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Alhilfi, H.O. (2018) Enhancing the developmental competence of bovine oocytes and embryos. PhD thesis, University of Nottingham.

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Abstract

Enhancing the developmental competence of oocytes and quality of embryos is central to the success of in vitro embryo production (IVP) both in human and animal laboratories. However, there is currently no optimal protocol for IVP in either a commercial or clinical setting that leads to high yields of developmentally competent embryos and pregnancy outcomes. The current series of studies were performed to establish the effects of nitric oxide donor (Sodium nitroprusside (SNP)) and the proteasome inhibitor (MG132) on nuclear maturation and mitochondrial activity of bovine oocytes. Also, to establish the effects of Transforming Growth Factor B1 (TGFB1) and Colony Stimulating Factor 2 (CSF2) added to embryo culture (IVC) on the development of bovine embryos beyond morulation.

In the first series of experiments, 10 µM sodium nitroprusside delayed polar body (PB) extrusion (P=0.013). Also, 10 µM MG132 reduced PB extrusion (P=0.003), ATP content in oocytes (P=0.003) and the proportion of zygotes reaching the blastocyst stage (P=0.001). However, neither SNP nor MG132 had any effect on mtDNA copy number. MG132 but not SNP reduced (P=0.001) the proportion of cleaved zygotes reaching blastocyst at D8 of culture.

In the second series of experiments, utilizing different concentrations of MG132 revealed that 10 µM MG132 reduced (P=0.003) the proportion of ≥4-cell embryos at D2 of IVC. On the other hand, truncated (8 h) incubation of cumulus-oocyte complexes (COCs) with sperm reduced (P=0.003) the number of cleaved zygotes but increased (P<0.001) ≥4-cell embryos at D2 of IVC. Treatment with MG132 late in maturation followed by 8 h incubation period of gametes resulted in increase (P=0.013) of cleaved zygotes at D2 of IVC and increases (P=0.007) blastocysts of inseminated oocytes, and (P=0.004) of cleaved zygotes, in comparison with 18 h IVF counterpart.

In the third series of experiments, a combination of TGFB1 and CSF2 reduced (P=0.016) the proportion of cleaved zygotes reaching the blastocyst stage but had no effect on total cell number in the resultant blastocysts. Also, TGFB1 from D5 eliminated (P=0.025) the positive effect of CSF2 on embryonic development. Furthermore, only CSF2 added separately increased (P=0.004) the proportion of blastocysts and cell within the ICM for early and late blastocysts. However, treatment of embryos with either 10 or 50 ng ml-1 TGFB1 from D2 of IVC had no effect on SMAD signalling in resultant D7 embryos.

In the fourth and final series of experiments, embryonic development were enhanced when MG132 was included during late maturation followed by 8 h IVF and CSF2 from D5 of IVC (P=0.039). On the other hand, applying the latter treatment regime skewed (P=0.05) blastocyst sex ratio in favour of females.

Accordingly, utilizing MG132 during late maturation has beneficial effects but only when followed by a short period of incubation of gametes during fertilization that eliminates the residual negative effect of this reagent. Also, CSF2 increases blastocyst development together with the quality of the resultant blastocysts when added at D5 of culture. Moreover, MG132 during late maturation followed by 8 h IVF and CSF2 from D5 of culture improves the development of embryos and skews the blastocyst sex ratio towards female. This new approach of IVP may have important implications in the dairy cattle industry and opens up area for further investigation in human assisted reproduction.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Sinclair, K.D. Alberio, R.
Keywords:	IVP; in vitro embryo production; oocytes
Subjects:	Q Science > QL Zoology > QL951 Embryology S Agriculture > SF Animal culture
Faculties/Schools:	UK Campuses > Faculty of Science > School of Biosciences
Item ID:	55506
Depositing User:	Alhilfi, Haitham
Date Deposited:	05 Apr 2019 13:33
Last Modified:	11 Dec 2020 04:30
URI:	https://eprints.nottingham.ac.uk/id/eprint/55506

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