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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
,2
* Department of Plant, Animal and Soil Sciences, School of Natural Resources and Agricultural Sciences, University of Alaska, Fairbanks 99775;
and
Department of Allied Health, Tanana Valley Campus, University of Alaska, Fairbanks 99775; and
and
Center for Reproductive Biology, Washington State University, Pullman 99164
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Key Words: estrogen • gestation length • ovulatory cycle • pregnancy • progesterone • reindeer
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Initial information on endocrine profiles in reindeer appeared in the late 1970s and early 1980s. Although they provided an important baseline for reproductive investigations, those studies were limited by infrequent and opportunistic sampling, samples from slaughtered animals, and small sample sizes (McEwan and Whitehead, 1980
; Rehbinder et al., 1981; Ringberg and Aakvaag, 1982). Furthermore, the majority of the information pertained to extensively ranched or wild populations. Management objectives of free-ranging and intensively farmed reindeer are different. Recently, reports on seasonal endocrine function in Alaskan reindeer (Bubenik et al., 1997) as well as the endocrinology of the estrous cycle (Ropstad et al., 1995) and pregnancy (Ropstad et al., 2005) in semidomestic Norwegian reindeer have been published.
Despite the accumulated information, large discrepancies in the most fundamental aspects of reindeer reproduction still exist. Estrous cycle length varies from 10 to 33 d (Dieterich and Luick, 1971; Ropstad et al., 1995) and gestation length from 198 to greater than 240 d (Dott and Utsi, 1973; Shipka et al., 2002). This variability may stem from environmental and latitude effects, different genetic stocks, or the constraints of the studies themselves.
Here we report seasonal endocrine changes in 5 non-bred and 10 pregnant Alaskan reindeer from September through May, a necessary prerequisite for developing protocols specific to Alaska reindeer farming.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). Yearlings were housed as a group at the R. G. White Large Animal Research Station. They were housed in a 950-m2 pen, separate from males, beginning in July.
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Blood Sampling
Blood sample collection began on September 3 in both groups of females. The 10 yearlings were placed with 2 fertile males on September 9 and remained there until October 22, being removed on sampling days for blood collection for less than 1 h. Blood samples were collected into 10-mL heparinized tubes (Becton Dickinson, Franklin Lakes, NJ) via jugular venipuncture from all 15 reindeer 3 times/wk during the breeding season (September 1 to October 22) and then 1 time/wk throughout the winter (October 29 to March 18). To characterize hormonal changes in the yearlings as parturition approached, the sampling intensity increased to 3 times/ wk from March 24 until each individual calved. In the nonbred, 2-yr-old females, blood sampling continued once weekly (October 29 to May 7). Samples were collected between 0900 and 1200, centrifuged for 15 min at 2,000 x g within an hour of collection, and the plasma was separated and frozen (–20°F) until assayed.
RIA
For all hormones assayed, samples from an individual animal were run in the same assay.
Progesterone.
Samples were analyzed for progesterone (P4) by RIA using commercial kits (Coat-A-Count, Diagnostics Products Co., Los Angeles, CA). Samples were run in 5 assays. The assay sensitivity was 0.02 ng/mL. High, medium, and low reference pools of reindeer and human plasma were used to calculate the CV. The intraassay CV averaged 5.9% for reindeer standards and 6.1% for human standards. Averages for the interassay CV were 8.7, 3.7, and 13.4% (reindeer) and 6.0, 7.8, and 8.6% (human) for the high, medium, and low pools, respectively.
Estrogens.
Estradiol-17ß, estrone, and estrone sulfate were analyzed at the Center for Reproductive Biology core laboratory, Washington State University, Pullman, according to the following protocols.
Before assaying for estradiol-17ß, plasma was extracted twice using anhydrous ethyl ether (Hotchkiss et al., 1971). Extraction efficiency was >98%. Extracted samples were reconstituted in assay buffer from Diagnostic Systems Laboratories Inc. (DSL), Webster, TX (DSL-39101) and assayed using a double antibody RIA from DSL (DSL-39100). Assay sensitivity was 0.38 pg/ mL, intraassay CV was 2.0%, and interassay CV was 9.3% over 3 assays. The antibody had the following cross-reactivities determined by DSL: estrone, 6.9%; equilenin, 0.4%; 17ß-estradiol-3-glucuronide, 0.27%.
Before assaying for estrone, samples were extracted twice using anhydrous ethyl ether (Hotchkiss et al., 1971). Extraction efficiency was >98%. Extracted samples were reconstituted in assay buffer (DSL-8701) and assayed using a double antibody RIA from DSL (DSL-8700). Assay sensitivity was 3.75 pg/mL, intraassay CV was 2.8%, and interassay CV was 5.8% over 4 assays. The antibody had the following cross-reactivities determined by DSL: estrone sulfate, 2.02%; 17ß-estradiol, 1.25%; 16
-hydroxyestrone, 0.46%; epriestriol, 0.3%; estriol, 0.22%; 17-OH progesterone, 0.1%.
A double antibody RIA from DSL (DSL-5400) was used to measure estrone sulfate concentrations directly in plasma. There was parallelism of plasma dilutions to the standard curve, so plasma samples were not extracted. The assay sensitivity was 0.05 ng/mL, intraassay variation was 1.4%, and interassay variation was 5.2% over 3 assays. The antibody had the following cross-reactivities determined by DSL: estrone, 4.9%; estrone glucuronide, 3.4%; 17ß-estradiol 3-sulfate, 1.0%; 17ß-estradiol, 0.3%; 17ß-estradiol glucuronide, 0.1%.
Definitions
Ovulatory Cycle.
During periods of frequent sampling (3 times/wk), the ovulatory cycle was calculated from the last low P4 concentration (
0.5 ng/mL; a low threshold value equivalent to the 1.6 nmol/L used by Ropstad et al., 1995) followed by a sustained rise resulting in elevated P4 concentrations (>0.5 ng/mL) lasting 
14 d.
Weekly blood sampling did not always detect baseline P4 values, especially in mid to late winter. In such cases (n = 8), if the previous or following sample was more than twice the low concentration, the low P4 point was taken as indicative of a new cycle.
Short Cycle.
Elevated P4 concentrations, calculated from the last low P4 concentration (<0.5 ng/mL) and lasting 14 d were considered indicative of a short cycle.
Pregnancy.
Elevated P4 concentrations calculated from the last low P4 concentration followed by a sustained rise lasting 42 d were considered indicative of pregnancy.
Statistical Analysis
Statistical analyses were accomplished using SigmaStat (Systat Software Inc., Richmond, CA). Differences in ovulatory cycle length over the season and between individuals were analyzed in a 2-way ANOVA, using GLM to compare the mean cycle length between individuals and between the first to the sixth cycle over the season. Peak P4 concentrations attained during each cycle were similarly analyzed (2-way ANOVA and GLM) comparing differences between individuals and between the first to sixth cycle of the season. During pregnancy, P4 concentrations were normalized to the last low P4 concentration that was followed by a sustained rise (estimated conception), so that mean values were based on the same approximate stages of gestation. Differences in mean P4 from wk 1 to 30 were analyzed by using a 1-way repeated measures ANOVA and GLM. These data were transformed to their natural logarithm to accommodate the assumption of normality of the parametric test.
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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reported the end of August as the beginning of rut in captive reindeer based on an elevation in LH (0.84 ng/mL), but previous reports from Fairbanks identify mid September as the beginning of rut (Dieterich and Luick, 1971; Rowell et al., 1999; Shipka et al., 2002). This is still approximately 1 mo earlier than published accounts on Norwegian reindeer (Ropstad et al., 1995). More detailed information on the endocrinology of reindeer entering the breeding season, with and without males present, is needed before conclusions on initiation of the breeding season can be made.
Among the 2-yr-old reindeer, elevated plasma P4 at the beginning of September was followed in 3 instances by a short cycle (8 to 11 d) and in 2 instances by a full-length cycle. Silent ovulation (a preovulatory LH peak followed by a short luteal cycle at the onset of the breeding season) has been characterized in Norwegian reindeer (Eloranta et al., 1994), and a similar transient rise in P4 has been previously documented in our reindeer (Shipka et al., 2002). Median date for the onset of the first recorded full-length ovulatory cycle was September 23 (range September 5 to October 2). Reindeer left open (not bred) over the winter experienced 6 to 8 consecutive full-length ovulatory cycles after the initiation of blood sampling (median = 7 ovulatory cycles). Ovulatory cycle length did not vary between individuals (P = 0.170) or between consecutive cycles (P = 0.244). Mean ovulatory cycle length, using only data from the period of more frequent sampling (3 times/wk), was 22 d ± 0.5 d (range 20 to 25, n = 8) and did not differ (P = 0.095) from mean ovulatory cycle length for the pooled data over the winter (24 d ± 0.6 d; range 18 to 29 d, n = 36). This ovulatory cycle length is consistent with an earlier report in reindeer (Dott and Utsi, 1973) and with estrous cycles in most cervid species (see Asher, 1985), but differs from reports in primaparous Norwegian reindeer where the first cycle of the season was longer than subsequent cycles and estrous cycle length varied from 13 to 33 d (Ropstad et al., 1995). It also differs from numerous early reports of a short 10 to 12 d estrous cycle detected in reindeer and caribou (Dieterich and Luick, 1971; McEwan and Whitehead, 1972; Bergerud, 1975). The latter 2 authors describe 2 types of estrous cycle: a short cycle lasting 10 to 12 d (differentiated from the silent, short cycle at the onset of the breeding season, McEwan and Whitehead, 1972) and a longer cycle lasting 18 to 25 d. Bergerud (1975) speculated that long estrous cycles were an artifact of captivity, but this is not supported by observations of 10 to 12 d cycles among captive reindeer (Dieterich and Luick, 1971). It is interesting to note that these early observations came from free-ranging populations where males and females commingle or from captive herds that used vasectomized males to identify estrus. The male reindeer has a marked effect on the initiation of ovarian activity (Shipka et al., 2002), and Bergerud (1975) stresses the potential synchronizing role of male biostimulation in female caribou. An investigation into the male effect on estrous cycle length could resolve some of these discrepancies.
During the ovulatory cycle, mean peak P4 concentration was 4.4 ± 0.19 ng/mL (range 2.4 to 7.6 ng/mL; n = 36). Peak P4 concentrations varied between individual reindeer (P = 0.015) but did not differ (P = 0.870) between cycles over the season. Bubenik et al. (1997) documented similar mean P4 concentrations in 4 nonpregnant reindeer from September until May, although the 3-wk sampling interval was inadequate to characterize individual ovulatory cycles. In this study, P4 did not return to baseline (0.5 ng/mL) in 8 of 42 instances but fell to 0.8 to 1.6 ng/mL (mean 1.1 ± 0.29 ng/mL). This occurred after the sampling interval was extended to once weekly and may reflect lower resolution of weekly sampling intervals.
The onset of the anovulatory period presented 2 different physiological pictures. In 2 females, P4 returned to baseline following cycles of normal length and remained at baseline, inferring a cessation of ovulation and luteal function (Figure 2a). This is similar to fallow deer (Asher et al., 1988), Père David’s deer (McLeod et al., 1991), and red deer hybrids with Père David’s deer or Wapiti (Asher et al., 2000) where the transition into anestrus is characterized by an abrupt cessation of luteal activity. In the remaining 3 reindeer, P4 apparently failed to return to baseline at the end of an ovulatory cycle and remained elevated for a variable length of time (Figure 2b). In 1 female, P4 returned to baseline after 53 d, whereas in the other 2 females, P4 concentrations had been elevated for 93 and 57 d, respectively, and were 2.5 ng/mL when the study ended on May 7. Although it is possible the extended sampling interval (1 time/wk) failed to detect baseline P4 for consecutive ovulatory cycles, the P4 profiles at the end of the breeding season did not exhibit the magnitude of P4 fluctuations characteristic of repeat estrous cycling and were suggestive of a persistent corpus luteum (CL). The formation of persistent CL is consistent with a previous study in which we documented elevated P4 in a female reindeer sampled from July through August (Shipka et al., 2002) and with a study by Bubenik et al. (1997) who reported a second P4 peak in nonpregnant reindeer between February and April. Apparent, persistent CL have been described in red deer, occurring during anestrus in 4 of 9 hinds (Asher et al., 2000) and 2 of 8 hinds (Meikle and Fisher, 1996). In the former study a putative, persistent CL was identified in 1 female at the beginning of the breeding season and again at the onset of anestrus (Asher et al., 2000). Asher et al. (2000) suggest that a partial or complete failure of the luteolytic process at the transition to anestrus may be responsible for persistent CL in red deer, an explanation that is equally plausible in reindeer. Because it is possible for a persistent CL to continue into the next breeding season, with potential impacts on breeding synchrony and timing of mating (Shipka et al., 2002), this phenomenon warrants further investigation.
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). However, those authors note that the adrenal cortex secretes large quantities of cortisol in response to most stressful stimuli, and only under conditions of extreme stress are high concentrations of P4 produced. It is important to reiterate that the reindeer in this study were born and raised in captivity, halter trained as calves, and fully acclimated to the sampling procedures, especially by mid to late winter. Furthermore, using weekly jugular blood collection from reindeer, Lincoln and Tyler (1994) demonstrated a significant drop in peripheral concentrations of P4 following ovariectomy. This supports the premise that jugular venipuncture in acclimated animals is unlikely to stimulate significant adrenal release of P4.
Pregnancy
Breeding occurred between September 10 and October 2. The animal bred on October 2 was being treated for a minor infection. Breeding in the remaining 9 yearlings occurred from September 10 to 24 (median date September 20), a 14-d interval. The 6 smaller yearlings, purchased 2 mo before the onset of the study, were placed on the same pelleted ration, fed ad libitum, as the other 4 reindeer were receiving. It is possible that the different nutritional plane created a flushing effect, but it is equally possible that once pubertal BW had been attained (60 kg; Ropstad et al., 1995), differences in BW had little effect on the seasonal onset of ovarian activity. This latter concept is exemplified by the observation that purchased and farm-raised yearling reindeer bred within a 2-wk period regardless of differences in BW.
Concentrations of P4 rose from baseline to a mean of 4.9 ± 0.36 ng/mL at 4 wk postconception and remained elevated throughout gestation. Individual P4 values ranged between 2.5 to 14 ng/mL, and the pattern of P4 secretion fluctuated with peaks and troughs varying by 3 to 9 ng/mL, but no clear pattern linked to a stage of gestation emerged. Mean P4 values for wk 1 to 3 and wk 29 to 31 differed (P = 0.05) from all other weeks. However, mean P4 between 4 to 28 wk of gestation did not vary (P = 0.104), contrary to a previous report (Ropstad et al., 2005). Between wk 28 and 31, P4 declined from a mean of 6.04 to 1.7 ng/mL (Figure 3). As P4 began declining, the estrogens coincidentally rose sharply until parturition (Figure 3). Although the endocrine profile during pregnancy is broadly consistent with previous reports in reindeer (McEwan and Whitehead, 1980; Bubenik et al., 1997; Ropstad et al., 2005), there was no evidence in this study or that of Bubenik et al. (1997) of a significant drop in P4 concentration during mid pregnancy, or any indication of an early or late peak, as was recently reported in Norwegian reindeer (Ropstad et al., 2005). Individual variation in P4 profiles during pregnancy precludes generalizations on the pattern of secretion. The normalized data for Alaskan reindeer produced a pattern characteristic of many species that produce luteal P4 throughout gestation. In this study, mean P4 concentrations during pregnancy were greater than those observed during the ovulatory cycle, but there was continual overlap throughout the winter in the magnitude of P4 concentrations observed in cyclic and pregnant females (Figure 4). The overlap in peripheral P4 concentrations between pregnant and nonpregnant females precludes the use of this hormone in a single blood sample for pregnancy determination in captive reindeer. This differs from previous studies on free-ranging reindeer where P4 concentrations during pregnancy are reported to be greater than P4 concentrations in nonpregnant reindeer, especially in mid to late winter (Gerhart et al., 1997; Flood et al., 2005; Ropstad et al., 2005), and elevated P4 concentrations in single samples have frequently been used to infer pregnancy (Russell et al., 1998). In general, nonpregnant females in free-ranging populations have failed to achieve pubertal BW or were in poor body condition entering the winter, or both (Russell et al., 1998). Both physiological states prevent or stop continued estrous cyclicity. However, among farmed reindeer, females are frequently separated from the male following mating and are capable of maintaining successive ovulatory cycles well into the spring, producing luteal P4 concentrations comparable with those of pregnancy. Although we cannot address the fertility of ovulatory cycles in mid to late winter, new pregnancies have been reported as late as January and February in Norway (Reimers, 1983) and Alaska (M. Shipka, unpublished data). In general, criteria used to distinguish between pregnant and nonpregnant free-ranging reindeer are not directly applicable to intensively farmed reindeer.
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; Bubenik et al., 1997; Ropstad et al., 2005), white-tail deer (Plotka et al., 1977), and red deer (Kelly et al., 1982). Because estrone sulfate is of feto-placental origin, the presence and concentrations of this hormone have been associated with pregnancy detection, fetal number and viability, and placental health in goats (Refsal et al., 1991), fallow deer (Willard et al., 1998), red deer (Barrell and Bos, 1989), and cattle (Dobson et al., 1993; Hirako et al., 2002). Collectively these studies demonstrate a marked species specificity regarding the appearance and concentrations of estrone sulfate in maternal peripheral circulation. In the reindeer, significant concentrations of estrone sulfate are not quantifiable until the final weeks of gestation, rendering the hormone of little use for pregnancy determination or as an indirect measure of placental health over gestation.
Calving occurred between April 8 and May 2. Mean gestation length was 211 ± 2.24 d and ranged from 198 to 221 d. Mean birth weight was 5.9 ± 0.17 kg with no difference (P = 0.100) in birth weight between males (n = 7) and females (n = 3). The 3 shortest gestation lengths, 198, 204, and 205 d, produced 1 female and 2 male calves, respectively, of normal birth weight (range 5.45 to 6.59 kg), although all 3 calves died within 72 h of birth. The association of neonatal loss with the shortest gestation lengths makes it tempting to speculate on the possibility of calf prematurity, but a 203-d gestation, recorded in a previous study, produced an apparently healthy calf (Shipka et al., 2002).
Mean gestation length reported here is comparable with the 216-d gestation reported by Ropstad et al. (2005) and 217 d for 2 artificially inseminated reindeer (Dott and Utsi, 1973), but considerably shorter than previous reports of 225 to 235 d (Bergerud, 1975; Blom et al., 1983). However, gestation length in this study, based on known breeding dates, ranged from 198 to 221 d. This range is equivalent to a full ovulatory cycle and greater than that reported by Ropstad et al. (2005). Recent reports of variable gestation lengths where conception dates are supported by endocrine data (Shipka et al., 2002; Ropstad et al., 2005) concur with earlier reports based solely on observational data (Dott and Utsi, 1973). More importantly, there appears to be a negative correlation between conception date and gestation length (Ropstad et al., 2005; Shipka and Rowell, 2006). A negative correlation was not identified in the current study, which is not surprising considering the narrow (14 d) range of conception dates. More information is needed on the variability in gestation length, its impact on managing the breeding season, and its association with calf viability and growth.
This study provides an accurate estimate in gestation length in Alaskan reindeer and demonstrates a continual overlap in the magnitude of P4 values observed in pregnant and cyclic females during the winter. The breeding potential of farmed reindeer is far less constrained by season than are free-ranging reindeer. Caution should be exercised when extrapolating information from wild conspecifics.
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Footnotes |
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2 Corresponding author: ffmps{at}uaf.edu
Received for publication August 31, 2006. Accepted for publication November 8, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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in Norwegian semi-domestic reindeer (Rangifer tarandus tarandus) during their first reproductive season. J. Reprod. Fertil. 105:307–314.[Abstract]
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–) was substantially heavier than the other 3 (–
–). The x-axis represents the weeks from September 10 to October 22.
