481 The procedures use for the in vitro production (IVP) of embryos have been described previously by Scenna

et al. (2004) and Lawrence et al.(2004). Sperm concentration was determined after Percoll purification using a haemocytometer and motility was also determined. Oocytes were cultured for 18-22 h with 375 000 motile sperm collected from bulls within each each group in 500L IVF Tyrodes albumin lactate pyruvate(one bull per well). A minimum of 50 oocytes per well was used foe each bull on each collection date. Approximately 18-22 h after fertilization, putative zygotes (PZ) were denuded of cumulus cells before transferring groups of approximately 50 zygotes to four-well plates containing 500 L potassium simplex optimized medium-bovine serum albumin per well. Zygotes were placed in a humidified atmosphere of 5.5% CO2, 7% O2, and 87,5% N2 at 38,5oC. The ability of PZ to cleave and develop to the blastocyst stage was evaluated on Days 3 and 8, respectively (where 0 is the day of IVF). The ability of the oocyte to cleave after IVF was assessed by recording the number of one-, two-, four- and eight- to 16 cell embryos present on Day 3 (70 75 h after insemination). Blastocyst stage embryos (Day 8; 187 195 h after insemination) were stained with Hoechst 33342 (5 g ml1; 15 min), mounted on a glass slide (50% glycerol) and exposed to ultraviolet (UV) light directed through a DAPI filter (excites at awavelenght of 330 380 nm) to enumerate the total number of nuclei contained within each embryo. Blood collection and radioimmunoassays Blood samples were centrifuged at 2000g for 30 min and serum decanted all stores at 20oC until assay for prolactin an testosterone. Radioimmunoassays (Coat-A-Count; Diagnostic Product, Los Angeles, CA, USA) were performed to determine the concentration of testosterone (Schuenemann et al. 2005). The sensitivity of the testosterone assay was 0.04 ng mL1, with intra- and interassay coefficients of variation (CV) of 10% and 2%, respectively. Radioimmunoassays to determine the concentration of prolactin assay was determined to be 0.05% ng mL1. The intra- and interassay CV of the prolactin assay were 11% and 10%, respectively. Statistical analyses The experiment was divided in two different analyses for optimal evaluation of treatment differences. Data were arranged in a complete randomized-design split-plot and repeated-meansures for Analysis I. A randomized block design was arranged for IVF assessment in Analysis II. The experimental period was divided into the two seasons winter and spring. Because no differences were observed in any variables analysed between new and old tall fascue stands, both pestures were combined for stastical analysis. Analysis I The performance and fertility parameters (animals per treatment based upon Berndtson 1990) of bulls in all pastures (E+ and NTE) were evaluated. Differences in average daily gain (ADG), SC, ST, serum concentrations of testosterone and prolactin and the motility and morphology of semen were analyses using SAS proc Mixed (SAS 2000). Data are presented as the least-squares mean s.e.m. A mixed model procedure that included treatments. Time was a repeated-meansures factors and animal (treatment pasture) was included as random effect. The time of each St was recorded and included as a

covariate in the model. Differences in individual least-squares means were evaluated using least significant differences (l.s.d.). Analysis II The fertilisation potential of semen from yearling beef bulls grazing NTE (n = 3 per year) and E+ (n = 3 per year) pastures was evaluated. Differences between E+ nad NTE for IVF procedures were analysed using SAS Proc. Mixed (SAS 2000) in two different replicates (5 May and 28 June) per year, using well as the experimental unit. For these IVF data, the key biological component was the oocyte and the experimental design addressed the large variation among oocytes. Thus, the experimental unit was a well, containing a group of similar oocytes, and treatments (semen) were applied to the wells. Bulls were simply acting as a source of treated material. We do recognize the potential risk in bulls responding differently, but our experience suggest that bulls show a consistent response to E+. In short, the IVF experiment need to be viewed as an assay, where biological material was collected from somesource and the actual experiment was under carefully controlled laboratory conditions. Distributional requirements of normality and equal varience were verified for percentage data. Data are presented as the least-squares mean s.e.m. A mixed-model procedure that included treatment, time, replicate and all interactions as fixed effects was used to compare differences amoung treatments. Replicates were a blocked factor and were includes as random effect. Differences in individual least-squares means were evaluated using l.s.d. A value of P < 0.05 was considered statistically significant for all analyses. Results Analysis I rectal temperature, growth, productin and hair coat scores Troughouth the experimental period, bulls grazing E+ pastures had decreased overall ADG compared with bulls grazing NTE pastures (0.530.01 v. 0.710.01 kg day 1, respectively; P < 0.01; Fig.1). No significant differences in treatment year interaction andpa sture (treatment) were observed (P > 0.10). However, an interaction in year animal (0.720.01 v. 0.600.01 kg days 1 for 2002 v. 2003, respectively; P < 0.01) was observed.

Fig. 1. Average daily gain (ADG) in bulls grazing Jezub/MaxQTM (NTE) and Kentucky 31 tall fescue (E+). Bulls grazing tall fescue E+ pastures. Arrow boxes illustrate the time of semen collection for in vitro fertilisation. E+, endophyte-infected tall fescue pastures; NTE, non toxic endophyte-infected tall fescue pastures; Week 0, November through to Week 32, June.

482

Fig. 2. Overall concentration of prolactin in bulls grazing Jesup/MaxTM (NTE) and Kentucky 31 tall (E+). Bulls grazing tall fescue E+ pastures had decreased prolactin compared with bulls grazing NTE pastures. Arrow boxes illustrate the time of semen collection for in vitro fertilisation. E+, endophyte-infected tall fescue pastures; NTE, non-toxic endophyte-infected tall fescue pastures; Week 0, November through to Week 32, June.

Bull grazing E+ tall fescue treatments had decreased overall serum concentrations of prolactin compared with bulls grazing NTE pastures (347 v. 102 7 ng mL 1, respectively; P < 0.01; Fig.2). In addition, a significant decrease in serum concentrations of pleractin was observed according to season (winter v. spring) in bulls grazing NTE pasture (25 11 v. 119 11 ngmL 1, respectively; P < 0.01; Fig. 2), illustrating seasonal variation in prolactin. Moreover, concentrations of ploractin in bulls grazing E+ tall fescue did not differ between winter and spring (P > 0.01; Fig. 2). Bulls in E+ tall fescue treatments had increased overall RT compared with animals grazing NTE pastures (39.020.03 v. 38.810.03oC, respectively; P < 0.01; Fig. 3), indicating that bulls were severely affected by the consumption of ergovaline-producing fungal endophyte tall fescue. In addition, bulls had a significant increase in RT according to season (spring v. winter) within E+ treatment pastures (P < 0.01; Fig. 3). However, no differences in RT of bulls grazing E+ were observed compared with bulls grazing NTE during the winter (P >0.10; Fig. 3). Coversely , bulls grazing E+ had increased RT compared with bulls grazing NTE pastures during the spring (P > 0.01; Fig. 3). Overall hair coat scores were higher in bulls grazing E+ compared with animals grazing NTE (1.61 0.08 v. 1.03 0.08, respectively; P < 0.01), indicating that bulls were affected by the caonsumption og ergovaline-producing fungal endophyte tall fescue.

Testicular responses Overall ST were decreased in bulls grazing E+ tall fescue compared with bulls grazing NTE pastures (32.5 0.4 v.

Fig. 3. Overall rectal temperatures (RT) in bulls grazing Jesup/MaxTM (NTE) and Kentucky 31 tall (E+). Bulls grazing tall fescue E+ pastures had decreased RT compared with bulls grazing NTE pastures. Arrow boxes illustrate the time of semen collection for in vitro fertilisation. E+, endophyte-infected tall fescue pastures; NTE, non-toxic endophyte-infected tall fescue pastures; Week 0, November through to Week 32, June.

33.5 0.4oC, respectively; P < 0.01). Scrotal temperatures were not different according to date (5 May v. 28 June) within each treatment group (P > 0.10). Neither average daily growth in SC (0.034 0.002 cm day 1; P > 0.10) nor concentrations of testosterone (8.4 1.1 ng mL 1; P > 0.10) were affected by the consumption of E+ tall fescue compared with NTE pasture. Immediately after semen collection (January, March, May and June) sperm motility and morphology were assessed. progeressive motility was not different between bulls grazing E+ tall fescue and bulls grazing NTE tall fescue (P > 0.10; Table 2). In addition, sperm morphology (primary and secondary abnormalities) was not differ amoung treatment groups (P > 0.10; Table 2). Moreover, sperm motility and morphology did not differ according to season (winter v. spring) among treatment groups ( P > 0.10). Owing to the age of bulls at first collection (January), the motility of the first ejaculate was significantly impaired compared with the motility of sperm collected at later dates (P < 0.01).

Analysis II Fertilisation potential of semen The motility of sperm at collection and immediately before and after Percoll preparation was not different between bulls grazing E+ and NTE pastures ((P > 0.10; Table 3). Thus, changes between motility at collection (at the farm) and motility before Percoll (24 h later) (24.6 5.7 and 21.7 46%, respectively; P > 0.10; Table 3) did not differ between bulls grazing E+ or NTE pasture. Cleavage rates were longer following IVF with semen from E+ bulls (P < 0.01; Table 3) caompared with cleavage after IVF using semen from NTE bulls. Development to the eight-cell and blastocyst stage and nuclei number of cleaved embryos did not differ between treatments (P > 0.10; Table 3). No