All animal treatments and procedures were approved by the
Animal Care and Use Committee at the Kyushu Okinawa
Agirucultral Research Center. Experiments were conducted
from January to March in 2009 (winter) and from July to
September in 2008 and 2009 (summer). A total of 13 nonlactating
Japanese Black cows were used for experiments
(4 cows in winter, 32–63 months old; 9 cows in summer,
26–73 months old). All cows exhibited regular signs of
estrus (average 21 days) and no reproductive problems were
observed prior to the experiment.
Cows were kept in a group of four or five animals in a
concrete-floored paddock without a roof. Cows were fed 1 kg
concentrated feed once a day (08.30 hour) and Italian
rye grass hay twice a day (08.30 to 09.00 and 16.00 to
16.30 hours), with water ad libitum.