Changes in production performance, antioxidant capacity and immune status of meat ducks under different rearing systems

As potential substitutes of traditional free-range rearing, oor rearing system (FRS) and net rearing system (NRS) are currently predominant dryland rearing systems. In this study, a total of 720 Nonghua ducks were assigned to a 2 × 3 factorial arrangement with two rearing systems (FRS and NRS) and three ages (4w, 8w, and 13w) to study the effects of FRS and NRS on production performance, antioxidant capacity and immune status. NRS ducks might have better antioxidant capacity at the early stage of breeding, while GSH-Px activity was increased for scavenging excess free radicals at the later one. NRS increased the levels of IFN-γ, IL-1β, IL-4, immunoglobulins in serum and promoted the development of thymus and spleen to improve duck immune function. These results revealed the physiological impacts of FRS and NRS on ducks and provided a reliable reference for rearing system selection.


Background
China, as the major producer and consumer of broiler ducks in the world [1], has embarked on the evolution from the free-range rearing to intensive rearing in order to improve production e ciency [2], which is also a transformation of duck husbandry from water-associated to terrestrial [3]. Compared with the demand of extensive waters in traditional rearing system [1,4], dryland rearing systems have common merit in minimizing the need of waterbodies to alleviate environmental issues, such as rivers and lakes, which could also prevent ducks from the incidence of intestinal diseases caused by pathogens in waters [5].
Dryland rearing systems are generally classi ed into the cage-rearing system (CRS), oor-rearing system (FRS) and net-rearing system (NRS). Though CRS makes full use of the breeding house space, ducks were vulnerable as individual activities are much constrained [1]. Whereas, FRS is usually to lay a thick pad of 5-10 cm by straw, sawdust or wood shavings on a cement oor [6][7][8], which could absorb excess moisture but has defects in rearing management. Plastic oor net-rearing and wire oor net-rearing are national wide applied NRS. Due to the materials, stainless wires are more durable than plastic oors though sharp edges should be avoided. Both of them can create better hygiene than other rearing systems as ducks have less contact with their excreta [2,9]. Floor-type had either positive or negative correlations with the environment and physiological characteristics of ducks during rearing [10]. Previous studies on this variable indicated multifaceted effects on ducks. Study on Pekin ducks showed that dryland rearing ducks had higher body weight (BW) than equipped with a swimming pool in intensive rearing system [11]. Study on NRS and FRS also demonstrated production performance was signi cantly affected [9,10,12]. Based on higher levels of nal BW, average daily gain (ADG), average daily feed intake (ADFI) and lower feed conversion rate (FCR) in NRS ducks, Zhang et al. [9] concluded Chaohu ducks had better growth performance in NRS than in FRS. By comparing plastic oor net-rearing and litter oor-rearing, Pekin ducks were also found higher ADG in NRS [10]. However, study on Cherry Valley ducks reported higher ADG and lower FCR in FRS, and concluded FRS was one of the best duck rearing systems compared with NRS [12].
Furthermore, animal health status is another important aspect of modern poultry production, especially in intensive rearing systems [13,14]. Health status is generally associated with individual immunity and antioxidant capacity. Research has reported that the rearing system could signi cantly in uence duck immune response to avian in uenza vaccine of different breeds [15], and alter the key immune genes expression, such as toll-like receptor 7 (TLR7) in bursa, lung and intestinal tissues of indigenous ducks [16]. Study on Shaoxing ducks also reported an impact of NRS on duck health [5]. Meanwhile, waterfowls in FRS suffered from uctuated temperature [9,12], either heat or cold stress caused by temperature change would possibly lead to oxidative stress of ducks causing the changes in antioxidant defense system and a threat to health [17][18][19]. However, few studies had been conducted to investigate the potential physiological effects of FRS and NRS on duck immune status and antioxidant capacity.
Therefore, the objectives of this paper were to study the effects of FRS and NRS on production performance, antioxidant capacity and immune status of Nonghua ducks, which is one of the most economically valuable broiler duck breeds with superior meat quality and strong disease resistance in Southwest China. Comparisons were made at different ages to explore general changes on Nonghua ducks, which would reveal the physiological impacts of predominant dryland rearing systems on meat ducks, and provide a reliable reference for rearing system selection in modern production.

Animals and experiment design
The present study was performed at Sichuan Agricultural University (Sichuan, China). This study constituted a 2 × 3 factorial arrangement with 2 different rearing systems (FRS and NRS) and 3 different slaughtering ages (4w, appropriate time for meat-type ducks breeding; 8w, market age; 13w, market age).
Nonghua duck is a new high-quality meat-type Chinese local Sheldrake crossbreed, which has been bred through ve generations. This Sheldrake strain has a merit of roughage tolerance, excellent growth performance and carcass traits. A total of 1000 one-day-old Nonghua ducks from one commercial hatchery were wire oor brooded with a commercial starter diet (Table 1) for two weeks. At 14-day-old, 720 ducks with a male/female ratio of 1:1 were selected by similar initial BW, then ducks were randomly allocated into FRS and NRS (10 replicates of 18 ducks each) with males and females separated (namely, 180 ducks in each group). Ducks in FRS were reared on concrete oor with sawdust bedding of 5 cm, bedding materials were added appropriately during the experiment period and cleaned weekly. Ducks in NRS were reared on stainless wire mesh bed with 1.0 cm diameter mesh holes at a height of 50 cm above the ground, the excreta was cleaned weekly. All groups of ducks were reared in a well-ventilated house with same environmental conditions of natural light in the experimental waterfowl breeding farm of Sichuan Agricultural University (Sichuan, China). The temperature was set at 31℃ at rst week and gradually decreased to 25℃ until 14 d of age. Afterward, it was kept at approximately 15 to 20℃. The stocking density was kept at a consistent 5 ducks/m 2 . The experimental diets (Table 1) were formulated in accordance with NY/T 2122 − 2012 [20]. All diets and water were provided ad libitum to ducks during the experimental period. After slaughtered on the same day, a total of 10 carcass traits were measured according to NY/T 823-2004 [21], including carcass weight (CW), semi-eviscerated weight (SEW), eviscerated weight (EW), sebum fat weight (SFW), abdominal fat weight (AFW), carcass yield (CY), semi-eviscerated yield (SEY), eviscerated yield (EY), sebum fat yield (SFY), abdominal fat yield (AFY). After separating the visceral organs and immune organs, thymus, spleen, and bursa of Fabricius were collected to evaluate duck immune status, while visceral organs parameters of heart weight (HW), heart index (HI), liver weight (LW), liver index (LI), gizzard weight (GW), gizzard index (GI), proventriculus weight (PW) and proventriculus index (PI) were measured as part of production performance, in which organs indexes was calculated as organ weight divided by BW. After the determination of production performance, 20 g of liver samples of male ducks were collected for gene expression analysis.
Determination of serum biochemical parameters and antioxidant capacity indicators 7 out of 30 serum samples from each group were used for serum parameters analysis. The biochemical parameters including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total proteins (TP), albumin (ALB) and globulin (GLOB) [22] were measured by automatic biochemical analyzer (Chemray 240, Shenzhen Rayto Life and Analytical Sciences Co., Ltd., Shenzhen, China). The serum antioxidant capacity indicators including the content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were measured by using commercial kits (Nanjing Jincheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. Brie y, MDA content was measured by using thiobarbituric acid (TBA) colorimetry. SOD activity was determined by using xanthine oxidase method (hydroxylamine method) with 10 µL of each sample, the corresponding amount of SOD when the SOD inhibition rate reached 50% per mL of reaction solution was de ned as one SOD activity unit (U). As for GSH-Px activity, after the sample was diluted three times, 200 µL of each sample was used for enzymatic reaction and following color reaction. After deducting the non-enzymatic reactions, the unit of enzyme activity (U) of GSH-Px was de ned as the GSH concentration reduced by 1 µmol/L in each reaction system involved in 0.1 mL of serum.  µL of forward/reverse primer. PCR conditions consisted of 3 min pre-denaturation at 95℃; followed by 40 cycles of 10 sec denaturation at 95℃, 30 s annealing at primers optimum reaction temperature, and 30 s extension at 72℃. Melting curve was conducted to verify the ampli ed PCR product was single. Every sample was tested for 3 times and adopted with standard deviations of threshold cycle (CT) below 0.5.

Determination of immune status
Quanti cation data was calculated by using the 2 −ΔΔCT method against reference genes.

Statistical analysis
The experimental data was analyzed by using general linear model (GLM) procedure in SPSS 22.0. The statistical model comprised the main effects of rearing system (RS), age, as well as their interactions.

Effects of FRS and NRS on growth performance, carcass traits and visceral organs
Results of growth performance and carcass traits were shown in Table 3, BW and ADG were affected by RS and the interactions between RS and age (P < 0.05). BW increased with ages, and together with ADG were higher in NRS ducks than FRS ducks at 8w (P < 0.05). As for carcass traits, CY, EY and SEY of Nonghua ducks exhibited a consistent tendency of higher than 85%, 70% and nearly 80% at different ages in both FRS and NRS. CW, SFW, SFY, AFW and AFY were affected by RS (P < 0.05). CY, EW, EY, SEW, SFW, SFY, AFW and AFY were affected by the interactions between RS and age (P < 0.05). CY was lower in NRS ducks at 4w and 8w (P < 0.05), while CW, EW and SEW were higher in NRS ducks at 8w (P < 0.05).
SFW, SFY, AFW and AFY were signi cantly higher in NRS ducks at 8w (P < 0.05), and SFY, AFW, AFY were also higher at 13w (P < 0.05).  The data was displayed as means (n = 60) 1 HW, heart weight; HI, heart index; LW, liver weight; LI, liver index; GW, gizzard weight; GI, gizzard index; PW, proventriculus weight; PI, proventriculus index; FRS, oor rearing system; NRS, net rearing system; RS, rearing system 2 When P-values were below 0.001, the speci c values were omitted and uniformly expressed as < 0.001 abc Different lowercase letters indicated that the difference between NRS and FRS corresponding group was signi cant (P < 0.05). Table 4, LI, GW and GI were affected directly by RS (P < 0.05), while HW, LW, LI, GW and GI were affected by the interactions between RS and age (P < 0.05). Notably, LW and LI were signi cantly higher in FRS ducks at 4w (P < 0.05). GW was higher in FRS ducks at 4w and 13w (P < 0.05), and GI was higher in FRS ducks at all ages (P < 0.05). The data was displayed with means (n = 14)

Results of visceral organs were shown in
1 GSH-Px, glutathione peroxidase; SOD, superoxide dismutase; MDA, malondialdehyde; FRS, oor rearing system; NRS, net rearing system; RS, rearing system 2 When P-values were below 0.001, the speci c values were omitted and uniformly expressed as < 0.001 abc Different lowercase letters indicated the difference between NRS and FRS corresponding group was signi cant (P < 0.05)

Effects of FRS and NRS on serum biochemical parameters and antioxidant capacity indicators
Results of serum biochemical parameters was depicted in Fig. 1, where ALT, ALP, TP, ALB and GLOB were affected by RS (P < 0.05), while TP, ALB and GLOB were also affected by the interactions between RS and age (P < 0.05). As for liver health biomarkers, ALP was signi cantly lower in NRS ducks at 4w and 8w (P < 0.05), while ALT was lower in NRS ducks at 8w and 13w (P < 0.05). TP and GLOB were signi cantly higher but ALB was lower than FRS ducks at 13w (P < 0.05). According to Table 5, though neither RS nor the interactions indicated a signi cant impact on antioxidant capacity indicators, a trend of lower MDA content but higher GSH-Px and SOD activities were found in NRS ducks at 4w (P > 0.05). The activity of SOD was decreased in NRS ducks at 8w (P < 0.05), but the activity of GSH-Px was increased in NRS ducks at 13w (P < 0.05).  The data was displayed as means (n = 60) 1

Effects of FRS and NRS on immune organ development and serum immune cytokines pro les
Results of immune organs and immune cytokines were shown in Table 6 and Fig. 2, respectively. As for immune organs, SW, BFW and BFI were signi cantly affected by RS (P < 0.05), while SW, SI and TW were signi cantly affected by the interactions (P < 0.05). BFI was signi cantly lower in NRS ducks at 4w and 8w (P < 0.05), while TW was signi cantly higher in NRS ducks at 8w (P < 0.05). SW and SI were signi cantly higher than FRS ducks at 13w (P < 0.05). In terms of immune cytokines, all indicators we detected were signi cantly affected by RS (P < 0.05), and IgG was also affected by the interactions (P < 0.05). Notably, IL-1β exhibited a consistent trend of higher pro les in NRS ducks at all ages (P < 0.05). IL-4 was signi cantly higher but IL-6 was lower in NRS ducks at 4w and 13w (P < 0.05), while IFN-γ, IgA and IgG were only higher than FRS ducks at 4w (P < 0.05).

Effects of FRS and NRS on liver gene expression
The results of gene expressions were depicted in Fig. 3. ALB was affected directly by RS (P < 0.05), and exhibited a consistent trend of lower expression in NRS ducks at different ages. Other genes were neither affected by RS, nor affected by the interactions (P > 0.05). ALB in FRS ducks was signi cantly higher than NRS ducks at 4w (P < 0.05), while ALB in 4w-FRS was also higher than 8w-FRS and 13w-FRS (P < 0.05). ALP in FRS ducks was signi cantly higher than NRS ducks at 13w (P < 0.05), while ALP in 13w-FRS was also higher than 4w-FRS and 8w-FRS (P < 0.05). IFN-γ was higher in 13w-FRS than 4w-FRS and 8w-FRS (P < 0.05).

Discussion
Production performance is one of the major concerns in poultry production as it is closely associated with the pro ts of animal products, while increasing focus on poultry health status, a re ection of physiological status, is not only due to its impact on production performance but also because of the raising attention to animal welfare issues. In this study, we compared the effects of FRS and NRS on production performance and health status of Nonghua ducks at different ages. Based on the higher nal BW, ADG and lower FCR of waterfowls in NRS, studies on Yangzhou geese and Chaohu ducks reported waterfowls in NRS exhibited better growth performance than in FRS [9,24]. Similarly, in Moulard ducks, though FCR was not signi cantly affected, the higher nal BW and ADG in NRS in comparison to sawdust-FRS and sand-FRS also revealed that NRS resulted in a better growth performance [25]. However, with a lower ADG but a higher FCR of Cherry Valley ducks in NRS, Chen et al. [12] reached an opposite conclusion of ducks in FRS exhibited better growth performance. These inconsistent conclusions may result from different breeds and slaughtering ages. To clarify the effects of FRS and NRS on meat ducks in different breeding stages, the current study selected three key weeks of age to study the interactions between RS and age. Results showed that BW and ADG were higher in NRS at 8w while no signi cant results were found at 4w and 13w, indicating a better growth performance of NRS ducks before the market age.
As for carcass traits, the average CY, EY and SEY of Nonghua ducks at 8w and 13w under either FRS or NRS were higher than 85%, 75% and 80%, indicating outstanding carcass traits compared with Pekin ducks, Cherry Valley ducks, White Muscovy ducks and Jingjiang ducks (a native Sheldrake breed in China) at similar ages [26][27][28][29]. Previous study on Chaohu ducks noticed the lower EY but higher AFY in NRS [9]. Similar to the current study, results showed that CY of NRS ducks was lower than FRS ducks at 4w and 8w, while CW, EW, SEW, SFW, AFW, SFY, and AFY were higher in NRS at 8w, SFY and AFY were also higher in NRS at 13w, indicating that NRS was conducive to carcass traits to some extent. With the age increasing, the majority of signi cances in carcass traits faded, but the lipid deposition was enhanced consistently in NRS. This was in agreement with Liu et al. [24], who reported Yangzhou geese in NRS exhibited higher subcutaneous fat thickness and higher AFY than in FRS. The visceral organ development is closely associated with duck growth, development, and health [30]. Results showed GW and GI were generally lower in NRS at different ages, indicated that FRS ducks had better gizzard development than NRS ducks. This was in agreement with Wang et al. [6], who also found a better gizzard development of birds in FRS compared with CRS and NRS. The reason can be concluded as NRS ducks cannot intake any grain of sand from the oor, because adding large particles and structural components to diets could stimulate poultry gizzard function and development [31,32]. Moreover, LW and LI of NRS ducks were lower than FRS ducks at 4w, indicating a liver developmental retardation at the early stage of breeding in NRS. Therefore, NRS improved the carcass weight before the market age but had defects in carcass yield. FRS ducks exhibited better gizzard and liver development at the early stage of breeding, while NRS enhanced the lipid deposition at the later one.
Since the liver is the major site of metabolism and detoxi cation, this retardation in NRS ducks may in uence the serum biochemical parameters of Nonghua ducks. Serum biochemical parameters of AST, ALT, and ALP were biomarkers to evaluate the health status of liver, because hepatocytes would secrete these enzymes into the blood once the liver was damaged [33,34]. Results showed these parameters were coherently lower in NRS ducks at different ages, demonstrating NRS was better for duck liver health despite the lipid deposition enhancement and liver developmental retardation. And this also showed an advantage over CRS, as ducks suffered liver injury once they were just put into cages [35]. Furthermore, antioxidant capacity prevents cell damage from free radicals and other reactive oxygen species (ROS) to avoid oxidative stress (the imbalance between ROS and antioxidants) [36,37]. The concentration of MDA and activities of SOD, GSH-Px are effective indicators to re ect the antioxidant status of ducks [38,39]. A study on Shaoxing ducks showed rearing systems did not have signi cant impacts on the activity of MDA, SOD, CAT, T-AOC, and GSH-Px in the liver [35]. Another study though only determined the total antioxidant capacity (T-AOC) in serum, results showed no signi cant difference between FRS and NRS [5].
Similarly in the current study, rearing system did not signi cantly affect MDA, SOD, and GSH-Px, however, it exhibited a trend of lower MDA content and higher activities of SOD and GSH-Px in NRS at 4w, which indicated that NRS ducks might have a better antioxidant capacity than FRS ducks at this early stage of breeding. The activity of SOD decreased in NRS at 8w and maintained relatively lower than FRS ducks at 13w, while GSH-Px activity was signi cantly higher in NRS and MDA content was also relatively higher than FRS ducks at 13w. These results implied GSH-Px was responsible for scavenging excess free radicals at the later stage of breeding.
As for immune function, Xi et al. [40] reported that there was no signi cant difference in immune organ indexes of Cherry Valley ducks between NRS and FRS. Differently in current study, NRS ducks were higher in TW at 8w as well as SW and SI at 13w, which indicated a promotion on immune organ development in NRS. Whereas, the decreased BFI in NRS ducks at 4w and 8w implied a faster degradation of bursa. Current study also showed a trend of higher TI at 4w and 8w though the results were not signi cant, which was similar to Zhao et al. [5], who reported NRS ducks had lower mortality rate and higher TI than FRS ducks. Previous study also reported IL-1β and IgG pro les of Shaoxing ducks were not signi cantly affected by NRS [5], while immune cytokines were extensively affected by RS in current study. Generally, the tendency of higher pro les of IFN-γ, IL-1β, IL-4, immunoglobulins in NRS ducks indicated a better immune status, IL-1 [41] and IL-4 [42] contribute to T helper type-2 (Th2) cell activity, whereas IFN-γ contributes to T helper type-1 (Th1) cell activity [43]. Higher levels of IFN-γ, IL-1β, and IL-4 during the experiment suggested the activity of Th1 and Th2 cells may be promoted in NRS. The enlargement of thymus at 8w may be associated with escalated IL-1β because IL-1 can act as a growth factor for thymocytes [41]. Faster degradation of bursa and lower IL-6 content suggested NRS ducks may have lower immunological requirements. This could be explained by the direct dropping of duck feces in NRS, which makes ducks less opportunity to contact with their excreta. Wang et al. [44] and Almeida et al. [8] reported that FRS had higher microbial contamination and higher concentrations of NH 3 and CO 2 in the air than NRS, hence the better air quality in NRS also helped reduce the burden on duck immune system.
Moreover, the elevation of immunoglobulins in NRS may be associated with escalated IL-1β, due to its promotion on IL-2 to enhance the secretion of immunoglobulins. In terms of serum proteins, NRS ducks showed higher GLOB and TP but lower ALB at 13w, this was in agreement with the study on Chaohu ducks [9], which also reported serum TP content was improved by NRS in comparison to FRS. Serum proteins change can be concluded as follows: First, FRS ducks took more exercise during rearing, which would enhance protein synthesis causing TP loss in blood [14]. Second, due to the enhancement of lipid deposition in NRS, ALB would be used for lipoprotein synthesis leading to the lower ALB serum pro le in NRS [45]. Third, higher levels of immunoglobulins in NRS contributed to the change of GLOB in serum, especially the consistent elevation in IgA, IgG, and IgM in NRS at 13w. Based on the liver functions of synthesizing serum proteins including albumins [46], a coherent trend of lower ALB expression in NRS ducks at different ages might be responsible for the signi cantly lower serum ALB pro les at 13w. There was no signi cant change in SOD1, indicating this antioxidant was not signi cant in the change of two different rearing systems. IFN-γ is a pro-in ammatory cytokine, which participated in multiple in ammatory responses [43]. Hence, the higher expression of IFN-γ as well as ALP in 13w-FRS than 4w-FRS and 8w-FRS indicated a potential more intense environmental stress in liver at the later stage of breeding in FRS. However, the speci c regulatory mechanism needs further study to elucidate. Therefore, NRS could improve duck immune function by increasing the levels of certain immune cytokines pro les and promoting the development of immune organs, while liver gene expression indicated that ducks might suffer from a potential higher environmental stress in FRS.

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In conclusion, compared with FRS, NRS was conducive to improve growth performance and carcass traits to some extent but it had defects in visceral organ development and lipid deposition. According to lower serum pro les of ALT, AST, and ALP as well as liver gene expression in NRS, NRS could be better for duck liver health compared with FRS. Antioxidant capacity was not signi cantly affected by rearing system, but indicators showed NRS ducks may have better antioxidant capacity at the early stage of breeding, while at the later one, GSH-Px activity was increased for scavenging excess free radicals in NRS. NRS increased the levels of IFN-γ, IL-1β, IL-4, immunoglobulins in serum and promoted the development of thymus and spleen to improve duck immune function, which would promote the activities of Th1 and Th2 cells.

Consent for publication
Not applicable.

Availability of data and materials
The datasets analyzed in the present study are available from the corresponding author on reasonable request.  Effects of FRS and NRS on serum immune cytokines of Nonghua ducks at different ages. Data was displayed as "Means ± SEM" in gures, the P-values of main effects of RS, age and their interactions were displayed in the table. Abbreviations: FRS, oor rearing system; NRS, net rearing system; RS, rearing system. 1 When P-values were below 0.001, the speci c values were omitted and uniformly expressed as <0.001. abc Different lowercase letters indicated the difference between NRS and FRS corresponding group was signi cant (P<0.05), for both FRS and NRS corresponding groups, n=14.