This study aimed to investigate the effects of oxidative stress and antioxidant activity in plasma and uterine fluid during early postpartum on the reproductive performance of Japanese Black cows. A total of 17 Japanese Black cows were included in this study. Plasma and uterine fluid samples were collected at days 7 and 14 of the estrus cycle (n = 17), between 47–67 days postpartum, and diacron-reactive oxygen metabolites (d-ROMs) and biological antioxidant potential (BAP) were measured. d-ROMs, BAP, and oxidation stress index (OSI: d-ROMs/BAP × 100) were compared between pregnant and nonpregnant cows at first postpartum artificial insemination (AI), cows pregnant less than 120 days postpartum (<120 days postpartum), and cows pregnant more than 120 days postpartum (≥120 days postpartum) regardless of AI number. The uterine fluid OSI on day 7 of the estrus cycle was significantly higher in cows pregnant ≥ 120 days postpartum (p < 0.05). d-ROMs, BAP, and OSI of uterine fluid during early postpartum were useful indicators for determining the subsequent reproductive performance of Japanese Black cows.

The objectives of this study were to assess the sequential dynamics of the endometrial polymorphonuclear cells (PMN) after calving by endometrial cytology, and clarify the factors that cause prolonged endometrial inflammation in lactating dairy cows. A total of 33 lactating Holstein dairy cows were used from −4 to 8 wk relative to calving (0 wk: the calving week). Endometrial samples were obtained sequentially from 2 to 8 wk. Body condition score and backfat thickness were obtained weekly from −4 to 8 wk. Blood samples collected from −4 to 8 wk were analyzed for indicators of energy status, hepatic function, systemic inflammation, and calcium. Blood amino acids were measured at 2 wk. Daily milk production was determined between 5 and 65 d postpartum. Based on the sequential cytological analysis, the endometrial inflammation threshold was set at ≥5.0% PMN, and the median wk of PMN% lower than 5.0% was 4.5 wk in this study; therefore, we classified the cows into the early group (cows with endometrial inflammation converged within 4 wk: n = 17) and the late group (cows with endometrial inflammation converged at or after 5 wk: n = 16). There were no differences in daily milk production, energy status, hepatic function, blood calcium concentration, and systemic inflammatory response. The late group had lower body condition scores and backfat thickness during the experimental period, and a higher blood concentration of 3-methyl histidine, indicating muscle breakdown, was observed in the late group at 2 wk. Our findings indicated that the lack of body fat reservation during the peripartum period and the increased muscle breakdown after calving were risk factors for prolonged endometrial inflammation.

The combined effects of human chorionic gonadotropin (hCG) treatment on Day 5 (Day 0 = the day of artificial insemination: AI) and intravaginal progesterone device (IVPD) treatment from Day 5 to 19 on the conception rate and plasma sex steroid hormone were examined in lactating dairy cows. In Experiment 1, cows were divided into a non-treatment group (n = 181), untreated; an IVPD group (n = 74), with an IVPD inserted into the vagina from Day 5 to 19; and an hCG + IVPD group (n = 87), with 3000 IU hCG administered intramuscularly on Day 5 and an IVPD inserted into the vagina from Day 5 to 19. The conception rates of the hCG + IVPD group, which underwent ≤3 AIs (52.8%) and >3 AIs (73.3%), and the IVPD group, which underwent >3 AIs (63.0%), were significantly higher than that of the non-treatment group, which underwent ≤3 AIs (34.2%). In Experiment 2, blood samples were collected from the non-treatment group (n = 6), the IVPD group (n = 6), and the hCG + IVPD group (n = 7) on days 5, 8, 12, and 15 from estrus to measure plasma progesterone (P₄) and total estrogen (E) concentrations. The plasma P₄ concentration of the hCG + IVPD group tended to be higher than that of the non-treatment group on Day 15; however, plasma E concentrations were not different among groups. These results suggest that the conception rate was improved by hCG + IVPD treatment regardless of AI number because of higher plasma P₄ concentrations later in the estrous cycle.

Repeat breeder (RB) cows fail to conceive after three or more breeding attempts, yet the uterine factors contributing to this condition remain unclear. Here, we compared uterine fluid composition, endometrial cell responses to uterine fluid, and uterine microbiota between normal pregnant (Normal) and RB cows. Uterine fluid was collected from multiparous Holstein cows classified as Normal (n = 5; conception within three breeding attempts) or RB (no conception after four attempts); RB cows were classified as RB-Rec (n = 4; conceived after uterine lavage) or RB-NoRec (n = 3; did not conceive after uterine lavage). Uterine fluids were applied to bovine endometrial cells, and microbiota composition was analyzed. Concentrations of lipopolysaccharide in the uterine fluid did not differ between the three groups, and uterine fluid from both RB groups significantly increased C-X-C motif chemokine ligand 8 secretion relative to the Normal group. RNA-seq revealed distinct endometrial cell responses to RB-NoRec uterine fluid, with enrichment of immune-related pathways including phagosome, ferroptosis, and cellular senescence. Uterine microbiota profiles differed among groups, with short-chain fatty acid-producing genera (e.g., Fusicatenibacter and Dorea) predominant in Normal cows and inflammation-associated genera (e.g., Trueperella and Helicobacter) predominant in RB-NoRec cows. These data indicate that uterine fluid properties and microbiota composition vary with reproductive status and may modulate endometrial immune activation associated with fertility outcomes.