Enhancing semen quality, brain neurotransmitters, and antioxidant status of rabbits under heat stress by acacia gum, vitamin C, and lycopene as dietary supplements: an in vitro and in silico study

Abstract

In the current study, the effects of enhancing semen quality, brain neurotransmitters, and antioxidant status in rabbits under heat stress with acacia gum, vitamin C, and lycopene as dietary supplements were estimated in vitro and in silico. A total of 40 males from New Zealand White rabbits aged 3 months were equally divided into four groups (n = 10). The first group was a control. The other three groups were the heat stress group (oral acacia gum, 100 mg/kg body weight), the ascorbic acid group (30 mg/kg body weight), and the lycopene group (50 mg/kg body weight). All semen showed physical characteristics in terms of increasing sperm motility, motility index, sperm normality, and live sperm as compared to the control group. On the other hand, sperm abnormalities, and dead sperm significantly (p < 0.001) decreased compared with the control group. It is of interest to show that most semen traits were significantly (p < 0.001) better for acacia gum than vitamin C and lycopene. The expression of neurotransmitters (5-HT, DA, Glu, Asp) was enhanced in all treatments compared to the control. The results of acacia gum were better (p < 0.001) than those obtained with vitamin C and lycopene. The acacia gum treatment had a lower plasma semen MDA and NO (p < 0.001) and higher GSH, TAC, SOD, CAT, L-Car, Na⁺-K⁺ ATPase, ATP, and total calcium content (p < 0.001) than in other treatments and controls. These results were confirmed by the prediction of the binding energy, several conventional and carbon H-bonds, hydrophobicity, and SAS through the in silico docking analysis results. It can be concluded that acacia gum, vitamin c, and lycopene are used for enhancing semen quality, brain neurotransmitters, and the antioxidant status of rabbits under heat stress.

HIGHLIGHTS

• One of the most significant influences on animal metabolism and productivity is heat stress.

• Fertility is negatively impacted by heat stress because the level of reactive oxygen species rises.

• Acacia gum, lycopene, and ascorbic acid were used to improve the quality of sperm, brain neurotransmitters, and antioxidant status of rabbits under heat stress in vivo.

Keywords:

• Acacia gum
• vitamin C
• lycopene
• semen quality
• brain neurotransmitters

Introduction

Heat stress is one of the most important factors that impact the metabolism and productivity of animals. Rabbits are affected by high ambient temperatures because they cannot sweat and must rely on vasomotor control and panting to dissipate excess body heat, which impacts feed intake, live weight gain, feed efficiency, meat quality, mortality, semen quality, and health (Cheeke 1987; Marai and El-Kelawy 1999; Marai et al. 2002). Heat stress has a negative impact on fertility because the level of reactive oxygen species increases (Aitken and De Iuliis 2007). High ambient temperature stimulates the hypothalamo–pituitary–adrenal axis activation evoking the functions of the sympathetic system, which increase levels of free radicals and imbalances in the antioxidant-defense system (Agarwal et al. 2007; Ahmad et al. 2012). Accumulations of free radicals have been associated with a significant decrease in sperm motility and sperm plasma membrane integrity and a significant increase in sperm abnormality and DNA damage leading to infertility (Potts et al. 2000; Surai et al. 2017). Ambient temperature initiates lipid oxidation in cell membranes, and the negative impact of high ambient temperatures may be partly due to oxidative stress (Bollengier-Lee et al. 1998; Sahin and Kucuk 2003). Therefore, diets supplemented with natural antioxidants that protect cells and tissues from lipoperoxidation damage caused by excess free radicals could be used to reduce the negative effects of high ambient temperature (Hussein 1996; Tuzcu et al. 2008). Various natural antioxidants can protect DNA and other molecules from oxidation-induced cell damage, improve sperm quality and increase male reproductive efficiency (Yang et al. 2006). Several natural antioxidants, such as acacia gum and lycopene (Mangiagalli et al. 2010; Patel and Goyal 2015) or folic acid (Tolba et al. 2015), are effective antioxidant defense components that protect the plasma membrane against lipid peroxidation and play an important role in the metabolism of amino acids and DNA.

Dried gummy exudate from the stems and branches of the Acacia senegal tree is known as Arabic gum (AG), and it can be eaten. The abundance of non-viscous soluble fibres makes it unique. In the pharmaceutical and food industries, it is frequently used as an emulsifier and stabiliser (Ali et al. 2009). Arabic gum is one of many natural products with a long history of being used as a preventative supplement in Arabic folk medicine. Recently, it has gained more attention because it protects against the effects of environmental and chemical exposure to dangers (Bliss 1992; Daoub et al. 2018). Because it did not cause genotoxicity or cancer when taken orally or intraperitoneally, acacia gum (AG) is considered a safe food additive (Johnson 2005). AG has powerful antioxidant activity that protects animals under heat stress. Fouda and Ismail (2018) reported that adding 1 kg/ton of acacia gum to the diet improves reproductive performance and increases the antioxidant activity under heat stress in blood plasma. Carotenoids, which are thought to be an important class of natural antioxidants, have been the subject of numerous studies (Rao and Rao 2007). Lycopene, a carotenoid found in tomatoes (Lycopersicon esculentum) and several ripe fruits and vegetables like watermelon, pink grapefruit, and carrots, has gained popularity in the last ten years (Tadmor et al. 2005), has been taken into account in a few studies about human and animal health (Mangiagalli et al. 2012; Amer et al. 2020), and the biology of reproduction (Mendiola et al. 2010). Lycopene is a natural antioxidant synthesised by plants or microorganisms but not by animals. Adding lycopene, which maintains oxidative balance in animals through increasing superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) enzymes (Blokhina et al. 2003; Martínez et al. 2008), synthesised it. Still, some experiments have suggested that lycopene supplementation increases sperm count and immunity and decreases lipid peroxidation and DNA damage (Durairajanayagam et al. 2014).

Low-molecular-mass antioxidant ascorbic acid (AA) fights free radicals. It is an efficient reducing agent and is highly soluble in water. It prevents lipid peroxidation caused by peroxyl radicals, making it one of the most potent antioxidants and free radical scavengers (El-Demerdash et al. 2005). Ascorbic acid, also known as vitamin C (VC) has antioxidant activity due to its action as an electron donor that protects the molecules from being oxidised (Padayatty et al. 2003). Selim et al. (2008) concluded that adding VC enhances growing rabbits’ growth performance, antioxidant status, and immunity traits. In the current study, the effects of enhancing semen quality, brain neurotransmitters, and antioxidant status in rabbits under heat stress with acacia gum, vitamin C, and lycopene as dietary supplements were estimated in vitro and in silico.

Materials and methods

Materials and chemicals

Lycopene was purchased from DM-Pharma (assay: 100%). All other chemicals, solvents, and reagents were purchased from Sigma and were of HPLC grade.

Animal and treatments

A total of 40 3-month-old male rabbits (Oryctolagus cuniculus) with similar body weights (2.100–2.350 kg) were randomly divided into four groups of equal number (n = 10) with the same average weight. Each buck served as a replicate and was individually in cages in batteries (45 × 55 × 30 cm) under summer conditions. Animals were supplied from the local farm in Nahea, Giza, Egypt. The animals were fed ad libitum and allowed to acclimate to the environment for two weeks before the experiment. The study lasted for 90 days. Bucks were divided into four groups: control (fed with a basal diet without additional). In the ascorbic acid group, rabbits received vitamin C at 30 mg/kg body weight; the Lycopene group had lycopene at 50 mg/kg body weight; the Acacia gum group had acacia gum at 100 mg/kg body weight. Supplementations were given orally daily during the experiment period.

For each selected group, a total of 675, 1125, and 2250 mg/10 mL of vitamin C, lycopene, and acacia gum were freshly prepared each day. Using a nozzle syringe, we administered 1 mL of the prepared solution to each rabbit by gently inserting it into the side of their mouth. Every day, the preparation was made at 7 a.m., just before the feed started. The method of administration ensures that the solution reaches the middle of the mouth rather than the throat. Additionally, ensure that the rabbit’s head remains straight throughout the application of the solution.

The basal diet compound and the computational analysis followed the nutritional requirements of rabbits from the National Research Council guidelines (Smith et al. 1966), as shown in Table 1.

Table 1. Composition and calculated analyses of the basal diet (g/kg, as-fed basis) of weaned rabbits (Atallah et al. 2021).

Ingredients	Content
Ingredients, g/kg
Alfalfa hay	350.0
Yellow corn	200.0
Soybean meal	96.0
Wheat bran	300.0
Corn Stover	30.0
Di-calcium phosphate	12.5
L-Lysine HCl	2.1
DL-Methionine	2.0
Sodium chloride	5.0
Vitamin/mineral premix*	1.5
Total	1000.0
Calculated analysis, g/kg on dry matter basis
Digestible energy, MJ/kg	11.6
Crude protein, g/kg	179.0
Crude fibre, g/kg	125.0
Crude fat, g/kg	32.0
Ca, g/kg	10.9
Available P, g/kg	5.9
Methionine, g/kg	4.2
Lysine, g/kg	9.0

*Minerals and vitamins mixture supply/kg of diet: vit. A, 20,000 IU; vit. D3, 15,000 IU; vit. E, 8.33 g; vit. K, 0.33 g; vit. B1, 0.33 g; vit. B2, 1.0 g; vit. B6, 0.33 g; vit. B5, 8.33 g; vit. B12, 1.7 mg; pantothenic acid,3.33 g; biotin, 33 mg; folic acid, 0.83 g; choline chloride, 200 g.

Average air temperature and relative humidity were recorded daily every 60 min during the experimental period from July to September using data loggers (Table 2). The temperature-relative humidity index (THI) was determined in accordance with the following equation: $$\mathrm{THI}=t-\left[\right(0.31-0.31(\mathrm{RH}/100)\left)\right(t-14.4\left)\right]$$ where: t° C = dry bulb temperature in degrees Celsius, and RH = relative humidity percentage/100 (Ogunjimi et al. 2008). Marai et al. (2000) protocols for the measurement of the rabbit heat stress index (HSI) were applied where temperature values <27.8 °C indicate lack of heat stress, 27.8–28.9 °C indicate moderate heat stress, 29.0–30.0 °C indicate extreme heat stress, >30.0 °C indicate very extreme heat stress.

Table 2. Average values of air temperature (T), relative humidity (RH), and temperature humidity index (THI) at the experimental site.

Measurements	Period, days
	15	30	45	60	75	90
T, db°C	30.20	32.6	33.40	34.00	34.4	35.90
RH%	62.30	63.1	64.20	64.70	65.1	65.80
THI	28.30	30.5	31.31	32.04	32.4	33.75

T, db°C: dry bulb temperature in degrees Celsius; RH: relative humidity percentage; THI: relative temperature humidity index.

Sample collection

Semen was collected after 60 days of treatments daily along with one month at 4th intervals each week. Indeed, rabbits were decapitated 30 days after the end of treatment and then brains were immediately collected and stored at −20 °C until amino acids and monogamies were analysed.

Determination of brain amino acids and monoamines

Each brain tissue was homogenised in 75% aqueous HPLC-grade methanol (10% w/v) (Arafa et al. 2010). The homogenate was spun at 4000 rpm for 10 min., and the supernatant was divided into two halves; the first was dried using a vacuum (70 Millipore) at room temperature and used for GABA (gamma aminobutyric acid) determination according to the method of Heinrikson and Meredith (1984) and glutamate determination, whereas the second half was used for monoamine determination according to the method described by Pagel et al. (2000).

Determination of antioxidant variables, nitrites, and nitrates

The concentration of reduced glutathione (GSH) was measured in semen samples by HPLC method (Agilent HP 1200 series HPLC apparatus, Midland, ON, Canada), according to Jayatilleke and Shaw (1993). Standard (GSH: 200-725-4) was obtained from Sigma-Aldrich Chemie GmbH Export Co. Ltd., Taufkirchen, Germany. As well, the semen activates of superoxide dismutase (SOD) and catalase (CAT) were obtained by the method of Marklund and Marklund (1974) at 420 nm for 1 min on a UV-Vis Shimadzu spectrophotometer (2450). The concentration of MDA was measured as a marker of cell membrane degradation by HPLC methods (Agilent HP 1100 series HPLC apparatus (USA)) according to Karatas et al. (2002). Nitrites and nitrate were determined according to the method of Papadoyannis et al. (1999) by HPLC.

Determination of adenosine tri-phosphate content (ATP, ADP, and AMP) semen plasma by HPLC

The detection of ATP by HPLC was done according to the method of Teerlink et al. (1993).

Determination of Na, K-ATPase, calcium, and TAC

Semen plasma was analysed for L-Carnitine (Khoshkam and Afshar 2014) via RP-HPLC method); Na,K-ATPase according to method (Sweadner et al. 2016); Calcium was colorimetrically determined according to Gindler and King (1972) and TAC was colorimetrically determined in accordance to Koracevic et al. (2001).

Determination of phosphatide in acacia gum

Major phospholipids were extracted after minor modifications of the method previously reported by Folch et al. (1957). The extracted phospholipid was dissolved in a mobile phase solvent containing 20% chloroform before HPLC analysis. The isocratic high-performance liquid chromatographic separation of different phospholipids was performed by the HPLC system (Agilent 1200 Series equipped with the computerised solvent delivery system and UV detector, Santa Clara, CA, USA) using a Porasil silica gel column (10-lm particle size). Samples (20 µL) were injected for HPLC analysis and eluted by the degassed mobile phase [acetonitrile–methanol–85% phosphoric acid (96:3:1, v/v/v)] that was delivered at a flow rate of 0.80 mL/min. The effluent was monitored at a 203 nm wavelength, and the concentration of each sample was detected using corresponding phospholipid standards. Standard (GSH: 200-725-4) was obtained from Sigma Chemical Company (St. Louis, MO, USA) (Figure 1).

Figure 1. HPLC chromatogram of Acacia gum.

In silico docking investigation

The in silico docking was initiated by a survey of each enzyme and component. Sequences of catalases (CAT), glutathione peroxidase (GSH), and superoxide dismutase (SOD) were obtained from the following databases: NCBI, Protein Data Bank, and UniProtKB. The CAT enzyme (PDB ID: XP_002709091.1), GSH enzyme (PDB ID: NP_001078913.1), and SOD enzyme (UniProtKB ID: G1TKH3) were used as templates for homology modelling with Swiss-Model to perform the 3D structure for each enzyme (Ritika et al. 2021). Lycopene, vitamin C, Phosphatidylserine, and phosphatidylcholine structures were obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/), followed by the performance of energy minimisation for each component of them using Swiss PDB Viewer software (spdbv). Finally, Open Babel (Version 2.3.1, http://openbabel.org) was used for converting the format from PDB to pdbqt. The modifications for each enzyme before the docking process were done by the Discovery Studio 2019 software, such as adding the hydrogen atoms for the domain and removing water molecules (Umar et al. 2021). Finally, AutoDock Vina was used for the performance of the grid box for each domain at 1.00 Å spacing, with points of numbers of 70 X × 80 Y × 74 Z Å grid focus dimensions for CAT domain, 44 X × 48 Y × 46 Z Å for GSH domain and 56 X × 40 Y × 62 Z Å for SOD domain (Labib et al. 2022).

Statistical analysis

All data were expressed as means with a standard error mean (SEM) and were subjected to analysis of variance (ANOVA) in a one-way analysis of variance, using statistical analysis system (SAS) software version 2004 (SAS 2004). Multiple comparisons were made using Duncan’s multiple-range tests to determine the differences between treatments. The applied static model is as follows: (1) $$y_{ij} = \mu + T_i + e_{ij}\mathrm{ }$$(1) where: y_ij is the observations, µ = general mean, T_i: effect of treatment, e_ij: random error.

Results

Semen quality

Nutritional impacts of acacia gum, vitamin C, and lycopene on semen quality (sperm motility, motility index, sperm normality %, sperm abnormality %, live sperm %, and dead sperm) for New Zealand rabbits under heat stress are presented in Table 3. Table 3 depicts the positive effects that acacia gum, vitamin C, and lycopene on semen quality (sperm motility, motility index, sperm normality %, sperm abnormality %, live sperm %, and dead sperm) had on New Zealand rabbits under heat stress. Acacia gum, vitamin C, and lycopene administration significantly improved (p < 0.001) all semen physical characteristics in terms of increasing sperm motility, motility index, Sperm normality, and live sperm as compared to the control group. On the other hand, sperm abnormalities, and dead sperm significantly (p < 0.001) decreased compared with the control group. It is of interest to show that most semen traits were significantly (p < 0.001) better for acacia gum than vitamin C and lycopene (Table 3).

Table 3. Nutritional impacts of acacia gum, vitamin C, and lycopene on semen quality New Zealand rabbits under heat stress.

Treatment	Semen quality
	Sperm motility	Motility index	Sperm normality, %	Sperm abnormality, %	Live sperm, %	Dead sperm, %
Control	2.28^c	48.06^c	57.09^c	43.08^a	59.60^c	37.66^a
Vitamin C	3.61^ab	64.66^b	72.17^b	28.41^c	64.94^b	33.14^b
Lycopene	3.34^b	59.12^b	62.95^c	36.85^b	63.85^bc	34.38^b
Acacia gum	3.87^a	71.31^a	80.13^a	17.70^d	94.15^a	9.33^c
SEM	0.07	1.45	1.63	0.73	1.29	0.69
p-Value	0.0001	0.0001	0.0001	0.0001	0.0001	0.0001

^a–dMean values within a column with different superscript letters are significantly different.

Brain neurotransmitter

Nutritional impacts of acacia gum, vitamin C, and lycopene on brain neurotransmitters (5-HT, DA, Glu, Asp) in New Zealand rabbits under heat stress are presented in Figure 2. Data presented in Figure 2 indicated that oral administration of acacia gum, vitamin C, and lycopene significantly increased 5-HT compared to the control. The highest value was recorded for acacia gum (0.61), vitamin C (0.52), and lycopene (0.47), respectively. The highest amount of dopamine was recorded in the case of acacia gum, and non-significant differences were observed between vitamin C and lycopene. On the other hand, Asp significantly (p < 0.001) decreased for treated groups (acacia gum, vitamin C, and lycopene) compared to the control. Glu significantly (p < 0.001) decreased for the acacia gum group compared to other treatments (vitamin C, and lycopene) and control.

Figure 2. Nutritional impacts of acacia gum, vitamin C and lycopene on brain neurotransmitter New Zealand rabbits under heat stress. ^a–dMean values within the same box with different superscript letters are significantly different. p < 0.001.

Semen antioxidants

When compared to the control group, the levels of antioxidant enzymes (SOD, CAT, and GSH) increased significantly (p < 0.001) when acacia gum, vitamin C, and lycopene were administered orally (Table 4). For GSH and CAT, non-significant differences were observed between acacia gum and vitamin C. Acacia gum, vitamin C, and lycopene had positive effects on TAC, L-Car, ATP, MDA, and NO compared to the control. Acacia gum and vitamin C had positive effects on Na⁺-K⁺ ATPase, and non-significant differences were observed between the vitamin-treated group and the control. For total calcium, all treatments had positive effects compared to the control, while the lycopene-treated group recorded non-significant differences with the control.

Table 4. Nutritional impacts of acacia gum, vitamin C, and lycopene on semen antioxidants New Zealand rabbits under heat stress.

	Semen
Treatment	GSH, μmol/mL	TAC	SOD, Ul/mµL	CAT, μmol of H2O2/mL	L-Car	Na^+-K^+ ATPase, nmol/g tissue	ATP	Total calcium, mmol/L	MDA, nmol/mL	NO
Control	0.34^c	19.90^b	36.08^c	13.45^c	315.18^c	402.01^b	0.30^c	2.98^b	38.79^a	0.67^a
Vitamin C	0.61^a	26.04^a	44.16^a	17.23^ab	344.65^ab	467.23^a	0.54^b	3.49^a	31.74^b	0.45^c
Lycopene	0.48^b	22.19^b	40.94^c	16.58^b	324.36^bc	403.55^b	0.54^b	3.19^ab	18.58^c	0.52^b
Acacia gum	0.64^a	27.20^a	46.13^b	18.15^a	372.80^a	439.48^a	0.63^a	3.41^a	14.01^d	0.41^c
SEM	0.01	0.63	0.78	0.38	7.12	10.76	0.01	0.08	0.71	0.01
p-Value	0.0001	0.0001	0.0001	0.0001	0.0001	0.0001	0.0001	0.0009	0.0001	0.0001

^a–dMean values within a column with different superscript letters are significantly different.

In silico molecular docking investigation and analysis

The homology modelling of GSH, SOD, and CAT enzymes was performed using Swiss-model and then visualised through the visualisation module of the Discovery Studio. The molecular docking and virtual screening for each domain showed each protein’s predicted ligand-domain complex and the binding affinities’ scores and rankings. The in silico docking analysis showed each domain-ligand complex’s aromatic interactions and 2D chemical interactions (Table 5 and Figures 3–5). The binding energy, number of conventional H-bond, carbon H-bond, hydrophobicity, and solvent-accessible surface (SAS) were shown in Table 5 for each domain-ligand complex. On the other hand, the H-bond acceptor in green and doner in pink colours, the positive charges with blue and negative with red colours, a hydrophobic area with a brown and hydrophilic area with blue colours, ionizability with basic in blue and acidic in red colours and solvent accessible surface (SAS) in blue colour through the interactions of the docked complexes between each domain and ligands were shown in Figures 6–8. The superior ligand in the binding energy results was the lycopene-catalase complex while the superior ligands in the 2D chemical interactions were the phosphatidylcholine and phosphatidylserine components. Hydrophobicity area and SAS interactions were increased through all the docked ligands-enzymes complexes and the H-bonds as donors, acceptors, charges, and ionizability. However, phosphatidylcholine and Phosphatidylserine were preferable in all the docking analysis parameters.

Figure 3. The aromatic interaction and the 2D chemical interactions of the docked complexes between catalase enzyme and (A,B) lycopene, (C,D) vitamin-C, (E,F) phosphatidylserine, and (G,H) phosphatidylcholine.

Figure 4. The aromatic interaction and the 2D chemical interactions of the docked complexes between glutathione peroxidase enzyme and (A,B) lycopene, (C,D) vitamin-C, (E,F) phosphatidylserine, and (G,H) phosphatidylcholine.

Figure 5. The aromatic interaction and the 2D chemical interactions of the docked complexes between superoxide dismutase enzyme and (A,B) lycopene, (C,D) vitamin-C, (E,F) phosphatidylserine, and (G,H) phosphatidylcholine.

Figure 6. The H-bond acceptor in green and doner in pink colours, charges positive with blue and negative with red colours, a hydrophobic area with a brown and hydrophilic area with blue colours, ionizability with basic in blue and acidic in red colours, and solvent accessible surface (SAS) in blue colour through the interactions of the docked complexes between catalase enzyme and (A–E) lycopene, (F–J) vitamin-C, (K–O) phosphatidylserine, and (P–T) phosphatidylcholine, respectively.

Figure 7. The H-bond acceptor in green and doner in pink colours, charges positive with blue and negative with red colours, a hydrophobic area with a brown and hydrophilic area with blue colours, ionizability with basic in blue and acidic in red colours, and solvent accessible surface (SAS) in blue colour through the interactions of the docked complexes between glutathione peroxidase enzyme and (A–E) lycopene, (F–J) vitamin-C, (K–O) phosphatidylserine, and (P–T) phosphatidylcholine, respectively.

Figure 8. The H-bond acceptor in green and doner in pink colours, charges positive with blue and negative with red colours, a hydrophobic area with a brown and hydrophilic area with blue colours, ionizability with basic in blue and acidic in red colours, and solvent accessible surface (SAS) in blue colour through the interactions of the docked complexes between superoxide dismutase enzyme and (A–E) lycopene, (F–J) vitamin-C, (K–O) phosphatidylserine, and (P–T) phosphatidylcholine, respectively.

Table 5. The docking analysis results of binding energy, No. of H-bond, No. of carbon H-bond, hydrophobicity, and SAS interactions between GSH, SOD, and CAT enzymes against vitamin C, lycopene, phosphatidylcholine, and phosphatidylserine.

Component	Binding energy			No. of conventional H-bond			No. of carbon H-bond			Hydrophobicity			SAS
	GSH	SOD	CAT	GSH	SOD	CAT	GSH	SOD	CAT	GSH	SOD	CAT	GSH	SOD	CAT
Vitamin C	−5.1	−5.8	−6.6	3	3	4	0	0	0	High	High	Moderate	Moderate	High	Moderate
Lycopene	−6.4	−7.1	−8.6	0	0	0	0	0	0	High	High	Moderate	High	Moderate	Low
Phosphatidylcholine	−4.2	−4.3	−6.9	3	3	1	3	1	0	High	High	High	High	Moderate	High
Phosphatidylserine	−4.4	−5.0	−5.2	4	3	4	0	3	1	Low	High	Moderate	High	High	Moderate

GSH: glutathione peroxidase; TAC: antioxidant capacity; SOD: superoxide dismutase; CAT: catalase; L-Car: L-Carnitine; ATP: adenosine triphosphate; MDA: malondialdehyde; NO: nitric oxide.

Discussion

The alleviation of the negative impacts of heat stress on livestock is a major challenge in subtropical regions, especially when high temperatures combine with high humidity. The temperature–humidity index obtained in this study found that rabbit bucks had extreme heat stress (28.9 to <30.0) (Marai et al. 2002, 2008). In the current study, the effects of enhancing semen quality, brain neurotransmitters, and antioxidant status in rabbits under heat stress with acacia gum, vitamin C, and lycopene as dietary supplements were estimated in vitro and in silico. Heat stress had negative effects on semen quality. Our results indicated that oral administration of acacia gum, vitamin C, and lycopene enhanced semen quality (sperm motility, motility index, sperm normality %, sperm abnormality %, live sperm %, and dead sperm) in New Zealand rabbits under heat stress. This effect could be attributed to an improvement in testosterone synthesis [a hormone necessary to complete the spermatogenesis process (Salem et al. 2002; Walker 2009)] or to the antioxidant activity of acacia gum, vitamin C, and lycopene, which may protect the different stages of spermatocytes from apoptosis, leading to an increase in sperm production. Generally, previous studies have shown that natural products with antioxidant activity, such as lycopene and acacia gum (Levy et al. 1995; Patel and Goyal 2015) and β-carotene (Rao and Agarwal 1999; Hashem et al. 2013) could improve the concentration of sperm in rabbits under heat stress conditions. High ambient temperature decreased hormonal activity, possibly due to over-reactive oxygen species (ROS) that oxidise and damage cell biological molecules, inhibit some activity of ATPases (Na⁺K⁺-ATPase, Ca²⁺-ATPase, and Mg²⁺-ATPase) and finally cause a variety of tissue disorders in the testes (Hunt et al. 1988; Hayashi et al. 1994; Payne and Southern 2005; Zhao and Shen 2005; Josephine et al. 2008).

In the current study, the positive impact of acacia gum, vitamin C, and lycopene on brain neurotransmitters (5-HT, DA, Glu, Asp) in New Zealand rabbits under heat stress was observed. In addition, when animals are exposed to heat stress, the hypothalamic-pituitary-adrenal axis is stimulated, and glucocorticoids are released from the adrenal cortex, which raises the level of free radicals and imbalances in the antioxidant defense system (Klemcke et al. 1989; Agarwal et al. 2007; Ahmad et al. 2012). Excessive concentrations of glucocorticoids result in catabolic effects in the testes that affect the quality of semen’s rabbit (Tomas et al. 1979; Odedra et al. 1983). Heat stress influences the excitatory and inhibitory neurotransmitters of amino acids in the central nervous system. Our tests showed a significant increase in 5-hydroxytryptamine receptors 5-HTP and Dopamine DA. Increased levels of these neurotransmitters could be due to their important role in temperature regulation. Noradrenaline and adrenaline are chemical transmitters at most sympathetic post-ganglionic endings, stored in the synaptic knobs of the adrenergic neurons. Corticosterone is the terminal product of the hypothalamic-pituitary–adrenal HPA axis and is one of the adrenal cortical synthetic glucocorticoids (Chanclón et al. 2012). Dopamine is the noradrenaline precursor found in the dopaminergic neurons of the brain’s hypothalamus. Our results appeared to be parallel with the literature (Faisal et al. 2008). Glutamate and aspartate are major excitatory neurotransmitters in mammalian brains and play important roles in the nervous system, immunity, and nutrition in animals and humans (Yanni et al. 2010; Khropycheva et al. 2011; Lin et al. 2014; Wang et al. 2017). Our tests showed a significant decrease glutamate and aspartate compared with control. Previous studies have shown that dietary supplementation with acacia gum and lycopene decreases oxidative stress-induced growth suppression (Mangiagalli et al. 2012; Patel and Goyal 2015).

Environmental stress, especially high temperatures, induces oxidative stress by increasing the development of malondialdehyde (Halliwell 1989; Sahin and Kucuk 2003). Using malondialdehyde as an oxidative stress index, lipid peroxidation in the plasma of semen has been studied. The results showed that Acacia gum, vitamin C, and lycopene significantly lowered malondialdehyde and nitric oxide levels in plasma semen. In the current study, Acacia gum, vitamin C, and lycopene had a positive effect on antioxidant enzymes, TAC, L-Car, ATP, MDA, and NO compared to the control.

Lee et al. (2013) reported that phosphatidylcholine has an antioxidant effect and prevents oxidative stress. Thus, due to the phosphatidylcholine, the major compound in Acaci gum as shown from HPLC, it was reasonable that acacia gum is more powerful strength of antioxidation. Also, natural antioxidants have been recorded to prevent the formation of superoxide and lipid peroxidation (Lau and King 2003). Hagerman et al. (1998) found that high antioxidant diets can reduce aortic malonaldehyde and cholesterol hydroperoxides in rabbits. Several endogenous antioxidant enzymes, such as SOD and GSH-Px, can convert ROS into less damaging compounds (Masella et al. 2005). Superoxide can be first degraded to hydrogen peroxide by SOD and then catalysed and converted to water by a series of enzymes, including GSH-Px (Blokhina et al. 2003). This could be helpful for rabbits reared under heat stress, as increased antioxidant activity ensures proper and rapid removal of ROS that could be produced at high ambient temperatures. Acacia gum, vitamin C, and lycopene have also increased GSH, TAC, CAT, SOD, L-Car, Na⁺-K⁺ ATPase and ATP plasma content, and total calcium content in this study. Our results are similar to those reported by Krajka-Kuźniak and Baer-Dubowska (2003), which observed that the activity of antioxidant enzymes was induced by tannic acid in the liver and kidneys of rats. Based on these results, it is proposed that there are two possible mechanisms: first, acacia gum, vitamin C, and lycopene may be used to compensate for the decline in the activity of antioxidant enzymes by responding directly to free radicals; second, acacia gum, vitamin C, and lycopene may selectively induce antioxidant gene expression, possibly through the up-regulation of gene transcription. Previously, Yeh and Yen (2006) and Patel and Goyal (2015) found that the mRNA levels of hepatic Cu-Zn SOD, GSH-Px, and catalase in the liver tissues of phenolic acid-supplemented rats were higher than those in the control group, and the Nrf2 protein may play a key role in the activation of phenolic acid-induced antioxidant genes.

The in silico investigation was performed by docking to ensure the in-vitro studies and to enhance the analysis results with more data about each interaction between domains and components. According to the in-vitro results, acacia gum (phosphatidylcholine and phosphatidylserine) was the best component, which resulted in a significant increase in sperm concentration. The in silico docking was performed to confirm those results and to predict the chemical interactions, hydrophobicity, charges, ionizability, and all other changes in each docked enzyme-ligand complex (Abishad et al. 2021). The binding energy of the CAT-lycopene complex was superior, whereas that of the GSH-phosphatidylcholine complex was the lowest. However, the conventional H-bond and carbon H-bond analyses illustrated that phosphatidylcholine and phosphatidylserine were the best and most robust in the chemical interactions with the residues that constituted the major binding site which ensured that those complexes were highly stable in their interactions (Wanandi et al. 2020). The conventional H-bonds and carbon H-bonds were absent through all the docked lycopene complexes with the three enzymes. However, docked complexes of vitamin C, phosphatidylcholine, and phosphatidylserine with the three enzymes showed the opposite. Vitamin C showed conventional H-bond with GSH domain in the amino acids residues GLN 110, HIS 19, and SER 120, with SOD domain in the amino acids residue VAL 148, and with CAT domain in the amino acids residues HIS 362, IEU 332, PHE 334, and ARG112. The carbon H-bonds were absent in all the docked vitamin C complexes with all three domains. Several conventional H-bonds and carbon H-bonds appeared in the interaction’s complexes with phosphatidylcholine and phosphatidylserine ligands through all three domains (Chen et al. 2016). In the phosphatidylcholine-GSH complex interactions, there were three conventional H-bonds with the amino acid’s residues ALA 22, LEU 21, and LEU 108, and there were three carbon H-bonds in the same interaction with the amino acid’s residues ALA 22, GLY 23, and GLU 25. The phosphatidylcholine-SOD complex interactions showed there were three conventional H-bonds with the amino acid’s residues GLY A:108, GLY B:108, and THR 3 when one bond of carbon H-bonds appeared in the same interaction with the amino acid’s residue SER 107 (Pantsar and Poso 2018). Also, the phosphatidylcholine-CAT complex interactions showed one conventional H-bond with the amino acid residue HIS 166. On the other hand, the phosphatidylserine- GSH complex interactions showed four conventional H-bond with the amino acid’s residues ARG 178, ASP 136, and two conventional H-bonds interactions with THR 142. The phosphatidylserine-SOD complex interactions showed three conventional H-bonds with the amino acid’s residues VAL 8, VAL 148, and ASN 53, when three bonds of carbon H-bonds appeared in the same interaction with the amino acids residues CYS 146, GLY A:147, and GLY B:147. Also, the phosphatidylserine-CAT complex interactions showed that there were four conventional H-bonds with the amino acid’s residues TYR 325, ALA 76, SER 120, and GLY 78 when one bond of carbon H-bonds appeared in the same interaction with the amino acid’s residues GLU 330. Also, there were interactions with van der Waals in all the docked complexes interactions (Zarezade et al. 2021). The H-bond was shown in each complex interaction with green colour for the acceptor and pink colour for the donor. In contrast, the ionizability was shown in blue for the basic and red colour for the acidic complex. Likewise, the charges were shown in blue for the positive and red for the negative charges, and most of the interaction’s areas were neutral. The hydrophobic area in the brown colour increased in the three enzymes complexes with phosphatidylserine and phosphatidylcholine ligands in comparison with the resulting complexes from lycopene, and vitamin C. Solvent accessible surface (SAS) was shown through each interaction of the docked complexes in blue colour which almost increased in all the docked complexes (Ray and Maunsell 2011).

Conclusions

Acacia gum, vitamin C, and lycopene may have a chemopreventive effect on heat stress by modulating reactive oxygen species, sustaining the antioxidant defense system in the brain, and improving the quality of rabbit sperm.

Author contributions

T.I., W.H., G.A., M.L., A.O., and O.F.: conceptualisation. T.I., W.H., G.A., M.L., A.O., A.E.ِِ.ُ., , A.Q., and O.F.: methodology. A.O., ِA.E., A.Q., and O.F.: software. T.I., W.H., G.A., M.L., A.O., A.Q., and O.F.: validation. A.T., W.H., G.A., M.L., A.O., A.E. A.Q., and O.F.: formal analysis. A.Q., and O.F.: investigation. T.I., W.H., G.A., M.L., A.O., A.Q., and O.F.: data curation. T.I., W.H., G.A., M.L., A.O., and O.F.: writing-original draft preparation. T.I., W.H., G.A., M.L., A.O., A.Q., and O.F.: writing-review and editing. A.T., W.H., G.A., M.L., A.O., A.Q., and O.F.: visualization. A.O., A.Q., and O.F.: supervision. All authors have read and agreed to the published version of the manuscript.

Animal welfare and ethics approval

The Scientific Ethics Committee, Animal Production Department, Faculty of Agriculture, Benha University, EG. approved (BUAPD-20196) the study's experimental procedures. The experiment was carried out at the Rabbit Research Unit, Benha University.

Acknowledgement

We would like to thank the authors Hanin A. Bogari and Mohammed M. Aldurdunji and their effort in guidance during the study.

Disclosure statement

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

Additional information

Funding

This research work was funded by Institutional Fund Projects under grant no (IFPIP:1938-249-1443). The authors gratefully acknowledge the technical and financial support provided by the Ministry of Education and King Abdulaziz University, DSR, Jeddah, Saudi Arabia.