Use of internal-external diversion stent in open pyeloplasty in patients under one year of age. / Uso de catéter de derivación interno-externo en la pieloplastia abierta en menores de un año.

INTRODUCTION: There are various alternatives available for renal pelvis drainage following pyeloplasty. One of them is the use of an internal-external diversion stent, which according to our protocol, is knotted 48 hours following surgery, prior to discharge, and removed 7 days later on an outpatient consultation basis, with no sedation or analgesia required. OBJECTIVE: To analyze the results of patients under one year of age who underwent open pyeloplasty associated with an outpatient internal-external diversion stent. MATERIALS AND METHODS: A retrospective, descriptive analysis of 28 patients (31 renal units) undergoing surgery from 2011 to 2021 was carried out. Diagnostic methods, indications, surgical approach, and postoperative progression were assessed. RESULTS: 28 patients (23 male) prenatally diagnosed with hydronephrosis confirmed by ultrasonography and/or renogram underwent pyeloplasty at a median age of 3 months (15 days-11 months). Pyeloplasty was conducted according to the Anderson-Hynes technique or dismembered pyeloplasty in 28 renal units, and according to the Culp-DeWeerd technique or spiral flap in 3. In all cases, an internal-external diversion stent was used according to our protocol. Mean hospital stay was 3.5 days (2-7 days), with a good postoperative progression. 2 patients had complications (urinary infection requiring intravenous antibiotics, and pyonephrosis requiring re-pyeloplasty). CONCLUSIONS: Using an internal-external diversion stent following pyeloplasty in patients under 1 year of age with ureteropelvic junction obstruction is a simple and safe option that allows for early discharge with outpatient management. It also avoids a second general anesthesia for drainage catheter removal purposes.

INTRODUCCION: Existen diversas alternativas para el drenaje de la pelvis renal tras una pieloplastia. Una de ellas es la utilización de un catéter de derivación interno-externo que, según nuestro protocolo, se anuda a las 48 horas posoperatorias previas al alta y se retira a los siete días de forma ambulatoria en consulta, sin necesidad de sedoanalgesia. OBJETIVO: Analizar los resultados de los pacientes menores de un año intervenidos mediante pieloplastia abierta, asociando un catéter de derivación interno-externo de manejo ambulatorio. MATERIAL Y METODOS: Análisis descriptivo retrospectivo de 28 pacientes (31 unidades renales) intervenidos entre los años 2011 y 2021. Se evaluaron métodos diagnósticos, indicaciones, abordaje quirúrgico y evolución posoperatoria. RESULTADOS: Veintiocho pacientes (23 varones) con diagnóstico prenatal de hidronefrosis confirmado con ecografía y/o renograma, fueron intervenidos mediante pieloplastia a una mediana de edad de tres meses (15 días-11 meses). Se realizó pieloplastia según técnica de Anderson-Hynes o pieloplastia desmembrada en 28 unidades renales y según técnica de Culp-DeWeerd o colgajo en espiral en 3. En todos los casos se utilizó un catéter de derivación interno-externo según protocolo. El tiempo medio de ingreso fue 3,5 días (2-7 días) con buena evolución posoperatoria. Dos pacientes presentaron complicaciones (infección urinaria que requirió antibioterapia intravenosa y pionefrosis que requirió repieloplastia). CONCLUSIONES: Asociar un catéter de derivación interno-externo a la pieloplastia en pacientes menores de un año con estenosis de la unión pieloureteral es una opción sencilla y segura que permite un alta precoz con manejo ambulatorio y evita una segunda anestesia general para la retirada del catéter de drenaje.

Uso de catéter de derivación interno-externo en la pieloplastia abierta en menores de un año / Use of internal-external diversion stent in open pyeloplasty in patients under one year of age

Introducción: Existen diversas alternativas para el drenaje de la pelvis renal tras una pieloplastia. Una de ellas es la utilización de un catéter de derivación interno-externo que, según nuestro protocolo, se anuda a las 48 horas posoperatorias previas al alta y se retira a los siete días de forma ambulatoria en consulta, sin necesidad de sedoanalgesia. Objetivos: Analizar los resultados de los pacientes menores de un año intervenidos mediante pieloplastia abierta, asociando un catéter de derivación interno-externo de manejo ambulatorio. Material y métodos: Análisis descriptivo retrospectivo de 28 pacientes (31 unidades renales) intervenidos entre los años 2011 y 2021. Se evaluaron métodos diagnósticos, indicaciones, abordaje quirúrgico y evolución posoperatoria. Resultados: Veintiocho pacientes (23 varones) con diagnóstico prenatal de hidronefrosis confirmado con ecografía y/o renograma, fueron intervenidos mediante pieloplastia a una mediana de edad de tres meses (15 días-11 meses). Se realizó pieloplastia según técnica de Anderson-Hynes o pieloplastia desmembrada en 28 unidades renales y según técnica de Culp-DeWeerd o colgajo en espiral en 3. En todos los casos se utilizó un catéter de derivación interno-externo según protocolo. El tiempo medio de ingreso fue 3,5 días (2-7 días) con buena evolución posoperatoria. Dos pacientes presentaron complicaciones (infección urinaria que requirió antibioterapia intravenosa y pionefrosis que requirió repieloplastia). Conclusiones: Asociar un catéter de derivación interno-externo a la pieloplastia en pacientes menores de un año con estenosis de la unión pieloureteral es una opción sencilla y segura que permite un alta precoz con manejo ambulatorio y evita una segunda anestesia general para la retirada del catéter de drenaje.(AU)

Introduction: There are various alternatives available for renal pelvis drainage following pyeloplasty. One of them is the use of an internal-external diversion stent, which according to our protocol, is knotted 48 hours following surgery, prior to discharge, and removed 7 days later on an outpatient consultation basis, with no sedation or analgesia required. Objective: To analyze the results of patients under one year of age who underwent open pyeloplasty associated with an outpatient internalexternal diversion stent. Materials and methods: A retrospective, descriptive analysis of 28 patients (31 renal units) undergoing surgery from 2011 to 2021 was carried out. Diagnostic methods, indications, surgical approach, and postoperative progression were assessed. Results: 28 patients (23 male) prenatally diagnosed with hydronephrosis confirmed by ultrasonography and/or renogram underwent pyeloplasty at a median age of 3 months (15 days-11 months). Pyeloplasty was conducted according to the Anderson-Hynes technique or dismembered pyeloplasty in 28 renal units, and according to the Culp-DeWeerd technique or spiral flap in 3. In all cases, an internal-external diversion stent was used according to our protocol. Mean hospital stay was 3.5 days (2-7 days), with a good postoperative progression. 2 patients had complications (urinary infection requiring intravenous antibiotics, and pyonephrosis requiring re-pyeloplasty). Conclusions: Using an internal-external diversion stent following pyeloplasty in patients under 1 year of age with ureteropelvic junction obstruction is a simple and safe option that allows for early discharge with outpatient management. It also avoids a second general anesthesia for drainage catheter removal purposes.(AU)

Common surgical training program: standardization of learning quality. / Programa de formación quirúrgica común: uniformidad en la calidad del aprendizaje.

INTRODUCTION: The various surgical specialties in our center have used the simulation and experimental surgery resources available for their training tasks in minimally invasive surgery (MIS) in an individualized manner. With this learning model, a great dispersion of effort and expense was observed, so it was decided to create a unified program based on the following: shared learning, synergy among specialties, moderation of the economic cost, and rational use of the facilities. OBJECTIVE: To describe and assess our consensually designed training program in order to consolidate a shared learning strategy that will enable our residents to acquire and perfect surgical skills in MIS. MATERIALS AND METHODS: The program consists of various increasingly complex phases implemented on a continuous basis throughout the period of specialized training in the virtual laboratory and experimental operating room. The assessment methods were based on quantifiable criteria: percentage of efficiency and completion time of the "McGill Inanimate System for Training and Evaluation of Laparoscopic Skills" (MISTELS) exercises at the beginning and end of the program. An economic study was also conducted. RESULTS: 20 residents have completed the program. Mean times show a significant reduction in each of the exercises. The efficiency percentages at the end of the program were higher than at the beginning (p < 0.001). The cost of the program represented a saving of 67.89%. CONCLUSION: The new MIS training program improved the quality of learning in a safe environment, establishing common criteria among the different specialties and an improved use of resources.

INTRODUCCION: Las diferentes especialidades quirúrgicas de nuestro centro han usado los recursos de simulación y cirugía experimental para sus tareas de formación en cirugía mínimamente invasiva (CMI) de manera individualizada. Con este modelo de aprendizaje se detectó una gran dispersión de esfuerzos y gasto, por lo que se decidió crear un programa unificado basado en: aprendizaje compartido, sinergia entre especialidades, moderación del coste económico y uso racional de las instalaciones. OBJETIVO: Describir y evaluar nuestro programa de entrenamiento diseñado por consenso de cara a la consolidación de una estrategia de aprendizaje compartido que permita a nuestros residentes adquirir y perfeccionar habilidades quirúrgicas en CMI. MATERIAL Y METODOS: El programa consta de diferentes fases con complejidad creciente desarrolladas durante todo el periodo de formación especializada de forma continuada en laboratorio virtual y quirófano experimental. Los criterios de evaluación se basaron en criterios cuantificables: porcentaje de eficiencia y tiempo de realización de los ejercicios de McGill Inanimate System for Training and Evaluation of Laparoscopic Skills (MISTELS) al inicio y final del programa. Se realizó también el estudio económico. RESULTADOS: Han completado el programa 20 residentes. Los tiempos medios demuestran una reducción significativa en cada uno de los ejercicios. Los porcentajes de eficiencia al final fueron mayores que al inicio del programa (p < 0,001). El coste del programa supuso un ahorro del 67,89%. CONCLUSION: El nuevo programa de entrenamiento en CMI mejoró la calidad de aprendizaje en un entorno seguro, estableciendo criterios comunes entre las diferentes especialidades y un mayor aprovechamiento de los recursos.

Programa de formación quirúrgica común: uniformidad en la calidad del aprendizaje / Common surgical training program: standardization of learning quality

Introducción: Las diferentes especialidades quirúrgicas de nuestrocentro han usado los recursos de simulación y cirugía experimentalpara sus tareas de formación en cirugía mínimamente invasiva (CMI)de manera individualizada. Con este modelo de aprendizaje se detectóuna gran dispersión de esfuerzos y gasto, por lo que se decidió crear unprograma unificado basado en: aprendizaje compartido, sinergia entreespecialidades, moderación del coste económico y uso racional de lasinstalaciones. Objetivo: Describir y evaluar nuestro programa de entrenamientodiseñado por consenso de cara a la consolidación de una estrategia deaprendizaje compartido que permita a nuestros residentes adquirir yperfeccionar habilidades quirúrgicas en CMI. Material y métodos: El programa consta de diferentes fases concomplejidad creciente desarrolladas durante todo el periodo de forma-ción especializada de forma continuada en laboratorio virtual y quiró-fano experimental. Los criterios de evaluación se basaron en criterioscuantificables: porcentaje de eficiencia y tiempo de realización de losejercicios de McGill Inanimate System for Training and Evaluation ofLaparoscopic Skills (MISTELS) al inicio y final del programa. Se realizótambién el estudio económico. Resultados: Han completado el programa 20 residentes. Los tiem-pos medios demuestran una reducción significativa en cada uno de losejercicios. Los porcentajes de eficiencia al final fueron mayores queal inicio del programa (p < 0,001). El coste del programa supuso unahorro del 67,89%. Conclusión: El nuevo programa de entrenamiento en CMI mejoróla calidad de aprendizaje en un entorno seguro, estableciendo criterioscomunes entre las diferentes especialidades y un mayor aprovechamientode los recursos.(AU)

Introduction: The various surgical specialties in our center haveused the simulation and experimental surgery resources availablefor their training tasks in minimally invasive surgery (MIS) in anindividualized manner. With this learning model, a great dispersionof effort and expense was observed, so it was decided to create aunified program based on the following: shared learning, synergyamong specialties, moderation of the economic cost, and rationaluse of the facilities. Objective: To describe and assess our consensually designed train-ing program in order to consolidate a shared learning strategy that willenable our residents to acquire and perfect surgical skills in MIS. Materials and methods: The program consists of various increas-ingly complex phases implemented on a continuous basis throughout theperiod of specialized training in the virtual laboratory and experimentaloperating room. The assessment methods were based on quantifiablecriteria: percentage of efficiency and completion time of the “McGillInanimate System for Training and Evaluation of Laparoscopic Skills”(MISTELS) exercises at the beginning and end of the program. Aneconomic study was also conducted. Results: 20 residents have completed the program. Mean timesshow a significant reduction in each of the exercises. The efficiencypercentages at the end of the program were higher than at the begin-ning (p < 0.001). The cost of the program represented a saving of67.89%.(AU)

Oocyte quality and in vivo embryo survival after ovarian stimulation in nulliparous and multiparous rabbit does.

Superovulation treatments aim to stimulate multifollicular recruitment, maximizing the number of oocytes or transferable embryos produced. Factors associated with the superovulation protocol, female characteristics and many other factors are determinants in the number and quality of oocytes obtained. An accurate way to assess oocyte quality more precise than morphological appearance is genetic expression. The present study aims to compare the response of nulliparous and multiparous females to superovulatory stimulation, studying its effect on the expression of some genes associated with the activation, growth, development and oocyte-embryo transition of oocytes, as well as its impact on in vivo embryonic development and viability rate at birth. In a first experiment, the effect of stimulation treatment on the ovulation response and the expression of the MSY2, MATER, ITPR1, ITPR2, ITPR3, eIF4E, PAR1, PAPOL-A, PAPOL-G, ZAR1 and YY1 genes in nulliparous and multiparous females were determined. In a second experiment, the implantation and viability at birth of embryos from superovulated nulliparous and multiparous females were analysed. The ovulation rate was significantly higher in the superovulation groups than in the control groups. The ovulation rate was significantly increased in nulliparous females compared with multiparous does. From the eleven genes analysed, only the expression of MATER, PAPOL-A, PAPOL-G and ZAR-1 genes was shown to be different among experimental groups. Finally, in terms of implantation rate and viability at birth, the nulliparous control group showed better results than the rest of the groups. Both hyperstimulation treatment and reproductive female's history seem to alter the transcriptome of important genes related to oocyte maturation and competence acquisition, affecting in vivo embryo viability.

π-Extended push-pull azo-pyrrole photoswitches: synthesis, solvatochromism and optical band gaps.

A new family of push-pull biphenyl-azopyrrole compounds 3b-g and 4b-d was efficiently obtained via a Suzuki cross-coupling reaction between 2-(4'-iodophenyl-azo)-N-methyl pyrrole (1a) or 3-(4'-iodophenyl-azo)-1,2,5-trimethyl pyrrole (2a) and 4'-substituted phenyl boronic acids in excellent yields. The influence of the π-biphenyl backbone and pyrrole pattern substitution was correlated with their optical properties. Solvatochromic studies via UV-visible spectrophotometry revealed that the inclusion of a 4'-nitro-biphenyl fragment favors a red-shift of the main absorption band in these azo compounds compared with their non-substituted analogues. Likewise, optical band-gaps were estimated by means of electronic absorption spectra and correlated with TD-DFT studies. The pyrrole pattern substitution and the π-conjugated backbone exhibit a clear influence on their thermal isomerization kinetics at room temperature. In all cases, biphenylazo-pyrrole compounds lead to the formation of J-type aggregates in binary MeOH : H2O solvents. Under these conditions, compounds 3b-c undergo a water-assisted cis-to-trans isomerization at room temperature.

[Hydrostatic enema outcome in patients with long history of ileocolic intussusception]. / Resultados del enema hidrostático en pacientes con clínica prolongada de invaginación ileocólica.

PURPOSE: Hydrostatic enema is the gold standard treatment for ileocolic intussusception in stable patients without radiologic complication. There is no consensus about the influence of long history of symptoms in the outcome of this treatment. The aim of this study is to determine whether hydrostatic enema is effective and safe in patients with history of ileocolic intussusception of over 24 hours. MATERIAL AND METHODS: Retrospective review of all patients with ileocolic intussusception admitted to our hospital between 2014 and 2017. We divided the patients in whom enema was attempted on into two groups according to the length of history: over or under 24 hours. Statistical analysis was done by using the Fisher's exact test. RESULTS: In this study period 59 children presented with ileocolic intussusception. Duration of symptoms was variable (range 2 hours-7 days). Of the total of patients, initial hydrostatic enema was attempted on 49, with a success rate of 91.8% (45 patients). In the group of history <24 hours (33 patients, 67.3%) we observed a 93.9% effectiveness and in the group >24 hours (16 patients, 32.7%) effectiveness was 87.5%. Success rate showed no significant difference when comparing both groups (p=0.588). No complications were seen after enema reduction. CONCLUSIONS: The use of hydrostatic enema in patients with long history of intussusception is successful and safe. We consider it to be the first-line treatment in stable patients with no radiologic complication, regardless the duration of symptoms.

OBJETIVOS: El gold standard para el tratamiento de la invaginación ileocólica en el paciente estable sin complicación radiológica es el enema hidrostático. No hay unanimidad sobre si la sintomatología prolongada de invaginación influye en los resultados de dicho tratamiento. El objetivo de este estudio es determinar si el enema hidrostático es efectivo y seguro en pacientes con clínica de invaginación ileocólica mayor de 24 horas. MATERIAL Y METODOS: Estudio retrospectivo de los pacientes diagnosticados de invaginación ileocólica en nuestro hospital entre 2014 y 2017. Hemos dividido en 2 grupos a los pacientes a los que se realizó enema en función del tiempo de clínica, mayor o menor de 24 horas. El análisis estadístico se realizó mediante el test exacto de Fisher. RESULTADOS: En este periodo se atendieron 59 niños con invaginación ileocólica con duración variable de los síntomas (2 horas a 7 días). Del total de pacientes, en 49 se realizó enema hidrostático inicial con una efectividad del 91,8% (45 pacientes). En el grupo de clínica <24 horas (33 pacientes, 67,3%) se objetivó una efectividad del 93,9% y en el grupo de clínica >24 horas (16 pacientes, 32,7%) se objetivó una efectividad del 87,5%. No hubo diferencias significativas al comparar la efectividad entre ambos grupos (p=0,588). En ningún paciente hubo complicaciones tras el enema. CONCLUSIONES: El empleo del enema hidrostático en pacientes con clínica prolongada de invaginación es efectivo y seguro. Consideramos que debería ser la primera herramienta terapéutica en el paciente estable sin complicación radiológica, independientemente del tiempo de evolución.

Protection of GnRH analogue by chitosan-dextran sulfate nanoparticles for intravaginal application in rabbit artificial insemination.

The present study was designed to prove new rabbit insemination extenders containing aminopeptidase inhibitors (AMIs) with or without chitosan (CS)-dextran sulfate (DS) nanoparticles entrapping the GnRH analogue. In addition, different hormone concentrations were tested in these extenders, evaluating their in vivo effect on rabbit reproductive performance after artificial insemination. A total of 911 females were inseminated with semen diluted with the four experimental extenders (C4 group: 4 µg buserelin/doe in control medium (Tris-citric acid-glucose supplemented with bestatin 10 µM and EDTA 20 mM), C5 group: 5 µg of buserelin/doe in control medium, Q4 group: 4 µg of buserelin/doe into CS-DS nanoparticles in control medium, Q5 group: 5 µg of busereline/doe into CS-DS nanoparticles in control medium). Results showed that fertility was significantly lower in C4 group compared to C5, Q5 and Q4 groups (0.7 versus 0.85, 0.85 and 0.82, respectively). On the contrary, prolificacy was similar in the four experimental groups studied (P > 0.05). We conclude that the CS-DS nanoparticles prepared by a coacervation process as carrier for buserelin acetate allows reducing the concentration of hormone used in extenders supplemented with bestatin and EDTA without affecting the fertility and prolificacy of rabbit females.

Insemination extender supplementation with bestatin and EDTA has no effect on rabbit reproductive performance.

The addition of aminopeptidase inhibitors (AMIs) to rabbit semen extenders could be a solution to decrease the hormone degradation (GnRH) by the aminopeptidases existing in the seminal plasma. Therefore, the quantity of GnRH needed to induce ovulation in doe would be comparable with the amount administered intramuscularly (i.m.). This study was conducted to evaluate the effects of two AMIs (bestatin and EDTA) on rabbit semen quality parameters, ß nerve growth factor (ß-NGF) degradation and reproductive performance after artificial insemination. Results showed that seminal quality was not affected by the incubation with AMIs; the values of motility, acrosome integrity and sperm viability were not significantly different between the AMIs and the control groups (positive i.m. and negative intravaginally without AMIs). In addition, the aminopeptidase activity of seminal plasma was inhibited in a 55.5% by the AMIs as well as ß-NGF degradation. On the other hand, regarding the effect of AMIs on reproductive performance, our results showed that the presence of bestatin and EDTA did neither affect fertility (85.3 vs. 88.6%), nor the prolificacy rate (10.12 vs. 10.51 kits per delivery), comparing AMIs group to positive control group, respectively. We conclude that the addition of specific AMIs in the rabbit semen extender has no effect on reproductive performance. Therefore, due to the fact that AMIs inhibit part of the aminopeptidase activity that degrades the GnRH analogue and ß-NGF, they could be used to develop new extenders with less hormone concentration.

Chitosan-dextran sulphate nanoparticles for GnRH release in rabbit insemination extenders.

This study was designed to develop chitosan (CS)-dextran sulphate (DS) nanoparticles containing a GnRH analogue and to study their effect on rabbit (Oryctolagus cuniculus) semen quality. Six experimental extenders were tested as follows: (control) Tris-citric acid-glucose (TCG), (1) 0.05% CS-0.05% DS (4:1), (2) 0.1% CS-0.05% DS (4:1), (3) 0.05% CS-0.05% DS (3:1), (4) 0.1% CS-0.05% DS (3:1), (5) 0.1% CS-0.05% DS (2:1). CS and DS were dissolved in TCG medium, and nanoparticles were obtained through magnetic stirring. Rabbit seminal samples were incubated up to 5 hr at 37°C in the extenders, and seminal quality was evaluated. The entrapment efficiency was 40%-50%. After 5 hr at 37°C, a 20% of the hormone was released. Results showed that the presence of CS-DS nanoparticles did not affect rabbit semen motility, viability and membrane functionality; however, acrosome integrity was significantly higher versus control (p < .001).

Can the Genetic Origin Affect Rabbit Seminal Plasma Protein Profile along the Year?

The study was designed to evaluate the influence of genetic origin on rabbit seminal plasma protein profile variation along the year. Seminal plasma of rabbits from line A (maternal line) and R (paternal line) collected during a natural year was subjected to polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoretic profile of rabbit seminal plasma resulted in multiple protein bands of different intensity ranging from 9 to 240 kDa. Results showed that seven protein bands were significantly different between genetic lines, and among these, three protein bands were significantly different between seasons. The differentially expressed proteins were identified by MALDI-TOF/TOF or LC-MS/MS analysis and were the following ones: FAM115E-like (220, 113 and 59 kDa), ectonucleoside triphosphate diphosphohydrolase 3 isoform X2 (72 kDa), annexin A5 (32 kDa), lipocalin allergen Ory c 4 precursor (19 kDa), and haemoglobin subunit zetalike (13 kDa) between genetic lines and FAM115E-like (113 kDa), haemoglobin subunit zetalike (13 kDa) and ß-nerve growth factor (12 kDa) between seasons. These results indicate that proteins from rabbit seminal plasma are under both seasonal control and genetic control. Furthermore, the differential presence of these proteins could be one of the causes explaining the differences observed in fertility and seminal parameters between these two lines in earlier studies.

Does the inclusion of protease inhibitors in the insemination extender affect rabbit reproductive performance?

The bioavailability of buserelin acetate when added to the seminal dose appears to be determined by the activity of the existing aminopeptidases. Thus, the addition of aminopeptidase inhibitors to rabbit semen extenders could be a solution to decrease the hormone degradation. This study was conducted to evaluate the effect of the protease activity inhibition on rabbit semen quality parameters and reproductive performance after artificial insemination. Seminal quality was not affected by the incubation with protease inhibitors, being the values of motility, viability, and acrosome integrity not significantly different between the protease inhibitors and the control group. In addition, seminal plasma aminopeptidase activity was inhibited in a 55.1% by the protease inhibitors. On the other hand, regarding the effect of protease inhibitors on reproductive performance, our results showed that the presence of protease inhibitors affected the prolificacy rate (9.2 ± 0.26 and 9.3 ± 0.23 vs. 8.2 ± 0.22 total born per litter for negative control, positive control, and aminopeptidase inhibitors group, respectively; P < 0.05), having this group one kit less per delivery. We conclude that the addition of a wide variety of protease inhibitors in the rabbit semen extender negatively affects prolificacy rate. Therefore, the development of new extenders with specific aminopeptidase inhibitors would be one of the strategies to increase the bioavailability of GnRH analogues without affecting the litter size.

Effect of luteinizing hormone on rabbit ovarian superstimulation and embryo developmental potential.

Assisted reproduction technologies require ovarian stimulation to increase the number of oocytes and embryos. Currently, superstimulation is achieved by gonadotropin treatment, but the embryo yield and quality are highly variable. Commonly, commercial preparations derived from pituitary and urinary origin are used to superovulate. Hence, ovarian superstimulation protocols have usually included both FSH and LH. The appearance of recombinant gonadotropins manufactured by genetic engineering techniques has ensured high quality and batch-to-batch consistency. Moreover, this enables us to assess the importance of LH in the ovarian stimulation. The main aim of this study was to evaluate the effect of recombinant human LH supplementation (10%) on embryonic development produced by rabbit does superovulated with low or high concentration (18.75 or 37.50 IU) of recombinant human FSH (rhFSH). Females treated with rhFSH increased the ovulation rate, and it was significantly higher when the high FSH dose was supplemented with LH. The superstimulation treatment used did not significantly affect in vitro development rate until the expanded blastocyst stage. The results of this study seem to suggest that, in terms of superovulatory response, when rabbit does are treated with 37.5-IU rhFSH, the use of LH supplementation allows an increase in the number of follicles recruited and the quality of embryos, in terms of ability to develop in vitro until blastocyst, and the expression profile of OCT4, NANOG, and SOX2 genes is not affected.

Role of embryonic and maternal genotype on prenatal survival and foetal growth in rabbit.

The aim of this work was to evaluate the influence of maternal and embryonic genotype on prenatal survival and foetal growth during pregnancy. Embryos were recovered at 48 h of gestation from two different donor lines (R = 46 and A = 40) and transferred to nulliparous recipient does (26 R and 24 A). Each recipient doe received six embryos into one oviduct from line R, and six embryos form line A into the other. Laparoscopy was performed at Day 14 to determine implantation rate. Recipient females were slaughter at Days 14, 24 and 30 (12, 24, and 14, respectively) to determine the number of live foetuses and the weight of live foetuses, foetal placenta and maternal placenta. A transcriptome analysis was performed to search for differences between foetal placentas at Days 14 and 24 of development. Prenatal survival at Days 14, and 24 was affected by embryonic genotype and determined by maternal genotype at Day 30. Foetal weight at Day 14 was influenced by both genotypes, being the weight higher for group A/A (0.29 ± 0.01 g vs 0.19 ± 0.01 g, for group R/R). However, both genotypes were determinant for foetal placenta weight at Day 24, while those genotypes affected maternal placenta weight at Day 30. Nevertheless, no differences in foetal placenta at transcriptome level and progesterone and IGF-I plasma levels in recipient does were found. In conclusion, results indicate that the influence of embryo and maternal genotype on the prenatal survival and growth seems to be changing over gestation.

Effect of different freezing velocities on the quality and fertilising ability of cryopreserved rabbit spermatozoa.

The freezing step of the cryopreservation protocol negatively influences the quality and fertilising ability of rabbit spermatozoa. This study determines the effect of different rates of freezing on the quality and fertilising ability of rabbit spermatozoa cryopreserved with dimethylsulfoxide (DMSO) (1.75M) and sucrose (0.05M). Ejaculates from meat rabbit line males (n=12) were pooled and each pool (n=7) was split into four aliquots. One group of straws (control, C) was frozen in static liquid nitrogen vapour (5cm above the liquid nitrogen, 10min) and the other groups were frozen at different freezing rates (°Cmin(-1)) from -6°C to -100°C using a programmable freezer: slow (-15°Cmin(-1), S), medium (-40°Cmin(-1), M) or fast (-60°Cmin(-1), F). After thawing (50°C, 12s), the quality was highest (P<0.05) in C and M samples and lowest in S and F samples. F samples presented the lowest litter sizes (P≤0.05) and fertility whilst M samples exhibited the highest values. In conclusion, the freezing rate affects both the quality and the fertilising ability of frozen-thawed rabbit spermatozoa, with both slow (-15°Cmin(-1)) and fast (-60°Cmin(-1)) freezing rates being detrimental for the quality and fertilising ability.

Reducing the time rabbit sperm are held at 5 °C negatively affects their fertilizing ability after cryopreservation.

Cooling sperm to and equilibrating the sperm at 5 °C require the most time in any sperm cryopreservation protocol. Reducing the time required for these phases would simplify sperm freezing protocols and allow greater number of ejaculates to be processed and frozen in a given time. This study determined how holding rabbit sperm at 5 °C for different lengths of time (0, 10, 15, 20, 30, or 45 minutes) affected the quality of rabbit sperm, measured by in vitro assays, and if reducing the cooling time to only 10 minutes affected the fertilizing ability of the sperm. Reducing the time sperm were held at 5 °C to 10 minutes did not affect the in vitro quality of the sperm (percent motile and with intact plasma membranes), although eliminating the cooling phase completely (directly freezing the sperm from room temperature) decreased in vitro assessed sperm quality (P<0.01). However, reducing the time sperm were held at 5 °C, from 45 to 10 minutes, negatively affected the fertilizing ability of sperm in vivo (P<0.05). In conclusion, completely eliminating cooling rabbit sperm to 5 °C before freezing is detrimental for rabbit sperm cryosurvival, and although shortening the time sperm are held at 5 °C to 10 minutes does not reduce in vitro sperm quality, it does reduce the fertility of rabbit sperm. Therefore, the length of time rabbit sperm equilibrate at 5 °C is crucial to the fertilizing ability of rabbit sperm and must be longer than 10 minutes. Currently, it is not known if holding rabbit sperm at 5 °C for less than 45 minutes will affect sperm fertilizing ability.

Direct comparison of the effects of slow freezing and vitrification on late blastocyst gene expression, development, implantation and offspring of rabbit morulae.

This study aimed to assess the effect of different cryopreservation procedures (slow freezing vs vitrification) on the gene expression in pre-implantation embryos and its implication in post-implantation embryo losses in rabbit. For this purpose, rabbit morulae were recovered at Day 3 of development, frozen or vitrified and transferred to recipients. Then, embryos were recovered on Day 3 post-transfer (Day 6 of development) or kept until the end of gestation. Apart from the gene expression analysis at Day 6, we also studied the pre-implantatory and foetal development ability of both cryopreserved embryo types by evaluating late blastocyst development at Day 6, embryo implantation at Day 11 post-transfer (Day 14 of development) and birth rate. We reported that slow freezing and vitrification have similar effects on embryo developmental ability till Day 6, but the distribution of losses changes during implantation and further development. These similarities at Day 6 of development were also reflected in gene expression patterns, and transcriptome analysis showed no differences between frozen and vitrified embryos. Our results confirm that vitrification provides better implantation and birth rates than slow freezing for rabbit embryos. As both the techniques are commonly used in human assisted reproduction, further experiments must be conducted to clarify the causes that may hinder foetal development and their impact on adulthood.

Aminopeptidase activity in seminal plasma and effect of dilution rate on rabbit reproductive performance after insemination with an extender supplemented with buserelin acetate.

Ovulation induction in artificially inseminated rabbits by adding GnRH synthetic analogues in the seminal doses is a welfare-orientated method to induce ovulation in rabbits and could have some advantages in field practice. This study was conducted to determine the effect of male genotype on the aminopeptidase activity in rabbit seminal plasma and the effects of dilution rate of semen on availability and reproductive performance when buserelin acetate is added to the seminal dose. To study the aminopeptidase activity, 12 mature bucks belonging to a paternal line and 12 from a maternal line were used. The bucks from the paternal line were used to study the effect of dilution rate on the availability of buserelin acetate after 2 hours of dilution and on the reproductive performance of the doses after artificial insemination of 389 commercial crossbreed does. Aminopeptidase activity in seminal plasma is dependent on the male genotype. The paternal line resulted 27% more aminopeptidase activity than the maternal line (P < 0.05). On the other hand, semen diluted 1:20 exhibited a marked increase in the availability of buserelin acetate and the fertility in this group was significantly higher than females from dilution rate 1:5 group, which showed similar results to that of the negative control group (does inseminated with semen diluted 1:20 in non-GnRH-supplemented extender). We conclude that the bioavailability of buserelin acetate when added to the seminal dose appears to be determined by the activity of the existing aminopeptidases and is consequently affected by the dilution rate used to prepare the artificial insemination doses.

Effect of freezing extender composition and male line on semen traits and reproductive performance in rabbits.

This study was conducted to elucidate the effect of different freezing extenders on two lines selected for hyperprolificacy and longevity (H and LP, respectively). In extender A, dimethyl sulphoxide (Me2SO) and sucrose were used as cryoprotectants. In extenders B and C, the sucrose was replaced by 20% egg yolk, and in extender C the Me2SO was substituted by acetamide. Semen was packaged in 0.25 ml plastic straws and cooled at 5°C for 45 min, and then was frozen in liquid nitrogen vapour for 10 min before being plunged into the liquid nitrogen. Thawing was carried out by immersing the straws in a water bath at 50°C for 10 s. Frozen-thawed semen characteristics and reproductive parameters were affected by freezing. Extender C showed significantly lower post-thawing quality traits than any of the three extenders. Acrosome integrity was significantly improved when Me2SO was used as cryoprotectant. Sucrose replacement by 20% egg yolk had no effect on acrosome integrity but provided significantly lower sperm motility and viability. Freezing extender affected fertility rate, total born, number of implantation sites and gestational losses, obtaining better results when extender A was used. The acrosomal integrity after frozen-thawed process showed a significant correlation with fertility at 12th day and also at birth, indicating that an increase in acrosomal integrity leads to an increase in both fertilities (12th day and at birth). A positive correlation between motility of semen and implantation sites was found. The post-thawing quality traits of semen were not affected by the genetic line, although LP line showed higher total born and lower foetal and gestational losses. The findings of this study suggest that freezing extender composition has a significant effect on the success of rabbit sperm for preservation, and when Me2SO was used as permeable cryoprotectant sucrose provided better protection compared with egg yolk and improved reproductive traits, and, on the other hand, the male genotypes used in the present study had no effect on frozen-thawed sperm parameters but negatively affected some of the reproductive parameters.

Vitrification alters rabbit foetal placenta at transcriptomic and proteomic level.

Although numerous studies have demonstrated that cryopreservation alters gene expression, less is known about those embryos that implanted successfully and continued in gestation. To raise the question of the neutrality of this technique, we examine the effects of vitrification through gestation in rabbit before and after the implantation. We monitored the distribution of losses of 569 vitrified morulae, observing that embryos which reach the last pre-implantatory stage are able to implant. However, we found that not all implanted embryos had the ability to continue with their gestation. The results reveal that vitrification decreased foetus and maternal placenta weights at mid-gestation, but led to a higher offspring birth weight. A novel finding is that while no differences in gene expression were detected in pre-implantatory embryos at day 6, vitrification affects a gene and protein expression in the placenta at day 14. Our results for first time reveal strong evidence of modifications in implanted embryos subjected to vitrification, suggesting that the crucial step that vitrified embryos must overcome is the placenta formation. On the basis of these findings, our work leaves the question open as to whether the effects we observed that cause vitrification during foetal development could give rise to some type of physiological or metabolic alteration in adulthood.