contemporaries. The cows, which did not ovulate after treatment, were also characterised according to LH pulse frequency and plasma oestradiol concentrations.
2. Materials and methods
2.1. Animals Ž
. Primiparous cows 17 Friesian, F; and 19 Jersey, J calved in July and August, 1995
Ž .
mean calving date: 20 July 2 days at 2 years of age were used. The cows used in the trial were anovulatory and did not have detectable pathology of the reproductive tract.
The pre-partum ration was solely pasture with feed intake managed by regulating the area of grazing. Liveweight was measured weekly and BCS assessed to manage the feed
Ž .
intake so as the cows calved with a BCS of F 4.5 Macdonald and Macmillan, 1993 . The serum copper, magnesium and selenium levels of each animal were monitored
Ž .
Alpha Scientific, Hamilton, New Zealand . Those with marginal levels were treated at least 8 weeks pre-partum with sustained release rumen capsules. Experimental use of
animals was approved by the Animal Ethics Committees of both AgResearch, Ruakura, and the University of Waikato, Hamilton, New Zealand.
2.2. Ultrasonography The ovaries of each cow were observed daily by trans-rectal ultrasonography with a
7.5 MHz transducer. These observations were commenced from 8 to 10 days post-partum so as to characterise the growth pattern of large ovarian follicles. The description of all
follicles by their size and location, and the definitions of a DF were as described by Ž
. McDougall et al. 1995a . If ultrasonographic examination commenced after emergence
Ž .
Ž of the first DF post-partum DF1 , the largest follicle present that was G 7 mm in
. diameter and at least 2 mm larger than the second largest follicle was defined as the
Ž .
DF1. Subsequent DFs were described as the second DF post-partum DF2 , and the third Ž
. DF post-partum DF3 if they emerged before the first post-partum ovulation.
Ž .
Cows were diagnosed as anovulatory at the commencement of treatment day 0 if a Ž
. new corpus luteum CL could not be identified. This definition incorporated the need to
monitor the regressing CL of pregnancy. Daily ultrasonography continued until ovula- tion was confirmed by the presence of a newly formed CL, or until the identification of
Ž .
a recently emerged DF 7 mm after the completion of the treatment period. 2.3. Experimental design
Ž .
This study had a factorial design with main effects of breed J and F
and Ž
. progesterone treatment. Half the cows, within breed, were treated T; day 0 with an
Ž .
intravaginal progesterone-releasing device CIDR-Be, InterAg, Hamilton, New Zealand for 5 days commencing 12–18 days post-partum. The remaining cows served as
Ž . untreated controls C . A jugular vein was cannulated on day y2 to facilitate regular
blood sampling. Changes in LH secretion were measured in samples taken at 15-min
Ž .
intervals for 8 h during the day before progesterone treatment day y1 , the day after Ž
. insertion of the progesterone device
day 1 , and the day before removal of the Ž
. progesterone device day 4 . Blood samples were centrifuged at 1500 = g for 15 min
and plasma stored at y208C. Daily blood samples were collected at the time of Ž
. ultrasonography at 06:00 h to determine plasma progesterone concentrations. A single
blood sample was collected on days 6 and 8 to measure plasma oestradiol concentra- tions. Additional samples were collected from T-group cows at 4-h intervals from 16 to
84 h after device removal to detect a possible pre-ovulatory surge in LH secretion. Every control cow was blood-sampled daily at 06:00 h.
Tailpaint and a contrasting aerosol spray-paint were applied to the tailhead on days 0 Ž
. and 5, respectively as described by Macmillan et al., 1988 to aid the detection of
oestrus. Changes in the coverage of paint were noted every 4 h from 06:00 h on day 6 to 18:00 h on day 9.
2.4. Hormone assay Ž
. A commercial kit Coat-a-count, DPC, CA, USA was used to measure progesterone
directly in a solid phase,
125
I-labelled radioimmunoassay as previously validated in our Ž
. laboratory McDougall et al., 1995b . Intra- and interassay coefficients of variance in
standards of 0.4, 2.0 and 4.4 ngrml were 11, 8 and 6, and 15, 11 and 12, respectively. The sensitivity of the assay was 0.08 ngrml.
Concentrations of LH were measured in duplicate plasma samples by direct heterolo- Ž
. gous RIA using a second antibody precipitation technique McDougall et al., 1995b .
The assay measured ovine LH concentrations with a primary antibody raised in rabbits Ž
. against ovine LH Ra2 antisera, AgResearch, Invermay, NZ and a secondary antibody
Ž .
of sheep against rabbit gammaglobulins AgResearch, Ruakura . Ovine LH for standards Ž
. and iodination was given by NIH NIH Lot No. AFP-7071B . Intra- and interassay
coefficients of variance in standards of 0.3, 0.9, 2.8 and 10.6 ngrml were 17, 7, 8 and 8, and 19, 9, 9 and 9, respectively. The sensitivity of the assay was 0.14
ngrml.
Ž .
Plasma oestradiol-17b PE concentrations were determined by a modified commer-
2
Ž .
cial kit RIA Estradiol MAIA, Biodata Diagnostics, Rome, Italy . The kit utilised a double antibody magnetic separation technique where rabbit antisera were raised against
oestradiol-17b as a primary antibody; and a secondary antibody consisted of goat antisera raised against rabbit gammaglobulins coupled to magnetic particles. Freeze-dried
125
I-labelled oestradiol-17b and oestradiol-17b standards were also provided. The Ž
. modified assay involved a similar protocol to Mann et al. 1995 and Prendiville et al.
Ž .
1995 . Intra- and interassay coefficients of variance in standards of 1.3, 5.7 and 16.0 pgrml were 22, 18 and 8, and 24, 23 and 10, respectively. The sensitivity
of the assay was 0.35 pgrml. 2.5. Statistical analysis
LH data from serial sampling on days y1, 1 and 4 were analysed for pulsatile characteristics of mean and pulse frequency using a specialised software package
Ž .
Pulster, Objective Software, Sydney, Australia . This package used the pulsar algorithm Ž
. described by Merrium and Wachter 1982 . The ‘‘G parameters’’ were set as G1 s 3.8,
G2 s 2.6, G3 s 1.9, G4 s 1.5, G5 s 1.2. The precision profile was optimised with the Ž
. Ž
. Ž
. coefficient of variation CV option as LH CV s 19.4–18.9 LH Mean .
Breed differences in liveweight and BSC at calving were analysed by Student’s t-test. Categorical data were analysed by chi-square methods, both in an Excel spreadsheet
Ž .
Ž .
Microsoft, USA and by the SAS CATMOD procedure SAS Institute, Cary, USA . Ž
. Continuous data were analysed by the general linear model GLM option of SAS v6.10
Ž .
Ž .
SAS Institute, Cary, USA , or Minitab v10.51Xtra Minitab, PA, USA . If measure- ments were repeated over time, a split plot GLM was used. Each result is presented as
mean SEM, unless stated.
3. Results
