Dominant follicle removal by ultrasound guided transvaginal aspiration and superovulatory response in Nellore cows

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

In vivo CYP2E1 phenotyping as a new potential biomarker of occupational and experimental exposure to benzene

Assessing CYP2E1 phenotype in vivo may be important to predict individual susceptibility to those chemicals, including benzene, which are metabolically activated by this isoenzyme. Chlorzoxazone (CHZ), a specific CYP2E1 substrate, is readily hydroxylated to 6-OH-chlorzoxazone (6-OH-CHZ) by liver CYP2E1 and the metabolic ratio 6-OH-CHZ/CHZ in serum (MR) is a specific and sensitive biomarker of CYP2E1 activity in vivo in humans. We used this MR as a potential biomarker of effect in benzene-treated rats and, also, in humans occupationally exposed to low levels of benzene. Male Sprague-Dawley rats (375-400 g b.w.) were treated i.p. for 3 days with either a 0.5 ml solution of benzene (5 mmol/kg b.w.) in corn oil, or 0.5 ml corn oil alone. Twenty-four hours after the last injection, a polyethylene glycol (PEG) solution of CHZ (20 mg/kg b.w.) was injected i.p. in both treated and control animals. After 2, 5.10,15, 20,30,45, 60, 90, 120, 180, and 240 min from injection, 0.2 ml blood was taken from the tip tail and stored at -20 degrees C until analysis. A modified reverse phase HPLC method using a 5 mu m Ultrasphere C18 column equipped with a direct-connection ODS guard column, was used to measure CHZ and its metabolite 6-OH-CHZ in serum. No statistically significant difference in the MR was observed, at any sampling time, between benzene-treated and control rats. The concentration-versus-time area under the curve (AUC), however, was lower (p < 0.05, Mann-Whitney test), whereas the systemic clearance was higher (p < 0.05) in treated than in control rats. Eleven petrochemical workers occupationally exposed to low levels of airborne benzene (mean +/- SD, 25.0 +/- 24.4 mu g/m(3)) and 13 non-exposed controls from the same factory (mean +/- SD, 6.7 +/- 4.0 mu g/m(3)) signed an informed consent form and were administered 500 mg CHZ p.o. Two hours later a venous blood sample was taken for CHZ and 6-OH-CHZ measurements. Despite exposed subjects showed significantly higher levels of t,t-MA and S-PMA, two biomarkers of exposure to benzene, than non-exposed workers, no difference in the MR mean values +/- SD was found between exposed (0.59 +/- 0.29) and non-exposed (0.57 +/- 0.23) subjects. So, benzene was found to modify CHZ disposition, but not CYP2E1 phenotype in benzene-treated rats, nor in workers exposed to benzene, probably due to the levels of exposure being too low. (C) 2009 Elsevier B.V. All rights reserved.

First detection of the equine herpesvirus 1 neuropathogenic variant in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The use of transmission electron microscopy and oocyte transfer to evaluate in vitro maturation of equine oocytes in different culture conditions

The current study evaluates the ability of equine oocytes matured in different conditions to undergo nuclear and cytoplasmic maturation.. After oocyte transfer, embryonic development was diagnosed at 1.5 and 90 days of gestation. For each group, immature oocytes obtained from slaughterhouse ovaries were matured in vitro (5 replicates). In experiment I, three different media were tested. HTF:BME, SOFaa, and TCM 199. In experiment 11, the HTF:BME was chosen as maturation medium containing pFSH, eFSH, or eFSH + eGH. Nuclear maturation was estimated after stripping the oocytes and staining with Hoechst 33342. The evaluation of cytoplasmic maturation was performed by transmission electron microscopy. For oocyte transfer, six non-cycling recipient mares were used, and 8 to 15 oocytes were transferred in each mare. In experiment I, the results showed no differences (P > .05) in nuclear maturation (MII) among experimental groups. The percentage of MII was 29.3 ( +/- 9.6), 23.4 ( +/- 8.4), and 13.5 ( +/- 12.4) for HTF:BME, SOF, and TCM, respectively. In experiment II, all media tested were efficient in inducing metaphase II. Also, no statistical differences (P > .05) were observed in percentages of nuclear maturation rates when porcine (37.1 +/- 22.4) or equine (25.8 +/- 8.2) FSH were used, or when eFSH + eGH was added to HTF:BME (29.4 +/- 12.3). The analysis of cytoplasmic morphology of oocytes cultured in TCM 199 and SOFaa showed signs of incomplete cytoplasmic maturation and premature cortical reaction. Meanwhile, oocytes cultured in HTF:BME medium presented cytoplasmic characteristics similar to those described by others for in vivo-matured oocytes. The addition of eFSH to the HTF:BME medium resulted in an improvement of cytoplasmic morphology. After oocyte transfer, two mares became pregnant, one from pFSH group and one from eFSH+eGH group. These results indicate that although in vitro matured equine oocytes are capable of fertilization and embryonic development, the percentage of competent oocytes is still low.

Induction of luteolysis in mares utilizing a micro-dose of prostaglandin F-2 alpha in the sacral lumbar space

An experiment was conducted to examine the luteolytic effectiveness of using low or micro doses of PGF(2 alpha) when administered at the (BAI HUI) acupuncture point, which is frequently used to treat ovarian disturbances in Veterinary Acupuncture. The results indicate that PGF(2 alpha) given at a very low micro dose of 0.5mg (one tenth the conventional recommended dose) when administered at the BAI HUI acupuncture point located at the sacral lumbar space is equally effective at inducing luteolysis in mid-luteal phase mares as conventional PGF(2 alpha) i.m. treatment using a tenfold higher dose. Therefore, based on the results of the present study we suggest that the BAI HUI sacral lumbar route somehow provides an extremely efficient pathway for the drug to the ovarian level.

The efficacy of a single chain recombinant equine luteinizing hormone (reLH) in mares: Induction of ovulation, hormone profiles, and inter-ovulatory intervals

The objectives of this study were to determine the efficacy of recombinant equine luteinizing hormone (reLH) in shortening the time to ovulation in cycling mares and to determine the effects of treatment on endogenous hormones and inter-ovulatory intervals. In study 1, mares of light horse breeds (3-20 years) were treated with either a vehicle, various doses of reLH, or human chorionic gonadotropin (hCG). Cycling mares were examined by palpation and ultrasound per rectum daily or every 12 h from the time of treatment to ovulation. In studies 2 and 3, jugular blood samples were collected daily or every 12 h from the time of treatment to ovulation for analysis of LH, follicle stimulating hormone (FSH), estradiol-17 beta (E-2), and progesterone (P-4) by radioimmunoassays (RIA). Increasing doses of reLH (0.3, 0.6, 0.75, and 0.9 mg) showed increasing effectiveness at inducing ovulation within 48 h of treatment. Treatments with the 0.75 and 0.9 mg doses of reLH resulted in 90% and 80% ovulation rates, which were similar to hCG treatment (85.7%). Except for the early rise in LH after treatment with 0.5, 0.65, and 1.0 mg of reLH, hormone profiles appeared to be similar between control and treated cycles. Inter-ovulatory intervals were similar between control and treatment cycles. In conclusion, reLH is a reliable and effective ovulatory agent that does not significantly alter endogenous hormone profiles or affect inter-ovulatory intervals.(c) 2007 Elsevier B.V. All rights reserved.

Ultrasonographic evaluation of the conceptus from days 10 to 60 of pregnancy in jennies

Daily ultrasound examinations were conducted from Days 10 to 60 (ovulation = Day 0) of pregnancy to monitor the conceptus in jennies (n = 12). The embryonic vesicle was first detected on Day 11.5 +/- 0.9 (mean +/- SD; range 10 to 13d) and was mobile until movement ceased (fixation) on Day 15.5 +/- 1.4 (range, 13 to 18d). The vesicle was spherical from Days 10 to 18 (mean growth rate, 3.2 mm/d), non spherical (irregular) with a reduced growth rate (0.5 mm/d) from Days 19 to 29, and then grew at a moderate rate (1.6 mm/d) up to Day 46. on average, detection of the embryo proper (consistently located on the ventral aspect of the yolk sac) and embryonic heartbeat were Days 20.7 +/- 1.2 and 23.5 +/- 1.3, respectively. Formation of the allantoic sac was first detected on Day 24.4 +/- 1.7 and was complete on Day 36.8 +/- 1.6. Descent of the fetus (and formation of the umbilical cord) began on Day 37.9 +/- 1.7 and was complete on Day 44.1 +/- 2.1. Crown-ramp length averaged 3.7, 15.4, 22.7, 37.5 and 59.6 mm on Days 20, 30, 40, 50 and 60, respectively. In general, morphologic features and dates of occurrence were similar to those reported previously in the mare. (C) 1998 by Elsevier B.V.

Induction of double ovulation in mares using deslorelin acetate

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Aspiration of equine oocytes from immature follicles after treatment with equine pituitary extract (EPE) alone or in combination with hCG

This study examined the effect of treating mares with equine pituitary extract (EPE) alone or in combination with hCG on the recovery rate of immature follicles by transvaginal follicular aspiration (ovum pick-up; OPU). Ten normally cycling crossbred mares aged 3-15 years and weighing 350-400 kg were subjected to each of three treatments in a random sequence with each exposure to a new treatment separated by a rest cycle during which a spontaneous ovulation occurred. The treatments were (1) superovulated with 25 mg EPE and treated with 2500 IU hCG, (2) superovulation with 25 mg EPE, and (3) control (no exogenous treatment). Treatments 7 days after spontaneous ovulation; and all the follicles > 10 mm were aspirated 24 h after the largest follicle achieved a diameter of 27-30 mm for control group, and most follicles reached 22-27 mm for the EPE alone treatment. To the group EPE+hCG, when the follicles reached 22-27 mm, hCG was administered, 24 h before OPU. Superovulation increased the number of follicles available for aspiration. The total number of follicles available for aspiration was 61 in the EPE/hCG group. 63 in the EPE group and 42 in the control. The proportion of follicles aspirated varied from 63.5% to 73.8%. Oocyte recovery rate ranged from 15.0% to 16.7% and the proportion of mares that yielded at least one oocyte was 70% (7/10) in the EPE/hCG, 60% (6/10) in the EPE alone and 50% (5/10) in control group. The EPE/hCG treatment had a higher proportion of follicles with expanded granulose cells (64.4%) than the control (3.3%: p < 0.05) and the EPE treatment (25.0%). The intervals from spontaneous ovulation to aspiration were similar for all treatments (11-12 days). However, superovulatory treatment significantly increased the aspiration to ovulation interval from 15 +/- 4 days for control to 27 +/- 15 days for EPE (p < 0.05) and to 23 +/- 13 days for EPE/hCG treatment with commensurate increases in the time between spontaneous ovulations. (c) 2008 Elsevier B.V. All rights reserved.

Improvement of embryo production by the replacement of the last two doses of porcine follicle-stimulating hormone with equine chorionic gonadotropin in Sindhi donors

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Use of a Low Dose of Equine Purified FSH to Induce Multiple Ovulations in Mares

The effects of a low dose of equine purified FSH (eFSH) on incidence of multiple ovulations and embryo recovery rate in mares were studied. During the physiological breeding season in Brazil (19 degrees 45'45'S), 14 Mangalarga Marchador donor mares were used in a crossover study and another 25 mares of the same breed, between 3 years and 12 years of age were used as recipients for the embryo transfers. Donors were monitored during two consecutive oestrus cycles, an untreated control cycle followed by a treated cycle, when eFSH was administered. In both cycles, after an embryo collection attempt on day 8 post-ovulation all mares received 7.5 mg dinoprost and had their two largest follicles tracked daily by ultrasonography until the period of ovulation. Mares were inseminated every 48 h with extended fresh semen from a single stallion after the identification of a 35-mm follicle until the period of ovulation. Ovulations were induced by intravenous administration of 2.500 IU of human chorionic gonadotropin, upon detection of a 35- to 40-mm follicle. In the treated cycle, 5 mg eFSH was given intramuscularly once a day, from day 8 post previous ovulation until at least one follicle reached 35 mm in diameter. Embryo flushes were performed on day 8 of dioestrus (day 0 = ovulation). Treatment with eFSH resulted in higher (p < 0.05) ovulation rate and incidence of multiple ovulations compared to the control (1.6 vs 1.0 and 50% vs 0%, respectively - one mare had triple ovulation). However, embryo recovery rates in the control and treated cycles were similar (0.8 and 1.0, respectively; p > 0.05). Pregnancy rates in the recipient mares following embryo transfer were similar for the control and eFSH cycles (11/11 and 10/14, respectively). Additional studies are necessary in order to develop a low-dose protocol for the use of eFSH that brings a more consistent contribution to the efficiency of commercial equine embryo transfer programs.

Embryo transfer in Bos indicus cattle

In the present short review superovulation treatments commonly used for Bos taurus and/or Bos indicus will be addressed with emphasis in recent superstimulation protocols associated with pharmacological manipulation of the follicular dynamics to improve donor management and potentially embryo yield. Results obtained after superovulation treatments in which the time of LH surge is selectively delayed as an attempt to improve embryo yield are presented and discussed. (C) 2001 by Elsevier B.V.

Use of knowledge regarding LH receptors to improve superstimulatory treatments in cattle

Embryo transfer is a biotechnology that has been used worldwide to increase the production of offspring from female bovines. Treatments to induce multiple ovulations (superovulation) have evolved from superstimulatory protocols that depended upon detection of oestrus to treatments that synchronise follicle growth and ovulation, allowing for improved donor management and fixed-timed AI (FTAI). The protocols associated with FTAI facilitate animal handling and produce at least as many viably embryos as conventional treatment protocols that required detection of oestrus. Recent knowledge regarding LH receptors (LHR) and follicular development can be applied to improve embryo transfer protocols. In fact, improvements in the superstimulatory treatment called the 'P-36 protocol', which include hormones that stimulate LHR, indicate that adjustments related to LHR availability may increase bovine embryo yield compared with conventional protocols based on the detection of oestrus.

Ovulation synchronization following commercial application of ultrasound-guided follicle ablation during the estrous cycle in mares

A regimen of progesterone plus estradiol (P&E) was used as a standard for ovarian synchronization to test the efficacy and evaluate the commercial application of ultrasound-guided follicle ablation as a non-steroidal alternative for ovulation synchronization in mares. Recipient mares at a private embryo transfer facility were at unknown stages of the estrous cycle at the start of the experiment on Day 1 when they were randomly assigned to an ablation group (n = 18-21 mares) or to a ME group (n = 20-21 mares). In the ablation group, mares were lightly sedated and all follicles > 10 mm were removed by transvaginal ultrasound-guided follicle aspiration. In the ME group, a combination of progesterone (150 mg) plus estradiol (10 mg) prepared in safflower oil was given daily (im) for 10 d. Two doses of prostaglandin FZ, (PGF, 10 mg/dose, im) were given 12 h apart on Day 5 in the ablation group, or a single dose on Day 10 in the ME group. Human chorionic gonadotropin (hCG, 2500 IU/mare, im) was given at a fixed time, 6 and 10 d after PGF treatment in the ablation and ME groups, respectively, with the expectation of a follicle > 30 mm at the time of treatment. In both the ablation and P&E groups, transrectal ultrasonography was done at the start of the study (Day 1) and again on the day of hCG treatment and daily thereafter to determine the presence of a CL, measure diameter of the largest follicle and detect ovulation. The mean interval from the start of the study and from PGF treatment to ovulation was shorter (P < 0.0001) in the ablation group (13.7 and 9.7 d, respectively) compared to the P&E group (22.3 and 13.2 d, respectively). Following fixed-day treatment with hCG after PGF treatment, the degree of ovulation synchronization was not different (P > 0.05) between the ablation and P&E groups within a 2-d (56 and 70%) or 4-d (83% and 90%) period. Although ultrasound-guided follicle ablation may not be practical in all circumstances, it excluded the conventional 10-d regimen of progesterone and estradiol and was considered an efficacious and feasible, non-steroidal alternative for ovulation synchronization in mares during the estrous cycle. (C 2007 Elsevier B.V. All rights reserved.

Cholangiocarcinoma and Squamous Cell Carcinoma of the Stratified Epithelial Portion of the Stomach in a Horse: A Case Report

Both cholangiocarcinoma and squamous cell carcinoma of the stomach stratified squamous epithelial portion are rarely found in equine medicine despite being more common in human beings, dogs, and cats. The objective of the present article was to report the simultaneous occurrences of these two types of neoplasias in an 11-year-old mare. Numerous firm, whitish nodules were distributed throughout the liver parenchyma and those protruding over its surface were umbilicated in their appearance. It was verified that the nodules adhered to the peritoneum and omentum, diaphragm, spleen, and stomach serosa compressed the adjacent structures. The stomach stratified squamous epithelial portion, particularly originating in the margo plicatus toward the cardia, was covered by numerous smooth, whitish spherules. Microscopic examination allowed the liver, tumor, and the abdominal implants to be identified as a cholangiocarcinoma, and the stomach neoplasia as a carcinoma of its stratified squamous epithelial portion. Considering this as an uncommon finding, although when considered individually, the presence of a cholangiocarcinoma and a squamous cell carcinoma of the stratified squamous epithelial portion of the equine stomach in the same specimen is worthy of reporting. (C) 2011 Elsevier B.V. All rights reserved.