| 1. |
- Becerro-Rey, Laura, et al.
(författare)
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Aging of stallion spermatozoa stored in vitro is delayed at 22C using a 67 mm glucose-10 mm pyruvate-based media
- 2023
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Ingår i: Andrology. - : WILEY. - 2047-2919 .- 2047-2927.
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Tidskriftsartikel (refereegranskat)abstract
- Background: Most commerce of equine seminal doses is carried out using commercial extenders under refrigeration at 5 degrees C.Objectives: To determine if 10 mM pyruvate in a 67 mM glucose extender and storage at 22 degrees C could be the basis of an alternative storage method to cooling to 5 degrees C.Material and methods: Stallion ejaculates were extendedin: INRA96 (67 mM glucose, non-pyruvate control), modified Tyrodes (67 mM glucose-10 mM pyruvate), supplemented with 0, 10, 50, and 100 mu M itaconate. As itaconate was vehiculated in DMSO, a control vehicle was also included. Sperm motility, viability, mitochondrial membrane potential, and production of reactive oxygen species were measured after collection and again after 48 and 96 h of storage at 22 degrees C. To disclose molecular metabolic changes, spermatozoa were incubated up to 3 h in modified Tyrodes 67 mM glucose-10 mM pyruvate and modified Tyrodes 67 mM glucose, and metabolic analysis conducted.Results: After 96 h of storage aliquots stored in the control, INRA96 had a very poor total motility of 5.6% +/- 2.3%, while in the 67 mM glucose-10 mM pyruvate/10 mu M itaconate extender, total motility was 34.7% +/- 3.8% (p = 0.0066). After 96 h, viability was better in most pyruvate-based media, and the mitochondrial membrane potential in spermatozoa extended in INRA96 was relatively lower (p < 0.0001). Metabolomics revealed that in the spermatozoa incubated in the high pyruvate media, there was an increase in the relative amounts of NAD(+), pyruvate, lactate, and ATP.Discussion and conclusions: Aliquots stored in a 67 mM glucose-10 mM pyruvatebased medium supplemented with 10 mu M itaconate, maintained a 35% total motility after 96 h of storage at 22 degrees C, which is considered the minimum acceptable motility for commercialization. Improvements may be related to the conversion of pyruvate to lactate and regeneration of NAD(+).
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| 2. |
- Gaitskell-Phillips, Gemma, et al.
(författare)
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Seminal plasma AnnexinA2 protein is a relevant biomarker for stallions which require removal of seminal plasma for sperm survival upon refrigeration
- 2020
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Ingår i: Biology of Reproduction. - : OXFORD UNIV PRESS INC. - 0006-3363 .- 1529-7268. ; 103:6, s. 1275-1288
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Tidskriftsartikel (refereegranskat)abstract
- Some stallions yield ejaculates that do not tolerate conservation by refrigeration prior to artificial insemination (AI), showing improvement after removal of most of the seminal plasma (SP) by centrifugation. In this study, the SP-proteome of 10 different stallions was defined through high-performance liquid chromatography with tandem mass spectrometry and bioinformatic analysis in relation to the ability of the ejaculates to maintain semen quality when cooled and stored at 5 degrees C. Stallions were classified into three groups, depending on this ability: those maintaining good quality after direct extension in a commercial extender (good), stallions requiring removal of seminal plasma (RSP) to maintain seminal quality (good-RSP), and stallions, unable to maintain good semen quality even after RSP (poor). Pathway enrichment analysis of the proteins identified in whole equine SP using human orthologs was performed using g: profiler showing enriched Reactome and the Kyoto Encyclopedia of Genes and Genomes pathways related to hexose metabolism, vesicle mediated transport, post translational modification of proteins and immune response. Specific proteins overrepresented in stallions tolerating conservation by refrigeration included a peroxiredoxin-6 like protein, and transcobalamin-2, a primary vitamin B12-binding, and transport protein. Also, the protein involved in protein glycosylation, ST3 beta-galactoside alpha-2,3-sialyltransferase 1 was present in good stallions. These proteins were nearly absent in poor stallions. Particularly, annexinA2 appeared as to be the most powerful discriminant variable for identification of stallions needing RSP prior to refrigeration, with a P = 0.002 and a q value = 0.005. Overall this is the first detailed study of the equine SP-proteome, showing the potential value of specific proteins as discriminant bio-markers for clinical classification of stallions for AI. Summary sentence The seminal plasma protein Annexin A2 identifies ejaculates needing removal of seminal plasma prior to conservation in refrigeration.
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| 3. |
- Ortiz-Rodriguez, Jose M., et al.
(författare)
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Transcriptome analysis reveals that fertilization with cryopreserved sperm downregulates genes relevant for early embryo development in the horse
- 2019
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Ingår i: PLOS ONE. - : PUBLIC LIBRARY SCIENCE. - 1932-6203. ; 14:6
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Tidskriftsartikel (refereegranskat)abstract
- Artificial insemination with cryopreserved spermatozoa is a major assisted reproductive technology in many species. In horses, as in humans, insemination with cryopreserved sperm is associated with lower pregnancy rates than those for fresh sperm, however, direct effects of sperm cryopreservation on the development of resulting embryos are largely unexplored. The aim of this study was to investigate differences in gene expression between embryos resulting from fertilization with fresh or cryopreserved sperm. Embryos were obtained at 8, 10 or 12 days after ovulation from mares inseminated post-ovulation on successive cycles with either fresh sperm or frozen-thawed sperm from the same stallion, providing matched embryo pairs at each day. RNA was isolated from two matched pairs (4 embryos) for each day, and cDNA libraries were built and sequenced. Significant differences in transcripts per kilobase million (TPM) were determined using (i) genes for which the expression difference between treatments was higher than 99% of that in the random case (P amp;lt; 0.01), and (ii) genes for which the fold change was amp;gt;= 2, to avoid expression bias in selection of the candidate genes. Molecular pathways were explored using the DAVID webserver, followed by network analyses using STRING, with a threshold of 0.700 for positive interactions. The transcriptional profile of embryos obtained with frozen-thawed sperm differed significantly from that for embryos derived from fresh sperm on all days, showing significant down-regulation of genes involved in biological pathways related to oxidative phosphorylation, DNA binding, DNA replication, and immune response. Many genes with reduced expression were orthologs of genes known to be embryonic lethal in mice. This study, for the first time, provides evidence of altered transcription in embryos resulting from fertilization with cryopreserved spermatozoa in any species. As sperm cryopreservation is commonly used in many species, including human, the effect of this intervention on expression of developmentally important genes in resulting embryos warrants attention.
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