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• physical examination of the egg exterior (eggshell) for the ‘white-spot’
• candling of the egg to visualize the embryo and/or extra- embryonic membranes
• physical examination of the egg contents for signs of embryonic development (e.g., embryo, blood, other tissues)
• microscopy or molecular methods to detect signs of sperm penetration of the ovum or syngamy

The decision tree and comparison table (Table 1) below complement Phillott & Godfrey (2020) and will help researchers who need to distinguish between fertile and infertile eggs. However, conclusions about infertility in a population should only be made with targeted studies utilizing rigorous sampling (see Phillott & Godfrey 2020).

Definitions of terms used:

Amplification of DNA - creating copies of genetic material (DNA) using laboratory techniques such as quantitative polymerase chain reaction (PCR) to allow analysis (see sequencing of DNA, below) of even very small samples.

Blastodisc - or germinal disc; the embryo-forming portion of an egg, which sits on the outer surface of the yolk.

Blastoderm - the first layer of cells that form as the fertilized blastodisc undergoes cleavage (division of cells).

Candling - a method of observing embryonic development by shining a bright light source through the egg from behind or below to illuminate the embryo, extra-embryonic membranes, and yolk. Candles were originally used as the light source and gave the process its name; torches/flashlights are more commonly used now.

Extra-embryonic membranes - membranes formed as tissue outgrowths of the embryo itself during development; the vitelline membrane or yolk sac, allantois, amnion, and chorion.

False positive – an error in which a positive result is mistakenly generated, e.g., microbe nuclei may be stained with the dye Hoechst 33342 and incorrectly identified as sperm heads. This could lead the researcher to wrongly conclude that eggs had been fertilized. The risk of a false positive can be overcome by use of multiple techniques, examination by multiple experts, and a suitable sample size.

Intra-oviducal - occurring in the oviduct, e.g., intra-oviducal embryo mortality.

Oviposition - the process of laying eggs.

Post-oviposition - after oviposition; after eggs are laid.

Perivitelline membrane (PVM) - the inner PVM encloses the yolk and the blastodisc. Sperm penetration of the inner PVM triggers formation of the outer PVM, so that polyspermy (fertilization of an egg by multiple sperm) does not occur. Sperm heads that are in contact with, but have not penetrated, the inner PVM when the outer PVM forms can become trapped between the two membranes.

Sequencing of DNA - any of a number of laboratory techniques can be used to determine the order in which nucleic acids (adenine, guanine, thymine, and cytosine) occur within DNA; a DNA sequence can then be compared within a genetic sequence database to confirm the species.

Shell membrane - the membrane located on the inner surface of the eggshell.

Stage of development - embryo development has been described by Miller et al. (2017) as a series of stages (1-31). Development in stages 1-5 occurs in the oviduct, and stages 6-21 in the nest after oviposition. Illustrations of the developmental stages are available in Miller et al. (2017), while photographs of stages 19-31 can be seen in Bladow & Milton (2019).

Syngamy - fusion of the sperm and ovum genetic material.

White-spot - a chalk-white spot that forms at the north pole of a fertile egg within 7 days of oviposition. When they are laid, eggs are usually translucent. After oviposition, the vitelline membrane comes in contact with the shell membrane at the top of the egg and draws water from it. Dehydration of the eggshell changes its appearance from translucent to chalk-white. As the embryo develops, the white spot grows in size to encompass the entire egg within ~20 days.

ABELLA, E., R.M. GARCÍA-CERDÁ & A. MARCO. 2017. Estimating the fertilization rate of sea turtle nests: comparison of two techniques. Basic Applied Herpetology 31: 33-44.

BIRKHEAD, T.R., J. HALL, E. SCHUT & N. HEMMINGS. 2008. Unhatched eggs: Methods for discriminating between infertility and early embryo mortality. Ibis 150: 508-517.

BLADOW, R.A. & S.L. MILTON. 2019. Embryonic mortality in green (Chelonia mydas) and loggerhead (Caretta caretta) sea turtle nests increases with cumulative exposure to elevated temperatures. Journal of Experimental Marine Biology and Ecology 518: 151180.

CROYLE, K., P. GIBBONS, C. LIGHT, E. GOODE, B. DURRANT & T. JENSEN. 2016. Chelonian perivitelline membrane-bound sperm detection: a new breeding management tool. Zoo Biology 35: 95-103.

EWERT, M.A. 1985. Embryology of turtles. In: Gans C., F. Billett & P.F.A. Maderson (Eds.). Biology of the Reptilia, Volume 14A. Development. Wiley-Interscience, New York. pp. 75-267.

MILLER, J.D. 1985. Embryology of marine turtles. In: Gans, C., F. Billett & P.F.A. Maderson (Eds.). Biology of the Reptilia, Volume 14A. Development. Wiley-Interscience, New York. pp. 269-328.

MILLER, J.D., J.A. MORTIMER & C.J. LIMPUS. 2017. A field key to the developmental stages of marine turtles (Cheloniidae) with notes on the development of Dermochelys. Chelonian Conservation & Biology 16: 111-122.

PHILLOTT, A.D. & M.H. GODFREY. 2020. Assessing the evidence of ‘infertile’ sea turtle eggs. Endangered Species Research 41: 329-338.

PHILLOTT, A.D. & C.J. PARMENTER. 2007. Deterioration of green sea turtle (Chelonia mydas) eggs after known embryo mortality. Chelonian Conservation & Biology 6: 262-266.

SAHOO, G., B.K. MOHAPATRA & S.K. DUTTA. 2009. Structural changes in olive ridley turtle eggshells during embryonic development. Herpetological Journal 19: 143-149.

Table 1a. Comparison of techniques to assess egg fertility at or shortly after oviposition.

Table 1b. Comparison of techniques to assess egg fertility during or after the incubation period.