Control of protein degradation in the rumen for improving protein efficiency and reducing polluting emissions

Resumen

Ruminants have the capacity of using fibrous feeds and low quality proteins to produce high-quality foods for humans, but they also have a low efficiency of nitrogen (N) utilization that is usually below 30%. As a consequence, a large part of the dietary N is excreted to the environment and contributes to soil and water eutrophication, being this problem especially marked in high-producing ruminants. Rumen metabolism is the most important factor contributing to the inefficient use of N in ruminants, especially in situations of an imbalance between the NH3-N generated in the degradation of protein and the use of NH3-N by the microbiota for microbial protein synthesis. In addition, it has been shown that protein degradation in the rumen generates CH4, thus contributing to greenhouse gases emissions. Therefore, reducing the ruminal degradation of protein cannot only increase the amount of feed protein reaching the small intestine, but also can reduce polluting emissions such as N and CH4. The general objective of this Doctoral Thesis is to investigate some aspects of the protein degradation in the rumen that are directly related to N losses and CH4 production, and in order to achieve this objective four Experiments (two in vitro and two in vivo) were conducted. The objective of Experiment 1 was to evaluate the inclusion of sunflower seed (SS) and sunflower meal (SM) protected against ruminal degradation in high-cereal diets on in vitro ruminal fermentation and CH4 production. Samples of SS and SM were sprayed with a solution of malic acid 1 M (400 ml/kg sample) and dried at 150°C for 1 h as a protective treatment. Four diets were formulated to contain either 13 (low) or 17 (high) g of crude protein (CP)/100 g dry matter (DM), and included SS and SM either untreated (13CON and 17CON diets) or treated as before described (13TR and 17TR diets). Diets were incubated in vitro with rumen fluid from sheep for 8 and 24 h. The treatment did not affect (p ≥ 0.57) total volatile fatty acid (VFA) production at any incubation time, but it reduced (p < 0.05) NH3-N concentrations by 19.2 and 12.5% at 8 and 24 h respectively. Both CH4 production and CH4/VFA ratio were lower (p < 0.02) in TR than in CON diets at 8 h, but differences disappeared (p > 0.05) at 24 h. The treatment increased the molar proportion of propionate (p = 0.001) and reduced that of isovalerate (p = 0.03) at 8 h compared with CON diets, but only a reduction of isovalerate proportion (p = 0.03) was detected at 24 h. There were no treatment x crude protein level interactions (p > 0.05) in any parameter, but high-protein diets had greater NH3-N concentrations (p < 0.001) and lower VFA production (p < 0.001) than low-protein diets at 24 h. The treatment reduced protein degradation, and CH4 production was decreased by 4.6 and 10.8% for low-and high-protein diets, respectively, at short incubation times without affecting VFA production, thus improving fermentation efficiency and decreasing polluting emissions. The objectives of Experiments 2 and 3 were to analyze the efficacy of a treatment combining malic acid and heating (MAH) to protect the protein in SS and SM against rumen degradation and to improve the growth, carcass characteristics and meat quality of lambs. Two high-cereal concentrates, either including untreated SS and SM (control concentrate) or MAH-treated SS and SM (MAH concentrate), were formulated. The Experiment 2 was conducted to analyze the 12-h in vitro fermentation of untreated and MAH-treated SS and SM samples, as well as of both concentrates, using ruminal fluid from sheep as inoculum. The results indicated that the MAH treatment modified the rumen fermentation pattern of SS, SM and the concentrate, increasing molar proportions of propionate. In addition, MAH treatment reduced (p = 0.009) the NH3-N concentrations for SM and tended (p = 0.065) to reduce them for the concentrate. However, no effects (p ≥ 0.100) on CH4 production were observed for any incubated feed. In Experiment 3, two homogeneous groups of 12 Lacaune lambs each (14.2  0.35 Kg body weight) were fed either the control or the MAH concentrate. Lambs were fed concentrate and barley straw ad libitum for 40 days (until reaching about 26 kg body weight). Feed intake and growth of lambs were recorded, blood samples were taken on days 0, 20, and the slaughter day for analysis of urea-N and amino acid-N, diet digestibility was determined, and ruminal and cecal samples were collected after slaughter. In addition, carcass characteristics and quality and fatty acid profile of meat were assessed. There were no significant effects of the MAH treatment on feed intake and growth of lambs. Organic matter digestibility tended to be greater (p = 0.07) in MAH-fed lambs than in the control group, but there were no differences (p ≥ 0.33) in the digestibility of CP, neutral detergent fiber (NDF) and acid detergent fiber (ADF). The hot carcasses of the lambs fed the MAH-treated concentrate were 7.9% heavier than those of the control group, but differences did not reach the significance level (p = 0.212). No differences between groups were detected in plasma concentrations of urea-N and amino acid-N (p = 0.755 and 0.500, respectively). There were no effects (p ≥ 0.172) of MAH treatment on ruminal papillae characteristics and post-mortem VFA concentrations and profile in the rumen and the cecum. The color of the ruminal epithelium was darker (p = 0.003) in the lambs fed the MAH-concentrate compared with the control group, which was attributed to either to a corrosive action of malic acid or to a greater abrasion of the MAH-treated sunflower husks. Compared with control lambs, those fed the MAH-concentrate showed greater (p = 0.016) amount of dorsal fat and greater (p ≤ 0.016) values of the color parameters a* (redness) and C* (chromaticity) in the rectus abdominis muscle. However, there were no differences (p > 0.05) in carcass measurements and in water-holding capacity, chemical composition, pH or color of longissimus dorsi muscle. Fatty acid profile of longissimus dorsi muscle was not affected (p > 0.117) by feeding the MAH-treated concentrate, with the exception of a trend (p = 0.055) to greater concentrations of C14:0 in the MAH-fed lambs. In conclusion, the MAH treatment increased the in vitro fermentation of SS, reduced the in vitro protein degradability of SM, and modified the VFA profile towards greater propionate production. However, under the conditions of the present study the inclusion of MAH-treated SS and SM in a concentrate for growing lambs did not influenced significantly feed intake, diet digestibility, growth performance and meat fatty acid profile, although it increased hot carcass weight by 7.9% and the amount of dorsal fat. In Experiment 4, the effects of spraying a solution of malic acid (1 M; 400 ml/kg) and ulterior heating to protect against ruminal degradation the proteins of SS and SM, as well as of a mixture of these both feeds (SSM; 45:55) were studied using in vivo, in situ and in vitro methods. Four rumen-fistulated sheep were fed two mixed diets composed of oat hay and concentrate (40:60) and differing only in the concentrate, that was either the control or the MAH concentrate used in Experiments 2 and 3. Sheep were fed the diets at 40 g DM/kg body weight0.75 into six equal meals per day using automatic dispensers. A crossover design with two 24-day experimental periods was used, and each period included successively 10 days of diet adaptation, 9 days for performing in situ incubations of SS, SM and SSM, one day for measuring ruminal parameters, and two days for rumen empting. From day 6 onwards a solution of (15NH4)2SO4 was continuously infused into the rumen of each sheep to label ruminal bacteria. Feeding the MAH diet did not affect either ruminal pH or concentrations of NH3-N and total VFA, but decreased (p ≤ 0.009) molar proportions of acetate and propionate and increased those of butyrate (p < 0.001). In addition, organic matter (OM) and lipid contents in ruminal bacteria were lower in sheep fed the MAH diet compared with the control diet, whereas both N content and 15N enrichment were greater (p ≤ 0.037). Estimates of effective degradability (ED) of different feed fractions in SS, SM and SSM were obtained considering the ruminal rates of particle comminution and passage and correcting the values for the microbial contamination measured by using the 15N infusion technique. The MAH treatment decreased the ED of most fractions in all tested feeds, increasing the supply of by-pass crude protein (CP) by 19.1 and 120% for SS and SM, respectively, and by 34% for crude fat in SS. The MAH treatment also increased the in vitro intestinal digestibility of the by-pass CP both for SS (from 60.1 to 75.4%) and SM (from 83.2 to 91.0%). The simultaneous heating of both feeds performed in SSM reinforced the protective effect increasing the by-pass CP without altering its intestinal digestibility. As a result, the intestinal digested CP content in SSM increased by 15.6% compared with the value estimated from the results obtained for SS and SM incubated independently. The results also confirm the previous observation that the effectiveness of MAH treatment is greater for high-CP feeds.