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Validation of Energy Estimates of Mixed Diets Obtained from RIVGPT through In-Vivo Digestibility Trials in Growing Ram Lambs

T. Thirumalesh U. Krishnamoorthy
Vol 2(2), 102-110

The experiment was conducted in growing ram lambs to validate energy estimates of mixed diets obtained from in vitro incubation for gas production technique (RIVIGPT) through in vivo digestibility trials. Twelve Bannur ram lambs were divided in to four groups of three each. Four mixed diets comprised of rhodes hay (RH) and concentrate supplements (CS) in the ratio of 50:50 were tested in four periods in a switch over design. Each period lasted for four weeks with an adjustment period of three weeks and digestion trial(DT) for one week. The metabolisable energy (ME, MJ kg-1 DM) content of mixed diets at maintenance level of intake obtained by DT for diets-1,2,3 and 4 were 9.3, 8.8, 8.9 and 8.1 respectively. The corresponding ME values obtained from RIVIGP were 9.8, 9.0, 9.3 and 8.7 respectively. The mean ME output (Maintenance + weight gain) for the diets-1,2,3 and 4 were 4.4, 4.2, 4.3 and 4.2 respectively. The ME intake estimated from ME obtained from DT and RIVIGP for the diets-1,2,3 and 4 were 4.5 and 5.0, 4.2 and 4.7, 4.3 and 4.9, 3.9 and 4.6 respectively. Hence, the observed performance of growing ram lambs expressed in terms of the total ME output was accountable to the extent of 98 and 90% of ME intake estimates obtained from ME values derived by DT and RIVIGP respectively.

Keywords : RIVIGPT digestion trial validation of ME ram lambs


The determination of intake and digestibility of feedstuffs in vivo is time consuming, laborious, expensive, requires large quantities of feed and is unsuitable for large scale feed evaluation (Coelho et al.,1988; Carro et al., 1994). Hence, many alternative methods to evaluate the ruminant feedstuffs for energy content were proposed to conventional digestion trials (AFRC,1987). Amongst such methods, the Hohenheim in vitro gas test is widely accepted and employed in routine feed evaluation to predict in vivo digestibility and metabolizable energy contents of feeds (Menke and Steingas ,1988) because these are  not only of being less expensive and less time consuming, but they allow one to maintain experimental conditions more precisely than do in vivo trials. But the values developed from temperate region feed stuffs may not be applicable to tropical or semi tropical region feedstuffs (Ranjhan, 1993). In this regard, similar in vitro studies were conducted to evaluate the ruminant feedstuffs of tropical regions and used such data in diet formulation (Khan, 1991; Krishnamoorthy, 1995). Even though, energy estimates for ruminant feedstuffs are available, the literature on their validation through in vivo digestion trials for Indian ruminant feedstuffs are scarce. Hence, the study was undertaken to validate energy estimates of mixed diets obtained from in vitro incubation for gas production technique (RIVIGPT) through in vivo digestibility trials.

Materials and methods

Twelve Bannur ram lambs (age – 4 to 10 months; Body wt.-12.2 to 18.6 kg ) were selected and divided into four groups of three each. The feeding trial was carried out in four periods in a switch over design. Each period lasted for four weeks with an adjustment period of three weeks and  collection period of one week. The ram lambs were housed in a open protected sheep shed in a single row and provided similar management care. All the ram lambs were individually dewormed  before starting the feeding trial. The diet comprised of rhodes hay (RH) and concentrate supplement (CS) were fed at the rate of 1.5 % of body weight each so as to provide energy and protein required for maintenance and a weight gain of 50g  per day  according to ARC (1984). The CS was offered once in a day at 8.30 a.m. Daily requirement of chaffed RH was offered in small quantities 6 times between 10 a.m. and 8 p.m. Clean drinking water was provided twice a day, at 10 a.m. and 3.30 p.m. The feeding trial lasted for a total of 16 weeks. The lambs were weighed once in a week in a salter spring balance of 25 kg, on three consecutive days between  7.30 a.m. to 8.30 a.m. before having access to feed and water.

The samples of RH and CS that offered were collected weekly once for determination of dry matter by drying samples at 105oC to a constant weight. The digestion trial lasted for 7 days during which daily intake of CS and RH and out put of dung were recorded. Samples of feed offered and faeces were collected every day for dry matter estimation. Dry matter in faeces were determined by drying at 60oC to a constant weight (72h). Dried samples obtained for 7 days from each animal were pooled, ground through 1mm sieve and preserved for analysis except for nitrogen. For nitrogen estimation, the faeces samples were preserved in 30 per cent (v/v)  sulfuric acid.

Chemical Analyses

The samples of RH, CS and faeces were subjected to proximate analysis (AOAC, 1995). NDF, ADF and ADL were determined as per the method described by Goering and Van Soest (1970). The NDF in concentrate supplements was estimated by the method of Van Soest and Robertson (1991) using amylase enzyme.

In vitro Studies

The samples of RH and CS fed to ram lambs were subjected to in vitro rumen incubation studies to evaluate for metabolisable energy content (Menke and Steingass, 1988).

Donor Cow and Collection of Rumen Fluid

A lactating dairy cow producing 3.0 kg of milk per day, fitted with a flexible rumen canula of large diameter (Bar Diamond, Inc. USA), receiving a basal diet consisting of finger millet straw (FMS) and CS ( Maize-56 % , GNC-20 % , WB-20 % , Mineral mixture-1.5 % , Salt-1 % , Urea-1 % , Sodium sulfate-0.5 % , Vit-A-28050 IU/kg feed) was used as the donor cow for rumen fluid. The FMS and CS were fed separately, FMS was offered 7 kg in small portions 6 times in a day, starting at 9.30 a.m. The CS was offered  3.0 kg per day  in two equal portions at 6 a.m. and 1.30 p.m. Rumen fluid was collected in the morning between 9 a.m. and 9.30 a.m. before offering  FMS.


The samples of RH and CS collected from digestion trials were subjected to in vitro rumen incubation by incubating 200mg air equilibrated feed samples with 30ml buffered rumen fluid in 100ml calibrated syringes placed in a water bath at 39oC. Each time the necessary corrections were made for the difference in the activity of rumen liquor by parallel incubation of reference standards. Incubations were run for 24h with recording of gas production at 8 and 24h.

Calculation of Energy Value

Digestion trial: Using digestion coefficient of proximate principles, total digestible nutrients (TDN) was calculated. TDN was converted to ME by a factor of 0.15 (1 kg TDN=18.45 MJ of DE, ME=0.82 DE, i.e. 1 kg TDN=15.1 MJ of ME).

RIVIGP: Using proximate composition and in vitro net gas production (corrected for blanks and reference standards) at 24h incubation, ME in RH and CS were calculated by equations (1) and (2) according to Menke and Steingass (1988).

Roughages                                                                                                                   (1)

ME  =  2.2 + 0.1357 GP + 0.0057 CP + 0.0002859 EE

Concentrate supplements                                                                                             (2)                                ME  =  1.06 + 0.1570 GP + 0.0084 CP + 0.022 EE – 0.0081 TA

Where, ME is metabolisable energy in MJ kg-1 DM, GP the Gas production (ml/200 mg DM);  CP, EE, TA are crude protein, ether extract and total ash, respectively, in g/kg DM.

ME of total diets was calculated  by using ME of diet components obtained by RIVGP (Eq.-3).

ME of total diet = [DMIRH/DMIT]MERH + [DMICS/DMIT]MECS                                               (3)

Where, DMIRH is the dry matter from Rhodes hay, DMICS the dry matter intake from concentrate supplement(CS), DMIT  total dry matter intake (g/day); MERH the ME content of rhodes hay and MECS the ME content of CS (MJ/kg).

Comparison of Energy Values obtained by RIVGPT and Digestion Trial

The ME in RH and CS determined by RIVGPT and the energy output by lambs in the form of maintenance and gain in body weight were computed for the observed body weight by using ARC (1984) requirements were compared and the extent to  which these estimates would account for the observed energy output in growing ram lambs were examined.

The energy output in the form of maintenance and gain in body weight were computed for, the observed body weight and gain in body weight averaged for the entire feeding trial using ARC (1984) requirements [Maintenance, MJ/day= 1.1 + (0.14xBody weight) ; weight gain = 16 MJ/kg gain for 17.5 kg body weight) and compared with energy intake. Energy intake is obtained from multiplying DMI from RH and CS for the entire feeding trial by the corresponding energy estimates obtained by digestion trial and RIVGP. The difference among energy intake estimates obtained by two methods in accounting for the observed energy output was tested by the analysis of variance of the difference.

Results and Discussion

The chemical composition, in vitro gas production and energy content predicted from RIVGPT for rhodes hay and concentrate supplements used in the experiment are presented in Table 2. The ME content (MJ/kg of DM) of RH was 6.0 and the CS were ranging from 11.3 to 13.5. The mean body weights, DMI from RH and CS and ME obtained from digestion trial (% TDN x 0.15), ME calculated at maintenance level of intake and ME obtained from RIVGPT are presented in Table 3. The relationship (Fig.1) of ME obtained from DT at maintenance level of intake with ME determined through RIVGPT was y=1.0x-0.3750 (r=0.94, p≤0.05). The difference between ME (DT) and ME (RIVGPT) was found to be 4.1%. The difference achieved in this study

Fig. 1  Relationship between ME content (MJ/kg DM) of mixed diets (D-1, D-2, D-3

   and D-4)  obtained by  RIVGPT and digestion trial


Table 1 Ingredient composition (g kg-1 as is) of diets used in ram lambs experiment.

Ingredient Mixed diets
1 2 3 4
Concentrate supplement 500 500 500 500
    Cotton seed extraction
40 40
      Ground nut cake 60
      Sunflower extraction 135 113
      Rape seed meal 37.5
      Gingely cake 75
      Soya bean meal 37.5
      Maize 307 220 225 160
      De-oiled rice bran 92.5
      Rice polish 135
     Wheat bran 97 70 85
     Urea 3.5 2.5 2.5 2
     Mineral mixture 10 10 10 10
     Salt 3.25 3.25 3.25 3.25
     Sodium sulphate 1.63 1.63 1.63 1.63
Rhodes hay 500 500 500 500

Ventrimix (AB2D3K) (Each gram contained Vit-A- 82,500 IU; Vit-B2– 50 mg; Vit-D3– 12,000 IU;

Vit-K- 10mg ) was added 40mg /kg feed.

Table 2  Chemical compositiona (g kg-1 DM) and ME (MJ kg-1 DM) predicted from RIVIGPT for concentrate supplements and rhodes hayb  used in experiment.

Constituents CS-1 CS-2 CS-3 CS-4 RH
Organic matter 947 917 937 897 924
Crude protein 198 208 209 203 61
Ether extract 21 20 18 44 13
Total ash 53 83 63 103 77
Neutral detergent fibre 312 399 384 420 890
Acid detergent fibre 93 172 161 230 539
Acid detergent Lignin 23 43 48 66 74
GP-24 334.2 284.1 306.5 254.9 126.1
ME 13.5 11.9 12.5 11.3 6.0

a Mean of four samples of two replicates. b ME values are mean of four  samples of three replicates.

CS, concentrate supplement; RH, rhodes hay ; GP-24, gas production at 24h , ml g-1 DM .


Table 3 Mean body weights (kg)a, DMI (g day-1) and ME (MJ kg-1) obtained from digestion trial (DT) and RIVIGP for the diets used in digestion trialb.

Diet Body weight DMI


ME RIVIGP % difference






ME       (observed) ME

intake (Mx)


at 1x










































3 17.5 267 260 527 8.8 1.2 8.9 9.3 3.8










































a Mean of 12 animals; b Mean of 12 observations; Mx= Multipe of maintenance;

ME at 1x = [ME observed + (Mx-1)0.6].


Table 4 Mean body weight (kg), DMI (kg day-1), gain (g day-1) and calculated ME output

             (MJ  day-1) compared with ME intake obtained from energy estimates from DT

              and RIVIGP for four diets used in feeding trial.

Diet Body DMI     ME ME intake  
  weight RH CS Gain output DT RIVIGP


17.5 0.265 0.255 50.6 4.4  




17.6 0.268 0.260 42.6 4.2 4.2 4.7


17.5 0.267 0.260 44.3 4.3 4.3 4.9


17.5 0.267 0.262 38.1 4.2 3.9 4.6


17.5 0.267 0.259 43.9 4.3 4.2 4.8
±SE ±0.0 ±0.0 ±0.0 ±2.6 ±0.0 ±0.1 ±0.1


was less than the difference (±15%) reported by Krishnamoorthy et al.(1995) for the varieties of Indian feedstuffs. Similarly, Nataraja et al. (1998) noticed that the ME determined by RIVGPT for mixed diets formulated by using locally available feed ingredients and straw were comparable to those obtained in digestion trial in cow. The relationship observed in the present study (r=0.94) showed that the ME predicted from RIVGPT for the four diets agreed well with the ME1x obtained in vivo.

The intake by the ram lambs in this experiment varied from 1.1 to 1.3 times the maintenance as intake was restricted to provide energy and protein for maintenance and a weight gain of 50g per day. The energy values of the diets predicted through RIVGPT refers to the ME values of the diets at maintenance level of intake, hence, the expected ME content of the diets at the maintenance level of intake was calculated in order to compare these values with ME determined by RIVGPT (Table 3).

Energy values of diets determined by RIVGPT were comparable to ME obtained by digestion trial indicating the reliability of RIVGPT to derive energy values for the diets used in this study. The reliability of RIVGPT for feed evaluation for their energy content should be based on the extent to which those data can account for the observed production performance. In this regard, Alderman (1979) suggested that an overall accuracy of ±10% on the relevant production parameter or ±15% of total energy input can be regarded as satisfactory.

The observed performance of growing ram lambs expressed in terms of total energy out put (Maintenance+weight gain) was accountable to the extent of 98% of the energy intake obtained from energy estimates derived from DT. Whereas the energy intake estimates obtained from the energy values derived from RIVGP, the energy out put accounted for 90% of the energy intake (Table 4). Similarly, Nataraja et al. (1998) reported that the observed performance in lactating cow was accountable to the extent of 92% of the energy intake obtained from DT, whereas 97% of the energy intake obtained from RIVGP.

The ME values obtained by RIVGP were comparable to ME values obtained by DT in accounting for the observed energy out put with in ±10% indicates the potential for application of this method for tropical feedstuffs in diet formulation for ruminants.

However, the number of feedstuffs and the diets tested in this experiment were small, the findings of this experiment can further be utilized for application of this method to evaluate the wider range of ruminant feedstuffs for their energy content and such data in turn can be used to formulate diets for ruminants. Further, the observed performance in this study was restricted only to weight gain in ram lambs, hence, this method may be extended and the reliability of this method can be tested in different species for other physiological status like milk production and pregnancy.


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