J. Anim. Sci. 2002. 80:2476-2484
© 2002 American Society of Animal Science
Appearance and disappearance of swainsonine in serum and milk of lactating ruminants with nursing young following a single dose exposure to swainsonine (locoweed; Oxytropis sericea)1,2
J. B. Taylor1,3 and
J. R. Strickland1,4
Department of Animal and Range Science, New Mexico State University, Las Cruces 88003
4 Correspondence:
MSC 3-1 Box 30003 (E-mail:
jistrick{at}nmsu.edu).
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Abstract
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A series of experiments were conducted to investigate the elimination of swainsonine in the milk of lactating ruminants following a single dose oral exposure to swainsonine (locoweed; Oxytropis sericea) and to assess subsequent subclinical effects on the mothers and their nursing young. In a preliminary experiment, lactating ewes were gavaged with locoweed providing 0.8 mg swainsonine/kg BW (n = 4; BW = 75.8 ± 3.6 kg; lactation = d 45) and lactating cows were offered up to 2.0 mg swainsonine/kg BW free choice (n = 16; BW = 389.6 ± 20.9 kg; lactation = d 90). Serum and milk were collected at h 0 (before treatment), 3, 6, 12, and 24 for ewes, and h 0 (before treatment), 6, 12, 18, and 24 for cows. Swainsonine was highest (P < 0.05) by h 6 in the serum and milk of ewes. Consumption of at least 0.61 mg swainsonine/kg BW induced consistent (> 0.025 µg/mL) appearance of swainsonine in cow serum and milk. In response to the results obtained in the preliminary experiment, a subsequent experiment utilizing lactating ewes (n = 13; BW = 74.8 ± 6.4 kg; lactation = d 30) and cows (n = 13; BW = 460.8 ± 51.9 kg; lactation = d 90) was conducted. Each lactating ruminant was gavaged with a locoweed extract to provide 0 (control), 0.2, or 0.8 mg swainsonine/kg BW and individually penned with her nursing young. Serum and milk from the mothers and serum from the nursing young were collected at h 0 (before treatment), 3, 6, 9, 12, 24 and 48 (an additional sample was obtained at h 72 for ewes and lambs). Serum and milk swainsonine was higher (P < 0.05) in the 0.8 mg treated groups and maximal (P < 0.05) concentrations occurred from h 3 to 6 for ewes and h 6 to 12 h for cows (P < 0.05). Rises in alkaline phosphatase activity indicated subclinical toxicity in the treated ewes (P < 0.05). Following a single dose oral exposure to 0.2 and 0.8 mg swainsonine/kg BW provided by a locoweed extract, swainsonine was detected in the serum and milk of lactating ewes and cows, and rises in serum alkaline phosphatase activity were observed in the ewes. Neither swainsonine nor changes in alkaline phosphatase activity was detected in the serum of the lambs and calves nursing the ewes and cows dosed with swainsonine.
Key Words: Cows • Ewes • Milk • Oxytropis • Swainsona • Lactation
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Introduction
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Based on producer testimonials, James and Hartley (1977) investigated the potential of locoweed toxicity to occur in lambs and calves ingesting milk from mothers consuming moderate to high amounts of locoweed. In both species, subclinical and clinical symptoms of toxicity were observed within 7 and 21 d, respectively. Since these studies were conducted prior to the isolation of swainsonine from locoweed (Molyneux and James, 1982), the estimated levels of swainsonine exposure and amount eliminated in the milk were not determined.
Previously, Taylor et al. (2000) suggested that short-term (< 28 d) consumption of 0.2 mg swainsonine/(kg BW•d) or less to be a potential minimum effective level in yearling wethers. This seems to agree with Stegelmeier et al. (1999) who reported minimal changes in some serum constituents (
-mannosidase, aspartate aminotransferase, alkaline phosphatase) and localized (rather than widespread) vacuolization of tissues (pancreas, kidney, thyroid) of wethers consuming less than 0.2 mg swainsonine/(kg BW•d) for 30 d. When considering the findings of James and Hartley (1977), coupled with the normal route of swainsonine elimination occurring in the urine (Stegelmeier et al., 1995a), this potential minimum effective level could be different for lactating ruminants due to an added route of elimination, the mammary system. However, the mammary system as an elimination route of swainsonine possibly poses a problem to the nursing young as observed by James and Hartley (1977), especially when the young animals are also allowed free access to the locoweed. Therefore, the objectives of this study were to investigate the elimination of swainsonine in the milk of lactating ewes and cows following single dose oral exposure to swainsonine (locoweed; Oxytropis sericea) and assess subsequent subclinical toxicity occurring in the mothers and their nursing young.
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Materials and Methods
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Animals, Treatments, Sampling Intervals
Preliminary Experiment 1.
Four lactating ewes (BW = 75.8 ± 3.6 kg; lactation = d 45), given ad libitum access to an alfalfa-based diet, were each placed in an individual pen with her single lamb. Locoweed (Oxytropis sericea; 0.614 mg swainsonine/g of plant matter) intake for each ewe was estimated based on a target swainsonine dosage of 0.8 mg swainsonine/kg BW. Milk and blood samples were obtained at h 0 (prior to treatment), and 3, 6, 12, and 24 h after treatment. A single dose locoweed treatment was delivered by gavage immediately following h 0 of sampling.
Preliminary Experiment 2.
Sixteen lactating cows (BW = 389.6 ± 20.9 kg; lactation = d 90), receiving a sudan grass hay diet fed at 1.9% BW and supplemented with 908 g/d of a 46.8% CP supplement, were each placed in an individual pen with her single calf. Based on a maximum intake of 2.0 mg swainsonine/kg BW, a predetermined amount of locoweed (Oxytropis sericea; 0.614 mg swainsonine/g of plant matter) was provided individually and free choice to each cow (calves were denied access); the locoweed was immediately offered after obtaining blood and milk samples at h 0. Cows were allowed 2 h to consume locoweed treatment before removal, and refusals were collected and weighed for estimation of swainsonine intake. Milk and blood sampling were conducted at h 0 (prior to treatment), and 6, 12, 18, and 24 h after treatment.
Experiment 3.
In two separate trials, 13 ewes (74.8 ± 6.4 kg; lactation = d 30) with single lambs and 13 cows (460.8 ± 51.9 kg; lactation = d 90) with single calves were individually penned with their nursing young and randomly assigned to one of three treatments: 0 (n = 3; control), 0.2 (n = 5), or 0.8 (n = 5) mg swainsonine/kg BW. Prior to treatment (1 d), a 24-h milk production estimate was conducted for ewes and cows. The estimate was based on the total amount of milk collected (mL) at the end of a 3-h period immediately following complete evacuation (milk let-down induced by 2 mL oxytocin i.v.) of milk from the udder and separation from the nursing young. Swainsonine treatments were delivered by gavage to each animal as a locoweed extract (Oxytropis sericea). For both ewes and cows, blood and milk samples were collected at h 0 (prior to treatment) and 3, 6, 9, 12, 24, and 48 h following treatment. Additional samples were collected at h 72 for the ewes. Blood samples were also obtained from the nursing young at each corresponding sampling hour. Animal use for all experiments was approved and followed the guidelines of the Institutional Animal Care and Use Committee (#98-0011, 98-0026).
Extract, Samples, and Analyses
Locoweed Extract Preparation.
The extract was prepared by boiling ground locoweed (1 mm) in methanol for 24 h. The liquid fraction was filtered and methanol was removed by evaporation. Resulting contents were vigorously mixed 1 to 9 with distilled water and centrifuged at 1500 x g for 30 min to separate the lipid and water soluble fractions. The water soluble fraction was decanted and analyzed for total swainsonine (extract = 5.85 and 9.13 mg swainsonine/mL for ewe and cow gavage, respectively) as described below. The fraction was then mixed 1 to 1 with molasses for treatment delivery (gavage). For the control groups, a 1 to 1 water and molasses solution was prepared.
Sample Collection and Processing.
Approximately 7 mL of venous blood (jugular) was collected from ewes, cows, lambs, and calves at the designated sampling times. Ten milliliters of milk was collected from each ewe (first 5 mL per half) and 16 mL from each cow (first 4 mL per quarter) at the designated sampling times. Blood was allowed to clot, then centrifuged at 1500 x g, and serum was decanted into 2-mL storage vials. Samples from all experiments were stored at -20°C for subsequent analyses. Serum samples, milk samples, and methanol extracts of locoweed from all experiments were analyzed for swainsonine using a modified -mannosidase inhibition assay (lower detection limit = 0.025 µg/mL; Li, 1967) described below.
Swainsonine and Alkaline Phosphatase Analyses.
For swainsonine analysis, samples were boiled in a water bath for 30 min and centrifuged for 30 min at 11,900 x g. In triplicate, 20 µL of the supernate was transferred to a 96-well plate (well volume = 320 µL) with 100 µL of citrate buffer (79.2 mM, pH = 4.5) and 20 µL of -mannosidase enzyme (0.025 U/mL; Sigma Chemical Co., St. Louis, MO), then incubated for 15 min at 37°C. Following incubation, 20 µL of 
-nitrophenyl -D-mannopyranoside (20 mM; Sigma Chemical Co.) was added to each well and then incubated for an additional 90 min. The reaction was stopped and color was developed with 80 µL of borate buffer (200 mM, pH = 9.8) added to each well. Optical density was determined at 405 nm (MRX Microtiter Plate Reader; Dynatech Laboratories Inc., Chantilly, VA). Additionally, serum obtained from the ewes, cows, lambs, and calves in Exp. 3 were analyzed for alkaline phosphatase activity (Procedure # 245, Sigma Chemical Co.) in order to detect symptoms of subclinical swainsonine toxicity.
Statistics and Calculations
Preliminary Experiments 1 and 2.
Swainsonine concentrations in serum and milk of ewes were subjected to analysis of variance (SAS 8.0; SAS Institute Inc., Cary, NC) and differences between sample hour means were identified using preplanned pairwise comparisons. To visualize potential changes in cow serum and milk swainsonine over the sampling period, arithmetic means and standard deviations of the swainsonine concentrations within each sampling hour were generated using only cows with detectable swainsonine in the serum and milk. Additionally, an arithmetic mean and standard deviation for total swainsonine intake was calculated, which included only cows that consumed a measurable amount of locoweed.
Experiment 3.
All data were analyzed as a completely randomized design with unequally spaced repeated measures (spatial power law; Littell et al., 1996) using mixed models procedure of SAS (8.0). Treatment, sampling hour, and corresponding interaction were included in the model. When treatment by sampling hour interactions were detected, preplanned pairwise comparisons were estimated to test for difference between treatments within sampling hour. To estimate change in alkaline phosphatase activity and swainsonine concentration over the sampling period (h 0 to 72) within each treatment, preplanned pairwise comparisons were conducted for each sampling hour vs h 0 for alkaline phosphatase and initial hour of detection for swainsonine. Linear, quadratic, and cubic effects of swainsonine treatment on serum and milk swainsonine concentrations were also estimated; when an effect was detected, solutions for the slope of highest hierarchical significance (cubic > quadratic > linear) and best representing the observed biological effect were generated. To estimate the amount of swainsonine per unit of BW potentially consumed by the nursing young, the estimated mean swainsonine content of the milk ((g/mL) was multiplied by the 24-h milk production estimate (L/d) and further divided by the BW of the corresponding nursing young.
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Results and Discussion
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Preliminary Experiment 1
Swainsonine was detected in the serum and milk of the ewes at all sampling points following initial swainsonine exposure. Mean serum swainsonine concentrations were 0.402, 0.568, 0.227, and 0.140 ± 0.045 µg/mL for h 3, 6, 12, and 24, respectively. All sampling hours were different (P < 0.02) from the others, and a maximal serum swainsonine concentration occurred at h 6 (P < 0.02). Mean milk swainsonine concentrations were 0.127, 0.256, 0.174, and 0.086 ± 0.03 (g/mL for h 3, 6, 12, and 24, respectively. As with serum, maximal swainsonine concentrations occurred in the milk at h 6 (h 6 > h 3 and 24; P < 0.05). Swainsonine rapidly declined in the serum (h 24 < h 3, 6 and 12; P < 0.02) and milk (h 24 < h 3, 6, 12; P < 0.06) over the 24-h sampling period after maximal concentrations were obtained. Although not as pronounced, disappearance of swainsonine from the milk was similar to that from serum.
Preliminary Experiment 2
The success of the ewe experiment prompted the potential use of the locoweed gavage in the preliminary lactating cow study. However, the large volumes of slurry to be prepared/dosed, coupled with the number and disposition of cows available, circumvented the use of this technique. Therefore, cows were offered a moderate portion of locoweed, free choice, providing up to 2 mg swainsonine/kg BW for 2 h. Only 13 of 16 cows consumed a measurable amount of swainsonine with the arithmetic mean amount consumed during the 2-h exposure period being 0.744 mg swainsonine/kg (Table 1
). A standard deviation of nearly 60% of the mean intake and a 4.7-fold difference between the lowest and highest amount consumed suggest much variation exists in the level of swainsonine voluntarily ingested when offered free choice to lactating cows not having been previously exposed to locoweed. Subsequent concentrations of swainsonine were detected in the serum of 13, 7, 6, and 6 cows for h 6, 12, 18, and 24, respectively (Table 1). Only one cow had detectable (> 0.025 µg/mL) concentrations of swainsonine in the serum at h 36 and 48. Swainsonine was also detected in the milk of 7, 8, 12, 13, and 12 cows at h 6, 12, 18, 30, and 36, respectively; following h 36, swainsonine was no longer detected in the milk of any cow. Interestingly, swainsonine was not present in the milk at h 24 even though most animals that consumed locoweed had detectable levels of swainsonine in the milk at the preceding and following sampling hours. Whether or not this was an actual event has yet to be determined; the lower limit of the swainsonine assay describe earlier is 0.025 µg/mL; therefore, swainsonine may have been present but not at a detectable concentration. At least 0.61 mg swainsonine/kg BW (provided by locoweed free choice) appeared to be the minimal intake level to induce consistent and detectable concentrations of swainsonine in the serum and milk of cows acutely exposed to locoweed. Regardless of the variation in intake, swainsonine was identified in the milk, detected early (h 6) following exposure, and seemed to require more time to disappear from the milk than the serum. The wide range of locoweed intake seems to be the reason for the variation in number of cows with detectable concentrations of swainsonine in the serum and milk; cows consuming more locoweed generally had detectable levels at more sampling hours and more swainsonine present in milk and serum.
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Table 1. Mean (arithmetic) swainsonine intake and concentrations in the serum and milk of lactating cows allowed 2 h access to locoweed (Oxytropis sericea), providing up to 2.0 mg swainsonine/kg BW
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Experiment 3
Due to some problems in preparing and orally delivering a locoweed slurry as in Exp. 1 and the variation in locoweed intake observed in Exp. 2, alternate methods to administer swainsonine in an inexpensive, rapid, concentrated way were explored. By using methanol to extract large quantities of swainsonine from ground plant matter followed by a series of concentration procedures (e.g., evaporation, centrifugation), an end product was produced that was concentrated and easy to deliver to both ewes and cows. Subsequently, this method was employed for Exp. 3.
Ewes.
A treatment by sampling hour interaction was detected (P < 0.001) for ewe serum and milk swainsonine. From h 3 to 24, swainsonine was detected in ewe serum (Figure 1) and milk (Figure 2) of both swainsonine treated groups. No swainsonine was detected in the serum or milk of the control ewes at any sampling hour and in the serum or milk of the treated ewes beyond h 24 (h 48 and 72). At sampling h 3, 6, 9, and 12, ewes receiving 0.8 mg swainsonine/kg BW had higher (P < 0.05) serum swainsonine concentrations than the 0.2 mg treated ewes. Serum swainsonine in ewes dosed with 0.2 mg swainsonine/kg BW remained fairly constant with minimal (P > 0.40) fluctuation from initial hour of detection (h 3) to h 24. Maximal (P < 0.001) concentrations of serum swainsonine in the 0.8 mg treated ewes were observed at h 3 and 6 (h 3 and 6 > h 9 to 24; P < 0.05); following h 6, serum swainsonine rapidly declined (h 6 > h9; P < 0.05) as indicated by the quadratic (P < 0.001) disposition of swainsonine over time. The near maximal concentrations of swainsonine occurring at h 3 indicated that much of the swainsonine absorption from the gut had occurred between dosing and h 3. Although describing the pharmacokinetics of swainsonine in lactating animals is beyond the scope of this study, the subsequent disposition of swainsonine in the 0.8 mg treatment group over the sampling period appears to be consistent with the results of Stegelmeier et al. (1995a), in which the half-life of swainsonine was suggested to be 20.3 ± 7.2 h. However, the lack of change in serum swainsonine over time in the ewes treated with 0.2 mg swainsonine/kg BW does not appear to support a T1/2 estimate of less than 24 h. Furthermore, no difference (P = 0.47) was detected between the 0.2 and 0.8 mg treatment groups at h 24. Taylor et al. (2000) reported that wethers consuming 0.8 or 1.6 mg swainsonine/kg BW had higher levels of serum swainsonine 24-h post locoweed exposure than wethers exposed to 0.2 and 0.4 mg swainsonine/kg BW. This apparent contrast in the disposition of serum swainsonine between the current experiment and Taylor et al. (2000) could be due to the effect of lactation on the movement of swainsonine away from the serum.
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Figure 1. Disposition of swainsonine (SW) in serum of lactating ewes following single dose exposure (gavage) to a locoweed extract (Oxytropis sericea; 5.85 mg SW/mL) at h 0. **Indicates difference (P < 0.05) between treatments within hour. xyzIndicates difference (P < 0.05) between sampling hours within treatment. Quadratic fit and solution were generated using individual responses of experimental units within treatment over time.
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Figure 2. Disposition of swainsonine (SW) in milk of lactating ewes following single dose exposure (gavage) to a locoweed extract (Oxytropis sericea; 5.85 mg SW/mL) at h0. **Indicates difference (P < 0.05) between sampling hours within treatment. Linear fit and solution were generated using individual responses of experimental units within treatments over time.
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No differences (P > 0.94) in estimated 24-h milk yield were detected between the 0 (control), 0.2, or 0.8 mg swainsonine/kg BW treated ewes (1.49, 1.56, and 1.58 ± 0.20 L, respectively). The 24-h mean swainsonine concentration of the milk was lower (P < 0.003) in the 0.2 than the 0.8 mg treated ewes (0.134 and 0.222 ± 0.015 µg/mL, respectively). The appearance and disappearance of swainsonine in the milk of the treated ewes was visually similar to the serum (Figure 2
). The 0.8 mg swainsonine/kg BW treated ewes had higher (P < 0.05) levels of swainsonine in milk at h 3, 6, and 9 than did the lower 0.2 mg treated ewes. Swainsonine in the milk of the 0.8 mg group throughout the first 24 h declined linearly (P < 0.002) with maximal swainsonine concentration occurring from h 3 to 6 (h 6 > h 9 to 24; P < 0.05). The rapid appearance of swainsonine in the milk of both treated groups establishes that the mammary system in lactating ewes is a route readily available and utilized for the elimination of swainsonine and, therefore, could have a major effect on T1/2 of swainsonine compared to nonlactating sheep.
A treatment by sampling hour interaction was detected (P = 0.01) for serum alkaline phosphatase in the ewes. Increased activity (P < 0.01) in serum alkaline phosphatase (Figure 3) was observed in ewes receiving 0.2 mg swainsonine/kg BW at h 24 and at h 6, 12, and 24 for those receiving 0.8 mg. Ewes dosed with 0.8 mg swainsonine had greater (P < 0.03) alkaline phosphatase activity than those dosed with 0.2 mg at h 6 and 12, and control ewes at h 6, 12, and 24; this response is similar to the reported alkaline phosphatase activity changes in the serum of wethers (when compared to a control group) 24 h following initial exposure to locoweed (0.8 and 1.6 mg swainsonine/kg BW; Taylor et al., 2000). Alkaline phosphatase activity in the 0.2 mg treated ewes was higher (P = 0.009) than control and not different (P = 0.59) from the 0.8 mg treated ewes by h 24. Alkaline phosphatase activity of the treated ewes promptly returned (P > 0.35) to the activity observed at h 0, and did not differ (P = 0.53) from control by h 48. Taylor et al. (2000) hypothesized that short-term exposure of wethers to 
0.2 mg swainsonine/kg BW was a minimal effective level and where irreversible damage potentially does not occur. This hypothesis was based on the lack of change in some serum constituents, specifically alkaline phosphatase, of wethers daily consuming 0.2 mg swainsonine/kg BW for 28 d. When considering this, the observation that alkaline phosphatase activity at h 24 for 0.2 mg ewes in the current study was increased and higher than control ewes was surprising and unexpected. This immediate response, during acute exposure ( 24 h), has not been observed before in sheep treated with less than 0.8 mg swainsonine/kg BW and, hence, suggests some toxicity of swainsonine at this very low level in lactating ewes. By d 15, Stegelmeier et al. (1999) did observe significant rises in alkaline phosphatase activity in wethers daily treated with locoweed providing 
0.1 mg swainsonine/kg BW. The rapid return of serum alkaline phosphatase activity of both swainsonine treated groups to normal by h 48 suggests no irreversible damage occurred in the lactating ewes. The source of increased alkaline phosphatase activity in swainsonine treated sheep has yet to be fully described. Vacuolization of cells induced by swainsonine inhibition of lysosomal acid -mannosidase does not appear to occur in sheep until 4 d following commencement of daily locoweed consumption (James et al., 1968). As such, the increased in alkaline phosphatase activity observed in the lactating ewes was probably due to other effects of swainsonine, such as alteration of glycoprotein processing by inhibition of golgi mannosidase II.
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Figure 3. Activity of alkaline phosphatase in serum of lactating ewes following single dose exposure (gavage) to a locoweed extract (Oxytropis sericea; 5.85 mg SW/mL) at h 0. **Indicates difference (P < 0.05) from control within hour.  Indicates difference (P < 0.05) from 0.2 mg SW/kg BW treatment within hour. xIndicates difference (P < 0.05) from all other sampling hours within treatment.
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Cows.
A treatment by sampling hour interaction was observed (P < 0.001) for cow serum and milk swainsonine. No swainsonine was detected in the serum or milk of control cows. Swainsonine was detected in the serum (Figure 4) of the cows receiving 0.8 mg swainsonine/kg BW at all sampling hours (except h 48) following treatment and was highest from h 9 to 12 (h 9 and 12 > h 3 and 24; P < 0.05). In comparison to the lactating ewes, appearance of swainsonine was delayed approximately one sampling interval (3 h). Disappearance of serum swainsonine following maximal levels is similar to that of the ewes and seems to support the T1/2 of 16.4 ± 3.1 h in nonlactating cows estimated by Stegelmeier et al. (1995a). The cubic response (instead of quadratic as observed with the ewes) obtained in the cows is likely due to the potential capturing of the absorption phase occurring sometime between h 3 and 6. Serum swainsonine was only detected at h 6, 9, and 12 in cows dosed with 0.2 mg swainsonine/kg BW, and as with the lactating ewes, no difference (P > 0.41) occurred between sampling hours. Additionally, serum swainsonine in the 0.2 mg treated group was lower (P < 0.05) than the values observed in the 0.8 mg treated group at h 6, 9, and 12. The lack of change in the lower treated ewes and cows suggests that the level of swainsonine intake could affect the T1/2 of swainsonine in the serum.
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Figure 4. Disposition of swainsonine (SW) in serum of lactating cows following single dose exposure (gavage) to a locoweed extract (Oxytropis sericea; 9.13 mg SW/mL) at h 0. **Indicates different (P < 0.05) between treatments within hour. xyzIndicates difference (P < 0.05) between sampling hours within treatment. Cubic fit and solution were generated using individual responses of experimental units within treatments over time.
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No differences (P > 0.52) in the estimated 24-h milk yield were detected between 0 (control), 0.2, and 0.8 mg swainsonine/kg BW treated cows (9.06, 9.26, and 7.02 ± 1.98 L, respectively). The 24-h mean swainsonine concentration of the milk was lower (P < 0.003) in the 0.2 than the 0.8 mg treatments (0.103 and 0.227 ± 0.021 µg/mL, respectively). In both treated groups, swainsonine was detected by h 3 in the milk (Figure 5). Peak milk swainsonine concentrations were obtained between h 9 and 12 (h 9 and 12 > h 3, 6, 24; P < 0.05) in cows treated with 0.8 mg swainsonine/kg BW and by h 12 (h 12 > h 3 to 9; P < 0.05) in those treated with 0.2 mg. For the first 24 h, the behavior of swainsonine over the sampling period was cubic (for comparison to the serum, h 48 was not included in regression estimate). Swainsonine was detected in both treated groups at h 48; this effect was expected due to the number of cows in the preliminary experiment that had detectable swainsonine in the milk at h 36 (Table 1). As with the lactating ewes, the similarity in the appearance and disappearance of swainsonine in the serum and milk suggest that swainsonine rapidly moves to the mammary system and the mammary gland serves as a major elimination route.
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Figure 5. Disposition of swainsonine (SW) in milk of lactating cows following single dose exposure (gavage) to a locoweed extract (Oxytropis sericea; 9.13 mg SW/mL) at h 0. **Indicates difference (P < 0.05) between treatments within hour. wxyz/uvIndicates difference (P < 0.05) between sampling hours within treatment. Quadratic fit and solution were generated using individual responses of experimental units within treatment over time.
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In contrast to the ewes, serum alkaline phosphatase activities of the cows were not affected (P = 0.53; Table 2) by swainsonine treatment. No data are available describing the acute response of serum alkaline phosphatase activity in the serum of cattle exposed to swainsonine. The choice to use alkaline phosphatase in the cow study was based on the acute response of this serum marker in sheep consuming locoweed (Taylor et al., 2000) and elevated alkaline phosphatase levels observed in cattle by d 7 after commencement of daily locoweed consumption (Bachman et al., 1992).
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Table 2. Serum alkaline phosphatase activitya of ewes and cows and their nursing young (lambs and calves), following a single dose exposure (gavage) of the ewes and cows to 0 (control), 0.2, or 0.8 mg swainsonine/kg BW provided by a locoweed extract (Oxytropis sericea)
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Lambs and Calves.
Body weight did not differ (P > 0.67) between the 0 (control), 0.2, or 0.8 mg swainsonine/kg BW treatment groups for the nursing lambs (14.03, 13.46, and 13.68 ± 1.26 kg, respectively) or calves (101.07, 100.50, and 91.48 ± 7.96 kg, respectively). No swainsonine (P > 0.05) were detected in the serum of lambs or calves nursing mothers in the control or treated groups. For the treated groups, this was unexpected due to the isolation of swainsonine in the milk of both the treated ewes and cows. James and Hartley (1977) observed onset subclinical toxicosis (elevated serum aspartate aminotransferase activity) by 7 d in lambs daily ingesting milk from either cows (via bottle) or ewes consuming 454 and 227 g of locoweed per day, respectively. Moreover, calves nursing the same cows daily also developed subclinical symptoms of toxicity by d 7. As mention earlier, the lower detection limit of the swainsonine assay used for this study is 0.025 µg/mL. Although it is obvious that the nursing young did ingest swainsonine by consuming milk (Figures 2 and 5) from their mothers, further dilution of the swainsonine by the blood and tissues of the lamb or calf coupled with the repeated exposure over the 24-h period apparently reduced swainsonine concentrations to less than the 0.025 µg/mL detection limit. In support of this, the estimated 24-h swainsonine intake by the nursing young of the 0.2 and 0.8 mg swainsonine/kg BW treatments was 0.016 and 0.026 ± 0.002 mg/kg BW for the lambs and 0.009 and 0.017 ± 0.002 mg/kg BW for the calves, respectively. This estimated intake by the nursing young is 12- to 48-fold lower than consumed by the corresponding mothers. Furthermore, the lambs and calves exposure to swainsonine via the milk occurred multiple times over a 24-h period unlike the single dose administered to the lactating ewes and cows.
In addition to the lack of swainsonine being detected, no changes in alkaline phosphatase activities (P > 0.05) were detected in the serum of lambs or calves nursing mothers in the treated or control groups (Table 2). The equivalent locoweed (based on 0.614 mg swainsonine/g plant matter) intake for the lactating animals treated with 0.8 mg swainsonine/kg BW was approximately 98.6 ± 11.4 and 583.1 ± 36.7 g for the ewes and cows, respectively. When compared to the study of James and Hartley (1977), the ewes and cows in the current study consumed an estimated 56% less and 22% more locoweed, respectively. Due to the high variability in swainsonine content of locoweeds (0.15 to 1.2 mg/g plant matter), caution must be exercised in assuming that lower intakes (compared to James and Hartley, 1977) were a reason for the lack of effect on alkaline phosphatase or no swainsonine being identified in the lambs. The lack of noticeable rises in serum alkaline phosphatase activities in the calves and lambs nursing treated mothers would suggest that either no significant levels of swainsonine were transferred to the nursing young to induce measurable subclinical or clinical symptoms of acute swainsonine toxicity or, as with the cows, serum alkaline phosphatase may not be an effective marker for subclinical swainsonine toxicity in young animals. Perhaps other markers, such as serum -mannosidase (Stegelmeier et al., 1995a, 1999) or serum iron (Taylor et al., 2000), could prove to be better markers of acute swainsonine exposure in young nursing ruminants.
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Implications
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Swainsonine appeared in the serum and milk of lactating ewes and cows in a dose-dependent fashion following a single dose oral exposure; thus, confirming the mammary system to be a route of swainsonine elimination and source of swainsonine exposure to the nursing young. However, detectable levels of swainsonine and(or) subclinical toxicity were not observed in the serum of nursing lambs or calves. Therefore, in order to transfer a sufficient amount of swainsonine to lambs and calves via the milk to subsequently induce detectable levels of swainsonine (>0.025 µg/mL) and(or) subclinical toxicity in the serum, a single oral dose of swainsonine (locoweed extract) greater than 0.8 mg/kg BW to the lactating mothers must occur. Based on this and results of others, the greater risk of swainsonine toxicity seems to be when nursing ruminants repeatedly (daily; subacute exposure) select a diet containing locoweed in addition to ingesting milk contaminated with swainsonine.
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Footnotes
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1 Portions of these data were presented at and published in the Proceedings of the Western Section of American Society of Animal Science, Bozeman, MT, 2001; 52:170–173. 
2 Research was funded by the Agriculture Experiment Station, New Mexico State University, Las Cruces, NM.
3 Current address: USDA, ARS, HC 62 Box 2010, Dubois, ID 83423 (E-mail: joshtay1{at}dcdi.net).
Received for publication February 14, 2002.
Accepted for publication April 26, 2002.
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Abstract
Introduction
