Development of ovum pickup and in vitro embryo production to assess fertility responses for mineral intervention studies

Black, David H. (2018) Development of ovum pickup and in vitro embryo production to assess fertility responses for mineral intervention studies. DVM thesis, University of Nottingham.

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As nutrition is of central importance to cattle fertility, this study sought to assess how veterinarians and nutritional advisers manage trace element imbalance in the UK; diagnosis and treatment. The study also sought to develop a robust system for oocyte recovery (ovum pick-up (OPU)) and in vitro embryo production (IVP) for commercial use, and to identify key factors influencing success, including oestrus synchrony and ovarian stimulation prior to OPU. The intention originally was to use OPU/IVP to investigate the impact of mineral imbalances on bovine oocyte quality, early embryo development and pregnancy establishment following embryo transfer (ET).

In the first survey of its kind in the UK, the understanding and approach of advisers to mineral nutrition on farms was investigated. Of the 173 respondents, 78% were vets in practice. The overall importance of minerals was recorded by vets as low 33%, medium 37%, and high 30%, while non-vets scored importance as low 17%, medium 48%, and high 35%. There was little consensus amongst the advisers, or within the vet and non-vet subgroups about mechanisms and interactions associated with deficiency, and particularly of copper responsive conditions. The most frequently identified deficiencies were selenium, copper and iodine, while the most commonly identified toxicity was molybdenum. For copper responsive conditions, all of the listed treatments were used at least “occasionally”; the most frequently being glass boluses, in-feed supplementation, matrix boluses, and then copper injections. While there was a diverse choice of treatments, altering the ration was relatively rarely selected.

This thesis also provides the first large-scale retrospective analysis of factors influencing the establishment of a commercially robust ovum pick up (OPU) and in vitro embryo production (IVP) platform in the UK. Over a 5-year period, a system was developed and validated for use in the UK with 2,138 cycles of OPU. These cycles were analysed as four sets of data and included two IVP laboratories and 6 OPU teams. Factors in these analyses included OPU team, IVP laboratory, ovarian stimulation protocol and semen type (unsorted vs sex-sorted).

The mean number of follicles aspirated by the OPU teams ranged from 6.5 to 14.9 (P<0.001), while the number of oocytes collected was between 4.0 and 12.4 (P<0.001). There was an indication (P=0.055) that the blastocyst per oocyte rate varied between teams. The proportion of blastocysts from oocytes that cleaved was higher (P=0.01) for unsorted than sexed semen. Two commercial products containing different ratios of follicle stimulating hormone (FSH) to luteinising hormone (LH) (Folltropin® and Pluset®) were compared in ovarian stimulation programs. The addition of ‘coasting’ (short-term (typically 48h) hormonal withdrawal after FSH stimulation), prior to OPU was also investigated. Pluset® resulted in a greater (P<0.001) mean number of follicles aspirated, more (P=0.003) blastocysts per oocyte matured and more (P<0.001) embryos per cycle (2.45), compared with Folltropin® (1.17) or with no stimulation (1.24). Throughout the study there was a steady improvement in blastocyst production per OPU cycle.

In a separate analysis, Grade 1 cumulus oocyte complexes (COCs) as a proportion of COCs recovered, oocytes that cleaved as a proportion of total COCs, and blastocysts as a proportion of total COCs, were all greater (P<0.05) for stimulated than non-stimulated cycles, irrespective of FSH/LH product. A composite score of oocyte quality and quantity was proposed (sCOC); Log Total Mean sCOC was correlated (P<0.001) with both the proportion of blastocysts per oocyte collected, and the total number of embryos produced per cycle.

Finally, twelve peri-pubertal heifers (approximately 10 months old) participated in a crossover trial which compared PRID® (Delta®) vs CIDR® progesterone releasing intravaginal devices for use in OPU/IVP cycles. Vaginoscopic examination found higher vaginal inflammation grades for PRID® than CIDR® (P<0.001). There was evidence of vaginal inflammation continuing for at least 2 weeks after device withdrawal. The proportion cleaved of oocytes inseminated was higher for PRID® than CIDR® (P<0.05). Numerically but not significantly there was a higher proportion of blastocysts per cycle and a higher Log Total Mean sCOC score per cycle with PRID® than CIDR® treatments, but blastocyst yield was low throughout, suggesting a need to repeat the trial.

Data collection and analyses are ongoing, to identify other key performance indicators within the OPU/IVP embryo transfer (ET) system, with a view to refining the sCOC composite score model. A robust OPU/IVP/ET system has been developed and this could be used to investigate further how mineral imbalances impact oocyte competence and blastocyst yield.

Item Type: Thesis (University of Nottingham only) (DVM)
Supervisors: Kendall, Nigel R.
Sinclair, Kevin D.
Robinson, R.S.
Keywords: bovine, fertility, ovum pickup, OPU, in vitro embryo production, IVP, minerals
Subjects: Q Science > QL Zoology > QL951 Embryology
S Agriculture > SF Animal culture
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Veterinary Medicine and Science
Item ID: 52598
Depositing User: Black, David
Date Deposited: 13 Dec 2018 04:40
Last Modified: 08 Feb 2019 08:46

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