Inflammatory response in repeat breeder buffaloes

The aim of this study was to investigate the repeat breeding condition in Italian Mediterranean buffaloes that failed to conceive after at least 300 days in milk. The trial was carried out on 40 pluriparous Italian Mediterranean buffaloes with more than 300 days in milk. All the animals underwent ultrasound examination to assess endometrial thickness and oestrus synchronization by the Ovsynch-TAI Program. On the day of oestrus, blood samples were collected for the haemocytometric cell count and biochemical assay, and the animals were slaughtered in a local abattoir. A post-mortem uterine flushing was performed using sterile saline for microbiological analyses. Furthermore, uterine biopsies were carried out for histopathological assessment. Finally, endometrial samples were used for real-time PCR (RT-PCR) analysis to evaluate the expression of genes involved in innate immune recognition of pathogens and the inflammatory response, such as Toll-like receptor (TLR)1, TLR8, interleukin (IL)-1β, IL-6, IL-8, COL4A2, connective tissue growth factor (CTGF), and cysteine-rich angiogenic inducer 61 (CYR61). Statistical analysis was performed by one-way ANOVA. Based on the infiltration of lymphocytes and plasma cells or endometrial gland, lymphatic, and blood vessel ectasia recorded in the histopathological examination, the animals were classified into three groups: healthy (H Group; n = 5), moderate endometritis (M Group; n = 25), and severe endometritis (S Group; n = 10). A significantly greater (P < 0.01) endometrial thickness was recorded in the S Group compared to that in the H and M Group (1.07 ± 0.03 vs. 0.70 ± 0.07 and 0.81 ± 0.04 cm in the S, H, and M Group, respectively). The white blood cell count was lower in the H Group compared to that in the M and S Group (6.3 ± 0.6 vs. 9.3 ± 0.4 and 10.5 ± 0.5 in the H, M, and S Group, respectively). To perform RT-PCR analysis, five animals from groups M and S were randomly selected in order to have balanced results. A higher (P < 0.01) expression of TLR1, together with a lower expression of COL4A2, IL-1β, IL-6, IL-8, and CYR61, was recorded in the H Group, compared to both the M and S Groups. In conclusion, about 90% of repeat breeder buffaloes show moderate or severe endometritis, associated with an altered histopathological endometrial profile and altered mRNA expression of pro-inflammatory and fibrotic factors.