This study, which took place between January and August of 2022, 40 local Holstein cross-breed cows in the Salah-Din region, ranging in age from 3 to 8 years, suffered from retained placenta, while 11 animals had normal placenta expulsion served as the control group. When a cow fails to evacuate her fetal membranes more than 12 hours after giving birth, it is said that she has retained placenta [11]. Every animal underwent a clinical examination to determine whether it was suffering from a metabolic disease or a systemic illness [12,13].

Each experiment animal's jugular vein was used to draw five milliliters of blood, which was then separated into serum using a centrifuge and stored at -20 C until analysis. The following were measured using ELISA kits from United States Biological Company: interleukin-10, IL-6, IL-1β, CRP and TNF-α.two genes used (GAPDH and SDHA) were utilized to assess the integrity of RNA and normalize RT-PCR levels. For GAPDH and SDHA, in specific cow primers (Table 1).

Statistical analysis

The data was presented as mean ± SD. Data evaluated using a one-way ANOVA were deemed significant using the Student's −test if the A value was ≤ 0.01.

Figure 1 shows that in RP cows, the mean values of IL10, IL6 and IL1β were considerably (P<0.01) higher compared to healthy cows (2.31±0.11 versus 1.41±0.07 pg per ml), (13.6±2.1 versus 4±0.9 pg per ml) and (10.48±0.24 versus 5.40±0.19 pg per ml), respectively.

Figures 2 and 3 show that when RP cows were compared to healthy cows, the mean value of CRP was lower (P ≤ 0. 01) in RP cows (0.9±0.02 vs. 0.32±0.04 mg/L, respectively), whereas the mean value of TNF-α was higher (P ≤ 0. 01) in RP cows (60.1 ± 12.79 vs. 29.5 ± 16.58 pg/ml, respectively).

Gene Expression in Retained Placenta in cattle

Semi quantitative RT-PCR could detect mRNA expression of SDHA in both paternal and fetal tissue samples , to assess the expression of SDHA polarization markers in mother and fetal cells in the cattle term placenta the level of expression was determined separately in fetal and mother tissues. The results revealed that SDHA expression was considerably higher in both tissues mother than in all other genes (Figure 4).

Figures & Tables

Discussion

Our investigation found that cows with RP had noticeably higher levels of IL-10, IL-6 and IL-1β than did healthy cows. These differences could be the result of an inflammatory insult that occurred in the mammary gland eight weeks prior to birth. Comparing the results to those of other studies, Trevisi et al. [14] and Sheldon et al. [15] found that elevated levels of inflammatory markers or cytokines in cows during the dry period were linked to inflammatory events or a high incidence of mastitis in the vicinity of parturition. Islam et al. [16] revealed that the IL-10 concentration can be used to identify cows that are prone to reproductive illnesses that may arise after calving, and they proposed that the IL-1 level decline during the two weeks prior to or at parturition can be utilized to do so. According to Ishikawa et al. [17], some retained fetal membranes were linked to higher levels of interleukin 6 prior to calving as opposed to later. C-reactive protein is a pentraxin family plasma protein that is produced by hepatocytes. Its expression is controlled by inflammatory cytokines produced by adipocytes, such as interleukin 6 (IL-6), and it serves primarily as an anti-inflammatory and pro-inflammatory molecule [18, 19]. By attaching phospholipids, phosphocholine, chromatin, fibronectin, and histone, it plays a crucial role in the identification and elimination of infections and damaged cells [20, 21].

In the present study, retained placental cows' CRP levels were considerably lower than those of normal cows. Acute phase proteins, primarily CRP, are more concentrated in retained fetal membranes when mastitis and endometritis are present. This is because lymphocytes and macrophages in retained fetal membranes secrete more IL-6, which causes CRP to bind to the surface of dying cells and make it easier for macrophages to phagocytose these cells, reducing the risk of infection and inflammation in the placental tissue [22, 23]. Activated macrophages, natural killer cells, and T lymphocytes create tumor necrosis factor-alpha (TNF-α), a pleiotropic cytokine that regulates inflammation and is elevated in serum and placental tissue [24]. Several cancers are susceptible to the growth-inhibitory and cytotoxic actions of tumor necrosis factor-alpha. An essential factor in initiating the host’s immune response is the rising levels of tumor necrosis factor-alpha in the serum of cows with retained fetal membranes, which triggers the release of interleukins 1 and 6 [25, 26].

The high level of SDHA expression in fetal and Mother tissues shows that the majority of macrophages are M1 type. In cow, it has been shown that during the second half of pregnancy, the proportion of M2 macrophages increases, contributing to the anti-inflammatory milieu that is essential to prevent fetal rejection [27].

Determining the blood concentrations of interleukins IL-10, IL-6, IL-1β, and TNF-α is a useful method for predicting placental retention in cows since these molecules have a significant role in the development of placental retention. The high expression of SDHA suggests a role for this transcription factor in the overexpression of cytokines.

Acknowledgments

The laboratory facilities were provided by College of Veterinary Medicine, University of Baghdad and Tikrit for which the author is appreciative.

Conflict of Interest

The authors declare that there is no conflict of interest.

Author Contributions

Nawaf Nooraldeen and Manar Sabah conceived the project. Hayder Al-Mutar  and Maythem Abdulealah supervised the project and provided guidance. All authors wrote, edited, and approved the final manuscript.

References