1. POTENTIAL HORMONAL AND NUTRITIONAL STRATEGIES TO IMPROVE EMBRYO SURVIAL IN DAIRY CATTLE J.E.P. Santos, R. Cerri, S. Juchem, K. Galvão and R. Chebel Veterinary Medicine Teaching and Research Center University of California - Davis

2. INTRODUCTION  Reproductive performance affects the profitability of dairy operations.  Pregnancy rates is the most meaningful measurement of reproductive efficiency (Ferguson and Galligan, 1993)  Pregnancy rate is the product of estrous detection and conception rates (Macmillan, 1992).  PR  Speed at which eligible cows become pregnant

3. Pregnancy Rate (Survival Graph Analysis)  Examine effective VWP  Area above & below the curve  Estimate PR  Estimate proportion remaining open Pregnant Open Slope = PR VWP 30% open Overton, 2002

4. Value/Cow and Pregnancy Rate Different combinations of Heat and CR for a given PR. Galligan, Penn. Vet Conf., 1999 Annuity Value - The accumulated value corrected for the time value of money.

5. Costs Associated with Changes in Reproductive Efficiency Note: 8% PR ~ 18,000 RHA, Milk is per milking cow/day Overton, 2002

6. Control of Reproductive Indices  Voluntary waiting period

7. Effect of TAI on DIM at First Postpartum AI in a Dairy Herd TAI VWP

8. Control of Reproductive Indices  Voluntary waiting period  Estrus detection rate

9. High Estrus Detection Rate Can be Achieved with Aggressive Breeding Protocols

10. Control of Reproductive Indices  Voluntary waiting period  Heat detection rate  Conception rate Pregnancy Rate

11. High Estrus Detection Rate Can be Achieved with Aggressive Breeding Protocols CR = 35%

12. Control of Reproductive Indices  Voluntary waiting period  Heat detection rate  Conception rate  Pregnancy loss Pregnancy Rate

13. Rate of Late Embryonic Loss is Greater between Days 27 and 45 ReferenceCowsDays 1 st Check Days last Check DaysLoss % Loss/ Day Chebel et al., 2002a19528421417.91.28 Moreira et al., 2000a13927451820.71.15 Chebel et al., 2002b1,50331451413.20.94 Stevenson et al., 200020328451715.80.93 Santos et al., 2002b36031451411.10.79 Santos et al., 2002a220274114100.71 Cerri et al., 20021763145149.70.70 Juchem et al., 200216728391111.41.03 Fricke et al., 19988928562813.50.48 Moreira et al., 2000b21132744213.70.33 Santos et al., 200017128906217.40.28 Vasconcelos et al., 19971,60128987019.60.28 Pursley et al., 199828530280250220.09 0.9%

14. Oocyte Quality Progesterone Secretion Embryonic Development IFN-t secretion Successful Implantation  PGF 2  secretion hCG Uterine Environment PUFAs bST Gossypol

15. Effects of bST on Reproductive Functions in Dairy Cows  Follicle development  CL differentiation/P4 secretion  Fertilization/embryo development  Uterine environment PGF 2α secretion (bst vs IGF-I) Oviduct vs Endometrium

16. Estrus d 0 Recipient Cows Superovulation Artificial Insemination bSTControl Flushing Record results d 0 d 7 Donor Cows Freeze embryos Experimental Design Pregnancy diagnosis (rectal palpation) Embryo Transfer d 7 bSTControl d 1 Treat recipients

17. Unfertilized Degenerate P < 0.05 * Total 9.4 per flush 9.3 per flush Transferable 5.4 per flush 7.4 per flush 0 1 2 3 4 5 6 7 8 9 10 Control bST Number of Ova/Embryos per Flush for Control (n = 26) and bST (n = 26) Treated Donors (LSM + SE).

18. Exp Blast Blast Early Blast Morula bST Control 60 50 40 30 20 10 0 60 50 40 30 20 10 Frequency of distribution of transferable embryos among stages of development P < 0.001

19. Control 56.4 % (150/266) bST 77.2 % (206/267) 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 Percentage of transferable embryos relative to total ova/embryos flushed P < 0.001

20. 20 30 40 50 60 Control embryobST embryo Pregnancy rates (%) Control recipientbST recipient 25.6 % (11/43) 43.2 % (16/37) 43.3 % (26/60) 56.1 % (23/41) Pregnancy rates following embryo transfer for treatment groups

21. Oocytes Maturation Fertilization IgG (10  g/ml) Anti-IGF-I Control Control Trt 1 Trt 4 Trt 7 GH (100 ng/ml) Trt 2 Trt 5 IGF-I (100 ng/ml) Trt 3 Trt 6 Control GH (100 ng/ml) Potential Zygotes Experimental Design

22. 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 Control IgG GHIGFAnti-IGF GHIGF Anti-IGFIgG Percentage of cleaved oocytes that developed to blastocyst at d 8 of embryo culture P > 0.10 P < 0.02 P < 0.01 P < 0.05

23. FLORIDA STUDY DESIGN GnRH PGF 2  TAI 63 DIM 70 DIM 72 DIM 73 DIM 147 DIM bST-63bST-73Control Pregnancy diagnosis Moreira et al. (2001)

24. 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 No PresynchPresynch bST-63bST-73 34.2% 33.7% Control 25.3% bST-63bST-73 58.1% 56.1% Control 42.6% Pregnancy Rates at 74 d after TAI for Cyclic Cows (LSM + SE; n = 375).

25. California Study Design PGF 2α PGF 2α GnRH 25 mg 25 mg 100μg d 37 d 51 d 63 Presynch Blood P 4 Ovsynch / TAI PGF 2α 25 mg bST +/- 500 mg/14 d d 70 d 72 d 73 AI/Detected Estrus d 70 d 77 PGF 2α GnRH TAI 25 mg 100 µg Blood P 4

26. Timing of the Ovsynch/Select Synch Injections GnRHPGF 2  TAI 2 4 6 8 0 P 4, ng/ml P4P4 d 5 to d 12 GnRH +7 d +9.5 d+10 d DIESTRUS COWS HIGH Estrus Detection

27. Pregnancy Diagnosis and Reinsemination AI Ultrasound d 0 d 31 PGF 2α AI Open Pregnant Palpate End Estrus Detection / AI

28. Effect of Reproductive Management (RM) and bST on Conception at day 45 after AI in Cycling Cows bST: P < 0.04 RM: P < 0.03 bST * RM: P < 0.30

29. Effect of Reproductive Management (RM) and bST on Pregnancy Rate at day 45 after AI in Cycling Cows bST: P < 0.04 RM: P < 0.03 bST * RM: P < 0.30

30. Effect of Reproductive Management (RM) and bST on Losses of Pregnancy between 31 and 45 d after First Postpartum AI in Cycling Dairy Cows bST: P < 0.07 RM: P < 0.32 bST*RM: P < 0.17

31. Study in Mexico with Cows Considered to Have Low Fertility Effect of bST treatment on pregnancy rates in dairy cows Treatment No. AIControlbST 3 to 431.5 a (n = 206)37.4 a (n = 117) 5 to 715.2 b (n = 76)25.3 ab (n = 54) > 83.9 b (n = 27)25.3 ab (n = 30) Overall16.9 b (n = 309)29.3 a (n = 201) a, b Different superscripts in the same row differ (P < 0.05) Morales-Roura et al.(2001)

32. 0 3 6 9 12 0 5000 10000 15000 20000 0 5000 10000 15000 20000 Day of cycle Large embryo Small embryo Day 16 interferon Progesterone Concentration and Embryo Production of Interferon-  ** P<0.01 Progesterone (ng.ml -1 ) Interferon-  (units per uterus) Mann et al., 1999

33. EXPERIMENTAL DESIGN GnRH, 100  g PGF 2 , 25 mg AI on morning of detected estrus D 0 D 7……..……….…………………..12 Experimental day Estrus detection hCG, 3,300 IU (i.m.) or 3 ml of saline + BCS Blood for P4 + ultrasound for CL # and location Preg. by ultrasound + BCS Preg. by palpation + BCS D 5 D 11…………16 D 28 D 45 and 90 Days after AI

35. Effect of Treatment on Conception Rates at days 28, 45 and 90 post AI 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 % d 28d 45d 90 ControlhCG 7.1 % 4.1 % 6.5 % Conception rates (%) hCG: P < 0.05

37. Nutritional Manipulation to Improve Embryo Survival

38.  Supplementation with fatty acids Caloric High energy content (often improves milk production) Extra-caloric effects Acetyl CoA for cholesterol synthesis Alters steroid clearance Influences plasma progesterone concentrations alters ovarian follicle dynamics, CL function and uterine secretion of PGF 2  Track Record of Fat Supplementation Effects on Lactating Dairy Cows

39. Size of Dominant follicle 16.5 mm 18.7 mm Diet 1.68 Mcal/kg DM 0% Ca-LCFA 1.68 Mcal/kg DM 2.2% Ca-LCFA Lucy et al., 1993 JDS 76:1014

40. Effect of Fat Supplementation on Plasma Progesterone Concentration

41. Targeting Specific FA

42. Ca Salts of Long Chain Fatty Acids Ca FA Scott and Ashes (1993)

43. Manipulating PGF 2  Synthesis  Effects on the uterus and corpus luteum Beneficial effects early postpartum on uterine immunity and involution Stimulation early postpartum might improve uterine involution and resumption of cyclicity Inhibition during maternal recognition of pregnancy might improve embryo survival

45. Cyclooxygenase Peroxidase PGHS Synthesis of PGF 2α Arachidonic acid PGE 2 PGF 2  PGES PGFS

46. Filey S.J.et al, 2000 Prostaglandin F 2  Response in Postpartum Beef Heifers Fed Rumen Bypass Fat  Fed Calcium Salts of Fatty Acids (CSFA) at 3% of estimated dry matter intake (.23 kg/heifer/d) vs isocaloric control supplement (.72kg ground barley) from time of calving.  12 heifers per treatment  Blood samples collected every other day from day 1-15 postpartum.

47. Filey S.J.et al, 2000 * *

48. Concentrations of PGFM in Holstein Cows Seals et al., 2002

49. Embryo Survival Improving Embryo Survival

52. PGF 2  CL Ovary Pregnancy, day 12 to 19 Pregnant Uterus 0 1 2 3 4 5 6 7 8 9 10 1516171819202122232425 Progesterone Day Embryo IFN-  

53. series  PGHS

54. 0 20 40 60 80 100 120 140 -60-300306090120150180210240 Time (min) ControlFish Meal * * * * P<0.05 Oxytocin (100 IU iv) Responses of PGFM in Cows Fed or Not Fed Fish Meal -240 min: Estradiol (3 mg, iv)

55. Experimental Design Extraction of RNA Northern blot analysis of PGHS-2 Wash Cells Add PDBu (100 ng/ml) >90% Confluence Add EPA 0, 20, 100 µM 24 h Starvation 0 3 6h 24 h Starvation 0 3 6h IFN-  : 0, 0.5 or 50 ng/ml Cells Grown to Confluence

56. Effect of EPA and IFN-tau on Secretion of PGF 2α from Bend Cells Treated with PDBU IFN-tau: P < 0.01 EPA: P < 0.01

57. PGHS-2 mRNA 18S rRNA Effect of EPA and IFN-tau on Concentrations of PGHS- 2 mRNA in Bend Cells Treated with PDBU EPA: P>0.1 IFN-  : P<0.01

58. PGF 2  CL Ovary Pregnancy, day 12 to 19 Pregnant Uterus 0 1 2 3 4 5 6 7 8 9 10 1516171819202122232425 Progesterone Day Embryo IFN-  

59. 24 h Starvation 0 3 6h Remove EPA Add PDBu and IFN-  3 x 3 Factorial Add EPA 0, 3 or 20 µM IFN-  : 0, 50 or 100 pg/ml Determine PGF 2α in medium Experimental Design

60. EPA (µM) IFN-  (pg/ml) PGF 2α (pg/ml) Effect of Combined Treatment with IFN-  and EPA on Secretion of PGF 2α from BEND Cells Stimulated with PDBu EPA: <0.01 IFN-  : <0.01 EPAxIFN -  : NS

61. PGF 2  CL Ovary Pregnancy, day 12 to 19 Pregnant Uterus 0 1 2 3 4 5 6 7 8 9 10 1516171819202122232425 Progesterone Day Embryo IFN-   EPA +

62. Fatty Acid Study - California  710 multiparous Holstein cows  Commercial dairy farm in Central CA (RHA = 12,026 kg)  September 2001 to October 2002  Two treatments: 400 g of fatty acids from tallow 400 g of fatty acids from palm and fish oil in a Ca salt form

63. 3.015.0 DHA, g 6.1 Crude fat, % 39.239.0 NFC, % 3.015.0 EPA, g 30.5 NDF, % 40.6 RUP, 1 % CP 18.1 CP, % 1.611.62 NE L, 1 Mcal/kg TallowCa SaltsNutrient Treatment Nutrient content of experimental diets 1 Based on NRC (2001) and adjusted for 26 kg of DM intake.

64. Reproductive Management for First Postpartum AI PGF 2α PGF 2  GnRH PGF 2α GnRH TAI 25 mg 25 mg 100μg 25 mg 100μg d 33 d 47 d 61 d 68 d 70 d 71 Presynch Ovsynch / TAI Blood P 4 Ovsynch / TAI

65. Effect of Source of Dietary FA on Pregnancy Rate at Day 28 After TAI in Dairy Cows TRT: P < 0.15 Season: P < 0.74 TRT * Season: P < 0.29 N= 172 N=50 N=94

67. Feeding Fish Meal Improved Pregnancy Rates of Beef Cows Experimental Methods  82 lactating, primiparous beef cows  Cows were penned; fed a corn silage-based diet; diets were isonitrogenous & isocaloric  Dietary Supplements: 5% fish meal or 8.7% corn gluten meal  Diets fed 25 d prior to and thru a 90-d breeding season over a 2-year study Bonnette et al., 2000

68. Feeding Fish Meal Effects on Pregnancy Rates of Beef Cows P < 0.14 Bonnette et al. (2000)

69. Inhibits PGHS-2 Reduced PGF 2α AA PGHS-2 Reduced PGF 2α AA DHA, EPA High PGF 3α (inactive) (exogenous) High IFN-  Low IFN-  EmbryoEndometrium CL and Pregnancy Maintained Low IFN-  Active PGHS-2 High PGF 2α CL Lysed Pregnancy Lost CL and Pregnancy Maintained Outcome D 17 Embryo Endometrium Successful Early Pregnancy Embryonic loss due to abnormally high secretion of PGF 2α Successful Early Pregnancy EPA and DHA compensate for low secretion of IFN- 

70. Cottonseed and Gossypol  Cottonseed is generally fed to lactating dairy cows at 10 to 15% of the diet  Provides a unique blend of protein, fat and fiber for inclusion in diets of dairy cows  Cottonseed contains gossypol (0.5 to 1.2%), which is a toxin present throughout the cotton plant that helps to control pests  However, gossypol can be toxic to mammals and it disrupts reproduction in males and possibly in female ruminants

71. Experimental Protocol Diets: 10% WUP vs 10% of a 1:2 blend of WUP & cracked Pima 813 cows day postpartum D 3 - 24 PP30 ± 344 ± 363 ± 393 ± 3183 BCSPGF Blood + BCS BCS Heat detection & AI Pregnancy Diagnosis - 3 dairy farms - 5 pens/trt

72. Nutrient Composition of Diets Cottonseed ItemWUPBWUPCPSEMP < Samples, n15 -- OM, %91.191.30.450.43 NE L, Mcal/kg1.701.710.040.36 CP, %18.418.50.340.87 Fat, %7.06.90.130.48 ADF, %21.8 0.930.99 NDF, %33.132.30.320.05 TG, mg/kg7431,000--

73. Effect of Type of Cottonseed on Performance of Dairy Cows Cottonseed ItemWUPBWUPCPSEMP < Cows, n402401-- DMI, kg/d24.424.30.710.91 FG, g/d17.823.40.700.01 Milk, kg/d39.939.70.350.78 3.5% FCM, kg/d40.040.20.360.61 Santos et al. (2002)

74. Plasma Total Gossypol in Primiparous and Multiparous Cows Fed WUP or a Blend of WUP and Cracked Pima Santos et al. (2002)

75. Effect of Type of Cottonseed and Gossypol Intake on Conception and Pregnancy Rates of Lactatin Dairy Cows P < 0.72 P < 0.01 Santos et al. (2002)

76. Effect of Type of Cottonseed and Gossypol Intake on Days Open in Lactating Dairy Cows Whole Upland: Blend of cracked Pima and WUP: Cottonseed: P < 0.01

77. P < 0.05 Effect of Plasma Gossypol Concentrations on Days Open in Dairy Cows

78. Plasma Gossypol Concentration in Lactating Dairy Cows P < 0.01 P < 0.04 Santos et al. (2002)

79. Effect of Free Gossypol Intake on Embryo Quality in Superovulated Holstein Dairy Heifers Gossypol: P < 0.04

80. Oocyte Quality Progesterone Secretion Embryonic Development IFN-t secretion Successful Implantation  PGF 2  secretion hCG Uterine Environment PUFAs bST Gossypol

81. Thank you