WO1990009188A1

WO1990009188A1 - In ovo administration of growth factors alters physiological development and response of poultry

Info

Publication number: WO1990009188A1
Application number: PCT/US1989/005131
Authority: WO
Inventors: Pamela S. Hargis; Samuel L. Pardue
Original assignee: The Texas A&M University System
Priority date: 1989-02-08
Filing date: 1989-11-16
Publication date: 1990-08-23
Also published as: AU5166590A; AU5102690A; WO1990009190A1

Abstract

The invention relates to a procedure for the in ovo introduction of biologically active compounds into fertile eggs of poultry to manipulate the physiological development and response of the birds after hatching.

Description

IN OVO ADMINISTRATION OF GROWTH FACTORS

ALTERS PHYSIOLOGICAL DEVELOPMENT AND RESPONSE OF POULTRY

The present invention relates to the production of meat producing poultry. It further relates to the in ovo introduction of biologically active substances into the fertile eggs of the poultry to manipulate physiological development and response of the poultry after hatching.

It further relates to the use of certain food additives to trigger or enhance certain altered characteristics in the maturing poultry. As part of an effort to increase the human food supply, investigators have explored numerous means of manipulating tissue development in commercial meat producing animals. Most of the work has centered on genetic manipulation, nutrition therapy and pharmaceutical

therapy. These teachings have been somewhat successful but they present various problems.

For example, genetic selection programs for improved growth rates in chickens have led to the development of increasingly fat broiler stocks. Excessive lipid deposition results in increased production costs for the broiler producer as well as poor consumer acceptability of the broiler product. Nutritional and pharmaceutical therapy of hatched chickens is extremely labor and capital intensive and is often unsuccessful.

The object of this invention is to provide a less expensive and more efficient and effective method of producing better poultry. This invention meets this goal by providing for a simple process of in ovo manipulation of the physiological development and response of the poultry. The method involves the introduction of various growth factors into the incubating fertilized eggs. The altered poultry are allowed to hatch and mature. This results in altered growth, development and physiological responses in the maturing poultry without further

treatment with the growth factors. Some of these altered characteristics of the maturing poultry may be triggered or enhanced by administering to the poultry a betaadrenergic agonist and this invention further provides for this production step. The invention further provides for a product, poultry, which have been altered by in ovo introduction of growth factors. More specifically, the invention provides for a method of enhancing the growth and meat production of poultry by injecting fertilized poultry eggs, particularly chickens or turkeys, with a growth factor. Specific growth factors which may be injected include, but are not limited to, bovine, ovine or porcine growth factor, thyroid hormones (i.e. T₃ and T₄) and isoproterenol. It further provides that the eggs be injected during the time period from and including the first day of the second half of the incubation period up to and including the day the eggs are transferred from the incubator to the hatcher. More specifically, it provides that the eggs be injected on the first day of said time period or on the day the eggs are transferred to the hatchery. The physiologically altered poultry are then allowed to hatch and mature. The invention further provides for the optional step of administering a beta-adrenergic agonist to the maturing poultry to enhance or trigger the altered physiological development and response.

Most specifically, the invention provides for a method of enhancing the growth and meat production of poultry by injecting an effective amount of ovine growth hormone or thyroid hormone into the albumin of incubating fertilized chicken eggs. The eggs are injected on the first day of the second half of the incubation period of the eggs. Thereafter, the altered poultry is allowed to hatch and mature. Isoproterenol may then be fed to the altered poultry to enhance or trigger certain altered characteristics. Figure 1 shows the effect of in ovo injection of oGH on shank length of broilers at 7 weeks of age. As the figure indicates, shank length increased significantly in males treated with either 2.5 or 250 μg of oGH. Figure 2 shows the adipocyte volume distribution at 7 weeks of age following in ovo oGH administration. Both male and female broilers treated with 250 μg oGH possessed higher percentages of large volume adipocytes than the controls.

Figure 3 shows the effect of in ovo injections of oGH on subsequently induced lipolysis of adipocytes in vitro. Lipolysis was measured as a function of glycerol release. The data indicate that oGH inhibits lipolysis by

increasing phosphodiesterase activity. Figure 4 shows the effect of in ovo injection of T₄ and T₃ and feeding of isoproterenol on body weights of male broilers. The data indicates that T₃ increased male body weight over the control and that T⁴ decreased body weight compared to the control.

Figure 5 shows the effect of in ovo injection of T₄ and T₃ and feeding of isoproterenol on body weights of female broilers. The data indicates that T₃ increased body weight through week 4 and that after week 4 both T₃ and T₄ decreased body weight compared to the control.

Figure 6 shows the effect of in ovo injection of T₄ and T₃ and feeding of isoproterenol on carcass protein composition. The data indicates that T₃ produced a significant increase in protein content in male broilers compared to controls.

Figure 7 shows the effect of in ovo injection of T₄ and T₃ and feeding of isoproterenol on carcass fat percentage. The data indicates that T₃ tends to decrease carcass fat compared to controls.

Figure 8 shows the effect of in ovo injection of T₄ and T₃ and feeding of isoproterenol on abdominal fat pad weight as a percentage of body weight. The data indicates that T₄ decreased percentage of fat pad weight compared to controls. The discussion and examples which follow detail the best known method for performing the invention. The method provides for the introduction of biologically active substances into the eggs of poultry to alter the physiological development and responses of the hatched birds. This method may be used for all poultry (i.e. domesticated birds kept for eggs or meat), including chickens and turkeys.

The in ovo manipulation can cause many desired changes in the physiological development and response of maturing birds after hatching. Some of these effects appear to be sex dependent. Growth factors can increase body weight, skeletal development and feed efficiency. This is especially notable with the use of mammalian growth hormones. Growth factors also affect adipose tissue development, altering both the size of adipose cells and the lipolytic responsiveness of such cells.

Some of the altered physiological changes are

phenotypically expressed in response to dietary changes. Injecting a growth factor in ovo and then administering to the poultry after hatching a beta-adrenergic agonist has resulted in increased growth rate, more lean tissue deposition and decreased fat accretion. It is believed that other alterations of physiological development and response will result in improved immunological response. Additionally it is believed that reproductive performance and response to heat stress may be enhanced.

The growth factors which may be utilized are various substances which affect the growth and development of tissue, and which affect physiological responses. The preferred growth factors are growth hormones and the thyroid hormones, T₃ and T₄. The most preferred growth hormones are mammalian hormones, particularly ovine and bovine. Other possible growth factors include, but are not limited to other hormones (e.g. androgens), various steroids, and beta-adrenergic agonists such as

isoproterenol. The amount of growth factor used depends upon the type and size of the egg, the species of poultry, and the type of growth factor used. For example, a range of 2.5 μg to 250 μg of growth homone is preferred for broilers with 250 μg most preferred, but 375 μg were administered to turkeys. For the thyroid hormones, the preferred amount for administration to broilers is .01 μg of T₃ and 1.0 μg of T₄. The preferred dosage was chosen because the amount is biologically effective and results in the highest rate of hatchability. These examples are not intended to limit the amounts of growth factor which may be used.

The growth factors are introduced into the eggs of the poultry during incubation. The growth factor may be introduced into the albumin or onto the chorioallantoic membrane. Those skilled in the art will appreciate that the site of introduction may also impact the necessary dosage. It is believed, although the inventors are not limited to this theory, that a smaller dosage will be effective if introduced onto the membrane. The preferred method of introduction is injection, although other methods such as spraying or dipping may be used if the growth factor is of sufficiently small size.

The preferred time window for injection is from and including the first day of the second half of the

incubation period up to and including the day of transfer from the incubator to the hatcher e.g. days 11 to 18 for broilers. This does preclude the injection of growth factor earlier in the incubation period. The incubation period is the time period from the day the eggs are placed in the incubator until they hatch. This includes the time period the eggs are in the hatcher. It is believed that the day the eggs are transferred from the incubator to the hatcher will be the most efficient day for introduction of the growth factor. Additionally, it appears that the hatchability rate is higher when the eggs are injected later in the incubation period. The inventors also believe, but are not limited to this theory, that later introduction of growth factors may be more effective as certain tissues mature and become more active. For example, growth hormones may have a greater effect on body weight when injected after day 17 of the broiler

incubation period as the anterior pituitary becomes more active. At this later stage of incubation the growth factor must be injected onto the chorioallantoic membrane. It is preferred for efficiency purposes that the eggs be injected only one time, however it is not precluded that a number of injections may be utilized if necessary for the desired result. Some of the changes in growth development and

response, such as, but not limited to, lipolysis may be enhanced or triggered by administration of certain food additives to the maturing birds. A preferred class of additives is the beta-adrenergic agonists, in particular isoproterenol. While these compounds are characterized as food additives it is not precluded that they could be administered to the birds in a different fashion.

Additionally, the time period of administration and the amounts used may differ depending on the size and needs of the poultry species and the desired result.

Example 1; The affect of in ovo administration of growth hormone on broiler chickens. Administration of Growth Hormone

Two trials were conducted. In trial one, 100 fertile eggs from a commercial broiler were injected into the inferior end of the egg on day 11 of embryonic development with 100 μℓ of either vehicle alone as a control or vehicle containing 0.25, 2.5, 25 or 250 μg of ovine GH (oGH). In trial 2, 600 eggs were injected with either vehicle, 2.5 or 250 μg of oGH on day 11 of the incubation. The vehicle consisted of the following: 0.03 M NaHCO₃ (Sodium Bicarbonate) and 0.15 M NaCl (Sodium Chloride), pH 8.3. Injections were performed manually by first piercing a hole in the shell with an 18-gauge needle and then injecting the solution into the albumin using a 25-gauge needle. The injection holes were sealed with paraffin. Determination of Growth

And Feed Efficiencies

At hatch, chicks from both trials were wingbanded, weighed and sexed. Chicks were randomly assigned to floor pens according to sex and treatment. Birds were fed diets ad libitum. From 0 to 3 weeks of age the birds were fed a 22% protein, 3125 kcal energy starter diet. From 3 to 7 weeks of age the birds were fed a 19% protein, 3284 kcal energy grower ration. Body weight and feed consumption were recorded weekly. Shank length was measured at 3, 5 and 7 weeks of age.

Determination of Carcass Composition Birds chosen for carcass analysis were sacrificed by cervical dislocation at 7 weeks. Fat pad weights were recorded. Frozen carcasses (including feathers) were ground twice in a commercial meat grinder to obtain an homogenous mixture. Carcass fat content was determined by ether extraction and protein content was determined by the Kjedahl method. Methods of Analysis, Association of

Official Analytic Chemists, (1984). Cellularity Studies

A sample of the fat pad of the broilers used for carcass analysis was obtained for adipose cellularity studies. Cells were prepared for sizing and counting by a modification of the procedure of Hirsh and Gallain, J. Lipid Res. 9:110-119 (1968). A 50-100mg sample of each fat pad was immediately placed in a 3% osmium tetroxide solution: 1.5 ml of 50 mM collidine, pH 7.4 plus 2.5 ml of 3% osmium tetroxide (diluted in the collidine buffer) for 72 hrs at 41°C for fixation. Following fixation, samples were washed with 0.9% saline (pH 7.4). Salinewashed samples were then incubated in 8 M urea for 48 hrs at 25°C. Adipocytes were then separated into two cell size ranges by sequential filtration through 240, 64 and 25 micron nylon screens. Debris collected on the 240 micron screen was discarded. Cells were washed from the remaining screens with 0.1% Triton X-100 in saline, pH 10. Cell size and number were determined by use of an electronic particle counter (Coulter Electronics, Inc., Model ZM) and channelizer (Coulter Electronics, Inc., Model C256). A 0.5 ml sample of the continuously stirring cell suspension was aspirated through a 400 micron aperature tube for cell sizing and counting. The distribution of cell diameters was calculated by totalling the sum of each 10-micron increment of cell sizes. Cell number was calculated using the specific density of fat (0.901) and the average cell volume. Average cell diameters and volumes were calculated using the standard geometrical formulas for a sphere.

In Vitro Lipolysis

Adipocytes were isolated from the abdominal fat pads of 7-week old broilers by a modification of the procedure of Rodbell, J. Biol. Chem. 239:375-380 (1964). Adipose tissue was placed in warmed, oxygenated Krebs-Ringer bicarbonate buffer (KRB) containing 0.1% collagenase (Sigma, lot 126F-6735), 4% albumin and 1 mM glucose for approximately 45 min in a shaking waterbath at 41°C.

During this time, samples were gently agitated using wide-mouthed siliconized pasteur pipettes to mechanically disrupt clumps of connective tissue. Following enzymatic disruption of the tissue, the samples were washed 3x in the above KRB solution, collagenase-free. Samples were then filtered through a 240 micron nylon-mesh filter and counted as previously. Dilutions of 3.0 x 10 cells/ml were made and 0.5 ml of the cell suspension was added to 0.5 ml of warmed KRB buffer. Fifty microliters of buffer containing each lipolytic compound at specified concentrations were added to the appropriate culture tubes.

Following an 1 hr incubation at 41°C in a shaking waterbath, culture tubes were placed on ice and 1 ml of trichloroacetic acid was added immediately to stop the reaction and precipitate all protein. Samples were then centrifuged at 5°C for 20 min. The supernatant was decanted and extracted 3 times with 5 volumes of diethyl ether. Following the third extraction, the aqueous phase was stored at -20°C for subsequent glycerol analysis following the method of Buccolo and David, Clin. Chem. 19:476-482 (1973).

Statistical Analysis

Data were analyzed using the GLM procedure of the Statistical Analysis Systems Institute. Barr, A.J., J.H. Goodnight, J.P. Sall, W.B. Blair, and D.M. Chilko. SAS User's Guide. Raleigh, N.C. SAS Institute. 1979.

Analyses were conducted within sex and age groups testing for treatment effects. If significant treatment effects (P<.05) were observed, differences between means were determined using Duncan's multiple range test. Determination of Growth

and Skeletal Development

Growth was determined as increase in body weight and skeletal development (which was determined by increase in shank length). In ovo administration significantly increased body weights of male broilers during the high protein phase of the grow out trial (0-3 wks). Body weights did not differ between control- and 2.5μg oGH-injected male broilers from 4-7 weeks of age. Males injected with 250μg oGH were significantly heavier than either of the other two treatments at 7 weeks of age.

(Table 1). Body weights of female broilers were not significantly altered. (Table 2). Similarly, the in ovo hormone treatment did not influence shank length of females, however injection of either 2.5 or 250μg of oGH resulted in significantly greater shank lengths in male broilers. (Figure 1). Measurements for shank length were taken at 7 weeks of age.

Table 1. Effect of in ovo ovine growth hormone injection on body weight (gms) and cumulative feed conversion ratios of male broilers from 1 to 7 weeks of age.

Dosage (μg/egg)

Age (weeks)* Vehicle 2.5 250

1 95 ± 2.8^a 102 ± 1.7^ab 103 ± 2.1^b 2 256 ± 6.5³ 271 ± 3.4^b 275 ± 5.8^b

3 519 ± 7.5^a 548 + 13.7^ab 553 ± 10.7^b

Feed/Gain 1.88 ± 0.07^a 1.70 ± 0.06^b 1.68 ± 0.04^b

(week 0-3)

4 920 ± 12.5^a 914 ± 8.9^a 936 ± 9.6^a

5 1346 ± 26.8^a 1323± 23.4^a 311 + ± 30.9^a

6 1873 ± 14.1^a 1884 ± 21.4^a 1889 ± 12.7^a

7 2411 ± 19.6^a 2413 ± 17.0^a 2464 ± 18.2^b

Feed/Gain 2.36 ± 0.06^a 2.36 ± 0.02^a 2.30 ± 0.03^a

(week 4-7)

^*Values are means ± SEM. Means within age groups differ significantly (P<.05) if superscripts differ.

Feed to Gain Ratio

Feed to gain ratio determinations were made on the broilers. Male broilers injected with oGH exhibited significantly improved feed conversion ratios from hatch to 3 weeks of age. No difference in feed to gain ratios was observed during the 4-7 week period. (Table 1). The feed to gain ratio in female broilers was not

significantly altered during either time frame by

injection of oGH. (Table 2).

Table 2. Effect of in ovo ovine growth hormone injection on body weight (gm) and cumulative feed conversion ratios of female broilers from 1 to 7 weeks of age.

Dosage (μg/egg)

Age (weeks)* Vehicle 2.5 250

1 98 ± 1.8 98 ± 2.8 100 ± 2.2 2 247 ± 7.1 256 ± 6.9 259 ± 5.1

3 498 + 11.3 513 ± 12.8 497 ± 9.5

Feed/Gain 1.88 ±0.05 1.72 ±0.07 1.77 ± 0.03 (week 0-3)

4 834 ± 7.1 820 ± 16.6 823 ± 12.2

5 1152 ± 11.5 1172 ± 30.2 1159 ± 20.5

6 1579 ± 22.0 1578 ± 30.4 1588 ± 19.4

7 2024 ± 14.9 2022 ± 13.3 2024 ± 13.8

Feed/Gain 2.44 ± 0.05 2.44± 0.02 2.43 ± 0.02 (week 4-7)

^*Values are means ± SEM. Means within age groups did not significantly (P<.05) differ.

Carcass Composition

In ovo hormone treatment did not statistically alter fat or protein content of the carcasses of either selected male or female broilers. (Table 3). Neither abdominal fat pad weights or fat pad as a percent of body weight was significantly altered in either of the sexes. (Table 4).

Table 3. Effect of in ovo administration of ovine growth hormone on percent carcass fat and protein of 7-week-old broilers.

Dosage* Male Female

(μg/egg)

Carcass Fat (%)

Vehicle 14.7 ± 0.5 15.7 ± 0.3

2.5 13.1 ± 0.5 15.5 ± 0.5

250 13.9 ± 0.6 16.4 ± 0.4

Carcass Protein (%)

Vehicle 16.0 ± 0.2 15.2 ± 0.2

2.5 15.8 ± 0.2 15.5 ± 0.2

250 15.5 ± 0.2 15.3 ± 0.2

^*Values are means ± SEM. Means within sexes did not significantly (P>.05) differ.

Table 4. Effect of in ovo administration of ovine growth hormone on abdominal fat pad weights of 7-week-old broilers.

Dosage* Male Female

(μg/egg)

Fat Pad (gms)

Vehicle 42.3 ± 1.7 50.3 ± 1.7

2.5 47.5 ± 2.9 49.2 ± 2.2

250 45.1 ± 3.4 51.7 ± 2.8

Percent Fat Pad

Vehicle 1.77 ± 0.07 2.47 ± 0.08 2.5 1.90 ± 0.11 2.41 ± 0.12

250 1.79 ± 0.13 2.45 ± 0.11

^*Values are means ± SEM. Means within sexes did not significantly (P>.05) differ. Adipose Cellularity

Adipose cellularity data on selected broilers suggested that in ovo oGH administration altered adipose tissue development. Both male and female broilers treated with 250 μg of oGH exhibited larger adipocytes with a corresponding decrease in the number of cells per gram tissue, as determined at 7 weeks of age. (Table 5).

Birds treated with oGH had a greater proportion of fat cells from large volume classes than did the controls. (Figure 2).

Table 5. Effect of in ovo injection of ovine growth hormone on abdominal fat pad cellularity of broilers.

Mean Mean

Dosage* Adipocyte Number Adipocyte Volume Adipocyte Diameter

(μg/egg) (x 10 /10^-1gm) (pL) (μm)

Male

Vehicle 8.7 ± 0.5^a 63.1 ± 5.5^a 44.9 ± 1.3

2.5 9.6 ± 2.6^ab 63.4 ± 4.5^a 45.1 ± 1.1^ab

250 6.2 ± 0.6^b 76.7 ± 4.5^b 48.8 ± 1.2^b

Female

Vehicle 11.6 ± 2.6^a 58.9 ± 5.5^a 44.3 ± 1.4^a

2.5 8.9 ± 1.6^ab 60.5 ± 5.7^a 44.8 ± 0.9^a

250 6.5 ± 0.8^b 79.4 ± 3.8^b 49.0 ± 0.7^b

*Values are means ± SEM. Parameter means differ significantly (P<.05) within sexes if superscripts differ. In Vitro Lipolysis Studies

An increase in adipocyte cell size suggests a decrease in lipolysis. Studies were done to determine if lypolysis is effected by in ovo injection of oGH, and if so how.

Glycerol release was used as an indicator of

lipolysis. Three compounds which induce lipolysis by differing mechanisms were tested: cholera toxin (enhances adenylate cyclase through a receptor-independent

mechanism), theophylline (inhibits phosphodiesterase), and dibutyryl cAMP ( dcAMP - - not degraded by phosphodiesterase). A combination of cholera toxin and theophylline was also used. The results are shown in Figure 3.

The adipocytes exhibited decreased sensitivity to cholera toxin or theophylline-induced lipolysis and increased lipolytic response to dcAMP in vitro, suggesting that oGH injected in ovo decreased lipolysis either by diminishing adenylate cyclase activity or enhancing phosphodiesterase activity. The combined addition of cholera toxin and theophylline significantly improved the lipolytic response over that of the addition of either compound alone, further implicating high phosphodiesterase activity in adipocytes of broilers injected with oGH in ovo. Example 2: Determination of Growth and Skeletal

Development of Broilers in Response to Vatious Sources of Mammalian GHs.

Four hundred fertile eggs from a commercial broiler strain were injected with either vehicle (control injection) or 250 μg of either bovine or porcine GH on day 11 of incubation. (See Example 1 for basic procedures.) Growth was determined as an increase in body weight and skeletal development was determined by an increase in shank length. Males injected with bGH were significantly heavier than control males, at 3, 5 and 7 weeks (Table 6). Shank length was significantly greater in bGH-treated males versus controls (Table 7).

Table 6. Effect of in ovo administration of mammalian GHs on body weights of male broilers.

Body Weight (gm)¹

Age Control Bovine Porcine

(weeks)

0 39 ± 0.2^a 39 ± 0.4^a 39 ± 0.3^a 1 121 ± 1.2^a 121 ± 1.8^a 123 ± 1.6^a 3 597 ± 4.7^a 615 ± 6.8^b 600 ± 5.9^ab

5 1277 ± 9.3^a 1345 ±14.2^b 1296 ±12.6^a

7 1869 ±10.6^a 1966 ±18.7^b 1901 ±16.6^a ¹Treatment means ± SEM significantly (P>.05) differ within ages if superscripts differ.

Table 7. Effect of in ovo administration of mammalian GHs on carcass parameters of male broilers at 7 weeks of age.

Parameter Control Bovine Porcine

Abdominal Fat 27.1 ± 1.39^a 30.5 ± 1.98^a 32.3 ± 1.46^a

Pad Weight (gms)

Shank Length 61 ± 0.56^a 63 ± 0.53^b 60 ± 0.46^a (mm)

¹Parameter means ± SEM significantly (P<.01) differ if superscripts differ.

Example 3: Summary of Trials Examining the Effects of In Ovo GH Administration on Seven Week Body Weights of Broilers.

Four trials (See Example 1 for general procedures) have been conducted to assess the influence of in ovo GH (250 μg/egg at 11 days of incubation) administration on subsequent growth of broilers. In each trial, in ovo GH resulted in improved 7 week body weights of male broilers (Table 8).

Table 8. In ovo mammalian growth hormone administration

significantly improves 7 week body weights of male broilers.

Weight (gms)

Trial 1

Control 2217± 76

GH* 2408 ± 59

(P<.05)

Trial 2

Control 2411 ± 20

GH* 2464 ± 18

(P<.05)

Trial 3

Control 2010 ± 22

GH* 2068 ± 22

(P<.04)

Trial 4

Control 1869 ± 11

GH** 1966 ± 19

(P<.0001)

*Ovine GH (250 μg/egg) was injected into the inferior end of fertile broiler eggs at day 11 of embryogenesis.

**Bovine GH (250 μg/egg) was injected into the inferior end of fertile broiler eggs at day 11 of embryogenesis. Example 4 : Determination of Growth and Skeletal

Development of Turkeys In Response to

In Ovo Administration of oGH.

Six hundred and eighty fertile eggs from a commercial turkey strain were injected in the inferior end with either vehicle or 375 μg oGH on day 15 of incubation. Injections were performed manually by first piercing a hole in the shell with an 18-gauge needle and then injecting the solution into the albumin using a 25-gauge needle. The injection holes were sealed with paraffin. At hatch, poults were wingbanded, weighed and sexed.

Birds were randomly assigned to floor pens according to sex and treatment and were fed conventional diets.

Growth was determined as increase in body weight.

Preliminary results indicate that the in ovo

administration of oGH significantly increased hatch and 10 week body weights of females (Table 9). Numerical differences among female treatment groups occurred at all other ages. Body weights of male turkeys were not significantly altered.

Table 9. Effect of in ovo ovine growth hormone injection on body weight (gms of turkeys from hatch of 10 weeks of age.

Dosage (μg/egg)

Age Vehicle 375

(weeks)*

Male

0 62.0 ± .6^a 61.7 ± .7^a

1 132.1 ± 1.9^a 133.2 ± 2.1^a

3 574.5 ± 9.6^a 571.9 ± 9.2^a

5 1430.1 ± 19.5^a 1421.5 ± 20.0^a

10 5198.8 ± 72.4^a 5265.9 ± 57.0^a

Female

0 60.5 ± .5^b 62.0 ± .5^a

1 120.8 ± 1.9^a 125.7 ± 1.8^a

3 518.9 ± 7.4^b 522.2 ± 8.4^a"

5 1236.8 ± 16.8^a 1275.3 ± 18.8^a

10 4350.4 ± 55.2^b 4535.7 ± 62.7^a

* Values are means + SEM. Means within age and sex differ significantly (P<.05) if superscripts differ. Example 5: Effect of In Ovo Administration of Thyroid Hormone On Lipolytic Responsiveness. Eggs (N=100/treatment) were incubated and injected in the inferior end with 100 μℓ, of phosphate buffered saline (pH 7.4) containing 0.1% BSA or .01 μg T₃ (Sigma). At hatch, birds were sexed, wingbanded, weighed and randomly assigned to floor pens by sex and treatment.

Body weights and feed consumption were recorded weekly and at 35 and 49 days of age adipocytes were isolated from abdominal fat pads by a modification of the method of Rodbell (1964). Fat pads from four birds per in ovo treatment were excised and rinsed in warm 0.9% saline (pH 7.4) to remove free lipid droplets. Fat tissue was then minced and a 4 gram sample from each in ovo treatment group placed into Kreb's Ringer bicarbonate buffer containing 0.1%

collagenase (Sigma, lot #126F-6735), 4% albumin, and 1 mM of glucose for 45 minutes in a shaking water bath at 41°C. Following enzymatic digestion, samples were washed three times in oxygenated collagenase-free Krebs-Ringer bicarbonate buffer containing 4% albumin and 1 mM glucose (pH 7.4). Adipocytes were then filtered through a 243 micron nylon-mesh filter to remove debris and cell clumps and counted by use of an electronic particle counter (Coulter, model ZM). Dilutions of 3-4 x 10 cells per ml were made and 0.5 ml of the cell dilution was added to 0.5 ml of warmed Krebs-Ringer bicarbonate buffer (pH

7.4). Fifty μℓ of a specific lipolytic agent was added to each culture tube. Treatments consisted of 0, 10, 100, 1000, and 10,000 ng/ml glucagon; 0, 1 and 10 mM theophylline; 0, 5, 10 and 20 μg of cholera toxin; or 10 mM of dibutyrl cAMP. Following a one-hour incubation at 41°C in a shaking water bath, culture tubes were placed on ice and 1 ml of 10% trichloroacetic acid immediately added to terminate the culture. The supernatant was removed and extracted three times with 5 volumes of diethyl ether. The aqueous phase was stored at -20°C for subsequent glycerol

analysis (Bucolo and David, 1973).

Data were analyzed using the GLM procedure of

Statistical Analysis Systems Institute (SAS, 1982).

Significant treatment effects (P<.05) were further separated using Duncan's multiple range test (Duncan, 1955). Effect On In Vitro Lipolysis

Glycerol release was used as an indicator of

lipolysis. Concentrations of 1 and 10 mM of theophylline significantly increased lipolysis at 35 and 49 days of age in cells of T₃-treated birds (Table 10). Adipocytes from control birds exhibited significant increases in lipolysis at concentrations of 10 mM of theophylline only (Table 10). The addition of 2 or 10 mM dcAMP to

adipocytes isolated from both control and T₃-treated birds resulted in significant increases in glycerol release at both 35 and 49 days (Table 11). Glycerol release, however, was significantly greater in response to 10 mM dcAMP in cells from T₃-treated birds as compared to controls (Table 11). No significant differences in glycerol release in response to cholera toxin were observed between control of T₃-treated birds at 35 or 49 days of age (Table 12). Cells from both in ovo

treatments responded maximally to the same dosage of cholera toxin (5 μg/ml). Increased lipolytic sensitivity to glucagon was seen in adipocytes from T₃-treated birds at both 35 and 49 days of age as compared to controls, although maximal stimulation of glycerol release was not statistically different.

Table 10. In vitro lipolytic response of adipocytes isolated from broilers injected in ovo with T₃ to theophylline.

Theophylline Control T₃

(mM)

35-Days of Age²

0 1.22 ± 0.09^a 1.25 ± 0.07^a

1 1.55 ± 0.23^a 2.40 ± 0.35^b

10 1.83 ± 0.07^b 2.60 ± 0.37^b

49-Days of Age"

0 1.90 ± 0.20^a 2.02 ± 0.65^a

1 1.95 ± 0.58^a 4.15 ± 0.23^b

10 3.72 ± 0.09^b 4.04 ± 0.25^b

Means within in ovo treatments significantly differ

(P≤.05) from basal if superscripts differ.

2Glycerol release (μg/1.8 x 10⁵ cells).

3Glycerol release (μg/2 x 10⁵ cells). Table 11. In vitro lipolytic response of adipocytes isolated from broilers injected in ovo with T₃ to dibutryl cAMP (dcAMP).

dcAMP¹ Control T₃

(mM)

35-Days of Age²

0 0.86 ± 0.05^a 0.98 ± 0.03^a

2 2.03 ± 0.20^b 2.08 ± 0.08^b

10 1.79 ± 0.18^b 2.55 ± 0.16^b

49-Days of Age³

0 1.90 ± 0.20^a 2.02 ± 0.65^a

2 6.42 ± 0.46^b 5.84 ± 0.65^b

10 7.60 ± 0.50^b 8.19 ± 0.24^c

¹Means within in ovo treatments significantly differ

(P≤.05) from basal if superscripts differ.

2Glycerol release (μg/1.8 x 10⁵ cells).

3Glycerol release (μg/2 x 10⁵ cells). Table 12. In vitro lipolytic response of adipocytes isolated from broilers injected in ovo with T₃ to cholera toxin.

Cholera toxin¹ Control T₃

(μg/ml)

35-Days of Age²

0 0.86 ± 0.05^a 0.98 ± 0.03^a

5 1.38 ± 0.18^b 1.61 ± 0.06^b

10 1.72 ± 0.17^b 1.62 ± 0.14^b

20 1.32 ± 0.12^b 1.26 ± 0.08^b

49-Days of Age³

0 1.90 ± 0.20^a 2.02 ± 0.23^a

5 3.99 ± 1.34^b 5.69 ± 1.02^b

10 4.57 ± 0.76^b 3.76 + 1.81^ab

20 3.03 ± 0.93^b 4.91 ± 0.62^b

Means within in ovo treatments significantly differ

(P≤.05) from basal if superscripts differ.

²Glycerol release (μg/1.8 x 10⁵ cells).

³Glycerol release (μg/2 x 10⁵ cells). Example 6 : The effect of in ovo injection of thyroid hormone and feeding of dietary betaadrenergic agonist on broiler chickens.

Two thousand Hubbard x Hubbard broiler eggs were conventionally incubated. At day 11 of incubation, eggs were candled and fertile eggs were injected into the albuminous end of the egg using a 25-gauge needle with a 3mm needle guard. Treatments consisted of 0.1 ml of either 0.01 μg of T₃ or 1.0 μg of T₄ in phosphatedbuffered saline (PBS) or PBS alone. At day of hatch, birds were sexed, wingbanded, weighed and randomly assigned to floor pens by treatment group and sex. For the first three weeks, the birds were fed a conventional broiler starter diet (22% protein, 3125 kcal/kg) ad libitum. From 3 to 8 weeks of age, a conventional broiler grower diet (19% protein, 3285 kcal/day) was fed ad libitum. Both starter and grower diets contained 1 ppm isoproterenol (ISP).

Feed consumption and body weights were recorded weekly throughout the grow-out trial. At the conclusion of experiment two, 10 randomly selected birds from each in ovo treatment group and sex were selected at 8 weeks of age and killed via cervical dislocation. Abdominal fat pads were removed and weighed and a sample was taken for cellularity studies. Carcasses were frozen at -20°C for subsequent carcass analysis. (See Example 1 and 5 for basic procedures).

Effect On Growth

Body weights of male broilers injected in ovo with T₃ and fed 1 ppm ISP in the feed were significantly greater than control male weights from 5 to 7 weeks of age (Figure 4). The T₄-treated males exhibited

significantly lower body weights than control or T₃-treated males from 1 to 4 weeks of age, however, by 7 weeks of age, body weights of T₄-treated males were not significantly different from control.

Body weights of female broilers did not

significantly differ between treatment groups from 2 to 6 weeks of age. As shown in Figure 5, T₃-treated females exhibited significantly greater body weights than T₄-treated birds at week one. At week 7, body weights of T₄-treated females were significantly less than weights of control females. Effect On Feed Conversion

No consistent differences in feed conversion ratios were observed for either male or female broilers due to in ovo hormone administration (Table 13). Feed

conversion ratios of male T₃-treated birds were

significantly improved versus control at week 5; whereas the feed to gain ratio of T₄-treated male birds were significantly lower than control at week 1. Feed

conversion ratios of T₄-treated females were

significantly increased at week 7 in comparison to control or T₃-treated females.

Effect On Carcass Composition A significant increase in carcass protein deposition was observed at 8 weeks of age in T₃-treated male

broilers fed 1 ppm isoproterenol in comparison to control or T₄-treated males. There were no significant treatment differences in carcass protein of female broilers (Figure 6). Although a trend (P<0.1) was observed for the T₃-treated broilers to exhibit less carcass fat than controls (Figure 7), no significant differences in percent lipid at 8 weeks of age were observed due to in ovo treatment for either male or female broilers.

Effect On Adipose Cellularity

Abdominal fat pad weights at 7 weeks of age

expressed as a percent of body weight are shown in Figure 8. In ovo injection of T₄ resulted in a significant reduction in fat pad weights when expressed as a percent of body weight in comparison to controls.

TABLE 13

Feed Conversion of In Ovo Thyroid

Hormone Injection and Dietary Isoproterenol.

Feed Conversion

MALE

Week Control T3 T4

1 1.89 + 0.09 1.76 + 0.06 1.66 + 0.07

2 1.97 + 0.06 1.91 + 0.04 1.87 + 0.07

3 2.08 + 0.06 1.97 + 0.03 1.97 + 0.06

4 1.82 + 0.03 1.80 + 0.02 1.79 + 0.04

5 2.47 + 0.08 2.16 + 0.08 2.23 + 0.08

6 2. 67 + 0.08 2.72 + 0.06 2.64 + 0.04

7 3.32 + 0.27 3.18 + 0.16 2.73 + 0.08

FEMALE

1 1.82 + 0.06 1.78 + 0.06 1.71 + 0.10

2 1.98 + 0.03 2.02 + 0.04 1.94 + 0.07

3 1.83 + 0.22 2.00 + 0.38 2.01 + 0.08

4 1.83 + 0.02 1.84 + 0.02 1.83 + 0.03

5 2.39 + 0.06 2.61 + 0.13 2.27 + 0.04

6 2.86 + 0.06 2.84 + 0.06 3.01 + 0.13

7 3.11 + 0.06 3.11 + 0.13 3.67 + 0.24

Means within week s differ (P<.05) if superworipts differ

Claims

CLAIMS :

1. A method of enhancing the physiological development and responses of poultry comprising the steps of: incubating fertilized eggs of the desired poultry species; introducing a biologically effective amount of

growth factor into the incubating fertilized egg; and thereafter allowing the altered poultry to hatch and mature.

2. The method of claim 1 comprising the added step of administering to the hatched poultry a biologically effective amount of a beta-adrenergic agonist.

3. The method of claim 2 wherein the beta-adrenergic agonist is isoproterenol.

4. The method of claim 1 or 2 wherein the growth factor is injected.

5. The method of claim 1 or 2 wherein the growth factor is a growth hormone.

6. The method of claim 5 wherein the growth factor is mammalian growth hormone.

7. The method of claim 6 wherein the growth factor is ovine growth hormone.

8. The method of claim 6 wherein the growth factor is bovine growth hormone.

9. The method of claim 1 or 2 wherein the growth factor is a thyroid hormone.

10. The method of claim 1 or 2 wherein the growth factor is isoproterenol.

11. The method of claim 1 or 2 wherein the step of injecting the eggs occurs during the time period from and including the first day of the second half of the

incubation period up to and including the day the eggs are transferred from the incubator to the hatcher.

12. The method of claim 1 or 2 wherein the step of injecting the eggs occurs on the first day of the second half of the incubation period of the egg.

13. The method of claim 1 or 2 wherein the step of injecting the eggs occurs on the day the eggs are

transferred from the incubator to the hatcher.

14. The method of claim 1 or 2 wherein the poultry are chickens.

15. The method of claims 1 or 2 wherein the poultry are turkeys.

16. A method of enhancing the growth and meat production of poultry comprising the steps of: incubating fertilized eggs of the desired poultry species; injecting a biologically effective amount of growth factor selected from the group consisting of ovine, bovine, or porcine growth hormone, or T₃ or T₄ into the incubating fertilized eggs during the time period from and including the first day of the second half of the incubation period up to and including the day the eggs are transferred from the incubator to the hatcher; and thereafter allowing the altered poultry to hatch and mature.

17. The method of claim 16 comprising the added step of administering to the hatched poultry a biologically active amount of a beta-adrenergic agonist.

18. The method of claim 17 wherein the beta-adrenergic is isoproterenol.

19. The method of claim 16 or 17 wherein the eggs are injected on the day the eggs are transferred from the incubator to the hatcher.

20. The method of claim 16 or 17 wherein the step of injection occurs on the first day of the second half of the incubation period of the eggs.

21. The method of claim 16 or 17 wherein the poultry are chickens.

22. The method of claim 16 or 17 wherein the poultry are turkeys.

23. A method of enhancing the growth and meat production of chickens comprising the steps of: incubating fertilized chicken eggs; injecting a biologically effective amount of ovine growth hormone into the albumin of the incubating fertilized eggs on the first day of the second half of the

incubation period of the eggs; and thereafter allowing the altered chickens to hatch and mature.

24. A method of enhancing the growth and meat production of chickens comprising the steps of: incubating fertilized chicken eggs; injecting a biologically effective amount of thyroid hormone into the albumin of the incubating fertilized eggs; on the first day of the second half of the

incubation period of the eggs; and thereafter allowing the altered chickens to hatch and develope.

25. The method of claim 23 or 24 comprising the added step of administering to the chickens a biologically active amount of isoproterenol.

26. A product comprising poultry produced by the method as in any of claims 1, 2, 3, 16, 17, 18, 23 or 24.