Clinical Theriogenology 2022; 14: 82 Immunoglobulin G, white blood cell, and fibrinogen concentrations among dairy cows with and without endometritis during transition period Ali Bazzazan,a Nelson Cardenas,a Dominic Dolbec,b Mariela Segura,b Réjean Lefebvreab aDepartment of Clinical Sciences, bGREMIP, Faculty of Veterinary Medicine, University of Montreal Saint-Hyacinthe, Quebec, Canada Abstract Postpartum reproductive performance closely depends on uterine health. Under normal circumstances, almost 100% of cows have uterine contamination within first 2 weeks after calving. Whereas the innate immune and reproductive systems usually eliminate most offending microbes, persistent infection still reportedly occurs in ~ 20% of postpartum cows. Our purpose was to estimate concentrations of systemic immune indicators (IgG, white blood cells, and fibrinogen) during the transition period in dairy cows with and without endometritis. Fifty-nine multiparous cows were systematically and consecutively enrolled during the dry period and examined 6 times from 40 days before to 40 days after calving. Cows in the diseased group (n = 11) were identified based on 4 criteria (presence of Trueperella pyogenes grade > 2 in the uterus, clinical endometritis, subclinical endometritis, and cervicitis). Cows in the control group (n = 11) were negative for all 4 criteria. Prevalence of Trueperella pyogenes, clinical endometritis, subclini- cal endometritis, and cervicitis was 25, 16, 23, and 31%, respectively. Concentrations of IgG, white blood cells, and fibrinogen did not change over the period or vary among control and diseased cows. In conclusion, systemic indicators of inflammation were not good markers for diagnosing or monitoring endometritis in postpartum dairy cows. Keywords: Diary cattle, inflammatory markers, uterine disease Introduction Reproductive health of cows is the foundation of productivity in the dairy industry. Although entire transition period is critical for dairy cows, postpartum reproductive performance is closely dependent on uterine health. For cattle, the transition period is essentially a period of stress management that requires met- abolic adjustments in the face of a substantial negative energy balance and immune system modulations.1 These adjustments enable a smooth transition from pregnancy state to restoration of uterine condition, ovarian cyclicity, and establishment of subsequent pregnancy.2-4 Under normal circumstances, almost 100% of cows have uterine contamination within the first 2 weeks after calving.5,6 Although the innate immune system usu- ally eliminates all offending microbes, persistent infection or inflammation reportedly occurred in 15 - 40% of postpartum cows.6 Postpartum uterine disease is commonly associated with Escherichia coli (E. coli), Trueperella pyogenes (T. pyogenes), Fuso- bacterium necrophorum (F. necrophorum), and Prevotella species.4 Numerically, the most common pathogens were E. coli (37% of pathogenic bacteria isolated) and T. pyogenes (49%).7 When contamination of the uterus persists beyond 4 weeks postpar- tum, the uterine infection is labelled as endometritis. Clinical and subclinical endometritis are prevalent conditions in post- partum dairy cows, causing economic losses due to decreases in both milk production and fertility.8 Clinical endometritis is defined as inflammation of the endometrium with purulent vaginal discharge (PVD) and in the absence of systemic clinical disease at > 21 days postpartum,4 whereas subclinical endome- tritis is defined as the presence of > 18% polymorphonuclear neutrophils (PMNs) in uterine cytosmears (21 - 33 days post- partum) or > 10% PMNs (34 - 47 days postpartum).9 Cellular and humoral mechanisms of nonspecific and specific immunity have important roles in the resolution of postpar- tum endometritis. First and most substantial cell type recruited during uterine inflammation is the PMN.10 These cells, along with monocytes, are recruited from the blood circulation into uterine lumen to eliminate bacteria.11 Like PMNs and macro- phages, endometrial cells also have Toll-like receptors (TLRs) that recognize pathogens present in the uterus. For exam- ple, TLR4 recognizes lipopolysaccharides in the cell wall of gram-negative bacterial species, initiating an innate immune re- sponse that triggers the release of antibacterial substances from responding cells.12 This inflammatory response results in other inflammatory cells attracting PMNs, amplifying an inflamma- tory loop.13 Endometrial cells also secrete diverse molecules, including cytokines, whose presence in uterine secretions re- flected the intensity of endometrial inflammation.14 Fibrinogen response is a useful indicator of the presence of inflammation, bacterial infection, and surgical trauma in cat- Clinical Theriogenology 2022; 14: 83 tle. Fibrinogen binds specifically to CD11/CD18 integrins on the cell surface of migrated phagocytes, triggering a cascade of intracellular signals that leads to degranulation, phagocytosis, cellular cytotoxicity, and delayed apoptosis. Integrins, a group of adhesion molecules that are expressed on leucocytes, have an important role in immune function.15 Plasma fibrinogen con- centrations were higher in cows with subclinical and clinical mastitis than in control cows.14 Immunoglobulins (Ig) also have a protective role against pathogens in the bovine uterus.16 However, the specific func- tions of the isotopes IgA, IgG, and IgM in the genital tract are still not clear. Innate and adaptive immunity, both local and systemic, have an essential role in defending the uterus against postpartum infection. We hypothesized that clinical endome- tritis is associated with an increase in systemic indicators of inflammation in dairy cows. The hypothesis was tested by com- paring concentrations of IgG, white blood cells (WBC), and fi- brinogen during the transition period among dairy cows with and without endometritis. Materials and methods Animals Procedures were carried out in compliance with Canadian Council on Animal Care Guidelines, and the animal care com- mittee of the Université de Montréal approved the experimen- tal protocol. Multiparous cows (n = 59) were selected based on sequential date of calving (June 2016 - February 2017) from 3 commercial dairy herds in Quebec (Canada). Herd size was from 70 - 130 lactating cows. Reproductive and health data were compiled in a databank using health record management soft- ware (DSAHR, Saint-Hyacinthe, Québec, Canada). Rolling herd average milk production was ~ 9000 kg. Cows were housed in tie stall barns and milked twice daily. Cows were fed a total mixed ration consisting of mainly corn silage or hay silage for- mulated to meet the dietary requirements of lactating dairy cows at each stage of production.17 Farms were visited weekly by the same veterinarians. Cows were vaccinated intramuscu- larly against E. coli on day 40 and on day 26 before calving (2 ml, J-VAC, Merial Inc., Athens, GA), and against types 1 and 2 BVD, IBR, PI-3, and BRSV on day 15 - 40 after calving (2 ml, Bovi-Shield GOLD® FPTM 5 L5, Zoetis, Parsippany, NJ). Cows were injected with 5 ml of selenium on day 60 after calving (MU-SE, Intervet Canada Corp., a subsidiary of Merck and Co. Inc, Kirkland, QC, Canada). Experimental design This was an observational prospective cohort study in which cows (n = 59) were systematically and consecutively enrolled during the dry period and then examined 6 times from 40 days before to 40 days after calving. Six examinations (EXAM 1 - 6) were performed: on days 40 ± 4, 26 ± 4 and 12 ± 4 before calv- ing (DBC), and on days 7 ± 4, 21 ± 4 and 35 ± 4 after calving (DAC). Examinations included assessment for lameness, cyclic- ity, and body condition. Milk, blood, and endometrial sam- ples (via cytobrush) were collected, and transrectal and vaginal examinations were performed. Before calving, each cow had a transrectal examination to confirm pregnancy. After calving, the reproductive tract was examined transrectally and ultrasono- graphically to determine cervical and uterine horns’ diameters, assess fluid in uterus, and characterize ovarian structures (cor- pus luteum, dominant follicle, and follicular cyst). Addition- ally, a uterine endometrial cytobrush examination (described below) and vaginoscopy were performed. Cows were enrolled in a systematic synchronization protocol with 2 injections of prostaglandins given at a 14-day interval starting at the end of the voluntary waiting period (60 DIM) with estrus observation. Reproductive data were collected until at least 300 days in milk (DIM). Throughout the sampling pe- riod, none of the 22 cows in the control and diseased groups received any antibiotics. Diseased cows (n = 11) were identified based on 4 criteria. In decreasing order of importance, these were: 1) cows with T. pyogenes grade ≥ +2 in the uterus (cul- ture-based method only on EXAM 5); 2) cows with PVD of grade ≥ 2;18 3) cows with > 18% PMNs on endometrial cytology (number of PMNs/number of total cells);19 and 4) cows with grade 2 cervicitis.20 Control cows (n = 11) were negative for all 4 criteria (T. pyogenes grade < 2, PVD < 2, PMNs < 5%, and cer- vicitis grade < 2). Vaginal and transrectal examinations Cervicitis was assessed20 and vaginal discharge was examined.18 Following transrectal palpation, vulva was cleaned of feces with a wet paper towel and then wiped with gauze soaked in iso- propyl alcohol. For vaginoscopy, a multiple-use vaginal spec- ulum (50 cm long) was inserted through the vulva and into vagina up to the outer cervical os. No lubricant was used. With a light source, the vaginal cavity and cervical os were examined visually for discharge and categorized using a 4-point classifica- tion system.18 Vaginal content was classified as follows: 0 = no discharge, 1 = clear and translucent mucus, 2 = cloudy mucus with or without flecks of pus (< 50% pus), 3 = mucopurulent discharge (> 50% pus), and 4 = purulent discharge with fetid smell.7 Transrectal examination was performed first for the sake of con- venience. However, detection of vaginal discharge by vaginos- copy was not enhanced by a preceding transrectal palpation of the uterus.9 Cervix was assessed and classified20 as follows: C0 = cervix without abnormality; C1 = second cervical fold swollen with no redness, and C2 = second cervical fold swollen with redness. Blood sampling At each examination, blood samples were collected from the coccygeal vein using 10-ml Vacutainer K2 EDTA blood collec- tion tubes for hematology analysis, and without an anticoag- ulant for biochemistry analysis (Becton, Dickinson and Com- pany, Franklin Lakes, NJ). Collected samples were immediately placed on ice for further processing at the laboratory within < 3 hours. Once clotting was complete in the red top tube, the serum was separated by centrifugation (3,000 revolutions per minute for 10 minutes). Beta-hydroxybutyrate concentration was then measured using a Freestyle Precision NEO Kit (Abbott Laboratories Ltd, Abbott Park, IL). Finally, 3.0-ml aliquots of serum were labelled and placed at – 80°C for long-term storage and further analysis. After 30 minutes at room temperature and gentle agitation, the anticoagulant-containing tube was subject Clinical Theriogenology 2022; 14: 84 to a complete hematology analysis (VetScan HM5 hematology analyzer, Abaxis Global Diagnostics, Union City, CA). Quantification of serum IgG and fibrinogen concentrations Bovine IgG concentrations were measured21 by ELISA using a commercial Bovine IgG ELISA Quantitation Set (E10-118, Beth- yl Laboratories, Montgomery, TX). Endometrial cytology sampling Uterine cytobrush samples (n = 59) were collected for routine endometrial cytology and bacteriology assessment. Briefly, a sterile cytobrush (CytoSoft, Camarillo, CA) was screwed onto a stainless-steel rod (65 cm x 4 mm) and was then protected within a sterile stainless-steel tube. The rod-guard apparatus was inserted into a hard protective plastic sheath (IMV Technol- ogies, L’Aigle, France) before being inserted into a second pro- tective sheath (30-cm long Sani-Shield Rod Protector, Agtech, Manhattan, NY) to reduce the risk of vaginal and cervical con- tamination. Instrument was pushed through the cervix and the sterile cytobrush gently rotated clockwise (360 degrees) on the endometrium to collect cellular material. Cytobrush was then retracted into the stainless-steel tube and hard plastic protective sheath, and withdrawn. The bristles of the brush were rolled over a sterile microscope slide (Fischer Scientific, Toronto, ON, Canada), and then the brush tip was cut off and packaged for bacterial culturing. Slide preparation and staining The smear on the slide was allowed to dry at room temperature for 10 -15 minutes. Cytology smears were stained with modified Wright Giemsa stain using a Hematek automated slide stainer (Miles Scientific, Napierville, IL). Slides were examined under the microscope at 250 and 400 x magnifications and various inflammatory cell types (PMNs, lymphocytes, and monocytes), and endometrial epithelial cells were counted. For each slide, a total of 300 cells were counted by a single observer. Cows were classified as endometritis positive if > 18% PMNs was present at 5 weeks after calving. Uterine bacterial culturing and identification Uterine cytobrush samples for bacteriology testing were collect- ed only at 21 days after calving (EXAM 5) for routine bacterial culturing (aerobic and anaerobic) using standard methods for bacteriological testing (API system, bioMérieux, Marcy l’Étoile, France). Cytobrush samples were stored in a culture tube (Star- plex Scientific Inc., Etobicoke, ON, Canada) and transported at room temperature to the Faculty of Veterinary Medicine’s diag- nostic laboratory within 3 hours. For microbiological analysis, the brushes were scraped onto sheep blood agar (soy agar with 5% sheep blood; Becton, Dickinson and Company, Sparks, MD). Plates were incubated for 48 hours at 35°C under aero- bic conditions and then examined. When growth was observed, colony types were identified based on morphology, pigmenta- tion, and hemolytic patterns. Tiny beta-hemolytic, catalase-neg- ative colonies demonstrating coliform gram-positive rods were identified as T. pyogenes, F. necrophorum and P. melaninogenicus. These were then isolated using the standard procedure at the Faculty of Veterinary Medicine’s diagnostic laboratory (PON- BAC-019). For other bacterial species, cytobrush samples were scraped directly onto Brucella agar containing neomycin (100 g/ml) and incubated anaerobically at 35°C for 5 days. When gram-negative rods were observed, colonies were examined us- ing the API 20 A gallery system for identifying F. necrophorum and P. melaninogenicus. For isolation of E. coli, cytobrush sam- ples were scraped directly on blood agar and MacConkey agar (Oxoid Inc., Ottawa, ON, Canada) at 37°C. At the OIE Refer- ence Laboratory for Escherichia coli (EcL; Faculty of Veterinary Medicine, University of Montreal), 5 typical lactose-positive E. coli colonies from the MacConkey agar plates were streaked with blood agar for isolation and further identification. Isolates were submitted to 3 biochemical tests (indole spot, Simmon’s citrate, and motility) for confirmation of E. coli. Isolates of E. coli were stored in tryptic soy broth containing 30% glycerol at – 80°C (Becton, Dickinson and Company, Sparks, MD). Data analyses Reproductive data were obtained from the health records da- tabank. We calculated descriptive statistics and then analyzed the data using SAS v. 9.2. The model used herd as the random effect. Statistical power and sample size calculations were per- formed before analysis. Based on reports,18,21 the following values were deemed indicative of endometritis: 18% PMNs on endometrial cytology, > grade 2 PVD, cervicitis score > 2, and 15 - 35 mg/ml of IgG in the serum. We determined that 11 cows per group were required based on a 10% difference between the control and disease groups, and using a one-sided test with 95% confidence interval and 80% power (G*Power, version 3.1.9.2, Germany).22 Data were not normally distributed and so were transformed using logarithm base 10 to normalize the distribution. A linear mixed model was used, with farm and cow nested within farm as random effects, and health status (control and diseased ani- mals) and period (1 - 6) as fixed effects. Means for each health status at each time were compared. Alpha level was adjusted downward using the Benjamin-Hochberg procedure. For ordi- nal scores, the Cochran-Mantel-Haenszel test was used to ex- amine potential differences in the distribution of score values between control and diseased cows. Significance was set at p ≤ 0.05. Results Descriptive statistics for the study population Initial potential sample size was 250 multiparous (2nd - 5th lac- tation) cows. From the original group of cows (n = 83), individ- uals were eliminated from the dataset because of culling (n = 5), use of uterine antibiotics (n = 4), missing data (n = 10), and metabolic diseases (n = 5). Mean day of sampling for EXAMS 1 - 6 were: 40 (45 - 37 DBC), 28 (31 - 24 DBC), 13 (17 - 10 DBC), 4 (0 - 7 DIM), 17 (14 - 21 DIM), and 31 (28 - 35 DIM), respec- tively. The remaining 59 cows had the following prevalence: clinical endometritis (EXAM 6) 23%; cytological endometritis (EXAM 6) 16%; T. pyogenes positive on culture (EXAM 5) 25%; and cervicitis (EXAM 6) 31%. There were no differences (p > 0.5) among 6 sampling times in blood concentrations of WBCs (Figure 1) and fibrinogen (Figure 2). Clinical Theriogenology 2022; 14: 85 Descriptive statistics for the study groups (control versus diseased cows) Body condition score, lameness score, and cyclicity status were not different (p > 0.05) between control (n = 11) and diseased groups (n = 11). Control cows were free of T. pyogenes (score ≤ 1) at EXAM 5; clinical Figure 1. WBC (c/ml) in cows (n = 59) overtime of examination; note the lack of difference at examinations. Figure 2. Serum fibrinogen (g/dl) in cows (n = 59) over time of examination; note the lack of difference at examinations. -6 wkbc -4 wkbc -2 wkbc 0 +1 wkbc +3 wkbc +5 wkbc Weeks before and after calving W BC c /m l CA LV IN G -6 wkbc -4 wkbc -2 wkbc 0 +1 +3 wkbc +5 wkbc Weeks before and after calving Se ru m fi br in og en (g /d l) -6 wkbc -4 wkbc -2 wkbc 0 +1 wkbc +3 wkbc +5 wkbc Weeks before and after calving Se ru m fi br in og en (g /d l) CA LV IN G Figure 1. WBC (c/ml) in cows (n = 59) overtime of examination; note the lack of difference at examinations. Figure 1. WBC (c/ml) in cows (n = 59) overtime of examination; note the lack of difference at examinations. Figure 2. Serum fibrinogen (g/dl) in cows (n = 59) over time of examination; note the lack of difference at examinations. -6 wkbc -4 wkbc -2 wkbc 0 +1 wkbc +3 wkbc +5 wkbc Weeks before and after calving W BC c /m l CA LV IN G -6 wkbc -4 wkbc -2 wkbc 0 +1 +3 wkbc +5 wkbc Weeks before and after calving Se ru m fi br in og en (g /d l) -6 wkbc -4 wkbc -2 wkbc 0 +1 wkbc +3 wkbc +5 wkbc Weeks before and after calving Se ru m fi br in og en (g /d l) CA LV IN G Figure 2. Serum fibrinogen (g/dl) in cows (n = 59) over time of examination; note the lack of difference at examinations. endometritis (score ≤ 1); subclinical endometritis (< 5% PMNs on endometrial cytology) at EXAM 5; and cervicitis (score ≤ 1) at EXAM 6. In diseased cows, 100% (n = 11) had T. pyogenes (score ≥ 2) on EXAM 5, whereas 65% (n = 7) had clinical endometritis (score ≥ 2 of PVD), 36% (n = 4) had sub- clinical endometritis (≥ 18% PMNs on endometrial cytology), and 55% (n = 6) had cervicitis (score of 2) at EXAM 6. Dis- eased cows met at least 3 of 4 criteria. Prevalence of T. pyogenes at EXAM 5 (n = 15) was 25%. In control cows, the distribution of PVD scores shifted to low- er values (p = 0.04) from EXAM 4 to EXAM 6. However, this effect was not observed in diseased cows. By contrast, there was more vaginal discharge in diseased cows at EXAM 5 and EXAM 6 compared to EXAM 4 (p < 0.04 and p < 0.002, respec- tively). At EXAM 6, diseased cows had more (p = 0.008) PVD than the control cows. Mean PMN counts on endometrial cy- tology were not different (p > 0.05) from EXAM 4 to EXAM 6 in control cows. In diseased cows, mean PMN counts on the endometrial cytology at EXAM 4 were lower (p < 0.0005) than at EXAMs 5 and 6. Although mean PMN count at EXAM 4 was Clinical Theriogenology 2022; 14: 86 different (p > 0.05) between control and diseased cows, it was higher at EXAM 5 and EXAM 6 for diseased cows compared to the control group (p < 0.0001 and p < 0.02, respectively). Cows with PVD score ≥ 2 had a higher (p = 0.0001) mean PMN count on endometrial cytology than those with lower scores. Cows with T. pyogenes had a higher mean PMN count on endometrial cytology at both EXAMs 5 and 6 compared to cows without this bacterial species (p = 0.0001 and p = 0.02, respectively). Similarly, cows with T. pyogenes were more likely to have clinical endometritis (n = 11; 73%) and subclinical en- dometritis (n = 10; 67%) at EXAM 5 (Table). Cervicitis scores were higher in diseased group compared to control group at EXAMs 5 and 6 (p = 0.04 and p = 0.002, respectively). Of the 22 diseased and control cows, 8 of 11 control cows and 4 of 11 diseased cows were pregnant after the first artificial insem- ination. Concentrations of IgG, WBC components (PMNs, mono- cytes, lymphocytes, and eosinophils), and fibrinogen did not change (p > 0.05) over period or vary among control and dis- eased cows at any examination (Figures 3a and 3b, Figures 4a and 4b, and Figures 5a and 5b). Farm did not explain any vari- ation (p = 0.23) in the data. A lack of variation (p > 0.05) over period (transition) for diseased and control groups and be- tween groups at various examinations for all 3 inflammation indicators was also observed when the postpartum diseases (clinical and subclinical endometritis, presence of T. pyogenes, and cervicitis) were analyzed individually. Discussion Postpartum endometritis may reflect a generalized systemic inflammatory environment that can be attributed to a sys- temic metabolic condition that is common in the transition period.23 Alternatively, it may be the direct result of impaired immune defences. To distinguish between these 2 possibilities and cast more light on the pathogenesis of postpartum endo- metritis, we sampled at 6 time points during the transition period. This study was also unique in defining the diseased animal based on several conditions. These are, in decreasing order of importance: 1) T. pyogenes (≥ 2 grade); 2) clinical endometritis (≥ 2 purulent vaginal discharge); 3) subclinical endometritis (≥ 18% PMNs), endometritis (≥ 18% PMNs), and 4) cervicitis (score of 2). T. pyogenes is associated with in- flammation and infection of the endometrium in vivo and in vitro. It is the most prevalent uterine bacterial species at 3 weeks postpartum and is associated with a substantial reduc- tion in pregnancy rate.24 Disease definition used in our study was based on combining various chronic postpartum diseas- es, assuming that 2 conditions (clinical and Table. Temporal changes in polymorphonuclear cells (PMN), purulent vaginal discharge (PVD), and cervicitis in control and diseased cows EXAM 4 EXAM 5 EXAM 6 PMN ≥ 18% Control 11 (24%) 14 (30%) 6 (13%) PMN ≥ 18% Diseased 2 (13%) 10 (67%) 4 (27%) PVD ≥ 2 Control 20 (43%) 21 (46%) 7 (15%) PVD ≥ 2 Diseased 4 (27%) 11 (73%) 7 (47%) Cervicitis (score = 2) Control 14 (30%) 14 (30%) 12 (26%) Cervicitis (score = 2) Diseased 8 (53%) 11 (73%) 7 (47%) Table. Temporal changes in polymorphonuclear cells (PMN), purulent vaginal discharge (PVD), and cervicitis in control and diseased cows Figure 3a. Blood IgG concentrations in control (n = 11) cows; note the lack of difference at examinations EXAM 4 EXAM 5 EXAM 6 PMN ≥ 18% Control 11 (24%) 14 (30%) 6 (13%) PMN ≥ 18% Diseased 2 (13%) 10 (67%) 4 (27%) PVD ≥ 2 Control 20 (43%) 21 (46%) 7 (15%) PVD ≥ 2 Diseased 4 (27%) 11 (73%) 7 (47%) Cervicitis (score = 2) Control 14 (30%) 14 (30%) 12 (26%) Cervicitis (score = 2) Diseased 8 (53%) 11 (73%) 7 (47%) -6wkbc -4wkbc -2wkbc 0 +1wkac +3wkac +5wkac Weeks before and after calving CA LV IN G Bl oo d Ig G c on ce nt ra tio n (m g/ m l) Figure 3a. Blood IgG concentrations in control (n = 11) cows; note the lack of difference at examinations Clinical Theriogenology 2022; 14: 87 Figure 3b. Blood IgG concentrations in diseased cows (n = 11); note the lack of difference at examinations Figure 4a. WBC in control (n =11) cows; note the lack of difference at examinations -6wkbc -4wkbc -2wkbc 0 +1wkac +3wkac +5wkac Bl oo d Ig G c on ce nt ra tio n (m g/ m l) Weeks before and after calving -6wkbc -4wkbc -2wkbc +1wkac +3wkac +5wkac Weeks before and after calving W BC c /m l CA LV IN G CA LV IN G Figure 3b. Blood IgG concentrations in diseased cows (n = 11); note the lack of difference at examinations Figure 3b. Blood IgG concentrations in diseased cows (n = 11); note the lack of difference at examinations Figure 4a. WBC in control (n =11) cows; note the lack of difference at examinations -6wkbc -4wkbc -2wkbc 0 +1wkac +3wkac +5wkac Bl oo d Ig G c on ce nt ra tio n (m g/ m l) Weeks before and after calving -6wkbc -4wkbc -2wkbc +1wkac +3wkac +5wkac Weeks before and after calving W BC c /m l CA LV IN G CA LV IN G Figure 4a. WBC in control (n =11) cows; note the lack of difference at examinations Weeks before and after calving Figure 4b. WBC in diseased cows (n = 11); note the lack of difference at examinations Figure 5a. Fibrinogen concentration in control cows (n = 11); note the lack of difference at examinations W BC c /m l -4wkbc -2wkbc +1wkac +3wkac +5wkac -6wkbc CA LV IN G -6wkbc -4wkbc -2wkbc +1wkac +3wkac +5wkac Weeks before and after calving S er um fi br in og en (g /d l) CA LV IN G Figure 4b. WBC in diseased cows (n = 11); note the lack of difference at examinations Clinical Theriogenology 2022; 14: 88 Weeks before and after calving Figure 4b. WBC in diseased cows (n = 11); note the lack of difference at examinations Figure 5a. Fibrinogen concentration in control cows (n = 11); note the lack of difference at examinations W BC c /m l -4wkbc -2wkbc +1wkac +3wkac +5wkac -6wkbc CA LV IN G -6wkbc -4wkbc -2wkbc +1wkac +3wkac +5wkac Weeks before and after calving S er um fi br in og en (g /d l) CA LV IN G Figure 5a. Fibrinogen concentration in control cows (n = 11); note the lack of difference at examinations Figure 5b. Fibrinogen concentration in diseased cows (n = 11); note the lack of difference at examinations -6wkbc -4wkbc -2wkbc +1wkac +3wkac +5wkac Weeks before and after calving CA LV IN G Se ru m fi br in og en (g /d l) Figure 5b. Fibrinogen concentration in diseased cows (n = 11); note the lack of difference at examinations subclinical endometritis) are more detrimental to fertility than is a single disease.25 Proportion of PMNs at 5 and 7 weeks postpartum has been associated with concomitant bacterial infection24 and with impaired reproductive performance.18,27 Since the most suitable threshold for the diagnosis of subclin- ical endometritis is still being debated, we used a threshold of 18% PMNs on endometrial cytology28 for samples obtained between 20 and 33 DIM. Definition of disease chosen was meant to increase the likelihood of finding differences be- tween control and diseased groups. Sample size calculation and using a systematic and consecutive enrollment design strengthened the experimental design. As less is known about the pathogenesis of postpartum endometritis, especially the role of systemic inflammatory markers, our study provided new insights into the systemic immune response of dairy cows to postpartum endometritis throughout the entire transition period. Prevalence of clinical and subclinical endometritis and of cer- vicitis at EXAM 5 (n = 59) were similar to reported values for a similar time point during the postpartum period.7,18,20 We observed a decrease in prevalence from EXAM 5 to EXAM 6, similar to the prevalence of clinical and subclinical endome- tritis that declined from 25.4% at 3 weeks to 14.7% at 8 weeks postpartum.29 Similarly, prevalence of cervicitis throughout the early part of the postpartum period decreased. Like endo- metritis, this was most likely due to spontaneous cure that is expected to decrease with number of days postpartum. Contrary to previous studies, the prevalence of T. pyogenes at EXAM 5 was 4 times greater than the prevalence reported at 28 Clinical Theriogenology 2022; 14: 89 DIM (6.3%),30 and 1.2 times less than the prevalence reported at 21 days postpartum (30.2%).31 However, the sample size in our study limited this comparison. On day 21 postpartum, < 40% of cows had T. pyogenes in uterine samples obtained using low-volume uterine lavage.31 As prevalence of T. pyo- genes was not greater than studies that had only 1 sampling, multiple sampling over the whole transition period probably did not affect results in our study. Definitively associated with endometrial lesions and fertility, T. pyogenes is the most recog- nized etiological agent of postpartum endometritis.32 Howev- er, prevalence of T. pyogenes varies with herd, type of facility, management, and location. Cows with T. pyogenes (score ≥ 2) exhibited a greater probability of having clinical and/or sub- clinical endometritis (p = 0.008 and p = 0.02, respectively), but not cervicitis (p > 0.05). We are the first group to assess systemic immune indicators of inflammation over the entire transition period (day – 40 to day + 35) in dairy cows with and without endometritis. There was no difference in IgG concentrations between EXAM 1 and EXAM 6, and among diseased and control cows at any exam- ination throughout the entire transition period. Immunoglob- ulins have an important role in immune defence by serving as opsonin that enhance phagocytosis, and by stimulating the classical complement pathway.14 As IgG is produced in part from the endometrium (mainly IgG1), with the balance of IgG1 derived from peripheral circulation and all IgG2 derived from peripheral circulation,33,34 one would expect variations in IgG concentrations in the blood stream of cows during the transition period.35 Decreased IgG concentrations in the pe- ripheral circulation around calving could be explained by the immunosuppressant status of pregnancy-associated physio- logical phenomena, the effect of cortisol at calving, and the stress associated with high-producing cows in the postpartum period.36 Some researchers have suggested that postpartum uterine diseases are related to an immunosuppressant status during the postpartum period, but they have provided no de- tails about what specific features of this status could be in- volved. In our study, control and diseased cows did not have any locomotor, mammary gland or metabolic diseases that could have been potential sources of inflammation causing the changes in inflammatory indicators. Therefore, our chanc- es of observing differences among control and diseased cows due to uterine anomalies alone was improved. Based on the results, and assuming that a large portion of total IgG in the uterine lumen is synthesized systemically, estimation of IgG serum concentrations is not a good inflammatory indicator to predict or monitor postpartum endometritis in dairy cows.37 In addition to measuring variation in IgG concentrations, sev- eral researchers also reported a decrease in peripheral blood lymphocyte counts.21,38 By contrast, as was the case for IgG, we did not observe variation in WBC numbers. Peripheral blood concentrations of total leukocytes or individual types (PMNs, monocytes, lymphocytes, and eosinophils) in the 2 groups of cows did not vary throughout the transition period, or differ between control and diseased cows at examinations. In nor- mal cows, the prepartum period is usually accompanied by peripheral leukocytosis,39 followed by leucopenia during the first week after calving.40 Cows with subclinical endometritis had higher blood leukocyte counts and elevated PMN concen- trations compared to control postpartum cows between days 45 and 55.41 Fibrinogen concentrations were not different between control and diseased cows at examinations during the transition peri- od. Fibrinogen is a secreted plasma protein that is the precur- sor of fibrin and is used to form secondary hemostatic plugs at sites of vascular injury, in several inflammatory and trau- matic conditions in cattle.42 Analyses of metabolic blood pa- rameters, serum inflammatory mediators, antibodies, and the cellular composition of cow blood for concentrations of ele- ments like beta-hydroxybutyrate, haptoglobin and sialic acid around calving have been proposed, but none was satisfactory for disease prediction.43 Increased fibrinogen concentrations (hyperfibrinogenemia) may be associated with inflammation or with dehydration. Although the ratio of plasma proteins/ fibrinogen may help to distinguish dehydration from inflam- mation, dehydration was not evident in our study. Conclusion Concentrations of systemic inflammatory indicators (IgG, white blood cells, and fibrinogen) did not change over time nor did they differ between control and diseased postpartum cows at examinations. 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