Yolk vitamin E prevents oxidative damage in gull hatchlings

Oxidative stress experienced during early development can negatively affect diverse life-history traits, and organisms have evolved complex defence systems against its detrimental effects. Bird eggs contain maternally derived exogenous antioxidants that play a major role in embryo protection from oxidative damage, including the negative effects on telomere dynamics. In this study on the yellow-legged gull (Larus michahellis), we manipulated the concentration of vitamin E (VE) in the egg yolk and analysed the consequences on oxidative status markers and telomere length in the hatchlings. This study provides the first experimental evidence that, contrary to the expectation, a physiological increase in yolk VE concentration boosted total antioxidant capacity and reduced the concentration of pro-oxidant molecules in the plasma, but did not reduce telomere attrition or ameliorate oxidative damage to proteins and lipids in the early postnatal period.


Introduction
Early-life development is characterized by rapid growth requiring high metabolic activity and oxygen consumption, which imposes notable reactive oxygen species (ROS) production [1], even if there is still ambiguity regarding the relationship between oxygen consumption and ROS production [2]. To efficiently counteract the detrimental effects of oxidizing molecules, organisms have evolved a complex antioxidant machinery, which relies on enzymatic and non-enzymatic defence. In oviparous species, embryo protection against ROS largely depends on egg exogenous non-enzymatic antioxidants of maternal origin [3]. However, a 2017 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.

Material and methods
The experiment was performed during March-May 2014 in a large breeding colony in the Comacchio lagoon (NE Italy, 44°20 N-12°11 E). Full details of the experiment are reported in [15] and in the electronic supplementary material. We aimed at increasing the yolk VE concentration (α-and γtocopherol mixture) by 1 standard deviation of that measured in eggs of gulls from the same colony [14] through a previously validated injection method. We adopted a within-clutch design whereby the VE dose due to be injected was tuned according to egg size at laying and position in the laying sequence. After VE injection, the nests were visited every day. At hatching a blood sample was collected for molecular sexing, oxidative status markers and TL analyses. Total antioxidant capacity (TAC) and the amount of pro-oxidant molecules (i.e. TOS) were measured according to colorimetric methods [16]. Protein carbonylation was assessed by western immunoblotting [16], while lipid peroxidation through the thiobarbituric acid reactive substances (TBARS) method [17]. It  compounds, apart from the main lipid peroxidation by-product malondialdehyde (MDA). Thus, TBARS results should be interpreted with caution because they may overestimate lipid peroxidation (LPO). TL was measured using the monochrome multiplex quantitative PCR method (MMQPCR) [18] and expressed as the ratio between the amount of telomeric repeats in the sample (T) and that of a single copy gene (S), relative to a reference sample (relative telomere length, RTL). All methods are fully described in the electronic supplementary material. We also verified the effectiveness of VE injection by measuring VE concentration in the yolk of some VE-injected eggs, which was always higher than that of sham-injected eggs (see electronic supplementary material for details). The effect of VE on investigated endpoints was analysed in linear mixed models (LMMs), including clutch identity as a random intercept effect. Egg treatment, embryo sex and egg-laying order were included as fixed-effect factors along with their two-way interactions. Egg mass at laying was included as a covariate in all models. All non-significant (p > 0.05) interaction terms were removed from the models in a single step. The effect of clutch identity was tested by the likelihood ratio test. Five chicks could not be sexed and were therefore excluded from all the analyses. Oxidative damage and TL analyses could not be assessed in some (2)(3)(4)(5)(6)(7)(8)(9) hatchlings. In all the analyses, we always used the largest sample available. Statistical analyses were performed by using SAS 9.3 PROC MIXED. Statistics are presented as estimated marginal means (EMMs) ± standard error (SE).

Results
An LMM showed that VE treatment caused a statistically significant increase in TAC in the hatchlings from VE-injected eggs compared to controls (EMM: controls: 1.27 (0.02); VE-treated: 1.36 (0.02); figure 1). Sex and laying order did not significantly predict TAC of the hatchlings (table 1). TOS levels were significantly lower in the plasma of chicks hatched from VE-injected eggs with respect to controls (control eggs: 0.429 (0.03); VE-injected eggs: 0.192 (0.03); figure 1). Both TAC and TOS significantly varied among broods (likelihood ratio test; χ 2 1 > 5.6, p < 0.017). No effect of VE treatment, sex, laying order and their interactions was found for protein carbonylation and lipid peroxidation in blood samples from hatchlings (table 1). Finally, VE supplementation did not significantly affect RTL, after controlling for the potentially confounding effects of sex and laying order (table 1). Separate LMMs of RTL where we included the markers of oxidative status as covariates did not reveal any significant effect (F < 2.04, p > 0.157 in all cases).

Discussion
The experimental increase in yolk VE concentration within physiological limits ameliorated plasma TAC and TOS, but this was not mirrored in a reduction in oxidative damage to proteins and lipids. In addition, VE supplementation did not affect TL, contrary to the expectation, stemming from the hypothesis of a negative effect of pro-oxidants on TL.
VE supplementation significantly increased plasma TAC and reduced TOS, confirming its crucial antioxidant role (figure 1). Similar effects were found in the plasma of hen chicks supplemented via the diet with supra-physiological VE doses [19], but are not consistent with those found in great tit nestlings, where neither plasma TAC nor TOS differed between experimental groups after administration of VE-enriched food [20]. Although our previous studies showed that VE supplementation exerted  positive effects on morphological traits of chicks hatched from third-laid VE-injected eggs [15], the significant effect on TAC and TOS was independent of egg laying order, suggesting that all chicks benefited from VE supplementation. However, we did not detect any effect of yolk VE increase on oxidative damage to proteins and lipids according to previous studies of wild birds [1,21]. Contrary to the expectation, VE treatment had no effect on TL in red blood cells, despite having positive effects on oxidative status. Oxidative stress has been often invoked as a determinant of telomere attrition, but no experimental study to date has capitalized on the advantages of the avian eggs as a cleidoic environment amenable to controlled manipulation of the level of antioxidants in the prenatal environment. While in ovo corticosterone injection caused ROS overproduction and telomere shortening in domestic chickens at 21 days [22], no experimental study of birds has tested for the effect of prenatal antioxidants on TL at the end of the embryonic stage, when telomere attrition is believed to have already progressed. These findings are the first experimental evidence that VE egg supplementation, mimicking physiological variation in maternal transfer to the egg, does not affect TL at hatching. Postnatal dietary supplementation of VE and vitamin C in the yellow-legged gull has also been shown to have no effect on TL of 7-day-old chicks [23]. These results combined suggest that availability of egg maternal and dietary VE has little influence on telomere dynamics in early life stages. However, such effects may become apparent at a later life stage, as shown for blue tit nestlings where the positive effect of a oneshot treatment with VE and methionine via subcutaneous injection on TL could be recorded 1 year after treatment [24].
Our study shows that a physiological increase in VE yolk concentration has positive effects in terms of plasma TAC and reduction in TOS but has no effect on oxidative damage or TL at hatching. This suggests that maternal allocation of VE to the egg is not limiting to protection from oxidative damage and any reduction of TL during prenatal life. However, we cannot exclude that the improvement of oxidative status of hatchlings due to the increase of VE concentration may result in positive effects in later life stages. Although TL at birth is considered an important predictor of fitness-related traits, telomere attrition can be more intense during postnatal growth. Thus, the availability of maternally transferred dietary antioxidants during early life may have long-term consequences by alleviating the costs of stressful conditions experienced during growth and preventing telomere attrition and the subsequent age-related risk factors for disease and increased risk of mortality.