only one previous study has experimentally examined whether avoidance of faeces by Ž
. ruminants results in a reduction in the number of parasites consumed. Michel 1955
compared the number of Dictyocaulus ÕiÕiparus larvae isolated from herbage immedi- ately adjacent to where cattle grazed with the number isolated from random samples of
pasture herbage. He concluded that cattle grazed in a manner that resulted in the consumption of fewer larvae than if grazing was random and that selective grazing was
a parasite avoidance strategy. However, this conclusion was based only on estimates of the numbers of larvae that cattle ingested. In the study reported here we used direct
measurements of consumed parasite larvae to lend weight to our findings.
Environmental and internal factors, other than simply the presence of faeces on herbage, may affect the ability of ruminants to distinguish between clean and contami-
nated areas. Selection of alternate foods by sheep is known to be influenced by the scale Ž
. of aggregation of patches Edwards et al., 1994 . Hence, in this study the influence of
the scale of aggregation of faeces-contaminated patches had on diet selection was investigated. Factors, internal to the animal, which play a part in diet selection include
Ž .
parasitism. Hutchings et al. 1998, 1999 conducted a series of small-scale diet-choice experiments and observed that parasite-infected sheep avoid faeces contaminated swards
more than uninfected sheep. Although demonstrative, these studies were conducted in artificial conditions. The current study approaches more natural conditions, as the effect
of parasite infection on faeces avoidance behaviour by sheep on pasture was investi- gated.
This paper presents two experiments, the aims of which were to investigate the cues used by sheep to avoid faecal contaminated patches, how avoidance of faecal-contamina-
tion and ingestion of parasite larvae by sheep on pasture was influenced by the spatial distribution of contaminated patches, and how parasite infection status influences
grazing by animals.
2. Materials and methods
This paper reports on two choice experiments: in experiment 1, the presence of faeces as an environmental cue in parasite avoidance was investigated. In experiment 2 we
determined the extent to which avoidance of faecal-contaminated patches of pasture was affected by the parasite infection status of grazing sheep and how avoidance behaviour
was influenced by the spatial distribution of the patches. Experiment 2 also sought to investigate the relationship between the parasite infection status of the animal, patch
distribution pattern, and the numbers of parasite larvae which were actually consumed by the animal whilst grazing on pasture. Experiments were conducted during August
Ž .
Ž .
1993 Experiment 1 and September 1995 Experiment 2 at the Macaulay Land Use Ž
. Research Institute’s Glensaugh Research Station Kincardineshire, Scotland .
2.1. Animals Ž
y1
. All animals were dosed orally with ivermectin Oramec drench; 200 mg kg
to remove internal parasites prior to the start of the experiments. Animals were housed
individually in indoor pens and fed a daily ration of 0.75 kg of a standard lamb and ewe Ž
. dry feed North Eastern Farmers, Aberdeen .
2.1.1. Experiment 1 Ž
Five, Scottish Blackface, castrate male sheep, approximately 1-year-old mean live .
weight 34.5 kg; range 31.5–38.0 kg were used in the study. To mimic a light,
sub-clinical parasite infection, animals were trickle-dosed daily with 1500 Ostertagia circumcincta infective larvae for 15 days. O. circumcincta is an abomasal nematode
Ž frequently associated with outbreaks of parasitic gastroenteritis in lambs Coop et al.,
. 1982 . Dosing ended 23 days before commencement of the grazing trials to ensure that
all parasites had matured. One week before the start of the grazing trials animals were Ž
. moved to an outdoor paddock and grazed on a perennial ryegrass
Lolium perenne pasture. The pasture had not been grazed for a year and a silage cut had been taken prior
to the experiment to minimise the number of viable O. circumcincta larvae present. The number of O. circumcincta larvae on the pasture was not determined but was assumed
to be low.
2.1.2. Experiment 2 Ž
Twenty, Scottish Blackface, castrate male sheep, approximately 1-year-old mean live .
weight 35.0 kg; range 30.0–42.5 kg , were weighed and ranked by live weight and then divided into four treatment groups, each of five animals, balanced for weight.
To enable the collection of ingested herbage, animals in Groups 1 and 3 were Ž
. fistulated at the oesophagus Pfister et al., 1989 and allowed to recover for a period of 3
weeks. Animals were monitored twice daily following recovery. One animal in Group 1 became dehydrated and lost condition due to loss of saliva during the recovery period
and was euthanased. To mimic a light, sub-clinical parasite infection, animals in Groups 1 and 2 were trickle-dosed daily with 1500 O. circumcincta infective larvae. Dosing
began after the fistulated animals’ recovery period and lasted for 15 days. Dosing ended 23 days before commencement of the grazing trials. One week before the start of the
grazing trials infected and uninfected animals were moved to seperate paddocks and grazed on perennial ryegrass pasture. The pasture had not been grazed for a year and a
silage cut had since been taken to minimise the number of viable O. circumcincta larvae present. The number of O. circumcincta larvae on the pasture was not determined but
was assumed to be low.
2.2. Plot layout and contamination In both studies, the experimental areas consisted of a 1-year-old perennial ryegrass
pasture, which had not been grazed for a year and that was divided into eighteen 10 m = 10 m fenced plots. A silage cut was taken immediately prior to the establishment of
the plots to minimise the number of viable O. circumcincta infective larvae contaminat- ing the treatments from previous grazings. Three weeks before the start of the grazing
trials, a predominantly nitrogen-based fertilizer was applied to the plots at a rate of 30 kg N ha
y1
to minimise any effect the faecal contamination might have on the chemical
composition of the herbage. At the same time, plots were cut to ensure a uniform sward height of 6–8 cm at the start of the experiment. Plots were divided into a grid of patches
that were marked with wooden pegs, painted white to enhance their detection on video tape.
2.2.1. Experiment 1 Plots were divided into four patches, each patch measuring 5 m = 5 m. Six replicates
of three different types of contamination were created on the 18 plots in the experimen- tal area. Plots were contaminated with either faeces from animals infected with O.
Ž .
Ž . circumcinta FP , faeces from worm-free animals F , or infective O. circumcincta
Ž . Ž
. larvae only P . Six kg of faeces wet weight was spread on each of two of the four
patches within a plot to give a contamination rate of 240 g m
y2
. Contaminated plots were diagonally opposite each other within the plot. Plots treated with faeces were
contaminated 3 weeks before they were to be grazed. To minimise larval losses due to desiccation or heavy rain, plots treated only with larvae were contaminated the day
before they were to be grazed. Larvae were applied using a watering can at a rate of 46,000 larvae per contaminated patch in a volume of 500 ml of water. This was
equivalent to the number of larvae isolated from 6 kg of faeces from a sheep whose faeces had also been used to contaminate the FP plots.
2.2.2. Experiment 2 Plots were divided into a grid of 16 patches, each patch measuring 2.5 m = 2.5 m.
Quantities of faeces were obtained from animals which had patent O. circumcincta infections. Three weeks before the plots were to be grazed, quantities of these faeces
were distributed across the patches within plots to create three distribution patterns of Ž
. Ž . Ž .
faecal contamination Fig. 1 : i Dispersed D , faecal pellets evenly spread over all Ž .
Ž . Ž .
squares; ii Small patch S , faecal pellets spread on every other square; iii Large Ž .
patch L , faecal pellets spread on half the squares in such a way so as to create four Ž
. patches. Faeces were spread at a level of 1.5 kg wet weight per 2.5 m = 2.5 m
contaminated square to give a contamination rate of 240 g m
y2
. Six replicates of each of the three patterns were created within the 18 plots.
Fig. 1. Patterns of larva distribution. Shaded areas indicate patches contaminated with faecesrparasites.
2.3. Grazing trials Animals were without food for 1 h prior to each trial to encourage grazing.
Throughout each trial, animals were fitted with faeces collection bags to avoid unwanted faecal contamination of plots.
2.3.1. Experiment 1 Two trials were conducted each day for 9 days. Each day, one trail was run in the
morning and the second trial was run in the afternoon. Animals were randomly rotated around plots from trial to trial. In each trial, sheep grazed an experimental plot for 30
min.
2.3.2. Experiment 2 Ž
. Four trials one for each treatment group were run daily for a period of nine
consecutive days. Two trials were run each morning and two were run each afternoon. Ž
. Ž
. Each day, infected Groups 1 and 2 and uninfected Groups 3 and 4 groups of animals
were randomly allocated to graze one of the three different distribution treatments. Allocation of infected animals to morning or afternoon trials was alternated daily to
eliminate any influence that time of day might have on collected data. This whole allocation procedure was replicated until each treatment group had grazed each of the
Ž .
three larva distribution plots three times nine trials per treatment group . Fistulated Ž
. animals Group 1 or 3 were introduced to a plot and allowed to graze the pasture for 20
min. During this time, ingested herbage samples were collected in polythene bags. Occasionally samples were contaminated with regurgitated material and these were
discarded. Fistulated animals on each plot were then replaced by unfistulated animals of the same infection status, i.e. Group 1 replaced by Group 2, Group 3 replaced by Group
4. Unfistulated animals grazed for 90 min.
2.4. Herbage measurements In both experiments herbage samples were taken by cutting all grass from within a
1.25 m = 0.20 m quadrant immediately before each trial. In experiment 1, one sample Ž
. was taken from each patch of the plot four samples per plot . O. circumcincta larvae
were isolated from each sample and counted using the technique described by Thomas Ž
. 1959 to give the mean larval count per patch type. In experiment 2, three samples were
Ž . taken from plots composed of only contaminated squares
D . Where plots were Ž .
Ž . composed of contaminated and uncontaminated squares, S and L , two samples were
Ž .
taken from each patch type contaminated and uncontaminated . Mean larval counts were then calculated for each plot.
In each experiment, subsamples of pasture herbage were freeze-dried and analysed to determine their nitrogen content by a Carlo Erba NA1500 elemental analyser, using an
Ž .
automated Dumas combustion system. In vitro organic matter digestibility OMD of the Ž
herbage was determined using a standard technique Alexander and McGowan, 1961; .
Tilly and Terry, 1963 .
2.5. Animal measurements Faecal egg counts were conducted on each animal using a modification of the method
Ž .
of Jackson 1974 on the first day of each experiment to ensure that infections had been established in all challenged animals and that unchallenged animals were uninfected.
All grazing trials were recorded using a GYYR time lapse video cassette recorder Ž
y1
. Ž
tape speed s 37 frames min , with the recorder mounted on a vertical tower 11 m
. tall located at the centre of the plot system. The video tapes were used to estimate the
total proportion of time each sheep spent grazing in uncontaminated patches of a plot and the duration of each discrete foraging bout in an uncontaminated patch. This was
achieved by determining the grazing location of each animal at 60-s intervals. For the first experiment, the proportion of time spent in each patch type was calculated for the
Ž .
Ž .
first grazing period 1 and last grazing period 2 30 min of the 90-min grazing trial involving unfistulated animals. Data from these two grazing periods were compared to
see whether the behaviour of the experimental animals changed during the time course of the trial. Where animals were seen to be obviously resting, data were excluded from
the analysis.
Larvae from extrusa samples collected from fistulated animals were recovered using Ž
. the technique used by Thomas 1959 for pasture herbage.
2.6. Statistical analysis Ž
Unless stated otherwise, statistical analyses were performed using Unistat 4 Unistat, .
1997 . Sheep graze in a synchronised manner and so individual animals should not be Ž
. treated as replicates in grazing experiments Rook and Penning, 1991 . Consequently,
although data was available for all individuals, mean values were calculated for each grazing trial and these mean values were used in the analysis. Hence, the grazing trial
Ž .
was the unit of replication and not the individual animals . Each experiment involved a total of 18 grazing trials. Hence, results from experiment 1, which involved infected
animals grazing plots with three different sources of contamination, are based on six replicates. The results from experiment 2, which involved both uninfected and infected
animals grazing plots with three different patch distributions, are based on three replicates.
Unless otherwise stated, data was analysed using standard one-way analysis of Ž
. variance ANOVA or t-tests. Analysis of data obtained from video tapes was restricted
to trials involving unfistulated animals grazing plots with both contaminated and uncontaminated patches. The mean proportion of time sheep spent in uncontaminated
plots was calculated for each trial replicate as was the duration of each grazing bout in an uncontaminated patch. Proportional data were subjected to an angular transformation
prior to analysis to stabilise variance. In the second experiment, data obtained from the
Ž .
video was analysed using an analysis of variance ANOVA procedure for a split unit design with trial as the main experimental unit and grazing period as the split unit. The
effects of parasite infection and pattern of contamination together with appropriate interactions was investigated. The structure of the ANOVA table is given in Table 1.
Ž These analyses were carried out using Genstat 5, Release 3.1 Lawes Agricultural Trust,
. 1994 .
Table 1 Structure of the ANOVA table used for analysis of data obtained from video tapes. Data were analysed using
an ANOVA procedure for a split unit design with trial as the main experimental unit and grazing period as the split unit. The effects of infection status, contamination pattern and appropriate interactions were investigated
Ž .
Source of variation Degrees of freedom missing values
Main residual stratum Contamination pattern
1 Grazing period
1 Contamination pattern=grazing period
1 Residual
8 Main residual units stratum
Infection status 1
Infection status=contamination pattern 1
Infection status=grazing period 1
Ž . Residual
8 1 Ž .
Total 22 1
Log transformations were taken of larval count data prior to analysis. The difference between larval counts from pasture and ingested herbage was determined and this
difference was subjected to a two-way ANOVA to investigate the effects of infection status and contamination pattern.
3. Results
