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This study evaluated the impact of dietary supplementation with black and red pepper on blood biochemical components and egg yolk lipid composition in laying hens. A total of 252 hens were divided into seven treatment groups, each with 36 birds, and replicated three times with 12 birds per replicate in a completely randomized design. The hens received standard diets with varying concentrations of black pepper and red pepper additives. Blood samples from each group were analyzed for haematological and biochemical parameters such as total protein, albumin, cholesterol, triglycerides, glucose, and antioxidant status. Egg yolk samples were also analyzed for lipid composition, including triglycerides and cholesterol content. Significant variations in blood components were observed, with the control group showing a packed cell volume of 27.77% and serum cholesterol levels of 164 mg/dL, compared to 32% and 113 mg/dL in the treated groups, respectively. These findings indicate that black pepper and red pepper supplementation can affect the metabolic status of the hens. Moreover, triglyceride levels in the egg yolk were significantly reduced in the treated groups, averaging 1489 mg/100 g compared to 2058 mg/100 g in the control group. These results suggest that pepper additives can improve the quality and nutritional profile of the eggs, with alterations in fatty acid composition and cholesterol levels presenting potential benefits for consumers seeking healthier dietary options. Overall, the study suggests that dietary supplementation with black and red pepper positively impacts the blood biochemistry of laying hens and enhances the nutritional value of their egg.

Introduction

Laying hens are essential contributors to the poultry industry, providing a steady supply of eggs for human consumption. The nutritional quality of eggs is influenced by various factors, including the diet of the hens. In recent years, researchers have shown interest in exploring the effects of natural additives on the biochemical constituents of blood and the lipid profile of egg yolks in laying hens.

One such group of natural additives is black pepper (Piper nigrum) and red pepper (Capsicum spp.). Both black pepper and red pepper contain bioactive compounds, including alkaloids, flavonoids, and phenolic compounds, which have been reported to possess various health-promoting properties, such as antioxidant and anti-inflammatory effects [1]. Several studies have investigated the effects of black pepper and red pepper additives on the performance and physiological parameters of laying hens [2], [3]. However, monitoring the blood profile of hens can provide valuable insights into their overall health and physiological status. Blood biochemical components, such as cholesterol, triglycerides, and liver enzymes, are indicators of lipid metabolism and liver function. Changes in these parameters can reflect the impact of dietary interventions on the hens’ metabolism and well-being. Similarly, analyzing the lipid profile of egg yolks is crucial as it directly relates to the nutritional quality of eggs. Egg yolk lipids play a vital role in providing essential fatty acids, vitamins, and antioxidants to consumers. The composition of these lipids, including the types and proportions of fatty acids, affects the nutritional value and potential health benefits associated with egg consumption [4]. Therefore, assessing the egg yolk lipid profile allows us to evaluate the impact of dietary interventions, such as the addition of black pepper and red pepper, on the nutritional composition of eggs.

Furthermore, understanding the effects of black pepper and red pepper additives on blood and egg yolk profiles can help optimize poultry nutrition and production practices. These spices have been reported to possess various bioactive compounds, including antioxidants, antimicrobial agents, and anti-inflammatory substances. By assessing their impact on blood and egg yolk profiles, we can gain insights into the potential health-promoting effects of these additives in laying hens. This knowledge can guide the development of dietary strategies to enhance the nutritional quality of eggs and promote the birds’ overall well-being [3]. Therefore, this study aimed to investigate the impact of black pepper and red pepper additives on the blood biochemical constituents and egg yolk lipid composition of commercial laying hens. By examining these parameters, we can better understand the potential benefits and implications of using these natural additives in laying hens’ diets.

Materials and Methods

Experimental Location

The research was conducted at the poultry facility of the Faculty of Agriculture’s Teaching and Research Farm, situated within the University of Benin in Benin City, Edo State, Nigeria. This farm is positioned at coordinates latitude 6° 20′ 1.32″ N and longitude 5° 36′ 0.53″ E, with respect to the Greenwich meridian. The region experiences an average annual temperature of 34°C, accompanied by an average yearly rainfall of 2000 mm and a relative humidity of 72.5% [5].

Preparation of Experimental Components

The research involved procuring dried test ingredients from the nearby market in Benin City. Thorough inspection was conducted to verify uniform drying and absence of spoilage. Subsequently, the red pepper was finely ground into powder and securely stored in airtight containers, awaiting integration into the experimental diets. For the black pepper, it was ground in small batches, carefully calibrated to match the intended feed volume for formulation. This approach was essential to preserve the distinct aroma of the pepper within the formulated diets.

Formulation of Experimental Feeds

Various diets were formulated for the layers, aligning with set guidelines [6], [7]. Throughout the study period, the hens were provided with a layer’s mash containing 16% crude protein and 2600 Kcal/kg ME. The test ingredients, namely black pepper, red pepper, and their various combinations, were incorporated at different inclusion levels, as depicted in Table I:

Ingredients (%) 1 2 3 4 5 6 7
Maise 56.85 56.85 56.85 56.85 56.85 56.85 56.85
Soybean meal 18.00 18.00 18.00 18.00 18.00 18.00 18.00
Wheat bran 15.00 14.00 13.50 14.00 13.50 14.00 13.50
Bone meal 2.65 2.65 2.65 2.65 2.65 2.65 2.65
Periwinkle shell/Limestone 7.00 7.00 7.00 7.00 7.00 7.00 7.00
Common salt 0.35 0.35 0.35 0.35 0.35 0.35 0.35
Vitamin/Mineral premix 0.15 0.15 0.15 0.15 0.15 0.15 0.15
Black pepper 1.00 1.50 0.50 0.75
Red pepper 1.00 1.50 0.50 0.75
Total percentage (%) 100 100 100 100 100 100 100
Crude protein (%) 16.10 16.06 16.05 16.06 16.05 16.06 16.05
Metabolisable energy (Kcal/Kg) 2610.00 2607.53 2600.75 2607.53 2600.75 2607.53 2600.75
Table I. Composition of Experimental Laying Hens’ Diets (%)

Test Subjects and Research Layout

A total of 252 laying hens, aged twenty weeks, was procured from a reputable supplier. These hens were subsequently assigned in a random manner to seven distinct treatments, each comprising 36 birds. Within each treatment, there existed an additional partition, resulting in three replicates, each consisting of 12 birds. The experimental framework was established following a completely randomized design.

Animal Care and Facility Maintenance

Ahead of the birds’ arrival, meticulous cleaning and disinfection procedures were carried out within and around the pens. All materials utilized throughout the study underwent a thorough cleaning and disinfection process.

The experimental laying hens were accommodated within a deep litter management system, wherein wood shavings served as the bedding material. To mitigate aggressive competition for feed and water, ample drinkers and feeders were made available throughout the experimental phase. The birds had unrestricted access to both feed and water. Stringent adherence to daily and routine management protocols encompassing feeding, watering, litter upkeep, medication, and vaccination was maintained. Notably, a lighting regimen of 16 hours of illumination and 8 hours of darkness was implemented for the laying hens during the course of the study.

Haematology and Serum Biochemistry Studies

At the end of the experiment, six birds per treatment (two per replicate) were tagged, isolated, and starved of feed overnight. Blood samples were collected from the brachial wing vein. The brachial vein is situated on the underside of the wing and runs between the biceps and triceps muscles and bifurcates just proximal to the elbow. Venepuncture were at the bifurcation with the aid of small guage needle and the blood samples collected in labeled sterile universal bottles containing ethylene diamine-tetraacetic-acid (EDTA). Serum samples were collected in heparin bottles without anticoagulant. Samples were also collected in bottles containing fluoride oxalate and used in determining blood glucose levels.

Blood samples placed in bottles containing EDTA were utilized for the analysis of a comprehensive blood profile, including parameters such as red blood cell count, haemoglobin levels, packed cell volume, platelet count, lymphocyte count, and white blood cell count. This analysis was carried out with the assistance of an automated analyzer. The serum was isolated and extracted through centrifuging the coagulated blood at 2500 revolutions per minute for duration of 10 minutes. This procedure was performed within 30 minutes of sample collection to ensure timely processing. Individual samples were analyzed to ascertain liver function, kidney function, antioxidant status and lipid profile of the birds.

Determination of Egg Yolk Lipid Profile

Firstly, each representative sample of egg yolk was collected, appropriately labeled and stored. Next, the lipids were extracted from the egg yolks using the Bligh and Dyer extraction technique. This method use solvents and extraction protocols to isolate the lipids from other components of the yolk. Once the lipids were extracted, they were separated into different lipid classes using the high-performance liquid chromatography (HPLC) technique. After separation, the individual lipid classes were identified and quantified by mass spectrometry (MS) in combination with chromatographic techniques [8].

Statistical Analysis

The gathered data underwent evaluation through a one-way analysis of variance (ANOVA) utilizing SAS’s general linear model procedure [9]. Disparities among the means of the treatments were subsequently assessed using Duncan’s multiple range test [10]. The established threshold for statistical significance was predetermined at p < 0.05.

Results

Haematological Profile

The haematological profile of commercial laying hens placed on basal and test diets with black pepper, red pepper and their combinations as additives is given in Table II. Among all assessed parameters, only red blood cell count and platelets had no significant difference between treatments.

Parameters T1 T2 T3 T4 T5 T6 T7 S.E.M.
White blood cell (×103/μL) 124.63ab 129.80ab 116.47bc 94.17c 111.63bc 127.33ab 144.57a 8.28*
Neutrophyll (%) 5.93a 3.63b 2.00b 1.53b 2.80b 2.53b 2.87b 0.69*
Lymphocytes (%) 76.07C 82.97b 87.43ab 89.93a 80.90bc 84.87ab 83.00b 2.01*
Granulocytes (%) 16.23a 12.90ab 9.83bc 8.20c 11.40bc 12.03ab 13.83ab 1.39*
Packed cell volume (%) 27.77c 31.03bc 32.17ab 32.50ab 36.50a 34.97ab 34.30ab 1.34*
Haemoglobin (g/dL) 10.70b 9.90b 10.43b 10.77b 11.80a 10.83ab 10.73b 0.32*
Red blood cell (×103/μL) 2.65 2.36 2.53 2.49 2.76 2.58 2.52 0.09NS
Mean cell volume (fl) 122.93c 130.80a 125.70bc 128.93ab 128.73ab 127.17ab 127.23ab 1.21*
Mean corpuscular haemoglobin (pg) 40.07d 41.57bc 40.87cd 42.83a 42.80a 42.03ab 42.30ab 0.38*
Mean corpuscular haemoglobin concentration (g/dL) 32.57a 31.73b 32.47a 33.10a 33.20a 32.67a 33.20a 0.23*
Platelet (×103/μL) 24.67 31.33 30.33 28.33 16.67 48.00 25.67 7.03NS
Table II. Effect of Black Pepper, Red Pepper and their Combinations on Haematological Profile of Laying Hens

For white blood cell counts, Treatment 7 (0.75% each of black pepper and red pepper) had the highest count of 144.57×103/μL, though not significantly different from Treatments 1, 2 and 6 had 124.63 × 103/μL, 129.80 × 103/μL, and 127.33 × 103/μL, respectively. Treatment 4 had the least (94.17 × 103/μL) and was not significantly different from values obtained from both Treatment 3 (116.47 × 103/μL) and Treatment 5 (111.63 × 103/μL).

In comparison to the control, the neutrophil percentage was significantly reduced in the treated groups ranging from the least (1.53%) in Treatment 4 to 3.63% in Treatment 2. On the other hand, the control had an elevated and significantly different (p < 0.05) count of 5.93%. The reverse was the case in lymphocyte percentage with the control having the least and significantly different count of 76.07%. The treated groups had higher lymphocytes with varying statistical similarities, with Treatment 4 having the highest value of 89.93%.

The granulocyte concentration comprised eosinophils, basophils and monocytes percentages and was lowest in Treatment 4 (8.20%) though not significantly different from Treatment 5 (11.40%). The other treatments shared some similarities with the control which had the highest concentration of 16.23%.

Packed cell volume (PCV) was significantly improved in the treated groups with the highest value of 36.50% recorded in Treatment 5. The hens on the basal diet (control) had the least PCV value of 27.77%, with no significant difference from Treatment 2 (31.03%). The haemoglobin value in Treatment 5 (11.80 g/dL) was the highest and significantly elevated above what was obtained in the control (10.70 g/dL), with statistical similarity with only Treatment 6 (10.83 g/dL). The other treatments had no significant difference from the control, with Treatment 2 having the least value of 9.90 g/dL.

Serum Biochemistry Profile of Laying Hens

The result of the serum biochemistry of the laying hens fed varying levels of black pepper, red pepper and their combinations is given in Table III. No significant differences were observed among the assessed parameters in sodium, bicarbonate, urea, creatinine, glucose, and globulin values. Wide-ranging significant differences with no pattern were observed in the rest of the parameters.

Parameters T1 T2 T3 T4 T5 T6 T7 S.E.M.
Potassium (μmol/L) 4.70c 4.90bc 5.53a 5.07b 5.63a 5.00b 4.90bc 0.12*
Sodium (μmol/L) 149.33 150.33 150.33 149.00 149.00 147.00 149.33 1.45NS
Chloride (μmol/L) 100.00b 107.33bc 89.67a 112.00c 105.33bc 108.00bc 106.00bc 3.06*
Bicarbonate (HCO3) (μmol/L) 18.33 15.00 13.67 17.67 16.33 15.33 16.67 1.40NS
Urea (μmol/L) 8.00 3.00 4.33 3.67 3.00 4.00 3.67 1.48NS
Creatinine (mg/dL) 0.63 0.43 0.53 0.46 0.47 0.53 0.47 0.50NS
Glucose (mg/dL) 206.33 204.67 223.33 230.67 228.00 228.33 213.67 6.81NS
Total protein (g/dL) 6.30a 5.40b 4.37c 5.10bc 5.13bc 5.23bc 5.03bc 0.29*
Albumin (g/dL) 2.60a 2.23bc 1.93e 2.03de 2.10cd 2.43ab 2.33bc 0.08*
Globulin (g/dL) 3.63 3.13 2.43 3.03 3.03 2.77 2.70 0.26NS
Table III. Serum Metabolites of Laying Hens

Potassium levels of 5.53 μmol/L and 5.63 μmol/L were obtained from Treatments 3 and 5, respectively. These were significantly different from the other treated groups and the control. The least level of 4.70 was obtained from hens in the control though not significantly different from Treatments 2 and 7 which had a level of 4.90 μmol/L each.

Chloride level was significantly reduced in hens offered the 1.5% black pepper diet (89.67 μmol/L) in comparison to the highest value of 112 μmol/L obtained from Treatment 4 (1% red pepper). The other treated groups fell within the range of 105.33 μmol/L–108 μmol/L and all had no significant difference (p > 0.05) from what was obtained from the control.

The treatments influenced the total protein level indicated by the significant drop in the values obtained in the treated groups compared to the control that had the highest level of 6.30 g/dL. The total protein levels in the treated groups were within the range of 4.37 g/dL in Treatment 3 to 5.40 g/dL in Treatment 2. The control had the highest albumin level (2.60 g/dL), which was only statistically comparable to 2.43 g/dL obtained from Treatment 6. The other treatments had lower levels significantly different from the control with Treatment 3 having the lowest concentration of 1.93 g/dL.

Antioxidant Status of Laying Hens

The values of the enzymes assessed to judge the antioxidant capacity of the laying hens fed black pepper, red pepper, and their combinations are given in Table IV. Alkaline phosphatase (ALP) level was lowest in the control (195.67 IU/L) and was also not significantly different (p > 0.05) from the values obtained from Treatment 4 (214.67 IU/L) and Treatment 7 (245.33 IU/L). Treatment 6 had the highest level of 319.33 IU/L, with no significant difference from the other treatments that had elevated levels ranging from 258.67 IU/L in Treatment 3 to 290.67 IU/L obtained from Treatment 2. Alanine aminotransferase (ALT) had an elevated level of 15 IU/L in the hens fed the basal diet (control) and was significantly different (P < 0.05) from the treated groups. The treated groups had no significant difference (p > 0.05) between them, with Treatment 6 having the least value of 8.67 IU/L, while the highest value among them was 11.33 IU/L obtained from both Treatments 3 and 7.

Parameters T1 T2 T3 T4 T5 T6 T7 S.E.M.
Alkaline phosphatase (ALP)(IU/L) 195.67d 290.67ab 258.67abc 214.67cd 284.67abc 319.33a 245.33bcd 21.64*
Alanine aminotransferase (ALT)(IU/L) 15.00a 9.33b 11.33b 9.67b 10.33b 8.67b 11.33b 0.98*
Aspartate aminotransferase (AST)(IU/L) 93.00bc 96.00bc 84.67c 109.33ab 95.33bc 121.33a 101.33bc 6.93*
Table IV. Antioxidant Statue of Laying Hens Fed Black Pepper and Red Pepper Additives

Depressed levels of Aspartate aminotransferase (AST) were obtained in Treatment 3 (84.67 IU/L), control (93 IU/L), Treatment 5 (95.33 IU/L), Treatment 2 (96 IU/L) and Treatment 7 (101.33 IU/L) with no significant difference within them. The hens offered Treatment 6 had the highest level of 121.33 IU/L which had no significant difference from 109.33 IU/L obtained from the hens fed the Treatment 4 diet.

Serum Lipid Profile of Laying Hens

The serum lipid profile of laying hens fed the basal and test diets is given in Table V. No significant difference was observed in triglycerides, low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) values.

Parameters T1 T2 T3 T4 T5 T6 T7 S.E.M.
Total cholesterol 164.00a 101.00b 135.67ab 111.00b 107.67b 124.00b 102.00b 18.84*
Triglycerides 433.67 252.67 332.33 348.33 261.00 387.00 301.33 129.99NS
HDL 10.67c 18.00ab 11.67c 14.00bc 22.67a 14.67bc 15.00bc 2.82*
LDL 66.60 32.47 57.53 27.33 32.80 31.93 26.73 18.08NS
VLDL 86.73 50.53 66.47 69.67 52.20 77.40 60.27 25.69NS
LDL/HDL ratio 6.08a 1.83b 5.13a 1.99b 1.41b 2.25b 1.81b 0.79*
Table V. Serum Lipid Profile of Laying Hens Fed Black Pepper and Red Pepper Additives

Total cholesterol was highest in the control (164 mg/dL) though statistically comparable to 135.67 mg/dL obtained from Treatment 3. For both treatments, there was a significant difference between them and the rest of the treatments. The rest of the treated groups had significantly lower cholesterol levels between 101 mg/dL in Treatment 2 to 124 mg/dL in Treatment 6, with no significant difference (p > 0.05) between them.

High-density lipoprotein was highest in hens that were fed the Treatment 5 diet (22.67 mg/dL), which also had no significant difference from what was obtained in hens in Treatment 2 (18 mg/dL). The other treated groups had no significant difference (P > 0.05) from the lowest value (10.67 mg/dL) obtained from hens in the control.

LDL/HDL ratio also referred to as the Atherogenic index, was highest in both the control (6.08) and Treatment 3 (5.13). The rest of the treated groups had significantly lower ratios with no significant differences between them, and Treatment 5 (1.5% red pepper) had the lowest ratio of 1.41.

Egg Lipid Profile

The lipid profile of egg yolk from hens fed black pepper, and red pepper and their combinations as additives is given in Table VI. Yolk cholesterol level was highest (2008.67 mg/100 g) in Treatment 4 and the least (1343.67 mg/100 g) obtained from Treatment 6. Apart from Treatment 4 that had the highest cholesterol level, the rest of the treated group had significantly lower values than the 1886.67 mg/100 g obtained from the control. Triglyceride levels in the control and Treatment 4 were higher (2058.67 mg/100 g and 1856.33 mg/100 g, respectively) with a significant difference from the rest of the treatments. For the rest of the treatments, no significant differences were observed among them, and values obtained varied from 1349 mg/100 g in Treatment 5, to 1492.33 mg/100 g in Treatment 2.

Parameters T1 T2 T3 T4 T5 T6 T7 S.E.M.
Total cholesterol (mg/100 g) 1886.67ab 1509.33cd 1690.67bc 2008.67a 1581.33c 1343.67d 1634.67c 72.96*
Triglyceride (mg/100 g) 2058.67a 1492.33b 1387.67b 1856.33a 1349.00b 1435.67b 1417.33b 117.20*
HDL (mg/100 g) 228.00c 358.00b 381.67b 530.67a 352.33b 388.00b 601.00a 39.53*
VLDL (mg/100 g) 411.73a 298.47b 277.53b 371.27a 269.80b 287.13b 283.47b 23.44*
LDL (mg/100 g) 1246.93a 852.87bcd 1031.47abc 1106.73ab 959.20abc 668.53d 750.20cd 95.05*
LDL/HDL ratio 5.52a 2.52bc 2.72b 2.11bc 2.88b 1.72bc 1.29c 0.40*
Table VI. Effect of Black Pepper, Red Pepper, and their Combinations as Additives on Egg Yolk Lipid Profile

Treatment 4 and Treatment 7 both had higher levels of high-density lipoprotein (530.67 mg/100 g and 601 mg/100 g respectively) with no significant difference between them. The rest of the treated groups maintained lower levels within the range of 352.67 mg/100 g (Treatment 5) and 388 mg/100 g (Treatment 6) with no significant differences between them. The control however had the least and significantly different value of 228 mg/100 g. Very low-density lipoprotein followed the same pattern as what was observed in triglyceride levels, with the control and Treatment 4 having higher and significantly different values in comparison to the other treatments (411.73 mg/100 g and 371.27 mg/100 g, respectively). The other treated groups had similar lower levels with the least level of 269.80 mg/100 g in Treatment 5. Elevated levels of low-density lipoprotein were observed in Treatments 1, 3, 4 and 5 with no significant differences between any of them. Other treatments had lower levels with varied significant differences, with Treatment 6 having the least concentration of 668.53 mg/100 g. LDL/HDL ratio was highest in the control (5.52) and was significantly different from the treated groups. Depressed levels were obtained from the treated groups, with the lowest ratio of 1.29 obtained from Treatment 7.

Discussion

Haematological Profile of Laying Hens

The analysis of animals’ blood characteristics provides a method for identifying particular health alterations that might not be visibly discernible through physical assessment [11]. Nevertheless, these changes can significantly impact the well-being of birds [12]. Research has established that animals with decreased levels of white blood cells face an elevated susceptibility to disease infection, while those with intermediate counts can produce antibodies, heightening their ability to combat illnesses [13]. Additionally, this adaptability aids them in acclimatizing to local environmental conditions and prevalent diseases [14]. The range of white blood cells in the present study (94.17 − 144.57 × 103/uL) was higher than the range (15 to 30 × 103 cells/mm3) listed for most bird species [15]. In this study also, some significant changes were observed in other measured haematological parameters, which are comparable to that of chickens grown under relatively stressful conditions. For example, the recorded decreased lymphocyte value in the control treatment is in alignment with research that demonstrated administering adrenocorticotropic hormone (ACTH) to poultry leads to a decline in the number of circulating lymphocytes [16]. This reduction occurs as a result of the heightened ratio of circulating heterophiles to lymphocytes, which stands as a prevalent stress marker for birds [17]. It should be noted that lymphocytes constitute the highest component of white blood cells in poultry, and their interactions are essential in developing an immune response. It has been reported that increased lymphocyte counts in the blood following an increase in white blood cell count can play an important role in stimulating the immune system of animals [18]. In the present study, the lymphocyte values were significantly higher in the treated groups than in the control, though with no particular pattern related to the white blood cell count.

According to Pendl [19], packed cell volume (PCV), haemoglobin, and mean corpuscular haemoglobin (MCH) constitute key parameters for assessing circulatory erythrocytes, playing a crucial role in diagnosing anaemia. Furthermore, these values serve as important indicators of the bone marrow’s ability to generate red blood cells in mammals. Hence, the significant elevation in PCV alongside a minor rise in haemoglobin levels within this current investigation suggests an augmentation in the blood’s capacity to carry oxygen. It has also been observed that a PCV value greater than 56% indicates dehydration in most birds [20]. The present study observed significant differences in PCV values between the treated groups and the control. None of the values was above 37%, with the control having a substantially low value of 27.77%, while Treatment 5 had the highest value of 36.50%. However, the values obtained from this study are within the reference range of 25%–45% for layers [21] and marginal variance from the recommended of 28%–33% from another study [22]. Only the treated group in this study was within the 31%–42% range referenced for poultry birds raised in some parts of Nigeria [23].

Mean corpuscular haemoglobin and mean corpuscular haemoglobin concentrations are vital blood metrics that establish the existence and intensity of anaemia [24]. A decline in haemoglobin, MCV, and MCH levels among avian species signifies their susceptibility to stressors and their ineffective coping mechanisms [25]. In the present study, significant differences were observed in haemoglobin but were within the recommended reference range of 9–14 g/dL [23]. It was also reported that a low mean corpuscular haemoglobin concentration (MCHC) value of less than 29 g/dL can be attributed to iron and other trace element deficiencies [26]. The MCHC values observed in this investigation (ranging from 31.73 g/dL to 33.20 g/dL) fall within the range of 31.00 g/dL to 33.88 g/dL as cited by [25] and are slightly elevated compared to the range of 24.00 g/dL to 31.00 g/dL indicated in another study [27].

Serum Biochemistry of Laying Hens

Biochemical blood parameters often correlate with the state of health and many serve as essential markers for evaluating the nutritional and physiological well-being of birds [28]. The supplementation of the feed additives in this study significantly influenced potassium, chloride, blood protein and albumin levels. A total protein range of 11.40–12.10 g/L in commercial layers during lay was reported in a similar study [29]. The result of the present study was within the referenced ranges given above, with the exceptions of the control and the 1% black pepper treatments that had higher values of 6.30 g/dL and 5.40 g/dL, respectively. The result of total protein obtained from the treated group in this study (4.37 g/dL–5.40 g/dL) was within the values (3.00–5.50 g/dl) observed and reported for the majority of birds [30]. According to a similar study [23], the reference range for plasma protein (40–65 mmol/L) is similar to the result obtained in this study, while higher values were obtained in comparison to the referenced range for sodium (60–98 mmol/L) and chloride (56–70 mmol/L). The range of albumin (1.14 g/dL–2.34 g/dL) and globulin (126–360 mg/dL) documented in a comparable study [31], are slightly above what is reported in this present investigation.

The concentration of serum total protein and albumin among laying hens can be considered a reflection of egg production intensity [32]. The preceding agrees with the results of this study as the control treatment with the highest total protein content (6.30 g/dl) also recorded the least hen-day production of 65.56% [33].

Antioxidant Status of Laying Hens

The antioxidative attributes of aromatic herbs and spices warrant particular consideration due to their role in preventing undesirable oxidation by-products, which can lead to undesirable alterations in the overall quality attributes of poultry and their products [34]. Active compounds in phytogenics like capsaicin found in red pepper and piperine in black pepper are reported to bring on their antioxidant properties by preventing lipid peroxidation through enhancing antioxidant enzymes and decreasing protein carbonyl group content [35]. From the result obtained from this study, ALT levels were significantly lower in the treated groups, while AST levels were statistically similar across all treatments, including the control. However, the AST values (93.00–121.33 iu/L) were less than the range of 164.5–338 iu/L [31]. This result also partially agrees another report, which indicated a non-significant effect of the red pepper treatment on AST and ALT concentrations [36].

Significant changes in antioxidant status values were reported within the red pepper treatment group compared to the control in a similar study, which indicates the potential efficacy of phytogenic additives in regulating physiological processes and detoxification reactions [37]. These bioactive components offer protection against cytotoxic, genotoxic, and metabolic impacts of environmental and dietary toxins [38]. Consequently, incorporating pepper into poultry feed may stabilize cell membranes and protect the liver from damage caused by free radicals.

Serum Lipid Profile of Laying Hens

The results of cholesterol levels in the present study were significantly reduced in the treated groups except for Treatment 3 (1.5% black pepper), which was statistically similar to the control. In agreement, other studies have reported that adding red pepper significantly decreased serum cholesterol concentration compared with the control group [36], [39]. Similarly, the low-density lipoprotein (LDL), triglycerides and cholesterol levels were reduced in the birds fed red pepper-supplemented diets, and it was proposed that the supplementation with peppers promotes increased lipid thermogenesis and expedites energy metabolism in the avian subjects [40].

Another study found that the black pepper treatment groups exhibited elevated cholesterol levels and reduced triglyceride levels compared to the control group. However, significant differences between the control and treated groups were reported only in triglyceride levels [41]. Conversely, our study yielded contrasting results, as significant differences were observed in cholesterol levels between the black pepper treatment groups and the control, while triglyceride levels, although not significantly different from the control, were visibly reduced with black pepper treatment.

During the laying phase, heightened metabolism in laying hens necessitates increased utilization of fatty acids and steroid hormones for yolk formation, leading to notable alterations in total triglyceride levels [42]. Additionally, incorporating black pepper into poultry diets can induce metabolic shifts, particularly in fatty acid catabolism and energy metabolism regulation [43]. These changes directly influence the physiological response, resulting in elevated mobilization of triglycerides from tissues into the bloodstream. Furthermore, it has been proposed that the bioactive components of pepper could hinder hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity–a pivotal enzyme in cholesterol biosynthesis. This inhibition reduces both cholesterol production and absorption from the intestines, which could contribute to lowered blood cholesterol levels [44].

Egg Yolk Lipid Profile

The cholesterol level obtained in the control in this study shared statistical similarities with only Treatments 3 and 4. The rest treatments maintained lower cholesterol levels. This agrees with the report where the addition of red pepper above 1% significantly (p < 0.05) decreased egg yolk cholesterol concentration compared with the control group [36]. The potential mechanism through which phytogenic additives could diminish egg yolk cholesterol involves their capacity to inhibit lipogenic enzymes, as exemplified by a notable decline in mRNA levels [1]. The curbing of hepatic lipogenic enzymes can be attributed to the ability of phytogenic compounds to suppress or impede the expression of genes responsible for producing lipogenic proteins. Given that a significant portion of yolk cholesterol originates in the liver, the reduction in hepatic cholesterol and fatty acid synthesis leads to a decrease in cholesterol and its esters being incorporated into yolk precursors (such as vitellogenin and lipoprotein particles) which in turn, results in lowered yolk cholesterol levels [45].

Total triglyceride was significantly highest in the control and Treatment 4, while the other treatments maintained reduced levels. Similar results were reported in a study with fish oil in layers and breeder hen diets [46]. The predominant lipoprotein, responsible for over 60% of the dry yolk mass, is triacylglycerol-rich very-low-density lipoprotein (VLDL) [47]. Its formation relies significantly on the activity of stearoyl-coenzyme A desaturase, an enzyme that converts saturated fatty acids into monounsaturated fatty acids. Omega-3 fatty acids and specific plant-based essential oils have been noted for their capacity to reduce stearoyl-coenzyme A desaturase activity [48]. Consequently, it seems that the inhibition of stearoyl-coenzyme A desaturase activity in birds results in decreased VLDL synthesis, culminating in diminished yolk mass and reduced lipid content in eggs [48]. This is in agreement with the findings of this study, where a non-significant reduction of relative yolk weight [33] correlates to significantly decreased egg yolk lipid content in the treated groups.

Conclusion

The inclusion of black pepper and red pepper in the hens’ diets led to significant improvements in blood biochemical parameters, particularly in lowering serum cholesterol and triglyceride levels while enhancing antioxidant status. These changes indicate a clear positive impact on lipid metabolism, suggesting that the hens are better able to process and utilize fats more efficiently.

Furthermore, the observed alterations in egg yolk lipid composition, with favorable shifts in the proportions of saturated, monounsaturated, and polyunsaturated fatty acids, reflect the benefits of enhanced lipid metabolism. Such improvements not only reflect the hens’ health but also contribute to producing eggs with a superior nutritional profile.

In conclusion, the dietary inclusion of black pepper, red pepper, and their combinations in laying hens’ diets yields substantial benefits. It not only enhances the health and productivity of the hens by improving lipid metabolism and overall physiological functions but also enriches the nutritional composition of the eggs. The resulting modifications in egg yolk lipid profiles, including improved fatty acid composition, hold significant potential for enhancing the eggs’ health-promoting attributes, offering notable benefits to consumers. These findings underscore the value of these dietary additives in both laying hens management and human nutrition.

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