Prediction of relative feed value of alfalfa hays harvested at different maturity stages using in vitro gas production

O Canbolat, A Kamalak, C O Ozkan, A Erol**, M Sahin* E Karakas***and E Ozkose*

Bursa Uludag University, Faculty of Agriculture, Department of Animal Science. Bursa, Turkey^*Kahramanmaras Sutcu Imam University, Faculty of Agriculture, Department of Animal Nutrition, Kahramanmaras, Turkey^Kahramanmaras Sutcu Imam University, Faculty of Agriculture, Department of Crop Science, Kahramanmaras, Turkey^**Bursa Uludag University, Yenisehir Vocational School, Bursa, Turkey*.
akamalak@ksu.edu.tr

Abstract

In this study the effect of maturity stage on chemical composition, relative feed value (RFV), dry matter digestibility (DMD), dry matter intake (DMI) and in vitro gas production, of alfalfa hays (Medicago sativa) was investigated. The potential of gas production technique to predict relative feed value (RFV) of alfalfa hays was also evaluated. Gas production was measured at 3, 6, 12, 24, 48, 72 and 96 hour incubation times.

The maturity had a significant effect on chemical composition, relative feed value and in vitro gas production, of alfalfa hays. With increasing maturity NDF, ADF and ADL increased and CP content, RFV, DMD and DMI decreased. The variation of RFV explained by the gas production ranged from 87.6 to 93.0. The gas production at 12 and 24 h incubation times explained 93.0 and 92.2 % of variation of RFV respectively. The gas production constants "a" and "b", alone or in combination, explained frpm 88 to 99.7% of the variation of RFV. The gas production rate constant "c" explained only 16.5 % of the variation of RFV.

Key words: Alfalfa hays, chemical composition, feed value, maturity stage

Introduction

Forages represent a major component of ruminant diets. Nutritive value of forages depends on their dry matter digestibility and voluntary dry matter intake. Relative feed value (RFV) is a widely accepted forage quality index in the marketing of hays in the United States of America. It was developed by the Hay Marketing Task Force of American Forage and Grassland Council (Rohweder et al 1978). RFV combines the estimates for forage digestibility and intake into a single number. RFV is calculated from estimation of acid detergent fiber (ADF) and neutral detergent fiber (NDF) (Caddel 2005). Hay producers and purchasers also use RFV in price discovery, especially in hay auctions (Undersander 2001). Forage seed producers use RFV to indicate variety improvement (Moore and Undersander 2002). Reference to RFV and the equations used to predict it appear in extension documents and textbooks (Kellems and Church 2002).

On the other hand, Menke et al. (1979) and Menke and Steingass (1988) developed the in vitro gas production technique to evaluate the nutritive value of forages and estimate the rate and extent of DM degradation indirectly using the gas production (CO₂) produced during fermentation.The in vitro gas production technique is claimed to be well correlated with animal performance (Ørskov 1989), food intake (Blummel and Ørskov 1993; Kamalak et al 2005a), microbial protein synthesis (Krishnamoorthy et al 1991) and in vivo digestibility (Khazaal et al 1993; Kamalak et al 2005a). However, there is no previous research about the relationship between RFV and in vitro gas production parameters.

The aim of this study was to: (i) determine the effect of maturity stage on the chemical composition, RFV, DMD and DMI and in vitro gas production of alfalfa hay (Medicago sativa); and (ii) evaluate the potential of the gas production technique to predict relative feed value of alfalfa hays.

Materials and Methods

Forage samples

The commercially available alfalfa variety (Elci) was sown in three replicate plots (5*1.2 m) for each harvesting stage in 2005 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 hays were harvested before flowering, at flowering and late maturity stages. The hay 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 stage were used.

Chemical analysis

Dry matter (DM) was determined by drying the samples at 105 ^°C overnight and ash by igniting the samples in a muffle furnace at 525 ^°C for 8 h. Content of nitrogen (N) was measured by the Keldahl method (AOAC 1990). The crude protein (CP) was calculated as N*6.25. Contents of neutral detergent fiber (NDF) and acid detergent fiber (ADF) of leaves were determined by the method of Van Soest et al. (1991). ADL contents of samples were determined following the method of Van Soest (1963).

Relative Feed Value

RFV was calculated from the estimates of DDM and DMI (Rohweder et al 1978).

% DDM = 88.9 - (0.779 * %ADF)

DMI % of BW = 120 / %NDF

RFV = (%DDM * %DMI) /1.29

DDM = Dry matter digestibility, ADF = acid detergent fibre (% of DM), DMI = Dry matter intake (% of BW), RFV = Relative feed value

Table 1. Legume, grass and legume-grass mixture quality standards
Quality standard ^a	CP, % of DM	ADF, % of DM	NDF, % of DM	RFV ^b
Prime	>19	<31	<40	>151
1	17-19	31-40	40-46	151-125
2	14-16	36-40	47-53	124-103
3	11-13	41-42	54-60	102-87
4	8-10	43-45	61-65	86-75
5	<8	>45	>65	<75
^a standard assigned by Hay Market Task Force of American Forage and Grassland Council^b Relative Feed Value(RFV )- Reference hay of 100 RFV contains 41 % ADF and 53 % NDF

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 alfalfa hay samples were incubated in the rumen fluid in calibrated glass syringes following the procedures of Menke and Steingass (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 pre-warmed at 39°C before injecting 30 ml rumen fluid-buffer mixture into each syringe followed by incubation in a water bath at 39 °C. 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'

Statistical Analysis

One-way analysis of variance (ANOVA) was carried out to compare the chemical composition, RFV, DMD and DMI and estimated parameters of in vitro gas production with harvesting maturity as the main factor using the General Linear Model (GLM) (Statistica 1993). Significance between individual means was identified using 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. Regression analysis was used to establish the relationship between RFV and in vitro gas production or estimated parameters of in vitro gas production.

Results and Discussion

The maturity stage had a significant (P<0.001) effect on the chemical composition and some estimated parameters of alfalfa hays (Table 2). With increasing maturity NDF, ADF and ADL increased and CP content, RFV, DMD and DMI decreased.

Table 2. The effect of maturity stage on the chemical composition (%), relative feed value, and predicted dry matter digestibility (%) and dry matter intake (% BW)
	Maturity stages
Parameters	Before flowering	Flowering	Late maturity	SEM	Sig.
NDF	30.95^a	44.70^b	54.15^c	0.495	***
ADF	20.20^a	31.15^b	36.25^c	0.430	***
ADL	7.80^a	9.35^b	13.40^c	0.184	***
CP	19.75^c	17.25^b	13.85^a	0.221	***
RFV	225^c	135.^b	1056^a	3.014	***
DMD	73.70^c	64.65^b	60.90^a	0.246	***
DMI	3.95^c	2.65^b	2.25^a	0.040	***
DM: Dry matter, NDF: Neutral detergent fiber, ADF: Acid detergent fiber, ADL: Acid detergent lignin, CP: Crude protein, RFV: Relative feed value, DMD: Dry matter digestibility, Dry matter intake*** P<0.001, Means within the same row without superscript in common are different. Sig: Significance level, SEM: Standard error of mean

This result is in agreement with the report of Kamalak et al (2005b) who found that NDF and ADF content of tumbleweed increased and CP content, DMD and DMI decreased with increased maturity. The RFV of alfalfa hays harvested at different maturity stages ranged from 106 to 225. As can be seen from the quality standards (Table 1), the grade of alfalfa hay harvested before flowering was prime whereas those of alfalfa hay harvested at flowering and late maturity were 1 and 2. These values are in agreement with those reported by Stallings (2005).

The decline in protein concentration with advancing maturity occurs both because of decrease in protein in leaves and stems, and because stems, with their lower protein concentration, make up a larger portion of the herbage in more mature forage (Buxton 1996). The average decreases in crude protein concentration with advance in maturity for several forages averaged 1 g kg^-1 d^-1 in data reported by Minson (1990). The decrease in DMD, DMI, RFV and gas production parameters are possibly associated with increased NDF and ADF contents (Wilson et al 1991) and increased lignification and decreased leaf: stem ratio (Hides et al 1983) as the plant matures. At early stages of growth, all parts of plants are highly digestible, but during stem elongation and flowering there is a more rapid decline in the digestibility of stem than of leaf (Terry and Tilley 1964).

Gas production decreased with increased maturity (Figure 1). The gas production of samples of alfalfa hay harvested before flowering was significantly (P<0.001) higher than those of alfalfa hays harvested at flowering and late maturity stages. This result is in agreement with findings of Zinash et al (1996), Lee et al (2000) and Kamalak et a (2005 b). They also found a decrease in gas production as the forage growing period was prolonged.

Figure 1. The effect of maturity on the gas production of alfalfa hays

The estimated gas production parameters of alfalfa hay harvested before flowering were significantly (P<0.001) higher than those of alfalfa hays harvested at flowering and late maturity stages, except for the estimate of gas production rate (c)

Table 3. The effect of maturity on the estimated parameters of in vitro gas production of alfalfa hays
	Maturity stages
Parameters	Before flowering	Flowering	Late maturity	SEM	Sig.
c	0.066	0.063	0.063	0.002	NS
a	3.75^c	2.91^b	2.15^a	0.205	***
b	71.40^c	67.17^b	62.34^a	0.491	***
a+b	75.15^c	70.21^b	64.49^a	0.407	***
c = the gas production rate constant for the insoluble fraction (b), a = the gas production from the immediately soluble fraction (ml), b = the gas production from the insoluble fraction (ml), a+b: Potential gas production, Means within the same row without superscript in common are different SEM: Standard error mean, Sig: Significance level

This result is in agreement with findings of Kamalak et al. (2005b) who also found a decrease in estimated parameters ("b" and "a+b") with increased maturity. There were significant relationships between relative feed value and in vitro gas production and estimated parameters (Table 4). . The relationships between RFV and gas production at 3 and 6 h incubation times were a little bit lower than those obtained at 12, 24 and 48 h incubation times. After 48 h incubation times the relationship between these parameter decreased. The variation of RFV explained by the gas production ranged from 87.6 to 93.0%. The gas production at 12 and 24 h incubation times explained 93.0 and 92.2 % of the variation of RFV, respectively.

Table 4. Prediction of relative feed value from in vitro gas production and estimated parameters
Equations and factors used	R²	RSD	P value
RFV = -246 + 22.5gas_3h	90.0	19.6	0.004
RFV = -398 + 21.4gas_6h	90.1	19.5	0.004
RFV = -499 + 16.6gas_12h	93.0	16.5	0.002
RFV = -578 + 13.3gas_24h	92.2	17.3	0.002
RFV = -664 + 13.1gas_48h	91.2	18.5	0.003
RFV = -571 + 10.4gas_72h	87.6	21.9	0.006
RFV = -611 + 10.5gas_96h	89.4	20.2	0.004
RFV = -257 + 6367c	16.5	56.8	0.425
RFV = -61.4 + 72.7a	88.1	21.4	0.006
RFV = -714 + 13.0b	89.7	19.9	0.004
RFV = -615 + 11.0ab	89.5	20.1	0.004
RFV = -8782 +426c – 909a + 173b	99.7	4.73	0.000
RFV = -8825 – 916a + 175b	99.6	4.26	0.001
RFV = -754 +923c + 12.7b	90.0	22.6	0.031
RFV: relative feed value, c = the gas production rate constant for the insoluble fraction (b), a = the gas production from the immediately soluble fraction (ml), b = the gas production from the insoluble fraction (ml),

As can be seen from Table 4, equations predicting RFV from in vitro gas production showed that constants of the exponential equation alone or in various combinations were well correlated with RFV. The rate constant "c" alone was a poor predictor of RFV (R²= 16.5) and while "a" and "b" alone explained 88.1 and 89.7% of variation of the RFV, respectively. Adding "c" to equations containing "a" and "b" did not improve the R² values.

Support for the superiority of the immediately soluble fraction of forages as an indicator of digestibility can be seen in data reported for tropical grasses in Cameroon. The coefficient of determination (R²)between potential DM digestibility (in sacco 72 hour degradability) was 0.74 for water soluble DM compared with 0.31 for NDF (Enoh et al 2005). A close relationship between water-extractable DM and in vivo DM digestibility in buffaloes was reported by Nguyen Van Thu (2005). Russell and Karsli (2002) observed that the water soluble "a" fraction determined by the in sacco method was comparable with NDF as predictors of whole tract digestibility in cattle (R² of 0.83 and 0.85, respectively) and better than NDF for the prediction of voluntary feed intake (R² of 0.80 and 0.69, respectively).

Conclusions

The nutritive value of alfalfa hay, as estimated from the relative feed value (RFV) coefficient, was impaired with increased maturity.
There were high coefficients of determination between RFV and the "a" and "b" constants, alone or in combination, derived from the gas production technique, and a poor non-significant relationship with the rate constant "c".

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