Speed associated with plasma pH, oxygen content, total protein and urea in an 80 km race.

To test the hypothesis that endurance performance may be related quantitatively to changes in blood, we measured selected blood variables then determined their reference ranges and associations with speed during an 80 km race. The plan had 46 horses in a 2 x 2 factorial design testing a potassium-free electrolyte mix and a vitamin supplement. Blood samples were collected before the race, at 21, 37, 56 and 80 km, and 20 min after finishing, for assay of haematocrit, plasma pH, pO2, pCO2, [Na+], [K+], [Ca++], [Mg++], [Cl-], lactate, glucose, urea, cortisol, alpha-tocopherol, ascorbate, creatine kinase, aspartate amino transferase, lipid hydroperoxides, total protein, albumin and creatinine, and erythrocyte glutathione and glutathione peroxidase. Data from 34 finishers were analysed statistically. Reference ranges for resting and running horses were wide and overlapping and, therefore, limiting with respect to evaluation of individual horses. Speed correlations were most repeatable, with variables reflecting blood oxygen transport (enabling exercise), acidity and electrolytes (limiting exercise) and total protein (enabling then, perhaps, limiting). Stepwise regressions also included plasma urea concentration (limiting). The association of speed with less plasma acidity and urea suggests the potential for fat adaptation and protein restriction in endurance horses, as found previously in Arabians performing repeated sprints. Conditioning horses fed fat-fortified and protein-restricted diets may not only improve performance but also avoid grain-associated disorders.

Antioxidant status and muscle cell leakage during endurance exercise.

Antioxidant status of 35 endurance horses was studied during an 80 (OD80) or 160 km (OD160) race. Packed cell volume (PCV), total plasma protein (TPP), plasma ascorbic acid (VIT C), plasma alpha-tocopherol (VIT E) and erythrocyte glutathione (GSH) concentrations, erythrocyte glutathione peroxidase (GPX), plasma aspartate aminotransferase (AST) and plasma creatine kinase (CK) activities were measured at 0, 40, 80 km and 60 min of recovery (REC) at OD80, and 0, 64, 106, 142, 160 km and REC at OD160. In both races, no changes were found in plasma VIT E concentration, but VIT C and GSH concentrations decreased (P<0.05), and mean GPX, AST and CK activities increased from 0 km (P<0.05). Indices of muscle cell leakage (plasma AST and CK) were correlated (r = 0.36 to 0.67; P<0.03) with indices of antioxidant status (VIT C, GSH and GPX). Associations between increased muscle leakage and decreased antioxidant status may, in part, reflect oxidative stress and suggest the testing of antioxidant supplements in endurance horses to improve performance and welfare.

Alkanes as internal markers to estimate digestibility of hay or hay plus concentrate diets in horses.

Dry matter intake (DMI), dry matter digestibility (DMD), and fecal output (FO) are difficult to measure directly in the field, and indirect methods using external and internal markers have thus been developed. An experiment was conducted consisting of two digestion trials with two periods in each trial to examine the use of five odd-chain alkanes (C25 to C33) of plant cuticular wax as internal markers to estimate DMD of hay or hay plus concentrate diets in horses. Eight mature Thoroughbred geldings were housed in 4- x 4-m stalls and randomly assigned to one of two mixed grass/legume hays (Diets 1 and 2) in Trial 1 and to mixed grass/legume hay plus one of two concentrates (Diets 3 and 4) in Trial 2. After the first 12-d period was conducted, dietary assignments for each group were switched for the second period in each trial. Each period consisted of a dietary accommodation from d 1 to 7 and total fecal collection from d 8 to 11. Results indicated that fecal recoveries of odd-chain alkanes were 88 to 90% for Diet 1, 75 to 92% for Diet 2, 71 to 81% for Diet 3, and 71 to 82% for Diet 4. Alkane recoveries were not related to alkane chain lengths. Digestibilities calculated from alkane concentration data adjusted using the mean fecal recovery of individual odd-chain alkanes (DA1) were not significantly different from the digestibilities estimated from total collection (DTC) for Diets 1 and 2 in Trial 1 and Diets 3 and 4 in Trial 2. When adjustment was based on the mean recovery of all alkanes (DA2; estimated by linear regression), all DA2 estimates for horses offered all diets were similar to DTC. Results indicate that accurate mean estimates of DMD can be obtained by using plant wax alkane markers and adjusting for the mean recovery of five odd-chain alkanes in a diet.

Sarcoplasmic reticulum responses to repeated sprints are affected by conditioning of horses.

Sarcoplasmic reticulum (SR) responses to repeated sprints and to physical conditioning were studied in 10 Quarter Horses. Exercise tests (four repeated sprints on a treadmill) were conducted before and after 12 wk of sprint conditioning. Muscle samples from the middle gluteal muscle were taken before and after each exercise test, and SR vesicles were isolated. Calcium uptake was determined spectrophotometrically using antipyrylazo III, and Ca2+-ATPase activity was determined using an enzyme-linked optical assay. Conditioning increased calcium uptake rate and Ca2+-ATPase activity by 14 and 38%, respectively, before exercise and by 25 and 26% after exercise. Exercise decreased calcium uptake rate and Ca2+-ATPase activity by 37 and 27%, respectively, before conditioning and by 28 and 21% after conditioning. Decreases in calcium uptake and Ca2+-ATPase activity of SR have been associated with fatigue during exercise, and this association is strengthened by the moderating effect of conditioning.

Vitamin A repletion in thoroughbred mares with retinyl palmitate or beta-carotene.

Forty-five Thoroughbred mares used in an 8-mo depletion study were kept for an additional 20 mo on the same three forage diets (15 mares each): 2-yr-old orchardgrass hay and vitamin A-free concentrate on a drylot (HC); pasture, orchardgrass/alfalfa hay, and vitamin A-free concentrate (PHC); or pasture and orchardgrass/alfalfa hay only (PH). Each diet group was divided into three subgroups, and mares (n = 5) in each group were given either retinyl palmitate (A) at twice the NRC (1989) recommended daily intake, the equivalent amount of vitamin A in the form of water-dispersible beta-carotene (B), or the vehicle (C). Vitamin A status was monitored with serum retinol and a relative dose response (RDR) test every 60 d. In the C subgroups, retinol concentration was 18.65 +/- .84 micrograms/dL (mean +/- SE) and the RDR was 16.26 +/- 1.72% over the 20 mo. Retinol and RDR fluctuated seasonally regardless of supplementation. Vitamin A status, based on serum retinol (P = .001) and RDR (P < .001) values, was lower in the HC than in the PH and PHC. Vitamin A status, based on retinol (P = .05) and RDR (P = .013) values, was improved by retinyl palmitate supplementation in all diet groups, but not by water-dispersible beta-carotene supplementation. Supplementation of the HC mares with vitamin A matched the serum retinol, but not the RDR, of the two pasture, control subgroups. Thus, replete vitamin A status in previously depleted mares was barely obtained by supplementation with twice the currently recommended daily intake of vitamin A.

The promoter for the ribosomal RNA genes of Leishmania chagasi.

A promoter for the rRNA genes of Leishmania chagasi was found to be located about 1 kb upstream of the 18S rRNA coding region and immediately downstream of 64 bp tandem repeats. Its approximate boundaries and corresponding transcription start site were determined by transient transfections and primer extension assays. This promoter for RNA polymerase I has differing activities when transfected into various Leishmania species and no activity in Trypanosoma cruzi. Its sequence has no obvious similarities with other known rRNA promoters in Trypanosomatids. Depending on the species, this promoter can be used to increase expression of a protein from a plasmid in Leishmania by as much as 45-fold over that from a plasmid lacking a promoter.

Point mutations are associated with a gene duplication leading to the bloodstream reexpression of a trypanosome metacyclic VSG.

African trypanosomes evade the immune response of their hosts by sequentially expressing different variant surface glycoproteins (VSGs). We isolated a bloodstream trypanosome clone of Trypanosoma brucei rhodesiense that expresses a VSG normally present during the metacyclic stage of the parasite in the insect vector. Associated with the bloodstream reexpression of this metacyclic VSG is a gene conversion in which the duplicated, expressed gene of 1650 nt contains 11 scattered point mutations when compared with its donor gene. Analysis of an uncloned population of bloodstream trypanosomes revealed another VSG reexpressor of the same donor gene in which the coding region had undergone 24 point mutations. The mutations are unique to the duplicated gene and appear to be nontemplated. The generation of these mutations provides a way for the trypanosome to increase further its antigenic diversity.

African trypanosomes express an immunogenic protein with a repeating epitope of 24 amino acids.

Infection with intracellular protozoan parasites such as Plasmodium, Leishmania and Trypanosoma cruzi induces a strong antibody response against proteins containing tandem repeats, suggesting that these repetitive epitopes may camouflage vulnerable parasite antigens from a 'protective' immune response. We tested this theory by immunoscreening a cDNA expression library of African trypanosomes, extracellular parasites that evade their hosts' immune response by antigenic variation, and found that the most frequently detected trypanosome protein contains more than 40 tandem copies of a 24-amino acid repeat with a consensus sequence of A-M-E-D-E-L-D-S-L-R-A-L-N-E-Q-Y-E-A-L-Q-R-T-N-A (net charge = -4). This protein is encoded on an mRNA of more than 20 kb and has slight sequence similarities with cytoskeletal, intermediate filament proteins in other organisms. Thus, protozoan proteins with tandemly repeating epitopes do not exist solely to divert the humoral immune response; they have other specific physiological functions for the parasites and affect the overall parasite-host interaction in unknown and perhaps different ways.

Studies on the role of actin's aspartic acid 3 and aspartic acid 11 using oligodeoxynucleotide-directed site-specific mutagenesis.

One or more of the five acidic amino-terminal residues of skeletal muscle actin have been implicated as being important in a number of actin-related processes. We have constructed a series of actins containing mutations at Asp3 and Asp11 and tested these mutant proteins for their ability to bind to DNase I-agarose, polymerize with rabbit skeletal muscle actin, undergo amino-terminal processing, and bind to the myosin-S1 subfragment. The mutant actins were expressed in vitro using a coupled transcription/translation system which involves the synthesis of mutant RNAs with SP6 RNA polymerase followed by their translation in a rabbit reticulocyte lysate. When Asp3 was changed to Ala, His, or Asn there was no difference in the tested properties as compared to wild type actin. These results suggest that an acidic residue at position 3 is not critical for the actin functions measured. When Asp11 was changed to Glu, Asn, or His or if the conserved Asp-Asn sequence at positions 11 and 12 was reversed, the mutants were able to copolymerize with rabbit skeletal muscle actin and be cross-linked to myosin-S1 to nearly the same extent as wild type actin. However, the amount of in vitro-synthesized actin capable of binding to DNase I-agarose with high affinity or undergoing amino-terminal processing was reduced significantly relative to the wild type actin synthesized in vitro. The Asp11 mutants ran anomalously on native polyacrylamide gels suggestive of a conformational change induced in the actin. Together, these results suggest that Asp11 may be important in proper actin folding and function.