Browsing by Author "Alvarado Ulloa, Jhonatan Marcelo"

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Análisis morfométrico y funcional de ovocitos bovinos obtenidos de ovarios de matadero y por aspiración folicular transvaginal en vacas criollas del altiplano ecuatoriano
(2020) Alvarado Ulloa, Jhonatan Marcelo; Perea Ganchou, Fernando Pedro; Soria Parra, Manuel Elías; Méndez Álvarez, María Silvana; Bueno Leon, Hernan Patricio; Iñiguez Gutierrez, Carlos Ulises; Argudo Garzón, Daniel; Galarza Lucero, Diego Andrés
This study assessed the morphometric and functional characteristics of oocytes from 10 native cows to the Ecuadorian Andes, collected by transvaginal follicular aspiration (OPU) and oocytes collected post mortem (PM) from abattoir ovaries. For this purpose, 1157 cumulus-oocyte complexes (CCOs) of the OPU group (n=271) and PM group (n=886) were recovered in 10 repetitions per group by follicular aspiration at 90 mm Hg pressure and classified as A, B or C, according to the characteristics of the cytoplasm and cumulus cells. The activity of the enzyme glucose 6 phosphate dehydrogenase (G6PD) and the integrity of the plasma membrane was determined by staining two groups of CCOs with cresyl brilliant blue (BCB +) and trypan blue, respectively. The size (diameter and volume) of the oocytes was evaluated using CaptaVisio® software v. 5.1. The data were analysed by logistic regression and by the SAS general linear model, and the means were compared by the least square method. The results showed that the oocytes of the PM group had a larger diameter (126.0 ± 0.48 vs. 122.7 ± 0.79 μm; p<0.01) and integrity of the plasma membrane (86.0 vs. 76.8%; p<0.05) than the OPU group. However, quality A and B oocytes did not show significant differences between groups with respect to plasma membrane integrity and BCB + values. The results suggest that, although the fact that the size and viability were more affected in the oocytes aspirated by OPU, those of quality A and B from both sources showed similar values regarding the integrity of the plasma membrane and metabolic condition to continue maturation.
Biotecnologías de la reproducción en ganado bufalino
(2012) Alvarado Ulloa, Jhonatan Marcelo; Soria Parra, Carlos Alonso
Bovine corpus luteum affects embryo developmental competence and production
(CSIRO, 2019) Alvarado Ulloa, Jhonatan Marcelo; Tenemaza Cumbe, Milton Alejandro; Merchan Vargas, Shirley Lissette; Argudo Garzón, Daniel; Méndez Álvarez, María Silvana; Soria Parra, Manuel Elías; Galarza Alvarez, Luis Rodrigo; Ayala Guanga, Luis Eduardo; Hernandez Fonseca, Hugo Jose; Perea Ganchou, Fernando Pedro; Merchan Vargas, Shirley Lissette
This study was conducted with the aim to determine the effect of bovine corpus luteum on in vitro embryo production. Immature cumulus–oocyte complexes (COCs) were aspirated from abattoir ovaries from cows with (ipsilateral; CL+) and without (contralateral; CL-) corpus luteum (CL), and from cows without CL in any of the ovaries. The experiment was completed within 12 replicates (100 ovaries per group). COCs were cultured in maturation medium (TCM-199 supplemented with 10% fetal bovine serum, 100 µg/ml sodium pyruvate, 0.75 mg/ml L-glutamine, 4 µg/ml FSH-p, 2 µM cysteamine and 250 µg/ml gentamicin) followed by in vitro fertilization (hepes synthetic oviductal fluid medium supplemented with 10 µg/ml heparin) and in vitro culture (citrate synthetic oviductal fluid medium). On day 7 after in vitro fertilization the embryos were evaluated and classified into morulae (M), early blastocysts (EB), blastocysts (B), expanded blastocysts (ExB) and hatched blastocysts (HB), and the embryos with better quality were recorded. Data were analyzed by logistic regression and general linear model of the SAS, and means compared by least-squares means method. Results of cleavage, embryo rate at day 7, and rates of M + EB, B, ExB and HB are shown in Table 1. The number of embryos per ovary was greater (P < 0.01) in CL+ (1.16 ± 0.11) than in CL- (0.45 ± 0.15) and C (0.55 ± 0.15). Also, the number of embryos per cultured oocyte was significantly greater in CL + than in CL- and C (0.27 ± 0.02 vs 0.14 ± 0.03 and 0.15 ± 0.03 respectively, P < 0.01). The results of this study reveal that the presence of the corpus luteum in the ovary at the time of the oocytes recovery affects the developmental capacity of the bovine embryos, and such influence probably occur through intraovarian interactions.
Evaluación de la calidad de ovocitos provenientes de vaquillas criollas y ovarios de matadero para formar bancos de germoplasma
(2018) Alvarado Ulloa, Jhonatan Marcelo; Argudo Garzon, Daniel Ernesto; Méndez Álvarez, María Silvana; Perea Ganchou, Fernando Pedro; Soria Parra, Manuel Elías; Soria Parra, Manuel Elías
El bovino criollo americano descendiente del ganado ibérico, se adaptó fenotípica y genotípicamente a la región andina, generando biotipos que se caracterizan por rusticidad, eficiencia reproductiva y baja producción lechera, provocando la sustitución por otras razas de alta producción Holstein y Brown-Swiss. En términos biológicos, ocasiona pérdida importante del material genético, patrimonio del país, por esto se promueve la conservación de gametas y embriones mediante biotécnicas reproductivas creando bancos de germoplasma. El objetivo fue evaluar la calidad de ovocitos recuperados a partir de aspiración folicular transvaginal guiada por ultrasonografía (OPU) en ganado criollo y aspiración folicular en ovarios de matadero. Se emplearon 10 novillas criollas, cíclicas, condición corporal 3 a 3,5; en pastoreo con suministro de minerales; y 200 ovarios de matadero. Se recuperaron ovocitos por OPU y aspiración folicular, con agujas 18G, a 90mmHg de presión. Se clasificaron en tres categorías A, B, C. La morfometría se determinó con una cámara de alta definición colocada sobre un microscopio invertido con lente 10X y equipado con software AmScope V.3.7. La viabilidad se evaluó con azul tripán y la actividad enzimática con test de azul de cresilo brillante (BCB). El análisis estadístico utilizó la prueba de Chi-cuadrado y estadístico “U de Mann Withney” (P<0,05). En relación a la calidad de ovocitos, se observaron diferencias significativas según la procedencia (P<0,05), siendo mejores los obtenidos de ovarios de matadero. Mocha & Quezada (2017) obtuvieron ovocitos de mejor calidad por OPU con presión de 65mmHg, factor que probablemente incidió en la característica de las células recuperadas en nuestra investigación. La viabilidad y actividad enzimática de los ovocitos A y B de OPU y Camal no expresan diferencias significativas (P>0,05); se observó que las hembras bovinas criollas producen mayor porcentaje de ovocitos clase A capacitados para el desarrollo in vitro (82,9%) que de matadero (70,6%), diferencia explicable por el estado de los ovarios al momento de la colecta y tiempo transcurrido hasta la aspiración. Mota (2008) evaluó actividad enzimática mediante BCB en ovocitos de matadero y obtuvo 60,37% BCB (-) de respuesta a la prueba. Se concluye que los ovocitos recuperados de calidad A y B de ovarios de matadero por aspiración folicular representan un mayor porcentaje con relación a los A y B obtenidos por OPU. La viabilidad y actividad enzimática no muestra diferencia, siendo útiles para programas FIV y formación de bancos de germoplasma.
Evaluación de la calidad de ovocitos provenientes de vaconas criollas y ovarios de matadero
(2017) Alvarado Ulloa, Jhonatan Marcelo; Soria Parra, Manuel Elías
This research work was conducted at the experimental farm "Irquis", Faculty of Agricultural Sciences at the University of Cuenca, the aim of this study was to evaluate the quality of oocytes in cattle recovered from two sources: transvaginal follicular aspiration guided by ultrasound (OPU) in native cattle and follicular aspiration in abattoir ovaries. 10 cyclical indigenous heifers from 3 to 3.5 CC maintained on pasture and supply of mineral salts were used; and 200 slaughterhouse ovaries. Oocyte recovery OPU and follicular aspiration was performed with disposable needles 18G and a vacuum pressure of 90 mmHg. The oocytes obtained were classified into three categories according to Hawk & Wall (1994). The morphometry was made using a high definition camera mounted on an inverted lens 10X and equipped with software V.3.7 AmScope microscope. The viability was assessed with trypan blue and enzyme activity with brilliant cresyl blue test (BCB). For statistical analysis the Chi-square and statistical "Mann Whitney" (P <0.05) was used. The results show significant differences for sources of oocytes (P <0.05) while for the viability and enzyme activity no significant differences (P> 0.05). It was concluded that oocyte recovery sources produce different qualities but oocyte viability and similar enzyme activity
Uso del azul brillante de cresilo en la selección de ovocitos competentes para la producción in vitro de embriones
(2019) Duma Pauta, José Mauricio; Argudo Garzón, Daniel Ernesto; Ochoa Calderón, René Rafael; Alvarado Ulloa, Jhonatan Marcelo; Ayala Guanga, Luis Eduardo
The objective was to evaluate the Brilliant Cresyl Blue (BCB) in the selection of oocytes for the production of embryos in vitro (PIV). Three treatments were evaluated. G1 = Witness without BCB exposure; G2 = staining positive oocytes (BCB +) and G3 = staining negative oocytes (BCB-). Morphometry, maturation (IVM), meiotic configuration, cleavage and embryos were determined. 2,340 cumulus oocyte complexes (COCs) were obtained, classified as suitable and not suitable for IVP. The 2/3 parts were exposed for 90 min. To BCB and the rest incubated for 90 min in H-SOF. 25% of COCs from each group were taken to assess the morphometry. The remaining 75% was placed in maturing medium for 24h. After IVM, 25% of COCs were used for morphometry and meiotic progression. The remaining 50% was in vitro fertilized (IVF) and cultivated. Cleavage was evaluated 48h after IVF and embryos on day 8. 62.3% of G2 oocytes were BCB + and had a larger diameter before (135, 9 μm) and after (134, 8 μm) of IVM. (P <0.05) vs group 3 BCB- (128, 1 and 127, 6 μm) and the Witness (129, 0 and 128, 2 μm). Maturation and meiotic configuration were not different between groups (P> 0.05). Cleavage was higher (P <0.05) in the Control (43.4%) than the BCB + group (33.5%). Finally, the global percentage of embryos was higher in BCB + (52.6%) than the Controlling (32.6%) and BCB- (33.5%). It is concluded that the BCB allowed to select oocytes with a larger diameter, although it did not allow to increase the percentage of maturation and cleavage, it contributed to improving the overall percentage of embryos obtained in vitro.