Examinando por Materia "Cryopreservation"

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Effect of extender and freezing rate on quality parameters and in vitro fertilization capacity of alpaca spermatozoa recovered from cauda epididymis
(Mary Ann Liebert, Inc., 2019-02-13) Mamani Mango, Guiulfo; Moina Gonzales, Milagros; Ramos Hidalgo, Martín; Mendoza Mallma, José; Ruiz Béjar, Jaime; Rivas Palma, Victoria Esther; Mellisho Salas, Edwin
In alpacas, improvement of reproductive efficiency of male camelids is limited by the small testicular size, low spermatozoa production, and low quality of semen. In this study we aim to evaluate the effect of two extenders and two freezing rates on post-thaw quality of sperm recovered from alpaca epididymis with two methods (flushing and mincing), and to evaluate the in vitro fertilization (IVF) capacity of frozen sperm selected with two different selection methods (washing and swim-up). Sperm samples were processed with Tris-egg yolk or Bioxcell® extenders and frozen with slow freezing and fast freezing. The oocytes were coincubated with spermatozoa for 72 hours, and cleavage rates were recorded afterward. The results indicated that the recovery method did not influence sperm quality (∼70%). However, total sperm recovery was significantly lower for the flushing method than the mincing method. The sperm quality was influenced by the freezing extender (23.3% vs. 33.2%) and freezing rate (20.9% vs. 35.7%). When comparing different methods of sperm selection for IVF, no differences were observed on cleavage rate except for the fact that the concentration of sperm from swim-up method (20.6%) was significantly lower than the one obtained from the washing method (78.7%). The recovery technique of sperm does not affect sperm quality and the method of fast freezing was shown to be the most effective for cryopreservation of alpaca sperm.
Effects on the quality of frozen-thawed alpaca (Lama pacos) semen using two different cryoprotectants and extenders
(Wolters Kluwer Medknow Publications, 2005-09-01) Santiani, Alexei; Huanca López, Wilfredo; Sapana Valdivia, Rómulo; Huanca Mamani, Teodosio; Sepúlveda, Néstor; Sánchez, Raúl
Aim: To evaluate two extenders and two cryoprotectant agents (CPA) for alpaca semen cryopreservation. Methods: Semen samples were obtained from four adult alpacas (Lama pacos) and frozen using extender I (TRIS, citrate, egg yolk and glucose) or extender II (skim milk, egg yolk and fructose), each containing either glycerol (G) or ethylene glycol (EG) as CPA. Consequently, four groups were formed: 1) extender I-G; 2) extender I-EG; 3) extender II-G; and 4) extender II-EG. Semen was diluted in a two-step process: for cooling to 5 °C (extenders without CPA), and for freezing (extenders with CPA). Viability and acrosome integrity were assessed using trypan blue and Giemsa stains. Results: When compared, the motility after thawing was higher (P < 0.05) in groups II-EG (20.0 % ± 6.7 %) and II-G (15.3 % ± 4.1 %) than that in groups I-G (4.0 % ± 1.1 %) and I-EG (1.0 % ± 1.4 %). Viable spermatozoa with intact acrosomes in groups II-EG (18.7 % ± 2.9 %) and II-G (12.7 % ± 5.9 %) were higher than that in groups I-G (5.7 % ± 1.5 %) and I-EG (4.0 % ± 1.0 %). Conclusion: The skim milk- and egg yolk-based extenders containing ethylene glycol or glycerol to freeze alpaca semen seems to promote the survival of more sperm cells with intact acrosomes than the other extenders.
Use of Androcoll-ETM to Separate Frozen-Thawed Llama Sperm From Seminal Plasma and Diluent
(Universidad Austral de Chile, 2021-01-21) Guillén Palomino, Crissthel Yverlin; Fumuso, Fernanda Gabriela; Bertuzzi, Mariana Lucía; Giuliano, Susana María; Velásquez González, Nicolás; Bariani, María Victoria
It is not easy to separate frozen-thawed South American camelid sperm from seminal plasma (SP) and diluents to be used for in vitro embryo production. The objective of this study was to evaluate Androcoll-E™ (AE) efficiency to separate llama sperm from SP and freezing extender in frozen-thawed semen. A total of 22 ejaculates from five Lama glama males were collected using electroejaculation. After performing semen analysis (sperm motility, concentration, viability, membrane function, and acrosome integrity), samples were cryopreserved with a diluent containing lactose, ethylenediaminetetraacetic acid (EDTA), egg yolk, and 7% dimethylformamide. After thawing, samples were divided in aliquots, one of which was used as a control and the others processed by AE. Experiment 1 (12 ejaculates): 100 μl of frozen-thawed semen was placed on top of 1,000 μl AE column and centrifuged at 800 g for 10 min. Experiment 2 (10 ejaculates): two samples of 100 μl of frozen-thawed semen were placed on two columns of 500 μl AE each, and both were centrifuged at 800 g for 10 and 20 min, respectively. Pellets were resuspended in Tyrode's albumin lactate pyruvate (TALP) medium, and sperm parameters were evaluated. A significant decrease in all sperm parameters was observed in thawed samples compared to raw semen. AE allowed the separation of frozen-thawed sperm from SP and freezing extender independently from the height of the column used and time of centrifugation assayed. Although no significant differences were found between AE columns, higher sperm recovery was observed with 500 μl of AE coupled with 20 min of centrifugation. Despite the significant decrease observed in sperm motility in AE samples, no changes in sperm viability, membrane function, and acrosome integrity were observed when comparing control thawed semen with the sperm recovered after AE (p > 0.05). The use of AE columns, either 500 or 1,000 μl, allows the separation of frozen-thawed llama sperm from SP and freezing extender, preserving the viability, membrane function, and acrosome integrity. Of the protocols studied, 800 g centrifugation during 20 min using a 500 μl column of AE would be the method of choice to process frozen-thawed llama semen destined for reproductive biotechnologies.
Vitrificación de embriones de alpacas: estudio preliminar
(Asociación Latinoamericana de Producción Animal, 2007-10-31) Vásquez, M.; Cervantes, M.; Cordero, A.; Cárdenas Minaya, Oscar Efraín; Huanca Mamani, Teodosio; Huanca, W.
El objetivo del presente trabajo fue evaluar el efecto de un protocolo de vitrificación de embriones de alpacas sobre la morfología embrionaria In Vitro post descongelamiento y viabilidad In Vivo post transferencia. Dos alpacas hembras donadoras recibieron un tratamiento de superovulación con eCG y fueron montadas naturalmente. Siete días después se realizó el lavado uterino no quirúrgico. Se recuperaron 9 embriones en estadío de blastocisto y clasificados según lo señalado en el manual de la IETS (1998). Los embriones fueron expuestos por 5 minutos a la solución de vitrificación 1 (SV1) (0.1 M de sucrosa, 0.125 M de glucosa y 10 % de glicerol); 5 minutos en la Solución de Vitrificación 2 (SV2 ) (0.2 M de sucrosa, 0.25 M de glucosa, 10 % de glicerol y 10 % de etilenglicol) y finalmente transferidos a la Solución de Vitrificación 3 (SV3), (0.3 M de sucrosa, 0.375 M de glucosa, 20 % de glicerol y 20 % de etilenglicol), por 1 minuto, antes de ser cargados en pajillas de 0.25 ml y ser sumergidos en nitrógeno líquido. Cuarenta y ocho horas después, los embriones fueron descongelados y evaluados. Ocho embriones descongelados de buena calidad morfológica, fueron transferidos a 8 alpacas receptoras previamente sincronizadas; sin embargo, a la evaluación ecográfica ninguna hembra fue diagnosticada preñada. Estos resultados nos sugieren que posiblemente existan factores que no afectan la calidad morfológica pero si la viabilidad de los embriones transferidos.
Vitrificación de embriones de alpacas: estudio preliminar
(Sitio Argentino de Producción Animal, 2007-10-31) Vásquez, M.; Cervantes, M.; Cordero, A.; Cárdenas Minaya, Oscar Efraín; Huanca Mamani, Teodosio
El objetivo del presente trabajo fue evaluar el efecto de un protocolo de vitrificación de embriones de alpacas sobre la morfología embrionaria In Vitro post descongelamiento y viabilidad In Vivo post transferencia. Dos alpacas hembras donadoras recibieron un tratamiento de superovulación con eCG y fueron montadas naturalmente. Siete días después se realizó el lavado uterino no quirúrgico. Se recuperaron 9 embriones en estadío de blastocisto y clasificados según lo señalado en el manual de la IETS (1998). Los embriones fueron expuestos por 5 minutos a la solución de vitrificación 1 (SV1) (0.1 M de sucrosa, 0.125 M de glucosa y 10 % de glicerol); 5 minutos en la Solución de Vitrificación 2 (SV2 ) (0.2 M de sucrosa, 0.25 M de glucosa, 10 % de glicerol y 10 % de etilenglicol) y finalmente transferidos a la Solución de Vitrificación 3 (SV3), (0.3 M de sucrosa, 0.375 M de glucosa, 20 % de glicerol y 20 % de etilenglicol), por 1 minuto, antes de ser cargados en pajillas de 0.25 ml y ser sumergidos en nitrógeno líquido. Cuarenta y ocho horas después, los embriones fueron descongelados y evaluados. Ocho embriones descongelados de buena calidad morfológica, fueron transferidos a 8 alpacas receptoras previamente sincronizadas; sin embargo, a la evaluación ecográfica ninguna hembra fue diagnosticada preñada. Estos resultados nos sugieren que posiblemente existan factores que no afectan la calidad morfológica pero si la viabilidad de los embriones transferidos.