Effect of sugarcane fiber digestibility, conservation method and concentrate level on the ruminal ecosystem of beef cattle

Feedlot diets have high inclusion of concentrate and low roughage, usually resulting in low ruminal pH that directly affects the digestibility of the fiber, and may also lead to metabolic disorders (Anderson et al. 2016). Fibrobacter succinogenes, Ruminococcus flavefaciens and Ruminococcus albus are considered the main bacteria responsible for roughage fiber degradation (Forsberg et al. 1997), and the proliferation of these microorganisms in the rumen is directly correlated with the quality and quantity of dietary fiber. Replacing fiber with non-fibrous carbohydrates (NFC), such as starch and sugars, influences the development of cellulolytic microorganisms, changing the dynamics of fermentation in the ruminal ecosystem (Tajima et al. 2001).

As the concentration of fermentable carbohydrates in the rumen increases, microbial growth is stimulated, increasing ruminal fermentation rate. On the other hand, increasing the digestibility of carbohydrates in the rumen results in pH to drop below 6.0 due to the greater production of short-chain fatty acids (SCFA). In this situation, there is a reduction in the activity of fibrolytic bacteria, affecting the digestibility of fiber, while there is increased activity of amylolytic bacteria, such as Streptococcus bovis and Ruminococcus amylophilus, and lactate users, as Megasphaera elsdenii (Tajima et al. 2001; Nagaraja and Titgemeyer 2007; Petri et al. 2012). The excess of ruminal fermentation of NFC, combined with low inclusion of roughage in the diet, can lead to acidosis, decreasing fiber digestibility, dry matter intake and, consequently, causing a reduction in weight gain of animals (Gonzalez et al. 2012).

Therefore, one alternative would be to use better quality roughages, reducing the diet concentrate content and favoring the proper development of cellulolytic microorganisms. Inclusion of high quality forage in the diet is positively related to growth of cellulolytic bacteria populations in the rumen (Fernando et al. 2010; Koike and Kobayashi 2001). Among the roughages, sugarcane is widely used as forage in diets for ruminants, either as freshly cut or ensiled (Freitas et al. 2006). However, beside low protein content, poor fiber digestibility is the main limitation of animal performance in sugarcane-based diets (Pereira et al. 2001). Moreover, sugarcane has high sucrose content, and despite the importance of fiber digestibility for animal performance, the sugarcane breeding programs are focused on total production of sucrose, with little attempt to evaluate quality parameters for animal nutrition. Forage breeding demonstrates that small increases in dry matter (DM) digestibility result in significant improvement in animal performance (Casler 2001).

In this context, it is important to understand how neutral detergent fiber digestibility (NDFD) of sugarcane genotypes, the level of concentrate, and the method of conservation affects the population dynamics of ruminal microorganisms. The ensiling process changes the chemical composition of sugarcane, increasing fiber and reducing the sucrose content (Kung and Stanley 1982; Daniel et al. 2013), altering the ruminal fermentation (Hungate 1966; Vallimont et al. 2004; Singh et al. 2014). These factors directly affect the mechanisms involved in fiber degradation by ruminal microorganism. Therefore, the objectives of this study were to evaluate the effect of NDFD and conservation method of sugarcane, and the concentrate level in the diet on the population of ruminal bacteria of cannulated beef cattle.

All experimental procedures were in agreement with the Guide for Care and Use of Agricultural Animals in Agricultural Research and Teaching (FASS 1999), with all animal procedures approved by the University of São Paulo Animal Bioethics Committee (Protocol number 7632051113).

For this experiment, samples of rumen contents coming from two previous experiments were used (Mesquita 2013; Sousa et al. 2014), which presented the results of performance and ruminal kinetics. The experiments were conducted at the College of Veterinary Medicine and Animal Science, University of São Paulo Beef Cattle Research Laboratory (Pirassununga, SP, Brazil). In both developed experiments, rumen contents samples were collected from cannulated animals for quantification on the ruminal microorganisms of interest.

Forage quality is an important factor regulating intake and efficiency of diet utilization, as well as in reducing the amount of concentrate in the diet of ruminants (Tafaj et al. 2005). Sugarcane has a particular physiological maturation process, as with the advance of maturity there is a decrease in fiber digestibility and in CP content. However, because of sucrose accumulation, there is an increase in NFC content leading to increase the total DM digestibility as maturity progress (Kung and Stanley 1982; Carvalho et al. 2010). However, the effect of sugar, like sucrose, on ruminal microorganisms has not been extensively studied (Sun et al. 2015). Therefore, low fiber digestibility is usually the main limiting factor for high performance beef cattle fed with sugarcane based diets, as NDF digestibility in sugarcane can be about half of that of corn silage (Corrêa et al. 2003).

The proportion of high quality forage in the diet exerts a positive influence on growth of cellulolytic bacteria in the rumen (Fernando et al. 2010), increasing intake and, consequently, animal performance. Thus, we hypothesized that modulation of animal performance by diet could be explained by modulation of the population of ruminal microorganisms. In the present study, the use of a sugarcane genotype with high-NDF digestibility favored growth of fibrolytic bacteria in the rumen, likely by substrate availability and maintenance of optimum rumen pH. To our knowledge, no other study evaluated the change in ruminal bacteria population to differing fiber digestibility of the roughage.

It is important to consider that in the present study, both liquid and solid fractions were analyzed together, which could potently mask important population changes happening in each of the fractions separately. Also, the method of DNA extraction can also influence the results, although the variation between DNA extraction methods is usually smaller than the variation caused by changes in diet (Henderson et al. 2013).

In the present study, the two different sugarcane genotypes had different chemical composition. The lignin and indigestible FDN contents were lower for the high-NDFD genotype, with greater DM and NDF digestibility (Sousa et al. 2014; Mesquita 2013). In the first study, the effect of greater NDF digestibility of sugarcane on F. succinogenes population was dependent on the method of conservation. F. succinogenes was increased with high-NDF digestibility, when sugarcane was offered as freshly cut. The R. albus population followed a similar pattern, although not significant (P = 0.12). The silage process alters the nutritional quality of roughages, where part of the NFC is consumed, and causing increase in the NDF and lignin concentration (Kung and Stanley 1982). Mode of conservation changed the chemical composition of sugarcane, with greater lignin and iNDF in the ensiled sugarcane, while DM and NDF digestibility were greater for the freshly-cut sugarcane. Therefore, when sugarcane is offered as freshly cut there is more soluble sugar arriving in the rumen environment. Consequently, mode of conservation influenced rumen pH, with greater rumen pH when sugarcane was offered as silage than as freshly cut (6.69 vs. 6.37 ± 0.08; P < 0.01). There was no effect of sugarcane genotype on rumen pH levels (Sousa et al. 2014). According to Tafaj et al. (2005), a moderate quantity of NFC in the diet can stimulate fiber digestibility because of better supply of fermentable organic matter, nitrogen, and energy for the ruminal bacteria.

Among the cellulolytic bacteria present in the rumen, F. succinogenes, R. flavefaciens, and R. albus are considered the main species responsible for fiber degradation (Varel and Dehority 1989). Therefore, proliferation of cellulolytic bacteria in the rumen would be correlated with the amount of digestible fiber in the diet, and the substitution of fiber for soluble carbohydrates would influence its growth and alter the dynamic of the rumen ecosystem (Tajima et al. 2001). In the second experiment, the effect of high-NDF digestibility on the population of these three species of fibrolytic bacteria was dependent on the level of concentrate in the diet; where the population of F. succinogenes and R. albus responded to high-NDF digestibility only at the lower level of concentrate inclusion. However, R. flavefaciens population responded to high-NDFD only in the diet with 80% concentrate. In study 2, rumen pH was influenced only by concentrate level, where the 60% concentrate diet had greater mean rumen pH than the 80% concentrate diet (6.38 vs. 6.12 ± 0.11, P < 0.05, Mesquita 2013). Although the three studied species F. succinogenes, R. flavefaciens, and R. albus are considered important rumen cellulolytic bacteria, their mechanism of action, ability to adhere to particles and enzymatic profiles are different (Mosoni et al. 1997). When cultured with excess cellulose, the number of cells for these three species was similar (Shi et al. 1997). However, when cultured with limited amount of cellulose, R. flavefaciens predominated over the other two, demonstrating the superior adhesion capacity for this microorganism (Shi et al. 1997). In the context of study 2, diets with the high-NDFD sugarcane genotype increased the total intake, NDF rumen passage rate and body growth of the animals, but only in the diet with 80% concentrate (Mesquita 2013). Also, the diet with 80% concentrate reduced rumen ammonia levels (11.66 vs. 7.18 mg/dL for the 60 and 80% concentrate diets, respectively—Mesquita 2013).

With the increment of fermentable carbohydrates in the rumen, there is an overall stimulus for microbial fermentation. On the other hand, rapidly fermentable carbohydrates and the accumulation of SCFA in the rumen forces a decline in ruminal pH, usually for values bellow 6.0. In these situations, there is a reduction in the activity of fibrolytic bacteria, hindering fiber digestibility (Weimer 1996; Owens et al. 1998; Russell and Rychlik 2001) while simultaneously stimulating amylolitic and lactate utilizing-bacteria (Tajima et al. 2001; Nagaraja and Titgemeyer 2007). Besides direct inhibition of cellulolytic bacteria with low ruminal pH, there is also a decline in attachment of bacteria to the substrate, caused by the lack of positive effectors, such as the ion bicarbonate, and by the excess of attachment inhibitors, such as soluble starch (Owens and Goetsch 1993).

Petri et al. (2012) observed that great inclusion of concentrate in the diet, in substitution for the roughage, reduces the populations of the fibrolytic bacteria F. succinogenes, R. flavefaciens, and R. albus. The substitution of roughage for concentrate, rich in rapidly fermentable carbohydrates, promoted a decline in particle size of the diet, with less physically effective NDF, reducing salivation and rumen motility and, consequently, rumen buffering. Several other studies demonstrate the reduction in cellulolytic bacteria due to the increase in concentrate in the diet (Tajima et al. 2001; Singh et al. 2014; Granja-Salcedo et al. 2016).

Different from the fibrolytic bacteria, the amilolytic bacteria (represented in this study by R. amylophilus and S. bovis) have preference for NFC and are more tolerant to low pH. In the present study, there was an increase in the population of R. amylophilus with greater concentrate inclusion in the diet, reflecting the increase in starch and total NFC in the diet (Schwartzkopf-Genswein et al. 2003; Petri et al. 2012). However, the S. bovis population was reduced with greater concentrate in the diet, and was increased with freshly cut sugarcane compared to sugarcane conserved as silage. It is important to highlight that the roughage used in this study was sugarcane, with high level of soluble sugars (mainly sucrose). Also, sugarcane as silage has lower sugar content than freshly cut sugarcane. Therefore, in the present study, the treatments that provided more soluble sugar and less starch to the rumen (lower concentrate inclusion and freshly cut sugarcane) favored growth of S. bovis. Hence it can be suggested that S. bovis prefer NFC sources other than starch, such as sucrose.

Supporting this hypothesis, Golder et al. (2014) reported that S. bovis became more prevalent in heifers fed with fructose than with starch. Moreover, the relative abundance of the Streptococcaceae and Veillonellaceae families was increased when heifers received fructose. Other studies found similar results, where the population of S. bovis was not increased with the increment of concentrate in the diet of Nellore steers, using corn silage as the roughage source (Granja-Salcedo et al. 2016) or freshly-cut sugarcane (Ribeiro Junior et al. 2016). Sun et al. (2015) report an increase of S. bovis population by replacing cornstarch in a high-concentrate diet (forage to concentrate ratio = 40:60) with 3% sucrose after 24 h in vitro incubation.

The ruminal bacteria S. bovis is a facultative anaerobe and tolerant to low pH, and is known to be prevalent during lactic acidosis, proliferating in the rumen of cattle fed high levels of concentrate (Owens et al. 1998; Russell and Hino 1985; Russell and Rychlik 2001). However, the fast growth of S. bovis was not observed in animals adapted to starch rich diets, but in animals with lactic acidosis (Nagaraja and Titgemeyer 2007). Fernando et al. (2010) reported that the population of S. bovis increased at the beginning of the adaptation regimen to high grain diet, whereas at the end of the step-up adaptation, the S. bovis population decreased, and did not show a significant change in population size compared to control, where animals received only hay during the adaptation phase.

Therefore, whether the animals are adapted or not to a high-grain diet seems to influence the effect of addition of grains in the S. bovis population in the rumen. The abrupt change in the diet in non-adapted animals favors the rapid growth of S. bovis, lactate and SCFA accumulation, decline in rumen pH, and metabolic disorders (Russell and Hino 1985; Hernandez et al. 2014). In contrast, when animals are adapted to high-grain diets, the S. bovis population can be controlled favoring the growth of lactate utilizers, such as M. elsdenii and Selenomonas ruminantium, and the maintenance of rumen pH (Fernando et al. 2010). Hence, the importance of removal of fermentation acids, specially lactate, so as not to cause the decline in rumen pH.

Some microorganism present in the rumen and tolerant to low pH can utilize lactate, therefore avoiding lactate accumulation in the rumen environment. The lactate utilizing bacteria measured in the present study was M. elsdenii, which population increased when sugarcane was offered as freshly cut as opposed to sugarcane silage. This microorganism is able to grow on sucrose or glucose, although from these substrates the end product is butyrate, not propionate which comes from lactate (Hino et al. 1994). In the study 2, there was greater proportion of butyric acid in the rumen of animals receiving diets with 60% concentrate in comparison to 80% of concentrate (15.67 vs. 13.28% of total SCFA, P = 0.01—Mesquita 2013), and this can be explained by the action of M. elsdenii. This increase in molar proportion of butyrate in response to the partial substitution of starch with sugar was consistent to previous studies (Vallimont et al. 2004; Sun et al. 2015).

Khafipour et al. (2009) reported that S. bovis was the prevalent species during severe acidosis and that M. elsdenii was the dominant species during mild acidosis caused by excess of grain in the diet. Also, strains of M. elsdenii has been used to reduce lactate accumulation and increases rumen pH, consequently preventing ruminal acidosis (Long et al. 2014). In the present study, the diet with fresh sugarcane provided more sucrose to the rumen, favoring rumen fermentation and more acid accumulation, including lactate. The transient increase in lactate would favor growth of M. elsdenii.

It is clear that NFC supply to the rumen alter the fermentation profile and therefore the ruminal microorganism populations. This fact becomes evident when the different sampling times are considered. When rumen samples were collected shortly after feeding, when there is a great supply of NFC to the rumen, there was a reduction in the population of F. succinogenes and an increase in the populations of R. amylophilus and M. elsdenii. Similar results have been reported in ovine by Mosoni et al. (2007), with a reduction in the populations of fibrolytic bacteria (F. succinogenes, R. albus, and R. flavefaciens) when the rumen was sampled 3 h after feeding. The population of another lactate utilizing bacteria, S. ruminantium, was also increased 2 h after feeding (Singh et al. 2014). The rumen is a complex and dynamic environment where the microorganisms must constantly adapt to changes in diet composition, amount and frequency of feeding.

Therefore, the supply of sugarcane with greater NDFD favored the growth of fibrolytic bacteria, but this effect was dependent on the conservation method and the concentrate level. The genotype with higher NDFD favored the growth of F. succinogenes only when sugarcane was offered as freshly cut. Furthermore, on a diet with less addition of concentrate, the population of fibrolytic bacteria F. succinogenes and R. albus increased in response to increasing NDFD. When sugarcane was offered as freshly cut, the population of S. bovis and M. elsdenii were increased in response to greater supply of readily fermentable sugars. In addition, the increase of dietary concentrate increased the population of R. amylophilus and reduced F. succinogenes, R. albus and S. bovis, highlighting the hypothesis that S. bovis has a higher affinity for sugar (Additional file 1) than the for starch itself.