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 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 Pregnancy Rate (Survival Graph Analysis) Examine effective VWP VWP Area above & below the curve Slope = PR Pregnant Estimate PR Estimate proportion remaining open Open 30% open Overton, 2002 Value/Cow and Pregnancy Rate Annuity Value - The accumulated value corrected for the time value of money. Anuity Value/cow/year 1000 900 Different combinations of Heat and CR for a given PR. 800 y = 232.06Ln(x) + 1063 700 2 R = 0.9554 600 500 400 300 0 0.2 0.4 0.6 0.8 1 Pregnancy Rate Galligan, Penn. Vet Conf., 1999 Costs Associated with Changes in Reproductive Efficiency Costs of Improved Reproduction 90 Milk (lbs) and Cost ($) 85 80 75 70 65 60 55 50 45 40 8% 10% 12% 14% 16% 18% 20% 22% 24% 26% 28% 30% Pregnancy Rate Cost per cow Cost per preg Note: 8% PR ~ 18,000 RHA, Milk is per milking cow/day Overton, 2002 Control of Reproductive Indices Voluntary waiting period Effect of TAI on DIM at First Postpartum AI in a Dairy Herd VWP VWP TAI Control of Reproductive Indices Voluntary waiting period Estrus detection rate High Estrus Detection Rate Can be Achieved with Aggressive Breeding Protocols Control of Reproductive Indices Voluntary waiting period Heat detection rate Conception rate Pregnancy Rate High Estrus Detection Rate Can be Achieved with Aggressive Breeding Protocols CR = 35% Control of Reproductive Indices Voluntary waiting period Heat detection rate Conception rate Pregnancy loss Pregnancy Rate Rate of Late Embryonic Loss is Greater between Days 27 and 45 Cows Days 1st Check Days last Check Days Loss % Loss/ Day Chebel et al., 2002a 195 28 42 14 17.9 1.28 Moreira et al., 2000a 139 27 45 18 20.7 1.15 Chebel et al., 2002b 1,503 31 45 14 13.2 0.94 Stevenson et al., 2000 203 28 45 17 15.8 0.93 Santos et al., 2002b 360 31 45 14 11.1 0.79 Santos et al., 2002a 220 27 41 14 10 0.71 Cerri et al., 2002 176 31 45 14 9.7 0.70 Juchem et al., 2002 167 28 39 11 11.4 1.03 Fricke et al., 1998 89 28 56 28 13.5 0.48 Moreira et al., 2000b 211 32 74 42 13.7 0.33 Santos et al., 2000 171 28 90 62 17.4 0.28 1,601 28 98 70 19.6 0.28 285 30 280 250 22 0.09 Reference Vasconcelos et al., 1997 Pursley et al., 1998 0.9% Oocyte Quality Uterine Environment Embryonic Development bST Progesterone Secretion hCG bST Gossypol Gossypol IFN-t secretion PGF2 secretion Successful Implantation PUFAs Effects of bST on Reproductive Functions in Dairy Cows Follicle development CL differentiation/P4 secretion Fertilization/embryo development Uterine environment PGF2α secretion (bst vs IGF-I) Oviduct vs Endometrium Experimental Design Donor Cows Superovulation d0 Artificial Insemination bST d7 Recipient Cows d 0 Estrus d1 Treat recipients bST Control Control Flushing Record results Freeze embryos d7 Embryo Transfer Pregnancy diagnosis (rectal palpation) Number of Ova/Embryos per Flush for Control (n = 26) and bST (n = 26) Treated Donors (LSM + SE). 10 Control 9 bST 8 7 6 * P < 0.05 5 4 3 2 1 9.4 per flush 9.3 per flush 5.4 per flush 7.4 per flush 0 Total Unfertilized Degenerate Transferable Frequency of distribution of transferable embryos among stages of development 60 60 50 50 40 40 30 30 20 20 10 10 0 Morula Early Blast Blast Exp Blast bST Control P < 0.001 Percentage of transferable embryos relative to total ova/embryos flushed 80 70 60 50 40 30 20 10 0 56.4 % (150/266) 77.2 % (206/267) Control bST P < 0.001 Pregnancy rates following embryo transfer for treatment groups Pregnancy rates (%) Control recipient 60 bST recipient 56.1 % (23/41) 50 40 30 43.2 % (16/37) 43.3 % (26/60) 25.6 % (11/43) 20 Control embryo bST embryo Experimental Design Oocytes Maturation Control GH (100 ng/ml) Fertilization Potential Zygotes IgG Anti-IGF-I (10 g/ml) (10 g/ml) Control Trt 1 Trt 4 GH (100 ng/ml) Trt 2 Trt 5 IGF-I (100 ng/ml) Trt 3 Trt 6 Control Trt 7 Percentage of cleaved oocytes that developed to blastocyst at d 8 of embryo culture 40 P < 0.05 P < 0.01 35 30 P < 0.02 P > 0.10 25 20 15 10 5 0 Control IgG GH IgG IGF Anti-IGF GH Anti-IGF IGF FLORIDA STUDY DESIGN 63 DIM 70 DIM 72 DIM 73 DIM 147 DIM GnRH PGF2 GnRH TAI Pregnancy diagnosis bST-63 bST-73 Control Moreira et al. (2001) Pregnancy Rates at 74 d after TAI for Cyclic Cows (LSM + SE; n = 375). 70 58.1% 56.1% 60 42.6% 50 40 34.2% 33.7% 25.3% 30 20 10 0 bST-63 bST-73 Control No Presynch bST-63 bST-73 Control Presynch California Study Design bST +/500 mg/14 d PGF2α 25 mg PGF2α PGF2α GnRH 25 mg 25 mg 100μg AI/Detected Estrus d 70 PGF2α Presynch 25 mg d 37 d 51 d 77 GnRH TAI 100 µg d 63 Blood P4 Ovsynch / TAI d 70 d 72 d 73 Blood P4 Timing of the Ovsynch/Select Synch Injections Estrus Detection 8 6 4 P4 2 0 d 5 to d 12 GnRH HIGH PGF2 +7 d HIGH GnRH TAI +9.5 d +10 d DIESTRUS COWS Pregnancy Diagnosis and Reinsemination AI Palpate Ultrasound End Pregnant Estrus Detection / AI d0 d 31 Open PGF2α AI 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 55 CR d 45, % 50 45 40 35 30 25 20 ED OV bST Control 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 55 PR d 45, % 50 45 40 35 30 25 20 ED OV bST Control Effect of Reproductive Management (RM) and bST on Losses of Pregnancy between 31 and 45 d after First Postpartum AI in Cycling Dairy Cows Preg. Loss, % 20 bST: P < 0.07 RM: P < 0.32 bST*RM: P < 0.17 16 12 8 4 0 ED bST OV Control Study in Mexico with Cows Considered to Have Low Fertility Effect of bST treatment on pregnancy rates in dairy cows Treatment No. AI Control bST 3 to 4 31.5a (n = 206) 37.4a (n = 117) 5 to 7 15.2b (n = 76) 25.3ab (n = 54) >8 3.9b (n = 27) 25.3ab (n = 30) 16.9b (n = 309) 29.3a (n = 201) Overall a, b Different superscripts in the same row differ (P < 0.05) Morales-Roura et al.(2001) 12 20000 9 15000 6 10000 3 5000 ** 0 0 2 4 6 8 10 Day of cycle Mann et al., 1999 12 14 16 Interferon- (units per uterus) Progesterone (ng.ml-1) Progesterone Concentration and Embryo Production of Interferon- 0 Large Small embryo embryo Day 16 interferon ** P<0.01 EXPERIMENTAL DESIGN GnRH, 100 g D0 Experimental day hCG, 3,300 IU (i.m.) or 3 ml of saline + BCS D5 Days after AI AI on morning of detected PGF 2 , estrus 25 mg D 7……..……….…………………..12 Estrus detection Blood for P4 + ultrasound for CL # and location D 11…………16 Preg. by ultrasound + BCS D 28 Preg. by palpation + BCS D 45 and 90 Conception % rates (%) Effect of Treatment on Conception Rates at days 28, 45 and 90 post AI 46 45 44 43 42 41 40 39 38 37 36 35 34 33 32 31 30 7.1 % 4.1 % 6.5 % d 28 hCG: P < 0.05 d 45 Control hCG d 90 Nutritional Manipulation to Improve Embryo Survival Track Record of Fat Supplementation Effects on Lactating Dairy Cows 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 PGF2 Diet 1.68 Mcal/kg DM 0% Ca-LCFA Size of Dominant follicle 16.5 mm 1.68 Mcal/kg DM 2.2% Ca-LCFA 18.7 mm Lucy et al., 1993 JDS 76:1014 Effect of Fat Supplementation on Plasma Progesterone Concentration Reference Time of measurement Control (ng/ml) Fat (ng/ml) Carroll et al., 1990 d 9 - 15 of cycle 6.6a 7.7b a b Lucy et al., 1993 d 1 - 12 of cycle 4.2 5.2 a,b Sklan et al.,1991 d 8 - 20 of cycle Greater accumulation 4.5a 4.2a 6.0b 4.8b Spicer et al.,1993 wk 5 - 12 PP Son et al., 1996 wk 2 - 12 PP Garcia et al., 1998 wk 1 - 7 PP Greater accumulation a,b Adams, 1998 wk 2 - 9 PP Greater accumulation a,b Targeting Specific FA Ca Salts of Long Chain Fatty Acids Ca Ca Ca FA FA FA FA Ca Ca Ca Scott and Ashes (1993) Manipulating PGF2 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 n-6 Family n-3 Family Linolenic acid (C18:3) Linoleic acid (C18:2) 6 desaturase Linolenic acid (C18:3) Stearidonic acid (C18:4) Elongase Dihomo- Linolenic acid (C20:3) Eicosatetraenoic acid (C20:4) 5 Desaturase Arachidonic Acid (C20:4) Eicosapentaenoic acid (C20:5) Synthesis of PGF2α Arachidonic acid Cyclooxygenase PGHS Peroxidase PGES PGE2 PGFS PGF2 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. Filey S.J.et al, 2000 3000 2500 * 2000 * 1500 1000 500 0 1 3 5 7 9 11 Day postpartum Control 13 15 CSFA Filey S.J.et al, 2000 Concentrations of PGFM in Holstein Cows 3 Control PGFM, ng/mL 2.5 Infected and Treated 2 Infected and Not Treated 1.5 1 0.5 0 0 10 Seals et al., 2002 20 30 40 Days postpartum 50 60 Improving Embryo Survival Pregnancy, day 12 to 19 r Ovary IFN - CL PGF 2 Embryo Progesterone Pregnant Uterus 10 9 8 7 6 5 4 3 2 1 0 15 16 17 18 19 20 Day 21 22 23 24 25 PGHS series Responses of PGFM in Cows Fed or Not Fed Fish Meal 140 -240 min: 120 Estradiol (3 mg, iv) 100 80 Oxytocin (100 IU iv) 60 * * * * P<0.05 40 20 0 -60 -30 0 30 Control 60 90 120 Time (min) 150 Fish Meal 180 210 240 Experimental Design >90% Confluence 24 h Starvation 0 3 Add EPA 0, 20, 100 µM Cells Grown to Confluence 6h Extraction of RNA Northern blot analysis of PGHS-2 Wash Cells Add PDBu (100 ng/ml) IFN-: 0, 0.5 or 50 ng/ml 24 h Starvation 0 3 6h Effect of EPA and IFN-tau on Secretion of PGF2α from Bend Cells Treated with PDBU 7000 IFN-tau: P < 0.01 EPA: P < 0.01 PGF2α (pg/ml) 6000 5000 4000 3000 2000 1000 0 EPA 0 EPA 20 EPA 100 IFN 0 Treatment IFN 0.5 IFN 50 Effect of EPA and IFN-tau on Concentrations of PGHS2 mRNA in Bend Cells Treated with PDBU 1.4 EPA: P>0.1 IFN-: P<0.01 1.2 1 0.8 0.6 0.4 0.2 0 0 20 EPA (µM) PGHS-2 mRNA 18S rRNA 100 0 0.5 50 IFN- (ng/ml) Pregnancy, day 12 to 19 r Ovary IFN - CL PGF 2 Embryo Progesterone Pregnant Uterus 10 9 8 7 6 5 4 3 2 1 0 15 16 17 18 19 20 Day 21 22 23 24 25 Experimental Design Determine PGF2α in medium Remove EPA Add PDBu and IFN- IFN-: 0, 50 or 100 pg/ml 24 h Starvation Add EPA 0, 3 or 20 µM 0 3 6h 3 x 3 Factorial Effect of Combined Treatment with IFN- and EPA on Secretion of PGF2α from BEND Cells Stimulated with PDBu PGF2α (pg/ml) 3000 EPA: <0.01 IFN-: <0.01 EPAxIFN - : NS 2500 2000 EPA (µM) 0 3 20 1500 1000 500 0 0 50 IFN- (pg/ml) 100 Pregnancy, day 12 to 19 + EPA Ovary r IFN - CL PGF 2 Embryo Progesterone Pregnant Uterus 10 9 8 7 6 5 4 3 2 1 0 15 16 17 18 19 20 Day 21 22 23 24 25 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 Nutrient content of experimental diets Treatment Nutrient Ca Salts Tallow NEL,1 Mcal/kg 1.62 1.61 CP, % 18.1 18.1 RUP,1 % CP 40.6 40.6 NDF, % 30.5 30.5 NFC, % 39.0 39.2 Crude fat, % 6.1 6.1 EPA, g 15.0 3.0 DHA, g 15.0 3.0 1Based on NRC (2001) and adjusted for 26 kg of DM intake. Reproductive Management for First Postpartum AI PGF2α PGF2 25 mg 25 mg GnRH PGF2α GnRH 100μg 25 mg 100μg Presynch d 33 Ovsynch / TAI d 47 d 61 Ovsynch Blood /PTAI 4 d 68 d 70 d 71 TAI 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 Pregnancy Rate, % 44 41 38 35 N=50 N= 172 N=94 32 29 26 N=50 23 20 TN HS Season Ca Salts Tallow Effect of Source of Dietary FA on Losses of Pregnancy from 28 to 39 d after TAI During Thermoneutrality in Multiparous Dairy Cows Pregnancy loss, % 14 12 TRT: P < 0.09 10 8 N=75 6 4 N=68 2 0 Ca Salts Tallow 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 Feeding Fish Meal Effects on Pregnancy Rates of Beef Cows 80 P < 0.14 75 PR, % 70 65 60 55 50 Fish Meal CGM Bonnette et al. (2000) D 17 Embryo Successful Early Pregnancy Embryonic loss due to abnormally high secretion of PGF2α High IFN- Endometrium Inhibits PGHS-2 Outcome Reduced PGF2α CL and Pregnancy Maintained High PGF2α CL Lysed Pregnancy Lost AA Low IFN- Active PGHS-2 AA Successful Early Pregnancy EPA and DHA compensate for low secretion of IFN- Low IFN- PGHS-2 DHA, EPA Reduced PGF2α CL and Pregnancy Maintained High PGF3α (inactive) (exogenous) Embryo Endometrium Outcome 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 Experimental Protocol Diets: 10% WUP vs 10% of a 1:2 blend of WUP & cracked Pima 813 cows day postpartum D 3 - 24 PP 30 ± 3 44 ± 3 63 ± 3 93 ± 3 183 BCS PGF PGF Blood + BCS Blood + BCS BCS Heat detection & AI Pregnancy Diagnosis - 3 dairy farms - 5 pens/trt Nutrient Composition of Diets Cottonseed Item WUP BWUPCP SEM P< 15 15 -- -- OM, % 91.1 91.3 0.45 0.43 NEL, Mcal/kg 1.70 1.71 0.04 0.36 CP, % 18.4 18.5 0.34 0.87 Fat, % 7.0 6.9 0.13 0.48 ADF, % 21.8 21.8 0.93 0.99 NDF, % 33.1 32.3 0.32 0.05 TG, mg/kg 743 1,000 -- -- Samples, n Effect of Type of Cottonseed on Performance of Dairy Cows Cottonseed Item WUP BWUPCP SEM P< Cows, n 402 401 -- -- DMI, kg/d 24.4 24.3 0.71 0.91 FG, g/d 17.8 23.4 0.70 0.01 Milk, kg/d 39.9 39.7 0.35 0.78 3.5% FCM, kg/d 40.0 40.2 0.36 0.61 Santos et al. (2002) Plasma Total Gossypol in Primiparous and Multiparous Cows Fed WUP or a Blend of WUP and Cracked Pima Plasma gossypol, ug/ml 14 12 10 8 6 4 2 0 10 33 WUP-P Santos et al. (2002) 61 WUP-M 91 DIM BUPCP-P 120 BUPCP-M 152 Effect of Type of Cottonseed and Gossypol Intake on Conception and Pregnancy Rates of Lactatin Dairy Cows P < 0.01 34 31 28 25 22 19 16 13 10 % % P < 0.72 First AI, CR WUP BWUPCP Santos et al. (2002) 90 85 80 75 70 65 60 55 50 PR, 170 DIM WUP BWUPCP Effect of Type of Cottonseed and Gossypol Intake on Days Open in Lactating Dairy Cows Whole Upland: Blend of cracked Pima and WUP: 1.0 Cottonseed: P < 0.01 Proportion of non-pregnancy 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 20 40 60 80 100 Days Open 120 140 160 180 Effect of Plasma Gossypol Concentrations on Days Open in Dairy Cows Q1 Q2 Q3 Q4 1.0 P < 0.05 0.9 0.8 Probability 0.7 0.6 0.5 0.4 0.3 0.2 0 20 40 60 80 100 Time to Failure 120 140 160 180 Plasma Gossypol Concentration in Lactating Dairy Cows P < 0.04 9 9 8 8 7 7 6 6 ug/ml ug/ml P < 0.01 5 5 4 4 3 3 2 2 Total Gossypol Pregnant Santos et al. (2002) Open Total Gossypol No Abortion Abortion Trasferable embryos, % Effect of Free Gossypol Intake on Embryo Quality in Superovulated Holstein Dairy Heifers 50 45 40 35 30 25 20 15 10 Gossypol: P < 0.04 0 mg/kg 20 mg/kg 40 mg/kg FG intake (mg/kg of BW) Oocyte Quality Uterine Environment Embryonic Development bST Progesterone Secretion hCG bST Gossypol Gossypol IFN-t secretion PGF2 secretion Successful Implantation PUFAs Thank you