Effects of injected gluconeogenic supplementation on the performance of broilers from young breeders12

ENVIRONMENT, WELL-BEING, AND BEHAVIOR Effects of Injected Gluconeogenic Supplementation on the Performance of Broilers from Young Breeders1,2 E. D. Peebles,*3 W. D. Berry,§ R. W. Keirs,† L. W. Bennett,† and P. D. Gerard‡ *Poultry Science Department, †College of Veterinary Medicine, and ‡Experimental Statistics Unit, Mississippi State University, Mississippi State 39762; and §Poultry Science Department, Auburn University, Auburn, AL 36849 ABSTRACT Previous research has shown that administering carbohydrates to late-term embryos increases chick hatching weight and liver glycogen content and that supplementing broiler chicks from young hens at day of hatch with subcutaneously injected hydrolyzed casein and thiamine enhances their early performance. It was hypothesized that other practical and readily available gluconeogenic energy sources, including hydrolyzed casein, may similarly be given to hatchlings from immature breeder hens to increase the availability of liver glycogen reserves and augment growth. In addition to physiological saline (sham) and hydrolyzed casein treatments, 2 other treatments containing practical gluconeogenic energy sources (chicken egg crude albumin or albumin hydrolysate) were tested in the current study using

hatchlings that were subsequently provided adequate brooding and nutrition. Added biotin was included in the crude albumin treatment. There were no treatment effects on mortality, BW gain, feed or water consumption, feed conversion, body temperature, hematocrit, plasma refractive index, relative liver weight, or liver glycogen content at any of the ages or age intervals examined through d 16 posthatch. These results suggest that under proper brooding conditions and timely feed provision, growth is not facilitated by injected casein hydrolysate, chicken egg crude albumin, or chicken egg albumin hydrolysate during the early transition from fat to carbohydrate-based nutrient uptake in posthatch chicks from young breeder hens.

Key words: breeder, brooding, chick, gluconeogenesis, growth 2006 Poultry Science 85:371–376

chine combinations and 378,000 hatching egg residue analyses lists early embryo mortalities of 6% at 25 wk, 3.8% at 30 wk, 3% at 33 wk, and an average of 3.07% through 71 wk (Keirs et al., 1996). Broiler chick hatching weight is approximately 68% of egg weight (Wilson, 1991), and hatching egg weight is influenced by breeder hen age (Clark, 1940). Therefore, chicks from pullet flocks are usually smaller than those from older hens. The pathology of such chick morbidity and mortality with loss of fat and muscular regression and nephritis with increased nitrogenous waste (present as urates) also suggest that there may be problems of increased metabolic gluconeogenesis, the metabolic pathway for the synthesis of glucose from other molecules. These lesions are commonly observed in association with improper brooding management (Peebles et al., 2005). At hatch, a chick is essentially poikilothermic and has few energy reserves, depending on its limited glycogen deposits, as it attempts to assimilate feed and water (Donaldson, 1995; Hazelwood, 2000). Liver glycogen reserves are converted to blood glucose and provide the chick with an immediate energy source. Early feeding or the expeditious provision of optimal nutrition to the hatchling has been shown to improve early chick livability and growth (Uni and Ferket, 2004). However, when dietary glucose proves insufficient to meet metabolic demands, amino acids in the diet

INTRODUCTION Management changes during the pullet and breeder periods, such as restricted feeding and dark-out lighting, have resulted in lighter weight hens and an earlier age at onset of egg production. Common field observations and production records strongly suggest that excessive mortality and poor growth performance of broiler chicks from young breeder hens 30 wk of age or younger can occur during the first 7 d of brooding. These same observations also appear to be correlated with seasonal changes, unexpected cold weather fronts, and improper brooding. In eggs from immature breeders, increases in early embryo mortality between d 1 and 12 of incubation (Keirs et al., 1996) may parallel increases in subsequent chick mortality during the first 7 d of brooding. A computerized database representing >4,000 separate flock-ma-

2006 Poultry Science Association, Inc. Received June 11, 2005. Accepted November 4, 2005. 1 Journal article no. J-10696 from the Mississippi Agricultural and Forestry Experiment Station supported by MIS-329090. 2 Use of trade names in this publication does not imply endorsement by the Mississippi Agricultural and Forestry Experiment Station of these products or of similar ones not mentioned. 3 Corresponding author: [email protected]

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are utilized in the process of gluconeogenesis (Klasing, 1998). The digestive tracts of hatchlings may be limited in their ability to digest and utilize diets rich in proteins and carbohydrates (Uni and Ferket, 2004). Furthermore, during the transition from fat to protein and carbohydrate-based nutrition, chicks may have limited reserves of glycogen (Donaldson, 1995). Without expeditious nutrient intake, an energy imbalance can be created, and chicks may catabolize their own body tissues for use in the conversion to glucose (Donaldson, 1995; Hazelwood, 2000). In the fasted state, gluconeogenic (GNG) amino acids are mobilized from body tissues (Klasing, 1998). In general, the preference of GNG precursors by the chicken hepatocyte is lactate or glycerol > pyruvate > Ala > Ser (Klasing, 1998). Depressed blood glucose concentrations and subsequent starvation may result from the prolonged catabolism of body tissue. Yolk and albumen provide insufficient carbohydrate levels to meet the metabolic demands of the embryo or chick (Klasing, 1998). However, glucose, as a nutritional supplement added to drinking water, has been noted to suppress GNG enzyme activity (Donaldson, 1995). External GNG substances may more aptly supplement natural GNG tissue reserves and facilitate GNG activity during the critical transitional period in energy source utilization. Therefore, external GNG substances were used in this study to supplement natural GNG tissue reserves and to facilitate GNG activity during a critical transitional period in energy source utilization. It has been reported that GNG supplementation using casein hydrolysate (CSH) and added thiamine augmented the early performance of broilers from a single young breeder flock at 29 wk of age (Keirs et al., 2002). In an effort to further explore this pragmatic concept in the current study, the effects of various other GNG energy sources, in addition to CSH, that are readily available and that could be practically applied were determined in broilers from a single young breeder flock. The other GNG energy sources utilized included chicken egg crude albumin (AL) and chicken egg albumin hydrolysate (ALH).

MATERIALS AND METHODS General Unnecessary discomfort to the birds was avoided by using proper housing and handling techniques (National Research Council, 1996). In addition, this experiment was conducted under an approved Auburn University Institutional Animal Care and Use protocol. Approximately 400 Ross 508 × Ross 508 broiler hatching eggs from a single young (27 wk old) breeder flock were obtained from a local integrator. Eggs were set, incubated, and hatched under standard conditions in a single Natureform NMC2000 incubator (Natureform Hatchery Systems, Jacksonville, FL) in the hatchery of Auburn University. Eggs were transferred on d 18. At 21 d of incubation, a total of 320 non-sexed (straight run) broiler chicks were pulled and injected at random with 1 of 4 treatment solutions before

Table 1. Composition of N-Z-Amine YT hydrolyzed casein used in injected gluconeogenic solution1,2 Nitrogen and mineral content Total nitrogen Sodium Potassium Calcium Magnesium Chloride Sulfate Phosphate Amino acid content Ala Arg Asp Glu Ile Leu Lys Phe Pro Ser Val All others

(%) 13.2 2.03 0.14 0.04 0.03 0.80 0.07 2.81 (% of total amino acids) 3.2 3.6 7.3 20.2 5.2 8.7 7.9 4.8 10.1 5.8 6.5 16.7

1

Supplied by Sheffield Products, Norwich, NY. Determined analysis of product provided by manufacturer.

2

being individually identified by numerical wing banding. Chicks were then placed and brooded according to treatment in 1 of 16 environmentally controlled and isolated floor pens at the same facility. There were 4 replicate pens per treatment with 20 birds per pen. The group of birds designated as sham-injected controls were injected with 0.2 mL of physiological saline. Physiological saline injections were included as sham controls primarily to rule out a possible negative response caused by the stress of injection and handling. Three other treatment groups were injected with 0.2 mL of 1 of 3 different solutions containing a GNG energy source. Solutions contained CSH, ALH, or AL at 200-g/L concentrations. The CSH, which contained GNG amino acids, was an enzymatic digest of casein (N-Z-Amine YT hydrolyzed protein, Sheffield Products, Norwich, NY; Table 1). Calculated as a percentage of total amino acids, the CSH contained 3.2% Ala and 5.8% Ser. The ALH also contained GNG amino acids (Table 2). Calculated as a percentage of total amino acids, the ALH contained 5.0% Ala and 5.5% Ser. The AL was a nonhydrolyzed spray-dried whole egg white product (Nepco Egg of Georgia LLC, Social Circle, GA). Because a specific component of AL, avidin, is known to render dietary biotin unavailable, 2.0 mg of biotin (Sigma-Aldrich Co., St. Louis, MO) was added to each injected solution of AL to prevent the possibility of a biotin deficiency in the treated birds. Injections were made subcutaneously in the back of the neck on day of hatch, which was d 0 of grow out. On d 16, one chick per pen was weighed and sampled for histological evaluation of the injection site. Approximately 3 h after the hatch was pulled from the incubator through the completion of the trial, broiler chicks were provided a broiler starter diet that was formulated to meet

INJECTED GLUCONEOGENIC SUPPLEMENTATION

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Table 2. Composition of chicken egg albumin hydrolysate used in injected gluconeogenic solution1,2

Table 3. Ingredient percentages and calculated analysis of broiler starter diet

Nitrogen and mineral content

Ingredient

Total nitrogen Sodium Potassium Calcium Magnesium Chloride Sulfate Phosphate Amino acid content Ala Arg Asp Glu Ile Leu Lys Phe Pro Ser Val All others

(%) 12.6 1.30 1.20 0.08 0.08 1.30 1.30 0.20 (% of total amino acids) 5.0 4.7 8.2 10.5 4.3 6.8 5.1 4.7 3.1 5.5 5.6 36.5

1

Supplied by Sigma-Aldrich Co., St. Louis, MO. 2 Determined analysis of product by manufacturer indicates complete hydrolysis by P5147 protease to individual amino acids and a total nitrogen content of 11.5 to 13.0%. Other information provided (nitrogen, mineral, and amino acid content) was based on previous determined analysis of chicken egg albumen (Watkins, 1995).

or exceed National Research Council (1994) specifications (Table 3). Birds were provided with continuous lighting throughout the trial. Loss of liver glycogen stores can be further augmented in birds raised under reduced environmental temperatures (Siegel, 1980). Therefore, a properly heated brooding environment, similar to that described by Keirs et al. (2002), was provided to determine the singular effects of nutrient-supplemented treatments on chick livability and growth. The ambient temperature of the house was maintained at 24°C, and the floor (litter) temperature under the brooder hoods was maintained at 32 to 35°C between d 7 and 10. Mortality was recorded daily, and total pen BW and bird numbers were recorded at d 0, 4, 8, 12, and 16 of grow out. Added and consumed feed and water were measured by weight in each pen on d 0, 4, 8, 12, and 16 and on d 0, 4, 8, 11, 14, 15, and 16, respectively, for total pen feed and water consumption determinations. Body weight gain, feed consumption, and feed conversion were calculated between 0, 4, 8, 12, and 16 d. Water consumption was calculated between 0, 4, 8, 11, 14, 15, and 16 d. Body weight gain, feed consumption, and water consumption were calculated and expressed as grams per bird per day, and feed conversion was calculated and expressed as grams of feed intake per gram of BW gain per bird. On d 16, the individual BW and rectal temperatures of 5 birds per pen were determined. Each of those birds was then bled for determination of hematocrit (HCT) and plasma refractive index (RI). A total volume of approximately 120 ␮L of blood from each chick was collected

Corn Soybean meal (48% CP) Feed-grade poultry by-product meal Dicalcium phosphate Limestone Poultry oil DL-methionine L-Lys Vitamin premix1 Trace mineral premix2 Salt Coccidiostat3 Calculated analysis CP (%) ME (kcal/kg) Total fat (g/kg) Calcium (%) Available phosphorus (%) TSAA (%) Lysine (%)

(%) 57.8 29.6 6.00 1.20 1.10 2.80 0.24 0.10 0.50 0.25 0.39 0.08 22.5 3,080 5.83 0.94 0.44 0.95 1.27

1 Supplied the following per kilogram of finished feed: vitamin A, 7,350 IU; cholecalciferol, 2,200 IU; vitamin E, 8 IU; riboflavin, 5.5 mg; d-pantothenic acid, 13.0 mg; niacin, 36 mg; choline, 500 mg; vitamin B12, 0.02 mg; menadione, 2.0 mg; folic acid, 0.5 mg; thiamine mononitrate, 1.0 mg; pyridoxine, 2.2 mg; d-biotin, 0.05 mg. Supplied by Roche Vitamins, Inc., Parsippany, NY. 2 Supplied the following per kilogram of finished feed: copper, 6.0 mg; iron, 54.8 mg; iodine, 1.0 mg; manganese, 65.3 mg; selenium, 0.3 mg; zinc, 55.0 mg. Supplied by Roche Vitamins, Inc. 3 Coban-60, Elanco Animal Health, Indianapolis, IN.

into heparinized capillary tubes after decapitation for determination of the 2 aforementioned parameters. Livers from each of the 5 chicks were weighed and then used for determination of respective liver glycogen content (LGLY).

Relative Liver Weight and LGLY Quantitation The weights of whole fresh livers from each chick were expressed as a percentage of total wet BW. Liver samples (approximately 0.25 g) from the same lobe of each liver were used for determination of LGLY (mg of glycogen/ g of wet liver). Briefly, livers were homogenized in 10% perchloric acid and then centrifuged, leaving the glygogen in suspension. After a second extraction, supernatants were combined. Subsequently, glycogen was precipitated by ethanol addition and recovered by centrifugation. The resulting glycogen pellet was resuspended and assayed by the iodine-binding method of Dreiling et al. (1987). Separate standard curves for each batch of color reagent used each day were generated to minimize batch-to-batch variation. The use of 2 extractions was based on a preliminary experiment involving 5 extractions that showed that an approximate 95% extraction efficiency was achieved with 2 extractions.

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PEEBLES ET AL. Table 4. Average BW gain per bird per day for d 0 to 4, 4 to 8, 8 to 12, 12 to 16, and 0 to 16 and d-16 BW in sham control-, casein hydrolysate- (CSH), egg albumin hydrolysate- (ALH), and crude egg albumin- (AL) injected chicks1 Treatment Control CSH ALH AL SEM P>F

d 0 to 4

d 4 to 8

10.9 10.5 10.8 10.6 0.4 0.91

23.9 23.7 23.7 23.0 0.4 0.46

d 8 to 12

d 12 to 16

BW gain (g per bird per d) 38.3 48.3 39.0 47.7 39.2 47.6 39.4 47.7 0.7 0.9 0.73 0.94

d 0 to 16

d-16 BW

30.3 30.2 30.3 30.1 0.5 0.99

(g) 523 521 523 520 7 0.99

1 No statistical differences were detected between treatment means within each column. n = 4 replicate units for the mean of each parameter within each treatment.

Rectal Temperature, HCT, and RI Determinations Rectal temperature (°C) was determined by inserting a YSI series 400 temperature probe (YSI, Inc., Yellow Springs, OH), connected to a Nist Traceable digital thermometer (Control Co., Friendswood, TX), approximately 1 cm into each chick via the rectum. Hematocrit, expressed as percentage blood packed cell volume, was determined via the use of capillary tubes that were centrifuged for 5 min in an IEC MB centrifuge (Damon/IEC Division, Needham Heights, MA) and read with a microcapillary reader (YSI, Inc.). Refractive index (g of protein/ dL) was determined by optical observation after dispensing 25 ␮L of plasma between the measuring prism and cover plate of a Model 10406 TS meter (American Optical Co., Scientific Instrument Division, Buffalo, NY).

Statistical Analysis Individual sample data within each replicate pen were averaged prior to analysis. Angular transformations (arc sine of the square root of the proportion affected) were performed on all percentage data prior to analysis. A completely randomized experimental design was employed, and data were analyzed separately for each specified age and age interval by one-way analysis of variance using the GLM procedure of SAS, Version 8.1 (2000). Least squares means were compared in the event of significant global effects (Steel and Torrie, 1980). Statements of significance were based on P ≤ 0.05, unless otherwise indicated.

RESULTS AND DISCUSSION In the current investigation, there was no bird mortality in any treatment group, and examination of the sham and treatment injection sites in birds on d 16 revealed no local histological reaction. (All lesions were mild with minimal inflammation.) Therefore, subcutaneous injection in the hatchery can be a practical means by which to provide GNG supplementation to chicks. Subcutaneous injections of GNG substances were provided in this study in an effort to prevent an energy imbalance and the catabolism of body tissues of posthatch broiler chicks from young breeder hens. However, injected saline, AL, CSH,

or ALH did not significantly affect growth (Table 4), feed consumption, feed conversion, or water consumption at any of the age intervals examined from hatch through d 16. Furthermore, none of the metabolic indices investigated on d 16 (rectal temperature, HCT, RI, relative liver weight, or LGLY; Table 5) were impacted by any of these treatments. Uni et al. (2005) reported that in ovo injection of carbohydrates and β-hydroxy-β-methylbutyrate into the amnion of late-term embryos (17.5 d of incubation) increased BW by 5 to 6%, improved liver glycogen 2- to 5-fold, and elevated relative breast muscle size by 6 to 8% in posthatch broiler chicks. Also, in a previous report (Keirs et al., 2002), the early performance of posthatch broiler chicks from a single young breeder flock at 29 wk of age and under adequate brooding conditions was shown to respond favorably to subcutaneously injected saline. Nevertheless, subcutaneously injected CSH and added thiamine also provided limited (d 1 and 6 of grow out) growth facilitation over saline. It was concluded in that study, that under adequate brooding conditions, chick growth may be facilitated by saline and further augmented by supplemental GNG nutrients during the early transition from fat to protein and carbohyrate-based nutrient uptake in posthatch broiler chicks. A hydration effect from the saline was suggested but not experimentally tested. However, the added growth response observed in the GNG substance-treated birds might have occurred as a result of a sparing effect on tissue catabolism. In one of the 2 trials of that study, 85% of the negative control chicks gained 10% BW within the first 24 h, confirming adequate brooding conditions. The BW of these same chicks nearly doubled at d 4 and nearly tripled at d 6. Because the negative control birds achieved normal growth when provided adequate brooding, it was also suggested that under less than adequate brooding conditions, the growthpromoting effect of saline or GNG substances might be increased. Although AL was provided with added biotin, its effective utilization by chicks after subcutaneous injection was questionable because it was not predigested. However, based on the previous results of Keirs et al. (2002) and the comparable Ala and Ser percentages of ALH and CSH, it was anticipated that these 2 treatments would have facilitated growth in the newly hatched chicks. The

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INJECTED GLUCONEOGENIC SUPPLEMENTATION Table 5. Rectal temperature (RT), hematocrit (HCT), plasma refractive index (RI), relative liver weight (RLW), and liver glycogen (LGLY) content at d 16 in sham control-, casein hydrolysate- (CSH), egg albumin hydrolysate(ALH), and crude egg albumin- (AL) injected chicks1 Treatment Control CSH ALH AL SEM P>F

RT

HCT

RI

RLW

LGLY

(°C) 41.7 41.7 41.6 41.7 0.1 0.80

(%) 29.4 29.8 30.8 29.4 0.5 0.17

(g/dL) 87.8 87.2 89.2 85.7 1.6 0.52

(%) 2.74 2.69 2.77 2.59 0.07 0.28

(mg/g) 10.91 9.22 8.11 5.80 3.39 0.76

1 No statistical differences were detected between treatment means within each column. n = 4 replicate units for the mean of each parameter within each treatment.

successful use of carbohydrates in late-term embryos by Uni et al. (2005) suggests that carbohydrates in addition to or in place of amino acid-based supplementation in hatchlings may facilitate growth. Reasonably, the use of carbohydrate supplementation in hatchlings and GNG amino acid supplementation in embryos as a means by which to potentiate posthatch growth in broiler chicks should be further investigated. Nevertheless, the contrasting effects of the CSH in the current and previous study by Keirs et al. (2002) may be related to the use of added thiamine. Keirs et al. (2002) added thiamine to CSH at approximately 4 times the thiamine requirement for each bird for the first week of posthatch life. Thiamine was added to prevent the possibility of a thiamine deficiency and a subsequent decrease in chick appetite (Hawk et al., 1949). The lack of differences in feed consumption and growth among treatments in the current study confirmed that added thiamine was not essential for birds injected with the GNG supplements. However, the contrasting effects of CSH with and without added thiamine in the 2 studies does suggest that thiamine may facilitate the utilization of hydrolyzed GNG nutrient sources injected into posthatch chicks. Thiamine is essential for the growth and metabolism of all animals and plays an important role in cellular metabolism of carbohydrates and proteins. The synthesis of fats from carbohydrates requires thiamine, and supplemental thiamine has been shown to dramatically increase the survival rate of animals fed diets consisting only of glucose or casein (Hawk et al., 1949). Hamano et al. (1999) further reported that absolute rate of liver oxygen consumption and relative breast muscle weight were increased, and that plasma glucose and triglyceride concentrations were decreased, in broiler chicks at 7 d of age after having been injected intramuscularly with thiamine at 2, 4, and 6 d of age. In conclusion, upon consideration of the results from this and earlier studies, it is suggested that under adequate brooding conditions and the timely provision of external nutrition, injected GNG hydrolysates do not alone improve LGLY or facilitate early growth in posthatch broiler chicks from young hens. However, further research concerning the use of supplemental carbohydrates and thiamine alone or as means by which to augment the utilization and potential growth-promoting

effects of injected GNG nutrient digests in embryos and hatchlings may be justified. The possible benefits of these injected substances in chicks from young parents that are subjected to less-than-adequate brooding conditions and prolonged delays in nutrition provision also deserve further consideration.

ACKNOWLEDGMENTS This work was funded through Hatch Projects MISV2993 and MIS-329090 and funds from the College of Veterinary Medicine and the Mississippi Agricultural and Forestry Experiment Station at Mississippi State University. The authors appreciate the expert technical assistance of Sharon Whitmarsh (Mississippi State University, Mississippi State).

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