Browsing by Author "Tenemaza Cumbe, Milton Alejandro"

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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.
Efecto del cuerpo lúteo sobre la producción in vitro de embriones bovinos y su posterior tolerancia a la criopreservación
(2019-05-13) Tenemaza Cumbe, Milton Alejandro; Merchán Vargas, Shirley Lissette; Soria Parra, Manuel Elías
This research was conducted to determine the effect of the corpus luteum on the rate of cleavage of bovine oocytes and the subsequent competence to develop to blastocysts and to tolerate the cryopreservation. The ovaries selected for this study were recovered from the municipal slaughterhouse of Cuenca, from non-pregnant cows with a corpus luteum (CCL) in one ovary (ipsilateral, group 1, CCL) and without CL in the contralateral ovary (group 2; SCL), and of cows without CL in both ovarys (control, group 3; C). The cumulus-oocyte complexes (COCs) were categorized and only those with apt (A and B) quality were included for maturation for 18-24 hours. The COCs were fertilized in vitro and the blastocysts obtained were cryopreserved by vitrification or freezing. From 348 ovaries, 2532 oocytes were obtained in 13 replicates, which, according to quality, 63.8% corresponded to categories (A and B) apt and 36.2% to (C and D) not apt. The proportion of AB oocytes was grater (P <0.05) in CCL (66.6%) than in SCL (61.9%) and C (62.7%). A greater proportion of oocytes from the CCL group cleaved in comparison to the SCL and C (68.3 versus 43.8 and 46.6% respectively; P<0.01). Likewise, a greater proportion of total embryos was obtained in CCL (39.0%) than in SCL (29.2%) and C (29.8%) (P <0.05). From the total of clivated embryos, a greater percentage (P <0.01) of embryos attained the blastocyst stage on day 7 and were cryopreserved, in the CCL group (68.2%) than in SCL (39.8%) and C (38.7%). Embryonic survival after vitrification or freezing determined at 2, 24 and 48 hours was statistically similar among the three study groups. General, the presence of a corpus luteum in the ovary at the time of recovering the oocytes affected the percentage of cleavage and embryo production on day 7, but did not influence the cryotolerance of the blastocysts. In addition, according to these results it is presumed that the effect of the corpus luteum occurred trough local mechanisms.
Intraovarian influence of bovine corpus luteum on oocyte morphometry and developmental competence, embryo production and cryotolerance.
(2020) Argudo Garzon, Daniel Ernesto; Tenemaza Cumbe, Milton Alejandro; Merchan Vargas, Shirley Lissette; Balvoa Tenelema, Jose Agustin; Méndez Álvarez, María Silvana; Soria Parra, Manuel Elías; Galarza Alvarez, Luis Rodrigo; Ayala Guanga, Luis Eduardo; Hernandez Fonseca, Hugo Jose; Perea Brugal, Mariana Sofia; Perea Ganchou, Fernando Pedro
Three experiments were conducted to determine influence of the bovine corpus luteum (CL) on morphometric and functional characteristics of oocytes, and subsequent embryonic development. Cumulus-oocyte complexes were aspirated from two types of cows: 1) with a CL in one ovary (CL+) and without a CL in the contralateral ovary (CL-), 2) and from cows without CL in either ovary (C). Intracellular activity of the enzyme glucose-6-phosphate dehydrogenase (G6PDH), oocyte diameter and thickness of the zona pellucida were determined (Experiment 1). Then, the rate of in vitro oocyte maturation for each ovarian category was evaluated and oocyte diameter and zona pellucida thickness were measured after maturation (Experiment 2). In Experiment 3, in vitro embryo production and cryotolerance were assessed. The oocyte diameter was greater (P 0.05) or C (P = 0.0131) ovaries. Activity of G6PDH was lower in oocytes from CL+ than CL- (P < 0.01) and C (P = 0.0148) ovaries. Rate of oocyte maturation, oocyte diameter and thickness of the zona pellucida after maturation did not differ among groups. Rate of cleavage was greater in zygotes from CL+ than from CL- or C (P < 0.01); and CL+ ovaries produced more total embryos on day 7 (P < 0.05) and more blastocysts (P < 0.01) than CL- and C ovaries. Rate of expansion and hatching of day-7 vitrified-warmed blastocysts at 24 and 48 h of culture did not differ among groups. In conclusion, oocytes collected from CL+ ovaries were larger and metabolically more prepared to continue maturation than those from ovaries lacking a CL. Also, rates of cleavage and yield of blastocysts were greater for oocytes from CL+ ovaries than from CL- and C ovaries. These findings indicate that a CL influenced oocyte developmental competence and embryonic development, presumably through intraovarian interactions.
La presencia de un cuerpo lúteo activo modifica la tasa de maduración pero no afecta la morfometría de los ovocitos bovinos
(Asociación Latinoamericana de Producción Animal, 2018) Argudo Garzon, Daniel Ernesto; Balvoa Tenelema, Jose Agustin; Tenemaza Cumbe, Milton Alejandro; Méndez Álvarez, María Silvana; Soria Parra, Manuel Elías; Galarza Alvarez, Luis Rodrigo; Ayala Guanga, Luis Eduardo; Perea Ganchou, Fernando Pedro; Argudo Garzon, Daniel Ernesto
En la actualidad, la información publicada sobre la influencia que tiene un cuerpo lúteo activo o funcional sobre la capacidad de maduración y morfometría de los ovocitos bovinos presentan aún discrepancias. Se estableció como objetivo de investigación determinar el efecto de la presencia de un cuerpo lúteo activo sobre la tasa de maduración y la morfometría de ovocitos bovinos obtenidos de ovarios de matadero. El estudio se llevó a cabo en el Laboratorio de Biotecnología de la Reproducción Animal de la Universidad de Cuenca, Ecuador. Se colectaron ovarios con cuerpo lúteo activo o funcional (CCL; n=30) y sin estructura lútea alguna (SCL; n=60) del matadero municipal de la ciudad de Cuenca. Con una jeringa desechable y una aguja calibre 18x1.5 pulgadas se puncionaron todos los folículos antrales de 2-8mm de cada ovario. Posteriormente, del líquido folicular obtenido se recuperaron y seleccionaron los mejores complejos cúmulos ovocitos (Coc’s) de cada grupo de ovarios: CCL (n=242) y SCL (n=523), los cuales fueron incubados por 24hs en un medio de maduración a base de TCM199, suero fetal bovino, FSH-p, estradiol, piruvato de sodio, L-glutamina y gentamicina en una estufa con 5% de CO2, 38.5°C y 90%rh. Luego de madurados, los Coc´s fueron denudados por pipeteo en una solución de hialuronidasa 0.1% (p/v) e inmediatamente teñidos con Hoechst 33342 y observados en un microscopio de fluorescencia. Aquellos que habían expulsado el primer corpúsculo polar se consideraron maduros, y este resultado fue expresado en porcentaje. Consecutivamente los ovocitos fueron fotografiados con una cámara acoplada a un microscopio provisto con un software (AmScope V.3.7) calibrado para mediciones microscópicas, cuyas medidas fueron expresadas en micrómetros. Los resultados fueron analizados con el software estadístico SAS. Los Coc´s provenientes de ovarios CCL tuvieron un espesor estadísticamente similar que los de SCL (16,0±0,18 y 15,8±0,22 µm). Asimismo, en las dos categorías de ovarios (CCL y SCL) el diámetro (125,9±0,49 y 126,5±0,57 µm) y el volumen (1´060.254,6±12502,0 y 1´072.845,0±11748,3µm3) fue también similar. Sin embargo, la tasa de maduración fue mayor (P<0,0001) en los ovocitos provenientes de ovarios CCL que en los SCL (62,8 ± 1.3 y 47,3 ± 1.6%). Se concluye que los Coc´s que fueron recuperados de ovarios que tenían un cuerpo lúteo activo o funcional presentaron una mayor tasa de maduración, pero iguales características morfométricas.