Improved Bovine Embryo Production Using Novel In...
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Improved Bovine Embryo Production Using Novel In Vitro Culture Systems J. H. Pryor 1 , J. F. Hasler 2 , L. Strøbech 3 , B. Avery 3 , N. Hashem 3 , S. Menges 1 , C. R. Long 4 , G. Shewfelt 5 , C. R. Looney 1 1 Ovagenix, Bryan, TX; 2 Vetoquinol, Fort Worth, TX; 3 Embryo Trans Biotech, Frederiksberg, Denmark; 4 Texas A&M University, Department of Veterinary Physiology and Pharmacology, College Station, TX; 5 Partnar Animal Health, Port Huron, Michigan INTRODUCTION • Testing new media conditions for embryo production is an essential component for improving in vitro development • The objective of this study was to compare media used in two bovine embryo production systems (Control and Embryo Trans Biotech: ETB). MATERIALS & METHODS Experiment 1: Abattoir-procured oocytes were matured, fertilized and cultured in the following media under a 5% CO 2 , 5% O 2 , 90% N 2 in a 38.5 o C humidified atmosphere: Control: • Maturation (IVM) - Medium 199 with Earles salts supplemented with 10% fetal bovine serum, 1% Penicillin/Streptomycin, 0.2 mM sodium pyruvate, 2 mM L-Glutamine, and 5.0 μg mL -1 of Folltropin ® -V. • Fertilization (IVF) - 500 μl pre-equilibrated modified Tyrode-lactate medium (Pryor et al. 2011, Therio. 75, 24-33). • In vitro Culture (IVC) - Seventeen hours post- insemination, presumptive zygotes were cleaned of cumulus cells and cultured in Bovine Evolve supplemented with 4 mg mL -1 of Probumin BSA under oil for 7 days (8 days post-IVF) where cleavage and viability rates were assessed (Table 1, Fig. 1 & 2). ETB: • IVM – ETB BO-IVM • IVF – ETB BO-IVF • IVC – ETB BO-IVC Experiment 2: Same protocol as above except a modified ETB BO-IVC was used (ETB mod.). Cell Counts: Embryos were fixed in cold methanol, washed in PBS/0.1% Tween 20 and mounted in 10 μg mL -1 Hoechst/glycerol to stain the nuclei. Cell counts were performed manually at 200X using UV light microscopy (Fig. 3 & 4). Statistical Analysis: Percentage data were transformed using arcsine square root function prior to analysis and means compared using a Student’s t- test; alpha = 0.05. • Additionally, we also evaluated the media effects on C quality oocytes (1 layer of cumulus cells; n=205), which were evenly divided between Control vs. ETB over 5 replicates. Due to the lower numbers, Chi Square analysis was performed to compare outcomes (Fig. 2). RESULTS • Experiment 1: No differences in rates of cleaved or viable embryos were observed between treatment groups (Fig. 1, Table 1). • Experiment 2: • The modified version of ETB produced an increase in viable embryos compared to Control (Fig. 1, Table 1). • Mean cell counts for viable embryos were significantly different following culture in modified ETB (Fig. 3). • Embryo viability decreased in the Control media but were maintained using the modified ETB between experiments with seasonal temperatures ranging from 23.8 o C (Exp1) to 33.8 o C (Exp2). • Viability rates for poor quality oocytes were significantly higher for ETB than Control across both experiments (Fig. 2). CONCLUSION • ETB’s modified BO-IVC media produced more higher quality embryos under varying conditions, produced higher cell counts for BL and enhanced the rate of development compared to Control. • Continued research is underway to ascertain pregnancy rates following fresh or frozen/thaw embryo transfer. ACKNOWLEDGMENTS The authors acknowledge and appreciate the financial support from Vetoquinol. Fig. 4 Representative day 8 hatched blastocysts stained for nuclei counts (200X magnification). A). Control, 150 nuclei. B). ETB, 250 nuclei. A B 40 μm 40 μm Table 1. In vitro development of bovine embryos at d8 post-IVF for Control vs. ETB systems. a,b,c Values in a column and within experiment with different superscripts were different, P<0.05. Experiment Treatment (n) % Cleaved % Viable % H BL % HBL/Expand ed BL 1 Control 193 81.0 a 42.9 a 9.3 a 39.3 a 1 ETB 206 80.5 a 48.4 a 11.6 a 36.8 a 2 Control 211 80.5 b 29.2 b 5.8 b 20.5 b 2 ETBmod 216 82.8 b 51.9 c 23.9 c 45.8 c 127.0 ±6.7 n = 49 162.7 ±5.7 n = 107 Fig. 3 Total nuclei counts for d8 post-IVF bovine embryos with ±SEM. *** Indicates significant difference, P<0.05 Fig. 1 Comparison of bovine embryo cleavage and viability rates at d8 post-IVF in Experiments 1 & 2. a,b Comparisons within experiment with different superscripts were different, P<0.05. 0 10 20 30 40 50 60 70 80 90 Control (n=193) ETB (n=206) 81 81 43 48 % Percentage Experiment 1 0 10 20 30 40 50 60 70 80 90 Control (n=211) ETB mod. (n=216) 81 83 29 52 % Percentage Experiment 2 % Cleaved % Viable Fig. 2 Comparison of bovine embryo cleavage and viability rates from poor quality oocytes (1 layer of cumulus cells) at d8 post-IVF in Experiments 1 & 2. a,b Comparisons within experiment with different superscripts were different, P<0.05. 0 10 20 30 40 50 60 70 Control (n=43) ETB (n=41) 62 44 19 44 % Percentage Experiment 1 0 10 20 30 40 50 60 70 80 90 Control (n=59) ETB mod. (n=62) 73 84 24 52 % Percentage Experiment 2 % Cleaved % Viable a a a a a b a a b a a a a a b a IETS 2016
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Transcript of Improved Bovine Embryo Production Using Novel In...
Improved Bovine Embryo Production Using Novel In Vitro Culture Systems
J. H. Pryor1, J. F. Hasler2, L. Strøbech3, B. Avery3, N. Hashem3, S. Menges1, C. R. Long4,G. Shewfelt5, C. R. Looney1
1Ovagenix, Bryan, TX; 2Vetoquinol, Fort Worth, TX; 3Embryo Trans Biotech, Frederiksberg, Denmark; 4Texas A&M University, Department of Veterinary Physiology and Pharmacology, College Station, TX; 5Partnar Animal Health, Port Huron, Michigan
INTRODUCTION• Testing new media conditions for embryo
production is an essential component forimproving in vitro development
• The objective of this study was to compare mediaused in two bovine embryo production systems(Control and Embryo Trans Biotech: ETB).
MATERIALS & METHODSExperiment 1: Abattoir-procured oocytes werematured, fertilized and cultured in the followingmedia under a 5% CO2, 5% O2, 90% N2 in a 38.5oChumidified atmosphere:
Control:• Maturation (IVM) - Medium 199 with Earles
salts supplemented with 10% fetal bovineserum, 1% Penicillin/Streptomycin, 0.2 mMsodium pyruvate, 2 mM L-Glutamine, and 5.0 µgmL-1 of Folltropin®-V.
• Fertilization (IVF) - 500 µl pre-equilibratedmodified Tyrode-lactate medium (Pryor et al.2011, Therio. 75, 24-33).
• In vitro Culture (IVC) - Seventeen hours post-insemination, presumptive zygotes werecleaned of cumulus cells and cultured in BovineEvolve supplemented with 4 mg mL-1 ofProbumin BSA under oil for 7 days (8 dayspost-IVF) where cleavage and viability rateswere assessed (Table 1, Fig. 1 & 2).ETB:
• IVM – ETB BO-IVM• IVF – ETB BO-IVF• IVC – ETB BO-IVC
Experiment 2: Same protocol as above except amodified ETB BO-IVC was used (ETB mod.).
Cell Counts: Embryos were fixed in cold methanol,washed in PBS/0.1% Tween 20 and mounted in 10μg mL -1 Hoechst/glycerol to stain the nuclei. Cellcounts were performed manually at 200X using UVlight microscopy (Fig. 3 & 4).
Statistical Analysis: Percentage data weretransformed using arcsine square root function priorto analysis and means compared using a Student’s t-test; alpha = 0.05.
• Additionally, we also evaluated the media effectson C quality oocytes (1 layer of cumulus cells;n=205), which were evenly divided betweenControl vs. ETB over 5 replicates. Due to thelower numbers, Chi Square analysis wasperformed to compare outcomes (Fig. 2).
RESULTS• Experiment 1: No differences in rates of cleaved
or viable embryos were observed betweentreatment groups (Fig. 1, Table 1).
• Experiment 2:• The modified version of ETB produced an
increase in viable embryos compared toControl (Fig. 1, Table 1).
• Mean cell counts for viable embryos weresignificantly different following culture inmodified ETB (Fig. 3).
• Embryo viability decreased in the Controlmedia but were maintained using the modifiedETB between experiments with seasonaltemperatures ranging from 23.8oC (Exp1) to33.8oC (Exp2).
• Viability rates for poor quality oocytes weresignificantly higher for ETB than Control acrossboth experiments (Fig. 2).
CONCLUSION• ETB’s modified BO-IVC media produced more
higher quality embryos under varyingconditions, produced higher cell counts for BLand enhanced the rate of developmentcompared to Control.
• Continued research is underway to ascertainpregnancy rates following fresh orfrozen/thaw embryo transfer.
ACKNOWLEDGMENTSThe authors acknowledge and appreciate thefinancial support from Vetoquinol.
Fig. 4 Representative day 8 hatched blastocysts stained
for nuclei counts (200X magnification). A). Control, 150
nuclei. B). ETB, 250 nuclei.
A B40
μm40
μm
Table 1. In vitro development of bovine embryos at d8 post-IVF for
Control vs. ETB systems. a,b,c Values in a column and within experiment with different
superscripts were different, P<0.05.
Experiment Treatment (n) % Cleaved % Viable % HBL
%
HBL/Expand
ed BL
1 Control 193 81.0a 42.9a 9.3a 39.3a
1 ETB 206 80.5a 48.4a 11.6a 36.8a
2 Control 211 80.5b 29.2b 5.8b 20.5b
2 ETBmod 216 82.8b 51.9c 23.9c 45.8c
127.0
±6.7
n = 49
162.7
±5.7
n = 107
Fig. 3 Total nuclei counts for d8 post-IVF bovine
embryos with ±SEM. *** Indicates significant difference,
P<0.05
Fig. 1 Comparison of bovine embryo cleavage and viability rates at d8 post-IVF in Experiments 1 & 2.a,b Comparisons within experiment with different superscripts were different, P<0.05.
0
10
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80
90
Control (n=193) ETB (n=206)
81 81
4348
% P
erc
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tag
e
Experiment 1
% Cleaved
% Viable
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Control (n=211) ETB mod. (n=216)
81 83
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% P
erc
en
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e
Experiment 2
% Cleaved
% Viable
Fig. 2 Comparison of bovine embryo cleavage and viability rates from poor quality oocytes (1 layer of cumulus
cells) at d8 post-IVF in Experiments 1 & 2.a,b Comparisons within experiment with different superscripts were different, P<0.05.
0
10
20
30
40
50
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70
Control (n=43) ETB (n=41)
62
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44
% P
erc
en
tag
e
Experiment 1
% Cleaved
% Viable
0
10
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70
80
90
Control (n=59) ETB mod. (n=62)
73
84
24
52
% P
erc
en
tag
e
Experiment 2
% Cleaved
% Viable
a a
a
a
a b
a
a
b
aa
a
a
a
b
a
IETS 2016
