229 The presence of an embryo in the uterine horn of llamas produces a variation in the concentration of inflammatory cytokines of uterine fluid

Llamas are induced ovulators, and 98% of pregnancies occur in the left uterine horn—on Day 9 after fertilisation, an embryonic migration from the right uterine horn to the left uterine horn occurs. The objective of this work was to determine the inflammatory cytokine profile of each uterine horn before embryonic migration from the right uterine horn to the left uterine horn. Llamas around 6–8 years of age and 130 to 150 kg of weight were used for this study. Ovaries were examined daily by transrectal ultrasonography using a 7.5 MHz lineal array transducer (Esaote, Mediclinic). When a growing follicle ≥ 8 mm in diameter was detected, llamas were randomly assigned to the following groups: Llamas without embryo (follicular phase [n = 5]; luteal phase induced by an i.m. dose of 50 µg gonadorelin acetate [gonadotrophin-releasing hormone, Ovalyse, Pfizer Chile SA] [n = 5]; luteal phase induced by an intrauterine infusion of 5 mL of seminal plasma [n = 5)]); Llamas with embryo (mating with a proved fertile male [n = 5]). Females were submitted to a nonsurgical uterine flushing using a 16 F Foley catheter with 5 mL of PBS and the fluid of both uterine horns was recovered separately. To evaluate the concentration of inflammatory mediators, the analysis of IL-1β, IL-6, IL-8, IFN γ, TNF-α, and PGE2 was carried out through ELISA kits. 100 μL of uterine fluid from both uterine horns was thawed and used to estimate the concentration of all inflammatory mediators according to the manufacturer’s instructions, with some modifications. Samples were analysed in an automatic Varioskan Flash Reader (Thermo Fisher Scientific). Since our data do not show normal distribution and variance homogeneity according to Shapiro-Wilk and Bartlett’s test, respectively, a Mann-Whitney test for nonparametric data was used. A P-value < 0.05 was considered as significant. Data were depicted in bar graph as mean ± s.e.M. All analyses were performed using the GraphPad Prism^® software (v 5.0, Graphpad Software). The uterine fluid obtained from the right uterine horn of the llamas who presented an embryo showed a higher concentration of IL-1β, IL-6, IL-8, and IFN γ in comparison with the left uterine horn of the llamas who did not present an embryo. TNF-α and PGE2 concentration was higher in the left uterine horn of llamas who produced an embryo when compared to llamas that did not produce an embryo. The profile of inflammatory cytokines of each uterine horn varies according to the presence or absence of an embryo. The greatest inflammatory response observed in animals with embryos may be due to interactions with the endometrium to prepare the futures stages of embryonic development during the pregnancy.

This study was supported by FONDECYT postdoctoral grant N° N° 3190504.