Embryo quality characteristics from superovulated cows receiving a blend of bioactive peptides and oligosaccharides to s...

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Embryo quality characteristics from superovulated cows receiving a blend of bioactive peptides and oligosaccharides to support immune function (Grade One)
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Creator:
Marquezini, Guilherme
Mercadante, Vitor
Ward, Mindy
Spell, Audy
Carter, Joel
Paton, Neil
Lamb, G. Cliff
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Institute of Food and Agricultural Sciences, University of Florida
Place of Publication:
Gainesville, Fla.
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University of Florida
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Embryo Quality Characteristics from Superovulated Cows Receiving a
Blend of Bioactive Peptides and Oligosaccharides to Support Immune
Function (Grade OneTM)


Guilherme Marquezini, Vitor Mercadante, Mindy Ward, Audy Spell, Joel Carter, Neil Paton,
G. Cliff Lamb1


Treatment of embryo donor cows with NHG1 (Nutrition Horizons Grade OneTM) during the super-
stimulation protocol did not alter the absolute number of embryos collected but it increased the quality of
the embryos by increasing the percentage of grade 1 embryos relative to the number of transferable
embryos harvested.


Summary
We determined whether supplementation of a
blend of bioactive peptides and oligosaccharides
to support immune function (Nutrition Horizons
Grade OneTM; Brookville, OH; US Patent no.
6,962,718) would alter quality, stage, and
fertilization rate of embryos recovered after
superovulation. Cows were superovulated using
follicle ',iiill,, ,i ig hormone as NIH-FSH-P1
and were stratified by breed before random
assignment to treatment: 1) donors received six
Grade OneTM boluses (13 g/capsule) containing
a blend of bioactive peptides and
oligosaccharides (NHG1; n = 35); or 2) donors
received 6 placebo capsules (13 g/capsule;
Control; n 37). Superovulation of donors was
initiated by insertion of a controlled internal
drug release device (CIDR) on d 0, eight
injections of follicle stimnilting hormone (FSH)
administered at 12 h intervals initiated on d 4,
plus two injections of prostaglandin F2a (PGF)
12 h apart on d 7. At 0 and 12 h after detected
estrus cows received an artificial insemination
(AI). Boluses were inserted into the esophagus
utilizing a balling gun. Cows received two
boluses at CIDR insertion (d 0), at the first (d 4),
and third (d 5) injection of FSH. Embryos were
collected 7 d after first detected estrus and were
recovered by nonsurgical embryo collection
procedure. The embryos were evaluated and
classified by stage and quality. Total ova (14.1
1.8) and transferable embryos (5.2 1.1) per


flush for NHG1 and Control did not differ.
Mean number of grade 1 (2.5 0.8) and 2 (2.7
0.5) were similar between treatments and no
differences existed between treatments for
degenerate (1.9 0.4) or unfertilized ova (7.0
1.4). However, the percentage of grade 1
embryos collected compared to recovered
transferable embryos was greater (P = 0.06) for
NHG1 (39.4%) than Control (23.4%). In
addition, the percentage of grade 2 embryos
collected compared to recovered transferable
embryos was greater (P < 0.05) for Control
(76.6%) than NHG1 (59.9%). We conclude that
the number of transferable embryos collected
per flush did not differ between treatments;
however, the quality of transferable embryos
was improved after embryo donor cows received
NHG1 prior to embryo collection.

Introduction
Embryo transfer has been developed to increase
the number of offspring from a superior genetic
donor. The technique enhances the opportunity
to create multiple offspring from a genetically
superior cow rather than a single offspring per
year. The results are accomplished by using
recipients (regular genetic cow/heifers with good
maternal ability) that serve as the surrogate for
the embryo collected from the donor cow.
Donors are selected based on an outstanding
characteristic that results in more value such as


'North Florida Research and Education Center, University of Florida, Marianna, FL










greater fertility, milk production, heavier calves,
disease resistance, etc. Recipients usually are
selected for their maternal ability such as milk
production and ability to nurture their young,
along with good temperament. Physiologically,
donors are super-stimulated with natural
hormones that act to recruit more than one
regular follicle, increasing the likelihood that
multiple follicles become dominant, ovulate, and
may be fertilized at the same time. Donors are
flushed 7 d after insemination to remove the
fertilized embryos. Embryos are then either
frozen or transferred fresh to recipients that were
previously synchronized to be at the same stage
of the estrous cycle of the donor. The
development of the non-surgical embryo transfer
technique, pushed the embryo transfer (ET)
industry to significantly increase the number
cattle derived from ET. Conception rates of
more desirable quality embryos (excellent or
good) are greater than embryos qualified as fair
or poor quality (JETS, Savoy, IL); therefore,
conception rates would increase if a greater
percentage of grade 1 (excellent) embryos are
harvested and transferred after collection.

Research in the field of ET is very expensive
and many of the basic procedures were
established some years ago. Consequently, little
academic research currently is being conducted
that is likely to immediately benefit the
commercial ET industry. Because ET success
rates in well-managed cattle herds are generally
quite high, most recent improvements involve
rather small, albeit important, increments. In a
1992 review article, Hasler (1992) stated "There
have been no significant improvements in
techniques for the superovulation of cattle in the
last 15 years". This statement remains largely
true today (Hasler, 2010). In fact, data provided
by both the American and Canadian Embryo
Transfer Associations indicate that mean embryo
yields per donor range from 5 to 7 and basically
have not changed for many years. These
embryo means are a composite average of
individual means of both beef (6.6) and dairy
cattle (5.7) flushed by the certified members of
the AETA.


A product, Nutrition Horizons Grade OneTM
(NHG1), was originally developed to provide
formulations of immune factors in combination
with minerals, antioxidants, amino acids, and
other nutraceuticals administered orally to treat
animals exhibiting disease symptoms but also to
lower general morbidity. Due to its ability to
improve immune function, NHG1 may possibly
improve uterine health and embryo quality.

Materials and Methods
The experimental was designed as a completely
randomized design. Seventy-two cows
underwent a super-stimulation protocol with
follicle stimulating hormone (FSH) prior to
embryo collection. Cows were superovulated
using FSH as NIH-FSH-P1 and were stratified
by breed before random assignment to
treatment: 1) donors received 6 Grade OneTM
boluses (13 g/capsule) containing a blend of
bioactive peptides and oligosaccharides (NHG1;
n = 35); or 2) donors received 6 placebo
capsules (13 g/capsule; Control; n = 37).
Superovulation of donors was initiated by
insertion of a CIDR on d 0, eight injections of
FSH administered at 12 h intervals initiated on d
4, plus two injections of PGF 12 h apart on d 7.
At 0 and 12 h after detected estrus cows received
an AI. Boluses were inserted into the esophagus
utilizing a balling gun. Cows received two
boluses at CIDR insertion (d 0), at the first (d 4),
and third (d 5) injection of FSH (Figure 1).
Embryos were collected 7 d after first detected
estrus and were recovered by nonsurgical
embryo collection procedure. Embryos were
evaluated under a stereomicroscope and
classified by stage and quality according to
standards set forth by the International Embryo
Transfer Society (developmental stage codes
were: 3 = early morula; 4 = morula; 5 = early
blastocyst; 6 = blastocyst; and 7 = expanded
blastocyst). Quality codes were: 1 = symmetrical
and spherical embryo mass with individual
blastomeres that were uniform in size, color, and
density with at least 85% of the cellular material
intact (excellent or good); 2 = moderate
irregularities in overall shape of embryonic mass
or in size, color and density of individual cells









with at least 50% of the cellular material intact
(fair); 3 = major irregularities in shape of the
embryonic mass or size, color and density of
individual cells with at least 25% of the cellular
material intact (poor); 4 = dead or degenerating;
and 5 = unfertilized.

Results
Table 1 represents the nutrients analysis of the
placebo and NHG1 boluses. Overall mean total
ova (14.1 + 1.8) and transferable embryos (5.2
1.1) per flush for NHG1 and Control did not
differ (P > 0.05; Table 2). Mean number of
grade 1 (2.5 0.8) and 2 (2.7 0.5) were similar
(P > 0.05) between treatments and no
differences (P > 0.05) existed between


treatments for degenerate (1.9 0.4) or
unfertilized ova (7.0 1.4; Figure 2). However,
the percentage of grade 1 embryos collected
compared to recovered transferable embryos
tended (P = 0.06) to be greater for NHG1
(39.4%) than Control (23.4%; Figure 2). In
addition, the percentage of grade 2 embryos
collected compared to recovered transferable
embryos was greater (P < 0.05) for Control
(76.6%) than NHG1 (59.9%; Figure 3). We
conclude that the number of transferable
embryos collected per flush did not differ
between treatments; however, the quality of
transferable embryos was improved after
embryo donor cows received NHG1 prior to
embryo collection.


Literature Cited
Hasler. 1992. J. Dairy Sci. 75:2857.
Hasler. 2010. Proc. Applied Reproductive Strategies in Beef Cattle, Nashville, TN pp. 265.


Acknowledgements
We thank Nutrition Horizons (Lewisburg, OH) for contributions of product and financial support for this
project. Appreciation also is expressed to Lamont and Leigh Ann Ennis from Southern Cattle Company for
providing the cows for the experiment.















Table 1. Nutrients analysis for placebo (Control) and NHG1 boluses
used to treat donor cows prior to super-stimulation.
Treatment'
Components NHG1 Control
--------- DM --------
Crude fat, % 40.2 4.1
Calcium, % 0.32 0.13
Phosphorous, % 0.94 0.97
Potassium, % 1.93 1.01
Sodium, % 0.335 0.035
Sulfur, % 0.24 0.15
Iron, ppm 145 271
Zinc, ppm 1,050 122
Manganese, ppm 19 152
'Donors received six Grade OneTM boluses (13 g/capsule) containing a
blend of bioactive peptides and oligosaccharides (NHG1) or donors
received 6 placebo capsules (Control).


Table 2. Effect of nutrient supplementation on embryo recovery characteristics in
superovulated cows.
Treatments'
Item NHG1 Control SEM P-value
Number of donors 35 37
Total embryos/ova, no.2 15.0 12.4 1.80 0.30
Transferable embryos, no. 5.2 4.5 1.09 0.64
Grade 1 embryos, no. 2.8 1.7 0.80 0.34
Grade 2 embryos, no. 2.5 2.8 0.47 0.58
'Donors received six Grade OneTM boluses (13 g/capsule) containing a blend of bioactive
peptides and oligosaccharides (NHG1) or donors received 6 placebo capsules (Control). SEM =
standard error mean.
2Total unfertilized, degenerate, and transferable structures recovered.











Detect estrus and Al
at estrus and 12 h
later 0


t t t t
d4 d5 d6 d7+(2x
PGF (2x)


Embryo
collection
O


d 15 or 16


FSH decreasing dose 12 h apart


STwo placebo boluses or boluses containing NHG1 were inoculated
directed into the rumen: each application contained 2 boluses (total = 6
boluses per cow)


Figure 1. Schematic of experimental design for cows treated with or without NHG1 on d 0, 4
and 5.


60

50

w 40
(u
OW 30
20

10


0 -1


T T


Transferable
embryos


Unfertilized

Category


*NHG1
* Control


Degenerate


Figure 2. Effect of nutritional supplement on embryo classification


t
dO
EB + P4




















100


80


60


40


20


0


* NHG1
* Control


Grade 1 Grade 2
Transferable embryos


Figure 3. Effect of supplement on embryo grade. abPercentage tended to differ
(P = 0.06) cdPercentage differs (P < 0.05).


I




Full Text

PAGE 1

Summary We determined whether supplementation of a blend of bioactive peptides and oligosaccharides to support immune function (Nutrition Horizons 6,962,718) would alter quality, stage, and fertilization rate of embryos re covered after superovulation. Cows were superovulated using follicle stimulating hormone as NIH FSH P1 and were stratified by breed before random assignment to treatment: 1) donors received six a blend of bioact ive peptides and oligosaccharides (NHG1; n = 35); or 2) donors received 6 placebo capsules (13 g/capsule; Control; n = 37). Superovulation of donors was initiated by insertion of a c ontrolled internal drug release device (CIDR) on d 0, eight injections of follicle stimulating hormone (FSH) administered at 12 h intervals initiated on d 4, plus two injections of prostaglandin F (PGF) 12 h apart on d 7. At 0 and 12 h after detected estrus cows received an artificial insemination (AI). Boluses were inserted i nto the esophagus utilizing a balling gun. Cows received two boluses at CIDR insertion (d 0), at the first (d 4), and third (d 5) injection of FSH. Embryos were collected 7 d after first detected estrus and were recovered by nonsurgical embryo collection p rocedure. The embryos were evaluated and classified by stage and quality. Total ova (14.1 1.8) and transferable embryos (5.2 1.1) per flush for NHG1 and Control did not differ. Mean number of grade 1 (2.5 0.8) and 2 (2.7 0.5) were similar between treatments and no differences existed between treatments for degenerate (1.9 0.4) or unfertilized ova (7.0 1.4). However, the percentage of grade 1 embryos collected compared to recovered transferable embryos was greater (P = 0.06) for NHG1 (39.4%) than Control (23.4%). In addition, the percentage of grade 2 embryos collected compared to recovered transferable embryos was greater (P < 0.05) fo r Control (76.6%) than NHG1 (59.9%). We conclude that the number of transferable embryos collected per flush did not differ between treatments; however, the quality of transferable embryos was improved after embryo donor cows received NHG1 prior to embryo collection. Introduction Embryo transfer has been developed to increase the number of offspring from a superior genetic donor. The technique enhances the opportunity to create multiple offspring from a genetically superior cow rather than a single offspring per year. The results are accomplished by using recipients (regular genetic cow/heifers with good maternal ability) that serve as the surrogate for the embryo collected from the donor cow. Donors are selected based on an outstanding characteristi c that results in more value such as Embryo Quality Characteristics from Superovulated Cows Receiving a Blend of Bioactive Peptides and Oligosaccharides to Support Immune Guilherme Marquezini, Vitor Mercadante, Mindy Ward, Audy Spell, Joel Carter Neil Paton, G. Cliff Lamb 1 Treatment of embryo donor cows with NHG1 stimulation protocol did not alter the absolute number of embryos collected but it increased the quality of the embryos by increasing the percentage of grade 1 embryos relative to the number of transferable embryos harvested. 1 North Florida Research and Education Center, University of Florida, Marianna, FL

PAGE 2

greater fertility, milk production, heavier calves, disease resistance, etc. Recipients usually are selected for their maternal ability such as milk production and ability to nurture their young, along with good tempera ment. Physiologically, donors are super stimulated with natural hormones that act to recruit more than one regular follicle, increasing the likelihood that multiple follicles become dominant, ovulate, and may be fertilized at the same time. Donors are flu shed 7 d after insemination to remove the fertilized embryos. Embryos are then either frozen or transferred fresh to recipients that were previously synchronized to be at the same stage of the estrous cycle of the donor. T he development of the non surgica l embryo transfer technique, pushed the embryo transfer (ET) industry to significantly increase the number cattle derived from ET. Conception rates of more desirable quality embryos (excellent or good) are greater than embryos qualified as fair or poor qua lity (IETS, Savoy, IL); therefore, conception rates would increase if a greater percentage of grade 1 (excellent) embryos are harvested and transferred after collection. Research in the field of ET is very expensive and many of the basic procedures were established some years ago. Consequently, little academic research currently is being conducted that is likely to immediately benefit the commercial ET industry. Because ET success rates in well managed cattle herds are generally quite high, most recent im provements involve rather small, albeit important, increments. In a have been no significant improvements in techniques for the superovulation of cattle in the This statement remains largely true today (Hasler, 2010) I n fact, d ata provided by both the American and Canadian Embryo Transfer Associations indicate that mean embryo yields per donor range from 5 to 7 and basically have not changed for many years. The se embryo means are a composit e average of individual means of both beef (6.6) and dairy cattle (5.7) flushed by the certified members of the AETA. (NHG1), was originally developed to provide formulations of immune factors in combination with minerals, antioxidants, amino acids, and other nutraceuticals administered orally to treat animals exhibiting disease symptoms but also to lower general morbidity. Due to its ability to improve immune function, NHG1 may possibly improve uterine health and embryo quality. Materials and Methods The experimental was designed as a completely randomized design. Seventy two cows underwent a super stimulation protocol with follicle stimulating hormone ( FSH ) prior to embryo collection. Cows were superovulated using FSH as NIH FSH P1 and were stratified by breed before random assignment to treatment: 1) donors received 6 boluses (13 g/capsule) containing a blend of bioactive peptides and oligosaccharides (NHG1; n = 35); or 2) donors received 6 placeb o capsules (13 g/capsule; Control; n = 37). Superovulation of donors was initiated by insertion of a CIDR on d 0, eight injections of FSH administered at 12 h intervals initiated on d 4, plus two injections of PGF 12 h apart on d 7. At 0 and 12 h after det ected estrus cows received an AI. Boluses were inserted into the esophagus utilizing a balling gun. Cows received two boluses at CIDR insertion (d 0), at the first (d 4), and third (d 5) injection of FSH (Figure 1). Embryos were collected 7 d after first d etected estrus and were recovered by nonsurgical embryo collection procedure. Embryos were evaluated under a stereomicroscope and classified by stage and quality according to standards set forth by the International Embryo Transfer Society (d evelopmental s tage codes were: 3 = early morula; 4 = morula; 5 = early blastocyst; 6 = blastocyst; and 7 = expanded blastocyst). Quality codes were: 1 = symmetrical and spherical embryo mass with individual blastomeres that were uniform in size, color, and density with at least 85% of the cellular material intact (excellent or good); 2 = moderate irregularities in overall shape of embryonic mass or in size, color and density of individual cells

PAGE 3

with at least 50% of the cellular material intact (fair); 3 = major irregul arities in shape of the embryonic mass or size, color and density of individual cells with at least 25% of the cellular material intact (poor); 4 = dead or degenerating; and 5 = unfertilized. Results Table 1 represents the nutrients analysis of the placebo and NHG1 boluses. Overall mean total ova (14.1 1.8) and transferable embryos (5.2 1.1) per flush for NHG1 and Control did not differ ( P > 0.05; Table 2). Mean number of grade 1 (2.5 0.8) and 2 (2.7 0.5) were similar ( P > 0.05) between treat ments and no differences ( P > 0.05) existed between treatments for degenerate (1.9 0.4) or unfertilized ova (7.0 1.4; Figure 2). However, the percentage of grade 1 embryos collected compared to recovered transferable embryos tended ( P = 0.06) to be greater for NHG1 (39.4%) than Control (23.4%; Figure 2) In addition, the percentage of grade 2 embryos collected compared to recovered transferable embryos was greater ( P < 0.05) for Control (76.6%) than NHG1 (59.9%; Figure 3). We conclude that the number of transferable embryos collected per flush did not di ffer between treatments; however, the quality of transferable embryos was improved after embryo donor cows received NHG1 prior to embryo collection. Literature Cited Hasler. 1992. J. Dairy Sci. 75:2857. Hasler. 2010. Proc. Applied Reproductive Strategies in Beef Cattle, Nashville, TN pp. 265. Acknowledgements We thank Nutrition Horizons (Lewisburg, OH) for contributions of product and financial support for this project. Appreciation also is expressed to Lamont and Leigh Ann Ennis from Southern Cattle Company for providing the cows for the experiment.

PAGE 4

Table 1. Nutrients analysis for placebo (Control) and NHG1 boluses used to treat donor cows prior to super stimulation. Treatment 1 Components NHG1 Control --------DM -------Crude f at, % 40.2 4.1 Calcium, % 0.32 0.13 Phosphorous, % 0.94 0.97 Potassium, % 1.93 1.01 Sodium, % 0.335 0.035 Sulfur, % 0.24 0.15 Iron, ppm 145 271 Zinc, ppm 1,050 122 Manganese, ppm 19 152 1 blend of bioactive peptides and oligosaccharides (NHG1) or donors received 6 placebo capsules (Control). Table 2. Effect of nutrient supplementation on embryo recovery characteristics in superovulated cows Treatments 1 Item NHG1 Control SEM P value Number of donors 35 37 Total embryos/ova, no. 2 15.0 12.4 1.80 0.30 Transferable embryos, no. 5.2 4.5 1.09 0.64 Grade 1 embryos, no 2.8 1.7 0.80 0.34 Grade 2 embryos, no. 2.5 2.8 0.47 0.58 1 peptides and oligosaccharides (NHG1) or donors received 6 placebo capsules (Control). SEM = standard error mean. 2 Total unfertilized, degenerate, and transferable structures recovered.

PAGE 5

Figure 1. Schematic of experimental design for cows treated with or without NHG1 on d 0, 4 and 5. Figure 2. Effect of nutritional supplement on embryo classification

PAGE 6

Figure 3. Effect of supplement on embryo grade. ab Percentage tended to differ ( P = 0.06) cd Percentage differs ( P < 0.05).