Haematological and biochemical restoration following E. coli lipopolysaccharide and silver nanoparticle therapy in cytological endometritic cows

Introduction

Cytological endometritis also known as subclinical endometritis is one of the most important postpartum uterine disorders affecting reproductive efficiency in dairy cows. The condition is characterized by chronic inflammation of the endometrium without evident clinical signs and is diagnosed by the presence of increased polymorphonuclear neutrophils (PMNs) in endometrial cytology. Cytological endometritis has been associated with reduced conception rates, prolonged calving-to-conception intervals and substantial economic losses in dairy herds (Sheldon et al., 2009; Nanda et al., 2024) [31] . Following parturition, the uterus is frequently exposed to bacterial contamination. Failure of uterine defence mechanisms to eliminate invading pathogens predisposes cows to persistent uterine infections and endometrial inflammation. Among the bacterial pathogens involved, Escherichia coli is considered one of the major organisms implicated in postpartum uterine disease and altered uterine physiology (Ahmad et al., 2003; Ahmad et al., 2004) [1,2] . Persistent uterine inflammation adversely affects reproductive performance and induces systemic physiological alterations in affected animals (Sarkar et al., 2016) [28] . Haematological and biochemical parameters are valuable indicators of the health, metabolic metabolic and reproductive status of dairy cows. Alterations in haemoglobin concentration, packed cell volume, leukocyte profile, serum proteins and metabolic biomarkers have been reported in cows affected with uterine infections (Heidarpour et al., 2012; Perumal et al., 2020; El-Sayed et al., 2024a) [10,11,13,24] . Furthermore, the proportion of PMNs within the postpartum uterus has been associated with ovarian activity and metabolic status which further highlights the importance of evaluating systemic responses during uterine disease (Krause et al., 2014) [16] . Conventional treatment of endometritis has largely relied on antimicrobial therapy. However, increasing concerns regarding antimicrobial resistance and drug residues in animal products have stimulated interest in alternative therapeutic approaches. Immunomodulatory agents such as Escherichia coli lipopolysaccharide (LPS) have been shown to enhance uterine defence mechanisms and facilitate clearance of pathogenic microorganisms. Likewise, silver nanoparticles possess broad-spectrum antimicrobial activity and have emerged as promising non-antibiotic alternatives for the management of uterine infections (Salzano et al., 2020; Nanda et al., 2024) [27] . Assessment of haematological and biochemical changes following therapy can provide valuable information regarding the physiological recovery of affected animals and the efficacy of therapeutic interventions. Therefore, the present study was undertaken to evaluate the effect of intrauterine administration of E. coli lipopolysaccharide and silver nanoparticles on selected haematological and biochemical parameters in cows affected with cytological endometritis.

Materials and methods

The present study was conducted on postpartum cows presented for reproductive examination at the Department of Veterinary Gynaecology and Obstetrics, NTR College of Veterinary Science, Gannavaram, Andhra Pradesh. A total of 86 postpartum cows were screened for cytological endometritis using endometrial cytology by the cytotype technique. Endometrial cytological smears were stained and examined Microscopically for determination of polymorphonuclear neutrophil (PMN) percentage. Cows showed more than 5 per cent PMNs in endometrial cytology were diagnosed as positive for cytological endometritis and selected for the study. Twenty-four cytological endometritic cows were randomly allocated into two treatment groups comprising 12 animals each. Cows in Group I received 100 µg of Escherichia coli lipopolysaccharide (LPS) administered intrauterinely at estrus. Cows in Group II were treated with 4 mg of silver nanoparticles administered intrauterinely for three consecutive days. Blood samples were collected from the jugular vein of all animals immediately before treatment and again at the subsequent estrus following treatment. Samples were collected into EDTA vacutainers for estimation of haematological parameters and into clot activator vacutainers for harvesting serum used in biochemical analysis. Haematological parameters including haemoglobin (Hb), packed cell volume (PCV), neutrophil, lymphocyte, monocyte, eosinophil and basophil counts were estimated using standard laboratory procedures. Biochemical parameters including total protein (TP), blood urea nitrogen (BUN), creatinine, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were estimated using commercially available diagnostic kits following the manufacturer's instructions. The data obtained were expressed as Mean ± SE. Statistical analysis was performed to compare the pretreatment and post-treatment values within each treatment group. Differences were considered significant at p<0.05.

Results and discussion

Haematological parameters

The haematological parameters recorded in cows affected with cytological endometritis before treatment and at the subsequent estrus following treatment with Escherichia coli lipopolysaccharide (LPS) and silver nanoparticles (AgNPs) are presented in Table 1. [13] , Perumal et al. (2020) [24] , Kumar et al. (2023), Singh et al.

(2023) [33] and El-Sayed et al. (2024a) [10,11] , who documented improvement in haemoglobin values following successful treatment of uterine disorders. The packed cell volume (PCV) increased significantly (p<0.05) following treatment in both groups. In the LPS-treated group, PCV increased from 25.91 ± 0.74 to 29.75 ± 0.69 per cent, while in the AgNP-treated group it increased from 25.00 ± 0.53 to 30.25 ± 0.66 per cent. Similar findings were reported by Heidarpour et al. (2012) [13] , Sarkar et al. (2016) [28] , Cui et al. (2019) [9] , Perumal et al.

(2020) [24] , Thangamani (2022) [35] and [1] , Kekan et al. (2005) [15] , Sarma (2007) [29] , Kumar et al. (2017) [17] , Perumal et al. (2020) [24] and El-Sayed et al. (2024a) [10,11] . The mean lymphocyte percentage increased significantly (p<0.05) following treatment in both groups. In the LPStreated group, lymphocyte count increased from 38. 25 [15] , Kumar et al. (2017) [17] , Cui et al.

(2019) [9] , Thangamani (2022) [35] and Singh et al. (2023) [33] .

The monocyte percentage decreased significantly (p<0.05) following treatment in both groups. The values declined from 5.25 ± 0.13 to 4.25 ± 0.13 per cent in the LPS-treated group and from 5.83 ± 0.11 to 4.83 ± 0.11 per cent in the AgNPtreated group. The reduction in monocyte count may be associated with regression of inflammatory processes and normalization of immune responses following successful treatment. The eosinophil and basophil counts remained within normal physiological limits and did not differ significantly before and after treatment in either group. Similar findings were reported by Ahmad et al. (2003) [1] , Sahoo et al. (2014) [26] and Singh et al. (2023) [33] , who observed no significant alterations in eosinophil and basophil counts following treatment of uterine infections.

Biochemical parameters

The biochemical parameters recorded in cows affected with cytological endometritis before treatment and at the subsequent estrus following treatment with Escherichia coli lipopolysaccharide (LPS) and silver nanoparticles (AgNPs) are presented in Table 2. The mean total protein concentration increased significantly (p<0.05) following treatment in both groups. In cows treated with E. coli LPS, total protein concentration increased from 6.96 ± 0.19 to 7.48 ± 0.13 g/dL, whereas in AgNP-treated cows it increased from 7.30 ± 0.13 to 7.50 ± 0.09 g/dL. Similar findings were reported by Srivastava and Sahni (2000) [34] , Pandey et al. (2009), Amle et al. (2014) [3] , Patel et al. (2020) [22] and El-Sayed et al. (2024b) [10,11] , who observed lower serum total protein concentrations in endometritic animals compared to healthy animals. The increase in total protein concentration following treatment may be attributed to restoration of uterine health, improved metabolic activity and reduction in inflammatory stress. The mean blood urea nitrogen (BUN) concentration increased slightly following treatment in both groups. In the LPS-treated group, BUN concentration increased from 22.16 ± 0.19 to 22.91 ± 0.22 mg/dL, whereas in the AgNP-treated group it increased significantly (p<0.05) from 21.39 ± 0.06 to 22.66 ± 0.22 mg/dL. The increase in BUN concentration may be associated with restoration of normal protein metabolism and improvement in nutritional status following recovery from uterine infection. Similar observations were reported by Ghanem et al. (2016) [12] , Jan et al. (2021) [14] and Safak et al. (2023) [25] . The mean creatinine concentration decreased following treatment in both groups. In the LPS-treated group, the reduction was significant (p<0.05), declining from 1.68 ± 0.09 to 1.19 ± 0.07 mg/dL, whereas in the AgNP-treated group it decreased from 1.52 ± 0.05 to 1.40 ± 0.07 mg/dL. Elevated creatinine concentrations prior to treatment may be associated with metabolic disturbances and inflammatory stress accompanying uterine infection. The reduction observed following treatment indicates improvement in metabolic status and physiological recovery. Similar findings were reported by Shabunin et al. (2020) [30] , Torres et al. (2020) [36] , Shinde (2022) [32] and El-Sayed et al. (2024b) [10,11] . The mean alanine aminotransferase (ALT) activity showed minimal variation following treatment. In the LPS-treated group, ALT activity increased slightly from 23.19 ± 0.13 to 23.75 ± 0.27 IU/L, whereas in the AgNP-treated group it decreased significantly (p<0.05) from 24.67 ± 0.82 to 22.91 ± 0.31 IU/L. The values remained within normal physiological limits, suggesting absence of severe hepatic damage. Similar observations were reported by Boonprong et al. (2007) [6] , Mondal et al. (2012) [19] , Biswal et al. (2013) [5] and Chaudhari et al. (2020) [7] . The mean aspartate aminotransferase (AST) activity decreased significantly (p<0.05) following treatment in both groups. In the LPStreated group, AST activity declined from 89.41 ± 0.26 to 84.41 ± 1.58 IU/L, while in the AgNP-treated group it decreased from 88.97 ± 0.26 to 80.50 ± 2.63 IU/L. Elevated AST levels prior to treatment may be associated with tissue damage and inflammatory stress resulting from uterine infection. The reduction following treatment indicates recovery of tissue integrity and improvement in physiological status. Similar findings were reported by Azawi et al. (2008) [4] , Heidarpour et al. (2012) [13] , Chaurasia et al. (2016) [8] , Chaudhari et al. (2020) [7] and Shinde (2022) [32] . Overall, the biochemical findings of the present study indicate that successful treatment of cytological endometritis with either E. coli lipopolysaccharide or silver nanoparticles was associated with improvement in metabolic status, reduction in inflammatory stress and restoration of normal physiological functions.

Conclusion

The present study demonstrated that cytological endometritis is associated with significant alterations in haematological International Journal of Veterinary Sciences and Animal Husbandry https://www.veterinarypaper.com and biochemical parameters, reflecting the inflammatory and metabolic disturbances accompanying uterine infection in cows.

Treatment with either Escherichia coli lipopolysaccharide (LPS) or silver nanoparticles resulted in favourable changes in haematological indices, including significant increases in packed cell volume and lymphocyte percentage, along with significant reductions in neutrophil and monocyte counts. Similarly, biochemical evaluation revealed significant increases in total protein concentration and reductions in creatinine and aspartate aminotransferase activity following treatment. These changes indicate improvement in immune competence, metabolic status and physiological recovery of affected animals. The findings suggest that both E. coli lipopolysaccharide and silver nanoparticles are effective non-antibiotic therapeutic alternatives for the management of cytological endometritis and contribute to restoration of normal haematological and biochemical profiles. Therefore, these therapeutic approaches may be considered promising options for improving uterine health and reproductive performance in postpartum dairy cows.

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