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Evaluation of Egg Injection Method of Pantothenic Acid in Turkey Eggs and Effect of Supplemental Pantothenic Acid on Hatchability
Evaluation of Egg Injection Method of Pantothenic Acid in Turkey Eggs and Effect of Supplemental Pantothenic Acid on Hatchability EDWARD J. ROBEL USDA, Agricultural Research Service, Livestock and Poultry Sciences Institute, Germplasm and Gamete Physiology Laboratory, Beltsville, Maryland 20705-2350 ABSTRACT Two experiments were conducted with a commercial strain cross of 120 Large White British United Turkeys of America to determine the effect of pantothenic acid egg injections and dietary pantothenic acid on hatchability. The hens were housed individually in cages in a conventional house. In Experiment 1, three dietary treatments were used: 1) an unsupplemented practical corn-soybean meal basal diet; 2) the basal diet supplemented with 37.4 mg pantothenic acid/kg; and 3) the basal diet supplemented with 74.8 mg pantothenic acid/kg. Incremental dietary supplemental pantothenic acid levels increased the transfer of pantothenic acid in eggs, but did not result in a hatchability increase over the unsupplemented pantothenic acid basal diet. The response patterns from dietary pantothenic acid for the reproductive variables were similar whether the data were analyzed on a production period basis using all of the hens or on a subset of hens producing eggs in each production period. In Experiment 2, with hens fed 37.4 mg supplemental pantothenic acid/kg of diet, hatchability did not increase in eggs injected with 1,800 ng pantothenic acid per egg as compared with uninjected eggs and eggs injected with the vitamin carrier solution. The results of the study indicate that hatchability was not increased in turkey eggs from hens fed supplemental pantothenic acid or with egg pantothenic acid injections, which suggests that pantothenic acid is not limiting for hatchability in commercial turkey hen diets that contain 10.5 mg/kg or more of pantothenic acid. (Key words: pantothenic acid, turkey eggs, hatchability, embryonic mortality, egg injections) 1993 Poultry Science 72:1740-1745
turkey breeder diets is 16 mg pantothenic acid/kg of diet. In recent years, intensive In the past, emphasis for determining selection for rapid growth, body conforpantothenic acid requirements for hatcha- mation, and increased feed efficiency may bility was placed on the use of dietary have changed the requirement of pansupplemental pantothenic acid (Gillis et tothenic acid for hatchability. al., 1942; Dawson et al., 1962; Beer et at, A positive relationship was demon1963; Scott et al, 1982), and the results strated between incremental dietary panwere controversial. This procedure does tothenic acid levels and pantothenic acid not directly consider pantothenic acid in concentrations in eggs, which suggests the egg as related to embryonic survival. that hatchability may be responsive to The National Research Council's (1984) recommendation for pantothenic acid in supplemental dietary pantothenic acid (Snell et al, 1941; Gillis et al, 1942). Robel (1983) reported that pantothenic acid content of eggs from Large and Small White turkey hens fed 32 mg pantothenic acid/ Received for publication January 28, 1993. kg of diet did not change significantly Accepted for publication May 24, 1993. INTRODUCTION
1740
PANTOTHENIC ACID FOR HATCHABILITY
1741
with maternal age, which suggests that the transport mechanism of pantothenic acid to the egg in the hen is stable with maternal age. This study was conducted to evaluate the affect of dietary supplemental pantothenic acid and pantothenic acid egg injections on the hatchability of turkey eggs.
based on egg counts during the production period. Early-dead, late-dead, and hatchability of fertile eggs are percentages based on egg counts. Egg weight, poult weight, and feed consumption are averages for the periods. Eggs per hen is the number of eggs set, except in a few cases in which a small number of eggs were taken for analysis. To determine whether there was a positive relationship between dietary panMATERIALS AND METHODS tothenic acid and pantothenic acid in the egg, eggs for pantothenic acid assays were collected from one hen in each treatment at Experiment 1 12 and 16 wk of production. Egg yolk and A total of 120 hens of a commercial strain albumen from each hen were separated, cross of Large White British United Turkeys blended separately, and each was freezeof America (BUTA1) 30 wk of age was dried and assayed independently. Microbiassigned randomly to individual wire- ological determinations for pantothenic bottomed cages (61.25 cm deep x 45 cm wide x 60 cm high) equipped with cup waterers in a conventional house. Feed and water were available for ad libitum access. TABLE 1. Composition of the basal diet The control treatment consisted of feeding the basal diet without supplemental pan- Ingredients and tothenic acid (Table 1). The basal diet calculated analyses Percentage contained 10.5 mg pantothenic acid/kg 62.00 based on analysis. The other two treatments Ground yellow corn meal (49% CP) 16.90 consisted of supplementing the basal diet Soybean Alfalfa meal (17% CP) 5.00 with two levels of exogenous d-pantothenic Meat and bone meal (50% CP) 5.00 2 acid (37.4 and 74.8 mg pantothenic acid/kg Soybean oil 3.00 .10 of diet), which contained 47.8 and 83.4 mg DL-methionine 1 .50 pantothenic acid/kg of diet based on analy- Vitamin premix 2 Trace mineral premix .10 sis. The photoperiod was 14 h light:10 h Iodized salt .30 dark. The hens were photostimulated at 30 Selenium premix3 .10 4 wk of age and assigned to dietary treat- Antibiotic premix .50 4.50 ments at that time. The hens were fed the Ground limestone 2.00 basal diet containing 37.4 mg supplemental Dicalcium phosphate Total 100.00 pantothenic acid/kg of diet at photostimulation. The hens were assigned in a com- Calculated analyses 17.00 pletely randomized design to the three Protein Calcium 2.71 treatment groups containing 40 hens per Phosphorus (total) .76 group. ME, kcal/kg 2,960 Hatching variables were recorded at Vitamin premix supplied the following per kiloweekly intervals. The weekly data gram of diet: riboflavin, 11 mg; folacin, 2.6 mg; 4.4 mg; niacin, 66 mg; d-biotin, .22 mg; responses averaged across 5 or 6 wk were thiamine, pyridoxine, 6.6 mg; vitamin B12,17.6 jtg; choline, 881 used to create three time periods (0 to 5,6 to mg; vitamin K, 3.0 mg; d-a-tocopherol acetate, 66 IU; 10, and 11 to 16 wk) of production. The cholecalciferol, 6,608 ICU; vitamin A acetate, 19,823 IU; variables eggs laid, and poults hatched are ethoxyquin, 150 mg. 2
British United Turkeys of America, Lewisburg, WV 24901. 2 Calcium d-pantothenate in a dry premix containing 7,496 jig d-pantothenate/g of premix; contribution of Hoffmann-La Roche, Inc., Nutley, NJ 07110-1199.
Trace mineral premix supplied the following in milligrams per kilogram of diet: manganese (sulfate), 100; iron (sulfate, carbonate, oxide), 100; copper (oxide), 10; cobalt (carbonate), 1; iodine (potassium iodide), 3; zinc (sulfate), 100. SSelenium premix supplied .1 mg sodium selenite/ kg of diet. 4 A n t i b i o t i c premix s u p p l i e d 110 mg chlortetracycline/kg of diet.
1742
ROBEL
acid in diets, injection solution, and eggs were completed by a commercial laboratory using methods described by the Association of Official Analytical Chemists (1990). Statistical Analysis The data were analyzed separately for each period, whereby all hens were used, including those not laying eggs in all three production periods (Table 2). The variables were analyzed using a one-factor analysis of variance model, with pantothenic acid treatments as the factor. If diagnostic techniques showed that the assumptions of the general linear model were not met for a particular variable, a suitable transformation was determined and the variable was reanalyzed; the one exception to this was hatchability, for which no suitable transformation existed. Hatchability was analyzed using the nonparametric median sign test (Conover, 1980). The data were also analyzed for the entire production cycle on a restricted subset of hens that produced eggs across the three production periods. This was done in an attempt to better isolate the pantothenic acid effect that might presumably be masked in the separate period by period production analysis. Because the results of the two analyses did not differ, the data are presented on a production period basis (Table 2). The egg analysis data were transformed using the inverse of the square root of the egg albumen and egg yolk transformation (SAS Institute, 1988). Experiment 2 Egg injection trials were conducted at 5, 10, 11, 12, 13, 14, 15, 16, 17, and 18 wk of production. Three eggs of good shell quality were collected from each of 10 hens in each trial; these hens laid eggs most consistently during the trials. Each one of the
3 D-pantothenic acid (hemi-calcium salt) crystalline, Number P-2250, Sigma Chemical Co., St. Louis, MO 63178-9916. 'Millipore Corp., Bedford, MA 01730. SFujisawa USA, Inc., Deerfield, IL 60015. *Becton Dickinson, Inc., Rutherford, NJ 07070.
three eggs from each hen was randomly assigned to a different treatment. There was a total of 10 eggs per treatment per trial. The eggs were injected on the 25th day of incubation in each trial. The hens in production were selected from a flock of 120 turkeys of a commercial strain cross of Large White BUTA turkeys. The hens were fed a practical corn-soybean meal basal diet (Table 1) supplemented with 37.4 mg pantothenic acid/kg (a level generally added as a safety measure for maintaining hatchability)—the same diet used for one of the treatments in Experiment 1. Egg Injection Treatments The treatments were the following: 1) eggs injected with .2 mL of saline solution containing 1,800 /tg pantothenic acid/.2 mL based on analysis; 2) eggs injected with .2 mL of the saline solution; and 3) control (uninjected eggs). Egg Injection and Solution Preparation Environment Egg injections and pantothenic acid solution preparation were completed under Gold Fluorescent (not ultraviolet) light in a particle-free environment with filtered high efficiency particulate air. D-Pantothenic Acid Solution The hemi-calcium salt of d-pantothenic acid3 (900 mg) was dissolved in 100 mL of pyrogen-free saline solution until the solution was clear and colorless at pH 6.96. A portion of the solution was withdrawn into a sterile 60-mL syringe. The syringe was equipped with a .22-/tm sterile nonpyrogenic filter unit (Millex-GV).4 The solution was dispensed into 10-mL empty sterilized vials5 from which .2-mL aliquots were withdrawn using a microfine III, 28-gauge sterile needle locked to a 1-mL sterile insulin syringe.6 For the egg injections, .2 mL of solution was injected per egg at 25 days of incubation, using a separate syringe with needle for each individual egg. Aseptic Egg Injections Egg injections were performed in a sanitized clean room with a particle-free
235 ± 2.v> 217 ± 3.0c 228 ± 2.0b
217 ± 2.0" 209 ± 3.0b 221 ± 2.0"
01 37.4 74.8
01 37.4
56.4 ± .8 57.0 ± .8 55.8 + .8
54.0 ± .6 53.6 ± .6 53.2 ± .6
52.4 ± .6 51.3 ± .6 52.8 ± .5
W
Poult weight
86.8 ± 1.1 87.6 ± .8 86.0 ± .8
84.5 ± .8 85.4 ± .8 85.2 ± .8
80.9 ± .7 80.3 ± .6 81.9 ± .7
Egg weight (no.) to 5 wk of production 27.4 ± .6 26.1 ± .9 25.3 ± .8 to 10 wk of production 21.2 ± 1.0 19.1 ± 1.1 18.4 ± 1.0 11 to 16 wk of production 16.3 ± 1.9 13.9 ± 1.8 14.3 ± 1.7
Eggs per hen
66. 57. 64.
77. 66. 59.
81. 81. 79.
Ha fer
"Means ± SE in a column within each production period with no common superscripts differ signific !Basal diet without supplemental pantothenic acid contained 10.5 mg pantothenic acid/kg based on ana
74.8
207 ± 2.0 207 ± 2.0 206 ± 1.0
Feed consumption per hen per day
01 37.4 74.8
(mg/kg)
Supplemental D-pantothenic acid
TABLE 2. Performance of turkey hens fed a basal diet supplemented with two levels of pan of the production cycle, Experiment 1
1744
ROBEL TABLE 3. Effect of dietary supplemental pantothenic acid on the transfer of pantothenic acid in turkey eggs, Experiment 1
Supplemental D-pantothenic acid (mg/kg) 02 37.4 74.8
Pantothenic acid in dried egg yolk1
Pantothenic acid in dried egg albumen1 ,
,„^
11.2 13.3 16.4
43.4b 101.3" 116.2"
a b
- Means in a column with no common superscripts differ significantly (P < .05). Levels of pantothenic acid based on analysis of eggs from the same hen per treatment, which produced eggs at 12 and 16 wk of production. 2 6asal diet (without supplemental pantothenic acid) contained 10.5 mg pantothenic acid/kg of diet based on analysis. 1
environment using filtered, high efficiency particulate air, and personnel were aseptically equipped. A 2% iodine tincture was applied to the injection site on the large end of the egg. A very slight indentation was made in the swabbed shell area using a sharp sterile steel punch. The force used to make the indentation was of a minor degree to avoid the formation of hairline cracks in the shell. The punch site exactly accommodated the microfine III, 28-gauge sterile needle that was locked to a 1-mL sterile insulin syringe. The needle was carefully inserted through the indentation site to a depth of 6 to 8 mm, and .2-mL of solution was injected over the inner eggshell membrane. The puncture was sealed with a small drop of fast-drying Duco cement.7 Statistical Analysis of Egg Injection Trials The egg injection trials were completed at 5,10,11,12,13,14,15,16,17, and 18 wk of production. The percentage hatchability of fertile eggs was analyzed as a one-way randomized block design in which the type of injection or no injection was the treatment and trial was the block. Diagnostic techniques showed that this model met the general linear model assumptions.
TDevcon Corp., Wood Dale, IL 60191.
RESULTS AND DISCUSSION Experiment 1 The means for the reproductive variables which were analyzed within each single period of production (0 to 5,6 to 10, and 11 to 16 wk) are presented in Table 2. Although there were statistical differences in feed consumption among treatments for 6 to 10 wk and 11 to 16 wk, the differences are not of practical importance; in fact, the largest difference between treatments in the period of 6 to 10 wk was 7.7% and from 11 to 16 wk was 5.4%. No obvious relationship can be made between the statistically significant differences for average dietary intakes and the results for average hatchability from 6 to 10 wk. Furthermore, these hatchability differences did not occur between 0 to 5 wk and 11 to 16 wk of production. Also, there were no differences in 7-day or 28-day embryonic mortality (components of hatchability) among treatments in any of the production periods. Dawson et ah (1962) reported high embryonic mortality in the 1st wk of incubation with few late survivors of eggs from turkey hens fed a diet deficient in pantothenic acid. Early embryonic mortality was low in the present experiment. The basal diet contained 10.5 mg pantothenic acid/kg based on analysis; however, its bioavailability level is expected to be less. The National Research Council's (1984) recommendation for pantothenic acid in turkey breeder diets is 16 mg pantothenic acid/kg of diet. There seems, therefore, to have been an ample margin for
PANTOTHENIC ACID FOR HATCHABILITY
1745
TABLE 4. Effect of pantothenic acid egg injections on hatchability of turkey eggs, Experiment 2
injections. There was no significant difference for the average percentage hatchability treatment means of the 10 pantothenic acid injection trials (Table 4). Injection of panTotal tothenic acid did not increase hatchability Injection fertile Hatchability of treatment eggs set fertile eggs1 over the control (uninjected eggs) or salineinjected eggs. The lack of hatchability (no.) (%) response suggests that there was more than Control (uninjected) 100 85.8 ± 1.4 adequate pantothenic acid in the egg for Pantothenic acid in saline 100 87.0 ± 1.8 embryo survival; therefore, pantothenic Saline solution 100 85.8 ± 1.4 acid egg injections did not increase hatcha'Means ± SE that represent results of 10 egg bility. Robel (1983) reported the effect of injection trials comprising 5,10,11,12,13,14,15,16,17, dietary pantothenic acid (32 mg/kg of diet) and 18 wk of production, were not significantly on the transfer of pantothenic acid in turkey different (P < .05). Three eggs of good shell qualitywere collected from each of 10 hens per trial; these hens eggs to be stable with maternal age. Therelaid eggs most consistently during the trials. Three fore, in the egg injection trials, pantothenic eggs from each hen was randomly assigned to a acid in the egg would not be expected to different treatment. There was a total of 10 eggs per vary greatly between egg injection trials. treatment per trial. The results of injecting pantothenic acid into eggs support the lack of hatchability response observed with the use of dietary supplemental pantothenic acid in the turhatchability increase with the dietary sup- key hen diet, because there was a positive plemental pantothenic acid treatments over relationship between incremental panthe basal dietary treatment; however, this tothenic acid levels and the transfer of did not occur. The interpretation from these pantothenic acid in the egg. findings is that pantothenic acid is not limiting for hatchability, especially as there REFERENCES was a positive relationship between incremental dietary pantothenic acid levels Association of Official Analytical Chemists, 1990. Official Methods of Analysis. Association of and the transfer of pantothenic acid to the Official Analytical Chemists 15th ed. Methods egg (Table 3) and 10.5 mg or more of 960.46-945.74, Arlington, VA. pantothenic acid/kg of diet supported Beer, A. E., M. L. Scott, and M. C. Nesheim, 1963. The hatchability. effects of graded levels of pantothenic acid on the Experiment 2 Eggs used for 10 pantothenic acid injection trials at 5, 10,11,12, 13,14,15, 16,17, and 18 wk of production were from hens fed 37.4 mg dietary supplemental pantothenic acid/kg of diet—a level generally added as a safety measure in maintaining hatchability. However, in recent years intensive selection for rapid growth, body conformation, and feed efficiency may have augmented the safety level required to sustain hatchability. The 25th day of incubation for the injections is unique and convenient for hatcheries because this is when eggs are transferred from incubators to hatchers. Also, embryonic mortality due to pantothenic acid deficiency at this time would be expected to be decreased by the
breeding performance of White Leghorn pullets. Br. Poult. Sci. 3:243-253. Conover, W. J., 1980. Practical Nonparametric Statistics. 2nd ed. John Wiley and Sons, New York, NY. Dawson, E., T. M. Ferguson, C. W. Deyoe, and J. R. Couch, 1962. Pantothenic acid deficient turkey embryos. Poultry Sci. 41:1639.(Abstr.) Gillis, M. B., G. F. Henser, and L. C. Norris, 1942. Quantitative requirement of the hen for pantothenic acid. Poultry Sci. 21:470.(Abstr.) National Research Council, 1984. Nutrient Requirements of Poultry. 8th rev. ed. National Academy Press, Washington, DC. Robel, E. J., 1983. The effect of age of breeder hen on levels of vitamins and minerals in turkey eggs. Poultry Sci. 62:1751-1756. SAS Institute, 1988. SAS/STAT® Guide: For Personal Computers. Version 6.03 Edition. SAS Institute Inc., Cary, NC. Scott, M. L., M. C. Nesheim, and R. J. Young, 1982. Nutrition of the Chicken. Scott and Associates, Ithaca, NY. Snell, E. E., E. Aline, J. R. Couch, and P. B. Pearson, 1941. The effect of diet on the pantothenic acid content of eggs. J. Nutr. 21:201-205.
turkey breeder diets is 16 mg pantothenic acid/kg of diet. In recent years, intensive In the past, emphasis for determining selection for rapid growth, body conforpantothenic acid requirements for hatcha- mation, and increased feed efficiency may bility was placed on the use of dietary have changed the requirement of pansupplemental pantothenic acid (Gillis et tothenic acid for hatchability. al., 1942; Dawson et al., 1962; Beer et at, A positive relationship was demon1963; Scott et al, 1982), and the results strated between incremental dietary panwere controversial. This procedure does tothenic acid levels and pantothenic acid not directly consider pantothenic acid in concentrations in eggs, which suggests the egg as related to embryonic survival. that hatchability may be responsive to The National Research Council's (1984) recommendation for pantothenic acid in supplemental dietary pantothenic acid (Snell et al, 1941; Gillis et al, 1942). Robel (1983) reported that pantothenic acid content of eggs from Large and Small White turkey hens fed 32 mg pantothenic acid/ Received for publication January 28, 1993. kg of diet did not change significantly Accepted for publication May 24, 1993. INTRODUCTION
1740
PANTOTHENIC ACID FOR HATCHABILITY
1741
with maternal age, which suggests that the transport mechanism of pantothenic acid to the egg in the hen is stable with maternal age. This study was conducted to evaluate the affect of dietary supplemental pantothenic acid and pantothenic acid egg injections on the hatchability of turkey eggs.
based on egg counts during the production period. Early-dead, late-dead, and hatchability of fertile eggs are percentages based on egg counts. Egg weight, poult weight, and feed consumption are averages for the periods. Eggs per hen is the number of eggs set, except in a few cases in which a small number of eggs were taken for analysis. To determine whether there was a positive relationship between dietary panMATERIALS AND METHODS tothenic acid and pantothenic acid in the egg, eggs for pantothenic acid assays were collected from one hen in each treatment at Experiment 1 12 and 16 wk of production. Egg yolk and A total of 120 hens of a commercial strain albumen from each hen were separated, cross of Large White British United Turkeys blended separately, and each was freezeof America (BUTA1) 30 wk of age was dried and assayed independently. Microbiassigned randomly to individual wire- ological determinations for pantothenic bottomed cages (61.25 cm deep x 45 cm wide x 60 cm high) equipped with cup waterers in a conventional house. Feed and water were available for ad libitum access. TABLE 1. Composition of the basal diet The control treatment consisted of feeding the basal diet without supplemental pan- Ingredients and tothenic acid (Table 1). The basal diet calculated analyses Percentage contained 10.5 mg pantothenic acid/kg 62.00 based on analysis. The other two treatments Ground yellow corn meal (49% CP) 16.90 consisted of supplementing the basal diet Soybean Alfalfa meal (17% CP) 5.00 with two levels of exogenous d-pantothenic Meat and bone meal (50% CP) 5.00 2 acid (37.4 and 74.8 mg pantothenic acid/kg Soybean oil 3.00 .10 of diet), which contained 47.8 and 83.4 mg DL-methionine 1 .50 pantothenic acid/kg of diet based on analy- Vitamin premix 2 Trace mineral premix .10 sis. The photoperiod was 14 h light:10 h Iodized salt .30 dark. The hens were photostimulated at 30 Selenium premix3 .10 4 wk of age and assigned to dietary treat- Antibiotic premix .50 4.50 ments at that time. The hens were fed the Ground limestone 2.00 basal diet containing 37.4 mg supplemental Dicalcium phosphate Total 100.00 pantothenic acid/kg of diet at photostimulation. The hens were assigned in a com- Calculated analyses 17.00 pletely randomized design to the three Protein Calcium 2.71 treatment groups containing 40 hens per Phosphorus (total) .76 group. ME, kcal/kg 2,960 Hatching variables were recorded at Vitamin premix supplied the following per kiloweekly intervals. The weekly data gram of diet: riboflavin, 11 mg; folacin, 2.6 mg; 4.4 mg; niacin, 66 mg; d-biotin, .22 mg; responses averaged across 5 or 6 wk were thiamine, pyridoxine, 6.6 mg; vitamin B12,17.6 jtg; choline, 881 used to create three time periods (0 to 5,6 to mg; vitamin K, 3.0 mg; d-a-tocopherol acetate, 66 IU; 10, and 11 to 16 wk) of production. The cholecalciferol, 6,608 ICU; vitamin A acetate, 19,823 IU; variables eggs laid, and poults hatched are ethoxyquin, 150 mg. 2
British United Turkeys of America, Lewisburg, WV 24901. 2 Calcium d-pantothenate in a dry premix containing 7,496 jig d-pantothenate/g of premix; contribution of Hoffmann-La Roche, Inc., Nutley, NJ 07110-1199.
Trace mineral premix supplied the following in milligrams per kilogram of diet: manganese (sulfate), 100; iron (sulfate, carbonate, oxide), 100; copper (oxide), 10; cobalt (carbonate), 1; iodine (potassium iodide), 3; zinc (sulfate), 100. SSelenium premix supplied .1 mg sodium selenite/ kg of diet. 4 A n t i b i o t i c premix s u p p l i e d 110 mg chlortetracycline/kg of diet.
1742
ROBEL
acid in diets, injection solution, and eggs were completed by a commercial laboratory using methods described by the Association of Official Analytical Chemists (1990). Statistical Analysis The data were analyzed separately for each period, whereby all hens were used, including those not laying eggs in all three production periods (Table 2). The variables were analyzed using a one-factor analysis of variance model, with pantothenic acid treatments as the factor. If diagnostic techniques showed that the assumptions of the general linear model were not met for a particular variable, a suitable transformation was determined and the variable was reanalyzed; the one exception to this was hatchability, for which no suitable transformation existed. Hatchability was analyzed using the nonparametric median sign test (Conover, 1980). The data were also analyzed for the entire production cycle on a restricted subset of hens that produced eggs across the three production periods. This was done in an attempt to better isolate the pantothenic acid effect that might presumably be masked in the separate period by period production analysis. Because the results of the two analyses did not differ, the data are presented on a production period basis (Table 2). The egg analysis data were transformed using the inverse of the square root of the egg albumen and egg yolk transformation (SAS Institute, 1988). Experiment 2 Egg injection trials were conducted at 5, 10, 11, 12, 13, 14, 15, 16, 17, and 18 wk of production. Three eggs of good shell quality were collected from each of 10 hens in each trial; these hens laid eggs most consistently during the trials. Each one of the
3 D-pantothenic acid (hemi-calcium salt) crystalline, Number P-2250, Sigma Chemical Co., St. Louis, MO 63178-9916. 'Millipore Corp., Bedford, MA 01730. SFujisawa USA, Inc., Deerfield, IL 60015. *Becton Dickinson, Inc., Rutherford, NJ 07070.
three eggs from each hen was randomly assigned to a different treatment. There was a total of 10 eggs per treatment per trial. The eggs were injected on the 25th day of incubation in each trial. The hens in production were selected from a flock of 120 turkeys of a commercial strain cross of Large White BUTA turkeys. The hens were fed a practical corn-soybean meal basal diet (Table 1) supplemented with 37.4 mg pantothenic acid/kg (a level generally added as a safety measure for maintaining hatchability)—the same diet used for one of the treatments in Experiment 1. Egg Injection Treatments The treatments were the following: 1) eggs injected with .2 mL of saline solution containing 1,800 /tg pantothenic acid/.2 mL based on analysis; 2) eggs injected with .2 mL of the saline solution; and 3) control (uninjected eggs). Egg Injection and Solution Preparation Environment Egg injections and pantothenic acid solution preparation were completed under Gold Fluorescent (not ultraviolet) light in a particle-free environment with filtered high efficiency particulate air. D-Pantothenic Acid Solution The hemi-calcium salt of d-pantothenic acid3 (900 mg) was dissolved in 100 mL of pyrogen-free saline solution until the solution was clear and colorless at pH 6.96. A portion of the solution was withdrawn into a sterile 60-mL syringe. The syringe was equipped with a .22-/tm sterile nonpyrogenic filter unit (Millex-GV).4 The solution was dispensed into 10-mL empty sterilized vials5 from which .2-mL aliquots were withdrawn using a microfine III, 28-gauge sterile needle locked to a 1-mL sterile insulin syringe.6 For the egg injections, .2 mL of solution was injected per egg at 25 days of incubation, using a separate syringe with needle for each individual egg. Aseptic Egg Injections Egg injections were performed in a sanitized clean room with a particle-free
235 ± 2.v> 217 ± 3.0c 228 ± 2.0b
217 ± 2.0" 209 ± 3.0b 221 ± 2.0"
01 37.4 74.8
01 37.4
56.4 ± .8 57.0 ± .8 55.8 + .8
54.0 ± .6 53.6 ± .6 53.2 ± .6
52.4 ± .6 51.3 ± .6 52.8 ± .5
W
Poult weight
86.8 ± 1.1 87.6 ± .8 86.0 ± .8
84.5 ± .8 85.4 ± .8 85.2 ± .8
80.9 ± .7 80.3 ± .6 81.9 ± .7
Egg weight (no.) to 5 wk of production 27.4 ± .6 26.1 ± .9 25.3 ± .8 to 10 wk of production 21.2 ± 1.0 19.1 ± 1.1 18.4 ± 1.0 11 to 16 wk of production 16.3 ± 1.9 13.9 ± 1.8 14.3 ± 1.7
Eggs per hen
66. 57. 64.
77. 66. 59.
81. 81. 79.
Ha fer
"Means ± SE in a column within each production period with no common superscripts differ signific !Basal diet without supplemental pantothenic acid contained 10.5 mg pantothenic acid/kg based on ana
74.8
207 ± 2.0 207 ± 2.0 206 ± 1.0
Feed consumption per hen per day
01 37.4 74.8
(mg/kg)
Supplemental D-pantothenic acid
TABLE 2. Performance of turkey hens fed a basal diet supplemented with two levels of pan of the production cycle, Experiment 1
1744
ROBEL TABLE 3. Effect of dietary supplemental pantothenic acid on the transfer of pantothenic acid in turkey eggs, Experiment 1
Supplemental D-pantothenic acid (mg/kg) 02 37.4 74.8
Pantothenic acid in dried egg yolk1
Pantothenic acid in dried egg albumen1 ,
,„^
11.2 13.3 16.4
43.4b 101.3" 116.2"
a b
- Means in a column with no common superscripts differ significantly (P < .05). Levels of pantothenic acid based on analysis of eggs from the same hen per treatment, which produced eggs at 12 and 16 wk of production. 2 6asal diet (without supplemental pantothenic acid) contained 10.5 mg pantothenic acid/kg of diet based on analysis. 1
environment using filtered, high efficiency particulate air, and personnel were aseptically equipped. A 2% iodine tincture was applied to the injection site on the large end of the egg. A very slight indentation was made in the swabbed shell area using a sharp sterile steel punch. The force used to make the indentation was of a minor degree to avoid the formation of hairline cracks in the shell. The punch site exactly accommodated the microfine III, 28-gauge sterile needle that was locked to a 1-mL sterile insulin syringe. The needle was carefully inserted through the indentation site to a depth of 6 to 8 mm, and .2-mL of solution was injected over the inner eggshell membrane. The puncture was sealed with a small drop of fast-drying Duco cement.7 Statistical Analysis of Egg Injection Trials The egg injection trials were completed at 5,10,11,12,13,14,15,16,17, and 18 wk of production. The percentage hatchability of fertile eggs was analyzed as a one-way randomized block design in which the type of injection or no injection was the treatment and trial was the block. Diagnostic techniques showed that this model met the general linear model assumptions.
TDevcon Corp., Wood Dale, IL 60191.
RESULTS AND DISCUSSION Experiment 1 The means for the reproductive variables which were analyzed within each single period of production (0 to 5,6 to 10, and 11 to 16 wk) are presented in Table 2. Although there were statistical differences in feed consumption among treatments for 6 to 10 wk and 11 to 16 wk, the differences are not of practical importance; in fact, the largest difference between treatments in the period of 6 to 10 wk was 7.7% and from 11 to 16 wk was 5.4%. No obvious relationship can be made between the statistically significant differences for average dietary intakes and the results for average hatchability from 6 to 10 wk. Furthermore, these hatchability differences did not occur between 0 to 5 wk and 11 to 16 wk of production. Also, there were no differences in 7-day or 28-day embryonic mortality (components of hatchability) among treatments in any of the production periods. Dawson et ah (1962) reported high embryonic mortality in the 1st wk of incubation with few late survivors of eggs from turkey hens fed a diet deficient in pantothenic acid. Early embryonic mortality was low in the present experiment. The basal diet contained 10.5 mg pantothenic acid/kg based on analysis; however, its bioavailability level is expected to be less. The National Research Council's (1984) recommendation for pantothenic acid in turkey breeder diets is 16 mg pantothenic acid/kg of diet. There seems, therefore, to have been an ample margin for
PANTOTHENIC ACID FOR HATCHABILITY
1745
TABLE 4. Effect of pantothenic acid egg injections on hatchability of turkey eggs, Experiment 2
injections. There was no significant difference for the average percentage hatchability treatment means of the 10 pantothenic acid injection trials (Table 4). Injection of panTotal tothenic acid did not increase hatchability Injection fertile Hatchability of treatment eggs set fertile eggs1 over the control (uninjected eggs) or salineinjected eggs. The lack of hatchability (no.) (%) response suggests that there was more than Control (uninjected) 100 85.8 ± 1.4 adequate pantothenic acid in the egg for Pantothenic acid in saline 100 87.0 ± 1.8 embryo survival; therefore, pantothenic Saline solution 100 85.8 ± 1.4 acid egg injections did not increase hatcha'Means ± SE that represent results of 10 egg bility. Robel (1983) reported the effect of injection trials comprising 5,10,11,12,13,14,15,16,17, dietary pantothenic acid (32 mg/kg of diet) and 18 wk of production, were not significantly on the transfer of pantothenic acid in turkey different (P < .05). Three eggs of good shell qualitywere collected from each of 10 hens per trial; these hens eggs to be stable with maternal age. Therelaid eggs most consistently during the trials. Three fore, in the egg injection trials, pantothenic eggs from each hen was randomly assigned to a acid in the egg would not be expected to different treatment. There was a total of 10 eggs per vary greatly between egg injection trials. treatment per trial. The results of injecting pantothenic acid into eggs support the lack of hatchability response observed with the use of dietary supplemental pantothenic acid in the turhatchability increase with the dietary sup- key hen diet, because there was a positive plemental pantothenic acid treatments over relationship between incremental panthe basal dietary treatment; however, this tothenic acid levels and the transfer of did not occur. The interpretation from these pantothenic acid in the egg. findings is that pantothenic acid is not limiting for hatchability, especially as there REFERENCES was a positive relationship between incremental dietary pantothenic acid levels Association of Official Analytical Chemists, 1990. Official Methods of Analysis. Association of and the transfer of pantothenic acid to the Official Analytical Chemists 15th ed. Methods egg (Table 3) and 10.5 mg or more of 960.46-945.74, Arlington, VA. pantothenic acid/kg of diet supported Beer, A. E., M. L. Scott, and M. C. Nesheim, 1963. The hatchability. effects of graded levels of pantothenic acid on the Experiment 2 Eggs used for 10 pantothenic acid injection trials at 5, 10,11,12, 13,14,15, 16,17, and 18 wk of production were from hens fed 37.4 mg dietary supplemental pantothenic acid/kg of diet—a level generally added as a safety measure in maintaining hatchability. However, in recent years intensive selection for rapid growth, body conformation, and feed efficiency may have augmented the safety level required to sustain hatchability. The 25th day of incubation for the injections is unique and convenient for hatcheries because this is when eggs are transferred from incubators to hatchers. Also, embryonic mortality due to pantothenic acid deficiency at this time would be expected to be decreased by the
breeding performance of White Leghorn pullets. Br. Poult. Sci. 3:243-253. Conover, W. J., 1980. Practical Nonparametric Statistics. 2nd ed. John Wiley and Sons, New York, NY. Dawson, E., T. M. Ferguson, C. W. Deyoe, and J. R. Couch, 1962. Pantothenic acid deficient turkey embryos. Poultry Sci. 41:1639.(Abstr.) Gillis, M. B., G. F. Henser, and L. C. Norris, 1942. Quantitative requirement of the hen for pantothenic acid. Poultry Sci. 21:470.(Abstr.) National Research Council, 1984. Nutrient Requirements of Poultry. 8th rev. ed. National Academy Press, Washington, DC. Robel, E. J., 1983. The effect of age of breeder hen on levels of vitamins and minerals in turkey eggs. Poultry Sci. 62:1751-1756. SAS Institute, 1988. SAS/STAT® Guide: For Personal Computers. Version 6.03 Edition. SAS Institute Inc., Cary, NC. Scott, M. L., M. C. Nesheim, and R. J. Young, 1982. Nutrition of the Chicken. Scott and Associates, Ithaca, NY. Snell, E. E., E. Aline, J. R. Couch, and P. B. Pearson, 1941. The effect of diet on the pantothenic acid content of eggs. J. Nutr. 21:201-205.