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Plasma concentrations of 15-ketodihydro-PGF2α, progesterone, oestrone sulphate, oestradiol-17β and cortisol during late gestation, parturition and the early post partum period in llamas and alpacas
Animal Reproduction Science 50 Ž1998. 111–121
Plasma concentrations of 15-ketodihydro-PGF2 a , progesterone, oestrone sulphate, oestradiol-17b and cortisol during late gestation, parturition and the early post partum period in llamas and alpacas M.A. Aba
a,d,)
, J. Sumar b, H. Kindahl L.-E. Edqvist a
c,d
, M. Forsberg
a,d
,
a
Department of Clinical Chemistry, Faculty of Veterinary Medicine, Swedish UniÕersity of Agricultural Sciences, P.O. Box 7038, SE-750 07, Uppsala, Sweden b Instituto Veterinario de InÕestigaciones Tropicales de Altura, UniÕersidad Mayor de San Marcos, Lima, Peru c Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, Swedish UniÕersity of Agricultural Sciences, P.O. Box 7039, SE-750 07, Uppsala, Sweden d Centre for ReproductiÕe Biology, Swedish UniÕersity of Agricultural Sciences, Uppsala, Sweden Accepted 23 September 1997
Abstract Plasma concentrations of 15-ketodihydroprostaglandin ŽPG. F2 a , progesterone, oestrone sulphate, oestradiol-17b and cortisol during late gestation, parturition and the early post-partum period were measured in six llamas and five alpacas. During the last 100 days of pregnancy, 15-ketodihydro-PGF2 a concentrations increased steadily until the day of parturition when a massive release was detected Ž P - 0.01. concomitant with a decrease in progesterone concentrations Ž P - 0.01.. The highest PGF2 a metabolite concentrations Ž159 " 35 nmol ly1 and 92 " 29 nmol ly1 in llamas and alpacas respectively. were detected in the sample collected during the morning on the day of parturition. Basal concentrations were registered by day 3 after delivery. Plasma concentrations of oestrone sulphate started to increase 80 days before parturition and reached peak concentrations immediately before parturition Ž15 " 3 nmol ly1 in llamas and 18 " 5 nmol ly1 in alpacas.. Oestrone sulphate concentrations dropped sharply Ž P - 0.01. on the day of parturition in llamas and one day later in alpacas, whereupon they remained relatively unchanged until at least 20 days postpartum. Oestradiol-17b concentrations were higher than 180 pmol ly1 during the last 45 days of pregnancy, began to decrease on the day of parturition and
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Corresponding author.
0378-4320r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 7 8 - 4 3 2 0 Ž 9 7 . 0 0 0 8 4 - 5
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reached very low concentrations within the following two days. High oestradiol-17b concentrations were registered 7 days postpartum in all alpacas Ž P - 0.05. and within 10 days of parturition in five of six llamas Ž P - 0.01.. No significant cortisol peaks were observed around parturition, but mean concentrations were increased in both species. q 1998 Elsevier Science B.V. Keywords: Llama; Alpaca; Pregnancy; Parturition; Postpartum period; Prostaglandin; Oestradiol; Progesterone
1. Introduction Endocrine changes after copulation, luteolysis and early pregnancy in llamas Ž Lama glama. and alpacas Ž Lama pacos . have been well described elsewhere ŽSumar et al., 1988; Bravo et al., 1990; Bravo et al., 1991b; Aba et al., 1995; Bravo et al., 1996.. Conversely, information about hormonal changes associated with late gestation, parturition and the early post-partum period is poorly defined. It has been shown that the corpus luteum is necessary for the maintenance of pregnancy throughout the gestation period in both species ŽSumar, 1988.. In llamas, plasma progesterone concentrations remain elevated throughout pregnancy, begin to decline between 15 days and 1 day prepartum and finally drop to low levels from day 1 prepartum until parturition ŽLeon et al., 1990; Bravo et al., 1996.. Oestrogens Žcombined oestrone, oestradiol-17a and oestradiol-17b . start to increase 9 months after mating and remain high until the end of pregnancy, with peak concentrations during the last week prepartum ŽLeon et al., 1990.. Only a slight increase in plasma cortisol concentrations has been observed in association with parturition in llamas ŽLeon et al., 1990.. Plasma concentrations of oestrogen and progesterone have been reported to be low following parturition and development of follicles beginning on 5–6 days after parturition ŽSumar et al., 1972; Leon et al., 1990; Bravo et al., 1991a,b.. Prostaglandin ŽPG. F2 a plays a major role during parturition in most domestic species ŽEdqvist et al., 1978; Barnes et al., 1978; Mitchell et al., 1979; Kindahl et al., 1982a.. To our knowledge there are no reports describing the plasma concentrations of PGF2 a metabolite and oestrone sulphate during this period in llamas or alpacas. The aim of this study was to document the secretory profiles of PGF2 a metabolite, progesterone, oestrone sulphate, oestradiol17b and cortisol during late gestation, parturition and the early postpartum period in llamas and alpacas.
2. Materials and methods 2.1. Animals and blood samples In total, six llamas and five alpacas were mated once, with a fertile male, for an average period of 20 min. The day of mating was registered in order to calculate the gestation length. Vasectomized males were used for sexual receptivity detection in both species on days 12, 24, 40, 60 and 90 after mating. The animals were kept on natural pastures at the La Raya Research Station, San Marcos University, Cuzco, Peru, ´ located
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at 158S, 708W and 4250 m altitude. Blood samples were obtained every second week from day 7 until the end of the 8th month and at 7-day intervals between 9 and 10.5 months of pregnancy. Thereafter, samples were collected twice a day Žmorning and afternoon. until parturition. Starting on the day of parturition, we collected four samples per day during the first 10 days and two a day during the following 10 days except for one llama in which sampling was discontinued after day 12 post partum. Blood was collected by jugular venipuncture into heparinized vacutainer tubes except for the 20 days post partum period, when the collections were made using permanent catheters placed in the jugular vein. In some cases where the catheters were obstructed, blood sampling was continued by venipuncture. Plasma was separated by centrifugation and stored at y208C until analyzed. 2.2. Hormone assays Progesterone was determined by an enhanced luminescence immunoassay technique ŽAmerlite; Kodak Clinical Diagnostics, UK. previously validated for llama and alpaca plasma ŽAba et al., 1995.. The intra-assay coefficient of variation calculated from the precision profiles was below 7% for concentrations between 2 and 160 nmol ly1 . The inter-assay coefficients of variation for three quality-control samples were 8% Ž2 nmol ly1 ., 8% Ž18 nmol ly1 . and 5% Ž54 nmol ly1 .. The lowest amount of progesterone detectable Ždefined as the intercept of maximal binding—2 SD. was 0.2 nmol ly1 . The plasma metabolite of PGF2 a , 15-ketodihydro-PGF2 a , was analyzed by RIA according to Kindahl et al. Ž1976. and Granstrom ¨ and Kindahl Ž1982.. The intra-assay coefficients of variation for two control samples were 8.5% Ž240 pmol ly1 . and 4.2% Ž485 pmol ly1 .. The corresponding inter-assay coefficients of variation in three assays were 15.5% and 8.4%, respectively. The practical detection limit of the assay was 30 pmol ly1 . Oestradiol-17b was determined using an RIA previously validated for use in bovine plasma ŽSirois and Fortune, 1990., with the following modification: the standard curve was prepared with standards supplied with the radioimmunoassay kit ŽDiagnostic Products, Los Angeles, CA, USA.. Serially diluted plasma from female llamas and alpacas containing high concentrations of oestradiol-17b produced displacement curves parallel to the standard curve. The intra-assay coefficients of variation calculated from the precision profile of seven assays were 13.4% at 6 pmol ly1 , 10.2% at 19 pmol ly1 and below 10% up to 180 pmol ly1 . The inter-assay coefficients of variation for three control samples were 25% Ž13 pmol ly1 ., 6% Ž39 pmol ly1 . and 10% Ž84 pmol ly1 .. The lowest amount of oestradiol-17b detectable Ždefined as the intercept of maximal binding—2 SD. was 3 pmol ly1 . Oestrone sulphate concentrations were determined by radioimmunoassay ŽWright et al., 1978; Kindahl et al., 1982b. using antiserum against oestrone-glucosiduronate-bovine thyroglobulin raised in rabbits. Serially diluted plasma from female llamas and alpacas containing high concentrations of oestrone sulphate produced displacement curves parallel to the standard curve. The sensitivity of the assay was 1 nmol ly1 . The intra-assay coefficient of variation for oestrone sulphate concentrations in the range of 2.0 to 100 nmol ly1 was below 15%.
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Cortisol was determined using a luminescence immunoassay according to a method previously described for pig plasma ŽMagnusson et al., 1994.. Serial dilutions of llama and alpaca plasma with high concentrations of cortisol produced displacement curves parallel to the standard curve. The sensitivity of the assay was 7 nmol ly1 , and the intra-assay coefficient of variation, calculated based on six assays, was below 15% for concentrations of cortisol between 9.1 and 435 nmol ly1 . The corresponding inter-assay coefficients of variation for two quality control samples were 27% Ž15 " 4 nmol ly1 . and 12% Ž147 " 18 nmol ly1 .. Hormone concentrations are expressed in SI units. To convert from pmol ly1 to pg y1 ml and from nmol ly1 to ng mly1 the following factors should be used: PGF2 a metabolite: 2.8; oestradiol-17b : 3.7; oestrone sulfate: 2.7; cortisol: 2.7 and progesterone: 3.2. 2.3. Analysis of data A completely randomized analysis of variance was performed to detect differences in hormone concentrations. Due to the variability in the length of pregnancy, samples obtained in different animals during the last 100 days of pregnancy, parturition and early post partum were normalized by day of parturition. Cortisol concentrations in samples obtained weekly between days y38 and y10 were pooled and therefore compared with concentrations measured in daily samples from day y9 to day q5 Žday 0 s day of parturition.. A least significant difference test ŽLSD. was used to determine significant differences between means. Results are expressed as mean " S.E.M.
3. Results The mean gestation length was 351.5 " 1.9 days Žrange 343–353. and 335.6 " 2.8 days Žrange 327–343. in llamas and alpacas respectively. All animals had normal unassisted deliveries, and none of the animals showed any signs of uterine disorders in the postpartum period studied. Mean progesterone concentrations remained relatively constant around 20 nmol ly1 Žllamas. or slightly increased from 11 to 15 nmol ly1 Žalpacas. over the last three months of pregnancy. During the last 7–10 days of pregnancy, progesterone concentrations dropped drastically Ž P - 0.01. and remained low until at least 20 days post partum ŽFig. 1a and b.. During the last 100 days of pregnancy, plasma concentrations of 15-ketodihydroPGF2 a increased steadily until the day of parturition when a massive release was detected in both species ŽFig. 1a and b. in connection with the declining concentrations of progesterone. In llamas, mean PGF2 a metabolite concentrations during the parturition day were significantly higher Ž P - 0.05. in the morning sample Ž159 " 35 nmol ly1 . than in the sample collected in the afternoon of the same day Ž21 " 6 nmol ly1 .. Although differences were not statistically significant, the same tendency was observed in alpacas Ž92 " 29 and 45 " 24 nmol ly1 respectively.. Thereafter, plasma PGF2 a metabolite concentrations sharply declined Ž P - 0.01., reaching around 150 pmol ly1 by
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Fig. 1. Plasma concentrations of 15-ketodihydro-PGF2 a Žmean, logarithmic scale. and progesterone wmean" S.E.M. Žshaded area.x during the last 100 days of pregnancy in llamas Ža. and alpacas Žb.. Day 0 sday of parturition.
day 4 and remaining relatively constant thereafter until at least day 20 post partum ŽFig. 2a and b.. No significant peaks were detected during this period. In both species, plasma oestrone sulphate concentrations rose steadily towards the end of gestation, with the highest values recorded during the last 30 days of pregnancy Ž15 " 3 nmol ly1 and 18 " 5 nmol ly1 in llamas and alpacas respectively.. In llamas, oestrone sulphate plasma concentrations declined on the day of parturition. In alpacas the decline was registered one day post partum. Thereafter, concentrations remained relatively unchanged until at least 20 days post partum ŽFig. 3a and b..
Fig. 2. Mean plasma concentrations of 15-ketodihydro-PGF2 a and progesterone w"S.E.M. Žshaded area.x from the day of parturition Žday 0. to day 20 post partum.
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Fig. 3. Plasma oestrone sulphate concentrations Žmean"S.E.M.. during the last 100 days of pregnancy and 20 days post partum in llamas Ža. and alpacas Žb..
Plasma oestradiol-17b concentrations exceeded 180 pmol ly1 during the last 45 days of pregnancy. The first significant decrease Ž P - 0.05. in oestradiol concentrations was observed during the day of parturition, whereupon they continued to decline, reaching concentrations just above the detection limit of the assay Žaround 8 pmol ly1 . within the following two days. A significant rise in oestradiol-17b concentrations was registered within 7 days post partum in all alpacas Ž P - 0.05. and within 10 days post partum in five of six llamas Ž P - 0.01.. The llama with no oestradiol increase was the one for which sampling had been discontinued after day 12. In Fig. 4, the oestradiol-17b secretory profile after parturition in a representative llama Ža. and alpaca Žb. are shown. In llamas, slight, but non-significant, increases in mean cortisol concentrations were observed during the last week of pregnancy and the first three days post partum. No
Fig. 4. Oestradiol-17b secretory profile after parturition in a representative llama Ža. and alpaca Žb..
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Fig. 5. Cortisol concentrations Žmean"S.E.M.. during the last 45 days of pregnancy and the first 7 days post partum in llamas Ža. and alpacas Žb..
significant peaks were detected in connection with parturition ŽFig. 5a.. In alpacas, cortisol concentrations were elevated Ž P - 0.05. during the day of parturition and the day before, as compared with concentrations recorded during late pregnancy Žday y38 to day y10. and the first five days postpartum ŽFig. 5b.. 4. Discussion The mean gestation length in llamas registered in this study is in agreement with previous reports ŽJohnson, 1989; Leon et al., 1990., while the gestation period observed in alpacas is slightly shorter than values reported by others ŽSan-Martın ´ et al., 1968; Knight et al., 1995.. The progesterone secretory pattern during late gestation, parturition and early postpartum observed in this study is in agreement with patterns previously reported in South American camelids ŽAdam et al., 1989; Leon et al., 1990; Adams et al., 1991; Bravo et al., 1991b; Bravo et al., 1996. and in Old World camels ŽAgarwall et al., 1987; Musa et al., 1993.. To our knowledge, this is the first report describing the PGF2 a metabolite secretory profile during gestation, parturition and early postpartum in domesticated South American camelids. The pattern of prostaglandin metabolite secretion registered during the last 3 months of pregnancy does not differ from the patterns reported in several other mammals Žcow: Edqvist et al., 1978; mare: Allen and Pashen, 1981; goat: Fredriksson et al., 1984 and ewe: Fredriksson, 1985.. In accordance with findings in llamas reported by Leon et al. Ž1990., no decline in plasma progesterone concentrations was observed during the period of high PGF2 a metabolite concentrations. The sharp increase and peak concentrations registered the day before parturition and on the day of parturition, respectively, and the drop in progesterone concentrations correlate well with the pattern shown by the same authors. It is widely accepted that in llamas and alpacas almost 100% of the parturitions occur during the early hours of the day. No births occur between 1700 and 0400, when temperatures are low throughout the year in the natural habitat ŽSumar, 1983.. The
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finding that peak concentrations of PGF2 a metabolite were registered during the morning of the day of parturition in both species suggests that in llamas and alpacas, like in the mare, the second stage of labour is accompanied by a massive rise in PGF2 a metabolite concentrations as the newborn passes through the cervix and vagina. Our previous report of an intense release of PGF2 a in connection with mating in llamas and alpacas ŽAba et al., 1995. further supports the hypothesis that mechanical stimulation of the cervix andror vagina may induce prostaglandin release in these species. The increased oestrone sulphate concentrations observed during late gestation correlate with earlier studies in cows ŽThatcher et al., 1980; Abdo et al., 1991. and ewes ŽTsang, 1978; Mohamed et al., 1987. showing that maximum oestrone sulphate concentrations are observed during the last third of the gestation period when the fetus rapidly increases in weight and size. However, Bravo et al. Ž1996. have reported increasing plasma concentrations of oestrone sulfate later in gestation Žaround month 11.. Although there is no clear explanation for the different patterns registered between studies, it could be speculated that different cross reactivity of the antibodies with other steroids and steroid conjugates from the foeto-placental unit, may be responsible. Thus, from our results, it may be concluded that in llamas and alpacas oestrone sulphate analysis during the last 90 days prepartum can be used for monitoring aimed at making sure that pregnancy is progressing normally. The secretory pattern of oestradiol-17b recorded during the last two weeks of pregnancy, parturition and first week postpartum in both species is in agreement with a previous study in llamas ŽLeon et al., 1990.. Similar oestradiol-17b secretory pattern during late gestation has also been reported in the dromedary camel ŽAgarwall et al., 1987.. The finding that oestradiol-17b concentrations started to rise Žindicating that follicular development was initiated. within the first 10 days postpartum in five of six llamas and within 7 days postpartum in all five alpacas correlates well with the observation that South American camelids usually become pregnant within the first 2–3 weeks postpartum. Bravo et al. Ž1991b. reported that urinary oestrone sulphate concentrations accurately reflect ovarian follicle development in the postpartum period in llamas. The disclosure that in the present study, follicular activity was not reflected as elevated plasma oestrone sulphate concentrations may be attributable to the low sensitivity of the assay. Like in the mare ŽAllen and Pashen, 1981; Stewart et al., 1984. and sow ŽKindahl et al., 1982a., prostaglandin concentrations returned to basal concentrations 1–2 days postpartum in llamas and alpacas. Placentation in these species has been classified as diffuse and epitheliochorial. Conversely, the prolonged release of PGF2 a for several weeks postpartum has been reported in the cow ŽEdqvist et al., 1978; Kindahl et al., 1992., water buffalo ŽPerera et al., 1981., goat ŽFredriksson et al., 1984. and ewe ŽFredriksson, 1985.. All these ruminants have a similar type of cotyledonary placenta, and there might be some relationship between prostaglandin release pattern and type of placentation. The physiological significance of this difference in postpartum prostaglandin release between species is not fully understood, but as long as the release is prominent ovulations do not occur. In most mammalian species, the fetus dominates the mechanism stimulating the onset of parturition. A significant increase in the fetal plasma concentration of cortisol occurs
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during the final stages of gestation in sheep, goat, cattle and pigs ŽThorburn et al., 1977.. Although some increase in adrenal activity probably occurs in the equine fetus near term, no spectacular rise in fetal plasma cortisol concentration occurs before birth ŽNathanielsz et al., 1975.. In the present study, only slightly elevated cortisol concentrations were recorded around the day of parturition in the llamas and alpacas. Similarities between prepartum and postpartum stable cortisol concentrations in the maternal circulatory system have been reported in the rhesus monkey ŽScott et al., 1984. and in the llama, although a low-frequency sampling schedule was used in the latter study ŽLeon et al., 1990.. The secretory pattern reported in this study resembles that observed in the mare but any role of cortisol in the induction of parturition in llamas and alpacas remains to be determined. In brief, plasma concentrations of PGF2 a metabolite, oestradiol-17b and oestrone sulphate, progressively increase during the last third of pregnancy in llamas and alpacas, with a sudden rise registered at parturition, in connection with a drop in progesterone concentrations and a slight increase in cortisol concentrations. Thereafter, plasma concentrations of PGF2 a metabolite, progesterone and oestrone sulphate remains low until at least day 20 postpartum. Meanwhile, oestradiol-17b plasma concentration starts to increase during this period, indicating follicular activity as early as day 7 and day 10 postpartum in alpacas and llamas respectively. Acknowledgements The Swedish Agency for Research in Developing Countries ŽSAREC., the Swedish International Development Agency ŽSIDA., the Swedish Council for Forestry and Agricultural Research and the Department of Physiopathology, Faculty of Veterinary Sciences, UNCPBA, Tandil, Argentina, are acknowledged for financial support. References Aba, M.A., Forsberg, M., Kindahl, H., Sumar, J., Edqvist, L.-E., 1995. Endocrine changes after mating in pregnant and non-pregnant llamas and alpacas. Acta Vet. Scand. 36, 489–498. Abdo, G.A., Njuguna, O.M., Fredriksson, G., Madej, A., 1991. Levels of oestrone sulphate during pregnancy in different breeds of cows and its possible association with retained foetal membranes. Acta Vet. Scand. 32, 183–188. Adam, C.L., Moir, C.E., Shiach, P., 1989. Plasma progesterone concentrations in pregnant and non-pregnant llamas Ž Lama glama.. Vet. Rec. 125, 618–620. Adams, G.P., Sumar, J., Ginther, O.J., 1991. Form and function of the corpus luteum in llamas. Anim. Reprod. Sci. 24, 127–138. Agarwall, S.P., Khanna, N.D., Agarwall, V.K., Dwaraknath, P.K., 1987. Circulating levels of estrogen and progesterone in female camel Ž Camelus dromedarius. during pregnancy. Theriogenology 28, 849–859. Allen, W.R., Pashen, R.L., 1981. The role of prostaglandins during parturition in the mare. Acta Vet. Scand. ŽSuppl.. 77, 279–298. Barnes, R.J., Comline, R.S., Jeffcott, L.B., Mitchell, M.D., Rossdale, P.D., Silver, M., 1978. Foetal and maternal plasma concentrations of 13,14-dihydro-15-oxo-prostaglandin F2 a in the mare during late pregnancy and at parturition. J. Endocrinol. 78, 201–215. Bravo, P.W., Fowler, M.E., Stabenfeldt, G.H., Lasley, B.L., 1990. Endocrine responses in the llama to copulation. Theriogenology 33, 891–899.
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Sumar, J., 1988. Removal of the ovaries or ablation of the corpus luteum and its effect on maintenance of gestation in the alpaca and llama. Acta Vet. Scand. 83, 133–141. Sumar, J., Novoa, C., Fernandez-Baca, S., 1972. Fisiologıa ´ reproductiva post-partum en la alpaca Žin spanish.. Revista de Investigaciones Pecuarias 1, 21–27. Sumar, J., Fredriksson, G., Alarcon, V., Kindahl, H., Edqvist, L.-E., 1988. Levels of 15-keto-13,14-dihydroPGF2 a progesterone and oestradiol-17b after induced ovulations in Llamas and Alpacas. Acta Vet. Scand. 29, 339–346. Thatcher, W.W., Wilcox, C.J., Collier, R.J., Eley, D.S., Head, H.H., 1980. Bovine conceptus maternal interactions during the pre and postpartum periods. J. Dairy Sci. 63, 1530–1540. Thorburn, G.D., Challis, J.R.C., Currie, W.B., 1977. Control of parturition in domestic animals. Biol. Reprod. 16, 18–27. Tsang, C.P.W., 1978. Plasma levels of estrone sulfate, free estrogens and progesterone in the pregnant ewe throughout gestation. Theriogenology 10, 97–110. Wright, K., Collins, D.C., Musey, P.I., Preedy, J.R.K., 1978. A specific radioimmunoassay for estrone sulphate in plasma and urine without hydrolysis. J. Clin. Endocrinol. Metab. 47, 1092–1098.
Plasma concentrations of 15-ketodihydro-PGF2 a , progesterone, oestrone sulphate, oestradiol-17b and cortisol during late gestation, parturition and the early post partum period in llamas and alpacas M.A. Aba
a,d,)
, J. Sumar b, H. Kindahl L.-E. Edqvist a
c,d
, M. Forsberg
a,d
,
a
Department of Clinical Chemistry, Faculty of Veterinary Medicine, Swedish UniÕersity of Agricultural Sciences, P.O. Box 7038, SE-750 07, Uppsala, Sweden b Instituto Veterinario de InÕestigaciones Tropicales de Altura, UniÕersidad Mayor de San Marcos, Lima, Peru c Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, Swedish UniÕersity of Agricultural Sciences, P.O. Box 7039, SE-750 07, Uppsala, Sweden d Centre for ReproductiÕe Biology, Swedish UniÕersity of Agricultural Sciences, Uppsala, Sweden Accepted 23 September 1997
Abstract Plasma concentrations of 15-ketodihydroprostaglandin ŽPG. F2 a , progesterone, oestrone sulphate, oestradiol-17b and cortisol during late gestation, parturition and the early post-partum period were measured in six llamas and five alpacas. During the last 100 days of pregnancy, 15-ketodihydro-PGF2 a concentrations increased steadily until the day of parturition when a massive release was detected Ž P - 0.01. concomitant with a decrease in progesterone concentrations Ž P - 0.01.. The highest PGF2 a metabolite concentrations Ž159 " 35 nmol ly1 and 92 " 29 nmol ly1 in llamas and alpacas respectively. were detected in the sample collected during the morning on the day of parturition. Basal concentrations were registered by day 3 after delivery. Plasma concentrations of oestrone sulphate started to increase 80 days before parturition and reached peak concentrations immediately before parturition Ž15 " 3 nmol ly1 in llamas and 18 " 5 nmol ly1 in alpacas.. Oestrone sulphate concentrations dropped sharply Ž P - 0.01. on the day of parturition in llamas and one day later in alpacas, whereupon they remained relatively unchanged until at least 20 days postpartum. Oestradiol-17b concentrations were higher than 180 pmol ly1 during the last 45 days of pregnancy, began to decrease on the day of parturition and
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Corresponding author.
0378-4320r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 7 8 - 4 3 2 0 Ž 9 7 . 0 0 0 8 4 - 5
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reached very low concentrations within the following two days. High oestradiol-17b concentrations were registered 7 days postpartum in all alpacas Ž P - 0.05. and within 10 days of parturition in five of six llamas Ž P - 0.01.. No significant cortisol peaks were observed around parturition, but mean concentrations were increased in both species. q 1998 Elsevier Science B.V. Keywords: Llama; Alpaca; Pregnancy; Parturition; Postpartum period; Prostaglandin; Oestradiol; Progesterone
1. Introduction Endocrine changes after copulation, luteolysis and early pregnancy in llamas Ž Lama glama. and alpacas Ž Lama pacos . have been well described elsewhere ŽSumar et al., 1988; Bravo et al., 1990; Bravo et al., 1991b; Aba et al., 1995; Bravo et al., 1996.. Conversely, information about hormonal changes associated with late gestation, parturition and the early post-partum period is poorly defined. It has been shown that the corpus luteum is necessary for the maintenance of pregnancy throughout the gestation period in both species ŽSumar, 1988.. In llamas, plasma progesterone concentrations remain elevated throughout pregnancy, begin to decline between 15 days and 1 day prepartum and finally drop to low levels from day 1 prepartum until parturition ŽLeon et al., 1990; Bravo et al., 1996.. Oestrogens Žcombined oestrone, oestradiol-17a and oestradiol-17b . start to increase 9 months after mating and remain high until the end of pregnancy, with peak concentrations during the last week prepartum ŽLeon et al., 1990.. Only a slight increase in plasma cortisol concentrations has been observed in association with parturition in llamas ŽLeon et al., 1990.. Plasma concentrations of oestrogen and progesterone have been reported to be low following parturition and development of follicles beginning on 5–6 days after parturition ŽSumar et al., 1972; Leon et al., 1990; Bravo et al., 1991a,b.. Prostaglandin ŽPG. F2 a plays a major role during parturition in most domestic species ŽEdqvist et al., 1978; Barnes et al., 1978; Mitchell et al., 1979; Kindahl et al., 1982a.. To our knowledge there are no reports describing the plasma concentrations of PGF2 a metabolite and oestrone sulphate during this period in llamas or alpacas. The aim of this study was to document the secretory profiles of PGF2 a metabolite, progesterone, oestrone sulphate, oestradiol17b and cortisol during late gestation, parturition and the early postpartum period in llamas and alpacas.
2. Materials and methods 2.1. Animals and blood samples In total, six llamas and five alpacas were mated once, with a fertile male, for an average period of 20 min. The day of mating was registered in order to calculate the gestation length. Vasectomized males were used for sexual receptivity detection in both species on days 12, 24, 40, 60 and 90 after mating. The animals were kept on natural pastures at the La Raya Research Station, San Marcos University, Cuzco, Peru, ´ located
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at 158S, 708W and 4250 m altitude. Blood samples were obtained every second week from day 7 until the end of the 8th month and at 7-day intervals between 9 and 10.5 months of pregnancy. Thereafter, samples were collected twice a day Žmorning and afternoon. until parturition. Starting on the day of parturition, we collected four samples per day during the first 10 days and two a day during the following 10 days except for one llama in which sampling was discontinued after day 12 post partum. Blood was collected by jugular venipuncture into heparinized vacutainer tubes except for the 20 days post partum period, when the collections were made using permanent catheters placed in the jugular vein. In some cases where the catheters were obstructed, blood sampling was continued by venipuncture. Plasma was separated by centrifugation and stored at y208C until analyzed. 2.2. Hormone assays Progesterone was determined by an enhanced luminescence immunoassay technique ŽAmerlite; Kodak Clinical Diagnostics, UK. previously validated for llama and alpaca plasma ŽAba et al., 1995.. The intra-assay coefficient of variation calculated from the precision profiles was below 7% for concentrations between 2 and 160 nmol ly1 . The inter-assay coefficients of variation for three quality-control samples were 8% Ž2 nmol ly1 ., 8% Ž18 nmol ly1 . and 5% Ž54 nmol ly1 .. The lowest amount of progesterone detectable Ždefined as the intercept of maximal binding—2 SD. was 0.2 nmol ly1 . The plasma metabolite of PGF2 a , 15-ketodihydro-PGF2 a , was analyzed by RIA according to Kindahl et al. Ž1976. and Granstrom ¨ and Kindahl Ž1982.. The intra-assay coefficients of variation for two control samples were 8.5% Ž240 pmol ly1 . and 4.2% Ž485 pmol ly1 .. The corresponding inter-assay coefficients of variation in three assays were 15.5% and 8.4%, respectively. The practical detection limit of the assay was 30 pmol ly1 . Oestradiol-17b was determined using an RIA previously validated for use in bovine plasma ŽSirois and Fortune, 1990., with the following modification: the standard curve was prepared with standards supplied with the radioimmunoassay kit ŽDiagnostic Products, Los Angeles, CA, USA.. Serially diluted plasma from female llamas and alpacas containing high concentrations of oestradiol-17b produced displacement curves parallel to the standard curve. The intra-assay coefficients of variation calculated from the precision profile of seven assays were 13.4% at 6 pmol ly1 , 10.2% at 19 pmol ly1 and below 10% up to 180 pmol ly1 . The inter-assay coefficients of variation for three control samples were 25% Ž13 pmol ly1 ., 6% Ž39 pmol ly1 . and 10% Ž84 pmol ly1 .. The lowest amount of oestradiol-17b detectable Ždefined as the intercept of maximal binding—2 SD. was 3 pmol ly1 . Oestrone sulphate concentrations were determined by radioimmunoassay ŽWright et al., 1978; Kindahl et al., 1982b. using antiserum against oestrone-glucosiduronate-bovine thyroglobulin raised in rabbits. Serially diluted plasma from female llamas and alpacas containing high concentrations of oestrone sulphate produced displacement curves parallel to the standard curve. The sensitivity of the assay was 1 nmol ly1 . The intra-assay coefficient of variation for oestrone sulphate concentrations in the range of 2.0 to 100 nmol ly1 was below 15%.
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Cortisol was determined using a luminescence immunoassay according to a method previously described for pig plasma ŽMagnusson et al., 1994.. Serial dilutions of llama and alpaca plasma with high concentrations of cortisol produced displacement curves parallel to the standard curve. The sensitivity of the assay was 7 nmol ly1 , and the intra-assay coefficient of variation, calculated based on six assays, was below 15% for concentrations of cortisol between 9.1 and 435 nmol ly1 . The corresponding inter-assay coefficients of variation for two quality control samples were 27% Ž15 " 4 nmol ly1 . and 12% Ž147 " 18 nmol ly1 .. Hormone concentrations are expressed in SI units. To convert from pmol ly1 to pg y1 ml and from nmol ly1 to ng mly1 the following factors should be used: PGF2 a metabolite: 2.8; oestradiol-17b : 3.7; oestrone sulfate: 2.7; cortisol: 2.7 and progesterone: 3.2. 2.3. Analysis of data A completely randomized analysis of variance was performed to detect differences in hormone concentrations. Due to the variability in the length of pregnancy, samples obtained in different animals during the last 100 days of pregnancy, parturition and early post partum were normalized by day of parturition. Cortisol concentrations in samples obtained weekly between days y38 and y10 were pooled and therefore compared with concentrations measured in daily samples from day y9 to day q5 Žday 0 s day of parturition.. A least significant difference test ŽLSD. was used to determine significant differences between means. Results are expressed as mean " S.E.M.
3. Results The mean gestation length was 351.5 " 1.9 days Žrange 343–353. and 335.6 " 2.8 days Žrange 327–343. in llamas and alpacas respectively. All animals had normal unassisted deliveries, and none of the animals showed any signs of uterine disorders in the postpartum period studied. Mean progesterone concentrations remained relatively constant around 20 nmol ly1 Žllamas. or slightly increased from 11 to 15 nmol ly1 Žalpacas. over the last three months of pregnancy. During the last 7–10 days of pregnancy, progesterone concentrations dropped drastically Ž P - 0.01. and remained low until at least 20 days post partum ŽFig. 1a and b.. During the last 100 days of pregnancy, plasma concentrations of 15-ketodihydroPGF2 a increased steadily until the day of parturition when a massive release was detected in both species ŽFig. 1a and b. in connection with the declining concentrations of progesterone. In llamas, mean PGF2 a metabolite concentrations during the parturition day were significantly higher Ž P - 0.05. in the morning sample Ž159 " 35 nmol ly1 . than in the sample collected in the afternoon of the same day Ž21 " 6 nmol ly1 .. Although differences were not statistically significant, the same tendency was observed in alpacas Ž92 " 29 and 45 " 24 nmol ly1 respectively.. Thereafter, plasma PGF2 a metabolite concentrations sharply declined Ž P - 0.01., reaching around 150 pmol ly1 by
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Fig. 1. Plasma concentrations of 15-ketodihydro-PGF2 a Žmean, logarithmic scale. and progesterone wmean" S.E.M. Žshaded area.x during the last 100 days of pregnancy in llamas Ža. and alpacas Žb.. Day 0 sday of parturition.
day 4 and remaining relatively constant thereafter until at least day 20 post partum ŽFig. 2a and b.. No significant peaks were detected during this period. In both species, plasma oestrone sulphate concentrations rose steadily towards the end of gestation, with the highest values recorded during the last 30 days of pregnancy Ž15 " 3 nmol ly1 and 18 " 5 nmol ly1 in llamas and alpacas respectively.. In llamas, oestrone sulphate plasma concentrations declined on the day of parturition. In alpacas the decline was registered one day post partum. Thereafter, concentrations remained relatively unchanged until at least 20 days post partum ŽFig. 3a and b..
Fig. 2. Mean plasma concentrations of 15-ketodihydro-PGF2 a and progesterone w"S.E.M. Žshaded area.x from the day of parturition Žday 0. to day 20 post partum.
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Fig. 3. Plasma oestrone sulphate concentrations Žmean"S.E.M.. during the last 100 days of pregnancy and 20 days post partum in llamas Ža. and alpacas Žb..
Plasma oestradiol-17b concentrations exceeded 180 pmol ly1 during the last 45 days of pregnancy. The first significant decrease Ž P - 0.05. in oestradiol concentrations was observed during the day of parturition, whereupon they continued to decline, reaching concentrations just above the detection limit of the assay Žaround 8 pmol ly1 . within the following two days. A significant rise in oestradiol-17b concentrations was registered within 7 days post partum in all alpacas Ž P - 0.05. and within 10 days post partum in five of six llamas Ž P - 0.01.. The llama with no oestradiol increase was the one for which sampling had been discontinued after day 12. In Fig. 4, the oestradiol-17b secretory profile after parturition in a representative llama Ža. and alpaca Žb. are shown. In llamas, slight, but non-significant, increases in mean cortisol concentrations were observed during the last week of pregnancy and the first three days post partum. No
Fig. 4. Oestradiol-17b secretory profile after parturition in a representative llama Ža. and alpaca Žb..
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Fig. 5. Cortisol concentrations Žmean"S.E.M.. during the last 45 days of pregnancy and the first 7 days post partum in llamas Ža. and alpacas Žb..
significant peaks were detected in connection with parturition ŽFig. 5a.. In alpacas, cortisol concentrations were elevated Ž P - 0.05. during the day of parturition and the day before, as compared with concentrations recorded during late pregnancy Žday y38 to day y10. and the first five days postpartum ŽFig. 5b.. 4. Discussion The mean gestation length in llamas registered in this study is in agreement with previous reports ŽJohnson, 1989; Leon et al., 1990., while the gestation period observed in alpacas is slightly shorter than values reported by others ŽSan-Martın ´ et al., 1968; Knight et al., 1995.. The progesterone secretory pattern during late gestation, parturition and early postpartum observed in this study is in agreement with patterns previously reported in South American camelids ŽAdam et al., 1989; Leon et al., 1990; Adams et al., 1991; Bravo et al., 1991b; Bravo et al., 1996. and in Old World camels ŽAgarwall et al., 1987; Musa et al., 1993.. To our knowledge, this is the first report describing the PGF2 a metabolite secretory profile during gestation, parturition and early postpartum in domesticated South American camelids. The pattern of prostaglandin metabolite secretion registered during the last 3 months of pregnancy does not differ from the patterns reported in several other mammals Žcow: Edqvist et al., 1978; mare: Allen and Pashen, 1981; goat: Fredriksson et al., 1984 and ewe: Fredriksson, 1985.. In accordance with findings in llamas reported by Leon et al. Ž1990., no decline in plasma progesterone concentrations was observed during the period of high PGF2 a metabolite concentrations. The sharp increase and peak concentrations registered the day before parturition and on the day of parturition, respectively, and the drop in progesterone concentrations correlate well with the pattern shown by the same authors. It is widely accepted that in llamas and alpacas almost 100% of the parturitions occur during the early hours of the day. No births occur between 1700 and 0400, when temperatures are low throughout the year in the natural habitat ŽSumar, 1983.. The
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finding that peak concentrations of PGF2 a metabolite were registered during the morning of the day of parturition in both species suggests that in llamas and alpacas, like in the mare, the second stage of labour is accompanied by a massive rise in PGF2 a metabolite concentrations as the newborn passes through the cervix and vagina. Our previous report of an intense release of PGF2 a in connection with mating in llamas and alpacas ŽAba et al., 1995. further supports the hypothesis that mechanical stimulation of the cervix andror vagina may induce prostaglandin release in these species. The increased oestrone sulphate concentrations observed during late gestation correlate with earlier studies in cows ŽThatcher et al., 1980; Abdo et al., 1991. and ewes ŽTsang, 1978; Mohamed et al., 1987. showing that maximum oestrone sulphate concentrations are observed during the last third of the gestation period when the fetus rapidly increases in weight and size. However, Bravo et al. Ž1996. have reported increasing plasma concentrations of oestrone sulfate later in gestation Žaround month 11.. Although there is no clear explanation for the different patterns registered between studies, it could be speculated that different cross reactivity of the antibodies with other steroids and steroid conjugates from the foeto-placental unit, may be responsible. Thus, from our results, it may be concluded that in llamas and alpacas oestrone sulphate analysis during the last 90 days prepartum can be used for monitoring aimed at making sure that pregnancy is progressing normally. The secretory pattern of oestradiol-17b recorded during the last two weeks of pregnancy, parturition and first week postpartum in both species is in agreement with a previous study in llamas ŽLeon et al., 1990.. Similar oestradiol-17b secretory pattern during late gestation has also been reported in the dromedary camel ŽAgarwall et al., 1987.. The finding that oestradiol-17b concentrations started to rise Žindicating that follicular development was initiated. within the first 10 days postpartum in five of six llamas and within 7 days postpartum in all five alpacas correlates well with the observation that South American camelids usually become pregnant within the first 2–3 weeks postpartum. Bravo et al. Ž1991b. reported that urinary oestrone sulphate concentrations accurately reflect ovarian follicle development in the postpartum period in llamas. The disclosure that in the present study, follicular activity was not reflected as elevated plasma oestrone sulphate concentrations may be attributable to the low sensitivity of the assay. Like in the mare ŽAllen and Pashen, 1981; Stewart et al., 1984. and sow ŽKindahl et al., 1982a., prostaglandin concentrations returned to basal concentrations 1–2 days postpartum in llamas and alpacas. Placentation in these species has been classified as diffuse and epitheliochorial. Conversely, the prolonged release of PGF2 a for several weeks postpartum has been reported in the cow ŽEdqvist et al., 1978; Kindahl et al., 1992., water buffalo ŽPerera et al., 1981., goat ŽFredriksson et al., 1984. and ewe ŽFredriksson, 1985.. All these ruminants have a similar type of cotyledonary placenta, and there might be some relationship between prostaglandin release pattern and type of placentation. The physiological significance of this difference in postpartum prostaglandin release between species is not fully understood, but as long as the release is prominent ovulations do not occur. In most mammalian species, the fetus dominates the mechanism stimulating the onset of parturition. A significant increase in the fetal plasma concentration of cortisol occurs
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during the final stages of gestation in sheep, goat, cattle and pigs ŽThorburn et al., 1977.. Although some increase in adrenal activity probably occurs in the equine fetus near term, no spectacular rise in fetal plasma cortisol concentration occurs before birth ŽNathanielsz et al., 1975.. In the present study, only slightly elevated cortisol concentrations were recorded around the day of parturition in the llamas and alpacas. Similarities between prepartum and postpartum stable cortisol concentrations in the maternal circulatory system have been reported in the rhesus monkey ŽScott et al., 1984. and in the llama, although a low-frequency sampling schedule was used in the latter study ŽLeon et al., 1990.. The secretory pattern reported in this study resembles that observed in the mare but any role of cortisol in the induction of parturition in llamas and alpacas remains to be determined. In brief, plasma concentrations of PGF2 a metabolite, oestradiol-17b and oestrone sulphate, progressively increase during the last third of pregnancy in llamas and alpacas, with a sudden rise registered at parturition, in connection with a drop in progesterone concentrations and a slight increase in cortisol concentrations. Thereafter, plasma concentrations of PGF2 a metabolite, progesterone and oestrone sulphate remains low until at least day 20 postpartum. Meanwhile, oestradiol-17b plasma concentration starts to increase during this period, indicating follicular activity as early as day 7 and day 10 postpartum in alpacas and llamas respectively. Acknowledgements The Swedish Agency for Research in Developing Countries ŽSAREC., the Swedish International Development Agency ŽSIDA., the Swedish Council for Forestry and Agricultural Research and the Department of Physiopathology, Faculty of Veterinary Sciences, UNCPBA, Tandil, Argentina, are acknowledged for financial support. References Aba, M.A., Forsberg, M., Kindahl, H., Sumar, J., Edqvist, L.-E., 1995. Endocrine changes after mating in pregnant and non-pregnant llamas and alpacas. Acta Vet. Scand. 36, 489–498. Abdo, G.A., Njuguna, O.M., Fredriksson, G., Madej, A., 1991. Levels of oestrone sulphate during pregnancy in different breeds of cows and its possible association with retained foetal membranes. Acta Vet. Scand. 32, 183–188. Adam, C.L., Moir, C.E., Shiach, P., 1989. Plasma progesterone concentrations in pregnant and non-pregnant llamas Ž Lama glama.. Vet. Rec. 125, 618–620. Adams, G.P., Sumar, J., Ginther, O.J., 1991. Form and function of the corpus luteum in llamas. Anim. Reprod. Sci. 24, 127–138. Agarwall, S.P., Khanna, N.D., Agarwall, V.K., Dwaraknath, P.K., 1987. Circulating levels of estrogen and progesterone in female camel Ž Camelus dromedarius. during pregnancy. Theriogenology 28, 849–859. Allen, W.R., Pashen, R.L., 1981. The role of prostaglandins during parturition in the mare. Acta Vet. Scand. ŽSuppl.. 77, 279–298. Barnes, R.J., Comline, R.S., Jeffcott, L.B., Mitchell, M.D., Rossdale, P.D., Silver, M., 1978. Foetal and maternal plasma concentrations of 13,14-dihydro-15-oxo-prostaglandin F2 a in the mare during late pregnancy and at parturition. J. Endocrinol. 78, 201–215. Bravo, P.W., Fowler, M.E., Stabenfeldt, G.H., Lasley, B.L., 1990. Endocrine responses in the llama to copulation. Theriogenology 33, 891–899.
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