Composition and method for increasing the hatchability of turkey eggs

The hatchability of fertile turkey eggs is increased by injection of the eggs with an effective amount of exogenous pyridoxine. The eggs are preferably injected between the outer and inner membranes following up to 25 days of incubation. Hatchability increases up to 5% have been observed.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to a treatment to increase the hatchability of 
fertile domestic turkey eggs. In particular, the present invention relates 
to a composition and a method of injecting fertile turkey eggs with 
exogenous pyridoxine to enhance the hatchability thereof. 
2. Description of the Prior Art 
The national average hatch rate of turkey eggs is about 20% lower than that 
of chicken eggs. Many variables contribute to this low hatch record in 
turkeys, but it is well recognized in the turkey industry that one factor 
is a deficiency of vitamins in the diet of breeder turkey hens. To cure 
this deficiency, modern commercial diets are fortified with a variety of 
synthetic vitamins. However, the relationship between vitamins and 
hatchability in the turkey remains complex and unresolved. Preliminary 
investigations have revealed that pyridoxine drops to low levels in the 
egg with maternal age of the hen [Robel, Fed. Amer. Soc. Exp. Biol. Med. 
41: 1129 (1982); Robel, Poult. Sci. 62: 1751-1756 (1983)]. Environmental 
stress and the presence of vitamin binding proteins may also be factors in 
pyridoxine deficiency. 
Robel [Comp. Biochem. Physiol., B: Comp, Biochem. 84B: 265-267 (1987)] 
reported that, as a result of certain field stress factors, certain 
breeder turkey hens experience hormonal imbalance which causes 
significantly high levels of avidin to be deposited into the albumenous 
portion of the egg. Avidin, which is a glycoprotein secreted in the magnum 
region of the oviduct of the hen, is hormonally induced and binds with 
biotin, rendering the nutrient unavailable for use. During the initial 
stages of development, the embryos are not adversely affected by the high 
levels of avidin because the yolk, which is protected by the yolk sac, 
provides a sufficient supply of free biotin for normal development. 
Following the second quarter of incubation, approximately 15-16 days of 
incubation, the yolk sac ruptures, and the avidin-rich albumen passes 
progressively into the yolk. At this point, a biotin deficiency occurs 
within the eggs as the high concentration of avidin makes biotin 
unavailable to the embryo by forming an avidin-biotin complex. 
Robel et al. [Poult. Sci. 66: 1429-1430 (1987)] found that by injecting the 
avidin-rich eggs with exogenous d-biotin in an inert liquid carrier after 
at least 23 days of incubation, the eggs were replenished with the supply 
of free biotin necessary for embryonic survival. The result was an 
increase in hatch rate up to 5%. 
SUMMARY OF THE INVENTION 
I have now surprisingly found that a similar increase in hatch rate is 
obtained when turkey eggs are injected with pyridoxine (vitamin B.sub.6) 
during incubation. Unlike biotin, pyridoxine is not known to form a 
complex with avidin which would make the vitamin nutritionally 
unavailable. Therefore, the benefits of in ovo supplementation of 
pyridoxine are not predictable from the results observed for biotin. 
In accordance with this discovery, it is an object of the invention to 
provide a method for increasing the amount of available pyridoxine in 
fertile turkey eggs, particularly in eggs which are pyridoxine-deficient 
as a result of aging hens or in eggs of flocks which have not responded 
sufficiently to dietary supplementation of pyridoxine. 
It is also an object of the invention to provide a pyridoxine composition 
useful to increase the batch rate of fertile turkey eggs. 
Another object of this invention is to provide a method of increasing the 
hatch rate of fertile turkey eggs by providing them with free pyridoxine 
during the incubation period without adversely affecting embryonic 
survival. 
Other objects and advantages of this invention will become obvious from the 
ensuing description. 
DETAILED DESCRIPTION OF THE INVENTION 
The pyridoxine composition useful in this invention comprises exogenous 
pyridoxine dissolved in an inert liquid carrier. For purposes of this 
invention, the term "pyridoxine" is defined as a substituted pyridine 
having the structural formula 
##STR1## 
Pyridoxine, also known as vitamin B.sub.6, is available from synthetic 
sources. It is most commonly produced as the hydrochloride, but other 
soluble pharmacological salts are also envisioned for use herein. The term 
"exogenous," as used herein, is meant to refer to pyridoxine produced 
externally from the egg. 
The liquid carrier may be any inert liquid, such as water or isotonic 
saline solution, that is physiologically compatible with the egg tissue 
and developing embryo. 
An "effective amount" of pyridoxine injected into each egg is defined as 
that amount which is nontoxic but effective to provide sufficient 
pyridoxine for survival of the embryo. Amounts in the range of about 
200-1,000 .mu.g pyridoxine per egg, and especially about 600 .mu.g per egg 
are preferred. To avoid infecting the egg contents with undesirable 
pathogens, care must be exercised to assure that the pyridoxine and liquid 
carrier are sterile and free of pyrogens (bacterial toxins). As a 
precautionary measure, it is preferred that sterilization and bottling of 
the pyridoxine be performed under strict asceptic conditions. 
The specific mechanism of the injection is not critical provided that it 
does not unduly damage the shell and underlying shell membranes. It is 
important to avoid the formation of hairline cracks in the shell and 
disruption in the physiological functioning of the maternal egg package. 
Prior to injection, a pilot hole or cavity may be formed through the shell 
in any suitable manner which will not crack the shell. A 21- to 28-gauge 
sterile needle locked to a sterile hypodermic syringe is suitable for the 
purpose. The needle should pierce the shell 3-5 mm on the shell end of the 
egg or 6-8 mm on the large end. Known and commercially used automatic 
injectors may also be used provided that stated parameters of caution are 
maintained. Following injection, the shell may be sealed with paraffin, a 
fast-drying cement, or other suitable sealant. 
For optimum protection of the embryo and to avoid upsetting the 
physiological stability of the egg, the pyridoxine solution should be 
injected into the interior of the eggs in a manner which allows the least 
amount of intrusion without compromising desired results. Underlying shell 
membranes consist of an outer egg membrane which is directly adjacent to 
the interior surface of the shell and an inner egg membrane which 
underlies the outer membrane and surrounds the albumen of the egg. 
Preferably, the pyridoxine is injected over the inner egg membrane; that 
is, between the outer and inner egg membranes. The injection is usually 
made along the longitudinal axis through either the large or small end of 
the egg shell. The most preferred site is into the air cell enclosed 
within the outer and inner shell membranes at the large end of the shell. 
In accordance with the invention, eggs may be injected any time before 
depletion of the natural supply of pyridoxine necessary for normal 
development of the embryo. Preferably, the time for injection of the 
pyridoxine is at about 25 days of incubation. Advantages of injection on 
about day 25 are twofold: (1) in a commercial hatchery, eggs are normally 
transferred from incubation chambers to the hatchers at 25 days and are 
therefore conveniently available for injection, and (2) embryo failure is 
highest after 25 days of incubation.

The following examples are intended only to further illustrate the 
invention and are not intended to limit the scope of the invention which 
is defined by the claims. 
EXAMPLE 1 
Preparation of Pyridoxine Solution 
Exogenous pyridoxine hydrochloride (USP-FCC, Hoffmann-La Roche, Inc., 
Nutley, N.J.), 3 g, was dissolved in 1 l of pyrogen-free saline solution 
(Abbott Laboratories, North Chicago, Ill.). The solution was passed 
through 0.2-.mu.m filters into empty sterile evacuated bottles (Abbott 
Laboratories, supra). The bottles were placed in amber bags (Associated 
Bag Co., Milwaukee, Wis.) to protect the solution from ultraviolet light. 
If evacuated bottles are not used, the headspace over the solution is 
filled with nitrogen after passing through a 0.2-.mu.m filter unit, 50 mm 
(Millipore Products Division, Molsheim, France). The sterilization and 
solution bottling functions were done under strict sanitized conditions in 
a dn-class 100 clean room environment with filtered HEPA (high efficiency 
particulate air), acceptably used to prepare pharmacological solutions for 
human use. 
EXAMPLE 2 
Manual Injection of Eggs 
Fertile eggs (900) were obtained from a single flock of 240 Nicholas Large 
White turkey hens for three separate laboratory trials at 8 wks, 12 wks, 
and 15 wks of egg production. For each trial, a total of 300 eggs were 
set: 100 for injection with 600 .mu.g exogenous pyridoxine (0.2 ml of the 
solution described in Example 1), 100 for injection with 0.2 ml saline 
solution (the carrier for the pyridoxine), and 100 were not injected. At 
17 days of incubation, injection through the large end of each egg was 
performed as follows: The shell area surrounding the injection site was 
swabbed with 2% iodine tincture. A slight indentation was made in the 
swabbed area, using a small sharp steel punch that was flamed before 
contacting the shell. The force used on the punch was slight, to avoid 
forming hairline cracks in the shell. The punch site exactly accommodated 
a micro-fine III, 28-gauge sterile needle that was locked to a 0.5-ml 
sterile insulin syringe (Becton Dickinson, Inc., Rutherford, N.J.). The 
needle was carefully inserted through the punch site to a depth of about 
6-8 mm, and 0.2 ml of the desired solution was injected over the inner 
shell membrane. The puncture was sealed with a small drop of fast-drying 
"Duco Cement" (Devcon Corp., Wood Dale, Ill.) which was lightly layered 
over the puncture site. The injections were done under a strictly 
sanitized, clean-room environment with HEPA, and personnel were 
asceptically equipped for handling the experimental solutions. The data 
reported in Table I show that pyridoxine injection over the three trials 
effected in average 4.5% increase in hatchability and that injection with 
the carrier saline solution had no effect on hatchability. 
EXAMPLE 3 
Automatic Injection of Eggs 
Fertile eggs were obtained from two flocks (6,000 hens each) of Nicholas 
Large White turkeys for three separate field trials at 16 wks, 20 wks, and 
24 wks of egg production. For each trial, 544 eggs were set for injection 
with exogenous pyridoxine (at the same level as in Example 2) and another 
544 eggs were not injected. At 25 days of incubation, injection was 
performed as follows: The eggs were dipped in antiseptic solution and 
injected at the small end with 0.2 ml of exogenous pyridoxine solution 
(supra) using an automatic egg injector. The injection needles were 
approximately 21 gauge (flat ends), and the eggs were pierced to a depth 
of approximately 3-5 mm. After injection, the needle sites were sealed 
with wax, and incubation was resumed. 
The data was analyzed as a two-factor block analysis of variance. Trial and 
treatment were the factors, and flock was the block. The main effects, 
trial and treatment, were each significant (P&lt;0.0021); trial x treatment 
was not. In all trials, pyridoxine-injected eggs gave significantly higher 
(P&lt;0.002) hatchability over uninjected eggs (Table II). There was a 4.6% 
increase in hatchability of injected eggs, compared to the control for the 
six trials combined using the two flocks. The results of the individual 
trials for flock 1 and flock 2 (6,000 hens each) are reported in Table II. 
Statistical significance of the six trials combined is reported in Table 
III. The results in Examples 2 and 3 demonstrate that it made no 
difference whether the eggs were injected at the large or the small end, 
nor whether they were injected at 17 or 25 days incubation time. 
It is understood that the foregoing detailed description is given merely by 
way of illustration and that modification and variations may be made 
therein without departing from the spirit and scope of the invention. 
TABLE I 
______________________________________ 
Results of Manual Injection 
Hatchability (%) 
At 8 wk At 12 wk At 15 wk 
production 
production 
production 
Pooled 
Treatment (Trial 1) (Trial 2) (Trial 3) 
means 
______________________________________ 
Pyridoxine- 
93.8 84.0 89.8 89.2.sup.a 
injected 
Saline- 88.0 81.0 85.0 84.7.sup.b 
injected 
Not injected 
89.7 81.0 83.8 84.8.sup.b 
______________________________________ 
.sup.a,b Values with different superscripts are significantly different ( 
&lt;0.05). 
TABLE II 
______________________________________ 
Results of Automatic Injection, 
Individua1 Trials 
Hatchabi1ity (%).sup.1 
At 16 wk At 20 wk At 24 wk 
Treatment production production production 
______________________________________ 
Flock l Trial 1 Trial 2 Trial 3 
______________________________________ 
Pyridoxine-injected 
91.4 90.6 91.1 
Not injected 
88.0 88.2 84.4 
______________________________________ 
Flock 2 Trial 4 Trial 5 Trial 6 
______________________________________ 
Pyridoxine-injected 
88.0 87.5 89.0 
Not injected 
82.1 85.8 82.5 
______________________________________ 
.sup.1 In all trials, pyridoxineinjected eggs gave significantly higher ( 
&lt;0.002) hatchability over uninjected eggs. 
TABLE III 
______________________________________ 
Results of Automatic Injection, 
Combined Trials 
Eggs set.sup.1 
Hatchability.sup.2 
Treatment (No.) (%) 
______________________________________ 
Pyridoxine-injected 
3264 89.8 
Not injected 3264 85.2 
______________________________________ 
.sup.1 Eggs set represent six trials from two different turkey flocks 
(three trials per flock, 544 eggs per treatment in each trial, incubated 
at 16, 20, and 24 wks of production). 
.sup.2 Hatchability was significantly higher (P &lt;0.002) in 
pyridoxineinjected eggs over noninjected eggs in the six trials.