1. Prevalence and Risk Factors for Postpartum Anovulation in Dairy Cows Robert Walsh November 30, 2006

2. Outline Resumption of Ovarian Activity –Identification of at Risk cows –Cow-level risk factors –Herd-level risk factors Questions

3. Outline Resumption of Ovarian Activity

4. 051015202530 35 Calving Ovulation DIM 16 14 12 10 8 6 4 2 0 Follicle diameter (mm) Resumption of Ovarian Activity All follicles present at birth Follicle development divided into 3 stages Stage SizeDuration Recruitment< 0.13mmbirth - Slow phase0.16 – 3 mm10 – 40 days Fast phase> 4 mm8 - 10 days (Lussier et al. 1987)

5. 051015202530 35 Calving Ovulation DIM 16 14 12 10 8 6 4 2 0 Follicle diameter (mm) Resumption of Ovarian Activity Atresia or ovulation IGF-1, IGFBP, E 2, …. Deviation Luteinizing Hormone (LH) Emergence Follicle Stimulating Hormone (FSH)

6. 051015202530 35 Calving Ovulation DIM 16 14 12 10 8 6 4 2 0 Follicle diameter (mm) Resumption of Ovarian Activity 33.8 days (1106 Lactations; Petersson et al. 2005) 27.4 days (>1200 Records; Royal et al. 2004) 28.7 ± 14.6 days (2503 Lactations, Lamming and Darwash 1998) 29 days Control line vs. 43 days Select (Lucy et al. 2001) 43 days First Estrus (1398 Lactations, Thatcher and Wilcox, 1973)

7. Resumption of Ovarian Activity Reference Location DIMDOV1ALL Lopez et al. 2004 Wisconsin 7028.5% Cerri et al. 2004California6525.7% Moreira et al. 2001Florida6323% Opsomer et al. 2000 Belgium 5021.2%49% Lamming and Darwash 1998 England 10.9% 38% Shrestha et al. 2004 Japan24.1%57% Archbald et al. 1990Florida3030% McDougal and Compton 2005NZ20.9% Thatcher and Wilcox 1973 Florida6025.9% (Estrus)

8. 5 10 15 20 25 Days In Milk 16 14 12 10 8 6 4 2 0 Follicle diameter (mm) (Wiltbank et al. 2002) Anovulation Follicular Growth to Deviation Consecutive waves of follicle growth initiated by FSH Insufficient LH to support deviation Communication failure between ovary and hypothalamus

9. 5 10 15 20 25 Days In Milk 16 14 12 10 8 6 4 2 0 Follicle diameter (mm) (Wiltbank et al. 2002) Anovulation Follicles Larger than Ovulatory Size LH release supports deviation Elevated E 2 concentration Failure of ovulation The presence of large follicles does not preclude Normal ovarian activity and function

10. Outline Resumption of Ovarian Activity –Identification of at Risk cows

11. Objectives Determine prevalence of anovulation Assess consequences of anovulation Identify cow-level risk factors –herd-level risk factors

12. Observational Protocol Milk Ketotest ™ BCS HH visit BCS, Lameness Milk P 4 Collect Reproduction and Culling Data 0 4 11 38 52 60 Record peripartum disease Data presented from 1341 cows Anestrus Both samples <1ng/ml Calving

13. Descriptive Data Tiestall Freestall Herds Cows Herds Cows Herd Size < 100 cows9 404 3 178 > 100 cows1 65 5 694 Milk Frequency 2 Times/day9 404 4 218 3 Times/day1 65 4 654 TAI First Service > 90 %5 236 1 61 0 to 50% 5 233 7 775 Rumensin CRC2 82 2 427 Premix6 292 3 308 Both2 95 3 137

14. Study Population Enrolled 20 herds (lost 2) –Total calving 2645 –Culled prior to 52 DIM 277 –Total eligible2368 –Two Milk Samples 1 1575 –Met Inclusion criteria1341 (56.7%) Herd specific inclusion ranged 33 – 90% Disease under-reported in cows not sampled No significant difference in parity distribution, first milk projection, days dry between included and excluded cows Excluded cows more likely culled (P < 0.001) 1 Excluded if milk sample protocol violated or use of hormones

15. Cow Level Prevalence of Anovulation 19.5% (95% CI 17.4 – 21.7%)

16. VWP 45-60 d Calving No Estrus Activity Ovarian Activity ?? Impact of Anovulation 1 in 5 cows Herd Repro Management Heat Detection Timed Insemination protocols

17. Time to First Insemination Herd Repro ProgramFactor AIHR P 95% CI HeatAnovular80 b 0.7 <0.0001 0.6 to 0.8 Detection 1 Cycling 72 a Referent TimedAnovular78 ab 0.67 0.19 0.1 to 1.2 AI 2 Cycling76 b Referent Multiparous0.84 0.04 0.7 to 0.9 Retained Placenta0.75 <0.001 0.6 to 0.8 1 Insemination at Observed Estrus in >50% of herd (12 herds 79% of cows) 2 Timed Insemination in > 90% of herd (6 herds, 21% of cows)

18. Probability of Pregnancy after First Insemination Herd Repro ProgramFactor FSCROR P 95% CI HeatAnovular20.3 b 0.6 0.007 0.4 to 0.6 Detection 1 Cycling 30.5 a Referent TimedAnovular29.7 ab 0.67 0.19 0.1 to 1.2 AI 2 Cycling35.9 a Referent Multiparous 0.73 0.02 0.6 to 0.9 1 Insemination at Observed Estrus in >50% of herd (12 herds 79% of cows) 2 Timed Insemination in > 90% of herd (6 herds, 21% of cows)

19. Time to Pregnancy 156 126 Time Adjusted HR 810.56 1230.74 1650.89 Adjusted HR = HR*TVC ln(t)

20. Diagnosis of Anovulation Rectal Palpation 1 (2 palpations 14-days apart) Sensitivity 65.4% Specificity 68.6% Positive Predictive value 31.9% 1 gold standard circulating progesterone

21. Diagnosis of Anovulation using Pedometry (HDR ~ 60%) Sensitivity 83.0% Specificity 68.0% Positive Predictive value 34.0%

22. Diagnosis of Anovulation Assumption 100 cows, True Prevalence of 20% 1 Walsh et al. (Unpublished) 2 Sprecher et al. 1989; Theriogenology 3 McDougal and Rhodes, 1999; NZVJ (Apparently Anestrus cows) 4 Core et al. (Unpublished)

23. Outline Resumption of Ovarian Activity –Identification of at Risk cows –Cow-level risk factors

24. Univariable Risk Factor Analysis Controlling for fresh season (* P < 0.05; ** P < 0.01; *** P < 0.001)

25. Final Risk Factor Model (* P < 0.05; ** P < 0.01; *** P < 0.001)

26. Prevalence of anovulation by Parity Parity is an inconsistent risk factor for anovulation

27. Lameness 238 Animals, 11% DOV Lameness (Median day of diagnosis = 15 DIM) –42% Moderately Lame –17% Lame Relative to animals never classified as lame controlling for calving season, lactation, ketotsis*and Milk Production (305ME)* –Moderate Lameness 2.14 odds of DOV (95%CI = 0.7, 6.14) –Lame 3.5 increased odds of DOV (95% CI = 1.0, 12.7) Calving Lameness Score (1-5) Milk P4 Garbarino et al. 2004

28. Lameness and Reproductive Performance 65 Cases, 130 Controls Controlling for parity, calving season, and milk yeild (305ME) –Non-lame cows 4.22 times more likely to conceive at first service (95% CI 1.59, 11.2) –Lame cows 2.63 times more likely to have an occurrence of an ovarian cyst Using Survival analysis –Lame cows HR 0.43 (95% CI 0.28, 0.66) Melendez et al. 2003

29. Body condition Loss Lopez-Gatius et al. Theriogenology 2003 4529 cows from 11 studies Relative to BCS 2.5-3.5 <2.5 at parturition + 6 DOPN >3.5 at parturition – 6 DOPN Relative to BCS change <0.5 units between parturition and first AI Loss >1 +10.6 DOPN Not significant in all studies Ruegg et al. 1995 Domecq et al. 1999

30. Milk Production StudyMilk Measure Delayed Ovulation Opsomer et al. 2000100d & 305d FCMns Shrestha et al. 2004305d FCMns Santos et al. 2004Proj 305 ME 0.21(0.05 to 0.98) high vs. Med Time to Pregnancy Gröhn and Rajala-Schultz 200060d MilkHR0.92 (highest vs. lowest)

31. Outline Resumption of Ovarian Activity –Identification of at Risk cows –Cow-level risk factors

32. Cow Level Prevalence of Subclinical Ketosis Week 1 26.2% (95% CI 23.5 to 28.9 %)

33. Energy Balance and DOV 54 Multiparous HF cows calculated energy balance ItemERLRNR Number25 (46%)14 (26%)15 (28%) 4% FCM (kg/d)33.431.728.5 DMI(kg/d)18.817.715.2 Day to Ovulation 21.943.1 Days Open13388200 Calving7 14 21 28 35 42 49 56 63 Progesterone Profile ER LR NR Staples et al. 1990

34. Estimating Energy Status Significant correlation between ketone body concentration and CLA (Reist et al. 2000) Milk Acetone week 3-6 not associated with FSCR (Plym-Forshell, 1991) Increased DOPN, Increased culling risk (Cook et al. 2000) 1400  mol/ml  HBA cutpoint used for disease status

35. Impact of subclinical ketosis on the probability of pregnancy at first service and time to pregnancy Objectives Is SCK early in lactation associated with the probability of pregnancy at first insemination? Which week postpartum has the most impact? Does the duration of SCK impact probability of pregnancy at first insemination or time to pregnancy ?

36. Impact of subclinical ketosis on the probability of pregnancy at first service and time to pregnancy Serum Sample Store samples and analyze for  HBA BCS Observe for estrus and breed PD -3 0 1 2 3 6 9 Calving BCS  25 Herds (25-160 cows)  1010 cows  Exclusion criteria  806 records for analysis  Rumensin CRC study 1995-1996 (Duffield et al. 1999)

37. Distribution of serum BHBA 16% week 1 ( 132/806 cows ) 19% Week 2 ( 152/806 cows )

38. Mean Serum BHBA Sorted by Pregnancy Diagnosis to First Insemination

39. Investigation of Cutpoints

40. Impact of Subclinical Ketosis in the Second Week Postpartum on the Probability of Pregnancy at First Insemination 0.1.2.3.4 Probability of Pregnancy 02000400060008000 Week 2 serum BHBA concentration (mmol/L)

41. Effect of Prolonged Elevation of Serum BHBA on the Probability of Pregnancy at First Insemination Number of Weeks Above BHBA Cutpoint Probability of Pregnancy (%) OR = 0.83 P = 0.2 OR = 0.47 P = 0.003

42. Impact of duration of subclinical ketosis on time to pregnancy 0.00 0.25 0.50 0.75 1.00 Survival Function (Proportion Not Pregnant) 0100200300400 Days in Milk Not Ketotic Ketotic week 1 or week 2 Ketotic both week 1 and 2 Time Adjusted HR 810.620.63 1230.750.84 1650.851.02 Adjusted HR = HR*TVC ln(t) Median time to Pregnancy 108 124 130

43. Cow-level Summary Diagnosis is problematic Calving history / periparturient disease Subclinical ketosis

44. Dry Period Length >77days vs. <63 days 2.9 times more likely to experience DOV (Opsomer et al. 2000) DD associated with increase risk of longer DOPN (Moss et al. 2002) (Gümen et al. 2005)

45. Milking Frequency 3X vs 2X majority of studies show no difference (Weiss et al. 2004; Barnes et al. 1990; Amos et al. 1985*) 3X vs 2X associated with 6d increase DOPN (Smith et al. 2002) 3X vs. 6X for 3 weeks increased DOPN (Bar- Peled et al. 1995)

46. Herd-Season Anovulation Prevalence 34 herd-seasons from 18 herds (Fall vs. Other) Linear regression of herd-level prevalence Relative to Tie-Stall barns the prevalence of anestrus ↓ 7.2% in 3-Row Freestalls (P = 0.1) ↓ 11.0 % in 2-Row Freestalls (P = 0.007) ↑ 2.1% for every 10% ↑in herd-season prevalence of SCK

47. Herd-Season Anovulation Prevalence Things to Consider –Stocking density –Number of ration or group changes –Dry period length

48. Questions ?

49. Acknowledgement Advisors Drs. Leblanc, Leslie, Duffield, Kelton and Walton Veterinarians Almonte, Bay of Quinte Veterinary Services, Embrun, Ferguson, Grenville-Dundas, Kirkton, Linwood, Listowel, Navan, New Hamburg, Tavistock AABP Research Assistantship