Theriogenology 83 (2015) 408–414
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Bulls grazing Kentucky 31 tall fescue exhibit impaired growth, semen quality, and decreased semen freezing potential S.L. Pratt a, *, H.M. Stowe a, B.K. Whitlock b, L. Strickland c, M. Miller a, S.M. Calcatera a, M.D. Dimmick a, G.E. Aiken d, F.N. Schrick c, N.M. Long a, S.K. Duckett a, J.G. Andrae a a
Department of Animal and Veterinary Sciences, Clemson University, Clemson, South Carolina, USA Large Animal Clinical Sciences, College of Veterinary Medicine, The University of Tennessee, Knoxville, Tennessee, USA c Department of Animal Science, University of Tennessee, Knoxville, Tennessee, USA d USDA-ARS-Forage-Animal Production Research Unit, University of Kentucky Campus, Lexington, Kentucky, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 December 2013 Received in revised form 17 September 2014 Accepted 1 October 2014
Serum prolactin (PRL) and testosterone concentrations, body weight, body composition, semen quality, and semen freezing potential for bulls grazing the toxic tall fescue (Lolium arundinaceum [Schreb.] Darbysh. ¼ Schedonorous arundinaceum [Schreb.] Dumort.) cultivar Kentucky 31 (Eþ) compared with a novel endophyte cultivar lacking ergot alkaloids (E) were evaluated. Angus bulls were allotted to treatment (Day 0) and grazed Eþ or E for 155 days. Treatment-by-day interaction was significant (P < 0.05) for serum PRL concentrations with Eþtreated bulls exhibiting reduced PRL values compared with E control bulls, but no differences were observed for serum testosterone concentrations (P > 0.05). Further, bulls on the Eþ treatment exhibited decreased total gain, average daily gain, and body weight by Day 140 (P < 0.05) compared with the E bulls. Rump muscle depth was lower because the treatment in bulls grazing Eþ compared with E (P < 0.05) and intramuscular fat in the E bulls compared with the Eþ group was higher by Day 155 (P < 0.05). Analysis of ejaculates showed significant treatment day effects for sperm concentration with lower values observed for bulls on the Eþ treatment (P < 0.05). The percent normal morphology was reduced in ejaculates from Eþ bulls compared with E bulls (P < 0.05), and the difference was due to an increase in abnormal sperm present in the Eþ ejaculates from Day 84 to 140 (P < 0.05). In addition, spermatozoa motility and progressive motility were decreased on thawing in semen samples from Eþ bulls compared with E bulls (P < 0.05). Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Computerized sperm-quality analysis Scrotal circumference Fescue toxicosis Breeding soundness examination Semen freezing
1. Introduction Tall fescue (Lolium arundinaceum [Schreb.] Darbysh. ¼ Schedonorous arundinaceum [Schreb.] Dumort.) is present on approximately 16 million ha in the MidAtlantic and Southern regions of the United States [1] and
* Corresponding author. Tel.: þ1 864 656 3135; fax: þ1 864 656 3131. E-mail address: [email protected] (S.L. Pratt). 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.10.001
serves as a forage for approximately 8.5 million cattle, making this species the dominant, cool-season, perennial grass in the region. The vast majority of this forage is infected with the endophyte, Neotyphodium coenophialum. The grass exists in a mutualistic relationship with the ergot alkaloid–producing endophyte that confers disease, drought, grazing tolerance, and insect resistance to tall fescue [1]. However, ergot alkaloids negatively impact growth and/or reproductive performance of animals grazing tall fescue containing the wild-type endophyte.
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Accumulation of the ergot alkaloids within the animal’s system results in the syndrome known as fescue toxicosis, which is characterized by rough hair coat, increased core body temperature, decreased blood flow to body extremities, and poor growth and reproductive performance [2]. Previous estimates for lost beef production due to fescue toxicosis were $600 million [1], and more recent reviews estimate that total to be approaching $1 billion lost per year for cattle and small ruminants [2,3]. The most studied aspects of fescue toxicosis are the effects on animal performance [4–7]. However, there is only limited documentation of the effects of fescue toxicosis on reproductive function [8–11] and very little, if any, of fescue toxicosis on bovine male reproduction [12–16]. In an effort to begin filling this gap, our previous research evaluated the effects of fescue toxicosis on semen characteristics when bulls were fed a constant amount of ergot alkaloid in conjunction with a high-concentrate ration to delineate effects of the ergot alkaloid from possible effects of reduced body condition and plane of nutrition [16]. Little, if any, effects were noted on breeding soundness examinations (BSE) or semen characteristics. The objectives of this study were to determine the effects of grazing endophyte-infected ergot alkaloid–producing Kentucky 31(KY31) or nonergot alkaloid–producing novel endophyte pasture on body composition, semen quality, and semen freezing potential for yearling beef bulls. 2. Materials and methods 2.1. Experimental design All animal research was approved by the Clemson University Institutional Animal Care and Use Committee (IACUC protocol #ARC2010–68). All reagents were purchased from Sigma Scientific (St. Louis, MO, USA) unless stated otherwise. Angus bulls (n ¼ 21) aged between 13 and 16 months exhibiting scrotal circumference (SC) of 30 cm or greater, a minimum of 30% motility, and 70% normal sperm BSE were stratified by body weight (BW) and body condition score (BCS) and allotted to one of the two treatments. Two bulls did not meet these minimum requirements and were not used for further SC measurements or semen analysis but were maintained on study for growth and body composition estimates. Stocking rates were one bull per 0.4 hectares for bulls grazing novel endophyte non-toxic fescue (E-) and 1.1 bulls per acre for the bulls grazing the ergot alkaloid producing KY31 tall fescue (Eþ). Bulls were rotationally grazed using two 4.0hectare paddocks. All bulls were evaluated at specific intervals for reproductive and growth parameters across a 155-day grazing period. The study duration was designed to monitor bulls through two full spermatogenic cycles. 2.2. Treatment Dietary treatments consisted of grazing the ergot alkaloid–producing KY31 or a nonergot alkaloid–containing tall fescue possessing a novel endophyte. The enzyme-linked immunosorbent assay test for ergot alkaloids (Agrinostics, LTC. Co, Watkinsville, GA, USA) was conducted on 50 tillers per pasture, and the Eþ pasture exhibited a 98% infection
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rate. Two weeks before the start of the study, all bulls were adjusted to a forage diet by grazing E pasture. At the start of the test (Day 0), bulls were weighed, BCS was evaluated, SC and semen quality were assessed, stratified, and allotted to Eþ or E treatments and remained on treatment for 155 days (April 2012 to August 2012). 2.3. Blood collection and RIA Blood samples were obtained via the coocygeal vein at the start of test and on Days 35, 84, 114, and 140 and assayed for serum prolactin (PRL) and testosterone. Blood was allowed to clot and placed at 4 C overnight, and serum was harvested by centrifugation at 2000g for 15 minutes at 4 C. Serum was placed in vials and stored at 20 C until used in RIA. Prolactin assays were performed by the F. Neal Schrick laboratory as previously described [17] with mean interassay and intraassay coefficients of variation of 9.7% and 6.0%, respectively. Concentrations of testosterone were determined in a single assay using the Coat-A-Count testosterone RIA with a sensitivity of 6 ng/dL (Siemens Medical Solutions Diagnostics, Los Angeles, CA, USA) with an intra-assay coefficient of variation of 6.3%. The assay was validated by generating two composite pools of bull plasma containing 494 and 855 ng/dL of testosterone, and recovery was 98.1 1.7%, 99.9 2.9%, and 101.4 2.5%, respectively. Additionally, when three different dilutions (one, two, and threefold dilutions) of each of the pooled samples were assayed and the recovered values were plotted, the slopes of the inhibition curves were similar to that of the standard curve (P ¼ 0.89). 2.4. Semen evaluation Bulls were restrained in standard animal handling chutes and subjected to electroejaculation using the Pulsator IV electroejaculator (Agtech, Manhattan, KS, USA) on the preprogrammed collection mode. The ejaculate volume was recorded, and semen quality parameters were estimated using a computerized sperm-quality analyzer (SQA-Vb; A-Tech, Los Angeles, CA, USA). Each sample had computerized sperm-quality analysis performed in duplicate, and if motility and/or morphology were below 30% and 70%, respectively, a second collection was obtained within 7 days of the first sample. If both samples were below 30% and 70%, respectively, the highest rated collection for the bull was used in subsequent statistical analysis. Parameters evaluated by computerized sperm-quality analysis were as described by Stowe et al. [16]. In addition, each ejaculate was subjected to a manual morphologic examination by standard staining and microscopic evaluation. All samples were independently evaluated by two technicians. Bulls failed BSE if their semen samples from both collections exhibited less than 30% motility, less than 70% normal morphology, or an SC lower than recommended for their respective age range using the 1993 Society for Theriogenology guidelines for BSE [18]. 2.5. Semen extension and freezing Semen collection was conducted on bulls grazing Eþ pasture (n ¼ 5) and bulls grazing E pasture (n ¼ 7) on Day
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50 or greater after the start of treatment for semen freezing. Bulls were selected whose semen exhibited acceptable motility, morphology, and with enough ejaculate volume and sperm concentration to extend and freeze 20 to 40 0.5mL doses. Semen was extended per manufacturer instructions (Andromed One-step; Minitube of America, Verona, WI, USA) to approximately 30 million motile sperm per milliliter (assuming an approximately 30% loss in motility due to freezing as empirically determined in the lab) to yield 10 million motile sperm per 0.5 mL, loaded into 0.5-mL straws, and frozen in liquid N2 vapors for 10 minutes. Straws were then plunged directly into liquid N2, transferred into goblets, and stored in liquid N2 until used for analysis. Forty-eight hours after freezing, straws were thawed at 37 C for 60 seconds (three straws per bull) and evaluated using computerized sperm-quality analysis.
the classic indicator for the presence of ergot alkaloids in the diet resulting in the induction of fescue toxicosis. Serum PRL concentrations were affected by TRT-by-day interactions (P < 0.05), and data are shown in Figure 1. Serum PRL concentrations were lower in Eþ-treated animals compared with bulls grazing E forage on Days 35, 84, and 140 (P < 0.05), and PRL concentrations were different within TRT for bulls grazing Eþ forage with Day 0 exhibiting higher serum concentration compared with all other days (P < 0.05). Testosterone serum concentrations (Fig. 1) did not differ because of TRT, day, or TRT-by-day interactions. Scrotal circumference differed due to day effect (P < 0.05; mean range, 34.9–36.8 cm). Previous data show
A
2.6. Real-time carcass ultrasound Intramuscular fat (IMF) and fat thickness were measured longitudinally over the 12th rib and top of the rump using real-time ultrasonography. Muscle depth was measured over the longissimus dorsi and the rump. Measurements were taken using an Aloka 500V with a 17-cm 3.5-mHz probe. Scanned images were saved and analyzed using Biotronics software (Ames, IA, USA). 2.7. Statistical analysis The JMP program (SAS Institute Inc., Cary, NC, USA) was used to verify normal distribution of the data and perform multivariate ANOVA and adjusted for repeated measures to test for the main effects and main effect interactions for all semen parameters, carcass evaluations, and BCS. Main effects were treatment (TRT; Eþ vs. E), day, and TRT by day. Bulls were stratified by BW and BCS and allotted to treatment as described by Stowe et al. [16]; however, BW was not used as a covariate in the analysis. This was deemed necessary as in contrast to Stowe et al. [16] in which no differences were observed in BW because of treatment, BW was significantly impacted by treatment in this study, and using BW in the model would negate any effect observed for other parameters because of treatment. The JMP program and LSMeans procedures were used to assess differences in BCS, BW, total gain, and ADG (average daily gain). Treatment-by-day combination means for all parameters were generated and compared using the Fisher LSD test. Furthermore, computerized sperm-quality analysis data for frozen-thawed semen and prefreezing were subjected to analysis using LSMeans procedures as stated with treatment as the main effect. The chi-square analysis was conducted within the day of TRT to determine differences in the percentage of bulls passing a BSE. 3. Results 3.1. Hormonal profiles and scrotal circumference Blood samples collected throughout the study were used to determine serum concentration of PRL and testosterone. Decreased serum concentrations of PRL are
B
C
Fig. 1. Serum concentration of serum prolactin (PRL) and testosterone and scrotal circumference (SC) of bulls grazing Eþ or E fescue. Prolactin serum concentrations are shown (A). Differences due to day effect within the E (a, b) and Eþ (x, y) treatments are denoted with different superscripts (P < 0.05). The numerals 1 and 2 denote differences in PRL concentrations due to within day due to treatment. No differences in testosterone levels were observed (B). Changes in SC across the 140 study with different superscripts (a, b) denoting difference in SC due to day effect (C; P < 0.05).
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Table 1 Effects of grazing Eþ fescue on mean weight gain and body condition score with standard error of the mean. Parameter
Body condition (1–9) Body weight (kg) Total gain Average daily gain (kg)
Day 0
Day 140
Eþ
E
Eþ
5.4 0.2 510.6 17.8a NA NA
5.4 0.2 490.2 17.8a NA NA
5.3 544.5 33.8 0.2
E
0.2 16.5a 8.1a 0.05a
5.5 595.9 105.8 0.8
0.2 16.5b 8.1b 0.05b
a,b Significant due to treatment day interactions or treatment (P < 0.05). Abbreviation: NA, not applicable.
with that in Eþ bulls, respectively. No TRT, day, or TRT-byday interactions were significant for percent motility or progressive motility, although both exhibited a statistical trend (P < 0.1) showing elevated values on Days 112 and 140 for E treated bulls. When conducting manual assessment of sperm morphology, we observed bulls grazing Eþ forage exhibited decreased percent normal cells by Day 84 of the grazing treatment, which continued up to Day 140 (P < 0.05; Fig. 2). The lower percent normal cells in the Eþ group was due to an increase in the number of primary abnormalities (P < 0.05) beginning on Day 84. The decrease in the number of normal sperm resulted in a significant decrease in the number of bulls grazing Eþ forage passing a BSE compared with the E group on Days 112 and 140 (60% vs. 100% passing for Eþ and E, respectively for both Days 112 and 140; P < 0.05).
that feeding a ration containing ergovaline and ergoalanine at a dose of 0.8 mg/kg DM for 126 days resulted in decreased SC [16]. Because of the decrease in SC, we decided to evaluate serum testosterone levels from this previous study. No differences were observed in serum testosterone concentrations because of ergot alkaloid consumption, day of treatment, or interactions. Testosterone concentrations ranged from 389.7 to 582.3 and 462.6 to 808 ng/dL (standard error of the mean ¼ 161.8), for the control and bulls consuming ergot alkaloid–containing seed, respectively. 3.2. Growth and real-time carcass ultrasound Body weight, total gain, ADG, and BCS are given in Table 1. Bulls grazing Eþ forage exhibited decreased BW by Day 140 (P < 0.05) compared with the bulls grazing E forage. The total weight gain was increased in bulls on the E TRT compared with the bulls grazing Eþ (P < 0.05), and the increased gain was indicative of higher ADG for the bulls grazing E forage (P < 0.05) by Day 140. Interestingly, no difference in the BCS was exhibited (Table 1). Further, subcutaneous fat depth did not differ because of TRT or TRT-by-day interaction (Table 2) for either site evaluated. Rump muscle depth differed because of TRT (136.7 2.0 and 126.7 2.3 mm, for E and Eþ respectively; P < 0.05). The only significant TRT-by-day interaction observed for carcass evaluation was IMF (P < 0.05), which was higher for the E bulls by the end of the study compared with Eþ bulls.
3.4. Semen extension and freezing Semen parameters of collections from E and Eþ bulls used for extension and freezing are provided in Table 3. No differences were observed for motility, progressive motility, or other parameters estimated by computerized sperm-quality analysis before extension. The percentage of normal cells in the ejaculates for the Eþ group was lower compared with that of bulls in the E group; however, the average was still above the 70% normal range required for passing the BSE. In contrast, treatment had a large effect on multiple semen parameters on thawing (Table 3); however, total sperm concentration did not differ because of treatment (P > 0.05) verifying the extension of the semen to a similar concentration before freezing.
3.3. Semen evaluation Semen quality parameters are shown in Figure 2. Primary semen parameters evaluated by computerized sperm-quality analysis that exhibited significant TRT-byday interactions were sperm concentration and sperm velocity (P < 0.05). Sperm velocity was elevated on Days 84 and 112 (P < 0.05), and sperm concentration of the ejaculate was greater on Day 84 (P < 0.05) for E bulls compared
4. Discussion Serum PRL concentrations are a classical method to evaluate the induction of fescue toxicosis. The serum PRL concentrations observed for the treatments in this study
Table 2 Effect of fescue toxicosis on mean carcass characteristics with standard error of the mean as measured by real-time ultrasonography. Parameter
Day 8
Day 155
Eþ Longissimus dorsi intramuscular fat (%) Longissimus dorsi subcutaneous fat thickness (mm) Longissimus dorsi muscle depth (mm) Rump subcutaneous fat thickness (mm) Rump muscle depth (mm) a,b
3.0 4.3 88.3 7.4 125.2
Denote difference due to treatment day interaction (P < 0.05).
E
0.3a 0.6 2.5 1.0 2.5
2.2 3.0 76.2 4.6 133.4
Eþ
0.3a 0.6 2.5 1.0 2.5
3.5 2.5 60.5 2.5 87.1
E
0.3a 0.6 1.0 1.0 2.5
4.9 2.5 59.2 2.5 142.5
0.3b 0.6 1.0 1.0 2.5
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A
B
C
D
Fig. 2. Impact of fescue toxicosis on semen quality. Concentration (A), percent motility (B), percent normal cells (C), and percent primary abnormalities (D) are shown as graphs with the E and Eþ treatments denoted within each graph. For all graphs, differences due to day within the E (a, b) and Eþ (x, y) are denoted with different superscripts (P < 0.05). Differences within day due to treatment are denoted with the numerals 1 and 2 (P < 0.05).
are consistent with our previous data [16] and that of several groups [13–15] and support the experimental design in that fescue toxicosis was induced by Day 35 of treatment. In contrast to our previous report, PRL concentrations were numerically lower and decreased over time in both treatment groups [16]. The primary differences between Stowe et al. [16] and other previous data were the energy levels of the diet [13–15]. One could extrapolate that the numerical differences between the studies for PRL serum concentrations may be due to differences in the diets as the bulls in the study of Stowe et al. [16] were fed a total mixed ration to meet nutrient requirements of young beef bulls to gain 1 kg/d in BW, and the bulls in this study were grazed on forage only without supplementation. Our previous study [16] showed that prolonged exposure to ergot alkaloids fed at 0.8 mg of ergovaline and ergovalanine/g DM resulted in reduced SC [16]; therefore, we wished to evaluate serum testosterone concentrations. In contrast to our previous report [16] and that of Jones et al. [12], our current data show no decrease in SC due to TRT. No differences in testosterone were observed between treatments, which supports several previous observations [13–15]. Interestingly, we evaluated testosterone serum concentrations in the samples from Stowe et al. [16] and did not observe any difference for serum testosterone values because of
treatment in that study. Jones et al. [12] did not report testosterone concentrations. As a whole, these data would support other observations in which PRL serum concentrations do not appear to influence testosterone production in situ [13,15], although in vitro data using rodent models show a direct correlation of PRL concentration to steroid production [19,20]. However, PRL serum concentrations may affect accessory gland function as implicated for several animal models [21–23]. The differences in the effect of fescue toxicosis on SC observed by Stowe et al. [16] and Jones et al. [12] compared with these findings and other reports could be because of the levels of circulating ergot alkaloids present within the animals. Using a rodent model, Dirami et al., 1998 [24]. observed that treatment with dopamine agonists resulted in a reduction of testicular fluid and hypertrophy of the Leydig cells. Unfortunately, no studies have yet addressed circulating levels of the ergot alkaloids within individual animals to assess differences in animal growth or reproductive response; whereas two studies have reported effects of diet on SC [12,16], a measurement indicative of testicular volume and sperm production [25], none of the studies reported to date shows dramatic impacts on semen quality parameters [12–16].
Table 3 Means with standard error of the mean of parameters evaluated by computerized semen-quality assessment of ejaculates before freezing procedures and postthaw. Parameter
Prefreezing Eþ
Sperm concentration (million/mL) Motility (%) Total sperm number (106) Total motile sperm number (106) a,b
362.7 92.8 8.9 8.2
Postthaw E
73.5 3.0 2.1 1.7
284.1 91.1 5.1 4.3
Eþ
68.0 2.8 1.9 1.6
Denote difference due to treatment (P < 0.05) between Eþ and E samples prefreezing and postthaw.
89.7 6.4 44.9 2.5
E
10.8 3.2a 5.4a 1.1a
101.3 21.7 50.7 9.5
10.8 3.2b 5.4a 1.1b
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Further supporting the induction of fescue toxicosis in the present study, we observed differences in off-test BW, total gain, and ADG (P < 0.05). This observation is in contrast to Stowe et al. [16]; however, it was expected as the previous study used feeding a high-energy concentrate ration in the presence or absence of a defined amount of ergot alkaloids formulated for bulls to gain 1.0 kg/d. The gains reported here for Eþ and E bulls are highly similar to those reported for British breed bulls grazing Eþ and non-Eþ fescue during the spring and summer months reported previously [14] and somewhat higher than Brahman influence bulls (1/8–3/16 of the individuals genetics) grazing E and reduced for bulls grazing Eþ fescue [15]. Carcass composition of bulls suffering from fescue toxicosis has not previously been reported. Bulls grazing Eþ and E pastures were monitored by evaluating BCS and performing real-time carcass ultrasound. Bulls’ BCS did not differ between treatments, and this would support the lack of detectable differences in subcutaneous fat thickness measurements taken over the longissimus dorsi and the rump. Interestingly, IMF was higher in the E group compared with that of Eþ group by Day 155 (TRT day; P < 0.05), and rump muscle depth differed because of treatment with lower values observed in the Eþ group (P < 0.05). As with other studies, modest differences in some semen quality parameters were detected by computerized spermquality analysis [12,15]. In this study, the computerized sperm-quality analysis revealed differences because of TRTby-day interactions between bulls grazing Eþ and E forage for sperm concentration and sperm velocity. Furthermore, motility and progressive motility tended to be lower in bulls in the Eþ treatment. These data, in general, would support those reported by Looper et al. [15] in which subtle differences in semen quality using computerized sperm-quality analysis were also observed. Stowe et al. [16] observed no difference in semen quality between bulls being fed a constant concentration of ergot alkaloids in a high-energy concentrate ration using computerized sperm-quality analysis. When conducting manual assessment of sperm morphology, bulls grazing Eþ forage exhibited decreased morphology by Day 84 of treatment. A decrease of this magnitude in morphology due to treatment has not been observed previously. In general, the data presented here are somewhat inconsistent with those reported by Schuenemann et al., 2005 [14] for bulls grazing KY31. The reasons for these differences could be due to genetics of the bulls, ergot alkaloid concentrations of the forage during the season, or any combination of other environmental factors. What is consistent across studies of Looper et al. [15] and the Schrick Lab (personal communication with F. Neal Schrick; University of Tennessee) is that sperm velocity is altered when bulls graze Eþ KY31. Stowe et al. [16] did observe a tendency for decreased sperm velocity because of treatment beginning by Day 84 of consuming Eþ seed and extending to the end of the study on Day 126. Our original intent was to freeze semen obtained from bulls grazing Eþ or non-Eþ pastures and assess in vitro fertility as described by Schuenemann et al., 2005 [13,14], but early attempts indicated an issue with freezing semen collected from bulls in the Eþ treatment. Subsequently, we evaluated multiple ejaculates obtained from bulls either on
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the Eþ or on the E treatments. Although morphology was lower in bulls on the Eþ grazing treatment compared with that on the E group at the time of freezing, both groups exhibited over 75% normal sperm morphology at the time of freezing. Furthermore, computerized sperm-quality analysis confirmed no difference in the other quality estimates of samples chosen to be extended and frozen between the treatment groups. Computerized spermquality analysis of semen postthaw revealed that semen of bulls in the Eþ treatment possessed a greater than threefold reduction in motility, progressive motility, motile sperm concentration, progressive motile sperm concentration, total motile sperm, and total progressive motile sperm. In contrast, the total sperm number observed postthaw was the same between treatments verifying that final extended concentration was not the reason for the observed reduction in the number of motile and total motile sperm. The computerized sperm-quality analysis data of frozen-thawed semen would indicate a structural and/or physiological change in the spermatozoa from bulls grazing endophyte-infected fescue, which is consistent with the decreased percentage of normal sperm present in bulls grazing Eþ forage. Physical changes of this magnitude could explain the differences observed for sperm velocity reported here and those reported by other groups [15] and explain lower fertility of semen from bulls consuming ergot alkaloids in vitro [13,14]. 4.1. Conclusions Fescue toxicosis results in subtle alterations in carcass composition and semen characteristics in yearling beef bulls. Furthermore, grazing the pasture containing ergot alkaloids alters sperm morphology and semen freezing potential of ejaculates suggesting an altered sperm structure/physiology which would explain data showing alterations in semen quality and in vitro fertility. Acknowledgments This material is based on work supported by National Institute of Food and Agriculture/United States Department of Agriculture, under project number SC-1700376 and was supported by National Research Initiative Competitive grant number 2010-38942-20745 from the USDA National Institute of Food and Agriculture and the Cooperative State Research, Education, and Extension Service and has been assigned Technical Contribution number 6194 of the Clemson University Experiment Station. The author would also like to thank Johanna E. Johnson with assistance in preparing this article. References [1] Hoveland CS. Importance and economic significance of the Acremonium endophytes to performance of animals and grass plant. Agric Ecosyst Environ 1993;44:3–12. [2] Strickland JR, et al. Board-invited review: St. Anthony’s fire in livestock: causes, mechanisms, and potential solutions. J Anim Sci 2011; 89:1603–26. [3] Allen VG, Segarra E. Anti-quality components in forage: overview, significance, and economic impact. J Range Management 2001;54: 409–12.
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[4] Browning Jr R. Effects of endophyte-infected tall fescue on indicators of thermal status and growth in Hereford and Senepol steers. J Anim Sci 2004;82:634–43. [5] Gunter SA, Beck PA. Novel endophyte-infected tall fescue for growing beef cattle. J Anim Sci 2004;82(E-Suppl):E75–82. [6] Watson RH, et al. Productivity of cow-calf pairs grazing tall fescue pastures infected with either the wild-type endophyte or a nonergot alkaloid-producing endophyte strain, AR542. J Anim Sci 2004;82: 3388–93. [7] Johnson JM, et al. Steer and pasture responses for a novel endophyte tall fescue developed for the upper transition zone. J Anim Sci 2012; 90:2402–9. [8] Burke JM, Rorie RW. Changes in ovarian function in mature beef cows grazing endophyte infected tall fescue. Theriogenology 2002; 57:1733–42. [9] Burke JM, et al. Reproductive responses to grazing endophyteinfected tall fescue by postpartum beef cows. Theriogenology 2001;56:357–69. [10] Jones KL, et al. Domperidone can ameliorate deleterious reproductive effects and reduced weight gain associated with fescue toxicosis in heifers. J Anim Sci 2003;81:2568–74. [11] Jones KL, King SS, Iqbal MJ. Endophyte-infected tall fescue diet alters gene expression in heifer luteal tissue as revealed by interspecies microarray analysis. Mol Reprod Dev 2004;67:154–61. [12] Jones KL, et al. Consumption of toxic fescue impairs bull reproductive parameters. The Prof Anim Scientist 2004;20:437–42. [13] Schuenemann GM, et al. Effects of administration of ergotamine tartrate on fertility of yearling beef bulls. Theriogenology 2005;63: 1407–18. [14] Schuenemann GM, et al. Fertility aspects in yearling beef bulls grazing endophyte-infected tall fescue pastures. Reprod Fertil Dev 2005;17:479–86.
[15] Looper ML, et al. Influence of toxic endophyte-infected fescue on sperm characteristics and endocrine factors of yearling Brahmaninfluenced bulls. J Anim Sci 2009;87:1184–91. [16] Stowe HM, et al. Effects of fescue toxicosis on bull growth, semen characteristics, and breeding soundness evaluation. J Anim Sci 2013; 91:3686–92. [17] Bernard JK, et al. Effects of prepartum consumption of endophyteinfested tall fescue on serum prolactin and subsequent milkproduction of Holstein cows. J Dairy Sci 1993;76:1928–33. [18] Spitzer JC, Hopkins FM. Breeding soundness evaluation of yearling bulls. Vet Clin North Am Food Anim Pract 1997;13:295–304. [19] Barkey RJ, et al. Prolactin and antiprolactin receptor antibody inhibit steroidogenesis by purified rat Leydig cells in culture. Mol Cell Endocrinol 1987;52:71–80. [20] Weiss-Messer E, Ber R, Barkey RJ. Prolactin and MA-10 Leydig cell steroidogenesis: biphasic effects of prolactin and signal transduction. Endocrinology 1996;137:5509–18. [21] Lotti F, et al. Clinical implications of measuring prolactin levels in males of infertile couples. Andrology 2013;1:764–71. [22] Hair WM, et al. Prolactin receptor expression in human testis and accessory tissues: localization and function. Mol Hum Reprod 2002; 8:606–11. [23] Steger RW, et al. Neuroendocrine and reproductive functions in male mice with targeted disruption of the prolactin gene. Endocrinology 1998;139:3691–5. [24] Almquist JO, Amann RP. Reproductive capacity of dairy bulls. II. Gonadal and extra-gonadal sperm reserves as determined by direct counts and depletion trials; dimensions and weight of genitalia. J Dairy Sci 1961;44:1668–78. [25] Pratt SL, et al. Comparison of methods for predicting yearling scrotal circumference and correlations of scrotal circumference to growth traits in beef bulls. J Anim Sci 1991;69:2711–20.
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Bulls grazing Kentucky 31 tall fescue exhibit impaired growth, semen quality, and decreased semen freezing potential S.L. Pratt a, *, H.M. Stowe a, B.K. Whitlock b, L. Strickland c, M. Miller a, S.M. Calcatera a, M.D. Dimmick a, G.E. Aiken d, F.N. Schrick c, N.M. Long a, S.K. Duckett a, J.G. Andrae a a
Department of Animal and Veterinary Sciences, Clemson University, Clemson, South Carolina, USA Large Animal Clinical Sciences, College of Veterinary Medicine, The University of Tennessee, Knoxville, Tennessee, USA c Department of Animal Science, University of Tennessee, Knoxville, Tennessee, USA d USDA-ARS-Forage-Animal Production Research Unit, University of Kentucky Campus, Lexington, Kentucky, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 December 2013 Received in revised form 17 September 2014 Accepted 1 October 2014
Serum prolactin (PRL) and testosterone concentrations, body weight, body composition, semen quality, and semen freezing potential for bulls grazing the toxic tall fescue (Lolium arundinaceum [Schreb.] Darbysh. ¼ Schedonorous arundinaceum [Schreb.] Dumort.) cultivar Kentucky 31 (Eþ) compared with a novel endophyte cultivar lacking ergot alkaloids (E) were evaluated. Angus bulls were allotted to treatment (Day 0) and grazed Eþ or E for 155 days. Treatment-by-day interaction was significant (P < 0.05) for serum PRL concentrations with Eþtreated bulls exhibiting reduced PRL values compared with E control bulls, but no differences were observed for serum testosterone concentrations (P > 0.05). Further, bulls on the Eþ treatment exhibited decreased total gain, average daily gain, and body weight by Day 140 (P < 0.05) compared with the E bulls. Rump muscle depth was lower because the treatment in bulls grazing Eþ compared with E (P < 0.05) and intramuscular fat in the E bulls compared with the Eþ group was higher by Day 155 (P < 0.05). Analysis of ejaculates showed significant treatment day effects for sperm concentration with lower values observed for bulls on the Eþ treatment (P < 0.05). The percent normal morphology was reduced in ejaculates from Eþ bulls compared with E bulls (P < 0.05), and the difference was due to an increase in abnormal sperm present in the Eþ ejaculates from Day 84 to 140 (P < 0.05). In addition, spermatozoa motility and progressive motility were decreased on thawing in semen samples from Eþ bulls compared with E bulls (P < 0.05). Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Computerized sperm-quality analysis Scrotal circumference Fescue toxicosis Breeding soundness examination Semen freezing
1. Introduction Tall fescue (Lolium arundinaceum [Schreb.] Darbysh. ¼ Schedonorous arundinaceum [Schreb.] Dumort.) is present on approximately 16 million ha in the MidAtlantic and Southern regions of the United States [1] and
* Corresponding author. Tel.: þ1 864 656 3135; fax: þ1 864 656 3131. E-mail address: [email protected] (S.L. Pratt). 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.10.001
serves as a forage for approximately 8.5 million cattle, making this species the dominant, cool-season, perennial grass in the region. The vast majority of this forage is infected with the endophyte, Neotyphodium coenophialum. The grass exists in a mutualistic relationship with the ergot alkaloid–producing endophyte that confers disease, drought, grazing tolerance, and insect resistance to tall fescue [1]. However, ergot alkaloids negatively impact growth and/or reproductive performance of animals grazing tall fescue containing the wild-type endophyte.
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Accumulation of the ergot alkaloids within the animal’s system results in the syndrome known as fescue toxicosis, which is characterized by rough hair coat, increased core body temperature, decreased blood flow to body extremities, and poor growth and reproductive performance [2]. Previous estimates for lost beef production due to fescue toxicosis were $600 million [1], and more recent reviews estimate that total to be approaching $1 billion lost per year for cattle and small ruminants [2,3]. The most studied aspects of fescue toxicosis are the effects on animal performance [4–7]. However, there is only limited documentation of the effects of fescue toxicosis on reproductive function [8–11] and very little, if any, of fescue toxicosis on bovine male reproduction [12–16]. In an effort to begin filling this gap, our previous research evaluated the effects of fescue toxicosis on semen characteristics when bulls were fed a constant amount of ergot alkaloid in conjunction with a high-concentrate ration to delineate effects of the ergot alkaloid from possible effects of reduced body condition and plane of nutrition [16]. Little, if any, effects were noted on breeding soundness examinations (BSE) or semen characteristics. The objectives of this study were to determine the effects of grazing endophyte-infected ergot alkaloid–producing Kentucky 31(KY31) or nonergot alkaloid–producing novel endophyte pasture on body composition, semen quality, and semen freezing potential for yearling beef bulls. 2. Materials and methods 2.1. Experimental design All animal research was approved by the Clemson University Institutional Animal Care and Use Committee (IACUC protocol #ARC2010–68). All reagents were purchased from Sigma Scientific (St. Louis, MO, USA) unless stated otherwise. Angus bulls (n ¼ 21) aged between 13 and 16 months exhibiting scrotal circumference (SC) of 30 cm or greater, a minimum of 30% motility, and 70% normal sperm BSE were stratified by body weight (BW) and body condition score (BCS) and allotted to one of the two treatments. Two bulls did not meet these minimum requirements and were not used for further SC measurements or semen analysis but were maintained on study for growth and body composition estimates. Stocking rates were one bull per 0.4 hectares for bulls grazing novel endophyte non-toxic fescue (E-) and 1.1 bulls per acre for the bulls grazing the ergot alkaloid producing KY31 tall fescue (Eþ). Bulls were rotationally grazed using two 4.0hectare paddocks. All bulls were evaluated at specific intervals for reproductive and growth parameters across a 155-day grazing period. The study duration was designed to monitor bulls through two full spermatogenic cycles. 2.2. Treatment Dietary treatments consisted of grazing the ergot alkaloid–producing KY31 or a nonergot alkaloid–containing tall fescue possessing a novel endophyte. The enzyme-linked immunosorbent assay test for ergot alkaloids (Agrinostics, LTC. Co, Watkinsville, GA, USA) was conducted on 50 tillers per pasture, and the Eþ pasture exhibited a 98% infection
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rate. Two weeks before the start of the study, all bulls were adjusted to a forage diet by grazing E pasture. At the start of the test (Day 0), bulls were weighed, BCS was evaluated, SC and semen quality were assessed, stratified, and allotted to Eþ or E treatments and remained on treatment for 155 days (April 2012 to August 2012). 2.3. Blood collection and RIA Blood samples were obtained via the coocygeal vein at the start of test and on Days 35, 84, 114, and 140 and assayed for serum prolactin (PRL) and testosterone. Blood was allowed to clot and placed at 4 C overnight, and serum was harvested by centrifugation at 2000g for 15 minutes at 4 C. Serum was placed in vials and stored at 20 C until used in RIA. Prolactin assays were performed by the F. Neal Schrick laboratory as previously described [17] with mean interassay and intraassay coefficients of variation of 9.7% and 6.0%, respectively. Concentrations of testosterone were determined in a single assay using the Coat-A-Count testosterone RIA with a sensitivity of 6 ng/dL (Siemens Medical Solutions Diagnostics, Los Angeles, CA, USA) with an intra-assay coefficient of variation of 6.3%. The assay was validated by generating two composite pools of bull plasma containing 494 and 855 ng/dL of testosterone, and recovery was 98.1 1.7%, 99.9 2.9%, and 101.4 2.5%, respectively. Additionally, when three different dilutions (one, two, and threefold dilutions) of each of the pooled samples were assayed and the recovered values were plotted, the slopes of the inhibition curves were similar to that of the standard curve (P ¼ 0.89). 2.4. Semen evaluation Bulls were restrained in standard animal handling chutes and subjected to electroejaculation using the Pulsator IV electroejaculator (Agtech, Manhattan, KS, USA) on the preprogrammed collection mode. The ejaculate volume was recorded, and semen quality parameters were estimated using a computerized sperm-quality analyzer (SQA-Vb; A-Tech, Los Angeles, CA, USA). Each sample had computerized sperm-quality analysis performed in duplicate, and if motility and/or morphology were below 30% and 70%, respectively, a second collection was obtained within 7 days of the first sample. If both samples were below 30% and 70%, respectively, the highest rated collection for the bull was used in subsequent statistical analysis. Parameters evaluated by computerized sperm-quality analysis were as described by Stowe et al. [16]. In addition, each ejaculate was subjected to a manual morphologic examination by standard staining and microscopic evaluation. All samples were independently evaluated by two technicians. Bulls failed BSE if their semen samples from both collections exhibited less than 30% motility, less than 70% normal morphology, or an SC lower than recommended for their respective age range using the 1993 Society for Theriogenology guidelines for BSE [18]. 2.5. Semen extension and freezing Semen collection was conducted on bulls grazing Eþ pasture (n ¼ 5) and bulls grazing E pasture (n ¼ 7) on Day
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50 or greater after the start of treatment for semen freezing. Bulls were selected whose semen exhibited acceptable motility, morphology, and with enough ejaculate volume and sperm concentration to extend and freeze 20 to 40 0.5mL doses. Semen was extended per manufacturer instructions (Andromed One-step; Minitube of America, Verona, WI, USA) to approximately 30 million motile sperm per milliliter (assuming an approximately 30% loss in motility due to freezing as empirically determined in the lab) to yield 10 million motile sperm per 0.5 mL, loaded into 0.5-mL straws, and frozen in liquid N2 vapors for 10 minutes. Straws were then plunged directly into liquid N2, transferred into goblets, and stored in liquid N2 until used for analysis. Forty-eight hours after freezing, straws were thawed at 37 C for 60 seconds (three straws per bull) and evaluated using computerized sperm-quality analysis.
the classic indicator for the presence of ergot alkaloids in the diet resulting in the induction of fescue toxicosis. Serum PRL concentrations were affected by TRT-by-day interactions (P < 0.05), and data are shown in Figure 1. Serum PRL concentrations were lower in Eþ-treated animals compared with bulls grazing E forage on Days 35, 84, and 140 (P < 0.05), and PRL concentrations were different within TRT for bulls grazing Eþ forage with Day 0 exhibiting higher serum concentration compared with all other days (P < 0.05). Testosterone serum concentrations (Fig. 1) did not differ because of TRT, day, or TRT-by-day interactions. Scrotal circumference differed due to day effect (P < 0.05; mean range, 34.9–36.8 cm). Previous data show
A
2.6. Real-time carcass ultrasound Intramuscular fat (IMF) and fat thickness were measured longitudinally over the 12th rib and top of the rump using real-time ultrasonography. Muscle depth was measured over the longissimus dorsi and the rump. Measurements were taken using an Aloka 500V with a 17-cm 3.5-mHz probe. Scanned images were saved and analyzed using Biotronics software (Ames, IA, USA). 2.7. Statistical analysis The JMP program (SAS Institute Inc., Cary, NC, USA) was used to verify normal distribution of the data and perform multivariate ANOVA and adjusted for repeated measures to test for the main effects and main effect interactions for all semen parameters, carcass evaluations, and BCS. Main effects were treatment (TRT; Eþ vs. E), day, and TRT by day. Bulls were stratified by BW and BCS and allotted to treatment as described by Stowe et al. [16]; however, BW was not used as a covariate in the analysis. This was deemed necessary as in contrast to Stowe et al. [16] in which no differences were observed in BW because of treatment, BW was significantly impacted by treatment in this study, and using BW in the model would negate any effect observed for other parameters because of treatment. The JMP program and LSMeans procedures were used to assess differences in BCS, BW, total gain, and ADG (average daily gain). Treatment-by-day combination means for all parameters were generated and compared using the Fisher LSD test. Furthermore, computerized sperm-quality analysis data for frozen-thawed semen and prefreezing were subjected to analysis using LSMeans procedures as stated with treatment as the main effect. The chi-square analysis was conducted within the day of TRT to determine differences in the percentage of bulls passing a BSE. 3. Results 3.1. Hormonal profiles and scrotal circumference Blood samples collected throughout the study were used to determine serum concentration of PRL and testosterone. Decreased serum concentrations of PRL are
B
C
Fig. 1. Serum concentration of serum prolactin (PRL) and testosterone and scrotal circumference (SC) of bulls grazing Eþ or E fescue. Prolactin serum concentrations are shown (A). Differences due to day effect within the E (a, b) and Eþ (x, y) treatments are denoted with different superscripts (P < 0.05). The numerals 1 and 2 denote differences in PRL concentrations due to within day due to treatment. No differences in testosterone levels were observed (B). Changes in SC across the 140 study with different superscripts (a, b) denoting difference in SC due to day effect (C; P < 0.05).
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Table 1 Effects of grazing Eþ fescue on mean weight gain and body condition score with standard error of the mean. Parameter
Body condition (1–9) Body weight (kg) Total gain Average daily gain (kg)
Day 0
Day 140
Eþ
E
Eþ
5.4 0.2 510.6 17.8a NA NA
5.4 0.2 490.2 17.8a NA NA
5.3 544.5 33.8 0.2
E
0.2 16.5a 8.1a 0.05a
5.5 595.9 105.8 0.8
0.2 16.5b 8.1b 0.05b
a,b Significant due to treatment day interactions or treatment (P < 0.05). Abbreviation: NA, not applicable.
with that in Eþ bulls, respectively. No TRT, day, or TRT-byday interactions were significant for percent motility or progressive motility, although both exhibited a statistical trend (P < 0.1) showing elevated values on Days 112 and 140 for E treated bulls. When conducting manual assessment of sperm morphology, we observed bulls grazing Eþ forage exhibited decreased percent normal cells by Day 84 of the grazing treatment, which continued up to Day 140 (P < 0.05; Fig. 2). The lower percent normal cells in the Eþ group was due to an increase in the number of primary abnormalities (P < 0.05) beginning on Day 84. The decrease in the number of normal sperm resulted in a significant decrease in the number of bulls grazing Eþ forage passing a BSE compared with the E group on Days 112 and 140 (60% vs. 100% passing for Eþ and E, respectively for both Days 112 and 140; P < 0.05).
that feeding a ration containing ergovaline and ergoalanine at a dose of 0.8 mg/kg DM for 126 days resulted in decreased SC [16]. Because of the decrease in SC, we decided to evaluate serum testosterone levels from this previous study. No differences were observed in serum testosterone concentrations because of ergot alkaloid consumption, day of treatment, or interactions. Testosterone concentrations ranged from 389.7 to 582.3 and 462.6 to 808 ng/dL (standard error of the mean ¼ 161.8), for the control and bulls consuming ergot alkaloid–containing seed, respectively. 3.2. Growth and real-time carcass ultrasound Body weight, total gain, ADG, and BCS are given in Table 1. Bulls grazing Eþ forage exhibited decreased BW by Day 140 (P < 0.05) compared with the bulls grazing E forage. The total weight gain was increased in bulls on the E TRT compared with the bulls grazing Eþ (P < 0.05), and the increased gain was indicative of higher ADG for the bulls grazing E forage (P < 0.05) by Day 140. Interestingly, no difference in the BCS was exhibited (Table 1). Further, subcutaneous fat depth did not differ because of TRT or TRT-by-day interaction (Table 2) for either site evaluated. Rump muscle depth differed because of TRT (136.7 2.0 and 126.7 2.3 mm, for E and Eþ respectively; P < 0.05). The only significant TRT-by-day interaction observed for carcass evaluation was IMF (P < 0.05), which was higher for the E bulls by the end of the study compared with Eþ bulls.
3.4. Semen extension and freezing Semen parameters of collections from E and Eþ bulls used for extension and freezing are provided in Table 3. No differences were observed for motility, progressive motility, or other parameters estimated by computerized sperm-quality analysis before extension. The percentage of normal cells in the ejaculates for the Eþ group was lower compared with that of bulls in the E group; however, the average was still above the 70% normal range required for passing the BSE. In contrast, treatment had a large effect on multiple semen parameters on thawing (Table 3); however, total sperm concentration did not differ because of treatment (P > 0.05) verifying the extension of the semen to a similar concentration before freezing.
3.3. Semen evaluation Semen quality parameters are shown in Figure 2. Primary semen parameters evaluated by computerized sperm-quality analysis that exhibited significant TRT-byday interactions were sperm concentration and sperm velocity (P < 0.05). Sperm velocity was elevated on Days 84 and 112 (P < 0.05), and sperm concentration of the ejaculate was greater on Day 84 (P < 0.05) for E bulls compared
4. Discussion Serum PRL concentrations are a classical method to evaluate the induction of fescue toxicosis. The serum PRL concentrations observed for the treatments in this study
Table 2 Effect of fescue toxicosis on mean carcass characteristics with standard error of the mean as measured by real-time ultrasonography. Parameter
Day 8
Day 155
Eþ Longissimus dorsi intramuscular fat (%) Longissimus dorsi subcutaneous fat thickness (mm) Longissimus dorsi muscle depth (mm) Rump subcutaneous fat thickness (mm) Rump muscle depth (mm) a,b
3.0 4.3 88.3 7.4 125.2
Denote difference due to treatment day interaction (P < 0.05).
E
0.3a 0.6 2.5 1.0 2.5
2.2 3.0 76.2 4.6 133.4
Eþ
0.3a 0.6 2.5 1.0 2.5
3.5 2.5 60.5 2.5 87.1
E
0.3a 0.6 1.0 1.0 2.5
4.9 2.5 59.2 2.5 142.5
0.3b 0.6 1.0 1.0 2.5
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A
B
C
D
Fig. 2. Impact of fescue toxicosis on semen quality. Concentration (A), percent motility (B), percent normal cells (C), and percent primary abnormalities (D) are shown as graphs with the E and Eþ treatments denoted within each graph. For all graphs, differences due to day within the E (a, b) and Eþ (x, y) are denoted with different superscripts (P < 0.05). Differences within day due to treatment are denoted with the numerals 1 and 2 (P < 0.05).
are consistent with our previous data [16] and that of several groups [13–15] and support the experimental design in that fescue toxicosis was induced by Day 35 of treatment. In contrast to our previous report, PRL concentrations were numerically lower and decreased over time in both treatment groups [16]. The primary differences between Stowe et al. [16] and other previous data were the energy levels of the diet [13–15]. One could extrapolate that the numerical differences between the studies for PRL serum concentrations may be due to differences in the diets as the bulls in the study of Stowe et al. [16] were fed a total mixed ration to meet nutrient requirements of young beef bulls to gain 1 kg/d in BW, and the bulls in this study were grazed on forage only without supplementation. Our previous study [16] showed that prolonged exposure to ergot alkaloids fed at 0.8 mg of ergovaline and ergovalanine/g DM resulted in reduced SC [16]; therefore, we wished to evaluate serum testosterone concentrations. In contrast to our previous report [16] and that of Jones et al. [12], our current data show no decrease in SC due to TRT. No differences in testosterone were observed between treatments, which supports several previous observations [13–15]. Interestingly, we evaluated testosterone serum concentrations in the samples from Stowe et al. [16] and did not observe any difference for serum testosterone values because of
treatment in that study. Jones et al. [12] did not report testosterone concentrations. As a whole, these data would support other observations in which PRL serum concentrations do not appear to influence testosterone production in situ [13,15], although in vitro data using rodent models show a direct correlation of PRL concentration to steroid production [19,20]. However, PRL serum concentrations may affect accessory gland function as implicated for several animal models [21–23]. The differences in the effect of fescue toxicosis on SC observed by Stowe et al. [16] and Jones et al. [12] compared with these findings and other reports could be because of the levels of circulating ergot alkaloids present within the animals. Using a rodent model, Dirami et al., 1998 [24]. observed that treatment with dopamine agonists resulted in a reduction of testicular fluid and hypertrophy of the Leydig cells. Unfortunately, no studies have yet addressed circulating levels of the ergot alkaloids within individual animals to assess differences in animal growth or reproductive response; whereas two studies have reported effects of diet on SC [12,16], a measurement indicative of testicular volume and sperm production [25], none of the studies reported to date shows dramatic impacts on semen quality parameters [12–16].
Table 3 Means with standard error of the mean of parameters evaluated by computerized semen-quality assessment of ejaculates before freezing procedures and postthaw. Parameter
Prefreezing Eþ
Sperm concentration (million/mL) Motility (%) Total sperm number (106) Total motile sperm number (106) a,b
362.7 92.8 8.9 8.2
Postthaw E
73.5 3.0 2.1 1.7
284.1 91.1 5.1 4.3
Eþ
68.0 2.8 1.9 1.6
Denote difference due to treatment (P < 0.05) between Eþ and E samples prefreezing and postthaw.
89.7 6.4 44.9 2.5
E
10.8 3.2a 5.4a 1.1a
101.3 21.7 50.7 9.5
10.8 3.2b 5.4a 1.1b
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Further supporting the induction of fescue toxicosis in the present study, we observed differences in off-test BW, total gain, and ADG (P < 0.05). This observation is in contrast to Stowe et al. [16]; however, it was expected as the previous study used feeding a high-energy concentrate ration in the presence or absence of a defined amount of ergot alkaloids formulated for bulls to gain 1.0 kg/d. The gains reported here for Eþ and E bulls are highly similar to those reported for British breed bulls grazing Eþ and non-Eþ fescue during the spring and summer months reported previously [14] and somewhat higher than Brahman influence bulls (1/8–3/16 of the individuals genetics) grazing E and reduced for bulls grazing Eþ fescue [15]. Carcass composition of bulls suffering from fescue toxicosis has not previously been reported. Bulls grazing Eþ and E pastures were monitored by evaluating BCS and performing real-time carcass ultrasound. Bulls’ BCS did not differ between treatments, and this would support the lack of detectable differences in subcutaneous fat thickness measurements taken over the longissimus dorsi and the rump. Interestingly, IMF was higher in the E group compared with that of Eþ group by Day 155 (TRT day; P < 0.05), and rump muscle depth differed because of treatment with lower values observed in the Eþ group (P < 0.05). As with other studies, modest differences in some semen quality parameters were detected by computerized spermquality analysis [12,15]. In this study, the computerized sperm-quality analysis revealed differences because of TRTby-day interactions between bulls grazing Eþ and E forage for sperm concentration and sperm velocity. Furthermore, motility and progressive motility tended to be lower in bulls in the Eþ treatment. These data, in general, would support those reported by Looper et al. [15] in which subtle differences in semen quality using computerized sperm-quality analysis were also observed. Stowe et al. [16] observed no difference in semen quality between bulls being fed a constant concentration of ergot alkaloids in a high-energy concentrate ration using computerized sperm-quality analysis. When conducting manual assessment of sperm morphology, bulls grazing Eþ forage exhibited decreased morphology by Day 84 of treatment. A decrease of this magnitude in morphology due to treatment has not been observed previously. In general, the data presented here are somewhat inconsistent with those reported by Schuenemann et al., 2005 [14] for bulls grazing KY31. The reasons for these differences could be due to genetics of the bulls, ergot alkaloid concentrations of the forage during the season, or any combination of other environmental factors. What is consistent across studies of Looper et al. [15] and the Schrick Lab (personal communication with F. Neal Schrick; University of Tennessee) is that sperm velocity is altered when bulls graze Eþ KY31. Stowe et al. [16] did observe a tendency for decreased sperm velocity because of treatment beginning by Day 84 of consuming Eþ seed and extending to the end of the study on Day 126. Our original intent was to freeze semen obtained from bulls grazing Eþ or non-Eþ pastures and assess in vitro fertility as described by Schuenemann et al., 2005 [13,14], but early attempts indicated an issue with freezing semen collected from bulls in the Eþ treatment. Subsequently, we evaluated multiple ejaculates obtained from bulls either on
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the Eþ or on the E treatments. Although morphology was lower in bulls on the Eþ grazing treatment compared with that on the E group at the time of freezing, both groups exhibited over 75% normal sperm morphology at the time of freezing. Furthermore, computerized sperm-quality analysis confirmed no difference in the other quality estimates of samples chosen to be extended and frozen between the treatment groups. Computerized spermquality analysis of semen postthaw revealed that semen of bulls in the Eþ treatment possessed a greater than threefold reduction in motility, progressive motility, motile sperm concentration, progressive motile sperm concentration, total motile sperm, and total progressive motile sperm. In contrast, the total sperm number observed postthaw was the same between treatments verifying that final extended concentration was not the reason for the observed reduction in the number of motile and total motile sperm. The computerized sperm-quality analysis data of frozen-thawed semen would indicate a structural and/or physiological change in the spermatozoa from bulls grazing endophyte-infected fescue, which is consistent with the decreased percentage of normal sperm present in bulls grazing Eþ forage. Physical changes of this magnitude could explain the differences observed for sperm velocity reported here and those reported by other groups [15] and explain lower fertility of semen from bulls consuming ergot alkaloids in vitro [13,14]. 4.1. Conclusions Fescue toxicosis results in subtle alterations in carcass composition and semen characteristics in yearling beef bulls. Furthermore, grazing the pasture containing ergot alkaloids alters sperm morphology and semen freezing potential of ejaculates suggesting an altered sperm structure/physiology which would explain data showing alterations in semen quality and in vitro fertility. Acknowledgments This material is based on work supported by National Institute of Food and Agriculture/United States Department of Agriculture, under project number SC-1700376 and was supported by National Research Initiative Competitive grant number 2010-38942-20745 from the USDA National Institute of Food and Agriculture and the Cooperative State Research, Education, and Extension Service and has been assigned Technical Contribution number 6194 of the Clemson University Experiment Station. The author would also like to thank Johanna E. Johnson with assistance in preparing this article. References [1] Hoveland CS. Importance and economic significance of the Acremonium endophytes to performance of animals and grass plant. Agric Ecosyst Environ 1993;44:3–12. [2] Strickland JR, et al. Board-invited review: St. Anthony’s fire in livestock: causes, mechanisms, and potential solutions. J Anim Sci 2011; 89:1603–26. [3] Allen VG, Segarra E. Anti-quality components in forage: overview, significance, and economic impact. J Range Management 2001;54: 409–12.
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[4] Browning Jr R. Effects of endophyte-infected tall fescue on indicators of thermal status and growth in Hereford and Senepol steers. J Anim Sci 2004;82:634–43. [5] Gunter SA, Beck PA. Novel endophyte-infected tall fescue for growing beef cattle. J Anim Sci 2004;82(E-Suppl):E75–82. [6] Watson RH, et al. Productivity of cow-calf pairs grazing tall fescue pastures infected with either the wild-type endophyte or a nonergot alkaloid-producing endophyte strain, AR542. J Anim Sci 2004;82: 3388–93. [7] Johnson JM, et al. Steer and pasture responses for a novel endophyte tall fescue developed for the upper transition zone. J Anim Sci 2012; 90:2402–9. [8] Burke JM, Rorie RW. Changes in ovarian function in mature beef cows grazing endophyte infected tall fescue. Theriogenology 2002; 57:1733–42. [9] Burke JM, et al. Reproductive responses to grazing endophyteinfected tall fescue by postpartum beef cows. Theriogenology 2001;56:357–69. [10] Jones KL, et al. Domperidone can ameliorate deleterious reproductive effects and reduced weight gain associated with fescue toxicosis in heifers. J Anim Sci 2003;81:2568–74. [11] Jones KL, King SS, Iqbal MJ. Endophyte-infected tall fescue diet alters gene expression in heifer luteal tissue as revealed by interspecies microarray analysis. Mol Reprod Dev 2004;67:154–61. [12] Jones KL, et al. Consumption of toxic fescue impairs bull reproductive parameters. The Prof Anim Scientist 2004;20:437–42. [13] Schuenemann GM, et al. Effects of administration of ergotamine tartrate on fertility of yearling beef bulls. Theriogenology 2005;63: 1407–18. [14] Schuenemann GM, et al. Fertility aspects in yearling beef bulls grazing endophyte-infected tall fescue pastures. Reprod Fertil Dev 2005;17:479–86.
[15] Looper ML, et al. Influence of toxic endophyte-infected fescue on sperm characteristics and endocrine factors of yearling Brahmaninfluenced bulls. J Anim Sci 2009;87:1184–91. [16] Stowe HM, et al. Effects of fescue toxicosis on bull growth, semen characteristics, and breeding soundness evaluation. J Anim Sci 2013; 91:3686–92. [17] Bernard JK, et al. Effects of prepartum consumption of endophyteinfested tall fescue on serum prolactin and subsequent milkproduction of Holstein cows. J Dairy Sci 1993;76:1928–33. [18] Spitzer JC, Hopkins FM. Breeding soundness evaluation of yearling bulls. Vet Clin North Am Food Anim Pract 1997;13:295–304. [19] Barkey RJ, et al. Prolactin and antiprolactin receptor antibody inhibit steroidogenesis by purified rat Leydig cells in culture. Mol Cell Endocrinol 1987;52:71–80. [20] Weiss-Messer E, Ber R, Barkey RJ. Prolactin and MA-10 Leydig cell steroidogenesis: biphasic effects of prolactin and signal transduction. Endocrinology 1996;137:5509–18. [21] Lotti F, et al. Clinical implications of measuring prolactin levels in males of infertile couples. Andrology 2013;1:764–71. [22] Hair WM, et al. Prolactin receptor expression in human testis and accessory tissues: localization and function. Mol Hum Reprod 2002; 8:606–11. [23] Steger RW, et al. Neuroendocrine and reproductive functions in male mice with targeted disruption of the prolactin gene. Endocrinology 1998;139:3691–5. [24] Almquist JO, Amann RP. Reproductive capacity of dairy bulls. II. Gonadal and extra-gonadal sperm reserves as determined by direct counts and depletion trials; dimensions and weight of genitalia. J Dairy Sci 1961;44:1668–78. [25] Pratt SL, et al. Comparison of methods for predicting yearling scrotal circumference and correlations of scrotal circumference to growth traits in beef bulls. J Anim Sci 1991;69:2711–20.
