Introduction Materials and methods

amplitude changing from 494 to 676, 388 and 547 pmol l y1 , respectively. The transient decrease and subsequent recovery in progesterone concentrations was observed to occur in connection with prostaglandin release during early pregnancy. Oestradiol-17b plasma peak concentrations attained Ž after luteolysis were significantly higher than those recorded in early pregnant animals around 30 y1 y1 . pmol l and ll pmol l . Concentrations of PGFM decreased rapidly after the first administra- tion of FM and remained low throughout the first 2 days of treatment. Thereafter, pulsatile release of prostaglandins started, and luteolysis proceeded; but a delay of 1–1.5 days in the progesterone decline was observed. Thus, it might be suggested that a higher dose andror a more intensive injection schedule is required in llamas than in other ruminants to prevent luteolysis. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Llama; Progesterone; Prostaglandins; Flunixin meglumine; Corpus luteum; Pregnancy

1. Introduction

Ž . In non-pregnant llamas Lama glama a dramatic fall in progesterone concentrations is observed between days 9 and 11 post-mating, in connection with repeated surge Ž . release of PGF from the uterus Sumar et al., 1988; Aba et al., 1995 . In pregnant 2 a animals, a transient decrease and subsequent recovery in progesterone concentrations has been reported during the period when maternal recognition of pregnancy is expected to Ž . occur Adams et al., 1991; Aba et al., 1995 . In these studies, no pulsatile release of PGF has been reported in association with the transitory decline in progesterone 2 a Ž . concentrations in llamas, but in the closely related alpaca L. pacos , prostaglandin metabolite peaks have been detected from days 8 to 13 of pregnancy, indicating a temporal relationship between the progesterone decline and PGF pulsatile release 2 a Ž . Aba et al., 1997 . Ž . Flunixin meglumine FM , a non-steroidal anti-inflammatory drug, has the ability to reduce prostaglandin biosynthesis by inhibiting the enzyme cyclo-oxygenase. In conse- quence, when used intensively, the drug has shown the ability to inhibit the synthesis of PGF to the extent that luteolysis is prevented in cattle, without interfering irreversibly 2 a Ž . with the pulsatile mechanism of prostaglandin Aiumlamai et al., 1990 . The objective of the present study was twofold: first, to investigate the plasma concentrations of progesterone in relation to concentrations of 15-ketodihydro-PGF 2 a Ž . PGFM during the period of luteolysis and maternal recognition of pregnancy in llamas and, second, to evaluate the ability of FM to inhibit luteolysis in non-pregnant llamas.

2. Materials and methods

2.1. Animals Ž . Eleven sexually mature female llamas L. glama , 3–5 years old with an average Ž . weight of 105 kg range 95–120 , were used. The animals were kept in a natural pasture Ž X at the Faculty of Veterinary Sciences, UNCPBA, Tandil, Argentina, 37817 S.L., sea . level and were supplemented with hay twice a day. Since several animals were assigned to successive trials, a complete rest period of 2 months was provided to the animals between treatments in order to circumvent possible carry-over effects of the previous treatment to the outcome of the following. 2.2. Treatments Ž . Ž Intra-vaginal sponges containing medroxyprogesterone acetate MPA 120 mg, . Syntex, Buenos Aires, Argentina were inserted and left in the vagina for 9 days. Six Ž . days after removal of the sponges day 15 , 10 animals were bred with an intact male, while 11 llamas were mated with a vasectomized male. Five of the animals mated with Ž w the vasectomized male were treated with FM Finadyne vet., Schering-Plough, Union, . Ž . NJ, USA . FM was injected intravenously every 6 h 06:00, 12:00, 18:00 and 24:00 h at a dose of 2.2 mgrkg from days 6 to 12 post-copulation. 2.3. Blood sampling Blood samples were collected daily from days 3 to 6 post-mating. Thereafter, samples Ž . were obtained every second hour during the day 08:00–20:00 h and every fourth hour Ž . during the night 20:00–08:00 h until day 16 post-mating. In animals treated with FM, the first sample was collected on day 5 post-mating, and the following samples were collected at 2-h intervals from 06:00 to 24:00 h from days 6 to 12 after mating. All blood samples were collected by venipuncture and immediately drawn into heparinized tubes. Plasma was separated by centrifugation and stored at y208C until analysed. In order to minimise possible damage to the jugular veins from the sampling protocol, puncture was performed according to a schedule at high, medium and low positions, on both the left and right sides of the neck. 2.4. Hormone assays Progesterone was assayed with an enhanced luminescence immunoassay technique Ž . Amerlite; Kodak Clinical Diagnostics, England previously validated for llama plasma Ž . Aba et al., 1995 . The intra-assay coefficient of variation calculated from the precision profiles was below 8 for concentrations between 2 and 160 nmol l y1 . The inter-assay Ž y1 . coefficients of variation, for three quality-control samples were 7 2 nmol l , 8 Ž y1 . Ž y1 . y1 18 nmol l and 6 54 nmol l . The sensitivity of the assay was 0.2 nmol l . The plasma metabolite of PGF , PGFM, was analysed by RIA according to Kindahl 2 a Ž . Ž . et al. 1976 and Granstrom and Kindahl 1982 . The intra-assay coefficients of variation ¨ were below 8 for samples containing 240 and 485 pmol l y1 . The corresponding inter-assay coefficients of variation were 5.5 and 8.4, respectively. The practical detection limit of the assay was 30 pmol l y1 . Ž Oestradiol-17b concentration was determined using RIA kit Diagnostic Products, . Ž Los Angeles, CA, USA , reported for use with bovine plasma Sirois and Fortune, . Ž 1990 , and validated for use with llama plasma after minor modifications Aba et al., . 1995 . The intra-assay coefficients of variation calculated from the precision profiles were 18 at 6 pmol l y1 , and below 11 for concentrations between 19 and 180 pmol y1 Ž l . The inter-assay coefficients of variation for three control samples were 22 13 y1 . Ž y1 . Ž y1 . pmol l , 6 39 pmol l and 10 84 pmol l . The lowest amount of Ž . oestradiol-17b detectable defined as the intercept of maximal binding y2 SD was 4 pmol l y1 . Hormone concentrations are expressed in SI units. To convert from pmol l y1 to pg ml y1 and from nmol l y1 to ng ml y1 the following factors should be used: PGF 2 a metabolite: 2.8; oestradiol-17b: 3.7 and progesterone: 3.2. 2.5. Analysis of data Basal concentrations of PGF metabolite were calculated by averaging the values 2 a obtained on each particular day and removing values higher than two standard devia- tions from the mean value, until the baseline remained unchanged. Concentrations that exceeded the baseline by more than two standard deviations between days 7 and 12 were defined as peaks. The amount of PGF released during each peak was estimated by 2 a calculating the areas under the release curve according to the formula: PGF release s Ý PGFM q PGFM r2 = 120 min, Ž . Ž . 2 a i iq120 where i s 0, 120, 240, . . . , min and where PGFM s basal concentration was consid- i ered equal to 0. Daily progesterone concentrations were estimated by averaging the concentrations measured in the sample collected at 08:00, 16:00 and 24:00 h for each Ž . individual animal. Analysis of variance, using a repeated measures within-SS design, was applied to detect differences in hormone concentrations. In all cases, a least-signifi- Ž . cant difference test LSD was used to determine differences between means. Because of variations in the time required to attain peak progesterone concentrations in pregnant animals, values were normalised against the highest concentration during days 7, 8 and 9. The mean peak value was further compared with mean concentrations attained during the following days. All statistical analyses were carried out using the StatisticarW, Ž . release 4.0, software package Statsoft, USA Statistica for Windows, 1993 . Results are expressed as mean SEM.