Associations between days in the close-up group and milk production, transition cow diseases, reproductive performance, culling, and behavior around calving of Holstein dairy cows

The objective of experiment I was to evaluate the association between days in the close-up group (DINCU) and milk production, early lactation diseases, reproductive performance, and culling. In experiment II behavioral changes associated with DINCU were evaluated using a neck-mounted sensor (Smarttag neck, Nedap Livestock Management, Groenlo, the Netherlands). Cow-lactations of 28,813 animals from 14,155 individual cows of 2 farms in northern Germany and western Slovakia, calving between January 2015 and December 2020, were included in the study. After exclusion of cows with a gestation length <262 and >292 d and cows with >42 DINCU data from 8,794 and 19,598 nulliparous and parous cows, respectively, were available for final statistical analyses. To analyze the association between DINCU and second test-d 305-d mature-equivalent milk projection, linear mixed models were calculated. Binary data (i.e., clinical hypocalcemia, hyperketone-mia, retained placenta [RP], acute puerperal metritis [APM], mastitis, left displaced abomasum [LDA], first service pregnancy risk) were analyzed using logistic regression models. To analyze the association between DINCU and culling or death during the first 300 DIM Cox proportional hazards were used. To analyze the association between DINCU and behavior 7 d before to 7 d after calving (i.e., activity, inactivity, eating, ruminating time), linear mixed models were calculated. Nulliparous cows with a short (<10 DINCU) and a long stay (>30 DINCU) in the close-up group had a lower milk production an increased risk for hyperketo-nemia, RP, and APM compared with nulliparous cows with DINCU between 21 to 28 d. Parous cows with a short (<10 DINCU) and a long stay (>30 DINCU) in the close-up group had a lower milk production, an increased risk for RP and mastitis, a reduced first service


INTRODUCTION
The transition from late gestation to early lactation is subject to hormonal, behavioral and metabolic changes (Chebel, 2021).In the transition period, typically defined as the last 3 wk of gestation and the first 3 wk of lactation (Grummer, 1995), more than 90% of production diseases occur (Ingvartsen et al., 2003).To adjust for reduced DMI during the last days of gestation and to reduce susceptibility to diseases, dry cows are fed with different diets during the dry period (Grummer, 1995;Drackley, 1999;Van Saun and Sniffen, 2014).Whereas far-off diets are typically fed for the first 4 to 6 wk after dry off, close-up diets are provided starting around d 255 of gestation (Vieira-Neto et al., 2021).Far-off diets are prepared to feed high forage and high NDF content in combination with low energy density to avoid gain of adipose tissue (Drackley, 1999).During Associations between days in the close-up group and milk production, transition cow diseases, reproductive performance, culling, and behavior around calving of Holstein dairy cows the last weeks of gestation DMI decreases, reaching a nadir on the day of calving (Soriani et al., 2012;Pérez-Báez et al., 2019a).This is accompanied by a reduction in rumination time (Soriani et al., 2012).To account for the change in DMI, close-up diets have a higher energy density compared with far-off diets.Cows in the closeup period need more energy and protein in response to synthesis of mammary tissue and colostrum as well as for fetal and uterine growth (Van Saun and Sniffen, 2014).Furthermore, a 2-phase dry period offers the possibility of feeding a more aggressive negative dietary anion cation difference (DCAD) to cows in the closeup pen to reduce the risk for subclinical and clinical hypocalcemia (CH; Block, 1984).The ideal duration of feeding such a diet, however, is still being discussed.Recent studies provide evidence that a certain duration of days in the close-up group (DINCU) should be achieved to improve milk production and reduce susceptibility to early lactation diseases.There is a quadratic association between DINCU and milk production of parous cows (Chebel, 2021;Vieira-Neto et al., 2021;Venjakob et al., 2022) with a maximum milk production when cows stayed 21 DINCU (Venjakob et al., 2022).Vieira-Neto et al. (2021) also evaluated the association between DINCU and disease incidence until 60 DIM.In parous cows, there was a quadratic association between morbidity and DINCU.Cows with a short (i.e., <10 d) or a long (i.e., >30 d) stay in the closeup group had a greater risk for diseases until 60 DIM.Also, Chebel (2021) found an increased risk for uterine diseases such as retained placenta (RP), metritis and acute puerperal metritis (APM) in cows with a short or long stay in the close-up group.One reason for the detrimental effects of a short and long stay in the closeup group might be attributed to behavioral changes due to the need to establish a new social hierarchy close to calving (Nordlund et al., 2006) and an altered feeding behavior (Grant and Albright, 2001).On the contrary, a long stay in the close-up group may increase the risk for metabolic diseases caused by an increased risk for excessive body condition and reduced DMI (Olagaray et al., 2020).
The objective of experiment I was to evaluate the association between DINCU and milk production, early lactation diseases, reproductive performance, and culling.The objective of experiment II was to evaluate the association between DINCU and time spent eating, ruminating, active, and inactive.Our hypotheses were that cows with a short and a long stay in the closeup group have a reduced milk production, a greater risk for diseases after calving, a decreased reproductive performance, and ultimately an increased culling risk.We also hypothesized that cows with a short stay in the close-up group have an altered behavior.

MATERIALS AND METHODS
The current study is a retrospective data analysis.No human or animal subjects were used, so this analysis did not require approval by an Institutional Animal Care and Use Committee or Institutional Review Board.

Study Design
This observational retrospective cohort study used the data from 2 farms of 6 calendar years between January 2015 and December 2021.In experiment I, we used health and performance data from both farms from January 2015 to December 2020.In experiment II, we collected data on time spent eating, ruminating, active, and inactive 7 d before calving to 7 d after calving.The data were generated by a commercial sensor system (Smarttag neck, Nedap Livestock Management, Groenlo, the Netherlands; Borchers et al., 2021) in farm 2 between January 2020 and December 2021.
Farm 1 was located in northern Germany and farm 2 was located in western Slovakia.Both farms kept exclusively Holstein Friesian cows.During the study period, farm 1 and farm 2 had an average of 2,872 (±110; SD) and 2,823 (±216; SD) calvings per year, respectively.Inclusion criteria were participation in a DHIA testing system and a 2-phase dry cow period consisting of a far-off and a close-up period with feeding of a negative DCAD diet (<0 mEq/100g of DM).Furthermore, farms were required to record cow movements from the far-off to the close-up group in their herd management software (Dairy Comp 305, Valley Ag Software, Tulare, CA).
Close-up cows of both farms were fed a TMR once daily to meet or exceed minimum nutritional requirements (Table 1; NRC, 2001) and feed was pushed up 10 times a day.The TMR samples from the close-up diets were analyzed in certified commercial laboratories (farm 1: Landwirtschaftliche Kommunikations-und Servicegesellschaft mbH, Lichtenwalde, Germany; farm 2: Rock River Laboratory Europe, Heiddorf, Germany).

Transition Cow Management
In farm 1 and 2, nulliparous cows were moved once weekly to the close-up pen at d 255.7 ± 4.5 (SD) and 256.5 ± 5.2 (SD) of gestation, respectively.Parous cows were dried-off approximately 55 d before expected parturition and moved to the far-off group.Transfer of parous cows from the far-off to the close-up group was conducted on a weekly basis at d 257.0 ± 4.5 (SD) and 254.6 ± 5.2 (SD) of gestation on farm 1 and 2, respectively.On farm 1, nulliparous and parous cows were kept separately in sand-bedded freestall pens during the close-up period.On farm 2 nulliparous and parous cows were housed together in pens with deep-straw bedding.Prepartum nulliparous and parous cows were monitored every hour to detect signs of imminent parturition (i.e., restlessness, vaginal discharge with bloody traces, lying lateral with abdominal contractions, a visible or broken amniotic sac, or feet of the emerging calf outside the vulva).Animals were moved into an individual maternity pen (3.5 × 3.5 m on farm 1 and 8 × 8 m on farm 2) bedded with fresh straw when the amniotic sac was visible or broken outside the vulva, or appearance of feet of the emerging calf were detected outside the vulva.If the cow had not delivered the calf within 1 h calving assistance was provided (Schuenemann et al., 2011).All parous cows received prophylactic calcium supplementation (Bovikalc, Boehringer Ingelheim, Ingelheim am Rhein, Germany) at parturition and 12 to 24 h later.After parturition, cows on both farms were milked twice daily.Postpartum cows were examined daily by the farm personnel until 10 DIM following standard operating procedures created by the herd manager.These examinations have been described in detail, previously (Venjakob et al., 2021).Between 11 and 30 DIM cows were screened for health disorders based on visual observations performed by the farm personnel every morning at 0800 h and on deviations in their daily milk production.

Disease Definitions
For the purpose of this study, the following diseases were recorded during the first 30 DIM: CH, RP, APM, hyperketonemia (KET), left displaced abomasum (LDA) and mastitis.Because both of the farms had a long-term history in collaborating with the Clinic for Animal Reproduction, disease definitions were consistent between both farms: when cows were recumbent during one of the first 5 DIM and rose in response to an intravenous Ca infusion, cows were considered as having CH; hyperketonemia was defined as serum BHB concentrations ≥1.2 mmol/L using a cow-side BHB test (Precision Xtra, Abbott Laboratories, Abbott Park, IL; on farm 1 cows between 2 and 14 DIM were checked for ketosis every Monday and Friday; on farm 2 all cows were tested on 5 and 10 DIM); when percussion of the left flank resulted in tympanic resonance auscultated with a stethoscope, cows were considered to suffer from LDA; when fetal membranes were not expelled by 24 h after calving, cows were diagnosed with RP; signs of systemic illness (e.g., decreased milk production, dullness, or other signs of toxemia) in combination with an animal with an abnormally enlarged uterus, fetid watery red-brown uterine discharge and fever >39.5°C was considered as APM (Sheldon et al., 2006); clinical mastitis was defined as visible signs of inflammation in an affected mammary gland (i.e., redness, swelling, pain, or heat) and alterations such as clots, flakes, discoloration, or abnormal consistency of secretions (Vasquez et al., 2017).Udder health was evaluated 2 times daily by farm personnel during regular milking.

Reproductive Management
Farm 1. From January 2015 until February 2016, cows were inseminated for first service using a combination of AI after estrus detection or timed AI (TAI) with the Presynch-Ovsynch protocol (PGF 2α , 14 d later PGF 2α , 12 d later GnRH, 7 d later PGF 2α , 56 h later GnRH, and 16 to 18 h later TAI; Moreira et al., 2001).Cows underwent AI after estrus detection if detected in estrus after the second PGF 2α treatment of the Presynch portion of the protocol given at 50 ± 3 DIM.Cows not detected in estrus completed the Ovsynch portion of the protocol and received TAI at 72 ± 3 DIM.Estrus detection was conducted by farm personnel, using a combination of visual observation and tail Farm 2. Cows were inseminated after estrus detection according to the alert of the automated activity monitoring system (i.e., 2015 to 2019: CowManager SensOor, Agis, Harmelen, the Netherlands; 2020 and 2021: Nedap Livestock Management, Groenlo, the Netherlands) that was confirmed by a technician via transrectal palpation of a highly contractile uterus or visualization of clear, stringy vaginal discharge or received timed AI after hormonal intervention.Voluntary waiting period was 50 DIM (January 2015 to June 2020) and 60 DIM (July 2020 to December 2021), respectively.The sensor system generated a list of cows eligible for breeding, based on the activity value.If cows were confirmed being in estrus, insemination was conducted on the same day.Cows that were not bred until 80 ± 3 DIM received timed AI using a modified 7-d Ovsynch protocol including a second PGF 2α treat-ment on d 8 (GnRH, 7 d later PGF 2α , 24 h later PGF 2α , 32 h later GnRH, and 16 to 18 h later TAI; Wiltbank et al., 2015).Cows received second and subsequent AI services after detected estrus any time after a previous insemination.Cows not re-inseminated at detected estrus underwent pregnancy diagnosis through transrectal ultrasound (Easi-Scan:GO, IMV Imaging, Bellshill, Scotland) 39 ± 3 d after AI.At 32 ± 3 d after AI, all cows received a GnRH treatment.Open cows with at least one CL ≥15 mm received 2 PGF 2α treatments 24 h apart, GnRH 32 h after the second PGF 2α , and TAI 16 to 18 h later.Cows that did not meet the criteria to be included in the CL group received a modified Ovsynch protocol (i.e., 2 PGF 2α treatments; GnRH, 7 d later PGF 2α , 24 h later PGF 2α , 32 h later GnRH, and 16 to 18 h later TAI).All pregnancy diagnoses were conducted by trained farm personnel.A second pregnancy diagnosis was performed 80 d after AI.

Behavioral Data
Time spent eating, ruminating, active, and inactive was recorded with a 3-dimensional accelerometer (Nedap Smarttag Neck, Nedap, city) validated for behavioral monitoring previously (Borchers et al., 2021).The sensor distinguishes between for-and backward, left and right and up and down movements.Rumination measurements are based on the periodic movement of the jaw during regurgitation and subsequent chewing of the bolus in both lying and standing positions.Eating measurements are based on the specific head movements of the cow during the intake of feed.Inactive time measurement occurs when the cow is not actively moving and can occur in either standing or lying positions (Borchers et al., 2021).The sensor classifies specific behavior-associated movements performed for the majority of each minute.Data were obtained in records containing observations for each cow in 1-min periods and summed into 24 h periods starting 7 d before parturition until 7 d after parturition using a Python script (Plenio et al., 2021).Only complete data sets were considered for this analysis.

Statistical Analyses
Experiment I. Individual cow data of cows that calved between January 2015 and December 2020 were transferred from Dairy Comp 305 (Valley Ag Software, Tulare, CA) to Microsoft Excel (Office 2013, Microsoft Deutschland Ltd., Munich, Germany).Statistical analyses were performed using SPSS for Windows (version 25.0, SPSS Inc., Chicago, IL).
During the study period 34,170 calvings occurred.2017) gestation length (GL) is associated with health and milk production in early lactation.Because GL is highly correlated with DINCU (r = 0.565; P = 0.001), we initially considered GL in all models as a potential confounder; however, both farms consistently moved cows once weekly based on their GL from the far-off to the close-up group, leading to potential collinearity among the predictor variables in the statistical models.Therefore, we recalculated all models excluding GL.As the results changed marginally, GL was not included in the final models to avoid autocorrelation.We excluded, however, cows with a GL longer (>292 d, n = 64) and shorter (<262 d, n = 239) than 3 standard deviations (SD) from the mean.In addition, cows with DINCU >42 DINCU (n = 118) were excluded from the study.

After exclusion of records with missing information
To analyze the association between DINCU and second test-d 305-d mature-equivalent milk projection (2nd305ME; mean ± SD DIM at second test was 56.5 ± 14.5 d) linear mixed models were calculated using the GENLINMIXED procedure of SPSS.This outcome was chosen as it allows to include cows that were culled after the second test day.Furthermore, waiting for a complete 305-d milk production record would bias the estimate due to missing data of cows that did not complete the period of 305 d (McCarthy and Overton, 2018).Cow within year was the experimental unit.To account for the fact that observations from cows that were enrolled repeatedly in different years were not independent from each other, parity was used as a repeated measure in the models of parous cows.Separate models were built for nulliparous and parous cows.As described by Dohoo et al. (2009) each variable considered for the mixed model was analyzed in a univariate model, including the variable as a fixed factor (i.e., categorical variable) or covariate (i.e., continuous variable).The final mixed model included all variables resulting in univariate models with P ≤ 0.100.We included the year of calving (2015 to 2020), farm (farm 1 vs. farm 2), season of calving (winter from December 1 to February 28, spring from March 1 to May 31, summer from June 1 to August 31, and autumn from September 1 to November 30), sex of the calf (female vs. male calf), calving ease (0 = not observed, 1 = unassisted calving, 2 = calving assisted by 1 person, 3 = calving assisted by more than 1 person), stillbirth (yes vs. no), twins (yes vs. no), DINCU and the interaction DINCU by DINCU (DINCU × DINCU) as explanatory variables.In addition, the model for nulliparous cows contained age at first calving (continuous) and the model of parous cows contained parity (second vs. third vs. ≥fourth lactation) and 305-d milk production of the previous lactation (continuous).
Binary data on diseases (CH, RP, APM, KET, LDA, mastitis) and first service pregnancy risk (FSPR; yes vs. no) were analyzed using logistic regression models using the GENLINMIXED procedure of SPSS.As in nulliparous cows, only 5 of 8,798 cows were suffering from CH, no model was calculated to investigate the relation between DINCU and CH in these cows.Continuous variables were analyzed as covariates and categorical variables were analyzed as fixed factors in univariable models.Variables with P ≤ 0.100 were considered for the mixed model.In the model of FSPR, farm was forced to remain in the model as there have been considerable differences in reproductive management.As described above, cow within year was the experimental unit.To account for the fact that observations from cows that were enrolled repeatedly in different years were not independent from each other, parity was used as a repeated measure in the model of parous cows.Separate models were built for nulliparous and parous cows.The initial models for nulliparous and parous cows contained the same variables as described above for the linear mixed models.To account for multiple comparisons, the P-value was adjusted using a Bonferroni correction.Variables were declared significant when P < 0.050.
Collinearity among variables was explored for all GENLINMIXED models developed using the collinearity diagnostics in SPSS that provide the variance inflation factor (VIF) and tolerance.In each model, a correlation matrix is produced for all predictor variables.The VIF indicates whether a predictor has a strong linear relationship with the other predictor(s) and the tolerance statistics, which is its reciprocal (1/VIF).If more than one strongly collinear variable qualified for entry into a model, one variable was chosen based on biological significance.Collinearity was not detected based on the VIF (<1.5).
Using the SURVIVAL procedure of SPSS, 2 different Cox proportional hazards were calculated to model the time to event outcomes for culling or death during the first 300 DIM of nulliparous and parous cows, respectively.The variables year of calving, season of calving, farm, GL, DINCU and DINCU × DINCU were tested as risk factors.In addition, the model for nulliparous cows contained age at first calving and the model for parous cows contained parity and milk yield in previous lactation.The proportional hazard assumption was checked using Schoenfeld residuals.
Experiment II.Data on time spent eating, ruminating, active, and inactive were obtained 7 d before calving until 7 d after calving from the Nedap system between January 2020 and December 2021.In total, 4,977 observations from 723 cows of farm 2 were included in the analyzes.Data were processed using a Python script and transferred to Microsoft Excel (Office 2013, Microsoft Deutschland Ltd., Munich, Germany) and analyzed using R (Version 4.0, R Core Team).
The GAMLj procedure of R was used to analyze the association between DINCU and time spent eating, ruminating, active, and inactive around calving, linear mixed models were calculated separately for the time period before (−7 to −1 d) and after (+1 to +7 d) calving.For both periods, separate models were built for time spent eating, ruminating, active, and inactive.To evaluate the effect of DINCU, only cows were considered that spent exactly 7, 14, 21, 28, and 35 DINCU.Each variable considered for the mixed model was analyzed in a univariate model, including the variable as a fixed factor (i.e., categorical variable) or covariate (i.e., continuous variable; Dohoo et al., 2009).All variables resulting in univariate models with P ≤ 0.10 were included in the final mixed model.We included parity (nulliparous vs. parous), time (−7, −6, −5, −4, −3, −2, −1 or 1, 2, 3, 4, 5, 6, 7 d relative to calving), DINCU (7, 14, 21, 28, or 35 d), and the interaction of time by DINCU.To account for multiple comparisons, the P-value was adjusted using a Bonferroni correction.

5
Acute puerperal metritis was defined as sign of systemic illness (e.g., decreased milk production, dullness, or other signs of toxemia) in combination with an animal with an abnormally enlarged uterus, fetid watery red-brown uterine discharge, and fever >39.5°C.
After calving, we detected an association between DINCU and time spent inactive (P < 0.001), eating (P = 0.005), and ruminating (P = 0.008).Cows with 28 DINCU spent less time eating compared with cows with 14 (P = 0.008) and 21 DINCU (P = 0.024).Furthermore, cows with 28 DINCU spent less time ruminating than cows with 14 DINCU (P = 0.008).Moreover, we detected an association between the interaction of time by DINCU and time spent inactive (P = 0.030).Associations between behavior around calving and DINCU, GL, time relative to calving, parity and time by DINCU are described in Table 4.

DISCUSSION
The frequency distribution of DINCU in nulliparous and parous cows is displayed in Figure 1.Compared with a previously published study (Venjakob et al., 2022) cows were more consistently transferred from the far-off to the close-up group on a weekly basis on both farms.As can be derived from Figure 1, this led to a relatively small variation in DINCU.In nulliparous and parous cows, we observed an association between DINCU and milk production with an optimum milk production at 25 and 22 DINCU, respectively.In nulliparous cows with a short (<10 d) and a long (>30 d) stay in the close-up group, DINCU was associated with a higher risk for KET and RP compared with cows with DINCU between 21 to 28 d.Nulliparous cows with a short stay in the close-up group (<10 d) had an increased risk for APM compared with nulliparous cows with a longer stay in the close-up group.In parous cows, a long stay (>30 d) in the close-up group was associated with a higher risk for CH, KET, LDA, RP, mastitis and culling and a short stay in the close-up group (<10 d) was associated with an increased risk for RP, APM, mastitis and culling in comparison to cows with DINCU between 21 to 28 d.Whereas we detected no association between FSPR and DINCU in nulliparous cows, FSPR of parous cows was lower when staying <10 and >30 DINCU compared with parous cows with DINCU between 21 to 28 d.In nulliparous cows, DINCU was not associated with the risk for LDA.In contrast, parous cows with ≥35 DINCU had an increased risk for LDA compared with parous cows ).The risk for left displaced abomasum was associated with the year of calving (P = 0.021), farm (P < 0.001), season (P = 0.014), parity (P < 0.001), calving ease (P < 0.001), twins (P < 0.001), and DINCU (P < 0.027).
with <35 DINCU.Moreover, DINCU was associated with behavioral changes around calving.Before calving, cows with 7 and 35 DINCU were more inactive and spent less time eating and ruminating than cows with 14, 21, and 28 DINCU.

Association with Milk Production
In the present study, nulliparous and parous cows with a short (<10 DINCU) and long (>30 DINCU) stay in the close-up group had a reduced 2nd305ME compared with cows with 21 to 28 DINCU (Figure 2).Evaluating the association between DINCU and 2nd-305ME, we observed an association with an optimum milk production at 25 and 22 DINCU for nulliparous and parous cows, respectively.A similar observation was made by Vieira-Neto et al. (2021); although liparous cows were not exposed to a negative DCAD diet in their study, these authors also found a quadratic association between 300-d milk production and DINCU in nulliparous and parous cows with an optimum at 21 and 24 DINCU, respectively.In a previous multisite study, including 18 farms, we also observed a quadratic association between DINCU and milk production at first test day for parous cows (Venjakob et al., 2022).In this study however, DINCU were linearly associated with milk production of nulliparous cows.Especially, nulliparous cows exceeding 28 DINCU had an increased milk production.A comparable association was found by Degaris et al. (2008), who investigated the association of DINCU with milk production in grazing Holstein and Jersey × Holstein cows.These authors did not differentiate between nulliparous and parous cows and found a plateau in milk production when DINCU were between 17 and 35 d.

Association with Diseases
Parous cows with >30 DINCU had an increased risk for CH (Figure 3) compared with cows with ≤30 DINCU.Lean et al. (2006) speculated that feeding of acidogenic diets for a long time period might increase the risk for hypocalcemia as long-term activation of Ca metabolism could deplete Ca stores around calving.In .Association between days in the close-up group (DINCU) and the predicted risk of retained placenta (RP) using least square estimates (mean ± SEM) from the generalized linear mixed model.In nulliparous cows (n = 8,798, blue) the risk of RP was associated with the year of calving (P < 0.001), farm (P < 0.147), season (P = 0.005), calving ease (P < 0.001), stillbirth (P < 0.001), sex of the calf (P < 0.001), DINCU (P < 0.001) and DINCU × DINCU (P < 0.001).In parous cows (n = 19,641; yellow), the year of calving (P < 0.001), farm (P < 0.001) season (P = 0.016), parity (P < 0.001), calving ease (P < 0.001), stillbirth (P < 0.001), sex of the calf (P < 0.001), DINCU (P < 0.001), and DINCU × DINCU (P < 0.001) were associated with the risk of RP. contrast, this effect was not observed by Weich et al. (2013) who found a higher postpartum Ca concentration in cows fed with a negative DCAD diet for 42 d, compared with cows fed with a negative DCAD diet for the last 21 d of gestation.In a previous study colostrum yield increased with increasing DINCU (1 d increase in DINCU was associated with 60 g more colostrum; Borchardt et al., 2022).As colostrum contains high amounts of calcium (i.e., 2.3 g/kg; Goff, 2014) an increased colostrum yield in cows with a long stay in the close-up group might increase the Ca demand after calving, which may explain a greater risk for CH in parous cows.This can, however, not be confirmed in the present study, as data on colostrum yield were not available for the whole study period.Nulliparous and parous cows with a short (<10 DINCU) and a long stay (>30 DINCU) in the close-up group had an increased risk for KET compared with cows with DINCU between 21 to 28 d (Figure 4).Especially, an increased risk for KET in cows with a long stay in the close-up group seems plausible, as it might be attributed to increased gain of adipose tissue.For example, Chebel (2021) found increased BHB and nonesterified fatty acid concentrations in cows with a long stay in the close-up group.Likewise, we observed an increased risk for LDA in parous cows with ≥35 DINCU, compared with parous cows with <35 DINCU.As KET is a risk factor for LDA (Ospina et al., 2013) it seems plausible that the risk for LDA is increased alongside KET.In nulliparous cows no association between LDA and DINCU was observed.This might be attributed to the low incidence of LDA (0.42%; 37/8,798 cows) in nulliparous cows during the study period (Table 3).
In nulliparous and parous cows, we detected a quadratic association between DINCU and the risk of incurring RP (Figure 5).This is in agreement with Vieira-Neto et al. (2021).In their study, the incidence of RP was highest in cows with <10 DINCU.Similar to our study, the risk for RP was also increased when cows stayed >30 DINCU.One might speculate that cows with a short stay in the close-up group experience more stress due to regrouping shortly before calving and therefore have a higher risk for RP.In contrast, a long stay in the close-up group may be associated with .Association between days in the close-up group (DINCU) and the predicted risk of acute puerperal metritis (APM) using least square estimates (mean ± SEM) from the generalized linear mixed model of nulliparous cows and parous.In nulliparous cows (n = 8,798, blue), the risk for APM was associated with the year of calving (P < 0.001), farm (P < 0.001), calving ease (P < 0.001), stillbirth (P < 0.001), sex of the calf (P < 0.001), and DINCU (P = 0.020).In parous cows (n = 19,641; yellow), the year of calving (P < 0.001), farm (P < 0.001) season (P = 0.003), parity (P < 0.057), calving ease (P < 0.001), stillbirth (P < 0.001), sex of the calf (P < 0.001), milk production in previous lactation (P = 0.066), and DINCU (P < 0.001) were associated with the risk of RP.
excessive body condition, which in turn increased the risk for dystocia, vulvovaginal laceration and uterine diseases.It is well accepted that vulvovaginal laceration is associated with postpartum uterine diseases (Vieira-Neto et al., 2016).In nulliparous and parous cows, a short stay (<10 DINCU) in the close-up group was also associated with an increased risk for APM compared with cows with DINCU >10 d (Figure 6).Although cows with a short GL (<262 d) were excluded from the study, it is possible that some cows had a short stay in the close-up group due to biological reasons, as for example heat stress and twin birth may lead to a shortened GL.It has been shown, that short GL is a risk factor for postpartum uterine diseases (Vieira-Neto et al., 2017).Accordingly, Olagaray et al. (2020) observed detrimental effects of shortened dry periods due to biological reasons (i.e., short GL) in comparison to shortened dry periods due to management reasons.
In parous cows, a short (<10 DINCU) and a long stay (>30 DINCU) in the close-up group was associated with an increased risk for mastitis until 30 DIM compared with cows with DINCU between 21 to 28 d (Figure 7).These findings contradict results of Vieira-Neto et al. (2021).In their study, the same association was found in nulliparous cows.In parous cows, however, an increase from 7 DINCU to 28 DINCU led to a 2.3% increase in mastitis incidence until 90 DIM.These authors observed the lowest risk for mastitis, when parous cows stayed in the close-up group for 1 d.As the risk for mastitis increased until 25 DINCU and cows with 27 DINCU had the highest milk production, the authors speculated that these cows might have had increased risk factors for mastitis, such as milk leakage and longer exposure to bacteria during the milking process.Degaris et al. (2010) also observed an increased risk for mastitis until 150 DIM with increasing DINCU.Per day increase of DINCU the hazard for mastitis increased by 2%.These authors also suggested an association between the risk of mastitis and higher milk production as a cause for udder infections.Vieira-Neto et al. (2021) and Degaris et al. (2010) evaluated relatively long risk periods for mastitis (i.e., 90 and 150 d, respectively).The different observation periods (i.e., 30 vs. 90 or 150 DIM) might explain the controversial findings.This assumption is supported by results of Chebel (2021) who found similar results to ours, when evaluating the association between DINCU and mastitis by 30 and 60 DIM. .Association between days in the close-up group (DINCU) and the predicted risk for mastitis within 30 DIM (n = 19,641) using least square estimates (mean ± SEM) from the generalized linear mixed model.The risk for mastitis was associated with the year of calving (P < 0.001), farm (P < 0.001), parity (P = 0.014), calving ease (P < 0.001), stillbirth (P < 0.001), 305-d milk production in previous lactation (P = 0.078), DINCU (P < 0.001), and DINCU × DINCU (P < 0.001).

Association with Reproduction
In parous cows we detected an association between DINCU and FSPR and DINCU × DINCU and FSPR (Figure 9).Chebel (2021) and Vieira-Neto et al. (2021) also found an associations between DINCU and reproductive outcomes.Chebel (2021) observed an association between DINCU and pregnancy by 305 DIM.The chance of being pregnant was highest when nulliparous and parous cows stayed 28 DINCU.In parous cows, the probability of pregnancy was increased by 18.8% when comparing cows with ≤10 DINCU to cows with 28 DINCU.Vieira-Neto et al. (2021) observed a linear association between DINCU and FSPR.An increase from 7 to 28 DINCU was associated with a 4.4% reduced FSPR and cows had the least predicted FSPR when DINCU was the greatest.

Association with Culling Risk
In nulliparous and parous cows, we observed an increased culling risk in cows with a short (<10 DINCU) and a long stay (>30 DINCU) in the close-up group, compared with cows with DINCU between 21 to 28 d (Figure 10).This result is plausible as nulliparous and parous cows with short and long DINCU hand an increased risk for downstream outcomes, such as disease or culling.The result on culling is in agreement with Vieira-Neto et al. (2021).In their study the same observation was made for nulliparous cows.Chebel (2021) and Venjakob et al. (2022) observed an increased culling risk, predominantly in cows with a short stay in the close-up group.

Association with Behavioral Changes
This is the first study evaluating the association between DINCU and time spent eating, ruminating, active, and inactive around parturition.Cows with 7 DINCU were highly active on the day of movement from the far-off to the close-up group (Figure 11A).According to Nordlund et al. (2006), a cow that is moved into a new group experiences stress, as she has to establish her rank within the social group of the pen.Compared with new arrivals, cows resident within the pen tend to maintain their social position.Kondo and Hurnik (1990) suggested that reestablishment of rank within a group takes approximately 2 d.This can .Association between days in the close-up group (DINCU) and the predicted first service pregnancy risk (FSPR) using least square estimates (mean ± SEM) from the generalized linear mixed model of parous cows (n = 19,585; yellow).The year of calving (P < 0.001), season (P = 0.003), parity (P < 0.001), stillbirth (P = 0.007), calving ease (P < 0.001), DINCU (P = 0.003), and DINCU × DINCU (P = 0.001) were associated with the predicted FSPR.To account for the different reproductive management on both farms, farm (P = 0.941) was forced to remain in the model.be confirmed by our data on activity, as cows with 7 DINCU also had an increased activity on d −6 (Figure 11A).Nordlund et al. (2006) suggested that pen moves close to calving do have adverse effects, as cows were involved in more interactions per hour.It has been shown that pen moves may alter feeding behavior for several days (Grant and Albright, 2001).The increase in social interactions indicative of the establishment of the rank within the hierarchy of the pen in cows with 7 DINCU might explain, why time spent eating (Figure 11C) and ruminating (Figure 11D) were reduced and inactivity increased in these cows (Figure 11B) before calving (Cook and Nordlund, 2004).Cows with 35 DINCU were also more inactive and had a reduced feeding and ruminating time before calving.A reduction in pre-partum feeding behavior may be associated with an increased risk for certain diseases as observed in our study, but also by others (Huzzey et al., 2007;Pérez-Báez et al., 2019a, 2019b).The relation between a reduced feeding behavior can, however, be confounded by DINCU, as cows with a long stay (35 d) in the close-up group spent less time eating.Extended dry period length is associated with increased body condition at calving (Chebel et al., 2018), which may lead to metabolic stress and enhanced lipolysis at calving (Weber et al., 2015) and decreased DMI (Roche et al., 2013).Because rumination time is reduced alongside feeding time (Beauchemin, 2018), it seems plausible that cows with a reduced feeding time, spend less time ruminating.It has been shown that prepartum intake is associated with diseases after calving (Huzzey et al., 2007;Pérez-Báez et al., 2019a, 2019b).Behavioral changes associated with a short and long stay in the close-up group may explain the increased risk for negative downstream outcomes in these cows.

Study Limitations
When evaluating the association of DINCU and early lactation health and performance, it is difficult to differentiate the effect of DINCU from the effect of GL using observational studies.In the present study, we addressed this issue by including GL into all initial models.As cows were moved very consistently to the close-up group in these 2 farms, we assumed a strong correlation among GL and DINCU and recalculated all   4. models excluding GL.Because exclusion of GL did not lead to major changes in the results, the results of the models without GL are presented herein.Future studies should be conducted in a randomly controlled fashion, specifically evaluating the effect of short DINCU on health and performance.
Even though it has been shown that the 3-dimensional sensor accurately monitored time spent eating, ruminating, active, and inactive (Borchers et al., 2021), we caution to extrapolate these results to actual dry matter intake, as the relationship between time spent eating and ruminating and DMI has not been sufficiently investigated.Future studies should investigate whether the behavioral changes observed in this study have a lasting effect on health and performance.Moreover, only 723 cows from one of the 2 farms were included in experiment II, limiting the validity of the results.
We are aware that disease identification on different farms for 6 years is prone to variability and bias among different observers, which might lead to false-negative or false-positive diagnoses (Kelton et al., 1998;Sannmann et al., 2012).As both farms had a long-term history of collaboration with the Clinic for Animal Reproduction and standard written operating procedures were provided by the herd manager in coordination with the researchers, we are confident that the risk of misclassification is low and that diseases have been recorded consistently over the course of this study.Evaluating the association between DINCU and APM we caution however that only severe cases of metritis were included in the statistical analyzes.A broader definition of the condition might have altered the results (McCarthy and Overton, 2018).
On the 2 participating farms, transfer from the faroff to the close-up group was conducted consistently based on the d of GL on a weekly basis.Only few cows stayed <10 d and longer than 30 DINCU.Moreover, nulliparous and parous cows both received a negative DCAD diet before calving.Therefore, the external validity of this study is limited.

CONCLUSIONS
The present study provides further evidence that DINCU were associated with productive, reproductive and disease outcomes.Moreover, DINCU were associated with time spent eating, ruminating, active, and inactive around the time of calving.Nulliparous cows with a short and long stay in the close-up group had a reduced milk production and an increased risk for RP, KET and APM compared with nulliparous cows with DINCU between 21 to 28 d.In parous cows, an increased risk for culling was observed in addition to increased disease incidences, a reduced FSPR, and a reduced milk production compared with parous cows with DINCU between 21 to 28 d.The results of our study suggest that pen moves during the last 10 d of gestation should be avoided.The reduced time spent eating and ruminating might lead to lower DMI increasing the risk for subsequent diseases.A similar effect was observed for cows that stayed for a longer period (30 DINCU) in the close-up group.Based on the observed GL (277 ± 4.8 d) we recommend to move cows from the far-off to the close-up group on d 254 of gestation to achieve an average stay in the closeup group of 3 wk.Randomized controlled trials are needed to further elucidate the importance of DINCU as a risk factor for health and performance in transition dairy cows.
Venjakob et al.: ASSOCIATIONS BETWEEN DAYS IN THE CLOSE-UP GROUP about DINCU, 28,856 lactations from 14,173 cows were remaining.As shown by Vieira-Neto et al. ( Figure 1.Frequency distribution of days in the close-up group of 8,798 nulliparous (blue) and 19,641 parous cows (yellow).
Venjakob et al.: ASSOCIATIONS BETWEEN DAYS IN THE CLOSE-UP GROUP

Table 1 .
Venjakob et al.: ASSOCIATIONS BETWEEN DAYS IN THE CLOSE-UP GROUP Chemical composition (% unless otherwise noted) of the close-up diets from both farms enrolled 1

Table 2 .
Multiple comparison among cows that stayed 7, 14, 21, 28, or 35 d in the close-up group and the association with 305-d milk projection (2nd305ME), retained placenta (RP), acute puerperal metritis (APM), hyperketonemia (KET), clinical hypocalcemia (CH), left displaced abomasum (LDA), mastitis, first service pregnancy risk (FSPR), and survival during the first 300 DIM (CULL300) 1 1All comparisons are based on estimates from the GENLINMIXED model or the Cox proportional hazard model.Outcomes were only included in the table when associated (P < 0.050) with days in the close-up group (DINCU) or the interaction of DINCU × DINCU.2 305-d milk projection based on second test day milk production.3

Table 4 .
Venjakob et al.: ASSOCIATIONS BETWEEN DAYS IN THE CLOSE-UP GROUP The least squares means and SEM as derived from the linear mixed model for time spent eating, ruminating, active, and inactive in minutes per day, considering days in the close-up group (DINCU)