Optimal dietary energy and protein levels for breeding pigeons in the winter "2 + 3" lactation pattern

Abstract

The nutritional requirements of breeding pigeons depend on their physiological period, breeding pattern, and environmental conditions. Despite works on reduced litter size in winter production to combat high mortality and the poor welfare of squabs, there are few studies on the related nutritional requirements of these pigeons. A total of 432 pairs of European Mimas pigeons were randomly divided into 9 groups in which 3 crude protein (CP) levels (15, 16.5, and 18%) and 3 metabolizable energy (ME) levels (12.2 MJ/kg, 12.4 MJ/kg, and 12.6 MJ/kg) were tested to determine the optimal energy and protein requirements of breeding pigeons in the winter "2 + 3" breeding pattern. The results showed that ME and CP levels had little effect on the body weight, feed intake, and egg quality of breeding pigeons during the lactation period. An 18% CP diet significantly increased the laying rate and hatchability (P < 0.05), but there was no difference in the laying rate with 18% CP and 16.5% CP during the whole reproductive cycle (P > 0.05). There was a significant interaction between ME and CP levels, and the laying interval of breeding pigeons in group 9 (18% CP; 12.6 MJ/kg) was significantly shortened (P < 0.05). For squabs, the ME level had no effect on growth performance, slaughter performance, or meat quality. The body weight of 21-day-old squabs in the 18% CP group increased by 3.16% compared with that of the 15% CP group, but there was no difference between the 18% CP and 16.5% CP groups. Compared with other experimental groups, group 7 (18% CP; 12.2 MJ/kg) had the fastest growth rate in squabs (P < 0.05), and the corresponding slaughter weight was also the heaviest (P < 0.05). We further found that the height of the squab intestinal epithelium was significantly increased in both the 16.5% CP and 18% CP groups of squabs (P < 0.01), but male breeding pigeons showed a certain degree of oxidative stress with an increase in CP level. In conclusion, the effects of 15 to 18% CP levels and 12.2 to 12.6 MJ/kg ME levels on the reproductive metabolism of breeding pigeons and the growth and development of squabs in the "2 + 3" breeding pattern during winter are small. For economic efficiency, we suggest that the CP level can be reduced to 16.5% while the ME level should not be less than 12.2 MJ/kg in practical production.

INTRODUCTION

The market consumption level of meat pigeons is increasing and China has become the largest producer and consumer of meat pigeons in the world (Jiang et al., 2019; Kokoszynski et al., 2020). Nowadays, most of the pigeon breeding industry uses machine incubation instead of natural incubation and adopts the "2 + 3" or "2 + 4" breeding pattern (i.e., a pair of breeding pigeons nursing 3 to 4 squabs at the same time) to further improve production efficiency (Chen, 2020). However, the main nutritional source of squabs (until they reach adult weight) is heavily dependent on the "crop milk" synthesized and fed by male and female pigeons (Dong et al., 2012; Gillespie et al., 2012). During lactation, breeders’ food intake also increases significantly and their metabolic utilization of nutrients differs greatly from that during the resting period (Xie et al., 2016). Thus, providing a reasonable nutritional diet for breeding pigeons at this stage is an important technical obstacle for the current meat pigeon industry (Xie et al., 2019).

The poultry production sector, faced with a significant increase in the cost of feed ingredients, has focused on improving feed conversion by providing more precise nutrition, thus achieving economic and environmental sustainability (Willems et al., 2013; Geng et al., 2022). Metabolizable energy (ME) and crude protein (CP) are the most important factors affecting feed costs, as these are essential nutrients for an animal's survival (Wu, 2007; Singh et al., 2015). The balance between ME and CP levels in poultry diets is also important for maximizing growth performance and improving reproduction (Ye et al., 2015; Candrawati, 2020; Chen et al., 2021). However, current research on the nutritional requirements of pigeons during lactation is lagging, with limited precision feeding studies. Previous studies also focused singularly on examining growth performance and carcass quality of squabs, while less attention was paid to breeder reproduction and metabolic stress, etc. (Bu et al., 2015; Gao et al., 2016a; Wang et al., 2018; Zhu et al., 2021; Li et al., 2022).

In addition to the direct influence of diet nutrient levels, poultry feed intake is also regulated by complex physiological mechanisms that include a series of internal stimuli (such as hormones) and external factors (such as climate) (Classen, 2017). Most commercial generations of meat birds are more sensitive to temperature changes than their wild counterparts due to genetic selection for growth rate and feed conversion (Piestun et al., 2015). Additionally, cold attacks after hatching can also negatively affect offspring welfare (Nyuiadzi et al., 2020). In particular, squabs have inadequate feather cover during the early stages of development and experience compromised thermoregulatory capacity, thus relying on the shelter provided by the breeding pigeons in colder temperatures (Shan et al., 2019). Previous work also showed that the mortality rate of squabs was significantly higher in winter when the litter size was too high, and the growth rate and intestinal development of squabs were hindered (Zhang et al., 2023). This finding suggests that the "2 + 3" breeding pattern is more suitable for practical production during harsher winter months.

Unfortunately, there are few studies on the nutritional requirements of breeding pigeons during winter in the corresponding breeding pattern. Therefore, based on previous experiments that explored the energy/protein ratio of diets in the high production pattern during summer (Peng et al., 2023), we assessed the effects of feeding diets with different ME and CP levels on the reproductive performance, egg quality, and plasma biochemical indices of breeding pigeons, as well as the growth performance, slaughter performance, and jejunal mucosa histomorphology of squabs during winter. In turn, the optimal ME and CP requirements of pigeon diets in the winter "2 + 3" breeding pattern were defined to provide theoretical references for the precise feeding of lactating breeding pigeons.

MATERIALS AND METHODS

All experimental procedures adhered to the Institutional Animal Care Guidelines and obtained approval from the Institutional Animal Ethics Committee of Zhongkai University of Agricultural Engineering (ethics code: ZHKUMO-2021-121).

Animals and Experimental Design

A total of 432 pairs of 12-mo-old white European Mimas breeding pigeons with similar weights (female pigeons weighed approximately 617.64 ± 24.5 g and male pigeons weighed approximately 659.11 ± 23.91 g) and reproductive cycles were provided by Meizhou Golden Green Modern Agricultural Development Company, located in Guangdong Province. All breeding pigeons were randomly divided into 9 treatment groups with 8 replicates and 6 pairs each. The experiment followed a 3 × 3 two-way design, with 3 levels of CP (15, 16.5, and 18%) and 3 levels of ME (12.2 MJ/kg, 12.4 MJ/kg, and 12.6 MJ/kg) in the diet setup. This experimental period consisted of 21-d of lactation and 7-d of the resting period for 28 d, counting from the breeding pigeons nursed the newly hatched squabs. The dovecote maintained an average daily temperature of 11°C ± 3°C during the winter test period and followed a standardized 16L:8D light cycle. Each pigeon pair was housed individually in a cage measuring 50 × 65 × 65 cm³, with ad libitum access to food and water. These experimental cages were evenly distributed across different cage levels (upper, middle, and lower) to minimize the impact of cage location. Throughout the experiment, self-prepared health sand (with a shell-to-gravel ratio of 1: 2) was continuously added to the diet to aid digestion. The 100% full-priced pellets used in this experiment were all produced by Guangdong Province Huabao Feed Co., Ltd. and had a diameter of about 4.0 mm, length of about 1.0 cm, and hardness of about 40 to 50 N. Further details of the specific dietary and nutritional compositions can be found in Table 1.

Table 1.

Nutrient content of experimental diets of breeding pigeons during winter lactation period.

Items	Ⅰ	Ⅱ	Ⅲ	Ⅳ	Ⅴ	Ⅵ	Ⅶ	Ⅷ	Ⅸ
Corn	43.00	49.50	50.50	40.00	46.00	51.80	42.50	45.20	47.50
Soybean meal	17.90	18.50	18.90	22.20	22.80	23.40	26.70	27.20	27.70
Wheat	33.80	26.20	23.90	31.50	24.40	17.50	24.00	20.00	16.50
Soybean oil	0.00	0.50	1.40	1.00	1.50	2.00	1.50	2.30	3.00
Salt	0.40	0.40	0.40	0.40	0.40	0.40	0.40	0.40	0.40
SID Lys	0.30	0.29	0.29	0.19	0.19	0.18	0.09	0.08	0.08
Zeolite powder	0.00	0.01	0.02	0.11	0.11	0.12	0.21	0.22	0.22
CaHPO4	3.60	3.60	3.60	3.60	3.60	3.60	3.60	3.60	3.60
Premix2	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
Total	100.00	100.00	100.00	100.00	100.00	100.00	100.00	100.00	100.00
Nutrient levels1
CP (%)	14.99	14.98	15.00	16.53	16.49	16.48	17.97	17.99	18.02
EE (%)	2.57	3.02	3.94	3.12	3.94	4.58	3.87	4.73	5.48
Moisture (%)	11.68	11.67	11.57	11.66	11.58	11.55	11.61	11.52	11.45
Ash (%)	2.14	2.14	2.14	2.35	2.35	2.36	2.55	2.56	2.57
Ca (%)	0.90	0.90	0.90	0.91	0.91	0.91	0.92	0.92	0.92
TP (%)	0.98	0.98	0.97	0.99	0.99	0.98	1.00	1.00	0.99
ME (MJ/kg)	12.23	12.38	12.58	12.19	12.39	12.58	12.20	12.40	12.58
ME/CP (kcal/g)	19.50	19.75	20.04	17.63	17.96	18.24	16.23	16.47	16.69
SIDAA (%)
Asp	1.31	1.30	1.31	1.49	1.48	1.50	1.66	1.67	1.69
Glu	3.02	3.05	3.02	3.27	3.27	3.17	3.50	3.45	3.40
Ser	0.72	0.72	0.72	0.80	0.79	0.79	0.86	0.86	0.87
His	0.41	0.41	0.41	0.44	0.44	0.44	0.48	0.48	0.48
Gly	0.40	0.40	0.40	0.42	0.42	0.41	0.44	0.44	0.43
Thr	0.55	0.55	0.55	0.61	0.60	0.61	0.66	0.66	0.67
Arg	0.93	0.93	0.94	1.05	1.05	1.05	1.16	1.16	1.16
Ala	0.72	0.72	0.72	0.78	0.77	0.79	0.83	0.83	0.84
Tyr	0.22	0.22	0.22	0.23	0.23	0.23	0.24	0.24	0.24
Val	0.63	0.63	0.63	0.70	0.70	0.71	0.77	0.78	0.78
Met	0.29	0.29	0.30	0.34	0.34	0.35	0.39	0.39	0.40
Phe	0.72	0.72	0.72	0.80	0.79	0.79	0.86	0.86	0.87
Ile	0.57	0.57	0.57	0.64	0.64	0.65	0.71	0.71	0.72
Leu	1.34	1.33	1.34	1.42	1.42	1.44	1.50	1.51	1.53
Lys	0.97	0.97	0.97	0.97	0.97	0.97	0.97	0.97	0.97

¹Nutrient levels were calculated values.

SIDAA, standardized ileal digestible amino acid.

²The premixes provided the following per kg of diet: Vitamin A 4,000 IU, vitamin E 20 mg, vitamin B1 5 mg, vitamin B2 12 mg, vitamin B6 2 mg, vitamin B12 25 μg, niacin 20 mg, folic acid 0.4 mg, D-pantothenic acid 20 mg, biotin 0.15 mg, choline chloride 450 mg, copper 5 mg, iron 35 mg, manganese 50 mg, zinc 55 mg, iodine 0.2 mg, selenium 0.2 mg.

Sample Collection and Performance Measurement

Growth Performance

The fasting weight of each pair of breeding pigeons and the squabs were measured at 0, 7, 14, and 21 d of the experiment. Following the marketing sale of the 21-day-old squabs, the breeding pigeons were retained and weighed again after a 7-day rest period to monitor their recovery. Throughout the experiment, the survival conditions of the squabs were observed and recorded. The weekly feed intake of the breeding pigeons was recorded during the trial and combined with the litter weight gain to calculate the feed-to-weight ratio (F/G).

Reproductive Performance

The laying interval and laying rate of the breeding pigeons were recorded for 2 laying cycles (following the commencement of the lactation period). Subsequently, pigeon eggs were collected daily, marked, and placed in the incubator. The fertilization rate, hatchability, and birth weight were recorded for each treatment group.

Egg Quality

Egg quality was assessed based on the method described by Zhang et al. (2021). Twenty-four eggs were randomly selected from each treatment group during the 2 laying cycles (after the onset of lactation) and stored at 4°C. The weights of the egg and yolk were determined using a precision electronic analytical balance (AL104). The egg shape index was calculated by measuring the long and short diameter of the egg with vernier calipers (egg shape index = long diameter/short diameter). The eggshell strength at the blunt end of the egg was measured using a strength meter (EFR-01, ORKA Food Technology Ltd., Ramat Hasharon, Israel). The Haugh unit and protein height was determined using a multifunctional egg analyzer (EA-01, robotmation Ltd., Tokyo, Japan). After rinsing and drying, the eggshells were weighed using a precision electronic analytical balance (AL104). Finally, the eggshell thickness was measured using a digital micrometer after removing the shell membrane (Model 1061).

Slaughter Performance and Meat Quality

Twenty-four 21-day-old squabs were randomly selected from each treatment group for slaughter. They underwent a 12-h, fasting period before being assessed for 10 carcass traits, including slaughter weight, semieviscerated weight, eviscerated weight, pectoral muscle weight, abdominal fat weight, slaughter rate, semieviscerated rate, eviscerated rate, pectoral muscle rate, and abdominal fat rate. These measurements were conducted by the NY/T 823-2020 standard (Ministry of Agriculture of the People's Republic of China, 2020). The internal organs of the squabs were dissected and weighed to evaluate their developmental status. The parameters for internal organ measurements included the liver index, heart index, kidney index, glandular stomach index, gizzard index, pancreas index, spleen index, thymus index, and bursae index. Finally, pectoral muscle samples were stored in sealed bags at 4°C and the meat color (a*, b*, and L* values) and pH value of the right pectoral muscle and the drip loss of the left pectoral muscle were measured at 45 min and 24 h, respectively, according to the method of Petracci et al. (2013) and Yang et al. (2016).

Morphological Analysis of Jejunum Mucosa

Twelve squabs were randomly selected from each group of previously slaughtered squabs. This selection process involved carefully opening the abdominal cavity and extracting a 1 cm segment from the proximal 1/3 of the jejunum. These intestinal segments were gently rinsed with phosphate-buffered saline (PBS) and preserved in 4% paraformaldehyde phosphate buffer (BL539A, Biosharp, Hefei City, Anhui Province, China). We replaced the fixative with a new one after 24 h of fixation and performed subsequent embedding operations within a week. Then, referring to the steps of Agazzi et al. (2020), the fixed jejunal tissue blocks were first washed in running water, dehydrated in a graded series of ethanol, cleared with xylene, and finally embedded in paraffin. Next, 4-μm-thick paraffin sections were obtained from a microtome and stained according to the instructions of the hematoxylin-eosin staining kit (c0105m, Shanghai Biyun Tian Biotechnology Co., Ltd., Shanghai, China). After first dewaxed with xylene and rehydrated with anhydrous ethanol, these paraffin sections were stained with hematoxylin for 5 min and rinsed with running water. Subsequently, these sections were fractionated with 1% hydrochloric acid ethanol for 5 s, rinsed with running water to return to blue for 10 min and stained with eosin for 2 min. Following dehydration again in gradient ethanol, these sections were cleared with xylene, sealed in neutral balsam and finally observed with a microscope. Image-Pro Plus 6.0 analysis software was used to measure villus height, crypt depth, villus width, epithelium height, muscular layer thickness, and goblet cell number, and the ratio of the villus height to crypt depth (VH/CD) was calculated.

Determination of Plasma Biochemical Indexes

At 14 d of lactation, 12 female and 12 male pigeons were randomly selected from each treatment group. These pigeons underwent a 12-h fasting period and had unrestricted access to drinking water. Subsequently, a 5 mL blood sample was collected from the subwing vein. Similarly, 12 squabs were randomly selected from each treatment group at 21 d of lactation and blood was collected before slaughter. All collected blood samples were centrifuged at 3,000 r/min for 15 min after 30 min of resting. The resulting plasma was separated and stored at −40°C. Subsequently, the plasma was examined for contents of total cholesterol (T-CHO), glucose (GLU), uric acid (UA), albumin (ALB), and triglycerides (TG) using a Varioskan LUX multifunctional enzyme marker (Thermo Fisher Scientific Inc., Waltham). Additionally, the total antioxidant capacity (T-AOC), catalase (CAT) and superoxide dismutase (SOD) activities, hydrogen peroxide (H₂O₂), and malondialdehyde (MDA) contents in the plasma were measured by the colorimetric method. The necessary kits for these measurements were purchased from the Nanjing Jiancheng Institute of Biological Engineering, and the methods for determining the above indexes were strictly adhered to based on the instructions provided by the manufacturer.

Statistical Analysis

Preliminary statistical data were acquired using Excel and SPSS 26.0 software. The significance of differences between groups was first compared by Duncan's method, followed by 2-way ANOVA with interaction using a general linear model (GLM), and the significance test was performed by the LSD method, and P < 0.05 was considered significant.

RESULTS

Weight Change of Breeding Pigeons

In this experiment, there was no significant difference in the weight loss of female and male breeding pigeons from different ME and CP level groups during the lactation period (P > 0.05), and the interaction between the CP and ME levels also had little effect on the body weight changes of breeding pigeons during winter lactation in the "2 + 3" breeding pattern (Table 2).

Table 2.

Effect of dietary energy and protein levels on the weight change of breeding pigeons during lactation in winter¹.

Treatments (n = 8)	CP/%	ME/MJ/kg	Male pigeon weight loss/g	Female pigeon weight loss/g	Total weight loss/g
I	15.0	12.2	63.875	53.150	117.038
II	15.0	12.4	65.787	46.250	112.038
III	15.0	12.6	65.700	58.313	124.000
IV	16.5	12.2	67.075	50.188	117.275
V	16.5	12.4	70.687	53.625	124.325
VI	16.5	12.6	63.712	51.638	114.288
VII	18.0	12.2	69.525	54.838	124.375
VIII	18.0	12.4	72.150	55.438	127.575
IX	18.0	12.6	70.325	49.813	120.113
SEM			1.294	1.393	2.005
Main effect
CP (n = 24)	15.0		65.121	52.571	117.692
	16.5		67.158	51.817	118.629
	18.0		70.667	53.363	124.021
ME (n = 24)		12.2	66.825	52.725	119.563
		12.4	69.542	51.771	121.313
		12.6	66.579	53.254	119.467
P values	CP		0.231	0.906	0.396
	ME		0.600	0.911	0.917
	C*M		0.762	0.686	0.647

¹All results are presented as mean ± SEM (n = 8).

The absence or the same letter in the same column of data indicates no significant difference (P > 0.05).

C*M, the interaction of the crude protein with metabolic energy; total weight loss, (male + female) pigeon weight loss.

Reproductive Performance of Breeding Pigeons

Results showed that the effect of dietary ME level on breeding pigeons’ reproduction was small, but that increasing the CP level improves the breeding pigeons' reproductive efficiency after lactation. In the first laying cycle after lactation, breeding pigeons fed an 18% CP level laid significantly earlier, where the laying rate reached 42.36% at 35 d (P < 0.01). Near the end of the cycle, the average laying rate of the breeding pigeons fed on the 16.5% CP and 18% CP levels was significantly higher than that of the 15% CP level (P < 0.05). But there was no significant difference in the overall laying rate between the 16.5% CP and the 18% CP levels (P > 0.05) (Table 3). This experiment also revealed that the CP level continued to influence the second laying cycle, and the hatchability of breeding eggs was also significantly higher in the 16.5% CP and 18% CP level groups than in the 15% CP level group (P < 0.01) (Table 4). The interaction between the CP and ME levels also had a significant effect on reproductive performance, with breeding pigeons in test group 9 (18% CP, 12.6 MJ/kg) laying earlier thereby optimizing their reproductive efficiency (P < 0.05). Combined throughout the cycle, the breeding advantage of this treatment group was less compared to the other test groups.

Table 3.

Effect of dietary energy and protein levels on the reproductive performance of breeding pigeons in winter (first laying cycle)¹.

Treatments (n = 8)	CP/%	ME/MJ/kg	Average laying interval/d	Fertility rate/%	Hatchability/%	Average birth weight/g	35-day laying rate/%	40-day laying rate/%	50-day laying rate/%
I	15.0	12.2	39.896	97.301	94.141	17.504	22.917c	50.000	97.917a
II	15.0	12.4	39.313	93.068	90.193	15.863	25.000bc	70.833	97.917a
III	15.0	12.6	40.104	95.322	85.385	16.522	16.667c	50.000	89.583b
IV	16.5	12.2	38.908	92.910	86.730	15.829	25.000bc	60.417	97.917a
V	16.5	12.4	39.229	89.899	88.223	16.262	29.167bc	58.333	100.000a
VI	16.5	12.6	38.354	95.392	86.203	16.530	35.417abc	66.667	100.000a
VII	18.0	12.2	38.688	98.958	88.621	16.477	33.333abc	64.583	97.917a
VIII	18.0	12.4	38.500	92.955	86.929	16.569	43.750ab	68.750	97.917a
IX	18.0	12.6	37.312	92.301	82.186	15.863	50.000a	75.000	100.000a
SEM			0.263	0.861	0.933	0.180	2.399	2.748	0.759
Main effect
CP (n = 24)	15.0		39.771a	95.230	89.906	16.630	21.528B	56.944	95.139b
	16.5		38.831ab	92.734	87.052	16.207	29.861B	61.806	99.306a
	18.0		38.167b	94.738	85.912	16.303	42.361A	69.444	98.611ab
ME (n = 24)		12.2	39.164	96.390	89.831	16.604	27.083	58.333	97.917
		12.4	39.014	91.974	88.448	16.231	32.639	65.972	98.611
		12.6	38.590	94.338	84.591	16.305	34.028	63.889	96.528
P values	CP		0.047	0.453	0.189	0.606	0.001	0.176	0.044
	ME		0.649	0.115	0.058	0.664	0.405	0.500	0.480
	C*M		0.327	0.328	0.181	0.450	0.019	0.337	0.035

¹All results are presented as mean ± SEM (n = 8).

Different lowercase letters in the data of the same column indicate significant differences (P < 0.05), and different capital letters indicate extremely significant differences (P < 0.01).

C*M, the interaction of the crude protein with metabolic energy.

Table 4.

Effect of dietary energy and protein levels on the reproductive performance of breeding pigeons in winter (second laying cycle)¹.

Treatments (n = 8)	CP/%	ME/MJ/kg	Average laying interval/d	Fertility rate/%	Hatchability/%	Average birth weight/g	10-day laying rate/%	12-day laying rate/%	14-day laying rate/%
I	15.0	12.2	12.371	93.889	81.101	15.245	10.417	58.333	85.416
II	15.0	12.4	13.508	92.361	83.563	15.596	10.417	43.750	72.917
III	15.0	12.6	13.194	98.438	75.347	15.623	2.083	43.750	77.083
IV	16.5	12.2	12.937	96.007	94.006	15.279	12.917	49.167	80.833
V	16.5	12.4	13.000	92.424	88.122	15.708	18.750	54.167	83.333
VI	16.5	12.6	12.908	94.975	92.438	15.766	8.333	62.500	85.417
VII	18.0	12.2	13.135	90.852	91.482	15.970	8.333	50.000	77.083
VIII	18.0	12.4	13.087	89.741	92.659	16.310	8.333	48.333	75.833
IX	18.0	12.6	12.342	94.375	85.178	15.458	14.584	52.083	93.750
SEM			0.156	0.969	1.660	0.091	1.419	2.512	2.055
Main effect
CP (n = 24)	15.0		13.024	94.896	80.004B	15.488	7.639	48.611	78.472
	16.5		12.949	94.469	91.592A	15.584	13.333	55.278	83.194
	18.0		12.855	91.656	89.773A	15.913	10.417	50.139	82.222
ME (n = 24)		12.2	12.815	93.583	88.883	15.498	10.556	52.500	81.111
		12.4	13.199	91.509	88.114	15.871	12.500	48.750	77.361
		12.6	12.815	95.929	84.321	15.616	8.333	52.778	85.417
P values	CP		0.910	0.347	0.010	0.145	0.259	0.541	0.611
	ME		0.529	0.191	0.471	0.219	0.482	0.774	0.281
	C*M		0.759	0.582	0.123	0.156	0.289	0.722	0.380

¹All results are presented as mean ± SEM (n = 8).

^A–BDifferent capital letters in the data of the same column indicate extremely significant differences (P < 0.01).

C*M, the interaction of the crude protein with metabolic energy.

Egg Quality of Breeding Pigeons

The test results of egg quality in the first 2 laying cycles of breeding pigeons after lactation are shown in Tables 5 and 6. Data revealed that the eggshell weight of pigeons in 16.5 and 18% CP level groups was significantly lower than that of the 15% CP level group in the first laying cycle (P < 0.01). In contrast, the different ME and CP levels of the diets had little effect on egg weight, yolk weight, egg shape index, eggshell strength, Hastelloy units, eggshell weight and eggshell thickness during the second laying cycle (P > 0.05).

Table 5.

Effect of dietary energy and protein levels on egg quality of breeding pigeons during lactation in winter (first laying cycle)¹.

Treatments (n = 24)	CP/%	ME/MJ/kg	Egg weight/g	Relative yolk weight/g	Relative shell weight/g	Egg shape index	Shell strength/Pa	Shell thickness/mm	Haugh unit
I	15.0	12.2	23.679	4.641	1.635	1.379	11.143	0.211	76.741
II	15.0	12.4	23.311	4.443	1.712	1.391	12.135	0.222	75.465
III	15.0	12.6	23.984	4.656	1.632	1.372	11.453	0.214	78.985
IV	16.5	12.2	23.241	4.544	1.569	1.375	11.404	0.211	76.100
V	16.5	12.4	23.363	4.657	1.576	1.387	10.599	0.210	76.821
VI	16.5	12.6	23.706	4.435	1.587	1.389	10.674	0.222	76.886
VII	18.0	12.2	23.906	4.755	1.553	1.357	10.583	0.206	74.800
VIII	18.0	12.4	24.215	4.826	1.589	1.369	11.176	0.210	79.459
IX	18.0	12.6	23.233	4.517	1.601	1.389	11.343	0.216	75.971
SEM			0.141	0.037	0.012	0.003	0.144	0.002	0.417
Main effect
CP (n = 72)	15.0		23.658	4.580	1.660A	1.381	11.577	0.216	77.064
	16.5		23.437	4.545	1.577B	1.384	10.893	0.214	76.602
	18.0		23.785	4.699	1.581B	1.372	11.028	0.211	76.743
ME (n = 72)		12.2	23.609	4.647	1.586	1.370	11.037	0.209	75.880
		12.4	23.630	4.642	1.626	1.383	11.303	0.214	77.248
		12.6	23.641	4.536	1.607	1.383	11.157	0.217	77.280
P values	CP		0.607	0.212	0.009	0.243	0.132	0.408	0.895
	ME		0.996	0.366	0.414	0.157	0.753	0.123	0.282
	C*M		0.691	0.189	0.119	0.150	0.240	0.272	0.121

¹All results are presented as mean ± SEM (n = 24).

^A–BDifferent capital letters in the data of the same column indicate extremely significant differences (P < 0.01).

C*M, the interaction of the crude protein with metabolic energy.

Table 6.

Effect of dietary energy and protein levels on egg quality of breeding pigeons during lactation in winter (second laying cycle)¹.

Treatments (n = 24)	CP/%	ME/MJ/kg	Egg weight/g	Relative yolk weight/g	Relative shell weight/g	Egg shape index	Shell strength/Pa	Shell thickness/mm	Haugh unit	Albumen height/mm
I	15.0	12.2	23.956	4.634	1.589	1.380	11.096	0.208	78.163	3.947
II	15.0	12.4	23.368	4.252	1.609	1.374	12.600	0.214	76.186	3.636
III	15.0	12.6	23.549	4.469	1.610	1.385	11.586	0.209	75.875	3.550
IV	16.5	12.2	23.253	4.426	1.596	1.385	11.885	0.211	77.555	3.864
V	16.5	12.4	23.700	4.420	1.599	1.375	11.310	0.206	76.991	3.826
VI	16.5	12.6	24.075	4.542	1.622	1.381	11.455	0.211	77.813	3.896
VII	18.0	12.2	24.141	4.557	1.693	1.376	12.253	0.216	77.979	3.963
VIII	18.0	12.4	24.034	4.576	1.592	1.376	11.201	0.204	77.662	3.900
IX	18.0	12.6	23.319	4.422	1.582	1.386	11.252	0.210	77.848	3.887
SEM			0.118	0.034	0.010	0.003	0.148	0.001	0.341	0.047
Main effect
CP (n = 72)	15.0		23.624	4.451	1.603	1.380	11.761	0.210	76.742	3.711
	16.5		23.676	4.463	1.605	1.380	11.550	0.210	77.453	3.862
	18.0		23.831	4.518	1.622	1.380	11.568	0.210	77.829	3.917
ME (n = 72)		12.2	23.783	4.539	1.626	1.380	11.745	0.212	77.899	3.925
		12.4	23.700	4.416	1.600	1.375	11.704	0.208	76.946	3.787
		12.6	23.648	4.478	1.605	1.384	11.431	0.210	77.178	3.778
P values	CP		0.757	0.696	0.711	0.991	0.812	0.951	0.434	0.216
	ME		0.894	0.337	0.555	0.429	0.639	0.349	0.520	0.413
	C*M		0.451	0.271	0.401	0.961	0.226	0.170	0.757	0.478

¹All results are presented as mean ± SEM (n = 24).

The absence or the same letter in the same column of data indicates no significant difference (P > 0.05).

C*M, the interaction of the crude protein with metabolic energy.

Growth Performance of Squabs

The survival status of squabs during the lactation period can be seen in Figure 1. The mortality rate of squabs in all the treatment groups gradually slowed down in the middle and late stages of lactation (Figure 1B), with the lowest mortality of 6.86% in group 8 compared to the other treatment groups for the whole period (Figure 1A). Diets with different CP and ME levels had little effect on the survival rate of squabs during the winter lactation period (P > 0.05), and there was no significant interaction.

Figure 1.

Effect of dietary energy and protein levels on the winter survival of 808 squabs. (A) Full-term total mortality of squabs; (B) Survival curves of squabs.

In the early stage of winter lactation (1–7 d), the different levels of ME and CP in the diet had little effect on the growth and development of the squabs, and there was no significant difference in body weight between all groups (P > 0.05). Bodyweight gain was positively correlated with CP level during the middle and late stages of lactation (7–21 d). The average litter weight at 21 d of age was 1302.7 g in the 18% CP group, with the fastest growth rate (P < 0.05), but the difference was not significant compared to the 16.5% CP level group (P > 0.05). The interaction between CP and ME had a significant effect on the growth performance of squabs, and experimental group 7 (18% CP, 12.2 MJ/kg) had the greatest weight gain over the whole lactation period (P < 0.05) (Table 7). Further analysis of feed remuneration during lactation revealed that the 12.2 MJ/kg ME level groups had a high F/G in the late lactation period (14–21 d) (P < 0.01). Compared with other experimental groups, the lowest F/G was seen in group 6 (16.5% CP, 12.6 MJ/kg) and group 2 (15% CP, 12.4 MJ/kg) during late lactation (P < 0.01), which was beneficial to short-term breeding. However, during the whole experimental period, there were no significant differences in the feed intake and feed conversion efficiency of squabs between all groups. Overall, different ME and CP levels in the diet had little effect on the production performance of squabs under the "2 + 3" breeding pattern in winter (Table 8).

Table 7.

Effect of dietary energy and protein levels on the growth performance of squabs in winter¹.

Treatments (n = 8)	CP/%	ME/MJ/kg	0-day squab weight/g	7-day squab weight/g	14-day squab weight/g	21-day squab weight/g	0-day litter weight/g	7-day litter weight/g	14-day litter weight/g	21-day litter weight/g
I	15.0	12.2	15.975	98.610	269.474c	414.135E	47.920	295.830	808.420c	1242.408d
II	15.0	12.4	15.766	99.572	271.960c	432.656ABC	47.294	298.716	815.042c	1299.189abc
III	15.0	12.6	15.778	98.605	272.521c	416.555DE	47.336	295.815	817.565c	1255.210cd
IV	16.5	12.2	15.965	99.402	273.480c	419.869CDE	47.898	298.209	820.443c	1264.454bcd
V	16.5	12.4	15.840	103.285	274.503bc	429.132ABCDE	47.523	309.854	823.505bc	1287.396abcd
VI	16.5	12.6	15.803	105.235	277.844abc	431.303ABCD	47.403	315.700	833.535abc	1297.789abc
VII	18.0	12.2	15.314	102.680	290.098a	440.870A	45.938	308.044	868.161a	1320.930a
VIII	18.0	12.4	15.876	102.664	277.490abc	425.339BCDE	47.633	307.994	832.470abc	1276.021abcd
IX	18.0	12.6	16.170	102.214	287.066ab	437.062AB	48.514	306.642	861.195ab	1311.187ab
SEM			0.077	0.770	1.685	1.976	0.222	2.352	5.009	6.208
Main effect
CP (n = 24)	15.0		15.840	98.929	271.318B	421.115b	47.517	296.787	813.676B	1265.602b
	16.5		15.869	102.641	275.275B	426.768ab	47.607	307.921	825.828B	1283.213ab
	18.0		15.787	102.519	284.885A	434.424a	47.361	307.560	853.942A	1302.713a
ME (n = 24)		12.2	15.751	100.231	277.684	424.958	47.252	300.694	832.341	1275.930
		12.4	15.827	101.840	274.651	429.043	47.483	305.521	823.672	1287.535
		12.6	15.917	102.018	279.144	428.307	47.751	306.052	837.432	1288.062
P values	CP		0.905	0.089	0.003	0.013	0.899	0.097	0.003	0.039
	ME		0.678	0.581	0.501	0.614	0.654	0.593	0.488	0.630
	C*M		0.428	0.401	0.040	0.008	0.366	0.430	0.046	0.032

¹All results are presented as mean ± SEM (n = 8).

Different lowercase letters in the data of the same column indicate significant differences (P < 0.05), and different capital letters indicate extremely significant differences (P < 0.01).

C*M, the interaction of the crude protein with metabolic energy.

Table 8.

Effect of dietary energy and protein levels on the production performance of squabs in winter¹.

Treatments (n = 8)	CP/%	ME/MJ/kg	First week feed intake/g	Second week feed intake/g	Third week feed intake/g	Total feed intake/g	First week F/G	Second week F/G	Third week F/G	Full-term F/G
I	15.0	12.2	514.660	1394.835	1895.054	3804.545	2.169	2.753	4.550AB	3.219
II	15.0	12.4	533.916	1409.970	1866.071	3809.958	2.176	2.758	3.988C	3.059
III	15.0	12.6	495.753	1372.198	1850.736	3718.685	2.060	2.672	4.437AB	3.102
IV	16.5	12.2	479.376	1350.472	1921.691	3751.539	1.987	2.660	4.660A	3.131
V	16.5	12.4	508.731	1393.373	1893.129	3795.234	2.062	2.761	4.193BC	3.088
VI	16.5	12.6	526.195	1345.058	1771.640	3642.896	2.045	2.656	3.976C	2.939
VII	18.0	12.2	474.035	1393.683	1900.999	3768.715	1.857	2.539	4.338ABC	2.992
VIII	18.0	12.4	469.814	1397.889	1822.868	3690.568	1.885	2.711	4.323ABC	3.035
IX	18.0	12.6	517.830	1451.844	1880.189	3849.861	2.066	2.678	4.295ABC	3.070
SEM			7.309	11.700	14.426	22.350	0.040	0.029	0.049	0.021
Main effect
CP (n = 24)	15.0		514.776	1392.334	1870.620	3777.729	2.135	2.728	4.325	3.127
	16.5		504.767	1362.967	1862.153	3729.890	2.031	2.693	4.276	3.053
	18.0		487.226	1414.472	1868.018	3769.715	1.936	2.643	4.319	3.032
ME (n = 24)		12.2	489.357	1379.663	1905.915	3774.933	2.004	2.651	4.516A	3.114
		12.4	504.154	1400.410	1860.689	3765.253	2.041	2.744	4.168B	3.061
		12.6	513.259	1389.700	1834.188	3737.148	2.057	2.669	4.236B	3.037
P values	CP		0.297	0.212	0.970	0.649	0.134	0.495	0.890	0.131
	ME		0.402	0.774	0.126	0.775	0.861	0.395	0.006	0.274
	C*M		0.329	0.589	0.336	0.487	0.569	0.745	0.007	0.087

¹All results are presented as mean ± SEM (n = 8).

^A–CDifferent capital letters in the data of the same column indicate extremely significant differences (P < 0.01).

C*M, the interaction of the crude protein with metabolic energy; F/G feed-to-gain ratio.

Slaughter Performance and Meat Quality of Squabs

We can see from Table 9 that the different ME and CP levels in the diets had little influence on the slaughter performance of commercial squabs. Among them, group 7 (18% CP, 12.2 MJ/kg) had the highest slaughter weight of squabs compared to other experimental groups (P < 0.01). Further evaluation of the organ development index of the squabs showed that the kidney index of the 16.5% CP level squab groups was significantly higher than other CP level groups (P < 0.05). The combined analysis showed that different CP and ME levels also had a nonsignificant effect on the organ development of winter squabs (Table 10). The results of the meat quality testing of the slaughtered squabs’ pectoral muscles are shown in Table 11. It can be seen that CP and ME levels did not significantly affect meat color (a*, b*, and L* values), pH, or drip loss in slaughtered squabs, and there was no significant interaction among groups (P > 0.05).

Table 9.

Effect of dietary energy and protein levels on the slaughter performance of squabs in winter¹.

Treatments (n = 24)	CP/%	ME/MJ/kg	Live body weight/g	Slaughter weight/g	Semieviscerated weight/g	Eviscerated weight/g	Pectoral muscle weight/g	Abdominal fat/g	Slaughter rate/%	Semieviscerated rate/%	Eviscerated rate/%	Pectoral muscle rate/%	Abdominal fat rate/%
I	15.0	12.2	464.000BCD	372.050BC	323.773	247.613	46.534	1.722	80.158	87.029	66.457	18.803	0.679
II	15.0	12.4	462.375BCD	375.333AB	326.583	243.752	49.449	2.119	81.184	87.084	65.002	20.258	0.859
III	15.0	12.6	460.565CD	370.891BC	319.583	239.322	50.550	2.329	80.581	86.509	64.836	21.148	0.973
IV	16.5	12.2	453.229D	355.204C	318.238	239.466	48.570	1.723	78.389	89.893	67.682	20.257	0.730
V	16.5	12.4	474.109ABC	379.065AB	330.065	248.877	66,917	2.323	80.020	86.992	65.538	27.167	0.942
VI	16.5	12.6	473.063ABC	387.646AB	335.208	253.898	53.895	1.943	81.963	86.513	65.482	21.110	0.752
VII	18.0	12.2	489.935A	391.891A	337.835	254.019	58.305	1.741	80.120	86.198	64.795	22.853	0.681
VIII	18.0	12.4	468.630BCD	372.065BC	323.609	246.190	51.244	1.742	79.341	87.113	66.134	20.866	0.720
IX	18.0	12.6	480.804AB	383.935AB	327.561	246.576	53.912	1.910	79.890	85.332	64.253	21.910	0.766
SEM			2.327	2.293	2.023	1.862	1.662	0.071	0.323	0.323	0.351	0.658	0.028
Main effect
CP (n = 72)	15.0		462.313B	372.758	323.313	243.562	48.844	2.057	80.641	86.874	65.432	20.070	0.837
	16.5		466.800B	373.972	327.837	247.414	56.460	1.997	80.124	87.799	66.234	22.845	0.808
	18.0		479.790A	382.630	329.668	248.928	54.487	1.798	79.784	86.215	65.061	21.876	0.722
ME (n = 72)		12.2	469.055	373.048	326.615	247.032	51.136	1.729	79.556	87.706	66.311	20.638	0.696
		12.4	468.371	375.488	326.752	246.273	55.870	2.061	80.181	87.063	65.558	22.764	0.840
		12.6	471.477	380.824	327.451	246.598	52.785	2.061	80.811	86.118	64.857	21.389	0.830
P values	CP		0.005	0.146	0.420	0.485	0.149	0.301	0.554	0.126	0.378	0.214	0.224
	ME		0.839	0.346	0.984	0.986	0.493	0.091	0.286	0.126	0.243	0.405	0.067
	C*M		0.006	0.007	0.301	0.505	0.141	0.206	0.331	0.096	0.440	0.178	0.102

¹All results are presented as mean ± SEM (n = 24).

^A–DDifferent capital letters in the data of the same column indicate extremely significant differences (P < 0.01).

C*M, the interaction of the crude protein with metabolic energy.

Table 10.

Effect of dietary energy and protein levels on the organ index of squabs in winter¹.

Treatments (n = 24)	CP/%	ME/MJ/kg	Liver/%	Spleen/%	Thymus/%	Pancreas/%	Glandular stomach/%	Gizzard/%	Kidney/%	Bursa/%	Heart/%
I	15.0	12.2	3.641	0.132	0.668	0.558	0.429	1.770	1.390	0.145	0.977
II	15.0	12.4	3.399	0.131	0.674	0.567	0.463	1.741	1.399	0.191	1.029
III	15.0	12.6	3.311	0.146	0.708	0.558	0.439	1.770	1.449	0.167	1.069
IV	16.5	12.2	3.312	0.128	0.627	0.534	0.437	1.706	1.465	0.161	0.985
V	16.5	12.4	3.309	0.148	0.690	0.534	0.448	1.653	1.365	0.188	1.027
VI	16.5	12.6	3.293	0.170	0.566	0.585	0.430	1.678	1.460	0.166	1.023
VII	18.0	12.2	3.309	0.141	0.612	0.526	0.417	1.682	1.381	0.194	1.000
VIII	18.0	12.4	3.178	0.121	0.707	0.542	0.428	1.673	1.282	0.175	1.039
IX	18.0	12.6	3.274	0.148	0.674	0.538	0.438	1.641	1.324	0.185	1.017
SEM			0.037	0.006	0.017	0.007	0.010	0.005	0.018	0.017	0.005
Main effect
CP (n = 72)	15.0		3.451	0.137	0.683	0.561	0.444	1.760	1.413ab	0.168	1.025
	16.5		3.305	0.148	0.627	0.551	0.438	1.679	1.430a	0.172	1.012
	18.0		3.254	0.137	0.665	0.535	0.428	1.665	1.329b	0.184	1.019
ME (n = 72)		12.2	3.421	0.134	0.636	0.539	0.428	1.719	1.412	0.167	0.987
		12.4	3.295	0.133	0.690	0.548	0.446	1.689	1.349	0.185	1.032
		12.6	3.293	0.155	0.649	0.560	0.435	1.696	1.411	0.173	1.036
P values	CP		0.084	0.700	0.383	0.315	0.462	0.076	0.041	0.331	0.869
	ME		0.280	0.303	0.388	0.458	0.369	0.784	0.233	0.289	0.102
	C*M		0.263	0.804	0.513	0.547	0.705	0.637	0.184	0.248	0.578

¹All results are presented as mean ± SEM (n = 24).

^a–bDifferent lowercase letters in the data of the same column indicate significant differences (P < 0.01).

C*M, the interaction of the crude protein with metabolic energy.

Table 11.

Effect of dietary energy and protein levels on the meat quality of squabs in winter¹.

Treatments (n = 24)	CP/%	ME/MJ/kg	45 min L*	45 min a*	45 min b*	24 h L*	24 h a*	24 h b*	pH 45 min	pH 24 h	Drip loss/%
I	15.0	12.2	43.717	20.711	6.394	50.903	13.548	6.513	6.204	5.814	3.150
II	15.0	12.4	43.648	18.795	6.177	53.082	13.503	6.400	6.278	5.878	3.573
III	15.0	12.6	44.678	18.916	6.251	52.031	14.239	6.041	6.126	5.790	3.308
IV	16.5	12.2	44.098	19.769	6.366	51.708	14.322	6.504	6.437	5.819	3.744
V	16.5	12.4	42.493	20.559	6.098	52.553	14.161	6.391	6.230	5.784	3.538
VI	16.5	12.6	42.853	19.834	6.358	52.614	14.815	5.934	6.135	5.826	3.779
VII	18.0	12.2	44.484	18.458	5.829	54.001	13.378	6.078	6.267	5.806	2.836
VIII	18.0	12.4	42.134	20.032	6.791	51.690	14.870	6.665	6.279	5.848	2.969
IX	18.0	12.6	42.411	19.846	6.091	52.732	14.021	6.076	6.270	5.790	3.255
SEM			0.246	0.302	0.150	0.474	0.172	0.151	0.026	0.015	0.119
Main effect
CP (n = 72)	15.0		44.014	19.474	6.274	52.005	13.763	6.318	6.203	5.827	3.344
	16.5		43.148	20.054	6.274	52.292	14.433	6.276	6.267	5.810	3.687
	18.0		43.010	19.446	6.237	52.807	14.089	6.273	6.272	5.814	3.020
ME (n = 72)		12.2	44.100	19.646	6.196	52.204	13.749	6.365	6.302	5.813	3.244
		12.4	42.758	19.795	6.355	52.442	14.178	6.485	6.262	5.837	3.360
		12.6	43.314	19.532	6.233	52.459	14.358	6.017	6.177	5.802	3.448
P values	CP		0.193	0.659	0.994	0.793	0.287	0.991	0.468	0.892	0.077
	ME		0.073	0.940	0.901	0.972	0.348	0.455	0.125	0.649	0.784
	C*M		0.136	0.644	0.953	0.921	0.348	0.965	0.165	0.904	0.547

¹All results are presented as mean ± SEM (n = 24).

The absence or the same letter in the same column of data indicates no significant difference (P > 0.05).

C*M, the interaction of the crude protein with metabolic energy; L* stands for brightness; a* stands for redness; b* stands for yellowness.

Histological Appearance of the Squab Jejunum Mucosa

After feeding diets with different levels of ME and CP, significant differences were observed in the morphology-related indexes of the jejunum mucosa of the various test groups of squabs. Among them, the 16.5 and 18% CP level groups showed significantly increased height of the jejunal epithelium (P < 0.01). The interaction between the CP and ME levels had a significant impact on the VH/CD and the mucosal epithelium height of the squab jejunum. The VH/CD of the squab jejunum in group 8 (18% CP, 12.4 MJ/kg) was significantly higher than that of other experimental groups (P < 0.01), but there was little difference between groups 2, 3, and 6. The jejunal mucosal epithelium height in group 1 was significantly thinner than that of the other experimental groups (P < 0.01) (Table 12).

Table 12.

Effect of dietary energy and protein levels on jejunal intestinal histomorphology of squabs in winter (HE staining)¹.

Treatments (n = 8)	CP/%	ME/MJ/kg	Villus width/μm	Epithelium height/μm	VH/CD	Muscle layer thickness/μm	Goblet cells
I	15.0	12.2	146.260	14.899C	23.936CD	86.764	10.675
II	15.0	12.4	170.047	18.178B	27.649AB	87.950	9.043
III	15.0	12.6	142.288	19.915AB	28.296AB	88.469	11.206
IV	16.5	12.2	165.934	22.781A	26.764BC	82.164	11.567
V	16.5	12.4	162.211	20.963AB	20.630E	88.026	13.918
VI	16.5	12.6	171.450	20.665AB	27.308AB	86.287	8.567
VII	18.0	12.2	175.259	20.211AB	26.674BC	98.444	8.366
VIII	18.0	12.4	139.122	19.658AB	30.020A	82.405	9.797
IX	18.0	12.6	154.196	20.456AB	22.529DE	86.326	8.905
SEM			3.464	0.418	1.613	0.462	0.447
Main effect
CP (n = 24)	15.0		152.865	17.664B	26.627	87.728	10.308
	16.5		166.532	21.470A	24.901	85.492	11.350
	18.0		156.192	20.108A	26.408	89.058	9.023
ME (n = 24)		12.2	162.484	19.297	25.791	89.124	10.202
		12.4	157.127	19.599	26.100	86.127	10.919
		12.6	155.978	20.345	26.044	87.027	9.559
P values	CP		0.234	<0.001	0.105	0.666	0.127
	ME		0.690	0.482	0.928	0.738	0.480
	C*M		0.071	0.001	<0.001	0.463	0.148

¹All results are presented as mean ± SEM (n = 8).

^A–EDifferent capital letters in the data of the same column indicate extremely significant differences (P < 0.01).

C*M, the interaction of the crude protein with metabolic energy; VH/CD = villus height (μm)/crypt depth (μm).

Plasma Biochemical Indices of Breeding Pigeons and Squabs

After feeding diets with different levels of ME and CP, the female and male breeding pigeons have varying effects on their plasma biochemistry indices, and significant interactions exist between ME and CP levels (Tables 13 and 14). Male breeding pigeons who consumed 12.4 MJ/kg of ME showed significantly enhanced SOD activity and reduced H₂O₂ content compared to other ME levels (P < 0.05). Additionally, male pigeon plasma exhibited a significant decrease in the GLU content, Alb content, and SOD activity with increasing CP levels (P < 0.05), while contents of UA and H₂O₂ significantly increased (P < 0.05). Moreover, compared to the other experimental groups, male pigeons from group 7 (18% CP, 12.2 MJ/kg) showed significantly lower T-CHO content and SOD activity and significantly higher UA and H₂O₂ content (P < 0.05), with a lower Alb content. Female breeding pigeons who consumed 18% of CP showed significantly increased H₂O₂ content and CAT activity compared to the other tested CP levels (P < 0.05). Compared to other experimental groups, group 8 (18% CP, 12.4 MJ/kg) significantly increased T-CHO content and significantly decreased SOD activity in female pigeon plasma (P < 0.05). We also found that different ME and CP levels in the diet affected the plasma biochemical indices of 21-day-old squabs, with a highly significant increase in TG content and T-AOC activity as ME and CP levels rose (P < 0.01) (Table 15).

Table 13.

Effect of dietary energy and protein levels on plasma biochemical indices of female breeding pigeons in winter (the 14th day of lactation)¹.

Treatments (n = 10)	CP/%	ME/MJ/kg	T-CHO	GLU	UA	Alb	TG	T-AOC	H2O2	MDA	SOD	CAT
I	15.0	12.2	6.732bc	15.363	43.310	11.858	23.047	5.958	315.784	34.315	70.475abc	6.560ab
II	15.0	12.4	4.469c	14.441	59.829	10.348	16.745	3.199	144.638	13.535	67.463abc	3.852bc
III	15.0	12.6	8.960ab	15.044	57.356	11.903	21.177	3.398	205.073	11.241	45.996c	1.590c
IV	16.5	12.2	7.132bc	14.337	52.725	12.963	17.584	3.727	167.094	12.629	94.150ab	5.752abc
V	16.5	12.4	6.965bc	16.414	51.156	12.308	22.841	4.567	284.008	16.188	57.891bc	5.414abc
VI	16.5	12.6	7.278bc	17.352	48.583	10.687	18.592	3.779	270.094	15.215	77.261abc	9.703a
VII	18.0	12.2	8.150ab	16.513	51.997	13.526	24.244	6.090	455.936	19.479	41.254c	5.678abc
VIII	18.0	12.4	10.232a	17.073	47.945	13.036	24.735	5.225	406.682	23.674	45.623c	5.744abc
IX	18.0	12.6	6.753bc	16.565	49.670	11.088	18.048	5.151	298.540	14.722	100.906a	8.503a
SEM			0.363	0.359	1.607	0.367	1.397	0.356	30.324	2.181	4.916	0.562
Main effect
CP (n = 30)	15.0		6.721	14.949	53.498	11.370	20.323	4.185	221.832	19.697	61.311	4.001
	16.5		7.125	16.035	50.821	11.986	19.673	4.024	240.399	14.678	76.419	6.956
	18.0		8.379	16.717	49.870	12.550	22.342	5.489	387.053	19.292	62.594	6.642
ME (n = 30)		12.2	7.338	15.404	49.344	12.782	21.625	5.258	312.938	22.141	68.627	5.997
		12.4	7.222	15.976	52.977	11.897	21.440	4.330	278.442	17.799	56.992	5.003
		12.6	7.664	16.320	51.870	11.226	19.272	4.109	257.902	13.726	74.706	6.599
P values	CP		0.117	0.133	0.633	0.428	0.733	0.189	0.052	0.566	0.331	0.051
	ME		0.856	0.573	0.639	0.230	0.760	0.378	0.751	0.278	0.280	0.498
	C*M		0.017	0.368	0.397	0.444	0.844	0.401	0.233	0.267	0.023	0.036

¹All results are presented as mean ± SEM (n = 10).

^a–cDifferent lowercase letters in the data of the same column indicate significant differences (P < 0.01).

C*M, the interaction of the crude protein with metabolic energy; T-CHO total cholesterol, mmol/L; GLU glucose, mmol/L; UA uric acid, mmol/L; Alb albumin, g/L; TG total triglycerides, mmol/L; T-AOC total antioxidant capacity, U/mL; H₂O₂ hydrogen peroxide, mmol/L; MDA malondialdehyde, nmol/L; SOD superoxide dismutase, U/mL; CAT catalase, U/mL.

Table 14.

Effect of dietary energy and protein levels on plasma biochemical indices of male breeding pigeons in winter (the 14th day of lactation)¹.

Treatments (n = 10)	CP/%	ME/MJ/kg	T-CHO	GLU	UA	Alb	TG	T-AOC	H2O2	MDA	SOD	CAT
I	15.0	12.2	6.508a	16.791	42.245bcd	15.113AB	1.559	2.971	36.014A	7.147	81.873AB	6.129bc
II	15.0	12.4	5.470bc	16.327	39.424d	14.725ABC	1.429	2.279	21.826C	4.846	81.438AB	3.659bc
III	15.0	12.6	4.859c	16.551	45.516bcd	16.202A	1.095	2.044	27.890BC	3.228	94.317A	4.716bc
IV	16.5	12.2	5.474bc	16.012	40.591cd	16.362A	1.306	2.131	32.621AB	5.694	80.629B	2.047c
V	16.5	12.4	5.720abc	16.250	50.371abc	15.381AB	1.266	2.366	33.566AB	6.055	92.425AB	5.954bc
VI	16.5	12.6	5.150bc	13.900	52.501ab	13.885ABCD	1.224	2.829	38.965A	3.228	35.354D	13.251a
VII	18.0	12.2	4.938c	15.471	58.395a	12.657BCD	1.175	2.766	35.271A	4.005	39.087D	9.251ab
VIII	18.0	12.4	5.532bc	14.158	45.915bcd	12.030CD	1.116	2.304	34.390AB	4.060	53.910C	7.633abc
IX	18.0	12.6	5.926ab	13.941	48.958abcd	11.770D	1.313	2.258	38.823A	4.480	56.167C	6.240bc
SEM			0.121	0.322	1.387	0.380	0.039	0.091	0.972	0.371	2.906	0.789
Main effect
CP (n = 30)	15.0		5.612	16.556a	42.395b	15.347A	1.361	2.431	28.610B	5.074	85.876A	4.834
	16.5		5.448	15.387ab	47.941ab	15.209A	1.266	2.442	35.051A	4.993	69.469B	7.084
	18.0		5.465	14.523b	51.089a	12.152B	1.201	2.443	36.161A	4.182	49.721C	7.708
ME (n = 30)		12.2	5.640	16.091	47.077	14.710	1.347	2.623	34.635a	5.615	67.196B	5.809
		12.4	5.574	15.578	45.357	14.046	1.270	2.316	29.927b	4.987	75.924A	5.749
		12.6	5.311	14.797	48.992	13.952	1.211	2.377	35.259a	3.645	61.946B	8.069
P values	CP		0.807	0.034	0.024	<0.001	0.240	0.998	0.001	0.536	<0.001	0.274
	ME		0.457	0.241	0.513	0.625	0.356	0.330	0.017	0.080	0.002	0.342
	C*M		0.032	0.149	0.017	0.010	0.140	0.153	<0.001	0.174	<0.001	0.055

¹All results are presented as mean ± SEM (n = 10).

Different lowercase letters in the data of the same column indicate significant differences (P < 0.05), and different capital letters indicate extremely significant differences (P < 0.01).

C*M, the interaction of the crude protein with metabolic energy; T-CHO, total cholesterol, mmol/L; GLU, glucose, mmol/L; UA, uric acid, mmol/L; Alb, albumin, g/L; TG, total triglycerides, mmol/L; T-AOC, total antioxidant capacity, U/mL; H₂O₂, hydrogen peroxide, mmol/L; MDA, malondialdehyde, nmol/L; SOD, superoxide dismutase, U/mL; CAT, catalase, U/mL.

Table 15.

Effect of dietary energy and protein levels on plasma biochemical indices of 21-day squabs in winter¹.

Treatments (n = 10)	CP/%	ME/MJ/kg	T-CHO	GLU	UA	Alb	TG	T-AOC	H2O2	MDA	SOD
I	15	12.2	6.687	14.877	49.646	11.539	1.394c	2.208CD	34.513	5.936	74.482
II	15	12.4	8.192	14.592	44.536	11.146	1.921bc	2.427CD	42.741	5.401	66.227
III	15	12.6	6.894	13.746	41.634	15.105	2.143ab	2.016D	37.983	5.968	42.266
IV	16.5	12.2	6.461	15.412	44.253	11.180	2.131ab	2.051D	41.804	5.635	36.168
V	16.5	12.4	7.061	15.059	40.479	13.521	2.182ab	2.607BCD	43.647	5.308	39.634
VI	16.5	12.6	6.340	13.461	40.878	10.058	2.668a	3.746AB	44.011	4.401	85.432
VII	18	12.2	7.292	13.834	38.585	13.340	2.298ab	2.69BCD	39.324	5.343	69.312
VIII	18	12.4	6.880	15.210	42.860	12.005	2.475ab	3.365ABC	41.504	4.102	70.566
IX	18	12.6	7.053	18.071	44.619	10.894	2.554ab	4.529A	42.294	4.265	51.862
SEM			0.202	0.432	0.941	0.425	0.087	0.167	1.422	0.344	5.112
Main effect
CP (n = 30)	15.0		7.257	14.405	45.272	12.597	1.819B	2.217B	38.412	5.768	60.992
	16.5		6.620	14.644	41.870	11.586	2.327A	2.801B	43.154	5.115	53.744
	18.0		7.075	15.705	42.021	12.080	2.442A	3.528A	41.041	4.570	63.913
ME (n = 30)		12.2	6.813	14.708	44.161	12.020	1.941b	2.316B	38.547	5.638	59.987
		12.4	7.377	14.954	42.625	12.224	2.193ab	2.800AB	42.631	4.937	58.809
		12.6	6.762	15.093	42.377	12.019	2.455a	3.430A	41.429	4.878	59.853
P values	CP		0.429	0.425	0.243	0.604	0.005	0.002	0.421	0.392	0.697
	ME		0.400	0.934	0.695	0.973	0.039	0.010	0.508	0.626	0.994
	C*M		0.618	0.350	0.235	0.120	0.020	0.001	0.860	0.886	0.233

¹All results are presented as mean ± SEM (n = 10).

Different lowercase letters in the data of the same column indicate significant differences (P < 0.05), and different capital letters indicate extremely significant differences (P < 0.01).

C*M, the interaction of the crude protein with metabolic energy; T-CHO, total cholesterol, mmol/L; GLU, glucose, mmol/L; UA, uric acid, mmol/L; Alb, albumin, g/L; TG, total triglycerides, mmol/L; T-AOC, total antioxidant capacity, U/mL; H₂O₂, hydrogen peroxide, mmol/L; MDA, malondialdehyde, nmol/L; SOD, superoxide dismutase, U/mL.

DISCUSSION

It is known that the nutrients in poultry feed will only be transferred to offspring production after satisfying the breeder's nutritional requirements (Maharjan et al., 2021). Dietary CP and ME levels also not only influence weight changes in breeding pigeons but are closely related to their reproductive cycles (Bu et al., 2015), where diet can significantly affect the quality of pigeon eggs, including albumen height, yolk color, and shell thickness (Ding et al., 2016; Geng et al., 2022). Thus, given that feed costs generally account for 60 to 70% of total production costs (Singh et al., 2015), formulating an optimal ME and CP levels diet for breeding pigeons is a crucial aspect of efficient farming. We found that raising CP levels resulted in a shortened laying interval during a reproductive cycle and improved breeding efficiency, consistent with prior findings (Gao et al., 2016a; Peng et al., 2023). It is noteworthy that increasing the CP level of the diet in this experiment reduced the eggshell weight during the first laying cycle. We reasonably assume that the significantly increased egg production for a short period in the high CP test group may have led to a limited calcium intake and accumulation in the body, resulting in a calcium deficiency that caused the decreased eggshell quality. Unlike other poultry, the parental pigeon needs both nursing and nesting after squabs are 12 to 14-days old, where the reproductive periods before and after nursing are closely interrelated (Gao et al., 2011). Even after the end of the feeding trial, we found that the effect of diet CP levels on the breeding pigeons remained, with the groups fed 16.5 and 18% CP showing significantly improved hatchability of breeding eggs during the second laying cycle. Throughout the experimental cycle, different ME and CP levels did not result in statistically significant differences in the egg shape index, eggshell strength, or other egg quality indicators, nor did it affect the weight loss of breeding pigeons during lactation, unlike the results of Chen et al. (2016) and Peng et al. (2023). This may be due to the "2 + 3" breeding pattern adopted in winter which helps relieve the nutritional burden on breeding pigeons during lactation. Finally, we observed no significant differences in total laying rates between the 18% CP and 16.5% CP level groups. Therefore, ME and CP levels of breeding pigeon diets in winter "2 + 3" breeding pattern of production could be appropriately reduced to save costs and reduce nitrogen emissions.

The nutritional regulation of the parental pigeon's diet can affect the "crop milk" secretion, which in turn affects the growth and development of their offspring (Xie et al., 2019; Jin and Tulake, 2020). While this study did not directly measure the effect of dietary nutritional levels on the "crop milk" of breeding pigeons, it can be inferred from the growth and slaughter performance of their offspring. Previous studies have shown that low ME diets affect the marketable live weight of offspring (Peebles et al., 2002), as well as nutritional metabolism (Enting et al., 2007) and muscle quality (Xu et al., 2010). Insufficient protein in the diet can also reduce the growth performance of squabs, subsequently affecting the development of their organs and their slaughter performance (Gao et al., 2016b). Therefore, optimal levels of ME and CP in the diet can significantly improve the growth performance and carcass quality of poultry (Lin et al., 2020). This study found that parental dietary ME levels only affect the F/G in the later stages of lactation (14–21 d), which is inconsistent with the findings of Sahin et al. (2003). This may be due to the incomplete development of the digestive system and absorption abilities of the squabs in early development, making them less sensitive to the ME and CP contents in their diet. We did find that the body weight gain of squabs was significantly and positively correlated with parental dietary CP levels in the middle and later stages of development. The 18% CP group showed an additional increase in body weight of 3.16% in the squabs compared to the 15% CP group, with no significant difference compared to the 16.5% CP group. However, a previous study has shown that high dietary protein levels cause elevated serum uric acid, body fat deposition, and thus impaired kidney function in squabs (Costantini, 2010). We found that the kidney index of the squabs in the different CP level groups in this experiment fell within the normal range, with limited studies on the correlation between dietary crude protein levels and kidney index, making it difficult to determine the precise impact of high protein levels on the kidney index. Overall, different ME and CP levels did not result in significant statistical differences in the slaughter performance and meat quality of the winter squabs throughout the experiment, and the feeding and body weight differences between the groups were not significant. From the perspective of economic benefit and low-nitrogen environmental protection, we recommend reducing the CP level in the diet to 16.5% when the dietary ME level is kept above 12.2 MJ/kg after reducing the litter size in winter.

The small intestine serves as the foundation for nutritional intake by livestock and poultry, and the maintenance of the normal morphology of the intestinal mucosa is crucial for the growth, development, and immune defense of young animals (Ray et al., 2002; Taylor, 2009; Yada et al., 2021). Intestinal epithelial cells (IECs) are responsible for nutrient absorption and the secretion of digestive enzymes, and their selective permeability characteristics are beneficial for maintaining immune homeostasis and intestinal barrier integrity (Hooper, 2015). Dietary protein levels that are too low can be detrimental to the proliferation and repair of jejunal intestinal epithelial cells and affect the integrity of the intestinal barrier function (Bodiga et al., 2005). At the same time, providing an adequate energy supply can also support the needs of cellular metabolism and proliferation, which helps to maintain normal intestinal physiological functions (Wan et al., 2019). This study found that increasing the dietary CP levels significantly increased the height of the jejunal epithelial cells in squabs, and the interaction between CP and ME levels was closely related to the height of these epithelial cells in squabs. However, excessive proliferation of jejunal epithelial cells may affect intestinal motility and nutrient absorption capacity, while low epithelial cell height may make the intestinal mucosa vulnerable to damage (Litvak et al., 2018; Beumer and Clevers, 2021). Although preliminary studies have indicated that a more balanced supply of amino acids in piglet diets promotes jejunal epithelial cell proliferation (Wu et al., 2012), there is currently limited research in this area, especially in poultry. It is difficult to ascertain whether high protein intake-induced changes in squab intestinal IECs are beneficial, and further studies are needed. The VH/CD is also an important indicator of intestinal health and function, as an increase in this ratio can effectively enhance the digestion and utilization of nutrients (Sen et al., 2012; Ma et al., 2018). Research on broilers has shown that decreasing dietary CP levels significantly reduces villus height, crypt depth, and VH/CD of the intestinal (Ding et al., 2016; Macelline et al., 2020). Conversely, feeding pregnant sows high-energy diets significantly increases the jejunal villus height in piglets and reduces the impact of stress on digestion and absorption (Liu et al., 2016). In contrast, in this experiment, there were no differences in the villus height, crypt depth, and VH/CD ratio of the jejunal intestinal in squabs between the different ME and CP level groups. However, we found that the interaction between dietary CP and ME significantly affected the VH/CD of the jejunal intestinal in squabs. These results suggested that rational regulation of protein and energy levels in diets may promote intestinal morphology development and jejunal absorption in squabs.

Plasma biochemical indicators can sensitively reflect the digestion, absorption, and metabolism of nutrients in the animal body (Graugnard et al., 2012; Alves-Bezerra and Cohen, 2017). We found that increasing the levels of ME and CP in the diet increased the TG content of squab plasma, which supports the results of Qiao (2013) and Chen et al. (2021). This also suggests that high protein levels can affect the utilization and conversion of amino acids in squabs, thereby increasing the TG content in the plasma. Furthermore, as increased TG content was within the normal range, the increased energy levels in the diet had a beneficial effect on the fat metabolism of squabs. Furthermore, previous studies conducted by Guan et al. (2017) and Ozek et al. (2004) have demonstrated that an increase in dietary protein levels promotes enhanced protein synthesis within organisms. Conversely, it has been reported by Darsi et al. (2012) and Liu et al. (2016) that excessive dietary protein intake can lead to the decomposition of protein metabolism, increasing UA synthesis or excretion. The present study revealed a positive correlation between the plasma UA content of male pigeons during the winter lactation period and the CP level in their diet. However, when the CP level in the diet was excessively high, the Alb content decreased. The changes in the content of UA and Alb in this experiment indicate that the crude protein level in the diet was too low or too high to facilitate the utilization of the protein by the pigeons. The interaction between the ME and CP levels in the diet also significantly affected the UA content of male pigeons, suggesting that dietary energy may interfere with the metabolic process of animal proteins.

Some studies have shown that excessive energy intake in the diet not only leads to metabolic surplus but may also impair the function of the body's antioxidant system, thereby increasing the oxidative stress of the animal (Pedernera et al., 2010; Tan and Norhaizan, 2019). Oxidative stress refers to a series of stress responses induced by various endogenous or exogenous factors stimulating the body (Tan et al., 2018; Biobaku et al., 2019). Common exogenous factors in meat pigeon production include environmental temperature and rearing management, while endogenous factors are closely related to the pigeon's immune status or nutritional metabolism. Some studies have also pointed out that moderate protein intake can increase the activity of antioxidant enzymes in the body and alleviate oxidative stress (Kitada et al., 2019). However, high protein intake in livestock and poultry diets may lead to metabolic disorders, excessive production of free radicals, and weakened antioxidant capacity, thereby increasing the degree of oxidative stress (Machín et al., 2004; Mahmoud et al., 2021). This experiment systematically evaluated the effect of dietary nutrient levels by detecting antioxidant substances (such as SOD, CAT, etc.) and oxidative damage markers (such as lipid peroxidation product MDA) in the plasma. We found that the energy and protein levels in the diet had little effect on the oxidative stress indicators in female pigeons, but as the dietary CP level increased, the H₂O₂ content in male pigeon plasma increased significantly, and the SOD activity decreased significantly, indicating that male pigeons may experience a certain degree of oxidative stress due to excessive protein intake. At the same time, this study found that the antioxidant capacity of squabs was significantly improved at higher protein levels, which may be related to the higher protein requirements and tolerance of squabs in early development. Most young animals in a period of rapid growth have much higher protein requirements than adults for cellular maintenance and reproduction differentiation (Maliwan et al., 2019; Tokach et al., 2019). Studies specifically focusing on artificial pigeon milk for squabs have also shown that a dietary CP level of 18 to 20.0% is required to meet the needs of squabs for feather development during the late lactation period (Chen et al., 2018). Since squabs cannot feed independently before 21 d of age, it may be challenging to simultaneously meet the antioxidant needs of both parents and offspring pigeons in terms of the nutrition level in the lactation diet. This necessitates the supplementation of additional antioxidant nutrients in the pigeons’ diets.

CONCLUSIONS

In conclusion, the ME level in the feed had a relatively small impact on the production of lactating breeding pigeons during winter, but an increase in the CP level favored the reproductive metabolism of breeding pigeons and the growth performance of squabs. Nevertheless, the overall performance improvement of each group after feeding different CP and ME levels was not much different. We recommend that in practical production under the "2 + 3" breeding pattern in winter, the CP level can be reduced to 16.5%, while the ME level should not be lower than 12.2 MJ/kg to maintain production while also saving on feed costs and improving economic efficiency.