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Filter paper tests were used to examine the palatability of fractions. Of six pieces of filter paper (5 x 8 cm, 1 g), two
were treated with the extract of A4 and two with that of N4; the remaining two were left untreated as a control. The amount
of fractions applied to each piece of filter paper was a one-gram dry-leaf equivalent. These were offered to hares kept in
cages (80 x 80 x 80 cm) or pens (1.8 x 3.6 m), for one night and the amount (area) of filter paper left uneaten was measured.
Commercial food pellets were always available during the trials, hence the intake of leaves or filter paper by hares
was entirely voluntary.
Seasonal Stability of Palatability and Solvent Extracts
Seasonal stability of the palatability of fresh leaves of A4, U36, and N4 was investigated by feeding trials every 3
months from May 1988 to May 1989. Leaves were taken from the trees and cut into approximately 5 to 10cm lengths. The
leaves of each clone were put on two trays, which were set at opposite positions in a pen to prevent biased consumption due
to tray position.
For the same materials, solvent extracts were investigated: essential oils were taken by steam distillation for 10 h;
dichloromethane and methanol extracts were consecutively obtained using a Soxhlet extractor. Leaves were cut into small
pieces (< 3 mm in length) with pruning shears before the analysis.
Effects of Extraction on Palatability
Two series of consecutive extractions were made: (a) steam distillation for I0 h followed by methanol extraction; and
(b) extraction by n-hexane and then methanol. The latter was done in a Soxhlet extractor. Leaves were cut into small pieces
(ca. 3 mm in length) with pruning shears before extraction.
Hot-water extraction was made using a common kitchen pot (capacity: 5 1) and a large steam-heated stainless pot for
mass extraction (capacity: 50 1). Leaves of 10 to 20 cm in length were used. Leaves in the kitchen pot were boiled for 7 days
(6 h per day) with water changed twice a day. In the steam-heated pot, leaves were boiled for 8, 16, or 24 h and once for 5
days with water changed daily.
The palatability of residual leaves were investigated by cafeteria test. Comparisons of palatability were made among
the clones after each extraction.
Effects of Methanol Extracts on Fresh and Residual Leaves
The fresh leaves and residue after extraction of A4 and N4 were treated with the methanol extracts of A4 and N4 and
feeding trials were made to compare the palatability before and after treatment. Methanol extracts were taken by soaking
dried fresh leaves (600 g wet weight) first in 36 1 methanol for 13 days and then 18 1methanol for 8 days. The extracts were
combined and concentrated in a vacuum evaporator to a small volume. The concentrate was then sprayed on fresh leaves or
leaf residue with the amount that was originally included in the fresh leaves.
Palatability of Extracts
Fresh leaves of 10 to 20 cm in length (260 g wet weight) were put in large flask (capacity: 10 1)with methanol and
heated in a hot water bath. The 2 h extraction was repeated twice for the same material each time with 7 1 methanol. The
extracts were then combined (MW).
The combined extract (MW) was added to 500 ml water and then evaporated to approximately 400 ml, and the waterinsoluble
material was precipitated. Water-soluble (W) and -insoluble (M) fractions were separated by decanting. The water'
soluble fraction was then consecutively extracted with n-hexane (Wh), ethyl acetate (We), and butanol (Wb) to leave a
residual water fraction (Ww). The water-insoluble fraction was dissolved with methanol and then extracted with n-hexane
(Mh) to leave a residual methanol fraction (Mm).
The palatability of the various fractions was examined by filter paper tests. Each fraction was tested in pairs of A4
and N4 ....
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