Results 2024

Simple Summary: Microplastics have emerged as a pressing global environmental issue and have been alarmingly detected in both the feed and feces of ruminants. However, their effects on the ruminal and intestinal digestive tract have not yet been studied. Our study represents the first investigation of the effects of polyethylene terephthalate microplastics on the ability of the ruminal-gastro-intestinal system to degrade and digest mixed hay. Our findings reveal that polyethylene terephthalate significantly reduce the digestibility of crude protein in mixed hay. Notably, low levels of PET MPs negatively impacted the intestinal phase, while medium and high levels disrupted the ruminal phase. Moreover, medium and high concentrations of polyethylene terephthalate hindered the degradation of fiber fractions, specifically of neutral detergent fiber. These insights underscore the potential risks posed by polyethylene terephthalate microplastics to ruminal–gastro-intestinal functionality and highlight the urgent need for innovative strategies to combat this emerging environmental challenge, ensuring the sustainability and productivity of ruminant farming.

Abstract - Plastics and, in particular, microplastics (MPs) (<5 mm) are emerging environmental pollutants responsible for interconnected risks to environmental, human, and animal health. The livestock sector is highly affected by these contaminants, with 50–60 % of the foreign bodies found in slaughtered domestic cattle being recognized as plastic-based materials. Additionally, microplastics were recently detected inside ruminant bodies and in their feces. MPs presence in ruminants could be explained by the intensive usage of plastic materials on farms, in particular to store feeds (i.e. to cover horizontal silos and to wrap hay bales). Although feed could be one of the main sources of plastics, especially of microplastics, a specific protocol to detect them in ruminant feeds is not actually present. Hence, the aim of this study was to optimize a specific protocol for the extraction, quantification, and identification of five microplastic polymers (high-density polyethylene, low-density polyethylene, polyamide fibers/particles, polyethylene terephthalate and polystyrene) from feeds typically used in ruminant diets (corn silage, hay, high protein feedstuff and total mixed ration). Several combinations of Fenton reactions and KOH digestion were tested. The final extraction protocol involved a KOH digestion (60 ◦C for 24 h), followed by two/three cycles of Fenton reactions. The extraction recoveries were of 100 % for high-density, low-density polyethylene, polyamide particles, and polystyrene and higher than 85 % for polyethylene terephthalate and polyamide fibers. Finally, the optimized protocol was successfully applied in the extraction of microplastics from real feed samples. All the feeds contained microplastics, particularly polyethylene, thus confirming the exposure of ruminants to MPs.

Abstract: This study aims to evaluate the efficacy of Texture Profile Analysis on homogenized cooked patties, referred to as TPAH, in accurately predicting patty texture and distinguishing between various patty types, similar to traditional TPA. Eight types of patties (96 samples), comprising one meat patty, one commercial pea protein-based patty, and six homemade pea protein-based patties, underwent analysis for hardness, cohesiveness, gumminess, chewiness, resilience, and springiness. All parameters measured by both methods exhibited similar variations, except for springiness. Homogenization maintained the ability to differentiate between different types of patties and generally reduced the load, albeit in a manner dependent on the type of product. For instance, the hardness decreased from 32.6 to 29.6N for the meat patty, from 18.7 to 10.0N for the homemade patties, and from 17.4 to 5.0N for the commercial patty. The meat patty exhibited a greater consistency with a 9.2% drop, while the homemade vegetable patties experienced a drop of 46.5%, and the commercial patty even more so with 71.3%. Canonical Discriminant Analysis applied to the two methods demonstrated the superior performance of TPAH compared to TPA. The TPAH method has proven to be valuable in predicting and comparing the texture of cooked patties.

ABSTRACT - The presence of microplastics in the forage and feedstuffs of domestic animals represents an imminent threat to the entire food chain that may reach humans since the particles could be transferred into the intestinal barriers and contaminate blood and animal products. Until now, there is no simple, rapid, sustainable, and reliable method to detect microplastics in animal feed. The objective of this study was to investigate the ability of near-infrared spectroscopy (NIRS) to detect microplastics in ruminant feeds. Two types of instruments were tested using four feeds (corn silage, mixed hay, rye grass silage, soybean meal) and a total mixed ration. Two types of crumbled contaminants, low-density polyethylene and polystyrene, were accurately mixed at ratios of 0, 1, 3, and 5 mg g-1. The pool of the five matrices examined by the benchmark instrument (714-3333 nm) yielded an accuracy of approximately 0.8 mg g-1 and a detection limit of about 1 mg g-1, however, the errors could be halved in separate calibrations. A short wavelength range (714-1070 nm) or a smart NIRS instrument proved an acceptable discrimination of the concentrations. Following these preliminary results, any validation on other samples with different and powerful NIRS tools is encouraged.

Abstract - The adverse effects of Polyamide microplastics  on lamb rumen activity and concentrate degradability were dose-dependent. Increasing the dose of Polyamide microplastics ingested led to a significant linear decrease in the efficiency of rumen microbiota.

Summary - Microplastics (MPs) enter in the animal digestive system through contaminated feeds creating a unique interface within the rumen with microbiota leading to some polymers degradation. The study highlighted an interaction between PET and ruminal microbiota, demostrating their influence on rumen activity. Particularly, these findings highlight the potential negative impacts of PET contamination of feed on the efficiency of ruminant fermentation and degradability. Further research is needed to explore the effect of PET on gastrointestinal activity and to develop strategies to mitigate these effects.

Summary - The primary plastic utilized in farms is low-density polyethylene (LDPE), often identified as a foreign object in the rumen of slaughtered cattle and in the form of microplastic (MPs, <5mm) both in ruminant feeds and their body tissues. Despite its widespread presence in farms, there is a notable absence of studies examining its potential impact on the ruminal degradability of feeds. This research endeavored to examine the influence of LDPE as MPs, on the in vitro rumen degradability of feeds. In 3 farms, 3 most used feeds were collected: mixed hay (Hy), corn silage (Cs), and concentrate (Cn). Feeds, dried at 60°C for 24h and ground (1mm sieve), were prepared in bags containing 0.5g on dry weight of feed, and incubated in a rumen fluid derived from Piemontese bulls. Three runs were performed. Four jars were added with 4 levels of LDPE (0, 0.6, 1.2, and 1.8% on feed dry weight basis). Each jar contained 3 bags for each feed and each farm, for a total of 27 bags. They were incubated for 48h in the Ankom DaisyII with 400mL of filtered rumen fluid, and 1600mL of buffer solutions for jar. Results showed that LDPE at all levels did not affect feed degradabilities. Ruminal degradabilities (%), for 0 to 1.8% of LDPE, were: 47.6±8.92, 47.7±8.96, 49.2±8.72, and 48.5±8.76% in Hy; 59.1±3.97, 59.1±5.13, 59.1±4.86, and 59.7±4.24% in Cs; 69.2±5.80, 70.0±4.38, 71.6±5.28, and 71.6±6.35% in Cn. These results provide valuable insights into the resilience of the rumen degradation process in the face of exposure to LDPE. Future studies are needed to investigate the effects of other MP polymers to provide a deep understanding of the impacts of MPs pollution on ruminant nutrition.

Conclusions - Rubus fruticosus leaves provide a valuable and sustainable alternative feed for goats, particularly in mountain regions, especially during spring. This presents an opportunity to diversify feed resources, reduce dependence from conventional feeds and mitigate the impact of feed shortages in mountain areas. It can also improve the sustainability of the mountain goat production systems.

Abstract - The alarming presence of microplastics (MPs) in the digestive system of ruminants highlights the urgent need for precise assessment methods to comprehend their effects specifically on rumen function, an integral aspect of overall animal health. This study aimed to validate the applicability of an in vitro gas box system that mimics the rumen environment for exploring the effects of introduction of varying levels of MPs (ranging from 2 to 200 mg) into the rumen environment under controlled laboratory conditions. The results successfully demonstrated the effectiveness of this technique in detecting the effect MPs on dynamic of gas produce by rumen fermentation including asymptotic gas production, the constant gas production rate, the average fermentation rate, the time to the onset of rumen gas production and the time to half-maximum gas production. Moreover, this system detected the effect of MPs on concentration of the rumen ammonia nitrogen and the level of rumen protozoa. Lastly, the study highlighted the ability of the gas box system to discriminate not only the presence of MPs but also the dose-dependent effects of MPs across all these parameters.

Abstract - Offspring health can be affected by maternal nutrition during pregnancy. For instance, a low-protein diet may pose a greater risk to their physical and neurological development. Therefore, dietary supplements such as soybeans, which are rich in phytoestrogens, particularly Genistein (GEN), are recommended to address deficiencies in maternal diets during pregnancy. Phytoestrogens, particularly Genistein (GEN), are classified as endocrine disruptors due to their ability to bind to estrogen receptors, affecting various estrogen-sensitive neural systems, including the stress axis.

In this study, we examined the effects on the HPA axis both directly of the mothers and in the offspring in Sprague Dawley rats of a chronic maternal diet low in protein (8%) with and without GEN. 

Molecular analysis by RT-PCR of the brains of mothers sacrificed at the end of lactation demonstrate a strong alteration of the stress axis, mainly with reduced expression of glucocorticoid receptors at both hypothalamic and hippocampal levels in all treated groups. The brain is particularly sensitive to changes in energy production that occur during sustained stress signaling, analysis by WB revealed alterations in ATP production within the treated groups in the hypothalamus and hippocampus. Moreover, analysis of breast milk revealed lower protein and fat content in two experimental groups, pups born to these mothers from birth are smaller and have never resumed normal development, remaining consistently underweight. The HPA axis analysis at PND1 of these pups demonstrated a sexually dimorphic effect with altered stress axis, especially in females, as well as reduced energy production. In adulthood, these animals were subjected to behavioral tests (Open Field and Elevated Plus Maze), and the females showed anxiety-like behaviors, particularly in females born to mothers on a low-protein diet supplemented with GEN.

Summary - The livestock sector faces a significant impact from microplastics (MPs), with their detection in various components of the ruminant production chain, including feeds, faeces, follicular fluid, blood, milk, and meat [1-4]. One potential origin of MPs in the livestock sector arises from the extensive use and breakdown of plastics in farming practices, coupled with environmental pollution from urban and industrial sources facilitated by atmospheric agents such as wind and rain. Notably, staple livestock feed like hay, a fundamental component of the total mixed ration for dairy cows, exhibits substantial plastic contamination acquired from meadows during harvesting and packaging. Despite these pervasive issues, the contamination of mixed hay (MH) by MPs has not been investigated. Hence, the objective of this study was to pioneer an investigation into the presence of MPs in MH originating from three farms in N-W Italy (farm A, B, and C). The MH samples underwent a specific plastic-free process, including collection, overnight drying at 60°C, and grinding. A specially optimised method for this complex matrix was employed for MPs extraction, involving KOH digestion, Fenton reactions, and successive recovery and quantification of MPs using a stereomicroscope, each step being separated by filtration through a 30m sieve. Triplicate analyses, including an environmental control blank, were conducted, and the resulting data were statistically analysed using SAS 9.2. The findings revealed the contamination of MH by MPs, in the shape of particles and fibres. In the three farms (A, B, and C), 17.3±3.06, 18.7±4.04, and 13.3±3.06 particles and fibres of MPs/g dry matter of MH were detected respectively (Fig. 1). Farms exibited no significant variance. Between MPs, the dominant form were fibres in all farms (16.7, 18.7, and 13.3 fibres of MPs/g dry matter of MH respectively). These results underscore the potential contribution of the use of plastic in hay storage and atmospheric plastic transport to field contamination as a pollutant sources. Mitigation efforts have the potential to decrease plastic usage in agriculture, yet addressing pollution stemming from urban and industrial human activities poses ongoing challenges. It is imperative to extend these investigations to all livestock feeds to understand the extent of MPs presence in cattle diets. Expanding this comprehension will enable the creation of robust countermeasures focused on reducing the entry of MPs into the human food chain.

Summary - The proliferation of microplastics (MPs) in ruminant digestive system [1] and feeds has raised significant concerns [2], necessitating urgent attention and precise assessment techniques to understand their impact on rumen function. This study was conducted to explore the capacity of the in vitro gas production (GP) technique, commonly used for rumen fermentation assessment, to investigate the influence of low-density polyethylene (LDPE), the predominant plastic in agriculture [3], on rumen activity and degradability. Rumen fluid was collected from five lambs before morning meal using a rubber stomach tube inserted into the rumen through the oesophagus. Rumen samples were prepared according to the official method [4]. Samples of 200 mg of concentrate fortified with LDPE at varying concentrations (0, 0.6, 1.2 and 1.8%) were inoculated with 30 mL of buff-ered rumen fluid in triplicate into serum bottles. Blanks containing only buffered rumen fluid were also prepared in triplicate to correct gas production from the buffered rumen. All bottles were immediately sealed and incubated for 96 hours in a shaking-water bath at 39 °C and 120 rpm. Gas production was measured after 2, 4, 6, 8, 12, 24, 48, 72, and 96 hours, and data were fitted with an exponential model [5]. Additionally, post fermentation assessments were con-ducted to evaluate concentrate dry matter degradability, rumen ammonia-nitrogen levels, rumen protozoa population, microbial crude protein and microbial efficiency. The in vitro GP technique successfully detected and quantified the significant impact of LDPE contamination on gas production (Fig. 1), rumen ammonia-nitrogen levels, fermentation rates and microbial efficiency across all doses. Par-ticularly at the highest LDPE dose, gas emissions, rumen ammonia-nitrogen levels and fermentation rates increased by 15, 12 and 18%, respectively, while microbial efficiency decreased by 16%. The effect of LDPE contamination on mi-crobial crude protein, rumen protozoa population, and concentrate dry matter degradability was noted only at the high-est LDPE dose, resulting in decreases of 8.1, 16.4 and 4.6%, respectively. These findings underscore the potential of the in vitro GP technique in studying the impact of LDPE on rumen func-tionality and concentrate utilization in controlled laboratory conditions by mimicking rumen fermentation processes. Further research on the utilization of in vitro GP technique can be conducted to understand the impact of different MP polymers present in feeds on the digestive physiology of ruminants.

Summary - Microplastics (MPs) were detected in ruminant feeds,  faeces, follicular fluid, blood, milk, and meat. Ruminants themselves may provide an approach for tackling these pollutants, given the presence of microorganisms in rumens possessed hydrolytic enzymes capable of degrading natural polyesters such as cutin. The similarities between plant-cell molecules (e.g., cutin) and plastics (e.g., polyethylene terephthalate, PET) propel researchers to investigating the capacity of rumen microbiota to degrade plastics. However, a reliable methdology to evaluate this ability has not been established. This study aimed to assess the use of ANKOM DaisyII Incubator as a potential instrument for measuring the ability of rumen microbiota, in rumen environment, to degrade two common polymers in farms, PET and low-density polyethylene (LDPE). Rumen fluid from Piemontese bulls was collected from a slaughterhouse. Three jars were allocated to different incubation periods; 24 (jar 1), 48 (jar 2), and 72 (jar 3) hours, each containing 0.5 g of each type of MPs weighed in filter bags in six replicates, two empty bags as a blank, 20 g of ground Total Mixed Ration, and 2000 mL of rumen fluid- buffer mixiture. All the jars were placed in a modified ANKOM DaisyII Incubator [6] at 39 °C to determine degradability of MPs calculated as weight change before and after incubation with adjustments made based on the weight change in the blanks. The experiment was triplicated. The results demonstrated that the ANKOM DaisyII Incubator identified various levels of MP degradability, ranging from 0.08 to 0.97 % (p-value < 0.05, Wilcoxon), among different MPs and incubation periods. The lowest degradability of 0.08 %, shown in LDPE with 24 hours incubation, was also succesfully distinguished from 0 (p-value = 0.0038, Wilcoxon). Significant differences were noted within both MP groups (p-value < 0.05, Kruskal-Wallis), indicating that this application provided insight into the alteration of MP degradability during the incubation. Moreover, this technique revealed that the levels of degradability statistically differed by the type of MPs, proving the polymer-specific activity of rumen microbiota. This study using the ANKOM DaisyII Incubator underscore its reliability as a method for evaluating the degradability of MPs by rumen microbiota in rumen environment. We anticipate our study to be a starting point for more sophisticated methodology for understanding this promissing ability of ruminants. Future efforts to refine this technique and explore new methods will be crucial to develop sustainable strategies to combat MPs pollution in agriculture.

Defending Against the Imperceptible: Micro and Nanoplastics as an Environmental Emergency?

Micro and nanoplastics represent an invisible but pervasive threat to the planet, even more insidious than the plastic from which they derive. Almost imperceptible, they silently spread through ecosystems, penetrating living organisms and triggering a vicious cycle that crosses soil, plants, animals, and people. Defending against them is arduous, as these microscopic particles cannot be easily collected. During the scientific café, researchers committed to finding solutions to this problem will present their multidisciplinary expertise, analyzing the critical aspects of this environmental challenge and discussing possible strategies to counter it."

Discover the world of microplastics in a fun and playful way.

Abstract

Cosa sono

• Le microplastiche (MPs) sono particelle di plastica con dimensioni tra 5 mm a 1 µm.

• Le nanoplastiche (NPs) sono particelle di plastica < 1 µm.

• MPs e NPs hanno due tipi di origine: fonti primarie e fonti secondarie.

• Da fonti primarie vengono prodotte direttamente e si trovano in cosmetici, dentifrici, make-up, etc.

• Da fonti secondarie derivano dalla frammentazione di plastiche più grandi (es. ossidazione, luce UV, vento).

• Perchè MPs e NPs sono un problema? 1.Potrebbero avere effetti negativi sulla salute e sull’ambiente 2.Sono

troppo piccole da individuare 3.Sono troppo piccole per essere raccolte 4.Non si degradano facilmente.

Dove sono?

• Le MPs sono ovunque dal Monte Everest alla Fossa delle Marianne.

• Negli oceani sono circa 51 mila miliardi.

• Nell’aria sono: indoor 1583±1181 MPs/m3, outdoor urbano 224±70 MPs/m3, outdoor rurale: 101±47 MPs/m3.

• Nei suoli coltivati 3535 MPs/kg e nei suoli urbani 2809 MPs/kg.

• Nell’uomo sono state trovate in capelli, saliva, polmoni, cuore, sangue, cervello, placenta, etc. Le fonti per l’uomo sono l’inalazione, l’ingestione (es: acqua fino a 2277 MPs/L, cozze 360-470 MPs/kg) e il contatto dermico.

• Negli animali da allevamento sono state trovate nel sangue, nella carne e nel latte. Le fonti sono l’inalazione e l’ingestione (es. da 8.3 a 39.4 MPs/g di alimento).

• Danni salute (es. stress ossidativo, danni sistema immunitario, problemi respiratori) da investigare.

Quali soluzioni?

• Evitare l'uso di plastica come primo passo per ridurre le MPs e NPs (es. smetti di acquistare prodotti in plastica e opta per alternative, acquista prodotti senza imballaggio, non utilizzare plastica monouso come cannucce e bottiglie, acquista in stock, usa abiti organici, utilizza filtri che catturano le MPs (es. per aria o lavatrice), opta per camminare e utilizzare i mezzi pubblici, evita di scaldare il cibo nei contenitori di plastica nel forno a microonde).

• L'incenerimento di plastica non è una soluzione perchè genera sottoprodotti volatili cancerogeni (es. furani, diossine).

• Il riciclaggio è una soluzione perché riduce i rifiuti di plastica e abbassa l'impatto ambientale della produzione di nuove materie plastiche.

• I microorganismi sono una soluzione perché alcuni sono in grado di degradare le MPs e NPs (es. batteri, funghi, insetti).

• Direttive e regolamenti dell’Unione Europea per affrontare il problema dell'inquinamento da plastica (es. Direttiva (UE) 2019/904 (giugno 2019), Regolamento (UE) 2023/2055 (settembre 2023).