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The aim of this study was to determine the effects of the cumulative gain in expertise in carrying out handmade cloning (HMC) procedures on embryo yield and pregnancy outcome in cattle. Results from in vitro and in vivo embryo development after HMC during three periods of 7 months, separated by 3-month intervals, were compiled and designated as P1, P2 and P3. Blastocyst yield, morphological quality and stage of development, and pregnancy per embryo transfer (ET) on Day 30 of gestation were compared. Zona-intact oocytes were activated chemically in each experiment replicate, and development of parthenogenetic blastocysts was used as a control measurement of oocyte quality and in vitro culture conditions. A total of 21,231 cumulus-oocyte complexes (COCs) were in vitro-matured, with 5,432, 10,721 and 5078 COCs used in 16, 18 and 10 replicates for P1, P2 and P3, respectively. Cloned blastocyst yields on Day 7 increased from 15.5% (124/798) in P1 to 21.6% (309/1428) and 36.6% (280/764) in P2 and P3, respectively. No differences were observed in blastocyst development of parthenogenetic embryos, which average 30.0, 37.6, and 36.4% in P1, P2, and P3, respectively. A 10-fold higher probability of obtaining cloned blastocysts at more advanced stages of development and of higher morphological grade was seen during P3 compared with P1. Pregnancy per ET on Day 30 also increased with gain in expertise, being 6.7% (2/30), 20.8% (10/48) and 40.0% (24/60) for P1, P2 and P3, respectively. The relative efficiency for the establishment of pregnancies (per total COC) increased from 0.04% (1:2716) in P1 to 0.22% (1:460) in P2, reaching 0.47% (1:212) in P3. Results demonstrated a gradual improvement in in vitro and in vivo embryo development over time after establishment of HMC procedures in the laboratory, highlighting the importance of gaining experience and technical skills on the overall cloning efficiency., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The bovine IGF2 locus is a genomic region with alternative transcripts controlled by five promoters (P0, P1, P2, P3 and P4). As transcriptional regulation can affect messenger RNA (mRNA) stability and translation, and thus, subsequent biological effects, this study evaluated the bovine IGF2 promoter‐specific expression patterns in oocytes and pre‐implantation embryos produced in vitro by our standard IVP procedures. Immature and matured oocytes, and pre‐implantation embryos at the 1‐, 2‐, 4‐, 8‐ and 16‐cell, and at early morula, compact morula, blastocyst and expanded blastocyst stages were collected in three pools of five structures per stage, in four replicates. Total RNA was extracted and subjected to RT‐qPCR, using four sets of IGF2 promoter‐specific primers covering transcripts driven by promoters P0/P1, P2, P3 and P4, with fragments sequenced for confirmation. Expression of P2‐ and P4‐derived transcripts showed an initial peak between immature (P4) or matured (P2/P4) oocytes and 2‐cell embryos, gradually falling until embryo genome activation (EGA), rising again at compaction and cavitation. P0/P1‐derived transcripts were identified after EGA, during compaction, whereas P3 activity was not detected at any stage. Our findings suggest that P0/P1 and P2 likely have secondary roles during early stages, whereas P3 may be more relevant later in development. P4 seems to be the main pathway for bovine IGF2 expression during oocyte maturation and embryo development and, therefore, the main target to influence IVP in modulation of embryo growth and in studies in developmental biology. [ABSTRACT FROM AUTHOR]

The present study was conducted to decipher the proteome of in vivo‐produced pre‐implantation ovine embryos. Ten locally adapted Morana Nova ewes received hormonal treatment and were inseminated 12 hr after ovulation. Six days later, 54 embryos (morula and blastocyst developmental state) were recovered from eight ewes and pooled to obtain sufficient protein for proteomic analysis. Extracted embryo proteins were analysed by LC‐MS/MS, followed by identification based on four database searches (PEAKS, Proteome Discoverer software, SearchGUI software, PepExplorer). Identified proteins were analysed for gene ontology terms, protein clusters and interactions. Genes associated with the ovine embryo proteome were screened for miRNA targets using data sets of TargetScan (http://www.targetscan.org) and mIRBase (http://www.mirbase.org) servers. There were 667 proteins identified in the ovine embryos. Biological processes of such proteins were mainly related to cellular process and regulation, and molecular functions, to binding and catalytic activity. Analysis of the embryo proteins revealed 49 enriched functional clusters, linked to energy metabolism (TCA cycle, pyruvate and glycolysis metabolism), zona pellucida (ZP), MAPK signalling pathway, tight junction, binding of sperm to ZP, translation, proteasome, cell cycle and calcium/phospholipid binding. Sixteen miRNAs were related to 25 pre‐implantation ovine embryo genes, all conserved in human, bovine and ovine species. The interaction network generated by miRNet showed four key miRNAs (hsa‐mir‐106b‐5p; hsa‐mir‐30‐5p; hsa‐mir‐103a‐5p and hsa‐mir‐106a‐5p) with potential interactions with embryo‐expressed genes. Functional analysis of the network indicated that miRNAs modulate genes related to cell cycle, regulation of stem cell and embryonic cell differentiation, among others. Retrieved miRNAs also modulate the expression of genes involved in cell signalling pathways, such as MAPK, Wnt, TGF‐beta, p53 and Toll‐like receptor. The current study describes the first major proteomic profile of 6‐day‐old ovine embryos produced in vivo, setting a comprehensive foundation for our understanding of embryo physiology in the ovine species. [ABSTRACT FROM AUTHOR]

• The dietary supplements influenced the follicular environment. • The dietary supplements influenced oocyte mitochondrial gene expression. • Lipid or glucose-rich diet supplementations did not compromise oocyte quality. • Lipid or glucose-rich diet supplementations did not compromise embryo yield. This study aimed to verify the effects of short-term supplementation of diets rich in lipids or glycogen precursors on the follicular environment, oocyte mitochondrial gene expression, and in vitro embryo development. Thirty adult Anglo-Nubian multiparous crossbred goats were fed for 28 days with a total mixed ration (TMR) to satisfy the requirements of breeding for adult non-dairy goats. Seven days prior to the end of the feeding period, either ground flaxseed on a 10.5 % DM basis (Flaxseed Group - FG, n = 10), or a 200-mL/goat/day solution of crude glycerin in water at a 9:1 ratio (80 % Glycerin Group - GG, n = 10) were added to the TMR diet. The Control Group (CG) was maintained at the TMR diet (n = 10). Ovaries were collected upon slaughter for collection of follicular contents. Glucose, cholesterol and triglyceride concentrations and the glutathione peroxidase activity were determined in the follicular fluid, and the expression of Cox-1, NRF-1, PPARGC1-A and GDF-9 genes performed in granulosa cells and in oocytes by qPCR. Viable COCs were in vitro -matured for 24 h, to be parthenogenetically activated and in vitro -cultured for seven days up to the blastocyst stage. Intra-follicular glucose concentration was higher in the Glycerin (20.2 ± 2.5 mgdL GG vs. 5.6 ± 1.7 mgdL FG vs. 14.2 ± 3.5 mgdL CG; P < 0.01) and glutathione peroxidase activity in Flaxseed group (432.0 ± 70.2 UL FG vs. 376.9 ± 63.1 UL GG; P < 0.05), (432.0 ± 70.2 UL FG vs. 355.0 ± 59.3 UL CG: P < 0.01). All genes were expressed in granulosa cells and in oocytes, with higher NRF-1 expression in oocytes from the Glycerin group (P < 0.001). Treatments did not affect cleavage rates or embryo yields, with the Control and Flaxseed groups displaying higher (P < 0.05) proportion of blastocysts (76.9 % CG and 77.1 % FG) of better morphological quality (50 % excellent embryo; P < 0.001) than Controls. In summary, lipid or glucose-rich diet supplementations for brief periods did not compromise oocyte quality and embryo yield in goats. [ABSTRACT FROM AUTHOR]