Is maternal thyroid hormone deposition subject to a trade-off between self and egg because of iodine? An experimental study in rock pigeon

Non-genetic inheritance is defined as the transmission of information between generations beyond coding genes (Danchin et al., 2011). Parental effects are included in this non-genetic inheritance and may be considered adaptive (Moore et al., 2019; Mousseau and Fox, 1998), although the strength and ubiquity of adaptive parental effects is still under debate (e.g. Sánchez-Tójar et al., 2020; Yin et al., 2019; Zhang et al., 2020). Parental effects of maternal origin, i.e. maternal effects, have received increasing attention since the 1990s (Bernardo, 1996; Mousseau and Fox, 1998). Hormones of maternal origin can be transferred to the offspring and constitute a potential pathway for mothers to influence their offspring's phenotype (Groothuis et al., 2019). Hormone allocation to offspring could be costly for mothers as it could induce a trade-off between allocating hormones to their own metabolism or to their offspring's. Steroid hormones, the most studied hormones in the context of hormone-mediated maternal effects, may not be that costly to produce as they are derived from cholesterol, which is abundant in the organism (Groothuis and von Engelhardt, 2005). In contrast, thyroid hormones (THs) may be considered costly, as their molecular structure includes iodine, a trace element that cannot be synthesised by organisms and must therefore be found in the environment.

THs are metabolic hormones that are present in two main forms: thyroxine (T₄) that contains four atoms of iodine, and triiodothyronine (T₃) that contains three atoms of iodine. Iodine is concentrated in the thyroid gland and incorporated into tyrosines that will be combined to form T₄ and T₃ (McNabb and Darras, 2015). The thyroid gland produces mostly T₄ and lesser amounts of T₃. In the peripheral tissues (e.g. liver, kidney, muscle), T₃ is mostly obtained from T₄ via removal of an iodine atom by deiodinase enzymes (McNabb and Darras, 2015). TH action mainly depends on TH receptors that have a greater affinity for T₃ than for T₄ (Zoeller et al., 2007). This is why T₄ is mostly seen as a precursor of T₃, the biologically active form. THs play important roles in growth, reproduction, metamorphosis and thermoregulation, and the TH signalling pathway is well conserved throughout the animal kingdom, from invertebrates (Holzer et al., 2017; Taylor and Heyland, 2017) to vertebrates (reviewed by Ruuskanen and Hsu, 2018).

The main sources of iodine for terrestrial animals are via uptake of food, where plants absorb it from the soil, and via drinking water (Anke, 2004). Because iodine availability differs across environments and food sources (Anke, 2004; Boggs et al., 2011), THs may be costly to produce when iodine availability is limited. Low iodine diet in rodents under laboratory conditions generally decreases circulating THs (e.g. Santisteban et al., 1982; Zhimei et al., 2014; but see Bocco et al., 2020). Furthermore, in wild alligators (Alligator mississippiensis), overall higher plasma THs were reported in populations exposed to higher iodine concentrations compared with those in a low iodine habitat (Boggs et al., 2011, 2013). In breeding animals, this potential cost may induce trade-offs between allocating resources (i.e. iodine and THs) to themselves or to their progeny because embryos rely exclusively on THs of maternal origin during early development (humans: Stepien and Huttner, 2019; fish: Castillo et al., 2015; birds: Darras, 2019). In humans, TH deficiency arising from iodine deficiency can severely impair brain development and increase fetus mortality (reviewed by Zimmermann, 2009). Although the importance of THs has long been acknowledged, such a potential trade-off has never been studied.

Birds provide an excellent model organism to study hormone-mediated maternal effects and the associated trade-offs (Groothuis et al., 2019). Unlike mammals, avian embryos develop in eggs, outside of the mother's body, which facilitates the measurement of the actual embryonic exposure to maternal hormones deposited in egg yolks. It has been demonstrated that early avian embryos have all the necessary machinery (i.e. transporters, receptors, deiodinases) to make use of the THs deposited by mothers (reviewed by Darras, 2019). Maternally transferred thyroid hormones play crucial roles in the development of the central nervous system (Darras, 2019) and several other tissues and organs (e.g. muscles, heart, eye, bones; McNabb and Darras, 2015). In addition, recent studies in birds have shown that physiological variation in prenatal THs can affect hatching success, growth and metabolism (Hsu et al., 2017; Hsu et al., 2019; Ruuskanen et al., 2016; Sarraude et al., 2020a).

Three older studies investigated the effect of different dietary iodine concentrations on the thyroid function in different species and life stages. In egg-laying Japanese quails (Coturnix japonica), McNabb and colleagues (1985a) found that circulating and yolk iodine were proportional to dietary iodine. In addition, thyroid T₄ was decreased by restricted dietary iodine but plasma THs were not affected (McNabb et al., 1985a), suggesting the presence of regulatory mechanisms maintaining normal circulating TH concentrations. In contrast, McNichols and McNabb (1987) found that both circulating iodine and T₄ concentrations decreased in response to low dietary iodine in ring doves (Streptopelia risoria). This discrepancy indicates that species may respond differently to limited iodine availability. In quails, embryos and hatchlings from eggs with low iodine concentrations suffered from thyroid gland hypertrophy, but their circulating TH levels were not different from those of eggs with moderate or high yolk iodine concentrations (McNabb et al., 1985b). Thus, developing embryos may be able to cope with limited iodine resources and maintain TH production. However, none of these studies measured yolk THs, which is necessary information to investigate the potential trade-offs between the mother's circulation and embryonic exposure to THs.

The aim of our study was twofold. First, to investigate whether limited iodine may constrain TH production, and second, to ascertain whether mothers can prioritise TH allocation to either self or their eggs. To this end, we provided breeding pairs of rock pigeons (Columba livia) with a diet restricted in iodine (hereafter, I− group), to create a potential trade-off between circulating and yolk THs, and compared this group with a reference group provided with ample iodine (hereafter, I+ group), removing such a trade-off in the female. Based on the literature, we did not expect our supplemented diet to be toxic (see Materials and Methods, ‘Iodine-restricted and -supplemented diet’, below). If restricted dietary iodine limits TH production, we would expect a decrease in circulating THs in the mother, as found in ring doves, and/or a decrease in yolk THs. To cope with limited TH production due to iodine limitations, mothers may have developed regulatory mechanisms allowing them to trade off THs for self or for the egg. This would be reflected in a different distribution of TH concentration between the mother's circulation and the egg in the iodine-restricted compared with the iodine-supplemented group. Alternatively, mothers may prioritise circulating and yolk TH concentrations by limiting egg production and thereby the total amount of TH allocated to yolks, thus prioritising quality over quantity of offspring. If this is true, we may expect iodine-restricted females to lay fewer eggs (i.e. smaller clutch sizes) than iodine-supplemented females. This would be consistent with two studies that showed that hypothyroidism ceased egg laying in Japanese quails (Wilson and McNabb, 1997), and reduced egg production in chickens (Van Herck et al., 2013). We also expected the effect of iodine restriction to be more pronounced as the exposure duration to the treatment becomes longer. For example, iodine stores may deplete with time in birds on the I− diet, thus decreasing circulating and/or yolk iodine and THs with time in this group but not in the I+ group. In addition, laying several clutches under limited iodine availability may further deplete iodine and TH stores. We therefore expected yolk iodine and THs to decrease with clutch order in the I− diet. In general, and in line with previous studies (McNabb et al., 1985b; McNichols and McNabb, 1987), we would expect limited dietary iodine to have a stronger effect on T₄ than on T₃, as T₄ needs one more atom of iodine and is much less biologically active.

The experiment was conducted in 2018 on 38 pairs of wild-type rock pigeons (Columba livia Gmelin 1789). Rock pigeons lay two eggs per clutch, with a 48 h interval between the two eggs. In addition, rock pigeons can lay multiple clutches in a single breeding season (Johnston and Janiga, 1995). The birds were identified by unique ring code and combination, and were housed in a large outdoor aviary (45 m long×9 m wide×4 m high) in Groningen, The Netherlands, divided into four equal compartments (two compartments per treatment, n=9–10 breeding pairs per compartment, see below). The aviary included enough nest boxes and nesting material for all the breeding pairs. Before the experiment, all birds were fed a standard diet for pigeons (seed mixture Kasper™ 6721+seed mixture Kasper™ 6712+pellets P40 Kasper™ 6700). Standard food, water and grit were provided ad libitum. Before the experiment, 18 eggs were collected from unidentified females under standard diet and used for analyses of yolk iodine (see ‘Statistical analysis’, below).

All the procedures were approved by the Centrale Commissie Dierproeven (AVD1050020185444) and the Animal Welfare Body of the University of Groningen (18544-01-001).

We provided the experimental birds with either an iodine-restricted diet (I−, n=19 pairs) or an iodine-supplemented diet (I+, n=19 pairs) until all eggs and blood samples were collected. Egg collection was ended around 3 weeks after the initiation of second clutches, leading to a total period of approximately 10 weeks (see below for more details). The restricted diet contained 0.06 mg iodine kg⁻¹ food (Altromin™ C1042) and the supplemented diet was the same but supplemented with 3 mg iodine kg⁻¹ by the manufacturer. Therefore, the two diets had exactly the same composition of all essential micronutrients and macronutrients except iodine. The restricted treatment corresponds to about 10% of the iodine content in the standard pigeon diet (0.65 mg kg⁻¹), and approximately 20% of the minimum iodine requirement for ring doves (0.30 mg iodine kg⁻¹) according to Spear and Moon (1985). In addition, this restricted treatment corresponds to a low iodine treatment (0.05 mg kg⁻¹) used in a previous experiment on Japanese quails (McNabb et al., 1985a) that induced a significant decrease in circulating and yolk iodine. The supplemented treatment (3 mg kg⁻¹) corresponds to 10 times the minimum requirements estimated by Spear and Moon (1985). As rock pigeons are 2–3 times larger than ring doves, our supplemented treatment was actually 3–5 times the minimum requirement of our study species, after correcting for body mass. In McNabb and colleagues (1985a), the maximal dietary iodine (1.2 mg iodine kg⁻¹ feed) was ca. 8 times the sufficient iodine concentration required for Japanese quails (0.15 mg iodine kg⁻¹ feed) and the authors observed no detrimental effects of this high dose. As our supplemented treatment was lower than that of McNabb and colleagues (1985a), we expected no detrimental effect of our supplemented treatment either. Food, water and grit were provided ad libitum throughout the experiment.

Nest boxes were opened and nesting material was provided 2 weeks after the experimental diet was introduced to stimulate egg laying. Egg laying usually starts within a week of nest-box opening. Based on Newcomer (1978), who fed hatchling chicken with low iodine diet (0.07 mg iodine kg⁻¹ feed), we could expect thyroid iodine content to be lowest from 10 days onwards after introducing the experimental diet. The first eggs (i.e. from the first experimental clutches) were collected 3 weeks after the introduction of the experimental diet and were collected over 12 days. On average, eggs from the first clutches were laid 26.4±2.9 days (mean±s.d.) after the onset of the experimental diet. Freshly laid eggs were collected, and replaced by dummy eggs to avoid nest desertion. Second clutches were initiated by removing dummy eggs approximately 2 weeks after the completion of the first clutch (i.e. 5 weeks after the start of the experimental diet), and eggs were collected over a period of 18 days. On average, eggs from the second clutches were laid 53.5±3.3 days after the onset of the experimental diet. We also collected some late first clutches (on average 52.6±6.5 days after the onset of the experimental diet).

Table 1 summarises the number of samples collected. We collected eggs from both the first and second clutches of females fed with restricted or supplemented iodine diets. We also collected blood samples after clutch completion from the two experimental groups (I− and I+). The second set of samples (i.e. eggs and blood) was collected to test for the effect of exposure duration of the treatment (see time line above). Eggs and blood samples were collected in the exact same manner for the first and second clutches. Freshly laid eggs were collected and stored in a −20°C freezer. Not all females laid complete clutches of 2 eggs, and several females did not lay an egg at all. Females were captured during incubation in the nest boxes, and blood samples (ca. 400 µl) were taken from the brachial vein after clutch completion (mean±s.d. 4±4.7 days after clutch completion, range 0–21 days). Unfortunately, we could not sample blood from females that did not lay eggs, as this would have caused serious disturbance to all the birds in the same aviaries as we had to catch them by hand netting in the large aviary. Half of the blood sample (ca. 200 µl) was taken with heparinised capillaries for plasma extraction (for TH analysis) and stored on ice until centrifugation. The other half of the sample was taken with a sterile 1 ml syringe (BD Plastipak™) and allowed to coagulate for 30 min at room temperature before centrifugation for serum extraction (for iodine analyses). Previous studies measured iodine in serum samples (McNabb et al., 1985a; McNichols and McNabb, 1987); therefore, we decided to measure iodine in the serum for comparable results. Whole-blood samples were centrifuged at 3500 RPM (ca. 1164 g) for 5 min to separate plasma from the red blood cells (RBCs), and at 5000 RPM (ca. 2376 g) for 6 min to separate serum from the RBCs. After separation, all samples (plasma, serum and RBCs) were stored in a −80°C freezer for analysis of THs and iodine.

Eggs were thawed, the yolks were separated and homogenised in MilliQ water (1:1), and a small sample (ca. 50 mg) was used for TH analysis. Yolk and plasma THs were analysed using nano-LC-MS/MS, following Ruuskanen et al. (2018, 2019). TH concentrations, corrected for extraction efficiency, are expressed as pg mg⁻¹ yolk or pg ml⁻¹ plasma.

Yolk and serum iodine (ICP-MS, LOD of 3 ng g⁻¹ of yolk and 1.5 ng ml⁻¹ of serum) analysis was conducted by Vitas Analytical Services (Oslo, Norway). Yolk iodine was measured in a ca. 1 g sample of yolk, and serum iodine was measured in a ca. 0.2 ml sample of serum.

Data were analysed with the software R version 4.0.2 (http://www.R-project.org/). To test for the effect of iodine restriction on egg laying, we compared the number of females that laid first clutches in both groups, and the total number of eggs laid in first clutches with two Pearson's chi-squared tests. The rest were fitted with linear mixed models (LMMs) using the R package lme4 (Bates et al., 2015) and P-values of the predictors and interactions were calculated by model comparison using the Kenward–Roger approximation with the package pbkrtest (Halekoh and Højsgaard, 2014). The response variables were concentrations of plasma TH (T₃, T₄), serum iodine, yolk THs and yolk iodine. Relevant interactions between predictors were added in a full model and removed when non-significant to estimate the main effects. Post hoc tests of interactions were performed with the package phia (https://CRAN.R-project.org/package=phia). Model residuals were inspected for normality and homogeneity with the package DHARMa with 1000 simulations (https://CRAN.R-project.org/package=DHARMa). When either of the assumptions was violated, the response was ln-transformed (see Tables 1 and 3) and in these cases the model residuals showed the required distributions. Estimated marginal means (EMMs) were calculated from the models using the package emmeans (Lenth, 2019). When the response was transformed, the EMMs were calculated on the back-transformed data. When presenting the results, we used the language of ‘statistical clarity’ instead of ‘statistical significance’, as the latter may interfere with the meaning of ‘biological significance’ (Dushoff et al., 2019).

Although the treatment started for all females on the same date, each female, at the time of egg laying, was exposed to the experimental diet for different durations because of the varying laying dates between females. This may influence the effects of iodine manipulation on circulating and yolk iodine and THs. However, because the second clutches were laid after a longer exposure to the treatments than were first clutches (mean±s.d. exposure duration: second clutches 53.5±3.3 days; first clutches 30.9±10.6 days), clutch order and exposure duration (i.e. the number of days between the onset of the experimental diet and laying date) were confounded. Therefore, we used two separate models, one with exposure duration and another with clutch order. We controlled for egg order (i.e. first or second egg in a clutch) in our models for yolk THs initially because a previous study in rock pigeons showed a non-significant trend for higher yolk T₃ concentrations in the second eggs (Hsu et al., 2016). However, we detected no such effect in our models (all F<0.37, all P>0.54) and thus egg order was excluded from the final models.

Circulating iodine (ln-transformed) and T₃ and T₄ concentrations were analysed by fitting LMMs with treatment (I− or I+), exposure duration and completeness of a clutch (complete or incomplete, i.e. to further test for the effect of number of eggs laid) as a categorical variable, and the two-way interaction between treatment and exposure duration or completeness as fixed factors. Female identity (for iodine and T₄, but not for T₃ because of singularity: variance estimate collapsed to 0) and hormone extraction batch (for T₃ and T₄) were added as random intercepts. Estimates and P-values of the main effects for T₄ were obtained using the lmerTest package (Kuznetsova et al., 2017) using the Kenward–Roger method for denominator degrees of freedom. However, this package presents t-values instead of F-values (provided by the pbkrtest package); hence, Table 2 shows F-statistics for circulating iodine and T₃, and t-statistics for circulating T₄.

Yolk components (iodine, T₃, T₄) were analysed in two different sets of models. The first set of models, for yolk iodine only, compared untreated eggs (collected before the start of the treatment) with the eggs collected in the two treatments (I+ and I−). This way, we could test whether yolk iodine differed between untreated and experimental eggs. Here, we only used experimental first clutches as only one clutch of eggs per untreated female was collected. The model only included treatment (a three-level categorical predictor: untreated, I+ and I−) as the predictor.

The second set of models tested the effect of iodine treatment, exposure duration to the treatment or clutch order on yolk iodine and THs. Here, we included both first and second clutches from iodine treatments, but no eggs from untreated females. Yolk iodine (ln-transformed) was first analysed in a LMM that included treatment as a categorical variable, exposure duration (days since the start of the experiment), completeness of a clutch (complete or incomplete) and the two-way interactions between treatment and exposure duration or completeness, and female identity as a random intercept. This LMM somewhat violated the assumption of homogeneity of variances between the groups because of the larger variance in yolk iodine in the I+ group. Nevertheless, such a violation should not undermine our results as a recent paper demonstrated that LMMs are fairly robust against violations of distributional assumptions (Schielzeth et al., 2020). Yolk iodine was also analysed in a similar model in which exposure duration was replaced with clutch order (first or second clutch, categorical variable), both as a main effect and in the interaction with treatment. Yolk T₃ and T₄ (ln-transformed) were analysed using the same models (with exposure duration or clutch order) as for yolk iodine. Hormone extraction batch was added as a random intercept for yolk T₃ and T₄.

In line with our expectations, there was a clear effect of iodine treatment on circulating iodine concentrations: serum iodine was about 75% lower in the I− group than in the I+ group (raw data means±s.e.m., I− 1.1±1.2 ng ml⁻¹ serum, I+ 44.0±4.7 ng ml⁻¹ serum; Table 2, Fig. 1). The effects of clutch completeness, exposure duration and their interactions with treatment on serum iodine were statistically unclear (Table 2).

Plasma T₃ was not affected by supplementation or restriction of iodine, nor by exposure duration (Table 2; Fig. 2A). There was a statistically significant interaction between iodine treatment and exposure duration of the treatment on plasma T₄ (Table 2). A post hoc test of the interaction showed that plasma T₄ increased with time in the I+ group but not in the I− group (adjusted slope±s.e.m. I+ 0.24±0.07, χ²=12.9, Holm-adjusted P<0.001; adjusted slope±s.e.m. I− 0.05±0.06, χ²=0.56, Holm-adjusted P=0.46; Fig. 2B). Yet, the large confidence intervals warrant due caution in interpreting this interaction. There were no clear effects of clutch completeness and its interaction with treatment on plasma THs (Table 2).

In line with our prediction, eggs from the I− group had ca. 87% lower iodine levels than eggs from the I+ group and 50% lower iodine levels than untreated eggs; eggs from the I+ group had ca. 4 times higher iodine levels than untreated eggs (back-transformed EMMs±s.e.m., untreated 29.7±4.1 ng g⁻¹ yolk, I− 16.1±2.0 ng g⁻¹ yolk, I+ 123.4±11.9 ng g⁻¹ yolk; overall test: LM, F=91.3, P<0.001; post hoc Tukey comparisons, all |t|>3.38 and all P<0.004; Fig. 3).

Also in this dataset, yolk iodine concentration was 87% lower in eggs of I− treated females than in those of I+ females (mean±s.e.m., I− 16.03±0.52 ng g⁻¹ yolk, I+ 117.48±12.36 ng g⁻¹ yolk; Table 3, Fig. 4), but longer exposure duration, clutch completeness or clutch order had no clear effect on yolk iodine concentration (Table 3).

Yolk T₃ was not affected by iodine treatment (EMMs±s.e.m. T₃: I− 2.67±0.20 pg mg⁻¹ yolk, I+ 2.95±0.20 pg mg⁻¹ yolk), but showed a slight increase over the exposure duration and with clutch order (Table 3, Fig. 5A). Yolk T₄ was not affected by iodine treatment (back-transformed EMMs±s.e.m. T₄: I− 7.13±0.59 pg mg⁻¹ yolk, I+ 7.48±0.60 pg mg⁻¹ yolk), exposure duration of the treatment, or clutch order (Table 3, Fig. 5B). Finally, there were no clear effects of clutch completeness and its interaction with treatment on yolk THs (Table 3).

Overall, fewer females in the I− group laid their first clutches (complete or incomplete) than those in the I+ group (I− 11 out of 19 females, I+ 18 out of 19 females; χ²=5.24, P=0.02; Table 1). This resulted in fewer eggs produced in the I− group than in the I+ group (I− 17 eggs, I+ 30 eggs; χ²=8.03, P=0.005; Table 1). Focusing on the complete clutches, half as many females in the I− group laid complete clutches compared with those in the I+ group, yet the difference between the two groups was unclear (I+ 12/19 females, I− 6/19 females; χ²=2.64, P=0.10; Table 1).

In this study we tested whether dietary iodine limits the mother's circulating TH concentration, TH transfer to the yolk and egg production. To our knowledge, our study is the first to investigate the potential trade-off between circulating and yolk THs induced by low dietary iodine. We found that fewer females laid first clutches under the iodine-restricted diet compared with those under the iodine-supplemented diet, resulting in a lower total number of eggs laid. The iodine-restricted diet decreased circulating and yolk iodine levels, though circulating and yolk THs were unaffected. Longer exposure to restricted iodine had no clear effect on circulating or yolk iodine and THs. Finally, we observed a slight increase in plasma T₄ (in the I+ group only) and in yolk T₃ across time that was unrelated to the dietary iodine and is probably explained by seasonal changes or clutch order effects. However, because exposure duration to the treatment and clutch order are partly confounded, our experimental design does not allow us to fully disentangle the two variables.

Our iodine-restricted diet successfully decreased circulating iodine concentrations compared with the supplemented diet. Despite this effect, we observed no differences in circulating TH concentrations. This is consistent with a previous study that showed that Japanese quails under limiting iodine availability maintained normal circulating TH concentrations (McNabb et al., 1985a). However, a similar study on ring doves found a decrease in circulating T₄ concentrations with no changes in circulating T₃, suggesting increased peripheral conversion of T₄ to T₃ to maintain normal T₃ levels (McNichols and McNabb, 1987). The causes for the discrepancies between our study and the dove study (McNichols and McNabb, 1987) are not clear. One potential explanation is that, in our study, we could only sample the females that laid eggs and thus apparently managed to maintain normal circulating THs despite restricted iodine whereas those that did not lay eggs may have suffered from low circulating TH concentrations.

In the yolk, as in the circulation, restricted dietary iodine decreased yolk iodine but not yolk TH concentrations, in contrast to our predictions. The result of decreased yolk iodine is in line with a previous study on quails, which found that mothers fed with low dietary iodine produced eggs with low iodine, but that study did not report yolk TH concentrations (McNabb et al., 1985a). Low egg iodine concentration in turn disturbs thyroid function in embryos and hatchlings (McNabb et al., 1985b; Stallard and McNabb, 1990). Circulating TH concentrations of embryos, however, were not affected by low egg iodine (McNabb et al., 1985b; Stallard and McNabb, 1990).

Contrary to previous studies that manipulated dietary iodine, we found that limited iodine availability hampered egg production, with 40% fewer females producing eggs in the I− group compared with the I+ group. However, females that managed to maintain normal circulating THs were also able to lay eggs with normal yolk THs, similar to the study by McNabb and colleagues (1985a). At the moment it is unclear why some females were affected and others not, but a potential explanation may be individual differences in the ability to store iodine. Two other studies found that administration of methimazole, a TH-production inhibitor, caused egg laying to cease in Japanese quails (Wilson and McNabb, 1997), and reduced egg production in chickens (Van Herck et al., 2013). These results suggest that our restricted diet might have induced hypothyroidism in some females, thus preventing them from laying eggs.

Therefore, we did not show evidence for a cost of restricted iodine in females that managed to lay eggs. Those females did not appear to face any trade-off between allocating iodine and THs to either self or their eggs. Yet, 40% of the females under the restricted diet paid a cost in terms of egg production. Whether this effect is due to limited production of THs is as yet unclear, but these females may have faced a trade-off between maintaining normal circulating THs and yolk TH deposition. Our results suggest that females would favour investing in eggs with normal yolk THs, but when facing suboptimal environmental conditions they may prefer to reduce egg production.

The fact that some females did not lay eggs, supposedly to maintain normal circulating THs, suggests that mothers are not able to regulate yolk TH deposition independently from their own circulating THs, as recently proposed by Sarraude et al. (2020b). This is contrary to previous studies showing evidence of independent regulation (Van Herck et al., 2013; Wilson and McNabb, 1997). However, those two studies induced supraphysiological hypothyroidism or hyperthyroidism in the birds, which may explain such discrepancies.

Interestingly, we found that our restricted diet reduced egg production. As discussed above, this may be due to a hypothyroid condition that prevented females from laying eggs. There is, to our knowledge, no evidence that iodine is directly involved in egg production (e.g. follicle maturation, yolk formation, shell formation). Still, previous studies have shown detrimental effects of limited iodine availability on embryos and hatchlings. In breeding hens, restricted dietary iodine can decrease egg hatchability (Rogler et al., 1959, 1961b) and retard embryonic development (McNabb et al., 1985b; Rogler et al., 1959, 1961b), and can induce thyroid gland hypertrophy in embryos and hatchlings (Rogler et al., 1961a; McNabb et al., 1985b; but see Stallard and McNabb, 1990). As producing such low-quality eggs and offspring is a waste of resources, decreasing egg production under low iodine availability seems adaptive. Thus, such regulation may have evolved to protect embryos from exposure to too low an iodine and/or TH concentration. Overall, our results suggest that mothers in the iodine-restricted group appear to prioritise self-maintenance and offspring quality over offspring quantity.

Our low-iodine diet (0.06 mg iodine kg⁻¹ food) is comparable to what birds may sometimes experience in the wild. Although relevant data are scarce, estimates of iodine content in food items such as barley and maize grains, wheat or rye are highly variable, ranging from 0.06 to 0.4 mg iodine kg⁻¹ (Anke, 2004). Insectivorous species may also encounter iodine deficiency as the iodine content in insects varies from <0.10 up to 0.30 mg iodine kg⁻¹ (Anke, 2004). As such low iodine availability can also occur in the wild, it is therefore relevant to study whether mothers may face trade-offs in iodine or TH allocation during the breeding season, or whether it influences egg laying itself. However, our study did not show evidence for the existence of a trade-off between circulating and yolk THs when environmental iodine was limited. Nevertheless, mothers may face a trade-off between allocating resources to themselves or producing eggs of sufficient quality.

In conclusion, we found that restricted dietary iodine did not decrease circulating or yolk THs despite reduced circulating and yolk iodine, and thus we did not find evidence of a trade-off between allocating THs to self or to the eggs. Restricting iodine intake even further might help to reveal such a trade-off. However, reducing iodine too much would render mothers hypothyroid, probably stopping egg production. Our results provide evidence that restricted availability of iodine induces a cost in terms of egg production. Thus, mothers may not be able to regulate yolk TH transfer, but may be able to regulate egg production when facing limited iodine. Our results also indicate that females under limited iodine availability may prioritise their own metabolism over reproduction, or avoid exposing their offspring to detrimentally low iodine and/or THs. These explanations serve as interesting hypotheses for future research to further explore the consequences of limited iodine in wild populations.

We thank Martijn Salomons and the animal caretakers for helping us maintain the colony. We are also grateful to Gerard Overkamp and Asmoro Lelono for their help with blood sampling. We also thank Bonnie de Vries and Tiphaine Bailly for their help with egg dissection, and Ido Pen for statistical help.