THE EFFECT OF IN OVO PROBIOTICS ON HATCHABILITY AND
DEVELOPMENT OF POULTS WITH OR WITHOUT DIETARY
RESTRICTIONS
By
Robert Charles Van Wyhe

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Animal Science-Doctor of Philosophy
2014

ABSTRACT
THE EFFECT OF IN OVO PROBIOTICS ON HATCHABILITY AND DEVELOPMENT OF
POULT WITH OR WITHOUT DIETARY RESTRICTIONS
By
Robert Charles Van Wyhe
Turkeys have to overcome several challenges early in life, including exposure
to pathogens and difficulty with the transition from yolk as a nutrient source to CHO
dietary sources. Probiotics are thought to help alleviate these symptoms in the posthatch poult, but any beneficial effects, if given prior to hatch are unknown.
Additionally, the optimal level of probiotic that can be injected into the egg must be
established. Once optimal levels of hatch are established, the effects post hatch must
be known. A series of experiments were performed to determine the benefits of in
ovo probiotics. The overall objective was to evaluate different probiotic
concentrations injected into turkey eggs prior to hatch on hatchability and intestinal
bacterial concentrations. Secondary objectives were to determine the impact of in
ovo probiotics on poult hatchability and first week mortality. Finally, whether a
probiotic injection could mitigate poult growth ramifications due to delayed access
to feed or a 20% dietary reduction in Ca and P. To study these objectives a series of
experiments were conducted. The general design of the study was for groups of
fertile turkey eggs to be incubated. The eggs were candled on d25 and allocated to
different treatment groups: (1) Non-injected eggs hatched in a separate hatcher
(Negative Control), (2) eggs injected with 1ml of saline and placed in the hatcher
with probiotic injected eggs (Sham Control), (3) eggs injected with various amounts
of probiotic solution (Probiotic). The most commonly used concentration was
106cfu/ml. An additional experimental factor of delayed placement on feed was

added to experiment 2. Placement of eggs in the incubator was offset by 24h, and
the hatched poults were all placed on feed at the same time. Hatch counts were
taken for each replicate and cecal contents collected from two birds per replicate to
determine bacterial counts on d28. Cecal contents were plated on media plates with
LB agar, MRS agar, Bifidobacteria agar or Enterococcusel agar. Intestinal contents
were collected to measure nutrient transporters and intestinal morphology in trial
two. Birds from experiments two and three were placed on feed for one (experiment
2) or three (experiment 3) wks. At DOH of exp3, half of the hatched poults were
given access to a diet with a 20% reduction in calcium and phosphorus. Tibia and
femur samples were collected for morphology and ash content from birds during
exp3. The injection of 106cfu of bacteria or lower into the egg did not alter
hatchability of eggs from trials one and two. Overall, hatchability was reduced by
almost 20% in exp3. Bacteria load in the intestine was two to three logs higher in
probiotic injected birds compared to control for every trial. The first week livability
of probiotic injected birds was not significantly different than controls in exp2, but
was almost double in exp3. Sham control poults from exp2 had the highest BW for
the entire trial. Probiotic injected birds had improved FCR through d3 in exp2 but
the effect on FCR was lost by d7. Probiotic injected birds had lower BW and had
shorter, narrower tibias and femurs at d7 and d21 compared to controls. The results
indicate that in ovo probiotics can negatively influence poult growth and
performance in the first 21d of life, if the bacteria reduce hatchability. When in ovo
probiotics are injected into eggs and increase bacterial load in the intestine without
altering hatchability, the benefits are improved FCR through the first 3d of life. The
results from the series of experiments are a promising insight into the effects in ovo
bacteria can have on the early post hatch poult.

ACKNOWLEDGEMENTS

I have so many people to thank for their extraordinary work that assisted me along
the way.

Dr. Karcher. Thank you for making me your first Ph.D. student. I have grown so
much over the years. Though at the time I didn’t always appreciate it, thanks for
sending me all over the country to learn and improve as a scientist. Your faith in my
ability to coordinate and run projects allowed me to confidence to succeed. I thank
you for all the help.

Dr. Hill. Your mentorship has always been appreciated. I was always excited to talk
with you in your office. Thank you for letting me teach Intro and experiment with
my methodology. I have grown appreciably as an instructor under you tutelage.

Dr, Applegate Dr. Balander, and Dr. Britton. My committee has always been a
source of knowledge and guidance. I appreciate all the tough honest advice I
received from everyone over the years. I always felt better prepared for whatever
grad school would throw at me once we were done talking. Thank you for being on
my committee.

Cara, Prafulla, Salah, and Joe- There have been long sample days and long days
period. Despite my best efforts to stay positive you guys were always there to pick
iv

me up when I was down. Thanks as well for letting me give advice even though most
of the time it wasn’t needed or asked for.
I thank all of the graduate (former and current) and the undergraduate
students that helped me in the sample collection, processing, and other lab work
during my project. Without your help I wouldn’t be where I am today.
Jessica and CeCe Charlotte, though you will probably have no memory of this, thank
you for hanging out with me during the final long nights of writing. I am glad you
could sleep on me while I worked. This thank you is mostly for my beautiful and
kind wife. I can’t truly express how much I need your support and how effortlessly
you cheer me up. Thanks for all the laughs and fun times. Thanks for helping with
our daughter. I am so excited to see what the future holds.

TABLE OF CONTENTS

LIST OF TABLES ..................................................................................................................... viii
LIST OF FIGURES ....................................................................................................................... x
LITERATURE REVIEW.............................................................................................................. 1
Incubation ............................................................................................................................................. 1
Fasting or delayed access to feed: effects on the intestine .................................................. 2
In ovo technologies ............................................................................................................................ 3
Bacterial colonization of the poultry intestine ........................................................................ 4
The effect of age on bacterial colonization................................................................................ 5
Non-host factors that effect intestinal microbiota ................................................................. 6
Probiotics in a non-challenge setting .......................................................................................... 7
Lactic acid bacteria ............................................................................................................................ 8
Lactobacillus......................................................................................................................................... 8
Bifidiobacteria .................................................................................................................................. 10
Enterococcus ..................................................................................................................................... 11
Pediococcus ....................................................................................................................................... 12
Bone development and probiotics ............................................................................................ 13
Glucose Transporters..................................................................................................................... 16
Peptide transporters ...................................................................................................................... 18
Conclusion .......................................................................................................................................... 19
REFERENCES ...................................................................................................................................... 20

CHAPTER 1: TURKEY POULT HATCHABILITY IN RESPONSE TO VARIOUS
LEVELS OF IN OVO PROBIOTICS ........................................................................................ 32
ABSTRACT .......................................................................................................................................... 32
INTRODUCTION ................................................................................................................................ 33
MATERIALS AND METHODS ......................................................................................................... 35
Probiotic preparation ................................................................................................................................ 35
Bird incubation and treatments ............................................................................................................ 35
Sampling ......................................................................................................................................................... 37
Cecal content plating ................................................................................................................................. 38
Statistical Analysis ...................................................................................................................................... 38
RESULTS AND DISCUSSION ........................................................................................................... 39
APPENDIX ........................................................................................................................................... 43
REFERENCES ...................................................................................................................................... 47

CHAPTER 2: TURKEY POULT HATCHABILITY AND LIVABILITY: THE EFFECT OF
IN OVO PROBIOTICS .............................................................................................................. 51
ABSTRACT .......................................................................................................................................... 51
INTRODUCTION ................................................................................................................................ 52
MATERIALS AND METHODS ......................................................................................................... 54
Probiotic preparation ................................................................................................................................ 54
Birds and Husbandry ................................................................................................................................. 55
Sample Collection........................................................................................................................................ 57
Cecal content plating ................................................................................................................................. 58
Nutrient Transporters............................................................................................................................... 59

vi

Morphology samples.................................................................................................................................. 60
Excreta samples ........................................................................................................................................... 61
Calcium assay................................................................................................................................................ 62
Phosphorus assay ....................................................................................................................................... 62
Energy .............................................................................................................................................................. 63
Dry Matter ...................................................................................................................................................... 64
RESULTS .............................................................................................................................................. 65
DISCUSSION ........................................................................................................................................ 68
APPENDIX ........................................................................................................................................... 68
REFERENCES ...................................................................................................................................... 94

CHAPTER 3: BONE CHARACTERISTICS AND MINERAL DIGESTION IN YOUNG
TURKEY POULTS: THE COMBINED EFFECT OF IN OVO PROBIOTICS AND LOW
CALCIUM AND PHOSPHORUS DIETS ................................................................................ 97
ABSTRACT .......................................................................................................................................... 97
INTRODUCTION ................................................................................................................................ 98
MATERIALS AND METHODS ......................................................................................................... 99
Probiotic preparation ................................................................................................................................ 99
Bird incubation and treatments ............................................................................................................ 99
Sampling ...................................................................................................................................................... 100
Bone Ash ...................................................................................................................................................... 102
Cecal content plating .............................................................................................................................. 102
Excreta samples ........................................................................................................................................ 103
Calcium assay............................................................................................................................................. 104
Phosphorus assay .................................................................................................................................... 104
Energy ........................................................................................................................................................... 105
Dry Matter ................................................................................................................................................... 106
Statistical Analysis ................................................................................................................................... 107
RESULTS ............................................................................................................................................ 107
Performance ............................................................................................................................................... 107
Cecal Bacterial Counts ............................................................................................................................ 108
Nutrient Digestibility .............................................................................................................................. 108
Bone parameters ...................................................................................................................................... 108
DISCUSSION ...................................................................................................................................... 110
APPENDIX ......................................................................................................................................... 117
REFERENCES .................................................................................................................................... 135

SUMMARY AND FUTURE STUDIES.................................................................................. 140
Alternative strains ........................................................................................................................................ 140
Delivery method ............................................................................................................................................ 141
Combination with current vaccinations .............................................................................................. 141

vii

LIST OF TABLES

Table 1.1: Hatchability of turkey poults administered in ovo probiotics ....................... 44
Table 1.2: The hatch weight of turkey poults administered in ovo probiotic............... 45
Table 1.3: Total cecal bacterial populations (log10cfu/ml) as determined by
cultivable plate count on four different medias from poults administered
various levels of in ovo probiotics........................................................................................ 46
1

Table 2.1: Primers used for real-time PCR on turkey intestinal samples .................... 75
Table 2.2: Hatchability of turkey poults administered in ovo probiotics administered
on d25 of incubation1................................................................................................................ 76
Table 2.3: Total cecal bacterial populations (log10cfu/ml) from poults administered
in ovo injections on d25 of incubation as determined by cultivable plate count
on four different medias1 ........................................................................................................ 77
Table 2.4: Body weight of turkey poults with either a delayed placement on feed
and/or an in ovo probiotic injection administered at 25d of incubation1 ............ 78
Table 2.5: Body weight of turkey poults with either a delayed placement on feed
and/or an in ovo probiotic injection administered at 25d of incubation1............ 79
Table 2.6: Apparent energy retention of poults who experienced either delayed
placement on feed and/or an in ovo probiotic injection administered at 25d of
incubation1 .................................................................................................................................... 80
Table 2.7: The main effect of in ovo probiotic injection administered at 25d of
incubation or time delay to feed on duodenal morphology of young poults1 .... 81
Table 2.8: The main effect of in ovo probiotic injection administered at 25d of
incubation or time delay to feed on ileal morphology of young poults1 ............... 82
Table 2.9: The interaction between placement on feed and in ovo injection on the
relative gene expression of PepT1 and GLUT2 in the duodenum of young turkey
poults1 ............................................................................................................................................. 83
Table 2.10: The interaction between placement on feed and in ovo injection on the
relative gene expression of PepT1 and GLUT2 in the ileum of young turkey
poults1 ............................................................................................................................................. 84

viii

Table 3.1: Composition, calculated, and analyzed nutrient content of diets fed from
0-21d of age ................................................................................................................................118
Table 3.2: Hatch performance of turkey poults administered in ovo probiotics at 25d
of incubation1 .............................................................................................................................119
Table 3.3: Performance of 0 to 7d old poults fed control or Ca and P deficient diets
with or without in ovo probiotic1 administration at 25d of incubation ..............120
Table 3.4: The performance poults from 8 to 21d post hatch fed control or Ca and P
deficient diets with or without in ovo probiotic administration............................121
Table 3.5: Total cecal bacterial populations (log10cfu/ml) as determined by
cultivable plate count on four different medias from poults administered
various amounts of in ovo probiotics1 administered at 25d of incubation ........122
Table 3.6: Apparent Ca and P retention in 7 and 21d old poults fed either control or
Ca and P deficient diets with or without in ovo probiotic1 injection at 25d of
incubation....................................................................................................................................123
Table 3.7: The main effects of control or Ca and P deficient diets and in ovo
probiotic1 injection at 25d of incubation on tibial and femoral morphology of
DOH, 7 and 21d old poults fed .............................................................................................124

ix

LIST OF FIGURES

Figure 2.1: The BW of poults who experienced a delayed placement on feed and with
or without an in ovo probiotic injection A.) Poults at d0 from either negative
control (non-injected eggs), control (sterile saline injected eggs at d25 of
incubation) or probiotic (eggs injected with 1 ml of probiotic inoculations at
106 cfu/ml at d25 of incubation) eggs than subjected to a 48h, 24h or 0h delay
placement at hatch. B.) Poults at d3 from either negative control (non-injected
eggs), control (sterile saline injected eggs at d25 of incubation) or probiotic
(eggs injected with 1 ml of probiotic inoculations at 106 cfu/ml at d25 of
incubation) eggs than subjected to a 48h, 24h or 0h delay placement at hatch.
C.) Poults at d7 from either negative control (non-injected eggs), control (sterile
saline injected eggs at d25 of incubation) or probiotic (eggs injected with 1 ml
of probiotic inoculations at 106 cfu/ml at d25 of incubation) eggs than subjected
to a 48h, 24h or 0h delay placement at hatch. All treatment groups were in their
own separate hatcher. 0h-poults not held in the hatcher following hatch; 24hpoults held in the hatcher for 24h prior to feed and water access; 48h-poults
held in the hatcher for 48h prior to feed and water access. Turkey eggs were
offset to ensure all poults were placed on feed and water at the same time. a,b
Bars within same group with no common superscript differ significantly
(P<0.05) ......................................................................................................................................... 85
Figure 2.2: The FCR of poults who experienced a delayed placement on feed and
either an in ovo probiotic injection A.) FCR from d0 to 3 of poults from either
negative control (non-injected eggs), control (sterile saline injected eggs at d25
of incubation) or probiotic (eggs injected with 1 ml of probiotic inoculations at
106 cfu/ml at d25 of incubation) eggs than subjected to a 48h, 24h or 0h delay
placement at hatch. B.) FCR from d3 to 7 of poults from either negative control
(non-injected eggs), control (sterile saline injected eggs at d25 of incubation) or
probiotic (eggs injected with 1 ml of probiotic inoculations at 106 cfu/ml at d25
of incubation) eggs than subjected to a 48h, 24h or 0h delay placement at hatch.
C.) FCR from d0 to 7 of poults from either negative control (non-injected eggs),
control (sterile saline injected eggs at d25 of incubation) or probiotic (eggs
injected with 1 ml of probiotic inoculations at 106 cfu/ml at d25 of incubation)
eggs than subjected to a 48h, 24h or 0h delay placement at hatch. All treatment
groups were in their own separate hatcher. 0h-poults not held in the hatcher
following hatch; 24h-poults held in the hatcher for 24h prior to feed and water
access; 48h-poults held in the hatcher for 48h prior to feed and water access.
Turkey eggs were offset to ensure all poults were placed on feed and water at
the same time. a,b Bars within same group with no common superscript differ
significantly (P<0.05)................................................................................................................ 87

x

Figure 2.3: Apparent calcium and phosphorus retention in 3 or 7d old poults who
experienced a delayed placement on feed with or without an in ovo probiotic
injection A. Calcium digestibility of in ovo injected poults (P=0.679). B.
Phosphorus digestibility of in ovo injected poults (P=0.179). C. Calcium
digestibility of poults with delayed placement of feed (P=0.679). D. Phosphorus
digestibility poults with delayed placement of feed (P=0.679). 0h-poults not
held in the hatcher following hatch; 24h-poults held in the hatcher for 24h prior
to feed and water access; 48h-poults held in the hatcher for 48h prior to feed
and water access. Turkey eggs were offset to ensure all poults were placed on
feed and water at the same time. Negative Control - non-injected eggs; control sterile saline injected eggs; probiotic - eggs injected with 1ml of probiotic
inoculations at 106cfu/ml. All treatment groups were in their own separate
hatcher. a,b Bars within same group for each item with no common superscript
differ significantly (P<0.05) ................................................................................................... 89
Figure 2.4: Ileal villus height to crypt depth ratio from A.) d0 turkeys whose eggs
were injected with probiotics on d25 of incubation and with or without a
delayed placement on feed. B.) d7 turkeys whose eggs were injected with
probiotics on d25 of incubation and with or without a delayed placement on
feed. Negative Control - non-injected eggs; control - sterile saline injected eggs;
probiotic - eggs injected with 1ml of probiotic inoculations at 106 cfu/ml. All
treatment groups were in their own separate hatcher. 0h-poults not held in the
hatcher following hatch; 24-poults held in the hatcher for 24h prior to feed and
water access; 48h-poults held in the hatcher for 48h prior to feed and water
access. Turkey eggs were offset to ensure all poults were placed on feed and
water at the same time. a,b Bars within same group with no common superscript
differ significantly (P<0.05) ................................................................................................... 91
Figure 2.5: Duodenal villus height to crypt depth ratio from A.) d0 turkeys whose
eggs were injected with probiotics on d25 of incubation and with or without a
delayed placement on feed. B.) d7 turkeys whose eggs were injected with
probiotics on d25 of incubation and with or without a delayed placement on
feed.. Non-injected control - non-injected eggs; control - sterile saline injected
eggs; probiotic - eggs injected with 1ml of probiotic inoculations at 106 cfu/ml.
All treatment groups were in their own separate hatcher. 0h-poults not held in
the hatcher following hatch; 24h-poults held in the hatcher for 24h prior to feed
and water access; 48h-poults held in the hatcher for 48h prior to feed and
water access. Turkey eggs were offset to ensure all poults were placed on feed
and water at the same time. a,b Bars within same group for each item with no
common superscript differ significantly (P<0.05) ........................................................ 93

xi

Figure 3.1: Tibial and femoral morphology of DOH, 7 and 21d old poults fed either
control or Ca and P deficient diets with or without in ovo probiotic1 injection at
25d of incubation. A.) Femur length. B.) Tibia length. C.) Femur width. D.) Tibia
width. Non-injected are non-injected eggs in a separate hatcher; probiotic are
eggs injected with 1 ml of probiotic inoculations at 106cfu/ml in a separate
hatcher. Control are birds fed diet containing 100% of NRC (1994) Ca and P
requirements; deficient are birds fed diet formulated to contain 80% of NRC
(1994) Ca and P requirements. Results are expressed the means ± SE of 10
replicate pens. a,b,c Bars with no common letter at same age are different
(P<0.05)........................................................................................................................................125
Figure 3.2: The main effect of in ovo probiotic injection and either standard or
deficient nutrient diets on tibial ash (%) in turkey DOH, d7 and d21 turkey
poults. A.) The effect of probiotics administered at 25d of incubation on the
tibial epiphyseal ash (25% of each end of the tibia) of turkeys. B.) The effect of
standard or a nutrient deficient diet on the tibial epiphyseal ash (25% of each
end of the tibia) of turkeys. C.) The effect of probiotics administered at 25d of
incubation on the tibial diaphyseal ash (middle 50% of the bone) of turkeys. D.)
The effect of standard or a nutrient deficient diet on the tibial diaphyseal ash
(middle 50% of the bone) of turkeys. Non-injected are non-injected eggs in a
separate hatcher; probiotic are eggs injected with 1 ml of probiotic inoculations
at 106cfu/ml in a separate hatcher. Control are birds fed diet containing 100%
of NRC (1994) Ca and P requirements; deficient are birds fed diet formulated to
contain 80% of NRC (1994) Ca and P requirements. Results are expressed the
means ± SE of 10 replicate pens. a,b Bars with no common letter at same age are
different (P<0.05). ....................................................................................................................127
Figure 3.3: The main effect of in ovo probiotic injection and either standard or
deficient nutrient diets on femoral ash (%) in turkey DOH, d7 and d21 turkey
poults. A.) The effect of probiotics administered at 25d of incubation on the
femoral epiphyseal ash (25% of each end of the tibia) of turkeys. B.) The effect
of standard or a nutrient deficient diet on the femoral epiphyseal ash (25% of
each end of the tibia) of turkeys. C.) The effect of probiotics administered at 25d
of incubation on the femoral diaphyseal ash (middle 50% of the bone) of
turkeys. D.) The effect of standard or a nutrient deficient diet on the femoral
diaphyseal ash (middle 50% of the bone) of turkeys. Non-injected are noninjected eggs in a separate hatcher; probiotic are eggs injected with 1 ml of
probiotic inoculations at 106cfu/ml in a separate hatcher. Control are birds fed
diet containing 100% of NRC (1994) Ca and P requirements; deficient are birds
fed diet formulated to contain 80% of NRC (1994) Ca and P requirements.
Results are expressed the means ± SE of 10 replicate pens.a,b Bars with no
common letter at same age are different (P<0.05). ....................................................129

xii

Figure 3.4: The interaction of in ovo probiotic injection at 25d of incubation and
either standard or deficient nutrient diets on (A) tibial ash from DOH turkeys,
(B) tibial ash from 7d old turkeys and (C) tibial ash from 21d old turkeys.
Epiphyseal is 25% of each end of the tibia. Diaphyseal is the middle 50% of the
bone. Non-injected are non-injected eggs in a separate hatcher; probiotic are
eggs injected with 1 ml of probiotic inoculations at 106cfu/ml in a separate
hatcher. Control are birds fed diet containing 100% of NRC (1994) Ca and P
requirements; deficient are birds fed diet formulated to contain 80% of NRC
(1994) Ca and P requirements. ...........................................................................................131
Figure 3.5: The interaction of in ovo probiotic injection at 25d of incubation and
either standard or deficient nutrient diets on (A) femoral ash from DOH
turkeys, (B) femoral ash from 7d old turkeys and (C) femoral ash from 21d old.
Epiphyseal is 25% of each end of the femur. Diaphyseal is the middle 50% of
the bone. Non-injected are non-injected eggs in a separate hatcher; probiotic
are eggs injected with 1 ml of probiotic inoculations at 106cfu/ml in a separate
hatcher. Control are birds fed diet containing 100% of NRC (1994) Ca and P
requirements; deficient are birds fed diet formulated to contain 80% of NRC
(1994) Ca and P requirements. ...........................................................................................133

xiii

LITERATURE REVIEW
The early development sets the poult up for optimal performance later in life.
Problems at the breeder farm or hatchery can lead to problems shortly after hatch,
which further leads to either early mortality or low performance numbers. If
optimal conditions are met before hatch, then enhanced performance and reduced
mortality can be seen early in life. Producers have used many different types of
dietary additives over the years to improve performance. The increasing complexity
of dietary additives recently has resulted in the need for producers to better
understand intestinal development and the role these additives play.
Incubation
The time difference between the first and last hatched chick or poult is called
the hatch window (HW). The variability in HW is approximately 24 to 48h
(Decuypere et al., 2001). Hatch window times and the ability of the embryo to hatch
are affected by the air quality and temperature. One reason for varying incubation
length relates to the ability of the egg shell to diffuse oxygen (Rahn et al., 1974). The
levels of 1 to 3% CO2 during the first 8d of incubation and over 6% for the 9 to 12d
of incubation can adversely affect hatch (Tona et al., 2007). Concentration of CO2
during the last few days of incubation helps stimulate hatching. When the egg
cannot meet the oxygen demands of the embryo, pipping will begin. Excessive CO2
during development can be lethal to the embryo (Tona et al., 2007).
Temperature plays an important role as well. High temperatures early in
incubation causes embryonic mortality (French, 2000). Overheating in the 2nd and
1

3rd quarters of incubation causes a head malposition, which reduces hatchability.
Turkey eggs incubated at a consistent reduced temperature hatched later than those
incubated at industry recommended temperature (French et al., 1994). Individual
eggs will experience different temperatures in the incubator (French et al., 1997).
The slight difference in incubation temperature for each egg contributes to the
variability of the HW.
Fasting or delayed access to feed: effects on the intestine
The main issue with HW is that early hatched chicks often have no access to
feed or water up to 72h after hatch by considering the spread of the HW, chick
handling, and transport time (Romanini et al., 2013). Simulation of shipping times as
short as 4 or 10h reduced BW compared to 0h shipping times (Bergoug et al., 2013).
This reduction in BW was maintained for the first 21d in broilers. Fasting at any age
for a period of 48h reduces the BW of birds (Geyra et al., 2001b). Additionally,
fasting during the first 48h post-hatch retards intestinal growth and reduces the
number of total cells and percentage of proliferating cells in the crypt (Geyra et al.,
2001a). In turkeys, a 48h delayed access to feed at the start of life reduces BW by
over 10% (Pchavov and Noy, 1993). Poults with a 48h delayed access to feed have
fewer bacteria in the digesta after 24h on feed (Potturi et al., 2005). In poults that
had a 54h delayed access to feed, the pancreatic amylase and trypsin activities were
reduced (Corless and Sell, 1999). The mucin layer thickens in chickens whose feed is
withheld for 72h (Smirnov et al., 2004). Additionally, the overall weight of the
intestine is reduced by 30%, and the villus surface area is decreased by over 25%

2

after 72h of fasting. In contrast, feeding in the hatcher or later in transport boxes
improves intestinal development (Noy et al., 2001).

In ovo technologies
The recent development of in ovo technologies has allowed researchers and
producers to introduce medicinal and nutritional factors into the egg prior to hatch.
In ovo vaccination is a practice used in over 90% of broiler production (Williams
and Zedek, 2009). Vaccines are injected into both the air cell and the amnion
without reducing hatchability. The vaccine technologies could be used for the
introduction of probiotics before hatch, but it is unclear if in ovo probiotics would
reduce hatchability. Hatcheries strive to keep the egg as clean as possible to prevent
pathogenic bacterial contamination that would reduce hatchability. One milligram of
competitive exclusion cultures injected into the air cell decreased hatchability
(Meijerhof and Hulet, 1997). Inoculation of diluted bacteria (103 cfu) from adult
cecal droppings into the air cell reduced hatchability as well, but the birds that did
hatch were more resistant to Salmonella (Cox et al., 1992). Injection into other parts
of the egg may not yield the same results. Currently, industry belief is that any
bacterial contamination of the egg could reduce hatchability.
Previous studies have focused on in ovo nutrition as a way to optimize the
start in life in chicks or poults. In ovo nutrition can come in several forms such as
injectable carbohydrates, amino acids, etc. The injectable carbohydrates could be
glucosamine, glucose, or other simple sugars. The exogenous injection of
carbohydrates into chicken eggs increased BW and intestinal villi area due to
increased villus height (Tako et al., 2004). There was an increased number of goblet
3

cells in the intestine of chickens given maltose, dextrose and dextrin at 17.5d of
incubation (Smirnov et al., 2006). In ovo injection of 19 different amino acids did not
increase serum amino acid levels in E19 embryos (Ohta et al., 2001). The use of
mannanoligosaccarides in chicken eggs at 17d of incubation increased villi area 20
to 32% (Cheled-Shoval et al., 2011). Goblet cells in the villi were increased 20 to
50% higher. Brush border peptidases and isomaltase activity was also increased.
These nutritive additives have demonstrated that intestinal development can be
increased prior to hatch.
Bacterial colonization of the poultry intestine
The egg has several antibacterial defenses. Ovotransferrin is a an Fe binding
protein that has antibacterial properties (Giansanti et al., 2005). Lysozyme, the
other major defensive protein, exhibits antibacterial properties against grampositive and gram-negative bacteria (Pellegrini et al., 1997). After the bird hatches,
these defenses are lost and the bird must rely on passive immunity before
developing adaptive. As a result, the bacterial profile of the intestine is in flux as the
bird ages (Lu et al., 2003). Diet, age of host, disease challenge, and antibiotic
administration are factors that can influence the intestinal community.
Born without a mature immune system, chickens must rely on maternal
antibodies for protection against pathogens early in life. Exposure to pathogens
begins at the hatchery, where birds consume debris and dust that contain E. coli. and
other disease causing bacteria (Cortés et al., 2004). Once exposed to harmful
bacteria, a young bird’s growth is reduced (Vila et al., 2009). Antibiotic use, the
traditional method of control, is being phased out due to consumer preferences and
4

concern of disease resistant bacteria (Vila et al., 2009). An alternative to mitigate the
effects of pathogenic bacteria is the use of competitive exclusion (CE) cultures. The
CE cultures are often called probiotic bacteria or direct fed microbials. Research
shows that not all bacteria are harmful to birds and that beneficial bacteria can
increase energy utilization from the diet (Muramatsu et al., 1991; Muramatsu et al.,
1994). Birds raised in sterile conditions, thus reducing the amount of microflora in
the gut, have a reduced intestinal weight and villus length (Maisonnier et al., 2003).
Intestinal development is not the only beneficial effect of early establishment of
bacteria, as immune function is upregulated as well (Kelly et al., 2007).
The effect of age on bacterial colonization
The intestine of a newly hatched chick or poult is thought to be sterile, but by
24h post hatch, a bacterial population is present in the intestinal tract (Naqi and
Lewis, 1970). Traditionally, the amount of bacteria is determined by plating.
However, modern molecular techniques have demonstrated that not all bacteria
species can be plated (McCracken, 2001). The use of PCR and other modern
methods of molecular sampling have demonstrated that a greater diversity of
bacteria species exists in the intestine than previously thought (Apajalahti et al.,
2004). These modern methods have shown conflicting results as to whether there
are bacteria in the intestine of chicks at hatch (Pedroso et al., 2005) or not (Wielen
et al., 2002). Though the population diversity and density is unclear at hatch, as the
bird ages, the bacterial load increases. Bacterial species diversity reaches a peak, in
the poult, between week 9 and 18 (Lu and Domingo, 2008). In chickens, bacterial
density peaks at 35d with a concentration of approximately 1011 cfu/g of digesta in
5

the ceca and ileum (Apajalahti et al., 2004). In younger birds, there are predominate
bacterial species throughout the duodenum, jejunum, and ileum (Wielen et al.,
2002). As the bird ages, these bacteria become more section specific because the
microenvironments throughout the gastrointestinal tract are different. These
environments affect the bacterial load in each section. The diversity and density of
bacteria increases toward the distal parts of the gastrointestinal tract (Brisbin et al.,
2008). In the proximal end of the small intestine, the bacterial load is 103 to 105
cfu/g while the distal portion of the intestine has 108 to 109 cfu/g. The ceca has
bacteria as high as 1012 cfu/g (Gong et al., 2002).
Non-host factors that effect intestinal microbiota
There are several factors beyond the host or intestinal section that can
influence the bacterial community in the intestine. The diet provides nutrients not
only to the host, but to the intestinal bacteria as well. The passage rate of the diet
will effect the bacterial community composition (Rozee et al., 1982). When 0 to 21d
chickens from organic or conventional production settings were compared, the
gastrointestinal tract microbes were not different suggesting that the nutrient
intake was more important than management practices (Wise and Siragusa, 2007).
Antibiotic growth promoters have been used in the poultry industry to reduce
pathogenic bacteria in the intestine. Virginiamycin increases the number of
commensal bacteria in the proximal small intestine, whereas there is a large
population shift in the distal intestine (Dumonceaux et al., 2006). Antibiotics
increase the percentage of commensal population mostly influencing gram-positive
bacteria (Knarreborg et al., 2002). Several broad spectrum antibiotics increase the
6

total amount of Lactobacillus in the intestine and alter the overall bacteria
population in the ileum (Torok et al., 2011). Antibiotic use not only reduces the
pathogenic bacteria load, but may also select for bacteria that are able to confer a
health benefit to the host. With these subtherapeutic uses of antibiotics being
phased out, viable alternatives need to be identified.
Probiotics in a non-challenge setting
Lactobacilli and Enterococci are often used in probiotics (Patterson and
Burkholder, 2003) with some strains having been shown to improve BW and FCR in
poultry (Cavazzoni et al., 1998; Jin et al., 1998; Khan et al., 2007). Day of hatch
broiler chicks given a probiotic containing Lactobacillus acidophilus, Lactobacillus
bifidus, and Enterococcus faecalis, had significantly increased BW compared to those
not given a probiotic by 42d although FCR was not different (O’Dea et al., 2006). In a
comparison study when chicks were given either Lactobacilli or Enterococci, both
improved BW and FCR compared to control birds, but feed conversion in birds given
Enterococci was significantly better than the Lactobacilli fed birds (Awad et al.,
2009). The performance improvements with probiotics may be due to improved
intestinal health. Villus height, crypt depth, and villus height to crypt depth ratio
(VCR) were improved in the jejunum and ileum of broilers fed probiotics
(Chichlowski et al., 2007).
The mechanism behind this increased performance may differ. Probiotics
increase the amylase activity levels in broilers when fed for the first 40d of life (Jin
et al., 2000). Undigested polysaccharides such as cellulose, xylan, and undigested
starches are further broken down by the gut microbiota (Resta, 2009). Microbiota
7

can ferment these carbohydrates and produce SCFA that can be used by the
enterocytes as a primary source of energy (Resta, 2009). Broilers supplemented
with Lactobacillus cultures for 28d, were found to have reduced abdominal fat, LDL,
and total serum cholesterol (Kalavathy et al., 2003). Additionally, total carcass
cholesterol of the broiler was reduced 13 to 19% by Lactobacillus (Kalavathy et al.,
2006).
Lactic acid bacteria
Lactic acid bacteria (LAB) are a large group of bacteria across many species
that share many of the same characteristics. Lactic acid bacteria are generally
accepted to be gram-positive, usually catalase negative, non-spore forming cocci,
and coccibacilli or rods with a DNA base composition of less than 55 molar percent
G+C (Klein et al., 1998). Bifidiobacteria is an exception to these rules with a G+C
content over 60%. All LAB grow anaerobically, but unlike most anaerobes, they
grow in the presence of O2 as "aerotolerant anaerobes". The term lactic acid bacteria
comes from the ability of the group to ferment glucose primarily to lactic acid or to a
combination of lactic acid, CO2 and ethanol. Lactic acid production as a result of
fermentation is a characteristic of many bacteria, but the term LAB is reserved for
genera in the order Lactobacillales and Streptococcus.
Lactobacillus
Lactobacilli are one of the most frequently used bacteria in probiotics and
have demonstrated competitive exclusion properties with pathogenic bacteria. They
also attach to epithelia cells in the intestine and enhance the immune response
(Heravi and Kermanshahi, 2011). Lactobacillus and other bacteria are often species
8

specific and inhabit a small area in the gastrointestinal. These bacteria colonize in
the small intestine and caeca of chickens, a week after hatch (Heravi and
Kermanshahi, 2011). Over 10 strains of Lactobacillus have been shown to adhere to
the crop and the intestinal epithelium (Jin et al., 1996) of chickens. Dietary inclusion
of Lactobacillus has been shown to have multiple effects in several avian species. In
combination with Enterococcus and Bifidobacteria, Lactobacillus has been shown to
increase the villus height in the intestine of broilers (Chichlowski et al., 2007).
Whether as a single strain or as a combination of 12 strains, Lactobacillus increased
amylase activity in the small intestine of broilers without effecting proteolytic and
lipolytic activities (Jin et al., 2000). Lactobacillus can be as effective as antibiotics
from a growth promoter perspective (Kalavathy et al., 2008). Lactobacillus salivarius
promoted butyric acid producing bacteria in the broiler ceca, which in turn reduced
the Salmonella population (Meimandipour et al., 2010).
L. reuteri is of great interest as a probiotic due its production of reuterin, a
bacteriocin where it gets its name (Talarico et al., 1988). Purified reuterin reduced
Listeria and E. coli numbers in contaminated meat (El-Ziney et al., 1999). The
membrane-anchored ubiquitin-fold protein allow L. reuteri to adhere to the mucus
layer in the intestine (Roos and Jonsson, 2002). Once adhered, the bacteria can block
receptor sites from pathogenic bacteria. L. reuteri has developed host specificity, so
producers using it as a probiotic must select a strain from the host species or a
closely related species to ensure maximum effect (Frese et al., 2011).

9

Bifidiobacteria
Bifidobacterium are gram-positive anaerobic, non-motile, non-spore forming
bacteria (Petr and Rada, 2001). Bifidobacteria are found in the intestinal tracts of
humans and many other animal species and have history of safe consumption. The
ubiquitous nature of the bacteria has led to many companies and researchers to use
it as a probiotic (Picard et al., 2005). The bacteria has been proven to be safe for
human consumption even in immune-compromised scenarios (Borriello et al.,
2003). Bifidobacteria can be found in several places throughout the gastrointestinal
tract of poultry. In laying hens, Bifidobacteria has been found in the crop (Petr and
Rada, 2001) and in the ceca of poultry (Rada, 2000; Thitaram et al., 2005b).
Bifidobacteria can become dormant during storage, allowing for better
shipping and storage (Saarela et al., 2005). The actions of Bifidobacteria are varied.
Several researchers have demonstrated that Bifidobacteria is capable of adhering to
intestinal cells (Crociani et al., 1995; Gopal et al., 2001; Servin, 2004). The adhesion
to intestinal cells allows for a barrier to prevent pathogenic bacteria from
interacting with intestinal receptor sites. The bacteria may also bind to the mucus
secreted from intestinal cells (Servin, 2004). Adhesion comes from several forces
including passive, electrostatic interactions, hydrophobic steric forces, lipoteichoic
acids; and specific structures, such as lectin-covered external appendages (Servin,
2004). Bifidobacteria may alter the immune response of chickens. Depending on the
age of bird and route of administration, research has demonstrate that the systemic
immune response can be altered by Bifidiobacteria inclusion in the diet (Haghighi et
al., 2005).
10

In poultry, the population of Bifidiobacteria within the intestinal tract can be
altered though several mechanisms. The most effective method is to supplement the
diet or drinking water with the bacteria themselves. Dietary additions of certain
sugars, such as oligosaccharides and isomaltooligosaccharide increased
Bifidobacteria in the ceca of chickens (Thitaram et al., 2005a). The bacteria have the
ability to ferment and digest these sugars as a food source. Since these
carbohydrates are indigestible to the host, breakdown of the sugars is a benefit to
both the host and the commensal bacteria (Williams et al., 2009). Certain pathogenic
bacteria can utilize indigestible starches as a food source as well, thus by increasing
the commensal bacteria load within the intestine of the host, there is a reduction in
food sources for pathogenic bacteria.
Enterococcus
Enterococcus has a long history of study and undergone several name
changes. The name "enterocoque" was first used by Thiercelinina paper published
in 1899; the name was used to emphasize the intestinal origin of this gram-positive
diplococcus (Murray, 2003). The name Streptococcus faecalis (faecalis, relating to
feces) was given by Andrewes and Horder (Murray, 2003). They isolated the
organism from the intestine of a patient with endocarditis and renamed the bacteria
Streptococcus faecalis. More modern DNA techniques have distinguished
enterococcus from streptococcus and thus a new genus was formed.
Enterococcus is another lactic acid bacteria which are non-spore forming, but
tolerant of a wide variety of temperatures, pH conditions, and high salt
concentrations. Enterococcus species have been used in several animal species as a
11

probiotic to offset the effects of several pathogenic bacteria. Camplyobacter spp. and
Clostridium spp. were found in dogs fed E. faecium (Vahjen and Männer, 2003).
Inclusion in the diet reduced sow mortality during an E. coli infection (Taras et al.,
2006). Enterococcus can be ingested and survive in the gastrointestinal tract of
humans (Lund et al., 2002)
In poultry, Enterococcus has mainly been used to combat the effect of
Salmonella in several species of poultry. Enterococci are effective against Salmonella
pullorum if administered prior to infection but not as a therapeutic agent (Audisio et
al., 2000). E. faecium produces bacteriocins that inhibit the growth of Salmonella on
media (Audisio et al., 1999). When fed to broilers, villus height was increased and
FCR was improved (Samli et al., 2007). In turkey poults, dietary supplementation
with Enterococcus increased the amount of lactic acid bacteria in the small intestine
(Vahjen et al., 2002). In combination with other probiotic species, Enterococcus
improved growth and antibody production (Kabir et al., 2004).
Pediococcus
Pediococcus, another potential probiotic strain, is a gram-positive bacteria,
able to grow in various environmental pH ranges, temperatures, and osmotic
pressures, and thus able to colonize and inhabit digestive tracts (Klaenhammer,
1993). A common Pediococcus strain used is acidilacti. This has been demonstrated
in several studies to reduce the harmful effect of a coccidial infection (Lee et al.,
2007; Taheri et al., 2010). P. acidilacti has been used in the production of meat
products, such as sausage, to reduce microbial contamination of food (BaccusTaylor et al., 1993; Yousef et al., 1991). There are several bacteriocins that can be
12

isolated from Pediococcus. The bacteriocin, Pediococcus acidilacti pediocin is the
most studied, but there are other bacteriocins that not only reduce the growth of
listeria, but can cause harm to host cells as well (Villarante et al., 2010). Though
bacteriocins harm host cells, the bacteria has been demonstrated to increase
survivability of tilapia (Ferguson et al., 2010). In poultry, P. acidilacti in a
combination with other bacteria improved the growth of broilers (Mountzouris et
al., 2007). In laying hens, P. acidilacti supplementation decreased the amount of
broken eggs and reduced yolk cholesterol (Mikulski et al., 2012).
Bone development and probiotics
Understanding the skeletal system’s development is important for any
poultry operation. Losses from skeletal development cost the industry millions of
dollars (Cook, 2000). Each avian species must be managed differently due to age and
BW when marketed. The turkey doesn’t reach full tibia length until around 130d
whereas the broiler reaches full tibia length in under 50d (Lilburn, 1994). Just as
bone length is different between species, mineralization can be different as well. The
amount of bone mineralization regardless of species or age determines the stiffness
and flexibility (Seeman, 2008). Bone mineralization responds to load potential,
therefore, each bone mineralizes differently depending on the applied loads. The
bones of poultry species are approximately 70% mineral (Rath et al., 2000). A
reduction in mineralization or mineralization rates can be the cause of several
skeletal problems in poultry. Ossification of bone in poultry species begins in ovo,
but primarily takes place after hatch (Bain and Watkins, 1993) Mineralization in
turkeys, as represented by femoral ash, continues to increase from DOH to 20wk,
13

with a slight decrease from 12 to 16wk (Zhong et al., 2012). Mineralization of
tibiotarsus in meat type chickens increases rapidly between 4 and 11d (Williams et
al., 2000). Research suggests that the rate of mineralization throughout life is
constantly changing.
There are several factors that influence mineralization and bone
development. Genetics play an important role in bone mineralization rates in
turkeys and chickens with birds selected for the fastest growth tending to have the
highest number of skeletal problems (Dibner et al., 2007; Talaty and Katanbaf, 2009;
Zhong et al., 2012). Higher stocking densities increase lameness and tibial
dyschondroplasia in broilers (Sanotra et al., 2001; Sorensen et al., 2000). The most
important factor in bone development that can be altered by producers is diet.
Calcium and P are the two most important minerals in bone development. The ratio
of Ca:P is important to monitor as variations in the Ca:P ratio of bone will cause
alteration in bone mineral crystal structure and consequently mechanical properties
(Thorp and Waddington, 1997). If either Ca or P is too high, the availability of the
other is reduced (Williams et al., 2004). Calcium deficient diets lead to osteoporosis,
especially in laying hens or older birds (Whitehead and Fleming, 2000). Large
particulate Ca increases bone mineralization in laying hens (Saunders-Blades et al.,
2009). The form and amount of Ca and P can affect the availability of these nutrients
to the bird.
A probiotic bacteria effect on Ca metabolism is important for producers to
consider when adding probiotics to the diet. Potential mechanisms for probiotic
alterations of Ca metabolism maybe an increase in absorptive surface by
14

proliferation of enterocytes, increased expression of Ca binding proteins, increased
releasing of bone modulating factors like phytoestrogen from food or degrading of
phytic acid (Peacuterez et al., 2008). A yogurt containing several Lactobacillus
species increased apparent Ca absorption and bone mineral content in rats (ScholzAhrens et al., 2007). In vitro studies with Caco-2 cells demonstrated that
Lactobacillus salivarius increased transepithelial Ca transport into the cells (Gilman
and Cashman, 2006). Feeding a combination of Clostridium butyricum and Bacillus
subtilis in a low Ca diet (75% of recommended) resulted in similar bone health
(length, weight, ash percentage, and bone strength of tibia) for broiler chickens fed
the control diet (100% of recommended). Thus, ameliorating the effects of a low Ca
diet (Houshmand et al., 2011). Further studies with Bacillus licheniformis and
subtilis in broiler diets, resulted in an increase in thickness of lateral and medial wall
of the tibia, tibiotarsal index, and ash percentage (Mutus et al., 2006).
Common practice is to use bacteria derived phytase in the diet to improve P
digestibility or bioavailability. There are several bacterial sources for microbial
phytase that have proven effective and thus usage has increased over the last 20
years (Kiarie et al., 2013). Bacterial phytase improved P retention and bone
mineralization in turkeys (Applegate et al., 2003). The addition of pure strains of
bacteria could alter P digestion in the same way. In laying hens, diets supplemented
with molasses and Lactobacillus had increased phytase activity and P retention
(Nahashon et al., 1994). Inclusion of phytase-producing Mitsuokella jalaludinii from
rumen of cattle increased tibial ash in broiler chickens (Lan et al., 2002). Due to the
variety of strains and implementation method used, which strains or time of
15

administration would be ideal to improve bone health in poultry is unclear.
Glucose Transporters
Feed is the highest cost to any livestock operation, and understanding
nutrition is key to optimal performance. Understanding the body’s ability and
capacity to absorb nutrients is a key factor in designing and implementing diets.
Nutrient transporters affect the ability of the body to carry nutrients from the lumen
into the blood stream. Glucose is an almost ubiquitous energy source for all animals.
The poult embryo begins to break down the yolk early in development. Compared to
mammals, birds have a higher blood glucose concentration throughout life (Akiba et
al., 1999; Tokushima et al., 2003). Glucose can be detected as early as E4, in
chickens; therefore, nutrient transporters must exist at that time to allow for
absorption into the body (Hazelwood, 1971). Further evidence of the embryo’s
ability to digest sugars is in the levels of digestive enzymes. Detectable levels of
disaccharidases are seen as early as E9. Other digestive enzymes such as
carboxypeptidase A and chymotrypsin are seen at E16 (Brisbin et al., 2008;
Marchaim and Kulka, 1967). The enzyme activity level of sucrase, maltase and
aminopeptidase increases two days prior to hatch in chickens (Uni et al., 2003). As
the body develops methods to breakdown simple sugars, the body must also
develop nutrient transporters to allow these nutrients into the cell. In chicken
embryos, aminopeptidase, sodium-glucose cotransporter-1 (SGLT-1), is found on
E19 whereas detectable levels of mRNA expression was detected as early as E15
(Haller et al., 2000; Uni et al., 2003). Though there are several detectable nutrient
transporters seen throughout development, the one class that is most essential to
16

glucose is the GLUT or solute carrier(SGLT) family. Glucose transport is done
through several mechanisms but is mostly facilitated by GLUT and SGLT
transporters. There are several GLUT isoforms in poultry (GLUT 2, 3, and 8) (Kono
et al., 2005). The most common GLUT transporter is GLUT1. The GLUT4 glucose
transporter in other species has not been identified in birds.
The diet plays an important role in regulation of GLUT2. The ability of GLUT2
to translocate to the plasma membranes of islet cells is inhibited in rats fed high
energy diets for a week (Reimer and Ahrén, 2002). In rats, mice, and sheep,
intestinal glucose transport increases in about 1 to 3d after a switch to a highcarbohydrate diet (Ferraris, 2001). Low salt diets reduced the presence of GLUT2 in
the enterocyte membranes of White male leghorns (Garriga et al., 2000). Dietary
ingredients such as flavonoids in fruit inhibit the expression of GLUT2 in vitro
(Kwon et al., 2007). Dietary contaminates such as the mycotoxin Cytochalasin B
inhibits GLUT2 expression (Ferraris, 2001).
There are several environmental factors that affect GLUT expression. The age
of the birds affects expression. The expression of GLUT2 in the intestine of broilers
increases linearly from E20 to d14 (Gilbert et al., 2007). The body’s physiological
reaction to a meal regulates GLUT2, regardless of age. Insulin causes GLUT2 to
retreat from the apical and basolateral membrane of enterocytes, thus reducing
blood sugar (Kellett et al., 2008). In challenge conditions, expression is altered.
Bacterial infection of brush border cells reduces GLUT5 expression in vitro (LievinLe Moal et al., 2002). The previous studies on glucose transport highlight the
understanding of glucose transport in other species, yet there is a need to better
17

understand transport in turkeys.
Peptide transporters
Protein is a key nutrient in the building of muscle and other body tissues. The
source of protein and amino acids mainly comes from the diet. A large portion of the
dietary amino nitrogen is absorbed as oligopeptides rather than as free or single
amino acids (Ganapathy et al., 1994). A key oligopeptide transporter is PepT1,
which is H+-dependent. PepT1 is the main peptide transporter in the intestine of
many animals and potentially can transport all 400 di and 8000 tripeptides that
combine to form 20 different dietary AA (Daniel, 2004). In poultry, PepT1 is located
in multiple tissues, but distribution is greatest in the small intestine, kidney, and the
ceca (Chen et al., 1999).
There are several factors that influence the presence of PepT1 in the
intestine. Age is important as the expression of PepT1 is seen as early as E23 in
turkeys, and there is roughly a 3-fold increase in expression beginning 5d prior to
hatch (Van et al., 2005). Based on PepT1 expression patterns in turkeys, small
peptides can be transported across the lumen at hatch (de Oliveira et al., 2009).
Expression of intestinal PepT1 increases with enterocyte maturation in mammals
(Meredith and Boyd, 2000). Dietary and intestinal environment play an important
role as well. PepT1 is pH dependent, and Na and K independent (Van et al., 2005). As
crude protein levels increase in the diet, the levels of PepT1 remain unchanged
(Frazier et al., 2008). In vitro studies demonstrated that specific amino acids or
dipeptdies decrease PepT1 expression (Adibi, 2003). Increase in quality of protein
or decrease of feed intake, increases the amount of PepT1 in broiler chicks in the
18

first week (Gilbert et al., 2008). A 24h fast increased the levels of PepT1 in the
intestine of rats (Adibi, 2003). These results indicate that the maturing intestine will
alter PepT1 regulation depending on both the age and dietary status of the host.
Conclusion
The increased emphasis on early life development of turkeys and the
understanding that bacteria play an important role in that development, in ovo
probiotics need to be explored. Previous work on in ovo bacteria has thus far been
unable to find a safe and effective bacterial load. There is most likely a bacteria
concentration that can be injected to help establish the intestinal microflora without
harming the host. The benefits or negative ramifications need to be further studied
and understood to determine application to the poultry industry. Therefore, the
potential exists for probiotics and commensal bacteria to benefit the host, which
could help the bird’s transition to feed and resist pathogenic bacteria during the
important first week post hatch.

19

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31

CHAPTER 1: TURKEY POULT HATCHABILITY IN RESPONSE TO VARIOUS
LEVELS OF IN OVO PROBIOTICS
ABSTRACT
Turkeys have to overcome several challenges early in life, including exposure
to pathogens and failure to transition from yolk nutrition to dietary sources.
Probiotics are thought to help alleviate these symptoms in the post-hatch poult, but
any beneficial effects, if given prior to hatch are unknown. Additionally, the optimal
level of probiotic that can be injected into the egg must be established. The overall
objective was to evaluate different probiotic concentrations injected into turkey
eggs prior to hatch. Specific objectives were to measure hatchability differences and
determine bacterial load in the poult ceca at hatch. Fertile turkey eggs were candled
on d25 and allocated to one of six treatment groups: (1) Non-injected eggs hatched
in a separate hatcher (positive control), (2) non-injected eggs housed with hatcher
with probiotic injected eggs (negative control), (3) eggs injected with 1 ml of saline
and placed in the hatcher with probiotic injected eggs (sham control), (4) eggs
injected with 104 cfu/ml probiotic solution, (5) eggs injected with 106 cfu/ml
probiotic solution or (6) eggs injected with 108 cfu/ml probiotic solution with 9
replicates per treatment and 15 eggs per replicate. All injections were in the amnion.
Hatch counts were taken for each replicate and cecal contents collected from two
birds per replicate to determine bacterial counts on d28. Cecal contents were plated
on media plates with LB agar, MRS agar, Bifidobacteria agar or Enterococcusel agar.
Plates were incubated for 24h and colony counts were recorded. Probiotics injected
into turkey eggs at 108 cfu or lower did not affect hatchability (P<0.05). The
32

bacterial counts for Bifidobacteria were highest among all other bacteria and the
injected treatment’s bacterial counts were higher compared to the negative control
treatment. The bacterial number increased in the negative control birds compared
to the positive control suggests that hatchery debris impacts the bacterial load in
the intestine. The injection of probiotic bacteria into the egg increased bacterial
content in the intestine without reducing hatchability.

INTRODUCTION
The EU implementation of an antibiotic ban has left the poultry industry
searching for an efficacious and consumer accepted alternative (Pugh, 2002).
Probiotics have demonstrated the ability to improve performance in during a
disease challenge and because of their help during a challenge may be a suitable
alternative to antibiotics (Grimes et al., 2008; Guarner and Malagelada, 2003;
Higgins et al., 2010). Probiotics optimal administration period is reported as a 24 h
minimum prior to the onset of the challenge (Audisio et al., 2000). The initial
challenge for a newly hatched chick or poult can occur on the day of hatch (Craven
et al., 2001; Rodgers et al., 1999); therefore, the probiotic administration should
occur in ovo.
The poultry industry has used in ovo technologies since 1980 with the
introduction of Marek’s vaccine (Sharma, 1987). The technology is widely used to
vaccinate embryos prior to hatch, thereby improving the viability or health status of
the chick post-hatch (Goyal and Patnayak, 2004; Johnston et al., 1997; Ricks et al.,
1999). More recently, in ovo nutrition has improved intestinal development and
33

energy stores in the first week chick or poult (Uni et al., 2005). Although the studies
demonstrate an impact at day of hatch, the effect does not always improve the first
week livability.
One main factor in early poult mortality is the transition from a
gluconeogenic state relying primarily on lipid based yolk stores to a carbohydrate
and protein dense diet post-hatch and an immature intestine that is not equipped to
handle it (de Oliveira et al., 2013; 2009). Commensal bacteria have been shown to
improve intestinal maturation and are instrumental in the development of the
intestine (Grimes et al., 2008; Guarner and Malagelada, 2003; Higgins et al., 2010;
Hooper et al., 2002). Probiotic bacteria, which often originate from commensal
bacteria, have the ability to improve intestinal maturation post-hatch and alter
intestinal colonization (Audisio et al., 2000; Matur and Eraslan, 2012). The early
maturing intestine, in turn, could adapt to feed quicker, improving performance. The
injection of in ovo probiotics could hasten the maturation of the intestine or could
overwhelm the embryo and reduce hatchability. The commercial bird’s immune
system isn’t functional until after the first week (Bar-Shira et al., 2003; Craven et al.,
2001; Rodgers et al., 1999). The added bacteria, though non-pathogenic, wouldn’t be
recognized and the host and bacteria interaction could result in death or the
bacteria could reduce the pathogenic bacteria in the intestine. Previous work has
shown that injection of bacteria or bacterial contamination in the egg, reduce
hatchability or increase mortality in the first week post hatch (Cortés et al., 2004;
Sharma and Burmester, 1982). Therefore, the study’s objective was to determine
the effect of a commercial probiotic injected in ovo on poult hatchability and cecal
34

bacterial load at hatch. The first hypothesis is that in ovo probiotic injection will
maintain or increase hatchability in turkey eggs compared to control eggs. The
second hypothesis is that in ovo probiotic injection will increase poult cecal bacterial
counts at DOH compared to control birds.

MATERIALS AND METHODS
All procedures were approved by Michigan State University Institutional Animal
Care and Use Committee. The approval number was AUF # 11/10-190-00.
Probiotic preparation
The microencapsulated probiotic contained 2 strains of Lactobacillus reuteri,
and a single strain of Enterococcus faecium, Bifidobacteria animalis and Pediococcus
acidilacti (Biomin, Herzogenburg, Austria). The probiotic mixture was suspended in
sterile PBS, and LB media was plated prior to solutions being made to determine
concentration. The probiotic mixture was weighed and added to sterile PBS (25 ml)
the day of the sample injection (E25) and diluted to create solutions of 108 cfu/ml,
106 cfu/ml and 104 cfu/ml. Solutions were stored on ice (~4h) and vortexed for 15s
immediately prior to egg injection.
Bird incubation and treatments
One thousand turkey fertile eggs were individually weighed, set in an
incubator (Petersime Model 5; Petersime Incubator Co., Gettysburg, PA), and
incubated for 25d. Egg weights were within 0.5 g average per replicate. Incubator
conditions (37.5 °C dry bulb and 29.2°C wet bulb) were monitored throughout the
trial using General Tools H10 data loggers (General tools, New York, NY). All eggs
35

were candled on E24; infertile eggs were removed, and a line was drawn at the
lowest point of the air cell. On injection day, E25, externally pipped eggs were
removed from the study. The remaining eggs were randomly assigned, using the
experimental animal allotment program, to six treatments based on initial egg
weight (EAAP, 2009). The treatments were: (1) Non-injected eggs hatched in a
separate hatcher (positive control), (2) non-injected eggs housed in the hatcher with
probiotic injected eggs (negative control), (3) eggs injected with 1 ml of saline and
placed in the hatcher with probiotic injected eggs (sham control), (4) eggs injected
with 104 cfu/ml probiotic solution, (5) eggs injected with 106 cfu/ml probiotic
solution or (6) eggs injected with 108 cfu/ml probiotic solution with 9 replicates per
treatment and 15 eggs per replicate. A treatment replicate was removed from the
incubator, and the large/blunt end of each egg wiped with an alcohol disinfectant
wipe. A Dremel stylus rotary tool (Model # 1100-01: Dremel, Racine, WI) was used
to drill a hole into the lowest point of the egg’s air cell as marked on the previous
day. An Allflex MR2 repeater syringe (Allflex USA INC) equipped with a 22 ga 1”
needle was used to inject each egg with 1 ml of probiotic solution (104, 106, or 108
cfu/ml) or a sham injection of sterile saline. The repeater syringe was rinsed with
sterile saline between each replicate and the needle replaced. All drilled holes were
sealed with paraffin wax, and each replicate was assigned to one of two hatchers.
Two treatments (negative control and sham control) were not drilled or injected but
rather each replicate was removed from the incubator, wiped with an alcohol wipe
and left at room temperature for the same amount of time as the other replicates.
The rest of the treatments were placed in pedigree baskets and randomly placed in
36

one of two Surepip (Agro Environmental Systems Inc., Dallas, Georgia, USA)
hatchers.
Sampling
The hatchers were opened and hatch counts recorded on E28. A poult was
considered hatched if the poult had completely cleared the shell; pipped if any
portion of the shell was cracked but the poult failed to clear the shell; unhatched if
the shell was completely intact. All poults were removed from the hatcher one
replicate at a time and euthanized. Body weights were recorded on each hatched
poult. For cecal content collection and yolk sac collection, two birds per replicate
were euthanized one at a time and the whole body of each bird was dipped in an
antiseptic solution. The bird was removed from the antiseptic solution and placed
on a clean disinfected cutting board. The abdominal cavity was opened with scissors
that had been dipped in alcohol and flame dried. Gloves were changed after the
abdominal cavity was opened to prevent contamination from the outside of the bird.
Using two sets of forceps, the yolk sac and both ceca were extracted from the bird.
The yolk sac was placed in a weigh boat and the difference between yolk weight and
total body weight was yolk-free body weight. Cecal contents were collected and
pooled by gently squeezing the cecal contents into 1.5 mL microcentrifuge tubes,
using the forceps to prevent contamination from gloves. Sterile forceps and gloves
were used for each bird to prevent cross contamination. Samples were immediately
chilled on ice until ¼ of the poults had been sampled at which point they were taken
back to the lab to make dilution series. This was done to prevent prolonged
exposure to the ice and to ensure viability of samples for plating.
37

Cecal content plating
Each cecal sample was a pool from two birds. For the six serial dilution
series, 30 µl of each cecal sample was pipetted into 270 µl of sterile PBS. The
mixture was vortexed for 15s, and the pipetting procedure repeated to create the
next dilution. Next, 50 µl of each serial dilution and the pure sample were pipetted
onto the center of each of four different media plates and spread using a L-shaped
glass spreader rod. Rods were flamed in alcohol between each use. The four medias
used were: Luria-Bertani agar (Acumedia, Lansing MI) for general gram-negative
bacteria, Bifidiobacteria agar (HiMedia Laboratories Ltd. Mumbai, India) for
Bifidobacteria spp, MRS agar (HiMedia Laboratories Ltd. Mumbai, India) for
Lactobacillus spp. plus Pediococcus spp. and Enterococcsel agar (a modified esculin
bile agar; Becton Dickinson and Company, Franklin Lakes, NJ) for Enterococcus spp.
All medias were made according to manufacturer instructions and stored at 15°C
prior to being used. Plates were incubated for 24h at 37°C in an incubator (Precision
Industries model# 30M,Chicago, IL), removed, and colony counts were made. Only
counts from a single plate from the dilution series with approximately 25 to 300
total colonies was selected for counting. The plate was placed on a lightbox and a
marker was used to mark counted colonies, and a lab cell counter was used to keep
track of the data counts.
Statistical Analysis
All parameters were analyzed using the PROC MIXED analysis of SAS (v 9.3)
with the LSMeans procedure. Differences between means were tested using the
pdiff option of the LSMeans statement with significance accepted at P<0.05.
38

RESULTS AND DISCUSSION
In ovo administration of probiotics did not affect poult hatchability except the
hatchability of 108 cfu injected eggs were lower compared to the saline sham
treatment (Tables 1, 2). The hatchability of the saline sham treatment was the
highest and significantly higher than the 108 treatment (P<0.05). This might be a
result of the sham poults not being as dehydrated as the control poults. If the poult
is dehydrated, and needs liquid to enhance hatchability, this might be a simple
solution. There were differences in pipping numbers. The saline treatment had
lower pipping rates than all the injected treatments (P<0.05). The higher percent of
pipping may indicate that the bacteria injection shifts the hatching window slightly
compared to saline sham treatment. Reason for this shift and if the injection is
involved is unknown. There were no differences in number of unhatched eggs,
though there was a trend where 104 cfu injected eggs to be lower than 108 cfu
injected eggs (P=0.08).
The hatch results of the current study support previous work involving in ovo
technology showing that in most chicken eggs hatchability was not altered (Goyal
and Patnayak, 2004; Johnston et al., 1997; Ricks et al., 1999). Other studies
examining in ovo nutrition found the that injection of nutrients into the egg will not
alter hatchability (Bailey and Line, 2001; Tako et al., 2004; Uni et al., 2005). The
confirmation that probiotic injections does not alter hatchability adds a potential
reason to use in ovo technology.

39

The injection site may be more important than the substance injected. Most
in ovo research has been done with injection of bacteria into the air cell through the
top of the egg, rather than into the amnion. Injection with a 19 mm needle compared
to a 13 mm needle reduced hatchability of broiler breeder eggs (de Oliveira et al.,
2013; 2009; Ohta and Kidd, 2001). Contrary to the current study, competitive
exclusion cultures injected into the air cell reduced hatchability and injection into
the embryo resulted in nearly a complete loss of hatch (Meijerhof and Hulet, 1997).
However, E. coli contaminated eggs given a Lactobacillus reuteri injection had
increased hatchability (60%) over controls (46%) (Edens et al., 1997). A single
108cfu dose of Lactobacillus into the amniotic fluid did not alter hatchability (89%;
(Edens et al., 1997). The current study’s hatchability numbers were lower than
expected, but much higher than the studies where cultures were injected into the
small end of the egg, 76% vs. 50%, respectively. Hatch result differences across the
studies where bacteria were injected into the amniotic fluid may be due individual
strains of bacteria or combinations used in each study.
Table 3 lists the bacterial counts from the cecal contents. Overall, bacterial
counts across all treatments were highest in the Bifidobacteria, though individual
treatments, such as 108, may have had higher counts of other bacteria.
Bifidobacteria had higher counts than both the Enterococcus and Lactobacillus
(P<0.05). The general gram-negative bacteria had an intermediate count between
Bifidobacteria and Enterococcus and were not different from any of the other
bacterial types. Studies have found that Bifidobacteria is present but not the most
predominate species in the intestine shortly after hatch (Lu et al., 2003; Scupham,
40

2007). The Lactobacillus and Pediococcus spp. are higher than Bifidobacteria in the
intestine of normal production birds and were expected to be higher in this study
(Scupham, 2007). The higher Bifidobacteria numbers in this study indicate that
normal succession of bacteria in the poult intestine is altered by probiotic injection.
The change in bacterial makeup of the intestinal population and the probiotic
bacteria presence in the intestine could be indicative of competitive exclusion
properties that would benefit the host during an early challenge setting. Further
studies are needed to determine if these properties are useful to the poultry
industry.
There were treatment differences in the bacteria counts. Bacterial counts
were lowest in the negative control treatment compared to all other treatments
with an average count of 6.87 log10cfu/ml (P<0.001). All other treatments had an
approximate value of 9 log10cfu/ml. The 104 and 106 injected birds had the highest
Bifidobacteria counts (P<0.05). This was true for all injected bacteria expect
Lactobacillus where 106 and 108 had the highest numerical counts. There may have
been environmental sources of bacteria, besides the injected bacteria and these
bacteria could account for the level of bacteria seen in the control birds. In chickens,
bacteria can be as high as 106 to 108 cfu/g of digesta after 24h (Apajalahti et al.,
2004). The current results show that levels of 106 cfu may be present at hatch or
obtained shortly thereafter as all poults had at least 6 log10cfu/ml bacteria on each
of the media. The environment is known to influence bacteria population and
colonization in poultry (Apajalahti et al., 2004). One source of environmental
bacteria may be the hatcher. The negative control treatment birds, hatched in a
41

separate hatcher, had bacterial growth though less than the sham saline or probiotic
injected birds (P<0.001). Sham control treatment birds were in the same hatcher as
the probiotic and saline injected birds. The difference in the bacterial levels of birds
from these differing types of controls indicates that there was an environmental
source of bacteria. Hatchery debris and dander floats throughout the hatcher while
the birds wait to be transferred. These particles are inhaled or ingested and might
be the source for bacteria. The amount of birds or debris needed to increase bacteria
in non-injected birds should be determined with future studies. Producers may
want to investigate ways to reduce bacterial loads in the hatcher. Fumigation or
other aerosol antibiotic treatments may help control bacteria in the environment.
Air filtration may be able to remove hatchery debris as birds hatch, prevent access
to the birds.
This study demonstrated that probiotics could be injected into the egg
without reducing hatchability while increasing bacterial presence in the intestine.
These bacteria have shown to improve the health and intestinal function post-hatch
(Ghareeb et al., 2012; Giannenas et al., 2014; Mountzouris et al., 2007; 2010). If an
increased amount of bacteria from the current study will benefit performance or
health of the host is unclear. Future studies are warranted to monitor the in ovo
injection’s effect on performance. This study’s results did not support current
convention that all bacteria in the egg are harmful to the embryo. Also, this study
further provides a basis for future work to fully elucidate the effect of beneficial
bacteria have after hatch.

42

APPENDIX

43

Table 1.1: Hatchability of turkey poults administered in ovo probiotics
SE
Pip (%)3
SE
Unhatch (%)4
SE
Treatment1 Hatch (%)2
Positive
3.20
4.50ab
1.50
14.97
3.16
79.79ab
Control
Negative
3.20
2.33ab
1.50
15.13
3.16
82.54ab
Control
Sham
3.20
0.79b
1.50
12.15
3.16
87.06a
Control
104cfu/ml
85.61ab
3.20
5.29a
1.50
9.10
3.16
6
ab
a
83.51
3.20
5.10
1.50
11.40
3.16
10 cfu/ml
8
b
a
10 cfu/ml
76.80
3.20
6.20
1.50
17.00
3.16
a,b Means within column for each treatment with no common superscript differ
significantly (P<0.05)
1 Mean of 9 replicate groups of 15 eggs. The treatments were: (1) Positive control
with non-injected eggs in a separate hatcher; (2) Negative Control with non-injected
eggs in the same hatcher as the injected treatments; (3) Sham Control with sterile
saline injected eggs placed in the same hatcher as the injected treatments; (4) eggs
injected with 104cfu/ml of probiotic solution (5) eggs injected with 106cfu/ml of
probiotic solution or (6) eggs injected with 108cfu/ml of probiotic solution
2 Percentage of poults that had hatched (completely cleared the shell) at time of
count.
3 Percentage of poults that had started to hatch (cracked the shell) at time of count.
4 Percentage of poults that had not started to hatch (shell completely intact) at time
of count.

44

Table 1.2: The hatch weight of turkey poults administered in ovo probiotic
Treatments1

BW (g)

SE

Yolk Wt
(g)

SE

Yolk
Free BW
(g)

SE

Yolk
(%)

SE

Positive
Control
Negative
Control
Sham
Control

62.59

1.11

7.03

0.48

55.56

0.98

11.24

0.63

64.54

1.11

8.52

0.48

56.02

0.98

13.20

0.63

64.50

1.11

7.75

0.48

56.75

0.98

12.00

0.63

104 cfu/ml

63.67

1.11

6.89

0.48

56.78

0.98

10.86

0.63

106 cfu/ml

63.53

1.11

7.55

0.48

55.98

0.98

11.87

0.63

108 cfu/ml

64.34

1.11

8.09

0.48

56.25

0.98

12.43

0.63

1 Mean

of 9 replicate groups of 15 eggs. The treatments were: (1) Positive control with
non-injected eggs in a separate hatcher; (2) Negative Control with non-injected eggs in
the same hatcher as the injected treatments; (3) Sham Control with sterile saline injected
eggs placed in the same hatcher as the injected treatments; (4) eggs injected with 104
cfu/ml of probiotic solution (5) eggs injected with 106 cfu/ml of probiotic solution or (6)
eggs injected with 108 cfu/ml of probiotic solution

45

Table 1.3: Total cecal bacterial populations (log10cfu/ml) as determined by
cultivable plate count on four different medias from poults administered various
levels of in ovo probiotics
Treatment1
Positive
Control
Negative
Control
Sham
Control

BIF2

SE

ENT3

SE

GN4

SE

LAC5

SE

6.87b

0.23

5.52b

0.15

8.09b

0.20

5.36b

0.15

8.97a

0.16

8.45a

0.15

8.64a

0.16

8.58a

0.15

8.85a

0.26

8.71a

0.16

8.83a

0.18

8.73a

0.15

104 cfu/ml

8.92a

0.17

8.75a

0.15

8.79a

0.17

8.69a

0.15

106 cfu/ml

9.04a

0.16

8.85a

0.16

8.84a

0.17

8.88a

0.15

108 cfu/ml

8.65a

0.23

8.61a

0.15

8.52a

0.23

8.68a

0.15

a,b Means

within column for each treatment with no common superscript differ
significantly (P<0.05)
1 Mean of 9 replicate groups of 15 eggs The treatments were: (1) Positive control
with non-injected eggs in a separate hatcher; (2) Negative Control with non-injected
eggs in the same hatcher as the injected treatments; (3) Sham Control with sterile
saline injected eggs placed in the same hatcher as the injected treatments; (4) eggs
injected with 104 cfu/ml of probiotic solution (5) eggs injected with 106 cfu/ml of
probiotic solution or (6) eggs injected with 108 cfu/ml of probiotic solution
2BIF- Bifidobacteria
3ENT-Enterococcosel
4GN- Gram-negative bacteria
5LAC-Lactobacillus

46

REFERENCES

47

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50

CHAPTER 2: TURKEY POULT HATCHABILITY AND LIVABILITY: THE EFFECT
OF IN OVO PROBIOTICS

ABSTRACT
The injection of probiotics into the egg could provide advantages to the poult
post hatch. The primary objectives of this study were to determine the effect of in
ovo probiotics on poult hatchability and investigate the ability of a probiotic
injection to mitigate the impact of delayed feed access on poult performance. Nine
hundred ninety fertile commercial turkey eggs were weighed, assigned to one of
three treatments, and incubated under standard conditions for 24 days. The first
treatment was placed in the incubator on d0 and treatments 2 and 3 were placed 24
and 48h after treatment 1. At E24 for each treatment, eggs were removed from the
incubator and assigned to one of three injection treatments: (1) eggs not injected
(Negative Control), (2) eggs injected with 1 ml of saline (Control), (3) eggs injected
with 106 cfu/ml probiotic solution (Probiotic). At E28, for each incubation
treatment, hatch counts were recorded within a specific hatch window (12h). On d0,
two birds per treatment were euthanized and intestinal contents and tissue
collected. All remaining poults in all treatments were housed in one of 40 pens in a
brooder and placed on a starter diet for 1wk. Intestinal samples and excreta
contents were collected from each replicate pen on d3 and d7. The hatchability and
yolk-free hatch weights were not affected by probiotic injection compared to
negative control. Poult BW was higher in the control poults at d0 maintaining the
difference through d7 (P<0.05). Feed conversion for the probiotic poults was
51

improved through d3 of life over negative control birds (P<0.05), but this effect was
lost by d7. While FCR was improved, apparent Ca and P retention was not altered at
d3 and d7 by probiotic injection and there was no main effect of probiotic injection
on intestinal morphology. In conclusion, in ovo probiotics can be safely injected into
turkey eggs and improve feed efficiency through the first day three of life.
INTRODUCTION
The United States has approximately 30 million turkey eggs in incubators
each month (NASS, 2014). Even with the large demand for poults, the number of
commercial hatcheries within the United States is decreasing. Therefore,
commercial hatcheries must ship poults farther distances, increasing the time
before poults are provided feed and water post-hatch. The main issue is that birds
often have no access to feed or water up to 72h after hatch when considering the
hatch window, bird handling, and transport time (Belo et al., 1976; Moran, 1989;
1990; Romanini et al., 2013). Fasting at any age for a period of 48h reduces the BW
of birds, crypt proliferation and villus area in the duodenum (Geyra et al., 2001a).
Fasting during the first 48h post-hatch has been shown to slow intestinal growth,
reduce the number of total cells and percentage of proliferating cells in the crypt
while increasing numbers of apoptotic enterocytes up to d5 post-feeding (Geyra et
al., 2001a; Gaffga et al., 2012). Pchavov and Noy (1993) reported post-hatch poults
experiencing a 48h fast had greater than a 10% reduction of BW. Also, poults with a
similar delay in access to feed had fewer bacteria in the digesta (Geyra et al., 2001b;
Potturi et al., 2005), intestine weight was reduced by 30%, and villus surface area
decreased by over 25% after a 72h fast (Smirnov et al., 2004).
52

Body weight reduction and the regression of the intestine occur
simultaneously with other challenges to homeostasis. Soon after the poult arrives on
the farm, the poult must transition to a carbohydrate dense diet from the yolk that
provided a lipid dense nutrient source. In preparation for the dietary switch the
intestine begins to mature. The absorptive surface area increases during this
maturation as the villi start to elongate at the end of incubation, and continue to
grow after hatch (Uni et al., 2003). Enterocytes in the villi, begin to produce nutrient
transporters and digestive enzymes, which facilitates increased nutrient absorption
in the intestine (Uni et al., 2003). There are several detectable nutrient transporters
seen throughout intestinal development, but the class that is most essential to
glucose is the GLUT or solute carrier family. Glucose transport is done through
several mechanisms but is mostly facilitated by glucose transporter (GLUT) and
sodium glucose linked transport (SGLT) transporters. There are several GLUT
isoforms (GLUT1, 2, 3, and 8) in poultry (Kono et al., 2005). The expression of
GLUT2 in broiler intestines increased linearly from E20 to d14 (Gilbert et al., 2007).
Nutrient transporters help facilitate the absorption of peptides as well as glucose.
Age is important in the expression of the peptide transporter, PepT1, reported to be
seen as early as E23 in turkeys with an approximate threefold increase in
expression observed five days prior to hatch (Van et al., 2005). Based on PepT1
expression patterns in turkeys, small peptides can be transported across the lumen
at hatch (de Oliveira et al., 2009).
Commensal bacteria can assist in the development of the intestine. Bacteria
in the digestive tract of poultry often adhere to the epithelium helping to prevent
53

them from being removed from the intestine. In chickens, the mucosal secretions
are a nutrient source for commensal bacteria (Smirnov et al., 2005). If bacteria
persist for an adequate period of time, they can develop a biofilm on the intestine,
thus helping maintain immune homeostasis (Maol and Servin, 2006). Dietary
supplementation with commensal or probiotic bacteria benefits the host as well.
When both Lactobacillus spp. and Bifidobacteria spp. were supplemented in the
feed, an increase in intestinal villi length and villus height:crypt depth ratio was
observed (Altekruse et al., 2006; Chichlowski et al., 2007; Rahimi et al., 2009). Most
studies have examined these benefits post hatch. Probiotic bacteria administered
prior to hatch may benefit the bird in the same way. Therefore, the objectives of this
study were to determine the effect of in ovo probiotics on poult hatchability and to
investigate the ability of a probiotic injection to mitigate the impact of delayed feed
access on poult performance.
MATERIALS AND METHODS
This research was approved by the Animal Care and Use Committee at Michigan
State University. The approval number was AUF #05/13-103-00.
Probiotic preparation
The microencapsulated probiotic contained two strains of Lactobacillus
reuteri, a single strain of Enterococcus faecium, Bifidobacteria animalis and
Pediococcus acidilacti (Biomin, Herzogenburg, Austria). To determine the
concentration of organisms, the probiotic mixture was suspended in sterile PBS and
plated. The four medias used were: Luria-Bertani agar (Acumedia, Lansing, MI) for
general gram-negative bacteria, Bifidiobacteria agar (HiMedia Laboratories Ltd.
54

Mumbai, India) for Bifidobacteria spp, de Man, Rogosa and Sharpe agar (HiMedia
Laboratories Ltd. Mumbai, India) for Lactobacillus spp. plus Pediococcus spp. and
Enterococcsel agar (a modified esculin bile agar; Becton Dickinson and Company,
Franklin Lakes, NJ) for Enterococcus spp. To create solutions of 106cfu/ml, the
probiotic mixture was weighed (0.423g) and suspended in PBS to create 108cfu/ml
solutions. The solutions were then diluted to 106cfu/ml in sterile PBS (45ml) on the
day of the sample injection (E25). Solutions were stored on ice (~4h) and vortexed
for 15s immediately prior to egg injection.
Birds and Husbandry
Three groups of 330 turkey eggs (990 eggs total) were individually weighed,
set in an incubator (Petersime Model 5; Petersime Incubator Co., Gettysburg, PA),
and incubated for 24d. The placement of each group of eggs in the incubator was
offset by 24h, so that the last group of 330 eggs went in the incubator 48h after the
first. All groups were obtained from the same breeder farm and placed in an egg
cooler (15°C) for storage prior to incubation; no group was stored longer than 4d.
The first group placed in the incubator was labeled 48h, the second group 24h and
the last group 0h. Incubator conditions (37.5°C dry bulb and 29.2°C wet bulb) were
monitored throughout the trial using General Tools H10 data loggers (General Tools,
New York, NY). All eggs were candled on E24, and infertile eggs were removed from
the study. The remaining eggs had the lowest point of the air cell marked. On the day
of injection (E25 for each group: 25d, 26d, 27d of the trial) any externally pipped
eggs were removed from the study. The remaining eggs (n ~ 300/group) were
returned to the incubator and randomly assigned, using the experimental animal
55

allotment program, to three treatments based on initial egg weight (EAAP, 2009).
The treatments were: Negative Control (NC; non-injected), Control (CON; 1 mL
injection of sterile saline), Probiotic (PRO; 1 mL of probiotic solution at 106 cfu/mL).
There were five replicates per treatment and 19 eggs per replicate. After
randomization, eggs were removed from the incubator one replicate at a time. The
large end of the egg that was to be injected was wiped with an alcohol disinfectant
wipe. Negative control treatment eggs were left out of the hatcher for the same
period of time as eggs in the other two treatments. A Dremel stylus rotary tool
(Model # 1100-01: Dremel, Racine, WI), was used to drill a hole into the lowest
point of the egg’s air cell. An Allflex MR2 repeater syringe (Allflex USA INC)
equipped with a 22 ga 1” needle was used to inject each egg with 1 ml of probiotic
solution (106 cfu/ml). Needles were changed and the repeater syringe was rinsed
with sterile saline between each replicate. Following injection, the drilled holes
were sealed with paraffin wax. The PRO and NC eggs were placed in pedigree
baskets and randomly placed in one of two Surepip hatchers (Agro Environmental
Systems Inc., Dallas, Georgia, USA). The CON eggs were placed in a hatcher portion
of the Petersime incubator. Hatcher conditions were monitored with data loggers to
ensure consistent conditions. Replicates were randomized within the respective
hatchers.
Beginning on d26 of the trial, hatch counts were recorded every 12h. The
following definitions were used: a hatched poult had completely cleared the shell; a
pipped poult had any portion of the shell cracked but failed to clear the shell; an
unhatched poult had a completely intact shell. Hatched birds were divided in
56

separate portions of the pedigree baskets to prevent crossover of the hatch window.
Only the birds from the hatch window with the most birds (E27 to E27.5) from all
treatments were selected for the trial, all other hatch birds were removed from the
study. Therefore, 60 birds per injection treatment/placement combination and 12
birds per individual replicate were used during the trial. At E28 for each placement
group (d28, d29, d30 of the trial), final hatch counts were recorded. The birds from
the three placement groups were not the same age and hatched on different days.
Due to offset placement of the eggs, birds from the 48h and 24h were left in the
hatcher for 48h and 24h without food or water to mimic hatch window and shipping
conditions. Once placed in brooder battery cages, all birds were given ad libitum
access to feed and water for seven days. Poults were provided a standard industry
starter diet that met or exceeded NRC nutrient requirements (NRC, 1994).
Sample Collection
Hatch day for the 0h eggs, (at d0, d3 and d7 on feed), four poults per replicate
were euthanized by cervical dislocation for intestinal sample collection. For cecal
content and yolk sac collection, the euthanized bird’s whole body was dipped in an
antiseptic solution one bird at a time. The bird was removed from the antiseptic
solution and placed on a clean disinfected cutting board. The abdominal cavity was
opened with sterile scissors, dipped in alcohol and flame dried between each poult.
Gloves were changed after the abdominal cavity was opened to prevent
contamination from the poult exterior. Using two sets of forceps, the yolk sac and
both cecal horns were extracted from the bird. The yolk sac was placed in a weigh
boat and yolk-free BW was calculated based on difference between total BW and
57

yolk weight. Cecal contents, from two of the four poults, were pooled by gently
squeezing the contents into a 1.5 mL sterile microcentrifuge tube (Denville
Scientific) using the forceps to prevent glove contamination. Sterile forceps and
gloves were used for each bird to prevent cross contamination. Samples were
immediately chilled on ice until 25% of the poults were collected. Samples were
taken to the laboratory to prevent prolonged exposure to the ice and ensure
viability of samples for the dilution series.
Intestinal sections approximately 2.5 cm in length were collected from the
ascending portion of the duodenal loop and the midway point between Meckel’s
diverticulum and the ileo-cecal junction of the ileum. Intestinal contents were
washed with chilled sterile PBS then placed in 10% neutral buffered formalin and
stored until processed. Samples (30 mg) were collected from each intestinal section
(duodenum and ileum) proximal to the previous sampling point and placed in
RNAlater (Qiagen, Valencia CA) filled microcentrifuge tubes. The microcentrifuge
tubes were placed on ice for 4h and afterwards transferred to a -80°C freezer until
processed. Two sets of whole pen excreta samples were collected for a 24h period
starting on d2 and d6 and ending on d3 and d7, respectively. Excreta contents were
immediately placed on ice, and then transported to the laboratory for storage at 0°C.
Cecal content plating
Each cecal sample was a pool from two birds. For the six serial dilution
series, 30 µl of each cecal sample was pipetted into 270 µl of sterile PBS and the
mixture vortexed for 15s. 30 µl of the resulting mixture was pipetted into 270 µl of
sterile PBS and the mixture vortexed for 15s. The pipetting procedure repeated until
58

the dilution series was complete. After the dilution series was made 50 µl of each
serial dilution and the pure sample were pipetted onto the center of each of four
different media plates and spread using a L-shaped glass spreader rod. Rods were
dipped in alcohol and passed through the flame between each use. The four medias
used were: Luria-Bertani agar (Acumedia, Lansing MI) for general gram-negative
bacteria, Bifidiobacteria agar (HiMedia Laboratories Ltd. Mumbai, India) for
Bifidobacteria spp, de Man, Rogosa and Sharpe agar (HiMedia Laboratories Ltd.
Mumbai, India) for Lactobacillus spp. plus Pediococcus spp. and Enterococcsel agar
(a modified esculin bile agar; Becton Dickinson and Company, Franklin Lakes, NJ)
for Enterococcus spp. All medias were made according to manufacturer instructions
and stored at 15°C prior to being used. Plates were incubated for 24h at 37°C in an
incubator (Precision Industries model# 30M,Chicago, IL), removed, and colony
counts were made. Only counts from a single plate from the dilution series with
approximately 25 to 300 total colonies was selected for counting. The plate was
placed on a lightbox and colonies were marked and a lab cell counter was used to
record counts.
Nutrient Transporters
Intestinal samples from each poult were thawed on ice, lysed, and
homogenized using the Qiashredder homogenization kit (Qiagen, Valencia, CA). The
RNA was isolated using the Qiagen RNeasy kit (Qiagen, Valencia, CA) following
manufacturer’s instructions. The RNA was eluted in 50 μl of nuclease-free water,
and a quality check representative sample (12%) of all the RNA was loaded onto
nanochips and run on an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto,
59

Calif) to determine purity. Samples had a RIN number above 8. The cDNA was
synthesized from 500 ng of RNA using the high-capacity cDNA reverse transcriptase
kit (Applied Biosystems, Foster City, CA). Real-time PCR reactions were performed
with SYBR Green Master mix and an Applied Biosystems 7300 system at 50°C for
2min, 95°C for 10min, 40 cycles of 95°C for 15s, and 60°C for 1min. The RNA test
samples were run with each primer set (Table 1) to test for genomic DNA
contaminations. Samples were run in triplicate and a Ct value of seven or more
cycles fewer than the cDNA sample were accepted. Relative gene expression was
calculated using the 2−ΔΔCt method with β-actin and TATA binding protein as an
endogenous control (Livak and Schmittgen, 2001). The average ΔCt of the control
samples was used to calculate ΔΔCt values. ΔΔCT was calculated as follows:
ΔΔCT=(CT(target,control)−CT(ref,control)−(CT(target,treatment or
placement)−CT(ref, treatment or placement)). Samples were only compared to
samples from birds of the same age.
Morphology samples
Each fixed intestinal tissue was cut into 10 mm sections and placed into a
tissue cassette. The tissues were processed by dehydration through a series of
graded alcohols, cleared with xylene, and embedded in paraffin. A paraffin section
from each bird (5 μm thickness) was mounted onto slides. Slides were stained with
hematoxylin and eosin (HE) stain. Measurements of villi height (VH: villus tip to
crypt opening) and crypt depth (CD: crypt opening to the base of the crypt) were
made using Nikon NIS Elements software. The intestinal section from each bird was
measured, with three villi and three crypt measurements taken on each of the
60

sections. The ratio of VH to CD (VCR) was calculated from these measurements. The
pen average of the group of four birds was used for statistical analysis.
Excreta samples
On d2 and d6, feed and excreta pans from each pen were removed, cleaned,
lined with paper, and replaced. Twenty-four hours later, excreta was collected, and
placed in plastic bags. The total collection during this time period was used for
determination of apparent nutrient and energy retention. All samples were freezedried and ground using the Cyclotec Sample Mill 1093 (FOSS North America, Eden
Prairie, MN) with a 1 mm screen to achieve a uniform grind. Approximately 0.4 g of
dried ground sample was weighed into a digestion vessel to which 10 mL of 70%
nitric acid (Omni Trace, EMD Chemicals, Inc., Gibbstown, NJ) was added. The sample
and acid solution was allowed to sit overnight (~16h) at room temperature to
predigest the sample. The following day, vessels were placed in the microwave
digester, with the digestion program: 1200 watts with a 30min ramp to 160°C under
pressure of 190 PSI, hold samples for 10min, and cool down for 5min. After
additional cooling in a fume hood for 10min, vessels were vented, and 2 ml of 30%
hydrogen peroxide (Sigma-Aldrich, St. Louis, MO) was added. The sample digest was
then transferred to a 25 ml volumetric flask and allowed to cool completely; double
deionized (ddi) H2O was added to bring the volume to 25 ml. Samples were
transferred to 50 ml polypropylene tubes and stored at room temperature until Ca
and P analysis was performed. All glassware used in the digestion process was acid
washed using 30% nitric acid and rinsed with ddi H2O to remove any residual
minerals.
61

Calcium assay
Calcium concentration was determined by atomic absorption spectroscopy.
The microwave digested samples were diluted 1:100 in a 1% LaCl3 (lanthanum
chloride, Sigma-Aldrich, St. Louis, MO) solution. The LaCl3 acts as an ionization
suppressant to eliminate the phosphate interference, which occurs in an airacetylene flame. Excreta and feed samples were analyzed for Ca using an AA7000
Series atomic absorption spectrophotometer (Shimadzu Scientific Instruments, Inc.,
Columbia, MD). These samples were analyzed against a five point, matrix-matched
standard curve (Ca standard source: VWR International, West Chester, PA) ranging
in concentration from 1 to 5 μg/ml Ca. Peach leaves, the organic standard, (NIST,
Gaithersburg, MD) were simultaneously analyzed to maintain instrument accuracy.
Apparent calcium retention was reported as mg of Ca in excreta per mg of Ca in feed.
Phosphorus assay
Spectrophotometery was used to determine P in the microwave digested
samples. Phosphate ions react with molybdate complexes, which in the presence of
a reducing agent (Elon), are converted to molybdenum-blue to be measured in a
spectrophotometer (Kaplan and Pesce, 1989). The MS solution contained 2.5 g of
molybdate (MS), 7 ml of sulfuric acid, and ddi H2O to bring the final volume to 500
ml. The Elon solution contained 1.5 g of sodium bisulfate, 0.5 g of Elon, and ddi H2O
to make the final volume 50 ml (Gomori, 1942). To analyze the microwave digested
sample, two mililiters of sample were diluted 1:10 with ddi H2O. Standards were
made by diluting 15 mg/dL stock solution of P (Phosphate (P) Standard, 1000 ppm,
LabChem, Inc., Pittsburgh, PA) with ddi H2O to make 5, 4, 3, 2, 1.5, 1, and 0 mg/dL
62

(Gomori, 1942). Into a 96-well microplate, 50µl of each standard and sample were
pipetted, in duplicate. Two hundred and fifty microliters of MS solution and 25 µL of
Elon were pipetted into each well. The plate was incubated for 45 min at room
temperature on a plate shaker (Brinkmann TiterMix 100) at 600 rpm and read at
700 nm on the plate reader (SpectraMax 384, Molecular Devices). The SpectraMax
determined the P concentration of all samples. The measurement was then adjusted
by DM and amount of sample used in the microwave digestion to yield the final
concentration. Apparent phosphorus retention was reported as mg of P in excreta
per mg of P in feed.
Energy
Ground feed and excreta samples were formed into 1.0 g pellets using a Parr
pellet press modified with a hydraulic jack, and the amount of energy in each sample
was determined via bomb calorimetry following manufacturer instructions (Parr
Instrument Co). The sample pellets were placed into a capsule that was placed in the
loop of the bomb head. A Parr 45C10 Fuse Wire (34 B&S gage, nickel-chromium
resistance wire) approximately 10 cm in length was attached to the two fuses of the
bomb head and bent to touch the sample pellet. The bomb head was secured into
the bomb cylinder. The bomb was filled with oxygen to bring the pressure to 32 atm.
The bomb was placed into a calorimeter bucket filled with 2000 ml of diH2O and
placed into the calorimeter. The two ignition wires were pushed into the terminal
sockets on the bomb head. The water temperature of the calorimeter was allowed to
reach equilibrium and the temperature recorded. An electrical surge was sent down
the terminals into the bomb igniting the oxygen and combusting the sample. A
63

second temperature reading was taken approximately six min later, after a
stabilized temperature had been reached. The remaining portion of the fuze wire
was measured and a calorie correction determined from the length. A benzoic acid
pellet was used to confirm the standardized values of each bomb. Therefore, the
energy was calculated based on the formula below:
Heat of Combustion ሺcal/gሻ
ൌ

ሺFt െ Itሻ ൈ energy standard of bomb െ ሺcalories of wire burnedሻ
sample dry wt. ሺgሻ

Ft= Final temperature
It= Initial temperature
Standard= benzoic acid

Dry Matter
Empty crucibles were placed in a constant temperature oven (Yamato DNF
600) at 105°C at least 2h prior to hot weighing. Each crucible was hot weighed and
0.5g of sample was added and weighed, in duplicate, and placed into the crucible.
The dry matter content of the feed and excreta was measured by drying the samples
at 105°C for 24h.
Statistical Analysis
All parameters were analyzed using the PROC MIXED analysis of SAS (v 9.3)
with the LSmeans procedure PROC MIXED in SAS PC Windows Version 9.2 software,
(SAS Institute Inc., Cary, NC). The model (‫ ݕ‬ൌ ߤ ൅ ߬௜ ൅ ߚ௝ ൅ ߬ߚ௜௝ ൅ ߳௜௝௞ ሻ included the
main effects of treatment (τ) and delayed access to feed (β) as well as the interaction

64

between treatment and delay (τβ). Interactions are presented as a comparison of
single treatments across various placements. Comparisons across time were not
used in the model. Pen or pedigree basket was used as the experimental unit for all
measurements, except nutrient transporters which were analyzed on a per bird
basis. Differences between means were tested using the Pdiff option of the LSmeans
statement with significance accepted at P<0.05.

RESULTS
There were no differences in hatchability except in the 48h delayed
treatment, where the control treatment had a higher hatchability than negative
control and probiotic birds (Table2; P <0.05). There were no differences in
hatchability between negative control or probiotic poults at any time point. As
expected, the percent of unhatched and pipped eggs mirrored the hatchability, with
the control treatment having a lower percent of unhatched eggs than probiotic and
negative control treatments at 48h. Though there was no difference in the
percentage of hatched or unhatched, the 0h control injected birds had a higher pip
count than the negative control and probiotic injected birds (P<0.05). There was
also no difference in mortality (data not shown).
The counts for all bacteria were one to two logs higher in the probiotic
treated eggs compared to the other treatments (Table 3; P<0.001). The 48h-delayed
poults had higher Bifidobacteria and Lactobacillus counts than the 0h birds (P<0.05).
The 48h-Control birds had higher Bifidobacteria and Lactobacillus counts than the
0h- and 24h-Control birds. The 48h-Probiotic treatment combination had higher
65

number of Enterococcus colonies than the 0h- and 24h- times with probiotic
injections.
The yolk weight of the 48h-negative control poults were not significantly
lower than the 24h-negative control poults, whereas the 48h-control and probiotic
birds were compared to their respective injections (data not shown). The 48hcontrol and 48-probiotic birds had a larger increase in BW from d0 to d3 than the
48h-negative control birds (Figure 1; P<0.05). There was a main effect of delay on
BW as the highest BW was measured in the 0h delayed treatment on d0 (Table 4;
P<0.05). The BW was influenced by yolk disappearance, as the yolk was reduced by
approximately 3g/d across the delayed placement treatments. The control injected
birds had a higher BW through d7 posthatch (P<0.05). There was no difference in
BW between negative control and probiotic injected birds throughout the trial. The
percent yolk and the overall residual yolk or yolk free BW was not (P>0.05)
different amongst any of the injection treatments. The yolk disappearance was
higher in probiotic (6.8g) and control injected birds (7.2g) over the 48h in the
hatcher compared to negative control (4.8g).
Feed conversion from d0 to 3 was highest in birds from the non-injected
negative control eggs compared to birds from eggs given a probiotic injection
(Figure 2; P<0.05), but there were no difference from d3 to7. The overall FCR (d0 to
7) was improved in the 24h- and 48h-negative control poults compared to the 0hnegative control poults. This was the same in the control, but there were no
differences amongst the various placement groups for the probiotic poults. The 24h

66

and 48h delayed placement poults had better FCR than the non-delayed birds (Table
5; P<0.001) at d3 and d7.
There was no interaction between injection and placement on apparent Ca
and P retention in young poults (Figure 3; P=0.81), and there were no main effects
of injection (P=0.77). The delayed placement on feed by 48h increased P
digestibility over 0h poults at 3d of age (P=0.03), but was lost by d7. The results
were not compared overtime but the numerical mean average retention for both
minerals were higher in the d7 birds. There were no differences in energy at either
d3 or d7 (Table 6).
There were differences in duodenal and ileal morphology (Table 7, 8). There
were no differences in villus height, crypt depth or ratio due to in ovo injection in the
duodenum or ileum at d0 and d7. However, birds placed on feed immediately after
hatch had shorter villi and as a result lower villus height crypt depth ratio (VCR) in
the duodenum and ileum than those offered feed at 24h or 48h (P<0.02). The
increased VCR, but not height, was maintained in the duodenum of the 48h and 24h
birds on d7 (P=0.006). These differences were not seen on d7 in the ileum. The ileal
VCR was higher in the 48h-probiotic birds compared to the 0h- or 24h-probiotic
birds on d0 and 0h-probiotic birds on d7 (Figure 4; P<0.05). The 0h-negative control
VCR was the lowest at d7 in the duodenum (Figure 5; P<0.05). The 24h-negative
control was highest whereas the 24h-probiotic and 24h-control groups were
intermediary to the delay treatments treated with the same injection.
Nutrient transporter levels were highly variable through the study. In the
duodenum, birds in the 24h placement treatment that were not injected (negative
67

control) had a 16-fold increase in PepT1 on d3 compared to 0h-negative control
birds (Table 9; P<0.001). This increase was not seen at d0 or d7. GLUT2 was
increased in both the 0h-control and 0h-probiotic birds over 0h-negative controls in
the d0 birds(P<0.001). There were no differences in duodenal GLUT2 expression on
d3 or d7. Both the 0h-control birds had increased ieal PepT1 expression compared
to 0h-negative control birds regardless of placement at d0 (Table 10; P <0.05). The
24h-probiotic birds had higher ileal PepT1 expression than the 24h-negative control
birds. The 0h-probiotic birds had a higher ileal GLUT2 expression than the other
treatments on d0 (P<0.05), but there were no difference on 3 or d7.

DISCUSSION
There was a varied response to the probiotic injections in the parameters
measured. The injection improved performance of the host in some parameters, but
did not affect overall performance of the poult. Though the overall hatchability in
the current study was lower than expected, the hatchability was still within average
for commercial turkey eggs (Schaal and Cherian, 2007). The opening and closing of
the hatcher could have contributed to these results, as incubating eggs are sensitive
to temperature and humidity fluctuations, which could have contributed to a
reduction in overall hatchability. Treatment means were lower than expected but
the lack of difference among treatments supports previous research (Chapter 1)
demonstrating that probiotics can be safely injected into the egg.
The increased bacterial load in the intestine of the probiotic treated poults
indicates that the birds were able to ingest the bacteria and the bacteria remained
68

viable until DOH. These results were similar to the first study (Chapter 1), where
each of the four bacteria types had a 1 to 2 log increase. The performance
parameters provide some insight to the overall effect the increased bacteria had on
the body. Poults in the control treatment had heavier BWs throughout the trial, yet
the reason for this is unknown. One possible cause that can be ruled out is egg
weight. Egg weight has been reported to be related to hatch weight and
performance (Moran, 1990). Eggs were initially weighed, and egg weights were
evenly distributed across all treatments and replicates, so that egg weight was
uniform (within 0.5 g) across treatments. The impact of saline on BW is still unclear.
With no difference in yolk or percent yolk, and yolk absorption does not appear to
be a contributing factor to the differences in BW observed here. The reduction in
BW among the delayed placement poults irrespective of injection are in agreement
with previous studies (Belo et al., 1976; Moran, 1989; 1990) and validates the 24h
difference in age and time off feed. In Chapter 1, there were no differences in yolk
weight or yolk free BW. Bacteria were injected into the amnion so that the bacteria
could not enter the yolk, thus bacteria should not be able to utilize the yolk as a
nutritive source. Turkey poults begin swallowing the amnion late in incubation just
prior to internal pipping and should swallow the bacteria (Uni and Ferket, 2004).
The injection site and swallowing by the embryo are factors that could contribute to
observations that the presence of bacteria do not alter yolk weight or yolk
metabolism.
Control poults may have had higher BW because these birds had increased
fluid in the egg. The increase in fluid would reduce moisture loss resulting in
69

increased BW. As previously mentioned, the multiple door openings may have
contributed to increased moisture loss of all the eggs. Injection of saline potentially
reduced dehydration, however, the probiotic was suspended in sterile PBS and this
did not alter d0 BW. Poults injected with 1.5 ml of saline and various levels of
protein and carbohydrates had increased BW at DOH in three different studies, yet
these BW differences were no longer apparent by d3 or d7 (Geyra et al., 2001a; Foye
et al., 2006; Gaffga et al., 2012). These studies contrast the current study where the
saline birds maintained BW differences through d7. The injection of nutrients or
probiotics could alter metabolism and thus account for these differences, whereas
saline may only alter hydration status.
Birds can spend 24 to 48h without access to feed from the time of hatch, but
usually this results in dehydrated birds upon arrival at the farm. One concern in
regards to long shipment are the decreases in both metabolism and yolk reserves.
This could lead to decreased BW and higher mortality. The BW and the yolk
reserves were lower in the 48h delay treatment. The decrease in BW should
correlate with a decreased VCR as the villus should regress in response to the fast,
but in the current study the VCR response was just the reverse. In laying hens, a fast
of 24h reduced villus length (Yamauchi et al., 1996; Geyra et al., 2001b; Potturi et al.,
2005). Contrastingly in broilers, ileal villus height was unaffected by a 24h fast
(Thompson and Applegate, 2006; Kellett et al., 2008). The 48h delayed access to
feed reduced BW, but this difference was lost by d7. The increase in VCR for 48h
birds in both the duodenum and ileum on 0d may be the reason the poults were able
to have a higher BW by d3.
70

The probiotic egg injection in this study was only able to influence
performance through the first three days of life. In the few studies that examined the
effect of a single dose of probiotics, no intestinal development post hatch was
investigated. The transit time of the bacteria through the intestine could be the
reason that bacterial injections had a short duration of effectiveness. In rats, single
doses of Lactobacillus casei, showed that bacteria have a short adhesion time to the
intestine (de Oliveira et al., 2009; Saxami et al., 2012). The bacterial population in
the intestine is influenced by diet as well as environment. Though the bacterial
counts at d3 or d7 were not examined, the diet could have shifted bacterial
populations after the injection.
Poults rely on dietary sources for Ca once the poult hatches. After hatch,
there is a tremendous need for Ca to maintain skeletal growth. The Ca and P
digestibility was expected to increase in the probiotic injected birds. The poults may
have absorbed adequate amount of Ca from the egg and coupled with adequate
dietary amounts, intake of Ca may have been at near optimal levels. The amount of
Ca in the diet may have been adequate for bird development. Little research has
been done on probiotic and Ca in optimally fed animals. Calcium absorption from
rats was not altered by probiotics when fed adequate Ca levels (Moran, 1990;
Scholz-Ahrens et al., 2007). Body stores of Ca and P were not factored into the
digestion calculations, and adequate reserves may have limited Ca absorption.
Calcium and P are influenced by one another and the ratio between the two is
important. A ratio that is too high or too low can reduce Ca absorption. Our ratio
(1.87) as prescribed by the NRC (1994) and the genetic company should be optimal
71

for the poults at that age. A shift in the ratio may influence the effect of probiotics on
P and Ca absorption.
The probiotic treated birds had greater villus height to crypt depth ratio after
a 48h delay, therefore, duration of bacterial exposure may impact probiotic efficacy.
The extra two day exposure to the bacteria may have been enough to significantly
impact the intestine. Four week old female broilers given an intragastric injection of
107 cfu of Lactobacillus spp. had increased weight d8 later and improved feed
conversion over a d29 period (Gilbert et al., 2007; Khan et al., 2007). Chickens and
ducks given a single dose of 4 x 1010 cfu of Lactobacillus spp. at d4 of age had
increased BW d56 later (Van et al., 2005; Angelakis and Raoult, 2010). The amount
of Lactobacillus spp. in the intestine was higher at d60 of age as well. The increase in
villi length would result in an increase in absorptive surface area for the intestine.
This could explain why a single dose of probiotics can positively influence the
performance of poultry.
Nutrient transporters were highly variable amongst all of the sample
replicates, but there was an increase in the GLUT2 transporter in the duodenum and
ileum of the 0h-probiotic poults over the 0h-negative control birds. An increase in
GLUT2 could have led to an increase in glucose from the diet. Increases in GLUT2
expression lead to an increase in energy absorption from the diet in weanling pigs
(Gabler et al., 2007; Schaal and Cherian, 2007). Gilbert (2007) demonstrated that
GLUT2 increases linearly with age in chickens. The probiotic poults had an
improved efficiency through d3 of life. These factors examined together indicate a
more mature and more functional intestine in the turkey poult administered
72

probiotic bacteria in ovo. These increases in development could ease the transition
to feed and reduce overall feed costs in turkey production.
The influence of probiotic bacteria on the intestine is beneficial to
performance. Probiotics improve intestinal morphology after a delayed access to
feed; and a single injection into the egg appears to improve FCR for up to three day
post injection. Future studies looking at the combined effect of probiotics in ovo and
probiotics in the feed could produce an even greater benefit. The results indicate
that probiotics can safely be injected into the egg and consequently, produce a
better performing poult after hatch. In ovo probiotics may be a beneficial
management practice to adopt since the supplementation of probiotics pre-hatch
appears to improve performance early in life.

73

APPENDIX

74

1

Table 2.1: Primers used for real-time PCR on turkey intestinal samples
Gene
Sequence
Pept1 F

5’-GTTTCTAGCTTGCGGTCGGA-3’

Pept1 R

5’-TCCAAAATCCGTGTCACCCA-3’

GLUT2 F

5’-AAAGAGAGTGTCCATCGGGC-3’

GLUT2 R

5’-CGTTGATGCCTGAGAACTGC-3’

Beta-actin F

5’-CTGGCCGTGACCTGACGGAC-3’

Beta-actin R

5’-GCCTCGGGGCACCTGAACCT-3’

Tm (°C)
60.4

Amplicon length (bp)
120

59.5
59.8

104

59.6
65.2

238

66.8

60.0
107
TATA Binding
5’-CACGTGTGTGGCCATTCTTG-3’
protein F
TATA Binding
60.0
5’-CCACGTTGAAGAGCTCTGGT-3’
protein R
1Primers designed by the NCBI Primer Blast software program (http://www.ncbi.nlm.nih.gov/tools/primer-blast/).

75

Table 2.2: Hatchability of turkey poults administered in ovo probiotics administered on d25 of incubation1
Pipped3
Hatched2
Unhatched4
Negative
Negative
Negative
Control Probiotic
Control Probiotic
Control Probiotic
Control5
Control
Control
0h6
62.1
56.8
62.1
11.6b
19.0a
12.6b
26.3
24.2
25.3
24h
70.3
70.5
64.2
8.4
5.3
10.5
21.2
24.0
25.3
b
a
b
b
a
a
a
b
48h
74.7
86.1
68.4
2.1
9.5
9.5
23.2
4.2
22.1a
SEM
4.9
4.9
4.9
2.3
2.3
2.3
4.9
4.9
4.9
a,b Means within row for each treatment with no common superscript are different (P<0.05)
1 A probiotic mixture of L. reuteri, B. animalis, E. faecium, and P. acidilacti.
2Percentage of poults that had hatched - completely cleared the shell at 27.5d of incubation.
3 Percentage of poults that had started to hatch - cracked the shell at 27.5d of incubation.
4 Percentage of poults that had not started to hatch - shell completely intact at time of count.
5 Mean of 5 replicates of 19 eggs each. Negative Control - non-injected eggs; control - sterile saline injected eggs; probiotic eggs injected with 1 ml of probiotic inoculations at 106cfu/ml. All treatment groups were in their own separate hatcher.
6 0h-poults not held in the hatcher following hatch; 24h-poults held in the hatcher for 24h prior to feed and water access; 48hpoults held in the hatcher for 48h prior to feed and water access. Turkey eggs were offset to ensure all poults were placed on
feed and water at the same time.

76

Table 2.3: Total cecal bacterial populations (log10cfu/ml) from poults administered in ovo
injections on d25 of incubation as determined by cultivable plate count on four different
medias1
Bacteria
Placement2
0h
24h
48h
P-value
Injection3
Negative Control
Control
Probiotic
P-value
a,b Means

BIF4

ENT5

LP6

GN7

8.24b±0.29

7.56±0.29

7.28b±0.3

7.96±0.27

8.40ab±0.27
8.81a±0.29
0.05

7.88±0.27
8.54±0.29
0.21

7.77ab±0.3
8.48a±0.3
0.05

8.62±0.26
8.91±0.26
0.23

7.69b±0.27
8.24b±0.29
9.52a±0.29
<0.001

7.69b±0.27
7.14b±0.29
9.15a±0.29
<0.001

7.65b±0.27
8.32b±0.26
9.52a±0.26
<0.001

6.99b±0.3
7.16b±0.3
9.39a±0.3
<0.001

and SEM within column for each item with no common superscript are different

(P<0.05)
1 A probiotic mixture of L. reuteri, B. animalis, E. faecium, and P. acidilacti.
2Five replicate groups with a two-bird pool of cecal contents. 0h-poults not held in the
hatcher following hatch; 24h-poults held in the hatcher for 24h prior to feed and water
access; 48h-poults held in the hatcher for 48h prior to feed and water access. Turkey eggs
were offset to ensure all poults were placed on feed and water at the same time.
3Negative Control - non-injected eggs; control - sterile saline injected eggs; probiotic - eggs
injected with 1 ml of probiotic inoculations at 106cfu/ml. All treatment groups were in their
own separate hatcher.
4BIF- Bifidobacteria count
5ENT-Enterococcosel count
6LP-Lactobacillus and Pediococcus count
7GN- Gram-negative bacteria count

77

Table 2.4: Body weight of turkey poults with either a delayed placement on feed and/or an in ovo probiotic injection
administered at 25d of incubation1
-------------------------------d0---------------------------

----------------------Total BW (g)--------------------------

Yolk Free BW (g)

Yolk Wt (g)

Yolk4 (%)

d05

d3

d7

0h

53.40±1.70

16.47a±0.57

20.62a±1.08

69.95a±0.43

77.01c±1.82

137.6b±6.91

24h

55.37±1.70

13.61b±0.57

16.45b±1.08

65.15b±0.43

82.38b±1.74

149.77ab±7.17

48h

56.71±1.70

10.53c±0.57

12.12c±1.08

59.49c±0.43

88.09a±1.93

160.15a±7.34

0.23

<0.001

<0.001

0.003

0.011

0.78

Negative Control

57.30±1.70

12.98±0.57

15.24±1.08

64.24b±0.43

78.74b±1.93

146.71b±7.58

Control

53.33±1.70

13.54±0.57

16.74±1.08

66.44a±0.43

87.09a±1.82

153.28a±6.91

Probiotic

54.85±1.70

14.09±0.57

17.22±1.08

63.91b±0.43

81.64b±1.74

147.54ab±6.91

0.21

0.45

0.38

<0.001

<0.001

0.05

Placement2

P-value
Treatment3

P-value
a,b Means

and SEM within column for each item with no common superscript are different (P<0.05)
probiotic mixture of L. reuteri, B. animalis, E. faecium, and P. acidilacti.
2Mean of 5 replicate groups of a two bird pool of cecal contents. 0h-poults not held in the hatcher following hatch; 24h-poults
held in the hatcher for 24h prior to feed and water access; 48h-poults held in the hatcher for 48h prior to feed and water
access. Turkey eggs were offset to ensure all poults were placed on feed and water at the same time.
3Negative - non-injected eggs; control - sterile saline injected eggs; probiotic - eggs injected with 1ml of probiotic inoculations
at 106cfu/ml. All treatment groups were in their own separate hatcher.
4Yolk weight as a percentage of body weight
5 Days on feed
1A

78

Table 2.5: Body weight of turkey poults with either a delayed placement on feed and/or an in ovo probiotic injection
administered at 25d of incubation1
----------------------------------d0------------------------------

-----------------------------Total BW (g)------------------------

Yolk Free BW (g)

Yolk Wt (g)

Yolk3 (%)

d04

d3

d7

55.98±2.94

15.90a±0.98

19.28a±1.91

69.58a±0.75

73.66±3.02

136.53±11.49

58.26±2.94

11.92b±0.98

13.71b±1.91

64.48b±0.75

80.94±3.02

148.00±12.85

48hNegative

57.65±2.94

11.13b±0.98

12.72c±1.91

58.66c±0.75

81.63±3.90

155.59±14.84

P-value

0.33

0.05

0.001

0.001

0.09

0.31

0h-Sham

52.88±2.94

17.66a±0.98

20.37a±1.91

70.25a±0.75

78.80b±3.38

132.56b±12.85

24h-Sham

54.01±2.94

14.2b±0.98

18.21a±1.91

67.12b±0.75

87.21b±3.02

156.29a±11.49

48h-Sham

57.66±2.94

10.43c±0.98

11.63b±1.91

61.94c±0.75

95.04a±3.02

170.98a±11.49

P-value

0.38

0.05

0.001

0.001

0.001

0.05

0h-Pro

51.35±2.94

15.86a±0.98

22.22a±1.91

70.03a±0.75

78.56b±3.02

143.72±11.49

24h-Pro

53.85±2.94

14.73b±0.98

17.43±1.91

63.85b±0.75

78.75b±3.02

145.02±12.85

48h-Pro
P-value

54.81±2.94
0.47

10.04c±0.98
0.05

12.01±1.91
0.001

57.86c±0.75
0.001

87.60a±3.02
0.05

153.86±11.49
0.49

Interaction
0h-Negative
24hNegative

a,b Means

and SEM within column for each item with no common superscript are different (P<0.05)
probiotic mixture of L. reuteri, B. animalis, E. faecium, and P. acidilacti.
2Mean of 5 replicate groups of a two bird pool of cecal contents. 0h-poults not held in the hatcher following hatch; 24h-poults
held in the hatcher for 24h prior to feed and water access; 48h-poults held in the hatcher for 48h prior to feed and water
access. Turkey eggs were offset to ensure all poults were placed on feed and water at the same time. Negative - non-injected
eggs; control - sterile saline injected eggs; probiotic - eggs injected with 1ml of probiotic inoculations at 106cfu/ml. All
treatment groups were in their own separate hatcher.
3Yolk weight as a percentage of body weight
4 Days on feed
1A

79

Table 2.6: Apparent energy retention of poults who experienced either delayed placement
on feed and/or an in ovo probiotic injection administered at 25d of incubation1
--------------------------------Apparent energy retention (%)-------------------------------0h
d3 4.3±1.0

Placement2
24h
48h
5.9±1.0

3.7±1.0

P-value

Negative

0.17

4.2±0.4

Injection3
Control Pro
5.0±0.4

4.9±0.4

P-value
0.77

d7 5.9±1.5 8.3±1.5 8.2±1.5
0.21
6.1±1.0 7.6±1.0 7.1±1.0
0.45
1 A probiotic mixture of L. reuteri, B. animalis, E. faecium, and P. acidilacti.
2Mean of 5 replicate groups of a 12 birds per pen each. 0h-poults not held in the hatcher
following hatch; 24h-poults held in the hatcher for 24h prior to feed and water access; 48h
– poults held in the hatcher for 48h prior to feed and water access. Turkey eggs were offset
to ensure all poults were placed on feed and water at the same time
3Negative - non-injected eggs; control - sterile saline injected eggs; probiotic - eggs injected
with 1ml of probiotic inoculations at 106cfu/ml. All treatment groups were in their own
separate hatcher

80

Table 2.7: The main effect of in ovo probiotic injection administered at 25d of incubation or time delay to feed on duodenal
morphology of young poults1
Placement

Injection2
Negative Control2

Control

Probiotic

P-value

02

24

48

P-value

Villus Height

661.4±29.6

682.8±29.6

661.2±29.6

0.84

597.9b±32.8

723.3a±32.8

684.1a±32.8

0.02

Crypt Depth

64.0±3.3

71.4±3.3

73.6±3.3

0.08

70.7±3.3

68.8±3.3

69.5±3.3

0.91

Ratio

11.1±0.5

10.0±0.5

9.4±0.5

0.08

8.9b±0.6

11.2a±0.6

10.3a±0.6

0.01

Villus Height

1310.2±64.1

1253.7±64.1

1203.0±64.1

0.46

1139.5±61.6

1336.2±61.6

1291.2±61.6

0.06

Crypt Depth

72.4±3.6

78.0±3.6

74.3±3.6

0.51

80.9±3.5

74.9±3.5

68.9±3.5

0.05

Ratio

20.3±1.2

17.2±1.2

17.7±1.2

0.16

15.2b±1.2

19.2a±1.2

20.8a±1.2

0.006

Duodenum4 d0

Duodenum d7

a,b Means

within column within same group for each item with no common superscript are different (P<0.05)
probiotic mixture of L. reuteri, B. animalis, E. faecium, and P. acidilacti.
2 Mean of 5 replicate groups of 12 birds per pen each. Negative Control - non-injected eggs; control - sterile saline injected
eggs; probiotic - eggs injected with 1 ml of probiotic inoculations at 106cfu/ml. All treatment groups were in their own
separate hatcher.
30h-poults not held in the hatcher following hatch; 24h-poults held in the hatcher for 24h prior to feed and water access; 48hpoults held in the hatcher for 48h prior to feed and water access. Turkey eggs were offset to ensure all poults were placed on
feed and water at the same time.
4 All measurement in µm
5 Ratio of villus height to crypt depth
1A

81

Table 2.8: The main effect of in ovo probiotic injection administered at 25d of incubation or time delay to feed on ileal
morphology of young poults1
Injection2

Placement

Negative
Control3

Control

Probiotic

P-value

03

24

48

P-value

Villus Height

278.1±26.2

329.9±26.2

323.7±26.2

0.20

254.4b±26.2

316.5a±26.2

360.8a±26.2

0.01

Crypt Depth

56.9±3.6

58.9±3.6

55.0±3.6

0.70

57.2ab±3.6

62.6a±3.6

51.1b±3.6

0.05

Ratio5

5.2±0.6

6.1±0.6

6.5±0.6

0.22

4.5b±0.6

5.4b±0.6

7.9a±0.6

0.001

Villus Height

484.2±19.5

503.9±19.5

471.7±19.5

0.46

480.5±20.4

488.2±20.4

491.1±20.4

0.92

Crypt Depth

69.6±2.7

69.8±2.7

74.1±2.7

0.41

74.5±2.9

67.8±2.9

71.1±2.9

0.21

Ratio

7.4±0.5

7.6±0.5

6.8±0.5

0.41

6.9±0.5

7.5±0.5

7.3±0.5

0.21

Ileum4 d0

Ileum d7

a,b Means

within column within same group for each item with no common superscript are different (P<0.05)
probiotic mixture of L. reuteri, B. animalis, E. faecium, and P. acidilacti.
2 Mean of 5 replicate groups of 12 birds per pen each. Negative Control - non-injected eggs; control - sterile saline
injected eggs; probiotic - eggs injected with 1 ml of probiotic inoculations at 106cfu/ml. All treatment groups were
in their own separate hatcher.
30h-poults not held in the hatcher following hatch; 24h-poults held in the hatcher for 24h prior to feed and water
access; 48h-poults held in the hatcher for 48h prior to feed and water access. Turkey eggs were offset to ensure all
poults were placed on feed and water at the same time.
4 All measurement in µm
5 Ratio of villus height to crypt depth
1A

82

Table 2.9: The interaction between placement on feed and in ovo injection on the relative gene expression of PepT1 and GLUT2
in the duodenum of young turkey poults1
PepT1
Interaction2

GLUT2

d0

d3

d7

d0

d3

d7

Mean3± St.Dev

Mean±St.Dev

Mean±St.Dev

Mean±St.Dev

Mean±St.Dev

Mean±St.Dev

1.00±1.63
1.00b±1.43
1.00±1.70
1.00c±1.67
1.00±1.46
1.00±1.75
24h-Negative
1.10±1.57
16.26a±1.7
0.15±2.32
1.00±1.75
1.00±2.43
0.53c±1.61
48h-Negative
0.90±1.49
0.56b±2.32
0.32±1.99
1.00±2.43
1.00±2.06
0.47c±1.52
0h-Control
2.25±1.57
1.99b±1.7
0.55±1.81
19.21a±1.61
1.99±1.75
1.06±1.87
24h-Control
2.22±1.52
0.75b±1.49
1.02±1.70
0.50c±1.56
0.05±1.52
2.68±1.75
48h-Control
1.18±1.52
0.84b±1.52
0.35±1.52
0.49c±1.56
0.90±1.56
1.07±1.56
0h-Pro
2.04±1.57
1.11b±1.41
0.18±1.81
8.39b±1.61
1.11±1.44
0.36±1.87
24h-Pro
2.08±1.52
1.80b±1.52
0.19±2.32
0.41c±1.56
0.11±1.56
1.24±2.43
48h-Pro
0.66±1.57
0.60b±1.49
0.67±1.57
0.16c±1.61
0.44±1.52
2.08±1.61
0.35
<0.001
0.36
<0.001
0.49
0.51
P-value
a,b Means within column within with no common superscript are different (P<0.05)
1 Relative gene expression was calculated using the 2ି∆∆஼௧ method
2 Interaction between duration of delayed placement on feed and in ovo injection treatment. Negative Control - non-injected
eggs; control - sterile saline injected eggs; probiotic - eggs injected with 1ml of probiotic inoculations at 106 cfu/ml. All
treatment groups were in their own separate hatcher.
3 0h-poults not held in the hatcher following hatch; 24h-poults held in the hatcher for 24h prior to feed and water access; 48hpoults held in the hatcher for 48h prior to feed and water access. Turkey eggs were offset to ensure all poults were placed on
feed and water at the same time.
4 Mean of 5 replicate groups of 4 birds per pen each.
0h-Negative

83

Table 2.10: The interaction between placement on feed and in ovo injection on the relative gene expression of PepT1 and
GLUT2 in the ileum of young turkey poults1
PepT1

GLUT2

d0

d3

d7

d0

d3

d7

Interaction2

Mean3± St.Dev

Mean±St.Dev

Mean±St.Dev

Mean±St.Dev

Mean±St.Dev

Mean±St.Dev

0h-Negative
24h-Negative

1.00b±1.49
1.87b±1.52

1.00±1.52
1.95±1.67

1.00±2.21
1.34±2.21

1.00b±1.64
1.04b±1.68

1.00±1.58
1.34±1.76

1.00±2.34
1.79±2.34

48h-Negative

1.40b±1.52

0.56±1.88

6.23±2.65

1.55b±1.68

0.66±1.99

1.05±2.84

0h-Control

4.78a±1.39

2.92±2.07

0.63±2.65

2.36b±1.51

2.40±2.22

0.28±2.84

24h-Control

4.13ab±1.52

1.13±1.61

2.52±1.75

1.39b±1.68

0.76±1.68

0.27±1.83

48h-Control

2.31b±1.47

0.27±1.52

1.26±1.99

1.94b±1.61

0.38±1.58

0.82±2.09

0h-Pro

2.95ab±1.60

1.12±1.49

0.72±2.21

7.90a±1.79

0.56±1.55

0.28±2.34

24h-Pro

6.12a±1.43

0.43±1.67

2.52±1.99

3.06b±1.55

0.60±1.76

1.93±1.93

48h-Pro

1.98b±1.43
0.05

0.60±1.61
0.28

2.08±1.99
0.35

2.61b±1.55
0.05

0.48±1.68
0.61

0.25±2.09
0.58

P-value
a,b Means

within column within with no common superscript are different (P<0.05)
Relative gene expression was calculated using the 2ି∆∆஼௧ method
2 Interaction between duration of delayed placement on feed and in ovo injection treatment. Negative Control - non-injected
eggs; control - sterile saline injected eggs; probiotic - eggs injected with 1ml of probiotic inoculations at 106cfu/ml. All
treatment groups were in their own separate hatcher.
3 0h-poults not held in the hatcher following hatch; 24h-poults held in the hatcher for 24h prior to feed and water access; 48hpoults held in the hatcher for 48h prior to feed and water access. Turkey eggs were offset to ensure all poults were placed on
feed and water at the same time.
4 Mean of 5 replicate groups of 4 birds per pen each
1

84

Figure 2.1: The BW of poults who experienced a delayed placement on feed and with
or without an in ovo probiotic injection A.) Poults at d0 from either negative control
(non-injected eggs), control (sterile saline injected eggs at d25 of incubation) or
probiotic (eggs injected with 1 ml of probiotic inoculations at 106 cfu/ml at d25 of
incubation) eggs than subjected to a 48h, 24h or 0h delay placement at hatch. B.)
Poults at d3 from either negative control (non-injected eggs), control (sterile saline
injected eggs at d25 of incubation) or probiotic (eggs injected with 1 ml of probiotic
inoculations at 106 cfu/ml at d25 of incubation) eggs than subjected to a 48h, 24h or
0h delay placement at hatch. C.) Poults at d7 from either negative control (noninjected eggs), control (sterile saline injected eggs at d25 of incubation) or probiotic
(eggs injected with 1 ml of probiotic inoculations at 106 cfu/ml at d25 of incubation)
eggs than subjected to a 48h, 24h or 0h delay placement at hatch. All treatment
groups were in their own separate hatcher. 0h-poults not held in the hatcher
following hatch; 24h-poults held in the hatcher for 24h prior to feed and water
access; 48h-poults held in the hatcher for 48h prior to feed and water access. Turkey
eggs were offset to ensure all poults were placed on feed and water at the same
time. a,b Bars within same group with no common superscript differ significantly
(P<0.05)
A
80

a

b

c

a

b

c

a

d0 Body Weight (g)

70
60
50
40
30
20
10
0

85

b

c

Figure 2.1 (cont’d)
B

d3 Body Weight (g)

120

b

a

a

100
80
60
40
20
0

d7 Body Weight (g)

C
200
180
160
140
120
100
80
60
40
20
0

86

Figure 2.2: The FCR of poults who experienced a delayed placement on feed and
either an in ovo probiotic injection A.) FCR from d0 to 3 of poults from either
negative control (non-injected eggs), control (sterile saline injected eggs at d25 of
incubation) or probiotic (eggs injected with 1 ml of probiotic inoculations at 106
cfu/ml at d25 of incubation) eggs than subjected to a 48h, 24h or 0h delay
placement at hatch. B.) FCR from d3 to 7 of poults from either negative control (noninjected eggs), control (sterile saline injected eggs at d25 of incubation) or probiotic
(eggs injected with 1 ml of probiotic inoculations at 106 cfu/ml at d25 of incubation)
eggs than subjected to a 48h, 24h or 0h delay placement at hatch. C.) FCR from d0 to
7 of poults from either negative control (non-injected eggs), control (sterile saline
injected eggs at d25 of incubation) or probiotic (eggs injected with 1 ml of probiotic
inoculations at 106 cfu/ml at d25 of incubation) eggs than subjected to a 48h, 24h or
0h delay placement at hatch. All treatment groups were in their own separate
hatcher. 0h-poults not held in the hatcher following hatch; 24h-poults held in the
hatcher for 24h prior to feed and water access; 48h-poults held in the hatcher for
48h prior to feed and water access. Turkey eggs were offset to ensure all poults
were placed on feed and water at the same time. a,b Bars within same group with no
common superscript differ significantly (P<0.05)

A
a

b

b

a

b

b

3

FCR from d0 to 3

2.5
2
1.5
1
0.5
0

87

Figure 2.2 (cont’d)
B

FCR from d0 to 7

2.5
2
1.5
1
0.5
0

C
1.8

a

b

b

a

ab

FCR from d3 to 7

1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

88

b

Figure 2.3: Apparent calcium and phosphorus retention in 3 or 7d old poults who
experienced a delayed placement on feed with or without an in ovo probiotic
injection A. Calcium digestibility of in ovo injected poults (P=0.679). B. Phosphorus
digestibility of in ovo injected poults (P=0.179). C. Calcium digestibility of poults with
delayed placement of feed (P=0.679). D. Phosphorus digestibility poults with delayed
placement of feed (P=0.679). 0h-poults not held in the hatcher following hatch; 24hpoults held in the hatcher for 24h prior to feed and water access; 48h-poults held in
the hatcher for 48h prior to feed and water access. Turkey eggs were offset to
ensure all poults were placed on feed and water at the same time. Negative Control non-injected eggs; control - sterile saline injected eggs; probiotic - eggs injected with
1ml of probiotic inoculations at 106cfu/ml. All treatment groups were in their own
separate hatcher. a,b Bars within same group for each item with no common
superscript differ significantly (P<0.05)

A.

Ca retention (%)

60
55
50
45
40
35
30
d3
0

d7
24

48

89

Figure 2.3 (cont’d)

B.

70

b

ab

a

P retention (%)

65
60
55
50
45
40
35
d3

d7

0

24

48

70

C.
Ca retention (%)

65
60
55
50
45
40
35
d3
Negative

D.

Control

d7
Probiotic

P retention (%)

60
55
50
45
40
35
d3
Negative

Control

90

d7
Probiotic

Figure 2.4: Ileal villus height to crypt depth ratio from A.) d0 turkeys whose eggs
were injected with probiotics on d25 of incubation and with or without a delayed
placement on feed. B.) d7 turkeys whose eggs were injected with probiotics on d25
of incubation and with or without a delayed placement on feed. Negative Control non-injected eggs; control - sterile saline injected eggs; probiotic - eggs injected with
1ml of probiotic inoculations at 106 cfu/ml. All treatment groups were in their own
separate hatcher. 0h-poults
poults not held in tthe hatcher following hatch; 24-poults
poults held
in the hatcher for 24h priorr to feed and water access; 48h
48h-poults
poults held in the hatcher
for 48h prior to feed and water access. Turkey eggs were offset to ensure all poults
were placed on feed and water at the same time. a,b Bars within same group with no
common superscript
ipt differ significantly ((P<0.05)
A.
b

14

b

a

Ileal Villus Height (mm)

12
10
8

0
24

6

48

4
2
0
Control

Non-injected

Sham

Probiotic

Control

Probiotic

91

Figure 2.4 (cont’d)

B.
10

b ab a

Ileal Villus Height (mm)

9
8
7
6
0

5

24

4

48

3
2
1
0
Control
Non-injected
injected

Sham
Control

92

Probiotic
Probiotic

Figure 2.5: Duodenal villus height to crypt depth ratio from A.) d0 turkeys whose eggs were
injected with probiotics on d25 of incubation and with or without a delayed placement on
feed. B.) d7 turkeys whose eggs were injected with probiotics on d25 of incubation and
with or without a delayed placement on feed.
feed.. Non-injected control - non-injected
injected eggs;
control - sterile saline injected eggs; probiotic - eggs injected with 1ml of probiotic
inoculations at 106 cfu/ml. All treatment groups were in their own separate hatcher. 0h0h
poults not held in the hatcher following hatch; 24h
24h-poults
poults held in the hatcher for 24h prior
to feed and water access; 48h-poults
poults held in the hatcher for 48h prior to feed and water
access. Turkey eggs were offset to ensure all poults were placed on feed aand
nd water at the
a,b
same time. Bars within same group for each item with no common superscript differ
significantly (P<0.05)

Duodenal Villus Height (mm)

A. 16
14
12
10
0

8

24
6

48

4
2
0
Control
Non-injected

Sham
Control

Probiotic

B.
30

b

a

a

Duodneal Villus height (mm)

25
20
0

15

24
10

48

5
0
Control
Non-injected

Sham
Control

Probiotic
Probiotic
Probiotic

93

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94

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96

CHAPTER 3: BONE CHARACTERISTICS AND MINERAL DIGESTION IN YOUNG TURKEY
POULTS: THE COMBINED EFFECT OF IN OVO PROBIOTICS AND LOW CALCIUM AND
PHOSPHORUS DIETS
ABSTRACT
The use of probiotics can increase intestinal maturity, increase BW, improve FCR,
and mineral absorption. In ovo probiotics may increase the performance of the host in the
same way as those presented in the diet. Therefore, the objective of this study was to
determine the impact of in ovo probiotics on performance and bone mineralization in
turkeys fed a diet with or without a 20% dietary reduction in Ca and P. Fertile turkey eggs
were candled on 25d and allocated to one of two treatments: non-injected eggs (Control) or
eggs injected with one ml of probiotic solution (concentration of 106 cfu/ml). Each
treatment had 20 replicates that contained 23 eggs per replicate. Eggs were transferred
into two separate hatchers, and hatch counts were recorded on 28d. Four poults per
replicate were sampled for bone measurements, and the ceca contents from two birds were
pooled to assess bacterial counts. The remaining poults were allocated to brooder battery
pens and assigned to a control diet (100% NRC, 1994) or a Ca and P deficient diet (80%
NRC, 1994) for 21d. Four poults per pen were euthanized for bone and intestinal samples
on 7d and 21d after hatch. The probiotic injection reduced the hatchability of birds, (87%
vs. 61%), however, the bacterial load in the intestine was increased by over 3 log10 cfu/ml
at DOH by probiotic injection. Probiotic injected birds had lower BW and shorter, narrower
tibias and femurs at 7 and 21d post hatch. Poults that received the in ovo injection had a
two-fold increase in mortality vs. non-injected poults (P<0.001). Poults fed the mineral
deficient diets had a 12% higher rate of mortality compared to control birds (P<0.05).
There were no differences in bone mineralization due to dietary treatment. These results
97

indicate that in ovo probiotics (106 cfu/ml) have negative effects on poult growth and
performance in the first 21d post hatch especially with a nutritional challenge.
INTRODUCTION
Calcium is the most abundant mineral in the turkey’s body, and an adequate intake
is important to build maximal bone strength. Mixing errors or variation in feedstuff
nutrient concentrations can lead to skeletal issues resulting in higher mortality or
underperforming birds (Oviedo-Rondón et al., 2006). Diets deficient in Ca cost producers
millions of dollars each year (Cook, 2000). Dietary feed additives, such as phytase, are often
incorporated in the diet to help offset or reduce nutrients that must be added to the diet
(Atia et al., 2000; Applegate et al., 2003). Many feed additives are derived from bacteria.
This process normally involves A. niger, Peniophora lycii and E. coli bacteria that are
selected and grown, and the additive is isolated from the bacteria (Selle and Ravindran,
2007). Feeding live bacteria (probiotics) could potentially provide the same benefits as the
purified metabolites derived from them.
Probiotic bacteria have been studied over the years and have been found to improve
the intestinal health of the host (Haghighi et al., 2006). The bacteria are often selected from
naturally present commensal strains that originate from the host species (Walker, 2008).
Patterson (2003) demonstrated that probiotic bacteria could help offset the effects of
several disease challenges because the bacteria can alter the gut microbiota and immune
system of the host. Probiotics are most effective given prior to the onset of a disease
challenge to allow the bacteria time to reach the intestine and alter microbiota before the
pathogenic bacteria reach the gut from the environment (Audisio et al., 2000). The earliest
exposure to pathogenic bacteria is in the hatchery, and in order to deliver commensal
98

bacteria prior to hatch, the probiotics need to be presented in ovo. Previous research trials
(Chapter 2) demonstrated that in ovo probiotics can benefit poults early in life in a nonchallenge setting, but the impact during a nutritional challenge is unclear. Therefore, the
objective of the study was to determine the impact of in ovo probiotics on performance and
bone mineralization in turkeys fed a diet with a nutritional challenge of a 20% dietary
reduction in Ca and P.

MATERIALS AND METHODS
The Michigan State University Institutional Animal Care and Use Committee approved all
procedures. The approval number was AUF #05/13-103-00.
Probiotic preparation
The microencapsulated probiotic contained two strains of Lactobacillus reuteri, a
single strain of Enterococcus faecium, Bifidobacteria animalis and Pediococcus acidilacti
(Biomin, Herzogenburg, Austria). To determine the concentration of organisms, the
probiotic mixture was suspended in sterile PBS and plated. To create solutions of 106
cfu/ml, the probiotic mixture was weighed (0.423 g) and suspended in PBS to create 108
cfu/ml solutions. The solutions were then diluted to 106 cfu/ml in sterile PBS (25 ml) the
day of the sample injection (E25). Solutions were stored on ice (~4h) and vortexed for 15s
immediately prior to egg injection.
Bird incubation and treatments
One thousand turkey eggs were individually weighed, set in an incubator (Petersime
Model 5; Petersime Incubator Co., Gettysburg, PA), and incubated for 24d. Incubator

99

conditions (37.5°C dry bulb and 29.2°C wet bulb) were monitored throughout the trial
using General Tools H10 data loggers (General tools, New York, NY). All eggs were candled
on E24, infertile and externally pipped eggs removed, and the lowest point of the air cell
marked. The remaining eggs were returned to the incubator and randomly assigned, using
the experimental animal allotment program to one of two treatments based on initial egg
weight (EAAP, 2009). The average difference in egg weight in each replicate was less than
0.5 g. There were 20 replicates per treatment with 23 eggs per replicate. The large end of
the egg was wiped with an alcohol disinfectant wipe. A Dremel stylus rotary tool (Model #
1100-01: Dremel, Racine, WI) was used to drill a hole into the lowest point of the egg’s air
cell. An Allflex MR2 repeater syringe (Allflex USA INC) equipped with a 22ga 1” needle was
used to inject each egg with 1 ml of probiotic solution (106 cfu/ml). Needles were changed,
and the repeater syringe was rinsed with sterile saline between each replicate. Following
injection, the drilled holes were sealed with paraffin wax. The control eggs were not drilled
or injected, but were removed from the incubator for the same amount of time as the
probiotic treatment. Each replicate was placed in a pedigree basket and assigned to one of
two Surepip hatchers (Agro Environmental Systems Inc., Dallas, Georgia, USA) resulting in
separate hatchers for each egg treatment. The pedigree baskets were randomly placed
throughout the assigned hatcher.
Sampling
Hatch counts were recorded on E28 using the following definitions: a hatched poult
had completely cleared the shell; a pipped poult had any portion of the shell cracked but
failed to clear the shell; an unhatched poult had a completely intact shell. All poults were
removed from the hatcher one replicate at a time, and total BW recorded. For cecal content
100

and yolk sac collection, four birds per replicate were euthanized by cervical dislocation and
dipped in an antiseptic solution. Each bird was removed from the antiseptic solution and
placed on a clean disinfected cutting board. The abdominal cavity was opened with sterile
scissors, which were dipped in alcohol, and flame dried between each poult. Gloves were
changed after the abdominal cavity was opened to prevent contamination from the poult
exterior. Using two sets of forceps, the yolk sac and both cecal horns were extracted from
the bird. The yolk sac was placed in a weigh boat and yolk-free BW was calculated based on
difference between total BW and yolk weight. Cecal contents, from two of the four poults,
were pooled by gently squeezing the contents into a sterile 1.5 mL microcentrifuge tube
(Denville Scientific) using the forceps to prevent glove contamination. Sterile forceps and
gloves were used for each bird to prevent cross contamination. Samples were immediately
chilled on ice until 25% of the poults had been collected, at which point, samples were
taken to the laboratory to prevent prolonged exposure to the ice and ensure viability of
samples for the dilution series.
The remaining birds (n = 8/replicate) were placed in brooder batteries by treatment
and assigned to one of two diets. The control diet was formulated to meet or exceed all
nutrient NRC requirements (NRC, 1994). The Ca and P deficient diet was formulated with a
20% reduction in Ca and P concentrations (Table 1). One kilogram feed samples were
collected from each diet for determination of Ca and P. Four poults per pen representing
the average weight were selected at 7d and 21d for a total of 40 birds per diet and injection
combination. Femurs and tibias were collected from each bird for the determination of
bone morphology and ash content.

101

Bone Ash
Muscle tissue was removed from the bone using a #10 scalpel blade, and the length
and width (measured at 50% of length) of each bone were measured. The bones were
divided with a scapel blade into epiphyseal and diaphyseal sections with the epiphyseal
section defined as the proximal and distal 25% of the bone, and the diaphyseal was defined
as the middle 50% of the bone. Bones were placed in a soxhlet and the fat removed via
ethyl ether (Avantor Performance Materials) extraction for 48h (AOAC International
method 920.39. 2006). Following fat extraction, bones were placed in crucibles and put
into a drying oven (American Scientific Products Model DN-81) for a minimum of 24h at
105°C. To determine percent ash, dried bones were removed, weighed while hot in a
crucible, and placed in an ashing oven (Thermolyne Furnace Type 30400) for
approximately 36h at 600°C. Crucibles were removed from the ashing oven and hot
weighed. To determine percent ash of the bone, the ash weight was divided by the ether
extracted dry weight.
Cecal content plating
Each cecal sample was a pool from two birds. For the six serial dilution series, 30 µl
of each cecal sample was pipetted into 270 µl of sterile PBS. The mixture was vortexed for
15s, and the pipetting procedure repeated to create the next dilution. Next, 50 µl of each
serial dilution and the pure sample were pipetted onto the center of each of four different
media plates and spread using a L-shaped glass spreader rod. Rods were flamed in alcohol
between each use. The four medias used were: Luria-Bertani agar (Acumedia, Lansing MI)
for general gram-negative bacteria, Bifidiobacteria agar (HiMedia Laboratories Ltd.
Mumbai, India) for Bifidobacteria spp, de Man, Rogosa and Sharpe agar (HiMedia
102

Laboratories Ltd. Mumbai, India) for Lactobacillus spp. plus Pediococcus spp. and
Enterococcsel agar (a modified esculin bile agar; Becton Dickinson and Company, Franklin
Lakes, NJ) for Enterococcus spp. All medias were made according to manufacturer
instructions and stored at 15°C prior to being used. Plates were incubated for 24h at 37°C
in an incubator (Precision Industries model# 30M, Chicago, IL), removed, and colony
counts were made. Only counts from a single plate from the dilution series with
approximately 25 to 300 total colonies was selected for counting. The plate was placed on a
lightbox and a marker was used to mark counted colonies, and a lab cell counter was used
to keep track of the counts.
Excreta samples
On 6d and 20d of the trial, excreta pans were removed, cleaned, lined with paper,
and replaced. Twenty-four hours later, all excreta was collected and placed in plastic bags
for apparent Ca and P retention. All samples were freeze-dried (FTS Systems, Inc,
Warminster, PA USA. Model: TRI9C0T002) and ground using the Cyclotec Sample Mill 1093
(FOSS North America, Eden Prairie, MN) with a 1 mm screen to achieve a uniform grind.
Ground samples were digested in nitric acid (OmniTrace, 67-70%, EMD Millipore, Billerica,
MA.) using a MARS 5 microwave digestion system (CEM Corporation, Matthews NC) in
accordance with procedures of Spears and Lloyd (2001). All glassware used in the
digestion process was acid washed using 30% nitric acid and rinsed in distilled de-ionized
(ddi) H2O in order to remove any residual minerals. Approximately 0.4 g of sample was
weighed into a digestion vessel and 10 mL of 70% nitric acid (Omni Trace, EMD Chemicals,
Inc., Gibbstown, NJ) was added. The sample and acid solution was allowed to sit overnight
(~16h) at room temperature to predigest the sample. The following day, vessels were
103

placed in the microwave digester, with the digestion program: 1200 watts with a 30min
ramp to 160°C under pressure of 190 PSI, hold samples for 10min, and cool down for 5min.
After additional cooling in a fume hood for 10min, vessels were vented, and two milliliters
of 30% hydrogen peroxide (Sigma-Aldrich, St. Louis, MO) was added. The sample digest
was then transferred to a 25 ml volumetric flask and allowed to cool completely; ddi H2O
was added to bring the volume to 25 ml. Samples were transferred to 50 ml polypropylene
tubes and stored at room temperature until Ca and P were determined.
Calcium assay
Calcium concentration was determined by atomic absorption spectroscopy. The
microwave digested samples were diluted 1:100 in a 1% LaCl3 (lanthanum chloride, SigmaAldrich, St. Louis, MO) solution. The LaCl3 acts as an ionization suppressant to eliminate the
phosphate interference, which occurs in an air-acetylene flame. Excreta and feed samples
were analyzed for Ca using an AA7000 Series atomic absorption spectrophotometer
(Shimadzu Scientific Instruments, Inc., Columbia, MD). These samples were analyzed
against a five point, matrix-matched standard curve (Ca standard source: VWR
International, West Chester, PA) ranging in concentration from 1 to 5 μg/ml Ca. Peach
leaves, the organic standard, (NIST, Gaithersburg, MD) were simultaneously analyzed to
maintain instrument accuracy. Apparent Ca retention reported as mg of Ca in excreta per
mg of Ca in feed.
Phosphorus assay
Spectrophotometery was used to determine P in the microwave digested samples.
Phosphate ions react with molybdate complexes, which in the presence of a reducing agent

104

(Elon), are converted to molybdenum-blue to be measured in a spectrophotometer (Kaplan
and Pesce, 1989). The MS solution contained 2.5 g of molybdate (MS), 7 ml of sulfuric acid,
and ddi H2O to bring the final volume to 500 ml. The Elon solution contained 1.5 g of
sodium bisulfate, 0.5 g of Elon, and ddi H2O to make the final volume 50 ml (Gomori, 1942).
To analyze the microwave digested sample, two ml of sample was diluted 1:10 with ddiH20.
Standards were made by diluting 15 mg/dL stock solution of P (Phosphate Standard, 1000
ppm, LabChem, Inc., Pittsburgh, PA) with ddi H2O to make 5, 4, 3, 2, 1.5, 1, and 0 mg/dL
(Gomori, 1942). Into a 96-well microplate, 50 µl of each standard and sample were
pipetted, in duplicate. Two hundred and fifty microliters of MS solution and 25 µL of Elon
were pipetted into each well. The plate was incubated for 45min at room temperature on a
plate shaker (Brinkmann TiterMix 100) at 600 rpm and read at 700 nm on the plate reader
(SpectraMax 384, Molecular Devices). The SpectraMax determined the P concentration of
all samples. The measurement was then adjusted by DM and amount of sample used in the
microwave digestion to yield the final concentration. Apparent phosphorus retention was
reported as mg of P in excreta per mg of P in feed.
Energy
Ground feed and excreta samples were formed into 1.0 g pellets using a combination
Parr pellet press modified with a hydraulic jack, and the amount of energy in each sample
was determined via bomb calorimetry following manufacturer instructions (Parr
Instrument Co). Briefly, sample pellets were placed into a capsule that was placed in the
loop of the bomb head. A Parr 45C10 Fuse Wire (34 B&S gage, nickel-chromium resistance
wire) approximately 10 cm in length was attached to the two fuses of the bomb head and

105

bent to touch the sample pellet. The bomb head was secured into the bomb cylinder. The
bomb was filled with oxygen to bring the pressure to 32 atm. The bomb was placed into a
calorimeter bucket filled with 2000 ml of diH2O and placed into the calorimeter. The two
ignition wires were pushed into the terminal sockets on the bomb head. The water
temperature of the calorimeter was allowed to reach equilibrium and the temperature
recorded. An electrical surge was sent down the terminals into the bomb igniting the
oxygen and combusting the sample. A second reading was taken approximately six min
later, after a stabilized temperature had been reached. The bomb was disassembled and the
remaining portion of the wire was measured and a calorie correction determined from the
length. A benzoic acid pellet was used to confirm the standardized values of each bomb.
Therefore, the energy was calculated based on the formula below:
Heat of Combustion ሺcal/gሻ
ൌ

ሺFt െ Itሻ ൈ energy standard of bomb െ ሺcalories of wire burnedሻ
sample dry wt. ሺgሻ

Ft= Final temperature
It= Initial temperature
Standard= benzoic acid

Dry Matter
Empty crucibles were placed into a constant temperature oven (Yamato DNF 600)
at 105°C at least 2h prior to hot weighing. Each crucible was hot weighed and 0.5 g of
sample were weighed, in duplicate, and placed into the crucible. The dry matter content of
the feed and excreta was measured by drying the samples at 105°C for 24h.
106

Statistical Analysis
All parameters were analyzed using the PROC MIXED analysis of SAS (v 9.3) with the
LSmeans procedure PROC MIXED in SAS PC Windows Version 9.2 software, (SAS Institute
Inc., Cary, NC). The model (‫ ݕ‬ൌ ߤ ൅ ߬௜ ൅ ߚ௝ ൅ ߬ߚ௜௝ ൅ ߳௜௝௞ ሻ included the main effects of
treatment (τ) and diet (β) as well as the interaction between treatment and diet (τβ).
Interactions are presented as a comparison of single treatments across various placements.
Comparisons across time were not used in the model. Pen or pedigree basket was used as
the experimental unit for all measurements. Differences between means were tested using
the Pdiff option of the LSmeans statement with significance accepted at P<0.05.

RESULTS
Performance
In ovo probiotic administration reduced hatchability of turkeys (P<0.05), but not
hatch day BW (Table 2). Probiotic injection increased the percent of poults pipped
compared to controls (P<0.05), and increased the percentage of poults unhatched by 125%
(P<0.05). Residual yolk was 4.28% higher and percent yolk was 2.97% higher in the
probiotic injected treatment compared to the control (P<0.05).
There were no differences in BW at DOH and 21d between diets (Table 3, 4). There
were interactions between injection and diet on BW, FI, and BWG. The deficient treated
birds had increased BW, FI, and BWG compared to control for the probiotic birds, but were
decreased in the non-injected birds. Mortality was 28% higher in the probiotic injected
eggs through the first week of life (P<0.01, Table 3) with no effect on mortality from 8 to
21d (Table 4). The poult mortality was greatest when the Ca and P deficient diet was fed to
107

probiotic injected birds (P<0.05). The probiotic injection reduced feed intake until 7d
(P=0.04), however, FCR was not affected by diet or injection throughout the trial (Tables 3,
4). There were no interactions between diet and injection on performance from 8d to 21d
(Table 4).
Cecal Bacterial Counts
The probiotic injection increased cecal bacteria load for all types of determined
bacteria (Table 5). Bifidobacteria and general gram-negative bacteria had a 3-log increase
in the probiotic injected birds compared to control poults (P<0.001). Lactobacillus and
Enterococcus were 4-logs higher in the probiotic injected birds than the controls (P<0.001).
Nutrient Digestibility
Birds fed the control diet had higher apparent retention of Ca and P than birds fed
the deficient diet (Table 6). 7d, the poults fed a control diet and received the in ovo
probiotic injection had higher apparent Ca retention (58%) than birds in any of the other
treatment combinations (P<0.05), but the group was not significantly higher than the noninjected control group. However, these differences were not seen on 21d (P<0.05). The
apparent retention of P was higher in the control diet fed birds than those fed the Ca and P
deficient diet (P<0.05) at both 7d and 21d. There were no significant diet by injection
interactions on P retention. There were no differences in apparent energy retention for the
injection treatment or diet.
Bone parameters
All morphology is reported on whole bone measurements, whereas the ash content
is separated into epiphyseal and diaphyseal sections. Poult femur length and the width of
108

tibia and femur bones did not respond to probiotic injection at DOH (Table 7). Tibial length
was reduced in the birds injected with probiotics compared to the non-injected birds
(P<0.05) on all sample days. There was a reduction of femur length on 7d and 21d in
probiotic injected birds compared to the non-injected (P<0.05). Probiotic injection
narrowed the tibia and femur width compared to birds not injected with the probiotic on
7d and 21d, but no effect was seen in DOH (P<0.05). There was no difference in tibial or
femoral morphology for the poults fed the Ca and P deficient diet compared to control fed
birds for any of the sample days. There were significant (P<0.05) interactions between the
injection and dietary treatments with birds in both the tibia and femur (Figure 1). Femur
length was longer in the probiotic deficient fed birds and the non-injected control birds on
DOH, and this carried through for all sample days (Figure 1A). There were no differences in
tibia length amongst the treatments at DOH. However, on 7d and 21d tibia length was
reduced when the probiotic was injected regardless of dietary treatment (Figure 1B;
P<0.05). The injected and deficient fed poults had numerically higher tibia length on d21,
whereas the non-injected deficient birds’ tibias were numerically shorter. Femur widths
were not affected by any treatment or interaction on any of the sample days (Figure 1C).
Tibia width was significantly lower on 21d when the probiotic was injected regardless of
dietary treatment (Figure 1D; P<0.05). The greater length was observed when the diet had
adequate Ca and P and the birds were not injected compared to the other treatments
(P<0.05).
Tibial epiphyseal ash was not changed by injection (Figure 2A). The diaphyseal ash
in the tibia was higher in the probiotic injected birds than non-injected birds on DOH, but
not on 7d and 21d (Figure 2C; P<0.05). Diet did not alter the epiphyseal or diaphyseal ash
109

content in the tibia (Figure 2B,D). There were no differences in ash content at 7d or 21d
due to dietary treatment or in ovo injection for either the diaphyseal or epiphyseal portion
of the femur or tibia (Figure 3). There was an increase in epiphyseal ash content of the
femur at DOH (Figure 3A; P<0.05) There were no differences in either section of bone’s ash
as a total ash percentage for any treatment (Figure 4, 5).

DISCUSSION
In the previous studies (Chapter 1, 2), injection of in ovo probiotics into turkey eggs
at 25d did not alter hatchability, whereas there was a 25% reduction in the current study.
The exact reason for the inconsistent results across trials is unknown, but there are
multiple possibilities as to why the hatchability was different in this trial compared to
other. The equipment, technique and personnel were the same for each trial and can be
ruled out as reason for the change. Sample plating confirmed the probiotic product was the
same concentration in each trial. Initial egg weight was measured and balanced across all
treatments. The eggs came from the same commercial operation, though the eggs could not
be confirmed to have come from the same hens or hens of the same age. A difference in
breeder hen age could explain the difference in the number of pipped eggs. Incubation in
eggs from younger hens can take longer that that of older hens (Applegate, 2002). The
injection could have shifted the hatch window thus decreasing the percent of poults
hatched but increasing the percent of pipped eggs. The eggs that were pipped in the
probiotic injected eggs contained embryos that appeared viable. The combination of pipped
and hatched probiotic poults would translate into numbers more closely aligned to control
poults (92% vs 82%).
110

Temperature plays an important role in development. A short reduction in
temperature before 10d can reduce growth rate of the avian embryo, but through
compensatory gain, these eggs will hatch as soon as their peers (French, 1997). A
temperature reduction late in incubation as a result of the injection could slow poult
development compared to the control eggs. The single probiotic injection could have
chilled the embryo enough to reduce hatchability. The probiotic solution was stored on ice
to prevent growth of the bacteria. The temperature of the solution was approximately 1°C,
considerably lower than hatcher temperature (~37°C). The dramatic reduction in
temperature of the internal environment for a short period of time seems to be enough to
delay hatch, where smaller temperature reduction from longer period of time was not.
The amount of residual yolk measured in the probiotic injected birds further points
to a developmental delay. The yolk sac begins to rapidly disappear as the poult approaches
hatch (Sell et al., 1991). In broilers, the yolk sac is approximately 12% of BW at hatch and
disappears at a rate of approximately 2.5 g/d (Huang et al., 2008). Yolk sac disappearance
was roughly 3 g over a 36h period in turkey poults held without feed for the first 48h of life
(Moran and Reinhart, 1980). These studies demonstrate a relatively consistent yolk
disappearance of 3 g across avian species in the first few days of life, which is consistent
with the difference in yolk weight between control and probiotic birds at hatch. A
developmental delay equaling one day would explain the increase in residual yolk.
The cecal bacterial load were approximately one-half to one log higher in the
current trial compared to the previous trials (Chapters 1, 2). An error in the probiotic
solution concentration was not an issue as plating confirmed concentrations before and
after the trial. In a previous study (Chapter 1), a 108 cfu injection reduced hatchability. The
111

results from that study indicate that a high level of embryonic intestinal bacteria correlates
with a low hatchability. The large amounts of commensal bacteria in the current study are
analogous to a necrotic enteritis outbreak. Necrotic enteritis is caused when the naturally
present commensal bacteria, Clostridium perfringenes, over proliferates and causes disease
(Lyhs et al., 2013). The data from the current experiment suggests a large commensal
bacteria load actually prevents the absorption of the yolk and reduces development by a
day. The previous c results (Chapters 1, 2) coupled with the current study indicate that 104
to 106 cfu of bacterial injection resulting in ~107 cfu of bacteria in the intestine is the
optimal amount of bacteria that the intestine can handle when administered in ovo.
The average hatch BW was not altered by probiotic injection. However, by 7d, BW
was higher in the in ovo control birds. Though the BW of the birds fed the control diet was
higher, FCR was not different between the treatments. Both injection treatments were less
than Hybrid performance goals. The FCR results are not consistent with the previous study
(Chapter 2) or published literature. Broilers and turkeys given Lactobacillus cultures had
improved BW gain and feed conversion (Kalavathy et al., 2003; Torres-Rodriguez et al.,
2007). The ability of in ovo probiotic bacteria to assist the performance of the host could be
limited to a non-nutrient challenged setting.
Previous studies on reduced nutrient diets effect on performance are more
numerous but results are variable. Turkeys fed low Ca diets did not have a change in FCR
(Sanders and Rowland, 1992; Roberson, 2004). A 20% reduction in NRC recommended
dietary Ca, increased the BW of turkeys but did not alter FCR (Atia et al., 2000). A 25 to
40% dietary reduction in Ca reduced broiler performance (Sebastian et al., 1997;
Houshmand et al., 2011). Broilers fed low P diets had reduced feed intake through the first
112

three weeks of life (Sebastian et al., 1997). A 25% reduction in dietary Ca reduced feed
intake and growth in broilers (Houshmand et al., 2011). In the current study, a reduction in
performance was seen in the low Ca and P fed birds. The poorer performance could be a
result of the diets or could be the result of an unknown condition that was exacerbated by
the reduced nutrient diets.
The dietary retention of Ca and P was higher in the control diets rather than the
reduced nutrient diets. In broilers, low P diet had a higher retention and retention
increased when Ca decreased (Ravindran et al., 1995) Bacteria in the intestine go through
a natural progression and change as the bird ages (Danzeisen et al., 2013). Altering this
progression may hinder absorption of nutrients early in life. The inability to retain
nutrient may have contributed to mortality. In both the second and third experiment
chapter the diets did not alter mortality, but the mortality was higher than expected. Bone
strength was reduced in low P diets (Ravindran et al., 1995). This could reduce mobility of
turkeys and result in fewer feeding. The inability of turkeys to transition to a commercial
diet is due to an underdeveloped intestine as another possible reason for early life
mortality (Lilburn, 1998). The probiotic injection into the egg may have exacerbated the
difficulty in transitioning to feed, causing increased mortality.
Probiotic injections into the egg increased the diaphyseal ash levels of both the
femur and tibia at DOH, though not significantly higher in the femur. The effect on ash was
short in duration, as there were no differences at 7d. Two strains of Bifidobacteria
increased Ca content of the tibia and femur of rats (Pérez-Conesa et al., 2007). Laying hens
fed combinations for Lactobacillius and Bifidobacteria and broilers fed a combination of
Bacillus strains had increased bone ash as well (Panda et al., 2003; Mutus et al., 2006). The
113

increase of bone ash in turkey poults could potentially be a benefit. The increased ash
percentage could help offset skeletal problems later in life though the potential reason for
increased mineralization at hatch is unclear. Probiotic bacteria can reduce pH in the
intestine through production of short chain fatty acids (Fooks and Gibson, 2007) allowing
for increased Ca absorption (Bronner and Pansu, 1999). The birds did not appear to have
increased Ca absorption so the exact mechanism is unclear. Dietary and in ovo
administration of probiotics could have further increased bone mineralization and
provided a longer duration of benefits.
The length of the femur and tibia increased with age within birds of the same
treatment in the current study, which is in accordance with studies by Bond et al. (1991),
Skinner and Waldroup (1995) and Bruno et al. (2007). The length of the femur and tibia
was shorter in the probiotic injected birds compared to the control birds on 7d and d21. In
rats, Bifidobacteria Longum in combination with a prebiotic and Lactobacillus improved
bone length and mineral absorption (Kruger et al., 2009; Rodrigues et al., 2012). In broilers,
probiotics did not alter bone length or width in birds fed diets with nutrients levels at NRC
recommendation or higher (Mutus et al., 2006). Turkeys are more closely related to
chickens, rather than rats, and the underlying mechanism for probiotic’s effects on bone
are not understood. Avian species may not respond to probiotics in the same manner as
mammalian species.
The probiotic injection, while influencing bone length and ash, did not alter
excretion of Ca or P in the diet. The probiotic injection was expected to decrease excreta
level of both minerals. Serum Ca levels were increased in production laying hens when
their diets were supplemented with 200 mg of probiotics containing two Lactobacillus spp.
114

and one Bifidobacteria spp. (Panda et al., 2003). Commensal and probiotic bacteria alter
vitamin D receptors in the host, and have been shown to improve Ca digestibility through
this mechanism (Resta, 2009). Another possible mechanism is the bacteria binding of
phytic acid rather than Ca. With less bound Ca, Ca absorption would increase (ScholzAhrens et al., 2007). In the current study, additional diets with prolonged use of probiotics
could have altered digestibility.
Calcium and P digestibility was higher in birds fed the control diets. In poultry
studies, the response to deficient Ca and P diet has varied. In 45wk old laying hens, a 10%
reduction in P decreased Ca digestibility (Jalal and Scheideler, 2001). While non-phytate
diets increased P and N digestibility in broilers (Ravindran et al., 2000). A greater reduction
in Ca and P in the diet may have affected birds and altered excreta mineral concentrations.
The recommended amount of Ca and P may have been adequate for early life growth needs
in turkeys. The tibia did have increased length in low Ca and P fed birds. However, femurs
were shorter in birds fed deficient diets, indicating that bones respond differently to
dietary deficiencies. Though, there was no interaction between the diet and injection
treatments, the difference in tibia and femur length between the non-injected control and
probiotic was greater in the 21d birds compared to the 7d. The differences observed may
have continued through the production period and warrant further investigation.
A common practice in poultry is to use bacteria derived phytase in the diet to
improve P digestibility. There are several bacterial sources of microbial phytase that have
proven effective, and thus usage has increased over the last 20 years (Kiarie et al., 2013).
Bacterial phytase improved P retention and bone mineralization in turkeys (Applegate et
al., 2003). The addition of the bacteria strains, which phytase is isolated from, could alter P
115

digestion in the same way as phytase addition to the diet, but this is unclear. Laying hen
diets supplemented with molasses and strains of Lactobacillus had increased phytase
activity and P retention (Nahashon et al., 1994). While the total Ca and P in the deficient
diet of the current study was less than the control diet, the ratio of Ca:P was approximately
the same for both diets (1.4, 1.5). Microbial phytase effectiveness was reduced in turkeys
from hatch to 21d when the ratio increased to 2.0 (Qian et al., 1996). In the current study,
the Ca:P ratio might have been too high for the microbes to effect digestibility. In the
current experiment, the single injection of probiotics was not enough to alter digestibility,
whereas continued use might have been. In rats, supplementation with probiotics for 30d
improved both Ca and P digestibility (Pérez-Conesa et al., 2006). The author is not aware of
a study examining a single dose probiotic in poultry and the effects on digestibility of Ca
and P. The effect of daily feeding is difficult to compare to a single dose.
The trial further demonstrates that a single injection of probiotics (106 cfu) has a
long lasting effect on the host. A shift in bacterial population in the intestine influences
skeletal morphology and body size. Further research could determine if the long lasting
effects could be beneficial. The current results show that even commensal bacteria can be
harmful if in too large amount. Producers must protect poults from high cecal bacterial
concentrations of any kind. The trial further demonstrates the potential of a single event to
alter performance early in life and for that shift in production to be maintained through the
first three weeks post hatch.

116

APPENDIX

117

Table 3.1: Composition, calculated, and analyzed nutrient content of diets fed from 0-21d of
age
Diet (as fed)
Ingredients (%)
Corn
Soybean meal, 48% CP
Soy oil
NaCl
Copper sulfate
Sodium bicarbonate
DL Methionine
Lysine HCl
Threonine
Limestone
Monocalcium phosphate
Vitamin/mineral premix2
Celite
Chromic (III) Oxide
Calculated values (DM)
ME (kcal/kg)
Crude protien
Ca (g/kg)
nPP (g/kg)
Ca:P ratio (g/g)
Analyzed values (DM)
Ca (g/kg)
Total P (g/kg)
Ca:P ratio (g/g)
1

Control 1

Deficient

41.63
48.47
3.00
0.25
0.05
0.20
0.28
0.34
0.13
2.10
2.70
0.35
0.00
0.50

41.63
48.47
3.00
0.25
0.05
0.20
0.28
0.34
0.13
1.65
2.00
0.35
1.15
0.50

2875
27.7
14.0
7.5
1.87

2875
27.7
11.3
6.1
1.85

15.76
11.17

11.17
9.89

1.41

1.51

Control are birds fed diet containing 100% of NRC Ca and P requirements; Deficient are
birds fed diet containing 80% of NRC Ca and P requirements.
2 Supplied per kilogram of diet: vitamin A, 13,233 IU; vitamin D3, 6,636 IU; vitamin E, 44.1
IU; vitamin K, 4.5 mg; thiamine, 2.21 mg; riboflavin,
6.6 mg; pantothenic acid, 24.3 mg; niacin, 88.2 mg; pyridoxine, 3.31 mg; folic acid, 1.10 mg;
biotin, 0.33 mg; vitamin B12, 24.8 mg; choline, 669.8
mg; iron from ferrous sulfate, 50.1 mg; copper from copper sulfate, 7.7 mg; manganese
from manganese oxide, 125.1 mg; zinc from zinc oxide,
125.1 mg; iodine from ethylene diamine dihydroidide, 2.10 mg; selenium from sodium
selenite, 0.30 mg.

118

Table 3.2: Hatch performance of turkey poults administered in ovo probiotics at 25d of
incubation1
Probiotic
In ovo control2
Mean ± SE
Mean ± SE
Hatchability3
87.21a±1.4
61.33b±1.4
Pipped4
4.99a±1.57
21.01b±1.57
Unhatched5
7.80a±1.4
17.66b±1.4
Hatch weight (g)
62.95±0.88
64.12±0.88
b
DOH Yolk weight (g)
15.52 ±0.80
18.49a±0.80
Yolk 6(%)
24.64b±1.25
28.92a±1.25
a,b Means and standard error within row with no common superscript are different
(P<0.05)
1 A probiotic mixture of L. reuteri, B. animalis, E. faecium, and P. acidilacti.
2 Mean of 20 replicate groups of 23 eggs each. Control are non-injected eggs in a separate
hatcher; probiotic are eggs injected with 1ml of probiotic inoculations at 106cfu/ml in a
separate hatcher.
3 Percentage of poults that had hatched - completely cleared the shell at time of count
4 Percentage of poults that had started to hatch - cracked the shell at time of count.
5 Percentage of poults that had not started to hatch - shell completely intact at time of count
6 Yolk weight as a percentage of hatch weight

119

Table 3.3: Performance of 0 to 7d old poults fed control or Ca and P deficient diets with or without in ovo probiotic1
administration at 25d of incubation
BW (g)
Mean ± SE

FI (g)
Mean ± SE

BWG (g)
Mean ± SE

FCR(g/g)
Mean ± SE

Mortality(%)
Mean ± SE

Injection2
In ovo control
Probiotic

121.73a±2.67
109.45b±2.67

72.91a±4.05
61.08b±4.05

59.17a±2.88
44.95b±2.88

1.26±0.24
1.70±0.24

10.86b±2.71
38.21a±2.71

Diet3
Control
Deficient

116.20±2.67
114.98±2.67

67.19±4.05
66.80±4.05

52.05±2.88
52.07±2.88

1.45±0.24
1.51±0.24

18.97b±2.71
30.10a±2.71

124.15a±3.78
119.31a±3.78
108.24b±3.78
110.65b±3.78

74.69a±5.73
71.12a±5.73
59.68b±5.73
62.48b±5.73

61.98a±4.07
56.35a±4.07
42.11b±4.07
47.78b±4.07

1.23±0.34
1.29±0.34
1.68±0.34
1.73±0.34

08.91c±3.83
12.82c±3.83
29.02b±3.83
47.39a±3.83

<0.001
0.75
0.02

0.04
0.94
0.58

0.01
0.96
<0.001

0.20
0.86
0.98

<0.001
0.006
<0.001

Item

Interaction
In ovo control x Control
In ovo control x Deficient
Probiotic x Control
Probiotic x Deficient

P-values
Injection
Diet
Interaction
a,b Means

and standard error within column for each item with no common superscript are different (P<0.05)
probiotic mixture of L. reuteri, B. animalis, E. faecium, and P. acidilacti.
2Mean of 10 replicate pens. Non-injected eggs were in a separate hatcher; probiotic are eggs injected with 1 ml of probiotic
inoculations at 106cfu/ml and placed in a separate hatcher.
3Control are birds fed diet containing 100% of NRC Ca and P requirements; deficient are birds fed diet formulated to contain
80% of NRC Ca and P requirements.
1A

120

Table 3.4: The performance poults from 8 to 21d post hatch fed control or Ca and P deficient diets with or without in ovo
probiotic administration
Item
Injection2
In ovo control
Probiotic
Diet3
Control
Deficient
Interaction

BW (g)
Mean ± SE

FI (g)
Mean ± SE

BWG (g)
Mean ± SE

FCR (g/g)
Mean ± SE

Mortality
Mean ± SE

525.91a±31.61
441.95b±35.34

545.99±13.66
474.03±15.27

396.71±22.31
330.27±24.95

1.44±0.14
1.53±0.15

0.20±0.15
0.25±0.15

488.63±33.53
479.23±33.53

508.52±14.49
511.5±14.49

346.86±23.67
380.12±23.67

1.63±0.14
1.35±0.15

0.35±0.15
0.10±0.15

0.40±0.15
In ovo control x Control
539.14±44.70
550.78±19.32
387.8±31.55
1.51±0.19
0.00±0.15
In ovo control x Deficient
512.68±44.70
541.19±19.32
405.61±31.55
1.37±0.20
0.30±0.15
Probiotic x Control
438.12±49.98
466.25±21.60
305.92±35.28
1.74±0.21
0.20±0.15
Probiotic x Deficient
445.79±49.98
481.82±21.60
354.63±35.28
1.33±0.22
P-value
Injection
0.001
0.08
0.06
0.64
0.94
Diet
0.88
0.84
0.32
0.18
0.82
Interaction
0.54
0.72
0.64
0.52
0.79
a-b Means and standard error within column for each item with no common superscript are different
1 A probiotic mixture of L. reuteri, B. animalis, E. faecium, and P. acidilacti.
2Mean of 10 replicate pens (8 birds/replicate). Non-injected are non-injected eggs in a separate hatcher; probiotic are eggs
injected with 1 ml of probiotic inoculations at 106cfu/ml in a separate hatcher.
3 Control are birds fed diet containing 100% of NRC Ca and P requirements; deficient are birds fed diet formulated to contain
80% of NRC Ca and P requirements.
* (P<0.05)

121

Table 3.5: Total cecal bacterial populations (log10cfu/ml) as determined by cultivable
plate count on four different medias from poults administered various amounts of in ovo
probiotics1 administered at 25d of incubation
Injection2

BIF3
Mean ± SE

ENT4
Mean ± SE

GN5
Mean ± SE

LAC6
Mean ± SE

In ovo control

7.15b±0.21

5.74b±0.21

7.13b±0.21

6.43b±0.21

Probiotic
10.42a±0.21
10.37a±0.22
10.22a±0.21
10.40a±0.22
within column for each treatment with no common superscript are different
(P<0.001)
1 A probiotic mixture of L. reuteri, B. animalis, E. faecium, and P. acidilacti.
2Mean of 20 replicate groups of a two-bird pool of cecal contents. Non-injected are
control eggs in a separate hatcher; probiotic are eggs injected with 1ml of probiotic
inoculations at 106cfu/ml in a separate hatcher
3 BIF- Bifidobacteria
4ENT-Enterococcocus
5GN- Gram-negative bacteria
6LAC-Lactobacillus and Pediococcus
a,b Means

122

Table 3.6: Apparent Ca and P retention in 7 and 21d old poults fed either control or Ca and P deficient diets with or without in
ovo probiotic1 injection at 25d of incubation
Item

Calcium4 (%)
day 7
day 21
Mean ± SE
Mean ± SE

Phosphorus (%)
day 7
day 21
Mean ± SE
Mean ± SE

Energy (%)
day 7
day 21
Mean ± SE
Mean ± SE

Injection2
In ovo control
52.34±1.34
42.59±1.95
67.11±0.84
56.35±2.61
8.5±1.07
13.8±0.8
6.5±1.07
15.2±0.8
Probiotic
54.33±1.47
44.18±2.13
66.38±0.93
54.58±2.78
3
Diet
Control
56.52a±1.34
42.09±2.13
68.17a±0.84
58.61a±2.78
6.7±1.06
14.6±0.6
b
b
b
Deficient
50.14 ±1.47
44.68±1.95
65.32 ±0.93
52.33 ±2.61
8.4±1.06
14.8±0.6
Interaction
In ovo control x Control
54.45a±1.89
39.93±2.91
67.49±1.19
59.53±3.80
8.4±1.02
14.0±1.04
45.24±2.60
66.73±1.19
53.17±3.58
8.2±1.02
13.5±1.04
In ovo control x Deficient
50.22b±1.89
a
Probiotic x Control
58.59 ±1.89
44.25±3.11
68.84±1.19
57.68±4.06
6.2±1.02
15.4±1.04
Probiotic x Deficient
50.06b±2.26
44.12±2.91
63.91±1.42
51.48±3.80
7.2±1.02
15.2±1.04
P-value
0.20
0.23
Injection
0.32
0.58
0.56
0.65
0.17
0.87
Diet
0.003
0.37
0.03
0.11
Interaction
0.29
0.35
0.10
0.98
0.28
0.77
a,b Means within column for each main effect or interaction with no common superscript are different (P<0.05).
1 A probiotic mixture of L. reuteri, B. animalis, E. faecium, and P. acidilacti.
2 Mean of 10 replicate cages. Non-injected are non-injected eggs in a separate hatcher; probiotic are eggs injected with 1ml
of probiotic inoculations at 106cfu/ml in a separate hatcher.
3 Control are birds fed diet containing 100% of NRC Ca and P requirements; deficient are birds fed diet formulated to contain
80% of NRC Ca and P requirements.
4 Apparent total tract digestibility of Ca, P, and energy as a percentage of total amount in ration

123

Table 3.7: The main effects of control or Ca and P deficient diets and in ovo probiotic1 injection at 25d of incubation on tibial
and femoral morphology of DOH, 7 and 21d old poults fed
Parameter4

Mean ± SE

Injection2
Mean ± SE

P-value

Mean ± SE

Diet3
Mean ± SE

Control

Deficient

P-value

In ovo control

Probiotic

36.11a±0.42
46.90a±0.34
78.31a±0.78

34.09b±0.40
44.16b±0.34
75.98a±0.66

<0.001
<0.001
<0.001

34.82±0.4
45.29±0.33
75.97±0.71

35.38±0.56
45.28±0.42
75.93±0.82

0.32
0.28
0.96

Tibia Width (mm)
DOH
d7
d21

1.98±0.02
2.56a±0.03
4.82a±0.05

1.94±0.02
2.42b±0.03
4.44b±0.06

0.09
0.002
<0.001

1.95±0.02
2.50±0.03
4.65±0.06

1.97±0.02
2.48±0.03
4.60±0.06

0.59
0.61
0.58

Femur Length (mm)
DOH
d7
d21

23.99±0.27
31.77a±0.19
51.93a±0.37

23.24±0.29
29.95b±0.22
48.82b±0.49

0.06
<0.001
<0.001

23.74±0.28
31.04±0.2
50.41±0.42

23.49±0.28
30.68±0.21
50.34±0.45

0.53
0.21
0.91

Femur Width (mm)
DOH
d7
d21

1.93±0.02
2.58a±0.03
4.66a±0.05

1.90±0.02
2.39b±0.03
4.34b±0.07

0.11
<0.001
<0.001

1.91±0.02
2.52±0.03
4.55±0.06

1.91±0.02
2.46±0.03
4.45±0.06

0.98
0.11
0.24

Tibia Length (mm)
DOH
d7
d21

a,b

Means and standard errors within row for each treatment with no common superscript are different (P<0.05).
probiotic mixture of L. reuteri, B. animalis, E. faecium, and P. acidilacti.
2 Mean of 10 replicate pens. Non-injected are non-injected eggs in a separate hatcher; probiotic are eggs injected with 1ml of
probiotic inoculations at 106cfu/ml in a separate hatcher
3 Control are birds fed diet containing 100% of NRC (1994) Ca and P requirements; deficient are birds fed diets formulated
to contain 80% of NRC Ca and P requirements

1A

124

Figure 3.1: Tibial and femoral morphology of DOH, 7 and 21d old poults fed either control or Ca and P deficient diets with or
without in ovo probiotic1 injection at 25d of incubation. A.) Femur length. B.) Tibia length. C.) Femur width. D.) Tibia width
width.
Non-injected are non-injected
injected eggs in a separate hatcher; probiotic are eggs injected with 1 ml of probiotic inoculations at
106cfu/ml in a separate hatcher. Control are birds fed diet containing 100% of NRC (1994) Ca and P requirements; deficient
are birds fed diet formulated
rmulated to contain 80% of NRC (1994) Ca and P requirements. Results are expressed the means ± SE of
a,b,c
10 replicate pens.
Bars with no common letter at same age are different ((P<0.05).

A.

B.
a b

b a

a b

b a

a

b

b

a

a

60

a

b

b

a

a

b

80
50
Tibia Length (mm)

Femur Length (mm)

70
60
50
40
30
20

40
30
20
10

10
0
DOH

d7

0

d21

DOH

125

d7

d21

b

Figure 3.1 (cont’d)

C.

D.
6
a

b

c

5

5

Tibia Width (mm)

Femur Width (mm)

6

4
3
2
1

4
3
2
1

0
DOH

d7

0

d21

DOH

126

d7

d21

c

Figure 3.2: The main effect of in ovo probiotic injection and either standard or deficient nutrient diets on tibial ash (%) in
turkey DOH, d7 and d21 turkey poults. A.) The effect of probiotics administered at 25d of incubation on the tibial epiphyseal
ash (25% of each end of the tibia) of turkeys. B.) The effect of standard or a nutrient deficient diet on the tibial epiphyseal ash
(25% of each end of the tibia) of turkeys. C.) The effect of probiotics administered at 25d of incubation on the tibial diaphyseal
ash (middle 50% of the bone) of turkeys. D.) The effect of standard or a nutrient deficient diet on the tibial diaphyseal ash
(middle 50% of the bone) of turkeys. Non-injected are non-injected eggs in a separate hatcher; probiotic are eggs injected with
1 ml of probiotic inoculations at 106cfu/ml in a separate hatcher. Control are birds fed diet containing 100% of NRC (1994) Ca
and P requirements; deficient are birds fed diet formulated to contain 80% of NRC (1994) Ca and P requirements. Results are
expressed the means ± SE of 10 replicate pens. a,b Bars with no common letter at same age are different (P<0.05).

B.
40

40

35

35

Epiphyseal Ash (%)

Epiphyseal Ash (%)

A.

30
25
20
15
10
5
0

30
25
20
15
10
5
0

DOH
In ovo control

d7

d21

DOH

Probiotic

d7
Control

127

Deficient

d21

Figure 3.2 (cont’d)

C.

D.
b

60

a
Diaphyseal Ash (%)

Diaphyseal Ash (%)

60
50
40
30
20
10

50
40
30
20
10
0

0
DOH
In ovo control

d7

DOH

d21

Control

Probiotic

128

d7
Deficient

d21

Figure 3.3: The main effect of in ovo probiotic injection and either standard or deficient nutrient diets on femoral ash (%) in
turkey DOH, d7 and d21 turkey poults. A.) The effect of probiotics administered at 25d of incubation on the femoral epiphyseal
ash (25% of each end of the tibia) of turkeys. B.) The effect of standard or a nutrient deficient diet on the femoral epiphyseal
ash (25% of each end of the tibia) of turkeys. C.) The effect of probiotics administered at 25d of incubation on the femoral
diaphyseal ash (middle 50% of the bone) of turkeys. D.) The effect of standard or a nutrient deficient diet on the femoral
diaphyseal ash (middle 50% of the bone) of turkeys. Non-injected are non-injected eggs in a separate hatcher; probiotic are
eggs injected with 1 ml of probiotic inoculations at 106cfu/ml in a separate hatcher. Control are birds fed diet containing 100%
of NRC (1994) Ca and P requirements; deficient are birds fed diet formulated to contain 80% of NRC (1994) Ca and P
requirements. Results are expressed the means ± SE of 10 replicate pens.a,b Bars with no common letter at same age are
different (P<0.05).

A.

B.
b

a

40

35

Epiphyseal Ash (%)

Epiphyseal Ash (%)

40
30
25
20
15
10

35
30
25
20
15
10

5

5

0

0
DOH
In ovo control

d7

DOH

d21
Probiotic

d7
Control

129

Deficient

d21

Figure 3.3 (cont’d)

D.

60
50

60
Diaphyseal Ash (%)

Diaphyseal Ash (%)

C.

40
30
20
10
0

50
40
30
20
10
0

DOH
In ovo control

d7

d21

DOH

Probiotic

d7
Control

130

Deficient

d21

Figure 3.4: The interaction of in ovo probiotic injection at 25d of incubation and
either standard or deficient nutrient diets on (A) tibial ash from DOH turkeys, (B)
tibial ash from 7d old turkeys and (C) tibial ash from 21d old turkeys. Epiphyseal is
25% of each end of the tibia. Diaphyseal is the middle 50% of the bone. Non-injected
are non-injected eggs in a separate hatcher; probiotic are eggs injected with 1 ml of
probiotic inoculations at 106cfu/ml in a separate hatcher. Control are birds fed diet
containing 100% of NRC (1994) Ca and P requirements; deficient are birds fed diet
formulated to contain 80% of NRC (1994) Ca and P requirements.

Ash

A.
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%

Diaphyseal
Epiphyseal

In ovo
control x
Control

In ovo
control x
Deficient

Probiotic x
Control

131

Probiotic x
Deficient

Figure 3.4 (cont’d)

Ash

B.
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%

Epiphyseal

In ovo
control x
Control

C.

Ash

Diaphyseal

In ovo
control x
Deficient

Probiotic x
Control

Probiotic x
Deficient

100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%

Diaphyseal
Epiphyseal

In ovo
control x
Control

In ovo
control x
Deficient

Probiotic x
Control

132

Probiotic x
Deficient

Figure 3.5:. The interaction of in ovo probiotic injection at 25d of incubation and
either standard or deficient nutrient diets on (A) femoral ash from DOH turkeys, (B)
femoral ash from 7d old turkeys and (C) femoral ash from 21d old. Epiphyseal is
25% of each end of the femur. Diaphyseal is the middle 50% of the bone. Noninjected are non-injected eggs in a separate hatcher; probiotic are eggs injected with
1 ml of probiotic inoculations at 106cfu/ml in a separate hatcher. Control are birds
fed diet containing 100% of NRC (1994) Ca and P requirements; deficient are birds
fed diet formulated to contain 80% of NRC (1994) Ca and P requirements.

Ash

A.
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%

Diaphyseal
Epiphyseal

In ovo
control x
Control

In ovo
control x
Deficient

Probiotic x
Control

133

Probiotic x
Deficient

Figure 3.5 (cont’d)

Ash

B.
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%

Ash

C.

Diaphyseal
Epiphyseal

In ovo
control x
Control

In ovo
control x
Deficient

Probiotic x
Control

Probiotic x
Deficient

100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%

Diaphyseal
Epiphyseal

In ovo
control x
Control

In ovo
control x
Deficient

Probiotic x
Control

134

Probiotic x
Deficient

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135

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139

SUMMARY AND FUTURE STUDIES
The current studies focused on the effect of in ovo probiotic injection on early
life development of the poult. In ovo probiotics can be injected at approximately 106
cfu without altering hatchability. The injection improved FCR in turkey poults that
were fed NRC recommended diets and increased diaphyseal ash levels in the femur
early in life. The studies had some conflicting results as well; the injection of 106 cfu
reduced hatchability and livability in the third study. Though the injections had the
same amount of bacteria in the egg, there is some possible mechanism or
unmeasured condition in the bird or the egg that allowed the amount of bacteria in
the intestine to be higher. Any studies in the future should try to tease out the
mechanism of growth of the bacteria in the egg. The current studies demonstrated
that the effect of probiotic injections appears to last approximately three days. What
is not clear is how this alteration can influence the birds later in life, specifically the
increase in bone mineralization.
Alternative strains
The performed studies examined a single commercially available probiotic
that was a mixture of several strains of bacteria. If the beneficial effects were a
result of a single strain or a combination of all of the strains is unclear. There are
also multiple commercial strains available, and these variable strains are expected
to yield different results. Futures studies could identify the bacteria that are most
beneficial to turkeys and which ones have little effect even though they are a part of
normal gut microflora population.

140

Delivery method
Future studies should compare in ovo delivery to more common delivery
methods, such as spray or drip delivery at the hatchery. These commonly used
delivery methods may work to improve the effect of in ovo probiotics if used in
concert with one another. Delivery in the diet or drinking water may assist in ovo
injections as well. Turkeys showed improved FCR in study two, and the birds may
have continued to improve FCR if prolonged dosage of bacteria were used. In ovo
probiotics may increase the benefits of probiotics post hatch.
Combination with current vaccinations
Vaccines are injected into millions of eggs each year. Vaccines are given at a
different location and a different dosage than the probiotic injections of the current
studies. Producers are normally hesitant to invest in expensive new equipment. This
makes the importance of studying how in ovo probiotics can work with current
vaccination techniques. The egg can only hold a finite volume of liquid, normally
believed to be one to one and one half milliliters. If injection machines can be
repurposed to inject both vaccines and bacteria, the practically of the bacterial
injections improves.
In ovo probiotics can be a benefit to turkeys, but are a long way from
commercial application. The technology exists to inject the eggs. If several of the
proposed studies were completed and shown to be beneficial, in ovo probiotics
could become and important industrial practice in the future.

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