close

Вход

Забыли?

вход по аккаунту

?

493

код для вставкиСкачать
J Sci Food Agric 1997, 75, 273È280
Maintenance of Rumen Protozoa Populations in a
Dual Outflow Continuous Fermenter
Laurent-Philippe Broudiscou,1* Yves Papon,1 Michel Fabre1 and
Anne F Broudiscou2
1 Station de Recherches sur la Nutrition des Herbivores, Institut National de la Recherche Agronomique,
Centre de Clermont-Theix, 63122 St Genès Champanelle, France
2 Laboratoire de Methodologie de la Recherche Experimentale, Universite dÏAix-Marseille III, 52 av
Escadrille Normandie Niemen, 13013 Marseille, France
(Received 27 September 1996 ; accepted 3 April 1997)
Abstract : Using a 25h2 fractional factorial design, Ðve factors were screened for
their ability to promote rumen protozoa growth in a dual outÑow fermenter and
on their inÑuence on fermentation. The tested factors were the addition of bovine
serum (BS, 20 ml litre~1) and of a 100 g litre~1 yeast extract solution (20 ml
litre~1) to artiÐcial saliva, the addition of diethystilbestrol (15 mg day~1) to fermentation broths, the reduction of shear stresses by lowering the stirring speed
from 260 rpm to 230 rpm, and the improvement of ciliate sequestration by the
use of a polyurethane foam belt. In the fermentors, the ciliate population density
ranged from 10 kl~1 to 58 kl~1. The genus Isotricha was rarely observed, with a
population density estimated at \0É7 kl~1 while Dasytricha was not maintained.
All the experimental factors markedly inÑuenced total protozoa numbers. Lowering the stirring speed was the sole beneÐcial treatment. Nutritional supplements,
in particular BS, were all detrimental to Entodinium. Their association was characterised by a signiÐcant antagonism. The population size of Polyplastron and
Eudiplodinium was lowered only by BS addition, possibly through the changes
induced in the fermentation pattern.
J Sci Food Agric 75, 273È280 (1997)
No. of Figures : 0. No. of Tables : 5. No. of References : 18
Key words : rumen protozoa, continuous culture system, factors of maintenance
INTRODUCTION
ditions. Their ability to support a varied protozoal
ciliate population, however, is uneven (MansÐeld et al
1995). As protozoa are major agents in the rumen
microbial community, investigating the best set of
general operating conditions to maintain their population at high levels is an important issue. The present
work was designed to screen Ðve factors for their ability
to promote protozoal growth in a dual outÑow fermentor supplied with an intermediate diet made of
750 g kg~1 pelleted orchard-grass hay and 250 g kg~1
ground barley, and on their inÑuence on fermentation.
Three nutritional factors were investigated : the addition
of yeast extract, bovine serum and diethylstilbestrol.
Two physical factors were also considered : the
reduction of shear stresses by lowering the stirring
For many years in Europe, the need for reliable alternative techniques to animal experimentation has been
urged by ethical and legal prescriptions (Mahouy 1995).
In ruminant nutrition, several continuous culture
devices have been developed since the 1960s to study
the metabolism of rumen microbes. Mastering an artiÐcial situation and closely simulating rumen physiological mechanisms were the main trends in validating in
vitro systems. Dual effluent stirred-tank reactors
(Hoover et al 1976 ; Merry et al 1987) have proved to be
suitable for the accurate control of environmental con* To whom correspondence should be addressed.
273
( 1997 SCI.
J Sci Food Agric 0022-5142/97/$17.50.
Printed in Great Britain
L -P Broudiscou et al
274
speed, and the improvement of ciliate sequestration by
the use of a polyurethane foam belt.
EXPERIMENTAL
The study was conducted in dual outÑow continuous
fermenters adapted from Hoover et al (1976). In these
discontinuously stirred reactors, the fermentation broth
is characterised by a di†erential turnover of particulate
and liquid phases due to the presence of an overÑow
and an outlet equipped with a Ðlter (in our apparatus, a
100 km pore size stainless-steel gauze). Intact fermenter
contents leave the vessel by the overÑow, mainly during
the periodic mixings, while only liquids and small particles can be pumped through the Ðltering outlet. These
effluent fractions are currently called displaced effluent
and Ðltered effluent, respectively. The fermenters differed from the original apparatus in the daily collection
and analysis of fermentation gases, the supply of solid
substrates in separate meals rather than on a semicontinuous mode, the use of a marine impeller
(Applikon Dependable Instruments, Schiedam, The
Netherlands) and of baffles rather than Ñat-blade
turbine impellers for fermentation broth mixing. The
working volumes of vessels, which are adjustable in our
system, were set to 1100 ^ 10 ml.
Four wethers, equipped with a rumen cannula and
kept indoors in individual boxes, were used as donors of
rumen contents. The animals were fed on 900 g day~1
chopped hay and 300 g day~1 ground and pelleted
barley, distributed in two meals, and they had free
access to water and mineral supplement. Four litres of
rumen contents were withdrawn after a 24 h fast. They
were immediately strained through a 1 mm stainlesssteel screen and kept at 39¡C under a continuous Ñow
of N . Within minutes, the vessels, previously Ðlled with
2
700 ml of artiÐcial saliva and 20 g of pelleted feed, were
inoculated with 400 ml of strained rumen Ñuid, Ñushed
with N then closed and started up. The fermenters,
2
maintained at 39¡C, were continuously infused with an
artiÐcial saliva containing Na HPO 12 H O (6É157 g
2
4
2
litre~1) NaHCO (5É268 g litre~1), KHCO (0É597 g
3
3
litre~1), NaCl (0É305 g litre~1), HClÈcysteine (0É4 g
litre~1) and adjusted to pH 10É0 with 1É63 g litre~1
NaOH. The fermentation broth was separately complemented with CaCl
(31É7 mg day~1), MgCl
2
2
(47É5 mg day~1) and (NH ) SO (0É755 g day~1). A pel42 4
leted diet made of 750 g kg~1 pelleted orchard-grass
hay and 250 g kg~1 ground barley was supplied to the
fermenters at the rate of 10 g at 10 :00 and 20 g at 18 :00.
The saliva and the Ðltered effluent were pumped to
obtain dilution rates of 0É03 h~1 and 0É06(^0É002) h~1,
respectively, for particle and liquid phases. All effluents
were separately collected in a container kept at ]4¡C.
The screening of environmental conditions favouring
the maintenance of a wide range of protozoal genera in
vitro considered three nutritional factorsÈthe addition
of bovine serum (BS, 20 ml litre~1) and of a 100 g
litre~1 yeast extract solution (YE, 20 ml litre~1) to the
artiÐcial saliva and the addition of diethystilbestrol
(DES, 15 mg day~1) to fermentation brothsÈand two
physical factorsÈthe stirring speed (SS, from 230 to
260 rpm) and the presence of a polyurethane foam belt
(F) disposed on the inner side of the culture vessel. All
chemicals were purchased from SIGMA. The polyurethane belt was organised into open cells of 300 km
diameter on average. It was 8 cm high, 5 mm thick, for
a volume of around 150 ml. We intended to estimate
independently all the main e†ects plus the two-way
interaction e†ects YE ] BS and YE ] DES. Therefore,
we selected a two-level fractional factorial design 25h2
(Haaland 1989) using generators I 4 124 4 235 4 1345.
Table 1 shows the corresponding experimental worksheet made of eight combinations of experimental treatments. The run K8 represented the presumed best set of
conditions for protozoa maintenance and it was applied
Ðve times to give an estimation of experimental error.
The 12 runs were randomly assigned to six independent
fermenters, identically assembled, which were operated
for two seven-day contiguous experimental periods. The
fermentation gas volumes and the amount of collected
effluents and delivered saliva were measured every day.
TABLE 1
Worksheet for the 25h2 experimental design
Run
Y east extract
Stirring speed
(rpm)
DES
Foam
Bovine serum
1
2
3
4
5
6
7
8
No
Yes
No
Yes
No
Yes
No
Yes
260
260
230
230
260
260
230
230
No
No
No
No
Yes
Yes
Yes
Yes
Yes
No
No
Yes
Yes
No
No
Yes
Yes
Yes
No
No
No
No
Yes
Yes
Maintenance of rumen protozoa in a continuous fermenter
After a 5-day adaptation period, the displaced and Ðltered effluents collected on days 6 and 7 were pooled
and kept at [20¡C prior to subsampling for volatile
fatty acids (VFA) and ammonia nitrogen (NH -N)
3
analysis. On day 6, the fermentation broths were
sampled at 16 :00 for pH, Eh, VFA and NH -N determi3
nation. On days 6 and 7 the fermentation gas composition was determined. At the end of the experimental
period, 16 h after the last input of solid substrate, the
fermenter contents were weighed, an aliquot was Ðltered
through a 200 km pore size nylon gauze, then the residues were washed in saline and Ðltered twice. Filtrates
were pooled, and a sample was Ðxed by addition of
three volumes of 13É3 ml litre~1 glutaraldehyde, 666 ml
litre~1 glycerol solution, for determination of the protozoa population in the vessels. In addition to total protozoa counting, the genera Entodinium, Polyplastron,
Eudiplodinium, Isotricha and Dasytricha were identiÐed
according to Ogimoto and Imai (1981) and enumerated
in a Jessen cell on a microscope (Nikon Labophot).
VFA were determined as described by Jouany (1982).
NH -N was determined as described by Davies and
3
Taylor (1965). Fermentation gases were analysed by gas
chromatography (column Ðlled with Carboxen 1000
(Supelco) at 100¡C). Individual gas molar concentration
was calibrated using a certiÐed standard (relative accuracy of 2%, Alphagaz no 073562.00). The hydrogen
balance was calculated as described by Demeyer and
Van Nevel (1975).
275
The results were submitted to linear regression by a
SAS procedure (SAS 1990), Data were Ðtted to the following model :
Y \ b ] b . Y E ] b . SS ] b . DES ] b . F
0
1
2
3
4
] b . BS ] b . Y E . DES ] b . Y E . BS (1)
5
13
15
for coded variables, Y E, DES, F, BS : [1 \ absent,
]1 \ present ; SS : [1 \ 260 rpm, ]1 \ 230 rpm. The
coefficient estimates of the model were compared to null
by StudentÏs t-test.
RESULTS
The mean ciliate population density in the rumen Ñuid
of donor animals was equal to 395É6 kl~1, 16 h after
feeding a meal. The population densities of the genera
Entodinium, Polyplastron, Eudiplodinium, Isotricha and
Dasytricha were respectively 368É0 kl~1 (93É0% of the
entire population), 5É8 kl~1 (1É5%), 8É0 kl~1 (2É0%),
3É4 kl~1 (0É9%), 10É4 kl~1 (2É6%). In the fermentors, the
ciliate population density ranged from 10 kl~1 to
58 kl~1. The genus Dasytricha was never observed. All
the experimental factors markedly inÑuenced total
protozoa numbers, mainly by a†ecting Entodinium
maintenance (Table 2). Nutritional supplements, in particular BS, were all detrimental to Entodinium. Their
association was characterised by a signiÐcant antagonism, as shown by the two-way interaction coefficient
TABLE 2
Regression analysis for protozoa population dataa
Response
Source
DF
T otal (]106)
SS
Model
Error
Total
R2
Adj R2
Exp error
7
4
11
2304
39É1
2343
Prob [ F
0É002
0É98
0É95
3É13
Factor
Coe†
Intercept
YE
SS
DES
F
BS
Y E ] DES
Y E ] BS
27É0
[4É7
3É1
[2É1
[6É0
[11É3
4É2
4É8
Entodinium (]106)
SS
2046
35É7
2082
Prob [ F
0É002
0É98
0É95
2É99
P-value
0É01
0É04
0É012
0É005
0É0004
0É017
0É010
Coe†
24É0
[4É8
2É8
[2É0
[5É5
[10É5
3É9
4É6
Poly ] Eudiplo (]106)
SS
10É01
2É53
12É54
Prob [ F
0É22
0É80
0É45
0É79
P-value
0É009
0É049
0É12
0É005
0É0005
0É018
0É010
Coe†
2É89
0É08
0É37
0É05
[0É33
[0É91
0É36
0É04
P-value
0É77
0É24
0É85
0É28
0É027
0É25
0É88
a Abbreviations : Adj R2 : adjusted R2 ; P-value : conÐdence level for t-test of null-hypothesis ; the
model coefficients are calculated for coded variables, Y E, DES, F, BS : [1 \ absent, ]1 \ present ;
SS : [1 \ 260 rpm, ]1 \ 230 rpm.
276
TABLE 3
Regression analysis for fermentation pattern dataa
Response
Source
Model
Error
Total
DF
pH
SS
7
4
11
0É0363
0É0119
0É0482
R2
Adj R2
Exp error
0É75
0É32
0É055
Factor
Coe†
Intercept
YE
SS
DES
F
BS
Y E ] DES
Y E ] BS
7É00
[0É06
[0É01
0É01
0É02
0É02
[0É01
0É01
Eh (mv)
Prob [ F
0É31
SS
371É5
61É7
433É2
Prob [ F
0É12
0É86
0É61
3É93
P-value
0É034
0É60
0É72
0É30
0É41
0É78
0É63
Coe†
[380É4
1É8
[0É2
[0É4
[2É7
[3É6
4É3
2É0
N-NH (mg litre~1)
3
SS
Prob [ F
155826
2298
158124
0É0016
0É99
0É96
24É0
P-value
0É25
0É89
0É77
0É11
0É052
0É032
0É20
Coe†
311É5
41É6
4É4
[6É7
0É4
104É3
[4É6
3É2
Acetate (mM
SS
7783
572
8355
M~1
V FA)
Prob [ F
0É033
0É93
0É81
12É0
P-value
0É007
0É61
0É45
0É96
0É0002
0É59
0É71
Coe†
625
0
[4
[1
[6
[23
0
2
Propionate (mM
SS
3077
1785
4863
M~1
V FA)
Prob [ F
0É54
0É63
0
6É7
P-value
0É96
0É36
0É74
0É20
0É004
0É95
0É71
Coe†
179
[2
[1
0
5
1
[1
1
Butyrate (mM
SS
1031
64
1095
M~1
V FA)
Prob [ F
0É025
0É94
0É84
4É0
P-value
0É33
0É77
0É98
0É078
0É79
0É72
0É77
Coe†
115
[1
5
[2
[2
8
2
[1
P-value
0É63
0É029
0É24
0É31
0É004
0É27
0É53
a Abbreviations : Adj R2 : adjusted R2 ; P-value : conÐdence level for t-test of null-hypothesis.
L -P Broudiscou et al
Response
Source
DF
Acetate
(mmol d~1)
SS
Model
Error
Total
R2
Adj R2
Exp error
7
4
11
423É8
78É8
502É6
Prob [ F
0É15
0É84
0É57
4É44
Factor
Coe†
Intercept
YE
SS
DES
F
BS
Y E ] DES
Y E ] BS
76É45
5É41
1É21
0É48
[1É51
1É93
[0É51
[0É14
Propionate
(mmol d~1)
SS
65É7
3É8
69É5
Prob [ F
SS
Prob [ F
0É022
4É44
0É07
4É52
0É002
0É94
0É85
0É98
P-value
0É022
0É46
0É76
0É37
0É27
0É75
0É93
Coe†
21É79
1É31
0É38
0É03
0É13
1É42
0É05
0É25
Isobutyrate
(mmol d~1)
0É98
0É96
0É136
P-value
0É016
0É31
0É94
0É71
0É013
0É89
0É49
Coe†
1É66
0É20
0É07
[0É01
[0É05
0É53
0É02
0É05
Butyrate
(mmol d~1)
SS
42É8
4É1
46É9
Prob [ F
SS
Prob [ F
0É052
9É061
0É078
9É138
0É0006
0É91
0É76
1É01
P-value
0É012
0É18
0É89
0É32
0É0003
0É63
0É37
Coe†
15É22
0É80
0É68
[0É04
0É09
1É35
[0É01
[0É19
Isovalerate
(mmol d~1)
0É99
0É98
0É139
P-value
0É080
0É12
0É91
0É80
0É017
0É98
0É60
Coe†
2É08
0É29
0É07
[0É03
[0É08
0É77
0É05
0É06
V alerate
(mmol d~1)
SS
6É896
0É118
7É014
Prob [ F
SS
Prob [ F
0É002
1É982
0É192
2É174
0É053
0É98
0É95
0É172
P-value
0É003
0É21
0É55
0É16
0É0001
0É31
0É24
Coe†
2É83
0É38
0É07
0É07
0É02
0É55
0É00
0É02
Caproate
(mmol d~1)
0É91
0É76
0É219
P-value
0É003
0É30
0É30
0É71
0É0007
0É98
0É74
Coe†
1É75
0É13
0É04
0É23
0É06
0É21
[0É07
0É00
Carbon
(mmol d~1)
SS
16820
1001
17821
Prob [ F
0É023
0É94
0É85
15É8
P-value
0É16
0É62
0É033
0É46
0É045
0É39
0É98
Coe†
323É2
22É8
7É5
2É7
[2É4
23É6
[1É0
0É3
Maintenance of rumen protozoa in a continuous fermenter
TABLE 4
Regression analysis for VFA production data
P-value
0É013
0É23
0É64
0É68
0É01
0É86
0É95
Abbreviations : Adj R2 : adjusted R2 ; P-value : conÐdence level for t-test of null-hypothesis.
277
L -P Broudiscou et al
278
TABLE 5
Regression analysis for gas production dataa
Response
Source
Model
Error
Total
R2
Adj R2
Exp error
Factor
Intercept
YE
SS
DES
F
BS
Y E ] DES
Y E ] BS
DF
7
4
11
V olume (litres d~1)
SS
Prob [ F
2É927
0É461
3É388
0É12
0É86
0É63
0É339
Coe†
3É85
0É35
0É21
0É05
[0É05
0É03
0É05
0É10
CH (mmol d~1)
4
CO (mmol d~1)
2
SS
SS
219É2
48É3
267É5
Prob [ F
0É19
0É82
0É50
3É48
P-value
0É037
0É14
0É69
0É71
0É83
0É68
0É42
Coe†
38É9
3É42
1É67
0É40
[1É30
[0É63
0É73
0É98
2945
416
3362
Prob [ F
0É098
0É88
0É66
10É2
P-value
0É043
0É23
0É75
0É33
0É62
0É57
0É45
Coe†
115É1
10É6
6É6
1É5
[0É5
1É7
1É3
3É0
P-value
0É036
0É12
0É68
0É90
0É65
0É73
0É43
a Abbreviations : Adj R2 : adjusted R2 ; P-value : conÐdence level for t-test of null-hypothesis.
estimates. The use of a polyurethane foam belt appeared
to be disadvantageous as well, while lowering the stirring speed to 230 rpm was slightly beneÐcial. The population size of larger protozoa (Polyplastron and
Eudiplodinium, of the subfamily Diplodininae) was
merely lowered by BS addition. The genus Isotricha was
observed in the experimental runs K2 and K3 (Table 1)
with a population estimated at less than 8 ] 105. Table
3 shows regression analysis for fermentation pattern
data. The pH and redox potential were not signiÐcantly
modiÐed by any experimental factor. BS and YE supplementation greatly increased NH -N concentration, with
3
no signiÐcant interaction.
The VFA concentration in the fermentation broth
averaged 78 mM. The variation in VFA concentration
was poorly explained by the statistical model (F-value
of 0É79, R2 of 0É47, experimental error of 7É5). In the
absence of nutritional supplements, the molar proportions of acetate, propionate and butyrate were, respectively, 654, 175 and 116 mM M~1 VFA. The addition of
BS tended to decrease acetate proportion, in relation
with an increase of butyrate. As shown by Table 4, the
factors Y E and BS clearly increased the amount of VFA
produced per day, with no signiÐcant interaction. Both
additives led to the same increase in the amount of
carbon recovered in effluents in the form of fermentation end-product. BS, however, had an e†ect mainly on
the production of VFA containing four or more carbon
atoms, in particular branched-chain VFA, while Y E
acted more on smaller VFA (acetate and propionate).
This di†erence was conÐrmed by the regression analysis
of fermentation gas production data (Table 5). Supple-
mentation with Y E was the most active factor and it led
to a 20% increase in the volume of fermentation gases
and in the amount of methane and carbon dioxide collected per day. The variation in the amount of hydrogen
collected was poorly explained by the statistical model.
The hydrogen balance was satisfactory in all experiments, with a high recovery rate estimated at 98%.
DISCUSSION
The physical characteristics of fermentation broths were
favourable for ciliate growth in all experimental runs. A
pH of 7É0 and a redox potential lower than [350 mv
were optimal values to maintain protozoa (Quinn et al
1962 ; Clarke and Hungate 1966). In a previous experiment with the same diet and artiÐcial saliva, the
osmotic pressure of the broth was equal to 210 mOsmol
litre~1. Anaerobiosis was satisfactory, with very low
proportions of oxygen detected in fermentation gases.
All experimental factors markedly inÑuenced the
ciliate population size in fermenters. The expected
antagonisms between nutritional factors were also
observed. Williams and Coleman (1991) stressed the
poor biosynthetic ability of ciliates and the need for a
number of organic compounds in the medium to maintain these microorganisms in vitro. YE supplies aminoacids and water-soluble vitamins and it is a common
component of culture medium. BS is a complex mixture
of albumins, globulins and growth promoters (Blood et
al 1989). According to Clarke and Hungate (1966) the
addition of BS or protozoa extract to holotrich culture
Maintenance of rumen protozoa in a continuous fermenter
medium was necessary. The e†ect of DES on ciliates in
ruminants is still uncertain (Williams and Coleman
1991). However, in dairy cows fed on alfalfa hay, barley
and soya, complementing the diet with DES (8 mg
litre~1) increased rumen ciliate population density
three-times (Ibrahim et al 1970). Subsequently, Itabashi
and Kurihara (1972) noted a favourable action of BS
(20 ml litre~1) and DES (10 mg litre~1) addition to
saliva on protozoa growth in single-outÑow stirred-tank
fermentors supplied with alfalfa hay, crushed barley and
concentrate pellets. Using similar incorporation rates,
we did not observe such e†ects in our experimental conditions.
All the nutritional additives tested hindered ciliate
growth, in accordance with the observations of Williams and Coleman (1991) on pure cultures of protozoa.
We can primarily conclude that the substrates supplied
by the diet and the bacterial biomass in our fermentors
efficiently supported protozoal growth. The mechanisms
by which BS and Y E were detrimental are unclear.
They can be attributed to a number of abiotic or biotic
factors. The amounts of carbon atoms recovered in the
form of VFA when Y S or BS was used imply that both
supplements were extensively fermented. Individual
VFA and gas production data indicate that they were
metabolised in two distinct patterns. BS fermentation
was characteristic of aminoacid catabolism while the
addition of Y E also led to end-products from carbohydrate catabolism. The speciÐc e†ect of BS on the
genera Eudiplodinium and Polyplastron could be related
to this di†erence. Ammonia and VFA concentration in
the fermentation broths, though, did not reach detrimental values (Williams and Coleman 1991). The supply
of bacteria with soluble organic substrate may also have
increased bacterial competitiveness for energy resources,
leading to a limitation of ciliate growth.
The choice of a resolution III fractional factorial
design limited the number of experiments but it allowed
major e†ects to be identiÐed with a number of two-way
interaction e†ects and with higher order interaction
terms. In particular, the main e†ect of SS was identiÐed
with the two-way interaction between BS and DES. The
main e†ect of F was similarly identiÐed with the threeway interaction between Y E, BS and DES. The F and
SS coefficient estimates reÑect, to some degree, the
complex interplay of nutritional factors. Nevertheless,
one can reach the conclusion that a synthetic foam belt
did not improve Entodinium maintenance. The foam
belt was supposed to enhance ciliate entrapment in the
vessels, as advocated by Abe and Kurihara (1984). In
our fermentors, the solid phase turn-over rate was low
enough to allow the development of a particulate bulk
and prevent an extended ciliate washout, in accordance
with Crawford et al (1980). Lowering the stirring speed
appeared slightly beneÐcial, probably by limiting the
disruption of shear-sensitive protozoa. In a dual effluent
culture system equipped with an overÑow and discon-
279
tinuously agitated, both factors are partly determined
by the stirring device properties. The calculation of
solid phase turn-over rate assumes that the distribution
of solids is homogeneous within the vessel. In practice,
the stirring device must satisfactorily mix the particulate
upper layer formed after the last agitation cycle, before
solids Ñow out of the fermentor. A marine propeller
engenders a better axial Ñow, leading to a better initial
mixing of solids, and creates lower shear than the Ñatblade turbines commonly used in similar fermenters.
The best combination of our experimental factors, ie
a stirring speed of 230 rpm with neither supplement nor
foam belt, allowed the maintenance of the major protozoal genera, in numbers large enough to signiÐcantly
compete with and prey on bacteria. Taking into
account the di†erences in substrate inÑows, the protozoan population in the fermentors represented approximately a third of the initial population in the rumen of
sheep. A subsequent test run under similar operating
conditions in all the fermentors conÐrmed the present
results : a mean ciliate population density of 49É3 kl~1
(n \ 12, SD \ 8É3) was observed, compared to a predicted value of 57É8 kl~1.
CONCLUSIONS
Applying a 25h2 fractional factorial design provided
enough information to conclude that, in our dual
outÑow continuous fermentors, the use of a synthetic
foam belt and supplementation with Y E, BS, DES were
unnecessary and that lowering the stirring speed from
260 to 230 rpm promoted protozoa maintenance. These
operating conditions were chosen for future in vitro
essays.
ACKNOWLEDGEMENTS
The authors are grateful to Mrs J Rohlion for her
skilled technical support.
REFERENCES
Abe M, Kurihara Y 1984 Long-term cultivation of certain
rumen protozoa in a continuous fermentation system supplemented with sponge materials. J Appl Bacteriol 56 201È
213.
Blood D C, Radostits O M, Arundel J H, Gay C C 1989 V eterinary Medicine. Baillière Tindall, London, UK, pp 1463È
1464.
Clarke R T J, Hungate R E 1966 Culture of the rumen holotrich ciliate Dasytricha ruminantium Schuberg. Appl Microbiol 14 340È345.
Crawford R J, Hoover W H, Knowlton P H 1980 E†ects of
solids and liquid Ñows on fermentation in continuous cultures. I. Dry matter and Ðber digestion, VFA production
and protozoa numbers. J Anim Sci 51 975È985.
280
Davies A W, Taylor K 1965 Application of the autoanalyser
in a river authority laboratory. Symposium T echnicon pp
294È300.
Demeyer D I, Van Nevel C J 1975 Methanogenesis, an integrated part of carbohydrate fermentation and its control.
In : Digestion and Metabolism in the Ruminant, eds McDonald I W & Warner A C I. University of New England
Publishing, Armidale, pp 366È382.
Haaland P D 1989 Experimental Design in Biotechnology.
Marcel Dekker Inc, New York, USA, pp 85È112.
Hoover W H, Crooker B A, Sni†en C J 1976 E†ects of di†erential solidÈliquid removal rates on protozoa numbers in
continuous cultures of rumen contents. J Anim Sci 43 528È
534.
Ibrahim E A, Ingalls J R, Stanger N E 1970 E†ect of dietary
diethylstilbestrol on populations and concentrations of
ciliate protozoa in dairy cattle. Can J Anim Sci 50 101È106.
Itabashi H, Kurihara Y 1972 Maintenance of rumen microbial
populations in an improved artiÐcial rumen. Jap J Ecol 22
262È265.
Jouany J-P 1982 Volatile fatty acid and alcohol determination
in digestive contents, silage juices, bacterial cultures and
anaerobic fermentor contents. Sci Aliments 2 131È144.
L -P Broudiscou et al
Mahouy G 1995 Legislation et reglementation de
lÏexperimentation
animale.
In :
L ivre
Blanc
sur
lÏExpe rimentation Animale, eds Carnaud C, Mahouy G,
Marsac J, Maurin-Blanchet H, Schmoll E & Tambourin P.
Les editions INSERM, CNRS Editions, Paris, France, pp
1È12.
MansÐeld H R, Endres M F, Stern M D 1995 Comparison of
microbial fermentation in the rumen of dairy cows and dual
Ñow continuous culture. Anim Feed Sci T echnol 55 47È66.
Merry R J, Smith R H, McAllan A B 1987 Studies of rumen
function in an in vitro continuous culture system. Arch Anim
Nutr 37 475È488.
Ogimoto K, Imai S 1981 Atlas of Rumen Microbiology. Japan
ScientiÐc Societies Press, Tokyo, Japan.
Quinn L Y, Burroughs W, Christiansen W C 1962 Continuous culture of ruminal microorganisms on chemically
deÐned medium. II. Culture medium studies. Appl Microbiol
10 583È592.
SAS 1990 UserÏs Guide, Release 6.04. SAS Institute Inc, Cary,
NC, USA.
Williams A G, Coleman G S 1991 T he Rumen Protozoa.
Springer-Verlag, New York, USA, pp 145È164.
Документ
Категория
Без категории
Просмотров
2
Размер файла
191 Кб
Теги
493
1/--страниц
Пожаловаться на содержимое документа