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The association of subclinical hypocalcemia at 4 days in milk with reproductive outcomes in multiparous Holstein cows

Open AccessPublished:December 14, 2022DOI:https://doi.org/10.3168/jdsc.2022-0279

      Highlights

      • Cows with SCH at 4 DIM had lower odds of becoming pregnant to first service than their normocalcemic counterparts.
      • SCH at 4 DIM was negatively associated with hazard of pregnancy by 150 DIM.
      • The negative association of SCH at 4 DIM and reproductive success suggests that the negative ramifications of delayed or prolonged reductions in blood Ca persist beyond the immediate postpartum period.

      Abstract

      Delayed or persistent episodes of subclinical hypocalcemia (SCH) have been associated with reduced milk production and feed intake and they put cows at risk for additional disease development. However, little is known about the effect of delayed or persistent SCH on reproductive outcomes. The objectives of our study were to describe the association between SCH status at 4 d in milk (DIM) with the odds of pregnancy to first service and time to pregnancy through 150 DIM. Data were collected from multiparous Holstein cows (n = 697) from 4 farms in New York State that did not receive supplemental Ca during the first 5 DIM. Cows were classified into 1 of 2 SCH groups based on blood total Ca (tCa) at 4 DIM: normocalcemic (NC; n = 515) if tCa >2.2 mmol/L at 4 DIM or SCH (n = 182) if tCa ≤2.2 mmol/L at 4 DIM. We created a multivariable logistic regression model to assess the association between SCH group and pregnancy to first service and a time-to-event analysis to evaluate the effect of SCH group on the hazard of pregnancy by 150 DIM. Cows that were SCH at 4 DIM had lower odds of pregnancy to first service [odds ratio = 0.75, 95% confidence interval (CI) = 0.61 to 0.93, incidence = 18.1%] compared with their NC counterparts (incidence = 27.4%). Similarly, SCH cows tended to have a lower hazard of pregnancy by 150 DIM (hazard ratio = 0.82, 95% CI = 0.67 to 1.01, incidence = 65.4%) than NC cows (incidence = 70.7%). Our results indicate that delayed or prolonged reductions in blood Ca in the early postpartum period, resulting in SCH at 4 DIM, were associated with reduced reproductive outcomes in multiparous cows. Our findings, in conjunction with previous reports of the negative associations of delayed or persistent SCH on health and production, suggest that reductions in blood Ca beyond the first day of lactation might be indicative of maladaptation to lactation, and the ramifications can persist beyond the immediate postpartum period.

      Graphical Abstract

      Figure thumbnail fx1
      Graphical AbstractSummary: Subclinical hypocalcemia (SCH) is a common threat to multiparous dairy cows, and reductions in blood Ca occurring at 4 DIM are associated with decreased production and overall cow health and well-being. We explored the association of SCH occurring at 4 DIM with odds of pregnancy to first service and hazard of pregnancy by 150 DIM. Cows that were SCH at 4 DIM had lower odds of pregnancy to first service and hazard of pregnancy by 150 DIM compared with their normocalcemic (NC) counterparts.
      Subclinical hypocalcemia (SCH), a decline in blood Ca unaccompanied by physical signs of the disease, affects nearly 50% of multiparous dairy cows as they transition to lactation (
      • Reinhardt T.A.
      • Lippolis J.D.
      • Mccluskey B.J.
      • Goff J.P.
      • Horst R.L.
      Prevalence of subclinical hypocalcemia in dairy herds.
      ). Decreased blood Ca concentrations in the early lactation period have been associated with the development of additional adverse health events such as metritis and displaced abomasum, as well as decreased milk production (
      • Chapinal N.
      • LeBlanc S.J.
      • Carson M.E.
      • Leslie K.E.
      • Godden S.
      • Capel M.
      • Santos J.E.P.
      • Overton M.W.
      • Duffield T.F.
      Herd-level association of serum metabolites in the transition period with disease, milk production, and early lactation reproductive performance.
      ;
      • Neves R.C.
      • Leno B.M.
      • Bach K.D.
      • McArt J.A.A.
      Epidemiology of subclinical hypocalcemia in early-lactation Holstein dairy cows: The temporal associations of plasma calcium concentration in the first 4 days in milk with disease and milk production.
      ). It has generally been accepted that blood Ca reaches a nadir within the first 24 h after calving. Nonetheless, recent work suggests that there is a stronger association between reductions in blood Ca at 3 and 4 DIM and decreased production and DMI and increased risk of adverse health events compared with cows that only experience a transient decline in blood Ca following calving (
      • Venjakob P.L.
      • Pieper L.
      • Heuwieser W.
      • Borchardt S.
      Association of postpartum hypocalcemia with early-lactation milk yield, reproductive performance, and culling in dairy cows.
      ;
      • McArt J.A.A.
      • Neves R.C.
      Association of transient, persistent, or delayed subclinical hypocalcemia with early lactation disease, removal, and milk yield in Holstein cows.
      ;
      • Seely C.R.
      • Leno B.M.
      • Kerwin A.L.
      • Overton T.R.
      • McArt J.A.A.
      Association of subclinical hypocalcemia dynamics with dry matter intake, milk yield, and blood minerals during the periparturient period.
      ). Multiparous cows with Ca ≤2.2 mmol/L at 4 DIM were nearly twice as likely to experience an adverse event during the first 60 DIM and had 3 kg/d lower DMI compared with cows that had Ca >2.2 mmol/L by 4 DIM (
      • McArt J.A.A.
      • Neves R.C.
      Association of transient, persistent, or delayed subclinical hypocalcemia with early lactation disease, removal, and milk yield in Holstein cows.
      ;
      • Seely C.R.
      • Leno B.M.
      • Kerwin A.L.
      • Overton T.R.
      • McArt J.A.A.
      Association of subclinical hypocalcemia dynamics with dry matter intake, milk yield, and blood minerals during the periparturient period.
      ).
      Despite the largely accepted negative association between SCH and overall cow health and production (
      • Rodríguez E.M.
      • Arís A.
      • Bach A.
      Associations between subclinical hypocalcemia and postparturient diseases in dairy cows.
      ;
      • Neves R.C.
      • Leno B.M.
      • Bach K.D.
      • McArt J.A.A.
      Epidemiology of subclinical hypocalcemia in early-lactation Holstein dairy cows: The temporal associations of plasma calcium concentration in the first 4 days in milk with disease and milk production.
      ), the effect of SCH on reproductive outcomes is less consistent. Although
      • Venjakob P.L.
      • Pieper L.
      • Heuwieser W.
      • Borchardt S.
      Association of postpartum hypocalcemia with early-lactation milk yield, reproductive performance, and culling in dairy cows.
      observed that cows with SCH had lower odds of pregnancy at first service and increased time to conception,
      • Chamberlin W.G.
      • Middleton J.R.
      • Spain J.N.
      • Johnson G.C.
      • Ellersieck M.R.
      • Pithua P.
      Subclinical hypocalcemia, plasma biochemical parameters, lipid metabolism, postpartum disease, and fertility in postparturient dairy cows.
      reported no difference in estrus expression by 60 DIM, services per pregnancy, or odds of pregnancy between cows that had SCH and those that did not. The lack of consensus between studies may be due in part to the differing times that blood was collected relative to calving, as well as varying blood Ca cutpoints used to diagnose SCH. Although several studies have explored the association of SCH on reproductive outcomes (
      • Ribeiro E.S.
      • Lima F.S.
      • Greco L.F.
      • Bisinotto R.S.
      • Monteiro A.P.A.
      • Favoreto M.
      • Ayres H.
      • Marsola R.S.
      • Martinez N.
      • Thatcher W.W.
      • Santos J.E.P.
      Prevalence of periparturient diseases and effects on fertility of seasonally calving grazing dairy cows supplemented with concentrates.
      ;
      • Wilhelm A.L.
      • Maquivar M.G.
      • Bas S.
      • Brick T.A.
      • Weiss W.P.
      • Bothe H.
      • Velez J.S.
      • Schuenemann G.M.
      Effect of serum calcium status at calving on survival, health, and performance of postpartum Holstein cows and calves under certified organic management.
      ;
      • Mahen P.J.
      • Williams H.J.
      • Smith R.F.
      • Grove-White D.
      Effect of blood ionised calcium concentration at calving on fertility outcomes in dairy cattle.
      ), they have diagnosed cows with SCH based on blood Ca measured within the first 24 h of calving and have not considered the impact that delayed or prolonged reductions in blood Ca may have on reproductive outcomes.
      Therefore, the objectives of our study were to describe the association of SCH, diagnosed at 4 DIM as serum total Ca ≤2.2 mmol/L based on the findings of
      • Neves R.C.
      • Leno B.M.
      • Bach K.D.
      • McArt J.A.A.
      Epidemiology of subclinical hypocalcemia in early-lactation Holstein dairy cows: The temporal associations of plasma calcium concentration in the first 4 days in milk with disease and milk production.
      , with the odds of pregnancy to first service and time to pregnancy. We hypothesized that cows experiencing SCH at 4 DIM would have decreased odds of pregnancy to first service and be less likely to be pregnant by 150 DIM compared with cows that experienced only a transient decline in blood Ca after calving or that remained normocalcemic.
      All animal protocols were approved by the Cornell University Institutional Animal Care and Use Committee (protocol nos. 2019-0025 and 2020-0102). Our analysis was conducted as a retrospective cohort design and was written following the STROBE-VET guidelines (
      • Sargeant J.M.
      • O'Connor A.M.
      • Dohoo I.R.
      • Erb H.N.
      • Cevallos M.
      • Egger M.
      • Ersbøll A.K.
      • Martin S.W.
      • Nielsen L.R.
      • Pearl D.L.
      • Pfeiffer D.U.
      • Sanchez J.
      • Torrence M.E.
      • Vigre H.
      • Waldner C.
      • Ward M.P.
      Methods and processes of developing the strengthening the reporting of observational studies in epidemiology − veterinary (STROBE-Vet) statement.
      ) for strengthening the reporting of observational studies in epidemiology-veterinary extension.
      Data were collected from 4 farms in northeastern (farms A and B) and central (farms C and D) New York State from September 1 to November 1, 2020, and July 15 to September 25, 2021 (farms C and D) and from June 2 to July 10, 2021 (farms A and B). Although sampling was conducted during 2 time periods for farms C and D, no cow contributed more than 1 lactation of data to this analysis. To be selected, farms had to meet the following criteria: milk at least 1,000 Holstein cows 3 times a day, have headlocks in the fresh cow pens, feed a TMR and prepartum negative DCAD diet, record daily milk weights using DairyComp 305 (Valley Agricultural software), and allow the research team access to computer records for 6 mo after the enrollment of the last cow. On all farms, cows were milked within 8 h of calving. Cows entering their second or greater lactation that did not receive supplemental Ca during the first 5 DIM were eligible for inclusion. A total of 697 cows were eligible for our analysis (farm A; n = 65, farm B; n = 30, farm C; n = 87, farm D; n = 515).
      Farms were visited daily throughout the study period at the same time each day, and blood samples were collected from all cows at 4 DIM from the coccygeal vessels using a 20-gauge Vacutainer needle (Becton Dickinson) into a 10-mL evacuated tube with no anticoagulant (Becton Dickinson). Serum was separated from whole blood following centrifugation at 2,000 × g for 20 min at 15°C and was aliquoted into 1.7-mL microfuge tubes and stored at −20°C until analysis. Serum samples were analyzed for total Ca (tCa) using a commercially available kit (Calcium Gen.2, Roche Diagnostics) at the New York State Animal Health and Diagnostic Center (Ithaca, NY) on an automated analyzer (Hitachi Modular P800, Roche Diagnostics). Inter- and intraassay coefficients of variation were 1.1 and 0.9%, respectively.
      On farms A and B, cows were enrolled in a breeding program on Thursdays and Fridays, respectively. Cows received their first injection of PGF at 36 to 42 DIM followed by a second injection 14 d later, at 50 to 56 DIM. The following week, at 64 to 70 DIM, cows received an injection of GnRH. One week later at 71 to 77 DIM, 72 h before breeding, cows received a third injection of PGF, with a fourth PGF administered again 48 h before breeding. Finally, 16 h before breeding, at 73 to 79 DIM, cows received a final injection of GnRH and were inseminated via AI at 74 to 80 DIM. Three employees bred cows on farm A, and 2 employees bred cows on farm B.
      Cows on farms C and D were enrolled in a breeding program on Mondays and Tuesdays, respectively, and were given an injection of PGF at 40 to 46 DIM (farm C) and at 33 to 39 DIM (farm D), with a second injection given at 14 d later (54 to 60 DIM on farm C; 47 to 53 DIM on farm D). Cows were bred via heat detection using activity collars (SCR Dairy and Afimilk; farms C and D, respectively), after a voluntary waiting period of 55 and 47 d for farms C and D, respectively, and given 14 d to be detected in estrus via activity before enrollment into a timed AI program. On both farms, 90% of cows were bred via heat detection during the study period. The remaining 10% of cows were started on an Ovsynch program at 68 to 73 DIM (farm C) and 61 to 67 DIM (farm D), in which cows were given an injection of GnRH on d 0 of the synchronization program, followed by an injection of PGF on d 7, an injection of GnRH on d 9, and inseminated via timed AI on d 10. Four farm employees bred cows on farm C and 7 employees on farm D.
      Before statistical analysis, cows were classified as being normocalcemic (NC; n = 515) if tCa >2.2 mmol/L at 4 DIM or having SCH (n = 182) if tCa ≤2.2 mmol/L at 4 DIM. A multivariable logistic regression model was created using the LOGISTIC procedure of SAS version 9.4 (SAS Institute Inc.) to assess the association between SCH group and pregnancy to first service. The initial model included the fixed effects of SCH group, farm, parity group (2 and ≥ 3), disease status (presence of displaced abomasum, metritis, or both, before 14 DIM), and all 2- and 3-way interactions. Regardless of statistical significance, the effects of SCH group, farm, and parity remained in the model and the remaining effects and interactions were removed via manual backward stepwise elimination if P > 0.1. No interactions or the effect of disease status were statistically significant at the P = 0.1 level, and the final model included only the fixed effects of SCH group, farm, and parity.
      Time-to-event analysis was conducted to evaluate the effect of SCH group on the hazard of pregnancy by 150 DIM by creating a Cox semiparametric proportional hazard model using the PHREG procedure of SAS (SAS Institute Inc.). The outcome variable was the DIM at which cows became pregnant, and right-censoring occurred for cows that were sold or died before 150 DIM. The time-to-event analysis was modeled as described above, and the final model included only the fixed effects of SCH group, farm, and parity. We then constructed a Kaplan-Meier survival analysis curve to illustrate the rate at which NC and SCH cows became pregnant until 150 DIM and the median time to pregnancy for NC and SCH cows using the LIFETEST procedure of SAS (SAS Institute Inc.).
      The adjusted odds ratio (OR) or hazard ratio (HR) and 95% confidence intervals (CI) were calculated for all models, and statistical significance was declared when P ≤ 0.05, with a tendency for a difference when was 0.05 < P ≤ 0.10.
      The mean tCa concentration at 4 DIM was 2.30 ± 0.18 mmol/L, a concentration similar to previous reports of tCa for multiparous cows at 3 and 4 DIM, which ranged from 2.15 to 2.20 (
      • Martinez N.
      • Risco C.A.
      • Lima F.S.
      • Bisinotto R.S.
      • Greco L.F.
      • Ribeiro E.S.
      • Maunsell F.
      • Galvão K.
      • Santos J.E.P.
      Evaluation of peripartal calcium status, energetic profile, and neutrophil function in dairy cows at low or high risk of developing uterine disease.
      ;
      • Neves R.C.
      • Leno B.M.
      • Bach K.D.
      • McArt J.A.A.
      Epidemiology of subclinical hypocalcemia in early-lactation Holstein dairy cows: The temporal associations of plasma calcium concentration in the first 4 days in milk with disease and milk production.
      ). Parity 2 cows had a mean tCa of 2.30 ± 0.17 mmol/L and parity ≥3 cows had a mean tCa of 2.30 ± 0.18 mmol/L. We analyzed data on a total of 697 cows, of which 74% (n = 515) were classified as NC at 4 DIM and 26% (n = 182) were classified as SCH at 4 DIM. The distribution of cows across parity and farms contributing to this analysis can be found in Table 1. During the first 14 DIM, in which we recorded disease events, there were 43 recorded cases of metritis, of which 16 cows were NC and 27 were SCH. There were 2 recorded cases of displaced abomasum, both of which occurred in SCH cows. Although the incidence of disease in our study was low, this could be attributed in part to poor recording of disease on farm B. Perhaps with a larger data set powered to detect a difference in disease, we would observe a more apparent effect of disease status on reproductive success as it relates to blood Ca.
      Table 1Parity group, subclinical hypocalcemia (SCH) proportions, and reproductive management protocols for 697 Holstein cows from 4 farms in New York State
      Cows were normocalcemic (NC; n = 515) if serum total Ca (tCa) was >2.2 mmol/L at 4 DIM or SCH (n = 182) if tCa ≤2.2 mmol/L at 4 DIM.
      CharacteristicFarm
      ABCD
      Parity, % (n)
       232 (21)60 (18)41 (36)43 (222)
       ≥368 (44)40 (12)59 (51)57 (293)
      SCH group, % (n)
       NC77 (50)80 (24)64 (56)75 (385)
       SCH23 (15)20 (6)36 (31)25 (130)
      Farm total653087515
      Time of first AI,
      Raw mean ± SD.
      DIM
      78 ± 278 ± 367 ± 262 ± 1
      Reproductive management
       Voluntary waiting period, DIM74745547
       First service managementOvsynchOvsynchHeat detection and OvsynchHeat detection and Ovsynch
       ≥ Second service managementResynchResynchResynchResynch
      1 Cows were normocalcemic (NC; n = 515) if serum total Ca (tCa) was >2.2 mmol/L at 4 DIM or SCH (n = 182) if tCa ≤2.2 mmol/L at 4 DIM.
      2 Raw mean ± SD.
      Cows that were SCH at 4 DIM had lower odds of pregnancy to first service (OR = 0.75; 95% CI = 0.61–0.93, incidence = 18.1%; P = 0.01) compared with NC cows (incidence = 27.4%; Table 2). An effect of farm and parity on the odds of pregnancy to first service were not detected (P = 0.5 and 0.4, respectively). The mean time of first AI was 71.6 ± 1.6 DIM for SCH cows and 70.6 ± 1.2 DIM for NC cows.
      Table 2Reproductive outcomes for multiparous Holstein cows (n = 697) from 4 farms in New York State
      Cows were classified into subclinical hypocalcemia (SCH) groups and were normocalcemic (NC; n = 515) if serum total Ca (tCa) was >2.2 mmol/L at 4 DIM or SCH (n = 182) if tCa ≤2.2 mmol/L at 4 DIM. Time to first AI was analyzed through a univariable ANOVA. The final logistic regression model for the odds of pregnancy to first service included the fixed effects of SCH group, farm, and parity. The final Cox proportional hazard model to evaluate the time to pregnancy included the fixed effects of SCH group, farm, and parity.
      VariableIncidence
      Mean DIM of first AI and incidence (%) for pregnancy to first service and pregnant by 150 DIM.
      (%)
      OR
      Odds ratio of pregnancy to first service.
      /HR
      Hazard ratio of pregnancy by 150 DIM.
      95% CIP-value
      Time of first AI, DIM
       NC64.162.3–65.40.28
       SCH65.163.4–66.8
      Pregnant to first service
       NC27.4Referent
       SCH18.10.750.61–0.930.01
      Pregnant by 150 DIM
       NC70.7Referent
       SCH65.40.820.67–1.010.06
      1 Cows were classified into subclinical hypocalcemia (SCH) groups and were normocalcemic (NC; n = 515) if serum total Ca (tCa) was >2.2 mmol/L at 4 DIM or SCH (n = 182) if tCa ≤2.2 mmol/L at 4 DIM. Time to first AI was analyzed through a univariable ANOVA. The final logistic regression model for the odds of pregnancy to first service included the fixed effects of SCH group, farm, and parity. The final Cox proportional hazard model to evaluate the time to pregnancy included the fixed effects of SCH group, farm, and parity.
      2 Mean DIM of first AI and incidence (%) for pregnancy to first service and pregnant by 150 DIM.
      3 Odds ratio of pregnancy to first service.
      4 Hazard ratio of pregnancy by 150 DIM.
      • Caixeta L.S.
      • Ospina P.A.
      • Capel M.B.
      • Nydam D.V.
      Association between subclinical hypocalcemia in the first 3 days of lactation and reproductive performance of dairy cows.
      also found that cows with chronic SCH, defined as cows with Ca ≤2.15 mmol/L for the first 3 DIM, had lower odds of pregnancy to first service compared with normocalcemic cows. The reduction in pregnancy to first service may be explained in part by the increase in uterine involution time and increased time to return to cyclicity previously reported in cows with SCH (
      • Jonsson N.N.
      • Pepper P.M.
      • Daniel R.C.W.
      • McGowan M.R.
      • Fulkerson W.
      Association between non-parturient post-partum hypocalcaemia and the interval from calving to first ovulation in Holstein-Friesian cows.
      ). Prolonged reductions in blood Ca have also been associated with less active ovaries, thereby reducing the efficiency of cows' responses to ovulation synchronization protocols (
      • Santos J.E.P.
      • Rutigliano H.M.
      • Filho M.F.S.
      Risk factors for resumption of postpartum estrous cycles and embryonic survival in lactating dairy cows.
      ;
      • Bisinotto R.S.
      • Chebel R.C.
      • Santos J.E.P.
      Follicular wave of the ovulatory follicle and not cyclic status influences fertility of dairy cows.
      ;
      • McNally J.C.
      • Crowe M.A.
      • Roche J.F.
      • Beltman M.E.
      Effects of physiological and/or disease status on the response of postpartum dairy cows to synchronization of estrus using an intravaginal progesterone device.
      ).
      The SCH cows tended to have a lower hazard of pregnancy by 150 DIM (HR = 0.82; 95% CI = 0.67–1.01, incidence = 65.4%; P = 0.06) compared with NC cows (incidence = 70.7%; Table 2 and Figure 1). The median time to pregnancy for the SCH cows was 119 ± 16 d and 103 ± 11 d for the NC cows. There was an effect of farm on the hazard of pregnancy by 150 DIM (P = 0.01) and a tendency for a difference between parity groups (P = 0.08). We collected data from 4 farms; however, 74% of the study population came from farm D. This may introduce bias and could partially explain the effect of farm on the hazard of pregnancy by 150 DIM. Furthermore, farms A and B were sampled during the summer months and farms C and D were sampled during both the summer and fall months. Although we did not include an effect of season in our statistical analysis, cows bred in summer months have been shown to have decreased reproductive success compared with cows bred in cooler months (
      • De Rensis F.
      • Marconi P.
      • Capelli T.
      • Gatti F.
      • Facciolongo F.
      • Franzini S.
      • Scaramuzzi R.J.
      Fertility in postpartum dairy cows in winter or summer following estrus synchronization and fixed time AI after the induction of an LH surge with GnRH or hCG.
      ;
      • De Rensis F.
      • Scaramuzzi R.J.
      Heat stress and seasonal effects on reproduction in the dairy cow: A review.
      ). To address these potential biases, future studies may consider sampling cows more equally distributed across farms and throughout multiple seasons.
      Figure thumbnail gr1
      Figure 1Kaplan-Meier curves for time to pregnancy for multiparous Holstein cows (n = 697) from 4 farms in New York State. Cows were classified into subclinical hypocalcemia (SCH) groups and were normocalcemic (NC; n = 515) if serum total Ca (tCa) was >2.2 mmol/L at 4 DIM or SCH (n = 182) if tCa ≤2.2 mmol/L at 4 DIM. The median time to pregnancy was 119 ± 16 d for SCH cows and 103 ± 11 d for the NC cows. Shaded regions represent 95% confidence intervals.
      Several homeorhetic and homeostatic adaptations must successfully work in unison to maintain blood Ca concentrations as the cow adjusts to the demands of lactation (
      • Goff J.P.
      Macromineral physiology and application to the feeding of the dairy cow for prevention of milk fever and other periparturient mineral disorders.
      ). Disruptions in these adaptations, such as inflammation and other metabolic disturbances (
      • Horst E.A.
      • Mayorga E.J.
      • Al-Qaisi M.
      • Abeyta M.A.
      • Portner S.L.
      • McCarthy C.S.
      • Goetz B.M.
      • Kvidera S.K.
      • Baumgard L.H.
      Effects of maintaining eucalcemia following immunoactivation in lactating Holstein dairy cows.
      ), or reductions in DMI, have the potential to result in SCH at 4 DIM, which might explain the overall reduction in reproductive success that we observed in SCH cows compared with NC cows. Subclinical hypocalcemia has long been associated with reduced immune capacity because reductions in blood Ca limit Ca availability to immune cells, thereby impairing neutrophil function (
      • Kimura K.
      • Reinhardt T.A.
      • Goff J.P.
      Parturition and hypocalcemia blunts calcium signals in immune cells of dairy cattle.
      ). Similarly, Ca is essential for smooth muscle contraction, a necessary function of the uterus to expel uterine contents during and after parturition (
      • Hansen S.S.
      • Blom J.Y.
      • Ersbøll A.
      • Jørgensen R.J.
      Milk fever control in Danish dairy herds.
      ). As such, cows with SCH are at greater risk for developing uterine disease than their normocalcemic herdmates (
      • Martinez N.
      • Risco C.A.
      • Lima F.S.
      • Bisinotto R.S.
      • Greco L.F.
      • Ribeiro E.S.
      • Maunsell F.
      • Galvão K.
      • Santos J.E.P.
      Evaluation of peripartal calcium status, energetic profile, and neutrophil function in dairy cows at low or high risk of developing uterine disease.
      ), and the negative association between uterine disease, such as metritis, and reproductive success is well documented (
      • Fourichon C.
      • Seegers H.
      • Malher X.
      Effect of disease on reproduction in the dairy cow: A meta-analysis.
      ;
      • Giuliodori M.J.
      • Magnasco R.P.
      • Becu-Villalobos D.
      • Lacau-Mengido I.M.
      • Risco C.A.
      • De la Sota R.L.
      Metritis in dairy cows: Risk factors and reproductive performance.
      ).
      With the onset of lactation, not only does a cow's Ca requirement more than double, but so does her overall energetic and nutritional demand. The increases in energy and nutritional requirements are not often met with an equal increase in DMI, leaving the cow in a state of energy deficit (
      • Bell A.W.
      Regulation of organic nutrient metabolism during transition from late pregnancy to early lactation.
      ). It has also been shown that cows with persistent or delayed SCH consume less feed in early lactation compared with their normocalcemic counterparts (
      • Seely C.R.
      • Leno B.M.
      • Kerwin A.L.
      • Overton T.R.
      • McArt J.A.A.
      Association of subclinical hypocalcemia dynamics with dry matter intake, milk yield, and blood minerals during the periparturient period.
      ). The reduction in intake by these cows may exacerbate the state of energy deficit, thereby further limiting the energy available to immune cells and augmenting immune dysfunction and susceptibility to additional diseases.
      Individual cow intakes were not available for the cows included in our analysis, but we can surmise that cows with SCH at 4 DIM were simultaneously experiencing an exacerbated state of energy deficit compared with their normocalcemic herdmates. As ovarian follicles develop over an extended period, it has been hypothesized that follicles developing during a state of metabolic disruption, such as early lactation, sustain long-lasting damage and have diminished capacity to become a viable conceptus (
      • Britt J.H.
      Impacts of early postpartum metabolism on follicular development and fertility.
      ).
      Although the biological mechanisms responsible for the reduced reproductive success of cows with SCH cannot be elucidated from our analysis, our study reports the impacts of delayed or persistent episodes of SCH on reproductive outcomes. As mentioned previously, cows with SCH at 4 DIM are more likely to produce less milk and consume less feed and are at greater risk for additional disease development (
      • Neves R.
      • Leno B.
      • Thomas M.
      • Overton T.
      • McArt J.
      Association of immediate postpartum plasma calcium concentration with early-lactation clinical diseases, culling, reproduction, and milk production in Holstein cows.
      ;
      • McArt J.A.A.
      • Neves R.C.
      Association of transient, persistent, or delayed subclinical hypocalcemia with early lactation disease, removal, and milk yield in Holstein cows.
      ;
      • Seely C.R.
      • Leno B.M.
      • Kerwin A.L.
      • Overton T.R.
      • McArt J.A.A.
      Association of subclinical hypocalcemia dynamics with dry matter intake, milk yield, and blood minerals during the periparturient period.
      ). The reduction in pregnancy to first service and the overall decrease in the number of cows pregnant by 150 DIM with SCH at 4 DIM support previous observations that reductions in blood Ca occurring at 4 DIM present a homeostatic disruption, threatening the cow's productive and reproductive potential.

      Notes

      This work was supported by the Animal Health and Production and Animal Products-Improved Nutritional Performance, Growth, and Lactation of Animals grant no. 2019-67015-29446/project accession no. 1018890 from the USDA National Institute of Food and Agriculture (Washington, DC).
      The authors thank the participating farms, the staff at the Miner Institute (Chazy, NY), and Katie Callero, Kaitlyn Fang, Ian Frost, Annaliesse Hines, Sadie Huebler, Hannah McCray, Abby Reid, Jackson Seminara, and Cassandra Wilbur (Cornell University) for their assistance with sample collection.
      The authors have not stated any conflicts of interest.

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