3.1. Alkaloid Profiles
Similar to previous studies of cereal ergot [
4,
11], ergocristine was the most prevalent
R, and ergocristinine the most prevalent
S epimer across all diets. Ergometrine, an alkaloid commonly used to stimulate uterine contractions in humans [
12], was the second-most predominant alkaloid, with ergotamine third or fourth in prevalence. Alkaloid profiles in the present study differed from those of Coufal-Majewski et al. [
8], as different sources of ergot-contaminated screenings were used in each study. In our previous study, ergocristine accounted for > 50% of
R epimers, and was approximately 3 times greater in concentration than the next-most concentrated alkaloid, ergocryptine. In the present study, ergocristine was approximately equal in concentration to ergometrine for 930 and 1402 ppb diets and was 1.8 times the concentration of ergometrine in the 2447 ppb diet. Consequently, 1402 and 2447 ppb diets in the present study were the only diets to have concentrations of ergocristine exceeding that previously evaluated by Coufal-Majewski et al. [
8]. Potentially, the concentration of ergocristine in the 930 ppb diet was insufficient to produce negative impacts, even though total
R epimers in the 930 ppb diet were 1.6 times that evaluated in the previous study.
The importance of relative concentrations of individual alkaloids as compared to absolute concentration of total alkaloids is poorly understood. Above a certain threshold, the liver can no longer detoxify alkaloids, leading to clinical symptoms such as tissue necrosis or death [
13], although both concentration of total alkaloids and their proportion to each other will influence the negative effects observed [
14]. Ergot alkaloids are thought to have additive effects [
15], but Dänicke and Diers [
14] found evidence of interactions among individual alkaloids in studies of liver function in piglets, where a diet with a lower total concentration had enhanced toxicity compared to a diet with a higher total alkaloid concentration. The reverse may also possibly be true, where certain relatively abundant alkaloids may modulate overall impacts by blocking metabolism of other, potentially more toxic alkaloids.
Ergocristine, ergocornine, ergocryptine and ergotamine all cause blood vessels to contract, with ergotamine eliciting the most potent response in terms of intensity and duration [
16]. Approximately 10 molecules of ergocristine or ergocornine would equal the vasoconstrictive potential of ergotamine [
17]. Ergotamine and ergocryptine exhibit similar binding properties to D2-dopamine receptors in cell culture, with their affinity being greater than ergometrine [
18]. Ergoamide derivatives such as ergometrine mainly promote psychotropic effects, while vasomotor effects and the reduction in serum prolactin concentration are less pronounced [
15]. In the present study, as ergocristine was approximately 2.5 times the concentration of ergotamine or ergocryptine; it is likely that ergocristine was responsible for the majority of the reduced growth performance. However, due to the complexity of evaluating interactions among ergot alkaloids, Dänicke [
1] suggested reporting animal responses to alkaloids solely on the basis of total dietary ergot alkaloids. Alternatively, Craig et al. [
17] has suggested conversion of alkaloids to “ergotamine equivalents” based on the vascular potency of individual alkaloids, although ergotamine equivalents for ergometrine and ergosine would need to be determined before this technique could be applied to cereal ergot.
3.2. Alkaloids and Their Epimers
Ergot alkaloids are relatively stable; however, epimerization can be induced with high or low pH, different solvents, and upon exposure to strong light [
19]. Consequently, storage conditions (best below −20 °C), handling and analysis need to be consistent to avoid altering alkaloid epimerization [
6]. While ergot alkaloids produce a positive charge in acid solutions and are neutral in alkali [
19], the actual mechanisms leading to conversion between epimeric forms are unknown [
20]. In addition, epimerization can be bi-directional in ergopeptines (i.e., ergometrine, ergotamine, ergosine, ergocristine, ergocryptine and ergocornine), either forming toxic C8-(
R) isomers or more inert C8-(
S) isomers referred to as ergopeptinines [
21], although
S epimers may also produce toxic effects [
22], and the toxicity of
S epimers requires additional investigation. Similar to the study of Mainka et al. [
23], which evaluated contaminated rye, concentration of
S epimers in our study averaged approximately 1/3 that of
R epimers across all diets (
Table 1). However, the ratio of
R/S epimers did vary among alkaloids. For example, the
S epimer only accounted for 1% of ergometrine, suggesting that further conversion of ergometrinine to ergometrine was unlikely. In contrast, if conversion of the
S epimer to the
R epimer was favored for ergocorninine or ergocristinine, the concentration of
R epimers of these alkaloids could increase by an average of 1.7 times. Further investigation of the factors that promote conversions among epimers is necessary to determine if allowable limits should be defined as total alkaloids (
R +
S epimers), or solely as the
R epimers in feed.
3.3. Nutrient Digestibility and Alkaloid Recovery in Feces
Microbial digestion may enhance ruminal ergot alkaloid solubility through the metabolism of ergovaline alkaloids to lysergic acid [
24]. However, it is unknown whether alkaloid solubility is strictly a function of particle size reduction, or a function of conversion from low-soluble alkaloid species to alkaloids with greater solubility. Moyer et al. [
25] found that less than 50% of ergovaline was soluble in in vitro ruminal fermentations, with almost 100% of the soluble components being transformed into an ergot alkaloid derivative within 46 h. Schumann et al. [
26] determined that approximately 30% of cereal ergot alkaloids were metabolized prior to the duodenum in dairy cows, although the toxicity of alkaloid metabolites produced in the rumen and elsewhere in the digestive tract requires additional study.
The current study used a 2-week adaption period between each digestibility collection period to reduce possible carry-over effects of alkaloids. Others have observed that alkaloids were undetectable in urine within 96 h after steers were switched from grazing endophyte-infected tall fescue to endophyte-free tall fescue [
27]. Acknowledging that alkaloids have the ability to suppress prolactin production, Aiken et al. [
28] found that prolactin returned to pre-exposure concentrations 3 d after the removal of ergot-contaminated feed. Schumann et al. [
26] used a 2-week adaptation period to cereal ergot alkaloid-contaminated diets before collection of feces and urine. These studies suggest that the clearance phase chosen (2 weeks) was adequate for lambs to avoid carry-over of alkaloids although residence time of alkaloids in tissues is a current subject of study [
29].
Increasing ergot content in feed has reduced nitrogen (N) intake and digestibility in livestock fed endophyte-infected hay [
30] and in pigs fed ergot-contaminated rye [
23,
31]. Many of these observations are likely associated with a reduction in feed intake [
32]. In the current study and that of Coufal-Majewski et al. [
8], ergot alkaloid concentrations did not impact any measure of nitrogen metabolism, and there were no reductions in feed intake. Some measures of nitrogen digestibility were affected by period, likely due to increasing N intake with growth of the lambs. Concentrations of ergot alkaloids 1.7 times greater than the highest concentration fed to lambs in our study were required to reduce N digestibility in pigs [
23], although allowable concentrations for ergot alkaloids in diets for swine are double those of sheep or cattle (from 4 to 6 ppm) [
9]. Consequently, greater concentrations of alkaloids, or an alkaloid profile that promotes a reduction in feed intake by lambs, may be necessary before reduced N metabolism is observed. It is important to note that the most adverse effects of ergot alkaloids are typically observed during periods of heat stress [
33] and cold stress [
34], conditions that were not encountered in the present study.
Between 14% and 18% of total alkaloids were recovered in feces of lambs receiving diets containing added alkaloids. Although some alkaloids would be excreted in urine, alkaloid recoveries in the present study were similar to the approximately 24% of ingested cereal alkaloids recovered from the feces of dairy cows [
26]. Ergot alkaloids were not analyzed in urine in the present study, as the urine was acidified (pH 2) to prevent loss of ammonia, and the low pH would have influenced epimerization and analysis of the alkaloids [
6]. Recovery of ergometrine and ergometrinine in feces increased with increasing dietary concentrations, possibly as proportionally less of these alkaloids were excreted in urine [
27]. In contrast to ergometrine and ergometrinine, concentrations of other alkaloids in feces either remained constant, or tended to decrease with increasing dietary concentrations.
All alkaloids in the present study had similar ratios of recovery of
R and
S epimers, although Dänicke [
29] found greater recoveries of
R than
S epimers in the excreta of chickens. Mechanisms for metabolism of ergot alkaloids likely differ across livestock species [
35]. Accordingly, alkaloid concentrations of diets fed to chickens in the study of Dänicke [
29] were up to 7-fold greater than those of the current study. Individual animals of the same species are also known to differ in their tolerance to ergot alkaloids [
7]. In the present study, with the exception of ergocornine and ergocryptine, recovery of alkaloids in feces significantly varied with period, suggesting that the metabolism of other alkaloids may have differed among individual lambs. Excretion of alkaloids in feces also increased during each subsequent period of the digestibility study, suggesting that lambs may have adapted to alkaloids over time through increasingly efficient metabolic pathways [
27]. Similarly, Dänicke and Diers [
14] observed that adverse effects of cereal ergot exposure in piglets became less as the experimental period progressed, likely due to metabolic adaptation to alkaloids.
Metabolic responses to dietary ergot are undoubtedly complex, as NDF digestibility by lambs decreased at 433 ppb
R epimers [
8], with no impacts of dietary treatments on fiber digestibility noted in the current study. The source of contaminated screenings and alkaloid profiles differed between studies, demonstrating the difficulty of determining the concentration of ergot alkaloids where no adverse effects are observed. In the study of Coufal-Majewski et al. [
8], the maximum concentration at which no adverse effects were noted for nutrient digestibility of pelleted diets was 185 ppb
R epimers, as compared to 1790 ppb
R epimers in the present study. Dänicke [
1] noted the difficulty in predicting the net effect of an ergot alkaloid mixture due to dose- and proportion-associated interactive effects. Further investigation to determine and evaluate the most common alkaloid profiles present in a region or country could be useful in determining the lower limit at which that profile has a negative impact on digestibility, growth performance, and animal health.
3.4. Effects of Ergot Alkaloid Concentrations on Growth Performance
Behavior of some lambs receiving diets with added alkaloids was judged to be unusual by barn staff (abnormal calm while being herded to the weigh crate), potentially due to the psychotropic effects of ergometrine [
15]. Alterations in behavior would have had limited impact on performance of individually penned sheep with no need to flee from predators and readily accessible ad libitum feed and water. Although not quantified in the present study, impacts of ergot alkaloids on animal behavior are an important consideration due to the potential for reduced animal fitness, especially under commercial management conditions.
Koontz [
32] noted that ruminal volatile fatty acid profile was altered by alkaloid ingestion, which could cause a reduction in nutrient absorption due to reduced feed intake. In contrast, the alkaloid concentration of diets did not impact DMI in the current study or that of Coufal-Majewski [
8], providing evidence that secondary effects of alkaloid ingestion may have a greater impact on lamb performance than alterations in feed intake. A reduction in feed intake has been identified as the most sensitive endpoint for determining the adverse effect concentration of ergot alkaloids in broiler chickens [
1], but based on the results of the present study, adverse effects in lambs are likely to occur before a reduction in feed intake would be noted.
Coufal-Majewski et al. [
8] observed only a minor reduction in the ADG in lambs receiving diets containing 433 ppb
R epimers; whereas in the present study, ADG was reduced by 27% as compared to Controls in lambs fed 1790 ppb
R epimers. All diets in the present study were pelleted, with alkaloids measured after pelleting, which may have helped to moderate alkaloid impacts on performance, as reductions in ADG in the study of Coufal-Majewski et al. [
8] were more pronounced with mash diets. However, different sources of ergot with varying alkaloid profiles makes comparison of the two studies difficult. Additionally, alkaloid analyses in the two studies were performed by different labs to facilitate analysis of both
R and
S epimers in the present study. As 2447 ppb is within the maximum allowable alkaloid range in diets for sheep and cattle in Canada (from 2 to 3 ppm) [
9], it was expected that a diet with this concentration of alkaloids would impact performance of the lambs, although current Canadian regulations do not distinguish between
R and
S epimers. More surprising was the lack of impact of the 930 and 1402 ppb diets on ADG, as lower concentrations of
R epimers in a previous study led to a significant reduction in ADG [
8].
Ergot alkaloids are known to impact lipid metabolism in sheep and other livestock, with increasing concentrations causing observable changes in fat cover and liver health [
36,
37]. In the current study, carcass dressing percentage was reduced for lambs fed the 1402 ppb diet compared to other treatments, possibly because of differing proportions of alkaloids, as relative concentrations of ergocornine and ergocryptine were elevated for this diet. Similarly, nutrient digestibility measures in a separate group of lambs tended to be depressed for lambs receiving the 1402 as compared to the 2447 ppb diet (
Table 2 and
Table 3). Although all diets were prepared from the same source of contaminated screenings, due to variations in alkaloid concentrations and profiles in individual ergot bodies [
1], it was not possible to achieve identical alkaloid profiles across diets.
Additional study is required to evaluate impacts of individual or combinations of alkaloids on lipid metabolism as effects of ergot-contaminated feed on livestock have been inconsistent [
14]. Lambs fed all diets contaminated with ergot had dressing percentages <48%, below the range for lambs to receive maximum market value at the abattoir (from 48 to 57.9%) [
38]. This would cause a reduction in overall revenue from feeding ergot-contaminated diets, in addition to that of the reduced ADG noted for the 2447 ppb diet
3.5. Effects of Ergot on Serum Prolactin and Rectal Temperature
Ergot alkaloids can act as either an agonist or antagonist for physiological functions depending on what type of alkaloid is present, frequently affecting the dopamine receptor [
39]. Administration of ergocristine, a dopamine agonist, can instantaneously block release of prolactin [
39,
40]. Ergocristine was the most prevalent alkaloid in feed in the present study, which may explain the significant linear reduction in serum prolactin concentration with increasing alkaloid concentration. Similarly, Coufal-Majewski et al. [
8] observed a decline in serum prolactin concentrations of lambs even at dietary alkaloid concentrations as low as 170 ppb. Rams grazing endophyte-infected pastures have also exhibited reduced serum prolactin concentrations and testicle weight, but the fertility of rams was unaffected one year after exposure to alkaloids [
41].
Rectal temperatures recorded for control lambs were within a normal range (38.5 to 39.9 °C) [
42], but lambs receiving diets with added alkaloids exhibited slightly greater rectal temperatures. Gadberry et al. [
43] found that dietary ergot alkaloids increased susceptibility of sheep to heat stress. However, to induce heat stress, ambient temperatures typically need to exceed 31 °C [
30,
44]. As the average daily ambient temperature never exceeded 27.4 °C in the current study, heat stress was not induced. Future studies are required to address impacts of temperatures above or below the thermo-neutral zone on toxicity of ergot alkaloids.