Laterality of Ovulation and Presence of the Embryo Do Not Affect Uterine Horn Blood Flow During the First Month of Gestation in Llamas.

We determined if laterality of ovulation and intrauterine embryo location differentially induces changes in the mesometrial/endometrial vascularization area (MEVA) between uterine horns, during and after embryo migration, elongation and implantation in llamas. Adult, non-pregnant and non-lactating llamas (n = 30) were subjected to daily B-mode ultrasound scanning of their ovaries. Llamas with a growing follicle ≥8 mm in diameter in the left (n = 15) or right (n = 15) ovary were assigned to a single mating with an adult fertile or vasectomized male. Power-doppler ultrasonography was used to determine the MEVA in a cross section of the middle segment of both uterine horns. MEVA was determined by off-line measurements using the ImageJ software. MEVA measurements were performed before mating (day 0) and on days 5, 10, 15, 20, 25, and 30 after mating in pregnant [llamas with left- (n = 6) or right-sided (n = 6) ovulations] and non-pregnant [llamas with left- (n = 6) or right-sided (n = 6) ovulations] females. Ovulation was confirmed by the disappearance of a follicle (≥8 mm) detected previously. Pregnancy was confirmed by the presence of the embryo proper. MEVA was analyzed by one-way ANOVA for repeated measures using the MIXED Procedure in SAS. If significant (P ≤ 0.05) main effects or interactions were detected, Tukey's post-hoc test for multiple comparisons was used. Ovulation rate did not differ (P = 0.4) between females mated to an intact or vasectomized male and between right- or left-sided ovulations. Three females mated to the intact and 3 to the vasectomized male did not ovulate and were excluded of the study. First observation of fluid inside the gestational sac and of embryo proper, were made exclusively in the left uterine horn, on day 15.8 ± 3.8 and 22 ± 2.7, and 16.7± 2.6 and 27.5 ± 2.8 for pregnant llamas ovulating in the right and left ovary, respectively. Although the MEVA of both uterine horns was affected by time (P < 0.05), it was not affected by physiological status (pregnant vs. non-pregnant; P = 0.9) or laterality of ovulation (P = 0.4). Contrary to expectations, regardless of the laterality of ovulation, in pregnant llamas the left horn did not display a greater MEVA before or after embryo arrival, a trend that was observed during the first 30 days of gestation.

Evaluation of the effect of mating, intrauterine deposition of raw seminal plasma or seminal plasma purified ß-NGF on endometrial vascularization in llamas.

The aim of this study was to evaluate the endometrial vascularization area (EVA) of both uterine horns in llamas subjected to different intrauterine treatments resembling physiological conditions after a single mating. Llamas with a growing follicle (≥8 mm) were randomly assigned to: a) single mating with a fertile male (mating; positive control; n = 6); b) intramuscular administration of 50 µg of gonadorelin acetate plus an intrauterine infusion of 4 ml of PBS (GnRH; negative control; n = 4); c) intrauterine infusion of 4 ml of raw llama seminal plasma (SP; n = 4) or d) intrauterine infusion of 10 mg of ß-NGF purified from llama semen diluted in 4 ml of PBS (spß-NGF; n = 6). Females in GnRH, SP and NGF group received 50% of treatment volume into each horn by guiding an insemination pipet through the cervix. Ovaries were examined by ultrasonography every 12 h until Day2 (Day 0 = Day of treatment) to determine ovulation. Power-Doppler ultrasonography evaluation of EVA in a cross-section of the middle segment of each horn was conducted at 1 h before and 1, 3, 6, 12 and 24 h (intensive evaluation) and 2, 4, 6 and 8 days (long-term evaluation) after treatment administration. Serial EVA data was analyzed as a 2-by-2 factorial design for repeated measures using the MIXED procedure. The analysis included main effects of treatment (mating, SP, spß-NGF or GnRH), uterine horn (left vs right), time, and their interactions. According to the 2 by 2 analysis there was no effect of uterine horn on EVA during the first 24 h and from Day 2 to Day 8 after treatment; therefore, data were grouped based on treatment type regardless of uterine horn for both periods of observation. Thus, EVA was affected by time (P < 0.04) and treatment by time interaction (P < 0.02) and tended (P = 0.07) to be influenced by type of treatment during the intensive evaluation period. Females on mating and spß-NGF group showed a significant increase in EVA at 3 and 12 h after treatment compared to GnRH and SP groups. However, no effect of treatment, time or their interaction was observed during the long-term evaluation period. In spite of the limited number of animals used in this study, our results allow us to concluded that natural mating and intrauterine deposition of 10 mg of spß-NGF induce a symmetrical increase in endometrial vascularization of both uterine horns during the first 24 h post treatment administration in llamas; however, this effect did not persist beyond that period.

Uterine endometrial vascularization during ovarian follicular growth in llamas: The effect of estradiol plasma concentration.

The aim of this study was to evaluated changes in endometrial vascularization area (EVA) between the left and right uterine horn: a) during the ovarian follicular growth in intact llamas, and b) after exogenous estradiol administration of estradiol benzoate in ovariectomized (OVX) llamas. In experiment 1 follicle wave emergence was synchronized (n = 5 llamas) by follicle ablation (Day 0). Females were examined every other day from Day 1 to Day 27, using B mode ultrasonography to evaluate dominant follicle growth profile. Also, EVA was evaluated in each horn using Color-Power Doppler. Blood samples were taken every other day from Day 1 to Day 27 to measure estradiol (E2) plasma concentration by RIA. In experiment 2 OVX llamas (n = 4 llamas/group) were given a single im administration of: a) 1 mg of estradiol benzoate (EB) or b) 1 mL of saline. Females were subjected to ultrasound examinations every 48 h from Day -4 until treatment (Day 0), every 12 h from Day 0 to Day 4, and again every 48 h from Day 5 to Day 11. Evaluation of EVA in both uterine horns was performed as described for experiment 1. Blood sampling for the measurement of E2 was carried out at the same time points indicated for the ultrasound examinations. Serial data were analyzed by one way ANOVA for repeated measures using the MIXED Procedure in SAS. Also, Pearson's correlation was used to determine the relationship between variables. In intact llamas there was an effect of day on the dominant follicle growing profile (P < 0.01) and estradiol plasma concentration (P < 0.05). Dominant follicle diameter positively correlated (r = 0.4; P < 0.017) with estradiol plasma concentration. Also, EVA of right and left uterine horn did not differ (P = 0.89) during the evaluation period; however, it was affected by time (P < 0.05). In ovariectomized llamas estradiol concentration was significantly (P < 0.001) affected by treatment, time and their interaction. Accordingly, treatment with EB (P < 0.0001), time (P < 0.05) and their interaction (P < 0.01) affected EVA of both uterine horns; however, this variable did not differ between horns (P = 0.98). In conclusion, circulating concentrations of estradiol determined an increase in uterine vascularization, during the phase of follicular growth in intact llamas and after the exogenous administration of EB to ovariectomized females; however, no differential effect in endometrial vascularization area between right and left uterine horn was observed.