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247

Aust. J. Exp. Agric., 1987, 27, 247-51

Recovery of pasture seed ingested by ruminants.


2. Digestion of seed in sacco and in vitro
M. Simao NetoAB and R. M. JonesA

* CSIRO Division of Tropical Crops and Pastures, 306 Carmody Road, St Lucia, Qld 4067, Australia.
B Present address:

EMBRAPAKPATU, C.P. 48, 66.000 Belem-PA, Brasil.

Summary. Seeds of the grasses Brachiaria decumbens


(signal grass) and Axonopus afinis (carpet grass), and
the legumes Neonotonia wightii cv. Tinaroo, Trifolium
semipilosum cv. Safari, Stylosanthes hamata cv.
Verano and S. scabra cv. Seca were suspended in nylon
bags in the rumen ofcattle (in sacco) and also subjected
to in vitro digestion techniques. Legume seeds were
evaluated in 3 categories: seed as supplied (mixture of
hard and soft), 100% soft and 100% hard. Seeds were
either placed in the rumen of cattle (using nylon bags)
for 24, 48 or 96 h or subjected to in vitro digestion (in
pepsin, and in rumen liquor or cellulase either with or

Introduction
In the first experiment of this study (Simao Neto et al.
1987) seeds of 6 pasture species were subjected to the
complete digestion process in penned cattle, sheep and
goats. It was not possible from that study to identify how
each site of the digestive tract, particularly the rumen,
affected the viability of seeds. The variable hard seed
content of the legume seeds fed to the ruminants, and the
digestion of many seeds during passage, made it difficult to
document dormancy breakdown and the degree of digestion of soft seeds. Specific studies on these aspects were not
found in the literature.
One possible way of studying the role of the ruminal site
on digestion of seed is by using the nylon bag technique. If
ruminal digestion alone is responsible for the major
changes to seed viability during the passage through the
digestive tract of ruminants, then rumen fistulated
animals could be used as a quick way to assess the effect of
passage on small quantities of seeds of a wide range of
species. Other possible methods include the use of in vitro
digestion techniques.
The aim of this experiment was to ascertain how in vivo
exposure in the rumen (using nylon bags) and in vitro
digestion affected germination, hard seed content and
viability of pasture seeds that were (i) unselected, (ii) all
soft, or (iii) all hard. The unselected seed had been

without subsequent digestion in pepsin). Other seed of


the same seed lots had been previously fed to penned
cattle, sheep and goats and the recovery in faeces had
been measured.
Soft legume seed were destroyed by the digestion
treatments whereas hard seeds were largely resistant to
digestion. Average effects of digestion in vitro on
viability were similar to average effects of digestion in
nylon bags, but there were large differences between
different treatments and between seed lots. The percentage of hard seed in the seed sample was the best
guide to the resistance of legume seed to digestion.

previously fed to penned cattle, sheep and goats (Simao


Neto et al. 1987).
Materials and methods
The experiment was carried out at the CSIRO Samford
Pasture Research Station and Cunningham Laboratory,
Brisbane, from July to September 1982.

Pasture seeds
Seeds of Brachiaria decumbens (signal grass), Axonopus
afinis (carpet grass), Neonotonia wightii cv. Tinaroo
(glycine), Trifolium semipilosum cv. Safari (Kenya white
clover), Stylosanthes hamata cv. Verano (Caribbean stylo)
and S. scabra cv. Seca (shrubby stylo) were treated as
follows.
(i) Unselected grass and legume seeds. From each
species, samples of 250 seeds were picked out without
any selection.
(ii) Selected soft legume seeds. Legume seeds were
scarified by using a constant air flow (pressure 1.25 kg
cm-2) through a small cylindrical container, whose
inner surface was covered with emery paper, for 5 min.
Verano seeds were only scarified for 3 min to avoid seed
breakdown. The scarified seed was placed on wet paper.
After 4-8 h, swelling soft seeds of each species were

248

M. Simao Neto and R. M. Jones

picked out and then dried at room temperature (about


20C).
(iii) Selected hard legume seeds. Unscarified legume
seeds were placed on wet paper for 48-52 h. Samples of
200 hard seeds of each species were then picked out and
dried out at room temperature.
Seed from the groups (i) and (ii) were taken 1 day and 2
days, respectively, before the start of the digestion treatments. Seeds of the stylos were depodded before scarification and selection for hard seed.

and viability of seeds. Differences between means were


assessed by Duncan's Multiple Range Test (Steel and
Torrie 1960). The germination characteristics of seed were
expressed as a percentage of seeds placed in the nylon bags
for in sacco and in the tubes for in vitro digestion.

Results
Results are presented separately for the selected and
unselected seeds as there were important differences in the
effects of digestion between these groups of seeds.

Digestion treatments
Seeds of each of the groups (i), (ii) and (iii) from each
legume and of group (i) for grasses were enclosed in nylon
bags (used for animal nutrition studies): and concurrently
placed in the rumen of cattle. The animals were 2 adult
Hereford steers, 1 for each replication, fitted with rumen
fistulae. The seeds were exposed in the rumen for periods
of 24, 48 and 96 h. This nylon bag technique was adapted
from Lowrey (1969) and is designated in sacco in this
experiment. Seeds exposed for 48 h were subsequently
either immersed or not immersed in pepsin for 24 h immediately after ruminal digestion (to simulate conditions
in the abomasum). The cattle were fed with the same diet
(80% lucerne hay and 20% milled wheat grain) used in the
previous pen feeding study (Simao Neto et al. 1987).
After treatment, the seeds were washed and then dried
for 24 h at 40C in an oven, with forced air draught. The
seeds were counted and tested for percentage germination
and hardseededness, as described previously (Simao Neto
et al. 1987). The percentage of viable legume seed was
taken as the sum of seed able to germinate (percentage
germination) and that with the potential to germinate
(percentage hardseededness). To save space in tables,
percentage hardseededness is not listed but can be found
by subtraction. The difference between percentage
viability and 100% represents the percentage of soft dead
seeds.
Seeds of each groups (i), (ii) and (iii) were also digested in
vitro in each of the following media: P, pepsin, for 24 h; C,
cellulase, for 48 h; R, ruminal liquor, for 48 h (ruminal
liquor taken from cattle used for in sacco digestion); CP,
cellulase, for 48 h, followed by pepsin for 24 h; and RP,
ruminal liquor, for 48 h, followed by pepsin for 24 h.
These in vitro methods were used as described by
Minson and McLeod (1972), for R and RP; and adapted
from Jones and Hayward (1973) and Goto and Minson
(1977), for C and CP.
After the in vitro digestion treatments were completed
the seeds were tested as for the in sacco digestion.

Experimental design
The experiment was a completely randomised block
design with 2 replicates for each treatment. The data
analysed were percentage germination, hardseededness

C'nselected seed
There were no dormant seeds of signal grass and carpet
grass except for zn sacco seed of signal grass digested for
24 h. In sacco digestion for 24 h reduced viability of signal
grass by 18%, but resulted in little change in viability of
carpet grass (Table 1). Digestion for longer periods or in
pepsin significantly reduced viability of both grasses.
Apart from pepsin alone. all zn vztro treatments drastically
reduced the viability of grass seeds. Overall, the zn vitro
treatments had a more severe effect than the zn sacco treatments.
In sacco digestion for 24 h decreased the percentage
germination and viability of all legume seeds (Table 2) and
these were reduced further by longer periods of digestion,

Table 1. Percentage viability of grass seeds digested in sacco and


in vitro
Values followed by the same letter do not differ significantly
between different in succo treatments (letter A, B, C, D) or
different in vitro treatments (letters E, F, G) for each species

Method of digestion

Viability of grasses
Signal
Carpet

In sacco

24 h
48 h
48 h + pepsin
96 h
In vitvo

Pepsin
Cellulase
Rumen liquor
Cellulase + pepsin
Rumen liquor pepsin

Untreated seeds
Seeds recovered from cattleA
*Data are from Simao Neto et al. (1987) on the same seed
samples; viable seed recovered as a percentage of total seed
input.

Recovery of seed ingested by ruminants. 2.


Table 2. Percentage germination (G) and viability (V) of unselected legume seeds
digested in sacco and in vitvo
Values followed by the same letter do not differ significantly. Capital letters
represent comparisons between periods of in sacco or between methods of in vltro
digestion, for G (letters A, Band C, or D, E, F and G) and V (letters X, Y and Z, or T,
U and V) (Pt0.05)

Method of
digestion

Legume seed:
Tinaroo
Safari

Verano

Seca

24.5A
64.4X
4.2B
38.5Y
2.1B
37.6Y
2.OB
36,OY

10.OA
79.4x
3.0B
62.8Y
2.7B
63.4Y
1.9B
51.5Y

4.4A
69,OX
2.8A
7 1,OX
2.4A
65.0X
2.OA
66,OX

12.7D
55.97
5.7DE
31.7U
3.8DE
36.3U
0.6E
33.7u
6.6DE
34.7u

0.2E
55.2T
1.2DE
50.2T
4.9D
55.21
1.2DE
52.OT
3.5DE
53.8T

6.3D
70.0T
3.OD
68.0T
3.1D
68.2T
2.7D
67.6T
1.6D
67.0T

54.0
86.0
9.1
39.2

28.0
93.0
8.2
18.3

16.0
83.0
12.7
38.9

In sacco
24 h

In vitro
Pepsin
Cellulase
Rumen liquor
Cellulase
+pepsin
Rumen liquor
+pepsin
Untreated seeds
Seeds recovered
from cattleA

AData are from Simao Neto et al. (1987) on the same seed samples; viable
seed recovered as a percentage of total seed input.

except for Seca. Digestion for any of the periods did not
cause important changes in hardseededness.
Pepsin alone usually had the least effect of the in vitro
treatments, and the percentage germination, viability and
hardseededness of seed from the remaining treatments
were similar. The average viability of Safari and Tinaroo
seed after in sacco or in vitro treatment was similar to that
recovered from cattle in a feeding experiment (base of
Table I), but average seed viability of Seca and Verano
was much less affected by in sacco or in vitro treatments
than by feeding.
The average percentage composition of the unselected
seed samples from the 4 legumes prior to treatment
(untreated seed) and after in sacco and in vitro treatments
was as follows:

Hard seed
Soft, germinable
Soft, dead or disintegrated
Viable (hard germinable)

Untreated
53
33
14
86

In sacco
52
7
41
59

In vitro
47
7
46
54

Thus, as there was little change in hardseededness, most


of the seed loss during digestion was from the seed which
was able to germinate prior to digestion.

Selected seed
None of the selected soft legume seeds were viable after
any of the digestion treatments. Nearly all seeds disintegrated, and those digested in sacco left only fragments
of their coats inside the nylon bags.

M. Simao Neto and R. M. Jones


Table 3. Percentage germination (G) and viability (V) of selected hard legume
seeds digested in sacco and in vitro
Values followed by the same letter do not differ significantly. Capital letters
represent comparisons between periods of in sacco or between methods of in vitro
digestion, for G (letters A, Band C, or D, E, Fand G) and V (letters X, Y and Z, or T,
U a n d V)(P<0,05)

Method of
digestion

Legume seed:
Tinaroo
Safari

Verano

Seca

In sacco
24 h

In vitro
Pepsin
Cellulase
Rumen liquor
Cellulase
+pepsin
Rumen liquor
+pepsin

All the selected hard legume seeds had some reduction


of hardseededness and some damage after being digested
either in sacco or in vitro. The resDonses of seed of the
different species to digestion treatments were different, as
shown in Table 3.
As the period of digestion increased from 24 to 48 h or
more, in sacco digestion of hard seeds decreased
germination and viability of Safari and Verano seeds.
Additional pepsin digestion had the same effect. The in
sacco treatments had less effect on seeds of Tinaroo and
Seca.
Percentage germination and viability were higher for
Tinaroo and Safari seeds digested in pepsin alone than in
the other in vitro treatments. Although significant, the
differences between the other treatments were small.
There was a good agreement between the average of in
sacco and of in vitro treatments for each species, but the
values for each treatment varied considerably.

Discussion
The results of using selected hard, selected soft and
unselected seed indicate the resistance ofhard seed and the

susceptibility of soft seed to digestion during passage. The


same finding was reported by Gardener et al. (1 983) who
tested a wider range of legume species in sacco.
All seeds from the group 'selected soft seeds' were
completely digested, as was also reported by Yamada and
Kawaguchi (1 97 1). It is probable that the light scarification of the seeds to select the soft ones increased their
susceptibility to digestion. In addition, the soft seeds of
this experiment (which took only 4-8 h to swell) were the
most permeable of seeds that are normally classified as
'soft' in standard 21 day germination tests. The average
viability of the unselected legume seeds after in vitro digestion (59%) was 6% higher than the average hard seed
content of the control seeds (53%), which suggests that a
small proportion of the unselected soft seeds were still
viable after digestion. A similar finding was reached when
animals were fed in pens (Simao Neto et al. 1987).
In sacco and in vitro digestion softened a variable
portion, usually less than 20/0, ofthe 'selected hard legume
seeds'. The 48 h taken to select the hard seeds was not long
enough to cause the swelling ofall potentially soft seed that
would occur in a standard 21 day test. This would explain

Recovery of seed ingested by ruminants. 2.

the minor differences between the 'hard seed' and 'unselected seed treatments'.
The viability of grass seeds was reduced with longer
periods of time in the rumen, particularly for signal grass
seeds which were all dead after 96 h. This is in agreement
with the pen feeding study (Simao Neto et al. 1987).
It is difficult to define which in sacco or in vitro
technique best relates to the results of the pen feeding
experiment. This is mainly because, in this study,
germination tests before and after treatment are comparable, whereas in pen studies the test after passage is
based on a different sample as some seed is digested during
passage. The best basis for comparison is to express the
viable seed recovered in pen studies as a percentage of
total seed ingested, and relate this to the percentage
viability of seed after in sacco or in vitro treatments. On
this basis, 48 h in sacco plus pepsin or 96 h in sacco gave a
reasonable prediction of damage to Safari and Tinaroo
seeds ingested by cattle and, to a lesser extent, goats.
Damage to Verano and Seca was much more severe in the
feeding experiment. This could be caused by Seca and
Verano pods being more readily caught up in a bolus,
leading to more damage during rumination. The in sacco
and in vitro techniques were poor quantitative predictors
for signal and carpet grasses but could be adequate to
distinguish seeds resistant to passage from seeds that were
quickly digested during passage (Gardener et al. 1983).
Comparison of the results of the in sacco studies and pen
feeding experiments suggest that much of the damage to
ingested seed will occur in the rumen. However, the losses
in viability with pepsin digestion indicate that some
damage occurs in the abomasum. Simao Neto and Jones
(1986) have also documented loss of viability when seed is
retained in faeces, so it is reasonable to assume that some
further damage to seed will occur during passage through
the intestine and after defaecation. Added to this is
damage caused by mastication, probably more severe with
smaller ruminants, and the effect of differing seed or pod
size on rate of passage (Simao Neto et al. 1987). Consequently it is not surprising that no single in sacco or in
vitro procedure could consistently predict the effect of
passage on seed of contrasting plant species, let alone with
different animal species.
These results confirm that if legume seed is to be
deliberately fed to ruminants so that it can be
disseminated, it should have a high content of hard seed. It
is doubtful whether there is any further advantage in
attempting to screen legume seed for resistance to
digestion by using an in vitro or an in sacco procedure.
There is also evidence that prediction of the effect of

25 1

passage may be different for seed which is enclosed in


pods.

Acknowledgments
We acknowledge the assistance of Mr H. Kiers, CSIRO
Division of Tropical Crops and Pastures and the advice of
Dr L. R. Humphreys, University of Queensland and Dr D.
Ratcliff, CSIRO Division of Mathematics and Statistics.
Thanks are also due to EMBRAPA (Brazilian Agricultural
Research Organization) and IIE (International Institute of
Education) for the provision and administration of
scholarship funds for the senior author.
References
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Survival of seeds in the digestive tract and faeces of cattle.
Annual Report 1982-83, pp. 120-1. (CSIRO Division of
Tropical Pastures: Brisbane.)
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digestibility of tropical grasses using a pepsin-cellulase assay.
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cattle dung on viability of tropical pasture seeds. Tropical
Grasslands 20, 132-50.
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of pasture seed ingested by ruminants. 1. Seed of six tropical
pasture species fed to cattle, sheep and goats. Australian
Journal of Experimental Agriculture 27: 239-46.
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Received 6 March 1986, accepted 29 September 1986