The effects of betaine and nano-emulsified plant-oil supplementation on growth performance and serum biochemistry indices of heat-stressed broiler chickens

Abstract

This study aimed to investigate the effects of water-added betaine (BET) and Magic oil, (MAGO) on growth performance and blood biochemistry of broilers exposed to constant heat stress (32 ± 1 °C) during 21–35 d. On day 21, 320 chicks (Ross 308) were straight run to one of four groups based supplements with drinking water (DW): control, BET (1 g/L DW), and/or MAGO (1 ml/L DW), respectively). Each treatment consisted of eight replicates with ten birds each. The performance data were obtained at 21, 28, and 35 d. Serum biochemical data of the birds were estimated at day 35. The results showed that body weight gain (BWG) and feed intake did not differ significantly (p > .05) between the treatments. However, Bet alone or in combination with MAGO has improved feed conversion ratio (FCR) during the 28–35 d and 21–35 d periods; also, BET lowered (p < .05) creatinine compared to control. The addition of MAGO alone or in combination with BET increased (p < .05) total protein, albumin, globulin, and low-density lipoprotein-cholesterol. In addition, MAGO decreased total lipids, and creatinine, whereas it increased (p < .05) cholesterol, especially good cholesterol, and triglycerides compared with the control. In conclusion, water-BET-supplements alone or in combination with MAGO has improved the FCR under heat stress conditions. Therefore, it is recommended to use Bet alone or both supplements under high-temperature conditions in broiler houses. However, further studies on the use of MAGO and/or BET at different doses are needed to maximise BWG and maintain the productivity of the birds.

Highlights

• Water-enriched magic oil and betaine had higher added value than feeding alone by improving the feed conversion ratio during 21–35 d.

• Compared with the other groups, water-supplemented magic oil produced some benefits, such as lower serum glucose, higher serum high-density lipoprotein ‘good cholesterol’ and lower creatinine levels.

• The added value of the combination of magic oil and betaine for growth performance and serum health biomarkers needs further investigation at various levels.

Keywords:

• Betaine and magic oil
• blood biochemistry
• heat stress
• growth performance
• broiler

Introduction

Heat stress (HS) is one of the most significant problems broilers face and can lead not only to lower welfare but also to lower productivity (Archer 2023). The presence of HS negatively affects performance, carcass weight, and poultry internal organ weight (Liu et al. 2022). HS reduces the antioxidant capacity and developmental status of immune organs in broilers (Wen et al. 2021; Sun et al. 2023). The poultry industry is working to mitigate the detrimental effects of HS on broiler growth and welfare. Recently, the use of nutritional supplements to achieve this goal has gained popularity. In addition, a number of water/feed supplements have been used in modern poultry production as antibiotic replacements to improve growth, FCR, and health status (Khan et al. 2022; Khan et al. 2023). Several researchers have shown that adding natural additives to feed or DW is a better method than other strategies because it allows animals to distribute the substance more effectively in the body and to ingest a tolerable and safe amount of the compound (Govaris et al. 2004; Bou et al. 2009; Keshavamurthy et al. 2013; Ishola 2021). Phytogenic feed supplements, such as essential oils and nano-emulsified essential oils, are viable options for increasing the efficiency of broilers and animal productivity (Namdeo et al. 2020; Abdelhadi et al. 2022). Essential oils (EOs) can be used medicinally in a wide range of broiler situations due to their antimicrobial, antiparasitic, and antiviral activities (Abd El-Hack et al. 2022). Consequently, the use of nanoemulsions appears to be one of the possible technologies for promoting the activity of EOs as growth promoters and their economic benefit (Baskara et al. 2020; Irawan et al. 2021).

In this regard, Betaine (BET) has numerous metabolic and osmoregulatory functions, and it plays a vital role in alleviating HS in poultry and monogastric animals (Ratriyanto and Mosenthin 2018; Pradista et al. 2022). BET, also known as tri-methyl-glycine, is a natural stable compound originating from plants, animals, and microorganisms. As an organic osmolyte, BET is essential for maintaining osmotic pressure in intestinal epithelial cells during osmotic stress and drought (Figueroa-Soto and Valenzuela-Soto 2018; Pradista et al. 2022; Yang et al. 2022). Such osmotic protection regulates the water balance and volume of intestinal cells, facilitating the secretion of digestive enzymes and nutrient uptake, and improving growth performance (Ratriyanto and Mosenthin 2018; Wang et al. 2020). BET supplementation improves growth performance, immunity and breast yield in broilers housed under HS condition (Chand et al. 2017; Ratriyanto and Mosenthin 2018). BET supplementation improves immune indices, egg production, and quality in layers (Ratriyanto and Mosenthin 2018).

It is hypothesized that water supplementation with BET and/or magic oil is believed to reduce stress by improving growth performance and biochemical indices in the blood of birds under HS. However, the role of BET in combination with magic oil in mitigating the negative effects of HS in broiler chickens has not yet been studied and should be investigated to identify any benefit or synergistic effect between them. In addition, broilers at older ages are more HS resistant than those at younger ages because the birds at younger ages have the incomplete thermoneutral system and immune systems. Therefore, in the current study, broilers were exposed to HS during the final fattening period (20–35 d), and at the same time, all stressed birds received water supplements of Magic oil, BET, and a combination of Magic oil and betaine to alleviate the adverse effects of HS in broilers. Thus, the aim of this study was to determine the effects of water-added BET and/or magic oil on the performance, blood proteins, liver and kidney functions, and serum lipids profile of broilers exposed to HS during 21–35 d of age.

Materials and methods

Ethics

The experiment was carried out in line with the guidelines for ethical conduct in the care and use of animals in research and the work protocol was permitted by the Ethics Committee, King Saud University (KSU), approval number: KSU-SE-21-02.

Bird’s management and treatments

At arrival, 320 commercial broiler chicks (Ross 308) were obtained with a body weight (BW) of around 46.32 ± 0.04 g. All of the birds were in good condition and there was no mortality between the ages of 1 and 21 d, so the birds in each group were the same number from day 0 to day 21. At day 21, 320 straight-run birds with similar BW (1002 ± 0. 84 g). The experiment contains four groups. Each group has eight replicates, each with ten birds. The location of the research was at the Animal Production Department, KSU, Saudi Arabia. The chicks were raised in flour cages under comparable management and hygienic situations. The lighting protocol was set to 23 h on 1 h off per day throughout the experiment, according to Abudabos et al. (2021) and Qaid et al. (2021). Chicks were kept in a room with different experimental units at recommended environmental temperatures; the temperature was set at 33 °C on day 0 and was gradually reduced daily until it reached 24 °C on day 20. Birds were raised under constant HS for the rest of the period (32 ± 1 °C; the temperature controlled by a Smart thermostat Schneider electric- network control relay). Using data loggers (HOBO Pro Series, Cape Cod, MA, USA), the ambient temperatures of the trial room was constantly monitored.

All birds were fed a control diet up to 21 d old, then afterward up to slaughter (35 d) they were distributed into four treatment groups that were provided with water-added supplements as follows: (1) Control, no supplement . (2) Magic oil (1 ml/L DW) (MAGO). (3) Betaine (1 g/L DW) (BET). (4) Magic + Betaine (a mixture of 1 ml Magic oil and 1 g betaine/L DW) (MAGOBET). The concentrations of 1 ml/L or 1 g/L were selected based on other researches (Suliman, Hussein, Al-Owaimer, et al. 2023; Suliman, Hussein, Alsagan, et al. 2023). Betaine and/or EOs were supplied to broiler chickens through different routes (such as feeding or DW) as a natural antistressor to solve the problem of HS. Therefore, based on others (Saeed et al. 2017; Al-Sagan et al. 2020; Suliman, Hussein, Al-Owaimer, et al. 2023; Suliman, Hussein, Alsagan, et al. 2023), supplementation of BET and/or essential nano-oil in DW was performed in this study. Magic oil^TM consists of polysorbate-80, 98.5% nano-emulsified crude soybean oil, and vitamin E (ATCO Pharma, Egypt). The natural betaine is an anhydrous betaine (91%) called commercially Betafin^® S6 (Danisco Animal Nutrition, Marlborough, Wiltshire SN81XN, United Kingdom). Because Betafin powder dissolves easily in DW, it is ideal for drinking water applications. Previous research has described the product’s other ingredients (Suliman, Hussein, Al-Owaimer, et al. 2023).

A standard starter, grower, and finisher diet (from 0 to 10, 11 to 20, 21 to 35 d; respectively) based on the corn-soybean meal (CSBM) was offered in pelleted (for starter and grower diets) and mashed (for finisher diets) forms (Table 1).

Table 1. Composition of experimental diets and nutrients analysis as a fed %

INGREDIENTS	Starter (0-10 d)	Grower (11-20 d)	Finisher (21-35 d)
Corn Grain	57.650	61.320	60.640
Soybean Meal48 % CP*	35.440	31.790	30.480
Palm Oil	2.060	2.610	4.630
Dicalcium phosphate	1.920	1.730	1.420
Limestone	1.060	0.960	1.420
Common Salt	0.360	0.360	0.350
L-Threonine	0.300	0.120	0.070
Methionine	0.380	0.330	0.28
L Lysine HCl	0.280	0.220	0.15
Choline Cl70	0.060	0.060	0.060
Minivet premix 0.5%**	0.500	0.500	0.500
Nutrients	Analysis
Dry Matter %age	89.614	89.576	85.664
ME kcal/kg	3000	3070	3200
Crude Protein %	22.400	20.700	19.500
Arginine %	1.520	1.399	1.273
Lysine %	1.435	1.290	1.131
Methionine %	0.711	0.641	0.566
Methionine + Cysteine %	1.080	0.990	0.882
Threonine %	1.143	0.900	0.803
Tryptophan %	0.277	0.254	0.228
Valine %	1.017	0.951	0.901
Ether extract	4.601	5.256	7.236
Linoleic acid %	1.597	1.714	4.496
Crude Fiber%	2.650	2.589	2.523
Calcium %	0.960	0.870	0.937
Total Phosphorus	0.713	0.663	0.625
Available Phosphorus %	0.480	0.440	0.400
Sodium %	0.160	0.160	0.160

*CP: crude protein ** Minivet Premix 0.5% provided Mineral and vitamins as the following per kg of the diets: Vitamin A 12000IU/Kg, Vitamin D3 3000 IU/Kgm vitamin E, 80,000 IU; vitamin K3, 3,200 mg; vitamin B1, 3,200 mg; vitamin B2, 8,600 mg; vitamin B3, 65,000 mg; vitamin B6, 4,300 mg; pantothenic acid, 20,000 mg; biotin 220 mg; vitamin B12, 17 mg; vitamin B9, 2,200 mg; antioxidant (BHT + BHA  ), 50,000 mg, calcium 0.94-1%, Sodium 0.16-0.24%, Phophourse(Min) 0.5% .; copper, 16,000 mg; iodine, 1,250 mg; manganese, 120,000 mg; iron, 20,000 mg; and zinc, 110,000 mg; and selenium, 300 mg.

Performance measurements

Bird BW and feed intake in each treatment were weighed at 21, 28, and 35 d of age on a replicate basis and daily by subtracting a quantity of rejected feed from the offered feed. Average daily feed intake (DFI, g), daily gain (ADG, g), and FCR; g:g were computed during days 21–28 or 28–25, and overall period days 21–35. Day 21 is used as the start of treatment and indicates normal condition.

Blood biochemistry measurements

At the end of the experiment, 16 blood samples (2 birds from each replicate) were collected from the wing vein of birds (3 mL collected per sample). Then blood samples collected were centrifuged at 3000 rpm x g for 10 min. The serum was gathered and stored at −20 °C until further investigation. Total protein (TP), albumin, glucose, triglycerides, cholesterol, alanine transaminase (ALT) aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were measured by enzymatic calorimetric kits in accordance with manufacturer’s directions (RANDOX, U.K) via a Spectrophotometric analyser (UDICHEM 310, K.S.A). According to Al-Mufarrej et al. (2019), serum globulin was calculated by subtracting albumin levels from serum TP. All the parameters were performed in duplicate.

Statistical analysis

Statistical analysis were conducted in a completely randomised design and performed using the general linear model of the Statistical Analysis System (SAS 2012). Mean separation was performed with Duncan’s multiple range test. The overall level of significant was set at p < .05. All values were expressed as means ± standard error of the mean (SEM).

In this study, the following model was used: $$\text{Yij }=\mu +\text{ Ti }+\text{ eij},$$ where Y_ij observation, μ is the general mean. T_i is the effect of treatment (i) and e_ij is the random error.

Results

Growth performance

Performance parameters (BW, ADG, DFI, and FCR) of birds subjected to HS are presented in Table 2. The results for the periods (21–28 and 28–35 d) revealed that the treatments’ effect was not significant for any performance parameter (p > .05). However, when compared to the control group, FCR was improved by BET and MAGOBET (p = .018) on days 28–35.

Table 2. Effects of water-supplementation of magic oil (MAGO), betaine (BET), and combination of the two (MAGOBET) on performance indices of heat-stressed broiler chickens at 21–35 d.

			Treatments			Standard error	p-value
Parameters		Control	BET	MAGO	MAGOBET
	ADG (g)	66.540	65.850	65.350	65.430	1.940	.971
Days 21–28	DFI (g)	128.400	126.200	129	123.700	2.080	.306
	FCR (g: g)	1.930	1.920	1.980	1.900	0.050	.643
	ADG (g)	74.360	86.340	77.750	88.010	4.350	.116
Days 28–35	DFI (g)	140.800	139.100	140.800	141	4.960	.992
	FCR (g: g)	1.910^a	1.610^b	1.730^a,b	1.610^b	0.070	.018
	ADG (g)	75.640	82.120	77.620	82.880	2.870	.256
Days 21–35	DFI (g)	134.600	132.600	134.900	132.400	3.250	.922
	FCR (g: g)	1.790^a	1.620^b,c	1.740^a,b	1.600^c	0.050	.026
Day 35	BW (g)	2090.100	2121.600	2101.600	2184.800	46.370	.497

ADG: average daily gain; DFI: daily feed intake; FCR: feed conversion ratio; BW: body weight.

^a,bMeans with different superscripts in the same row differ significantly (p < .05).

The results for the overall period (21–35 d) revealed no substantial effect of experimental groups (p > .05). However, the control had a significantly worse FCR than the MAGOBET and BET (p = .026), but it was insignificant for MAGO. BET and MAGOBET obtained insignificantly higher ADG than the control (p > .05) under the HS condition.

Serum biochemistry measurements

The effects of water-supplemented magic oil, BET, and a combination of the two on serum blood proteins and glucose are shown in Table 3. The treatments had significant (p < .05) effects on the TP, albumin, globulin, and glucose. When compared to the control and BET groups, MAGO and MAGOBET supplementation increased serum TP (p = .0001), albumin (p = .0002), and globulin. Albumin: globulin ratio was not affected by experimental treatments. The birds in the MAGO group had lower significant (p = .035) serum glucose concentrations compared to that in the control and MAGOBET groups, but not in the BET group.

Table 3. Effects of water-supplementation of magic oil (MAGO), betaine (BET), and combination of the two (MAGOBET) on serum blood proteins and glucose levels of heat-stressed broiler chickens at day 35.

Parameters	Treatments				Standard error	p-value
	Control	BET	MAGO	MAGOBET
Total protein (g/dL)	2.9000^b	2.8400^b	4.0100^a	3.7000^a	0.1700	<.0001
Albumin (A) (g/dL)	1.6500^b	1.5300^b	2.0400^a	2.0900^a	0.1000	.0002
Globulin (G) (g/dL)	1.2500^c	1.3100^c	1.9600^a	1.6100^b	0.1000	<.0001
Albumin: globulin ratio	1.4100	1.3400	1.0700	1.3300	0.1200	.1950
Glucose (mg/dL)	139.5500^a	132.0500^a,b	122.9700^b	141.0400^a	4.7300	.0350

^a,b,cMeans with different superscripts in the same row differ significantly (p < .05).

The serum lipids profile of broiler birds is shown in Table 4. When compared to the other groups, the birds in the MAGO group had higher serum cholesterol, specifically high-density lipoprotein (HDL-C) ‘good’ cholesterol. When compared to the control, MAGOBET supplementation increased low-density lipoprotein ‘undesirable cholesterol’ (LDL-C). When compared to the control, total lipid decreased in the BET-supplemented group but not in the MAGO and MAGOBET-supplemented groups. Serum triglycerides were lower in the BET-supplemented group than in the MAGO and MAGOBET-supplemented groups.

Table 4. Effects of water-supplementation of magic oil (MAGO), betaine (BET), and combination of the two (MAGOBET) on lipids profile of heat-stressed broiler chickens at day 35.

Parameters (mg/dL)	Treatments				Standard error	p value
	Control	BET	MAGO	MAGOBET
Total lipids	322.9000^♣,a	292.4000^a,b	245.3000^b	251.9000^b	19.1600	.0180
Triglycerides	83.7300^a,b	77.4800^b	87.7600^a	88.5300^a	2.8500	.0320
Cholesterol	129.5300^b	122.5900^b	167.6400^a	141.4100^b	8.0600	.0010
HDL-C	80.5400^b	69.3400^b	102.9800^a	69.8900^b	5.6800	.0002
LDL-C	32.2400^b	37.7500^a,b	47.1100^a,b	53.8200^a	5.5700	.0390

HDL-C: high-densitylipoprotein-cholesterol; LDL-C: low-density lipoprotein-cholesterol; ^♣ n = 16 birds.

^a,bMeans with different superscripts in the same row differ significantly (p < .05).

The effects of experimental treatments on kidney function (serum urea and creatinine concentrations) and liver enzymes (alanine transaminase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels) in heat-stressed broiler chickens at day 35 are shown in Table 5. There was no effect of the treatments on serum urea or the enzymes AST, ALT, and ALP (p > .05). Creatinine levels were significantly lower (p = .004) in the BET or MAGO groups alone, but not in the combined groups when compared to the control.

Table 5. Effects of water-supplementation of magic oil (MAGO), betaine (BET), and combination of the two (MAGOBET) on kidney function and liver enzymes of heat-stressed broiler chickens at day 35.

Parameters	Treatments				Standard error	p-value
	Control	BET	MAGO	MAGOBET
Kidney function
Urea (mg/dL)	7.860	7.140	7.150	8.320	0.360	.061
Creatinine (mg/dL)	0.820^a	0.590^b	0.510^b	0.660^a,b	0.060	.004
Liver enzymes
AST (U/L)	22.750	21.490	21.820	21.130	0.510	.151
ALT (U/L)	206.720	209.890	208.600	213.260	5.570	.865
ALP (U/L)	427.290	431.150	416.280	401.980	9.620	.148

ALT: alanine transaminase; AST: aspartate aminotransferase; ALP: alkaline phosphatase. n = 16 birds.

^a,bMeans with different superscripts in the same row differ significantly (p < .05).

Discussion

The main findings of this investigation were that the water fortification of BET, MAGO, or MAGOBet alleviate HS effects by improving FCR in broilers subjected to continuous HS. Heat-stressed animals experience various physiological changes. Many studies have found that cyclic high-temperature has a negative impact on broiler performance (Wang et al. 2018; Shakeri et al. 2019; Abudabos et al. 2021). In the current study, daily FI and ADG of broiler chickens did not differ between treatment groups, whereas MAGOBET and BET improved FCR during days 28–35 and the overall period (21–35). Bartlett and Smith (2003) and Abudabos et al. (2021), found lower DFI in birds with chronic and cyclic HS when compared to broilers with thermoneutral. They attributed the drop in DFI could be due to birds attempting to reduce heat generated by feed metabolism. On fortunately, FCR was improved by combining magic oil and BET higher than separately during the 21–35 d when compared to the control group.

Dietary emulsifier blend and/or BET supplementation relief unfavourable effects of HS on growth performance, intestinal health, immune status of birds (Yousefi et al. 2023). According to Shakeri et al. (2019), BET improved overall growth rates in broilers under both thermoneutral and HS conditions, as demonstrated by lower rectal temperature and respiration rate. In agreement with (Zhan et al. 2006) who found that BET was similar to the control diet, our current study found that the ADG was comparable to the control. When comparing the control to BET, MAGO, or MAGOBET combination, only numerical improvements in ADG were reported. According to Abudabos et al. (2021), water supplementation with BET and Nano emulsified vegetable oil could significantly improve performance parameters. EOs can decrease the amount of pathogens due to their antimicrobial activity, increase the digestive tract enzymes activity and absorption strength, as well as maintaining gastrointestinal health (Ma et al. 2022; Moharreri et al. 2022). In addition, BET influences physiological improvement that begins in the gastrointestinal system and progresses to the metabolic level, thereby enhancing growth performance. Due to BET's chemical structure and nutritional properties, this improvement is given special consideration. According to Ratriyanto and Mosenthin (2018), BET in poultry diets may be beneficial in mitigating bodily responses to HS, as evidenced by increased nutrient digestibility. BET is a promising antioxidant for mitigating various stress conditions, such as HS. Therefore, adding BET or in combination with vitamin C improved the performance and antioxidant capacity of immune organs in heat-stressed broilers (Sun et al. 2023). According to Al-Sagan et al. (2020), BET enrichment attenuates the stressful effects of water deprivation on the genes expression of proopiomelanocortin in the brain and glucocorticoids in the liver and maintains cellular osmosis via interactions with the variable expression of aquaporins, particularly aquaporin 1 and aquaporin 2. In addition, Betaine fed to poultry helps to reduce stress by increasing blood electrolyte levels and total short-chain fatty acid concentrations, which then promotes poultry growth (Park and Park 2017). A broiler diet with 1000 mg/kg of BET improves FCR and BWG and decreases intra-muscular saturated fatty acids, while increasing total mono and poly unsaturated fatty acids, and has an antioxidant effect (Yang et al. 2022). This might be because the HS group used their feed more efficiently because of better osmolality, villi length restoration, and water balance in a hypertonic environment. Klasing et al. (2002) demonstrated that including BET at 0.05% and 0.1% enhanced osmolality and increased villus length of the duodenum. Furthermore, 0.2% BET supplementation was revealed to help the duodenum maintain water equilibrium (Kettunen et al. 2001).

Blood biochemical indicators in serum can rapidly reflect the nutritional and health status of animals and the metabolic functions of the body (Raju et al. 2019; Zheng et al. 2023). MAGO and MAGOBET increased TP by 38.28% and 27.59%, respectively, when compared to the control. Furthermore, MAGO and MAGOBET increased albumin by 23.64% and 26.67%, respectively, and MAGO increased glucose by 56.80, indicating that MAGO can improve the protein anabolism of the chicken body. These data agree with Ghanima et al. (2021), who stated that NEVO supplementation in a rabbit’s diet improved serum protein and globulin levels. In this study, the BET group did not differ from the control group. In contrast to Attia et al. (2005) and to Al-Sagan et al. (2020), they found that that BET incorporation increased blood albumin by contributing methyl groups, which is required for protein metabolism. The MAGO group reduced the concentration of glucose in the serum. This decrease could be attributed to sodium-glucose transporter 1 in the intestine inhibiting glucose uptake (Nasir et al. 2016; Al-Baadani et al. 2022). Researchers demonstrated cutting-edge nano-systems for phytocompounds in type 2 diabetes mellitus treatment, demonstrating nanotechnologies’ dominance and potential (Nie et al. 2020). However, MAGOBET raised blood glucose levels when compared to MAGO alone. This is consistent with the findings of Bogin et al. (1996), who found that elevated blood glucose aids in the ‘fight or flight’ response during HS exposure may increase the survivability rate of chickens. In comparison to their counterparts, Attia et al. (2016) found plasma glucose decreased in laying hens raised under HS and fed a diet with no supplementation. According to Al-Sagan et al. (2020), glucose levels were higher in stressed birds given BET at 0.15%, but similar in birds given BET at 0.0, 0.075, and 0.10%. The increase observed in blood glucose and total lipids levels in stressed broilers in the control group indicates alterations in carbohydrate and lipid metabolism (Ghasemi and Nari 2020). Contradictory Attia et al. (2009) found that there was a decline in plasma glucose and total protein due to HS.

MAGO reduced total lipids by 24.03% while increasing cholesterol by 29.42%. In agreement with Ghanima et al. (2021) on total lipids but disagreed on cholesterol, they found that increasing NEVO levels reduced serum total lipids and total cholesterol levels. Fortunately, MAGO increased HDL-C by 27.86%, and MAGOBET increased LDL-C by 66.94%, this indicated that adding BET to MAGO elevated undesirable cholesterol, while MAGO supplementation alone increased desirable cholesterol compared to control. Citrus aurantifolia leaf EOs improved dyslipidemia: serum total cholesterol levels, triacylglycerol, and LDL-C were significantly reduced, while HDL-C was increased (Ibrahim et al. 2019). When compared to the control, BET decreased triglycerides by 7.46%, whereas MAGO and MAGOBET increased triglycerides by 4.81% and 5.73%, respectively. Uzunoğlu and Yalçın (2019) reported similar results, stating that dietary BET supplementation reduced triglycerides. However, El-Shinnawy (2015) found that broilers fed betaine-supplemented diets had higher triglyceride levels than those in the control group. MAGO reduced total lipids while increasing cholesterol levels, especially HDL-C, which could be attributed to increased absorption of bile acid and distribution in the intestine, resulting in lower serum total lipids and higher cholesterol. MAGO may also increase the viscosity of duodenum contents, reducing intestinal lipid absorption. The third possibility is that MAGO stimulated the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A-reductase (HMG-CoA), which is the rate-limiting enzyme in cholesterol synthesis, in the liver of birds. HMG-CoA reductase, malic, and acetyl-CoA carboxylase enzymes activity can all be affected by phytosterols (Chandra 2014).

BET and MAGO were reduced creatinine by 28.05% and 37.80%, respectively compared to control indicating that BET and MAGO improved renal functions. The fact that BET and/or MAGO supplementation had no effect on liver enzymes indicated that the birds were healthy, and BET and/or MAGO is a non-toxic as water additives. These findings are similar to those reported by Attia et al. (2016) who found significant differences in these biochemicals as a result of HS. Dietary BET supplementation had no effect on the blood biochemicals tested, with the exception of triglycerides (Ghasemi and Nari 2020). Furthermore, Attia et al. (2009) found that supplementing 1 g of betaine/kg diet resulted in complete relief of plasma glucose but only partial relief of triglycerides and total protein.

In most blood biochemistry, BET was lower than MAGO, but their combination had a synergistic effect. More researches are needed to determine betaine’s ability to mitigating HS in poultry. Moreover, additional studies are needed to determine MAGOBET's ability to reduce BET or MAGO supplementation alone and make MAGOBET supplementation cost-effective, particularly when combined with different levels of BET and MAGO.

Conclusions

In conclusion, birds in the magic oil group had lower serum glucose and creatinine levels and higher cholesterol levels, especially high-density lipoprotein, to the control. In addition, the administration of aqueous magic oil alone or in combination with betaine produced some benefits: the birds in both groups had lower total lipids and higher blood protein levels compared to the control group. Water BET supplements alone or in combination with MAGO improved feed conversion efficiency under HS conditions during 28–35 and 21–35 d compared to the control. However, the additional effects of the MAGO, BET and MAGOBET combination need to be further investigated, especially in terms of their added value for growth performance under different conditions.

Acknowledgement

The authors extend their appreciation to the Deputyship for Research and Innovation, Ministry of Education in Saudi Arabia for funding this research work through project No. (IFKSURG-2-592).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The results and analyses presented in this paper freely available upon request.

Additional information

Funding

This work was supported by Deputyship for Research and Innovation, Ministry of Education in Saudi Arabia [IFKSURG-2-592].