EFFECT OF SUPPLEMENTING TWO DIFFERENT COMMERCIAL STRAINS OF YEAST CULTURES ON RUMEN FERMENTATION, NUTRIENT DIGESTIBILITY AND BIO-CHEMICAL PROFILE IN KANKREJ COWS

B M Bhanderi 1* , Subhash Parnerkar 2 , Ashish Aggarwal 2 , Sachin Shankhpal 2 , Harshala Thube 2 and S Pathan 2 . 1. Animal Nutrition Group, National Dairy Development Board, Anand-388001, Gujarat, India. 2. Animal Nutrition Research Department, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand-388001, Gujarat, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

The Kankrej is one of the largest and heaviest Indian breeds of cattle and are prized as powerful draft animals and are moderate milk producers. In Asia, they are more created in the Western and Northern India (Joshi and Phillips, 1953). Guzerat cattle breed is also developed in Brazil from Kankrej cattle imported from India. In United States, this race with Nellore and Gir, are three major Indian breeds that have had the most important impact on American cattle breeding. Moreover, Kankrej is the most important zebu breed for the formation of American Brahman cattle (Mason, 1996). They show the usual advantages of Zebu cattle in the tropical and semi-tropical environment, good heat tolerance and pest resistance (Garg et al., 2012). The use of yeast cultures to improve production efficiency and the underlying mechanisms for such improvement have attracted increasing attention during recent years (Williams and Newbold, 1990). Yeast cells are known to be a rich source of vitamins, enzymes and some unidentified cofactors that are helpful in increasing microbial activity in the rumen (Dawson et al., 1990 andWilliams et al., 1991); hence, yeast culture supplementation has been shown to improve the growth rate (Panda et al., 1995) and feed conversion efficiency (Mir and Mir, 1994). Several workers (Williams, 1989;Williams et al., 1991;Singh et al., 1998 andLila et al., 2004) have reported that dietary yeast culture supplements produce a range of effects in the rumen including increased pH, increased ruminal concentration of volatile fatty acids and acetate: propionate ratio (Alshaikh et al., 2002), decreased methane production and increased total number of microorganisms and cellulolytic bacteria, others have demonstrated no effect of yeast culture supplementation on ruminal pH, ammonia-N and VFA patterns (Adams et al., 1995; Garrett, 1999 andChaudhary et al., 2008). The objectives of the following study were to examine the effect of supplementing yeast culture from two different commercial sources on the rumen fermentation, nutrient digestibility and bio-chemical profile in Kankrej cows.

Subjects and methods:-
All experimental procedures were approved by the Institutional Animal Ethics Committee (IAEC) of the Faculty of Veterinary and Animal Science, Anand Agricultural University, Anand, Gujarat, India and were carried out by experienced technical experts.

Selection, feeding and management of experimental animals:-
Based on the results of in vitro studies, probiotic strains YC-1026 and YC-1077 were incorporated @ 15 and 1.5g/day, respectively in TMR, to study the effect of supplementing these strains of probiotics on ruminal fermentation, digestibility of nutrients and bio-chemical profile in Kankrej cows. The feeding trial was conducted for 40 days (excluding 10 days pre-experimental feeding) at Animal Nutrition Research Station farm. Fifteen Kankrej cows were divided into three equal groups of five animals in each group, based on age and body weight: i.e. T 0 (Control) group with no probioics, T 1 group (YC-1026) and T 2 group (YC-1077). Animals in control group were fed TMR without probiotics and those in experimental groups were fed TMR formulated with probiotics level selected on the basis of results of in vitro studies. All the animals were fed TMR with or without probiotics to meet their nutrients requirement per NRC (2001) standard. Individual feeding of all the animals was followed. The animals were let loose for exercise for two hours in the morning and one hour in the afternoon under controlled conditions, during which they had free access to fresh, wholesome drinking water. De-worming of all the animals were carried out using broad spectrum anthelmintic before initiation of the experiment.

Results and discussion:-
The chemical composition and fiber fractions (NDF, ADF), cellulose and hemi-cellulose of wheat straw based total mixed ration offered to experimental animals are summarized i n Table 1. It was within the normal range.

Effect of YC supplementation on rumen parameters:-
Average values for ruminal pH, total nitrogen, ammonia nitrogen, non protein nitrogen, TCA-N and total volatile fatty acid of experimental animals during different hours of post feeding are summarized in Table 3. 760

Ruminal pH:-
The average pH in SRL of T 0 , T 1 and T 2 groups was 6.40, 6.50 and 6.52, respectively, and were statistically significant between treatments (Table 3). The pH of SRL decreased up to 4 th h of post feeding, again increased at 6 th h post feeding indicating that the pH of SRL during periods differed significantly (P<0.05). Pandey (2009) found no differences among treatment groups in rumianl pH on yeast supplementation. It is likely that the yeast strain's positive effects on rumen pH were due to inhibiting the growth of lactate-producing bacteria while stimulating the growth of lactate-utilizing bacteria, thus leading to an overall decrease in lactate accumulation. Live yeast has been shown to increase rumen pH in a number of studies with varying levels of starch in the diets. Guedes et al. (2008) found that live yeast reduced rumen pH variation and increased average rumen pH from 6.41 to 6.55 in non-acidotic cows.

Ammonia nitrogen:-
The average ammonia nitrogen in SRL in T 0 , T 1 and T 2 groups was 12.87, 10.30 and 11.12 mg/dl, respectively. The treatment differed significantly (P<0.05) from each other in this respect. Also, the concentration of ammonia nitrogen differed significantly during different periods. The control group T 0 recorded significantly (P<0.05) higher ammonia nitrogen than T 1 group. The animals under T 2 group had an intermediate position between T 0 and T 1 groups. Ammonia nitrogen levels attained peak at 4 h post feeding and then declined afterwards up to 8 h post feeding with the same pattern in all treatment groups. NH 3 -N was depressed significantly (P<0.05) due to supplementation of live yeast in T 1 and T 2 groups. In present study, the yeast supplementation in animal's diets changed the pattern of the end products of rumen fermentation, suggesting a shift in metabolic activities of rumen microflora. The results of the present investigation are corroborated by the findings of Lascano Laborde (2008) and Tripathi et al. (2008) found non-significant differences among treatment groups but NH 3 -N levels tend to be lower in yeast supplemented group. Thus, it could be inferred that live yeast (Saccharomyces cerevisiae) supplementation in ration decreased NH 3 -N level because of its utilization for microbial protein synthesis.

Total nitrogen:-
The average total nitrogen in SRL was 92.85, 108.36 and 111.55 mg/dl in T 0 , T 1 and T 2 groups , respectively. There was significant (P<0.05) higher total nitrogen in T 1 and T 2 groups, as compared to T 0 (control) group. Pandey and Agrawal (2001a) found significantly higher total nitrogen values for animals fed probiotics. It was evident that group T 0 , T 1 and T 2 groups manifested same pattern of total-N concentration showing no difference within the groups. The peak concentration was found at 4 h post feeding and then declined up to 8 th h post feeding. The periodical changes in total nitrogen were found to be significant (P<0.05).

Non protein nitrogen:-
The result revealed that average non protein nitrogen in SRL of T 0 , T 1 and T 2 groups was 28.78, 31.36 and 31.53 mg/dl, respectively. The highest concentration was found in T 2 group, but the treatment groups did not differ from each other. Kumar et al. (1997) reported the similar trend, whereas, Pandey and Agrawal (2001a) found significantly higher values in the group of animals supplemented with probiotics. The treatment and interaction between treatments and intervals did not differ significantly. Maximum non protein nitrogen concentration was recorded at 4 h and then declined up to 8 h post feeding with the same pattern in all the three treatment groups. Pandey and Agrawal (2001a) and Chaudhary et al. (2008) reported significantly (P<0.05) higher rumen pH, total N and NPN in yeast supplemented group.

TCA precipitable nitrogen:-
The results revealed that average TCA nitrogen in SRL of T 0 , T 1 and T 2 groups was 54.67, 64.64 and 66.82 mg/dl, respectively (Table 3). There was significant higher TCA-N in T 1 and T 2 groups, as compared to T 0 group. Pandey and Agrawal (2001a) reported significantly higher values in the group of animals supplemented with probiotics, whereas, Kumar et al. (1997) found no effect of TCA-N values in the animals supplemented with live yeast. The treatment and interaction between treatments and intervals found differed significantly (P<0.05). Maximum TCA-N concentration was recorded at 2 h and then declined up to 8 h post feeding with the same pattern in all the three treatment groups.

Total volatile fatty acids (TVFA):-
The maximum TVFA concentration was found at 4 h and then declined up to 8 h of post feeding with the same pattern in all treatment groups. The average TVFA concentration in SRL was 9.92, 13.65 and 14.03 mM in T 0 , T 1 and T 2 groups , respectively. The highest concentration was found in T 2 group. The treatment and the periodical changes in TVFA were found to be significant (P<0.05). These results agreed with the review of Robinson and Garret (1999), which showed an average increase in pH (1.6%) and an overall increase in TVFA (5.4%). Kumar 2008) reported that concentration of TVFA was not altered in the continuous cultures and rumen of steers and buffalo calves. Thus, the role of live yeast cell is as a rumen microbial activity enhancer and capable of influencing some aspects of rumen fermentation processes to increase the outcome of TVFA's production fortifying the total number of anaerobic bacteria, particularly cellulolytic bacteria and to plunge NH 3 -N. Thus, inclusion of live yeast (Saccharomyces cerevisiae) in the ration had positive influence on ruminal TVFA concentration. It may be due to ability of yeast to provide soluble growth factors that stimulate growth of cellulolytic bacteria and cellulose digestion (Callaway and Martin, 1997).

Molar proportion of volatile fatty acids (VFA) in SRL:-
The results revealed that average molar proportion of acetate, propionate and butyrate, respectively was 59.17±0.76, 23.34±0.41 and 12.18±0.33 mM in T 0 group, 54.21±0.23, 28.90±0.63 and 11.69±0.48 mM in T 1 group and 54.50±0.62, 29.14±0.81 and 11.23±0.16 mM in T 2 group (Table 4). There was significant (P<0.05) reduction in acetate and butyrate concentrations and increase in propionate concentration in T 1 and T 2 groups, as compared to T 0 group. Acetate to propionate (A:P) ratio was also decreased in the yeast supplemented groups, as compared to control group. The dietary yeast culture strains YC 1026 and YC-1077 supplementation to experimental animals resulted in 762 decreased molar proportion of acetate and butyrate, acetate to propionate ratio and increased molar proportion of propionate. Harrison et al. (1988) found decreased molar proportion of acetate and acetate to propionate ratio and increased molar proportion of propionate in rumen fluid of Holstein cows supplemented with a yeast culture containing S. cerevisiae. The yeast supplementation to animals changed the pattern of the end products of ruminal fermentation, suggesting a shift in metabolic activities of ruminal microflora (Kobayashi et al., 1995). Total anaerobic bacteria and cellulolytic bacteria counts in SRL:-Total anaerobic bacteria concentrations (log 10 ml -1 ) in SRL were 8.71, 10.40 and 11.01 in T 0 , T 1 and T 2 groups, respectively. Concentrations of cellulolytic bacteria were 7.79, 8.98 and 9.15 in T 0 , T 1 and T 2 groups, respectively (Table 4) Cellulolytic bacterial activity accounts for the majority of fibre digestion in the rumen. These bacteria capture most of the energy from fibre when pH is maintained at or above pH 6.0. By nurturing a healthy, dynamic population of cellulolytic or fibre digesting bacteria, yeast culture helps increase fibre digestibility.

Haemato-biochemical and enzymatic profile of experimental animals:-
Blood metabolites are frequently used to monitor the metabolic health status of dairy cows (Ametaj et al., 2009). The average haemato-biochemical and enzymatic profile of experimental animals in T 0 , T 1 and T 2 groups are presented in Table 5.

Total protein:-
The serum protein level indicates the balance between anabolism and catabolism of proteins in the body. The serum protein level at any given time in turn is a function of hormonal balance, nutritional status, water balance and other factors affecting the state of health. The average total protein (g/dl) concentration of experimental animals in T 0 , T 1 and T 2 groups was 6.36±0.22, 6.80±0.13 and 6.94±0.19, respectively, among which T 1 and T 2 groups differed significantly (P<0.05) from T 0 group.  Albumin:-The albumin is synthesized by liver and catabolized by wide variety of tissues. Albumin supplies a readily available pool of amino acids to meet the tissue needs depending on nutritional status as its synthesis is diminished during fasting or mal-nutrition, hormonal imbalance and general poor condition of liver (Jain, 1993). The average albumin (g/dl) concentration in experimental animals in T 0 , T 1 and T 2 groups was 2.

Aspartate amino-transferase (AST) / Sreum glutamate oxaloacetate transaminase (SGOT):-
The SGOT activity is commonly seen in many tissues and it is a good marker of soft tissue. SGOT is both cytoplasmic and mitochondrial enzyme which is released even during mild degenerative changes that increase membrane 764 permeability (Evans 1988). SGOT level is raised during acute and chronic disorders of liver and muscle damage (Cornelius, 1980 andPensent, 1983). Because of the presence of AST (SGOT) activity in a number of tissues its serum level will be good marker of soft tissue damage, but precludes its use as an organ specific enzyme (Boyd, 1983). Marked elevation of SGOT preceded by lowered creatinine kinase activity could serve as an indicator of muscle damage (Kramer, 1989). In the present study, the average AST concentration of experimental animals in T 0 , T 1 and T 2 groups was 59.40±6. 71

Serum creatinine:-
The average creatinine concentration of experimental animals in T 0 , T 1 and T 2 groups was 1.42±0.08, 1.40±0.06 and 1.38±0.04 mg/dl, respectively. The three groups did not differ significantly from each other. The levels were within the normal range as reported by Kaneko et al. (1997). Thus, inclusion of live yeast (Saccharomyces cerevisiae) in the ration had no influence on serum creatinine concentration.

Haemoglobin (Hb):-
The average Hb (g/dl) concentration of experimental animals in T 0 , T 1 and T 2 groups was 10.36±0.1, 11.44±0.46 and 12.00±0.20, respectively, among which T 1 and T 2 groups differed significantly (P<0.05) from T 0 group. The levels were within the normal range as reported by Kaneko et al. (1997).

Packed cell volume (PCV):-
The PCV (%) content of experimental animals in T 0 , T 1 and T 2 groups was 31.22±1.24, 33.60±1.24 and 33.70±1.24, respectively. The three groups did not differ significantly from each other.

White blood cells (WBCs):-
The average WBCs (x10 3 ) content of experimental animals in T 0 , T 1 and T 2 groups was 7.40±0.41, 8.56±0.48 and 7.90±0.16, respectively. The three groups did not differ significantly from each other.

Digestible nutrients:-CP and TDN content of TMRs:-
The data on content of CP and total digestible nutrients (%) in TMRs is presented in Table 6. The average content of CP was 11.39±0.09, 11.76±0.07 and 11.61±0.11% and TDN was 50.72±1.10, 53.21±1.11 and 54.89±0.51% in T 0 , T 1 and T 2 , respectively. The CP content of TMRs was significantly higher (P<0.05) in T 1 group as compared to T 0 and T 2 groups, whereas, the TDN content of the T 2 group was significantly (P<0.05) higher than T 0 and T 1 groups.

CP and TDN intake (as the % of requirement) of animals:-
The average daily CP intake as per cent of requirement (NRC, 2001) of experimental animals in T 0 , T 1 and T 2 groups during digestion trial was 120.96±5.46, 121.14±6.39 and 119.63±7.41%, respectively ( Table 6). The treatment groups did not differ statistically (P<0.05) among themselves. The CP intake was enough to support the performance of experimental animals. The average daily TDN intake as per cent of requirement of experimental animals in T 0 , T 1 and T 2 groups during digestion trial was 100.14±0.43, 113.64±5.98 and 115.73±5.38%, respectively. The TDN intake as per cent of requirement in treatment groups was also significantly (P<0.05) similar. The data on TDN intake as per cent of requirement indicates more than enough intake of TDN to support the performance of experimental animals 765

CP and TDN intake of animals:-
The average daily crude protein intake (CPI) and total digestible nutrients intake (TDNI) of experimental animals of T 0 , T 1 and T 2 groups, during digestion trial are presented in Table 6. The daily CP intake of experimental animals of T 0 , T 1 and T 2 groups, during digestion trial was 818.11±28.03, 848.76±34.52 and 860.24±48.48 g/head and 281.38±14.53, 293.46±15.23 and 292.64±10.42 in terms of g/100 kg body weight and the same when expressed as g/ kg W 0.75 it was 11.60±0.44, 13.10±0.48 and 12.08±0.27. The CP intake of all the three groups was statistically (P<0.05) similar. Pandey and Agrawal (2001b) reported similar CP intake in growing animals given Saccharomyces cerevisiae in the ration. The average daily TDN intake of experimental animals of T 0 , T 1 and T 2 groups during digestion trial was 3.69±0.15, 4.03±0.20 and 4.33±0.15 kg/head, 1.27±0.05, 1.39±0.08 and 1.49±0.11 kg/100 kg body weight and in terms of g/kg W 0.75 ; it was recorded as 52.22±1.45, 57.34±2.51 and 61.40±3.40, respectively. The treatment groups T 1 and T 2 recorded statistically higher (P<0.05) TDN intake in terms of g/kg W 0.75 than T 0 group.

Average daily digestible dry matter intake:-
The average daily digestible dry matter intake (DDMI) of the experimental animals (Table 6) in T 0 , T 1 and T 2 groups was 4.90±0.31, 5.14±0.21 and 5.32±0.30 kg/d, respectively and the same when expressed as kg/100kg body weight was 1.67±0.01, 1.77±0.04 and 1.81±0.06 and in terms of g/kg W 0.75 was recorded as 68.90±1.27, 72.95±0.76 and 74.66±1.82, respectively. The DDMI of animals in T 1 and T 2 groups was significantly higher (P<0.05) as compared to T 0 group. It is evident that inclusion of live yeast (Saccharomyces cerevisiae) in the ration had an influence on the digestible dry matter intake in terms of kg/day, kg/100 kg body weight and metabolic body weight in the experimental animals.

Average daily digestible organic matter intake:-
The average daily digestible organic matter intake (DOMI) of the experimental animals in T 0 , T 1 and T 2 groups was 3.44±0.15, 4.11±0.17 and 4.18±0.19 kg/d, respectively and the same when expressed as kg/100kg body weight was 1.21±0.04, 1.42±0.05 and 1.43±0.05 and in terms of g/kg W 0.75 was recorded as 48.66 ± 1.78, 58.38 ± 1.05 and 58.86 ± 1.06, respectively. The DOMI (g/kg W 0.75 ) of animals fed T 1 and T 2 groups were significantly higher (P<0.05) as compared to T 0 group. The intake of digestible organic matter of rations in terms of metabolic body weight was influenced by inclusion of live yeast (Saccharomyces cerevisiae) in the ration. The digestibility coefficients of TMRs for DM, OM, CP, NFE, EE, CF, NDF, ADF, cellulose and hemi-cellulose  have been depicted in Table 7.   Kumar and Reddy (2004) observed improvement in EE digestibility on account of feeding yeast culture.

Conclusion:-
From the present study, it could be concluded that supplementation of yeast culture (Saccharomyces cerevisiae) strains YC-1026 and YC-1077 @ 15 and 1.5 g/animal/day, respectively, in the ration of Kankrej cows was found to be beneficial in improving the rumen fermentation and digestibility of nutrients without affecting bio-chemical profile.