Nutrient and energy digestibility, mineral status and antioxidant defense in goat wethers and periparturient does supplemented with poplar and willow leaves

1 Introduction

Goats as intermediate types between browsers and grazers possess an intrinsic ethology to consume not only grass but also foliage (Hofmann, 1989). Recent studies showed that goats satisfy up to 60% of their nutritional needs through the consumption of foliage when given the choice (Rahmann, 2004; Chuluunbat et al., 2025). European and German guidelines recommend grazing for goats (Ganter et al., 2012; European Parliament and Council, 2018), but access to trees and shrubs is not required. One practical approach to better meet the ethological and nutritional requirements could be the inclusion of foliage derived from agroforestry systems (e.g., alley cropping, short rotation plantations, or silvopasture) into the ration of goats.

Fast-growing poplar (Populus spp.) and willow (Salix spp.) trees are widely used for cultivation in agroforestry systems in areas with sufficient rainfall. The use of foliage from these trees as feed is widespread, particularly in New Zealand, China, North America and some regions in Europe (Eichhorn et al., 2006; Smith, 2010; Wangui et al., 2025). In Germany, the total area of poplar and willow short rotation plantations currently amounts to 6, 600 hectares yielding an average of 9 t per hectare of aboveground woody dry mass annually (Umweltbundesamt, 2019; Liesebach and Schneck, 2024) or up to 3 t foliage dry mass per hectare in the second year after planting (FuLaWi – Merkblätter, 2026).

The fodder value of black poplar and willow foliage is comparable to meadow hay, especially when harvested in the summer (Becker and Nehring, 1965; Rahmann, 2004). The crude protein (CP) content of various poplar and willow leave species is remarkable [>14% of dry matter (DM)] (Rahmann, 2004), while the lignin (14.5%) and tannin (0.7% to 1.5%) contents are relatively low (Becker and Nehring, 1965). Moreover, the mineral content, (e.g., Ca, Zn, and Se) is even greater in leaves than grass (Becker and Nehring, 1965; McWilliam et al., 2005; Rahmann, 2004). These numbers suggest that poplar and willow foliage could be a suitable feed component for goats, but little is known about their feed value.

The periparturient period of dairy goats is characterized by increased energy and nutrient demands for fetal growth, colostrum and milk production, often resulting in negative energy and Ca balances due to insufficient intake (Simões and Margatho, 2024). These balances become even more negative in does with multiple fetuses, severing the demand for energy and Ca (Simões and Margatho, 2024). The increased need for Ca is compensated by mineral mobilization from bones which can be assessed by the ratio between osteocalcin (OC), a bone formation marker, and the C-terminal telopeptide of collagen type I (CTX), a bone resorption marker (Wilkens et al., 2014; Camassa et al., 2017). A recent study demonstrated that the level of dietary Ca intake antepartum (ap) influences serum OC concentrations and thus bone metabolism postpartum (pp) (Brugger and Liesegang, 2025). If the increased energy requirements are not met by intake, goats mobilize body fat and protein reserves, which is reflected by increased plasma non-esterified fatty acids (NEFA) and urea concentrations (Radin et al., 2015). This high metabolic burden around parturition is associated with increased oxidative stress (Rodríguez Patiño et al., 2018) activating the defense machinery such as glutathione peroxidase (GPx), which produces the antioxidant glutathione (Radin et al., 2015). Feeding periparturient goats a diet high in antioxidants, such as phenolic and flavonoid compounds, increases the plasma concentrations of antioxidants and reduces oxidative stress and metabolic burden (Afzal et al., 2022).

Poplar and willow leaves contain not only substantial amounts of Ca and metabolizable energy (ME) but also numerous secondary plant metabolites with high antioxidant potential (Pobłocka-Olech et al., 2025). In this role, flavonoids and polyphenols from these plants could account for the improved reproductive performance observed in ewes fed poplar and willow trimmings (McWilliam et al., 2004, 2005). However, if willow or poplar foliage feeding ameliorates the oxidative and metabolic stress and stabilizes Ca homeostasis in the period of lambing has not been tested so far.

The first aim of the present study was to determine the macronutrient and energy digestibility and the usability of minerals of poplar and willow foliage in goats. To determine the extent to which these feeds are suitable for meeting the increased nutritional requirements of breeding animals, the second objective of this study was to examine whether supplementing the diet with the highest feed quality identified in the first aim would improve antioxidant defense status, calcium and metabolic homeostasis during the periparturient period in dairy goats.

2 Materials and methods

2.1 Ethical considerations

The feeding studies were conducted in 2024 at the Research Institute for Farm Animal Biology (FBN), Dummerstorf, Germany in accordance with the ARRIVE guidelines (Percie du Sert et al., 2020), the German Animal Welfare Act and approved by the ethics committee of the State Department for Agriculture, Food Security and Fisheries Mecklenburg-Western Pomerania, Rostock, Germany (Registration No. LALLF M-V 7221.3-1-012/23). Persons involved in sample preparation and analyses were blinded to the experimental design. Persons who collected blood samples, prepared the feed, and fed the goats were not blinded.

2.2 Animal feeding and housing

Branches from black poplar hybrids (P. nigra × P. deltoides), balsam poplar hybrids MAX (P. nigra × P. maximowiczii) and willow hybrids (Salix triandra × viminalis) were harvested in September 2023 in Saxony (09603 Großschirma and 09600 Langhennersdorf), stacked in a container and subsequently dried using heat of a biogas plant at 60 to 70 °C for 1 to 2 d. After drying, the leaves with petioles were stripped from the branches and stems and mixed to one batch before feeding. As a further forage, first-cut, air-dried meadow hay (German ryegrass, meadow grass and red fescue) was used.

Experiment 1. Four male castrated, 1.5-year-old Saanen goats were habituated to the experimental facilities, including individual pens (2.25 × 1.35 × 1.25 m), metabolic cages (2.37 × 1.06 × 1.68 m) and respiration chambers (4.00 × 2.00 × 2.00 m) (Derno et al., 2009), over a period of 14 d prior to the start of the experiment. Habituation was considered successful when the animals lay down, ate, drunk and ruminated regularly. The animals had an average body weight of 51.8 ± 1.3 SD kg at the beginning. The study was conducted in a 4×4 Latin square design based on the recommendations of Schiemann (1981). The experiment consisted of four blocks, each lasting 28 d, including an 18-d feeding period followed by a 10-d wash-out phase on pasture. During the feeding period, goats were kept individually first in pens for 14 d and received daily one of the 4 diets: hay in combination with either 650 g black poplar foliage (PNIG), 650 g balsam foliage (PMAX), 650 g willow foliage (SVIM), or without any foliage (HAY). The foliage was offered in two equal portions at 08.00 and 15.30, while hay was offered ad libitum. Foliage not eaten in the morning was added to the afternoon portion. The individual hay and foliage intake was measured daily. Following the first 14 d in pens, wethers were equipped with a feces collection bag and transferred into a metabolic cage which was inserted into a respiration chamber. Housing was at 20 °C and a light-dark cycle from 06.00 to 20.00 from d 15 to 18. After overnight equilibration, gas exchange measurement started on d 16 at 08.00 and ended at d 19 at 08.00. The body weight (BW) was determined on d 1, d 14 and d 19 of each period using a scale, and the metabolic BW (mBW^0.75) was calculated based on the mean.

Experiment 2. Sixteen one-year old Saanen goats in the third trimester of pregnancy were adapted to their new group housing at least four 4 weeks before parturition. Goats had ad libitum access to mineral lick (KULMIN Mineralleckstein OLB, Bergophor, Kulmbach, Germany) and hay and were supplemented with 0.2 to 0.3 kg/d wheat. After birth, does and their lambs (Supplementary Table 1) were moved to individual pens (1.3 × 4 m) and assigned to one of two groups. One group was fed 700 g of black poplar hybrid foliage plus wheat (100 g/d in the first week, 200 g/d in the second week, and 250 g/d wheat in week 3 to 5 pp) and hay ad libitum (POPLAR; n = 9). Black poplar was chosen because it revealed the highest acceptance assessed as voluntary intake, and feed quality as indicated by the highest Ca plus Se concentration, ME and CP digestibility compared to the other foliage species analyzed in Experiment 1. The other group received only wheat as described above and hay ad libitum (CON; n = 7). Individual foliage and hay intakes were recorded daily and does were weighed weekly.

2.3 Animal samplings

Experiment 1. Feces collection bags were emptied at 08.00 and 15.30 to determine daily fecal excretions. The feces were weighed, wetted with 3% formic acid and stored at 4 °C. After pooling at the end of each block, feces were mixed, sampled and frozen at -20 °C until further processing. The excreted urine was drained through a dedicated tub and collected in a canister. From d 15 to 16, the urine was collected for 24 h without acidification to measure the volume and weight allowing the calculation of density. A sample was taken and stored at -20 °C until further analyses. From d 16 to 19, the canister was prefilled daily at 08.00 with 125 ml 25% sulphuric acid and placed on a magnetic stirrer for immediate urine acidification. Total urine output was subjected to volume and weight measurement, and an additional sample was stored at -20 °C until further analyses. On d 19, blood samples were taken from the jugular vein of the wethers into 9-mL tubes to obtain whole blood, serum and EDTA plasma. After centrifugation (1, 573 × g, 20 min, 4 °C) whole blood, buffy coat, plasma and serum samples were frozen at -20 °C or -80 °C until further analysis.

Experiment 2. Dairy goats were blood sampled as described above starting 2 weeks before calculated lambing. The real sampling dates ap ranged between d -14 to -7 for week 2 ap and d -7 to 0 for week 1 ap. Whole blood, buffy coat, plasma and serum were frozen at -20 °C and -80 °C until further analysis.

2.4 Organic nutrient and nitrogen analyses

For the determination of dry matter, feed and fecal samples were dried at 60 °C for 24 h and ground to 1 and 0.7 mm. Ground samples were dried at 103 °C for 4 h to determine the DM content according to the methodological guidelines of the Association of German Agricultural Analytic and Research Institutes (VDLUFA, 2012). The concentrations of crude ash, CP, crude fat, crude fiber, acid detergent fiber (ADF), amylase-treated neutral detergent fiber (aNDF_OM), acid detergent lignin (ADL) were analyzed by the accredited laboratory of Landwirtschaftliche Untersuchungs- und Forschungsanstalt (LUFA GmbH, Rostock, Germany) according to VDLUFA (2012). Hemicellulose and cellulose were determined by LUFA GmbH (Kirchgeßner et al., 2014). For the determination of the N content, urine and dried feces were weighed into tin capsules, dried overnight at 55 °C and subjected to an element analyzer (FlashEA 1112 NC analyzer, Thermo Fisher, Bremen, Germany). The water N concentration was measured according to DIN EN 12260 (H34) and P concentration to DIN EN ISO 11885/E22 by LUFA GmbH. The gross energy (GE) of feed, feces, and urine samples was measured by a C 200 bomb calorimeter (IKA-Werke GmbH & Co. KG, Staufen, Germany). The ME for Experiment 2 was determined according to GfE, 1995-01(1995) for wheat and poplar leaves and GfE, 2008-01 (2008) for hay by LUFA GmbH.

2.5 Mineral analyses

Experiment 1. The mineral concentrations in water were analyzed by BioCheck GmbH (Leipzig, Germany) using an inductively coupled plasma mass spectrometry (ICP-MS) (iCAP RQ ICP-MS, Thermo Fisher) and are shown in Supplementary Table 2. Feed and fecal Ca, P, Na, Mg, K, Cu, Zn, Mn, Fe, and Se concentrations were analyzed after pressure decomposition on an ICP-MS (iCAP RQ ICP-MS) by SGS Analytics Germany GmbH (Jena, Germany). Urinary mineral concentrations were analyzed by the same method except for Na, K and Cl, which were analyzed via an indirect ion-selective electrode (ISE; SGS Analytics). The concentrations of Cr, Co, Ni, Mo, Ba, Hg, and Pb in plant material as well as P, K, Ca, Cr, Mn, Fe, Co, Ni, Cu, Zn, Se, Mo, Ba, Hg, and Pb in plasma were measured using an Agilent 8900 inductively coupled plasma triple quadrupole MS (ICP-QQQ; Agilent Technologies) equipped with an octopole collision/reaction cell and operated in single MS or MS/MS mode (German Federal Institute for Risk Assessment, BfR, Berlin, Germany).

Experiment 2. Feed Ca and Se and plasma Se concentrations were analyzed as described above by BioCheck GmbH. Serum Ca concentrations were analyzed using the ABX Pentra Calcium AS CP Kit for the Pentra C400 (HORIBA Europe GmbH, Oberursel, Germany).

2.6 Secondary plant metabolite analyses

Phenol and tannin concentrations in feed samples were determined according to the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture (2000), and condensed tannins according to Porter et al. (1985) with minor modifications described by Schwerdtfeger et al. (2025). Briefly, after the plant material was extracted with 50% acetone, 0.5-ml extract was treated with 0.25-ml Folin–Ciocalteu and the absorbance was measured at 700 nm to determine the phenol concentration. For the analysis of total tannin concentration, 0.5-ml extract was mixed with 100-mg polyvinylpolypyrrolidone, centrifuged, and 0.5-ml of the supernatant mixed with 0.25-ml Folin–Ciocalteu reagent to measure the absorbance at 700 nm. Condensed tannins were analyzed by incubating 100-µl plant extract with 600-µl butanol–HCl reagent and 20-µl 2%NH₄Fe (SO₄)₂ before the absorbance was measured at 550 nm. All chemicals were purchased from Sigma.

2.7 Antioxidant analyses

The ferric-reducing antioxidant power (FRAP) in feed was analyzed according Wojtunik-Kulesza (2020) and in plasma according to Benzie and Strain (1996) with minor adjustments described by Schwerdtfeger et al. (2025). In brief, an ethanol extract of plant material or plasma was incubated with a mixture of 0.3 M acetate buffer, 0.01 M 2, 4, 6-tris-(2-pyridyl)-s-triazine (TPTZ) in 0.04 M HCl and 0.02 M FeCl₃ × 6 H₂O and the absorbance was measured at 550 nm. The Trolox Equivalent Antioxidative Capacity (TEAC) was determined using the 2, 2-diphenyl-2-picrylhydrazyl radical (DPPH) assay according to Brand-Williams et al. (1995) or the 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assay according to Re et al. (1999). For the measurement of GPx activity, EDTA whole blood samples were thawed on ice, spun at 900 × g (2 min, 4 °C), diluted 1:2 to 1:7 to meet the kit’s measurement range and applied to the RANSEL Glutathione Peroxidase kit for PENTRA 400 from Randox Laboratories Ltd. (Crumlin, United Kingdom) according to the manufacturer’s instructions. For the analysis of 8-iso-prostaglandin F2α (8-iso-PGF2α), a marker of lipid peroxidation, plasma samples were prepared and loaded on the 8-Isoprostane Oxidative Stress ELISA Kit (Detroit R&D Inc., Detroit, USA) according to the manufacturer’s instructions.

2.8 Metabolites and bone mineralization markers

The plasma concentrations of glucose, urea and NEFA in does were analyzed by Pentra C400 (HORIBA Europe GmbH, Oberursel, Germany) using the kits ABX Pentra Glucose HK CP, ABX Pentra Urea CP and Wako NEFA-HR. Further, plasma was applied to the Serum Crosslaps^® (CTX) ELISA kit (ids immunodiagnostic systems, Frankfurt am Main, Germany) or to the MicroVue Bone Osteocalcin EIA kit (Quidel, San Diego, USA) and processed according to the manufacturer’s instructions.

2.9 Indirect calorimetry

The used respiration chambers were described by Derno et al. (2009) but the volume was reduced by 1.37 m³ by inserting airtight bags. The CO₂ and CH₄ concentrations were measured by non-dispersive infrared spectroscopy and O₂ concentrations measured at a paramagnetical analyzer (SIDOR, SICK AG, Waldkirch, Germany) at 6-minute intervals. The airflow through the chamber was adjusted to approximately 7 m³/h and recorded using a V differential pressure cone flow meter (McCrometer, Hemet, CA). The CO₂ recovery rate for each chamber averaged 99.7%. Heat production (HP) was calculated according to Brouwer (1965):

HP(kJ)=16.18×O2 (L)+5.02×total CO2 (L)–2.17×CH4(L)–5.99×Nu (g)

where Nu is the average urinary N excretion.

Metabolizable energy intake (MEI) and ME were calculated using the following formula:

MEI (MJ)=GE feed (MJ)–GE urine (MJ)–GE feces (MJ)–GE CH4 (MJ)

and

ME(MJkg)=MEDMI

where DMI is the dry matter intake. The energy for hoof and hair growth was not considered in ME calculation.

2.10 Digestibility, balances and microbial protein production

The digestibility (D) of OM, CP and aNDF_OM was calculated as D=feed intake−fecal outputfeed intake according to Meyer and Kamphues (2014). The mineral and N balances (B) were calculated as

B=intake (water+feed)−excretion (feces+urine)

For the determination of microbial protein production (MPP), urinary urea and non-urea N metabolite concentrations were analyzed on a high-performance liquid chromatography (HPLC) (1200/1260 infinity Series; Agilent Technologies) as described by Schwerdtfeger et al. (2025). The MPP was calculated as

MPP(gd)=(28×BW113.1×(allantoin (mM)+uric acid (mM))creatinine (mM))−(0.385×BW0.75)0.85×70×6.250.13×0.83×1000

published by Janicek et al. (2008) and Chen and Gomes (1992) for cattle.

2.11 Statistical analysis

All statistical analyses were performed using R version 4.3.2 (R Core Team, 2023). Data processing and visualization were conducted using the tidyverse package (v2.0.0; Wickham et al., 2019). Estimated marginal means (EMMs) and their standard errors (SEs) were calculated using the emmeans package (v1.8.9; Lenth, 2023). Pairwise comparisons were adjusted using the Tukey–Kramer method. Differences were considered statistically significant at p< 0.05 and as a tendency at p< 0.10.

Sample size determination. For Experiment 1, no formal a priori power analysis was performed. The number of animals was chosen according to established guidelines for digestion trials (GfE, 1991; Schiemann, 1981). Based on the percent of feed stuff implementation to the basal diet of approximately 30%, the use of four goat wethers was considered appropriate.

For Experiment 2, a formal a priori sample size calculation was performed. The expected effect size was derived from published studies (Trávníček et al., 2008; Pavlata et al., 2011; Pechova et al., 2012; Wilkens et al., 2016; Nedelkov et al., 2020) reporting treatment effects on selenium status, GPx activity, mineral concentrations in blood, milk, and urine. To avoid relying on the largest published effects, a conservative effect size of Cohen’s d = 1.5 was used for the sample size calculation. Using a two-sided two-sample t-test with α = 0.05 and 80% power, the required sample size was estimated as 8 animals per group. The study was initially planned with balanced group sizes. However, due to logistical constraints during animal allocation, the final group sizes were n = 9 for the POPLAR group and n = 7 for the CON group.

Experiment 1 and 2. Normality of residuals was assessed using check_model and check_normality from the performance package (Lüdecke et al., 2021).

Experiment 1. Data were analyzed using a linear model appropriate for a 4 × 4 crossover design:

Yijk=μ+αi+βj+γk+ϵijk,

where Y_ijk is the dependent variable for the diet i, period j and animal k; μ is the overall mean; α_i is the fixed effect of diet (four levels: HAY, PNIG, PMAX, SVIM); β_j is the fixed effect of experimental period (four levels: block 1 to 4); γ_k is the fixed effect of the individual animal (four levels, one per animal) and ϵ_ijk is the random residual error.

Experiment 2. Raw data were examined for normal distribution using the Shapiro-Wilk test (shapiro.test, stats package) and visual inspection of residual plots. If normality assumption was violated, data were log-transformed (OC/CTX ratio, FRAP, 8-iso-PGF2α) and reassessed. If log-transformed data deviated from normality distribution, potential outliers were identified using the boxplot.stats function or values exceeding mean ± 2 × SD. Identified outliers were excluded from further analysis. The number of goats included in the analysis of each parameter is shown in Supplementary Table 3.

Data referring to a single time point (ap: Se, Ca, glucose, NEFA, urea, CTX, OC, GPx, 8-iso-PGF2α, log-transformed OC/CTX ratio; pp: log-transformed 8-iso-PGF2α), were analyzed using a linear model:

Yik=μ+αi+ϵik,

where Y_ik is the dependent variable of group i and animal k; μ is the overall mean; α_i is the fixed effect of group i (two levels: CON and POPLAR) and ϵ_i is the random residual error.

Variables measured at multiple time points (ap: TEAC_ABTS, log-transformed FRAP; pp: TEAC_ABTS, glucose, GPx; pp log-transformed: Se, FRAP, CTX, OC/CTX ratio, OC), data were evaluated by the following linear mixed model (LMM, lmerTest package (Kuznetsova et al., 2017):

Yijk=μ+αi+βj+(αβ)ij+υk+ϵijk,

where Y_ijk is the dependent variable for group i, time point j and animal k; µ is the overall mean; α_i is the fixed effect of group i; β_j is the fixed effect of time point j; (αβ)_ij is the interaction between group i and time point j; υ_k the random effect of the kth animals and ϵ_ij is the random error term.

For pp plasma urea and NEFA concentrations, singularity issues occurred in the mixed effects model. Therefore, a simplified linear model without random effects was fitted:

Yijk=μ+αi+βj+υk+ϵijk,

where Y_ijk is the value of the dependent variable for group i, time point j of the kth animal k; µ is the overall mean; α_i is the effect of the group i; β_j is the effect of time point j; υ_k is the fixed effect of the animals k and ϵ_ijk is the random residual error.

3 Results

Experiment 1

Contents of nutrients, GE, secondary plant metabolites and antioxidants in hay and foliage are presented in Table 1. The CP content (black poplar +19%) and the crude ash content (black poplar +30%, balsam poplar +21%) were higher in foliage compared to hay, while the content of the latter was similar to willow leaves. The contents of crude fiber, aNDF_OM, and cellulose in leaves were about half of those in hay. However, leaves exhibited 1.5- to 3.2-fold higher ADL, 2.3- to 2.8-fold higher condensed tannin, and 1.5- to 2.0-fold higher phenol concentrations than hay. Antioxidant capacity, assessed by ABTS and FRAP assays, was 3-times higher in black poplar and balsam poplar leaves and even 6- to 10-times higher in willow leaves than in hay. The highest GE concentration was found in willow leaves. Black poplar leaves were particularly rich in Ca, Mg, Cu, Zn and Fe, whereas willow leaves had the highest Mn and P concentrations (Table 2). All foliage species revealed 2- to 6-fold greater Se concentrations than hay, but hay possessed the highest K and Na concentrations.

Table 1. Concentrations of nutrients, energy, and secondary plant compounds as well as antioxidant characteristics of hay and leaves fed in experiments 1 and 2, respectively.

Hybrids: black poplar – P. nigra × deltoides, balsam poplar – P. nigra × maximowiczii,.

willow – S. triandra × viminalis.

Determined according to GfE (2008) for hay and GfE (1995) for wheat and leaves.

ABTS, 2,2'-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt), DPPH, 2,2-diphenyl-2-picrylhydrazyl radical; FRAP, ferric-reducing antioxidant power; GE, gross energy; ME, metabolizable energy; LOD, limit of detection; n.d., not determined; TEAC, Trolox Equivalent Antioxidative Capacity.

Table 2. Concentrations of minerals, trace elements and heavy metals of hay and leaves fed in experiments 1 and 2, respectively.

Hybrids: black poplar – P. nigra × deltoides, balsam poplar – P. nigra × maximowiczii,.

willow – S. triandra × viminalis.

Analysed by SGS (Jena, Germany).

Analysed by Federal Institute for Risk Assessment (BfR, Berlin, Germany).

n.d. - not determined.

The composition of the HAY, PNIG, PMAX and SVIM diets and the nutrient intakes are shown in Supplementary Table 4. The daily crude ash, ADL, hemicellulose, condensed and total tannins, phenol, TEAC_(ABTS), and FRAP intake was greater in the foliage groups (p< 0.05; Supplementary Table 4). The greater crude ash intake was reflected by a higher intake of all minerals in foliage supplemented groups, except for P, K and Cr (p< 0.01; Supplementary Table 5).

The plasma mineral concentrations in goat wethers are shown in Table 3. Goats fed the PNIG (72.6 µg/L) diets had higher Se concentrations than those fed the PMAX (59.4 µg/L; p< 0.01) and HAY (61.4 µg/L; p< 0.05) diets. SVIM goats (67.6 µg/L) showed higher Se concentrations than those fed the PMAX (59.4 µg/L; p< 0.05). The remaining plasma element concentrations were either found not significantly different between groups or were below the detection limit.

Table 3. Plasma mineral concentrations in goat wethers fed hay or leaf-containing diets – Experiment 1 (emmean ± SEM)¹.

Different lower-case letters within a row indicate significant differences among diets (P< 0.05).

HAY diet: 100% hay.

PNIG diet: 38% dried black poplar leaves (P. nigra × deltoides hybrid) and 62% hay.

PMAX diet: 35% dried balsam poplar leaves (P. nigra × maximowiczii hybrid) and 65% hay.

SVIM diet: 36% dried willow leaves (S. triandra × viminalis hybrid) and 64% hay.

Standard error of the mean.

Analytical method for Pb in plasma not certified< LOD - below limit of detection.

n.d. - not detected.

The mean daily DMI (1.4 kg/d; p = 0.34), water intake (4.3 L/d; p = 0.96), fecal DM (570 g/d; p = 0.07) and urine (1.3 L/d; p = 0.52) excretions did not differ between diets (Table 4). However, the OM digestibility of PMAX (64.0%) was significantly higher compared to HAY (59.0%; p< 0.04) and SVIM (56.1%; p< 0.01). Furthermore, the SVIM diet revealed the lowest aNDF_OM (60.0%) and CP digestibility (52.9%) compared to HAY (73.1% and 62.0%, respectively), PMAX (70.6% and 63.6%, respectively), and PNIG (69.6% and 64.5%, respectively) (p = 0.01). Although foliage supplementation increased Ca, Mg, Cu, Zn, Mn, Fe, and Se intake and reduced Na intake compared to HAY feeding (Supplementary Table 5), there were no significant differences in the mineral balances between groups, except for Mg and Fe (Table 4). The Mg balance tended to be lower on the PMAX (0.09 g/d) compared to PNIG and SVIM diet (each 0.38 g/d; p< 0.09) but did not differ from HAY (0.20 g/d; p = 0.65). The Fe balance was positive for PNIG (58.5 mg/d), and was significantly higher compared to HAY (-12.7 mg/d), PMAX (-21.9 mg/d) and SVIM (-30.5 mg/d) feeding (p< 0.04).

Table 4. Nutrient and energy digestibility and element balances in goat wethers fed hay or leaf-containing diets – experiment 1 (emmean ± SE)¹.

Different lower-case letters within a row indicate significant differences among diets (P< 0.05).

HAY diet: 100% hay.

PNIG diet: 38% dried black poplar leaves (P. nigra × deltoides hybrid) and 62% hay.

PMAX diet: 35% dried balsam poplar leaves (P. nigra × maximowiczii hybrid) and 65% hay.

SVIM diet: 36% dried willow leaves (S. triandra × viminalis hybrid) and 64% hay.

Standard error of the mean.

on the dry matter base.

The GE excreted with feces was highest on the SVIM diet (11.6 MJ/d) and urinary GE excretion (2.2 MJ/d) was greatest during PMAX feeding (each p< 0.03; Table 5). The GE released as methane was similar between groups, but the ME of the PNIG diet tended to be higher than that of the SVIM diet (9.2 vs 8.4 MJ/kg; p = 0.08). Methane emission per DMI was higher in HAY than in all foliage diets (p< 0.02). The fecal N excretion was higher on the SVIM (17.9 g/d) than HAY (13.0 g/d) or PMAX and PNIG diets (each 14.2 g/d, p< 0.01). Although the urinary N excretion was not different between feeding groups, urinary urea excretion was higher under the influence of PNIG (499 g/d) compared to HAY (301 g/d) or SVIM (322 g/d; p< 0.05) feeding. Moreover, urinary hippuric acid excretion was significantly higher during feeding PNIG (112.0 g/d), PMAX (102.0 g/d) and SVIM (96.6 g/d) than HAY (53.3 g/d; p< 0.01). However, urinary allantoin and uric acid excretions as well as MPP were comparable between the HAY and foliage groups.

Table 5. Faecal and urinary energy and nitrogen excretions, and calculated metabolizable energy and microbial protein synthesis in goat wethers fed hay or leaves-containing diets – experiment 1 (emmean ± SE)¹.

Different lower-case letters within a row indicate significant differences among diets (P< 0.05).

HAY diet: 100% hay.

PNIG diet: 38% dried black poplar leaves (P. nigra × deltoides hybrid) and 62% hay.

PMAX diet: 35% dried balsam poplar leaves (P. nigra × maximowiczii hybrid) and 65% hay.

SVIM diet: 36% dried willow leaves (S. triandra × viminalis hybrid) and 64% hay.

Standard error of the mean.

MPP, microbial protein production (Chen and Gomes, 1992; Janicek et al., 2008); MPP(gd)=(28×BW113.1×(allantoin (mM)+uric acid (mM))creatinine (mM))−(0.385×BW0.75)0.85×70×6.250.13×0.83×1000.

Experiment 2

Per design, the intake of black poplar foliage was higher and the intake of hay lower in POPLAR than CON goats postpartum (p< 0.01; Supplementary Table 6). However, total DMI did not differ between POPLAR and CON groups (p = 0.81), but increased from parturition to week 5 after lambing (p< 0.01; Figure 1; Supplementary Table 6). The POPLAR goats had lower aNDF_OM, ADL, hemicellulose and cellulose intake but greater phenol, tannin, TEAC_(ABTS), FRAP, Ca, and Se intake than CON goats (p< 0.5; Supplementary Table 7). The BW tended to be higher in the CON than POPLAR group (p = 0.03) and declined over time (p< 0.01; Supplementary Table 8).

Plasma Se concentrations were not different between groups ap (Supplementary Table 9; Figure 2), but declined from gestation until the fifth week of lactation in both feeding groups (p< 0.01), and the decline was more pronounced in the CON compared to the POPLAR group (p = 0.03; Supplementary Table 6). Pairwise comparison revealed higher plasma Se concentrations in the POPLAR compared to the CON group in week 5 pp (p = 0.01; Figure 2; Supplementary Table 6), with a tendency over all (p = 0.052). Plasma FRAP, TEAC_(ABTS), GPx and 8-iso-PGF2α concentrations were not significantly different between dietary groups, but FRAP and GPx increased (each p = 0.01) and TEAC_(ABTS) tended to increase (p = 0.09) in the pp period.

Serum Ca concentrations were comparable between feeding groups ap (p = 1.0; Figure 3; Supplementary Table 9), but were lower in POPLAR than CON goats in week 2 pp, whereas this difference was absent in other lactation weeks, resulting in a group × time interaction (p = 0.02, Figure 3; Supplementary Table 6). CON goats had a higher Ca concentration than POPLAR goats in week 2 pp, this difference was absent in other lactation weeks, resulting in a group × time interaction (p = 0.02). Plasma OC concentrations were not different between groups ap (Figure 3; Supplementary Table 9), but tended to be lower in the POPLAR group (p = 0.07) and changed over time pp (p = 0.01, Figure 3; Supplementary Table 6). Plasma CTX concentrations were comparable in both groups ap (Figure 3; Supplementary Table 9), greater in the POPLAR group in the week of lambing, and declined in both groups pp (p< 0.01, Figure 3; Supplementary Table 6). The OC/CTX ratio was also not different ap (Figure 3; Supplementary Table 9), but changed over time (p< 0.01) and seemed to be lower in the POPLAR group in weeks 1 and 2 pp (Figure 3; Supplementary Table 6). However, this could not be verified statistically.

Although the plasma glucose concentrations were lower in the POPLAR than CON group ap (p = 0.046), no difference were detected in the pp period (Figure 4; Supplementary Table 6). Plasma urea and NEFA concentrations were not different between groups neither ap nor pp, but a significant group × time interaction was observed for plasma NEFA concentrations pp (p = 0.039).

4 Discussion

4.1 Experiment 1

4.1.1 Nutrient composition and secondary compounds

4.1.1.1 Crude nutrients

Feed analysis revealed that all foliage species had higher concentrations of CP compared to hay. Different contents of foliage OM, crude ash, CP, CL, CF, aNDF_OM, and ADF were reported earlier (Becker and Nehring, 1965; McCabe and Barry, 1988; Luske and van Eekeren, 2018; Bonizzi et al., 2025). However, the crude nutrient concentrations in leaves are influenced by harvest time, e.g., the CP concentration declines whereas the crude fiber concentration increases across season in Canadian poplar (Becker and Nehring, 1965). Thus, the different harvest times and varieties of species may explain most of the differences in crude nutrients of poplar and willow in our study compared to literature.

4.1.1.2 Minerals

The contents of K (15–20 g/kg DM) and Ca (18–22 g/kg DM) in poplar were slightly lower than reported by Rahmann (2004) (K: 41 g/kg DM; Ca: 27 g/kg DM) or Maillard et al. (2015) (K: 8.9 g/kg DM; Ca: 14.3 g/kg DM) Becker and Nehring (1965) (K: 26 g/kg; Ca: 31 g/kg DM). Furthermore, the willow foliage concentrations of K, Na and Cu were lower but the Mn (688 mg/kg DM) and Se (0.15 mg/kg DM) concentrations remarkably higher than mentioned in earlier studies (Mn: 56 mg/kg DM; Se: 2 µg/kg DM) (Kacálková et al., 2015; Luske and van Eekeren, 2018; Bonizzi et al., 2025). The Mg, P, Fe, Co and Zn concentration of poplar and willow foliage fell within literature ranges (Luske and van Eekeren, 2018; Bonizzi et al., 2025). Interestingly, Zn concentrations may vary to quite some extent (60 – 1015 mg /kg DM) (Kacálková et al., 2015; Bonizzi et al., 2025). Although local conditions, such as soil conditions, can also affect mineral levels (Della Maggiora et al., 2023), it was reported that the age of leaves, species and the variety could explain more of the variation than soil composition (Kacálková et al., 2015; Maillard et al., 2015).

4.1.1.3 Secondary compounds

The concentrations of condensed tannins in poplar and willow foliage were 2- to 3-times higher than in hay and ranged between 5 and 116 g/kg DM, which is similar to earlier reports (McCabe and Barry, 1988; McWilliam et al., 2005). The total phenol concentration in our willow foliage (73 g/kg DM) was almost the same as for phenolic acid concentrations in willow leaves (72 g/kg DM) (Müller-Kiedrowski et al., 2025), but only half as much (149 g/kg DM) as reported by Piątczak et al. (2020). The latter may be explained by the different methods applied: Piątczak et al. (2020) analyzed phenolic acids via UPLC-PDA-Q/TOF-MS, whereas a colorimetric approach was used in our study. However, Pobłocka-Olech et al. (2025) reported a total phenolic content in poplar foliage of 85 g/kg DM by using gallic acid as a standard, which is similar to 78–96 g/kg DM determined by us using tannic acid as a reference. The antioxidant capacity of both poplar leaves was higher for FRAP (57 vs. 0.15 mg Fe²⁺eq/g DM) and lower for DPPH (14 vs. 910 µmol Trxeq/g DM) and ABTS (5 vs. 2510 µmol Trxeq/g DM) compared to the Pobłocka−Olech et al. study (2025). To compare our results for willow to those of Piątczak et al. (2020), we converted the units according to Polak and Bartoszek (2018) and found a DPPH value for their Salix alba (190 µmol/L) comparable to ours (135 µmol/L), whereas their ABTS value (83 µmol/mL) was roughly two-fold lower than in this study (163 µmol/mL). The differences in ABTS values could be owed to different drying processes. Shahin et al. (2025) have shown that drying the leaves and flowers of Paulownia spp. at high temperatures for an extended period can reduce their total antioxidant potential.

4.1.2 Crude nutrient digestibility, MPP, and CH₄

4.1.2.1 Poplar

The OMD (~63%) was comparable, but the CPD (~64%) for both PNIG and PMAX clearly exceeded previously reported data (OMD 66%, CPD 52%) for pure but not further specified poplar foliage fed to goats (Masson et al., 1980). The greater CPD found in our study can be explained by the lower foliage inclusion level (38% in PNIG and 36% in PMAX). Furthermore, the variety and the harvest time, both not specified in the Masson et al. (1980) study, may account for the different CPD. However, the poplar OMD and CPD matched the OMD (58–63%) and CPD (56–80%) found for Canadian and black poplar harvested in July and October, respectively, and fed pure to sheep (Becker and Nehring, 1965). Furthermore, the CPD of aspen foliage harvested in July in Germany amounted to 65% (Rahmann, 2004) and is thus comparable to our CPD data. Our results also align with the OMD of 66% measured in sheep supplemented with poplar foliage during pasture grazing between February and April in Australia (McWilliam et al., 2005).

4.1.2.2 Willow

The SVIM diet showed a lower OMD (56%) than the poplar diets but was comparable to the 59% OMD reported for goats fed pure Salix viminalis (McCabe and Barry, 1988). However, Azim et al. (2002) described for 50% Salix babylonia leaves a CPD of 65% and a crude fiber digestibility of 53% in goats, while Näsi (1984) identified an OMD for S. aquatica pulp harvested in September of 42–61% and a CPD of 36–52% in rams. In an experiment with calves, Müller-Kiedrowski et al. (2025) found an aNDF_OM digestibility of 54% for a ration containing 36% willow leaves and 46% grass clippings, which was slightly lower than the 60% aNDF_OM digestibility in our study. One reason for this difference could be the more efficient chewing activity of goats (Domingue et al., 1991) and the lower ADL concentration of SVIM in our study (49 vs 54 g/kg DM). On the other hand, Näsi (1984) formulated two rations for rams containing 94% whole crushed S. dasyclados or S. aquatica including stems or 96% S. aquatica leaf pulp, both mixed with 6% or 4% wheat straw, respectively. The higher lignin concentration in the ration containing stems (29–34%) and the higher portion of foliage (94%) in the Näsi (1984) study may be the reason for the lower fiber digestibility (42–61%) compared to our rations. Besides, the CPD of these willow-containing diets was in a range between 37 and 52 % (Näsi, 1984) and thus was lower than in our study (53%), likely because we used willow foliage and no stems and included greater amounts of hay in the diet. In contrast, Azim et al. (2002) feed 50% concentrate with the leaves, which resulted in a diet with a lower fiber content explaining the higher digestibility. In an in vitro study, Bonizzi et al. (2025) determined the digestibility for Salix babylonica leaves harvested in September and found an OMD of ~58%, which is in line with our results. However, the aNDF_OM digestibility was only 41% (Bonizzi et al., 2025), whereas it was 60% in our study, presumably because Bonizzi et al. fed 100% leaves. Overall, SVIM exhibited a slightly poorer OMD, CPD, and aNDF_OM digestibility than the poplar diets. This discrepancy was likely due to the 22-fold greater condensed tannin concentration in willow foliage. Condensed tannins form complexes with nutrients in the rumen, thereby hindering the digestibility of CP and fiber (Woodward and Reed, 1997; Min et al., 2015; Besharati et al., 2022). As a consequence, the amount of rumen−undegradable protein and fecal N losses increases (Min et al., 2015; Besharati et al., 2022), which could also be observed when tannin intake increased in the present study. However, the amount of urinary N, creatinine and purine excretion remained unchanged, suggesting that the MPP was not affected by foliage intake. Nevertheless, MPP is also influenced by ruminal N availability and feed energy (Reynolds and Kristensen, 2008). Finally, the bacteriostatic action of tannins causing the highest reduction in aNDF_OM digestibility (Min and Solaiman, 2018; Besharati et al., 2022), together with the suppressive effect of tannins on methanogenic archaea (Moraes et al., 2014) probably caused the lowest methane yield on the willow diet. Similarly, in vitro fermentation of willow leaves harvested in June produced less methane relative to total gas production than fermentation of other leaf species such as Robinia or Ulmus (Bonizzi et al., 2025).

4.1.3 Mineral balances

Mineral requirements of goats were previously deduced from the mineral content of the pregnant uteri of goats (Härter et al., 2017) or from the occurrence of clinical symptoms in response to mineral undersupply (Haenlein and Anke, 2011). Little is known about mineral balances (retentions) under conditions of tree or bush product feeding. Singh et al. (1998) found that feeding goats 2.5 kg of fresh oak leaves induced negative Ca and P balances (-0.9 and -0.4 g/d, respectively). By contrast, our PNIG, PMAX and SVIM numerically increased the Ca balance by 0.3 to 1.6 g/d relative to HAY. In addition, the P balance remained unaffected by poplar and willow feeding, likely because of their higher Ca and P concentrations compared to oak leaves (Becker and Nehring, 1965; Singh et al., 1998; Rahmann, 2004). Min et al. (2015) investigated the digestibility of various minerals after supplementing ground pine bark (Pinus taeda L.; 30% of DM) to a straw- and hay-based diet and found increased digestibility for Ca, P, Mg, Mn, Zn, Cu and Fe. The authors suggested that condensed tannins may account for this result but the mechanism remained unknown (Min et al., 2015). However, tannins and other anti-nutritional factors were reported to reduce the bioavailability of minerals (Guo et al., 2025). Feeding PMAX numerically reduced Mn, Cu and Fe balances, whereas SVIM numerically increased Mn and Se balances, and PNIG numerically increased Fe, Na and K balances. The Fe, Zn, Mn, Co and Cu balances also increased 1.2 to 4.1-fold when goats fed Pennisetum clandestinum- grass were supplemented with 70% Quercus spp. leaves (Raju et al., 2018). While the different mineral intakes may explain numerical balance differences, the high standard deviation makes the results difficult to interpret, thus more research is needed to elucidate which foliage substances support or inhibit mineral bioavailability.

4.1.4 Absorption of heavy metals

Foliage of various tree species can accumulate heavy metals (Kacálková et al., 2015; Hard et al., 2019). Kacálková et al. (2015) found accumulating Cd (willow: up to 8.2 mg/kg; poplar: up to 3.0 mg/kg), Zn (willow: up to 1, 050 mg/kg; poplar: up to 561 mg/kg) and Cu (willow: up to 12.3 mg/kg; poplar: up to 8.4 mg/kg) concentrations. Indeed, the heavy metal concentrations in foliage fed in the present study had higher Zn, Mn, Ni, and Pb concentrations than hay, highlighting a potential danger for the animals. However, the plasma concentrations of these heavy metals were not different between HAY and foliage fed goats, suggesting no health risk for the animals. We are not aware of any other study dealing with the uptake of heavy metals from poplar and willow foliage by goats. Some studies showed that goats grazing on pastures near petroleum industry (Ajarem et al., 2023) or mining areas (Bataa et al., 2022) had higher blood heavy metal concentrations than goats that grazed on pastures further away. However, the increased concentrations can also be due to high heavy metal concentrations in the drinking water in these areas (Bataa et al., 2022).

Except for Se, plasma mineral concentrations (Ca, P, Na, Mg, K, Cu, Zn, Fe, Se, Mo, Ba, and Pb) did not differ between the groups. The higher plasma Se concentrations in goats fed the PNIG compared to HAY diet can be attributed to the higher Se content of black poplar foliage. However, balsam poplar leaves had the highest Se content, but goats fed this diet had the lowest plasma Se concentration. This effect cannot fully be explained by differences in Se intake (PMAX: 0.25 mg/d; PNIG: 0.23 mg/d), suggesting that the absorption of Se from balsam poplar is lower than from black poplar. Differences in Se bioavailability could also explain the higher plasma Se concentrations in SVIM wethers despite the lower intake (0.13 mg/d) compared to PMAX wethers. To verify this hypothesis, more research is needed to elucidate the inorganic and organic Se sources in poplar and willow leaves.

4.2 Experiment 2

4.2.1 Antioxidative status of early lactating goats

In contrast to our hypothesis, supplementing goats in early lactation with black poplar leaves did not alter the antioxidative stress status, although goats doubled the leaf intake in the first 5 weeks pp. Goats fed 3.5% Moringa oleifera leaf powder in the periparturient period had an 18–44% higher TEAC_(ABTS) than the control group (Afzal et al., 2022). Furthermore, feeding a 1:1 mixture of eucalyptus and poplar leaves powder (150 g/d) halved the concentration of malonyl dialdehyde, a lipid peroxidation maker, in erythrocytes of buffalo calves (Kumar et al., 2022). However, Schwerdtfeger et al. (2025) observed no differences in plasma TEAC_(ABTS) and FRAP between dairy cows supplemented or not with 7.4% dried ground hemp leaves. These and our results show that not only the variety and portion of leaves in the diet, the degree of grinding, and the antioxidant content in leaves determine the plasma antioxidant concentration. The antioxidant profile and the absorption of individual antioxidants are further contributors, whereby black-poplar foliage fed in our study revealed a rather low bioavailability of antioxidants. However, POPLAR feeding resulted on average in a 2.7-fold higher Se intake and tendency towards higher plasma Se concentrations compared to the CON group. This highlights the potential of black poplar to meet some of the Se demand and to spare inorganic Se originating from fossil sources. Our results further show a direct correlation between plasma Se concentrations and blood GPx activity during the pp period, which reflects the role of Se as co-factor for GPx. Interestingly, the inclusion of 20% mulberry leaf silage to the diet of goats for 80 days also increased GPx activities (Wang and Luo, 2021), but if this effect was caused by a higher Se intake remained unclear. There may be a delay in GPx activity increase in response to Se intake, because GPx is mostly present in erythrocytes (Knowles et al., 1999), which have a lifespan of 120 days while erythropoiesis takes 5 to 7 days (Glenn and Armstrong, 2019). If this delay explains the lack of GPx activity increase during the 5-week feeding period in our study remains elusive.

4.2.2 Calcium and bone mobilization in the periparturient period

In the pp period, serum Ca concentrations averaged approximately 2.2 ± 0.1 mmol/L and did not change over time, which is consistent with stable plasma total Ca concentrations for 10 days pp in goats fed hay (2.4 ± 0.1 mmol/L; Wilkens et al., 2014). A nadir in Ca occurring at or up to 4 days after parturition of goats has been reported (Brugger and Liesegang, 2025), which unfortunately could not be detected here due to our weekly sampling scheme. However, we found a group × time effect with lower Ca concentrations in the POPLAR than CON group in week 2 pp despite POPLAR goats having a higher Ca intake than CON goats (13.4 g/d vs 5.5 g/d). It was shown that tannins may reduce Ca absorption, in rats (Chang et al., 1994) and rabbits (Al-Mamary et al., 2001), whereas its effect in ruminants remains elusive (Naumann et al., 2017). Yet, Ca enhances the binding between tannins and proteins, which presumably reduces the absorption of Ca itself (Naumann et al., 2017). On the other hand, the CON group could have adapted to the lower dietary Ca supply by increased intestinal Ca absorption (Wilkens et al., 2012), explaining the higher serum Ca concentrations in week 2 pp, as the Ca homeostasis is maintained by an interplay of gastrointestinal absorption, renal excretion and accretion and mobilization from the skeleton (Wilkens et al., 2020).

The significant decline in plasma OC together with the increase in CTX from week 1 ap until week 2 pp suggest reduced bone formation and intensified bone mineral mobilization in both dietary groups. But there were no differences in plasma OC and CTX concentrations between groups, likely because a higher dietary Ca intake, at least ap, does not affect serum OC and CTX concentrations (Brugger and Liesegang, 2025). However, the OC/CTX ratio, reflecting the balance of bone formation and resorption (Wilkens et al., 2014), was lower in the POPLAR compared to the CON group in the first 2 weeks pp, suggesting a more intense bone mobilization and greater challenge of Ca homeostasis in the POPLAR group. Taken together, tannin-rich poplar supplementation should be avoided, at least in the first 2 weeks pp, to prevent challenges to serum Ca levels and bone mineral mobilization.

4.2.3 Energy and protein status of early lactating goats

There were no statistical differences in NEFA concentrations between the dietary groups. Plasma NEFA concentrations increased from week 1 ap to week 1 pp in response to parturition-induced endocrine changes stimulating lipolysis (Bell and Bauman, 1997). In the pp period, NEFA correlated inversely with DMI, declined until week 5 pp but did not reach the level of the ap period. This result indicates that the ME intake was not sufficient to meet the energy requirements of lactation, resulting in an increase in body fat mobilization and a negative energy balance. The NEFA course observed in the present study is comparable to multiparous goats fed a diet containing 10.4 MJ per kg of DM (Magistrelli and Rosi, 2014) or hay and concentrate (Radin et al., 2015).

Plasma glucose concentrations sharply increased on the day of delivery or shortly before due to parturition-induced endocrine changes stimulating gluconeogenesis (Bell and Bauman, 1997). Similarly, we found greater glucose concentrations in week 1 ap compared to the pp period. Further, plasma glucose concentrations in week 1 ap were greater in the CON than POPLAR group, but this effect could also be due to variations in sampling times within week 1 ap, which were 7 to 0 days before lambing for the POPLAR group and 6 to 0 days before lambing for the CON group. In addition, 4 out of 7 animals of the CON group and only 2 out of 9 animals of the POPLAR were sampled in a period from 3 to 0 days before lambing, the period with the strongest increase in glucose concentration (Zamuner et al., 2020). Thus, different sampling days and numbers of animals sampled between day 3 and 0 before lambing may further explain the difference in ap glucose concentrations.

4.3 Limitations of the study

The crossover design in Experiment 1 was used following the guidelines for the planning of digestibility trials by Schiemann (1981), which allowed each animal to serve as its own control, thereby reducing between-animal variability. However, the limited number of animals restricts statistical power, particularly for outcomes with high biological variability such as mineral balance and methane emissions. Consequently, non-significant results should be interpreted with caution, as the study lacked sufficient power to detect moderate treatment effects. Future studies involving a larger number of animals per tree species would allow us to refine and validate the current findings. Further, the nutrient concentrations of foliage depend on many factors, e.g. the age of the leaves, the location of the plants, and the compositions of the soil and water, which do not allow for the generalization of our findings. To improve the comparability between studies, future works should document the environmental conditions influencing foliage constituents. Due to the design of Experiment 2, the effects on milk production, reproductive performance, and long-term mineral homeostasis could not be addressed but should be analyzed in the future.

4.4 Conclusion

Willow, balsam, and black poplar foliage exhibited superior CP, crude ash, lignin and tannin concentrations and antioxidant capacity, whereas fiber content was lower than in hay. Compared to hay, none of the leaves increased plasma heavy metal concentrations, but black poplar supplementation increased plasma selenium levels, numerically improved CP and organic matter digestibility and reduced methane yield, whereas these effects were less or not prominent with willow or balsam poplar supplementation. These results demonstrate that black poplar foliage is a valuable feed resource for non-lactating goats. For early lactating goats, black poplar feeding also increased plasma selenium concentrations but did not ameliorate the antioxidant status; rather, it compromised plasma calcium concentration and bone metabolism, likely due to the tannin content of the leaves. Thus, black poplar supplementation is not recommended in early lactation. The long-term effects on growth, milk production, reproductive performance, and mineral homeostasis remain unclear and need still further investigation.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fanim.2026.1861647/full#supplementary-material

Supplementary material Supplementary material at the source

Footnotes

Edited by: Matheus Ramalho De Lima, Federal University Rural Semi-Arid, Brazil

Reviewed by: Nathaniel Ogunkunle, Alabama Agricultural and Mechanical University, United States

Attapol Tiantong, Silpakorn University, Thailand