Quality Attributes of Eggs from Laying Hens Fed Diets Supplemented with Varying Inclusion Levels of Ascorbic Acid and Cholecalciferol

Bamidele Samuel Adedeji¹, Folasade O. Jemiseye², Adeyemi Ayodeji Afolabi¹, Sabur O. Oladimeji³, Funmilayo Grace Adebiyi¹, Bukola Christiana Majekodunmi^4,5, Babatunde F. Adebayo⁵, T. V. Abokede¹, Sherifat Omowunmi Olufeko⁶ and Olugbenga Adeniran Ogunwole^1*
*Correspondence: Olugbenga Adeniran Ogunwole, Agricultural Biochemistry & Nutrition Unit, Department of Animal Science, University of Ibadan, Ibadan, Nigeria, Email:

Keywords

Supplemental vitamins • Egg quality characteristics • Shell thickness • Haugh unit • Albumen height

Introduction

Vitamins are previously believed to be useful only for the treatment and correction of deficiency diseases in livestock. According to report [1], dietary recommendation of vitamins and minerals in livestock was with respect to age, sex, breed, environment, season, health among others. These are however, now outdated and inadequate for the present day more highly productive commercial stock. The importance of vitamins is presently beyond prophylactic and curative treatments of diseases; rather, it has become an essential production tool in livestock management.

More recently, the diets of livestock, especially, poultry are fortified with supplemental vitamins to promote their wellbeing. The use of Ascorbic Acid (AA) also known as vitamin C, for example, has been prominent for stress management [2,3], performance [3-8], immunity, while vitamin D₃ has mostly been implicated in the improvement of immunity [9-12], optimization of skeletal integrity [13-17], egg nutrient deposition [18] and eggshell strength [19,20] in chickens.

In spite of these reports on the use of AA and D₃ in poultry, attention so far has been so much on performance attributes without similar emphases on the quality attributes of poultry produce. Few reports on egg quality attributes [4,21] from hens fed diets supplemented with combined AA and D₃, were inconsistent. This study was therefore aimed at assessing the implications of the sole and combined dietary supplement of AA and D₃ on hen egg quality characteristics.

Materials and Methods

Experimental location

The experimental site was the Poultry Unit, Teaching and Research Farm, University of Ibadan, Ibadan, Nigeria. The study area lies between longitude 7°27.05 north and 3°53.74 of the Greenwich Meridian east at an altitude 200 m above sea level. Average temperature and relative humidity of the location is between 23-42 ℃ and 60-80%, respectively. Laboratory procedures were conducted at the Animal Products and Processing Laboratory and the Nutritional Biochemistry Laboratory, Department of Animal Science, University of Ibadan, Ibadan, Nigeria.

Experimental pullets and management

Bovan Brown pullets (n=384) with a track record of medication, vaccination schedule and productive performance from one day-old were used for this experiment. The pullets were raised in a conventional battery house. Each cage in the three-tier cage which measured 40 × 41 × 32 cm. The pullets were allotted to an isonitrogenous and isocaloric basal diet supplemented with three levels of AA (0, 300 and 600 mg) and four levels of D₃ (0 IU, 1000 IU, 2000 IU and 300 IU) in a 3 × 4 factorial arrangement of a completely randomized design to produce 12 treatments. Each treatment was replicated four times. Details of dietary experimental layout have been published [17].

Sample collection

Freshly laid eggs (n=96) (2 per replicate and 8 per treatment) were each collected at weeks 26, 30 and 34 in the early laying period (week 22-38) as well as weeks 43, 47 and 51 in mid laying phase (week 39-55). The eggs collected were assessed for internal and external egg characteristics.

Parameters monitored

Egg weight (g) was measured with a sensitive scale. Egg length and diameter (mm) were obtained using digital Vernier caliper. Egg shell thickness was assessed at three respective locations (broad, middle and small ends) with a micrometer screw gauge and the average taken [22] while, shell weight was measured with a sensitive scale after drying the shell at room temperature [23]. Each egg was broken on a flat surface for internal egg quality indices measurement. Yolk was carefully separated from the albumen for measurement. Yolk diameter and albumen diameter were monitored using a Vernier caliper. Yolk was placed on a petri-dish of known weight and measured using electronic digital scale. Yolk height was measured by dipping the pointed tip of the Vernier caliper through the middle of the yolk while for albumen height it was measured 1 cm away from the edge of the yolk and the height of the sticky fluid on the tip was adjusted to read the height on the digital screen. Yolk and shell weights were deducted from the weight of the egg to obtain albumen weight. The relationship between albumen height and egg weight was used to calculate the Haugh unit (Hu) as described by Haugh RR from the equation [24];

Hu = 100 log₁₀ (h - 1.7W^0.37 + 7.6); where

Hu = Haugh unit;

h = height of the albumen (mm)

W = egg weight (g)

Data were pooled for each monitored parameter in this study and the mean from the pool was further processed statistically.

Experimental design

The experiment was a factorial arrangement in a completely randomized design with the experimental model is as follows:

Y_ijk = μ+ α_i + β_j + α_iβ_j + e_ijk

Where;

Y_ijk = Observation k^th in i^th level of factor A and j^th level of factor B

μ = Overall population mean

α_i = Effect of i^th level of ascorbic acid supplementation

β_j = Effect of j^th level of cholecalciferol supplementation

α_iβ_j = Interaction effect of ascorbic acid and cholecalciferol supplementation

e_ijk = random error with mean 0 and variance σ²

Statistical analysis

Data were subjected to two-way ANOVA of the GLM procedure of SAS [25] and the means separated using Tukey’s HSD option of the same software at α_0.05.

Results

Egg quality attributes of laying hens at the early laying phase (22-38 weeks)

The external characteristics of eggs from hens fed supplemental AA and D₃ at the early phase of laying are shown in Table 1. The influence of AA was not observed on all external egg parameters monitored at this phase (p>0.05). Egg weight (55.85-57.10 g), egg length (54.26-56.20 mm), egg diameter (42.80- 43.04 mm), shell thickness (0.37-0.40 mm), shell weight (5.13-5.59 g) and egg shell percentage (9.18-9.82%) were not significantly affected (p>0.05) by AA supplementation. Similarly, dietary D₃ supplementation had no significant influence (p>0.05) on egg weight (55.04-56.88 g), egg length (54.21-56.05 mm), egg diameter (42.34-43.43 mm), shell thickness (0.37-0.38 mm), shell weight (5.17-5.55 g) and shell percentage (9.06-9.61%).

The internal characteristics of eggs from hens fed supplemental AA and D₃ at the early laying phase are shown in Table 2. Supplemental AA had no significant effect (p>0.05) on all monitored internal egg parameters except yolk weight and yolk %. Albumen weight, albumen length, albumen diameter, albumen height, yolk diameter ranged from 32.75 to 34.94 g, 93.54 to 100.92 mm, 70.20 to 79.90 mm, 6.28 to 6.79 mm and 37.72 to 40.45 mm, respectively. Others were 13.50 to 13.98, 1.10 to 1.36 mm, 58.67 to 61.63% and 78.73 to 82.96, respectively for yolk colour, yolk height, albumen % and Hu. Yolk weight of eggs from pullets on 0 mg/kg AA (13.56) was lower (p<0.05) than 14.79 and 14.58 g in those on 300 and 600 mg/kg supplemental AA, while yolk (%) of pullets on 0 mg/kg (24.78%) was lower than 26.12% in those on 300 and 600 mg/kg supplemental AA, respectively.

The D₃ had no influence (p>0.05) on all internal egg parameters observed at this phase. Albumen weight, albumen length, albumen diameter, albumen height and albumen % ranged from 33.32 to 34.34, 81.87 to 105.26, 71.64 to 78.25, 6.01 to 7.44 and 59.08 to 60.28, respectively. Yolk weight ranged from 12.93 to 14.19, yolk diameter from 37.45 to 40.58, yolk colour from 12.93 to 14.19, yolk height from 1.00 to 1.33, yolk % from 25.06 to 25.93 and Hu from 77.95 to 86.75. Effect of interaction of ascorbic acid and cholecalciferol were not significant (p>0.05) except for the yolk %.

Effects of dietary supplement of AA and D₃ on egg quality attributes of hens at the mid-laying phase (week 39-55)

The external characteristics of egg from hens given diets supplemented with AA and D₃ at the mid phase of lay are shown in Table 3. The dietary supplemental AA had no significant effect (p>0.05) on EW (63.0 to 65.22), EL (56.66 to 57.46), ED (44.14 to 44.60), SW (6.30 to 6.84) and S% (10.04 to 10.48). Shell thickness of eggs from hens given 600 mg/kg supplemental AA (0.41) was similar (p>0.05) to those on 300 mg/kg AA (0.39) but thicker (p<0.05) than 0.37 in those on 0 mg/kg AA. Similarly, supplemental D₃ resulted in significantly thicker (p<0.05) egg shell in hens on 3000 IU/kg (0.42) compared to 0.37 in those on 0 IU/kg supplemental D₃. However, shell thickness of eggs from hens on 3000 IU/kg was similar (p>0.05) to 0.39 in hens fed 1000 and 2000 IU/kg D₃.

The internal characteristics of eggs from hens fed supplemental AA and D₃ at the mid phase of lay are shown in Table 4. Supplemental AA had no influence on albumen weight (39.60 to 39.74), albumen length (85.06 to 90.79), albumen diameter (70.98 to 75.51), albumen height (7.44 to 7.94) and albumen % (62.51 to 63.00). Yolk weight (14.88 to 15.22), yolk diameter (38.96 to 39.39), yolk height (13.16 to 14.89), yolk colour (5.90 to 6.50), yolk % (22.92 to 24.03) and Hu (84.54 to 88.03) also followed the same pattern. However, dietary supplement of D₃ on the other hand, had no significant influence (p>0.05) on monitored parameters except for albumen height, yolk diameter, yolk weight and HU. Albumen height of eggs from hens on 2000 IU/kg D₃ (8.37) was similar (p>0.05) to those on 1000 IU/kg (7.55) and 3000 IU/kg D₃ (7.78) but significantly higher (p<0.05) than 7.07 in those on 0 IU/kg D₃. Yolk weight of eggs from hens offered 2000 IU/kg (15.09) was significantly higher (p<0.05) than 14.37 in those on 1000 IU/kg but similar (p>0.05) to 15.09 and 14.87 in those on 0 and 3000 IU/kg D₃ supplementation. The Hu of eggs from hens on 2000 IU/kg (90.27) was higher than 85.50, 86.11 and 86.58 in eggs from hens on 0, 1000 and 3000 IU/kg D₃, respectively. Effects of interaction of AA and D₃ dietary supplementation was however significant (p<0.05) for yolk % and diameter.

Discussion

Early laying phase

Egg Weight (EW) is a parameter that could be determined without breaking an egg [26,27] and a vital egg characteristic which impacts highly on quality and grading [27,28]. Egg weight from this study was similar irrespective of whether they were obtained from hens fed vitamin supplemented diets individually or in combination. The eggs were however, within medium to large sized category (55.04-57.10 g) in line with Canadian egg size standards [29,30]. It was earlier noted that egg weight is directly proportional to the size of the albumen, yolk and shell [31]. Since egg weight is similar across treatments, it is expected that quality parameters for albumen, yolk and shell be the same.

The similarity in egg weight may also be an indication that the dietary supplementations did not confer any negative influence on the productivity of the hens in this study. Earlier reports showed improvement in production parameters with supplemental AA or D₃ in poultry [3,6,8,9,15-17,20]. Other reports also showed that AA did not proffer any beneficial influence on egg production parameters in chickens under normal environmental conditions [20,32,33]. Conversely, dietary AA supplementation did not influence observation in this study, in spite the hens were raised under temperature above thermoneutrality. The plausible reason for the insignificant effect of dietary supplement of AA on measured egg parameters would be that the effects of the vitamins on physical egg qualities at this stage were not pronounced as the stress of laying was mitigated with the adequate dietary D₃ levels. Since egg weight was reported to be directly proportional to albumen, yolk and shell [32,34], the similarity in albumen and yolk attributes monitored in vitamin treated and non-treated groups was unexpected. Yolk weight and yolk percentage showed a different trend as they were influenced by AA supplementation. Similarly, Adesola AA, et al. observed changes in albumen, yolk and shell weight in Venda hens fed AA supplemented diets [35].

Though, eggshell thickness at this phase of the study was similar across treatments, the values were higher than approximated 0.33 mm tagged suitable to withstand normal handling chance of conditions for at least 50% without breakage [36]. This suggests that the eggs were strong enough to withstand considerable handling or transportation pressures, contrary to observed low egg HU when diets of hens had no supplemental AA [37]. At week 28 of the birds’ age, authors observed that albumen index and Hu increased with AA supplementation, with noticeable changes in HU values at week 35. Yolk weight and % were the only internal egg parameters which improved with dietary supplement of AA in this study.

Dietary AA supplement has been implicated in the uptake of nutrients especially non-heme iron in plant-based feed to a more absorbable form for use by animals [38]. This attribute of supplemental AA in the feeds of animals may account for the observed increase in yolk weight and yolk percentage. The observed improvement in some yolk indices due to AA supplementation conformed to earlier reports on improved yolk attributes [39,40] though this did not translate to increase in deposition of calcium, iron and phosphorus as earlier reported by OA Ogunwole [18]. The scanty reports by Saki AA, et al. [41] on supplemental AA effect on egg yolk were inconsistent with earlier study of Çıftçı M, et al. [39] that hens offered supplemental AA, α-tocopherol or their combination had higher deposition of yolk in the egg compared to those on the control diet. Other authors [33,37] however, did not report such deposition in the egg.

Mid-laying phase

Although the influence of dietary vitamin supplements was not observed in the early laying phase of the hens. Howbeit, their roles were obvious at this stage as shown in Table 3. Amidst all egg assessed external parameters, egg shell thickness was the only parameter that was improved at this stage [42]. The influence of hen age on the observed increase in eggshell thickness compared to the early laying phase may not be totally erased as there has been documentation on changes in egg shell weight with the age of laying flock [43]. Shell thickness in this study (0.37-0.42 mm) were higher than 0.29 to 0.34 mm reported [44]; and 0.354 mm [45] in 20-24 weeks old hen but similar to 0.372 mm in 56-60 weeks old hens [45]. Another author [46] observed decreased shell thickness with increased hen age. Observed improvement in eggshell thickness at this phase of study compared to the early stage of lay could be that the hens had developed or improved in their efficient use and mobilization of calcium and phosphorus for shell production. This further explained the importance of vitamin C and D on egg shell calcification.

According to Animal Nutrition (7^th edition) [47] improved shell thickness in hens given AA could be adduced to importance of the vitamin in collagen metabolism sine qua non for the normal structure of egg shells. Also, dietary AA has the capacity to maintain dietary metallic ions in reduced state for enhance rapid absorption and utilization [48]. Thus, AA perhaps stimulated the mobilizing potential of D₃ for calcium and phosphorus in egg shell calcification. Also, the cofactor D₃, is imperative for the synthesis of calbindin required for active calcium uptake across intestinal membrane may facilitate Ca availability for shell synthesis [49].

Similarly, studies [4] and [50], shown that AA improved eggshell thickness while other reports [4,50,51] showed improved shell quality in hens given vitamin D₃ compared to the control group. Other authors [33,39,41,51], conversely, reported no effect of AA and D₃ [52-58] on monitored egg shell quality parameters.

The nutrition of the hen does not appear to have any great effect on albumen quality [59,60]. Authors [61] ascertained earlier that any nutrient which improves the rate of production would affect the quality of the eggs. Supplemental dietary AA had no influence on albumen, yolk and Hu at this phase. The Hu, yolk and albumen are vital indices of egg quality [41]. Haugh unit is a measure of the integrity or protein quality in the egg [24] and is a numerical expression of the quality of egg albumen [41]. Contrary to observations in this study, albumen quality and HU, were reportedly improved with AA supplementation [20,38]. Also, albumen height or HU or both were reportedly improved by adding AA to the diet of laying hens [59,60]. Other reports on effects of AA on albumen quality were inconsistent [33,39-41,60,61]. However, in this study, supplemental D₃ at 2000 IU/kg was observed to increase albumen height, yolk weight and diameter and HU. This suggests that supplemental vitamin D₃ increased the quality of protein in eggs of the hens when diets were supplemented with D₃. There was progressive increase in HU with higher dietary D₃ supplementation. Beyond 2000 IU/kg dietary supplementation with D₃, the HU diminished, thus suggesting the quality of protein may be affected by higher supplemental levels of D₃. Contrary to findings in this study, Observations [62] did not find any effect of D₃ intake on internal egg quality of second productive cycle hens which allied with other findings [63] on non-observable effect of D₃ administration in the diets of hens on yolk parameters and HU. Interaction effect of AA and D₃ was consistently positive on egg yolk % at the early and mid-laying stages. This suggests that both vitamins had synergistic effect and would positively enhance yolk percentage when combined in the diets of hens. Previous study had earlier demonstrated the synergistic relationship between AA and D₃ on increased phosphorus deposition in the yolk as reported [18]. Perhaps the increase in yolk percentage observed in this study may be due to increased nutrient deposition in the egg.

Conclusion

The influence of ascorbic acid and cholecalciferol on egg quality attributes of hens may be age related. Supplemental ascorbic acid and cholecalciferol improved eggshell thickness while supplemental cholecalciferol beyond 2000 IU/kg in hens’ diets lowered the quality of egg albumen and Haugh unit. Interaction of dietary supplement of ascorbic acid and cholecalciferol in the diets of laying hens enhanced egg yolk percentage and diameter.

It is recommended that further studies be conducted on the relationship between vitamin D and internal egg parameters.

Acknowledgement

None.

Funding Statement

Authors hereby express our unflinching appreciation to the Tertiary Education Funds (TETFUND) for the institutional grant availed to Prof. O. A. Ogunwole from which the research was funded.

Conflicts of Interest

The authors declare no conflict of interest.

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