Progress 01/01/24 to 12/31/24
Outputs
Target Audience:Targeted audience would include university faculty members, graduate students from primarily Animal Science Departments, as well as veterinarians and veterinary students. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training and professional development have been a component for two graduate students, two interns, and one post doctoral candidate. How have the results been disseminated to communities of interest?Three abstracts have been written and presented at the 2023 and 2024 Society for the Study of Reproduction Annual meetings held in Ottawa, Canada (w0w3) and Dublin, Ireland (2024). An abstract was also submitted and presented at the National Association of Animal Breeders Annual Meeting in Madison, Wisconsin in 2024. Invited presentations have been given to faculty and students at Montana State Universitity, veterinarians at the Montana Veterinary Association Symposium and producers at the Centenial Celebration for USDA-ARS, Fort Keogh. What do you plan to do during the next reporting period to accomplish the goals?An additional study evaluating ability ofnanopurification of frozen-thawed semen samples to improve fertilization success, embryo cleavage and blastocyst formation in vitro. Also, we plan to conduct a heterospermic field trial using nanopurified semen to assess field fertility.
Impacts
What was accomplished under these goals?
While the first objective of the proposal was to field test the improvement in fertility obtained by nano-purification of semen using peanut agglutinin (PNA) as the lectin coating on magnetic nanoparticles to remove acrosome damaged sperm from the ejaculate, we have chosen to expand our evaluation of lectins that may be able to remove additional damaged sperm from an ejaculate before proceeding to the heterospermic field trial. We have evaluated a new set of lectin-conjugated magnetic nanoparticles targeting different surface glycans, both alone and in combination. The lectins PNA, LBA, LCA, and UEA1 were included in our evaluations. Nanopurification with PNA coated nanoparticles targets galactose, LBA targets N-acetylgalactosamine, LCA targets mannose, and UEA-1 targets fucose moieties on sperm membranes. We aimed to investigate how selective removal of spermatozoa based on their surface glycan motifs can influence pre- and post-cryopreservation bull sperm parameters. Post-sort cell recovery ranged from 71 to 78% of pre-sort sperm numbers for the 4 individual lectin and 2 mixed lectin nanoparticles. Mix 1 included LBA, UEA1 and PNA nanoparticles and Mix 2 included LBA and UEA1 nanoparticles. Twelve mature bulls (2.5-4 years old) were used for bull semen purification. Semen was collected by electroejaculation, and two ejaculates were pooled per collection for each bull. Semen was assessed fresh and post thaw for sperm viability, membrane integrity, mitochondrial membrane potential, oxidative potential, and capacitation status (zinc signatures) using conventional flow cytometry at three timepoints (neat semen, post purification and post thaw of cryopreserved samples). Motility and morphology were also assessed at all three timepoints by using an AndroScope computer-assisted semen analysis (CASA) and light microscopy, respectively. Nanopurification with Mix2 increased (P = 0.002) the percentage of bovine spermatozoa that were able to effectively respond to an oxidative challenge before cryopreservation when compared to CON, suggesting a greater potential to mitigate oxidative stress. Nanopurification with Mix2 (P = 0.018) increased and Mix1 (P = 0.083) tended to increase the proportion of morphologically normal sperm. Upon further examination, nanopurification with UEA-1 alone or in Mix2 decreased (P < 0.02) the percentage of sperm head abnormalities in samples. Nanopurification using PNA (P = 0.022), Mix1 (P = 0.041) and Mix2 (P = 0.022) decreased the proportion of bovine spermatozoa with disrupted acrosome and plasma membranes post thaw when compared to non-sorted control. Additionally, purification using PNA (P = 0.031) and Mix1 (P = 0.044) improved post thaw motility. Subsequent evaluation of a Mix3 cocktail of nanoparticles (different proportions of Mix1 nanoparticles) revealed more encouraging results for further experiments. Finally, PNA, LBA, and UEA-1 nanopurification (individually and in mixtures) of spermatozoa from commercial semen straws post thaw significantly improved (P < 0.05) sperm progressive motility, viability and acrosome integrity. Thus, seminal plasma in fresh ejaculates may interfere with, or greater concentrations of nanoparticles may be necessary for removal of damaged sperm from fresh ejaculates. We have identified Mix3 as the optimal nanoparticle cocktail to proceed with purification of spermatozoa for our heterospermic field trial to assess pregnancy rates. Overall, this work will advance our knowledge of sperm surface glycans, their impact on bull sperm survival, fertilization, and potential use as bull sperm fertility biomarkers to improve bovine reproductive efficiency.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Awaiting Publication
Year Published:
2025
Citation:
Zezeski, A. L., L.E. Hamilton, and T. W. Geary. 2025. Improved evaluation of mitochondrial membrane potential in bovine spermatozoa using JC-1 with flow cytometry. Biol. Reprod. (accepted, awaiting proof).
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Progress 01/01/23 to 12/31/23
Outputs
Target Audience:Targeted audience would include university faculty members, graduate students from primarily Animal Science Departments, as well as veterinarians and veterinary students. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training and professional development have been a component for one post doctoral candidate, one graduate student, and two interns. How have the results been disseminated to communities of interest?An abstract was written and presented as a poster at the 2023 Society for the Study of Reproduction Annual Meeting held in Ottawa, Canada in July, 2023. Two additional abstracts have been submitted for presentation at the 2024Society for the Study of Reproduction Annual Meeting to be held in Dublin, Ireland this summer. What do you plan to do during the next reporting period to accomplish the goals?An additional study is planned with nanopurification of another cocktail of lectin-coated nanoparticles before proceeding to our first field trial that will evaluate the best lectin-coated nanoparticles identified for removing subfertile and dead populations of sperm from ejaculates before creating straws for artificial insemination.
Impacts
What was accomplished under these goals?
While the first objective of the proposal was to field test the improvement in fertility obtained by nano-purification of semen using peanut agglutinin (PNA) as the lectin coating on magnetic nanoparticles to remove acrosome damaged sperm from the ejaculate, we have chosen to expand our evaluation of lectins that may be able to remove additional damaged sperm from an ejaculate before proceeding to the heterospermic field trial. We have now evaluated a new set of lectin-conjugated magnetic nanoparticles targeting different surface glycans, both alone and in combination. The lectins PNA, LBA, LCA, and UEA1 were included in our evaluations. We aimed to investigate how selective removal of spermatozoa based on their surface glycan motifs can influence pre- and post-cryopreservation bull sperm parameters. Post-sort cell recovery ranged from 71 to 78% of pre-sort sperm numbers for the 4 individual lectin and 2 mixed lectin nanoparticles. Mix 1 included LBA, UEA1 and PNA nanoparticles and Mix 2 included LBA and UEA1 nanoparticles. Twelve mature bulls (2.5-4 years old) were used for bull semen purification. Semen was collected by electroejaculation, and two ejaculates were pooled per collection for each bull. Semen was assessed for sperm viability, membrane integrity, mitochondrial membrane potential, oxidative potential, and capacitation status (zinc signatures) using conventional flow cytometry at three timepoints (neat semen, post purification and post thaw of cryopreserved samples). Motility and morphology were also assessed at all three timepoints by using an AndroScope computer-assisted semen analysis (CASA) and light microscopy, respectively. Nanopurification using lectin PNA (targets galactose), or a mixture of lectins UEA1 (targets fucose) and LBA (targets N-acetylgalactosamine); Mix2 decreased (P < 0.05) the proportion of bovine spermatozoa with disrupted membranes post thaw when compared to non-sorted control (CON). Purification with Mix2 also increased (P < 0.01) the percentage of bovine spermatozoa that were able to effectively respond to an oxidative challenge post thaw when compared to CON, suggesting a greater potential to mitigate oxidative stress. Additionally, purification using Mix1 tended (P = 0.08) to improve post thaw motility. We are considering evaluating one more additional mixture of differently coated nanoparticles before conducting a heterospermic field trial with bull semen from the best performing lectin-coated nanoparticles to assess the impact of the nanopurification on bovine pregnancy rates. Overall, this work will advance our knowledge of sperm surface glycans, their impact on bull sperm survival, fertilization, and potential use as bull sperm fertility biomarkers to improve bovine reproductive efficiency.
Publications
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Progress 01/01/22 to 12/31/22
Outputs
Target Audience:The initial target audience will be university researchers and industry focused on improving fertility of semen doses used with artificial insemination in cattle. Beef and dairy producers using artificial insemination will be the next target audience followed by university researchers and veterinarians that will develop and test new assessments of fertility among bulls used with natural service. The beef and dairy producers will be the target audiences of these improvements leading to more efficient beef and dairy production that could be passed along to the consumer. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training was provided to technicians/graduate students participating in the research. The PostDoc participating in this research, as well as all project directors participated in the 2022 NIFA Project Directors Meeting and attended Society for the Study of Reproduction annual meeting. How have the results been disseminated to communities of interest?Only preliminary results, in the form of oral presentations, have been provided to university and veterinarian staff. What do you plan to do during the next reporting period to accomplish the goals? Further evaluation of different lectin coated nanoparticles will be evaluated individually and in cocktails to determine the optimum combinations for removing damaged sperm from the ejaculates of bulls. Primary criteria evaluated will be via computer assisted sperm analyses and flow cytometry measures of known fertility biomarkers. Field studies of nanoparticle sorted sperm will begin using artificial insemination of cows/heifers usingheterospermic straws of semen containing purified and/or non-purified sperm from two bulls (A/B) per straw. these studies will include inseminations of five bull A/B combinations (10 bulls total). Fertility from artificial inseminations will be determined but sire parentage won't be known until the next reporting period. Additional laboratory studies will be conducted to identify additional sperm fertility biomarkers using various methods.
Impacts
What was accomplished under these goals?
While the first objective of the proposal was to field test the improvement in fertility obtained by nano-purification of semen using peanut agglutinin (PNA) as the lectin coating on magnetic nanoparticles to remove acrosome damaged sperm from the ejaculate, we have chosen to expand our evaluation of lectins that may be able to remove additional damaged sperm from an ejaculate before proceeding to the heterospermic field trial. The lectins evaluated and their binding on sperm are listed in Table 1. Table 1. Lectins evaluated for sperm binding. Lectin acronym Lectin name Target glycan PNA Peanut Agglutinin Galactose CAA Caragana Arborescens Complex glycans LBA Phaseolus Limensis N-Acetylgalactosamine (NAcGal) LFA Limax Flavus Sialic acids LCA Lens Culinaris Mannose UEA1 Ulex Europaeus Agglutinin 1 Frucose GSL1 Griffonia Simplicifolia 1 Galactose For each additional lectin candidate, the sperm cell localization of binding and its ability to remove abnormal morphology sperm rather than normal morphology sperm from fresh bull ejaculates was determined (Table 2). In addition, the percentage of PNA negative sperm bound by each lectin was determined from fresh ejaculates of random bulls to estimate the additional amount of sperm that could be removed with PNA & lectin coated nanoparticles (Table 2). PNA has been shown to bind exclusively to the acrosomal region of spermatozoa and is known to label spermatozoa with compromised membrane integrity. Therefore, our objective was to find an additional lectin that also targeted defective sperm but showed differential sperm binding than PNA. Table 2. Localization of lectin binding of sperm, lectin binding morphology agreement (% unlabeled normal morphology sperm + % labeled abnormal morphology sperm) and the potential benefit of each lectin when combined with PNA sorting (PNA-/Lectin+ labeled fresh spermatozoa). Lectin Binding localization Lectin binding-morphology agreement PNA-/Lectin+ binding of fresh sperm CAA Head & Tail 36.6% 1.25% LBA Postacrosomal sheath 91.9% 2.01% LFA Midpiece 81.0% 0.98% LCA Head & tail 79.4% 5.17% UEA1 Postacrosomal sheath 76.7% 3.61% GSL1 Head 68.8% 4.01% From these finding we decided to proceed testing the effectiveness of sperm sorting using magnetic nanoparticles conjugated to LBA, LCA, & UEA1, based on their differential binding patterns from PNA and high lectin binding-morphology agreements. Our preliminary sorting experiments (3 replicates) suggest that both LBA and UEA1 sorting result in improved post-thaw viability, membrane integrity, and capacitation (zinc signatures) compared to non-sorted controls. We plan to perform 3-4 additional single lectin sorting experiments before proceeding to combined lectin conjugated sorting experiments to ensure the universality of our lectin conjugated sorting procedures across bulls. These experiments are schedule to proceed over the course of Summer 2023.
Publications
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