J.E. Correa

Superovulation in llamas (Lama glama) with pFSH and equine chorionic gonadotrophin used individually or in combination

The purpose of this experiment was to evaluate the efficacy of pFSH and/or equine chorionic gonadotrophin (eCG) for inducing superovulation in llamas. Sixteen adult llamas weighing on average 130 kg (range 110-140) and which had been showing signs of oestrus for 5 days were randomly allocated to three treatment groups and one control group (n = 4). Llamas in Group A received eCG (500 IU, i.m.) once daily for 3 days, those in Group B received pFSH i.m., in decreasing doses every 12 h for 5 days for a total of 220 mg, while those in Group C received eCG (500 IU, i.m.) once, and pFSH (total of 156 mg, i.m.) in decreasing doses for the next 4 days. Llamas in Group D (control) received saline (5 ml, i.m.) every 12 h for 5 days. All llamas were allowed to be mated on the evening of Day 5 and were given hCG (750 IU, i.m.) at that time; a second mating was carried out 12 h later. A non-surgical ova/embryo collection technique was performed 7 days after the first mating and then the ovarian response was evaluated by way of laparoscopy. All 16 llamas were mated successfully. The mean (SEM) number of ovulations (7.3 +/- 3.1) in Group B was significantly higher (P < 0.05) than in the other groups (1.5 +/- 0.5, 2.0 +/- 0.7, and 0.3 +/- 0.3 for groups A, C and D, respectively). The number of follicles > 10 mm at the time of ova/embryo collection was significantly higher (P < 0.05) in the groups treated with eCG. A total of 21 ova/embryos was recovered from the all flushed llamas, corresponding to 47.7% of corpora lutea observed. Of the 21 ova, 15 were fertilised; 13 of those were classified as excellent blastocysts and the remaining 2 were classified as dead or degenerating. Results demonstrate that llamas can be successfully ovarian superstimulated while expressing behavioural oestrus and suggest that pFSH is more effective than eCG to induce superovulation.

Timing of mating and ovarian response in llamas (Lama glama) treated with pFSH

The effect of the timing of mating on ovarian response in llamas was evaluated using 20 adult llamas weighing 90-120 kg which had been in oestrus for 5 days and were treated with 20 mg pFSH every 12 h for the following 5 days (total dose: 200 mg of FSH-NIH-P1). They were randomly allocated to Group A (N = 10) and mated immediately at the end of pFSH treatment or to Group B (n = 10) and mated 36 h after the end of pFSH treatment. Llamas of both groups were given hCG (750 iu, i.m.) immediately after mating. A second mating was allowed 12 h later. Ova and embryos were recovered by non-surgical uterine flushing 7 days after the first mating. Ovarian response was immediately evaluated afterwards via laparoscopy. The mean ovulation rate of 4.5 corpora lutea for Group A was significantly lower (P < 0.01) than the mean of 13.8 observed for Group B. The total ovarian response (number of corpora lutea + follicles > 10 mm) was also significantly higher (P < 0.01) in Group B than in Group A. Twenty-seven ova were recovered in each group, corresponding to 60% and 20% (P < 0.01) of the corpora lutea observed in Groups A and B, respectively; however, no significant difference (P > 0.05) in fertilisation rate was observed. The results show that pFSH induces superovulation in llamas treated during oestrus and that a 36-h interval between the end of FSH treatment and mating increases ovulation rate and the total ovarian response but does not affect the number of ova/embryos recovered.

Transvaginal ultrasound-guided cumulus oocyte complexes aspiration and in vitro embryo production in suckled beef and lactating dairy cattle on pasture-based management conditions

This study was conducted to determine the use of repeated transvaginal ultrasound-guided cumulus oocyte complex (COC) aspiration on COC recovery rate, in vitro embryo production (IVP) and subsequent pregnancy rates in Holstein Friesian (HF) and Aberdeen Angus (AA) cows (Experiment 1), and in pregnant and non-pregnant Holstein Friesian cows (Experiment 2). Cycling, non-pregnant HF (n=17) and AA (n=32) cows with 40-70 days postpartum, between 3 and 5 years of age were used in the Experiment 1. All cows were submitted to repeated transvaginal ultrasound-guided COC aspiration twice a week for 5-7 weeks. Cumulus ooctye complexes (COC) were in vitro matured, fertilized and cultured for 8 days. An overall of 100 and 350 embryos from HF and AA cows respectively were cryopreserved using a conventional slow freezing (Experiment 1). A total of 81 and 285 frozen-thawed embryos from HF and AA cows respectively were transferred to recipient cows. Pregnancy diagnosis was performed at 60 and 150 days of gestation using transrectal ultrasonography. In Experiment 2, cycling non-pregnant (n=9) and pregnant (n=8) HF cows were submitted to repeated ultrasound-guided COC aspiration and COC were in vitro matured, fertilized and cultured as in Experiment 1, except that embryos were cryopreserved but not thawed and transferred as described for Experiment 1. The results of this study indicate that COC recovery rate and blastocyts production are affected by the breed of the donor cow. The quality of blastocyts produced from both breed did not differ in terms of pregnancy and calving rates (Experiment 1). The physiologic state of pregnancy did not affect COC recovery rate and blastocysts production per donor/session (Experiment 2). The use of ultrasound-guided COC aspiration and IVP could be a powerful technique to improve the genetic of beef and dairy cattle managed under pasture-based conditions management in the southern Chile.

Vitrification of in vitro mature alpaca oocyte: effect of ethylene glycol concentration and time of exposure in the equilibration and vitrification solutions.

The effect of different ethylene glycol concentrations, times of exposure and vitrification procedure on viability, cleavage and blastocyst rate of in vitro matured alpaca oocytes chemically activated after vitrification was analyzed. In Experiment 1, oocytes were incubated for 12-15 min with different concentrations of ethylene glycol (EG) in the equilibration solution (ES) followed by chemical activation and in vitro cultured for 8 days to determine oocyte viability, cleavage and blastocyst rates. In Experiment 2, oocytes were incubated in the equilibration solution containing 4% of EG for 12-15 min and then randomly assigned to vitrification solutions containing 25, 35 or 45% of EG for 30s, vitrified and stored at -196°C. In Experiment 3, oocytes were incubated in the equilibration solution containing 4% of EG for 12-15 min and then randomly assigned to the vitrification solution containing 35% of EG for 15, 30 or 45s, vitrified and stored at -196°C. For Experiments 2 and 3, non-vitrified and vitrified oocytes were activated and cultured in vitro. In Experiment 1, oocyte viability was lowest at concentrations of 6 or 8%, intermediate at 2 or 4% and highest at 0% of EG. Oocyte viability and cleavage rate were affected by EG concentration, time of exposure in the vitrification solution or vitrification procedure in Experiment 2 and 3. Alpaca oocytes were viable after vitrification, given that oocyte viability, cleavage and blastocyst rate were affected by the vitrification procedure, EG concentration and time of exposure in the equilibration and vitrification solutions.

Fertilization rate in sheep unilaterally inseminated with frozen semen

In order to evaluate a simple surgical technique for artificial insemination (AI) with frozen semen, 40 adult ewes were synchronised with vaginal sponges impregnated with 60 mg of medroxyprogesterone acetate for 12 d and a single injection of 400 IU PMSG at sponge withdrawal. Semen from fertile rams was pooled and frozen in straws, each containing 3 × 108 spermatozoa. All ewes were inseminated 60 h after PMSG injection. Twenty ewes (group A) were unilaterally inseminated by a small midventral laparotomy and 20 (group B) were cervically inseminated. Fertilization rate was measured by flushing of uteri and oviducts throughout a laparotomy carried out 3–4 d after insemination. The 83.7% fertilization rate in group A was significantly different (P < 0.0001) than the 19.1 % fertilization rate of group B. This surgical technique seems to be useful when no laparoscopic equipment is available.

Vitrificación de ovocitos bovinos y su uso en el desarrollo partenogenético de embriones

El objetivo de este trabajo fue evaluar el efecto de la vitrificacion en la viabilidad de ovocitos activados quimicamente para la produccion de embriones partenogeneticos bovinos. Ovocitos bovinos aspirados de ovarios obtenidos en el matadero fueron madurados in vitro por 20-22 horas y se distribuyeron en los siguientes grupos. I (n=76): ovocitos vitrificados/descongelados, II (n=119): ovocitos expuestos a crioprotectantes sin vitrificacion y III (n=142): ovocitos control. Los ovocitos fueron vitrificados en microgotas sobre un papel de aluminio preenfriado flotando en nitrogeno liquido, utilizando una solucion de equilibrio con 4% de etilenglicol y una solucion de vitrificacion con 35% de etilenglicol 5% de polivinilpirrolidona y 0,4 M de trehalosa. Las microgotas vitrificadas fueron almacenadas en nitrogeno liquido y fueron descongeladas 1-3 dias despues del almacenamiento. Los tres grupos de ovocitos se activaron partenogeneticamente por exposicion de 4 minutos a 5 µM de ionomicina de Ca a temperatura ambiente seguido de una incubacion por 5 horas en 6-dimetilaminopurina a 38,5 oC en una atmosfera humeda con 5% CO2. Los embriones se cultivaron en medio mSOF durante 8-9 dias. Las tasas de ovocitos sobrevivientes fueron 55,1% y 93,7% para ovocitos vitrificados/descongelados (I) y expuestos (II) respectivamente. Las tasas de segmentacion de 55,3%, 72,3% y 74,6%, y de desarrollo hasta blastocistos fueron 7,1%, 17,4% y 21,7% en los grupos I, II y III respectivamente. Estos resultados demuestran que la tecnica de vitrificacion ha quedado establecida y permite la produccion de embriones partenogeneticos bovinos.

Gestaciones producidas con embriones bovinos clonados por transferencia nuclear

SUMMARY With the aim of obtaining pregnancies from nuclear transfer embryos reconstituted with somatic cells and enucleated oocytes, bovine oocytes from slaughterhouse were matured in vitro and enucleated by micromanipulation. Nuclear donor cells were obtained from the ear of an adult cow, cultured for 9 to 14 days and cryopreserved in liquid nitrogen. Confluent cells were inserted individually in the perivitelline space of enucleated oocytes and treated with 2 electric pulses to induce fusion. The reconstituted zygotes were then cultured for 2 hours and treated with ionomycin and 6-dimethylaminopurine for their activation. The embryos were cultured in synthetic oviduct fluid for 8 to 9 days to obtain the blastocyst stage. These blastocysts were transferred to recipient heifers 7 to 9 days after oestrus. The ultrasound pregnancy diagnosis was done about 30 days after recipient oestrus. Five pregnancies were obtained from 25 transfers (20%). Two of them were lost at 42 days and a third at 120 days. This seems to be the first report on pregnancies obtained by cloned embryos in Chile.

Inducción de mellizos mediante la transferencia de un segundo embrión ipsilateral o contralateral al cuerpo lúteo en vacas cubiertas

El objetivo de este trabajo fue comparar el efecto del sitio de deposito (ipsi o contralateral al CL) de un segundo embrion en vacas cubiertas sobre la tasa de prenez y de gestaciones dobles. Treinta y dos vacas receptoras recibieron un embrion congelado-descongelado ipsilateralmente a su cuerpo luteo y 28 contralateralmente, 7 dias posterior a su cubierta. En el grupo de transferencias ipsilaterales 21 receptoras se prenaron al primer servicio (65, 6%), mientras que en el grupo contralateral 21 de las 28 receptoras se diagnosticaron prenadas (75%), no existiendo diferencias entre ambos grupos. Sin embargo, si se observo diferencias en la tasa de prenez doble. Entre los 60 y 90 dias post servicio en ambos grupos (57, 1 v/s 14, 3% y 45 v/s 4, 8% respectivamente). Contrariamente a algunos antecedentes que senalan la mayor sobrevida de embriones transferidos ipsilateralmente al ocurrir la muerte del embrion nativo. No se observo diferencias entre los porcentajes de prenez en ambos grupos. La diferencia observada en el numero de gestaciones dobles entre el grupo de transferencia ipsilateral y el contralateral es mayor a la descrita en la literatura, pero confirma antecedentes de que la existencia de dos embriones en el cuerno uterino ipsilateral no aumenta la tasa de mortalidad embrionaria ni fetal.