Mean Number Of Blastomeres Research Articles

Morphokinetic changes and apoptotic cell death in vitrified and non-vitrified in vitro-produced ovine embryos.

This article addresses morphokinetic changes and the extent of apoptosis in vitrified and non-vitrified in vitro-derived ovine blastocysts. Cumulus-oocyte complexes were collected after ovarian scarification obtain after slaughter and in vitro maturation was performed in TCM 199 medium supplemented with Earle's Salt, 10 % of FBS, and 5µg/mL of LH/FSH at 38°C for 24h. After maturation, the oocytes were co-incubated with thawed ram semen (IVF) for 19h.Embryo development was monitored with the aid of the Primo Vision Time-Lapse (TL) system. Twenty-five out of thirty-one ovine blastocysts that were vitrified using the Cryotop system at the early blastulation stage of development subsequently re-expanded. Both the vitrified (n=25) and non-vitrified (control group: n=28) blastocysts were examined for detection of apoptosis (TUNEL assay) and total blastomere counts at the time they attained the expanded blastocyst stage. Blastocyst formation occurred earlier in non-vitrified than in vitrified ovine embryos (147:49±20:23 compared with 156:46±19:24; hours:minutes post-insemination; mean ± SD; P<0.05). The average number of blastocyst collapses was greater (2.45±1.64 compared with 1.45±1.64), but the number of weak contractions was less for vitrified than non-vitrified ovine blastocysts (P<0.05). The mean number of blastomeres was greater (131.8±38.6 compared with 91.5±18.3; P<0.05) while the number of TUNEL-positive cells (4.4±1.6 compared with 6.3±2.3) and apoptotic index (3.4±1.2 % compared with 6.9±2.6 %) were less (P<0.05) in non-vitrified compared with vitrified blastocysts. Vitrification of ovine embryos was associated with a delayed blastocyst formation, greater numbers of apoptotic cells, significant reduction in the number of blastomeres, and higher/lower incidence of blastocyst collapse/weak contractions.

Cross-species efficacy of a chemically-defined, soy lecithin-based cryomedium for semen banking in imperiled wild felids

Felid semen has historically been frozen using an egg yolk-based cryopreservation medium. However, the use of egg introduces several potential concerns, such as variability in composition, microbial contamination, and regulatory issues. In the present study, our aim was to compare a chemically-defined, soy-based medium (SOY) to a commercial egg yolk-based medium (TEY) for the cryopreservation of sperm in four imperiled small cat species. Semen was collected from adult male cats (n = 6 black-footed cats; n = 6 sand cats; n = 4 fishing cats; and n = 7 Pallas’ cats) via electroejaculation, split into two aliquots, and cryopreserved in SOY or TEY. Frozen-thawed samples were evaluated for sperm motility and rate of progressive motility (up to 24 h post-thaw) and acrosome status (0 and 6 h). No difference in post-thaw traits were observed between treatments in all four species. Heterologous IVF using oocytes collected laparoscopically from domestic cats demonstrated no difference among freezing treatments in percentage of mature oocytes that cleaved or the mean number of blastomeres at 48 h post-insemination. More spermatozoa frozen with SOY were bound to the zona pellucida in the sand cat (P = 0.018), but no treatment effect was observed in the other three species. These findings collectively demonstrate that SOY may be a preferable alternative to TEY for sperm cryopreservation in these four wild felid species.

154 COMPARISON OF DIFFERENT CULTURE MEDIA AND INCUBATION METHODS ON CULTURING MURINE EMBRYOS IN VITRO USING STRAW AS A RECEPTACLE

In vitro fertilization in the straw system might increase the efficiency of fertilization and the quality of blastocyst formation as compared with micro-drops-IVF systems. The aim of the study was to in vitro fertilize mouse oocytes and culture the resulting zygotes in bi-gas incubator and in a goat vagina and compare the in vitro embryo developmental stages in TCM-199 and Ham’s F10 culture media until the blastocyst-stage of development. F1 generations (Balb C × C57) were used to harvest oocytes and spermatozoa. The fresh sperm were capacitated in different incubation methods (bi-gas incubator and in the vagina of a goat). A volume of 2–4 µL of Ham’s F10 containing capacitated sperm (~8 × 106 per mL) were placed into Ham’s F10 fertilization drops under the oil, containing 10 oocytes and penicillamine, hypotaurine, and epinephrine for enhancing sperm motility and penetration of oocytes. The same procedure was used with the TCM-199 medium and IVF drops without oil (both TCM-199 and Ham’s F10) for straw filling. The presumptive embryos in Ham’s F10 and TCM-199 were divided into different groups: first group were cultured in micro-drops, second group the embryos were aspirated in semen straws and placed in the incubator (incubator straws) for culture, and other straws were covered with a sponge and inserted in the vagina of a goat (vaginal straws) for culture. The resulted blastocysts were stained using Hoechst 33528 solution and blastomeres were counted on a fluorescent UV light inverted microscope at 400× magnification (Nikon Eclipse TI, Narishige Co., Ltd., Amityville, NY, USA). The results were analysed by 2 × 2 factorial designs and Student’s t-test was used to separate the mean. There was no statistical difference (P &gt; 0.05) between the media and incubators on the stage of murine embryo development. The overall fertilization rate was 94 to 99%. The incubator straws with Ham’s F10 (80.5%) had the highest rate of embryos that reached the blastocyst stage, followed by incubator straws with TCM-199 (77.0%), and vaginal straws with Ham’s F10 (60.0%) had the lowest rate of embryos that reached the blastocyst stage. The overall mean number of blastomeres in the blastocyst stage of the embryos ranged from 85 ± 9 to 90 ± 9 cells in all receptacles and incubators. It was concluded that the fertilization and culturing of murine embryos are possible in straws incubated in a bi-gas incubator and in the goat vagina as an alternative method of fertilizing oocytes and culturing murine embryos. In addition, Ham’s F10 and TCM-199 can both be used to fertilize oocytes and culture murine embryos until blastocyst formation embryo in vitro, incubated in a bi-gas incubator or in the vagina.

Reduced oxygen tension improves embryo quality but not clinical pregnancy rates: a randomized clinical study into ovum donation cycles

To investigate the effect of low O2 tension during in vitro culture in terms of ongoing pregnancy rates in ovum donation cycles. Randomized trial. Private university-affiliated IVF center, university-based hospital. A total of 1,125 cycles of ovum donation. Embryo culture in an atmosphere of 5.5% CO2, 6% O2, and 88.5% N2 versus a dual-gas system of 5.5% CO2 in air. Ongoing clinical pregnancy rates per intention-to-treat (ITT) patients. The use of low O2 tension achieved a 41.3% ongoing pregnancy rate per ITT compared with a 40.8% rate obtained for 5% CO2 in air. The mean number of blastomeres and the percentage of top-quality embryos were significantly higher after lower O2 concentration during in vitro culture (7.1 ± 3.6 and 28.6% vs. 7.3 ± 8.4 and 32.1%, respectively). In the ovum donation cycles undergoing day-3 embryo transfers, the use of low O2 tension did not improve ongoing pregnancy rates per cycle and per transfer. However, it benefited embryo quality, demonstrating the potential negative impact of high O2 tension on the in vitro embryo development.

The effects of cryopreservation on early development and chromosome constitution in Chinese hamster embryos.

The effects of cryopreservation on early embryonic development were investigated in Chinese hamster embryos. Embryos were randomly divided into 3 groups, as follows: the control group, embryos which were simply cultured; the DMSO group, embryos which were exposed to dimethyl sulfoxide (DMSO) and then cultured; the cryopreservation group, embryos which were cryopreserved and then cultured. The percentages of embryos which developed into blastocysts after 40 hours of cultivation were high in all groups. However, there were significant differences in the mean number of blastomeres with lower values after an exposure to DMSO and cryopreservation (73.2 in the control group, 62.0 in the DMSO group, and 40.2 in the cryopreservation group). No significant differences in chromosome abnormality rate were evident and there was no distinct tendency for variation in karyotype among the 3 groups. These results indicate that DMSO adversely affects the division of blastomeres, and that cryopreservation with DMSO as a cryoprotectant might aggravate these adverse effects.

A study of the effect of an extremely low oxygen concentration on the development of human embryos in assisted reproductive technology.

To determine whether embryos cultured with a low oxygen level (2%) brought about beneficial effects on the outcome of ART. This is a sequential case-control embryo-culture study. Embryos were cultured either with a gas mixture containing 2% O2, 5% CO2, and 93% N2 (low-oxygen group) or 5% O2, 5% CO2, and 90% N2 (conventional group). From January 2008 to September 2008, 873 fertilized oocytes were obtained from 250 patients in the low-oxygen group and from October 2008 to March 2009, 730 fertilized oocytes were obtained from 213 patients in the conventional group. The outcomes of ART were compared between two groups. The cleavage rate in the low-oxygen group (94.4%) was similar to that (94.7%) in the conventional group. The mean number of blastomeres on Day 3 in the low-oxygen group (mean ± SE) was 6.5 ± 1.9, and this was significantly lower than in the conventional group (6.8 ± 1.9, p < 0.05). Moreover, the low-oxygen group produced worse quality embryos, on the basis of the significantly higher embryo grade 2.1 ± 0.6 versus 1.9 ± 0.6, p < 0.001, in 5% oxygen. The pregnancy and miscarriage rates in the low-oxygen group were 22.3 and 20.8%, respectively, which were statistically similar to the outcomes in the conventional group. Overall, culture of embryos at the low oxygen level did not significantly improve ART results compared with embryos grown in 5% oxygen. The study suggests that a low oxygen level worsens embryo morphology but does not impair embryo viability.

Developmental Potential of Human Oocytes After Slow Freezing or Vitrification: A Randomized In Vitro Study Based on Parthenogenesis

The aim of the this study was to compare the in vitro developmental competence of parthenogenetically activated oocytes cryopreserved with slow-freezing or vitrification. Supernumerary metaphase II oocytes obtained during in vitro fertilization procedures were randomized to slow freezing or vitrification procedure. After thawing or devitrification, oocytes were parthenogenetically activated and cultured. Survival, activation, development rate, and cell number during culture were compared. The 2 groups showed no significant differences between the rates of parthenogenetic activation, development, good quality parthenotes and blastomere number on day 2 of culture. However, parthenotes from the devitrified oocytes continued cleaving till day 3 in a significantly low proportion (27% vs. 42%). On day 3, the mean number of blastomeres was also lower in vitrification group compared to slow-freezing (4.8 + 1.9 vs. 5.8 + 1.7). In conclusion, parthenogenesis highlights a reduced potential of vitrified oocytes to cleave on day 3 compared with oocytes from slow-freezing.

Do cytokine levels in the supernatant from conditioned media (CM) of embryos grown in autologous endometrial coculture (AECC) correlate with pregnancy outcomes after in vitro fertilization (IVF)?

AECC has been shown to improve outcomes in patients with multiple failures after IVF. AECC works to enhance in vitro conditions in several ways including the elaboration of cytokines by endometrial cells. IL-2, IL-12p70, IL-4, IL-5 are known to be produced by the endometrium. The aim of this study was to determine the association of these cytokines with IVF outcomes. In 92 IVF patients, supernatant from AECC was tested for the presence of IL-2, IL-12p70, IL-4 and IL-5. From a luteal endometrial biopsy performed prior to IVF, glandular and stromal cells were isolated, separated, then plated in a 50/50 combination. After one week in which the culture medium was changed every 2–3 days, the cells were cryopreserved to be thawed 3 days prior to embryo placement. If more than 6 oocytes fertilized normally, embryos were randomly grown on AECC cells (75%) and conventional media (25%), otherwise, all embryos were grown on AECC. Immediately after embryo removal before transfer, the CM was collected and cryopreserved. Only CM after embryo exposure was tested as previous research has shown that the presence of embryos does not alter cytokine levels from CM with AECC. Cytokine levels were measured utilizing commercially available enzyme linked immunoassays. The average age of the patients was 37.2 ± 4.4 years. Mean numbers of MIIs, 2PNs, and embryos transferred were 12.0 ± 6.6, 7.3 ± 4.8, and 3.3 ± 1.4 respectively. The clinical pregnancy rate was 44.6% and implantation rate was 22.9%. Embryos grown on AECC demonstrated a significant improvement in the mean number of blastomeres and mean fragmentation rate when compared to embryos grown in conventional media (5.8 ± 1.2 vs. 5.0 ± 1.9; 10.5% ± 7.1 vs. 17.6% ± 13.8; P<0.01). The presence of IL-2, IL-12p70, IL-4 and IL-5 was detected in 84.8%, 94.6%, 98.9%, and 95.7% of samples, respectively. Although cytokine levels in the CM did not correlate with either mean number of blastomeres or mean fragmentation rate, IL-12p70 levels were decreased in CM from embryos of good quality in comparison to levels in embryos of poor quality (1.0 ± 1.0 vs. 2.0 ± 3.1; P<0.05). No association was found between cytokine levels and pregnancy outcomes. Significant improvement in blastomere number and fragmentation rate was achieved with AECC. All of the evaluated cytokines were expressed by cells in the AECC. Cytokine levels did not correlate with pregnancy outcomes. However, IL-12p70 levels were decreased in CM from good quality embryos.

Simple pronuclear analysis: relation to embryo cleavage on day 3

Selection of embryos for transfer can be based on pronuclear score, early cleavage, cell number and fragmentation on day of transfer, or blastocyst score following extended culture. Whether these morphological observations relate to one another is questionable. We aimed to record differences in embryo pronuclei symmetry, nucleoli distribution, and the combination of these criteria; and investigate overall pronuclear morphology vs. mean number of cells with subsequent cleavage and embryo morphology on day 3. Prospective study. Two hundred and twenty five patients, age 36.2 ± 5.36 (mean ± SD) years, presenting for fertility treatment with ICSI between October 2006 and January 2007 were included in this study. Oocytes (n = 1146) were inseminated according to standard ICSI techniques and zygotes were evaluated after 16 to 18 hours of culture. Pronuclei (PN) were evaluated for position and symmetry on an inverted microscope with contrast optics at 400× magnification. Pronuclei were categorized as: A – central PN with equal size, B – non-centered PN with equal size, and C – different PN sizes independent of their position. Additionally, nucleoli alignment was classified as: 1 – male and female aligned juxtaposed independent of number and size, 2 – equal distribution throughout the PN, independent of number, and 3 – other patterns. Combination of these criteria resulted in nine classes. Mean number of blastomeres on day 3 were compared between these nine classes by ANOVA followed by Tukey's test for means. There were significant differences in mean number of embryonic cells on day 3 according to the pronuclear symmetry (P<0.01), nucleoli alignment (P<0.01) and combined criteria (P<0.01). Data shown in Table. Table 1Mean (± SD) number of blastomeres on day 3 according to PN symmetry, nucleoli alignment and combined criteriaPN symmetryCell NumberNucleoli alignmentCell NumberCombined criteriaCell NumberA5.5a ± 2.215.6a ± 2.322A15.6a ± 2.3B5.3a ± 2.325.3a ± 2.2A25.6a ± 2.0C4.6b ± 2.32635.0b ± 2.371A35.4ab ± 2.2B15.7ab ± 2.2B25.2abc ± 2.2B35.1abc ± 2.4C15.5abc ± 2.1C24.6bc ± 2.2C34.5bc ± 2.3Differences indicated by letters. Open table in a new tab Differences indicated by letters. Our data indicates that PN morphology is related to blastomere cell number on day 3. Zygotes with asymmetric PN, disorganized nucleoli, and their combination had significantly fewer blastomeres compared to other classifications. Pronuclear assessment may provide a simple, rapid, and important parameter in the arsenal of morphologic evaluation of preimplantation embryos.

Does sperm morphology affect the outcome of ICSI (intracytoplasmic sperm injection) cycles?

Till now, there is no unanimity about the impact of the use of abnormal spermatozoa on the success rate after intracytoplasmic sperm injection (ICSI). However, later experience has shown that pregnancy and implantation rates are lower in cases in which only morphologically abnormal spermatozoa are available for ICSI, as compared with those in which morphologically normal spermatozoa can be found. Therefore, and aware of the lack of consensus, we conducted the present study in order to assess the effects of the use of abnormal sperm on the outcome of ICSI in our clinic. A retrospective study of 282 ICSI cycles conducted as a consecutive case series in a 2 year period of patients that use their own oocytes. We excluded TESE (testicular sperm extraction) cycles and donor sperm samples. In 241 cycles, all oocytes were injected with spermatozoa with normal morphology (group A) and in 41 cycles, all oocytes were injected with sperm with abnormal morphology (group B). ICSI was performed with an optical magnification of about ×400 in an inverted microscope. Fertilization rate, embryo development and pregnancy, implantation and miscarriage rates were compared between the two groups. Statistical analysis has been performed using STATA™. The mean age of the female partners in group A was 35.3 years (95% CI 24.8–35.8) and in group B 34.8 years (95% CI 33.8–35.8). The mean number of retrieved oocytes (9.1 in group A vs. 9.0 in group B), fertilization rate (76% in group A vs. 73% in group B), mean number of blastomeres per embryo replaced (7.4 in group A vs. 7.1 in group B) and number of replaced embryos (1.6 in group A vs. 1.8 in group B) was not different among both groups. The pregnancy rate was 60.5% in group A vs. 50% group B (P=0.25). The implantation rate was significantly higher in group A (38.9%) compared to group B (25.5%) (P=0.004). The miscarriage rate was slightly lower in group A (14.95%) than in group B (17.65%) but the difference did not reach statistical significance. Individual sperm morphology assessed at the moment of ICSI does not seem to affect the fertilization rate or embryo development. However, the implantation rate was significantly lower when only embryos resulting from the injection of an abnormal spermatozoon were available. Although, the present study based on a limited number of cycles should be extended, it already emphases the need of careful examination of the morphology of the sperm that will be use for the ICSI procedure.

129 IN VITRO PRODUCTION OF EQUINE EMBRYOS FROM YOUNG AND OLD MARES BY INTRACYTOPLASMIC SPERM INJECTION

High merit mares obtain their utmost productive value at the same time their reproductive soundness diminishes. The aim of our study was to compare the developmental competence of equine oocytes from young and old mares after intracytoplasmic sperm injection (ICSI) and in vitro culture. Ovaries from young and old mares were obtained from a pool of slaughterhouse animals that have been previously selected by overall good body condition, reproductive status, and age. Young mares were 3 to 8 years old, and old mares were more than 15 years old. The age of all mares was determined by teeth observation and reproductive status by ultrasonography. Oocytes were obtained from ovaries 1 h postmortem by individual dissection of follicles between 10 and 25 mm and scraping of the follicle wall with a bone curette. Recovered oocytes were matured in vitro for 24–30 h, and all oocytes with an intact cytoplasm and a visible polar body were subject to ICSI and cultured for 7.5 days in SOFm. The maturation rate, cleavage, and embryo development rate and mean number of blastomeres at 7.5 days of culture were compared between oocytes and embryos from young and old mares. Maturation, cleavage, and developmental rates were analyzed by Chi Square and Fisher exact test, whereas the mean number of blastomeres at 7.5 days of culture was compared by one way ANOVA and t-test. A total number of 54 oocytes from young mares and 37 oocytes from old mares were obtained. There were no significant differences between the maturation, cleavage, or embryo development rates between young (79.63, 56.41, and 18.18%) and old (91.89, 63.33, and 15.79%) mares. In addition, the mean number of cells on embryos from each group did not differ significantly (57.75 v. 81; young v. old). Our preliminary results show that similar in vitro rates are achieved when oocytes from young or old mares are matured and fertilized in vitro by ICSI. This does not correlate with the reproductive senescence in old mares and the result obtained with other reproductive techniques. Further studies will determine if pregnancies are equally achieved using in vitro produced embryos from both age groups.

Development to the blastocyst stage of parthenogenetically activated in vitro matured porcine oocytes after solid surface vitrification (SSV)

The effects of solid surface vitrification (SSV) on viability and parthenogenetic development of in vitro matured (IVM) porcine oocytes was investigated in the present study. Cumulus-free IVM porcine oocytes were subjected either to SSV or SSV combined with a cytochalasin B (CB) pre-treatment (SSV + CB) or all steps of SSV but without cooling (toxicity control = TC; toxicity control with CB pre-treatment = TC + CB). Oocyte viability was evaluated by plasma membrane integrity and esterase activity measured by a combined staining with fluorescein diacetate, propidium iodide and Hoechst 33342. Surviving oocytes were parthenogenetically activated then cultured in vitro (IVC) for 6 days. The proportion of live oocytes after vitrification was significantly lower than that of the TC, TC + CB and the control groups, regardless of the CB pre-treatment. Treatment of oocytes with cryoprotectants did not decrease the rates of surviving oocytes. After activation of oocytes, the proportion of cleaved embryos was significantly higher in the SSV + CB ( P < 0.05) than that of the SSV group. Nevertheless, significantly more oocytes cleaved ( P < 0.05) in the TC, TC + CB and the control groups. On Day 6, the rate of blastocysts in the SSV and SSV + CB groups did not differ significantly. The number of oocytes developing to blastocyst and the mean number of blastomeres per embryo were significantly higher ( P < 0.05) in the TC, TC + CB and the control compared with that of the SSV and SSV + CB groups. To our knowledge, this is the first report on parthenogenetic development to blastocysts of porcine oocytes vitrified at the metaphase-II stage. Results indicate that the high concentrations of cryoprotectants were not harmful for in vitro development, and that CB pre-treatment may increase survival and development of SSV vitrified porcine oocytes.

Autologous endometrial co-culture in patients with repeated failures of implantation after in vitro fertilization-embryo transfer.

Our purpose was to evaluate the effect of coculture on preembryo development and clinical outcome. Enrolled patients underwent a luteal-phase endometrial biopsy. The tissue was then enzymatically digested (collagenase) and the stromal and glandular cells were separated by differential sedimentation rates. These cells were cultured to confluence, released, and then cryopreserved until the patient's in vitro fertilization (IVF)-embryo transfer (ET) cycle. All normally fertilized oocytes were then placed on the co-cultured cells until transfer on day 3. Preembryo development on co-culture was compared to that in the patient's noncocultured previous cycle. Implantation and clinical pregnancy rates were compared to those in a control group of patients undergoing IVF during the study period who were matched for age, stimulation protocol, number of oocytes retrieved, and preembryos transferred. Twenty-nine women underwent 31 cycles of IVF-ET. On day 3 the overall mean number of blastomeres per preembryo on co-culture compared to that in the patient's previous cycle was 6.3 +/- 1.8 vs. 5.6 +/- 1.2 (P = 0.04). The average percentage of cytoplasmic fragments on co-culture compared to the previous cycle was 16 +/- 9% vs. 19 +/- 9% (P = 0.32). At transfer, after preembryo selection, the mean number of blastomeres per preembryo on co-culture compared to that in the patient's previous cycle was 6.8 +/- 1.6 vs. 6.6 +/- 1.3 (P = 0.5). The implantation and clinical pregnancy rates between co-culture and the matched control group were 15% (14/93) vs. 13% (16/124) (P = 0.79) and 29% (9/31) vs. 25% (10/40) (P = 0.45). There was a significant improvement in the average number of blastomeres per preembryo on co-culture compared to that in the patient's previous noncoculture cycle. The overall implantation and clinical pregnancy rates between co-culture and a matched control group were not significantly different.

Effect of neonatal blood serum on the early stages of mouse embryogenesisin vitro

The effect of blood sera from newborns delivered after complicated pregnancy and abnormal labor (CPAL) on 4-cell mouse embryos is studied. The embryos develop normally in the control sera, 78–100% of them reach the blastocyst stage, the mean number of blastomeres per embryo varies from 68 to 88 cells, and the mortality rate is 3–7%. The CPAL sera have an almost 100% embryolethal effect. The development of mouse embryos is markedly delayed. The most potent pathological effect is elicited by the sera of newborns whose mothers suffered from hypoxia.

Endocrine changes in the late-follicular and postovulatory intervals as determinants of the in vitro fertilization pregnancy rate

This investigation examines the hormone pattern in in vitro fertilization (IVF) cycles from the time of human chorionic gonadotropin (hCG) administration through embryo transfer to ascertain whether the absolute levels or secretory patterns of the major reproductive hormones affect the IVF pregnancy rate. Thirty-one women who underwent IVF treatment were enrolled in the study. All patients received clomiphene citrate/human menopausal gonadotropin for ovulation induction. Significant elevations in serum estradiol (E2) levels in the pregnant group were found throughout the cycle interval studied. After hCG administration the serum hCG levels were not different between the groups. Significant elevations in serum progesterone (P) concentrations were found in the pregnant group from the day after laparoscopy through embryo transfer. Embryos obtained from the pregnant group appeared to be different in that the mean number of blastomeres per embryo transferred was significantly greater. Therefore for achievement of an IVF pregnancy the optimal hormone pattern employing combination ovulation induction in the ovulation to transfer interval is a relatively high E2 level in ovulation followed by a high P level at transfer and into the luteal phase. These elevated hormone levels do not depend on the response to exogenous hCG.

Pregnancy rates at Days 2 and 14 and estimated embryonic loss rates prior to day 14 in normal and subfertile mares

Pregnancy rates at Days 2 and 14 postovulation were determined for 15 normal mares and 15 subfertile mares. Embryonic loss rates were estimated by the difference in the Day 2 and Day 14 pregnancy rates. Mares were artificially inseminated with the pooled ejaculates from three stallions, and the embryonic vesicle was detected with ultrasonography at Days 9, 10, 12 and 14. Mares were short-cycled with prostaglandin F 2 alpha (PGF 2α) and rebred to the same stallions, and the Day 2 pregnancy rates were determined by recovery of cleaved ova (embryos) from the surgically excised oviducts. Significantly more (P < 0.01) normal versus subfertile mares were pregnant at Day 14 ( 12 15 vs 3 15 ). There was no significant difference in the Day 2 pregnancy rate for normal versus subfertile mares ( 10 14 vs 11 14 ). There were no significant differences (P > 0.5) in the mean number of blastomeres per embryo or in the mean diameter of embryos recovered at Day 2 from normal or subfertile mares. The estimated embryonic loss rate was significantly lower (P < 0.01) for normal verusus subfertile mares ( 0 10 vs 8 11 ). Fertilization rates were similar for normal and subfertile mares; however, subfertile mares had a higher embryonic loss rate prior to Day 14 postovulation.