DOI: 10.5061/dryad.ttdz08m32
title: Dietary fat supplements influence weight gain and egg production but not offspring sex ratios in Japanese quail

description: Sex ratio theory suggests that females should bias offspring sex ratios
 based on maternal condition and the availability of critical food
 resources. Work in birds indicates that females do, indeed, bias sex
 ratios according to maternal condition and food quality and quantity. Yet
 it is unknown whether these sex ratio skews occur due to fluctuations in
 particular micro- or macro-nutrients, caloric content overall, or even the
 perception of food availability. We hypothesized that dietary fats may
 drive biases in offspring sex ratios, because measures of maternal
 condition often reflect fat reserves, and fats are critical for the
 process of egg-laying in birds. To test this, we provided breeding
 Japanese quail, a species that biases sex ratios in response to maternal
 condition, with either a control breeding diet or a diet supplemented with
 two oils (safflower oil and flaxseed oil). These oils were chosen for
 their high omega-3 and omega-6 fatty acid content as well as their
 importance in mammalian sex allocation. We then measured influences of
 these diets on the sex ratio of offspring, the change in maternal weight,
 and the laying rates of female quail. The dietary oil supplements
 increased weight gains in quail but decreased the number of eggs laid
 during the experiment. There was no influence of the oil supplements on
 offspring sex ratios. This indicates that fat may not be a macro-nutrient
 involved in the process of sex ratio adjustment in quail.
description: Housing and Bird Care Sexually mature
 Japanese quail (<em>Coturnix coturnix japonica</em>) (n=65
 males and 65 females) were pair-housed in wire cages (6”x12”x10”) with one
 male and one female per cage. Quail had ad libitum access to water and
 feed throughout the entire experiment through nipple drinkers and trough
 feeders. The quail cages were housed in a single, climate-controlled room
 with a light clock schedule of 14:10 hours of light to dark. These quail
 were part of a breeding colony maintained by the University of Georgia and
 were available for inclusion in this experiment when they were in peak
 lay, at approximately 24 weeks old. Japanese quail lay one egg per day,
 and clutch sizes range from 10–14 eggs per clutch (Lukanov and Pavlova
 2020) with an average lag of 21.6h between successive clutches (Aggrey et
 al. 1993). In our breeding colony, we have noted similar clutch sizes, but
 rarely, we have observed females lay more than one egg per day, a
 phenomenon that has never been officially reported on in quail but has
 been observed previously in chickens (Navara and Wrobel 2019). The average
 fertility rates in previous studies of domestic quail were around 87%
 (reviewed in Lukanov and Pavlova 2020), though it is not uncommon in our
 breeding colony to see lower fertility rates, between 60 and 70%, in
 unmanipulated birds. It is still unknown whether wild Japanese quail are
 monogamous or polygynous; instead, it is likely that they can exhibit
 either mating strategy, but we maintain our birds in monogamous pairs, and
 female quail conduct all parental care in both wild and domestic
 situations. Design and Dietary Treatments
 The control diet was the standard quail layer diet used at the
 University of Georgia Poultry Research Facility (Table 1). Our high-fat
 diet was formulated by a poultry nutritionist at the University of Georgia
 (Dr. Woo Kim); it included 5% safflower seed oil (Hollywood® Safflower
 Oil) and 5% flaxseed oil (Puritan’s Pride® Natural Organic Flaxseed Oil)
 by weight and a reduction of carbohydrate content to account for the
 increased caloric load of the two added oils. We chose these amounts
 because it was the largest change in dietary fat that we could achieve
 without reducing a majority of the other critical nutrients in the diet
 other than carbohydrates. Overall, the formulation effectively elevated
 the fat content of the diet while simultaneously decreasing the
 carbohydrate content. We chose to decrease the carbohydrate content rather
 than increase the total caloric content of the diet because it would have
 been impossible to determine whether any effects caused were because of
 caloric content or a particular macronutrient. Unfortunately, it was
 impossible to adjust fat content by itself, so results will be interpreted
 with the understanding that carbohydrate content was reduced in this study
 as well, with the assumption that any effects we saw would need to be
 further examined to ensure that the decrease in carbohydrate content was
 not playing a role. We allocated 30 quail pairs to the fat-supplemented
 group and 35 pairs were allocated to stay on the control diet. The two
 groups remained on these diets for the remainder of the experiment. After
 two weeks on the dietary treatments, eggs were then collected for 14 days
 (Figure 1). We waited this two-week period because quail eggs can take
 anywhere from 4–7 days to complete rapid yolk deposition (Bacon and Koontz
 1971); we wanted to be sure that all birds had acclimated to the treatment
 for at least two weeks and all eggs we measured were influenced by the
 dietary supplementation. We collected a total of 496 eggs from control
 females and 312 eggs from females on the experimental diet. Female body
 weights were measured both before and at the end of the experiment using a
 digital scale (accuracy 0.01g). Sexing of
 Offspring After collecting eggs, we stored them in a
 cooler at 4℃ for a maximum of seven days before transferring them to an
 incubator at 37.5℃ at 58% relative humidity for four days. The incubated
 eggs were then removed and frozen at -50℃. While some suggest that sexing
 unincubated eggs is a better method of detecting primary sex ratios (Klein
 et al., 2003), there have been questions about whether contamination with
 maternal granulosa cells may influence the results (Arnold et al. 2003a).
 We opted to incubate for four days, as we have in previous studies,
 because this provided ample embryonic tissue for DNA extraction (Gam et
 al. 2011, Pinson et al. 2015). A total of 332 eggs from control females
 and 187 eggs from females on the experimental diet were fertile, and
 embryos were collected from these eggs. The remaining 164 eggs from
 control females and 125 eggs from females on the experimental diet were
 infertile and did not yield embryonic material for sexing.
 To extract DNA from embryos, we used a standard salt extraction
 according to procedures described in Lambert et al. (2000). While eggs
 were still frozen, we removed their eggshells and weighed out 10–20mg of
 embryonic tissue. DNA amplification was focused around the CHD-1 alleles
 to visualize male and female sex chromosomes (Fridolfsson and Ellegren
 1999). PCR primers and reaction concentrations were the same as specified
 in Pinson et al. (2015). Reaction times and temperatures were as described
 in Fridolfsson and Ellegren (1999). Primers used were 2550F
 (5'-GTTACTGATTCGTCTACGAGA-3') and 2718R
 (5'-ATTGAAATGATCCAGTGCTTG-3'). PCR products were visualized
 utilizing ethidium bromide staining of a 3% agarose gel. Male products
 presented as a single band while female products presented as two bands.
 Eggs for which there was no evidence of embryonic development were deemed
 to be infertile. Statistical Analyses
 To test whether the treatment influenced the sex ratio of embryos
 produced by females, embryos were coded as “1” for female and “0” for
 male. We then conducted a generalized linear mixed effects model,
 including dietary treatment, the change in weight, the log-transformed
 value of initial weight, and the interactions of these variables as fixed
 factors and female ID as a random effect. We conducted a similar analysis
 to test the effects of treatment on whether the eggs laid were fertile
 (fertile eggs were coded as “1” while infertile eggs were coded as “0”),
 and whether an egg was laid on a given day, since quail generally lay one
 egg per day (egg laid was coded as “1” while egg not laid was coded as
 “0”). Because quail in our population occasionally lay more than one egg
 per day, we also tested whether treatment influenced the incidence of
 double eggs using a logistic regression analysis; females that laid 2 eggs
 in one day were coded as “1” and females that did not were coded as “0”.
 Next, we tested whether the initial weight and/or change in weight of the
 females was related to the number of eggs they laid using general linear
 models. We tested whether female body weights were
 different between the two treatment groups at the beginning and the end of
 the experiment using a repeated measures ANOVA. The residuals of the
 initial and final weights were both non-normally distributed based on
 Shapiro-Wilks tests and needed to be log-transformed for analysis. We also
 tested whether treatment influenced the change in weight over the duration
 of the experiment using a general linear model with dietary treatment as
 the predictor variable and change in weight as the dependent
 variable. Differences were considered significant at p
 &lt; 0.05 and results are reported below with means ± standard
 deviations. Statistical analyses were carried out using RStudio (version
 4.2.1), using the <em>lmer</em> package for sex ratio
 analyses.
description: Microsoft Excel