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Vasoconstrictive responses of the testicular and caudal arteries in bulls exposed to ergot alkaloids from tall fescue1
The Professional Animal Scientist 31 (2015):130–136; http://dx.doi.org/10.15232/pas.2014-01373 ©2015 American Registry of Professional Animal Scientists
Vasoconstrictive responses of the testicular and caudal
arteries in bulls exposed to ergot alkaloids from tall fescue1 G. E. Aiken,*2 PAS, M. G. Burns,† H. M. Stowe,† J. G. Andrae,† and S. L. Pratt† *USDA-ARS Forage-Animal Production Research Unit, University of Kentucky Campus, Lexington 40546; and †Department of Animal and Veterinary Sciences, Clemson University, Clemson, SC 29634
ABSTRACT Color Doppler ultrasonography was used to evaluate vasoconstrictive responses of the testicular artery in yearling bulls to ergot alkaloids (Neotyphodium coenophialum) produced by a fungal endophyte that infects tall fescue [Lolium arundinaceum (Schreb.) Darbysh.]. Alkaloid-induced constriction of the testicular artery could disrupt thermoregulation. Luminal areas of the testicular artery were monitored and analyzed for 2 experiments that were designed to evaluate fertility responses of bulls to toxic endophyte-infected tall fescue. Experiment 1 (pen experiment) compared diets containing toxic endophyte-infected or endophyte-free tall fescue seed, and Exp. 2 (grazing experiment) compared grazing diets consisting of toxic endophyte-
1 Mention of trade names or commercial products in the article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. The USDA is an equal opportunity employer. 2 Corresponding author: glen.aiken@ars. usda.gov
infected and nontoxic endophyte-infected tall-fescue pasture. In Exp. 1 ultrasound images were acquired on 3 dates during the 126-d of feeding and in Exp. 2 on 4 dates during the 155-d of grazing. Luminal area of the caudal artery also was monitored as an indicator of alkaloidinduced vasoconstriction. Caudal and testicular arteries for the toxic treatment responded similarly between experiments. Across imaging dates for Exp. 1, caudal artery lumens for the toxic diet averaged 42% less (P < 0.01) area than the nontoxic diet, and testicular arteries for the toxic diet averaged 40% less (P < 0.05) area than the nontoxic diet. For Exp. 2, there were interactions (P < 0.05) between treatment and imaging date on luminal areas of both arteries. Differences between treatments were not detected until the last 2 image dates, with luminal areas of caudal and testicular arteries on the last date for the toxic tall fescue being 46 and 41%, respectively, less than the nontoxic counterpart. Results indicated that ergot alkaloids can induce constriction of blood flow to the testes that may adversely affect bull fertility. Key words: bull fertility, ergot alkaloids, fescue toxicosis, tall fescue, vasoconstriction
INTRODUCTION Tall fescue [Lolium arundinaceum (Schreb.) Darbysh.] is a persistent and productive cool-season perennial grass that is the most predominant grass used for pasture in the eastern United States. Unfortunately, ergot alkaloids produced by an endophyte (Neotyphodium coenophialum) that infects most tall-fescue plants can cause a toxicosis in cattle that reduces growth and thriftiness (Schmidt and Osborn, 1993) and reproductive performance (Porter and Thompson, 1992). From a review, Paterson et al. (1995) reported pregnancy rates and weaning weights on endophyte-infected tall fescue were reduced an average of 32 and 14%, respectively, as compared with animals on endophyte-free tall-fescue pastures Limited research has shown that bulls exposed to endophyte-infected tall fescue have elevated scrotal temperature (Jones et al., 2004), smaller scrotal circumference (Jones et al., 2004; Stowe et al., 2013), and lower percentages of motile and progressive sperm with high ambient temperatures (Looper et al., 2009). Although reports have been inconsistent in detection of which bull fertility char-
Alkaloid-induced constriction of the bovine testicular artery
acteristics were affected by ergot alkaloids, the extent of an adverse effect on these characteristics likely depends on genetics, environment, and amount of bioaccumulation of ergot alkaloids in the vasculature (Pratt et al., 2015). Ergot alkaloids bind biogenic amine receptors in the vasculature (Oliver, 2005; Strickland et al., 2011) to constrict blood flow to peripheral tissues (Rhodes et al., 1991; Aiken et al., 2007; Klotz et al., 2007) and reduce the ability of the animal to thermoregulate body temperature. Thermoregulation of the testes is critical to maintaining fertility in bulls (Kastelic et al., 1997; Brito et al., 2004) and constriction of blood flow through the testicular artery could affect production of morphologically sound and viable sperm. Ergot alkaloid–induced vasoconstriction of the testicular artery in bulls has not been documented. Therefore, an experiment was conducted using color Doppler ultrasonography to compare cross-sectional areas of the testicular artery between bulls fed diets with or without toxic endophyteinfected fescue seed in a feeding experiment (Exp. 1), and also between bulls grazing either toxic or nontoxic fescue pastures (Exp. 2). The caudal artery also was measured as a marker of alkaloid-induced vasoconstriction because it has been documented to be sensitive to ergot alkaloids (Aiken et al., 2007, 2009b).
MATERIALS AND METHODS Animals and Treatments Vasoconstrictive responses were measured for 2 groups of bulls between 13 and 16 mo of age that were used in separate experiments with objectives to compare fertility traits and semen characteristics between bulls consuming diets with or without ergot alkaloids. A feeding experiment fed either toxic or nontoxic seed as the treatment (Exp. 2), and a grazing experiment was conducted to make comparisons between bulls grazing either toxic or nontoxic endophyte-
infected tall fescue pastures (Exp. 2). Both experiments were conducted at the Simpson Research and Education Center in Pendleton, South Carolina. All animal research followed procedures approved by the Clemson University Institutional Animal Care and Use Committee.
Feeding Experiment (Exp. 1) Seven Angus and 6 Hereford Bulls were stratified by breed, BW, and BCS for random assignment to feeding treatments that consisted of concentrate containing either toxic (E+, n = 6) or nontoxic (E−, n = 7) seed. Quantity of seed in the diet was similar between the 2 treatments and set by the quantity of toxic seed needed to provide a diet concentration of 0.8 μg of ergovaline and ergovalanine per gram of DM. Seed was assayed for ergovaline and ergovalanine using HPLC with fluorescence detection following procedures of Yates and Powell (1988) that were modified as described by Carter et al. (2010). A 2-wk diet adjustment period was done by feeding the concentrate with E− seed to all bulls. A 126-d test period (April 6 to August 10, 2011) with ad libitum feeding followed the adjustment period. Details of the experimental design and procedures are described by Stowe et al. (2013). Following the 126-d test period, 3 bulls on the toxic treatment were switched to the nontoxic diet, and 3 bulls remained on the nontoxic diet until October 19, 2011, to determine whether there was recovery from ergot alkaloid–induced vasoconstriction.
Grazing Experiment (Exp. 2) Angus bulls were stratified by BW and visual BCS for allocation to single pastures of Kentucky 31 toxic endophyte-infected (n = 11) and MaxQ II [Texoma (Pennington Seed Inc., Madison, GA) infected with the nontoxic endophyte, AR584; n = 10] tall-fescue pastures. Although ergot alkaloid concentrations in grazed forage were not quantified, serum
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prolactin concentrations were used as indicators of fescue toxicosis (Strickland et al., 1993). Blood samples were collected from the caudal arteries on d 35, 84, 114, and 140 for assaying serum prolactin following procedures of Bernard et al. (1993). Pastures were grazed for 155 d from April to August 2012. Experimental design and procedures are described by Pratt et al. (2015).
Doppler Ultrasound Imaging Color Doppler ultrasound images of the cross-sections of the testicular and carotid arteries were collected on 3 dates for Exp. 1 (May 26, July 28, October 19) and 4 dates for Exp. 2 (May 10, June 28, August 16, and October 10). Baseline measures were not collected because logistical limitations of the sonographers caused wide time periods between imaging dates, and baselines would have been taken during cooler ambient temperatures that could have confounded comparisons between baselines and those when the bulls were on treatment diets. Images were collected using a Classic Medical TeraVet 3000 Ultrasound Unit (Classic Universal Ultrasound, Tequesta, FL) with a 12L5-VET (12 MHz) linear array transducer. Crosssections of the medial caudal artery were imaged with a ventral, transverse orientation of the transducer on the tail at the fourth coccygeal (Cd4) vertebrae (Aiken et al., 2007, 2009b), and cross-sections of the testicular artery were imaged with a caudal, transverse orientation on the scrotum approximately 2 cm above the dorsal testes. Five images were collected for each artery using a frequency of 5.0 MHz and a pulse repetitive frequency that ranged between 3.0 to 4.5 kHz. Scan depth was set at 3 cm for both arteries. Following freezing of an individual scan, frames stored in the cine memory of the unit were searched to store the image exhibiting the maximum flow signal and assumed to be at peak systolic phase. The flow signal was traced to estimate luminal area (Aiken et al., 2009a).
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Figure 1. Cross-sectional B-mode (A) and color Doppler (B) ultrasound images of the intertwined testicular artery and pampiniform plexus (i.e., testicular vein). The arrow is pointing to a cross-section of a testicular artery (a) with color that delineates blood flow within the lumen.
Statistical Analysis Testicular and caudal artery luminal areas were analyzed using mixed models of SAS (SAS Institute Inc., Cary, NC) as repeated measures with the autoregressive covariance structure (Littell et al., 1996). The analysis used animal as the experimental unit. Treatment (toxic vs. nontoxic), imaging session, and the interaction between treatment and imaging session were analyzed as fixed effects for both experiments. In the presence of a significant (P ≤ 0.05) treatment– by–imaging session interaction, simple effects between treatments within image sessions were determined using the PDIFF option of SAS (Littell et al., 1996).
RESULTS AND DISCUSSION Cross-Sectional Luminal Areas for Assessing Alkaloid-Induced Vasoconstriction Blood flow under normal conditions is regulated by vascular tone of vessels that are controlled by the autonomic nervous system. Blood-flow volume is proportional to the fourth power of luminal radius, such that large changes in blood flow can occur with small changes in cross-sectional luminal area from vasoconstriction or vasorelaxation and vasodialation
(Carter, 2000). Therefore, responses of cross-sectional areas of vessel lumens can provide a direct measure of vasoconstriction or vasorelaxation that has a direct bearing on bloodflow volume. Color Doppler ultrasonography has been used to assess ergot alkaloid– induced vasoconstriction in cattle (Aiken et al., 2007, 2009b), horses (McDowell et al., 2013), and sheep (Aiken et al., 2011). Luminal area of the medial caudal artery, which supplies blood to the tail, has been demonstrated to be useful in determining vasoconstriction in cattle when comparisons are made between diets that are with or without ergot alkaloids. There are challenges in imaging cross-sections of the testicular artery. The testicular artery lies within the spermatic cord and exhibits substantial coiling and winding and is intertwined with the pampiniform plexus (Polguj et al., 2010); therefore, these images can contain a multitude of vessel cross-sections and slight to full longitudinal artery and vein orientations (Figure 1A). A cross-section of the testicular artery with a true circular lumen was chosen to image and measure (Figure 1B).
Caudal Arteries Caudal arteries in bulls fed the toxic diet during Exp. 1 exhibited vaso-
constrictive responses (P < 0.05) to ergot alkaloids that were consistent (treatment by imaging date, P > 0.10; Figure 2A) across imaging dates. This indicated that caudal arteries in bulls on the toxic treatment therefore remained constricted for 70 d after they were switched to the nontoxic diets. Averaged across imaging dates, caudal luminal areas for bulls on the toxic and nontoxic seed treatments were 4.8 ± 0.7 and 8.3 ± 0.7 mm2, respectively. For Exp. 2, there was a treatment–by–imaging date interaction (P < 0.01) on caudal artery luminal area (Figure 3A). Differences in luminal areas between treatments were not detected (P > 0.27) on the first and second imaging dates, but bulls grazing toxic tall fescue had smaller luminal areas (P ≤ 0.05) than those grazing nontoxic fescue on the third and fourth dates. The caudal arteries in bulls consuming diets containing ergot alkaloids in Exp. 1 and those grazing toxic endophyte-infected fescue in Exp. 2 had smaller luminal areas, indicating presence of ergot alkaloid–induced vasoconstriction. Difference between the 2 experiments was that the vasoconstriction in Exp. 1 was consistent across imaging dates, whereas alkaloid-induced vasoconstriction was not detected in Exp. 2 until the latter 2 imaging dates. Aiken et al. (2007, 2009b) reported a vasoconstrictive response by the caudal arteries in heifers on diets with the same ergovaline concentration as in Exp. 1 in less than 28 h. Alkaloid-induced vasoconstriction in Exp. 1 likely occurred before the first imaging date. Ergot alkaloid concentrations in tall fescue generally increase in the spring as plants mature but decline during the summer (Carter et al., 2010). Diet concentrations of ergot alkaloids may have been lower for the grazing bulls in Exp. 2 than for those in Exp. 1, but ergot alkaloids could have bioaccumulated in the vasculature (Klotz et al., 2009) to elicit vasoconstriction in the latter days of grazing. The rationale in measuring crosssectional areas of the caudal artery was that this artery had previously
Alkaloid-induced constriction of the bovine testicular artery
Figure 2. Mean ± SE for luminal areas of the medial caudal (A) and testicular (B) arteries for 3 imaging dates in bulls fed toxic endophyte–infected (Neotyphodium coenophialum; n = 6) or endophyte-free tall fescue [Lolium arundinaceum (Schreb.) Darbysh.] seed (n = 7). Probability values are provided below the regression lines for predicted differences among least squares means for pasture treatments. The dotted vertical line designates the date when bulls fed toxic endophyte–infected seed were switched to endophyte-free seed.
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been reported to be responsive to ergot alkaloids. Prolactin concentration in serum or plasma has been accepted as a marker of toxicosis because reductions in prolactin concentrations have consistently been determined for cattle exposed to endophyte-infected tall fescue. For Exp. 1, Stowe et al. (2013) reported that depressed serum prolactin concentration in bulls on the toxic treatment as compared with those on the nontoxic treatment was not observed until 63 d after the starting of treatments, but vasoconstriction was detected in the bulls on the toxic treatment on the first imaging date (d 50). Differences not being detected until after the d-42 collection indicated that ergot alkaloids may not have bioaccumulated to an extent to elicit prolactin or caudal artery lumen-area responses. Trends in prolactin and luminal areas in Exp. 2 were somewhat different. Vasoconstriction was not detected until the last 2 imaging dates, whereas Pratt et al. (2015) reported bulls on the toxic treatment have lower prolactin concentrations than those on the nontoxic treatment on the first bloodcollection day (d 35) after treatments were imposed. Ergot alkaloids are agonists to D-2 receptors on the anterior pituitary that inhibit secretion of prolactin into the circulatory system (Strickland et al., 2011), whereas their influence on blood flow is due to their being agonists to α-adrenergic receptors that induce contraction of vascular smooth muscle (Oliver, 2005; Strickland et al., 2011). The mechanisms employed by ergot alkaloids to depress prolactin and blood flow apparently are different. Therefore, for studying physiological responses to ergot alkaloids, prolactin might be a better indicator of alterations in the endocrine systems, and measures of vessel luminal areas or blood-flow characteristics are needed for reliable determination of altered blood flows.
Testicular Arteries Over the first 2 imaging dates, luminal areas of testicular arteries for bulls in Exp. 1 appeared larger
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Figure 3. Mean ± SE for luminal areas of the medial caudal (A) and testicular (B) arteries for 4 imaging dates in bulls grazing either Kentucky 31 toxic endophyte– infected (Neotyphodium coenophialum; n = 11) or MaxQ II [Texoma (Pennington Seed Inc., Madison, GA) infected with the non-ergot-alkaloid-producing endophyte, AR584; n = 10] tall fescues [Lolium arundinaceum (Schreb.) Darbysh.]. Probability values are provided below the regression lines for predicted differences among least squares means for pasture treatments.
than those for bulls in Exp. 2. This could have been due to the higher ambient temperatures recorded on imaging dates in Exp. 1 that caused some vascular relaxation and dilation. Maximum/minimum air temperatures (°C) for imaging dates in Exp. 1 were 33°/18° on 26 May, 37°/24° on 28 July, and 18°/9° on 19 October, and in Exp. 2 they were 24°/12° on 10 May, 37°/18° on 28 June, 31°/21° on 16 August, and 28°/22° on 4 October (Weather Source, 2015). Testicular arteries of bulls on the toxic treatment in both experiments responded similarly to those of the caudal artery. Testicular lumens in Exp. 1 were smaller (P < 0.05) in bulls on the toxic diet than those on the nontoxic diet across all imaging dates (treatment by imaging dates, P > 0.46; Figure 2B), which indicated that the testicular artery in bulls on the toxic treatment remained constricted for 70 d after being switched to the nontoxic treatment. Across imaging dates, testicular artery areas for bulls on the toxic diet averaged 9.8 ± 1.6 mm2, and for those on the nontoxic treatment they averaged 16.4 ± 1.8 mm2. Similar to Exp. 1, there was a treatment–by–imaging date interaction (P < 0.05) on testicular-artery luminal area (Figure 3B) in Exp. 2. There were no differences (P > 0.64) in luminal areas between treatments on the first and second imaging dates, but bulls grazing toxic tall fescue on the third imaging date had a tendency (P < 0.10) and on the fourth date they had significantly (P < 0.01) smaller luminal areas. Similar to caudal arteries, responses of testicular arteries to ergot alkaloids were detected across imaging dates for Exp. 1, but responses in Exp. 2 were not determined until the last 2 imaging dates. This supports the previous explanation that ergot alkaloids in bulls grazing toxic endophyte-infected fescue accumulated in the testicular artery to reach a threshold above which induced measurable constriction. For both experiments, luminal areas of testicular arteries of bulls on toxic treatments ranged from 40
Alkaloid-induced constriction of the bovine testicular artery
to 46% less than those on nontoxic treatments, which indicated similar vasoconstrictive responses between the 2 arteries. Although vasoconstrictive responses of the testicular artery were detected in both experiments, scrotal circumference in Exp. 1 was less for bulls on the toxic treatment, and sperm characteristics were not affected (Stowe et al., 2013). However, even though scrotal circumference in Exp. 2 was not affected by grazing toxic tall fescue, sperm concentration, motile and progressive sperm concentrations, and sperm velocity were less in bulls grazing toxic pastures (Pratt et al., 2015). Both experiments provided long-term exposure to ergot alkaloids, but ergot alkaloid concentrations in diets for Exp. 1 were constant, whereas these concentrations in diets of bulls in Exp. 2 would have been inconsistent because of changes in ergot alkaloid concentrations in tall fescue within a grazing season (Carter et al., 2010). It was interesting that the caudal and testicular arteries in bulls on the toxic diet in Exp. 1 remained constricted for 70 d after they were switched to the nontoxic diet. Aiken et al. (2013) observed that caudal arteries in steers grazed on toxic endophyte–infected tall fescue tended to relax over a 30-d period but did not expand to a luminal area that was measured for those that had grazed endophyte-free pasture. It is plausible that the vasculature of bulls fed the toxic diet in Exp. 1 was saturated with ergot alkaloids and, therefore, required a longer time for ergot alkaloids to clear from their tissues than those that obtain ergot alkaloids from a pasture source (Exp. 2). However, actual clearance of ergot alkaloids from animal tissues has not been documented.
Any effects of alkaloid-induced vasoconstriction of the testicular artery on thermoregulation of the testes are probably combined with the effects of bull genetics, environment, ergot alkaloid concentrations in the diet, or any combination to decrease bull fertility. Nonetheless, ineffective heat regulation and reduction in nutrient supply from alkaloid-induced vasoconstriction of the testicular artery would likely be a major factor in adversely altering scrotum circumference and sperm characteristics.
IMPLICATIONS
Bernard, J. K., A. B. Chestnut, B. H. Erickson, and F. M. Kelly. 1993. Effects of prepartum consumption of endophyte-infected tall fescue on serum prolactin and subsequent milk production. J. Dairy Sci. 76:1928–1933.
Results of the color Doppler ultrasound imaging provide clear evidence that ergot alkaloids circulating in the vasculature of bulls can induce constriction of the testicular artery.
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Relationship with scrotal, testicular vascular cone and testicular morphology, and effects on semen quality and sperm production. Theriogenology 61:511–528. Carter, J. M., G. E. Aiken, C. T. Dougherty, and F. N. Schrick. 2010. Steer responses to feeding soybean hulls and steroid hormone implantation on toxic tall fescue pasture. J. Anim. Sci. 88:3759–3766. Carter, S. A. 2000. Hemodynamic considerations in peripheral vascular and cerebrovascular disease. Pages 3–16 in Introduction to Vascular Ultrasonography. W. J. Zwiebel, ed. W. B. Saunders Co., Philadelphia, PA.
ACKNOWLEDGMENTS
Jones, K. L., C. R. McCleary, S. S. King, G. A. Apgar, and K. E. Griswold. 2004. Case study: Consumption of toxic fescue impairs bull reproductive parameters. Prof. Anim. Sci. 20:437–442.
The authors express appreciate appreciation to Tracy Hamilton, USDAARS agricultural research technician, for his effort and technical support in the collection of ultrasound images.
Kastelic, J. P., R. G. Cook, and G. H. Coulter. 1997. Contribution of the scrotum, testes, and testicular artery to scrotal/ testicular thermoregulation in bulls at two ambient temperatures. Anim. Reprod. Sci. 45:255–261.
LITERATURE CITED Aiken, G. E., B. H. Kirch, J. R. Strickland, L. P. Bush, M. L. Looper, and F. N. Schrick. 2007. Hemodynamic responses of the caudal artery to toxic tall fescue in beef heifers. J. Anim. Sci. 85:2337–2345. Aiken, G. E., J. L. Klotz, J. M. Johnson, and J. R. Strickland Jr. 2013. Postgraze assessment of toxicosis symptoms for steers grazed on toxic endophyte-infected tall fescue pastures. J. Anim. Sci. 91:5878–5884. Aiken, G. E., J. L. Klotz, B. H. Kirch, J. R. Strickland, and D. L. Boykin. 2009a. Technical note: Comparison between two tracing methods with ultrasonography to determine lumen area of the caudal artery in beef cattle. J. Anim. Sci. 87:371–374. Aiken, G. E., J. R. Strickland, M. L. Looper, L. P. Bush, and F. N. Schrick. 2009b. Hemodynamics are altered in the caudal artery of beef heifers fed different ergot alkaloid concentrations. J. Anim. Sci. 87:2142–2150. Aiken, G. E., B. L. Sutherland, and L. R. Fletcher. 2011. Haemodynamics of lambs grazing perennial ryegrass (Lolium perenne L.) either infected with AR6 novel, wild-type endophyte or endophyte-free. NZ J. Vet Sci. 59:179–184.
Brito, L. F., A. Silva, T. Barbosa, and J. Kastelic. 2004. Testicular thermoregulation in Bos indicus, crossbred and Bos taurus bulls:
Klotz, J. L., L. P. Bush, D. L. Smith, W. D. Shafer, L. L. Smith, and B. C. Arrington. 2007. Ergovaline induced vasoconstriction in an isolated bovine lateral saphenous vein bioassay. J. Anim. Sci. 85:2330–2336. Klotz, J. L., B. H. Kirch, G. E. Aiken, L. P. Bush, and J. R. Strickland. 2009. Bioaccumulation of ergovaline in bovine lateral saphenous veins in vitro. J. Anim. Sci. 87:2437–2447. Littell, R. C., G. A. Milliken, W. W. Stroup, and R. D. Wolfinger. 1996. SAS System for Mixed Models. SAS Inst. Inc., Cary, NC. Looper, M. L., R. W. Rorie, C. N. Person, T. D. Lester, D. M. Hallford, G. E. Aiken, C. A. Roberts, G. E. Rottinghaus, and C. F. Rosenkrans Jr. 2009. Influence of toxic endophyteinfected fescue on sperm characteristics and endocrine factors of yearling Brahman-influenced bulls. J. Anim. Sci. 87:1184–1191. McDowell, K. J., E. S. Moore, A. G. Parks, L. P. Bush, D. W. Horohov, and L. M. Lawrence. 2013. Vasoconstriction in horses caused by endophyte-infected tall fescue seed is detected with Doppler ultrasonography. J. Anim. Sci. 91:1677–1684. Oliver, J. W. 2005. Pathophysiologic response to endophyte toxins. Pages 291–301 In Neotyphodium in Cool-Season Grasses. C. A. Roberts, C. P. West, and D. E. Spiers, ed. Blackwell Publ., Ames, IA. Paterson, J., C. Forcherio, B. Larson, M. Samford, and M. Kerley. 1995. The effects of fescue toxicosis on beef cattle productivity. J. Anim. Sci. 73:889–898. Polguj, M., K. S. Jedrzejewski, and M. Topol. 2010. Arterial supply of human and bovine testes: A topographical and morphometric
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Schmidt, S. P., and T. G. Osborn. 1993. Effects of endophyte-infected tall fescue on animal performance. Agric. Ecosyst. Environ. 44:233–262.
Pratt, S. L., H. M. Stowe, M. Miller, S. M. Calcatera, M. D. Owens, G. E. Aiken, F. N. Schrick, N. M. Long, S. K. Duckett, and J. G. Andrae. 2015. Bulls grazing toxic tall fescue exhibit impaired growth, semen quality and decreased semen freezing potential. Theriogenology 83:408–414. Rhodes, M. T., J. A. Paterson, M. S. Kerley, H. E. Garner, and M. H. Laughlin. 1991. Reduced blood flow to peripheral and core body tissues in sheep and cattle induced by
Stowe, H. M., M. Miller, M. G. Burns, S. M. Calcatera, J. M. Andrae, G. E. Aiken, F. N. Schrick, T. Cushing, W. C. Bridges, and S. L. Pratt. 2013. Effects of fescue toxicosis on bull growth, semen characteristics, and breeding soundness evaluation. J. Anim. Sci. 91:3686–3692. Strickland, J. R., M. L. Looper, J. C. Matthews, C. F. Rosenkrans Jr., M. D. Flythe, and K. R. Brown. 2011. Board-Invited Re-
view: St. Anthony’s fire in livestock: Causes, mechanisms, and potential solutions. J. Anim. Sci. 89:1603–1626. Strickland, J. R., J. W. Oliver, and D. L. Cross. 1993. Fescue toxicosis and its impact on animal agriculture. Vet. Hum. Toxicol. 35:454–464. Weather Source. 2015. US National Service Archive Data for Clemson, SC. Accessed Feb. 17, 2015. http://weathersource.com/ past-weather/weather-history-reports/ free?gclid=CI. Yates, S. G., and R. G. Powell. 1988. Analysis of ergopeptine alkaloids in endophyteinfected tall fescue. J. Agric. Food Chem. 36:337–340.
Vasoconstrictive responses of the testicular and caudal
arteries in bulls exposed to ergot alkaloids from tall fescue1 G. E. Aiken,*2 PAS, M. G. Burns,† H. M. Stowe,† J. G. Andrae,† and S. L. Pratt† *USDA-ARS Forage-Animal Production Research Unit, University of Kentucky Campus, Lexington 40546; and †Department of Animal and Veterinary Sciences, Clemson University, Clemson, SC 29634
ABSTRACT Color Doppler ultrasonography was used to evaluate vasoconstrictive responses of the testicular artery in yearling bulls to ergot alkaloids (Neotyphodium coenophialum) produced by a fungal endophyte that infects tall fescue [Lolium arundinaceum (Schreb.) Darbysh.]. Alkaloid-induced constriction of the testicular artery could disrupt thermoregulation. Luminal areas of the testicular artery were monitored and analyzed for 2 experiments that were designed to evaluate fertility responses of bulls to toxic endophyte-infected tall fescue. Experiment 1 (pen experiment) compared diets containing toxic endophyte-infected or endophyte-free tall fescue seed, and Exp. 2 (grazing experiment) compared grazing diets consisting of toxic endophyte-
1 Mention of trade names or commercial products in the article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. The USDA is an equal opportunity employer. 2 Corresponding author: glen.aiken@ars. usda.gov
infected and nontoxic endophyte-infected tall-fescue pasture. In Exp. 1 ultrasound images were acquired on 3 dates during the 126-d of feeding and in Exp. 2 on 4 dates during the 155-d of grazing. Luminal area of the caudal artery also was monitored as an indicator of alkaloidinduced vasoconstriction. Caudal and testicular arteries for the toxic treatment responded similarly between experiments. Across imaging dates for Exp. 1, caudal artery lumens for the toxic diet averaged 42% less (P < 0.01) area than the nontoxic diet, and testicular arteries for the toxic diet averaged 40% less (P < 0.05) area than the nontoxic diet. For Exp. 2, there were interactions (P < 0.05) between treatment and imaging date on luminal areas of both arteries. Differences between treatments were not detected until the last 2 image dates, with luminal areas of caudal and testicular arteries on the last date for the toxic tall fescue being 46 and 41%, respectively, less than the nontoxic counterpart. Results indicated that ergot alkaloids can induce constriction of blood flow to the testes that may adversely affect bull fertility. Key words: bull fertility, ergot alkaloids, fescue toxicosis, tall fescue, vasoconstriction
INTRODUCTION Tall fescue [Lolium arundinaceum (Schreb.) Darbysh.] is a persistent and productive cool-season perennial grass that is the most predominant grass used for pasture in the eastern United States. Unfortunately, ergot alkaloids produced by an endophyte (Neotyphodium coenophialum) that infects most tall-fescue plants can cause a toxicosis in cattle that reduces growth and thriftiness (Schmidt and Osborn, 1993) and reproductive performance (Porter and Thompson, 1992). From a review, Paterson et al. (1995) reported pregnancy rates and weaning weights on endophyte-infected tall fescue were reduced an average of 32 and 14%, respectively, as compared with animals on endophyte-free tall-fescue pastures Limited research has shown that bulls exposed to endophyte-infected tall fescue have elevated scrotal temperature (Jones et al., 2004), smaller scrotal circumference (Jones et al., 2004; Stowe et al., 2013), and lower percentages of motile and progressive sperm with high ambient temperatures (Looper et al., 2009). Although reports have been inconsistent in detection of which bull fertility char-
Alkaloid-induced constriction of the bovine testicular artery
acteristics were affected by ergot alkaloids, the extent of an adverse effect on these characteristics likely depends on genetics, environment, and amount of bioaccumulation of ergot alkaloids in the vasculature (Pratt et al., 2015). Ergot alkaloids bind biogenic amine receptors in the vasculature (Oliver, 2005; Strickland et al., 2011) to constrict blood flow to peripheral tissues (Rhodes et al., 1991; Aiken et al., 2007; Klotz et al., 2007) and reduce the ability of the animal to thermoregulate body temperature. Thermoregulation of the testes is critical to maintaining fertility in bulls (Kastelic et al., 1997; Brito et al., 2004) and constriction of blood flow through the testicular artery could affect production of morphologically sound and viable sperm. Ergot alkaloid–induced vasoconstriction of the testicular artery in bulls has not been documented. Therefore, an experiment was conducted using color Doppler ultrasonography to compare cross-sectional areas of the testicular artery between bulls fed diets with or without toxic endophyteinfected fescue seed in a feeding experiment (Exp. 1), and also between bulls grazing either toxic or nontoxic fescue pastures (Exp. 2). The caudal artery also was measured as a marker of alkaloid-induced vasoconstriction because it has been documented to be sensitive to ergot alkaloids (Aiken et al., 2007, 2009b).
MATERIALS AND METHODS Animals and Treatments Vasoconstrictive responses were measured for 2 groups of bulls between 13 and 16 mo of age that were used in separate experiments with objectives to compare fertility traits and semen characteristics between bulls consuming diets with or without ergot alkaloids. A feeding experiment fed either toxic or nontoxic seed as the treatment (Exp. 2), and a grazing experiment was conducted to make comparisons between bulls grazing either toxic or nontoxic endophyte-
infected tall fescue pastures (Exp. 2). Both experiments were conducted at the Simpson Research and Education Center in Pendleton, South Carolina. All animal research followed procedures approved by the Clemson University Institutional Animal Care and Use Committee.
Feeding Experiment (Exp. 1) Seven Angus and 6 Hereford Bulls were stratified by breed, BW, and BCS for random assignment to feeding treatments that consisted of concentrate containing either toxic (E+, n = 6) or nontoxic (E−, n = 7) seed. Quantity of seed in the diet was similar between the 2 treatments and set by the quantity of toxic seed needed to provide a diet concentration of 0.8 μg of ergovaline and ergovalanine per gram of DM. Seed was assayed for ergovaline and ergovalanine using HPLC with fluorescence detection following procedures of Yates and Powell (1988) that were modified as described by Carter et al. (2010). A 2-wk diet adjustment period was done by feeding the concentrate with E− seed to all bulls. A 126-d test period (April 6 to August 10, 2011) with ad libitum feeding followed the adjustment period. Details of the experimental design and procedures are described by Stowe et al. (2013). Following the 126-d test period, 3 bulls on the toxic treatment were switched to the nontoxic diet, and 3 bulls remained on the nontoxic diet until October 19, 2011, to determine whether there was recovery from ergot alkaloid–induced vasoconstriction.
Grazing Experiment (Exp. 2) Angus bulls were stratified by BW and visual BCS for allocation to single pastures of Kentucky 31 toxic endophyte-infected (n = 11) and MaxQ II [Texoma (Pennington Seed Inc., Madison, GA) infected with the nontoxic endophyte, AR584; n = 10] tall-fescue pastures. Although ergot alkaloid concentrations in grazed forage were not quantified, serum
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prolactin concentrations were used as indicators of fescue toxicosis (Strickland et al., 1993). Blood samples were collected from the caudal arteries on d 35, 84, 114, and 140 for assaying serum prolactin following procedures of Bernard et al. (1993). Pastures were grazed for 155 d from April to August 2012. Experimental design and procedures are described by Pratt et al. (2015).
Doppler Ultrasound Imaging Color Doppler ultrasound images of the cross-sections of the testicular and carotid arteries were collected on 3 dates for Exp. 1 (May 26, July 28, October 19) and 4 dates for Exp. 2 (May 10, June 28, August 16, and October 10). Baseline measures were not collected because logistical limitations of the sonographers caused wide time periods between imaging dates, and baselines would have been taken during cooler ambient temperatures that could have confounded comparisons between baselines and those when the bulls were on treatment diets. Images were collected using a Classic Medical TeraVet 3000 Ultrasound Unit (Classic Universal Ultrasound, Tequesta, FL) with a 12L5-VET (12 MHz) linear array transducer. Crosssections of the medial caudal artery were imaged with a ventral, transverse orientation of the transducer on the tail at the fourth coccygeal (Cd4) vertebrae (Aiken et al., 2007, 2009b), and cross-sections of the testicular artery were imaged with a caudal, transverse orientation on the scrotum approximately 2 cm above the dorsal testes. Five images were collected for each artery using a frequency of 5.0 MHz and a pulse repetitive frequency that ranged between 3.0 to 4.5 kHz. Scan depth was set at 3 cm for both arteries. Following freezing of an individual scan, frames stored in the cine memory of the unit were searched to store the image exhibiting the maximum flow signal and assumed to be at peak systolic phase. The flow signal was traced to estimate luminal area (Aiken et al., 2009a).
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Figure 1. Cross-sectional B-mode (A) and color Doppler (B) ultrasound images of the intertwined testicular artery and pampiniform plexus (i.e., testicular vein). The arrow is pointing to a cross-section of a testicular artery (a) with color that delineates blood flow within the lumen.
Statistical Analysis Testicular and caudal artery luminal areas were analyzed using mixed models of SAS (SAS Institute Inc., Cary, NC) as repeated measures with the autoregressive covariance structure (Littell et al., 1996). The analysis used animal as the experimental unit. Treatment (toxic vs. nontoxic), imaging session, and the interaction between treatment and imaging session were analyzed as fixed effects for both experiments. In the presence of a significant (P ≤ 0.05) treatment– by–imaging session interaction, simple effects between treatments within image sessions were determined using the PDIFF option of SAS (Littell et al., 1996).
RESULTS AND DISCUSSION Cross-Sectional Luminal Areas for Assessing Alkaloid-Induced Vasoconstriction Blood flow under normal conditions is regulated by vascular tone of vessels that are controlled by the autonomic nervous system. Blood-flow volume is proportional to the fourth power of luminal radius, such that large changes in blood flow can occur with small changes in cross-sectional luminal area from vasoconstriction or vasorelaxation and vasodialation
(Carter, 2000). Therefore, responses of cross-sectional areas of vessel lumens can provide a direct measure of vasoconstriction or vasorelaxation that has a direct bearing on bloodflow volume. Color Doppler ultrasonography has been used to assess ergot alkaloid– induced vasoconstriction in cattle (Aiken et al., 2007, 2009b), horses (McDowell et al., 2013), and sheep (Aiken et al., 2011). Luminal area of the medial caudal artery, which supplies blood to the tail, has been demonstrated to be useful in determining vasoconstriction in cattle when comparisons are made between diets that are with or without ergot alkaloids. There are challenges in imaging cross-sections of the testicular artery. The testicular artery lies within the spermatic cord and exhibits substantial coiling and winding and is intertwined with the pampiniform plexus (Polguj et al., 2010); therefore, these images can contain a multitude of vessel cross-sections and slight to full longitudinal artery and vein orientations (Figure 1A). A cross-section of the testicular artery with a true circular lumen was chosen to image and measure (Figure 1B).
Caudal Arteries Caudal arteries in bulls fed the toxic diet during Exp. 1 exhibited vaso-
constrictive responses (P < 0.05) to ergot alkaloids that were consistent (treatment by imaging date, P > 0.10; Figure 2A) across imaging dates. This indicated that caudal arteries in bulls on the toxic treatment therefore remained constricted for 70 d after they were switched to the nontoxic diets. Averaged across imaging dates, caudal luminal areas for bulls on the toxic and nontoxic seed treatments were 4.8 ± 0.7 and 8.3 ± 0.7 mm2, respectively. For Exp. 2, there was a treatment–by–imaging date interaction (P < 0.01) on caudal artery luminal area (Figure 3A). Differences in luminal areas between treatments were not detected (P > 0.27) on the first and second imaging dates, but bulls grazing toxic tall fescue had smaller luminal areas (P ≤ 0.05) than those grazing nontoxic fescue on the third and fourth dates. The caudal arteries in bulls consuming diets containing ergot alkaloids in Exp. 1 and those grazing toxic endophyte-infected fescue in Exp. 2 had smaller luminal areas, indicating presence of ergot alkaloid–induced vasoconstriction. Difference between the 2 experiments was that the vasoconstriction in Exp. 1 was consistent across imaging dates, whereas alkaloid-induced vasoconstriction was not detected in Exp. 2 until the latter 2 imaging dates. Aiken et al. (2007, 2009b) reported a vasoconstrictive response by the caudal arteries in heifers on diets with the same ergovaline concentration as in Exp. 1 in less than 28 h. Alkaloid-induced vasoconstriction in Exp. 1 likely occurred before the first imaging date. Ergot alkaloid concentrations in tall fescue generally increase in the spring as plants mature but decline during the summer (Carter et al., 2010). Diet concentrations of ergot alkaloids may have been lower for the grazing bulls in Exp. 2 than for those in Exp. 1, but ergot alkaloids could have bioaccumulated in the vasculature (Klotz et al., 2009) to elicit vasoconstriction in the latter days of grazing. The rationale in measuring crosssectional areas of the caudal artery was that this artery had previously
Alkaloid-induced constriction of the bovine testicular artery
Figure 2. Mean ± SE for luminal areas of the medial caudal (A) and testicular (B) arteries for 3 imaging dates in bulls fed toxic endophyte–infected (Neotyphodium coenophialum; n = 6) or endophyte-free tall fescue [Lolium arundinaceum (Schreb.) Darbysh.] seed (n = 7). Probability values are provided below the regression lines for predicted differences among least squares means for pasture treatments. The dotted vertical line designates the date when bulls fed toxic endophyte–infected seed were switched to endophyte-free seed.
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been reported to be responsive to ergot alkaloids. Prolactin concentration in serum or plasma has been accepted as a marker of toxicosis because reductions in prolactin concentrations have consistently been determined for cattle exposed to endophyte-infected tall fescue. For Exp. 1, Stowe et al. (2013) reported that depressed serum prolactin concentration in bulls on the toxic treatment as compared with those on the nontoxic treatment was not observed until 63 d after the starting of treatments, but vasoconstriction was detected in the bulls on the toxic treatment on the first imaging date (d 50). Differences not being detected until after the d-42 collection indicated that ergot alkaloids may not have bioaccumulated to an extent to elicit prolactin or caudal artery lumen-area responses. Trends in prolactin and luminal areas in Exp. 2 were somewhat different. Vasoconstriction was not detected until the last 2 imaging dates, whereas Pratt et al. (2015) reported bulls on the toxic treatment have lower prolactin concentrations than those on the nontoxic treatment on the first bloodcollection day (d 35) after treatments were imposed. Ergot alkaloids are agonists to D-2 receptors on the anterior pituitary that inhibit secretion of prolactin into the circulatory system (Strickland et al., 2011), whereas their influence on blood flow is due to their being agonists to α-adrenergic receptors that induce contraction of vascular smooth muscle (Oliver, 2005; Strickland et al., 2011). The mechanisms employed by ergot alkaloids to depress prolactin and blood flow apparently are different. Therefore, for studying physiological responses to ergot alkaloids, prolactin might be a better indicator of alterations in the endocrine systems, and measures of vessel luminal areas or blood-flow characteristics are needed for reliable determination of altered blood flows.
Testicular Arteries Over the first 2 imaging dates, luminal areas of testicular arteries for bulls in Exp. 1 appeared larger
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Figure 3. Mean ± SE for luminal areas of the medial caudal (A) and testicular (B) arteries for 4 imaging dates in bulls grazing either Kentucky 31 toxic endophyte– infected (Neotyphodium coenophialum; n = 11) or MaxQ II [Texoma (Pennington Seed Inc., Madison, GA) infected with the non-ergot-alkaloid-producing endophyte, AR584; n = 10] tall fescues [Lolium arundinaceum (Schreb.) Darbysh.]. Probability values are provided below the regression lines for predicted differences among least squares means for pasture treatments.
than those for bulls in Exp. 2. This could have been due to the higher ambient temperatures recorded on imaging dates in Exp. 1 that caused some vascular relaxation and dilation. Maximum/minimum air temperatures (°C) for imaging dates in Exp. 1 were 33°/18° on 26 May, 37°/24° on 28 July, and 18°/9° on 19 October, and in Exp. 2 they were 24°/12° on 10 May, 37°/18° on 28 June, 31°/21° on 16 August, and 28°/22° on 4 October (Weather Source, 2015). Testicular arteries of bulls on the toxic treatment in both experiments responded similarly to those of the caudal artery. Testicular lumens in Exp. 1 were smaller (P < 0.05) in bulls on the toxic diet than those on the nontoxic diet across all imaging dates (treatment by imaging dates, P > 0.46; Figure 2B), which indicated that the testicular artery in bulls on the toxic treatment remained constricted for 70 d after being switched to the nontoxic treatment. Across imaging dates, testicular artery areas for bulls on the toxic diet averaged 9.8 ± 1.6 mm2, and for those on the nontoxic treatment they averaged 16.4 ± 1.8 mm2. Similar to Exp. 1, there was a treatment–by–imaging date interaction (P < 0.05) on testicular-artery luminal area (Figure 3B) in Exp. 2. There were no differences (P > 0.64) in luminal areas between treatments on the first and second imaging dates, but bulls grazing toxic tall fescue on the third imaging date had a tendency (P < 0.10) and on the fourth date they had significantly (P < 0.01) smaller luminal areas. Similar to caudal arteries, responses of testicular arteries to ergot alkaloids were detected across imaging dates for Exp. 1, but responses in Exp. 2 were not determined until the last 2 imaging dates. This supports the previous explanation that ergot alkaloids in bulls grazing toxic endophyte-infected fescue accumulated in the testicular artery to reach a threshold above which induced measurable constriction. For both experiments, luminal areas of testicular arteries of bulls on toxic treatments ranged from 40
Alkaloid-induced constriction of the bovine testicular artery
to 46% less than those on nontoxic treatments, which indicated similar vasoconstrictive responses between the 2 arteries. Although vasoconstrictive responses of the testicular artery were detected in both experiments, scrotal circumference in Exp. 1 was less for bulls on the toxic treatment, and sperm characteristics were not affected (Stowe et al., 2013). However, even though scrotal circumference in Exp. 2 was not affected by grazing toxic tall fescue, sperm concentration, motile and progressive sperm concentrations, and sperm velocity were less in bulls grazing toxic pastures (Pratt et al., 2015). Both experiments provided long-term exposure to ergot alkaloids, but ergot alkaloid concentrations in diets for Exp. 1 were constant, whereas these concentrations in diets of bulls in Exp. 2 would have been inconsistent because of changes in ergot alkaloid concentrations in tall fescue within a grazing season (Carter et al., 2010). It was interesting that the caudal and testicular arteries in bulls on the toxic diet in Exp. 1 remained constricted for 70 d after they were switched to the nontoxic diet. Aiken et al. (2013) observed that caudal arteries in steers grazed on toxic endophyte–infected tall fescue tended to relax over a 30-d period but did not expand to a luminal area that was measured for those that had grazed endophyte-free pasture. It is plausible that the vasculature of bulls fed the toxic diet in Exp. 1 was saturated with ergot alkaloids and, therefore, required a longer time for ergot alkaloids to clear from their tissues than those that obtain ergot alkaloids from a pasture source (Exp. 2). However, actual clearance of ergot alkaloids from animal tissues has not been documented.
Any effects of alkaloid-induced vasoconstriction of the testicular artery on thermoregulation of the testes are probably combined with the effects of bull genetics, environment, ergot alkaloid concentrations in the diet, or any combination to decrease bull fertility. Nonetheless, ineffective heat regulation and reduction in nutrient supply from alkaloid-induced vasoconstriction of the testicular artery would likely be a major factor in adversely altering scrotum circumference and sperm characteristics.
IMPLICATIONS
Bernard, J. K., A. B. Chestnut, B. H. Erickson, and F. M. Kelly. 1993. Effects of prepartum consumption of endophyte-infected tall fescue on serum prolactin and subsequent milk production. J. Dairy Sci. 76:1928–1933.
Results of the color Doppler ultrasound imaging provide clear evidence that ergot alkaloids circulating in the vasculature of bulls can induce constriction of the testicular artery.
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Relationship with scrotal, testicular vascular cone and testicular morphology, and effects on semen quality and sperm production. Theriogenology 61:511–528. Carter, J. M., G. E. Aiken, C. T. Dougherty, and F. N. Schrick. 2010. Steer responses to feeding soybean hulls and steroid hormone implantation on toxic tall fescue pasture. J. Anim. Sci. 88:3759–3766. Carter, S. A. 2000. Hemodynamic considerations in peripheral vascular and cerebrovascular disease. Pages 3–16 in Introduction to Vascular Ultrasonography. W. J. Zwiebel, ed. W. B. Saunders Co., Philadelphia, PA.
ACKNOWLEDGMENTS
Jones, K. L., C. R. McCleary, S. S. King, G. A. Apgar, and K. E. Griswold. 2004. Case study: Consumption of toxic fescue impairs bull reproductive parameters. Prof. Anim. Sci. 20:437–442.
The authors express appreciate appreciation to Tracy Hamilton, USDAARS agricultural research technician, for his effort and technical support in the collection of ultrasound images.
Kastelic, J. P., R. G. Cook, and G. H. Coulter. 1997. Contribution of the scrotum, testes, and testicular artery to scrotal/ testicular thermoregulation in bulls at two ambient temperatures. Anim. Reprod. Sci. 45:255–261.
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