Refinement of a physiologically relevant IVM/IVF system on oocyte developmental competency in New Zealand dairy cows
Authors:   , Murray La’tarsha Anne 
Published: May 2023

<p>Fertility of high-producing dairy cows world-wide has declined over the last 50 years, with a decline observed in New Zealand (NZ) dairy cows during the 1990s and early 2000s. This decline is thought to be largely due to metabolic strain experienced during lactation, when peak milk production and mating coincide. It has been demonstrated that the major loss of pregnancy in NZ dairy cows across the 282 days of gestation occurs within the first 7 days following conception. This evidence suggests that the conditions associated with early lactation result in compromised oocyte quality and low conception rate. Experiments in which oocytes are obtained from dairy cows for research purposes are very expensive limiting the amount of research in this area. Thus, traditional bovine in vitro embryo production is utilised which produces embryos from oocytes obtained from abattoir ovaries. This process involves extracting immature oocytes from follicles and maturing them in vitro prior to fertilisation and embryo culture. One major limitation to this system is that it uses an in vitro maturation (IVM) media that is unreflective of the in vivo microenvironment in which the oocyte matures. Therefore, in order to better understand the factors affecting oocyte quality in the NZ dairy cow, a physiologically relevant IVM system needs to be developed.</p>
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<p>The overall objective of my PhD study was to develop a refined IVM culture system that better resembles the follicular microenvironment; and in comparison, to the gold-standard IVM system, test its impact on oocyte developmental competency by measuring known biomarkers of oocyte quality, blastocyst rate and indicators of embryo quality. New cumulus cell, oocyte and embryo-derived gene candidates were also associated with embryonic outcome in order to identify novel molecular biomarkers that could be used to predict oocyte quality. A bi-phasic physiologically-relevant (PR) IVM media system was developed to mimic the changing concentrations of specific constituents that had been measured within subordinate antral (‘Early’ media) and pre-ovulatory (‘Late’ media) follicles of NZ dairy cows. Additionally, each media were further divided into two media that consisted of either median (‘Good-PR’ media) or extreme-percentile (‘Bad-PR’ media; either 10th or 90th -percentil dependent on constituent) concentration values of each constituent, that was anticipated to represent good or poor quality follicles, respectively. Optimization experiments were conducted to test additional supplement requirements, as well as optimal timing of media changes and addition of sperm (fertilization) within the IVP protocol. All results were generated from two large IVP experiments.</p>
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<p>In brief, for all experiments, COCs were matured in individual culture drops containing either the Good-PR, Bad-PR or traditional (gold standard) IVM media to avoid cross-sample contamination and enable sample tracking. All COCs underwent media changes at 6 and 7.5 hours and after 22 hours of culture in conditions of 38°C and 5% CO2, were graded by morphology and biopsies of cumulus cells were collected for gene expression analyses. A group culture control was included in the first IVP experiment for a more accurate comparison to the traditional bovine IVP system. Following 22 hours in culture, remaining cumulus cells were dislodged, and all oocytes then followed traditional in vitro embryo production procedures. Resultant embryos were graded at Day 7-8 and any blastocysts were harvested and collected for either gene expression analyses or counting of total, healthy or pyknotic inner cell mass (ICM) and trophectoderm (TE) cell numbers.</p>
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<p>Overall, my results identified genes related to oocyte quality including four cumulus cell-derived (BMPR2, PDE8A, GJA1 and GATM) and four blastocyst-derived (ASNS, CDX2, PTGS2 and MSO1) genes. In addition, four genes related to cumulus cell expansion were also identified in cumulus cells (VCAN, PDE8A, BMPR2, GATM) and in the oocyte (HSP90B1). Interestingly, despite only minor differences in media constituent concentrations between the two PR IVM media, maturation of oocytes in the Bad-PR media consistently resulted in lower blastocyst rates. Intriguingly, there were no significant differences in blastocyst rate between the nutrient-rich Commercial and Good-PR IVM media. The Bad-PR IVM media also resulted in blastocysts with lower cell numbers than either the Good-PR or Commercial IVM media. Additionally, there was no differences in the number of cells of embryos created from oocytes matured in the Good-PR or Commercial IVM media. Critically however, there were fewer pyknotic cells present in embryos produced from oocytes matured in the Good-PR media compared to both the Bad-PR and Commercial media. Overall, the percentage of pyknotic ICM cells of embryos created from oocytes matured in the Good-PR treatment was lower than those created in the Commercial and Bad-PR IVM media.</p>
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<p>In summary, my results suggest that maturation of oocytes in concentrations of amino acids and metabolites at values within the outer extremes (i.e. 10th and 90th percentiles) of which are present in subordinate and pre-ovulatory follicles result in the production of low-quality oocytes associated with increased incidence of poor embryological development and reduced embryo quality. Importantly, this study also suggests that maturation of oocytes in concentrations of amino acids and metabolites at median values present in subordinate and pre-ovulatory follicles results in the production of high-quality oocytes associated with high quality embryos with fewer pyknotic ICM cells. Overall, this suggests that the small variations in constituent concentrations observed in follicular fluid of NZ dairy cows affects the quality of the oocytes developed. Further work should be undertaken in order to identify the specific constituent(s) (e.g. amino acids, glucose, cholesterol, fatty acids), and their threshold concentrations, which result in the alterations in oocyte quality. This information will allow a better understanding of the deficiencies within ovarian follicles that compromise oocyte quality which may lead to an intervention such as targeted feed management.</p>