Interaction of Endophyte-Infected Fescue and Heat Stress on Ovarian Function in the Beef Heifer 1

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1 BIOLOGY OF REPRODUCTION 65, (2001) Interaction of Endophyte-Infected Fescue and Heat Stress on Ovarian Function in the Beef Heifer 1 J.M. Burke, 2,3 D.E. Spiers, 4 F.N. Kojima, 4 G.A. Perry, 4 B.E. Salfen, 4 S.L. Wood, 4 D.J. Patterson, 4 M.F. Smith, 4 M.C. Lucy, 4 W.G. Jackson, 3 and E.L. Piper 5 United States Department of Agriculture, 3 Agricultural Research Service, Booneville, Arkansas Department of Animal Science, 4 University of Missouri, Columbia, Missouri Department of Animal Science, 5 University of Arkansas, Fayetteville, Arkansas ABSTRACT The objective of the experiment was to examine the interaction of endophyte-infected tall fescue and environmental temperature on follicular and luteal development and function in beef heifers. Heifers were fed endophyte-free or endophyte-infected tall fescue seed at thermoneutral or heat stress temperatures (n 6/treatment) 4 wk before and 3 wk after synchronized ovulation. All heifers were subjected to thermoneutral conditions (19 C, 50% relative humidity) from Days 7 to 2; temperature increased incrementally from Days 1 to 0 and cycled between 25 C and 31 C between Days 1 and 20 for heatstressed heifers. Serum was collected and ovaries monitored every other day after induced luteolysis between Days 1 and 23 or until ovulation. Size and location of follicles 4 mm and corpora lutea were recorded. Serum concentrations of prolactin were reduced in heat-stressed heifers fed infected seed and both heat stress and infected seed decreased total cholesterol. Rectal temperature and respiration rate were greatest in heifers fed the infected seed when exposed to maximal temperatures. Heat stress led to reduced diameter of the corpus luteum and serum progesterone compared with thermoneutral conditions. Progesterone was reduced more so in heifers fed infected seed. The combination of infected seed and heat stress was associated with reduced diameter of the preovulatory dominant follicle, and consumption of infected seed led to fewer large follicles during the estrous cycle. Both stressors led to reduced serum estradiol. Impaired follicle function may explain reduced pregnancy rates commonly observed in heifers grazing infected tall fescue pasture. corpus luteum function, environment, follicle, ovary, stress FIG. 1. Experimental time line depicting dietary and heat stress treatment and estrus synchronization protocol. Diet of endophyte-free (EF) or endophyte-infected (EI) tall fescue seed began on Day 27 and ended on Day 20. Heifers were exposed to thermoneutral (TN) environmental chambers between Days 7 and 2. Temperature for heat-stressed (HS) heifers increased between Days 1 and 0 and were maximal (cycled between 25 and 31 C) between Days 1 and 20. plemental diets [2]. In addition, follicle number was decreased in beef heifers grazing endophyte-infected fescue [8, 9], as well as ecg-induced or GnRH agonist-induced estradiol production [8 10]. During summer months, when core body temperature is INTRODUCTION Grazing endophyte-infected tall fescue leads to negative responses in breeding livestock, such as decreased circulating prolactin, pregnancy and calving rates [1], impaired luteal function in heifers [2, 3], and delayed conception [4, 5]. Serum cholesterol was also reduced [6, 7], which may be important because it is the precursor to progesterone produced by the corpus luteum. In fact, progesterone production was reduced in heifers fed endophyte-infected fescue [2, 3], although this was prevented by high energy sup- 1 Mention of trade names or commercial products in this manuscript is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. 2 Correspondence: J.M. Burke, 6883 South State Highway 23, Booneville, AR FAX: ; jmburke@spa.ars.usda.gov Received: 4 December First decision: 29 December Accepted: 1 March by the Society for the Study of Reproduction, Inc. ISSN: FIG. 2. Feed intake (kg d 1 ) for heifers consuming endophyte-free seed diet at thermoneutral (open circles, EF-TN; n 5) or heat-stressed environment (open squares, EF-HS; n 6) and endophyte-infected seed diet at thermoneutral (black circles, EI-TN; n 6) or heat-stressed temperatures (black squares, EI-HS; n 6). The least squares means are presented for each day from Days 6 to 20 and with an estimated standard error of 0.64 kg d 1. Heat stress conditions were initiated on Day 1 as arrow indicates.

2 FESCUE HEAT STRESS INTERACTIONS IN BEEF HEIFERS 261 FIG. 3. Respiration rate (breaths/min 1 ) and rectal temperature ( C) determined at 0600 h (A, C) and 1600 h (B, D) for heifers consuming endophytefree seed diet at thermoneutral (open circles, EF-TN; n 5) or heat-stressed environment (open squares, EF-HS; n 6) and endophyte-infected seed diet at thermoneutral (black circles, EI-TN; n 6) or heat-stressed temperatures (black squares, EI-HS; n 6). The least squares means ( SEM) are presented for each day from Days 6 to 20. Heat stress conditions were initiated on Day 1 as arrow indicates. elevated [11 14], fescue toxicosis elicits its greatest effect on grazing animals. The effect of heat stress on reproduction in beef heifers affected by fescue toxicosis is not understood. Aside from the effects of endophyte-infected fescue on reproductive responses, heat stress may cause other problems, such as decreased pregnancy and conception rates [15, 16], and changes in follicular dynamics [12 14]. In an attempt to understand the interaction between heat stress and endophyte-infected fescue on reproductive responses during the estrous cycle, heifers in this study were exposed to thermoneutral temperatures or controlled heat stress while consuming endophyte-free or endophyte-infected fescue seed. Follicular and luteal dynamics were examined using ultrasound for a synchronized estrous cycle. Serum progesterone and estradiol were measured on days of ultrasound scanning. MATERIALS AND METHODS Animals and Treatments All experimental procedures were reviewed and accepted by the Agricultural Research Service Animal Care and Use Committee in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Pain and stress to animals was minimized throughout the experimental period. Twenty-four Angus or Angus Hereford heifers between 10 and 18 mo of age, weighing approximately 350 kg, determined by ultrasound to have a corpus luteum, received either endophyte-free seed (EF) or endophyte-infected seed (EI) in a mixed feed ration and thermoneutral (TN) or heat stress (HS) temperatures resulting in four treatment combinations (EF- TN, EF-HS, EI-TN, and EI-HS; n four Angus and two Angus Hereford heifers per treatment). Dietary treatments were initiated 28 days before synchronized ovulation. In individual tie stalls inside the Brody Climatic Laboratory at the University of Missouri, all heifers underwent TN treatment (19 C, 50% relative humidity) from Days 7 to 2. For heifers in the HS treatment, temperature increased incrementally between Days 1 and 0 and cycled between 25 C and 31 C (minimum and maximum temperatures were maintained for a period of 4 h each day) from Days 1 to 20, whereas the TN-treated heifers remained at 19 C (Fig. 1). TABLE 1. Mean serum concentrations of prolactin and total cholesterol in beef heifers fed endophyte-free or endophyte-infected fescue seed diets and exposed to thermoneutral or heat stress conditions. Item Prolactin, ng ml Total cholesterol, mg 100 ml EF-TN EF-HS EI-TN EI-HS Pooled (n 5) (n 6) (n 6) (n 6) SEM a b 7.8 a Diet temperature interaction, P 0.003; diet, P 0.001; temperature, P b Diet, P 0.04; temperature, P

3 262 BURKE ET AL % vitamin E premix) and daily feed intake was measured. Initially, all heifers were fed 1.75% of body weight (on a dry-matter basis), which was formulated for a 0.68-kg gain day 1. However, because 30% of the heifers had lost weight after 19 days of feeding, heifers were then offered 2.5% of body weight of feed (dry matter basis). Once heifers entered the heat stress/thermoneutral period, feed offered to EF heifers was reduced to the average intake of EI heifers, as a percentage of body weight, from the previous day for TN and HS treatments, so that intake between diets was similar. Estrus Synchronization and Reproductive Responses Seven days after dietary treatment was initiated, melengestrol acetate (0.5 mg MGA-200 day 1 ; Pharmacia Inc., Kalamazoo, MI) was fed for 7 days. An injection of PGF 2 (25 mg lutalyse i.m.; Pharmacia Inc.) was administered on the last day of MGA feeding followed by injections of GnRH (100 g cystorelin i.m.; Rhone Merieux Inc., Athens, GA) 4 days and PGF 2 11 days later ([18]; Fig. 1). Ovaries were monitored by transrectal ultrasonography (Aloka SSD 500V ultrasound scanner equipped with a 7.5 MHz linear array transrectal transducer; Aloka Co. Ltd, Japan) on each Monday, Wednesday, and Friday beginning on Day 2 and daily from Days 17 to 23 or until ovulation (Day 0 day of expected ovulation; Fig. 1). The size and number of ovarian follicles 4 mm, their position, and position and size of corpora lutea were recorded. Ovulation was determined by the disappearance of the largest follicle and subsequent development of a corpus luteum in the respective location on the ovary. Blood was collected for serum concentrations of progesterone and estradiol on days of ultrasound scanning and daily from Days 17 to 23 or until ovulation. In addition, serum concentrations of progesterone were determined on the day of GnRH and second PGF 2 treatment (Days 10 and 3, respectively, relative to the day of experiment). Two heifers (one EF-TN, one EI-TN) were not included in analyses of reproduction responses because they were acyclic (serum concentrations of progesterone were 1 ngml 1 on days measured throughout the study). FIG. 4. Diameter of the corpus luteum for heifers at thermoneutral (open triangles, TN; n 9) or heat stress temperatures (black stars, HS; n 12) analyzed by heterogeneity of regression with predicted values presented for each day before final ovulation (A) or mixed models procedure with least squares means presented for each day before final ovulation and an estimated standard error for dietary and temperature treatment of 2.8 mm (B). The laboratory contains a set of four identical environmental chambers with a capacity of six heifers per chamber. One heifer from the EF-TN group was removed because of aggressive behavior in the environmental chambers. Respiration rate and rectal temperature were measured daily at 0600 and 1600 h. Serum prolactin and total cholesterol were analyzed from blood collected on Day 17 (heifers were expected to be in luteal phase of estrous cycle) of the experiment to confirm toxicity from endophyte-infected fescue diet. Diet EI tall fescue seed (Grande variety; Seed Research of Oregon, Corvallis, OR) contained 1.9 g g 1 of ergovaline, and EF seed (Teton variety; Cascade International Seed Company, Aumsville, OR) contained 0 g g 1 of ergovaline. Ergovaline concentration in seed was determined by highperformance liquid chromatography (HPLC) [17]. Heifers consumed a total mixed ration throughout the experiment (as a percentage of dry matter: 39.4% corn; 20.5% soybean hulls; 18.4% fescue seed; 10% cotton seed hulls; 4.5% soybean meal; 6% molasses; 0.25% trace mineralized salt; 0.25% salt; 0.6% limestone; 0.035% vitamins A, D, and E premix; and Assays Blood was collected from a coccygeal vessel on days of ultrasound monitoring. Samples were allowed to clot at room temperature (outside of chambers) for 60 min and then were centrifuged (3000 g for 20 min at 4 C). Serum was collected and stored at 20 C until analyzed. Serum concentrations of prolactin were determined in a single assay, using a modification of Henson et al. [19]. The intraassay coefficient of variation (CV) was 16.8%. Serum concentrations of total cholesterol were analyzed in a single assay by the procedure of Wybenga et al. [20]. The intraassay CV was 1.9%. Serum concentrations of progesterone run in a single assay (Coat-A- Count Progesterone; Diagnostic Products Corp., Los Angeles, CA, validated by Srikandakumar et al. [21]) and estradiol [22] were determined from blood collected after ultrasonography. The intraassay CV for progesterone was 9.8% and intraassay and interassay CVs for estradiol were 3.4 and 8.5%, respectively. Statistical Analysis Data were analyzed by least squares ANOVA and mixed models procedures of the Statistical Analysis System [23]. The experiment was designed as a 2 2 factorial repeated measures with diet and environmental temperature as the main effects. The mathematical model that was used to analyze respiration rate, rectal temperature, and feed intake included the following: diet, temperature and breed, the interactions of diet by temperature, diet by breed, temperature by breed, and diet by temperature by breed and heifer within diet by temperature by breed as the error term. Day and time (0600 h vs h) were repeated effects. The model for serum concentrations of prolactin and total cholesterol included diet, temperature, breed, and their interactions. Regression analysis [23] was used to determine whether treatments affected the relationship between a response variable (respiration rate, rectal temperature, feed intake) and day of experiment to the order of significance [24]. These models were adjusted for the appropriate independent variables described previously. Four heifers (two EF-HS; one EI-TN; one EI-HS) did not respond to synchronization and ovulated between Days 9 and 16. All but three of the heifers that did synchronize ovulated between Days 18 and 22. The remaining three heifers (two EF-HS; one EF-TN) had not ovulated by Day 22, but the uterus had estrus tone. Therefore, analysis of reproduction responses considered 1) day of experiment (Fig. 1), as well as 2) days before final ovulation as Day 1 for all heifers. Further, because day of

4 FESCUE HEAT STRESS INTERACTIONS IN BEEF HEIFERS 263 FIG. 5. Serum concentrations of progesterone for heifers consuming endophytefree seed diet at thermoneutral (open circles, EF-TN; n 4) or heat-stressed environment (open squares, EF-HS; n 6) and endophyte-infected seed diet at thermoneutral (black circles, EI-TN; n 5) or heat-stressed temperatures (black squares, EI-HS; n 6). Regression lines are presented for each treatment from Days 19 to 1 relative to day of final ovulation (A) or for each day of experiment (B) with predicted values presented for each day. Serum progesterone expressed as least squares means for each day of experiment for all cyclic heifers with an estimated standard error of 0.88 ng ml 1 (C) and with two heifers with aberrant cycles removed with an estimated standard error of 0.99 ng ml 1 (D). final ovulation was determined retrospectively, there were missing heiferday cells for the latter. Heterogeneity of regression [23] was used to examine treatment effects on the relationship between response variables (number or diameter of follicles or corpora lutea and serum concentrations of progesterone or estradiol) and days before ovulation (determined retrospectively) or day of experiment (Days 0 until ovulation; [24]). These models were adjusted for the appropriate independent variables described previously. For the analysis with days before ovulation as the covariate, data included up to Day 21 to Day 1 for diameter of the corpus luteum, serum concentrations of progesterone, and numbers of medium and large follicles. Because we were interested in increasing serum concentrations of estradiol in the presence of the preovulatory follicle, Days 10 to 1 were included for estradiol and preovulatory follicle diameter analyses when days before ovulation was the covariate. Heterogeneity of regression and mixed model analyses as described previously were performed for reproductive responses after removal of two EI-HS heifers with aberrant cycles to compare responses with and without these animals. RESULTS Feed intake was reduced under HS conditions (P 0.001; Fig. 2). Respiration rate and rectal temperature were similar among treatments before heat stress was imposed. Respiration rate increased for heifers consuming EI diets under heat stress conditions (diet temperature day time [0600 vs h], P 0.001; Fig. 3, A and B). By the end of the experiment, respiration rate was similar between dietary treatments for both TN and HS heifer groups. Rectal temperature increased in HS heifers exposed to EI relative to EF diets at 0600 and 1600 h (diet temperature day time, P 0.001; Fig. 3, C and D). Serum concentrations of prolactin were reduced in EI-treated heifers under HS, but not TN conditions (diet temperature, P 0.003; Table 1). Both EI diet and HS treatment led to a reduction in serum concentrations of total cholesterol (diet, P 0.04; temperature, P 0.003; Table 1). There was no effect of diet on diameter of the corpus luteum, but diameter was reduced in HS heifers (x day before ovulation: temperature day, P 0.07, R 2 model 0.13, Fig. 4A; x day of experiment: temperature day, P 0.05, R 2 model 0.15; least squares means: temperature day, P 0.33, Fig. 4B). Similarly, serum progesterone was reduced in HS heifers and to a greater extent in EI-HS heifers (x days before final ovulation: diet temperature day, P 0.004, R 2 model 0.34, Fig. 5A; x day of experiment: diet temperature day, P 0.004, R 2 model 0.22, Fig. 5B; least squares means: diet day, P 0.08, Fig. 5C). Serum concentrations of progesterone were below the detection level during the experimental estrous cycle in two of six EI-HS heifers for all but one sample (1.5 ng ml 1 in the early luteal phase) and each had a corpus luteum that appeared normal by ultrasound examination. Because serum concentrations of progesterone were greater than 1 ng ml 1 for one of two samples collected 7 days apart before heat stress conditions were initiated and characteristics of the preovulatory follicle and serum estradiol appeared normal, these heifers were considered cyclic. However, data were analyzed with and without these animals for reproductive responses. Serum concentrations of progesterone were not different among treatments when these heifers were removed (regression: diet temperature day, P 0.16; mixed models: diet temperature day, P 0.66, Fig. 5D). One heifer, interestingly, ovulated between Days 18 and 20 of the study (Day 0 was the day of expected ovulation) and the other heifer had not ovulated by Day 22.

5 264 BURKE ET AL. Serum concentrations of estradiol for the two heifers with negligible progesterone in the blood ranged between 0.8 and 3.1 pg ml 1 and 1.9 and 2.2 pg ml 1, similar to that of other EI or HS heifers. After these heifers were removed from the data, the diameter of the preovulatory dominant follicle was not different among treatments (Fig. 8A). However, serum concentrations of estradiol were still decreased for stressed heifers (x days before final ovulation: diet temperature day, P 0.005, R 2 model 0.32; least squares means: diet day, P 0.003, Fig. 8B; x day of experiment: diet temperature day, P 0.001, R 2 model 0.18). The number of large follicles was reduced in heifers that consumed EI diets whether or not the two heifers with negligible progesterone concentrations were removed from data (x day before final ovulation with all cyclic heifers: P 0.07, R 2 model 0.05, Fig. 9A; least squares means: diet day, P 0.02, temperature day, P 0.04, Fig. 9B; x day of experiment: P 0.04, R 2 model 0.12; x day before final ovulation with two heifers removed: P 0.05, R 2 model 0.15; least squares means with two heifers removed: P 0.10, Fig. 9C), but large follicle numbers were not altered by heat stress. Diet or temperature did not affect number of medium follicles during monitored estrous cycle. FIG. 6. Diameter of the preovulatory dominant follicle analyzed by heterogeneity of regression with regression line presented for each treatment from Days 10 to 1 relative to day of final ovulation and predicted values presented for each day (A). Diameter of the preovulatory dominant follicle analyzed by mixed models procedure with least squares means presented for each day before final ovulation and an estimated standard error of 1.4 mm (B) for heifers consuming endophyte-free seed diet at thermoneutral (open circles, EF-TN; n 4) or heat-stressed environment (open squares, EF-HS; n 6) and endophyte-infected seed diet at thermoneutral (black circles, EI-TN; n 5) or heat-stressed temperatures (black squares, EI-HS; n 6). The diameter of the preovulatory dominant follicle was reduced in EI-HS heifers relative to other treatments (x days before ovulation: diet temperature, P 0.008, R 2 model 0.52, Fig. 6A; x day of experiment: diet temperature, P 0.11, R 2 model 0.22; least squares means: diet temperature, P 0.27, temperature, P 0.08, Fig. 6B). Heat stress led to decreased serum concentrations of estradiol and under TN conditions, estradiol was reduced for EI-treated compared with EF-treated heifers (x days before final ovulation: diet temperature day, P 0.01, R 2 model 0.28, Fig. 7A; least squares means: diet day, P 0.05, Fig. 7B; x day of experiment: diet temperature day, P 0.001, R 2 model 0.17, Fig. 7C; least squares means: temperature day, P 0.001, Fig. 7D). DISCUSSION By reducing the intake of EF-HS treated heifers to that of EI-HS treated heifers, the effects of decreased intake typically observed for endophyte-infected fescue-fed animals [25, 26] were minimized in the current study. Daily feed consumption by cattle is approximately 2 3% of body weight. The concentration of ergovaline, an indicator of endophyte infection, in endophyte-infected tall fescue seed and forage ranges from 0.1 to 6.0 g g 1 [27 29]. Based on a 350-kg heifer consuming 1.75% of body weight, a normal range of ergovaline consumption would be g kg 1 of body weight per day. In the current study, heifers that weighed 350 kg and fed endophyte-infected fescue diets received 6.1 (feed intake at 1.75% body weight) to 8.7 (feed intake at 2.5% body weight) g kg 1 body weight of ergovaline per day. Mizinga et al. [30] administered g ergovaline kg 1 body weight per day to 338-kg heifers by feeding endophyte-infected fescue seed during the months of April and May, before animals likely were heat stressed, and observed no difference in serum prolactin between these heifers and those fed endophytefree fescue seed. However, serum prolactin decreased in endophyte-infected, fescue-treated heifers after ergovaline administration was increased above 9.75 g kg 1 body weight. Although we would have liked to have administered a higher dose of ergovaline through diet in the current study, greater than 19% seed in the ration may have reduced intake or palatability for all heifers. It was not surprising then that serum concentrations of prolactin in the current study were not different between endophyte-free and endophyte-infected, fescue-fed cows at thermoneutral conditions. In contrast, concentrations of prolactin were reduced in endophyte-infected, fescue-fed heifers under heat stress conditions. Similarly, other signs of fescue toxicosis were present in endophyte-infected, fescue-fed cows only under heat stress conditions. Rectal temperature and respiration rate increased in endophyte-infected fescue-fed heifers under heat stress conditions. These observations correspond with that of Hemken et al. [25] who observed an increase in rectal temperature and respiration rate in steers fed endophyte-infected tall fescue under heat stress condi-

6 FESCUE HEAT STRESS INTERACTIONS IN BEEF HEIFERS 265 FIG. 7. Serum concentrations of estradiol analyzed by heterogeneity of regression (A, C) with regression line (predicted values presented for each day) for each treatment from Days 10 to 1 relative to day of final ovulation (A) or for each experimental day (C). Serum concentrations of estradiol analyzed by mixed models procedure (B, D) with least squares means presented for each day before final ovulation and an estimated standard error of 1.1 pg ml 1 (B) or for each experimental day with an estimated standard error of 0.9 pg ml 1. Treatments include heifers consuming endophyte-free seed diet at thermoneutral (open circles, EF-TN; n 4) or heat-stressed environment (open squares, EF-HS; n 6) and endophyte-infected seed diet at thermoneutral (black circles, EI-TN; n 5) or heat-stressed environment (black squares, EI-HS; n 6). tions, but not cooler temperatures. Interestingly, heatstressed heifers may have adjusted to the effects of the endophyte-infected fescue in the diet, as evidenced by similar respiration rate between dietary groups of heifers by Day 20 of the study. The greatest decrease in serum concentrations of progesterone occurred in heifers fed the endophyte-infected fescue under heat stress conditions, although heifers with aberrant cycles may have contributed to this. Others reported a decrease in circulating progesterone in heifers fed endophyte-infected fescue [2, 3]. A decrease in total cholesterol in the blood can lead to decreased progesterone [31, 32]. In ruminants, the cholesterol used in steroidogenesis by the corpus luteum is derived from serum low-density lipoproteins, high-density lipoproteins, or both [33 35]. In the current study, there was indeed a decrease in serum concentrations of total cholesterol in endophyte-infected, fescue-fed heifers, as others have observed [5 7], which may have led to decreased steroidogenesis. There was a greater frequency of heat-stressed, endophyte-infected fescue-fed heifers observed with dysfunctional corpora lutea (a corpus luteum that failed to produce progesterone) in the present study. Similarly, others reported a greater frequency of dysfunctional corpora lutea in heifers grazing endophyte-infected fescue pastures [2, 3, 36]. Development of the corpus luteum appears normal, but function may be impaired in endophyte-infected fescue-fed animals. In the two endophyte-infected fescue-treated heifers with undetectable concentrations of progesterone, circulating estradiol was not increased and the life span of the dominant follicle appeared normal. This is surprising, because typically, in the presence of low progesterone, circulating estradiol increases relative to that of cows with normal luteal function [37]. This may suggest altered hormonal feedback mechanisms between the ovary and the hypothalamus and or pituitary. In addition, circulating concentrations of LH may be reduced in endophyte-infected fescue-treated cows [38], which in turn, could lead to decreased luteal function. On the other hand, others reported no changes in LH concentration in heifers or cows exposed to endophyte-infected fescue [30, 39], but the concentration of ergovaline administered to these animals in these studies may not have been great enough to observe a change in LH activity. There appeared to be an earlier rise in serum progesterone in both thermoneutral and heat-stressed heifers exposed to endophyte-infected fescue seed, although heifers that did not respond to estrus synchronization may have contributed to this anomaly. Similarly, production of progesterone may have ceased earlier in heat-stressed heifers fed infected fescue seed, even after heifers with aberrant cycles were removed from data set. In addition, in the presence of the preovulatory follicle, estradiol production of infected fescue-fed heifers may peak and then decline at least 1 day after that of endophyte-free fescue-fed heifers. There have been no other reports, to the authors knowledge, of potential reproductive hormonal asynchrony in association with endophyte-infected fescue. In the thermoneutral environment, preovulatory serum estradiol was decreased in endophyte-infected fescue-fed heifers in the present study, in agreement with McKenzie and Erickson [8] and McLane et al. [10]. Similarly, estradiol also was decreased in heat-stressed heifers. However, endophyte-infected fescue seed did not lead to a further decrease in estradiol under heat stress conditions, suggesting that the two stressors were not additive to follicular

7 266 BURKE ET AL. FIG. 8. Diameter of the preovulatory dominant follicle (A) and serum concentrations of estradiol (B) with least squares means presented for each day before final ovulation for heifers consuming endophyte-free seed diet at thermoneutral (open circles, EF-TN; n 4) or heat-stressed environment (open squares, EF-HS; n 6) and endophyte-infected seed diet at thermoneutral (black circles, EI-TN; n 5) or heat-stressed environment (black squares, EI-HS; n 4). Heifers with aberrant cycles were removed from data analysis. Estimated standard errors are 1.48 mm and 1.01 pg ml 1, respectively. function. During chronic [40] or acute heat stress [13, 14, 40], follicular or serum estradiol production was reduced relative to thermoneutral conditions. Decreased estradiol production may have been due to impaired follicle growth in endophyte-infected fescue-fed heifers in the current study as evidenced by decreased diameter and number of large follicles, which agrees with McKenzie and Erickson FIG. 9. Number of large follicles for heifers consuming endophyte-free seed diet (open triangles, EF; n 10) or endophyte-infected seed diet (black hexagons, EI; n 12; A). Regression lines are presented for each dietary treatment from Days 21 to 1 relative to day of final ovulation with predicted diameter presented for each day. Numbers of large follicles analyzed by mixed models procedure for heifers consuming endophytefree seed diet at thermoneutral (open circles, EF-TN; n 4) or heatstressed environment (open squares, EF-HS; n 6) and endophyte-infected seed diet at thermoneutral (black circles, EI-TN; n 5), or heatstressed environment (black squares, EI-HS; n 6); least squares means are presented for each day before final ovulation with an estimated standard error of 0.41 mm for all cycling heifers (B) and 0.41 mm for heifers with aberrant cycles removed (C).

8 FESCUE HEAT STRESS INTERACTIONS IN BEEF HEIFERS 267 [8, 9]. Others reported no changes in follicle diameter or number of large follicles in heifers fed ergotamine [41], but these heifers were not exposed to the combination of ergot alkaloids present in endophyte-infected fescue. Consistent with Seals et al. [41], there was no observed change in medium follicle numbers in heifers fed endophyte-infected fescue seed (current study) or ergotamine vs. control diet. Beef heifers and cows are typically bred during late spring and early summer months, often on fescue-based pastures in the southeastern and midwestern United States. Lower fertility in heifers that graze endophyte-infected fescue could be explained by decreased luteal function resulting in early embryonic loss, which has been observed in sheep [5]. Alternatively, there could be an asynchrony of reproductive hormones between the dam and the conceptus as evidenced by the earlier rise in serum progesterone and delayed rise in estradiol production in endophyte-infected fescue-fed heifers in the current study. Altered follicular function occurs when heifers are heat-stressed and exposed to endophyte-infected fescue, which contributes to poorer fertility. The inability to regulate body temperature, triggered by fescue toxicosis, predisposes the animal to heat stress, which by itself, is associated with reduced conception rates. Lack of a negative response of endophyte-infected fescue-fed heifers under thermoneutral conditions, with the exception of decreased circulating estradiol, indicates that signs of fescue toxicosis are certainly less severe when heifers are not heat stressed. This may explain lack of consistent results to treatment with endophyte-infected fescue as reported in the literature. Some researchers reported no decrease in reproduction responses in association with endophyte-infected fescue (similar conception rates [42], increased progesterone [43], similar pregnancy rates; unpublished data). This strongly suggests that optimal conditions to breed heifers grazing endophyte-infected fescue pastures would be in the absence of heat stress. The level of heat stress that induces signs of fescue toxicosis leading to decreased fertility warrants further investigation. ACKNOWLEDGMENTS The authors thank J. Cherry, G. Robson, and M. Leonard for technical assistance; and D. Jones, T. Preston, J. Bader, and University of Missouri student aid for help with handling animals. Thanks to T. Popham for statistical advice, G. Rottinghaus for ergovaline analysis, and K. Coffey for ration formulation. Appreciation is extended to Pharmacia Inc. for supplying Lutalyse and MGA-200. REFERENCES 1. Gay N, Boling JA, Dew R, Miksch DE. Effects of endophyte-infected tall fescue on beef cow-calf performance. Appl Agric Res 1988; 3: Estienne MJ, Schillo KK, Fitzgerald BP, Hielman SM, Bradley NW, Boling JA. Effects of endophyte-infected fescue on puberty onset and luteal activity in beef heifers. J Anim Sci 1990; 68(suppl 1):468 (abstract). 3. Mahmood T, Ott RS, Foley GL, Zinn GM, Schaeffer DJ, Kesler DJ. Growth and ovarian function of weanling and yearling beef heifers grazing endophyte-infected tall fescue pastures. Theriogenology 1994; 42: Bond J, Hawk HW, Lynch GP, Jackson C Jr. Lower fertility in ewes grazing tall fescue pastures. J Anim Sci 1982; 55(suppl 1):46 (abstract). 5. Bond J, Lynch GP, Bolt DJ, Hawk HW, Jackson G Jr, Wall RJ. Reproductive performance and lamb weight gains for ewes grazing fungus-infected tall fescue. Nutr Rep Int 1988; 37: Bond J, Powell JB, Undersander DJ, Moe PW, Tyrrell HF, Oltjen RR. Forage composition and growth and physiological characteristics of cattle grazing several varieties of tall fescue during summer conditions. J Anim Sci 1984; 59: Stuedemann JA, Rumsey TS, Bond J, Wilkinson SR, Bush LP, Williams DJ, Caudle AB. Association of blood cholesterol with occurrence of fat necrosis in cows and tall fescue summer toxicosis in steers. Am J Vet Res 1985; 46: McKenzie PP, Erickson BH. The effects of fungal-infected fescue on hormonal secretion and ovarian development in the beef heifer. J Anim Sci 1989; 67(suppl 2):58 (abstract). 9. McKenzie PP, Erickson BH. Effects of fungal-infested fescue on gonadotrophin secretion and folliculogenesis in beef heifers. J Anim Sci 1991; 69(suppl 1):387 (abstract). 10. McLane HL, Rohrbach NR, Erickson BH, Schrick FN. Follicular population of beef heifers exposed to endophyte-infected tall fescue in utero. J Anim Sci 1998; 76(suppl 1):220 (abstract). 11. Wolfenson D, Bartol FF, Badinga L, Barros CM, Marple DN, Cummins K, Wolfe D, Lucy MC, Spencer TE, Thatcher WW. Secretion of PGF 2 and oxytocin during hyperthermia in cyclic and pregnant heifers. Theriogenology 1993; 39: Trout JP, McDowell LR, Hansen PJ. Characteristics of the estrous cycle and antioxidant status of lactating Holstein cows exposed to heat stress. J Dairy Sci 1998; 81: Wilson SJ, Kirby CJ, Koenigsfeld AT, Keisler DH, Lucy MC. Effects of controlled heat stress on ovarian function of dairy cattle. 2. Heifers. J Dairy Sci 1998; 81: Wilson SJ, Marion RS, Spain JN, Spiers DE, Keisler DH, Lucy MC. Effects of controlled heat stress on ovarian function of dairy cattle. 1. Lactating cows. J Dairy Sci 1998; 81: Badinga L, Collier RJ, Thatcher WW, Wilcox CJ. Effects of climatic and management factors on conception rate of dairy cattle in a subtropical environment. J Dairy Sci 1985; 68: Wolfenson D, Flamenbaum I, Berman A. Hyperthermia and body energy store effects on estrous behavior, conception rate, and corpus luteum function in dairy cows. J Dairy Sci 1988; 71: Rottinghaus GE, Garner GB, Cornell CN, Ellis JL. HPLC method for quantitating ergovaline in endophyte-infested tall fescue: seasonal variation of ergovaline levels in stems with leaf sheaths, leaf blades, and seed heads. J Agric Food Chem 1991; 39: Kojima FN, Salfen BE, Bader JF, Ricke WA, Lucy MC, Smith MF, Patterson DJ. Development of an estrus synchronization protocol for beef cattle with short-term feeding of melengestrol acetate: 7 11 synch. J Anim Sci 2000; 78: Henson MC, Piper EL, Daniels LB. Effects of induced fescue toxicosis on plasma and tissue catecholamine concentration in sheep. Domest Anim Endocrinol 1987; 4: Wybenga DR, Pileggi VJ, Dirstine PH, Di Giorgio J. Direct manual determination of serum total cholesterol with a single stable reagent. Clin Chem 1970; 16: Srikandakumar A, Ingraham RH, Ellsworth M, Archbald LF, Liao A, Godke RA. Comparison of a solid-phase, no-extraction radioimmunoassay for progesterone with an extraction assay for monitoring luteal function in the mare, bitch, and cow. Theriogenology 1986; 26: Kirby CJ, Smith MF, Keisler DH, Lucy MC. Follicular function in lactating dairy cows treated with sustained-released bovine somatotropin. J Dairy Sci 1997; 80: SAS/STAT User s Guide, release 6.03 ed. Cary, NC: Statistical Analysis System Institute, Inc.; Wilcox CJ, Thatcher WW, Martin FG. Statistical analysis of repeated measurements in physiology experiments. In: Livestock Reproduction in Latin America. Vienna, Austria: International Atomic Energy Agency; 1990: Hemken RW, Boling JA, Bull LS, Hatton RH, Buckner RC, Bush LP. Interaction of environmental temperature and anti-quality factors on the severity of summer fescue toxicosis. J Anim Sci 1981; 52: Paterson J, Forcherio C, Larson B, Samford M, Kerley M. The effects of fescue toxicosis on beef cattle productivity. J Anim Sci 1995; 73: Belesky DP, Stuedemann JA, Plattner RP, Willkinson SR. Ergopeptine alkaloids in grazed tall fescue. Agron J 1988; 80: Hill NS, Parrott WA, Pope DD. Ergopeptine alkaloid production by endophytes in a common tall fescue genotype. Crop Sci 1991; 31: Porter JK. Analysis of endophyte toxins: fescue and other grasses toxic to livestock. J Anim Sci 1995; 73: Mizinga KM, Thompson FN, Stuedemann JA, Kiser TE. Effects of

9 268 BURKE ET AL. feeding diets containing endophyte-infected fescue seed on luteinizing hormone secretion in postpartum beef cows and in cyclic heifers and cows. J Anim Sci 1992; 70: Talavera F, Park CS, Williams GL. Relationship among dietary lipid intake, serum cholesterol, and ovarian function in Holstein heifers. J Anim Sci 1985; 60: Burke JM, Carroll DJ, Rowe KE, Thatcher WW, Stormshak F. Intravascular infusion of lipid into ewes stimulates production of progesterone and prostaglandin. Biol Reprod 1996; 55: Savion N, Laherty R, Cohen D, Lui D, Gospodarowicz D. Role of lipoproteins and 3-hydroxy-3-methylglutaryl coenzyme A reductase in progesterone production by cultured bovine granulosa cells. Endocrinology 1982; 110: O Shaughnessy PJ, Wathes DC. Role of lipoproteins and de-novo cholesterol synthesis in progesterone production by cultured bovine luteal cells. J Reprod Fertil 1985; 74: Wiltbank MC, Flores MG, Niswender GD. Regulation of the corpus luteum by protein kinase C. II. Inhibition of lipoprotein-stimulated steroidogenesis by prostaglandin F 2. Biol Reprod 1990; 42: Ahmed NM, Schmidt SP, Arbona JR, Marple DN, Bransby DI, Carson RL, Coleman DA, Rahe CH. Corpus luteum function in heifers grazing endophyte-free and endophyte-infected Kentucky-31 tall fescue. J Anim Sci 1990; 68(suppl 1):468 (abstract). 37. Taft R, Sayre B, Inskeep EK. Early increases in estradiol in follicular fluid from cattle with low peripheral concentrations of progesterone. J Anim Sci 1999; 77(suppl 1):123 (abstract). 38. Browning R Jr, Schrick FN, Thompson FN, Wakefield T Jr. Reproductive hormonal responses to ergotamine and ergonovine in cows during the luteal phase of the estrous cycle. J Anim Sci 1998; 76: Christopher GK, Salfen BE, Schmidt SP, Arbona JR, Marple DN, Sartin JL, Bransby DI, Carson RL, Rahe CH. Effects of grazing Kentucky-31 tall fescue infected with Acremonium coenophialium on endocrine function in ovariectomized beef heifers. J Anim Sci 1990; 68(suppl 1):469 (abstract). 40. Wolfenson D, Lew BJ, Thatcher WW, Graber Y, Meidan R. Seasonal and acute heat stress effects on steroid production by dominant follicles in cows. Anim Reprod Sci 1997; 47: Seals RC, Schrick FN, Hopkins FM, Waller JC, Fribourg HA. Follicular dynamics in heifers administered ergotamine tartrate (ET) as a model of endophyte-infected tall fescue consumption. J Anim Sci 1996; 74(suppl 1):11 (abstract). 42. Rorie RW, Hazlett WD, Kreider DL, Piper EL. Effect of feeding endophyte-infected fescue seed on conception rate and early embryonic development in beef heifers. J Anim Sci 1998; 76(suppl 1):229 (abstract). 43. Rorie RW, Hazlett WD, Randel RD, Lewis AW, McNew RW. Prostaglandin secretion in response to oxytocin challenge in heifers fed either orchardgrass or endophyte-infected fescue hay. Biol Reprod 1998; 58(suppl 1):137 (abstract).