Influence of Rumen Inoculum Source on In Vitro Dry Matter Digestibility of Pasture

The Professional Animal Scientist 21 (2005):45–49

TInoculumNSource: Influence of Rumen on In Vitro Dry ECHNICAL

OTE

Matter Digestibility of Pasture K. J. SODER1, PAS USDA/Agricultural Research Service, Pasture Systems and Watershed Management Research Unit, University Park, PA 16802-3702

Abstract The objective of this study was to evaluate the effects of rumen inoculum donor source [pasture diet vs total mixed ration (TMR) or individual cow effects] on in vitro DM digestibility (IVDMD) of pasture. Pasture samples (four levels of pasture diversity from a larger study) were digested in vitro (Tilley and Terry method) using various rumen inoculum sources. Rumen inoculum was collected from four fistulated lactating dairy cows grazing pasture (one cow per pasture diversity treatment). Additionally, rumen inoculum was collected from a confined lactating dairy cow fed a TMR. Samples digested in rumen inoculum from the TMR donor cow diet resulted in lesser (P<0.05) IVDMD values than samples digested in rumen inoculum from pastured cows. Cow-to-cow variation was also significant (P<0.05). When applied to a pasture DMI equation, IVDMD values obtained from a TMR-fed cow resulted in a 19.3% decrease in estimated pasture DMI as opposed to when IVDMD values obtained from pastured cows were inserted into the DMI equation. These results emphasize the importance of researching laboratory and scientific methods used in obtaining IVDMD values for

1

To whom correspondence should be addressed: [email protected]

pastures. Use a donor cow that is consuming a diet similar to that being tested whenever possible. Extra caution should be taken when interpreting the IVDMD results obtained on pasture samples from outside labs, especially if the donor cow diet is unknown. Ingredient and nutrient composition of the donor cow diet should be described when reporting IVDMD results in the literature. (Key Words: In Vitro Digestibility, Inoculum, Pasture, Rumen.)

Introduction The in vitro DM digestibility (IVDMD) technique is used extensively to analyze feeds because of the high degree of correlation with in vivo DM digestibility (Tilley and Terry, 1963; Marten and Barnes, 1980; AOAC, 1990). Many factors can influence IVDMD, including source and activity of inoculum; however, results are mixed. Some researchers found no effect of rumen inoculum source on IVDMD of a given forage (Quicke et al., 1959; Marinucci et al., 1992), whereas other studies reported significant effects (Church and Petersen, 1960; Bezeau, 1965; Grant et al., 1974; Holden, 2000). Most of these studies utilized either different animals and/or stored forage diets. Information is scarce, however, on the effects of rumen inoculum source on

pasture diets. In many pasture research trials, pasture samples are collected, stored, and then later analyzed for IVDMD, usually in wintertime after the grazing season has ended. In these cases, rumen inoculum is obtained from a cow consuming stored feeds and/or high levels of concentrate, such as a total mixed ration (TMR), because of seasonality of pasture. The effects of obtaining rumen inoculum from a cow fed a TMR on the IVDMD of pasture samples is unknown when compared with rumen inoculum obtained from a pastured cow. In addition, limited and conflicting information exists on the effect of individual cow as a rumen inoculum source on IVDMD. Therefore, the objectives of this study were to determine the influence of donor source (pasture vs TMR diet or individual cow effects) on IVDMD of pasture diets.

Materials and Methods Pasture samples were collected from a larger grazing research study (Soder et al., 2004). The pasture treatments consisted of a two-species mix containing orchardgrass (Dactylis glomerata L.) and white clover (Trifolium repens L.) (2SP); a three-species mix containing 2SP plus chicory (Cichorium intybus L.) (3SP); a six-species mix containing orchardgrass, chicory,

46

red clover (Trifolium pratense L.), tall fescue (Festuca arundinacea Schreb.), perennial ryegrass (Lolium perenne L.), and birdsfoot trefoil (Lotus corniculatus L.) (6SP); and a nine-species mix containing 6SP plus white clover, Kentucky bluegrass (Poa pratensis L.), and alfalfa (Medicago sativa L.) (9SP). The pasture samples were dried in a forced-air oven for 48 h at 55°C and ground through a 1-mm screen in a Wiley mill (Arthur H. Thomas, Philadelphia, PA) prior to analysis for IVDMD. Pasture samples were also analyzed for CP (AOAC, 1990), ADF, and NDF (ANKOM200 Fiber Analyzer威; ANKOM Technology Corporation, Fairport, NY). Five donor cows (midlactation multiparous Holstein cows) fitted with rumen cannulas were used as rumen inoculum donors. Four of the donor cows were on a pasture diet; one fistulated cow was on each of the four pasture treatments. The pastured cows were supplemented twice daily with a corn-based supplement at a rate of 9 kg DM/d. The fifth fistulated cow was fed a free-choice TMR in confinement. For the pastured cows, a randomized block design was used with four periods, including 9 d for an adjustment period and rumen sampling on d 10. These cows were part of a larger experiment evaluating the effects of pasture diversity on productivity of lactating dairy cows (Soder et al., 2004). Rumen inoculum was collected 4 h after supplementation and turnout on pasture (or 4 h after morning feeding in the case of the TMR-fed cow). Rumen inoculum was collected from six areas of the rumen by use of a vacuum pump and was then filtered through four layers of cheesecloth. Carbon dioxide was passed into the flask to displace air from the rumen inoculum. Pasture samples were incubated for 48 h following the procedures of Tilley and Terry (1963). The reagents used were solution A (10 g of KH2PO4, 0.5 g of MgSO4ⴢ7H2O, 0.5 g of NaCl, 0.1 g of CaCl2ⴢH2O, and 0.5 g of urea dissolved in 1 L of de-

Soder

ionized H2O) and Solution B (15 g of Na2CO3 and 1 g of Na2Sⴢ9H2O dissolved in 100 mL of deionized H2O). A buffer solution was made just prior to each of the four digestion runs by warming Solutions A and B to 39°C and adding 20 mL of Solution B to 1 L of Solution A. The pH of the buffer solution was adjusted to 6.8 (if needed) by adding small amounts of solution B (1 to 2 mL). Each pasture treatment was incubated using the rumen inoculum from the cow that was consuming that particular treatment. In addition, rumen inoculum from the TMR cow was used to incubate all four pasture treatments each period to conduct a head-on comparison of donor cow diet. Concentrate samples were also incubated in each source of inoculum. Each of the pasture and concentrate samples was digested in triplicate for each source of inoculum. To each 50-mL Nalgene tube was added 0.25 g of sample, 15 mL of buffer solution, and 5 mL of rumen inoculum from an individual cow. The tube was then flushed with CO2, stoppered with a Bunsen valve, and incubated for 48 h at 39°C. Tubes were swirled by hand at 0, 2, 4, 6, and 24 h of incubation. At the end of the 48-h incubation, 1 mL of Solution C (6.5 g of pepsin added to 100 mL 6N HCl) was added to each tube. Tubes were then incubated for an additional 24 h without stoppers and filtered through #54 Whatman filter paper (Whatman Limited, Maidstone, England). Filter paper was dried at 100°C for 24 h, and blank tubes containing filter paper only were used to correct for bacterial contamination (Robertson et al., 1972). The statistical design was a randomized block design with the following model: IVDMD = source + period + pasture treatment + source × treatment + error. Source was either individual donor cow or donor diet (pasture vs TMR). Pasture treatments were 2SP, 3SP, 6SP, and 9SP mixes. Data were analyzed using the GLM procedure of SAS威 (1999). Means separa-

TABLE 1. Nutrient composition of donor cow diets [pasture diet vs total mixed ration (TMR); DM basis].

CP, % of DM ADF, % of DM NDF, % of DM NEl, Mcal/kg

Pasture dieta

TMR

19.9 18.0 29.8 1.66

16.5 22.0 33.7 1.68

a

Pasture diet: mean value across all pasture diversity treatments; pasture (24.5% CP, 21% ADF, 30.6% NDF, and 1.67 Mcal/kg of NEl) plus concentrate (14.8% CP, 14.7% ADF, 28.9% NDF, and 1.65 Mcal/kg of NEl).

tion was by the least squares means procedures of SAS威 (SAS Institute, Cary, NC).

Results and Discussion Nutrient Content of Diet. The nutrient content of the donor cows diets are shown in Table 1. The pasture diet had greater CP and less ADF and NDF than the TMR diet, which is typical of a high quality pasture. Despite the differences in pasture species diversity, CP, NDF, and ADF of the pasture treatments (2SP, 3SP, 6SP, and 9 SP) were not significantly different (P>0.05) and averaged 21.8, 33.6, and 44.5% for CP, NDF, and ADF on a DM basis, respectively (data not shown). These pastures were in their second year of an intensive grazing study, and it was noted that pasture diversity had decreased compared with the first year and that the pastures were becoming more homogeneous (Matt Sanderson, USDA/Agric. Res. Serv., University Park, MD, unpublished data). Therefore, it is not surprising that nutrient content was similar among pasture treatments. Inoculum Source. Pasture IVDMD was affected (P<0.05) by in-

TECHNICAL NOTE: Diet Influences on Pasture In Vitro Digestibility

TABLE 2. Mean in vitro DM digestibility (IVDMD) of pasture and concentrate as affected by rumen inoculum source (% of DM). Inoculum source Item Pasture IVDMDd Concentrate IVDMD

Pasture dieta,b

TMRc

SE

65.3e 68.8

59.3f 66.1

0.89 0.89

a

Pasture diet: pasture (24.5% CP, 21% ADF, 30.6% NDF, and 1.67 Mcal/kg of NEl) plus concentrate (14.8% CP, 14.7% ADF, 28.9% NDF, and 1.65 Mcal/kg of NEl). b Mean value across all fistulated cows on pasture diets. c TMR = Total mixed ration. d Mean value across all pasture diversity treatments. e,f Means within the same row with different superscripts differ (P<0.05).

oculum source; IVDMD values were approximately 10% greater when the donor diet was pasture-based compared with TMR. Concentrate IVDMD tended (P=0.06) to be greater when pastured cows were used as a donor source compared with the inoculum from the TMRfed cow (Table 2). This is in disagreement with some studies (Quicke et al., 1959; Marinucci et al., 1992) that reported similar IVDMD values regardless of donor diet. However, other researchers (Church and Petersen, 1960; Bezeau, 1965; Cherney et al., 1993; Holden, 2000) supported these results by showing that source of inoculum did have a significant effect on IVDMD of feeds. Individual Cow. Individual cow affected IVDMD (P<0.05; Table 3).

Specifically, one particular cow consuming the pasture diet (designated as cow “Pasture A” in Table 3) had greater (P<0.05) IVDMD than the other pasture cows (Pasture B, C, D cows) and the TMR cow. It is interesting to note that although not significantly different in all cases, all pastured cows tended to have greater IVDMD than the cow consuming TMR, which supports the data shown in Table 2. Bezeau et al. (1965) reported a significant difference in the activity of the inoculum from two different donor animals, a conclusion contrary to results reported by others (Donefer et al., 1960; Mabjeesh et al., 2000), who observed no difference in IVDMD because of source of inoculum.

TABLE 3. Mean in vitro DM digestibility (IVDMD) of pasture and concentrate as affected by individual donor cow (% of DM). Individual pasture cows Item Pasture IVDMDb Concentrate IVDMD a

A

B

C

D

TMRa

SE

71.5c 70.4

62.9d 69.8

63.3d 68.3

63.0d 68.8

59.3d 66.1

2.30 2.29

TMR = Total mixed ration. Mean value across all pasture diversity treatments. c,d Means within the same row with different superscripts differ (P<0.05). b

47

Nelson et al. (1969) reported that when glucose and urea were added to the buffer, there was a significant difference in IVDMD between donor animals. Church and Peterson (1960) reported that different sources of rumen liquor had marked effects on substrate digestion, with significant differences observed between twin steers. Pasture Diversity. The IVDMD of the four pasture treatments was significantly affected (P<0.05) by source of rumen inoculum (Table 4). The 2SP and 3SP treatments had greater (P<0.05) IVDMD when rumen inoculum was used from the pastured cows compared with rumen inoculum from a TMR-fed cow. Although not statistically significant for the 6SP and 9SP pasture treatments, IVDMD tended (P=0.06) to be greater when digested in the pasture-based rumen inoculum. IVDMD Ranking. In addition to differences in IVDMD caused by source of inoculum, the ranking of feeds from greatest to least IVDMD also changed (Table 4). This result differs from Ayres (1991), who reported little to no change in relative ranking of IVDMD of various feeds despite source of rumen inoculum. It should be noted, however, that both previous studies used forages that typically exceeded 40% NDF, and neither study used rumen inoculum from a cow consuming a pasture diet. Holden (2000) noted that the rankings remained unchanged in that study except when pasture was included in the rankings, which suggests that pasture diets may react differently than harvested feeds that have been processed or dried. DMI Equations. The significance of obtaining accurate IVDMD values becomes clear in developing and using estimated pasture DMI equations, both for research and in practical applications, such as ration balancing. Numerous pasture studies have estimated pasture DMI of lactating dairy cows (Holden et al., 1994; Kolver and Muller, 1998; Soder and Holden, 1999; Bargo et al., 2002; De-

48

Soder

TABLE 4. Mean in vitro DM digestibility (IVDMD) and relative ranking of pasture diversity treatments as affected by rumen inoculum source (% of DM). IVDMD Rankinga

Inoculum source Itemb 2SP 3SP 6SP 9SP

Pasture dietc e

66.7 69.2e 60.2 64.4

TMRd

SEM

Pasture diet

TMR

f

2.28 2.30 2.29 2.30

2 1 4 3

3 2 4 1

58.4 60.6f 57.2 61.0

a

Relative ranking from highest to lowest IVDMD within inoculum source (pasture vs TMR). 2SP = orchardgrass + white clover; 3SP = 2SP + chicory; 6SP = orchardgrass + red clover + chicory + tall fescue, perennial ryegrass, and birdsfoot trefoil; 9SP = 6SP + Kentucky bluegrass, white clover, and alfalfa. c Pasture diet: pasture (24.5% CP, 21% ADF, 30.6% NDF, and 1.67 Mcal/kg of NEl) plus concentrate (14.8% CP, 14.7% ADF, 28.9% NDF, and 1.65 Mcal/kg of NEl). d TMR = Total mixed ration. e,f Means within the same row with different superscripts differ (P<0.05). b

lahoy et al., 2003; Soder et al., 2004). In those studies, cows were orally dosed with chromic oxide, fecal samples were collected, and the concentration of Cr was determined in the feces to estimate fecal output using the equation: fecal output = (g Cr/d)/(g Cr/g fecal DM). Pasture DMI was determined using the equation: pasture DMI = fecal output/(1 − IVDMD) (Holden et al., 1994). If concentrate was fed during the experi-

ment, the equation used was pasture DMI = [fecal output − concentrate DMI × (1 − IVDMD of concentrate)]/ (1 − IVDMD of pasture). It is important to note the relevance of IVDMD in these equations. As an example of how rumen inoculum source influences results at the applied level, the IVDMD values obtained in the current study for both the pasture-based and TMR-based rumen inoculum were individually sub-

stituted into the second pasture DMI equation (including concentrate) for the larger grazing study (Soder et al., 2004; Table 5). Using IVDMD values obtained from the TMR-fed cow resulted in a 19.3% (2-kg DMI per head) mean decrease across treatments in estimated pasture DMI when compared with IVDMD values obtained from the pastured cows. This level of error could have profound effects on interpretation of re-

TABLE 5. Mean estimated pasture DMI values as affected by rumen inoculum sourcea,b. Inoculum source Itemc

Pasture dietd

Total mixed ration

Difference

SEM

Percentage decrease

9.0 7.7 9.1 8.2

2.1 1.9 2.1 2.0

1.7 1.7 1.7 1.7

19.1 19.6 19.0 19.4

(kg of DM) 2SP 3SP 6SP 9SP

11.0 9.5 11.2 10.1

a Pasture DMI = [fecal output − concentrate DMI × (1− IVDMD of concentrate)]/(1 − IVDMD of pasture); fecal output = (g of Cr/d)/(g of Cr/g of fecal DM). b Data set (except IVDMD) obtained from Soder et al. (2004). c 2SP = orchardgrass + white clover; 3SP = 2SP + chicory; 6SP = orchardgrass + red clover + chicory + tall fescue, perennial ryegrass, and birdsfoot trefoil; 9SP = 6SP + Kentucky bluegrass, white clover, and alfalfa. d Pasture diet: pasture (24.5% CP, 21% ADF, 30.6% NDF, and 1.67 Mcal/kg of NEl) plus concentrate (14.8% CP, 14.7% ADF, 28.9% NDF, and 1.65 Mcal/kg of NEl).

TECHNICAL NOTE: Diet Influences on Pasture In Vitro Digestibility

Kolver, E. S., and L. D. Muller. 1998. Performance and nutrient intake of high producing cows consuming pasture or a total mixed ration. J. Dairy Sci. 81:1403.

search results and ration balancing, resulting in nutrient balance and supplementation recommendations for pasture-based diets.

Implications Pasture IVDMD was affected by both rumen inoculum source (pasture vs TMR) and by individual donor cow. These results emphasize the importance of using a donor cow that is consuming a diet similar to that being tested, particularly in research trials. Often times in pasture research trials, pasture samples are collected and stored until after the grazing season and analyzed in the wintertime, when a pastured donor cow would not be available. Feed ingredients of the diet of the donor cow, as well as chemical composition, should be described when reporting IVDMD in research publications. Maintaining a pastured fistulated cow is not an economically feasible option for commercial laboratories, and nutritionists, researchers, and others who use pasture IVDMD values that were analyzed by outside labs should be aware of this when interpreting the IVDMD results.

Acknowledgments The assistance of J. L. Stack (The Pennsylvania State University), M. D. Rubano, and C. U. Coiner (USDA-ARS) is gratefully acknowledged in conducting this study.

49

Literature Cited AOAC. 1990. Official Methods of Analysis. 15th ed. AOAC, Arlington, VA. Ayres, J. F. 1991. Sources of error with in vitro digestibility assay of pasture feeds. Grass Forage Sci. 46:89. Bargo, F., L. D. Muller, J. Delahoy, and T. Cassidy. 2002. Performance of high producing dairy cows with three different feeding systems combining pasture and total mixed rations. J. Dairy Sci. 85:2948. Bezeau, L. M. 1965. Effect of source of inoculum on digestibility of substrate in in vitro digestion trials. J. Anim. Sci. 65:823. Cherney, D. J. R., J. Siciliano-Jones, and A. N. Pell. 1993. Technical note: Forage in vitro dry matter digestibility as influenced by fiber source in the donor cow diet. J. Anim. Sci. 71:1335. Church, D. C., and R. G. Petersen. 1960. Effect of several variables on in vitro rumen fermentation. J. Dairy Sci. 43:81. Delahoy, J. E., L. D. Muller, F. Bargo, T. W. Cassidy, and L. A. Holden. 2003. Supplemental carbohydrate sources for lactating dairy cows on pasture. J. Dairy Sci. 86:906. Donefer, E., E. W. Crampton, and L. E. Lloyd. 1960. Prediction of the nutritive value index of a forage from in vitro rumen fermentation data. J. Anim. Sci. 19:545. Grant, R., P. J. Van Soest, and R. E. McDowell. 1974. Influence of rumen fluid source and fermentation time on in vitro true dry matter digestibility. J. Dairy Sci. 57:1201. Holden, L. A. 2000. Comparison of methods of in vitro dry matter digestibility for ten feeds. J. Dairy Sci. 82:1791. Holden, L. A., L. D. Muller, and S. L. Fales. 1994. Estimation of intake in high producing Holstein cows grazing grass pasture. J. Dairy Sci. 77:2332.

Mabjeesh, S. J., M. Cohen, and A. Arieli. 2000. In vitro methods for measuring the dry matter digestibility of ruminant feedstuffs: Comparison of methods an inoculum source. J. Dairy Sci. 83:2289. Marinucci, M. T., B. A. Dehority, and S. C. Loerch. 1992. In vitro and in vivo studies of factors affecting digestion of feeds in synthetic fiber bags. J. Anim. Sci. 70:296. Marten, G. C., and R. F. Barnes. 1980. Prediction of energy digestibility of forages with in vitro rumen fermentation and fungal enzyme systems. In Standardization of Analytical Methodology for Feeds. W. J. Pigden, C. C. Balch, and M. Graham, Eds. p 61. Int. Dev. Res. Center, Ottawa, Canada. Nelson, B. D., H. D. Ellzey, C. Montgomery, and E. B. Morgan. 1969. Factors affecting the variability of an in vitro rumen fermentation technique for estimating forage quality. J. Dairy Sci. 55:358. Quicke, G. V., O. G. Bentley, H. W. Scott, and A. L. Moxon. 1959. Cellulose digestion in vitro as a measure of the digestibility of forage cellulose in ruminants. J. Anim. Sci. 18:275. Robertson, J. B., P. J. Van Soest, and F. Torres. 1972. Substitution of filter paper for crucibles in the in vitro rumen true digestibility determination. J. Dairy Sci. 55:1305. SAS User’s Guide: Statistics, Version 8.01 Edition. 1999. SAS Inst., Inc., Cary, NC. Soder, K. J., and L. A. Holden. 1999. Use of anionic salts with grazing prepartum dairy cows. Prof. Anim. Sci. 15:278. Soder, K. J., Sanderson, M. A., Stack, J. L., and Muller, L. D. 2004. Effect of forage diversity on intake and productivity of grazing lactating dairy cows over two grazing seasons. J. Dairy Sci. 87(Suppl. 1):30. (Abs.) Tilley, J. M. A., and R. A. Terry. 1963. A two-stage technique for the in vitro digestion of forage crops. Br. Grassl. Soc. 18:104.