Annick Bertrand

Alfalfa Cut at Sundown and Harvested as Baleage Improves Milk Yield of Late-Lactation Dairy Cows

Alfalfa (Medicago sativa L.) cut at sundown has been shown to contain greater concentration of total nonstructural carbohydrates (TNC) than that cut at sunup. Fourteen multiparous (8 ruminally cannulated) and 2 primiparous lactating dairy cows were randomly assigned to 2 treatments in a crossover design (2 periods of 24 d) to investigate the effects of alfalfa daytime cutting management on ruminal metabolism, nutrient digestibility, N balance, and milk yield. Half of each alfalfa field (total of 3 fields) was cut at sundown (PM) after a sunny day, whereas the second half was cut at sunup (AM) on the following day. Both PM and AM cuts were field-wilted and harvested as baleage (531 +/- 15.0 g of dry matter/kg of fresh matter). Bales (PM and AM) were ranked according to their concentrations of TNC, paired, and each pair of PM and AM baleages was then assigned to each experimental day (total of 48 d). The difference in TNC concentration between PM and AM baleages fed during the 10 d of data and sample collection varied from -10 to 50 g/kg of dry matter. Each pair of baleage was fed ad libitum to cows once daily with no concentrate. Ruminal molar proportion of acetate and total volatile fatty acid concentration were greater in animals fed the AM baleage, whereas the proportion of valerate was greater with PM baleage; no other significant changes in ruminal molar proportions of volatile fatty acids were observed between forage treatments. Digestible organic matter intake, organic matter digestibility, and plasma Lys concentration were significantly greater in cows fed PM alfalfa, suggesting that more nutrients were available for milk synthesis. Significantly lower body weight gain and retained N as a proportion of N intake were observed in cows fed PM alfalfa, thus suggesting that nutrients were channeled to milk synthesis rather than to body reserves. Intake of dry matter (+1.0 kg/d), and yields of milk (+1.0 kg/d), milk fat (+70 g/d), and milk protein (+40 g/d) were significantly greater in cows fed PM vs. AM alfalfa. Concentration of milk urea N and excretion of urea N as a proportion of total urinary N were significantly reduced, and milk N efficiency was increased when feeding PM vs. AM alfalfa, indicating an improvement in N utilization. Increasing the TNC concentration of alfalfa by shifting forage cutting from sunup to sundown improved N utilization and milk production in late-lactation dairy cows.

Alfalfa cut at sundown and harvested as baleage increases bacterial protein synthesis in late-lactation dairy cows.

Alfalfa (Medicago sativa L.) cut at sundown (p.m.) has been shown to have a greater concentration of total nonstructural carbohydrates (TNC) than when cut at sunup (a.m.). Eight ruminally cannulated Holstein cows that were part of a larger lactation trial were used in a crossover design (24-d periods) to investigate the effects of alfalfa cutting time on digestibility and omasal flow of nutrients. Alfalfa was cut at sundown or sunup, field-wilted, and harvested as baleage (530 +/- 15.0 g of dry matter/kg of fresh matter). The difference in TNC concentration between p.m. and a.m. alfalfa within each pair of bales fed daily during the 10 d of data and sample collection varied from -10 to 50 g/ kg of dry matter. Each pair of bales was fed for ad libitum intake to cows once daily with no concentrate. During the 3 d of omasal sampling, intake (+0.8 kg/d) and omasal flow of organic matter (OM; +0.42 kg/d) tended to be greater when cows were fed p.m. vs. a.m. alfalfa, but no differences were found for ruminal and postruminal digestion of this nutrient. Similarly, N apparently digested ruminally and postruminally did not differ when feeding p.m. vs. a.m. alfalfa. However, N truly digested in the rumen, as a proportion of N intake, was significantly greater in cows fed p.m. (79%) vs. a.m. alfalfa (74%), thus suggesting that longer wilting time of alfalfa cut at sundown increased forage proteolysis. Supply of rumen-degradable protein did not change (2,716 g/d) when averaged across treatments, whereas omasal flow of non-NH(3) nonbacterial N was significantly decreased (-29 g/d) when feeding p.m. vs. a.m. alfalfa. Omasal flow of total bacterial non-NH(3)-N (NAN) increased (+21 g/d) significantly when cows were fed p.m. vs. a.m. alfalfa possibly because bacteria from cows fed p.m. alfalfa captured significantly more NH(3) than those from cows fed a.m. alfalfa. Therefore, greater availability of fermentable energy as TNC appears to increase the capacity of microbes to uptake NH(3)-N and convert it to microbial protein. Enhanced OM intake can also explain the observed increase in bacterial protein synthesis with p.m. alfalfa. Efficiency of bacterial protein synthesis, expressed on a fermented OM basis or as grams of bacterial NAN per gram of rumen-degradable N, did not differ between p.m. and a.m. alfalfa. Conversely, bacterial efficiency, as grams of bacterial NAN per gram of N intake, was significantly increased when cows were fed p.m. baleage. No significant difference between forage treatments was found for the omasal flow of total AA from omasal true digesta, suggesting no benefit of daytime cutting management on the passage of total AA to the lower gastrointestinal tract. Enhancing energy intake and TNC concentration of alfalfa by shifting forage cutting from sunup to sundown increased protein synthesis and NH(3) uptake by ruminal bacteria indicating an improvement in N utilization.

Winter damage to perennial forage crops in eastern Canada : Causes, mitigation, and prediction

Harsh winter climate results in frequent losses of stands and yield reduction in many forage-growing areas of Canada and other parts of the world. Climatic conditions and crop management both affect the winter survival of perennial forage crops. In this review, we present the main causes of winter damage in eastern Canada and we discuss crop management practices that help mitigate the risks of losses. Predictive tools available to assess the risks of winter damage both spatially and temporally are also presented. Our understanding of the causes of winter damage and of the plant adaptation mechanisms to winter stresses, particularly the role of N and C organic reserves, has improved. Forage species commonly grown in eastern Canada differ in their tolerance to subfreezing temperatures and to anoxia caused by the presence of ice on fields. Some improvement in winter hardiness of forage legume species has been achieved through breeding in eastern Canada but new technologies based on laboratory freezing tests ...

MicroRNA156 as a promising tool for alfalfa improvement

A precursor of miR156 (MsmiR156d) was cloned and overexpressed in alfalfa (Medicago sativa L.) as a means to enhance alfalfa biomass yield. Of the five predicted SPL genes encoded by the alfalfa genome, three (SPL6, SPL12 and SPL13) contain miR156 cleavage sites and their expression was down-regulated in transgenic alfalfa plants overexpressing miR156. These transgenic plants had reduced internode length and stem thickness, enhanced shoot branching, increased trichome density, a delay in flowering time and elevated biomass production. Minor effects on sugar, starch, lignin and cellulose contents were also observed. Moreover, transgenic alfalfa plants had increased root length, while nodulation was maintained. The multitude of traits affected by miR156 may be due to the network of genes regulated by the three target SPLs. Our results show that the miR156/SPL system has strong potential as a tool to substantially improve quality and yield traits in alfalfa.

SRAP polymorphisms associated with superior freezing tolerance in alfalfa (Medicago sativa spp. sativa)

Sequence-related amplified polymorphism (SRAP) analysis was used to uncover genetic polymorphisms among alfalfa populations recurrently selected for superior tolerance to freezing (TF populations). Bulk DNA samples (45 plants/bulk) from each of the cultivar Apica (ATF0), and populations ATF2, ATF4, ATF5, and ATF6 were evaluated with 42 different SRAP primer pairs. Several polymorphisms that progressively intensified or decreased with the number of recurrent cycles were identified. Four positive polymorphisms (F10-R14, Me4-R8, F10-R8 and F11-R9) that, respectively, yielded 540-, 359-, 213-, and 180-bp fragments were selected for further analysis. SRAP amplifications with genotypes within ATF populations confirmed that the polymorphisms identified with bulk DNA samples were reflecting changes in the frequency of their occurrence in response to selection. In addition, the number of genotypes cumulating multiple polymorphisms markedly increased in response to recurrent selection. Independent segregation of the four SRAP polymorphisms suggests location at unlinked loci. Homology search gave matches with BAC clones from syntenic Medicago truncatula for the four SRAP fragments. Analysis of the relationship with low temperature tolerance showed that multiple SRAP polymorphisms are more frequent in genotypes that maintain superior regrowth after freezing. These results show that SRAP analysis of bulk DNA samples from recurrent selections is an effective approach for the identification of genetic polymorphisms associated with quantitative traits in allogamous species. These polymorphisms could be useful tools for indirect selection of freezing tolerance in alfalfa.

Effects of nonstructural carbohydrate concentration in alfalfa on fermentation and microbial protein synthesis in continuous culture

Insufficient readily fermentable energy combined with extensive degradation of proteins in alfalfa (Medicago sativa L.) may result in poor forage N utilization by ruminants. Using the inherent genetic variability and differences between harvests, our objective was to compare the effect of contrasting concentrations of nonstructural carbohydrates (NSC) in alfalfa on rumen fermentation and microbial protein synthesis. Individual genotypes of the alfalfa cultivar AC Caribou grown near Québec City, Québec, Canada, were harvested at the vegetative and early flowering stages, dried at 55 degrees C, ground, and analyzed for soluble carbohydrates (fructose + sucrose + glucose + pinitol) and starch. Approximately 20 genotypes having, respectively, the highest and lowest NSC concentrations were pooled to constitute 2 contrasted 1-kg forage samples. Samples of high- (17.9% DM) and low- (7.4% DM) NSC alfalfa were respectively allocated to separate dual-flow fermenters in a completely randomized design with 3 replications. Rumen inoculum was obtained from 4 ruminally fistulated cows in early lactation that were fed a TMR with a 50:50 forage to concentrate ratio. A 10-d incubation period was used, with the first 6 d serving as an adaptation period followed by 4 d of sampling with solid and liquid dilution rates in the fermenters set at approximately 2.0 and 4.3%/h, respectively. High versus low NSC concentration in alfalfa significantly enhanced the apparent digestibility of OM (59.1% for high-NSC alfalfa vs. 54.4% for low-NSC alfalfa) and DM (60.0 vs. 54.3%) and the true digestibility of DM (74.1 vs. 64.7%). Increasing NSC concentration in alfalfa (high vs. low) significantly decreased ruminal pH (6.85 vs. 7.08) and NH(3)-N concentration (26.0 vs. 33.6 mg/dL) and increased total VFA concentration (94.9 vs. 83.0mM). Molar proportions of acetate, isobutyrate, and isovalerate significantly decreased, whereas molar proportions of propionate and butyrate significantly increased with high-NSC alfalfa, resulting in a more glucogenic fermentation. More importantly, microbial N flow (263 vs. 230 mg/d) and bacterial N efficiency (41.1 vs. 29.6% of available N), measured using (15)N as a microbial marker, both significantly increased with the high-NSC alfalfa. These results indicate that increasing the concentration of NSC in alfalfa promotes a glucogenic fermentation and enhances microbial N synthesis in the rumen.

Comparative analysis of the cold acclimation and freezing tolerance capacities of seven diploid Brachypodium distachyon accessions

Background and Aims Cold is a major constraint for cereal cultivation under temperate climates. Winter-hardy plants interpret seasonal changes and can acquire the ability to resist sub-zero temperatures. This cold acclimation process is associated with physiological, biochemical and molecular alterations in cereals. Brachypodium distachyon is considered a powerful model system to study the response of temperate cereals to adverse environmental conditions. To date, little is known about the cold acclimation and freezing tolerance capacities of Brachypodium. The main objective of this study was to evaluate the cold hardiness of seven diploid Brachypodium accessions. Methods An integrated approach, involving monitoring of phenological indicators along with expression profiling of the major vernalization regulator VRN1 orthologue, was followed. In parallel, soluble sugars and proline contents were determined along with expression profiles of two COR genes in plants exposed to low temperatures. Finally, whole-plant freezing tests were performed to evaluate the freezing tolerance capacity of Brachypodium. Key Results Cold treatment accelerated the transition from the vegetative to the reproductive phase in all diploid Brachypodium accessions tested. In addition, low temperature exposure triggered the gradual accumulation of BradiVRN1 transcripts in all accessions tested. These accessions exhibited a clear cold acclimation response by progressively accumulating proline, sugars and COR gene transcripts. However, whole-plant freezing tests revealed that these seven diploid accessions only have a limited capacity to develop freezing tolerance when compared with winter varieties of temperate cereals such as wheat and barley. Furthermore, little difference in terms of survival was observed among the accessions tested despite their previous classification as either spring or winter genotypes. Conclusions This study is the first to characterize the freezing tolerance capacities of B. distachyon and provides strong evidence that some diploid accessions such as Bd21 have a facultative growth habit.

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.

Alleviation of Drought Stress and Metabolic Changes in Timothy (Phleum pratense L.) Colonized with Bacillus subtilis B26

Drought is a major limiting factor of crop productivity worldwide and its incidence is predicted to increase under climate change. Drought adaptation of cool-season grasses is thus a major challenge to secure the agricultural productivity under current and future climate conditions. Endophytes are non-pathogenic plant-associated bacteria that can play an important role in conferring resistance and improving plant tolerance to drought. In this study, the effect of inoculation of the bacterial endophyte Bacillus subtilis strain B26 on growth, water status, photosynthetic activity and metabolism of timothy (Phleum pratense L.) subjected to drought stress was investigated under controlled conditions. Under both drought-stress and non-stressed conditions, strain B26 successfully colonized the internal tissues of timothy and had a positive impact on plant growth. Exposure of inoculated plant to a 8-week drought-stress led to significant increase in shoot and root biomass by 26.6 and 63.8%, and in photosynthesis and stomatal conductance by 55.2 and 214.9% respectively, compared to non-inoculated plants grown under similar conditions. There was a significant effect of the endophyte on plant metabolism; higher levels of several sugars, notably sucrose and fructans and an increase of key amino acids such as, asparagine, glutamic acid and glutamine were recorded in shoots and roots of colonized plants compared to non-colonized ones. The accumulation of the non-protein amino acid GABA in shoots of stressed plants and in roots of stressed and unstressed plants was increased in the presence of the endophyte. Taken together, our results indicate that B. subtilis B26 improves timothy growth under drought stress through the modification of osmolyte accumulation in roots and shoots. These results will contribute to the development of a microbial agent to improve the yield of grass species including forage crops and cereals exposed to environmental stresses.

Accelerated Growth Rate and Increased Drought Stress Resilience of the Model Grass Brachypodium distachyon Colonized by Bacillus subtilis B26

Plant growth-promoting bacteria (PGB) induce positive effects in plants, for instance, increased growth and reduced abiotic stresses susceptibility. The mechanisms by which these bacteria impact the host plant are numerous, diverse and often specific. Here, we studied the agronomical, molecular and biochemical effects of the endophytic PGB Bacillus subtilis B26 on the full life cycle of Brachypodium distachyon Bd21, an established model species for functional genomics in cereal crops and temperate grasses. Inoculation of Brachypodium with B. subtilis strain B26 increased root and shoot weights, accelerated growth rate and seed yield as compared to control plants. B. subtilis strain B26 efficiently colonized the plant and was recovered from roots, stems and blades as well as seeds of Brachypodium, indicating that the bacterium is able to migrate, spread systemically inside the plant, establish itself in the aerial plant tissues and organs, and is vertically transmitted to seeds. The presence of B. subtilis strain B26 in the seed led to systemic colonization of the next generation of Brachypodium plants. Inoculated Brachypodium seedlings and mature plants exposed to acute and chronic drought stress minimized the phenotypic effect of drought compared to plants not harbouring the bacterium. Protection from the inhibitory effects of drought by the bacterium was linked to upregulation of the drought-response genes, DREB2B-like, DHN3-like and LEA-14-A-like and modulation of the DNA methylation genes, MET1B-like, CMT3-like and DRM2-like, that regulate the process. Additionally, total soluble sugars and starch contents increased in stressed inoculated plants, a biochemical indication of drought tolerance. In conclusion, we show a single inoculation of Brachypodium with a PGB affected the whole growth cycle of the plant, accelerating its growth rates, shortening its vegetative period, and alleviating drought stress effects. These effects are relevant to grasses and cereal crops.