Albanian j. agric. sci. 2017; (Special edition)

Agricultural University of Tirana

(Open Access)

RESEARCH ARTICLE

Chemical content and in vitro digestibility of successive cuts o fresh alfalfas and its hay. NERTILA MALUSHI1, 2*, MARGARIDA R.G. MAIA2, ANA R:J. CABRITA2, LUMTURI PAPA1, HUGO M. OLIVEIRA2, 3, ANTÓNIO J.M. FONSECA2, ASLLAN CELAMI4, 1

Department of Animal Production, Agricultural University of Tirana, Koder Kamez 1001, Albania

2

REQUIMTE, LAQV, ICBAS, Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Rua Jorge Viterbo

Ferreira, 228, 4050-313 Porto, Portugal 3 INL, International Iberian Nanotechnology Laboratory, Avenida Mestre José Veiga s/n, 4715-330 Braga, Portugal 4

Center of Agricultural Technology TransferFushë-Krujë

*Corresponding author; E-mail: [email protected]

Abstract Chemical analyses, enzymatic and Tilley & Terry digestibility were used to describe the feeding values of eight alfalfa samples. Six fresh alfalfa samples of different successive cuts and two alfalfa hays of two successive cuts of local varieties grown in Albania were used in this study. All samples were analysed for their chemical content of dry matter (DM), crude protein (CP), ash, crude fat (CF), neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL), and digestibility of organic matter (OMD). Fresh alfalfa samples had significant variation on chemical content for main Weende parameters. Early and late cut alfalfas have lower values for NDF, ADF and ADL content compering to medium cut. The values for CP of fresh alfalfas ranged from (17.0-22.7%) and for alfalfa hays (12.2-19.7%), the content of CF ranged from (28.2-40.0%) for fresh alfalfas and (26.8-29.6%) for hays. Ash content varied from (8.60-10.8%) and (8.90-10.2%), respectively for fresh and hays alfalfas. The NDF, ADF and ADL content of alfalfas varied respectively (39.6-52.4%), (30.543.1 %), (5.70-10.3%) for fresh alfalfas and (38.0-47.5%), (31.4-33.4%), (6.40-7.60%) for hays. The OMD digestibility values determined with Tilley and Terry method for all feeds included in the study resulted higher than OMD determined with enzymatic method respectively (66.9 vs 51.3%). The results of OMD determined by two “in vitro” methods were strongly correlated. According to R 2-value (0.89) the OMD determined by Tilley and Terry method could be predicted from enzymatic test as most convenient one since it does not need animals. Key words: Fresh alfalfa, hay, cut, chemical content, in vitro digestibility, ruminant feeding

Introduction

3]. New varieties have been introduced in Albania and their continuous study about agronomic performances

High feed quality is important for optimum

and feeding value estimation for animals is still in

animal performance. Highly digestible feed like

focus of research. In addition of chemical content

alfalfas promotes high animal intake. Alfalfa is

their differences according to cutting regime and

considered a premium feed for livestock due to its

preservation

high protein content and digestibility being very

digestibility with in vitro methods have been in centre

valuable for ruminant animal nutrition. Alfalfa forage

of many researchers of animal nutrition. Traditionally,

promotes high animal growth rates, milk production

feed value has been measured by “in vivo” trials to

and reproduction and for this reason they have been

asses a better digestibility evaluation but these

one of the most important forage crops in various

methods are time consuming and expensive. A lot of

countries of the world. Being as the most used forage

work has been made to standardize an in vitro method

for ruminant nutrition both fresh or hay all-round the

that can be commonly used, offering a much quicker,

year its feeding value evaluation for some local

cheaper and accessible method, that can predict the

varieties has been determined in pervious study [1, 2,

”in vivo”organic matter digestibility of feeds 11

method

the

prediction

of

their

from

Malushi et al., 2017

”in vitro” digestibillity method. In addition to

Digestibility triels

previous studies [4, 5], our aim is to determine the

Two “in vitro” methods were used to

nutritional value and the organic matter digestibilityof

determine the organic matter digestibility of selected

some alfalfas through Tilley and Terry and Pepsincellulase

in

vitro

methods

and

undertake

feeds: enzymatic (cellulase and pepsin) and Tilley and

a

Terry method.

comperative study for the determination of organic matter digestibility.

Tilley and Terry method

Materials and methods

Organic matter digestibility of alfalfa samples was determined in vitro using a modification by [15]

Samples

of the two-stage technique ofTilley&Terry [16]. Rumen fluid was obtained from twonon-pregnant and

Six warm seasons of fresh alfalfa with

non-lactating Holstein cows after a two weeks

different harvest date grown in Albania as the most

adaptation period to the diet with continuous access to

common feedstuffs used in Albania on ruminant

fresh drinking water, approximately 2 h after feeding.

nutrition and twoalfalfa hay of two successive cuts,

50 mL buffer-inoculum mixture as described by [17]

were selected to evaluate chemical content and in

was added to each of 50 mL conical centrifuge tubes

vitroorganic matter digestibility. The harvest year for

(Corning Inc., New York, NY, USA) containing 0.25

all alfalfas (Medicago sativa) was 2015. The collected

g DM of each experimental sample ground at 1 mm,

samples previously air-dried have been brought from

under purging with CO2. Blanks and samples were

Albania in labelled vacuum bags and milled according

incubated anaerobically for 48 h in a water-bath at 39

to the procedure for its analysis at Animal Science

◦C, in duplicate/inoculum/incubation.After the rumen

Laboratory of Abel Salazar Biomedical Sciences

fluid digest, the contents of each incubation tube were

Institute (ICBAS), University of Porto, Portugal.

filtered into glass crucibles under vacuum, dried incinerated in a muffle furnace at 500 °C at least 5 Chemical analysis

h,for the calculations of OMD.

All faba seeds samples were ground through a

Pepsin-cellulase procedure

1 mm screen and analyzed for main Weende The three stage procedure for assessing the in

parameters according to AOAC 2000 [6], for dry

vitro enzymatic digestion of alfalfa samplesreported

matter (DM) at 103 ºC, ash at 500 °C for at least 5 h

by [18], was carried outby adding 300mg DM of

[7], crude fiber (CF) treated with sulfuric acid

sample (ground to pass a 1 mm screen)to 50 mL

followed by potassium hydroxide [8], ether extract

capped conical centrifuge tubes (Corning Inc., New

(EE) was extracted for 3 h with petroleum ether[9],

York, NY, USA). Fifty millilitres pre-heated solution

whereas the Kjeldahl procedure was used to determine

of

nitrogen (N) [10, 11]. CP was calculated as N × 6.25.

Hemicellulose

1:10.000,

39 ºC for 24 h shaking some times. After 24 h

lignin (ADL). NDF and ADF were expressed with the ash.

(Pepsin

Thereafter the tubes were incubated in a water bath at

detergent fiber (ADF) and [13] for acid detergent residual

solution

Parkway) was added and the tubes were shaken.

determine neutral detergent fiber (NDF), acid

of

pepsin-HCl

Biotechnology, VWR AMRESCO, LLC, Fountain

Van Soest detergent system [12] was used to

inclusion

2%

incubation, the tubes were transferred in another water

was

bath for an acid hydrolysis at 80°C for 30 min. The

calculated as NDF – ADF and cellulose as ADF –

residue was then washed with 300 mL hot distilled

ADL [14]. Duplicate samples were analyzed for each

water at 40°C and vacuum filtered in a Dosi-Fiber

forage.

equipment (JP Selecta S.A., Spain) to glass crucibles

12

Chemical content and in vitro digestibility of successive cuts o fresh alfalfas and its hay.

that were then incubated for another 24 h in a water

The organic matter OM ranged from 89.2-

bath at 39 °C with 50 mL pre-heated cellulase-buffer

91.4% for fresh alfalfas respectively for alfalfa I and

from Trichoderma viride (Onozuka R-10, Yakult

III and for hays the OM content ranged from 89.8-

Pharmaceutical, Japan). At the end of second

91.1%, respectively for alfalfa second and third cut,

incubation the residue was washed again with 300 mL

similar with the values found by [22].The EE ranged

hot distilled water and vacuum filtrated in the Dosi-

from 0.90% for fresh alfalfa III to 1.60% for fresh

Fiber, dried for 48 h in an air-forced ventilated oven at

alfalfa VI and 1.20-1.50% for alfalfa hay second and

103°C, and ashed at 500°C for estimation of OMD.

third cut respectively, in harmony with the ones found

Blanks and samples were incubated in triplicate.

by [2, 23]. Among fresh alfalfas the highest content of

Statistical analysis

NDF was recorded for alfalfa IV(52.4%) while alfalfa The statistical analyses for

obtain the

VI presented the lowest content with 39.6%, fresh

correlation between two methods were carried out

alfalfa III showed higher ADF (43.1%) and ADL

using Minitab 18 software.

(10.3%) content and alfalfa I showed the lowest value

Results and discussions

of ADF (30.5%) and ADL (5.70%). Through alfalfa hays the highest NDF (47.5%) and ADF (33.4%)

Chemical composition of alfalfa samples:

content was found for alfalfa second cut while higher

The chemical composition of alfalfa samples is shown

ADL (7.60%) content for alfalfa third cut, comparable

in Table 1. For fresh alfalfas the ash content ranged

with the one obtained by[24].

from 8.60-10.8% respectively for alfalfa III and

The differences in cell wall content may be

alfalfa I. Meanwhile for alfalfa hays the ash content

due

ranged from8.90-10.2% respectively for alfalfa third

25]environmental conditions,agronomic factors[26,

and second cut. The values for ash content of our

27] and leaves to stem ratio [21].

alfalfa hays are in accordance with the values reported

to

differences

in

maturity

[19,

21,

The values of CP and CF content in our

by [19].

samples of fresh alfalfas reflect the fact that the early The highest protein content in fresh alfalfas

stage of maturity was generally higher in protein and

was found for alfalfa VI (22.7%) followed by alfalfa

lower in crude fiber, in agreement with the findings of

II (20.2%) while alfalfa III contain less crude protein

numerous workers [28, 29, 30, 31]. The chemical

(17.0%). The crude protein in alfalfa hays ranged

parameters for alfalfa hays in this study showed low

from 12.2% for second cut to 19.7% for third cut. The

quality for alfalfa hay second cut compering to alfalfa

values of CP for fresh alfalfa are similar with those of

hay third cut, in agreed in general with [32] early-cut

[20] and our findings for CP in alfalfa hays are

hays was equivalent to low quality.

comparable with [19, 21].

Digestibility of alfalfa samples: The organic

The results of crude fiber CF content obtained

mater digestibility data (OMD) with two “in vitro”

for fresh alfalfasextended from 28.2% for alfalfa I

methods are presented in Table 3.These data show

presented the lowest value followed by alfalfa VI with

that the extent of OMD obtained by the TT assay was

28.9% meanwhile alfalfa III and IV presented the

greater than that measured by the PC method with

highest and equal value of CF content with 40.0%.

average values respectively 66.9 vs. 51.3% of DM.

For alfalfa hay the alfalfa II showed the highest

Among fresh alfalfa samples the alfalfa I showed the

content with 29.6% and alfalfa III presented the

highest value of digested organic matter determined

lowest with 26.8%, comparable with the values found

by both methods, 74.1% by TT and 56.9% by PC

in[2].

method and for alfalfa hay the third cut presented the higher digested organic matter for TT and PC,

13

Malushi et al., 2017

respectively 71.9% and 60.5%. Tilley and Terry in

(Figure 1), had a R2 value of 0.89, suggesting

vitro method produced constituently higher values of

acceptable accuracy and precision. The values for

digested organic matter than pepsin-cellulase method

organic matter digestibility obtained by TT method

as more similar to in vivo digestibility.The Pearson

were regressed against the values obtained by PC

correlation coefficient (r) for all alfalfa samples in

method indicating that PC method can replace TT

these study (n= 8) were 0.945 showed a strong

method to assess the determination of organic matter

relationship for OMD determined by two in vitro

digestibility of alfalfa samples.In this situation

methods indicating that the enzymatic method was at

regression equation (Table 4) for alfalfa samples can

least good as Tilley&Terry method in predicting

be used to predict the expected value of OMD by TT

OMD. The obtained linear regression equation of

method having the result of PC method as more

Tilley and Terry and pepsin-cellulase procedure

convenient method since it does not need animals.

Table 1.Description of the species, variety, date of harvest and country of origin of some of the most common feedstuffs used in Albania. Harvest Nr.

Samples

Variety

Species

number

Harvest date

Year

Country

Fresh alfalfas 1

Alfalfa

BelaCampagnola

Medicago sativa

I

24/04/15

2015

Albania

2

Alfalfa

BelaCampagnola

Medicago sativa

II

22/05/15

2015

Albania

3

Alfalfa

BelaCampagnola

Medicago sativa

III

8/6/2015

2015

Albania

4

Alfalfa

BelaCampagnola

Medicago sativa

IV

13/07/15

2015

Albania

5

Alfalfa

BelaCampagnola

Medicago sativa

V

25/07/15

2015

Albania

6

Alfalfa

BelaCampagnola

Medicago sativa

VI

14/08/15

2015

Albania

Alfalfa hays 7

Alfalfa

BelaCampagnola

Medicago sativa

II

2015

Albania

8

Alfalfa

BelaCampagnola

Medicago sativa

III

2015

Albania

Table 2.Chemical content of alfalfacuts(%DM). Harvest Nr.

number

DM

Ash

OM

CP

CF

EE

NDF

ADF

ADL

NDIN

ADIN

Fresh alfalfas 1

I

92.1

10.8

89.2

19.6

28.2

1.2

41.9

30.5

5.7

0.35

0.19

2

II

92.3

10.6

89.4

20.2

32.6

1.1

44.5

36.2

7.9

0.38

0.25

3

III

92.3

8.6

91.4

17

40

0.9

51.5

43.1

10.3

0.3

0.23

4

IV

92.6

8.7

91.3

18.2

40

1.1

52.4

40.6

9

0.34

0.22

5

V

92.7

9.2

90.8

17.9

38

1.4

48

39.3

8.9

0.2

0.2

6

VI

92.6

10.4

89.6

22.7

28.9

1.6

39.6

32.2

7

0.18

0.21

Alfalfa hays 7

II

91.8

10.2

89.8

12.2

29.6

1.2

47.5

33.4

6.4

0.58

0.21

8

III

91.2

8.9

91.1

19.7

26.8

1.5

38

31.4

7.6

0.4

0.23

14

Chemical content and in vitro digestibility of successive cuts o fresh alfalfas and its hay. Table 3. In vitro organic matter digestibility (IVOMD) with enzymatic and Tilley and Terry methods of alfalfa samples (%DM). Nr.

Harvest number

IVOMDTT

IVOMDPC

Fresh alfalfas 1

I

74.1

56.9

2

II

67.5

49.9

3

III

58.1

43.7

4

IV

60.5

43

5

V

63.8

48.8

6

VI

71.9

56.6

Alfalfa hays 7

II

67.5

51.3

8

III

71.9

60.5

66.9±5.76

51.3±6.29

Mean ±SD

Tabe 4. Regression equations describing the relation between OMD estimated with Tilley&Terryand Pepsincellulase method. Feeds

Regression equation

r

R2

Standart

Propability

error

level (P)

Alfalfa

Y=0.8662 + 22.4348 * X1

0.94

0.89

2.03

0.01

X1 = OMD determined by Pepsin-cellulase. Y= OMD determined by Tilley&Terry. r= correlation coefficient of OMD-s determined by two “in vitro” methods. R2= determination coefficient. 70.0

y = 1.031x - 17.66 R² = 0.893

60.0

PC (%DM)

50.0 40.0 30.0 20.0 10.0 0.0 0.00

20.00

40.00

60.00

80.00

TT (%DM)

Figure 1.Relationship between Tilley-Terry and Pepsin-cellulase digestibility values on alfalfa samples.

15

Malushi et al., 2017

Biological Sciences 2008, 3 (10): 12271241.

Conclusions The results of the present study for chemical

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parameters are in agreement with the findings of the latter workers for alfalfa samples. The chemical composition of alfalfa samples reflects that they can be a good feed resource for ruminant animal’s nutrition. Among fresh alfalfas the alfalfa VIreflected good chemical parameters and she can be an excellent

3. Papa L, Kolaneci V, Daija A: The evaluation of organic matter digestibility of feeds for ruminat animals through two in vitro methods. Buletini i Shkencave Natyrore. Numer special 2011: 423-430.

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the alfalfa third cut produced the highest value of digested organic matter determined with both methods. Even if the Tilley and Terry in vitro method produced constituently higher values of digested organic matter than Pepsin-cellulase method, the

5. Cismileanu A, Dragomir C, Toma S, Grosu H, Mircea E: Comparison between the Tilley-Terry and Pepsin-Cellulase Methods, used to Predict the Digestibility of some Minor By-Products in Ruminants. Animal Science and Biotechnologies 2015, 72.

results of this study showed a strong relationbetween two methods, indicating that the pepsin-cellulase digestion procedure can be used to predict the digested organic matter of Tilley and Terry method. Anyway, more feed samples must be analysed with both methods in order to have anidealstatistical model

6. AOAC: Official Methods of Analysis: Arlington, VA, Association of Official Analytical Chemists, 2000.

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Acknowledgment

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