Effect of variety on chemical composition, in vitro gas production, metobolizable energy and organic matter digestibility of alfalfa hays

A Kamalak, O Canbolat, A Erol^,C Kilinc^,M Kizilsimsek ^**, C O Ozkan and E Ozkose

Kahramanmaras Sutcu Imam University, Faculty of Agriculture, Department of Animal Nutrition, Kahramanmaras, Turkey^Bursa Uludag University, Faculty of Agriculture, Department of Animal Science. Bursa, Turkey^Kahramanmaras Sutcu Imam University, Faculty of Agriculture, Department of Crop Science, Kahramanmaras, Turkey^ Kahramanmaras Agricultural Experimental Station, Kahramanmaras, Turkey
akamalak@ksu.edu.tr

Abstract

In this study the effect of variety on chemical composition, in vitro gas production, organic matter digestibility (OMD) and metobolizable energy (ME) of alfalfa hays (Medicago sativa) were investigated. Gas production was measured at 3, 6, 12, 24, 48, 72 and 96 hours, and gas production kinetics were estimated using the equation y = a + b (1 - e^-ct).

Variety had a significant effect on chemical composition, in vitro gas production, OMD and ME. Crude fibre (CF) contents varied between 25.57 and 31.28 %. Kayseri had the highest CF content. Crude protein (CP) contents ranged from 15.05 to 21.39 %. Prista-2 had the highest CP content. Gas produced after 96 h incubation time ranged between 49.8 and 58.6 ml per 0.200 g of dry matter. OMD and ME of alfalfa hays ranged from 59.15 and 66.33% and 8.65 to 9.76 (MJ/kg Dry matter (DM)) respectively.

Linfa, X-1313 and Elci with higher in vitro gas production, OMD and ME can be recommended for hay production to meet livestock forage requirement. In addition to chemical analysis in vitro gas production technique is very useful and simple to determine the variety differences in terms of in vitro gas production, OMD and ME.

Key words: alfalfa hay, digestibility, gas production, metabolizable energy, variety

Introduction

In southern Turkey hay production from alfalfa to meet ruminant forage requirements has gained popularity lately since alfalfa is considered to be higher in crude protein and metabolizable energy than other forages (Marten et al 1998). Alfalfa produces more protein per hectare than grain and oilseed crops. Alfalfa forage is used to meet livestock's requirements especially dairy cows (Mir et al 1996).

The introduction of new alfalfa varieties has provided new raw materials with a range of nutritional characteristics. The newest varieties of alfalfa are evaluated by farmers and plant breeders for their dry matter yield for the selection of suitable variety to be used for hay production. In addition to dry matter yield, OMD and ME are very important parameters in ration preparation in practice (Lee et al 2000). More information about alfalfa varieties is required by the farmers to make more sound decision.

Digestibility experiments provide an important method of determining the nutritive value of ruminant feeds. They are, however, expensive and time consuming to perform. The use of animals to measure dry matter degradability, OMD and ME requires large quantities of food. (Minson 1998). It is not always possible to evaluate the nutritional characteristics of the individual feed components in a complex feeding experiment. For these reason there have been a number of attempts to develop rapid simple, reliable and cheap in vitro methods which can be used to screen a large numbers of raw feed materials and predict their digestibility and metabolizable energy content. Menke et al (1979) developed the in vitro gas production technique to evaluate the nutritive value of feedstuffs. In vitro gas production is a method that detects small differences among feedstuffs and allows more frequent sampling than in vitro digestibility (DePeters et al 2003).The gas produced during fermentation can be used as an indirect measure of dry matter degradability. The gas produced in 24 h from incubating 200 mg food dry matter can be used together with the concentration of crude protein and ash to estimate metabolizable energy and organic matter digestibility (Menke et al (1979). Gas production is associated with volatile fatty acid production following fermentation of substrate so the more fermentation of a substrate the greater the gas production (Blummel and Orskov 1993). The in vitro gas production technique has been found to reliably predict the nutritive value of forages (Makkar et al 1996). In vitro gas production method has been used to compare sorghum grain hybrids that differed in endosperm colour (Streeter et al 1993), to evaluate effects of varieties, growing sites, and grain species (Opatpatanakit et al 1994) and compare forage quality (Siaw et al 1993, Ronguillo et al 1998).

The aim of this study was to determine the effect of alfalfa species variety on chemical composition, gas production, OMD and ME of alfalfa hays.

Materials and Methods

Forage Samples

Fourteen commercially available alfalfa varieties were sown in four replicate plots (5X1.2 m) in 2002 at the seed rate of 30 kg ha^-1. Fifty kg/ha^-1 N and 150 kg/ha^-1 P₂O₅ fertilizers were used. Each plot was irrigated as required for optimal growth. Alfalfa varieties in each plot were harvested at beginning of flowering (~ 10 %) which is common practice in Turkey for hay production. The samples were shade-dried. Representative samples for each variety from each plot were taken to the University laboratory for chemical analysis and in vitro fermentation. Three samples for each variety were used.

Chemical Analysis

DM was determined by drying the samples at 105 ⁰C overnight and ash by igniting the samples in muffle furnace at 525 ⁰Cfor 8 h. Nitrogen (N) content was measured by the Kjeldahl method (AOAC 1990). CP was calculated as N X 6.25. CF and ether extract (EE) were determined by the methods described by the AOAC (1990). All chemical analysis was carried out in duplicate.

In vitro gas production

Rumen fluid was obtained from two fistulated sheep fed twice daily with a diet containing alfalfa hay (60%) and concentrate (40%). The samples were incubated in the rumen fluid in calibrated glass syringes following the procedures of Menke et al (1988) as follows. 0.200 g dry weight of the sample was weighed in triplicate into calibrated glass syringes of 100 ml. The syringes were prewarmed at 39 ⁰C before injecting 30 ml rumen fluid-buffer mixture into each syringe followed by incubation in a water bath at 39 ⁰C. The syringes were gently shaken 30 min after the start of incubation and every hour for the first 10 h of incubation. Gas production was measured as the volume of gas in the calibrated syringes and was recorded before incubation (0) and 3, 6, 12, 24, 48, 72 and 96 hours after incubation. Total gas values were corrected for blank incubation which contains only rumen fluid. Cumulative gas production data were fitted to the model of Ørskov and McDonald (1979)

y= a + b (1-exp^-ct)

Where
a = the gas production from the immediately soluble fraction (ml)
b = the gas production from the insoluble fraction (ml)
c = the gas production rate constant for the insoluble fraction (b)
t = incubation time (h)
y = gas produced at time't'

The OMD of forages was calculated using equations of Menke et al (1979) as follows:

OMD (%) = 14.88 + 0.889 GP + 0.45 CP + XA

Where
GP is 24 h net gas production (ml / 200 mg),
CP = Crude protein (%)
XA = Ash content (%)
ME (MJ/kg DM) content of forages was calculated using equations of Menke et al (1979) as follows:

ME (MJ/kg DM) = 2.20 + 0.136 GP + 0.057 CP + 0.0029CP²

Where
GP is 24 h net gas production (ml/200 mg),
CP = Crude protein

Statistical Analysis

One-way analysis of variance (ANOVA) was carried out to compare chemical composition, gas production kinetics, OMD and ME values using General Linear Model (GLM) of Statistica for windows (1993). Significance between individual means was identified using the Tukey's multiple range test (Pearse and Hartley 1966). Mean differences were considered significant at P<0.05. Standard errors of means were calculated from the residual mean square in the analysis of variance.

Results and Discussion

Significant (P<0.001) differences in the chemical composition of the varieties of alfalfa tested were found (Table 1). CF concentrations of hays ranged from 25.57 to 31.28 %. CF content of Kayseri was significantly (P<0.001) higher than the others except for Prista-2 and Elci. CF contents reported in this experiment are consistent with those reported by Zervas et al (1996). CP contents of Prista-2 and Sital were significantly (P<0.001) higher than the others. CP content varied between 15.05 and 21.39 % which is in agreement with those reported by Mir et al (1996) and Giger-Riverdin (2000). It is well known that CP of forages is highly influenced by the fertilization and maturity (Giger-Riverdin 2000). EE content of alfalfa hays varied between 1.20 and 2.29 %. EE contents of Linfa was significantly (P<0.001) higher than the others except for Belia, Prista-2 and Sital. The ash contents of alfalfa hays ranged from 10.33 to 11.65 %, which are in agreement with that reported by Giger-Riverdin (2000).

Table 1. Chemical composition (%) of alfalfa hays from 14 different varieties (N=84)
Variety	DM	CF	CP	EE	Ash
Kayseri	93.23	31.28^g	16.04^abc	1.90^cd	10.33^a
Belia	92.99	27.34^abcd	17.20^bcde	2.03^de	11.49^cd
Resis	92.90	27.39^abcd	17.99^e	1.86^cd	11.14^bcd
Prista-2	93.39	29.42^efg	21.39^f	2.03^de	10.78^ab
P.5683	93.05	27.26^abcd	17.95^de	1.96^d	11.65^d
Sital	92.78	26.00^ab	20.37^f	2.03^de	11.14^bcd
Daisy	92.77	25.57^a	17.05^bcde	1.63^bc	11.46^cd
Labo	92.79	28.20^cde	17.23^bcde	1.59^bc	11.40^cd
X-1313	92.85	26.98^abc	17.32^cde	1.61^bc	10.99^bc
Bilensoy	92.94	29.08^def	17.33^cde	1.43^ab	10.69^ab
Marina	93.20	28.01^cde	16.59^bcd	1.50^ab	11.07^bc
Elci	93.87	31.07^fg	15.05^a	1.20^a	10.66^ab
Eagle	93.48	27.95^bcde	15.87^ab	1.88^cd	11.16^bcd
Linfa	93.32	27.56^abcde	16.87^bcde	2.29^e	10.45^ab
SEM	0.265	0.387	0.268	0.060	0.099
Sig.	NS	***	***	***	***
Means within the same column wit differing superscripts are significantly different. DM: Dry matter, CF: Crude fibre, CP: Crude protein, EE: Ether extracts, NS- Non-significant. SEM: Standard Error Mean, *** P<0.001, Sig: Significance level

Data of gas production during the fermentation period were given in Figure 1. The cumulative volume of gas production increased with increasing time of incubation. Gas produced after 96 h incubation ranged between 49.8 and 58.6 ml per 0.200 g of dry matter.

There were considerable variations among alfalfa varieties in terms of gas production at all incubation times. As can be seen from Figure 1 Linfa, X-1313 and Elçi had considerable higher gas production.

Figure 1. Gas production during the fermentation period

The gas production at 48 h incubation time is in consistent with those reported by Getachew et al (2002). On the other hand the gas production volumes at all incubation times except at 3 h incubation time was 5-10 ml lower than those reported by Filya et al (2002).

There were significant (P<0.001) differences among varieties in terms of estimated degradation and calculated nutritional parameters (Table 2). X-1313 and Linfa had a significantly (P<0.001) higher gas production from quickly soluble fraction (a) than those of Eagle, Marina, Labo, P.5683 and Belia. There were no significant (P>0.05) differences among varieties in terms of gas production rate (c). As can be seen from Table 2 the gas production from slowly degradable fraction (b) and potential gas production (a+b) values of X-1313 and Linfa were significantly higher than the others except for Elci.

The intake of a feed is mostly explained by the rate of gas production (c) which affects the passage rate of feed through the rumen, whereas the potential gas production (a +b), is associated with degradability of feed (Khazaal et al 1995). Therefore the higher values obtained for the potential gas production in Linfa, Elci and X1313 might indicate a better nutrient availability for rumen microorganisms.

The gas production (a) from quickly soluble fraction is in agreement with those reported by Filya et al 2002 whereas gas production (b) from slowly fermentable fraction and potential gas production (a+b), and gas production rate (c) was lower than those reported that by Filya et al (2002). The reason why the gas production and the estimated parameters obtained in this experiment are lower than those reported by Filya et al (2002) is possibly due to differences in rumen fluid content and chemical composition of hays used. CF contents of alfalfa hays used in the experiment carried out by Filya et al (2002) was considerably lower than alfalfa hays used in this experiment. It is well known that there was a negative correlation between cell wall constituents and gas production (Abdulrazzak et al 2000)

Table 2. The parameters estimated from the gas production of alfalfa hays
Variety	a	b	a+b	c	OMD	ME
Kayseri	2.8^bcd	48.5^de	51.4^de	0.08	61.45^bcd	9.02^cde
Belia	1.6^a	48.2^cd	49.8^abcd	0.08	62.67^def	9.19^de
Resis	2.4^abcd	44.6^a	47.0^a	0.08	59.37^ab	8.68^ab
Prista-2	2.7^bcd	45.4^abc	48.1^abc	0.08	61.61^cde	8.98^abcd
P.5683	1.8^ab	48.6^de	50.5^bcd	0.09	63.62^efg	9.32^ef
Sital	2.6^abcd	47.6^bcd	50.2^bcd	0.08	62.62^def	9.14^cde
Daisy	2.6^abcd	47.4^abcd	50.1^abcd	0.08	61.15^abcd	8.95^abcd
Labo	2.0^abc	48.9^de	50.9^cde	0.08	62.06^cde	9.09^cde
X-1313	3.5^d	52.0^f	55.6^f	0.08	64.97^gh	9.54^fg
Bilensoy	2.4^abcd	44.9^ab	47.4^ab	0.07	59.15^a	8.65^a
Marina	2.1^abc	47.7^bcd	49.9^abcd	0.08	61.36^bcd	9.00^bcde
Elci	2.6^abcd	51.5^ef	54.1^ef	0.08	64.75^fgh	9.54^fg
Eagle	2.2^abc	47.5^abcd	49.8^abcd	0.08	60.29^abc	8.84^abc
Linfa	3.1^cd	53.2^f	56.3^f	0.08	66.33^h	9.76^g
SEM	0.12	0.57	0.61	0.005	0.418	0.063
Sig.	***	***	***	NS	***	***
Means within the same column with differing superscripts are significantly different. a = the gas production from the immediately soluble fraction (ml), b = the gas production from the insoluble fraction (ml), c = the gas production rate constant for the insoluble fraction (b), a+b: Potential gas production, ME: Metabolizable energy, OMD: Organic matter digestibility, *** P<0.001, NS- Non-significant. SEM: Standard error mean, Sig: Significance level

OMD and ME contents of Linfa was significantly higher than others except for Elci and X1313. OMD values of alfalfa hays from 14 different varieties varied between 59.15 and 66.33% which is consistent with those reported by Iantcheva et al (1999)

ME contents of alfalfa hays from 14 different varieties ranged from 8.65 to 9.76 (MJ/kg DM) which are consistent with those reported by Filya et al (2002) and Getachew et al (2002).

Chemical analysis is essential for understanding the nutritional potential of forages but it is not sufficient. Sital might appear to be an excellent variety when evaluated using only proximate analyses, however, OMD and ME calculations based on gas production show Sistal to be lower nutritive value than Linfa, Elci and X-1313. Choosing the best variety of alfalfa using the OMD and ME values seems to be sound since these parameters were calculated the some parameters related to chemical compositions and gas productions. OMD and ME are very important parameters to evaluate and compare forages. OMD and ME estimates for forage can be applied in developing countries because it is not only rapid, convenient and cost saving but also suitable for practical application. It can be adapted to evaluate various sources of forage with different compositions and provide data for the most important nutrients and energy sources for proper ration formulation in practice.

Conclusion

The tested varieties of alfalfa differed significantly with respect to chemical composition, in vitro gas production, OMD and ME. Linfa, X-1313 and Elci had higher in vitro gas production OMD and ME can be therefore recommended for hay production to meet livestock forage requirement.
In addition to chemical analysis in vitro gas production technique is very useful and simple to determine the variety differences in terms of in vitro gas production, OMD and ME.

References

Abdulrazzak S A, Fujihara T, Ondiek J K and Ørskov E R 2000 Nutritive evaluation of some acacia tree leaves from Kenya. Animal Feed Science and Technology 85: 89-98.

Association of Official Analytical Chemists (AOAC) 1990 Official Method of Analysis. pp.66-88. 15^th edition Washington, DC. USA.

Blummel M. and Ørskov E R 1993 Comparison of in vitro gas production and nylon bag degradability of roughages in predicting feed intake in cattle. Animal Feed Science and Technology 40:109-119.

De Peters E J, G Getachew G, Fadel J G Zinn R A, Taylor S J, Pareas J W, Hinders R G and Aseltine M S 2003 In vitro gas production as a method to compare fermentation characteristics of steam-flaked corn. Animal Feed Science and Technology 105:109-122.

Filya I, Karabulut A, Canbolat O, Degirmencioglu T and Kalkan H 2002 Investigations on determination of nutritive values and optimum evaluation conditions by animal organisms of the foodstuffs produced at bursa province by in vivo and in vitro methods. Uludag Universitesi Ziraat Fakültesi Bilimsel Arastirmalar ve Incelemeler Serisi. No: 25, Bursa, pp.1-16.

Getachew G, Crovetto G M Fondevila M, Krishnamoorthy U, Singh B, Spanghero M, Steingass H, Robinson P H and Kailas M M 2002 Laboratory variation of 24 h in vitro gas production and estimated metabolizable energy values of ruminant feeds. Animal Feed Science and Technology 102:169-180.

Giger-Reverdin S 2000 Characterization of feedstuffs for ruminants using some physical parameters. Animal Feed Science and Technology 86:53-69.

Iantcheva N, Steingass N and Payloy D 1999 A comparison of in vitro rumen fluid and enzymatic methods to predict digestibility and energy value of grass and alfalfa hay. Animal Feed Science and Technology 81:333-344.

Khazaal K A, Dentino M T, Ribeiro J M and Ørskov E R 1995 Prediction of apparent digestibility and voluntary intake of hays fed to sheep: comparison between using fibre components, in vitro digestibility or characteristics of gas production or nylon bag degradation. Animal Science 61:527-538.

Lee M Y, Hwang S Y, Chiou P W S 2000 Metabolizable energy in Taiwan. Small Ruminant Research 36:251-259.

Makkar H P S, Goodchild A V, El-Monein A.A and Becker K 1996 Cell-constituents, tannin levels by chemical and biological assays and nutritional value of some legume foliage and straw. Journal of Food and Agriculture 71:129-136.

Marten G C, Buxton D R and Barne R F 1998 Feeding value (forage quality). A.A In: Alfalfa and Alfalfa improvement. (Editors Hanson, D K Barnes and R R Hill Jr). American Society of Agronomy. Madison, WI. pp.463-491.

Menke K H, Raab L, Salewski A, Steingass H, Fritz D and Schneider W 1988 Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Animal Research Development, 28:7-55.

Menke K H, Raab L, Salewski A, Steingass H, Fritz D and Schneider W 1979 The estimation of digestibility and metabolizable energy content of ruminant feedstuffs from the gas production when they incubated with rumen liquor in vitro. Journal of Agricultural Science (Cambridge) 92:217-222.

Minson D J 1998 A history of in vitro techniques. In: In vitro techniques for measuring nutrient supply to ruminants. (Editors E R Deaville, E Owen, A T Adesogan, C Rymer, J A Huntington and T L J Lawrence. Occasional Publication. British Society, Animal Science. No: 22. pp. 13-19.

Mir Z, Acharya S N, Mir P S, Taylor W G, Zaman M.S, Mears G J and Goonewardene L A 1996 Nutrient composition, in vitro gas production and digestibility of fenugreek (Trigonella foenum-graecum) and Alfalfa forages. Canadian Journal of Animal Science 77:119-124.

Opatpatanakit Y, Kellawya R, Clean I J, Annison G and Kirby A 1994 Microbial fermentation of cereal grains in vitro. Australian Journal of Agricultural Research 45:1247-1263.

Ørskov E R and McDonald P 1979 The estimation of protein degradability in the rumen from incubation measurements weighed according to rate of passage. Journal of Agricultural Science 92:499-503.

Pearse E S and Hartley H O 1966 Biometrica tables for statisticians. Volume 1. Cambridge University Press

Ronguillo M G, Fondevilla M, Urdenata A B and Newman Y 1998 In vitro gas production from buffel grass fermentation in relation to the cutting interval, the level of nitrogen fertilization and the season of growth. Animal Feed Science Technology 72:19-32.

Siaw D E K A, Osuji P O and Nsahlai I V 1993 Evaluation of multipurpose tree germplasm: the use of gas production and rumen degradation characteristics. Journal Agricultural Science (Cambridge) 120:319-330.

Stastica 1993 Stastica for windows release 4.3, StatSoft, Inc. Tulsa, OK

Streeter M N, Hill G M, Wagner D G, Hibbert C A and Owens F N 1993 Chemical and physical properties and in vitro dry matter and starch digestion of eighth sorghum grain hybrids and maize. Animal Feed Science Technology 44:45-58.

Zervas G, Fegeros K and Papadopoulos G 1996Feeding system of sheep in a mountainous area of Greece. Small Ruminant Research 21:11-17.