Evaluation of in vitro gas production and nutrient digestibility of complete diets supplemented with different levels of thermotolerant yeast in Nellore rams

Aim: The objective of the present study was to know the effect of dietary supplementation of varied levels of thermotolerant yeast to determine best levels for sheep diets by in vitro gas production. An in vivo study on Nellore rams was used for further evaluation of diets with three best levels of yeast (obtained from in vitro data) to determine diet with optimum yeast level for growing lambs by assessing nutrient digestibility, plane of nutrition and nitrogen balance. Materials and methods: A complete diet was formulated and supplemented with five levels (0 g/kg (D ); 1 g/kg (D ); 2 g/kg 1 2 8 (D ); 3 g/kg (D ); 4 g/kg (D ) and 5 g/kg (D ) of thermotolerant yeast (Saccharomyces cerevisiae, OBV-9) @ 5x10 cfu/g to 3 4 5 6 determine best levels for sheep diets by IVGP technique. An in vivo study was conducted on Nellore rams (39.75 0.24 kg body weight, aged 3 years) in a 4 x 4 latin square design for further evaluation of diets with three best yeast levels based on in vitro data, to determine optimum yeast level for diets of growing lambs by assessing nutrient digestibility, plane of nutrition and nitrogen balance. The rams were housed individually in metabolic cages that allowed separation of urine and faeces to evaluate digestibility of nutrients and N balance. Animals were given 10 days adaptation period followed by 7-day collection period, feed intake and refusals were recorded. During the digestibility and N balance study, feed, refusals and faeces were analyzed for dry matter (DM), organic matter (OM) and crude protein (CP) as per AOAC, USA, while fibre fractions like neutral detergent fibre (aNDF) and acid detergent fibre (ADF) were analyzed. Data were analyzed as per the procedures suggested by Snedecor, G. W. and Cochran, W. G. (1994) and the difference between treatment means was tested for significance by Duncan's multiple-range and F Test. Results: Higher (P<0.01) IVGP volumes, in vitro organic matter degradability, metabolizable energy (ME) and total degradable organic matter were recorded for rations D to D over control, whereas the difference among D to D diets was not 2 6 2 4 significant. Significant (P<0.05) partitioning factor and efficiency of microbial biomass synthesis (EMBP) values for rations D and D and significant (P<0.01) microbial biomass production for D to D diets, recorded. Significant (P<0.01) nitrogen 2 3 2 6 (N) retention was recorded in yeast supplemented rations with high EMBP than control. Dry matter intake (DMI), DMI (% b. 0.75 wt.), DMI/kg W and the digestibility of dry matter, organic matter, crude protein, crude fibre, ether extract (P<0.05), nitrogen free extract and fiber fractions were significant (P<0.01) on rations D to D over control, while the difference among 2 4 rations D to D was not significant. All rams were in positive N balance and was higher (P<0.01) on rations D to D over 2 4 2 4 control. The digestible crude protein (DCP) and ME were higher (P<0.01) on rations D to D indicating all rams were 2 4 adequately met with DCP and ME requirements as suggested by ICAR, India. Conclusions: The present study demonstrated the potential of theromotolerant yeast at 1 g/kg to 3g/kg level in improving digestibility of nutrients, intake of DCP and ME and N retention without affecting health of rams under study. Thus, incorporation of 1 g/kg level of thermotolerant probiotic yeast in complete diet for lambs appears to be beneficial for livestock producers.


Introduction
grazing lands are dwindling fast because they are mostly diverted for cultivation of cereal and Inadequate availability of feed and fodder is a commercial crops to meet the urgent human needs major constraint to the prevalent small ruminant resulting in decreased land for fodder cultivation and production system [1].Due to population growth, To cite this article : Harikrishna Ch., Mahender M, Ramana Reddy Y, Gnana Prakash M, Sudhakar K, Pavani M (2012) Evaluation of in vitro gas production and nutrient digestibility of complete diets supplemented with different levels of thermotolerant yeast in Nellore rams, Vet World 5(8): 477-485, doi: 10.5455/vetworld.2012.477-485forcing livestock to depend on alternate feed able to survive these harsh conditions before resources.Large number of feed ingredients of colonizing the gut.A researcher [8] isolated thermo, variable chemical composition, digestibility and acid, bile and osmo tolerant strain of yeast (S. o nutrient quality derived from agriculture, forest, cerevisiae, OBV-9), which grows at >42 C marine and industrial sources are being utilized as temperature, pH 2, 2% Ox bile and 30% sugars, that animal feed.Since livestock subsist mainly on poor can be used as a feed additive in livestock for better quality roughages, several new technologies are being productivity.tried to improve their digestibility and utilization.One Therefore, the objective of the present study was such effort in recent years is supplementation of yeast to know the effect of dietary supplementation of varied to livestock rations to improve the utilization of levels of thermotolerant yeast to determine best levels cellulosic materials, health, productivity and for sheep diets by in vitro gas production.An in vivo reproduction.
study on Nellore rams was used for further evaluation Probiotic yeast (Saccharomyces cerevisiae) used of diets with three best levels of yeast (obtained from to enhance animal performance by improving the in vitro data) to determine diet with optimum yeast balance of microbial flora in the gastrointestinal tract level for growing lambs by assessing nutrient and nutrient utilization [2].Several studies reported digestibility, plane of nutrition and nitrogen balance.changes in the stimulation of ruminal digestion in

goats, cattle and sheep when fed yeast supplemented diets [3] and increased concentration of specialized
Experiments were conducted at the Livestock bacteria associated with fiber digestion and lactic acid Experimental Station, Livestock Research Institute, utilization in rumen on yeast supplementation [4,5].Hyderabad.The lyophilized thermotolerant yeast 8 But the yeast used so far, was mesophilic in nature and (5x10 cfu/g) obtained from DBT project on may not exert more beneficial action due to the harsh "Development and Application of Thermotolerant environmental (temperature, variation in pH and bile Probiotic Yeast for Enhanced Animal Productivity", level) conditions under which they have to survive in College of Veterinary Science, Hyderabad was used in the gastrointestinal (GI) tract of animal.Furthermore, the present study.it was reported that, stimulation of rumen bacteria by yeast was different with specific strains and many Ethical approval: The study was approved by th improvements in strain selection and stability have Institutional Animal Ethics Committee on 8 April, resulted from research in the past 15 years but strains 2009.with thermo, acid, bile and osmo tolerance need to be In vitro gas production experiment: Complete developed in order to explore the full potential activity diets (Table 1) supplemented with (0 g/kg (D ); 1 g/kg temperature in the gut of animals makes the limitation (D ) thermotolerant yeast (S. cerevisiae, OBV-9) used of using mesophilic strain of lactobacilli as probiotic 6 for in vitro study.In vitro gas production (IVGP) as reported [7].Acid and bile levels in GI tract of technique [9,10] was used to describe the extent of gas animals vary considerably and dietary probiotic are production from treatment diets.Rumen liquor was which is calculated as the ratio of substrate truly obtained with the help of a stomach tube fitted with degraded to gas volume produced [14].The Efficiency vacuum pump from Nellore rams that were fed of microbial biomass production (EMBP) of experimental rations was determined by measuring complete diets before offering the morning feed.
the ratio of TDOM and gas production as described Approximately 350 ml of rumen liquor was siphoned [15].from different depths and directions of reticulo rumen and transferred into pre heated thermos flask, strained Undigested residues were recovered and freeze dried These diets were formulated to have 12% CP (DM o for 6 h and oven dried at 60 C for 24 h and weighed to basis) to meet requirements of animals according to determine apparent undigested residue, which was [16].Diets were mixed biweekly during the study and extracted in 100 ml of NDS by boiling for one hour, were sampled upon mixing to ensure consistency in followed by filtration on preweighed gooch crucibles, their chemical composition (Table 2).Animals were and washing in hot distilled water and acetone to kept in hygienic, well ventilated individual metabolic recover true undigested residue [12].Crucibles with cages with feeding and watering arrangement during o undigested residue were dried at 100 C overnight and the trial.Rams were injected with ivermectin (Ivectin weighed to determine true undigested residue.1%) for treatment and control of GI and ecto-parasites.
Residue was ashed at 500 C for 3 h to determine true Animals were shifted to metabolic cages that allowed undigested OM, which was corrected for the separate collection of urine and faeces to evaluate appropriate blanks.The TDOM was calculated as the digestibility of nutrients and N balance, 10 days prior difference between OM incubated and the undigested to collection period to acclimatize the cage OM recovered in the residue of ND extraction.environment.Animals were offered weighed Determination of the truly undigested substrate quantities of respective diets and clean drinking water reveals the amount of substrate that was totally ad libitum for the duration of the study.A 10 d available to fermentation and the gas volume indicates preliminary period and a 7 d collection period was the proportion of this substrate used for the SCFA.A followed during the digestion trial.During the variation in the relationship reflects the variation in collection period, feed intake and refusals were microbial yield per unit SCFA produced [13].This recorded, feed samples and refusals were sampled for variation can be expressed as Partitioning factor (PF), further analysis.Daily faecal output was collected, weighed and recorded, and then 10% was kept for difference in gas production for test diets in current subsequent analyses.Similarly using glass bottles, study might be due to the suppressing effect of high cell wall and lignin present in feeds resulting in urine was collected, weighed and recorded, and then decreased attachment of ruminal microbes to feed 5% was aliquoted for N estimation.Each bottle had 50 particles [24].Gas production is basically the result of ml of 6N HCl to prevent nitrogen losses.All samples o fermentation of carbohydrates to acetate, propionate were dried at 55 C in a forced-air oven to reach a and butyrate and gas from protein is relatively small, constant weight, air equilibrated, and then ground to compared to carbohydrate fermentation [11].Higher pass 1 mm screen and stored in air tight bottles for gas values obtained for the diets D to D , indicating a further analysis.better nutrient availability for rumen microorganisms The feed and faecal samples were analysed for [25].These results were also in agreement with the proximate constituents [17] and fibre fractions [12].In reports of [5].Higher (P<0.01)IVOMD, ME and vitro gas production (ml/200 mg DM) was measured TDOM were recorded for yeast supplemented diets as per the formula specified [10,18].Metabolizable compared to D , and there was no difference among energy (ME, MJ/kg DM) value was calculated from 1 diets with yeast level up to 3 g/kg, whereas the total digestible nutrients (TDN) using the factors difference was significant (P<0.01) with D and D suggested [19].Data on in vivo and in vitro studies 5 6 diets except for TDOM.Higher (P<0.01)IVOMD, were analysed according to the procedures suggested ME and TDOM values recorded for yeast [20] and the difference between treatment means was supplemented diets, indicating addition of yeast tested for significance by Duncan's multiple-range and stimulated microbial metabolism and content of F Test [21].

Results and Discussion
a better nutrient availability for rumen microorganisms [24][25][26].The findings of current study were In vitro gas production (IVGP) profiles: There in agreement with the results of [27,28], who reported was a significant (P<0.01)difference in gas higher IVOMD values on yeast based rations.IVOMD production for control and test diets (D to D ), and the 2 6 values recorded were higher than the in vivo OMD difference was not significant among diets with yeast values of the present study, possibly due to differences level up to 3 g/kg, whereas the difference was in rumen fluid content in which the test feeds were significant (P<0.01) with diets D and D (Table 3), 5 6 incubated.showing the use of dietary yeast in sheep rations for Similarly, higher (P<0.05)PF and EMBP for desired effects.These results were in agreement with diets D and D , and higher (P<0.01)MBP for yeast 2 3 [22], who reported, total gas was increased with the supplemented diets were recorded in the current study.yeast supplementation that might have resulted from There was no difference for PF and EMBP among the increased production of propionate, because diets that contain yeast level up to 3 g/kg, whereas the carbon dioxide is produced when propionate is made difference was lower (P<0.05)for D and D diets, by some ruminal bacteria via the succinate to 5 6 which were also comparable with control, while there propionate pathway.A researcher [23]  Evaluation of in vitro gas production and nutrient digestibility of complete diets supplemented with yeast in Nellore rams mented diets compared to D , indicating higher feed higher EMBP. 1 In addition to chemical analysis, the IVGP intake.Similarly, higher (P<0.01) in vivo DMI technique applied in the present study proved to be recorded for the diets having higher PF in the present study, is consistent with the reports of [29,30], who simple to determine the difference in IVGP, OMD and defined PF as an index of the distribution of truly ME of diets with yeast, which can be more rapidly degraded substrate between microbial biomass and applied in developing countries because of its fermentation waste products.When less gas is convenience, cost saving and practical applicability produced per unit weight of substrate truly degraded, [31].Further, diets producing higher ME and OMD proportionately more substrate is converted into were having potential to be used as protein and energy microbial biomass, which means that, a higher PF supplements for ruminants as reported by [32], would reflect higher conversion of truly degraded though, the predictive ME values (8.78 to 9.58) were substrate into microbial biomass and vice versa.In the found within the range of reported values for a large current study, diets having higher IVGP volumes were number of feedstuffs [33].It is opined that, addition of having a low MBP value, showing an inverse yeast up to 3 g/kg level in complete diet is appropriate relationship between IVGP and MBP.The results for adult sheep, as the difference between 1 g/kg to 5 obtained were also in agreement with the findings of g/kg level of yeast was meager.
[13].The intake of aNDF and ADF recorded in Nellore

Nutrient digestibility and plane of nutrition:
rams in digestibility study was lowest for the rations Least square means of DM intake, digestibility of having lowest PF values, and these results were also nutrients and plane of nutrition of rams fed complete consistent with findings of [30].The average diets with 1 g/kg to 3 g/kg level of thermotolerant yeast efficiency of microbial biomass production (EMBP, are presented in (Table-4).Higher (P<0.01)DMI, g/kg) from present study indicated, different contents 0.75 DMI (% b. wt.) and DMI/kgW were recorded in of fermentable carbohydrates and available nitrogen rams fed yeast supplemented diets, might be due to to the rumen microbes.The in vivo digestibility study more intake of diets and might also due to yeast.on rams has also shown higher (P<0.01)nitrogen Although differences existed in the acceptability of retention for the diets with yeast over control with  Evaluation of in vitro gas production and nutrient digestibility of complete diets supplemented with yeast in Nellore rams experiential diets, all the animals met with DMI present study corroborate with the findings of [40], requirements as suggested by [16] indicating, these who reported similar results on dietary incorporation test diets were adequately palatable (due to blending of of yeast in sheep.increased aNDF degradability due to roughages and concentrates in correct proportions) addition of yeast in rations under incubation trials was and inclusion of yeast might have influenced the DMI, reported [49,50].On contrary, [51,52] reported no on par with [34] recommendations for adult sheep.difference in digestibility of aNDF, when sheep fed These results were in accordance with the findings of rations with or without yeast.Higher (P<0.01)ADF [35-39] in rams and [3] in goats on yeast based digestibility for yeast supplemented diets, recorded in complete diets.
the present study was in agreement with findings of Digestibility of DM, OM, CP, EE (P<0.05),CF, [39] in cows and [53] in buffalo calves, who reported NFE, NDF, ADF, Hemicellulose and Cellulose was increased ADF digestibility due to dietary supplehigher (P<0.01) on yeast supplemented diets, with no mentation of yeast, whereas [54] recorded no impact difference among them.Higher DM digestibility was of yeast addition in diets on ADF digestibility in sheep.also reported by [35,40,41,42] with dietary addition of Higher hemicellulose and cellulose digestibility yeast in sheep.Higher digestibility of OM and CP by recorded in the present study was consistent with feeding yeast supplemented diets was reported by [40-findings of [53], who observed higher (P<0.05)44] in sheep and [3] in goats.The results indicated that, hemicellulose digestibility due to dietary supplemenyeast supplementation might have exerted selective tation of yeast in buffalo calves.stimulatory effect on specific rumen bacteria responsible Similarly, higher (P<0.01)intake and intake per 0.75 for fibre degradation and microbial protein synthesis unit/kgW of DCP and ME was also recorded in the in rams [45,46].The EE digestibility was increased by current study on three test diets over control, might be around 3.5-5.0% in yeast supplemented diets, than due to higher energy density as well as higher control.Higher EE digestibility on complete diets in digestibility of CP and other nutrients.Further, all the Barbari goats was also reported [47].Significantly rams were adequately met with the DCP and ME higher CF digestibility on yeast supplemented diets requirements as suggested by [16].The results of the might be due to complementary effect of yeast by present study were in agreement with findings of [41], providing favourable rumen environment for stimulating who reported higher (P<0.05)DCP intake in sheep fed cellulolytic and hemicellulolytic bacteria along with yeast based complete diets.Higher (P<0.05)ME concentrates.The results were consistent with values in calves fed diets with different levels of findings of [35,41,42,48], who reported significant thermotolerant yeast was also reported by [53].(P<0.05)improvement in CF digestibility in sheep and Nitrogen balance: The N intake, N balance (% [3] in goats fed feeds with yeast.The results were also intake) and N balance (% absorbed) of yeast supplein accordance with [9], who reported higher CF mented diets were significantly (P<0.01)higher than digestibility with dietary yeast supplementation in control, while there was no difference recorded among calves.NFE digestibility was increased by 3.85 -5.01 these diets (Table 5).Higher daily average N intake percentage units on yeast supplemented diets than and balance on yeast supplemented diets, reflecting control.Improved efficiency in feed utilization, better the daily average intake and digestibility pattern of CP CF digestion and higher DM digestibility might have among the test diets.Higher N balance on these rations resulted in significantly higher NFE digestibility.The compared to control might be due to addition of results were in agreement with the findings of [41,42] thermotolerant yeast.Positive N balance was recorded on yeast supplemented diets in sheep. in all test diets indicating, all the rations met with the N The increased aNDF digestibility recorded in the

2
In vivo experiment through a fourfold muslin cloth and flushed with CO .Experimental design, animals and feeding:A "4 Rumen fluid-medium mixture (inoculum) is prepared x 4 Latin Square Designed digestion trial" was under continuous flushing with Co . 2 conducted on four Nellore rams (39.75 0.24 kg body Three replications of 200 mg dry weight of feed weight, aged 3 years) chosen randomly from the Sri samples were weighed into 100 ml calibrated syringes Venkateswara Veterinary University farm.Test diets and incubated with 30 ml of mixed rumen inoculum at with three best performing levels based on in vitro data o 39 C with parallel incubation of blanks [11].(D D and D ) were further evaluated by in vivo study.

Table - 2
. Chemical composition and nutritive value of complete diets

Table - 3
. Least square means for in vitro gas production (IVGP) profiles of test diets

Table - 5
. Least square means for N retention in rams fed test diets a, b : means with different superscripts row wise differ significantly (P<0.01).Evaluation of in vitro gas production and nutrient digestibility of complete diets supplemented with yeast in Nellore rams Leucaena leucocephala, Morus alba and Tectona requirements of sheep, might be because of optimum grandis.Small Rumin.Res.51: 47-56.utilization of dietary N by microbes due to matching 2. Moallem, U., Lehrer, H., Livshitz, M., Zachut, M. and supply of energy [55].Positive nitrogen balance in Yakoby, S. (2009).The effects of live yeast sheep fed complete diets was reported by [56].The supplementation to dairy cows during the hot season results of the present study were consistent with the on production, feed efficiency and digestibility.J. findings of [57], who reported N degradability and N Dairy Sci.92: 343-351.balances were increased by the addition of yeast in 3. Rohilla, P. P., Patil, N. V. and Bohra, H. C. (2009).sheep.Higher (P<0.05)N retention in sheep fed yeast Effects of probiotics and nutri mix on kids and goats.based complete diets was also reported by [41].Pahlavan, M., Afzalzadeh, A. and Conclusion Rezaeian, M. (2007).Effects of live yeast S. cerevisiae on fermentation parameters and microbial The results of the in vitro gas production study populations of rumen, total tract digestibility of diet indicated that the theromotolerant yeast level up to 3 nutrients and on the in situ degradability of alfalfa hay g/kg can be incorporated in adult sheep rations and in in Iranian Chall sheep.Pakistan J. Biol.Sci.10(4): vivo study on Nellore rams demonstrated the potential 590-597.for supplementing yeast at 1 g/kg level without much 5. Malik, R. and Singh, R. (2009).Effect of yeast and economic impact for improvement in digestibility of fungi culture on in vitro ruminal fermentation.Indian nutrients, intake of DCP and ME and positive N J. Anim.Nutri.26(1): 40-45.balance without any effect on the health of sheep.6.New Bold, C. J., Wallance, R. J. and McIntosh, F. M. Thus, incorporation of 1 g/kg level of thermotolerant (1995).Different strains of Saccharomyces cerevisiae differ in their effects on ruminal bacterial numbers in probiotic yeast in the straw based complete diet shall vitro and in sheep.J. Anim.Sci.73: 1811-1818.be recommended for economical rearing of ram lambs 7. Lankaputra, W. E. V. and Shah, N. P. (1995).Cultured with fattening diets that may be beneficial for dairy products.Tamil Nadu J. Anim.Vet.Sci.30: 2-7.