Expression of genes associated with fertility in the uterus and oviduct of heifers challenged with lipopolysaccharide

Summary Lipopolysaccharide (LPS) endotoxemia has been negatively associated with fertility. This study aimed to investigate the effect of LPS-induced inflammation on gene expression associated with bovine fertility in the uterus and oviduct. Sixteen healthy heifers were divided into two groups. The LPS group (n = 8) received two intravenous (i.v.) injections of 0.5 µg/kg of body weight of LPS with a 24-h interval, and the control group (n = 8) received two i.v. injections of saline solution with the same interval of time. All the animals had the follicular wave synchronized. Three days after the second injection of LPS, all animals were slaughtered and uterine and oviduct samples were collected. Gene expression associated with inflammatory response, thermal and oxidative stresses, oviduct environment quality, and uterine environment quality was evaluated. Body temperature and leucogram demonstrated that LPS induced an acute systemic inflammatory response. In the uterus, the expression of PTGS2 and NANOG genes was downregulated by the LPS challenge. However, no change in expression was observed in the other evaluated genes in the uterus, nor those evaluated in the oviduct. In conclusion, the inflammatory process triggered by LPS did not persist in the uterus and oviduct 3 days after challenge with LPS. Nonetheless, reduction in PTGS2 and NANOG expression in the uterus suggested that, indirectly, LPS may have a prolonged effect, which may affect corpus luteum and endometrial functions.

STRATEGIES TO INCREASE FERTILITY DURING FIXED-TIME ARTIFICIAL INSEMINATION IN BEEF CATTLE

The application of Fixed Time Artificial Insemination (FTAI) protocols has strongly boosted artificial insemination in Brazilian bovine herds. The control of the estrous cycle with protocols based on progesterone and estrogen involves synchronizing the emergence of a follicular wave, controlling the progesterone phase, and inducing ovulation in a synchronized manner. The increase in pregnancy rates starts with the preparation of the breeding season and the choice of protocols and females. Thereafter, based on the characteristics of the property and the lots of animals, small adjustments can be made to the hormonal protocols as needed. Finally, resynchronization can increase usability by allowing multiple inseminations within short periods. The search for new strategies is of paramount importance for the development of the FTAI protocol and the increase in final pregnancy rates. Thus, this review sought to highlight the main critical points and adjustments of the FTAI protocols and their applicability in the reproduction of beef cattle.

Ovarian dynamics, hormone profiles, and characterization of ovarian and uterine blood flow in cycling Sahiwal cows

The objectives were to investigate the ovarian dynamics and hormonal profiles during the oestrus cycle, and to characterize the ovarian and uterine blood flow (OBF versus UBF) indices on the extent of vascular perfusion towards the dominant follicle (DF) or corpus luteum (CL) during the peri-ovulatory follicular wave in Sahiwal cows. In experiment 1, cyclic cows (n = 21) were selected at their spontaneous oestrus (day 0) and subjected to B-mode ultrasonography throughout the oestrus cycle. In experiment 2, cyclic cows (n = 9) were randomly selected at day 0, and categorized according to the intra-ovarian patterns as ovaries with DF (n = 3), CL (n = 3) or both (DF+CL; n = 3) to characterize the OBF and UBF indices using Doppler ultrasonography. The length of the oestrus cycle (days), number of follicular waves (n) and size (mm) of DF and CL were 20.1 ± 0.9, 2.1 ± 0.2, 14.7 ± 0.7 and 15.9 ± 2.5, respectively. The concentrations (ng/mL) of plasma progesterone increased linearly, and a peak was attained at day 12.2 ± 1.0. However, it reduced linearly with the onset of luteolysis at day 16.4 ± 0.3 of the oestrus cycle. The plasma progesterone (P4) concentrations and the diameter of CL correlated throughout the oestrus cycle. The mean OBF and UBF indices did not differ between intraovarian patterns. Taken together, this information on reproductive physiological parameters could be used to develop synchronization protocols to improve reproductive management in Sahiwal cows.

P–674 Development of a predictive model indicating the population of poor responders benefiting from luteal phase oocyte retrieval

Study question Can successful implementation of luteal phase oocyte retrieval (LuPOR) following conventional follicular phase oocyte retrieval (FoPOR) be predicted for poor ovarian response (POR) patients? Summary answer Antral follicle count (AFC), number of small follicles recorded in FoPOR, and estradiol (E2) levels on FoPOR and LuPOR trigger days, predict successful LuPOR application. What is known already A second follicular wave in the same menstrual cycle was first observed in domestic animals such as horses and cattle and thenceforth in women. The second follicular wave has been introduced as an encouraging means towards optimizing the context of in vitro fertilization (IVF) success rates for infertile women and especially for POR patients. Double ovarian stimulation coupled with two oocyte retrievals in the same menstrual cycle has been proposed, and encouraging results have been reported. However, the high heterogeneity characterizing POR patients dictates that studies should focus on factors indicating efficient LuPOR application. Study design, size, duration This retrospective observational study included 1688 women diagnosed with POR, undergoing natural IVF cycles between 2012–2020 including two oocyte retrievals in the same menstrual cycle. Patients’ age, body mass index (BMI), number of previous POR incidences, basal hormonal levels, AFC, E2 evaluated on both trigger days and number of small follicles (8–13 mm) were evaluated on their predictive power regarding retrieval of at least one MII oocyte following LuPOR, being regarded as successful LuPOR implementation. Participants/materials, setting, methods A diagnosis of POR according to Bologna criteria served as the inclusion criterion for this single center study. All other infertility etiologies were excluded. Patient dataset was stratified according to age in quantiles. A random 20% of each quantile was employed to validate the model. The remaining 80% was employed to develop this model. The predictive value was determined employing the Area Under the Curve (AUC) of the Receiver Operating Characteristics, employing Youden’s index. Main results and the role of chance Patients’ age, BMI, number of previous failed IVF attempts, basal levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin and progesterone failed to be predictive of a successful LuPOR as the AUC was below 0.6. AFC with a threshold value of 4.47, was found to be predictive of an effective LuPOR with an AUC of 0.86, sensitivity 0.8, specificity 0.75, and accuracy 0.79. E2 levels evaluated on the FoPOR trigger day, with a threshold value of 232.66 pg/ml, were similarly predictive of an effective LuPOR presenting with an AUC 0.86, specificity 0.75, sensitivity 0.86 and accuracy 0.82. Similarly, E2 evaluated on the LuPOR trigger day, with a threshold value of 200.89 pg/ml, presented with an AUC 0.89, specificity 0.85, sensitivity 0.95 and accuracy 0.92. The number of small follicles during FoPOR also appeared to be predictive of the presence of at least one MII oocyte during LuPOR, with a threshold value of 2.94. The AUC was 0.82, specificity 0.75, sensitivity 0.76 and accuracy 0.75. When combining the above characteristics into a single predictive model the AUC was 0.88, specificity 0.73, sensitivity 0.94 and accuracy 0.89. The positive and negative predictive value of the model were 93.5% and 46.8%, respectively. Limitations, reasons for caution Employment of natural cycles may present as a limitation when examining the value of this study, as the cut-off values reported herein may be altered when stimulation is employed. Since internal validation may be confounded by the fact that this was a single center study, external validation is required. Wider implications of the findings: The clinical end-point of this study reporting back to the practitioner, is the development of a predictive model identifying the optimal POR population for whom LuPOR practice is valuable. The high positive predictive value of this model may assist clinicians in identifying poor responders who will benefit from this approach. Trial registration number Not applicable

A Prepubertal Mice Model to Study the Growth Pattern of Early Ovarian Follicles

Early folliculogenesis begins with the activation of the follicle and ends with the formation of the follicular antrum, which takes up most of the time of folliculogenesis. In this long process, follicles complete a series of developmental events, including but not limited to granulosa cell (GC) proliferation, theca folliculi formation, and antrum formation. However, the logical or temporal sequence of these events is not entirely clear. This study demonstrated in a mouse model that completion of early folliculogenesis required a minimum of two weeks. The oocyte reached its largest size in the Type 4–5 stage, which was therefore considered as the optimum period for studying oogenesis. Postnatal days (PD) 10–12 were regarded as the crucial stage of theca folliculi formation, as Lhcgr sharply increased during this stage. PD13–15 was the rapid growth period of early follicles, which was characterized by rapid cell proliferation, the sudden emergence of the antrum, and increased Fshr expression. The ovarian morphology remained stable during PD15–21, but antrum follicles accumulated gradually. Atresia occurred at all stages, with the lowest rate in Type 3 follicles and no differences among early Type 4–6 follicles. The earliest vaginal opening was observed at PD24, almost immediately after the first growing follicular wave. Therefore, the period of PD22–23 could be considered as a suitable period for studying puberty initiation. This study objectively revealed the pattern of early folliculogenesis and provided time windows for the study of biological events in this process.

Synchronization with estradiol benzoate in the presence of the corpus luteum in Wagyu cows increases the number of medium follicles but does not interfere with in vitro production of embryos

Reproductive biotechnologies are emerging as an important element for livestock; however, some strategies must be modified to adapt to different breeding systems, such as the use of follicular synchronization protocols. This study aimed to evaluate follicular synchronization using estradiol benzoate (EB), in the presence of the corpus luteum (CL) from Wagyu oocyte donors in in vitro embryo production (IVEP). Rounds of IVEP were performed in heifers and cows (n=19) that were classified into three groups: G1/CL - animals with CL, G2/WCL - animals without CL, and G3/CL + EB - animals with CL that were subjected to follicular synchronization with EB at D0. The groups G1/CL and G2/WCL were considered the control and undertook the natural process of follicular dynamics. The results showed that the synchronization of the follicular wave with the application of EB in the presence of CL, presented a smaller number of small (6.05 ± 0.55) and large follicles (0.45 ± 0.15), but increased (P < 0.05) the number of medium-sized follicles (16.20 ± 0.90). However, the results of ovum pick up showed that regardless of whether or not EB was applied, and regardless of the presence or absence of CL in the Wagyu donor, there was no difference among the groups (P > 0.05) concerning the number of viable oocytes and the viability rate. It was concluded that follicular synchronization using EB in Wagyu oocyte donors that presented a CL, increased the number of medium-sized follicles. However, there was no improvement in the efficiency of ovum pick up, in vitro embryo production, and pregnancy rate.