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Research Article

Agricultural Science Digest, volume 41 issue 2 (june 2021) : 295-300

Relationship between In vivo, In vitro Parameters and Chemical Composition to Predict the Nutritive Value of Some Legume Forages

A. Aïssa1,*, C. Ben Mustapha2, M.R. Alvir3, M. Hadj Ayed1, I.E.A. Znaïdi1, N. Moujahed2, K. Kraiem1
1Département de Ressources Animales, Agroalimentaires et Développement Rural, Institut Supérieur Agronomique de Chott-Mariem, BP 47, 4042 Chott-Mariem. Université de Sousse, Tunisie.
2Département des Sciences Animales, Institut National Agronomique de Tunisie. 43 Avenue Charles Nicolle, Tunis 1082. Université de Carthage, Tunisie.
3Departamento de Produccion Animal E.T.S.I.A Universidad Politécnica de Madrid 28040, Madrid (Espagne).
Cite article:- Aïssa A., Mustapha Ben C., Alvir M.R., Ayed Hadj M., Znaïdi I.E.A., Moujahed N., Kraiem K. (2021). Relationship between In vivo, In vitro Parameters and Chemical Composition to Predict the Nutritive Value of Some Legume Forages . Agricultural Science Digest. 41(2): 295-300. doi: 10.18805/ag.D-252.

ABSTRACT

Background: Many in vivo and laboratory methods have been used to evaluate ruminant feeds. The objective of this experiment was to determine feed intake, digestibility, kinetics of gas production, in vitro organic matter digestibility (OMDv) and metabolizable energy (ME) of Trifolium alexandrinum (T. alexandrinum) and Medicago sativa (M. sativa) at different maturity stages through in vivo and in vitro methods to elaborate predictive equations from chemical constituents. 

Methods: In vivo assay was carried out with two groups of five rams male kipped in metabolism cage. Samples of forage, refusal and feces were collected and processed for chemical analysis. In vitro gaz production technique was performed on forage samples.

Result: The bud and early bloom stage of M. sativa recorded the highest digestibility values of the chemical component (P<0.05). The OMDv of M. sativa was 60.65 and 68.26% (P<0.01) for early bloom stage and bud stage, respectively. Crude protein digestibility (CPD) was positively correlated with crude protein rate (R2=0.83, P<0.05). The gas production from the insoluble fraction “b” fraction showed a positive correlation with acid detergent fiber (ADF) (R2=0.999, P<0.001). However, cumulative gas production at 24h and 48 h of incubation were negatively correlated (P<0.05) with the ADF rate (R2= -0.98 and -0.97, respectively). 

INTRODUCTION

Chemical analysis allows to determine fodder chemical constituents which are used to evaluate the nutritive value of forages. But, this is not sufficient or accurate enough. To ensure the evaluation of digestibility of forages and to predict the nutritive value of feeds, in vivo and in vitro techniques were made available to livestock (Karabulut et al., 2007).
       
Trifolium alexandrinum L. (T. alexandrinum) and Medicago sativa (M. sativa) are popular among livestock farmers as “King and queen of Fodder Crops” and are cultivated on all the continents (Elif and Veysel, 2016; Singh et al., 2020). They provide a large amount of soluble protein and enrich the soil by fixing atmospheric nitrogen (Elif and Veysel, 2017). So, they allow an improvement of the productivity and the quality of final products.
       
The aim of this study was to determine (i) chemical parameters and nutritive value of Tunisian green chopped legume forages (T. alexandrinum, M. sativa) at different maturity stages, through in vivo and in vitro methods and (ii) to establish predictive equations of nutritive value parameters using chemical components.

MATERIALS AND METHODS

Forage samples
Trifoluim alexandrinum L. (variety Meskaoui) and Medicago sativa L. (variety of Gabes) were cultivated on small plots of 1000 m2 in the experimental station of the Agronomic Higher Institute of Chott-Meriem (35°56'17"N, 10°33'18"E) during winter and spring 2015. These forages were hand-harvested at different maturity stages: Bloom for T. alexandrinum and bud, early bloom and seed for M. sativa.
 
In vivo assay
 
For each trial, two groups of five rams male Black of Thibar with an average age of nine months and an average initial body weight of 33.2 ± 1.62 kg were kept in metabolism cage. The digestibility test includes a seven day adaptation period, a seven day preliminary period during which ingestion where the animals were fed ad libitum and a seven day collection period.
       
Intake was evaluated daily. Samples of the offered forage, the refusals and feces were collected in plastic bags and were stored at -20°C for further analysis. At the end of each period, those samples were combined homogeneously to determine their chemical composition and to evaluate feed intake and digestibility of dietary nutrients. Urine is collected for each animal in a 10 L plastic container containing 200 mL of a 10% solution of H2SO4. At the end of the collection period, the total amount of urine was measured and 20% of the total volume was stored in plastic bottles. At the beginning and at the end of each test, the animals are weighted.
 
Chemical analysis
 
Representative samples of forage, refusal and feces were dried at 60°C for 72 h and then grounded at 1 mm screen for chemical analysis composition. Ash, crude protein (CP) and crude fiber (CF) contents were quantified according to AOAC (2000) and acid detergent fiber (ADF), neutral detergent fibre (NDF) and acid detergent lignin (ADL) were analysed following the procedures described by Van Soest et al., (1991). 
 
In vitro assay
 
In vitro gas production was carried out using the method described by Menke and Steingass (1988). Rumen fluid was collected from four fistulated Black of Thibar sheep, combined, homogenized, filtered four layers of cheese-cloth and purged with CO2.The artificial saliva was prepared (Table 1). The inoculum fluid was prepared of rumen fluid and artificial saliva (1:2 v/v). This mixture (pH = 6.9± 0.1) was then kept under CO2 at 39°C water bath and stirred using a magnetic stirrer.
 

Table 1: Components of artificial saliva and their concentrations (Menke and Steingass, 1988).


       
200 mg of air-dry feedstuffs are incubated in 4 graduated glass syringes, preheated to 39°C, with 30 ml of the prepared mixture. The syringes are closed and the starting volume (V0) is recorded at time t0. Then, they were placed in the incubator. The syringes were gently shaken every 30 minutes during the first 8 to 10 hours of incubation. The volume of gas was recorded after 2, 4, 6, 12, 24, 36, 48, 72 and 96 hours of incubation. Three control syringes in each manipulation were incubated and considered as the blanks.
       
Total gas values were corrected for the blank incubation. Cumulative gas production data were fitted to the model of Ørskov and McDonald (1979):
 
y = a + b (1- e-ct)
 
Where
y =  Gas produced at the time “t”.
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 (h).
a+b =  The potential gas production (ml).
t = Incubation time (h).
       
Menke and Steingass (1988) have developed equations to calculate the in vitro digestibility of organic matter (OMDv) and the metabolizable energy (ME) of greens:
 
OMDv = 14.51 + 0.8490 × GP + 0.0653 × CP + 0.0686 × Ash
 
Where
GP = 24 h net gas production (ml/200 mg)
 
ME (kcal) = (1.14 + 0.1439 × OMDv - 0.0134 × Ash) × (1000/4.18)
 
Statistical analysis
 
The data were analyzed according to the General linear Model (GLM) procedure of the SAS software (2002) studying the effect of a one factor: maturity stage. Significance between individual means was identified using the Student test. Correlations between the in vivo, in vitro parameters and the chemical composition components were established then the Stepwise procedure was used to establish prediction equations.

RESULTS AND DISCUSSION

Chemical composition
 
The chemical composition of legume forages is shown in Table 2. The CP and CF of T. alexandrinum were founded to be 14.22% DM and 21.52% DM, respectively. Its CF content was higher but its CP content was lower than the values founded by Nefzaoui and Chermiti (1989).
 

Table 2: Chemical composition of legume forages.


       
The DM and CF content showed a gradual increase throughout the evolution of the maturity stage; the highest value is recorded at the final stage (P<0.01). However, the CP content noted a gradual decrease (P<0.001). Indeed, the highest CP content was founded at bud stage. The budding stage CP content was higher than the values reported by Nefzaoui and Chermiti (1989) and the tables of INRA (2007). Early bloom stage showed the highest NDF and ADF content (P<0.05). Its CF rate was lower than that founded by Nefzaoui and Chermiti (1989) and the tables of INRA (2007). The NDF and ADF contents were relatively identical with the values of the INRA (2007) and lower than that reported by Hollis et al., (2020).
 
Feed intake, nitrogen balance and nutrient digestibility
 
T. alexandrinum exhibited the highest value of DM intake, OM intake and intake/W0.75 (P<0.001) amounts (Table 3). These results were confirmed by Minson (1990) who mentioned that temperate legumes are ingested in large quantities because they offer less resistance to their reduction. M. sativa showed a decrease in DM intake, OM intake and intake/W0.75 between the bud and early bloom stage but at the pod stage the situation reversed.
 

Table 3: Feed intake, nitrogen balance, retained nitrogen and nutrient digestibility of legume forages.


       
The maturity stage had an effect on the N intake, N balance and retained N (P <0.001). The highest amount of N intake, N balance and retained N has been registered at bud stage of M. sativa.
       
The in vivo digestibility of DM, OM, CF, ADF (P <0.05) and CF (P <0.05) were affected by the maturity stage. Indeed, the highest digestibility values of the chemical component were recorded at bud and early bloom stage (Table 3).
       
The M. sativa CPD value was higher than the value reported in the INRA tables (2007). The results of the nitrogen balance followed the same trend as the digestibility of CPD. At the bud stage, the INRA tables (2007) reported values around 52% and 50% for the CFD and ADFD, respectively. The maturity stage has a very sensitive effect on the in vivo digestibility of the different chemical components, so the youngest maturity stage is the most digestible. During the plant aging, the cell walls thicken and become encrusted with lignin. This is not only indigestible, but it also constitutes a barrier to the walls digestion’s of the forage by the rumen microbes. This is confirmed by the low digestibility of forage fibers in the last maturity stage, which reflects a significant degree of lignification (Jarrige, 1988).
 
Kinetics of gas production
 
The Kinetics of gas production of T. alexandrinum and M. sativa, corrected with blanks are showed in Fig 1. The cumulative gas production profiles of legume forages increased during incubation period. A constant level of gas production was almost reached around 48 h and 72 h of incubation.
 

Fig 1: Kinetics of gas production of legume forages.


       
The M. sativa gas production kinetics curves for the three maturity stages were very close to each other with a slight superiority of the bud stage. At 72 h of incubation, the three curves were intertwined. The GP48 was slightly lower than those reported by Lantcheva et al., (1999) and similar to those reported by Karabulut et al., (2007).
       
The GP48 of T. alexandrinum was higher than that reported by Sallam et al., 2007 and Nasser et al., 2009).
 
Estimated kinetic parameters model, in vitro organic matter digestibility and energy content
 
« a » of T. alexandrinum showed an average value of 2.18 mL. It was higher than the values reported by Borba et al., (2001) and Nasser et al., (2009). All M. sativa “a” values were negative (P<0.05) (Table 4). Karabulut et al., (2007) reported a positive value of « a ». The absence of production means the incapacity of microorganism to develop in the medium (Rakotoarison, 2005) so there is a lag time preceding the start of the fermentations (Sallam et al., 2007; Elif and Veysel, 2017).
 

Table 4: Estimated kinetics parameters model, in vitroorganic matter digestibility and energy content of legume forages.


       
« b » ranged from 59.02 to 63.50 (P>0.05). « a+b » ranged from 54.55 to 65.68 and followed almost the same trend of «b» (Table 4). The « b » value of T. alexandrinum was higher than the values reported by Borba et al., (2001) and by Nasser et al., (2009). The « b » and the « a+b» of M. sativa was lower than the value reported by Karabulut et al., (2007).
       
« c » of T. alexandrinum was equivalent to 0.073 mL / h (P<0.05) and was lower than values reported by Borba et al., (2001) and Nasser et al., (2009). The rate of gas production was similar between the three maturity stages of M. sativa. It was higher than that founded by Borba et al., (2001) but lower than that noted by Karabulut et al., (2007). The high and fast gas production recorded for these legume forages means the presence of immediately and easily fermentable substances in this forage.
       
The OMDv showed the highest value (P <0.01) at the earliest maturity stage (Bud stage of M. sativa) (Table 4). For the calculated ME, the same trend was observed than that of OMDv (P<0.05).The OMDv of M. sativa was lower than that reported by Karabulut et al., (2007). The OMDv of T. alexandrinum was higher than that reported by Nasser et al., (2009) and Sallam et al., (2007).
       
The ME of M. sativa was lower than that founded by Kamalak et al., (2005b) and Karabulut et al., (2007).
 
Correlation among in vivo, in vitro parameters and chemical composition
 
Only, CPD was positively correlated with CP rate (P<0.05) (Table 5). It was also predicted by CP content in the French system but with better precision (Andriew et al., 1979).
 

Table 5: Correlation coefficient among feed intake, nutrient digestibility, estimated kinetic parameters, cumulative gas production, in vitro organic matter digestibility, metabolizable energy and chemical constituents of legume forages.


       
A few in vitro parameters have been found to have high and significant correlations with ADF content. “b” was positively correlated with ADF (P<0.001). GP24 and GP48 were negatively correlated (P<0.05) with the ADF rate. OMDv and ME were positively correlated with CP and negatively correlated with fiber content (Table 5). This result is consistent with the findings of Parissi et al., (2005), Kamalak et al., (2005a) and Karabulut et al., (2007).
 
Prediction of in vivo and in vitro parameters among chemical composition
 
The best prediction of CPD was obtained with CP (R2=0.69, P<0.01). The ADF was the best variable predictor since its R2 varies from 0.96 (P<0.05) for GP24 to 0.999 (P<0.001) for “b” (Table 6). This result is in agreement with the findings of Karabulut et al., (2007).
 

Table 6: In vivo and in vitro parameter prediction equations based on chemical composition.

CONCLUSION

It may be concluded that the maturity stage affected DM, CP, CF, NDF, ADF content, feed intake, in vivo digestibility, estimated parameters such as “a”, “c” fractions, OMDv and the ME. The ADF content had a determinent effect on the prediction of some gas production parameters. Therefore, further studies are needed to evaluate the nutritive value of other Tunisian green forages, hays and by-products to be more informative about ruminant nutrition.

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