Leonardite (Humic and Fulvic Acid Complex) Long-Term Supplementation in Lambs Finished Under Subtropical Climate Conditions: Growth Performance, Dietary Energetics, and Carcass Traits

Simple Summary

Leonardite (LEO), a microbial derived product rich in humic and fulvic acids, has been extensively used as a fertilizer and lately used as a feed additive, mainly for non-ruminant species. The reasons why LEO is used as a feed additive is because of its protective, anti-inflammatory, antimicrobial, antioxidant, and anti-toxicity properties. Because diets containing high soluble carbohydrates and lower fiber can cause inflammatory processes in the gastrointestinal tract in the ruminant, and the adverse climatic conditions such high ambient temperatures can affect the efficiency of energy utilization by different metabolic processes, all the properties of LEO can be advantageous to ruminants when they are fed with high-energy diets and undergo fattening under high ambient temperatures. However, supplementing for 130 days with 0.20, 0.40, or 0.60% LEO to finishing lambs did not show improvements in growth, feed efficiency, or carcass traits compared to unsupplemented lambs. It is concluded that supplementation with up to 0.60% leonardite does not improve lamb productive performance when they were fattening under high ambient temperatures.

Abstract

Leonardite (LEO), a microbial derived product rich in humic and fulvic acids, has been tested, due to its beneficial properties for health and well-being, as a feed additive, mainly in non-ruminant species. Although there are some reports of LEO supplementation in ruminants fed with high-to medium-forage based diets, there is no information available of the potential effects of LEO in ruminants fed, under sub-tropical climate conditions, with high-energy diets during long-term fattening. For this reason, the objective of the present experiment was to evaluate the effects of LEO levels inclusion in diets for feedlot lambs finished over a long-term period. For this reason, 48 Pelibuey × Katahdin lambs (initial weight = 20.09 ± 3.55 kg) were fed with a high-energy diet (88:12 concentrate to forage ratio) supplemented with LEO (with a minimum of 75% total humic acids) for 130 days as follows: (1) diet without LEO, (2) diet supplemented with 0.20% LEO, (3) diet supplemented with 0.40% LEO, and (4) diet supplemented with 0.60% LEO. For each treatment, Leonardite was incorporated with the mineral premix. Lambs were blocked by weight and housed in 24 pens (2 lambs/pen). Treatment effects were contrasted by orthogonal polynomials. The average climatic conditions that occurred during the experimental period were 31.6 ± 2.4 °C ambient temperature and 42.2 ± 8.1% relative humidity (RH). Those values of ambient temperature and RH represent a temperature humidity index (THI) of 79.07; thus, lambs were finished under high heat load conditions. The inclusion of LEO in diet did not affect dry matter intake (p ≥ 0.25) and average daily gain (p ≥ 0.21); therefore, feed to gain ratio was not affected (p ≥ 0.18). The observed to expected dietary net energy averaged 0.96 and was not affected by LEO inclusion (p ≥ 0.26). The lower efficiency (−4%) of dietary energy utilization is an expected response given the climatic conditions of high ambient heat load presented during fattening. Lambs that were slaughtered at an average weight of 49.15 ± 6.00 kg did not show differences on the variables measured for carcass traits (p ≥ 0.16), shoulder tissue composition (p ≥ 0.59), nor in visceral mass (p ≥ 0.46) by inclusion of LEO. Under the climatic conditions in which this experiment was carried out, LEO supplementation up to 0.60% in diet (equivalent to 0.45% of humic substances) did not did not help to alleviate the extra-energy expenditure used to dissipate the excessive heat and did not change the gained tissue composition of the lambs that were fed with high-energy diets during long-term period under sub-tropical climate conditions.

1. Introduction

The final phase of lamb production can be stressful since the lambs are pushed to gain large amounts of weight via the consumption of greater amounts of high-soluble carbohydrates (and lower concentrations of fiber). The low-fiber and high-energy concentrates in diets offered over a long period of time increase the risk of digestive disturbances, i.e., inflammatory processes in enteric cells that decrease the rate of nutrient absorption, which can affect cattle productivity [1]. This situation can be aggravated during the final stage of production, where there are high ambient heat load conditions, since climatic conditions of this type alter energy consumption and ruminal motility, affecting dry matter intake, passage rate, and the digestion of nutrients [2]. Furthermore, recent studies have shown that heat stress disrupts free radical concentrations, causing oxidative damage to both mitochondria and cells, which affects energy efficiency [3]. Thus, in warm regions, the efficient fattening of cattle is an ever-present challenge. Strategies such as the control of environment conditions (e.g., the use of shade, fans, etc.) are commonly used to diminish the negative impact of hot environments [4,5]. However, in recent years, feed additives have gained interest as an additional tool that can be used to decrease the negative impact of hot environments on livestock productivity. Regarding this, several feed additives (both synthetic and natural) have been used to improve weight gain rates or feed efficiency in lambs [6,7,8] and steers [9,10] during the final period of production under hot environmental conditions. Leonardite (LEO) is a fossil mineraloid that is produced via the oxidation of lignite; it is characterized by its high content of humic and fulvic acids, which are produced during the decomposition of organic matter. The natural binding of mineral clay with humic and fulvic acids (HFAs) creates conditions that result in a compound with a high content of organic matter, an efficient ion exchange capacity, and characteristics of stabilizers and colloidal agents [11]. In addition to nitrogen and sulfur molecules, humic substances have phenolic hydroxyl and quinone groups that neutralize free radicals due to their heterogeneous compositions and supramolecular structures. In this way, humic substances act as antioxidant agents [12]. A further benefit of humic substances is that they inhibit the release of inflammatory-related cytokines and components of the complement system, thereby ensuring an effective anti-inflammatory effect [13]. Another property of HFAs is their ability to block or reduce the production of stress-causing hormones, subsequently making animals more productive [14]. Finally, as a natural surfactant, HFAs act as antimicrobial agents, being more toxic for certain types of non-beneficial Gram-Positive microorganisms [15]. Several studies in non-ruminant species have corroborated the anti-inflammatory, antimicrobial, antioxidant, and anti-toxic properties of HFAs [16,17,18]. In ruminants, certain studies attribute benefits to the use of HFAs in ruminal fermentation and digestion, indicating that HFAs stimulate the growth of diverse fungi and microorganisms, changing the ruminal fermentation patterns; as a result, the most consistent effect is the reduction in NH₃-N ruminal with an increase in microbial protein synthesis [19,20]. One study reports increases in ruminal OM digestibility, mainly due to increases in fiber and starch fractions [21]. In theory, all these properties could be beneficial in ruminants that are being fattened under high heat load ambient conditions and that are fed with high-energy diets. However, these effects have not been consistent. Some reports indicate that ruminants fed with medium-to-high forage diets and receiving HFAs reported an improved productivity and ruminal fermentation when HFAs were supplemented at concentrations ranging from 1.8 to 3.6 g HFAs/kg of diet [20,22,23]. However, reports relating to the effects of HFAs in ruminants fed with high-energy diets are very limited and contradictory [24,25,26]. According to the information reviewed, most studies indicate that cattle supplemented with HFAs in high-energy diets do not show advantages in growth rate or feed efficiency [25,26]. Due to associative effects, feed composition has substantial effects on ruminal fermentation and digestibility. High-concentrate diets change the microbial population, which, in turn, changes ruminal fermentation patterns (resulting in a high organic acid production and a low ruminal pH), ruminal kinetics, and the nutrient absorption rate. Therefore, these conditions could affect the potential benefits of humic substances on the digestive processes of ruminants that are fed with high-energy diets. However, it should be considered that high-energy diets that are consumed over a long period of time can cause inflammatory processes in enteric cells, as well as digestive disturbance and stress [1]. In the same way, hot climatic conditions can affect these parameters, in addition to increasing oxidative damage [2]. As such, the anti-inflammatory, antioxidant, and stress-relieving properties of HFAs could be advantageous for the ruminants that are subject to long-term fattening with a high-energy diet under high ambient temperature conditions. For this reason, the objective of this study was to evaluate the effect of leonardite inclusion in high-energy diets (~2.12 Mcal EN_m/kg) offered to lambs feeding under subtropical conditions for a duration of 130 days. The variables measured were growth performance, dietary energetics, carcass traits, shoulder tissue composition, and visceral mass.

2. Materials and Methods

2.1. Location of the Study and Ethical Statement

This experiment was conducted at the Universidad Autónoma de Sinaloa Feedlot Lamb Research Unit, located in the Culiacán, México (24°46′13″ N and 107°21′14″ W). Culiacán is about 55 m above sea level and has a subtropical climate.

All animal management and care procedures were conducted in accordance with the guidelines approved by the Universidad Autónoma de Sinaloa Animal Use and Care Committee (Protocol #23012024).

2.2. Climatic Variables and Temperature Humidity Index (THI) Calculation

Climatic variables (ambient temperature and relative humidity) were obtained every hour from two on-site pieces of portable weather equipment (Thermo-hygrometer Avaly, Mod. DTH880, Mofeg S.A., Zapopan, Jalisco, Mexico). The temperature humidity index (THI) was calculated using the following formula: THI = 0.81 × T + (RH/100) × (T − 14.40) + 46.40. Here, T = temperature expressed in Celsius grade, while RH = relative humidity [27].

2.3. Animals, Diets, and Experimental Design

To evaluate the treatment effects of LEO supplementation, 48 Pelibuey × Katahdin male intact lambs (age: 142 ± 14 d; initial BW at the start of the experiment: 20.09 ± 3.55 kg) were used in a randomized complete block design experiment in a growth-performance trial that lasted 130 d. Four weeks before the initiation of the experiment, the lambs were treated for parasites (Albendaphorte 10%, Animal Health and Welfare, México City, Mexico), injected with 1 × 10⁶ IU vitamin A (Synt-ADE^®, Fort Dodge, Animal Health, México City, Mexico), and vaccinated for Mannheimia haemolytica (One shot Pfizer, México City, Mexico). After health management, lambs were then allocated to communal pens for one week with a specific diet (50:50 forage-to-concentrate ratio). Three weeks before the initiation of the experiment, lambs were adapted to the experimental facilities and to the total mixed finishing basal diet (

Table 1. Composition of a total mixed basal diet and treatments.

	Leonardite Inclusion, % of Diet DM
Item	0.00	0.20	0.40	0.60
Ingredient composition (%)
Sudangrass hay 1	12.00	12.00	12.00	12.00
Cracked corn 2	60.00	59.80	59.60	59.40
Soybean meal	14.00	14.00	14.00	14.00
Leonardite 3	0.00	0.20	0.40	0.60
Molasses cane	8.00	8.00	8.00	8.00
Yellow grease 4	3.50	3.50	3.50	3.50
Mineral-protein premix 5	2.50	2.50	2.50	2.50
Chemical composition (%DM basis) 6
Crude protein	14.67	14.64	14.62	14.61
Neutral detergent fiber	16.45	16.47	16.43	16.43
Calcium 7	0.71	0.73	0.73	0.73
Phosphorus 7	0.34	0.34	0.34	0.33
Net energy (Mcal/kg) 7
Maintenance	2.12	2.11	2.11	2.10
Gain	1.45	1.44	1.44	1.44

¹ Sudangrass hay was ground in a hammer mill (Azteca 20, Molinos Azteca, Guadalajara, Mexico) with a 3.81-cm screen before incorporation into total mixed ration. ² Corn grain cracked was prepared by passing corn through rollers (46 × 61 cm, corrugated) that had been adjusted so that kernels were broken to obtain a final density approximately of 0.52 kg/L. ³ Leonardite used was a commercial product soft-sandy presentation its characteristics are: purity = 93–96%; density = 1.2 g/cm³; pH = 9.0; water solubility = 99%; minimum of total humic acids (43% humic acid, 32% fulvic acids) = 75%; nitrates = 3.10 ppm; phosphorus = 12.9 ppm, potassium = 20 ppm; C; N ratio = 28.7%; macro minerals = 5.08% (Leonardita; Terra Forte, San Luis Potosí, Mexico). ⁴ Yellow grease (restaurant grease) containing moisture, 1.56%; impurities, 0.80% and total fatty acids, 88.50%. ⁵ Mineral-protein premix contained: Total crude protein (as NNP) 50%, Calcium, 20%; CoSO₄, 0.010%; CuSO₄, 0.15%; FeSO₄, 0.528%; ZnO, 0.111%; MnSO₄, 0.160%; KI, 0.007%; and NaCl, 16%. ⁶ Dietary composition of crude protein and NDF were determined by analyzing subsamples collected and composited throughout the experiment. Accuracy was ensured by adequate replication with acceptance of mean values that were within 5% of each other. ⁷ Based on tabular net energy (NE) and nutrient composition values (Ca and P) for individual feed ingredients [28].

) that was offered during the experiment. The basal diet was formulated to meet the nutrient requirements of lambs to reach a daily gain of 200–300 g [28].

Upon the initiation of the experiment, all lambs (n = 48) were individually weighed before the morning meal (electronic scale; TORREY TIL/S: 107 2691, TORREY Electronics Inc., Houston TX, USA). Lambs were blocked by initial weight and were randomly assigned to 1 of 6 weight groupings in 24 pens (2 lambs/pen, 6 replicas/treatment, and 12 observations within each treatment). Treatments were randomly assigned to the pens within weight blocks. Pens were 6 m² with overhead shade, a soil floor (tucuruguay–sand mix), bucket waterers, and 1 m fence-lined feed bunks. Although there is no scientific information regarding the optimal levels of LEO inclusion in finishing diets for final-stage ruminants, the available scientific literature [19,21,22,24] indicated that inclusions ranging from 1.8 to 3.6 g HFAs/kg of diet have shown positive effects on ruminal fermentation and productivity in ruminants that are fed with a dairy-type diet (approximately 50:50 concentrate-to-forage ratio). The LEO used in the current experiment contained 75% HFAs; therefore, treatments consisted of a total mixed-ration corn-based diet with an 88:12 concentrate-to-forage ratio (Sudangrass hay as the only source of forage; Table 1), which was supplemented as follows: (1) basal finishing diet without LEO, (2) basal finishing diet supplemented with 2 g LEO/kg diet (equivalent to 1.5 g HFAs/kg diet), (3) basal finishing diet supplemented with 4 g LEO/kg diet (equivalent to 3 g HFAs/kg diet), and (4) basal finishing diet supplemented with 6 g LEO/kg diet (equivalent to 4.5 g HFAs/kg diet). The proportions of LEO inclusion in each treatment directly replaced cracked corn grain in the basal diet (Table 1). To facilitate the proper mixing of LEO in the final diet, the LEO proportion of each treatment was added to 25 kg of a mineral–protein premix. Each combination (2 kg LEO plus 25 kg premix; 4 kg LEO plus 25 kg premix; and 6 kg LEO plus 25 kg premix) was premixed using a 2.5 m³ capacity paddle mixer (model 30910-7, Coyoacán, Mexico). Finally, the resulting premix comprising LEO and the mineral–protein mix was incorporated (as the second step of the final diet) during the elaboration of the final diet, according to each respective treatment. After mixing, the elaborated experimental diets were stored in 120 kg capacity bags and were carefully labeled for later use. To prevent cross-contamination between treatments, the mixer underwent thorough cleaning between each batch. The source of LEO was a commercial product with a soft-sandy presentation containing 75% minimum of total humic acids (Leonardita; Terra Forte, San Luis Potosí, Mexico).

Clean water was available at all times. Fresh feed was provided twice daily at 0800 and 1400 h, in a 40:60 proportion of the total daily feed consumption. While the amount of feed provided in the morning feed was constant, the feed offered in the afternoon feed was adjusted daily, allowing for a residual ~50 g/kg daily feed offering. Residual feed was collected daily between 0740 and 0750 h each morning, before being weighed. Feed intake was calculated as the difference between quantities offered minus refusals.

Lambs were weighed just prior to the morning feed on days 1, 56, 112, and on the final day (day 130). The initial and intermediate body weight (BW) measures were converted to shrunk body weight (SBW) by multiplying LW by 0.96 to adjust for the gastro-intestinal fill [29]. All lambs were fasted (from feed, but not from drinking water) for 18 h before being individually weighed to determine final fasted BW (FFBW).

Feed samples and feed refusal were collected daily and were composited weekly. The composited feed and feed refusal samples were subject to DM analysis (method 930.15) [30]. Experimental diets were elaborated weekly, and a sample of each batch was sampled and subject to CP analysis (N × 6.25; method 984.13) according to AOAC [30]. Neutral detergent fiber (NDF) was determined following procedures described by Van Soest et al. (corrected for NDF–ash, incorporating heat-stable α-amylase using Ankom Technology, Macedon, NY, USA) [31].

2.4. Calculations of Productive Performance

Estimates of the average daily weight gain (ADG) and dietary net energy (NE) are based on initial SBW and fasted final weight (FFBW). The average daily gain was computed by subtracting the initial SBW from final SBW and dividing the result by the number of days on feed. Average dry matter intake (DMI) was calculated by dividing the total intake during the experiment by 130 (total days on feed). Feed efficiency was computed as ADG/average DMI observed during the 130 days of the experiment. One approach for the evaluation of the efficiency of dietary energy utilization in growth-performance trials is the ratio of observed-to-expected DMI and observed-to-expected dietary NE. Based on estimated diet NE concentration and measures of growth performance, there is an expected energy intake. This estimation of expected DMI is performed based on observed ADG, average SBW, and NE values of the diet, as follows: expected DMI, kg/d = (EM/NE_m diet) + (EG/NE_g diet), where EM (energy required for maintenance, Mcal/d) = 0.056 × SBW^0.75, EG (energy required for gain, Mcal/d) = 0.276 × ADG × SBW^0.75, and the NE_m and NE_g values used were 2.12 and 1.45 Mcal, respectively. These energy values were calculated based on the ingredient composition in the diet offered to the lambs (Table 1). The coefficient (0.276) was taken from NRC [32], assuming a mature weight of 113 kg for Pelibuey × Katahdin male lambs. The observed dietary net energy was calculated using EM and EG values, as well as the DMI observed during the experiment, by means of the following quadratic formula:

$$x={\displaystyle \frac{-b\pm \sqrt{{{\displaystyle b}}^2-4ac}}{2c}}$$

where x = observed dietary NE_m, Mcal/kg; a = −0.41 EM; b = 0.877 EM + 0.41 DMI + EG; and c = −0.877 DMI [33].

2.5. Carcass Characteristics and Visceral Organ Mass

All the lambs were harvested on the same day. The lambs were skinned and the gastrointestinal organs were separated and weighed. After carcasses (with kidneys and internal fat included) were chilled in a cooler at −2 to 1 °C for 24 h, the following measurements were obtained: (1) fat thickness perpendicular to the m. longissimus thoracis (LM), measured over the center of the ribeye between the 12th and 13th rib; (2) LM surface area, measured using a grid reading of the cross sectional area of the ribeye between the 12th and 13th rib; and (3) kidney, pelvic, and heart fat (KPH). The KPH was manually removed from the carcass, weighed, and reported as a percentage of the cold carcass weight (CCW) according to the USDA [34]. The dressing percentage (DP) was calculated by dividing HCW by final fasted BW. The tissue composition of the shoulder was assessed using physical dissection following the procedure described by Luaces et al. [35]. The components of the gastro-intestinal tract (GIT), including the tongue, esophagus, stomach (rumen, reticulum, omasum, and abomasum), pancreas, liver, gallbladder, small intestine (duodenum, jejunum, and ileum), and large intestine (caecum, colon, and rectum), were removed and weighed. The GIT was then washed, drained, and weighed to obtain empty weights. The difference between full and washed digesta-free GIT was subtracted from the SBW to determine the empty body weight (EBW). All tissue weights were reported on a fresh tissue basis. Organ mass was expressed as grams of fresh tissue per kilogram of final EBW, where final EBW represents the final full live weight minus the total digesta weight. The full visceral mass was calculated by the summation of all visceral components (stomach complex + small intestine + large intestine + liver + lungs + heart), including digesta. The stomach complex was calculated as the digesta-free sum of the weights of the rumen, reticulum, omasum, and abomasum.

2.6. Statistical Analyses

The number of pen replicates (6) and animals (12) within treatments was enough to determine the statistical differences on performance, carcass, and visceral mass variables of feedlot lamb. Based on power analysis and SD for measure, we had a power of 0.915 for detecting a 5% difference. All the data were tested for normality using the Shapiro–Wilk test. Performance data (DMI, gain, gain efficiency, observed dietary NE, and observed-to-expected dietary NE ratio), carcass characteristics, and visceral organ mass data were analyzed as a randomized complete block design using the MIXED procedure of SAS software ver. 9.3 [36], considering the initial shrunk weight as the blocking criterion and the pen as the experimental unit, with treatment and block as fixed effects and the experimental unit (pen) within treatment as a random effect. LEO level supplementation was partitioned into linear, quadratic, and cubic orthogonal polynomials, considering four equally spaced levels (0.00, 0.20%, 0.40%, and 0.60%). Treatment effects were considered significant when the p-value was ≤ 0.05.

3. Results

There were no deaths or morbidities observed during the experiment, and no lambs had to be retired.

The average minimum and maximum estimated THI were 69.50 and 87.10, respectively. During the first 7 weeks of the experiment, the daily THI averaged 74.20. From week 8 to the end of the study, the daily THI averaged 82.13. The overall daily THI averaged 79.07, corresponding to “alert” conditions [27]. Because intermediate weighing was performed (at 56 and 112 days), the statistical analysis for ADG as a response variable was performed using a linear model with the components of general mean, block, treatments, periods, treatment by period interaction effects and the random error component. As a result, no statistical differences were observed for the treatment by period interaction (p = 0.28), for the treatment effect (p = 0.35), and for the period effect (p = 0.43). As a result, the ADG general average value was 230 g in period 1–56 and 218 g in period 56–112 (p = 0.43). Therefore, the analysis strategy for ADG was to consider 1–112 d and use a linear model that included the general mean, block and treatment effects and random error. In this way, the constructed tables of results were not overloaded with data that did not provide information different from the global average data.

The effects of treatments on growth performance and dietary energetics are shown in

Table 2. Effect of Leonardite (LEO) as feed additive on growth performance and dietary energetics in feedlot lambs.

	Leonardite Level (%) of Diet DM				p-Value
Item	0.0	0.20	0.40	0.60	SEM	Linear	Quadratic	Cubic
Days on test	130	130	130	130
Number of lambs	12	12	12	12
Pen replicates	6	6	6	6
Live weight, kg 1
Initial	20.27	20.13	19.99	19.97	0.125	0.15	0.61	0.84
Final	50.70	47.63	48.36	49.92	1.769	0.84	0.21	0.71
Average daily gain, kg	0.234	0.212	0.218	0.231	0.013	0.95	0.21	0.70
Dry matter intake, kg/d	1.084	0.987	1.023	1.064	0.058	0.92	0.25	0.63
Gain to feed, kg/kg	0.216	0.214	0.213	0.217	0.002	0.84	0.18	0.41
Observed dietary NE, Mcal/kg
Maintenance	2.04	2.03	2.01	2.03	0.019	0.63	0.26	0.40
Gain	1.38	1.37	1.35	1.37	0.012	0.63	0.26	0.40
Observed to expected dietary NE ratio
Maintenance	0.96	0.96	0.95	0.97	0.007	0.63	0.26	0.40
Gain	0.95	0.95	0.93	0.96	0.008	0.63	0.26	0.40

DM = dry matter; SEM = standard error of mean, NE = net energy. ¹ Initial live weight (LW) was reduced by 4% to adjust for the gastrointestinal fill. Final LW was obtained following an 18-h fast without access to feed (access to drinking water was not restricted).

. The average daily dry matter intake (average = 0.231 ± 0.03 kg) was very similar (p ≥ 0.25) between treatments; in the same manner, compared to controls, the average daily gain was not different (p ≥ 0.21) in lambs supplemented with LEO. Therefore, the feed-to-gain ratio was very similar (p ≥ 0.18) between controls and LEO-supplemented lambs (p ≥ 0.18). The observed-to-expected dietary net energy averaged 0.96 and was not affected by LEO inclusion (p ≥ 0.26). The lower efficiency (−4%) of dietary energy utilization shown by all lambs in this trial is an expected response that is caused by both the climatic conditions of high ambient heat load presented during the experiment and by the number of days of fattening.

Due to the extension (130 days) of the fattening period, lambs were slaughtered at an average weight of 49.15 ± 6.00 kg. The late phase of finishing is characterized by a greater fat deposition instead of protein accretion in the muscle tissue. In this sense, LEO did not promote changes (p ≥ 0.59) in tissue gain composition (i.e., a smaller amount of fat and a greater muscle accretion;

Table 3. Effect of Leonardite (LEO) as feed additive on carcass characteristics and shoulder tissue composition in feedlot lambs.

	Leonardite (%) of Diet DM				p-Value
Item	0.0	0.20	0.40	0.60	SEM	Linear	Quadratic	Cubic
Number of lambs	12	12	12	12
Pen replicates	6	6	6	6
Hot carcass weight, kg	29.80	27.97	28.09	29.24	1.001	0.73	0.16	0.83
Dressing percentage	58.75	58.75	58.14	58.59	0.472	0.60	0.65	0.44
Cold carcass weight, kg	29.52	27.73	27.84	28.99	0.991	0.74	0.16	0.85
Longissimus m. area, cm2	16.54	16.28	16.12	16.23	1.003	0.81	0.86	0.97
Fat thickness, mm	3.35	3.26	3.32	3.22	0.120	0.56	0.98	0.58
Kidney-pelvic fat, %	4.26	4.28	4.22	4.19	0.262	0.81	0.95	0.93
Shoulder composition,%
Lean	63.21	63.62	63.20	63.37	0.581	0.98	0.84	0.59
Fat	18.38	18.26	18.23	18.46	0.502	0.93	0.74	0.93
Lean to fat ratio	3.45	3.49	3.50	3.43	0.118	0.94	0.67	0.89

DM = dry matter; SEM = standard error of mean.

). In the same manner, lambs supplemented with LEO did not show differences (p ≥ 0.16) in the variables measured for carcass traits (dressing percentage, LM area, and fat deposits; Table 3). Visceral mass (expressed as g/kg of empty BW) was not affected (p ≥ 0.46) by treatments (

Table 4. Effect of Leonardite (LEO) as feed additive on visceral mass in feedlot lambs.

	Leonardite Level (%) of Diet DM				p-Value
Item	0.0	0.20	0.40	0.60	SEM	Linear	Quadratic	Cubic
Number of lambs	12	12	12	12
Pen replicates	6	6	6	6
Organs, g/kg EBW
Stomach complex	23.76	24.32	24.18	23.75	0.562	0.95	0.40	0.87
Intestines	40.48	39.82	39.52	39.68	0.816	0.46	0.62	0.97
Hearth + lungs	19.82	20.19	20.09	19.75	0.644	0.91	0.59	0.95
Liver + spleen	17.53	17.23	17.03	17.15	0.650	0.62	0.73	0.86
Kidney	2.62	2.54	2.47	2.55	0.098	0.52	0.44	0.73
Omental fat	37.66	37.83	38.01	37.80	0.803	0.87	0.81	0.92
Mesenteric fat	19.24	19.29	18.81	18.86	1.604	0.82	0.99	0.89
Visceral fat	56.90	57.12	56.82	56.65	1.833	0.90	0.92	0.94

DM = dry matter; SEM = standard error of mean; EBW = Empty body weight.

).

4. Discussion

Previous studies have shown that compounds with anti-inflammatory, antioxidant, antimicrobial, and stress-reducing properties are useful as animal feed additives, both to alleviate the negative effects of high environmental heat load and the potential risks of the prolonged consumption of diets containing high-soluble carbohydrates and low levels of effective fiber, which are typical characteristics of finishing diets. In this regard, antimicrobials such as ionophores and virginiamycin [9,37], plant-derived products such as isoquinoline derivatives and essential oils [6,38], chelated minerals [39], and direct-fed microbials [40] have proven effective in improving the productivity and/or efficiency of ruminants raised under these conditions. The main extra-ruminal effects are primarily associated with intestinal improvements in health (microbiota, epithelial integrity, and oxidative status). These same properties have been attributed to HFAs [14]; therefore, the objective of this trial was to test the effects of including HFAs in finishing diets in lambs that are fattened under high ambient heat load conditions, primarily assessing growth and energy efficiency aspects as well as some characteristics of the carcass and visceral mass.

It is important to note that this is the first report regarding the evaluation of the mineraloid leonardite as a feed additive used over a long period of time in finishing-stage lambs that are fed with a high-energy diet under hot environmental conditions. The impact of leonardite on dietary energetics and visceral mass in ruminants is evaluated for the first time in this type of trial.

Because the products tested in the different studies have a large variation in the net concentration of HFAs [14], to standardize the comparison of our results with those studies, we made a comparison based on the net amount of HFAs used in those reports and not according to the level of the product used.

Based on the average DM intake registered during the experiment, the daily net intake of LEO averaged 1.9, 4.1, and 6.4 g/lamb. Considering that LEO contains 75% of humic acids, the net daily intake of HFAs corresponded to 1.4, 3.1, and 4.8 g (equivalent to 0.04, 0.09, and 0.18 g HFAs/kg BW, respectively). This is within, or slightly above, the intakes ranging from 0.09 to 0.12 g of HFAs/kg BW, which have been shown to exert positive effects on rumen fermentation and productivity in ruminants fed with a medium-energy diet [19,21,22,24].

The absence of the effects on the DMI and productive performance (ADG and/or gain-to-feed ratio) of supplemental LEO in feedlot cattle fed with high-energy diets (>2.10 Mcal NE_m/kg) over short-term periods has been previously reported by McMurphy [25] and by Chirase et al. [41]. These researchers fed crossbred steers for a duration of 56 days with high-energy diets containing anywhere from 4.5 up to 13.5 g of humic acids. The inclusion of humic acids did not affect DMI, growth performance, or feed efficiency. They concluded that although feeding humic acids did not adversely affect beef cattle, it also did not improve animal health or performance. Unfortunately, environmental conditions are not reported in those studies. Although some reports indicate that ruminants fed with medium-to-high forage diets that received HFAs (supplemented from 1.8 to 3.6 g HFAs/kg of diet) demonstrate an improved productivity mainly by positive changes in ruminal fermentation and through the better N utilization of the diet [20,22,23], the results for the effects of HFAs in ruminants fed with high-energy diets do not seem to follow this pattern. In this sense, there were no changes in ruminal fermentation patterns (VFA or NH₃-N) or in ruminal microbial populations when feedlot lambs (supplemented with 5.5 g HFAs/kg diet) [26] or Holsteins steers (supplemented with up to 13.5 g HFAs/kg diet) [42] received HFAs in high-concentration diet during a 21 to 27 d period. Contrary to our results, beef cattle that were supplemented with a complex of humic and fulvic acids enriched with trace minerals at a level of 1.40 g HFAs/kg showed improvements of 12.8% ADG and 11% feed efficiency [24]. It is important to note that the duration of supplementation was 69 days under favorable ambient conditions, as well as the fact that the finished diet used by Cusack [24] contained 10% less energy than the experiment by McMurphy et al. [25], as well as a 4% lower energy concentration than in the current experiment. In any case, the positive effect on the growth rate observed by Cusack [24] was imputed as humic substances were able to increase the absorption of the supplemental trace minerals. Several researchers claim that due to the significant variability in the chemical composition (i.e., the presence of phenolic, organic compounds, as well as macro and micro minerals in variable proportions) of humic and fulvic acid complexes causes inconsistencies in the results of HFAs used to finish cattle; therefore, making comparisons of generic HFAs can be meaningless [24,43].

The primary reason for decreased ADG is the reduced feed intake as a result of hot climates [44]. Based on NRC [45], DMI for control lambs and supplemented lambs in the present study was lower (−7.2 and −12.4%, respectively) than expected. Studies conducted in similar climatic conditions have reported a similar reduction in DM intake [46,47]. However, hot climates not only decrease energy intake and the rate of weight gain, but also affect the efficiency with which dietary energy is allocated to tissue accretion. The above findings are consistent with the lower observed-to-expected dietary energy ratio (0.96), which means that lambs show a 4% lower efficiency in energy utilization destined for growth. As has been previously explained by Estrada-Angulo et al. [7], by comparing diet energy density based on tabular values for individual feed ingredients with estimated diet energy density based on growth performance, we can assess dietary energy utilization. According to a ratio of observed-to-expected dietary NE of 1.00, the observed ADG matches the expected ADG from DM intake and formulated dietary energy density. A ratio of less than 1.00 indicates a lower-than-expected energetic efficiency, and vice versa for a ratio greater than 1.00. The lower efficiency (−4%) of dietary energy utilization shown by all lambs in this trial is an expected response given the climatic conditions of high ambient heat load presented during fattening. It is well known that sustained high environmental heat load conditions can affect dietary energy utilization even in well-adapted cattle such as Pelibuey sheep and their crosses [48]. According to the above findings, similar reductions (3–4%) in dietary energy utilization have been reported for Pelibuey lambs that are being fattened in similar environmental ambient conditions and fed with a similar type of diet without supplemental feed additives [8,49]. The negative effects of tropical and subtropical climatic conditions (which are characterized by high temperatures and relative humidities) are primarily related to increased energy requirements for maintenance, which are mediated by the extra-energy costs that are mediated by physiological mechanisms for body heat dissipation (changes in hemodynamic and respiratory rate, among others) under these environmental conditions [44]. Pooling the observed average of DMI and BW of all treatments, it is possible to estimate the changes in the coefficient of maintenance requirement (MQ) of the lambs using the equation described by Estrada-Angulo et al. [7]. In this manner, the coefficient of MQ is estimated to be 0.062, which is 11.1% greater than the MQ specified (0.056) for lambs under favorable environmental conditions [28]. In addition, recent studies have shown that environments with high ambient heat load conditions disrupt free radical concentrations in animals, causing oxidative damage to both mitochondria and cells, subsequently affecting the energy efficiency [3]. Several studies suggest that HFAs protect the intestinal mucosa; exert anti-inflammatory, antimicrobial, antioxidant, and anti-toxic properties; and act as abiotic and biotic stress relievers in non-ruminant species [14,16,17,18]; therefore, we hypothesized that LEO can alleviate the negative impact of adverse environment conditions, improving energy efficiency utilization in ruminants when compared to non-supplemented animals. However, in the current study, it was observed that LEO supplementation did not help to alleviate the extra-energy expenditure used to dissipate excessive heat.

Additionally, the lower efficiency of energy utilization in the long-term finishing phase is expected. The greater proportion of fat relative to protein in weight gain during the late finishing phase results in a lower gain efficiency (i.e., a greater energy intake/kg of body weight gain), as fat accretion requires nearly twice as much energy as muscle accretion [50]. Compounds that promote changes in gain composition (i.e., lowering fat and increasing protein) improve performance during the finishing phase [51]. Information about the effect of HFAs in body fat depots is scarce. Even so, in crossbred pigs (Landrace × Yorkshire × Duroc) that were fed for a duration of 56 d, the inclusion of 6.7 g HFAs/kg in the diet significantly decreased the backfat thickness [52]. At the same line, broilers that were supplemented for a duration of 42 days with 0.05 g HFAs/L in drinking water showed a reduction of 15.9% of the body fat content [53]; this effect could have partially contributed to the improvements in growth and feed efficiency in these studies. However, in feedlot cattle, the carcass or meat composition was not modified by HFA inclusion in the diet at levels of 1.40 g HFAs/kg for a duration of 69 days [24] or at levels of 9.9 g HFAs/kg for a duration of 100 days [54]. Likewise, supplementation with 4.5 g HFAs (obtained from potassium humate source)/kg in the diet failed to affect carcass fat composition in yearling steers that were fattened for a duration of 112 d with a diet containing 1.93 Mcal NE_m/kg [55]. Apparently, the absence of the effects of HFAs on the carcass composition of ruminants is usual and in agreement with our results, since no effects of LEO were noted in tissue composition and in visceral fat depot (Table 3 and Table 4).

Lambs that were slaughtered at an average weight of 49.15 ± 6.00 kg did not show differences in carcass traits. Since the rate of weight gain and gain composition were not affected by treatments, similarities in carcass characteristics (hot carcass weight, longissimus area, dressing percentage, and fat thickness) are to be expected [56,57]. The lack of effects on carcass characteristics in ruminants supplemented with HFAs has already been reported. In this sense, supplementations (ranging from 1.40 up to 9.9 g HFAs/kg in the diet) for anywhere between 69 and 112 days had no effect on the carcass characteristics evaluated (i.e., dressing percentage, LM area, subcutaneous fat, and kidney–pelvic–heart fat) [24,54,55]. Information regarding LEO supplementation effects on the relative weight (g organ/kg BW) of the visceral organ mass of animals is very limited. Some reports indicate that LEO supplementation did not affect the relative organ mass in broilers [16,53]. No information in relation to this subject is available in ruminants; however, similarly to reports for non-ruminant species, LEO did not affect the relative organ mass of the lambs in the current study (Table 3).

It is beyond our reach as to exactly why the finishing-stage lambs that are being fattened under high heat environment conditions and that have been supplemented with HFAs do not have the same positive responses on productive parameters as ruminants fed with low-to-moderate-energy diets. Even so, the absence of the effects of HFAs observed here is in close agreement with previous studies performed with ruminants fed with high-energy diets and supplemented with HFAs at different levels. The lack of significant effects across all parameters may also indicate that the doses of LEO tested so far are ineffective under the challenging conditions of heat stress (or in the case of diets containing high amounts of soluble carbohydrates). However, it is important to note that the absence of measurements of ruminal parameters, intestinal health, and oxidative status limits the interpretation of HFAs’ mechanisms of action, or lack thereof. In the same way, it is necessary to mention the limited number of replicates per treatment used in this experiment; therefore, larger-scale trials are needed to assess the extrapolation potential of the experimental results to feedlot systems.

5. Conclusions

It is concluded that under the conditions in which this experiment was carried out, supplementation up to 4.5 g HFAs/kg in the diet did not show any advantages on growth performance and carcass characteristics in lambs fed with a high-energy diet. However, marked variability in the chemical composition of humic and fulvic acid complexes makes it difficult to precisely evaluate the benefits of HFAs as feed additives, because the variation in composition may be related to the variation in physiological responses to their administration. In addition, the mechanisms by which HFAs could provide benefits to cattle during this fattening stage require further investigation, as well as studies addressing the physiological and metabolic variables involved in animal growth and welfare. This information is needed to more accurately determine the potential use of LEO as a feed additive in finishing diets for lambs.