Silver nanoparticles (AgNano) as carrier of available oxygen (O2) and with high surface reactivity may increase O2 consumption, enhance fat uptake (FU), and stimulate growth and development. The objective was to investigate the effects of in ovo injection of AgNano on the metabolic rate (O2 consumption, CO2 production, and heat production, HP), fat uptake, and the development of broiler and layer hatchlings. AgNano concentrations (50, 75, and 100 mg/kg) were injected in ovo at day 1 of incubation to different breeds of broiler and layer chicken embryos. Oxygen consumption and subsequently FU did not increase linearly following AgNano treatment. FU was lower in hatchlings treated with 50 and 100 mg AgNano/kg, but surprisingly not in hatchlings treated with 75 mg AgNano/kg. Interestingly, the difference in FU between treatments was not reflected in hatchling development. The results indicated that AgNano affected metabolic rate and FU; however, it did not influence the development of hatchlings. This suggests that in ovo injection of AgNano reduces the need to use yolk fat as an energy source during embryonic development and consequently the remaining fat in the residual yolk sac may provide a potent source of nutritional reserves for chicks of few days after hatching.
During the latter part of incubation, fat in the yolk provides the main source of energy for the developing embryo. Glucose from carbohydrates and protein provides an important source of energy during the first two weeks of embryonic development, and from then until 2 or 3 days before internal piping, fatty acids from the yolk supply 90% of the energy requirement of the embryo. Towards the end of incubation, a few days before piping, energy metabolism switches to glucose through gluconeogenesis and glycogenesis [
Oxygen (O2) is an important factor for the effective retrieval of fat and
Several techniques are under investigation to mitigate this effect and facilitate growth before and after hatching. In ovo nutrient supply, for example, is a technique that involves the injection of a nutrient solution few days prior to piping to provide an external source of energy to the embryo and to the hatchlings [
Recently, we reported that the metabolism of layer embryos can be modified with the in ovo injection of silver nanoparticles (AgNano) [
A 2 × 3 factorial experiment with two breeder strains (Ross × Ross 308 and Lohmann) and three AgNano concentrations (50, 75 and 100 mg/kg) was used.
584 fertile chicken eggs from Lohmann (layer-type) and Ross × Ross 308 (broiler-type) breeder strains of the same age (37 weeks old) were obtained from a commercial hatchery. In each breeder strain, 292 eggs were randomly grouped into two batches and stored in a refrigerator (10°C) for 1–3 days before being placed in the incubator. In each batch, 140 eggs were distributed into 5 treatment groups, with 28 eggs per treatment from which 12 eggs from each treatment were used for the measurement of O2 consumption and CO2 production, 5 eggs for the evaluation of hatchling development, and the rest were used as reserves. In addition, 12 eggs per breeder strain were opened at day 1 and the yolk samples were collected. At day 1 of incubation, the eggs from the first batch were numbered and injected according to the following treatment descriptions: (1) control (no injection), (2) sham control (placebo), (3) 50 mg AgNano/kg, (4) 75 mg AgNano/kg, and (5) 100 mg AgNano/kg. The eggs were injected with 0.3 mL of a hydrocolloidal AgNano and phosphate buffered saline solution (placebo) into the air sac using a sterile 27 gauge, 20 mm needle. Immediately after injection, the hole was sealed with hypoallergenic tape and the eggs were placed into an incubator. The eggs were incubated for 21 days under standard conditions (temperature 37.8°C, 55% humidity, eggs turned once per hour for the first 18 days, and temperature 37°C, and 60% humidity from day 19 until hatching). The same procedure was repeated for the eggs in batch 2 on the following day.
The O2 consumption and CO2 production were measured at day 10, 13, 16, and 19 of incubation, according to the procedure described by Chwalibog et al. [
The eggs were weighed and candled prior to measurements to check for the presence of embryos. Eggs without an embryo were discarded and replaced with eggs of the same age from the same treatment kept in the incubator as reserves. Six eggs from each treatment were placed in the respiration chambers and measured for 3 h from 9:00 to 12:00, followed by another six eggs from the same treatment measured from 13:00 to 16:00. After each measurement, the eggs were put back into the incubator. The procedure was repeated over the following day with the other batch of eggs; thus, a total of 24 eggs were measured from each treatment per breeder strain. All gas exchange and heat production (HP) measurements were standardised to a 50 g egg mass in order to account for differences in weight during each measurement.
HP was calculated from O2 consumption and CO2 production in accordance with Brouwer [
Hydrocolloid AgNano solutions were obtained from Nano-Tech (Warsaw, Poland) and were produced by a patented nonexplosive high voltage method (Polish Patent 3883399) from high purity metals (99.9999%) and high purity demineralised water. The concentrations of nanoparticles in the hydrocolloids were 50, 75, and 100 mg/kg AgNano with a particle size ranging from 2 to 35 nm based on TEM evaluations as described by Chwalibog et al. [
All eggs were weighed at day 1 of incubation to determine the egg weight at setting. Twelve eggs from each breeder strain, representing the average weight of the eggs at set, were selected at day 1 for fresh yolk collection. At 24 h after hatching, 10 chicks from each treatment were euthanised by decapitation and the yolk sac (YS) was removed from the abdominal cavity and then weighed, stored at −20°C and freeze-dried prior to analysis.
On the day of hatching, newly hatched chicks were kept without feed and water in a temperature-controlled (32°C) brooder box furnished with a heat lamp for 24 h. Chick weight (CW;
The experimental procedures followed Danish National Legislation.
Yolk samples collected at day 1 (
Yolk-free body weight (YFBW) was determined as the difference between CW and the residual YS. Liver, heart, and intestine weights relative to YFBW (as % of YFBW) were used to calculate relative organ weights (g of organ weight/g of YFBW) × 100. The absolute weights of the YS, yolk fat content (YF), and fat uptake (FU) and their weights relative to YFBW (as % of YFBW) were both calculated, and in case the trend is the same, only the absolute values are shown.
YF at day 1 (
Data were analysed using the GLM procedure of SAS (SAS Institute Inc., 2009) considering the main effects of treatments (50, 75, and 100 mg/kg AgNano), breeder strain (Ross or Lohmann), and the interactions between these variables. The Tukey-Kramer honestly significant difference test was used to test the separation of the means at a significance level of
The residual YS weight was larger in hatchlings treated with AgNano (
Comparison of (a) yolk sac weight (YS), (b) yolk fat content (YF), (c) fat uptake (FU), and (d) yolk-free body weight (YFBW) of hatchlings in ovo injected with different concentrations of AgNano (50, 75, and 100 mg/kg) and the control (no injection) at 24 h after hatching. Mean values and standard errors of 4 treatments, each containing 10 chicks. a,b,cSignificant difference (
The residual YS of hatchlings treated with 100 mg AgNano/kg contained more fat (
Fresh yolk weight (18.6 versus 15.8 g) was larger in broiler eggs at day 1 of incubation compared with layer eggs. Expectedly, the YF content (5.97 versus 4.82 g) was also higher in broiler than in layer eggs. The weight of residual YS was 0.37 g higher (
Comparison of (a) yolk sac weight (YS), (b) yolk fat content (YF), (c) fat uptake (FU), and (d) yolk-free body weight (YFBW) of Ross (broiler-type) and Lohmann (layer-type) hatchlings in ovo injected with different concentrations of AgNano (50, 75, and 100 mg/kg) and the control (no injection) at 24 h after hatching. Mean values and standard errors of 4 treatments, each containing 10 chicks. a,bSignificant difference (
There was no significant interaction effect between treatment and breeder strain on the YS, YF, and FU (data not shown).
AgNano concentrations did not affect egg weight at setting (
The weight of eggs at setting was 5% heavier in broilers than in layers (63.0 versus 60.0;
No interaction effect was noted between treatment and breeder strain on YFBW and the relative weights of intestine, liver, and heart (
An effect of treatment and breeder strain was observed on gas exchange and HP. The hatchlings treated with 50 and 100 mg AgNano/kg had the lowest metabolic rate (O2 consumption, CO2 production, and HP) during incubation (
Total oxygen consumption (O2), carbon dioxide production (CO2), and heat production (HP) of layer (Lohmann-Lo) and broiler (Ross-Ro) hatchlings in ovo injected with different concentrations of AgNano (50, 75, and 100 mg/kg) and the control (no-injection, C) measured at embryonic days 13 to 191.
O2 |
CO2 |
HP |
|
---|---|---|---|
Treatment | |||
100 | 13.2a | 8.4a | 255a |
75 | 13.7b | 8.9b | 267b |
50 | 13.2a | 8.3a | 255a |
C | 14.3b | 9.3b | 279b |
Breeder strain | |||
Lohmann | 12.7a | 8.0a | 245a |
Ross | 14.6b | 9.6b | 284b |
SE2 | 0.121 | 0.124 | 2.45 |
|
|||
Treatment | 0.001 | <0.0001 | <0.0001 |
Breeder strain | <0.0001 | <0.0001 | <0.0001 |
Treatment |
0.64 | 0.06 | 0.06 |
Within columns: means with different superscripts differed significantly (
1Mean values of 4 treatments, each containing 24 embryos per breeder strain.
2Pooled standard error.
Broiler embryos consumed 13% more O2, produced 17% more CO2, and expended 14% more heat compared with layer embryos (
There was no interaction effect recorded between treatment and breeder strain on the metabolic rate (
This study revealed that AgNano reduced FU, indicating a reduction in fat oxidation, as demonstrated by higher weights of the YS and YF 24 h after hatching. The results are unexpected and contrary to our hypothesis that AgNano, as carrier of available O2 with high surface reactivity, would increase O2 consumption and accelerate fat oxidation. In the current study, we demonstrated that FU was lower in hatchlings treated with 50 and 100 mg AgNano/kg, but surprisingly not in hatchlings treated with 75 mg AgNano/kg, which suggested that the pattern of oxidation did not respond linearly to the increase in AgNano concentration. The reason for this response is unknown, but perhaps the rates of O2 consumption could explain this pattern of fat oxidation. O2 supply is essential for the
Several studies have demonstrated that at hatching, the weights of the residual YS, YF, and FU are higher in broiler compared to layer hatchlings because of the higher metabolic rate in the former, which favours the oxidation of fat and explains the higher rates of fat consumption [
No significant interaction effect between treatments and breeder strain was noted on YS, YF, and FU of hatchlings. This can be explained by the lack of an interaction effect on the metabolic rate exhibited by both breeder strains in response to AgNano treatments.
In comparison with the control (no injection) group, a negative effect on the metabolic rate was recorded in hatchlings injected with 50 and 100 mg AgNano/kg. This finding is contrary to our previous work [
In agreement with other studies [
The lack of a significant interaction effect between the treatment and breeder strain on the metabolic rate was not in complete agreement with our previous study. In conformity with previous experiments [
In the current study, the YFBW and relative organ weights of chicks were used as an indicator of hatchling development. The YFBW was higher for broiler than for layer hatchlings, which is in agreement with the documented higher BW and faster development in broiler versus layer chicken embryos reported previously [
The present results also demonstrate a difference in the relative intestine weight between breeder strains. We recorded a higher relative weight of the intestine in broiler compared to layer hatchlings, which may indicate more advanced maturation of the intestine in broiler hatchlings [
Hatchling development was not affected by the AgNano concentration. This lack of treatment effect is consistent with findings in quail, pigs, and chickens and supports the conclusion that AgNano do not affect growth [
The results demonstrate that AgNano at 50 and 100 mg/kg affected the metabolic rate and fat uptake; however, it did not influence the development of hatchlings. In ovo injection of AgNano at 50 and 100 mg/kg reduces the need to use yolk fat as an energy source, and it can be speculated that the remaining fat in the residual YS can be a potent source of posthatching nutritional reserves for hatchlings. However, further investigation is necessary to establish its potential.
This work was supported by the Danish Agency for Science Technology and Innovation (Grant no. 2106-08-0025).