Embryonic Vesicle Research Articles

129 EFFECT OF DEVELOPMENTAL STAGE OF ICSI-PRODUCED EQUINE EMBRYOS ON PREGNANCY RATES

Intracytoplasmic sperm injection (ICSI) can be used to produce offspring from mares or stallions with fertility problems. Early embryos can be transferred into recipients’ oviducts or embryos can be cultured for nonsurgical transfer into recipients’ uteri. The aim of this research was to evaluate the optimal time to transfer ICSI-produced embryos into recipients’ uteri. The objective was to compare pregnancy rates after the nonsurgical transfer of early morulae, compact morulae, and blastocysts. Oocytes were collected by transvaginal, ultrasound-guided follicular aspirations between 20 and 24 h after administration of deslorelin (1.5 mg, i.v., Francks Pharmacy, Ocala, FL, USA) to donors. Oocytes were cultured for 16 to 18 h in M199 (Invitrogen, San Jose, CA, USA) with 10% FCS (HyClone, Logan, UT, USA), 0.2 mm pyruvate (Sigma, St. Louis, MO, USA) and 25 μg mL–1 gentamycin (Sigma, St. Louis, MO, USA) at 38.5°C and 6% CO2. Cumulus cells were denuded by gentle pipetting, after oocytes were placed into hyaluronidase (500 U mg–1, Sigma, St. Louis, MO, USA). Oocytes were injected with a single sperm from one of two stallions, with sperm being frozen or sex-sorted and refrozen (Squires EL et al. 2008). Forty-six of 62 (74%) injected oocytes cleaved. The presumptive zygotes were cultured in DMEM/F12 (Sigma, St. Louis, MO, USA) with 10% FCS at 38.5°C and an atmosphere of 5% CO2, 5% O2 and 90% N2. Embryos were placed in fresh medium every 3 days. Injected oocytes were observed for cleavage at 2 days, and embryos were assigned to a transfer group. Embryos were transferred as early morulae (EM, 8-cell to precompaction stage, n = 14), compact morulae (CM, postcompaction, n = 10) or blastocysts (B, observed blastocoele, n = 9) into recipients at 3 to 5 days (EM), 3 to 6 days (CM) or 5 to 6 days (B) after the recipient’s ovulation or follicle aspiration. Pregnancy scans were performed on Day 12, 14, and 16 after ICSI, and pregnant recipients were examined until 30 days to detect the embryo proper and heartbeat. Number of embryonic vesicles detected per transferred embryo was determined by Fisher’s Exact Test. Pregnancy rates differed (P = 0.0017) among groups (EM, 1/14, 7%; CM, 4/10, 40%; B, 7/9, 78%), with fewer (P = 0.001) EM than B resulting in embryonic vesicles; however, pregnancy rates were not significantly different between CM and other embryo stages. An embryo proper with heartbeat was observed for all pregnancies, with the exception of one pregnancy resulting from the transfer of a blastocyst. In this study, all blastocysts were transferred prior to the embryo attempting to hatch from the zona pellucida, but after the appearance of a distinct blastoceole. In our study, pregnancy rates were higher after the transfer of later v. earlier stages of embryo development.

Early effects of equine FSH (eFSH) treatment on hormonal and reproductive parameters in mares intended to carry their own pregnancy

Superovulatory treatment may potentially increase the embryo recovery rate and the per-cycle pregnancy rate in normal or subfertile mares that are managed properly. However, some studies suggest a possible negative effect of superovulatory treatment on ovarian follicular maturation and embryo viability. Objectives of the present study were to investigate the early effects of eFSH treatment in reproductively normal mares in terms of: folliculogenesis, pregnancy rate, early embryonic development, reproductive tract parameters (tone and edema), and serum estradiol-17β and progesterone concentrations. Reproductively sound mares ( n = 26) were evaluated daily by transrectal palpation and ultrasonography. Five days after spontaneous ovulation, mares were randomly assigned to one of two treatment groups. In the eFSH group, mares ( n = 16 estrous cycles) were administered eFSH twice daily; beginning when a follicle ≥20 mm was detected, and continuing until at least one follicle reached a diameter of ≥35 mm. PGF2α was administered 2 days following initiation of eFSH therapy, and hCG was administered approximately 36 h after cessation of eFSH therapy. In the control group, mares ( n = 26 estrous cycles) were administered PGF2α 7 days after spontaneous ovulation, and hCG when a follicle ≥35 mm was detected. All mares were bred with fresh semen, monitored for ovulation (Day 0), and evaluated for pregnancy on Days 11–16. Serum estradiol-17β and progesterone concentrations were analyzed using radioimmunoassay on the Day of hCG administration, and Days 8, 11 and 16. Mares treated with eFSH had more follicles ≥30 mm at the time of hCG administration (2.6 ± 0.4 compared with 1.1 ± 0.1; P < 0.01), and more ovulations (2.3 ± 0.5 compared with 1.1 ± 0.3; P < 0.01). However, pregnancy rates were not significantly different between groups (50%; 8/16 compared with 62%; 16/26). Mean overall daily growth rate of embryonic vesicles from Day 11 to 16 was not statistically different between the two groups (3.3 ± 0.3 compared with 3.7 ± 0.1 mm/day) ( P = 0.2); however, was more variable ( P < 0.01) in the eFSH group (95%CI: 2.6–3.8 mm/day) than in the control group (95%CI: 3.5–3.9 mm/day). Administration of eFSH modified the reproductive tract variables and serum concentrations of progesterone and estradiol-17β on the days that oocyte maturation, fertilization, and early embryonic development are expected to occur. These alterations may be related to the greater incidence of non-ovulatory follicles (25% compared with 0%), fewer embryos per ovulation rate (0.3 ± 0.1 compared with 0.6 ± 0.1), and the lesser than expected pregnancy rates in the eFSH-treated mares.

Early pregnancy diagnosis and embryonic and fetal development using real time B mode ultrasound in goats

Real time B mode ultrasound scanning was used for the diagnosis of early pregnancy and monitoring of embryonic and fetal development in goats. Estrus in 8 cyclic cross-bred does was synchronized using a single injection of PGF2α followed by mating with a buck. All the does were subjected to weekly transrectal (from day 14 of mating) and transabdominal ultrasonography (from day 28) up to the 4th month of gestation and once during the 5th month of gestation. Of the 8 animals, 6 does were confirmed pregnant. The embryonic vesicle was detected on day 21 and day 28 and the embryo proper on day 28, day 35 using the transrectal and transabdominal methods, respectively. Heart beat was observed as early as day 21 of gestation and it was recordable by day 28 using the transrectal approach. However, via transabdominal scanning the detection and recording of the heart beat was possible only on day 35 of gestation. Singles and twins were differentiated on day 35 and day 42 by the transrectal and transabdominal approaches, respectively. Placentomes as a circular ‘C’ shape structure was detectable on day 42 with an average diameter of 0.97 cm, using the rectal probe. Using the abdominal probe, placentomes were detectable on day 50 (average diameter of 1 cm of gestation). Skeletal structures such as the skull, rib cage and vertebral column were first viewed on day 56 in both approaches. A significant ( P < 0.001) high correlation ( r = 0.99) was recorded between the gestational age and head, thoracic and placentome parameters. It may concluded that the earliest identification of an embryonic vesicle (day 21) by transrectal scanning and confirmation of its viability by fetal heart beat (day 28) were the most reliable ultrasonographic features for early pregnancy diagnosis in goats. Transabdominal ultrasonographic measurements of placentome, head and thoracic diameter may be of use in the assessment of fetal age.

Estimation of gestational age in Egyptian native goats by ultrasonographic fetometry

The main aim of the present study was to estimate the gestational age of Egyptian goats by B-mode ultrasound measurement of embryonic or fetal parts throughout pregnancy. Trans-rectal (TR) ultrasonography (7 MHz) was carried out on 15 pregnant Egyptian does at Day 10 post mating on alternate days until Day 25 and then once at 3–5-day intervals until Day 50. Trans-abdominal (TA) ultrasonography (3.5–5 MHz) was carried out on the same animals from Days 25 to 130 at 3–5-day intervals. After imaging the embryo or the fetus, the following parameters were measured: length of the embryo or fetus (CRL), heart rate (FHR), biparital diameter (BPD), trunk diameter (TD), placentome size (PS), umbilical cord diameter (UCD) and femur length (FL). The average of days at which the embryonic vesicle was first determined by TR and TA ultrasonography was 16.98 ± 1.97 and 27.87 ± 3.48, respectively. The embryo proper with a beating heart was first determined by TR and TA ultrasonography at an average of 22.36 ± 2.66 and 30.36 ± 4.75 days, respectively. All the fetal measures were significantly ( P < 0.0001) correlated with the gestational age. With the exception of fetal heart rate ( R 2 = 0.551), all the measured fetal structures were highly correlated ( R 2 ≥ 90) with the gestational age. In conclusion, the age of embryo or fetus in Egyptian does can be estimated by ultrasound measuring the crown rump length, biparital diameter, trunk diameter, placentome size, umbilical cord diameter and femur length.

In vitro and in vivo developmental competence of dromedary ( Camelus dromedarius) oocytes following in vitro fertilization or parthenogenetic activation

Parthenogenetic activation of the oocyte represents an important step in the somatic cloning. The aim of the present study was to evaluate the effectiveness (in term of in vitro development) of different methods of parthenogenetic activation of dromedary oocytes. Selected cumulus–oocytes-complexes ( n = 1264) collected by follicular aspiration from ovaries obtained postmortem were matured in vitro (IVM) for 30 h then divided randomly into seven groups and submitted to artificial activation. Two groups were preactivated with 25 μM of calcium ionophore (CaI) for 20 min then incubated for 4 h with either 2 mM 6-dimethylaminopurine (6-DMAP) (group 1, n = 202) or with 10 μg/mL cycloheximide (CHX) (group 2, n = 194). Group 3 ( n = 172) and group 4 ( n = 184), oocytes were pretreated with 5 μM ionomycin (Iono) for 5 min then incubated with either 2 mM 6-DMAP or 10 μg/mL cycloheximide for 4 h, respectively. Group 5 ( n = 161) and group 6 ( n = 155) oocytes were preactivated with electrical stimulation (ES) then activated with either 2 mM 6-DMAP or 10 μg/mL cycloheximide for 4 h, respectively. Group 7 ( n = 196) oocytes were submitted to in vitro fertilization (IVF) and served as a control. All groups containing oocytes were cultured in vitro following activation or IVF, at 38.5 °C under 5% CO 2 in air with >95% humidity. The in vitro development rates of dromedary oocytes exposed to 6-DMAP after CaI (61%), ES (74%) and the IVF group (71%) were similar and significantly greater ( P < 0.05) than other treatments (10% for group 2, 47% for group 3, 27% for group 4 and 41% for group 6). The blastocyst developmental rate was better ( P < 0.05) in parthenotes following activation with Iono/6-DMAP (21%) compared to activation with Iono/CHX (12%). However, all were less than that achieved in the IVF group (35%). We conclude that parthenogenetic activation of camel oocytes with 6-DMAP is more effective than activation with CHX for all pre-treatments tested (CaI, Iono or ES). The viability of activated ( n = 15) or IVF ( n = 10) hatched-dromedary embryos was examined by transfer to synchronized recipients. An embryonic vesicle was seen by ultrasonography at 15 days post transfer in four females (CaI/6-DMAP: 1/5; 20%, IVF: 3/10; 30%). The only pseudopregnancy obtained with an activated embryo resorbed at 25 days. One of the females receiving the IVF produced embryos aborted at 2 months and the other two females carried to term and gave birth to healthy calves (one female and one male). This study shows that artificial activation of dromedary oocytes with CaI/6-DMAP or ES/6-DMAP is more effective than other treatments in terms of in vitro embryo development. This provides efficient activation conditions which may lead to the development of the somatic cell nuclear transfer procedure in dromedary.

Pregnancy rates and semen usage in rectally guided and endoscopic inseminations

Reducing sperm numbers in the inseminate increases the number of mares bred per ejaculate, maximizes the use of frozen semen from dead stallions, increases the fertility of some stallions with sub-optimal semen quality, aids in the use of epididymal sperm harvested from dead stallions and reduces the incidence of post-breeding endometritis in mares with delayed uterine clearance. Two techniques have been used for reducing sperm numbers in the inseminate: (1) rectally guided insemination (RGI) and (2) hysteroscopic insemination (HI). The objective of this study was to analyze frozen semen data from three breeding seasons in a large commercial farm in order to compare the two insemination techniques, RGI or HI. Semen frozen in 0.5-ml straws and concentrations of 50 to 150× 106 per straw was available from 88 stallions whose fertility with frozen semen was between 0 and 100%; 2544 inseminations were performed. Fertility was assessed by embryo recovery on days 7 or 8 post ovulation in 48% of the mares or by the ultrasonographic presence of an embryonic vesicle at 14 days post insemination in 51% of the mares. RGI (n= 1279) resulted in 43% fertility rate and 5.1 straws/pregnancy. Fertility of the mares bred by the HI technique was 45%. Although there was a consistent advantage across years for the HI technique over the RGI, the difference was not statistically significant (p> 0.05). However, there was a significant difference (p< 0.05) in the number of straws used per cycle 2.2 and 1.4 for RGI and HI respectively resulting in a significant reduction in the number of straws required to establish a pregnancy 5.1 vs 3.1 straws/preg for RGI and HI respectively). Although it would appear that the HI results in a higher fertility and requires approximately 40% less semen to establish a pregnancy than the RGI technique, it is difficult to justify the routine use of HI considering the additional expense in personnel and equipment needed to conduct the procedure. Our results indicate that although there is a slight and consistent advantage of the HI technique, veterinarians must weigh the advantages and disadvantages as well as semen availability before deciding what technique to use. However, if the volume is ≤250 l, it is strongly suggested to deposit the semen by HI. The results of this analysis agree with previous reports in which the HI resulted in a 4% increase in fertility compared to the RGI procedure. In conclusion, the two techniques described in the present study are comparable and can result in the establishment of pregnancies on a commercial basis. It is clear that the two techniques are a viable option to maximize the use of frozen semen, and with proper equipment, technique and skills the hysteroscopic insemination technique further maximizes the use of semen. This could prove to be economically beneficial when using semen of very valuable or deceased stallions.

Non-invasive ultrasonographic characterization of phenotypic changes during embryo development in non-anesthetized mice of different genotypes

This study aimed to determine phenotypic changes during embryo development in the mouse, and the influence of genetic background, by non-invasive real-time ultrasonography. Serial scannings were performed from Day 4.5 after the appearance of the vaginal plug in a total of 34 adult mice of different strains (9BALB/c, 10C57BL/6 and 15 CD1). Embryonic vesicle diameter was measurable from Day 4.5 of pregnancy. Images of embryos were obtainable from Day 5.5 of gestation onwards, and crown-rump length and trunk parameters (diameter and area) were measured. At more advanced stages, the positions of fetuses prevented accurate measurement of crown-rump length; therefore, head diameters (occipito-snout length and biparietal diameter) were used as an alternative index of fetal size. All measurements correlated highly with gestational age (P<0.0005). No significant differences were observed between genotypes in early pregnancy, but during the last week of gestation trunk measurements were larger in CD1 embryos (P<0.05) while head diameters were larger in C57BL/6 conceptuses (P<0.05). There was a significant effect of genetic background on heart rate throughout pregnancy; although heart rate was similar in CD1 and C57BL/6 embryos (154.5+/-2.8 and 147.8+/-4.5 beats/min, respectively), it was significantly lower in BALB/c mice (127.0+/-2.1; P<0.005 vs. CD1 and C57BL/6).

Effect of exogenous insulin administration on ovarian function, embryo/fetal development during pregnancy in goats

The effect of insulin was investigated on ovarian follicle population, ovulation rate, hormonal profiles and embryo/fetal development during pregnancy using transrectal ultrasonography in goats. Twelve goats synchronized in estrus were selected for the experiment. They were divided into two groups, viz. (untreated control, n = 6) and (insulin treated, n = 6). In treated group long acting bovine insulin was administered @ 0.2 IU/kg body weight subcutaneously for three consecutive days, i.e. days 7–9 of estrous cycle. Thereafter, weekly single injection of insulin was continued for rest of the experiment. However, in control group only normal saline was injected as placebo. Breeding was allowed by natural service in both the groups. The does were subjected to B-mode transrectal ultrasound scanning of ovary and uterus weekly up to 120 and 98 days of gestation, respectively. Blood samples were collected weekly up to 135 days of gestation for the estimation of estradiol 17β and progesterone (P4). The result revealed no difference in mean number of total follicles between the control and insulin treated groups. The diameter of medium follicle did not differ where as diameter of large follicle was comparatively higher in treated than control goats. The average number of corpus luteum (CL) was higher in insulin treated group as compared to control (1.66 vs. 1.16). However, the number as well as mean diameter of CL did not differ significantly between treated and control group. Serum concentrations of estradiol 17β and progesterone were significantly ( P < 0.01) higher in treated than control goats. Embryonic vesicle was detected by day 21 in both the groups, however, its diameter did not differ significantly (0.73 and 0.72 cm) between the groups. The twinning percentage was higher (50 vs. 16%) in insulin treated than the control goats. Placentome diameter was also higher ( P > 0.05) in treated animals. The results demonstrated beneficial effect of exogenous administration of insulin on ovarian function and twinning percentage in goats.

AVALIAÇÃO ULTRA–SONOGRÁFICA DO DESENVOLVIMENTO EMBRIONÁRIO-FETAL DE OVINOS DA RAÇA SANTA INÊS

This study was evaluated, by transretal ultrasound, the embryonic-fetal development from days 15th to 45th of gestation visualizing the first signs of gestation in 60 Sant Ines ewes. The identification of the earliest and the latest parameters of evaluation were performed between days 15th to 19th (16.7 ± 1,3) of pregnancy for intra-uterine fluid, . embryonic vesicle between days 16th to 22th (18.6 ± 1.4), embryo between 18th to 26th (22.8 ± 1.9), placentoms between 20th to 29th (25.1 ± 2.0), heartbeat between days 24th to 29th (25.9 ± 1.4), amniotic membrane between 24th to 32th (27.4 ± 1.8), head and upper body differentiation between 30th to 37th (33.4 ± 2.2), fetus movement between 30th to 38th (34.2 ± 2.0), umbilical cord between 32th to 39th (35.1 ± 1.5), button of the anterior and posterior members between 34th to 39th (36.7 ± 1.5) and ocular globe between days 39th to 43th (40.9 ± 1.2). It was concluded that the first signs of gestation can be identified as soon as day 15th days of gestation. However, it is wise diagnosing pregnancy after day 24th, when it is possible to visualize the fetus and the heart beat.

Early embryo development in the elephant assessed by serial ultrasound examinations

The elephant has an extraordinary long pregnancy, lasting 21 months. However, knowledge on embryo development is limited. To date, only single morphological observations of elephant embryo development associated with placentation are available, all lacking correlation to gestational age. The present study describes morphological characteristics of early embryo development in the elephant with exact biometric staging. Six pregnancies in five Asian and one African elephants with known conception dates were followed by 2D and 3D ultrasound, covering the embryonic period from ovulation to day 116 post-ovulation. The embryonic vesicle was earliest observed was on day 50 p.o. The proper embryo was not detected until day 62 p.o. Embryonic heartbeat was first observed on day 71 p.o. The allantois, which became visible as a single sacculation on day 71 p.o. was subdivided in four compartments on day 76 p.o. By day 95 p.o., head, rump, front and hind legs were clearly distinguished. Between days 95 and 103 p.o. the choriovitelline placenta was replaced by the chorioallantoic placenta. A physiological midgut herniation was transiently present between days 95 and 116 p.o. On the basis of the late appearance of the embryonic vesicle, delayed implantation in the elephant is discussed. The study provides a coherent description of elephant embryonic development, formation of the extraembryonic organs and their role in placenta formation, all of which are of interest for both comparative evolutionary studies and the improvement of assisted reproduction techniques.

Intrauterine Growth Retardation in Endothelial Nitric Oxide Synthase-Deficient Mice Is Established from Early Stages of Pregnancy1

The current study aimed to determine effects of deficiencies in nitric oxide synthase (NOS) 3 on embryo and fetal development by in vivo, noninvasive, real-time ultrasonographic assessment of phenotypic changes in Nos3-knockout pregnant mice and their wild-type counterparts. From Day 4.5 of pregnancy onwards, embryonic vesicle diameters, crown-rump lengths, and trunk diameters were obtained by serial scanning of seven adult pregnant female mice, strain B6.129P2-Nos3(tm1Unc)/J, N9 generation backcrossing with C57BL/6J mice, homozygous for the disruption of the endothelial NOS gene (group Nos3(-/-)), and 12 pregnant, wild-type C57BL/6J mice (group Nos3(+/+)). All the measurements increased in both genotypes throughout gestation. However, embryo length and width were significantly larger in Nos3(+/+) than in Nos3(-/-) mice from Day 8.5, and both longitudinal and transverse diameters of the entire gestational sacs were larger in Nos3(+/+) mice from Day 10.5. Assessment of the relative growth of embryos/fetuses and gestational annexes showed different patterns among Nos3(-/-) and Nos3(+/+) mice. Throughout pregnancy, the distance between the external limit of the gestational sac and the embryo in Nos3(+/+) mice diminished in longitudinal sections, or remained unaffected in transverse sections. In Nos3(-/-) mice, there were significant increases (P < 0.005) in the differences between embryo and gestational vesicle measurements in both longitudinal and transversal curves from Days 5.5 to 14.5, but from Day 14.5 of pregnancy onward, the changes were not significant. The results demonstrate that the processes of fetal growth retardation in the Nos3(-/-) mice are established from early pregnancy stages.

USO DE EXAMES ULTRA-SONOGRÁFICOS PARA DETERMINAR A DATA DE PARTURIÇÃO EM CADELAS DA RAÇA YORKSHIRE

, ,²Oobjetivo deste trabalho foi verificar a eficácia do método ultra-sonografico visando prever a data departo em cadelas da raça Yorkshire. Também foi objetivo determinar um padrão de mensurações de vesículagestacional, comprimento do feto, diâmetro biparietal, diâmetro do corpo, diâmetro do tórax, diâmetro deabdome e comprimento do fêmur, além de estabelecer uma fórmula de regressão linear para ser utilizada poroutros veterinários ultra-sonografistas nesta raça.Aduração da gestação a partir da data de primeira cópula atéos primeiros sinais de parto resultou numa média de 63,57 dias. Para poder predizer a data de gestação foirealizada uma análise de regressão linear multivariada. Foi possível determinar uma fórmula para prever a datade parturição em cadelas da raça Yorkshire utilizando o comprimento fetal diâmetro biparietal e comprimentodo fêmur obtendo-se significativa correlação (R =0,998).

Estimation of fetal age in sheep by measurement of the embryonic vesicle diameter and umbilical cord diameter by using real-time ultrasonography

This experiment includes 72 local Awassi ewes aged between 2 to 5 years old. The aim of this study was to estimate the fetal age by the measurement of the embryonic vesicle and diameter of umbilical cord diameters during the different periods of gestation by using 5MHz real-time ultrasonography. Results of this study indicated that the embryonic vesicle could be seen at 14 days post breeding, and this can be the first sign of early pregnancy diagnosis. The embryo could be successfully seen at 20 days post breeding. The umbilical cord was first diagnosed at 23 days post breeding. Linear, quadratic and cubic equations were described, which adjust the relationship between the fetal age and diameter of embryonic vesicle and umbilical cord. In a conclusion, linear equation of the relationship between diameter of embryonic vesicle and fetal age was the best. equation to estimate the fetal age, while the cubic equation was the best equation to estimate the relationship between fetal age, and diameter of the umbilical cord.

Use of a Compounded Long-Acting Progesterone Formulation for Equine Pregnancy Maintenance

Abstract The objective of this study was to test the efficacy of a compounded long-acting progesterone formulation (BioRelease P4 LA 150; BETPHARM, Lexington, KY) containing 150 mg progesterone/ml for pregnancy maintenance in mares after prostaglandin (PG) F 2α -induced luteolysis. On day 18 of gestation, mares were randomly assigned to one of four groups (n = 7/group): (1) saline-treated control (Saline); (2) PGF 2α -treated control (PGF); (3) PGF 2α - and Regu-Mate-treated (Regu-Mate); and (4) PGF 2α - and BioRelease P4 LA 150-treated (BioRelease). On day 18, Saline mares received 1 ml sterile saline IM, whereas PGF, Regu-Mate, and BioRelease mares received 250 μg cloprostenol IM. Beginning on day 18, Regu-Mate mares received 10 ml Regu-Mate orally once daily and BioRelease mares received 10 ml BioRelease P4 LA 150 containing 150 mg/ml progesterone IM once every 7 days; treatments were continued until day 45 or until pregnancy loss occurred. Pregnancy diagnosis was performed every 3 days between days 18 and 45 (or until pregnancy loss). Pregnancy loss was defined as complete absence of a discernible embryonic vesicle as determined with transrectal ultrasonography. Pregnancy loss rates between days 18 and 45 were: Saline, 1/7; PGF, 7/7; Regu-Mate, 1/7; and BioRelease, 0/7. The pregnancy loss rate was higher ( P 2α -treated control mares compared with the other groups. There were no differences ( P > .1) in pregnancy loss rates among the saline-treated control, Regu-Mate-treated, and BioRelease P4 LA 150-treated mares. These results indicate that intramuscular administration of BioRelease P4 LA 150 containing a total of 1.5 g progesterone every 7 days provided a sufficient level of progesterone to maintain pregnancy between days 18 and 45 of gestation in mares that lacked an endogenous source of progesterone; therefore, this long-acting formulation of progesterone appears to be an efficacious and suitable alternative to currently available progesterone formulations that require daily administration.

Factors Concerning Early Embryonic Death in Thoroughbred Mares in South Korea

A total of 384 Thoroughbred mares were investigated to determine and evaluate the features of early embryonic death at nine equine farms on Jeju Island, South Korea, from 2001 to 2003. Overall, 771 matings for 384 mares resulted in 376 pregnancies 15 days after ovulation. Subsequently, 12.2% (46/376) of these early conceptuses were lost within 45 days after ovulation. Furthermore, about three quarters of the 46 embryonic deaths occurred between 16 and 25 days after ovulation. The incidence of embryonic death was highest in the barren (17.2%), more than 15 years old (15.4%), and more than 10 parities (18.2%) groups compared with the other groups (9.1-16.9%). Mares mated in March, April, and during the first estrus postpartum had higher embryonic death rates, (19.6%, 17.2%, and 24.6%, respectively). Mares with abnormal fluid and cysts in the uterus (18.0%) or multiple embryonic vesicles (23.1%) had higher embryonic death rates than those with normal uterine conditions (9.8%) or single embryonic vesicles (10.5%). The condition of mares, such as breeding condition and the environment of the embryo or fetus, are the major factors in relation to occurrence of early embryonic death.

Very Early Pregnancy

This is a transverse view of the uterine body containing a 4 week 2 day gestational age embryonic vesicle. The gestational sac was first identified in the anterior endometrial wall at 16 days after ovulation (4 weeks and 2 days from last menstrual period) and appears to be fully nidated. The sac measures 1.8 mm in diameter. Embryonic development was subsequently documented with twice weekly scans as part of a study designed to refine our knowledge of the earliest stages of human gestation. The embryonic vesicle is seen as a dark grey circle located within the more hyperechoic oval representing decidualized endometrial tissue. The deeper grey myometrium surrounds the endometrium. Consent to publish this image was obtained from the patient.

Incidence and nature of disorientation of the embryo proper and spontaneous correction in mares

Summary Orientation of the embryo proper at the periphery of the equine embryonic vesicle is normally antimesometrial or on the ventral aspect of the embryonic vesicle (6 o'clock relative to 12 o'clock at the center of the mesometrial attachment). An early ultrasonographically detectable vascular endometrial indicator of the future position of the embryo proper has been reported previously and was first detected in a mean 2.5 days before detection of the embryo proper. In the current study, four occurrences of disorientation of the embryo proper were found in a group of 30 mares (incidence, 13%). When first detected, the early indicator of the clock-face position of the embryonic pole for the disorientation and normal orientation group, respectively, was 1.3 ± 0.3 and 0.4 ± 0.1 hours from the 6 o'clock position ( P

Application of ultrasonography in the study of the reproductive system of tropical jennies (shape Equus asinus)

The use of high-frequency (5 MHz) ultrasonography was studied in 11 jennies (7 non-pregnant and 4 pregnant) to characterize the reproductive organs and follicular activities at different stages of reproduction. The result showed close similarity with the mare. The visibility of endometrial folds increased towards ovulation. A positive correlation (p < 0.001; tau = 0.79) was found between the score of the folds and the size of the dominant follicle. The diameter of the uterus and the size of the dominant follicle were significantly correlated (p < 0.001; r = 0.80). In pregnant jennies, an embryonic vesicle was detectable at 14 days. Follicular growth was characterized by more than one wave. The smallest ovarian follicle was 2 mm and the largest 40mm. Depending on the reproductive stage, up to 13 follicles were detected per ovary. After monitoring 84 cycles, a mean (+/- SD) diameter of 34.4 +/- 3.6 mm (27.5-40.2 mm) of the preovulatory follicle and 67.85% incidence of single ovulation were found. The mean (+/-SD) interovulatory interval was 25.7 +/- 6 days. This study proved that high-frequency ultrasonography is highly effective in characterizing the reproductive organs and follicular activity of jennies and could be useful in the reproductive management of donkeys.

Using “Uterine Index” to diagnose embryonic death in mares

Embryonic death plays an important role in infertility in mares, and most of embryonic mortality occurs at an early stage (before day 40) of pregnancy. Previous investigations focused on B-mode sonographic images to determine abnormal pregnancy; however, no study has provided a quantitative and objective method to diagnose embryonic death. Here we developed a new index, “Uterine Index,” using color Doppler sonography to describe alterations of uterine blood flow in physiological and pathological gestations during early gestation. Six Trotter mares were used in this study. The pregnancies were interrupted with a prostaglandin F2α (PGF 2α) analog (tiaprost) at days 25, 30, and 40. Three gestations were investigated in each stage. After the PGF 2α administration, these mares were examined every 8 hours until the embryonic vesicles were not visible on ultrasound. In the control group, three pregnancies were examined daily from days 12 to 60. The embryo size was measured in B-mode sonography, and the blood flow in uterine arteries was examined in Doppler ultrasound. The resistance index (RI) and uterine index (UI) were calculated. In the control group, the RI values of uterine artery were lower in pregnant side compared with that of the non-pregnant side from day 15 on, and the difference became greater as the pregnancy advanced ( P < .05). The UI was close to 0 before week 3 and increased to approximately 10 after week 4. In the PGF 2α-treated group, the UI values reduced from pregnancy level to approximately 0. As early as 32 hours before embryonic death, we observed a significant difference of UI values between control and treated mares. On the contrary, the embryo size of treated mares was not different from that of normal mares. Through the analysis of all recorded UI values, we suggest that the UI values 10 and 5 are good values for distinguishing normal pregnancy (UI >10) from embryonic death (UI < 5). If the UI values are located between 10 and 5, a reexamination at intervals of 1 to 3 days is recommended. Our findings demonstrate that the UI is a useful method to diagnose and predict embryonic death in mares by providing reliable and objective information.

217 A NEW APPROACH TO CRYOPRESERVATION OF LARGE EQUINE EMBRYOS BY VITRIFICATION AFTER BLASTOCOEL MICROMANIPULATION

Vitrified large equine embryos &amp;gt;800 �m recovered on Day 8 after ovulation have not been successfully transferred yet. In this study, we examined the effects of reduction of the blastocoelic fluid and microinfusion of a cryopreservative prior to vitrification on pregnancy outcome. In 2006, 33 embryos, recovered at the expanded blastocyst stage, were transferred fresh with an average pregnancy rate of 91% (30/33). However, suitable recipients are not always available. The sizes of embryos used for this vitrification project were 805 �m (embryo #1), 820 �m (#2), 1120 �m (#3), 1286 �m (#4), and 979 �m (#5). They were all morphologically graded excellent (according to IETS guidelines). These embryos were assigned to either no micromanipulation (embryos #1–#3) or microinfusion of VS1 (1.4 M glycerol in PBS; embryos #4 and #5) after additional aspiration of the blastocoelic fluid and before microinfusion for embryo #5. To facilitate aspiration and microinfusion, a laser system (XYclone; Hamilton Thorne Biosciences, Beverly, MA, USA) was applied. Approximately 20% of the total blastocoelic fluid was removed using a standard micromanipulator and microcapillary system (Eppendorf). All embryos were then vitrified as previously described (Eldridge-Panuska et al. 2005 Theriogenology 63, 1308–1319). In brief, embryos were exposed to VS1 and VS2 (1.4 M glycerol + 3.6 M ethylene glycol in PBS) for 5 min, and VS3 (3.4 M glycerol + 6.6 M ethylene glycol in PBS) for 1 min. Embryos in VS3 were then individually loaded into 0.25-mL straws, separated by 2 air bubbles from columns of 0.5 M galactose. Then straws were placed for 1 min into a cooled plastic goblet surrounded by liquid nitrogen. The goblet was finally plunged into liquid nitrogen. Digital images of all embryos were taken prior to and during the vitrification procedure, and also after thawing prior to embryo transfer. All expanded blastocysts initially decreased in size. After transfer to VS2 and VS3, they lost their spherical shape and blastocoels collapsed. Four vitrified embryos were transfered to recipients on Day 8 after ovulation. After thawing in air at room temperature for 5 s and then in water at 30�C for 15 s, straw contents were emptied into a Petri dish and mixed. After 5 min, single embryos were loaded into an AI pipette and nonsurgically transferred to recipients. The blastocoel of only one embryo re-expanded during the 5 min after thawing (#3) and one embryo was split into two halves (#4). One week after transfer of embryos, recipients were examined by ultrasonography. None of the control embryos nor the split embryo in the treatment group led to the formation of an embryonic vesicle. However, the blastocyst, which had undergone both aspiration of blastocoelic fluid and microinfusion of VS1 (#5), had formed an embryonic vesicle at Day 15 after ovulation. During a further exam on Day 28, the uterine tone in the recipient was still increased, but ultrasonography revealed resorption of the embryo, which was probably caused by heat stress. Nevertheless, we will test this protocol for the cryopreservation of large equine embryos on a larger scale during the next breeding season.