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Influence of in ovo inoculation with various nutrients and egg size on broiler performance
©2010 Poultry Science Association, Inc.
Influence of in ovo inoculation with various nutrients and egg size on broiler performance T. T. dos Santos,* A. Corzo,† M. T. Kidd,† C. D. McDaniel,† R. A. Torres Filho,* and L. F. Araújo‡1 *Globoaves Agro Avícola Ltda, Chapecó, Brazil 89.815-280; †Department of Poultry Science, Mississippi State University, Mississippi State 39762; ‡Faculty of Animal Science, University of São Paulo, Pirassununga, Brazil 13.635-900
SUMMARY This study evaluated the influence of inoculation of different nutrients into eggs of 18-d-old broiler embryos. On d 18 of incubation, before transferring, eggs were injected with a solution containing either maltose, a multivitamin supplement, zinc-glycine, glutamine, a mixture containing all these elements, or sodium chloride (control). After hatching, 2,400 male broiler chicks were placed in an experimental broiler house and distributed into 60 floor pens in a completely randomized design comprising a 2 × 6 factorial arrangement of treatments (2 egg weights × 6 solutions), for a total of 12 treatments. Birds and feed consumption were measured weekly. At 42 d of age, 3 birds per replicate pen were randomly selected for processing. Birds derived from heavier eggs had greater hatchability and hatching weight. At 42 d of age, birds from heavier eggs had higher BW, carcass weight, and breast meat weight. The livability of birds derived from heavy eggs was higher at 7 and 14 d of age. The in ovo inoculation of the nutrients to 18-d-old embryos did not influence live performance or carcass traits. The technique of in ovo inoculation of certain nutrients may be used in industrial poultry production, but further studies are required to define the best solutions or mixture of nutrients to be used. Key words: in ovo nutrition, broiler, maltose, glutamine, zinc 2010 J. Appl. Poult. Res. 19:1–12 doi:10.3382/japr.2009-00038
DESCRIPTION OF PROBLEM Early intake after hatch of exogenous feed is accompanied by rapid development of the gastrointestinal tract and associated organs [1]. During the last day of incubation, the fastest developing organ is the intestinal tract, representing 1% of the weight of the 18-d embryo and reaching 3.5% at hatch [2]. Hatching drastically changes the way chicks retrieve nutrients. During incubation, energy 1
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and nutrients are provided via the yolk, which is rich in lipids but has relatively low protein and carbohydrate concentrations. In the embryo, yolk lipids are directly transported to the blood by endocytosis [3], but after hatching, yolk content is absorbed through both the yolk sack membrane and Meckel’s diverticulum, and is digested and absorbed by the intestinal tract. Noy and Sklan [3] observed that the passage of the yolk content to the intestinal tract is higher when the bird is fed as compared with when it is fast-
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Primary Audience: Nutritionists, Veterinarians
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nal development. Uni and Ferket [8] inoculated the same carbohydrate cocktail and observed increases in liver glucose at hatch and BW at hatch that continued up to 35 d of age. Uni et al. [9] observed that the inoculation of β-hydroxy β-methylbutyrate and carbohydrates improved BW at hatch, improved breast yield at hatch and at 10 and 25 d of age, and increased liver glycogen content in chicks. According to these authors, the supplied energy reduced muscle protein degradation destined to serve as the energy source at hatch, resulting in higher BW and breast percentage at hatch. Other factors, such as broiler breeder genetic line, breeder age, and egg size, may affect initial development and therefore final performance. As the breeder flock ages, egg weight tends to increase [14], and increasing egg weight and breeder age can affect hatchling performance. Vieira and Moran [15] observed that heavier eggs produced larger chicks and that this difference was maintained until market age. Heavier eggs also contained higher protein content in the yolk and albumen [16]. The objective of this study was to observe any effect in the use of various nutrient solutions inoculated at 18 d of age, via in ovo administration, in broiler eggs of 2 different weights.
MATERIALS AND METHODS This study used a completely randomized design comprising a 2 × 3 factorial arrangement of treatments. Factors involved were egg weight and in ovo solutions. Eggs were sorted by weight into either light or heavy eggs. At d 18 of incubation, eggs were injected in ovo with one of various nutrient solutions. Solutions were delivered using a saline solution (stock solution). These treatment solutions were as follows: 0.5 mL of a 50% maltose syrup solution (MALT); 0.5 mL of a multivitamin solution (VIT); 0.5 mL of a 0.5% zinc-glycine solution (ZG); 0.5 mL of a 10% glutamine solution (GLU); 0.5 mL of a 50% maltose (12% dextrose, 42% maltose, 10% maltotriose, and 23 to 28% other sugars), 0.5% zinc-glycine, 10% glutamine, and multivitamin solution (MIX); and a control group containing 0.5 mL of 0.4% saline solution. The composition of the multivitamin solution used in the VIT and MIX diets is shown in Table 1.
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ed. According to these authors, this difference may be due to the physical presence of feed, the peristaltic movements of the intestine, the negative pressure in the abdominal cavity, or their combination, thereby stimulating passage of the intestinal content. The yolk sac energy reserves present at hatch may not be sufficient to supply their maintenance energy requirements [4], and fasting effects may occur before the flock is removed from the hatchery. However, despite being capable of ingesting feed, the intestinal tract of the chick is still immature [2]. Weight loss between hatching and removal from the hatcher is approximately 0.18 g/h while fasting [5]. Halevy et al. [6] observed lower BW and breast meat yield values at 41 d when broilers were fasted for 24 h after hatching when compared with those immediately fed, likely caused by a change in satellite cell activity, leading to subsequent changes in hyperplasia and an associated delay in muscle maturation. Several alternatives have been proposed to improve performance during this initial phase, such as feeding at the hatchery [4, 5] and in ovo feeding [7–10]. However, despite having shown promising results in formal experimentation, these techniques are rarely put into practice by broiler companies for complex logistical reasons. Concerning in ovo nutrient inoculation, Ohta et al. [11] suggested that the increase in hatching weight of 7-d-old embryos injected with amino acids may have been due to a higher content of amino acids in the yolk or to better utilization of amino acids by the embryo. In geese, AlMurrani [12] observed that in ovo injection of amino acids at 7 d of incubation increased BW at hatch, and the BW difference was maintained up to 52 d of age when compared with noninoculated embryos. Foye et al. [10] observed higher BW and breast weight of 1-d-old turkeys when these were inoculated at 23 d of incubation with egg-derived protein. The inoculation with carbohydrates (maltose, sucrose, and dextrose) in the amniotic fluid of broilers between 17 and 18 d of incubation was studied [13]. These authors observed an increase in jejunal villi surface area from hatching up to 3 d of age. As the embryo ingests the amniotic fluid at the end of incubation, the inoculation of carbohydrates would be an extra source of nutrients, stimulating intesti-
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virus. These embryos were subsequently killed with carbon dioxide and weighed. Yolk sac, intestinal tract (between the proventriculus and the rectum), and liver were dissected and weighed, and their percentages relative to embryo weight were calculated. At 21 d of incubation, hatched chicks were removed from the hatcher and weighed to determine BW loss at hatch. Nonhatched eggs were opened to determine the percentage of infertile eggs and embryo age at death. The calculation of mortality postinoculation considered only pipped eggs and mortality after 18 d of incubation. Chicks were vaccinated against infectious bronchitis approximately 12 h before transport to the experimental house. The day-old male chicks (3,360) were selected and housed in a 50 × 12 m experimental broiler house, divided into 60 pens (3.12 m2/pen), covered with new litter (softwood shavings). The treatments (2 egg weights × 6 solutions) were each replicated 5 times, with each replicate consisting of 40 birds, for a total of 60 experimental units. Birds were offered feed and water ad libitum. All birds received common feeds (Table 1) from placement until the end of the experiment. Birds were weighed weekly until 42 d of age. Body weight gain, feed intake, and livability were measured,
Table 1. Composition and nutritional levels of experimental diets (%, as is) Item Ingredient Corn, % Soybean meal, % Soybean oil, % Sodium chloride, % Limestone, % Dicalcium phosphate, % Choline chloride, % dl-Methionine, % Vitamin-mineral premix,1 % Calculated composition CP, % AME, kcal/kg Calcium, % Available phosphorus, % Sodium, % Lysine, % TSAA, % 1
Prestarter, 1 to 7 d
Starter, 7 to 21 d
Grower, 21 to 35 d
Finisher, 35 to 42 d
55.00 38.00 2.40 0.40 1.30 1.60 0.60 0.30 0.40
60.00 33.00 2.70 0.30 1.30 1.60 0.45 0.25 0.40
64.00 28.00 4.00 0.30 1.15 1.60 0.35 0.20 0.40
66.00 25.50 4.80 0.30 1.10 1.40 0.30 0.20 0.40
22.0 2,950 1.00 0.50 0.25 1.28 0.94
20.0 3,050 0.95 0.48 0.20 1.10 0.84
18.0 3,175 0.90 0.45 0.20 1.00 0.80
17.0 3,250 0.85 0.43 0.20 0.95 0.77
Multivitamin solution composition (per L): vitamin A, 3,000 IU; vitamin D3, 1,500 IU; vitamin E, 25 IU; vitamin K3, 1.50 mg; vitamin B1, 1.00 mg; vitamin B2, 2.50 mg; vitamin B6, 1.50 mg; vitamin B12, 6.00 mg; nicotinic acid, 20.00 mg; pantothenic acid, 5.00 mg; biotin, 75.00 μg; folic acid, 0.50 mg; vitamin C, 125.00 mg.
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Two liters of each solution was produced and used as a vaccine diluent in the in ovo injection solution [17]. Osmolality and pH of the solutions were not measured. Eggs from 6 trays per weight group were inoculated with each solution. The vaccination equipment automatically injected a dose of 0.5 mL into the amniotic fluid of each egg. Before each solution was applied, the equipment was cleaned by flushing air through the tubes, and the absence of solution was confirmed by injecting a test plate. This procedure was repeated between all solutions. Nutrients were injected in ovo in the following sequence: MALT, VIT, ZG, control, MIX, and GLU. This sequence was followed because of the lower solubility of GLU, particularly when diluted only in saline solution. A total of 9,600 eggs derived from 30-wkold Cobb 500 breeders [18] were separated into heavy or light eggs by using a scale (Table 2). Eggs were placed in 100 incubation trays with a capacity of 96 eggs each. Eggs were incubated in a single incubator up to 18 d. Before transferring to the hatcher, eggs were weighed again to monitor egg weight loss at transfer. At the same time, 5 embryos from each egg weight group were randomly removed for collection of blood from the cardiac artery to determine antibody levels against infectious bursal disease and reo-
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Table 2. Characteristics of broiler eggs injected with nutritional solutions at 18 d of incubation Item
Incubation weight, g 54.0 59.9
47.9 53.0
Δ Weight, %
Hatch, %
18-d livability, %
88.61 88.51
89.17 91.30
98.58 98.76
37.7 41.9
89.04 89.67 91.25 90.49 90.39 90.56
98.41 98.22 99.02 98.49 98.85 99.02
39.9 39.7 40.4 39.7 39.8 39.3
0.001
0.001
0.310
3.1
2.8
0.39
0.005 0.59 0.10 3.29
0.50 0.60 0.70 1.34
Hatch BW, g
0.001 0.23 0.70 1.9
1
Treatment solutions: maltose = 0.5 mL of a 50% maltose syrup solution; vitamins = 0.5 mL of a multivitamin solution; zinc = 0.5 mL of a 0.5% zinc-glycine solution; control = 0.5 mL of a 0.4% saline solution; mix = 0.5 mL of a 50% maltose (12% dextrose, 42% maltose, 10% maltotriose, and 23 to 28% other sugars), 0.5% zinc-glycine, 10% glutamine, and multivitamin solution; glutamine = 0.5 mL of a 10% glutamine solution. 2 Significance level according to ANOVA.
and feed conversion corrected for mortality was calculated based on performance data. At placement, 1 bird per replicate was randomly selected, killed with carbon dioxide, and weighed. The yolk sac, intestinal tract (from the beginning of the proventriculus until the end of the rectum), and liver were removed and weighed, and their percentages relative to embryo weight were calculated. This procedure was repeated when chicks were 7 and 21 d old. At 42 d of age, 3 birds per replicate pen were randomly removed and leg banded. The birds were weighed, submitted to electrical stunning, and killed by exsanguination. After the neck, feet, and offal were removed, hot carcasses were weighed to calculate carcass yield. Breasts (with bones and skin) and legs and thighs (with bones and skin) were cut and weighed to determine their yields. The collected data were analyzed by the SAEG statistical package as a 6 × 2 factorial arrangement [19]. Data were tested by 2-way interactions (P < 0.05), and in their absence, only main effects are shown. Experimentation was approved by the Universidade de São Paulo Institutional Animal Care and Use Committee.
RESULTS AND DISCUSSION The difference in egg weights after being classified immediately following lay was main-
tained at the time eggs were transferred to the hatcher and injected with the treatments (18 d of incubation), and also when eggs hatched (Table 2). However, weight loss percentage from the start of incubation until transferring to the hatcher was similar between the 2 groups. Solution inoculation did not affect weight at hatch (Table 2). The hatchability of heavy-weight eggs was higher as compared with light eggs (Table 2), but this was not caused by late embryo mortality (after 18 d of incubation) because livability in this period was similar for both weight categories. Inoculation with the treatments did not affect hatching and livability between d 18 and 21 of incubation. These results are different from those observed by Pedroso et al. [20], who reported greater embryo mortality when a glucose solution was injected into the amniotic fluid of 16-d-old embryos. According to those authors, this may have been caused by the high osmolarity of the inoculated solution, which was not measured in this trial. Conversely, there was no effect on hatchability of the inoculation of GLU in the amniotic fluid of 16-d-old embryos [21], which is consistent with the data obtained in the present experiment. Gore and Qureshi [22] observed lower hatchability when 25-d-old turkey embryos were inoculated with 20 and 30 IU of vitamin E. Ohta et al. [23] reported lower hatch-
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Eggs Light Heavy Solution1 Maltose Vitamins Zinc Control Mix Glutamine P-value2 Egg Solution Egg × solution SEM
18-d weight, g
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Lopes et al. [30] injected glutamine to 16-d-old embryos and did not find BW gain differences in 10- to 21-d-old broilers. The addition of 10 IU of vitamin E did not influence turkey and broiler BW at hatch and at 35 d of age [22]. On the other hand, the inoculation of carbohydrates, carbohydrates and proteins, hydroxy-methylbutyrate, or a mixture of these compounds increased chick BW at hatch and subsequent chick development [7–10]. According to these authors, inoculation with these substances enhanced intestinal development and enzyme expression at hatch, thereby allowing more efficient posthatch development. Moreover, the supply of these nutrients improved bird energy status, which spared energy used for metabolism and development [10]. In ovo inoculation with amino acids at 7 d of incubation in the yolk sac increased hatching weight as a percentage of egg weight [11, 23, 24]. Ohta et al. [11] reported that this increase in hatching weight might be due to an increase in yolk amino acid content or in amino acid utilization by the embryo. However, in geese, Al-Muranni [12] showed that in ovo inoculation of amino acids at 7 d of incubation increased hatching weight, and this difference was maintained up to 52 d of age. Cumulative feed intake and feed conversion are shown in Tables 4 and 5, respectively. Chicks from heavy eggs consumed more feed during the entire experimental period. This higher feed intake capacity may explain the higher BW of the broilers derived from heavier eggs as compared with those originating from lighter ones. Broilers that originated from heavy eggs had superior feed conversion at d 7 but poorer feed conversion at d 21, 28, and 35. At market age (d 42), these differences had dissipated. No nutrient solution inoculation effect was observed for feed intake or feed conversion in the present study. Pedroso et al. [31] did not observe any differences in feed intake or feed conversion of chicks inoculated with glucose or GLU at 16 d of incubation. In agreement with the present results, Vieira and Moran [15] reported no FCR differences between chicks derived from light or heavy eggs at market age. Chicks derived from heavy eggs had greater livability up to 14 d of age (Table 6). Turkeys derived from young breeders and small eggs were less capable of coping with the metabolic chal-
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ability when injecting amino acids at d 0 of incubation. However, when injection was carried out in the yolk of 7-d-old embryos, hatchability was not affected. In ovo injection must be made in the extraembryonic cavity or yolk sac to prevent a reduction in hatchability, as observed when amino acids were inoculated in the amniotic cavity or in the chorioallantoic membrane [24]. It must be noted that Ohta and Kidd [24] inoculated 7-d-old embryos, whereas in the present study, embryos were 18 d old. Jochemsen and Jeurissen [25] reported that age at inoculation may influence where the product is applied. In the present study, chicks derived from heavier eggs were heavier at hatch. This result is consistent with those observed by Vieira and Moran [15, 16] in broiler chicks and those observed by Applegate and Lilburn [26] in turkey poults. Inoculation of the different treatment solutions at 18 d of incubation did not influence hatch weight (Table 2). Body weight of chicks from heavier eggs was observed to be higher throughout the study (Table 3). There was no influence of the nutrient solutions applied via in ovo injection on BW other than at placement (Table 3). Note that BW on d 0 was lower than BW at hatch. This is probably due to fasting during the 24-h interval between hatching and placement, during which birds were removed from the hatcher, selected, vaccinated, sexed, and transported in temperature-controlled trucks to the experimental farm. Vieira and Moran [27] observed that younger broiler breeders produced lighter eggs and, consequently, lighter broilers at slaughter as compared with older breeders [28]. However, according to Sklan et al. [14], breeder age does not influence bird performance. When working with eggs of different weights derived from breeders of similar BW, Sklan et al. [14] observed that heavier eggs produced chicks with heavier hatching weights and heavier broilers at market age (41 d). This is consistent with the results obtained in the present experiment, in which eggs of different weights derived from breeders of the same age exhibited different performance, with heavier eggs producing heavier chicks and heavier broilers at market age. Leitão et al. [29] did not observe BW gain differences between 0 and 10 d in chicks supplemented with glucose at 16 d of incubation.
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Table 3. Body weight (g) of broilers derived from light and heavy eggs inoculated with nutritional solutions at 18 d of incubation Age, d Item
7
14
21
28
35
42
35.8 39.1
159 170
408 424
831 856
1,377 1,408
2,041 2,084
2,685 2,740
38.2a 37.9a 37.4ab 37.3ab 37.2ab 36.6b
165 166 165 165 166 160
417 415 420 415 420 408
844 847 853 843 842 830
1,401 1,402 1,400 1,392 1,379 1,381
2,063 2,064 2,070 2,071 2,048 2,059
2,710 2,711 2,709 2,723 2,708 2,712
0.001 0.001 0.003 2.9
0.001 0.12 0.14 7.4
0.001 0.25 0.25 14.4
0.001 0.24 0.90 23.5
0.002 0.44 0.86 35.5
0.001 0.85 0.74 50.6
0.001 0.99 0.93 70.2
a,b
Values within a column with different superscripts differ significantly (P < 0.05). Treatment solutions: maltose = 0.5 mL of a 50% maltose syrup solution; vitamins = 0.5 mL of a multivitamin solution; zinc = 0.5 mL of a 0.5% zinc-glycine solution; control = 0.5 mL of a 0.4% saline solution; mix = 0.5 mL of a 50% maltose (12% dextrose, 42% maltose, 10% maltotriose, and 23 to 28% other sugars), 0.5% zinc-glycine, 10% glutamine, and multivitamin solution; glutamine = 0.5 mL of a 10% glutamine solution. 2 Significance level according to ANOVA. 1
lenges that occur during the first days posthatch [26], and this may explain the higher mortality of this group of birds because the capacity to produce and metabolize carbohydrates is essen-
tial for survival during this period [32]. The in ovo inoculation of nutritional solutions at 18 d of incubation did not influence livability (Table 6). After birds were processed, we observed that
Table 4. Cumulative feed intake (g) of broilers derived from light and heavy eggs inoculated with nutritional solutions at 18 d of incubation Age, d Item Eggs Light Heavy Solution1 Maltose Vitamins Zinc Control Mix Glutamine P-value2 Egg Solution Egg × solution SEM 1
7
14
21
28
35
42
137 143
495 512
1,132 1,178
2,046 2,112
3,202 3,282
4,513 4,607
139 142 140 141 141 137
500 508 505 502 509 496
1,144 1,179 1,159 1,153 1,161 1,136
2,062 2,112 2,082 2,078 2,083 2,056
3,227 3,273 3,248 3,248 3,243 3,216
4,544 4,572 4,557 4,572 4,575 4,538
0.001 0.44 0.71 5.7
0.001 0.20 0.11 16.8
0.001 0.06 0.62 39.2
0.001 0.16 0.78 58.8
0.001 0.59 0.95 82.3
0.001 0.95 0.94 123.1
Treatment solutions: maltose = 0.5 mL of a 50% maltose syrup solution; vitamins = 0.5 mL of a multivitamin solution; zinc = 0.5 mL of a 0.5% zinc-glycine solution; control = 0.5 mL of a 0.4% saline solution; mix = 0.5 mL of a 50% maltose (12% dextrose, 42% maltose, 10% maltotriose, and 23 to 28% other sugars), 0.5% zinc-glycine, 10% glutamine, and multivitamin solution; glutamine = 0.5 mL of a 10% glutamine solution. 2 Significance level according to ANOVA.
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Eggs Light Heavy Solution1 Maltose Vitamins Zinc Control Mix Glutamine P-value2 Egg Solution Egg × solution SEM
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Table 5. Cumulative FCR, corrected for mortality, of broilers derived from light and heavy eggs inoculated with nutritional solutions at 18 d of incubation Age, d Item
14
21
28
35
42
1.109 1.087
1.341 1.343
1.428 1.450
1.552 1.573
1.615 1.625
1.717 1.720
1.103 1.101 1.091 1.102 1.089 1.101
1.332 1.358 1.331 1.340 1.342 1.348
1.425 1.461 1.424 1.436 1.446 1.441
1.540 1.576 1.555 1.565 1.580 1.560
1.612 1.633 1.616 1.618 1.630 1.611
1.716 1.724 1.717 1.716 1.726 1.713
0.02 0.90 0.50 0.036
0.80 0.45 0.56 0.047
0.02 0.13 0.41 0.036
0.02 0.11 0.80 0.034
0.04 0.40 0.49 0.021
0.47 0.58 0.61 0.018
1
Treatment solutions: maltose = 0.5 mL of a 50% maltose syrup solution; vitamins = 0.5 mL of a multivitamin solution; zinc = 0.5 mL of a 0.5% zinc-glycine solution; control = 0.5 mL of a 0.4% saline solution; mix = 0.5 mL of a 50% maltose (12% dextrose, 42% maltose, 10% maltotriose, and 23 to 28% other sugars), 0.5% zinc-glycine, 10% glutamine, and multivitamin solution; glutamine = 0.5 mL of a 10% glutamine solution. 2 Significance level according to ANOVA.
broilers derived from heavy eggs had higher carcass and breast meat weights as compared with those originating from light eggs (Table 7). No differences, however, were observed for any of the monitored yields (Table 7). The lack of differences in carcass trait yields helps explain that the observed differences in carcass and breast meat weights were associated solely with BW improvements. The inoculated nutrient solutions did not influence carcass trait absolute or relative weights. Vieira and Moran [15] found that heavier carcasses in broilers were derived from heavier eggs, but there were no differences in carcass and parts yields, which is consistent with findings from the present experiment. On the other hand, Foye et al. [10] observed higher breast weights at hatch in turkey poults injected with a protein solution at 23 d of incubation as compared with control birds. The injection of a carbohydrate solution in 18-d-old broiler embryos promoted higher breast meat yields at hatching and at 7 d of age [8]. Uni et al. [9] observed higher breast meat weights and yields at hatch and at 10 and 25 d of age in chicks that were supplied with an in ovo solution of carbohydrates and hydroxy-methylbutyrate at 18 d of incubation. Those results are different from the
ones observed in the present experiment. One of the possible reasons for the higher breast meat yield is the supply of nutrients before hatch, which helps alleviate any possible fasting effects between hatching and first feeding. Sklan et al. [14] mentioned that posthatching skeletal muscle growth is determined by muscle fiber hypertrophy and accumulation. Halevy et al. [6] reported lower BW gain and breast meat yield in 41-d-old broilers when these birds were fasted for 24 h after hatch as compared with chicks fed immediately after hatch. Reduction in BW gain and muscle hypertrophy are caused by changes in satellite cells, leading to subsequent changes in their hyperplasia and a delay in muscle maturation [6]. Mozdziak et al. [33] observed a lower muscle fiber diameter and a higher number of apoptotic cells in 7-d-old broilers fasted for 3 d as compared with fed birds. Absolute and relative embryo weights at 18 d of incubation are presented in Table 8. Embryos from heavy-weight eggs were heavier than those from light eggs, but there were no differences in embryo weight relative to egg weight, suggesting that all embryos presented a similar developmental stage. When eggs were submitted to different storage times, Christensen et al. [32]
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Eggs Light Heavy Solution1 Maltose Vitamins Zinc Control Mix Glutamine P-value2 Egg Solution Egg × solution SEM
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Table 6. Cumulative livability (%) of broilers derived from light and heavy eggs inoculated with nutritional solutions at 18 d of incubation Age, d Item
14
21
28
35
42
95.00 97.58
94.46 96.88
94.20 95.91
94.20 95.51
94.11 95.37
93.74 94.55
95.75 94.75 97.00 97.25 98.00 95.00
95.48 93.93 95.69 96.72 97.49 94.46
95.48 93.12 95.43 95.95 96.69 93.65
95.21 93.12 95.16 95.40 96.69 93.65
95.21 92.85 95.16 95.14 96.69 93.36
94.11 92.58 94.32 94.60 96.16 93.09
0.02 0.25 0.58 3.92
0.09 0.29 0.48 3.93
0.17 0.35 0.44 3.85
0.21 0.26 0.55 3.84
0.43 0.41 0.60 3.82
0.007 0.24 0.82 3.72
1
Treatment solutions: maltose = 0.5 mL of a 50% maltose syrup solution; vitamins = 0.5 mL of a multivitamin solution; zinc = 0.5 mL of a 0.5% zinc-glycine solution; control = 0.5 mL of a 0.4% saline solution; mix = 0.5 mL of a 50% maltose (12% dextrose, 42% maltose, 10% maltotriose, and 23 to 28% other sugars), 0.5% zinc-glycine, 10% glutamine, and multivitamin solution; glutamine = 0.5 mL of a 10% glutamine solution. 2 Significance level according to ANOVA.
observed that somatic growth was slower as the storage period increased, and this did seem to occur in embryos derived from eggs with different weights. Embryos from heavy eggs had higher yolk sac and intestinal weights at 18 d of incu-
bation as compared with those from light eggs; however, relative viscera weight was not affected. Liver absolute and relative weights at 18 d of incubation were not influenced by egg weight. Yolk sac, liver, and intestinal absolute and rela-
Table 7. Carcass traits (weight and % of live BW) of broilers derived from light and heavy eggs inoculated with nutritional solutions at 18 d of incubation Item Eggs Light Heavy Solution1 Maltose Vitamins Zinc Control Mix Glutamine P-value2 Egg Solution Egg × solution SEM 1
Carcass, g
Carcass, %
Legs, g
Legs, %
Breast, g
Breast, %
2,127 2,167
80.7 80.7
646 653
24.5 24.4
680 695
25.8 25.9
2,143 2,138 2,155 2,167 2,149 2,114
80.9 80.8 80.7 81.0 80.9 79.9
648 650 651 657 655 639
24.4 24.5 24.4 24.6 24.7 24.2
686 684 696 699 683 678
25.9 25.8 26.1 26.1 25.7 25.6
0.04 0.59 0.50 70.0
0.87 0.16 0.29 1.06
0.25 0.64 0.41 24.0
0.44 0.59 0.65 0.65
0.04 0.43 0.44 30.4
0.54 0.57 0.28 0.74
Treatment solutions: maltose = 0.5 mL of a 50% maltose syrup solution; vitamins = 0.5 mL of a multivitamin solution; zinc = 0.5 mL of a 0.5% zinc-glycine solution; control = 0.5 mL of a 0.4% saline solution; mix = 0.5 mL of a 50% maltose (12% dextrose, 42% maltose, 10% maltotriose, and 23 to 28% other sugars), 0.5% zinc-glycine, 10% glutamine, and multivitamin solution; glutamine = 0.5 mL of a 10% glutamine solution. 2 Significance level according to ANOVA.
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Eggs Light Heavy Solution1 Maltose Vitamins Zinc Control Mix Glutamine P-value2 Egg Solution Egg × solution SEM
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Table 8. Absolute and relative embryo weight (% of egg weight), and absolute and relative weights (% of BW) of yolk sac, intestine, and liver of 18-d-old embryos derived from light and heavy eggs Item
Embryo, g
Weight, %
Yolk, g
Yolk, %
Liver, g
Liver, %
Intestine, g
Intestine, %
40.8 45.9 0.001 3.59
83.4 84.0 0.33 1.42
8.5 10.2 0.009 1.50
20.9 22.3 0.26 2.60
0.6 0.7 0.23 0.11
1.45 1.42 0.77 0.25
2.6 2.9 0.04 0.37
6.35 6.37 0.95 0.81
Eggs Light Heavy P-value1 SEM 1
Significance level according to ANOVA.
explain the higher yolk weight and yolk percentage in birds inoculated with MALT. Light eggs inoculated with GLU displayed intermediate relative yolk weights. The gastrointestinal tract undergoes rapid development during the final period of incubation, and its size increases proportionally at a faster rate than the rest of the body. Uni et al. [2] observed an increase in intestinal weight from 1% of the embryo weight at 18 d of incubation to 3.5% at hatch. In the present study, this increase in gastrointestinal weight relative to BW was also observed (Table 11). However, these values are different from those of Uni et al. [2] because, in the present study, the intestinal tract was weighed along with the gizzard and the proventriculus, whereas in the study by Uni et al., only the small and the large intes-
Table 9. Absolute and relative weight (% of BW) of yolk sac, intestine, and liver of 0-d-old chicks derived from light and heavy eggs, and inoculated with nutritional solutions at 18 d of incubation Item Eggs Light Heavy Solution1 Maltose Vitamins Zinc Control Mix Glutamine P-value2 Egg Solution Egg × solution SEM 1
Yolk, g
Yolk, %
Liver, g
Liver, %
Intestine, g
Intestine, %
2.3 2.6
6.58 6.82
1.04 1.13
2.94 2.93
4.9 5.2
13.89 13.50
3.0 2.4 2.5 2.1 2.5 2.5
7.82 6.64 6.59 5.89 6.38 6.90
1.12 1.05 1.13 1.05 1.10 1.06
3.00 2.93 3.00 2.91 2.85 2.92
5.2 5.0 5.2 5.0 5.2 4.8
13.79 13.94 13.69 14.02 13.55 13.19
0.15 0.38 0.04 0.87
0.64 0.42 0.04 2.12
0.001 0.23 0.67 0.11
0.88 0.73 0.37 0.24
0.04 0.52 0.75 0.52
0.26 0.75 0.72 1.26
Treatment solutions: maltose = 0.5 mL of a 50% maltose syrup solution; vitamins = 0.5 mL of a multivitamin solution; zinc = 0.5 mL of a 0.5% zinc-glycine solution; control = 0.5 mL of a 0.4% saline solution; mix = 0.5 mL of a 50% maltose (12% dextrose, 42% maltose, 10% maltotriose, and 23 to 28% other sugars), 0.5% zinc-glycine, 10% glutamine, and multivitamin solution; glutamine = 0.5 mL of a 10% glutamine solution. 2 Significance level according to ANOVA.
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tive weights are displayed in Table 9. At hatch, chicks derived from heavy eggs had heavier livers and intestines, but their relative weights were similar to those derived from chicks from light eggs. An interaction was observed between egg weight and nutritional solutions (Table 10) for yolk sac absolute and relative weights, whereas liver and intestine weights were not influenced by the inoculation of nutritional solutions. The inoculation of nutritional solutions did not influence the yolk absolute or relative weight of eggs regardless of size, with the exception of heavy eggs inoculated with MALT. These eggs had increased absolute and relative yolk weights as compared with those inoculated with all other solutions. It is likely that the supplementation of energy as carbohydrates helps reduce the need to use yolk content as an energy source, and may
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Table 10. Effects between egg weight and inoculation of nutritional solutions at 18 d of incubation on total yolk and relative yolk weight (%) of 0-d-old chicks Total yolk weight, g Item
Light eggs
Heavy eggs
Light eggs
Heavy eggs
2.06b 2.56b 2.22b 2.06b 2.68b 2.48b
3.86a 2.28b 2.76b 2.20b 2.24b 2.56b
5.84b 7.14b 6.17b 6.02b 7.08b 7.24ab
9.80a 6.14b 7.02b 5.76b 5.68b 6.55b
0.04 0.36
0.04 0.90
a,b
Values within comparisons with different superscripts differ significantly (P < 0.05). Treatment solutions: maltose = 0.5 mL of a 50% maltose syrup solution; vitamins = 0.5 mL of a multivitamin solution; zinc = 0.5 mL of a 0.5% zinc-glycine solution; control = 0.5 mL of a 0.4% saline solution; mix = 0.5 mL of a 50% maltose (12% dextrose, 42% maltose, 10% maltotriose, and 23 to 28% other sugars), 0.5% zinc-glycine, 10% glutamine, and multivitamin solution; glutamine = 0.5 mL of a 10% glutamine solution. 2 Significance level according to ANOVA. 1
tines were collectively weighed. Heavier eggs present higher protein content in the yolk and albumen [16]. Eggs from older breeders present higher yolk percentages, and the difference in lipid transference from the yolk at the end of incubation may influence intestinal maturation [26]. The higher development of turkey poults derived from older breeders is due to the higher
phospholipid content in the yolk [26], which is consistent with the findings of Vieira and Moran [27], who found lower phosphorus content in the yolk sac of chicks derived from young breeders as compared with those from old breeders. At 7 d of age, chicks from heavy eggs had higher liver weights (Table 11). However, liver weight relative to BW was not different. Ab-
Table 11. Absolute and relative weight (% of BW) of liver and intestines of 7-d-old chicks derived from light and heavy eggs, and inoculated with nutritional solutions at 18 d of incubation Item Eggs Light Heavy Solution1 Maltose Vitamins Zinc Control Mix Glutamine P-value2 Egg Solution Egg × solution SEM a–c
Liver, g
Liver, %
Intestine, g
6.2 6.6
3.74 3.76
30.4 31.7
18.42 18.06
6.5 6.5 6.8 6.0 6.1 6.4
3.81 3.82 3.92 3.65 3.63 3.69
32.6 29.6 29.4 30.0 31.5 33.1
19.19a 17.25bc 17.02c 18.20abc 18.75ab 19.03a
0.03 0.17 0.31 0.8
0.85 0.61 0.99 0.41
0.15 0.07 0.12 3.8
Intestine, %
0.23 0.001 0.49 1.43
Values within a column with different superscripts differ significantly (P < 0.05). Treatment solutions: maltose = 0.5 mL of a 50% maltose syrup solution; vitamins = 0.5 mL of a multivitamin solution; zinc = 0.5 mL of a 0.5% zinc-glycine solution; control = 0.5 mL of a 0.4% saline solution; mix = 0.5 mL of a 50% maltose (12% dextrose, 42% maltose, 10% maltotriose, and 23 to 28% other sugars), 0.5% zinc-glycine, 10% glutamine, and multivitamin solution; glutamine = 0.5 mL of a 10% glutamine solution. 2 Significance level according to ANOVA. 11
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Solution1 Maltose Vitamins Zinc Control Mix Glutamine P-value2 SEM
Relative yolk weight, %
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Table 12. Absolute and relative weight (% of BW) of liver and intestines of 21-d-old chicks derived from light and heavy eggs, and inoculated with nutritional solutions at 18 d of incubation Item
Liver, %
Intestine, g
Intestine, %
23.2 23.5
2.80 2.79
100.5 102.8
12.16 12.29
22.0 24.0 23.5 23.1 23.3 23.9
2.64 2.89 2.76 2.77 2.89 2.81
104.7 102.9 108.4 99.2 97.6 97.2
12.57 12.30 12.72 12.13 12.16 11.46
0.73 0.81 0.81 3.3
0.84 0.31 0.25 0.27
0.52 0.39 0.25 13.5
0.76 0.56 0.22 1.51
1
Treatment solutions: maltose = 0.5 mL of a 50% maltose syrup solution; vitamins = 0.5 mL of a multivitamin solution; zinc = 0.5 mL of a 0.5% zinc-glycine solution; control = 0.5 mL of a 0.4% saline solution; mix = 0.5 mL of a 50% maltose (12% dextrose, 42% maltose, 10% maltotriose, and 23 to 28% other sugars), 0.5% zinc-glycine, 10% glutamine, and multivitamin solution; glutamine = 0.5 mL of a 10% glutamine solution. 2 Significance level according to ANOVA.
solute and relative intestinal weights were not influenced by egg weight. Nutritional solutions did affect absolute (P = 0.07) and relative intestinal weights at 7 d of age. Despite the lack of difference relative to the control, the in ovo inoculation with MALT, GLU, and MIX increased relative intestinal weight as compared with ZG. At 21 d of age, there was no influence of egg weight or inoculated solutions on absolute and relative liver and intestinal weights (Table 12). Chicks derived from heavier eggs have heavier livers because liver metabolism at hatch is highly active in absorbing yolk lipids by endocytosis, remodeling them into lipoproteins, and then releasing them into the blood circulation by exocytosis; therefore, the liver weight reflects the higher yolk weight [14]. This may explain the higher liver weight observed in chicks from heavy eggs at 0 and 7 d of age in the present experiment because these chicks presented higher yolk weights at 18 d of incubation. The administration of zinc-methionine in the amniotic fluid of the embryo was previously shown to enhance intestinal development [34]. In addition, it has been suggested that in ovo inoculation of nutrients improves gastrointestinal traits such as enzyme activity, protein expression, and intestinal development [7, 8, 13, 34]. Uni et al. [35] observed that vitamin A deficiency interferes with
intestinal cell proliferation and maturation in broilers. However, in the present experiment, no influence of vitamin inoculation was observed on intestinal tract weight, possibly because of the correct supply of these vitamins.
CONCLUSIONS AND APPLICATIONS Under the conditions of the present experiment, it is possible to conclude that
1. Heavy eggs resulted in higher 42-d BW compared with those derived from light eggs. 2. The use of MALT may have resulted in partial egg yolk energy sparing but this was also dependent on egg size. 3. The choice of products or compounds to be inoculated in ovo warrants further study, but the use of a readily available energy source has shown some promise. 4. In ovo feeding was associated with no effect on live performance, breast yield, or carcass yield.
REFERENCES AND NOTES 1. Yegani, M., and D. R. Korver. 2008. Factor affecting intestinal health in poultry. Poult. Sci. 87:2052–2063. 2. Uni, Z., E. Tako, O. Gal-Garber, and D. Sklan. 2003. Morphological, molecular, and functional changes in the
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Eggs Light Heavy Solution1 Maltose Vitamins Zinc Control Mix Glutamine P-value2 Egg Solution Egg × solution SEM
Liver, g
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breeders’ embryos mortality submitted to glucose in ovo inoculation. Braz. J. Avian Sci. Camp. 8:44. 21. Pedroso, A. A., L. S. Chaves, M. B. Café, N. S. M. Leandro, J. H. Stringhini, and J. F. M. Menten. 2006. Glutamine as broilers embryos nutrient. Braz. J. Avian Sci. Camp. 8:43. 22. Gore, A. B., and M. A. Qureshi. 1997. Enhancement of humoral and cellular immunity by vitamin E after embryonic exposure. Poult. Sci. 76:984–991. 23. Ohta, Y., N. Tsushima, K. Koide, M. T. Kidd, and T. Ishibashi. 1999. Effect of amino acid injection in broiler breeder eggs on embryonic growth and hatchability of chicks. Poult. Sci. 78:1493–1498. 24. Ohta, Y., and M. T. Kidd. 2001. Optimum site for in ovo amino acids injection in broiler breeders eggs. Poult. Sci. 80:1425–1429. 25. Jochemsen, P., and S. H. M. Jeurissen. 2002. The location and uptake of in ovo injected soluble and particulate substances in the chicken. Poult. Sci. 81:1811–1817. 26. Applegate, T. J., and M. S. Lilburn. 1999. Effect of turkey (Meleagridis gallopavo) breeder hen age and egg size on poult development. 1. Intestinal growth and glucose tolerance of the turkey poult. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 124:371–380. 27. Vieira, S. L., and E. T. Moran Jr. 1998. Eggs and chicks from broiler breeders of extremely different age. J. Appl. Poult. Res. 7:372–376. 28. Vieira, S. L., and E. T. Moran Jr. 1998. Broiler yields using chicks from extremes in breeder age and dietary propionate. J. Appl. Poult. Res. 7:320–327. 29. Leitão, R. A., N. S. M. Leandro, A. A. Pedroso, J. H. Stringhini, J. R. Oliveira Neto, and M. B. Café. 2006. Effect of glucose in ovo supplementation at starter performance of broilers. Braz. J. Avian Sci. Camp. 7:69. 30. Lopes, K. L. A. M., A. A. Pedroso, N. S. M. Leandro, J. H. Stringhini, and C. E. Barbosa. 2006. Glutamine in ovo inoculation effect at the starter performance of broilers. Braz. J. Avian Sci. Camp. 8:103. 31. Pedroso, A. A., L. S. Chaves, K. L. A. M. Lopes, N. S. M. Leandro, M. B. Café, and J. H. Stringhini. 2006. Nutrient inoculation in eggs from heavy breeders. Braz. J. Anim. Sci. 35:2018–2026. 32. Christensen, V. L., M. J. Wineland, G. M. Fasenko, and W. E. Donaldson. 2001. Egg storage effects on plasma glucose and supply and demand tissue glycogen concentrations of broiler embryos. Poult. Sci. 80:1729–1735. 33. Mozdziak, P. E., J. J. Evans, and D. W. McCoy. 2002. Early posthatch starvation induces myonuclear apoptosis in chickens. J. Nutr. 132:901–903. 34. Tako, E., P. R. Ferket, and Z. Uni. 2005. Changes in chicken intestinal zinc exporter mRNA expression and small intestinal functionality following antra-amniotic zinc-methionine administration. J. Nutr. Biochem. 16:339–346. 35. Uni, Z., G. Zaiger, O. Gal-Garber, M. Pines, I. Rozenboim, and R. Reifen. 2000. Vitamin A deficiency interferes with proliferation and maturation of cells in the chicken small intestine. Br. Poult. Sci. 41:410–415.
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chicken small intestine of the late-term embryo. Poult. Sci. 82:1747–1754. 3. Noy, Y., and D. Sklan. 2001. Yolk and exogenous feed utilization in the posthatch chick. Poult. Sci. 80:1490– 1495. 4. Dibner, J. J., C. D. Knight, M. L. Kitchell, C. A. Atwell, A. C. Downs, and F. J. Ivey. 1998. Early feeding and development of the immune system in neonatal poultry. J. Appl. Poult. Res. 7:425–436. 5. Careghi, C., K. Tona, J. Buyse, E. Decuypere, and V. Bruggeman. 2005. The effects of the spread of hatch and interaction in delayed feed access after hatch on broiler performance until seven days of age. Poult. Sci. 84:1314–1320. 6. Halevy, O., A. Geyra, M. Barak, Z. Uni, and D. Sklan. 2000. Early posthatch starvation decrease satellite cell proliferation and skeletal muscle growth in chicks. J. Nutr. 130:858–864. 7. Tako, E., P. R. Ferket, and Z. Uni. 2004. Effects of in ovo feeding of carbohydrates and β-hydroxy-βmethylbutyrate on the development of chicken intestine. Poult. Sci. 83:2023–2028. 8. Uni, Z., and P. R. Ferket. 2004. Methods for early nutrition and their potential. World’s Poult. Sci. J. 60:103– 113. 9. Uni, Z., P. R. Ferket, E. Tako, and O. Kedar. 2005. In ovo feeding improves energy status of late-term chicken embryos. Poult. Sci. 84:764–770. 10. Foye, O. T., Z. Uni, and P. R. Ferket. 2006. Effect of in ovo feeding egg white protein, β-hydroxy-β-methylbutyrate, and carbohydrates on glycogen status and neonatal growth of turkeys. Poult. Sci. 85:1185–1192. 11. Ohta, Y., M. T. Kidd, and T. Ishibashi. 2001. Embryo growth and amino acid concentration profiles of broiler breeder eggs, embryos, and chicks after in ovo administration of amino acids. Poult. Sci. 80:1430–1436. 12. Al-Murrani, W. K. 1982. Effect of injecting amino acids into the egg on embryonic and subsequent growth in the domestic fowl. Br. Poult. Sci. 23:171–174. 13. Smirnov, A., E. Tako, P. R. Ferket, and Z. Uni. 2006. Mucin gene expression and mucin content in the chicken intestinal goblet cells are affected by in ovo feeding of carbohydrates. Poult. Sci. 85:669–673. 14. Sklan, D., S. Heifetz, and O. Halevy. 2003. Heavier chicks at hatch improves marketing body weight by enhancing skeletal muscular growth. Poult. Sci. 82:1778–1786. 15. Vieira, S. L., and E. T. Moran Jr. 1998. Broiler yields using chicks from egg weight extremes and diverse strains. J. Appl. Poult. Res. 7:339–346. 16. Vieira, S. L., and E. T. Moran Jr. 1998. Broiler chicks hatched from egg weight extremes and diverse breeder strains. J. Appl. Poult. Res. 7:392–402. 17. Injection system, 96-head model, Inovoject do Brasil, Chapecó, Santa Catarina, Brazil. 18. Guapiaçu, São Paulo, Brazil. 19. Universidade Federal de Viçosa. 1995. Sistema de anpalises estatísticas e genetic—SAEG. Univ. Fed. Viçosa, Viçosa, Minas Gerais, Brazil. 20. Pedroso, A. A., V. T. Barbosa, M. B. Café, N. S. M. Leandro, J. H. Stringhini, and J. F. M. Menten. 2006. Broiler
JAPR: Research Report
Influence of in ovo inoculation with various nutrients and egg size on broiler performance T. T. dos Santos,* A. Corzo,† M. T. Kidd,† C. D. McDaniel,† R. A. Torres Filho,* and L. F. Araújo‡1 *Globoaves Agro Avícola Ltda, Chapecó, Brazil 89.815-280; †Department of Poultry Science, Mississippi State University, Mississippi State 39762; ‡Faculty of Animal Science, University of São Paulo, Pirassununga, Brazil 13.635-900
SUMMARY This study evaluated the influence of inoculation of different nutrients into eggs of 18-d-old broiler embryos. On d 18 of incubation, before transferring, eggs were injected with a solution containing either maltose, a multivitamin supplement, zinc-glycine, glutamine, a mixture containing all these elements, or sodium chloride (control). After hatching, 2,400 male broiler chicks were placed in an experimental broiler house and distributed into 60 floor pens in a completely randomized design comprising a 2 × 6 factorial arrangement of treatments (2 egg weights × 6 solutions), for a total of 12 treatments. Birds and feed consumption were measured weekly. At 42 d of age, 3 birds per replicate pen were randomly selected for processing. Birds derived from heavier eggs had greater hatchability and hatching weight. At 42 d of age, birds from heavier eggs had higher BW, carcass weight, and breast meat weight. The livability of birds derived from heavy eggs was higher at 7 and 14 d of age. The in ovo inoculation of the nutrients to 18-d-old embryos did not influence live performance or carcass traits. The technique of in ovo inoculation of certain nutrients may be used in industrial poultry production, but further studies are required to define the best solutions or mixture of nutrients to be used. Key words: in ovo nutrition, broiler, maltose, glutamine, zinc 2010 J. Appl. Poult. Res. 19:1–12 doi:10.3382/japr.2009-00038
DESCRIPTION OF PROBLEM Early intake after hatch of exogenous feed is accompanied by rapid development of the gastrointestinal tract and associated organs [1]. During the last day of incubation, the fastest developing organ is the intestinal tract, representing 1% of the weight of the 18-d embryo and reaching 3.5% at hatch [2]. Hatching drastically changes the way chicks retrieve nutrients. During incubation, energy 1
Corresponding author: [email protected]
and nutrients are provided via the yolk, which is rich in lipids but has relatively low protein and carbohydrate concentrations. In the embryo, yolk lipids are directly transported to the blood by endocytosis [3], but after hatching, yolk content is absorbed through both the yolk sack membrane and Meckel’s diverticulum, and is digested and absorbed by the intestinal tract. Noy and Sklan [3] observed that the passage of the yolk content to the intestinal tract is higher when the bird is fed as compared with when it is fast-
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Primary Audience: Nutritionists, Veterinarians
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nal development. Uni and Ferket [8] inoculated the same carbohydrate cocktail and observed increases in liver glucose at hatch and BW at hatch that continued up to 35 d of age. Uni et al. [9] observed that the inoculation of β-hydroxy β-methylbutyrate and carbohydrates improved BW at hatch, improved breast yield at hatch and at 10 and 25 d of age, and increased liver glycogen content in chicks. According to these authors, the supplied energy reduced muscle protein degradation destined to serve as the energy source at hatch, resulting in higher BW and breast percentage at hatch. Other factors, such as broiler breeder genetic line, breeder age, and egg size, may affect initial development and therefore final performance. As the breeder flock ages, egg weight tends to increase [14], and increasing egg weight and breeder age can affect hatchling performance. Vieira and Moran [15] observed that heavier eggs produced larger chicks and that this difference was maintained until market age. Heavier eggs also contained higher protein content in the yolk and albumen [16]. The objective of this study was to observe any effect in the use of various nutrient solutions inoculated at 18 d of age, via in ovo administration, in broiler eggs of 2 different weights.
MATERIALS AND METHODS This study used a completely randomized design comprising a 2 × 3 factorial arrangement of treatments. Factors involved were egg weight and in ovo solutions. Eggs were sorted by weight into either light or heavy eggs. At d 18 of incubation, eggs were injected in ovo with one of various nutrient solutions. Solutions were delivered using a saline solution (stock solution). These treatment solutions were as follows: 0.5 mL of a 50% maltose syrup solution (MALT); 0.5 mL of a multivitamin solution (VIT); 0.5 mL of a 0.5% zinc-glycine solution (ZG); 0.5 mL of a 10% glutamine solution (GLU); 0.5 mL of a 50% maltose (12% dextrose, 42% maltose, 10% maltotriose, and 23 to 28% other sugars), 0.5% zinc-glycine, 10% glutamine, and multivitamin solution (MIX); and a control group containing 0.5 mL of 0.4% saline solution. The composition of the multivitamin solution used in the VIT and MIX diets is shown in Table 1.
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ed. According to these authors, this difference may be due to the physical presence of feed, the peristaltic movements of the intestine, the negative pressure in the abdominal cavity, or their combination, thereby stimulating passage of the intestinal content. The yolk sac energy reserves present at hatch may not be sufficient to supply their maintenance energy requirements [4], and fasting effects may occur before the flock is removed from the hatchery. However, despite being capable of ingesting feed, the intestinal tract of the chick is still immature [2]. Weight loss between hatching and removal from the hatcher is approximately 0.18 g/h while fasting [5]. Halevy et al. [6] observed lower BW and breast meat yield values at 41 d when broilers were fasted for 24 h after hatching when compared with those immediately fed, likely caused by a change in satellite cell activity, leading to subsequent changes in hyperplasia and an associated delay in muscle maturation. Several alternatives have been proposed to improve performance during this initial phase, such as feeding at the hatchery [4, 5] and in ovo feeding [7–10]. However, despite having shown promising results in formal experimentation, these techniques are rarely put into practice by broiler companies for complex logistical reasons. Concerning in ovo nutrient inoculation, Ohta et al. [11] suggested that the increase in hatching weight of 7-d-old embryos injected with amino acids may have been due to a higher content of amino acids in the yolk or to better utilization of amino acids by the embryo. In geese, AlMurrani [12] observed that in ovo injection of amino acids at 7 d of incubation increased BW at hatch, and the BW difference was maintained up to 52 d of age when compared with noninoculated embryos. Foye et al. [10] observed higher BW and breast weight of 1-d-old turkeys when these were inoculated at 23 d of incubation with egg-derived protein. The inoculation with carbohydrates (maltose, sucrose, and dextrose) in the amniotic fluid of broilers between 17 and 18 d of incubation was studied [13]. These authors observed an increase in jejunal villi surface area from hatching up to 3 d of age. As the embryo ingests the amniotic fluid at the end of incubation, the inoculation of carbohydrates would be an extra source of nutrients, stimulating intesti-
JAPR: Research Report
dos Santos et al.: IN OVO NUTRITION
virus. These embryos were subsequently killed with carbon dioxide and weighed. Yolk sac, intestinal tract (between the proventriculus and the rectum), and liver were dissected and weighed, and their percentages relative to embryo weight were calculated. At 21 d of incubation, hatched chicks were removed from the hatcher and weighed to determine BW loss at hatch. Nonhatched eggs were opened to determine the percentage of infertile eggs and embryo age at death. The calculation of mortality postinoculation considered only pipped eggs and mortality after 18 d of incubation. Chicks were vaccinated against infectious bronchitis approximately 12 h before transport to the experimental house. The day-old male chicks (3,360) were selected and housed in a 50 × 12 m experimental broiler house, divided into 60 pens (3.12 m2/pen), covered with new litter (softwood shavings). The treatments (2 egg weights × 6 solutions) were each replicated 5 times, with each replicate consisting of 40 birds, for a total of 60 experimental units. Birds were offered feed and water ad libitum. All birds received common feeds (Table 1) from placement until the end of the experiment. Birds were weighed weekly until 42 d of age. Body weight gain, feed intake, and livability were measured,
Table 1. Composition and nutritional levels of experimental diets (%, as is) Item Ingredient Corn, % Soybean meal, % Soybean oil, % Sodium chloride, % Limestone, % Dicalcium phosphate, % Choline chloride, % dl-Methionine, % Vitamin-mineral premix,1 % Calculated composition CP, % AME, kcal/kg Calcium, % Available phosphorus, % Sodium, % Lysine, % TSAA, % 1
Prestarter, 1 to 7 d
Starter, 7 to 21 d
Grower, 21 to 35 d
Finisher, 35 to 42 d
55.00 38.00 2.40 0.40 1.30 1.60 0.60 0.30 0.40
60.00 33.00 2.70 0.30 1.30 1.60 0.45 0.25 0.40
64.00 28.00 4.00 0.30 1.15 1.60 0.35 0.20 0.40
66.00 25.50 4.80 0.30 1.10 1.40 0.30 0.20 0.40
22.0 2,950 1.00 0.50 0.25 1.28 0.94
20.0 3,050 0.95 0.48 0.20 1.10 0.84
18.0 3,175 0.90 0.45 0.20 1.00 0.80
17.0 3,250 0.85 0.43 0.20 0.95 0.77
Multivitamin solution composition (per L): vitamin A, 3,000 IU; vitamin D3, 1,500 IU; vitamin E, 25 IU; vitamin K3, 1.50 mg; vitamin B1, 1.00 mg; vitamin B2, 2.50 mg; vitamin B6, 1.50 mg; vitamin B12, 6.00 mg; nicotinic acid, 20.00 mg; pantothenic acid, 5.00 mg; biotin, 75.00 μg; folic acid, 0.50 mg; vitamin C, 125.00 mg.
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Two liters of each solution was produced and used as a vaccine diluent in the in ovo injection solution [17]. Osmolality and pH of the solutions were not measured. Eggs from 6 trays per weight group were inoculated with each solution. The vaccination equipment automatically injected a dose of 0.5 mL into the amniotic fluid of each egg. Before each solution was applied, the equipment was cleaned by flushing air through the tubes, and the absence of solution was confirmed by injecting a test plate. This procedure was repeated between all solutions. Nutrients were injected in ovo in the following sequence: MALT, VIT, ZG, control, MIX, and GLU. This sequence was followed because of the lower solubility of GLU, particularly when diluted only in saline solution. A total of 9,600 eggs derived from 30-wkold Cobb 500 breeders [18] were separated into heavy or light eggs by using a scale (Table 2). Eggs were placed in 100 incubation trays with a capacity of 96 eggs each. Eggs were incubated in a single incubator up to 18 d. Before transferring to the hatcher, eggs were weighed again to monitor egg weight loss at transfer. At the same time, 5 embryos from each egg weight group were randomly removed for collection of blood from the cardiac artery to determine antibody levels against infectious bursal disease and reo-
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Table 2. Characteristics of broiler eggs injected with nutritional solutions at 18 d of incubation Item
Incubation weight, g 54.0 59.9
47.9 53.0
Δ Weight, %
Hatch, %
18-d livability, %
88.61 88.51
89.17 91.30
98.58 98.76
37.7 41.9
89.04 89.67 91.25 90.49 90.39 90.56
98.41 98.22 99.02 98.49 98.85 99.02
39.9 39.7 40.4 39.7 39.8 39.3
0.001
0.001
0.310
3.1
2.8
0.39
0.005 0.59 0.10 3.29
0.50 0.60 0.70 1.34
Hatch BW, g
0.001 0.23 0.70 1.9
1
Treatment solutions: maltose = 0.5 mL of a 50% maltose syrup solution; vitamins = 0.5 mL of a multivitamin solution; zinc = 0.5 mL of a 0.5% zinc-glycine solution; control = 0.5 mL of a 0.4% saline solution; mix = 0.5 mL of a 50% maltose (12% dextrose, 42% maltose, 10% maltotriose, and 23 to 28% other sugars), 0.5% zinc-glycine, 10% glutamine, and multivitamin solution; glutamine = 0.5 mL of a 10% glutamine solution. 2 Significance level according to ANOVA.
and feed conversion corrected for mortality was calculated based on performance data. At placement, 1 bird per replicate was randomly selected, killed with carbon dioxide, and weighed. The yolk sac, intestinal tract (from the beginning of the proventriculus until the end of the rectum), and liver were removed and weighed, and their percentages relative to embryo weight were calculated. This procedure was repeated when chicks were 7 and 21 d old. At 42 d of age, 3 birds per replicate pen were randomly removed and leg banded. The birds were weighed, submitted to electrical stunning, and killed by exsanguination. After the neck, feet, and offal were removed, hot carcasses were weighed to calculate carcass yield. Breasts (with bones and skin) and legs and thighs (with bones and skin) were cut and weighed to determine their yields. The collected data were analyzed by the SAEG statistical package as a 6 × 2 factorial arrangement [19]. Data were tested by 2-way interactions (P < 0.05), and in their absence, only main effects are shown. Experimentation was approved by the Universidade de São Paulo Institutional Animal Care and Use Committee.
RESULTS AND DISCUSSION The difference in egg weights after being classified immediately following lay was main-
tained at the time eggs were transferred to the hatcher and injected with the treatments (18 d of incubation), and also when eggs hatched (Table 2). However, weight loss percentage from the start of incubation until transferring to the hatcher was similar between the 2 groups. Solution inoculation did not affect weight at hatch (Table 2). The hatchability of heavy-weight eggs was higher as compared with light eggs (Table 2), but this was not caused by late embryo mortality (after 18 d of incubation) because livability in this period was similar for both weight categories. Inoculation with the treatments did not affect hatching and livability between d 18 and 21 of incubation. These results are different from those observed by Pedroso et al. [20], who reported greater embryo mortality when a glucose solution was injected into the amniotic fluid of 16-d-old embryos. According to those authors, this may have been caused by the high osmolarity of the inoculated solution, which was not measured in this trial. Conversely, there was no effect on hatchability of the inoculation of GLU in the amniotic fluid of 16-d-old embryos [21], which is consistent with the data obtained in the present experiment. Gore and Qureshi [22] observed lower hatchability when 25-d-old turkey embryos were inoculated with 20 and 30 IU of vitamin E. Ohta et al. [23] reported lower hatch-
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Eggs Light Heavy Solution1 Maltose Vitamins Zinc Control Mix Glutamine P-value2 Egg Solution Egg × solution SEM
18-d weight, g
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Lopes et al. [30] injected glutamine to 16-d-old embryos and did not find BW gain differences in 10- to 21-d-old broilers. The addition of 10 IU of vitamin E did not influence turkey and broiler BW at hatch and at 35 d of age [22]. On the other hand, the inoculation of carbohydrates, carbohydrates and proteins, hydroxy-methylbutyrate, or a mixture of these compounds increased chick BW at hatch and subsequent chick development [7–10]. According to these authors, inoculation with these substances enhanced intestinal development and enzyme expression at hatch, thereby allowing more efficient posthatch development. Moreover, the supply of these nutrients improved bird energy status, which spared energy used for metabolism and development [10]. In ovo inoculation with amino acids at 7 d of incubation in the yolk sac increased hatching weight as a percentage of egg weight [11, 23, 24]. Ohta et al. [11] reported that this increase in hatching weight might be due to an increase in yolk amino acid content or in amino acid utilization by the embryo. However, in geese, Al-Muranni [12] showed that in ovo inoculation of amino acids at 7 d of incubation increased hatching weight, and this difference was maintained up to 52 d of age. Cumulative feed intake and feed conversion are shown in Tables 4 and 5, respectively. Chicks from heavy eggs consumed more feed during the entire experimental period. This higher feed intake capacity may explain the higher BW of the broilers derived from heavier eggs as compared with those originating from lighter ones. Broilers that originated from heavy eggs had superior feed conversion at d 7 but poorer feed conversion at d 21, 28, and 35. At market age (d 42), these differences had dissipated. No nutrient solution inoculation effect was observed for feed intake or feed conversion in the present study. Pedroso et al. [31] did not observe any differences in feed intake or feed conversion of chicks inoculated with glucose or GLU at 16 d of incubation. In agreement with the present results, Vieira and Moran [15] reported no FCR differences between chicks derived from light or heavy eggs at market age. Chicks derived from heavy eggs had greater livability up to 14 d of age (Table 6). Turkeys derived from young breeders and small eggs were less capable of coping with the metabolic chal-
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ability when injecting amino acids at d 0 of incubation. However, when injection was carried out in the yolk of 7-d-old embryos, hatchability was not affected. In ovo injection must be made in the extraembryonic cavity or yolk sac to prevent a reduction in hatchability, as observed when amino acids were inoculated in the amniotic cavity or in the chorioallantoic membrane [24]. It must be noted that Ohta and Kidd [24] inoculated 7-d-old embryos, whereas in the present study, embryos were 18 d old. Jochemsen and Jeurissen [25] reported that age at inoculation may influence where the product is applied. In the present study, chicks derived from heavier eggs were heavier at hatch. This result is consistent with those observed by Vieira and Moran [15, 16] in broiler chicks and those observed by Applegate and Lilburn [26] in turkey poults. Inoculation of the different treatment solutions at 18 d of incubation did not influence hatch weight (Table 2). Body weight of chicks from heavier eggs was observed to be higher throughout the study (Table 3). There was no influence of the nutrient solutions applied via in ovo injection on BW other than at placement (Table 3). Note that BW on d 0 was lower than BW at hatch. This is probably due to fasting during the 24-h interval between hatching and placement, during which birds were removed from the hatcher, selected, vaccinated, sexed, and transported in temperature-controlled trucks to the experimental farm. Vieira and Moran [27] observed that younger broiler breeders produced lighter eggs and, consequently, lighter broilers at slaughter as compared with older breeders [28]. However, according to Sklan et al. [14], breeder age does not influence bird performance. When working with eggs of different weights derived from breeders of similar BW, Sklan et al. [14] observed that heavier eggs produced chicks with heavier hatching weights and heavier broilers at market age (41 d). This is consistent with the results obtained in the present experiment, in which eggs of different weights derived from breeders of the same age exhibited different performance, with heavier eggs producing heavier chicks and heavier broilers at market age. Leitão et al. [29] did not observe BW gain differences between 0 and 10 d in chicks supplemented with glucose at 16 d of incubation.
5
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Table 3. Body weight (g) of broilers derived from light and heavy eggs inoculated with nutritional solutions at 18 d of incubation Age, d Item
7
14
21
28
35
42
35.8 39.1
159 170
408 424
831 856
1,377 1,408
2,041 2,084
2,685 2,740
38.2a 37.9a 37.4ab 37.3ab 37.2ab 36.6b
165 166 165 165 166 160
417 415 420 415 420 408
844 847 853 843 842 830
1,401 1,402 1,400 1,392 1,379 1,381
2,063 2,064 2,070 2,071 2,048 2,059
2,710 2,711 2,709 2,723 2,708 2,712
0.001 0.001 0.003 2.9
0.001 0.12 0.14 7.4
0.001 0.25 0.25 14.4
0.001 0.24 0.90 23.5
0.002 0.44 0.86 35.5
0.001 0.85 0.74 50.6
0.001 0.99 0.93 70.2
a,b
Values within a column with different superscripts differ significantly (P < 0.05). Treatment solutions: maltose = 0.5 mL of a 50% maltose syrup solution; vitamins = 0.5 mL of a multivitamin solution; zinc = 0.5 mL of a 0.5% zinc-glycine solution; control = 0.5 mL of a 0.4% saline solution; mix = 0.5 mL of a 50% maltose (12% dextrose, 42% maltose, 10% maltotriose, and 23 to 28% other sugars), 0.5% zinc-glycine, 10% glutamine, and multivitamin solution; glutamine = 0.5 mL of a 10% glutamine solution. 2 Significance level according to ANOVA. 1
lenges that occur during the first days posthatch [26], and this may explain the higher mortality of this group of birds because the capacity to produce and metabolize carbohydrates is essen-
tial for survival during this period [32]. The in ovo inoculation of nutritional solutions at 18 d of incubation did not influence livability (Table 6). After birds were processed, we observed that
Table 4. Cumulative feed intake (g) of broilers derived from light and heavy eggs inoculated with nutritional solutions at 18 d of incubation Age, d Item Eggs Light Heavy Solution1 Maltose Vitamins Zinc Control Mix Glutamine P-value2 Egg Solution Egg × solution SEM 1
7
14
21
28
35
42
137 143
495 512
1,132 1,178
2,046 2,112
3,202 3,282
4,513 4,607
139 142 140 141 141 137
500 508 505 502 509 496
1,144 1,179 1,159 1,153 1,161 1,136
2,062 2,112 2,082 2,078 2,083 2,056
3,227 3,273 3,248 3,248 3,243 3,216
4,544 4,572 4,557 4,572 4,575 4,538
0.001 0.44 0.71 5.7
0.001 0.20 0.11 16.8
0.001 0.06 0.62 39.2
0.001 0.16 0.78 58.8
0.001 0.59 0.95 82.3
0.001 0.95 0.94 123.1
Treatment solutions: maltose = 0.5 mL of a 50% maltose syrup solution; vitamins = 0.5 mL of a multivitamin solution; zinc = 0.5 mL of a 0.5% zinc-glycine solution; control = 0.5 mL of a 0.4% saline solution; mix = 0.5 mL of a 50% maltose (12% dextrose, 42% maltose, 10% maltotriose, and 23 to 28% other sugars), 0.5% zinc-glycine, 10% glutamine, and multivitamin solution; glutamine = 0.5 mL of a 10% glutamine solution. 2 Significance level according to ANOVA.
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Eggs Light Heavy Solution1 Maltose Vitamins Zinc Control Mix Glutamine P-value2 Egg Solution Egg × solution SEM
0
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Table 5. Cumulative FCR, corrected for mortality, of broilers derived from light and heavy eggs inoculated with nutritional solutions at 18 d of incubation Age, d Item
14
21
28
35
42
1.109 1.087
1.341 1.343
1.428 1.450
1.552 1.573
1.615 1.625
1.717 1.720
1.103 1.101 1.091 1.102 1.089 1.101
1.332 1.358 1.331 1.340 1.342 1.348
1.425 1.461 1.424 1.436 1.446 1.441
1.540 1.576 1.555 1.565 1.580 1.560
1.612 1.633 1.616 1.618 1.630 1.611
1.716 1.724 1.717 1.716 1.726 1.713
0.02 0.90 0.50 0.036
0.80 0.45 0.56 0.047
0.02 0.13 0.41 0.036
0.02 0.11 0.80 0.034
0.04 0.40 0.49 0.021
0.47 0.58 0.61 0.018
1
Treatment solutions: maltose = 0.5 mL of a 50% maltose syrup solution; vitamins = 0.5 mL of a multivitamin solution; zinc = 0.5 mL of a 0.5% zinc-glycine solution; control = 0.5 mL of a 0.4% saline solution; mix = 0.5 mL of a 50% maltose (12% dextrose, 42% maltose, 10% maltotriose, and 23 to 28% other sugars), 0.5% zinc-glycine, 10% glutamine, and multivitamin solution; glutamine = 0.5 mL of a 10% glutamine solution. 2 Significance level according to ANOVA.
broilers derived from heavy eggs had higher carcass and breast meat weights as compared with those originating from light eggs (Table 7). No differences, however, were observed for any of the monitored yields (Table 7). The lack of differences in carcass trait yields helps explain that the observed differences in carcass and breast meat weights were associated solely with BW improvements. The inoculated nutrient solutions did not influence carcass trait absolute or relative weights. Vieira and Moran [15] found that heavier carcasses in broilers were derived from heavier eggs, but there were no differences in carcass and parts yields, which is consistent with findings from the present experiment. On the other hand, Foye et al. [10] observed higher breast weights at hatch in turkey poults injected with a protein solution at 23 d of incubation as compared with control birds. The injection of a carbohydrate solution in 18-d-old broiler embryos promoted higher breast meat yields at hatching and at 7 d of age [8]. Uni et al. [9] observed higher breast meat weights and yields at hatch and at 10 and 25 d of age in chicks that were supplied with an in ovo solution of carbohydrates and hydroxy-methylbutyrate at 18 d of incubation. Those results are different from the
ones observed in the present experiment. One of the possible reasons for the higher breast meat yield is the supply of nutrients before hatch, which helps alleviate any possible fasting effects between hatching and first feeding. Sklan et al. [14] mentioned that posthatching skeletal muscle growth is determined by muscle fiber hypertrophy and accumulation. Halevy et al. [6] reported lower BW gain and breast meat yield in 41-d-old broilers when these birds were fasted for 24 h after hatch as compared with chicks fed immediately after hatch. Reduction in BW gain and muscle hypertrophy are caused by changes in satellite cells, leading to subsequent changes in their hyperplasia and a delay in muscle maturation [6]. Mozdziak et al. [33] observed a lower muscle fiber diameter and a higher number of apoptotic cells in 7-d-old broilers fasted for 3 d as compared with fed birds. Absolute and relative embryo weights at 18 d of incubation are presented in Table 8. Embryos from heavy-weight eggs were heavier than those from light eggs, but there were no differences in embryo weight relative to egg weight, suggesting that all embryos presented a similar developmental stage. When eggs were submitted to different storage times, Christensen et al. [32]
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Eggs Light Heavy Solution1 Maltose Vitamins Zinc Control Mix Glutamine P-value2 Egg Solution Egg × solution SEM
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Table 6. Cumulative livability (%) of broilers derived from light and heavy eggs inoculated with nutritional solutions at 18 d of incubation Age, d Item
14
21
28
35
42
95.00 97.58
94.46 96.88
94.20 95.91
94.20 95.51
94.11 95.37
93.74 94.55
95.75 94.75 97.00 97.25 98.00 95.00
95.48 93.93 95.69 96.72 97.49 94.46
95.48 93.12 95.43 95.95 96.69 93.65
95.21 93.12 95.16 95.40 96.69 93.65
95.21 92.85 95.16 95.14 96.69 93.36
94.11 92.58 94.32 94.60 96.16 93.09
0.02 0.25 0.58 3.92
0.09 0.29 0.48 3.93
0.17 0.35 0.44 3.85
0.21 0.26 0.55 3.84
0.43 0.41 0.60 3.82
0.007 0.24 0.82 3.72
1
Treatment solutions: maltose = 0.5 mL of a 50% maltose syrup solution; vitamins = 0.5 mL of a multivitamin solution; zinc = 0.5 mL of a 0.5% zinc-glycine solution; control = 0.5 mL of a 0.4% saline solution; mix = 0.5 mL of a 50% maltose (12% dextrose, 42% maltose, 10% maltotriose, and 23 to 28% other sugars), 0.5% zinc-glycine, 10% glutamine, and multivitamin solution; glutamine = 0.5 mL of a 10% glutamine solution. 2 Significance level according to ANOVA.
observed that somatic growth was slower as the storage period increased, and this did seem to occur in embryos derived from eggs with different weights. Embryos from heavy eggs had higher yolk sac and intestinal weights at 18 d of incu-
bation as compared with those from light eggs; however, relative viscera weight was not affected. Liver absolute and relative weights at 18 d of incubation were not influenced by egg weight. Yolk sac, liver, and intestinal absolute and rela-
Table 7. Carcass traits (weight and % of live BW) of broilers derived from light and heavy eggs inoculated with nutritional solutions at 18 d of incubation Item Eggs Light Heavy Solution1 Maltose Vitamins Zinc Control Mix Glutamine P-value2 Egg Solution Egg × solution SEM 1
Carcass, g
Carcass, %
Legs, g
Legs, %
Breast, g
Breast, %
2,127 2,167
80.7 80.7
646 653
24.5 24.4
680 695
25.8 25.9
2,143 2,138 2,155 2,167 2,149 2,114
80.9 80.8 80.7 81.0 80.9 79.9
648 650 651 657 655 639
24.4 24.5 24.4 24.6 24.7 24.2
686 684 696 699 683 678
25.9 25.8 26.1 26.1 25.7 25.6
0.04 0.59 0.50 70.0
0.87 0.16 0.29 1.06
0.25 0.64 0.41 24.0
0.44 0.59 0.65 0.65
0.04 0.43 0.44 30.4
0.54 0.57 0.28 0.74
Treatment solutions: maltose = 0.5 mL of a 50% maltose syrup solution; vitamins = 0.5 mL of a multivitamin solution; zinc = 0.5 mL of a 0.5% zinc-glycine solution; control = 0.5 mL of a 0.4% saline solution; mix = 0.5 mL of a 50% maltose (12% dextrose, 42% maltose, 10% maltotriose, and 23 to 28% other sugars), 0.5% zinc-glycine, 10% glutamine, and multivitamin solution; glutamine = 0.5 mL of a 10% glutamine solution. 2 Significance level according to ANOVA.
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Eggs Light Heavy Solution1 Maltose Vitamins Zinc Control Mix Glutamine P-value2 Egg Solution Egg × solution SEM
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Table 8. Absolute and relative embryo weight (% of egg weight), and absolute and relative weights (% of BW) of yolk sac, intestine, and liver of 18-d-old embryos derived from light and heavy eggs Item
Embryo, g
Weight, %
Yolk, g
Yolk, %
Liver, g
Liver, %
Intestine, g
Intestine, %
40.8 45.9 0.001 3.59
83.4 84.0 0.33 1.42
8.5 10.2 0.009 1.50
20.9 22.3 0.26 2.60
0.6 0.7 0.23 0.11
1.45 1.42 0.77 0.25
2.6 2.9 0.04 0.37
6.35 6.37 0.95 0.81
Eggs Light Heavy P-value1 SEM 1
Significance level according to ANOVA.
explain the higher yolk weight and yolk percentage in birds inoculated with MALT. Light eggs inoculated with GLU displayed intermediate relative yolk weights. The gastrointestinal tract undergoes rapid development during the final period of incubation, and its size increases proportionally at a faster rate than the rest of the body. Uni et al. [2] observed an increase in intestinal weight from 1% of the embryo weight at 18 d of incubation to 3.5% at hatch. In the present study, this increase in gastrointestinal weight relative to BW was also observed (Table 11). However, these values are different from those of Uni et al. [2] because, in the present study, the intestinal tract was weighed along with the gizzard and the proventriculus, whereas in the study by Uni et al., only the small and the large intes-
Table 9. Absolute and relative weight (% of BW) of yolk sac, intestine, and liver of 0-d-old chicks derived from light and heavy eggs, and inoculated with nutritional solutions at 18 d of incubation Item Eggs Light Heavy Solution1 Maltose Vitamins Zinc Control Mix Glutamine P-value2 Egg Solution Egg × solution SEM 1
Yolk, g
Yolk, %
Liver, g
Liver, %
Intestine, g
Intestine, %
2.3 2.6
6.58 6.82
1.04 1.13
2.94 2.93
4.9 5.2
13.89 13.50
3.0 2.4 2.5 2.1 2.5 2.5
7.82 6.64 6.59 5.89 6.38 6.90
1.12 1.05 1.13 1.05 1.10 1.06
3.00 2.93 3.00 2.91 2.85 2.92
5.2 5.0 5.2 5.0 5.2 4.8
13.79 13.94 13.69 14.02 13.55 13.19
0.15 0.38 0.04 0.87
0.64 0.42 0.04 2.12
0.001 0.23 0.67 0.11
0.88 0.73 0.37 0.24
0.04 0.52 0.75 0.52
0.26 0.75 0.72 1.26
Treatment solutions: maltose = 0.5 mL of a 50% maltose syrup solution; vitamins = 0.5 mL of a multivitamin solution; zinc = 0.5 mL of a 0.5% zinc-glycine solution; control = 0.5 mL of a 0.4% saline solution; mix = 0.5 mL of a 50% maltose (12% dextrose, 42% maltose, 10% maltotriose, and 23 to 28% other sugars), 0.5% zinc-glycine, 10% glutamine, and multivitamin solution; glutamine = 0.5 mL of a 10% glutamine solution. 2 Significance level according to ANOVA.
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tive weights are displayed in Table 9. At hatch, chicks derived from heavy eggs had heavier livers and intestines, but their relative weights were similar to those derived from chicks from light eggs. An interaction was observed between egg weight and nutritional solutions (Table 10) for yolk sac absolute and relative weights, whereas liver and intestine weights were not influenced by the inoculation of nutritional solutions. The inoculation of nutritional solutions did not influence the yolk absolute or relative weight of eggs regardless of size, with the exception of heavy eggs inoculated with MALT. These eggs had increased absolute and relative yolk weights as compared with those inoculated with all other solutions. It is likely that the supplementation of energy as carbohydrates helps reduce the need to use yolk content as an energy source, and may
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Table 10. Effects between egg weight and inoculation of nutritional solutions at 18 d of incubation on total yolk and relative yolk weight (%) of 0-d-old chicks Total yolk weight, g Item
Light eggs
Heavy eggs
Light eggs
Heavy eggs
2.06b 2.56b 2.22b 2.06b 2.68b 2.48b
3.86a 2.28b 2.76b 2.20b 2.24b 2.56b
5.84b 7.14b 6.17b 6.02b 7.08b 7.24ab
9.80a 6.14b 7.02b 5.76b 5.68b 6.55b
0.04 0.36
0.04 0.90
a,b
Values within comparisons with different superscripts differ significantly (P < 0.05). Treatment solutions: maltose = 0.5 mL of a 50% maltose syrup solution; vitamins = 0.5 mL of a multivitamin solution; zinc = 0.5 mL of a 0.5% zinc-glycine solution; control = 0.5 mL of a 0.4% saline solution; mix = 0.5 mL of a 50% maltose (12% dextrose, 42% maltose, 10% maltotriose, and 23 to 28% other sugars), 0.5% zinc-glycine, 10% glutamine, and multivitamin solution; glutamine = 0.5 mL of a 10% glutamine solution. 2 Significance level according to ANOVA. 1
tines were collectively weighed. Heavier eggs present higher protein content in the yolk and albumen [16]. Eggs from older breeders present higher yolk percentages, and the difference in lipid transference from the yolk at the end of incubation may influence intestinal maturation [26]. The higher development of turkey poults derived from older breeders is due to the higher
phospholipid content in the yolk [26], which is consistent with the findings of Vieira and Moran [27], who found lower phosphorus content in the yolk sac of chicks derived from young breeders as compared with those from old breeders. At 7 d of age, chicks from heavy eggs had higher liver weights (Table 11). However, liver weight relative to BW was not different. Ab-
Table 11. Absolute and relative weight (% of BW) of liver and intestines of 7-d-old chicks derived from light and heavy eggs, and inoculated with nutritional solutions at 18 d of incubation Item Eggs Light Heavy Solution1 Maltose Vitamins Zinc Control Mix Glutamine P-value2 Egg Solution Egg × solution SEM a–c
Liver, g
Liver, %
Intestine, g
6.2 6.6
3.74 3.76
30.4 31.7
18.42 18.06
6.5 6.5 6.8 6.0 6.1 6.4
3.81 3.82 3.92 3.65 3.63 3.69
32.6 29.6 29.4 30.0 31.5 33.1
19.19a 17.25bc 17.02c 18.20abc 18.75ab 19.03a
0.03 0.17 0.31 0.8
0.85 0.61 0.99 0.41
0.15 0.07 0.12 3.8
Intestine, %
0.23 0.001 0.49 1.43
Values within a column with different superscripts differ significantly (P < 0.05). Treatment solutions: maltose = 0.5 mL of a 50% maltose syrup solution; vitamins = 0.5 mL of a multivitamin solution; zinc = 0.5 mL of a 0.5% zinc-glycine solution; control = 0.5 mL of a 0.4% saline solution; mix = 0.5 mL of a 50% maltose (12% dextrose, 42% maltose, 10% maltotriose, and 23 to 28% other sugars), 0.5% zinc-glycine, 10% glutamine, and multivitamin solution; glutamine = 0.5 mL of a 10% glutamine solution. 2 Significance level according to ANOVA. 11
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Solution1 Maltose Vitamins Zinc Control Mix Glutamine P-value2 SEM
Relative yolk weight, %
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Table 12. Absolute and relative weight (% of BW) of liver and intestines of 21-d-old chicks derived from light and heavy eggs, and inoculated with nutritional solutions at 18 d of incubation Item
Liver, %
Intestine, g
Intestine, %
23.2 23.5
2.80 2.79
100.5 102.8
12.16 12.29
22.0 24.0 23.5 23.1 23.3 23.9
2.64 2.89 2.76 2.77 2.89 2.81
104.7 102.9 108.4 99.2 97.6 97.2
12.57 12.30 12.72 12.13 12.16 11.46
0.73 0.81 0.81 3.3
0.84 0.31 0.25 0.27
0.52 0.39 0.25 13.5
0.76 0.56 0.22 1.51
1
Treatment solutions: maltose = 0.5 mL of a 50% maltose syrup solution; vitamins = 0.5 mL of a multivitamin solution; zinc = 0.5 mL of a 0.5% zinc-glycine solution; control = 0.5 mL of a 0.4% saline solution; mix = 0.5 mL of a 50% maltose (12% dextrose, 42% maltose, 10% maltotriose, and 23 to 28% other sugars), 0.5% zinc-glycine, 10% glutamine, and multivitamin solution; glutamine = 0.5 mL of a 10% glutamine solution. 2 Significance level according to ANOVA.
solute and relative intestinal weights were not influenced by egg weight. Nutritional solutions did affect absolute (P = 0.07) and relative intestinal weights at 7 d of age. Despite the lack of difference relative to the control, the in ovo inoculation with MALT, GLU, and MIX increased relative intestinal weight as compared with ZG. At 21 d of age, there was no influence of egg weight or inoculated solutions on absolute and relative liver and intestinal weights (Table 12). Chicks derived from heavier eggs have heavier livers because liver metabolism at hatch is highly active in absorbing yolk lipids by endocytosis, remodeling them into lipoproteins, and then releasing them into the blood circulation by exocytosis; therefore, the liver weight reflects the higher yolk weight [14]. This may explain the higher liver weight observed in chicks from heavy eggs at 0 and 7 d of age in the present experiment because these chicks presented higher yolk weights at 18 d of incubation. The administration of zinc-methionine in the amniotic fluid of the embryo was previously shown to enhance intestinal development [34]. In addition, it has been suggested that in ovo inoculation of nutrients improves gastrointestinal traits such as enzyme activity, protein expression, and intestinal development [7, 8, 13, 34]. Uni et al. [35] observed that vitamin A deficiency interferes with
intestinal cell proliferation and maturation in broilers. However, in the present experiment, no influence of vitamin inoculation was observed on intestinal tract weight, possibly because of the correct supply of these vitamins.
CONCLUSIONS AND APPLICATIONS Under the conditions of the present experiment, it is possible to conclude that
1. Heavy eggs resulted in higher 42-d BW compared with those derived from light eggs. 2. The use of MALT may have resulted in partial egg yolk energy sparing but this was also dependent on egg size. 3. The choice of products or compounds to be inoculated in ovo warrants further study, but the use of a readily available energy source has shown some promise. 4. In ovo feeding was associated with no effect on live performance, breast yield, or carcass yield.
REFERENCES AND NOTES 1. Yegani, M., and D. R. Korver. 2008. Factor affecting intestinal health in poultry. Poult. Sci. 87:2052–2063. 2. Uni, Z., E. Tako, O. Gal-Garber, and D. Sklan. 2003. Morphological, molecular, and functional changes in the
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Eggs Light Heavy Solution1 Maltose Vitamins Zinc Control Mix Glutamine P-value2 Egg Solution Egg × solution SEM
Liver, g
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