Author: Xiaochen Zhu
Zhu, Xiaochen, 2024 The role of lipids and antioxidants in cryopreservation of bivalve oocytes and larvae, Flinders University, College of Science and Engineering
Terms of Use: This electronic version is (or will be) made publicly available by Flinders University in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. You may use this material for uses permitted under the Copyright Act 1968. If you are the owner of any included third party copyright material and/or you believe that any material has been made available without permission of the copyright owner please contact copyright@flinders.edu.au with the details.
Pacific oysters (Magallana gigas) and Mediterranean mussels (Mytilus galloprovincialis) are economically important aquaculture species in Australia and the world. Reliable techniques for gamete and embryo cryopreservation are crucial for germplasm biobanking and sustainable aquaculture development as they can be used to address issues in off-season productions, cross breeding between and within species, and long-term preservation of genetic resources. However, cryopreservation of oocytes, embryos, and larvae is more challenging than sperm in aquatic species. Lipids are one of the cellular components most susceptive to cryopreservation, and their compositions are closely related to the level of cryoresistance in most organisms. Some exogenous lipids and antioxidants have also shown enhancement in cryoresistance in recent studies, mainly in some livestock species. This thesis aims to improve the existing mussel oocyte and oyster larval cryopreservation protocols by including exogenous lipids and antioxidants in cryoprotectant agents (CPAs). Their potential cryoprotective mechanisms were revealed through investigating fatty acid (FA) profiles, DNA integrity, and gene expression.
To improve the existing oocyte and larval cryopreservation protocols, this thesis first examines the effects of exogenous lipids and α-tocopherol (TOC; an antioxidant) on the post-thaw performance of Pacific oyster larvae and Mediterranean mussel oocytes. The lipids evaluated include three polyunsaturated fatty acids [PUFAs; arachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)], one phospholipid [1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)] and the lipid extract from Atlantic salmon (Salmo salar; mussel oocyte experiments only). All three PUFAs exhibited dose-dependent cytotoxicity and showed no cryoprotective benefits in all treatments. In the larval cryopreservation experiment in Pacific oysters, the relative survival rates at D-stage and spat stages have been improved significantly and doubled, respectively by adding 0.25 mg/mL POPC and 1 mg/mL TOC into the existing oyster CPA [or Base CPA; 10 % (v/v) ethylene glycol (EG), 5 % (w/v) Ficoll® PM 70 (FIC), and 0.2 % (w/v) polyvinylpyrrolidone (PVP)]. In the oocyte experiment in Mediterranean mussels, the addition of 0.5 mg/mL POPC and 0.5 mg/mL TOC or 0.5 mg/mL salmon lipid extract into the existing mussel CPA [or Base CPA; 10% (v/v) EG and 7.5% (w/v) FIC] increased the relative survival rate of D-stage larvae by approximately 100%. The salmon lipid extract treatment produced twice the number of spat compared with the Base CPA.
Next, the roles of both endogenous FAs and exogenous lipids and antioxidants in the larval cryopreservation were examined. The former was investigated by comparing the FA profiles between species in the larvae cryopreserved with the Base CPA (without POPC and TOC), as mussel larvae exhibited better cryotolerance than oysters in published papers. The roles of exogenous lipids and antioxidants were studied by comparing the FA profiles between Pacific oyster larvae cryopreserved by two different CPAs [Base CPA vs LA CPA (Base CPA plus POPC and TOC)]. Fresh mussel larvae had a higher proportion of total PUFAs (42.11% vs 35.33%) and a lower proportion of total monounsaturated fatty acids (MUFAs; 20.54% vs 27.04%) than fresh oyster larvae. Importantly, FA profiles did not differ significantly between post-thaw and pre-freeze mussel larvae. On the contrary, a significant increase in saturated FAs and a significant decrease in PUFAs were detected in oyster larvae cryopreserved with either CPAs. The abundance of PUFAs in fresh mussel larvae, especially DHA (C22:6), and the high resistance of FA profiles to cryopreservation were probably related to better post-thaw survivability in mussels than oysters. As FA profiles did not differ between larvae cryopreserved by either CPAs in Pacific oysters, the supplementation of POPC and TOC did not affect the endogenous FA profiles during the cryopreservation in this species, although the post-thaw larval survivability has been improved significantly.
Finally, the thesis explores the DNA integrity and gene expression of post-thaw larvae cryopreserved by Base and LA CPAs to evaluate the effects of supplementing POPC and TOC in CPA on the post-thaw oyster larvae at both cellular and molecular levels. Although no difference was found between Base and LA CPA treatments in the global DNA damage or lesions on DNA regions evaluated, genes expressed differently at the trochophore and umbo larvae stages among treatments. In comparison with controls, upregulated expression was detected in post-thaw trochophore larvae in genes related to antioxidant defence, energy metabolism, unfolded protein response (UPR), and apoptosis in either CPAs, whereas downregulation of genes associated with UPR and apoptosis in post-thaw umbo larvae. At the umbo larvae stage, the expression of genes related to FA synthesis, stress resistance, and apoptosis became similar between the LA CPA treatment and fresh controls. In contrast, these genes were significantly downregulated in the Base CPA treatment. These results indicate that exogenous lipids and antioxidants could alleviate cryopreservation impacts and improve post-thaw larval performance.
Overall, this thesis has refined the current cryopreservation protocols to improve the post-thaw performance in Pacific oyster larvae and Mediterranean mussel oocytes. The results also indicate that including exogenous lipids and antioxidants in CPA would provide a new approach for developing oocyte and larval cryopreservation techniques in bivalve species. This thesis has deepened our understanding of the cryodamage mechanism from the perspectives of FA profile response, DNA integrity, and gene expression.
Keywords: Magallana gigas; Mytilus galloprovincialis; oocyte; larva; cryopreservation; lipid; antioxidant; cryoprotectant agent; fatty acid profile; DNA damage; gene expression
Subject: Aquaculture thesis
Thesis type: Doctor of Philosophy
Completed: 2024
School: College of Science and Engineering
Supervisor: Professor Jianguang Qin