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Endocrinology of pregnancy and early pregnancy detection by reproductive hormones in reindeer (Rangifer tarandus tarandus)
Theriogenology 63 (2005) 1775–1788 www.journals.elsevierhealth.com/periodicals/the
Endocrinology of pregnancy and early pregnancy detection by reproductive hormones in reindeer (Rangifer tarandus tarandus) E. Ropstada,*, V. Veibergb, H. Sa¨kkinenc, E. Dahla, H. Kindahld, Ø. Holande, J.F. Beckersf, E. Elorantag a
Department of Production Animal Clinical Sciences, Norwegian School of Veterinary Science, P.O. Box 8146 Dep., NO-0033 Oslo, Norway b Department of Biology, The University Centre in Svalbard, P.O. Box 156, NO-9171 Longyearbyen, Norway c Department of Biology, University of Oulu, P.O. Box 3000, FI-90014 Oulu, Finland d Department of Obstetrics and Gynaecology, Centre for Reproductive Biology in Uppsala, Swedish University of Agricultural Sciences, SE-75007 Uppsala, Sweden e Department of Animal Science, Agricultural University of Norway, Box 5025, NO-1432 A˚s, Norway f Department of Physiology of Reproduction, Faculty of Veterinary Medicine, University of Lie`ge, Bd de Colonster No. 20, B41, BE-4000 Sart-Tilman, Belgium g Department of Physiology, University of Oulu, Kajaanintie 52 A, FI-90570 Oulu, Finland Received 24 May 2004; received in revised form 3 August 2004; accepted 25 August 2004
Abstract The endocrinology was studied throughout pregnancy in reindeer (Rangifer tarandus tarandus) located in Oulu, Finland (658N, 258E) with 13 captive, semi domestic adult females. Blood samples were analyzed for plasma progesterone (P4), estradiol (E2) and estrone sulphate (E1SO4), 15ketodihydro-PGF2a (PG-metabolite) and pregnancy associated glycoproteins (PAG). The mean plasma P4 concentration peaked twice during gestation: at around 24 and three weeks prior to calving. In pregnant females the plasma PAG concentration increased over basal concentrations 21–30 days after the estimated day of conception and peaked at the time of calving. The concentrations of E2 and E1SO4 remained low until 60 days before calving when a rapid increase was found for both hormones. The mean plasma concentration of PG-metabolite increased throughout pregnancy to a maximum at parturition. The estimated mean (range) gestation length was 216 (212– 220) days. * Corresponding author. Tel.: +47 22 96 45 00; fax: +47 22 59 70 81. E-mail address: [email protected] (E. Ropstad). 0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2004.08.003
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Judged from measures on reproductive organs collected from 86 free-ranging, semi-domestic female reindeer of unknown age presented for slaughter at Røros, Norway (638N, 118E) in the second week of December 1999, it was concluded that the breeding season lasted from early September until the end of November. The results also showed that plasma PAG concentration could provide a tool for detection of pregnancy in reindeer. # 2004 Elsevier Inc. All rights reserved. Keywords: Estradiol; Pregnancy associated glycoproteins; 15-Ketodihydro-PGF2a; Estrone sulphate; Progesterone; Pregnancy diagnosis
1. Introduction Reindeer, like most temperate cervids, are seasonal breeders, with mating coinciding with the decreasing photoperiod in the autumn, and with fawning in the spring. The fact that reindeer normally are not subject to intensive farming conditions and generally have a high fecundity explains why little interest has been shown in their reproductive physiology. Semi-domestic reindeer are kept mainly for meat production. One of the most important factors for productivity is the proportion of fertile females that have a calf at foot in the autumn. In the future, it is likely that semi-domestic reindeer will be managed and herded more intensively, which implies that there will be an increased need for knowledge on reproduction in this species. Thus there is a need to expand our knowledge of normal reproductive physiology during the breeding season and pregnancy. Also there may be benefits from development of methods to detect pregnancy and to control reproduction. The reproductive endocrinology of pregnancy in reindeer is poorly investigated. Only a few animals have been blood sampled repeatedly throughout pregnancy, and detailed individual hormone profiles for hormones other than progesterone (P4) are not available [1]. P4 secretion is associated with pregnancy in all mammals as this hormone is produced by the corpus luteum and, in some species, by the placenta as well [2]. There are large differences between ruminant species both to the time course of changes in the P4 concentration [3], and to the range of which P4 varies during pregnancy [4]. The fact that females of equivalent species and reproductive status show considerable variation in P4 plasma concentration has also been documented in reindeer [3,5,6]. The general pattern of P4 variation during pregnancy in reindeer is an increase from conception until around 1–3 months of pregnancy. After this the concentration stabilizes or a slight decrease is seen. Prior to parturition a further increase takes place [1] followed by a rapid drop just around the time of calving [3]. Maternal plasma levels of estrogens, either as free steroids or their conjugates are shown to increase during parts of gestation in most mammals [2,3,7–9]. Increased concentration of estrone and especially of E1SO4 in maternal blood has been associated with the presence of a conceptus in pigs [8], sheep [10], cattle [11] and red deer (Cervus elaphus) [2], and has been used as a method for pregnancy detection in these species. The level of E1SO4 reflects primarily the condition of the placenta and its continued production represents a gross index of foetal-placental wellbeing [12]. Lowered levels of E1SO4 indicate that the endocrine foetal-placental function is disturbed [13].
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In ewes [14], a rise in estradiol (E2) precedes parturition by only a couple of days, whereas in goats [15] and cows [7,8] there is a more gradual rise during the last part of gestation. Similar results are also reported in white-tailed deer [16,17], red deer [9] and reindeer [3]. At parturition there is a drastic decline in E2 in all the above-mentioned ruminants. Pregnancy-specific glycoproteins (PSPB) [18], and pregnancy-associated glycoproteins (PAG) [19] are products of the feto-placental unit and therefore specific for pregnancy [20]. Both PSPB and PAG are synthesized in the outer layers of the trophoblast and secreted in the maternal circulation [20,21]. Molecular cloning studies have shown that their primary structure is closely related [22]. Such conceptus-derived proteins are suspected to signal to the maternal system that pregnancy has occurred and to prevent luteal regression [23–25]. Both substances normally appear in the maternal circulation from about 30 days after conception, and increase to maximum levels around parturition [21,26]. Assays for measuring PSPB (cattle; 20, sheep; 27, elk Cervus elaphus and moose Alces alces; 28) and PAG (cattle: 21; goat: 29) concentrations have only been developed for a few species. However, these assays have proved successful for pregnancy diagnosis in a number of other species although there have been difficulties with precise quantification of PSPB/PAG concentrations across species [30,31]. Prostaglandin (PGF2a) of uterine origin, measured as the PG-metabolite, has been suggested as the main luteolytic factor in female reindeer [5]. Normal pregnancy depends upon the early embryo signaling its presence to the maternal system. In many species in which secretion of PGF2a from the uterus is the primary signal for luteolysis, an obvious potential strategy for blocking luteolysis is the inhibition of this secretion in pregnant animals [32]. High levels of the PG-metabolite have been seen in many species during late pregnancy (e.g. sheep: 33; guinea pig: 34; goat: 35; horse: 36). The reason for a high PG production during pregnancy is not known but can most likely be related to placenta development. However, the production as such seems to be non-harmful for the luteal function. To our knowledge, measurement of PGF2a or its metabolite has not been reported for the gestational period in reindeer. Information about endocrine processes related to reproduction provides basic tools for studies of reproductive success and improvement of reproductive performance. In this context precise methods for early pregnancy diagnosis are essential. In domestic and wild ruminants common pregnancy tests include rectal palpation [20], ultrasonographic detection of pregnancy [20,37] and measurement of pregnancy related biochemical markers in blood (e.g. [9,17,38]), faeces [39,40] or milk [41], mainly including measurement of progesterone and E1SO4 [20]. In reindeer management there is a need for cost-efficient methods for early and accurate pregnancy diagnosis that would allow removal and slaughter of barren, unproductive females, so that reindeer herds can be managed more efficiently [6]. The first objective of the present study was to describe the endocrinology of pregnancy in reindeer by repeated measurement of reproductive hormone concentrations (P4, E2, E1SO4, PG-metabolite and PAG) from the time of conception until delivery in adult females kept in a controlled environment. Secondly, we wanted to assess the potential of measured blood constituents for early pregnancy diagnosis. This objective was achieved by studying experimental animals as well as animals from the field slaughtered after the rut.
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2. Materials and methods 2.1. Animals and sampling 2.1.1. Captive animals The pregnancy of thirteen pluriparus female reindeer (Rangifer tarandus tarandus) were corralled outdoors at the Zoological Gardens of Oulu University, Finland (658N, 258E), and fed commercial pelleted reindeer feed (Poron Herkku, Raisio Group, Finland), 1.8–2.0 kg/animal and day, and with about 400 g lichens (Cladonia spp.) every day in addition to the pelleted feed. A reindeer stag was brought together with the females on 1st October 1997 and was allowed to mate freely with them. Pregnancies were also verified by transrectal ultrasonography. The scanner was equipped with a 5 MHz linear probe (Scanner 100, Pie Medical, Netherlands) at 4–6 weeks in gestation [42]. Based on previous detailed studies of P4 profiles during the estrous cycle [5], the date of conception was set at 2 days before plasma progesterone showed persistent levels above 1 nmol/L followed by increased concentration of PAG (2 ng/mL). Jugular venous blood samples were collected with evacuated heparinized tubes (Venoject1, Leuven, Belgium) from September 1997 until May 1998. Blood samples were collected by hand three times a week without using sedatives or medication. Plasma was separated by centrifugation at 3000 g within 2 h and stored at 20 8C until analysis. Samples were analyzed for P4, E2, E1SO4, PG-metabolite and PAG. The study was performed with permission from the Committee on Animal Experiments of Oulu University. 2.1.2. Culled animals In the second part of the study, 86 free ranging semi-domestic female reindeer of varying, but unknown age were slaughtered in the second week of December 1999 at Røros, Norway (638N, 118E). Heparinized blood samples were collected when the animals were bled immediately after slaughter. Plasma was separated and stored as described previously, until analyzed for P4 and PAG. Dressed weight was recorded and reproductive organs were collected and stored at 20 8C until examined. Pregnancy was verified after visual detection of a fetus in the uterine lumen. Uteri and ovaries from all slaughtered females were weighed. Fetuses were weighed and the crown-rump length (CRL) measured. 2.2. Hormone analyses 2.2.1. Progesterone (P4) Plasma levels of total progesterone were determined by a solid-phase radioimmunoassay kit (Spectria1 Progesterone 125I Coated Tube RIA, Orion Diagnostica, Espoo, Finland). The assay was validated for use with reindeer plasma by demonstrating parallelism between dilutions of reindeer plasma samples and the standard curve, and by recovery of unlabeled ligand. No modifications of the standard procedures were needed. The detection
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limit of the assay was 0.3 nmol/L. Intra-assay coefficient of variation was less than 5%. Inter-assay coefficients of variation were 8.8% (1.25 nmol/L), 7.6% (19.1 nmol/L) and 5.9% (44.1 nmol/L) (N = 18), respectively. 2.2.2. Pregnancy-associated glycoproteins (PAG) A heterologous double antibody radioimmunoassay derived from the method previously described by Zoli et al. [21] was used to determine PAG concentrations in plasma. Bovine PAG (bPAG) was used as the standard and tracer, anti-caprine PAG55,59 as the first antibody (dilution 1:500,000 [29] and sheep anti-rabbit IgG as the second antibody for precipitation. The standard curve ranged from 0.2 to 25 ng bPAG equivalents/mL. Bovine zero-plasma was added to all standard tubes. Samples and standards were incubated with antibody overnight at room temperature. The next day, labelled bPAG (25,000 cpm) was added, and the mixture was further incubated for 4 h. The free and bound fractions were separated by centrifugation at 3400 g for 15 min after addition of sheep anti-rabbit IgG. The precipitates were counted in a 1470 Wizard Gamma Counter (Wallac, Turku, Finland). The interassay coefficients of variation were 11.6% (2.6 ng bPAG equivalents/mL) and 5.0% (10.3 ng bPAG equivalents/mL), respectively. Assay sensitivity was 0.8 ng bPAG equivalents/mL. 2.2.3. Estradiol-17b (E2) Estradiol concentrations were determined by a 125I-estradiol-17b double antibody radioimmunoassay kit, ‘‘Coat-a-count1, Estradiol’’ (Diagnostic Products Corporation, Los Angeles, USA). To construct the standard curve, estradiol standards from the kit were diluted in human zero plasma also provided by the kit (5.7–180 pmol/L). Briefly 250 ml aliquots of plasma or standards were extracted in duplicate before assay with 2.5 mL diethyl ether and evaporated under dry air in glass tubes. The extracted plasma was reconstituted in 200 ml phosphate buffer (pH 7.0) and stored overnight at 4 8C. Antiserum (30 ml) was added to each tube, which was vortexed and incubated for 2 h in room temperature before addition of 75 ml of 125I-estradiol-17b with further 1 h incubation in room temperature. Separation of free from antibody bound hormone was performed by addition of 1 mL precipitating solution from the kit. The contents of the tubes were centrifuged for 15 min (3000 g). After decantation, radioactivity of the tubes were counted in a gamma counter for 1 min. Serial dilutions of reindeer plasma with high concentrations of estradiol-17b produced inhibition curves parallel to the standard curve. The intraassay coefficients of variation calculated from nine precision profiles were 21% at 5.6 pmol/L, 13% at 11.3 pmol/L and below 10% for concentrations exceeding 22.5 pmol/L. The corresponding interassay coefficients of variation were 11.4% (13 pmol/L), 6.6% (42 pmol/L) and 4% (86 pmol/L), respectively. The detection limit was 2.1 pmol/L. 2.2.4. Estrone sulphate (E1SO4) The plasma was analyzed for E1SO4 by a commercial radioimmunoassay kit ‘‘Estronesulfate DSL-54001’’ (Diagnostic Systems Laboratories, Inc. Webster, Texas, USA) modified for use with reindeer plasma. Modification was done as follows: The standard curve was replaced by E1SO4 diluted in pooled plasma from male reindeer calves of
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approximately 30 kg live weight (zero-plasma). Dilutions of plasma samples with varying concentrations of E1SO4 produced inhibition curves parallel with the standard curve. Inter-assay coefficients of variation in samples containing 3.3, 7.7 and 22.8 ng/mL were 10.9, 8.9 and 3.8%, respectively. Minimum detection limit in the assay was 0.01 ng/mL. 2.2.5. 15-Ketodihydroprostaglandin F2a (PG-metabolite) Plasma samples were analyzed for the prostaglandin F2a metabolite, 15-ketodihydroPGF2a [43,44]. The relative cross-reactions of the anti-15-ketodihydro-PGF2a antibody were 16% with 15-keto-PGF2a, 4% with 13,14-dihydro PGF2a, 0.5% with PGF2a and 1.7% with the corresponding metabolite of prostaglandin E2. Serial dilutions of reindeer plasma with high concentrations of 15-ketodihydro-PGF2a produced inhibition curves parallel to the standard curve. The lower limit of detection was 75 pmol/L for a 200 ml plasma sample. The intra-assay coefficient of variation was 14% (114 pmol/L). In samples with concentrations exceeding the upper range of the standard curve (1.2 nmol/L), the plasma was diluted and reassayed to get exact concentrations. 2.3. Data analysis Since the exact dates of conception were not known in the captive animals, the mean (S.D.) plasma concentrations of reproductive hormones during pregnancy were expressed relative to the time of calving. Differences between individual mean concentrations of reproductive hormones during specific time periods of pregnancy were compared by student’s t-test. In addition, correlation analysis (Spearman’s correlation coefficient, rs) and linear regression analysis was used to study the relationship between selected variables. Based on previous detailed studies of P4 profiles during the estrous cycle [5], the date of conception was set at 2 days before plasma progesterone showed persistent levels above 1 nmol/L followed by increased concentration of PAG. Plasma concentrations of P4, E2, E1SO4, PG-metabolite and PAG were related to days relative to calving.
3. Results 3.1. Captive animals The females calved between 14th and 29th of May. Median calving date was 18th of May, and mean (S.D.) estimated gestation length was 216 (3.2) days (Table 1). The median estimated day of conception was 13 October. A negative relationship was found between gestation length and estimated Julian day of conception (gestation length = 0.3398 (estimated Julian day of conception) + 221.1; R2 = 0.70, P < 0.01). The onset of cyclic ovarian activity was preceded by short but regular periods of increased concentrations of plasma P4 (Fig. 1). Four of the 13 females cycled twice before successful conception. After conception, the mean P4 concentration peaked twice during pregnancy. Even though plasma concentrations fluctuated widely both within and between
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Table 1 Individual and mean pregnancy data in 13 captive reindeer kept at Oulu University, Finland (658N, 258E) 1997– 1998 ID
6 7 14 15 27 28 51 52 59 95 100 1586 2055 Mean S.D.
Estimated date of conception
8 Oct. 13 Oct. 27 Oct. 15 Oct. 13 Oct. 11 Oct. 11 Oct. 29 Oct. 11 Oct. 8 Oct. 20 Oct. 6 Oct. 27 Oct.
Days before calving
P4 PAG differencec
Days after calving PAG < 5 (ng/mL)
Gestation length (days)d
P4 1a (nmol/L)
PAG 2b (ng/mL)
217 216 208 215 217 215 214 210 218 213 211 216 210
196 190 189 191 191 188 191 184 195 185 183 193 189
21 26 19 24 26 27 23 26 23 28 28 23 21
19 17 15 15 29 13 19 19 10 – 18 21 21
219 218 210 217 219 217 216 212 220 215 213 218 212
213.9 3.2
189.6 4.0
24.2 2.9
18 4.8
215.9 3.2
Number of days elapsing between establishment of pregnancy and calving indicated by (a) increased progesterone (P4; cut off value: 1 nmol/L) and (b) pregnancy associated glycoprotein (PAG; cut off value: 2 ng/mL); (c) the time difference between (a) and (b) ((a) (b); days); and (d) the time from calving until PAG < 5 ng/mL and gestation length ((a) + 2 days).
animals, the general variation pattern was consistent in all animals. The first peak was found 24 weeks prior to calving (mean (S.D.) = 16.9 (3.7) nmol/L). Significantly lower P4 levels were found during mid-pregnancy than in early pregnancy (means (S.D.) were: 16.1 (0.9) nmol/L, 24–27 weeks before calving, versus 10.5 (0.9) nmol/L, 16–21 weeks before calving; P < 0.001]. The second peak occurred at three weeks before calving (mean (S.D.) = 23.0 (10.8) nmol/L). The mean P4 concentration thereafter decreased towards calving, after which it declined to baseline levels (Fig. 2a). PAG proved to be 100% accurate for pregnancy diagnosis when a discriminatory level of 2 ng PAG/mL plasma was used. PAG concentration exceeded 2 ng/mL between 183 and 196 days prior to calving, or alternatively 21 and 30 days after the estimated conception date (median date: 10th November). Thereafter, PAG increased gradually to a mean (S.D.) level of 14.6 (3.4) ng/mL at the time of parturition. After calving, PAG concentrations decreased to baseline level within 5 weeks (Fig. 2b). The concentrations of E2 and E1SO4 were low until the last 6 weeks of gestation. Concentrations of both steroids then increased rapidly and peaked one week prior to calving (means (S.D.) were for E2: 70.1 (25.5) pmol/L and for E1SO4: 3.1 (1.2) ng/mL), before decreasing to basal levels within 1 week after parturition (Fig. 2c and d). Mean plasma concentrations of the PG-metabolite were at the baseline level around 0.3 nmol/L during the rut and early pregnancy. From 170 days prior to calving the mean
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Fig. 1. Plasma concentration of progesterone (~) and PAG (&) for a selected female related to the time of calving. A points to the small rise in progesterone concentration at initiation of regular oestrous cyclicity. B is the progesterone maximum of a normal oestrous cycle not resulting in conception. C is the mid-pregnancy decrease in P4 concentration between week 16 and 21 pre-partum.
concentration increased gradually and reached 12 nmol/L the week before calving. Four females blood sampled on the day of calving showed PG-metabolite concentrations ranging between 47.7 and 83.5 nmol/L. About 3 weeks after parturition plasma concentrations of PG-metabolite had returned to baseline level (Fig. 2e). 3.2. Culled animals Forty-one of the 86 culled females were verified as pregnant. Dressed weights of the slaughtered animals varied from 25.8 to 42.6 kg (mean = 33.4, S.D. = 4.2, N = 41). The majority of the non-pregnant females were calves, and had not yet become fertile. Results from measurements of reproductive organs and analyses of blood samples from pregnant females are presented in Table 2. A measurable fetus was detected from all but one of the females verified as pregnant. In this female the fetus was too small to be measured. Fetal weights ranged from 1.5 to 53.3 g (Fig. 3), and correlated significantly with CRL (rs = 0.90, N = 40, P < 0.001; range 2.7–11.4 cm). PAG concentrations in the blood samples ranged from 0.2 to 9.2 ng/mL. None of the females verified as non-pregnant, had plasma PAG concentrations exceeding the cut-off value of 2 ng/mL. Among the pregnant animals, the only one with a PAG concentration below 2 ng/mL, was the one without a measurable fetus, verifying that this female was in an early stage of gestation. Except ovarian weight (rs = 0.57, N = 40, P < 0.001) none of the measured variables were significantly correlated to female bodyweight. However, a positive correlation was found between the maternal plasma PAG concentration and fetal weight (rs = 0.42, P = 0.008, N = 40; Fig. 4).
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Fig. 2. Mean (+S.D.) plasma concentrations of (a) progesterone (P4), (b) pregnancy associated glycoproteins (PAG), (c) estradiol (E2), (d) estrone sulphate (E1SO4) and (e)15-ketodihydro-PGF2a (PG-metabolite) relative to calving in 13 reindeer kept at Oulu University, Finland (658N, 258E) 1997–1998.
4. Discussion The captive females showed a temporary increase in plasma P4 concentration at the onset of the reproductive season. This probably indicates ovulations or LH peaks not preceded by estrous symptoms [5]. Reindeer are seasonally polyestrous, and are, as other
Table 2 Measurements of reproductive organs and mean plasma hormone concentrations in pregnant females slaughtered at Røros, Norway (638N, 118E) in the second week of December 1999 Parameter
Mean
S.D.
Range
N
Dressed weight (kg) Uterus weight (g) Ovary weight (g) Crown-rump length (cm) Foetal weight (g) P4 (nmol/L) PAG (ng/mL)
33.4 491.32 2.79 8.04 21.82 13.67 5.89
4.2 153.48 0.85 1.94 12.19 4.88 1.09
25.8–42.6 61.6–766.0 1.3–4.4 2.7–11.4 1.48–53.3 7.5–26.7 3.2–9.2
41 40 40 40 40 41 41
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Fig. 3. Individual weight distribution (range: 1.5–53.3 g; N = 40) of foetuses collected from slaughtered females in the second week of December 1999 at Røros, Norway (638N, 118E).
ruminants, dependent of temporary increases in P4 concentration for the initiation of regular estrous cyclicity [5,45]. The corpus luteum is the main source of P4 in early pregnancy. The temporary decrease in plasma P4 concentration between weeks 9–19 in gestation (Fig. 2a), most likely reflected a regression of this structure [46]. Later in pregnancy P4 production in reindeer is gradually taken over by the feto-placental tissues [47]. Probably, the significant drop in P4 observed in weeks 16–21, prior to calving is associated with a shift in the main production site for P4, from the corpus luteum to the placenta. Though plasma P4 concentrations remained elevated during gestation they only occasionally exceeded the maximum levels of the luteal phase of the estrous cycle (Figs. 1 and 2a). Because cyclic ovarian activity may continue until at least mid February [5], plasma P4 measurement can only be used as non-pregnancy test until cyclic estrous activity has ceased in late winter [6]. In animals that are blood sampled repeatedly over an extended period of time, P4 measurements can on the other hand serve as a reliable indicator of early pregnancy, although they are not pregnancy specific (Figs. 1 and 2a). In this study both plasma E1SO4 and E2 concentrations were low through early and mid gestation (Fig. 2c and d). As indicated by Blom et al. [3] increased
Fig. 4. Plasma PAG concentration by foetal weight in mid December 1999 (N = 40) at Røros, Norway (638N, 118E).
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concentrations of estrogens were first registered at the end of pregnancy, from approximately 50 days pre-partum. A nearly similar secretion pattern was found in whitetailed deer where both E1SO4 and E2 remained at low levels until 12 weeks prior to parturition [2,16]. Increased concentration of estrogens, especially E1SO4 in maternal blood, have been associated with the presence of conceptus in pigs [8], sheep [10], cattle [11] and red deer [2] and has been used as methods for detection of pregnancy in these species. However, the secretion pattern varies between species. In cattle, E1SO4 is detectable by day 72 after conception, rising through day 147 and remains elevated until delivery [11]. In red deer, E1SO4 peaks between days 60 and 80 after conception. A gradual decrease then takes place until a new increase occurs at around 6 weeks prior to calving [2,9]. The secretion patterns of E1SO4 and E2 reported in the present study showed that these hormones cannot be used for early pregnancy detection in reindeer. The mean PG-metabolite concentration increased gradually from about Day 170 until parturition (Fig. 2e). The concentrations in late pregnancy were very high and far exceeded the release associated with luteolysis [5]. High levels of PG-metabolite have been seen in many species during late pregnancy (e.g. sheep: 33; guinea pig: 34; goat: 35; horse: 36). The reason for a high prostaglandin production during pregnancy is not known but can most likely be related to development of the placenta. However, the production as such does not seem to be harmful for the luteal function. The high levels seen at the time of parturition is also similar to the situation in all studied species. Prostaglandin production seems to be a prerequisite for myometrial contractions resulting in the labor process (for review, see [48]). After parturition the PG-metabolite concentration decreased to baseline levels at about 3 weeks postpartum. A similar pattern is found in all ruminants and is correlated to uterine involution [49]. The estimated gestation length of 216 days differs from earlier reports in reindeer and caribou of approximately 225–235 days [3,50,51]. Contrary to the referred studies, where results are based on registered mating and calving, our estimation of gestation length was based on the time between calving and a defined time of conception. Detailed knowledge about P4 profiles during the estrous cycle [5] and the fact that frequent sampling for P4 analysis was done in this study indicate that the given estimates reflected the real gestation length. The fact that late conceptions in the captive animals were associated with shorter duration of pregnancy deserves further attention in future studies. Investigating the variation in timing of conception and calving would provide new insight to mechanisms important for survival and reproductive success in reindeer. Measurement of plasma PAG concentrations proved to be an accurate method for pregnancy diagnosis in reindeer provided that females were more than one month pregnant and that pregnancy testing could be carried out successfully on the herd level in mid December. The stage in gestation at which pregnancy detection was possible by PAG measurement corresponded to that reported for white-tailed deer [52] and goats [38]. The PAG concentration increased gradually with advancing pregnancy (Fig. 4). The same was found in white tailed deer [52], and suggests that PAG could possibly be used for estimation of gestational age in cervid species that usually carry singleton fetuses. However, the results indicate that PAG is a better predictor of fetal age within than between animals (Figs. 1, 2 and 4).
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The wide range of fetal weights shown in Fig. 3 indicates a considerable variation in time of conception. The reasons for this variation could be natural variation in timing of ovulation and/or unsuccessful mating. The lack of an apparent peak in the distribution of fetal weights suggests that the rutting period in reindeer is less synchronized than earlier believed [46,53,54]. However, we think it is premature to draw general conclusions on this point because this study was based on animals presented for slaughter, which probably do not represent the general population of females with regard to reproductive performance. Also the experimental animals showed a rather narrow estimated conception date. The fact that the smallest foetus was hardly detectable by the naked eye indicates that conception must have taken place during late November or early December. Except for this case, all females classified as pregnant had PAG concentrations exceeding the cut-off point of 2 ng/ml, indicating that conception had occurred more than three weeks prior to slaughter (i.e. prior to 23 November). PAG concentrations measured in females carrying the biggest fetuses indicated that conception had taken place 11–14 weeks earlier (Fig. 4; i.e. early or mid September). Although Wood et al. [30] and Huang et al. [31] pointed to limitations in the PAG assay regarding quantitative measurements in other species, the consistent patterns found in captive reindeer in the present study encourages further use. Earlier morphological studies reporting fetal growth curves in reindeer fetuses support this conclusion [46]. In domestic ruminants high PAG concentrations have been reported in maternal circulation two to three months after calving [21]. In non-seasonal, poly-oestrous animals, high concentrations of PAG in the postpartum period may thus cause false positive pregnancy diagnoses. This problem is not likely to occur in reindeer because the rut is more than four months after calving. In the present study the postpartum PAG concentrations returned to baseline levels within one month after calving. In conclusion, the variation in reproductive hormone concentrations during pregnancy in reindeer showed great similarities with other ruminant species. Plasma PAG concentration provided a reliable pregnancy detection method when measured >30 days post conception and on herd level when used in mid December. Measured foetal weights and plasma PAG concentrations from culled females indicate that the breeding season of reindeer extends from early September until late November. No synchronized peak in matings was found in the present study. Further studies are needed to characterise the breeding season and time of conception in reindeer. References [1] Ropstad E. Reproduction in female reindeer. Anim Reprod Sci 2000;60–61:561–70. [2] Barrell GK, Bos S. Changes in serum estrone sulphate and progesterone levels of red deer hinds during pregnancy. N Z Vet J 1989;37:1–3. [3] Blom AK, Sjaastad ØV. Jacobsen E. Plasma levels of progesterone and estradiol-17b in reindeer (Rangifer tarandus tarandus) during pregnancy. Acta Vet Scand 1983;24:287–94. [4] Bulman DC, Lamming GE. The use of milk progesterone analysis in the study of oestrus detection, herd fertility and embryonic mortality in dairy cows. Br Vet J 1979;135:559–67. [5] Ropstad E, Forsberg M, Sire JE, Kindahl H, Nilsen T, Pedersen Ø, et al. Plasma concentrations of progesterone, estradiol, LH and 15-ketodihydro-PGF2a in Norwegian semi-domestic reindeer (Rangifer tarandus tarandus) during their first reproductive season. J Reprod Fert 1995;105:307–14.
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[6] Ropstad E, Johansen O, King C, Dahl E, Albon SD, Langvatn R, et al. Comparison of plasma progesterone, transrectal ultrasound and pregnancy specific proteins (PSPB) used for pregnancy diagnosis in reindeer. Acta Vet Scand 1999;40:151–62. [7] Edqvist L-E, Ekman L, Gustafsson B, Johansson EDB. Peripheral plasma levels of oestrogens and progesterone during late bovine pregnacy. Acta Endocr 1973;72:81–8. [8] Robertson HA, King GJ. Plasma concentrations of progesterone, estrone, estradiol-17b and of estrone sulphate in the pig at implantation, during pregnancy and at parturition. J Reprod Fert 1974;40:133–41. [9] Kelly RW, McNatty KP, Moore GH, Ross D, Gibb M. Plasma concentrations of LH, prolactin, estradiol and progesterone in female red deer (Cervus elaphus) during pregnancy. J Reprod Fert 1982;64:475–83. [10] Carnegie JA, Robertson HA. Conjugated and unconjugated estrogens in fetal and maternal fluids of the pregnant ewe: a possible role for estrone sulphate during early pregnancy. Biol Reprod 1978;19:202–11. [11] Robertson HA, King GJ. Conjugated and unconjugated oestrogens in fetal and maternal fluids of the cow throughout pregnancy. J Reprod Fert 1979;55:463–70. [12] McArthur CP, Geary A. Field evaluation of a pregnancy immunoassay for the detection of oestrone sulphate in goats. J Endocrinol 1986;110:133–6. [13] Engeland IV, Ropstad E, Kindahl H, Andresen Ø, Waldeland H, Tverdal A. Foetal loss in dairy goats: function of the adrenal glands, corpus luteum and the foetal-placental unit. Anim Reprod Sci 1999;55:205–22. [14] Challis JRG. Sharp increase in free circulating oestrogens immediately before parturiton in sheep. Nature 1971;229:208. [15] Challis JRG, Linzell JL. The concentrationof total unconjugated oestrogens in plasma of pregnant goats. J Reprod Fert 1971;26:401–4. [16] Harder JD, Woolf A. Changes in plasma levels of estrone and estradiol during pregnancy and parturition in white-tailed deer. J Reprod Fert 1976;47:161–3. [17] Plotka ED, Seal US, Verme LJ, Ozoga JJ. Reproductive steroids in the white-tailed deer (Odocoileus virginianus borealis). II. Progesterone and estrogen levels in peripheral plasma during pregnancy. Biol Reprod 1977;17:78–83. [18] Butler JE, Hamilton WC, Sasser RG, Ruder CA, Hass GM, Williams RJ. Detection and partial characterization of two bovine pregnancy-specific proteins. Biol Reprod 1982;26:925–33. [19] Zoli AP, Beckers J-F, Closset J, Ballman-Wouters P, Falmagne P, Ectors F. Purification and characterization of a bovine pregnancy-associated glycoprotein. Biol Reprod 1991;45:1–10. [20] Sasser RG, Ruder CA. Detection of early pregnancy in domestic ruminants. J Reprod Fert Suppl 1986;34:261–71. [21] Zoli AP, Guilbault LA, Delahaut P, Oritz WB, Beckers J-F. Radioimmunoassay of a bovine pregnancyassociated glycoprotein in serum: its application for pregnancy diagnosis. Biol Reprod 1992;46:83–92. [22] Lynch KA, Alexander RM, Sasser RG. The cloning and expression of the bovine pregnancy specific protein B (bPSPB) gene. Biol Reprod Suppl 1992;46: (abstract 73). [23] Godkin JD, Cote´ C, Duby RT. Embryonic stimulation of ovine and bovine corpora lutea. J Reprod Fert 1978;54:375–8. [24] Northey DL, French LR. Effect of embryo removal and intrauterine infusion of embryonic homogenates on the lifespan of the bovine corpus luteum. J Anim Sci 1980;50:298–302. [25] Xie SB, Low BG, Nagel RJ, Kramer KK, Anthony RV, Zoli AP, et al. Identification of the major pregnancyspecific antigens of cattle and sheep as inactive members of the aspartic proteinase family. Proc Natl Acad Sci USA 1991;88:10247–51. [26] Sasser RG, Crock J, Ruder CA. Characteristics of pregnancy-specific protein B in cattle. J Reprod Fert Suppl 1989;37:109–13. [29] Garbayo JM, Remy B, Alabart JL, Folch J, Wattiez R, Falmagne P, et al. Isolation and partial characterization of a pregnancy-associated glycoprotein (PAG) family from the goat placenta. Biol Reprod 1998;58:109–15. [30] Wood AK, Short RE, Darling AE, Dusek GL, Sasser RG, Ruder CA. Serum assays for detecting pregnancy in mule and white-tailed deer. J Wildl Manage 1986;50:684–7. [31] Huang F, Cockrell DC, Stephenson TR, Noyes JH, Sasser RG. A serum pregnancy test with a specific radioimmunoassay for moose and elk pregnancy-specific protein. Br J Wildl Manage 2000;64(2):492–9. [32] Niswender GD, Juengel JL, McGuire WJ, Belfiore CJ, Wiltbank MC. Luteal function: the estrous cycle and early pregnancy. Biol Reprod 1994;50:239–47.
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[37] Buckrell BC, Bonnett BN, Johnson WH. The use of real-time ultrasound rectally for early pregnancy diagnosis in sheep. Theriogenology 1986;25:665–73. [38] Gonza´ lez F, Sulon J, Garbayo JM, Batista M, Cabrera F, Calero P, et al. Early pregnancy diagnosis in goats by determination of pregnancy-associated glycoprotein concentrations in plasma samples. Theriogenology 1999;52:717–25. [39] Knox WM, Miller KV, Collins DC, Bush PB, Kiser TE, Marchinton RL. Serum and urinary levels of reproductive hormones associated with the estrous cycle in white-tailed deer (Odocoileus virginianus). Zoo Biol 1992;11:121–31. [40] Stoops MA, Anderson GB, Lasley LB, Shideler SE. Use of fecal steroid metabolites to estimate the pregnancy rate of a free-ranging herd of tule elk. J Wildl Manage 1999;63:561–9. [41] Glossop CE, Foulkes JA, Whitwoth A, Cornwell E. Use of an on-farm progesterone assay kit to determine pregnancy in sows. Vet Rec 1989;124:115–7. [42] Vahtiala S, Sa¨ kkinen H, Dahl E, Eloranta E, Ropstad E. Ultrasonography in early pregnancy diagnosis and measurements of fetal growth in reindeer (Rangifer tarandus tarandus). Theriogenology 2004;61:785–95. [43] Kindahl H, Edqvist L-E, Granstro¨ m E, Bane A. The release of prostaglandin F2a as reflected by 15-keto13,14-dihydro prostaglandin F2a in peripheral circulation during normal luteolysis in heifers. Prostaglandins 1976;11:871–8. [44] Granstro¨ m E, Kindahl H. Radioimmunoassay of the major plasma metabolite of PGF2a 15-keto-13,14dihydro prostaglandin F2a. Methods Enzymol 1982;86:320–39. [45] Ryan KD, Goodman RL, Karsch FJ, Legean SJ, Foster DL. Patterns of circulating gonadotropins and ovarian steroids during the first periovulatory period in the developing sheep. Biol Reprod 1991;45:471–7. [46] Roine K. Studies on Reproduction in Female Reindeer–with Special Reference to Morphological and Physiological Conditions and Fetal Development. Thesis. Department of Obstetrics and Gynaecology, College of Veterinary Medicine, Helsinki: Finland; 1974. p. 57. [47] Sjaastad Ø, Blom AK, Austad R, Øen EO. Plasma progesterone in reindeer in relation to ovariectomy and hysterectomy. Acta Vet Scand 1990;31:45–51. [48] Mitchell MD. Regulation of eicosanoid biosynthesis during pregnancy and parturition. In: Hillier K, editor. Advances in Eicosanoid Research Eicosanoids and Reproduction, vol 1. Lancaster: MTP Press; 1987. p. 108–27. [49] Kindahl H, Odensvik K, Aiumlamai S, Fredriksson G. Utero-ovarian relationships during the bovine postpartum period. Anim Reprod Sci 1992;28:363–9. [50] Skjenneberg S, Slagsvold L. Reindriften og dens naturgrunnlag (The basics of reindeer management). Universitetsforlaget, Oslo. 1968. p. 332. [51] Bergerud AT. The reproductive season of Newfoundland caribou. Can J Zool 1975;53:1213–21. [52] Osborn DA, Beckers J-F, Sulon J, Gassett JW, Muller LI, Murphy BP, et al. Use of glycoprotein assays for pregnancy diagnosis in white-tailed deer. J Wild Manage 1996;60:388–93. [53] Lenvik D. Utvalgsstrategi i reinflokken: 6. Brunsttopp og brunstsynkronisme hos tamrein i Trøndelag. (Selection strategy in domestic reindeer: 6. Oestrous peak and oestrous synchronization in domestic reindeer in Trøndelag County Norway) Nor Landbruksforsk 1988;22:163–174. [54] Hirotani A. Social organization of reindeer (Rangifer tarandus), with special reference to relationships among females. Can J Zool 1990;68:743–9.
Endocrinology of pregnancy and early pregnancy detection by reproductive hormones in reindeer (Rangifer tarandus tarandus) E. Ropstada,*, V. Veibergb, H. Sa¨kkinenc, E. Dahla, H. Kindahld, Ø. Holande, J.F. Beckersf, E. Elorantag a
Department of Production Animal Clinical Sciences, Norwegian School of Veterinary Science, P.O. Box 8146 Dep., NO-0033 Oslo, Norway b Department of Biology, The University Centre in Svalbard, P.O. Box 156, NO-9171 Longyearbyen, Norway c Department of Biology, University of Oulu, P.O. Box 3000, FI-90014 Oulu, Finland d Department of Obstetrics and Gynaecology, Centre for Reproductive Biology in Uppsala, Swedish University of Agricultural Sciences, SE-75007 Uppsala, Sweden e Department of Animal Science, Agricultural University of Norway, Box 5025, NO-1432 A˚s, Norway f Department of Physiology of Reproduction, Faculty of Veterinary Medicine, University of Lie`ge, Bd de Colonster No. 20, B41, BE-4000 Sart-Tilman, Belgium g Department of Physiology, University of Oulu, Kajaanintie 52 A, FI-90570 Oulu, Finland Received 24 May 2004; received in revised form 3 August 2004; accepted 25 August 2004
Abstract The endocrinology was studied throughout pregnancy in reindeer (Rangifer tarandus tarandus) located in Oulu, Finland (658N, 258E) with 13 captive, semi domestic adult females. Blood samples were analyzed for plasma progesterone (P4), estradiol (E2) and estrone sulphate (E1SO4), 15ketodihydro-PGF2a (PG-metabolite) and pregnancy associated glycoproteins (PAG). The mean plasma P4 concentration peaked twice during gestation: at around 24 and three weeks prior to calving. In pregnant females the plasma PAG concentration increased over basal concentrations 21–30 days after the estimated day of conception and peaked at the time of calving. The concentrations of E2 and E1SO4 remained low until 60 days before calving when a rapid increase was found for both hormones. The mean plasma concentration of PG-metabolite increased throughout pregnancy to a maximum at parturition. The estimated mean (range) gestation length was 216 (212– 220) days. * Corresponding author. Tel.: +47 22 96 45 00; fax: +47 22 59 70 81. E-mail address: [email protected] (E. Ropstad). 0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2004.08.003
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Judged from measures on reproductive organs collected from 86 free-ranging, semi-domestic female reindeer of unknown age presented for slaughter at Røros, Norway (638N, 118E) in the second week of December 1999, it was concluded that the breeding season lasted from early September until the end of November. The results also showed that plasma PAG concentration could provide a tool for detection of pregnancy in reindeer. # 2004 Elsevier Inc. All rights reserved. Keywords: Estradiol; Pregnancy associated glycoproteins; 15-Ketodihydro-PGF2a; Estrone sulphate; Progesterone; Pregnancy diagnosis
1. Introduction Reindeer, like most temperate cervids, are seasonal breeders, with mating coinciding with the decreasing photoperiod in the autumn, and with fawning in the spring. The fact that reindeer normally are not subject to intensive farming conditions and generally have a high fecundity explains why little interest has been shown in their reproductive physiology. Semi-domestic reindeer are kept mainly for meat production. One of the most important factors for productivity is the proportion of fertile females that have a calf at foot in the autumn. In the future, it is likely that semi-domestic reindeer will be managed and herded more intensively, which implies that there will be an increased need for knowledge on reproduction in this species. Thus there is a need to expand our knowledge of normal reproductive physiology during the breeding season and pregnancy. Also there may be benefits from development of methods to detect pregnancy and to control reproduction. The reproductive endocrinology of pregnancy in reindeer is poorly investigated. Only a few animals have been blood sampled repeatedly throughout pregnancy, and detailed individual hormone profiles for hormones other than progesterone (P4) are not available [1]. P4 secretion is associated with pregnancy in all mammals as this hormone is produced by the corpus luteum and, in some species, by the placenta as well [2]. There are large differences between ruminant species both to the time course of changes in the P4 concentration [3], and to the range of which P4 varies during pregnancy [4]. The fact that females of equivalent species and reproductive status show considerable variation in P4 plasma concentration has also been documented in reindeer [3,5,6]. The general pattern of P4 variation during pregnancy in reindeer is an increase from conception until around 1–3 months of pregnancy. After this the concentration stabilizes or a slight decrease is seen. Prior to parturition a further increase takes place [1] followed by a rapid drop just around the time of calving [3]. Maternal plasma levels of estrogens, either as free steroids or their conjugates are shown to increase during parts of gestation in most mammals [2,3,7–9]. Increased concentration of estrone and especially of E1SO4 in maternal blood has been associated with the presence of a conceptus in pigs [8], sheep [10], cattle [11] and red deer (Cervus elaphus) [2], and has been used as a method for pregnancy detection in these species. The level of E1SO4 reflects primarily the condition of the placenta and its continued production represents a gross index of foetal-placental wellbeing [12]. Lowered levels of E1SO4 indicate that the endocrine foetal-placental function is disturbed [13].
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In ewes [14], a rise in estradiol (E2) precedes parturition by only a couple of days, whereas in goats [15] and cows [7,8] there is a more gradual rise during the last part of gestation. Similar results are also reported in white-tailed deer [16,17], red deer [9] and reindeer [3]. At parturition there is a drastic decline in E2 in all the above-mentioned ruminants. Pregnancy-specific glycoproteins (PSPB) [18], and pregnancy-associated glycoproteins (PAG) [19] are products of the feto-placental unit and therefore specific for pregnancy [20]. Both PSPB and PAG are synthesized in the outer layers of the trophoblast and secreted in the maternal circulation [20,21]. Molecular cloning studies have shown that their primary structure is closely related [22]. Such conceptus-derived proteins are suspected to signal to the maternal system that pregnancy has occurred and to prevent luteal regression [23–25]. Both substances normally appear in the maternal circulation from about 30 days after conception, and increase to maximum levels around parturition [21,26]. Assays for measuring PSPB (cattle; 20, sheep; 27, elk Cervus elaphus and moose Alces alces; 28) and PAG (cattle: 21; goat: 29) concentrations have only been developed for a few species. However, these assays have proved successful for pregnancy diagnosis in a number of other species although there have been difficulties with precise quantification of PSPB/PAG concentrations across species [30,31]. Prostaglandin (PGF2a) of uterine origin, measured as the PG-metabolite, has been suggested as the main luteolytic factor in female reindeer [5]. Normal pregnancy depends upon the early embryo signaling its presence to the maternal system. In many species in which secretion of PGF2a from the uterus is the primary signal for luteolysis, an obvious potential strategy for blocking luteolysis is the inhibition of this secretion in pregnant animals [32]. High levels of the PG-metabolite have been seen in many species during late pregnancy (e.g. sheep: 33; guinea pig: 34; goat: 35; horse: 36). The reason for a high PG production during pregnancy is not known but can most likely be related to placenta development. However, the production as such seems to be non-harmful for the luteal function. To our knowledge, measurement of PGF2a or its metabolite has not been reported for the gestational period in reindeer. Information about endocrine processes related to reproduction provides basic tools for studies of reproductive success and improvement of reproductive performance. In this context precise methods for early pregnancy diagnosis are essential. In domestic and wild ruminants common pregnancy tests include rectal palpation [20], ultrasonographic detection of pregnancy [20,37] and measurement of pregnancy related biochemical markers in blood (e.g. [9,17,38]), faeces [39,40] or milk [41], mainly including measurement of progesterone and E1SO4 [20]. In reindeer management there is a need for cost-efficient methods for early and accurate pregnancy diagnosis that would allow removal and slaughter of barren, unproductive females, so that reindeer herds can be managed more efficiently [6]. The first objective of the present study was to describe the endocrinology of pregnancy in reindeer by repeated measurement of reproductive hormone concentrations (P4, E2, E1SO4, PG-metabolite and PAG) from the time of conception until delivery in adult females kept in a controlled environment. Secondly, we wanted to assess the potential of measured blood constituents for early pregnancy diagnosis. This objective was achieved by studying experimental animals as well as animals from the field slaughtered after the rut.
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2. Materials and methods 2.1. Animals and sampling 2.1.1. Captive animals The pregnancy of thirteen pluriparus female reindeer (Rangifer tarandus tarandus) were corralled outdoors at the Zoological Gardens of Oulu University, Finland (658N, 258E), and fed commercial pelleted reindeer feed (Poron Herkku, Raisio Group, Finland), 1.8–2.0 kg/animal and day, and with about 400 g lichens (Cladonia spp.) every day in addition to the pelleted feed. A reindeer stag was brought together with the females on 1st October 1997 and was allowed to mate freely with them. Pregnancies were also verified by transrectal ultrasonography. The scanner was equipped with a 5 MHz linear probe (Scanner 100, Pie Medical, Netherlands) at 4–6 weeks in gestation [42]. Based on previous detailed studies of P4 profiles during the estrous cycle [5], the date of conception was set at 2 days before plasma progesterone showed persistent levels above 1 nmol/L followed by increased concentration of PAG (2 ng/mL). Jugular venous blood samples were collected with evacuated heparinized tubes (Venoject1, Leuven, Belgium) from September 1997 until May 1998. Blood samples were collected by hand three times a week without using sedatives or medication. Plasma was separated by centrifugation at 3000 g within 2 h and stored at 20 8C until analysis. Samples were analyzed for P4, E2, E1SO4, PG-metabolite and PAG. The study was performed with permission from the Committee on Animal Experiments of Oulu University. 2.1.2. Culled animals In the second part of the study, 86 free ranging semi-domestic female reindeer of varying, but unknown age were slaughtered in the second week of December 1999 at Røros, Norway (638N, 118E). Heparinized blood samples were collected when the animals were bled immediately after slaughter. Plasma was separated and stored as described previously, until analyzed for P4 and PAG. Dressed weight was recorded and reproductive organs were collected and stored at 20 8C until examined. Pregnancy was verified after visual detection of a fetus in the uterine lumen. Uteri and ovaries from all slaughtered females were weighed. Fetuses were weighed and the crown-rump length (CRL) measured. 2.2. Hormone analyses 2.2.1. Progesterone (P4) Plasma levels of total progesterone were determined by a solid-phase radioimmunoassay kit (Spectria1 Progesterone 125I Coated Tube RIA, Orion Diagnostica, Espoo, Finland). The assay was validated for use with reindeer plasma by demonstrating parallelism between dilutions of reindeer plasma samples and the standard curve, and by recovery of unlabeled ligand. No modifications of the standard procedures were needed. The detection
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limit of the assay was 0.3 nmol/L. Intra-assay coefficient of variation was less than 5%. Inter-assay coefficients of variation were 8.8% (1.25 nmol/L), 7.6% (19.1 nmol/L) and 5.9% (44.1 nmol/L) (N = 18), respectively. 2.2.2. Pregnancy-associated glycoproteins (PAG) A heterologous double antibody radioimmunoassay derived from the method previously described by Zoli et al. [21] was used to determine PAG concentrations in plasma. Bovine PAG (bPAG) was used as the standard and tracer, anti-caprine PAG55,59 as the first antibody (dilution 1:500,000 [29] and sheep anti-rabbit IgG as the second antibody for precipitation. The standard curve ranged from 0.2 to 25 ng bPAG equivalents/mL. Bovine zero-plasma was added to all standard tubes. Samples and standards were incubated with antibody overnight at room temperature. The next day, labelled bPAG (25,000 cpm) was added, and the mixture was further incubated for 4 h. The free and bound fractions were separated by centrifugation at 3400 g for 15 min after addition of sheep anti-rabbit IgG. The precipitates were counted in a 1470 Wizard Gamma Counter (Wallac, Turku, Finland). The interassay coefficients of variation were 11.6% (2.6 ng bPAG equivalents/mL) and 5.0% (10.3 ng bPAG equivalents/mL), respectively. Assay sensitivity was 0.8 ng bPAG equivalents/mL. 2.2.3. Estradiol-17b (E2) Estradiol concentrations were determined by a 125I-estradiol-17b double antibody radioimmunoassay kit, ‘‘Coat-a-count1, Estradiol’’ (Diagnostic Products Corporation, Los Angeles, USA). To construct the standard curve, estradiol standards from the kit were diluted in human zero plasma also provided by the kit (5.7–180 pmol/L). Briefly 250 ml aliquots of plasma or standards were extracted in duplicate before assay with 2.5 mL diethyl ether and evaporated under dry air in glass tubes. The extracted plasma was reconstituted in 200 ml phosphate buffer (pH 7.0) and stored overnight at 4 8C. Antiserum (30 ml) was added to each tube, which was vortexed and incubated for 2 h in room temperature before addition of 75 ml of 125I-estradiol-17b with further 1 h incubation in room temperature. Separation of free from antibody bound hormone was performed by addition of 1 mL precipitating solution from the kit. The contents of the tubes were centrifuged for 15 min (3000 g). After decantation, radioactivity of the tubes were counted in a gamma counter for 1 min. Serial dilutions of reindeer plasma with high concentrations of estradiol-17b produced inhibition curves parallel to the standard curve. The intraassay coefficients of variation calculated from nine precision profiles were 21% at 5.6 pmol/L, 13% at 11.3 pmol/L and below 10% for concentrations exceeding 22.5 pmol/L. The corresponding interassay coefficients of variation were 11.4% (13 pmol/L), 6.6% (42 pmol/L) and 4% (86 pmol/L), respectively. The detection limit was 2.1 pmol/L. 2.2.4. Estrone sulphate (E1SO4) The plasma was analyzed for E1SO4 by a commercial radioimmunoassay kit ‘‘Estronesulfate DSL-54001’’ (Diagnostic Systems Laboratories, Inc. Webster, Texas, USA) modified for use with reindeer plasma. Modification was done as follows: The standard curve was replaced by E1SO4 diluted in pooled plasma from male reindeer calves of
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approximately 30 kg live weight (zero-plasma). Dilutions of plasma samples with varying concentrations of E1SO4 produced inhibition curves parallel with the standard curve. Inter-assay coefficients of variation in samples containing 3.3, 7.7 and 22.8 ng/mL were 10.9, 8.9 and 3.8%, respectively. Minimum detection limit in the assay was 0.01 ng/mL. 2.2.5. 15-Ketodihydroprostaglandin F2a (PG-metabolite) Plasma samples were analyzed for the prostaglandin F2a metabolite, 15-ketodihydroPGF2a [43,44]. The relative cross-reactions of the anti-15-ketodihydro-PGF2a antibody were 16% with 15-keto-PGF2a, 4% with 13,14-dihydro PGF2a, 0.5% with PGF2a and 1.7% with the corresponding metabolite of prostaglandin E2. Serial dilutions of reindeer plasma with high concentrations of 15-ketodihydro-PGF2a produced inhibition curves parallel to the standard curve. The lower limit of detection was 75 pmol/L for a 200 ml plasma sample. The intra-assay coefficient of variation was 14% (114 pmol/L). In samples with concentrations exceeding the upper range of the standard curve (1.2 nmol/L), the plasma was diluted and reassayed to get exact concentrations. 2.3. Data analysis Since the exact dates of conception were not known in the captive animals, the mean (S.D.) plasma concentrations of reproductive hormones during pregnancy were expressed relative to the time of calving. Differences between individual mean concentrations of reproductive hormones during specific time periods of pregnancy were compared by student’s t-test. In addition, correlation analysis (Spearman’s correlation coefficient, rs) and linear regression analysis was used to study the relationship between selected variables. Based on previous detailed studies of P4 profiles during the estrous cycle [5], the date of conception was set at 2 days before plasma progesterone showed persistent levels above 1 nmol/L followed by increased concentration of PAG. Plasma concentrations of P4, E2, E1SO4, PG-metabolite and PAG were related to days relative to calving.
3. Results 3.1. Captive animals The females calved between 14th and 29th of May. Median calving date was 18th of May, and mean (S.D.) estimated gestation length was 216 (3.2) days (Table 1). The median estimated day of conception was 13 October. A negative relationship was found between gestation length and estimated Julian day of conception (gestation length = 0.3398 (estimated Julian day of conception) + 221.1; R2 = 0.70, P < 0.01). The onset of cyclic ovarian activity was preceded by short but regular periods of increased concentrations of plasma P4 (Fig. 1). Four of the 13 females cycled twice before successful conception. After conception, the mean P4 concentration peaked twice during pregnancy. Even though plasma concentrations fluctuated widely both within and between
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Table 1 Individual and mean pregnancy data in 13 captive reindeer kept at Oulu University, Finland (658N, 258E) 1997– 1998 ID
6 7 14 15 27 28 51 52 59 95 100 1586 2055 Mean S.D.
Estimated date of conception
8 Oct. 13 Oct. 27 Oct. 15 Oct. 13 Oct. 11 Oct. 11 Oct. 29 Oct. 11 Oct. 8 Oct. 20 Oct. 6 Oct. 27 Oct.
Days before calving
P4 PAG differencec
Days after calving PAG < 5 (ng/mL)
Gestation length (days)d
P4 1a (nmol/L)
PAG 2b (ng/mL)
217 216 208 215 217 215 214 210 218 213 211 216 210
196 190 189 191 191 188 191 184 195 185 183 193 189
21 26 19 24 26 27 23 26 23 28 28 23 21
19 17 15 15 29 13 19 19 10 – 18 21 21
219 218 210 217 219 217 216 212 220 215 213 218 212
213.9 3.2
189.6 4.0
24.2 2.9
18 4.8
215.9 3.2
Number of days elapsing between establishment of pregnancy and calving indicated by (a) increased progesterone (P4; cut off value: 1 nmol/L) and (b) pregnancy associated glycoprotein (PAG; cut off value: 2 ng/mL); (c) the time difference between (a) and (b) ((a) (b); days); and (d) the time from calving until PAG < 5 ng/mL and gestation length ((a) + 2 days).
animals, the general variation pattern was consistent in all animals. The first peak was found 24 weeks prior to calving (mean (S.D.) = 16.9 (3.7) nmol/L). Significantly lower P4 levels were found during mid-pregnancy than in early pregnancy (means (S.D.) were: 16.1 (0.9) nmol/L, 24–27 weeks before calving, versus 10.5 (0.9) nmol/L, 16–21 weeks before calving; P < 0.001]. The second peak occurred at three weeks before calving (mean (S.D.) = 23.0 (10.8) nmol/L). The mean P4 concentration thereafter decreased towards calving, after which it declined to baseline levels (Fig. 2a). PAG proved to be 100% accurate for pregnancy diagnosis when a discriminatory level of 2 ng PAG/mL plasma was used. PAG concentration exceeded 2 ng/mL between 183 and 196 days prior to calving, or alternatively 21 and 30 days after the estimated conception date (median date: 10th November). Thereafter, PAG increased gradually to a mean (S.D.) level of 14.6 (3.4) ng/mL at the time of parturition. After calving, PAG concentrations decreased to baseline level within 5 weeks (Fig. 2b). The concentrations of E2 and E1SO4 were low until the last 6 weeks of gestation. Concentrations of both steroids then increased rapidly and peaked one week prior to calving (means (S.D.) were for E2: 70.1 (25.5) pmol/L and for E1SO4: 3.1 (1.2) ng/mL), before decreasing to basal levels within 1 week after parturition (Fig. 2c and d). Mean plasma concentrations of the PG-metabolite were at the baseline level around 0.3 nmol/L during the rut and early pregnancy. From 170 days prior to calving the mean
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Fig. 1. Plasma concentration of progesterone (~) and PAG (&) for a selected female related to the time of calving. A points to the small rise in progesterone concentration at initiation of regular oestrous cyclicity. B is the progesterone maximum of a normal oestrous cycle not resulting in conception. C is the mid-pregnancy decrease in P4 concentration between week 16 and 21 pre-partum.
concentration increased gradually and reached 12 nmol/L the week before calving. Four females blood sampled on the day of calving showed PG-metabolite concentrations ranging between 47.7 and 83.5 nmol/L. About 3 weeks after parturition plasma concentrations of PG-metabolite had returned to baseline level (Fig. 2e). 3.2. Culled animals Forty-one of the 86 culled females were verified as pregnant. Dressed weights of the slaughtered animals varied from 25.8 to 42.6 kg (mean = 33.4, S.D. = 4.2, N = 41). The majority of the non-pregnant females were calves, and had not yet become fertile. Results from measurements of reproductive organs and analyses of blood samples from pregnant females are presented in Table 2. A measurable fetus was detected from all but one of the females verified as pregnant. In this female the fetus was too small to be measured. Fetal weights ranged from 1.5 to 53.3 g (Fig. 3), and correlated significantly with CRL (rs = 0.90, N = 40, P < 0.001; range 2.7–11.4 cm). PAG concentrations in the blood samples ranged from 0.2 to 9.2 ng/mL. None of the females verified as non-pregnant, had plasma PAG concentrations exceeding the cut-off value of 2 ng/mL. Among the pregnant animals, the only one with a PAG concentration below 2 ng/mL, was the one without a measurable fetus, verifying that this female was in an early stage of gestation. Except ovarian weight (rs = 0.57, N = 40, P < 0.001) none of the measured variables were significantly correlated to female bodyweight. However, a positive correlation was found between the maternal plasma PAG concentration and fetal weight (rs = 0.42, P = 0.008, N = 40; Fig. 4).
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Fig. 2. Mean (+S.D.) plasma concentrations of (a) progesterone (P4), (b) pregnancy associated glycoproteins (PAG), (c) estradiol (E2), (d) estrone sulphate (E1SO4) and (e)15-ketodihydro-PGF2a (PG-metabolite) relative to calving in 13 reindeer kept at Oulu University, Finland (658N, 258E) 1997–1998.
4. Discussion The captive females showed a temporary increase in plasma P4 concentration at the onset of the reproductive season. This probably indicates ovulations or LH peaks not preceded by estrous symptoms [5]. Reindeer are seasonally polyestrous, and are, as other
Table 2 Measurements of reproductive organs and mean plasma hormone concentrations in pregnant females slaughtered at Røros, Norway (638N, 118E) in the second week of December 1999 Parameter
Mean
S.D.
Range
N
Dressed weight (kg) Uterus weight (g) Ovary weight (g) Crown-rump length (cm) Foetal weight (g) P4 (nmol/L) PAG (ng/mL)
33.4 491.32 2.79 8.04 21.82 13.67 5.89
4.2 153.48 0.85 1.94 12.19 4.88 1.09
25.8–42.6 61.6–766.0 1.3–4.4 2.7–11.4 1.48–53.3 7.5–26.7 3.2–9.2
41 40 40 40 40 41 41
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Fig. 3. Individual weight distribution (range: 1.5–53.3 g; N = 40) of foetuses collected from slaughtered females in the second week of December 1999 at Røros, Norway (638N, 118E).
ruminants, dependent of temporary increases in P4 concentration for the initiation of regular estrous cyclicity [5,45]. The corpus luteum is the main source of P4 in early pregnancy. The temporary decrease in plasma P4 concentration between weeks 9–19 in gestation (Fig. 2a), most likely reflected a regression of this structure [46]. Later in pregnancy P4 production in reindeer is gradually taken over by the feto-placental tissues [47]. Probably, the significant drop in P4 observed in weeks 16–21, prior to calving is associated with a shift in the main production site for P4, from the corpus luteum to the placenta. Though plasma P4 concentrations remained elevated during gestation they only occasionally exceeded the maximum levels of the luteal phase of the estrous cycle (Figs. 1 and 2a). Because cyclic ovarian activity may continue until at least mid February [5], plasma P4 measurement can only be used as non-pregnancy test until cyclic estrous activity has ceased in late winter [6]. In animals that are blood sampled repeatedly over an extended period of time, P4 measurements can on the other hand serve as a reliable indicator of early pregnancy, although they are not pregnancy specific (Figs. 1 and 2a). In this study both plasma E1SO4 and E2 concentrations were low through early and mid gestation (Fig. 2c and d). As indicated by Blom et al. [3] increased
Fig. 4. Plasma PAG concentration by foetal weight in mid December 1999 (N = 40) at Røros, Norway (638N, 118E).
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concentrations of estrogens were first registered at the end of pregnancy, from approximately 50 days pre-partum. A nearly similar secretion pattern was found in whitetailed deer where both E1SO4 and E2 remained at low levels until 12 weeks prior to parturition [2,16]. Increased concentration of estrogens, especially E1SO4 in maternal blood, have been associated with the presence of conceptus in pigs [8], sheep [10], cattle [11] and red deer [2] and has been used as methods for detection of pregnancy in these species. However, the secretion pattern varies between species. In cattle, E1SO4 is detectable by day 72 after conception, rising through day 147 and remains elevated until delivery [11]. In red deer, E1SO4 peaks between days 60 and 80 after conception. A gradual decrease then takes place until a new increase occurs at around 6 weeks prior to calving [2,9]. The secretion patterns of E1SO4 and E2 reported in the present study showed that these hormones cannot be used for early pregnancy detection in reindeer. The mean PG-metabolite concentration increased gradually from about Day 170 until parturition (Fig. 2e). The concentrations in late pregnancy were very high and far exceeded the release associated with luteolysis [5]. High levels of PG-metabolite have been seen in many species during late pregnancy (e.g. sheep: 33; guinea pig: 34; goat: 35; horse: 36). The reason for a high prostaglandin production during pregnancy is not known but can most likely be related to development of the placenta. However, the production as such does not seem to be harmful for the luteal function. The high levels seen at the time of parturition is also similar to the situation in all studied species. Prostaglandin production seems to be a prerequisite for myometrial contractions resulting in the labor process (for review, see [48]). After parturition the PG-metabolite concentration decreased to baseline levels at about 3 weeks postpartum. A similar pattern is found in all ruminants and is correlated to uterine involution [49]. The estimated gestation length of 216 days differs from earlier reports in reindeer and caribou of approximately 225–235 days [3,50,51]. Contrary to the referred studies, where results are based on registered mating and calving, our estimation of gestation length was based on the time between calving and a defined time of conception. Detailed knowledge about P4 profiles during the estrous cycle [5] and the fact that frequent sampling for P4 analysis was done in this study indicate that the given estimates reflected the real gestation length. The fact that late conceptions in the captive animals were associated with shorter duration of pregnancy deserves further attention in future studies. Investigating the variation in timing of conception and calving would provide new insight to mechanisms important for survival and reproductive success in reindeer. Measurement of plasma PAG concentrations proved to be an accurate method for pregnancy diagnosis in reindeer provided that females were more than one month pregnant and that pregnancy testing could be carried out successfully on the herd level in mid December. The stage in gestation at which pregnancy detection was possible by PAG measurement corresponded to that reported for white-tailed deer [52] and goats [38]. The PAG concentration increased gradually with advancing pregnancy (Fig. 4). The same was found in white tailed deer [52], and suggests that PAG could possibly be used for estimation of gestational age in cervid species that usually carry singleton fetuses. However, the results indicate that PAG is a better predictor of fetal age within than between animals (Figs. 1, 2 and 4).
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The wide range of fetal weights shown in Fig. 3 indicates a considerable variation in time of conception. The reasons for this variation could be natural variation in timing of ovulation and/or unsuccessful mating. The lack of an apparent peak in the distribution of fetal weights suggests that the rutting period in reindeer is less synchronized than earlier believed [46,53,54]. However, we think it is premature to draw general conclusions on this point because this study was based on animals presented for slaughter, which probably do not represent the general population of females with regard to reproductive performance. Also the experimental animals showed a rather narrow estimated conception date. The fact that the smallest foetus was hardly detectable by the naked eye indicates that conception must have taken place during late November or early December. Except for this case, all females classified as pregnant had PAG concentrations exceeding the cut-off point of 2 ng/ml, indicating that conception had occurred more than three weeks prior to slaughter (i.e. prior to 23 November). PAG concentrations measured in females carrying the biggest fetuses indicated that conception had taken place 11–14 weeks earlier (Fig. 4; i.e. early or mid September). Although Wood et al. [30] and Huang et al. [31] pointed to limitations in the PAG assay regarding quantitative measurements in other species, the consistent patterns found in captive reindeer in the present study encourages further use. Earlier morphological studies reporting fetal growth curves in reindeer fetuses support this conclusion [46]. In domestic ruminants high PAG concentrations have been reported in maternal circulation two to three months after calving [21]. In non-seasonal, poly-oestrous animals, high concentrations of PAG in the postpartum period may thus cause false positive pregnancy diagnoses. This problem is not likely to occur in reindeer because the rut is more than four months after calving. In the present study the postpartum PAG concentrations returned to baseline levels within one month after calving. In conclusion, the variation in reproductive hormone concentrations during pregnancy in reindeer showed great similarities with other ruminant species. Plasma PAG concentration provided a reliable pregnancy detection method when measured >30 days post conception and on herd level when used in mid December. Measured foetal weights and plasma PAG concentrations from culled females indicate that the breeding season of reindeer extends from early September until late November. No synchronized peak in matings was found in the present study. Further studies are needed to characterise the breeding season and time of conception in reindeer. References [1] Ropstad E. Reproduction in female reindeer. Anim Reprod Sci 2000;60–61:561–70. [2] Barrell GK, Bos S. Changes in serum estrone sulphate and progesterone levels of red deer hinds during pregnancy. N Z Vet J 1989;37:1–3. [3] Blom AK, Sjaastad ØV. Jacobsen E. Plasma levels of progesterone and estradiol-17b in reindeer (Rangifer tarandus tarandus) during pregnancy. Acta Vet Scand 1983;24:287–94. [4] Bulman DC, Lamming GE. The use of milk progesterone analysis in the study of oestrus detection, herd fertility and embryonic mortality in dairy cows. Br Vet J 1979;135:559–67. [5] Ropstad E, Forsberg M, Sire JE, Kindahl H, Nilsen T, Pedersen Ø, et al. Plasma concentrations of progesterone, estradiol, LH and 15-ketodihydro-PGF2a in Norwegian semi-domestic reindeer (Rangifer tarandus tarandus) during their first reproductive season. J Reprod Fert 1995;105:307–14.
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