FROM POLLUTION TO SOLUTION: MICROPLASTIC CONTAMINATION AND GREENHOUSE GAS EMISSIONS IN RUMINANT LIVESTOCK

Ruminant livestock contribute significantly to global food systems, providing meat, milk, hides, wool, and energy, and supporting farmer livelihoods. With the world population projected to rise from 8.2 billion in 2025 to 9.7 billion by 2050, the demand for ruminantderived products is expected to grow exponentially. However, livestock production poses environmental challenges - most notably greenhouse gas (GHG) emissions. Less explored but increasingly relevant is the issue of microplastic (MP) contamination in ruminant livestock, which could affect animal health, food safety, and environmental sustainability. This PhD project addressed critical aspects of MPs in ruminant systems, focusing on: 1) development of MP extraction protocol for ruminant feeds; 2) application of this protocol to ruminant feeds; 3) evaluation of MP effects on ruminant digestion; 4) assessment of rumen microbiota ability to biodegrade MPs. Moreover, during my PhD mandary period in the company Nutristar S.p.A. it was investigated the effects of essential oils (EOs) on ruminal fermentation and GHGs. For the optimization of MPs extraction protocol for ruminant feeds (corn silage (CSG), hay (HAY), high protein feedstuff (HPF) and total mixed ration (TMR)) several combinations of Fenton reactions and KOH digestion were tested. The final extraction protocol involved a KOH (10% w/w) digestion (60 °C for 24 h), followed by two/three cycles of Fenton reactions (two for HAY and TMR; three for CSG and HPF). The extraction recoveries were all above 85% for all feeds and MPs (low- and high-density polyethylene (LDPE, HDPE), polyamide (PA) fibers and particles, polyethylene terephthalate (PET) and polystyrene (PS)). Applying this protocol revealed that CSG had the highest MP contamination (37.9 ± 7.94 items/g), followed by HAY and HPF (both ~16 items/g). LDPE, transparent, fiber-shaped MPs were the most abundant, with lengths from 0.03 to 5 mm. To evaluate MP effects on ruminant digestion, PET MPs were added to in vitro ruminal-gastro-intestinal incubations at 0, 5, 10, and 15 g/L. PET MPs significantly reduced crude protein degradation in the rumen (-9% at 10 g/L, -16% at 15 g/L) and gastric-intestinal digestibility (-8% at 5 g/L). Neutral detergent fiber degradation also declined (-9% at 10 g/L, -13% at 15 g/L), indicating negative impacts on nutrient utilization. Rumen microbiota potential to biodegrade LDPE, PET, and PA was investigated in vitro over 24, 48, and 72 h. PET showed the highest degradation (up to 0.96% at 72 h), followed by LDPE (up to 0.56% at 72 h) and PA (up to 0.10% at 24 h). PA showed browning over incubation time. Lastly, EO effects on ruminal fermentation and GHGs were tested using an in vitro incubation with TMR substrate. Treatments included lemongrass and oregano EOs (LEO and OEO): 1) control only TMR (0 % EOs) 2) 0.07% LEO 3) 0.07% OEO 4) 0.035% LEO + 0.035% OEO 5) 0.07% LEO + 0.07% OEO. EO blends significantly reduced total gas production (-9%) and EOs alone and in blends CO₂ emissions (-5 to -12%), although no antimethanogenic effect was observed. High-dose EO blends also slightly altered volatile fatty acid profiles. Canonical discriminant analysis highlighted distinct effects among EOs, suggesting their potential as natural feed additives to mitigate ruminant environmental impacts. To conclude, this PhD thesis has addressed critical challenges and potential solutions for improving ruminant livestock sustainability, transitioning from pollution to solution. It highlighted the issue of MP contamination in ruminant feeds, demonstrating its negative impact on nutrient degradability and digestibility, while also identifying the rumen microbiota partial ability to biodegrade MPs as a promising solution. Finally, it also investigated the potential of LEO and OEO to affect ruminal fermentation and reduce GHG emissions, supporting more sustainable ruminant production systems.