Effects of stocking density on performance, egg quality, reproductive hormones, and antioxidant capacity in egg-laying ducks

ABSTRACT

This study was conducted to evaluate the effects of stocking density on productive performance, egg quality, and antioxidant capacity in laying ducks. A total of 720 20-week-old Jinding laying ducks were randomly assigned to 5 stocking densities (4, 5, 6, 7, and 8 birds/m²) with 8 replicate pens each treatment. The results showed that increasing stocking density linearly increased egg production and egg mass and linearly decreased FCR of laying ducks (P < 0.05). The eggshell strength and thickness decreased linearly (P < 0.05) and quadratically (P < 0.05) with an increase in stocking density. Increased stocking density linearly decreased concentrations of estradiol-17β and follicle-stimulating hormone, activities of superoxide dismutase, glutathione peroxidase, and total antioxidant capacity in plasma (P < 0.05), but linearly increased plasma and hepatic malondialdehyde content (P < 0.05). The results suggested that high stocking density adversely influenced laying performance and egg quality of ducks, which is associated with impaired antioxidant capacity. Under our experimental conditions, we recommend that the stocking density of laying ducks should be kept to 4 or fewer birds/m² to avoid the negative effects of high stocking density on performance and egg quality of laying ducks.

KEYWORDS:

• Egg production
• egg mass
• egg components
• antioxidant enzymes
• malondialdehyde

1. Introduction

With the increased demand for preserved eggs, such as alkalized eggs and salted duck eggs, the laying duck production has been gradually changed from conventional free range and open water outdoors to confinement in birdhouses in Southwest China. To pursue higher economic benefit, the highest possible density in confined laying duck production was commonly adopted by producers. A high stocking density has been demonstrated to cause adverse effects, such as reduced body weight, feed intake, and feed conversion efficiency, and higher incidence of foot-pad dermatitis in broilers (Estevez 2007). Many researchers have indicated that increasing stocking density negatively influences the productive performance and health of laying hens (Lay et al. 2011; Guo et al. 2012). Ducks are social animals and live in flocks. Flock size and stocking density have been shown to affect the performance and welfare of meat ducks (Rodenburg et al. 2005). Previous study investigated the effects of stocking density on meat ducks and found that high stocking density could decrease the growth performance of Pekin ducks without affecting carcass yield and foot pad lesions (Xie et al. 2014). In addition, flock size can significantly affect welfare of meat ducks, as larger group more nervous and panic reactions can cause serious damage and increased mortality (Rodenburg et al. 2005). However, few studies have investigated the effects of stocking density on production performance of laying ducks, and few recommendations have been provided for the maximal stocking density of laying ducks reared in confined buildings.

In addition, previous studies have clearly showed that broilers stocked at high density exhibited higher levels of oxidative stress indicators (Beloor et al. 2010; Simitzis et al. 2012). Likewise, high stocking density has been shown to compromise the antioxidant capacity of meat ducks, and dietary supplementation with antioxidants could alleviate the detrimental effects that high stocking density had on performance and meat quality of Pekin ducks (Liu et al. 2015). Moreover, previous study adopting proteome analysis revealed that higher stocking density was accompanied with imbalanced redox status in liver (Wu et al. 2018). However, it is unclear that whether stocking density could influence antioxidant parameters of laying ducks.

Therefore, the present study was conducted to investigate the effects of stocking density on the production performance, egg quality, reproductive hormones concentrations, and antioxidant parameters in plasma and liver of laying ducks.

2. Materials and methods

2.1. Birds, diets and experimental design

This study was conducted at Xichang Huanong Poultry Industry Co., Ltd. (Xichang, Sichuan, China) from September 2018 to February 2019. All of the experimental procedures were approved by the Animal Care and Use Committee of Sichuan Animal Science Academy, China. A total of 720 Jinding ducks, which is a typical breed of egg-laying ducks in South China, were selected at 20 weeks of age and randomly assigned to 40 plastic wire-floor pens according to 5 different stocking densities. All ducks were kept in the same room with the same ventilation and lighting protocol with 18 h of incandescent lighting at 15 l× from 0500 to 2300 and 6 h of dark during the whole experiment. The mean room temperature was 18.5 (±1.2) °C and mean relative humidity was 62 (±6)% during the 20-week experimental period. The pen size was 3 m² (2.5 m × 1.2 m), and the stocking densities were set as 4, 5, 6, 7, and 8 birds per m² corresponding to 12, 15, 18, 21, 24 ducks per pen, respectively, during the period from 20 weeks to 40 weeks of age. Each treatment has 8 replicates. The ducks were all fed the same diet with composition included in Table 1 and were free access to feed and water throughout the experiment.

Table 1. Ingredients and nutrient composition of experimental diet (as-fed basis).

Items	Experimental diet
Ingredients (%)
Corn	35.97
Corn starch	16.80
Wheat bran	10.00
Soybean meal	10.00
Peanut meal	16.00
Limestone	8.48
Calcium hydrogen phosphate	1.20
Sodium chloride	0.30
Premix^a	1.00
L-Lys•HCl (98.5%)	0.22
DL-Met (99%)	0.03
Total	100.00
Calculated nutritional value
Metabolic energy (MJ/kg)	10.46
Crude protein (%)	17.17
Ca (%)	3.60
Total P (%)	0.56
Available P (%)	0.35
Lys (%)	0.85
Met (%)	0.51
Met + Cys (%)	0.86

^aThe premix provided per kilogram diet: vitamin A, 3.96 mg; vitamin D₃, 0.066 mg; vitamin E, 20 mg; vitamin K₃, 2.75 mg; vitamin B₁, 4.4 mg; vitamin B₂, 9.0 mg; vitamin B₆, 4.4 mg; vitamin B₁₂, 0.02 mg; choline, 500 mg; nicotinic acid, 30 mg; pantothenic acid, 22 mg; folic acid, 1.1 mg; biotin, 0.22 mg; Fe, 60 mg; Cu, 8.0 mg; Mn, 100 mg; Zn, 90 mg; iodine, 0.5 mg; Se, 0.2 mg; Co, 0.26 mg.

2.2. Sample collection

At the end of the experiment (after 20 weeks of trial), two ducks from each replicate were selected at random for sampling. Heparinized blood samples were collected from the wing vein and then were centrifuged at 3000 r/min for 20 min (LDZ4-1.2 centrifuge, Beijing Jingli Centrifuge Co., Ltd, Beijing, China) to obtain plasma sample. The selected ducks were then stunned and exsanguinated, liver samples were collected and stored at −20°C pending further analysis.

2.3. Determination of production performance

The feed intake was recorded and the eggs produced were individually weighed daily on a per replicate basis. Egg production (%) (100 × number of eggs/number of laying ducks), egg weight (g), egg mass (g/d), average daily feed intake and FCR (grams of feed per gram of egg mass) were calculated daily, on a per replicate basis, and the data were then pooled before analysis.

2.4. Egg quality

At the end of the experiment, five eggs (excluding obvious outliers such as excessively large or small, broken, misshapen, cracked or dirty eggs) were randomly selected from each replicate to determine egg components and egg quality traits. The eggs were weighed, and then the yolks were separated, weighed and expressed as percentages of egg weight. The eggshells with membrane were weighed after drying at 105°C to calculate egg shell proportion. The albumen weight was then calculated as egg weight minus the sum of yolk and eggshell weight, and expressed as percentages of egg weight. The yolk colour, albumen height, and Haugh unit were measured using an egg multi tester (EMT-5200, Robotmation Co., Ltd., Tokyo, Japan). The eggshell strength was measured using an eggshell force gauge model II (Robotmation Co., Ltd., Tokyo, Japan). A digital micrometer was used to determine the eggshell thickness at three different points (large end, equatorial region, and small end) of each egg. The egg shape index was determined with a digital calliper.

2.5. Measurement of reproductive hormones in plasma

The plasma concentrations of estradiol-17β (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and progesterone were determined by radioimmunoassay using commercially available kits purchased from Beijing North Institute of Biology Technology (Beijing, China) according to the manufacturer’s instructions. Each sample was assayed in triplicate.

2.6. Determination of antioxidant indices in plasma and liver

The content of malondialdehyde (MDA) and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and total antioxidant capacity (T-AOC) in plasma and liver were measured in triplicate using commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.

2.7. Statistical analysis

Data were analyzed by one-way ANOVA procedure in SAS software (version 9.0, SAS Institute Inc.) with the pen being the experimental unit. Differences between treatments were further assessed by using Tukey post hoc test when the main effect was significant. The linear and quadratic effects of stocking density were estimated by orthogonal comparisons. Results are expressed as means with standard error of means (SEM). The differences were considered significant when value of P < 0.05.

3. Results

3.1. Production performance

Stocking density significantly affected (P < 0.05) the egg production, egg mass, and FCR (Table 2). The egg production decreased in a linear and quadratic manner with an increase in stocking density (P < 0.05). The egg mass and feed intake decreased in a linear (P < 0.05) manner as stocking density increased. Increased stocking density linearly and quadratically increased FCR of laying ducks (P < 0.05). There was no significant effect (P > 0.05) of increasing stocking density on the egg weight.

Table 2. Effects of stocking density on egg production performance (20–40 weeks) of laying ducks.

Item	Stocking density (birds/m^2)					SEM^2	P-value
	4	5	6	7	8		SD^3	Linear	Quadratic
Egg production (%)	88.07^a	85.18^a,b	80.61^b,c	76.04^c	67.14^d	1.246	<0.001	<0.001	0.015
Egg weight (g)	65.46	64.61	64.64	65.60	65.46	0.736	0.784	0.673	0.405
Egg mass (g/d)	57.67^a	55.03^a,b	52.13^b,c	49.86^c	43.96^d	1.030	<0.001	<0.001	0.135
Feed intake (g/d)	163.35^a,b	170.75^a	157.39^b	162.15^a,b	148.00^c	2.235	<0.001	<0.001	0.005
FCR^1	2.84^c	3.11^a,b,c	3.03^b,c	3.25^a,b	3.38^a	0.067	<0.001	<0.001	0.988

¹FCR, feed/egg conversion ratio.

²SEM, standard error of means, n = 8.

³SD, stocking density.

^a–dMean values within the same row with different superscripts differ significantly (P < 0.05).

3.2. Egg quality

As shown in Table 3, the eggshell strength and eggshell thickness decreased in a linear (P < 0.05) and quadratic (P < 0.05) manner as stocking density increased. There were no significant effects (P > 0.05) of increasing stocking density on the other egg components and egg quality indexes.

Table 3. Effects of stocking density on egg components and quality of laying ducks during the laying period from 20 to 40 weeks of age.

Item	Stocking density (birds/m^2)					SEM^1	P-value
	4	5	6	7	8		SD^2	Linear	Quadratic
Egg weight (g)	64.35	64.71	64.68	65.05	64.82	0.541	0.927	0.460	0.706
Yolk weight (g)	19.38	19.24	19.82	19.51	19.67	0.224	0.393	0.241	0.729
Albumen weight (g)	36.97	37.69	36.99	37.77	37.42	0.507	0.704	0.551	0.727
Eggshell weight (g)	8.00	7.78	7.87	7.77	7.73	0.107	0.434	0.119	0.659
Yolk (%)	30.13	29.73	30.68	30.00	30.35	0.375	0.468	0.558	0.930
Albumen (%)	57.43	58.25	57.16	58.05	57.72	0.412	0.344	0.778	0.843
Eggshell (%)	12.44	12.02	12.16	11.95	11.93	0.171	0.239	0.056	0.511
Egg shape index	1.38	1.36	1.37	1.43	1.36	0.023	0.189	0.681	0.603
Eggshell strength (N)	53.41^a	52.22^a,b	54.14^a	51.45^a,b	48.77^b	0.975	0.005	0.003	0.045
Eggshell thickness (mm)	39.66^a,b	40.30^a	40.09^a	38.54^b,c	38.18^c	0.318	<0.001	<0.001	0.008
Albumen height (mm)	6.10	5.87	6.20	5.99	5.97	0.129	0.443	0.737	0.845
Yolk colour	4.85	4.76	4.84	4.79	4.81	0.096	0.963	0.847	0.795
Haugh unit	75.36	72.49	75.61	73.51	75.55	1.087	0.169	0.685	0.266

¹SEM, standard error of means, n = 8.

²SD, stocking density.

^a–cMean values within the same row with different superscripts differ significantly (P < 0.05).

3.3. Reproductive hormones in plasma

Table 4 showed that the concentrations of E2 and FSH in plasma decreased linearly (P < 0.05) with an increase in stocking density. No significant effects (P > 0.05) of stocking density on plasma concentrations of LH and progesterone were observed.

Table 4. Effects of stocking density on plasma reproductive hormones concentrations of laying ducks.

Item	Stocking density (birds/m^2)					SEM^1	P-value
	4	5	6	7	8		SD^2	Linear	Quadratic
E2 (pg/mL)	193.44	159.81	125.93	120.85	108.34	25.070	0.131	0.012	0.454
LH (mIU/mL)	2.22	1.96	2.18	2.06	1.75	0.281	0.774	0.360	0.674
FSH (mIU/mL)	4.32^a	3.67^a,b	3.63^a,b	2.56^a,b	2.08^b	0.501	0.022	0.001	0.720
Progesterone (pg/mL)	408.55	398.61	366.74	361.07	323.03	34.913	0.452	0.067	0.820

E2, estradiol-17β; LH, luteinizing hormone; FSH, follicle-stimulating hormone.

¹SEM, standard error of means, n = 8.

²SD, stocking density.

^a,bMean values within the same row with different superscripts differ significantly (P < 0.05).

3.4. Antioxidant indices in plasma and liver

Among the antioxidant parameters (Table 5), the activities of SOD, GSH-Px, and T-AOC in plasma decreased in a linear (P < 0.05) manner as stocking density increased, whereas the plasma MDA content increased linearly (P < 0.05) as stocking density increased. In addition, the hepatic MDA content increased in a linear (P < 0.05) manner as stocking density increased. No significant effects (P > 0.05) of stocking density on activities of SOD, GSH-Px, and T-AOC in liver were observed.

Table 5. Effects of stocking density on antioxidant indices in plasma and liver.

Item	Stocking density (birds/m^2)					SEM^1	P-value
	4	5	6	7	8		SD^2	Linear	Quadratic
Plasma
SOD (U/mL)	165.09^a	154.94^a,b	153.55^a,b	145.01^a,b	132.12^b	7.062	0.029	0.002	0.636
GSH-Px (U/mL)	381.18^a	369.82^a,b	361.11^a,b	341.07^a,b	291.63^b	19.663	0.024	0.002	0.242
T-AOC (U/mL)	13.26	11.68	11.32	9.39	7.13	1.697	0.127	0.010	0.648
MDA (nmol/mL)	11.05^b	11.55^b	13.09^a,b	13.10^a,b	16.86^a	0.991	0.002	<0.001	0.188
Liver
SOD (U/mg prot)	183.34	188.60	185.68	178.77	164.17	16.447	0.848	0.361	0.482
GSH-Px (U/mg prot)	69.62	70.03	67.49	65.73	55.00	6.018	0.397	0.087	0.347
T-AOC (U/mg prot)	1.15	0.81	0.81	0.84	0.74	0.133	0.236	0.072	0.297
MDA (nmol/mg prot)	0.68^b	0.81^a,b	0.88^a,b	0.95^a,b	1.02^a	0.080	0.049	0.003	0.758

SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; T-AOC, total antioxidant capacity; MDA, malondialdehyde.

¹SEM, standard error of means, n = 8.

²SD, stocking density.

^a,bMean values within the same row with different superscripts differ significantly (P < 0.05).

4. Discussion

4.1. Productive performance and egg quality

Although a large number of studies have been conducted on broilers, meat ducks, and laying hens (Guo et al. 2012; Simitzis et al. 2012; Xie et al. 2014), few studies have evaluated the effects of stocking density on the productive performance of laying ducks (Tao et al. 2020). The effects of stocking density on performance of laying hens have been extensively explored, but the results were inconclusive. Some reports contended that higher stocking density (7 birds vs 5 birds per cage, 1800 square centimetres) decreased hen-day egg production and egg mass, whereas other reports (586 cm² per hen vs 398 cm² per hen) have found little or no effect (Guo et al. 2012). These contradictory findings might stem from different cage systems adopted by previous studies. Previous study showed that egg production was higher in ducks housed at the low feeding density (12 ducks per net, 2.2 square metres) compared to those housed at the high feeding density (18 ducks per net) (Tao et al. 2020). Likewise, in the present study, the egg production and egg mass of laying ducks decreased as stocking density increased (five densities, 4, 5, 6, 7, or 8 ducks/m²). Previous study showed that increasing stocking density decreased the feed efficiency of laying hens (Guo et al. 2012; Mirfendereski and Jahanian 2015). In agreement, in this study, laying ducks in higher stocking density group exhibited higher FCR. In addition, the effects of stocking density on egg weight of birds were also inconsistent. Some studies have indicated that stocking density (four cage space allowance, 342, 413, 516, and 690 cm²/bird) has no effect on egg weight of birds (Jalal et al. 2006). Others demonstrated that eggs produced by birds kept at high stocking density (20 vs 15 hens per cage measuring 3 square metres) were heavier than those produced by their counterparts (Mtileni et al. 2007). In the present study, stocking density did not affect the egg weight of laying ducks. These inconsistent results might stem from the differences in the setting of stocking densities between studies. Birds reared at high stocking density are more difficult to dissipate their heat than those reared at lower stocking density (El-Tarabany et al. 2015). Previous study has shown that high stocking density results in higher rectal temperature of laying hens, indicating the birds raised in high stocking density tend to expose to a low-degree heat stress (Guo et al. 2012). High stocking density has been reported to adversely affect the egg quality of birds (El-Tarabany et al. 2015; Campbell et al. 2017). In the present study, eggshell strength and eggshell thickness decreased as stocking density increased. The reduction in eggshell quality associated with heat stress is a well-known phenomenon in domestic birds (Ebeid et al. 2012). Previous study indicated that heat stress in ducks decreased eggshell strength and thickness, yolk colour and Haugh unit of eggs (Ma et al. 2014). Therefore, the poorer eggshell quality in ducks reared at high stocking density might be attributed to the exposure of low-degree heat stress. Taken together, laying ducks raised in high stocking density had poor laying performance and eggshell quality than those raised in low stocking density.

4.2. Reproductive hormones

The reproductive hormones, which include E2, LH, FSH, and progesterone, were found to be associated with the development of reproductive organs, thereby playing an essential role in regulating laying performance of domestic birds (Zhang et al. 1997; Long et al. 2016). Birds reared at high stocking density are generally exposed to chronic stress that impair performance and health status (Kang et al. 2011). Stressful situations experienced by the bird have been shown to decrease reproductive hormones (Henriksen et al. 2011). In addition, the decreased levels of E2, LH, FSH, and progesterone in plasma of laying hens subjected to thermal stress were associated with decreased egg production (Elnagar et al. 2010; González-Morán 2016). In the present study, high stocking density decreased plasma E2 and FSH concentrations of laying ducks, which is consistent with the findings in laying performance, implying that laying ducks kept at high stocking density suffered a considerable stress. Indeed, the plasma corticosterone level and the expression of stress-related genes were increased in birds when raised in high stocking density (Beloor et al. 2010; Kang et al. 2016).

4.3. Redox status in plasma and liver

Previous study indicated that high stocking density caused chronic stress of birds (Kang et al. 2011). Moreover, accumulating evidences suggested that the most of stresses in poultry production were associated with oxidative stress at the cellular level (Surai 2016). Increasing stocking density has been reported to induce oxidative stress in broilers (Simsek et al. 2009; Simitzis et al. 2012). The MDA is the main final product of lipid peroxidation and serves as a biomarker of oxidative stress (Aengwanich et al. 2010). Previous study showed that the serum MDA level in high stocking density group was significantly higher than that in low density group (Li et al. 2019). In agreement, in this study, MDA content in plasma and liver of laying ducks increased as stocking density increased, suggesting higher lipid peroxidation in birds reared at high stocking density. Crowding has been shown to increase conflicts between birds and cause stress, thereby resulting in enhanced redox imbalance(Simsek et al. 2009). In the current study, the activities of plasma antioxidant enzymes, including SOD, GSH-Px, and T-AOC, reduced as stocking density increased. These results implied that the increased lipid peroxidation in birds reared at high stocking density might result from the decreased activities of antioxidant enzymes (Li et al. 2019). It revealed that high stocking density induced redox imbalance in laying ducks. The imbalanced redox status has been associated with reduced egg production and poor egg quality (Eid et al. 2008). Therefore, high stocking density-induced loss in egg production might be associated with the reduction in antioxidant capacity of laying ducks. However, it is imperative to explore the mediatory role of redox status in the effects of stocking density on laying performance of ducks.

5. Conclusion

In conclusion, increasing stocking density reduced egg production, egg mass, eggshell strength, and eggshell thickness of laying ducks, which is associated with changes in reproductive hormones concentrations and impaired antioxidant capacity. Therefore, under the condition of this study, we suggest raising laying ducks in 4 or fewer birds/m² to obtain optimal laying performance and egg quality.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the National Key Research and Development Program of China [grant number 2016YFD0500510] and College Students’ Innovative Entrepreneurial Training Plan Program of Sichuan Province [grant numner S201910619063].