The aim of this study was to determine the combined relationship of heat stress and pre-ovulatory follicular size estimated by rectal palpation at fixed-time AI (FTAI) following a progesterone-based estrous synchronization protocol and the subsequent conception rate (CR). The experiment was performed in a commercial dairy herd located in North-Eastern Spain since July to December 2016. A single inseminator palpated the ovaries during AI and assigned the cow to 3 different possible follicle groups: (S) Small (estimated follicular diameter (EFD) < 12mm), (M) Medium (12–16 mm) and (L) Large (>16mm).The sizes were verified by ultrasound within 30 min. The 3 follicle groups had significant different diameters S (n = 56, 11.5^a ± 6mm), M (n = 114, 14.73^b ± 3) and L (n = 72, 18.3^c ± 5mm) according to ANOVA and Tukey’s tests (P < 0.001). Pregnancy diagnosis was performed by ultrasonography on Day 28 post-AI. The mean CR was 58/268 = 21.56%. The single factor affecting CR in the final logistic regression model was the interaction between heat stress (HS) (temperature humidity index (THI) > 72 at AI) and the EFD. Cows in group L with THI ≤72 at AI had a greater likelihood of becoming pregnant (odd ratio = 4.185; P = 0.013) than the remaining cows (Table 1). Our results suggest that cows with large follicles under HS could suffer ovulation failure resulting in a low CR, and that estimation of pre-ovulatory follicular size at AI may be a good predictor of subsequent CR. The clinical implication of this study is that a trained inseminator can identify which cows are more likely to become pregnant at FTAI. Table 1. Odds ratios of the variables included in the final logistic regression model for factors affecting the conception rate 28 d post-AI

Factor		n	CR (%)	Odds ratio	95% CI	P-value
EFD × HS	S without HS	39	12.8	Reference
	M without HS	70	27.1	2.53	2.53-0.86	0.091
	L without HS	42	38.1	4.18	1.356-12.9	0.013*
	S with HS	23	8.7	0.64	0.115-3.645	0.622
	M with HS	57	21.1	1.81	0.583-5.683	0.304
	L with HS	37	10.8	0.82	0.203-3.340	0.787

EFD = estimated follicular diameter (S: <12mm, M: 12-16mm, L:>16mm); HS = heat stress; CR = conception rate.