Use of Saccharomyces cerevisiae -based products and effects on rumen environment and performance of sheep subjected to dietary changes

: Natural additives in ruminant diet optimizes animal performance by controlling or modifying the rumen fermentation pattern. The aim of this study was to evaluate the effects of using Saccharomyces cerevisiae products during abrupt changes in dietary starch concentration on ruminal pH maintenance, performance and metabolism of confined sheep. Twenty adult ewes, crossbred Texel x Corriedale(44.7±6.97 Kg BW) were divided into three groups: Control (n=6), Cultron X (yeast culture; n=7) and Cultron Pro (hydrolyzed yeast; n=7), administered at a dose of 5 g/sheep/day (10x10 10 CFU/g DM). The experiment lasted 20 days divided into four periods, alternating between 40 and 60% concentrate. Blood and ruminal fluid were collected at the end of each period, and daily dry matter intake was measured to determine the average daily gain (ADG). Blood tests included blood count and biochemical parameters such as glucose, urea, total plasma proteins (TPP), liver enzymes, acute phase proteins and minerals. There was an upward trend in ADG in group Cultron X compared to control in period 3. Although, ruminal pH did not differ between groups, protozoan motility was higher in animals supplemented with hydrolyzed yeast. The Cultron Pro group showed an increase in TPP in periods 3 and 4, and AST in periods 2 and 3 compared to the control group. There was no difference in other metabolites. Yeast supplementation is a potential tool in times of feeding challenge, in which yeast culture is the most effective for improving performance of confined sheep being in improving performance of confined sheep.


INTRODUCTION
Intensification of animal production seeking to maximize the performance of ruminants is closely linked to the increase in the use of foods with high energy content in the diet for these animals (ALVES et al., 2003;YANG et al., 2012). However, the use of this tool can develop changes in the rumen environment, making the animal more susceptible to the development of metabolic disorders (RUSSEL & RYCHLIK, 2001;MCCANN et al., 2016).
Due to the importance of the microbial population in the feeding efficiency of ruminants, the use of natural additives in the diet is a practice that optimizes the performance of animals, mainly those that act in the rumen, controlling or modifying the rumen fermentation pattern (WALLACE & NEWBOLD, 1993;ENJALBERT et al., 1999). Among the additives used, supplementation with Saccharomyces cerevisiae strains has stood out in modulating the rumen environment and improving the health and performance of animals (ÖZTÜRK et al., 2015;CHAUCHEYRAS-DURAND et al., 2008).
There are different types of yeast for use in ruminant nutrition and, among them, hydrolyzed additives. They are composed of β-glucans and mannan oligosaccharides (MOS), main components of the cell wall of yeast cells (NOCEK et al., 2011), which act as immunomodulators (FRANKLIN et al., 2005). Glucans are stimulators of macrophages, increasing the response to pathogens (MORAN et al., 2004), while MOS act as high-affinity ligands for pathogenic bacteria that affect the gastrointestinal tract of ruminants, inhibiting local colonization (BOUDERGUE et al., 2009;OFEK et al., 1977). Active yeasts, in turn, are characterized by a high concentration of viable cells (above 10 billion CFU/g), while yeast culture consists of a fermentation product containing cell wall, live cells, among a series of compounds, such as vitamins and amino acids, which provide an ideal growth medium for yeasts (NOCEK et al., 2011).
To date, there are few studies investigating ruminal modulation strategies in times of abrupt changes in diet, as well as the use of supplementation in order to reduce potential negative impacts of these changes. In this sense, the hypothesis of this research was that animals supplemented with yeast present pH stabilization and consequent modulation of the rumen environment when diet was changed, with positive effects on performance and metabolism. Thus, the objective of the study was to evaluate the effects of using two products based on Saccharomyces cerevisiae in periods of sudden change in the amount of concentrate in the diet on the maintenance of ruminal pH, performance and metabolism of confined sheep.

Animals and experimental groups
Twenty non-lactating crossbred ewes, Texel x Corriedale, aged between 24 and 36 months, average weight of 44.7 ± 6.97, were confined throughout the experimental period and assigned to pens of two and three animals, three pens for each treatment. The animals were divided into three groups according to body weight: Control Group (no supplementation; n = 6), Cultron X ® Group (Aleris, São Paulo, Brazil), supplemented with Saccharomyces cerevisiae fermentation products containing live yeast in its formulation (2 x 10 10 CFU/g DM) (n = 7) and Cultron Pro ® Group (Aleris, São Paulo, Brazil), supplemented with a product based on enzymatically hydrolyzed yeast (n = 7). Ewes belonging to the treatment groups received 5 g of the products daily, supplied in the feeders before morning meal.

Experimental design and diets
The experimental period was a period of 20 days, characterized by changes in the proportion of diet components every 5 days, alternating the amount of forage and concentrate, without prior adaptation. The study was divided into four experimental periods without prior adaptation, namely: P1, referring to 60% forage; P2, referring to 60% concentrate; P3, referring to 60% forage and P4, referring to 60% concentrate. The supplemented animals were subjected to a previous adaptation for 20 days, with the appropriate dose of each product. The sheep had individual feeders; the total diet was homogenized manually and offered twice a day, in the morning at 8:30 am and in the afternoon at 4:30 pm, consisting of corn silage, wheat meal, with water ad libitum. The diet was calculated at a proportion of 3% body weight DM. The nutritional composition of the ingredients is listed in table 1.

Blood samples and hematological and biochemical analyses
Blood was collected every five days (days 5, 10, 15 and 20) by puncturing the jugular vein into a vacutainer system, before the morning meal at the end of each experimental period, totaling five collections per animal. The blood was drawn into three separate tubes, one containing 10% EDTA for complete blood count, one with sodium fluoride for serum glucose analysis, and a third with clot activator used for biochemical analysis. After collection, the samples containing 10% EDTA were properly homogenized and analyzed in a hematology analyzer (BC Vet 2800 Mindray), for erythrogram and leukogram, and used for blood smears for leukocyte differentiation.
Blood samples for biochemical analyses were centrifuged at 3,000 rpm for 15 minutes, and the serum aliquots were poured into Eppendorf tubes and frozen. Measurements were performed for glucose, urea and total plasma protein (TPP) to assess energyprotein metabolism; gammaglutamyltransferase (GGT) and aspartate aminotransferase (AST) enzymes to evaluate liver function; acute phase proteins albumin, fibrinogen and paraoxonase (PON1), in addition to the minerals calcium (Ca), magnesium (Mg), sodium (Na) and potassium (K). The analyses were performed using a LabmaxPlenno ® automated analyzer (Labtest, Minas Gerais, Brazil) and the PON1 activity was quantified by the rate of phenol extinction at 270 nm in serum samples, as described by Pradiee et al. (2017).

Ruminal fluid collection
Before the experimental period, a pilot experiment was conducted, in which it was decided to collect ruminal fluid two hours after feeding, taking into account the population results obtained. Thus, the fluid was collected on the days referring to blood collections, two hours after feeding, through an orogastric tube. The collected liquid was filtered through 4 layers of gauze, avoiding contamination of the sample with saliva, as described by DEHORITY (1984). Upon collection, ruminal pH was immediately measured with a portable digital pH meter (Hanna ® , Brazil), then the physical aspects of the fluid, such as color, odor and consistency, were evaluated. The color of the liquid was classified as olive green (1), pure green (2), yellowish brown (3), milky gray (4) and blackish green (5), while the grades for odor assessment were considered aromatic (1) , acid (2), putrid (3), ammoniacal (4) and odorless (5). Liquid consistency was classified into different degrees of viscosity, including watery, slightly viscous and viscous (grades 1, 2 and 3, respectively). The organoleptic evaluations were adapted from DIRKSEN (1993). Afterwards, the analyses were performed, starting with the visualization of ruminal protozoa motility under an optical microscope according to DIRKSEN (1993). Bacterial activity was indicated by the methylene blue reduction test and the sedimentation and fluctuation analysis were performed according to the methodology described by ROSENBERGER (1993).

Performance analysis
All animals were weighed on the days referring to collections, every five days, on a digital scale (Tru-Test ® , Brazil), before the morning meal. Thus, it was possible to determine the average daily gain (ADG) of the animals in each of the experimental periods. The total diet and leftovers were weighed twice a day, in order to measure the dry matter intake (DMI) in each period.

Chemical analysis
Samples of Total Mixed Ration (TMR) and leftover in the troughs were collected. The samples were analyzed for dry matter (DM), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (FDA) and ashes, according to SILVA & QUEIROZ (2002). Parameters of dry matter digestibility (DMD) and total digestible nutrients (TDN) were determined according to the following Table 1 -Nutritional composition of the total diet used during the experimental period.

Statistical analysis
The experimental design was completely randomized, in which pens were the experimental units. Statistical analyses were run using the SAS statistical program (SAS Institute Inc., Cary, USA, 2016). The mean values were analyzed using the MIXED MODELS method, considering the group fixed effects (Control, Cultron Pro and Cultron X), the time of collection and their interactions, and the random effect of animal. Data were analyzed according to the following model: Y ijk = µ + T i + P j + S k + TP ij + e ijk where Y ijk is the dependent variable, µ is the overall mean, T i is the fixed effect of treatment (i = Control, Cultron Pro and Cultron X), P j is the fixed effect of period (j = 1; 2; 3; 4), S k is the random effect of animal (k = 1 to 20), TP ij is the interaction between treatment and period, and e ijk is the experimental error. Means were compared by Tukey-Kramer test. Effects of treatments were significant when P-values <0.05 and trends were discussed when 0.05<P<0.10. The results are presented as mean ± standard error of the mean.

RESULTS
Ruminal pH values did not differ between groups and experimental periods, as illustrated in figure 1. All animals presented pH values between 6.0 and 7.0, considered a physiological value for the species (FEITOSA, 2008). It was possible to observe an increasing trend in ruminal pH values in period 4 in relation to period 3 (6.28 x 6.13, respectively) (P=0.06) in all experimental groups.
The ruminal fluid showed two distinct colors, olive green and brownish yellow. The difference was detected only in the Cultron X group in period 1, with 85.71% animals showing olive green fluid and 14.29%, brownish yellow. The aromatic odor observed in all groups during the experiment indicated that the animals in the study had conditions compatible with normal physiological parameters (GONZÁLEZ et al., 2000). Regarding the consistency of the fluid, during the entire experimental period, 42.8% animals in the Cultron Pro group had ideal viscosity, compared to 39.3% in the Cultron X group, and 33.3% in the control group. In period 2, there was a high number of animals with ruminal liquid with watery consistency in the three treatments, with the animals in the control group showing 100% abnormal consistency, followed by 85% in the Cultron X group, and 71% in the Cultron Pro group. With respect to motility of ruminal protozoa, there was a significant difference between the groups (P <0.001). The animals in the control group had higher cases of reduced motility (20.8%) compared to the Cultron X (3.57%) and Cultron Pro (0%) groups. The Cultron Pro group presented superior results (P<0.05) compared to the others, with 64.3% animals showed excellent motility of protozoa, compared to 39.3% and 20.8% in the Cultron X and control groups, respectively.
In the present study, there was no effect of supplementation on the fermentation activity of bacteria, all groups were within the normal range. Results of pH, PRAM and TSF are listed in tables 2 and 3.
As for the zootechnical parameters analyzed, supplementation did not influence the DMI of the animals, with a significant difference only between the Cultron Pro and Cultron X groups (P=0.04).
Differences in ADG were detected between periods in all groups (P <0.001), especially in period 2 compared to the others. The group supplemented with Cultron X showed a more intense reduction in this second period; however, there was a notable increase in this group in period 4 compared to period 2 (-0.6 x 0.4 ± 0.09 kg, respectively) (P=0.003), characterized by the same diet. In period 3, there was an upward trend in the Cultron X group compared to the control group (P=0.1). The means of DMI and ADG are presented in tables 2 and 3.
In relation to the energy metabolism, there was a difference in serum glucose levels between the control and Cultron X groups (53.7 x 68.0 ± 2.0 mg/ dL) and between control and Cultron Pro (56.7 x 68.0 ± 2.0 mg/dL) in period 1 (P=0.02) ( Figure 2).
As for protein metabolism, there was a significant difference in the concentration of total plasma proteins between treatments, with a noticeable increase in the Cultron Pro group compared to the control group in period 3 (6.64 x 7.26 ± 1.18 g/ dL; P=0.02) and in period 4 (7.07 x 6.67 ± 0.09 g/ dL; P=0.01), indicating increased concentrations in animals supplemented with hydrolyzed yeast.    In this study, TPP concentrations were higher in the Cultron Pro group and this increase may be related to the increase in protein content in the diet, since this product contains hydrolyzed yeast,     which provides a widely available protein source, in addition to favoring stimulatory factors for rumen microorganisms, increasing their concentration in the rumen environment and thus increasing microbial protein (MAO et al., 2013). In addition, these proteins play a key role in immunity, as indicators of infectious processes (GONZÁLEZ et al., 2000). Hydrolyzed yeast, conversely, contains β-glucans and mannan oligosaccharides (MOS) in the cell wall, which basically act in the increase of function phagocytic cells, mainly macrophages, and can stimulate the response to antigens (CZECH et al., 2018), showing that the results of TPP associated with yeast components are configured as having great potential to increase quality and quantity of the immune response.
There was a difference in serum urea levels between experimental periods (P<0.001), with a significant increase in the first challenge with a higher proportion of concentrate in the diet. A difference was detected between the Cultron X group and the control group (P = 0.03), with higher values in the supplemented group (36.39 x 32.16±1.94 mg/dL).
The Cultron Pro group showed higher levels of the AST enzyme compared to the control group in periods 2 and 3 (P=0.002), but the levels of the GGT enzyme were not altered by supplementation (P>0.05). The other metabolic markers showed no significant difference between experimental groups, as listed in tables 2 and 3.

DISCUSSION
The rumen pH is one of the variables that directly interfere with the microbial population and the activity of protozoa (DAYANI et al., 2007). The pH of ruminal fluid can vary between 5.5 and 6.2 in diets with a higher level of concentrate, while in diets with a greater participation of forage the values are between 6.2 and 7.0 (ODENYO et al., 1997). In this case, the pH fluctuations were observed in small proportions, keeping within the physiological values for the species, which points out the maintenance of the rumen environment and a balance of microbial activity.
According to FEITOSA (2008), the fluid consistency should be slightly viscous, indicating the presence of supernatant particles of nutrients and microorganisms in an adequate amount.
The high number of animals with ruminal fluid of abnormal consistency in the control group may be related to microbial inactivity, demonstrating that animals supplemented with yeast culture or hydrolyzed yeast may have less negative consequences Tavares et al.
in the rumen environment when challenged with diets with higher starch content. Protozoa play an important role in controlling the ruminal fluid pH, being able to easily engulf starch granules. These microorganisms are responsible for up to 45% amylolytic activity in the rumen, significantly increasing their population in animals supplemented with high-concentrate diets (BORGES et al., 2002). In the present study, a greater number of cases with active protozoan motility were identified in the first challenge period with a more concentrated diet, corroborating HUHTANEN (1992), who reported a greater number of protozoa when there was a replacement of fiber with grain in the diet. This result is because these microorganisms digest starch more slowly than bacteria, indirectly helping to control pH and leading to a decrease in the number of amylolytic bacteria through competition for the substrate, also participating in the fermentation of lactate present in the rumen.
When comparing the supplemented groups to the control group, it is possible to observe that both supplemented groups had a greater number of animals with good to excellent protozoan motility, which demonstrates the effectiveness of supplementation in modulating the fermentation, directly reflecting on the activity of ruminal protozoa.
The methylene blue reduction test (PRAM) reflects the anaerobic fermentative metabolism of the microbial population (FEITOSA, 2008). This author stated that the reduction time of up to 3 minutes is the result of an active microbiota, which can be reduced in a highly concentrated diet, which did not occur in the present study, since, regardless of the energy content of the diet, all groups remained within the physiological values.
The methylene blue reduction test (PRAM) reflected the anaerobic fermentative metabolism of the microbial population (FEITOSA, 2008). This author stated that the reduction time of up to 3 minutes is the result of an active microbiota, which can be reduced with a high-concentrate diet, which did not occur in the present study, since, regardless of the diet energy content, all groups remained within the physiological values.
Although, supplementation did not influence DMI, animals supplemented with yeast culture showed better feeding efficiency compared to the control group, in period 3. This corroborated DING et al. (2008), in which sheep supplemented with live yeast at a dose of 10x10 9 CFU/day showed 7.3% improvement in feed efficiency compared to the control group, but without influence on DMI. In contrast, STELLA et al. (2007), evaluating a diet with 47% concentrate DM, and ABD EL-GHANI (2004), with 60% concentrate DM, observed an increase in the DMI of these animals, resulting in an increase in milk production in goats. DING et al. (2008) evaluated the digestibility of hemicellulose, which increased from 55.7% to 62.4% in diets supplemented with live yeast, which, according to SALINAS-CHAVIRA et al. (2017), would have a beneficial effect on the regulation of DMI as a result of greater fiber degradation, and microbial protein synthesis in the rumen. In a study by DIAS et al. (2018), DMI values were similar between the non-supplemented group receiving a low starch diet and the group supplemented with yeast fed a more energetic diet, which makes it possible to verify that yeast has a beneficial effect on the rumen environment, minimizing the effects from the concentrates supply.
Another study conducted by DIAS et al. (2018) supports this statement, demonstrating that supplementation increased fiber digestibility in dairy cows fed a high starch diet, which could positively influence DMI rates. This is not consistent with the results obtained in the present study, in which a reduction in DMI was reported in periods with a higher proportion of concentrate in the diet, corroborating DESNOYERS et al. (2009), who stated that supplementation with S. cerevisiae increases the digestibility of organic matter with beneficial results in more fibrous diets compared to energy diets.
The difference in ADG values of the Cultron X group in periods 2 and 4 can be explained by the better adaptation at the rumen level of animals supplemented with yeast culture, since the abrupt change in feeding without prior adaptation does not guarantee an appropriate digestion and ruminal fermentation (FARENZENA et al., 2016), indicating that these individuals go through feeding challenge periods less severely.
According to WALLACE & NEWBOLD (1992), the effects of yeast in the rumen trigger a modulation in the microbial population, contributing to improve feeding efficiency in dairy cows, which is supported by ZHU et al. (2017), who identified a significant increase in the number of cellulolytic bacteria and a reduction in lactateproducing species in response to supplementation. HADDAD & GOUSSOUS (2005) stated that supplementation with yeast culture has a positive effect on nutrient digestibility, resulting in greater ADG and higher feeding efficiency in sheep fed a diet with 80% concentrate.
The increase in serum glucose levels was expected due to the greater energy supply, as reported by DIAS et al. (2018). According to the authors, diets with high-concentrate stimulated the concentrations of propionate in the rumen, which favors gluconeogenesis and increases glucose levels.
According to KHALID et al. (2011), the lower concentration of plasma glucose in animals receiving supplementation can be ascribed to greater fiber digestion as a consequence of the modulation in the rumen environment provided by the action of yeast. This statement can be seen in the study by YUAN et al. (2015), who identified that supplementation with two different doses of yeast tended to reduce blood glucose levels in cows. Contrary to the results obtained, ABO EL-NOR & KHOLIF (1998) reported a higher level of glucose in supplemented cows. However, HRISTOV et al. (2010) andOLAGARAY et al. (2019) reported no effect of supplementation on plasma glucose levels in dairy cows. It is worth mentioning that the control group has already started the experimental period with high levels of glucose, thereby maintaining during periods with low energy intake. This demonstrated that, probably, supplemented animals may have an improved starch metabolization efficiency in cases of greater energy supply, since at that time, the glucose levels between groups were similar, even with the control group starting from higher glycemic index.
Total plasma proteins (TPP) are produced in the liver and their synthesis is related to the protein content of the diet and the nutritional status of the animal (GONZÁLEZ & SCHEFFER, 2003). In this study, TPP levels were higher in the Cultron Pro group and this increase may be related to the increase in protein content in the diet, since this product contains hydrolyzed yeast, which provides a highly available protein source, in addition to favoring stimulatory factors for rumen microorganisms, increasing their concentration in the rumen and thus increasing microbial protein (MAO et al., 2013).
Further, these proteins play an important role in immunity, as indicators of infectious processes (GONZÁLEZ et al., 2000). In turn, hydrolyzed yeast contains β-glucans and mannan oligosaccharides (MOS) in the cell wall, which basically act in the increase of phagocytic cells, mainly macrophages, and can stimulate the response to antigens (CZECH et al., 2018), showing that the TPP results associated with yeast components are configured as having great potential to increase the quality and quantity of the immune response.
Urea is a metabolite produced by the liver by recycling rumen ammonia from the fermentation of nitrogen ingested in the diet, and part of it is metabolized in the liver and can be excreted via urine or return to the rumen via saliva or bloodstream (BERCHIELLI et al., 2011). In the present study, the concentrations of this metabolite in the first challenge were higher than the reference values set by GONZÁLEZ et al. (2000). This may suggest that the high levels may be related to the protein content of the diet, since there was an increase in the percentage of crude protein when switching the diet based on forage to a higher proportion of concentrate.
An increase in the level of AST enzyme was observed after the period with the highest energy supply, which remained until the end of the following period. The increase of this metabolite in animals fed a diet rich in energy was also reported by SCHWEGLER et al. (2014), demonstrating that there was some liver overload in both periods. In addition, the authors observed that the increase in AST levels was maintained even after a period of recovery from induction of acidosis. TABELEÃO et al. (2008) identified that the inclusion of yeast-based additives in a balanced diet promotes changes in liver function, which were evidenced by the increased activity of GGT enzyme during the adaptation period, which did not occur in the present study, but demonstrates that these products may be related to the increase in the activity of hepatocytes. NASROLLAHI et al. (2019) andXU et al. (2016) reported an increase in the serum activity of AST in cows with lower ruminal pH, indicating that there is a relationship between the drop in pH and the liver efficiency of these animals, partially explaining the results reported in the present study. Still, NASROLLAHI et al. (2019) suggested that AST serum activity may be a possible indicator of susceptibility to low ruminal pH values.
Additives containing viable cells tend to modulate the rumen environment in a more direct way (CHAUCHEYRAS-DURAND et al., 2008), which is closely related to health; and consequently, to liver function. Thus, the use of hydrolyzed yeasts (absence of viable cells) may not act directly on rumen maintenance, reflecting changes in liver metabolism due to changes in the rumen fermentation pattern, especially during feeding challenge, corroborating the results reported at both liver and rumen levels in the present study.

CONCLUSION
Saccharomyces cerevisiae yeast-based products are a potential tool in times of dietary changes, with direct positive effects on animal performance and metabolism, with yeast culture showing greater effects on the performance of confined sheep.