In-vitro produced embryos (IVP) differ greatly from in-vivo derived embryos (IVD) in gene expression, metabolism, development, and cryotolerance which limit the widespread use of this technology. In-vitro maturation (IVM) is one of the most important components for successful in-vitro embryo development. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) have been found to be essential during oocyte maturation and thus female fertility. These proteins are oocyte secreted factors (OSFs) and are produced with pro- and mature protein regions where the pro-regions are thought to aid in protein folding and dimerization where heterodimerization the two proteins has been termed cumulin (Motterhead et al., 2015). Cumulin was found to have significant effects on oocyte maturation, blastocyst rate and hatching rates. However, only recombinant mature forms of these proteins are available commercially and making pro-mature GDF9 and BMP15 as well as pro- and mature cumulin is problematic. A few studies have evaluated the mature versions finding slight, although non-significant effects on oocyte maturation and blastocyst rates. However, none have studied the effects of using both mature GDF9 and BMP15 on bovine oocytes; thus, it was tested in this thesis. For the first experiment we hypothesized cumulus–oocyte complexes (COCs) matured for 23 h in maturation media supplemented with the mature proteins would increase blastocyst development, decreased lipid levels, and increased mitochondrial activity. Additionally, cryopreservation of embryos induces oxidative damage. However, studies have shown adding individual antioxidants to cryopreservation medium help alleviate post-thaw oxidative stress by reducing the accumulation of reactive oxygen species (ROS) and detoxifying lipid peroxidation (Tarin and Trounson, 1993; Lane et al., 2002; Takahashi et al., 2013). Few studies have evaluated effects using combinations of antioxidants supplemented in slow-freezing media. For the second experiment we further hypothesized blastocysts slow frozen with antioxidants would have increased cryotolerance compared to controls. For the first experiment, bovine embryos were IVP in three treatment groups, J: a commercial IVM media, T: control (TCM 199 supplemented with gonadotrophins), and TGB: control supplemented with GDF9 (200 ng/µL) and BMP15 (100 ng/µL). For experiment 2, only IVM groups T and TGB were used. Embryos were produced in five then four replicates, respectively, from abattoir ovaries, oocytes were matured, fertilized with frozen-thawed semen from one of three bulls, and presumptive zygotes were cultured for 7-8 days. For experiment 1, stage 6–9 blastocysts were stained with Nile Red or Mitotracker Red CMX-Rosamine to evaluate lipid content and mitochondrial polarity, respectively, utilizing confocal microscopy at ×40. Five slices per embryo were evaluated and averaged for fluorescence. Blastocyst rates, Nile Red (sqrt transformed, outliers removed), and Mitotracker data were analyzed by an ANOVA and means separated by Tukey HSD. For experiment 2 stage 6–9 blastocysts were slow frozen then thawed in a 2x2 factorial and evaluated for re-expansion 24 hours post-thaw. Results indicate that there was no difference for blastocyst rates for experiment 1 and 2 (J: 26.7 0.02%, T: 26.9 0.02%, TGB: 24.2 0.031%, P > 0.1; and T: 22.0 0.020%, TGB: 21.8 0.024%, P > 0.1; respectively). TGB’s Nile Red fluorescence intensity was significantly lower (5.09 2.16 AFU, P < 0.0001) than T (12.0 2.11) and J (11.05 2.18). MitoTracker fluorescence was similar among all treatments (P > 0.05). There was no significant interactions or main effects seen between cryopreservation groups; however, T/AO (52.9 0.05%, n = 37) and T/C (39.8 0.05%, n =38) having on average a 13.1% higher re-expansion rate and AO overall had on average 6.2% higher re-expansion rates. There was no difference seen between TGB/AO and TGB/C. These results suggest that the mature forms of GDF9 and BMP15 supplemented during oocyte maturation can lower lipid content of resulting embryos, however they do not increase blastocyst rates, mitochondrial activity, or re-expansion rates after cryopreservation.
| Identifer | oai:union.ndltd.org:CALPOLY/oai:digitalcommons.calpoly.edu:theses-4270 |
| Date | 01 June 2023 |
| Creators | Thompson, Jamie |
| Publisher | DigitalCommons@CalPoly |
| Source Sets | California Polytechnic State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Master's Theses |
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