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Effects of administration of ergotamine tartrate on fertility of yearling beef bulls
Theriogenology 63 (2005) 1407–1418 www.journals.elsevierhealth.com/periodicals/the
Effects of administration of ergotamine tartrate on fertility of yearling beef bulls Gustavo M. Schuenemanna, J. Lannett Edwardsa, Mark D. Davisa, Heather E. Blackmona, Fernando N. Scennaa, Nancy R. Rohrbacha, Arnold M. Saxtona, H. Stephen Adairb, Fred M. Hopkinsb, John C. Wallera, F. Neal Schricka,c,* a
Department of Animal Science, University of Tennessee, Knoxville, USA Department of Large Animal Clinical Sciences, University of Tennessee, Knoxville, USA c 205 C Brehm Animal Science Building, University of Tennessee, Knoxville, TN 37996-4574, USA b
Received 31 December 2003; accepted 4 July 2004
Abstract Sixteen yearling bulls were utilized to investigate administration of ergotamine tartrate on semen parameters, fertilization, and endocrinology. Bulls were allotted to a control diet of cracked corn, corn silage, and soybean meal (CON, n = 8) or a diet supplemented daily with 40 mg/kg body weight of ergotamine tartrate (ET, n = 8). Blood samples, average daily gain, scrotal circumference and rectal temperatures were collected every 14 day. Semen samples were obtained every 60 day and evaluated for motility and morphology. Scrotal temperatures were obtained by thermography immediately before electroejaculation. Semen from a subset of bulls from each treatment was also evaluated for in vitro fertilization. Administration of ET increased rectal temperature and resulted in lower scrotal temperatures compared to CON bulls (P < 0.05). However, prolactin, scrotal circumference, testosterone, and semen motility and morphology did not differ between groups throughout the experimental period (224 day). Cleavage rates of embryos derived from in vitro fertilization (IVF) with semen of bulls, fed with ET, were reduced compared to CON (P < 0.05); however, development of cleaved embryos to blastocyst did not differ between treatments. In conclusion, extended exposure of bulls to ET appeared to reduce fertilization potential of sperm. # 2004 Elsevier Inc. All rights reserved. Keywords: Fescue toxicosis; Ergotamine tartrate; Bull; Spermatozoa; Fertility
* Corresponding author. Tel.: +1 865 974 3147; fax: +1 865 974 7297. E-mail address: [email protected] (F.N. Schrick). 0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2004.07.014
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1. Introduction Fescue toxicosis results from ingestion of tall fescue infested with the endophyte, Neotyphodium coenophialum. Prevailing signs such as elevated body temperature, vasoconstriction of capillaries, and reproductive problems associated with fescue toxicity have contributed to massive declines in overall productivity. Mechanisms by which fescue toxicosis reduces cattle performance are not clearly understood but appear to involve various alkaloid compounds [1]. Consumption of endophytic tall fescue lowered nutrient intake and digestibility [2]. Fescue toxicosis affects reproductive performance, growth, and lactation [1]. Acute exposure of steers to an elevated dose of systemically delivered ergotamine elevated plasma concentrations of cortisol and triiodothyronine [3]; these hormones are involved in mediating metabolic processes and subsequent nutrient utilization. Fescue toxicosis has also been linked to reduced LH concentrations [4], compromised embryonic development [5], and reductions in pregnancy rates [6]. Hoveland [7] calculated that producers lose $ 609 million annually due to decreased calf gains and lowered conception rates associated with fescue toxicosis. While research has clearly documented how fescue toxicity affects female reproduction, few studies have examined the effects of fescue toxicosis on male reproductive performance. The objective of the present study was to investigate effects of simulated fescue toxicosis (administration of ergotamine tartrate(ET)) on semen parameters, endocrine profiles, and developmental competence of oocytes fertilized in vitro with semen from bulls fed with ET.
2. Materials and methods 2.1. Animals and treatments Sixteen Angus crossbred bulls were utilized to determine effects of ET administration on semen parameters, endocrine profiles and in vitro fertilization (IVF). Bulls, approximately 350 kg body weight and 270 day of age, were assigned to one of two groups. Breed composition, weaning weight, hip height, scrotal circumference (SC), and age were used to allot bulls to treatment groups. Prior to experimental period (224 day), bulls were weaned at the beginning of September and fed corn silage. From mid-November to the end of June in central Tennessee, bulls were fed a diet of corn silage, cracked corn, and soybean meal formulated to meet NRC [8] requirements for gain. Both groups of bulls were offered a diet of 2.5% body weight of feed (on a dry matter basis), which was formulated for a 0.850 kg gain/day and to be isonitrogenous and isocaloric throughout the experimental period. Bulls limited to this diet served as controls (CON, n = 8). The base diet was supplemented daily with 40 mg/kg body weight of ET for the treatment group (ET, n = 8; provided by Dr. Miroslav Flieger, Institute of Microbiology, Czech Republic). In order to maintain a dose of 40 mg/kg body weight, the amount of ET administered was increased as body weight (BW) increased. Water and minerals were available ad libitum. ET was administered to simulate fescue toxicity, since it mimics the action of the natural
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endophyte, ergovaline, but has only 10% of the potency of ergovaline [9]. A guideline for the ET dose was derived from previous studies [5,6]. Bulls were housed in pens with four bulls per pen. Bulls were fed ET at 12:00 h and blood samples and other data were collected from 06:00 to 09:00 h. The ambient temperature ranged from 4 8C in January to 17 8C in June. Every 14 day, average daily gain (ADG), SC (Lane Manufacturing Co. Denver, CO, USA), and rectal temperature (RT; GLA Agricultural Electronics; San Luis, Obispo, CA, USA) were measured. Blood samples (10 mL) were also collected via caudal venipuncture and placed on ice until centrifuged. Backfat thickness was measured (at initiation, middle, and end of the experimental period) by a certified technician utilizing ultrasonography (Aloka 500 V with 3.5 MHz backfat transducer; Corometrics Medical Systems Inc., Wallingford, CT, USA). Scrotal temperatures were determined prior to semen collection in May and June using an infrared thermography [10] camera (Emerge Vision DTIS 500, Emerge International Inc., Sebastian, FL, USA). The camera had an opaque chopper and internal calibration that allowed determination of absolute temperatures. Recorded images were analyzed by EResearch software. In brief, images were imported into the software and a region of interest (ROI) was drawn on each testis. The same region was taken on each testis and each ROI contained the same number of pixels. This information allowed the program to calculate absolute temperature for each ROI. 2.2. Semen collection Semen was evaluated approximately 60 day from initiation of ET administration and at 60-day intervals. Bulls were collected individually, with technician and semen evaluator having no knowledge of treatment. Semen was collected using an electroejaculator (Lane Manufacturing Co., Denver, CO, USA) and placed in a 10 mL sterile conical tube. An estimate of progressively motile sperm was obtained using a light microscope at 400. Approximately 25 mL of sperm was placed on a warmed slide and covered with a cover slip to assess motility. Another sample of sperm (approximately 25 mL) was placed on a slide, mixed with eosin–nigrosin stain, smeared and allowed to dry. Morphology was evaluated under oil immersion at 1000 using a light microscope [11]. Spermatozoa were classified as having normal morphology or as having primary or secondary abnormalities [11]. After motility and morphology examinations, semen was diluted with Bioxcell extender solution. Semen extender and straws were provided by Dr. Gustave Hansen (IMV International Technologies, France). Extender (100 mL) and sterile water (400 mL) were warmed to approximately 32 8C in a water bath for 10 min prior to use. Once the temperature of water had equilibrated, 100 mL of extender was added to the water and the solution mixed thoroughly. Semen was extended at a 1:1 mL semen/ extender ratio. Diluted semen remained at 32 8C for 10 min and then at room temperature (25 8C) for 15 min. Extended semen was packaged in Bioxcell straws horizontally and placed in a cooler with cold packs for transport. At the laboratory, straws of extended semen were refrigerated at 4 8C until IVF of oocytes, approximately 26 h after semen collection.
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2.3. In vitro evaluation of semen From both groups, collected semen from the same bulls (n = 2/trt) having the largest and smallest scrotal circumference were extended (two straws/bull) at the location and returned to the laboratory for further semen assessment. Semen was collected from the same bulls (one bull/pen) for both collecting days (May 5 and June 28). Mean SC from bulls used for in vitro evaluation of semen were 39.4 and 39.5 cm at the end of the experimental period (June 28) for CON and ET, respectively. The procedures utilized for in vitro production (IVP) of embryos were modifications of procedures previously described by Edwards and Hansen [12]. Medium 199, gentamicin, and penicillin–streptomycin were purchased from Specialty Media, Inc. (Phillipsburg, NJ, USA). Fetal bovine serum (FBS) was obtained from BioWhittaker (Walkersville, MD, USA). Folltropin-V was provided by Vetrepharm Canada, Inc. (London, Ontario, Canada) and luteinizing hormone (LH) was obtained from the United States Department of Agriculture (Beltsville, MD, USA). Media [13] (HEPESTALP, IVF-TALP, and SPERM-TALP) and KSOM [14], with modifications provided by Dr. John Hasler (personal communication), were prepared in the laboratory or purchased from Specialty Media. Ovaries obtained from an abattoir (Brown’s Packing Company; Gaffney, SC, USA) were packaged in a thermos and contained within a cooler during air transport to the laboratory (on the day of collection). Upon arrival, ovaries were immediately washed with warm water, equilibrated to arrival temperature of ovaries (generally 28–30 8C). Extraneous tissue surrounding the ovaries was removed and ovaries were washed with water one additional time. For oocyte recovery performed within 4 h of ovary collection at the abattoir, ovaries were held firmly by clamping the base of the ovary with a hemostat and checkerboard incisions were made (with a scalpel) across follicles (approximately 3–8 mm in diameter). Ovaries were washed vigorously in oocyte collection medium (OCM) to remove cumulus oocyte complexes (COC). The collection medium was filtered and rinsed using an Emcon Filter unit (Vet Concepts, Spring Valley, WI, USA) until the medium appeared clean. Medium containing COC was poured into a gridded culture dish to facilitate oocyte retrieval. Cumulus oocyte complexes were transferred to a plate with OCM and washed four times to eliminate cellular debris. Cumulus oocyte complexes of good quality were washed in oocyte maturation medium (OMM) and placed in groups of approximately 50 COC per well in a 4-well plate containing 500 mL OMM. Maturation of oocytes was performed in an incubator at 5.5% CO2 in air at 38.5 8C until fertilization (approximately 22 h after placement in OMM). Maturation medium was equilibrated in the incubator (5.5% CO2 in air at 38.5 8C) for a minimum of 5 h before oocyte collection. At 22–24 h after oocyte collection, maturation medium from each well was removed and 25 mL of penicillamine/hypotaurine/epinephrine (PHE) and 500 mL of fertilization medium (IVF-TALP) were added per well. Two straws of semen from two bulls known to have high fertility were used to fertilize oocytes for every replicate of the study (served as a laboratory control). Briefly, semen straws were removed from liquid nitrogen tank and placed in water at 36.7 8C for 45 s. Straws with collected semen and straws from laboratory control were then emptied on top of a discontinuous percoll density gradient (2 mL of 45% percoll over 2 mL of 90% percoll contained in a 15 mL conical tube) and semen was
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centrifuged at 2200 rpm for 15 min to remove excess extender, debris, and non-motile sperm prior to fertilization. The sperm pellet present at the bottom of the 90% fraction was collected and transferred to10 mL SPERM-TALP [13] and centrifuged at 1100 rpm for 8 min. The supernatant was removed and the sperm pellet re-suspended in 500 mL of modified IVF-TALP. After percoll purification, sperm concentration was determined with a hemacytometer and motility was also determined. A sperm concentration of 375,000/ 500 mL IVF-TALP collected from bulls within each group was added to the oocytes, one bull/well. A minimum of 50 oocytes was used for each bull on each collection date. Approximately 18–22 h post-fertilization, putative zygotes (PZ) were denuded of cumulus cells by vortexing. Putative zygotes were transferred to a 15 mL conical tube containing 500 mL of HEPES-TALP and vortexed for 4 min. Recovered putative zygotes were washed four times in HEPES-TALP and once in KSOM-BSA before transferring groups of approximately 50 zygotes to 4-well plates containing 500 mL of KSOM-BSA per well. Zygotes were placed in a humidified atmosphere of 5.5% CO2, 7% O2, and 87.5% N2 at 38.5 8C. The ability of PZ to cleave and develop to the blastocyst stage was evaluated on days 3 and 8, respectively (day 0 = day of IVF). Ability of the oocyte to cleave after IVF was assessed by recording the number of 1-, 2-, 4-, and 8 to 16 cell embryos present on day 3 (70–75 h post-insemination). 2.4. Blood collection and radioimmunoassays Blood samples were centrifuged at 2000 g for 30 min and serum decanted and stored at 20 8C until assayed for prolactin and testosterone. Radioimmunoassays (Coat-ACount; Diagnostic Products Corporation; Los Angeles, CA, USA) were performed to determine concentrations of testosterone [15]. Sensitivity of the testosterone assay was 0.04 ng/mL, with intra- and inter-assay coefficients of variation (CV) of 10 and 2%, respectively. Radioimmunoassay for concentrations of prolactin was performed as described by Moura and Erickson [16]. Sensitivity of prolactin assay was determined to be 0.05 ng/mL. Intra- and inter-assay CV were 11 and 10%, respectively. 2.5. Statistical analyses Differences in ADG, backfat, RT, plasma concentrations of prolactin and testosterone, SC, scrotal temperatures, and motility and morphology of semen were analyzed by SAS Proc Mixed [17]. Data are presented as the least squares means (S.E.M.). A mixed model procedure that included treatment, time, pen, and all interactions as fixed effects, was used to compare differences among treatments. Time was a repeated measures factor and animal (treatment pen) was included as a random effect. Differences among least squares means were evaluated using L.S.D. Comparisons between ET and CON treatments for IVF studies were made with Proc Mixed [17] in two different replicates (May 5 and June 28), using well as the experimental unit. For these IVF data, the key biological component was the oocyte, and experimental design addressed the large variation among oocytes. Thus, the experimental unit was a well, containing a group of similar oocytes, and treatments (semen) were applied to the wells. Bulls were simply acting as a source of treated material. We do recognize the potential risk in bulls responding differently, but our experience
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suggests that bulls show a consistent response to ET. In short, the IVF experiment needs to be viewed as an ‘assay’, where biological material was collected from some source, and the actual experiment was under carefully controlled laboratory conditions. Distributional requirements of normality and equal variance were verified for the percentage data. A value of P < 0.05 was considered significant.
3. Results 3.1. Backfat thickness, rectal temperature, ADG, and prolactin Throughout the duration of experimental period, average daily gain did not differ between groups (1.05 + 0.04 kg/day versus 1.1 + 0.04 kg/day; P > 0.10). Furthermore, there were no differences in backfat thickness between ET and CON bulls (5.3 0.12 mm versus 5.3 0.12 mm; P > 0.10). An overall increase in RT was observed in ET bulls (39.4 + 0.06 8C) compared to CON bulls (39.0 + 0.04 8C; P < 0.05), indicating that bulls were affected by administration of ET to simulate fescue toxicosis. Moreover, rectal temperatures were elevated at the time of semen collection in bulls given ET compared to CON (P < 0.05; Fig. 1). Bulls given ET (70.8 + 7.8 ng/mL) had similar plasma concentrations of prolactin compared to CON bulls (65.9 + 7.8 ng/mL; P > 0.10). 3.2. Testicular response Neither average daily growth in SC (0.034 + 0.002 cm/day; P > 0.10) nor concentrations of testosterone (8.38 + 1.1 ng/mL; P > 0.10) were affected by ET
Fig. 1. Rectal temperatures at semen evaluation. Rectal temperatures were elevated in bulls given ergotamine tartrate (ET) compared to control (CON) animals at each time of semen collection (a,bleast squares means differ within treatments; P < 0.05).
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Fig. 2. Sperm motility measured at the time of semen collection did not differ between bulls given ergotamine tartrate (ET) and control (CON) bulls. However, there was a collection date sperm motility interaction attributed to age of the bulls (a,bleast squares means differ within times of collections; P < 0.05).
administration. Furthermore, sperm motility was not compromised by ET (P > 0.10). However, due to the age of bulls at first collection, motility of first ejaculate was impaired compared to later collection dates (P < 0.05; Fig. 2). Morphological evaluation of semen indicated that extended exposure to ET did not alter the percentage of normal sperm or the percentage of primary and secondary abnormalities (P > 0.10; Table 1). Bulls exposed to ET had lower scrotal temperatures (31.6 + 0.3 8C) compared to CON bulls (33.0 + 0.2 8C; P < 0.05; Fig. 3); however, a date effect (May 5 and June 28) was not observed. Moreover, oocytes fertilized with semen from bulls administered ET had reduced cleavage rates compared to those fertilized with sperm from CON bulls (P < 0.05; Table 2); however, ability of cleaved embryos to develop to an 8–16-cell stage was not different (P > 0.10; Table 2). The percentage of cleaved embryos that continued in development to blastocyst did not differ between treatments (P > 0.10; Table 2). Replicates also did not differ between ET and CON bulls (P > 0.10). As stated previously, SC from bulls used for in vitro evaluation of semen did not differ (39.4 and 39.5 cm) at the end of the experimental period (June 28) for CON and ET, respectively.
Table 1 Sperm (%) with normal and abnormal morphology in control (CON) bulls and those treated with ergotamine tartrate (ET) Sperm
Normal
Primary abnormalitiesa
Secondary abnormalitiesb
CON ET P-value
76.9 3 73.9 3 0.51
13.5 2 16.8 2 0.33
9.5 1.4 10.9 1.4 0.54
a b
Abnormal head shape, midpiece, or presence of proximal protoplasmic droplets. Detached heads, distal protoplasmic droplets, or bent tails.
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Fig. 3. Scrotal thermography at semen collection (May 5 and June 28). Scrotal temperatures were lower in bulls given ergotamine tartrate (ET) compared to control (CON) animals (a,bleast squares means differ within treatments; P < 0.05).
Table 2 Ability of sperm collected from bulls fed a control (CON, n = 2) or ergotamine tartrate-supplemented (ET, n = 2) diet to fertilize bovine oocytes in vitro TRT
REP (n)
Motility after percoll
COC (n)
PZ (n)
Cleavage (%)
8–16 (%)
Blast (%)
CON ET P-value Lab Con
2 2
59.5 5.4 59.3 5.9 0.99 NA
200 200
169 143
100
86
69.2 3.3a 51.1 3.3b 0.001 74.4
75.2 6.3 64.4 6.3 0.11 67.2
22.2 3.1 22.0 3.1 0.96 43.3
2
REP: total number of replications per bull (Replicate 1, May 5; Replicate 2, June 28); Motility after percoll: proportion of motile sperm after percoll purification; COC: cumulus oocyte complexes; PZ: number of putative zygotes placed in culture after denuding COC; Cleavage: number of putative zygotes cleaved; 8–16 cell: number of cleaved embryos developing to an 8–16 cell stage; Blast: blastocyst, percentage of cleaved embryos developing to the blastocyst stage. a,b Least squares means differ within a column.
4. Discussion 4.1. Backfat thickness, average daily gain, rectal temperature, and prolactin Fescue toxicosis is derived from ingestion of over 40 different alkaloids produced when tall fescue is infected with N. coenophialum; therefore, administration of one synthetic compound (ET) may not be indicative of the full spectrum of effects of fescue toxicosis on male fertility. Common signs of fescue toxicosis include elevated RT, reduction in average daily gain, vasoconstriction of blood vessels, and lowered prolactin concentrations during endophyte exposure. Bulls in the current study given ET exhibited signs of fescue toxicosis including elevated RT and vasoconstriction (reduced scrotal temperature). However, neither prolactin nor testosterone concentrations declined during the experimental period
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in bulls receiving ET compared to control bulls. Moreover, recent data from bulls grazing endophyte-infected pastures also indicated little change in concentrations of prolactin compared to severe reductions observed in companion steers grazing the same pasture (unpublished data, Waller JC, Schrick FN). A consistent measurable effect of fescue toxicosis is decreased serum prolactin [3,18– 20]. However, during ET administration, prolactin secretion did not decline in bulls receiving ET. Concentrations of prolactin have been shown to vary among studies utilizing ET to simulate effects of toxic fescue on plasma concentrations of prolactin [4–6,21–23]. Species and mode of ET administration have been shown to influence the ability of ET to inhibit prolactin secretion [4,21]. While plasma prolactin concentrations did not differ between ET and CON bulls, other effects of ET such as elevated rectal temperatures and vasoconstriction of extremities were still apparent. Weight loss associated with fescue toxicosis impaired reproductive performance in livestock [24,25]. During administration of ET, bulls were offered a diet of 2.5% body weight of feed (on a dry matter basis), which was formulated for a 0.850 kg gain/day throughout the experimental period. However, neither average daily gain nor backfat thickness differed between treatment groups. Rectal temperatures increased during exposure to ET, apparently due to impaired ability to dissipate excess heat to extremities and altered thyroid function [18]. Under normal physiological conditions, mammals attempt to dissipate heat by vasodilatation of capillary beds. By increasing blood flow to the extremities, core temperatures decline, reducing overall body temperature. Vasodilatation, however slight, is the normal response to elevated temperature. In vitro studies confirmed that treatment with ergot alkaloids induced vasoconstriction of the dorsal pedal veins [26]. Animals affected by fescue toxicosis have decreased ability to shuttle blood away from the body core. Elevated RT observed in ETtreated bulls were similar to increased RT reported in steers given ET [19]. 4.2. Testicular response Receiving a diet supplemented with ET did not alter average daily growth in SC. Blood flow and SC are highly correlated with testicular weight and sperm production [27,28]. When considering that SC did not differ between treatments, it is not surprising that serum concentrations of testosterone did not significantly decline during administration of ET [29]. In addition, concentrations of testosterone paralleled those of Evans et al. [30]; they found no reduction in testosterone concentrations in bull calves fed with endophyteinfected fescue hay. Scrotal temperature of bulls fed a diet containing ET were 1.4 8C cooler than control bulls. Vasoconstriction occurs under the influence of ergot alkaloids [31]; thus, possibly inhibiting blood flow to the testicles resulting in cooler testicular temperatures in those bulls receiving ET. Clark et al. [31] found ergotamine-restricted normal blood flow within capillaries by as much as 10 times that of normal flow. Constriction of peripheral capillary beds would reasonably explain a decrease in peripheral temperatures, including scrotal temperatures. Effects of ET on scrotal temperatures are of significant importance in spermatogenesis. Impaired thermoregulation due to reduced blood flow of the scrotum and testicles can cause infertility [32].
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Vasoconstriction has been attributed to an interaction of the ergot alkaloids with adrenergic, dopaminergic, and sertonergic receptors. Tall fescue also produces a chemical associated with the endophyte fungus known as lysergamide that also acts as a further agonist or antagonist to induce vasoconstriction [33]. Oliver et al. [34] determined that lysergamide-produced vasoconstriction in vitro; however, the degree of contractility was dose dependent. Testicular temperature must be maintained between 30 and 34 8C, i.e. between 2 and 4 8C cooler than body cavity temperature, in order not to impair spermatogenesis [10,35]. Bulls given ET had significantly lowered scrotal temperatures than that of control bulls; however, scrotal temperature remained within parameters. While numerous studies have illustrated the detrimental effects of elevated temperatures on spermatozoa [26,36], few studies have reported effects of lowered testicular temperatures on sperm integrity and subsequent fertilization. Moreover, the molecular aspects of the sperm affected by lowered testicular temperature are yet to be examined in cattle. 4.3. Semen morphology and fertilization Utilization of IVF provided an optimal environment to determine fertilization potential associated with administration of ET. Gross motility and morphology of spermatozoa were not different in ET bulls compared to CON during the experimental period; however, ability of oocytes to cleave after IVF with sperm exposed to ET may have been compromised. That oocytes fertilized with sperm from bulls exhibiting signs of simulated fescue toxicosis did not cleave as well as those fertilized with CON sperm implies that ET could produce ‘ultrastructural’ damage to sperm that were not manifest as gross morphological diagnostics. It is important to note that IVF was carried out on a limited number of animals, and further investigation is warranted and being conducted. Further illustrating that sperm damage may occur without recognition, Austin [37] reported that sperm have the ability to remain motile much longer than their ability to maintain fertilization capacity; thus, motility may remain high but sperm may be unable to penetrate the zona pellucida. The ability of putative zygotes (PZ) that cleaved to further progress to blastocyst suggested that while administration of ET may compromise cleavage, sperm integrity was not completely destroyed. 4.4. Summary and implications Bulls given ET exhibited symptoms of fescue toxicosis. Rectal temperatures were elevated in ET-treated bulls; however, concentrations of prolactin did not differ between ET-treated and CON bulls. Scrotal temperature declined with administration of ET, suggesting that supplementation with ET induced two signs of fescue toxicosis measured in the current study: altered body temperature and vasoconstriction, but not the reduction in serum prolactin concentrations. Furthermore, gross motility and morphology of semen from bulls given ET were not compromised. However, cleavage rates of putative zygotes were depressed when utilizing sperm from bulls exposed to ET for IVF; subsequent developmental competence of embryos was not compromised. It was noteworthy that IVF
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was carried out on a limited basis, and further investigation is warranted. We inferred that while gross sperm motility and morphology remained unchanged, perhaps undetected ‘ultrastructural’ damage to sperm components may have been caused by ET administration. In conclusion, although fertility was only slightly affected, ET may damage sperm in ways undetectable with the methods used in the present study.
Acknowledgments Authors wish to thank the UT Highland Rim Experiment Station for assistance in conducting the current study. Authors also want to thank Dr. Gustave Hansen for providing Bioxcell extender and semen storage straws for the study.
References [1] Porter JK. Analysis of endophyte toxins: fescue and other grasses toxic to livestock. J Anim Sci 1995;73:871–80. [2] Paterson J, Forcherio C, Larson B, Samford M, Kerley M. The effects of fescue toxicosis on beef-cattle productivity. J Anim Sci 1995;73:889–98. [3] Browning Jr R, Leite-Browning ML, Smith HM, Wakefield Jr T. Effects of ergotamine and ergonovine on plasma concentrations of thyroid hormones and cortisol in cattle. J Anim Sci 1998;76:1644–50. [4] Browning Jr R, Thompson FN, Sartin JL, Browning ML. Plasma concentrations of prolactin, growth hormone, and luteinizing hormone in steers administered ergotamine or ergonovine. J Anim Sci 1997;75:796–802. [5] Hockett ME, Towns TM, Edwards JL, Rohrbach NR, Schrick FN. Embryonic development in beef cattle administered ergotamine tartrate to stimulate fescue toxicosis (abstract). J Anim Sci 2000; 78(Suppl 1):199. [6] Seals RC, Schrick FN, Hopkins FM, Waller JC, Fribourg HA. Follicular dynamics in beef heifers consuming ergotamine tartrate as a model of endophyte-infected tall fescue consumption (abstract). J Anim Sci 1996;74(Suppl 1):11. [7] Hoveland CS. Economic importance of Acremonium endophytes. Agric Ecosyst Environ 1993;44:3. [8] NCR. Nutrient requirements of beef cattle. 7th ed. Washington DC: National Academy Press; 1996. [9] Fluckinger EE, Del Pozo E, Richardson BP. Influence of endocrine system. In: Schild IB, Berde B, editors. Handbook of experimental pharmacology. Berlin: Springer; 1978. p. 615–90. [10] Purohit RC, Hudson RS, Riddell MG, Carson RL, Wolfe DF, Walker DF. Thermography of the bovine scrotum. Am J Vet Res 1985;46:2388–92. [11] Spitzer JC, Hopkins FM, Webster HW, Kirkpatrick FD, Hill HS. Breeding soundness examination in yearling beef bulls. J Am Vet Med Assoc 1988;193:1075–9. [12] Edwards JL, Hansen PJ. Elevated temperature increases heat shock protein 70 synthesis in bovine two-cell embryos and compromises function of maturing oocytes. Biol Reprod 1996;55:340–6. [13] Parrish JJ, Suskoparrish J, Winer MA, First NL. Capacitation of bovine sperm by heparin. Biol Reprod 1988;38:1171–80. [14] Biggers JD, McGinnis LK, Raffin M. Amino acids and preimplantation development of the mouse in proteinfree potassium simplex optimized medium. Biol Reprod 2000;63:281–93. [15] Towns TM, Schrick FN, Hopkins FM, Kirkpatrick FD, Saxton AM, Thompson KW, et al. Relationship of leptin and puberty in performance-tested bulls (abstract). J Anim Sci 2000;78(Suppl 1):204. [16] Moura AA, Erickson BH. Age-related charges in peripheral hormone concentrations and their relationships with testis size and number of Sertoli and germ cells in beef bulls. J Reprod Fertil 1997;111:183–90. [17] SAS/STAT User’s Guide. Cary, NC; 2000.
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[18] Oliver JW. Physiology manifestations of endophyte toxicosis in ruminant and laboratory species. In: Bacon CW, Hill NS, editors. Proceedings of the Third International Symposium on Neotyphodium/Grass Interaction1997. p. 311–46. [19] Osborn TG, Schmidt SP, Marple DN, Rahe CH, Steenstra JR. Effect of consuming fungus-infected and fungus-free tall fescue and ergotamine tartrate on selected physiological variables of cattle in environmentally controlled conditions. J Anim Sci 1992;70:2501–9. [20] Porter JK, Thompson FN. Effects of fescue toxicosis on reproduction in livestock. J Anim Sci 1992;70:1594– 603. [21] Rice RL, Blodgett DJ, Schurig GG, Swecker WS, Thatcher CD, Eversole DE. Oral and parental vaccination of mice with protein–ergotamine conjugate and evaluation of protection against fescue toxicosis. Vet Immunol Immunopath 1997;61:305–16. [22] Browning Jr R. Physiological responses of Brahman and Hereford steers to an acute ergotamine challenge. J Anim Sci 2000;78:124–30. [23] Browning Jr R, Thompson FN. Endocrine and respiratory responses to ergotamine in Brahman and Hereford steers. Vet Hum Toxicol 2002;44:149–54. [24] Hemken RW, Bull LS, Boling JA, Kane E, Bush LP, Buckner RC. Summer fescue toxicosis in lactating dairy cows and sheep fed experimental strains of ryegrass-tall fescue hybrids. J Anim Sci 1979;49:641–6. [25] Washburn SP, Green Jr JT, Johnson BH. Effects of endophyte presence in tall fescue on growth, puberty, and conception in Angus heifers. In: Tall Fescue Toxicosis Workshop; 1989. p. 80. [26] Solomons RN, Oliver JW, Linnabary RD. Dorsal pedal vein of cattle: reactivity to selected alkaloids associated with Acremonium coenophialum-infected fescue grass. Am J Vet Res 1989;50:235–8. [27] Coulter GH, Foote RH. Bovine testicular measurements as indicators of reproductive-performance and their relationship to productive traits in cattle. Theriogenology 1979;11:297–311. [28] Wang YH, Huang TS, Lin MC, Yeh CS, Lien IN. Scrotal temperature in spinal-cord injury. Am J Physic Med Rehab 1993;72:6–9. [29] Veeramachaneni DN, Ott RS, Heath EH, McEntee K, Bolt DJ, Hixon JE. Pathophysiology of small testes in beef bulls: relationship between scrotal circumference, histopathologic features of testes and epididymides, seminal characteristics, and endocrine profiles. Am J Vet Res 1986;47:1988–99. [30] Evans KL, Zavos PM, Hemken RW, Jackson JA. Effects of feeding endophyte-infected (Acremonium coenophialum) KY-31 fescue hay on the reproductive development of Holstein bulls. Theriogenology 1988;30:169–79. [31] Clark BJ, Chu D, Aellig WH. Actions on the heart and circulation. In: Berde B, Schild IB, editors. Handbook of Experimental Pharmacology. Berlin: Springer; 1978. p. 321–420. [32] Gazvani MR, Wood SJ, Thomson AJM. Kingsland CR, Lewis-Jones DI. Assessment of testicular core temperatures using microwave thermography. Hum Reprod 2000;5:1723–6. [33] Bacon CW, De Battista J. Endophytic fungi of grasses. In: Arora DK, Rai BR, Mukerji KG, Knudsen GR, editors. Handbook of applied mycology. New York: Marcel Dekker; 1991. p. 231–56. [34] Oliver JW, Abney LK, Strickland JR, Linnabary RD. Vasoconstriction in bovine vasculature induced by the tall fescue alkaloid lysergamide. J Anim Sci 1993;71:2708–13. [35] Harrison R, Weiner J. Abdominal–testicular temperature gradients. J Physiol 1948;107:48–58. [36] Setchell BP, Bergh A, Widmark A, Damber JE. Effect of testicular temperature on vasomotion and bloodflow. J Androl 1995;18:120–6. [37] Austin CR. Sperm fertility, viability and persistence in the female tract. J Reprod Fertil 1975;22:75–89.
Effects of administration of ergotamine tartrate on fertility of yearling beef bulls Gustavo M. Schuenemanna, J. Lannett Edwardsa, Mark D. Davisa, Heather E. Blackmona, Fernando N. Scennaa, Nancy R. Rohrbacha, Arnold M. Saxtona, H. Stephen Adairb, Fred M. Hopkinsb, John C. Wallera, F. Neal Schricka,c,* a
Department of Animal Science, University of Tennessee, Knoxville, USA Department of Large Animal Clinical Sciences, University of Tennessee, Knoxville, USA c 205 C Brehm Animal Science Building, University of Tennessee, Knoxville, TN 37996-4574, USA b
Received 31 December 2003; accepted 4 July 2004
Abstract Sixteen yearling bulls were utilized to investigate administration of ergotamine tartrate on semen parameters, fertilization, and endocrinology. Bulls were allotted to a control diet of cracked corn, corn silage, and soybean meal (CON, n = 8) or a diet supplemented daily with 40 mg/kg body weight of ergotamine tartrate (ET, n = 8). Blood samples, average daily gain, scrotal circumference and rectal temperatures were collected every 14 day. Semen samples were obtained every 60 day and evaluated for motility and morphology. Scrotal temperatures were obtained by thermography immediately before electroejaculation. Semen from a subset of bulls from each treatment was also evaluated for in vitro fertilization. Administration of ET increased rectal temperature and resulted in lower scrotal temperatures compared to CON bulls (P < 0.05). However, prolactin, scrotal circumference, testosterone, and semen motility and morphology did not differ between groups throughout the experimental period (224 day). Cleavage rates of embryos derived from in vitro fertilization (IVF) with semen of bulls, fed with ET, were reduced compared to CON (P < 0.05); however, development of cleaved embryos to blastocyst did not differ between treatments. In conclusion, extended exposure of bulls to ET appeared to reduce fertilization potential of sperm. # 2004 Elsevier Inc. All rights reserved. Keywords: Fescue toxicosis; Ergotamine tartrate; Bull; Spermatozoa; Fertility
* Corresponding author. Tel.: +1 865 974 3147; fax: +1 865 974 7297. E-mail address: [email protected] (F.N. Schrick). 0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2004.07.014
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1. Introduction Fescue toxicosis results from ingestion of tall fescue infested with the endophyte, Neotyphodium coenophialum. Prevailing signs such as elevated body temperature, vasoconstriction of capillaries, and reproductive problems associated with fescue toxicity have contributed to massive declines in overall productivity. Mechanisms by which fescue toxicosis reduces cattle performance are not clearly understood but appear to involve various alkaloid compounds [1]. Consumption of endophytic tall fescue lowered nutrient intake and digestibility [2]. Fescue toxicosis affects reproductive performance, growth, and lactation [1]. Acute exposure of steers to an elevated dose of systemically delivered ergotamine elevated plasma concentrations of cortisol and triiodothyronine [3]; these hormones are involved in mediating metabolic processes and subsequent nutrient utilization. Fescue toxicosis has also been linked to reduced LH concentrations [4], compromised embryonic development [5], and reductions in pregnancy rates [6]. Hoveland [7] calculated that producers lose $ 609 million annually due to decreased calf gains and lowered conception rates associated with fescue toxicosis. While research has clearly documented how fescue toxicity affects female reproduction, few studies have examined the effects of fescue toxicosis on male reproductive performance. The objective of the present study was to investigate effects of simulated fescue toxicosis (administration of ergotamine tartrate(ET)) on semen parameters, endocrine profiles, and developmental competence of oocytes fertilized in vitro with semen from bulls fed with ET.
2. Materials and methods 2.1. Animals and treatments Sixteen Angus crossbred bulls were utilized to determine effects of ET administration on semen parameters, endocrine profiles and in vitro fertilization (IVF). Bulls, approximately 350 kg body weight and 270 day of age, were assigned to one of two groups. Breed composition, weaning weight, hip height, scrotal circumference (SC), and age were used to allot bulls to treatment groups. Prior to experimental period (224 day), bulls were weaned at the beginning of September and fed corn silage. From mid-November to the end of June in central Tennessee, bulls were fed a diet of corn silage, cracked corn, and soybean meal formulated to meet NRC [8] requirements for gain. Both groups of bulls were offered a diet of 2.5% body weight of feed (on a dry matter basis), which was formulated for a 0.850 kg gain/day and to be isonitrogenous and isocaloric throughout the experimental period. Bulls limited to this diet served as controls (CON, n = 8). The base diet was supplemented daily with 40 mg/kg body weight of ET for the treatment group (ET, n = 8; provided by Dr. Miroslav Flieger, Institute of Microbiology, Czech Republic). In order to maintain a dose of 40 mg/kg body weight, the amount of ET administered was increased as body weight (BW) increased. Water and minerals were available ad libitum. ET was administered to simulate fescue toxicity, since it mimics the action of the natural
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endophyte, ergovaline, but has only 10% of the potency of ergovaline [9]. A guideline for the ET dose was derived from previous studies [5,6]. Bulls were housed in pens with four bulls per pen. Bulls were fed ET at 12:00 h and blood samples and other data were collected from 06:00 to 09:00 h. The ambient temperature ranged from 4 8C in January to 17 8C in June. Every 14 day, average daily gain (ADG), SC (Lane Manufacturing Co. Denver, CO, USA), and rectal temperature (RT; GLA Agricultural Electronics; San Luis, Obispo, CA, USA) were measured. Blood samples (10 mL) were also collected via caudal venipuncture and placed on ice until centrifuged. Backfat thickness was measured (at initiation, middle, and end of the experimental period) by a certified technician utilizing ultrasonography (Aloka 500 V with 3.5 MHz backfat transducer; Corometrics Medical Systems Inc., Wallingford, CT, USA). Scrotal temperatures were determined prior to semen collection in May and June using an infrared thermography [10] camera (Emerge Vision DTIS 500, Emerge International Inc., Sebastian, FL, USA). The camera had an opaque chopper and internal calibration that allowed determination of absolute temperatures. Recorded images were analyzed by EResearch software. In brief, images were imported into the software and a region of interest (ROI) was drawn on each testis. The same region was taken on each testis and each ROI contained the same number of pixels. This information allowed the program to calculate absolute temperature for each ROI. 2.2. Semen collection Semen was evaluated approximately 60 day from initiation of ET administration and at 60-day intervals. Bulls were collected individually, with technician and semen evaluator having no knowledge of treatment. Semen was collected using an electroejaculator (Lane Manufacturing Co., Denver, CO, USA) and placed in a 10 mL sterile conical tube. An estimate of progressively motile sperm was obtained using a light microscope at 400. Approximately 25 mL of sperm was placed on a warmed slide and covered with a cover slip to assess motility. Another sample of sperm (approximately 25 mL) was placed on a slide, mixed with eosin–nigrosin stain, smeared and allowed to dry. Morphology was evaluated under oil immersion at 1000 using a light microscope [11]. Spermatozoa were classified as having normal morphology or as having primary or secondary abnormalities [11]. After motility and morphology examinations, semen was diluted with Bioxcell extender solution. Semen extender and straws were provided by Dr. Gustave Hansen (IMV International Technologies, France). Extender (100 mL) and sterile water (400 mL) were warmed to approximately 32 8C in a water bath for 10 min prior to use. Once the temperature of water had equilibrated, 100 mL of extender was added to the water and the solution mixed thoroughly. Semen was extended at a 1:1 mL semen/ extender ratio. Diluted semen remained at 32 8C for 10 min and then at room temperature (25 8C) for 15 min. Extended semen was packaged in Bioxcell straws horizontally and placed in a cooler with cold packs for transport. At the laboratory, straws of extended semen were refrigerated at 4 8C until IVF of oocytes, approximately 26 h after semen collection.
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2.3. In vitro evaluation of semen From both groups, collected semen from the same bulls (n = 2/trt) having the largest and smallest scrotal circumference were extended (two straws/bull) at the location and returned to the laboratory for further semen assessment. Semen was collected from the same bulls (one bull/pen) for both collecting days (May 5 and June 28). Mean SC from bulls used for in vitro evaluation of semen were 39.4 and 39.5 cm at the end of the experimental period (June 28) for CON and ET, respectively. The procedures utilized for in vitro production (IVP) of embryos were modifications of procedures previously described by Edwards and Hansen [12]. Medium 199, gentamicin, and penicillin–streptomycin were purchased from Specialty Media, Inc. (Phillipsburg, NJ, USA). Fetal bovine serum (FBS) was obtained from BioWhittaker (Walkersville, MD, USA). Folltropin-V was provided by Vetrepharm Canada, Inc. (London, Ontario, Canada) and luteinizing hormone (LH) was obtained from the United States Department of Agriculture (Beltsville, MD, USA). Media [13] (HEPESTALP, IVF-TALP, and SPERM-TALP) and KSOM [14], with modifications provided by Dr. John Hasler (personal communication), were prepared in the laboratory or purchased from Specialty Media. Ovaries obtained from an abattoir (Brown’s Packing Company; Gaffney, SC, USA) were packaged in a thermos and contained within a cooler during air transport to the laboratory (on the day of collection). Upon arrival, ovaries were immediately washed with warm water, equilibrated to arrival temperature of ovaries (generally 28–30 8C). Extraneous tissue surrounding the ovaries was removed and ovaries were washed with water one additional time. For oocyte recovery performed within 4 h of ovary collection at the abattoir, ovaries were held firmly by clamping the base of the ovary with a hemostat and checkerboard incisions were made (with a scalpel) across follicles (approximately 3–8 mm in diameter). Ovaries were washed vigorously in oocyte collection medium (OCM) to remove cumulus oocyte complexes (COC). The collection medium was filtered and rinsed using an Emcon Filter unit (Vet Concepts, Spring Valley, WI, USA) until the medium appeared clean. Medium containing COC was poured into a gridded culture dish to facilitate oocyte retrieval. Cumulus oocyte complexes were transferred to a plate with OCM and washed four times to eliminate cellular debris. Cumulus oocyte complexes of good quality were washed in oocyte maturation medium (OMM) and placed in groups of approximately 50 COC per well in a 4-well plate containing 500 mL OMM. Maturation of oocytes was performed in an incubator at 5.5% CO2 in air at 38.5 8C until fertilization (approximately 22 h after placement in OMM). Maturation medium was equilibrated in the incubator (5.5% CO2 in air at 38.5 8C) for a minimum of 5 h before oocyte collection. At 22–24 h after oocyte collection, maturation medium from each well was removed and 25 mL of penicillamine/hypotaurine/epinephrine (PHE) and 500 mL of fertilization medium (IVF-TALP) were added per well. Two straws of semen from two bulls known to have high fertility were used to fertilize oocytes for every replicate of the study (served as a laboratory control). Briefly, semen straws were removed from liquid nitrogen tank and placed in water at 36.7 8C for 45 s. Straws with collected semen and straws from laboratory control were then emptied on top of a discontinuous percoll density gradient (2 mL of 45% percoll over 2 mL of 90% percoll contained in a 15 mL conical tube) and semen was
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centrifuged at 2200 rpm for 15 min to remove excess extender, debris, and non-motile sperm prior to fertilization. The sperm pellet present at the bottom of the 90% fraction was collected and transferred to10 mL SPERM-TALP [13] and centrifuged at 1100 rpm for 8 min. The supernatant was removed and the sperm pellet re-suspended in 500 mL of modified IVF-TALP. After percoll purification, sperm concentration was determined with a hemacytometer and motility was also determined. A sperm concentration of 375,000/ 500 mL IVF-TALP collected from bulls within each group was added to the oocytes, one bull/well. A minimum of 50 oocytes was used for each bull on each collection date. Approximately 18–22 h post-fertilization, putative zygotes (PZ) were denuded of cumulus cells by vortexing. Putative zygotes were transferred to a 15 mL conical tube containing 500 mL of HEPES-TALP and vortexed for 4 min. Recovered putative zygotes were washed four times in HEPES-TALP and once in KSOM-BSA before transferring groups of approximately 50 zygotes to 4-well plates containing 500 mL of KSOM-BSA per well. Zygotes were placed in a humidified atmosphere of 5.5% CO2, 7% O2, and 87.5% N2 at 38.5 8C. The ability of PZ to cleave and develop to the blastocyst stage was evaluated on days 3 and 8, respectively (day 0 = day of IVF). Ability of the oocyte to cleave after IVF was assessed by recording the number of 1-, 2-, 4-, and 8 to 16 cell embryos present on day 3 (70–75 h post-insemination). 2.4. Blood collection and radioimmunoassays Blood samples were centrifuged at 2000 g for 30 min and serum decanted and stored at 20 8C until assayed for prolactin and testosterone. Radioimmunoassays (Coat-ACount; Diagnostic Products Corporation; Los Angeles, CA, USA) were performed to determine concentrations of testosterone [15]. Sensitivity of the testosterone assay was 0.04 ng/mL, with intra- and inter-assay coefficients of variation (CV) of 10 and 2%, respectively. Radioimmunoassay for concentrations of prolactin was performed as described by Moura and Erickson [16]. Sensitivity of prolactin assay was determined to be 0.05 ng/mL. Intra- and inter-assay CV were 11 and 10%, respectively. 2.5. Statistical analyses Differences in ADG, backfat, RT, plasma concentrations of prolactin and testosterone, SC, scrotal temperatures, and motility and morphology of semen were analyzed by SAS Proc Mixed [17]. Data are presented as the least squares means (S.E.M.). A mixed model procedure that included treatment, time, pen, and all interactions as fixed effects, was used to compare differences among treatments. Time was a repeated measures factor and animal (treatment pen) was included as a random effect. Differences among least squares means were evaluated using L.S.D. Comparisons between ET and CON treatments for IVF studies were made with Proc Mixed [17] in two different replicates (May 5 and June 28), using well as the experimental unit. For these IVF data, the key biological component was the oocyte, and experimental design addressed the large variation among oocytes. Thus, the experimental unit was a well, containing a group of similar oocytes, and treatments (semen) were applied to the wells. Bulls were simply acting as a source of treated material. We do recognize the potential risk in bulls responding differently, but our experience
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suggests that bulls show a consistent response to ET. In short, the IVF experiment needs to be viewed as an ‘assay’, where biological material was collected from some source, and the actual experiment was under carefully controlled laboratory conditions. Distributional requirements of normality and equal variance were verified for the percentage data. A value of P < 0.05 was considered significant.
3. Results 3.1. Backfat thickness, rectal temperature, ADG, and prolactin Throughout the duration of experimental period, average daily gain did not differ between groups (1.05 + 0.04 kg/day versus 1.1 + 0.04 kg/day; P > 0.10). Furthermore, there were no differences in backfat thickness between ET and CON bulls (5.3 0.12 mm versus 5.3 0.12 mm; P > 0.10). An overall increase in RT was observed in ET bulls (39.4 + 0.06 8C) compared to CON bulls (39.0 + 0.04 8C; P < 0.05), indicating that bulls were affected by administration of ET to simulate fescue toxicosis. Moreover, rectal temperatures were elevated at the time of semen collection in bulls given ET compared to CON (P < 0.05; Fig. 1). Bulls given ET (70.8 + 7.8 ng/mL) had similar plasma concentrations of prolactin compared to CON bulls (65.9 + 7.8 ng/mL; P > 0.10). 3.2. Testicular response Neither average daily growth in SC (0.034 + 0.002 cm/day; P > 0.10) nor concentrations of testosterone (8.38 + 1.1 ng/mL; P > 0.10) were affected by ET
Fig. 1. Rectal temperatures at semen evaluation. Rectal temperatures were elevated in bulls given ergotamine tartrate (ET) compared to control (CON) animals at each time of semen collection (a,bleast squares means differ within treatments; P < 0.05).
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Fig. 2. Sperm motility measured at the time of semen collection did not differ between bulls given ergotamine tartrate (ET) and control (CON) bulls. However, there was a collection date sperm motility interaction attributed to age of the bulls (a,bleast squares means differ within times of collections; P < 0.05).
administration. Furthermore, sperm motility was not compromised by ET (P > 0.10). However, due to the age of bulls at first collection, motility of first ejaculate was impaired compared to later collection dates (P < 0.05; Fig. 2). Morphological evaluation of semen indicated that extended exposure to ET did not alter the percentage of normal sperm or the percentage of primary and secondary abnormalities (P > 0.10; Table 1). Bulls exposed to ET had lower scrotal temperatures (31.6 + 0.3 8C) compared to CON bulls (33.0 + 0.2 8C; P < 0.05; Fig. 3); however, a date effect (May 5 and June 28) was not observed. Moreover, oocytes fertilized with semen from bulls administered ET had reduced cleavage rates compared to those fertilized with sperm from CON bulls (P < 0.05; Table 2); however, ability of cleaved embryos to develop to an 8–16-cell stage was not different (P > 0.10; Table 2). The percentage of cleaved embryos that continued in development to blastocyst did not differ between treatments (P > 0.10; Table 2). Replicates also did not differ between ET and CON bulls (P > 0.10). As stated previously, SC from bulls used for in vitro evaluation of semen did not differ (39.4 and 39.5 cm) at the end of the experimental period (June 28) for CON and ET, respectively.
Table 1 Sperm (%) with normal and abnormal morphology in control (CON) bulls and those treated with ergotamine tartrate (ET) Sperm
Normal
Primary abnormalitiesa
Secondary abnormalitiesb
CON ET P-value
76.9 3 73.9 3 0.51
13.5 2 16.8 2 0.33
9.5 1.4 10.9 1.4 0.54
a b
Abnormal head shape, midpiece, or presence of proximal protoplasmic droplets. Detached heads, distal protoplasmic droplets, or bent tails.
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Fig. 3. Scrotal thermography at semen collection (May 5 and June 28). Scrotal temperatures were lower in bulls given ergotamine tartrate (ET) compared to control (CON) animals (a,bleast squares means differ within treatments; P < 0.05).
Table 2 Ability of sperm collected from bulls fed a control (CON, n = 2) or ergotamine tartrate-supplemented (ET, n = 2) diet to fertilize bovine oocytes in vitro TRT
REP (n)
Motility after percoll
COC (n)
PZ (n)
Cleavage (%)
8–16 (%)
Blast (%)
CON ET P-value Lab Con
2 2
59.5 5.4 59.3 5.9 0.99 NA
200 200
169 143
100
86
69.2 3.3a 51.1 3.3b 0.001 74.4
75.2 6.3 64.4 6.3 0.11 67.2
22.2 3.1 22.0 3.1 0.96 43.3
2
REP: total number of replications per bull (Replicate 1, May 5; Replicate 2, June 28); Motility after percoll: proportion of motile sperm after percoll purification; COC: cumulus oocyte complexes; PZ: number of putative zygotes placed in culture after denuding COC; Cleavage: number of putative zygotes cleaved; 8–16 cell: number of cleaved embryos developing to an 8–16 cell stage; Blast: blastocyst, percentage of cleaved embryos developing to the blastocyst stage. a,b Least squares means differ within a column.
4. Discussion 4.1. Backfat thickness, average daily gain, rectal temperature, and prolactin Fescue toxicosis is derived from ingestion of over 40 different alkaloids produced when tall fescue is infected with N. coenophialum; therefore, administration of one synthetic compound (ET) may not be indicative of the full spectrum of effects of fescue toxicosis on male fertility. Common signs of fescue toxicosis include elevated RT, reduction in average daily gain, vasoconstriction of blood vessels, and lowered prolactin concentrations during endophyte exposure. Bulls in the current study given ET exhibited signs of fescue toxicosis including elevated RT and vasoconstriction (reduced scrotal temperature). However, neither prolactin nor testosterone concentrations declined during the experimental period
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in bulls receiving ET compared to control bulls. Moreover, recent data from bulls grazing endophyte-infected pastures also indicated little change in concentrations of prolactin compared to severe reductions observed in companion steers grazing the same pasture (unpublished data, Waller JC, Schrick FN). A consistent measurable effect of fescue toxicosis is decreased serum prolactin [3,18– 20]. However, during ET administration, prolactin secretion did not decline in bulls receiving ET. Concentrations of prolactin have been shown to vary among studies utilizing ET to simulate effects of toxic fescue on plasma concentrations of prolactin [4–6,21–23]. Species and mode of ET administration have been shown to influence the ability of ET to inhibit prolactin secretion [4,21]. While plasma prolactin concentrations did not differ between ET and CON bulls, other effects of ET such as elevated rectal temperatures and vasoconstriction of extremities were still apparent. Weight loss associated with fescue toxicosis impaired reproductive performance in livestock [24,25]. During administration of ET, bulls were offered a diet of 2.5% body weight of feed (on a dry matter basis), which was formulated for a 0.850 kg gain/day throughout the experimental period. However, neither average daily gain nor backfat thickness differed between treatment groups. Rectal temperatures increased during exposure to ET, apparently due to impaired ability to dissipate excess heat to extremities and altered thyroid function [18]. Under normal physiological conditions, mammals attempt to dissipate heat by vasodilatation of capillary beds. By increasing blood flow to the extremities, core temperatures decline, reducing overall body temperature. Vasodilatation, however slight, is the normal response to elevated temperature. In vitro studies confirmed that treatment with ergot alkaloids induced vasoconstriction of the dorsal pedal veins [26]. Animals affected by fescue toxicosis have decreased ability to shuttle blood away from the body core. Elevated RT observed in ETtreated bulls were similar to increased RT reported in steers given ET [19]. 4.2. Testicular response Receiving a diet supplemented with ET did not alter average daily growth in SC. Blood flow and SC are highly correlated with testicular weight and sperm production [27,28]. When considering that SC did not differ between treatments, it is not surprising that serum concentrations of testosterone did not significantly decline during administration of ET [29]. In addition, concentrations of testosterone paralleled those of Evans et al. [30]; they found no reduction in testosterone concentrations in bull calves fed with endophyteinfected fescue hay. Scrotal temperature of bulls fed a diet containing ET were 1.4 8C cooler than control bulls. Vasoconstriction occurs under the influence of ergot alkaloids [31]; thus, possibly inhibiting blood flow to the testicles resulting in cooler testicular temperatures in those bulls receiving ET. Clark et al. [31] found ergotamine-restricted normal blood flow within capillaries by as much as 10 times that of normal flow. Constriction of peripheral capillary beds would reasonably explain a decrease in peripheral temperatures, including scrotal temperatures. Effects of ET on scrotal temperatures are of significant importance in spermatogenesis. Impaired thermoregulation due to reduced blood flow of the scrotum and testicles can cause infertility [32].
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Vasoconstriction has been attributed to an interaction of the ergot alkaloids with adrenergic, dopaminergic, and sertonergic receptors. Tall fescue also produces a chemical associated with the endophyte fungus known as lysergamide that also acts as a further agonist or antagonist to induce vasoconstriction [33]. Oliver et al. [34] determined that lysergamide-produced vasoconstriction in vitro; however, the degree of contractility was dose dependent. Testicular temperature must be maintained between 30 and 34 8C, i.e. between 2 and 4 8C cooler than body cavity temperature, in order not to impair spermatogenesis [10,35]. Bulls given ET had significantly lowered scrotal temperatures than that of control bulls; however, scrotal temperature remained within parameters. While numerous studies have illustrated the detrimental effects of elevated temperatures on spermatozoa [26,36], few studies have reported effects of lowered testicular temperatures on sperm integrity and subsequent fertilization. Moreover, the molecular aspects of the sperm affected by lowered testicular temperature are yet to be examined in cattle. 4.3. Semen morphology and fertilization Utilization of IVF provided an optimal environment to determine fertilization potential associated with administration of ET. Gross motility and morphology of spermatozoa were not different in ET bulls compared to CON during the experimental period; however, ability of oocytes to cleave after IVF with sperm exposed to ET may have been compromised. That oocytes fertilized with sperm from bulls exhibiting signs of simulated fescue toxicosis did not cleave as well as those fertilized with CON sperm implies that ET could produce ‘ultrastructural’ damage to sperm that were not manifest as gross morphological diagnostics. It is important to note that IVF was carried out on a limited number of animals, and further investigation is warranted and being conducted. Further illustrating that sperm damage may occur without recognition, Austin [37] reported that sperm have the ability to remain motile much longer than their ability to maintain fertilization capacity; thus, motility may remain high but sperm may be unable to penetrate the zona pellucida. The ability of putative zygotes (PZ) that cleaved to further progress to blastocyst suggested that while administration of ET may compromise cleavage, sperm integrity was not completely destroyed. 4.4. Summary and implications Bulls given ET exhibited symptoms of fescue toxicosis. Rectal temperatures were elevated in ET-treated bulls; however, concentrations of prolactin did not differ between ET-treated and CON bulls. Scrotal temperature declined with administration of ET, suggesting that supplementation with ET induced two signs of fescue toxicosis measured in the current study: altered body temperature and vasoconstriction, but not the reduction in serum prolactin concentrations. Furthermore, gross motility and morphology of semen from bulls given ET were not compromised. However, cleavage rates of putative zygotes were depressed when utilizing sperm from bulls exposed to ET for IVF; subsequent developmental competence of embryos was not compromised. It was noteworthy that IVF
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was carried out on a limited basis, and further investigation is warranted. We inferred that while gross sperm motility and morphology remained unchanged, perhaps undetected ‘ultrastructural’ damage to sperm components may have been caused by ET administration. In conclusion, although fertility was only slightly affected, ET may damage sperm in ways undetectable with the methods used in the present study.
Acknowledgments Authors wish to thank the UT Highland Rim Experiment Station for assistance in conducting the current study. Authors also want to thank Dr. Gustave Hansen for providing Bioxcell extender and semen storage straws for the study.
References [1] Porter JK. Analysis of endophyte toxins: fescue and other grasses toxic to livestock. J Anim Sci 1995;73:871–80. [2] Paterson J, Forcherio C, Larson B, Samford M, Kerley M. The effects of fescue toxicosis on beef-cattle productivity. J Anim Sci 1995;73:889–98. [3] Browning Jr R, Leite-Browning ML, Smith HM, Wakefield Jr T. Effects of ergotamine and ergonovine on plasma concentrations of thyroid hormones and cortisol in cattle. J Anim Sci 1998;76:1644–50. [4] Browning Jr R, Thompson FN, Sartin JL, Browning ML. Plasma concentrations of prolactin, growth hormone, and luteinizing hormone in steers administered ergotamine or ergonovine. J Anim Sci 1997;75:796–802. [5] Hockett ME, Towns TM, Edwards JL, Rohrbach NR, Schrick FN. Embryonic development in beef cattle administered ergotamine tartrate to stimulate fescue toxicosis (abstract). J Anim Sci 2000; 78(Suppl 1):199. [6] Seals RC, Schrick FN, Hopkins FM, Waller JC, Fribourg HA. Follicular dynamics in beef heifers consuming ergotamine tartrate as a model of endophyte-infected tall fescue consumption (abstract). J Anim Sci 1996;74(Suppl 1):11. [7] Hoveland CS. Economic importance of Acremonium endophytes. Agric Ecosyst Environ 1993;44:3. [8] NCR. Nutrient requirements of beef cattle. 7th ed. Washington DC: National Academy Press; 1996. [9] Fluckinger EE, Del Pozo E, Richardson BP. Influence of endocrine system. In: Schild IB, Berde B, editors. Handbook of experimental pharmacology. Berlin: Springer; 1978. p. 615–90. [10] Purohit RC, Hudson RS, Riddell MG, Carson RL, Wolfe DF, Walker DF. Thermography of the bovine scrotum. Am J Vet Res 1985;46:2388–92. [11] Spitzer JC, Hopkins FM, Webster HW, Kirkpatrick FD, Hill HS. Breeding soundness examination in yearling beef bulls. J Am Vet Med Assoc 1988;193:1075–9. [12] Edwards JL, Hansen PJ. Elevated temperature increases heat shock protein 70 synthesis in bovine two-cell embryos and compromises function of maturing oocytes. Biol Reprod 1996;55:340–6. [13] Parrish JJ, Suskoparrish J, Winer MA, First NL. Capacitation of bovine sperm by heparin. Biol Reprod 1988;38:1171–80. [14] Biggers JD, McGinnis LK, Raffin M. Amino acids and preimplantation development of the mouse in proteinfree potassium simplex optimized medium. Biol Reprod 2000;63:281–93. [15] Towns TM, Schrick FN, Hopkins FM, Kirkpatrick FD, Saxton AM, Thompson KW, et al. Relationship of leptin and puberty in performance-tested bulls (abstract). J Anim Sci 2000;78(Suppl 1):204. [16] Moura AA, Erickson BH. Age-related charges in peripheral hormone concentrations and their relationships with testis size and number of Sertoli and germ cells in beef bulls. J Reprod Fertil 1997;111:183–90. [17] SAS/STAT User’s Guide. Cary, NC; 2000.
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G.M. Schuenemann et al. / Theriogenology 63 (2005) 1407–1418
[18] Oliver JW. Physiology manifestations of endophyte toxicosis in ruminant and laboratory species. In: Bacon CW, Hill NS, editors. Proceedings of the Third International Symposium on Neotyphodium/Grass Interaction1997. p. 311–46. [19] Osborn TG, Schmidt SP, Marple DN, Rahe CH, Steenstra JR. Effect of consuming fungus-infected and fungus-free tall fescue and ergotamine tartrate on selected physiological variables of cattle in environmentally controlled conditions. J Anim Sci 1992;70:2501–9. [20] Porter JK, Thompson FN. Effects of fescue toxicosis on reproduction in livestock. J Anim Sci 1992;70:1594– 603. [21] Rice RL, Blodgett DJ, Schurig GG, Swecker WS, Thatcher CD, Eversole DE. Oral and parental vaccination of mice with protein–ergotamine conjugate and evaluation of protection against fescue toxicosis. Vet Immunol Immunopath 1997;61:305–16. [22] Browning Jr R. Physiological responses of Brahman and Hereford steers to an acute ergotamine challenge. J Anim Sci 2000;78:124–30. [23] Browning Jr R, Thompson FN. Endocrine and respiratory responses to ergotamine in Brahman and Hereford steers. Vet Hum Toxicol 2002;44:149–54. [24] Hemken RW, Bull LS, Boling JA, Kane E, Bush LP, Buckner RC. Summer fescue toxicosis in lactating dairy cows and sheep fed experimental strains of ryegrass-tall fescue hybrids. J Anim Sci 1979;49:641–6. [25] Washburn SP, Green Jr JT, Johnson BH. Effects of endophyte presence in tall fescue on growth, puberty, and conception in Angus heifers. In: Tall Fescue Toxicosis Workshop; 1989. p. 80. [26] Solomons RN, Oliver JW, Linnabary RD. Dorsal pedal vein of cattle: reactivity to selected alkaloids associated with Acremonium coenophialum-infected fescue grass. Am J Vet Res 1989;50:235–8. [27] Coulter GH, Foote RH. Bovine testicular measurements as indicators of reproductive-performance and their relationship to productive traits in cattle. Theriogenology 1979;11:297–311. [28] Wang YH, Huang TS, Lin MC, Yeh CS, Lien IN. Scrotal temperature in spinal-cord injury. Am J Physic Med Rehab 1993;72:6–9. [29] Veeramachaneni DN, Ott RS, Heath EH, McEntee K, Bolt DJ, Hixon JE. Pathophysiology of small testes in beef bulls: relationship between scrotal circumference, histopathologic features of testes and epididymides, seminal characteristics, and endocrine profiles. Am J Vet Res 1986;47:1988–99. [30] Evans KL, Zavos PM, Hemken RW, Jackson JA. Effects of feeding endophyte-infected (Acremonium coenophialum) KY-31 fescue hay on the reproductive development of Holstein bulls. Theriogenology 1988;30:169–79. [31] Clark BJ, Chu D, Aellig WH. Actions on the heart and circulation. In: Berde B, Schild IB, editors. Handbook of Experimental Pharmacology. Berlin: Springer; 1978. p. 321–420. [32] Gazvani MR, Wood SJ, Thomson AJM. Kingsland CR, Lewis-Jones DI. Assessment of testicular core temperatures using microwave thermography. Hum Reprod 2000;5:1723–6. [33] Bacon CW, De Battista J. Endophytic fungi of grasses. In: Arora DK, Rai BR, Mukerji KG, Knudsen GR, editors. Handbook of applied mycology. New York: Marcel Dekker; 1991. p. 231–56. [34] Oliver JW, Abney LK, Strickland JR, Linnabary RD. Vasoconstriction in bovine vasculature induced by the tall fescue alkaloid lysergamide. J Anim Sci 1993;71:2708–13. [35] Harrison R, Weiner J. Abdominal–testicular temperature gradients. J Physiol 1948;107:48–58. [36] Setchell BP, Bergh A, Widmark A, Damber JE. Effect of testicular temperature on vasomotion and bloodflow. J Androl 1995;18:120–6. [37] Austin CR. Sperm fertility, viability and persistence in the female tract. J Reprod Fertil 1975;22:75–89.