Primary sex ratios in birds: problems with molecular sex identification of undeveloped eggs

Abstract

Sex allocation studies seek to ascertain whether mothers manipulate offspring sex ratio prior to ovulation. To do so, DNA for molecular sexing should be collected as soon after conception as possible, but instead neonates are usually sampled. Here, we aim to identify and quantify some of the problems associated with using molecular techniques to identify the sex of newly laid avian eggs. From both fertilized and unfertilized chicken (Gallus gallus) eggs, we sampled (1) the blastoderm/disc, (2) vitelline membrane and (3) a mixture of (1) and (2). Thus, we replicated scenarios under which contaminated samples are taken and/or unfertilized eggs are not identified as such and are sampled. We found that two commonly used molecular sexing tests, based on the CHD-1 genes, differed in sensitivity, but this did not always predict their ability to sex egg samples. The vitelline membrane was a considerable source of maternal and probably paternal contamination. Fertile eggs were regularly assigned the wrong sex when vitelline membrane contaminated the blastoderm sample. The membrane of unfertilized eggs was always female, i.e. maternal DNA had been amplified. DNA was amplified from 47 to 63% of unfertilized blastodiscs, even though it was highly unlikely that DNA from a single haploid cell could be amplified reliably using these polymerase chain reaction (PCR) techniques. Surprisingly, the blastodiscs were identified as both males and females. We suggest that in these cases only maternal DNA was amplified, and that 'false' males, Z not ZZ, were detected. This was due to the reduced ability of both sets of primers to anneal to the W chromosome compared to the Z chromosome at low DNA concentrations. Overall, our data suggested that estimates of primary sex ratios based on newly laid eggs will be appreciably inaccurate.

Sensitive males: inbreeding depression in an endangered bird

“…An additional consideration in using visually identified failed embryos was to obtain enough reliable DNA for amplification. Even if early death embryos could be identified, there is a risk of contamination from parental DNA in the vitelline membrane (Arnold et al 2003). Each category was analysed separately using a generalized linear mixed model with a binomial response variable (dead/ alive), and inbreeding (continuous variable), sex, clutch size at laying point, maternal inbreeding level and maternal age as potential explanatory variables.…”

Fluorescent in situ hybridization for sex chromosome determination before and after fertilization in mice

“…Detection of DNA sequences specific to X and Y chromosomes through the use of PCR has been employed frequently as a method of gender determination in isolated preimplantation or postimplantational conceptuses [14][15][16]. This technique is particularly effective for processing large numbers of samples, but the potential exists for erroneous sex identification due to selective amplification of X chromosome genes from maternal uterine tissue that inadvertently occurs in species with highly invasive placentation, such as in rodents and humans [17,18] or where preferential amplification of one allele occurs relative to the other [19].…”

Manipulation of primary sex-ratio: an updated review

“…Badyaev et al (2005) were able to demonstrate that pre-ovulation oocytes producing males or females can differ in their growth rates. Using house finches (Carpodacus mexicanus), they analysed the temporal pattern of yolk deposition by the measurement of the lipid layers present in the yolk (see description in Young and Badyaev, 2004) and also carrying out early embryo sexing (but see Arnold et al, 2003a for limits of early sexing techniques). Their results also challenged the traditional view that a strict hierarchical order of oocyte sequestration into the RYD phase exists, and that such order should be maintained at the time of ovulation because growth rate would not differ among oocytes ( Figure 1A; King, 1973).…”

“…However, many of these eggs could have contained embryos that had died at the first stages of development. Sexing undeveloped embryos is very difficult, leading to errors due to contamination from maternal tissues (see Arnold et al, 2003a). In vitro experiences could overcome this limitation.…”