摘要Background Heat stress(HS)is posing as a tremendous threat to the swine industry,due to the thermos-sensitive gonads of boars.Testes are immune-privileged organs in which spermatogenesis needs to remain undisturbed,whereas immune cells are thermo-sensitive,especially macrophages,which are the most abundant testicular immune cells.Our study aimed to unveil the underlying immune responses and assess their consequences on the semen quality of boars under HS.The results will aid in addressing environmental temperature-related seasonal infertility and in selecting the best boar for use in artificial insemination.Methods The 3-week experiment assigned 26 8-week-old Rongchang male pigs into thermal neutral pair-feed(TN-PF)and HS groups.During the last 2 weeks,which served as the HS period,the HS group was subjected to 14-day 35±1 ℃,while the TN-PF group was kept at 26±1 ℃.Pig gonad tissues were sampled at the end of HS period for assessments and measurements.Results Our findings confirmed HS-related reactions such as elevated respiration rate(P＜0.05)and elevated heat shock protein 60(HSP60;P＜0.05)and heat shock protein 90(HSP90;P＜0.05)expression levels.Sperm motil-ity(P=0.06)and progressive sperms(P=0.08)were decreased under HS as was a significant reduction in average straight-line velocity(VSL;P＜0.05).Additionally,total abnormality levels increased(P＜0.05).Fibrosis,caspase-3 expression,and accumulations of tumor necrosis factor-α(TNF-α;P＜0.05)and interleukin-1 β(IL-1 β;P＜0.05),along with an elevated macrophage composition(P＜0.05)characterized the orchitis under HS.Single cell RNA sequencing(scRNA-seq)revealed fluctuations in engulfing and inflammatory signals in testicular macrophages(TMs).In particular,the complement cascade was promoted by CD163+macrophages,resulting in membrane attack com-plex(C5b-9)assembly(P＜0.05).Linear regressions further revealed a negative correlation between C5b-9 and sperm motility(P＜0.05),as well as near-negative correlations between the C5b-9 and both progressive motility(P=0.08)andVSL(P=0.06).Conclusions Our findings highlighted the relationship between HS,the onset of orchitis,and the activation of the complement system,all of which decreased the boar semen quality.