Skip to main content
Chemical and Biological Technologies in Agriculture
Download PDF
Download ePub

Targeted metabolomics analysis of fatty acids in lamb meat for the authentication of paper mulberry silage as a substitute for alfalfa silage

Chemical and Biological Technologies in Agriculture volume 11, Article number: 160 (2024) Cite this article

Abstract

Background

The paper mulberry (Broussonetia papyrifera L.) is a valuable source of woody forage that can be used for ruminants, such as goat and lambs. However, there is limited knowledge about how paper mulberry silage affects carcass characteristics, meat physicochemical attributes, amino acid and unsaturated fatty acids in Hu lambs compared to alfalfa silage. The objective of this experiment was to investigate the impact of substituting alfalfa silage with paper mulberry silage on the slaughter performance, meat quality, free amino acid and fatty acid composition in muscles of Hu lambs.

Results

Thirty 60-day-old male Hu lambs with 17.13 ± 0.26 kg body weight were randomly allocated into paper mulberry silage (30% of total mixed ration) and alfalfa silage (30% of total mixed ration) treatment, and the feeding trial lasted 70 days. The results indicated no significant differences in all measurements (P > 0.05). However, compared to the group fed with alfalfa silage, the group fed with paper mulberry silage exhibited a reduction in meat drip loss (P < 0.05) without any notable effect on meat nutrients (P > 0.05). Targeted metabolomics analysis revealed that feeding paper mulberry silage led to decreased levels of certain bitter amino acids, such as valine, leucine, isoleucine, tryptophan, and phenylalanine (P < 0.05). Furthermore, the consumption of paper mulberry silage significantly augmented the levels of monounsaturated and polyunsaturated fatty acids, particularly n6-polyunsaturated fatty acids (C18:2n6, C20:3n6, C20:4n6, etc.), in meat.

Conclusions

Substituting paper mulberry silage for alfalfa in the daily diet of Hu lambs not only has no detrimental effect on animal performance but also improves meat unsaturated fatty acid composition.

Graphical Abstract

Introduction

Forage plays a crucial role in meeting the health and nutritional needs of herbivores. It not only helps in avoiding competition for resources among feed, food, and fuel [1], but also has positive effects on animal growth and improves the quality of meat products [2, 3]. Alfalfa (Medicago sativa L.) is a widely grown forage crop that is known for its high nutritional value and easy digestibility, making it a popular choice for feeding different herbivorous animals [4]. However, the increasing demands of animal husbandry in China have exceeded the domestic production capacity of alfalfa. As a result, pastoralism faces an annual average of 82 million tons of forage-livestock conflict in domestic [5], which hampers the development of the lamb breeding industry [6]. Therefore, there is an urgent need to explore forage resources to compensate for the scarcity of high-quality forage.

Globally, particularly in developing nations, there is a growing trend toward using climate-resilient, high biomass, and nutrient-dense plants to improve animal performance and meat quality [7, 8]. One such promising forage source is paper mulberry (Broussonetia papyrifera L.), which offers high adaptability to diverse environmental conditions and can be cultivated in both northern and southern regions of China [9]. It has a high biomass yield of over 45 tons per hectare and can be harvested for multiple years [10]. The crude protein content of paper mulberry leaves exceeds 20%, similar to that of alfalfa. Furthermore, paper mulberry is particularly rich in essential nutrients such as fiber, crude fat, minerals and vitamins. It also contains bioactive and antibacterial compounds (tannin, oxalic acid, saponin, etc.) that provide additional benefits to animal health and animal products [11, 12].

The paper mulberry is a valuable source of woody forage that can be used for ruminants such as goats and lambs [13]. However, there is limited knowledge about how paper mulberry silage affects carcass characteristics, meat physicochemical attributes, amino acids and unsaturated fatty acids in Hu lambs compared to alfalfa silage. Therefore, this study was conducted with the hypothesis that incorporating paper mulberry silage into animal feed could enhance meat quality, amino acid and fatty acid composition, especially potentially enhance the unsaturated fatty acids content of their meat. The study focused on various aspects, including carcass characteristics and meat physicochemical attributes.

Methods

Ethics statement

The experiment of feeding Hu lambs with paper mulberry silage instead of alfalfa silage was carried out Rongcheng Gouyang Modern Agriculture Co., Ltd, Chongqing, China (N29°53′, E105°48′). The experimental protocols and procedures for Hu lambs were conducted in accordance with the Chinese Guidelines for Animal Welfare and Experimental Protocol, which received approval from the Animal Care and Use Committee of China Agricultural University (AW62011202-1-1).

Lambs experimental design

A total of 30 Hu lambs (Hu × Hu, male), all healthy and possessing comparable genetic backgrounds, were selected for the study. These lambs were approximately 60 days and had similar live weight (17.13 ± 0.26 kg). The experiment in feedlot lasted for 70 days, which included a 2-week pre-feeding phase followed by an 8-week experimental feeding phase. Throughout the entire duration of the experiment, the Hu lambs were feed ad libitum and clean drinking water. Tannins contents of paper mulberry were 25.90 g/kg DM. The lambs were randomly and equally allocated paper mulberry silage group (PS, 30% of total mixed ration) and alfalfa silage group (AS, 30% of total mixed ration), and each diet had three replicates with five lambs in a pen (3 m*5 m). The nutrients of two diets are shown in Table 1. The silage making was conducted accordance with our previously study [3]. Chemical composition of paper mulberry silage and alfalfa silage is shown in Table S1. Throughout the experiment, Hu lambs were fed twice a day, at 8:00 am and 5:00 pm, respectively.

Table 1 Composition and nutrient levels of TMR of Hu lambs (%DM)
Full size table

Performance parameters, Carcass traits and sampling

Following the 70-day feeding trial, the weights of all Hu lambs were measured after a fasting period of 12 h. All lambs were subjected to electric shock for humane slaughter, followed by bloodletting and subsequent processes of peeling, evisceration, and midline splitting performed by professionals at Rongcheng Gouyang Modern Agriculture Co., Ltd (Chongqing, China). The paper mulberry silage did not affect the growth performance of Hu lambs when replaced alfalfa silage, there were no significant difference in average daily feed intake (1072.3 vs. 1090.2 g/day, P > 0.05), average daily gain (226.7 vs. 241.31 g/day, P > 0.05) and feed/ gain ratios (4.75 vs. 4.53, P > 0.05) between two treatments, as reported in our previous study [3]. The slaughter performance of Hu lambs was determined after a half-day fasting period prior to slaughter, during which the live weight before slaughter (LWBS) was measured and recorded. Carcasses were weighed immediately after slaughter, and the determination of meat quality and preservation of samples were initiated. Then organs, pericardial fat, perirenal fat and tail fat of Hu lambs were separated and calculated immediately. The eye muscle area of Hu lambs was defined as the cross-sectional area of the longissimus thoracis et lumborum (LTL) muscle, located between the 12th and 13th ribs of the carcass. The eye muscle area was meticulously traced on acetate paper to accurately measure the eye muscle area. 50 g of LTL muscle from the left side of lamb carcasses was used to analyze the meat chemical composition. In addition, approximately 20 g of LTL muscle from the right side of lamb carcasses was stored at −80 ℃ for targeted metabolic analysis of amino acids and fatty acids. After 24 h of storage at 4 ℃, the drip loss, Warner–Bratzler shearing force, and cooking loss of the meat were measured. Finally, the dressing percentage within 45 min after slaughter was calculated using the following formula:

\(\text{Dressing percentage of Hu lambs }(\text{\%})\hspace{0.17em}=\hspace{0.17em}(\text{Hot carcass weight}/\text{LWBS})\hspace{0.17em}\times \hspace{0.17em}100.\)

Chemical composition and meat quality

The moisture content of LTL muscle (three replicates) was determined as weight loss of each sample after drying based on Labconco Benchtop freeze dryer (Labconco, USA). Crude protein content (CP) was calculated by an OPSIS KD 625 automatic kjeldahl nitrogen analyzer (OPSIS, Sweden). The intramuscular fat (IMF) content in the LTL muscle was accurately weighed to 0.2 g and subsequently packed into a specialized fat filter bag for detection using the Soxhlet extraction method with an AnkomXT15 crude fat extractor (ANKOM Technology, USA). Chemical compositions were analyzed according to the methods by the Association of Official Analytical Chemists [14, 15]. Both crude protein content and intramuscular fat were quantified as the weight percentage of each fresh muscle sample.

The segmented muscle samples (three replicates) were placed in an air-filled plastic bag and suspended in a low-temperature storage room at 4 ℃ for 24 h, while the drip loss was quantified (approximately 30 mm × 50 mm × 20 mm) [16]. Meanwhile, cooking loss was detected by the method previously reported [17]. In brief, the muscle samples were exposed to a temperature of 80 ℃ using a steamer until their central temperature reached 75 ℃. Subsequently, the samples were promptly extracted from the steamer and their weight was reassessed after they had cooled down to the surrounding temperature. The cooked samples were used to determine Warner–Bratzler shear force (WBSF) by the method previously described [7].

Analysis of amino acids

The HPLC–MS platform of Bionovogene Co., ltd. (Jiangsu, China) was utilized to detect the amino acid content in meat [18]. To begin, 50 mg muscle samples were extracted using a mixture of 10% formic acid in methanol–water (1:1, v/v), adding two steel balls and vortexing for 30 s. Afterward, the tissue grinder was used to grind the samples at a frequency of 55 Hz for a duration of 90 s. Subsequently, the mixture underwent centrifugation at 12,000 revolutions per minute and at a temperature of 4 ℃ for a period of 5 min. Next, an appropriate volume of supernatant was taken and diluted by 50 times using a mixture consisting of 10% formic acid in methanol–water (1:1, v/v), followed by vortexing for a time span of 30 s. Furthermore, 100 μL of the resulting supernatant was combined with 100 μL of Trp-d3 (10 ng/mL) and vortexed for 30 s. The obtained supernatant was then filtered through a 0.22 μm filter and transferred to the LC–MS bottle for analysis. The LC analysis was executed utilizing an AB SCIEX Jasper HPLC Liquid chromatography system (USA), while the detection of metabolites was conducted employing an AB4500MD mass spectrometer (AB SCIEX, USA). A ZORBAX Eclipse XDB-C18 chromatographic column (4.6 × 150 mm, Agilent, USA) was implemented with an injection volume of 5 μL and the column temperature was maintained at 40 ℃. The mobile phase was composed of two constituents: A-10% methanol water (containing 0.1% formic acid) and B-50% methanol water (with 0.1% formic acid). The gradient elution conditions were as follows: from 0 to 6.5 min, 10–30% B; from 6.5 to 7 min, 30–100% B; from 7 to 18 min, 100% B; from 18 to 18.5 min, 100–10% B; and finally from 18.5 to 21 min, 10% B. The flow rate was set at 0.3 mL/min for 0–8 min and increased to 0.4 mL/min for 8.5–21 min. For ionization, the positive ionization mode was employed utilizing an electrospray ionization (ESI) source. The ion source temperature, ion source voltage, collision gas, curtain gas, atomizing gas, and auxiliary gas were set to 500 °C, 5500 V, 6 psi, 30 psi, 50 psi, and 50 psi, respectively. Scans were conducted utilizing multiple reaction monitoring (MRM).

Analysis of fatty acids

The gas chromatography–mass spectrometry (GC–MS) was utilized to detect the fatty acid composition in LTL muscle tissue [19]. A mixed solution containing 51 types of fatty acid methyl esters (4000 μg/mL) was prepared by diluting with n-hexane to concentrations of 1, 5, 10, 25, 50, 100, 250, 500, and 1000 μg/mL. Ten concentration gradients of mixed standard solutions were prepared at a total concentration of each component set at 2000 μg/mL. The mother liquor is stored at -20 ℃, while the working standard solution is readily available for use. The process of extracting samples from LTL muscle involved the following steps: First, 50 mg of muscle samples were placed in a 2 mL centrifuge tube. To make the grinding process easier, a glass bead and 1 mL of extract were added to the tube. The tube was then centrifuged at 12,000 rpm for 5 min at 4 ℃. After centrifugation, the resulting supernatant was mixed with 2 mL of a solution containing 1% sulfuric acid and methanol. This mixture was then subjected to methylation in water at a temperature of 80 ℃ for a duration of 30 min. Following methylation, the mixture was extracted using 1 mL of n-hexane and washed with 5 mL of pure water. As an internal standard, 25 μL of methyl salicylate was added to the supernatant, along with 500 μL of the mixture. This mixture was then mixed and transferred to a sample bottle for analysis using GC–MS (Thermo Trace 1310, Thermo ISQ 7000).

The gas chromatographic conditions for analysis were as follows: A Thermo TG–FAME capillary column with the dimensions of 50 m × 0.25 mm ID × 0.20 μm was used. The sample size used was 1 μL and a shunt ratio of 8:1 was maintained. The inlet temperature was set at 250 ℃, while the ion source and transmission line temperatures were set at 300 and 280 ℃, respectively. The heating procedure started at an initial temperature of 80 ℃ and was held for 1 min. The temperature was then increased to 160 ℃ at a rate of 20 ℃/min and maintained for 1.5 min. Subsequently, the temperature was further increased to 196 ℃ at a rate of 3 ℃/min and held for 8.5 min. Finally, the temperature was raised to 250 ℃ at a rate of 20 ℃/min and maintained for 3 min. The carrier gas used in the system was helium, which flowed at a rate of 0.63 mL/min. To ensure the stability and repeatability of the system, a quality control (QC) sample was included in the sample queue, along with the same number of experimental samples, for detection purposes. The mass spectrometry analysis was performed using the GC–MS system (Trace 1310/ ISQ 7000, Thermo) in the electron bombardment ionization (EI) source. The SIM scan mode was utilized with an electron energy of 70 eV.

Statistic analysis

The data used a completely randomized design analyzed with the mixed model procedure of SPSS 24.0 software (SPSS, Chicago, USA) with diet as the fixed effect and pen as a random effect. A significance level of P < 0.05 was deemed as significant, while P ≤ 0.10 indicated a tendency. Amino acids and fatty acids of LTL muscle discriminate analysis (OPLS–DA), KEGG pathway and enrichment analysis were performed by online platform MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/). Significant enrichment of differential metabolites was assessed through the application of KEGG pathway analysis and metabolite set enrichment analysis. A threshold of P < 0.05 was employed to determine the significance of enrichment.

Results

Slaughter performance

As shown in Table 2, no significant differences (P > 0.05) were detected in LWBS, dressing percentage, organ weight and eye muscle area between AS and PS treatments. However, compared with AS treatment, adding PS significantly decreased the hot carcass weight (P = 0.047), perirenal fat (P = 0.040) of Hu lambs, and the pericardial fat showed a decreasing trend (P = 0.064) in PS treatment.

Table 2 Effect of PS and AS diets on slaughter performance of Hu lambs
Full size table

Meat quality

According to the data presented in Table 3, the meat moisture content (P = 0.440), protein levels (P = 0.743), intramuscular fat (P = 0.675) composition and cooking loss (P = 0.486) of the LTL muscle showed no significant difference in Hu lambs when subjected to either PS or AS treatments. In addition, PS treatment significantly increased WBSF (P = 0.030) and decreased drip loss (P = 0.036).

Table 3 Effect of PS and AS diets on chemical composition and meat quality of LTL muscle in Hu lambs
Full size table

Amino acid composition

The free amino acid content of LTL muscle is shown in Table 4. Compared with the AS treatment, the contents of methionine (Met) (P = 0.009), lysine (Lys) (P = 0.001), valine (Val) (P = 0.014), isoleucine (Ile) (P = 0.009), phenylalanine (Phe) (P = 0.014), leucine (Leu) (P = 0.007), tryptophan (Trp) (P = 0.003), threonine (Thr) (P = 0.012), glutamic acid (Glu) (P = 0.014), aspartic acid (Asp) (P = 0.044), arginine (Arg) (P = 0.004), serine (Ser) (P = 0.017), tyrosine (Tyr) (P = 0.006) significantly decreased in PS treatment. However, PS and AS treatments had no significant difference (P = 0.082) in glycine (Gly) (P = 0.624), alanine (Ala) (P = 0.219), proline (Pro) (P = 0.432), asparagine (Asn) (P = 0.651), glutamine (Gln) (P = 0.938), histidine (His) (P = 0.532) total amino acid (TAA) (P = 0.082).

Table 4 Effect of PS and AS diets on free amino acid composition (μg/g) and meat quality of LTL muscle in Hu lambs
Full size table

Fatty acid composition

As shown in Table 5, the contents of polyunsaturated fatty acid and n6-PUFA in LTL muscle of lambs significantly higher (P < 0.05) in PS treatment compared with AS treatment. There was no significant difference (P > 0.05) on total fatty acids, SFA, UFA, MUFA, n3-PUFA and n6/n3 ratio between PS and AS treatments.

Table 5 Effects of PS and AS diets on fatty acid composition (mg/100g) of LTL muscle in Hu lambs
Full size table

PS treatment increased (P < 0.05) C20:0, C22:0 content, also had a tendency to increase the content of C18:0 of LTL muscle compared with AS treatment. Meanwhile, PS treatment significantly increased (P < 0.05) the levels of C14:0, C18:1n9t, C18:2n6t, C20:3n6, C20:4n6, C20:5n3, C22:1n9, and C22:4n6 compared to AS treatment. In addition, PS treatment also exhibited a tendency to elevate the levels of C18:3n6 and C20:2n6 in LTL muscle.

Discrimination analysis of metabolomics

Orthogonal partial least squares–discrimination analysis (OPLS–DA) was powerful in differentiating variables and separating treatments. The OPLS–DA score plots revealed clearly distinctions in amino acids and fatty acids between the treatments (Fig. 1).

Fig. 1
figure 1

Orthogonal partial least squares–discrimination analysis (OPLS–DA) scores plot of amino acids (A) and fatty acids (B) in LTL of Hu lamb fed PS and AS group

Full size image

Enrichment and metabolomic pathway analyses

Metabolite set enrichment analysis and KEGG pathway analysis were both processed by MetaboAnalyst 5.0 on the differential metabolites between PS group and AS group. In the results of the analysis on metabolite set enrichment (Fig. 2A), it was observed that both lysine degradation and biotin metabolism showed significant enrichment (P < 0.05). In addition, the KEGG pathway analysis highlighted the significant enrichment (P < 0.05) of phenylalanine, tyrosine, and tryptophan biosynthesis, as well as biotin metabolism (Fig. 2B). The inclusion of lysine was found to characterize biotin metabolism in both of these methods.

Fig. 2
figure 2

Functional analysis for the differential metabolites of lambs. A Metabolite set enrichment analysis of differential amino acids (AS/PS). B KEGG pathway enrichment of differential metabolites related to amino acids (AS/PS). C Metabolite set enrichment analysis of differential fatty acids (PS/AS). D KEGG pathway enrichment of differential metabolites related to fatty acids (PS/AS)

Full size image

Differential metabolites set enrichment analysis between PS and AS groups revealed a significant enrichment (P < 0.05) in linoleic acid metabolism (Fig. 2C). The KEGG pathway analysis also indicated a significant enrichment in both linoleic acid metabolism and arachidonic acid metabolism (Fig. 2D). The identified compounds associated with linoleic acid metabolism included C18:2n6, C18:3n6, C20:3n6, and C20:4n6.

Discussion

The similarity in lamb hot carcass weight, dressing percentage of Hu lambs suggests that incorporating paper mulberry silage into the diet is comparable to using alfalfa silage. Previous study has shown that substituting paper mulberry for alfalfa hay does not affect the goat hot carcass weight and dressing percentage [20]. These consistent findings can be attributed to the similar nutritional composition between the two treatments, which is supported by an earlier study involving dairy cattle fed with paper mulberry silage [21]. Therefore, paper mulberry offers a promising alternative forage option when there is a limited supply of high-quality alfalfa.

Limited documented studies have investigated the effect of paper mulberry silage diet on perirenal fat in lambs. In this study, treatment with paper mulberry silage resulted in a decrease in perirenal fat weight compared to alfalfa silage treatment, thereby promoting lamb fattening [22]. Previous research has also shown that increasing the supplement of alfalfa in lamb diet leads to an increase in perirenal fat deposition which aligns with our findings [23]. Flavonoids derived from mulberry leaves also exhibit inhibitory effects on adipose tissue and enhance the distribution of fatty acids in adipose tissue in finishing pigs [24]. There has been limited research conducted on the impact of mulberry leaves on visceral fat in meat sheep, but a study involving rabbits also demonstrated their potential to reduce perirenal fat [25]. Perirenal fat refers to a visceral adipose tissue depot located adjacent to the kidneys [26]. Previous studies have indicated that the weight of perirenal fat in animals is influenced by both the quantity and composition of dietary energy [27, 28]. It has been suggested that visceral adipose tissue, compared to subcutaneous fat, has stronger associations with metabolic and cardiovascular risk factors in human [29, 30]. Therefore, incorporating paper mulberry silage into the diet of Hu lambs may potentially have health benefits by reducing visceral fat deposition. However, there few investigate the effects of paper mulberry on visceral fat. The confirmation of these findings and the elucidation of the underlying mechanisms necessitate further research.

Lamb meat represents a valuable source of food, with its texture playing a pivotal role in determining consumer acceptability. In this study, the protein content of meat treated with paper mulberry silage falls within the reported ranges for Tibetan lambs fed diets containing alfalfa powder (22.17% of diets) [16], and even surpasses that observed in a previous study where Dohne Merino lambs were fed alfalfa hay (16.2% of diets) [8]. Water-holding capacity (WHC) is a critical attribute of meat that determines its ability to retain moisture, which plays an essential role in longevity of meats and their impact on sales [31]. Su reported that natural antioxidant functional components in feedstuff may result lower drip loss to some extent [16]. In current study, meat of PS group showed a reduction in drip loss, which indicated that PS had positive effect on improving Hu meat quality. Warner–Bratzler shear force (WBSF) could indicate the tenderness of LTL muscle [32]. Lamb of PS group showed an increase in WBSF value, which may decrease the tenderness of meat. In addition, it is important to consider that WBSF values could be influenced by metabolites in lamb. Previous study on Tibetan sheep have shown the significant impact of muscle metabolites like linoleic acid and arachidonic acid on muscle tenderness, because arachidonic acid was correlated positively with shear force [33]. Consistent with current findings, our fatty acid-targeted metabolomics analysis revealed elevated levels of linoleic acid and arachidonic acid, which may contribute to the higher WBSF value observed in lambs fed with paper mulberry silage. In future investigations, it is crucial to integrate Targeted metabolomics analysis to better understand the complex associations between meat quality and metabolite deposition.

This study observed that the lamb from the alfalfa silage treatments had a higher concentration of free amino acids. The presence of amino acids is important for determining the distinct aroma and flavor profiles of lamb [34]. These free amino acids contribute to various tastes in meat, such as sweetness, freshness, and bitterness. For example, threonine (Thr), serine (Ser), alanine (Ala), and glycine (Gly) contribute to sweetness, while umami taste intensity is enhanced by umami amino acids like glutamic acid (Glu) and aspartame (Asp) [35]. On the other hand, tryptophan (Trp), phenylalanine (Phe), valine (Val), isoleucine (Ile) and leucine (Leu) contribute to bitterness [36]. The lower content of bitter amino acids such as Val, Ile, Leu, arginine (Arg), and tyrosine (Tyr) in meat treated with paper mulberry silage could result in a more favorable flavor compared to alfalfa silage treatment.

Currently, there has been limited scientific investigation conducted to examine the effects of utilizing paper mulberry silage as opposed to alfalfa silage on the composition of fatty acids found in lamb. The findings of the current study demonstrate that the utilization of paper mulberry silage leads to a higher content of polyunsaturated fatty acids (PUFAs) in lamb, particularly n6 fatty acids. The quantity of fatty acids observed in this study falls within the range reported by previous study [37]. Fatty acids play a crucial role as precursors for fat synthesis and are indispensable for human metabolism [38]. The World Health Organization has reported that modifying the dietary content of saturated fatty acids and increasing the presence of unsaturated fatty acids can effectively reduce the risk of chronic diseases [39]. Given the richness of lamb in saturated fatty acids, there has been a focus on enhancing the levels of unsaturated fatty acids in high-quality meat production through regulated feeding strategies. It is worth noting that alfalfa, which is abundant in saponin but lacks condensed tannin [40, 41], and paper mulberry silage with over 17.72 g/kg saponin content and more than 30.74 g/kg condensed tannin content [12] have been investigated for their potential to increase the content of unsaturated fatty acids in LTL muscle of Hu lambs [13]. Previous studies have shown that tannin-containing forage can lead to desirable increases in polyunsaturated fatty acid (PUFA) levels [42]. Maintaining a balanced ratio of n6/n3 fatty acid intake has been supported by evidence to protect against cancer, cardiovascular disease and metabolic syndrome [43]. Our study revealed that lamb from the paper mulberry silage group had an n6/n3 ratio close to the recommended value of four for optimal cardiovascular health in human diets [44]. These findings suggest that the higher levels of condensed tannins present in paper mulberry silage may contribute to high quality lamb-meat with improved unsaturated fatty acid composition.

The addition of paper mulberry silage to lamb diet resulted in increased levels of linoleic acid (C18:2n6) and arachidonic acid (C20:4n6) in the LTL muscle. Research has shown that C18:2n6 has a positive impact on reducing blood cholesterol and preventing atherosclerosis [45]. Arachidonic acid C20:4n6 plays various roles in human health, including tissue regeneration and disease progression diagnosis [46, 47]. Previous studies have found that including tannin extracts in the diet increases the levels of C18:2n6 in meat, which aligns with our current findings from feeding Hu lambs a tannin-rich silage diet [48]. And, the C18:2n6 content in the paper mulberry silage diet was found to be lower compared to that in the alfalfa silage diet. Another relatively recent finding on the benefits of tannins is their ability to modulate ruminal biohydrogenation and, consequently, the fatty acids meat [49]. It is hypothesized that incorporating a paper mulberry silage diet into Hu lambs’ feeding regimen could potentially enhance the C18:2n6 content of their meat through ruminal biohydrogenation. In addition, quantitative results (Table 5) of fatty acids, along with the KEGG analysis of differential metabolites (Fig. 2), particularly substantiate that the up-regulation of the arachidonic acid metabolic pathway is responsible for the observed increase in n6 fatty acids in Hu lambs following paper mulberry silage treating. Therefore, the observed increase in C20:4n6 with a paper mulberry silage diet could contribute to a similar trend consist with C18:2n6 levels in lamb, as C18:2n6 acts as a precursor for C20:4n6 [50].

The attention toward monounsaturated fatty acids persists due to its association with reduced cardiovascular disease risk, enhanced insulin sensitivity, decreased susceptibility to certain cancers, and positive impact on cognition [51]. In this study, a noteworthy discovery was the higher presence of Nervonic acid (C24:1) in the PS group, which aligns with our previous investigation on lamb subjected to a 30% paper mulberry silage treatment [13]. Nervonic acid (C24:1) is a very long-chain fatty acid whose name originated from the original discovery in mammalian nerve tissues. The nutrient plays a vital role in human health, especially for the brain [52]. Generally, synthesis of C24:1 involves the elongation of the oleic acid (C18:1n9) carbon chain, wherein two carbon units donated by malonyl-CoA are cyclically added to the acyl chain [53], higher level of C24:1 in lamb from the PS group may attributed to the fact that C18:1n9 content was higher in paper mulberry silage compared to alfalfa silage in the current study (Table S1). In addition, significant deposition of neuronic acid in the muscle may also attributed to the substantial escape of oleic acid from rumen biohydrogenation and its subsequent entry into the long chain unsaturated fatty acid synthesis pathway (n9 fatty acid) for carbon chain elongation, resulting in the accumulation of C24:1 [53].

Although the World Health Organization recommends that the intake of n3 fatty acids is beneficial to health, the n6/n3 ratio of lamb in the paper mulberry silage group in this study was still in a reasonable range (<5) [54, 55]. The increase in n6 fatty acids, found in Hu lambs fed with paper mulberry silage, was not meant to be an undesired effect, because the n3 and n6 fatty acids and their long chain derivatives are both involved in important functions [56]. Besides, feeding paper mulberry silage brought more C24:1 in lamb, which characterized by an exceptionally long chain and plays a key role in prevention of immune regulation, and metabolic diseases, and has anti-inflammatory properties [57]. Therefore, the comprehension of the silage diet impact of monounsaturated, n6 and n3 fatty acids in ruminants nutrition is crucial for a comprehensive understanding [58].

Conclusion

In current study, it was demonstrated that substituting alfalfa silage diets with paper mulberry silage diets in Hu lambs effectively reduces perirenal fat, meat drip loss, and bitter amino acid content. Moreover, it improves the composition of unsaturated fatty acids without any negative impact on lamb slaughter performance or meat chemical composition. These findings contribute to our comprehension of the impacts of paper mulberry on Hu lamb growth and meat quality, particularly regarding polyunsaturated fatty acid profiles. Furthermore, this study supports the potential application of paper mulberry as an efficient woody forage in the lamb industry.

Availability of data and materials

No datasets were generated or analysed during the current study.

References

  1. Halmemies-Beauchet-Filleau A, Rinne M, Lamminen M, Mapato C, Ampapon T, Wanapat M, et al. Review: alternative and novel feeds for ruminants: nutritive value, product quality and environmental aspects. Animal. 2018;12:s295–309.

    Article  PubMed  CAS  Google Scholar 

  2. Kao PT, Darch T, McGrath SP, Kendall NR, Buss HL, Warren H, et al. Chapter Four - Factors influencing elemental micronutrient supply from pasture systems for grazing ruminants. In: Sparks DL, editor. Advances in Agronomy, vol 164, p. 161–229. 2020; London: Academic Press.

  3. Xiong Y, Wang X, Li X, Guo L, Yang F, Ni K. Exploring the rumen microbiota of Hu lambs in response to diet with paper mulberry. Appl Microbiol Biotechnol. 2023;107(15):4961–71.

    Article  PubMed  CAS  Google Scholar 

  4. Garrett JL. Role of Alfalfa in Animal Diets. 1995:26–31.

  5. Yang M, Liang S, Zhou H, Li Y, Zhong Q, Yang Z. Consumption in non-pastoral regions drove three-quarters of forage-livestock conflicts in China. Environ Sci Technol. 2023;57(20):7721–32.

    Article  PubMed  CAS  Google Scholar 

  6. Yang T, Dong J, Huang L, Li Y, Yan H, Zhai J, et al. A large forage gap in forage availability in traditional pastoral regions in China. Fundam Res. 2023;3(2):188–200.

    Article  Google Scholar 

  7. Mahachi LN, Chikwanha OC, Katiyatiya CLF, Marufu MC, Aremu AO, Mapiye C. Meat production, quality, and oxidative shelf-life of Haemonchus-parasitised and non-parasitised lambs fed incremental levels of Sericea lespedeza substituted for lucerne. Meat Sci. 2023;195: 109009.

    Article  PubMed  CAS  Google Scholar 

  8. Uushona T, Chikwanha OC, Katiyatiya CLF, Strydom PE, Mapiye C. Production and meat quality attributes of lambs fed varying levels of Acacia mearnsii leaf-meal as replacement for Triticum aestivum bran. Meat Sci. 2023;196: 109042.

    Article  PubMed  CAS  Google Scholar 

  9. Yang F, Chen C, Ni K, Zhang Q. Research progress of new forage and woody forage. In: Yang F, Guo X, Ni K, editors. Research progress on forage production, processing and utilization in China. Singapore: Springer; 2022. p. 209–30.

    Chapter  Google Scholar 

  10. Guo L, Wang X, Lin Y, Yang X, Ni K, Yang F. Microorganisms that are critical for the fermentation quality of paper mulberry silage. Food Energy Security. 2021;10(4): e304.

    Article  CAS  Google Scholar 

  11. Wu C, Sun W, Huang Y, Dai S, Peng C, Zheng Y, et al. Effects of different additives on the bacterial community and fermentation mode of whole-plant paper mulberry silage. Front Microbiol. 2022;13:904193. https://doi.org/10.3389/fmicb.2022.904193.

  12. Wang N, Xiong Y, Wang X, Guo L, Lin Y, Ni K, et al. Effects of Lactobacillus plantarum on fermentation quality and anti-nutritional factors of paper mulberry silage. Fermentation. 2022;8(4):144.

    Article  CAS  Google Scholar 

  13. Xiong Y, Guo C, Wang L, Chen F, Dong X, Li X, et al. Effects of paper mulberry silage on the growth performance, rumen microbiota and muscle fatty acid composition in Hu lambs. Fermentation. 2021;7(4):286.

    Article  CAS  Google Scholar 

  14. Kong F, Gao Y, Tang M, Fu T, Diao Q, Bi Y, et al. Effects of dietary rumen-protected Lys levels on rumen fermentation and bacterial community composition in Holstein heifers. Appl Microbiol Biotechnol. 2020;104(15):6623–34.

    Article  PubMed  CAS  Google Scholar 

  15. Official methods of analysis of the association of offical analytical chemists. 2012; Gaithersburg: AOAC International.

  16. Su YY, Sun X, Zhao SM, Hu ML, Li DF, Qi SL, et al. Dietary alfalfa powder supplementation improves growth and development, body health, and meat quality of Tibetan sheep. Food Chem. 2022;396:133709. https://doi.org/10.1016/j.foodchem.2022.133709.

  17. Honikel KO. Reference methods for the assessment of physical characteristics of meat. Meat Sci. 1998;49(4):447–57.

    Article  PubMed  CAS  Google Scholar 

  18. Thiele B, Hupert M, Santiago-Schübel B, Oldiges M, Hofmann D. Direct analysis of underivatized amino acids in plant extracts by LC-MS/MS (improved method). Methods Mol Biol. 2019;2030:403–14.

    Article  PubMed  CAS  Google Scholar 

  19. Beccaria M, Franchina FA, Nasir M, Mellors T, Hill JE, Purcaro G. Investigation of mycobacteria fatty acid profile using different ionization energies in GC–MS. Anal Bioanal Chem. 2018;410(30):7987–96.

    Article  PubMed  CAS  Google Scholar 

  20. Zhang JP, Wei QX, Li QL, Liu RF, Tang LQ, Song YX, et al. Effects of hybrid Broussonetia papyrifera silage on growth performance, visceral organs, blood biochemical indices, antioxidant indices, and carcass traits in dairy goats. Anim Feed Sci Technol. 2022;292: 115435.

    Article  CAS  Google Scholar 

  21. Wu Z, Liang C, Huang R, Ouyang J, Zhao L, Bu D. Replacing alfalfa hay with paper mulberry (Broussonetia papyrifera L.) silage in diets do not affect the production performance of the low lactating dairy cows. Anim Feed Sci Technol. 2022;294:115477.

  22. Nie C, Hu Y, Chen R, Guo B, Li L, Chen H, et al. Effect of probiotics and Chinese medicine polysaccharides on meat quality, muscle fibre type and intramuscular fat deposition in lambs. Ital J Anim Sci. 2022;21(1):811–20.

    Article  CAS  Google Scholar 

  23. Devincenzi T, Prunier A, Meteau K, Nabinger C, Prache S. Influence of fresh alfalfa supplementation on fat skatole and indole concentration and chop odour and flavour in lambs grazing a cocksfoot pasture. Meat Sci. 2014;98(4):607–14.

    Article  PubMed  CAS  Google Scholar 

  24. Liu Y, Peng Y, Chen C, Ren H, Zhu J, Deng Y, et al. Flavonoids from mulberry leaves inhibit fat production and improve fatty acid distribution in adipose tissue in finishing pigs. Anim Nutr. 2024;16:147–57.

    Article  PubMed  CAS  Google Scholar 

  25. Martínez M, Motta Ferreira W, Cervera C, Pla M. Feeding mulberry leaves to fattening rabbits: effects on growth, carcass characteristics and meat quality. Anim Sci. 2005;80:275–80.

    Article  Google Scholar 

  26. Liu B-X, Sun W, Kong X-Q. Perirenal fat: a unique fat pad and potential target for cardiovascular disease. Angiology. 2019;70(7):584–93.

    Article  PubMed  Google Scholar 

  27. Li HQ, Wang B, Li Z, Luo HL, Zhang C, Jian LY, et al. Effects of maternal and post-weaned rumen-protected folic acid supplementation on slaughter performance and meat quality in offspring lambs. Br J Nutr. 2021;126(8):1140–8.

    Article  PubMed  CAS  Google Scholar 

  28. Limea L, Alexandre G, Berthelot V. Fatty acid composition of muscle and adipose tissues of indigenous Caribbean goats under varying nutritional densities. J Anim Sci. 2012;90(2):605–15.

    Article  PubMed  CAS  Google Scholar 

  29. Huang N, Mao E-W, Hou N-N, Liu Y-P, Han F, Sun X-D. Novel insight into perirenal adipose tissue: a neglected adipose depot linking cardiovascular and chronic kidney disease. World J Diabetes. 2020;11(04):115–25.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Drackley JK, Wallace RL, Graugnard D, Vasquez J, Richards BF, Loor JJ. Visceral adipose tissue mass in nonlactating dairy cows fed diets differing in energy density. J Dairy Sci. 2014;97(6):3420–30.

    Article  PubMed  CAS  Google Scholar 

  31. Álvarez-Rodríguez J, Urrutia O, Lobón S, Ripoll G, Bertolín JR, Joy M. Insights into the role of major bioactive dietary nutrients in lamb meat quality: a review. J Anim Sci Biotechnol. 2022;13(1):20.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Karumendu LU, Ven Rvd, Kerr MJ, Lanza M, Hopkins DL. Particle size analysis of lamb meat: effect of homogenization speed, comparison with myofibrillar fragmentation index and its relationship with shear force. Meat Sci. 2009;82(4):425–31.

  33. Zhang X, Han L, Hou S, Raza SHA, Wang Z, Yang B, et al. Effects of different feeding regimes on muscle metabolism and its association with meat quality of Tibetan sheep. Food Chem. 2022;374: 131611.

    Article  PubMed  CAS  Google Scholar 

  34. Wang B, Zhao X, Zhang B, Cui Y, Nueraihemaiti M, Kou Q, et al. Assessment of components related to flavor and taste in Tan-lamb meat under different silage-feeding regimens using integrative metabolomics. Food Chem X. 2022;14: 100269.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Watkins PJ, Frank D, Singh TK, Young OA, Warner RD. Sheepmeat flavor and the effect of different feeding systems: a review. J Agric Food Chem. 2013;61(15):3561–79.

    Article  PubMed  CAS  Google Scholar 

  36. Fan W, Tan X, Xu X, Li G, Wang Z, Du M. Relationship between enzyme, peptides, amino acids, ion composition, and bitterness of the hydrolysates of Alaska pollock frame. J Food Biochem. 2019;43(4): e12801.

    Article  PubMed  Google Scholar 

  37. Zhong RZ, Li HY, Fang Y, Sun HX, Zhou DW. Effects of dietary supplementation with green tea polyphenols on digestion and meat quality in lambs infected with Haemonchus contortus. Meat Sci. 2015;105:1–7.

    Article  PubMed  CAS  Google Scholar 

  38. Flakemore AR, Malau-Aduli BS, Nichols PD, Malau-Aduli AEO. Omega-3 fatty acids, nutrient retention values, and sensory meat eating quality in cooked and raw Australian lamb. Meat Sci. 2017;123:79–87.

    Article  PubMed  CAS  Google Scholar 

  39. Gao H, Zhang Y, Liu K, Fan R, Li Q, Zhou Z. Dietary sodium butyrate and/or vitamin D3 supplementation alters growth performance, meat quality, chemical composition, and oxidative stability in broilers. Food Chem. 2022;390: 133138.

    Article  PubMed  CAS  Google Scholar 

  40. Niyigena V, Coffey KP, Philipp D, Savin MC, Zhao J, Naumann HD, et al. Intake, digestibility, rumen fermentation and nitrogen balance in sheep offered alfalfa silage with different proportions of the tannin-rich legume Sericea lespedeza. Anim Feed Sci Technol. 2024;308: 115863.

    Article  CAS  Google Scholar 

  41. Liu C, Qu Y, Guo P, Xu C, Ma Y, Luo H. Effects of dietary supplementation with alfalfa (Medicago sativa L.) saponins on lamb growth performance, nutrient digestibility, and plasma parameters. Anim Feed Sci Technol. 2018;236:98–106.

  42. Girard M, Dohme-Meier F, Wechsler D, Goy D, Kreuzer M, Bee G. Ability of 3 tanniferous forage legumes to modify quality of milk and Gruyère-type cheese. J Dairy Sci. 2016;99(1):205–20.

    Article  PubMed  CAS  Google Scholar 

  43. Yang Y, Xia Y, Zhang B, Li D, Yan J, Yang J, et al. Effects of different n-6/n-3 polyunsaturated fatty acids ratios on lipid metabolism in patients with hyperlipidemia: a randomized controlled clinical trial. Front Nutr. 2023;10:1166702.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Molendi-Coste O, Legry V, Leclercq IA. Why and how meet n-3 PUFA dietary recommendations? Gastroenterol Res Pract. 2011;2011: 364040.

    Article  PubMed  Google Scholar 

  45. Lee JM, Lee H, Kang S, Park WJ. Fatty acid desaturases, polyunsaturated fatty acid regulation, and biotechnological advances. Nutrients. 2016;8(1):23.

    Article  PubMed  PubMed Central  Google Scholar 

  46. Hadley KB, Ryan AS, Forsyth S, Gautier S, Salem N. The essentiality of arachidonic acid in infant development. Nutrients. 2016;8(4):216.

    Article  PubMed  PubMed Central  Google Scholar 

  47. Hanna VS, Hafez EAA. Synopsis of arachidonic acid metabolism: a review. J Adv Res. 2018;11:23–32.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Kamel HEM, Al-Dobaib SN, Salem AZM, López S, Alaba PA. Influence of dietary supplementation with sunflower oil and quebracho tannins on growth performance and meat fatty acid profile of Awassi lambs. Anim Feed Sci Technol. 2018;235:97–104.

    Article  CAS  Google Scholar 

  49. Vasta V, Daghio M, Cappucci A, Buccioni A, Serra A, Viti C, et al. Invited review: Plant polyphenols and rumen microbiota responsible for fatty acid biohydrogenation, fiber digestion, and methane emission: experimental evidence and methodological approaches. J Dairy Sci. 2019;102(5):3781–804.

    Article  PubMed  CAS  Google Scholar 

  50. Díaz MT, Cañeque V, Sánchez CI, Lauzurica S, Pérez C, Fernández C, et al. Nutritional and sensory aspects of light lamb meat enriched in n−3 fatty acids during refrigerated storage. Food Chem. 2011;124(1):147–55.

    Article  Google Scholar 

  51. Powell L, Wallace EC. 79 - Fatty acid metabolism. In: Pizzorno JE, Murray MT, editors. Textbook of natural medicine. 5th ed. St. Louis (MO): Churchill Livingstone; 2020. p. 584- 92.e4.

    Chapter  Google Scholar 

  52. Tanaka K, Shimizu T, Ohtsuka Y, Yamashiro Y, Oshida K. Early dietary treatments with Lorenzo’s oil and docosahexaenoic acid for neurological development in a case with Zellweger syndrome. Brain Develop. 2007;29(9):586–9.

    Article  Google Scholar 

  53. Li Q, Chen J, Yu X, Gao J-M. A mini review of nervonic acid: source, production, and biological functions. Food Chem. 2019;301: 125286.

    Article  PubMed  CAS  Google Scholar 

  54. Wang B, Wang Y, Zuo S, Peng S, Wang Z, Zhang Y, et al. Untargeted and targeted metabolomics profiling of muscle reveals enhanced meat quality in artificial pasture grazing tan lambs via rescheduling the Rumen Bacterial Community. J Agric Food Chem. 2021;69(2):846–58.

    Article  PubMed  CAS  Google Scholar 

  55. Raes K, De Smet S, Demeyer D. Effect of dietary fatty acids on incorporation of long chain polyunsaturated fatty acids and conjugated linoleic acid in lamb, beef and pork meat: a review. Anim Feed Sci Technol. 2004;113(1):199–221.

    Article  CAS  Google Scholar 

  56. Husted KS, Bouzinova EV. The importance of n-6/n-3 fatty acids ratio in the major depressive disorder. Medicina. 2016;52(3):139–47.

    Article  PubMed  Google Scholar 

  57. Yuan S-N, Wang M-x, Han J-L, Feng C-Y, Wang M, Wang M, et al. Improved colonic inflammation by nervonic acid via inhibition of NF-κB signaling pathway of DSS-induced colitis mice. Phytomedicine. 2023;112:154702.

  58. dos Santos Neto JM, Worden LC, Boerman JP, Bradley CM, Lock AL. Long-term effects of abomasal infusion of linoleic and linolenic acids on the enrichment of n-6 and n-3 fatty acids into plasma lipid fractions of lactating cows. J Dairy Sci. 2024;107(10):7996–8008.

Download references

Acknowledgements

Not applicable.

Funding

This study was financially supported by the National Key Research and Development Program of China (2022YFD1300900), and National Natural Science Foundation of China (No.32171686). In addition, all authors thank the faculty and staff from ChongQing Academy of Animal Sciences for their assistance in sampling.

Author information

Authors and Affiliations

  1. College of Grassland Science and Technology, China Agricultural University, Beijing, 100193, China

    Xiong Yi, Zhou Hongzhang, Wang Ruhui, Li Xiaomei, Lin Yanli, Ni Kuikui & Yang Fuyu

  2. Frontier Technology Research Institute of China Agricultural University in Shenzhen, Shenzhen, 518124, China

    Xiong Yi & Yang Fuyu

  3. Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, 518000, China

    Xiong Yi

  4. Comprehensive Convenience Service Center in Fengzhou Town of Wuxiang County, Changzhi, 046300, China

    Shi Yue

  5. College of Animal Science, Guizhou University, Guiyang, 550025, China

    Yang Fuyu

Authors
  1. Xiong Yi
    View author publications

    Search author on:PubMed Google Scholar

  2. Zhou Hongzhang
    View author publications

    Search author on:PubMed Google Scholar

  3. Wang Ruhui
    View author publications

    Search author on:PubMed Google Scholar

  4. Li Xiaomei
    View author publications

    Search author on:PubMed Google Scholar

  5. Lin Yanli
    View author publications

    Search author on:PubMed Google Scholar

  6. Shi Yue
    View author publications

    Search author on:PubMed Google Scholar

  7. Ni Kuikui
    View author publications

    Search author on:PubMed Google Scholar

  8. Yang Fuyu
    View author publications

    Search author on:PubMed Google Scholar

Contributions

Yi Xiong: Data Curation, Formal analysis, Methodology, Writing Original Draft. Hongzhang Zhou: Data Curation, Methodology, Visualization. Ruhui Wang: Data Curation. Xiaomei Li: Methodology. Yanli Lin: Investigation. Yue Shi: Formal analysis. Kuikui Ni: Project administration, Review & Editing. Fuyu Yang: Conceptualization, Funding acquisition.

Corresponding author

Correspondence to Yang Fuyu.

Ethics declarations

Ethics approval and consent to participate

The experimental protocols and procedures for Hu lambs were conducted in accordance with the Chinese Guidelines for Animal Welfare and Experimental Protocol, which received approval from the Animal Care and Use Committee of China Agricultural University (AW62011202-1-1).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yi, X., Hongzhang, Z., Ruhui, W. et al. Targeted metabolomics analysis of fatty acids in lamb meat for the authentication of paper mulberry silage as a substitute for alfalfa silage. Chem. Biol. Technol. Agric. 11, 160 (2024). https://doi.org/10.1186/s40538-024-00688-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s40538-024-00688-5

Keywords