Egg quality and coccidiosis infestation in three production systems for laying hens

A total of 240 white Shaver laying hens from 22 to 34 weeks of age were assigned to 3 treatments and 5 replications. The treatments included: i) Conventional (hens were kept in experimental building without access to outdoor area and fed with the conventional diet), ii) Semi-organic (hens were kept in experimental building with access to outdoor area and fed with the organic diet plus amino acids and vitamin-minerals supplement), and iii) Organic (hens were kept in experimental building with access to outdoor and fed with the organic diet). The results showed statistically significant differences in the feed intake, egg production, egg mass, egg weight, and change body weight(g) means among the treatments (p < 0.05). The hens kept in the organic treatment had fecal highest contain of oocytes coccidia (p < 0.05). The yolk color index and shell strength in the organic treatment significantly increased in comparison with that of other treatments (p < 0.05). The highest HDL was in the semi-organic and organic treatments (p < 0.05). The lowest egg yolk cholesterol concentration was found in hens kept in the semi-organic and organic treatments (p < 0.05). It is concluded that organic production system is useful for improving egg quality.


Introduction
In recent decades, with increasing public concern for poultry welfare, poultry friendly rearing systems are gaining popularity in Europe and many countries (Philippe et al., 2020;Geng et al., 2020). The consumers began to pay more and more attention to the food quality and poultry welfare and also have willing to pay for the poultry welfare products (Geng et al., 2020). The laying hen rearing system is main factor that impress egg production and quality. There has been a gradual increase in interest in more expensive eggs of free range origin. Consumers perceive these eggs as traditional food, because hens have access to a chicken run and a much more varied diet. This type of rearing has a substantial impact on the eggs composition. Consumers usually associate the quality of eggs with their freshness and the color of their yolk (Gałązka-Czarnecka et al., 2019). Consumer priority for poultry specialty products is related to greater quality and safety of products derived from such systems coupled with environmental standards and animal welfare. However, there is little scientific document supporting these perceptions (Napolitano et al., 2013). The result of some investigations haven't shown any significant difference in productivity traits of hens under different rearing systems (Neijat, House, Guenter, & Kebreab, 2011;Ahammed et al., 2014). On the other hand, many have shown a better productivity traits in conventional systems than in intermittent rearing system (Yakubu, Saleko, & Ige, 2007;Mugnai, Bosco, & Castellini, 2009). Some Opinions on egg quality are vague (Hidalgo, Rossi, Clerici, & Ratti, 2008). Some researchers observed higher Haugh units values from hens kept under organic or free-range systems (Minelli, Sirri, Folegatti, Meluzzi, & Franchini, 2007) whereas others reported higher Haugh unit values of eggs from hens housed in cages (Hidalgo et al., 2008). Under organic systems, hens have access to an outside area promoting foraging, feed selection, and activity and thus theoretically improve the welfare of the hens. Although outdoor access is intrinsic to the organic system, there are large variations concerning the amount and type of outdoor access provided in most of the organic system that are presently in practice in Europe and the United States. Therefore, despite the association between outside access and pasture and invertebrate consumption, the nutritional value derived from the intake of such products is unknown and varies dramatically with the system in use (Walker & Gordon 2003). Compared with conventional and organic systems, the pastured poultry alternative is likely to induce considerably greater levels of pasture consumption. Therefore, it can be considered as an ideal system to evaluate the nutritional impact of pasture intake in layer performance and egg quality. In addition, the presence of a range of bioactive compounds in the forage, such as xanthophylls and several hypocholesterolemic and anticarcinogenic compounds, may lead to improvements in products quality (Ponte et al., 2008). However, the high fiber content of pasture biomass may limit nutrient utilization and could reduce growth rates and feed efficiency. To our knowledge, the effects of pasture intake in layer performance and egg quality in organic systems remain largely unknown. (Walker & Gordon 2003). Since December 2004, when the EU derogation (Council Regulation (EEC) no. 1804/99) was removed, producers rearing hens under organic certification body standards are no longer allowed to use feed that includes synthetic amino acids. The impact of the inability to add synthetic amino acids is exacerbated by the need for an increasing proportion of organic materials in organic diets in the EU annually (Council Regulation (EEC) no. 2092/91, 85% in 2007. Although organic ingredients are in limited supply, a wide variety of ingredients is needed to achieve required sulfur amino acid levels in the hen's diet. Furthermore, even if a wide range of ingredients were available, the preparation of adequate diets is difficult to achieve in commercial systems because of the limited storage available to feed compounders. Hens fed conventional diets that provide methionine at suboptimal levels have been shown to be predisposed to health, welfare, and production-related problems (Friedman, Bar-Shira, & Sklan, 2003;Kidd Ferket, & Garlich, 1997;Klasing, 2006). It can, therefore, be postulated that poultry feed formulations that meet organic certification standards have the potential to adversely affect hen health and welfare, particularly where high-output genotypes are used. A good indicator of health status can be Serum biochemistery. Cholesterol and triglyceride are transported from the original place as lipoprotein particles LDL and HDL to their destination within blood circulation. Serum concentrations of cholesterol, triglyceride, LDL-C, and HDL-C are parameters used to measure serum lipid levels (Ma et al., 2014). Therefore, the primary objective of this experiment is to investigate the conventional, organic, and semi-organic conditions on performance and egg quality parameters and investigate the possibility of improved cholesterol and egg yolks color by organic and semiorganic conditions to shaver laying hen.

Experimental design and measurements
The experiment was designed based on a complete random design. Two hundred and forty 22-weeks old Shaver laying hens of the white Shaver were used in a complete random design experiment. Hens were assigned to three treatments with five replications. Each replication consisted of 16 hens and thus 80 birds per treatment were assigned. The trial was conducted in a windowless house at Agriculture Research and Education Center of Research Station in Khorasan Razavi, Iran, during April to July in 2018. Birds were grown in an indoor and outdoor area. Adaptation period was between 16 to 22 weeks of age in pens (150 × 300 cm dimensions and 2812 cm 2 per bird). Fifteen groups, each with 16 randomly selected birds were placed in plots (900 × 1,200 cm dimensions and 67,500 cm 2 per bird) cultivated with a mix of barley and alfalfa (previous two years without poultry), thus forming five replicates for each treatment combination.
Body weights and body weight changes were measured by weighting all hens individually at the onset and end of the experiment. The ingredients and chemical composition of the basal experimental diet are presented in Table 1.
The experimental treatments were as follows: i) Conventional floor (hens were kept in experimental building without access to outdoor area and fed with the conventional diet), ii) Semi-organic (hens were kept in experimental building with access to outdoor area and fed with the organic diet plus amino acids and vitamin-minerals supplement), and iii) Organic (hens were kept in experimental building with access to outdoor and fed with the organic diet). Birds in organic treatment had been transferred to the organic management system more than 6 weeks before the start of the experiment (Codex Alimentarius International Food Standards, 2013). Dietary treatments (in mash form) and water were provided ad libitum.In conventional group, a photoperiod of 14 hours per day was established. Light stimulation period extend into the peaking period. Achieve 16 hours of light at approximately 30 weeks. Birds in the two groups, semi-organic and organic, had access to daylight. Egg production were recorded on a weekly basis from 22 to 34 weeks of age (12 weeks). Each of the 15 groups had 4 individual laying nest (n = 60). During this period, 30 eggs treatment -1 day -1 were sampled randomly on two consecutive days every week. Eggs were weighed to determine the average egg weight. Feed intake was recorded on a weekly basis. Feed conversion was calculated as the ratio of a gram of feed consumed per gram of egg weight produced. The production variables such as feed intake and egg production were adjusted for hen mortalities and determined means of the whole period of the experiment (22-34 weeks).

Determination of parasite eggs excreted
Two individual birds from each of the 15 groups (n = 30) were monitored twice a week for excretion of coccidia oocysts (OPG). The coccidia oocysts were detected by using the Clayton-Lane method and their intensity was determined as number of oocysts per gram (OPG) of the litter (Hendrix, 1998) and counted by the modified McMaster method (Fakhri & Yakhchali, 2015).

Determination of egg weight, yolk index, haugh unit, shell weight, and shell strength
Sampling and the analysis of the egg's quality were performed after 4, 8, and 12 weeks of the experiment. Eggs were weighed using an electronic balance. A tripod micrometer is used to measure the height of the albumen midway between the yolk and the edge of the albumen. The Haugh unit score (Haugh, 1937) was calculated using the following formula: = 100 log 10 ( − 1.7 0.37 + 7.57) where: H = denotes the height of the albumen (mm) and W = is the weight of egg (g).
Eggshell strength was evaluated using a press meter (FHK, Japan) to gauge the force-producing cracking under longitudinal compression, and the shell maximum deformation was recorded (g cm -2 ).

Determination of serum and yolk cholesterol
Blood (about 5 mL) was collected (sampled from brachial wing vein) in a glass tube (16 mm × 100 mm) at the end of weeks 4, 8, and 12 of the experiment. Blood samples were kept at room temperature for clotting, and then the serum was obtained by centrifugation at 4,000 ×g for 3 min. Cholesterol was determined in serum by the spectrophotometric method using commercial kits (Pars Azmon, Iran) in auto-analyzer (Cobas, Japan). Yolk cholesterol was determined by the enzymatic method. A cholesterol reagent kit (Pars Azmon Cholesterol assay) was used for enzymatic determination using cholesterol esterase and cholesterol oxidase. The color intensity was determined photometrically at 540 nm, using a spectrophotometer (Hitachi U-2000, Japan). The cholesterol contents of the eggs were determined and expressed as milligram of cholesterol per egg.

Statistical analysis
Data were analyzed using the GLM procedure of SAS, and variances between groups were analyzed with SAS software (SAS, 1997). Differences between means were determined using least square difference (LSD) test contrasts among the means, according to the following model: where: Y denotes the dependent variable, µ denotes the mean, T is the treatment effect, and E ij is random residual error term.
All values were presented as means and SEM, with the significance level set at p < 0.05 (SAS, 1997). Egg production, egg weight, egg output, FCR, and feed consumption were analyzed with the mixed model procedure of SAS (1997). Significant treatment effects were detected by LSD multiple range tests. Percentage of egg production and death rates was also calculated.

Laying hen performance
Data analysis results show that the effect of experimental treatments on egg production(%) and feed conversion, egg mass, egg weight (g), and feed intake (g bird -1 day -1 ) in the treatments during the whole period of the experiment (Table 2) (p < 0.05). The hens in conventional treatment showed better performance than semi-organic and organic treatments. These traits have no significant difference between the organic and semi-organic treatments. However, there is a significant difference in feed conversion among treatments such that the better feed conversion efficiency was found in the semi-organic treatment (Table 2). Studies on the effect of changing from conventional cages to alternative rearing systems on production traits are continuing, especially on the ability of pure breeds to adapt to these alternate systems under certain climates. In the present study, the effect of the treatment on feed intake was found to be contrary to that reported in previous studies. Feed intake in an organic rearing system will be increased by higher activity, uncontrolled environmental temperatures and loss of feathers. (Leinonen, Williams, Wiseman, Guy, & Kyriazakis, 2012, Leenstra et al., 2012. The lower amount of feed intake was consumed in the organic and semi-organic groups due to the possible use of forage. The use of forages is limited due to their bulk and lack of bird fermentation ability. However, using fiber causes to normal digestive function (Wenk, 2001), stimulating gastrointestinal health, enhancing satiety, natural behavior, and animal comfort (Bach Knudsen, Hedemann, & Laerke, 2001;Wenk, 2001;Leeuw, Bolhuis, Bosch, & Gerrits, 2008). It should be noted, however, that the use of high levels of fiber in the diet results in reduced energy intake for the bird. The use of fiber in the bird diet reduces the behavior of cannibalism. It also has a positive effect on the metabolism of fats and carbohydrates in poultry. The bird can receive about 10 -30 grams of its dry matter daily through forage, but in another study the bird had access to the chicory about 50 -70 grams of dry matter per day (Horsted, Hammershoj, & Hermansen, 2006).
Egg production decreased in organic groups due to less feed intake, weight loss, and the possibility of egglaying in the pasture. As a result, egg production was higher in a conventional system than in other rearing systems (Yakubu et al., 2007). In the last 20 years, the typical cage system for laying hens has been largely criticized because birds are restricted in movement and space and are unable to exhibit their natural behaviors such as having a nest and bathing in soil. (Duncan & Fraser, 1997). Some investigators reported that the laying performance in laying hens (Ancona, a pure breed in Italy) in cages is superior to the ones reared under organic system (Mugnai et al., 2009). Sole purpose of conventional cage systems was to maximize profit and productivity with more hens being housed in a small area and higher egg production (Jones, Karcher, & Abdo, 2014). Lower egg production in the organic and semi-organic groups might be due to the concomitant lower energy and protein ingestion, which was diluted by grass intake as reflected by the lower feed intake. Thus, compared to the conventional group, the higher temperatures reached in the organic and semi-organic groups could also be attributed to its feed intake decrease and hence egg production.
Also, the hens in conventional group produced heavier eggs possibly due to the weight loss of the hens in organic and semi-organic groups. This might be partly due to the rearing system but mostly to the insufficient feed intake, which does not support the birds with adequate levels of protein needed to maximize their production. The decrease of feed intake in organic and semi-organic groups is caused by their higher activity, heat loss and ambient temperature (Leinonen et al., 2012;Leenstra et al., 2012).

Egg quality parameters
In this trial, although the internal or external egg quality characteristics were affected by the rearing system, yolk color index from hens reared in organic treatment were higher than those of eggs from hens reared in the conventional group. In all periods of sampling (p < 0.05), which is consistent with previous investigations, the yolk color index of organic treatment shows a significant difference with other treatments. Total carotenoid existing in alfalfa (Lutein) could be more than that of conventional treatment (Danish food composition databank-ed.7.01 1 ). Haugh unit data show that egg qualities were affected by the treatments in all periods (p < 0.05). Egg quality is largely controlled by the strength of egg white and jelly structure. Therefore, egg quality increases with an increase in white strength. Ovomucin is a protein that causes a jellylike structure in egg whites. The high content of this protein in egg white increases its quality, leading to an increase in the Haugh unit. There is a significant difference between the Haugh unit of conventional treatment in comparison with organic and semi-organic. The increase in liver metabolism may provide more ovalbumin for egg formation; as our results show a larger Haugh unit for conventional treatment.
The yolk index was different only in the organic treatment when compared with conventional and semi-organic treatments but Haugh unit was significantly higher for conventional treatment (p < 0.05) ( Table 3). The hens in organic treatment produced eggs with more shell strength than conventional treatment (Table 3). Hens in conventional treatment produced eggs with heavier shell than organic treatment (Table 3) (p < 0.05). Evaluation of cholesterol content in egg yolk and serum indicates that the effect of treatment was statistically significant (p < 0.05). The lowest yolk cholesterol and serum cholesterol were observed in organic and semi-organic treatments (Table 4). It was shown that the hens kept in the organic group had fecal contain highest oocytes coccidia (p < 0.05). (Table 5).
The results of experimental treatments on the relative weight of organs show that experimental treatments have a significant effect on oviduct and ovary relative weights, but no significant effect on relative weights of other internal organs. As shown in Table 5, the relative weight of oviduct and ovary (as reproductive organs) in conventional treatment is higher than that of organic and semi-organic treatments (p < 0.05). Shell strength in organic treatment is higher than other treatments (p < 0.05) that might be due lower egg mass and access to pasture (higher mineral supply)in the organic and semi-organic groups than conventional. Several authors reported variable results about the influence of the rearing systems on shell thickness. Leyendecker, Hamann, and Huartung (2005) reported thicker shells in free range eggs when compared to conventional cage and aviary systems and to conventional and furnished cages.
Moreover, serum cholesterol and triglyceride le vels can be used for stress indicators of hens. In this regard, some researchers have reported cage rearing as a stress factor that plays a major role in increasing serum cholesterol (Ma et al., 2014). In this study, hens kept in an organic group showed a decrease in cholesterol of their egg and serum. Sosin-Bzduca and Krawczyk (2012) showed that the use of plants supplements in the diet of hens can reduce contain cholesterol in the yolk. A similar effect was reported by the authors for plant supplements in the diet of chickens. Case, He, Mo, and Elson (1995) show a 9% reduction of serum cholesterol in leghorn chicks fed 15 ppm thyme essential oil. It seems that the reducing effect of plant supplements is not related to lignans compounds, because they have no effect on involved gene expression in cholesterol biosynthesis (i.e., HMG-CoA reductase, cholesterol 7_ hydroxylase, and acyl CoA oxidase); however, the effect of lignans on cholesterol metabolism is not rejected (Felmlee et al., 2009). Thus, here are two suggested mechanisms for hypolipidemic effects in organic treatment. First, pasture containing ~7% soluble fiber binds to cholesterol and makes a cholesterol-fiber complex that is unavailable to absorb from intestine. Second, this plant included many phytoestrogen compounds (lignans family) that affect cholesterol biosynthesis (Felmlee et al., 2009); nevertheless, the molecular mechanisms of flax-induced cholesterol reduction are unknown.
Reduction of cholesterol in serum and yolk of hens maintained in organic treatment may possibly be related to the active component of pasture. Both components may reduce the liver enzyme activity of 3hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase), which is actually a key enzyme in cholesterol synthesis (Abdulkarimi, Daneshyar, & Aghazadeh, 2011). There was a 2% reduction in poultry serum cholesterol in spite of a 5% reduction reported for HMG CoA reductase activity. The investigation on chickens kept in organic treatment have shown the same results; nevertheless, triglycerides and total phospholipids in chicken blood is reduced by forage and plant supplements significantly. In addition, it seems that essential oils in plants on pasture reduce the cause of cholesterol in organic and semi-organic treatments.

Parasite eggs excreted
In this experiment, the hens maintained in organic treatment produced excreta with the highest OPG. The conventional treatment showed significantly lower OPG. The possibility of exposure to the coccidia, leading to an increased incidence of coccidiosis is boosted by the higher levels of wheat in the organic treatment and access to the outdoor area. The results is similar to that reported in previous experimente (Williams, 2005).

Conclusion
Our data indicated that semi-organic and organic rearing systems decrease the concentrations of serum lipids, which might be beneficial to hens' health. Again, the lower serum lipids level may be attributed to the physical higher activity of hens from alternative rearing systems. Organic rearing system is useful for reducing egg cholesterol and improved yolk color index and shell strength.