Secreted metabolome of porcine blastocysts encapsulated within in vitro 3D alginate hydrogel culture systems undergoing morphological changes provides insights into specific mechanisms involved in the initiation of porcine conceptus elongation.

CONTEXT: The exact mechanisms regulating the initiation of porcine conceptus elongation are not known due to the complexity of the uterine environment. AIMS: To identify contributing factors for initiation of conceptus elongation in vitro , this study evaluated differential metabolite abundance within media following culture of blastocysts within unmodified alginate (ALG) or Arg-Gly-Asp (RGD)-modified alginate hydrogel culture systems. METHODS: Blastocysts were harvested from pregnant gilts, encapsulated within ALG or RGD or as non-encapsulated control blastocysts (CONT), and cultured. At the termination of 96h culture, media were separated into blastocyst media groups: non-encapsulated control blastocysts (CONT); ALG and RGD blastocysts with no morphological change (ALG- and RGD-); ALG and RGD blastocysts with morphological changes (ALG+ and RGD+) and evaluated for non-targeted metabolomic profiling by liquid chromatography (LC)-mass spectrometry (MS) techniques and gas chromatography-(GC-MS). KEY RESULTS: Analysis of variance identified 280 (LC-MS) and 1 (GC-MS) compounds that differed (P <0.05), of which 134 (LC-MS) and 1 (GC-MS) were annotated. Metabolites abundance between ALG+ vs ALG-, RGD+ vs RGD-, and RGD+ vs ALG+ were further investigated to identify potential differences in metabolic processes during the initiation of elongation. CONCLUSIONS: This study identified changes in phospholipid, glycosphingolipid, lipid signalling, and amino acid metabolic processes as potential RGD-independent mechanisms of elongation and identified changes in lysophosphatidylcholine and sphingolipid secretions during RGD-mediated elongation. IMPLICATIONS: These results illustrate changes in phospholipid and sphingolipid metabolic processes and secretions may act as mediators of the RGD-integrin adhesion that promotes porcine conceptus elongation.

Yearling bulls have reduced sperm concentration and increased seminal plasma interleukin-8 after a 28-day breeding season.

We hypothesized that yearling bulls selected for a 28-d breeding season would have reduced sperm concentrations and morphology, and have increased seminal plasma concentrations of pro-inflammatory cytokine interleukin-8 (IL-8). Yearling bulls were selected based on a breeding soundness examination (BSE) at approximately 415 d of age and contained at least 750 million sperm in the ejaculate, with 12 bulls randomly selected for breeding (BREEDERS) and 12 bulls not selected for breeding (NON-BREEDERS). After a 28-d breeding period, all bulls underwent a BSE. Plasma and seminal plasma were collected at each time point for analysis. Data were analysed utilizing either the MIXED or GLIMMIX procedures with repeated measures in SAS with breeding group, age and the interaction as fixed effects. Sperm concentration per ml of ejaculate was reduced (p < .05) in yearling bulls used for breeding compared with those not used for breeding at the end of the breeding season. Seminal plasma IL-8 concentrations in yearling bulls used for breeding were increased (p < .05) after the breeding season compared with bulls not used for breeding. Taken together, yearling bulls selected for a 28-d breeding season have reduced sperm production per ml of an ejaculate and increased inflammatory response in the seminal plasma that can lead to impaired breeding response if they are to be used for more than 30 d of breeding.

Global analysis of differential gene expression within the porcine conceptus transcriptome as it transitions through spherical, ovoid, and tubular morphologies during the initiation of elongation.

This study aimed to identify transcriptome differences between distinct or transitional stage spherical, ovoid, and tubular porcine blastocysts throughout the initiation of elongation. We performed a global transcriptome analysis of differential gene expression using RNA-Seq with high temporal resolution between spherical, ovoid, and tubular stage blastocysts at specific sequential stages of development from litters containing conceptus populations of distinct or transitional blastocysts. After RNA-Seq analysis, significant differentially expressed genes (DEGs) and pathways were identified between distinct morphologies or sequential development stages. Overall, 1898 significant DEGs were identified between distinct spherical and ovoid morphologies, with 311 total DEGs between developmental stages throughout this first morphological transition, while 15 were identified between distinct ovoid and tubular, with eight total throughout these second morphological transition developmental stages. The high quantity of DEGs and pathways between conceptus stages throughout the spherical to ovoid transition suggests the importance of gene regulation during this first morphological transition for initiating elongation. Further, extensive DEG coverage of known elongation signaling pathways was illustrated from spherical to ovoid, and regulation of lipid signaling and membrane/ECM remodeling across these early conceptus stages were implicated as essential to this process, providing novel insights into potential mechanisms governing this rapid morphological change.

Metabolic compounds within the porcine uterine environment are unique to the type of conceptus present during the early stages of blastocyst elongation.

The objective of this study was to identify metabolites within the porcine uterine milieu during the early stages of blastocyst elongation. At Days 9, 10, or 11 of gestation, reproductive tracts of White cross-bred gilts (n = 38) were collected immediately following harvest and flushed with Roswell Park Memorial Institute-1640 medium. Conceptus morphologies were assessed from each pregnancy and corresponding uterine flushings were assigned to one of five treatment groups based on these morphologies: (a) uniform spherical (n = 8); (b) heterogeneous spherical and ovoid (n = 8); (c) uniform ovoid (n = 8); (d) heterogeneous ovoid and tubular (n = 8); and (e) uniform tubular (n = 6). Uterine flushings from these pregnancies were submitted for nontargeted profiling by gas chromatography-mass spectrometry (GC-MS) and ultra performance liquid chromatography (UPLC)-MS techniques. Unsupervised multivariate principal component analysis (PCA) was performed using pcaMethods and univariate analysis of variance was performed in R with false discovery rate (FDR) adjustment. PCA analysis of the GC-MS and UPLC-MS data identified 153 and 104 metabolites, respectively. After FDR adjustment of the GC-MS and UPLC-MS data, 38 and 59 metabolites, respectively, differed (p < .05) in uterine flushings from pregnancies across the five conceptus stages. Some metabolites were greater (p < .05) in abundance for uterine flushings containing earlier stage conceptuses (i.e., spherical), such as uric acid, tryptophan, and tyrosine. In contrast, some metabolites were greater (p < .05) in abundance for uterine flushings containing later stage conceptuses (i.e., tubular), such as creatinine, serine, and urea. These data illustrate several putative metabolites that change within the uterine milieu during early porcine blastocyst elongation.

Development of pre-implantation porcine blastocysts cultured within alginate hydrogel systems either supplemented with secreted phosphoprotein 1 or conjugated with Arg-Gly-Asp Peptide.

Although deficiencies in porcine blastocyst elongation play a significant role in early embryonic mortality and establishment of within-litter developmental variation, the exact mechanisms of elongation are poorly understood. Secreted phosphoprotein 1 (SPP1) is increased within the uterine milieu during early porcine pregnancy and contains an Arg-Gly-Asp (RGD) peptide sequence that binds to cell surface integrins on the uterine endometrium and trophectoderm, promoting cell adhesion and migration. The aim of the present study was to evaluate the development of preimplantation porcine blastocysts encapsulated and cultured within alginate hydrogels either supplemented with SPP1 or conjugated with RGD. Blastocysts encapsulated within alginate hydrogels supplemented with SPP1 or conjugated with RGD had increased survival compared with non-encapsulated control blastocysts. In addition, the percentage of blastocysts encapsulated within RGD hydrogels that underwent morphological changes was greater than that of blastocysts encapsulated within standard alginate hydrogels or SPP1-supplemented hydrogels. Finally, only blastocysts encapsulated within RGD hydrogels had both increased expression of steroidogenic and immune responsiveness transcripts and increased 17ß-oestradiol production, consistent with blastocysts undergoing elongation in vivo. These results illustrate the importance of the integrin-binding RGD peptide sequence for stimulating the initiation of blastocyst elongation.