. Median age of intervention: 10.85 moths (ICR 86.8) in RALP and 23.30 moths (ICR 54.7) in MPLP. No significant differences were found in surgical time (p>0.05). Double J was left in 90% of the RALP and 52.4% of the MPLP. Median length of incision was 1.5 cm of the RALP and 3.0cm in MPLP(p<0.05). The Median hospital stay was 2.0 days and 3.0 respectively (p<0.05). We found surgical complications (restenosis and urinoma) in 9 patients of the RALP group and 1 in the mini lumbotomy group (p>0,05). The follow-up was performed using ultrasound in an average of 68,04 months of age (range 7.5-186.5) and diuretic renogram in an average of 50,25 months of age (range 6,6-173,8). The above parameters showed improvements in both groups of patients without significant differences. CONCLUSIONS: In our experience, the RALP is a technique with a greater rate of complications than the expected. Moreover, the MPLP, is a technique we consider safe and with adequate aesthetic and functional results.

OBJETIVO: Evaluar la pieloplastia asistida por retroperitoneoscopia, exteriorizando la unión pieloureteral (PAR) versus la pieloplastia por minilumbotomía posterior en ángulo costovertebral (PMLP). MATERIAL Y METODOS: Estudio retrospectivo de 77 pacientes diagnosticados de estenosis pieloureteral (2007-2013), se analizaron técnica, complicaciones y resultados, mediante la valoración del diámetro anteroposterior de la pelvis renal, grosor del parénquima renal, función renal y morfología de la curva del renograma. RESULTADOS: Realizamos 50 PAR y 21 PMLP. Edad mediana de intervención: 10,85 meses [rango intercuartílico (IC) 86,8] en PAR y 23,3 meses (rango IC 54,7) en PMLP. No hubo diferencias significativas en tiempo quirúrgico. Se dejó doble J en 90% de las PAR y en 52,4% de las PMLP. La incisión fue 1,5 cm en la PAR y 3,0 cm de mediana (rango IC 1,0) en la PMLP (p<0,05). La mediana de estancia fue 2,0 días y 3,0 respectivamente (p<0,05). Encontramos complicaciones (reestenosis y urinomas) en 9 pacientes del grupo PAR y en uno en el grupo de minilumbotomía (p>0,05). El seguimiento mediante ecografía en una media de 68,04 meses de edad (rango 7,5-186,5) y renograma diurético en una media de 50,25 meses de edad (rango 6,6-173,8), evidenciaron mejora en los parámetros arriba señalados en ambos grupos de pacientes, sin diferencias significativas. CONCLUSIONES: En nuestra serie, la PAR es un abordaje con un índice de complicaciones mayor al esperado. Por otra parte, la PMLP es un abordaje seguro y con resultados estéticos y funcionales adecuados.

[Endocrine disrupting chemicals exposure and other parental factors in hypospadias and cryptorchidism etiology]. / Exposición a disruptores endocrinos y otros factores paternos en la etiología del hipospadias y la criptorquidia.

AIM OF THE STUDY: To investigate the association between endocrine disrupting chemicals (EDC) exposure and other paternal factors in the etiology of hipospadias and cryptorchidism. METHODS: A case-control study. Cases were infants between 0 and 6 years of age diagnosed with hypospadias or cryptorchidism in our pediatric urology and general pediatric surgery services during a period of 6 months, and controls were infants with the same range of age attending the same services without any urological problem. Several variables were collected by face-to-face interviews with both parents. After data abstraction, we compared the characteristics of both groups using parametric statistical tests. MAIN RESULTS: A total of 180 patients were studied, 90 cases (45 hypospadias/45 cryptorchidism) and 90 controls with a mean age of 2,37 ± 1.50 years [range 0,5-6]. Median of mother´s age was significantly greater in case group (34,40 ± 5,64 versus 31,74 ± 5,05; p= 0,001). Significant differences were observed between cases and controls in regard to maternal occupational exposure to EDC (mainly phthalates), adjusted Odds ratio (OR) was 3.67 [95% confidence interval (CI): 1.28-10.51; p= 0,018] and regarding the paternal occupational exposure to EDC (mainly pesticides and herbicides), adjusted OR was 6.65 [95% CI: 2.60-17.02; p= 0,001]. Increased risk was also observed in smoking fathers and fathers who drink alcohol, adjusted Odds ratio were 2.36 [95% CI: 1.11. CONCLUSIONS: This study represents a little contribution to the possible etiologic factors of hypospadias and cryptorchidism, further studies with higher statistical power would be needed to prove it.

OBJETIVOS: Investigar la asociación entre la exposición a disruptores endocrinos (DE) y otros factores en el desarrollo del hipospadias y la criptorquidia. MATERIAL Y METODS: Estudio de casos y controles. Consideramos como casos a los niños de entre 6 meses y 6 años de edad diagnosticados de hipospadias y/o criptorquidia que acudieron a las consultas de Urología/Cirugía durante un período de estudio de 6 meses y como controles, a los niños con mismo rango de edad que acudieron a las mismas consultas con otros diagnósticos. Recogimos las variables de interés mediante una encuesta epidemiológica y comparamos los resultados obtenidos en cada grupo mediante tests estadísticos paramétricos. RESULTADOS: Estudiamos 180 pacientes, 90 casos (45 hipospadias/45 criptorquidias) y 90 controles, con edad media de 2,60 ± 1,72 años [rango 0,5-6]. Las medias de edad gestacional y peso al nacer fueron menores en el grupo-caso sin objetivarse diferencias significativas. La edad media materna fue significativamente mayor en el grupo-caso (34,40 ± 5,64 versus 31,74 ± 5,05; p= 0,001). Encontramos asociación significativa entre la exposición ocupacional materna a DE (ftalatos principalmente) y el grupo-caso siendo la Odds ratio (OR) de 3,67 (IC 95%: 1,28-10,51; p= 0,018) y también en la paterna (principalmente a pesticidas/herbicidas) con OR= 6,65 (IC 95%: 2,60-17,02; p= 0,001). Encontramos asociación significativa entre el consumo de tabaco y alcohol paternos y el grupo-caso: OR= 2,08 (IC 95%: 1,11-3,87; p= 0,029) y OR= 2,50 (IC 95%: 1,36-4,57; p= 0,003) respectivamente. CONCLUSIONES: Este estudio supone una pequeña aportación respecto a los posibles factores etiológicos del hipospadias y la criptorquidia, y demuestra la necesidad de estudios ulteriores con mayor potencia estadística para aumentar la evidencia científica de nuestros hallazgos.

Síndrome del babuino por exposición a mercurocromo en el curso de una varicela / Baboon syndrome after exposure to mercurochrome during chickenpox

No disponible

Nuestra experiencia con el uso de alfa-líticos en el tratamiento de la disfunción de vaciado / Our experience with the use of alpha-lithic therapy in the treatment of voiding dysfunction

Introducción. El vaciamiento disfuncional (VD) se debe a una disfunción en la musculatura del suelo pélvico, ocasionado por una hiperactividad del esfínter uretral y de la musculatura de esa zona durante la fase de vaciado del ciclo miccional. Los tratamientos utilizados son recomendar hábitos miccionales correctos, biofeedback y ejercicios de relajación pélvica. En la actualidad, también se utilizan fármacos, como alfalíticos e injección de toxina botulínica, pero su experiencia en niños es escasa. Objetivos. Mostrar nuestra experiencia con el uso de alfalíticos en pacientes con VD. Material y métodos. Revisión de los pacientes con VD tratados con alfa-líticos en nuestro servicio. Resultados. 6 pacientes con VD fueron tratados con alfa-líticos(Alfuzosina o Doxazosina). Niña (12 años) con síndrome de Wolfram (..) (AU)

Introduction. Dysfunctional voiding (DV) is due to a dysfunction in the pelvis floor muscles caused by hyperactivity of the urethral sphincter and the musculature of this zone during the voiding phase of the micturition cycle. The treatments used are recommending correct micturition habits, biofeedback and pelvic relaxation exercises. Currently, drugs are also used, such as alpha lithic drugs and injection of botulinic toxin, however its experience in children is limited. Objectives. Show our experience with the use of alpha-lithics inpatients with DV. Material and methods. Review of patients with DV treated with alpha-lithics in our institution .Results. 6 patients with DV were treated with alpha-lithics (Alfuzosin or Doxazosin). Girl (12 years) with Wolfram Syndrome with significant post-void residual urine, who initially rejected intermittent catéter. Residual urine decreases slightly with treatmente, but isn´t (..) (AU)

[Our experience with the use of alpha-lithic therapy in the treatment of voiding dysfunction]. / Nuestra experiencia con el uso de alfa-líticos en el tratamiento de la disfunción de vaciado.

INTRODUCTION: Dysfunctional voiding (DV) is due to a dysfunction in the pelvis floor muscles caused by hyperactivity of the urethral sphincter and the musculature of this zone during the voiding phase of the micturition cycle. The treatments used are recommending correct micturition habits, biofeedback and pelvic relaxation exercises. Currently, drugs are also used, such as alpha lithic drugs and injection of botulinic toxin, however its experience in children is limited. OBJECTIVES: Show our experience with the use of alpha-lithics in patients with DV. MATERIAL AND METHODS: Review of patients with DV treated with alpha-lithics in our institution. RESULTS: 6 patients with DV were treated with alpha-lithics (Alfuzosin or Doxazosin). Girl (12 years) with Wolfram Syndrome with significant post-void residual urine, who initially rejected intermittent catéter. Residual urine decreases slightly with treatment, but isn't enough. Boy (7 years) with uretral duplicity and difficulty urine flow without observable stenosis. He had initial improvement but then required continent bladder diversión and vasectomy due to recurrent orchiepididymitis. Boy (5 years) with ureteral-bladder stenosis and ureteral reimplantation with urinary tract infection and vesico-ureteral reflux that did not improve (an anatomic obstruction was subsequently found). Boy (12 years) with tethered cord and pyelonephritis, who rejected intermittent catheter. He hadn't improvement and he required continent bladder diversión. Boy (7 years) with Syringomyelia, operated in another center for vesico-ureteral reflux with ureteral reimplantation, with recurrent urine infections, who hadn't improvement and required continent bladder diversion. Boy (10 years) with resected urethra valves and normal cystoscopy, with episodes of urine retention, without improvement with treatment. CONCLUSION: The role of alpha-lithics in the treatment of dysfunctional voiding in children is limited and of doubtful efficacy. It may be useful in patients with significant post-micturition residue.

Día mundial del riñón y nefrologia en Cuba: tratar algo más que la enfermedad renal / The World Kidney Day and the nephrology in Cuba. Treat something more than the kidney disease

Las enfermedades renales son frecuentes, perjudiciales y tratables. Las jornadas internacionales por el Día del Riñón en su segundo año de celebración llama a la acción no sólo a los nefrólogos y pacientes renales, sino a médicos de todas las especialidades, enfermeras, científicos, expertos, administradores, gobiernos, para que estén conscientes de los desafíos que representa la Enfermedad Renal Crónica (ERC). En este artículo, se analiza el desarrollo de la Nefrología en Cuba y la relación de la ERC a otras enfermedades crónicas no transmisibles caracterizadas por el daño endotelial. Su prevención, diagnóstico y tratamiento es la vía común para contribuir a disminuir la morbimortalidad cardiovascular internacionalmente(AU)

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.

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.

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.

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.