Omega-3 polyunsaturated fatty acid (PUFA) and lutein are important value-added ingredients incorporated from diet and deposited to egg yolk in layers. Whereas numerous products are available, how birds transfer these ingredients to the egg as well as how multiple ingredients might interact during the enrichment process are much more poorly understood. For example, due to the antinutritive factors associated with ground or whole flax, it has been reported that the incorporation of omega-3 PUFA in diet may reduce transfer of lutein to the yolk. Strategies to improve incorporation into the yolk include modifying delivery of ingredients, or modifying the ingredients themselves. For example, the researchers attempted to improve delivery of lutein to the egg by feeding the omega-3 PUFA diet and lutein diet on alternate days. While this may not be a practical solution, the intent was to separate the enrichment ingredients in the gut while allowing them to still coexist in the final product.

Modification of dietary ingredients has more immediate relevance. An extruded form of flax was the omega-3 PUFA enrichment source in the diet in several of the experiments. There is minimal information about this type of product. For flax, the extrusion process breaks open the seed to provide better exposure of the fats, carbohydrates and proteins to the gut while disrupting the activity of the antinutritive factors. If flax was extruded alone, it would be an oily mess due to the high oil content of these seeds. The product used was co-extruded with peas, which provides carbohydrates to help provide structure and a dry, stable product. Unlike ground flaxseed, which can go rancid in a matter of months (or weeks in warm weather), this product is claimed to be stable for over a year.

* "Omega-3 enrichment process in eggs may be increased by optimising the metabolic efficiency and improving the absorptive efficiency by manipulating the gut condition through dietary treatment in birds"

Factors Affecting PUFA Transfer from Feed to Egg

The researchers explored the impact of other physiological factors on nutrient transfer to the egg. For example, the impact of individual hen energetic efficiency was determined and compared to the amount of omega-3 PUFA that ended up in the final egg product. Hens with increased energetic efficiency tend to be more effective at efficiency supporting protein turnover and are thought to suffer fewer losses to processes such as electron leakage during basic metabolism.

The Alberta group also believed these birds would have more efficient nutrient absorption. However, when it came to transfer of omega-3 PUFA from the ground flax to the egg, they did not see strong ties to energetic efficiency. Efficient birds had more omega-3 PUFA than the non-efficient birds at 14 days (289 versus 313mg per egg). The effect of difference in efficiency score of birds on enrichment may be masked due to higher inclusion of omega-3 PUFA for longer duration in diets.

Since they also examined other factors that could relate to the final omega-3 PUFA enrichment, they were still able to identify some interesting differences. There was significant difference in histomorphological parameters in birds scored on their energetic efficiency. The efficient hens had longer (P=0.01) and wider (P=0.04) villi, resulting in a greater absorptive surface area/villi (3.32 mm²) than in non-efficient birds (2.04 mm²; P=0.03). Also, the efficient birds had significantly higher villus length to crypt depth ratio.

The results for the gut morphometric clearly indicate that efficient birds were better suited for nutrient absorption than the non-efficient birds as they had greater duodenal surface area/villi, higher total duodenal surface area and higher villus length/crypt depth ratio. A shallow crypt in efficient hens possibly indicates a lower turnover rate of the intestinal epithelium, which results in a lower maintenance requirement and favors increased growth. The reduced impact on omega-3 PUFA enrichment suggest that lipid metabolism parameters are affected by more than just gut condition.

Building on what his group has found so far, Dr Renema says future work could include assessment of markers of lipid metabolism such as liver enzymes as 95 per cent of lipids in poultry are made in the liver.

Utilizing more energetically-efficient birds for the purpose of value-added egg enrichment might help in reducing the variability in end- product. Moreover, the uniformity in end-product can be achieved by targeting the absorption efficiency at gut level. In brief, Dr Renema concludes that omega-3 enrichment process in eggs may be increased by optimising the metabolic efficiency and improving the absorptive efficiency by manipulating the gut condition through dietary treatment in birds.

The omega-3 PUFA from the diet can be measured in the feed, in the blood plasma and in the final egg product. In the feed, levels are very consistent. In the blood, samples had the greatest amount of bird-to-bird variation. In the final egg product, omega-3 PUFA concentrations were more stable, with uniformity measures being on the scale of 50 per cent better than that recorded in the blood.

Keep in mind that blood measures would include both the omega-3 PUFA travelling from the gut to the liver, as well as the omega-3 PUFA that had been repackaged into the very low density lipoprotein (VLDL) particles travelling to the yolk for deposition, says Dr Renema. His group took blood samples at the same point each day to reduce time effects on plasma lipid measures but still saw differences that between birds that were much greater than that of the egg yolks.

When the fatty acid profiles were assessed, they noted that the level of omega-3 PUFA in the blood was much lower than that of the egg. Keep in mind that the egg yolk is a final product that loosely follows a 'recipe' that provides highly available lipids to the potential growing embryo. The VLDL particles targeted for the yolk have a different composition than the VLDL going to the rest of the body, with the yolk VLDL containing a higher proportion of the high-energy triglycerides than regular VLDL.

The Alberta group wants to see the long-chain omega-3 PUFA increase, as these fats (such as DHA and DPA) have stronger ties to the health benefits of omega-3 fatty acids than the medium chain omega-3 PUFA found in flax does (LNA). Interestingly, Dr Renema's group did not get measureable levels of long-chain omega-3 PUFA in the feed but did find it in the egg. Dietary LNA from flax increased yolk LNA and led to increased long-chain omega-3 PUFA while reducing omega-6 PUFA in yolk and blood plasma. The amount of omega-3 LC-PUFA increased with greater intensity in egg yolk as compared to plasma. Moderate and High diets resulted in 64 per cent and 70 per cent increases in yolk DHA, respectively, while in plasma the increase in DHA was only 13 per cent and eight per cent in these groups, respectively.

They have recently found that the long-chain omega-3 PUFA in the yolk are mostly in the phospholipid class of lipids than in the triglyceride fraction (Yuan Ren, unpublished data). This explains why they found a higher concentration of long-chain omega-3 PUFA in the egg than in the blood in the current study. The surface of the VLDL particles entering the yolk is covered by a phospholipid bi-layer.

In preliminary work, Dr Renema reports that the final enrichment level appeared to be less variable than the starting point, suggesting enrichment could be hitting a wall when using higher levels of enrichment. They added an experiment to look at the enrichment level to sort this out. The follow-up trial demonstrated that final enrichment could be just as variable in control, medium and high levels of enrichment.

They were unable to repeat results suggesting a decline in lutein levels when a ground flax source of omega-3 PUFA was used. Inclusion of lutein alone in diet did not affect the fatty acid composition of egg as compared to control. However, the protective antioxidant role of lutein was pronounced on long chain omega-3 PUFA in the egg stored for 30 days. These results were further substantiated through TBARS analysis. As the TBARS number was significantly higher in stored egg than fresh egg. TBARS value among different dietary treatment was similar in fresh eggs. However, among stored eggs, the lutein-enriched eggs had significant lower TBARS numbers than the control.

* "Increased lutein enrichment improves the overall carotenoid profile and also enhances stability during storage of egg"

The carotenoid analysis indicated that enrichment of lutein is positively correlated to canthaxanthin and beta-carotene pigments. The lutein-enriched yolk contained three-fold more lutein while alternate-day-fed hens had two-fold increase than hens on control and flax diets. Moreover, the lutein-enriched yolks had significant higher amount of canthaxanthin and beta-carotene. Further, the storage of eggs for 30 days had no affect on stability of lutein and beta-carotene in egg yolk whereas, significant reduction in canthaxanthin amount was observed. Ultimately, increased lutein enrichment improves the overall carotenoid profile and also enhances stability during storage of egg. The output of this experiment can be utilised for further studies for developing the feeding models targeted to multiple enrichments of egg.

Time-Course for Enrichment

Interestingly, there was a time course to the enrichment process that reached a plateau state much faster than expected. Laying hens have the ability to turn over stored lipids at about five times the rate that broiler stocks can do it. This would suggest that a longer term enrichment would be needed to properly enrich both the liver lipid and carcass lipid stores. However, the researchers found that enrichment plateaued in approximately six days on the enrichment diet. The total omega-3 PUFA in egg yolk achieved plateau of of 343.7mg per egg and 272.0mg per egg in 6.6 and 5.9 days, respectively, on the high and moderate diets. In plasma, the omega-3 PUFA concentrations reached saturation in 7.2 days with 0.93mg/ml and 0.67mg/ml with high and moderate diets, respectively. There was more variability in blood and egg omega-3 PUFA concentrations among hens early in the enrichment process, with peak variation found at the day 6 measurements.

The essential fatty acids LA (medium chain omega-6 PUFA) and LNA (medium chain omega-3 PUFA) compete with each other to utilise the desaturase and elongase enzyme for respective long-chain substrate synthesis. The calculated desaturase and elongase enzymatic activities for omega-6 PUFA (C20:4/C18:2) were negatively correlated with LNA (r= -0.59). Whereas, activity for same set of enzymes for omega-3 LC-PUFA (C20:5/C18:3 omega-3) was almost constant. The delta-9 desaturase enzyme activity (C16:1/C16:0) for de novo synthesis also decreased during the experiment. Excess dietary omega-3 fatty acid can reduce retention efficiency of LC-PUFA in egg yolk. Hence, these results indicates that mere increasing the LNA concentration in diets is not enough for increasing up-conversion to omega-3 LC-PUFAs in yolk. To maximise the conversion efficiency, it is necessary to improve the activity of rate-limiting factor, delta-6 desaturase. The delta-6 desaturase activity can be ameliorated with an efficient dietary plan (such as higher protein) and proper hormonal balance (control of glucagon, glucocorticoids and epinephrine) but is adversely affected by fasting and high temperature.

Effects on Calcium Absorption

In this study, Dr Renema reports they they also examined the impact of dietary treatment on calcium absorption as an indicator of gut condition. Early results did not indicate significant differences due to diets tested (data not reported) Previous work from their lab showed a negative impact of using oxidised fat (restaurant grease) on calcium uptake in the duodenum. As their diets did not appear to be causing any damage to the gut (as shown in the gut histology work), this is not surprising.

Conclusions and Further Work

There is huge potential for future work in this area through use of alternative omega-3 PUFA sources that contain longer chain omega-3 PUFA, or that solve the enzyme issue for up-conversion either by bypassing this step or adding the ability to the next step in the omega-3 PUFA biosynthesis pathway. While there has been some success in adding this enzyme to food animals, it may be more likely to achieve uptake of new products in this field by focusing on plant or bacteria based modification to achieve these goals. In the short term, we still have long-chain omega-3 PUFA sources such as fish oils that could be blended into hen enrichment diets.

The common value-added enrichment added to layers ration like omega-3 PUFA, lutein and vitamin E are fat-soluble ingredients. Fat from diet is digested and absorbed mainly in the small intestine. Further, the digestion is enhanced by emulsification of dietary fats with bile salts. The hen's age, intestinal microflora and gut microstructure are main factors affecting the utilisation of dietary fat. However, fat digestibility in layers is greatly influenced by the structural type, form, composition and amount of fat in dietary ration. Hence, the maintenance of gut health is very much essential for optimising digestion and hence improving the production potential of value-added enrichment in layers.

Dr Renema reports that his group achieved the target threshold of 300mg of total omega-3 PUFA per egg in just five days using a 15 per cent Linpro ration (extruded flax product). The results substantiate previous work suggesting that the fact that feeding of the extruded flaxseed improves the digestibility of flax because of the disruption of anti-nutritional factors (mucilage and lignans) associated with feeding ground flaxseed (Zuidhof et al., 2009). This is an important result, as one the main concerns with the use of flax in poultry diets is the potential for negative impact on growth and gut condition.

Hence, Dr Renema concludes that individual hen effects on omega-3 PUFA absorption in this project suggest further work to optimise egg enrichment through dietary strategies would be beneficial for the field of egg enrichment.

December 2010