Sperm preservation is a pivotal technique in reproductive science, facilitating the long-term preservation and utilization of valuable genetic material, particularly in animal breeding programs and assisted reproductive technologies (ART). However, the freezing-thawing process imposes significant physiological stress on sperm cells, primarily due to oxidative damage. This oxidative stress disrupts critical cellular functions, leading to reduced sperm motility, viability, DNA integrity, and membrane stability, thereby compromising overall reproductive potential. Among various antioxidant strategies, lycopene, a potent carotenoid, has emerged as a promising candidate for mitigating cryoinjury. This review provides a comprehensive overview of lycopene’s role in sperm cryopreservation and cold storage, emphasizing its effect on post-thaw sperm quality. Experimental evidence from animal studies indicates that lycopene supplementation effectively neutralizes reactive oxygen species, reduces lipid peroxidation, and preserves structural integrity by integrating into cell membranes. These protective effects contribute to enhancing sperm functionality post-thaw, potentially improving fertilization outcomes. Yet, this review critically highlights that the efficacy of lycopene is a double-edged sword: its effects are highly dose- and species-dependent, with excessive concentrations paradoxically impairing sperm performance. We conclude that a 'one-size-fits-all' approach is ineffective, and future investigations must move beyond simple supplementation to focus on optimizing species-specific formulations and validating in vitro benefits with in vivo fertility trials to fully harness lycopene's potential in reproductive applications.
{"title":"Lycopene as a protective antioxidant in sperm preservation","authors":"Mohsen Shayestehyekta , Azita Faramarzi , Zahra Rashidi , Mojtaba Moradi","doi":"10.1016/j.anireprosci.2025.108075","DOIUrl":"10.1016/j.anireprosci.2025.108075","url":null,"abstract":"<div><div>Sperm preservation is a pivotal technique in reproductive science, facilitating the long-term preservation and utilization of valuable genetic material, particularly in animal breeding programs and assisted reproductive technologies (ART). However, the freezing-thawing process imposes significant physiological stress on sperm cells, primarily due to oxidative damage. This oxidative stress disrupts critical cellular functions, leading to reduced sperm motility, viability, DNA integrity, and membrane stability, thereby compromising overall reproductive potential. Among various antioxidant strategies, lycopene, a potent carotenoid, has emerged as a promising candidate for mitigating cryoinjury. This review provides a comprehensive overview of lycopene’s role in sperm cryopreservation and cold storage, emphasizing its effect on post-thaw sperm quality. Experimental evidence from animal studies indicates that lycopene supplementation effectively neutralizes reactive oxygen species, reduces lipid peroxidation, and preserves structural integrity by integrating into cell membranes. These protective effects contribute to enhancing sperm functionality post-thaw, potentially improving fertilization outcomes. Yet, this review critically highlights that the efficacy of lycopene is a double-edged sword: its effects are highly dose- and species-dependent, with excessive concentrations paradoxically impairing sperm performance. We conclude that a 'one-size-fits-all' approach is ineffective, and future investigations must move beyond simple supplementation to focus on optimizing species-specific formulations and validating in vitro benefits with in vivo fertility trials to fully harness lycopene's potential in reproductive applications.</div></div>","PeriodicalId":7880,"journal":{"name":"Animal Reproduction Science","volume":"285 ","pages":"Article 108075"},"PeriodicalIF":3.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145754729","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}
High ewe density in group breeding enhances ram selectivity and favors the most attractive ewes. This study aimed to compare the sexual attractiveness of multiparous and nulliparous ewes. We hypothesize that multiparous ewes will be ranked as more attractive. Forty-two ewes, 21 multiparous and 21 nulliparous, Santa Ines × Dorper in estrus were tested. Each ewe was observed for five minutes, and behaviour was recorded from live and video observations. After each 5-minute test, the ewe courted for the longest time was removed and ranked as most attractive (rank 1), and this procedure continued until all six ewes in a group were ranked (1 = most to 6 = least attractive). Ewe ranking, ram behavior, and the odds of mounting acceptance were analyzed using the Friedman test, generalized mixed-effects models, and mixed-effects logistic regression, respectively. Mean attractiveness ranks were higher for multiparous than nulliparous ewes (2 ± 0.17 vs. 5 ± 0.17, P = 0.008). Rams also exhibited longer mean mounting duration (2.46 ± 0.91 s vs. 1.59 ± 0.91 s, P = 0.045) and a higher number of ejaculations (0.90 ± 0.31 vs. 0.14 ± 0.31, P = 0.0015) with multiparous ewes. While odds of accepting mounts were similar between categories (odds ratio = 0.45; 95 % CI: 0.08–2.75; P = 0.369). All first-ranked ewes were multiparous, and last two ranks were nulliparous. These results indicate multiparous estrous ewes as more attractive to rams, suggesting separating breeding groups per parity may improve fertilization rates in nulliparous ewes.
{"title":"Older yet more attractive: Multiparous ewes are preferentially courted, and elicit more mountings and ejaculations than nulliparous ewes in group breeding","authors":"Gustavo Dias , Rodolfo Ungerfeld , Aline Freitas-de-Melo , Adroaldo Zanella","doi":"10.1016/j.anireprosci.2025.108047","DOIUrl":"10.1016/j.anireprosci.2025.108047","url":null,"abstract":"<div><div>High ewe density in group breeding enhances ram selectivity and favors the most attractive ewes. This study aimed to compare the sexual attractiveness of multiparous and nulliparous ewes. We hypothesize that multiparous ewes will be ranked as more attractive. Forty-two ewes, 21 multiparous and 21 nulliparous, Santa Ines × Dorper in estrus were tested. Each ewe was observed for five minutes, and behaviour was recorded from live and video observations. After each 5-minute test, the ewe courted for the longest time was removed and ranked as most attractive (rank 1), and this procedure continued until all six ewes in a group were ranked (1 = most to 6 = least attractive). Ewe ranking, ram behavior, and the odds of mounting acceptance were analyzed using the Friedman test, generalized mixed-effects models, and mixed-effects logistic regression, respectively. Mean attractiveness ranks were higher for multiparous than nulliparous ewes (2 ± 0.17 vs. 5 ± 0.17, P = 0.008). Rams also exhibited longer mean mounting duration (2.46 ± 0.91 s vs. 1.59 ± 0.91 s, P = 0.045) and a higher number of ejaculations (0.90 ± 0.31 vs. 0.14 ± 0.31, P = 0.0015) with multiparous ewes. While odds of accepting mounts were similar between categories (odds ratio = 0.45; 95 % CI: 0.08–2.75; P = 0.369). All first-ranked ewes were multiparous, and last two ranks were nulliparous. These results indicate multiparous estrous ewes as more attractive to rams, suggesting separating breeding groups per parity may improve fertilization rates in nulliparous ewes.</div></div>","PeriodicalId":7880,"journal":{"name":"Animal Reproduction Science","volume":"285 ","pages":"Article 108047"},"PeriodicalIF":3.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145697909","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 : 2026-02-01Epub Date: 2025-12-13DOI: 10.1016/j.anireprosci.2025.108077
César Augusto Pinzón-Osorio , Thomaz Kranen Cunha , German Holguin-Sanabria , Marco Alves Machado , Favorino José de Freitas Collares , Luís Henrique de Aguiar , Paula Rodriguez-Villamil , Felipe Ledur Ongaratto , Eduardo Pradebom da Silva , Eduardo de Oliveira Sanguinet , Carolina Silveira da Silva , Daiane Mentz , Karine Campagnolo , Paula Viero Marchioretto , José Martin Klafke , Luís Fernando Schutz , Fernando Caetano de Oliveira , José Luiz Rodrigues , Marcelo Bertolini
Timed artificial insemination (TAI) protocols based on estradiol and progesterone (E2/P4) typically yield pregnancy rates (PR) near 50 % in beef cattle, falling short of the 65–70 % expected in fertile females. This study investigated physiological and procedural limitations that may compromise TAI success due to suboptimal synchrony between insemination and ovulation. Three experiments involving 840 suckled crossbred females evaluated follicular responses, semen type, and alternative reproductive strategies to enhance fertility. In Experiment I (n = 28), daily ultrasonography revealed that 46.4 % of females exhibited suboptimal ovarian responses, defined as no emergence/atresia of the new follicular wave, and ovulation delay or failure. In Experiment II (n = 348), cows with small follicles (<8.5 mm) at P4 removal had markedly higher PR when inseminated with cooled versus frozen semen (71.9 % vs 38.0 %), highlighting the relevance of sperm cell viability when ovulation is delayed. In Experiment III (n = 464), strategies combining TAI with estrus detection (ED), timed embryo transfer (TET), or both significantly increased PR compared to TAI alone (TAI: 53.6 %; TAI+TET: 67.9 %; ED-AI/TAI: 70.5 %; ED-AI/TAI+TET: 68.2 %), which was accompanied by increased twinning rates after TET, suggesting that many TAI failures were not due to uterine inadequacy or embryo mortality but to asynchrony in gamete encounter. Collectively, results suggest that most TAI failures stem from impaired synchrony between ovulation and sperm cell availability rather than uterine or embryonic limitations. Incorporating tools such as ultrasonography, semen selection, ED, or TET may enhance reproductive outcomes in commercial beef herds under E2/P4-based TAI protocols.
{"title":"Suboptimal responses to E2/P4-based timed AI protocols compromise pregnancy outcomes in fertile beef cattle: Roles of ovulation timing and semen viability","authors":"César Augusto Pinzón-Osorio , Thomaz Kranen Cunha , German Holguin-Sanabria , Marco Alves Machado , Favorino José de Freitas Collares , Luís Henrique de Aguiar , Paula Rodriguez-Villamil , Felipe Ledur Ongaratto , Eduardo Pradebom da Silva , Eduardo de Oliveira Sanguinet , Carolina Silveira da Silva , Daiane Mentz , Karine Campagnolo , Paula Viero Marchioretto , José Martin Klafke , Luís Fernando Schutz , Fernando Caetano de Oliveira , José Luiz Rodrigues , Marcelo Bertolini","doi":"10.1016/j.anireprosci.2025.108077","DOIUrl":"10.1016/j.anireprosci.2025.108077","url":null,"abstract":"<div><div>Timed artificial insemination (TAI) protocols based on estradiol and progesterone (E2/P4) typically yield pregnancy rates (PR) near 50 % in beef cattle, falling short of the 65–70 % expected in fertile females. This study investigated physiological and procedural limitations that may compromise TAI success due to suboptimal synchrony between insemination and ovulation. Three experiments involving 840 suckled crossbred females evaluated follicular responses, semen type, and alternative reproductive strategies to enhance fertility. In Experiment I (<em>n</em> = 28), daily ultrasonography revealed that 46.4 % of females exhibited suboptimal ovarian responses, defined as no emergence/atresia of the new follicular wave, and ovulation delay or failure. In Experiment II (<em>n</em> = 348), cows with small follicles (<8.5 mm) at P4 removal had markedly higher PR when inseminated with cooled versus frozen semen (71.9 % <em>vs</em> 38.0 %), highlighting the relevance of sperm cell viability when ovulation is delayed. In Experiment III (<em>n</em> = 464), strategies combining TAI with estrus detection (ED), timed embryo transfer (TET), or both significantly increased PR compared to TAI alone (TAI: 53.6 %; TAI+TET: 67.9 %; ED-AI/TAI: 70.5 %; ED-AI/TAI+TET: 68.2 %), which was accompanied by increased twinning rates after TET, suggesting that many TAI failures were not due to uterine inadequacy or embryo mortality but to asynchrony in gamete encounter. Collectively, results suggest that most TAI failures stem from impaired synchrony between ovulation and sperm cell availability rather than uterine or embryonic limitations. Incorporating tools such as ultrasonography, semen selection, ED, or TET may enhance reproductive outcomes in commercial beef herds under E2/P4-based TAI protocols.</div></div>","PeriodicalId":7880,"journal":{"name":"Animal Reproduction Science","volume":"285 ","pages":"Article 108077"},"PeriodicalIF":3.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145766934","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 : 2026-02-01Epub Date: 2025-12-09DOI: 10.1016/j.anireprosci.2025.108074
Darmawan Setia Budi , Agus Priyadi , Asep Permana , Ikhsan Khasani , Tamás Müller , Ahmad Shofy Mubarak , Imam Mustofa
Habitat degradation, overfishing, pollution, and climate change threaten many fish species, necessitating urgent conservation efforts. Captive breeding is essential for fish species recovery and genetic diversity preservation, with maturity determination being crucial for broodstock management, breeding success, and sustainable aquaculture practices. Standardized, species-specific protocols and non-invasive methods for accurately determining fish maturity are currently lacking. This review synthesizes current methodologies and practical applications of maturity determination in fish captive breeding, emphasizing how established knowledge can be applied to improve broodstock management and conservation. Traditional methods, like morphological and gonadal assessments, remain foundational, while histological and molecular techniques offer enhanced precision but are resource-intensive. Non-invasive imaging methods, including ultrasound, provide innovative alternatives for evaluating gonadal development. Behavioral cues, such as aggression, shoaling, and nesting, complement physiological assessments, enhancing accuracy. A multi-method approach is essential to address the diverse reproductive strategies of fish species. Advances in camera-based technologies and diagnostic kits for hormone detection offer novel, non-invasive, and scalable maturity assessments, reducing stress on fish. The integration of maturity determination with aquaculture facilitates broodstock selection, stock enhancement, and conservation efforts, promoting the long-term sustainability of aquatic ecosystems. This review highlights the need for standardized, species-specific protocols and technological advancements to support sustainable fish breeding. Future research should prioritize developing accessible tools and methodologies that integrate traditional and modern approaches, enhancing biodiversity conservation and promoting aquaculture growth.
{"title":"Maturity determination and its potential application for fish captive breeding","authors":"Darmawan Setia Budi , Agus Priyadi , Asep Permana , Ikhsan Khasani , Tamás Müller , Ahmad Shofy Mubarak , Imam Mustofa","doi":"10.1016/j.anireprosci.2025.108074","DOIUrl":"10.1016/j.anireprosci.2025.108074","url":null,"abstract":"<div><div>Habitat degradation, overfishing, pollution, and climate change threaten many fish species, necessitating urgent conservation efforts. Captive breeding is essential for fish species recovery and genetic diversity preservation, with maturity determination being crucial for broodstock management, breeding success, and sustainable aquaculture practices. Standardized, species-specific protocols and non-invasive methods for accurately determining fish maturity are currently lacking. This review synthesizes current methodologies and practical applications of maturity determination in fish captive breeding, emphasizing how established knowledge can be applied to improve broodstock management and conservation. Traditional methods, like morphological and gonadal assessments, remain foundational, while histological and molecular techniques offer enhanced precision but are resource-intensive. Non-invasive imaging methods, including ultrasound, provide innovative alternatives for evaluating gonadal development. Behavioral cues, such as aggression, shoaling, and nesting, complement physiological assessments, enhancing accuracy. A multi-method approach is essential to address the diverse reproductive strategies of fish species. Advances in camera-based technologies and diagnostic kits for hormone detection offer novel, non-invasive, and scalable maturity assessments, reducing stress on fish. The integration of maturity determination with aquaculture facilitates broodstock selection, stock enhancement, and conservation efforts, promoting the long-term sustainability of aquatic ecosystems. This review highlights the need for standardized, species-specific protocols and technological advancements to support sustainable fish breeding. Future research should prioritize developing accessible tools and methodologies that integrate traditional and modern approaches, enhancing biodiversity conservation and promoting aquaculture growth.</div></div>","PeriodicalId":7880,"journal":{"name":"Animal Reproduction Science","volume":"285 ","pages":"Article 108074"},"PeriodicalIF":3.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145735570","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 : 2026-02-01Epub Date: 2025-12-08DOI: 10.1016/j.anireprosci.2025.108071
J. Gómez-Delgado , B. Martínez-Madrid , A. Toledano-Díaz , C. Castaño , A. Gómez-Crespo , G. de Pedro Aguilar , D. Cubero , A. Kowalczyk , E. Łukaszewicz , J. Santiago-Moreno
The capercaillie (Tetrao urogallus L.) is a threatened forest bird with distinct Iberian and European ecotypes. This study examines the functional and morphometric differences between the sperm of these ecotypes. In a first experiment, semen volume, sperm concentration, motility, viability, and DNA integrity were assessed in 'Iberian' and 'European' capercaillies, both maintained at breeding centres in Spain. In a second experiment, morphometric traits were measured in Iberian capercaillies maintained at a breeding centre in Spain, and in European capercaillies also maintained at breeding centres in Spain (EmS birds) and in Poland (EmP birds). In the first experiment, the European males produced larger ejaculate volumes (39.80 ± 3.56 µL) than the Iberian males (29.68 ± 4.64 µL). However, the Iberian males returned significantly higher sperm concentrations (501.99 ± 83.90 × 10⁶ spz/mL vs. 77.66 ± 26.09 × 10⁶ spz/mL). In the second experiment, the origin of the birds also affected (P < 0.001) sperm head dimensions. These were smaller in the EmP birds compared to the EmS birds (P < 0.01), and compared to Iberian males (always maintained in Spain) (P < 0.001). Within each of these groups, three sperm subpopulations were identified according to head dimensions, with differences (P < 0.001) between these groups in terms of the proportion of each subpopulation. This is the first comprehensive study of sperm morphometric characteristics in these capercaillie ecotypes. These results may provide critical insights into the reproductive and evolutionary strategies of capercaillies and contribute to improving the success of reproductive technologies across different ecotypes and populations.
{"title":"Sperm functional and morphometric differences between Iberian and European ecotypes of capercaillie (Tetrao urogallus L.)","authors":"J. Gómez-Delgado , B. Martínez-Madrid , A. Toledano-Díaz , C. Castaño , A. Gómez-Crespo , G. de Pedro Aguilar , D. Cubero , A. Kowalczyk , E. Łukaszewicz , J. Santiago-Moreno","doi":"10.1016/j.anireprosci.2025.108071","DOIUrl":"10.1016/j.anireprosci.2025.108071","url":null,"abstract":"<div><div>The capercaillie (<em>Tetrao urogallus</em> L<em>.</em>) is a threatened forest bird with distinct Iberian and European ecotypes. This study examines the functional and morphometric differences between the sperm of these ecotypes. In a first experiment, semen volume, sperm concentration, motility, viability, and DNA integrity were assessed in 'Iberian' and 'European' capercaillies, both maintained at breeding centres in Spain. In a second experiment, morphometric traits were measured in Iberian capercaillies maintained at a breeding centre in Spain, and in European capercaillies also maintained at breeding centres in Spain (EmS birds) and in Poland (EmP birds). In the first experiment, the European males produced larger ejaculate volumes (39.80 ± 3.56 µL) than the Iberian males (29.68 ± 4.64 µL). However, the Iberian males returned significantly higher sperm concentrations (501.99 ± 83.90 × 10⁶ spz/mL vs. 77.66 ± 26.09 × 10⁶ spz/mL). In the second experiment, the origin of the birds also affected (P < 0.001) sperm head dimensions. These were smaller in the EmP birds compared to the EmS birds (P < 0.01), and compared to Iberian males (always maintained in Spain) (P < 0.001). Within each of these groups, three sperm subpopulations were identified according to head dimensions, with differences (P < 0.001) between these groups in terms of the proportion of each subpopulation. This is the first comprehensive study of sperm morphometric characteristics in these capercaillie ecotypes. These results may provide critical insights into the reproductive and evolutionary strategies of capercaillies and contribute to improving the success of reproductive technologies across different ecotypes and populations.</div></div>","PeriodicalId":7880,"journal":{"name":"Animal Reproduction Science","volume":"285 ","pages":"Article 108071"},"PeriodicalIF":3.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145735571","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 : 2026-02-01Epub Date: 2025-12-13DOI: 10.1016/j.anireprosci.2025.108076
Emily A. Lugo , James K. Graham , James D. Gillis , Jennifer P. Barfield
Domestic cats (Felis catus) serve as a model for studying non-domestic cat reproduction. In particular, assisted reproductive technologies, such as cryopreservation of gametes and embryos, often are developed in domestic species and then applied to non-domestic species. Membrane composition and osmotic tolerance impact a sperm’s ability to withstand the stresses of cryopreservation. In this study, the membrane cholesterol content and osmotic tolerance range of domestic field spermatozoa were investigated and considered when testing alternative cryoprotectants and methods (addition of cholesterol) for felid sperm cryopreservation. The most commonly used cryoprotectant for felid sperm, glycerol, was compared to methylformamide (MF), dimethylformamide (DMF), and combinations of glycerol with both MF and DMF. To verify osmotic tolerance range, domestic cat sperm were subjected to 12 different anisosmotic solutions, ranging from 0 to 1200 mOsm/kg. Using flow cytometry, > 65 % of the sperm maintained intact membranes between 50 and 600 mOsm/kg, verifying that epididymal domestic cat sperm have a wide osmotic tolerance range. In addition, epididymal cat sperm have a relatively high cholesterol:phospholipid ratio (0.70) and attempting to increase the cholesterol content did not benefit cryosurvival. The alternative cryoprotectant DMF, however, did improve post-thaw percentages of motile sperm (43 %) compared to glycerol (24 %; P < 0.05), with 8 % DMF resulting in higher post-thaw motility than sperm frozen with 5 % DMF (33 vs 54 %; P < 0.05). These findings highlight physiological properties of felid sperm that should be considered when updating protocols to improve cat sperm cryopreservation methods.
{"title":"Investigating the plasma membrane composition and osmotic tolerance of domestic felid (Felis catus) spermatozoa as a model for non-domestic felids","authors":"Emily A. Lugo , James K. Graham , James D. Gillis , Jennifer P. Barfield","doi":"10.1016/j.anireprosci.2025.108076","DOIUrl":"10.1016/j.anireprosci.2025.108076","url":null,"abstract":"<div><div>Domestic cats (<em>Felis catus</em>) serve as a model for studying non-domestic cat reproduction. In particular, assisted reproductive technologies, such as cryopreservation of gametes and embryos, often are developed in domestic species and then applied to non-domestic species. Membrane composition and osmotic tolerance impact a sperm’s ability to withstand the stresses of cryopreservation. In this study, the membrane cholesterol content and osmotic tolerance range of domestic field spermatozoa were investigated and considered when testing alternative cryoprotectants and methods (addition of cholesterol) for felid sperm cryopreservation. The most commonly used cryoprotectant for felid sperm, glycerol, was compared to methylformamide (MF), dimethylformamide (DMF), and combinations of glycerol with both MF and DMF. To verify osmotic tolerance range, domestic cat sperm were subjected to 12 different anisosmotic solutions, ranging from 0 to 1200 mOsm/kg. Using flow cytometry, <u>></u> 65 % of the sperm maintained intact membranes between 50 and 600 mOsm/kg, verifying that epididymal domestic cat sperm have a wide osmotic tolerance range. In addition, epididymal cat sperm have a relatively high cholesterol:phospholipid ratio (0.70) and attempting to increase the cholesterol content did not benefit cryosurvival. The alternative cryoprotectant DMF, however, did improve post-thaw percentages of motile sperm (43 %) compared to glycerol (24 %; <em>P</em> < 0.05), with 8 % DMF resulting in higher post-thaw motility than sperm frozen with 5 % DMF (33 vs 54 %; <em>P</em> < 0.05). These findings highlight physiological properties of felid sperm that should be considered when updating protocols to improve cat sperm cryopreservation methods.</div></div>","PeriodicalId":7880,"journal":{"name":"Animal Reproduction Science","volume":"285 ","pages":"Article 108076"},"PeriodicalIF":3.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145766983","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 : 2026-02-01Epub Date: 2025-12-18DOI: 10.1016/j.anireprosci.2025.108088
Paulo Victor dos S. Pereira , Ana Paula da S. Cupello , Lucas Francisco L. Correia , Walter Lilenbaum , Joanna M.G. Souza-Fabjan
Although a few studies have reported Leptospira spp. in cumulus-oocyte complexes (COCs) from experimentally infected cows, it remains unclear whether this bacterium can penetrate COCs in naturally infected animals. This study aimed to detect the presence of Leptospira spp. in COCs from naturally infected cows. Ovaries and uterine body fragments were collected from 40 cows after slaughter. Follicular fluid (FF) and uterine fragments (UF) were screened using conventional PCR (cPCR), and negative results were confirmed by quantitative PCR (qPCR). COCs were immunolabeled using a monoclonal anti-LipL41 antibody, followed by an Alexa Fluor 488-conjugated secondary antibody, and counterstained with propidium iodide. Detection of Leptospira spp. was performed by epifluorescence microscopy. Cows were then classified as positive (POS-FF, POS-UF, or both) or negative (NEG). In POS-FF animals (n = 8), 9.1 COCs/female were recovered, with 51.1 ± 12.7 % showing Leptospira presence (not significant; n.s.). In POS-UF cows (n = 12), 6.6 COCs/female were obtained, and 81.0 ± 5.5 % showed bacterial presence (n.s.). In animals positive for both FF and UF (n = 10), 5.4 COCs/female were collected, with 63.7 ± 12.9 % testing positive (n.s.). In NEG animals (n = 10), which were both cPCR- and qPCR-negative, 69.3 ± 9.7 % of COCs exhibited bacterial labeling, suggesting a low bacterial load (n.s.). These cows yielded 9.7 ± 3.7 COCs/female (n.s.). Although no difference was observed between the groups, this is the first report demonstrating the presence of Leptospira spp. in COCs from naturally infected cows, highlighting a mechanism contributing to reproductive failure in cattle.
{"title":"First detection of Leptospira spp. in cumulus-oocyte complexes from naturally infected cows","authors":"Paulo Victor dos S. Pereira , Ana Paula da S. Cupello , Lucas Francisco L. Correia , Walter Lilenbaum , Joanna M.G. Souza-Fabjan","doi":"10.1016/j.anireprosci.2025.108088","DOIUrl":"10.1016/j.anireprosci.2025.108088","url":null,"abstract":"<div><div>Although a few studies have reported <em>Leptospira</em> spp. in cumulus-oocyte complexes (COCs) from experimentally infected cows, it remains unclear whether this bacterium can penetrate COCs in naturally infected animals. This study aimed to detect the presence of <em>Leptospira</em> spp. in COCs from naturally infected cows. Ovaries and uterine body fragments were collected from 40 cows after slaughter. Follicular fluid (FF) and uterine fragments (UF) were screened using conventional PCR (cPCR), and negative results were confirmed by quantitative PCR (qPCR). COCs were immunolabeled using a monoclonal anti-<em>Lip</em>L41 antibody, followed by an Alexa Fluor 488-conjugated secondary antibody, and counterstained with propidium iodide. Detection of <em>Leptospira</em> spp. was performed by epifluorescence microscopy. Cows were then classified as positive (POS-FF, POS-UF, or both) or negative (NEG). In POS-FF animals (n = 8), 9.1 COCs/female were recovered, with 51.1 ± 12.7 % showing <em>Leptospira</em> presence (not significant; n.s.). In POS-UF cows (n = 12), 6.6 COCs/female were obtained, and 81.0 ± 5.5 % showed bacterial presence (n.s.). In animals positive for both FF and UF (n = 10), 5.4 COCs/female were collected, with 63.7 ± 12.9 % testing positive (n.s.). In NEG animals (n = 10), which were both cPCR- and qPCR-negative, 69.3 ± 9.7 % of COCs exhibited bacterial labeling, suggesting a low bacterial load (n.s.). These cows yielded 9.7 ± 3.7 COCs/female (n.s.). Although no difference was observed between the groups, this is the first report demonstrating the presence of <em>Leptospira</em> spp. in COCs from naturally infected cows, highlighting a mechanism contributing to reproductive failure in cattle.</div></div>","PeriodicalId":7880,"journal":{"name":"Animal Reproduction Science","volume":"285 ","pages":"Article 108088"},"PeriodicalIF":3.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145788243","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 : 2026-02-01Epub Date: 2025-12-18DOI: 10.1016/j.anireprosci.2025.108087
Diego A. Galarza , Mauricio Duma , Gissela Sánchez-Pacheco , María García-Pacheco , Camila Ramón-Barrera , Gilda Sanmartín-Ordóñez , Manuel Soria , Antonio J. Vallecillo
HSPA1A is a heat shock protein belonging to the HSP70 family that acts during cryopreservation by mitigating oxidative stress, stabilizing denatured proteins, preventing their aggregation, and maintaining proteostasis. This study evaluated the effect of recombinant HSPA1A protein on the cryosurvival of dog epididymal spermatozoa subjected to slow-freezing (SF) and kinetic vitrification (VIT). Twenty adult dogs were orchiectomized, and epididymal sperm from the left testes were cryopreserved with SF, while the right ones were with VIT. Four treatments were established according to the addition of 10 µg/ml (+HSPA1A) or 0 µg/ml (-Control) of recombinant bovine HSPA1A, produced using Escherichia coli Rosetta 2 (DE3): pET15b-HSPA1A strain and IMAC purified. The different cryopreservation treatments evaluated were SF+HSPA1A, SF-Control, VIT+HSPA1A, and VIT-Control (n = 20 samples per treatment). Samples from SF+HSPA1A and SF-Control treatments were frozen in 0.25 ml straws by exposing them to liquid nitrogen (LN₂) vapors. For VIT, samples were vitrified by direct immersion of 30 µl drops into LN₂ and stored in cryotubes. Results showed that SF+HSPA1A and SF-Control yielded significantly higher (P < 0.05) progressive motility, velocities, linearity and straightness, and beat cross frequency compared to VIT+HSPA1A and VIT-Control. Furthermore, sperm treated with HSPA1A (both SF and VIT) exhibited significantly improved (P < 0.05) motility, viability and DNA integrity compared to their respective controls. In conclusion, the addition of recombinant HSPA1A to both slow-freezing and vitrification media enhanced the post-cryopreserved quality of dog epididymal spermatozoa, demonstrating its protective effect on motility, viability, and integrity of acrosome and DNA.
{"title":"Recombinant heat shock protein HSPA1A enhances the cryosurvival of frozen-thawed or vitrified-warmed dog epididymal spermatozoa","authors":"Diego A. Galarza , Mauricio Duma , Gissela Sánchez-Pacheco , María García-Pacheco , Camila Ramón-Barrera , Gilda Sanmartín-Ordóñez , Manuel Soria , Antonio J. Vallecillo","doi":"10.1016/j.anireprosci.2025.108087","DOIUrl":"10.1016/j.anireprosci.2025.108087","url":null,"abstract":"<div><div>HSPA1A is a heat shock protein belonging to the HSP70 family that acts during cryopreservation by mitigating oxidative stress, stabilizing denatured proteins, preventing their aggregation, and maintaining proteostasis. This study evaluated the effect of recombinant HSPA1A protein on the cryosurvival of dog epididymal spermatozoa subjected to slow-freezing (SF) and kinetic vitrification (VIT). Twenty adult dogs were orchiectomized, and epididymal sperm from the left testes were cryopreserved with SF, while the right ones were with VIT. Four treatments were established according to the addition of 10 µg/ml (+HSPA1A) or 0 µg/ml (-Control) of recombinant bovine HSPA1A, produced using <em>Escherichia coli</em> Rosetta 2 (DE3): pET15b-HSPA1A strain and IMAC purified. The different cryopreservation treatments evaluated were SF+HSPA1A, SF-Control, VIT+HSPA1A, and VIT-Control (n = 20 samples per treatment). Samples from SF+HSPA1A and SF-Control treatments were frozen in 0.25 ml straws by exposing them to liquid nitrogen (LN₂) vapors. For VIT, samples were vitrified by direct immersion of 30 µl drops into LN₂ and stored in cryotubes. Results showed that SF+HSPA1A and SF-Control yielded significantly higher (P < 0.05) progressive motility, velocities, linearity and straightness, and beat cross frequency compared to VIT+HSPA1A and VIT-Control. Furthermore, sperm treated with HSPA1A (both SF and VIT) exhibited significantly improved (P < 0.05) motility, viability and DNA integrity compared to their respective controls. In conclusion, the addition of recombinant HSPA1A to both slow-freezing and vitrification media enhanced the post-cryopreserved quality of dog epididymal spermatozoa, demonstrating its protective effect on motility, viability, and integrity of acrosome and DNA.</div></div>","PeriodicalId":7880,"journal":{"name":"Animal Reproduction Science","volume":"285 ","pages":"Article 108087"},"PeriodicalIF":3.3,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145788275","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 : 2026-01-01Epub Date: 2025-12-02DOI: 10.1016/j.anireprosci.2025.108053
Soudabeh Rouzbehani , Asmita Shrestha , Nazia Haque , Syeda Afsana Nushrat , Teklu Tewoldebrhan Zeremichael , Anna Nordborg , Ann Helen Gaustad , Anne Hege Alm-Kristiansen
The objective of this study was to evaluate sperm quality of Duroc boars during sexual maturation and examine whether these parameters were related to metabolites in semen. Ejaculates were collected longitudinally from 28 Duroc boars at approximately 7 months of age (Age 1), 6 weeks later (Age 2) and 12 weeks later (Age 3). Motility characteristics, acrosome integrity, and viability were assessed on collection day and after five days storage at 18°C using computer-assisted sperm analysis and flow cytometry, while DNA fragmentation was measured on day five. Amino acid and amine concentrations in semen were measured by liquid chromatography-mass spectrometry and correlated with sperm quality parameters. Motility, rapid progressive motility, and acrosome intact live sperm increased significantly (adj P < 0.05) with boar maturation. Mature boars (Age 2 and Age 3) maintained superior sperm quality compared to younger boars after storage. The DNA fragmentation index was low in all samples but declined significantly from 1 % at Age 1 to 0.64 % at Age 3 (adj P < 0.05). Correlation analysis showed significant relationships between sperm quality and specific metabolites. Cystine, glutamate, aspartate, choline and taurine were inversely correlated with progressive and rapid progressive motility, while showing positive correlation with rapid non-progressive motility. These findings demonstrate that sperm quality continues improving beyond initial reproductive ability, especially between Age 1 and Age 3, with improvements in motility, viability, chromatin stability, and storage resilience. The observed relationships between metabolites and sperm quality parameters provide insights into biochemical mechanisms underlying sperm functionality during sexual maturation.
{"title":"Influence of sexual maturity on sperm quality in Duroc boars","authors":"Soudabeh Rouzbehani , Asmita Shrestha , Nazia Haque , Syeda Afsana Nushrat , Teklu Tewoldebrhan Zeremichael , Anna Nordborg , Ann Helen Gaustad , Anne Hege Alm-Kristiansen","doi":"10.1016/j.anireprosci.2025.108053","DOIUrl":"10.1016/j.anireprosci.2025.108053","url":null,"abstract":"<div><div>The objective of this study was to evaluate sperm quality of Duroc boars during sexual maturation and examine whether these parameters were related to metabolites in semen. Ejaculates were collected longitudinally from 28 Duroc boars at approximately 7 months of age (Age 1), 6 weeks later (Age 2) and 12 weeks later (Age 3). Motility characteristics, acrosome integrity, and viability were assessed on collection day and after five days storage at 18°C using computer-assisted sperm analysis and flow cytometry, while DNA fragmentation was measured on day five. Amino acid and amine concentrations in semen were measured by liquid chromatography-mass spectrometry and correlated with sperm quality parameters. Motility, rapid progressive motility, and acrosome intact live sperm increased significantly (adj <em>P</em> < 0.05) with boar maturation. Mature boars (Age 2 and Age 3) maintained superior sperm quality compared to younger boars after storage. The DNA fragmentation index was low in all samples but declined significantly from 1 % at Age 1 to 0.64 % at Age 3 (adj <em>P</em> < 0.05). Correlation analysis showed significant relationships between sperm quality and specific metabolites. Cystine, glutamate, aspartate, choline and taurine were inversely correlated with progressive and rapid progressive motility, while showing positive correlation with rapid non-progressive motility. These findings demonstrate that sperm quality continues improving beyond initial reproductive ability, especially between Age 1 and Age 3, with improvements in motility, viability, chromatin stability, and storage resilience. The observed relationships between metabolites and sperm quality parameters provide insights into biochemical mechanisms underlying sperm functionality during sexual maturation.</div></div>","PeriodicalId":7880,"journal":{"name":"Animal Reproduction Science","volume":"284 ","pages":"Article 108053"},"PeriodicalIF":3.3,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145676201","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 : 2026-01-01Epub Date: 2025-11-24DOI: 10.1016/j.anireprosci.2025.108049
Eduardo de Mercado , Sonia Sánchez-Rodríguez , Adrián Martín-San Juan , Helena Nieto-Cristóbal , María José Martinez-Alborcia , Alejandro Vicente-Carrillo , Manuel Álvarez-Rodríguez
The TRPC4 channel is related to the movement of calcium within cells, and therefore possibly to sperm motility, capacitation and the acrosome reaction. This study investigated the presence and localization of TRPC4 channels in frozen-thawed boar sperm by direct immunofluorescence. In addition, different concentrations of the specific inhibitor ML204 were tested to evaluate its effect on post-thaw sperm motility and multiple sperm quality parameters assessed by flow cytometry, under conditions that promote motility recovery, such as caffeine supplementation or incubation in a basic extender. Our results confirmed that the presence of TRPC4 in boar sperm is independent of the use of ML204 and located in the midpiece and post-acrosomal regions. At the highest concentrations (22 µM), channel inhibition with ML204 caused a significant reduction in total and progressive motility, as well as in mitochondrial membrane potential, without a clear detrimental effect on the measured cytometry parameters. The loss of motility was exacerbated under co-incubation conditions with caffeine (2 mM), possibly due to an imbalance between calcium influx and demand via the cAMP/PKA pathway activated by this methylxanthine. Our results, however, when the thawing medium had a basic pH (8.2), motility was comparable to the control, likely because other pH-dependent channels compensated for the reduced calcium and cation influx. In conclusion, the TRPC4 channel is present in porcine sperm and appears to play an important role in regulating motility and maintaining mitochondrial membrane potential.
{"title":"Transient receptor potential channel 4 (TRPC4) in boar sperm: Immunolocalization and functional analysis using the specific inhibitor ML204","authors":"Eduardo de Mercado , Sonia Sánchez-Rodríguez , Adrián Martín-San Juan , Helena Nieto-Cristóbal , María José Martinez-Alborcia , Alejandro Vicente-Carrillo , Manuel Álvarez-Rodríguez","doi":"10.1016/j.anireprosci.2025.108049","DOIUrl":"10.1016/j.anireprosci.2025.108049","url":null,"abstract":"<div><div>The TRPC4 channel is related to the movement of calcium within cells, and therefore possibly to sperm motility, capacitation and the acrosome reaction. This study investigated the presence and localization of TRPC4 channels in frozen-thawed boar sperm by direct immunofluorescence. In addition, different concentrations of the specific inhibitor ML204 were tested to evaluate its effect on post-thaw sperm motility and multiple sperm quality parameters assessed by flow cytometry, under conditions that promote motility recovery, such as caffeine supplementation or incubation in a basic extender. Our results confirmed that the presence of TRPC4 in boar sperm is independent of the use of ML204 and located in the midpiece and post-acrosomal regions. At the highest concentrations (22 µM), channel inhibition with ML204 caused a significant reduction in total and progressive motility, as well as in mitochondrial membrane potential, without a clear detrimental effect on the measured cytometry parameters. The loss of motility was exacerbated under co-incubation conditions with caffeine (2 mM), possibly due to an imbalance between calcium influx and demand via the cAMP/PKA pathway activated by this methylxanthine. Our results, however, when the thawing medium had a basic pH (8.2), motility was comparable to the control, likely because other pH-dependent channels compensated for the reduced calcium and cation influx. In conclusion, the TRPC4 channel is present in porcine sperm and appears to play an important role in regulating motility and maintaining mitochondrial membrane potential.</div></div>","PeriodicalId":7880,"journal":{"name":"Animal Reproduction Science","volume":"284 ","pages":"Article 108049"},"PeriodicalIF":3.3,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145610555","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}
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