FERTILISATION ASSAYS
Our block cross-classified design involved a series of 2×2 in vitro fertilisation trials (following the analogous block North Carolina II quantitative genetic breeding design described by Lynch and Walsh (1998)), each involving two males (one from a high density and one from a low density site from the same location) crossed with two females (one from a high and one from a low density site from the same location) in every pairwise combination. For each male-female cross, two replicate fertilisation assays were carried out across each of the six different sperm concentrations, giving a total of 48 fertilisation assays per block. A total of five blocks were carried out for each of the three locations sampled, giving a total of 15 blocks and 720 fertilisation assays across all three locations. We conducted fertilisation assays in sterile 24-well cell culture plates with a total fertilisation volume of 2 ml. This consisted of 1 ml of the stock egg solution (2000 eggs total), and 1 ml of sperm solution, resulting in a final egg concentration of 1000 eggs ml-1 and sperm concentrations of: 1 × 102; 1 × 103; 1 × 104; 1 × 105; 1 × 106; and 1 × 108 sperm ml-1. All fertilisation assays within a block were conducted immediately after gamete standardisation and within 1 minute of each other although we also ensured that the time from the start of spawning to fertilisation was recorded and included in the analysis to control for potential gamete aging effects (‘gamete age’ in the model). Fertilisation assays were left at room temperature for 3 hours before samples were fixed with 1% formalin. For each fertilisation assay, a random sub-sample of approximately 100 eggs were observed under an inverted microscope at 400× magnification and the number of fertilised eggs were distinguished from unfertilised eggs by counting the number of cells that had undergone cell division, or had a clearly visible fertilisation envelope. Unfertilised eggs were identified from the lack of cell division or the absence of a fertilisation envelope (Sherman et al 2015). Abnormal eggs were identified by signs of irregular cleavage, incomplete blastula development and by dissolution of the egg membrane resulting in deformed embryos and/or the breaking apart of the developing cell cluster (Lewis and Galloway 2009).