Effects of Dietary Supplementation with Peanut Skin and Bromelain on Egg Production, Blood Parameters and Liver Fat Accumulation in Middle-age and Aged Laying Hens

Laying hens play a crucial role in agriculture and the global economy because their eggs serve as a vital source of essential nutrients for humans. Eggs are among the most affordable sources of animal protein worldwide (Bain et al., 2016). However, the production of older laying hens is associated with an increased incidence of broken eggs, resulting in considerable economic losses for the animal husbandry industry. Aging is a complex process molded by various environmental factors and molecular pathways; however, the regulatory mechanisms remain poorly understood. The primary reasons for chicken flock replacement are a decline in egg production and deterioration of eggshell quality, a trend often observed at approximately 72 w of age (Bain et al., 2016). In addition to a reduction in egg quantity and quality, various organs such as the ovaries age during egg production, (Liu et al., 2018), increasing the likelihood of the chickens developing osteoporosis (Beck and Hansen, 2004), diminished liver function and impaired feather conditioning (Su et al., 2006).
       
Fatty liver, also known as fatty liver hemorrhagic syndrome (FLHS), is a metabolic disorder that interferes with fatty acid metabolism in the liver and the synthesis and transport of low-density lipoproteins. FLHS is characterized by the accumulation of excessive intra-abdominal fat, a pale and fragile liver that may lack structural integrity and, in severe cases, liver rupture leading to massive intra-abdominal bleeding (Rozenboim et al., 2016). This syndrome commonly affects laying hens and causes abnormal accumulation of fat in the liver. These consequences often include reduced egg production, liver rupture and, in extreme cases, mortality. The factors contributing to FLHS are diverse and include genetics, nutrition, hormones and the environment (Squires and Leeson, 1988).
       
Peanuts (Arachis hypogaea) are technically classified as legumes and belong to the family Fabaceae. Although peanuts are legumes, they are commonly classified as an oilseed because of their high oil content. Peanuts are rich in protein, oil and fiber. Peanut skin (PS), in particular, is rich in antioxidants, such as anthocyanidins, catechin, epicatechin and resveratrol, which can decrease the rate of free radical-induced oxidation. PS is composed of cellulose (40.5%), hemicellulose (14.7%) and lignin (26.4%) (Bharthare et al., 2014). (Toomer et al., 2021), showed that PS does not affect production performance or egg quality in 40-w-old Hy-Line W36 hens (Toomer et al., 2021).
       
Bromeliads (Ananas comosus) belong to the family Bromeliaceae and are known for their high bromelain content. Bromelain exhibits various beneficial properties including anticancer (Pillai et al., 2014), antithrombotic (Metzig et al., 1999) and anti-inflammatory effects (Sahbaz et al., 2015). Bromelain, derived mainly from pineapple stems, is a cysteine endopeptidase primarily associated with protein hydrolysis (Hale, 2004). Bromelain contains nine enzymes with proteolytic activities, including stem and fruit bromelain, bromelain phosphatase, glycosidase, peroxidase, cellulase, glycoprotease and ribonuclease, (Chobotova et al., 2010). Yenice et al., (2019) reported that bromelain does not affect laying performance or egg quality but could increase the blood concentration of antioxidant enzymes, such as superoxide dismutase and glutathione peroxidase, in laying hens (Yenice et al., 2019). Similarly, Yan et al., (2020) used microorganisms to ferment pear peel waste, which was then added to the diet of laying hens. They found that bromelain had no discernible effect on the egg quality of laying hens (Yan et al., 2020).
       
This study aimed to examine the effects of diets supplemented with PS and bromelain on egg production, blood characteristics and fatty acid accumulation in middle-aged and aged laying hens.

Animal treatment and experimental design
 
In Trial 1 and Trial 2, 120, 62-w-old and 96, 93-w-old Hisex Brown laying hens were purchased from a commercial breeding farm and housed in cages. In Trial 1, the hens were randomly assigned to five groups with different dietary treatments: normal control (NC) (Supplementary Table 1), high PS supplementation (HPS; 5 g/kg), low PS supplementation (LPS; 2.5 g/kg), high-bromelain (HBN; 50 g/kg) supplementation and low-bromelain (LBN; 25 g/kg) supplementation groups. In Trial 2, the hens were randomly assigned to four groups with different dietary treatments: basal diet (normal control, NC), high PS supplementation (HPS; 5 g/kg), high-bromelain (HBN; 50 g/kg) supplementation and a combination of PS powder (2.5 g/kg) and bromelain (25 g/kg) (PS and BN mixed; PSBNM) supplementation groups. Each treatment was performed in triplicate, with eight hens per replicate. The basal diet was purchased from a commercial feed mill and formulated according to the requirements of the laying hens. The hens had ad libitum access to food and water.


 
Egg production performance and quality
 
During the 8-w study period, feed consumption was recorded bi-weekly and egg production was documented daily. Egg production was expressed as the average daily production per hen, calculated by dividing the total number of eggs laid by the number of days and averaged per treatment. The egg production rate, feed-to-egg ratio and daily feed intake were calculated for each experimental phase and for the overall trial period. There were three replicates for each group and eight eggs per replicate were selected for egg quality analysis. The egg weight, Haugh unit (HU) and yolk color were determined using an egg multi-tester (Touhoku Rhythm Co. Ltd., Tokyo, Japan); eggshell breaking strength was assessed with an eggshell force gauge (Model II, Robotmation Co., Ltd., Tokyo, Japan); and eggshell thickness was measured using a dial pipe gauge (Ozaki MFG. Co., Ltd., Tokyo, Japan), considering the average thickness from the rounded, pointed and the middle ends and excluding the inner membrane.
 
Collection and examination of blood specimens
 
At the end of Trials 1 and 2, a 25G, 1.6-cm needle was used to collect blood samples from the wing veins of the 120 and 96 laying hens, respectively. The serum samples were separated by centrifugation at 3000 x g for 20 min at 4°C. Aliquots were stored at -80°C until analyzed for blood traits using colorimetric analysis on autoanalyzer (Automatic Biochemical Analyzer, RA-1000; Bayer Corp., Tarrytown, NY, USA).
 
Carcass traits and liver fat accumulation
 
At the end of Trials 1 and 2, six animals were randomly selected from each treatment group and their live weights were measured. Carcass data, including the weights of the carcass, wet liver, abdominal fat, ovaries, total reproductive tract, as well as the weight of other organs, were measured post-slaughter. Liver samples were scored using the fatty liver index (Supplementary Fig 1).


 
Statistical analysis
 
All groups were analyzed using a two-way analysis of variance, followed by Tukey’s post-hoc test for pairwise comparison. The results in all experiments were considered significantly different at a p-value <0.05. Pearson’s correlation coefficients were calculated to investigate the relationships between the physicochemical characteristics of peanut and bromelain and egg production, egg quality and blood biochemistry parameters of laying hens, with probabilities considered significant at the 5% level (p<0.05).

Production performance
 
The effects of dietary supplementation with PS and/or bromelain on the egg production of 62- and 93-w-old laying hens are presented in Table 1 and 2. HPS and HBN supplementation resulted in an increase (p<0.05) in the daily feed intake of the 62-w-old laying hens (Table 1). Additionally, LPS and HBN increased (p<0.05) egg production and the number of eggs laid daily. There were no differences in average feed intake (p>0.05) among the 93-w-old laying hens (Table 2). However, hens in the HPS (but not in the HBN) group, had higher feed-to-egg ratios and lower egg production and daily laying rates compared to those in the NC group (p<0.05). Supplementing dietary intake with 5 g/kg PS led to decreased performance in hens in certain cases; however, this decrease was not observed in the bromelain group.




 
Egg quality
 
The effects of a dietary supplemented with PS and/or bromelain on egg quality in middle-aged and aged laying hens are presented in Supplymentary Table 2 and Table 3. Compared to the control group, there were no differences in albumen height, Haugh unit, eggshell strength, eggshell thickness, yolk height, yolk diameter, or yolk index among all groups (p>0.05) of the 62-and 93-w-old laying hens. However, dietary supplementation with PS and bromelain resulted in yellower yolk color compared to the NC group (p<0.05) in 62- and 93-w-old laying hens. The egg weight of the HBN group decreased (p<0.05) in the 93-w-old laying hens. PS and bromelain had no effect on most egg quality parameters in middle-aged and aged laying hens but enhanced yolk color.



​
 
Blood biochemistry parameters
 
The effects of a diet supplemented with PS and/or bromelain on blood plasma biochemical parameters of middle-aged and aged laying hens are presented in Table 4 and Supplymentary Table 3. Compared with the control group, there were no differences in glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), total protein (TP), albumin, globulin, albumin/globulin ratio (A/G), creatinine and triglyceride (TG) among all groups (p>0.05) of the 62- and 93-w-old laying hens. However, the HBN and LBN group showed an increase in blood urea nitrogen (BUN) content and low density lipoprotein-cholesterol (LDL-C) levels in the 62-w-old laying hens (p<0.05).

​


 
Carcass traits and fatty liver
 
The effects of dietary supplementation with PS and/or bromelain on the carcass traits of middle-aged and aged laying hens are presented in Supplymentary Table 4 and  Table 5. There were no differences in liver, abdominal fat, carcass, ovarian, reproductive tract weights, or other organ weights among all groups (p>0.05) when compared to the control group. The highest wet liver weight was observed in the NBN group (in 93-w-old laying hens). Bromelain and PS have protective effects against non-alcoholic fatty liver disease. The 93-w-old laying hens treated with bromelain had a lower lesion score (1.48±1.12) compared to the NC (1.61±0.98) and PS (1.86±1.08) and PSBNM groups (1.87±1.03) (Table 6).



​

​
 
Effect of peanut skin and bromelain on egg production
 
In this study, the middle-aged laying hens had higher egg production in the LPS group than the NC group, whereas in the HPS group, middle-aged hens demonstrated better performance than aging hens. Because fat accumulation causes irreversible liver damage in aging laying hens, PS supplementation cannot improve the decline in egg production caused by fatty liver in these hens. A previous study investigated the enrichment of consumable food products by incorporating waste byproducts rich in polyphenolic compounds into poultry feed rations (Toomer et al., 2021). Enhancing the value of agricultural waste byproducts, such as incorporating PS into poultry feed, can foster sustainable agriculture and offer innovative solutions for utilizing these byproducts, which often has considerable residual nutritional value.
 
Effect of peanut skin and bromelain on egg quality
 
The decline in egg production is attributed to health after prolonged egg production in the late-laying stage (Vlèková  et al., 2019). The LPS and HBN group of 62-w-old laying hens had higher egg production than the control, HPS and LBN groups. Although overall egg quality parameters showed no substantial improvements over the control group, the PS and bromelain groups of 62- and 93-w-old laying hens showed enhancements in yolk color. These findings suggest that PS and bromelain supplementation might offer specific benefits to egg quality. Contrary to a previous report by (Toomer et al., 2021), egg yolk color in the current study was deeper in hens fed a diet containing PS and bromelain. β-carotene is a naturally occurring compound that serves as a precursor to vitamin A and functions as an antioxidant (Mezzomo and Ferreira, 2016). β-carotene has been found in bromelain (Ataide et al., 2017) and peanut oil (Pattee and Purcell, 1967). A previous report indicated that the supplementation of tomato powder to the diet of laying hens could sufficiently transfer carotenoids to the yolk, thereby improving both yolk color and oxidative stability (Akdemir et al., 2012). Although we did not evaluate the carotenoid content in the yolk in the current study, the diet supplemented with PS and bromelain substantially enhanced yolk color. This finding implies that PS and bromelain may transfer and retain β-carotene in the yolk, leading to improved yolk pigmentation.
 
Effect of peanut skin and bromelain on blood biochemistry parameters
 
Xiang et al., (2020) reported that PS extract could decrease inflammation and maintain gut microbiota homeostasis, thereby improving the symptoms of type 2 diabetes mellitus in mice (Xiang et al., 2020). Bromelain substantially decreases fasting blood glucose levels in mice with streptozotocin- induced type 1 diabetes (Abo El-Magd  et al., 2021). PS is rich in flavonoids, which mitigate glucose intolerance and enhance insulin sensitivity to regulate blood glucose. These findings suggest that although PS and bromelain supplementation did not affect most blood biochemical parameters, they were effective in lowering blood glucose content in aged laying hens, indicating their potential benefits for metabolic health.
 
Effect of peanut skin and bromelain on carcass traits and fatty liver
 
In this study, the results indicated a high prevalence of FLHS lesions among aged laying hens exposed to PS. Nevertheless, bromelain tended to promote the repair of fatty liver in aged laying hens. As a substantial portion of the lipids in egg yolk is synthesized by the liver, aging laying hens are more prone to developing fatty liver syndrome (FLS) (Squires and Leeson, 1988; Shini et al., 2019). Fatty liver disease in laying hens is induced by free fatty acids through the inhibition of AMPK signaling (Huang et al., 2022). Our results showed that PS increased, while bromelain decreased, the percentage of hepatic lipid accumulation in aging laying hens, respectively. Therefore, bromelain may be correlated with fatty acid formation in the liver via the AMPK signaling pathway.
       
Pineapple (Ananas comosus) is a short-stemmed, terrestrial bromeliad and an edible member of the Bromeliaceae family. Bromelain, a protease extracted from pineapple, is used as a medicinal herb in several indigenous cultures (Mondal et al., 2011). A mixture of PS extracts substantially improved hepatic lipid accumulation in mice (Yi et al., 2023). Our findings indicate that PS, but not bromelain, led to lower production (lower egg production, daily laying rate and feed-to-egg-ratio) and increased liver lipid accumulation in laying hens, which is inconsistent with previous reports (Toomer et al., 2021; Yi et al., 2023). Although the daily feed intake in the current study was similar in both groups, the high tannin and low calorie content of PS may have contributed to the downregulation of egg production in aged laying hens. However, the LPS group with low tannin content improved the production of middle-aged laying hens. In the past, tannins were considered antinutritional compounds in diets for monogastric animals. However, low concentrations of tannins can improve feed intake, nutrition and growth performance in monogastric animals (Schiavone et al., 2008). A combination of LPS and HBN might improve the performance of young and middle-aged hens.

In conclusion, this study demonstrated that PS and bromelain could improve egg production performance and enhance yolk color through β-carotene in middle-aged laying hens. This can be beneficial in increasing vitamin A supplementation in egg consumers. In future research, we will investigate the supplementation of new compounds in combination with PS or bromelain in the diet of laying hens.

The authors thank the Animal Research Farm of Agriculture College, National Chia-Yi University, Taiwan for providing the facilities and animal care required for this study.
 
Informed consent
 
All experimental procedures involving animals adhered to the guidelines of the Institutional Animal Care and Use Committee of National Chiayi University.

The authors declare no conflict of interest.