Skeletal Muscle Response to Deflazacort, Dexamethasone and Methylprednisolone.

Glucocorticoids represent some of the most prescribed drugs that are widely used in the treatment of neuromuscular diseases, but their usage leads to side effects such as muscle atrophy. However, different synthetic glucocorticoids can lead to different muscle effects, depending upon its chemical formulation. Here, we intended to demonstrate the muscle histologic and molecular effects of administering different glucocorticoids in equivalency and different dosages. Methods: Seventy male Wistar rats distributed into seven groups received different glucocorticoids in equivalency for ten days or saline solution. The study groups were: Control group (CT) saline solution; dexamethasone (DX) 1.25 or 2.5 mg/kg/day; methylprednisolone (MP) 6.7 or 13.3mg/kg/day; and deflazacort (DC) 10 or 20 mg/kg/day. At the end of the study, the animals were euthanized, and the tibialis anterior and gastrocnemius muscles were collected for metachromatic ATPase (Cross-sectional area (CSA) measurement), Western blotting (protein expression of IGF-1 and Ras/Raf/MEK/ERK pathways) and RT-PCR (MYOSTATIN, MuRF-1, Atrogin-1, REDD-1, REDD-2, MYOD, MYOG and IRS1/2 genes expression) experiments. Results: Muscle atrophy occurred preferentially in type 2B fibers in all glucocorticoid treated groups. DC on 10 mg/kg/day was less harmful to type 2B fibers CSA than other doses and types of synthetic glucocorticoids. In type 1 fibers CSA, lower doses of DC and DX were more harmful than high doses. DX had a greater effect on the IGF-1 pathway than other glucocorticoids. MP more significantly affected P-ERK1/2 expression, muscle fiber switching (fast-to-slow), and expression of REDD1 and MyoD genes than other glucocorticoids. Compared to DX and MP, DC had less of an effect on the expression of atrogenes (MURF-1 and Atrogin-1) despite increased MYOSTATIN and decreased IRS-2 genes expression. Conclusions: Different glucocorticoids appears to cause muscle atrophy affecting secondarily different signaling mechanisms. MP is more likely to affect body/muscles mass, MEK/ERK pathway and fiber type transition, DX the IGF-1 pathway and IRS1/2 expression. DC had the smallest effect on muscle atrophic response possibly due a delayed timing on atrogenes response.

Efficacy of the re-utilization of an ear implant impregnated with progestogen in estrus synchronization response and pregnancy in sheep.

The goal of this study was to assess the efficacy of the re-utilization of an ear implant impregnated with norgestomet on estrus synchronization response and pregnancy rates in sheep. Fifty-five Texel ewes were classified according to body condition (3.5) and live weight (65 kg), and randomly assigned to two experimental groups: NORN-new (n = 30) and NORU-used (n = 25). The dose of norgestomet used in the treatments was half of that recommended for bovine (1.5 mg). The synchronization protocol consisted of an ear implant inserted on day 0 (beginning of the experiment) and kept for 6 days. The removal of the implant on day 6 was followed by an injection of a prostaglandin analogue (0.263 mg) and eCG (250 IU). Rams with paint applied to their chest were used to facilitate estrus detection for 5 days following implant withdrawal. Estrus behavior was observed in 93.3% (28/30) and 90% for NORN and NORU, respectively. Pregnancy rates for NORN were 73.3% and for NORU were 68%. Estrus behavior and pregnancy rates between treatment groups did not differ statistically. Therefore, the re-utilization of ear implants impregnated with norgestomet, in addition to eCG and a prostaglandin analogue in short-term estrus synchronization protocols, allow acceptable estrus response and pregnancy rates in sheep.

Dual Therapy Deflazacort/Doxycyclyne Is Better Than Deflazacort Monotherapy to Alleviate Cardiomyopathy in Dystrophin-Deficient mdx Mice.

Cardiomyopathy related to the absence of dystrophin is an important feature in Duchenne muscular dystrophy (DMD) and in the mdx mouse. Doxycycline (DOX) could be a potential therapy for mdx skeletal muscles dystrophy. We investigated whether the corticoid deflazacort (DFZ) plus DOX could improve cardiac mdx dystrophy better than DFZ alone, later (17 months) in dystrophy. Mdx mice (8 months old) received DFZ/DOX or DFZ for 9 months. The combined therapy was greater than DFZ in reducing fibrosis (60% decrease with DFZ/DOX and 40% with DFZ alone) in the right ventricle and transforming growth factor ß levels (6.8 ± 3.2 in untreated mdx mice, 2.8 ± 1.4 in combined therapy, and 4.6 ± 1.7 in DFZ; P < .05). Combined therapy more effectively ameliorated cardiac dysfunction (electrocardiogram [ECG]) than DFZ. Improvements were seen in the cardiomyopathy index (0.8 ± 0.1 in combined therapy and 1.0 ± 0.2 in DFZ), heart rate (418 ± 46 bpm in combined therapy and 457 ± 29 bpm in DFZ), QRS interval (11.3 ± 2 in combined therapy and 13.6 ± 1 in DFZ), and Q wave amplitude (-40.7 ± 21 in combined therapy and -90.9 ± 36 in DFZ). Both therapies decreased markers of inflammation (tumor necrosis factor α, nuclear factor κB, and metalloproteinase 9). DFZ/DOX improved mdx cardiomyopathy at this stage of the disease, supporting further clinical investigations.

Long-Term Outcome of Interdisciplinary Management of Patients with Duchenne Muscular Dystrophy Receiving Daily Glucocorticoid Treatment.

OBJECTIVE: To evaluate clinical outcomes and steroid side effects in a cohort of patients with Duchenne muscular dystrophy (DMD) treated with long-term daily glucocorticoid therapy. Although daily glucocorticoid therapy has been shown to extend ambulatory function in DMD, less frequent dosing is often used because of side effect concerns. STUDY DESIGN: Retrospective study of 97 patients with DMD aged 10 to <16 years treated with daily glucocorticoid (89% on deflazacort) for a mean of 8.5 years. Outcome measures were motor, pulmonary, and cardiac function, and scoliosis. Side effects were growth failure and weight gain, facial fullness, blood pressure, bone health, cataracts, gastrointestinal symptoms, behavior, hypertrichosis, and need for medication interventions. RESULTS: For 13- to 16-year-old patients, 40% could rise from the floor and 50% could perform the 30-foot run test. Forced vital capacity for the entire cohort was well preserved. Thirteen percent of younger (10- to <13-year-old) and 21% of older patients had findings of left ventricle systolic dysfunction. Six percent (all aged 16 years) developed scoliosis (Cobb angle >20 degrees). Eighty-six percent had normal weight velocities; 30% had no increased facial fullness; 72% had short stature; and 19% had asymptomatic cataracts. Asymptomatic spine compression deformities were noted in 76% and long bone fractures in 30%. One patient stopped glucocorticoid because of behavioral concerns. CONCLUSIONS: With evidence for improved outcomes and manageable side effects, we recommend use of daily glucocorticoid therapy for patients with DMD with anticipatory management of side effects and a coordinated interdisciplinary care approach.

Corticosteroid Treatment and Growth Patterns in Ambulatory Males with Duchenne Muscular Dystrophy.

OBJECTIVES: To evaluate growth patterns of ambulatory males with Duchenne muscular dystrophy (DMD) treated with corticosteroids compared with ambulatory, steroid-naïve males with DMD and age-matched unaffected general-population males and to test associations between growth and steroid treatment patterns among treated males. STUDY DESIGN: Using data from the Muscular Dystrophy Surveillance, Tracking, and Research Network, we identified a total of 1768 height, 2246 weight, and 1755 body mass index (BMI) measurements between age 2 and 12 years for 324 ambulatory males who were treated with corticosteroids for at least 6 months. Growth curve comparisons and linear mixed-effects modeling, adjusted for race/ethnicity and birth year, were used to evaluate growth and steroid treatment patterns (age at initiation, dosing interval, duration, cumulative dose). RESULTS: Growth curves for ambulatory males treated with corticosteroids showed significantly shorter stature, heavier weight, and greater BMI compared with ambulatory, steroid-naïve males with DMD and general-population US males. Adjusted linear mixed-effects models for ambulatory males treated with corticosteroids showed that earlier initiation, daily dosing, longer duration, and greater dosages predicted shorter stature with prednisone. Longer duration and greater dosages predicted shorter stature for deflazacort. Daily prednisone dosing predicted lighter weight, but longer duration, and greater dosages predicted heavier weight. Early initiation, less than daily dosing, longer duration, and greater doses predicted greater BMIs. Deflazacort predicted shorter stature, but lighter weight, compared with prednisone. CONCLUSION: Prolonged steroid use is significantly associated with short stature and heavier weight. Growth alterations associated with steroid treatment should be considered when making treatment decisions for males with DMD.

Co-administration of deflazacort and doxycycline: a potential pharmacotherapy for Duchenne muscular dystrophy.

The standard therapy used in the treatment of Duchenne muscle dystrophy (DMD) is corticoids, such as deflazacort and prednisone. However, they have limited therapeutic value, and their combination with drugs already in use to treat other human diseases could potentially increase corticoid outcomes in DMD. In the present study, we evaluated whether a combined therapy of the corticoid deflazacort with doxycycline could result in greater improvement in mdx dystrophy than deflazacort alone. Deflazacort alone or deflazacort/doxycycline were administered for 36 days (starting on postnatal day 0) in drinking water. Histopathological, biochemical (creatine kinase), functional (forelimb muscle grip strength and fatigue) parameters and inflammatory markers (MMP-9, TNF-α, NF-kB) were evaluated in biceps brachii and diaphragm muscles of the mdx mice. The combined therapy was superior in improving the dystrophic phenotype compared to monotherapy. The primary results were observed in attenuating muscle fatigue, decreasing muscle total calcium and inflammatory markers and increasing ß-dystroglycan, a main component of the dystrophin-protein complex. Furthermore, the combined therapy was effective in preventing the loss of body mass observed with deflazacort alone at this very early stage of therapy. The present study offers preclinical data to support further studies with deflazacort/doxycycline combined therapy in DMD clinical trials.

Effects of progestagen exposure duration on estrus synchronization and conception rates of crossbreed ewes undergoing fixed time artificial insemination.

Synchronization of estrus and ovulation are of paramount importance in modern livestock improvement programs. These methods are critical for assisted reproduction technologies, including artificial insemination and embryo transfer, that can increase productivity. In the current study, subcutaneous implants containing norgestomet were placed for long (14 days), medium (9 days), and short (5 days) periods of time in 70 crossbred ewes undergoing fixed-time artificial insemination. The resulting effects on estrus synchronization and conception rates were subsequently evaluated. Among the synchronized ewes, 85.7% (60/70) underwent estrus over a period of 72 h after progestagen treatment ceased. The shortest mean interval between withdrawal of the device and onset of estrus (34.2 ± 8.9 h) was observed in the G14 days of P4 group (p < 0.05). The conception rate of the G14 days of P4 group was statistically higher than that of the other groups (83.3% vs. 60.9% vs. 47.8%; p < 0.05). In conclusion, 14 days of norgestomet treatment produced higher conception rates and a greater number of pregnancies at the beginning of the breeding season.

Timed artificial insemination should be performed early when used norgestomet ear implants are applied for synchronizing ovulation in beef heifers.

The present study evaluated the effect of the type of norgestomet ear implant (new vs. used) on the ovarian follicular response (experiment 1) and pregnancy per artificial insemination (AI) (P/AI; experiment 2) of beef heifers subjected to an estradiol plus progestin timed artificial insemination (TAI) program. In experiment 1, 57 cyclic beef heifers were randomly assigned to one of two groups according to the type (new or previously used for 9 days) of norgestomet ear (NORG) implant. At the time of NORG implant insertion, the heifers were treated with 2 mg of intramuscular estradiol benzoate. Eight days later, the NORG implants were removed, and the heifers received an intramuscular administration of 150 µg of d-cloprostenol, 300 IU of equine chorionic gonadotropin, and 0.5 mg of estradiol cypionate. The heifers had their ovaries scanned every 12 hours from the time of NORG implant removal to 96 hours after verifying the occurrence and timing of ovulation. No difference (P = 0.89) was observed in the ovulation rates between the two treatments (new = 80.0%; 24/30 vs. used = 81.5%; 22/27). However, the heifers treated with a used NORG implant had (P = 0.04) higher proportion (36.4%; 8/22) of early ovulation (between 36 and 48 hours after NORG implant removal) compared with the heifers treated with a new NORG implant (8.3%; 2/24). In experiment 2, at the beginning of the synchronization protocol, 416 beef heifers were randomly assigned into two groups, as described in the experiment 1. Two days after the NORG implant removal, the heifers were reassigned to be inseminated at 48 or 54 hours after NORG implant removal. There was an interaction (P = 0.03) between the type of NORG implant and the timing of TAI on P/AI. The timing of insemination only had an effect (P = 0.02) on the P/AI when the heifers were treated with a used NORG implant [(TAI 54 hours = 41.9% (44/105) vs. TAI 48 hours = 58.6% (58/99)]. In conclusion, beef heifers synchronized with a used NORG implant plus estradiol exhibited a higher proportion of earlier ovulations, and TAI in these heifers should be performed 48 hours after removal of used NORG implants.

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.

The effect of timing of the induction of ovulation on embryo production in superstimulated lactating Holstein cows undergoing fixed-time artificial insemination.

Two experiments evaluated the effects of timing of the induction of ovulation in superstimulated lactating Holstein donor cows that were fixed-time artificially inseminated. Secondary objectives were to evaluate the effects of the timing of progesterone (P4) device removal (Experiment 1) or the addition of a second norgestomet implant (Experiment 2) during superstimulation. In Experiment 1, 12 cows were allocated to one of four treatment groups with the timing of P4 device removal (24 or 36 h) and pLH treatment (48 or 60 h), after the first PGF as main factors, in a Latin Square (cross-over) design. There was an interaction (P = 0.03) between time of P4 device removal and time of pLH treatment. Mean (± SEM) numbers of transferable embryos were higher when the P4 device was removed at 36 h and pLH was administered at 60 h after the first PGF (P36LH60 =6.3 ± 1.4) compared to other treatments (P24LH60 =3.7 ± 1.1; P24LH48 =2.4 ± 0.8; or P36LH48 =2.2 ± 0.7). In Experiment 2, 40 cows were randomly allocated into one of four treatments with the number of norgestomet implants (one or two) and the time of induction of ovulation with GnRH relative to the first PGF (48 vs. 60 h) as main effects. The mean number of transferable embryos was higher (P = 0.02) when GnRH was administered at 60 h (4.2 ± 1.3) compared to at 48 h (2.7 ± 0.8), and the number of freezable embryos was increased (P = 0.01) in cows receiving two (3.0 ± 1.0) rather than one norgestomet implant (1.5 ± 0.5). In summary, embryo production in lactating Holstein cows was increased when the ovulatory stimulus (pLH or GnRH) was given 60 h after the first PGF, particularly when the P4 device was removed 36 h after the first PGF and when two norgestomet ear implants were used during the superstimulation protocol.

Induction of ovarian follicular wave emergence and ovulation in progestin-based timed artificial insemination protocols for Bos indicus cattle.

The present study aimed to evaluate the efficacy of different inducers of new follicular wave emergence (FWE) and ovulation in fixed-time artificial insemination (FTAI) synchronization protocols using norgestomet ear implants (NORG) in Bos indicus cattle. In Experiment 1, the synchronization of FWE was evaluated when two different estradiol esters in different doses [2mg estradiol benzoate (EB), 2.5mg EV or 5mg estradiol valerate (EV)] were administered with NORG implant insertion in B. indicus cattle (estrous cyclic heifers and cows with suckling calves; n=10 per treatment). After estradiol treatment, ovarian ultrasonic exams were performed once daily to detect the interval between treatment and FWE. There were significant treatment-by-animal category interaction (P=0.05) on the interval from the estradiol treatment to FWE. An earlier (P<0.0001) and less variable (P=0.02) interval from estradiol treatment to FWE was observed in heifers treated with EB (2.5±0.2; mean±SE) than in those treated with 2.5mg EV (4.2±0.3) or 5mg EV (6.1±0.6). Cows treated with 5mg EV (4.0±0.5) had longer (P=0.05) interval than cows receiving EB (2.5±0.2), however, there was an intermediate interval in those cows treated with 2.5mg EV (3.1±0.4). In Experiment 2, the number of uses of the NORG implant (new; n=305 or previously used once; n=314) and three different ovulation induction hormones [0.5mg estradiol cypionate (EC) at implant removal (n=205), 1mg EB given 24h after implant removal (n=219), or 100µg gonadorelin (GnRH) given at FTAI (n=195)] were evaluated in Nelore heifers (2×3 factorial design). Similar pregnancy per AI (P/AI; 30 days after FTAI; P>0.05) were achieved using each of the three ovulation induction hormones (EB=40.6%; EC=48.3%, or GnRH=48.7%) and with a new (47.2%) or once-used NORG implant (44.3%). In Experiment 3, the effect of different ovulation induction hormones for FTAI [1mg EC at NORG implant removal (n=228), 10µg buserelin acetate at FTAI (GnRH; n=212) or both treatments (EC+GnRH; n=215)] on P/AI was evaluated in suckled beef cows treated with a once-used NORG implant and EB to synchronize the FWE. Similar P/AI (P=0.71) were obtained using GnRH (50.9%), EC (51.8%) or both treatments (54.9%) as ovulation induction hormones. Therefore, both doses of EV (2.5 or 5.0mg) with NORG implant delayed and increased the variation of the day of new FWE compared with EB in B. indicus cattle. These effects were more pronounced in B. indicus heifers than cows. Synchronization protocols for FTAI with either a new or once-used NORG implant with EB at insertion to induce a new FWE and either the use of EB, EC or GnRH as ovulation induction hormones may be successful in B. indicus heifers. Also, when a once-used NORG implant was used, either the administration of EC, GnRH or both as ovulation inducers resulted in similar P/AI in suckled B. indicus cows, showing no additive effect of the combination of both ovulation induction hormones.

Ciclesonide, a hypotonic intranasal corticosteroid.

Intranasal corticosteroids (INCSs) are established as the first-line treatment of moderate to severe allergic rhinitis (AR) in both adults and children. Compared with other nasal allergy medications, INCSs are the most effective at providing symptom relief and increasing quality of life. Ciclesonide nasal spray is the most recently approved INCS. The formulation of ciclesonide does not contain benzylalkonium chloride or phenyl ethyl alcohol, excipients that have been associated with reduced mucociliary transport, and unpleasant sensory perceptions. Additionally, ciclesonide has been formulated in a hypotonic suspension that has been shown to optimize intranasal absorption and it has a lower volume of spray compared with most other INCS products. Systemic exposure to ciclesonide and its active metabolite desisobutyryl-ciclesonide is low after intranasal administration. High protein binding ( approximately 99%) and rapid first-pass clearance further reduce systemic exposure to the drug. Studies up to 1 year have shown that intranasal ciclesonide does not cause cortisol suppression as monotherapy and does not have an additive effect on the hypothalamic-pituitary-adrenal axis function when administered in combination with inhaled corticosteroids. The efficacy of ciclesonide, 200 microg/day, has been shown in pediatric, adolescent, and adult patients with moderate to severe seasonal AR and perennial AR treated for up to 1 year. Additionally, environmental exposure unit studies have established an onset of action as early as 1 hour after administration. Ciclesonide nasal spray has also been shown to have an acceptable safety profile in patients with AR as young as 2 years of age. Thus, intranasal ciclesonide appears to provide an additional effective treatment option for patients with AR.

Equine chorionic gonadotropin and gonadotropin-releasing hormone enhance fertility in a norgestomet-based, timed artificial insemination protocol in suckled Nelore (Bos indicus) cows.

Two experiments were conducted to investigate the effects of equine chorionic gonadotropin (eCG) at progestin removal and gonadotropin-releasing hormone (GnRH) at timed artificial insemination (TAI) on ovarian follicular dynamics (Experiment 1) and pregnancy rates (Experiment 2) in suckled Nelore (Bos indicus) cows. Both experiments were 2x2 factorials (eCG or No eCG, and GnRH or No GnRH), with identical treatments. In Experiment 1, 50 anestrous cows, 134.5+/-2.3 d postpartum, received a 3mg norgestomet ear implant sc, plus 3mg norgestomet and 5mg estradiol valerate im on Day 0. The implant was removed on Day 9, with TAI 54 h later. Cows received 400 IU eCG or no further treatment on Day 9 and GnRH (100 microg gonadorelin) or no further treatment at TAI. Treatment with eCG increased the growth rate of the largest follicle from Days 9 to 11 (means+/-SEM, 1.53+/-0.1 vs. 0.48+/-0.1mm/d; P<0.0001), its diameter on Day 11 (11.4+/-0.6 vs. 9.3+/-0.7 mm; P=0.03), as well as ovulation rate (80.8% vs. 50.0%, P=0.02), whereas GnRH improved the synchrony of ovulation (72.0+/-1.1 vs. 71.1+/-2.0 h). In Experiment 2 (n=599 cows, 40 to 120 d postpartum), pregnancy rates differed (P=0.004) among groups (27.6%, 40.1%, 47.7%, and 55.7% for Control, GnRH, eCG, and eCG+GnRH groups). Both eCG and GnRH improved pregnancy rates (51.7% vs. 33.8%, P=0.002; and 48.0% vs 37.6%, P=0.02, respectively), although their effects were not additive (no significant interaction). In conclusion, eCG at norgestomet implant removal increased the growth rate of the largest follicle (LF) from implant removal to TAI, the diameter of the LF at TAI, and rates of ovulation and pregnancy rates. Furthermore, GnRH at TAI improved the synchrony of ovulations and pregnancy rates in postpartum Nelore cows treated with a norgestomet-based TAI protocol.

Effect of fixed-time embryo transfer on reproductive efficiency in high-producing repeat-breeder Holstein cows.

The aim of the present study was to compare a synchronization of time of ovulation protocol for fixed-timed embryo transfer (FTET) with the usual administration of a single dose of prostaglandin associated with detection of estrus. Also, the effect of the presence of CL at the beginning of FTET protocol was evaluated. Lactating Holstein cows (n=651) with three previous artificial inseminations were classified according to presence or absence of a corpus luteum (CL). Cows with a CL were randomly assigned to two additional treatments and submitted to embryo transfer after detection of estrus (PGF-Estrus) or FTET (FTET-CL). Cows without CL were allocated to the FTET-NoCL treatment. On a random day of the estrous cycle (Day 0), cows in the PGF-Estrus treatment (n=229) were treated with 150 microg d-cloprostenol (PGF) i.m. followed by detection of estrus from Day 1 through Day 5 after PGF. Embryos were transferred 6-8 days after estrus detection. Cows in the FTET-CL (n=208; presence of CL) and FTET-NoCL (n=214; absence of CL) treatments received a norgestomet ear implant plus 2mg estradiol benzoate (EB) and 50mg progesterone i.m. on Day 0. On Day 8, the implant was removed and 400 IUeCG, 150 microg d-cloprostenol and 1mg estradiol cypionate i.m. were administered. No detection of estrus was performed and Day 10 was arbitrarily considered as the estrus day. Ultrasonographic exams were performed in all recipients and only cows with a single CL> or =15 mm or multiple CL received a fresh or frozen-thawed embryo on Day 17. Pregnancy was diagnosed by ultrasonography at 30 and 60 days of pregnancy. When FTET and PGF-Estrus were compared, the proportion of cows receiving an embryo (recipients transferred-to-treated rate) was greater in the FTET-CL (75.0% (156/208) than in PGF-Estrus (34.5%, 79/229; P<0.0001) treatment. Pregnancy rate (60 days) was also greater in FTET-CL (29.3%, 61/208) when compared to PGF-Estrus (16.2%, 37/229; P=0.001). However, no differences were found in pregnancy loss [PGF-Estrus=11.9% (5/42), FTET-CL=9.0% (6/67); P=0.62] and circulating progesterone concentration at embryo transfer [PGF-Estrus=4.02+/-0.52 ng/mL (n=25), FTET-CL=3.33+/-0.32 ng/mL (n=27); P=0.25] among these treatments. The presence of CL at the beginning of FTET protocol resulted greater transferred-to-treated rate [FTET-CL=75.0% (156/208) vs. FTET-NoCL=61.2% (131/214); P=0.003], but showed no effect on pregnancy rate at 60 days [FTET-CL=29.3% (61/208) vs. FTET-NoCL=22.9% (49/214); P=0.13], pregnancy loss [FTET-CL=9.0% (6/67) vs. FTET-NoCL=2.0% (1/50); P=0.15] and circulating progesterone concentration at ET [FTET-CL=3.33+/-0.32 ng/mL (n=27) compared to FTET-NoCL=3.44+/-0.40 ng/mL (n=2 9); P=0.82]. In conclusion, the protocol for synchronization of time of ovulation using norgestomet ear implant, EB and eCG increased recipients transferred-to-treated and pregnancy rates in high-producing repeat-breeder Holstein cows. Also, recipients without CL at the beginning of the time of ovulation synchronization treatment resulted in similar pregnancy rate as recipients with CL submitted to FTET protocol. Thus, the suggested protocol allowed the performance of FTET, without the need for detection of estrus, simplifying the reproductive management and increasing the reproductive efficiency in repeat-breeder Holstein recipients.

Effect of follicular wave synchronization on in vitro embryo production in heifers.

Aiming to achieve the ideal time of ovum pick-up (OPU) for in vitro embryo production (IVP) in crossbred heifers, two Latin square design studies investigated the effect of ovarian follicular wave synchronization with estradiol benzoate (EB) and progestins. For each experiment, crossbred heifers stage of estrous cycle was synchronized either with a norgestomet ear implant (Experiment 1) or a progesterone intravaginal device (Experiment 2) for 7d, followed by the administration of 150microg d-cloprostenol. On Day 7, all follicles >3mm in diameter were aspirated and implants/devices were replaced by new ones. Afterwards, implant/device replacement was conducted every 14d. Each experiment had three treatment groups. In Experiment 1 (n=12), heifers in Group 2X had their follicles aspirated twice a week and those in Groups 1X and 1X-EB were submitted to OPU once a week for a period of 28d. Heifers from Group 1X-EB also received 2mg EB i.m. immediately after each OPU session. In Experiment 2 (n=11), animals from Group 0EB did not receive EB while heifers in Groups 2EB and 5EB received 2 and 5mg of EB respectively, immediately after OPU. The OPU sessions were performed once weekly for 28d. Therefore, in both experiments, four OPU sessions were performed in heifers aspirated once a week and in Experiment 1, eight OPU sessions were done in heifers aspirated twice a week. Additionally, during the 7-d period following follicular aspiration, ovarian ultrasonography examinations were conducted to measure diameter of the largest follicle and blood samples were collected for FSH quantification by RIA. In Experiment 1, all viable oocytes recovered were in vitro matured and fertilized. Results indicated that while progestin and EB altered follicular wave patterns, this treatment did not prevent establishment of follicular dominance on the ovaries of heifers during OPU at 7-d intervals. Furthermore, the proposed stage of follicular wave synchronization strategies did not improve the number and quality of the recovered oocytes, or the number of in vitro produced embryos.

Equine chorionic gonadotropin improves the efficacy of a progestin-based fixed-time artificial insemination protocol in Nelore (Bos indicus) heifers.

A total of 177 Nelore heifers were examined by ultrasonography to determine the presence or absence of a corpus luteum (CL) and received a 3mg norgestomet ear implant plus 2mg of estradiol benzoate i.m. On Day 8, implants were removed and 150 microg of d-cloprostenol i.m. was administered. At the time of norgestomet implant removal, heifers with or without CL at the time of initiating treatment were assigned equally and by replicate to be treated with 0IU (n=87) or 400IU (n=90) eCG i.m. All heifers received 1mg of EB i.m. on Day 9 and were submitted to fixed-time artificial insemination (FTAI) 30-34h later. The addition of eCG increased the diameter of the largest follicle (LF) at FTAI (10.6+/-0.2mm vs. 9.5+/-0.2mm; P=0.003; mean+/-SEM), the final growth rate of the LF (1.14+/-0.1mm/day vs. 0.64+/-0.1mm/day; P=0.0009), ovulation rate [94.4% (85/90) vs. 73.6% (64/87); P=0.0006], the diameter of the CL at Day 15 (15.5+/-0.3mm vs. 13.8+/-0.3mm; P=0.0002), serum concentrations of progesterone 5 days after FTAI (6.6+/-1.0 ng/ml vs. 3.6+/-0.7ng/ml; P=0.0009), and pregnancy per AI [P/AI; 50.0% (45/90) vs. 36.8% (32/87); P=0.04]. The absence of a CL at the beginning of the treatment negatively influenced the P/AI [30.2% (16/53) vs. 49.2% (61/124); P=0.01]. Therefore, the presence of a CL (and/or onset of puberty) must be considered in setting up FTAI programs in heifers. In addition, eCG may be an important tool for the enhancement of follicular growth, ovulation, size and function of the subsequent CL, and pregnancy rates in progestin-based FTAI protocols in Bos indicus heifers.

Follicle deviation and ovulatory capacity in Bos indicus heifers.

The objectives of Experiment 1 were to determine the interval from ovulation to deviation, and diameter of the dominant follicle (DF) and largest subordinate follicle (SF) at deviation in Nelore (Bos indicus) heifers by two methods (observed and calculated). Heifers (n = 12) were examined ultrasonographically every 12 h from ovulation (Day 0) to Day 5. The time of deviation and diameter of the DF and largest SF at deviation did not differ (P>0.05) between observed and calculated methods. Overall, deviation occurred 2.5+/-0.2 d (mean +/- S.E.M.) after ovulation, and diameters for DF and largest SF at deviation were 6.2+/-0.2 and 5.9 +/- 0.2 mm, respectively. Experiment 2 was designed to determine the size at which the DF acquires ovulatory capacity in B. indicus heifers. Twenty-nine heifers were monitored every 24 h by ultrasonography, from ovulation until the DF reached diameters of 7.0-8.4 mm (n=9), 8.5-10.0 mm (n=10), or >10.0 mm (n=10). At that time, heifers were treated with 25 mg of pLH and monitored by ultrasonography every 12 h for 48 h. Ovulation occurred in 3 of 9, 8 of 10, and 9 of 10 heifers, respectively (P<0.05). In summary, there was no significant difference between observed and calculated methods of determining the beginning of follicle deviation. Deviation occurred 2.5 d after ovulation when the DF reached 6.2 mm, and ovulatory capacity was acquired by DF as small as 7.0 mm.