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