Impact of Selenium-Supplemented Diets on Egg Morphometry and Quality in Four Varieties of Indigenous Aseel Chicken

I Department of Poultry Science, Cholistan University of Veterinary and Animal Sciences, Bahawalpur-63100, Pakistan. II Department of Pathology, Cholistan University of Veterinary and Animal Sciences, Bahawalpur-63100, Pakistan. III Institute of Continuing Education and Extension, Cholistan University of Veterinary and Animal Sciences, Bahawalpur-63100; †Livestock and Dairy Development Department, Poultry Research Institute, Rawalpindi-46300, Pakistan.


Impact of Selenium-Supplemented Diets on Egg Morphometry and Quality in Four Varieties of Indigenous Aseel Chicken
and strains (Monira et al., 2003) of hens are among the significant factors that influence egg quality. The Aseel variety has heavy bodyweight (Ahmad et al., 2014) and strong muscular thighs (Dohner, 2001), can have the ability to withstand and survive in harsh tropical climatic conditions. Despite these excellent features, its potential regarding fertility and hatchability is not fully exploited because of poor egg physical characteristics and internal quality. Therefore, there is a dire need to improve the egg morphometric and quality traits of Aseel hens to achieve a reasonable progeny size. A balanced diet with dietary supplementation of selenium may improve egg morphometric and quality traits of Aseel eggs.
The appearance of compounds as nano-sized elements can result in unique properties, showing interactions with other ingredients, and change coordination with other compounds in biological systems, consistent with demonstration of compounds as larger elements or in solution (Gangadoo et al., 2020). Selenium is amongst the essential minerals compulsory for optimum development and production in birds. It maintains numerous functions linked to poultry rearing, production, and prophylaxis. Selenium is a fundamental portion of the enzyme glutathione peroxidase that works as an antioxidant enzyme and helps to govern levels of hydrogen peroxide and lipid peroxides (Ahmadi et al., 2018). Nowadays, selenium is known to modulate a wide spectrum of key biological processes and play a vital role in immune system functioning, protecting from oxidative injury, and carcinogenesis reticence as well. On the other hand, it is generally known that greater selenium quantities are dangerous causing several grave fitness complications (Ahmadi et al., 2019). The nanoselenium supplementation may improve feed intake, feed conversion, and weight gain in poultry. Moreover, nano-selenium stimulates immune system of broiler chickens in comparison with the other sources of selenium, as at nano-scales selenium shows novel and new potent properties. Consequently, supplementing poultry feed with nano-selenium is recommended to propagate poultry with enhanced performance (Shabani et al., 2019).
Dietary selenium (Se) is an essential trace mineral for poultry (NRC, 1994), identified to increase and maintain shell quality in older hens. Selenium, either inorganic as sodium selenite or organic such as selenocysteine, selenomethionine, and / or Se-enriched yeast (Foster & Sumar, 1997) is reported to have a positive effect on various egg characteristics. Organic Se being more bio-available (Cruz & Fernandez, 2011) has been preferred to improve shell quality and maintenance of egg freshness during storage (Papazyan et al., 2006). However, others still differ and state no difference in the performance of both organic and inorganic Se (Paton et al., 2000b). Very little work, so far, has been published on the evaluation of different egg physical and morphometric characteristics in Aseel chicken. Therefore, a study was designed to investigate the main and interactive effects of Se supplementation on egg morphometric and quality traits in different varieties of Aseel chicken. There is a hypothesis that dietary Se supplementation improves egg morphometric (egg length, breadth, volume, surface area, and shape index), and quality (egg weight, shell thickness, Haugh unit, and yolk index) characteristics in Aseel hens.

Experimental birds, housing, and ration
The present study was conducted at the Indigenous Chicken Genetic Resource Centre (ICGRC), Department of Poultry Production, University of Veterinary and Animal Sciences (UVAS), Lahore, for eight weeks duration. The ethical committee of the University of Veterinary and Animal Sciences, Lahore, approved the protocol of the trial. In total, 84 adult females from four varieties of indigenous Aseel were used. From each array, 21 hens were further divided into three groups (7 each) according to the type of Se-supplemented diets in a Randomized Complete Block Design. Each of the seven hens was kept in an individual cage to get a separate record of egg production. The experiment was conducted in a well-ventilated open housing system. A corn-soybean meal basal diet was formulated for females keeping in view their nutritional requirement (NRC, 1994). The basal diet was iso-nitrogenous and iso-caloric containing 15.1% CP and 2,682 ME Kcal/ kg, respectively. Before the initiation of the trial, the basal diet was analyzed for Se content. The quantity of Se in the basal diet was 0.04 mg/kg. Se from sodium selenite and Se-enriched yeast (Se-yeast Sel-plex® Alltech, Nicholasville, KY) was supplemented at 0.30 ppm into the basal diet. Control diet was without selenium supplementation (Table 1).

Measurements
In total, 84 eggs (21 from each variety), according to dietary Se treatments, were picked to study egg morphometric (egg length, egg breadth, shape index, egg surface area, and egg volume) and quality (egg weight, shell thickness with and without membrane,

Impact of Selenium-Supplemented Diets on Egg Morphometry and Quality in Four Varieties of Indigenous Aseel Chicken
Haugh unit, and yolk index) parameters. These eggs were weighed using electronic balance capable of measuring up to 0.1 g. In contrast, egg length and breadth were measured with the help of a digital vernier caliper capable of measuring up to 0.1 cm. The shape index was taken as the ratio between egg width and egg length (Anderson et al., 2004), whereas egg volume and egg surface area were determined using two separate formulae for each parameter and to take the average of the results (Etches, 1996). Eggs were then broken one by one, and the egg contents were carefully poured into a petri dish. Eggshell thickness was measured with and without vitelline membranes with the help of a dial pipe gauge and was taken as the average of three separate measurements from three different places without cracking the shell. Yolk index was chosen as the ratio between yolk height and yolk width whereas the Haugh unit score was measured using the egg weight and albumen height values in the formula H unit = 100 × log (H + 7.57 -1.7 × W 0.37 ), where H = albumin height (mm), and W = egg weight (g).
Data were analyzed using factorial ANOVA with the help of GLM procedure of Statistical Analysis System (SAS Institute Inc., 2002-04) and means were separated using Duncan's Multiple Range Test at 5% probability level, considering each bird as an experimental unit.

Egg morphometric traits
Mushki and Mianwali hens indicated significantly (p≤0.05) greater egg length than those of Lakha and Peshawari (Table 2). This greater egg length may be due to variations in genotype and bodyweight as different strains, or varieties lay eggs of varying weight and size because of different bodyweights (Bell & Weaver, 2005). Similarly, it was also observed that strains with varying bodyweight lay eggs of varying weight and size, which have their unique egg length (Arafa et al., 1982). Mianwali variety in interaction with inorganic Se supplemented diet revealed the highest egg length (56.04±0.7) indicating synergistic effect.
Similarly, significant differences (p≤0.05) were observed in egg breadth among varieties and their interaction with Se sources ( Table 2). Birds of Lakha variety exhibited significantly lower (p≤0.05) egg breadth than the rest of the three types, which may be due to genetic variations like different strains or varieties which lay eggs of varying egg weight and size (Bell & Weaver, 2005). Similar results were reported in a study on strains of varying bodyweights, which lay eggs of varying egg weight and size, having their unique egg breadth (Arafa et al., 1982). A study conducted on Aseel, likewise, indicated maximum egg breadth in Mushki Aseel (Ahmad, 2013). Egg breadth was the highest when Mushki was fed diet supplemented with inorganic Se showing a positive relationship between Mushki and inorganic Se. However, the Se sources alone could not produce any effect on egg breadth.
Mianwali variety demonstrated the highest egg volume followed by Mushki, Peshawari, and Lakha (Table 2) that may be the result of more top egg length and breadth in several ranges. Another study on Aseel, however, indicated higher egg volume in Lakha and Mushki than than in the other two types (Ahmad, 2013). Brown breeder hens laid eggs of significantly higher egg volume as compared to those of white egg layers (Rayan et al., 2010). Interaction of organic Se and Aseel varieties resulted in the highest egg volume in Mianwali variety, which may be due to its trend and synergistic effect with organic Se. However, Se sources could not produce any effect on egg volume. Results indicated the highest egg surface area in Mianwali variety followed by Mushki, Peshawari, and Lakha (Table 2). This may be due to strain variation (Anderson, 1996;Anderson et al., 2004) and comparatively higher egg length and breadth in that variety. Significant differences in egg surface area, likewise, have already

Impact of Selenium-Supplemented Diets on Egg Morphometry and Quality in Four Varieties of Indigenous Aseel Chicken
been reported in Aseel varieties (Ahmad, 2013). The highest egg surface area was observed again in Mianwali variety when coupled with organic Se whereas Se sources alone could not show any effect on the egg surface area. Among varieties, shape index was significantly higher (p≤0.05) in Lakha and Peshawari type compared with Mushki and Mianwali (Table 2), which may be due to differences in genotype. In contrast, another study on indigenous Aseel reported a higher shape index in Mianwali and Peshawari than the rest of the two varieties (Ahmad, 2013). Different strains also have clear indications of having a significant effect on egg shape index (Anderson, 1996;Ali et al., 2012), resulting in higher egg shape index in brown layers than white egg layers (Brand et al., 2004;Rayan et al., 2010). The interaction of Se sources and varieties showed the highest shape index in Lakha when fed diet containing inorganic Se, which might be due to positive relationship between Lakha variety and inorganic Se. Different Se sources alone could not show any effect on shape index. Shape index, likewise, remained unchanged despite supplementation of Se in diet (Pavlovic et al., 2010), showing no response of sodium selenite or selenium-enriched yeast on shape index (Invernizzi et al., 2013). Organic Se in the form of Sel-Plex™ also did not influence shape index (Renema, 2004;Hanafy et al., 2009).

Egg quality traits
Enhanced egg weight was observed in Mianwali variety followed by Mushki, Peshawari, and Lakha (Table 3), which might be due to higher bodyweight of the respective birds as speculated that bodyweight and egg weight are positively associated. Interaction of different treatments revealed that Mianwali variety produced eggs with an improved weight when diet was supplemented with organic Se, which might be attributed to Mianwali variety based on its higher bodyweight, further enhanced by supplementation of organic Se showing synergistic impact with Mianwali variety. However, Se sources alone could not exert any influence on egg weight. In a similar study on quail, no significant effect of different Se sources was reported on egg weight (Cruz & Fernandez, 2011), which clearly showed that egg weight remained unchanged despite dietary supplementation of Se in the diet (Attia et al., 2010;Arpasova et al., 2012).
Significantly lower eggshell thickness (p≤0.05; with and without membrane) was observed in birds of Lakha variety in comparison to the other three types (Table 3), which might be due to genetic variations.

Impact of Selenium-Supplemented Diets on Egg Morphometry and Quality in Four Varieties of Indigenous Aseel Chicken
Interaction of Se sources and types resulted in the lowest eggshell thickness with and without membrane in Lakha variety when fed with inorganic selenium supplemented diet. The Se sources, however, could not influence the eggshell thickness. Pavlovic et al. (2010) in laying hens and Cruz & Fernandez (2011) in quail breeders as well, reported a similar response of Se on eggshell thickness, concluding that there is no difference between SS and SY groups for eggshell thickness (Invernizzi et al., 2013). The shell thickness, likewise, remained unaffected in layers when fed with organic selenium supplemented diet (Correia et al., 2000;Paton et al., 2000a). Among varieties, the highest Haugh unit score was seen in Lakha, followed by Mushki, and then Mianwali (Table 3). It might be due to comparatively higher albumen height in that variety. A significant difference in Haugh unit score, likewise, was also found in a study on different breeds of poultry (Monira et al., 2003;Ali et al., 2012). Interaction of Se sources and varieties revealed maximum (81.04±1.1) Haugh unit score in Lakha hens using the diet supplemented with organic Se, which might be attributed to the trend of Lakha variety for higher Haugh unit score. The Se sources alone, however, could not affect Haugh unit score. Likewise, Haugh unit score in layers was not changed even if the diet was supplemented with Se from an organic source (Correia et al., 2000) or regardless of the source (Patton, 2000;Cruz & Fernandez, 2011). There is another claim that the administration of selenium (Mohiti-Asli et al., 2008), or organic trace minerals individually or in combination (Scatolini, 2007) did not affect Haugh unit score.
The highest yolk index was observed in Lakha when fed with a control diet. However, Se sources alone could not show any difference in yolk index (Table 3). Yolk index remained unaffected despite supplementation of Se in the diet (Mohiti-Asli et al., 2008;Attia et al., 2010;Cruz & Fernandez, 2011) either in the form of organic or inorganic (Arpasova et al., 2012;Invernizzi et al., 2013). Varieties alone also could not show any difference in the yolk index. Non-significant differences (p>0.05) were reported in the yolk index among different types of Aseel (Shafiq et al., 2013;Ahmad, 2013), and between Vanaraja and White Leghorn breeds of chicken (Haunshi et al., 2011).

CONCLUSIONS
From the findings, it can expediently be concluded that Mianwali variety, for its good egg physical characteristics and positive interaction with the

Impact of Selenium-Supplemented Diets on Egg Morphometry and Quality in Four Varieties of Indigenous Aseel Chicken
organic Se, can be selected for further improvement if uninterruptedly fed on organic Se supplemented diet. This novel approach might help the farmers in producing quality eggs and chickens, fetching premium returns from the elite class, reviving a prestigious agrobased rural poultry culture, and having livelihoods for their households.

DISCLOSURE OF POTENTIAL CONFLICT OF INTEREST
No competing conflict of interest was reported by the authors.