Optimización de las técnicas de evaluación y vitrificación de ovocitos de équidos

Equine oocyte cryopreservation is an assisted reproductive technology that allows to preserve female genetic material from valuable mares. The main objective of this thesis was to optimize the evaluation and vitrification of equine oocyte. In chapter 1, two concentrations of the fluorochrome Hoechst 33342 (HO) were compared to assess the nuclear chromatin stage of the oocyte after vitrification using nonpermeable and permeable cryoprotectant agents (CPAs). A higher percentage of oocytes showing upper visibility grade was found when using a lower concentration of fluorescent labels. The assessment of a vitrification solution free of permeable CPAs was performed for the first time in equine oocytes. Unfortunately, no cryoprotectant-free-vitrified oocytes reached metaphase II after warming, therefore cryoprotectant-free vitrification cannot be considered an alternative to conventional equine oocyte vitrification. Chapter 2 was designed to evaluate the DNA fragmentation of equine granulosa cells (GCs) as a biomarker to evaluate the developmental competence of the equine oocyte after cryopreservation. The use of cryopreservation protocols combining different storage temperatures (-80ºC/-196ºC) and CPAs (ethylene glycol or dimethyl sulfoxide), adequately preserved the DNA of equine GCs. The chromatin dispersion test was found as a reliable method to assess DNA fragmentation in equine GCs. The percentage of GCs-DNA fragmentation was higher in GCs from oocytes able to mature. In chapter 3, the effect of permeable cryoprotectant-free vitrification was evaluated on DNA fragmentation of equine cumulus cells. Low and total fragmentation rates of vitrified equine cumulus cells were higher when compared to control group. The use of sucrose as CPA increase the total DNA fragmentation rates of equine cumulus cells but protected them against high DNA fragmentation rates during equine oocyte vitrification. In conclusion, according to the results obtained in this Doctoral Thesis; a low concentration of Hoechst 33342 is recommended to assess the nuclear chromatin stage of equine oocytes; DNA fragmentation analysis of equine granulosa cells can be a valuable test to identify equine oocytes showing the best meiotic competence after in vitro maturation; and cryoprotectant-free vitrification cannot be considered an alternative to conventional vitrification solution for equine oocytes.La criopreservación de ovocitos equinos es una técnica de Reproducción Asistida que permite preservar el material genérico de yeguas valiosas. El objetivo de la presente tesis fue optimizar las técnicas de evaluación y vitrificación de ovocitos equinos. En el capítulo 1, se compararon dos concentraciones del fluorocromo Hoechst 33342 (HO) para evaluar el estado de la cromatina nuclear del ovocito tras la vitrificación usando agentes crioprotectores (CPAs) permeables y no permeables. Empleando menores concentraciones de sustancias fluorescentes, se obtuvo un mayor porcentaje de ovocitos mostrando mayor grado de visibilidad. Medios de vitrificación sin CPAs permeables se han empleado por primera vez en ovocitos equinos. Desafortunadamente, ningún ovocito consiguió alcanzar el estado de metafase II tras el calentamiento en ausencia de crioprotectores permeables, por lo que la vitrificación sin CPAs permeables no puede ser considerada una alternativa a la vitrificación convencional de ovocitos. El capítulo 2 fue diseñado para evaluar la fragmentación del ADN de las células de la granulosa (GCs) como biomarcador para el desarrollo de competencia de los ovocitos de équidos tras la crioconservación. El uso de cualquier protocolo de crioconservación combinando diferentes temperaturas (-80ºC/-196ºC) y CPAs (etilenglicol y dimetilsulfóxido) preservó adecuadamente el ADN de las GCs de équidos. El test de la dispersión de la cromatina resultó en una técnica válida para evaluar el ADN de las GCs de ovocitos equinos. Los ovocitos que consiguieron madurar mostraron mayor fragmentación del ADN de las GCs. En el capítulo 3, se evaluó el efecto de la vitrificación en ausencia de CPAs permeables sobre la fragmentación del ADN de las células del cúmulo de ovocitos equinos. La tasa de fragmentación baja y total de las células del cúmulo del ovocito equino fue mayor cuando se comparó con el grupo control. El uso de medios de vitrificación compuestos por sacarosa como único CPA, aumenta la tasa de fragmentación total del ADN de las células del cúmulo, pero las protege frente a la alta tasa de fragmentación del ADN durante la vitrificación de ovocitos equinos. En conclusión, de acuerdo a los resultados obtenidos en esta Tesis Doctoral; una concentración baja de fluorocromo Hoechst 33342 es recomendada para evaluar el estado nuclear de la cromatina de ovocitos equinos; el análisis de la fragmentación del ADN de las células de la granulosa de équidos puede ser un método valioso para identificar ovocitos equinos mostrando la mejor competencia meiótica tras ser madurados in vitro; la vitrificación sin agentes crioprotectores permeables no puede ser considerada una alternativa a la vitrificación convencional

Vitrification of Donkey Sperm: Is It Better Using Permeable Cryoprotectants?

Vitrification by direct exposure of sperm to liquid nitrogen is increasing in popularity as an alternative to conventional freezing. In this study, the effect of permeable cryoprotectant agents for donkey sperm vitrification was compared to an extender containing non-permeable cryoprotectants. First, three different concentrations of sucrose (0.1, 0.2, and 0.3 molar, M) and bovine serum albumin, BSA (1, 5, and 10%) were compared. Secondly, the concentration of non-permeable agents producing the most desirable results was compared to an extender containing glycerol as permeable agent. Vitrification was performed by dropping 30 μL of sperm suspension directly into LN2 and warming at 42 °C. Sperm motility (total, TM; and progressive, PM) and plasma membrane integrity, PMI (mean ± SEM) were statistically compared between treatments. Sucrose 0.1 M showed a significantly higher percentage of total sperm motility (21.67 ± 9.22%) than sucrose 0.2 M (14.16 ± 4.50%) and 0.3 M (8.58 ± 6.22%); and no differences were found in comparison to the control (19.71 ± 10.16%). Vitrification with sucrose 0.1 M or BSA 5% obtained similar results for TM (21.67 ± 9.22% vs. 19.93 ± 9.93%), PM (13.42 ± 6.85% vs. 12.54 ± 6.37%) and PMI (40.90 ± 13.51% vs. 37.09 ± 14.28); but both showed higher percentages than glycerol (TM = 9.71 ± 4.19%; PM = 5.47 ± 3.17%; PMI = 28.48 ± 15.55%). In conclusion, donkey sperm vitrification in spheres using non-permeable cryoprotectants exhibited better sperm motility and viability parameters after warming than sperm vitrification using extenders containing permeable cryoprotectants

Hormonal Management for the Induction of Luteolysis and Ovulation in Andalusian Jennies: Effect on Reproductive Performance, Embryo Quality and Recovery Rate

Two prostanglandins (luprostiol, LUP, and dinoprost, DIN) and two ovulation-inducing agents (human Chorionic Gonadotropin, hCG, and deslorelin, DES) were evaluated for luteolysis and estrus induction, and for ovulation induction, respectively, in embryo donor jennies. Twenty-six fertile Andalusian jennies were used. In Experiment 1, jennies (n = 112 cycles) were randomly treated with either LUP or DIN after embryo flushing. In Experiment 2, donors (n = 84 cycles) were randomly treated with either hCG or DES to induce ovulation. No differences were found between prostaglandins for all variables studied (prostaglandin–ovulation interval (POI), interovulatory interval (IOI), embryo recovery rate (ERR), positive flushing rate (PFR) and embryo grade (EG)). The ovulation rate was similar for hCG and DES (60.9% vs. 78.7%). However, the interval to ovulation (ITO) was affected (62.61 ± 7.20 vs. 48.79 ± 2.69 h). None of the other variables studied (ERR, PFR and EG) were affected (p > 0.05), except for embryo quality (p = 0.009). In short, both prostaglandins evaluated are adequate to induce luteolysis and estrus. Both ovulation-inducing agents hastened ovulation, but DES seems to be more effective than hCG. Follicular diameter affected the interval from treatment to ovulation, and high uterine edema was related to low embryo quality

Factors Affecting Embryo Recovery Rate, Quality, and Diameter in Andalusian Donkey Jennies

Embryo transfer and the vitrification of embryos could be used for the conservation and recovery of endangered donkey breeds. It is important to develop techniques that optimize recovery rates and the cryotolerance of donkey embryos. This study evaluates factors affecting the recovery rate, quality, and diameter of embryos obtained from donor jennies as a starting point for the use of vitrification and embryo transfer in the conservation of the Andalusian donkey. A total of 100 embryos were recovered out of 124 estrous cycles (80.6%). The donor jenny affected the rates of positive flushings (PFR; p = 0.040) and embryo recovery (ERR; p < 0.05) as well as embryo quality (p = 0.004). ERR was also affected by the number of flushings (p < 0.001), donor age (p < 0.05), successive cycle within donor (p < 0.001), and jacks (p < 0.05). Number of flushings (p < 0.001) and jack (p < 0.05) had a significant effect on PFR, whereas the day of flushing influenced the developmental stage (p < 0.001), embryo quality (p < 0.05), and diameter of embryos (p < 0.001). The number of flushings significantly influenced the diameter (p = 0.038) and embryo developmental stage (p = 0.001), whereas the developmental stage was statistically different between herds (p = 0.020). The factors influencing the success of this assisted reproductive technique were donor jenny, donor age, successive cycle within donor, day of flushing, number of flushings, and jack. The identification of these key points is crucial to achieve a higher efficiency of embryo transfer and vitrification processes, before considering their application in the conservation of endangered donkey breeds

Virtual self-evaluation english systems to promote plurilinguism in Reproduction and Obstetrics

En el presente proyecto se han elaborado de videos demostrativos de diferentes aspectos prácticos de la asignatura Reproducción y Obstetricia (“training clips”) con audio en inglés que completan las presentaciones, facilitando el aprendizaje de los estudiantes. Asimismo, estos videos forman parte de una plataforma de enseñanza interactiva, que incluye cuestionarios que permiten la búsqueda de información y autoevaluación del alumno. Esto permite disponer de una herramienta didáctica aplicable dentro de un marco de docencia plurilingüe.In this project, training clips showing practical activities of Reproduction and Obstetrics has been developed. These videos contain English audio and text and can be used to complete teaching presentations, making the learning process easier. Moreover, training clips also include questionnaires, which promote self-evaluation of the students and the search of external resources. All these contribute to obtain an important tool within a multilingualism program for teaching

Sistemas de autoevaluación virtual en inglés para fomentar el plurilingüismo en la asignatura de reproducción y obstetricia

In this project, training clips showing practical activities of Reproduction and Obstetrics has been developed. These videos contain English audio and text and can be used to complete teaching presentations, making the learning process easier. Moreover, training clips also include questionnaires, which promote self-evaluation of the students and the search of external resources. All these contribute to obtain an important tool within a multilingualism program for teaching.En el presente proyecto se han elaborado de videos demostrativos de diferentes aspectos prácticos de la asignatura Reproducción y Obstetricia (“training clips”) con audio en inglés que completan las presentaciones, facilitando el aprendizaje de los estudiantes. Asimismo, estos videos forman parte de una plataforma de enseñanza interactiva, que incluye cuestionarios que permiten la búsqueda de información y autoevaluación del alumno. Esto permite disponer de una herramienta didáctica aplicable dentro de un marco de docencia plurilingüe

DNA fragmentation of equine cumulus cells from Cumulus-Oocyte complexes submitted to vitrification and its relationship to the developmental competence of the oocyte

The objectives of this study were to evaluate the effect of vitrification on the DNA fragmentation rate of equine cumulus cells and to assess its relationship to oocyte in vitro maturation (IVM) after vitrification. Cumulus cells (CC) from 14 mares were recovered from COCs, previously submitted to vitrification (VIT) and IVM. The DNA fragmentation rate of the cumulus cells (CC-DF) was assessed using a chromatin dis-persion test. CC-DF rates between vitrified and control COCs were statistically com-pared by Student’s t- test. The rates of CC-DF from control COCs were lower than in vitrified COCs. The percentage of CC-DF was not significantly different (p> .05) be-tween groups of COCs able to reach metaphase II (MII > 0) and those in which oocyte maturation was not achieved (MII = 0). In conclusion, vitrification has a deleterious effect on the DNA fragmentation of equine cumulus cells; however, this parameter cannot be used as a predictor for IVM success after COCs vitrification

The Effect of different vitrification and staining protocols on the visibility of the nuclear maturation stage of equine oocytes

In this study, we compared two staining protocols assessing the nuclear chromatin stage of equine oocytes after vitrification using permeable and nonpermeable cryoprotectants. Slaughterhouse-derived oocytes (n = 155) were obtained from a total of 32 mares and in vitro matured in M199 medium for 42 hours at 38.5°C in 5% CO2. In the first experiment, two concentrations of Hoechst 33342 (HO) were tested (10 μg/mL; P1 and 2.5 μg/mL; P2) combined with 50 μg/mL of propidium iodide as staining protocols to evaluate the visibility of matured oocytes (n = 44). In the second experiment, 111 oocytes were evaluated using the staining protocol P2, before (C, control) and after vitrification following a two-step conventional protocol with (15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M sucrose; V1) or without (1 M sucrose; V2) using permeable cryoprotectants. Our results showed that P2 provided a higher percentage of oocytes with outstanding visibility of the nuclear chromatin stage (52.17%; P < .05) in comparison with P1 (19.04%). In the second experiment, no cryoprotectant-free vitrified oocytes reached the metaphase II maturation stage. This result was significantly lower (P < .05) than conventional vitrification (15.38%) and both lower in comparison with the nonvitrified control group (42.11%). In conclusion, permeable cryoprotectant-free vitrification of equine oocytes obtained poor results and therefore cannot be considered an alternative to vitrification using permeable cryoprotectants. In addition, a staining protocol with a low concentration of HO is recommended to evaluate the nuclear chromatin stage of equine oocytes after in vitro maturation.Fil: Pereira, Blasa C.. Universidad de Córdoba; EspañaFil: Ortiz, Isabel. Universidad de Córdoba; EspañaFil: Dorado, Jesús. Universidad de Córdoba; EspañaFil: Diaz Jimenez, Maria. Universidad de Córdoba; EspañaFil: Consuegra, Cesar. Universidad de Córdoba; EspañaFil: Demyda Peyrás, Sebastián. Universidad Nacional de La Plata; Argentina. Universidad de Córdoba; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Hidalgo, Manuel. Universidad de Córdoba; Españ

Effect of warming temperatures on donkey sperm vitrification in 0.5 mL straws in comparison to conventional freezing

Aim of study: There is little information about vitrification of sperm in large volumes (up to 0.5 mL). This study aimed to develop the vitrification technique in 0.5 mL straws in donkey sperm, evaluating the effect of three warming temperatures. Area of study: Cordoba, Spain. Material and methods: Ejaculates from five donkeys were divided in four groups: one control subjected to conventional slow freezing (C) and three vitrified in 0.5 mL straws and warmed using different protocols (W1: 37 degrees C/30s, W2: 43 degrees C/20s and W3: 70 degrees C/8s + 37 degrees C/52s). Sperm motility, kinematic parameters, plasma membrane and acrosome integrity were evaluated. Conventional freezing resulted in significantly higher values for total (42.7 +/- 19.6%), and progressive motility (30.3 +/- 16.7%), plasma membrane (49.1 +/- 10.4%) and acrosome integrity (39.6 +/- 14.5%) respect to vitrification method. Main results: Values after warming ranged between 0.2-2.8% for total motility; 0.2-2.1% for progressive motility; 5.5-20.0% for plasma membrane integrity and 14.5-29.8% for acrosome integrity in all warming protocols after sperm vitrification. However, no differences were found between W3 and C for kinematic parameters; and W3 resulted in significantly higher values for membrane integrity (20.0 +/- 11.0%) in comparison to W1 (5.5 +/- 3.6%) and W2 (9.3 +/- 8.4%). Research highlights: High warming rates seem to be better for donkey sperm vitrification in large volumes; but this methodology is still not an alternative to conventional sperm freezing

Evaluation of DNA Damage of Mare Granulosa Cells Before and After Cryopreservation Using a Chromatin Dispersion Test

DNA fragmentation of granulosa cells might be related to developmental competence of the equine oocyte. Granulosa cells are commonly stored before DNA fragmentation assessment, but the effect of preservation methods on this parameter remains unexplored. The aim of this study was to evaluate whether or not cryopreservation of granulosa cells affects the DNA damage. Equine oocytes were recovered from postmortem ovaries of five mares. Granulosa cells were washed by centrifugation and then analyzed (control) or stored in cryovials following four different protocols: P1 = directly plunged in liquid nitrogen (LN2) and then stored at −80°C; P2 = LN2/−80°C adding cryoprotectants (7.5% ethylene glycol + 7.5% dimethyl sulfoxide); P3 = −80°C; P4 = −80°C + cryoprotectants. Granulosa cell samples were processed with the prototype D3-Ovoselect, Halotech DNA, Spain), and DNA was visualized under fluorescence microscopy. High, low, and total DNA fragmentation percentages were compared among treatments by analysis of variance. Results were expressed as mean ± standard error. No significant differences (P >.05) were found among treatments and the control group. Therefore, the four conservation protocols could be considered equally efficient for DNA preservation of granulosa cells from mare oocytes. In conclusion, cryopreservation of granulosa cells in any of the four protocols used adequately preserved the DNA for further analysis.Fil: Pereira, Blasa C.. Universidad de Córdoba; EspañaFil: Ortiz, Isabel. Universidad de Córdoba; EspañaFil: Dorado, Jesús. Universidad de Córdoba; EspañaFil: Consuegra, Cesar. Universidad de Córdoba; EspañaFil: Jiménez Díaz, María Atilana. Universidad de Córdoba; EspañaFil: Demyda Peyrás, Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; ArgentinaFil: Gosalvez, Jaime. Universidad Autónoma de Madrid; EspañaFil: Hidalgo, Manuel. Universidad de Córdoba; Españ