Outbreak of aflatoxicosis in a dairy herd induced depletion in milk yield and high abortion rate in Pakistan.

This case report investigated the outbreak of aflatoxicosis in a dairy herd in Pakistan, which resulted in 30 abortions of 40 confirmed (75%) pregnant cows in a period of 35 days and in 18.8% depression of farm average milk production for the entire herd. The analysis of the concentrate feed of the total mixed ration (TMR), using enzyme-linked immunosorbent assay (ELISA) procedures from two different local laboratories, indicated concentrations of 60 µg/kg dry matter (DM) of aflatoxin B1 (AFB1) and 100 µg/kg DM of total aflatoxins (AFs: sum of B1, B2, G1 and G2). Subsequently, a confirmatory analysis with a more sensitive and validated multi-metabolite liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was performed. This analysis detected a concentration of total AFs in the TMR of 166 µg/kg DM ± 3.5 (AFB1:134, AFB2:17.4 and AFM1:14.9 µg/kg DM). The concentrate feed (55% of the TMR DM) was confirmed as a source of contamination, presenting a concentration >29 times higher than the EU-maximum limit value (5.68 µg/kg DM). Additionally, the multi-mycotoxin analysis evidenced the co-occurrence of 81 other toxic and potentially toxic fungal metabolites in the fed TMR. After replacing the contaminated concentrate feed with feedstuffs of the same formulation but from a new charge of ingredients, the abortion episodes ceased, and milk production increased significantly. In conclusion, the data of this case report suggest that AFs may be associated with pregnancy losses in dairy cattle and milk production depression. From the public health perspective, the data also indicate the need for a more careful examination of dairy animal feed in Pakistan. Since the high concentration of AFB1 detected in feed and considering the literature-reported transfer rates (1-6%) of this toxin to AFM1 (carcinogen for humans) in milk, the milk produced during the outbreak period is expected to be contaminated with AFM1, which raises public health concerns.

Co-occurrence of mycotoxins and other fungal metabolites in total mixed rations of cows from dairy farms in Punjab, Pakistan.

After India and the USA, Pakistan is the third country leading in global dairy production, a sector of very high socioeconomic relevance in Asia. Mycotoxins can affect animal health, reproduction and productivity. This study analysed a broad range of co-occurring mycotoxins and fungal secondary metabolites derived from Alternaria, Aspergillus, Fusarium, Penicillium and other fungal species. To complete this, a validated multi-metabolite liquid chromatography/electrospray ionization-tandem mass spectrometric (LC/ESI-MS/MS) method was employed, detecting 96 of > 500 tested secondary fungal metabolites. This first preliminary study demonstrated that total mixed rations (TMRs) (n = 30) from big commercial dairy cattle farms (> 200 lactating cows) in Punjab, Pakistan, presented ubiquitous contamination with mixtures of mycotoxins. The mean of mycotoxins per sample was 14, ranging from 11 to 20 mycotoxins among all TMR samples. Metabolites derived from other fungi and Fusarium spp. showed the highest levels, frequency and diversity among the detected fungal compounds. Among the most prevalent mycotoxins were Fusarium toxins like fumonisins B1 (FB1) (93%), B2 (FB2) (100%) and B3 (FB3) (77%) and others. Aflatoxin B1 (AFB1) was evidenced in 40% of the samples, and 7% exceeded the EU maximum limit for feeding dairy cattle (5 µg/kg at 88% dry matter). No other mycotoxin exceeds the EU guidance values (GVs). Additionally, we found that dietary ingredients like corn grain, soybean meal and canola meal were related to increased contamination of some mycotoxins (like FB1, FB2 and FB3) in TMR from the province of Punjab, Pakistan. Among typical forage sources, the content of maize silage was ubiquitous. Individually, the detected mycotoxins represented relatively low levels. However, under a realistic scenario, long-term exposure to multiple mycotoxins and other fungal secondary metabolites can exert unpredictable effects on animal health, reproduction and productivity. Except for ergot alkaloids (73%), all the groups of metabolites (i.e. derived from Alternaria spp., Aspergillus spp., Fusarium spp., Penicillium spp. and other fungi) occurred in 100% of the TMR samples. At individual levels, no other mycotoxins than AFB1 represented a considerable risk; however, the high levels of co-occurrence with several mycotoxins/metabolites suggest that long-term exposure should be considered because of their potential toxicological interactions (additive or synergistic effects).

Winery by-products as a feed source with functional properties: dose-response effect of grape pomace, grape seed meal, and grape seed extract on rumen microbial community and their fermentation activity in RUSITEC.

BACKGROUND: Grape and winery by-products have nutritional values for cattle and also contain functional compounds like phenols, which not only bind to protein but can also directly affect microbiota and their function in the rumen. We characterized the nutritional and functional effects of grape seed meal and grape pomace as well as an effective dosage of grape phenols on ruminal microbiota and fermentation characteristics using a rumen simulation technique. RESULTS: Six diets (each n = 8) were compared including a control diet (CON, no by-product), a positive control diet (EXT, CON + 3.7% grape seed extract on a dry matter (DM) basis), two diets with grape seed meal at 5% (GS-low) and 10% (GS-high), and two diets with grape pomace: at 10% (GP-low) and 20% (GP-high), on a DM basis. The inclusion of the by-product supplied total phenols at 3.4%, 0.7%, 1.4%, 1.3%, and 2.7% of diet DM for EXT, GS-low, GS-high, GP-low, and GP-high, respectively. Diets were tested in four experimental runs. All treatments decreased ammonia concentrations and the disappearances of DM and OM (P < 0.05) compared to CON. EXT and GP-high lowered butyrate and odd- and branch-chain short-chain fatty acids while increased acetate compared to CON (P < 0.05). Treatments did not affect methane formation. EXT decreased the abundance of many bacterial genera including those belonging to the core microbiota. GP-high and EXT consistently decreased Olsenella and Anaerotipes while increased Ruminobacter abundances. CONCLUSION: The data suggest that the inclusion of winery by-products or grape seed extract could be an option for reducing excessive ammonia production. Exposure to grape phenols at a high dosage in an extract form can alter the rumen microbial community. This, however, does not necessarily alter the effect of grape phenols on the microbial community function compared to feeding high levels of winery by-products. This suggests the dominant role of dosage over the form or source of the grape phenols in affecting ruminal microbial activity. In conclusion, supplementing grape phenols at about 3% of diet DM is an effective dosage tolerable to ruminal microbiota.

Moringa oleifera and Propolis in Cattle Nutrition: Characterization of Metabolic Activities in the Rumen In Vitro.

Moringa oleifera by-products such as seed cake and leaves are protein-rich ingredients, while raw propolis has the potential to influence ruminal protein metabolism. These substances are also known to be sources of functional compounds. With these properties, they could modulate ruminal fermentation activities. Using the rumen simulation technique, we investigated ruminal fermentation and the antioxidant properties of four dietary treatments. These included a control diet (CON) without supplementation; the CON diet top-dressed on a dry matter (DM) basis, either with moringa seed cake (MSC, containing 49% crude protein (CP)), moringa leaf powder (ML, containing 28% CP), or raw propolis (PRO, 3% CP). MSC, ML, and PRO accounted for 3.8, 7.4, and 0.1% of the total diet DM, respectively. Both ML and MSC resulted in 14 and 27% more ammonia concentration, respectively than CON and PRO (p < 0.05). MSC increased the propionate percentage at the expense of acetate (p < 0.05). Both ML and MSC decreased methane percentages by 7 and 10%, respectively, compared to CON (p < 0.05). The antioxidant capacity of the moringa seed cake, moringa leaf powder, and raw propolis were 1.14, 0.56, and 8.56 mg Trolox/g DM, respectively. However, such differences were not evident in the fermentation fluid. In conclusion, the supplementation of moringa seed cake desirably modulates rumen microbial activities related to protein and carbohydrate metabolism.

Changes in postmortem metabolites profile of atypical and typical DFD beef.

This study explored the changes in postmortem metabolites of atypical and typical DFD beef using UHPLC-Q-TOF/MS. The longissimus thoracis muscles were categorized into normal, atypical DFD and typical DFD groups. The L⁎, a⁎, b⁎, glycogen and lactic acid contents were lower (P < 0.05) in typical DFD than normal beef. A total of 240 compounds were significantly different among groups. Levels of glycerol-3-phosphate, d-ribose-5-phosphate, d-glucose-6-phosphate, d-fructose-6-phosphate, alpha-d-glucose-1-phosphate and dihydroxyacetone phosphate were higher in atypical DFD than those in the normal and typical DFD beef and the contents of 9 amino acids were elevated in typical DFD. KEGG analysis showed that metabolomic changes were primarily related with ATP-binding cassette transporters, metabolism of purine, central carbon, glycine, serine and threonine, biosynthesis of aminoacyl-tRNA, digestion and absorption of proteins, metabolism of amino sugar and nucleotide sugar, pyrimidine, bile secretion and galactose. In conclusion, development of atypical and typical DFD meat results in differences at level of postmortem metabolite concentrations of different metabolic pathways.

Effect of Animal Age, Postmortem Calcium Chloride Marination, and Storage Time on Meat Quality Characteristics of M. longissimus thoracis et lumborum of Buffalo Bulls.

This study investigated the effect of animal age, calcium chloride marination, and storage time on meat quality characteristics of buffalo bulls to suggest a cost-effective method of improving buffalo meat quality. The current study was designed considering the importance of buffalo meat and the usage of meat from spent buffalo animals in local markets of South Asian countries. A total of 36 animals comprised of 18 young and 18 spent buffalo bulls were selected. After slaughtering and 24 h of postmortem chilling, striploins were separated and cut into 16 steaks and equally divided into two groups, i.e., either marinated with calcium chloride or not. Meat quality characteristics were recorded on 0, 2, 4, 6, 8, and 10 days of storage. The results showed that the pH value of young animals was higher than the value of spent animals and pH was increased over the storage time. Color b*, C*, and h* values were higher in spent animals as compared with the young animals; however, values of colors L* and h* were higher and a* was lower in marinated samples than the values of non-marinated samples. Color a* and C* values were increased and h* was decreased with lengthening the storage time. The meat cooking loss was higher in marinated and the water-holding capacity was higher in non-marinated meat samples. Shear force values were lower in young animals and marinated samples than the values of spent animals and non-marinated meat samples, respectively. Sensory characteristic scores of marinated samples were better than the non-marinated samples. In conclusion, calcium chloride marination can be used to improve the quality characteristics of buffalo meat.

Betaine Modulates Rumen Archaeal Community and Functioning during Heat and Osmotic Stress Conditions In Vitro.

Rumen archaea play an important role in scavenging ruminal hydrogen (H2) and thus facilitate rumen fermentation. They require optimum temperature and osmolality for their growth and metabolism; however, a number of external factors may put archaea under heat and osmotic stress. Betaine is an osmolyte, molecular chaperone, and antioxidant; therefore, it bears potential to combat against these stressors. In this in vitro study, three betaine levels, namely, 0 (control), 51 (low), and 286 (high) ppm, were used. Each of these was subjected to two temperatures (39.5 and 42°C) and two osmolality conditions (295 and 420 mOsmol kg-1) with n = 6 per treatment. Sequencing analyses of the solid phase (which use solid materials containing primarily fibrous materials of low-density feed particles) and the liquid phase (rumen fermenter liquid) using 16S rRNA revealed that more than 99.8% of the ruminal archaea in fermenters belong to the phylum Euryarchaeota. At the genus level, Methanobrevibacter was the most prevalent in both phases, and Methanosaeta was only detected in the liquid phase. The genera Methanobrevibacter and Methanobacterium both showed a positive correlation with methane (CH4) formation in the liquid and solid phases, respectively (P < 0.05). Heat stress increased the relative abundance of genus Methanimicrococcus at the expense of candidate archaeal genus Vadin CA11 (P < 0.05). In the solid phase, osmotic stress significantly reduced the Shannon and Simpson indices of diversity, and relative abundance was higher for Methanobrevibacter at the expense of Methanimicrococcus. In the liquid phase, osmotic stress increased not only the abundance-based coverage estimator (ACE) and singles parameters of diversity but also the relative abundances of Methanosphaera and Methanobacterium. The overall decrease in all gas parameters and estimated metabolic hydrogen ([2H]) utilization was observed during osmotic stress conditions (P < 0.05). Betaine enhanced the diversity of solid phase archaea as indicated by the increase in ACE and singles during heat stress, and only a high dose improved all diversity parameters in the liquid phase during osmotic stress (P < 0.05). Thus, betaine alleviates the effects of heat stress and osmotic stress on the archaea community.

Physicochemical stressors and mixed alkaloid supplementation modulate ruminal microbiota and fermentation in vitro.

The drop of ruminal pH and heat are common physicochemical stressors challenging ruminal microbiota, nutrient digestion and cattle performance. We characterized the ruminal microbiota and digestive activity in response to different pH (6.0 and 6.6) and temperature (39.5 and 42 °C), as well as established the effective dose of alkaloid supplementation (0, 0.088 and 0.175% of feedstock DM) to modulate ruminal fermentation under these conditions. The acidotic condition decreased microbial diversity and abundances of minor bacterial families whereas most of the highly abundant families like Lactobacillaceae, Prevotellaceae, and Bifidobacteriaceae thrived under the stress. Abundances of all three methanogenic archaea taxa detected increased with heat, as did methane production. However, while Methanomassiliicoccaceae benefited from the low pH, Methanomicrobiaceae diminished and methane production decreased. The low dose of alkaloid addition shifted the fermentation to more propionate and less acetate and the high dose decreased methane and ammonia concentration under the low pH. In conclusion, physicochemical stressors shape the microbial community and function. Mixed alkaloid supplementation facilitates the activity of rumen microbial community under acidotic stress.

Betaine addition as a potent ruminal fermentation modulator under hyperthermal and hyperosmotic conditions in vitro.

BACKGROUND: Climatic and dietary shifts predispose ruminal microbes to hyperthermal and hyperosmotic stress, leading to poor fermentation and subsequently adverse effects on ruminant productivity. Betaine may function as substrate, osmolyte, antioxidant, and methyl donor for microbes. However, its effect depends on the extent of microbial catabolism. This study revealed the ruminal disappearance kinetics of betaine and its dose effect on ruminal fermentation during thermal and osmotic stress using a rumen simulation technique. RESULTS: Three different betaine doses were used: 0, 50, and 286 mg L-1 ; each was assigned to two incubation temperatures (39.5 and 42 °C) and two osmotic conditions (295 and 420 mOsmol kg-1 ). Betaine disappeared rapidly within the first 6 h of incubation; however, the rate was lower during hyperosmotic stress (P < 0.05), the stress condition that also suppressed the overall fermentation and degradation of organic nutrients and decreased the bacterial diversity (P < 0.001). During hyperosmotic stress, betaine shifted the fermentation pathway to more propionate (P < 0.05). Betaine counteracted the negative effect of hyperthermal stress on total short-chain fatty acid concentration (P < 0.05) without affecting the composition. Both stress conditions shifted the bacterial composition, but the effect of betaine was minimal. CONCLUSION: Despite its rapid ruminal disappearance, betaine modulated microbial fermentation in different ways depending on stress conditions, indicating the plasticity of the betaine effect in response to various kinds of physicochemical stress. Although betaine did not affect the abundance of ruminal microbiota, the enhanced fermentation suggests an improved microbial metabolic activity under stress conditions. © 2020 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.