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Supplementing IL6, IL11, and LIF to Improve Cultured Bovine Oocyte Competency

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dc.contributor.authorMcKinley, Endyaen
dc.contributor.committeechairEaly, Alan Daleen
dc.contributor.committeememberEdwards, Lannetten
dc.contributor.committeememberRhoads, Michelleen
dc.contributor.committeememberBiase, Fernando H.en
dc.contributor.departmentAnimal and Poultry Sciencesen
dc.date.accessioned2023-07-25T08:00:37Zen
dc.date.available2023-07-25T08:00:37Zen
dc.date.issued2023-07-24en
dc.description.abstractBovine embryos produced in vitro consistently display decreased quality in terms of their potential to reach the blastocyst stage as well as post-transfer survival. Media formulations, oocyte quality, and inferior expression of needed transcripts are all causes of this reduced developmental potential commonly present in in vitro-produced (IVP) bovine embryos. Recently our lab has confirmed interleukin-6 as an embryokine whose capabilities include increasing inner cell mass (ICM) numbers and promoting bovine blastocyst development. LIF is another family member of the IL6 cytokine family and has been shown to produce several positive effects when supplemented during oocyte in vitro maturation. IL6 has predominantly been studied as being supplemented post-fertilization. However, published transcriptomic work described receptors for IL6, IL11, and LIF as present in cumulus cells at the time COCs are removed from their follicles. Consequently, we wanted to investigate if supplementing 25 ng/ml of IL6, IL11, or LIF would improve IVM bovine oocyte competency. Several experiments were completed (4replicates/experiment; 30-60 cumulus-oocyte complexes (COCs)/replicate). In Experiment 1, COCs were in vitro matured for 16 or 22 hours, then meiotic stage was assessed after denuding, fixation, and DNA staining. No cytokine treatment influenced the percentage of oocytes that achieved metaphase II at either time point. In Experiment 2, COCs were in vitro matured for 4 hours before snap freezing. and processing to examine changes in five cumulus-expressing transcripts associated with oocyte competency (CX43, CX37, AREG, TNFAIP6, HAS2). Our chosen housekeeping gene, HPRT1, served as the internal control. An increased abundance of AREG occurred following exposure to LIF but not with the other treatments. Supplementation with IL6 and IL11 but not LIF tended to increase TNFAIP6 abundance (P<0.10). No other transcript differences were detected. In Experiment 3, we examined whether supplementing these cytokines during IVM affects subsequent fertilization and blastocyst rates. No effects were detected on cleavage rates but at day 8 increases in blastocyst yield were detected for LIF and IL11, but not IL6. LIF showed a tendency to increase hatching rates. In Experiment 4, we aimed to assess how the cytokine treatments affected cryosurvivability. Blastocysts (5-10/replicate, 9 replicate studies) were frozen at a rate of -0.6 degrees C/min until reaching -32 degrees C, then were stored in liquid nitrogen for 4-8 weeks before being thawed and incubated in conventional embryo culture medium (SOF-BE1) for 3 days. No treatment effects were noted for re-expansion, hatching, and overall survivability. In summary, these results implicate IL11 and LIF as potential mediators of oocyte competency. However, the evidence presented here suggest that IL6 and IL11 may function differently than LIF when provided during COC maturation.en
dc.description.abstractgeneralThe numerous similarities in the regulation of early embryonic development between human and cow has made bovine embryos an excellent model for exploring how to optimize assisted reproductive techniques (ARTs) and other methods for improving and preserving fertility in humans. Pregnancy loss is also very similar in both cattle and humans. In beef cattle, more than 50 percent of reproductive failures occur before day 16 of gestation. In women, approximately 15 percent of all clinically recognized pregnancies result in spontaneous loss, however, several more pregnancies fail prior to ever being clinically recognized. Various ARTs are used to treat sub-fertile conditions in cattle, and these technologies are generally deemed as a viable way to improve fertility. However, IVP embryos are inferior in their ability to properly fertilize and develop to the blastocyst stage, the stage when embryos are normally transferred. Furthermore, IVP-generated embryos are inferior at maintaining pregnancies. There are two primary restraints to the IVP process: a low percentage of oocytes that become fertilized and produce transferable embryos and transferred IVP embryos have decreased chances of maintaining a viable embryo than embryos produced in vivo. Very little is known about the various hormone and molecular factors that promote oocyte and embryo development. Therefore, a primary objective for bovine oocyte and embryo research is to classify these factors and implement them into their maturation and culture media to improve overall IVP efficiency. My lab studies members of the IL6 cytokine family as potential factors that might play a role in the development of oocytes and embryos. The aim of this work is to assess the capacity of three molecules within this family, IL6, IL11, and Leukemia inhibitory factor (LIF) to improve oocyte development, fertilization rate, and blastocyst yield when supplemented during in vitro maturation (IVM). This work revealed that both IL11 and LIF improved IVP bovine blastocyst development at day 8. Unfortunately, none of the treatments had any effect on fertilization rates. LIF increased the expression of a cumulus-specific transcript known to aid in cumulus expansion. Cumulus cells are the somatic cells immediately surrounding the oocyte. Cumulus expansion is a key indicator of proper oocyte maturation. We did not observe any treatment effect on post-thaw survival of cryopreserved bovine embryos. This indicates that our treatments did not help the embryos maintain viability after undergoing a slow-freeze cryopreservation protocol followed by thawing and culture. In summary, this work showed that IL11 and LIF have potential benefits to the in vitro production of bovine embryos when supplemented at IVM. However, future work is needed to assess how these molecules are causing these improvements. Our results indicate that IL11 and LIF may function differently from IL6 when supplemented during IVM.en
dc.description.degreeMaster of Scienceen
dc.format.mediumETDen
dc.identifier.othervt_gsexam:37372en
dc.identifier.urihttp://hdl.handle.net/10919/115837en
dc.language.isoenen
dc.publisherVirginia Techen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectEmbryoen
dc.subjectoocyteen
dc.subjectblastocysten
dc.subjectcytokineen
dc.subjectcryo-survivabilityen
dc.titleSupplementing IL6, IL11, and LIF to Improve Cultured Bovine Oocyte Competencyen
dc.typeThesisen
thesis.degree.disciplineAnimal and Poultry Sciencesen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.levelmastersen
thesis.degree.nameMaster of Scienceen

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