dr. jason ross - understanding the biology of seasonal infertility to develop mitigation strategies...
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Understanding the Biology of Seasonal Infertility in Swine to Develop Mitigation Strategies
Jason W. Ross, Aileen F. Keating and Lance H. BaumgardJune 8th, 2016
Iowa State UniversityIowa Pork Industry Center
Heat Stress and Seasonal Infertility
Infertility, reduction in reproductive efficiency Predictable dip in performance: Seasonal Infertility Coincides with increased environmental temps
Service, July through September Reduced farrow rates, November-December
Multiple proposed causes of seasonal infertility Environmental heat- heat stress Seasonality- melatonin and light sensitivity Other unrecognized factors or combination of factors
In Vitro Heat Stress Model
Heat Stress TreatmentsControl - 39°C for 42 hr
HS1 - 41°C for 42 hr
HS2 - 39°C for 21 hr, 41°C for 21 hr
HS3 - 41°C for 21 hr, 39°C for 21 hr
GV oocytes
MII Oocytes (42 hr) Denuded MII Oocyte
Understanding the Biology of Seasonal Infertility
Heat Stress Alters Oocyte Maturation Rates
P = 0.01
• 41°C had a greater negative impact during the first 21 hr of maturation.
Control = 39/39 HS1 = 41/41 HS2 = 39/41 HS3 = 41/39
Heat Stressed Oocytes Are Still Capable of Development to the 4-cell Stage
P = 0.33
• Ability to achieve 4-cell stage of embryonic development was not significantly effected by HS during IVM
Control = 39/39 HS1 = 41/41 HS2 = 39/41 HS3 = 41/39
Heat Stressed Oocytes Have Decreased Development to Blastocyst Stage
a
a
b
c
P < 0.05
Control = 39/39 HS1 = 41/41 HS2 = 39/41 HS3 = 41/39
• HS during IVM negatively impacts blastocyst formation rate
HS could indirectly impact ovarian function and fertility through…. Circulating insulin increased
during HS Insulin receptor (IR) is dysregulated
in the ovary and developing oocyte
Increased circulating endotoxin observed during HS Premature embryonic loss Could alter follicle
development through TLR4 signaling
Pearce 2014, Thesis
Nteeba et al., 2015Biology of Reproduction
Determine if physiological indicators of HS during WEI are associated with reproductive performance in commercial swine production facilities Wean to Estrus Interval (WEI) AI Service Rate Farrow Rate (FR) Litter Size
Experimental Objectives
Reduced reproductive efficiency during seasonal infertility is associated with HS during the WEI
Hypothesis
Materials and Methods
Commercial P1 Sows, n=869 Peak seasonal infertility
Summer: July-August 2013 (n=450) Calendar Year Weeks: 29-32 Outside Temp: Hi:34°C Lo:15°C
Peak reproductive performance Spring: March-April 2014 (n=419) Calendar Year Weeks: 12-15 Outside Temp: Hi:16°C Lo:-1.5°C
Materials and Methods
Physiological responses to HS Rectal Temperature (Tr)
Digital thermometer Skin Temperature (Ts)
Infrared surface temperature Respiratory Rate (RR)
Collected on sows at rest Flank movements per 15 s, x 4
Collected 5x daily, 7 d post wean Serum samples collected, d1 and 3 of WEI New group of sows each week of study
Production Data Collection
P1 gestation outcome litter data provided Values reviewed
P1 Litter Born alive Weaned
Wean Date Date Serviced Service Result Total and Live Born, P2 Stillborn and Mummies, P2
Lack of Correlation Between HS Indicators and Production
WEI Rectal Temp Skin Temp Resp.
Rate Total Born Live Born
WEI
Corr. Coeff.
0.0731 -0.12332 -0.12218 -0.07518 -0.06845
P value 0.0335 0.0003 0.0004 0.044 0.0668n 846 846 846 718 718
Rectal Temp
-0.03086 0.38907 0.01241 -0.00564 0.3644 <.0001 0.7405 0.8802 866 866 715 715
Skin Temp 0.00002 -0.04395 -0.02985 0.9995 0.2405 0.4254 866 715 715
Resp. Rate 0.10231 0.11912 0.0062 0.0014 715 715
Rectal Temperature during WEI Not Affected by Season
Respiration Rate during WEI Not Affected by Season
Skin Temperature during the WEI was Reduced in Summer
Reproductive Performance
Spring Summer SEM P
Farrowing Rate1 89.40% 79.95% 1.72 < 0.01
Total Born*, pigs 13.66 13.81 0.23 0.68
Born Alive*, pigs 12.62 12.66 0.23 0.90
Born Still*, pigs 0.67 0.79 0.06 0.23
Mummies*, pigs 0.37 0.36 0.06 0.891 Farrowing Rate, of all sows enrolled in trial, Spring n= 419, Summer n= 450*Litter data from sows with P2 litter data, Spring n= 371, Summer n= 347
Increased WEI during Summer
*All P1 sows enrolled in trial, Spring n = 419, Summer n= 450
Spring Summer
n 419 450
Av. WEI, ≤7 d 4.14 4.24
Serviced 88.3 % 76.7%n 370 345
Farrow Rate 91% 82%
Av. WEI, > 7 d 24.4 26.2
Serviced 10.7% 19.8%n 45 89
Farrow Rate 75.6% 70.8%
No Heat 1.0% 3.6%
WEI and FR by Season
Spring Summer
n 419 450
Av. WEI, ≤7 d 4.14 4.24
Serviced 88.3 % 76.7%n 370 345
Farrow Rate 91% 82%
Av. WEI, > 7 d 24.4 26.2
Serviced 10.7% 19.8%n 45 89
Farrow Rate 75.6% 70.8%
No Heat 1.0% 3.6%
WEI and FR by Season
Production Cost of Seasonal Infertility
Per 100 P1 Sows Weaned
Seasonal Infertility • 224 piglets are not produced from AI service
during a normal <7 d WEI
• Reduced to 140 pigs not produced• Recovered pregnancies via opportunity
sows but includes additional 210 non-productive sow days
Circulating Insulin and LBP during Seasonal Infertility Sample Selection Serum samples, d 1 and 3 of WEI
ELISA, circulating levels Insulin Lipopolysaccharide Binding Protein (LBP)
Sample Selection 40 sows per season
15 successful/farrowed (WEI 4-5 days) 25 unsuccessful
15 PCN, return, abort, etc. (WEI 4-5 days) 10 WEI>15 days (All farrowed)
No Difference in Circulating Insulin
No Difference in Circulating LBP
P > 0.1
Summary of Commercial Study
Significant differences in reproductive performance Wean to Estrus interval Farrowing Rate
No difference in physiological indicators of HS during WEI Body temperature regulation, Tr, RR No difference in circulating Insulin or LBP
Understanding the ‘tolerance’ of heat stress in gilts
OBJECTIVE: To determine if HS tolerance early in life
is predictive of reproductive success during HS
HYPOTHESIS:Gilts susceptible to HS are more likely to
demonstrate reduced reproductive success during HS
Phase I: Gilt Selection
24 hours 24 hours
Acclimation:TN (22±0.5°C, 62±13% RH), ad libitum
TN period:ad libitum
HS period:HS (30±1°C, 49±8% RH), ad libitum
Body Weights Body Weights & Blood Samples
Body Weights & Blood Samples
235 gilts (PIC maternal x Duroc terminal sire)
5 reps of 48 animals Daily feed intake (FI)
Thermal indices 32 time points per rep Respiration Rate (RR) Skin Temperature (TS) Rectal Temperature (TR)
TR by Hour
x
x
40.0
39.0
HS TR does not explain decreased FI
R2 = 0.001
R2 = 0.01
HS FI x = 1.77
TN FI x = 2.48
ΔBW is not associated with HS TR
R2 = 0.03
ΔBW is not explained by HS FI
R2 = 0.09
Tolerant and Susceptible Classification
Tolerant and Susceptible Classification
Tolerant and Susceptible TR
40.6
40.3
40.0
39.7
39.4
39.2
38.9
38.6
38.3
38.1
HS TN
*
** P < 0.05
Acclimation:
TN (21°C), 12 d
Gestational period:TN (21°C), 43-48 d
Start of Matrix Supplementation (220 d old)
Sacrifice & Uterine Tracts Removed
End of Matrix, start of HS & estrus detection/breeding
TN period:
2 dHS period:
Cyclical HS, 9 d
End of HS & breeding
Phase II: HS During the Follicular Phase
Tolerant (T; n=48) & Susceptible (S; n=48)
Estrus detection Beginning ~160 d of age Ending ~220 d of age
HS period Cyclical conditions
1st d (21 to 29°C) 2nd d (21 to 31°C) 3rd d (21 to 33°C) Remaining 6 d (21 to 35°C)
Limit fed 2.7 kg feed / dPregnancy Validation
Estrus detection 18-21 d later Ultrasound 36 d of pregnancy
Tissue and Fetal Analysis Total uterine tract weight Fetal count, weight, and crown-rump length Ovary weights & corpus lutea (CL) diameters
Statistical Analysis SAS 9.3; PROC CORR
Phase II: HS During the Follicular Phase
Variation in Thermoregulation between pigs is repeatable
3-4 months of age8 months of age
Phase I Phase II
Fetal count is not associated with HS TR during estrus and breeding
38.3 38.6 38.9 39.2 39.4 39.7 40.0 40.3 40.6
R2 = 0.01
Fetal size was increased in susceptible compared to tolerant gilts
P = 0.007 P = 0.002
Fetal size is correlated with HS TR during estrus and breeding
38.3 38.6 38.9 39.2 39.4 39.7 40.0 40.3 40.6
Phase II HS TR (°C)
Corpus lutea count is not associated with HS TR during estrus and breeding
38.3 38.6 38.9 39.2 39.4 39.7 40.0 40.3 40.6
Corpus luteum diameter is not explained by HS TR during estrus and breeding
38.3 38.6 38.9 39.2 39.4 39.7 40.0 40.3 40.6
Embryo survivability is not associated with HS TR during estrus and breeding
R2 = 0.003P = 0.31
38.3 38.6 38.9 39.2 39.4 39.7 40.0 40.3 40.6
Pre-pubertal TN TR is correlated with post-pubertal TN TR
38.6 38.9 39.2 39.4 39.7 40.0 40.3 40.6
38.938.838.738.6
38.338.2
37.9
38.4
37.8
38.0
38.1
Pre-pubertal HS TR is correlated with post-pubertal HS TR
40.6 40.8 41.1
39.7
39.4
38.9
38.6
39.2
38.1
38.3
38.3 38.6 38.9 39.2 39.4 39.7 40.0 40.3
Pre-pubertal TN TR is correlated with post-pubertal HS TR
38.3 38.6 38.9 39.2 39.4 39.7 40.0 40.3
39.7
39.4
38.9
38.6
39.2
38.1
38.3
Genome Wide Association Study
Conclusions
HS TR is not associated with FI and ΔBW during acute HS
HS TR does not explain decreased production
Fetal size is associated with HS TR during estrus and breeding
HS TR response in pigs following an exposure early in life is indicative of the HS TR response later in life
Prenatal Programming of Postnatal Problems
Does in utero heat stress impact productivity? Intrauterine Growth Retardation Models
Reduced blood flow/placental function during different phases of gestation has negative implications on a variety of postnatal parameters
Maternal Stress Alters the behavior of offspring Disrupts the Hypothalamic-Pituitary-Adrenal Axis
Endocrine Disruption during Pregnancy Pregnancy Loss Postnatal endocrine dysfunction Diethylstilbestrol (DES)
Does HS during Gestation Impact Postnatal Performance?
4 Gestational Treatments 2 Postnatal Treatments TN (21°C), HS (35°C) 24 h or 5 weeks of TN
or HS conditions beginning at 12 or 14 wks. of age
n=6 per gestational treatment * postnatal treatment combination
Treatment
1st Half of Gestation
2nd Half of Gestation
TNTN thermo-neutral thermo-neutral
TNHS thermo-neutral heat stress
HSTN heat stress thermo-neutral
HSHS heat stress heat stress
Effect of Gestational HS on Ultrasound Parameters at 12 Weeks of Age
P = 0.013 P = 0.39
Boddicker et al., 2014
Prenatal HS and Postnatal HS Impact Back Fat Thickness (19 weeks of age)
Postnatal Effect P = 0.03
Gestation Effect P = 0.04 Boddicker et al., 2014
Gestational Environment Impacts Postnatal Insulin Levels
P = 0.09P = 0.01
HS during first half of gestation results in elevated insulin during postnatal growth and development Boddicker et al., 2014
Experiment Two-Serial Slaughter General Procedure
2 trials: Lean (30 to 60 kg) and Lipid (60 to 90 kg) accretion
Pigs from in-utero TNTN and HSHS conditions Subjected to:
Postnatal TN (12 TNTN, 12 HSHS; 22 C) Postnatal HS (12 TNTN, 12 HSHS; 34 C)
Pigs sacrificed at 30, 60 , and 90 kg BW Carcasses ground and chemical composition (N, lipid,
ash, GE) determined Deposition rates/d determined
Johnson et al., 2015
Body Composition Trial:Lipid Accretion Phase
Carcass Analysis DataEnvironment P
Parameter TNTN-TN HSHS-TN TNTN-HS HSHS-HS G P G x PProtein % 17.1 16.6 17.6 16.8 0.01 0.04 0.46Fat % 19 20.8 19 20.4 0.11 0.81 0.82Protein/d (g) 173 155 141 110 0.01 0.03 0.57Fat/d (g) 248a 335a 192b 241b 0.09 0.03 0.55Fat : Protein 1.32a 2.54b 1.26a 2.43b 0.01 0.73 0.93
Johnson et al., 2015
Body Composition Trial:Lipid Accretion Phase
Carcass Analysis DataEnvironment P
Parameter TNTN-TN HSHS-TN TNTN-HS HSHS-HS G P G x PProtein % 17.1 16.6 17.6 16.8 0.01 0.04 0.46Fat % 19 20.8 19 20.4 0.11 0.81 0.82Protein/d (g) 173 155 141 110 0.01 0.03 0.57Fat/d (g) 248a 335a 192b 241b 0.09 0.03 0.55Fat : Protein 1.32a 2.54b 1.26a 2.43b 0.01 0.73 0.93
P = 0.01
P = 0.04P = 0.03
P = 0.01
Body Composition Trial:Lipid Accretion Phase
Carcass Analysis DataEnvironment P
Parameter TNTN-TN HSHS-TN TNTN-HS HSHS-HS G P G x PProtein % 17.1 16.6 17.6 16.8 0.01 0.04 0.46Fat % 19 20.8 19 20.4 0.11 0.81 0.82Protein/d (g) 173 155 141 110 0.01 0.03 0.57Fat/d (g) 248a 335a 192b 241b 0.09 0.03 0.55Fat : Protein 1.32a 2.54b 1.26a 2.43b 0.01 0.73 0.93
P = 0.11
P = 0.03
P = 0.09
Body Composition Trial:Lipid Accretion Phase
Carcass Analysis DataEnvironment P
Parameter TNTN-TN HSHS-TN TNTN-HS HSHS-HS G P G x PProtein % 17.1 16.6 17.6 16.8 0.01 0.04 0.46Fat % 19 20.8 19 20.4 0.11 0.81 0.82Protein/d (g) 173 155 141 110 0.01 0.03 0.57Fat/d (g) 248a 335a 192b 241b 0.09 0.03 0.55Fat : Protein 1.32a 2.54b 1.26a 2.43b 0.01 0.73 0.93
P = 0.01 Johnson et al., 2015
Prenatal HS Impact on Postnatal Gene Expression
Chr Start End Gene Description TNTN TNHS HSTN HSHS P Value Q Value14 53394004 53402671 ENSSSCG000000100
77ENSSSCG00000010077 1.00 2.34 4.11 4.75 1.68E-03 3.39E-02
4 464087 469224 SLC39A4 Solute carrier family 39 (zinc transporter), member 4
1.00 1.90 1.79 3.85 2.36E-05 2.29E-03
1 313854923 313859160 FAM69B Family with sequence similarity 69, member B 1.00 1.64 1.41 3.69 1.15E-05 1.53E-0312 54329874 54330888 VMO1 Vitelline membrane outer layer 1 homolog
(chicken)1.00 2.62 1.68 3.47 1.66E-04 8.62E-03
6 50296129 50296209 RPL13A Ribosomal protein L13a 1.00 1.66 1.67 3.38 2.87E-07 1.30E-042 77485667 77487843 CFD Complement factor D (adipsin) 1.00 1.67 1.53 3.38 2.49E-03 4.39E-027 122228313 122229025 IFI27L2 Interferon, alpha-inducible protein 27-like 2 1.00 1.07 1.68 3.31 1.22E-05 1.58E-035 66307078 66319269 LEPREL2 Leprecan-like 2 1.00 1.88 1.66 3.10 1.31E-04 7.66E-033 9925863 9927499 HSPB1 Heat shock 27kDa protein 1 1.00 1.48 1.36 3.04 9.48E-08 7.15E-05
Chr Start End Gene Description TNTN TNHS HSTN HSHS P Value Q Value8 522342 524469 WHSC2 Wolf-Hirschhorn syndrome candidate 2 1.00 3.50 1.62 4.91 2.58E-03 2.90E-022 142435 143584 TSPAN4 Tetraspanin 4 1.00 2.65 2.02 4.21 1.17E-04 6.75E-031 314009381 314012067 ENTPD8 Ectonucleoside triphosphate diphosphohydrolase 8 1.00 2.87 1.90 3.98 2.43E-03 2.84E-026 45233203 45234396 B9D2 B9 protein domain 2 1.00 2.31 1.85 3.88 6.89E-03 4.88E-026 45290992 45292185 BCKDHA Branched chain keto acid dehydrogenase E1, alpha
polypeptide1.00 2.31 1.85 3.88 6.89E-03 4.88E-02
3 9925863 9927499 HSPB1 Heat shock 27kDa protein 1 1.00 2.12 1.70 3.28 3.66E-04 1.11E-02
Chr Start End Gene Description TNTN TNHS HSTN HSHS P Value Q Value3 41631025 41633605 NUBP2 Nucleotide binding protein 2 (MinD
homolog, E. coli)1.00 2.36 0.92 5.08 2.98E-03 2.61E-02
1 314032464 314078885 EXD3 Exonuclease 3'-5' domain containing 3 1.00 2.01 1.73 4.87 3.34E-03 2.73E-022 18179200 18180435 CHST1 Carbohydrate (keratan sulfate Gal-6)
sulfotransferase 11.00 2.01 1.51 4.48 1.31E-04 6.35E-03
1 303105455 303107386 ZDHHC12 Zinc finger, DHHC-type containing 12 1.00 2.6 1.25 4.25 6.14E-06 2.82E-033 41659587 41660822 NME3 Non-metastatic cells 3, protein expressed
in1.00 2.01 0.88 3.82 8.38E-03 4.88E-02
3 40892304 40898426 MPG N-methylpurine-DNA glycosylase 1.00 2.83 1.19 3.67 4.36E-05 4.15E-033 42707012 42716475 AMDHD2 Amidohydrolase domain containing 2 1.00 1.78 1.61 3.38 4.34E-03 3.22E-022 5541055 5541663 FOSL1 FOS-like antigen 1 1.00 2.17 0.89 3.27 1.83E-03 2.04E-02
Longissimus Dorsi
434 differentially expressed
genes
Adipose
925differentially expressed
genes
Liver
418differentially expressed
genes
Gest*Sex P = 0.0011
Gest P = 0.01
Gest P = 0.0063
Post P = 0.0078
a
b
Gest*Post P = 0.05
Gest*SexP = 0.01
Gest*SexP = 0.03
a
bc bcbc bc
bcac
c
aa
ab abab
bc
a aa
c
a a
b
a
TNTN TNHS HSTN HSHSTN HS TN HS TN HS TN HS
HIF1α~150 kDa
HIF1αcleavage product25 kDa
HSF1~85 kDa
HSP7070 kDa
MEF2A55 kDa
MYOD140 kDa
170 kDa
130 kDa
35 kDa
15 kDa
100 kDa
70 kDa
100 kDa
55 kDa
70 kDa
40 kDa
55 kDa
35 kDa
Prenatal HS alters postnatal protein abundance
Summary Points Heat stress has direct negative effects on oocyte and early embryo
development in pigs. Despite a lack of physiological heat stress in P1 sows, seasonal infertility
significantly compromised production efficiency. The thermoregulatory response to heat stress is highly variable between gilts
but appears ‘fixed’. Prenatal exposure to heat stress could compromise the efficiency of pork
production. Reduced lean tissue accretion rate
Protein contributing to a lower percentage of whole body composition and increased ratio of lipid to lean accretion as a result of gestational HS environment
Acknowledgements
Jacob Seibert Beth Hines Kody Graves Rebecca Boddicker Jay Johnson Matt Romoser Aileen Keating Baumgard, Gabler and
Ross Lab Groups
AFRI PI Group Lance Baumgard Nick Gabler John Patience Steven Lonergan Joshua Selsby Rob Rhoads Matt Lucy Tim Safranski
Max Rothschild and Kwan-Suk Kim