Abundance and diversity of rumen protozoa in lambs fed Gliricidia sepium silage

The objective of this study was to evaluate changes in ruminal protozoa in lambs after partial replacement of feed concentrates in their diets with Gliricidia sepium silage. Twenty-four male Santa Ines lambs with an average initial weight of 14.5 kg were used. The experimental design was completely randomized, with four treatments and six replications. Treatments (given as a percentage of dry matter) were as follows: control corn silage (600 g kg−1 as fed) + concentrate (400 g kg−1 as fed); GS133 corn silage (600 g kg−1 as fed) + G. sepium silage (133 g kg−1 as fed) + concentrate (267 g kg−1 as fed); GS267 corn silage (600 g kg−1 as fed) + G. sepium silage (267g kg−1 as fed) + concentrate (133 g kg−1 as fed); and GS400 corn silage (600 g kg−1 as fed) + G. sepium silage (400 g kg−1 as fed). Samples of rumen contents were obtained at slaughter, and analysis revealed the presence of nine genera of rumen protozoa that were present in all animals, with the exception of Enoploplastron and Eremoplastron. There were no significant differences in the average total numbers of rumen ciliates or in the composition of species between lambs. Inclusion of up to 400 g kg−1 (as fed) G. sepium silage in the diet of lambs does not affect the diversity or density of rumen protozoa.


Introduction
In tropical and arid areas, the demand for lower cost, readily available alternative protein sources in ruminant feed has stimulated experimentation with different legumes.In addition to their protein value, some legumes have secondary compounds that can potentially act as defaunating agents.Defaunating agents reduce the density of or even eliminate protozoa in the rumen and have been considered an alternative strategy for reducing ruminant methane emissions (Delgado et al., 2012;Goel and Makkar, 2012).
Gliricidia sepium, provided in the form of hay or silage, has been used as a ruminant feed in many tropical countries because it can be stored for months or even years, provides dietary protein and solves the problem of feeding ruminants in the dry season (Avilés-Nieto et al., 2013;Oduguwa et al., 2013).
Several studies have examined the effects of G. sepium on different parameters of ruminant physiology and productivity (Avilés-Nieto et al., 2013;Oduguwa et al., 2013).However, studies evaluating the effects of G. sepium on ruminal microorganisms are preliminary and contradictory (Espinosa et al., 2006;Delgado et al., 2010).Therefore, the present study evaluates the effects of replacing a portion of concentrate feed with G. sepium silage on rumen protozoa in lambs.

Material and Methods
The experiment was conducted at the Embrapa Semi-Arid Experimental Station (10°13'06"N latitude, 37°25'13"W longitude) in the state of Sergipe, Brazil.Twenty-four Santa Ines lambs with an average initial weight of 14.6±2.9kg were used.The lambs were uncastrated and approximately four months old.The animals were housed in individual pens and distributed in a completely randomized design with four treatments and six replicates, with the lamb as the experimental unit.The animals were divided into four treatments (Table 1) which were provided at 08.00 h and 16.00 h, and the amounts were adjusted daily.Animals were given between 100 g kg −1 day -1 and 150 g kg −1 day -1 scraps and received water ad libitum and mineral salt.
The animals were adapted to the experimental diets for 15 days, and the total experimental period was 87 days.Weekly samples of the provided rations were collected to determine the dry matter (DM), crude protein (CP), neutral and acid detergent fiber (NDF and ADF, respectively) contents (Table 1).Dry matter and CP were analyzed according to Silva & Queiroz (2002).The cellwall fractions of neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined as described by Van Soest et al. (1991).
At the end of the experimental period, the animals were subjected to a period of fasting for 14 hours and were subsequently slaughtered.To analyze rumen protozoa, samples of rumen contents were collected from the rumen mass center during slaughter.Each sample consisted of 50 mL of rumen contents fixed in a 6.16 mol/m³ formol (v/v) solution (Dehority, 1984).The density of rumen protozoa per milliliter was obtained by using a Sedgewick-Rafter counting chamber (Dehority, 1984) with the modifications proposed by D' Agosto and Carneiro (1999), and the protozoa present in each sample were identified based on the criteria described by Ogimoto and Imai (1981).
The total and average densities of several protozoa genera were assessed.Data were tested for normality and were log base 10 (log10)-transformed when necessary.One-way ANOVA was performed on the data using the statistical analysis program R (R Development Core Team, 2010).Significant differences were declared at P<0.05.

Results and Discussion
The genera of protozoa present (Figure 1), the average density of protozoa and the relative abundance (%) in the rumen were not affected by the replacement of concentrate with G. sepium silage (P>0.05).The exception was Enoploplastron, which tended to have the highest density in the 267 g kg −1 G. sepium silage treatment (P = 0.06) (Table 2).
A  17.28 and 20.73 × 10 4 protozoa/mL of rumen contents in supplemented and unsupplemented animals, respectively.According to Espinosa et al. (2006), the effect of defaunatory G. sepium may be dependent on the associated feed, which may lead to an increase in ruminal protozoa populations.The higher density of protozoa observed in this study compared with that reported in Espinosa et al. (2006) corroborates this information, likely because the use of corn silage in the feed composition provides sufficient nutrients to stimulate growth and maintenance of protozoa.
The effects of G. sepium and other leguminous feeds on ruminal protozoa, and consequently on rumen physiology, may be related to the nature and concentration of secondary metabolic compounds.Secondary metabolites are influenced by various factors including local environmental and cultivation characteristics, plant maturity and form of processing and supply, among others (Goel and Makkar, 2012).Thus, understanding the factors previously mentioned and the chemical properties of G. sepium and other legumes used in animal feeding can aid in the understanding of their effects on the ruminal microbiota.

Conclusions
Inclusion of up to 400 g kg −1 of G. sepium silage in the diet of lambs replacing concentrate does not affect the protozoan communities, which show significant generic diversity and a high population density.

Table 1 -
Composition of treatments and chemical composition of treatments as a percentage of dry matter