Impact of Agro-industrial Byproducts on Bioconversion, Chemical Composition, in vitro Digestibility, and Microbiota of the Black Soldier Fly (Diptera: Stratiomyidae) Larvae.

The interest in using byproducts from agro-food industries as a rearing substrate for insects is increasing rapidly. We investigated the influence of byproducts of vegetal origin (okara-a byproduct of soy milk production, maize distillers with solubles, brewer's grains), used as rearing diet for black soldier fly larvae (BSFL), on the following parameters: biomass production, substrate reduction (SR), nutritional profile and in vitro digestibility, and larval gut microbiota. Hen diet was used as a control substrate. The highest larval biomass was collected on maize distillers, whereas the highest SR was observed on okara. The rearing substrate affected ash, ether extract, and chitin larval content. The BSFL reared on okara were characterized by a lower lauric acid content (17.6% of total fatty acids). Diets also influenced in vitro crude protein digestibility (%) for monogastrics, with the highest values for BSFL reared on maize distillers (87.8), intermediate for brewer's grains and okara BSFL, and the lowest for hen BSFL (82.7). The nutritive value for ruminants showed a lower Net Energy for lactation for BSFL reared on hen diet than okara and dried maize distillers BSFL. The different byproducts showed an influence on the larval gut microbiota, with a major bacterial complexity observed on larvae fed with the hen diet. The neutral detergent fiber concentration of dietary substrate was negatively correlated with Firmicutes and Actinobacteria relative abundance. Insects valorized byproducts converting them into high-value larval biomass to be used for feed production. The results evidenced the effects of the tested byproducts on the measured parameters, underling the chemical composition importance on the final insect meal quality.

Efficient milking hygiene reduces bacterial spore contamination in milk.

Clostridia in the milk can lead to late blowing, a cheese defect. Clostridia are ubiquitous, deriving from both the farm environment and the feed ingested by the cows, and are transferred into the milk through faecal contamination. Our aim was to investigate the effect of different in-parlour practices on the content of anaerobic spore-forming bacteria in milk, and to monitor the variation in spore content in the feed and environment. The experiment, conducted in an experimental dairy during autumn, was repeated in exactly the same way for two consecutive years. The experimental design applied three different milking routines in three consecutive 7-d periods: forestripping alone (F); forestripping and post-dipping (F+Post); pre-dipping, wiping, forestripping and post-dipping (Pre+F+Post). Teat skin swabs and samples of feed, faeces, bedding materials and milk were collected for microbiological analyses. The dietary forage of the lactating cows included maize silage, which, in both years, was found to have the highest level of clostridial spore contamination. Pre-dipping with a detergent/emollient solution, and drying with a disposable paper towel, proved much more efficient in reducing spore contamination than forestripping alone, both on the teats (1·30 vs. 2·20 log10 MPN/swab; P < 0·001) and in the milk (1·82 vs. 2·47 log10 MPN/L, P < 0·02), while post-dipping had little influence on spore count. The standard plate count in milk was significantly lower with Pre+F+Post treatment than with F (3·80 vs. 4·51 log10 CFU/mL, P < 0·01). The teat preparation procedure did not influence the lactic acid bacterial levels in the milk, which is very positive in that decreased lactic acid bacterial content can lessen raw milk cheese quality.

Chemical and ruminal in vitro evaluation of Canadian canola meals produced over 4 years.

To test the effects of year and processing plant on the nutritional value of canola meal (CM), 3 CM samples/yr were collected from each of 12 Canadian production plants over 4yr (total=144). Samples of CM were analyzed for differences in chemical composition and for in vitro ruminal protein degradability using the Michaelis-Menten inhibitor in vitro (MMIIV) method. In the MMIIV method, protein degradation rate (kd) was estimated by 2 methods: from net release (i.e., blank corrected) of (1) ammonia plus AA determined by o-phthaldialdehyde fluorescence (OPAF) assay or (2) ammonia, AA, plus oligopeptides determined by o-phthaldialdehyde absorbance (OPAA) assay; rumen-undegradable protein (RUP) was computed assuming passage rates of 0.16 and 0.06/h for, respectively, soluble and insoluble protein. Casein, solvent soybean meal (SSBM), and expeller soybean meal (ESBM) were included in all incubations as standard proteins. Differences among years and plants were assessed using the mixed procedures of SAS. Small but significant differences were found in CM among years for chemical composition, including N solubility; some of these differences may have been related to changes in our analytical methods over time. However, adjustment of degradation activity of individual in vitro incubations based on the mean degradation activity over all incubations yielded kd and RUP that did not differ by year using either assay. Simultaneously incubating CM samples from 2yr in the same in vitro runs confirmed that no year effects existed for kd or RUP. Differences existed in chemical composition of CM among the 12 processing plants over the 4yr of sample collection. Moreover, consistent differences in kd and RUP were observed among plants: kd ranged from 0.069 to 0.113/h (OPAA assay) and 0.075 to 0.120/h (OPAF assay), and RUP estimates ranged from 51 to 43% (OPAA assay) and 49 to 41% (OPAF assay). Regression of kd on insoluble N content of CM yielded correlation coefficients (R(2))=0.40 (OPAA assay) and 0.42 (OPAF assay), and regressions of kd on NDIN and N-fraction B3 yielded R(2)<0.02. Mean estimates from both OPAA and OPAF assays for casein, SSBM, ESBM, and CM were, respectively, kd=0.764, 0.161, 0.050, and 0.093/h and RUP=18, 33, 56, and 45%. A range of 8 percentage units from lowest to highest RUP suggests that substantial differences exist in metabolizable protein content of CM produced by different processing plants.

Management practices and forage quality affecting the contamination of milk with anaerobic spore-forming bacteria.

BACKGROUND: Anaerobic spore-forming bacteria (ASFB) in milk derive from the farm environment, and the use of silages and management practices are the main responsible of milk ASFB contamination. The aim of this study was to evaluate the relationships between feeding, milking routine and cow hygiene and milk and Grana Padano cheese (produced with and without lysozyme) ASFB contamination. RESULTS: The study involved 23 dairy farms. ASFB in corn silage were on average 2.34 ± 0.87 log10 MPN g(-1). For grass, Italian ryegrass and alfalfa, ASFB (log10 MPN g(-1)) were numerically higher for silages (3.22) than hays (2.85). The use of corn silages of high quality (high lactic and acetic acids concentrations) decreased the milk ASFB contamination, whilst the use of herbage silages did not affect it. The presence (>40%) of cows with dirty udders increased the ASFB contamination of milk, while forestripping had a positive effect (-9% ASFB). Ripened Grana Padano had an ASFB count below the analytical limit; Clostridium tyrobutyricum DNA was found only in wheels produced without lysozyme, which also showed late blowing. CONCLUSION: The factors increasing milk spore contamination were corn silage quality, cow udder hygiene and inadequate milking routine. Late blowing was present only in cheeses without lysozyme.

Evaluation of Camelina sativa (L.) Crantz meal as an alternative protein source in ruminant rations.

BACKGROUND: Camelina sativa (CS) is an oilseed crop used for biofuel production. By-products from oil extraction are high in protein and can be used in ruminant rations; more information about their nutritive value is required also considering the antinutrional factor content of the by-products. The aim of this study was to evaluate the nutritive value of CS meal genotypes in comparison with canola. RESULTS: Ten CS genotypes and one canola cultivar were evaluated. Meals were obtained from seeds after solvent oil extraction. CS average crude protein (CP) content (g kg⁻¹ dry matter) was 457. Numerical differences in lysine and sulfur amino acid content were observed among CS genotypes. Glucosinolate (mmol kg⁻¹) content was higher for CS (23.1) than canola (7.2). Sinapine content (g kg⁻¹) was lower for CS (2.79) than for canola (4.32). Differences were observed among CS genotypes for rumen undegraded protein (RUP). Average RUP (g kg⁻¹ CP) was 316 for CS and 275 for canola. CONCLUSIONS: CS meal has potential for use in ruminant rations as a high-quality protein source. In vivo studies are needed to compare CS with other protein sources used in cattle rations. Implementation of breeding programs for improved meal quality is recommend.

An In Vitro Study on the Role of Cellulases and Xylanases of Bacillus subtilis in Dairy Cattle Nutrition.

The administration of Bacilli to dairy cows exerts beneficial effects on dry matter intake, lactation performance, and milk composition, but the rationale behind their efficacy is still poorly understood. In this work, we sought to establish whether cellulases and xylanases, among the enzymes secreted by B. subtilis, are involved in the positive effect exerted by Bacilli on ruminal performance. We took advantage of two isogenic B. subtilis strains, only differing in the secretion levels of those two enzymes. A multi-factorial study was conducted in which eight feed ingredients were treated in vitro, using ruminal fluid from cannulated cows, with cultures of the two strains conveniently grown in a growth medium based on inexpensive waste. Feed degradability and gas production were assessed. Fiber degradability was 10% higher (p < 0.001) in feeds treated with the enzyme-overexpressing strain than in the untreated control, while the non-overexpressing strain provided a 5% increase. The benefit of the fibrolytic enzymes was maximal for maize silage, the most recalcitrant feed. Gas production also correlated with the amount of enzymes applied (p < 0.05). Our results revealed that B. subtilis cellulases and xylanases effectively contribute to improving forage quality, justifying the use of Bacilli as direct-fed microbials to increase animal productivity.

In vitro effects of different levels of quebracho and chestnut tannins on rumen methane production, fermentation parameters, and microbiota.

Both condensed and hydrolysable tannins (CTs and HTs, respectively) have the ability to reduce enteric CH4 production in ruminants. However, the precise mechanism of action is not fully understood. Among the proposed hypotheses are the reduction of ruminal digestibility, direct control action on protozoa, reduction of archaea, and a hydrogen sink mechanism. In this in vitro study, which simulated rumen fermentation, two additives, one containing CTs (70% based on DM) from quebracho and one with HTs (75% based on DM) from chestnut, at four levels of inclusion (2, 4, 6, 8% on an as-fed basis) were added to the fermentation substrate and tested against a negative control. Both types of tannins significantly reduced total gas (GP) and CH4 (ml/g DM) production during the 48 h of incubation. The lower GP and CH4 production levels were linked to the reduction in dry matter digestibility caused by CTs and HTs. Conversely, no significant differences were observed for the protozoan and archaeal populations, suggesting a low direct effect of tannins on these rumen microorganisms in vitro. However, both types of tannins had negative correlations for the families Bacteroidales_BS11 and F082 and positive correlations for the genera Prevotella and Succinivibrio. Regarding the fermentation parameters, no differences were observed for pH and total volatile fatty acid production, while both CTs and HTs linearly reduced the NH3 content. CTs from quebracho were more effective in reducing CH4 production than HTs from chestnut. However, for both types of tannins, the reduction in CH4 production was always associated with a lower digestibility without any changes in archaea or protozoa. Due to the high variability of tannins, further studies investigating the chemical structure of the compounds and their mechanisms of action are needed to understand the different results reported in the literature.

Evaluation of dietary addition of 2 essential oils from Achillea moschata, or their components (bornyl acetate, camphor, and eucalyptol) on in vitro ruminal fermentation and microbial community composition.

This study investigated the effects of 2 Achillea moschata essential oils extracted from plants collected in 2 different valleys of the Italian Alps and 3 pure compounds of oils - bornyl acetate (BOR), camphor (CAM), and eucalyptol (EUCA) - on in vitro ruminal fermentation and microbiota. An in vitro batch fermentation experiment (Exp. 1) tested the addition of all of the substances (2 essential oils and 3 compounds) in fermentation bottles (120 mL) at 48 h of incubation, whereas a subsequent in vitro continuous culture experiment (Exp. 2) evaluated the pure compounds added to the fermenters (2 L) for a longer incubation period (9 d). In both experiments, total mixed rations were incubated with the additives, and samples without additives were included as the control (CTR). Each treatment was tested in duplicate and was repeated in 3 and 2 fermentation runs in Exp. 1 and 2, respectively. Gas production (GP) in Exp. 1 was similar for all of the treatments, and short chain volatile fatty acid (SCFA) production was similar in both experiments except for a decrease of SCFA produced (P = 0.029) due to EUCA addition in Exp. 2. Compared to CTR, BOR and CAM reduced the valerate proportion (P = 0.04) in Exp. 1, and increased (P < 0.01) the acetate proportion in Exp. 2. All treatments increased (P < 0.01) total protozoa counts (+36.7% and +48.4% compared to CTR on average for Exp. 1 and 2, respectively). In Exp. 1, all of the treatments lowered the Bacteroidetes and Firmicutes and increased the Proteobacteria relative abundances (P < 0.05), whereas in Exp. 2, the EUCA addition increased (P = 0.012) the Ruminococcus. In Exp. 1, methane (CH4) as a proportion of the GP was lowered (P = 0.004) by the addition of CAM and EUCA compared to CTR, whereas in Exp. 2, EUCA reduced the amount of stoichiometrically calculated CH4 compared to CTR. Overall, essential oils extracted from A. moschata and the pure compounds did not depress in vitro rumen fermentation, except for EUCA in Exp. 2. In both experiments, an increase of the protozoal population occurred for all the additives.

Effect of Linseeds and Hemp Seeds on Milk Production, Energy and Nitrogen Balance, and Methane Emissions in the Dairy Goat.

The effect of whole linseeds or hemp seeds on milk production, energy and nitrogen balance, and methane emission was studied in 12 Alpine goats using respiration chambers. Diets tested were a control diet (C) and two diets supplemented with whole linseeds (L) or hemp seeds (H) at 9.3% on a dry matter (DM) basis. DM intake was similar among treatments, whereas DM and organic matter digestibility were lower for L compared to C. Milk yield (2.30 kg/d on average) and rumen fermentation profile were not affected by treatments. Treatment also did not affect the milk composition, with the exception of fat, which was higher in H and L compared to C (4.21, 3.94, and 3.20%, respectively). Oilseed supplementation caused a reduction in the concentration of de novo fatty acids (FA) (41.1, 48.8, and 64.1% of FA, for L, H, and C, respectively). Moreover, L and H diets reduced the sum of saturated FA, and increased monounsaturated FA, whereas only the L diet increased the concentration of polyunsaturated FA. Regarding methane production, and nitrogen and energy balances, no differences were registered among the diets. Our research indicates that including whole linseeds and hemp seeds in the dairy goat diet is an effective strategy for increasing milk fat content and positively modifying the milk FA composition, without a change in nitrogen and energy balances, but also without a reduction in enteric methane emission.

Use of Undigested NDF for Estimation of Diet Digestibility in Growing Pigs.

Undigested neutral detergent fibre (uNDF) is commonly used as an internal marker for the estimation of diet digestibility in ruminants. This work aimed to verify (i) whether the in vivo method with uNDF could be used to evaluate diet digestibility in growing pigs, and (ii) whether pre-treating the samples with neutral detergent solution (NDS) and α-amylase improves the accuracy of the estimates. Samples from a previously published work of two diets with known in vivo digestibility values estimated by the total faecal collection method and 16 individual samples of faeces were used. For each sample, four Ankom F57 bags were weighed. Before the incubation, two F57 bags were pre-treated with NDS and α-amylase. All the samples were incubated for 240 h in the Ankom DaisyII incubator and then analysed for their uNDF contents. Dry matter, organic matter, and neutral detergent fibre digestibilities were estimated using the uNDF contents, and the results were compared with those of the former study. The digestibility values obtained using the uNDF method with pre-treatment were not statistically different from those determined with the total faecal collection. On the contrary, the uNDF method without the pre-treatment could not satisfactorily predict the digestibilities of pig diets.

The Effects of Substituting Dietary Soybean Meal with Maize Grain on Milk Production in Dairy Goats.

In view of better environmental sustainability, livestock diets must not exceed protein requirements, as often happens with lactating goats reared in semi-intensive systems. The aim of this experiment was to verify in real-breeding conditions the influence of two diets with different protein contents (% crude protein (CP) on dry matter (DM)): 16.0 (high-protein diet; HP) vs 12.2 (low-protein diet; LP) on milk production in dairy goats. The diets differed only in the replacement-in the LP diet-of 250 g soybean meal with 250 g maize grain meal. Twenty-three Alpine goats were divided into two groups and used in a cross-over feeding trial for 2 months. Animals were weighed at the beginning of each month of the trial, and feed intake and milk yield and composition were recorded weekly. HP and LP did not differ statistically for milk yield and composition (3.32 vs 3.42 kg milk/d, 3.21% vs 3.27% fat, 3.31% vs 3.27% protein for HP and LP, respectively), but the HP diet determined a higher milk urea content (51.2 vs 36.6 mg/dL, p < 0.001) and a worse efficiency of nitrogen utilization (28.0% vs 37.2%). In conclusion, the LP diet resulted in a reduction of urinary nitrogen excretion by 28% and of the feed cost by about 10%.

Comparison of Faecal versus Rumen Inocula for the Estimation of NDF Digestibility.

Cow faeces have been investigated as alternative inoculum to replace rumen fluid to determine neutral detergent fibre (NDF) digestibility (NDFD). Aims of this study were to estimate: (1) the NDFD (48 h) of feed ingredients using a rumen inoculum in comparison with faecal inocula from cows fed diets with different forage basis; (2) the undigestible NDF (uNDF) at 240 and 360 h with ruminal fluid and faecal inocula from lactating cows fed two different diets. At 48 h incubation, the NDFD was affected both by feed and type of inoculum (p < 0.01) and by their interaction (p = 0.03). Overall, the mean NDFD was higher for rumen inoculum than for faecal inocula (585 vs. 389 g/kg NDF, p < 0.05), and faecal inoculum obtained from cows fed hay-based diets gave lower NDFD than those from cows fed maize silage (367 vs. 440 g/kg, p < 0.05). At long incubation times, the average uNDF was affected by substrate, inoculum and incubation time (p < 0.01), but not by their interactions. For each inoculum, significantly lower values were obtained at 360 than at 240 h. Regressions between uNDF with rumen and with the tested faecal inocula resulted in r2 ≥ 0.98. Despite the differences at 48 h, the uNDF showed that faecal inoculum could replace rumen fluid at longer incubation times.

Rearing of Hermetia Illucens on Different Organic By-Products: Influence on Growth, Waste Reduction, and Environmental Impact.

The aim of the study was to evaluate the use of three by-products as growing substrates for Hermetia illucens (Black Soldier Fly (BSF)) larvae: okara, maize distiller, brewer's grains, and a control hen diet. The study focused on larval growth and bioconversion performance, production of methane by larvae and environmental burden of larvae production, using Life Cycle Assessment (LCA) on a lab scale. Chemical composition of substrates differed: okara had the highest crude protein and ether extract contents, while brewer's grains showed the highest fiber content. Larvae fed on a hen diet and maize distiller exhibited the highest final weights (2.29 and 1.97 g, respectively). Larvae grown on okara showed the highest indexes for waste reduction and efficiency of conversion of the ingested feed. The BSF larvae did not produce any detectable traces of CH4. LCA evaluation showed that larvae production on a hen diet resulted in the most impact for most of environmental categories, for the inclusion of soybean meal in the diet (for climate change, 5.79 kg CO2 eq/kg dry larvae). Feed production activities resulted in the main contributions to environmental impact. In order to compare the larvae production obtained on all substrates, an environmental impact was attributed to okara and brewer's grain through a substitution method, and, by this approach, the best sustainable product resulted from the larvae grown on the maize distiller.

Rumen Inoculum Collected from Cows at Slaughter or from a Continuous Fermenter and Preserved in Warm, Refrigerated, Chilled or Freeze-Dried Environments for In Vitro Tests.

The utilization of animal donors of rumen fluid for laboratory experiments can raise ethical concerns, and alternatives to the collection of rumen fluids from live animals are urgently requested. The aim of this study was to compare the fresh rumen fluid (collected at slaughter, W) with that obtained from a continuous fermenter (RCF) and three methods of rumen fluid preservation (refrigeration, R, chilling, C, and freeze-drying, FD). The fermentability of different inoculum was evaluated by three in vitro tests (neutral detergent fiber (NDF) and crude protein (CP) degradability and gas production, NDFd, RDP and GP, respectively) using six feeds as substrates. Despite the two types of inoculum differed in terms of metabolites and microbiota concentration, the differences in vitro fermentability between the two liquids were less pronounced than expected (-15 and 20% for NDFd and GP when the liquid of fermenter was used and no differences for RDP). Within each in vitro test, the data obtained from rumen and from fermenter liquids were highly correlated for the six feeds, as well as between W and R (r: 0.837-0.985; p < 0.01). The low fermentative capacity was found for C and, particularly, FD for liquids. RCF could be used to generate inoculum for in vitro purposes and short-term refrigeration is a valuable practice to manage inoculum.

Effects of a selection of additives on in vitro ruminal methanogenesis and in situ and in vivo†NDF digestibility.

The effects of 18 essential oils, yeast, Quebracho tannin and Quillaja saponin on ruminal methane and gas production (GP) were studied in vitro. A lactating cow diet was incubated with rumen inoculum. Doses of the additives (mg/L) were: essential oils = 500; tannin = 67; yeast = 8.35 and 16.7; and saponin = 300. Lemongrass, estragole, eugenol, geraniol, limonen, thyme oil and thymol produced less gas (overall mean 33.8 mL/200 mg dry matter (DM)) than control (43.6 mL/200 mg DM; P < 0.001). Methane produced (mL/200 mg DM) by guaiacol (10.7), lemongrass (9.6), limonene (11.4), thyme oil (10.9) and thymol (2.1) was lower than control (12.5) (P < 0.001). Methane percentage on total GP was lower (P < 0.001) for cinnamic alcohol (25.4), guaiacol (24.5), thymol (19.7) and vanillin (26.3) than control (28.8). In a second experiment, thymol, guaiacol and yeast were added to the diet of dry fistulated cows to determine in situ neutral detergent fiber digestibility (NDFD) of six forages and in vivo dietary NDFD. Thymol and yeast decreased in situ NDFD after 24 h (33.9% and 33.5% vs. 38.1%; P = 0.008). Thymol decreased in vivo total tract NDFD (40.8% vs. 51.4%; P = 0.02). Differences in GP and methane levels were registered within classes of additives. A careful selection of additives may allow for the manipulation of ruminal fermentation.