Improving Delivery and Offspring Viability of In Vitro-Produced and Cloned Sheep Embryos1

Abstract

Recently developed, assisted reproductive technologies (e.g., in vitro embryo production and nuclear transfer) have encountered perinatal morbidity/mortality of the offspring produced, which are likely to hinder the application of these techniques. Consequently we have sought to develop a system of hormonal stimulation that will ensure the delivery of offspring more prepared for extrauterine life. Here we examine deliveries outcome in sheep carrying in vitro-produced and nuclear transfer (NT) embryos in comparison to artificially inseminated and naturally mated control ewes. All groups (excluding NT, which received one treatment) were subjected to one of two hormonal treatments for induction of delivery, whereas the third part of each group was left without any treatment. The first (commonly used for naturally mated ewes) dexamethasone treatment did not solve a majority of parturition disturbances, and actually the number of deliveries necessitating assistance was reduced (P < 0.05) by this treatment in the control group. On the other hand, combined estradiol plus betamethasone stimulation (E + B) solved a majority of complications regarding delivery performance such as lack of the preparation of the mammary gland, low myometrial contractility, insufficient cervical ripening, and impaired maternal behavior. Moreover, substantial reduction of neonatal mortality was observed following the combined treatment. In conclusion, the E + B induction of delivery overcame the majority of physiological and behavioral intrapartum failures of sheep foster mothers and increased the survival of offspring, and thus can be recommended as a safe method for inducing delivery in foster mothers carrying in vitro-generated embryos.

Birth of lambs of a pre-determined sex after in vitro production of embryos using frozen–thawed sex-sorted and re-frozen–thawed ram spermatozoa

“…Due to the large size of a proportion of the lambs and associated birthing difficulties and ewe and lamb mortality experienced during the lambing of recipient crossbred ewes in ET Trial 1, a combination of 5 ml dexame thasone (Dexadreson 2 mg/ml; Intervet) and 5 ml oestradiol benzoate (Mesalin 0.2 mg/ml; Intervet) was given at Day 143 of gestation to each of the Merino recipient ewes in ET Trial 2 to induce earlier births and smaller offspring (Ptak et al 2002).…”

Effect of vitamin A supplementation at different gaseous environments on in vitro development of pre-implantation sheep embryos to the blastocyst stage

“…Thus, maintaining an optimal level of ROS by adding antioxidants to the culture media seems to be absolutely essential for the proper production of gametes, successful interaction of gametes, fertilisation and development of embryos in vitro (Miesel et al, 1993;Sanocka and Kurpisz, 2004). Some authors have reported improvements in the overall outcome of IVF-IVEC -embryo transfer procedure (Ptak et al, 2002). Others have attempted to supplement the culture media with different sets of nutrients or co-culture the oocytes and embryos to obtain better results (Rexroad and Powell, 1993;Urdaneta et al, 2003;Karja et al, 2006).…”

Plasma membrane and acrosome loss before ICSI is required for sheep embryonic development

“…Oocytes having at least 2-3 layers of compact cumulus cells were selected for IVM in bicarbonate-buffered TCM-199 (Gibco) containing 2 mM glutamine, 0.3 mM sodium pyruvate, 100 μM cysteamine, 10 % fetal bovine serum (FBS) (Gibco), 5 μg/ml follicle stimulating hormone FSH (Ovagen, ICP, Auckland, New Zealand), 5 μg/ml luteinizing hormone (LH), and 1 μg/ml estradiol. Maturation was conducted in 4-well culture plates (Nunclon, Roskilde, Denmark) containing 0.4 ml/well of IVM medium, incubated in a humidified atmosphere of 5 % CO 2 in air at 38.5°C for 24 h [24]. After IVM, only selected MII oocytes with expanded cumulus and normal morphology were used for ICSI.…”

“…All nuclei were counterstained with propidium iodide. Scale bar = 10 μm Fertilization was carried out at a final concentration of 5 × 10 6 spermatozoa/ml and left overnight in a humidified atmosphere at 38.5°C, 5 % CO 2 , and 7 % O 2 [24].…”

“…The medium was renewed on day 3 (SOF supplemented with 0.27 mg/ml glucose (SOF+), 2 % EAA, 1 % NEAA), on day 5 (SOF+ with 10 % of charcoal stripped FBS (csFBS), 2 % EAA, 1 % NEAA), and on day 6 (1:1 MEM/M199 enriched with 10 % csFBS, 2.5 μg/ml gentamicin, and 1 % sodium pyruvate) until day 8. The in vitro development was recorded on day 1 (cleavage stage) and 7/8th day (blastocyst stage) as previously described in Ptak et al [24] and Iuso et al [12].…”