Comparing the effect of estradiol benzoate and 17ß-estradiol plus progesterone on follicular turnover and development, and pregnancy outcomes in a timed artificial insemination protocol.

The objective of this study was to compare the efficiency of estradiol benzoate (EB) and 17ß-estradiol (E2) associated with progesterone (P4) in a fixed-time artificial insemination (FTAI) protocol. We hypothesized that E2+P4 induces an earlier emergence of a new follicular wave (NFW), improving pre-ovulatory follicle diameter and pregnancy rates to FTAI (P/FTAI). In Exp.1, on Day 0 (D0), all Bos indicus cows (n = 12/group) received an intravaginal P4 device and a dose of PGF2α analogue. On D0, females were randomly assigned to receive EB or E2+P4. On D8.5, P4 intravaginal devices were removed and a dose of PGF2α and EB were administered in all females followed by fixed-timed AI on D10. Between D0 and D10, the dominant follicular growth was determined by ovary ultrasonography exams. On D8.5 and D10 the percentage of color power-Doppler signals in the dominant follicular wall was evaluated. In Exp. 2, 467 females (2-year-old nulliparous [n = 76], primiparous [n = 92] and pluriparous [n = 299]) were subjected to the similar FTAI and assigned to be treated with EB (n = 243) or E2+P4 (n = 224). Pregnancy diagnosis was performed 30 days after FTAI by ultrasonography. The day to emergence of NFW was similar between treatments (EB: 3.7 ± 0.37 vs. E2+P4: 3.3 ± 0.3, P = 0.76). Females treated with E2+P4 presented greater (P = 0.06) follicular growth between the emergence and D9 (1.18 ± 0.07) than those treated with EB (0.97 ± 0.08). There was also a positive effect (P < 0.05) of E2+P4 on diameter of the dominant follicle on D9 (13.0 ± 0.6 vs. 10.9 ± 0.55) and blood perfusion of the follicle wall on D8.5 (49 vs. 40%). There was a treatment by parity category interaction effect on P/FTAI (P < 0.05). Treatment with E2+P4 was advantageous to P/FTAI of primiparous cows (E2+P4: 58% and EB: 30%). However, for nulliparous and pluriparous cows, P/FTAI was similar between treatments (∼50%). In conclusion, in a E2/P4-based protocol for FTAI, E2+P4 is as efficient as EB in inducing new follicular emergence within a similar day range, but it results similar or greater P/FTAI.

Reproductive seasonality influences oocyte retrieval and embryonic competence but not uterine receptivity in buffaloes.

Since buffaloes are a seasonal, polyestrous species, optimizing reproduction during the non-breeding season is a key factor in increasing the reproductive and productive efficiency of herds. Ovum pick-up associated with in vitro embryo production and embryo cryopreservation is an alternative to reduce seasonal impacts. We studied the effects of seasonality in buffalo oocyte donors and embryo recipients during the favorable and non-favorable breeding seasons. Donors were evaluated for oocyte recovery and blastocyst production rate as dFBS (donors in favorable breeding season) or dNBS (donors in non-favorable breeding season). Embryos produced from dFBS or dNBS were cryopreserved by vitrification or the slow-freeze method for direct transfer and transferred to recipients in the favorable (rFBS) or non-favorable breeding season (rNBS). The heifers or cows were subjected to a fixed-time embryo transfer protocol and conception rates were determined on day 30 and on day 60. The oocyte recovery was lower in dFBS than in dNBS (7.6 vs. 10.0 oocyte/OPU, p = 0.0262); while no difference was found comparing blastocyst production rate (23.7% vs. 30.9% of blastocysts, respectively). Embryos from dFBS resulted in greater (p = 0.0013) conception rates on day 30 compared to dNBS (46.5% vs. 22.4%, respectively), despite the breeding season. The rFBS and rNBS treatments had similar (p = 0.6714) conception rates on day 30 (38.0% vs. 33.0%, respectively), indicating similar uterine receptivity. However, heifers on FBS had higher (p = 0.0003) conception rates on day 30 than cows (73.9% vs. 13.3%, respectively) when receiving embryos from dFBS. Vitrification and direct transfer had similar (p = 0.1698) conception rates on day 30 (30.4% vs. 41.4%, respectively). In conclusion, in vitro-produced embryos derived from dFBS were more competent in establishing pregnancy than dNBS counterparts, independent of recipients' reproductive seasonality. Heifers achieved better conception rates than cows during the favorable breeding season when the embryo came from dFBS. Cryopreserved in vitro produced embryos represent a reliable alternative to reduce seasonal variations in buffalo reproduction. The data elucidate the seasonal effects on embryo competence and on recipients' uterine receptivity, affording new strategies to implement ovum pick-up associated with in vitro embryo production programs in buffalo herds.

Cholesterol-loaded cyclodextrin is efficient in preserving sperm quality of cryopreserved ram semen with low freezability.

Semen freezability is positive correlated with the cholesterol content in the sperm cell. Freeze-thawing mainly cause temperature chock and change on media osmolarity, which can modify plasma membrane lipids content and sperm conformation, resulting in decreased fertility. Therefore, the aim of this study is to investigate the effect of adding cholesterol-loaded cyclodextrin (CLC) to the cryopreservation process of ram semen with low freezability. For that, two experiments were performed using 5 ejaculates of 6 rams, totalizing 30 samples. For experiment 1 the following treatments were tested: in natura (IN), Tris solution (CON), CLC + Tris solution (CLC), and pure methyl-ß-cyclodextrin + Tris solution (MCD). For experiment 2 treatments CON and CLC were tested in samples subdivided into three freezability classes: high (n = 10), intermediate (n = 10) and low (n = 10). Freezability classes were based on the variation of sperm motility between IN and CON groups from the first experiment. Sample analyzes included sperm motility, sperm morphology, plasma and acrosome membrane integrity, mitochondrial membrane potential, reactive oxygen species content, lipid peroxidation, and fluidity of plasma membrane. Results showed that CLC treatment was more efficient in maintaining sperm motility, integrity of plasma membrane, integrity of acrosome, and mitochondria membrane potential. In addition, CLC treatment in the groups with low and intermediate freezability showed improvement on progressive motility and percentage of rapid cells. In contrast, no difference was noted between CLC and CON treatments in the high freezability group. Therefore, the addition of CLC to semen extender improved sperm cryopreservation, especially in rams with low freezability.

Molecular detection of Leishmania spp. in cattle from Brazil by means of PCR using internal transcribed spacer 1.

Leishmania spp. are important agents of human and animal leishmaniases that have an important impact on public health. In this study, we aimed to detect the circulation of Leishmania spp. in cattle from a visceral leishmaniasis non-endemic area of the state of São Paulo, Brazil. DNA was extracted from blood samples from 100 heifers in the municipality of Pirassununga and was amplified using primers specific for the first internal transcriber spacer (ITS1), to assess the presence of trypanosomatids. The assays revealed that one sample presented bands of between 300 and 350 base pairs. In GenBank, this sample matched 100% with Leishmania infantum (314 base pairs). The results suggest that cattle can be infected by Leishmania infantum in Brazil.

Molecular detection of Leishmania spp. in cattle from Brazil by means of PCR using internal transcribed spacer 1 / Detecção molecular de Leishmania spp. em gado no Brasil por PCR com espaçador interno transcrito 1

Abstract Leishmania spp. are important agents of human and animal leishmaniases that have an important impact on public health. In this study, we aimed to detect the circulation of Leishmania spp. in cattle from a visceral leishmaniasis non-endemic area of the state of São Paulo, Brazil. DNA was extracted from blood samples from 100 heifers in the municipality of Pirassununga and was amplified using primers specific for the first internal transcriber spacer (ITS1), to assess the presence of trypanosomatids. The assays revealed that one sample presented bands of between 300 and 350 base pairs. In GenBank, this sample matched 100% with Leishmania infantum (314 base pairs). The results suggest that cattle can be infected by Leishmania infantum in Brazil.

Resumo Leishmania spp. são agentes causadores das leishmanioses em humanos e em animais, gerando grande impacto à saúde pública. Este estudo objetivou detectar a circulação de Leishmania spp. em área não endêmica para leishmaniose visceral de São Paulo, Brasil. Foram extraídas amostras de DNA de 100 novilhas da cidade de Pirassununga. Estas amostras foram amplificadas com os iniciadores específicos para tripanosomatídeos Internal Transcriber Spacer 1 (ITS1). Os ensaios revelaram uma amostra com bandas entre 300 e 350 pares de base (pb). A amostra demonstrou 100% de identidade com Leishmania infantum (314 pb). Os resultados sugerem que o gado pode ser infectado por L. infantum no Brasil.

Effect of hCG application at different moments of the estrous cycle on corpus luteum and uterine vascularization and serum progesterone concentration in mares.

Establishment of pregnancy after embryo transfer is the ultimate goal of an embryo transfer program and increasing pregnancy rates and reducing pregnancy loss are mandatory. The utilization of treatments to improve conception rates in recipient mares has been the focus of several research groups over the last years and the results are controversial. Some studies using human chorionic gonadotrophin (hCG) found promising results. Our hypothesis was that hCG administration would cause an additional stimulation on luteal function, uterine and luteal vascularization and progesterone concentration, and the mares would have increased uterine and cervix tone. Therefore, in the present study the effects of hCG administration to induce ovulation, on day 0 (day of ovulation) or day 5 postovulation were evaluated on corpus luteum characteristics, reproductive tract vascularization, and serum progesterone concentration from ovulation until day 15 postovulation. Groups were: G1: (control) - no hCG; G2: 2500 IU of hCG to induce ovulation when a follicle greater than 35mm and uterine edema were detected; G3: 2500 IU hCG on day 0; G4: 2500 IU hCG on day 5 postovulation. Twelve mares were randomly assigned to each group, during consecutive cycles, in a Latin Square experimental design, in a total of 48 cycles. Doppler ultrasound evaluations were performed daily from day 0 until day 15 postovulation, including mesometrial vascularity, endometrial vascularity and corpus luteum vascularity. Blood samples were collected for serum progesterone concentration. Data was analyzed using the Proc Glimmix SAS Procedure for nonparametric variables and Proc Mixed for parametric parameters. There was no treatment effect for all variables studied (P > 0.05). Characteristics were only affected by day (P < 0.05). It can be concluded that hCG administration at the time points suggested in the current study did not alter the characteristics evaluated.

Follicular dynamics, ovarian vascularity and luteal development in mares with early or late postpartum ovulation.

Follicular development and deviation processes during the postpartum period are not completely known in horses. Thus, we aimed to study the characteristics of follicular dynamics and ovarian vascular perfusion during the postpartum period in mares that demonstrated estrous behavior and had early (<10 days) or late (≥10 days) postpartum ovulation. Ten mares were scanned daily by transrectal ultrasonography from the first day postpartum (d1) to the sixteenth day after the first postpartum ovulation (D0 = ovulation). The animals were split in the early (n = 3) and late (n = 7) ovulation groups (average interval between parturition and ovulation: 8.0 ± 0.0 and 14.7 ± 1.2 days, respectively). For the follicular growth, no difference (P > 0.1) was detected between the groups when the data were normalized for the days preceding the first postpartum ovulation (from D-7 to D-1). However, when the data were normalized to days postpartum, the dominant follicle was larger (P < 0.05) in the early-ovulated group in all days during this period (d1 to d7). The number of follicles >25 mm diameter was greater (P < 0.05) in the early-ovulated group during the first 3 days postpartum, and the late-ovulated mares showed greater number of follicles with 20-25 mm during d4 to d7. For blood flow characteristics, no difference (P > 0.1) was detected between groups in vascular perfusion of the dominant follicle or in the ovarian pedicle ipsilateral to the largest follicle. Similarly, no difference (P > 0.1) was detected in progesterone concentrations, corpus luteum (CL) area and vascular perfusion of the CL. Pregnancy rate did not differ (P > 0.1) between the early (3/3; 100%) and late (5/7; 71.4%) groups. Therefore, the characteristics of the follicle growth on the preceding days of ovulation were similar between the early- and late-ovulated mares and were consistent with the follicular dynamics expected in non-pregnant and non-lactating mares. However, when the data were analyzed for the days relative to parturition, greater follicle development was present in mares that ovulate earlier during the postpartum period (<10 days). The results suggest that important events may occur before parturition, resulting in early follicle development, mainly in those mares that show estrus and ovulate within 10 days postpartum.