Effects of Adding Sodium Diacetate and Aspergillus oryzae to TMR Diets on Lactating Camel Production Performance, Milk Quality, and Fecal Microbiota

TL;DR

Adding sodium diacetate to camel feed improves milk quality by increasing vitamins and fatty acids without affecting milk yield.

Contribution

Demonstrates that sodium diacetate enhances bioactive milk components in lactating camels without altering yield.

Findings

SDA increased Vitamin E, Vitamin C, and unsaturated fatty acids in camel milk.

SDA treatment boosted the abundance of the fungal genus Melanocarpus in fecal microbiota.

Milk yield and routine components remained unaffected by SDA or AO supplementation.

Abstract

Low milk production and inconsistent milk quality in lactating camels are significant challenges in the camel breeding industry that require urgent attention. The use of feed additives to enhance both milk yield and quality represents an efficient and cost-effective approach. This study assessed the effects of two feed additives—sodium diacetate and Aspergillus oryzae—on lactating camels. The results suggest that these additives have the potential to improve the bioactive content in camel milk, offering a promising strategy for enhancing milk quality. These findings provide valuable insights into the future development of feed additives tailored for lactating camels, guiding the industry toward improved productivity and milk quality.

Camel milk is highly valued for its nutritional and therapeutic properties. However, extensive management systems often lead to inconsistent milk quality. This study aimed to evaluate the effects of sodium diacetate (SDA) and Aspergillus oryzae (AO) as dietary additives on the milk composition and fecal microbiota of lactating Bactrian camels. Thirty camels of similar parity were randomly assigned to three groups: a control group (TMR), an SDA group (1000 mg/kg DM), and an AO group (40 g/d). The trial lasted 45 days, including a 15-day acclimation period. Routine milk components were analyzed every 10 days, while fatty acids and fecal microbiota were assessed on day 30. Results showed that SDA supplementation significantly increased the concentrations of Vitamin E, Vitamin C, and unsaturated fatty acids (UFA) in milk without affecting milk yield or routine components. Microbiota analysis indicated that SDA treatment significantly increased the abundance of the fungal genus Melanocarpus, although no broad shifts in microbial community structure were observed across groups. In conclusion, dietary SDA (1000 mg/kg) effectively enhances bioactive substances in camel milk while maintaining stable milk quality. These findings suggest that SDA is a viable “green” additive for improving the functional value of camel milk in intensive production systems.

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1. Introduction

Due to its compositional similarity to human milk, camel milk serves as a vital source of nutrition and functional health benefits for populations in arid and semi-arid regions. Beyond its fundamental nutritional value, its unique bioactive profile endows it with diverse therapeutic properties [1]. Research has demonstrated its potential in managing autism spectrum disorder (ASD), regulating glycemic levels in diabetes, and preventing hepatic cirrhosis. Furthermore, camel milk serves as a safe alternative to bovine milk, effectively mitigating allergic reactions in infants [2]. Studies have demonstrated that camel milk is rich in medium-chain fatty acids (MCFAs), which are rapidly absorbed and metabolized by the human body, serving as an efficient energy source. Furthermore, the presence of omega-3 and omega-6 polyunsaturated fatty acids helps mitigate lipid deposition in the arteries, thereby promoting cardiovascular health. Another significant health benefit of camel milk is its high concentration of insulin-like proteins [3]. These proteins effectively facilitate glucose uptake and enhance the functional integrity of pancreatic β, contributing to the management of metabolic disorders [4].

In recent years, the consumer demand for camel milk has increased steadily; however, production levels remain insufficient to meet market needs. The prevalence of traditional extensive farming practices poses significant challenges to large-scale production and the standardization of milk quality [5]. In China, lactating camel husbandry is primarily concentrated in the arid desert grasslands of Inner Mongolia, Gansu, Xinjiang, Qinghai, and Ningxia. In Xinjiang, where the population consists mainly of Bactrian camels (Camelus bactrianus), milk yields are generally low. This low productivity is attributed to intrinsic factors, such as genetic and physiological traits, as well as extrinsic factors, including husbandry management, environmental conditions, and dietary nutrition. Among these, the role of feed additives is of critical importance. Current research in ruminant nutrition indicates that dietary supplementation with specific additives can effectively enhance production performance, rumen fermentation, and milk yield while reducing the reliance on antibiotics due to their significant antioxidant properties [6].

Sallam [7] demonstrated that supplementing camel diets with a mixture of Saccharomyces cerevisiae and Ruminococcus significantly increased milk yield and dry matter digestibility (DMD) while markedly reducing the rumen protozoa population. Similarly, Ghazzawy [8] reported that date seed-derived biochar could significantly mitigate methane CH_4_ emissions and enhance nutrient digestibility in camels.

Sodium diacetate (SDA) primarily functions by dissociating into acetic acid, which interferes with enzymatic interactions within fungal cell walls, thereby suppressing the activity of harmful microorganisms [9]. As a feed additive, SDA has been shown to improve milk fat conversion efficiency in dairy animals [10]. Specifically, fistula trials in Boer goats by Dai [11] revealed that SDA-supplemented silage significantly enhanced the apparent digestibility of dry matter (DM), crude protein (CP), and neutral detergent fiber (NDF).

On the other hand, Aspergillus oryzae (AO) is a prebiotic additive widely utilized in ruminant nutrition to improve feed intake and the digestibility of DM and fiber by promoting the proliferation of cellulolytic bacteria in the rumen [12]. Recent studies on Holstein cows indicated that AO extracts significantly increased milk yield [13], feed intake [14], and rumen fermentation efficiency [15] without adverse health effects. Furthermore, research in beef cattle [16] suggested that SDA supplementation increased the apparent digestibility of NDF and reduced CH_4_ emissions while maintaining consistent growth performance.

Despite their extensive application in traditional dairy livestock, the effects of SDA and AO on lactating camels remain unexplored. Current additives used in camel husbandry are often region-specific, and a universally applicable additive has yet to be identified. Therefore, investigating the application of SDA and AO in the daily production of lactating camels is significant for the industry.

In summary, this study selected sodium diacetate (SDA) and Aspergillus oryzae (AO) as dietary additives to evaluate their effects on milk yield and milk quality in lactating Bactrian camels. The objective was to determine whether SDA or AO can serve as a universally applicable feed additive, free from regional specificity, to facilitate the standardized and large-scale production of camel milk.

2. Materials and Methods

2.1. Test Materials

Sodium diacetate and Aspergillus oryzae were purchased from Dahan Enzyme Biotechnology Co., Ltd. (Beijing, China), and the Aspergillus oryzae had a live bacteria count of 12 billion/g.

2.2. Test Time and Location

The experiment was conducted from 1 July to 14 August 2023, at the camel breeding base of Keping County Livestock Technology Development Co., Ltd., Aksu City, Xinjiang Uygur Autonomous Region, China. The experiment was conducted in a single, unified camel housing facility. The camels were allocated into three designated pens, each measuring 15 m × 3 m, with 10 camels per pen. Each pen was equipped with an integrated outdoor exercise area and an automated watering system to ensure ad libitum access to water.

2.3. Experimental Animals and Experimental Design

Thirty lactating Bactrian camels (2 years old) with synchronized estrus, similar parity, and comparable lactation stages were selected for the study. The camels were randomly assigned to one of three groups (n = 10 per group): a control group (TMR), a sodium diacetate group (SDA), and an Aspergillus oryzae group (AO). The TMR group was fed a basal total mixed ration (TMR); the SDA group received the basal TMR supplemented with 1000 mg/kg of sodium diacetate (SDA); and the AO group received the basal TMR supplemented with 40 g/d of Aspergillus oryzae (AO).

2.4. Basal Diet and Nutritional Requirements

Since there are no feeding standards for lactating camels, based on the characteristics of Bactrian camels to tolerate roughage, and with reference to the NRC (2001) [17] standard for dairy cows, a diet for lactating camels was formulated. The roughage consisted of hydroponic wheat seedlings, wheat straw, and alfalfa, which were crushed to 2–3 cm using a TMR machine and then mixed with concentrate to form the TMR diet. The composition and nutritional levels of the diet are shown in Table 1. The dry matter (DM) of the feed was determined according to GB/T 6435-2014 [18] Determination of Moisture in Feed; crude protein (CP) was determined according to GB/T 6432 [19] Determination of Crude Protein in Feed-Kjeldahl Method; crude fat (EE) was determined according to GB/T 6433 [20] Determination of Crude Fat in Feed; crude ash (Ash) was determined according to GB/T 6438 [21] Determination of Crude Ash in Feed; calcium (Ca) was determined according to GB/T 13885 [22] Determination of Calcium, Copper, Iron, Magnesium, Manganese, Potassium, Sodium and Zinc Content in Feed-Atomic Absorption Spectrometry; phosphorus (P) was determined according to GB/T 6437 [23] Determination of Total Phosphorus in Feed-Spectrophotometry; neutral detergent fiber (NDF) was determined according to GB/T 20806 [24] Determination of Neutral Detergent Fiber (NDF) in Feed; and acid detergent fiber (ADF) was determined according to NY/T The determination was performed according to the “1459 Determination Method of Acidic Detergent Fiber in Feed”. The SOD addition amount is 1000 mg/kg according to EU standards [25] and the AO addition amount is 40 g/day based on previous research progress [26,27,28,29,30].

2.5. Feeding and Management

All experimental camels were housed in the same environment. Ambient temperature and relative humidity within the facility were recorded daily (Supplementary Table S1). The camels were managed in groups with ad libitum access to both feed (for diet composition, see Supplementary Table S2) and water. Feeding was conducted twice daily at 09:00 and 19:30. Daily feed intake for each pen was recorded, and the amount of feed offered was adjusted daily to ensure approximately 15% orts (refusals) to guarantee unrestricted intake.

2.6. Sample Collection and Methods

2.6.1. Lactation Performance

The milk yield of the experimental camels was measured at 0 d, 10 d, 20 d and 30 d during the trial period. On the 30th day of the trial period, 50 mL of milk sample was collected (n = 10). Milk protein, milk fat, lactose, non-fat, freezing point, ash content, conductivity and temperature were detected using a milk composition analyzer.

2.6.2. Detection of 16S and ITS in Fecal Microbiota

Fecal samples (n = 3) were collected from each camel for four consecutive days following the formal experimental period using the spot-sampling method. Sampling was conducted 16 times in total (four times daily at 6 h intervals). Approximately 10 g of fecal matter was collected via the rectum using PE gloves. To fix nitrogen, 5 mL of 10% sulfuric acid was added per 100 g of fresh feces and mixed thoroughly. Parallel samples from various time points for each camel were pooled into a single composite sample and stored at −80 °C. Sequencing of the bacterial 16S rRNA and fungal ITS regions was performed by Suzhou Norminket Biomedical Technology Co., Ltd. (Suzhou, China).

Total genomic DNA was extracted from the feces using the cetyltrimethylammonium bromide (CTAB) method, followed by PCR amplification using specific primers [31]. The resulting PCR products were quantified by fluorometry (Qubit 3.0) and sequenced on the Illumina PE300 platform. Raw sequencing data were quality-controlled using QIIME2 2020.6 software to remove low-quality sequences and potential contaminants. Sequence alignment was performed against the SILVA database. Operational Taxonomic Units (OTUs) were clustered at a 97.0% similarity threshold using Usearch drive5 software [32].

Alpha diversity indices were evaluated using QIIME2 (version 2020.6) [33]. Beta diversity was analyzed to compare similarities in species composition between samples, and Principal Component Analysis (PCA) plots were generated using R language [34]. Furthermore, PICRUSt2 was employed to align 16S rRNA feature sequences with the Integrated Microbial Genomes (IMG) database to construct phylogenetic trees. The “nearest species” of the feature sequences were identified to predict the genomic information of unknown taxa, which was further integrated with the KEGG pathway database to infer the functional potential of the microbial community [35]. No significant microbial differences were detected using LEfSe in this study. Fungal microbiota analysis via the ITS method followed the same procedures as the bacterial analysis.

2.6.3. Serum Biochemical Indicators and Detection Methods

After the initial testing period, fasting blood was collected from the anterior vena cava using a disposable vacuum blood collection device. After collection, the blood was centrifuged at 3500 rpm, and the serum was transferred to a 1.5 mL EP tube using a 1 mL pipette and stored in a refrigerator (−20 °C). The levels of SOD (superoxide dismutase), MDA (malondialdehyde), GSH-PX (glutathione peroxidase), CAT (catalase), and T-AOC (total antioxidant capacity) in serum were measured.

2.6.4. Dairy Quality Testing

On day 30, the sample was sent to Novogene Biotechnology Co., Ltd. (Suzhou, China). to determine the content of camel milk fatty acids, minerals (Na, Mg, K, Ca, Fe), vitamin E, insulin and vitamin C.

2.6.5. Fatty Acid Detection

Total fatty acids (FAs) were extracted from frozen meat samples following the procedure of Liang et al. FA separation was performed using gas chromatography (GC-450; Varian Co., Walnut Creek, CA, USA), with peaks identified based on retention time. Individual FA concentrations were quantified against standard curves prepared from a known methyl ester mixture (C4–C24; Sigma-Aldrich, St. Louis, MO, USA)

2.7. Data Analysis

The experimental data were initially processed using Excel 2016, and then a two-way ANOVA was performed using SPSS 26.0 statistical analysis software. Multiple comparisons were then performed using Duncan’s method. p < 0.01 indicated extremely significant differences, p < 0.05 indicated significant differences, and p > 0.05 indicated no significant differences.

3. Results and Analysis

3.1. Effects of Different Feed Additives on Milk Production Performance in Lactating Camels

As shown in Table 2, the addition of sodium diacetate and Aspergillus oryzae to the camel diet had no significant effect on the milk production of lactating camels (p > 0.05).

3.2. Effects of Different Feed Additives on the Quality of Milk from Lactating Camels

As shown in Table 3, the addition of sodium diacetate and Aspergillus oryzae to the diet had no significant effect on any of the indicators in the morning and evening milk of lactating camels after 30 days (p > 0.05).

3.3. Effects of Different Feed Additives on the Composition of Milk from Lactating Camels

As shown in Table 4, the vitamin E content in the milk of the sodium diacetate group was significantly higher than that of the basal diet TMR group and the Aspergillus oryzae group (p < 0.01), and the vitamin E content in the milk of the Aspergillus oryzae group was significantly higher than that of the basal TMR group (p < 0.01); the vitamin C content in the milk of the sodium diacetate group was significantly higher than that of the basal TMR group (p < 0.01); there was no significant difference in insulin content among the three groups (p > 0.05).

As shown in Table 5, there were no significant differences in the content of sodium, magnesium, potassium, calcium and iron in camel milk among the three groups (p > 0.05).

As shown in Table 6, the C21:0 content in the milk of the SDA group was significantly higher than that of the basal diet group and the Aspergillus oryzae group (p < 0.01), and the C22:6N3 content was significantly higher than that of the basal diet group and the Aspergillus oryzae group (p < 0.05). The C20:3N6 content in the milk of the sodium diacetate group and the basal TMR group was significantly higher than that of the Aspergillus oryzae group (p < 0.01), and the C20:5N3 content was significantly higher than that of the Aspergillus oryzae group (p < 0.05). The C24:1N9 content in the milk of the basal TMR group and the Aspergillus oryzae group was significantly higher than that of the sodium diacetate group (p < 0.01).

3.4. Effects of Different Feed Additives on Antioxidant Indices in Lactating Camels

As shown in Table 7, the addition of different types of additives to the feed had no significant effect on the antioxidant indicators in the serum of lactating camels (p > 0.05).

3.5. Effects of Different Feed Additives on the Fecal Microbiota of Lactating Camels

As shown in Figure 1a, there are 1514 unique bacterial microorganisms in the TMR group, 2122 in the SDA group, and 1992 in the AO group, for a total of 659 microorganisms across the three groups. As shown in Figure 1b, there are 90 unique fungal microorganisms in the TMR group, 67 in the SDA group, and 84 in the AO group, for a total of 11 fungal microorganisms across the three groups.

As shown in Figure 2, there were no significant differences in Alpha diversity among the three groups of bacteria and fungi (p > 0.05).

As illustrated in Figure 3a, the bacterial microbial communities of the AO and TMR groups exhibit a distinct clustering trend; similarly, Figure 3b shows a notable separation between the fungal microbial communities of the AO and SDA groups. While the sample size is limited, these PCA results suggest potential differences in community structures between the treatment groups.

As shown in Table 8 and Figure 4a, the Actinobacteria phylum in the TMR group was significantly higher than that in the other two groups (p < 0.01), while there were no significant differences among the groups in other bacterial phyla (p > 0.05). As shown in Table 9 and Figure 4b, there were no significant differences among the groups in any bacterial genus (p > 0.05). As shown in Table 10 and Figure 4c, there were no significant differences among the three groups in any fungal phylum (p > 0.05). As shown in Table 11 and Figure 4d, the Melanocarpus genus in the SDA group was significantly higher than that in the other two groups (p < 0.01), while there were no significant differences among the other fungal genera.

As shown in Figure 5a, compared with the SDA group, the Zeatin_biosynthesis pathway and Systemic_lupus_erythematosus pathway were significantly upregulated in the TMR group (p < 0.01); as shown in Figure 5b, compared with the AO group, the Photosynthesis_antenna-proteins pathway was significantly upregulated in the TMR group (p < 0.05), and the Polycyclic_aromatic_hydrocarbon_degeadation pathway was significantly downregulated (p < 0.001).

As shown in Figure 6a, at the bacterial level, there are significant positive correlations among the Firmicutes, Bacteroidetes, Verrucomicrobia, and Proteobacteria phyla, indicating a strong synergistic effect in the rumen of lactating camels. Figure 6b shows that at the fungal level, there are high levels of synergistic and antagonistic effects among the Ascomycota phyla. Ascomycota is significantly positively correlated with Neocallimastigomycota and Mucoromycota phyla, and significantly negatively correlated with Basidiomycota phylum.

As shown in Figure 7, the C24:1N9 content in camel milk was significantly positively correlated with the abundance of Tenericutes bacteria (p < 0.05).

4. Discussion

Due to their unique physiological adaptations, camels can thrive in arid conditions, providing a vital nutritional source for local pastoralists. Currently, common camel dietary additives primarily include enzyme preparations and probiotics, which significantly enhance production performance, optimize the rumen environment, and mitigate the risk of acidosis [7]. As a widely utilized feed additive and preservative, sodium diacetate (SDA) is extensively used in the production and preservation of ruminant silage, effectively improving its nutritional value and aerobic stability [36]. In the present study, dietary supplementation with SDA showed no significant impact on milk yield or routine milk parameters in lactating camels. This finding aligns with the results reported by Shockey in dairy cows, suggesting that at conventional nutritional levels, the role of SDA as an exogenous precursor of acetic acid may be constrained by the metabolic homeostasis of the host [37]. Aspergillus oryzae (AO), a fungal enzyme preparation, has been widely applied in dairy cow production. Previous research indicated that AO supplementation could significantly increase the population of cellulolytic bacteria in the rumen and improve apparent dry matter digestibility [28]. Zhang [38] further reported that AO increased milk yield, milk protein, and lactose content while modulating the hindgut microbiota. These findings differ partially from our results, as AO supplementation did not significantly alter milk yield or routine components in this trial. However, our microbial findings were consistent with the literature; distinct separation in fecal fungal communities was observed between groups, likely due to the fungal nature of AO. The lack of significant change in production performance may be attributed to the unique physiological structure and superior fiber digestion capacity of camels, which may reduce their reliance on external enzymatic additives [39].Interestingly, although milk yield and routine quality remained unaffected, both SDA and AO significantly increased the concentrations of Vitamin E (VE) and Vitamin C (VC) in camel milk, with significant differences also observed in polyunsaturated fatty acid (PUFA) profiles (Table 7). The most pronounced effects were observed in the SDA group. The elevated VE and VC levels in the SDA group echo previous findings regarding SDA’s role in preserving nutritional value and enhancing aerobic stability in silage. This suggests that SDA not only protects nutrients in vitro but may also promote vitamin deposition in milk in vivo by regulating cellular redox status.

In the present study, fecal samples for microbiota analysis were collected from only three camels per group. This limited sample size imposes certain constraints on the comprehensive prediction and generalization of the microbial profiles. Nonetheless, the preliminary analysis of the fecal microbiota still reveals the significant potential of these additives in modulating the intestinal metabolism of camels. Analysis of the fecal fungal microbiota indicated that the abundance of the genus Melanocarpus in the SDA group was significantly higher than that in the TMR and AO groups. Current research characterizes Melanocarpus as a xylanase-producing microorganism capable of effectively degrading arabinoxylans in the diet, thereby reducing feed viscosity and enhancing nutrient digestibility in ruminants [40,41]. This finding suggests that SDA supplementation may bolster the cellulolytic and hemicellulolytic capacity of the camel gastrointestinal tract, subsequently promoting overall dietary digestion and metabolism.

KEGG pathway enrichment analysis revealed that Zeatin biosynthesis and Systemic lupus erythematosus (SLE) pathways were significantly downregulated in the SDA group compared to the TMR group. The Zeatin biosynthesis pathway is closely associated with the microbial regulation of carbohydrate utilization [42], as zeatin-related derivatives function as signaling molecules that modulate bacterial metabolic flux. Furthermore, the SLE pathway, which in the context of microbial functional profiling reflects core immunomodulatory processes—such as complement activation and inflammatory signaling cascades—has been linked to gastrointestinal microbiota composition [43]. Microbial dysbiosis can trigger metabolic disturbances that influence this pathway, potentially compromising host immune homeostasis [44]. Consequently, these results suggest that dietary supplementation with sodium diacetate (SDA) effectively enhances the functional stability of the gastrointestinal microbiota in lactating camels and optimizes carbohydrate utilization efficiency.

Comparison between the AO and TMR groups revealed a significant downregulation of the Photosynthesis-antenna proteins pathway in the AO group, reflecting a shift in the metabolic niches of specific microbial taxa. Notably, the polycyclic aromatic hydrocarbon (PAH) degradation pathway was significantly upregulated in the AO group. PAHs are hazardous environmental pollutants and recognized carcinogens that pose severe threats to human health [45]. Previous research has indicated that dairy cows exposed to PAH-contaminated environments can transfer these residues into milk, thereby presenting a substantial risk via dairy consumption [46]. In the present study, the significant upregulation of the PAH degradation pathway indicates that dietary Aspergillus oryzae (AO) supplementation bolsters the enzymatic capacity of the gut microbiota to detoxify complex aromatic compounds. This finding is of significant importance for mitigating harmful residues in camel milk and enhancing the overall safety and quality of camel-derived dairy products.

Fecal microbial network analysis revealed that the bacterial interaction nodes were primarily concentrated within the phylum Firmicutes, suggesting that Firmicutes plays a central role as a “hub” taxon in the fecal microbiota of all three groups of lactating camels. Furthermore, a significant positive correlation was observed between Firmicutes and Bacteroidetes. In ruminant microbiology, these two phyla are the predominant taxa involved in lignin degradation and carbohydrate binding [47]. Our findings align with these established roles, indicating a high degree of synergistic interaction among the microbiota of lactating camels, which likely leads to the production of convergent metabolites. Regarding the fungal community, the phylum Ascomycota appeared to function as the primary core connector. Ascomycota plays a critical role in cellulose degradation [48] and, together with Basidiomycota, typically constitutes the dominant fungal community in the ruminant gastrointestinal tract. However, in our interaction network, a certain degree of antagonism was observed between Ascomycota and Basidiomycota. As both phyla are non-anaerobic fungi and traditionally considered dominant in the ruminant gut [49], the antagonism observed in this study may stem from competition for limited oxygen resources. Moreover, the dietary supplementation of sodium diacetate (SDA) and the fungal additive Aspergillus oryzae (AO) may have intensified this competitive relationship, leading to increased niche competition between these two fungal populations.

In this study, the effects of sodium diacetate (SDA) and Aspergillus oryzae (AO) on milk yield, routine milk composition, and fecal microbiota in lactating camels were evaluated. However, certain limitations remain. Specifically, the relatively small sample size for microbial detection restricts a comprehensive characterization of the influence exerted by SDA and AO on the gastrointestinal microbiota. Nevertheless, these preliminary findings provide valuable insights and directions for future research. Subsequent studies should focus on elucidating the precise metabolic mechanisms of SDA and AO and determining their optimal dosage to further enhance the quality of camel milk and support the development of intensive camel husbandry.

5. Conclusions

In summary, under identical dietary conditions, the supplementation of sodium diacetate (SDA) significantly enhances the concentrations of Vitamin C and Vitamin E in camel milk and optimizes the fatty acid profile without adverse effects on milk yield. Furthermore, the improvement in these bioactive components elicited by SDA was superior to that observed with Aspergillus oryzae (AO) supplementation.