Effect of the Replacement of Wheat Straw by Spent Mushroom Substrate in the Diet of Dairy Ewes during Late Lactation on Milk Production, Composition, Oxidation Stability and Udder Health

Abstract

The aim of the present study was to investigate the effects of different dietary levels of spent mushroom substrate (SMS) at the expense of wheat straw (WS) on milk characteristics in dairy sheep. Thirty ewes at their final stage of lactation (145 ± 5 days after parturition) were randomly assigned into three groups; control (C), provided with a diet consisting of concentrates, alfalfa hay and WS, and SMS1 and SMS2, where WS was replaced by SMS at 50 or 100%, respectively. The experiment lasted for 28 days, and milk yield, composition, somatic cell count (SCC) and oxidative stability were monitored weekly, while milk fatty acid and immune cell profile were also determined on day 28. No significant differences were found in produced milk quantity, fat, protein, lactose, total solids non-fat, SCC and fatty acid profile between the experimental groups. However, milk oxidative stability was significantly improved as an effect of SMS (p < 0.001). At the same time, milk polymorphonuclear leukocyte percentage was decreased in SMS2 group (p < 0.05). As indicated, SMS seems to be a promising agro-industrial by-product for ewes’ diet that could improve milk oxidative stability, without negatively affecting milk yield, composition and ewe health status, contributing in the context of sustainability, circular economy and protection of natural resources.

1. Introduction

Spent mushroom substrate (SMS) can be defined as the exhausted residual lignocellulosic biomass left after the harvest of mushrooms, containing large qualities of mycelium. Various decomposed lignocellulosic residues, e.g., straw, sawdust, wood chips and shavings, corn cobs, cottonseed hulls, livestock litter and manure, etc., not completely degraded by the fungi during the fermentation procedure, additives, such as lime, peat, and gypsum, nutrients, such as polysaccharides and polypeptides, phenolic compounds and an increased content of enzymes and organic matter were previously detected in SMS [1,2,3]. Particularly, SMS contained around up to 35% dry matter, 20% organic matter and 13% ash, and up to 39.8% lignin, 34% hemicellulose and 48.7% cellulose, depending on the mushroom strain and the cultivation substrate, as seen in previous studies [1,4,5]. Enzymes, such as laccase, lignin peroxidase, Mn-peroxidase, versatile peroxidase, xylanase, β-glucanase and phytase, were also active as a significant amount in SMS from various mushroom crops [5,6,7], as well as small numbers of vitamins and minerals. However, the composition of SMS usually differs as an effect of the raw materials, the location, the mushroom genus and the cultivation method; therefore. it is essential that the composition of each SMS is determined before its evaluation for re-use [1,5].

The upsurge in consumer demand for healthy and protein-rich foods has boosted cultivations of edible mushrooms [8]. Production of mushrooms and truffles has increased worldwide by 65% during the recent years, to 48.34 million metric tons (MTs) in 2022 from 31.78 million MTs in 2012 [9]. China was the prevailing mushroom producer country in 2022 with 45.43 million MTs, while the EU, France, Spain, Netherlands and Poland were the major producers with 102,000, 167,000, 235,000 and 257,000 MTs, respectively [9]. As Pleurotus is the second most widely cultivated mushroom species worldwide [10], and approximately 5 kg of SMS are generated per kilogram of fresh mushroom [11,12], it can be concluded that disposal of SMS wastes is a major problem, since accumulation of tons of an agricultural waste with high organic load pose a risk to the environment if not appropriately handled [13]. In the past, discarding by disposal, burying, or landfilling and incineration or burning methods were used that are nowadays not accepted in the context of sustainability, the circular economy and protection of natural resources [14]. Efficient re-utilization and proper evaluation of SMS is therefore necessary [15]. For example, there have been studies about the potential of using SMS to produce value-added products, such as lignocellulosic enzymes, i.e., hemicellulose, cellulose, xylanase, laccase and lignin peroxidase [7], and regarding its re-utilization in second-cycled mushroom crops [1].

SMS properties and synthesis are influenced by the raw materials and supplements used to compose the initial mushroom substrate. In general, it is a lignocellulosic biomass, i.e., beech wood shavings, coffee residue, rice bark, barley and oats straw, wheat straw and corncob that contain several useful nutrients, such as polysaccharides, i.e., cellulose and hemicellulose, proteins (as extra-cellular enzymes), vitamins and some trace elements, namely magnesium (Mg), zinc (Zn), calcium (Ca) and iron (Fe) [1,14,16], which make SMS suitable for ruminant feedstock [17]. According to Adamović et al. [18], the most important modification in SMS composition is the reduction of hemicelluloses, cellulose and lignin by 17, 15 and 4%, respectively, since they are broken down by enzymes secreted during mushroom production and mycelium growth, leading to an increase in the in vivo SMS dry matter digestibility in ruminants [19]. However, SMS is abundant in lignin that still diminishes feed digestibility, so biological treatments, such as fermentation, could be applied before its incorporation into animal diets to improve its nutritional value by enhancing ash, fat and crude protein content and lowering the amount of crude fiber, resulting in an increase in supplementation level into livestock diets [20].

The high cost of animal feed poses a significant challenge to livestock production. As a result, utilizing agro-industrial by-products as alternative feed resources is a practical approach in low-input systems. This strategy aims to overcome the issue of expensive conventional feedstuffs and provide a cost-effective solution for livestock farmers in these regions. By the application of agro-industrial by-products, such as crop residues or agricultural waste, farmers can reduce their reliance on costly feed options and improve the sustainability of their livestock production. This approach not only helps to mitigate the financial burden on farmers but also promotes the efficient use of available resources and contributes to the overall development of a resilient livestock sector in semi-arid and arid countries [21].

Utilization of SMS in ruminant diets has been steadily gaining ground, since they are herbivores and can digest large quantities of lignocellulosic biomass. However, although SMS is an emerging feed material, no clear requirements in the current feed hygiene standards and feed material catalogue exist [5]. Pleurotus ostreatus SMS has been already evaluated as an ingredient in cattle diets as raw, just dried and milled [22] or after fermentation with bacterial inoculation [22,23]. At the same time, SMS-based silage could reduce rumen protozoa populations leading to the mitigation of rumen methanogenesis in steers [24]. According to the literature, scarce data exist on the effects of using SMS as a dietary ingredient in lactating animals and especially small ruminants [25,26]. As a result, the objective of the present study was to investigate the effects of the replacement of wheat straw by spent mushroom substrate on milk production, composition, oxidative stability and udder health in dairy ewes during the final stage of lactation.

2. Materials and Methods

2.1. Animals

Thirty 3-years-old Karagouniko dairy ewes, with a mean weight of 59.2 ± 1.8 kg and comparable body condition score (2.5–3.0) during their second parity and 145 ± 5 days after parturition were selected at random from the sheep herd bred on the facilities of the Agricultural University of Athens. Synchronization of ewes was carried out by intravaginal progestogen sponges (Ovigest, Girona, Spain).

At the beginning of the experiment, all animals were provided with concentrates, alfalfa hay and wheat straw at a ratio of 1:0.6:0.4 (

Table 1. Dietary formula and analysis.

Ingredients (%)
Corn	23.4
Wheat	17.5
Barley	17.5
Soybean Meal (44%)	18.25
Sunflower Meal (28%)	5.0
Wheat Bran	15.0
Sodium Chloride (NaCl)	1.0
Limestone	1.85
Monocalcium Phosphate	0.4
Vitamins and Trace elements Premix *	0.1
Analysis †	Concentrates	Alfalfa hay	Wheat straw	SMS
Dry Matter (%)	86.0	93.5	95.2	94.0
Crude protein (%)	17.0	10.2	3.4	4.7
Crude Fiber (%)	6.0	34.2	51.7	26.7
Ash (%)	6.5	7.4	7.2	18.4
Fat (%)	2.1	2.3	1.0	1.4

* Premix contained per kg: 150 mg, 35 mg Mn, 50 mg Fe, 60 mg Zn, 0.8 mg Se, 0.75 mg Co, 1.25 mg I, 200 mg Mo, 15 kIU vitamin A, 2 kIU vitamin D3, 25 mg vitamin E (kIU: 1000 international units). ^† Dry matter was measured gravimetrically, after drying at 105 °C for 24 h [27], total nitrogen according to the Kjeldahl Method [28] and multiplied by 6.25 for protein determination, crude fiber based on the method of Goering and Van Soest [29], ash content according to Sparks [30], and fat levels gravimetrically using a mixture of chloroform/methanol 2:1 (v/v) as the extracting solvent [31,32].

) for a week, serving as an acclimation period. After this adaptation period, they were further assigned into three treatment groups and were offered the experimental diets for 4 weeks; one of the groups was designated as the control (C) and was fed with a diet consisting of concentrates, alfalfa hay and wheat straw at a ratio of 1:0.6:0.4, whereas in the other two groups wheat straw was replaced by SMS at 50% or 100% (SMS1 and SMS2, respectively). Groups were formed according to the body weight and milk yield of ewes at the beginning of the study (58.80 vs. 59.40 vs. 59.50 ± 1.67 kg and 820 vs. 800 vs. 815 ± 97 mL for C, SMS1 and SMS2, respectively). Commercial SMS deriving from industrial-scale experiments of Pleurotus ostreatus production in wheat straw as main substrate, conducted by Manitus S.A. (Athens, Greece), was used. Ewes were provided with approximately 2.0 kg of feed (concentrate and forage at a mean ratio of 50:50) in two meals. At 7:00 am, 500 g of concentrates and 500 g of alfalfa hay were provided to the three experimental groups while at 17:00 p.m., 500 g of concentrates, 100 g of alfalfa hay and (1) 400 g of wheat straw (C), or (2) 200 g of wheat straw and 200 g of mushroom spent (SMS1) or (3) 400 g of mushroom spent (SMS2) were offered.

Each group of dairy ewes was housed in an individual pen at the experimental station of the Agricultural University of Athens, which was divided into an outdoor and indoor area and had the same covered area (3 m²/ewe), similar orientation, and was equipped with 10 individual troughs for feeding. Animals had free access to water and the diet was formulated to marginally exceed ewes’ individual requirements, based on their milk yield and body weight [33]. Forage was offered to the ewes after the complete consumption of concentrate. No refusals of concentrates and/or forage were recorded, apart from 2 days during the first sampling of the main experimental period, when ambient temperature exceeded 35 °C.

2.2. Milk Yield, Composition and Oxidative Stability

Milking of ewes was carried out twice daily, at 6:00 a.m. and 18:00 p.m., in a 12-stall milking parlor (Westfalia, Radar-Wiedenbrück, Germany). Vacuum level was 37.5 kPa, while pulsation rate was 150 cycles min⁻¹. Milk yield, calculated as the total quantity of the collected milk during the morning and afternoon, was determined on day 1 prior to, and on days 7, 14, 21 and 28 after, SMS addition. Collection of individual milk samples in tubes appropriately labeled were also carried out on acclimation week and at weeks 1–4 after SMS dietary supplementation for the direct assessment of lactose, protein, fat and total solids-not-fat content, while pH and somatic cell count was also measured by using the Lactoscan COMBO Milk Cell Analyser (Lactosan, Nova Zagora, Bulgaria). Milk oxidative stability was determined by measuring malondialdehyde (MDA) concentration (ng/mL) [34].

2.3. Milk Fatty Acid Profile

Milk fatty acids composition was established by direct methylation of lyophilized milk samples as previously described by Massouras et al. [35]. Briefly, a quantity of 150 mg lyophilized milk was directly methylated with 2 mL of 0.5 M sodium methylate and 2 mL of 140 g L⁻¹ boron trifluoride in methanol (BF3) at 50 °C for 30 min. Fatty acid methyl esters (FAMES) were quantified using a Shimadzu gas chromatograph (model GC-17A, Kyoto, Japan) equipped with flame ionization detector (FID). A SP-2560 capillary column (60 m × 0.25 mm I.D., 0.20 µm; Supelco, Bellefonte, PA, USA) was used to separate FAMES. The split ratio was 1:50 and helium was used as the carrier with a flow rate at 1.8 mL min⁻¹. The temperatures of injector and detector were 250 °C and 270 °C, respectively. The injection volume was 1 μL. The operating conditions are described in detail by Massouras et al. [35]. Individual fatty acids were identified by comparing the retention times and areas of their peaks to those obtained from the FAME reference standards (Supelco 37 Component FAME Mix, Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). A GC Solution software version 2.30 (Shimadzu Corporation, Kyoto, Japan) was used for the integration of the peaks. The individual fatty acid (FA) content was expressed as a weight percentage (% w/w) of total FAMES. The different groups of FA were determined according to the following formulas:

Saturated fatty acids (SFA) = C4:0 + C6:0 + C8:0 + C10:0 + C12:0 + C14:0 + C16:0 + C18:0 + C20:0; Monounsaturated fatty acids (MUFA) = C14:1 + C16:1n-7 + C18:1n-9; Polyunsaturated fatty acids (PUFA) = CLA + C18:2n-6 + C18:3n-3.

2.4. Isolation of Milk Somatic Cells and Milk Somatic Cell Immunophenotyping

At the end of the experiment (day 28), 15 mL milk samples were collected from each ewe and were kept on ice until their transfer to the laboratory for immediate isolation of milk somatic cells. Isolation of milk somatic cells (MSC) was carried out following a modified protocol of Koess and Hamann [36] adapted for sheep milk as described in Karageorgou et al. [37]. Approximately 2 × 105 MSC per sample were then immunophenotyped using cell surface labelling and nuclear dies, in 50 μL staining buffer (PBS containing 0.01% NaAzide and 0.2% BSA) containing 0.002 mg/mL an-ti-CD11b-Fluorescein isothiocyanate (FITC) conjugated (OriGene Techologies, Inc., Rockville, MD, USA), 0.002 mg/mL anti-CD8-R-Phycoerythrin (R-PE) conjugated (OriGene Techologies, Inc., Rockville, MD, USA) and 0.25 μL 200× RedDotTM1 (Bio-tium, San Francisco, CA, USA). Cells were incubated for 30 min in the staining solution at room temperature in the dark. Following incubation, cells were washed with 2 mL dilution buffer and centrifuged at 300× g for 5 min. Cell pellets were resuspended in 10 μL dilution buffer containing 50 μg/mL PI, incubated for 10 min at room temperature in the dark, then 200 μL of dilution buffer were added and samples were analyzed by Flow Cytometry (Cytomics FC 500, Beckman Coulter Inc., Brea, CA, USA).

Instrument voltage/gain for detectors FS (Linear), SS (Linear), FL1 (Log), FL2 (Log), FL3 (Log), FL4 (Log) and AUX (FL4 Linear) were set at 700/2.0, 640/20.0, 550/1.0, 620/1.0, 580/1.0, 500/1.0, 650/20, respectively. Samples were run at maximum speed; 40,000 events were collected and data was stored as list mode files. Nucleated live cells were identified as events of appropriate size and granularity based on their FS/SS dotplot position, negative for PI and positive for RedDotTM1 staining. Classification of live, nucleated cells as polymorphonuclear granulocytes (P), monocytes/macrophages (M) and lymphocytes (L) was further performed as shown in Figure S1.

2.5. Statistical Analysis

A repeated measures analysis of variance was applied to data for milk yield, protein, fat, lactose, total solids-non-fat, pH, somatic cell count, and MDA values with the MIXED procedure of SAS software, version 9.3. The fixed factor was the dietary treatment and the repeated factor was the sampling week. SCC data were not normally distributed and therefore a log transformation was applied in order to meet the ANOVA assumptions. Data for immune cell profile and fatty acid profile were analyzed with the dietary treatment as fixed effect. The linear dose responses to dietary SMS were tested with orthogonal polynomials with the CONTRAST procedure. Multiple comparisons were applied after Bonferroni adjustment. Classification of milk samples according to the dietary treatment with SMS was evaluated by a discriminant analysis of the fatty acid profile. Fatty acids that were mainly responsible for the classification observed were identified by a stepwise discriminant analysis. The significance level was set at 0.05 and results are presented as least square means ± S.E.M.

3. Results

In general, mean daily feed intake was not different between the experimental groups (1.76 vs. 1.78 vs. 1.79 ± 0.05 kg for CONTROL, SMS1 and SMS2 group, respectively). A similar trend was also observed for daily DM intake (1.59 vs. 1.60 vs. 1.60 ± 0.05 kg), CP intake (0.216 vs. 0.220 vs. 0.223 ± 0.006 kg) and ME (15.14 vs. 14.99 vs. 14.70 ± 0.42 MJ) for CONTROL, SMS1 and SMS2 group, respectively (p > 0.05).

As shown, the milk yield (

Table 2. Effect of P. ostreatus spent mushroom substrate (SMS) dietary supplementation on milk yield (mL/day), fat (%), protein (%), lactose (%), total solids-not-fat (%), pH and oxidative stability (ng MDA/kg).

Parameter	Sampling Day	CONTROL 1	SMS1	SMS2	SEM	p-Value	P-Linear
Milk yield (mL/kg)	0	620	625	660	69	NS 2	NS
	7	840	875	875	105	NS	NS
	14	825	795	815	102	NS	NS
	21	795	870	920	98	NS	NS
	28	835	825	845	104	NS	NS
	p-value	NS	NS	NS
Fat (%)	0	5.49	5.37	5.39	0.38	NS	NS
	7	5.08	5.36	5.43	0.35	NS	NS
	14	6.29	6.18	6	0.25	NS	NS
	21	5.96	5.9	5.47	0.37	NS	NS
	28	5.7	5.83	5.75	0.23	NS	NS
	p-value	NS	NS	NS
Protein (%)	0	4.7	4.92	4.82	0.09	NS	NS
	7	4.64	4.62	4.49	0.06	NS	NS
	14	4.67	4.6	4.57	0.11	NS	NS
	21	4.74	4.71	4.69	0.06	NS	NS
	28	5.04	4.93	4.76	0.2	NS	NS
	p-value	NS	NS	NS
Lactose (%)	0	4.45	4.66	4.55	0.08	NS	NS
	7	4.39	4.37	4.25	0.06	NS	NS
	14	4.41	4.35	4.32	0.1	NS	NS
	21	4.48	4.46	4.44	0.06	NS	NS
	28	4.76	4.66	4.51	0.19	NS	NS
	p-value	NS	NS	NS
Total solids-not-fat (%)	0	10.04	10.37	10.14	0.16	NS	NS
	7	9.76	9.73	9.46	0.13	NS	NS
	14	9.82	9.68	9.62	0.22	NS	NS
	21	9.97	9.92	9.88	0.13	NS	NS
	28	10.6	10.37	10.04	0.42	NS	NS
	p-value	NS	NS	NS
pH	0	6.67 A	6.64 A	6.68 A	0.04	NS	NS
	7	6.66 A	6.68 A	6.72 A	0.03	NS	NS
	14	6.6 A	6.57 A	6.65 A	0.06	NS	NS
	21	6.62 A	6.49 A	6.59 A	0.04	NS	NS
	28	7.05 B	6.94 B	7.06 B	0.04	NS	NS
	p-value	<0.001	<0.001	<0.001
Log SCC 3	0	5.01	5.01	4.9	0.16	NS	NS
	7	4.93	4.82	5.21	0.16	NS	NS
	14	4.65	5.01	5.05	0.14	NS	NS
	21	4.54	4.82	5	0.16	NS	NS
	28	5.11	5.18	5.15	0.17	NS	NS
	p-value	NS	NS	NS
MDA 4 (ng/kg)	0	3.48	3.81 A	3.32	0.25	NS	NS
	7	3.59 a	2.32 bB	2.62 b	0.33	<0.05	<0.05
	14	3.75 a	2.99 bAB	2.79 b	0.4	<0.05	NS
	21	3.87 a	2.96 bAB	2.53 b	0.25	<0.01	<0.001
	28	3.68 a	3.24 bAB	2.88 b	0.2	<0.05	<0.01
	p-value	NS	<0.05	NS

¹ Control (C) group was fed with a standard diet, whereas in the other two groups wheat straw was replaced by SMS at 50 or 100% (SMS1 and SMS2, respectively). ² Not significant. ³ Somatic Cell count. ⁴ Malondialdehyde. a, b Values sharing dissimilar superscripts within a row and parameter are significantly different. A, B Values sharing dissimilar superscripts within a column and parameter are significantly different.

) was not significantly influenced by SMS dietary inclusion (p > 0.05). Milk composition was also not different among the experimental groups (p > 0.05). No discernible differences were shown for milk fat, protein, lactose and total solids-not-fat throughout the experiment (Table 2). Μilk pH was also not influenced by the replacement of wheat straw by SMS in the diets of dairy ewes (p > 0.05). The linear dose responses to dietary spent mushroom substrate were not significant for milk yield, composition and pH throughout the experimental period (p > 0.05).

However, milk oxidative stability was generally improved in SMS supplemented groups as indicated by the decreased MDA values. Significant differences were determined on weeks 1, 2, 3 and 4 between the C and the SMS1 and SMS2 groups (p < 0.05; Table 2). The respective MDA values (ng/g) were 3.59 vs. 2.32 and 2.62 (± 0.33) on week 1, 3.75 vs. 2.99 and 2.79 (±0.40) on week 2, 3.87 vs. 2.96 and 2.53 (±0.26) on week 3 and 3.68 vs. 3.24 and 2.88 (±0.20) on week 4 for the C vs. SMS1 and SMS2 groups, respectively. A linear dose–response effect was observed on weeks 1, 3 and 4 (P-linear < 0.05).

No significant effects of SMS on milk fatty acid profile were observed (Table S1; p > 0.05), except for palmitoleic acid (C16:1n7) and pentadecanoic acid (C15:1), which were significantly lower in SMS2 than in the other groups (p < 0.05), and undecanoic acid (C11:0), which increased with increasing levels of SMS (P-linear < 0.05). The proportions of SFA, MUFA and PUFA were not different among the control and SMS1 and SMS2 groups (

Table 3. Effect of P. ostreatus spent mushroom substrate dietary supplementation on milk SFA, MUFA and PUFA of dairy ewes.

Fatty Acid, g/100 g Fat	Treatment 2			SEM	p-Value	P-Linear
	CONTROL	SMS1	SMS2
SFA 1	71.3	72.9	72.0	1.00	0.558	0.642
MUFA	22.5	21.2	21.8	0.88	0.556	0.544
PUFA	3.87	3.64	4.05	0.16	0.196	0.411

¹ SFA: saturated fatty acids, MUFA: monounsaturated fatty acids, PUFA: polyunsaturated fatty acids. ² Control (C) group was fed with a standard diet, whereas in the other two groups wheat straw was replaced by SMS at 50% or 100% (SMS1 and SMS2, respectively).

; p > 0.05). The linear dose responses to dietary mushroom spent substrate were also not significant (P-linear > 0.05).

Stepwise discriminant analysis of classification according to diet type indicated that 9 out of 27 fatty acids contributed to the discrimination of milk samples in dietary treatment groups. These fatty acids were C11:0, C12:0, C15:1, C16:1n7, C17:1, C18:2n6, CLA, C18:3n3 and C20:4n6. Two discriminant functions were shown as significant (p < 0.01) for classification of samples among the different levels of dietary supplementation with SMS for ewes. The graph of the two discriminant functions, presented in Figure 1, shows that milk samples are clearly distinguished in separate clusters in accordance with the dietary treatment.

No significant differences were shown for milk somatic cell count (log SCC) (Table 2) throughout the experiment. The linear dose responses to dietary SMS were also not significant (p > 0.05). Proportions for lymphocyte (L) and the L/(M + P) ratio were also not significantly different among the experimental groups (

Table 4. Effect of dietary supplementation with P. ostreatus spent mushroom substrate on immune cell profile.

	Treatment 1			SEM	p-Value	P-Linear
	CONTROL	SMS1	SMS2
Lymphocyte (L) (%)	11.22	10.52	12.90	2.42	NS	NS
Macrophage (M) (%)	1.02	1.30	1.58	0.19	NS	<0.05
Polymorphonuclear leucocytes (P) (%)	45.94 a	42.73 a	22.82 b	6.52	<0.05	<0.05
L/(M + P)	0.41	0.37	0.78	0.17	NS	NS

¹ Control (C) group was fed with a standard diet, whereas in the other two groups wheat straw was replaced by SMS at 50% or 100% (SMS1 and SMS2, respectively). a, b Values sharing dissimilar superscripts within a row are significantly different.

). However, polymorphonuclear leukocytes (P) proportion decreased with increasing levels of SMS (P-linear < 0.05) with discernible differences observed between the SMS2 and the control and SMS1 groups (p < 0.05). Macrophage (M) proportion increased linearly with increasing SMS levels in the diet of ewes (P-linear < 0.05).

4. Discussion

While incorporation of SMS in lamb diet has been studied [38,39,40], this is the first study on its effects on the performance and other parameters of lactating ewes. Aldoori et al. [38] incorporated dried SMS into the ration of Awassi lambs at up to 20%. The animals also had free access to straw, while the amount of the provided barley was adjusted based on the offered amount of SMS so that all groups would have the same fixed amount of protein. After slaughter, lambs that were fed SMS at a percentage higher than 15% had lower values for body, hot and cold carcass weight, rib eye area and backfat thickness. The authors proposed that P. ostreatus SMS can be used in the nutrition of lambs and serve as a cost-effective solution, but only at a percentage less than 15%. Ethesham and Vakili [39] composted a wheat straw-based mixture with P. ostreatus and provided it to Kurdish male lambs and reported that the weight gain of the lambs decreased when the composted SMS was used at a percentage higher that 25%, since the nutritional value of the diet was reduced and, consequently the growth of the animals. Huang et al. [40] used composted P. eryngii SMS as feed supplement, again up to 45%, in the diet of Hu lambs and observed an improvement in feed conversion ratio and meat quality. As indicated in the present study, no effect on feed intake was observed as a result of SMS inclusion at 10–20% of the diet. In accordance with these findings, voluntary intake of sheep was not affected when they were fed with a diet containing 10–20% of SMS originated from wheat straw, while it was decreased at the level of 30% [41]. However, there are authors who recommend lower levels of SMS in the diet; Xu et al. [42] suggested a SMS dietary level of 6.5% for silage based TMR in wethers.

As indicated in Table 2 and Table 3, no effects of wheat straw hay replacement by SMS on milk yield, composition, fatty acid profile and pH were observed. However, as can be observed in Table 2, milk yield was lower for all groups during the first sampling day. This decrease could be attributed to the high ambient temperatures for two days during this period (>35 °C), which negatively affected feed intake and, as a result. milk yield of all groups. On the other hand, milk oxidative stability was ameliorated (Table 2), possibly due to SMS antioxidant properties attributed to its high phenolic content [43,44], while the count of polymorphonuclear leukocytes in milk decreased in the SMS2 group and the macrophage proportion linearly increased (Table 3). There are no studies in the literature examining the effects of dietary SMS on the udder health parameters of dairy ewes. SCC and percentages of leukocyte subsets of somatic cells in milk are used as udder health indicators in lactating ruminants and, in particular, elevated SCC and percentage of polymorphonuclear leukocytes are associated with clinical and subclinical mastitis [45,46]. Here, we observed a decrease in polymorphonuclear (P) percentage, which is indicative of a healthier mammary gland, since P are the principal leukocytes that increase upon microbial immune challenge and are positively associated with milk microbial load and high SCC [46] and mammary gland oxidative stress [47]. In contrast, an increase in macrophage (M) percentage was observed in response to SMS treatment, but percentages are low in all treatment groups, typical for ewe milk, indicating a minimal contribution of this cell type to phagocytosis in the udder [48]. No effect of SMS was observed on lymphocyte percentage. These cells typically make up a large percentage of milk leukocytes (approximately 40%) [49] and the absence of effect is consistent with the stability of the lymphocyte population in ewe milk [46].

Liu et al. [25] studied the effects of Ganoderma lucidum hot water extract (HWE) derived from SMS on milk and serum immunoglobulin levels and serum antioxidant capacity in dairy cattle. No discernible differences in milk IgA, IgG or IgM levels were observed as a result of the addition of HWE derived from SMS, but serum IgA concentration was increased in treated groups compared to the controls. At the same time, serum total antioxidant capacity was significantly higher in HWE groups (p < 0.05). Taking into consideration both findings, the authors concluded that HWE may boost immunity and improve antioxidant capacity in dairy cattle [25]. In dairy goats, dry matter intake and milk yield were improved as an effect of utilization of rice straw fermented with spent Pleurotus sajor-caju mushroom substrate at the level of 15% [26]. Kim et al. [23] also tried to examine inoculated SMS (combination of Lactobacillus plantarum Lp1’, Pediococcus acidilactici Pa193 and L. plantarum Lp2M) as postweaning feed for dairy calves. The calves that were fed 10% of the inoculated SMS displayed the highest growth performance, immunoglobulin A and G, hemoglobin, hematocrit, and red blood cell concentration. Therefore, the inoculated SMS was also recommended as a feed supplement that can strengthen the immune system and improve health status and postweaning growth performance. In growing sika deer, addition of SMS of Pleurotus ostreatus as a replacement of concentrated feed at 10–30% also increased serum IgM levels [50].

5. Conclusions

SMS appears as a potential unconventional feedstuff in the diets of dairy ewes in the context of sustainability, circular economy and protection of natural resources. As can be shown by the findings of the current study, replacement of wheat straw by SMS improves milk oxidative stability, without negatively affecting milk yield, composition and the health status of ewes during their final stage of lactation.