The effect of particle size on the in vitro fermentation of different ratios of high-temperature dried lucerne and sugar beet pulp incubated with equine faecal inocula

Animal Feed Science and Technology 162 (2010) 47–57

Contents lists available at ScienceDirect

Animal Feed Science and Technology journal homepage: www.elsevier.com/locate/anifeedsci

The effect of particle size on the in vitro fermentation of different ratios of high-temperature dried lucerne and sugar beet pulp incubated with equine faecal inocula Jo-Anne M.D. Murray a,b,∗ , Rachael K.T. Bice b , Meriel J.S. Moore-Colyer b a b

Division of Veterinary Clinical Sciences, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Roslin, Midlothian, EH25 9RG, UK Institute of Rural Sciences, Aberystwyth University, Llanbadarn Campus, Aberystwyth, SY23 3AL, Wales, UK

a r t i c l e

i n f o

Article history: Received 10 March 2009 Received in revised form 31 August 2010 Accepted 3 September 2010 Keywords: Particle size Sugar beet pulp Lucerne In vitro fermentation Equine faecal inoculum

a b s t r a c t An in vitro gas production technique, where equine faeces was the source of microbial inoculum, was used to determine the effect of particle size (ground vs. unground) on the in vitro fermentation of high-temperature dried lucerne (L) and molassed sugar beet pulp (SB). Two experiments were conducted; in experiment 1, unprocessed (U) L and SB or ground L and SB (G; to pass through a 1 mm dry mesh screen) were mixed in the following ratios: 100:0, 90:10, 80:20 and 70:30, L and SB, respectively. In experiment 2, unprocessed L or ground L, and ground SB were mixed in the following ratios: 100:0, 80:20, 60:40, 40:60, 20:80 and 0:100, L and SB, respectively. Substrate combinations were fermented in vitro with equine faecal inocula using in vitro gas production (GP). In both experiments, total gas pool was unaffected by particle size. Conversely, mathematical analysis of the cumulative gas production curves showed significantly different rates of fermentation in bottles containing ground substrates compared to unprocessed feedstuffs (P<0.001). Rate parameter values, fractional rate of degradation (FRGP) and time taken to produce 50 or 95% of the total gas production (T50 and T95 , respectively) were all increased (P<0.001) by grinding the substrates in both experiments. In experiment 2, total volatile fatty acid (TVFA) concentration in the culture fluid post-fermentation was also higher (P<0.001) in bottles containing U material compared to G (82.9 mmol/l vs. 64.0 mmol/l, respectively), with a marked change in the molar proportions of volatile fatty acids (VFA) present, with bottles containing G material containing more propionate and less acetate compared to the U substrates. In conclusion, particle size has a marked effect on the rate of substrate fermentation and TVFA concentration and VFA composition of the culture fluid, which has important implications for predicting in vivo digestibility from in vitro digestibility measurements; therefore, further work is required to determine optimal particle size of substrates evaluated in vitro using equine faecal inocula. © 2010 Elsevier B.V. All rights reserved.

Abbreviations: L, high-temperature dried lucerne; DML, dry matter loss; FRGP, fractional rate of gas production; G, ground substrates; GL, ground lucerne; GP, gas production; HT, high-temperature dried; LT , lag time; SB, sugar beet pulp; TG, total gas volume; T50 , time taken to reach 50% of the total gas production; T95 , time taken to reach 50% of the total gas production; TVFA, total volatile fatty acid; U, unground substrates; VFA, volatile fatty acid. ∗ Corresponding author at: Division of Veterinary Clinical Sciences, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Roslin, Midlothian, EH25 9RG, UK. Tel.: +44 0131 650 6259; fax: +44 0131 650 6588. E-mail address: [email protected] (J.-A.M.D. Murray). 0377-8401/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2010.09.001

48

J.-A.M.D. Murray et al. / Animal Feed Science and Technology 162 (2010) 47–57

1. Introduction In the majority of the published literature, in vitro gas production experiments for all species have been conducted using substrates that have been dried and finely ground, usually to pass through a 1 mm dry mesh screen, prior to fermentation (Pell and Schofield, 1993; Beuvink and Spoelestra, 1994; Theodorou et al., 1994). It is known that the processing, for example grinding, of feedstuffs can have a large effect on the rate and extent of their degradation in vitro (McAllister et al., 1993), which may not necessarily reflect the in vivo situation; for example, equine feeds (both grazed and conserved) are generally ingested intact and, as such, estimating the degradability of a feed by incubating ground samples may be misleading. However, grinding of samples enables representative, homogeneous samples to be evaluated whereas unprocessed samples present problems for analysis. For example, it is difficult and time consuming to grind the residue remaining in each culture bottle following gas production (GP). Lowman (1998) found differences in GP between fresh and dried substrates and between chopped and ground grass hay incubated with an ovine rumen fluid and concluded that, where possible, feedstuffs should be incubated in the state in which they are to be fed. Furthermore, Davies (2000) found no effect of enzyme treatment on silages ground prior to in vitro fermentation and incubated with bovine ruminal fluid, but a significant effect on both the rate and extent of GP when the same silages were left unprocessed. Therefore, when evaluating feedstuff/feed additives for ruminants, the physical state of the material prior to gas production appears to be important, this may also be the case for equids. The in vitro gas production technique of Theodorou et al. (1994) was originally developed to assess the fermentation kinetics of ruminant feedstuffs; however, more recently it has been used to evaluate the in vitro digestibility of feedstuffs incubated with an equine microbial inoculum (Murray et al., 2006). Although the gas production technique of Theodorou et al. (1994) initially relied upon ruminal fluid as the source of microbial inoculum, faecal inocula have been successfully used in gas production studies investigating the fermentation of feeds for ruminants (Akhter, 1994) and equids (Lowman, 1998). High-temperature dried lucerne (L) is a common component of horse diets in the UK due to its relatively high nutritive value in comparison to grass hays (Cuddeford, 1994). Furthermore, it is common practise in the UK to combine L with sugar beet pulp (SB), as this is regarded as an inexpensive and nutritious feed for horses. Previous in vitro results (Murray et al., 2006) have suggested that the substitution of lucerne with SB appears to have considerable potential to enhance the nutritive value of the total diet. Nevertheless, this was conducted using ground substrates, which may not be representative of the in vivo situation. Moreover, to the authors’ knowledge there is no information available in the literature assessing the effect of particle size on in vitro fermentation of feedstuffs incubated with an equine microbial inoculum. Consequently, the objectives of the experiments reported here were to examine the effect of particle size on the degradation kinetics of different ratios of L and molassed sugar beet pulp when incubated with equine faecal inocula, with the hypothesis that a reduction in particle size by grinding increases the rate and extent of substrate degradation. 2. Materials and methods The following studies were carried out following approval from Aberystwyth University’s ethical review committee. 2.1. Substrates used in experiments 1 and 2 L and shredded molassed sugar beet pulp (SB) were used as substrates in both experiments reported in this paper. L was produced from pre-bloom lucerne (Medicago sativa: variety: Daisy/Capri mix) that was mown with a 2-drum grass hopper mower and left to wilt overnight. The herbage was then chopped to 75 mm lengths by a precision-chop forage harvester (John Deere 5830) and immediately transported to the drying plant, where it was high-temperature dried at 800 ◦ C using a Van den Broek rotary dryer (Van den Broek International, Barneveld, The Netherlands) for 0.5 min. 2.2. Experiment 1 Four 160 ml identical series of serum bottles per treatment group/substrate combination were used to assess the fermentation characteristics of unprocessed (U) high-temperature dried lucerne and shredded molassed sugar beet pulp pellets or ground (G; to pass through a 1 mm dry mesh screen) L and SB mixed in the following ratios: 100:0, 90:10, 80:20, 70:30 and 0:100 L and SB, respectively, to give a total sample mass of 1 g (±0.5%) per bottle. Larger amounts (1 kg) of the substrates were combined, thoroughly mixed and then sub-sampled (1 g) for use in the GP experiment. The experiment was a factorial arrangement of treatments with the main effects being (i) grinding (none or through a 1 mm dry mesh screen) and (ii) the level of SB substitution of L (100:0, 90:10, 80:20, 70:30 and 0:100 L and SB, respectively); there were four replicate bottles for each treatment combination. In addition, 16 blanks (no substrate; 4 per inocula source) were included in the design. Substrate combinations were fermented in vitro with an equine faecal inoculum using the in vitro gas production (GP) technique of Theodorou et al. (1994). Methods employed for the GP technique were as described by Theodorou et al. (1994), with the exception of the preparation of the faecal inoculum, which was prepared as described previously (Murray et al., 2005). Faeces for the inoculum were collected from individual ponies fed L and SB in the ratios described above (with the

J.-A.M.D. Murray et al. / Animal Feed Science and Technology 162 (2010) 47–57

49

exception of 0:100 L:SB, which were inoculated with faeces from ponies fed 30:70 L:SB), processed individually, and used to inoculate bottles containing the respective substrate combination. Head-space gas pressure readings were taken at 2, 4, 6, 8, 11, 14, 17, 20, 28, 36, 47, 57, 72, 97 and 120 h post-inoculation, with the accumulated gas volume measured and then released to zero following each reading. After the final reading, bottles were refrigerated at 4 ◦ C to arrest fermentation. Following incubation, the culture fluid was separated from residual plant particles and adherent microbial biomass by vacuum filtration through sintered glass crucibles (porosity 1) and the residue rinsed with two volumes of distilled water. The washed residues were then lyophilised to constant weight for the determination of residual apparent dry matter. 2.3. Experiment 2 Three 160 ml identical series of serum bottles per treatment group/substrate combination were used to assess the fermentation characteristics of unprocessed (UL) or ground L (GL) and ground molassed sugar beet pulp (SB) mixed in the following ratios: 100:0, 80:20, 60:40, 40:60, 20:80 and 0:100 lucerne and SB, respectively, to give a total sample mass of 1 g (±0.5%) per bottle. Larger amounts (1 kg) of the substrates were combined, thoroughly mixed and then sub-sampled (1 g) for use in the GP experiment. The experiment was a factorial arrangement of treatments with the main effects being (i) grinding (none or through a 1 mm dry mesh screen) and (ii) the level of SB substitution of L (100:0, 80:20, 60:40, 40:60, 20:80 and 0:100 L and SB, respectively); there were three replicate bottles for each treatment combination. In addition, 4 blanks (no substrate) were included in the design. Substrate combinations were fermented in vitro with an equine faecal inoculum using the in vitro gas production (GP) technique as described for experiment 1. Faeces for the inoculum were collected from a horse maintained on a basal diet of ad libitum hay supplemented with 1 kg DM of molassed sugar beet pulp and 2 kg DM of molassed L. Head-space gas pressure readings were taken as described for experiment 1. After the final reading, bottles were refrigerated at 4 ◦ C to arrest fermentation. Vessel contents were subsequently analysed for DM, TVFA, VFA molar proportions and pH measurements. Aliquots (1.2 ml) of culture fluid (taken immediately following termination of the experiment) were acidified with orthophosphoric acid (5 ␮l) and stored at −20 ◦ C prior to VFA analysis according to the method of Merry et al. (1995). The remaining culture fluid was separated from residual plant particles and adherent microbial biomass by vacuum filtration through sintered glass crucibles (porosity 1) and the residue rinsed with two volumes of distilled water. The washed residues were then lyophilised to constant weight for the determination of residual apparent dry matter. 2.4. Statistical analyses 2.4.1. Mathematical modelling of gas production data The maximum likelihood programme (MLP: Ross, 1987) was used for non-linear regression, to fit curves to experimentally derived gas accumulation profiles using the model of France et al. (1993): y = A − BQ t Z

√

t

, √

where Q = e−b , Z = e−c , and B = AebT+c T . In this equation, y denotes cumulative gas production (ml), t is the incubation time (h), A is the predicted asymptotic value for gas pool size (ml), T is the lag time (h), and b (h−1 ) and c (h−0.5 ) are the rate constants. The mean control profiles for gas produced in inoculated culture bottles in the absence of substrate were subtracted prior to curve fitting analysis. The time dependant fractional rate FRGP (h−1 ) was also calculated: FRGP =

b+c . √ 2 T50

where T50 is the time taken to reach 50% of the total gas production. 2.4.2. Statistical analyses of modelled gas production parameters Values for the modelled gas production parameters (as described above with the addition of T95 ; the time taken to produce 95% of the total GP), and DM loss for experiment 1 following gas production were analysed for significant differences using two-way analysis of variance (grinding × level of SB substitution of L). Values for modelled gas production parameters in experiment 2, plus pH and TVFA concentration and VFA composition, were also analysed by two-way analysis of variance (grinding × level of SB substitution of L). All data were checked for normality and all data were normally distributed. Comparisons between treatment groups were made using Fisher’s least significant difference (LSD). All statistical analyses were carried out using in Genstat Release 9.1 (Lawes Agricultural Trust, Harpended, UK). For experiment 1, predicted rate parameter values for the different substrate combinations were estimated using the FRGP, T50 and T95 values for L and SB (U and G).

50

J.-A.M.D. Murray et al. / Animal Feed Science and Technology 162 (2010) 47–57

Table 1 Gas production curve fitted parameters for experiment 1; total gas volume (TG) and lag time (LT ) for unprocessed high-temperature dried lucerne and unmolassed sugar beet pulp (U) and ground high-temperature dried lucerne and unmolassed sugar beet pulp (G) mixed in the following ratios: 100:0, 90:10, 80:20 and 70:30, respectively and incubated with equine faecal inocula (n = 4). Lucerne:sugar beet pulp combination

Treatment mean

100:0

90:10

80:20

70:30

0:100

TG (ml g−1 DM) U G Substrate mean Substrate s.e.d. Treatment s.e.d. Substrate × treatment s.e.d.

131 136 134a 8.5 5.4 12.1

142 140 141a

167 161 164b

176 169 173c

226 228 227d

DML (mg g−1 ) U G Substrate mean Substrate s.e.d. Treatment s.e.d. Substrate × treatment s.e.d.

570 363 466a 24.8 15.7 35.1

590 324 457a

a–d

Significance (P)

169 167 <0.001 ns ns

605 321 463a

643 320 481a

787 530 658b

639 372 <0.001 <0.001 ns

Mean values in a row (substrate) with different superscripts differ significantly (P<0.05).

3. Results 3.1. Experiment 1 3.1.1. End-point measurements There was no interaction between substrate and processing treatment for total gas pool size (Table 1). Processing had no effect on total gas production from the experimental substrates (169 ml vs. 167 ml for U and G, respectively). There was no interaction between substrate and processing treatment for dry matter loss (DML; Table 1), which was greater (P<0.001) in the unground samples (639 mg/g vs. 372 mg/g for U and G, respectively). Moreover, greater DML was observed for SB as a sole substrate (0:100 L:SB) in comparison to all other substrate combinations (P<0.05). 3.1.2. Fermentation kinetics Mathematical analysis of the cumulative gas curves also showed grinding to have an effect on rate parameter values, e.g. fractional rate of degradation (FRGP) and time taken to produce 95% of the total gas production (T95 ) were both improved (P<0.001) by grinding (Table 2). Rate parameter values (FRGP and T95 ) were increased (P<0.001) by the substitution of lucerne with SB in both the unprocessed and ground material (Table 2). The shortest times to reach both T50 and T95 occurred in bottles containing the lucerne and SB combinations, whilst the longest times were observed in bottles containing L as the sole substrate, followed by SB as a single feedstuff (Table 2). Lag times were also affected by grinding; overall the ground substrates produced curves that had shorter (P<0.001) lag phases than the unprocessed samples (2.17 vs. 2.72, respectively) (Fig. 1). 3.1.3. Associative effects Considering the FRGP of L (0.0307 and 0.0560 for U and G, respectively) and SB (0.0361 and 0.0777 ml/h for U and G, respectively), theoretical values of the different ratios were estimated. Positive associate effects were observed for these two substrates (L and SB) when combined (Figs. 2 and 3). The FRGP for the unground substrate combinations was above those expected if a purely additive effect were to exist through SB substitution, with increases above predicted values of 37, 24 and 52% for 90:10, 80:20 and 70:30 substrate combinations, respectively. Similarly, increases above predicted values were also observed for the ground material, with increases above predicted values of 24, 17 and 25% for 90:10, 80:20 and 70:30 substrate combinations, respectively (Fig. 2). A similar effect was observed for T50 and T95 values (Fig. 3). 3.2. Experiment 2 3.2.1. End-point measurements There was an interaction between the main effects (grinding and level of SB substitution of L) for total gas pool size (P=0.013; Table 3). Therefore, it was not possible to consider the main effects in isolation. However, there was no interaction between the main effects for DML, which was greater (P<0.001) in the unground samples (747 mg/g vs. 651 mg/g for UA and GA, respectively; Table 3). Greater DML was also observed in bottles containing L substituted with SB in comparison to those containing L as a sole substrate (P<0.001) (Table 4). The pH values ranged from 6.61 to 6.82, with a similar interaction between the main effects (P<0.001; Table 5). Grinding had an effect (P<0.001) on total VFA concentration present in the culture fluid (Table 5). Overall, bottles containing ground

J.-A.M.D. Murray et al. / Animal Feed Science and Technology 162 (2010) 47–57

51

Table 2 Gas production curve fitted parameters for experiment 1; fractional rate of gas production (FRGP) and time taken to produce 50% (T50 ) and 95% (T95 ) of total gas production for unprocessed high-temperature dried lucerne and unmolassed sugar beet pulp (U) and ground high-temperature dried lucerne and unmolassed sugar beet pulp (G) mixed in the following ratios: 100:0, 90:10, 80:20, 70:30 and 0:100, respectively and incubated with equine faecal inocula (n = 4). Lucerne:sugar beet pulp combination 100:0 FRGP (h−1 ) U G Substrate mean Substrate s.e.d. Treatment s.e.d. Substrate × treatment s.e.d.

90:10

0.0307 0.0560 0.0434a 0.00617 0.00390 0.00872

80:20

0.0438 0.0762 0.0600b

Treatment mean 70:30

0.0416 0.0786 0.0601b

0.0527 0.0902 0.0715c

Significance (P)

0:100 0.0361 0.0777 0.0569b

0.0410 0.0758 <0.001 <0.001 ns

T50 U G Substrate mean Substrate s.e.d. Treatment s.e.d. Substrate × treatment s.e.d.

28.7 15.3 22.0 1.51 0.96 2.14

22.0 14.2 18.1

T95 U G Substrate mean Substrate s.e.d. Treatment s.e.d. Substrate × treatment s.e.d.

99.2 56.5 77.8d 3.36 2.13 4.76

69.8 40.7 55.2ab

21.7 14.0 17.4

20.0 12.2 16.1

25.2 14.6 19.9

23.5 13.9 <0.001 <0.001 0.001

72.9 39.4 56.1ab

62.0 34.5 48.3a

89.0 40.2 64.6c

78.6 42.2 <0.001 <0.001 ns

LT U G Substrate mean Substrate s.e.d. Treatment s.e.d. Substrate × treatment s.e.d. a–d

1.97 1.86 1.91a 0.559 0.353 0.790

2.64 2.18 2.41a

1.97 2.31 2.12a

2.87 1.97 2.42a

4.18 2.52 3.35b

2.72 2.17 <0.001 <0.001 ns

Mean values in a row (substrate) with different superscripts differ significantly (P<0.05).

material contained greater (P<0.001) concentrations of total VFA (82.9 mmol/1 vs. 64.0 mmol/1 for UL and GL, respectively). Analysis of the molar proportions of VFA and ratio of acetate plus butyrate:propionate present in the culture fluid showed an interaction between the main effects (P<0.001) (Table 6). 3.2.2. Fermentation kinetics Mathematical analysis of the cumulative gas production curves also showed an interaction (P<0.001) between the main effects (grinding and level of SB substitution of lucerne; Table 4) for total gas pool size (P=0.013), FRGP (P<0.001), T50 (P<0.001) Table 3 Gas production curve fitted parameters for experiment 1; total gas volume (TG) and lag time (LT ) for unprocessed (UL) or ground (GL) high-temperature dried lucerne and ground unmolassed sugar beet pulp mixed in the following ratios: 100:0, 80:20, 60:40, 40:60 and 100:0, respectively and incubated with an equine faecal inoculum (n = 3). Lucerne:sugar beet pulp ratio

Treatment mean

100:0

80:20

60:40

40:60

20:80

0:100

TG (ml) UL GL Substrate mean Substrate s.e.d. Treatment s.e.d. Substrate × treatment s.e.d.

188 148 168 8.1 4.7 11.4

201 186 193

210 216 213

227 244 235

286 267 276

269 281 275

DML (mg g−1 ) UL GL Substrate mean Substrate s.e.d. Treatment s.e.d. Substrate × treatment s.e.d.

682 548 615a 24.2 14.0 34.3

707 589 648ab

Significance (P)

230 224 <0.001 ns 0.013

737 638 687bc

748 678 713bc

779 698 738cd

828 756 792d

747 651 <0.001 <0.001 ns

52

J.-A.M.D. Murray et al. / Animal Feed Science and Technology 162 (2010) 47–57 Unprocessed

Cumulative gas production (ml)

250

200

150

100

50

0 0

20

40

60

80

100

120

Incubation time (h)

Cumulative gas production (ml)

100:0; L:SB

90:10; L:SB

80:20; L:SB

70:30; L:SB

0:100; L:SB

Ground

250

200

150

100

50

0 0

20

40

60

80

100

120

Incubation time (h) 100:0; L:SB

90:10; L:SB

80:20; L:SB

70:30; L:SB

0:100; L:SB

Fig. 1. Experiment 1: fitted cumulative gas production curves for unprocessed high-temperature dried lucerne and unmolassed sugar beet pulp (U) and ground high-temperature dried lucerne and unmolassed sugar beet pulp (G) mixed in the following ratios: 100:0, 90:10, 80:20, 70:30 and 0:100, respectively and incubated with equine faecal inocula (n = 4).

and T95 (P<0.001). However, the shortest times to reach both T50 and T95 occurred in bottles containing the ground feedstuffs (Table 4). Values for T50 and T95 were also shortest in bottles containing SB as the sole substrate, whilst the longest times were observed in bottles containing L as the sole feedstuff (Table 4). Lag times also showed an interaction between the main effects, with lower (P<0.001) lag phases observed in bottles containing the ground substrate combinations compared to those with a single substrate and the unprocessed feedstuff combinations (Fig. 4). 4. Discussion The aim of the experiments reported here was to examine the effect of particle size (ground vs. unground substrates) on the in vitro fermentation of different ratios of L and SB incubated with equine faecal inocula. Grinding had no effect on total gas production from any of the substrate combinations in both experiments reported herein; however, the ability to measure the fermentation patterns of these samples proved to be a useful tool. Extrapolation of data from the cumulative GP curves showed distinct differences in the fermentation kinetics of the ground and unground substrates. Rate parameter values (FRGP, T50 and T95 ) showed a significant increase in the rate of GP from ground substrates compared to the unground substrates in both experiments. This was likely attributable to the decrease in particle size leading to an increase in the surface area available for microbial attachment (Emanuele and Staples, 1988; Bowman and Firkins, 1993) and hence an increase in the rate of degradation. Moreover, the ground substrates also produced curves that had significantly shorter lag phases than the unground samples indicating a more rapid adherence of bacteria to particles, population density increasing with increased surface area (Uden, 1992). This may also explain the reduction in in vitro DML that occurred in bottles containing the ground substrates compared to the unground substrates. Particle size was also an important factor affecting the rate of fermentation in vivo and has been seen to influence ruminal function and hence animal performance (Cherney et al., 1988). Although reduction of particle size generally increases the rate of fermentation, as seen in the current study, it can decrease in vivo digestibility, particularly in ruminants, by increasing the rate of passage through the rumen (Jaster and Murphy, 1983). Consequently, if this passage rate exceeds the increase in

J.-A.M.D. Murray et al. / Animal Feed Science and Technology 162 (2010) 47–57

53

Unprocessed

0.1

Actual

0.09

Predicted

FRGP (h-1)

0.08 0.07 0.06 0.05 0.04 0.03 0.02 90:10

80:20

70:30

lucerne: SB combination Ground

0.1

Actual

0.09

Predicted

FRGP (h-1)

0.08 0.07 0.06 0.05 0.04 0.03 0.02 90:10

80:20

70:30

lucerne: SB combination Fig. 2. Experiment 1: actual and predicted fractional rate of gas production (FRGP) values for unprocessed high-temperature dried lucerne and unmolassed sugar beet pulp (U) and ground high-temperature dried lucerne and unmolassed sugar beet pulp (G) mixed in the following ratios: 100:0, 90:10, 80:20, 70:30 and 0:100, respectively and incubated with equine faecal inocula (n = 4).

120

30

110 100

25

90 20

80

T95 (h)

T50 (h)

Unprocessed

130

35

T50 Actual T50 Predicted T95 Actual T95 Predicted

70

15

60

10

50 90:10

80:20

70:30

lucerne: SB combination 20

Ground

60

50 45

15

40

T95 (h)

T50 (h)

55 T50 Actual T50 Predicted T95 Actual T95 Predicted

35 30

10 90:10

80:20

70:30

lucerne: SB combination Fig. 3. Experiment 1: actual and predicted T50 and T95 (time taken to produce 50% and 95% of total gas production, respectively) values for unprocessed high-temperature dried lucerne and unmolassed sugar beet pulp (U) and ground high-temperature dried lucerne and unmolassed sugar beet pulp (G) mixed in the following ratios: 100:0, 90:10, 80:20, 70:30 and 0:100, respectively and incubated with equine faecal inocula (n = 4).

54

J.-A.M.D. Murray et al. / Animal Feed Science and Technology 162 (2010) 47–57

Table 4 Gas production curve fitted parameters for experiment 2; fractional rate of gas production (FRGP) and time taken to produce 50% (T50 ) and 95% (T95 ) of total gas production for unprocessed (UL) or ground (GL) high-temperature dried lucerne and ground unmolassed sugar beet pulp mixed in the following ratios: 100:0, 80:20, 60:40, 40:60, 20:80 and 0:100, respectively and incubated with an equine faecal inoculum (n = 3). Lucerne:sugar beet pulp ratio 100:0 FRGP (h−1 ) UL GL Substrate mean Substrate s.e.d. Treatment s.e.d. Substrate × treatment s.e.d.

0.0584 0.0780 0.0682 0.00284 0.00164 0.00402

80:20 0.0655 0.0890 0.0773

60:40 0.0513 0.0907 0.0701

Treatment mean 40:60 0.0584 0.0859 0.0722

20:80 0.0619 0.0847 0.0733

Significance (P)

0:100 0.1054 0.0923 0.0988

0.0668 0.0868 <0.001 <0.001 <0.001

T50 UL GL Substrate mean Substrate s.e.d. Treatment s.e.d. Substrate × treatment s.e.d.

13.5 10.3 11.9 0.50 0.29 0.70

12.2 7.8 10.0

T95 UL GL Substrate mean Substrate s.e.d. Treatment s.e.d. Substrate × treatment s.e.d.

53.5 39.9 46.6 2.19 1.26 3.10

48.3 33.7 41.0

14.9 7.7 11.3

13.2 8.1 10.6

12.1 8.2 10.1

8.5 7.5 8.0

12.4 8.3 <0.001 <0.001 <0.001

59.8 33.1 46.4

53.0 34.9 43.9

49.3 35.4 42.4

30.3 32.5 31.4

50.0 34.9 <0.001 <0.001 <0.001

LT UL GL Substrate mean Substrate s.e.d. Treatment s.e.d. Substrate × treatment s.e.d.

1.52 1.36 1.44 0.131 0.075 0.185

1.30 0.58 0.94

1.35 0.34 0.84

1.23 0.16 0.70

0.89 0.18 0.53

1.89 1.18 1.54

1.37 0.63 <0.001 <0.001 0.031

fermentation rate, the overall extent of rumen fermentation can be reduced (Russell and Hespell, 1981). Therefore, feedstuff degradation is a function of both the time available for digestion and the rate of microbial degradation. Consequently, passage rate through the caecum and colon plays an important role in digestion in vivo, which is not simulated by batch culture techniques, such as the one used in this study. The extent of equine hind-gut digestion is therefore influenced by passage rate, which is affected by feed particle size. Reducing the variability of particle size by grinding to produce a more homogenous sample may not mimic in vivo conditions; nevertheless it does increase the accuracy of in vitro and in situ measurements (Lowman, 1998). Table 5 Experiment 2: dry matter loss (DML), pH and total volatile fatty acid (TVFA) concentration of the culture fluid following the in vitro fermentation of unprocessed (UL) or ground (GL) high-temperature dried lucerne and ground unmolassed sugar beet pulp mixed in the following ratios: 100:0, 80:20, 60:40, 40:60, 20:80 and 0:100, respectively and incubated with an equine faecal inoculum (n = 3). Lucerne:sugar beet pulp ratio 100:0 pH UL GL Substrate mean Substrate s.e.d. Treatment s.e.d. Substrate × treatment s.e.d. TVFA (mmol l−1 ) UL GL Substrate mean Substrate s.e.d. Treatment s.e.d. Substrate × treatment s.e.d. a–d

6.82 6.72 6.77 0.002 0.011 0.027 70.7 54.2 62.5a 2.80 1.62 3.96

Treatment mean

80:20

60:40

40:60

20:80

0:100

6.83 6.81 6.82

6.75 6.81 6.78

6.69 6.79 6.74

6.67 6.74 6.71

6.62 6.61 6.62

Significance (P)

6.73 6.75 <0.001 ns <0.001

81.7 60.0 70.8b

83.5 64.6 74.1bc

92.1 67.0 79.5cd

79.6 66.1 72.9b

Mean values in a row (substrate) with different superscripts differ significantly (P<0.05).

89.5 72.4 81.0d

82.9 64.0 <0.001 <0.001 ns

Cumulative gas production (ml)

J.-A.M.D. Murray et al. / Animal Feed Science and Technology 162 (2010) 47–57

55

Unprocessed

300 250 200 150 100 50 0 0

20

40

60

80

100

120

Incubation time (h) 100:0

80:20

60:40

40:60

20:80

0:100

Ground

Cumulative gas production (ml)

300 250 200 150 100 50 0 0

20

40

60

80

100

120

Incubation time (h) 100:0

80:20

60:40

40:60

20:80

0:100

Fig. 4. Experiment 2: fitted cumulative gas production curves for unprocessed or ground high-temperature dried lucerne and ground unmolassed sugar beet pulp mixed in the following ratios: 100:0, 80:20, 60:40, 40:60, 20:80 and 0:100, respectively and incubated with an equine faecal inoculum (n = 3). Table 6 Experiment 2: volatile fatty acid composition of the culture fluid following the in vitro fermentation of unprocessed (UL) or ground (GL) high-temperature dried lucerne and ground unmolassed sugar beet pulp mixed in the following ratios: 100:0, 80:20, 60:40, 40:60, 20:80 and 0:100, respectively and incubated with an equine faecal inoculum (molar proportions) (n = 3). Lucerne:sugar beet pulp ratio

Treatment mean

100:0

80:20

60:40

40:60

20:80

0:100

Acetate UL GL Substrate mean Substrate s.e.d. Treatment s.e.d. Substrate × treatment s.e.d.

0.67 0.69 0.68 0.005 0.003 0.007

0.66 0.70 0.68

0.66 0.70 0.68

0.65 0.71 0.68

0.62 0.68 0.65

0.57 0.58 0.57

Propionate UL GL Substrate mean Substrate s.e.d. Treatment s.e.d. Substrate × treatment s.e.d.

0.24 0.19 0.21 0.006 0.003 0.008

0.26 0.17 0.21

A + B:P UL GL Substrate mean Substrate s.e.d. Treatment s.e.d. Substrate × treatment s.e.d.

3.05 4.06 3.56 0.095 0.055 0.135

2.85 4.58 3.72

Significance (P)

0.64 0.68 <0.001 <0.001 <0.001

0.24 0.18 0.21

0.25 0.18 0.22

0.27 0.22 0.24

0.35 0.33 0.34

0.27 0.21 <0.001 <0.001 <0.001

3.01 4.37 3.73

2.87 4.16 3.51

2.66 3.49 3.08

1.80 1.96 1.88

2.72 3.77 <0.001 <0.001 <0.001

56

J.-A.M.D. Murray et al. / Animal Feed Science and Technology 162 (2010) 47–57

The extrapolating data from gas production studies to the animal is difficult due to the wide range of feed particles found in the animal, which are continually changing size and shape due to mastication and microbial fermentation. Therefore, the extent of particle size reduction is also an important consideration; for example, Firkins et al. (1986) reported higher digestibilities in the rumen of steers fed ground grass hay compared to those fed chopped hay. Since there was no significant differences in ruminal particulate dilution rate (both ground and chopped hay particles remaining in the rumen for the same period of time), this increased digestibility was attributed to the greater surface area available for colonisation by the ruminal microbial population and subsequently, more extensive fermentation of ground vs. chopped hay, which concurs with the findings reported here. Conversely, other studies have reported decreases in in vivo digestibility by reducing particle size, attributable to increased rate of passage through the rumen (Jaster and Murphy, 1983). These differences in results are likely due to variation in particle sizes used in the respective experiments, with Firkins et al. (1986) evaluating the difference between 10 mm vs. 50 mm particles and Jaster and Murphy (1983) using a wider size range; long hay (no actual measurement given) vs. finely chopped hay (approx. 2 mm long). The later concurs with earlier in vivo and in situ studies in ruminants (Shaver et al., 1986; Uden, 1988) whereby fine grinding was seen to cause a reduction in the rate and extent of substrate degradation, and/or an increased lag phase. This effect of particle size on rate of passage and in vivo digestibility has been reported in equids, with similar equivocal results reported (Cymbaluk, 1990; Tisserand and Faurie, 1995; Todd et al., 1995), mainly due to varying experiment conditions and in particular, particle size distribution and level of feed intake (Drogoul et al., 2000a). Where fibrous feedstuffs have been finely ground, mean retention time (MRT) is reportedly longer compared to chopped forages at similar levels of feed intake in some studies (Hintz and Loy, 1966; Wolter et al., 1974), with no difference reported in others (Drogoul et al., 2000a). However, there appears to be no affect of grinding on total tract digestibility (Hintz and Loy, 1966; Wolter et al., 1974; Tisserand and Faurie, 1995; Todd et al., 1995; Drogoul et al., 2000a). Conversely, in situ rate and extent of fibre degradation is reportedly reduced by fine grinding of forages (Drogoul et al., 2000b). This depression in fibre degradation resulting from fine grinding of forage has been related to adverse conditions in the rumen for fibre degradation and has been associated with low pH (Shaver et al., 1986; Uden, 1988); however, Drogoul et al. (2000b) reported no detrimental effects on caecal or colonic pH in ponies feed ground or chopped hay, which concurs with the current study where the pH of the culture fluid following fermentation did not differ between unprocessed or ground substrates. Conversely, TVFA concentration of the culture fluid following fermentation was affected by processing, with significantly higher levels found in bottles containing the ground substrates compared to the chopped feedstuffs. Moreover, inspection of the molar proportions of VFA found lower levels of acetate present and higher amounts of propionate in bottles containing ground substrates, with a consequential increase in the ratio of A + B:P. This increase in TVFA concentration and shift in VFA profiles is concurrent with ruminant studies whereby Shaver et al. (1986) reported a reduction in the ratio of acetate:propionate in the rumen of cows fed ground compared to chopped hay. This also concurs with in situ data from ponies, where acetate:propionate ratios decreased in animals fed ground vs. chopped hay (Drogoul et al., 2000b). The marked increase in fermentation rate with grinding substrates in the current study may have been further enhanced by the type of substrates utilised. The results of this study also highlight the differences in the extent and rates at which lucerne and SB are degraded. Sugar beet pulp is rich in readily degradable structural carbohydrates such as pectins and hemicelluloses (Longland et al., 1994) that are rapidly degraded by gut micro-organisms. Conversely, lucerne contains higher levels of secondary cell wall material (Longland et al., 1994) resulting in a slower and less extensive degradation. In combination, these two botanically diverse fibrous feeds produced positive associative effects. In experiment 1, the rates of fermentation for the substrate combinations were above those expected if a purely additive effect were to exist through SB substitution, which concurs with previous studies (Murray et al., 2006); consequently, further work is required to examine the effect of particle size using an array of single substrates. 5. Conclusions This study has demonstrated that sample preparation is an important factor in gas production studies with particle size having a significant effect on gas production profiles. Moreover, it has also highlighted the value of mathematical analysis of cumulative GP curves since end-point GP measurements showed no effect of particle size. Further work is required to assess various degrees of grinding of a range of substrates to ascertain the effect this has on their in vitro fermentation when incubated with an equine microbial inoculum. Acknowledgements The authors would like to acknowledge Dengie Crops Ltd. and BBSRC for financial support under the auspices of the Teaching Company Scheme, Alison Brooks for technical support and M.S. Dhanoa for expert statistical advice. References Akhter, S., Use of Cow Faeces to Provide Micro-organisms for the in Vitro Digestibility Assay of Forages. PhD Thesis, University of Reading, 1994. Beuvink, J.M.W., Spoelestra, S.F., 1994. In vitro gas production kinetics of grass silages treated with different cell wall-degrading enzymes. Grass Forage Sci. 49, 277–283.

J.-A.M.D. Murray et al. / Animal Feed Science and Technology 162 (2010) 47–57

57

Bowman, J.G.P., Firkins, J.L., 1993. Effects of forage species and particle size on bacterial cellulolytic activity and colonisation in situ. J. Anim. Sci. 71, 1623–1633. Cherney, J.H., Cherney, D.J.R., Mertens, D.R., 1988. Fibre composition and digestion kinetics in grass stem internodes as influenced by particle size. J. Dairy Sci. 71, 2112–2122. Cuddeford, D., 1994. Artificially dehydrated lucerne for horses. The Vet. Record 135, 426–429. Cymbaluk, N.F., 1990. Comparison of forage digestion by cattle and horses. Can. J. Anim. Sci. 70, 601–610. Davies, Z.S., Manipulation of Silage Fermentation Using Biological Additives and Genetic Algorithms. PhD Thesis, University of Wales, Aberystwyth, UK, 2000. Drogoul, C., Poncet, C., Tisserand, J.L., 2000a. Feeding ground and pelleted hay rather than chopped hay to ponies. 1. Consequences for in vivo digestibility and rate of passage of digesta. Anim. Feed Sci. Technol. 87, 117–130. Drogoul, C., Tisserand, J.L., Poncet, C., 2000b. Feeding ground and pelleted hay rather than chopped hay to ponies. 2. Consequences on fibre degradation in the cecum and the colon. Anim. Feed Sci. Technol. 87, 131–145. Emanuele, S.M., Staples, C.R., 1988. Effect of forage particle size on in situ digestion kinetics. J. Dairy Sci. 71, 1947–1954. Firkins, J.L., Berger, L.L., Merchen, N.R., Fahey, G.C., 1986. Effects of forage particle size, level of feed intake and supplemental protein degradability on microbial protein synthesis and site of nutrient digestion in steers. J. Anim. Sci. 62, 1081–1094. France, J., Dhanoa, M.S., Theodorou, M.K., Lister, S.J., Davies, D.R., Isac, D., 1993. A model to interpret gas accumulation profiles associated with in vitro degradation of ruminant feeds. J. Theor. Biol. 163, 99–111. Hintz, H.F., Loy, R.G., 1966. Effect of pelleting on nutritive value of horse rations. J. Anim. Sci. 25, 1059–1062. Jaster, E.H., Murphy, M.R., 1983. Effects of varying particle size of forage on digestion and chewing behaviour of dairy heifers. J. Dairy Sci. 66, 802–810. Longland, A.C., Carruthers, J., Low, A.G., 1994. The ability of piglets 4 to 8 weeks old to digest and perform on diets containing two contrasting sources of non-starch polysaccharides. Anim. Prod. 58, 405–410. Lowman, R.S., Investigations into the Factors Which Influence Measurements During In Vitro Gas Production Studies. PhD Thesis, University of Edinburgh, UK, 1998. McAllister, T., Doug, Y., Yanke, L.J., 1993. Cereal grain digestion by selected strains of ruminal fungi. Can. J. Microbiol. 39, 367–376. Merry, R.J., Dhanoa, M.S., Theodorou, M.K., 1995. Use of freshly cultured lactic-acid bacteria as silage inoculants. Grass Forage Sci. 50, 112–123. Murray, J.M.D., Longland, A.C., Moore-Colyer, M.J.S., 2006. In vitro fermentation of different ratios of high-temperature dried lucerne and sugar beet pulp incubated with an equine faecal inoculum. Anim. Feed Sci. Technol. 129, 89–98. Murray, J.M.D., Longland, A.C., Moore-Colyer, M.J.S., Dunnett, C., 2005. The effect of enzyme treatment on the in vitro fermentation of lucerne incubated with equine faecal inocula. Br. J. Nutr. 94, 771–782. Pell, A.N., Schofield, P., 1993. Computerised monitoring of gas production to measure forage digestion in vitro. J. Dairy Sci. 76, 1063–1073. Ross, G.J.S., 1987. MLP: Maximum Likelihood Programme (A Manual). Rothamsted Experimental Station, Harpenden, UK. Russell, J.B., Hespell, R.B., 1981. Microbial rumen fermentation. J. Dairy Sci. 64, 1153–1169. Shaver, R.D., Nytes, A.J., Satter, L.D., Jorgensen, N.A., 1986. Influence of amount of feed and forage physical form on digestion and passage of prebloom Alfalfa hay in dairy cows. J. Dairy Sci. 69, 1545–1559. Theodorou, M.K., Williams, B.A., Dhanoa, M.S., McAllen, A.B., France, J., 1994. A simple gas production method using a pressure transducer to determine the fermentation kinetics of ruminant feeds. Anim. Feed Sci. Techol. 48, 185–197. Tisserand, J.L., Faurie, F., 1995. Effet de la forme de presentation du foin son utilisation digestive chez le poney. Ann. Zootech. 44, 185. Todd, L.K., Sauer, W.C., Christopherson, R.J., Coleman, R.J., Caine, W.R., 1995. The effect of feeding different forms of alfalfa on nutrient digestibility and voluntary intake in horses. J. Anim. Physiol. Anim. Nutr. 73, 1–8. Uden, P., 1988. The effect of grinding and pelleting hay on digestibility, fermentation rate, digesta passage rate rumen and faecal particle size in cows. Anim. Feed Sci. Technol. 19, 145–157. Uden, P., 1992. The influence of leaf and stem particle size in vitro and of sample size in sacco on neutral detergent fibre fermentation kinetics. Anim. Feed Sci. Technol. 37, 85–97. Wolter, R., Durix, A., Letourneau, J.C., 1974. Influence du mode de presentation du fourrage sur la vitesse du transit digestif chez le poney. Ann. Zootech. 23, 293–300.