Pub Date : 2025-03-17DOI: 10.1016/j.theriogenology.2025.117395
Meicheng Li , Yifan Bai , Jiajun Zhang , Hongyang Wang , Junjie Li , Wenjun Wang
Cryopreservation of boar semen is widely applied in the conservation of genetic resources and animal breeding to enhance the utilization efficiency of superior boars. However, accurately identifying individuals with good freezing tolerance in boar sperm remains challenging. In this study, based on the differences in sperm motility before and after cryopreservation from 328 boars, we selected six boars each from the Duroc, Landrace, and Large White breeds, and categorized them into poor freezability ejaculates (PFE) and good freezability ejaculates (GFE) groups for sperm metabolomic analysis. A total of 1288 metabolites were identified using both positive and negative ion modes. There were 148 differentially expressed metabolites between the GFE and PFE groups, which were enriched in pathways such as alanine, aspartate and glutamate metabolism; arginine biosynthesis; D-amino acid metabolism; histidine metabolism; beta-alanine metabolism; citrate cycle (TCA cycle); pantothenate and CoA biosynthesis; and pyruvate metabolism. Further analysis, including ROC curve evaluation, identified seven potential biomarkers for sperm cryopreservation. Argininosuccinic acid, asparagine, L-aspartate, fumarate, D-ornithine, DL-serine and histidine were tightly interconnected in a series of amino acids metabolism. In conclusion, our findings imply that differences in certain amino acid biosynthetic pathways contribute to the variations in freezing tolerance of boar sperm.
{"title":"Sperm metabolomics identifies freezability markers in Duroc, Landrace, and Large White boars","authors":"Meicheng Li , Yifan Bai , Jiajun Zhang , Hongyang Wang , Junjie Li , Wenjun Wang","doi":"10.1016/j.theriogenology.2025.117395","DOIUrl":"10.1016/j.theriogenology.2025.117395","url":null,"abstract":"<div><div>Cryopreservation of boar semen is widely applied in the conservation of genetic resources and animal breeding to enhance the utilization efficiency of superior boars. However, accurately identifying individuals with good freezing tolerance in boar sperm remains challenging. In this study, based on the differences in sperm motility before and after cryopreservation from 328 boars, we selected six boars each from the Duroc, Landrace, and Large White breeds, and categorized them into poor freezability ejaculates (PFE) and good freezability ejaculates (GFE) groups for sperm metabolomic analysis. A total of 1288 metabolites were identified using both positive and negative ion modes. There were 148 differentially expressed metabolites between the GFE and PFE groups, which were enriched in pathways such as alanine, aspartate and glutamate metabolism; arginine biosynthesis; D-amino acid metabolism; histidine metabolism; beta-alanine metabolism; citrate cycle (TCA cycle); pantothenate and CoA biosynthesis; and pyruvate metabolism. Further analysis, including ROC curve evaluation, identified seven potential biomarkers for sperm cryopreservation. Argininosuccinic acid, asparagine, L-aspartate, fumarate, D-ornithine, DL-serine and histidine were tightly interconnected in a series of amino acids metabolism. In conclusion, our findings imply that differences in certain amino acid biosynthetic pathways contribute to the variations in freezing tolerance of boar sperm.</div></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":"240 ","pages":"Article 117395"},"PeriodicalIF":2.4,"publicationDate":"2025-03-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143654534","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-15DOI: 10.1016/j.theriogenology.2025.117393
Laura Becerro-Rey, Francisco Eduardo Martín-Cano, María Cruz Gil, Cristina Ortega-Ferrusola, Eloy Redondo, Javier Masot, Eva da Silva-Álvarez, Fernando J Peña
Modern flow cytometers enable the simultaneous analysis of an increasing number of parameters or dimensions, offering significant advantages for andrological assessments. However, relying on conventional procedures for data processing increases the complexity of the analysis and the time required to complete it. We propose a simplified and efficient protocol that leverages commercial software and dimensional reduction algorithms for multiparametric flow cytometry data analysis, to improve data visualization and speed up the analysis. Ejaculates from six stallions were split into two media, a modified Tyrode's containing 67 mM glucose and 10 mM pyruvate or a commercial media containing 67 mM glucose but not pyruvate and stored at room temperature for 48 h. A five-color panel was designed to assess key sperm parameters, including viability, membrane permeability, mitochondrial membrane potential, mitochondrial mass, and apoptotic changes. Data were uploaded to FlowJo 10.10 for Mac, where automatic compensation and conventional 2D plot analyses were performed on the compensated files. In a second analysis, individual files were concatenated (merged) in FlowJo, generating new.fcs files, which were subsequently downsampled from 50,000 to 3000 events (spermatozoa) per file. The downsampled, concatenated files were exported and analyzed in Cytobank (https://premium.cytobank.org). Preprocessing included Arcsinh transformation for improved population visualization, automated compensation, and the exclusion of non-sperm debris, clumps, and doublets. Initial 2D plots were generated for each condition, providing a baseline comparison. Dimensionality reduction and clustering analyses were then performed on the same merged files. Our results showed that the proportions of sperm subpopulations identified by advanced analytical approaches, such as downsampling, dimensional reduction, and clustering, were consistent with those obtained via traditional 2D dot-plot analysis. This study demonstrates the feasibility and accuracy of advanced methodologies for flow cytometric analysis of spermatozoa. These approaches enhance processing speed, improve data visualization, and offer deeper insights into the dynamics of sperm subpopulations under varying metabolic conditions.
{"title":"Multidimensional data analysis algorithms facilitate and speed the analysis, and provide improved visualization of flow cytometry data in spermatology.","authors":"Laura Becerro-Rey, Francisco Eduardo Martín-Cano, María Cruz Gil, Cristina Ortega-Ferrusola, Eloy Redondo, Javier Masot, Eva da Silva-Álvarez, Fernando J Peña","doi":"10.1016/j.theriogenology.2025.117393","DOIUrl":"https://doi.org/10.1016/j.theriogenology.2025.117393","url":null,"abstract":"<p><p>Modern flow cytometers enable the simultaneous analysis of an increasing number of parameters or dimensions, offering significant advantages for andrological assessments. However, relying on conventional procedures for data processing increases the complexity of the analysis and the time required to complete it. We propose a simplified and efficient protocol that leverages commercial software and dimensional reduction algorithms for multiparametric flow cytometry data analysis, to improve data visualization and speed up the analysis. Ejaculates from six stallions were split into two media, a modified Tyrode's containing 67 mM glucose and 10 mM pyruvate or a commercial media containing 67 mM glucose but not pyruvate and stored at room temperature for 48 h. A five-color panel was designed to assess key sperm parameters, including viability, membrane permeability, mitochondrial membrane potential, mitochondrial mass, and apoptotic changes. Data were uploaded to FlowJo 10.10 for Mac, where automatic compensation and conventional 2D plot analyses were performed on the compensated files. In a second analysis, individual files were concatenated (merged) in FlowJo, generating new.fcs files, which were subsequently downsampled from 50,000 to 3000 events (spermatozoa) per file. The downsampled, concatenated files were exported and analyzed in Cytobank (https://premium.cytobank.org). Preprocessing included Arcsinh transformation for improved population visualization, automated compensation, and the exclusion of non-sperm debris, clumps, and doublets. Initial 2D plots were generated for each condition, providing a baseline comparison. Dimensionality reduction and clustering analyses were then performed on the same merged files. Our results showed that the proportions of sperm subpopulations identified by advanced analytical approaches, such as downsampling, dimensional reduction, and clustering, were consistent with those obtained via traditional 2D dot-plot analysis. This study demonstrates the feasibility and accuracy of advanced methodologies for flow cytometric analysis of spermatozoa. These approaches enhance processing speed, improve data visualization, and offer deeper insights into the dynamics of sperm subpopulations under varying metabolic conditions.</p>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":"239 ","pages":"117393"},"PeriodicalIF":2.4,"publicationDate":"2025-03-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143670794","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Vitamin supplementation is commonly used for peri-conceptual consumption, but the long-term impacts are not yet understood. We examined the effects of adding vitamins riboflavin (B2) and pyridoxine (B6) to culture medium on embryo quality produced from in vitro fertilization (IVF) and in vivo fertilization (IVV). At the two-cell stage, IVF and IVV embryos were cultured in a standard medium, Chatot-Ziomel-Bavister (CZB) with (IVVB2B6 and IVFB2B6) or without adding vitamins B2 and B6 (IVVCZB and IVFCZB). For IVVB2B6, vitamin supplementation presented lowered total cell numbers by an elevated inner cell mass (ICM) and reduced trophectoderm (TE) cells (Total cell numbers: IVVCZB = 57 ± 1.6, IVVB2B6 = 44 ± 1.9, IVFCZB = 45 ± 1.6, and IVFB2B6 = 60 ± 4.0; P < 0.0001). In contrast, the IVFB2B6 group showed an inverse response by increased TE cells and reduced placental efficiency after embryo transfer, (fetal:placental ratio: natural mating = 22.7 ± 1.0, IVVCZB = 11.2 ± 0.7, IVVB2B6 = 12.8 ± 1.2, IVFCZB = 12.7 ± 0.5, and IVFB2B6 = 9.5 ± 0.4; P < 0.0002). To investigate the relationship of vitamin B2 and B6 with one-carbon metabolism, by immunostaining heterochromatin methylation levels with H3K27me3. This showed significantly increased intensities in the IVVB2B6 group but not in the IVFB2B6. Oral glucose tolerance test indicated an increased area under the curve (iAUC) in IVVB2B6 at the early adult stage. Our findings suggest that embryo development trajectory with vitamin supplementation is influenced by the method of fertilization, leading to diverse responses.
{"title":"Inverse embryonic responses of In vivo and In vitro fertilized mouse embryos to vitamin B supplementation during preimplantation period with limited long-term risks","authors":"Norermi Firzana Alfian , Masashi Hisamoto , Satoshi Kishigami","doi":"10.1016/j.theriogenology.2025.117392","DOIUrl":"10.1016/j.theriogenology.2025.117392","url":null,"abstract":"<div><div>Vitamin supplementation is commonly used for peri-conceptual consumption, but the long-term impacts are not yet understood. We examined the effects of adding vitamins riboflavin (B2) and pyridoxine (B6) to culture medium on embryo quality produced from <em>in vitro</em> fertilization (IVF) and <em>in vivo</em> fertilization (IVV). At the two-cell stage, IVF and IVV embryos were cultured in a standard medium, Chatot-Ziomel-Bavister (CZB) with (IVV<sub>B2B6</sub> and IVF<sub>B2B6</sub>) or without adding vitamins B2 and B6 (IVV<sub>CZB</sub> and IVF<sub>CZB</sub>). For IVV<sub>B2B6</sub>, vitamin supplementation presented lowered total cell numbers by an elevated inner cell mass (ICM) and reduced trophectoderm (TE) cells (Total cell numbers: IVV<sub>CZB</sub> = 57 ± 1.6, IVV<sub>B2B6</sub> = 44 ± 1.9, IVF<sub>CZB</sub> = 45 ± 1.6, and IVF<sub>B2B6</sub> = 60 ± 4.0; P < 0.0001). In contrast, the IVF<sub>B2B6</sub> group showed an inverse response by increased TE cells and reduced placental efficiency after embryo transfer, (fetal:placental ratio: natural mating = 22.7 ± 1.0, IVV<sub>CZB</sub> = 11.2 ± 0.7, IVV<sub>B2B6</sub> = 12.8 ± 1.2, IVF<sub>CZB</sub> = 12.7 ± 0.5, and IVF<sub>B2B6</sub> = 9.5 ± 0.4; P < 0.0002). To investigate the relationship of vitamin B2 and B6 with one-carbon metabolism, by immunostaining heterochromatin methylation levels with H3K27me3. This showed significantly increased intensities in the IVV<sub>B2B6</sub> group but not in the IVF<sub>B2B6</sub>. Oral glucose tolerance test indicated an increased area under the curve (iAUC) in IVV<sub>B2B6</sub> at the early adult stage. Our findings suggest that embryo development trajectory with vitamin supplementation is influenced by the method of fertilization, leading to diverse responses.</div></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":"239 ","pages":"Article 117392"},"PeriodicalIF":2.4,"publicationDate":"2025-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143642159","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-14DOI: 10.1016/j.theriogenology.2025.117374
Xiaolin Yang , Shuangyi Zhang , Bo Liu , Wei Mao , Pengfei Gong , Lili Guo , Jingze Wu , Yi Zhao , Yongfei Wang , Surong Hasi , Jinshan Cao
Endometritis in dairy cows is associated with pathogenic microorganisms, local inflammatory injuries, and uterine microecological disorders. Escherichia coli (E. coli) is the primary pathogen responsible for bovine endometritis onset; however, the underlying pathomechanisms remain unclear. In this study, we aimed to investigate E. coli-induced endometritis mechanisms in dairy cows using bovine bone marrow-derived macrophages and endometrial tissue. Following E. coli infection of macrophages, we observed a significant increase in the mRNA expression levels of innate immune recognition receptors, including toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), as well as prostaglandin D2 (PGD2)-related enzymes (cyclooxygenase-2 and hematopoietic prostaglandin D synthase). Furthermore, the secretion of PGD2, a major mediator of inflammation, was markedly upregulated. In E. coli-infected macrophages, TLR2, TLR4, and NLRP3 increased the secretion of inflammatory mediators, including PGD2, by activating mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB). This lead to enhanced inflammatory response. During early E. coli infection, PGD2 inhibitors reduced the secretion of inflammatory mediators by modulating MAPK and NF-κB pathway activation and enhancing macrophage bacterial killing, thereby alleviating endometrial tissue damage in dairy cows. In contrast, in the later stages of infection, PGD2 inhibitors exacerbated the inflammatory response and impaired the killing capacity of macrophages, which lead to increased endometrial tissue damage. Therefore, our findings highlight that TLR2, TLR4, and NLRP3 are pivotal in regulating PGD2 secretion during E. coli-induced endometritis in dairy cows. PGD2 had a pro-inflammatory effect in the early stages of E. coli infection and anti-inflammatory effects in the later stages. These findings can help develop strategies benefiting endometritis treatment.
{"title":"Dual roles of the TLR2/TLR4/NLRP3-H-PGDS-PGD2 axis in regulating the inflammatory response in Escherichia coli-infected bovine bone marrow-derived macrophages and endometrial tissue","authors":"Xiaolin Yang , Shuangyi Zhang , Bo Liu , Wei Mao , Pengfei Gong , Lili Guo , Jingze Wu , Yi Zhao , Yongfei Wang , Surong Hasi , Jinshan Cao","doi":"10.1016/j.theriogenology.2025.117374","DOIUrl":"10.1016/j.theriogenology.2025.117374","url":null,"abstract":"<div><div>Endometritis in dairy cows is associated with pathogenic microorganisms, local inflammatory injuries, and uterine microecological disorders. <em>Escherichia coli</em> (<em>E. coli</em>) is the primary pathogen responsible for bovine endometritis onset; however, the underlying pathomechanisms remain unclear. In this study, we aimed to investigate <em>E. coli</em>-induced endometritis mechanisms in dairy cows using bovine bone marrow-derived macrophages and endometrial tissue. Following <em>E. coli</em> infection of macrophages, we observed a significant increase in the mRNA expression levels of innate immune recognition receptors, including toll-like receptor 2 (<em>TLR2</em>), toll-like receptor 4 (<em>TLR4</em>), and nucleotide-binding oligomerization domain-like receptor protein 3 (<em>NLRP3</em>), as well as prostaglandin D<sub>2</sub> (PGD<sub>2</sub>)-related enzymes (<em>cyclooxygenase-2</em> and <em>hematopoietic prostaglandin D synthase</em>). Furthermore, the secretion of PGD<sub>2</sub>, a major mediator of inflammation, was markedly upregulated. In <em>E. coli</em>-infected macrophages, TLR2, TLR4, and NLRP3 increased the secretion of inflammatory mediators, including PGD<sub>2</sub>, by activating mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB). This lead to enhanced inflammatory response. During early <em>E. coli</em> infection, PGD<sub>2</sub> inhibitors reduced the secretion of inflammatory mediators by modulating MAPK and NF-κB pathway activation and enhancing macrophage bacterial killing, thereby alleviating endometrial tissue damage in dairy cows. In contrast, in the later stages of infection, PGD<sub>2</sub> inhibitors exacerbated the inflammatory response and impaired the killing capacity of macrophages, which lead to increased endometrial tissue damage. Therefore, our findings highlight that TLR2, TLR4, and NLRP3 are pivotal in regulating PGD<sub>2</sub> secretion during <em>E. coli</em>-induced endometritis in dairy cows. PGD<sub>2</sub> had a pro-inflammatory effect in the early stages of <em>E. coli</em> infection and anti-inflammatory effects in the later stages. These findings can help develop strategies benefiting endometritis treatment.</div></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":"239 ","pages":"Article 117374"},"PeriodicalIF":2.4,"publicationDate":"2025-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143644240","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-13DOI: 10.1016/j.theriogenology.2025.117390
Margherita Moncada , Martina Lo Sterzo , Luca Palazzese , Francesca Boffa , Debora Agata Anzalone , Domenico Iuso , Marta Czernik , Luisa Gioia , Federica Turri , Beatrice Mislei , Diego Bucci , Jacques Bonnet , Marthe Colotte , Sophie Tuffet , Michał Bochenek , Pasqualino Loi
Dry storage at room temperature (RT) could simplify spermatozoa banking. Here, we explored DNA stability and in vitro and in vivo development of embryos derived from vacuum-dried encapsulated (VDE) ram spermatozoa stored for four years or after accelerated aging. While some genomic damage was detected at time 0, DNA fragmentation increased from 3.32 ± 3 % (time 0) to 37.64 ± 4 % (4 years). A decrease in blastocyst rate was observed after four years of storage and 6.7 years of simulated storage (10.2 % and 9 % versus 13.16 % at time 0). Embryo quality, assessed based on Cdx2 and Inf-τ gene expression, declined over time. Only two of the 23 embryos transferred into synchronized ewes were implanted but were lost by day 40.
In conclusion, dry spermatozoa generated blastocysts after four years of RT storage, but their post-implantation development was impaired. Optimization of the water extraction and storage conditions could better preserve the spermatozoa's DNA integrity, resulting in improved embryo quality, compatible with development to term.
{"title":"Fertility preservation of vacuum-dried ram spermatozoa stored for four years at room temperature","authors":"Margherita Moncada , Martina Lo Sterzo , Luca Palazzese , Francesca Boffa , Debora Agata Anzalone , Domenico Iuso , Marta Czernik , Luisa Gioia , Federica Turri , Beatrice Mislei , Diego Bucci , Jacques Bonnet , Marthe Colotte , Sophie Tuffet , Michał Bochenek , Pasqualino Loi","doi":"10.1016/j.theriogenology.2025.117390","DOIUrl":"10.1016/j.theriogenology.2025.117390","url":null,"abstract":"<div><div>Dry storage at room temperature (RT) could simplify spermatozoa banking. Here, we explored DNA stability and <em>in vitro</em> and <em>in vivo</em> development of embryos derived from vacuum-dried encapsulated (VDE) ram spermatozoa stored for four years or after accelerated aging. While some genomic damage was detected at time 0, DNA fragmentation increased from 3.32 ± 3 % (time 0) to 37.64 ± 4 % (4 years). A decrease in blastocyst rate was observed after four years of storage and 6.7 years of simulated storage (10.2 % and 9 % <em>versus</em> 13.16 % at time 0). Embryo quality, assessed based on <em>Cdx2</em> and Inf-τ gene expression, declined over time. Only two of the 23 embryos transferred into synchronized ewes were implanted but were lost by day 40.</div><div>In conclusion, dry spermatozoa generated blastocysts after four years of RT storage, but their post-implantation development was impaired. Optimization of the water extraction and storage conditions could better preserve the spermatozoa's DNA integrity, resulting in improved embryo quality, compatible with development to term.</div></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":"239 ","pages":"Article 117390"},"PeriodicalIF":2.4,"publicationDate":"2025-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143632156","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-12DOI: 10.1016/j.theriogenology.2025.117389
Shanpeng Wang , Qi Wang , Xuejun Zeng , Lingjiang Min , Wenxian Zeng , Adedeji O. Adetunji , Zhendong Zhu
5-Aminolevulinic acid (5-ALA) is a crucial metabolic intermediate that affect mitochondrial function in somatic cells. However, the mechanisms by which 5-ALA regulates boar reproductive performance remain unclear. The effect of dietary supplementation of 5-ALA on boar semen quality and reproductive performance were investigated in this study. Forty-five boars were randomly assigned to a control group and four 5-ALA-treatment groups (125, 250, 500, and 1000 mg/kg/d). After nine weeks of treatment, serum and semen samples were collected from the boars for analysis. Results showed that the supplementation of 5-ALA to boar diet significantly (p < 0.05) increased semen volume, total sperm count, and sperm motility while reducing the proportion of abnormality (p < 0.05). Specifically, 250 mg/kg/d 5-ALA treatment significantly (p < 0.05) reduced sperm DNA oxidative damage and elevated serum testosterone levels. Metabolomic analysis revealed that 250 mg/kg/d 5-ALA supplementation significantly increased (p < 0.05) the levels of tricarboxylic acid (TCA) cycle intermediates such as malate and isocitrate, while significantly (p < 0.05) reducing harmful metabolites such as N-acetyl phenylalanine that negatively impact sperm quality and fertility. Notably, 250 mg/kg/d 5-ALA supplementation upregulated the protein levels of mitochondrial oxidative phosphorylation (OXPHOS) complex subunits (NDUFB8, SDHB, UQCRC2, MTCO2, and ATP5A1) and improved sperm mitochondrial membrane potential, adenosine triphosphate (ATP) levels, and complex IV activity (p < 0.05). Regarding reproductive performance, compared to the control, the percentage of piglets born alive increased by 2.97 %, the farrowing rate improved by 10.59 %, and the occurrence of mummified fetuses decreased by 1.07 % in the 5-ALA-treated groups (p < 0.05). Additionally, 250 mg/kg/d 5-ALA had a long-term beneficial advantage on boar semen quality parameters. Accordingly, dietary supplementation of 5-ALA, specifically, 250 mg/kg/d 5-ALA improved boar semen quality by enhancing seminal plasma metabolome, sperm mitochondrial function and overall reproductive performance.
{"title":"5-Aminolevulinate acid improves boar semen quality by enhancing the sperm mitochondrial function","authors":"Shanpeng Wang , Qi Wang , Xuejun Zeng , Lingjiang Min , Wenxian Zeng , Adedeji O. Adetunji , Zhendong Zhu","doi":"10.1016/j.theriogenology.2025.117389","DOIUrl":"10.1016/j.theriogenology.2025.117389","url":null,"abstract":"<div><div>5-Aminolevulinic acid (5-ALA) is a crucial metabolic intermediate that affect mitochondrial function in somatic cells. However, the mechanisms by which 5-ALA regulates boar reproductive performance remain unclear. The effect of dietary supplementation of 5-ALA on boar semen quality and reproductive performance were investigated in this study. Forty-five boars were randomly assigned to a control group and four 5-ALA-treatment groups (125, 250, 500, and 1000 mg/kg/d). After nine weeks of treatment, serum and semen samples were collected from the boars for analysis. Results showed that the supplementation of 5-ALA to boar diet significantly (p < 0.05) increased semen volume, total sperm count, and sperm motility while reducing the proportion of abnormality (p < 0.05). Specifically, 250 mg/kg/d 5-ALA treatment significantly (p < 0.05) reduced sperm DNA oxidative damage and elevated serum testosterone levels. Metabolomic analysis revealed that 250 mg/kg/d 5-ALA supplementation significantly increased (p < 0.05) the levels of tricarboxylic acid (TCA) cycle intermediates such as malate and isocitrate, while significantly (p < 0.05) reducing harmful metabolites such as N-acetyl phenylalanine that negatively impact sperm quality and fertility. Notably, 250 mg/kg/d 5-ALA supplementation upregulated the protein levels of mitochondrial oxidative phosphorylation (OXPHOS) complex subunits (NDUFB8, SDHB, UQCRC2, MTCO2, and ATP5A1) and improved sperm mitochondrial membrane potential, adenosine triphosphate (ATP) levels, and complex IV activity (p < 0.05). Regarding reproductive performance, compared to the control, the percentage of piglets born alive increased by 2.97 %, the farrowing rate improved by 10.59 %, and the occurrence of mummified fetuses decreased by 1.07 % in the 5-ALA-treated groups (p < 0.05). Additionally, 250 mg/kg/d 5-ALA had a long-term beneficial advantage on boar semen quality parameters. Accordingly, dietary supplementation of 5-ALA, specifically, 250 mg/kg/d 5-ALA improved boar semen quality by enhancing seminal plasma metabolome, sperm mitochondrial function and overall reproductive performance.</div></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":"239 ","pages":"Article 117389"},"PeriodicalIF":2.4,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143629494","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-12DOI: 10.1016/j.theriogenology.2025.117388
Jia Cheng , Qiqi Dong , Saiya Nie , Xu Hao , Sha Mo , Yixing Liu , Zhendong Zhu , Hongzhao Lu , Tao Zhang , Wenxian Zeng
Glucose metabolism is an essential pathway that indirectly supports cellular redox homeostasis by providing reducing equivalents, such as NADPH, particularly in the highly specialized sperm. Sperm exhibit higher progressive motility in low glucose extender. However, the underlying mechanisms remain unclear. The objective of the present study was to investigate effect of low glucose on sperm metabolism and lactylation modification. After 3 h of incubation, low glucose had an effect on the redox state of boar semen in vitro, particularly in terms of the concentration of reactive oxygen species (ROS) and reductive products. Furthermore, glucose-6-phosphate dehydrogenase (G6PD) activity was significantly increased at low glucose condition, accompanied by increased lactate accumulation extracellularly. Meanwhile, protein lactylation levels were enhanced, with G6PD identified as one of lactylation proteins. In conclusion, low glucose incubation induced lactylation of G6PD, resulting in increased enzymatic activity that enhanced the pentose phosphate pathway (PPP), which in turn increased antioxidant capacity and maintained sperm motility in a low glucose environment. The research results provide valuable insights into the adaptation mechanisms of sperm to their environment and offer new perspectives and opportunities for reproductive biology research.
{"title":"G6PD lactylation is involved in regulating redox balance of boar sperm in low glucose extender","authors":"Jia Cheng , Qiqi Dong , Saiya Nie , Xu Hao , Sha Mo , Yixing Liu , Zhendong Zhu , Hongzhao Lu , Tao Zhang , Wenxian Zeng","doi":"10.1016/j.theriogenology.2025.117388","DOIUrl":"10.1016/j.theriogenology.2025.117388","url":null,"abstract":"<div><div>Glucose metabolism is an essential pathway that indirectly supports cellular redox homeostasis by providing reducing equivalents, such as NADPH, particularly in the highly specialized sperm. Sperm exhibit higher progressive motility in low glucose extender. However, the underlying mechanisms remain unclear. The objective of the present study was to investigate effect of low glucose on sperm metabolism and lactylation modification. After 3 h of incubation, low glucose had an effect on the redox state of boar semen <em>in vitro</em>, particularly in terms of the concentration of reactive oxygen species (ROS) and reductive products. Furthermore, glucose-6-phosphate dehydrogenase (G6PD) activity was significantly increased at low glucose condition, accompanied by increased lactate accumulation extracellularly. Meanwhile, protein lactylation levels were enhanced, with G6PD identified as one of lactylation proteins. In conclusion, low glucose incubation induced lactylation of G6PD, resulting in increased enzymatic activity that enhanced the pentose phosphate pathway (PPP), which in turn increased antioxidant capacity and maintained sperm motility in a low glucose environment. The research results provide valuable insights into the adaptation mechanisms of sperm to their environment and offer new perspectives and opportunities for reproductive biology research.</div></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":"239 ","pages":"Article 117388"},"PeriodicalIF":2.4,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143644239","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-10DOI: 10.1016/j.theriogenology.2025.117384
Xiuwen Yuan , Hewei Ji , Yuhao Zhang , Huilin Peng , Neng-hao Cao , Jia-jun Ren , Xue-rui Yao , Xingwei Liang , Nam-hyung Kim , Yong-nan Xu , Ying-hua Li
Mangiferin (MGN), a flavonoid known for its anti-inflammatory and antioxidant properties, was evaluated in this study for its effects on porcine oocyte maturation in vitro, as well as its potential to modulate the mechanisms associated with aging oocytes. The inclusion of 0.1 μM MGN in the IVM culture medium significantly enhanced blastocyst development following parthenogenetic activation, while also notably upregulating the expression of key embryonic development genes, including SOX2, PCNA, POU5F1, and DNMT3A. MGN treatment improved the oocytes' antioxidant capacity and mitochondrial functionality, concurrently reducing cathepsin B activity and lowering LC3B protein expression (1.06 ± 0.09 vs. 0.55 ± 0.12). To investigate the underlying mechanisms, NRF2 expression was assessed, revealing a marked increase in NRF2 fluorescence and a significant elevation in both NRF2 mRNA and protein levels (1.00 ± 0.05 vs. 1.25 ± 0.09) following MGN treatment compared to the control group. Additionally, MGN treatment enhanced the early developmental potential of aged oocytes, elevating GSH levels and mitochondrial membrane potential and reducing ROS accumulation. Furthermore, MGN treatment upregulated antioxidant genes (SOD1, SOD2). Collectively, these findings suggest that MGN improves porcine oocyte maturation in vitro and enhances subsequent developmental potential through the activation of NRF2 signaling. Moreover, MGN may also delay postovulatory oocyte aging by boosting antioxidant defense and mitochondrial function in aged oocytes.
{"title":"Mangiferin promotes porcine oocyte maturation and delays the postovulatory aging process by up-regulating NRF2 levels","authors":"Xiuwen Yuan , Hewei Ji , Yuhao Zhang , Huilin Peng , Neng-hao Cao , Jia-jun Ren , Xue-rui Yao , Xingwei Liang , Nam-hyung Kim , Yong-nan Xu , Ying-hua Li","doi":"10.1016/j.theriogenology.2025.117384","DOIUrl":"10.1016/j.theriogenology.2025.117384","url":null,"abstract":"<div><div>Mangiferin (MGN), a flavonoid known for its anti-inflammatory and antioxidant properties, was evaluated in this study for its effects on porcine oocyte maturation <em>in vitro</em>, as well as its potential to modulate the mechanisms associated with aging oocytes. The inclusion of 0.1 μM MGN in the IVM culture medium significantly enhanced blastocyst development following parthenogenetic activation, while also notably upregulating the expression of key embryonic development genes, including <em>SOX2</em>, <em>PCNA</em>, <em>POU5F1</em>, and <em>DNMT3A</em>. MGN treatment improved the oocytes' antioxidant capacity and mitochondrial functionality, concurrently reducing cathepsin B activity and lowering LC3B protein expression (1.06 ± 0.09 vs. 0.55 ± 0.12). To investigate the underlying mechanisms, NRF2 expression was assessed, revealing a marked increase in NRF2 fluorescence and a significant elevation in both NRF2 mRNA and protein levels (1.00 ± 0.05 vs. 1.25 ± 0.09) following MGN treatment compared to the control group. Additionally, MGN treatment enhanced the early developmental potential of aged oocytes, elevating GSH levels and mitochondrial membrane potential and reducing ROS accumulation. Furthermore, MGN treatment upregulated antioxidant genes (SOD1, SOD2). Collectively, these findings suggest that MGN improves porcine oocyte maturation <em>in vitro</em> and enhances subsequent developmental potential through the activation of NRF2 signaling. Moreover, MGN may also delay postovulatory oocyte aging by boosting antioxidant defense and mitochondrial function in aged oocytes.</div></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":"239 ","pages":"Article 117384"},"PeriodicalIF":2.4,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143620835","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-03-10DOI: 10.1016/j.theriogenology.2025.117386
Tingting Xian , Yan Liu , Xin Cao , Tao Feng
Endometritis is a prevalent disease in sows that strongly reduces reproductive performance. Its causes are complex and multifaceted, making disease management challenging without targeted measures. To provide a scientific basis for developing targeted disease management strategies, we examined vaginal microbiota diversity and its correlation with serum pro-inflammatory cytokine levels in sows with endometritis and the main pathogenic bacterial species contributing to endometritis. Fourteen post-weaning sows selected at a pig farm in Beijing, China, were randomly divided into healthy (C) and endometritis (E) groups. Levels of cytokines including interleukin-1α (IL-1α), IL-1β, IL-6, IL-8, and tumor necrosis factor-α (TNF-α) were quantified by enzyme-linked immunosorbent assays. Vaginal microbiota species were identified using 16S rDNA sequencing. Concentrations of IL-1α and IL-6 in serum and vaginal microbial diversity were significantly increased in group E versus group C. The relative abundance of Firmicutes exhibited a marked increase in group E, whereas that of Fusobacteriota was significantly decreased. The composition and relative abundance of dominant bacterial genera differed between the two groups, and the dominant genera in group E were Clostridium_sensu_stricto_1, UCG-005, UCG-002, Terrisporobacter, and Christensenellaceae_R-7_group. IL-6 was significantly positively correlated with Clostridium_sensu_stricto_1 and Terrisporobacter in group E. These results suggest that vaginal microbiota abundance and diversity and serum pro-inflammatory cytokine levels are increased in sows with endometritis, and the latter are closely associated with vaginal microbiota abundance and composition. This study provides a foundation for investigating the pathogenesis of endometritis in sows and a theoretical basis for disease prevention and treatment.
{"title":"Alterations to the vaginal microbiota and their correlation with serum pro-inflammatory cytokines in post-weaning sows with endometritis","authors":"Tingting Xian , Yan Liu , Xin Cao , Tao Feng","doi":"10.1016/j.theriogenology.2025.117386","DOIUrl":"10.1016/j.theriogenology.2025.117386","url":null,"abstract":"<div><div>Endometritis is a prevalent disease in sows that strongly reduces reproductive performance. Its causes are complex and multifaceted, making disease management challenging without targeted measures. To provide a scientific basis for developing targeted disease management strategies, we examined vaginal microbiota diversity and its correlation with serum pro-inflammatory cytokine levels in sows with endometritis and the main pathogenic bacterial species contributing to endometritis. Fourteen post-weaning sows selected at a pig farm in Beijing, China, were randomly divided into healthy (C) and endometritis (E) groups. Levels of cytokines including interleukin-1α (IL-1α), IL-1β, IL-6, IL-8, and tumor necrosis factor-α (TNF-α) were quantified by enzyme-linked immunosorbent assays. Vaginal microbiota species were identified using 16S rDNA sequencing. Concentrations of IL-1α and IL-6 in serum and vaginal microbial diversity were significantly increased in group E versus group C. The relative abundance of Firmicutes exhibited a marked increase in group E, whereas that of Fusobacteriota was significantly decreased. The composition and relative abundance of dominant bacterial genera differed between the two groups, and the dominant genera in group E were <em>Clostridium_sensu_stricto_1</em>, <em>UCG-005</em>, <em>UCG-002</em>, <em>Terrisporobacter</em>, and <em>Christensenellaceae_R-7_group</em>. IL-6 was significantly positively correlated with <em>Clostridium_sensu_stricto_1</em> and <em>Terrisporobacter</em> in group E. These results suggest that vaginal microbiota abundance and diversity and serum pro-inflammatory cytokine levels are increased in sows with endometritis, and the latter are closely associated with vaginal microbiota abundance and composition. This study provides a foundation for investigating the pathogenesis of endometritis in sows and a theoretical basis for disease prevention and treatment.</div></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":"239 ","pages":"Article 117386"},"PeriodicalIF":2.4,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143629493","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Published knowledge about cat breeders' habits regarding breeding management and contraception is limited, but vital for efficient veterinary support of catteries. With an online questionnaire, we surveyed breeders’ origin, breed, number of breeding queens/tomcats in the cattery, age at first estrus, use of contraceptives and ovulation-inducing drugs or treatment in queens [progestin pills; proligestone injection; hCG; GnRH agonists as injection or deslorelin implants (DSRI); melatonin; vaginal stimulation], as well as the use of contraceptives in tomcats [progestins pills; proligestone injection; DSRI; melatonin]. The participating 404 breeders of 46 cat breeds originated from 24 countries (German (201, 49.8 %) and Bengal breeders (73, 15.8 %) overrepresented) with the majority having ≥10 years of experience (n = 195, 48.3 %). In average, 3–4 intact adult queens (n = 185, 45.8 %) and one intact tomcat (n = 149, 36.9 %) were kept per cattery. Breeders answered that 50.6 % (n = 896) of their queens were 7–10 months old at first heat, long-haired breeds significantly older than short-haired (p < 0.0001). Progestin pills (n = 235, 47.4 %) were most commonly administered in queens, followed by DSRI (n = 53, 10.7 %). Regarding tomcats, the slight majority of breeders (n = 229, 53.3 %) did not use contraceptives, next common were DSRI (n = 141, 32.8 %). Within the study population, contraceptives were most popular in (Northern) Europe, while least common in Northern America. Despite its limitations, this large worldwide study provides relevant insights into reproductive and contraceptive management of catteries. Limited access to contraceptives and dissatisfaction with local veterinary support as commented by several participants suggest the establishment of telephone consultation services and breeder seminars by specialized veterinarians.
{"title":"Insights into breeding management and contraception in catteries – What we learned from an online survey","authors":"Ricarda Schaper, Eva-Maria Packeiser, Sandra Goericke-Pesch","doi":"10.1016/j.theriogenology.2025.117385","DOIUrl":"10.1016/j.theriogenology.2025.117385","url":null,"abstract":"<div><div>Published knowledge about cat breeders' habits regarding breeding management and contraception is limited, but vital for efficient veterinary support of catteries. With an online questionnaire, we surveyed breeders’ origin, breed, number of breeding queens/tomcats in the cattery, age at first estrus, use of contraceptives and ovulation-inducing drugs or treatment in queens [progestin pills; proligestone injection; hCG; GnRH agonists as injection or deslorelin implants (DSRI); melatonin; vaginal stimulation], as well as the use of contraceptives in tomcats [progestins pills; proligestone injection; DSRI; melatonin]. The participating 404 breeders of 46 cat breeds originated from 24 countries (German (201, 49.8 %) and Bengal breeders (73, 15.8 %) overrepresented) with the majority having ≥10 years of experience (n = 195, 48.3 %). In average, 3–4 intact adult queens (n = 185, 45.8 %) and one intact tomcat (n = 149, 36.9 %) were kept per cattery. Breeders answered that 50.6 % (n = 896) of their queens were 7–10 months old at first heat, long-haired breeds significantly older than short-haired (p < 0.0001). Progestin pills (n = 235, 47.4 %) were most commonly administered in queens, followed by DSRI (n = 53, 10.7 %). Regarding tomcats, the slight majority of breeders (n = 229, 53.3 %) did not use contraceptives, next common were DSRI (n = 141, 32.8 %). Within the study population, contraceptives were most popular in (Northern) Europe, while least common in Northern America. Despite its limitations, this large worldwide study provides relevant insights into reproductive and contraceptive management of catteries. Limited access to contraceptives and dissatisfaction with local veterinary support as commented by several participants suggest the establishment of telephone consultation services and breeder seminars by specialized veterinarians.</div></div>","PeriodicalId":23131,"journal":{"name":"Theriogenology","volume":"239 ","pages":"Article 117385"},"PeriodicalIF":2.4,"publicationDate":"2025-03-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143609918","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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