L. G. B. Siqueira

Color Doppler flow imaging for the early detection of nonpregnant cattle at 20 days after timed artificial insemination.

The objective was to determine the accuracy of a pregnancy test for predicting nonpregnant cattle based on the evaluation of corpus luteum (CL) blood flow at 20 d (CLBF-d20) after timed artificial insemination (TAI). Crossbred Holstein-Gir dairy heifers (n=209) and lactating cows (n=317) were synchronized for TAI using the following protocol: intravaginal implant (1.0 g of progesterone) and 2mg of estradiol benzoate i.m. on d -10, implant removal and 0.526 mg of sodium cloprostenol i.m. on d -2, 1mg of estradiol benzoate i.m. on d -1, and TAI on d 0. On d 20, animals underwent grayscale ultrasonography (US) to locate the CL and color flow Doppler to evaluate CLBF-d20 using a portable ultrasound equipped with a 7.5-MHz rectal transducer. Based only on a visual, subjective CLBF evaluation, the animals were classified as pregnant or not pregnant. On d 30 to 35, blinded from results of the previous diagnosis, the same operator performed a final pregnancy diagnosis using US to visualize the fetal heartbeat (gold standard; US-d30). A second evaluator also analyzed the CLBF-d20 in the same animals by watching 7-s recorded videos. Blood samples were collected from a subset of 171 females to determine, by RIA, plasma progesterone (P4) concentrations, which indicate CL function. The final pregnancy outcome (US-d30) was retrospectively compared with the CLBF-d20 diagnoses and then classified either as correct or incorrect. The number of true positive, true negative, false positive, and false negative decisions were inserted into a 2 × 2 decision matrix. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the CLBF-d20 test were calculated using specific equations. Binomial variables (pregnancy rate and proportions) were analyzed using Fishers exact test for the effect of parity and to compare between evaluators and tests (CLBF-d20 vs. plasma P₄). The kappa values were calculated to quantify the agreement between CLBF-d20 and the gold standard (US-d30) and between evaluators. The performance parameters of CLBF-d20 test were as follows: sensitivity=99.0%, specificity=53.7%, positive predictive value=65.1%, negative predictive value=98.5%, and accuracy=74.8%. False negatives represented only 0.4% of the exams. No differences existed in these parameters between evaluators (no. 1 vs. no. 2) and tests (CLBF-d20 vs. plasma P4). Moreover, a high level of agreement was observed between evaluators (0.91). In conclusion, visual evaluation of CLBF-d20 represents a quick, reliable, and consistent diagnostic test that enables the early detection of nonpregnant cattle.

Ovarian follicular dynamics, follicle deviation, and oocyte yield in Gyr breed (Bos indicus) cows undergoing repeated ovum pick-up.

The objective of this study was to evaluate ovarian follicular dynamics during intervals between successive ovum pick-up (OPU) and determine its effects on the number and quality of recovered cumulus-oocyte complexes (COCs) in Zebu cows (Bos indicus). Pluriparous nonlactating Gyr cows (Bos indicus; n=10) underwent four consecutive OPU sessions at 96-h intervals. The dynamics of ovarian follicular growth between OPU sessions was monitored by twice-daily ultrasonographic examinations. A single dominant follicle (DF) or two codominant (CDF) follicles (>9mm) were present in 63.3% (19 of 30) of intervals studied, with follicle deviation beginning when the future dominant follicle (F1) achieved a diameter of 6.2+/-0.3mm. The phenomenon of codominance was observed in four (13.3%) of the inter-OPU intervals. The remaining intervals (36.6%, 11 of 30) were characterized by a greater follicular population, lower rate of follicular growth, and a smaller diameter F1 (P<0.0001). There was a tendency (P=0.08) toward an increase in the number of recovered COCs when dominant follicles were not present (NDF). The quality of COCs was not affected by the presence of a single dominant follicle, but codominant follicles resulted in recovery of a lower proportion of viable embryos (40.0%, 62.1%, and 63.6%; P<0.05) and higher proportions of degenerate COCs (56.0%, 30.3%, and 28.6%; P<0.05) for CDF, NDF, and DF respectively. We concluded that, in Zebu cows, (a) repeated follicle aspirations altered ovarian follicular dynamics, perhaps by increasing follicular growth rate; (b) follicular dominance could be established in cows undergoing twice-a-week OPU; and (c) the presence of a dominant follicle during short inter-OPU intervals may not affect COC quality, except when a codominant follicle was present.

Pregnancy rates and corpus luteum–related factors affecting pregnancy establishment in bovine recipients synchronized for fixed-time embryo transfer

The objective was to investigate the influence of corpora lutea physical and functional characteristics on pregnancy rates in bovine recipients synchronized for fixed-time embryo transfer (FTET). Crossbred (Bos taurus taurus x Bos taurus indicus) nonlactating cows and heifers (n=259) were treated with the following protocol: 2mg estradiol benzoate (EB) plus an intravaginal progesterone device (CIDR 1.9g progesterone; Day 0); 400 IU equine chorionic gonadotropin (eCG; Day 5); prostaglandin F(2alpha) (PGF(2alpha)) and CIDR withdrawal (Day 8); and 1mg EB (Day 9). Ovarian ultrasonography and blood sample collections were performed on Day 17. Of the 259 cattle initially treated, 197 (76.1%) were suitable recipients; they received a single, fresh, quality grade 1 or 2 in vivo-derived (n=90) or in vitro-produced (n=87) embryo on Day 17. Pregnancy rates (23 d after embryo transfer) were higher for in vivo-derived embryos than for in vitro-produced embryos (58.8% vs. 31.0%, respectively; P<0.001). Mean (+/-SD) plasma progesterone (P(4)) concentration was higher in cattle that became pregnant than that in nonpregnant cattle (5.2+/-5.0 vs. 3.8+/-2.4 ng/mL; P=0.02). Mean pixel values (71.8+/-1.3 vs. 71.2+/-1.1) and pixel heterogeneity (14.8+/-0.3 vs. 14.5+/-0.5) were similar between pregnant and nonpregnant recipients (P>0.10). No significant relationship was detected between pregnancy outcome and plasma P(4), corpus luteum area, or corpus luteum echotexture. Embryo type, however, affected the odds of pregnancy. In conclusion, corpus luteum-related traits were poor predictors of pregnancy in recipients. The type of embryo, however, was a major factor affecting pregnancy outcome.

Infertility in a beef bull due to a failure in the capacitation process

The objective of this case report was to identify the cause of apparent idiopathic infertility in a Red Angus (beef) bull. Semen was collected by electroejaculation and submitted to a series of assays, including evaluation of sperm motility by computer-assisted sperm analysis (CASA), sperm morphology and DNA integrity, semen cryopreservation, AI, IVF, induction of the acrosome reaction, and determination of the level of sperm proteins associated with bull fertility potential. Total (92 ± 2%) and progressive (79 ± 4%) sperm motility; sperm concentration (1647 ± 429 × 10(6) sperm/mL); proportions of morphologically normal sperm (83 ± 6%) and DNA integrity (96 ± 2), and acrosome-intact sperm (64 ± 4%) exceeded minimum acceptable values. Frozen sperm had good total (58.7 ± 6.7%) and progressive (43.9 ± 9.2%) motility immediately after thawing. However, AI of 16 heifers resulted in no pregnancies and blastocyst production rate (following IVF using sperm from this infertile bull) was nearly identical to that produced using dead sperm (a control of parthenogenesis; 2 ± 2 and 2 ± 3%; respectively P < 0.05). Treatment with a calcium ionophore (A23187) failed to induce the acrosome reaction in sperm from the infertile bull (P < 0.05). Evaluation of several proteins associated with the fertility potential of bulls revealed that the level of Binder Sperm Protein-1 (BSP1), known to be associated with the capacitation process, was much greater on sperm from the infertile bull compared to that of his sire. In conclusion, we inferred that the idiopathic infertility in this bull was caused by a failure to complete the capacitation process.

The use of PGF2α as ovulatory stimulus for timed artificial insemination in cattle

The objective of this study was to evaluate the effect of a PGF2α-analogue (PGF) on ovulation and pregnancy rates after timed artificial insemination (TAI) in cattle. In experiment 1, crossbred dual-purpose heifers, in a crossover design (3 × 3), were given an intravaginal progesterone-releasing insert (controlled internal drug release [CIDR]) plus 1 mg estradiol benzoate (EB) intramuscularly (im) and 250 μg of a PGF-analogue im on Day 0. The CIDR inserts were removed 5 days after follicular wave emergence, and the heifers were randomly divided into three treatment groups to receive the following treatments: (1) 1 mg of EB im (EB group, n = 13); (2) 500 μg of PGF im (PG group, n = 13); or (3) saline (control group, n = 13), 24 hours after CIDR removal. Ovulation occurred earlier in EB (69.81 ± 3.23 hours) and PG groups (73.09 ± 3.23 hours) compared with control (83.07 ± 4.6 hours; P = 0.01) after CIDR removal. In experiment 2, pubertal beef heifers (n = 444), 12 to 14 months of age were used. On Day 0, the heifers were given a CIDR insert plus 2 mg EB im. On Day 9, the CIDR was removed and the heifers were given 500 μg of PGF im. Heifers were randomly assigned into one of three treatment groups: (1) 1 mg of EB (EB group; n = 145); (2) 500 μg of PGF (PG group; n = 149), both 24 hours after CIDR removal; or (3) 600 μg of estradiol cypionate (ECP group; n = 150) at CIDR removal. Timed artificial insemination occurred 48 hours after CIDR removal in the ECP group and 54 hours in the PG and EB groups. The percentage of heifers ovulating was higher in the PG group compared with the other groups (P = 0.08). However, the pregnancy rates did not differ among groups (47.6%, 45%, and 46.6%, for EB, PG, and ECP, respectively; P = 0.9). In experiment 3, 224 lactating beef cows, 40 to 50 days postpartum with 2.5 to 3.5 of body condition score were treated similarly as described in experiment 2, except for the ECP group, which was excluded. The treatments were as follows: 1 mg EB (EB group; n = 117) or 500 μg PGF (PG group; n = 107), 24 hours after CIDR removal. The calves were temporarily separated from their dams from Days 9 to 11. No difference was detected on the pregnancy rate between the EB and PG groups (58.1% vs. 47.6%, respectively; P = 0.11). Taken together, the combined results suggested that PGF2α could be successfully used to induce and synchronize ovulation in cattle undergoing TAI, with similar pregnancy rates when compared with other ovulatory stimuli (ECP and EB).

Occurrence and characteristics of residual follicles formed after transvaginal ultrasound-guided follicle aspiration in cattle.

Ultrasound-guided transvaginal follicle aspiration is used to recover cumulus-oocyte complexes (for IVF) and to synchronize follicular wave emergence (ablation of dominant follicle). Although aspirated follicles are generally supposed to undergo immediate atresia, there are indications that they may remain active. The objective was to evaluate the occurrence and characteristics of residual follicles (RF) after transvaginal follicle aspiration in cattle. Ovarian follicular wave emergence was synchronized in Holstein cows (N = 13) in the presence (groups 1 and 3) or absence (groups 2 and 4) of norgestomet implants. The largest follicle was aspirated at a diameter of 8 mm (groups 1 and 2) or 12 mm (groups 3 and 4). Ovarian follicles were visualized (transrectal ultrasonography) every 12 h after wave emergence. Follicular fluid samples were collected from the largest follicle and from the ensuing RF and concentrations of estradiol and progesterone were determined. After aspiration, 73.2% (52/71) of the follicles refilled with fluid, and a new antrum was detected 12 to 24 h later. Norgestomet did not affect (P > 0.05) RF occurrence or diameter, but in RF from group 4, concentrations of estradiol decreased (-530.7 ± 133.9 ng/mL; P < 0.01) whereas progesterone increased (+429.6 ± 171.7 ng/mL; P < 0.05) relative to preaspiration. In RF, there were three steroidogenesis patterns: (1) high estradiol concentration and high estradiol:progesterone ratio (estradiol-active RF); (2) low estradiol, but high progesterone concentrations (luteinized RF); and (3) low estradiol and low progesterone concentrations (inactive RF). Estradiol-active RF were more likely (P < 0.05) from follicles with high estradiol concentrations (regardless of diameter). In conclusion, fluid-filled structures (RF) with variable steroid production patterns are frequently formed after ultrasound-guided follicle aspiration. The occurrence and features of these RF depended on the diameter and status of these follicles before aspiration.

In vivo imaging of cumulus-oocyte-complexes and small ovarian follicles in cattle using ultrasonic biomicroscopy

The objectives of this study were to: (1) evaluate the feasibility of ultrasonic biomicroscopy (UBM) for imaging ovarian follicles in vivo in cows and heifers, (2) compare the transvaginal to the transrectal approach, (3) compare the number of follicles detected by UBM to conventional ultrasonography (US), and (4) identify cumulus-oocyte-complexes (COC) within follicles by UBM. Mature cows (n=5) and peri-pubertal heifers (n=5) were examined once using conventional B-mode US (Aloka 900) with a 7.5 MHz transrectal, and a 5 MHz transvaginal transducers. A second series of examinations was performed using UBM (Visualsonics Vevo 660) equipped with a 30 MHz transrectal and a 40 MHz transvaginal transducer. A three- to four-fold increase in the number of small follicles (<3 mm) was detected using the transvaginal approach with UBM compared to conventional US in both heifers (32.4 ± 4.24 compared to 7.2 ± 1.4; P<0.0001) and cows (35.0 ± 13.8 compared to 10.7 ± 7.5; P=0.0013). However, fewer follicles (all sizes combined) were detected using the transrectal approach with UBM compared to conventional US in both heifers (8.6 ± 5.6 compared to 17.6 ± 4.9; P=0.02) and cows (5.3 ± 6.1 compared to 20.3 ± 7; P=0.04). In heifers, COC-like structures were identified in 39 of 202 (19.3%) follicles examined. In conclusion, UBM using a transvaginal approach is feasible and may be used for in vivo assessment of early antral follicles as small as 0.4 mm, and COC within follicles.

Assessment of harmonic distortions on power and energy measurements

This paper presents the results of a study aiming to assess the effects of harmonic distortion on power and energy measurements by watt-hour meters. The readings from several different meters installed in large customers have been compared in order to verify the effects on tariff parameters commonly used. Based on present tariff model and corresponding legal framework in Brazil, results show that moderate harmonic distortion has no significant effect on tariff parameters and invoice amount. Therefore, neither the utilities nor the customers seem to be paying high prices for not taking or taking harmonic distortions into account.

Embryo development and follicular status of Toggenburg does fed urea diet

Objetivou-se investigar os efeitos da ureia na producao, qualidade e estadio de desenvolvimento embrionario e status folicular ao inicio do tratamento superovulatorio de animais alimentados com ureia na dieta. Dezoito cabras da raca Toggenburg foram distribuidas equitativamente para receberem dietas: sem ureia (controle, n=8) e 2,4% de ureia na materia seca total da dieta (n=10) UMS. Os embrioes foram recuperados entre o 7o o 8o dia apos o estro pela tecnica transcervical e classificados pela qualidade e pelo estadio de desenvolvimento. O status folicular foi determinado por ultrassonografia transretal no dia da aplicacao da primeira dose de FSH. O numero e a qualidade dos embrioes nao foram influenciados pela concentracao de ureia na dieta. Embrioes em estadio avancado de desenvolvimento foram recuperados do tratamento com 2,4% de ureia. A concentracao de ureia testada (2,4%) diminui o numero de foliculos com menos de 5 mm de diâmetro, categoria que se correlaciona positivamente ao numero de embrioes coletados e ao numero e a porcentagem de embrioes excelentes e bons.

Intrafollicular oestradiol production, expression of the LH receptor (LHR) gene and its isoforms, and early follicular deviation in Bos indicus

The aim of the present study was to characterise the roles of intrafollicular oestradiol production and granulosa cell (GC) expression of the LH receptor (LHR) gene and its isoforms during follicular deviation in Bos indicus. Follicular wave emergence was synchronised in heifers from a Bos taurus dairy (Holstein; n=10) and a B. indicus dairy breed (Gir; n=10). Follicles were aspirated individually at sizes corresponding to the periods of predeviation, deviation and postdeviation. Intrafollicular oestradiol (IF-E2) and progesterone (IF-P4) concentrations were determined in the follicular fluid (FF) by radioimmunoassay, and relative expression of P450 aromatase (CYP19A1) and LHR forms was evaluated in GC using real-time quantitative-polymerase chain reaction. Despite differences in the size of the dominant follicle at deviation, changes in CYP19A1 expression and IF-E2 concentrations were similar in follicles of the same diameter in both breeds. A peak in total LHR expression occurred after follicular deviation in association with low expression of LHR isoforms. The results suggest that regulation of LHR function by sequential changes in the expression pattern of LHR isoforms may play a role in the early deviation of the dominant follicle, as observed in B. indicus breeds.