Progress 01/01/22 to 12/08/23
Outputs
Target Audience:The primary and initial target audience for the results of our work will be researchers and scientists interested in the application and improvement of Assisted Reproductive Technologies (ART). Those will be researchers examining different aspects of sperm function, fertilization in embryo development in large domestic species. Longer-term recipients of our work will be practitioners who routinely apply ART to facilitate the generation of high genetic impact. Once the practical aspects of our methods are well-established; our methods could be applied in the field to improve fertilization rates after in vitro fertilization or even increase the rates of conception post-artificial insemination. We will reach out to these audiences by presenting at meetings and publishing in society journals. Changes/Problems:The project director has established a new laboratory at an alternative institution, the University of Connecticut. Consequently, the project will transition to the new institution following the successful transfer of funds. A formal request for transfer of funds was sent to Mark Mirando in the NIFA/USDA awards Management Division on 11/21/23. What opportunities for training and professional development has the project provided?Undergraduate and graduate students The project director Dr. Maria G. Gervasi continued the training and supervision of the two undergraduate students that had started working on the project the previous year, Christina Eckenreiter and Riley Shaw. Both undergraduate students were further trained in their laboratory skills, in the analyses and presentation of results, and in refining critical thinking. This training ensured their significant contribution to the project, and it is reflected in their inclusion in the presentation of the work in scientific meetings. Both students graduated in the Spring semester 2023. In addition, during 6 months of the reported period, the project director trained and worked in close collaboration with a graduate student visiting scholar that came from the University of Buenos Aires, Argentina. The visiting scholar, Camila Arroyo-Salvo, had previous experience working with bull and horse sperm, and in the laboratory of Dr. Gervasi learned the application of the SER treatment and preparation of samples for NMR and mass spec analyses. She benefited from the laboratory and data analysis training, in addition to the preparation and presentation of posters in the Gordon Research Seminar and in the Gordon Research Conference on Fertilization and Activation of Development in July 2023. In addition, experiments performed during her stay are part of an ongoing collaboration with her home institution, and produced results that were included in a manuscript that has been recently submitted to the journal Andrology (currently under revision). Project director Dr. Maria G. Gervasi actively contributed to her career growth by engaging in academic conferences and collaborating with fellow researchers in her field. In July 2023, she delivered a presentation at the NIFA/AFRI Animal Reproduction annual project director meeting in Ottawa, Canada. Shortly thereafter, she participated in the prestigious Gordon Research Conference on Fertilization and Activation of Development held in New Hampshire, USA. During this event, the visiting graduate student who worked under Dr. Gervasi's supervision presented a poster showcasing data from the ongoing project led by Dr. Gervasi. Additionally, Dr. Gervasi secured a faculty position as an assistant professor in the Department of Animal Science at the University of Connecticut. The alignment between the ongoing project's prospects and the department's vision for advancing agriculture played a pivotal role in this significant career advancement. How have the results been disseminated to communities of interest?The findings have been shared at sicentific conferences and through casual discussions with pears. In addition, a manuscript is under revision in the journalAndrology, a second manuscript is ready for submission, and a third one is in preparation. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The use of in vitro production (IVP) of embryos for cattle production has been on the rise worldwide. This technique together with other assisted reproductive technologies have been fundamental for the improvement of reproductive performance and genetic gain in the cattle industry. Our research explores the manipulation of bovine sperm function to enhance assisted reproductive technologies, particularly in vitro fertilization which is central part of the IVP protocols. The obtained results indicate that manipulation of the sperm metabolism has a positive impact on sperm function which in turn could be applied for the IVP of embryos. Enhancing embryo production for healthier offspring could substantially benefit the agricultural sector, supporting producers, local economies, and consumers. Besides the direct benefits on cattle production, our exploration of manipulating bull sperm sets the stage for improved reproductive technologies in other large animal species with agricultural relevance. Award objectives: 1. To study how changes in [Ca2+]i and metabolism affect bull sperm function. 1.1 To determine the effect of SER treatment on [Ca2+]I levels in bovine sperm. In this second period of funding (Jan 2023 to Aug 2023, see changes for details), we continued and deepened the analyses of sperm motility and focused on the sperm energy restriction and recovery (SER) treatment. We tested the need for sperm capacitation conditions for the SER treatment by analyzing sperm motility with a computer assisted sperm analysis (CASA) system.We were able to establish that the best conditions for the SER treatment are in non-capacitating medium, and that there is a need for recovery of the sperm in the presence of both lactate and pyruvate as energy sources. These results were essential for the refinement of the SER treatment and its application prior to IVF. 1.2 To evaluate the OxPhos pathway response to SER and A23187 pulse treatments. We performed experiments to study bull sperm metabolism, particularly focusing on how this is affected by the SER treatment. We were able to find the proper experimental conditions for the identification of metabolites of bull sperm by nuclear magnetic resonance (NMR) and by mass spectrometry (mass spec). We successfully identified metabolites by NMR after the application of SER treatment and compared it with control samples. In addition, we pursued a pilot study and have determined the number of sperm necessary to identify metabolites in bull sperm by mass spec. These results stand as the steppingstone to extend our understanding of sperm metabolism and its impact on sperm function. The key outcomes for this first objective include the refinement of the SER treatment for the improvement of sperm function as we further established the sperm incubation conditions during the treatment. This will contribute to the development of a protocol for the application of this treatment prior to in vitro fertilization for the IVP of embryos. 2. To test the use of A23187 pulse and/or SER treatment for development of new ART in the bovine model. 2.1 To evaluate the effect of SER or a combination of SER and A23187 pulse on fertilization and early embryo development after IVF in the bovine model. Once the optimized SER conditions were established, we performed two preliminary IVF experiments in which SER and control sperm were used. The rates of cleavage to 2-cell/3-4-cellsdid not differ between the treatments (SER-NC: 52.1 %; Control: 58.9 %); however, the percentage of cleaved eggs that reached blastocyst stage was higher in the SER-derived embryos in comparison to controls (SER-NC: 43.1 %; Control: 19.1 %). These results support the hypothesis that sperm incubation conditions prior to fertilization have post-fertilization effects that affect early embryo development. 2.2 To test the application of SER treatment for the improvement of bovine ICSI. No activities were performed in this period regarding this particular objective. 2.3 To analyze sperm PLCz accessibility after SER treatment. Regarding this sub-aim, we developed a new research tool as we produced a custom antibody for the study of PLCz, the sperm factor that is delivered to the egg during fertilization that triggers development. We also proved the specificity of the developed antibody by western blot and immunofluorescence using sperm from male mice lacking PLCz (PLCz knockout animals). Then, we used the antibody for the detection of PLCz in bull and horse sperm by western blot and immunofluorescence. A band of approximately 70 kDa corresponding to PLCz was found in horse samples. In addition, when immunofluorescences of PLCz were performed in bovine sperm after SER treatment, no changes in the localization of the sperm factor were detected in comparison to controls. These results indicate that relocalization of PLCz is not part of the SER mechanism that facilitates egg activation after fertilization. However, the development of a PLCz antibody is a valuable tool that can be used for the study of sperm PLCz in diverse species. Overall, we deepened in the study of bull sperm function and metabolism when subject to SER treatment and determined that the effects observed of SER sperm on egg activation are not due to a re-localization of the sperm factor PLCz. In addition, we extended our investigations to the study cryopreserved horse sperm with the possibility of the application of the SER treatment for the improvement of horse IVF. While preliminary, the enhanced development of embryos following IVF with the refined SER treatment protocol establishes a foundation for investigating sperm contributions to early embryo development. This challenges the prevailing paradigm that assigns a minor role to sperm in this process.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Sperm energy restriction and recovery (SER) treatment improves sperm function in the bovine model. Arroyo-Salvo C, Aguila-Paredes L, Eckenreiter C, Shaw R, Navarrete FA, Perez-Martinez S, Fissore R, Visconti PE, and Gervasi MG. Gordon Research Conference (GRC), Fertilization & Activation of Development. Holderness, NH, United States. July 2023.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Sperm energy restriction and recovery (SER) treatment improves sperm function in the bovine model. Arroyo-Salvo C, Aguila-Paredes L, Eckenreiter C, Shaw R, Navarrete FA, Perez-Martinez S, Fissore R, Visconti PE, and Gervasi MG. Gordon Research Seminar (GRS), Fertilization & Activation of Development. Holderness, NH, United States. July 2023
- Type:
Other
Status:
Accepted
Year Published:
2023
Citation:
Manipulation of sperm metabolism improves sperm function revealing post-fertilization paternal effects: possible applications on assisted reproductive technologies . Invited speaker at the Department of Animal Science, University of Connecticut, Storrs. March 1, 2023.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
Effect of bicarbonate and polyvinyl alcohol on in vitro capacitation and fertilization ability of cryopreserved equine sperm. Arroyo-Salvo C, Rio S, Bogetti ME, Plaza J, Miragaya M, Yaneff A, Davio C, Fissore R, Gervasi MG, Gambini A, Perez-Martinez S. Andrology (under revision)
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Bovine sperm Ca2+ signaling and energy pathways in basic science and assisted reproductive technologies (ART). Gervasi MG. National Institute of Food and Agriculture, AFRI Animal Reproduction annual project director meeting. Ottawa, Canada. July 2023.
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Progress 01/01/22 to 12/31/22
Outputs
Target Audience:The primary and initial target audience for the results of our work will be researchers and scientists interested in the application and improvement of Assisted Reproductive Technologies (ART). Those will be researchers examining different aspects of sperm function, fertilization in embryo development in large domestic species. Longer-term recipients of our work will be practitioners who routinely apply ART to facilitate the generation of high genetic impact. Once the practical aspects of our methods are well-established; our methods could be applied in the field to improve fertilization rates after in vitro fertilization or even increase the rates of conception post-artificial insemination.? Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The Project Director Dr. Maria G. Gervasi and Dr. Pablo Visconti (co-PD), sponsored two summer internships for undergraduate students to work in the funded project. On one hand, the undergraduate student Christina Eckenreiter (junior, animal sciences major) was awarded a UMass CAFE Summer Scholars Undergraduate Internship Program scholarship to start her training in the manipulation of bull sperm and its application for the improvement of assisted reproductive technologies. This summer internship offered the opportunity for training a motivated undergraduate student and significantly contribute to the advancement of her career as well as the project. On the other hand, the undergraduate student Riley Shaw (senior, microbiology major) was awarded a Lee Science Impact Program (Lee SIP) fellowship to start her training in the manipulation of bull sperm and its application for the improvement of assisted reproductive technologies.The Project Director trained and worked directly in the laboratory with both students during the Summer. Christina focused her research on the application of calcium ionophore, and Riley focused her research on the application of the sperm energy restriction and recovery treatment. Both projects were presented to a more general scientific audience in poster sessions organized by the respective programs at the end of the Summer. The success in the training received during the Summer prompted both students to continue their scientific career by training in the laboratory during the Fall semester 2022 and continue working in the project as independent studies. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The cattle industry relies on the successful application of assisted reproductive technologies to produce animals with higher economic value. In recent years the use of in vitro production of cattle has been rising in the United States and worldwide. Our proposal is focused on the study of bovine sperm function and its use for the improvement of assisted reproductive technologies such as in vitro fertilization. The main goal of our research is the development of technologies that would increase the efficiency of animal production. Any improvement of embryo production thatincreases the number and/or quality of embryos able to generate healthy offspring would have a considerable financial impact on the agricultural industry. This would directly benefit producers, regional economies, and ultimately consumers. Our work studying the manipulation of bull sperm function provides the framework for the development of improved assisted reproductive technologies in large animal species. Once the practical aspects of our methods are well-established; our methods could be applied in the field to improve fertilization and early embryo development rates after in vitro fertilization. During this first year of funding, we have performed experiments of calcium manipulation of sperm and evaluation of sperm functional parameters such as motility, acrosome reaction and membrane integrity patterns. Overall, during this period we found that the manipulation of calcium intracellular concentration in the sperm significantly increases the occurrence of events related to the ability of sperm to fertilize in comparison with regular sperm incubation conditions. These results indicate that the developed sperm treatment has the potential to be applied for the improvement of the embryo production after in vitro fertilization in cattle. We have used the developed sperm calcium treatment before in vitro fertilization in the bovine model and evaluated fertilization rates and development. Our results indicate that the sperm treatment with calcium ionophore prior to in vitro fertilization increased fertilization rates by about a 15% margin and doubled the percentage of embryo development in vitro. We have also optimized the incubation conditions for a different sperm treatment that was developed in our laboratory: the sperm energy restriction and recovery (SER) treatment. SER consists in the incubation of bull sperm in the absence of nutrients (starvation period) followed by a recovery period in which nutrients are added back to the medium. We found that, similarly to the calcium ionophore treatment, SER improves sperm functional parameters. We had previously shown that this treatment also improves the activation of development. Then, we studied the cellular mechanism behind this effect by analyzing the occurrence of calcium oscillations in the formed embryo which are essential for activation ofdevelopment. We measured these calcium oscillations and found that 40% of the SER-sperm fertilized cow eggs present a greater number of calcium oscillations while only about 10% of the control-sperm fertilized eggs do. These results indicate that SER is facilitating the activation of development after fertilization. A specific sperm factor delivered by the sperm at fertilization is related to egg activation and initiation of development. We are interested in studying the localization of this sperm factor during the SER treatment. As there are no molecular tools available for the study of this factor in bovine species, we customized the production of a specific antibody that will recognize the bull sperm factor. We expect that the use of the developed tool will allow us to deepen in the understanding of the molecular mechanism elicited by SER. Overall, the progress attained during the first year of the funded project set the basis for the study and the application of two different sperm treatments that can be used for the improvement of the in vitro production of bovine embryos. We optimized the conditions of the treatments and showed that the application of these treatments can result in higher fertilization rates after in vitro fertilization and the production of larger numbers of embryos.
Publications
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