Increased dietary calcium inclusion in fully acidified prepartum diets improved postpartum uterine health and fertility when fed to Holstein cows

Journal Pre-proof Increased dietary calcium inclusion in fully acidified prepartum diets improved postpartum uterine health and fertility when fed to Holstein cows

K.T. Ryan, A.R. Guadagnin, K.M. Glosson, S.S. Bascom, A.A. Rowson, A.J. Steelman, F.C. Cardoso PII:

S0093-691X(19)30467-4

DOI:

https://doi.org/10.1016/j.theriogenology.2019.10.014

Reference:

THE 15209

To appear in:

Theriogenology

Received Date:

13 July 2019

Accepted Date:

13 October 2019

Please cite this article as: K.T. Ryan, A.R. Guadagnin, K.M. Glosson, S.S. Bascom, A.A. Rowson, A.J. Steelman, F.C. Cardoso, Increased dietary calcium inclusion in fully acidified prepartum diets improved postpartum uterine health and fertility when fed to Holstein cows, Theriogenology (2019), https://doi.org/10.1016/j.theriogenology.2019.10.014

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

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Increased dietary calcium inclusion in fully acidified prepartum diets improved postpartum uterine health and fertility when fed to Holstein cows

K. T. Ryana, A. R. Guadagnina, K. M. Glossona, b , S. S. Bascomb, A. A. Rowsonb, A. J. Steelmana, and F.C. Cardosoa*

aDepartment

bPhibro

of Animal Sciences, University of Illinois, Urbana, Illinois, USA;

Animal Health Corporation, Teaneck, New Jersey, USA

*Corresponding author: 1207 W. Gregory Drive, Urbana, IL 61801; phone: 217.300.2303; fax: 217.333.7088; email: [email protected] (F.C. Cardoso).

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ABSTRACT The objective of this study was to determine the effects of prepartum negative dietary cation-anion difference diet (DCAD) fed at two dietary Ca inclusion rates on postpartum uterine health and ovulation dynamics of multiparous Holstein cows (n = 76). Treatments began at 28 days before expected calving until parturition and were: CON: DCAD = +6 mEq/100g of DM with low dietary Ca (46.2 ±15.2 g Ca/d; 0.4% DM; n = 26); ND: DCAD = -24 mEq/100g of DM with low dietary Ca (44.1 ± 16.1 Ca/d; 0.4% DM; n = 24); NDCA: DCAD = -24 mEq/100g of DM with high dietary Ca (226.6 ± 96.0 g Ca/d; 2.0% DM; n = 26). Vaginal discharge was evaluated through the fresh period via Metricheck (MC) for presence of purulent material. Polymorphonuclear (PMN) cell concentration in the uterus was evaluated at 15 and 30 days relative to calving (DRC). Endometrial tissue was harvested at 30 DRC for glandular morphology, presence of tight-junctions and adheren-junctions proteins, as well as assessment of superoxide dismutase (SOD) and glutathione peroxidase (GPX) activity. Blood plasma and serum samples were harvested in the prepartum and postpartum phase and were assessed for concentrations of lipopolysaccharide binding protein (LBP), serum amyloid A (SAA), and haptoglobin (HP). Ovarian dynamics were assessed through the fresh period until first timed artificial insemination (TAI). Cows fed CON had a lower MC score (P = 0.06) than the average of cows fed ND and cows fed NDCA. Cows fed ND had a higher MC score than cows fed NDCA. Cows fed NDCA had greater uterine gland epithelial height (P = 0.02) than cows fed ND. Cows fed NDCA also had a greater number of epithelial cells per gland (P = 0.05) than cows fed ND. Cows fed NDCA had greater intensity of occludin expression (P = 0.15) than cows fed ND. Cows fed NDCA had increased 2

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activity of SOD (P = 0.05) and decreased activity of GPX (P < 0.001) than cows fed ND. Cows fed ND had higher plasma HP concentrations than cows fed NDCA in the prepartum (P = 0.01) and post-partum (P = 0.03) periods. Cows fed ND and NDCA had lower (P = 0.01) postpartum plasma HP concentration than cows fed CON. In conclusion, cows fed NDCA had an improved uterine environment most likely due to alleviation of oxidative stress, an enhanced immune response to parturition and uterine discharge comparable to cows fed CON.

Key words: Negative DCAD; uterine health; calcium; occludin; E-cadherin 1

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1.

INTRODUCTION During the transition period (3 weeks before calving through 3 weeks after

calving) the cow is subjected to various stressors that include metabolic disorders, prolonged inflammation, and most prevalently, subclinical hypocalcemia (SCH) [1–4]. As the periparturient cow transitions from a pregnant and non-lactating status, to lactation, her energy and nutrient demands increase dramatically [5,6]. In the first 3 days following parturition, the cow is often in a negative energy balance as well as negative Ca status [4,7,8]. If this status is chronic or prolonged, the cow becomes predisposed to numerous disorders observed around calving such as retained placenta, displaced abomasum, ketosis, metritis and mastitis [6,9,10]. If left untreated, hypocalcemia and SCH [11], can reduce the ability of immune cells to respond to stimuli [10], thus increasing susceptibility to infections such as metritis [11]. Many strategies have been employed to treat the negative Ca balance such as Ca boluses, or injections of Ca salts [9], however, these are often only treatments to the clinical signs of the disease. An effective preventative strategy in controlling SCH is to induce the pregnant cow into a state of compensated metabolic acidosis, achieved by feeding a negative dietary cation-anion difference (DCAD) diet in the 4 weeks before parturition [12,13]. This negative DCAD is achieved by an acidification of the diet and effectively reduces Ca absorption in the prepartum phase to maintain calciotropic hormones (parathyroid hormone; PTH) sensitivity, so that the cow has the capacity to mobilize and absorb Ca in the post-partum period [1,9,14,15]. The urine pH is a parameter normally used to monitor this degree of acidification and it may reflect blood pH status [16]. A fully 4

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acidified prepartum diet usually maintains urine pH between 5.5 and 6.0, while a partially acidified prepartum diet maintain the urine between 6 and 7 [14, 16]. Inflammation related blood metabolites, such as lipopolysaccharide binding protein (LBP), serum amyloid A (SAA), and haptoglobin (HP), are key indicators of systematic inflammation [17,18]. These indicators can be related to inflammation as acute phase proteins throughout the body of the cow. At parturition, the uterus of the dairy cow can be contaminated with pathogens [19] and this contamination can develop into severe uterine inflammation (i.e.; metritis). The immune response during the peripartum period is known to be suppressed due to the sudden change to a negative energy balance system [3]. The supression of the immune response can diminish the ability of the dairy cow to tolerate pathogens [20] and furthermore enhance the severity of inflammation within the uterus, thus, prolonging the return to normal ovarian cyclic activity [21]. The return of normal ovarian cyclic activity is dependent on the uterine involution process and return to a non-inflammatory state [22]. Uterine infection and inflammation can prolong the uterine involution process [22] with detrimental effects on ovarian function [23] as well as uterine function (hormone secretion and uterine adenogenesis). At the time of parturition, the dairy cow experiences physical trauma and bacterial contamination of the endometrium [19]. This damage and contamination of the endometrium initiates an immune response in the sites of infection and trauma, and results in reactive oxygen species (ROS) response [24]. Reactive oxygen species production, a common byproduct of cellular metabolism, is tightly controlled by antioxidant enzymes, primarily superoxide dismutase (SOD) and glutathione peroxidase 5

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(GPX), to maintain the reduction-oxygenation (redox) environment within the cell [25]. In high concentrations of ROS, such that it overwhelms the tissues antioxidant capacity, damage to cellular macromolecules (lipids and proteins) can happen and induce cell death (apoptosis) [25]. Pathogen associated molecular patterns (PAMPs), such as lipopolysaccharides from the cell wall of gram-negative bacteria, also produce ROS once bound to the cell PAMP receptors [26]. Kimura et al. [10] observed that cows diagnosed with hypocalcemia experienced a blunted immune cell response at the time of parturition, possibly due to the unavailable Ca, an important secondary signaling molecule in immune response pathways. The objective of this study was to determine the effects of feeding fully acidified negative DCAD prepartum diets with two different concentrations of dietary Ca on the reproductive health postpartum. Reproductive performance was evaluated by the cows’ ovarian development, likelihood of pregnancy, uterine health, and inflammatory blood metabolites.

2.

MATERIALS AND METHODS The following procedures were approved by the University of Illinois Institutional

Animal Care and Use Committee (IACUC; protocol # 16115). 2.1 Animals and experimental design A total of 76 multiparous Holstein cows entering their second or greater lactation [BW (mean ± SD) = 770.1 ± 13.9 kg; parity = 3.0 ± 1.4)] were enrolled in the experiment from 50 days before calving until 75 days postpartum. The experiment began in 6

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September 2016 and concluded in December 2017. Cows were blocked for treatment assignment based on parturition, previous lactation 305-d milk yield (12,219 ± 123.5 kg) and body condition score. Treatment began at 28 days before expected calving and ended at parturition. Treatments were CON (n = 26): DCAD = +6 mEq/100g of DM (urine pH = 8.12) with low dietary Ca (46.2 ±15.2 g Ca/d; 0.4% DM; n = 26); ND (n = 24): DCAD = -24 mEq/100g of DM (urine pH = 5.76) with low dietary Ca (44.1 ± 16.1 Ca/d; 0.4% DM; n = 24); and NDCA (n = 26): DCAD = -24 mEq/100g of DM (urine pH = 8.67) with high dietary Ca (226.6 ± 96.0 g Ca/d; 2.0% DM; n = 26). Cows were housed in a free stall style barn at the University of Illinois Dairy Research Farm during the dry period. Immediately after calving, cows were moved to a tie stall style barn until 30 days relative to calving (DRC) and then finally moved into free stall style lots for the remainder of the trial. A complete total mixed ration (TMR) was provided during the dry and lactation period that met but did not exceed the energy requirements of the cows. Dietary cation anion difference was calculated using: 𝐷𝐶𝐴𝐷 = (𝑁𝑎 + + 𝐾 + ) ―(𝐶𝑙 ― + 𝑆 ―2) [27]. 2.2 Vaginal discharge and evaluation Evaluations of vaginal discharge were performed at 4, 7, 10, 13, 15, 17, and 30 DRC via the Metricheck® device (MC, Simcro, New Zealand), following the procedure described by Skenandore et al [28]. The device is composed of a 50 cm long stainless steel rod with a 4 cm rubber hemisphere to collect vaginal contents. The MC device was disinfected before and after each use in a single cow with chlorhexidine diacetate disinfectant (Nolvasan Solution, Zoetis Animal Health, Florham Park, NJ). The evaluation began with cleaning the perineal region of the cow with a paper towel and disinfectant solution. The tail was then moved to the side and the MC was inserted into 7

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the vaginal canal until the cervix was reached. The device was then retracted and removed from the reproductive tract with the vaginal contents remaining in the rubber hemisphere. With the vaginal content in the rubber hemisphere, evaluation of smell was scored (smell 0 = no odor or smell 3 = fetid odor). The vaginal contents was then poured onto a paper towel for examination and scored on a scale of 0 – 3: score 0 = clear or translucent mucus; score 1 = mucus containing flecks of white or off-white pus; score 2 = discharge containing ≤ 50% white or off-white mucopurulent material; and score 3 = discharge containing ≥ 50% purulent material, may be white, yellow or sanguineous [20]. Rectal temperature (GLA M700 Thermometer, GLA Agricultural Electronics, San Luis Obispo, California) and ultrasonography (7.5-MHz linear array probe, E.I. Medical Imaging, Loveland, Colorado) of uterine content (yes = contained hyperchogenic material or no = contained no visible material) were also obtained after vaginal evaluation. The MC was not used if the cow had retained placenta or visible vulvar damage at the time of the metricheck. 2.3 Cytology of the uterine endometrium Cytology of the endometrium was performed using a cytology brush (Andwin Scientific, CA) at 15 and 30 DRC. The sterile cytology brush was mounted to a sterile stainless steel cytology rod and inserted into a larger sterile stainless steel rod (SSR) covered with a plastic sleeve for easy passage through the cervix and into the uterine body without contamination. Prior to the procedure, the cow was restrained and the vulva was cleaned with water and 70% ethanol. After passage of the cytology rod through the first ring of the cervix, the SSR was exposed through the plastic sleeve and was advanced into the uterine body. Once inside the uterine body, the outer SSR was 8

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pulled back to expose the cytology brush. The SSR that was mounted to the cytology brush was then rotated three times while the cytology brush remained in contact with the endometrium. Finally, the cytology brush was retracted back into the outer SSR and removed from the reproductive tract. The SSRs were washed and autoclaved between each day of use. If multiple samples were being taken during a single day, the SSRs were sanitized in a chlorhexidine diacetate disinfectant solution between each animal. Cytology slides were prepped immediately following the procedure previously described by Stella et al. [29], where the cytology brush was rolled onto a clean glass microscope slide and fixed using a cytology fixative (Cytoprep, Fisher Scientific, Pittsburg, PA). Once the fixative was dry, the samples were transported to the laboratory where they were stained with a differential stain (Camco Quik Stain 2 – Self Buffered Differential Wright-Giemsa Stain, Cambridge Diagnostic Products, FL). After being allowed to dry for 24 hours, the slides were covered using a mounting medium (Permount, Fisher Scientific, Pittsburg, PA) and dried for at least 48 hours before being scanned. All slides were scanned at the Institute for Genomic Biology at the University of Illinois with 20X magnification using whole slide imaging (Nanozoomer Digital Pathology System, Hamamatsu Photonics, Japan). Five areas were captured at 20X magnification from five separate locations, one image from each corner of the sample area of the slide and one image from the center, to represent the entire population of the slide (NDP-view software, Hamamatsu Photonics). A minimum of 100 cells were counted within the individual areas (ImageJ, National Institutes of Health, MD) and the proportion of PMN to epithelial cells was determined. Cell counting was performed by the same technician for all samples. 9

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2.4 Endometrium biopsy Endometrial tissue samples were collected transcervically from the body of the uterus from all eligible cows at 30 ± 1.32 DRC, as described elsewhere [29]. The biopsy was not performed if the cow had a MC score of 3, a smell score of 3, or if the body temperature, pulse or respiration rates were abnormally high (rectal temperature > 39.5º C; respiratory rate >50 breaths per minute; heart rate >84 beats per minute). The biopsy instrument (Aries Surgical, Davis, CA) was covered with a sanitary disposable sleeve and inserted into the vagina. The biopsy forceps was positioned at the cervical opening, and the sleeve was retracted to force the instrument through the end of the sleeve. The exposed biopsy forceps was then threaded through the cervix and into the uterine body. Endometrial tissue was collected at a location approximately 1 cm beyond the end of the cervix. The sample was placed into phosphate buffered saline (PBS) containing 4% paraformaldehyde for 24 h. The samples were then set in a block of paraffin wax at the University of Illinois Veterinary Diagnostic Lab for hematoxylin and eosin staining or placed in PBS containing 30% sucrose at 4°C for 24 h. After staying in sucrose solution for 24 h, samples were embedded in optimal cutting temperature (OCT) gel and frozen at -80 ºC until the fluorescent immunolabeling. 2.4.1 Hematoxylin and eosin, glutathione peroxidase 1, and superoxide dismutase staining of the uterine endometrium The staining performed by the histology lab included 1) hematoxylin and eosin, 2) glutathione peroxidase 1 (antibody dilution 1:100, GPX, ab59546, Abcam, Cambridge, UK), and 3) superoxide dismutase 1 (antibody dilution 1:100, SOD, ab13498, Abcam, Cambridge, UK). The slides were stained according to the protocols of the lab [30]. In 10

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brief, slides were deparaffinized in graded alcohols and steamed for 1 h in citrate buffer (pH = 6). Samples were then blocked using 3% hydrogen peroxide for 10 min and washed with tris-buffered saline (TBS). Background protein blocking occurred using background buster (NB306, Innovex Biosciences, Richmond, CA, USA) and the primary antibodies (either GPX for 1 h or SOD for 2 h) were incubated at room temperature. The slides were washed 3 times with TBS and incubated with the secondary antibody (Rabbit on Rodent HRP-Polymer, RMR 622, Biocare Medical, Pacheco, CA, USA) for 30 min. Finally, the samples were washed with TBS (3 times for 5 min each), stained with DAB (Innovex Biosciences, Richmond, CA, USA) and counter stained with hematoxylin before being dehydrated and mounted. Slides were scanned using whole slide imaging with a 20X objective (Nanozoomer Digital Pathology System, Hamamatsu Photonics, Japan). After whole image scanning, individual gland structures were labeled and images were captured (NDP.view2 Viewing software, Hamamatsu Photonics, Japan). Total glandular area and perimeter, glandular epithelial height, number of glandular epithelial cells, and number of glands per tissue sample were measured and calculated by the ImageJ software (version 1.47, National Institutes of Health, MD, USA). All glandular measurements were obtained by the same trained technician. Additionally, after whole image scanning samples were stained with SOD1 and GPX1 antibodies, then 10 images were captured with a 40X objective (NDP.view2 Viewing software, Hamamatsu Photonics, Japan) and the percentage of positive cells were obtained by counting the number of positively and negatively immunolabeled cells (ImageJ version 1.47, National Institutes of Health, MD, USA). All slides were counted by the same trained technician. 11

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2.4.2 Fluorescent immunolabeling of the uterine endometrium with occluding and Ecadherin 1 antibodies Uterine frozen samples embedded in OCT were sectioned (15 μm thickness) and placed into positive slides for the immunolabeling process. Slides containing tissue sections were washed three times in PBS and incubated with 5% goat serum [diluted 1:20in 0.3% PBST (PBS with 0.3% v/v Triton X-100)] for 1 h at room temperature. Cryosections were then washed three times with PBS and incubated with rabbit antihuman polyclonal occludin (diluted 1:200 with 0.3% PBST) overnight. Following that, samples were rinsed in PBS three times over 15 min and incubated with Alexa Fluor 488-labeled goat anti-rabbit IgG (diluted 1:1000 with 0.3% PBST) in the dark for 30 min. Samples were washed again with PBS and incubated with anti-CD324 E-cadherin 1 monoclonal antibody (clone: DECMA-1) (diluted 1:800 with 0.3% PBST) overnight. The sections were then rinsed with PBS three times over 15 min and incubated in the dark with Alexa Fluor 594-labeled goat anti-rat IgG (diluted 1:1000 with 0.3% PBST) for 30 min, washed three times with PBS then stained with Hoechst 3570 (diluted 1:2000 in PBS; Life Technologies) for 2 min then washed three additional times. Finally, the tissue was mounted on cover glass in Fluoromount-G™ medium. Sections were then imaged on a confocal fluorescence microscope (Carl Zeiss, LSM 710 Oberkochen, Germany). Verification of molecular identity was performed by assessing single-plane confocal images acquired using a constant 1.0-μm-thick optical section and a 20X and 63X oil-immersion objective lens. Each fluorescent channel was imaged in a separate track. Appropriate sets of laser beams and emission windows were used for Alexa Fluor 488 (emission 500-550 nm), Alexa Fluor 594 (emission 80012

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850 nm), and DAPI (emission 400-450 nm). Intensity of emission was analyzed with the image processing software AxioVision 4.6 (Carl Zeiss MicroImaging Oberkochen, Germany). 2.5 Ultrasonography of ovarian structures and synchronization protocols The first postpartum follicular growth cycle was monitored at 7, 9, 11-17, 20, and 30 DRC via transrectal ultrasonography (7.5-MHz linear array probe, E.I. Medical Imaging, Loveland, Colorado). Follicular growth continued to be monitored in tandem with the synchronization protocol until 75 DRC at which the cow was bred. Ultrasonographic videos of ovarian structures were recorded to allow drawing of ovarian structures and measurement of the follicles present. Once the dominant follicle was ≥ 5 mm in diameter in the recordings, measurements were continually recorded at all relevant time points until the end of the first follicular wave. Ovulation was classified as the disappearance of the previously identified dominant follicle and the appearance of a corpus luteum (CL) in the subsequent examinations. Synchronization protocol started on Day 30 ± 1 relative to calving. Estrous cycles were presynchronized with one injection of PGF2α (25 mg, intramuscular of dinoprost tromethamine; 5 mL of Lutalyse, Zoetis Animal Health, NJ, USA). On Day 41, cows received the first injection of GnRH (100 mg, intramuscular, of gonadorelin hydrochloride; 2 mL of Factrel, Zoetis Animal Health). On Day 48 cows received an injection of PGF2α. On Day 55 cows received an injection of GnRH. On Day 62 cows received an injection of GnRH and a controlled internal drug-release insert (CIDR; Eazi-Breed CIDR, Zoetis Animal Health) containing 1.38 g of progesterone. Seven days later (Day 69), cows received an injection of PGF2α, intramuscular, concurrent with the removal of the CIDR insert. On Day 71, cows 13

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received the last injection of GnRH. Cows were artificially inseminated at 12 hours (Day 72) after GnRH using commercially available Holstein sires. The same technician performed all artificial inseminations. 2.6 Blood sampling and analysis Blood was sampled from the coccygeal vein or artery at 21 and 7 days before expected calving and at 15 and 30 DRC from each cow for serum and plasma collection (BD Vacutainer; BD and Co., Franklin Lakes, NJ). Additional time points for blood samples were shared from the other portion of this study. These serum and plasma samples were obtained by centrifugation of the tubes at 699 × g for 15 min and stored at −80℃. Plasma lipopolysaccharide-binding protein (LBP; Human LBP Multispecies Reactive ELISA Kit, Cell Sciences, Newburyport, MA) and serum amyloid A (SAA; PHASETM Range Multispecies SAA ELISA kit, Tridelta Development LTD, Maynooth, Ireland) were assessed at 30, 21, and 7 days before expected calving and 15 and 30 DRC. Haptoglobin (HP; PHASETM Range Haptoglobin kit, Tridelta Development LTD, Maynooth, Ireland) was assessed at 14, 7, 4, 2, and 1 days before expected calving and 1, 2, 4, 7, and 14 DRC. 2.7

Statistical analysis Statistical analyses were performed using SAS 9.4 (SAS Institute Inc. Cary, NC,

USA). The MIXED procedure of SAS was used to model the fixed effects of treatment, day, and block using the following model: Yijk = μ + 𝑇𝑖 + 𝐷𝑗 + 𝑇𝑖 × 𝐷𝑗 + 𝐵𝑘 + εijk

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where Yijk = the observations for dependent variables; μ = the overall mean; 𝑇𝑖 = the fixed effect of the ith treatment; 𝐷𝑗 = the repeated measurement (DRC) effect; 𝑇𝑖 × 𝐷𝑗 = the interaction of treatment and repeated measurement; 𝐵𝑘 = effect of the kth block; and εijk = the random residual error. Cow was considered the experimental unit and included as a random effect. Residual distribution was evaluated for normality and homoscedasticity of variance in all analyses and transformation were used where appropriate. The estimation method was restrictive maximum likelihood (REML) and the degrees of freedom method was Kenward-Rogers [31]. Variables were subjected to 5 covariance structures: compound symmetry, autoregressive order 1, autoregressive heterogeneous order 1, unstructured, and toeplitz. The covariance structure that yielded the lowest corrected Akaike information criterion was compound symmetry and used in the model [28]. Cow was the experimental unit and considered as a random effect. Two contrasts were used: Contrast 1 (CONT1): CON compared with the average of NDCA and ND; and contrast 2 (CONT2): ND compared with NDCA. A logarithmic transformation was used for circulating LBP and HP concentrations, and for intensity of E-cadherin 1 and occludin for normality and homogeneity of residuals. A square root transformation was used for prepartum SAA concentration, and the number of uterine glandular epithelial cells for normality and homogeneity of residuals. Least squares means and standard errors shown for these variables were back transformed unless stated in tables and figures. Multivariable logistic mixed models (PROC GLIMMIX) considered the likelihood of endometritis (based on PMN cell concentration) at 15 and 30 DRC, retained placenta, development of CL at 30, 57, 64, follicle at 71 DRC, success of first TAI at 75 DRC, and 15

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high or low GPX or SOD concentration at 30 DRC. Association between treatments and days to first ovulation and pregnancy per first TAI (P/AI) were assessed using Kaplan Meier curves and Cox’s proportional hazard regression. Treatments were forced into the models, with cows considered as a random effect. Endometritis was classified if the proportion of PMN at 15 DRC was greater than 40% and at 30 DRC if the proportion was greater than 18% [29]. Likelihood analysis of being classified as having endometritis at 15 or 30 DRC was then determined based on these values. Tissue samples stained with GPX and SOD antibodies were evaluated for concentration of cells stained positive for their respective enzyme activity following the same protocol as the aforementioned PMN cell concentration from cytology samples. Samples were further evaluated to determine high concentration of cells with GPX and SOD activity (HGPX and HSOD; respectively), and low concentration of cells with GPX and SOD activity (LGPX and LSOD; respectively). High concentration of cells with GPX activity were classified when concentrations were ≥ 74%, high concentration of cells with SOD activity were classified when concentrations were ≥ 64%. Classification of high and low concentrations were determined from median values. Five cows were excluded from the overall analysis due to treatment of milk fever with intravenous Ca during the fresh period (CON: n = 1; ND: n = 3; NDCA: n = 0). Cows excluded from PMN cell evaluation (CON: n = 5; ND: n = 6; NDCA: n = 4). Cows were excluded from endometrial biopsy (CON: n = 9; ND: n = 6; NDCA: n = 8) due to high MC score (≥3). Cows excluded from fluorescent immunolabeling (CON = 12; ND = 6; NDCA = 11). Significance was declared at P ≤ 0.05 and trends at 0.05 < P ≤ 0.15.

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3.

RESULTS Cow performance data is described in Glosson, 2018 [32]. Briefly, cows did not

differ in dry matter intake (NDCA 18.80 kg/d; ND 18.38 kg/d; CON 17.65) or milk yield (NDCA 44.90 kg/d; ND 42.62 kg/d; CON 42.07 kg/d) during the early postpartum period. There was no difference in body weight (NDCA 690 kg; ND 669 kg; CON 691 kg) nor in body condition score (NDCA 3.45; ND 3.36; CON 3.47). Cows fed NDCA and ND had a lower prepartum urinary pH (5.71 and 5.79; respectively) than cows fed CON (8.11; P <0.01). Cows that were fed NDCA and ND had greater (P <0.01) ionized Ca concentration directly after calving (NDCA 1.11 mM; ND 1.10 mM) and 24 h after calving (NDCA 1.05 mM; ND 1.11) than cows fed CON (0.98 mM; and 0.98 mM; respectively). Treatments differed for MC (P = 0.02; Table 1), indicating that the average of cows fed ND and NDCA had higher a MC score than cows fed CON. There was a tendency (P = 0.11) for cows fed NDCA to have a lower MC score than cows fed ND. There was a tendency (P = 0.10) for the average of cows fed ND and NDCA to have a higher proportion of PMN cells in the uterus than cows fed CON. There was a tendency for a treatment effect (P = 0.06) for cows fed ND to have a higher MC score than cows fed CON and cows fed NDCA. There was a time (DRC) effect for MC score (P < 0.001), MC score + smell (P < 0.0001), and proportion of PMN cells in the uterus (P < 0.001). Ovulation dynamics (Table 2) differed in days to first ovulation (P = 0.05), indicating that cows fed ND and NDCA had less days to first ovulation of the dominant follicle of the first follicular wave than cows fed CON. A tendency was observed for cows fed ND (P = 0.12) to have a greater total growth of the first dominant follicle than cows 17

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fed NDCA, as well as to have more days to first ovulation when the same groups were compared (ND vs NDCA, P = 0.12). There was a tendency for a treatment x days interaction (P = 0.11) indicating that cows fed NDCA and ND had a faster rate of growth in the 4 days prior to ovulation of the first dominant follicle than cows fed CON. Regarding the likelihood of developing endometritis (Table 3), we observed a tendency for cows fed NDCA (P = 0.14) and for cows fed ND (P = 0.08) to be more likely (OR = 0.17; CON: 2 of 23 confirmed endometritis; NDCA: 5 of 23 confirmed endometritis; OR = 0.12; CON: 2 of 23 confirmed endometritis; ND: 5 of 23 confirmed endometritis; respectively) to develop endometritis at 30 DRC than cows fed CON. Cows fed NDCA (11/21 P/AI) tended to have greater P/AI (P = 0.11; 95Cl = 1.02 – 16.6) than cows fed CON (4/19 P/AI), but cows fed ND (8/20 P/AI) did not differ from cows fed CON. There was a tendency (Table 4; P = 0.09) for cows fed NDCA to be more likely (OR = 0.22; 95Cl = 0.06 – 1.22; NDCA: 10/19 confirmed pregnant) to become pregnant at the first TAI than cows fed CON (3/15 confirmed pregnant). There was also a tendency (P = 0.14) for cows fed NDCA to be more likely (23/24 had a follicle; OR = 0.18; Cl = 0.02 – 2.80) to have a follicle at 71 DRC than cows fed ND (21/26 had a follicle). Treatments did not differ for plasma LBP or SAA in both prepartum and postpartum periods (Table 5). Plasma HP concentrations was higher for cows fed ND than cows fed NDCA in both prepartum (P = 0.01) and postpartum (P = 0.03) periods. Postpartum HP concentrations were greater (P = 0.01) for cows fed CON than cows fed ND and cows fed NDCA. There was a DRC effect for HP (Figure 1) in both prepartum (P = 0.024) and postpartum (P < 0.001) periods. There was a tendency for treatment × DRC interaction (P = 0.06), indicating cows fed CON diet had increased circulating HP

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concentrations in the first 4 days postpartum. There was a tendency (P = 0.06) for HP concentration postpartum to be greater for cows fed CON than cows fed ND or cows fed NDCA. There was no treatment difference for endometrial glandular area and perimeter (Table 6); however, cows fed CON tended (P = 0.06) to have shorter glandular epithelial height at 30 DRC when compared to the average of cows fed ND and cows fed NDCA. Cows fed NDCA had (P = 0.02) greater glandular epithelial height than cows fed ND at 30 DRC. Cows fed NDCA had (P = 0.05) more epithelial cells per gland than cows fed ND. Cows fed NDCA had (P < 0.0001) lesser concentration of cells stained positive for GPX than cows fed ND, and cows fed NDCA had (P = 0.05) higher concentration of cells stained positive for SOD than cows fed ND. In addition, cows fed CON tended to have increased likelihood of being classified as having a HSOD than cows fed NDCA (P = 0.08, OR = 3.79; CON: HSOD = 12 of 17; NDCA: HSOD = 7 of 18) or cows fed ND (P = 0.06, OR = 4.42; ND: HSOD = 6 of 18). Cows fed NDCA tended (P = 0.15) to have greater intensity of occludin than cows fed ND (Table 5).

4.

DISCUSSION From a clinical standpoint nearly all cows are at risk of developing metritis or

endometritis, since 90% of all cow’s uteri can be contaminated with bacteria following calving [33, 34]. Since uterine infection is a known cause of infertility [22] due to inflammation, the delay in the uterine involution, and the histological lesions of the endometrium [19], it is of greatest importance to differentiate uterine contamination from uterine infection. Cows fed CON had lesser MC scores and a tendency for a higher 19

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PMN percentage for endometrial cytological in the early postpartum period than cows fed NDCA and ND. It was previously hypothesized that, in the early postpartum period, neutrophils are mobilized to the uterine lumen to assist with pathogen evasion as well as with placental tissue removal [34]. Although a 2.21 MC score (as seen for cows fed ND) indicates a mild case of endometritis, it is known that even when subclinically presented, endometritis is associated with lower conception rates to first service [35], which was also seen in our study. One could hypothesize that feeding ND may have as many negative effects on reproduction as a CON diet. Development of the dominant follicle of the first follicular wave postpartum is highly correlated with conception rates [36–38]. Butler [38] described a positive association of the early commencement of the ovulatory cycle and increased conception rates for the cow that is able to have multiple ovulatory cycles before the first timed artificial insemination. In the current study, the average days to first ovulation postpartum was shorter for the cows fed NDCA and cows fed ND than for the cows fed CON. Caixeta et al. [39] observed that cows that had abnormal Ca levels at the first three days after calving tended to take more time before returning to normal cyclicity. Martinez et al. [40] indicated that there were no differences in the likelihood of pregnancy at first TAI for cows that received either a positive DCAD (145 ± 11 mEq/kg DM) or a negative DCAD (-129 ± 11 mEq/kg DM) diets. However, in the present study we observed a tendency for increased likelihood of cows fed NDCA to be pregnant after the first TAI in comparison to CON. In the study reported by Martinez et al. [40] a partially acidified prepartum diet was used, what could explain the dissimilar outcomes,

20

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since it could have elicited different responses in the cow than a fully acidified prepartum diet. Caixeta et al. [39] reported that the development of SCH or HC in early postpartum can contribute to an increased incidence of metritis in the fresh period. Furthermore, Santos et al. [41] reported in a meta-analysis on the effect of acidified prepartum diets that the incidences of metritis, retained placenta, and overall health events was decreased with the increase in acidification. The authors attributed these findings to the increased serum total Ca concentrations as a consequence of acidifying prepartum diets, thus improving Ca homeostasis at the onset of lactation. Our findings on the likelihood of developing endometritis do not agree with the aforementioned theories. The lack of increased dietary Ca supplementation when feeding a fully-acidified diet prepartum can be as negative (i.e., suppressed immune response [11]) as feeding a positive DCAD diet. Cows in Caixeta et al. [39] did not receive an acidified diet prepartum. Unfortunately, authors did not report the dietary Ca concentration. In that study, Santos et al. [41] reported that prepartum diets, acidified or not, had a median dietary Ca concentration of 0.86% of DM. Cows in our experiment received a dietary Ca concentration of 2% of DM. Nonetheless, our findings may not be fully explanatory and need to be interpreted with caution. Research into matters such as this are still of major importance due to the biological relevance of dietary Ca concentration and acidification in prepartum diets. Haptoglobin, which is nearly undetectable in healthy animals, is an acute phase protein that is elevated in the blood in response to inflammation [42]. Parturition is a very critical period for all mammals and generates an immune response that can be 21

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classified as physiological, therefore haptoglobin can be found both in diseased and clinically healthy animals during the first week postpartum [43]. Le Blanc [44] hypothesized that an inhibited immune response in the peripartum can lead to a persistent infection, delayed conception, and that an increase in neutrophil infiltration during the first two weeks after parturition is an indicator of improved immune function. However, Nightingale et al. [45] reported that the intensity of this immune response, especially during the acute phase, in the early postpartum can decrease the reproductive efficiency of cows, suggesting that this immune response needs to be balanced to be efficient without presenting future damage. Our data supports this concept and highlights the importance of fully acidified prepartum diets in improving local immune function in the uterus during the early postpartum period, whilst decreasing the acute phase protein HP concentration in blood. Endometrial cells change their morphology and function throughout the estrous cycle in order to prepare the uterus to establish pregnancy [46]. There are several hormones and steroids involved in the regulation of these processes, including progesterone that has an antiproliferative role, and estrogen, which activates epithelial cell proliferation [47]. Overall health and nutritional status of the cow may also be involved in the endometrial change. Stella et al. [34] reported that cows with improved nutritional status (i.e.; supplemented with rumen-protected methionine) had greater perimeter of glands in the early postpartum, suggesting that these cows experienced improved uterine immunity with lower inflammation. In fact, authors have reported that cows that experienced severe inflammation had noticeable glandular atrophy [46]. In our study, we observed that the concentration of dietary Ca prepartum also plays an

22

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important role in the glandular morphology, as well as providing an acidified prepartum diet. Our findings corroborate the outcomes from Martinez et al. [40], which suggested that an improved Ca status around parturition can lead to lower incidences of endometritis, thus, reducing tissue damage that occurs with the disease. Instabilities regarding the redox balance intracellularly increase the susceptibility and, sometimes, directly contributes to the pathology of many diseases [48]. Superoxide dismutase catalyzes the dismutation of superoxide radical into hydrogen peroxides, which are later disposed by GPX [49]. Superoxide radicals are a class of reactive oxygen species (ROS) that can threat cell survival [50], and SOD plays an important role in the first line of antioxidant defense. In humans, members of SOD family constitute the first enzymatic defense pathway protecting the endometrial tissue from oxidative stress [51]. Our findings provide evidence to suggest that different dietary Ca concentrations can have an impact on the SOD activity in the endometrium when cows receive a fully acidified diet during the prepartum period. One possible explanation involves the link between Ca and the immune function, in this case, especially in the uterine tissue. This was evidenced by the greater SOD activity observed in the uteri of cows that were fed NDCA, which means that ROS could have being controlled by these antioxidant enzymes. Glutathione peroxidase is a crucial antioxidant enzyme involved in preventing intracellular accumulation of hydrogen peroxide, but it has also been studied for its modulatory effects in cellular growth and proliferative responses [48]. Even though it protects the cell environment against oxidative stress, there is now evidence suggesting that the excess GPX can also have deleterious effects due to a lack of essential cellular oxidants [52, 53]. This could result in what is called a “reductive stress” 23

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state [54]. Therefore, the greater activity of GPX detected in the uterine samples of cows that were fed ND could possibly result in a state of reductive stress to those cells, if maintained. Therefore, the combined results elicit the importance of Ca metabolism to the uterine immunity. The appropriate uterine environment (i.e.; that fulfills immunological requirements) needs to be maintained in order for the cow to effectively respond to infection, especially in the early post-partum period. The uterine epithelia serve as a barrier, and the junctional complexes within the epithelia maintain this unique environment [55]. Occludin is a transmembrane protein found at the tight-junctions and the amount of occludin, as well as other tight-junction proteins, present in a tissue is inversely related to the permeability of that tissue [56]. Cows fed NDCA tended to have greater intensity for occludin, therefore a less permeable uterine luminal tissue, than cows fed ND. Calcium is one of the major factors influencing the regulation of tightjunction-dependent transepithelial paracellular permeability [57]. Our results provide insight regarding the influence of Ca on the junctional complexes in the bovine uterine epithelia, which would help in the establishment and maintenance of a healthy uterine environment. An early study using Madin-Darby canine kidney (MDCK) cells culture reported that Ca plays an important role regarding the structure and function of tight-junctions [57]. However, it is difficult to achieve a low physiological extracellular value for Ca concentration. There are several stimulating signals influencing the mobilization of Ca to the extracellular space (i.e.; vasopressin and parathyroid hormones) [11, 8]. Therefore, if Ca is hypothetically removed from the extracellular space, the concentration gradient 24

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would naturally cause an influx of Ca from the endoplasmic reticulum of cells [58]. This helps to explain why there was no differences in intensity for occludin or E-cadherin 1 when comparing CON versus the average of NDCA and ND. However, we have, to the best of our knowledge, characterized the presence of occludin and E-cadherin 1 in the bovine uterine endometrium during this critical period by fluorescent immunolabeling for the first time. The identification of E-cadherin 1 is an indicative of the presence of adherens junctions between endothelial cells in the bovine endometrium, another type of intercellular junction. Further research in regard to the function and mechanism of action of Ca in the bovine uterine epithelia can increase the understanding of the regulation of junctional complexes in this tissue. In conclusion, a fully acidified prepartum diet formulated with a higher Ca concentration improved reproductive performance and uterine immune function in the postpartum period through the decrease of days to first ovulation, a tendency to decrease service per conception rate, improved glandular morphology, a tendency to increase polymorphonuclear neutrophil infiltration, and a tendency to increase the tightjunction protein occludin. Additionally, a fully acidified prepartum diet that does not provide enough Ca can have as many negative impacts on reproduction postpartum as a prepartum diet with a positive dietary cation-anion difference.

Acknowledgements This project was partially supported by Phibro Animal Health (USA) and by the USDA National Institute of Food and Agriculture (Washington, DC; NC-3112). Sincere appreciation is expressed to the Dairy Focus Team at the University of Illinois, along 25

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with the University of Illinois Dairy Research Unit staff for assisting with data collection and cow health; and Dr. Flavio Silvestre and Zoetis for providing CIDR, Factrel, and Lutalyse used in this project. REFERENCES [1]

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Table 1. Least squares means and associated SEM for metricheck (MC) evaluation of uterine health and proportion of polymorphonuclear (PMN) cells present in the uterus. Treatment1 P-value Contrasts2 CON ND NDCA SEM Variable n 1 2 MC Score3

76

1.82

2.21

1.94

0.12

0.02

0.11

MC Score + Smell4

76

2.07

2.38

2.26

0.21

0.29

0.70

PMN, %5

62

14.75

21.22

16.34

2.35

0.10

0.18

¹CON: Positive DCAD (+6 mEq/100g of DM; urine pH > 8.0) with low dietary Ca (0.40% of DM; 46.2 ±15.2 g Ca/d; n = 26); ND: Negative DCAD (-24 mEq/100g of DM; urine pH = 5.8) with low dietary Ca (0.40% of DM; 44.1 ± 16.1 Ca/d; n = 24); NDCA: Negative DCAD (-24 mEq/100g of DM; urine pH = 5.8) with high dietary Ca (2.0% of DM; 226.6 ± 96.0 g Ca/d; n = 26). 2Contrasts were CONT1 = CON vs. the average of NDCA and ND; CONT2 = ND vs. NDCA. There were no treatment or treatment × DRC interaction for all 3 variables. Only a DRC effect (P < 0.001, for all 3 variables) of decreasing scores and concentration of PMN cells. 3MC Score and MC Score + Smell: CON: n = 26; ND: n = 24; NDCA: n = 26. 4Number of cows that scored 3 for smell: 4 DRC: CON: n = 2; ND: n = 1; NDCA: n = 1; 7 DRC:CON: n = 4; ND: n = 2; NDCA: n = 1; 10 DRC: CON: n = 3; ND: n = 1; NDCA: n = 3; 13 DRC: CON: n = 2; ND: n = 2; NDCA: n = 3: 15 DRC: CON: n = 3; ND: n = 2; NDCA: n = 2; 17 DRC: CON: n = 1; ND: n = 2; NDCA: n = 3; 30 DRC:CON: n = 0; ND: n = 1; NDCA: n = 1 5Proportion of Polymorphonuclear cells: CON: n = 22; ND: n = 18; NDCA: n = 22.

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Table 2. Least squares means and associated SEM for ovulation dynamics of the dominant follicle of the first follicular wave Treatment1 P-value Contrasts2 Variable

CON

ND

NDCA

SEM

1

2

Diameter of dominant follicle at first measurement, mm3

8.29

8.42

8.79

0.38

0.81

0.46

Diameter of dominant follicle at last measurement before first ovulation, mm4

17.87 18.33

17.56

0.44

0.44

0.16

Growth rate of dominant follicle before first ovulation, mm/d5

1.48

1.59

1.69

0.09

0.54

0.40

Growth rate of dominant follicle over last four days before first ovulation, mm/d6

3.71

3.82

3.78

0.34

0.30

0.66

Total growth of dominant follicle from first measurement to last measurement, mm7

9.58

9.91

8.77

0.53

0.64

0.12

Days to first Ovulation, d8

18.93 17.93

16.30

0.78

0.05

0.12

1Treatments

were CON: Positive DCAD (+6 mEq/100g of DM; urine pH > 8.0) with low dietary Ca (0.40% of DM; 46.2 ±15.2 g Ca/d; n = 26); ND: Negative DCAD (-24 mEq/100g of DM; urine pH = 5.8) with low dietary Ca (0.40% of DM; 44.1 ± 16.1 Ca/d; n = 24); NDCA: Negative DCAD (-24 mEq/100g of DM; urine pH = 5.8) with high dietary Ca (2.0% of DM; 226.6 ± 96.0 g Ca/d; n = 26). 2Contrasts were CONT1 = CON vs. the average of NDCA and ND; CONT2 = ND vs. NDCA. 3First measurement was on average 7.21 ± 0.79 days relative to calving (DRC): CON (n = 23); ND (n = 22); NDCA (n = 25). 4Last measurement was on average 19.33 ± 4.31 DRC: CON (n = 23); ND (n = 22); NDCA (n = 25). 5Growth rate of the dominant follicle from first measurement of the dominant follicle of the first follicular wave post-partum. CON (n = 23); ND (n = 22); NDCA (n = 25). 6Growth rate of dominant follicle for the last 4 days of growth before first ovulation: CON (n = 23); ND (n = 22); NDCA (n = 25). 7Total growth of the dominant follicle from first ultrasound until first ovulation: CON (n = 23); ND (n = 22); NDCA (n = 25). 8Days of growth from first measurement until first ovulation of dominant follicle: CON (n = 23); ND (n = 22); NDCA (n = 25).

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Table 3. Odds ratio (OR) for animals classified1 as having endometritis from uterine cytology samples.

Variable Endometritis at 15 DRC4

Endometritis at 30 DRC5

Retained Placenta6

Treatments2

Level

OR

95% CI3

P-value

CON

CON-NDCA

0.53

0.08 - 3.73

0.39

–

CON-ND

0.40

0.06 – 2.56

0.27

ND

ND-NDCA

1.33

0.25 – 7.26

0.36

CON

CON-NDCA

0.17

0.02 – 1.82

0.14

–

CON-ND

0.12

0.01 – 1.29

0.08

ND

ND-NDCA

1.40

3.01 – 6.48

0.66

CON

CON-NDCA

1.00

0.05 – 18.44

0.99

–

CON-ND

0.96

0.92 – 17.05

0.96

ND

ND-NDCA

0.91

0.06 – 20.12

0.96

¹15 days relative to calving (DRC): animals were classified as having endometritis if proportion of polymorphonuclear cells was >40%; At 30 DRC animals were classified as having endometritis if proportion of polymorphonuclear cells was >18% [28]. 2CON: Positive DCAD (+6 mEq/100g of DM; urine pH > 8.0) with low dietary Ca (0.40% of DM; 46.2 ±15.2 g Ca/d; n = 26); ND: Negative DCAD (-24 mEq/100g of DM; urine pH = 5.8) with low dietary Ca (0.40% of DM; 44.1 ± 16.1 Ca/d; n = 24); NDCA: Negative DCAD (-24 mEq/100g of DM; urine pH = 5.8) with high dietary Ca (2.0% of DM; 226.6 ± 96.0 g Ca/d; n = 26). 3CI: confidence interval derived from a binomial regression. 4Likelihood of endometritis from at 15 ± 2 days relative to calving (DRC); CON (n = 25: Yes = 3, and No = 22); ND (n = 22: Yes = 4, and No = 18); NDCA (n = 24; Yes = 4, and No = 20). 5Likelihood of endometritis at 30 ± 2 DRC; CON (n = 23; Yes = 2, and No = 21); ND (n = 23; Yes = 5, and No = 18); NDCA (n = 23; Yes = 5, and No = 18). 6Likelihood of retained placenta; CON (n = 26; Yes = 2, and No = 24); ND (n = 24; Yes = 1, and No = 23); NDCA (n = 26; Yes = 1, and No = 25).

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Table 4. Odds ratio (OR) for the presence of a Corpus Luteum (CL) at 30, 57 and 64 days relative to calving (DRC), presence of a follicle at 71 DRC and pregnant at first timed artificial insemination (TAI) Variable1 CL at 30 DRC4

CL at 57

DRC5

CL at 64 DRC6

Follicle at 71

DRC7

Pregnant at First

TAI8*

Treatments2

Level

OR

95% CI3

P-value

CON

CON-NDCA

0.86

0.86– 3.18

0.82

–

CON-ND

1.00

0.28 – 3.57

0.99

ND

ND-NDCA

0.86

0.86– 3.18

0.82

CON

CON-NDCA

2.59

0.55 – 12.16

0.22

–

CON-ND

3.00

0.65 – 13.58

0.16

ND

ND-NDCA

0.86

0.248 – 3.09

0.83

CON

CON-NDCA

2.03

0.53 – 7.69

0.49

–

CON-ND

1.60

0.42 – 6.13

0.29

ND

ND-NDCA

1.27

0.38 – 4.27

0.70

CON

CON-NDCA

0.21

0.02 - 5.16

0.18

–

CON-ND

1.13

0.25 – 2.14

0.87

ND

ND-NDCA

0.18

0.02 – 2.80

0.14

CON

CON-NDCA

0.29

0.06 – 1.22

0.09

–

CON-ND

0.43

0.10 – 1.89

0.26

ND

ND-NDCA

0.67

0.19 – 2.38

0.53

1Variables

are CL at 30 (n = 76), 57 (n = 74), and 64 DRC (n = 74); Follicle at 71 DRC (n = 73); Pregnant at first TAI (n = 55). 2CON: Positive DCAD (+6 mEq/100g of DM; urine pH > 8.0) with low dietary Ca (0.40% of DM; 46.2 ±15.2 g Ca/d; n = 26); ND: Negative DCAD (-24 mEq/100g of DM; urine pH = 5.8) with low dietary Ca (0.40% of DM; 44.1 ± 16.1 Ca/d; n = 24); NDCA: Negative DCAD (-24 mEq/100g of DM; urine pH = 5.8) with high dietary Ca (2.0% of DM; 226.6 ± 96.0 g Ca/d; n = 26). 3CI: confidence interval derived from a binomial regression. 4CON (n = 26; Yes = 19, and No = 7); NDCA (n = 25; Yes = 19, and No = 6); ND (n = 25; Yes = 19, and No = 7). 5CON (n = 23; Yes = 20, and No = 3); NDCA (n = 25; Yes = 18, and No = 7); ND (n = 26; Yes = 18, and No = 8). 6 CON (n = 23; Yes = 18, and No = 5); NDCA (n = 25; Yes = 16, and No = 9); ND (n = 26; Yes = 18, and No = 8). 7CON (n = 23; Yes = 19, and No = 4); NDCA (n = 24; Yes = 23, and No = 1); ND (n = 26; Yes = 21, and No = 5). 8CON (n = 15; Yes = 3, and No = 12); NDCA (n = 19; Yes = 10, and No = 9); ND (n = 21; Yes = 10, and No = 11). 38

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Table 5. Least squares means and associated SEM for blood metabolites. Treatment¹ P-value Contrasts2 1 2 Blood3 CON ND NDCA SEM Prepartum 7.31 7.32 5.84 0.98 0.20 LBP4, µg/mL 0.47 5 SAA , µg/mL 36.07 37.09 35.29 0.89 0.65 0.84 HP6, µg/mL 160 220 150 0.02 0.26 0.01 Postpartum LBP4, µg/mL 8.71 7.82 8.76 0.48 0.33 0.40 5 36.69 36.36 35.05 0.32 0.40 SAA , µg/mL 0.96 6 HP , µg/mL 530 420 330 0.05 0.01 0.03 ¹CON: Positive DCAD (+6 mEq/100g of DM; urine pH > 8.0) with low dietary Ca (0.40% of DM; 46.2 ±15.2 g Ca/d; n = 26); ND: Negative DCAD (-24 mEq/100g of DM; urine pH = 5.8) with low dietary Ca (0.40% of DM; 44.1 ± 16.1 Ca/d; n = 24); NDCA: Negative DCAD (-24 mEq/100g of DM; urine pH = 5.8) with high dietary Ca (2.0% of DM; 226.6 ± 96.0 g Ca/d; n = 26). 2Contrasts were CONT1 = CON vs. the average of NDCA and ND; CONT2 = ND vs. NDCA. There was a tendency for treatment × DRC interaction (Figure 1) present prepartum and postpartum for HP concentration (P = 0.08 and P = 0.06, respectively). 3Blood samples for LBP and SAA concentration were harvested at -30, -21, -7, 15 and 30 DRC. Samples for SAA and LBP were analyzed on blood plasma. Samples analyzed for HP were harvested at -14, -7, -4, -2, -1, 0, 1, 2, 4, 7, and 14 DRC. 4LBP: Lipopolysaccharide Binding Protein. 5SAA: Serum Amyloid A. 6HP: Haptoglobin.

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Table 6. Least squares means and associated standard error for glandular morphology and immunolabeling for glutathione peroxidase 1 and superoxide dismutase 1 in endometrial tissue harvested from Holstein cows. P-value Treatment1,2 3 Contrasts 1 CON ND NDCA SEM 1 2 Item 2 4 Glandular Morphology Glandular Area, 𝜇m 890.25 8175.05 9316.83 7981.45 0.66 0.29 Glandular Perimeter, 𝜇m 18.92 348.13 384.48 376.71 0.17 0.77 Glandular Epithelial Height, 𝜇 18.01 18.67 22.47 1.08 0.06 0.02 m Number of Cells per Gland 23.58 22.93 25.93 1.07 0.51 0.05 Number of Glands 304.47 284.76 235.39 30.38 0.24 0.25 Immunolabeling Glutathione Peroxidase, %4 56.85 68.31 32.89 5.05 0.32 <0.0001 4 Superoxide Dismutase, % 78.50 69.49 73.50 2.83 0.31 0.05 E-cadherin 15 441.15 698.12 324.40 1.61 0.90 0.18 5 Occludin 128.50 125.01 128.46 1.91 0.46 0.15 1CON:

Positive DCAD (+6 mEq/100g of DM; urine pH > 8.0) with low dietary Ca (0.40% of DM; 46.2 ±15.2 g Ca/d); ND: Negative DCAD (-24 mEq/100g of DM; urine pH = 5.8) with low dietary Ca (0.40% of DM; 44.1 ± 16.1 Ca/d); NDCA: Negative DCAD (-24 mEq/100g of DM; urine pH = 5.8) with high dietary Ca (2.0% of DM; 226.6 ± 96.0 g Ca/d) fed from 28 days before expected calving until parturition. 2Due to sampling criteria, samples were not taken from every cow. Cows not sampled per treatment: CON: n = 9; ND: n = 6; NDCA: n = 8. 3Contrasts were CONT1 = CON vs. the average of NDCA and ND; CONT2 = ND vs. NDCA. 4Endometrial tissue was harvested at 30 ± 1.32 days after calving. 4 CON (n = 17); ND (n = 18); NDCA (n = 18). 5 CON (n = 14); ND (n = 18); NDCA (n = 15).

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Haptoglobin, µg/mL

700 600 500 400

Prepartum: Trt: P = 0.93 DRC: P = 0.054 Trt × DRC: P = 0.08

300

Postpartum: Trt: P = 0.06 DRC: P < 0.001 Trt × DRC: P = 0.06

200 100 0 -14

-7

-4

-2 -1 Calving 1 Days relative to calving

2

4

7

14

Figure 1. Least squares means and associated SEM for haptoglobin (HP) found in blood plasma samples of Holstein cows fed CON: Positive DCAD (+6 mEq/100g of DM; urine pH > 8.0) with low dietary Ca (0.40% of DM; 46.2 ±15.2 g Ca/d; n = 26); ND: Negative DCAD (-24 mEq/100g of DM; urine pH = 5.8) with low dietary Ca (0.40% of DM; 44.1 ± 16.1 Ca/d; n = 24); NDCA: Negative DCAD (-24 mEq/100g of DM; urine pH = 5.8) with high dietary Ca (2.0% of DM; 226.6 ± 96.0 g Ca/d; n = 26). There was a DRC effect prepartum (P = 0.024) for increasing concentrations of HP. A tendency for treatment × DRC interaction for HP concentration (P = 0.08) prepartum indicates that CON had a higher HP concentration in the last 2 days before calving than cows in NDCA and ND. A tendency for a treatment effect and a tendency for a treatment × DRC interaction in HP concentration postpartum indicates cows in CON had higher HP concentrations postpartum, especially in the first 4 days postpartum. The DRC effect postpartum HP (P < 0.001) indicates a decreasing concentrations of HP overtime.

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A

B

C

D

Figure 2. Optimal cutting temperature-embedded frozen bovine endometrial tissue samples of Holstein cows fed CON: Positive DCAD (+6 mEq/100g of DM; urine pH > 8.0) with low dietary Ca (0.40% of DM; 46.2 ±15.2 g Ca/d; n = 26); ND: Negative DCAD (-24 mEq/100g of DM; urine pH = 5.8) with low dietary Ca (0.40% of DM; 44.1 ± 16.1 Ca/d; n = 24); NDCA: Negative DCAD (-24 mEq/100g of DM; urine pH = 5.8) with high dietary Ca (2.0% of DM; 226.6 ± 96.0 g Ca/d; n = 26). Samples were stained with hematoxylin and eosin (A, scale bar is 200 μm) and fluorescent staining (B-D, scale bar is 20 μm). Samples were stained with Hoechst DNA stain (blue) to confirm the presence of a nucleus, Anti-E-cadherin 1 antibody (B) that was fluoresced with red to confirm the presence of E-cadherin 1, a protein of adherent-junctions. Samples were also stained with anti-occludin antibody (C) that fluoresced with green to reveal the presence of 42

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occludin, a protein of the tight-junctions. Together, the merged image (D) confirms the presence of E-cadherin 1 and occluding in the bovine endometrial tissue.

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HIGHLIGHTS 

Cows fed a negative DCAD diet with a higher Ca prepartum had an improved uterine environment postpartum.



Cows fed a negative DCAD diet with a higher Ca prepartum tended to have increased tight-junction protein occludin.



The presence of occludin and E-cadherin 1 in the bovine uterine endometrium were identified for the first time.