Impact of storage duration and short periods of incubation during egg storage on embryonic development and hatching traits of hybrid chicken strain

Figures & data

Figure 1. A Graphical representation of the temperature-time chart of egg storage and SPIDES treatment before incubation.

Table 1. Effect of cold storage duration on basic egg quality parameters.

				Percentage
Storage duration	Initial egg weight (g)	Final egg weight (g)^1	Final egg weight (g)^2	Weight loss	Wet yolk weight	Dry yolk weight	Wet shell weight	Dry shell weight	Albumen weight	Wet shell thickness (cm)	Blastoderm diameter (cm)
1	52.80^b	51.40^ab	52.30	2.67	25.40	22.73	10.94^b	8.97	63.65	0.62^a	0.34
4	50.20^b	46.87^b	50.31	6.49	25.85	19.36	11.71^ab	9.16	62.44	0.45^ab	0.36
7	53.50^b	49.46^b	49.69	7.76	28.04	20.28	11.301^ab	9.17	60.66	0.59^a	0.37
10	59.60^a	57.50^a	51.77	3.73	25.70	21.97	11.80^ab	9.53	62.50	0.58^a	0.37
14	54.00^ab	51.34^ab	51.08	4.97	26.56	21.58	12.78^ab	9.65	60.67	0.25^bc	0.41
17	55.00^ab	52.66^ab	51.42	4.18	27.04	22.87	13.39^a	9.98	59.57	0.22^c	0.30
21	51.00^b	48.73^b	51.39	4.30	26.77	22.47	12.87^ab	9.79	60.36	0.30^bc	0.35
SEM	1.390	1.493	0.656	1.250	1.226	1.141	0.503	0.367	1.328	0.054	0.028
p-Value	0.0003	0.0002	0.1009	0.0839	0.738	0.2366	0.0054	0.394	0.2713	<0.0001	0.3026

SEM: standard error of means, p-value: probability value. Means of different superscripts within a column differ significantly (p ≤ 0.05), Initial egg weight used as a covariate was significant (p < 0.05). ¹Final egg weight before covariate analysis, ²Final egg weight after covariate analysis to remove effect due to initial egg weight.

Table 2. Effect of short incubation period and different storage durations on egg weight loss of hybrid breeder ‘black-tailed’ eggs.

Treatment	Initial egg weight (g)	Final egg weight (g)	Weight loss (%)
SPIDES (SP)
0-h	54.31	53.60	1.08
6-h	53.91	53.26	1.23
SEM	0.423	0.439	0.067
p-Value	0.5027	0.5813	0.1188
Days of storage
14	54.33	53.83	0.71^b
21	53.89	53.03	1.60^a
SEM	0.423	0.439	0.067
p-Value	0.4697	0.2037	<.0001
Interaction
0–6*14	53.50^b	52.88^b	0.73^c
Co*21	55.12^a	54.32^a	1.42^b
SPIDES*14	55.16^a	54.77^a	0.68^c
SPIDES*21	52.66^b	51.75^b	1.77^a
SEM	0.598	0.623	0.095
p-Value	0.0007	0.0004	0.0320

SEM: standard error of means, p-value: probability value. Means of different superscripts within a column differ significantly (P ≤ 0.05), SPIDES: short period of incubation during egg storage. Initial egg weight used as a covariate was significant (p < 0.0001).

Figure 2. Representative profile of the impact of storage duration, SPIDES, and their interaction on egg weight (a) and average egg weight (b) during incubation. Data with bars having similar superscripts showed no significant difference (p > 0.05): ns: not significantly different; 0-h SPIDES – eggs that did not receive short period incubation during (control); 6-h SPIDES: eggs that received 6-h short period incubation during egg storage.

Figure 3. Comparative stages of embryonic development and chick quality at hatch for Non-SPIDES & SPIDES treated eggs after prolonged storage according to the procedures of Hamburger–Hamilton (1951) stages. KEY: Plates a, c, e, g, i (orange-arrows transition: Non-SPIDES or control treatments); Plates b, d, f, h, j (blue-arrows transition: SPIDES treatments); (a) Unincubated non–SPIDES egg. Blastoderm under-developed with unclear ‘donut appearance’. (b) Unincubated SPIDES egg. Blastoderm is well-developed with clear ‘donut-appearance’. (c) ‘donut-shape’ enlarged with no appearance of ‘blood islet’ (d) ‘donut-shape’ enlarged greatly with the appearance of ‘blood islets’ (e) ‘donut-shape’ enlarged greatly with only ‘blood islets’ in the outer ring of developing embryo. No appearance of the circulatory system and embryonic appendages at this stage (f) Clear appearance of an embryo with a well-developed circulatory system and embryonic appendage. (g) Protrated terminal hatching with few externally pipped chicks (h) rapid terminal hatching with more hatched and externally pipped chicks. (i) Poor quality chick (light, weak and passive) (j) good quality chick (heavy, thrifty, and active).

Table 3. Impact of storage duration, SPIDES, and their interaction on mean wet and dry embryo weight changes during incubation.

	Wet embryo weight(g)					Dry embryo weight(g)
Treatment	4	7	11	14	18	4*	7	11	14	18
SPIDES (SP)
0-h	0.061^b	0.523^b	3.41^b	10.9	22.9	–	0.042	0.611^b	2.95	9.73
6-h	0.234^a	0.984^a	5.11^a	11.2	25.8	–	0.071	0.742^a	3.26	9.25
SEM	0.004	0.107	0.116	1.13	1.32	–	0.024	0.013	0.432	0.851
p-Value	0.019	0.020	<.0001	0.857	0.177	–	0.457	<0.01	0.634	0.707
Storage duration (DS)
14	0.132	0.872	4.16	11.8	24.5	–	0.0721	0.682	3.61	9.80
21	0.144	0.641	4.26	10.3	24.2	–	0.0542	0.661	2.61	9.18
SEM	0.004	0.108	0.116	1.12	1.32	–	0.0241	0.019	0.444	0.851
p-Value	0.129	0.174	0.540	0.353	0.879	–	0.531	0.364	0.151	0.625
Interaction (SP*SD)
0-h*14	0.062	0.112	3.38	11.7	24.2	–	0.052	0.632	3.16	11.4
0-h*21	0.072	0.153	3.43	10.1	21.7	–	0.042	0.591	2.74	8.02
6-h14	0.221	2.22	4.94	11.9	24.8	–	0.092	0.742	4.05	8.17
6-h*21	0.223	0.112	5.09	10.4	26.7	–	0.062	0.073	2.47	10.3
SEM	0.005	0.147	0.164	1.48	1.82	–	0.032	0.026	0.602	1.18
p-Value	0.129	0.755	0.755	0.986	0.279	–	0.838	0.598	0.389	0.054

^a,bMeans of different superscripts within a column differ significantly (p < 0.05); SEM: standard error of means, p-value: probability value. SPIDES: short period of incubation during egg storage; 0-h SPIDES: eggs that did not receive short period incubation during egg storage (control); 6-h SPIDES: eggs that received 6-h short period incubation during egg storage; *data missing because samples could not be recovered after oven drying.

Table 4. Impact of storage duration, SPIDES, and their interaction on embryo length changes during incubation.

	Embryo Length (mm)
Treatment	4	7	11	14	18	Mean
SPIDES (SP)
0-h	0.881^b	2.061	5.78^b	9.41	12.7	6.16
6-h	1.21^a	2.94	6.75^a	9.42	13.4	6.74
SEM	0.077	0.342	0.0971	0.568	0.298	1.47
p-Value	0.019	0.119	<0.001	0.979	0.132	0.856
Storage duration (SD)
14	0.991	2.89	6.30	9.64	0.232	4.011
21	1.11	2.12	6.23	9.19	0.361	3.83
SEM	0.077	0.342	0.097	0.578	0.298	1.15
p-Value	0.368	0.159	0.564	0.599	0.335	0.933
Interaction (SP*SD)
0-h*14	0.873	2.48	5.78	9.42	13.2	6.31
0-h*21	0.912	1.65	5.79	9.42	12.3	6.01
6-h*14	1.12	3.30	6.84	9.88	13.5	6.93
6-h*21	1.33	2.59	6.66	8.97	13.3	6.56
SEM	0.107	0.467	0.137	0.785	0.411	1.011
p-Value	0.531	0.912	0.496	0.599	0.588	0.992

^a,bMeans of different superscripts within a column differ significantly (p < 0.05); SEM: standard error of means, p-value: probability value., SPIDES: short period of incubation during egg storage; 0-h SPIDES: eggs that did not receive short period incubation during egg storage (control); 6-h SPIDES: eggs that received 6-h short period incubation during egg storage.

Figure 4. Representative profile of the impact of storage duration, SPIDES treatment, and their interaction on hatchability. Data bars with similar superscripts show no significant difference (p > 0.05): 0-h SPIDES: eggs that did not receive short period incubation during egg stage (control); 6-h SPIDES: eggs that received 6-h short period incubation during egg storage; interaction = SPIDES × storage duration interaction.

Table 5. Impact of storage duration, SPIDES, and their interaction on hatching traits.

	Chick weight (g)		Chick length (mm)		Shank length (mm)		Residual yolk (g)		PASCAR score (%)
Treatment	Day 1	Day 7	Day 1	Day 7	Day 1	Day 7	Day 1	Day 7	Day 1	Day 7
SPIDES (SP)
0-h	36.9	64.7	173	215	66.8	98.5	5.83	0.542	50^b	100
6-h	36.4	70.9	174	220	69.8	95.5	5.041	0.251	100^a	100
SEM	4.48	4.34	2.89	5.76	1.29	1.92	1.16	0.286	0.00	–
p-Value	0.930	0.365	0.819	0.573	0.178	0.331	0.652	0.517	0.001	–
Storage duration (SD)
14D	37.4	69.1	174	221	68.2	96.8	5.48	0.333	75	100
21D	35.9	66.6	173	213	68.3	97.3	5.39	0.462	75	100
SEM	4.48	4.34	2.89	5.76	1.29	1.92	1.16	0.286	14.43	–
p-Value	0.827	0.708	0.819	0.409	1.000	0.863	0.962	0.761	1.000	–
Interaction (SP*SD)
0-h*14D	34.7	68.9	174	215	66.1	102	4.92	0.254	50	100
0-h*21D	39.2	60.1	172	214	67.5	95	6.76	0.824	50	100
6-h*14D	40.1	69.2	174	227	70.5	91.5	6.052	0.404	100	100
6-h*21D	32.6	72.8	174	213	69.2	99.5	4.031	0.101	100	100
SEM	6.35	6.14	4.093	8.15	1.83	2.72	1.64	0.405	0.00	–
p-Value	0.401	0.382	0.819	0.469	0.460	0.051	0.301	0.339	0.001	–

SEM: standard error of means, p-value: probability value; SPIDES: short period of incubation during egg storage. 0-h SPIDES: eggs that did not receive a short period of incubation during egg storage (control); 6-h SPIDES: eggs that received a 6-h short period of incubation during egg storage; Interaction: SPIDES × storage duration interaction; PASCAR scored measured ‘clean navel (2) or presence of navel button at various degrees (1)’. Most clean was 100% while the presence of the navel button was 0%.

Table 6. Impact of 6-h short period incubation during egg storage (SPIDES) on hematology of 7-day-old chicks.

Treatment	RBC (10^12/L)	HGB (g/dL)	HCT/ PCV (%)	MCV f/L	MCH (pg)	MCHC (g/dL)	PLT (10^9/L)	MPV (fL)	PDW (%)	PCT (%)
SPIDES (SP)
0-h	2.34	8.10	32.7	140.0	34.6	247.0	17.3	11.2	13.0	0.02
6-h	1.56	6.10	22.7	109.3	225.6	1509.2	17.0	11.3	11.6	0.01
SEM	0.378	0.871	5.45	25.7	135.2	899.4	2.55	0.233	1.40	0.003
p-Value	0.219	0.179	0.264	0.443	0.374	0.377	0.949	0.835	0.545	0.353
Storage duration (SD)
14D	2.16	7.48	31.3	145.3	34.6	238.3	16.5	11.2	13.3	0.01
21D	1.74	6.73	24.1	104.0	225.6	1518.0	17.8	11.4	13.3	0.01
SEM	0.378	0.871	5.45	25.6	135.2	899.5	2.58	0.233	1.40	0.003
p-Value	0.474	0.576	0.403	0.316	0.374	0.371	0.756	0.545	0.388	1
Interaction (SP*SD)
0-h*14D	2.28	8.10	32.8	144.1	35.6	247.0	15.0	11.7^a	11.8	0.02
6-h*14D	2.04	6.85	29.8	146.5	33.6	229.5	18.0	10.6^b	14.9	0.01
0-h*21D	2.41	8.10	32.6	135.9	33.1	247.0	19.5	10.8^b	14.2	0.02
6-h*21D	1.08	5.35	15.6	72.2	35.7	2789.0	16.0	12.0^a	8.40	0.01
SEM	0.535	1.23	7.71	36.2	19.2	1272.0	3.62	0.326	1.96	0.004
p-Value	0.372	0.576	0.576	0.413	0.370	0.371	0.441	0.038	0.111	1

^a,bMeans of different superscripts within a row differ significantly (p < 0.05); SEM: standard error of means, p-value: probability value. SD: cool storage duration (14 and 21 days); SPIDES (SP): short period of incubation during egg storage; 0-h SPIDES: eggs that did not receive a short period of incubation during egg storage (control); 6-h SPIDES: eggs that received 6-h short period incubation during egg storage; Interaction: SPIDES × storage duration interaction. Erythrocyte (RBC: red blood cells, HGB: hemoglobin, HCT: hematocrits, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin concentration); Thrombocyte (PLT: platelet count, MPV: mean platelet volume, PDW: platelets distribution width, PCT: platelet crit).

Data availability statement

The data that support the findings of this study are available and will be provided at the request of the corresponding author.