Superovulation in Cycling Mares Using Equine Follicle Stimulating Hormone (eFSH)

Superovulation would potentially increase the efficiency and decrease the cost of embryo transfer by increasing embryo collection rates. Other potential clinical applications include improving pregnancy rates from frozen semen, treatment of subfertility in stallions and mares, and induction of ovulation in transitional mares. The objective of this study was to evaluate the efficacy of purified equine follicle stimulating hormone (eFSH; Bioniche Animal Health USA, Inc., Athens, GA) in inducing superovulation in cycling mares. In the first experiment, 49 normal, cycling mares were used in a study at Colorado State University. Mares were assigned to 1 of 3 groups: group 1, controls (n = 29) and groups 2 and 3, eFSH-treated (n = 10/group). Treated mares were administered 25 mg of eFSH twice daily beginning 5 or 6 days after ovulation (group 2). Mares received 250 (of cloprostenol on the second day of eFSH treatment. Administration of eFSH continued until the majority of follicles reached a diameter of 35 mm, at which time a deslorelin implant was administered. Group 3 mares (n = 10) received 12 mg of eFSH twice daily starting on day 5 or 6. The treatment regimen was identical to that of group 2. Mares in all 3 groups were bred with semen from 1 of 4 stallions. Pregnancy status was determined at 14 to 16 days after ovulation. In experiment 2, 16 light-horse mares were used during the physiologic breeding season in Brazil. On the first cycle, mares served as controls, and on the second cycle, mares were administered 12 mg of eFSH twice daily until a majority of follicles were 35 mm in diameter, at which time human chorionic gonadotropin (hCG) was administered. Mares were inseminated on both cycles, and embryo collection attempts were performed 7 or 8 days after ovulation. Mares treated with 25 mg of eFSH developed a greater number of follicles (35 mm) and ovulated a greater number of follicles than control mares. However, the number of pregnancies obtained per mare was not different between control mares and those receiving 25 mg of eFSH twice daily. Mares treated with 12 mg of eFSH and administered either hCG or deslorelin also developed more follicles than untreated controls. Mares receiving eFSH followed by hCG ovulated a greater number of follicles than control mares, whereas the number of ovulations from mares receiving eFSH followed by deslorelin was similar to that of control mares. Pregnancy rate for mares induced to ovulate with hCG was higher than that of control mares, whereas the pregnancy rate for eFSH-treated mares induced to ovulate with deslorelin did not differ from that of the controls. Overall, 80% of mares administered eFSH had multiple ovulations compared with 10.3% of the control mares. In experiment 2, the number of large follicles was greater in the eFSH-treated cycle than the previous untreated cycle. In addition, the number of ovulations during the cycle in which mares were treated with eFSH was greater (3.6) than for the control cycle (1.0). The average number of embryos recovered per mare for the eFSH cycle (1.9 ± 0.3) was greater than the embryo recovery rate for the control cycle (0.5 ± 0.3). In summary, the highest ovulation and the highest pregnancy and embryo recovery rates were obtained after administration of 12 mg of eFSH twice daily followed by 2500 IU of hCG. Superovulation with eFSH increased pregnancy rate and embryo recovery rate and, thus, the efficiency of the embryo transfer program.

Description and genetic analysis of three sets of monozygotic twins resulting from transfers of single embryos to recipient mares

Case Description-3 sets of monozygotic twins resulting from transfers of single embryos to recipient mares were examined. Clinical Findings-In all 3 recipient mares with twin pregnancies, only 1 embryonic vesicle was detected before day 25 of gestation. In 1 recipient mare, 2 apparent adjacent vesicles, each containing an embryo with a heartbeat, were visualized on ultrasonographic examination on day 37 of gestation. The other 2 recipient mares underwent ultrasonographic examination on day 30 of gestation, at which time only 1 vesicle and embryo was identified. In these latter 2 recipient mares, however, a thorough ultrasonographic examination for a second conceptus on day 30 had not been performed, as only 1 embryo had been transferred and visualized on early ultrasonographic examination. Treatment and Outcome-All twin pregnancies resulted in death of both fetuses. Genetic analysis confirmed that each set of monozygotic twins originated from the transferred embryo. Clinical Relevance-Monozygotic twin pregnancy may occur after embryo transfer; thus recipient mares should be examined thoroughly for multiple conceptuses, especially between 25 and 30 days of gestation. At this time, the allantoides of monozygotic twins should be visible ultrasonographically and effective management may still be possible.

Doppler ultrasonography principles and methods of evaluation of the reproductive tract in mares

Background: Doppler ultrasonography is a non-invasive real time pulse-wave technique recently used for the transrectal study of the reproductive system hemodynamics in large animals. This technic is based in the Doppler Effect Principle that proposes the change in frequency of a wave for an observer (red blood cells) moving relative to the source of the respective wave (ultrasonic transducer). This method had showed to be effective and useful for the evaluation of the in vivo equine reproductive tract increasing the diagnostic, monitoring, and predictive capabilities of theriogenology in mares. However, an accurate and truthful ultrasonic exam requires the previous knowledge of the Doppler ultrasonography principles. Review: In recent years, the capabilities of ultrasound flow imaging have increased enormously. The current Doppler ultrasound machines offer three methods of evaluation that may be used simultaneously (triplex mode). In B-mode ultrasound, a linear array of transducers simultaneously scans a plane through the tissue that can be viewed as a two-dimensional gray-scale image on screen. This mode is primarily used to identify anatomically a structure for its posterior evaluation using colored ultrasound modes (Color or Spectral modes). Colored ultrasound images of flow, whether Color or Spectral modes, are essentially obtained from measurements of moving red cells. In Color mode, velocity information is presented as a color coded overlay on top of a B-mode image, while Pulsed Wave Doppler provides a measure of the changing velocity throughout the cardiac cycle and the distribution of velocities in the sample volume represented by a spectral graphic. Color images conception varies according to the Doppler Frequency that is the difference between the frequency of received echoes by moving blood red cells and wave frequency transmitted by the transducer. To produce an adequate spectral graphic it is important determine the position and size of the simple gate. Furthermore, blood flow velocity measurement is influence by the intersection angle between ultrasonic pulses and the direction of moving blood-red cells (Doppler angle). Objectively colored ultrasound exam may be done on large arteries of the reproductive tract, as uterine and ovary arteries, or directly on the target tissue (follicle, for example). Mesovarium and mesometrium attachment arteries also can be used for spectral evaluation of the equine reproductive system. Subjectively analysis of the ovarian and uterine vascular perfusion must be done directly on the corpus luteum, follicular wall and uterus (endometrium and myometrium associated), respectively. Power-flow imaging has greater sensitivity to weak blood flow and independent of the Doppler angle, improving the evaluation of vessels with small diameters and slow blood flow. Conclusion: Doppler ultrasonography principles, methods of evaluation and reproductive system anatomy have been described. This knowledge is essential for the competent equipment acquisition and precise collection and analysis of colored ultrasound images. Otherwise, the reporting of inconsistent and not reproducible findings may result in the discredit of Doppler technology ahead of the scientific veterinary community.

Ovarian response to porcine FSH in association with ablation-induced or spontaneous follicular wave development during the estrous cycle in crossbred and Brazilian Warmblood mares

The primary objective of this study was to examine the follicular and ovulatory responses following treatment with pFSH in association with ablation-induced or spontaneous follicular wave emergence or follicle deviation during diestrus in crossbred (Mangalarga × Arabian) and Brazilian Warmblood mares with a propensity for spontaneous multiple ovulations; secondary considerations were given to the collection of embryos In Experiment 1, crossbred mares were administered (im) saline (control, n= 7) or pFSH (25 mg) when the largest follicle of the ablation-induced follicular wave reached ≥13 mm (n= 7) or ≥20 mm (n= 7) or, after pre-treatment ovulation (Day 0) on Day 6 (n= 7) In Experiment 2, crossbred mares were administered (im) saline (control, n= 10) or a larger dose of pFSH (50 mg, n= 7) when the largest follicle of the ablation-induced follicular wave reached ≥13 mm In Experiment 3, Brazilian Warmblood mares were administered (im) saline (control, n= 7), pFSH (25 mg, n= 7 or 50 mg, n= 5) or EPE (12.5 mg, n= 7) as a positive control on Day 6 Ultrasonic technology was used to ablate all follicles ≥8 mm and to monitor follicular development and detect ovulation Treatment with pFSH or EPE was done twice daily until the largest follicle reached ≥32 mm; thereafter, hCG (2500 IU) was administered (iv) when the largest follicle reached ≥35 mm Artificial insemination was done 12 h after hCG and embryo collections were done 8 d after post-treatment ovulations In Experiments 1 and 2, treatment of crossbred mares with pFSH post-ablation in association with the expected time of wave emergence or follicle deviation did not (P> 0.05) enhance the follicular or ovulatory responses or collection of embryos compared to controls In Experiment 3, although the enhanced ovulatory response of mares to EPE at the expected time of spontaneous wave emergence was not different (P> 0.05) from controls, it was greater (P< 0.05) than the response to pFSH In conclusion, the novelty of using follicle ablation prior to pFSH treatment at the time of wave emergence or follicle deviation did not enhance the follicular or ovulatory responses or collection of embryos to treatment in crossbred mares In addition, the hypothesis that Brazilian Warmblood mares with a greater propensity for spontaneous multiple ovulations are as responsive to pFSH compared to EPE was not supported Thus, the combined experimental results of the present study continue to support the general consensus that pFSH is relatively ineffective for follicular superstimulation/superovulation in mares © 2012 Elsevier B.V.

Determining the gestational age of crioulo mares based on a fetal ocular measure

The practice of covering in herds is often used in Crioulo breeding farms, thus not allowing the determination of ovulation dates and the early detection of pregnancies. This fact complicates the estimation of gestational age after 70 days of conception. This study aimed to evaluate the diameter of the fetal orbits of Crioulo mares to develop a formula to estimate a gestational date. Measurements of 164 fetal ocular orbits were performed in 124 Crioulo mares with gestational ages ranging between 119 and 341 days. The current study observed a linear growth pattern of Crioulo fetal orbits up to 341 days of gestation. Thus, obtaining a suitable linear regression model to determine the gestational age of Crioulo mares based on fetal ocular orbit diameter measure is possible. The obtained model was y = 8.3756x + 11.90, where y represents the gestational age in days, and x represents the diameter of the fetal ocular orbit in millimeters. This formula had anr2 of 0.985 (P < .001). We conclude that this model allows researchers and veterinarians to accurately estimate the gestational age of Crioulo mares between 4 and 11months of pregnancy. © 2013 Elsevier Inc.

Decreasing pH of mammary gland secretions is associated with parturition and is correlated with electrolyte concentrations in prefoaling mares

The objectives of this study were to determine pH of the mammary gland secretions and the corresponding electrolyte concentrations in prefoaling mares. Pregnant mares (seven primiparous and seven multiparous) were monitored daily from 310.320 days of gestation until parturition. Prefoaling mammary gland secretions were collected, and pH was immediately determined with a pH meter and pH strip test. An aliquot of prefoaling mammary secretions was frozen and stored until further analyses. After parturition, samples from day .4 to 0 (day of foaling) were thawed and electrolyte concentrations (ie, Ca2+, Mg2+, Na+, K+ and Cl-) were determined with an automated analyser. Data were analysed via a mixed model with the mare as a random effect. Correlations were determined between pH and electrolyte concentrations by the Pearson product-moment for each pair. There was significant reduction in pH of mammary secretions on the day of foaling (P<0.0001), and most mares (11/14) with a pH .7 foaled within 24 hours. There was high correlation between the two pH methods (r=0.93). Additionally, there were significant (P<0.05) increases in Ca2+ and K+ concentrations, and significant decreases in Na+ and Cl- concentrations from one day before to the day of foaling. The pH of mammary secretions was highly and significantly (P<0.001) correlated with Na + (r=0.87), Cl- (r=0.85), Ca2+ (r=-0.88); and K+(r=.0.80) concentrations, and moderately correlated with Mg 2+ (r=-0.58). Daily evening pH measure of the mammary gland secretions can predict foaling in most mares.

Biochemical Profile of Amniotic and Allantoic Fluid During Different Gestational Phases in Mares

Fetal fluids have different vital functions that sustain both pregnancy and normal parturition. The biochemical composition of amniotic fluid during gestation is not well established; thus the purpose of the present study was to determine the biochemical profile of both amniotic and allantoic fluids from mares during initial, mid, and latter third phases of pregnancy. Samples were collected after slaughter, using allantocentesis and amniocentesis. Sixty samples of fetal fluids were analyzed. Alkaline phosphatase (AP), glucose, total protein (TP), urea, creatinine, Ca, chloride (Cl), Na, and K concentrations were measured using commercially available kits. The AP concentration in amniotic fluid was higher than that in allantoic fluid during the three gestational phases (P < .05). There were no differences between glucose mean values of allantoic and those of amniotic fluids (P < .05). However, glucose values were higher in the allantoic fluid in the last trimester of pregnancy. TP was higher in the amniotic fluid than in allantoic fluid (P < .05). Urea values varied among the phases; however, there were no differences between the amniotic and allantoic fluid values (P > .05). Creatinine values were higher in allantoic fluid (P < .05). Na and Cl concentrations were higher in amniotic fluid (P < .05). However, Ca and K concentrations were higher in the allantoic fluid. © 2013 Elsevier Inc. All rights reserved.

Efeito de dois períodos de tratamaneto com altrenogest sobre a manutenção da gestação em éguas receptoras acíclicas

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

Utilização do implante de progesterona intra-vaginal e acetato de deslorelina em éguas acíclicas associados ou não a luz artificial para o controle da sazonalidade repprodutiva

Pós-graduação em Medicina Veterinária - FMVZ

A comparison of microbiological and molecular detection of vaginal Lactobacillus spp. between mares and women

A possible explanation for endometritis in mares is ascendant contamination from the vagina. The presence of Lactobacillus spp. is considered to be important in women for a healthy vaginal environment; however, there are few studies in mares related to the presence of Lactobacillus in the vaginal flora of healthy mares. The present work aims to determine the occurrence of Lactobacillus spp. in the vaginal micro-environment of mares. A total of 35 crossbred multiparous mares, aged between 4 and 12 years, with no history of reproductive problems and with healthy reproductive tracts, were used. Two vaginal swabs were obtained from the mares during estrus for Lactobacillus isolation and PCR evaluation. Ten human female volunteers, aged between 24 and 35 years, sexually active, with no history of gynecological diseases and treatments in the past two years were used. Lactobacillus spp. were isolated from 5.7% of the mares' vaginal samples and from 90% of the women's vaginal samples. Lactobacillus DNA was detected by PCR in 22.9% of the mares' vaginal samples and in all of the vaginal samples from the healthy women. The primers used here were demonstrated to have in silico specificity for the detection of L. equi (AB425924.1), L. pantheris (DQ471798.1) and L. mucosae (DQ471799.1), but they did not anneal on Enterococcus faecalis (EU887827.1) or E. faecium (EU887814.1). In conclusion, this study showed a low occurrence of Lactobacillus spp. in mares, suggesting that this bacterium may not play a fundamental role in the equilibrium of the vaginal micro- environment of normal mares.

Supplementary corpora lutea monitoring allows progestin treatment interruption on day 70 of pregnancy in non-cyclic recipient mares

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