Equine maternal aging affects the metabolomic profile of oocytes and follicular cells during different maturation time points.

Introduction: Oocyte quality and fertility decline with advanced maternal age. During maturation within the ovarian follicle, the oocyte relies on the associated somatic cells, specifically cumulus and granulosa cells, to acquire essential components for developmental capacity. Methods: A nontargeted metabolomics approach was used to investigate the effects of mare age on different cell types within the dominant, follicular-phase follicle at three time points during maturation. Metabolomic analyses from single oocytes and associated cumulus and granulosa cells allowed correlations of metabolite abundance among cell types. Results and Discussion: Overall, many of the age-related changes in metabolite abundance point to Impaired mitochondrial metabolic function and oxidative stress in oocytes and follicular cells. Supporting findings include a higher abundance of glutamic acid and triglycerides and lower abundance of ceramides in oocytes and somatic follicular cells from old than young mares. Lower abundance of alanine in all follicular cell types from old mares, suggests limited anaerobic energy metabolism. The results also indicate impaired transfer of carbohydrate and free fatty acid substrates from cumulus cells to the oocytes of old mares, potentially related to disruption of transzonal projections between the cell types. The identification of age-associated alterations in the abundance of specific metabolites and their correlations among cells contribute to our understanding of follicular dysfunction with maternal aging.

Maturation and culture affect the metabolomic profile of oocytes and follicular cells in young and old mares.

Introduction: Oocytes and follicular somatic cells within the ovarian follicle are altered during maturation and after exposure to culture in vitro. In the present study, we used a nontargeted metabolomics approach to assess changes in oocytes, cumulus cells, and granulosa cells from dominant, follicular-phase follicles in young and old mares. Methods: Samples were collected at three stages associated with oocyte maturation: (1) GV, germinal vesicle stage, prior to the induction of follicle/oocyte maturation in vivo; (2) MI, metaphase I, maturing, collected 24 h after induction of maturation in vivo; and (3) MIIC, metaphase II, mature with collection 24 h after induction of maturation in vivo plus 18 h of culture in vitro. Samples were analyzed using gas and liquid chromatography coupled to mass spectrometry only when all three stages of a specific cell type were obtained from the same mare. Results and Discussion: Significant differences in metabolite abundance were most often associated with MIIC, with some of the differences appearing to be linked to the final stage of maturation and others to exposure to culture medium. While differences occurred for many metabolite groups, some of the most notable were detected for energy and lipid metabolism and amino acid abundance. The study demonstrated that metabolomics has potential to aid in optimizing culture methods and evaluating cell culture additives to support differences in COCs associated with maternal factors.

Autogenous transfer of intracytoplasmic sperm injection-produced equine embryos into the uterus of the oocyte donor during the same oestrous cycle.

The clinical use of intracytoplasmic sperm injection (ICSI) in horses usually involves the transfer of embryos into recipient mares, resulting in substantial cost increases. This is essential when subfertile mares are oocyte donors; but some donors are fertile, with ICSI compensating for limited or poor-quality spermatozoa. Fertile oocyte donors could carry pregnancies, eliminating the need for a recipient. We assessed the potential of using oocyte donors as recipients for their own ICSI-produced embryos during the same cycle. Donors in oestrus and with large dominant follicles were administered ovulation-inducing compounds to cause follicle and oocyte maturation. Maturing oocytes were collected, cultured and fertilised using ICSI. At 6 or 7 days after ICSI, developing blastocysts were transferred into respective donors' uteri, and pregnancy rates were determined. Twenty follicles were aspirated from nine mares and 12 oocytes were collected. After ICSI, 10 of the 12 oocytes (83%) cleaved, and eight (67% of injected oocytes) developed into blastocysts for transfer. Five pregnancies resulted from the eight transferred embryos (pregnancy rate 62% per embryo and 42% per sperm-injected oocyte). Following this synchronisation regime, ICSI-produced embryos can be transferred into oocyte donors' uteri during the same cycle, allowing donors to carry pregnancies after assisted fertilisation.

Experimental evaluation of two different types of reactors for CO2 removal from gaseous stream by bottom ash accelerated carbonation.

Low methane content landfill gas may be enriched by removing carbon dioxide. An innovative process, based on carbon dioxide capture and storage by means of accelerated carbonation of bottom ash is proposed and studied for the above purpose. Within this research framework we devoted a preliminary research activity to investigate the possibility of improving the way the contact between bottom ash and landfill gas takes place: this is the scope of the work reported in this paper. Two different types of reactors - fixed bed and rotating drum - were designed and constructed for this purpose. The process was investigated at laboratory scale. As the aim of this phase was the comparison of the performances of the two different reactors, we used a pure stream of CO2 to preliminarily evaluate the reactor behaviors in the most favorable condition for the process (i.e. maximum CO2 partial pressure at ambient condition). With respect to the simple fixed bed reactor concept, some modifications were proposed, consisting of separating the ash bed in three layers. With the three layer configuration we would like to reduce the possibility for the gas to follow preferential paths through the ash bed. However, the results showed that the process performances are not significantly influenced by the multiple layer arrangement. As an alternative to the fixed bed reactor, the rotating drum concept was selected in order to provide continuous mixing of the solids. Two operating parameters were considered and varied during the tests: the filling ratio and the rotating speed. Better performances were observed for lower filling ratio while the rotating speed showed minor importance. Finally the performances of the two reactors were compared. The rotating drum reactor is able to provide improved carbon dioxide removal with respect to the fixed bed one, especially when the rotating reactor is operated at low filling ratio values and slow rotating speed values. Comparing the carbon dioxide specific removal obtained by using the rotating reactor (35-37g/kgBA), in the best operating conditions, with that measured for the fixed bed reactor (21-23g/kgBA), an increase of about 61-66% is observed.

Validation of a heterologous fertilization assay and comparison of fertilization rates of equine oocytes using in vitro fertilization, perivitelline, and intracytoplasmic sperm injections.

IVF in horses is rarely successful. One reason for this could be the failure of sperm to fully capacitate or exhibit hyperactive motility. We hypothesized that the zona pellucida (ZP) of equine oocytes prevents fertilization in vitro, and bypassing the ZP would increase fertilization rates. Limited availability of equine oocytes for research has necessitated the use of heterologous oocyte binding assays using bovine oocytes. We sought to validate an assay using bovine oocytes and equine sperm and then to demonstrate that bypassing the ZP using perivitelline sperm injections (PVIs) with equine sperm capacitated with dilauroyl phosphatidylcholine would result in higher fertilization rates than standard IVF in bovine and equine oocytes. In experiment 1, bovine oocytes were used for (1) IVF with bovine sperm, (2) IVF with equine sperm, and (3) intracytoplasmic sperm injections (ICSIs) with equine sperm. Presumptive zygotes were either stained with 4',6-diamidino-2-phenylindole from 18 to 26 hours at 2-hour intervals or evaluated for cleavage at 56 hours after addition of sperm. Equine sperm fertilized bovine oocytes; however, pronuclei formation was delayed compared with bovine sperm after IVF. The delayed pronuclear formation was not seen after ICSI. In experiment 2, bovine oocytes were assigned to the following five groups: (1) cumulus oocyte complexes (COCs) coincubated with bovine sperm; (2) COC exposed to sucrose then coincubated with bovine sperm; (3) COC coincubated with equine sperm; (4) COC exposed to sucrose, and coincubated with equine sperm; and (5) oocytes exposed to sucrose, and 10 to 15 equine sperm injected into the perivitelline (PV) space. Equine sperm tended (P = 0.08) to fertilize more bovine oocytes when injected into the PV space than after IVF. In experiment 3, oocytes were assigned to the following four groups: (1) IVF, equine, and bovine COC coincubated with equine sperm; (2) PVI of equine and bovine oocytes; (3) PVI with equine oocytes pretreated with sucrose; and (4) ICSI of equine oocytes. Oocytes were examined at 24 hours for cleavage. No equine oocytes cleaved after IVF or PVI. However, ICSI conducted with equine sperm treated with dilauroyl phosphatidylcholine resulted in 85% of the oocytes cleaving. Sperm injected into the PV space of equine oocytes did not appear to enter the ooplasm. This study validated the use of bovine oocytes for equine sperm studies and indicates that failure of equine IVF is more than an inability of equine sperm to penetrate the ZP.

The effect of equine metabolic syndrome on the ovarian follicular environment.

Obesity in many species is associated with reduced fertility and increased risk of metabolic disorders and cardiovascular dysfunction in offspring. Equine metabolic syndrome (EMS) is associated with obesity and characterized by insulin resistance, decreased adiponectin, and elevated insulin, leptin, and pro-inflammatory cytokines. These alterations can potentially disrupt follicular development and impair fertility. We hypothesized that mares with EMS have an altered follicular environment when compared to their normal counterparts, affecting gene regulation for follicle and oocyte maturation. Samples were collected from light-horse mares (11 to 27 yr) in a clinical assisted reproductive program. Mares were screened based on phenotype. Insulin sensitivity was determined by using two proxies, the reciprocal of the square root of insulin (RISQI) and the modified insulin-to-glucose ratio (MIRG). Insulin resistant mares (RISQI < 0.32 and MIRG > 5.50) were allocated to the EMS group (n = 8), and the remaining mares were considered normal controls (CON, n = 12). Follicular fluid (FF) and granulosa cells (GC) from preovulatory follicles were aspirated 24 ± 2 h after administration of a GnRH analog (SucroMate, 0.9 to 1.4 mg, i.m.) and hCG (Chorion, 1500 to 2000 IU, i.v.). After an overnight fast, blood was collected on the morning of follicle aspiration to evaluate serum concentrations of insulin, leptin, adiponectin, and inflammatory cytokines. Expression of 32 genes related to metabolism, follicle maturation, and oocyte maturation were assessed in GC. Concentrations of insulin, leptin, adiponectin, and cytokines were highly correlated between serum and FF (P < 0.001). Insulin was lower (P < 0.001) in serum and FF of CON compared to EMS, but leptin and IL1ß tended (P = 0.07 and P = 0.10, respectively) to be lower in FF of CON than EMS. Tumor necrosis factor-α in serum and FF was lower (P < 0.07 and P < 0.05, respectively) in CON than EMS. Conversely, adiponectin was higher (P < 0.05) in serum and FF in CON versus EMS. In GC from CON when compared to EMS, gene expression for epiregulin was elevated (P < 0.05) and tissue inhibitor of matrix metalloproteinase-2 tended to be lower (P = 0.09). Our findings demonstrate that the intrafollicular environment in the mare is influenced by metabolic disease, consistent with findings in other species. Influences on follicular development, oocyte maturation, and subsequent offspring by perturbations due to metabolic disease need further study.

Effects of FSH and LH on ovarian and follicular blood flow, follicular growth and oocyte developmental competence in young and old mares.

Objectives of the experiment were to determine the effects of mare age and gonadotropin treatments on dominant follicle vascularity, ovarian blood flow and dominant follicle growth and to associate follicular vascularity with oocyte developmental capacity. Growing follicles >30 mm from young (4-9 years) and old (>20 years) mares were assessed for blood flow using color Doppler ultrasonography before maturation induction with recombinant equine LH (eLH) and immediately prior to oocyte collection at 20-24 h after eLH. Pulsed Doppler was used to obtain resistance indices of ovarian arteries ipsilateral to preovulatory follicles. For eFSH-treated estrous cycles, eFSH administration was started after detection of a cohort of follicles ≥20 to <25 mm and continued until a follicle >30 mm. Oocytes were harvested using transvaginal, ultrasonic-guided aspirations and cultured and injected with sperm at 40 ± 1 h after eLH. Presumptive zygotes were incubated, and rates of cleavage (≥2 cells) and blastocyst formation were obtained. Embryos were transferred nonsurgically into recipients' uteri, and pregnancy rates were assessed. Vascularity (number of color pixels per total pixels) was higher (P=0.003) in the follicles of old compared to young mares, with no significant interaction of eFSH or eLH. Effects of eFSH and time from eLH on follicle vascularity were not significant. The vascularity of follicles associated with oocytes that did compared to those that did not form blastocysts was greater (P=0.048), although follicular vascularity was less (P=0.02) for follicles associated with oocytes that did compared to those that did not develop into pregnancies. Resistance indices were not different for age, eFSH treatment, time after eLH administration and oocyte developmental potential. Growth of the dominant follicle was not associated with vascularity, although advanced age tended (P=0.09) to have a negative effect on follicle growth.

[Diagnosis of deficit and attention/hyperactivity disorders (ADHD) in patients with bipolar or unipolar depression: an experimental study]. / Diagnosi pregressa di Disturbo da Defi cit d'Attenzione/Iperattività (ADHD) in pazienti con Depressione Bipolare e Unipolare: uno studio sperimentale.

INTRODUCTION: The present research study starts up from the current scientific and academic interest concerning Deficit and Attention/Hyperactivity Disorders, which in this period seems to have an "epidemic" diffusion. Some authors have proved how the Deficit and Attention/Hyperactivity Disorder may predispose to the development of other psychopathological attitude in adulthood. A recent study has underlined a common comorbidity between ADHD in childhood and Bipolar Disorder. The aim of the present was to verify the existence of an ADHD diagnosis in patients with depression (Unipolar and Bipolar) and to verify if such syndrome overstays in the present psychopathological picture. Moreover there has been even the intention to investigate on a difference in ADHD symptomatology in patients with Bipolar and Unipolar Depression. MATERIALS AND METHODS: The study has been conducted on a sample of 67 patients with depression diagnosis (35 patients with bipolar depression diagnosis, 32 patients with depression unipolar diagnosis) enrolled at the Bipolar Disorders Unit of the Clinical Psychiatry and Drug Dependence Institute of the Policlinico Universitario A. Gemelli in Rome. The evaluation has been performed through the supply of the following psychometric tests: Neo Personality Inventory (Mole-pi-R), Brown Attention Deficit Disorder Scale (Brown ADD-Scale), Adult ADHD Self-Report Staircases (ASRS-v1.1), Criteria of the Deficit and Attention / Hyperactivity Disorder for childhood according to the DSM-IV-Tr. RESULTS: The achieved results point out that 42% of the sample has satisfied the ADHD Criterions during their childhood according to the DSM-IV-Tr and that symptomatology seems to remain in the present psychopathological picture. As to polarity of depression it has emerged that patients with Bipolar Depression diagnosis have satisfied with a greater frequency the ADHD criteria during their childhood than patients with Unipolar Depression. CONCLUSIONS: Our results seem to confirm the hypothesis that patients with bipolar depression diagnosis have more Deficit and Attention / Hyperactivity Disorders comorbidity diagnosis than others.

VURD Syndrome in a Female.

VURD syndrome has been repeatedly described as unilateral reflux into a nonfunctioning renal moiety. This syndrome is considered a pop-off mechanism dissipating pressure in lower urinary tract obstruction: it may be found in association with other protective mechanisms occurring in utero, such as ascites and/or urinomas, and has been exclusively described in male patients. A premature female baby with signs and symptoms of outflow obstruction underwent diagnostic workup revealing congenital urethral hypoplasia with unilateral reflux into a dysplastic kidney. Obstetrical history was positive for early onset, serologically negative ascites without cardiomegaly, which required serial aspirations. Reconstructive surgery was carried out with good results: ascites and VURD syndrome were both deemed to be perinatal protective mechanism against excess pressure in the urinary tract. Although rare, lower urinary tract obstruction in the female can lead to the same protective mechanisms seen in male fetuses/newborns. VURD syndrome and ascites should be interpreted as such and require perinatal specialist counselling.

Temporal gene expression in equine corpora lutea based on serial biopsies in vivo.

A biopsy procedure was developed to enable repeated sampling of a single equine corpus luteum (CL) over the course of an estrous cycle. The tissue collected was utilized in characterizing mRNA abundance for genes involved in luteal formation, function, and regression in the cyclic mare. Serial biopsies of CL in cyclic mares (2.7 to 27.5 mg per biopsy) were collected using an ultrasound-guided transvaginal technique. Biopsies were collected from each mare on d 2 and 5 (d 0 = ovulation) of the estrous cycle, and every other day from d 12 through luteolysis. Samples were obtained from 4 mares with normal estrous cycles and 1 mare with a retained CL. The biopsy procedure did not adversely affect luteal size or function, as measured by luteal area and serum concentrations of progesterone. Real-time reverse-transcription PCR was used to quantify steady state mRNA concentrations in each tissue sample obtained. Mean abundance of steroidogenic acute regulatory protein (StAR) mRNA was not different (P = 0.102 to 0.964) on any of the sampling dates, but a trend for mRNA encoding StAR to decrease between d 12 and 14 (P = 0.10) was observed. Values for mRNA encoding StAR were positively correlated to serum concentrations of progesterone on d 5 (R = 0.95; P = 0.05) and 14 (R > 0.99; P < 0.01). Steady-state abundance of mRNA for 3ß-hydroxysteroid dehydrogenase, Δ 5-Δ 4 isomerase (3ß-HSD) declined between d 12 and 14 (P = 0.15). There were positive correlations between mRNA for 3ß-HSD and concentrations of progesterone on d 5 (R = 0.94; P = 0.06) and 12 (R > 0.99; P = 0.05). No difference was detected in abundance of mRNA encoding cyclooxygenase-2 (cox-2; P = 0.340 to 0.840) or caspase-3 (P = 0.517 to 0.882) between any of the sampling dates. A successful luteal biopsy procedure was developed that did not negatively affect luteal function, and abundance of mRNA encoding StAR, 3ß-HSD, cox-2, and caspase-3 was characterized in luteal biopsy tissue collected on d 2, 5, 12, and 14 of the estrous cycle in the mare.

Fetal urinoma in females without obstructive uropathy.

OBJECTIVE: Prenatal diagnosis of urinomas has long been established with underlying obstructive uropathy generally responsible for urinary extravasation. Because urinoma formation represents a pop-off mechanism in cases of posterior urethral valves, the number of affected males greatly exceeds the number of females. Fetal urinoma has rarely been reported without obstruction and in females it has only been described as a consequence of a complicated amniocentesis. METHODS: Three cases of fetal urinoma in female fetuses without any dilatation of the urinary tract are described. Since the fetus remained healthy, they were all conservatively managed. RESULTS: Two urinomas resolved after birth and 1 exhibited significant regression. In the second case, a compressed kidney was visualized with fetal MRI. Renal function was impaired in cases 1 and 3 and absent in case 2 (the kidney was no longer visualized). CONCLUSIONS: Fetal urinomas can occur even in the absence of urinary tract obstruction and in a low-pressure system as is found in female fetuses. Fetal MRI may help both visualize the ipsilateral kidney and differentiate the mass from other conditions. In a healthy fetus, fetal urinomas can be conservatively managed, but renal function after birth is often absent or impaired. Whether or not in utero aspiration may be beneficial for the preservation of renal function remains unclear.

Effects of age and equine follicle-stimulating hormone (eFSH) on collection and viability of equine oocytes assessed by morphology and developmental competency after intracytoplasmic sperm injection (ICSI).

Young (4 to 9 yr) and old (>or=20 yr) mares were treated with equine follicle-stimulating hormone (eFSH), and oocytes were collected for intracytoplasmic sperm injections (ICSI). Objectives were to compare: (1) number, morphology and developmental potential of oocytes collected from young v. old mares from cycles with or without exogenous eFSH and (2) oocyte morphology parameters with developmental competence. Oocytes were collected from preovulatory follicles 20 to 24 h after administration of recombinant equine LH and imaged before ICSI for morphological measurements. After ICSI, embryo development was assessed, and late morulae or blastocysts were transferred into recipients' uteri. Cycles with eFSH treatment resulted in more follicles (1.8 v. 1.2) and more recovered oocytes (1.1 v. 0.8) than those without eFSH. Age and eFSH treatment did not effect cleavage, blastocyst and pregnancy rates. Treatment with eFSH had no effect on oocyte morphology, but age-associated changes were observed. In old mares, zona pellucidae (ZP) were thinner than in young mares, and perivitelline space and inner ZP volume (central cavity within the ZP) were larger and associated with oocytes that failed to develop. These results suggest that administration of eFSH can increase the number of oocytes collected per cycle. Oocyte morphology differed with age and was associated with developmental competence.

Vitrification of early-stage bovine and equine embryos.

The objectives of this study were to: (1) determine an optimal method and stage of development for vitrification of bovine zygotes or early embryos; and (2) use the optimal procedure for bovine embryos to establish equine pregnancies after vitrification and warming of early embryos. Initially, bovine embryos produced by in-vitro fertilization (IVF) were frozen and vitrified in 0.25mL straws with minimal success. A subsequent experiment was done using two vitrification methods and super open pulled straws (OPS) with 1- or 8-cell bovine embryos. In Method 1 (EG-O), embryos were exposed to 1.5M ethylene glycol (EG) for 5min, 7M ethylene glycol and 0.6M galactose for 30s, loaded in an OPS, and plunged into liquid nitrogen. In Method 2 (EG-DMSO), embryos were exposed to 1.1M ethylene glycol and 1.1M dimethyl sulfoxide (DMSO) for 3min, 2.5M ethylene glycol, 2.5M DMSO and 0.5M galactose for 30s, and loaded and plunged as for EG-O. Cryoprotectants were removed after warming in three steps. One- and eight-cell bovine embryos were cultured for 7 and 4.5 d, respectively, after warming, and control embryos were cultured without vitrification. Cleavage rates of 1-cell embryos were similar (P>0.05) for vitrified and control embryos, although the blastocyst rates for EG-O and control embryos were similar and higher (P<0.05) than for EG-DMSO. The blastocyst rate of 8-cell embryos was higher (P<0.05) for EG-O than EG-DMSO. Therefore, EG-O was used to cryopreserve equine embryos. Equine oocytes were obtained from preovulatory follicles. After ICSI, injected oocytes were cultured for 1-3 d. Two- to eight-cell embryos were vitrified, warmed and transferred into recipient's oviducts. The pregnancy rate on Day 20 was 62% (5/8) for equine embryos after vitrification and warming. In summary, a successful method was established for vitrification of early-stage bovine embryos, and this method was used to establish equine pregnancies after vitrification and warming of 2- to 8-cell embryos produced by ICSI.

Potential involvement of EGF-like growth factors and phosphodiesterases in initiation of equine oocyte maturation.

Human chorionic gonadotropin (hCG) was administered to mares in estrus with large, dominant ovarian follicles to initiate follicular and oocyte maturation. Follicular contents were collected at 0, 2, 4 and 6 h after hCG. Epiregulin, amphiregulin and phosphodiesterase (PDE) mRNA contents of granulosa cells (PDE 4D) were determined by reverse transcription and real-time PCR; PDE 3A mRNA content of single oocytes was determined similarly. Copy numbers of mRNA did not increase for PDE 3A or 4D over the time interval studied. Amounts of epiregulin and amphiregulin mRNA were correlated (r=0.98) when log transformed. Epiregulin and amphiregulin mRNA increased (P<0.01) from controls by 4 h after hCG administration, with amphiregulin increasing (P<0.01) by 2 h after hCG administration. Epiregulin and amphiregulin mRNA levels remained elevated (P<0.01) at 6h after hCG. These results indicate that EGF-like growth factors are likely paracrine mediators of the LH signal in the horse.

The mare model for follicular maturation and reproductive aging in the woman.

Reproductive aging and assisted reproduction are becoming progressively more relevant in human medicine. Research with human subjects is limited in many aspects, and consequently animal models may have considerable utility. Such models have provided insight into follicular function, oocyte maturation, and reproductive aging. However, models are often selected based on factors other than physiological or functional similarities. Although the mare has received limited attention as a model for reproduction in women, comparisons between these species indicate that the mare has many attributes of a good model. As the mare ages, cyclic and hormonal changes parallel those of older women. The initial sign of reproductive aging in both species is a shortening of the reproductive cycle with elevated concentrations of FSH. Subsequently, cycles become longer with intermittent ovulations and elevated concentrations of FSH and LH. Reproduction ceases with failure of follicular growth and elevated gonadotropins, apparently because of ovarian failure. In the older woman and mare, oocytes have been maintained in meiotic arrest for decades -- approximately four to five for the woman and two to three for the mare; in both species, reduced oocyte quality is the end factor identified in age-associated infertility. After induction of oocyte maturation in vivo, the timeline to ovulation is the same for the mare and woman, suggesting a comparable sequence of events. The mare's anatomy, long follicular phase and single dominant follicle provide a foundation for studies in oocyte and follicular development. The aim of this review is to evaluate the mare as an animal model to study age-associated changes in reproduction and to improve our understanding of oocyte and follicular maturation in vivo.

Factors affecting the success of oocyte transfer in a clinical program for subfertile mares.

Oocyte transfer is a potential method to produce offspring from valuable mares that cannot carry a pregnancy or produce embryos. From 2000 through 2004, 86 mares, 19.2 +/- 0.4 yr of age (mean +/- S.E.M.), were used as oocyte donors in a clinical program at Colorado State University. Oocytes were collected from 77% (548/710) of preovulatory follicles and during 96% (548/570) of cycles. Oocytes were collected 21.0+/-0.1h after administration of hCG to estrous donors and cultured 16.4 +/- 0.2 h prior to transfer into recipients' oviducts. At 16 and 50 d after transfer, pregnancies were detected in 201 of 504 (40%) and 159 of 504 (32%) of recipients, respectively, with an embryo-loss rate of 21% (42/201). Pregnancy rates were similar (P > 0.05) for cyclic and noncyclic recipients and for recipients inseminated with cooled, fresh or frozen semen. One or more recipients were detected pregnant at 16 and 50 d, respectively, for 80% (69/86) and 71% (61/86) of donors. More donors <20 than > or = 20 yr (mean ages +/- S.E.M. of 15.5 +/- 0.4 and 23.0 +/- 0.3 yr, respectively) tended (P = 0.1) to have one or more pregnant recipients at 50 d (36/45, 80%; 28/45, 62%, respectively). Results of the program confirm that pregnancies can consistently be obtained from older, subfertile mares using oocyte transfer.

Establishment of pregnancies after serial dilution or direct transfer by vitrified equine embryos.

Experiments were conducted to determine viability of equine embryos in vivo after vitrification. In a preliminary study (Experiment 1), embryos were exposed in three steps to vitrification solutions containing increasing concentrations of ethylene glycol and glycerol (EG/G); the final vitrification solution was 3.4 M glycerol + 4.6 M ethylene glycol in a base medium of phosphate-buffered saline. Embryos were warmed in a two-step dilution and transferred into uteri of recipients. No pregnancies were observed after transfer of blastocysts >300 microm (n = 3). Transfer of morulae or blastocysts < or = 300 microm resulted in four embryonic vesicles (4/6, 67%). In a second experiment, embryo recovery per ovulation was similar for collections on Day 6(28/36, 78%) versus Days 7 and 8(30/48, 62%). Embryos < or = 300 and >300 microm were vitrified, thawed and transferred as in Experiment 1. Some embryos < or = 300 microm were also transferred using a direct-transfer procedure (DT). Embryo development rates to Day 16 were not different for embryos < or = 300 microm that were treated as in Experiment 1(10/22, 46%) or transferred by DT (16/26, 62%). Embryos > 300 microm (n = 19) did not produce embryonic vesicles.

Oocyte transfer and gamete intrafallopian transfer in the mare.

Methods for the collection and transfer of equine oocytes have been developed, and uses of these techniques have resulted in new clinical and research possibilities. Because oocyte transfer avoids reproductive problems associated with the oviduct, uterus, and cervix, pregnancies can be produced from many mares that cannot carry a pregnancy or produce embryos. Oocytes for clinical transfers are usually collected from preovulatory follicles and cultured for a short interval or transferred directly into a recipient's oviduct. For oocyte transfer, the recipient is inseminated within the uterus. A large number (1 x 10(9) to 2 x 10(9)) of motile sperms are preferred for inseminations. In contrast, sperm and oocyte are transferred into the oviduct during gamete intrafallopian transfer (GIFT). Therefore, a lower number (1 x 10(5) to 2 x 10(5)) of sperm can be used. Potentially, GIFT could be used in situations where sperm numbers are limited. Use of oocyte transfer and GIFT in clinical and research settings will aid us in understanding the interactions between oocyte, sperm, and oviduct in the equine.

In vitro maturation and transfer of equine oocytes after transport of ovaries at 12 or 22 degrees C.

Transportation of equine ovaries would allow shipment of oocytes for research purposes or transfer after the death of a valuable mare. The objective of this study was to compare two temperatures for maintaining ovaries during a transport interval of 18-24 h. The goal was to obtain pregnancies after transport of ovaries, maturation of oocytes in vitro, and transfer of oocytes. Each shipment was composed of ovaries four to seven mares collected from an abattoir. From each mare, one ovary was packaged at approximately 12 degrees C, and the other was packaged at approximately 22 degrees C. Upon arrival at our laboratory, oocytes were collected and cultured for 24 h. For each transfer, between 9 and 15 oocytes from each group were placed into the oviducts of estrous mares through standing flank laparotomies. Recipients received human chorionic gonadotropin (hCG; 2000 IU, i.v.) 30-36 h before transfer (to synchronize ovulation). Recipients were inseminated 18-20 h before transfers with 2 x 10(9) progressively motile sperm. Uteri of recipients were examined with ultrasound to determine the number of developing embryos. On Day 16 ( ovulation = day 0), developing embryos were recovered by uterine lavage. Parentage verification was performed on recovered vesicles. Pregnancy rates were analyzed by Chi-square. The percentage of oocytes that developed into embryonic vesicles on Day 16 was not different between transport temperatures (22 degrees C, 13/73, 18% versus 12 degrees C, 11/73, 15%). In conclusion, pregnancies were obtained from in vitro matured oocytes that were recovered from ovaries transported for 18-24h at 12 or 22 degrees C.