Browsing by Author "Samaniego Campoverde, Jorge Xavier"

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Accuracy of ultrasound and visual inspection in antral follicular count in crossbred Holstein cows raised under grazing systems at high altitude
(2023) Astiz, Susana
The objective of this research was to investigate the diagnostic accuracy of post- mortem ultrasound in antral follicle count (AFC) determination and compare it with visual AFC in grazing crossbred Holstein cows, at high altitude in Ecuador. Pre- mortem blood from 80 cows was collected, and AFC and ovarian characteristics were ana-lysed post- mortem by ultrasound and visual techniques. AFC counts were stratified as high, medium or low by terciles. Mean AMH concentration in pre- mortem blood was 280.1 ± 15.53 pg/mL. The AFC obtained by visual inspection (26.9 ± 9.49 follicles) was 23.8% higher than by ultrasound (20.5 ± 7.53 follicles) in all ovaries. Body condi-tion score, age and weight of the cattle did not interact with the count technique. In the low AFC group, visual inspection and ultrasound provided similar AFC results. However, in the Medium- and High-AFC groups, AFC by ultrasound was 14.9% lower than AFC by visual inspection. We confirm that ultrasound can be used with great accuracy for AFC >3 mm (close to the resolution limit) in grazing crossbred Holstein cows at high altitude.
BoviPure® density-gradient centrifugation procedure enhances the quality of fresh and cryopreserved dog epididymal spermatozoa
(2022) Perea Ganchou, Fernando Pedro; Muñoz León, Esteban Mauricio; Santiago Moreno, Julián; Galarza Lucero, Diego Andrés; Jara Jimbo, Diana Isabel; Paredes Quito, Efraín Bernardo; Samaniego Campoverde, Jorge Xavier; Méndez Álvarez, María Silvana; Soria Parra, Manuel Elías
BoviPure® is a salt solution containing colloidal silica particles coated with silane used to select sperm (e.g., ruminants) by density-gradient centrifugation (DGC). This research assessed the suitability of the BoviPure-DGC and swim-up methods for selecting dog epididymal sperm in fresh, chilled and frozen-thawed samples on post-treatment sperm quality. Sperm samples (n = 60 epididymides) were recovered by retrograde flushing from thirty orchiectomized adult dogs. Thereafter, 20 sperm pools, containing sperm aliquots of three randomly selected animals, were used for chilling (at 5 ºC for 24 h) and freezing (in liquid nitrogen vapors). Sperm selection by BoviPure-DCG and swim-up was performed in both individual and pooled samples, including non- selected samples as controls. Overall, after BoviPure-DGC selection a higher sperm retrieval rate was obtained than the swim-up selection in both individual (P < 0.05) and pooled (P < 0.01) samples. BoviPure-DGC improved (P < 0.05) the total (TM) and progressive (PSM) sperm mo- tilities, curvilinear (VCL) and straight-line (VSL) velocities, linearity (LIN), wobble (WOB), beat- cross frequency (BCF), and integrity of plasmatic (IPM) and acrosomal (IAM) membranes of in- dividual samples in comparison with non-selected samples. In pooled samples, however, the BoviPure-DGC improved (P < 0.05) the PSM, VCL, WOB, and IPM of chilled and frozen-thawed samples. The swim-up method improved (P < 0.05) only some kinematic variables of the indi- vidual (VCL, WOB and BCF) and cryopreserved pooled samples (VCL and ALH) in comparison with non-selected samples. In conclusion, BoviPure-DGC was more effective for recovering and selecting both fresh and cryopreserved dog epididymal sperm than the swim-up procedure improving the kinematic variables, and membranes intactness
Capacidad de reinicio de la meiosis de ovocitos provenientes de folículos de varios tamaños obtenidos de ovarios de camal
(2019) Ayala Guanga, Luis Eduardo; Palacios Cedeño, Tito Emmanuel; Calle Ortiz, Guido Rigoberto; Rodas Carpio, Ermes Ramiro; Dutan Sanango, Jorge Bolivar; Murillo Apolo, Yury Agapito; Samaniego Campoverde, Jorge Xavier
The competence of oocytes according to follicle size to restart meiosis was evaluated. The follicles were measured and classified as Group 1 (G1 <4 mm), Group 2 (G2 4-8 mm) and Group 3 (G3 >8 mm). The aspiration was performed by group with a 21G needle connected to a vacuum pump, with a pressure of 65 mmHg. The complex oocyte clusters (COCs) recovered were classified as suitable and unsuitable to produce embryos in vitro, according to the characteristics of the cumulus and the cytoplasm. Eligible and unsuitable COCs were assessed with the Brilliant Cresyl Blue (BCB) and were classified into BCB+ and BCB-. In vitro maturation (IVM) was carried out in microdroplets, incubated in a 5% CO2 chamber, 38.5 °C and 90% humidity for 24 hours. The meiotic progression was determined by extrusion of the polar corpuscle by epifluorescence under an inverted microscope. Oocyte morphometry was established using a high definition camera (Excelis AU-600-HD) and software (AmScope v.3.7). The recovery percentage of oocytes was greater than 63%. The G2 follicles provided a higher percentage of eligible COCs (65.7%), where 59% of this group was classified as BCB+. The fit oocytes of G1 and G2 resumed meiosis by 75%. In addition, it was observed that oocytes after IVM reduced their diameter. It is concluded that follicle oocytes between 4-8 mm (G2) provide a higher percentage of mature COCs; however, 50% of follicles <4 mm (G1) are a promising source of viable oocytes, so they should be used for in vitro embryo production.
Caracterización cinemática de espermatozoides criopreservados de tres biotipos de gallos criollos ecuatorianos
(Asociación Latinoamericana de Producción Animal., 2022) Galarza Lucero, Diego Andrés; Moscoso Piedra, Andrés; Maldonado Cabrera, Manuel Efren; Alvarado Alvarado, Juan Carlos; Argudo Garzón, Daniel; Samaniego Campoverde, Jorge Xavier
Comparison of characterization of fighting rooster (gallus gallus) semen ejaculates recovered by electroejaculation and dorsal massage techniques
(2021) Alvarado, Juan Carlo; Galarza Lucero, Diego Andrés; Moscoso Piedra, Andrés; Muñoz Verdugo, Marco Antonio; Argudo Garzon, Daniel Ernesto; Samaniego Campoverde, Jorge Xavier; Maldonado Cornejo, Manuel Esteban; Cabrera Córdova, Bolívar Santiago
Implementing alternatives methods to dorsal massage (e.g., electroejaculation) for recovering semen from fighting rooster, known to be very stressful due to its aggressiveness, has become a priority for breeders of this cock breed in Ecuador. Therefore, the objective of the present study was to evaluate two semen collection techniques in fighting roosters, one by electroejaculation (EE) and another by dorsal massage (DM) on seminal quality parameters. For this purpose, thirty attempts of semen recovery from six adult Spanish fighting roosters were carried out using DM (n = 12) and EE (n = 18). Electroejaculation was performed previous sedation, applying five stimulation cycles (of 2 s) generated from a handmade electroejaculation probe (9 to 12 V). The results showed that the EE produced lower response (P < 0.01) to semen ejaculation than the DM (44.4 % vs. 100.0 %, respectively). However, semen samples obtained by EE had better (P < 0.05) spermatic kinetic with greater values of straight-line velocity (VSL, μm/s), average path velocity (VAP, μm/s), and beat-cross frequency (BCF, Hz) as well as higher percentages (P < 0.01) of wobble and linearity compared to DM, irrespective of sperm viability. In addition, the number of urates present in the ejaculates obtained by EE was lower (P < 0.05) than those obtained by DM. In conclusion, electrical stimulation with prior sedation produced a low semen ejaculation response in fighting cocks. However, EE yielded semen ejaculates with better spermatic kinetic compared with the conventional dorsal massage technique.
Competencia del ovocito bovino obtenido por ovum pick-up valorado mediante el azul brillante de cresilo
(2018) Ayala Guanga, Luis Eduardo; Samaniego Campoverde, Jorge Xavier; Nieto Escandón, Pedro Emilio; Rodas Carpio, Ermes Ramiro; Dután Sanango, Jorge Bolívar; Calle Ortiz, Guido Rigoberto; Murillo Apolo, Yury Agapito; Vázquez Mosquera, Juan Mesías; Argudo Garzón, Daniel Ernesto; Perea Ganchou, Fernando Pedro
The objective of the present study was to evaluate the bright blue cresyl (BCB) test as an indirect method to select competent oocytes for the in vitro production of embryos (IVP). The cumulus-oocyte complexes (COC) were obtained from two Creole heifers subjected to two treatments: T1 = COC recovered by OPU (ovum pick-up) previous stimulation with FSH-LH; T2 = COC recovered from non-stimulated animals (control).The two heifers were alternated in the two treatments and five repetitions were done. Recovered COCs were classified into types A, B, C and D. Then the BCB test was applied to each of the COC types to determine if they were BCB+ or BCB-. T1 allowed to recover 5.2 more COC than T2 (p<0.05). When applying the BCB test, it was determined that all type A oocytes of T1 and T2 were BCB+; that is, they finished their growth and were ready to start the process of in vitro maturation; however, about 50% of the type B, C and D COCs of T1 and T2 were BCB+. It is concluded that the selection of COC based on morphological characteristics is a reliable method only for type A and has a 50% error for COC type B, C and D and, therefore, the application of the BCB test allows to improve this selection non-invasively
Competencia ovocitaria en procesos de maduración in vitro y su relación con el tamaño del folículo
(ALPA, 2018) Ayala Guanga, Luis Eduardo; Nieto Escandón, Pedro Emilio; Dután Sanango, Jorge Bolívar; Calle Ortiz, Guido Rigoberto; Ortega Herrera, Vanessa Viviana; Samaniego Campoverde, Jorge Xavier; Estrella Bermeo, Carlos Adrián; Suconota Pizarro, Ana Gabriela; Ayala Guanga, Luis Eduardo
El objetivo fue valorar la competencia post maduración in vitro (MIV) de ovocitos bovinos provenientes de folículos de tres tamaños diferentes. Se realizó en el laboratorio de Biotecnología Animal de la Universidad de Cuenca a 2.650msnm. Previo a la aspiración, los folículos fueron medidos y clasificados en grupo 1 (<4mm); grupo 2 (4-8mm) y grupo 3 (>8mm). La punción folicular se realizó a cada grupo por separado con una aguja 21G conectada a una bomba de vacío, a presión de 65mmHg. Los complejos cumulus ovocitos (COC´s) recuperados de cada grupo fueron clasificados en base a las características del cumulus y citoplasma en aptos y no aptos para la producción de embriones in vitro (PIV). La determinación enzimática se realizó a través de la prueba del azul brillante de Cresilo (BCB). La maduración se realizó en microgotas colocadas en una cámara de CO2 al 5%; a 38,5°C y 90% de humedad, durante 24h, usando como medio TCM 199. La valoración de la progresión meiótica a metafase II (competencia) fue determinada con la prueba de Hoesch en base a la extrusión del primer corpúsculo polar, valorados mediante epifluorescencia bajo un microscopio invertido. Los resultados fueron analizados con el paquete estadístico IBM® SPSS® versión 22. Se realizaron 14 sesiones, aspirando 1.964 folículos en el grupo 1 (G1), grupo 2 (G2=1.073) y grupo 3 (G3=336). Se obtuvo un porcentaje de recuperación del 76,1% en G2, 64,7% en G1 y 63,4% en G3 con diferencia estadística (P<0,05). El grupo G2 presentó un mayor porcentaje de COC´s valorados como aptos (65,7%), seguido de G3 (54,9%) y finalmente de G1 (42,4%), con diferencia entre grupos (P<0,05). De los COC’s clasificados como aptos, en G2 el 59,0% fueron positivos al colorante (BCB+), en G1 (44.6%) y G3 (35.7%); es decir, habían terminado su crecimiento y estaban listos para continuar con el proceso de maduración (P>0,05). Sin embargo, al valorar el porcentaje de COC´s clasificados como aptos que fueron BCB+ y BCB- dentro de un mismo grupo, se estableció que G2 presentó más BCB+ que BCB- (P<0,05). Finalmente, los ovocitos clasificados como aptos de G1 y G2, reanudaron la meiosis y llegaron a metafase II en un 75% a diferencia de G3 (61%), (P<0,05). Se concluye que los COC's provenientes de folículos entre 4-8mm proporcionan mayor porcentaje de maduración; sin embargo, los folículos <4mm son una fuente interesante de ovocitos viables por lo cual deberían ser utilizados para la PIV. Palabras clave: tamaño, COC’s, aptos, meiosis
Criopreservación de espermatozoides de gallo de combate: efecto de diferentes concentraciones de glicerol
(2022) Moscoso Piedra, Andrés; Reinoso Leon, Jaime Marcelo; Samaniego Campoverde, Jorge Xavier; Argudo Garzon, Daniel Ernesto; Alvarado Alvarado, Juan Carlos; Galarza Lucero, Diego Andrés
Glycerol (Gly) is the cryoprotective agent (CPA) most widely used to cryopreserve rooster semen. However, its cryoprotection, toxicity, and efficacy may vary in different breeds of roosters (e.g., fighting rooster). In this sense, this investigation evaluated the effect of different concentrations of Gly added to the Lake-Ravie extender on the kinetic variables and plasma membrane integrity (PMI, equivalent to viability) of rooster spermatozoa. A total of 42 semen ejaculates from 6 Spanish fighting roosters (collected in 7 weekly sessions) by dorsal massage technique were used to conform 7-pools. Each pool was divided into 6-aliquots and then 6 treatments were formed according to Gly concentrations: 0 (control), 2% (Gly-2), 4% (Gly-4), 6% (Gly-6), 8% (Gly-8), and 10% (Gly-10). The samples were frozen in static liquid nitrogen vapors, and their post-thaw sperm quality was analyzed using the CASA system (SCA-2018) and Fluorescence (PI). Results after thawing showed that total (MT, %) and progressive (MP, %) motilities were higher (P<0.01) with the Gly-8 treatment compared to the control group and the Gly-4 and Gly-6 treatments. Regarding the post-thaw kinetics, the oscillation (WOB, %) and the beat-cross frequency (BCF, Hz) were higher (P < 0.05) in the Gly-6, Gly-8, and Gly-10 treatments compared to their control. Finally, the PMI (%) was greater with the Gly-8 treatment compared to the Gly-2 treatment (P<0.05) and the control group (P<0.01). In conclusion, the addition of 8% glycerol to the freezing medium produced better sperm cryosurvival based on higher kinetics and integrity of the plasma membrane of fighting rooster spermatozoa.
Cryopreservation of dog epididymal spermatozoa by conventional freezing or ultra-rapid freezing with nonpermeable cryoprotectant
(2021) Landi Loja, Blanca Gabriela; Santiago Moreno, Julián; Mejia Jara, Edisson Leonardo; Galarza Lucero, Diego Andrés; Castaño, Cristina; Sánchez Calabuig, María Jesús; Taboada Pico, Juan Wualverto; Soria Parra, Manuel Elías; Méndez Álvarez, María Silvana; Samaniego Campoverde, Jorge Xavier
This study was aimed to assess the effectiveness of two methods for cryopreservation of dog epididymal spermatozoa, one by conventional freezing (CF) with shortening both equilibration and cooling times, and the other by ultra-rapid freezing (URF) with nonpermeable cryoprotectant. Sixty epididymides were recovered from thirty orchiectomized adult dogs and the sperm samples were retrieved by retrograde flushing using TCG-EY (tris, citric acid, glucose + 20% egg yolk) extender and then 20 pools were conformed. Each pool was divided into 2 aliquots and then cryopreserved by CF and URF methods respectively. The CF method maintained the cooled-pool samples for 2h (1h without and 1h with 5% glycerol) and then were frozen by liquid nitrogen (LN2) vapors for 2 min. The URF method cryopreserved the cooled-pool samples using TCG-EY+250 mM sucrose, equilibrating during 30 min (5 ◦C) and submerging 30-μL drops directly in LN2. The results showed that the URF method produced a lower percentage of total and progressive motilities and acrosome integrity (P < 0.05) than the CF method. However, the kinetic variables (curvilinear and straight-line velocities, straightness, linearity, wobble, amplitude of lateral head displacement, and beat-cross frequency) and plasma membrane integrity did not differ (P > 0.05) between both cryopreservation methods. Unlike the URF method, the width, area and perimeter of sperm head were reduced after the CF method (P < 0.05). In conclusion, despite the low motility achieved after the ultra-rapid freezing method, the similar values of kinetic, viability and head morphometric dimensions to those obtained after conventional freezing, suggest that ultra-rapid freezing with sucrose may be a useful alternative for the cryopreservation of canine epididymal sperm.
Cryopreservation of Domestic and Wild Animal Spermatozoa: Update of Knowledge
(Intechopen, 2023) Samaniego Campoverde, Jorge Xavier; Galarza Lucero, Diego Andrés; Méndez Álvarez, María Silvana; Duma Pauta, José Mauricio
Current sperm cryopreservation protocols for domestic and wild mammals aim to minimize the cryogenic damage caused by cell dehydration, ice formation, and osmotic stress. The optimization of sperm cryopreservation include the use of different synthetic and nonsynthetic-based extenders supplemented with additives (e.g., egg yolk, coconut water, etc.) and antioxidants (e.g., melatonin, L-carnitine, caffeine, resveratrol, etc.) that protect the plasmalemma, acrosome, and mitochondria against the detrimental effects caused by the cryopreservation process. Furthermore, the use of penetrating (e.g., glycerol, ethylene glycol, dimethylformamide, etc.) and nonpenetrating (e.g., sucrose and trehalose) cryoprotectant agents (CPAs) or their combination should be investigated to protect sperm during the freezing process in slow and ultra-rapid freezing procedures. Finally, new cryopreservation protocols should focus on freezing curves and initial cooling rates that allow optimal dehydration during freezing and adequate hydration during thawing. The suitable interaction of all these factors will allow a sperm subpopulation to survive cryopreservation with integrity and fertilizing capacity, contributing to the improvement of the efficiency of genetic resource management and the development of germplasm banks that support the preservation of genetic diversity in domestic and wild animals.
Dinámica folicular de vaquillas Criollas, al pastoreo en la sierra ecuatoriana
(2018) Ayala Guanga, Luis Eduardo; Rodas Carpio, Ermes Ramiro; Dután Sanango, Jorge Bolívar; Murillo Apolo, Yury Agapito; Vázquez Mosquera, Juan Mesías; Nieto Escandón, Pedro Emilio; Ortega Herrera, Vanessa Viviana; Samaniego Campoverde, Jorge Xavier; Ayala Guanga, Luis Eduardo
El ganado bovino Criollo a nivel mundial está catalogado en peligro de extinción. En los últimos años este tipo de animales han sido reemplazados por ganado lechero especializado, sin considerar la rusticidad, adaptabilidad y resistencia a ciertas enfermedades, así como el desconocimiento de las características fisiológicas como la dinámica folicular, número de folículos reclutados por onda, concentraciones de hormonas esteroideas, entre otras; las cuales no han permitido la conservación y multiplicación de este material genético criollo, razón por lo cual el objetivo fue caracterizar el patrón de comportamiento de la dinámica folicular en vaquillas Criollas al pastoreo en la sierra Ecuatoriana. Se realizó en la granja experimental Irquis de la Universidad de Cuenca, a una altitud de 2650msnm durante el año 2016. Se evaluó el ciclo estral de 9 vaquillas Criollas con peso promedio de 243,3±45,0 kg; CC 2,5±0,4, escala 1-5 puntos y edad 19,9±4,8 meses. Se realizaron ecografías diarias durante un ciclo estral. Cada 48h se determinó los niveles de Progesterona (P4). Se estableció un patrón de dos y tres ondas foliculares (44,4 y 55,6% respectivamente). El promedio de duración del ciclo estral fue de 20,3±0,03 días (dos ondas) y de 23,6±0,02 días (tres ondas). El tamaño del folículo preovulatorio (FPO) fue de 15,3±0,04mm para animales de dos ondas y para las de tres ondas 13,8±1,48mm. El folículo subordinado (FS) alcanzó su máximo tamaño el día 4,0±0,04, con 8,0±0,04mm (dos ondas) y 4,8±0,03 días con 7,4±0,03mm (tres ondas). El desarrollo del cuerpo lúteo (CL) presentó tres fases: crecimiento (hasta el día 6), estática (6-18 días) y regresión (>18 días). En el día 12 el CL alcanzó su mayor tamaño 21,7±1,45mm y 23,5±0,61mm para animales de dos y tres ondas respectivamente. La P4 alcanzó niveles superiores a 1ng/ml a partir del día 4 (5,8±3,35ng/ml dos ondas y 5,1±1,15ng/ml tres ondas). Los niveles de P4 durante el ciclo estral fueron más altos que los reportados en razas diferentes. En conclusión, las vaquillas de genotipo Criollo poseen características propias, las cuales se ven influenciadas por el patrón de comportamiento folicular (dos o tres ondas).
Efecto de la grasa bypass sobre la reactivación ovárica postparto en vacas holstein friesian mestizas con condición corporal diferente
(2018) Ayala Guanga, Luis Eduardo; Aguilar Campoverde, Nelson Adrian; Nieto Escandón, Pedro Emilio; Rodas Carpio, Ermes Ramiro; Dután Sanango, Jorge Bolívar; Murillo Apolo, Yury Agapito; Vázquez Mosquera, Juan Mesías; Samaniego Campoverde, Jorge Xavier
The effect of bypass fat on productive and reproductive parameters in crossbred Holstein Friesian cows with different body condition (CC) was the objective of the study. Four treatments were analyzed: T1 (n=10) CC> 3.5 without fat bypass; T2 (n=10) CC> 3.5 with bypass grease; T3 (n=10) CC <2.5 without fat bypass and T4 (n=10) CC <2.5 with bypass fat. The CC and weight were evaluated on the 25th day before, at delivery and postpartum until day 65. Milk production was determined during the first two months of lactation. Ovarian reactivation was assessed by ultrasonography once a week. The first postpartum estrus (1CPP), preovulatory follicle (FPO), corpus luteum (CL) and progesterone levels (P4) at day 7 were evaluated. The first delivery interval (IPPS) and the number of inseminations per pregnancy (IA/P). Uterine health (SU) was determined at day 35 postpartum. A mixed linear model was used for the statistical analysis using the MIXED procedure. The T1 cows lost more CC than the other treatments (P <0.05); however, T2, T3 and T4 presented no difference (P> 0.05). Animals that received fat bypass (T2 and T4) lost less weight (P <0.05) compared to T1 and T3. T2 cows produced more milk (P <0.05) than T1, T3 and T4 animals. T2 presented less polymorph nuclear (8.12%), compared to T1, T3 and T4 (P <0.05). The number of IA/P used in T2 was lower (1.69) than T1, T3 and T4 (2.9, 2.5 and 2.5 respectively) (P <0.05). The CPI was reduced to 73.6 days in T2 and its P4 levels were higher (6.06 ng/ml), determining differences with the other treatments. The addition of fat bypass in the basal diet of lactating cows improved the productive and reproductive parameters.
Effect of resveratrol on cryopreservation of bull spermatozoa by conventional slow freezing or ultrarapid freezing
(Conference of the International Embryo Technology Society, 2024) Jaramillo López, Jennifer Alexandra; Campoverde Guailacela, Byron Alexander; Aguirre Narea, Brian Oswaldo; Amón Togra, Nicole Alexandra; Galarza Lucero, Diego Andrés; Samaniego Campoverde, Jorge Xavier
Resveratrol (RES) is a powerful antioxidant with free radical scavenger properties that result in a reduction in reactive oxygen species (ROS) generation and lipid peroxidation, these effects have been demonstrated in cryopreserved spermatozoa. This work evaluated the effect of RES (0 µM as control and 50 µM) on the cryopreservation of bull spermatozoa by conventional slow (CS) or ultrarapid (UR) freezing on post-thaw quality and fertility. Twenty-four ejaculates from four fertile bulls (six per bull) were collected using an artificial vagina and then diluted with TCG-EY (tris-citric acid-glucose + 6% egg yolk). Four treatments per ejaculation were conformed according to the freezing method and RES addition: CS-RES (n = 80), CS-0 (n = 80), UR-RES (n = 24), and UR-0 (n = 24). The CS freezing method was done by exposing the 0.25-mL sperm sample straws (plus 5% glycerol) to liquid nitrogen (NL) vapors, and the UR freezing was made using submerging 30-µL drops of samples (plus 100 mM sucrose) into NL. Initially, the kinetic parameters, plasma, and acrosome membranes integrity (IPIA), and oxidative stress were assessed using a CASA system (SCA®), PI/PNA-FICT double fluorescence test, and Cell ROX Deep Red test. After that, IVF based on cleavage and blastocyst rates from the four treatments spermatozoa were assessed using 200 in vitro-matured bovine oocytes in each treatment. Eight IVF sessions were performed using two cryopreserved semen per bull. Sperm samples from all treatments were selected by Percoll (45/90%) before IVF. A factorial ANOVA and Tukey test were used to assess the interactions between the “freezing method” and “RES dose.” The results showed significant (P < 0.05) differences between bulls regarding post-thaw sperm quality. The CS freezing treatments yielded a better sperm cryoresponse than UR freezing treatments, irrespective of RES addition. After CS-RES treatment, the total and progressive motilities, and IPIA percentages were higher (P < 0.05) than after CS-0, UR-RES, and UR-0 treatments. In addition, the UR-RES treatment produced greater (P < 0.05) values of the beat cross frequency and lower values (P < 0.05) of oxidative stress than the UR-0 treatment. The CS-RES treatment produced a higher (P < 0.05) cleavage rate (%) than the CS-0 and UR-RES treatments (64.0 ± 3.40 vs 34.7 ± 3.44 and 42.0 ± 4.96, respectively). Likewise, the CS-RES treatment produced a higher (P < 0.05) blastocyst rate (%) than all treatments (CS-RES: 22.5 ± 3.11 vs CS-0: 10.2 ± 2.29; UR-RES: 8.0 ± 3.88; and UR-0: 4.0 ± 2.8). In conclusion, RES improved the motility, plasmatic and acrosomal membrane integrity, and in vitro fertility of spermatozoa after the freezing-thawing process. However, after ultra-rapid freezing, RES reduced only oxidative stress.
Effect of resveratrol supplementation in conventional slow and ultra-rapid freezing media on the quality and fertility of bull sperm
(2024) Aguirre Narea, Brian Oswaldo; Amón Togra, Nicole Alexandra; Jaramillo López, Jennifer Alexandra; Galarza Lucero, Diego Andrés; Duma Pauta, José Mauricio; Samaniego Campoverde, Jorge Xavier; Taboada Pico, Juan Wualverto; Campoverde Guailacela, Byron Alexander
The study investigated the impact of resveratrol (RES) on bull sperm cryopreservation employing conventional slow (CS) and ultra-rapid (UR) freezing methods on sperm quality and in vitro fertility. Twenty-four ejaculates from four bulls were divided into four groups based on the cryopreservation method and RES addition: CS-RES (n = 80), CS-Co (n = 80), UR-RES (n = 24), and UR-Co (n = 24). The CS freezing involved exposing sperm straws with 5% glycerol to liquid nitrogen (LN2) vapors, while UR freezing submerged sperm drops with 100 mM sucrose directly into LN2. Overall, sperm kinematic parameters and integrity of plasma and acrosome membranes significantly decreased (P < 0.001) after cryopreservation. Post-thaw values of motilities (total [TM] and progressive [PSM]), velocities (curvilinear and straight-line), beat cross frequency (BCF), and sperm with intact plasma membrane/intact acrosome (PI-/PNA-) were higher (P < 0.05) with CS-RES and CS-Co treatments compared to UR-RES and UR-Co treatments. CS-RES treatment resulted in greater percentages (P < 0.05) of TM, PSM, PI-/PNA-, and fertility (blastocyst rate) than their control, CS-Co; while UR-RES showed higher BCF values (P < 0.05) than its control, UR-Co. Additionally, UR-RES treatment exhibited lower oxidative stress percentages than UR-Co (P < 0.05). This study presents the following conclusions: (1) the CS freezing resulted in better cryosurvival of bull sperm than UR freezing; (2) the RES supplementation to CS freezing medium improved sperm motility, membrane integrity, and fertility; and (3) despite low cryosurvival sperm and fertility, the RES addition to ultra-rapid freezing medium reduced oxidative stress.
Egg Yolk-Supplemented Tris–Citric Acid Extender Improves the Prefreezing and Post-Thaw Sperm Quality Indices of Guinea Pig (Cavia porcellus) Epididymal Spermatozoa
(2023) Samaniego Campoverde, Jorge Xavier; Duma Pauta, José Mauricio; Perea Ganchou, Fernando Pedro; Carrera Durazno, Rubén; León Machuca, Danny; Galarza Lucero, Diego Andrés; Santiago Moreno, Julián
This study aimed to assess the suitability of egg yolk (EY) supplementation to a tris–citric acid–based extender on cryosurvival of guinea pig (Cavia porcellus) epididymal spermatozoa. Two synthetic-based extenders, tris–citric acid–glucose plus 20% EY (TCG-EY) and tris–citric acid–fructose (TCF) both with 5% glycerol, were compared. Thirty-two epididymides were recovered from 16 adult guinea pig males by gonadectomy, and then the sperm samples were retrieved by retrograde flushing using TCG-EY and TCF extenders for left or right epididymis, respectively. TCG-EY and TCF sperm samples were frozen in static liquid nitrogen vapors through a two-step cooling procedure. Before freezing, the percentage of progressive sperm motility and sperm with intact plasma and acrosome membranes from TCG-EY sperm samples were higher (p < 0.05) than those diluted with TCF. Post-thaw sperm kinematic variables and membrane integrity were drastically reduced (p < 0.001) compared with prefreezing samples, regardless of extender type. The post-thaw plasmatic and acrosome membrane integrity from TCG-EY sperm samples was higher (p < 0.05) than those from TCF samples. Except for the length, the morphometric head dimensions of sperm diluted with TCG-EY or TCF did not vary (p > 0.05) after the freezing–thawing process compared with the prefreezing samples. In conclusion, despite greater cell cryoinjury with both extenders, the EY supplementation exerted greater cell membrane protection before and after the freezing–thawing process. This research shows an in-depth analysis of guinea pig sperm cryopreservation; however, more studies are recommended.
Evaluación de dos diluyentes en la congelación de espermatozoides epididimarios de perros (alcohol polivinílico y yema de huevo)
(2022) López Zhindón, Noelia Natali; Duma Pauta, José Mauricio; Samaniego Campoverde, Jorge Xavier; Galarza Lucero, Diego Andrés; Argudo Garzón, Daniel Ernesto
Egg yolk (EY) is a common component of semen extenders due to its effectiveness in protecting the sperm membrane from cold shock during cryopreservation and it is also rich in proteins, vitamins, antioxidants, and phospholipids. However, the EY is an undefined component and at the same time dangerous due to the risk of transmission of pathogenic agents. For this reason, it is necessary to evaluate the use of other components that reduce this problem. An understudied alternative is polyvinyl alcohol (PVA). For this reason, this investigation evaluated the effect of PVA and EY supplemented with TCG diluent (tris, citric acid, glucose) on the cryosurvival of dog epididymal spermatozoa (ES). For this, ES were recovered using the retrograde flow technique in 15 orchiectomized adult dogs to form five pools (4-6 epididymal samples/pool). Each pooled sample was divided into two aliquots, which were subsequently diluted and frozen using two additives supplemented to the TCG diluent: 1% (w/v) PVA [TCG–PVA] and 20% (v/v) egg yolk [TCG–EY]. After thawing, morphological abnormalities, vitality (eosin/nigrosin), plasma membrane integrity (propidion iodide), and kinematic variables (CASA system, SCA-2018 ) were analyzed. The results demonstrated that the vitality and integrity of the plasma membrane were superior (P<0.05) in samples frozen with TCG–EY, compared to those samples frozen with TCG–PVA. Likewise, the samples frozen with TCG–EY obtained higher post-thaw values (P<0.05) of kinematic variables such as total and progressive motility, average and rectilinear velocities, straightness index and flagellum beat frequency, compared to with frozen samples with TCG–PVA. In conclusion, enhancement of EY to TCG medium was effective in freezing dog ES, as it improved vitality, plasma membrane body, and sperm kinetics; however, the addition of 1% PVA did not improve the response to cryopreservation.
Evaluación de ovocitos recuperados por Ovum Pick Up (OPU) en tiempos diferentes, luego de la estimulación ovárica con FSH-LH (Pluset®) en vaquillas criollas
(2017) Samaniego Campoverde, Jorge Xavier; Ayala Guanga, Luis Eduardo
The present investigation was carried out in the experimental farm Irquis of the University of Cuenca. The objective of the present study was to evaluate the quality and quantity of oocytes recovered at two different times of OPU (40h and 48h) on the quality and quantity of COC’s recovered by Ovum pick up (OPU), after an ovarian stimulation protocol based FSH-LH (Pluset®) in heifers of Creole genotype. Twenty two OPU sessions were divided into four treatments. We analyzed 330 data in total and 15 variables, a general lineal model was used for the analysis of the data through the SAS version 9,3 and MEANS procedure in the determination of descriptive statistics of the variables. In addition, the Tukey-Kramer test was used for the comparison between the least squares media. The treatments OPU 40h and OPU 48h allowed to obtain twice as many COC’s (8,2 and 9,8 respectively), compared to those who did not receive ovarian stimulation (40h control and 48h control), presenting statistical difference (P<0,05). Three times more COC’s of quality A were obtained in the OPU 48 treatment and 100% of these structures reacted positively to the bright blue test of cresyl (BCB+), that is to say they finished their growth and they were ready to initiate the process of maturation in vitro. It is concluded that the OPU 48h treatment allows to obtain higher quality and quantity of COC’s recovered and suitable for the PIV process.
L-carnitine supplementation in conventional slow and ultra-rapid freezing media improves motility, membrane integrity, and fertilizing ability of dog epididymal sperm
(2024) Samaniego Campoverde, Jorge Xavier; Méndez Álvarez, María Silvana; Ramón López, Ángel Eduardo; Fernández Collahuazo, Jessica Paola; Duma Pauta, José Mauricio; Galarza Lucero, Diego Andrés; Galarza Alvarez, Luis Rodrigo; Soria Parra, Manuel Elías
This study aimed to assess the impact of L-carnitine (LC) supplementation in conventional-slow (CS) and ultra-rapid (UR) freezing media on post-thaw quality and fertilizing ability of dog epididymal spermatozoa. Sperm samples were collected from 60 epididymides obtained from 30 adult orchiectomized dogs via retrograde flushing. Twenty pooled sperm samples were then created (3 epididymal samples/pool). Four treatments were established according to the freezing method (CS and UR) and LC supplementation (5 and 0 mM [control, Co]): CS-LC5, CS-Co, URLC5, and UR-Co. The CS freezing involved exposing 0.25 mL straw to liquid nitrogen vapors (LN2), while UR freezing submerged 30-μL drops of sperm samples directly into LN2. Sperm kinematics, membrane integrity, and fertilizing ability (by heterologous in vitro fertilization using bovine oocytes) were evaluated for all treatments. Post-thaw results revealed that the CS freezing treatments resulted in significantly higher values (P < 0.05) of curvilinear and average-path velocities, and beat-cross frequency compared to the UR freezing treatments, regardless of LC supplementation. The CS-LC5 and UR-LC5 treatments cryoprotected the sperm by increasing (P < 0.05) the percentage of ‘live-sperm/intact-acrosome’ compared to their controls treatments CS-Co and UR-Co. Regarding fertilizing ability, the CS-LC5 treatment yielded a higher percentage (P < 0.05) of pronuclei formation compared to both UR treatments. The UR-LC5 treatment, however, obtained greater percentage (P < 0.05) than their control UR-Co. In conclusion, supplementation with L-carnitine in conventional-slow and ultra-rapid freezing improved sperm motility, plasma, and acrosome membranes integrity and fertilizing ability of dog epididymal spermatozoa
La suplementación de jalea real a diluyente de base sintética y no sintética ayuda a mantener la motilidad y cinética de esperma de toro refrigerado
(Universidad de Cuenca, 2022) Galarza Lucero, Diego Andrés; Guanga Tenempaguay, Jacqueline; Lucero Zhumi, Juan Ismael; Samaniego Campoverde, Jorge Xavier; Galarza Lucero, Diego Andrés; Galarza Lucero, Diego Andrés
Royal Jelly (RJ) is a substantial nutrient produced by the hypopharynx and mandibular glands of worker bees from 4 to 11 days of age, and its composition is mainly based on water (60–70%), protein (27–41% ) and carbohydrates (30%), lipids (8–19%), vitamins (A, B, C, E), salts, and amino acids. Due to its composition, JR has been successfully used in sperm cryopreservation at concentrations from 0.2 to 1% (w/v) supplemented with synthetic base extenders (eg TRIS or TALP). Its effect has been evidenced in the increase of motility, viability, functionality of the plasmatic membrane and fertilizing capacity of fresh and cryopreserved ram sperm. Conventional freezing requires the use of penetrating cryoprotectants (eg glycerol) to prevent damage caused by ice crystal formation, however, vitrification requires high concentrations (0.1 to 6 M) of non-penetrating cryoprotectants (eg sucrose or trehalose) and rapid cooling, passing rapidly to the glass transition without forming ice crystals. This research evaluated the effect of JR (0.2%) supplemented to freezing (TCG-YH + 5% glycerol) and vitrification (TCG-YH + 100 mM sucrose) media on cryosurvival of bull sperm.
Optimization of cryopreservation of arabian stallion sperm using dimethylformamide, glycerol, and different freezing protocols
(Wiley-Blackwell, 2022) Serpa Carangui, Erika Margoth; Samaniego Campoverde, Jorge Xavier; Galarza Lucero, Diego Andrés; Soria Parra, Manuel Elías; Ochoa Guillén, Juan Diego; Álvarez Palacios, David Alejandro; De la Cruz Tandazo, Francisco Javier; Galarza Lucero, Diego Andrés
This study evaluated the effect of penetrating cryoprotectant agents (CPA) and the cryosurvival of three freezing protocols on the kinematics and integrity of membranes of frozen-thawed stallion sperm. Twenty-four ejaculates of four adult Arabian horses were collected in six weekly sessions (six ejaculates/horse). Each ejaculate was divided into two aliquots. With the first aliquot, three CPA treatments were conformed: 5% glycerol (GLY), 5% dimethylformamide (DMF), and 3%–3% DMF–GLY combination, and the sperm samples were frozen exposing them to liquid nitrogen (LN2) vapors. The second aliquot was diluted with freezing medium plus 5% DMF and the sperm samples were frozen in three freezing protocols: (P1) Styrofoam cryo-box (30 × 29 × 31 cm of length, width, and height, respectively) with two ramps (at 17 and 7 cm above LN2); (P2) freezing unit® (Minitüb, Germany); and (P3) programmable TK 4000-freezer® (Compacta, Brazil). The DMF-GLY combination and DMF yielded higher (p<.05) post-thaw values than the GLY regarding the motility (SM: 54.2±2.25 and 50.2±1.80 vs. 41.4±2.35%, respectively), curvilinear velocity (VCL: 58.0±1.71 and 54.0±1.58 vs. 42.3±1.60 µm/s), and the proportion of sperm with intact plasma and intact acrosome (IPIA: 58.0±1.11 and 52.6±0.99 vs. 42.5±1.07%). Furthermore, the P1 protocol produced a similar (p>.05) post-thaw SM, VCL, and IPIA than the other protocols. Indeed, the P1 and P3 protocols yielded lower proportion (p<.05) of sperm with damaged plasma and damaged acrosome than the P2 protocol after thawing (3.7±0.18 and 3.1±0.18 vs. 6.1±0.44%, respectively). In conclusion, the addition of DMF or combined with GLY to freezing medium, and the freezing with Styrofoam cryo-box with two ramps increase the cryosurvival of Arabian stallion spermatozoa.