Superovulation with eCG alone or combined with the GnRH analogue buserelin improves transferable embryo production in weaned sows without affecting embryo quality
Superovulation with eCG alone or combined with the GnRH analogue buserelin improves transferable embryo production in weaned sows without affecting embryo quality
Embryos were recovered via midline laparotomy on Day 6 of the estrous cycle (D0=onset of estrus). Sows were sedated with azaperone (2 mg/kg body weight, intramuscular) and then give sodium thiopental (7 mg/kg body weight, intravenous) to induce general anesthesia and maintained with isoflurane (3.5-5%). The ovaries and uterine horns were exposed through a mid-ventral incision and the number of corpora lutea was immediately counted to assess the response to hormonal stimulation, and ovulatory response was quantified. Embryo collection was carried out by flushing the proximal 50 cm of each uterine horn with 30 mL of recovery medium (mTL-HEPES-PVA). After collection, the embryos were evaluated for morphological quality and stage of development, and only embryos at the blastocyst stage with appropriate morphology were selected for the experiment.  Treatment protocol eCG Multiparous sows (LandracexLarge-White) of parity 4 to 6 were used as embryo donors. Animals were housed in individual crates within a mechanically ventilated building and received a standard commercial diet twice daily with free access to water. In this protocol sows received 1000 IU of equine chorionic gonadotropin (eCG, Folligon®, Intervet International B.V., Boxmeer, The Netherlands) 24 hours post-weaning.  Treatment protocol eCG+BU Multiparous sows (LandracexLarge-White) of parity 4 to 6 were used as embryo donors. Animals were housed in individual crates within a mechanically ventilated building and received a standard commercial diet twice daily with free access to water. In this protocol sows received 1000 IU of equine chorionic gonadotropin (eCG, Folligon®, Intervet International B.V., Boxmeer, The Netherlands) 24 hours post-weaning, followed by 10 µg of BU (2.5 mL Receptal®, Merck Sharp & Dohme Animal Health, S.L., Salamanca, Spain) at the onset of estrus. Control treatment Multiparous sows (LandracexLarge-White) of parity 4 to 6 were used as embryo donors. Animals were housed in individual crates within a mechanically ventilated building and received a standard commercial diet twice daily with free access to water. In this case, control sows received no hormonal stimulation and were monitored for spontaneous estrus post-weaning Nucleic acid extraction protocol: Extraction of RNA from blastocyst samples was carried out by using a miRNeasy Micro Kit (Qiagen Ibérica, Madrid, Spain) according to the manufacturer´s instructions. RNA concentration and purity were measured on a NanoDrop 2000 (Thermo Fisher Scientific, Madrid, Spain). RNA integrity was then examined with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Only high-quality preparations (RIN > 8) were taken forward for library construction.  Strand-specific RNA-seq libraries were prepared using Total RNA Prep Ligation with Ribo Zero Plus kit (Illumina; Cat. No. 20040525). Library preparation included depletion of ribosomal RNA, fragmentation of the remaining RNA, and synthesis of double-stranded cDNA. Libraries were completed through end repair, adapter ligation, PCR enrichment, and a cleanup step to eliminate small fragments and adapter dimers. Library size distribution was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).