2. Materials and methods
2.1. Ethical considerations These studies were undertaken with the approval of the AgResearch Invermay
Ž Animal Ethics Committee, as required in New Zealand by the Animal Welfare Codes of
. Ethical Conduct Act 1987. All procedures were conducted by fully trained staff from
Ž the Invermay Agricultural Centre and in accredited facilities
NZQA accreditation .
scheme . 2.2. Animals and management
Ž .
This study was conducted in the year subsequent i.e., 1996 to that described by Ž
. Ž
Asher et al. 2000 using the same animals. The group was comprised of 25 adult 5–11 .
Ž . Ž
years old parous hinds of the three distinct genotypes : i Scottish red deer C. e. . Ž .
Ž .
Ž .
scoticus; n s 9 ; ii F hybrid wapiti C. e. nelsoni = red deer C. e. scoticus; n s 6 ;
1
Ž . Ž
. Ž
and iii maternal backcross hybrid PD deer E. daÕidianus; 25 = red deer C. e.
. scoticus; 75; n s 9 . However, following the deaths of one red deer hind and one PD
Ž .
deer hybrid hind from malignant catarrhal fever MCF in September 1996, replacement individuals of corresponding genotype were included in the study at this time. The hinds
were grazed on predominantly ryegrassrclover pasture as a single group in the continuous presence of a vasectomised red deer stag throughout the duration of the trial
from February 1996 to March 1997.
2.3. SuperoÕulation treatment At intervals of exactly 6 weeks starting in February 1996, all hinds received a
standardised treatment regimen used routinely to induce a superovulatory response in Ž
. red deer hinds Fennessy et al., 1994 . This treatment consisted of a 12-day placement of
Ž an intravaginal progesterone-releasing device Easi-breed CIDR type G; InterAg, Hamil-
. ton, NZ , with device replacement on the eighth day. A series of eight consecutive i.m.
Ž injections of 0.05 units ovine FSH Ovagen; Immuno-Chemical Products, Auckland,
. NZ; 0.04 units total dosage per animal equivalent to 72 units NIH-FSH-S
was
1
delivered approximately every 12 h from the device replacement to 12 h after the final device removal. The final injection also contained 250 i.u. equine chorionic go-
Ž .
nadotrophin eCG; Folligon; Intervet, Lane Cove, NSW, Australia . Additional treatment to ensure complete regression of all luteal tissues prior to the next superovulatory
Ž treatment involved the i.m. injection of 500 mg cloprostenol 2 ml Estrumate; Coopers
. Animal Health NZ, Upper Hutt, NZ 14 days after the removal of the CIDR device.
2.4. Assessment of oÕarian response All hinds underwent laparascopic ovarian examination 6–7 days after the device
Ž .
removal approximately 5–6 days from ovulation . Anaesthesia was induced, following the 12-h fasting, by an i.v. injection of an aqueous mixture containing 58.3 mg ml
y1
y1 y1
Ž xylazine hydrochloride, 3.2 mg ml
azaperone, and 0.4 mg ml fentanyl citrate 0.02
y1
. ml kg
liveweight Fentazin; Parnell Laboratories, Auckland, NZ . Uterine turgidity Ž
was reduced by an i.v. injection of 0.5 mg clenbuterol hydrochloride 5 ml Planipart; Ž
. .
Boehringer Ingelheim NZ , Auckland 2–3 min prior to laparoscopy. Each ovary was examined for the presence and the number of corpora lutea, luteinised follicles, and
non-luteinised follicles with a diameter greater than or equal to 8 mm. Upon the completion of the ovarian examination, the hinds were given an i.v. injection of 50 mg
Ž .
yohimbine hydrochloride 5 ml Recervyl; Aspiring Animal Services, Wanaka, NZ to reverse the effects of anaesthesia.
2.5. Measurement of oFSH antibody in plasma All hinds in the trial were blood-sampled by jugular venepuncture at the start, middle,
and end of the study. Plasma samples were diluted 1:100 in pH 7.0 phosphate buffered Ž
. Ž
. saline containing 0.1 BSA PBS BSA . Diluted samples 100 ml were incubated
overnight with 100 ml pre-precipitated sheep anti-rabbit second antibody, the samples were then incubated for 1 h and then centrifuged at 3200 rpm for 35 min after the
addition of 1.0 ml of 4 WrV PEG 6000. Samples were decanted and counted for 60 s. The oFSH binding activity of the plasma was determined from the ratio of bound to the
total counts following correction for non-specific binding controls.
2.6. Statistical analyses Ž
. Ovulation rate OR was assessed as the number of corpora lutea and the total
Ž .
follicular stimulation TFS was calculated as the sum of corpora lutea, luteinised Ž
qx
. follicles, and large non-luteinised follicles. OR and TFS data were log 1
trans- Ž
. Ž formed and analysed by residual maximum likelihood REML
Patterson and Thomp- .
son, 1971 with individuals as a random effect, and genotype, date, and their interaction as fixed effects. The assumption of individual randomness in ovarian response over time
was evaluated for each genotype independently by the non-parametric means test Ž
. Friedman’s Test based on ovulation rate rankings. This evaluation was conducted for
the first five consecutive treatments only due to missing values for some animals at subsequent dates.
3. Results
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