Guy Allard

PRODUCTION OF A DIAGNOSTIC MONOCLONAL ANTIBODY IN PERENNIAL ALFALFA PLANTS

The increasing use of monoclonal antibodies (mAbs) in diagnostic reagents necessitates efficient and cost-effective mAb production methods. In blood banks, one of the most routinely used reagents is the anti-human IgG reagent used for the detection of non-agglutinating antibodies. Here we report the production of a functional, purified anti-human IgG, through the expression of its encoding genes in perennial transgenic alfalfa. Transgenic plants expressing the light- and heavy-chain encoding mRNAs were obtained, and plants from crosses were found to express fully assembled C5-1. The purification procedure yielded mainly the H2L2 form with specificity and affinity identical to those of hybridoma-derived C5-1. The ability to accumulate the antibody was maintained both in parental F1 lines during repeated harvesting and in clonal material; the antibody was stable in the drying hay as in extracts made in pure water. Also, plant and hybridoma-derived C5-1 had similar in vivo half-lives in mice. These results indicate that plant C5-1 could be used in a diagnostic reagent as effectively as hybridoma-derived C5-1, and demonstrates the usefulness of perennial systems for the cost-effective, stable, and reliable production of large amounts of mAbs.

Genetic transformation of commercial breeding lines of alfalfa (Medicago sativa)

Bio-engineering technologies are now routinely used for the genetic improvement of many agricultural crops. However, breeding lines of Medicago sativa are not easily amenable to genetic transformation and therefore cannot benefit from the molecular tools that have been developed for genetic manipulations. This paper describes a strategy that has been developed to transfer DNA into commercially important breeding lines of winter-hardy alfalfa via Agrobacterium infection. Three highly regenerative genotypes have been selected from ca 1000 genotypes within 11 breeding lines. They have been used as basic material for an extensive genetic transformation trial. Combinations of genotypes (11.9, 8.8, 1.5) expression vectors (pGA482, pGA643, pBibKan) and bacterial strains (C58, A281, LBA4404) were tested for their ability to produce stable transgenic material. Putative transgenic plantlets were further screened by nptII-specific PCR amplification, Southern hybridization and recallusing assays. One genotype (1.5) gave only one transformant out of 432 individual trials. With the two other genotypes, efficiency of transformation (kanamycin-resistant calluses obtained/explant tested) ranged from 0 to 0.92 depending on the strain/vector combination used. Statistical interactions underline the possibility of obtaining good genotype-strain-vector combinations for alfalfa transformation. Predicted transformation probability indicates that with strain LBA4404 containing the vector pGA482 and genotype 11.9, transformation efficiency is above 60% and 10% or more of the calluses retain embryogenic potential. PCR amplification and Southern hybridization of randomly chosen regenerated plantlets demonstrated that all embryos developing on 50 μg ml-1 kanamycin had a stable genomic insertion of nptII. Sexual crosses with untransformed genotypes showed that segregation of the transgenic trait followed Mendelian heredity.

Near-infrared reflectance spectroscopy prediction of neutral detergent-soluble carbohydrates in timothy and alfalfa

Carbohydrates in forage crops can be divided into neutral detergent-insoluble fiber and neutral detergent-soluble carbohydrates (NDSC); the latter includes organic acids (OA), total ethanol:water-soluble carbohydrates (TESC), starch, and neutral detergent-soluble fiber (NDSF). The accurate and efficient estimation of NDSC in forage crops is essential for improving the performance of dairy cattle. In the present study, visible and near-infrared reflectance spectroscopy (NIRS) were applied to evaluate the feasibility of predicting OA, TESC, starch, NDSF, NDSC, and all related constituents used to calculate these 5 carbohydrate fractions in timothy and alfalfa. Forage samples (n = 1,008) of timothy and alfalfa were taken at the first and second harvests at 2 sites in 2007; samples were dried, ground, and then scanned (400 to 2,500 nm) using an NIRSystems 6500 monochromator. A calibration (n = 60) and a validation (n = 15) set of samples were selected for each species and then chemically analyzed. Concentrations of TESC and NDSC in timothy, as well as starch in alfalfa, were successfully predicted, but many other carbohydrate fractions were not predicted accurately when calibrations were performed using single-species sample sets. Both sets of samples were combined to form new calibration (n = 120) and validation (n = 30) sets of alfalfa and timothy samples. Calibration and validation statistics for the combined sets of alfalfa and timothy samples indicated that TESC, starch, and NDSC were predicted successfully, with coefficients of determination of prediction of 0.92, 0.89, and 0.93, and a ratio of prediction to deviation (RPD) of 3.3, 3.1, and 3.6, respectively. The NDSF prediction was classified as moderately successful The NIRS prediction of OA was unsuccessful All related constituents were predicted successfully by NIRS except ethanol-insoluble residual OM, with Our results confirm the feasibility of using NIRS to predict NDSC, its fractions, and other related constituents, except for OA and ethanol-insoluble residual OM, in timothy and alfalfa forage samples.

Hay to Reduce Dietary Cation-Anion Difference for Dry Dairy Cows

Timothy grass has a lower dietary cation-anion difference [DCAD = (Na + K) - (Cl + S)] than other cool-season grass species. Growing timothy on low-K soils and fertilizing it with CaCl2 could further decrease its DCAD. The objective of this study was to evaluate the effects of feeding low-DCAD timothy hay on dry dairy cows. Six nonpregnant and nonlactating cows were used in a replicated 3 x 3 Latin square. Treatments were as follows: 1) control diet (control; DCAD = 296 mEq/kg of dry matter); 2) low-DCAD diet based on low-DCAD timothy hay (L-HAY; DCAD = - 24 mEq/kg of dry matter); and 3) low-DCAD diet using HCl (L-HCl; DCAD = - 19 mEq/kg of dry matter). Decreasing DCAD with L-HAY had no effect on dry matter intake (11.8 kg/d) or dry matter digestibility (71.5%). Urine pH decreased from 8.21 to 5.89 when L-HAY was fed instead of the control. Blood parameters that decreased with L-HAY were base excess (- 0.4 vs. 3.8 mM) and HCO3- (23 vs. 27 mM), and blood parameters that increased were Ca2+ (5.3 vs. 5.1 mg/dL), Cl- (30.5 vs. 29.5 mg/dL), and Na+ (60.8 vs. 60.1 mg/dL). Compared with the control, L-HAY resulted in more Ca in urine (13.4 vs. 1.2 g/d). Comparing L-HAY with L-HCl, cow dry matter intake tended to be higher (11.5 vs. 9.8 kg/d), and blood pH was higher (7.37 vs. 7.31). Urine pH; total dry matter; Ca, K, P, and Mg apparent absorption; and Ca, K, Na, Cl, S, P, and Mg apparent retention were similar. Absorption as a percentage of intake of Na and Cl was lower for L-HAY as compared with L-HCl. In an EDTA-challenge test, cows fed L-HAY regained their initial level of blood Ca2+ twice as quickly as the control treatment (339 vs. 708 min); there were no differences between L-HAY and L-HCl. This experiment confirms that feeding low-DCAD hay is an effective means of decreasing the DCAD of rations and obtaining a metabolic response in dry dairy cows.

Nutritive Value of Timothy Fertilized with Chloride or Chloride-Containing Liquid Swine Manure

Chloride fertilization of timothy (Phleum pratense L.) decreases forage dietary cation-anion difference to an acceptable value [(<250 mmol(c)/kg of dry matter (DM)] for dry dairy cows (Bos taurus). However, high Cl concentrations in forages as a result of fertilization might affect nutritive value. Two experiments were used to evaluate the effects of chloride fertilization on timothy spring growth and summer regrowth by determining concentrations of crude protein and neutral detergent fiber (NDF), in vitro true digestibility of DM (IVTD), and in vitro digestibility of NDF (dNDF). In an inorganic fertilization experiment, forages grown at 4 locations were fertilized with CaCl(2) (0, 80, 160, and 240 kg of Cl/ha per yr) or NH(4)Cl (160 kg of Cl/ha per yr) in combination with 2 N application rates (70 and 140 kg of N/ha per yr). The increase in Cl fertilization rate affected forage NDF concentration (+1.4%), IVTD (-0.8%), and dNDF (-1.2%) only at the highest rate of N fertilization, but this effect was not of biological importance. Crude protein concentration was not affected by Cl fertilization. Both Cl fertilizer types had a similar impact on forage nutritive value. In an organic fertilization experiment, forages grown at 2 locations received 1 of 7 experimental treatments [unfertilized control, inorganic fertilizer, raw liquid swine manure (LSM), and liquid fractions of 4 pretreated LSM types (decanted, filtered, anaerobically digested, and flocculated)] that provided, respectively, 0, 60, 41, 44, 44, 36, and 101 kg of Cl/ha per yr. The last 6 fertilizer treatments also provided 140 kg of N/ha per yr. The IVTD, dNDF, and concentration of NDF in timothy forage were not affected by the Cl content of the different LSM types. Nitrogen fertilization increased concentration of forage NDF and decreased IVTD and dNDF, but this effect was not biologically important. In both experiments, soil types and harvests had a negligible effect on forage nutritive value. Organic or inorganic Cl fertilizers applied to decrease timothy dietary cation-anion difference have little or no effect on forage nutritive value.

Predicting timothy mineral concentrations, dietary cation-anion difference, and grass tetany index by near-infrared reflectance spectroscopy.

The mineral concentration of forage grasses plays a significant role in 2 metabolic disorders in dairy cattle production, namely, hypocalcemia (milk fever) and hypomagnesemia (grass tetany). Risks of occurrence of these 2 metabolic disorders can be evaluated by determining the dietary cation-anion difference (DCAD) and the grass tetany (GT) index of forages and specific rations. The objective of this study was to evaluate the feasibility of predicting timothy (Phleum pratense L.) mineral concentrations of Na, K, Ca, Mg, Cl, S, and P, the DCAD, and the GT index by near-infrared reflectance spectroscopy (NIRS). Timothy samples (n = 1,108) were scanned using NIRS and analyzed for the concentration of 7 mineral elements. Calculations of the DCAD were made using 3 different formulas, and the GT index was also calculated. Samples were divided into calibration (n = 240) and validation (n = 868) sets. The calibration, cross-validation, and prediction for mineral concentrations, the DCAD, and the GT index were performed using modified partial least squares regression. Concentrations of K, Ca, Mg, Cl, and P were successfully predicted with coefficients of determination of prediction (R(P)2) of 0.69 to 0.92 and coefficients of variation of prediction (CV(P)) ranging from 6.6 to 11.4%. The prediction of Na and S concentrations failed, with respective R(P)2 of 0.58 and 0.53 and CV(P) of 82.2 and 12.9%. The 3 calculated DCAD and the GT index were predicted successfully, with R(P)2 >0.90 and CV(P) <20%. Our results confirm the feasibility of using NIRS to predict K, Ca, Mg, and Cl concentrations, as well as the DCAD and the GT index, in timothy.

SHORT COMMUNICATION: Diurnal variations of nonstructural carbohydrates and nutritive value in timothy

Morin, C., Bélanger, G., Tremblay, G. F., Bertrand, A., Castonguay, Y., Drapeau, R., Michaud, R., Berthiaume, R. and Allard, G. 2012. Short Communication: Diurnal variations of nonstructural carbohydrates and nutritive value in timothy. Can. J. Plant Sci. 92: 883-887. Nonstructural carbohydrate (NSC) concentration in timothy (Phleum pratense L.) increased during the day by 16 to 41 g kg-1 DM (+23 to 137%) in spring and summer growth cycles and reached a maximum value between 12.8 and 13.2 h after sunrise in an area with 15 to 16 h of day length. This increase in NSC concentration was generally accompanied by a decrease in concentrations of neutral detergent fiber (-2 to 6%), acid detergent fiber (-1 to 7%), and N (-6 to 11%).

Timothy mineral concentration and derived indices related to cattle metabolic disorders: A review

Timothy (Phleum pratense L.) is grown in several countries, mainly for livestock forage. The nutritive value and dry matter (DM) yield of timothy is well established but less information is available on mineral concentration and related indices, like the dietary cation-anion difference (DCAD) and the grass tetany (GT) index. These indices predict the risk of a forage diet to cause metabolic disorders, i.e., milk fever for DCAD and grass tetany for the GT index. Improving our knowledge of factors affecting timothy mineral concentration is important for livestock production. This review paper compares timothy with other common cool-season grasses for mineral concentrations, DCAD, and GT index. Also assessed are variations in timothy mineral concentration affected by harvest and fertilization management, soil mineral availability, and environmental conditions. These results demonstrate the high potential for improvement and use of timothy forage to decrease the incidence of metabolic disorders within the dai...

Timothy silage with low dietary cation-anion difference fed to nonlactating cows

Decreasing the dietary cation-anion difference (DCAD) by using anion sources before calving reduces hypocalcemia in cows at calving. Reduced DCAD from CaCl2-fertilized timothy hay achieves similar results, but the effects of feeding low-DCAD forage as silage have not been determined. The objective of this study was to evaluate the effect of low-DCAD timothy silage on dry cows. Six nonlactating and nonpregnant Holstein cows were used in a replicated 3 x 3 Latin square. Treatments were 1) control diet (DCAD = 232 mEq/kg of dry matter, DM); 2) low-DCAD diet using a low-DCAD timothy silage (LDTS; DCAD = -21 mEq/kg of DM); and 3) low-DCAD diet using a fermentation by-product (LDBP; DCAD = -32 mEq/kg of DM). Differences between dietary treatments were considered statistically significant at P < or = 0.05 and tendencies were noted when 0.05 < P < 0.10. Compared with the control, feeding LDTS tended to decrease DM intake (10.6 vs. 12.5 kg/d) and decreased urinary pH (6.15 vs. 8.18) as well as apparent digestibility of DM (67 vs. 69%). Blood pH (7.37 vs. 7.42), HCO3- (25.3 vs. 27.5 mM), and base excess (0.4 vs. 3.1 mM) were decreased, and blood Cl- (29.6 vs. 29.1 mg/dL) was increased. Apparently absorbed Na and Cl were higher and apparently absorbed K, P, and digested ADF were lower for LDTS compared with the control. Both LDTS and LDBP resulted in similar DM intake. Urinary pH tended to be higher (6.15 vs. 5.98) and percentage of digested DM was lower (67 vs. 70%) with LDTS compared with LDBP. Blood ionized Ca (5.3 vs. 5.4 mg/dL) tended to be lower and blood Cl- (29.6 vs. 30.1 mg/dL) was lower, whereas blood pH (7.37 vs. 7.33), HCO3- (25.3 vs. 21.5 mM), and base excess (0.4 vs. -3.8 mM) were higher with LDTS compared with LDBP. Apparent absorption of Na, Cl, S, and P, as well as apparent digestion of acid detergent fiber, neutral detergent fiber, and N were lower, and K, Cl, S, P, Mg, and N were less retained with LDTS compared with LDBP. Results confirm that low-DCAD timothy silage can be used to produce a compensated metabolic acidosis by decreasing the DCAD of rations served to nonlactating dairy cows.

Dietary cation-anion differences in some pasture species, changes during the season and effects of soil acidity and lime amendment

The difference between cation and anion concentrations is an important property when assessing feed for dry dairy cows in order to avoid hypocalcaemia following calving. Dietary cation–anion difference (DCAD) is used to assess suitability of feed and predict the risk of milk fever; a value of –5 cmol(+)/kg dry matter (DM) or less is desirable. This work has examined the DCAD of 16 field-grown species found in pasture in southern Australia. The DCAD [cmol(+)/kg DM] at the flowering stage varied from 7 to 32 for grasses, 21 to 72 for legumes and 72 to 99 for dicot weeds. The average DCAD for legumes was 50 cmol(+)/kg DM, over 2-fold higher than the 20 cmol(+)/kg DM average for grasses. There was a substantial decline in DCAD of herbage as the season progressed. In a glasshouse experiment with five grass species in an acid soil, lime application increased yield and tended to lower the DCAD. Lime decreased uptake per unit root length of potassium and chlorine and increased uptake of calcium by phalaris and timothy. While DCAD is an important attribute of herbage for assessing its suitability for prepartum diets of dairy cows, the present data indicate that it would be prudent to also consider concentrations of calcium and other mineral nutrients in herbage, particularly when examining less familiar plant species or the effects of different cultural practices on the composition of herbage for such diets.