Histotroph and uterine receptivity

In cattle, before implantation, conceptus development depends on the uterine secretome (i.e., histotroph). Despite such pivotal role, little is known about the dynamics of histotroph synthesis and changes in composition throughout the early diestrus and the relevance of this entirely dynamic process for pregnancy establishment. Objective was to alter histotroph composition at early diestrus and verify the effect on (1) embryo survival and (2) timing to recover composition. Follicle growth of cycling, non-suckled, multiparous Nelore cows was synchronized with an estradiol/progesterone based protocol followed by estrus detection (d0) twice a day for 4 days. In Exp1, each uterine horn was flushed with 30 mL of D-PBS on d1 (n = 15), d4 (n = 17) or d7 (n = 16), or were not flushed (control, n = 16). On d7.5, 3 IVP embryos were transferred to the uterine horn ipsilateral to the CL. Pregnancy was checked on d25 by transrectal ultrasonography. In Exp2, each uterine horn was flushed thrice with 30 mL of D-PBS on d1 (n = 9), d4 (n = 9), d7 (n = 9), d1 + 4 + 7 (n = 9) or was not flushed (control, n = 9). A sham flushing (all procedures except delivering D-PBS to the uterus) was performed in each cow, in each experimental day when no flushing was scheduled. On d7.5, the uterine horns of all cows were flushed with 30 mL D-PBS and flushings were used to quantify total protein (BCA protein assay kit), abundance of albumin, a common serum protein (ALB; SDS-PAGE followed by Coomassie staining and densitometry) and abundance of SERPINA14, a pregnancy-related protein (SERP; Western Blotting). Preliminary data were analysed by SAS using Chi-square test for Exp1 and PROC MIXED for Exp2. In Exp1, pregnancy (P)/ET was similar between the control group, (60%; 9/15) and the flushing on d1 group (60%; 9/15) but compared to the control, the P/ET from flushing on d4 group tended to be lower (29.4%; 5/17; p = 0.06) and in the flushing on d7 group, it was numerically lower (37.5%; 6/16; p = 0.16). In Exp2, flushing increased total protein content. Compared to the control, flushing on d4 or d7 increased total protein 4.1 and 3.8 fold (P u003c 0.05), while flushing on d1 or d1 + 4 + 7 resulted in no changes or only a moderate increase (1.6 fold, P = 0.07), respectively. Similarly, flushings on d4 or d7 increased (P u003c 0.05) ALB abundance 5.8 and 5.2 fold, while flushing on d1 or d1 + 4 + 7 resulted in increases of 2.1 and 2.4-fold, respectively. For SERP, flushing on d4 or d7 resulted in increases of 9 and 14.5-folds (P u003c 0.01), respectively, and flushing on d1 or d1 + 4 + 7 did not change it. In conclusion, embryo retention was decreased by flushings conducted at time points approaching ET, highlighting the importance of an appropriate intrauterine milieu for the success of pregnancy. Uterine flushing at early diestrus successfully altered histotroph composition, sharply increasing the abundance of total, serum and pregnancy-related proteins.