The embryonated egg: a practical target for genetic based advances to improve poultry production.

The embryonated avian egg is an attractive target for applying technology-based advances to improve poultry production. There are a number of reasons for this. First, the egg is immobile and can be easily accessed by high-speed automated equipment such as the commercial egg injection system used for vaccination of broilers worldwide. Second, due to successful breeding techniques, the embryonic period now composes 30 to 40% of a broiler's total lifespan, underscoring the importance of this window in the bird production life cycle. Third, the period of incubation involves rapid development from a ball of 40,000 to 60,000 undifferentiated blastodermal cells to a fully formed chick and associated extra-embryonic compartments in 21 d, allowing development of novel approaches for improving poultry production. Some of these novel approaches will be discussed in this paper and include gender discrimination of embryos and the possibility of changing the breeding paradigm through introduction of undifferentiated cells such as avian blastodermal or embryonic stem cells.

In ovo vaccination technology.

More than 80% of the U.S. broiler industry has converted to the in ovo vaccination process for control of Marek's disease. Providing certain criteria are met, including timing and site of vaccine placement, vaccine mixing, machine sanitization, and hatchery management specifications, this has proven to be an efficacious and convenient method of vaccination. Efforts to extend the technology for other viral vaccines including Newcastle, bronchitis and bursal disease, and bacterial and parasitic vaccines are in progress. Collectively, these studies demonstrate that in ovo vaccination technology using approved vaccine is a safe, efficacious, and convenient method for vaccination of poultry.

Development of a highly quantitative, reproducible assay for determination of chicken T cell growth factor biological activity.

This report examines optimal culture conditions necessary for accurate and sensitive quantification of chicken T Cell Growth Factor (TCGF) activity. With this bioassay, TCGF is quantified by measuring its ability to cause proliferation of splenocytes prestimulated with mitogen. Proliferation is quantified by determining the optical density (OD) or "signal" of test samples in microtiter wells by measuring the incorporation of tetrazolium salt by live cells. To optimize assay conditions, systematic evaluation of the effects of cell culture variables was carried out with the constant aim of increasing signal to noise ratio in the assay. Higher signal to noise ratios were found when using Dulbecco's Modified Eagle's Medium (DMEM) rather than Roswell Park Memorial Institute Medium (RPMI) for basal tissue culture media containing the same supplements. The addition of lipid supplement to the assay system not only increased the proliferation signal, but also decreased the background OD. Incubation temperatures of 41 C rather than 37 C for both the mitogen prestimulation and proliferation phases of the assay also resulted in a higher signal to noise ratio. While incorporating the optimal experimental conditions, a finalized assay procedure employing test sample normalization with an internal assay standard was tested for accuracy. The assay can accurately detect 2 to 15 U/mL of TCGF activity. The within-assay variation ranged from 2 to 13% and the between-assay variation ranged from 11 to 22% depending upon the TCGF preparation being tested. The excellent reproducibility of this assay has facilitated investigations of TCGF production, processing, and purification.

Applications in in ovo technology.

By mid-August 1995, 55% of broiler embryos in North America were vaccinated for Marek's disease using the INOVOJECT system, with 201 INOVOJECT machines placed with 16 of the top 25 poultry producers, providing the industry with the capacity to inject in excess of 400 million eggs per month or about 5 billion eggs per annum. In ovo administration of a bursal disease antibody-infectious bursal disease virus (BDA-IBDV) complexed vaccine to specific-pathogen-free (SPF) embryos was safer and more potent than conventional IBDV vaccine alone because it delayed the appearance of bursal lesions, produced no early mortality, produced higher geometric mean antibody titers against IBDV, and generated protective immunity against challenge. In ovo administration of a BDA-IBDV complexed vaccine to broiler embryos generated antibody titers against IBDV sooner than conventional virus vaccinates, and generated protective immunity against challenge Direct DNA injection of plasmid DNA encoding beta-galactosidase into breast muscle in ovo and posthatch was an effective means to achieve both gene transfer and expression, with potential for the development of gene vaccines using plasmids encoding protective antigens from poultry pathogens. In ovo administration of 800 U chicken myelomonocytic growth factor (cMGF), a chicken hematopoietic cytokine for cells of the monocytic-granulocytic lineages, significantly reduced mortality associated with Escherichia coli exposure within the hatcher when compared to PBS controls (6.1 vs 12.4, P < or = 0.05), but not when compared to a yeast expression control. A procedure was developed enabling injection prior to the onset of incubation without compromising embryo viability. This in ovo injection process has opened up the window of embryo development during incubation for intervention, as illustrated by the 100% male phenotype produced in chicks hatching from eggs injected with aromatase inhibitor prior to incubation. These data illustrate some of the in ovo applications presently in use by the poultry industry, and under development or in research at EMBREX.

Efficacy of a novel infectious bursal disease virus immune complex vaccine in broiler chickens.

Two experiments were conducted to test the efficacy of a novel infectious bursal disease virus (IBDV) vaccine in broiler chickens with maternal IBDV immunity. The IBDV vaccine was formulated by mixing IBDV strain 2512 with bursal disease antibodies (BDA) to produce the IBDV-BDA complex vaccine. In Expt. I, 1-day-old Cobb x Cobb broiler chickens were vaccinated subcutaneously with either IBDV-BDA or commercial live intermediate IBDV vaccine (vaccine A) or were left unvaccinated. In Expt. 2, the vaccine A group was not included; instead, IBDV strain 2512 was included. Chickens were maintained in isolation houses. On day 28 (Expt. 1) and day 32 (Expt. 2) of age, chickens from each group were challenged with a standard USDA IBDV (STC strain) challenge. Challenged and unchallenged chickens were evaluated for their bursa/body weight ratios and antibody titers 3 days post-challenge. Bursae collected from Expt. 2 were examined histologically to evaluate bursal lesions and confirm gross examination. None of the unvaccinated chickens was protected against the challenge virus as evidenced by the presence of acute bursal lesions (edema/hemorrhage). All chickens receiving the IBDV-BDA complex or the IBDV strain 2512 (Expt. 2) were protected from the challenge virus as evidenced by no acute bursal lesions. Additionally, chickens receiving the IBDV-BDA complex vaccine or the IBDV strain 2512 had antibody titers to IBDV, indication the presence of an active immune response. In Expt. 1, chickens vaccinated with vaccine A and challenged had bursal lesions similar to those observed in the unvaccinated, challenged chickens. These chickens also showed no indication of active immunity against the virus. These results suggest that the 1-day-of-age-administered IBDV-BDA complex vaccine can induce active immunity and protection against a standard IBDV challenge in the face of variable levels of maternal IBDV immunity.

The effect of in ovo oocyst or sporocyst inoculation on response to subsequent coccidial challenge.

A trial was conducted to investigate the effects of in ovo Eimeria maxima inoculation on response to subsequent posthatch challenge with E. maxima. The in ovo treatments were arranged in a 4 x 2 factorial with four in ovo inoculation sites (air cell, amnion, yolk sac, and allantois) and two parasite forms (oocyst and sporocyst). Four control treatments included an uninoculated (naive) unchallenged group, a naive challenged group, and two posthatch inoculated challenged groups. Chicks were challenged by crop intubation with 50,000 sporulated E. maxima oocysts 10 d posthatch. On Day 8 postchallenge, feed intake was determined and birds were weighed and lesions scored. During the brooding period, oocysts were isolated from the fecal material of chicks receiving in ovo administration of sporocysts in the amnion and sporocysts or oocysts in the yolk sac. Posthatch inoculated chicks had gain and feed:gain ratios similar to those of naive unchallenged chicks. Gain, feed:gain ratio, lesion scores, and oocyst shedding of chicks inoculated in ovo were similar to those of naive, challenged chicks. Although there was some indication that parasites introduced in ovo may complete their life-cycle within the developing chick, this experiment provided no evidence that in ovo administration of either E. maxima oocysts or sporocysts will protect birds from subsequent coccidial challenge. Contrarily, inoculating chicks on day of hatch with a single oral dose of E. maxima oocysts provided significant protection against subsequent coccidial challenge.

Determination of optimum formulation of a novel infectious bursal disease virus (IBDV) vaccine constructed by mixing bursal disease antibody with IBDV.

A novel vaccine against infectious bursal disease virus (IBDV) has been developed. The new vaccine was constructed by mixing bursal disease antibody (BDA) contained in whole antiserum with live IBDV before lyophilization. To establish various formulations of BDA and IBDV, several BDA doses between 5 units and 80 units of BDA/50 microliters were mixed with 100 EID50/50 microliters of IBDV suspension in Expt. 1; in Expt. 2, several IBDV doses between 10 EID50/50 microliters and 977 EID50/50 microliters of IBDV suspension were mixed with 24 units of BDA/50 microliters. Vaccine preparations were administered subcutaneously to the nape of 1-day-old specific-pathogen-free (SPF) chicks. Safety, potency, and immunogenicity of the different vaccine formulations were evaluated using bursal weight, bursal gross examination, and IBDV antibody titer. Some bursae were examined histologically to confirm gross examinations. Several vaccine formulations were safe and efficacious and met the safety, potency, and immunogenicity criteria. A vaccine construct of 100 EID50 mixed with 24 units of BDA was selected as the release dose. When administered at 1 day of age, the novel vaccine allows for delayed infection of the bursa until after days 6-8 of age in SPF chicks, while initiating potency and immunogenicity to an IBDV challenge. The addition of BDA to the IBDV results in a complex vaccine that allows for safer immunization in SPF birds than under administration of the vaccine virus without BDA.

Adaptation of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay for the determination of virus-neutralizing antibodies using the virus-neutralization assay.

The tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] has been widely used for bioassays. Herein is described the use of the MTT dye with a virus-neutralization (VN) assay to titer infectious bursal disease virus (IBDV)-neutralizing antibodies. A standard VN assay using chicken embryo fibroblasts (CEFs) and IBDV was used for the assessment of IBDV-neutralizing antibodies. The percent of CEF killing due to IBDV was quantitated using MTT, and the absorbance (A) data were used to calculate the VN antibody titer. This method of calculation offers the expression of VN titer in terms of units of activity per unit of volume.

In vivo effects of biosynthetic chicken growth hormone in broiler-strain chickens.

Two experiments were conducted to examine the effects of growth hormone (GH) in growing chickens older than broiler age. In experiment 1, 8 week old male (heavy "broiler" type strain) chickens received daily injections of either biosynthetic chicken GH or an implant of biosynthetic bovine GH for 3 weeks. Neither growth rate (daily weight gain) nor the feed:gain ratio was influenced by either preparation of GH. However, an increase in breast muscle was observed in birds receiving the higher dose (250 micrograms/kg/day) of chicken GH. No changes were observed with chicken GH treatment on the weights of the fat pad, heart, gizzard and shank bone or on shank bone length or on plasma concentrations of free fatty acids. Experiment 2 employed 12 week old male, heavy strain, chickens with biosynthetic chicken GH being administered via Alzet osmotic pumps. In this study, GH (50 micrograms/kg/day) increased growth rate, body weight, pectoralis (breast) muscle weight, adipose tissue weight, bursa Fabricius weight and the plasma concentrations of free fatty acids. It is concluded that GH can influence carcass composition and growth in older chickens.

Efficacy of nemadectin, a new broad-spectrum endectocide, against natural infections of canine gastrointestinal helminths.

Nemadectin, a new broad-spectrum endectocide, was highly efficacious against natural infections of all the major canine gastrointestinal helminths. At single oral dosages of 0.2-0.4 mg kg-1 body weight (BW), a liquid formulation administered in gelatin capsules was 100% effective in eliminating natural infections of Toxocara canis, Toxascaris leonina, Ancylostoma caninum and Uncinaria stenocephala. Tablets (267 mg) containing 4.6% nemadectin given at a rate of 1/3 tablet per 20 kg BW (0.2 mg nemadectin kg-1) were 100% active against T. canis, A. caninum and U. stenocephala. With both formulations, an increase in the dose rate to 0.6-0.8 mg kg-1 BW resulted in 99-100% elimination of Trichuris vulpis infections. No adverse reactions were observed in any of the treated dogs.

Effects of cimaterol, a beta-adrenergic agonist, on protein metabolism in rats.

Fractional accretion rates of total body 3-methylhistidine containing proteins (actin and myosin) were elevated 40% to 120% in rats fed a high-carbohydrate diet containing 10 or 100 ppm cimaterol for 1 week. Fractional degradation and fractional synthesis rates of these proteins were examined by measuring total body 3-methylhistidine content and urinary excretion of 3-methylhistidine. Consumption of the diet containing 100 ppm cimaterol for 1 week caused a 25% reduction in fractional degradation rates and a concomitant 32% increase in fractional synthesis rates of 3-methylhistidine containing proteins. Effects of cimaterol on fractional accretion, degradation, and synthesis rates of 3-methylhistidine containing proteins diminished after 1 week. Cimaterol failed to influence plasma insulin, triiodothyronine, or corticosterone concentrations. The dramatic increase in accretion of 3-methylhistidine containing proteins observed during the first week rats are fed diets containing cimaterol is caused by reciprocal action on protein degradation and synthesis.

Effects of cimaterol, a beta-adrenergic agonist, on lipid metabolism in rats.

Rats fed a high carbohydrate diet containing 10 or 100 ppm cimaterol for 4 weeks gained 41% to 59% less fat and 70% to 76% more protein than controls, with no major changes in either energy gain or efficiency of energy retention. Effects of cimaterol on lipid metabolism in these rats were assessed. Cimaterol stimulated lipolysis in vivo and in vitro, but failed to influence rates of de novo fatty acid synthesis in either liver or white adipose tissue. Activities of fatty acid synthetase and malic enzyme in these tissues were also unaffected by cimaterol. Cimaterol administered in vivo failed to affect lipoprotein lipase activity in white adipose tissue, but elevated enzyme activity 67% to 75% in the extensor digitorium longus muscle. Lipoprotein lipase activity in the extensor digitorum longus muscle was also elevated by 66% during a 2 hour incubation with 1 mmol/L cimaterol. We conclude that cimaterol selectively stimulates both lipolysis in white adipose tissue and lipoprotein lipase activity in skeletal muscle, to direct energy away from adipose tissue deposition toward skeletal muscle accretion.

Effect of cimaterol on sheep adipose tissue lipid metabolism.

Effects of dietary cimaterol (5 mg/kg) on adipose tissue metabolism of wether lambs were studied. Lipogenesis, lipolysis, fatty acid composition and adipocyte size and number were measured. Cimaterol feeding increased lipogenesis; however, this effect was not statistically significant. Insulin (1,000 microU/ml) stimulated lipogenesis of adipose tissue from control sheep. However, this elevated rate was abolished by in vitro cimaterol. Insulin had no stimulatory effect on lipogenesis in cimaterol-fed sheep. Lipolysis was depressed by cimaterol feeding. However, 10(-4) M cimaterol stimulated lipolysis in the adipose tissue from both control and cimaterol-fed sheep. Insulin inhibited stimulated lipolysis in adipose tissue from control sheep but had no effect on the stimulated lipolysis in cimaterol-fed sheep. Mean adipocyte diameter was smaller (from 74 to 70 microns) and adipocyte size distribution also was changed in the cimaterol-fed sheep. Adipocyte number per gram of tissue was not affected by cimaterol. There was a significant increase in percentage of unsaturated fatty acids in adipose tissue from cimaterol-fed sheep. These results indicate that lipogenic and lipolytic responses to insulin and cimaterol in sheep adipose tissue were altered by cimaterol feeding. The carcass fat content decrease in cimaterol-fed sheep may be attributed to the reduction in adipocyte size.

Cimaterol-induced muscle hypertrophy and altered endocrine status in lambs.

The objectives of this study were 1) to determine how cellular growth of skeletal muscle is altered by the repartitioning agent cimaterol and 2) to determine if cimaterol alters endocrine status in association with its repartitioning effects. Thirty Dorset wether lambs were randomly assigned to a pre-treatment baseline group or received 0 or 10 ppm cimaterol in a complete, mixed, high-concentrate diet for 7 or 12 wk. Weights of biceps femoris (BF), semimembranosus (SM) and semitendinosus (ST) muscles were 32.8, 27.1 and 31.5% greater, respectively, in treated lambs at 7 wk, and were 22 to 24% greater at 12 wk. Longissimus (LD) cross-sectional area was 26 and 32% greater at these treatment intervals. Percent type I fibers declined significantly over the course of the experiment in ST, SM and LD, and cimaterol caused a small but significant reduction in percent type I fibers in the ST at 7 and 12 wk. Muscles from lambs fed cimaterol contained 50 and 75% more fibers that exhibited negative staining for phosphorylase activity. Mean cross-sectional area of type I and type II fibers in the combined portions of the ST were 30.4 and 29.3% greater, respectively, in cimaterol-fed lambs after 12 wk, while type I and type II fiber areas in the longissimus were only 13 and 15% greater, respectively. Cimaterol-induced hypertrophy of the ST resulted in both protein and RNA content being 30 to 35% greater (P less than .01) at 7 and 12 wk, while DNA concentration was 22% less (P less than .01) at 7 wk. DNA concentration returned to normal by 12 wk. These results indicate that cimaterol elicits a rapid increase in muscle RNA and protein accretion without concurrent incorporation of satellite cell nuclei. Plasma insulin and insulin-like growth factor-1 (IGF-1) concentrations were 55 and 34% lower, respectively, in cimaterol-fed lambs. Plasma somatotropin concentration and area under the curve were 2.3 times greater (P less than .01) in lambs fed cimaterol for 6 wk, while plasma cortisol, prolactin and glucose concentration were unaffected at 6 or 12 wk. The significant changes in endocrine status may be important in the mechanism(s) of cimaterol in altering muscle accretion.

A repartitioning agent to improve performance and carcass composition of broilers.

Clenbuterol [benzyl alcohol, 4-amino-alpha-(t-butylamino)methyl-3,5-dichloro] was tested in the finisher ration of broilers in three series of experiments for its effects on performance and carcass characteristics. In Experiment 1, administration of clenbuterol at feed levels of .25, .5, 1, 2, and 4 ppm from 28 to 49 days of age resulted in significant sex combined weight gain improvements at all levels except 4 ppm and significant feed efficiency improvements at all levels tested. Uneviscerated body composition analysis indicated that clenbuterol treatment significantly increased body protein and water content and decreased body fat in female birds. In males, body fat was significantly reduced by 1, 2, and 4 ppm clenbuterol. From these results the 1 ppm level was selected for further testing. In Experiments 2 and 3, clenbuterol fed from 28 to 49 days of age significantly improved sex combined weight gain and feed efficiency and both male and female 49-day weights. Carcass yield was significantly increased in males and females by 1.11 and 1.91 percentage points, respectively. Abdominal fat was significantly reduced by clenbuterol feeding only in females. Carcass analysis indicated that clenbuterol-fed birds had a significantly lower carcass fat content. In Experiments 4 to 7, clenbuterol was administered at 1 ppm in the finishing feed but was withdrawn 3 to 5 days prior to terminal performance and carcass evaluations. Clenbuterol significantly improved sex combined weight gain and feed efficiency, and 49-day weights for both sexes. Carcass yield was significantly increased in males and females by .54 and .98 percentage points, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of volatile fatty acid uptake by mitochondrial acyl CoA synthetases of bovine liver.

Mitochondria of bovine liver contain acyl CoA synthetases necessary for the uptake of propionate, butyrate, and valerate whereas acetate is bound only weakly. Purification of these enzymes separated a distinct propionyl CoA synthetase highly specific for propionate and acrylate and a butyrate-activating fraction with broad substrate specificity for short and medium chain fatty acids. Evidence from kinetic studies and sucrose density centrifugation suggested that this latter fraction was composed of two enzymes, a butyryl CoA synthetase and a valeryl CoA synthetase. The apparent molecular weights of the propionyl, butyryl, and valeryl CoA synthetases were 72,000, 67,000, and 65,000. The Michaelis-Menten constants of propionyl CoA synthetase for propionate, adenosine 5'-triphosphate, and coenzyme A were 1.3 x 10(-3)M, 1.3 x 10(-3)M, and 6.3 x 10(-4)M. Enzyme activity is regulated by the concentration of propionate in portal blood. Relative to propionyl, butyryl, or valeryl CoA synthetases little acetyl CoA synthetase could be demonstrated. In ruminants hepatic metabolism is such that use of acetate as an energy source is minimum. This ensures that an alternative energy source to glucose, as acetate units, will reach the extrahepatic tissues. Separation of a distinct propionyl CoA synthetase regulated by the concentration of propionate in portal blood is significant because a primary role of ruminant liver is to synthesize glucose from ruminally derived propionate.

Regulation of volatile fatty acid uptake by mitochondrial acyl CoA synthetases of bovine heart.

Purification of components of heart mitochondria activating short chain fatty acids prepared from tissue of lactating Holstein cows demonstrated predominantly one acyl CoA synthetase, acetyl CoA synthetase activating acetate, and propionate. Activity of butyryl CoA synthetase was low. Propionyl CoA synthetase characteristically in bovine liver and kidney tissue could not be demonstrated in heart mitochondria. Thus, of the ruminally derived volatile fatty acids only acetate can be used by heart mitochondria as a primary energy source because of small quantities of propionate in peripheral blood. Acetyl CoA synthetase was a glycoprotein composed of a single polypeptide chain of apparent molecular weight 67,500. The Michaelis-Menten constant for acetate was 1.8 x 10(-4)M. By comparison with literature for blood acetate concentration we concluded that enzyme is saturated with substrate at all physiological concentrations of acetate. These kinetic properties ensure a constant supply of acetate as an energy source for maintaining heart function in ruminants.

Partial purification of enzymes of bovine kidney mitochondria activating volatile fatty acids.

Mitochondria were prepared from kidney cortex tissue of lactating Holstein cows, and enzymes activating volatile fatty acids partially purified. Two fractions activating volatile fatty acids were obtained; one was similar to components isolated from bovine heart mitochondria activating acetate and propionate (acetyl CoA synthetase). The other was similar to components of bovine liver mitochondria and activated propionate, butyrate, and valerate but not acetate. On calcium phosphate gel chromatography this latter fraction could be separated into a distinct propionyl CoA synthetase (Michaelis-Menten constant for propionate 2.54 x 10(-3)M) and a fraction activating butyrate and valerate. The role of a distinct propionyl CoA synthetase in kidney mitochondria remains to be elucidated because propionate is not a major substrate in peripheral blood of ruminants. We concluded that volatile fatty acid activation by bovine kidney mitochondria is due to an acetyl CoA synthetase, a propionyl CoA synthetase, and a butyrate and valerate-activating fraction probably composed of separate butyryl and valeryl CoA synthetases.