Effects of parity on metabolic, oxidative status and reproductive responses in heat-stressed transition dairy cows supplemented with slow release boluses

Effects of parity on metabolic, oxidative status and reproductive responses in heat-stressed transition dairy cows supplemented with slow release boluses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of parity on metabolic, oxidative status and reproductive responses in heat-stressed transition dairy cows supplemented with slow release boluses Mohammad Choupani, Ahmad Riasi, Masoud Alikhani, Mohammad Reza Samadian, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7633697/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Heat stress negatively impacts transition cow health, metabolism, and fertility, while parity further influences these physiological responses. Antioxidant supplementation is proposed to mitigate oxidative and metabolic stress during this critical period. This study evaluated the effect of parity on metabolic, oxidative, and reproductive parameters in heat-stressed dairy cows receiving antioxidant boluses. Primiparous and multiparous Holstein cows were supplemented with antioxidant boluses at calving. Measurements included calving-related traits, body condition score (BCS) changes, metabolic indicators (glucose, BHBA), oxidative status (TAC, GPX, SOD, MDA), hematology, uterine health, and reproductive performance over 42 days postpartum. Parity significantly affected postpartum physiology despite antioxidant supplementation. Multiparous cows exhibited greater BCS loss (P < 0.05), higher BHBA concentrations (P < 0.05), and elevated oxidative stress, along with stronger inflammatory responses indicated by increased WBC and neutrophil counts (P < 0.01). In contrast, primiparous cows experienced more calving difficulties (P < 0.05) but maintained a relatively favorable metabolic profile. Days open tended to be shorter in primiparous cows (P = 0.09), suggesting improved reproductive recovery. Antioxidant boluses improved oxidative balance but did not fully offset parity-driven metabolic and immune challenges under heat stress. These findings highlight the need for integrated, parity-adjusted nutritional and management strategies to optimize transition cow health and fertility in thermal environments. Parity Heat stress Antioxidant bolus Oxidative stress Dairy cows Reproduction Figures Figure 1 Figure 2 Introduction The transition period, defined as the three weeks before and three weeks after calving, is a pivotal stage in the productive life of dairy cows. This period is characterized by profound metabolic, physiologic, and immunologic challenges (LeBlanc 2010 ). During the transition period, cows become vulnerable to multiple disorders, including negative energy balance (NEB), hypocalcemia, oxidative stress, uterine infections, and impaired fertility (Santos et al. 2016 ). It is reported that the challenges intensify under heat stress condition (Das et al. 2016 ). Primiparous cows, due to their ongoing body growth and lack of previous lactation experience, typically experience higher levels of stress. On the other hand, multiparous cows, because of greater metabolic load and a higher risk of systemic inflammation, are more prone to developing postpartum metabolic and reproductive disorders (Choupani et al. 2023a ; Choupani et al. 2023b ; Corset et al. 2024 ). Nutritional intervention with antioxidant supplements during the transition period has garnered increasing attention. Choupani et al. ( 2023a , b ) demonstrated that sustained-release antioxidant boluses administered around calving improved metabolic profiles, maintained body condition, stabilized hematological parameters, and supported uterine health in heat-stressed cows. Importantly, a recent study by Hao et al. ( 2025 ) provided further evidence in support of antioxidant strategies. They showed that supplementation with rumen-protected glutathione during the transition period reduced oxidative stress biomarkers, lowered inflammatory indices, enhanced antioxidant enzyme activity, and improved lactation outcomes in Holstein cows. Recent studies have indicated that multiparous dairy cows, due to their higher milk production, are generally exposed to greater oxidative stress (Yehia et al. 2020 ). They reported that malondialdehyde (MDA) concentrations were higher in multiparous cows than that the primiparous cows. Moreover, Alberghina et al. ( 2024 ) showed that heat stress further exacerbates the oxidative challenges by increasing the production of reactive oxygen species and imposing additional pressure on antioxidant defense systems in dairy cows. Multiparous cows, despite having a higher oxidative burden, exhibit a stronger antioxidant defense capacity, functioning as a compensatory mechanism to counteract oxidative stress (Corset et al. 2024 ). Based on these findings, we hypothesized that supplementation with antioxidant slow release boluses could enhance antioxidant capacity, particularly in multiparous cows, and help mitigate the negative effects of oxidative stress during the Summer season. Considering that multiparous cows generally experience greater metabolic and reproductive challenges compared to primiparous cows, the present study aimed to evaluate whether sustained-release antioxidant bolus supplementation could differentially influence transition cow health and performance between these two parity groups. Understanding these differential responses may help design targeted nutritional strategies to improve postpartum adaptation under heat stress conditions. Therefore, in the present study, the effects of slow-release oral bolus on metabolic, inflammatory, and reproductive status in primiparous and multiparous cows during heat stress were investigated. Materials and methods Animals and management The study was conducted during the summer season, under heat stress conditions (average THI max >72) in a large commercial dairy farm consisting of 5000 dairy cows (Ghiam Dairy Company, Isfahan, Iran). A total of 200 Holstein dairy cows, including 60 primiparous and 140 multiparous (parity = 3.87 ± 1.78), were allocated to completely randomized design. Therefore, two experimental treatments were defined in this study: multiparous cows (MB) and primiparous cows (PB), both of which received antioxidant boluses. All experimental procedures involving animals were approved by the Institutional Animal Care Committee of Isfahan University of Technology and carried out in accordance with the ethical guidelines established by the Iranian Council of Animal Care ( 2015 ). Slow release bolus specifications All cows orally received three slow release antioxidant boluses (Golpooneh Safahan, Knowledge-based Co., Isfahan, Iran) at calving using a bolus gun. Each bolus was manufactured in a cylindrical form (16 mm in length, 25 mm in diameter, weighing 15 g) and was completely coated with an inert polymer, except in the bottom surface, to facilitate a sustainable release of its active components. The active ingredients in each bolus were: copper (2.0 g Cu, copper oxide), selenium (0.063 g Se, sodium selenite), manganese (2.1 g Mn, manganese sulfate), zinc (0.75 g Zn, zinc oxide and zinc sulfate), and vitamin A (470 × 10³ IU). According to the manufacturer, these components were gradually released over 21 days as the bolus dissolved in reticulorumen. Based on the analysis of the boluses and their release rate profile, each cow received approximately 9 mg of selenium, 107.14 mg of zinc, 285.71 mg of copper, 300 mg of magnesium, and 67,142.85 IU of vitamin A per day. Housing and feeding All cows were housed in free-stall barns equipped with cooling systems and fed a total mixed ration (TMR) formulated according to NRC (2001) recommendations, with ad libitum access to water. The average dry matter intake (DMI) of primiparous and multiparous cows during the fresh period was 16.5 ± 2.4 kg/day and 18.2 ± 1.90 kg/day, respectively. During the entire lactation, the mean DMI was 24.76 ± 1.12 kg/day for primiparous cows and 27.21 ± 1.40 kg/day for multiparous cows. Cows were milked three times per day (08:00, 16:00, and 24:00). The ingredients and compositions of the fresh and lactation rations are presented in Table 1 . Feed samples were analyzed to determine dry matter (DM; AOAC method 934.01), crude protein (AOAC method 976.05), ether extract (EE; AOAC method 920.29), and ash (AOAC method 942.05) based the AOAC (2000) guidelines. Neutral detergent fiber (aNDF) and acid detergent fiber (ADF) concentrations were analyzed according to Van Soest et al. ( 1991 ). Table 1 Ingredients and chemical composition of diets fed to Holstein cows. Diets 1 Items Fresh Lactation Ingredients (% DM) Corn silage 19.81 17.36 Alfalfa hay 18.36 10.19 Straw 2.42 1.89 Whole cotton seed 4.35 1.89 Beet pulp 4.35 5.66 Barley grain 7.61 5.67 Corn grain 20.06 31.54 Corn bran - - Soybean meal 10.50 5.04 Canola meal 2.03 4.41 Full-fat soybean 3.55 4.10 Corn gluten - 1.26 Meat meal 3.04 4.73 Fat powder 0.25 1.83 Sodium bicarbonate 0.91 1.39 Calcium carbonate 0.76 0.69 Vitamin supplement 2 0.46 0.36 Mineral supplement 3 0.46 0.36 Magnesium oxide 0.25 0.35 Bio binder 0.20 - Dicalcium phosphate 0.25 0.33 Salt 0.38 0.44 Toxin binder - 0.28 Urea - 0.25 Magnesium sulfate - - Calcium chloride - - Chemical composition Dry matter (% as fed) 49 49.1 Crude protein (%) 16.50 16.28 Ether extract (%) 4.34 5.91 NE L 4 (MJ/kg DM) 6.8 7.4 aNDF 5 (%) 31.05 26.16 ADF (%) 19.05 15.01 Ash (%) 8.6 8.5 Selenium (mg/kg) 0.1 0.09 Zinc (mg/kg) 69.41 67.96 Copper (mg/kg) 7.94 6.71 Manganese (mg/kg) 28.03 28.31 Calcium (%) 1.08 1.13 Phosphorus (%) 0.53 0.61 Vitamin A (IU/d) 100,000 150,000 1 Fresh: the first 3 week of lactation, and Lactation: after 3 weeks of lactation. 2 Vitamin supplements contained: Vitamin A at 1,500,000 IU/kg; Vitamin D₃ at 400,000 IU/kg; and Vitamin E at 15,000 IU/kg. 3 Mineral supplement contained: Co, 120 mg/kg (cobalt sulfate); Cu, 5,000 mg/kg (copper sulfate); I, 200 mg/kg (calcium iodate); Fe, 800 mg/kg (ferrous sulfate); Mn, 15,000 mg/kg (manganese sulfate); Se, 100 mg/kg (sodium selenite); and Zn, 2,000 mg/kg (zinc sulfate). 4 Net energy for lactation was calculated according to NRC (2001) recommendations. 5 Neutral detergent fiber was analyzed using a heat-stable amylase and expressed inclusive of residual ash. Calving related parameters and BCS Calving related parameters including time to expulsion of fetal membranes, calving difficulty score (Score 1 - unassisted, 2- normal calving, 3- difficult calving (Eriksson et al. 2004 )), incidence of retained placenta, milk fever, and colostrum yield were recorded immediately after calving. The backfat thickness of each cow was measured using an Easi-Scan ultrasound scanner (BCF Technology Ltd., Livingston, UK) operating at a frequency range of 4.5–8.5 MHz, and BCS was assessed on a 1 to 5 scale at calving (day 0) and on days 28 and 42 after calving (Schröder and Staufenbiel 2006 ). Sampling and measurements Blood samples were collected by jugular venipuncture on days 0, 3, 14, 28, and 42 after calving. Two types of samples were obtained: Whole blood in EDTA tubes for hematological analysis including white blood cells (WBC), neutrophils, lymphocytes, and polymorphonuclear cells (Cell counter analyzer MS 9 − 3). Serum samples obtained after centrifugation at 3000 × g for 15 minutes and stored at − 20°C until analysis. Serum biochemical parameters measured included glucose (Pars Azmoon glucose Kit, Photometric method, Tehran, Iran), β-hydroxybutyrate (BHBA, Randox Kit, intra- and inter-CV 2.3% and 5.1%), total protein (photometric, biuret method, intra- and inter-CV < 2.1% and < 3.1%), albumin (photometric, bromocresol green method, intra- and inter-CV < 1.9% and < 3%). Oxidative stress and antioxidant status were evaluated by measuring total antioxidant capacity (TAC), glutathione peroxidase (GPX), superoxide dismutase (SOD) (Autoanalyzer, Alcyon 300, intra- and inter-CV < 2.4% and < 3.2%, intra- and inter-CV < 3.7% and < 3.7%, intra- and inter-CV < 8% and < 10%, respectively), and malondialdehyde (MDA, based on the colorful complex formed from the reaction of MDA with 2-thiobarbituric acid in acid environment). Liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (Pars Azmoon Kit, Tehran, Iran), and the serum calcium (photometric method, cresolphthalein complexone, Intra- and inter-CV < 8.9% and < 10.5%), were measured. Uterine health evaluation Uterine health was evaluated by assessing vaginal discharge scores (VDS) on days 14 and 28 after calving using the Metricheck device, with scores assigned on a 0–5 scale (Zero = no discharge, 1 = clear mucosa, 2 = infection inside mucosa not clear, 3 = mucosa with less than 50% infection, 4 = mucosa with more than 50% infection, 5 = mucosa with more than 50% infection with odor) as described by Kusaka et al. ( 2020 ). The cows with a score > 3 were considered as metritis disorder. Reproductive performance At 45 days postpartum, all cows were subjected to estrous synchronization employing the OvSynch-HeatSync protocol, as described by Borakhatariya et al. ( 2017 ). This protocol facilitates precise timing of ovulation to improve reproductive efficiency. Reproductive parameters including interval from calving to first service, first-service conception rate, number of services per conception, and days open were recorded during the study period. Statistical analysis Data were analyzed using the MIXED procedure of SAS software (version 9.4; SAS Institute Inc., Cary, NC, USA). The statistical model included parity (primiparous vs. multiparous), time, and their interaction as fixed effects, and cow nested within parity as a random effect. Repeated measures analysis was applied for longitudinal data. Least squares means (LSMeans) were compared using Tukey’s test at a significance level of P ≤ 0.05. Results are presented as LSMeans ± SEM. Result Calving related parameters The results related to calving are presented in Table 2 . The placental expulsion time tended to be shorter in primiparous cows compared with multiparous cows (P = 0.09). The incidence of retained placenta was not affected by parity (P > 0.05). However, the highest occurrence was observed in multiparous cows. The calving difficulty score was higher in primiparous cows (P < 0.01), which may indicate a greater physiological burden and stress in this group. No significant differences were observed between the two groups regarding the incidence of milk fever and concentration of serum calcium on day 3 postpartum (P > 0.05). Table 2 Interactive effects of parity and antioxidant bolus supplementation on calving-related parameters of Holstein cows under heat stress conditions. Parameters Experimental groups 1 MB PB SEM P-value 2 Placenta separation time 7.33 6.69 0.20 0.09 Retain placenta (ratio) 0.14 0.07 0.04 0.15 Calving difficulty score 3 0.51 b 0.93 a 0.07 < 0.01 Milk fever (%) 3.92 3.90 0.26 0.95 Ca 4 (mg/dL) 7.26 7.67 0.22 0.31 1 Multiparous cows (MB) and primiparous cows (PB), both groups received antioxidant boluses. Each bolus included copper (2.0 g Cu, copper oxide), selenium (0.063 g Se, sodium selenite), manganese (2.1 g Mn, manganese sulfate), zinc (0.75 g Zn, zinc oxide and zinc sulfate), and vitamin A (470 × 10³ IU). 2 The significant level was declared at P ≤ 0.05. 3 Score 1 - unassisted, 2- normal calving, 3- difficult calving (Eriksson et al. 2004 ). 4 The concentration of calcium at 3 d after calving. BCS changes Changes in BCS from calving time to day 42 after calving are showed in Table 3 . Both primiparous and multiparous cows experienced a decline in BCS during this period. The overall reduction was greater in multiparous cows compared with primiparous cows. The most pronounced BCS loss was observed in multiparous cows between calving time and day 28 after calving, as well as between calving time and day 42 after calving (P < 0.05). Table 3 Interactive effects of parity and antioxidant bolus supplementation on BCS changes in Holstein cows under heat stress conditions. BCS changes, After calving Experimental groups 1 MB PB SEM P-value 2 0 to 28 -0.29 -0.16 0.03 0.02 28 to 42 -0.34 -0.31 0.04 0.66 0 to 42 -0.63 -0.48 0.04 0.04 1 Multiparous cows (MB) and primiparous cows (PB), both groups received antioxidant boluses. Each bolus included copper (2.0 g Cu, copper oxide), selenium (0.063 g Se, sodium selenite), manganese (2.1 g Mn, manganese sulfate), zinc (0.75 g Zn, zinc oxide and zinc sulfate), and vitamin A (470 × 10³ IU). 2 The significant level was declared at P ≤ 0.05. Hematological parameters Hematological measurements, including WBC counts and differential leukocyte percentages, are shown in Table 4 . Multiparous cows exhibited higher WBC counts across all sampling times compared with primiparous cows (P < 0.05). Neutrophil and polymorphonuclear cells percentages were also higher in multiparous cows, especially on days 28 and 42 after calving (P < 0.05). Additionally, lymphocyte percentage was higher in multiparous cows on day 28 after calving (P < 0.05). Table 4 Interactive effects of parity and antioxidant bolus supplementation on white blood cells in Holstein cows under heat stress conditions. Parameters 2 Experimental groups 1 MB PB SEM P-value 3 WBC (10 9 /L) d 14 8.57 a 6.74 b 0.39 0.01 d 28 10.94 a 8.84 b 0.49 0.01 d 42 11.10 a 9.22 b 0.41 0.01 Entire period 10.18 a 8.27 b 0.35 < 0.01 Neutrophils (%) d 14 43.85 39.07 3.03 0.20 d 28 53.41 a 48.68 b 0.96 < 0.01 d 42 54.49 50.48 1.75 0.06 Entire period 50.59 a 46.06 b 1.09 0.02 Lymphocytes (%) d 14 54.56 54.51 2.78 0.98 d 28 42.48 a 37.63 b 1.16 0.02 d 42 37.01 37.90 1.70 0.67 Entire period 44.68 43.36 1.27 0.40 Polymorphonuclear cells (%) d 14 44.45 40.79 2.33 0.36 d 28 56.65 a 51.98 b 1.16 0.02 d 42 59.79 a 55.07 b 1.26 0.03 Entire period 53.66 a 49.21 b 1.16 0.03 1 Multiparous cows (MB) and primiparous cows (PB), both groups received antioxidant boluses. Each bolus included copper (2.0 g Cu, copper oxide), selenium (0.063 g Se, sodium selenite), manganese (2.1 g Mn, manganese sulfate), zinc (0.75 g Zn, zinc oxide and zinc sulfate), and vitamin A (470 × 10³ IU). 2 WBC: White blood cells. 3 The significant level was declared at P ≤ 0.05. Metabolic profile Metabolic parameters during the postpartum period are shown in Fig. 1 . The concentration of glucose in primiparous cows increased significantly on day 14 after calving (P < 0.05) and showed a tendency to remain higher on day 42 after calving (P = 0.09). On the other hand, multiparous cows exhibited the highest concentration of BHBA on day 42 after calving (P = 0.06). Concentrations of serum total protein and albumin were not affected by parity throughout the experimental period (P > 0.05). However, primiparous cows consistently maintained numerically higher concentration of albumin than that the multiparous cows across all sampling times. The highest concentration of ALT was recorded in multiparous cows on day 14 after calving (P 0.05). Antioxidant status Antioxidant-related parameters during the postpartum period are presented in Fig. 2 . No significant differences were observed in the concentration of TAC between treatments throughout the study period (P > 0.05). However, the concentration of MDA increased significantly in primiparous cows on day 42 after calving (P < 0.01). In addition, concentrations of GPX and SOD tended to increase in multiparous cows on day 14 after calving (P = 0.10). Uterine health Indicators of uterine health are summarized in Table 5 . The VDS on days 14 and 28 after calving, and the entire period, did not differ between the two groups (P > 0.05). Similarly, the incidence of metritis showed no significant differences between primiparous and multiparous cows (P > 0.05), although numerically occurrence was double in multiparous cows (8% vs. 4%). Table 5 Interactive effects of parity and antioxidant bolus supplementation on uterine health in Holstein cows under heat stress conditions. Parameters Experimental groups 1 MB PB SEM P-value 2 VDS 3 d 14 2.40 2.48 0.26 0.80 d 28 1.28 1.07 0.24 0.47 Entire period 1.84 1.77 0.21 0.79 Metritis (ratio) 0.08 0.04 0.03 0.36 1 Multiparous cows (MB) and primiparous cows (PB), both groups received antioxidant boluses. Each bolus included copper (2.0 g Cu, copper oxide), selenium (0.063 g Se, sodium selenite), manganese (2.1 g Mn, manganese sulfate), zinc (0.75 g Zn, zinc oxide and zinc sulfate), and vitamin A (470 × 10³ IU). 2 The significant level was declared at P ≤ 0.05. 3 Vaginal discharge score: Zero = no discharge, 1 = clear mucosa, 2 = infection inside mucosa not clear, 3 = mucosa with less than 50% infection, 4 = mucosa with more than 50% infection, 5 = mucosa with more than 50% infection with odor (Kusaka et al. 2020 ). Reproductive performance Reproductive performance data are shown in Table 6 . Parameters such as days to first service, number of inseminations per conception, and first-service conception rate were not affected by parity (P > 0.05). However, days open tended to be shorter in primiparous cows () than multiparous cows (85.51 d vs. 90.84 d) (P = 0.09). Table 6 Interactive effects of parity and antioxidant bolus supplementation on reproductive parameters in Holstein cows under heat stress conditions. Parameters Experimental groups 1 MB PB SEM P-value 2 Calving to first service (d) 70.82 68.86 1.19 0.17 Services per pregnancy 1.84 1.78 0.10 0.65 First service conception rate (ratio) 0.41 0.43 0.06 0.78 Open days (d) 90.84 85.51 1.77 0.10 1 Multiparous cows (MB) and primiparous cows (PB), both groups received antioxidant boluses. Each bolus included copper (2.0 g Cu, copper oxide), selenium (0.063 g Se, sodium selenite), manganese (2.1 g Mn, manganese sulfate), zinc (0.75 g Zn, zinc oxide and zinc sulfate), and vitamin A (470 × 10³ IU). 2 The significant level was declared at P ≤ 0.05. Discussion Calving related parameters The primiparous cows experienced higher calving difficulty scores than multiparous cows, despite identical antioxidant bolus supplementation under heat stress. This result was confirmed the previous findings that showed heifers are more prone to dystocia due to narrower pelvic dimensions and lack of previous calving experience (Wathes et al. 2008 ; Zaborski et al. 2009 ). Moreover, multiparous cows tended to have longer placental expulsion times, consistent with LeBlanc ( 2008 ) and Braga Paiano et al. ( 2019 ), who linked uterine atony and metabolic stress with impaired clearance of fetal membranes. Although antioxidant boluses may reduce oxidative stress around parturition (Choupani et al. 2023a ), parity related physiological constraints persist, confirming that structural and endocrine differences cannot be completely mitigated by antioxidant therapy under heat stress. BCS changes Greater BCS loss in multiparous cows during the first 42 days postpartum aligns with studies by Roche et al. ( 2009 ) and Martens ( 2023 ), who reported that the association of higher milk yield and metabolic demand in older cows. Yehia et al. ( 2020 ) reported similar trends, with multiparous cows experiencing deeper NEB than primiparous cows. Antioxidant supplementation did not affect the NEB decreasing, which aligns with the conclusions of Lopreiato et al. ( 2020 ) that showed nutraceutical interventions alone are insufficient to counteract parity-driven differences in nutrient partitioning, as the root cause lies in the elevated energy demands and profound metabolic adjustments occurring during the transition period. Furthermore, Chen et al. ( 2022 ) indicated that environmental heat stress exacerbates BCS loss by reducing feed intake and increasing energy demands. Hematological parameters Multiparous cows exhibited higher WBC counts and neutrophil percentages, particularly on days 28 and 42 after calving, indicating stronger inflammatory responses compared to primiparous cows. These findings align with da Silva et al. ( 2024 ), who demonstrated that multiparous cows display a more pronounced pro-inflammatory profile than primiparous. Interestingly, despite the administration of antioxidant boluses to both groups at calving, multiparous cows still showed a higher inflammatory status, suggesting that parity-related immune modulation may override the antioxidant effect. Metabolic Profile Primiparous cows exhibited higher concentrations of glucose on day 14, while multiparous cows tended to show increased concentrations of BHBA on day 42, reflecting differences in energy balance between parity groups. According to Saqib et al. ( 2018 ), multiparous cows maintained higher concentrations of glucose and cholesterol in later weeks postpartum, suggesting better metabolic adjustment, whereas primiparous cows showed higher cortisol early postpartum, reflecting greater stress. Similarly, Chen et al. ( 2022 ) observed that heat stress exacerbates ketogenesis in dairy cows, increasing BHBA levels under high metabolic demand. Although antioxidant supplementation improves systemic oxidative balance (Lopreiato et al. 2020 ), our results and previous evidence indicate that parity-related differences in metabolism are primarily driven by energy requirements rather than oxidative stress alone. Antioxidant status Although the concentration of TAC did not differ significantly between parity groups, multiparous cows tended to have higher concentrations of GPX and SOD on day 14 after calving, whereas primiparous cows exhibited a late increase in the concentration of MDA on day 42 after calving. These trends are consistent with Corset et al. ( 2024 ), who reported parity and time-dependent variations in oxidative stress biomarkers, and Yehia et al. ( 2020 ), who highlighted compensatory antioxidant responses under oxidative challenges in transition cows. Notably, these oxidative patterns correspond with other physiological findings from the present study: multiparous cows experienced greater negative energy balance, indicated by higher concentration of BHBA, and showed stronger inflammatory responses, as reflected by increased WBC counts and neutrophil percentage, compared with primiparous cows. These associations suggest that, despite supplementation with antioxidant boluses under heat stress, the elevated metabolic and immune demands in multiparous cows likely intensified oxidative stress earlier in the postpartum period, whereas primiparous cows faced a delayed oxidative challenge, possibly due to extended recovery under persistent thermal conditions. Reproductive Performance In the present study, reproductive parameters such as days to first service, number of inseminations per conception, and first-service conception rate were not significantly influenced by parity, which is consistent with previous reports indicating that reproductive outcomes are multifactorial and strongly influenced by metabolic and uterine health rather than parity alone (LeBlanc 2008 ; Walsh et al. 2011 ). However, days open tended to be shorter in primiparous cows compared with multiparous cows, suggesting a possible advantage for primiparous cows in terms of reproductive recovery. This trend likely reflects the more favorable metabolic status and lower systemic inflammation observed in primiparous cows during early lactation in the current trial, as evidenced by lower concentration of BHBA and reduced oxidative and inflammatory stress. Similar findings were reported by Butler ( 2003 ) and Walsh et al. ( 2011 ), who highlighted that severe NEB and systemic inflammation delay ovarian cyclicity and reduce conception rates, particularly in older cows. Furthermore, da Silva et al. ( 2024 ) demonstrated that multiparous cows exhibit a more pronounced pro-inflammatory immune profile during early pregnancy, which may compromise embryonic survival. Conclusion Our findings highlight that sustained-release antioxidant bolus supplementation influenced transition cows differently depending on parity. Multiparous cows experienced greater body condition loss, heightened inflammatory responses, and increased oxidative stress, whereas primiparous cows faced more frequent calving difficulties but maintained a relatively favorable metabolic balance. These results emphasize that parity continues to play a pivotal role in shaping postpartum physiology, even under antioxidant supplementation. Therefore, the administration of antioxidant boluses alone is insufficient to fully mitigate parity-associated metabolic and immunological challenges. Future management strategies should adopt an integrated, parity-specific approach, combining nutritional, environmental, and health interventions to optimize the health, reproductive performance, and overall transition success of dairy cows under heat stress conditions. Declarations Author contribution M. Choupani conducted and managed the study and served as a manuscript author. A. Riasi designed the study, supervised the project, and contributed to project management. M. R. Samadian and M. Alikhani acted as project supervisors and assisted in project management. N. Javani Javani contributed to manuscript writing and editing. All authors read and approved the final version of the manuscript. Data availability All data of this study are available to the corresponding author. All supporting data and conclusion of this study are given in the main text. Code availability Not applicable. Ethics approval The Animal Care Advisory Committee of Isfahan University of Technology approved all stages of the research project. Consent to participate All authors agreed to conduct this study. Consent for publication All authors agree to publish the relevant data in the journal. Conflict of interest The authors declare no competing interests. References Alberghina, D., Amato, A., Brancato, G., Cavallo, C., Liotta, L., and Lopreiato, V., 2024. Impact of Heat Stress on the Balance between Oxidative Markers and the Antioxidant Defence System in the Plasma of Mid-Lactating Modicana Dairy Cows. Animals, 14(14), 2034. Borakhatariya, D., Panchal, M., Dhami, A., Hadiya, K., and Kalasariya, R., 2017. Efficacy of estrus synchronization protocols during summer and winter seasons together with biochemical and minerals profile in anestrus cows. 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Comparison of diagnostic methods for uterine health in dairy cattle on different days postpartum. Veterinary Record, 186(3), 91–91. LeBlanc, S., 2010. Monitoring metabolic health of dairy cattle in the transition period. Journal of reproduction and Development, 56(S), S29-S35. LeBlanc, S. J., 2008. Postpartum uterine disease and dairy herd reproductive performance: a review. The Veterinary Journal, 176(1), 102–114. Lopreiato, V., Mezzetti, M., Cattaneo, L., Ferronato, G., Minuti, A., and Trevisi, E., 2020. Role of nutraceuticals during the transition period of dairy cows: a review. Journal of Animal Science and Biotechnology, 11(1), 96. Martens, H., 2023. Invited review: increasing milk yield and negative energy balance: a gordian knot for dairy cows? Animals, 13(19), 3097. NRC., 2001. Nutrient Requirements of Dairy Cattle. 7th revised ed. National Academic Science, Washington, DC, USA. Roche, J. R., Friggens, N. C., Kay, J. K., Fisher, M. W., Stafford, K. J., and Berry, D. P., 2009. Invited review: Body condition score and its association with dairy cow productivity, health, and welfare. Journal of Dairy Science, 92(12), 5769–5801. Santos, J., Bisinotto, R., and Ribeiro, E., 2016. Mechanisms underlying reduced fertility in anovular dairy cows. Theriogenology, 86(1), 254–262. Saqib, M. N., Qureshi, M. S., and Khan, R. U., 2018. Changes in postpartum metabolites and resumption of ovarian cyclicity in primiparous and multiparous dairy cows. Applied Biological Chemistry, 61(1), 107–111. Schröder, U., and Staufenbiel, R., 2006. Invited review: Methods to determine body fat reserves in the dairy cow with special regard to ultrasonographic measurement of backfat thickness. Journal of Dairy Science, 89(1), 1–14. Van Soest, P. v., Robertson, J. B., and Lewis, B. A., 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science, 74(10), 3583–3597. Walsh, S., Williams, E., and Evans, A., 2011. A review of the causes of poor fertility in high milk producing dairy cows. Animal reproduction science, 123(3–4), 127–138. Wathes, D., Brickell, J., Bourne, N., Swali, A., and Cheng, Z., 2008. Factors influencing heifer survival and fertility on commercial dairy farms. animal, 2(8), 1135–1143. Yehia, S. G., Ramadan, E. S., Megahed, E. A., and Salem, N. Y., 2020. Effect of parity on metabolic and oxidative stress profiles in Holstein dairy cows. Veterinary world, 13(12), 2780. Zaborski, D., Grzesiak, W., Szatkowska, I., Dybus, A., Muszynska, M., and Jedrzejczak, M., 2009. Factors affecting dystocia in cattle. Reproduction in domestic animals, 44(3), 540–551. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Choupani","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYHACNgjFzHwASEpABQ2I0sKWANbCQ7wWBh6IMh5CrtJtYH724GPbHXlzdp6PH78wWCTuZ2B++IGh4B5OLWYH2MwNZ7Y9M9zZzLtZWoZBIrGHgc1YgsGgGI8WHjZp3rbDjBsO826QlgBrYTAD+iWBoBb7DYd5Hv+GaGH/RpSWRKAWNskPYC08BGw5zGYmOePcs+QNQIY1g4GEcc9hnmKJBHxajjc/k/hQdsd2w/nDj2/+qKiTbW9v3/jhwx/cWhiYweQBCBscNSARPBpgAKKF8QdhlaNgFIyCUTACAQAlo0ny895vDgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-6306-2397","institution":"Isfahan University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Mohammad","middleName":"","lastName":"Choupani","suffix":""},{"id":532879347,"identity":"34606e01-423f-46b9-acfe-f0d6b3ce1dd2","order_by":1,"name":"Ahmad Riasi","email":"","orcid":"","institution":"Isfahan University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Ahmad","middleName":"","lastName":"Riasi","suffix":""},{"id":532879348,"identity":"a02050d1-7c21-417e-ac36-bcf9fec99665","order_by":2,"name":"Masoud Alikhani","email":"","orcid":"","institution":"Isfahan University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Masoud","middleName":"","lastName":"Alikhani","suffix":""},{"id":532879349,"identity":"bab07a0b-7a03-4425-aa53-b33bbff17776","order_by":3,"name":"Mohammad Reza Samadian","email":"","orcid":"","institution":"Isfahan University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Reza","lastName":"Samadian","suffix":""},{"id":532879350,"identity":"138a8741-f4f8-4fd5-9d2c-4b22c1689be1","order_by":4,"name":"Nazanin Javani Javani","email":"","orcid":"","institution":"Isfahan University of 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15:07:46","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":110060,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7633697/v1/8f4a9018b23eb1690cb9f747.html"},{"id":94871988,"identity":"3a812aa4-5b06-41ca-972b-a06b6b83a6c5","added_by":"auto","created_at":"2025-10-31 15:07:46","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":225471,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA-F \u003c/strong\u003eInteractive effects of parity and antioxidant bolus supplementation on some blood metabolites in Holstein cows under heat stress conditions. \u003csup\u003e1 \u003c/sup\u003eMultiparous cows (MB) and primiparous cows (PB), both groups received antioxidant boluses. Each bolus included copper (2.0 g Cu, copper oxide), selenium (0.063 g Se, sodium selenite), manganese (2.1 g Mn, manganese sulfate), zinc (0.75 g Zn, zinc oxide and zinc sulfate), and vitamin A (470 × 10³ IU). Significant differences are shown by different tags (‡P≤ 0.1; *P≤ 0.05; **P≤ 0.01). Values are expressed as mean ± SE\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7633697/v1/f24b948e2443da365e7b68dd.jpeg"},{"id":94986553,"identity":"45d6f01d-63e3-4520-9a86-5452bb92669f","added_by":"auto","created_at":"2025-11-03 07:00:26","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":174954,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA-D \u003c/strong\u003eInteractive effects of parity and antioxidant bolus supplementation on antioxidant status in Holstein cows under heat stress conditions. \u003csup\u003e1 \u003c/sup\u003eMultiparous cows (MB) and primiparous cows (PB), both groups received antioxidant boluses. Each bolus included copper (2.0 g Cu, copper oxide), selenium (0.063 g Se, sodium selenite), manganese (2.1 g Mn, manganese sulfate), zinc (0.75 g Zn, zinc oxide and zinc sulfate), and vitamin A (470 × 10³ IU). Significant differences are shown by different tags (‡P≤ 0.1; *P≤ 0.05; **P≤ 0.01). Values are expressed as mean ± SE\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7633697/v1/5a4ae18a9e5a0d4a6c527314.jpeg"},{"id":97135559,"identity":"bf2dc45d-7a03-49c2-9f1c-52107660b219","added_by":"auto","created_at":"2025-12-01 09:51:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1448331,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7633697/v1/96063689-d723-49d2-84fb-de008c58038a.pdf"}],"financialInterests":"","formattedTitle":"Effects of parity on metabolic, oxidative status and reproductive responses in heat-stressed transition dairy cows supplemented with slow release boluses","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe transition period, defined as the three weeks before and three weeks after calving, is a pivotal stage in the productive life of dairy cows. This period is characterized by profound metabolic, physiologic, and immunologic challenges (LeBlanc \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). During the transition period, cows become vulnerable to multiple disorders, including negative energy balance (NEB), hypocalcemia, oxidative stress, uterine infections, and impaired fertility (Santos et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). It is reported that the challenges intensify under heat stress condition (Das et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Primiparous cows, due to their ongoing body growth and lack of previous lactation experience, typically experience higher levels of stress. On the other hand, multiparous cows, because of greater metabolic load and a higher risk of systemic inflammation, are more prone to developing postpartum metabolic and reproductive disorders (Choupani et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e; Choupani et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e; Corset et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Nutritional intervention with antioxidant supplements during the transition period has garnered increasing attention. Choupani et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003eb\u003c/span\u003e) demonstrated that sustained-release antioxidant boluses administered around calving improved metabolic profiles, maintained body condition, stabilized hematological parameters, and supported uterine health in heat-stressed cows. Importantly, a recent study by Hao et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) provided further evidence in support of antioxidant strategies. They showed that supplementation with rumen-protected glutathione during the transition period reduced oxidative stress biomarkers, lowered inflammatory indices, enhanced antioxidant enzyme activity, and improved lactation outcomes in Holstein cows. Recent studies have indicated that multiparous dairy cows, due to their higher milk production, are generally exposed to greater oxidative stress (Yehia et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). They reported that malondialdehyde (MDA) concentrations were higher in multiparous cows than that the primiparous cows. Moreover, Alberghina et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) showed that heat stress further exacerbates the oxidative challenges by increasing the production of reactive oxygen species and imposing additional pressure on antioxidant defense systems in dairy cows. Multiparous cows, despite having a higher oxidative burden, exhibit a stronger antioxidant defense capacity, functioning as a compensatory mechanism to counteract oxidative stress (Corset et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Based on these findings, we hypothesized that supplementation with antioxidant slow release boluses could enhance antioxidant capacity, particularly in multiparous cows, and help mitigate the negative effects of oxidative stress during the Summer season. Considering that multiparous cows generally experience greater metabolic and reproductive challenges compared to primiparous cows, the present study aimed to evaluate whether sustained-release antioxidant bolus supplementation could differentially influence transition cow health and performance between these two parity groups. Understanding these differential responses may help design targeted nutritional strategies to improve postpartum adaptation under heat stress conditions. Therefore, in the present study, the effects of slow-release oral bolus on metabolic, inflammatory, and reproductive status in primiparous and multiparous cows during heat stress were investigated.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimals and management\u003c/h2\u003e\u003cp\u003eThe study was conducted during the summer season, under heat stress conditions (average THI\u003csub\u003emax\u003c/sub\u003e \u0026gt;72) in a large commercial dairy farm consisting of 5000 dairy cows (Ghiam Dairy Company, Isfahan, Iran). A total of 200 Holstein dairy cows, including 60 primiparous and 140 multiparous (parity\u0026thinsp;=\u0026thinsp;3.87\u0026thinsp;\u0026plusmn;\u0026thinsp;1.78), were allocated to completely randomized design. Therefore, two experimental treatments were defined in this study: multiparous cows (MB) and primiparous cows (PB), both of which received antioxidant boluses. All experimental procedures involving animals were approved by the Institutional Animal Care Committee of Isfahan University of Technology and carried out in accordance with the ethical guidelines established by the Iranian Council of Animal Care (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSlow release bolus specifications\u003c/h3\u003e\n\u003cp\u003eAll cows orally received three slow release antioxidant boluses (Golpooneh Safahan, Knowledge-based Co., Isfahan, Iran) at calving using a bolus gun. Each bolus was manufactured in a cylindrical form (16 mm in length, 25 mm in diameter, weighing 15 g) and was completely coated with an inert polymer, except in the bottom surface, to facilitate a sustainable release of its active components. The active ingredients in each bolus were: copper (2.0 g Cu, copper oxide), selenium (0.063 g Se, sodium selenite), manganese (2.1 g Mn, manganese sulfate), zinc (0.75 g Zn, zinc oxide and zinc sulfate), and vitamin A (470 \u0026times; 10\u0026sup3; IU). According to the manufacturer, these components were gradually released over 21 days as the bolus dissolved in reticulorumen. Based on the analysis of the boluses and their release rate profile, each cow received approximately 9 mg of selenium, 107.14 mg of zinc, 285.71 mg of copper, 300 mg of magnesium, and 67,142.85 IU of vitamin A per day.\u003c/p\u003e\n\u003ch3\u003eHousing and feeding\u003c/h3\u003e\n\u003cp\u003e All cows were housed in free-stall barns equipped with cooling systems and fed a total mixed ration (TMR) formulated according to NRC (2001) recommendations, with ad libitum access to water. The average dry matter intake (DMI) of primiparous and multiparous cows during the fresh period was 16.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4 kg/day and 18.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.90 kg/day, respectively. During the entire lactation, the mean DMI was 24.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12 kg/day for primiparous cows and 27.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.40 kg/day for multiparous cows. Cows were milked three times per day (08:00, 16:00, and 24:00). The ingredients and compositions of the fresh and lactation rations are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Feed samples were analyzed to determine dry matter (DM; AOAC method 934.01), crude protein (AOAC method 976.05), ether extract (EE; AOAC method 920.29), and ash (AOAC method 942.05) based the AOAC (2000) guidelines. Neutral detergent fiber (aNDF) and acid detergent fiber (ADF) concentrations were analyzed according to Van Soest et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1991\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eIngredients and chemical composition of diets fed to Holstein cows.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eDiets\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eItems\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFresh\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLactation\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIngredients (% DM)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCorn silage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e19.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17.36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAlfalfa hay\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e18.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.19\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStraw\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.89\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWhole cotton seed\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.89\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBeet pulp\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.66\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBarley grain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCorn grain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e31.54\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCorn bran\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSoybean meal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.04\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCanola meal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.41\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFull-fat soybean\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCorn gluten\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.26\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMeat meal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.73\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFat powder\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.83\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSodium bicarbonate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.39\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCalcium carbonate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.69\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVitamin supplement\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMineral supplement\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMagnesium oxide\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.35\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBio binder\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDicalcium phosphate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSalt\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.44\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eToxin binder\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUrea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMagnesium sulfate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCalcium chloride\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eChemical composition\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDry matter (% as fed)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e49.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCrude protein (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16.28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEther extract (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.91\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNE\u003csub\u003eL\u003c/sub\u003e\u003csup\u003e4\u003c/sup\u003e (MJ/kg DM)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eaNDF\u003csup\u003e5\u003c/sup\u003e (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e31.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e26.16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eADF (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e19.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAsh (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSelenium (mg/kg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.09\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eZinc (mg/kg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e69.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e67.96\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCopper (mg/kg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.71\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eManganese (mg/kg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e28.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e28.31\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCalcium (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhosphorus (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.61\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVitamin A (IU/d)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e100,000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e150,000\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Fresh: the first 3 week of lactation, and Lactation: after 3 weeks of lactation.\u003c/p\u003e\u003cp\u003e\u003csup\u003e2\u003c/sup\u003e Vitamin supplements contained: Vitamin A at 1,500,000 IU/kg; Vitamin D₃ at 400,000 IU/kg; and Vitamin E at 15,000 IU/kg.\u003c/p\u003e\u003cp\u003e\u003csup\u003e3\u003c/sup\u003e Mineral supplement contained: Co, 120 mg/kg (cobalt sulfate); Cu, 5,000 mg/kg (copper sulfate); I, 200 mg/kg (calcium iodate); Fe, 800 mg/kg (ferrous sulfate); Mn, 15,000 mg/kg (manganese sulfate); Se, 100 mg/kg (sodium selenite); and Zn, 2,000 mg/kg (zinc sulfate).\u003c/p\u003e\u003cp\u003e\u003csup\u003e4\u003c/sup\u003e Net energy for lactation was calculated according to NRC (2001) recommendations.\u003c/p\u003e\u003cp\u003e\u003csup\u003e5\u003c/sup\u003e Neutral detergent fiber was analyzed using a heat-stable amylase and expressed inclusive of residual ash.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eCalving related parameters and BCS\u003c/h3\u003e\n\u003cp\u003eCalving related parameters including time to expulsion of fetal membranes, calving difficulty score (Score 1 - unassisted, 2- normal calving, 3- difficult calving (Eriksson et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2004\u003c/span\u003e)), incidence of retained placenta, milk fever, and colostrum yield were recorded immediately after calving. The backfat thickness of each cow was measured using an Easi-Scan ultrasound scanner (BCF Technology Ltd., Livingston, UK) operating at a frequency range of 4.5\u0026ndash;8.5 MHz, and BCS was assessed on a 1 to 5 scale at calving (day 0) and on days 28 and 42 after calving (Schr\u0026ouml;der and Staufenbiel \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eSampling and measurements\u003c/h3\u003e\n\u003cp\u003eBlood samples were collected by jugular venipuncture on days 0, 3, 14, 28, and 42 after calving. Two types of samples were obtained: Whole blood in EDTA tubes for hematological analysis including white blood cells (WBC), neutrophils, lymphocytes, and polymorphonuclear cells (Cell counter analyzer MS 9\u0026thinsp;\u0026minus;\u0026thinsp;3). Serum samples obtained after centrifugation at 3000 \u0026times; g for 15 minutes and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until analysis. Serum biochemical parameters measured included glucose (Pars Azmoon glucose Kit, Photometric method, Tehran, Iran), β-hydroxybutyrate (BHBA, Randox Kit, intra- and inter-CV 2.3% and 5.1%), total protein (photometric, biuret method, intra- and inter-CV\u0026thinsp;\u0026lt;\u0026thinsp;2.1% and \u0026lt;\u0026thinsp;3.1%), albumin (photometric, bromocresol green method, intra- and inter-CV\u0026thinsp;\u0026lt;\u0026thinsp;1.9% and \u0026lt;\u0026thinsp;3%). Oxidative stress and antioxidant status were evaluated by measuring total antioxidant capacity (TAC), glutathione peroxidase (GPX), superoxide dismutase (SOD) (Autoanalyzer, Alcyon 300, intra- and inter-CV\u0026thinsp;\u0026lt;\u0026thinsp;2.4% and \u0026lt;\u0026thinsp;3.2%, intra- and inter-CV\u0026thinsp;\u0026lt;\u0026thinsp;3.7% and \u0026lt;\u0026thinsp;3.7%, intra- and inter-CV\u0026thinsp;\u0026lt;\u0026thinsp;8% and \u0026lt;\u0026thinsp;10%, respectively), and malondialdehyde (MDA, based on the colorful complex formed from the reaction of MDA with 2-thiobarbituric acid in acid environment). Liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (Pars Azmoon Kit, Tehran, Iran), and the serum calcium (photometric method, cresolphthalein complexone, Intra- and inter-CV\u0026thinsp;\u0026lt;\u0026thinsp;8.9% and \u0026lt;\u0026thinsp;10.5%), were measured.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eUterine health evaluation\u003c/h2\u003e\u003cp\u003eUterine health was evaluated by assessing vaginal discharge scores (VDS) on days 14 and 28 after calving using the Metricheck device, with scores assigned on a 0\u0026ndash;5 scale (Zero\u0026thinsp;=\u0026thinsp;no discharge, 1\u0026thinsp;=\u0026thinsp;clear mucosa, 2\u0026thinsp;=\u0026thinsp;infection inside mucosa not clear, 3\u0026thinsp;=\u0026thinsp;mucosa with less than 50% infection, 4\u0026thinsp;=\u0026thinsp;mucosa with more than 50% infection, 5\u0026thinsp;=\u0026thinsp;mucosa with more than 50% infection with odor) as described by Kusaka et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The cows with a score\u0026thinsp;\u0026gt;\u0026thinsp;3 were considered as metritis disorder.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eReproductive performance\u003c/h3\u003e\n\u003cp\u003eAt 45 days postpartum, all cows were subjected to estrous synchronization employing the OvSynch-HeatSync protocol, as described by Borakhatariya et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This protocol facilitates precise timing of ovulation to improve reproductive efficiency. Reproductive parameters including interval from calving to first service, first-service conception rate, number of services per conception, and days open were recorded during the study period.\u003c/p\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData were analyzed using the MIXED procedure of SAS software (version 9.4; SAS Institute Inc., Cary, NC, USA). The statistical model included parity (primiparous vs. multiparous), time, and their interaction as fixed effects, and cow nested within parity as a random effect. Repeated measures analysis was applied for longitudinal data. Least squares means (LSMeans) were compared using Tukey\u0026rsquo;s test at a significance level of P\u0026thinsp;\u0026le;\u0026thinsp;0.05. Results are presented as LSMeans\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/p\u003e\u003c/div\u003e"},{"header":"Result","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003eCalving related parameters\u003c/h2\u003e\u003cp\u003eThe results related to calving are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The placental expulsion time tended to be shorter in primiparous cows compared with multiparous cows (P\u0026thinsp;=\u0026thinsp;0.09). The incidence of retained placenta was not affected by parity (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, the highest occurrence was observed in multiparous cows. The calving difficulty score was higher in primiparous cows (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), which may indicate a greater physiological burden and stress in this group. No significant differences were observed between the two groups regarding the incidence of milk fever and concentration of serum calcium on day 3 postpartum (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eInteractive effects of parity and antioxidant bolus supplementation on calving-related parameters of Holstein cows under heat stress conditions.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eParameters\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eExperimental groups\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSEM\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eP-value\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePlacenta separation time\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.09\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRetain placenta (ratio)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCalving difficulty score\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.51\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.93\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMilk fever (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.95\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCa\u003csup\u003e4\u003c/sup\u003e (mg/dL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Multiparous cows (MB) and primiparous cows (PB), both groups received antioxidant boluses. Each bolus included copper (2.0 g Cu, copper oxide), selenium (0.063 g Se, sodium selenite), manganese (2.1 g Mn, manganese sulfate), zinc (0.75 g Zn, zinc oxide and zinc sulfate), and vitamin A (470 \u0026times; 10\u0026sup3; IU).\u003c/p\u003e\u003cp\u003e\u003csup\u003e2\u003c/sup\u003e The significant level was declared at P\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e\u003cp\u003e\u003csup\u003e3\u003c/sup\u003e Score 1 - unassisted, 2- normal calving, 3- difficult calving (Eriksson et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003csup\u003e4\u003c/sup\u003e The concentration of calcium at 3 d after calving.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eBCS changes\u003c/h2\u003e\u003cp\u003eChanges in BCS from calving time to day 42 after calving are showed in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Both primiparous and multiparous cows experienced a decline in BCS during this period. The overall reduction was greater in multiparous cows compared with primiparous cows. The most pronounced BCS loss was observed in multiparous cows between calving time and day 28 after calving, as well as between calving time and day 42 after calving (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eInteractive effects of parity and antioxidant bolus supplementation on BCS changes in Holstein cows under heat stress conditions.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eBCS changes,\u003c/p\u003e\u003cp\u003eAfter calving\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eExperimental groups\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSEM\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eP-value\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0 to 28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-0.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e28 to 42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-0.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.66\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0 to 42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-0.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-0.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Multiparous cows (MB) and primiparous cows (PB), both groups received antioxidant boluses. Each bolus included copper (2.0 g Cu, copper oxide), selenium (0.063 g Se, sodium selenite), manganese (2.1 g Mn, manganese sulfate), zinc (0.75 g Zn, zinc oxide and zinc sulfate), and vitamin A (470 \u0026times; 10\u0026sup3; IU).\u003c/p\u003e\u003cp\u003e\u003csup\u003e2\u003c/sup\u003e The significant level was declared at P\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eHematological parameters\u003c/h2\u003e\u003cp\u003eHematological measurements, including WBC counts and differential leukocyte percentages, are shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Multiparous cows exhibited higher WBC counts across all sampling times compared with primiparous cows (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Neutrophil and polymorphonuclear cells percentages were also higher in multiparous cows, especially on days 28 and 42 after calving (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, lymphocyte percentage was higher in multiparous cows on day 28 after calving (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eInteractive effects of parity and antioxidant bolus supplementation on white blood cells in Holstein cows under heat stress conditions.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eParameters\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eExperimental groups\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSEM\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eP-value\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWBC (10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ed 14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8.57\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.74\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ed 28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.94\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.84\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ed 42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11.10\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.22\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEntire period\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10.18\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.27\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNeutrophils (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ed 14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e43.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e39.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ed 28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e53.41\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e48.68\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ed 42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e54.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.06\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEntire period\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e50.59\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e46.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLymphocytes (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ed 14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e54.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e54.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.98\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ed 28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e42.48\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e37.63\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ed 42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e37.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e37.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.67\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEntire period\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e44.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e43.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.40\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePolymorphonuclear cells (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ed 14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e44.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ed 28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e56.65\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e51.98\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ed 42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e59.79\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e55.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEntire period\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e53.66\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e49.21\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Multiparous cows (MB) and primiparous cows (PB), both groups received antioxidant boluses. Each bolus included copper (2.0 g Cu, copper oxide), selenium (0.063 g Se, sodium selenite), manganese (2.1 g Mn, manganese sulfate), zinc (0.75 g Zn, zinc oxide and zinc sulfate), and vitamin A (470 \u0026times; 10\u0026sup3; IU).\u003c/p\u003e\u003cp\u003e\u003csup\u003e2\u003c/sup\u003e WBC: White blood cells.\u003c/p\u003e\u003cp\u003e\u003csup\u003e3\u003c/sup\u003e The significant level was declared at P\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eMetabolic profile\u003c/h2\u003e\u003cp\u003eMetabolic parameters during the postpartum period are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The concentration of glucose in primiparous cows increased significantly on day 14 after calving (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and showed a tendency to remain higher on day 42 after calving (P\u0026thinsp;=\u0026thinsp;0.09). On the other hand, multiparous cows exhibited the highest concentration of BHBA on day 42 after calving (P\u0026thinsp;=\u0026thinsp;0.06). Concentrations of serum total protein and albumin were not affected by parity throughout the experimental period (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, primiparous cows consistently maintained numerically higher concentration of albumin than that the multiparous cows across all sampling times. The highest concentration of ALT was recorded in multiparous cows on day 14 after calving (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), whereas the concentration of AST showed no significant differences between groups during the study period (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eAntioxidant status\u003c/h2\u003e\u003cp\u003eAntioxidant-related parameters during the postpartum period are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. No significant differences were observed in the concentration of TAC between treatments throughout the study period (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, the concentration of MDA increased significantly in primiparous cows on day 42 after calving (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In addition, concentrations of GPX and SOD tended to increase in multiparous cows on day 14 after calving (P\u0026thinsp;=\u0026thinsp;0.10).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eUterine health\u003c/h2\u003e\u003cp\u003eIndicators of uterine health are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The VDS on days 14 and 28 after calving, and the entire period, did not differ between the two groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Similarly, the incidence of metritis showed no significant differences between primiparous and multiparous cows (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), although numerically occurrence was double in multiparous cows (8% vs. 4%).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eInteractive effects of parity and antioxidant bolus supplementation on uterine health in Holstein cows under heat stress conditions.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eParameters\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eExperimental groups\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSEM\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eP-value\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVDS\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ed 14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.80\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ed 28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.47\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEntire period\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.79\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMetritis (ratio)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Multiparous cows (MB) and primiparous cows (PB), both groups received antioxidant boluses. Each bolus included copper (2.0 g Cu, copper oxide), selenium (0.063 g Se, sodium selenite), manganese (2.1 g Mn, manganese sulfate), zinc (0.75 g Zn, zinc oxide and zinc sulfate), and vitamin A (470 \u0026times; 10\u0026sup3; IU).\u003c/p\u003e\u003cp\u003e\u003csup\u003e2\u003c/sup\u003e The significant level was declared at P\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e\u003cp\u003e\u003csup\u003e3\u003c/sup\u003e Vaginal discharge score: Zero\u0026thinsp;=\u0026thinsp;no discharge, 1\u0026thinsp;=\u0026thinsp;clear mucosa, 2\u0026thinsp;=\u0026thinsp;infection inside mucosa not clear, 3\u0026thinsp;=\u0026thinsp;mucosa with less than 50% infection, 4\u0026thinsp;=\u0026thinsp;mucosa with more than 50% infection, 5\u0026thinsp;=\u0026thinsp;mucosa with more than 50% infection with odor (Kusaka et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eReproductive performance\u003c/h2\u003e\u003cp\u003eReproductive performance data are shown in Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Parameters such as days to first service, number of inseminations per conception, and first-service conception rate were not affected by parity (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, days open tended to be shorter in primiparous cows () than multiparous cows (85.51 d vs. 90.84 d) (P\u0026thinsp;=\u0026thinsp;0.09).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eInteractive effects of parity and antioxidant bolus supplementation on reproductive parameters in Holstein cows under heat stress conditions.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eParameters\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eExperimental groups\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePB\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSEM\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eP-value\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCalving to first service (d)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e70.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e68.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eServices per pregnancy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.65\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFirst service conception rate (ratio)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.78\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOpen days (d)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e90.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e85.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Multiparous cows (MB) and primiparous cows (PB), both groups received antioxidant boluses. Each bolus included copper (2.0 g Cu, copper oxide), selenium (0.063 g Se, sodium selenite), manganese (2.1 g Mn, manganese sulfate), zinc (0.75 g Zn, zinc oxide and zinc sulfate), and vitamin A (470 \u0026times; 10\u0026sup3; IU).\u003c/p\u003e\u003cp\u003e\u003csup\u003e2\u003c/sup\u003e The significant level was declared at P\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eCalving related parameters\u003c/h2\u003e\u003cp\u003eThe primiparous cows experienced higher calving difficulty scores than multiparous cows, despite identical antioxidant bolus supplementation under heat stress. This result was confirmed the previous findings that showed heifers are more prone to dystocia due to narrower pelvic dimensions and lack of previous calving experience (Wathes et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Zaborski et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Moreover, multiparous cows tended to have longer placental expulsion times, consistent with LeBlanc (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) and Braga Paiano et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), who linked uterine atony and metabolic stress with impaired clearance of fetal membranes. Although antioxidant boluses may reduce oxidative stress around parturition (Choupani et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e), parity related physiological constraints persist, confirming that structural and endocrine differences cannot be completely mitigated by antioxidant therapy under heat stress.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eBCS changes\u003c/h2\u003e\u003cp\u003eGreater BCS loss in multiparous cows during the first 42 days postpartum aligns with studies by Roche et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and Martens (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), who reported that the association of higher milk yield and metabolic demand in older cows. Yehia et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) reported similar trends, with multiparous cows experiencing deeper NEB than primiparous cows. Antioxidant supplementation did not affect the NEB decreasing, which aligns with the conclusions of Lopreiato et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) that showed nutraceutical interventions alone are insufficient to counteract parity-driven differences in nutrient partitioning, as the root cause lies in the elevated energy demands and profound metabolic adjustments occurring during the transition period. Furthermore, Chen et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) indicated that environmental heat stress exacerbates BCS loss by reducing feed intake and increasing energy demands.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eHematological parameters\u003c/h2\u003e\u003cp\u003eMultiparous cows exhibited higher WBC counts and neutrophil percentages, particularly on days 28 and 42 after calving, indicating stronger inflammatory responses compared to primiparous cows. These findings align with da Silva et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), who demonstrated that multiparous cows display a more pronounced pro-inflammatory profile than primiparous. Interestingly, despite the administration of antioxidant boluses to both groups at calving, multiparous cows still showed a higher inflammatory status, suggesting that parity-related immune modulation may override the antioxidant effect.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eMetabolic Profile\u003c/h2\u003e\u003cp\u003ePrimiparous cows exhibited higher concentrations of glucose on day 14, while multiparous cows tended to show increased concentrations of BHBA on day 42, reflecting differences in energy balance between parity groups. According to Saqib et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), multiparous cows maintained higher concentrations of glucose and cholesterol in later weeks postpartum, suggesting better metabolic adjustment, whereas primiparous cows showed higher cortisol early postpartum, reflecting greater stress. Similarly, Chen et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) observed that heat stress exacerbates ketogenesis in dairy cows, increasing BHBA levels under high metabolic demand. Although antioxidant supplementation improves systemic oxidative balance (Lopreiato et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), our results and previous evidence indicate that parity-related differences in metabolism are primarily driven by energy requirements rather than oxidative stress alone.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eAntioxidant status\u003c/h2\u003e\u003cp\u003eAlthough the concentration of TAC did not differ significantly between parity groups, multiparous cows tended to have higher concentrations of GPX and SOD on day 14 after calving, whereas primiparous cows exhibited a late increase in the concentration of MDA on day 42 after calving. These trends are consistent with Corset et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), who reported parity and time-dependent variations in oxidative stress biomarkers, and Yehia et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), who highlighted compensatory antioxidant responses under oxidative challenges in transition cows. Notably, these oxidative patterns correspond with other physiological findings from the present study: multiparous cows experienced greater negative energy balance, indicated by higher concentration of BHBA, and showed stronger inflammatory responses, as reflected by increased WBC counts and neutrophil percentage, compared with primiparous cows. These associations suggest that, despite supplementation with antioxidant boluses under heat stress, the elevated metabolic and immune demands in multiparous cows likely intensified oxidative stress earlier in the postpartum period, whereas primiparous cows faced a delayed oxidative challenge, possibly due to extended recovery under persistent thermal conditions.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eReproductive Performance\u003c/h2\u003e\u003cp\u003eIn the present study, reproductive parameters such as days to first service, number of inseminations per conception, and first-service conception rate were not significantly influenced by parity, which is consistent with previous reports indicating that reproductive outcomes are multifactorial and strongly influenced by metabolic and uterine health rather than parity alone (LeBlanc \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Walsh et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). However, days open tended to be shorter in primiparous cows compared with multiparous cows, suggesting a possible advantage for primiparous cows in terms of reproductive recovery. This trend likely reflects the more favorable metabolic status and lower systemic inflammation observed in primiparous cows during early lactation in the current trial, as evidenced by lower concentration of BHBA and reduced oxidative and inflammatory stress. Similar findings were reported by Butler (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) and Walsh et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), who highlighted that severe NEB and systemic inflammation delay ovarian cyclicity and reduce conception rates, particularly in older cows. Furthermore, da Silva et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) demonstrated that multiparous cows exhibit a more pronounced pro-inflammatory immune profile during early pregnancy, which may compromise embryonic survival.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur findings highlight that sustained-release antioxidant bolus supplementation influenced transition cows differently depending on parity. Multiparous cows experienced greater body condition loss, heightened inflammatory responses, and increased oxidative stress, whereas primiparous cows faced more frequent calving difficulties but maintained a relatively favorable metabolic balance. These results emphasize that parity continues to play a pivotal role in shaping postpartum physiology, even under antioxidant supplementation. Therefore, the administration of antioxidant boluses alone is insufficient to fully mitigate parity-associated metabolic and immunological challenges. Future management strategies should adopt an integrated, parity-specific approach, combining nutritional, environmental, and health interventions to optimize the health, reproductive performance, and overall transition success of dairy cows under heat stress conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM. Choupani conducted and managed the study and served as a manuscript author. A. Riasi designed the study, supervised the project, and contributed to project management. M. R. Samadian and M. Alikhani acted as project supervisors and assisted in project management. N. Javani Javani contributed to manuscript writing and editing. All authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data of this study are available to the corresponding author. All supporting data and conclusion of this study are given in the main text.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Animal Care Advisory Committee of Isfahan University of Technology approved all stages of the research project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agreed to conduct this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agree to publish the relevant data in the journal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlberghina, D., Amato, A., Brancato, G., Cavallo, C., Liotta, L., and Lopreiato, V., 2024. 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Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science, 74(10), 3583\u0026ndash;3597.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWalsh, S., Williams, E., and Evans, A., 2011. A review of the causes of poor fertility in high milk producing dairy cows. Animal reproduction science, 123(3\u0026ndash;4), 127\u0026ndash;138.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWathes, D., Brickell, J., Bourne, N., Swali, A., and Cheng, Z., 2008. Factors influencing heifer survival and fertility on commercial dairy farms. animal, 2(8), 1135\u0026ndash;1143.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYehia, S. G., Ramadan, E. S., Megahed, E. A., and Salem, N. Y., 2020. Effect of parity on metabolic and oxidative stress profiles in Holstein dairy cows. Veterinary world, 13(12), 2780.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZaborski, D., Grzesiak, W., Szatkowska, I., Dybus, A., Muszynska, M., and Jedrzejczak, M., 2009. Factors affecting dystocia in cattle. Reproduction in domestic animals, 44(3), 540\u0026ndash;551.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Parity, Heat stress, Antioxidant bolus, Oxidative stress, Dairy cows, Reproduction","lastPublishedDoi":"10.21203/rs.3.rs-7633697/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7633697/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHeat stress negatively impacts transition cow health, metabolism, and fertility, while parity further influences these physiological responses. Antioxidant supplementation is proposed to mitigate oxidative and metabolic stress during this critical period. This study evaluated the effect of parity on metabolic, oxidative, and reproductive parameters in heat-stressed dairy cows receiving antioxidant boluses. Primiparous and multiparous Holstein cows were supplemented with antioxidant boluses at calving. Measurements included calving-related traits, body condition score (BCS) changes, metabolic indicators (glucose, BHBA), oxidative status (TAC, GPX, SOD, MDA), hematology, uterine health, and reproductive performance over 42 days postpartum. Parity significantly affected postpartum physiology despite antioxidant supplementation. Multiparous cows exhibited greater BCS loss (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), higher BHBA concentrations (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and elevated oxidative stress, along with stronger inflammatory responses indicated by increased WBC and neutrophil counts (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In contrast, primiparous cows experienced more calving difficulties (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but maintained a relatively favorable metabolic profile. Days open tended to be shorter in primiparous cows (P\u0026thinsp;=\u0026thinsp;0.09), suggesting improved reproductive recovery. Antioxidant boluses improved oxidative balance but did not fully offset parity-driven metabolic and immune challenges under heat stress. These findings highlight the need for integrated, parity-adjusted nutritional and management strategies to optimize transition cow health and fertility in thermal environments.\u003c/p\u003e","manuscriptTitle":"Effects of parity on metabolic, oxidative status and reproductive responses in heat-stressed transition dairy cows supplemented with slow release boluses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-31 15:07:41","doi":"10.21203/rs.3.rs-7633697/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fc675686-dceb-45e4-b3ea-4345b6b5736f","owner":[],"postedDate":"October 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-27T14:24:43+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-31 15:07:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7633697","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7633697","identity":"rs-7633697","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}