J. Taponen et al. Animal Reproduction Science 64 2000 161–169 163
given slightly post-ovulation on the development and function of the corpus luteum and the length of the oestrous cycle in cattle.
2. Materials and methods
2.1. Animals Three cows 1–2 calvings and six heifers of the Finnish Ayrshire breed were used. The
cows had calved 9–13 months earlier. The heifers were between 12 and 15 months of age at the beginning of the experiment. All animals had shown normal oestrous cycles and were
clinically healthy. They were stanchioned and fed grass silage, dry hay, and corn according to Finnish standards. The experiment lasted 5 months, from March to July.
2.2. Experimental procedures The experiment included two different GnRH treatments T1, T2 and one control ma-
nipulation C. Every animal was assigned once to each of these three manipulations. These were performed in random order for each animal. In all cases a spontaneous ovulation
preceded the manipulation period. Oestrus was induced with 0.5 mg of cloprostenol PG Estrumat® 0.25 mgml, Mallinck-
rodt Veterinary Ltd., Harefield, Uxbridge, England administered intramuscularly i.m. in 18 cases. In nine cases, oestrus was induced with an intravaginal device containing 1.9 mg
of progesterone and 10 mg of encapsulated oestradiol benzoate, attached to the device EAZI-BREED
TM
CIDR® device, InterAg, Hamilton, New Zealand; Cidirol® capsules, InterAg, Hamilton, New Zealand. The device was inserted for 12 days. From the second
day after PG or device removal, the animals were examined daily by transrectal ultrasound to determine the occurrence of ovulation.
After detection of ovulation, the animals were given 250 mg of gonadorelin i.m. Fertagyl® 0.1 mgml, Intervet International B.V., Boxmeer, Holland as follows: 0–24 h after ovulation
T1, 24–48 h after ovulation T2, or no gonadorelin control manipulation, C. Ultrasonographic examinations were performed daily from the second day after PG treat-
ment or device removal until ovulation. After this, examinations were carried out on days 1, 4 or 5, 7 or 8, 11 or 12, 14 or 15 and daily from the beginning of the next oestrous signs
until ovulation day 0 = day of ovulation. Blood samples for hormone determinations were collected by vacuum puncture of a tail
blood vessel into silicone plain tubes Venoject by 20 G needles Vacutainer. Samples were taken daily from day 1 after ovulation until the next oestrus. Serum was harvested,
frozen and stored in plastic tubes at −20
◦
C until analysed. All procedures were performed in the morning, from 08.00 to 10.00 h.
2.3. Ovarian examinations A real-time B-mode ultrasound scanner Aloka SSD-210DXII, Aloka Co. Ltd., Japan
equipped with a 7.5 MHz rectal linear array transducer was used to follow the growth of
164 J. Taponen et al. Animal Reproduction Science 64 2000 161–169
the corpus luteum CL and of possible secondary corpora lutea, and to determine the day of ovulation and diameter of the ovulatory follicle as described by Assey et al. 1993. All
examinations were carried out by the same operator. The ovaries were scanned several times in lateromedial and dorsoventral planes to de-
termine the largest cross-section of follicles andor a CL. By freezing the image, the largest and the smallest diameters were measured and recorded, with the average diam-
eter calculated later. The central cavities of CLs were measured and recorded in the same way.
All follicles equal to or larger than 8 mm were measured. Follicles smaller than 8 mm were only counted and divided according to their size into groups of small less than
5 mm and medium 5–8 mm follicles. Day of ovulation day 0 was determined to be the last day when the follicle was intact according to ultrasound scanning in the morn-
ing before the subsequent examination the next morning when the follicle had disap- peared.
2.4. Hormone determinations The concentration of progesterone in the serum was measured in all samples by radioim-
munoassay by use of a commercial kit Coat-A-Count® Progesterone, Diagnostic Products Corporation, Los Angeles, USA. The intra-assay coefficient of variation for progesterone
was 6.31 when calculated from duplicates of measurements between 1.0 and 20.0 nmoll n = 380. The inter-assay coefficient of variation was 6.74 81.9 nmoll, n = 8. The
detection limit of the assay was 0.3 nmoll.
2.5. Statistical analysis Data were analysed using the statistical analysis system software SAS, 1987. Pro-
gesterone concentrations were analysed by repeated measures analysis of variance, with manipulation and day as the within-subject factors. Significance of day effects and day
by manipulation interaction effects was evaluated by use of Greenhouse–Geisser-adjusted P
-values. Effects of manipulations on interoestrous intervals and on size of CLs were anal- ysed by repeated measures analysis of variance, with manipulation as the within-subject
factor. Differences between animals in interoestrous intervals were tested by one-way anal- ysis of variance. Differences were considered significant at P 0.05.
3. Results
