Nutritional supplement based on spent substrate of the edible mushroom Pleurotus ostreatus for stable sheep

Abstract

Agro-industrial activities generate large volumes of organic residues that are frequently underutilized or improperly managed, contributing to environmental pollution, greenhouse gas emissions, and nutrient losses. Therefore, it is necessary to promote the valorization of these by-products through integrated production systems. One such strategy involves the use of agro-industrial wastes as substrates for cultivating Pleurotus ostreatus, followed by the reutilization of the resulting spent mushroom substrate (SMS) as animal feed. This study evaluated the feasibility of using SMS from P. ostreatus as a functional dietary component for sheep. The objective was to determine whether supplementation with SMS could maintain productive performance while contributing to the revalorization of agro-industrial residues. Twenty mixed-breed Dorper × Katahdin sheep were assigned to either a control diet or a diet supplemented with 20% SMS (dry matter basis) over 30 days (5 days of adaptation and 25 days of experimentation). Diets were chemically characterized, and response variables included voluntary dry matter intake, body weight, daily weight gain, feed conversion ratio, and body condition score. Data were analyzed using one-way ANOVA or non-parametric tests. The SMS-supplemented diet showed higher crude protein (5%) and ash content (6%), and lower fiber levels (6%) compared to the control. No significant differences were observed in intake, weight gain, or body condition. However, animals receiving SMS exhibited improved feed conversion efficiency, indicating enhanced nutrient utilization without compromising performance. These findings demonstrate that P. ostreatus SMS can be effectively integrated into sheep diets, providing a sustainable alternative feed ingredient and supporting circular economy strategies in agro-industrial systems.

Introduction

Agro-industrial activities generate an enormous quantity of organic residues and by-products worldwide, many of which remain underutilized or are disposed of inadequately. The continuous increase in food production and processing has intensified the generation of agricultural and agro-industrial wastes, creating important environmental, economic, and public health concerns (Chikezie and Nnaemeka 2023). Conventional disposal practices, such as landfilling or uncontrolled decomposition, contribute to greenhouse gas emissions, soil and water contamination, nutrient losses, and increased waste management costs (Gómez-García et al. 2021).Within this context, the circular economy has emerged as an alternative aimed at reducing waste generation and reintegrating agro-industrial by-products into productive systems through reuse, recycling, and valorization strategies (Chiaraluce et al. 2021). One key argument for circular economy is the reintegration of agro-industrial by-products into value chains to reduce waste, costs, and environmental footprint (Fernández-Ochoa et al. 2021; Barros et al. 2023). One underused source of food is spent mushroom substrate (SMS), which is generated in vast volumes after harvesting mushrooms (Green and Wösten 2018). Yet, the SMS still contain partially degraded lignocellulosic fiber and bioactive compounds (Green and Wösten 2018) that can be reintegrated to food chains. In the case of mushrooms, their enzymatic capacity to modify lignin and hemicellulose (Andlar et al. 2018) makes SMS a promising candidate for ruminant feeding, as the rumen microbiota can further valorize this carbon source, thereby aligning livestock productivity with sustainable residue management (Baptista et al. 2023).

In recent years, several studies have evaluated P. ostreatus SMS in sheep diets (e.g. Lu et al. 2024a; Crisostomo et al. 2025). For example, replacing a fraction of the forage (e.g., whole-plant corn silage) with 5–10% SMS improved apparent digestibility without adverse effects on liver integrity or on the structure of the duodenal microbial community in sheep (e.g., Hu breed) (Lu et al. 2024b). Because of its initial moisture and fermentative profile, SMS can be ensiled to stabilize it and improve palatability. In support of this, silages formulated with P. ostreatus SMS achieve fermentation parameters comparable to high-quality silages and favorable organoleptic traits, supporting its nutritional viability and practical applicability as a sustainable feed alternative (Lorenzana-Moreno et al. 2025). In dairy ruminants and goats, complementary strategies—such as enzyme extracts derived from SMS or co-ensiling with forages—have shown improved feed utilization and technological outcomes (e.g., cheese yield), pointing to positive externalities beyond a simple fiber contribution (Trejo et al. 2021).

In this sense, it was proposed to evaluate the efficiency of using Pleurotus ostreatus SMS as feed for sheep, under a practical supplementation scheme that accounts for acceptability, voluntary intake, weight gain, and body condition. Our working hypothesis is that moderate inclusion of SMS—consistent with the literature—will maintain or improve productive performance without compromising animal health, while valorizing a residue with economic and environmental potential for sheep production systems.

Methods

Location and animal subjects

The experimental site was the “Rancho Cuevas” located in the municipality of Xochitepec, in the town of Alpuyeca, Morelos, Mexico (18°45’43.8“N 99°16’24.5”W). The experimental sheep were selected at the discretion of the cooperating producer and consisted of females (2–4 years old) and males (< 1 year old) of mixed Dorper and Katahdin breeds. Animals were randomly allocated to both experimental and control groups (see below).

Production of spent substrate from Pleurotus ostreatus

Corn cobs (Zea mays), pangola grass (Digitaria eriantha), and coffee pulp (Coffea canephora) were used as the substrate in a 1:1:1 ratio, and sterilized at 121 °C for 30 min. The substrate was inoculated with 2.5% P. ostreatus spawn and kept in perforated bags for 15 days at 24 °C. Cultures were maintained at 85% relative humidity, 22 °C, under natural light and ventilation for 40 days (Avendaño-Hernández and Sánchez 2013). During this period, basidiomes were harvested and the substrate was shade-dried for 5 days to preferably reach an 8% moisture content. Two kilograms of the degraded substrate were reserved for in vitro assays and bioassay-guided chemical fractionation. Finally, the remaining substrate was milled using a 1 cm mesh screen for in vivo tests.

Supplementation with spent substrate from P. ostreatus in stabled sheep

To evaluate supplementation with the spent substrate from P. ostreatus, an amount of feed equivalent to 3% of each animal’s live weight on a dry matter (DM) basis was offered. Twenty animals were used, 10 per group. The control group (from now on, control) received a balanced feed diet, while the experimental group (from now on, experimental) received a diet supplemented with the spent substrate of P. ostreatus (Table 1). Inclusion of the degraded substrate in the diet was 20%, based on previous results showing the best acceptability (all authors’ unpub. data). The feeding period lasted 30 days and was divided into two phases: (1) 5 days of adaptation and (2) 25 days for measuring voluntary intake (experimental period; see also Can Celis et al. 2022; Lu et al. 2024a, b). For each group, live weight and body condition were obtained at the beginning and at the end of the experiment.

Table 1.

Food composition with and without supplementation of spent substrate from P. ostreatus. Quantities are given per 100 kg of feed

Components	Control group (kg)	Experimental group (with spent substrate) (kg)
Jicama peel	10.00	0.00
Cottonseed hulls	10.00	0.00
Balanced feed	20.00	20.00
Cracked corn	13.33	13.33
Ground ear corn	13.33	13.33
Soybean meal	6.67	6.67
Sequestering agent (binder)	0.83	0.83
Whole sorghum	15.00	15.00
Micronutrient premix	1.00	1.00
Molasses	5.67	5.67
Vegetal oil	0.83	0.83
Alfalfa	3.33	3.33
SMS of P. ostreatus	0.00	20.00

Analysis of the nutritional value of diets

Contents of dry matter (AOAC, 1980), crude protein (AOAC Method 990.03, 1980), neutral detergent fiber (ANKOM Method 6), acid detergent fiber (ANKOM Method 5), lignin (ANKOM Method 8), ether extract (AOAC Method 920.39, 1980), and ash (Method NMX-Y-093-2003) were determined. In addition, the total phenol content was determined using the Folin-Ciocalteau method, the total tannin content using the Folin-Ciocalteau + Polyvinylpyridone (PVPP) method, and the vanillin content of both diets using the vanillin method (Price et al. 1978).

Voluntary intake

During the experimental period, the food offered to and refused by each animal was weighed daily to calculate voluntary intake. This value, together with the DM content of each food, was used to estimate dry matter intake (DMI).

Weight gain

Animals were weighed weekly, and the amount of food offered was adjusted based on body weight. Daily weight gain and total weight gain were calculated using the following formulas:

Statistical analysis

Data on total weight gain and daily weight gain were analyzed using a one-way analysis of variance and post-hoc Tukey’s mean comparison test. For dry matter intake, the Kruskal-Wallis test was used, followed by Dunn’s test to determine statistical differences. For statistical analysis, R software version 3.6.3 was used.

Results

Nutritional value of diets

The experimental diet contained higher amounts of crude protein, ash, and ether extract, while the control diet showed a higher content of the other nutrients evaluated (Table 2).

Table 2.

Nutritional composition of the control and experimental diets (supplemented with SMS)

Analysis	Control diet (%)	Experimental diet (%)
Dry matter	94.4	90.1
Crude protein	14.41	19.43
Neutral detergent fiber	54.66	48.12
Acid detergent fiber	20.72	16.20
Lignin	6.91	6.03
Ashes	6.74	12.83
Ether extract	2.57	2.74
Total phenols	0.6	0.5
Total tanins	0.4	0.2

Supplementation with spent substrate from P. ostreatus in stabled sheep

There was no difference in weight gain, daily weight gain, and dry matter intake for both groups (Table 3). The average body condition score increased for both groups and also revealed no significant differences. However, note that feed conversion was more efficient in the experimental group.

Table 3.

Average of pre- and post-treatment, response variables in sheep with and without supplementation of spent substrate from P. ostreatus

Variable	Control group	Experimental group (with SMS)
Initial animal weight	40.77 ± 4.87	37.01 ± 8.37
Final animal weight	44.46 ± 5.70	41.54 ± 9.93
Total weight gain (kg)	3.69	4.53 (NS)
Daily weight gain (g)	153.83	188.91 (NS)
Consumption of DM (kg/día)	1.24	1.11 (NS)
Feed conversion	8.06	5.87
BC pre-treatment	3.1	3.0
BC post-treatment	3.6	3.4

BC: Body condition; DM: dry matter. NS = non significant differences between control and experimental groups

Discussion

From a circular economy perspective, the innovation of the present work lies in the cascading use of agro-industrial residues through a dual-purpose system: first as a substrate for Pleurotus ostreatus production and subsequently as a functional feed ingredient for ruminants. This approach aligns with biomass valorization strategies that prioritize waste minimization and resource efficiency by extending the lifecycle of organic materials (Geissdoerfer et al. 2017; Ravindran and Jaiswal 2016). The bioconversion of lignocellulosic residues by white-rot fungi not only generates edible mushrooms but also transforms low-value waste into nutritionally improved biomass suitable for animal feeding (Van Kuijk et al. 2015).

Importantly, agro-industrial residues are often associated with methane emissions and nutrient leaching when improperly disposed (FAO 2011). Moreover, their incorporation into livestock feeding systems reduces environmental burdens while simultaneously decreasing dependence on conventional feed resources, which are frequently linked to high economic and ecological costs (Makkar 2018). Thus, the use of SMS represents a practical and scalable strategy to close nutrient loops, reduce waste accumulation, and enhance sustainability in small-ruminant production systems. This integrative approach highlights the broader impact of the present study beyond animal performance, positioning SMS utilization as a key component in sustainable agro-industrial management.

This study showed that supplementation of sheep diets with SMS from Pleurotus ostreatus at a 20% inclusion rate maintained a productive performance comparable to a conventional balanced diet. Sheep receiving SMS supplementation had no significant differences in total weight gain, average daily gain, dry matter intake, or body condition score when compared to the control group, while exhibiting a numerically improved feed conversion ratio. These results support the hypothesis that P. ostreatus SMS can be incorporated into practical feeding strategies without detrimental effects on animal performance.

An absence in weight gain and intake is not new: previous studies evaluating SMS inclusion have found similar results using small ruminant diets. For instance, Lu et al. (2024a) reported that inclusion levels of 5–10% SMS in Hu sheep diets did not negatively affect growth performance or ruminal fermentation parameters, while Crisostomo et al. (2025) showed that SMS from P. ostreatus sustain adequate digestibility and fermentation profiles in vitro and in vivo. Although the inclusion rate used in our study was higher (20%), the lack of adverse effects suggests that sheep can efficiently utilize SMS when it is properly processed and integrated into a balanced ration. Of course, more studies are needed to confirm/reject our results.

The nutritional composition of the experimental diet helps explain these findings. The SMS-supplemented diet exhibited higher crude protein, ether extract, and ash content, alongside reduced fiber fractions (NDF and ADF), likely reflecting the partial lignocellulosic degradation mediated by P. ostreatus (Andlar et al. 2018). White-rot fungi are well known for their ability to selectively degrade lignin through extracellular oxidative enzymes, improving fiber accessibility to ruminal microorganisms (Zhao et al. 2015; Wang et al. 2023). This biochemical modification may enhance ruminal fiber utilization and contribute to the improved feed conversion observed in the experimental group, despite similar intake levels. Note that the relatively low concentrations of total phenols and tannins detected in both diets indicate that secondary plant metabolites were unlikely to exert anti-nutritional effects. This aligns with observations by Lorenzana-Moreno et al. (2025), who reported that phenolic compounds in SMS-based silages remained within ranges compatible with normal ruminal fermentation and digestibility. The absence of negative effects on intake and body condition further supports the notion that SMS from P. ostreatus is palatable and safe for sheep under short-term supplementation.

The improved feed conversion ratio observed in the SMS group, although not subjected to statistical testing, is biologically relevant. Similar trends have been reported in sheep and goats supplemented with SMS or SMS-derived products, where improved efficiency has been attributed to better nutrient synchronization in the rumen and increased microbial protein synthesis (Lu et al. 2024b; Trejo-López et al. 2021). From a production standpoint, improved feed efficiency is particularly relevant in extensive or semi-intensive systems, where feed costs represent a major economic constraint (FAO 2012).

Taken together, the results of this study reinforce the role of SMS as a functional feed ingredient rather than merely a low-cost fiber source. Its integration into sheep diets contributes not only to maintaining animal performance but also to advancing circular economy principles by revalorizing agro-industrial residues that would otherwise represent an environmental burden (Rinker 2017; Ravlikovsky et al. 2024).

Conclusions

Supplementation of sheep diets with spent substrate from Pleurotus ostreatus at a 20% inclusion level maintained body weight gain, daily weight gain, dry matter intake, and body condition comparable to those observed with a conventional balanced diet. The SMS-supplemented diet showed favorable nutritional characteristics, including higher crude protein content and reduced fiber fractions, and resulted in improved feed conversion efficiency.

Beyond its nutritional value, the innovation of the present study resides in the integration of agro-industrial waste valorization with animal production systems through a circular economy approach. By converting agricultural residues into mushroom biomass and subsequently reutilizing the degraded substrate as feed, this strategy reduces waste disposal, mitigates environmental impacts, and improves resource-use efficiency. Therefore, P. ostreatus SMS represents a viable, sustainable, and scalable feed ingredient capable of partially replacing conventional inputs while contributing to waste reduction and the development of more resilient and circular livestock production systems.

Author contributions

Conceptualization, T-AJFJ; P-AJA, and A-ML; writing—original draft preparation, P-AJA; C-AA and A-ML; Methodology, P-AJA, M-RL, A-GRH; writing—review and editing, P-AJA, C-AA, A-ML. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Agencia Mexicana de Cooperación Internacional para el Desarrollo (AMEXCID) and Agencia Chilena de Cooperación Internacional para el Desarrollo (AGCID), via the project "Intercambio de tecnología para el control de vectores y nemátodos gastrointestinales".

Data availability

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Declarations

Ethical approval

Animal handling and feeding protocol were carried out in accordance with the official Mexican standards NOM-024-ZOO-1995 (SAGAR 1995a) and NOM-051-ZOO-1995 (SAGAR 1995b).

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.