Effects of Salsola tragus as a Forage Source During Fattening on Productivity and Meat Metabolomics of Rambouillet Lambs

Simple Summary

The genus Salsola comprises salt-tolerant plants with a potential use as livestock fodder in arid regions. However, there is a lack of studies that characterize this potential utility of Salsola spp. In this study, Rambouillet lambs were fed Salsola tragus as a forage source to determine its effects during the fattening phase on lamb growth, in vitro dry matter degradability, gas production, carcass quality, and cellular damage from oxidative stress during the fattening phase, while also determining its impact on the metabolomic meat interaction in lambs. Overall, the use of Salsola tragus as a source of forage in arid regions for lambs during the fattening period is promising due to its positive impact on meat metabolites without inhibiting lamb growth performance and carcass quality.

Abstract

The aims of the current study were to characterize the natural compounds of Salsola tragus via GC-MS and determine its effects as a forage source on lamb growth, in vitro rumen fermentation kinetics, carcass quality, cellular damage, and metabolomic meat interaction. Twenty-one Rambouillet lambs were randomly assigned to one of three experimental diets (seven lambs per treatment): (1) a control diet (W/o-Salsola) containing 300 g/kg dry matter (DM) of sorghum stover; (2) a diet with a medium inclusion of Salsola tragus (15-Salsola), which contained 150 g/kg DM of Salsola tragus and 150 g/kg DM of sorghum stover; and (3) a diet comprising 300 g/kg of Salsola tragus (30-Salsola). The results showed there were no differences (p > 0.05) in lamb growth performance during the fattening phase. The in vitro gas analysis demonstrated that the 30-Salsola treatment increased lag time h⁻¹ (p < 0.05) and reduced gas production (p = 0.03). The metabolomic analysis findings suggest that the treatments that included Salsola tragus significantly positively affect the metabolomic composition of meat (p < 0.05). The use of 15-Salsola as a source of forage is promising for feeding lambs during the fattening phase.

1. Introduction

Across the American continent, arid zones exist in many countries, such as Canada, the United States of America, Mexico, Bolivia, Venezuela, Brazil, Argentina, Chile, and Peru [1]. In these countries, livestock are an important asset and an integral part of agriculture and farming systems [2]. Many pastoralist communities comprising small ruminants have been established in these arid regions and depend on local ranges for their livelihood [3]. However, current climatic volatility involves global warming, resulting in prolonged periods of dry weather and poor soil nutrient quality, affecting grassland plant species and threatening rangeland livestock production in arid zones [1,3]. Nevertheless, even though livestock production faces considerable challenges from climate change, more significant global threats to the future of humanity, such as food security, require the use of the entire range potential in arid zones [4].

Thus, in the last decade, research has endeavored to identify plant species in grasslands adapted to alkaline soils, highly porous soils, and drier conditions with comparatively higher leaf drought tolerance [5]. However, most plant species with these characteristics are identified as weedy or invasive; hence, grassland specialists recommend their elimination or removal [6]. Interestingly, one of these plant genera is Salsola, which comprises halophyte plants and annual semi-dwarf to dwarf shrubs, including woody trees. Murshid et al. [6] described Salsola as being rich in many classes of phytoconstituents, including flavonoids, phenolics, saponins, and volatile constituents. Nevertheless, the phytochemical composition and biological consequences of this genus have received little attention; only a few species from the genus Salsola have been examined for chemical and biological applications [6,7]. The most commonly reported species are Salsola vermiculata, Salsola cyclophylla, and Salsola komarovii [8]. Nevertheless, these species do not exist on the American continent, the most abundant being Salsola tragus [6]. However, government agencies describe little botanical information about this species [9,10]. Hence, the chemical characterization and biological applications of Salsola tragus on the American continent are unknown.

Little information on the genus Salsola has been linked to herbal medicine applications. El-Bassossy et al. [11] reported a pharmacological study that described the chemical constituents of the aerial parts of Salsola kali. The authors concluded that Salsola kali extract exhibited promise as a therapeutic source due to its increased antioxidant, anti-inflammatory, and cytotoxic activities. In addition, Arrekhi et al. [12] and Mohammed et al. [13] proposed that the metabolites and nutrients potentially convert the Salsola genus into an optimistic target for herbivore feed sources in arid zones. However, little information on animal performance in diets has been published using family species from the genus Salsola. Arrekhi et al. [12] evaluated the forage potential of Salsola turcomanica and found that the plant’s growth and developmental stage determines the total digestive nutrients, fiber content, and crude protein. They concluded that an acceptable forage quality occurs during the young plant phase when Salsola turcomanica has chemical values comparable with traditional forages such as Medicago sativa. Based on these descriptions, the genus Salsola has excellent potential for use in ruminant feed. Nevertheless, the chemical composition of each species in the genus is accompanied by phytoconstituents that might modify the rumen microbiota [14]. Therefore, the quality of meat or milk will depend on the chemical compounds and metabolites donated to the ruminant system by the particular species [15].

Dietary flavonoids and phenolic compounds have been reported to increase serum growth hormone levels [16], improve the immune system and antioxidant enzymes activity in the blood serum [13], and promote muscle tissue synthesis in ruminants [8,11]. Therefore, different studies have proposed that photogenic compounds or phytoconstituents have a wide range of applications to provide insights into the biochemical balance of muscle tissue [17]. This is because of the cascade of chemical pathways that involve proteins, carbohydrates, lipids, and minerals that impact meat color, tenderness, and flavor [13]. In recent years, muscle physiology and meat science research has increasingly incorporated metabolomic analysis of the longissimus muscle metabolome, establishing connections to the chemical pathways within muscle tissue [18]. Different scientific reports have characterized the effects of diet on the fattening phase and meat tastiness [19,20]. Nevertheless, few studies have employed metabolomics approaches to evaluate new sources of forage, such as arid plants that grow in saline soils, and their impact on meat production in small ruminants [15]. Thus, we hypothesized that the herbal compounds of Salsola tragus have specific metabolites with properties that can modify the metabolome during muscle growth. Thus, the objectives of the current study were (1) to characterize the natural compounds of Salsola tragus via gas chromatography coupled with mass spectrometry (GC-MS), (2) evaluate the effect of Salsola tragus as a source of forage on lamb growth and cellular damage from oxidative stress during the fattening phase, and (3) determine the impact of Salsola tragus on the metabolomic meat interaction in lambs.

2. Materials and Methods

2.1. Ethics

The procedures involving sheep and their ethical treatment were evaluated and sanctioned in accordance with the Animal Research Guidelines of the Committee for the Ethical Use of Animals in Experiments and Research at the Universidad Autónoma Metropolitana and Universidad Autónoma de San Luis Potosí (Project: BP-PA-20210513092803518-1069570). This approval aligns with the Mexican Government’s federal laws: (1) NOM-062-ZOO-1995, which outlines the technical specifications for the care and use of animal models in laboratory tests and on livestock farms [21]; (2) NOM-051-ZOO-1995, which pertains to the humane treatment and transportation of animals [22]; and (3) NOM-033-SAG/ZOO-2014, which addresses the humane slaughter of domestic and wild animals [23].

2.2. Animals, Experimental Design, and Treatments

This study was conducted at the Sheep Experimental Center of the Facultad de Agronomía y Veterinaria of Universidad Autónoma de San Luis Potosi (φ 22°14′0.58; λ 100°50′48.5). Twenty-one individually fed male Rambouillet lambs (initial body weight 29.66 ± 0.33) were randomly assigned to one of three experimental diets (n = 7 per treatment): (1) a control diet (W/o-Salsola) containing 300 g/kg dry matter (DM) of sorghum stover as the source of forage; (2) a diet with a medium inclusion of Salsola tragus (15-Salsola), which contained 150 g/kg DM of Salsola tragus and 150 g/kg DM of sorghum stover; and (3) a diet using only Salsola tragus (30-Salsola) as the source of forage with 300 g/kg DM (

Table 1. Ingredients and chemical composition of experimental basal diets.

Item	Treatments 1
	W/o-Salsola	15-Salsola	30-Salsola
	Ingredients, as Offered (DM Basis, %)
Salsola tragus	-	15.00	30.00
Sorghum stover	30.00	15.00	-
Corn flaked	28.00	28.00	28.00
Sorghum grain	28.00	28.00	28.00
Soybean meal	7.00	7.00	7.00
Molasses	5.00	5.00	5.00
Urea	0.50	0.50	0.50
Minerals	1.50	1.50	1.50
Chemical composition (DM basis, %) 2
Dry matter	88.50	89.00	90.00
Crude protein	14.10	14.30	14.99
Neutral detergent fiber	77.10	76.55	64.39
Acid detergent fiber	67.77	60.35	49.88
Ether extract	4.80	4.99	5.01
Ash	4.90	4.99	5.12

¹ Treatments: control diet (W/o-Salsola); diet with a medium inclusion of Salsola tragus (15-Salsola); diet using only Salsola tragus (30-Salsola). ² Values were determined using AOAC methods [24], and fiber fraction was determined according to Van Soest et al. [25].

).

The experimental diets were divided into two phases. In the first phase, the amount of Salsola tragus inclusion as a forage source was theoretically determined based on its nutrient values to assess its capacity as a substitute for conventional forage sources, characterizing whether the plant could indeed pose a less immediate risk to the intake and performance of lambs. Based on this approach, the amount of inclusion was established using the same amounts as traditional forage, with the relevance of using intermediate inclusion. The second step was to situate the Salsola tragus in the field by identifying communities in the growth phase. Salsola tragus identified in the seed-throwing phase was tracked. Later, the new plants were allowed to grow for a maximum of five months after germination and cut at a height of 45 cm. Cutting was carried out using a rear-mounted mower with pendulum suspension (DiscCutter^TM; cutting width of 2.77 m, New Holland, OK, USA) on an agricultural tractor (Ford-6600, 77 HP, Detroit, MI, USA). Once cutting was completed, the plants were sun-dried for 48 h and then packed using a forage baler (New Holland 570, Delano, CA, USA). Samples of Salsola tragus collected from different bales were stored for further bromatological analysis. The chemical composition of Salsola tragus was determined using the Association of Official Analytical Chemists (AOAC) methods for dry matter (method number 981.10), crude protein (CP; method number 967.03), ether extract (E.E; method number 963.15), and ashes (method number 942.05 [24]. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were measured as described by Van Soest et al. [25] (

Table 2. Chemical composition (% dry matter (DM) basis) of Salsola tragus used as a source of forage in the lamb fattening phase.

Item	Chemical Composition (DM Basis, %) 1
Dry matter	88.22
Crude protein	13.11
Neutral detergent fiber	51.20
Acid detergent fiber	32.81
Ether extract	1.42
Ash	4.50

¹ Values were determined using AOAC methods [24], and fiber fraction was determined according to Van Soest et al. [25].

).

Finally, experimental diets were formulated to meet the nutritional requirements for fattening lambs during the finishing period and were offered as a total mixed ration (Table 1) [26]. The lambs were housed in individual pens equipped with feeders and drinkers. Feed was provided at 0600 h, 1200 h, and 1800 h, while water was provided twice daily (0600 h and 1800 h) to guarantee ad libitum access. The seven-day adaptation period was when the lambs were adapted to individual pens and diets. Additionally, lambs received sanitary management through a single subcutaneous administration of 0.2 mg/kg of body weight of ivermectin (Iverfull^®; Laboratorios Aranda, Jalisco, México) and intramuscular administration of a source of vitamins (Vigantol ADE: 250,000 IU of vitamin A; 37,500 IU of vitamin D; 25 mg of vitamin E; Elanco Animal Health Korea Co., Ansan-si, Gyeonggi-do, Republic of Korea). The lambs participated in the experimental period of 27 days. During this time, the dry matter intake (DMI) increased to ensure ten percent feed refusal. The experimental diet samples were collected weekly and dried in an oven equipped with forced air at 65 °C for 24 h. The experimental diet samples were milled using a 2-mm screen and analyzed following the same procedure of the AOAC [24] for Salsola tragus: DM (981.10), CP (967.03), E.E (963.15), and ashes (942.05). Finally, NDF and ADF levels were determined according to the method described by Van Soest et al. [25].

2.3. Feedlot Performance and Blood Sampling

The dry matter intake (DMI) was calculated daily by the difference between the amount of feed offered in the feeder and feed refusal. The lambs were weighed at the start (day 1) and end (day 27) of the experimental period to calculate body weight (BW) and to evaluate the average daily gain (ADG). Feed conversion (G/F) was estimated as the ratio of DMI to ADG. On day 27 at 0500 h, blood samples (5 mL) were collected in sodium heparin tubes via venipuncture of the jugular vein (VacutainerTM; Becton Dickinson de México, México) for DNA comet assay analysis.

2.4. Carcass Characteristics and Meat Quality

Once the feedlot period was completed, lambs were fasted for 12 h and transported to the municipal slaughterhouse of Soledad de Graciano Sanchez. The lambs were bled, skinned, and eviscerated to measure the individual weights of the rumen fill and empty rumen. One hundred milliliters of ruminal liquid for each lamb was stored at 38 °C and immediately transported to the laboratory for in vitro dry matter digestibility (IVDMD) and gas production analysis.

The carcasses were chilled at 4 °C for 24 h to generate rigor mortis and obtain the carcass cold weight. Subsequently, carcasses were ribbed along the midline and the right half was ribbed between the 12th and 13th ribs to measure Longissimus thoracis (LT) eye width, depth, and fat thickness. The LT muscle was also used to determine pH using a pH meter device equipped with a penetration electrode (Hanna Instruments, model HI 98140, Woonsocket, RI, USA). The LT muscle was then cut into three sections. The first section was cooked (60 °C) in meat cubes and longitudinally oriented to the muscle fibers using a Warner–Bratzler force device (Salter 235, Manhattan, KS, USA). Finally, the last two sections of the LT muscle (ten grams each) were vacuum-packed, frozen using an ultra-freezer (Thermo Scientific TSX Universal Series, Waltham, MA, USA), and stored at −80 °C until chemical composition and metabolomic analyses.

2.5. In Vitro Fermentation Kinetics

As described previously, 100 mm of ruminal liquid was obtained from each lamb that received the three experimental diets. Each liquid sample was placed in three glass flasks with a capacity of 2 L to form a mixed sample for each treatment. The evaluation of in vitro dry matter degradability was started following the Tilley and Terry methodology [27], modified as suggested by Holden et al. [28], to the best performance of the Ankom system (Ankom Technology Corp., Macedon, NY, USA) for in vitro digestibility evaluation. Three bags (F57, Ankom Technology Corp., Macedon, NY, USA) that contained 0.5 g of sample from each experimental diet were incubated following one of the eight timetables (24 bags per flask, 16 blank filter bags for contamination correction): 0, 2, 4, 8, 12, 24, 48, and 72 h⁻¹. Flasks contained 1.6 L of buffer solution (solution A = (g/L), 10.0 g of KH₂PO₄, 0.5 g of MgSO₄-H₂O, 0.5 g of NaCl, 0.1 g of CaCl₂-H₂O, and 0.5 g of urea; solution B = (g/100 mL), 15.0 g Na₂CO₃, and 1.0 g Na₂S₉-H₂O). The final solution had an A/B ratio of 5:1 and a pH of 6.8. Subsequently, 400 mL of the mixture of ruminal fluid from each treatment was placed into each flask with the corresponding experimental diet treatment, which was purged with CO₂ for 5 s and incubated with the A/B solution for 72 h at 39 °C using programmable continuous shaking. A unique incubator was used to maintain all flasks. When one of the timetables was finished, three bags and one blank bag were removed from the flasks, washed with distilled water, and oven-dried at 65 °C for 48 h⁻¹. Blank bags were used to estimate a correction factor adjusted for weight changes in the sample bags [29]. The IVDMD was calculated from the difference between the amount of nutrients in the feed and the residue after incubation. These analyses were performed using the weight of the three bags per timetable to obtain repetitions of the in vitro degradability results [28].

From three identical flasks containing mixed ruminal fluid from the three treatments, 10 mL was transferred to the culture medium to achieve a final concentration of 10%. Subsequently, each of the three experimental diets (0.5 g) was introduced into a 120 mL vial containing 90 mL of solution A/B, as Theodorou et al. described [29]. Vials containing only 90 mL of the inoculum served as controls. The vials were incubated in a water bath maintained at 39 °C. Using the same methodology as Theodorou et al. [29], gas production was quantified as follows. The volume of gas produced was determined at various time intervals by using a hypodermic needle connected to a 0–1 kg/cm² gauge inserted into the vial plug. During incubation, pressure measurements were performed at 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 24, 36, and 48 h. The pressure (kg/cm²) was converted to volume and accumulated gas production using the model proposed by Menke and Steingass [30].

$$\mathrm{Y}=\mathrm{v}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{m}\mathrm{e}\left(1+\mathrm{e}\mathrm{x}\mathrm{p}\right)\times \left(2-4\right)\times \mathrm{g}\mathrm{a}\mathrm{s} \mathrm{p}\mathrm{r}\mathrm{o}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\times (\mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e}-\mathrm{l}\mathrm{a}\mathrm{g} \mathrm{p}\mathrm{h}\mathrm{a}\mathrm{s}\mathrm{e})$$

(1)

After 96 h of incubation (96 h), the contents of each serum bottle were filtered using sintered glass crucibles (coarse porosity no. 1, 100–160 μm pore size, Pyrex, Stone, UK) under vacuum. The fermentation residues were dried overnight at 105 °C to estimate the disappearance of DM. Applying the model proposed by Menke and Steingass [30], the metabolizable energy (ME, MJ/kg of DM) was calculated as follows:

$$\mathrm{M}\mathrm{E},\mathrm{M}\mathrm{J}/\mathrm{k}\mathrm{g} \mathrm{o}\mathrm{f} \mathrm{D}\mathrm{M}=2.20+0.1357\mathrm{G}\mathrm{P}24+0.0057\mathrm{C}\mathrm{P}+0.0002859\mathrm{E}\mathrm{E}$$

(2)

The concentration of short-chain fatty acids (mmol SCFA) was calculated according to the equation proposed by Miranda et al. [31]:

$$\mathrm{m}\mathrm{m}\mathrm{o}\mathrm{l} \mathrm{S}\mathrm{C}\mathrm{F}\mathrm{A}=-0.00425+0.0222(\mathrm{m}\mathrm{L} \mathrm{g}\mathrm{a}\mathrm{s} \mathrm{a}\mathrm{t} 24 \mathrm{h})$$

(3)

The volume and production of CO₂ were measured volumetrically after 4, 8, 12, 24, 36, 48, and 72 h⁻¹ of fermentation. The production of methane and other minor gases was estimated based on the differences observed in the CO₂ measurements [31]. In this study, we combined 30 milliliters of solution A/B with rumen fluid at a 2:1 ratio, adding it to 60 mL amber flasks that contained 0.25 g of each experimental diet. Gas production was evaluated at 0, 6, 12, and 24 h⁻¹ using a 150 mL glass syringe to quantify the levels of CO₂ and minor gases. The outflow from each tube was sequentially directed to an infrared gas analyzer (IRGA) (Li-6262, LiCor, Lincoln, NE, USA), allowing for real-time monitoring of CO₂ mixing ratios corrected for water vapor effects. The IRGA was calibrated using secondary standards traceable to the NOAA CMDL standards before and after each measurement. Span and zero drifts were maintained at less than 1 ppm, ensuring that the analytical accuracy was consistent with the NOAA’s Climate Monitoring and Diagnostics Laboratory (CMDL) standards.

2.6. Salsola tragus GC-MS Characterization

An ultrasonic processor (GEX130, 115 V 50/60 Hz) with a 3 mm titanium tip and mechanical stirrers (Cole-Parmer, Vernon Hills, IL, USA) was utilized to extract bioactive compounds. Salsola tragus (1 g) was sieved (2-mm screen) and combined with hexane (7.5 mL) and acetone (2.5 mL). The organic phase was then isolated, concentrated to 1 mL of the extract mixture, and evaporated (Zymark, Turbovap LV Concentration Evaporator, Hopkinton, MA, USA) for subsequent analysis.

Characterization of Salsola tragus was performed using gas chromatography (GC-HP 6890) linked to mass spectrometry (MSHP 5973), featuring a capillary column measuring 60 m in length, 0.255 mm in diameter, and 0.25 µm in film thickness (HP 5MS, Agilent, Santa Clara, CA, USA). The temperature program, based on previous studies describing metabolites in herbal compounds [32,33], involved setting the oven to 70 °C for 2 min, followed by increases to 250 °C at 20 °C/min, 290 °C at 5 °C/min, 300 °C at 1 °C/min, and 310 °C at 5 °C/min, with a final hold for 36 min. The injector was set to 250 °C in splitless mode, and helium flow was maintained at 1 mL/min. Compound identification was achieved using mass spectrometry in SCAN mode (50–500 m/z).

2.7. Comet Assay

A single-cell gel electrophoresis assay was performed following the method described by Singh et al. [34], with slight modifications as outlined by Shaposhnikov et al. [35]. In summary, blood samples (30 µL) were mixed with 225 µL of agarose solution and spread on slides pre-coated with 0.5% low melting point agarose (Sigma-Aldrich, St. Louis, MO, USA). Once solidified, the slides were submerged in lysis solution (10 mM TrisHCl, 2.5 M NaCl, and 0.1 M Na₂EDTA, pH 10, with 10% DMSO and 1% Triton X-100 added) and kept at 4 °C for 24 h. The slides were placed in an alkaline buffer for 5 min, followed by electrophoresis in the same buffer (pH > 13) for 5 min at 25 V and 300 mA. All steps were carried out under dim light at 4 °C. After electrophoresis, the slides were washed with Tris-HCl buffer (pH 7.5) and dehydrated using ethanol. Staining was performed with ethidium bromide (Sigma-Aldrich, St. Louis, MO, USA) at a 0.05 mM concentration. DNA damage assessment involved examining 100 cells (two sets of 50 randomly chosen cell nuclei) under an epifluorescence microscope (Nikon Eclipse E400, Tokyo, Japan) (Figure 1). Olive tail moment and length were measured using image analysis software (Kom-et, version 4; Kinetic Imaging Ltd., Bromborough, UK).

2.8. GC-MS Meat Compounds Analysis

The extraction of meat compounds was conducted following the method outlined by Pérez-Segura et al. [36]. In summary, an ultrasonic processor (GEX130, 115 V 50/60 Hz) equipped with a 3 mm titanium tip and mechanical stirrers (Cole-Parmer, Vernon Hills, IL, USA) were utilized for the extraction process. The procedure involved mixing 1 g of meat with 7.5 mL hexane and 2.5 mL acetone, followed by organic phase separation. The resulting organic phase was concentrated to 1 mL of the extracted mixture and subsequently evaporated (Zymark, Turbovap LV Concentration Evaporator, Hopkinton, MA, USA) for final analysis.

Meat characterization was performed using gas chromatography (GC-HP 6890) and mass spectrometry (MSHP 5973). The system employed a capillary column (HP 5MS, Agilent) with dimensions of 60 m length, 0.255 mm diameter, and 0.25 μm film thickness. The temperature program began at 70 °C for 2 min, then increased to 250 °C at 20 °C/min, 290 °C at 5 °C/min, 300 °C at 1 °C/min, and 310 °C at 5 °C/min, where it was held for 36 min. The injector was set to 250 °C in splitless mode, with helium flowing at 1 mL/min. Compound identification was carried out using mass spectrometry in SCAN mode (50–500 m/z).

2.9. Statistical Analysis

Data was analyzed using the GLM procedure in SAS 9.0 for a completely randomized design, with three treatments and seven replicates, considering each lamb as an experimental unit. A Shapiro–Wilk test was used to verify the normal distribution and variance homogeneity of the data; orthogonal contrasts were used to test the linear or quadratic effect of Salsola tragus addition on the response of the variables of interest (p < 0.05). The initial body weight was used as a covariate. For quadratic responses, the optimal Salsola tragus addition was estimated by linearizing the gamma function.

A multivariate statistical approach was utilized to analyze the GC-MS meat compounds, and principal component analysis (PCA) was performed considering the number of samples and replicates per group. PCA is an unsupervised automated machine learning algorithm that aims to reduce the dimensionality of data on a linear basis by transforming a large set of variables into a smaller one that preserves crucial information by searching for directions of maximum variance. This analysis comprises data dispersion directions through eigenvectors. This is accomplished by transforming the original variables into a new set of uncorrelated variables called principal components, ranked by the variance they explain, thus allowing for efficient feature selection. For this analysis, the data were normalized before processing by unit variance and mean centering. Cross-validation was performed using random forest analysis to avoid overfitting the model. The influential variables were also evaluated using random forest (RF), and p values (p < 0.05) from the univariate (ANOVA) statistical analysis were used to identify potential differential metabolites. Based on these results, a hierarchical clustering heat map was obtained to assess the compounds that exhibited an unusually high or low abundance within the clusters and explore whether this was related to influential variables. It is important to note that in the present study, only an unsupervised model was used because of the number of samples and replicates per group. Evaluating the data using a supervised model would lead to overfitting, and therefore, a misinterpretation of the results. Statistical analysis was performed using Orange Quasar^® and Metaboanalyst 6.0 (https://metaboanalyst.ca/ accessed on 20 June 2024) software.

3. Results

3.1. Chemical Composition of Salsola Tragus

The screening for bioactive compounds via GC-MS using hexane–acetone on Salsola tragus revealed the presence of secondary metabolites with well-known health-beneficial properties in ruminants (Figure 2). The results revealed 23 chemical compounds: alkanes, alcohols, siloxanes, esters, amides, organic acids, aromatic and polycyclic compounds, flavonoids, isoflavones, nitrogenous bases, and alkaloids.

3.2. Productive Performance and In Vitro Dry Matter Degradability

Table 3. Effect of Salsola tragus inclusion as a source of forage in the finishing diets of lambs during the fattening phase.

Item	Treatments 1			SEM 2	p Value
	W/o-Salsola	15-Salsola	30-Salsola		Linear	Quadratic
Initial BW, kg	29.83	29.66	30.16	0.68	0.85	0.83
Final BW, kg	37.58	37.08	38.66	1.11	0.63	0.61
Total weight. gain, kg	7.75	7.41	8.50	0.56	0.64	0.61
ADG, kg/d−1	0.310	0.315	0.340	0.02	0.63	0.61
DMI, kg/d−1	1.64	1.63	1.65	0.03	0.96	0.87
ADG/DMI ratio	5.11	5.09	4.95	0.30	0.58	0.67

¹ Treatments: control diet (W/o-Salsola); diet with a medium inclusion of Salsola tragus (15-Salsola); diet using only Salsola tragus (30-Salsola). ² SEM: standard error of the mean.

summarizes the growth, final weight, and ADG of lambs fed Salsola tragus as a forage source compared to the control treatment. Using Salsola tragus as a forage source did not affect DMI or total weight gain (p > 0.05) in lambs during the fattening phase.

As shown in Figure 3, using Salsola tragus as a forage source for fattening lambs did not result in measurable differences in in vitro dry matter degradability.

3.3. In Vitro Cumulative Gas Production and Estimated Kinetic Parameters

The in vitro gas analysis demonstrated that using the 30-Salsola treatment in finishing diets increased the lag time (h⁻¹) (p < 0.01) and the percentage of methane production at 48 h⁻¹ of the analysis (

Table 4. In vitro accumulative gas production and estimated kinetic parameter model for Salsola tragus as a source of forage during the fattening period of Rambouillet lambs.

Item	Treatments 1			SEM 2	p Value
	W/o-Salsola	15-Salsola	30-Salsola		Linear	Quadratic
The volume of gas produced (v), mL	418.3	352.4	332.05	4.51	0.07	0.03
Production rate (s), mL/g−1	0.018	0.019	0.019	0.01	0.98	0.30
Lag time (L), h−1	1.57	3.63	4.53	0.25	<0.01	0.35
% CO2 at 48 h−1	47.69	48.55	40.29	2.51	0.07	0.61
% CH4 at 48 h−1	52.30	51.44	59.70	5.27	0.03	0.98
ME; MJ/kg−1 DM	7.69	7.48	8.55	0.87	0.44	0.02
mmol SCFA 3	3.58	3.78	4.23	0.54	<0.01	0.05

¹ Treatments: control diet (W/o-Salsola); diet with a medium inclusion of Salsola tragus (15-Salsola); diet using only Salsola tragus (30-Salsola). ² SEM: standard error of the mean. ³ SCFA: short-chain fatty acids.

). Similarly, the SCFA concentration increased when the fattening diets included the 30-Salsola treatment as a forage source (p < 0.01). In addition, this increase was observed as a quadratic response to SCFA and the calculated metabolized energy (p < 0.05) in the kinetic parameter model when Salsola tragus was included in the diets of fattening lambs via the 30-Salsola treatment. Finally, the volume of gas produced showed a quadratic response (p = 0.03) because the 30-Salsola treatment reduced gas production in the lamb rumen model (Table 4).

3.4. Carcass and Meat Quality

Including Salsola tragus as a forage source in lamb fattening diets tended to increase cold carcass weight (p = 0.10) without negatively affecting postmortem pH at 24 h (

Table 5. Carcass and meat quality 24 h post-slaughter of lambs fed with Salsola tragus as a source of forage during the fattening phase.

Item	Treatments 1			SEM 2	p Value
	W/o-Salsola	15-Salsola	30-Salsola		Linear	Quadratic
Cold carcass weight (kg−1)	16.17	15.75	16.79	0.66	0.10	0.17
pH postmortem at 24 h−1	6.26	6.21	6.15	0.05	0.38	0.87
Fat thickness (cm)	0.30	0.28	0.28	0.03	0.73	0.94
Loin eye depth (cm)	6.23	6.64	7.15	0.23	0.05	0.65
Loin eye width (cm)	17.66	18.46	19.01	0.30	0.10	0.60
WBSF 3 (N)	20.73	26.6	23.87	2.68	0.41	0.20
Protein (%)	16.69	19.13	19.87	0.52	0.05	<0.01
Crude fat (%)	8.33	8.46	8.25	0.92	0.29	0.41
Ash (%)	1.00	1.46	1.25	0.16	0.72	0.30

¹ Treatments: control diet (W/o-Salsola); diet with a medium inclusion of Salsola tragus (15-Salsola); diet using only Salsola tragus (30-Salsola). ² SEM: standard error of the mean. ³ WBSF: Warner–Bratzler shear force.

). Notably, loin eye depth (cm) increased in lambs fed the 30-Salsola treatment (p = 0.05). Finally, the lamb-fed diets that included Salsola tragus exhibited a linear and quadratic effect (p < 0.05) on the protein content in the LT muscles compared to those fed the control treatment.

3.5. DNA Damage

As summarized in

Table 6. DNA damage in the blood of lambs fed with Salsola tragus as a source of forage during the fattening phase.

Item	Treatments 1			SEM 2	p Value
	W/o-Salsola	15-Salsola	30-Salsola		Linear	Quadratic
Tail DNA (%)	21.05	23.78	24.12	2.99	0.37	0.56
Olive tail moment	5.88	6.12	6.43	0.53	0.69	0.98
Tail length μm	29.52	31.92	32.97	1.81	0.46	0.86

¹ Treatments: control diet (W/o-Salsola); diet with a medium inclusion of Salsola tragus (15-Salsola); diet using only Salsola tragus (30-Salsola). ² SEM: standard error of the mean.

, the biomarkers of DNA damage determined via the comet assay in the blood of lambs fed Salsola tragus as a source of forage during the fattening phase did not present adverse effects.

3.6. Metabolomic Analysis of Meat

To establish the treatments’ metabolomics profiles, which showed variations in their chemical composition in LT, a principal component analysis (PCA) was performed to describe the composition of volatile organic compounds (VOCs) (Figure 4). The PCA showed that VOCs from the LT muscle tissue of lambs that received the different treatments formed specific groups according to their treatment. Thus, a separation was observed in Principal Component 1 (85.7%) between the 30-Salsola and W/o-Salsola treatments.

The top 15 VOCs discriminating between W/o-Salsola, 15-Salsola, and 30-Salsola are shown in Figure 5. Higher VIP values were associated with the 15-Salsola treatment. According to the VIP score, the essential VOCs differentiating treatments were octadecanoic acid, 9-octadecenoic acid, tetradecanal, cyclostrisiloxane, heptadecanoic acid, decanoic acid, hexadecanoic acid, nonanoic acid, thiosulfuric acid, hexadecane, and hexanedioic acid.

Finally, the correlation between the chemical composition of treatments and the VOCs in LT from lambs is shown as a heatmap in Figure 6. The heatmap depicts the relative abundance of organic compounds in LT under the three treatments. The organic compounds clustered hierarchically based on their response patterns, revealing distinct changes in the concentration levels depending on the treatment. Compounds such as Hexadecanoic acid exhibited higher concentrations in Salsola-treated groups, particularly the 30-Salsola, whereas others showed decreased levels. The dendrograms highlight the similarities between the treatments, indicating a dose-dependent effect on organic compounds. These findings suggest that treatments that include Salsola tragus significantly alter the metabolic composition in the LT of lambs, with more significant effects observed at higher doses.

4. Discussion

Twenty-three bioactive compounds were detected in Salsola tragus, some of which have potential implications for ruminant metabolism and muscle formation [6]. Previous studies [6,11,37] provide solid evidence that the identified metabolites, such as tricosane, heneicosane, and octacosane, have a potential role in modulating lipid metabolism due to their hydrophobic nature and low reactivity [38]. Additionally, sterol-based compounds such as cholest-5-en-3-ol (cholesterol derivative) might contribute to the modulation of membrane integrity and sterol biosynthesis pathways, which are critical for cellular function in muscle tissue [39]. Additionally, several phenolic derivatives, including N-[2′-(3″,4″-dihydroxyphenyl)-2′-hydroxyethyl]-3-(4‴-methoxyphenyl)prop-2-enamide and isorhamnetin-(3β)-d-diglucuronate dimethyl ester, were also detected. These compounds are known for their antioxidant properties in desert plants [40] and might support oxidative stress regulation, thereby influencing the redox status in muscle tissues [6,37]. Notably, several unique compounds, including 5H-isoindolo [1,2-b][3] benzazepin-5-one,7,8,13,13a-tetrahydro-10-hydroxy-3,4,12-trimethoxy-, and N,N-diisopropyl-4,6-bis-(methoxycarbonyl)-4,6-dimethylcyclohept-1-enecarboxamide, were identified for the first time in a forage context. These findings suggest a potential for novel metabolic pathways or bioactivities that warrant further exploration.

The presence of ferulic acid and its derivatives, such as S-(−)-trans-N-feruloyloctopamine and N-trans-feruloyltyramine, further highlights the functional benefits of Salsola tragus as a forage source. Ferulic acid derivatives have been associated with anti-inflammatory effects and improved metabolic health in ruminants, as documented in studies exploring other forage plants like alfalfa and ryegrass [41,42,43]. Specifically, N-trans-feruloyltyramine, an inhibitor of cyclooxygenases 1 and 2, mitigates inflammation, while its antioxidant and radical-scavenging properties contribute to cellular homeostasis [6,37]. Nevertheless, Soberón et al. [44] described how ferulic acid and its derivatives modulate the microbiota in the rumen through a complex formed with arabinoxylans and lignin in forage cell walls in ester and ether covalent forms. According to this, Wang et al. [42] reported that after ruminants ingest forages with ferulic acid conjugates, these cross-linkages form physical and chemical barriers to protect cell wall carbohydrates from microbial attack and enzymatic hydrolysis, increasing the capacity to break down the ester linkages within forage cell walls by secreting feruloyl and p-coumaroyl esterase, resulting in the release of free ferulic acid and an improvement in cell wall digestibility.

Concerning the productive performance of the lambs, no effect was observed on the ADG kg/d⁻¹ and DMI kg/d⁻¹ in lambs fed Salsola tragus as the source of forage compared to the lambs fed sorghum stover in their diet. This consistency suggests that, at the tested inclusion levels, Salsola tragus provided comparable or digestible nutrients relative to sorghum stover. This implies that the lambs were able to maintain an adequate dry matter intake. The unique nutritional profile of Salsola tragus was not negatively impaired and potentially even supported the metabolic processes crucial for muscle growth. Different studies have proposed that plants of the genus Salsola increase ruminant productivity based on their nutrient values. Nevertheless, there is a lack of data on the use of plants of the genus Salsola during the fattening phase in ruminants [6,15]. The few reported studies have been linked to extensive systems in rangelands. Osman et al. [45] evaluated the use of Salsola vermiculata as a source of forage in Awassi ewes, in which grazed rangelands cover different types of forage. The authors concluded that rangelands in the presence of Salsola vermiculata increased productive performance and economic benefits because Salsola vermiculata maintains an amount of nutrients comparable to traditional forages during the year, allowing ewes to intake quality forage. Hanif et al. [37] and ElNaggar et al. [8] also reported that in the case of Salsola vermiculata, the plant is highly palatable in the early stage of its growth for goats and sheep. These descriptions might be comparable to those of the current study because the DMI was maintained for the experimental period. In addition, the form of the Salsola tragus particles used in the current study was observed by Sokolowska-Krzaczek et al. [46], and Ali et al. [47], where the authors emphasized that the plant has to be dried in the early stage as hay bait straw and later ground without spines or hardening. Based on these recommendations, Asaadi et al. [48] concluded that in ruminants, Salsola arbusculiformis has relatively high digestibility and metabolic energy during vegetative growth. As per Murshid et al. [6], the benefits of animals using the genus Salsola are related to their secondary metabolites and phytochemical composition at different stages of growth.

In line with previous studies, the in vitro and estimated kinetics parameter model for lambs fed Salsola tragus as a source of forage showed a quadratic effect for the reduction in the volume of gas produced when lambs received the two treatments that included Salsola tragus. According to various studies, plants that contain ferulic acids might reduce the volume of gases emitted to the atmosphere to modulate microorganisms in the rumen, promoting sustainability and potential mitigation through adjusted inclusion rates of Salsola tragus [42,49]. Wang et al. [42] presented an extensive review of the potential relationship between esters or ethers linked to ferulic acid and fiber digestion in ruminants. The authors pointed out that, in conjunction with specific bacteria that naturally produce ferulic acid, gas production can be decreased by changes in the digestibility of the fiber, leading to a delay in the fermentation process in the rumen. These observations might be related to the increase in lag time in the 30-Salsola treatment. Wang et al. [42] and Rosa-Prates et al. [50] reported that different plant extracts prolong the lag time in the rumen by substantially depressing the activity of cellulolytic ruminal bacteria, such as Selenomonas ruminantium, which have been reported to be capable of metabolizing ferulic acid. Nevertheless, the findings in the current study are restricted and only permit theorizing whether the secondary metabolites of Salsola tragus have the capacity to increase lag time and prolong the period of microbial activity, ultimately producing greater methane emissions over a longer time frame. Finally, the quadratic effects observed in the estimated ME and mmol SCFA analysis might be explained by the greater concentration of methane at 48 h of gas accumulation and the lag time in the two treatments that included Salsola tragus as a source of forage compared to the treatment with sorghum stover. Beauchemin et al. [51] described that methane emissions and energy generated in the rumen by fermentation are commonly higher, partly because of greater fiber content, slow retention lag time, and in specific cases, the secondary metabolites in the source of forage. To our knowledge, from all the volatile components and chemical composition of Salsola tragus, only ferulic acid and its secondary metabolites have been reported as modifiers of fiber digestibility in the rumen, while also improving the energy rate to potentiate the development of muscle. Lynch et al. [52] concluded that ferulic acid could increase forage digestibility and the availability of cell wall carbohydrates to cellulolytic bacteria, potentially improving the substrate but increasing hydrogen production [53]. Despite these promising results, more species of the genus Salsola should be investigated to characterize their fermentation and gas production effects on different ruminants when the genus is utilized as a unique source of forage in arid regions.

Despite the relatively minor effects of treatments on productive performance, such as DMI and ADG, the 30-Salsola treatment promotes muscle tissue formation to increase loin eye depth and tends to grow wider loins. In addition, the 30-Salsola treatment augmented the percentage of protein in LT muscle tissue compared to the other treatments. Even though the potential of the genus Salsola to be used as a source of forage has been described [6,11,36], there is a lack of studies that have verified its effect on meat production. However, this response can be attributed to the increased CP in the diets where Salsola tragus was included and the fact that lambs fed with the same treatments maintained their DMI during the current study, generating, as we described previously, different fermentation parameters in the rumen. In addition, the greater amount of CP in both diets with Salsola tragus might increase the net energy to produce muscle growth by improving carbohydrate digestion and using secondary metabolites. Other halophyte plant species, such as Salsola tragus, have been identified as forage sources with positive effects similar to traditional forages on muscle development during different ruminant productive periods [54,55]. Ahmed et al. [56] concluded that Atriplex nummularia and Acacia saligna positively impact carcass weight and the CP content of the meat from Barki lambs when they were fed with a mix of both plants. The authors also concluded that changes in the CP of meat could be linked to the highest level of CP present in both plants compared to traditional forage.

In contrast to the increase in muscle development and CP content, fat thickness and crude fat content did not differ between treatments. These results support the diverse studies that have postulated that halophyte plants decrease carcass fat content and increase lean meat quantity compared to ruminants that are finished with a high-concentrate diet [55,56,57]. This indicates that meat from sheep fed Salsola tragus is nutritionally desirable, as this source of forage linearly reduces the amount of intramuscular fat and increases CP when it is included in the diet. These results could be further studied and used as commercial tools in arid systems of sheep meat production using Salsola tragus as forage. Some initiatives have already been implemented in some regions of the American continent, aiming at these objectives, which will be invaluable in fully ascertaining the financial benefits of incorporating Salsola tragus into livestock diets [54,58,59].

The single-cell gel electrophoresis or comet assay has become a valuable tool in livestock production for assessing DNA damage and genotoxicity [60]. In our study, this technique allowed us to evaluate the integrity of genetic material in individual cells, providing insights into the potential impacts of Salsola tragus as a source of forage for lambs during the fattening phase. In livestock, Pu et al. [61] proposed that the comet assay should be applied to farm animal models to investigate the effects of factors such as heat stress, mycotoxin exposure, and oxidative stress on DNA stability with respect to the type of ingredients in diets. Applying this analysis to lambs elucidates that Salsola tragus is effective in promoting animal health and productivity compared to sources of traditional forage. Nevertheless, further research is necessary to evaluate the inclusion of Salsola tragus as a forage source at different moments of livestock production.

The sources of nutrients in feed systems are pivotal in determining the quality of raw meat, particularly in relation to the composition of fatty acids in meat. As noted by Wang et al. [62], lipids and metabolites are key components that influence the taste, flavor, tenderness, and juiciness of cooked meat [63]. The volatile compounds present in raw meat contribute to both the odor of raw lamb meat and the flavor of cooked meat. Metabolomic analysis is employed to rapidly screen small molecular metabolites under specific conditions, such as in muscle tissue, muscular cells, and fat adipocytes, and has been utilized to assess muscle mass growth and meat quality [61]. Based on the GC-MS analysis, we found several important metabolites that showed not only a potential as biomarkers for discriminating the specific source of forage but also as indicators of meat quality, as illustrated in the PCA. The results obtained from the PCA also showed distinct metabolite profiles in the LT muscle of lambs fed the 15-Salsola treatment. This implies that the metabolomic analysis was able to authenticate all three sources of forage. In recent research, sheep meat from grazing or feedlots has been tested to understand the role of VOCs in determining flavor, tenderness, and shelf life [64,65]. The identified metabolite markers provide valuable information for future targeted studies. Subsequent research should aim to validate these VOCs in the metabolic pathways in meat modified by Salsola tragus in meat, using different ruminant animal models.

In our study, the 15-Salsola treatment showed the highest correlations between the 15 metabolites with the greatest importance index in LT tissue of lambs, mainly contributing to the discrimination between treatments. According to Strandvik [66], the detection of volatile compounds in lamb meat and their relationship with better flavor has been linked for the greater part to the source of forage, independent of the amount of concentrate fed. This is because forages reduce certain types of unpleasant odors in meat via the types of volatile compounds that translocate in the form of lipids in the muscle [62,65]. Despite the fact that few studies have focused on volatile compounds in sheep meat and their relationship with different sources of forage, different authors have postulated that different silage-, alfalfa-, or hay-feeding regimes could modify decanoic acid content in meat, which is a medium-chain fatty acid associated with fatty acid metabolic modulations [64,65]. The increased abundance of decanoic acid determined using GC–MS-based metabolomics might confirm an altered fatty acid metabolism based on other fatty acids with a higher correlation to the 15-Salsola treatment. Fatty acids (FAs), particularly octadecanoic acid, 9-octadecanoic acid, and hexadecanoic acid, play a significant role in meat composition and quality. Curiously, the 15-Salsola treatment was positively correlated with previous FAs. Tánori-Lozano et al. [49] found a source of ferulic acid might modify the fatty acid profile of the meat of lambs, such as octadecanoic acid and hexadecanoic acid, which are the primary saturated fatty acids found in meat. In addition, the 15-Salsola treatment increased its correlation with hexadecanoic acid, which is particularly prevalent and one of the most abundant fatty acids in various meat sources [62,65]. Finally, the utilization of the 30-Salsola or W/o-Salsola treatments as a forage source did not demonstrate a positive correlation with 9-Octadecanoic acid, a monounsaturated fatty acid that is also prevalent in meat. This compound is one of the primary substances released during cooking and contributes significantly to the distinctive aroma and flavor of cooked meat [65,67,68]. Various forage sources have the potential to influence the fatty acid composition and volatile profiles of lamb meat, as well as certain biochemical properties of muscle, including protein content, texture, and shear force [66,67,68]. The present study demonstrates that Salsola tragus can beneficially alter volatile compounds in meat, suggesting that its inclusion in diets is viable in arid regions where traditional forage production is challenging. Furthermore, the data indicate that lambs fed fresh Salsola tragus are capable of accumulating a greater quantity and diversity of plant-derived fatty acids or metabolites, such as ferulic acid, which may positively affect lipid metabolism. However, additional research is necessary to substantiate the essential roles and functions of these altered components in meat chemistry.

5. Conclusions

Salsola tragus was included as a partial or complete replacement for 30 percent of the sorghum stover as a source of forage in the diet of lambs during the fattening phase. It maintained productive performance and induced specific beneficial shifts in meat metabolomic profiles without adversely affecting animal health or feed intake. Notably, the absence of detectable genotoxic effects in our experimental animals is a highly significant finding, strongly supporting the safe and non-toxic nature of Salsola tragus as a potential forage source for ruminants at the tested inclusion levels. Although our metabolomic analysis was exploratory, future research should aim to externally validate the identified metabolic signatures in sheep at different life stages. These results collectively suggest that Salsola tragus is a promising, sustainable, and safe alternative forage source, particularly valuable in arid and semi-arid regions, where it can contribute to feed security and diversify agricultural practices. While we acknowledge limitations such as the study duration and sample size, our findings lay a robust foundation for future, more extensive investigations, including long-term feeding trials, in vivo methane assessments, and comprehensive analyses of the rumen microbial community structure, to fully elucidate the optimal and sustainable incorporation of Salsola tragus into ruminant production systems.