In this experiment, compared with the S or OM3 diets, the addition of pigments at high level L/Z (50+50 or 100+100) did not have any significant impact on the egg production, FI, FCR or final BW during the 5-week experimental period (P>0.05). Findings of these data are in line with previous studies which have been reported that pigments did not have any significant effects on feed intake or body weight (Pérez-Vendrell et al., 2001; Li et al., 2012, Karadas et al., 2016) or egg production data were not significantly affected by using different dose or type of carotenoids in laying feeds (Hasin et al., 2006; Zhang et al., 2011; Lu et al., 2013; Karadas et al., 2016). Leeson and Caston (2004) also reported that dietary 125, 250, 375, 500, 625, 750 and 1000 ppm lutein supplementation had no significant effect on egg production data is in line with the results of this experiment. However, it has been reported that inclusion of marigold flower extract at 150 mg/kg dose in hens fed provide the highest hen-day egg production (Skřivan et al., 2015) not in agreement with our results.
As seen in Table 4, the mean values for albumen index and Haugh Unit were parallel to each other, which were significantly increased (P<0.05) when laying hens fed with OM3 diet. These finding data are in line with previous studies (Al-Daraji et al., 2010; Promila et al., 2017) which have been reported that higher levels of linseed or linseed oil significantly increase these values when they added to layer diets. However, these finding data are not compatible with results of the studies (Jiang et al. 1991; Scheideler et al., 1998; Grobas et al., 2001) who found no significant effect on HU and albumen index. These differences between findings data could be due to a strain-diet interaction, as mentioned by Scheideler et al. (1998).
The accumulation of xanthophylls (lutein/zeaxanthin) in egg yolk is noteably affected by dietary factors including type and concentrations of carotenoids, type of dietary fat, extent of processing, etc. and non-dietary factors including management system and physiological status of laying hens such as stress, diseases, age and breed (Zaheer, 2017; Pitarque et al., 2019). Such dietary factors as the level and saturation of fat have important role in bioavailability of xanthophylls due to their transportation of low-density lipoprotein and high-density lipoprotein (Papadopoulos et al., 2019).
There are series of steps that are associated with the release of lutein and zeaxanthin from the dietary matrix, the transfer of lutein and zeaxanthin to micelles, the absorption of lutein and zeaxanthin by intestinal membrane, and the transport of lutein and zeaxanthin to the blood and egg yolk. There are several factors that affect the absorption, bioaccessibility or bioavailability of carotenoids (Goltz et al., 2012). The literature reported controversial data on the effect of fat source for bioavailability of carotenoids. Some researchers (Hu et al., 2000; Gleize et al., 2013) suggested in vitro studies that dietary fats rich in saturated fatty acids led to higher bioavailability of lutein and zeaxanthin. On the other hand, some researchers (Failla et al., 2014; Mashurabad et al., 2016) suggested in vitro studies that the extent of micellarization of carotenoids increased with dietary rich in unsaturated fatty acid when compared to saturated fatty acid.
In this experiment, dietary inclusion of L/Z (50+50 or 100+100) either S or OM3 diets significantly increased the individual carotenoid concentrations of egg yolks paralleled those in the feed. But interestingly there was significant different has been recorded for egg yolk lutein concentration between L/Z (50+50) and L/Z (100+100) added to S diet. However same significant was not recorded for zeaxanthin level of egg yolk. For standard egg yolk feed lutein transport to egg yolk were more efficient compare to zeaxanthin. Parallel results were recorded with 125 ppm lutein in the diet of layer even higher level of lutein is recorded in 500 ppm lutein in the diet there was not difference lutein concentration in egg yolk by different lutein inclusion of 375 to 1000 ppm (Leeson and Caston, 2004). Similar results were obtained by Steinberg et al. (2000), with maximum 120 ppm level of lutein added to layer diets. Skřivan et al. (2015) reported that the addition of marigold flower extracts (350 mg/kg) rich in lutein and zeaxanthin increased both the lutein and zeaxanthin content of egg yolk when compared to control diet, which are in agreement with our results.
These accumulation carotenoids were recorded opposite effect between L/Z (50+50 or 100+100) added to OM3 diet and it was seen that zeaxanthin more efficiently accumulate in egg yolk compare to lutein concentration (P<0.05). Leeson et al. (2007) indicated that when flaxseed added to layer diets the accumulation of lutein into the egg reduced are in agreement with our results. But this concern not seen by zeaxanthin supplement diets. Therefore, in commercial omega-3 enriched egg production could be better for zeaxanthin supplementation to layer diet.
Since zeaxanthin and lutein are both found in the retina to form yellow pigments to protect eye from light and retinal damage (Tapiero et al., 2004). But for enrichment carotenoids egg yolk as a functional food production, we should consider the cost of feed and accumulation of carotenoids in the egg yolk as well. Factors such as appropriate doses of carotenoids, storage conditions, and the combination of dietary carotenoids influence optimal health-promoting aspects of carotenoids (Merhan, 2017).
In this experiment, egg yolk colorimeter (L* and a*) measurement results (Table 5) showed that compared with the S or OM3 diet, the addition of pigments at high level L/Z (50+50) and L/Z (100+100) cause significant improvement of L* and a*. This result is in agreement with report of Alay and Karadas (2016) who investigated the same dose (10 mg/kg) of carotenoids pigments (apoester, canthaxanthin, paprika oleoresin and aztec marigold extract pigments addition to non-pigmented wheat-soybean based quail’s diet improve calorimeter results of L*, a* values compare non-supplemented control group. However, ISA brown’s corn-soybean basal diet supplemented with different concentration (120,180 and 240 ppm) of marigold extract or with 40, 60 and 80 ppm apoester did not significantly affect L* values (Sirri et al., 2007) result not in line with our L*data. But, Hy-line White strain egg yolk L* values getting decreased by higher concentration of marigold 120 and 240 ppm or 40, 60, 80 ppm supplement of apoester (Sirri et al., 2007) showed significant different results are in line with our results. Similarly, redness a* results are in line with our results since all concentrations significantly improved in both strain (Sirri et al., 2007). Our L* and a* values are in agreement with previous reports that using red and yellow sources of pigments in hen’s diets associate with increasing a* values but decreased L* values (Niu et al., 2008; Skřivan et al., 2015). Minolta b* values showed interesting results in our experiment, OM3 group significantly low values compare all other groups except OM3+L/Z (50+50) group. However, L/Z (50+50 or 100+100) added to S diet could not change b* values significantly (P>0.05), since higher concentration of pigments shaded yellowness and converted to redness. Similar results have been reported by (Sirri et al., 2007) using different concentration of Marigold extract (120, 180 and 240 ppm) or 40, 60, 80 ppm apoester added to ISA Brown’s or Hy-line white strain layer corn-soybean diets did not significantly changed yellowness (b* values). It is important underline that compare all standard groups b* values were significantly decreased when laying hens fed with OM3 diet, even this diet (OM3) supplemented with L/Z (50+50) some improved seen but reached at the level of control at the L/Z (100+100) added to OM3 diet. In commercial condition in case of omega-3 enriched eggs production b* value reduction has to be taken in consideration. Egg yolk Roche color fan score was not affected by high dose of lutein (between 250-1250 ppm) in diet of layer (Leeson and Caston, 2004) are in agreement with our results even we did not record Roche color fan score data in case of b* value. Loetscher et al. (2013) was reported as lutein and zeaxanthin are highly active as yolk colorants are in line with our results.
In this experiment, the total saturated fatty acid (SFA) composition remained constant while total PUFA and MUFA composition was higher when laying hens fed with OM3 or OM3+L/Z (50+50 or 100+100) diets. The present results are in agreement with the findings of some other studies using different doses of fish oil, flaxseed or its oil (Galobart et al., 2001; Basmacıoğlu et al., 2003; Souza et al., 2008). Also, the absorption of MUFA especially C18:1 and C20:1n-9 seems to be encouraged by the dietary OM3, OM3+L/Z (50+50) and OM3+L/Z (100+100). Selvaraj and Cherian (2004) reported that increasing the levels of dietary fat increased the circulation of fatty acids which are preferentially deposited into specific tissues. This could result in increased MUFA composition of egg yolk. However, such lutein or zeaxanthin effect has not previously reported, so the reason for high egg yolk MUFA and PUFA composition are not due to the effect of lutein and zeaxanthin.