Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
2004
Volume 7
Issue 2
Topic:
Veterinary Medicine
ELECTRONIC
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
Zawadzki W. , Kotecki A. , Czerski A. , Wincewicz E. , Kozak M. , Jeszka J. , Graczyk S. 2004. NUTRITIONAL VALUE AND FERMENTATION COURSE OF GROUND SEEDS OF YELLOW LUPIN OF MARKIZ AND TEO TYPES IN THE RUMEN FLUID IN CONDITIONS OF IN VITRO, EJPAU 7(2), #01.
Available Online: http://www.ejpau.media.pl/volume7/issue2/veterinary/art-01.html

NUTRITIONAL VALUE AND FERMENTATION COURSE OF GROUND SEEDS OF YELLOW LUPIN OF MARKIZ AND TEO TYPES IN THE RUMEN FLUID IN CONDITIONS OF IN VITRO

Wojciech Zawadzki, Andrzej Kotecki, Albert Czerski, Edyta Wincewicz, Marcin Kozak, Jan Jeszka, Stanisław Graczyk

 

ABSTRACT

The goal of the research has been to determine the influence of ground seeds of yellow lupin of MARKIZ and TEO types on the value of selected fermentation parameters of rumen fluid in vitro. The following fermentation parameters were analyzed: the concentration of ammonia, total protein, methane and lactic acid as well as pH of the contents. The samples for analysis were drawn in the 1st, 2nd, 4th, 6th and 24-th hour of fermentation. Fermentation was conducted in specially designed vessels. It was determined, basing on the conducted tests, that the addition of yellow lupin to the fermented rumen contents caused the increase of ammonia concentration in all samples (except a sample including TEO type seeds in the amount of 1g per vessel in the 24-th hour of fermentation). The highest increase of ammonia level was observed in the 24-th hour of incubation after addition of 5g of MARKIZ type lupin. The mean values of total protein in the fermented contents with the addition of both types of yell

Key words: fermentation, yellow lupin, sheep.

INTRODUCTION

The high nutritional value of yellow lupin is related to high protein content of high biological value and insignificant content of non-nutritional substances. The yellow lupin seeds include approx. 42-44% of general protein. The main protein consists of globulins, which constitute from 60% up to 75% of the general protein [13]. One negative property of protein included in the yellow lupin is its small amino-acid variety. The content of exogenous amino-acids is high, mainly methionine and cysteine, while the content of lysine and tryptophan is low. The lupin protein is digested well by pigs and poultry, and undergoes intensive decomposition in the rumen. Non-nutritional substances present in the yellow lupin seeds of high toxicity include lupinine and sparteine as well as tannin, whose content differs depending on genetic traits of the plant types [21]. There is no limit to lupin content in the correctly composed feeding of the ruminants. Lupin is a good fodder for fattened lambs, and can constitute 1/3 of the nutritional dose mass [6]. The addition of lupin seeds to the sheep fodder has a positive impact on their reproduction. It increases the levels of leptins, FSH, glucose and numerous amino-acids in the blood plasma [9]. The ovulation index in ewes also increases significantly [5, 16, 19]. Lupin constitutes a very good high-energy and high-protein fodder supplement for ruminants. Due to the introduction, in September of 2003, of a ban on using meals of animal origin in the feeding of ruminants [18], the protein included in the lupin seeds may become an alternative, filling the gap of high-protein fodder for animals. Intensively growing and intensively used animals (e.g. dairy cows) show particularly high demand for high-protein feed.

The aim of this research has been to examine the influence of the yellow lupin seeds on the selected rumen fermentation parameters in sheep in conditions of in vitro, for subsequent application of the obtained results in the in vivo research.

MATERIAL AND METHODS

The examination was conducted on 15 sheep, interracial hybrids (at the age from 2 to 4 and body mass of 40 up to 45 kg), from whom the rumen fluid and contents were taken 2.5 hours after morning feeding. The material for analysis was collected through the cannule of the dorsal rumen sack, made in accordance with Dejneka and Zięba method [4]. Incubation of the collected contents was conducted in the vessels (Fig. 1), made in accordance with Barnett and Reid method [2] with Zawadzki’s modification [22]. The incubation environment in the vessels included 50 ml of the collected rumen fluid contents, 50 ml of distilled water and 50 ml of McDougall’s artificial saliva of pH = 6.9 and the following composition (in 1 dm3 of distilled water): NaHCO3 – 9.60 g, KCl – 0.60 g, CaCl2 – 0.04 g, Na2HPO4·12H2O – 9.15 g, NaCl – 0.45 g, MgSO4·7H2O – 0.11 g, ZnSO4·H2O – 0.06 g, CaCl2·6H2O – 0.01 g, NH4HCO3 – 0.5 g. [2]. The control sample consisted of the vessels including 50 ml of the collected contents, 50 ml of distilled water and 50 ml of artificial saliva. Other vessels included 50 ml of the filtered rumen contents, 50 ml of distilled water and 50 ml of artificial saliva as well as ground seeds of the yellow lupin of MARKIZ and TEO types in three different doses: 1g, 2g and 5g per vessel. The total incubation volume of the sample in the vessels was 150 ml (with total vessel volume of 250 ml). The vessel leaktightness was secured by means of polished plugs and valves, covered with sealing silicon lubricant. Nitrogen was added to vessels under pressure to obtain anaerobic conditions. The gas was passed through the vessel content for 10-15 minutes. Then, all valves were closed. The maintaining of anaerobic conditions was checked with DO-5508 device (manufactured by Lutron). The vessels prepared in this way were then placed in the seat s of a shaker with water bath of 39-42°C temperature and amplitude of 4 cycles per minute [15]. The contents samples were collected in the 1st, 2nd, 4th, 6th and 24-th hour of incubation in order to determine the level of ammonia, lactic acid, total protein and methane as well as pH of the fermented rumen contents. The ammonia level was determined with Conway method [22], while the lactic acid concentration - with titration method. The total protein level in the sample was determined with the methods of Bradford [3] and Lowry et al. [14]. Methane concentration was measured by means of a methanometer of Barbara-3 type and method described by Varadyova and all [20]. The pH reaction of the collected samples of the incubated rumen contents was measured with pH-meter (pX-processor PM-600). The research results were elaborated statistically using the t-Student test and ANOVA variation analysis.

Fig. 1. The sheep rumen fluid incubation vessel with the supplement of yellow lupin grain

RESULTS AND DISCUSSION

The yellow lupin seeds include big amounts of plant protein, which is assimilated well by monogastric animals and constitutes a rich protein source for ruminants. They also include a number of macro- and microelements necessary for the organism [21]. Apart from the nutritive values of lupin, its cultivation contributes to soil fertilization by opening it, and atmospheric nitrogen bonding by nodule bacteria [10, 11, 17].

The addition of the yellow lupin to the fermented rumen contents resulted in the increase of ammonia concentration in all samples (except the sample with TEO type addition in the amount of 1g per vessel in the 24-th hour of fermentation) in comparison to the control sample. The highest increase (10.1 mmol/l, p<0.001) was observed in the 24-th hour of incubation after adding 5g of MARKIZ type lupin. An identical addition of TEO type caused the increase of ammonia concentration only by 2.72 mmol/l (table 1). As results from the obtained ammonia concentrations in the examined samples, the addition of MARKIZ type causes bigger production of ammonia (especially the doses of 2 and 5g) than the addition of TEO type (table 1).

As for the mean values of total protein in the fermented content, they were similar in the samples including both types of yellow lupin seeds (table 3). The high production of ammonia and total protein is the evidence of intensive fermentation processes of lupin protein by bacteria and protozoa existing in the rumen contents [12]. According to White et al. [21], the addition of lupin to sheep fodder results in the increase of total protein in the rumen contents. The ammonia concentration also increases, and pH value remains above 6.0, even with 70% addition of lupin seed to the nutritional dose [21]. The results of Allen et al. [1] also confirm the increase of ammonia concentration during sheep feeding with lupin. The addition of lupin seeds in the amount of over 2 kg/day may cause, as a result of a sudden change of diet, acidosis and necrosis of the rumen mucous membrane. It is connected with the increase of ammonia level (over 1400 mg/l) and lactic acid level (over 6 0 mmol/l) [1]. The above mentioned disorders should not occur during gradual introduction of lupin to the daily nutritional dose, especially in well fed sheep.

Table 1. The mean level of ammonia [mmol/l] during incubation of rumen fluid in in vitro research with the addition of 1g, 2g and 5g of MAZKIZ and TEO lupin grain

 

 

MARKIZ

TEO

Incubation time

Control group

1g

2g

5g

1g

2g

5g

1 hour

2.37

2.36

2.93

5.43***

2.79

2.19

2.94

2 hour

2.39

2.48

3.30*

6.33***

2.44

2.94

4.09**

4 hour

2.43

2.77

4.36**

7.07***

3.85**

2.39

7.33***

6 hour

2.79

3.19*

4.42**

7.66***

3.38*

3.49*

6.67***

24 hour

4.39

6.17*

10.11***

14.49***

2.29**

3.42*

7.11**

Essential statistical changes of ammonia concentration in comparison to ammonia concentration in the control group.
***p£ 0.001
  **p£0.01
    *p£0.05

Table 2. The mean level of lactic acid [mmol/l] during incubation of rumen fluid in in vitro research with the addition of 1g, 2g and 5g of MAZKIZ and TEO lupin grain

 

 

MARKIZ

TEO

Incubation time

Control group

1g

2g

5g

1g

2g

5g

1 hour

0.27

0.37

0.39

14.61**

1.35*

0.25

1.36*

2 hour

2.79

0.69*

1.59

3.99

1.95

0.49*

1.94

4 hour

3.79

0.78**

2.83

12.04**

0.47*

0.0043*

4.38

6 hour

4.99

1.11***

2.99

27.06***

1.07*

0.47*

20.92***

24 hour

5.79

2.99*

6.71

68***

4.14

41.07***

77.58***

Essential statistical changes of lactic acid concentration in comparison to lactic acid concentration in the control group.
***p£ 0.001
  **p£0.01
    *p£0.05

Table 3. The mean level of total protein [mmol/l] during incubation of rumen fluid in in vitro research with the addition of 1g, 2g and 5g of MAZKIZ and TEO lupin grain

 

 

MARKIZ

TEO

Incubation time

Control group

1g

2g

5g

1g

2g

5g

1 hour

4.65

6.84*

6.37*

10.04***

2.63

2.83

3.11

2 hour

4.72

5.52

5.96

10.34***

3.81

3.81

3.81

4 hour

6.61

5.95

6.21

9.47**

3.59

3.59

4.63

6 hour

5.67

5.41

6.8

7.75**

4.04

4.04

5.09

24 hour

6.61

5.52

7.07

6.83

4.73*

4.73*

8.26*

Essential statistical changes of total protein concentration in comparison to lactic acid concentration in the control group.
***p£ 0.001
  **p£0.01
    *p£0.05

The addition of lupin to the fermented rumen contents resulted in the increase of lactic acid concentration, particularly of the 5g dose (both MARKIZ and TEO type) in the 6-th and 24-th hour of fermentation. Smaller doses of lupin seeds did not cause statistically significant changes of the lactic acid level in the fermented rumen contents in comparison to the blind test (table 2). The addition of MARKIZ type in the amount of 5g caused the increase of lactic acid level by 8.24 mmol/l (in the 4-th hour of fermentation, p£0.01), 22.06 mmol/l (in the 6-th hour of fermentation, p£ 0.001) and 62.2 mmol/l (in the 24-th hour of fermentation, p£0.001) respectively in comparison to the values obtained from control samples. The addition of TEO type, on the other hand, in the amount of 2g (24-th hour of fermentation) resulted in the increase of the lactic acid level by 35.9 mmol/l of the fermented rumen content (p£0.001). The increase of lactic acid concentration was observed after the addition of 5g of TEO type in the 6-th and 24-th hour o fermentation – 15.92 mmol/l, 71.79 mmol/l of the fermented contents respectively. The lactic acid is mainly produced during the decomposition process of saccharides included in the fodder. The rumen bacteria include numerous enzymes enabling digestion and assimilation of both mono- and structural saccharides. The final product of complex carbohydrate decomposition are monosaccharides, mainly glucose. The produced glucose is absorbed by bacteria and transformed into lactic acid, which is then t ransformed into volatile fatty acids LKT. The lupin seeds include negligible amounts of starch (about 1.5%) and glucose. Lupin accumulates carbohydrates in the form of hemicellulose and oligosaccharides [7]. They are indigestible for monogastric animals, and are only digested in ruminants as a result of bacterial fermentation. The observed increase of lactic acid level proves the fermentation process of carbohydrates included in the lupin seeds.

During bacterial fermentation, gasses are also produced in the rumen. The most important ones include: carbon dioxide and methane [22]. The quantitative proportion of these two gases (CO2:CH4) is frequently used for the evaluation of the fermentation process correctness [22]. It increases with the introduction of nutritive fodder, and decreases when feeding with bulky feed [22, 23, 24, 25]. The production of large amounts of methane during fermentation causes considerable energy losses [22, 25]. The losses can be reduced through a modification of the nutritional dose or introduction of methanogenesis inhibitors into the fodder [25]. The addition of lupin of both MARKIZ and TEO type caused the reduction of methane production (table 4). It is very important from the point of view of animal production profitability, because it results in the better use of fodder by the animal. The reduction of methane production by lupin addition to the nutritional d ose is also of ecological importance [8]. Methane is classified as greenhouse gas, and its emission reduction through the ruminants diet modification has been the subject of numerous publications [8, 12, 22, 25].

Table 4. The mean level of methane [% vol.] during incubation of rumen fluid in in vitro research with the addition of 1g, 2g and 5g of MAZKIZ and TEO lupin grain

 

 

MARKIZ

TEO

Incubation time

Control group

1g

2g

5g

1g

2g

5g

1 hour

1.40

0.60**

0.58**

0.47***

0.55***

0.49***

0.54***

2 hour

1.30

1.05

1.00

0.90*

1.05

1.02

0.90*

4 hour

1.22

0.98

0.96

0.85*

1.10

0.95

0.84*

6 hour

1.25

0.96

0.92

0.98

0.90

0.98

0.86*

24 hour

1.35

0.85

0.83**

0.97

0.95

0.89

0.96

Essential statistical changes of methane concentration in comparison to methane concentration in the control group.
***p£ 0.001
  **p£0.01
    *p£0.05

The pH changes of the fermented rumen contents are shown in table 5. A statistically significant decrease of pH was observed, of the contents with addition of MARKIZ as well as TEO type seeds in the dose of 5g. The tendencies of pH changes are different in comparison to the changes of the lactic acid concentration.

Table 5. The mean value of pH during incubation of rumen fluid in in vitro research with the addition of 1g, 2g and 5g of MAZKIZ and TEO lupin grain

 

 

MARKIZ

TEO

Incubation time

Control group

1g

2g

5g

1g

2g

5g

1 hour

9.0

10.07

9.07

8.78

8.87

9.0

8.50

2 hour

8.50

10.21*

9.07

8.93

8.25

8.30

8.58

4 hour

8.50

10.07*

9.21

7.71

8.75

8.62

7.70

6 hour

8.30

10.12**

8.97

6.57**

8.62

8.55

6.28**

24 hour

8.0

8.07

8.50

4.79***

7.90

6.76**

5.50***

Essential statistical changes of pH rumen fluid in comparison to pH rumen fluid in the control group.
***p£ 0.001
  **p£0.01
    *p£0.05

CONCLUSIONS

The addition of the yellow lupin seeds to the sheep fodder has a positive influence on the fermentation process in their rumens. It is proved by a significant increase of the level of total protein, ammonia and lactic acid, especially after the introduction of higher doses of lupin (5g per vessel).

The lupin seeds may become a nutritional alternative which will substitute animal-origin fodder, withdrawn from ruminant feeding. The addition of lupin to the nutritional dose is also justified ecologically, since it reduces methanogenesis, thus contributing indirectly to the reduction of greenhouse gas emission.

SUMMARY

Lupin seeds constitute very good nutritive fodder (high-protein one) in the feeding of sheep. The research conducted in vitro, which demonstrated several-fold higher ammonia level with the addition of the yellow lupin seeds – particularly of MARKIZ type, confirms this fact. The increase of mean values of total protein in the rumen fluid was also observed, which was similar in case of both types of the yellow lupin and significantly higher than the values observed in case of control samples. The addition of both types of the yellow lupin seeds to the fermented rumen content samples also reduced the production of methane during fermentation.

The research has been conducted within the grant of the Committee of Scientific Research, No 53/4-R/01G.

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Wojciech Zawadzki
Institute of Animal Physiology
Agricultural University
Norwida 31, 50 – 375 Wrocław, Poland
e-mail: waza@ozi.ar.wroc.pl

Andrzej Kotecki
e-mail: kotecki@ekonom.art.wroc.pl

Albert Czerski
e-mail: albert-czerski@wp.pl

Edyta Wincewicz
e-mail: wince@ozi.ar.wroc.pl

Kozak Marcin
e-mail: kozak@ekonom.ar.wroc.pl

Jan Jeszka
e-mail:jeszkaj@au.poznan.pl

Stanisław Graczyk
e-mail: graczyk@ozi.ar.wroc.pl


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