Parental effects occur whenever the phenotype of parents or the environment that they experience inﬂuences the phenotype and ﬁtness of their offspring. In birds, parental effects are often mediated by the size and biochemical quality of the eggs in terms of maternally transferred components. Exogenous antioxidants are key egg components that accomplish crucial physiological functions during early life. Among these, vitamin E plays a vital role during prenatal development when the intense metabolism accompanying rapid embryo growth results in overproduction of pro-oxidant molecules. Studies of captive birds have demonstrated the positive effect of vitamin E supplemen- tation on diverse phenotypic traits of hatchling and adult individuals, but its effects on embryo phenotype has never been investigated neither in captivity nor under a natural selection regime. In the present study, we experimentally tested the effect of the in ovo supplementation of vitamin E on morphological traits and oxidative status of yellow-legged gull (Larus michahellis) embryos. The supplementation of vitamin E promoted somatic growth in embryos soon before hatching, but did not affect their oxidative status. Our results suggest that maternally transferred vitamin E con- centrations are optimized to prevent imbalances of oxidative status and the consequent raise of oxidative damage in yellow-legged gull embryos during prenatal development. Key words: Larus michahellis, maternal effects, morphological traits, oxidative status, prenatal period, vitamin E. Parents can maximize their Darwinian fitness by modulating the allo- Groothuis et al. 2006; Rubolini et al. 2011; von Engelhardt and cation of care to individual offspring according to their reproductive Groothuis 2011). Mothers may adaptively tune their investment, value. In oviparous organisms, mothers can adjust offspring pheno- including prenatal maternal effects via eggs to individual offspring. type via the modulation of the size and biochemical quality of their Exogenous antioxidants (e.g., vitamins and carotenoids) are eggs, which can widely vary not only among mothers but also among acquired with food, and may be available in limiting amounts, sibling eggs (Mousseau and Fox 1998; Saino et al. 2002; Groothuis implying that mothers may tune the amount of antioxidants they et al. 2005). In fact, the size and the concentration of quantitatively transfer to their eggs according to the reproductive value of their in- major (e.g., lipids, albumen) and minor (e.g., steroid hormones, vita- dividual offspring as determined, for example, by hatching order mins) maternally transferred components often vary within-clutch ac- (Grether et al. 2001; Catoni et al. 2008). The existence of reproduct- cording to laying order (Royle et al. 2001; Badyaev et al. 2006; ive trade-offs and the major role that antioxidants have in early-life V C The Author (2017). Published by Oxford University Press. 285 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact firstname.lastname@example.org Downloaded from https://academic.oup.com/cz/article-abstract/64/3/285/3848950 by Ed 'DeepDyve' Gillespie user on 17 June 2018 286 Current Zoology, 2018, Vol. 64, No. 3 offspring physiology have attracted considerable attention to the natural selection regimes is scanty and to date no study has investi- study of ecology and evolution of maternal effects mediated by egg gated the effects on embryonic growth or oxidative status in import- antioxidants. Egg antioxidants of maternal origin provide protection ant organs that are likely to be the target of the antioxidant activity of vitamin E. to the developing embryo against the detrimental effects of free rad- In a recent study, we have shown that a physiological increase of icals produced during early-life growth (Surai et al. 1996). Low lev- vitamin E concentration in yellow-legged gull Larus michahellis els of maternally transferred yolk antioxidants impair embryonic eggs enhanced postnatal body size of chicks from the last-laid eggs development (Wilson 1997), suggesting that they play a crucial role in a clutch (Parolini et al. 2015). However, information on the ef- in counteracting oxidative stress (Surai and Speake 1998; Blount fects of vitamin E during the prenatal period in free-living species is et al. 2000; McGraw et al. 2005). In fact, intense metabolic activity largely unavailable. For this reason, here we investigate the effect of during early developmental stages exposes the organism to oxidative a physiological increase in yolk vitamin E concentration on pheno- stress, resulting from the breakdown of the equilibrium between the typic traits of embryos shortly before hatching. We expected that production of pro-oxidants (reactive oxygen and nitrogen species, the supplementation of vitamin E would promote growth, positively ROS and RNS, respectively), and antioxidant defense and repair affect oxidative status, and reduce embryo oxidative damage. In mechanisms in favor of the former (Finkel and Holbrook 2000). The addition, because vitamin E concentration declines with laying order prenatal period is crucial to redox homeostasis because high meta- (Rubolini et al. 2011) and in our previous study we showed that it bolic rates during rapid growth stages can induce ROS overproduc- limits postnatal growth of chicks from third-laid eggs, we expected a tion (Rollo 2002), leading to oxidative damage to cellular decrease of pro-oxidant molecules accompanied by an increase of macromolecules (i.e., DNA, lipids, and proteins) and providing a total antioxidant capacity (TAC) mainly in embryos from last-laid potential mechanism for negative effects on fitness-related traits vitamin E-injected eggs. Lastly, although the concentration of vita- (Costantini 2014). Because of the adverse consequences of oxidative min E in the yolks of yellow-legged gull eggs does not vary according stress on phenotype, selection is expected to favor the evolution of to the sex of developing embryos (Rubolini et al. 2011), we also mechanisms for antioxidant defense and repair of oxidative damage tested if the effect of egg treatment depended on the sex of the em- (Costantini et al. 2010; Metcalfe and Alonso-Alvarez 2010; bryo because embryos of either sex may differ in their susceptibility Isaksson et al. 2011; Metcalfe and Monaghan 2013; Costantini to yolk antioxidants (Romano et al. 2008). Thus, we studied the ef- 2014). Variation in oxidative stress (Monaghan et al. 2009, fects of vitamin E on embryo morphology (body mass and tarsus Metcalfe and Alonso-Alvarez 2010) and in maternal transfer of anti- length) and oxidative status by measuring TAC, amount of pro-oxi- oxidants depending on environmental conditions experienced by the dant molecules (called as ‘TOS’ 128 according to the terminology by mother (Blount et al. 2002; Royle et al. 2003) suggests that the re- Erel 2005) lipid peroxidation (LPO) and protein carbonylation sponse to oxidative stress may be modulated by maternal effects. (PCO) in brain and liver explanted from the embryos. We focused Therefore, maternal allocation of exogenous antioxidants to egg on brain for 3 reasons; it is particularly sensitive to LPO because the yolk may constitute a strategy to minimize oxidative damage to de- phospholipids of the neuronal membranes contain large amounts of veloping embryos (Blount et al. 2002). highly polyunsaturated fatty acids, it generates free radicals at a In birds, vitamin E is one of the most important yolk antioxi- greater extent than other tissues as a consequence of high rates of dants (Surai et al. 2016). Vitamin E is transported from the yolk to energy metabolism and oxygen consumption, and the amount of the embryonic tissues during development (Surai et al. 1996; Cherian and Sim 2003) and protects embryos against the toxicity of many exogenous antioxidants is lower compared to other tissues free radicals (Khan et al. 2011). Vitamin E acts as chain-breaking (Surai et al. 1999b). Liver was chosen because it is the main reposi- lipid antioxidant and free radical scavenger in the membranes of tory of antioxidants, including vitamin E (Surai 2002), and it has a cells and subcellular organelles (Young et al. 2003), maintaining the crucial role in antioxidant defense. integrity and functioning of the reproductive, muscular, circulatory, nervous, and immune systems of vertebrates (Leshchinsky and Klasing 2001). The effects of egg vitamin E have been mostly investi- Materials and Methods gated by means of maternal dietary supplementation in captivity. Field and experimental procedures These studies have shown that vitamin E supplementation positively The yellow-legged gull is a monogamous species that breeds mostly affects growth, immune function, performance, and antioxidant colonially (Cramp 1998). Clutch size ranges between 1 and 3 eggs capacity of poultry (Gore and Qureshi 1997; Surai et al. 2001; (modal size¼ 3), which are laid at 1–4 (most frequently 2) days inter- Bhanja et al. 2012; Selim et al. 2012; Goel et al. 2013), as well as vals and hatch 27–31 days after laying. Hatching is asynchronous and the transcription and the expression of specific genes involved in di- spans over 1–4 days. The chicks are semi-precocial and are fed by verse metabolic pathways (Surai 2002). Experiments in captivity both parents and fledge at 35–40 days of age (Cramp 1998). We where egg vitamin E has been manipulated by injection have partly studied a large colony (>400pairs) breeding on anisland in the clarified its direct effects on offspring phenotype. Direct manipula- 0 0 Comacchio lagoon (NE Italy, 44 20 N–12 11 E) in March–May tion of yolk vitamin E levels improved hatchability, immune status, 2014. The colony was monitored every other day and when a new and both embryonic and post-hatch growth of Muscovy ducks nest was found the newly laid egg was temporarily removed and Cairina moschata (Selim et al. 2012), and reduced the production of replaced with an egg collected from a nest outside of the study colony ROS in tissues of hen chicks (Cherian and Sim 1997; Surai et al. (i.e., “dummy” egg) to avoid interference with parental incubation 1999a). Although these experiments are valuable to identify the ef- behavior. Nests that were found with more than 1 egg were con- fects and mechanisms behind the allocation of antioxidants to eggs, sidered, but egg order was estimated based on previously described the most insightful perspective for the interpretation of the evolution differences in egg mass for the species. The removed egg was marked of maternal effects rests on the experimental analysis of the conse- quences of egg quality manipulation under a natural selection re- and taken to a nearby tent for experimental manipulation. gime in the wild. However, information on yolk vitamin E effects The experimental design has been described in details by Parolini derived from yolk manipulation in free-ranging populations under et al. (2015) and in the Supplementary material, therefore it is only Downloaded from https://academic.oup.com/cz/article-abstract/64/3/285/3848950 by Ed 'DeepDyve' Gillespie user on 17 June 2018 PAROLINI et al. – Vitamin E effects on gull embryo phenotype 287 briefly summarized here. Our objective was to increase the concen- Analysis of vitamin E content in residual yolk sac, brain, tration of vitamin E by 1 standard deviation (SD) of the concentra- and liver of embryos tions measured in the egg yolk of individuals from the same colony The concentration of vitamin E in residual yolk sac, brain, and liver in a previous study (Rubolini et al. 2011). Since the concentration of from embryos was determined according to Karadas et al. (2006) vitamin E in the yolk of yellow-legged gull eggs varied according to using a high-performance liquid chromatography system (Shimadzu egg size and position in the laying sequence (Rubolini et al. 2011), Liquid Chromatography, LC-10AD, Japan Spectroscopic Co. Ltd.). we adjusted the injection dose according to these factors. We esti- Briefly, 100–150 mg of yolk and organs were homogenized with mated yolk mass based on total egg mass for each of the eggs in lay- 1 mL of ethanol plus 0.7 mL NaCl 5% and extracted twice by centri- ing sequence based on a Linear Mixed Model from previously fugation with 2 mL of hexane each. Then, hexane extracts were collected yellow-legged gull eggs (yolk mass¼ 0.227 (0.039 SE) egg pooled and evaporated at 60–65 C under nitrogen flow and the re- massþ 1.815 (3.461 SE); F ¼ 34.38, P< 0.001). Then, we 1,88 sidual was dissolved in 500 mL of a dichloromethane:methanol mix- grouped first (a-), second (b-), or third (c-) laid eggs into 3 classes ture (50:50 v/v). Vitamin E (a-, and c-tocopherol) concentrations (tertiles) of size according to egg mass and calculated the standard were detected with a Hypersil GOLD type 3 mm C18 reverse-phase deviation of vitamin E concentration in the yolk for each tertile. The column (150 4.6 mm Phase Separation, Thermo Fisher Scientific injection amount of vitamin E was computed as the product of the 81, Wyman, Street Waltham, MA USA) with a mobile phase of SD (in mgg ) of vitamin E concentration for each tertile and pos- methanol:distilled water (97:3 v/v) at a flow rate of 1.05 mL min ition in the laying sequence and the estimated yolk mass (see using fluorescence detection by excitation and emission wavelength Supplementary material). Corn oil was used as the carrier solvent of of 295 nm and 330 nm, respectively. Peaks of a-, and c-tocopherol vitamin E and it was used as a control treatment in the control group were identified and quantified by comparison with the retention of eggs. We adopted a within-clutch design, whereby both sham- time of standards of tocopherols at renown concentration (Sigma, (control) and vitamin E-injected eggs were established within each Poole, UK). According to Karadas et al. (2006), standard solutions clutch to minimize the confounding effects of environmental and a-tocopherol in methanol were used for instrument calibration, parental effects. The following treatment schemes were assigned se- while tocol was used as an internal standard to check for the reliabil- quentially to the clutches as follows: (nest, a-, b-, c-egg): nest 1, vita- ity of analytical process. min E injection (E), control injection (C), E; nest 2, C-E-C; nest 3, E- C-C; nest 4, C-E-E and so forth with the following nests. The injec- Oxidative stress methods tion procedure was performed according to a previously validated TAC, TOS, PCO and LPO were measured in liver and brain hom- method on eggs from the same species (Romano et al. 2008). ogenates. In addition, TAC was also measured in the residual yolk After the in ovo vitamin E supplementation and 5 days before from sampled eggs. the earliest expected hatching date, all the nests were visited daily to An appropriate amount of yolk (0.15 g), brain, and liver check for any sign of imminent hatching such as eggshell fractures (0.1 g) was homogenized in 100 mM phosphate buffer pH 7.4, (i.e., “cracking stage”). When eggshells were fractured, eggs were with 1 mM EDTA and 100 mM KCl, by an automatic homogenizer. weighed (to the nearest g), collected and frozen at 20 C within 3 h After 10 min centrifugation at 13,000 rpm, an aliquot of the super- from sampling. natant was immediately processed for the determination of protein Field collected eggs (n¼ 76 eggs) were transferred to the lab content according to the Bradford method (Bradford 1976) using where they were dissected. We focused on 26 clutches, 15 of which bovine serum albumin (BSA) as a standard, while the remainder was had 3 eggs, while the remaining 11 clutches had 2 eggs only. We first used for oxidative stress assays. A detailed description of applied removed and weighed the residual yolk sac from each egg, which methods is reported in Supplementary material. Briefly, TAC was was frozen at 80 C until the analysis of total vitamin E concentra- measured according to a colorimetric method based on the discolor- tion and TAC that we performed as a validation of the experimental ation of 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) rad- treatment. We expected that vitamin E injection would result in a ical cation (ABTS* ), adapted from Erel (2004). TOS was measured measurable increase in the yolk concentrations into late develop- according to a colorimetric method developed by Erel (2005), mental stages, as well as in an increase in TAC. Then, the embryos adapted to tissue homogenates. Carbonylated proteins were meas- were weighed (to the nearest g) and tarsus length was measured by ured with 2,4-dinitrophenylhydrazine (DNPH). Protein carbonyla- calipers before the dissection of liver and brain, which were immedi- tion (PCO) was measured by Western immunoblotting and ately weighed (to the nearest mg) and frozen at 80 C until bio- immunostained protein bands were visualized with enhanced chemi- chemical analyses. All the measurements were taken by the same luminescence detection. Carbonylated proteins were quantified by person to ensure consistency. Molecular sexing of embryos and densitometric analysis using Image J 1.40d software (Schneider et al. chicks was performed according to Saino et al. (2008). 2012). LPO was measured according to the thiobarbituric acid re- The study was carried out under permission of the Parco active substances (TBARS) method (Ohkawa et al. 1979), adapted Regionale del Delta del Po (#657, 4 February 2014), which allowed to tissue homogenates of embryos and were expressed as nmol both the manipulation and the collection of eggs when the eggshell TBARS g wet weight. showed signs of imminent hatch (eggshell fractures). According to the Guidelines for the Euthanasia of Animals by the American Veterinary Medical Association, physical methods of euthanasia Statistical analyses may be necessary in some field situations if other methods are im- The effect of vitamin E treatment on its concentration in residual practical or impossible to implement. We performed a field experi- yolk sac and embryo focal organs, embryo morphological traits and ment in which we could not euthanize embryos by methods such as oxidative status markers, was analyzed in Linear Mixed Models carbon dioxide (CO ), anesthetic agents, or decapitation. Thus, we (LMM; Normal as the distribution and Identity as the link function), euthanized embryos by placing eggs into a 20 C freezer within 3 h including clutch identity as a random intercept effect. Egg mass at from the collection. the time of laying was included as a covariate in all the models. Downloaded from https://academic.oup.com/cz/article-abstract/64/3/285/3848950 by Ed 'DeepDyve' Gillespie user on 17 June 2018 288 Current Zoology, 2018, Vol. 64, No. 3 Egg treatment (vitamin E versus sham-injection), embryo sex, and egg-laying order were included as fixed-effect factors along with their two-way interactions. All non-significant (P> 0.05) interaction terms were removed from the model in a single step. In all the mod- els, the effect of clutch identity was tested by a likelihood ratio test, by comparing the log-likelihood value of the model including or excluding the random effect of clutch identity. Mixed models with the same design, but assuming a binomial error distribution, were run to investigate the effects of vitamin E treatment on the propor- tion of eggs that reached the “cracking stage”, as well as on the sex ratio of embryos. A single embryo could not be dissected because of sample deterioration. All the statistical analyses were performed by using SAS 9.3 PROC MIXED and PROC GLIMMIX. Group statis- tics are presented as estimated marginal means (6 SE). Results Vitamin E concentration in residual yolk sac, embryo brain, and liver To assess the reliability of the injection procedure in causing an in- crease in vitamin E yolk concentration, and consequently on yolk TAC, we first analyzed whether the concentration of vitamin E and TAC in the residual yolk sac differed between sham- and vitamin E- injected eggs. We used the yolk sac samples from 66 embryos (n¼ 26 nests). As expected, vitamin E concentration was significantly higher in vitamin E treated eggs compared to controls (F ¼ 4.314; 1,44.5 P¼ 0.044) (Figure 1A). Neither sex (F ¼ 1.162; P¼ 0.286) nor lay- 1,38 Figure 1. Marginal means (þ SE) of (A) concentration of total vitamin E (mgg ing sequence (F ¼ 0.841; P¼ 0.438) affected yolk sac vitamin E 1,36 wet weight) and (B) total antioxidant capacity (TAC - mM Trolox Eq. g wet concentrations. In addition, we estimated the total amount of vitamin weight) in the residual yolk sac from the embryos at the cracking stage. E in the yolk as the product of vitamin E concentration (expressed in Sample sizes are reported. Signiﬁcant differences between vitamin E and con- mg/g) and the yolk mass estimated according to the relationship trol embryos are indicated by the asterisk (*P< 0.05). described above (see Materials and Methods Section). The total amount of yolk vitamin E was significantly higher in vitamin E treated (F ¼ 0.40, P ¼ 0.530) but significantly declined with laying order 1,36 eggs compared to controls (F ¼ 4.623; P¼ 0.037), while neither 1,44.3 (F ¼ 36.41, P< 0.001; estimated marginal means (SE): a-eggs: 2,36 sex (F ¼ 0.905; P¼ 0.346) nor laying sequence (F ¼ 0.899; 1,50.7 1,48.7 91.8 (1.25); b-eggs: 90.1 (1.21); c-eggs: 83.8 (1.28)). The sex ratio P¼ 0.413) affected mass of vitamin E in the yolk. Accordingly, vita- (proportion of males) did not differ significantly between the experi- min E supplementation caused a significant increase of TAC in re- mental groups (controls: 10/30¼ 0.333; 95% confidence inter- sidual yolk sac (Figure 1B; F ¼ 4.298; P¼ 0.045), while no 1,36 val¼ 0.164–0.502 and vitamin E: 19/36¼ 0.528; 95% confidence significant effect of embryo sex (F ¼ 0.01; P¼ 0.929) or laying 1,38 interval¼ 0.365–0.691; v ¼ 1.78, P ¼ 0.182). order (F ¼ 1.25; P¼ 0.298) was found. Since vitamin E is effi- 1,36 A LMM of embryo body mass showed no significant effect of ciently transferred from yolk to developing embryos, we also meas- the interactions among fixed-effect factors (Table 1; Figure 2). The ured its concentration in brain and liver. The concentrations of reduced model, retaining the main effects of sex, treatment and lay- vitamin E measured in focal organs from vitamin E-treated embryos ing order, showed a statistically significant difference in body mass soon before hatching were not significantly higher than controls in of embryos between the experimental groups (F ¼ 4.19, 1,38 the brain (F ¼ 1.707; P¼ 0.196) or in the liver (F ¼ 0.305; 1,59 1,35 P ¼ 0.048; control embryos: 43.2 (1.03) and vitamin E embryos P¼ 0.584), and did not vary according to sex (brain: F ¼ 0.083; 1,59 45.0 (0.99)). There was large among-clutch variation in body mass P¼ 0.775; liver: F ¼ 0.107; P¼ 0.746 for liver), laying order 1,38 (Likelihood ratio test; v ¼ 21.00, P< 0.001). No significant effect (brain: F ¼ 0.066; P¼ 0.936; liver: F ¼ 2.561; P¼ 0.089) or 1,35 2,42 on tarsus length was found (Table 1). LMM of embryo liver and their interactions (all P> 0.05), which were removed from the LMM. brain mass revealed no significant effect of vitamin E treatment (liver: F ¼ 0.17; P¼ 0.678; brain: F ¼ 0.08; P ¼ 0.784). 1,38 1,39 No significant effect of vitamin E treatment, embryo sex, laying Effect of vitamin E on embryo morphology and order, and their interactions was found for all the considered oxida- oxidative status tive stress endpoints in both the target organs, with the exception The sample included 26 clutches, 15 of which had 3 eggs while the for a significant effect of laying order on TOS in the liver, and of sex remaining 11 clutches had 2 eggs. The proportion of eggs that on TOS in the brain (Table 2). reached the cracking stage did not differ significantly between the control (proportion of eggs at cracking¼ 30/66¼ 0.455; 95% confi- dence interval¼ 0.335–0.575) and the vitamin E-injected eggs (36/ 2 Discussion 66¼ 0.545; 95% confidence interval¼ 0.424–0.575; v ¼ 0.76, P ¼ 0.384). In a LMM where clutch identity was included as a ran- We experimentally increased vitamin E concentrations within dom effect, egg mass did not differ between the experimental groups physiological limits in yolks of yellow-legged gull eggs and found Downloaded from https://academic.oup.com/cz/article-abstract/64/3/285/3848950 by Ed 'DeepDyve' Gillespie user on 17 June 2018 PAROLINI et al. – Vitamin E effects on gull embryo phenotype 289 Table 1. LMM of morphological traits of embryos at the cracking estimated yolk size and to position in the laying sequence. Thus, we stage in relation to vitamin E treatment, sex of the embryo, and lay- are confident that our vitamin E supplementation caused a post- ing order. Clutch identity was included in the model as a random manipulation concentration that did not exceed the upper limit of intercept effect. We controlled for egg mass at the time of laying by the natural range of variation, at least in the vast majority of the including it as covariate in the models. The non-signiﬁcant effects eggs. of the 2-way interactions were excluded from the ﬁnal model. The injection of a physiological dose of vitamin E into the yolk Signiﬁcant effects are reported in bold caused an increase in embryo body mass around hatching, independ- Morphological traits Body mass Tarsus length ently of egg laying order and mass of the original egg. Since the con- centration of vitamin E in yellow-legged gull eggs from the same F dfP F dfP colony declines with laying order, showing a 1.6-fold difference be- tween the second- and third-laid eggs (Rubolini et al. 2011), a more Final model Treatment 4.19 1, 38 0.048 1.54 1, 38 0.222 marked positive effect of vitamin E supplementation on somatic Sex 0.34 1, 41 0.565 0.64 1, 42 0.430 growth of embryos from third-laid eggs was expected. In fact, previ- Laying order 1.68 2, 47 0.198 2.01 2, 46 0.145 ous evidence showed that chicks hatched from third-laid eggs in- Excluded terms jected with vitamin E were heavier and had significantly longer tarsi Treatment sex 0.08 1, 48 0.777 0.37 1, 47 0.545 than controls, whereas vitamin E treatment had no effect on the size Treatment laying order 1.14 2, 51 0.329 0.49 2, 50 0.613 of chicks from first- or second-laid eggs (Parolini et al. 2015). These Sex laying order 2.83 2, 49 0.069 1.61 2, 50 0.210 positive effects on morphological traits of chicks from the third-laid eggs suggest that the concentration of vitamin E in first- and second- laid eggs at hatchling is close to optimal, whereas in the third-laid eggs is sub-optimal. In contrast, the results from embryos suggest that during pre-hatching development the concentration of vitamin E in the yolk might be sub-optimal for somatic growth, and the ad- ministration of an additional dose is beneficial to growth independ- ent of position in the laying sequence. From a functional perspective, maternal allocation of vitamin E to the eggs may serve to increase body size in late prenatal stages and to enhance post-hatching growth. Yet, the mechanisms underly- ing the positive effect of vitamin E supplementation on embryo (and chick) body size remains to be elucidated. Although no information is available for embryos of any bird species, vitamin E may increase the efficiency of conversion of egg materials into somatic tissues, as suggested for commercial Muscovy ducks during the first 2 weeks Figure 2. Marginal means (þ SE) of body mass (g) and tarsus length (mm) at after hatching (Selim et al. 2012). Alternatively, vitamin E supple- the cracking stage of embryos from control or vitamin E injected eggs. mentation may reduce the production of pro-oxidant molecules, pre- Sample sizes are reported. Signiﬁcant differences between vitamin E and venting oxidative stress. During early developmental stages, control embryos are indicated by the asterisk (*P< 0.05). embryos and chicks are particularly prone to suffering oxidative stress because of high metabolic rates and the onset of aerobic res- that the supplementation of this exogenous antioxidant promoted piration at hatching, implying that they need efficient antioxidant growth of embryos at late prenatal stages, while it did not affect oxi- protection particularly during the late embryo and the early post- dative status of their brains or livers. hatching stages (Panda and Cherian 2014). The overproduction of A number of studies of captive and free-living birds have shown pro-oxidants and the consequent oxidative imbalance should be det- that vitamin E supplementation via the maternal diet increased the rimental to developmental and growth processes (Smith et al. 2016). concentration of this antioxidant in the egg yolk and in embryonic The latter hypothesis is supported by a number of studies showing tissues, promoting somatic growth at hatching (Surai et al. 1999b; that vitamin E supplementation improves antioxidant defense Larcombe et al. 2010; Noguera et al. 2011; Surai and Fisinin 2013; increasing superoxide dismutase (SOD) and glutathione peroxidase Surai et al. 2016). However, these studies manipulated vitamin E (GPx) activity, preventing negative effects of LPO in broiler chicks availability to mothers and tested the effect of dietary vitamin E sup- (Sodhi et al. 2008; Tsai et al. 2008). Since oxidative stress has been plementation on the offspring. This approach integrates information suggested to limit growth rates (Alonso-Alvarez et al. 2007), on the direct effect of maternal vitamin E on progeny with indirect enhanced body mass in vitamin E-treated embryos (and hatchlings) effects mediated by the consequences of increased availability of likely reflects the antioxidant properties of tocopherols (Marri and dietary vitamin E on maternal physiology. In contrast, our in ovo in- Richner 2015).Thus, vitamin E may protect lipid membranes from jection approach reveals the direct effects of vitamin E on the off- the harmful effects of ROS, allowing increased lipid utilization for spring, independently of maternal physiology (Surai et al. 1998; energy production (Schaal 2008) to be used in somatic growth. Blount 2004). In addition, several experiments (e.g., Cherian and However, the present results on oxidative status markers do not sup- Sim 1997; Surai et al. 1999a; Selim et al. 2012; Goel et al. 2013), port this interpretation. While TAC was found to be larger in re- mainly in captivity, applied supra-physiological vitamin E doses, sidual yolk from vitamin E-injected eggs (Figure 1B), TAC, TOS, hampering the ecological and evolutionary interpretation of mater- and oxidative damage to lipids and proteins in the brain and in the nal effects mediated by egg vitamin E content. In designing our ex- liver were not affected by vitamin E supplementation (Table 2). The periment we, therefore, paid special attention to scale the injection lack of significant effects on oxidative status markers may depend amount of vitamin E to natural variation, as well as according to on the amount of residual yolk in eggs at the cracking stage. Thus, Downloaded from https://academic.oup.com/cz/article-abstract/64/3/285/3848950 by Ed 'DeepDyve' Gillespie user on 17 June 2018 290 Current Zoology, 2018, Vol. 64, No. 3 Table 2. LMM of oxidative status markers in the liver and brain of embryos at the cracking stage in relation to vitamin E treatment, sex of the embryo, and laying order. Clutch identity was included in the model as a random intercept effect. We controlled for egg mass at the time of laying by including it as covariate in the models. The non-signiﬁcant effects of the 2-way interactions were excluded from the ﬁnal model. Signiﬁcant effects are reported in bold Oxidative status markers TAC TOS PCO LPO F dfP F dfP F dfP F dfP Liver Final model Treatment 1.33 1, 37 0.257 0.16 1, 40 0.694 0.10 1, 38 0.750 1.45 1, 31 0.237 Sex 0.03 1, 40 0.861 0.15 1, 45 0.701 0.29 1, 41 0.595 0.68 1, 34 0.417 Laying order 1.07 2, 46 0.350 3.98 2, 48 0.025 0.25 2, 43 0.780 0.20 2, 39 0.816 Excluded terms Treatment sex 0.01 1, 45 0.938 0.75 1, 49 0.391 0.22 1, 42 0.642 0.62 1, 39 0.435 Treatment laying order 0.13 2, 49 0.880 0.14 2, 51 0.872 2.98 2, 46 0.061 0.36 2, 43 0.697 Sex laying order 0.65 2, 46 0.526 1.88 2, 52 0.163 1.98 2, 46 0.150 0.32 2, 38 0.729 Brain Final model Treatment 1.03 1, 37 0.316 0.26 1, 40 0.616 1.65 1, 41 0.206 2.30 1, 38 0.138 Sex 0.20 1, 41 0.657 6.24 1, 44 0.016 2.13 1, 42 0.152 0.20 1, 41 0.655 Laying order 0.01 2, 46 0.987 0.07 2, 48 0.932 0.73 2, 44 0.486 0.46 2, 47 0.636 Excluded terms Treatment sex 1.79 1, 47 0.187 0.96 1, 50 0.332 0.02 1, 49 0.902 0.32 1, 46 0.576 Treatment laying order 1.81 2, 50 0.174 1.91 2, 52 0.158 0.77 2, 46 0.468 0.12 2, 50 0.889 Sex laying order 2.11 2, 48 0.133 0.96 2, 53 0.390 0.24 2, 50 0.790 0.43 2, 48 0.650 the small amount of yolk adsorbed by the embryos up to the crack- concentration boosts embryonic somatic growth, consistent with pre- ing stage may have limited the transfer of an “effective” dose of vita- vious findings on hatchlings. The conspicuous differences in the ef- min E, able to affect the oxidative status and to reduce oxidative fects of maternal vitamin E on offspring phenotype occurring between damage of developing embryos. In fact, although the amount of vita- the prenatal and the early postnatal life stages, which may differ ac- min E was higher in the residual yolk sac of injected eggs compared cording to hatching order, should suggest that vitamin E is of primary to controls (Figure 1), no significant differences were measured in importance mainly during post-hatching periods. However, the par- brains or livers dissected from embryos. Indeed, our results may sug- tial inconsistency of the present results compared to some previous ex- gest that maternally transferred vitamin E concentration up to late perimental studies of birds suggests that further studies are required prenatal stages seems to be optimal in preventing the occurrence of to assess the role of this maternally transferred antioxidant during oxidative damage, and embryos may use the supplemental vitamin E early life periods under a natural selection regime. dose to promote somatic growth rather than to limit the detrimental consequences of oxidative stress. These findings are consistent with Supplementary material those reported in a study of red-winged blackbird Agelaius phoeni- ceus nestlings treated with an antioxidant-enriched diet (Hall et al. Supplementary material can be found at http://www.cz.oxfordjour 2010). The lack of positive consequences of increased vitamin E con- nals.org/. centration on oxidative status markers do not lessen the role of vita- min E in protecting embryo by oxidative stress during prenatal development. It simply suggests that vitamin E concentrations trans- Author contribution ferred from yolk to embryo tissues during pre-hatching development M. P. participated in field activity, performed biochemical analyses could show its beneficial effects after hatching. In fact, yolk vitamin and wrote the article; C. D. P. and G. C. performed biochemical E is effectively transferred to the embryo and its initial concentration analyses; F. K. performed analyses to assess vitamin E concentration determines the reserve of the chick at least for the first week post- in yolk eggs; M. R. and M. C. performed the field experiment; I. D. hatch (Surai et al. 1997). For instance, the highest concentrations of D. and A. M. supervised biochemical analyses and contributed writ- vitamin E in the liver occur at hatching and protect chicks from the ing the article; D. R. participated in field activity and helped to write adverse effects of oxidative stress for up to 2 weeks post-hatching the article; N. S. designed the experiment, helped to perform statis- (Surai et al. 1998). Thus, since newly hatched chicks are not able to tical analyses, and to write the article and supervised both field and effectively assimilate vitamin E from the diet and are dependent on laboratory work. their reserve built during embryonic development (Surai 2002), its accumulation in the embryo tissues, mainly in liver, is considered an adaptive mechanism providing antioxidant defense in the critical References time of hatching (Surai et al. 1996). Alonso-Alvarez C, Bertrand S, Faivre B, Sorci G, 2007. 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Current Zoology – Oxford University Press
Published: May 23, 2017
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