Effect of Alfalfa (Medicago sativa L.) supplementation on nutrient digestibility and meat quality of Grimaud ducks

Le Thi Thuy Hang^1,2, Tran Xuan Hien^1,2, Le Thi Thuy Loan^1,2 and Nguyen Tuyet Giang^1,2

¹ An Giang University, An Giang, Vietnam
ltthang@agu.edu.vn
² Vietnam National University Ho Chi Minh City, Vietnam

Abstract

The study investigated the effect of Alfalfa ( Medicago sativa L.) on the nutrient digestibility and meat quality of Grimaud duck. Altogether 64 Grimaud ducks were randomly assigned to 4 dietary treatments (A0, A2, A4 and A6, which corresponded to 4 levels of Alfalfa 0, 2, 4 and 6% in the diet). Each treatment had 4 replicates of 4 ducks each. The results showed that with 4% Alfalfa added to the diet, ducks enhanced feed intake (84.4 g/bird/day) as well as the digestibility of dry matter and crude protein, which were 76.1% and 73.6%, respectively and were significantly better than all other treatments. The inclusion of 4% Alfalfa improved fiber content, providing more nitrogen which improved nutrient retention and absorption. In addition, Alfalfa reduced total crude fat in duck meat from 3.77% in A0 to 3.38% in A4. It was also noted that 4% Alfalfa added was able to retain color and pH remarkably in duck breast meat stored in a fridge. These results demonstrate the potential of using Alfalfa as a natural feed additive for enhanced meat quality by lowering crude fat content and decreasing moisture loss during preservation and processing.

Keywords: Alfalfa, Nitrogen retention, duck meat, color, crude fat, storge

Introduction

Raising ducks is one of the most prominent livestock activities apart from providing nutrition within Mekong Delta. The Grimaud duck is becoming more popular because it grows quickly, uses feed efficiently and has a high yield of breast meat (Nguyen et al 2020). The search for alternative feeds that are cheaper and more nutritious has emerged due to increasing nutrition costs.

Protein content in Alfalfa (Medicago sativa L.) perennial legümes is high because it contains minerals as well as bioactive compounds such as saponins and flavonoids; hence, it is a functional forage. Alfalfa increases nutrient digestibility as well as growth in goats (Befekadu and Yunus 2015). Additionally, it improves intestinal structure, including increased villus height and crypt depth(Jha R et al 2021). Moderate levels of Alfalfa in poultry diets help to improve gut health and lower the need for antibiotics (Turra et al 2024, Buse et al 2024).

Nutrition research on ducks showed that supplementing Alfalfa to the diet at a rate of four percent improved body weight as well as carcass yield and meat quality, you get a reduction in abdominal fat along with a decrease in cholesterol (Nguyen et al 2022b; Le et al 2025). The effects of alfalfa on digestibility and meat quality have not been extensively studied. With this reasoning, the goal of this work is to study the effect of different rates of Alfalfa on feed intake, nutrient digestibility, meat quality of Grimaud ducks.

Materials and methods

Animal and experimental design

The experiment was conducted from April to June 2024 on an experimental farm located in Tri Ton district, An Giang province, Vietnam. A total of 64 birds with 21-day-old ducks with similar initial body weights were randomly allocated to one of four dietary treatments, corresponding to different levels of Alfalfa supplementation: A0 (0%), A2 (2%), A4 (4%) and A6 (6%). Each treatment included four replicates, with each replicate consisting of four ducks housed in individual metabolic cages. The cages, measuring 0.8 x 0.8 x 0.6 m, housed a mix of male and female ducks and were equipped with separate feeders and drinkers (B). Aluminum trays were placed beneath the cages to facilitate fecal collection. The experiment was divided into three distinct phases: an initial 7-day adaptation period to familiarize the ducks with the experimental diets, followed by 7 days of feed intake monitoring and concluding with 5 days for feed and waste sample collection.

Feed and feeding

Basal diets were formulated to meet the nutritional requirements for poultry, based on the NRC (2001) guidelines, with ingredient composition and proximate analysis detailed in Table 1. Drinking water was provided ad libitum, ensuring unrestricted access. Scheduled vaccinations, including those for Hepatitis, Duck Plague, H₅N₁, Colibacillosis and pasteurellosis, were administered in accordance with the Animal Science and Veterinary Medicine Department recommendations.

The feed used in the experiment was a custom-formulated powdered mash, referred to as the basal diet. Ingredients, including corn grain, rice bran, broken rice, soybean meal, fish meal and a vitamin–mineral premix, were purchased in a single batch prior to the start of the trial. All feed materials were ground and mixed in predetermined ratios to formulate a complete diet that met the nutritional requirements of ducks from 8 to 49 days of age, based on NRC (2001) recommendations.

The chemical composition of ingredients was analyzed following standard methods of AOAC (2005) and showed in table 2.

Alfalfa was cultivated at the experimental site in Tri Ton. The first harvest was carried out 60 days after planting, followed by a second regrowth harvest 45 days after the initial cutting. Based on the daily feed intake of ducks in each treatment group, the quantity of Alfalfa to be harvested was calculated to ensure a precise daily supply. For optimal freshness and nutrient preservation, the forage was harvested in the morning for afternoon feeding and in the afternoon for feeding the following morning

Sample collection and analysis

During the 5-day collection period, feed intake was recorded and representative samples were collected, All feed refusals and excreta were collected and weighed. Excreta were collected three times daily, then stored at -20°C. At the end of the collection period, the excreta were thawed, oven-dried at 60°C, ground to pass through a 0.5 mm screen and stored for subsequent analysis (Ravindran et al 1999).

Ileal digesta were collected after the excreta collection phase. Four hours after the final feeding at 42 days of age, birds were anesthetized and euthanized by cervical dislocation to collect ileal digesta (Sarmiento-Franco et al 2003). The ileal section, from Meckel’s diverticulum to the ileo-cecal junction, was excised immediately post-mortem and the digesta were collected within 5 minutes. Samples were then frozen and later oven-dried using the same procedure as for excreta samples (Ravindran et al 1999).

Intake and digestibility

Nutrient intake and digestibility: DM; OM; CP; EE; CF; Ash; and ME

Apparent nutrient digestibility coefficient (%)

Nutrient intake - Nutrient in waste

Where:

Nitrogen (N) accumulation: Determined as the difference between N intake and N excreted in waste.

The chemical composition of ingested feed, leftovers and ileal digesta was analyzed for parameters such as total minerals (Ash); crude protein (CP); crude fiber (CF); ether extract (EE). The analysis of moisture and other chemical components was performed according to AOAC standards (2005). Metabolizable energy (ME) was estimated based on (McDonald et al 2011) database.

Carcass characteristics

After 42 days of rearing, 4 average-weighted ducks in each treatment were individually weighed, labeled and subsequently subjected to a total feed withdrawal of 12 h, before slaughtered by severing the jugular vein. Subsequently, all carcasses were transferred to the Central laboratory of An Giang University using a cooler with ice. After removing skin, subcutaneous fat and visible connective tissue, both sides of the breast fillet ( Pectoralis major muscle) were kept in sealable polyethylene bags and stored at 2-4^oC for subsequent meat quality evaluation. Physicochemical traits such as pH, drip loss and cooking loss were determined in the left-side breast muscle while proximate composition and colour attributes were determined on the right-side muscle.

Physicochemical traits

At 24h postmortem, samples of breast meat were evaluated for the physicochemical traits. Drip loss was calculated as the percentage of weight loss during storage and cooking loss was measured by heating the same sample in a water bath (85°C, 25 min) and cooling to room temperature for 30 min.

Cooking loss was expressed as the percentage of weight loss after heating. The proximate composition of the breast meat (dry matter, ash, crude protein and crude fat) was determined according to the AOAC (2005) official standards. Each test was carried out in triplicate. The caloric value of breast meat was calculated according to Tashla et al (2019).

Caloric value (kcal/100g) = (crude protein (g/100g) × 4 kcal) + (crude fat (g/100g) × 9 kcal)

pH measurement

The meat color, pH, drip loss and shear force were determined using the left side of the breast and thigh muscles. The pH of the meat samples was measured using a pH meter (Extech 407228 Heavy Duty pH/mV/Temperature Meter Kit, Extech Instruments). The pH meter was calibrated with a pH 4.01 and 7.01 phosphate buffer before and during pH determinations. The average pH value was defined using 3 measurements.

The drip loss and shear force were determined using a meat quality pressure meter (Meat-1, Tenovo Food, Beijing, China) and a digital tenderness meter (C-LM3B, Tenovo Food, Beijing, China), respectively, following the methods of Liu et al (2011) and Tang et al (2009).

Colour measurement

Colour measurement was performed using a portable colourimeter (CR-20 Chromometer, Konica Minolta, Japan). The CIE (Commission Internationale de l'Éclairage) coordinates were lightness (L^*), redness (a^*), yellowness (b^*) and colour difference (∆E^*). The colourimeter was calibrated throughout the study using a white ceramic plate (CIE Standard Illuminant D65). Four readings were taken randomly from different locations of each sample of breast meat and areas selected for colour measurement of breast meat surface were free from obvious defects, such as bruises, discolourations, hemorrhages, damage, or any other condition that might have affected uniform colour reading (Petracci and Fletcher 2002). The colour was determined initially at 15 min. postmoterm (0h), 24, 48 and 72h postmortem. Colour difference was calculated as follows:

where,

represent the initial colour readings measured at 15 min. postmortem and other values represent colour parameters measured at each subsequent time

Statistical analysis

Statistical analysis of the results was performed using the Analysis of Variance (ANOVA) with the general linear model (GLM) procedure of Minitab 16.0. The significant differences among treatments were considered significant at p≤0.05.

Results and discussion

Chemical composition of diets

Feed can absorb and evaluate the quality of feed greatly depends on how the ingredients are composed nutritionally. The ingredients in the experiment are based on their chemical compositions shown in Table 3.

Table 3 showed that the experimental diets (A0–A6) were kept dry matter (DM) fixed at a range of 87.3% to 87.9%, which value is quite narrow. This finding was comparable to the study Suwignyo et al (2021), where the supplementation of Alfalfa in poultry feed has consistency and stability of dry matter which contributes positively to nutrient intake and feed efficiency.

The crude protein (CP) concentrations of duck growing feeds ranged from 17.0% to 17.2% and this met their nutritional needs according to NRC (2001). These levels also increased growth in ducks and feed efficiency in both ducks and chickens, which is in agreement with (Wu et al 2024, Cui et al 2022) that reported a level of CP of almost 17% produced beneficial responses in production outcomes in poultry fed meals.

Crude fiber (CF); Ether extract (EE) and Metabolizable energy (ME) were adequate for duck growth. Alfalfa, being inherently rich in structural carbohydrates, contributes significantly to the fiber content of diets. This is supported by the findings of (Xu et al 2023, Xu et al 2025) and further corroborated by Buse et al (2024), who observed improved gut morphology and digestibility in poultry when moderate amounts of Alfalfa or fiber-rich forages were included in the diet. Importantly, the CF levels in this study remain within the acceptable range for meat ducks and are not expected to compromise nutrient digestibility or voluntary feed intake.

Effect of supplement of Alfalfa on feed consumption and body weight of duck

According to Table 4, the addition of Alfalfa markedly affected the feed intake of Grimaud ducks. The highest average daily feed intake was recorded in group A4 with 89.4 g/head/day (p< 0.05) compared to other treatments. This indicates that 4% Alfalfa inclusion enhances the intake by improving palatability. The initial body weights of all treatments were statistically equal. The final body weights and total gains though, increased with Alfalfa inclusion up to A4, indicating improved nutrient metabolization. Also, animal studies have reported an increased final weight and carcass yield in ducks fed moderate levels of Alfalfa (Nguyen et al 2020, Bai et al 2022) which augments the current data.

Interestingly, while feed intake increased in A4, a further increase in Alfalfa content (A6) resulted in a slight reduction in intake. This may be due to higher fiber content, which can reduce voluntary intake, especially in non-ruminants like ducks.

From a nutritional perspective, Alfalfa provides valuable protein, minerals and bioactive compounds that may support gut health and immune function (He et al 2021). However, as supported by He et el (2021), over-supplementation with high-fiber ingredients may compromise feed efficiency and digestibility, reinforcing that moderation is key. The optimal Alfalfa supplementation level in Grimaud duck diets appears to be around 4%, as demonstrated in the A4 treatment. This level improves feed intake and likely enhances growth performance without introducing excessive fiber that may suppress intake.

Effect of supplement of Alfalfa on feed and nutrition digestibility

Based on Table 5, it showed that the digestibility of DM was highest in the A4 group (76.1%), followed by A2 (74.8%), A0 (73.7%) and lowest in A6 (72.1%) (p<0.05). For CP, the A4 group again exhibited superior digestibility (73.6%) compared to A2 (72.1%), A0 (71.8%) and A6 (70.5%) (p< 0.05). These findings are consistent with previous studies in poultry demonstrating that moderate Alfalfa supplementation can increase nutrient digestibility, while excessive supplementation may have adverse effects at different levels, Alfalfa at different levels significantly affected dry matter (DM) and crude protein (CP) in Grimaud ducks. For instance, Cui et al (2022) reported that laying hens fed diets containing up to 6% Alfalfa meal exhibited improved digestibility of dry matter and crude protein, along with enhanced intestinal morphology. However, higher levels beyond this threshold were associated with reduced nutrient absorption, likely due to increased indigestible fiber and lower energy density. Similarly, Buse et al (2024) found that moderate Alfalfa inclusion (around 4%) in broiler diets significantly improved CP digestibility and nitrogen retention, while excessive fiber in higher inclusion rates negatively affected gut efficiency. These studies suggest that the benefits of Alfalfa depend strongly on maintaining a balance between fiber enrichment and digestible nutrient supply and support the current findings that 4% Alfalfa is an optimal level for improving digestibility in meat ducks.

The inclusion of 4% Alfalfa in the diet of Grimaud ducks resulted in significantly higher feed intake (84.4 g/bird/day), increased dry matter (DM) digestibility (76%) and crude protein (CP) digestibility (73.6%) compared to other inclusion levels. Additionally, 4% Alfalfa supplementation promoted intestinal development by increasing villus length and width, thereby improving nitrogen utilization from the feed.

These findings are consistent with research by Suwignyo et al (2021), Cui et al (2022) who demonstrated that Alfalfa inclusion improved growth, intestinal morphology and nutrient digestibility in hybrid ducks. Alfalfa meal supplementation (up to 6%) improved villus height, crypt depth and gut wall thickness, which are closely linked to enhanced nutrient absorption and gut integrity. Another relevant report by Samur et al (2020) found that supplementing duck diets with 10% fresh Alfalfa led to significantly improved feed consumption, growth performance and meat quality. However, excessive inclusion (above 6%) tended to increase crude fiber levels, which can potentially impair digestibility, especially in monogastric animals like ducks.

From a mechanistic perspective, Alfalfa provides essential bioactive compounds such as flavonoids and saponins, which may enhance microbial balance and enzymatic efficiency in the intestines. This contributes to better nutrient absorption and overall performance in poultry and ducks. This suggests that 4% Alfalfa inclusion optimized nutrient utilization, possibly due to balanced fiber content that supports gut function without overwhelming the digestive capacity of the ducks.

Effect of Alfalfa supplementation on Nitrogen retention

The data in the Table 6, Alfalfa supplementation had a considerable effect on Grimaud duck nitrogen retention. Alfalfa had the greatest retention efficiency (retention/intake ratio) in A4 of 80.5%. But when the level of Alfalfa was increased to 6%, the N retention/N intake decreased significantly to 75.2% (p<0.05).

These findings corroborate what is available in the poultry nutrition literature. According to Jiang et al (2014) studying Muscovy ducks showed that Alfalfa inclusion into diets improved nitrogen metabolism and lowered nitrogen excretion by protein digestibility alongside microbial activity within the intestinal tract.

In the same with Englmaierová et al (2019), Zheng et al (2019) and Dong et al (2007) documented improved nitrogen retention in laying hens supplemented with Alfalfa meal, attributing these results to the high-quality protein and bioactive compounds that enhance nitrogen retention.

Effect of Alfalfa level on Physicochemical traits

Table 7 presents the physicochemical traits of duck breast meat at 24 h postmortem. According to Font-i-Furnols (2015); Tadeusz et al (2021), water holding capacity (WHC) of the meat is the ability to retain its moisture when exposed to external forces, such as heating, pressing, gravity force.

In meat, the majority of water is immobilized within the myofibrill fillaments of the muscle. Due to postmortem changes in pH, this entrapped water is released. Meat WHC is affected by various factors, including the inherent (muscle types and genetics) as well as external factors (rearing conditions and pre- and post-slaughter handling methods). The dietary inclusion of Alfalfa powder significantly influenced several meat quality parameters. As shown in Figure 1, drip loss decreased with increasing levels of Alfalfa, from 3.55% in the control group (A0) to 3.11% in the A6 group (p<0.05), suggesting an improvement in the water-holding capacity of the muscle. The levels of Alfalfa tend to reduce the percentage of water escaping from the meat due to drip and cooking with R²=0.94 (Figures 1 and 2). These changes indicate that Alfalfa inclusion may enhance the structural integrity of the muscle tissue, thereby retaining more moisture during storage and cooking. These findings are consistent with those reported by Addini et al (2020), who observed similar improvements in water retention properties in duck meat fed with Alfalfa-enriched diets.

Dry matter content also showed an increasing trend (Figure 3), from 27.0% to 28.5%, although the differences were not statistically significant (p>0.05). The ash content significantly increased (p<0.05), reflecting a higher mineral concentration in meat from ducks fed with Alfalfa. Crude fat content was significantly reduced (p<0.01), from 3.77% in A0 to 3.22% in A6, resulting in a corresponding decrease in caloric value (p<0.05). These results agree with Suwignyo et al (2021), who reported that Alfalfa-based diets modulated lipid metabolism and reduced fat deposition in ducks.

Figure 1. Effect of Alfalfa levels on the drip loss of duck breast meat	Figure 2. Effect of Alfalfa levels on the cooking loss of duck breast meat
Figure 3. Effect of Alfalfa levels on dry matter of duck breast meat	Figure 4. Effect of Alfalfa levels on the shear force of duck breast meat

Tenderness is considered the most important quality of meat and is defined by shear force. Here, the shear force of the breast was shown in Figure 4. The shear force value decreased in meat ducks supplemented with Alfalfa, indicating that meat tenderness was improved, but not significantly (p > 0.05). Ducks fed with 6% Alfalfa (A6) exhibited the lowest shear force, suggesting that bioactive compounds in Alfalfa may positively affect muscle fiber characteristics and postmortem proteolytic processes. This result aligns with findings from Zhang et al (2021), who demonstrated that dietary interventions such as calcium enrichment could reduce shear force by promoting muscle cell apoptosis and enhancing enzymatic degradation of muscle proteins. Moreover, Li et al (2015), Han et al (2020) also observed that dietary fiber sources like Alfalfa could alter muscle microstructure, including fiber diameter and density, which are closely related to meat tenderness.

Changes in pH and colour of the duck breast meat during cold storage

The finding presented in Table 8 describes the quality attributes of duck breast meat regarding the Alfalfa supplementation impacts. Alfalfa’s effect on duck meat quality characteristics can be observed in the results of Table 8. The initial pH values of duck meat ranged from 5.95 to 6.26 which means that it was still fresh and postmortem acidification had not started taking place. After 24 hours, the slaughter pH values slightly dropped to 5.75-6.09, indicative of glycolysis and lactate accumulation (Toldrá et al 2019). Additionally, the data indicated that at 72 hours the pH of meat from ducks given 6% Alfalfa (A6) pH 6.15 which is significantly higher than control (A0) (p<0.05). This may indicate that Alfalfa bioactive compounds could be responsible for the moderation of pH drop during chill storage.

However, Alfalfa is rich in saponins, flavonoids and polyphenols—bioactive compounds with antimicrobial and antioxidant properties (Raeeszadeh et al 2022). These compounds may inhibit the growth of spoilage microorganisms and limit the accumulation of organic acids by reducing protein breakdown, thereby helping to maintain or slightly elevate pH levels during storage (Wang et al 2021 ). This trend is consistent with findings by Besharati et al (2020), who reported improved meat pH stability in goats fed 4–6% Alfalfa.

Regarding meat color, the L* values (lightness) decreased as dietary Alfalfa levels increased, particularly at 48 and 72 hours. The A6 group showed significantly lower L* values compared to the control group (p<0.01), which may indicate better myoglobin stability due to the antioxidant effects of Alfalfa. Barbut and Leishman (2022), Xu et al (2025) noted that reduced L* values during storage could result from decreased oxidation of muscle pigments and minimized protein denaturation.

The a* (redness) at 0h showed a significant increase with higher levels of Alfalfa supplementation, with the highest value in the A6 group (12.6) and the lowest in A0 (11.3) (p<0.01). This suggests that antioxidant compounds in Alfalfa may delay myoglobin oxidation, thereby preserving the red color of the meat during storage. These results align with those reported by Narciso-Gaytán et al (2011), who demonstrated that herbal additives could preserve the redness of poultry meat under cold conditions. But the b* values (yellowness) showed no statistically significant differences among treatments, although there was a slight downward trend in the A6 group at 48h. This may reflect limited carotenoid accumulation in muscle tissues. According to Sugiharto et al (2020), longer feeding periods or higher Alfalfa inclusion rates may increase carotenoid deposition in subcutaneous fat or skin, which could affect yellowness values over time. According to Li et al (2022a) showed that the addition of alfalfa meal can increase the a*, b* value of muscle, as well as flavor amino acids and essential amino acids.

The overall colour difference (∆E*) did not significantly vary among treatments across storage periods, which suggests that the colour changes remained within an acceptable sensory threshold. However, the trend in ∆E* corroborates the gradual colour fading and browning of duck meat with time, in line with observations by Zhao et al (2024). These findings support the potential use of Alfalfa as a natural feed additive to improve the physicochemical stability of duck meat postmortem, aligning with current trends in safe and sustainable poultry production using dietary fiber and herbal ingredients.

Conclusions

From the analysis. it is concluded that:

• Digestibility of nutrients was significantly improved when Alfalfa was supplemented at 4% supplementation level.
  
  
• Adding 4% Alfalfa in the diet enhances nutrient utilization and improves meat quality in ducks. It effectively reduces crude fat content and minimizes water loss during storage and processing.
  
  
• Adding 4% Alfalfa supplementation has been shown to stabilize pH and retain color remarkably in duck breast meat when stored in a refrigerator. These findings underscore the use of Alfalfa as a natural feed supplement to improve meat quality and shelf life in ducks.

Acknowledgements

This research is funded by Vietnam National University Ho Chi Minh City (VNU-HCM) under grand number C2023-16-11. The support from our staff and students is gratefully acknowledged

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