Sex-dependent differences for larkspur ( barbeyi) toxicosis in yearling Angus cattle1

Benedict T. Green†,2, John W. Keele‡, Dale R. Gardner†, Kevin D. Welch†, Gary L. Bennett‡, , Daniel Cook†, James A. Pfister†, T. Zane Davis†, Clint A. Stonecipher†, Stephen T. Lee†, and Bryan L. Stegelmeier†

†USDA-ARS, Poisonous Research Laboratory, Logan, UT 84341; and ‡USDA-ARS, U.S. Meat Animal Research Center, Clay Center, NE 68933 Downloaded from https://academic.oup.com/jas/article/97/3/1424/5321883 by guest on 29 September 2021 ABSTRACT: Larkspur (Delphinium spp.) poi- data. The statistical methods (censReg and lmec) soning is a long-term problem for cattle grazing were used to adjust for data truncation or cen- on rangelands of western North America. Results soring. The heifers were only able to walk −8.9 ± from preliminary experiments have suggested that 3.9 min (65.5% censored on the left) compared differences in larkspur toxicity may exist between with 13.2 ± 3.7 min for bulls and 15.9 ± 2.7 min heifers and bulls. The objective of this study was for steers. When heifers were compared with bulls to compare the physiological responses of year- and steers together, heifers walked 23.4 ± 4.5 min ling Angus heifers, steers, and bulls with a stand- less (P < 0.0001). Serum alkaloid concentrations ardized dose of Delphinium barbeyi and to test were measured immediately before walking, and the hypothesis that the response is sex dependent. deltaline concentrations averaged 266 ± 28, 131 ± Clinical signs of intoxication, including muscle 20, and 219 ± 28 ng mL−1 for all heifers, steers, and coordination and function, were measured 24 h bulls, respectively, and serum methyllycaconitine after oral dosing with larkspur by walking the concentrations averaged 660 ± 46, 397 ± 32, and cattle at a pace of 5 to 6 km h−1 for up to 40 min 612 ± 34 ng mL−1 for all heifers, steers, and bulls, on an oval dirt track. Due to the experimental respectively. The relative risk of a zero walk time methods used, the variation in susceptibility to for yearling heifers is 330% that of yearling bulls larkspur was not quantifiable for walking times (P = 0.0008). These results suggest that yearling of 0 or 40 min or more. Larkspur susceptible ani- Angus heifers are more susceptible to larkspur mals that were not able to walk (0 min; 36% of intoxication and, when possible, heifers should be the animals) or larkspur resistant animals that kept from grazing larkspur-infested rangelands as walked the entire test period of 40 min (9% of a simple management tool to reduce the risk of the animals) resulted in censored or truncated fatal poisoning. Key words: Angus, Delphinium, methyllycaconitine, N-(methylsuccinimido) anthranoyllycocto- nine, sex dependent, toxicokinetic

Published by Oxford University Press on behalf of the American Society of Animal Science 2019. This work is written by (a) US Government employee(s) and is in the public domain in the US. J. Anim. Sci. 2019.97:1424–1432 doi: 10.1093/jas/skz002

INTRODUCTION Larkspurs (Delphinium spp.) have been a long- 1This research was supported by United States Depart- term problem for cattle grazing on rangelands ment of Agriculture, Agricultural Research Service. of western North America (Marsh et al., 1916). 2Corresponding author: ben.green @ars.usda.gov Annual livestock losses on larkspur-infested Received August 30, 2018. rangelands may vary greatly from 2% to 5% up Accepted January 2, 2019. to 15% (Pfister et al., 1999). A simple, practical 1424 Sex-dependent larkspur toxicity in cattle 1425 solution for larkspur poisoning is needed by west- Agricultural Animals in Research and Teaching, third ern U.S. cattle ranching operations. In an effort to edition, 2010, standards were followed. provide solutions for larkspur poisoning of cat- tle, a research program was developed to identify Plant Material the genetic basis for larkspur tolerance in cattle. Initial studies focused on steers, but more recently, Delphinium barbeyi in the early flowering stage the emphasis has shifted to intact bulls and heifers was collected during July, 2003 near Manti, UH to support selection of susceptible and resistant (N lat 39° 03.154, W long 111° 30.752′, collections parents to create lines of susceptible and resistant number 03-12) at an elevation of ~3,000 m above Angus cattle which resulted in the discovery of sea level. A voucher specimen was deposited at the sex-dependent effects. Larkspur and their State University, Intermountain Herbarium toxicity have been intensively studied in cattle, goat, (#237494). The plant material was processed and lizard, and rodent models, but none of the studies stored as previously described (Green et al., 2009a); have reported sex-dependent responses (Nation the composition of the dried plant ma- Downloaded from https://academic.oup.com/jas/article/97/3/1424/5321883 by guest on 29 September 2021 et al., 1982; Manners et al., 1991, 1995; Welch et al., terial is stable. The long-term storage of dried 2008, 2009, 2010, 2012, 2013, 2017; Cook et al., ground larkspur plant material does not modify al- 2011). Grazing studies have shown that naïve and kaloid compositions (Cook et al., 2009). young animals generally begin consuming larkspur before older, more experienced animals; however, no Animals studies have investigated sex-dependent differences (Pfister and Gardner 1999; Pfister et al., 2011). The Yearling Angus steer (n = 60; 366 ± 15 kg) observations of heifer susceptibility from the selec- responses to larkspur were tested during 2009, tion experiment have led to the hypothesis that there 2010, 2014, and 2016; some of the steer responses are sex-dependent differences in the responses of to larkspur have previously been published in Green yearling Angus cattle to larkspur poisoning. An et al. (2014, 2018). Yearling Angus bull (n = 33; incomplete block design consisting of steers, heif- 315 ± 6 kg) responses to larkspur were tested dur- ers, and bulls tested over multiple years was used ing 2016 and 2018, and yearling Angus heifers were to compare the responses of the three groups to tested during 2017 and 2018 (n = 30; 280 ± 11 kg). an 8 mg kg−1 BW dose of N-(methylsuccinimido) Experiment 1 (Figure 1) consisted of all animals anthranoyllycoctonine type (MSAL-type) alkaloids. tested before 2018. The 15 Angus bull calves and 15 Angus heifer calves used in Exp. 2 were born during the spring of 2017 and were obtained from the U.S. MATERIALS AND METHODS Meat Animal Research Center in Clay Center, NE. The experiments were performed at the A timeline of this work is presented in Figure 1. For Poisonous Plant Research Laboratory, in Logan, Exp. 2, the bulls and heifers were kept in the same Utah. All animal work was done under veterinary feedlot, fed the same diet, trained to walk using a supervision with the approval of the Utah State lead rope at the same time, and then tested for their University Institutional Animal Care and Use responses to larkspur during the spring of 2018. Committee, and the Guide for the Care and Use of One bull was lost to endotoxemia and was replaced

Figure 1. A timeline depicting the progression of research from Exp. 1, and Exp. 2 with Angus cattle. The Angus steer walk times from 2009, 2010, and 2014 were first reported in Green et al. (2014). The steer walk times from 2016 were reported in Green et al. (2018). These walk times were used in fixed and mixed model analyses as described in the Material and Methods section and presented in Table 1. 1426 Green et al. with a yearling Angus bull raised at our labora- their pen. This process continued until all animals tory to keep the group sizes equal. All cattle were exhibited clinical signs or they walked for 40 min. maintained on alfalfa hay with free choice mineral supplement (iOFIX T-M salt block, Morton Salt, Serum Alkaloid Analyses Chicago, IL). Approximately 7 mL of blood was collected by Dosing venipuncture from the heifers just before oral dos- ing and then 24 h after dosing, just before walking. Feed was withheld overnight before each The blood was allowed to clot at room tempera- animal was dosed with larkspur. For Exp. 1, the ture and the serum was separated from the clot cattle dosed as previously described (Green et al., by centrifugation at 2,000 × g for 20 min at 4 °C. 2014). For Exp. 2, at ~0700 hours. Three bulls and The serum was then stored frozen at −80 °C until three heifers were weighed, restrained in a squeeze chemical analysis. Chemical analysis of the serum chute, and orally gavaged with a standard alkaloid for norditerpenoid alkaloids found in larkspur Downloaded from https://academic.oup.com/jas/article/97/3/1424/5321883 by guest on 29 September 2021 dose of 8 mg kg−1 BW MSAL-type alkaloids in included: methyllycaconitine, and deltaline as pre- the form of dry, finely ground larkspur in ~7 to viously described and was performed at the same 10 L of tap water (Green et al., 2014). This dose time in the same laboratory (Welch et al., 2017). normally only elicits clinical signs of intoxication (muscle weakness and reluctance to walk) after Data Analysis moderately resistant cattle have been exercised (Green et al., 2014). Walking times and serum alkaloid concen- trations are expressed as the mean ± SE, and the Exercise Protocol threshold for significance was set atP < 0.05. For the analysis of serum alkaloid concentrations, a one- All of the cattle in this study were obtained way ANOVA with Tukey’s multiple comparisons as weanlings and were halter broke and trained to test in Prism version 6.03 (GraphPad Software, San walk using a lead rope as they matured to yearlings. Diego, CA) was used. For the analysis of weight This exercise protocol is similar to that of Cook differences between heifers and bulls, a Mann– et al. (2011) and Green et al. (2014). Twenty-four Whitney test of untransformed data in Prism was hours after oral dosing, groups of haltered year- used because the heifer weights were not normally lings (three bulls and three heifers) were tethered distributed. A comparison of the walking times be- with a lead rope to a fence rail before exercise. Some tween heifers and bulls for Exp. 2 was done with an cattle showed severe clinical signs of intoxication at unpaired t-test in Prism after a square root trans- this time such that they were not able to walk be- form of the data to equalize the variances caused hind the tractor. Therefore, they were given a zero by censoring of the walk times at 0 and 40 min. walk time, their halters removed and then released back into their original pen. The cattle not showing Mixed and Fixed Model Analyses severe clinical signs were then transferred to a spe- cially devised 2.5 m tow bar secured with a trailer Due to pen space limitations, all of the larkspur tie to their halter (the quick release panic snap was responses of the heifers, steers, and bulls were not attached to the halter and the trigger snap to the measured during the same year. The walk times of tow bar) and then walked behind a tractor at 5 to 6 the cattle from Exp. 1 and Exp. 2 were therefore km h−1 (3 to 4 mph) on a fenced dirt track until they analyzed as an incomplete block design using the exhibited larkspur-induced muscle weakness, or for R packages (R Core Team, 2013), lmec (mixed), a maximum of 40 min. Animals with a walk time of and censReg (fixed) were used to allow compari- 40 min were classified as resistant for the purposes son among bulls, heifers, and steers and adjusting of this study. If an animal exhibited larkspur-in- for censoring of animals with 0 and 40 min walk duced muscle weakness, the tractor was stopped, times (Tobin, 1958; Vaida et al., 2007; Greene, the time to clinical signs was recorded, and the 2008; Kleiber and Zeileis, 2008; Vaida and Liu, animal was quickly released using the panic snap 2009). Mixed model analysis with censoring (lmec) (elapsed time, ≤30 s), after which the exercise was was used to account for year of the observation continued for the remaining yearlings. Any yearling as a random nuisance factor. The use of censored which exhibited clinical signs was allowed to rest regression models like censReg allowed for the until the animal recovered sufficiently to return to accounting of censoring at 0 and 40 min and an Sex-dependent larkspur toxicity in cattle 1427 unbiased estimation of sex differences. Similar methods have been used to assess risk in drug tox- icity studies and human immunodeficiency virus studies (Matos et al., 2015; Berges et al., 2018).

Risk Analysis

Relative risk was quantified as the occurrence of a risk variable after exposure compared with the likelihood of its occurrence in a control group (Andrade, 2015), and was analyzed using the MedCalc Software online (MedCalc Statistical Software version 16.4.3 (MedCalc Software bvba,

Ostend, Belgium; https://www.medcalc.org)). For Downloaded from https://academic.oup.com/jas/article/97/3/1424/5321883 by guest on 29 September 2021 this analysis, the number of animals with a zero walk time was the bad outcome, and the number of animals with a >0 walk time was the good out- come. The heifers were considered the exposed group and the bulls the control group (https:// www.medcalc.org/calc/relative_risk.php, accessed 8/19/2018).

RESULTS AND DISCUSSION

Experiment 1

An ongoing experiment (Exp. 1) at this labora- tory to identify factors that affect larkspur sus- ceptibility in cattle has documented differences in susceptibility for cattle breed and age (Green et al. Figure 2. Walk times (a) and serum alkaloid concentrations (b) in 15 heifers and 15 bulls tested during Exp. 2. (a) A scatter plot 2014, 2018). When bulls and steers were tested dur- with the mean ± SE walk times 24 h after receiving a dose of dried ing 2016, there were no differences in walk times be- ground Delphinium barbeyi (8 mg kg−1 BW N-(methylsuccinimido) tween the 2 groups. The first observed Angus heifer anthranoyllycoctonine type alkaloids). To determine the walk times, responses to larkspur occurred in 2017 (Figure 1), the cattle were walked behind a tractor at 5 to 6 kph on a dirt track until they exhibited larkspur-induced exercise fatigue or for 40 min. the 15 heifers tested during that year walked 0.7 ± Walking times were compared with an unpaired t-test. (b) The serum 0.3 min. These observations led to the Exp. 2 with concentrations of deltaline and methyllycaconitine (ng mL−1) in year- age-matched heifers and bulls obtained from the ling cattle at 24 h after oral dosing and obtained just before the cattle being exercised. Serum alkaloid concentrations were compared using a U.S. Meat Animal Research Center. one-way ANOVA with Tukey’s multiple comparisons test. Heifer and bull serum methyllycaconitine, and deltaline concentrations were not Experiment 2 different.

In Exp. 2 with Angus heifers and bulls, the heif- Exp. 2: Serum Alkaloid Analysis ers were more severely affected, and most could not be walked, ~70% of the heifers had a zero walk time Serum samples were obtained at 24 h after (Figure 2a). Those that could walk had limited en- oral dosing from the heifers and bulls in Exp. 2 durance resulting in shorter walk times. Unadjusted and serum alkaloid concentrations were not dif- for censoring, the heifers in Exp. 2 walked an average ferent (Figure 2b). Serum deltaline concentrations of 4.1 ± 2.1 min, and the bulls walked an average of at 24 h post-dosing averaged 276 ± 45 and 291 ± 11.7 ± 3.3 min (Figure 2a). These walk times were 47 ng mL−1, for heifers and bulls, respectively, and different by t-test (P = 0.032). However, this analysis serum methyllycaconitine concentrations averaged does not take into account data censoring which is 799 ± 62, and 674 ± 54 ng mL−1, for heifers and why mixed and fixed model analyses accounting for bulls, respectively. However, interpretation of the censoring (lmec, censReg) were used and the results serum alkaloid concentrations must be carefully of these analyses are described below. considered due to the relatively small sample size 1428 Green et al. of Exp. 2, and could change if the number of ani- mals increased. When the methyllycaconitine and deltaline concentrations are compared within each sex, for Exp. 2, they were different (P < 0.01, heifer deltaline vs. heifer methyllycaconitine; P < 0.01, bull deltaline vs. bull methyllycaconitine). This was expected, previous work at this laboratory has reported similar observations from serum toxicoki- netics research. Specifically,Green et al. (2009b) have shown that the half-life of deltaline is shorter than methyllycaconitine and is eliminated from the body more quickly resulting in lower serum deltaline concentrations at 24 h after dosing (elimi- nation half-life; deltaline, 8 ± 1 h vs. methyllycaco- Downloaded from https://academic.oup.com/jas/article/97/3/1424/5321883 by guest on 29 September 2021 nitine, 21 ± 4 h, P < 0.05, N = 5 Angus steers). We were particularly interested in serum alkaloid concentrations in the heifers and bulls, because recent research has shown that Angus steers eliminate larkspur alkaloids from their serum more effectively as they age (Green et al., 2018). Specifically, for 2-yr old Angus steers, there was a 99% decrease in serum deltaline concentrations and a 75% decrease in serum methyllycaconitine concentrations at 24 h post-dosing compared to when they were yearlings. These changes in serum alkaloid concentrations were associated with walk- ing time changes from 16.0 ± 5.3 min as yearlings to 40 min as 2-yr olds. The lack of similar differ- ences in serum alkaloid concentrations in heifers 24 h after dosing compared to bulls suggests that toxicokinetic differences between the sexes did not Figure 3. Walk times (a) and serum alkaloid concentrations (b) in heifers, steers, and bulls sampled in Exp. 1 and Exp. 2 combined. (a) play a role in the reduced walk times of the heifers. A scatter plot with the mean ± SE walk times of 30 heifers, 60 steers, and 33 bulls, 24 h after receiving a dose of dried ground Delphinium −1 Mixed and Fixed Model Analyses barbeyi (8 mg kg BW N-(methylsuccinimido) anthranoyllycoctonine type alkaloids). Fixed model analysis with censoring (censReg) was used to compare heifer walk times (n = 30) to the consolidated walk Mixed and fixed model analyses were used to times of the bulls and steers combined (n = 93). (b) The serum concen- analyze the walking times data from cattle tested in trations of deltaline and methyllycaconitine (ng mL−1) in 30 heifers, 23 Exp. 1 and Exp. 2 together. This dataset included a steers, and 33 bulls at 24 h after oral dosing and serum obtained just before the cattle being exercised. Serum alkaloid concentrations were total of 123 Angus cattle. The non-adjusted walk compared using a one-way ANOVA with Tukey’s multiple comparisons times of the three groups were: 2.9 ± 1.2 min (30 test. The serum methyllycaconitine concentrations for both heifers and heifers), 16.6 ± 1.9 min (60 steers), and 15.5 ± bulls were significantly different from the steers P( < 0.01). Heifer and bull serum methyllycaconitine concentrations were not different. 2.4 min (33 bulls), and are displayed in Figure 3a for comparison. Mixed model analysis with censoring (R pack- Due to the experimental methods used cattle age, lmec) was used as an initial analysis (model 1, that were unable to be exercised were given a 0 walk Table 1). The variance in walk times due to year time, and resistant cattle were stopped from walk- was small, so we adjusted for both left and right ing after 40 min. However, not all animals which censoring with a fixed model (R package, cen- walked 0 min were the same. For example, some sReg) including only sex (heifers, bulls, and steers) animals exhibited clinical signs of intoxication in in the model (models 2 and 3, Table 1). The walk their pen at 24 h after dosing and were not dis- times between bulls and steers were not different turbed. Others walked to an area behind a squeeze in any of the three models used to compare walk chute where they exhibited severe clinical signs, and times so bulls and steers were combined for mod- some exhibited clinical signs while being haltered or els 2 and 3. led to the dirt track for exercise. All of these cattle Sex-dependent larkspur toxicity in cattle 1429

Table 1. Mixed and fixed model analysis of Angus cattle walk times from Exp. 1 and Exp. 2 combined, presented as adjusted walk times

Heifer walk Bull walk Steer walk Estimated differences P-value Estimated P-value bulls times1 (min) times1 (min) times1 (min) between heifers vs. heifers vs. bulls differences between bulls vs. steers Analysis n = 30 n = 33 n = 60 bulls and steers (min) and steers vs. steers (min) walk times Model 1 −7.26 ± 3.23 13.03 ± 3.35 14.89 ± 2.48 −21.22 ± 3.83 1.58 × 10−7 −1.86 ± 4.07 0.65 Model 2 −7.16 ± 3.35 12.77 ± 3.19 14.94 ± 2.35 −21.02 ± 3.83 1.90 × 10−7 −2.17 ± 3.95 0.58 Model 3 −8.89 ± 3.90 13.18 ± 3.68 15.93 ± 2.72 −23.44 ± 4.47 5.94 × 10−7 −2.75 ± 4.57 0.55

1The variation in walk times was not quantifiable for times of 0 or 40 min resulting in censored data. Censoring of data for extreme animals reduces variation, and adjusting for this censoring (censReg and lmec) increased variation causing negative mean walking times for the heifers. Model 1 left censored at zero; year fit as random, analyzed using lmec. Model 2 left censored at zero; year was not fit, analyzed using censReg. Model 3 left censored at zero, right censored at 40; year was not fit, analyzed using censReg. Downloaded from https://academic.oup.com/jas/article/97/3/1424/5321883 by guest on 29 September 2021 were classified as susceptible and scored 0 min for were no significant differences in serum alkaloid their walk time resulting in censored or truncated concentrations between the heifers and bulls. This data. Adjusting for this censoring increased the var- experiment was the first to orally dose large num- iation among animals as well as the means of the bers of heifers, steers, and bulls with dried ground groups, causing negative estimates of mean walking larkspur and measure their serum alkaloid concen- times for the heifers which were quantified in min- trations at 24 h after dosing. Previous work at this utes similar to the measured walking time (Table 1). laboratory has documented differences in methyl- Using these statistical methods, heifers were only lycaconitine elimination half-life values between able to walk −8.89 ± 3.90 min compared with Holstein steers (11.8 h, Green et al., 2013b) and 13.18 ± 3.68 min for bulls and 15.93 ± 2.72 min for Angus steers (15.6 h, Green et al., 2012). We have steers. Heifers walked 23.44 ± 4.47 min less than the no explanation as to why the serum alkaloid con- average for bulls and steers (P = 5.94 × 10–7), while centrations differ in Angus steers compared with walking times for bulls and steers did not differ Angus bulls and Angus heifers. (P = 0.55). Based upon these analyses, the negative walk times of the heifers indicated that they were Serum Alkaloid Concentrations and Sex very susceptible to larkspur intoxication. In previous larkspur experiments at this la- There were no differences in serum alkaloid boratory with Angus steers, Holstein steers, Line 1 concentrations between heifers and bulls from ei- Hereford steers, and mixed breed heifers, none of ther Exp. 1 or Exp. 2. There are many well-docu- the cattle exhibited the severe clinical signs of in- mented toxicokinetic differences between males toxication observed in this experiment with these and females in the literature. For example, there is Angus heifers (Green et al., 2009a, 2009b, 2011, sex-dependent expression of genes coding for xeno- 2012, 2013b, 2018). biotic metabolizing and transport enzymes, and dif- ferences in male and female BW, and body fat, all Exp. 1 and Exp. 2: Combined Serum Alkaloid of which affect xenobiotic toxicokinetics (Zanger Concentrations and Schwab, 2013). In this research, there was a dif- ference between the body weights of the heifers and To increase sample sizes for the statistical com- bulls (P = 0.0077, 30 heifers, 33 bulls). However, parison of serum alkaloid concentrations, heifers, this difference does not appear to have affected the bulls, and a subset of the steers from Exp. 1 and toxicokinetics of the larkspur alkaloids between Exp. 2 were analyzed together (Figure 3b). The cu- heifers and bulls because there were no differences mulative serum deltaline concentrations averaged in serum alkaloid concentrations between the sexes 266 ± 28, 131 ± 20, and 219 ± 28 ng mL−1, and (Figure 3b). This suggests that an alternative mech- serum methyllycaconitine concentrations averaged anism is responsible for the sex dependent decrease 660 ± 46, 397 ± 32, and 612 ± 34 ng mL−1 for 30 heif- in walking times and that larkspur alkaloid metab- ers, 23 steers, and 30 bulls, respectively (Figure 3b). olite concentrations in serum are not a factor in Heifers and bulls had larger serum methyllycaco- toxicosis. nitine concentrations than did steers (Figure 3b; One possible explanation for the differences P < 0.01). These combined results from Exp. 1 and other than toxicokinetics could be differential Exp. 2 with larger numbers of cattle suggests there expression of nicotinic acetylcholine receptors 1430 Green et al.

(nAChR), the receptors to which larkspur alkaloids palatability to cattle, plant growth stage, and the bind (Green et al., 2013a). For example, Welch et al. concentration of toxic plant alkaloids without con- (2009) identified nAChR involvement in differen- sideration of cattle sex, breed, age, or other suscep- tial responses to methyllycaconitine with 10 dif- tibility factors (Gardner et al., 2002, 1997; Gardner ferent inbred mouse strains. In that study, median and Pfister 2009; Green et al., 2014). Work by Pfister lethal dose (LD50) values for methyllycaconitine in et al. (1999) has identified a toxic window indicating the 10 strains were determined, with the A/J mouse when cattle should not be grazed on larkspur-in- −1 strain being most susceptible (LD50 = 3.3 mg kg ) fested rangelands (Figure 4). This window was and the 129 mouse strain being most resistant developed primarily based on grazing studies with −1 (LD50 = 5.8 mg kg ). When the protein expression steers, and also with the assumption that all cattle of nAChR subunits was compared, the 129 mouse respond to larkspur in a similar manner. The results strain had twice the amount of α7 nAChR subunit presented in this manuscript suggest that yearling expression and significantly greater (P < 0.05) ex- heifers must be more carefully managed than either

pression of α3 and α5 nAChR subunits than the bulls or steers. However, it is currently not known Downloaded from https://academic.oup.com/jas/article/97/3/1424/5321883 by guest on 29 September 2021 A/J strain. It was concluded that the susceptibility what might be the width (i.e., number of weeks) of of A/J mice to methyllycaconitine may be due to a the “toxic window” for heifers during which they lower expression of nAChR subunits. It is tempting should be removed from larkspur-containing pas- to speculate that there may have been differences tures and when late-season grazing would be safe in nAChR expression in the yearling heifers which to begin. In addition, plant factors such as matu- increased their sensitivity to larkspur alkaloids and rity and toxicity of the larkspur must be consid- that these receptor differences are estrogen depend- ered. Even so, it seems reasonable to assume that ent. Researchers have documented sex-dependent the time frame for the toxic window will expand differences in the vasodilatory effects of nicotine when considering grazing highly susceptible Angus in the kidneys of female rats which was abolished heifers on larkspur-infested rangelands. Clearly, the by the estrogen-receptor antagonist tamoxifen toxic window management recommendation needs and mediated by changes in the expression of α7 further refinement for specific populations of cattle nAChRs (El-Mas et al., 2011). There are also sex-de- such as replacement heifers. Increasing the speci- pendent differences in the expression of β2 nAChR ficity of larkspur management recommendations subunits in humans which may be responsible for using genetic markers is a long-term goal of this sex differences in nicotine and tobacco smoking use research, marker-based screening like that used in (Cosgrove et al., 2012). Further research is needed to determine whether the sex-dependent differences in cattle are due to estrogen-dependent modulation of nAChR expression or whether there is another mechanism behind the differences.

Risk Analysis

In addition to measuring the walk times and serum alkaloid concentrations, the relative risk of a zero walk time was calculated for heifers and bulls. There were 21 zero walk time heifers vs. seven zero walk time bulls (Figure 3a). Heifers relative to bulls were associated with a 3.3-fold increased relative risk of a zero walk time (P = 0.0008, 95% CI 1.6 to 6.6, 30 heifers vs. 33 bulls). This greater relative risk Figure 4. The Delphinium toxicity and palatability relationship in emphasizes the importance of careful management cattle from Pfister et al. (1999) which is used as management guide for needed when grazing heifers on larkspur-infested toxic larkspur-infested pastures. The toxic window is the growth stage of the plant when the concentration of toxic alkaloids is sufficient to rangelands. intoxicate cattle, and larkspur consumption by cattle begins to increase due to increased palatability. Currently, there are low-risk grazing win- dows early in the season before flowering and late in the grazing season Grazing Management after the seed pods shatter. It is not yet known what the toxic window would be for extremely susceptible cattle like yearling Angus heifers, Current management options for grazing lark- but it is reasonable to assume that the time period for the toxic window spur-containing pastures are based on the relative would expand for this highly susceptible population of cattle. Sex-dependent larkspur toxicity in cattle 1431 human personalized medicine (Abrahams, 2008), nicotinic cholinergic receptor/eNOS signaling. Life Sci. would allow for the identification of the right ani- 88:187–193. doi:10.1016/j.lfs.2010.11.009 mal, for the right larkspur-infested pasture, at the Gardner, D. R., G. D. Manners, M. H. Ralphs, and J. A. Pfister. 1997. Quantitative analysis of norditerpe- right time. noid alkaloids in larkspur (Delphinium spp.) by Fourier transform infrared spectroscopy. Phytochem. Anal. 8:55– Summary 62. doi:10.1002/(SICI)1099-1565(199703)8:2<55::AID- PCA338>3.0.CO;2-Q The results from this research suggest that Gardner, D. R., and J. A. Pfister. 2009. HPLC-MS analysis yearling Angus heifers are the most larkspur-sus- of toxic norditerpenoid alkaloids: refinement of tox- icity assessment of low larkspurs (Delphinium spp.). ceptible cattle of any tested to date. There were no Phytochem. Anal. 20:104–113. doi:10.1002/pca.1104 differences in serum alkaloid concentrations be- Gardner, D. R., M. H. Ralphs, D. L. Turner, and S. L. Welsh. tween the heifers and bulls at 24 h after oral dosing 2002. Taxonomic implications of diterpene alkaloids in which suggests that larkspur alkaloid toxicokinet- three toxic tall larkspur species (Delphinium spp.). Biochem. ics are similar between the two groups of animals. Syst. Ecol. 30:77–90. doi:10.1016/S0305-1978(01)00120-X Downloaded from https://academic.oup.com/jas/article/97/3/1424/5321883 by guest on 29 September 2021 This research indicates that Angus heifers should Green, B. T., D. R. Gardner, D. Cook, J. A. Pfister, K. D. Welch, and J. W. Keele. 2018. Age-dependent intoxication by be kept from larkspur-containing rangelands dur- larkspur (Delphinium) in Angus steers. Toxicon 152:57–59. ing the toxic window as a simple management tool doi:10.1016/j.toxicon.2018.07.020 to reduce the risk of larkspur poisonings. Green, B. T., J. A. Pfister, D. Cook, K. D. Welch, B. L. Stegelmeier, S. T. Lee, D. R. Gardner, E. L. Knoppel, ACKNOWLEDGMENT and K. E. Panter. 2009a. Effects of larkspur (Delphinium barbeyi) on heart rate and electrically evoked electromyo- The authors wish to thank Isabelle McCollum, graphic response of the external anal sphincter in cattle. Rex Probst, Jessie Roper, Kermit Price, Ed Knoppel, Am. J. Vet. Res. 70:539–546. doi:10.2460/ajvr.70.4.539 Chuck Hailes, and Scott Larsen for their expert Green, B. T., K. D. Welch, D. R. Gardner, B. L. Stegelmeier, T. Z. Davis, D. Cook, S. T. Lee, J. A. Pfister, and technical support of this research. K. E. Panter. 2009b. Serum elimination profiles of methyl- Conflict of interest statement. None declared. lycaconitine and deltaline in cattle following oral adminis- tration of larkspur (Delphinium barbeyi). Am. J. Vet. Res. 70:926–931. doi:10.2460/ajvr.70.7.926 LITERATURE CITED Green, B. T., K. D. Welch, D. R. Gardner, B. L. Stegelmeier, Abrahams, E. 2008. Right drug-right patient-right time: per- and S. T. Lee. 2013b. A toxicokinetic comparison of two sonalized medicine coalition. Clin. Transl. Sci. 1:11–12. species of low larkspur (Delphinium spp.) in cattle. Res. doi:10.1111/j.1752-8062.2008.00003.x Vet. Sci. 95:612–615. doi:10.1016/j.rvsc.2013.04.018 Andrade, C. 2015. Understanding relative risk, odds ratio, and Green, B. T., K. D. Welch, D. 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