Effect of Dietary Supplementation of Chromium and Yeast on in vitro Dry Matter Degradability and Rumen Fermentation Pattern

In the present scenario of scarcity of feed resources, dry roughages are mostly used for feeding ruminants in developing countries. Yeast (Saccharomyces cerevisiae), widely used probiotics, was found to increase the utilization of fiber and improve the production in ruminants (Khan et al., 2016). The concept of chromium (Cr) supplementation has also received a great deal of attention since past decade, as Cr could have supra-physiological benefits for domestic livestock. Chromium is biologically active as part of a biomolecule called chromodulin, which is part of an insulin-signaling pathway and appears to affect carbohydrate and lipid metabolism via the action of insulin (Vincent, 2000). The ability to increase glucose tolerance could lead to more efficient use of glucose and ultimately improvement in growth and feed efficiency. There is still lack of studies regarding the effect of chromium on nutrient utilization and rumen fermentation pattern in ruminants. Looking at the beneficial effects of yeast and chromium, the present in vitro experiment was conducted to investigate the effect of dietary chromium and yeast supplementation on in vitro dry matter degradability and rumen fermentation by rumen simulation technique.

Source of yeast and chromium
 
Chromium propionate or organic chromium propionate (4% Cr) and Yeast (Saccharomyces cerevisiae @ 50×109 CFU/g) were procured from Kemin Asia Pvt Ltd., Chennai, Tamil Nadu.
 
Dietary treatments
 
A basal diet (BD) was formulated with sorghum stover as the sole roughage source along with other concentrate ingredients for adult sheep. The ingredient composition is given in Table 1. From these 4 diets were formulated as given below:

Diet 1: BD: Negative Control (No yeast or Cr supplementation).
Diet 2: 1.5Cr: Chromium Propionate (1.5 mg/kg) supplemented to BD.
Diet 3: Y: Dry Yeast (5 billion CFU/Kg) supplemented to BD
Diet 4: 1.5Cr+Y: Yeast and chromium (1.5 mg/kg of Cr + 5 billion CFU/kg of yeast) supplemented to BD.

The study was conducted in the Department of Animal Nutrition, College of Veterinary Science, Rajendranagar, Hyderabad in 2019. About 10 kg of each experimental diet was prepared in triplicate at feed plant. The feed samples from each replicate were collected and were ground to uniform size to pass through 1 mm sieve with Wiley mill and finally stored for in vitro studies.
 
Rumen simulation technique
 
All the feed samples were analyzed for proximate constituents (AOAC, 2012) and neutral detergent fibre (Van Soest et al., 1991). The in vitro DM degradability, gas production and rumen fermentation pattern was assessed by Rumen simulation apparatus as per Czerkawski and Breckenridge (1977). About 500 ml of the pooled strained rumen liquor (collected from 5 freshly slaughtered sheep), 200 ml of artificial saliva (McDougall, 1948) and 100 ml of distilled water were added in each reaction vessel with continuous flushing of CO2. About 80 g cud was tied tightly in a nylon bag and placed in the feed container of reaction vessel before setting in the groove. The flow rate of saliva (0.55 ml/min) and temperature (39°C) was adjusted properly with the frequent observation on microbial density. After 24 h, 5 g of feed sample was incubated along with cud for next 24 h. Then the cud was replaced with another nylon bag containing the feed sample. So each feed sample was incubated for 48 h in their respective reaction vessels. After 48 h of incubation, the gas bags were analyzed quantitatively for the amount of gas produced by water displacement method and the quantity of effluent collected in the effluent vessels was recorded. At initial period, gas and effluent were found to be high and then became constant after 5-6 days as the digestibility of DM remained constant after 4 to 6 days of incubation (standardization) (Czerkawski and Breckenridge 1977). Then these bags were washed and dried in the spin drier and the samples were dried in a hot air oven at 65°C for a period of 3 days and the final weight was taken. Five grams ground dried sample were placed in separate nylon bags and incubated at 0, 3, 6, 12, 24 and 48 h in six reaction vessels of Rusitec (Czerkawski and Breckenridge, 1977). The feed samples were analyzed in triplicate for the in vitro study. The total volatile fatty acid (TVFA) (mmol/100 ml) was estimated using Markham’s distillation apparatus as per the method of (Barnett and Reid, 1957) and the ammonia nitrogen (NH3-N) (mg/100 ml) was estimated using Gerhardt, Germany by steam distillation procedure including blank (Makkar and Becker, 1996).

The DM disappearance was calculated at 0, 3, 6, 12, 24 and 48 h and the percentage of in vitro degradability of samples in Rusitec were calculated using the following formula:


The results of dry matter degraded at various time intervals were fitted to the exponential equation of Mc Donald (1981) using NAAWAY (1992) software.

P= a + b (1 - e-ct)

 Where;
P= Effective degradability.
t= Time of incubation.
a + b= Potential degradability.
c= Rate of degradability.
a, b and c are constants in exponential equations.
 
Statistical analysis
 
All the statistical procedures were done as per Snedecor and Cochran (1994) and Duncan (1955) with a significance at P<0.05.

The chemical composition of the four complete diets is given in Table 2. The CP and NDF content in all the diets varied from 10.51% to 10.76% and 53.13% to 54.52%, respectively. No significant (P>0.05) difference was observed in amount of gas produced among four dietary treatments (Table 4). The Cr supplementation did not affect the in vitro gas production and these results were in accordance with those obtained by Sarma (2013) and Keshri (2016) when added at the dose rate of 0 to 3 and 0 to 2.5 ppm, respectively. Sarma (2013) noticed no statistical difference in total gas production in range of 37.33 to 39 (ml/200 mg DM) in all Cr supplemented group (0-3 ppm). Supplementation of yeast (5×109 CFU/kg) also had no significant (P>0.05) effect on gas production compared to control (Table 4). which could be attributed to reduced CH4 production as a result of suppressed methanogenic bacteria in the rumen with yeast supplementation (Wang et al., 2016). Besharati (2015) also noticed similar gas production in vitro when S. cerevisiae was added @ 2.5 and 5 g/kg DM (355.82 and 327.52 ml/g DM respectively) in comparison to control (no supplementation) (340.61 ml/g DM), while a reduction in gas production was observed @ 7.5 g/kg DM (163.44 ml/g DM) on 48 hr of incubation. Similar results were also observed when Cr was added to yeast (1.5 Cr+Y) (Table 4). Chen et al., (2018) reported 13.23% higher gas production with yeast supplemented @ 0.25% and Cr 2000 ppm as chromium enriched yeast to maize stover than control, however no significant (P>0.05) difference was observed in rice straw based diet as the chromium level increased in the yeast.

The effect of yeast and Cr on cumulative DM degradation at 0 h, 3 h, 6 h, 12 h, 24 h and 48 h given in Table 3. Cumulative DM degradability at various intervals and effective DM degradability were not affected due to supplementation of only chromium @ 1.5 Cr and were comparable to control (Table 3 and 4). Similar results were reported by Sarma (2013) and Keshri (2016) with regard to Cr supplementation on in vitro DM and OM degradability. The in vitro DM and OM degradability ranges around 66 to 68% and 67% respectively (Sarma, 2013). In Rusitec, the yeast supplementation increased the cumulative DM degradation at 12 h, 24 h and 48 h interval and the effective degradability than BD (Table 3 and 4). Higher degradability of nutrients with yeast supplementation could be associated with stimulation of growth of rumen microbial population, attributed to its oxygen scavenging ability and maintaining optimum environment for anaerobic bacteria (Jouany, 2001). Secondly, S. cerevisiae could provide certain vitamins i.e. biotin and thiamine, required for the growth and activity of microbes in the rumen (Akin and Borneman, 1990). Significant (P<0.05) increase in IVDMD and IVOMD with yeast supplementation was also observed by Harikrishna et al., (2012), Elmasry et al., (2016) and Elanthamil et al., (2018). Elanthamil et al., (2018) noticed highest IVDMD in 0.5×108 CFU level (56.42%) against non- supplemented group (48.09%). Regarding the effect of added chromium to yeast (1.5 Cr+Y), higher cumulative degradability (at 12 h, 24 h and 48 hr) and effective DM degradability was observed in comparison to control and the values were comparable with yeast supplemented group (Y) (Table 3, 4). While Chen et al., (2018) did not notice any effect on IVDMD and IVNDFD when supplemented Cr enriched yeast (yeast @ 0.10, 0.25, 0.40 and 0.55% of fermentation medium and Cr at 2000 ppm). The reason may be regarding the variation in dose level of chromium and yeast as well as type of substrates used in the experiment.

The rumen fermentation metabolites viz. pH, NH3-N and TVFA due to supplementation of yeast, Cr and their combination to sorghum stover based complete diets assessed by Rusitec is presented in Table 4. In the present study, there was no significant variation in pH, TVFA and NH3-N level among Cr supplemented groups than BD (Table 4). Samanta et al., (1998) and Sarma (2013) also reported non-significant effect on in vitro TVFA concentration due to Cr supplementation (0, 0.25, 0.5, 0.75, 1.00, 1.50, 2.00, 2.50, 3.00 ppm Cr) and (10, 25, 50, 100 ppm Cr), respectively. In the Cr supplemented groups Rikhari et al., (2010) also observed no significant (P>0.05) effect on NH3-N level than control similar to present study. In contrast, Sarma (2013) and Samanta et al., (1998) found decreasing trend of NH3-N on higher level of Cr supplementation, however, the levels tried by Samanta et al., (1998) were higher upto 100 ppm.  In yeast supplemented diets (Y) no significant difference was observed in the levels of pH and TVFA when compared to control (Table 4). The possible explanation for constant pH could be the enhanced utilization of lactic acid by Selenomonas ruminantium by yeast supplementation (Martin and Nisbet, 1992). The present results were corroborative with results obtained by Rodriguez et al., (2015) who also found no effect of live cells and cell extract of yeast on pH level in the rumen. Lila et al., (2006) also reported no effect of twin strain of live cells of S. cerevisiae on rumen pH. However inconsistent results were observed regarding TVFA concentration with yeast supplementation compared to previous studies. Enjalbert et al., (1999) and Bayat et al., (2015) observed no effect of yeast on TVFA concentration and their molar proportions whereas higher concentration of TVFA was reported by Mao et al., (2013), Elghandour et al., (2014a) and Elanthamil et al., (2018) on forage based diet. As per Wallace and Newbold (1992), the variable response in VFA production and patterns is a consequence of the effects of yeast culture on microbial numbers in the rumen rather than a direct effect on ruminal fermentation. The substrates or diets influence the growth of different species of rumen microbes that are responsible for the VFA production and pattern when yeast culture was supplemented (Lascano and Heinrichs, 2009). A significant (P<0.01) decreased level of NH3-N was observed in Y group than BD (Table 4). This could be due to increased incorporation of ammonia into microbial protein which was reflected by the higher microbial protein synthesis in the concerned group. When supplemented with yeast by Chaucheyras-Durand and Fonty (2001) and Krizova et al., (2011) reported lower NH3-N at non-significant (P>0.05) level at all sampling times compared to control. The 1.5Cr +Y diet also did not affect in vitro pH and TVFA level and values were similar to BD and Y groups (Table 4). Chen et al., (2018) also observed similar pH level in vitro when chromium enriched yeast (0, 0.10, 0.25, 0.40 and 0.55% yeast and 2000 ppm Cr) was added to maize stover based diet whereas increased level of VFA was noticed for maize stover and decreased trend was observed in case of rice straw. Significantly (P<0.01) lower level of NH3-N was found in Cr+Y combination groups than control and the level was similar to yeast supplemented groups (Table 4). Similar to our experiment, Chen et al., (2018) also reported lower (P<0.05) NH3-N level due to addition of Cr enriched yeast in maize stover and rice straw diet which coincides with present results. The highest NH3-N concentration of maize stover obtained at the level of 0.1% of Cr+Y which was 24.85% higher than the lowest concentration level obtained at the level of 0.55% (P<0.05) (Chen et al., 2018).

From the results obtained from the present in vitro study, it could be inferred that the supplementation of Cr (1.5 mg/kg) did not affect the in vitro effective dry matter degradability, cumulative degradability and rumen fermentation pattern when compared with control. However, yeast supplementation positively affected the in vitro dry matter degradability and rumen fermentation pattern. The supplementation of yeast along with chromium at a concentration of 1.5 mg/kg resulted in higher in vitro rumen fermentation pattern as compared to control but the values were comparable with yeast supplementation.

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