Effect of Kalahari melon essential oil (Citrullus lanatus), butyric acid, and their blend in grower pig diets on blood hematology, nutritional status, liver enzyme activity, and oxidative stress

Abstract

The need for alternative growth promoters in pig production has arisen from the ban on the use of antibiotics as growth promoters. Important markers of an animal’s physiological and health state are blood biochemistry and hematology. Blood analysis is a useful tool for evaluating pig physiology and health. Therefore, the study assessed the effects of dietary supplementation of Kalahari essential oil, butyric acid and their blend based on blood hematology, serum biochemistry and state of immunity in growing pigs. Forty mixed sex Large White growing pigs were weighed and randomly allocated to five dietary treatments, NC-negative control (no growth promoter), PC- positive control growth promoter (Zinc Bacitracin), KEO- Kalahari melon essential oil (0.4%), BA- Butyric acid (0.6%) and KEBA—Kalahari melon essential oil (0.4%) + Butyric acid (0.6%). Each dietary treatment was allocated eight pigs and each pig was considered an experimental unit. The red blood cell (RBC) count for the NC and PC were within the normal reference range, while the white blood cell count (4.7 ± 1.16) for NC was below the normal range. The platelet count was significantly different (P < 0.05) from KEO, BA and KEBA. White blood cell count for NC (4.7 ± 1.16) was below the normal reference range and significantly different (P < 0.05) when compared to other dietary treatments. Kalahari melon essential oil, butyric acid and their blend had a significant effect (P < 0.05) on liver enzyme activity and oxidative stress and it resembled some association with liver functions. Calcium was normal for all dietary treatments. The results indicated that Kalahari melon essential oil, butyric acid had a positive influence on blood hematology, serum biochemistry and oxidative stress in growing pigs and can be used as a growth promoter.

Introduction

The ban on antibiotic growth promoters in pig production has increased mortality and compromised animal welfare (Monger et al. 2021). Antibiotics are administered to animals as precautionary measures against production-related diseases (Waluszewski et al. 2021). Pigs and poultry are produced in confined, crowded spaces hence increasing stress and risk of disease transmission (Saucier 2016). Antibiotics in pig production assist in different ways (Yang et al. 2019). The addition of antibiotics at sub-therapeutic levels in feed improves growth rates and reduces mortality and diseases. At intermediate levels antibiotics further prevent diseases and at therapeutic levels, they are used to treat diseases (Cromwell 2002; Cheng et al. 2014). To overcome the increase in mortalities and morbidity as a result of the antibiotic ban, alternative growth promoters have been proposed which include the use of phytogenic plant extracts, essential oils, organic acids and some probiotics (Seibel et al. 2021).

Nevertheless, the effectiveness of alternative growth promoters compared to antibiotics is still unclear (Cheng et al. 2014) and needs continuous interrogation. Approaches aimed at stimulating natural host defenses through the use of natural substances such as organic acids and essential oils to modulate immune function and improve the general health of animals have gained immense interest over the years (Gallois et al. 2009).

Blood analyses provide critical information on the physiological aspects of animal welfare assessment including the neuroendocrine and immune system responses, acute and long-term responses due to adverse husbandry activity, potential diseases and genetic predispositions (Seibel et al. 2021). Biochemical and haematological variables of blood are, generally, indicators of health, nutritional and physiological status in animals. Moreover, haematology and serum biochemistry are possible biomarkers used to measure the effectiveness of nutrient perturbations in animals (Washaya et al. 2020) as well as oxidative stress. The use of essential oils and organic acids in animal diets as alternatives to conventional antibiotics may have desirable influences on animal physiology. Butyric acid stimulates the development of the GALT (gut associated lymphoid tissues) which has an effect on the immune system of animals (Makowski et al. 2022). Kalahari melon essential oil is rich in phenolic compounds garlic acid, vanillic acid, caffeic acid and ferulic acid (Marume et al. 2020) which promote immunity by activating and signalling pathways that initiate immune response and inducing epigenetic changes in cells (Ding et al. 2019). Citrullus lanatus seeds are rich in fats and contain essential fats palmitic, stearic, oleoc and various linoleic acid isomers and linoleic acids in particular have anticarcinogenic, antiatherogenic and immune stimulatory effects (Marume et al. 2020). Essential oils and organic acids have been reported to increase glucose levels but do not affect cholesterol levels and antibody filter levels (Iqbal et al. n.d.). Abo Ghanima et al. (2020) also reported that dietary supplementation of essential oils improved immunity indices, serum calcium and phosphorus on layer chickens but decreased cholesterol, urea, creatinine and liver enzymes. Kalahari melon essential oils in layer hens had a positive effect on haematopoiesis and improved health status (Marume et al. 2020). In broilers, humic acids improved the haematological parameters and the general health of the birds (Disetlhe et al. 2018). Studies on the effects of essential oils and organics acids on blood haematology and biochemistry in pigs are, however, generally limited (Estienne et al. 2020). The study, therefore, evaluated the effect of Kalahari melon essential oil, butyric acid and their blend on blood haematology, serum biochemistry and immunity as well as oxidative stress in growing pigs.

Materials and methods

Experimental site

The study was carried out at the North-West University experimental farm (Molelwane) which is located in the North-West province of South Africa. The geographical coordinates are 25° 28′ 0″ South and 22° 28′ 0″ East. Average minimum temperatures of3 °C and average maximum of 38 °C with an annual rainfall range of 300—500 mm.

Animal management

A total of 40 mixed sex Large White growing pigs aged 8 weeks were used in a twelve-week feeding trial. The pigs were given two weeks for adaptation to the diet which was not part of the experimental period. All the pigs were allowed individual ad libitum access to feed and water through a self-feeder and nipple drinker throughout the experimental period. Experimental grower diets were offered from 0.5 kg per day on arrival and the amount was increased depending on average weights.

Experimental design and dietary treatments

Mixed sex Large White piglets were randomly allocated into one of the five treatments arranged in a completely randomized design and were fed individually. Each treatment had 8 replicates and each pig was considered an experimental unit. Dietary treatments were formulated with the inclusion of antibiotic growth promoter Zinc Bacitracin (PO), organic feed additives butyric acid (BA) and Kalahari melon essential oil (EO) as follows: negative control (no growth promoter), positive control (Zinc Bacitracin growth promoter), grower diet + EO (0.4%), grower diet + BA (0.6%) and grower diet + BA (0.4%) + EO (0.6%). The diets were analyzed for dry matter (DM), crude protein (CP), ether extract (EE), ash, neutral detergent fiber (NDF) and acid detergent fiber (ADF) using the protocol of the Association of Official Analytical Chemicals (AOAC). The diets and compositional analysis are shown in Table 1.

Table 1.

Feed ingredients for dietary treatments

	Dietary Treatments
Ingredient (%)	NC	PC	KEO	BA	KEBA
Maize	68.32	68.32	68.32	68.32	68.32
Soya bean meal (Local)	25	25	25	25	25
Soya bean crude oil	1	1	1	1	1
Salt-(fine)	0.5	0.5	0.5	0.5	0.5
Mono calcium phosphate	0.8	0.8	0.8	0.8	0.8
Calcium carbonate	0.9	0.9	0.9	0.9	0.9
L-Lysine HCI	0.32	0.32	0.32	0.32	0.32
DL-Methionine	0.13	0.13	0.13	0.13	0.13
L-Tryptophan	0.01	0.01	0.01	0.01	0.01
L-Threonine	0.13	0.13	0.13	0.13	0.13
Vitamin premix	0.1	0.1	0.1	0.1	0.1
Trace mineral premix	0.1	0.1	0.1	0.1	0.1
Copper sulphate, 25%	0.05	0.05	0.05	0.05	0.05
Antioxidant	0.02	0.02	0.02	0.02	0.02
Additives other	0	0	0.1	0.1	0.1
Zinc Bacitracin	0	0.1	0	0	0
Nutritional composition (%)
Dry matter	93.12	93.29	93.44	93.69	93.99
Moisture	6.87	6.70	7.55	7.10	7.00
Crude protein	18.34	18.47	18.17	18.49	18.84
Ether extracts	3.68	3.71	4.51	3.63	4.24
NDF	20.09	19.31	23.94	20.82	21.56
ADF	11.24	11.22	11.13	11.21	11.29

NC, Negative control (no antibiotic growth promoter); PC, Positive control (antibiotic growth promoter); KEO, Kalahari melon essential oil; BA, Butyric acid; KEBA, Kalahari melon essential oil butyric acid blend.

Antibiotic growth promoter Zinc Bacitracin was purchased from Animate Animal Health, Edenvale, Gauteng, South Africa. The essential oil, butyric acids were purchased from Nutrica Organics (Pvt Ltd), South Africa. The essential oil was extracted from C. Lanatus seed using cold pressing method (Marume et al. 2020). All other dietary components were purchased from Nutri feeds, Pretoria (SA). The experimental procedure and designs of the study were approved by the North-West University Animal Production Research Ethics committee (NWU-AnimProdREC) and it was granted ethics number NWU-02010–20-A5.

Blood collection

At week 12 each pig was bled via the jugular vein and blood samples were collected into EDTA purple vacuum tubes for blood haematology. Blood samples for serum analysis were placed in red-capped test tubes, centrifuged and separated for serum analysis.

Blood haematology

The IDEXX lasercyte was used to analyse blood haematology. Blood haematological indices analysed included red blood cells (RBC), white blood cells(WBC), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) and mean corpuscular volume (MCV), haemoglobin (Hb), haematocrit (HCT), red cell distribution width (RDW) and platelets (PLT).

Nutritional status and liver enzyme activity

IDEXX catalyst one veterinary chemistry analyser was used for serum metabolites and IDEXX chem17 CLIP. Serum total protein, albumin, total cholesterol, glucose, calcium, phosphorus, albumin and liver enzymes (Aspartate aminotransferase AST, Alanine transaminase ALT, Alkaline phosphatase ALKP, Gamma glutamyl transferase GGT) were analysed.

Oxidative stress

Serum antioxidant-related indices, including superoxide dismutase (T SOD), catalase (CAT) and glutathione (GSH) were determined using the Elabscience bioassay kit for serum antioxidant activity purchased from Biocom Africa (Pty) Ltd, Centurion, South Africa. When analysing for T SOD activity 1 ml of reagent 1 working solution and 0.1 mL sample were added to the sample tubes and 1 mL of reagent 1 working solution and 0.1 ml double distilled water was added to the control tubes. A volume of 0.1 mL of reagent 2, 0.1 mL of reagent 3 and 0.1 mL of reagent 4 working solution were successively added into the control and sample tubes mixed fully with a vortex mixer and incubated for 40 min at 37 ℃. 2 ml of a chromogenic agent was added into the control and sample tubes, mixed and allowed to stand for 10 min at room temperature. The OD values for each tube were measured at 550 nm with a 1 cm optical path quartz cuvette. T SOD activity was calculated using the formula:

$$\mathrm{T}-\mathrm{SOD}\mathrm{activity}(\mathrm{U}/\mathrm{mL})=\mathrm{i}÷50\%\times \mathrm{v}1/\mathrm{v}2\times \mathrm{f}$$

When analysing for CAT activity 1 ml of reagent 1 was added into the 5 ml EP tubes for control and 0.1 ml of sample was added together with 1 mL of Reagent 1 into the 5 mL EP tubes. Tubes were incubated at 37 ℃ for 5 min and 0.1 ml of reagent 2 was added into each tube, mixed fully and allowed to react at 37 ℃ for 5 min accurately. In the sample tube 1 mL of reagent 3 application solution was added and 0.1 mL of reagent 4 was added, mixed fully. In the control tube 1 ml of reagent 3 application solution, 0.1 mL of reagent 4 and 0.1 ml of sample were added and mixed fully. It was allowed to stand for10 min at room temperature and the OD values of each tube were measured at 405 nm with a 0.5 cm optical path cuvette. CAT activity was calculated using the formula

$$\text{CAT}\text{activity}(\text{U}/\mathrm{mL})=\frac{\mathrm{\Delta }A}{a}\times \frac{{0.02}^{*}}{1^{*}\times V}\times f$$

GSH activity was determined through a series of steps for the enzymatic reaction and the non-enzymatic reaction. In the non-enzymatic tubes and enzymatic tubes, 0.2 ml of 1 mmol/L GSH solution was added to 5 mL EP tubes. In the enzymatic tubes, 0.1 ml of serum sample was added. Both the enzymatic and non-enzymatic tubes were pre-heated at 37 ℃ in a water bath for 5 min. Some 0.1 mL of reagent 1 application solution was added to the tubes, mixed thoroughly and allowed to react at 37 ℃ for 5 min. In the non-enzymatic tube 2 mL of reagent 2 and 0.1 mL of a serum solution were added. Also, in the enzymatic tube 2 mL of reagent 2 was added. The tubes were mixed using a vortex mixer and centrifuged at 3100gx for 10 min and 1 mL of supernatant was collected and mixed with reagents for the chromogenic reaction step. After mixing the tubes were allowed to react at room temperature for 15 min and OD values for each tube were measured at 412 nm with 1 cm optical length cuvettes. GSH activity was calculated using the formula:

$$\mathrm{GSH}\mathrm{activity}(\mathrm{U})=\mathrm{\Delta }\mathrm{A}1÷\mathrm{\Delta }\mathrm{A}2\times \mathrm{c}\times \mathrm{f}1\times \mathrm{f}2$$

Statistical analysis

All data was subjected to statistical analysis (SAS 2010) and comparisons in particular the GLM procedures for once-off data. The PDIFF option was used for separation of means while contrasts were used to assess the specific effects of organic acids and essential oils on the different parameters. For all tests, the level of significance was set at (P < 0.05). The following model was used for data analysis of once-off data with diet as the main factor:

$$\text{Yij}=\mu +\text{Ti}+\epsilon \text{ij}$$

where: Yij = observation (blood haematology, serum metabolites, liver enzyme activity and oxidative stress).

µ = population mean constant common to all observations

Ti = effect of diet

εij = random error term.

Results

Blood haematology

Table 2 presents the impact of Kalahari melon essential oil, butyric acid, and their blend on erythrocyte composition. Dietary treatments had a significant (P < 0.05) effect on blood RBC, HCT, HGB, MCH, and RDW levels. Pigs fed the BA diet consistently had the highest levels of RBC, HCT, HGB, MCH, and RDW, followed by those fed the KEO and KEBA diets. Pigs fed BA had the highest HCT% (53.4 ± 2.97), which was above the normal range. Dietary treatment BA had normal MCV content (50.4 ± 4.12), while NC, PC, KEO, and KEBA blood MCV values were below normal ranges. The reticulocyte content for PC was the lowest (99.8 ± 25.90 K/µL) but it was within the normal reference range. Diet did not affect all leucocytes apart from the total WBC and lymphocytes concentrations as shown in Table 3. The white blood cell (WBC) content for NC (4.7 ± 1.16 × 109/L) was below the normal reference range and was significantly lower (P < 0.05) than those of all other dietary treatments. Neutrophil content was not significantly different (P < 0.05) for all dietary treatments and they were all below the normal reference range (4.9–7.5 × 109/L). Pigs fed the BA and KEO diets had the highest values for lymphocyte count while those fed NC (1.4 ± 6.78 × 109/L) had the lowest lymphocytes count and were below the normal reference range. KEO had the highest (14.9 ± 6.78 × 109/L) lymphocyte count and it was within the normal reference range (6.60–18.7 × 109/L). Monocyte content was not affected by dietary treatment. NP, PC and KEBA had monocyte values within the normal reference range. The ratios of neutrophils, monocytes, and lymphocytes did not differ significantly (P < 0.05) across all dietary treatments.

Table 2.

Effect of Kalahari melon essential oil and butyric acid blend on blood erythrocytes composition in growing pigs

Dietary treatments
Parameters	NC	PC	KEO	BA	KEBA	SEM	Significance	1Normal Range
Red blood cells (× 1012/L)	7.73a	7.66a	9.61b	10.71b	9.16b	0.832	*	5.30–8.48
Haematocrit (%)	25.43a	38.21b	48.19c	53.43c	38.21b	2.968	***	32–50
Haemoglobin (g/dL)	7.4a	7.08a	9.26b	12.43c	10.30b	1.172	*	10.70–16.70
Mean corpuscular volume (fL)	36.20	45.66	42.59	50.39	41.05	4.116	NS	50–68
Mean corpuscular haemoglobin(pg)	6.49a	8.62b	9.65b	11.76c	10.76c	1.212	*	16.3–20.2
Mean corpuscular haemoglobin concentration (g/dL)	6.52	6.72	9.15	9.18	12.18	4.358	NS	-
Red cell distribution width (%)	16.33a	21.66b	25.89c	28.71c	28.26c	1.958	***	30–34
Reticulocytes (K/µL)	124.59	99.77	156.98	149.43	101.65	25.901	NS	0–100

^a,b,cmeans in the same row with different superscripts are significantly different (P < 0.05). ***(P < 0.0005),** (P < 0.005),*(P < 0.05) and NS-no significant difference. NC, Negative control (without growth promoter); PC, Positive control (with growth promoter); KEO, Kalahari melon essential oil (0.4%); BA, Butyric acid (0.6%); KEBA, Kalahari melon essential oil (0.4%) and butyric acid (0.6%)blend. ¹IDEXX (2021)

Table 3.

Effect of Kalahari melon essential oil and Butyric acid blend on leukocytes composition in growing pigs

Dietary treatments
Parameters	NC	PC	KEO	BA	KEBA	SEM	Significance	1Normal Range
White blood cells (× 109/L)	4.68a	14.14b	15.60b	15.42b	16.08b	1.16	***	11–22
Neutrophils (× 109/L)	1.22	1.28	1.02	1.54	0.52	0.36	NS	4.48–7.52
Lymphocytes(× 109/L)	1.38a	2.80a	14.85a	33.79b	3.63a	6.78	**	6.60–18.70
Monocytes (× 109/L)	1.59	1.47	1.80	2.43	0.93	0.44	NS	0.30–1.25
Neutrophil/lymphocyte ratio	1.01	0.92	1.09	0.08	2.62	0.73	NS	-
Neutrophil/monocyte ratio	0.56	0.88	1.30	0.63	1.36	0.33	NS	-
Monocyte/lymphocyte ratio	1.22	0..93	0.59	0.15	0.99	0.27	NS	-

^a,bmeans in the same row with different superscripts are significantly different (P < 0.05). ***(P < 0.0005),** (P < 0.005),*(P < 0.05) and NS, no significant difference. NC, Negative control (without growth promoter); PC, Positive control (with growth promoter); KEO, Kalahari melon essential oil; BA, Butyric acid; KEBA, Kalahari melon essential oil and Butyric acid blend ¹IDEXX (2014)

Table 4 summarizes the effects of Kalahari melon essential oil, butyric acid, and their combination on thrombocyte composition. Dietary treatment significantly impacted PLT count (P < 0.05). KEBA had the lowest value (587.6 ± 150.35 K/µL) but was within the normal reference range. NC, PC, KEO, and BA were all above the normal reference range (300–700 K/µL). KEBA had the lowest PCT value (0.3 ± 0.07), while KEO had the highest PCT count (0.7 ± 0.07). Table 4 presents the effects of Kalahari melon essential oil, butyric acid and their blend on thrombocyte composition. Dietary treatment significantly affected the PLT count (P < 0.05). KEBA had the lowest (587.6 ± 150.35 K/µL) but was within the normal reference range. NC, PC, KEO and BA were above the normal reference range (300–700 K/µL). Dietary treatment KEBA also had the lowest PCT value (0. 3 ± 0.07) and EO the highest PCT count (0.7 ± 0.07).

Table 4.

Effect of Kalahari melon essential oil and butyric acid blend on thrombocytes composition in growing pigs

Dietary treatments
Parameter	NC	PC	KEO	BA	KEBA	SEM	Significance	1Normal Range
Platelets (K/µL)	1429.75b	1512.88b	2030.37a	1794.75b	587.62c	150.378	***	300–700
Mean platelet volume (fL)	2.48	3.81	3.40	3.60	3.45	0.453	NS	-
Platelet distribution width (%)	12.75	19.39	17.39	17.86	15.56	1.912	NS	-
Plateletcrit (%)	0.34c	0.55b	0.68b	0.62b	0.31c	0.067	**	-

^a,bmeans in the same row with different superscripts are significantly different (P < 0.05). ***(P < 0.0005),** (P < 0.005),*(P < 0.05) and NS, no significant difference. NC, Negative control (without growth promoter); PC, Positive control (with growth promoter); KEO, Kalahari melon essential oil; BA, Butyric acid; KEBA, Kalahari melon essential oil and Butyric acid blend ¹IDEXX (2014)

Liver enzyme activity

Table 5 shows the effect of Kalahari melon essential oil, butyric acid, and their blend on liver enzyme activity. Dietary treatments significantly influenced ALT, AST levels (P < 0.05). Pigs fed NC and PC diets had the highest levels of ALT, and was above the normal reference range (9–43µ/L). Pigs fed the NC diet had the highest AST content (239.0 ± 19.65 µ/L), which was greater than the normal range. Pigs fed the PC, KEO, and KEBA had AST levels within the normal reference range. Dietary treatment did not have a significant effect on alkaline phosphatase (ALP). However, the ALKP content in KEBA was lower than the normal reference range. GGT levels were lowest in NC (10.6 ± 6.28 µ/L), while PC and KEBA had normal values.

Table 5.

Effect of Kalahari melon essential oil and Butyric acid blend on liver enzyme activity in growing pigs

	Dietary treatments
Parameter	NC	PC	KEO	BA	KEBA	SE	Significance	IDEXX (2014)
Alanine aminotransferase (U/L)	211.25b	291.5b	24.38a	31.14a	21.56a	37.333	**	9–43
Aspartate aminotransferase (U/L)	239.00a	21.50b	20.25b	19.87b	17.78b	19.645	**	16–64
Alkaline phosphatase (U/L)	188.88a	95.63	143.13	114.86	54.33b	40.890	NS	92–294
Gamma-glutamyl transferase (U/L)	10.63	25.5	14.00	11.29	18.78	6.279	NS	16–30

^a,b,cmeans in the same row with different superscripts are significantly different (P < 0.05). ***(P < 0.0005),** (P < 0.005),*(P < 0.05) and NS, no significant difference. NC, Negative control (without growth promoter); PC, Positive control (with growth promoter); KEO, Kalahari melon essential oil; BA, Butyric acid; KEBA, Kalahari melon essential oil and Butyric acid blend

Nutritional status

Table 6 presents the effects of Kalahari melon essential oil, butyric acid and their blend on serum biochemistry. Diet had a significant effect on serum total protein and glucose (P < 0.05).Total protein was highest in the PC dietary treatment (109.5 ± 8.97 g/L) and lowest in the BA (78.7 ± 8.97 g/L) fed pigs. In the negative control, PC and KEO had total protein content above the normal reference range. Cholesterol levels were normal and were not affected by dietary treatments (P < 0.05). Low glucose levels were recorded in KEO (2.1 ± 0.66 µ/L) and the highest was in PC (6.3 ± 0.66 µ/L). Calcium was normal for all dietary treatments and the diets had no significant effect on calcium content. Phosphorous content was above the normal reference range for all dietary treatments.

Table 6.

Effect of Kalahari melon essential oil and butyric acid blend on nutritional status in growing pigs

Parameter	NC	PC	KEO	BA	KEBA	SE	SIG	IDEXX
Total protein (g/L)	103.88a	109.50a	99.63b	78.71c	60.33c	8.972	**	60–80
Cholesterol (mmol/L)	1.91	3.18	2.21	1.99	2.31	0.850	NS	0.46–2.04
Glucose (mmol/L)	4.00b	6.32a	6.03a	2.13c	3.56c	0.663	***	4.7–8.9
Calcium (mmol/L)	2.35	2.05	2.05	2.20	2.25	0.601	NS	1.63–2.85
Phosphorus (mmol/L)	3.90	5.20	5.02	4.46	4.62	0.562	NS	1.2–3.0

^a,b,cmeans in the same row with different superscripts are significantly different (P < 0.05). ***(P < 0.0005),** (P < 0.005),*(P < 0.05) and NS, no significant difference. NC, Negative control (without growth promoter); PC, Positive control (with growth promoter); KEO, Kalahari melon essential oil; BA, Butyric acid; KEBA, Kalahari melon essential oil and Butyric acid blend

Oxidative stress

Table 7 presents the effects of Kalahari melon essential oil, butyric acid and their blend on oxidative stress in pigs. Diet significantly affected T-SOD activity (P < 0.0005). T-SOD activity was highest in the KEBA (340.5 ± 26.03µ/mL) treatment and lowest in NC (112.9 ± 26.03µ/mL) treatment. Catalase activity (CAT) showed a significant difference among dietary treatments (P < 0.0005). The KEBA (102 6 ± 6.59 µ/mL) dietary treatment obtained the highest CAT activity whilst the PC (23.0 ± 6.59 µ/mL) treatment had the least CAT activity (P < 0.0005). GSH activity was not affected by dietary treatment with no significant differences observed among the treatments.

Table 7.

Effect of Kalahari melon essential oil, Butyric acid and their blend on serum antioxidant activity in growing pigs

Dietary treatments
Parameter	NC	PC	KEO	BA	KEBA	SE	Significance
T-SOD µ/mL	118.64c	112.86c	190.08b	170.52b	340.49a	26.032	***
CAT µ/mL	34.17c	23.02c	28.382c	86.94b	102.59a	6.591	***
GSH µ	289.17	326.05	346.76	291.79	319.62	19.873	NS

^a,b,cmeans in the same row with different superscripts are significantly different (P < 0.05). ***(P < 0.0005),** (P < 0.005),*(P < 0.05) and NS, no significant difference. NC, Negative control (without growth promoter); PC, Positive control (with growth promoter); KEO, Kalahari melon essential oil; BA, Butyric acid; KEBA, Kalahari melon essential oil and Butyric acid blend

Discussion

Blood haematology

Haematology references are useful components in determining the health status of animals (Perri et al. 2017). Haematological indices contribute to the early detection of diseases or poor growth (Marume et al. 2020). Reference intervals can be used to interpret results to better understand the health status of individuals in pigs (Perri et al. 2016). In this study high RBC levels in dietary treatment KEBA can be attributed to the high levels of a phenolic compounds (Marume et al. 2020) in Kalahari melon essential oil. Polyphenols when included in diets form an inactive redox Fe-Polyphenol complexes or chelates with iron on red blood cells protecting the RBC from lysis (Grinberg et al. 1997). According to Papatsiros et al. (2024) and Alagbe et al. (2023) phytogenic feed additives increased the red blood cell count in pigs and chickens respectively. Butyric acid induces the synthesis of foetal haemoglobin HbF and for this reason its presence can influence red blood cell count in growing pigs (Blau et al. 1993). White blood cell count below the reference interval could indicate leukopenia due to suppressed bone marrow functioning. It can also be attributed to an increased rate of cell migration from the peripheral blood beyond the rate of production. Low WBC count in NC can be attributed to high levels of infection which out-competed the rate of WBC production. Development of antibiotic resistance in pigs fed a diet with antibiotic growth promoters can also alter WBC level and composition in response to infection. Neutrophils and lymphocytes are associated with phagocytosis, humoral and cell-mediated immunity. Polyphenols in Kalahari melon essential oil increase lymphocyte proliferative responsiveness and modulation of the function of B cells (Shakoor et al. 2021). Pigs fed diets without antibiotics have a higher possibility of getting infections hence an effect on white blood cell counts. Papatsiros et al. (2024) state that studies on haematology in pigs are limited.

Platelets are involved in primary homeostasis between platelets and blood vessels. Increased consumption of platelets may be due to infection and is immune-mediated (Packirisamy et al. 2018). Papatsiros et al. (2024) states that inclusion of phytogenic feed additives in pig diets will increase the anticoagulant and or antiaggregatory action on platelets. However, in previous studies on poultry and pigs, the effects of essential oil and organic acids’ on blood haematology varied depending on age, type of metabolites and the inclusion levels (Marume et al. 2020; Adewole et al. 2021; Hofmann et al. 2022).

Liver enzyme activity

High levels of ALT in dietary treatment NC indicate high chances of infectious diseases and trauma in pigs whereas low levels are considered of no effect in pigs (IDEXX 2021). High ALT levels generally indicate liver damage or stress, which can be caused by various factors including infections, toxins or trauma. Diet and drug administration may affect ALT levels (Evans 2009). AST levels in the blood also are correlated to red blood cell count. The NC-fed pigs had a high level of AST and this is associated with liver malfunctioning and tissue injury perhaps due to a lack of immune protection. Alkaline phosphatase (ALP), Gamma-glutamyl transferase GGT and bilirubin levels are also important indicators of liver functioning (Fasuyi et al. 2013). Organic acids and essential oils could have the same function as conventional antibiotics in enhancing liver functioning as they recorded normal levels of liver enzymes compared to NC and PC-fed pigs.

Nutritional status

Total protein content was lowest in the NC dietary treatment. Total serum protein is generally used as an indicator of nutritional deficiency and intestinal malfunction (IDEXX Laboratories 2016). Organic acids and essential oils have a positive impact on intestinal morphology and intestinal permeability that may increase the efficiency of nutrient absorption (Liu et al. 2018; Upadhaya et al. 2018). Calcium and Phosphorous levels were in the normal range for all dietary treatments indicating that organic acid and essential oils inclusion in the diets had no negative effect on intestine, bone and kidney interaction as well as endocrine glands that regulate phosphorous and calcium homeostasis (IDEXX Laboratories 2016). Serum glucose levels were normal for all dietary treatments. Cholesterol levels for NC, KEO, BA and KEBA were all in the normal range except for NC. Cholesterol is an important component of the cell membrane and is a precursor for steroid hormones and bile acids (Fasuyi et al. 2013). Low cholesterol content can be attributed to low uptake or intestinal malabsorption. Reduction in cholesterol levels can be due to inhibition of Sterol regulatory element binding proteins (SREBPs) by herbal extracts (Castro-Torres et al. 2014). Active ingredients in essential oils can have an effect on lethal metabolites resulting in increased blood cholesterol (Rad et al. 2018).

Oxidative stress

Oxidative stress and inflammation are correlated physiological processes (Sierżant et al. 2019). In the current study, KEBA-fed pigs showed improved antioxidant activity and subsequently reduced oxidative stress which could be attributed to the complementarity effect of essential oil and organic acids in reducing the reactive oxygen species that stimulate oxidative stress. Nevertheless, Li et al. (2008) reported no changes in the inflammatory response of pigs fed organic acids. Studies on the influence of organic acids on antioxidant activity and inflammation remain limited (Xu et al. 2020). Plant extracts contain terpenes, phenols glycoside and aldehydes which have reducing activity potential (Li et al. 2022). Kalahari melon essential oil is rich in non-flavonoid polyphenols and are pro oxidants and antioxidants due to their chelating behaviour, solubility characteristics, metal reducing potential and pH at site of action hence protecting the body from severe oxidative stress (Rudrapal et al. 2022). T-SOD activity was high in EOOA and can be due to the influence of phenol groups in Kalahari melon essential oil which can act as donors of hydrogen interacting with peroxy radicals retarding the hydroxyl peroxide formation (Chaudhary et al. 2023). SOD can increase TAOC in weaned pigs contributing to sustained health status and growth rate (Ahasan et al. 2019). Su et al. (2018) reported an increase in antioxidant activity and antioxidant-related genes in weaned pigs fed plant essential oils.

Conclusion

The findings of the study show that Kalahari melon essential oil, butyric acid and their blend influence the physiological, immune and oxidative status of growing pigs. The high WBC count in pigs fed Kalahari melon essential oil and butyric acid diets indicate the potential they have in promoting animal health status of the pigs. High liver enzymes activity in pigs fed diet with no growth promoter was a confirmatory of the effectiveness of the essential oil, organic acid and their blend on liver function. Levels of total protein, cholesterol, glucose, calcium and phosphorous obtained in the study are indicative of the potential of the Kalahari melon essential oil and butyric acids in influencing nutrient uptake and utilisation. Lastly, Kalahari melon essential oil and butyric acid blend showed immense potential in improving the antioxidant activity hence improving the ability of the animals to cope with oxidative stress. Nevertheless, assessment of the effects of Kalahari melon essential oils and butyric acid blend on the prevalence of resistant E. coli, Salmonella in pigs may be critical.

Author contributions

All authors contributed to manuscript review, editing, final proofreading and approving for submission. Conceptualization: NRB &MU Methodology: NRB & MU Formal Analysis: MU Data Curation: MU Writing-Original Draft: NRB. Writing – Review and Editing: NRB & MU.

Funding

Open access funding provided by North-West University. The manuscript was extracted from the first author thesis that was financially supported by North West University School of Agricultural science, Department of Animal Science.

Data availability

Not applicable.

Declarations

Ethics approval

The experimental procedure and designs of the study were approved by the North-West University Animal Production Research Ethics committee (NWU-AnimProdREC) and it was granted ethics number NWU-02010–20-A5.

Consent to participate

Not applicable

Consent for publication

Not applicable.

Conflict of interest

The authors declare no competing interest.