Studies of the novel methods for recipient selection and ultra-early pregnancy diagnosis using color doppler ultrasonography in dairy cows

<p>　The bovine embryo transfer（ET）conception rate has not improved since the 1980s and is approximately 50%. The development of a novel selection method of recipient cows is required to increase the ET conception rate. We have always selected recipient cows on the basis of corpus luteum（CL）size because the CL size correlates with plasma progesterone（P₄） concentrations, which is necessary for pregnancy. From the 2000s, several reports indicate that the blood flow area（BFA）in a CL are closely associated with plasma P₄ concentrations. Color Doppler ultrasonography（CDUS）has provided us new information regarding blood flow in the CL, therefore, CDUS is a novel tool to evaluate CL function. Thus, the current study aimed to assess the applicability of luteal blood flow data obtained using CDUS for recipient selection and early pregnancy diagnosis. In Experiment 1, we demonstrated the changes in the luteal BFA and time-averaged maximum velocity（TAMV）of the spiral artery at the CL base as parameters of luteal blood flow before and after ET（Days 3, 5, 7 and 14; Day 0 = estrus）, then, compared these indexes between the pregnant and non-pregnant groups. On Days 7 and 14, luteal BFA was higher in the pregnant group than in the non-pregnant group. The TAMV of the pregnant group was higher than that of the non-pregnant group on Day 14. In addition, we investigated whether BFA and TAMV can predict conception and how accurate conception can be predicted by these indexes compared with the CL area and plasma P₄ concentrations. The best logistic regression model to predict pregnancy included scores for BFA on Day 7, BFA and TAMV on Day 14. A BFA cutoff value ≥0.43 cm² yielded the best sensitivity（79.4%）and specificity（75.0%）among all cutoff values for any data available on Day 7. Furthermore, setting the cutoff value at one obtained from a sample with BFA 0.63 cm² and TAMV 50.60 cm/s yielded the highest sensitivity（85.3%）and specificity（91.7%）on Day 14. Thus, the evaluations of BFA on Day 7, and paired BFA and TAMV on Day 14 represent reliable predictors of pregnancy in the cow. The secretions of follicle stimulating hormone（FSH）and luteinizing hormone（LH）increase acutely after the administration of a gonadotropin-releasing hormone（GnRH）agonist. These gonadotropins are involved in the regulation of angiogenic factors, and consequently in vascular development in the CL. There are high possibility that administration of GnRH agonist would increase vascularity of the CL and change the accuracy of pregnancy prediction using luteal BFA and TAMV. In Experiment 2, effects of GnRH agonist administration on luteal blood flow and the accuracy of pregnancy prediction were examined. The BFA values in the GnRH group（cows treated with 100μg of GnRH agonist on Day 5） were significantly higher than those in the control group （cows untreated with GnRH agonist） on Days 7 and 14. TAMV did not differ among these groups. In addition, the BFA of the GnRH pregnant group was higher than those of the control and GnRH non-pregnant groups on Days 7 and 14. According to receiver operating characteristic analyses to predict pregnancy, a BFA cutoff of 0.52 cm² yielded the highest sensitivity（83.3%）and specificity（90.5%）on Day 7, and BFA 0.94 cm² and TAMV 44.93 cm/s yielded the highest sensitivity（97.1%）and specificity（100%）on Day 14 in the GnRH group. The areas under the curve for the paired BFA and TAMV in the GnRH group were 0.058 higher than those in the control group（0.996 and 0.938, respectively, p < 0.05）.</p>