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Introduction
In tropical dry forests, small ruminant productions are limited by low rainfall and high temperatures, which considerably reduce the nutritional properties of forages (Ulukan, 2011). Therefore, it is necessary to look for alternative feedings, such as silvopastoral systems or the inclusion of tree leaves and fruits, that improve animal performance and mitigate the impact of the dry season (Castrejón-Pineda et al., 2016). In addition, Dichanthium spp. is one of the most used forages by small farmers in the Colombian tropical dry forest, and it is often used as a primary source for small ruminant feeding. However, its offer and nutritional quality are reduced during the dry season, limiting its performance.
Guazuma ulmifolia is an adaptable tree of the American tropic that can withstand adverse climatic conditions such as high temperatures or hydric stress (Calzavara et al., 2017; Villa et al., 2009). It is considered a multipurpose tree due to the wide variety of products and services that it provides to agroforestry, agriculture, livestock farming, and alternative medicine (Manríquez- Mendoza et al., 2011). Studies have shown the Guazuma ulmifolia as an alternative for ruminant feeding, either partially replacing soybean meal (Glycine max) in sheep production (Castrejón- Pineda et al., 2016) or being used as an alternative source of forage biomass in silvopastoral systems (Nicodemo et al., 2016; Villanueva et al., 2016). Nonetheless, there is a lack of information about the nutritional value, digestibility, and ruminal kinetic degradability of Guazuma ulmifolia leaves in hair lambs. Therefore, this research aimed to evaluate the effect of replacement Dichanthium spp. hay by G. ulmifolia leaves on in vivo and in vitro digestibility, ruminal kinetics, and blood metabolites in hair lambs.
Materials and methods
Location
This research was carried out in the experimental farm “Las Brisas” located in Ibague, Tolima province, Colombia (4°25' 35.5'' N 75°13'47.8'' W), at 1,285 m a.s.l., with an average temperature of 22 ºC, relative humidity of 94%, and annual rainfall of 1,620 mm. It is classified as tropical dry forest according to Holdridge (1978).
G. ulmifolia leaves were obtained from the farm “El Recreo” located in Guamo, Tolima, Colombia (4°00'18.0'' N 74°58' 47.2'' W), at 321 m a.s.l, with an average temperature of 28 ºC, relative humidity of 74.9%, and annual rainfall between 1,000 and 1,400 mm. Samples of adult trees were taken manually; the leaves were dried in the shade and then stockpiled in sealed containers for later use in the experimental diets.
Animals and experimental design
Twelve non-castrated hair lambs 22.0 ± 1.3 kg were assigned to a Latin square design (4 × 4) of four treatments, four periods, and three animals in each experimental group. The treatments consisted of increasing levels of Guacimo leaves (G. ulmifolia) by replacing Dichanthium spp. hay (aged 90 days) as follows: T1 = 100% Dichanthium spp. hay, T2 = 85% Dichanthium spp. hay and 15% G. ulmifolia leaves; T3 = 70% Dichanthium spp. hay and 30% G. ulmifolia leaves, and T4 = 55% Dichanthium spp. hay and 45% G. ulmifolia leaves. Each experimental period lasted 21 days: 17 days for adaptation to the experimental diet and four days for the sample collection. The animals were confined in individual metabolic cages (1 m in height, 0.8 m in width, and 1.5 m in length) equipped with collectors, feces and urine separators, individual feeders, and troughs.
Chemical properties of the diets
The assigned diets, feces, and orts were determined by proximal chemical analysis according to the methods established by the Association of Analytical Communities (AOAC) (2012) for dry matter (DM), organic matter (OM), crude protein (CP), and mineral matter (MM) content. The neutral detergent fiber (NDF) and acid detergent fiber (ADF) were found based on the protocol of Van Soest et al. (1991; Table 1).
Table 1. Nutrient composition of ingredients and experimental diets as DM %
Note.Diets formulated for the study based on the DM intake. Proportions were as follows: T1 = 100 % Dichanthium spp. hay; T2 = 85 % Dichanthium spp. hay, 15 % G. ulmifolia leaves; T3 = 70 % Dichanthium spp. hay, 30 % G. ulmifolia leaves; T4 = 55 % Dichanthium spp. hay, 45 % G. ulmifolia leaves.
Source: Prepared by the authors
Dry matter intake
The treatments were calculated based on 4% of the live weight of the animals. The feed was provided in two daily meals at 7:00 A.M. and 3:00 P.M. in a sufficient quantity to obtain 5–10% orts. The DM intake of each treatment was calculated during collection days, and orts were removed daily for each animal before the morning meal. This intake was expressed in animal grams per day (ING g/d-1) concerning live weight (ING % LW) and metabolic weight (kg W 0.75) (ING % MW).
In vivo digestibility
The animals had 17 days for adaptation to the experimental diets in individual pens per treatment, and they were offered 4% of the live weight in DM, daily adjusted to obtain between 5% and 10% of orts. After this period, each animal was housed in a metabolic cage for four days for sample collection. Samples of diets, orts, and feces were dried for chemical analysis of DM, OM, NDF, and CP digestibility according to the previous bromatologically described methods.
Degradation kinetics and in vitro digestibility
The in vitro DM digestibility (IVDMD) of the experimental diets was determined based on the Tilley and Terry (1963) technique adapted to the artificial rumen DAISY II®-ANKOM®. A Girolando breed was used to obtain the ruminal fluid (provided with a ruminal cannula) maintained in a Cynodon spp. pasture with 500 g/day of G. ulmifolia leaves. Next, 0.5 g samples of each diet (previously ground to 1 mm) were weighed and deposited in F57 ANKOM® filter bags distributed in four glass jars to which A and B buffer solutions and a ruminal inoculum were added. Then they were introduced in the DAISY II® incubator for 48 hours, guaranteeing a temperature of 39 ºC. At the end of this period, 40 ml of 6N HCI and 8 g of pepsin (EC3.4.23.1 Sigma®) were added, leaving the samples in the incubator for other 24 hours. Afterward, the bags were dried at 105 ºC for eight hours. IVDMD was calculated by the difference between the incubated feed and the residue after incubation. The degradation kinetics were determined parallel to the IVDMD using DAISY II®, incubating each diet at 3, 6, 12, 24, 36, 48, 72, and 108 hours. The in vitro ruminal degradation parameters of the DM were calculated using Equation 1, as described by Ørskovand Mcdonald (1979):
(1)
where p = rate of degradation at time t; α = water-soluble fraction; b = water-insoluble, potentially degradable fraction; c = rate of degradation of fraction b; t = incubation time.
The DM’s effective degradability (ED) was calculated using Equation 2:
(2)
where k is the speed of the passage of particles in the rumen.
The ED of the DM in vitro was estimated for each diet, considering the passage rates of 2.5 and 8%/hour (values that can be attributed to low, medium, and high intake levels), respectively. Potential degradability was calculated using Equation 3:
(3)
Protein degradation kinetics were obtained by quantifying the nitrogen in Dichanthium spp. hay samples and G. ulmifolia leaves;.5-gram samples (previously ground) were weighed and deposited in F57 ANKOM® filter bags. These samples were incubated in the DAISY II® at 3, 6, 12, 24, 36, 48, 72, and 108 hours.
Blood metabolites
The glucose, blood urea nitrogen (BUN), and beta-hydroxybutyric acid (βHBA) were determined for each animal on the last day of the experimental period utilizing a venipuncture of the jugular vein with Vacutainer™ tubes. The samples were refrigerated and analyzed using commercial Biosystems® kits (glucose and BUN and Randox® (βHBA) with the automated system of blood chemistry Biosystems A15® at the Veterinary Diagnostic Laboratory (LADIVE, for its acronym in Spanish) of the Universidad del Tolima.
Secondary metabolites in G. ulmifolia leaves
The G. ulmifolia leaf samples were analyzed in the LASEREX laboratory of the Chemistry Department of the Universidad del Tolima, where the total phenols were quantified using the Folin-Ciocalteu method (Singleton et al., 1999). Tannins, flavonoids, and terpenes content were determined using the guide techniques described by Terrill et al. (1992) and modified by Del Pino et al. (2005; Table 2).
Statistical analysis
The results were interpreted by regression study using the PROC REG package of the statistical program Statistical Analysis System, version 9.1 (SAS). The in vitro ruminal degradation parameters were estimated using the Gauss-Newton iterative process applying the SAS PROC NLIN package. Significance was set at P < 0.05.
Results
Intake and in vivo digestibility
There was a linear effect of DM, OM, NDF, and CP intake (p < 0.05; Table 3) with the increase of replacing Dichanthium spp. hay with G. ulmifolia leaf levels in the diets. The DM intake in g/day rose from 609.47 (T1) to 762.66 (T4), respectively. Live weight and metabolic weight varied from 2.17% to 2.99% and 4.87% to 6.57%, respectively. The fecal production of DM, OM, NDF, and CP increased (p < 0.05) with the G. ulmifolia leaf volume. The CP digestibility linearly increased (p < 0.05) with the G. ulmifolia leaf batch. Meanwhile, the DM and OM digestibility did not vary between treatments (p > 0.05); however, the NDF digestibility linearly decreased with the G. ulmifolia size replacing the hay in the diets, going from 58.6 (T1) to 55.4 (T4).
Table 3. Intake (INT), fecal excretion (FE), and digestibility (Dig) of dry matter (DM), organic matter (OM), neutral detergent fiber (NDF), and crude protein (CP) in lambs fed with G. ulmifolia leaves
Note. 1:Dichanthium spp. hay; 2: 85% Dichanthium spp. hay, 15% G. ulmifolia leaves; 3: 70% Dichanthium spp. hay, 30% G. ulmifolia leaves; 4: 55% Dichanthium spp. hay, 45% G. ulmifolia leaves. SEM: standard error mean; L: linear effect; Q: quadratic effect. Significance level < 0.05.
Source: Prepared by the authors
Degradation kinetics and in vitro digestibility
The CP degradation increased as the G. ulmifolia leaf supplementation increased (Figure 1).
Source: Prepared by the authors
Figure 1. In vitro degradation kinetics of protein in diets with different levels of G. ulmifolia leaves.
The in vitro dry matter degradability decreased linearly in each incubation period. This trend remained for all incubation schedules. From 36 hours of incubation, the control treatment had a degradability of 10% above T4 (45% G. ulmifolia). Additionally, we observed a linear decrease (P < 0.05) of the IVDMD, while the levels of G. ulmifolia leaves in the diets increased, obtaining values of 51.51% (T1), 48.8% (T2), 46.1% (T3), and 43.4% (T4), respectively (Table 4).
The ruminal degradation parameters are presented in Table 5. The water-soluble fraction (a) decreased (P < 0.01) as the level of inclusion of G. ulmifolia leaves increased, going from 13.31% (T1) to 9.83% (T4). However, the potentially degradable fraction (b) did not have significant differences (P > 0.05). In the meantime, the inclusion levels of G. ulmifolia leaves increased in the diets while the effective and potential degradability increased linearly (P < 0.05).
Table 4. In vitro degradability and digestibility of dry matter in diets with levels of G. ulmifolia leaves
Note. 1:Dichanthium spp. hay; 2: 85% Dichanthium spp. hay, 15% G. ulmifolia leaves; 3: 70% Dichanthium spp. hay, 30% G. ulmifolia leaves; 4: 55% Dichanthium spp. hay, 45% G. ulmifolia leaves; IVDMD %: in vitro dry matter digestibility; SEM: standard error mean
Source: Prepared by the authors
Table 5. In vitro ruminal degradation parameters in diets with different levels of G. ulmifolia leaves
Note. 1:Dichanthium spp. hay; 2: 85% Dichanthium spp. hay, 15% G. ulmifolia leaves; 3: 70% Dichanthium spp. hay, 30% G. ulmifolia leaves; 4: 55% Dichanthium spp. hay, G. ulmifolia leaves 45%; a (%): water soluble fraction; b (%): water insoluble, potentially degradable fraction; c (%).h-1: degradation rate of an “a” fraction; PD: potential degradation; ED: effective degradation; SEM: standard error mean.
Source: Prepared by the authors
Blood metabolites
There was an increasing linear effect on blood glucose values with the replacement levels of G. ulmifolia (P < 0.05; Table 6). Although the BUN was greater in T4, the other treatments did not differ. Regarding the blood βHBA in all the treatments with G. ulmifolia, the blood βHBA values were higher than the control treatment, but no linear effect was observed.
Table 6. Blood metabolites in lambs fed with different levels of G. ulmifolia leaves
Note. 1:Dichanthium spp. hay; 2: 85% Dichanthium spp. hay, 15% G. ulmifolia leaves; 3: 70% Dichanthium spp. hay, 30% G. ulmifolia leaves; 4: 55% Dichanthium spp. hay, 45% G. ulmifolia leaves; BUN: blood ureic nitrogen; βHBA: beta-hydroxybutyric acid; SEM: standard error mean.
Source: Prepared by the authors
Discussion
The increase in DM intake in diets replacing Dichanthium spp. hay with G. ulmifolialeaves is probably favored by its high palatability (Baumont, 1996). It is also reported by Villa et al. (2009), who observed a high G. ulmifolia intake in cattle. Likewise, a relationship exists with the lower NDF content in the G. ulmifolia leaves compared with the hay used in this study (Table 1), which would decrease the physical effect of the ruminal filling (Gebremariam et al., 2006). On the other hand, Nkosi et al. (2016) describe a direct relationship between digestibility and intake, also affecting growth; therefore, it can be deduced that the CP digestibility rose, favoring the intake of OM, NDF, and CP (Table 3). The hay used in the present study had an IVDMD% of 51.51% compared to 59.61% reported by Giraldo et al. (2007) for the same species, which is evidence of the loss of quality related to age. The NDF amount in G. ulmifolia leaves against the NDF of the 90-day Dichanthium spp. hay can be linked to improved CP digestibility by replacing the hay over the G. ulmifolia leaf percentage. In that sense, Slanac et al. (2011) reported that fiber lignification decreases the ruminal degradation level. Similarly, increasing age accentuates lignification. García-Castillo et al. (2008) showed DM degradability reductions between 7% and 13% in Parmentiera edulis fruit, as its maturity stage increases.
In the in vitro experiment, different tendencies than those in vivo were noted. The in vitro CP degradability was lower in G. ulmifolia leaves compared to Dichanthium spp. hay. Also, less IVDMD occurs with the G. ulmifolia leaf level compared to in vivo digestibility. This behavior could be explained by the tannin and phenol contents in the G. ulmifolia leaves, 4.12 mEq-tannic acid/g and 6.47 mEq-tannic acid/g, respectively (Table 2). Castro et al. (2006) reported tannin and phenol concentrations of 4.7% and 2.8%, respectively, in the G. ulmifolia leaves, and Rojas et al. (2015) showed a failure in the digestibility of G. ulmifolia leaves and Ayale (Crescentia alata) fruit related to high concentrations of secondary metabolites. Recent studies defend the importance of tannins in feeding lambs because they favor the animals’ health (García- Hernández et al., 2017; Peng et al., 2016). Nevertheless, they had been previously classified as anti-nutritional factors for decreasing protein bioavailability at the ruminal level (Huisman et al., 1990). This has also been reported by Otero and Hidalgo (2004), who explain that condensed tannins decrease the proteolysis the ruminal microflora performs due to its ability to form complexes with the proteins, preventing bacterial action. According to Min et al. (2003), when a pH of less than 3.5 is reached, the nutrients can be released from these complexes, allowing their absorption at the intestinal level.
Moreover, some proteins present in saliva as proline-rich proteins and histidine-rich proteins have a high affinity for condensed and hydrolyzable tannins, interacting with them and preventing the formation of tannin diet protein complexes (Shimada, 2006). According to Alonso-Díaz et al. (2012), lambs and goats have a high salivary protein concentration and are physiologically adapted to consume plants with high tannin levels. Also, the constant rumination action can increase the salivation and release of these proteins, which could explain the in vivo protein digestibility increase contrasted with the in vitro protein degradability by increasing the replacement with G. ulmifolia leaves.
The DM degradation kinetics showed a reduction in the water-soluble fraction (a) and a potentially degradable fraction (b) when the G. ulmifolia leaf content was increased in the diets. Probably for the G. ulmifolia leaves, the bacterial ruminal colonization phase described by Mertens (1993) would depend on external factors previously described (such as salivary proteins and pH intervention), which would delay the in vitro degradation kinetics.
The positive effect on blood glucose did not overcome the typical values that Galván Doria et al. (2014) reported in Colombian-bred lambs, where the glucose was 75.57 mg/dL in females and 83.70 mg/dL in males. However, the animals were fed low nutritional value diets in the present study, so these results were expected. The glucose increment can be correlated with DM and metabolizable energy intake, while the G. Ulmifolia levels increased in the diets (Przemysław et al., 2015). On the other hand, Knaus et al. (1998) affirm that the protein with surpassing properties (such as that found in G. ulmifolia) can increase glucose as an indirect effect of the increase in glucogenic precursors.
The high protein diets tend to raise the BUN concentration (Rufino et al., 2016), and it can be inferred that the BUN increase resulted from the more significant protein contribution in G. ulmifolia leaves, adding an insufficient DM intake. Nevertheless, the values in all treatments are below the reported standard by Singleton et al. (1999). βHBA has an inverse relationship to the blood glucose level, which has been reported in both cows (Erfle et al., 1974) and sheep (Knaus et al., 1998) in negative energy balance. Nonetheless, the βHBA values of the present study tended to increase with the replacement of hay with G. ulmifolia leaves in the diet (together with the blood glucose level), but this increase was below the standard (Galván Doria et al., 2014) (which reflects that all animals had low energy values); so βHBA was not stored in the adipose tissue of the animals, being free in the blood plasma. A similar phenomenon was observed by Penner et al. (2009) by increasing the amount of concentrate in the diet of cows in a positive energy balance. Steele et al. (2012) explain that the increase of βHBA in high levels of non- structural carbohydrate diets results from greater ketogenesis by the ruminal epithelium by having an alternative source of energy, which would increase hepatic gluconeogenesis and lower use of βHBA. Despite the increase described, the βHBA body values follow the standard for lambs reported by Russel et al. (1967), which was less than 0.71 mmol/L for nutrition without energy deficit.
Conclusions
The Guácimo (G. ulmifolia) leaf supplementation in hair lambs fed with low-quality hay can improve the in vivo DM, OM, NDF, and CP intake and their digestibility. Furthermore, some blood metabolites can increase. However, the in vitro digestibility and degradability are negatively affected by replacing hay with G. ulmifolialeaves. It is suggested to carry out more research that contributes to the understanding and analysis of the vegetal species of the tropical dry forest and the role of the secondary metabolites of plants in ruminal kinetics in small ruminants. These findings demonstrate that G. ulmifolia leaves can be used safely to replace part of hay in the rations of hair lambs.
Ethical implications
The experiment was conducted based on Universidad del Tolima’s bioethical regulations for animal experimentation (Academic Council Agreement Number 0171 dated October 29, 2008) and Committee of Bioethics Minutes 02/2017 and following the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978).
