J. Dairy Sci. 106:9150–9163 https://doi.org/10.3168/jds.2022-22811 © 2023, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). A short-term comparison of wheat straw and poplar wood chips used as litter in tiestalls on hygiene, milk, and behavior of lactating dairy cows M. Simoni,1* N. Mezzasalma,1 R. Pitino,1* C. L. Manuelian,2,3 C. Spadini,1 M. Iannarelli,1 S. L. Montanaro,1 A. Summer,1 C. S. Cabassi,1 M. De Marchi,2 and F. Righi1 1 Department of Veterinary Science, University of Parma, 43126 Parma, Italy 2 Department of Agronomy, Food, Natural resources, Animals and Environment (DAFNAE), University of Padova, 35020 Legnaro (PD), Italy 3 Group of Ruminant Research (G2R), Department of Animal and Food Sciences, Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Spain ABSTRACT (including discomfort signs), and the lowest dry matter intake. However, despite this apparent reduction in cow A short-term study was conducted to compare the comfort, no biologically important differences were ob- effect of using poplar wood chips (PWC) instead of served in this short-term study between cows on PWC wheat straw (WS) litter in dairy cows. A total of 38 and WS in milk production or hygiene. lactating Holstein cows (204 ± 119 days in milk, 26.9 ± Key words: milk composition, total bacterial count, 6.5 kg of milk yield [MY]) were housed in a tiestall farm bedding materials, organic for a 10-d trial including 5 d of adaptation followed by 5 sampling days (from d 5 to 10). Cows were divided INTRODUCTION into 2 homogeneous groups: one group was bedded with WS, and the second with PWC. Both litter materials The organic production system is looking for new were provided in the amount of 7 kg/stall per d. Each alternative litter materials that can fulfill the organic group was composed of 3 subgroups of 6 or 7 cows; the husbandry production rules and phase-out the use of subgroups were physically separated along the feeding conventionally-produced wheat straw (WS) litter that line by wooden boards. During the sampling days, fe- is, currently, still accepted in organic production (Eu- cal composition, used litter composition, and bacterial ropean Union, 2018). However, bedding materials and count (Clostridium spp., Salmonella spp., Escherichia resting areas have a huge effect on dairy cows’ welfare coli, Lactobacillus, and total bacterial count) were ana- and productivity (Bewley et al., 2017; Mondaca, 2019; lyzed by subgroup twice a day. On d 1 and from d 5 to Singh et al., 2020). It is, thus, fundamental to test the 10, udder hygiene score and cow cleanliness score were adequacy of every new litter product with the potential also evaluated individually twice a day. Meanwhile MY, to be certified as organic before it is proposed to farm- milk hygiene (total bacterial count [TBC], coliform ers as an alternative to WS. bacterial count [CBC], and spore-forming units [SFU]) When assessing a potential new litter material, the and quality were measured and analyzed from 9 ani- sustainability of its production process, its availabil- mals per group. Moreover, individual animal behavior ity, and its local cost (Kour, 2017; Singh et al., 2020) (body position and behavioral traits) and subgroup dry should be considered, along with how it is going to matter intake were measured on d 9 and 10. Fecal dry be used (e.g., resting area characteristics and type). matter did not differ between groups, PWC had the These aspects become even more important within the lowest used litter moisture and N content favoring the context of organic production. The use of by-products highest clean cow frequency, but also gave rise to the as bedding material on dairy farms—organic or not—is greatest used litter microbial contamination. The MY, a good option, as they are generally inexpensive and milk quality, TBC, SFU, and CBC were similar. The can contribute to reduction of industry waste (Kuipers lying behavior frequency was similar between groups. et al., 2022). Indeed, WS is commonly used as litter However, the PWC group showed the lowest sleeping material in southern Europe because it is a by-product frequency, the highest frequency of other behaviors of wheat production. In contrast, in regions with lim- ited grain harvest (e.g., in parts of northern Europe), wood chips are more commonly used (Johanssen et al., Received September 23, 2022. 2018), as they are either available as a by-product from Accepted May 21, 2023. the wood industry or obtained from dedicated crops *Corresponding authors: marica.simoni@​unipr​.it and rosario. pitino@​unipr​.it (Kunttu et al., 2020). 9150 Simoni et al.: POPLAR WOOD CHIPS AS DAIRY CATTLE LITTER 9151 If properly managed, the ideal litter should provide creto legislativo no. 26/2014) and Directive 2010/63/ a comfortable resting area and optimize general health EU on the protection of animals used for scientific pur- status, particularly hoof, hock and udder health and poses (European Parliament and Council. 2010). hygiene, while simultaneously supporting high milk production and quality (Singh et al., 2020; Alanis et al., Animals and Housing 2021; Li et al., 2021a). Used litter is the main source of pathogens responsible for environmental mastitis and The study was conducted in July 2021 in a tiestall a key source of bacteria and spores that contaminate farm located in the Parmigiano-Reggiano cheese pro- milk and lead to high bacterial counts in bulk milk duction area (Po Valley, northern Italy: Lat. 44.693080; (Hogan et al., 1989; Godden et al., 2008; Patel et al., Lon. 10.367579). The dairy barn included 2 rows of 19 2019; Cheng and Han, 2020; Alanis et al., 2021). This face-to-face stalls separated by a 5.5-m drive-through bacterial contamination and high microbiological load feed alley. Each tiestall had a smooth concrete floor can occur because of poor physical and biochemical with a 2% slope toward the rear end, covered by a layer characteristics of the litter material. of bedding material, which was cleaned and partially Among alternative bedding materials, wood products renewed daily. A channel fitted with flap scrapers for have been reported to be widely used in organic farms manure evacuation was also present. A 50-cm-wide (Smith et al., 2017) and specifically in organic ties- concrete feed bunk was located in the front end, 10 tall farms (Andrews et al., 2021) in the United States. cm over the floor level. The feed bunk was separated Among wood products, few studies have tested the from the floor of the stall by a 12-cm-high border. An effects of wood chips as a bedding material for dairy automatic drinker was placed at every other stall. The cattle. Johanssen et al. (2018) evaluated the effects of individual stalls were not separated. A total of 38 lac- wood chip bedding material from broadleaf trees on tating Holstein dairy cows (630 ± 102 kg BW; 3.03 heifers, and Ferraz et al. (2020) studied their effects on ± 0.43 BCS; 204 ± 119 DIM; 26.9 ± 3.5 kg/d MY), lactating cows. However, Ferraz et al. (2020) did not originally housed on long wheat straw, were allocated report the plant from which wood chips were produced. into 2 homogeneous (for productivity and parity) lit- It should be considered that the plant of origin and ter groups corresponding to the 2 separated stall rows, its structural anatomy may affect the physico-chemical namely WS and PWC. The WS was self-produced (Munir et al., 2019) and antibacterial properties (Re- and harvested as a wheat by-product by the farmer in neau et al., 2002) of the bedding material. Additionally, the summer of 2020 and stocked in the barn as round the available litter studies often focused on a specific bales. The WS was long WS (>50 cm). The PWC was aspect (e.g., behavior, milk contamination, or bedding produced and provided by the Leibniz Institute for characteristics) and consequently did not provide a Agricultural Engineering and Bioeconomy (ATB; Pots- general overview of the effects of the litter. Among suit- dam, Germany). The geometric mean particle size of able wood bedding materials, untreated poplar wood the PWC was 1.44 cm (ranging from <0.1 to 4.5 mm). chips (PWC)—a mixture of bark, sawdust, and post- The PWC was packed into waterproof plastic big-bags peelings—have the potential to be a useful litter mate- and transported to the farm in January 2021, where it rial based on the criteria proposed by Niraula et al. was stored in the barn together with the straw until (2018). It is, in fact, characterized by good availability, the beginning of the trial. The commercial values of easy handling and storage, cost-effectiveness, absence the products at the time, and in the area where the of bacteria or unhealthy and toxic products, high DM trial was performed, were approximately 75 and 50 €/t level, modular particle size, and good cleanliness. for WS and PWC, respectively. The trial lasted 10 d In this context, our short-term study aimed to com- and included an adaptation period of 5 d followed by pare the use of PWC as an alternative litter material a sampling period of 5 d. Each litter group was further to conventional WS in terms of used litter composition divided into 3 subgroups of 6 or 7 animals. The feeding and microbial load, cow milk hygiene, milk quality and line was divided through wooden boards into 3 sec- yield (MY), and animal behavior. tions per group, corresponding to the subgroups, for an accurate measurement of the related feed intake. No MATERIALS AND METHODS separation was created in the bedding area between the subgroups, to avoid effects on the behavioral traits. Due This study was approved by the Ethical Committee to difficulties with on-field individual measurements for the Care and Use of Experimental Animals of the for some traits and sampling procedures, the subgroup University of Parma, Italy (PROT. N.16/CESA /2021) instead of the individual animal was considered as the and was conducted in accordance with Italian law (De- experimental unit. The dirty and wet used litter was Journal of Dairy Science Vol. 106 No. 12, 2023 Simoni et al.: POPLAR WOOD CHIPS AS DAIRY CATTLE LITTER 9152 Table 1. Chemical composition of the wheat straw (WS) and poplar a pH meter Checker (HANNA instruments), and an wood chips (PWC) tested as litter materials aliquot was used to test microbiological contamination. Item PWC WS The analytical results of the litter and feed, expressed 1 Chemical composition, % DM on a DM basis, are reported in Table 1 and Table 2 DM, % as fed 99.8 90.7 respectively. Animals were sequentially fed grass hay, CP 2.01 3.58 alfalfa hay, and mixed hay twice a day (starting at 0700 Ether extract 0.34 0.72 and 1900 h). The concentrate was provided separately aNDFom 87.3 75.7 CP-NDF 0.97 1.24 from the forage every 3 h by automatic feeder. The esti- ADFom 71.3 47.0 mated chemical composition of the whole diet is shown Lignin 18.5 6.9 in Table 3 and was calculated using the software NDS Ash 8.7 11.8 pH 6.3 6.5 professional (version 3.9.10.01, Rumen S.a.s., Reggio LTBC,2 log10 (cfu × 103)/g 2.1 1.8 Emilia, Italy, based on the CNCPS model, version 6.55, Escherichia coli, log10 (cfu + 1)/g ND3 ND Cornell University, Ithaca, NY), based on the average Lactobacillus spp., log10 (cfu + 1)/g ND ND 1 amount of each ingredient supplied daily to the cows. aNDFom = amylase-treated NDF expressed as exclusive of residual ash; CP-NDF = CP bound to NDF; ADFom = ADF expressed as exclusive of residual ash. Measurement and Sampling 2 LTBC = litter or used litter total bacterial count. 3 ND = not detected. On the first day of the experiment (d 1) and ev- ery day during the 5 d of sampling (d 6–d 10), fecal removed daily from the back of the stall and placed in (FS), overall cleanliness (CS), and udder hygiene (US) the rear channel for automatic removal. Each stall was scores of each cow were evaluated before each milking provided with a constant amount of 7 kg/d of the cor- (0600 and 1800 h). The FS was evaluated as described responding clean litter material, whose volumes were by Hutjens (2010), with a 5-point scoring system where 5.43 and 22.72 dm3/kg for PWC and WS, respectively. 1 indicated a pea soup-like consistency and 5 indicating Before the beginning of the trial, both litter materi- fecal balls. The CS and US were evaluated according to als and feed ingredients were sampled and chemically Reneau et al. (2002) and Schreiner and Ruegg (2003) analyzed. The chemical composition analysis panel fol- also using a 5-point scoring system, where 1 signified a lowed the analyses described by Simoni et al. (2021a) very clean and 5 a very dirty body or udder. and Manuelian et al. (2021). It included DM, ash, ether At the same time as the recording of FS, CS, and extracts (EE), aNDFom, ADFom and lignin, CP, CP US, 1 kg of feces and 500 g of used litter were col- bound to NDF (CP-NDF) and ADF (CP-ADF), and lected from each stall per cow and pooled by subgroup. starch. The CP, CP-NDF, and CP-ADF were calcu- Feces were collected directly from the rectum of each lated as N × 6.25, and starch was determined only on cow, mixed thoroughly, stored at room temperature, feedstuffs. The pH of the litter was determined using and transported to the University of Parma. Used lit- Table 2. Chemical composition (mean ± SD) of the feeds supplied to the dairy cows Item1 Grass hay Alfalfa hay Mixed hay Feedstuff2 DM, % as fed 91.88 ± 3.26 86.26 ± 0.54 87.90 ± 4.45 92.67 ± 0.33 % DM CP 6.91 ± 0.09 17.54 ± 0.05 15.07 ± 5.81 16.85 ± 0.73 EE 0.95 ± 0.03 1.76 ± 0.15 1.38 ± 0.29 1.80 ± 0.63 aNDFom 67.74 ± 3.93 43.66 ± 5.26 59.87 ± 7.54 33.56 ± 0.86 CP-NDF 3.72 ± 0.21 6.32 ± 0.73 6.61 ± 1.94 9.93 ± 0.00 ADFom 43.61 ± 4.50 30.55 ± 2.84 39.58 ± 5.10 13.75 ± 0.74 CP-ADF 2.75 ± 0.17 3.64 ± 0.61 3.70 ± 0.75 6.31 ± 0.00 Lignin 7.61 ± 2.82 6.48 ± 0.39 8.97 ± 0.45 1.74 ± 0.43 Ash 8.06 ± 0.17 13.50 ± 1.54 9.96 ± 1.43 6.24 ± 0.19 Starch 28.79 ± 0.15 Crude fiber 12.63 ± 1.11 1 aNDFom = amylase-treated NDF expressed exclusive of residual ash; CP-NDF = CP bound to NDF; ADFom = ADF expressed as exclusive of residual ash; CP-ADF = CP bound to ADF. 2 Feedstuff: beet pulp 43%, corn meal 28%, soybean meal 11%, sorghum meal 10%, soybean hulls 3%, barley malt 1%, calcium carbonate and sulfate 0.8%, wheat middlings 0.8%, sodium bicarbonate and chloride 0.8%, dicalcium and monocalcium phosphate 0.7%, cane molasses 0.4%, magnesium sulfate, oxide, and carbonate 0.4%, vitamin 0.1%. Journal of Dairy Science Vol. 106 No. 12, 2023 Simoni et al.: POPLAR WOOD CHIPS AS DAIRY CATTLE LITTER 9153 Table 3. Proportion of feeds delivered to the animals and estimated Nitrogen was determined following the Dumas method chemical composition of the whole diet (AOAC 968.03; AOAC International, 2005) by combus- Item1 Amount tion digestion (Dumatherm, Gerhardt GmbH and Co., Ingredient proportion, % DM Königswinter, Germany), and CP was calculated as Grass hay 16 percentage of N × 6.25. The aNDFom was analyzed Alfalfa hay 30 according to Van Soest et al. (1991) and Mertens et Mixed hay 20 al. (2002) with the use of α amylase, but without the Feedstuff 34 Estimated chemical composition, % DM use of sodium sulfite, and corrected for ash. A semi- DM, % as fed 89.5 automated system was used for the boiling and filtering CP 15.1 phase (FIWE Raw Fiber Extractor, VELP Scientifica, Ether extract 1.6 aNDFom 47.3 Usmate Velate, Italy). ADFom 28.7 Used Litter Bacterial Count. Aliquots of 25 g Lignin 5.5 each, of fresh PWC and WS and each used litter sample, Ash 9.5 Starch 10.0 were withdrawn and stored at −20°C. For the analysis, Sugar 6.0 each aliquot was thawed and suspended in 225-mL ster- 1 Feedstuff: beet pulp 43%, corn meal 28%, soybean meal 11%, sorghum ile saline solution (NaCl 0.9%) in a stomacher bag and meal 10%, soybean hulls 3%, barley malt 1%, calcium carbonate and homogenized with a stomacher (Laboratory Blender sulfate 0.8%, wheat middlings 0.8%, sodium bicarbonate and chlo- ride 0.8%, dicalcium and monocalcium phosphate 0.7%, cane molasses 400, Seward Medical, London, UK) for 60 s at medium 0.4%, magnesium sulfate, oxide and carbonate 0.4%, vitamin 0.1%. speed (230 rpm). aNDFom = amylase-treated NDF, expressed exclusive of residual ash; From this 1/10 suspension, 1 mL was withdrawn and ADFom = ADF, expressed exclusive of residual ash. resuspended in 49-mL sterile saline in a sterile tube to achieve a 1/50 dilution. This was thereafter further ter samples were obtained by pooling 3 similar aliquots diluted to reach a final dilution of 1/25,000. From this collected with a scoop in the front, middle, and rear final dilution, 20 µL was plated onto MacConkey agar, areas of each stall. A different scoop was used for each Columbia blood agar and de Man, Rogosa, and Sharpe subgroup. The used litter samples were further divided (MRS) agar plates (Oxoid, Basingstoke, UK). All 3 into 2 composite samples, labeled, and stored at room plates were incubated for 24 h at 37°C in an aerobic temperature to investigate chemical composition, or at environment, except MRS agar, which was incubated −20°C to be transported to the microbiology labora- under microaerophilic conditions. For each sample, tory for bacterial culture determination. an additional blood agar plate was incubated for 48 Nine cows per group were chosen so that the mean h at 37°C under anaerobic conditions to evaluate the and standard deviation of MY per milking at the begin- growth of Clostridium spp. To evaluate the presence ning of the trial were similar and representative of the of Salmonella spp., 50 μL of the final dilution were entire herd (PWC = 13.6 ± 2.3 kg/milking per cow; inoculated in 5 mL of peptone water and incubated WS = 13.3 ± 1.9 kg/milking per cow) and allocated 3 aerobically for 24 h at 37°C. Subsequently, 1 mL of pep- per subgroup. On the first day of the experiment (d 1) tone water was transferred into Rappaport-Vassiliadis and every day during the 5 d of sampling (d 6–10), MY broth (BD Difco, Franklin Lakes, NJ) and incubated was measured on the selected cows twice daily using aerobically for 48 h at 42°C. Thereafter, an aliquot a 42-kg milk meter (Waikato MKV, Waikato Milking of 20 μL was plated onto a MacConkey agar plate Systems, Hamilton, New Zealand), and 2 aliquots of and incubated for 24 h at 37°C in aerobic conditions. 150 mL were sampled per cow and kept at 4°C until Lactose nonfermenting colonies were tested through a analysis. In the first milk aliquot, 0.3 mL/dL Bronopol micro-agglutination test with anti-Salmonella serum were added as a preservative for chemical composition (Salmonella O Poly-Gp A-S; Thermo Fisher Scientific, determination. The second aliquot was used to deter- Waltham, MA). Then, the positive lactose nonferment- mine total bacterial count (TBC), coliform bacterial ing colonies were identified with API 20E biochemical count (CBC), and spore-forming units (SFU). test systems (bioMérieux, France) and conventional biochemical tests (Quinn, 1994). Escherichia coli and Feces and Used Litter Analysis Lactobacillus spp. were identified through the morphol- ogy of bacterial colonies. Colony-forming unit counts Chemical Analysis. The DM content of feces and were performed directly on MacConkey agar plates used litter samples was determined by drying the sam- for E. coli and on MRS agar plates for Lactobacillus ples at 103°C overnight (European Commission, 2009). spp., and E. coli hemolytic activity was assessed on Ash content was obtained by ignition at 550°C for 4 h. Columbia blood agar. The presence of Clostridium spp. Journal of Dairy Science Vol. 106 No. 12, 2023 Simoni et al.: POPLAR WOOD CHIPS AS DAIRY CATTLE LITTER 9154 Table 4. Descriptions of body positions and general behaviors recorded during direct observations Observed variable Definition1 Body position Lying Without support of any leg and with the belly in contact with the floor Standing Standing with all 4 feet on the ground General behavior Ruminating Regurgitating or chewing on bolus that had been regurgitated Eating Head down close to forages or feedstuff, taking bites or chewing without regurgitating Drinking Cow engaged in water ingestion Sleeping Lying down on the side or on the belly, not ruminating, head up or turned to the side or stretched forward, eyes closed or half-closed Other All other behaviors not listed above, including grooming, licking itself or other animals, pushing other cows, observing, social interactions, vocalizing, stepping or kicking, urinating and defecating 1 The classification was based on the description reported by Pavlenko et al. (2011), with minor modifications. was evaluated through microscopic examination of the Calculations suspected colonies grown in an anaerobic environment and stained with Gram staining. The sample was con- Energy-corrected milk (3.140 MJ/kg) was calculated sidered positive for Clostridium spp. when oval, bulg- as described by Sjaunja et al. (1990) using the following ing spores located in terminal or subterminal positions equation: were detected. For each sample, 50 µL from the 1/10 suspensions ECM yield (kg) = MY (kg) were withdrawn and resuspended in 50 mL of sterile × [(38.3 × fat g/kg + 24.2 × protein g/kg saline in a sterile tube to determine the litter or used litter total bacterial count (LTBC). This step was re- + 15.7 × lactose g/kg + 20.7)/3,140]. peated until it reached a dilution of 10−3 for clean litter material and 10−7 for used litter material. One millili- Behavior and Feed Intake ter was withdrawn and plated onto a sterile Petri dish filled with plate count agar medium (PCA, BD Difco) Individual cow behavior was monitored on d 9 and and incubated for 24 h at 37°C. After incubation, the 10 (the last 2 sampling days) by 4 groups of paired colony-forming units were counted. trained operators (switched every 6 h) for each litter group. Every 15 min, for a time span of 48 h, the opera- tors recorded the frequency of standing or lying and Milk Quality and Hygiene behavioral traits, including ruminating, eating, drink- Milk composition was analyzed with Milkoscan FT7 ing, sleeping, or other (grooming, licking itself or other (Foss Electric A/S, Hillerød, Denmark) to determine animals, pushing other cows, observing, social interac- fat, protein, casein, and lactose percentages according tions, vocalizing, stepping or kicking, urinating, or def- to ISO 21543:2020 (ISO, 2020). Cheese coagulation ecating). The definitions adopted to describe the body properties were analyzed by FT6000 (Foss Electric positions and behaviors are those given by Pavlenko et A/S, Hillerød, Denmark; rennet coagulation time al. (2011) with minor modifications (Table 4) [RCT], curd firmness 30 min after rennet addition to On d 9 and 10, the amount of forage and concen- milk [A30], curd firming time [K20]; De Marchi et al., trate fed to the cows, as well as the feed refusals, were 2013) in the laboratory of the Breeders Association of weighed by subgroup on a daily basis and multiplied by Veneto Region (Padova, Italy). Somatic cell count was their DM content to calculate the DMI. The latter was determined using a Fossomatic (Foss Electric A/S, Hill- calculated as the difference between hay and concen- erød, Denmark) according to ISO 13366-2:2006 (ISO, trate supplied (kg of DM) and the refusals (which were 2006). also analyzed for DM content), divided by the number The TBC was determined through flow cytometry of cows of the subgroup. with BactoScan FC (Foss Electric A/S). The CBC was determined on polystyrene Petri dishes with VR-BA Statistical Analysis Agar ground after incubation at 37°C for 24 h (ISO 11866-1/IDF 170-1; ISO, 2005). The SFU was deter- The data were analyzed using the software SPSS v28 mined as the most probable number using the Weinzirl (IBM SPSS Statistics for Windows, Version 28.0; IBM method modified by Annibaldi (1969) Corp., Armonk, NY). Normality of continuous variables Journal of Dairy Science Vol. 106 No. 12, 2023 Simoni et al.: POPLAR WOOD CHIPS AS DAIRY CATTLE LITTER 9155 Table 5. Mean chemical composition of feces and litter from the dairy cows of the 2 litter groups (PWC = poplar wood chips, n = 30; WS = wheat straw, n = 30) Item1 PWC WS SEM P-value Feces Moisture, % 86.0 85.2 0.191 0.086 CP, % DM 15.1 15.0 0.121 0.869 aNDFom, % DM 62.9 61.0 1.128 0.445 Ash, % DM 14.1 14.5 0.128 0.041 Litter Moisture, % 59.1 67.9 1.736 0.006 CP, % DM 7.13 8.99 0.254 ≤0.001 aNDFom, % DM 79.6 68.8 0.471 ≤0.001 Ash, % DM 8.55 13.49 0.436 ≤0.001 pH 8.22 8.29 0.062 0.593 1 aNDFom: NDF obtained using heat-stable α amylase and expressed as exclusive of residual ash. was assessed by evaluating kurtosis, skewness, and the Q-Q plot. When necessary, data were transformed to ensure a normal distribution: a log10-transformation was applied to SCC and a log10 (cfu + 1) per gram of litter or used litter was applied to bacterial count (Rowbotham and Ruegg, 2016). Data on chemical Figure 1. Effect of the litter used (PWC = poplar wood chips; WS composition of fecal and used litter samples, used litter = wheat straw) on the absolute fecal score frequency evaluated with a pH, milk composition, milk clotting ability, ECM, and 5-point scoring system, where 1 indicated a pea soup-like consistency and 5 was descriptive of fecal balls. log10 SCC were analyzed by generalized linear models (GLM) using the least significant difference post hoc test. Differences between used litter bacterial counts 1. Moisture, CP, and aNDFom content, as well as fecal were tested by ANOVA. The MY was analyzed using consistency were similar between the groups. Mean fe- repeated measures GLM with the value recorded on d 1 cal ash content was greater in the WS than the PWC as a covariate. The FS frequency was analyzed by a chi- group (P = 0.041). squared test. Multinomial logistic regression was used to The WS used litter had a greater moisture, CP, and evaluate the effect of the bedding typology on CS and ash content than the PWC (P = 0.006, P ≤ 0.001, and US. Scores were divided into a multinomial response. In P ≤ 0.001, respectively) but a lower aNDFom content addition to bedding typology, the FS effect was tested (P ≤ 0.001). The pH was similar between the groups. as further explanatory variable in the model. Because Used Litter Bacterial Count. Higher counts of the FS variable had a significant effect (P < 0.05) was E. coli (P ≤ 0.001), Lactobacillus spp. (P = 0.017), and included in the final model. The frequency of each score LTBC (P ≤ 0.001) were observed in the PWC than was calculated and reported also as a percentage. The WS used litter (Table 6). Moreover, E. coli hemolytic TBC, CBC, and SFU milk data were normalized through activity, Clostridium spp., and Salmonella spp. were not a log-transformation and analyzed using repeated mea- present. sure GLM with the log10 transformed value collected Udder and General Cleanliness. Table 7 dis- before the start of the study as a covariate. The number played the relative score frequency of the US and CS. of observations of animals standing or lying as well as Most of the cows were scored in categories 1 and 2. the number of observations of each behavioral trait was Tables 8 and 9 report the results of the multinomial compared between groups using the Chi-squared test. logistic regression for the US and CS respectively. The The DMI of both groups was compared by a t-test. The FS was demonstrated to inversely affect the CS, with statistical significance was set a P ≤ 0.05. higher fecal score leading to cleaner cows. The prob- ability of having dirtier animals (for both CS and US) was higher for animals housed on WS than PWC. RESULTS Fecal and Used Litter Composition Milk Yield, Quality, and Hygiene Feces and litter mean chemical composition are Milk yield, milk composition, log10 SCC, and clot- shown in Table 5, and FS score is displayed in Figure ting ability were unaffected by the litter material as Journal of Dairy Science Vol. 106 No. 12, 2023 Simoni et al.: POPLAR WOOD CHIPS AS DAIRY CATTLE LITTER 9156 Table 6. Used litter microbiology according to the 2 litter groups (PWC = poplar wood chips, n = 30; WS = wheat straw, n = 30) Item PWC WS SEM P-value Escherichia coli, log10 (cfu + 1)/g 3.488 0.863 0.417 ≤0.001 Lactobacillus spp., log10 (cfu + 1)/g 5.185 2.922 0.481 0.017 LTBC,1 log10 (cfu + 1)/g 10.157 9.106 0.150 ≤0.001 1 LTBC = litter or used litter total bacterial count. shown in Table 10. The CBC tended to be greater in exception could be milk production, whose evaluation milk from cows on WS than PWC litter (P = 0.053), deserves further studies. whereas SFU and TBC were unaffected (Table 11). Fecal and Used Litter Composition Behavior Fecal composition and consistency were similar be- The body position frequency occurrences were tween the 2 groups and in line with values reported in similar between groups (Table 12). Similarly, drinking, literature for dairy cows (Thomsen et al., 2013; Righi eating, and ruminating frequencies were not affected et al., 2017). In the present short-term study some dif- by the litter material. Inversely, the WS group exhib- ferences were detected in the ash content, which was ited a greater sleeping frequency compared with PWC higher in the WS group. Even if not quantitatively (P ≤ 0.001). Meanwhile, the PWC group showed a measured, cows of the WS group were occasionally ob- higher frequency of “other” behaviors (P ≤ 0.001), served eating some litter material, and this could have which included several types of nervousness or dis- increased the fecal ash content of these animals. Part comfort behaviors such as stepping, vocalizing, licking of this effect may be related to the greater DMI of themselves or other animals, and kicking. these animals, which may have led to variations in the digestion process. Dry Matter Intake Although the animals of the 2 groups showed similar fecal composition and consistency, a lower moisture A 13.72% DMI reduction was observed in cows content was found in the PWC used litter, probably in housed on PWC in comparison to WS (19.5 vs. 22.6 relation to the higher lignin content of PWC compared kg; P = 0.008). with WS litter (Table 1), as observed also by Larney et al. (2008). Those authors reported that, due to its DISCUSSION physico-chemical characteristics, lignin makes wood chips less biodegradable and more hydrophobic. This Our study aimed to compare the short-term effect of may have improved urine percolation through the bed- PWC as an alternative litter material to conventional ding, facilitating its flowing on the smooth and sloped WS in terms of used litter composition and microbiology, surface of the stall, or reduced urine retention of the cows’ milk hygiene, milk quality and MY, and behav- bedding itself. This would also have facilitated evapora- ior. Considering the characteristics of the experimental tion of water. Indeed Ferraz et al. (2020) showed that setup, it should be noted that the results obtained can WS have a water-holding capacity more than double be considered specific to the tiestall barn type and are that of wood chips. Additionally, a lower N concentra- not necessarily transferable to other barn types. Addi- tion was observed in the PWC used litter. This could tionally, it needs to be highlighted that tiestall animals are in contact with the bedding material 24 h a day, and the trial was performed in the summer season, with Table 7. Frequency of udder and cleanliness scores (4-point scale) of possible effects on moisture and microbial proliferation. the animals bedded on the 2 litter groups (PWC = poplar wood chips; The experimental period was relatively short because WS = wheat straw) the trial was conducted as a small-scale exploratory Udder score, % Cleanliness score, % field study, testing a bedding material whose effects on animal welfare and productivity were substantially Score PWC WS PWC WS unknown. Despite the limited trial duration, all the 1 67.5 37.2 48.6 18.3 measured outcome results were stable and consistent 2 16.9 33.3 21.6 38.3 among the sampling days (data not shown), indicating 3 8.4 14.1 25.7 23.3 4 7.2 15.4 4.1 20.1 the adequacy of the adaptation period length. The only Journal of Dairy Science Vol. 106 No. 12, 2023 Simoni et al.: POPLAR WOOD CHIPS AS DAIRY CATTLE LITTER 9157 Table 8. Results of the multinomial logistic regression describing the factors influencing the udder cleanliness of cows housed on the 2 litter groups (PWC = poplar wood chips; WS = wheat straw) CI 95% per OR Udder score Factor Coefficient SE P-value OR1 Lower limit Upper limit 1 vs. 2 Intercept 0.949 1.064 0.372 Bedding WS Referent PWC −1.130 0.360 0.002 0.323 0.159 0.654 FS2 −0.119 0.326 0.714 0.887 0.468 1.682 1 vs. 3 Intercept 0.394 1.432 0.783 Bedding WS Referent PWC −1.174 0.484 0.015 0.309 0.120 0.798 FS −0.248 0.445 0.578 0.781 0.326 1.867 1 vs. 4 Intercept 1.146 1.660 0.490 Bedding WS Referent PWC −1.402 0.574 0.015 0.246 0.080 0.758 FS −0.593 0.527 0.261 0.553 0.197 1.553 2 vs. 3 Intercept −0.555 1.428 0.698 Bedding WS Referent PWC −0.044 0.480 0.927 0.957 0.374 2.450 FS −0.128 0.447 0.775 0.880 0.366 2.114 2 vs. 4 Intercept 0.197 1.647 0.905 Bedding WS Referent PWC −0.272 0.570 0.633 0.762 0.249 2.327 FS −0.474 0.527 0.369 0.623 0.222 1.749 3 vs. 4 Intercept 0.752 1.900 0.692 Bedding WS Referent PWC −0.228 0.654 0.727 0.796 0.221 2.870 FS −0.346 0.606 0.568 0.708 0.216 2.320 1 OR = odds ratio. 2 FS = fecal score. partially support the previous hypothesis of a more contrast to the results on other wood chip materials pronounced urine percolation, even if this result can be (e.g., pine products; Miller et al., 2003). affected by the initial difference in litter’s CP content. The lower moisture content of the PWC used litter con- Used Litter Microbiology tributed to maintaining the animals drier and cleaner. These results are, however, inconsistent with findings The microbial count was expressed in this study on showing a higher ability of the wood litter materials a weight basis because, considering the nature of the in lowering N losses, when composting, compared with tested bedding materials, the mass (weight) is more straw litter materials (Larney et al., 2008). However, representative of the quantity of bedding material that our short-term study was focused on used litter’s per- is in contact with the animal body when the animal formance over a limited time span without considering is lying. Furthermore, the weight is a more objective the composting phase that usually follows used litter measure than volume because in general the volume removal and lasts several months. Some differences of these materials may be affected by the length of the were observed for aNDFom and ash content between fiber, the particle size, and the pressure exerted on the the 2 materials, even if they are more likely due to mass while measuring the volume itself. their initial differences. Concerning used litter pH, no The higher LTBC found in PWC is partially ex- differences were found in this study between the 2 ma- plained by the initially higher microbial contamination terials tested (Table 5), in agreement with Spiehs et al. of this material, which also appeared dustier. However, (2013), who reported a similar used litter pH when WS contamination of the litter material may be affected and wood chips were compared. Overall, these results by the humidity, temperature, and managing condi- suggest that PWC does not exert an acidic effect, in tions during storage (Bramley and Neave, 1975). In the Journal of Dairy Science Vol. 106 No. 12, 2023 Simoni et al.: POPLAR WOOD CHIPS AS DAIRY CATTLE LITTER 9158 Table 9. Results of the multinomial logistic regression describing the factors influencing the general cleanliness of cows housed on the 2 litter groups (PWC = poplar wood chips; WS = wheat straw) CI 95% per OR Cleanliness score Factor Coefficient SE P-value OR1 Lower limit Upper limit 1 vs. 2 Intercept 3.402 1.394 0.015 Bedding WS Referent PWC −1.699 0.464 <0.001 0.183 0.074 0.454 FS2 −0.619 0.409 0.130 0.538 0.242 1.199 1 vs. 3 Intercept 4.842 1.443 <0.001 Bedding WS Referent PWC −1.273 0.483 0.008 0.280 0.109 0.723 FS −1.210 0.430 0.005 0.298 0.128 0.692 1 vs. 4 Intercept 5.249 1.732 0.002 Bedding WS Referent PWC −2.197 0.586 <0.001 0.111 0.035 0.350 FS −1.439 0.532 0.007 0.237 0.084 0.673 2 vs. 3 Intercept 1.439 1.212 0.235 Bedding WS Referent PWC 0.427 0.391 0.275 1.532 0.712 3.299 FS −0.591 0.380 0.120 0.554 0.263 1.167 2 vs. 4 Intercept 1.847 1.495 0.217 Bedding WS Referent PWC −0.498 0.510 0.329 0.608 0.224 1.650 FS −0.820 0.478 0.086 0.440 0.172 1.125 3 vs. 4 Intercept 0.407 1.498 0.786 Bedding WS Referent PWC −0.925 0.517 0.074 0.397 0.144 1.092 FS −0.229 0.484 0.636 0.795 0.308 2.052 1 OR = odds ratio. 2 FS = fecal score. present study, the tested litter materials were stored Despite the absence of E. coli and Lactobacillus spp. in in the same conditions, but PWC underwent a differ- the clean litter materials (Table 1), which was expected, ent process, had a longer transport duration, and were these bacteria were present in the used litter and were packed in waterproof plastic big-bags. These factors found to be greater in the PWC than in the WS. Vari- could have affected microbial growth in the biomass able results can be found in literature concerning this before its usage. topic, and in general the reported LTBC are higher in comparison to our findings. Spiehs et al. (2013) found initial differences in coliform and E. coli contamination of WS and wood chips, which disappeared after 6 wk Table 10. Milk yield (MY) and quality according to the 2 litter groups (PWC = poplar wood chips, n = 90; WS = wheat straw, n = 90) of use. However, some authors reported similar total bacteria, coliforms, and E. coli counts between WS and Item1 PWC WS SEM P-value wood chips (Miller et al., 2003; Ferraz et al., 2020). MY, kg/cow per d 27.5 26.4 0.24 0.483 Differences across studies may be attributable to the ECM, kg/cow per milking 12.0 12.7 0.427 0.123 Fat, % 4.11 4.13 0.101 0.932 Protein, % 3.48 3.68 0.052 0.465 Table 11. Milk hygiene parameters according to the 2 litter groups Lactose, % 4.65 4.63 0.042 0.568 (PWC = poplar wood chips; WS = wheat straw) Casein, % 2.74 2.93 0.047 0.517 A30, mm 24.7 27.3 2.054 0.719 Item1 PWC WS SEM P-value K20, min 4.65 4.01 0.335 0.856 RCT, min 20.5 20.3 0.707 0.952 CBC, log10 cfu/mL 2.87 3.07 0.083 0.053 Urea, mg/dL 35.9 35.0 0.698 0.549 SFU, log10 MPN/L 2.09 2.07 0.030 0.658 Log10 SCC × 103 cells/mL 1.827 1.974 0.157 0.657 TBC, log10 cfu/mL 1.64 1.77 0.040 0.102 1 1 A30 = curd firmness 30 min after rennet addition to milk; K20 = curd CBC = coliform bacterial count; SFU = spore-forming unit; TBC = firming time; RCT = rennet coagulation time. total bacterial count; MPN = most probable number. Journal of Dairy Science Vol. 106 No. 12, 2023 Simoni et al.: POPLAR WOOD CHIPS AS DAIRY CATTLE LITTER 9159 Table 12. Body position and general behaviors (number of observation and frequency) of the cows according to the 2 litter groups (PWC = poplar wood chips; WS = wheat straw)1 PWC, no. of WS, no. of Item observations PWC, % observations WS, % P-value Body position Standing 1,946 53.6 1,971 54.3 0.556 Lying 1,683 46.4 1,658 45.7 General behaviors Ruminating 1,069 29.5 1,098 30.3 0.457 Eating 1,077 29.7 1,074 29.6 0.938 Drinking 169 4.7 158 4.4 0.533 Sleeping 267 7.4 375 10.3 ≤0.001 Other 1,047 28.9 924 25.5 ≤0.001 1 No. of observations per group = 3,629. Behaviors were defined in Table 4 following the instruction of Pavlenko et al. (2011) with minor modifications. Behavior other than those cited were classified as “other” (including grooming, licking itself or other animals, pushing other cows, observing, social interactions, vocalizing, stepping or kicking, urinating, and defecating). plant species used to produce the wood-based materi- the potential for environmental mastitis by exposing als, with specific reference to their potential content of teats to the high level of bacteria present in the litter antimicrobial substances (Miller et al., 2003; Spiehs et material. However, due to the diet type, the animals al., 2013). Furthermore, because Lactobacillus spp. are in our study had firm feces, and this was beneficial to usually isolated in cow feces (Lin et al., 2020), the high- maintaining stalls and keeping animals clean (Ward et est contamination of the PWC used litter detected in al., 2002). this study, can be considered as indicative of a greater fecal contamination. Additionally, these results suggest Milk Yield, Quality, and Hygiene greater bacterial proliferation in the used litter as result of more favorable environmental conditions, including The mean MY and composition observed (Table 10) higher bulk density (Ferraz et al., 2020) with decreased are both similar to or higher than data obtained in the aeration. The higher bulk density also justifies the in- same geographic area and productive context in other creased E. coli presence in PWC used litter because in studies (Comino et al., 2015; Franceschi et al., 2020; this case it is associated with a smaller particle size and Simoni et al., 2021b). The mean of MY in the present consequently with a higher surface area for bacterial study was close to the lowest productivity (29.0 kg/d) colonization per weight unit of material. However, ac- reported by Simoni et al. (2021b), which may be due cording to Lin et al. (2020) autochthonous lactic acid to our study having hay and concentrates administered bacteria isolated from dairy cow feces seem to express separately instead of using a total mixed ration, which antibacterial activity against enteric bacterial patho- is a feeding system that notoriously improves ruminal gens of cattle. This could provide an explanation for fermentation and productivity (Schingoethe, 2017). the decreased values observed for milk CBC and TBC Despite the short duration of the present trial, the found in cows housed on PWC. This is consistent with similar MY and composition between the litter groups some evidence relating to the presence of Lactobacillus is consistent with findings from Tucker et al. (2009). spp. to a reduction of E. coli-induced mammary gland Those authors observed that MY was unaffected by the infections in both experimental animals (Zhao et al., different litters used (straw and wood shavings) after 2021) and dairy cattle (Li et al., 2021b, 2022) as well 7 d of treatment, and they argued that litter and time as in dairy cattle reproductive trait (Genís et al., 2016). devoted to lying behavior should not be considered limiting factors for MY. Moreover, the same authors Udder and General Cleanliness Score claimed that a 1-wk trial is too short to observe MY variations, which are more related to DMI. Because this As previously mentioned, the PWC used litter in- latter parameter was negatively affected by PWC in creased animal cleanliness. Indeed, cows housed on this our study, we speculate that changes in MY could have litter material were found to be more frequently clean occurred over a longer time span. Thus, any economic considering both US and CS (Tables 8 and 9). Similar evaluation should be performed only after a follow-up results were found by Johanssen et al. (2018) testing confirmatory experiment on this parameter. wood chip (from broadleaf trees) and straw (from bar- The literature shows a direct relation between coli- ley) as litter for heifers. Dirty used litter can exacerbate form count in litter and incidence of coliform mastitis Journal of Dairy Science Vol. 106 No. 12, 2023 Simoni et al.: POPLAR WOOD CHIPS AS DAIRY CATTLE LITTER 9160 in bovines. Bramley and Neave (1975) concluded that straw or wood shavings provided in a similar amount. sawdust, which is also included in wood chips, can pro- Accordingly, we found no differences in the lying be- duce high levels of contamination even if those levels havior frequency. It should be highlighted here that, are different among materials and year of production. although different quantities of litter were not tested In agreement with this finding, our wood product in the present study, the amount of litter provided per showed a greater coliform contamination. However, due stall per day was comparable to the middle–high levels to the lower moisture of the PWC used litter, and the tested by the latter-mentioned authors, who also found consequent greater cleanliness of the cows, PWC led a positive correlation between quantity of litter material to a trend of lower CBC of the milk, which was, along offered to the animals and lying behavior time. Norring with the TBC of the milk, lower than values reported et al. (2008) observed that animals spent a longer time by Franceschi et al. (2020) under the same productive lying when housed on straw than on sand, because cows conditions. prefer to lie down on soft litter materials (Singh et al., For proper dairy product processing and quality, it 2020). Therefore, the results of this study could indicate is essential to reduce the contamination of spores in that PWC is comparable to WS. However, looking into raw milk (Cook and Sandeman, 2000). In our study, other behavioral parameters such as sleeping frequency similar milk SFU were found between the WS and and “other behaviors” reported in Table 12, it appears PWC groups. To the best of our knowledge, there are that WS is preferred over PWC. In our case, a higher no data available in literature regarding the milk spore sleeping frequency was in fact seen in animals housed content related to cattle housed on PWC. However, on WS than PWC litter, suggesting a greater level of studies comparing straw with other litter typologies comfort. It is well known that the comfort level affects highlighted inconsistent results for this parameter. DMI in livestock animals. The reduction of DMI has, For example, Gagnon et al. (2020) found no difference in fact, been addressed as one of the most important between recycled manure and straw, whereas Driehuis indicators of reduced welfare (Leliveld and Provolo, et al. (2014) obtained higher numbers of thermophilic 2020). Both the reduction in DMI and reduced sleeping spore-forming bacteria in the bulk milk from farms frequency indicate impaired welfare attributable to the using biodegradable litter from composted materials bedding type. In contrast to our results, Fukasawa et compared with farms using straw. According to Gagnon al. (2019) indicated that sleeping parameters were un- et al. (2020) the discrepancy between studies may be affected by farm management practices including litter due to other external factors, including season or feed- type (i.e., rice husk, straw, and sawdust) when 12 dairy ing plans. Indeed, a study focusing on Bacillus cereus farms were evaluated. Additionally, we found a higher reported an increase in milk spore levels during summer frequency of behaviors classified as “other” in animals compared with other seasons (Vissers et al., 2007). housed on PWC. Indeed, these animals devoted more In our study the TBC of milk was not affected by time to stepping, kicking, licking the other animals or litter materials, in analogy to the findings of Robles their own legs and bellies, as well as observing the other et al. (2020), who conducted a survey on 70 farms in cows or the litter per se. As explained by Hedlund and Ontario (Canada) testing wood products and straw. Løvlie (2015), increased stepping behavior, which is However, it should be considered that the latter study positively correlated with milk cortisol and heart rate, was performed in the colder months, whereas our study along with other behaviors such as vocalization and was set in the middle of the summer, which potentially facing the herd, reflect nervousness or discomfort in exacerbated the used litter microbiological load (Hogan cows. In the latter study those traits were negatively et al., 1989). related, although not strongly, with MY. Conversely, a rested cow produces more saliva, which improve rumi- Behavior nal health and increases both udder blood supply and MY (Tucker et al., 2009; Sadiq et al., 2017; Ferraz et When optimal litter and welfare conditions are al., 2020) reached, dairy cows lie down between 8 and 16 h per day (Tucker et al., 2009), but inappropriate litter condi- Dry Matter Intake tions could reduce the lying time by 48% (O’Connor et al., 2019). Depriving cows of the possibility to lie down The overall DMI found in the present trial is consis- represents a stressor that leads cows to prioritize rest- tent with those reported in other studies performed in ing over other activities (Cooper et al., 2007; Norring et the Parmigiano-Reggiano area, where animals were also al., 2008) and increases plasma cortisol concentration fed hay-based diets (Cavallini et al., 2021; Simoni et (Fisher et al., 2002). Tucker et al. (2009) found no differ- al., 2021b). Moreover, it is similar to the one reported ences in the lying behavior time of animals bedded with by Umphrey et al. (2001) under analogous managerial Journal of Dairy Science Vol. 106 No. 12, 2023 Simoni et al.: POPLAR WOOD CHIPS AS DAIRY CATTLE LITTER 9161 and environmental conditions, namely tiestalls and tion of data; in the writing of the manuscript; nor in the summer season (DMI between 16.7 and 18.5 kg/d). decision to publish the results. None of the authors have However, in this short-term study, the PWC group a financial or personal relationship with other people or showed a lower DMI than the WS group, probably due organizations that could inappropriately influence or to greater discomfort or impaired welfare related to bias the content of this paper. The data presented in the former litter material. The reduction of DMI has, this study are available free of charge for any user upon in fact, been addressed as one of the most important reasonable request from the corresponding authors. indicators of reduced welfare (Leliveld and Provolo, The authors have not stated any conflicts of interest. 2020). This hypothesis is supported also by the lower sleeping frequency observed, along with the higher fre- REFERENCES quency of behaviors classified as “others” in the present study, which, according to literature (Rousing et al., Alanis, V. M., M. Zurakowski, D. Pawloski, T. Tomazi, D. V. Nydam, and P. A. Ospina. 2021. Description of the characteristics of five 2006; Pavlenko et al., 2011; Hedlund and Løvlie, 2015), bedding materials and association with bulk tank milk quality on includes manifestations of discomfort (e.g., stepping, five New York dairy herds. Front. Vet. Sci. 8:636833. https:​/​/​doi​ vocalizing, licking themselves or other animals, and .org/​10​.3389/​fvets​.2021​.636833. Andrews, T., C. E. Jeffrey, R. E. Gilker, D. A. Neher, and J. W. kicking behaviors). Additionally, based on the resource Barlow. 2021. Design and implementation of a survey quantifying allocation theory, if cows invested more time licking, winter housing and bedding types used on Vermont organic dairy kicking, observing, interacting, and stepping, then they farms. J. Dairy Sci. 104:8326–8337. https:​/​/​doi​.org/​10​.3168/​jds​ .2020​-19832. had less time for other activities (Hedlund and Løvlie, Annibaldi, S. 1969. Modificazione della prova di Weinzirl per la ricerca 2015). dei clostridi butirrici nel latte. Sci. Tec. Latt.-Casearia 20:75–79. AOAC International. 2005. Official Methods of Analysis of AOAC in- ternational. 18th ed. AOAC International. CONCLUSIONS Bewley, J. M., L. M. Robertson, and E. A. Eckelkamp. 2017. A 100- year review: Lactating dairy cattle housing management. J. Dairy Based on the results of this trial, productive per- Sci. 100:10418–10431. https:​/​/​doi​.org/​10​.3168/​jds​.2017​-13251. Bramley, A. J., and F. K. Neave. 1975. Studies on the control of coli- formance of the cows housed on WS and PWC litter form mastitis in dairy cows. Br. Vet. J. 131:160–169. https:​/​/​doi​ materials are comparable in the short-term period. The .org/​10​.1016/​S0007​-1935(17)35336​-8. PWC used litter showed lower moisture, leading to a Cavallini, D., L. M. E. Mammi, G. Biagi, I. Fusaro, M. Giammarco, A. Formigoni, and A. Palmonari. 2021. Effects of 00-rapeseed meal higher frequency of cleaner animals, and even though inclusion in Parmigiano Reggiano hay-based ration on dairy cows’ the used litter microbial contamination was higher, production, reticular pH and fibre digestibility. Ital. J. Anim. Sci. milk contamination was similar. However, PWC litter 20:295–303. https:​/​/​doi​.org/​10​.1080/​1828051X​.2021​.1884005. Cheng, W. N., and S. G. Han. 2020. Bovine mastitis: Risk factors, reduced the sleeping frequency, induced the frequency therapeutic strategies, and alternative treatments—A review. of behaviors including discomfort signs, and reduced the Asian-Australas. J. Anim. Sci. 33:1699–1713. https:​/​/​doi​.org/​10​ DMI of the cows in the short term. Larger-scale, confir- .5713/​a jas​.20​.0156. Comino, L., F. Righi, M. Coppa, A. Quarantelli, E. Tabacco, and matory studies are needed to evaluate the use of PWC G. Borreani. 2015. Relationships among early lactation milk fat litter material over a longer period, also considering depression, cattle productivity, and fatty acid composition on in- different physical forms of the poplar wood materials. tensive dairy farms in Northern Italy. Ital. J. Anim. Sci. 14:3656. https:​/​/​doi​.org/​10​.4081/​ijas​.2015​.3656. Cook, G.M., and R.M. Sandeman. 2000. Sources and characterisation ACKNOWLEDGMENTS of spore-forming bacteria in raw milk. Australian Journal of Dairy Technology 55:119. Cooper, M. D., D. R. Arney, and C. J. C. Phillips. 2007. Two- or four- This research was funded by the European Union’s hour lying deprivation on the behavior of lactating dairy cows. Horizon 2020 Research and Innovation Program, grant J. Dairy Sci. 90:1149–1158. https:​/​/​doi​.org/​10​.3168/​jds​.S0022​ agreement no. 774340 for the Organic-PLUS Project. -0302(07)71601​-6. De Marchi, M., V. Toffanin, M. Cassandro, and M. Penasa. 2013. The authors kindly acknowledge Costanza Ampollini Prediction of coagulating and noncoagulating milk samples using (University of Parma, Parma, Italy) and Luigi Tonelli mid-infrared spectroscopy. J. Dairy Sci. 96:4707–4715. https:​/​/​doi​ (Az.Agr.Tonelli&Buratti, Parma, Italy) for the techni- .org/​10​.3168/​jds​.2012​-6506. Driehuis, F., E. Lucas-van den Bos, and M. H. J. Wells-Bennik. 2014. cal assistance. Moreover, we thank all the volunteer Sporen van Thermofiele, Aerobe Sporenvormers in Compost en students that helped us in the data collection and Na- Andere Beddingmaterialien bij Melkveebedrijven Met een Vri- dia Cusack (Freelance, Parma, Italy) and Selina Moore jloop-of Ligboxenstal. Spores of thermophilic aerobic sporeformers in compost and other bedding materials used by dairy farmers (University of Padova, Padova, Italy) for the language with a bedded pack or freestall barn). NIZO-Report E, 45. revision of the manuscript. C. L. Manuelian is cur- European Commission. 2009. Commission regulation (EC) no 152/2009 rently a postdoctoral researcher funded with a María of 27 January 2009 laying down the methods of sampling and analysis for the official control of feed. Off. J. Eur. Union 54:1–130. Zambrano grant of the Spanish Ministry of Universities European Parliament and Council. 2010. Directive 2010/63/EU of 22 (Madrid, Spain). The funders had no role in the design September 2010 on the protection of animals used for scientific of the study; in the collection, analyses, or interpreta- purposes. Text with EEA relevance, 276. Journal of Dairy Science Vol. 106 No. 12, 2023 Simoni et al.: POPLAR WOOD CHIPS AS DAIRY CATTLE LITTER 9162 European Union. 2018. Regulation (EU) 2018/848, 30 May 2018. by-products in Finland. For. Policy Econ. 110:101946. https:​/​/​doi​ Regulation (EU) 2018/848 of the European Parliament and of .org/​10​.1016/​j​.forpol​.2019​.101946. the Council of 30 May 2018 on organic production and labelling Larney, F. J., A. F. Olson, J. J. Miller, P. R. DeMaere, F. Zvomuya, of organic products and repealing Council Regulation (EC) No and T. A. McAllister. 2008. Physical and chemical changes dur- 834/2007. Off. J. Eur. Union 150:1–92. ing composting of wood chip-bedded and straw-bedded beef cattle Ferraz, P. F. P., G. A. S. Ferraz, L. Leso, M. Klopčič, M. Barbari, and feedlot manure. J. Environ. Qual. 37:725–735. https:​/​/​doi​.org/​10​ G. Rossi. 2020. Properties of conventional and alternative bedding .2134/​jeq2007​.0351. materials for dairy cattle. J. Dairy Sci. 103:8661–8674. https:​/​/​doi​ Leliveld, L. M. C., and G. Provolo. 2020. A review of welfare indica- .org/​10​.3168/​jds​.2020​-18318. tors of indoor-housed dairy cow as a basis for integrated automatic Fisher, A. D., G. A. Verkerk, C. J. Morrow, and L. R. Matthews. welfare assessment systems. Animals (Basel) 10:1430. https:​/​/​doi​ 2002. The effects of feed restriction and lying deprivation on pitu- .org/​10​.3390/​ani10081430. itary-adrenal axis regulation in lactating cows. Livest. Prod. Sci. Li, H., X. Wang, Y. Wu, D. Zhang, H. Xu, H. Xu, X. Xing, and Z. Qi. 73:255–263. 2021a. Relationships among bedding materials, bedding bacterial Franceschi, P., M. Malacarne, P. Formaggioni, F. Righi, and A. Sum- composition, and lameness in dairy cows. Anim. Biosci. 34:1559– mer. 2020. Chemical composition, hygiene characteristics, and 1568. https:​/​/​doi​.org/​10​.5713/​a jas​.20​.0565. coagulation aptitude of milk for Parmigiano Reggiano cheese Li, K., M. Yang, M. Tian, L. Jia, J. Du, Y. Wu, L. Li, L. Yuan, and from herds yielding different milk levels. Rev. Bras. Zootec. Y. Ma. 2022. Lactobacillus plantarum 17-5 attenuates Escherichia 49:e20180113. https:​/​/​doi​.org/​10​.37496/​rbz4920180113. coli-induced inflammatory responses via inhibiting the activation Fukasawa, M., T. Komatsu, and Y. Higashiyama. 2019. Sleep and of the NF-κB and MAPK signalling pathways in bovine mammary lying behavior of milking Holstein cows at commercial tie-stall epithelial cells. BMC Vet. Res. 18:250. https:​/​/​doi​.org/​10​.1186/​ dairy farms. Anim. Sci. J. 90:1313–1319. https:​/​/​doi​.org/​10​.1111/​ s12917​-022​-03355​-9. asj​.13265. Li, Y., Y. Zhu, B. Chu, N. Liu, S. Chen, and J. Wang. 2021b. Lactoba- Gagnon, M., L. Hamelin, A. Fréchette, S. Dufour, and D. Roy. 2020. cillus rhamnosus GR-1 prevents Escherichia coli-induced apoptosis Effect of recycled manure solids as bedding on bulk tank milk through PINK1/Parkin-mediated mitophagy in bovine mastitis. and implications for cheese microbiological quality. J. Dairy Sci. Front. Immunol. 12:715098. https:​/​/​doi​.org/​10​.3389/​fimmu​.2021​ 103:128–140. https:​/​/​doi​.org/​10​.3168/​jds​.2019​-16812. .715098. Genís, S., À. Bach, F. Fàbregas, and A. Arís. 2016. Potential of lactic Lin, W. C., C. P. Ptak, C. Y. Chang, M. K. Ian, M. Y. Chia, T. H. acid bacteria at regulating Escherichia coli infection and inflam- Chen, and C. J. Kuo. 2020. Autochthonous lactic acid bacteria mation of bovine endometrium. Theriogenology 85:625–637. https:​ isolated from dairy cow feces exhibiting promising probiotic prop- /​/​doi​.org/​10​.1016/​j​.theriogenology​.2015​.09​.054. erties and in vitro antibacterial activity against foodborne patho- Godden, S., R. Bey, K. Lorch, R. Farnsworth, and P. Rapnicki. 2008. gens in cattle. Front. Vet. Sci. 7:239. https:​/​/​doi​.org/​10​.3389/​fvets​ Ability of organic and inorganic bedding materials to promote .2020​.00239. growth of environmental bacteria. J. Dairy Sci. 91:151–159. https:​ Manuelian, C. L., V. Vigolo, F. Righi, M. Simoni, S. Burbi, and M. de /​/​doi​.org/​10​.3168/​jds​.2007​-0415. Marchi. 2021. MIR and Vis/NIR spectroscopy cannot authenticate Hedlund, L., and H. Løvlie. 2015. Personality and production: Nervous organic bulk milk. Ital. J. Anim. Sci. 20:1810–1816. https:​/​/​doi​ cows produce less milk. J. Dairy Sci. 98:5819–5828. https:​/​/​doi​ .org/​10​.1080/​1828051X​.2021​.1954559. .org/​10​.3168/​jds​.2014​-8667. Mertens, D. R., M. Allen, J. Carmany, J. Clegg, A. Davidowicz, M. Hogan, J. S., K. L. Smith, K. H. Hoblet, D. A. Todhunter, P. S. Drouches, K. Frank, D. Gambin, M. Garkie, B. Gildemeister, D. Schoenberger, W. D. Hueston, D. E. Pritchard, G. L. Bowman, Jeffress, C. S. Jeon, D. Jones, D. Kaplan, G. N. Kim, S. Kobata, L. E. Heider, B. L. Brockett, and H. R. Conrad. 1989. Bacte- D. Main, X. Moua, B. Paul, J. Robertson, D. Taysom, N. Thiex, rial counts in bedding materials used on nine commercial dair- J. Williams, and M. Wolf. 2002. Gravimetric determination of am- ies. J. Dairy Sci. 72:250–258. https:​/​/​doi​.org/​10​.3168/​jds​.S0022​ ylase-treated neutral detergent fiber in feeds with refluxing in bea- -0302(89)79103​-7. kers or crucibles: Collaborative study. J. AOAC Int. 85:1217–1240. Hutjens, M. F. 2010. Manureology 101. Pages 59–61 in Proc. Four- Miller, J. J., B. W. Beasley, L. J. Yanke, F. J. Larney, T. A. McAl- State Dairy Nutrition and Management Conference, Dubuque, lister, B. M. Olson, L. B. Selinger, D. S. Chanasyk, and P. Hassel- Iowa. Cooperative Extension for Iowa State University, University back. 2003. Bedding and seasonal effects on chemical and bacterial of Illinois, University of Minnesota, and University of Wisconsin. properties of feedlot cattle manure. J. Environ. Qual. 32:1887– ISO. 2006. Milk—Enumeration of somatic cells—Part 2: Guidance on 1894. https:​/​/​doi​.org/​10​.2134/​jeq2003​.1887. the operation of fluoro-opto-electronic counters. ISO 13366-2:2006. Mondaca, M. R. 2019. Ventilation systems for adult dairy cattle. Vet. International Organization for Standardization. Clin. North Am. Food Anim. Pract. 35:139–156. https:​/​/​doi​.org/​ ISO. 2020. Milk and liquid milk products—Guidelines for the applica- 10​.1016/​j​.cvfa​.2018​.10​.006. tion of near infrared spectrometry. ISO 21543:2020. International Munir, M. T., C. Belloncle, M. Irle, and M. Federighi. 2019. Wood- Organization for Standardization. based litter in poultry production: A review. Worlds Poult. Sci. J. ISO. 2005. Milk and milk products—Enumeration of presumptive 75:5–16. https:​/​/​doi​.org/​10​.1017/​S0043933918000909. Escherichia coli—Part 1: Most probably number technique using Niraula, R., P. Eng, B. Lebeau, and P. Eng. 2018. Alternative bedding 4-methylumbelliferyl-beta-D-glucuronide (MUG). ISO 11866-1/ materials for livestock. Accessed on Feb. 15, 2023. http:​/​/​www​ IDF 170-1. International Organization for Standardization. .omafra​.gov​.on​.ca/​english/​environment/​facts/​18​-011​.pdf. Johanssen, J. R. E., K. M. Sørheim, T. Strøm, and E. I. Brunberg. Norring, M., E. Manninen, A. M. de Passillé, J. Rushen, L. Munks- 2018. Bedding hygiene, cleanliness, and lying behaviour for heifers gaard, and H. Saloniemi. 2008. Effects of sand and straw bedding housed on wood chip or straw deep bedding. Acta Agric. Scand. on the lying behavior, cleanliness, and hoof and hock injuries of A Anim. Sci. 68:103–111. https:​/​/​doi​.org/​10​.1080/​09064702​.2019​ dairy cows. J. Dairy Sci. 91:570–576. https:​/​/​doi​.org/​10​.3168/​jds​ .1601763. .2007​-0452. Kour, S. 2017. Bedding options for dairy cattle. Journal of Scientific O’Connor, C., S. Dowling, V. Cave, and J. Webster. 2019. Cow ly- Achievements 2:43–45. ing behaviour and bedding quality changes during five weeks on Kuipers, A., P. Galama, L. Leso, K. Bruegemann, and M. Klopčič. a stand-off pad. Animals (Basel) 9:257. https:​/​/​doi​.org/​10​.3390/​ 2022. A composting bedding system for animals as a contribution ani9050257. to the circular economy. Processes (Basel) 10:518. https:​/​/​doi​.org/​ Patel, K., S. M. Godden, E. Royster, B. A. Crooker, J. Timmerman, 10​.3390/​pr10030518. and L. Fox. 2019. Relationships among bedding materials, bedding Kunttu, J., E. Hurmekoski, H. Heräjärvi, T. Hujala, and P. Leskinen. bacteria counts, udder hygiene, milk quality, and udder health in 2020. Preferable utilisation patterns of wood product industries’ Journal of Dairy Science Vol. 106 No. 12, 2023 Simoni et al.: POPLAR WOOD CHIPS AS DAIRY CATTLE LITTER 9163 US dairy herds. J. Dairy Sci. 102:10213–10234. https:​/​/​doi​.org/​10​ of the Art. Proceedings of the 27th Biennial Session of the Interna- .3168/​jds​.2019​-16692. tional Committee for Animal Recording. Butterwords. Pavlenko, A., C. Bergsten, I. Ekesbo, T. Kaart, A. Aland, and L. Lid- Smith, M. M., C. L. Simms, and J. D. Aber. 2017. Case Study: Animal fors. 2011. Influence of digital dermatitis and sole ulcer on dairy bedding cost and somatic cell count across New England dairy cow behaviour and milk production. Animal 5:1259–1269. https:​/​/​ farms: Relationship with bedding material, housing type, herd doi​.org/​10​.1017/​S1751731111000255. size, and management system. Prof. Anim. Sci. 33:616–626. https:​ Quinn, P. J. 1994. Clinical Veterinary Microbiology. Wolfe Publishing /​/​doi​.org/​10​.15232/​pas​.2016​-01601. and Mosby-Year Book Limited. Spiehs, M. J., T. M. Brown-Brandl, D. B. Parker, D. N. Miller, E. Reneau, J. K., R. F. Bey, and R. J. Farnsworth. 2002. Bedding man- D. Berry, and J. E. Wells. 2013. Effect of bedding materials on agement and udder health. Pages 83–90 in American Association concentration of odorous compounds and Escherichia coli in beef of Bovine Practitioners, Proceedings of the Conv. Am. Assoc. Bo- cattle bedded manure packs. J. Environ. Qual. 42:65–75. https:​/​/​ vine Pract., Madison, WI. Am. Assoc. Bovine Pract. doi​.org/​10​.2134/​jeq2012​.0251. Righi, F., M. Simoni, G. Visentin, C. L. Manuelian, S. Currò, A. Quar- Thomsen, I. K., J. E. Olesen, H. B. Møller, P. Sørensen, and B. T. antelli, and M. de Marchi. 2017. The use of near infrared spectros- Christensen. 2013. Carbon dynamics and retention in soil after copy to predict faecal indigestible and digestible fibre fractions in anaerobic digestion of dairy cattle feed and faeces. Soil Biol. Bio- lactating dairy cattle. Livest. Sci. 206:105–108. https:​/​/​doi​.org/​10​ chem. 58:82–87. https:​/​/​doi​.org/​10​.1016/​j​.soilbio​.2012​.11​.006. .1016/​j​.livsci​.2017​.10​.006. Tucker, C. B., D. M. Weary, M. A. G. von Keyserlingk, and K. A. Robles, I., D. F. Kelton, H. W. Barkema, G. P. Keefe, J. P. Roy, M. Beauchemin. 2009. Cow comfort in tie-stalls: Increased depth A. G. von Keyserlingk, and T. J. DeVries. 2020. Bacterial concen- of shavings or straw bedding increases lying time. J. Dairy Sci. trations in bedding and their association with dairy cow hygiene 92:2684–2690. https:​/​/​doi​.org/​10​.3168/​jds​.2008​-1926. and milk quality. Animal 14:1052–1066. https:​/​/​doi​.org/​10​.1017/​ Umphrey, J. E., B. R. Moss, C. J. Wilcox, and H. H. van Horn. 2001. S1751731119002787. Interrelationships in lactating Holsteins of rectal and skin tempera- Rousing, T., J. H. Badsberg, I. C. Klaas, J. Hindhede, and J. T. tures, milk yield and composition, dry matter intake, body weight, Sørensen. 2006. The association between fetching for milking and feed efficiency in summer in Alabama. J. Dairy Sci. 84:2680– and dairy cows’ behaviour at milking, and avoidance of human 2685. https:​/​/​doi​.org/​10​.3168/​jds​.S0022​-0302(01)74722​-4. approach—An on-farm study in herds with automatic milk- Van Soest, P. J., J. B. Robertson, and B. A. Lewis. 1991. Methods for ing systems. Livest. Sci. 101:219–227. https:​/​/​doi​.org/​10​.1016/​j​ dietary fiber, neutral detergent fiber, and nonstarch polysaccha- .livprodsci​.2005​.11​.013. rides in relation to animal nutrition. J. Dairy Sci. 74:3583–3597. Rowbotham, R. F., and P. L. Ruegg. 2016. Bacterial counts on teat https:​/​/​doi​.org/​10​.3168/​jds​.S0022​-0302(91)78551​-2. skin and in new sand, recycled sand, and recycled manure solids Vissers, M. M. M., M. C. te Giffel, F. Driehuis, P. de Jong, and J. M. used as bedding in freestalls. J. Dairy Sci. 99:6594–6608. https:​/​/​ G. Lankveld. 2007. Minimizing the level of Bacillus cereus spores doi​.org/​10​.3168/​jds​.2015​-10674. in farm tank milk. J. Dairy Sci. 90:3286–3293. https:​/​/​doi​.org/​10​ Sadiq, M. B., S. Ramanoon, W. Shaik Mossadeq, R. Mansor, and S. .3168/​jds​.2006​-873. Syed-Hussain. 2017. Association between lameness and indicators Ward, W. R., J. W. Hughes, W. B. Faull, P. J. Cripps, J. P. Suther- of dairy cow welfare based on locomotion scoring, body and hock land, and J. E. Sutherst. 2002. Observational study of tempera- condition, leg hygiene, and lying behavior. Animals (Basel) 7:79. ture, moisture, pH, and bacteria in straw bedding, and faecal con- https:​/​/​doi​.org/​10​.3390/​ani7110079. sistency, cleanliness, and mastitis in cows in four dairy herds. Vet. Schingoethe, D. J. 2017. A 100-year review: Total mixed ration feeding Rec. 151:199–206. https:​/​/​doi​.org/​10​.1136/​vr​.151​.7​.199. of dairy cows. J. Dairy Sci. 100:10143–10150. https:​/​/​doi​.org/​10​ areZhao, C., X. Hu, L. Bao, K. Wu, L. Feng, M. Qiu, H. Hao, Y. .3168/​jds​.2017​-12967. Fu, and N. Zhang. 2021. Aryl hydrocarbon receptor activation by Schreiner, D. A., and P. L. Ruegg. 2003. Relationship between ud- Lactobacillus reuteri tryptophan metabolism alleviates Escherichia der and leg hygiene scores and subclinical mastitis. J. Dairy Sci. coli-induced mastitis in mice. PLoS Pathog. 17:e1009774. https:​/​/​ 86:3460–3465. https:​/​/​doi​.org/​10​.3168/​jds​.S0022​-0302(03)73950​-2. doi​.org/​10​.1371/​journal​.ppat​.1009774. Simoni, M., A. Canepa, R. Pitino, A. Quarantelli, and F. Righi. 2021b. Digestibility and sorting of hay-based total mixed rations employed in the Parmigiano-Reggiano area as affected by dietary ORCIDS particle size distribution. Large Anim. Rev. 27:123–132. Simoni, M., A. Goi, M. De Marchi, and F. Righi. 2021a. The use of M. Simoni https:​/​/​orcid​.org/​0000​-0003​-3920​-9744 visible/near-infrared spectroscopy to predict fibre fractions, fibre- N. Mezzasalma https:​/​/​orcid​.org/​0000​-0001​-5652​-2922 bound nitrogen, and total-tract apparent nutrients digestibility in R. Pitino https:​/​/​orcid​.org/​0000​-0003​-0195​-3172 beef cattle diets and faeces. Ital. J. Anim. Sci. 20:814–825. https:​/​ C. L. Manuelian https:​/​/​orcid​.org/​0000​-0002​-0090​-0362 /​doi​.org/​10​.1080/​1828051X​.2021​.1924884. C. Spadini https:​/​/​orcid​.org/​0000​-0003​-0211​-5293 Singh, A., T. Kumari, M. Rajput, A. Baishya, N. Bhatt, and S. Roy. M. Iannarelli https:​/​/​orcid​.org/​0000​-0003​-2234​-5563 2020. Review on effect of bedding material on production, repro- S. L. Montanaro https:​/​/​orcid​.org/​0000​-0002​-4139​-6166 duction, and health of dairy animals. Int. J. Livest. Res. 1:1. https:​ /​/​doi​.org/​10​.5455/​ijlr​.20200207073618. A. Summer https:​/​/​orcid​.org/​0000​-0002​-4833​-657X Sjaunja, L. O., L. Baevre, L. Junkkarinen, J. Pedersen, and J. Setala. C. S. Cabassi https:​/​/​orcid​.org/​0000​-0002​-1936​-9858 1990. A Nordic proposal for an energy corrected milk (ECM) for- M. De Marchi https:​/​/​orcid​.org/​0000​-0001​-7814​-2525 mula. Pages 156–157 in Performance Recording of Animals: State F. Righi https:​/​/​orcid​.org/​0000​-0001​-9274​-4143 Journal of Dairy Science Vol. 106 No. 12, 2023