Can injecting Vitamin-D into eggs result in a better hatch rate and weight

[beneduck14]

I've been working on a research project for the 2018 INTEL science fair for a college scholarship and I wanted to share with you my interesting results!

Keep in mind I am only in High school so this is not perfect

DISCLAIMER: THIS DOES NOT REPLACE ANY PROFESSIONAL WORK OR GIVE ANY ADVICE. DO NOT TRY THIS AT HOME UNLESS UNDER SUPERVISION OF A PROFESSIONAL FAMILIAR WITH THIS OR A VETERINARIAN.

*Procedure Approved by Board and Veterinarians prior*

Here it goes...
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Q: What is INOVO
A: The method of injecting a substance into an egg. Mareks vaccination is an example.
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Q: Why the heck am I studying this?
A: Scientists are trying to find an alternative of using antibiotics in broiler(meat) poultry because of it's harmful effects on the people who eat it. The perfect "formula" for an alternative is still yet to be found.

*Substances like glucose, vitamin A-C, magnesium, etc. have already been tested...

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Except for:

VITAMIN-D3 (Cholecalciferol) ON HATCH WEIGHT AND HATCH RATE

No one has done this before!!
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Abstract (Summary):

(Simplified)

Hypothesized that injecting Vitamin-D3 in eggs would increase hatch rate and weight. From results, there was no significantly different results and therefore Vitamin-D3 does not improve anything.

(Extensive)​

This experiment was conducted to evaluate the effects of in-ovo injection of cholecalciferol on broiler chickens (Cobb500) based on results of hatchability and hatch weight. This was done to determine if choleciferol is a reliable solution to improving hatch rates and birth-weight in the practice of in-ovo at various poultry farms as an alternative to antibiotics. The experiment consisted of five groups of fertile eggs that were injected on day seventeen and a half in the amnion with varying concentrations of cholecalciferol. Treatments were the following: 1) control group (without- injection), 2) group injected with 0.5 ml sterile water (sham group), 3) group injected with 15% of cholecalciferol in 0.5 sterile water , 4) group injected with 20% of cholecalciferol in 0.5 ml sterile water and 5) group injected with 25% of cholecalciferol in 0.5 ml sterile water. Upon each hatch, the hatch weight of newly hatched chicks was recorded. After five days, the final data was recorded and analyzed using the standard t-test method. The means of the groups injected with cholecalciferol and the groups without cholecalciferol were compared and found that they are not significantly different p < 0.05. From these results, it can be concluded that cholecalciferol is not significantly effective for use in in-ovo vaccinations for improving hatch success.

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Materials:

(Simplified)​

• 
  
  
  • 45 Cobb500 fertile eggs (Donated by Wincorp International)
  • Pure Vitamin-D3 liquid (100 IU per drop)
  • Two Styrofoam Circulated Air Incubators with additional thermometers and hygrometers
  • Sterile water
  • 4 Sterile Tubes
  • 40 Sterile Syringes with blunt tip needles (1 ml)
  • 45 Sterile syringes with 21Gx 1/2" hypodermic needles
  • Medical gloves and masks
  • Alcohol pads
  • Sterile medical tape
  • A sharp tool to drill a hole (I used a clean screw)

(Extensive)

The forty-five fertile eggs used in this experiment were obtained from a Cobb-500 broiler breeder flock donated by Wincorp International. Pure cholecalciferol liquid was manufactured by NOW FOOD Company (Item Number #0370). The eggs were incubated in Farm Innovators Model 2200 Circulated Air Incubator. Various amounts of solution were mixed with sterile water and solutions were transferred into sterile tubes (4; one for each group) by 1 ml syringes supplied by BSTEAN using blunt tip needles. Additional digital thermometers and hygrometers were added inside each incubator as supplied by Avianweb, model AV-1951HT. Forty-five sterile syringes and 21 Gx ½” sterile hypodermic needles were used for the procedure and medical gloves, medical masks, alcohol pads, and sterile medical tape were needed to maximize protection against contamination.

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Procedure:

(Simplified)​

1. 
   
   
   • Dry incubation method with 37.5'C as temperature and 30% relative humidity
   • Made injection solutions diluted in sterile water (0.5ml) a couple of hours before procedure and kept inside sterile tube
     
     
   • 5 groups in total: 15% of V-D3 / 20% of V-D3 / 25% V-D3 / Sham (just injected water, sham was included to see if stress of injection affected results!) / Control.
   • Day 14 candling of eggs and removed nonviable/deceased eggs
   • Day 17.5 day of procedure:

- Swabbed site of injection with alcohol pad (Broad end)
- Drilled a tiny hole using a sharp tipped screw (You can see tiny hole in pic)

- Put syringe through the hole, pass the air sac, and pierced the membrane. Let the needle drop until it stops from resting on top of embryo but not inside. Slowly inject 0.5ml of mixed solution per group in amnion sac.

- Swab hole again and tape the top with sterile medical tape.

- Each egg stays outside for ten minutes from the time taken out of incubator till the time put back inside after procedure
-Control group stays unutouched.

(Extensive)
​

All eggs were incubated at 37.5'C and 30% RH(relative humidity). The injection solutions were pre-made seven hours pre-procedure and stored in a sterile tube:

Table 1. Concentration (ml/0.5 ml of sterile water) of cholecalciferol used in in ovo injection
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15% 20% 25% Sham



0.625 0.9 11.25 0




The eggs were divided into five groups; 1) control group (without- injection), 2) group injected with 0.5 ml sterile water (sham group), 3) group injected with 15% of cholecalciferol in 0.5 ml sterile water , 4) group injected with 20% of cholecalciferol in 0.5 ml sterile water and 5) group injected with 25% of cholecalciferol in 0.5 ml sterile water. On day 14 of incubation, eggs were candled, and unfertilized eggs or those containing dead embryos were discarded. On day 17.5 of incubation, each egg was removed from the incubator for ten mins to perform cholecalciferol injections of various concentrates that were carried out in the amnion. A small injection hole was made by a sharp sterilized tool in the broad end of the egg and swabbed with alcohol pads prior to each injection. The syringe and needle was pierced through the membrane with force and were allowed to drop down until the embryo could be felt. The injection was done just above the embryo to insure it went into the amnion. Post injection, the injection site was swabbed with alcohol and covered with sterile tape. After each injection, the syringe and needle was replaced with a new one.The control group was untouched and kept inside the incubator during the treatment. Upon hatch, each chick per group was weighed in grams and transferred to a brooder.


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Results:

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Interesting that the less Vitamin-D3 was injected,the higher the group weight was
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Thank you all for looking at this! Yes this isn't all (Left out statistical evidence) I didn't want to bore you all with more talk. Yes I will keep testing substances that have never been experimented before

Total injection time for the 36 eggs was almost 6 hours so imagine sitting down and doing this without stopping the whole time!
This was done all manually due to no funding for auto-injectors or being able to use an actual lab (this was all done at home)

Thank you to Wincorp International for donating eggs and 3riverschick for giving the idea of this project in January of 2017.

The fair (round one of qualifyers) is next month, will update how it goes!

 

[Kenny_]

What made vitamin D3 be considered instead of antibiotics? (They aren't really comparable.)

great job!

 

[beneduck14]

What made vitamin D3 be considered instead of antibiotics? (They aren't really comparable.)

Vitamin D3 is not harmful to consume, cheaper to be used in poultry industries who use INOVO, and the list goes on to use it as an alternative

I chose to test this vitamin due to nobody trying it before and because I thought since VD3 increases bone mass, the broiler chickens would be naturally born built and weigh bigger.

Sorry if I didn't answer your question, I may not have understood it correctly

 

[BantyChooks]

Good job.

 

[Kenny_]

Vitamin D3 is not harmful to consume, cheaper to be used in poultry industries who use INOVO, and the list goes on to use it as an alternative

I chose to test this vitamin due to nobody trying it before and because I thought since VD3 increases bone mass, the broiler chickens would be naturally born built and weigh bigger.

Sorry if I didn't answer your question, I may not have understood it correctly

No, you answered it perfectly to me. Great rationales for the testing. Of course, the results won't really tell the tale at first but really more so at later intervals, if the bone mass continues to increase and support the added poundage that the chickens are putting on. (But would that mean it would need continued vitamin d3 supplementation?) Or would theoretically that one time injection be enough for the life of the bird? The half-life of that d3 should wear out very quickly and shouldn't stimulate a long enough infusion of bone growth, to me - but I am not an aviary veterinarian nor medical doctor. )

 

[beneduck14]

No, you answered it perfectly to me. Great rationales for the testing. Of course, the results won't really tell the tale at first but really more so at later intervals, if the bone mass continues to increase and support the added poundage that the chickens are putting on. (But would that mean it would need continued vitamin d3 supplementation?) Or would theoretically that one time injection be enough for the life of the bird? The half-life of that d3 should wear out very quickly and shouldn't stimulate a long enough infusion of bone growth, to me - but I am not an aviary veterinarian nor medical doctor. )

Oh I would love to have done that! Unfortunately Intel regulations does not give students that much of a time to do so. (We have a schedule to follow). The inovo professional studies that do look at the injected birds for weeks after do not give the supplement again because the intial substance injected would be the overall effect studied however. To truly see if the bone mass is the reason for the extra weight would need several birds to be culled and dissected. That is against the rules of Intel lol and I would not be able to bring myself to that

 

[lazy gardener]

Excellent study. I missed it. were the control group also swabbed with alcohol, and shell pierced?

I have heard that soaking in Vitamin C does improve viability. Much less labor intensive than injection.

In my own flock, I put my breeders on multi vitamins prior to collecting eggs. I figure it's easier to get the nutrients into the embryo by filtering them through the hen.

Have you read the studies related to raising chicks on used litter? That improves viability, as well as growth, and feed conversion rates.

In light of the above information, has Vitamin B been trialed for administration prior to hatch?

 

[beneduck14]

Excellent study. I missed it. were the control group also swabbed with alcohol, and shell pierced?

I have heard that soaking in Vitamin C does improve viability. Much less labor intensive than injection.

In my own flock, I put my breeders on multi vitamins prior to collecting eggs. I figure it's easier to get the nutrients into the embryo by filtering them through the hen.

Have you read the studies related to raising chicks on used litter? That improves viability, as well as growth, and feed conversion rates.

In light of the above information, has Vitamin B been trialed for administration prior to hatch?

Thank you! No the control group was not touched at all. I wanted to simulate all the other hatcheries that don't do inovo and keep them in. By creating a Sham group, I tested to see if stress of handling, not only injection, affected hatch rate.

While those are all good things to study, unfortunately I specifically chose the topic of Inovo injections and the effects different substances as independant variables to influence the hatcheries who are already using them.

I have previously done a study on something similar looking at medicated feed (amprollium) vs unmedicated feed to look at growth up to four weeks if that helps. Found no significant data and no statistical average weight difference!

If you mean Vitamin D, it has been trialed used before for inovo studies, but not for looking at hatch rate or weight but rather bone mass and meat quality. If that wasn't a typo, then i'm not sure about Vitamin B. It has been used quite extensively in inovo studies but I cannot answer as I have not done research on it.

Please let me know if you have anymore questions or if I did not answer you question. I am more then happy to answer anything!

 

[lazy gardener]

I specifically meant vitamin B! B/C the theory is that the benefit of being raised on used litter is that it is inoculated with beneficial bacteria and fungi that manufacture vitamin B, so wondering if injection of Vitamin B into the developing egg has been studied. Of course, using something as simplistic as raising them on litter that is inoculated with proper bacteria/fungi would preclude the need to do the labor intensive pre hatch treatment of the egg.

Keep up the good work. You have a bright future ahead of you.

 

[beneduck14]

I specifically meant vitamin B! B/C the theory is that the benefit of being raised on used litter is that it is inoculated with beneficial bacteria and fungi that manufacture vitamin B, so wondering if injection of Vitamin B into the developing egg has been studied. Of course, using something as simplistic as raising them on litter that is inoculated with proper bacteria/fungi would preclude the need to do the labor intensive pre hatch treatment of the egg.

Keep up the good work. You have a bright future ahead of you.

I would imagine! There was one study in Central Avian Research Institute (India) that tried all the Vitamins (Vitamin D was not included for some reason) and tested embryonic and post-growth performance. Intrestingly Vitamin-B (and E) did the worst. Vitamin-B caused early embryonic death!

I would like to study the litter method you are talking about as it sounds interesting. Thank you for the idea, all my experiments were inspired by people on this site, I will let you know what happens with it