Scientists have developed techniques like ovum pick up (OPU) and follicular ablation as a large source of oocytes for creating IVP bovine embryos. These techniques have allowed for more efficient dissemination of valuable female genetics compared to traditional artificial insemination or embryo flushing. IVP embryos have lower embryo development rates and quality, leading to lower pregnancy rates. Nerve growth factor-beta (NGF), however, has been previously shown to improve 48-hour cleavage rates and the number of hatching/ hatched blastocysts out of total presumptive zygotes. We hypothesize that NGF will improve IVP embryo development by positively influencing cleavage and blastocyst rates. The first two experiments' objectives were to determine the effect of recombinant bovine (60 or 90% purity) and human NGF (97% purity) supplementation during in vitro fertilization on 24- and 48-hour cleavage and day 8 blastocyst development rates. The objective of the third experiment was to assess the effect of the supplementation of bovine NGF (90% purity) on heat shocked and non-heat shocked in vitro-matured cumulus-oocyte complexes, assessing cleavage rates at 48 and 72 hours post insemination and blastocyst development rates. The results of experiment 1 show there were no differences between any of the three treatment groups (bNGF60, hNGF95, and control) for 24 hour (P = 0.66) or 48 hour (P = 0.33) embryonic cleavage rates. Additionally, there were no differences between treatments in the total percentage of blastocysts per oocyte (P = 0.91) or the percentage of blastocysts per cleaved embryo (P = 0.32). The results of experiment 2 also showed no differences between any of the three treatment groups (bNGF90, hNGF95, and control) for 24 hour (P = 0.16) or 48 hour (P = 0.18) embryonic cleavage rates. Additionally, there were no differences between treatments in the total percentage of blastocysts per oocyte (P = 0.42) or the percentage of blastocysts per cleaved embryo (P = 0.57). In the 3rd experiment, there was not a significant effect of treatment (P ≤ 0.05) at all stages of embryonic development assessed. On the contrary, in the third experiment, non-heat stressed NGF treatment had an interestingly detrimental effect on early cleavage rates of embryos compared to the non-treated control embryos. These results showed that NGF could not improve in vitro embryonic development rates in standard conditions; however, this negative impact of NGF on early cleavage was not observed in heat-shocked embryos. Suggesting that there could be a protectant factor in NGF that warrants further investigation. / Master of Science / Nerve growth factor (NGF) was initially thought to only play a role in nerve cell development, but research has since shown an influence on female reproduction in cattle. NGF and its receptors have been identified in the follicular fluid and reproductive cell types of females, contributing to egg maturation. Previous data on NGF supplementation with IVP embryos, which took place during the summer, showed that NGF positively affected in vitro-produced embryo development when added to fertilization media, specifically on cleavage rates (division without growth, must be two cells or greater) and blastocyst development. The actual role of NGF on embryo development is still unclear. Therefore, replication of this study is essential. First, we added either recombinant human nerve growth factor (90% pure) or bovine nerve growth factor (60% or 90% pure) to the IVF medium. The goal was to determine if NGF would have the same effects on cleavage rates as bovine purified NGF when supplemented during the fertilization stage, as well as to decide if protein purity and species affected how NGF influenced embryo development rates. For the second part of the study, we heat-shocked oocytes during maturation in a "hot incubator" and supplemented them with bovine 90% NGF. This was done to mimic the summer month heat stress that may have occurred in the abstract data. Our objective was to determine if NGF could mitigate the detrimental heat shock during development and potentially improve embryo developmenNerve growth factor (NGF) was initially thought to only play a role in nerve cell development, but research has since shown an influence on female reproduction in cattle. NGF and its receptors have been identified in the follicular fluid and reproductive cell types of females, contributing to egg maturation. Previous data on NGF supplementation with IVP embryos, which took place during the summer, showed that NGF positively affected in vitro-produced embryo development when added to fertilization media, specifically on cleavage rates (division without growth, must be two cells or greater) and blastocyst development. The actual role of NGF on embryo development is still unclear. Therefore, replication of this study is essential. First, we added either recombinant human nerve growth factor (90% pure) or bovine nerve growth factor (60% or 90% pure) to the IVF medium. The goal was to determine if NGF would have the same effects on cleavage rates as bovine purified NGF when supplemented during the fertilization stage, as well as to decide if protein purity and species affected how NGF influenced embryo development rates. For the second part of the study, we heat-shocked oocytes during maturation in a "hot incubator" and supplemented them with bovine 90% NGF. This was done to mimic the summer month heat stress that may have occurred in the abstract data. Our objective was to determine if NGF could mitigate the detrimental heat shock during development and potentially improve embryo development rates under these stressful conditions. The results of all experiments indicated that NGF could not influence development rates. positively. On the contrary, in the third experiment, non-heat stressed NGF treatment had an interestingly detrimental effect on early cleavage rates of embryos when compared to non-treated control embryos. This negative impact of NGF on early cleavage was not observed in heat-shocked embryos pointing to a possible protectant factor in NGF that needs further investigation.t rates under these stressful conditions. The results of all experiments indicated that NGF could not influence development rates. positively. On the contrary, in the third experiment, non-heat stressed NGF treatment had an interestingly detrimental effect on early cleavage rates of embryos when compared to non-treated control embryos. This negative impact of NGF on early cleavage was not observed in heat-shocked embryos pointing to a possible protectant factor in NGF that needs further investigation.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/111544 |
| Date | 17 August 2022 |
| Creators | Hellstern, Emily Anne |
| Contributors | Biomedical and Veterinary Sciences, Stewart, Jamie L., Rhoads, Michelle, Ealy, Alan Dale |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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