157 First in Vitro Embryo Production in Alpacas (Lama Pacos)

Abstract

The objective was to produce alpaca embryos in laboratory due to its potential role for the multiplication of genetically superior animals and for conservation purposes. Ovaries were collected from an alpaca abattoir located in the Central Highlands of Peru and transported in a thermos flask with warm saline and antibiotics to the laboratory located 200 km away on the coast. Alpaca epididymal sperm to be used for fertilization was previously frozen by diluting in a TRIS-Fructose based extender with 10% glycerol and frozen as pellets in liquid nitrogen vapor. From 31 ovaries, 262 cumulus–oocyte complexes (COCs) were collected (mean of 8.5 COCs per ovary) which were matured in TCM-199 supplemented with 10% heat inactivated FCS plus epidermal growth factor (EGF), FSH, LH, estradiol, and cysteamine for 30 h incubation at 38.5°C, 5% CO2 and 90% humidity. The selected oocytes post-maturation were fertilized with the frozen/thawed sperm that was subjected post-thawing to Percoll gradient (90 and 45% Percoll), centrifugation and resuspension in TALP-IVF medium supplemented with 20 μm D-penicillamine, 10 μm hypotaurine, 1 μm epinephrine and 1.1 μg mL–1 of heparin. The oocytes were inseminated with a concentration of 10 × 106 spermatozoa per drop of 100 mL of fertilization medium containing 30 oocytes each and incubated for 24 h at 38.5°C, 5% CO2 and 90% humidity. The presumptive zygotes were transferred to 200 μL drops (30 zygotes per drop) of SOFaa media supplemented with 5% heat-inactivated FCS which was replaced by SOFaa plus 1% heat-inactivated FCS on day 5 after fertilization. The incubation period post-fertilization was up to day 7 at 38.5°C, 5% CO2 and 90% humidity, when the embryos were inspected and graded. The cleavage rate was evaluated at 72 h post-fertilization and embryo development was evaluated on day 5 and 7 post-fertilization. The cleavage rate was 27.1% (71/262) and the percentage of oocytes that reached the stage of morula and blastocyst was 8.0% (21/262). The percentage of blastocyst that hatched when incubated after day 7 was 14.28% (3/21). The in vitro embryo production in alpacas was successful and suggests the possibility for application in intensive reproduction for conservation of South American camelids and for genetic improvement. Research was partially funded by contributions of BIONICHE and SAIS TUPAC AMARU, Junin, Peru.

PRODUCCIÓN Y TRANSFERENCIA DE EMBRIONES in vitro EN CAMÉLIDOS SUDAMERICANOS: NUEVAS OPORTUNIDADES Y DESAFIOS

“…Los primeros trabajos de investigación en la producción de embriones por FIV en camélidos sudamericanos se han realizado utilizando gametos recuperados de animales beneficiados (Del Campo et al, 1994;Gamarra et al, 2008;Mendoza et al, 2008). Estos trabajos han sido de valiosa ayuda para investigar en estandarizar protocolos de MIV y FIV.…”

“…Por otro lado, en la MIV de ovocitos de alpaca se han utilizado diferentes tiempos tales como: 24-26 horas (Ratto et al, 2007;Mendoza et al, 2008;Huamán et al, 2011), 27 horas (Ruiz y Correa 2007) y 30 horas (Gamarra et al, 2008). Sin embargo, Huanca et al (2009), indican que son necesarias más de 38 horas para la MIV y Santayana et al (2012), realizaron un estudio con el objetivo de determinar el tiempo óptimo (24, 28 y 32 horas) de maduración nuclear in vitro de ovocitos de alpaca y evaluar la influencia de este sobre el desarrollo embrionario luego de la FIV.…”

“…El uso del semen congelado es un aspecto limitante para la masificación de la inseminación artificial en camélidos sudamericanos. Sin embargo, en la producción in vitro de embriones se han fertilizado ovocitos de alpaca con semen congelado (Gamarra et al, 2008), pero las tasas de blastocistos obtenidas han sido menores a las reportadas con semen fresco . El uso del semen refrigerado se ha presentado como una alternativa para conservar y trasladar el semen para la inseminación artificial de alpacas y llamas.…”

“…Sin embargo, el éxito de esta biotecnología está supeditada a la colección y maduración in vitro (MIV) de los ovocitos, condiciones de cultivo de los embriones y sincronización del embrión producido in vitro con la receptora para obtener mejores tasas de preñez con la transferencia de estos embriones. Existen desde hace varios años, trabajos de investigación en la producción de embriones por FIV en camélidos sudamericanos (Del Campo et al, 1994;Conde et al, 2008;Gamarra et al, 2008;Mellisho et al, 2014;. Asimismo, se han reportado gestaciones tempranas en llamas (Mendoza et al, 2013;Trasorras et al, 2014) y alpacas (Mendoza et al, 2013), y el nacimiento de la primera llama en el mundo (Landeo et al, 2016a) con la aplicación de esta biotecnología.…”

Embryo Preservation and in Vitro Production of Embryos

“…23 Likewise, Sansinena et al did not use IVF in llamas because of the low percentages of spermatozoa with progressive motility. 44 If IVF is to become an acceptable method and use males with high genetic value, it is important to improve semen collection and preparation techniques to allow separation and selection of highly motile spermatozoa from seminal plasma. 44 If IVF is to become an acceptable method and use males with high genetic value, it is important to improve semen collection and preparation techniques to allow separation and selection of highly motile spermatozoa from seminal plasma.…”