By measuring several production and metabolic traits in purebred Muscovy or hybrid mule ducks of which the paternal grand-mother was fed a methionine-restricted diet, we showed that the mother diet is able to affect the offspring of her sons. The transmission of such effects over two generations through the father was observed in both genetic types for traits such as body weight before force-feeding and triglycerides plasma concentration, while other traits affected by the grand-mother diet were genetic type-specific.
GMMD effects in mule ducks vs. Muscovy ducks
The effects observed at G2 in mule ducks and in Muscovy ducks share common features: in both genetic types, GMMD decreased body weights during the growth phase from 4 to 12 weeks of age. In the purebred Muscovy ducks, this effect was however limited to the male progeny. A year effect was observed in G2 mule ducks, and particularly a sex by year interaction on liver-related traits. This year effect is likely to be multifactorial since factors influencing these zootechnical traits are numerous, including incubation conditions, temperature and hygrometry during the growth phase, and force-feeding conditions.
Surprisingly, some effects of GMMD were specific of the genetic type of the G2 offspring. The marked effect of GMMD on weight gain during force-feeding and on abdominal fat weight was observed in the mule duck progeny only. The effects of GMMD on magret weight and on carcass proportion of fatty liver were specific of the purebred Muscovy progeny.
The hybrid mule duck progeny and the purebred Muscovy progeny have thus specific responses to GMMD, particularly for growth traits (sex-specific response in Muscovy ducks only) and for FF traits (weight gain during FF in mule ducks only and specific localization of fat deposits).
The sex-specific effects on growth in Muscovy ducks may be linked to the sexual dimorphism of body weight which is particularly marked in this species, as compared to the common duck or to the mule duck [18]. This sexual dimorphism clearly appeared in our study, as for example in the control group: the 12 weeks body weight was 5501 g in males vs. 2941 g in females. The nutritional requirements of males and females are therefore very different, and the fact that GMMD affected particularly weight gain between the ages of 8 and 12 weeks, including the phase of preparation to FF with limited time for feed consumption, could involve sex-specific response group to nutritional restriction. Several studies have shown environmental sex-specific effects, including epigenetics modifications, during embryogenesis (see [19] for a review).
Grand-maternal Met restriction affected traits associated with lipid metabolism
In contrast with the depressive effect on body weight, GMMD increased traits associated with lipid metabolism such as weight gain during FF and abdominal fat weight in the mule duck progeny, and the carcass proportion of fatty liver in the Muscovy duck progeny. Thus, the diet seems to drive the localization of fat deposits towards abdominal fat in mule ducks, and towards liver in Muscovy ducks. This seems to reinforce the natural differences between these two genetic backgrounds, already observed [20–22]. In birds, the liver is the principal site of lipogenesis, which, in the case of force-feeding, consists of the synthesis of triglycerides from glucose, resulting itself from the digestion of the starch of the feed. In the liver, the unbalance between lipogenesis and lipid exportation leads to liver steatosis. The exported triglycerides are transported as VLDL (Very Low Density Lipoproteins) to peripheral tissues, forming subcutaneous, abdominal, and intra-muscular fat deposits. During FF, most of the body weight gain is made of lipids, not excluding the continuation of muscular growth. Liver weight is a good indicator of liver fat content, since lipids represent 61 % of fatty liver weight [22] and since the correlation between liver weight and liver fat content was found to be 0.95 in a population of mule ducks (X Fernandez, personal communication).
Beyond the lipid synthesis, Met restriction of the grand-mother influenced the body distribution of the triglyceride storage from lipogenesis. Indeed, in the mule duck progeny, abdominal fat weight was altered, but neither was the liver weight nor the subcutaneous fatness of the body after force-feeding. In the Muscovy duck progeny, the body distribution of the triglycerides storage was also altered by GMMD: the ducks from Met-restricted grand-mothers retained more triglycerides in the liver, at the expense of subcutaneous fatness which was decreased, however not significantly. Indeed, the indicator of the overall body subcutaneous fatness, the magret percentage of (skin + fat) was 9.07 ± 0.08 % in the Met-restricted group vs. 9.21 ± 0.08 % in the Control one.
Our data could be viewed in relation with what is observed in mammals where several studies showed that methyl donor deficient diets (MDD diets) affect hepatic metabolism in F0 and/or F1 generations. In rodents for example, Methionine and Choline deficiency favors hepatic steatosis [23, 24], through increased fatty acid uptake and decreased VLDL secretion by the liver [25]. In rats, maternal MDD diet induces hepatic steatosis [26] and changes in liver proteome [27]. In sheep, maternal MDD diet affects the gene methylation level in the liver of F1 offspring [28]. Epigenetic changes have been evoked to explain these effects since methyl donors such as folate, choline, methionine and vitamins B6 and B12, are involved in one-carbon metabolism which releases methyl groups (−CH3) used by methyltransferases to methylate DNA and histone proteins (see [29–31] for reviews). Furthermore, the deficiency of methyl groups may affect the transmethylation of co-regulators of nuclear receptors such as PGC1-α which is a master regulator of lipid metabolism and fatty acid oxidation [26, 29, 30]. More recently, Chen and coworkers identified modifications of DNA methylation in the promoter regions of 1032 genes in liver of F1 offspring from female rats fed with MMD diet [32]. To our knowledge, no such maternal MDD diet studies have been carried out in birds, and our work differs from the ones cited above in the fact that it focuses on the F2 generation and that the non genetic effects of the MMD diet are transmitted through the sons, and thus through their spermatozoa, to the F2 generation. Such effects of female diet transmitted to their grandchildren via their sons have already been reported in humans. Thus studying a cohort of grandchildren of women exposed to 1944–45 Duch famine, Veenendaal and coworkers reported that adult offspring (F2) of prenatally exposed F1 fathers were heavier and more obese than children of fathers who had not been prenatally exposed [9].
Epigenetic vs genetic effects
It cannot be excluded that part of the differences observed in G2 between the two grand-maternal diet groups, in both the mule duck and the purebred Muscovy progenies, can have a genetic origin. Sampling bias can have occurred in G0, due to the small number of founders, resulting in genetic differences between the two groups of founders. Again, the small number of G1 drakes may add some genetic drift, responsible for an additional error in the G2 progeny mean estimation [33]. Our tests for comparing met-deficient and control G2 offspring did not take the genetic drift into account, but the absence of differences in the average weights of the two groups of G1 males brings some reassurance concerning this putative bias. But the results observed are compatible with the existence of epigenetic effects. Indeed, it is now well documented in mammals that environmental exposures (e.g., toxins, stress or nutritional deprivation) of the G0 generation can influence gene regulations and the adult phenotypes of the G1, G2 (“multigenerational”) and G3 generations (“transgenerational”) through epigenetic mechanisms (e.g., DNA methylation, histone modifications or miRNA) [8, 34–38]. In birds, resources deposited in the egg (e.g., nutrients, hormones, carotenoids, vitamins or RNA transcripts) can also impact newborn fitness and later adult phenotypes of the G1 generation. But the egg composition can also directly affect the G2 generation since the developing G1 generation bears the primordial germ cells that will eventually form a G2 progeny. Hence, the maternal nutritional deficiency may have affected the epigenetic information carried by the G1 drake spermatozoa, as already reported in mammals [39]. This non-genetic inheritance may be partially involved in human metabolic diseases [40]. Epigenetics marks in the Primordial Germ Cells of the developing father may be influenced by the environment of his mother [7]. This transmission of information through the paternal germline may involve modifications of chromatin, small RNAs, or other mechanisms, yet to be deciphered [41, 42].
Contrary to maternal effects [43], paternal effects have received much less attention [44, 45]. Nevertheless, several studies have shown that the father may transmit non-genetic information through spermatozoa epigenetic marks [39, 46, 47], or sperm component factors [48]. As the father’s environment modification happened during the father’s embryonic development in our study, we hypothesize that epigenetic effects may be involved in the results observed here.
