This experimental study was performed following the guidelines of the Iranian Council of Animal Care (22) (protocol ID 19293). This study was carried out at the “Camel and Rangeland Research Station of South Khorasan,” located at 37.42° N and 57.31° E longitude, 1,491 m above sea level, in Birjand, Iran.

In total, 18 male 9–10-month-old Sindhi camel calves with a mean body weight (BW) of 115 ± 7 kg were randomly assigned to the three treatment diets in a completely randomized design with six animals per treatment, each kept in an individual shaded pen (3 m × 3 m) for 150 days preceded by an adaptation period of 14 days. During the adaptation period, all the camels were treated for external and internal parasites, vaccinated against enterotoxemia (Enteroprotect P 100, Razi Vaccine and Serum Research Institute, Iran), and fed the same diet (control diet). The diets were formulated by partial and total substitution of alfalfa hay with AM and included the following: (1) control, with no AM (CON), (2) AM-25, containing 25% AM, and (3) AM-50, containing 50% AM (DM basis) (Table 1). The whole AM plant was hand harvested from the Heydarabad (Nehbandan, South Khorasan, Iran) rangelands in June (at flowering growth stage) of 2021 and then dried and stored in a dark place until used. Diets consisting of 50 forage and 50% concentrate were formulated to meet energy and protein requirements of growing camels (23, 24), which were isocaloric (contain 2.47 Mcal/kg ME) and isonitrogenous (contain 114 g/kg CP). Throughout the experiment, the total mixed ration (TMR) diets were offered ad libitum, with feeding levels designed to ensure a daily refusal margin of 10% with free access to the water. During the experiment, camels were fed individually twice daily (0800 and 1700 h), and amounts of diet fed and refusals were weighed daily to determine DM intake (DMI) by subtracting the quantity fed minus refusals multiplied by the DM content of the TMR.

The TMR and refusal samples were taken during the trial (on days 0, 30, 60, 90, 120, and 150) before the morning feeding and frozen at −20°C for later analysis. The camels were weighed individually before the morning feeding on days 0, 30, 60, 90, 120, and 150 to determine the average daily gain (ADG). Feed conversion ratio (FCR) and feed efficiency (FI) was calculated as follows: FCR = [DMI, (kg/d)/ADG, (kg/d)]; FCR = [ADG, (kg/d)/DMI, (kg/d)]. An estimate was also made of the feeding cost per kg of body weight gain for the experimental groups. Blood samples were collected after overnight fasting through the jugular vein using tubes containing heparin lithium as an anticoagulant (Avapezeshk, Tehran, Iran) on days 0, 75, and 150. Plasma samples were obtained by centrifuging the blood tubes for 10 min at 3,000 × g and then were frozen at −20°C until analysis.

Samples of TMR and orts were separately pooled and grounded in a hammer mill with a 1-mm screen (Arthur Hill Thomas Corporation, Philadelphia, Pennsylvania, USA) and analyzed (three replicates) for DM (930.15), CP (Kjeldahl N × 6.25, 990.03), ether extract (EE) (945.16), and ash (967.05) according to Association of official analytical collaboration (AOAC) (25). The NDF and ADF content of samples were analyzed (Fibertec 1010, Tecator, Sweden) according to Van Soest et al. (26).

The plasma samples were analyzed for glucose (Glu) (mg/dl), total protein (TP) (g/dl), creatinine (Cr) (mg/dl), albumin (Alb) (g/dl), triacylglycerol (TG) (mg/dl), cholesterol (CHOL) (mg/dl), high-density lipoprotein (HDL) (mg/dl), blood urea nitrogen (BUN) (mg/dl), ALT (U/L), AST (U/L), and alkaline phosphatase (ALP) (U/L) using commercial kits (Pars Azmun Corporation Ltd., Tehran, Iran) by an autoanalyzer (BT 1,500, Biotecnica Instruments SpA, Rome, Italy) according to the manufacturer's instructions. Plasma total antioxidant capacity (TAC) (mmol/l) was measured using 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical formation with the commercially available Randox Kit (Randox Laboratories Ltd., Crumlin, County Antrim, UK). Plasma malondialdehyde (MDA) (nmol/ml) level was determined using the thiobarbituric acid reactive substance (TBARS) according to a study by Placer et al. (27). The enzymatic activities of glutathione peroxidase (GPx) (U/ml), superoxide dismutase (SOD) (U/ml), and catalase (CAT) (U/ml) were measured using the Randox Kit (Randox Laboratories Ltd., Crumlin, County Antrim, UK) according to the manufacturer's instructions.

A completely randomized design with three treatments (diets) and six replicates (camels) was used for this study. All the data were statistically analyzed using the MIXED procedure of SAS (version 9.2, SAS Institute Incorporation, Cary, North Carolina, USA) (28) for repeated measures. The fixed effects in the model were: the dietary treatment (diet), the time of storage (time), and their interaction (diet × time), while camel was included as a random factor. Initial body weight and blood parameters of camels were used as covariates in the model. Least-square means (LSM) were calculated and tested for differences by Tukey's test. A polynomial contrasts analysis was employed to determine the linear and quadratic effects of AM levels. Differences in LSM were significant at P ≤ 0.05 and P ≤ 0.10 was considered as a tendency.

The animal performance results are given in Table 2. DMI was increased with the inclusion of 25 and 50% (DM basis) AM to diet. The average initial BW (IBW) of the camels from each treatment had no difference (p = 0.652). The final BW (FBW) of animals fed AM diets tended to decrease (p = 0.096). The camels fed AM-25 and AM-50 had approximately 1.01 and 4.4% lower FBW compared to the CON group. Likewise, total weight gain (p = 0.048) and ADG (p = 0.043) decreased with AM-50; however, no differences were observed between the AM-25 and CON groups. In addition, higher FCR values were found in AM-50 treatment (p = 0.018). Feed cost per kg BW gain tended to decrease (p = 0.092) by the increasing inclusion levels of AM in the experimental diets. A tendency to increase (p = 0.087) in return per kg BW gain was also observed with AM-25 and AM-50 feeding.

No significant effects between treatments were observed regarding Glu, TP, Cr, hemoglobin (Hb), and BUN (Table 3). The TG (p = 0.046) and CHOL (p = 0.025) concentrations were lower for AM-25 and AM-50 compared with CON. The Alb levels were increased (p = 0.047) with the increasing AM inclusion in the growing camel's diet. Additionally, the AM50-fed camels showed the lowest concentration of AST (p = 0.008) and ALT (p = 0.0036), followed by AM-25. Likewise, a tendency (p = 0.008) to decrease was also observed for ALP with increasing AM inclusion to the diets.

The AM-25 and AM-50 treatments showed a 19.4 and 27.6% lower (p = 0.037; Table 4) plasma MDA content compared to CON. Moreover, the plasma TAC of growing camels was increased (p = 0.016) with increasing the AM inclusion to diets, which led by AM-50 following AM-25. Likewise, the inclusion of 25 and 50% (DM basis) AM to the diets led to higher (p = 0.004) GPx concentration. In addition, SOD (p = 0.075) and CAT (p = 0.094) concentrations were also tended to enhance by AM inclusion to the diets.

The DMI in this study is consistent with an earlier report (29) demonstrating a daily 4.36 kg DMI for growing camels aged 8 to 12 months (with an average 120 kg BW) during the 90-day feeding period. Also, it has been reported that camel DMI mainly depends on the type and availability of forages as well as their quality (19, 30). Although most camel studies have not reported DMI based on standard diets, the DMI of growing camels has been reported to be between 1.6 and 3 kg per 100 kg live weight (30, 31). Thus, the observed DMI values were within the normal range for dromedary growing camels. The inclusion of 25 and 50% AM (DM basis) to the diets resulted in 1.2 and 2.9% higher DMI compared to the CON group, respectively. Our results agree with a study by Kazemi and Bezdi (18) and Karamshahi Amjazi et al. (32) who found significantly higher DMI in ewes fed AM diets. In addition, it has been reported that there is a contraction between the type of pasture and feed behavior in dromedary camels (33), where plants with higher palatability have been associated with higher camels intake (19, 33). In line with the present data, a study (19) that aimed to investigate the nutritive value of some herbage for dromedary camels in the central arid zone of Iran reported that the AM classified as the most palatable plants for these animals. Camels have been shown to prefer prickly shrubs such as AM to other species (34). AM appears to enhance DMI, which is mainly associated with the higher palatability of AM for camel as well as camel preference for the consumption of thorny plants. Consistently, it has been well-documented that camels consume more prickly plants, which is related to the inherent adaptation of this animal and its grazing behavior, which make these plants more palatable (34, 35).

It has been shown (31, 36) that the growth rate of the camel calves is higher in the early stages of growth (up to 6 months old) and then decrease with puberty. Growth performance data presented in this study are similar to results reported in the literature using dromedary camels (37). ADG in dromedary camel calves is lower than cows and limited to 500 g/day (30). Wilson (36) reported a 326 g/day weight gain for 1-year-old camels that were fed diets containing 50 forage and 50% concentrate for 175 days, which is almost consistent with the present results.

Our results indicated that the inclusion of AM up to 25% DM did not affect ADG, FCR, and FE. In contrast, camels fed the greatest AM level (up to 50% DM of diet) had a reduction in total BW gain (~12.1% compared to the CON group and 10.6% compared to inclusion with 25% AM), resulting to lower ADG and higher FCR. It is well-accepted (38, 39) that the reduction in diet digestibility negatively affects weight gain. In this study, we did not investigate the digestibility of diets. However, it is known (18, 32, 34) that AM had lower digestibility compared to alfalfa hay. Similarly, Karamshahi Amjazi et al. (32) reported that the lower digestibility of AM compared to alfalfa hay may relate to its higher NDF, ADF, and lignin content. In this regard, Kazemi and Bezdi (18) showed that replacing the dietary forage with AM resulted in decreased nutrient digestion and decreased BW change of ewes. In this study, lower ADG in animal fed AM up to 50% DM of diet is probably attributed to decrease nutrient digestion that further increases FCR. In line with this, Karamshahi Amjazi et al. (32) reported that the apparent digestibility of DM and CP was decreased with AM silage inclusion in a sheep diet. Moreover, total BW gain and ADG in AM-25 were numerically higher and this could provoke different rates of digestion, which might enhance the efficiency of energy utilization and consequently enhance growth rate (40). Analysis of feeding costs showed that inclusion of AM to diet led to a sensible reduction of the feed cost per kg BW gain (7.6 and 6.9% reduction with the inclusion of 25 and 50% AM and DM basis). Altogether, our results indicated a favorable effect of AM inclusion on the DMI of growing camels, which was probably associated with its higher palatability as well as camel preferences for consume AM. The ADG was decreased with AM-50. However, there were no differences between AM-25 and CON in regard of ADG and FCR that along with lower feed cost per kg BW gain make AM-25 rationalization in growing camel diet.

The current values of blood parameters were in line with previously published data on fattening camels (20). The inclusion of AM in camel diets led to a reduction of TG and CHOL concentrations. In this regard, the inclusion of 1.5, 3.0, and 4.5% (DM basis) AM powder in laying hens diet reduced in plasma TG. Also, Riasi et al. (41) reported that the inclusion of halophyte plants in sheep diets reduced the plasma concentration of TG and CHOL. In support of earlier findings, it has been shown that the ethanolic extracts of AM (at a dose of 200 and 300 mg/kg) significantly reduce the level of blood CHOL and TG in diabetic Wistar rats (42). Some previous studies (15, 43) indicate that the lipid-lowering effects of AM may relate to its large number of active compounds such as alkaloid, peptidoglycan, terpenoid, amino acid, and, especially, flavonoids.

Overall, feeding of both the AM-25 and AM-50 mitigates the concentration of liver enzymes, namely, AST, ALT, and ALP, along with the increase in Alb. The activities of the aforementioned biomarkers were discussed together, as they are commonly used to study liver function (44). The reduction in AST, ALT, and ALP of the AM-containing groups implies the enhanced liver function in these camels. Consistent with our results, Kuerbanjiang et al. (45) showed that the AM significantly alleviated alcohol-induced liver injury in mice by reducing serum ALT and AST. Moreover, Alqasoumi et al. (15) have shown that the ethanolic extract of aerial parts of AM at a dose of 500 mg/kg decreased liver enzymes and increased plasma Alb in CCl₄-induced liver injury in rats. In agreement with our results, it has been shown (5, 46) that using AM extract reduced the liver enzymes (AST and ALT) in mice with liver injury, which is associated with high levels of flavonoids and bioactive polysaccharides in this plant. The AM is known to reduce the gene expression of Toll-like receptor 4 (TLR4), inhibiting the release of tumor necrosis factor-α (TNF-α) as well as preventing further signal transmission and reducing the efficiency of endotoxin signal transduction, which led to hepatoprotective effects (5). In addition, previous studies (5, 15) suggested that the hepatoprotective mechanism of AM has been partly attributed to the reduction of oxidative stress and inhibition of the expression of cytochrome P450 2E1 (CYP2E1). Our finding suggests that AM not only favors a plasma lipid profile, but it could also improve liver activity of growing camels.

The dynamic balance between oxidation and antioxidant activity in the circulatory system plays a critical role in the health and productive performance of livestock (47). The rich functional compounds in nature-derived feedstuffs make them an attractive component to improve the antioxidant status of animals, thereby improving the quality of products (47–49). Currently, consumers are interested in safe and natural foods of animal origin and in some cases, they are also willing to pay a premium price for them (49). TAC reflects the cumulative effects of enzymatic and non-enzymatic antioxidants present in plasma and body fluids (47, 48), whereas MDA is generated because of lipid peroxidation that could be measured as a biomarker of oxidative stress (47). Mammalian enzymatic antioxidation against free radical damage is mainly facilitated by the activities of SOD, GPx, and CAT (47). The results obtained regarding the antioxidant status in this study are consistent with previously published data on growing camels (23). Our results demonstrated that AM had positive effects on antioxidant status, as indicated by enhanced blood TAC, GPx, SOD, and CAT activity and depressed MDA levels. In confirmation, the antioxidant activity of AM has been proved by in-vitro and in-vivo studies (13, 15). In this regard, Peluso et al. (50) reported that the treatment of diabetic rats with AM extracts improved the plasma antioxidant status by increasing the activities of SOD, GPx, and glutathione transferase (GST). Likewise, AM extract (100 mg/kg body weight) decreased plasma MDA levels in rats (51). AM is rich in biologically active phytochemicals such as phenolics, flavonoids, alkaloids, and polysaccharides along with different essential minerals, proteins, and lipids, which has turned it into a powerful antioxidant (13, 15). Natural plant feeds containing phenolic, flavonoid, and alkaloid substances can prevent peroxidation by scavenging free radicals or by activation of antioxidant enzymes such as glutathione reductase, GPx, and SOD (49). A study (52) reported that the aqueous extract of AM was appraised by reducing MDA levels using thiobarbituric acid assay. Laghari et al. (12) demonstrated high antioxidant activity of AM plant using 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay, which can be related to the high radical-scavenging activities of its phenolic contents. In addition, the AM antioxidant potential has been proved by measuring ABTS radical cation scavenging and DPPH scavenging (13, 14).

In line with the current findings, a study (45) using rats with alcohol-induced liver damage found that the plasma MDA concentration decreased and the activity of the antioxidant enzymes including GPx and SOD enhanced following AM extract treatment. Also, the use of 50, 100, and 200 mg/kg of AM extract was associated with an increased SOD activity and decreased MDA levels in mice (53). Recently, Asghari Baghkheirati et al. (54) showed that the use of 10 and 20 g/kg of AM powder in broilers diet reduces the MDA concentration in meat.

It is well-documented that a higher intake of natural antioxidants led to improved antioxidant status through transferring of these molecules to animal tissues (10), which leads to high-value food products (e.g., meat) and contributes to the meeting customer desires over safety and quality of products (49). Hence, improved GPx, SOD, and CAT along with mitigated MDA concentration in camels receiving AM-containing diets demonstrated that it could be a useful tool against oxidative damage.