This article aims to explore various ruminant enteric methane mitigation strategies and delve into their underlying modes of action. Furthermore, it addresses the importance of ruminant enteric methane mitigation in the context of climate change and its potential impact on global warming. The article also highlights the need for interdisciplinary research and collaboration to develop comprehensive and practical solutions. By considering the ecological, economic, and social implications of these strategies, policymakers and stakeholders can make informed decisions to reduce methane emissions while ensuring the sustainability of livestock production systems.This review examines the current state of knowledge regarding the effectiveness of different dietary ruminant enteric methane mitigation strategies and their modes of action together with the issues discussed regarding the potential harms/risks and applicability of such strategies. By investigating these strategies, we can enhance our understanding of the mechanisms by which they influence methane production and identify promising approaches for sustainable mitigation of methane emissions. Out of all nutritional strategies, the use of 3-nitrooxypropanol, red seaweed, tannins, saponins, essential oils, nitrates, and sulfates demonstrates the potential to reduce emissions and receives a lot of attention from the scientific community. The use of certain additives as pure compounds is challenging under certain conditions, such as pasture-based systems, so the potential use of forages with sufficient amounts of plant secondary metabolites is also explored. Additionally, improved forage quality (maturity and nutrient composition) might help to further reduce emissions. Red seaweed, although proven to be very effective in reducing emissions, raises some questions regarding the volatility of the main active compound, bromoform, and challenges regarding the cultivation of the seaweed. Other relatively new methods of mitigation, such as the use of cyanogenic glycosides, are also discussed in this article. Together with nitrates, cyanogenic glycosides pose serious risks to animal health, but research has proven their efficacy and safety when control measures are taken. Furthermore, the risks of nitrate use can be minimized by using probiotics. Some of the discussed strategies, namely monensin or halogenated hydrocarbons (as pure compounds), demonstrate efficacy but are unlikely to be implemented widely because of legal restrictions. The liv4 in the atmosphere have shown an increase of 150% since 1750 [4 emissions constituting 39.1% of the total [2 and is subsequently fixed by plants through photosynthesis. Despite the relatively short lifetime of methane, it has approximately 28 times more global warming potential than carbon dioxide (CO2) on a 100-year timescale and is 84 times more potent on a 20-year timescale [4 [Reducing enteric methane emissions from ruminants has become a major concern in recent years due to its environmental impact and role in climate change. Concentrations of CHnce 1750 . The liv2 pressure, when even a slight increase in H2 in the rumen negatively affects the degradation of plant material [4 reduction strategy and found that an average increase of 386 g/kg DM [4 intensity (g/kg of product) by 10% for sheep, 27% for dairy cattle, and 31% for beef cattle.Increasing the amount of concentrates in animal diets has been a widely used strategy for many years, although this shift was more influenced not by the efforts to reduce emissions, but by the efforts to increase livestock productivity. By increasing the amount of concentrates  in the diet, the number of cellulolytic bacteria decreases and the number of amylolytic bacteria increases due to the changing ratio of substrates ,6. Amylomaterial ,12,13. Mmaterial ,15,16. Vmaterial  analyzed g/kg DM  in conceIt is worth noting that increasing the amount of concentrates in the diet up to 30–40% decreases methane emissions relatively linearly, but when the amount of concentrates exceeds 80%, a sharp drop in methane production is observed ,19. HoweThe use of concentrates can indirectly increase the total GHG emissions of farms as more intensive cultivation of cereal crops requires more herbicides, pesticides, and fertilizers, and the application of these substances requires heavy agricultural machinery, so the resulting upstream emissions could offset the GHG amount reduced through enteric fermentation while feeding such feedstuffs. One of the most important things worth mentioning is that by using diets with large amounts of concentrates, the physiological characteristics of the ruminant to convert indigestible fiber into high-quality protein sources available to humans, such as meat and milk, are not used as intensively, and due to climate change and the demographic explosion of the human population, the use of cereal crops will likely become more and more essential for the nutritional needs of mankind and not for ruminant feed ,23.4 production between different types of forages used [4 production per animal might increase but a decrease in CH4 yield g/kg DMI is evident, as well as a decrease in methane intensity due to increased animal productivity [4 production while decreasing animal productivity, thus elevating CH4 intensity [4 emissions than nutrient content alone. Apart from the nutritional approach described above, well-managed grazing systems could indirectly mitigate emissions through extensive carbon sequestration [Although methane production g/kg of dry matter intake (DMI) is higher in animals fed higher forage/concentrate ratios, forage remains a promising method to reduce methane emissions since there is a significant variation in CHges used . High-quges used . Consequuctivity ,25. As fntensity . While nntensity  proved tstration ,28,29.Secondary metabolites in plants  have a toxic effect on bacteria, protozoa, and methanogenic archaea. Due to commensal relationships, changes in bacterial and protozoan populations also affect the populations of methanogenic archaea, consequently decreasing methane production ,30.Fundamentally, it is practically impossible to optimize ruminant diets in such a way that roughage can be entirely removed without causing health problems to the animal. Secondly, in different parts of the world, due to agroecological and other factors, forages in ruminant diets are dominated by different plant species, and there is a considerable variation in the production of enteric methane between different types of forages. Thirdly, in the USA, Canada, and most European countries, the animal-safe use potential of concentrates has already reached or is approaching its maximum, and forages are the main source of energy for ruminants in many developing countries due to their availability and cost . For the4 emissions, but, to the author’s knowledge, the literature on this topic is limited. The intensity of methanogenesis tends to decrease when the feed is preserved, e.g., ensiled rather than dried. Such a trend can be explained by the fact that ensiled feeds are already fermented to a certain level, thus becoming easier and faster to digest [Different feed preservation technologies also affect CHo digest ,31,32.To reduce methane emissions with the help of roughages, it would be worthwhile to pay attention to improving the quality of forage and its availability by testing and applying different grazing strategies, choosing plant species with higher digestibility and nutritional value, and using early-cut forages for ensiling while enhancing the quality of the preservation process itself.3-nitrooxypropanol (3-NOP) is a synthetic organic compound that was first synthesized in 1990 and patented in 2012 as a potent methanogenesis inhibitor ,34. The Coenzyme M, so named because of its function, participates in methanogenesis as a carrier of a methyl group and is the smallest known cofactor. Methyl-CoM is the last intermediate compound that completes all pathways of methanogenesis  . The lasIt was found that the effectiveness of 3-nitrooxypropanol decreases as the amount of NDF in the feed increases  , while i4 emissions ranged from 29% to 47%.Vyas et al.  indicate4 emissions from cattle of this type. Van Gastelen et al. [4 production (g/d) by 28.2%, 37%, and 38% in animals fed a grass-silage-based diet, a grass silage and corn silage mixed diet, and a corn-silage-based diet, respectively. An amount of 80 mg/kg DM of 3-NOP was even more effective, reducing methane production by 31.4%, 42%, and 45.1% when feeding the aforementioned rations, respectively. The same 3-NOP dose of 60 mg/kg DM was used in the trial by Melgar et al. [Research conducted with dairy cattle suggests that smaller amounts of 3-NOP are enough to achieve substantial results in mitigating CHn et al.  noted thr et al. , resultiThe literature on the effect of 3-NOP on methanogenesis in sheep is very scarce with, to the author’s knowledge, only one publication on the aforementioned topic. Martinez-Fernandez et al.  reportedWhile there is some evidence suggesting that increasing amounts of 3-NOP negatively affect DMI, the data are inconsistent. For example, Alemu et al. , Vyas et4 released by different ruminant groups and species [4 emissions is also influenced not only by the species of the animal but also by the composition of its diet, the dose of the compound or substance, and the method and frequency of administration, and 3-NOP is no exception.Although the literature indicates efficiency variations between different groups of ruminants , 3-NOP, due to its specific and selective mechanism of action (participating in the last step of methanogenesis), can be useful for reducing the amount of CH species . The effStreptomyces cinnamonensis [Ionophores are a class of antimicrobial agents with bacteriostatic and coccidiostatic effects. These substances interfere with the intracellular and transmembrane ion movement of some bacteria and protozoa found in the digestive tract. The most widely used substance of this class in animal husbandry is monensin, which was first isolated in 1967 from monensis .The mechanism of action of this antimicrobial provides a competitive advantage to propionate-producing bacteria that use hydrogen for propionic acid synthesis, thus competing with methanogenic archaea for it .Monensin affects protozoa, which are the main producers of hydrogen in the rumen .Monensin in Europe is approved only for use in dairy cattle as a slow-release capsule for the prevention of ketosis and is used as a coccidiostat in the poultry industry. In the USA, this ionophore is widely used in ruminant feeding as a substance that improves ruminant energy metabolism and increases the efficiency of consumed feed. The effect of monensin on methanogenesis is indirect:4 production by an average of 19 g/d, while the results on dairy cattle were marginal (6 g/d). Such differences were speculated to arise from the fact that the DMI of dairy cattle was almost three times higher than that of beef cattle (18.6 vs. 7.2 kg/d) while the doses of monensin used were larger in beef cattle than in dairy cattle (32 vs. 21 mg/kg of DMI).The results of the studies with monensin vary widely in the literature. Appuhamy et al.  in their4 production. Almeida et al. [4 emission.Benchaar  reporteda et al. , after aa et al.  used mona et al.  used a h4 production in female lambs by 12.7%. However, more research on this topic is needed to elucidate the effects of monensin feeding in sheep production systems.There is very limited evidence in the literature regarding the effects of monensin on methanogenesis in sheep. Zhang et al.  found thIt could be hypothesized that due to the improved energy metabolism and feed efficiency, animals raised for meat could reach the target weight faster because of a possible increase in ADG , so the 1 hydrocarbons (HHC), also called halogenated methane analogs, are organic compounds composed of carbon and hydrogen atoms, with one or more halogen element  covalently bonded to the carbon backbone. This group includes such compounds as bromoform, chloroform (CF), and bromochloromethane (BCM). Due to the high redox potential of these compounds and their structural similarity to some intermediate products of methanogenesis, it is believed that they affect this process both directly and indirectly as elucidated by Yu et al. [Halogenated Cu et al. . The diru et al. .Knight et al.  during tThe studies conducted with halogenated hydrocarbons show that archaeal populations in the rumen might become at least partially resistant to such compounds. In addition, a significant number of halogenated hydrocarbons (including BCM) deplete the ozone layer and are, therefore, banned or strictly controlled by the Montreal Protocol in many countries. Other compounds, e.g., chloroform, are recognized as toxic and carcinogenic, so their use in livestock production systems raises both ethical and public health issues. For all these reasons, as well as legal difficulties in registering such substances for commercial use, halogenated hydrocarbons as pure compounds alone are unlikely to be used to reduce methane emissions from ruminant livestock.Chlorophyta (green), Phaeophyta (brown), and Rhodophyta (red). It is estimated that there are around 6200 different red macroalgae species and around 1800 species of brown and green macroalgae each, making red algae the most diverse [Asparagopsis taxiformis and Asparagopsis armata. Although partial mitigation of methane production can be attributed to various compounds  [A. taxiformis and A. armata is the halogenated compound bromoform (CHBr3) [Marine macroalgae, commonly known as seaweeds, are receiving more and more attention from the scientific community as a possible enteric methane emission mitigation option. Macroalgae, depending on their color, belong to three main phyla:  diverse . Researcs, etc.)  and inte (CHBr3) ,71,72. TA. armata at two different inclusion rates of 0.5% (low) and 1% (high) on an organic matter (OM) basis. Methane production (g/d) decreased by 26.4% and 67.2% for low- and high-inclusion groups, respectively. A 10.8% DMI reduction was observed in the low-inclusion group of A. armata with no significant changes in BW or milk composition. However, researchers observed negative effects on DMI (−38%), BW gain (9.72 kg less), and milk yield (−11.6%) in the high-inclusion group, compared to the control, while the adjusted feed conversion efficiency was significantly greater (0.95 kg milk/kg intake) in the high-inclusion group. Stefanoni et al. [A. taxiformis at inclusion rates of 0.25% (low AT) and 0.5% (high AT) on a DM basis. The authors reported an average of 34.4% CH4 emission reduction in the high AT group, but no significant changes in the low AT group were observed, compared to the control. Interestingly, the reduction in CH4 production was observed only in periods 1 and 2 of the experiment , while no effect was observed in later periods 3 and 4. Also, a DMI reduction of 7.11% and a milk yield decrease of 6.5% were observed in the high AT group, along with a decrease in lactose yield. Alvarez-Hess et al. [A. armata steeped in canola oil with (ASP1) and without (ASP2) seaweed biomass removed. Doses of ASP1 and ASP2 were 136 and 145 g/d, respectively. The authors reported a decrease in methane production by 44% and 39% for the ASP1 and ASP2 groups, respectively. No significant effect on DMI or milk yield was observed.Roque et al.  in theiri et al.  used A. s et al.  examinedA. taxiformis at inclusion rates of 0.25% (low) and 0.5% (high) on an OM basis. The authors reported a 45 and 68% reduction in methane yield for low- and high-inclusion groups, respectively. There was a tendency for DMI to decrease (8%) in the low-inclusion group and a statistically significant decrease of 14% in the high-inclusion group with no significant effect on ADG or carcass quality observed. Consequently, the researchers observed a tendency for feed conversion efficiency (FCE) to increase (7%) in the low-inclusion group and a significant increase (14%) in the high-inclusion group. Kinley et al. [A. taxiformis at inclusion rates of 0.05, 0.10, and 0.20% on an OM basis. While no significant effect on CH4 production was found in steers receiving 0.05% of alga, the authors reported a 40% and 98% reduction in methane production in the groups receiving 0.10% and 0.20%, respectively. No negative effect on DMI or FCE was observed, while the average daily weight gain (ADWG) increase by 26% and 22% was observed for 0.10% and 0.20% inclusion groups, respectively.In a study with beef cattle, Roque et al.  used frey et al.  used freA. taxiformis and reported a CH4 production decrease of up to 80% with an inclusion rate of 3% on an OM basis. However, the authors reported refusals of A. taxiformis in the groups with inclusion rates of 2% and 3%, respectively. The actual intake of added material was calculated over an 11-day period with the results of 1.0–1.5% and 1.2–3.0% for the 2% and 3% groups, respectively  with an average lifetime of 24 days [2 emissions resulting from such production systems should also be evaluated.Even though CHBr 24 days  and therAsparagopsis is a very promising strategy to reduce ruminant CH4 emissions, but currently more research is needed to determine the sustainability and viability of the implementation of this strategy on a large scale.In conclusion, the red seaweed genus 2 needed for methanogenesis [Tannins are water-soluble compounds of high molecular weight found in various parts of plants. These are plant secondary metabolites, the purpose of which is to protect the plant from insects, bacteria, or herbivores ,88. Tannogenesis ,58,98. S4 production (L/kg of DM consumed), respectively. The authors also observed a decrease in relative protozoa and methanogen abundance and consequently reduced OM digestibility at 26 g TA/kg DM. Crude protein digestibility was reduced by 5, 8.6, and 15.7% for 6.5, 13, and 26 g TA/kg DM, respectively. Consequently, 13 and 26 g TA/kg DM reduced ammonia nitrogen (N-NH3) in the rumen. Orzuna-Orzuna et al. [3 concentrations in rumen were decreased and a nitrogen shift from urine to feces was observed. This is a positive result since nitrogen excreted in urine is less stable and results in higher N2O emissions than nitrogen excreted in feces [4/day with the incorporation of Samanea saman pod meal (30% of DM) into a crossbred heifer ration without affecting DMI or apparent DM digestibility. The authors did not observe any reduction in protozoal count. However, a part of the significant reduction in CH4 emissions was attributed to the presence of saponin in S. saman pods. In their study with sheep, El-Zaiat et al. [Acacia saligna and Leucaena leucocephala and reported 12.3 and 10.5% reductions in CH4 production (L/d), respectively. Of the two plant species used, Acacia saligna increased fecal N, retention N, and reduced urinary N without negatively affecting DMI or apparent digestibility.The molecular weight, structure, activity, and concentration of tannins vary greatly depending on the species, age, and the part of the plant used, so the results of the tests carried out with tannins in the literature are variable. This is understandable considering the broad group of compounds that tannins are. Yang et al.  conductea et al.  includedin feces ,100. Valin feces  achievedt et al.  used AcaLotus corniculatus L.), seem most likely to be implemented in large operations. Tanniferous plant use is particularly attractive for pastoral production systems, as a limited number of strategies are adoptable in such systems. Furthermore, because of the anthelminthic properties of tannins, the use of specific tanniferous plants for grazing could additionally benefit small ruminant operations by improving animal productivity and would possibly decelerate the development of anthelmintic resistance. More research is needed to identify the most promising tanniferous plants and to evaluate their possibility to be used for CH4 mitigation in different regions of the world. In conclusion, the use of tannins or tannin-containing plants is a compelling and sustainable strategy, but due to their variable efficacy they are most likely to be used in conjunction with other methane emission mitigation options.Tannins in the rations containing the amount of CP above the requirement could be beneficial and increase animal productivity while also decreasing ammonia excretion, but in mid-quality or poor diets, their inclusion would probably be harmful . Compoun2 required for methanogenesis decreases accordingly. Kozlowska et al. [2 transfer.Saponins are another group of plant secondary metabolites consisting of various glycosides . It is ta et al.  found a Yucca schidigera), soap bark tree (Quillaja saponaria), and tea (Camellia sinensis). The authors reported that the addition of increasing levels of a saponin-rich source decreased methane emission per unit of substrate incubated as well as per unit of total gas produced. The proportion of propionate increased with the increasing levels of saponins and protozoal count decreased.Jayanegara et al.  carried 4 production from 6% up to 27%. Zhang et al. [Enterolobium cyclocarpum) to hair sheep. The amounts of ground pods fed were 0.15, 0.3, and 0.45 kg with saponin contents of 4.35, 8.70, and 13.05 g, respectively. The results showed a tendency for increased DMI with increasing doses of saponin. Methane production was reduced by 21.1, 36.6, and 26.1% with saponin inclusion rates of 4.35, 8.7, and 13.05 g/animal/day, respectively. However, the total methane reduction effect could not be fully attributed to saponins only, as nutrient composition and other plant secondary metabolites of ground pods could have affected methanogenesis. Part of the antimethanogenic effect could also be explained as a result of increased DMI as the inclusion rate of ground pods increased. Increased DMI may have consequently increased the digesta passage rate, thus reducing nutrient availability for rumen microbiota. Mao et al. [Goel and Makkar  analyzedg et al.  achievedg et al.  fed sapoo et al.  used teao et al.  fed tea Medicago sativa L.) could also be used in methane emission mitigation efforts. The efficacy of alfalfa was also proven in vivo by Kumar et al. [2 transfer between protozoa and methanogenic archaea. However, in a study by Kumar et al. [Kozlowska et al.  reportedr et al.  in a stur et al.  reportedr et al. , the lowr et al. ,115. As 4 emission at a tea saponin inclusion rate of 0.5% DM.Ramírez-Restrepo et al.  fed tea The results obtained with saponins, same as with tannins, vary greatly and can also be attributed to differences in the plant species used, variations in the rate of inclusion, different animal type, and diet composition. Nonetheless, as reported in some studies, saponins do have a potential for enteric methane emission mitigation and various species of saponin-rich plants can be used widely in different geographical locations. Moreover, the addition of pure saponin to rations could also be facilitated, as, for example, tea seed saponins are considered a byproduct of tea seed oil extraction . HoweverEssential oils (EOs) are the third group of plant secondary metabolites reviewed in this article. The main functions of EOs are to protect plants from abiotic stress, infections, damage, and pests . These m2 available for methanogenesis [The antimicrobial effect of essential oils is associated with membrane damage, changes in electron flow and ion gradient, protein translocation, and phosphorylation ,125. Altogenesis . Chao etogenesis  found th4 production (L/kg digestible organic matter), compared to control, at inclusion rates of 200 mg and 400 mg/kg DM, respectively. The enhanced effect on methanogenesis with an EO dose of 400 mg/kg DM could be partially attributed to a significantly decreased protozoa count, which occurred 1 week after the beginning of supplementation. No significant differences were observed on DMI or total tract nutrient digestibility. Both treatments also reduced the acetate/propionate ratio.As with other plant secondary metabolites, the vast diversity of EOs leads to different results. Soltan et al.  in theirp = 0.08) for anise oil to reduce methane emission was noted.Santos Torres et al.  analyzedTrachyspermum ammi), garlic (Allium sativum), and cinnamon (Cinnamomum verum) leaf oils at inclusion rates of 0.15 and 0.3 mL/kg DMI. The authors reported no effect on absolute methane emissions per day, but CH4 production expressed as L/kg DMI was reduced by 14.1 and 14.2% in both groups, respectively. No effect on nutrient utilization was observed, but the authors reported an increased DMI in both treatment groups with a tendency for increased body weight gain. It could be speculated that the increased DMI in the treatment groups resulted from an increased palatability/aromaticity of the feed when the EOs were added, as the oils used were not encapsulated. A tendency for an increased body weight gain could confirm this, as no effect on the digestibility of nutrients was observed. Conflicting results were published by Alemu et al. [4 production g/d when supplementing steers with an EO and pepper extract mix Activo® Premium. No effect on the average DMI, ADG, or gain-to-feed (G:F) ratio was observed. In their meta-analysis of 10 studies, Torres et al. [®  supplemented to beef cattle.In their study with buffaloes, Yatoo et al.  used a bu et al. , who reps et al.  reporteds et al.  found noOriganum vulgare) essential oil with carvacrol as the main active component and reported no effect on methane production in dairy cattle. Belanche et al. [® and found an average 8.8% reduction in methane production , certain clovers (Trifolium spp.), arrowgrasses (Triglochin spp.), and cassava (Manihot esculenta) [Cyanogenic glycosides (CGs) are a group of natural compounds found in certain plants and act as a defense mechanism against herbivores . Chemicaculenta) . These bculenta) . During culenta) . HCN binculenta) . HCN is culenta) .The toxicity of cyanide to methanogens has been known for a least 4 decades and was a problem encountered in anaerobic wastewater treatment systems ,146,147.The research focused on cyanide as a candidate for inhibiting enteric methane formation consists mostly of trials performed in vitro. Zavaleta et al.  performeCyanogenic glycosides can be effective in reducing enteric methane emissions, but this topic warrants more intense research to determine which CGs are the most suitable for such use, as well as their dose and mode of administration  because relatively small amounts of cyanide can be toxic ,151. Mos3−) and sulfate (SO42−), with the formation of ammonium ions (NH4+), nitrogen (N2), and hydrogen sulfide (H2S) as end products.Some of the rumen microorganisms are able to reduce nitrate , but the balance between these two compounds is highly affected by pH, with ammonia concentrations increasing as pH increases [2O) and the final product is nitrogen (N2). Although nitrous oxide is a very potent GHG, there are relatively few genes encoding denitrification in rumen metagenomics samples, so this process is not considered to be a very significant pathway for nitrate metabolism in the rumen, although it cannot be ruled out [Nitrate reduction in the rumen can take place via assimilatory and dissimilatory pathways . The finynthesis ,153. Ammncreases . Denitriuled out ,156.2S and incorporate sulfur (S) in the synthesis of S-containing amino acids and coenzymes [2S is dissociated into sulfidic anions (HS−) and remains in the rumen liquid phase; the other part is released as gas, which is expelled from the rumen via eructation.Bacterial sulfate reduction is also divided into assimilatory and dissimilatory . During oenzymes . During 2 as an electron donor for the reduction of both NO3− and SO42−. Nitrate and sulfate reduction are thermodynamically more favorable reactions than methanogenesis, so these compounds can act as hydrogen sinks, thus lowering the amount of H2 available for methanogenesis [Microorganisms can use Hogenesis .2S is known to be toxic [The reduction of nitrite to ammonium in the rumen is a slower process than the reduction of nitrate to nitrite. Nitrite is readily absorbed into the bloodstream from the rumen, so the accumulation of nitrite may put the animal at risk of methemoglobinemia . The potbe toxic , so its 4 production after the introduction of sulfate at 2.6% on a DM basis. The scientists also noticed that when nitrate and sulfate were used together, their anti-methanogenic effect was cumulative—methane production decreased by 47%. No effect on feed intake or average weight gain was observed. Feng et al. [4 production of 20.4 and 10.1% for dairy and beef cattle, respectively, with an average NO3− inclusion rate of 16.7 g/kg DM (3− (beef cattle mostly received slow-release nitrate).Almeida et al.  analyzedg et al.  included g/kg DM . It shou2− and H2S). One of the possible ways to reduce the risk of toxicity could be microencapsulating nitrate, which could help to provide more stable delivery to the rumen. For both nitrate and sulfate, the administration of nitrate-, nitrite-, and sulfate-reducing bacteria as probiotics would help to reduce the risk of toxicity for the animal. Different nitrate and sulfate salts and/or their combinations should be tested in vivo in search of the most effective mix . In addition to methane reduction potential, nitrate is a non-protein nitrogen (NPN) source and could benefit animal production systems where dietary CP content is insufficient. However, in the production systems where the dietary CP in rations is high, feeding nitrate can increase nitrogen concentration in manure/urine, which would result in increased N2O emissions. Furthermore, in animal diets where distillers grain is used (high S content), additional sulfate inclusion is likely to increase the risk of disorders such as polioencephalomalacia.Overall, the use of nitrate and sulfate seems like an effective methane emission mitigation strategy, but care should be taken when administering these substances as the metabolism of both compounds results in toxic intermediate or end products , are microbiological feed additives based on selected bacterial or yeast cultures . ProbiotSaccharomyces cerevisiae, can improve the general health of animals, feed consumption, and fiber digestibility, which results in an increase in milk production or body weight gain. Although the benefits of yeast for ruminants have been confirmed by a number of scientific studies, yeast is probably not the right strategy when it comes to methane emissions. Harper et al. [Saccharomyces cerevisiae and reported no significant effect on CH4 production, intensity, or yield. However, milk yield was increased by 2 kg/d in the treatment group, with no effect on DMI or feed digestibility. Darabighane et al. [4 production. Although no direct effect on methane emission was observed, improved animal production might result in lower methane intensity and, consequently, lower the total methane footprint of animals in certain production systems, e.g., faster-growing meat breeds could reach target weight faster. However, since no direct effect on methanogenesis was observed, this option will not be discussed further. Different species, strains, and doses could be used to screen for possible candidates for enteric methane mitigation.Yeast, such as r et al.  fed lacte et al.  performeLactobacillus, Enterococcus spp.) are often used in ruminant feeding. Due to constantly elevated levels, the microflora of the rumen adapts to the presence of lactic acid, thus also reducing the risk of acidosis [Bacteria or their metabolites shift rumen fermentation and consequently reduce methanogenesis;Bacteria or their metabolites directly inhibit rumen methanogens;Bacteria or their metabolites inhibit H2-producing microorganisms.Lactate-producing bacteria  such as Propionibacterium spp. and Megasphaera spp. can increase the production of propionate in the rumen, thereby increasing the amount of energy available to the ruminant and reducing the risk of acidosis by reducing the concentration of lactate in the rumen. Propionate production is also an alternative electron sink, diverting hydrogen flow from methanogenesis [4 mitigation. The increase in methane production could be due to the direct effect of the strain introduced into the diet on methanogens or an indirect effect through metabolites affecting the ruminal ecosystem [Jeyanathan et al.  used Lacn et al.  also repogenesis , so lactogenesis . Howeverogenesis ,175, no cosystem .2 by hydrogen to CH4 is a thermodynamically more favorable reaction than the reduction of CO2 to acetate [2 partial pressure in the rumen, so in this case, natural cultures of acetogens in the rumen act more as a protective mechanism, which is activated more intensively when methanogenesis stops and the hydrogen concentration increases rapidly. Such bacteria can be useful for reducing increased hydrogen concentration when effective methanogenic population control methods are applied. Because of the higher affinity of methanogenic archaea to H2 and a consequent need for higher hydrogen concentrations in order to be effective, reductive acetogenesis so far seems like an unlikely method to effectively reduce methane emissions.Naturally found in rumen cultures, acetogenic bacteria can use hydrogen to produce acetate during a process called reductive acetogenesis, which in theory can compete with methanogenesis. However, the reduction of CO acetate ,177. To 2-reducing bacteria. As discussed in the previous section, nitrate has the potential to reduce methane emissions by competing with methanogens for H2, but the toxic intermediate—nitrite—poses health risks for animals. An additional inclusion of nitrite-reducing bacteria in the rations of steers supplemented with nitrate was proven to be effective—Latham et al. [Paenibacillus 79R4 aided nitrite metabolism and prevented methemoglobin accumulation. Other classes of microorganisms, such as sulfate-reducing or lactate-utilizing bacteria, can help minimize the negative effects associated with certain mitigation strategies.Probiotics have the potential to aid in methane emission mitigation efforts when used in conjunction with other strategies. One such possibility is using NOm et al.  reportedBacillus licheniformis, can positively affect ruminal fermentation and reduce CH4 emissions via pathways that have not been clearly elucidated yet. Deng et al. [Bacillus licheniformis to sheep and reported an average 12% reduction in methane production when scaled to digestible DM for low- and medium-inclusion rates of the additive—2.5 × 108 and 2.5 × 109 colony-forming units (CFU) of B. licheniformis per head per day, respectively. A slightly smaller, 9.3%, reduction was observed in the high-inclusion group (2.5 × 1010 CFU)  . The add4 emissions and can be used together with other mitigation strategies.Due to the particularly wide variety of microorganisms and their different abilities to influence the fermentation process, the possibilities of using probiotics have a lot of potential not only in methane mitigation but in enhancing animal health and productivity in general. Probiotics, when used in tandem with other additives used for emission mitigation, demonstrate the ability to enhance their effects or reduce potential risks associated with before-mentioned additives. However, care should be taken to investigate the use of different species and strains of microbes, as some have been shown to increase methane emissions. Due to the wide possibilities of application, more research is needed to screen for such microorganisms and their interspecies connection. Overall, probiotics seem a very likely option to reduce CHAsparagopsis taxiformis and Asparagopsis armata are the main seaweeds studied.Currently, some of the most widely studied enteric methane mitigation strategies are the use of 3-NOP, nitrates, plant secondary metabolites (PSM), and macroalgae, out of which 3-NOP was proven to be an effective emission mitigation strategy, but it is likely to be applied mostly in confined systems such as feedlots where it can be incorporated into the animal feed. This strategy seems to be difficult to adopt in grazing-based production systems for the reason mentioned before. It could be speculated that the creation of a slow-release capsule or bolus might be one of the possible solutions. Furthermore, in the European Union, 3-NOP is authorized only for use for dairy cattle, but it is highly likely that authorization will include other ruminants, such as sheep and goats, in the near future.Although proven to be very effective, the use of seaweeds raises some issues. Firstly, two of the most effective seaweed species are found mostly in tropical waters, so their production in colder waters is questionable. Some companies are exploring possibilities to create inland seaweed production systems, but such systems are expected to be energy-consuming . Large-scale production of seaweed for ruminant feeding will result in additional upstream emissions from different production stages and care should be taken to estimate if the carbon footprint of such industry will not offset the reduction coming from feeding the seaweed. It should be stressed that the main active component of red seaweed, bromoform, although short-lived (24 days), is a compound with ozone-depleting potential and improper processing of seaweed can increase bromoform flux into the atmosphere. Furthermore, it has been proven that bromoform content in seaweed degrades substantially over time, so other processing methods than freeze-drying should be explored.The use of nitrates and sulfates, although effective, poses some risks to animal health. Nonetheless, current research suggests that these risks can be minimized or circumvented. No commercial products of these compounds formulated specifically for ruminants exist to the author’s knowledge. To help minimize potential risks, encapsulation or other techniques should be further explored to determine the safest and most effective methods of administration. The addition of specific direct-fed microbials has also been proven to alleviate the risks arising from the usage of such compounds.Other feeding strategies, such as the use of plant secondary metabolites, have also been proven to be effective. Regarding pasture-based systems, plants containing higher amounts of different secondary metabolites seem to be the most attractive methods to mitigate emissions from such systems. Additionally, the use of PSM also has the potential to contribute to the control of ruminant gastrointestinal parasitism and slow down the formation of anthelminthic resistance. Although the efficacy of such natural compounds on enteric methane formation is variable, their additive/synergistic effect warrants more research to find the most suitable plant or active compound combinations and doses. Furthermore, because of higher public acceptance, their natural origin, and the occurring shift towards ecological farming practices, certain plant species and their secondary metabolites will most likely become a part of a multilevel enteric emission mitigation plan.Due to various geographical and agro-ecological factors and adaptability issues, mass adoption of only one or two mitigation strategies is unlikely and a number of different strategies will probably be adopted in ruminant production systems across the world. In addition, the possibility of a combination of different mitigation strategies and their interactions should be assessed further.


The incorporation of different functional fillers has been widely used to improve the properties of polymeric materials. The polyhydroxy structure of PVA with excellent film-forming ability can be easily combined with organic/inorganic multifunctional compounds, and such an interesting combining phenomenon can create a variety of functional materials in the field of materials science. The composite membrane material obtained by combining MOF material with high porosity, specific surface area, and adjustable structure with PVA, a non-toxic and low-cost polymer material with good solubility and biodegradability, can combine the processability of PVA with the excellent performance of porous filler MOFs, solving the problem that the poor machinability of MOFs and the difficulty of recycling limit the practical application of powdered MOFs and improving the physicochemical properties of PVA, maximizing the advantages of the material to develop a wider range of applications. Firstly, we systematically summarize the preparation of MOF/PVA composite membrane materials using solution casting, electrostatic spinning, and other different methods for such excellent properties, in addition to discussing in detail the various applications of MOF/PVA composite membranes in water treatment, sensing, air purification, separation, antibacterials, and so on. Finally, we conclude with a discussion of the difficulties that need to be overcome during the film formation process to affect the performance of the composite film and offer encouraging solutions. Polymer is a traditional material with macromolecular structure that is widely used in our daily life due to its light weight, low cost, and chemical stability . The pro2, where MOF powder has the drawbacks of light scattering and being difficult to recycle after the reaction, which has limited its practical application  (H2O)2 changed due to the presence of label Bacillus anthracis , indicating an energy transfer from DPA to the Tb3+ and Eu3+ centers and a linear relationship between the emission intensity ratio (ITb/IEu) and DPA concentration for DPA present in methanol and water. Therefore, Othong et al. used PVA/[Tb0.43Eu1.573(H2O)] (H2O)2 composite films as a ratiometric luminescence sensor for the calibration of DPA. In the presence of DPA, the visible luminescence color of this composite film under UV light changes from red-orange in methanol to orange-yellow, and from red-orange in water to green , which has great potential for food freshness testing.In addition to combining Ln-MOFs with PVA to produce excellent membranes with sensing properties, Lou et al.  comparedetection B. Ma et etection C. In add2S, NH3) poses an alarming threat to public health and the environment, especially in some heavy industries and densely populated cities. Traditional adsorbents such as activated carbon and zeolite have been widely proven to be good candidates for removing toxic substances from the air, but most of these materials suffer from limited adsorption capacity and high regeneration energy consumption [Through risk factor studies, air pollution has been identified as the most significant contributor to disease and premature death in the world . Ambientsumption ,132. MOFsumption ,134,135.sumption ,137,138.sumption . For exasumption . Moreovesumption . MIL-101sumption .2 nanofiber membrane, the ZIF-8@SiO2 composite nanofiber membrane increased the electrostatic interaction with PM due to the increased surface area and the unbalanced metal ions and defects of MOF, and the removal efficiency of PM was as high as 99.96%, and the adsorption of formaldehyde also increased with the increase in ZIF-8 content on the composite nanofiber, up to 48.87 mg/g, while the adsorption of formaldehyde on the SiO2 nanofiber membrane was 1.49 mg/g [2.5 was as high as 96.5% with a quality factor (Qf) of 0.099 Pa−1  binding to the open metal sites of MOFs; (2) interacting with functional groups on MOFs or polymers; and (3) electrostatic interactions with MOF nanocrystals . In part.49 mg/g . The PP/.49 mg/g  had a po099 Pa−1 A. Kim etectively .x,y,z) depend on the permanent dipole moment of the polar molecule (μ) and on the electric field strength determined by the local space charge density of the MOF  [With the depletion of fossil fuels, the development of clean, renewable biofuels has entered the limelight because of their ability to alleviate the global energy crisis and environmental degradation, especially renewable clean biofuels such as bioethanol ,149. The .2 nanocrystals, added to PVA membranes for the total evaporation of 92.5 wt% ethanol. Due to interparticle interactions , the best MIL-53-NHCOH/PVA MMMs showed a 206% increase in water permeability and a 200% increase in selectivity, and the best MIL-53-NHCOC4H9/PVA MMMs showed a 340% increase in water permeability and a 170% increase in selectivity compared to the pristine PVA membrane , which is 483% and 567% higher than the water permeability and selectivity of the original PVA membrane, respectively  and Staphylococcus aureus (S. aureus) and concluded that Cu-BTC/PVA electrospun fibers have strong antibacterial properties due to the interaction between the released Cu2+ and the bacterial membrane. The antibacterial activity increased with increasing concentration of Cu-BTC and completely inhibited the growth of Staphylococcus aureus. Zhu et al. [2@PVA nanofiber membranes compared with LV@UiO-66-NH2@PVA nanofiber membranes loaded with the antibacterial drug levofloxacin (LV) against E. coli and S. aureus. Due to the long-term effective release of LV stored in the pores of the nanofiber membrane and UiO-66-NH2, the antibacterial performance of the nanofiber membrane was enhanced, especially the bactericidal efficiency of LV@UiO-66-NH2@PVA at 100μg/mL, which was more significant than 99.9% for both E. coli and S. aureus (2+ (In addition to the low toxicity, biodegradability, and good dispersion of MOF particles combined with strong interfacial interactions with non-toxic and non-hazardous PVA polymeric materials, MOF-based MMMs with high antimicrobial efficiency have great potential for applications in the healthcare sector. The three-dimensional network structure of the fiber membrane facilitates the adsorption of bacteria. Singbumrung et al.  studied u et al.  studied . aureus A. In addreus (2+ B. Thanks−3 S·cm−1 at 50 °C (The world’s demand for electric energy devices has accelerated the development of new energy storage systems with high energy density and long life . For theat 50 °C C. Yang eThe combination of MOF materials with high specific surface area, regular pore size, and tunable structure with PVA, a biodegradable material with low toxicity, cheap and easy availability, good mechanical properties, and biocompatibility, is an important material for the preparation of composite membranes with multiple application potentials. This paper summarizes the preparation of MOF/PVA composite films from PVA and MOFs using solution casting, electrostatic spinning, and other different methods. The simple and effective embedding of MOF particles in PVA membrane materials by these methods has led to widespread interest in MOF/PVA composite membrane materials for a wide range of applications such as water purification, sensing, air purification, antimicrobials, UV blocking, and so on. However, to further achieve the functionalization of MOF/PVA composite membranes, the prepared composite membranes still face some challenges in improving the dispersion of MOF particles in PVA, active sites, and specific surface area. In the future, the experimental methods can be further optimized, e.g., pre-dispersing MOF particles ultrasonically in solvents, preparing good quality MOF particles, designing organic ligands for specific functions, optimizing electrostatic spinning parameters, exploring the viscosity of the PVA solution, and incorporating dispersing agents or cross-linking agents, etc., to improve the uniform and stable loading of MOF particles on the composite membranes and to increase the specific surface area of the composite membranes with the active sites, which is crucial for evaluating the performances of the MOF/PVA composite membranes. In addition, this paper concludes that MOF/PVA composite membranes are found to have important application potential in utilizing their adsorptive separation properties; therefore, more optimization of the process of MOF/PVA composite membranes as an adsorptive separation application can be investigated in the future. Finally, to give more excellent properties to MOF/PVA composite membranes, MOFs or PVA can be pre-combined with different functional materials, and then the structure–property relationship of MOF/PVA composite membranes combined with functional materials can be investigated to explore more different applications, as different applications may require composites with different properties.


Polylactic acid (PLA) is considered a mature alternative to synthetic plastics made from petroleum by-products, possessing the advantages of good mechanical strength. However, it also has some disadvantages such as brittleness and low toughness. In order to overcome and improve some of these unfavorable properties, PLA/PBAT composites were prepared by blending PLA with Poly  (PBAT), and adding 4,4′-methylene diphenyl diisocyanate (MDI) and chitosan nanoparticles (ChNPs) as compatibilizers to investigate the effects of different compatibilizers on the properties of the composites. The main observations are as follows: FT-IR indicated that MDI did not add new groups, while the addition of ChNPs added a substantial amount of hydroxyl and methylene groups. The addition of both MDI and ChNPs did not have any effect on the crystalline shape of the composites, but could potentially reduce their crystallinity, increase the melt peak temperature, wet the boundary of the PLA and PBAT phases, decrease the size of the dispersed phases, reduce the number of dispersed phases, and improve interfacial compatibility. The incorporation of MDI increased the tensile strength from 13.02 MPa to 19.24 MPa, whereas the addition of ChNPs substantially enhanced the elongation at the break from 3.84% to 19.24%. Furthermore, the inclusion of MDI conferred enhanced moisture resistance, whereas the addition of ChNPs seemed to weaken the resistance to moisture. As the economy continues to grow, the rapid depletion of petroleum reserves due to the widespread use of petroleum-based products and the intensification of greenhouse gas emissions have prompted researchers to explore new materials that are biodegradable, renewable, and recyclable . BiodegrBiodegradable plastics can be broadly categorized based on their carbon source into two types, namely bio-based biodegradable plastics and petrochemical-based biodegradable plastics . The forPLA has excellent tensile properties, but its disadvantages are extreme brittleness , elongat4,4′-Methylene-diphenyl diisocyanate (MDI) is a widely used compatibilizer in two-component systems based on reactive polyurethane chemistry due to its possession of two isocyanate groups and high reactivity towards carboxyl and hydroxyl groups. Thus, MDI serves to enhance interfacial compatibility. Isocyanate groups readily react with hydroxyl and carboxylic acid groups to form carbamate groups . Pan et MDI is a commonly used compatibilizer, but MDI is toxic; we therefore wanted to find a new type of compatibilizer to replace MDI, and after preliminary research, we chose chitosan nanoparticles. Chitosan is a non-toxic, biodegradable, and biocompatible cationic polysaccharide derived from the partial deacetylation of chitin, which is isolated from the exoskeletons of natural crustaceans. Biocompatibility can have a significant impact on the human body as well as the environment ,30,31, sThe primary objective of this paper is to contrast the impacts of the commonly utilized compatibilizers MDI with the ChNPs for enhancing interfacial compatibility in PLA/PBAT composites. This will be done by examining changes in the chemical structure, crystalline structure, thermal properties, and mechanical properties resulting from the addition of these agents to the composites. The goal is to analyze the effects of incorporating ChNPs into the composites to provide a reference for future research and production.PLA  was purchased from the Nature Works Company .PBAT  was purchased from the Macklin Biochemical Co., Ltd. .MDI (Purity: 98%) was purchased from the Aladdin Biochemical Technology Co., Ltd. .Chitosan  was purchased from the Macklin Biochemical Co., Ltd. .Sodium Tripolyphosphate  was purchased from the Aladdin Biochemical Technology Co., Ltd. .Trichloromethane (AR) was purchased from the Chinasun Specialty Products Co., Ltd. Chitosan nanoparticles (ChNPs) were prepared using the ionic cross-linking method . ChitosaPLA and PBAT composite films were prepared using the solution casting method . PLA andField emission scanning electron microscope, regulus 8100, Hitachi, Ltd. .Differential scanning calorimetry analyzer, DSC8000, PerkinElmer, Ltd. .X-ray powder diffractometer, Rigaku Co., Ltd. .Fourier transform infrared microscope system, spotlight 200i, PerkinElmer, Ltd. (USA).Electronic universal testing machine, UTM4103, SUNS Technology Stock Co., Ltd. Xc, %) and melting temperature (Tm) of PLA composites were determined using differential scanning calorimetry (DSC). The samples were heated up to 300 °C and the melt heat absorption curve was recorded. Xc was calculated with Equation (1) [The crystallinity (tion (1) :(1)Xc=ΔH−1 with a resolution of 4 cm−1 and a scan number of 64.Fourier transform infrared spectroscopy (FTIR) was employed for the qualitative identification of components in the composites. The measured wavenumber range spanned from 4000 to 450 cmX-ray diffraction (XRD) was utilized for characterizing PLA/PBAT composites, using Cu-Kα rays, a Ni sheet filter, a wavelength of 0.15 mm, a radiation tube voltage of 40 kV, a tube current of 40 mA, a scan rate of 0.02°/s, and a diffraction angle of 2θ ranging from 10° to 60°.3× and an acceleration voltage of 1 kV. Additionally, the microscopic morphology of ChNPs was observed by SEM at magnifications between 150× and 104×, and an acceleration voltage of 1 kV.Scanning electron microscopy (SEM) was used to observe the surface and section morphology of PLA/PBAT composites at a magnification of 10Specimens were tested at room temperature using an electronic universal testing machine following GB/T1040.3-2006 Type 1B specifications with a sample length of 70 mm and a tensile rate of 2 mm/min. Each group of specimens was tested three times to obtain the average value.0. The samples were then placed in an oven at 90 °C for drying for 24 h, after which they were removed and weighed again as m1. Afterwards, the PLA/PBAT composites were immersed in distilled water for 24 h before being taken out to calculate the water content using Equation (2) and the swelling degree using Equation (3):Following the testing method by Zhang Zimo et al. , PLA/PBAIn comparison to the PLA/PBAT composites, the melting peaks of the composites containing compatibilizers demonstrated a propensity to shift to the right in the melting curves. Possibly, some hydroxyl groups and end groups in PLA participated in the chain elongation reaction, and the chain expansion through MDI addition augmented the molecular weight while reducing the number of end groups . FurtherFollowing the addition of ChNPs, the melting peak increased from 161.21 °C to 161.50 °C . This al−1, attributable to the stretching vibration of C=O, and another distinct peak at 1454 cm−1 caused by the distortion of the hydroxyl group; both peaks are typical of PLA transmission bands. Asymmetric vibrations of the ester group in PLA can also be observed at 1269 cm−1 and 1181 cm−1 [−1 corresponds to the C=O stretching peak of carbonyl and ester groups, which is typical of the IR transmission band of PBAT [−1, representing a symmetric stretching vibration of -COOH, a characteristic C-O-C absorption peak around 1083 cm−1, and a peak at 872 cm−1, signifying a C-H bending peak, can be observed. The peak at 727 cm−1 is the methylene stretching peak [181 cm−1 ,43,44. T−1 [Clearly, the characteristic peaks did not change significantly after the addition of MDI as a bulking agent, and no stretching band corresponding to the isocyanate group was found at 2270 cm−1 , indicat−1, symmetric stretching vibration peak of -COOH around 1360 cm−1, asymmetric vibration peaks of the ester group at 1270 cm−1 and 1181 cm−1, C-O-C characteristic absorption peak at 1083 cm−1, and interstitial substitution peak of the benzene ring at 872 cm−1 all decreased in intensity. Simultaneously, the distorted peak of the hydroxyl group at 1460 cm−1 and the telescopic peak of the methylene group at 720 cm−1 experienced an increase in intensity [−1 and 2848 cm−1 were distinctly observed. Miao et al. [Conversely, upon incorporating chitosan nanoparticles as compatibilizers, it can be observed that although most of the characteristic peaks maintained their positions, the intensities of the peaks altered. The C=O stretching peak at 1755 cmntensity ,46. Notao et al.  stated tAfter incorporating MDI and ChNPs, both the positions and numbers of diffraction peaks in the composites remained unchanged; however, the intensity of the peaks diminished. Notably, the intensity of the diffraction peak at 2θ = 16.9° decreased significantly after adding ChNPs ,49. ThisAfter increasing the magnification, compared to Polyester-based composites are widely used in flexible packaging applications, which require a good balance between flexibility and strength. For this purpose, information on the static mechanical properties of PLA/PBAT and composites with different compatibilizers added to PLA/PBAT were obtained by tensile tests, and the tensile strength and elongation at break of the materials are shown in Interestingly, the addition of chitosan nanoparticles (ChNPs) to the composites as a bulking agent had little effect on the tensile strength but significantly increased the elongation at break of the composites to 19.24%, with a tensile strength of 13.20 MPa. This is due to the fact that the tensile strength of the composites is mainly provided by PLA, and in the PLA/PBAT composites, the interfacial compatibility between PLA and PBAT is poor, with PLA being dominant. After the addition of ChNPs, the elongation at break reached 19.24%, an increase of 401%. The addition of ChNPs can significantly enhance the high toughness of PBAT and greatly improve the elongation at break of the composite due to ChNPs as a bulking agent enhancing the interfacial compatibility between PLA and PBAT, reducing the dispersed phase, promoting their interaction, and increasing the strength of the blended interface with a large amount of hydrogen bonding and entanglement.The water content and swelling of a membrane are dependent on the presence or absence of hydrogen bonding, ionization of amino or carboxyl groups, and re-relaxation of the membrane structure. These factors reflect the membrane’s ability to resist moisture from external sources . As indiIn this paper, the effects of MDI and ChNPs addition on the chemical structure, crystallization behavior, thermal properties, microscopic morphology, mechanical properties, and resistance to moisture of PLA/PBAT composites were investigated. The FTIR spectra demonstrated that the starting isocyanate groups reacted completely with PLA and PBAT, without adding any new groups. In contrast, the addition of ChNPs introduced a substantial number of -CH2 groups as well as hydroxyl groups. The addition of both MDI and ChNPs did not change the crystalline shape of the composites, but the crystallinity of PLA/PBAT/MDI and PLA/PBAT/ChNPs decreased from 18.03% to 16.78% and 17.12%, respectively. The addition of both substances was able to promote wetting at the boundary of the PLA and PBAT phases, reduce the size of the dispersed phases, and decrease their number. This confirmed that MDI and ChNPs could effectively improve interfacial compatibility. It was also observed that both components were uniformly distributed within the matrix without pores, indicating good compatibility and interfacial adhesion between the composites and ChNPs or MDI. Both MDI and ChNPs can enhance the mechanical properties of the composites. However, the addition of MDI primarily increases the tensile strength from 13.02 MPa to 19.24 MPa, while the addition of ChNPs mainly raises the elongation at break from 3.84% to 19.24%. The incorporation of MDI reacts with the -OH at both ends of PLA and PBAT, resulting in reduced hydroxyl groups in the composites, which leads to improved resistance to moisture. In contrast, the addition of ChNPs introduces a large quantity of hydroxyl groups, making the composites less resistant to moisture.From the experimental results, it is easy to see that although they are both compatibilizers, MDI and ChNPs have different effects on the chemical structure, mechanical properties and moisture resistance of the materials. We are studying the specific reactions of both in the materials to further investigate whether ChNPs as compatibilizers can further improve the performance of the composites and provide a good modification solution for PLA/PBAT composites. However, further studies are needed to determine the feasibility of applying these materials in industrial production.


Colorectal cancer (CRC) is the third most common cancer. It is a heterogeneous disease, including both hereditary and sporadic types of tumors. CRC results from complex interactions between various genetic and environmental factors. Inflammatory bowel disease is an important risk factor for developing CRC. Despite growing understanding of the CRC biology, preclinical models are still needed to investigate the etiology and pathogenesis of the disease, as well as to find new methods of treatment and prevention.The purpose of this review is to describe existing murine models of CRC with a focus on the models of colitis-associated CRC. This manuscript could be relevant for experimental biologists and oncologists.We checked PubMed and Google from 01/2018 to 05/2023 for reviews of CRC models. In addition, we searched PubMed from 01/2022 to 01/2023 for articles using the azoxymethane (AOM)/dextran sulfate sodium (DSS) CRC model.Existing murine models of CRC include spontaneous, genetically engineered, transplantation, and chemically induced models. For the study of colitis-associated cancer (CAC), the AOM/DSS model is predominantly used. This model is very similar in histological and molecular characteristics to the human CAC, and is highly reproducible, inexpensive, and easy to use. Despite its popularity, the AOM/DSS model is not standardized, which makes it difficult to analyze and compare data from different studies.Each model demonstrates particular advantages and disadvantages, and allows to reproduce different subtypes or aspects of the pathogenesis of CRC. Among malignant neoplasms, according to GLOBOCAN worldwide 2020 data, colorectal cancer (CRC) is the third most common (10% of new cases of cancer) and second in the structure of mortality (9.4% of deaths from cancer). CRC is more common among men than among women. Age-standardized incidence rate of CRC (ASR—age standardized incidence rates) in men is 23.4 per 100,000 population, and in women 16.2, and the mortality rate  is 11.0 and 7.2 per 100,000 population, respectively .About 3% of CRC cases are due to one of two autosomal dominant hereditary diseases: hereditary non-polyposis colorectal cancer (Lynch syndrome) and familial adenomatous polyposis. Approximately 90% of CRC cases are sporadic and occur in patients without genetic predisposition or family history of CRC .The colon is considered to be the organ with an inherently high rate of stem cell division, which leads to high risk of developing cancer throughout the life under the environmental and hereditary factors influence. According to existing concepts, CRC occurs as a result of a progressive accumulation of genetic and epigenetic changes that lead to the normal colon mucosa transformation into adenocarcinoma .MSH6, RNF43, ATM, TGFBR2, BRAF, PTEN, a high level of DNA methylation. In addition, this subtype is characterized by a pronounced infiltration of the tumor microenvironment by immune cells (mainly cytotoxic and activated Th1 lymphocytes), due to neoantigens resulting from hypermutability. CMS2, 3 and 4 subtypes demonstrate high degree of chromosomal instability (CIN) with the loss and/or acquisition of large chromosome regions, loss of heterozygosity and aneuploidy. They are characterized by an initial loss of the tumor suppressor gene APC followed by an activating mutation in KRAS and loss of TP53. Mutation frequency is significantly lower than in CMS1. 37% of CRC cases are CMS2 . It is characterized by activation WNT and MYC signaling pathways, high expression of EGFR and HFN4A, and poor immunogenicity. CMS3  is characterized by dysregulation of a number of metabolic pathways, including glutamine, fatty acid, and lysophospholipid metabolism, and frequent KRAS mutations. In comparison to CMS2 and CMS4, CMS3 demonstrates lower level of somatic copy number alterations (SCNA) and higher microsatellite instability. CMS4  is characterized by the increased expression of genes regulating inflammation, matrix remodeling, stromal invasion, and angiogenesis. CMS4 tumors demonstrate extremely low hypermutation rates, microsatellite stable status, and very high SCNA levels. CMS4 tumors are located in the distal colon, infiltrated by Treg, Th17 and innate immune cells, and demonstrate high levels of cytokines IL-23 and IL-17  and epigenetic  changes, followed by clonal expansion of somatic epithelial cells. In contrast to the sporadic form of CRC characterized by dysplastic changes  developing as discrete small lesions, IBD is characterized by extensive areas of chronically inflamed mucosa are prone to neoplastic transformation. This phenomenon was called “field cancerization”  . IBD, inization” . AccordiAPC, KRAS, TP53, PIK3CA, SMAD4, ARID1A, MYC, etc., but the time and frequency of their occurrence can differ. CAC reflects the “Big Bang” model of evolution, characterized by chronic inflammation causing rapidly, occurring molecular changes leading to neoplasia, rather than the gradual accumulation of lesions as in sporadic forms of CRC. There are no exact data on the position of CAC in the concept of the CMS classification of CRC. It is demonstrated that CAC is not characterized by CMS2 type. In CAC, there is an increase in the number of CD4+ T cells and monocytes, decrease in HNF4 expression and overexpression of OSMR, a tendency to epithelial-mesenchymal transition, which indicates a shift towards CMS4   .However, specific mechanisms for the development of CRC are still unknown, which makes difficult the early diagnostics and treatment of disease. The etiology and pathogenesis study of CRC on clinical material is complicated, since it is often almost impossible to establish the initial stages of the disease, the exact course and the presence of concomitant pathology and the use of drugs change the clinical manifestations of the disease. Studies of biopsy material provide information only about colon mucosa changes from several local colon areas. In its turn, the surgical material is represented only by the most severe cases of the disease and its late stages. The development of new drugs and treatments for CRC requires preclinical trials in experimental models. The current experimental models of CRC in laboratory animals include spontaneous, genetically engineered, transplant and chemically-induced models . Each moThe purpose of this review is to describe existing murine models of CRC with a focus on the models of colitis-associated CRC. This review can help experimental biologists to choose the most optimal colorectal cancer model for their purposes, and it will help oncologists to extrapolate experimental data more correctly.We first searched through PubMed and Google for reviews of CRC models. The query “colorectal cancer model” was used, selecting only reviews from the last 5 years that discussed all types of mouse and rat models. Seven reviews were analyzed . Based oSecondly, we searched PubMed for articles using the AOM/DSS model. The “AOM DSS” query was used.Inclusion criteria. Publication date between 01/2022 and 01/2023, free full text, original studies, AOM/DSS CAC model reproduced.Exclusion criteria. Publications with priority date to January 1st, 2022; ex vivo/in vitro studies; commentaries, summaries, reviews, editorials, and duplicate studies.Overall 91 articles were analyzed. The methodology of reproducing the AOM/DSS CAC model and the histological characteristics of the tumors were evaluated.Spontaneous tumors in the intestine of laboratory mice and rats develop rarely. For example, C57BL/6J mice developed colon cancer at 1% of cases. In colon cancer-prone rats substrain WF-Osaka the incidence of colon carcinoma varied from 30 to 40%. Spontaneous models are rarely used due to unpredictability and low reproducibility .Genetically engineered models of colorectal cancer make it possible to study the genetic predisposition to the development of colorectal cancer, interaction with environmental and modifying factors, tumor microenvironment, as well as systemic immune responses .KRAS, KIT, PIK3CA, MET and EGFR are among the most frequently mutated oncogenes, and TP53, APC, CDKN2A, STK11 and FBXW7 are the most frequently mutated tumor suppressor genes in patients with CRC. In GEMMs mutations in Apc, p53, K-ras genes and DNA mismatch repair genes (MMR) are the most oftenly used, but other genetic mutations are also taken  is an ethylnitrosourea (Enu)-induced mutation in the murine Apc (adenomatous polyposis coli) gene . As the sequence identity of the human and mouse APC proteins exceed 89%, the mouse represents a suitable model to study the involvement of APC truncations in intestinal cancer  gene . The lin of life . APC encl cancer . The Apchenotype . Througharcinoma . Moreovearcinoma .Apc gene, such as ApcMin/850, ApcΔ716, Apc1638N, Apc1638T, ApcΔ468 and ApcΔ474, were developed to allow reproduction of disease models closer to CRC in humans and study the role of certain regions of the APC gene in the development of cancer. Transgenic mice with different mutations of the APC gene differ in the number of tumors and their morphology  or Lynch syndrome. It is caused by autosomal dominant mutations in the DNA mismatch repair genes such as MLH1, MSH2, MSH6, and PMS2, that leads to the development of various cancers, including colorectal cancer. Developed tumors are not specific to the colon-rectum; they occur in other organs such as skin, lungs, lymphatic system, stomach, and small intestine  have regions of hyperplasia in the colon as well as aberrant crypt and ring cells. Associating the K-rasG12D and ApcMin mutations lead to increase in the number of lesions in the colon, as well as the presence of completely undifferentiated cells. Another mutation, K-rasv12, alone is not capable of inducing tumorigenesis, but once it is associated with mutations in Msh2 gene, it promotes a greater number of tumors in the small and in the large intestine, than Msh2-/- animals  Type of transplant. Either tumor cell suspensions or tumor tissue fragments are used. It is easier to work with a cell suspension, cheaper, and the tumor engraftment rate is higher. Cell lines of tumors are phenotypically and genetically quite homogeneous and do not reproduce the heterogeneity of real tumors; in addition, tumor cells due to (2) Source of the graft. Syngeneic tumor transplantation is characterized by the engraftment of tumor tissue or a tumor cell line within the same animal line; xenogeneic transplants are obtained from other animal lines or human donors. Patient-derived xenografts retain the pathological and molecular characteristics of individual patient CRC and are therefore the most reliable for preclinical drug development. However, in xenograft models, tumor cells or tumor fragments are implanted in immunocompromised animals, which makes it impossible to study the role of immune responses in carcinogenesis.e.g., ultrasound) to control cell implantation and animal anesthesia.(3) Site of the transplantation. The implantation of tumor could be various: directly into the colon or rectum (orthotopic models) or subcutaneously, into the renal capsule, spleen, bloodstream (heterotopic models). The most widely used method in case to initiate reproduction, provide access and fast tumor growth is subcutaneous inoculation, nonetheless, the tumor microenvironment is different from the colon and metastasis do not develop. In comparison to heterotopic models the orthotopic model, are technically more difficult to create, requiring the use of imaging techniques  heterocyclic amines (HCAs) such as 2-amino-3-methylimidazoquinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo  pyridine (PhIP);(2) aromatic amines such as 3,2-dimethyl-4-aminobiphenyl (DMAB);(3) alkylnitrosamide compounds such as N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and methylnitrosourea (MNU);(4) 1,2-dimethylhydrazine (DMH), azoxymethane (AOM), methylazoxymethanol (MAM).The introduction of chemical carcinogens to animals is possible with the free access to water and food or through a gastric tube, enema, or intraperitoneal or subcutaneous injection .Heterocyclic aminesHeterocyclic amines are among the most common genotoxic mutagens present in the environment, and the human body is exposed to them in daily life. Heterocyclic amines are formed in food when amino acids and proteins are heated. Throughout frying meat and fish, various imidazoquinoline, imidazoquinoxaline and imidazopyridine compounds are formed, which have a strong mutagenic effect.Among heterocyclic amines, 2-amino-3-methylimidazoquinolone (IQ) and 2-amino-1-methyl-6-phenylimidazopyridine (PhIP) are used to induce CRC in rodents. Rats given long-term IQ or PhIP develop tumors in the small and large intestine, mammary gland, and prostate. However, the incidence of colon tumors is low and ranges from 5% to 28% when these drugs are administered with food for up to 1 year. When adding 100 p.p.m. PhIP in rat diets takes approximately 2 years to reach a 50% incidence of colon cancer, and at 25 p.p.m. PhIP after 2 years no colon carcinomas were observed. It should be stated that the combination of PhIP with a high-fat diet leads to accelerated tumor formation.Thus, the model of CRC induced by heterocyclic amines is applicable for the development of methods for preventing the development of tumors under the action of food carcinogens. The disadvantages of this model are the relatively low incidence of tumor development and too long time of their development .Aromatic aminesSubcutaneous weekly administration of DMAB at 50 mg/kg body weight in male F344 rats provided multiple colon tumors in 27% animals fed low and 75% animals fed high fat diet, respectively . DMAB coOne of the key shortcommings of this model is that it suggests multiple injections of DMAB to induce colon tumors. On a molar basis, DMAB is considered to be less effective than AOM or DMH in rodent models. Another shortcoming of this model is the initiation of neoplasms in other organs: mammary adenocarcinomas in female rats, salivary gland sarcomas, squamous cell carcinomas of the ear canal and skin, squamous cell papillomas of the fundus of the stomach (forestomach), sarcomas and lymphomas, and bladder urothelial carcinomas .Alkyl nitrosamide substancesMethylnitrosourea (MNU) and N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) are direct alkylating agents that do not require metabolic activation and thus are potent local carcinogens.Intrarectal injection of MNU at 1–3 mg per rat weekly for 5 month provided colon tumors in 100% of male F344 rats, of which 43% of the tumors were adenocarcinomas and 57% were adenomas. All neoplasms were detected in rectum and distal colon, where MNU and MNNG were injected.When using alkylnitrosamide substances in rats and mice, most of the induced colon tumors were sessile or polypoid lesions that were well differentiated and often invaded the submucosa. Metastases are usually not detected.Since biochemical activation is not necessary, these carcinogens are perfect for initiation tumors of colon in animals and investigation the changing effects of xenobiotics without establishing the metabolism of the initiating carcinogen. MNU and MNNG administered intrarectally selectively initiate tumors in the rectum and distal colon, such models are widely spread.The main disadvantage of this model is that intrarectal injection is a significant technical problem, the reproducibility of such experiments depends on the skills of the experimenter, and the quantification of carcinogens administered intrarectally is difficult .DMH, AOM, MAMThe most commonly used chemicals to induce CRC are 1,2-dimethylhydrazine (DMH) and its metabolite azoxymethane (AOM). They are potent carcinogens, causing a wide range of mutations in key genes that code for components of multiple intracellular signaling cascades .DMH is oxidized in the liver to azomethane, which is then oxidized to AOM and then hydroxylated to methylazoxymethanol (MAM). MAM enters the intestine with bile or through the bloodstream in the form of glucuronides and glucosides.MAM is cleaved by the effect of colon and liver enzymes to form formaldehyde and a strong alkylating agent,—the methyldiazonium ion. DNA alkylation results in base mismatch and mutagenesis. Thus, guanine is methylated in DNA at the N-7 position. Alkylated guanine combines with thymidine instead of cytosine. In the course of further replication, a nucleotide replacement occurs .DMH is typically administered subcutaneously, but intraperitoneal and intrarectal administration were also stated in the literature. Dosing, number of injections and duration of the experiment vary significantly: 2–200 mg/kg body weight, 1–30 injections, 2–20 weeks . ProtocoAOM is administered, as a rule, once a week intraperitoneally at a dose of 10-15 mg/kg of animal body weight for 2-10 weeks, the development of tumors is assessed after 16-36 weeks from the start of the experiment . AccordiThe introduction of DMH or AOM leads to the development of epithelial neoplasia, which begins with the developing of abnormal crypts in the colon—the so-called aberrant crypt foci (ACF); ACF further progresses to adenoma and then to malignant adenocarcinoma .Different mice strains vary according to the AOM sensitivity. A/J and SWR/J mice are highly sensitive to AOM with high incidence of colon tumors. C57B/L6 and Balb/c mice are moderately sensitive with a relatively lower incidence of colon tumors in comparison to A/J and SWR/J mice, while administration of AOM to AKR/J and 129/SV mice does not induce colon tumors .The disadvantages of this model are the long tumor induction period, not very high reproducibility potential, and the need for multiple injections of carcinogen.As was mentioned above, in humans, the development of CRC is often associated with IBD. Therefore, This model proved to be very convenient, relatively inexpensive, and highly reproducible, and is widely used in studies of colitis-associated carcinogenesis. Investigations made on this model reflected the significance of the inflammation process in CRC development and revealed some of the mechanisms of inflammation-related colon carcinogenesis in the gut, with an emphasis on the function of pro- and anti-inflammatory cytokines . This moHistologically, dysplasia in human IBD often looks like dysplasia in sporadic adenomas, resembling tubular, tubulovillous or villous adenomas. Although a clear distinction cannot be relied upon, dysplastic cells in sporadic adenomas often occupy the upper portion of neoplastic crypts  whereas with colitis, the dysplastic cells tend to occupy the entire height of the crypts . In geneAs with AOM, different mice strains demonstrate different sensitivity to AOM/DSS. Sometimes DMH is used instead of AOM, and instead of DSS, another pro-inflammatory agent is 2,4,6-trinitrobenzenesulfonic acid (TNBS). Sometimes DMN is used instead of AOM, and instead of DSS, another pro-inflammatory agent is TNBS . Thus, tDespite the widespread use of AOM/DSS-induced CAC, the model has not been standardized. We analyzed AOM/DSS CAC induction techniques in 91 free access original studies published in the public domain between 01/2022 and 01/2023 in the PubMed database .The predominant majority of studies (90 out of 91 articles) were performed on mice, mainly C57BL/6, C57BL/6J, C57BL/6N (70% of works) and Balb/c (19% of works). In 52% of cases, the study was carried out on male animals. CRC was induced by 1-5 injections of AOM at a total dose of 7.4-60 mg/kg of animal body weight and 1-4 cycles of consumption of 1–5% DSS solution for 2-8 days with breaks of 7-30 days. AOM was administered, as a rule, intraperitoneally (89 out of 91 articles). In 91% of cases there was only one AOM injection on day 1 of the experiment. The most commonly used AOM doses are 10 mg/kg (57% of articles) and 12.5 mg/kg (19% of articles). Mostly DSS is added to drinkers for three cycles (80% of articles), lasting 7 (70%) or 5 (21%) days at a concentration of 2% (42% of articles) or 2.5% (28% of articles). In 71% of articles, the breaks between DSS cycles were 14 days. The first cycle of DSS starts on days 1-8 of the experiment, in 51% of articles—on day 8. The duration of the experiment ranges from 6 to 25 weeks, most often 10 (21%), 11 (13%) or 12 (19%) weeks. Unfortunately, in some articles the molecular weight of the DSS used is not indicated, but most of the authors use DSS with a molecular weight of 36–50 kDa.According to this data, the average scheme should look like stated below. Adult C57BL/6 mice on the 0th day of the experiment are injected intraperitoneally with AOM at a dose of 10 mg/kg of animal body weight, then 7 days leave them without exposure. Starting from the 8th, 29th and 50th days of the experiment, the water in the drinkers is replaced with 2% DSS for 1 week. Animals are withdrawn from the experiment at day 84 .Such experimental scheme is presented in four analyzed studies . In thesIn general, in most of the works analyzed, there is no pathomorphological characterization of tumors induced by AOM/DSS. The observed changes are most often characterized as low-grade and high-grade dysplasia, adenomas or adenomatous polyposis. The term “adenocarcinoma” was used to describe AOM/DSS-induced tumors in only 20 out of 91 analyzed papers. The development of adenocarcinomas was noted at 8-25 weeks of the experiment (most often 12 weeks) with an injection of 10-30 mg/kg AOM (most often 12.5 mg/kg) and 1-4 cycles of consumption of 1.5–5% DSS during 2–7 days (most often 3 cycles of 2.5% DSS for 7 days). However, the incidence of adenocarcinomas was assessed only in two studies. According to Min CAC mice develop fewer tumors when housed in germ-free conditions  can be used to reliably induce colorectal carcinogenesis measured as preneoplastic lesions in both B6 and A/J mouse strains while limiting severe symptoms. This study emphasizes the importance of adjusting the treatment regimen according to mice strain and study aims in future investigations employing the AOM/DSS model (Another shortcoming of this model should be noted. There are important differences in invasive and metastatic potentials of CRC between animal models and human disease. At diagnosis, at about 50% of CRC patients have lymphatic metastases and 33% have hematogenous metastases . HoweverSS model . TherefoThere are different types of CRC, differing in molecular mechanisms of occurrence and features of the course. Therefore, it is impossible to create one single model that reflects all the features of CRC. Development of spontaneous, genetically engineered, transplantation and chemically induced models allows to reproduce and study different types of CRC and certain aspects of CRC pathogenesis. Inflammatory bowel disease is an important risk factor for CRC. The AOM/DSS-induced CAC model is used to study carcinogenesis associated with chronic inflammation of the large intestine. However, this model is not standardized: according to different studies, the doses and exposure to AOM and DSS vary significantly, which makes it difficult to analyze and compare the obtained experimental data.


The training set was used for a sequential replacement regression and a simultaneous 10-fold cross-validation. The resulting model was improved by including a cut-off of 20 years, dividing the younger individuals with non-linear from the older individuals with linear dependence between age and methylation status. Sex-specific models were developed and improved prediction accuracy in females but not in males, which might be explained by a small sample set. We finally established a non-linear, unisex model combining the markers EDARADD, KLF14, ELOVL2, FHL2, C1orf132, and TRIM59. While age- and sex-adjustments did not generally improve the performance of our model, we discuss how other models and large cohorts might benefit from such adjustments. Our model showed a cross-validated MAD and RMSE of 4.680 and 6.436 years in the training set and of 4.695 and 6.602 years in the validation set, respectively. We briefly explain how to apply the model for age prediction.DNA methylation patterns change during human lifetime; thus, they can be used to estimate an individual’s age. It is known, however, that correlation between DNA methylation and aging might not be linear and that the sex might influence the methylation status. In this study, we conducted a comparative evaluation of linear and several non-linear regressions, as well as sex-specific versus unisex models. Buccal swab samples from 230 donors aged 1 to 88 years were analyzed using a minisequencing multiplex array. Samples were divided into a training set (The online version contains supplementary material available at 10.1007/s00414-023-02967-6. If the source of a biological trace cannot be identified by conventional DNA comparison, forensic DNA phenotyping (FDP) of biological traces might provide further investigative leads. Information on phenotypical aspects of the donor of a trace, such as skin, eye and hair color, height, or even male baldness patterns –3, mightBeside analyzing DNA traces, further potential fields of applying molecular age estimation comprise the identification of unknown bodies , 7 and tRecent studies revealed high estimation accuracy of age estimation models for blood with a MAD ranging from 3.16 to 10.33 years –13. BestSeveral recent studies focused on methylation patterns of young individuals , 21. WhiThe aim of this study was to develop an age estimation model and analyze whether or not considering varying methylation change rates in young versus older individuals improves the prediction model. Human oral mucosa samples were analyzed by minisequencing multiplex PCR. The eight markers used in the study  have been previously reported separately to show a correlation with age , 24, 25.Oral mucosa samples from 230 donors  aged 1 to 88 years (mean 38 years) were collected using sterile swabs. The Ethics Committee of the Hamburg Medical Association (Ethikkommission bei der Bundesärztekammer) approved the study protocol (PV6098) and all participants or their legal representatives provided written informed consent. DNA was extracted using the Casework Extraction Kit and Maxwell 16 (Promega) following manufacturer’s recommendations. DNA was quantified using the PowerQuant System (Promega) following manufacturer’s recommendations. Purified DNA samples were stored at 6 °C until further use.DNA samples were bisulfite converted and purified following the instructions of the EpiTect Fast DNA Bisulfite Kit (Qiagen) for high concentration samples. Depending on the determined concentration of each sample, up to 400 ng DNA was used for the treatment. Carrier RNA was not added to Buffer BL. Unmethylated cytosines were converted to uracils by bisulfite treatment, whereas methylated cytosines remained unconverted. To prove a successful bisulfite conversion, a second PowerQuant reaction was performed. The PCR primers should not bind to the converted DNA, meaning that a negative result would prove a complete conversion . BisulfiThe bisulfite converted DNA was amplified by PCR using the PyroMark PCR Kit (Qiagen). Each sample was set in three reactions with primers for eight different markers . Size standard 120 LIZ (Applied Biosystems); diluted 1:100 in HiDi formamide (Applied Biosystems) was used and results were evaluated using Gene Mapper ID (v3.2). The proportion of methylated cytosines of the samples was determined by calculating the relative peak heights for adenine and guanine or thymine and cytosine, respectively.r) and corresponding p values.Correlations between chronological age and methylation status of each CpG site was assessed calculating Pearson correlation coefficient  and the root-mean-square error (RMSE) were computed on the training set  as well as on the validation set. Statistical analyses were performed using R (version R-4.1.2) including the packages ggplot2 [gridExtra [Data was split into a training and validation set, comprising 161 and 69 samples, respectively. Model accuracy was tested for both, training and validation set using the coefficient of determination  ggplot2  and gridridExtra  for the To make it easier to follow, a detailed description of model development and validation regarding non-linear dependences and influence of the sex can be found in the “R2 , and corresponding p values (Supplementary Table r = 0.86). In this study, SST, ELOVL2, and TRIM59 revealed the strongest correlations with age, matching the results of previous studies [R2 values in our study, it had to be removed from further analysis due to missing values from two thirds of all samples.Examples of the multiplex results are shown in Supplementary Figure R2 Fig. , Pearson studies , 18, 26. studies . EDARADD studies , 19, SST studies . AlthougDue to missing values in the data set of SST, this marker was excluded. The rest of the markers showed moderate to strong correlations with chronological age; therefore, the seven CpG sites of the markers PDE4C, EDARADD, KLF14, ELOVL2, FHL2, C1orf132, and TRIM59 of 161 individuals (training set) were included in the regression analysis.yc and epigenetic age ye and improve the performance of subsequent regression analyses [It is well known that the relationship between methylation and chronological age is not necessarily linear , 29. Ouranalyses :\documenyc, adult = 20 on the right hand side of Fig. yc, adult lead to even steeper curves at the origin. For a fixed value of yc, adult, one may then conduct a regression analysis to estimate regression parameters β for the linear model:This transformation is also displayed for x1, …, xk denote methylation values from k different CpG sites. The estimated chronological age can be identified afterwards by application of the inverse of f for a fixed value of yc, adult, where where yc, adult was set to 20. Since a study on further choices of this value has not been described in the original work by Horvath, we investigated whether the choice of this cut-off value can be optimized. That is, we regard the cut-off yc, adult from Horvath’s transformation as a hyperparameter whose value shall be determined via a repeated 10-fold cross-validation on the training data. We repeated this cross-validation 100 times to exclude a dependence of the results from the choice of the folds.Upon first-time application of this transformation , the valyc, adult for women and men. The results of the cross-validation procedure on the training data set can be found in Fig. yc, adult = 20 is a good choice for a unisex model and a separate model for men, the RMSE for a separate model for women can be improved by choosing a larger cut-off value. This suggests that there are differences in the epigenetic aging pattern between men and women [Furthermore, we investigated potential sex-specific differences in epigenetic development. As sex-specific influences on age estimation models were discussed in previous studies , 19, 30,nd women  which giFinally, we fitted the different models on the training data resp. its sex-specific subsets and computed the RMSE on the validation set. While the observations from the cross-validation could be replicated for women, this was not possible for the unisex model (where the optimized model performed worse than the default choice) and men (where both transformations performed worse than the standard linear model). However, this may be due to the small sample sizes, especially for men with only 29 individuals in the validation set.As shown in Fig. As we are dealing with quite small sample sizes, the sex-specific models have a clear disadvantage in that they can only be fitted with half of the observations. This disadvantage should diminish with an increasing overall sample size . In thisr = 0.942) and a MAD and RMSE of 4.680 and 6.436 years, respectively. Within the training set, the seven CpG site model could explain 88.8% of the age variance .Age estimation of the training set revealed a strong correlation with age . Firstly, the linear prediction according to our estimation of the epigenetic age In the present situation, it appears most appropriate to apply the unisex model with the default cut-off value of If this value is positive, the chronological age can be transformed back to the chronological scale with the linear transformation:Otherwise , one obtains the estimated chronological age via:Prediction intervals for the chronological age can be computed from the same backtransformation procedure after computing prediction intervals on the epigenetic scale with the model’s residual standard error of 0.2905. One should note that the shape of the backtransformation function leads to shorter prediction intervals for subjects that are estimated to be young.R2 = 0.878, adj. R2 = 0.864). The age predictions of the resulting model have a strong correlation between chronological and predicted age (r = 0.937) with a MAD of 4.695 years and a RMSE of 6.602 years , cg09809672 (−12 bp), cg14361627, cg16867657 (−16 bp), cg06639320, cg10501210 (+6 bp), and cg07553761) in EDARADD, KLF14, ELOVL2, in FHL2, in C1orf132, and TRIM59 were included in this study. Validation of the final model revealed a cross-validated MAD and RMSE of 4.680 and 6.436 years in the training set and 4.695 and 6.602 years in the validation set, respectively, making this model likely to be useful in forensic investigations in the future. Regarding RMSE, sex-specific models did not outperform the unisex models in our limited data set. In larger sample sets, however, sex-specific modeling might increase prediction accuracy. DNA methylation analysis by minisequencing has the potential to become a tool in criminal investigation. Compared to massively parallel sequencing approaches, minisequencing has the benefit of being more flexible, less time consuming when analyzing small sample numbers, and easy to implement into forensic laboratories without the need for specified sequencing equipment.ESM 1(PDF 209 kb)ESM 2(XLSX 11 kb)ESM 3(XLSX 41 kb)