Irvine Scientific Research Articles

Development of the safety cytoplasmic exchange method in pronuclear stage oocyte; prevention of mitochondria coexistence in cytoplasmic that used the pronuclear microinjection

OBJECTIVE: The effectiveness of oocyte cytoplasmic exchange has already been demonstrated for the treatment of the infertility cause of defective cytoplasm and of the prevention of heredity of the mitochondrial diseases (Kuwayama, 58th ASRM meeting, 2002). However, mitochondria is brought in with the nuclear when the cell membrane fusion method (McGrath, et al.Science 1983) is used, and two kinds of mitochondria, derivation from both of donor and recipient mitochondria, exist together consequentially in the donor cytoplasm. Because mitochondria coexistence does not exist in nature, and the safety in the situation is uncertain, these two types of mitochondria coexistence is an obstruction in a clinical use of this technology application. To prevent mitochondria coexistence, we tried the development of the pronuclear microinjection method. DESIGN: Prospective in vitro study. MATERIALS AND METHODS: The pronuclear stage oocytes with informed consent were used for the experiment. A pronuclear was removed from the cell membrane crushed oocyte that was crushed by the pulse of the Piezo micromanipulator (the cell membrane crushing method) in modified P1 medium containing 60% of serum substitute supplement (SSS, Irvine Scientific). To remove the cytoplasm containing mitochondria, the removed pronuclear was washed by the repetitive pipetting with the small glass pipette assembled in the micromanipulator. The cytoplasm-free pronuclear was injected directly to the donor cytoplasm with the glass pipette of the inside diameter 15 or 25 μm. The pronuclear injected oocytes were cultured with Cleavage Medium (SAGE) containing 10% of SSS for 30 minutes 5% CO2 5% O2 and 90% N2 at 37C. The oocyte that the cell membrane had been maintained intact after 30 minutes from the pronuclear microinjection was judged survival and the oocyte that was able to confirm the pronuclear intact was judged the pronuclear microinjection success. RESULTS: The survival rate in the pronuclear micro injected oocyte using a pipette inner diameter 15 or 25 μm were 93% (37 / 40) and 50% (20 / 40) (P<0.01), and the pronuclear microinjection success rate were 78% (29 / 37) and 70% (14 / 20), respectively. CONCLUSIONS: The pronuclear that was removed from the oocyte without cytoplasm is able to inject into the enucleated cytoplasm directly. The result shows the possibility that this pronuclear microinjection method becomes the effective treatment of the infertility cause of defective cytoplasm, and of the prevention of heredity of the mitochondrial diseases.

The effect of embryo glue on clinical pregnancy rate in frozen embryo transfers

OBJECTIVE: The goal of this study was to investigate whether the use of EmbryoGlue® had any effect on the Clinical Pregnancy Rate (CPR) in Frozen Embryo Transfer (FET) cycles of cleavage stage embryos. EmbryoGlue® contains hyaluronan and product literature states that it contains nutrients that are required to support the embryo from transfer to implantation and the biomechanical interactions during implantation. We designed this study to test if the use of EmbryoGlue® in FET cycles increased Clinical Pregnancy Rates as compared with embryos transferred in conventional culture media, Multiblast (Irvine Scientific).

Comparison of day 3 embryo cryo-survival and morphology after slow-rate freeze/thaw or vitrification/warming

OBJECTIVE: Slow-rate freezing/thawing (SRF) and vitrification/warming (V) differ in concentration of cryoprotectants used, times of cryoprotectant exposure, rate of temperature decrease and increase, and presence or absence of intracellular and extracellular ice crystal formation. We aimed to compare cleavage-stage embryo SRF/thawing to V/warming in relation to number of degenerated embryos, blastomere loss and change in fragmentation grade after cryopreservation. DESIGN: Retrospective study. MATERIALS AND METHODS: Supernumerary embryos not transferred during a fresh IVF cycle were cryopreserved by either SRF or V between 01/04 to 02/08. Embryo cryopreservation by SRF was performed with propanediol and sucrose, with a programmable freezer. Rapid thawing was performed with stepwise dilutions of sucrose. Vitrification and warming were performed according to manufacturer's instructions (Vitrification Freeze and Thaw Kit, Irvine Scientific). Within 1 hour of thawing/warming embryos were microscopically assessed for survival (degenerate: < 50% of original blastomeres present), number of blastomeres, and percentage of fragmentation. Fragmentation grades were assigned: 1: ≤10%, 2: 11-20%, 3: 21-50%, 4: >50%. Results were represented as mean±standard error of the mean. Statistical comparisons were performed with Student's t-test and considered significant when p<0.05. RESULTS: Embryos thawed following SRF numbered 145 from 30 cycles, equating to 5±2 embryos thawed per cycle. Embryos warmed following V numbered 508 from 126 cycles, yielding 4.0±2 embryos warmed per cycle. The number of degenerated embryos was significantly higher after SRF/thawing (1.5±0.1) in comparison to V/warming (0.5±0.1), giving survival percentages of 69% (SRF) and 88% (V). Of embryos that survived there was no significant difference in numbers of blastomeres lost following thawing or warming. However, there was an increase in fragmentation following SRF/thawing compared to V/warming which resulted in a significant increase in fragmentation grade of embryos after thawing (0.3±0.05) compared to warming (0.1±0.01). CONCLUSIONS: Cryopreservation of cleavage-stage embryos by vitrification, followed by warming, results in enhanced embryo survival rates and better embryo morphology in comparison to SRF/thawing. Future prospective studies are required to determine which procedure is superior in yielding pregnancy rates, live-birth rates, and healthy offspring.

Validation of a simple and effective sterile vitrification system for embryos

OBJECTIVE: In a decade of escalated concern and awareness for good tissue handling practices (GTPs) and FDA regulations, cryopreservation methods that emphasize secure storage, technical ease/repeatability, and consistent outcomes are needed. DESIGN: Frozen-thawed 1-cell mouse embryos (Embryo-Tech) were used to validate a novel double container vitrification procedure. MATERIALS AND METHODS: The vitrified (VTF) embryos were pipette into shortened (cut: 1.5-2.0 cm) denuding pipettes (275-300 μm ID), detached, loaded tip-end first and then LN2 plunged /stored in sealed CBS™ High Security straws. Mouse 2-cell and blastocyst (BL) stage embryos (5/straw) were VTF and rewarmed using Irvine Scientific kits. A preliminary expt. (I) was performed to test BL survivability compared to conventional freezing. In expt.2, 2-cell embryos were VTF and cultured to the BL stage. In expt. 3, cooling and warming rate curves were validated using micro-thermocouples and a datalogging device. In expt. 4, BL survivability was reassessed using a modified warming step where the tip was immediately placed in warm H-HTF solution for 10 sec upon removal and then reattached to a Stripper™ device to decant embryos and pipette them serially. All embryos were cultured in LG medium + 5% HSA in 25μl microdroplets. RESULTS: In expt. 1, 67% re-expansion of re-frozen BL's occurred, similar to controls. In expt. 2, 16 to 20 VTF 2-cells produced ≥ expanded BLs (80%), similar to control cultured 2-cells (80% BL). In expt. 3, cooling and warming rates inside the CBS straws adjacent to, as well as inside, the contained VS-stripper tip itself were measured, warming in methanol or in air. The VTF-tip sealed in a CBS™ straw cooled at a rate of 1000 ± 100°C/ min between −10°C and −140°C. Warming rates were similar with both methods up to −70°C, but slowed beyond that in air. To better maintain high warming rates above −70°C, we placed the isolated tip in warm H-HTF for 10 sec before securing the tip for embryo removal. In expt. 4, 25 full to hatched mouse BLs were VTF and rewarmed using the latter method, with all 25 BLs recovered, surviving and developing at 24 hr (100%), being similar to LAH treated controls and VS-exposed BLs. CONCLUSIONS: The data support the promising effectiveness of a novel VTF procedure which offers safe, sterile LN2 storage and technical simplicity, with excellent recovery and survival rates. Additional studies with different solutions are needed to optimize the safety of this procedure for vitrifying human oocytes and embryos.

The value of repeat testicular sperm retrieval in azoospermic men

To determine the predictive value of a previous testicular biopsy to the chance of sperm retrieval in the next testicular sperm extraction (TESE) procedure, we retrospectively analyzed the outcome of past sperm collection procedures and histopathology diagnoses of patients with nonobstructive azoospermia. Repeated TESE ensured a high recovery rate (96%) when the first recovery procedure had been successful and when hypospermatogenesis was diagnosed (77%); when no spermatozoa were found on the first attempt, a repeat TESE procedure was successful in one-third of the patients.

A Novel Approach to Embryo Vitrification by LN 2 Vapor Storage of Stripper Tips in CBS High-Security Straws

Introduction: Embryo vitrification first succeeded in 1986 (Rall and Fahey). Since then various vitrification solutions (VS) and container types have been investigated. The combination of DMSO and EG (≥30% v/v) has been widely applied as the VS of choice. Owing to the sensitive nature of osmotic stress (“toxicity”) in high-molar VS, the ease of technique application and technician consistency are important factors. Additionally, there is increasing concern over pathogen cross-contamination and the safe long-term storage of gametes and embryos in LN2. Objective: To develop a vitrification procedure using FDA-approved devices/solutions that is effective, easily repeatable, and offers secure contamination-free LN2 storage, before we implement vitrification into our clinical egg and embryo cryopreservation plan. Methods: Frozen 1-cell mouse embryos (Embryo-Tech) were cultured to expanded/hatching blastocyst (BL) in tri-gas Sanyo mini-incubators for proficiency testing purposes. Fifty good-quality BL were randomly assigned to either a control group (Grp 1) or a vitrification treatment (Grp 2). In Grp 1, BL were diluted in 1.5 mol/L glycerol solution for 10 min + 0.2 mol/L sucrose for 5 min before being loaded and sealed in CBS straws and slow cooled from −7°C to −35°C at 0.5°C/min. Embryos were thawed using a standard Irvine Scientific BL Thaw kit. In Grp 2, BL were vitrified and thawed using the Irvine Scientific VS Cryo and Thaw kits as described, the primary difference being that the 275-μm denuding pipette (Stripper Tip) used for embryo handling was also used as the final VS-embryo storage container (approx. 2 μL volume). Each Stripper Tip was shortened about 1.5 cm to accommodate placement into a prelabeled/sealed CBS straw, tip-end first, and then sealed again (both sides securely sealed) using a Symm sealing device before direct placement into LN2 (plunged within 1 min of final VS exposure). Each straw contained 5 BL/straw. Results: No difference was observed between treatment groups, with 17 of 25 BL (68%) in both Grp 1 and Grp 2 re-expanding and continuing development 24 h after thaw. Conclusions: A recent report by Isachenko et al. (Cryobiol 54:305) indicated that a double-straw container system can reduce the ultra-rapid cooling rate of vitrification up to 25-fold (15,000°C/min in open container versus 600°C/min, respectively) without any effect on BL survival. In this study, the use of CBS straws to contain vitrified embryos within a denuding pipette has proven to be a simple and effective approach to contamination-free cryostorage.

Hyperglycosylated Human Chorionic Gonadotropin Production by Human Preimplantation Embryos In Vitro

Background: Hyperglycosylated hCG (HhCG) is produced by the cytotrophoblasts during implantation in higher quantities than the traditional form of hCG. It has been reported that embryos make hCG prior to implantation, but it has not been previously investigated whether or not they also secrete HhCG. We hypothesized that human preimplantation embryos produce HhCG and that this may be a useful marker of embryo development. Objective: To investigate whether preimplantation embryos produce HhCG. Materials and Methods: Female subjects under age 38, undergoing autologous oocyte IVF, were offered participation in this study. All oocytes were cultured separately in P1 media (Irvine Scientific, CA) and transfered to fresh media daily. On day 3, embryos were assessed for cell number and grade. Discarded culture media from embryos was collected daily starting 48 hours after aspiration until embryo transfer. HhCG was measured in culture media from transfered embryos by an electrochemiluminescence immunoassay (Quest Diagnostics Nichols Institute, CA). The assay had a sensitivity of 2 pg/mL and a CV of <12%. Pregnancies were diagnosed with serum hCG measurements and ultrasound. Results: Five subjects, aged 34.0 ± 1.3 yrs (mean ± SEM), were included. From each subject 23.4 ± 2.3 oocytes, which resulted in 14.2 ± 2.2 embryos, were obtained and 2.8 ± 0.4 embryos were subsequently transfered. 80% (4/5) of the subjects had a positive pregnancy test, of which 2 resulted in ongoing pregnancies. The implantation rate was 35.7%. The HhCG level on day 2 was 4.69 ± 0.93 pg/mL compared with 4.70 ± 0.70 pg/mL on day 3 (P=.86). HhCG levels in conception cycles were 5.02 ± 1.00 pg/mL on day 2 and 5.00 ± 0.70 pg/mL on day 3, whereas in the nonconception cycle they were 3.04 ± 3.04 pg/mL on day 2(P=.45) and 2.90 ± 2.90 pg/mL on day 3 (P=.31). Embryo morphology did not correlate with HhCG levels in culture media on day 2 or 3. Conclusions: 1) Human preimplantation embryos produce HhCG in culture media in small but measurable amounts. 2) This is the first report of HhCG production by embryos. 3) HhCG levels on day 2 or day 3 do not appear to correlate with embryo morphology or pregnancy potential.

CD117 Expression May Signify Enduring Proliferative Capacity of Amniotic Fluid-Derived Mesenchymal Stem Cells (MSC).

INTRODUCTION: Recent publications have suggested that a highly pluripotent stem cell population may be derived from the cellular fraction of amniotic fluid. During the surgical correction of twin to twin transfusion syndrome by fetoscopic laser ablation, large amounts of amniotic fluid are harvested, the cellular component of which could serve as a potentially rich source of amniotic fluid-derived pluripotent stem cells. To explore this hypothesis, we collected large amounts of amniotic fluid following fetoscopic laser ablation procedures and sought to generate and characterize amniotic fluid-derived stem cells (AFS) as described previously.1METHODS: Amniotic fluid was collected following fetoscopic laser ablation with IRB approval. The total cellular component was cultured in α-MEM (Hyclone) supplemented with 15% ES FBS (Invitrogen), 100 U/ml penicillin G/streptomycin, 2 mM L-glutamine, 18% Chang Basal Medium, and 2% Chang C Medium (both from Irvine Scientific). Adherent cells were expanded and selected with CD117 microbeads (Miltenyi Biotec). Cells were characterized by flow cytometry, and hematologic pluripotence was assessed by culture in 100 ng/ml GM-CSF, co-culture with OP-9 cells, or co-culture with primary human bone-marrow stroma. Proliferative capacity was assessed and doubling times were determined for all serial passages.RESULTS: Amniotic fluid derived cells were typically CD90posCD105posHLA-DRneg with minority populations that were HLA-DRlow and CD133pos. This phenotype is consistent with that of bone marrow-derived MSCs. Cellular morphology was also consistent with that of bone marrow-derived MSCs. We did not detect any expression of CD34, CD45, or CD38 either pre- or post-CD117 selection. Some samples were fully devoid of CD117pos cells; however, others did exhibit CD117-expressing cells which could be selected. Attempts at transdifferentiation of either CD117pos or CD117neg cells by ES cell methods were unsuccessful; the AFS cell phenotype remained consistent throughout the addition of exogenous cytokines or co-culture with bone marrow stroma. CD117neg AFS cell lines began to divide more slowly, exhibiting doubling times of 90 hours by post-selection passage 10. In contrast, CD117pos AFS cell lines maintained a doubling time of 25–30 hours through post-selection passage 10.CONCLUSIONS: The AFS cells derived from fetoscopic laser ablation procedures exhibited an MSC phenotype and did not appear to have pluripotent plasticity. We hypothesize that AFS cells with greater plasticity might exist at the time of diagnostic amniocentesis (week 14–16) but appear to be absent in the fluid samples that we obtained on weeks 24–26. The AFS MSCs had a proliferative potential greater than that exhibited by bone marrow MSCs, and the presence of surface CD117 (SCF receptor) seemed to be predictive of an enduring proliferative ability. Further study will be necessary to determine the validity and significance of this correlation.

Better ongoing pregnancy rates from frozen embryo transfers when vitrified blastocysts are used

Better ongoing pregnancy rates from frozen embryo transfers when vitrified blastocysts are used

New insights into immunological infertility testing for anti-sperm antibodies and sperm-zona pellucida binding ability by the immunobead assay and sperm-hyaluronic acid interaction

New insights into immunological infertility testing for anti-sperm antibodies and sperm-zona pellucida binding ability by the immunobead assay and sperm-hyaluronic acid interaction

Influence of semen source on early cleavage in ICSI cycles

Choosing the “best embryos” for transfer is one of the critical steps of in vitro fertilization. Early embryo cleavage (EC), defined as the presence of 2 cell embryo in less than 25 hours after ICSI, is one of the parameters that may be taken into account for selection. The influence of paternal/sperm factors related to IVF outcomes and to embryo development also remains poorly recognized. We evaluated the influence of semen source and quality on IVF outcomes with special attention to EC. Retrospective study involving 225 ICSI patients. We assessed the developmental characteristics of 1959 oocytes submitted to treatment with ICSI. The semen samples were divided in 5 groups, according to quality and origin. G1: Ejaculated normal semen (>20 × 106/ml); G2: Ejaculated abnormal semen (>2 × 106/ml and <20×106/ml); G3: Ejaculated semen with severe abnormality (<2 × 106/ml); G4: Semen from Percutaneous Epididymal Sperm Aspiration (PESA); and G5: Semen from Testicular Sperm Aspiration (TESA). Ovarian stimulation was performed by long protocol using Lupron and recombinant FSH. HCG was administered when at least 3 follicles reached 19mm diameter. The number of oocytes, fertilization, EC, transferred embryos, and pregnancies, were evaluated and analyzed. The culture media used was HTF (Irvine Scientific) plus 15% Synthetic Serum Substitute. Statistical analyses was performed using the Chi-square test and the One-way ANOVA test; significance was established at P<0.05. Mean age for female patients was 32.92 years. The percentage of embryos with EC was 53.2%, 51.6%, 51.6% in groups 1, 2 and 3, and 29.6% and 33.7% in groups 4 and 5, respectively. In the groups with ejaculated sperm, the presence of embryos with EC was higher but not significantly different among them. The proportion of embryos with EC in the PESA or TESA groups were lower but not significantly different among them, however, it was significantly lower compared to G1, G2 and G3. The overall pregnancy rates were 48.5%, 47.7%, 30.0% 53.3%, and 41.7% in groups 1 to 5 respectively (NS). Sperm derived from the ejaculate seems to be associated with higher frequency of EC, even in conditions of severe oligozoospermia, than sperm from PESA or TESA. Poorer outcomes in terms of EC when TESA and PESA sperm was used may be associated with sperm immaturity. Otherwise, sperm source and EC did not determine pregnancy outcome.

In vitro fertilization outcome using three types of culture media containing different concentrations of glucose for developing cleavage stage embroys. a comparative study

To compare the IVF outcome of cleavage stage embryos incubated in three different culture media: a) Universal IVF Medium (IVF-Med) – containing 5.55 mM glucose (Medi-Cult, Copenhagen, Denmark). b) ISM1 medium – containing 1 mM glucose (Medi-Cult, Copenhagen, Denmark). c) P1- glucose and phosphate free medium supplemented with 16% synthetic serum substitute (Irvine Scientific, Santa Ana, CA, USA).

Enzymes Responsible for Synthesis of Corneal Keratan Sulfate Glycosaminoglycans

Keratan sulfate glycosaminoglycans are among the most abundant carbohydrate components of the cornea and are suggested to play an important role in maintaining corneal extracellular matrix structure. Keratan sulfate carbohydrate chains consist of repeating N-acetyllactosamine disaccharides with sulfation on the 6-O positions of N-acetylglucosamine and galactose. Despite its importance for corneal function, the biosynthetic pathway of the carbohydrate chain and particularly the elongation steps are poorly understood. Here we analyzed enzymatic activity of two glycosyltransferases, beta1,3-N-acetylglucosaminyltansferase-7 (beta3GnT7) and beta1,4-galactosyltransferase-4 (beta4GalT4), in the production of keratan sulfate carbohydrate in vitro. These glycosyltransferases produced only short, elongated carbohydrates when they were reacted with substrate in the absence of a carbohydrate sulfotransferase; however, they produced extended GlcNAc-sulfated poly-N-acetyllactosamine structures with more than four repeats of the GlcNAc-sulfated N-acetyllactosamine unit in the presence of corneal N-acetylglucosamine 6-O sulfotransferase (CGn6ST). Moreover, we detected production of highly sulfated keratan sulfate by a two-step reaction in vitro with a mixture of beta3GnT7/beta4GalT4/CGn6ST followed by keratan sulfate galactose 6-O sulfotransferase treatment. We also observed that production of highly sulfated keratan sulfate in cultured human corneal epithelial cells was dramatically reduced when expression of beta3GnT7 or beta4GalT4 was suppressed by small interfering RNAs, indicating that these glycosyltransferases are responsible for elongation of the keratan sulfate carbohydrate backbone.

Molecular Changes in the Vasculature of Injured Tissues

We have explored molecular specialization of the vasculature of regenerating wound tissue in the skin and tendons to identify a different repertoire of markers from that obtained by studying tumor vasculature. We screened a phage-displayed peptide library for peptides that home to wounds in mice and identified two peptides that selectively target phage to skin and tendon wounds: CARSKNKDC (CAR) and CRKDKC (CRK). CAR is homologous to heparin-binding sites in various proteins and binds to cell surface heparan sulfate and heparin. CRK is similar to a segment in thrombospondin type 1 repeat. Intravenously injected CAR and CRK phage, as well as fluorescein-labeled CAR and CRK peptides, selectively accumulated at wound sites, where they partially co-localized with blood vessels. The CAR peptide showed a preference for early stages of wound healing, whereas the CRK favored wounds at later stages of healing. The CAR peptide was internalized into the target cells and delivered the fluorescent label into the cell nuclei. These results identify new molecular markers in wound tissues and show that the expression of these markers in wound vasculature changes as healing progresses. The peptides recognizing these markers may be useful in delivering treatments into regenerating tissues.

A Key Role for Orphan Nuclear Receptor Liver Receptor Homologue-1 in Activation of Fatty Acid Synthase Promoter by Liver X Receptor

Liver X receptor (LXR) activates fatty acid synthase (FAS) gene expression through binding to a DR-4 element in the promoter. We show that a distinct nuclear receptor half-site 21 bases downstream of the DR-4 element is also critical for the response of FAS to LXR but is not involved in LXR binding to DNA. This half-site specifically binds liver receptor homologue-1 (LRH-1) in vitro and in vivo, and we show LRH-1 is required for maximal LXR responsiveness of the endogenous FAS gene as well as from promoter reporter constructs. We also demonstrate that LRH-1 stimulation of the FAS LXR response is blocked by the addition of small heterodimer partner (SHP) and that FAS mRNA is overexpressed in SHP knock-out animals, providing evidence that FAS is an in vivo target of SHP repression. Taken together, these findings identify the first direct lipogenic gene target of LRH-1/SHP repression and provide a mechanistic explanation for bile acid repression of FAS and lipogenesis recently reported by others.

Ten-year experience with preimplantation genetic diagnosis (PGD) at the New York University School of Medicine Fertility Center

We describe our experience of over 300 cycles of preimplantation genetic diagnosis (PGD) and report clinical pregnancy rates (35%-67%) that support using this technology to screen for genetic disorders and chromosomal abnormalities. In clinical practice for over ten years, PGD offers couples the earliest form of genetic screening and may help improve ongoing pregnancy rates in poor-prognosis patients.

Positive expression of the immunoglobulin superfamily protein IZUMO on human sperm of severely infertile male patients

Recently, the immunoglobulin superfamily protein IZUMO on the sperm surface was identified as being essential for sperm-egg fusion. Although we examined the expression of IZUMO on sperm of 13 male infertile patients with severe oligozoospermia and/or athenozoospermia and 12 infertile patients with fertilization failure in previous conventional IVF to determine whether it is varied in infertility patients, we could not identify any patients with IZUMO negative by immunocytochemistry.

267 BIRTH OF DOMESTIC CAT KITTENS OF PREDETERMINED SEX AFTER TRANSFER OF EMBRYOS PRODUCED BY IN VITRO FERTILIZATION OF OOCYTES WITH FLOW-SORTED SPERMATOZOA

In cats, sex selection by fertilization of oocytes with sperm sorted into X- andY-chromosome-bearing populations has credible biomedical, commercial, and conservation connotations. Our objectives were (1) to evaluate the efficiency with which embryos could be produced by IVF of in vivo- and in vitro-matured oocytes with cooled sex-sorted sperm after overnight shipment to the sorting facility and overnight return delivery to an IVF laboratory, and (2) to determine if live kittens of predetermined sex (female) could be produced after transfer of embryos derived by IVF of in vivo-matured oocytes with X-chromosome-bearing sperm to recipient females. Semen samples (n = 5) collected from a single male using an artificial vagina were extended in electrolyte-free solution (glucose–BSA) and shipped overnight in an Equitainer (4�C) to the sorting facility. Upon arrival, sperm were stained (9 µm Hoechst 33 342; 75 � 106 sperm mL–1), adjusted to 50 � 106 sperm mL–1 with 4% egg yolk TALP containing 0.002% food dye, and sorted on an SX MoFlo� flow cytometer (Dako, Fort Collins, CO, USA). Purities from resort analysis averaged 94% (X) and 83% (Y). After sorting, sperm were concentrated by centrifugation, suspended in TEST-yolk buffer (Irvine Scientific, Santa Ana, CA, USA), and return-shipped to the IVF lab where they were received 48 h after collection. In vivo-matured oocytes recovered from gonadotropin-treated donors were inseminated in vitro with X-chromosome-bearing sperm. In vitro-matured oocytes obtained from ovaries donated by local clinics were inseminated in vitro with control (cooled/shipped/non-sorted) or X- orY-chromosome-bearing sperm. At 5 h or 18 h post-insemination, in vivo- and in vitro-matured oocytes, respectively, were rinsed and placed in IVC-1 medium (Pope et al. 2006 Theriogenology 66, 59–71). Embryos produced from in vitro-matured oocytes were allowed to develop until Day 8 in a three-step culture system (Pope et al. 2006). Cleavage frequency of in vitro-matured oocytes after insemination with X-, Y-, and control sperm was 33% (40/120), 35% (52/150), and 42% (48/115), and blastocyst development was 50% (11/22), 55% (23/42), and 53% (21/40), respectively (P &gt; 0.05). Incidence of cleavage after insemination of in vivo-matured oocytes with X-sperm was 62% (54/87). On Day 2, 45 embryos (9–16 per recipient) produced by in vitro insemination of in vivo-matured oocytes with X-sperm were transferred by laparoscopy to the oviducts of four Day 1 gonadotropin-treated recipients. Three recipients established pregnancies and delivered litters of one, four, and seven female kittens between Day 62 and Day 66 of gestation. We have demonstrated that sperm-sorting technology can be applied and used effectively in domestic cats and, potentially, should be relevant to the selective breeding of endangered cats.

213 IN VITRO DEVELOPMENT OF BOVINE EMBRYOS IN THE PRESENCE OF GROWTH HORMONE, INSULIN-LIKE GROWTH FACTOR-1, AND INSULIN IN OOCYTE MATURATION AND EMBRYO CULTURE MEDIA

The addition of hormones and growth factors to bovine IVM and IVC media has been reported to affect early embryonic development by enhancing the blastocyst formation rate and quality of embryos produced. The purpose of this study was to investigate the influence of adding growth hormone (GH), insulin-like growth factor-1 (IGF-1), and insulin to IVM and IVC media. Blastocyst production rate and blastocyst quality, as verified by the number of cells with DNA fragmentation, were evaluated. Ovaries from an abattoir were transported to the laboratory and COC were selected and cultured in IVM medium 199 (Earle's salts, Sigma, St. Louis, MO, USA), 10% fetal calf serum (Sigma), 50 µg mL–1 of sodium pyruvate, 1 µg mL–1 of estradiol (Sigma), 50 µg mL–1 of hCG (Profasi hp�, 5000 IU, Serono Inc., Rockland, MA, USA), 5 µg mL–1 of FSH (Folltropin�, Vetrepharm, Ontario, Canada), and 75 µg mL–1 of gentamicin sulfate for 24 h. After IVF (18 h), zygotes were partially denuded and transferred to IVC medium HTF (HTF�, Irvine Scientific, Santa Ana, CA, USA) and BME (BME�, Sigma), in a 1:1 proportion (HTF:BME), 0.6% BSA (Sigma), 0.01% myoinositol (Sigma), and 75 µg mL–1 of gentamicin sulfate, at 38.5�C, in a humidified atmosphere of 5% CO2 in air, supplemented with 10% fetal calf serum at Day 3 of culture. Three different experiments were performed. The first and second experiments were analyzed using the chi-square test (P &lt; 0.05). The third experiment was analyzed with the general linear model of SAS� (SAS Institute Inc., Cary, NC, USA) and the Tukey test (P &lt; 0.1). In the first experiment, oocytes were cultured in IVM medium supplemented with GH (10 ng mL–1), IGF-1 (100 ng mL–1), insulin (1 µg mL–1), or all 3 combined. In the second experiment, IVC medium was supplemented with GH, IGF-1, insulin, or all 3 combined (same concentrations as above). In the third experiment, the quality of the embryos produced in the first 2 experiments was determined by the percentage of cells with DNA fragmentation. After 96 h of culture, embryos were stained with orange acridin (100 µg mL–1) and propidium iodide (100 µg mL–1) and slides were evaluated by fluorescence microscopy (450 to 490 nm). Rates of blastocyst production (blastocysts/oocytes) in the first experiment (29, 28, 28, 26, and 28% for control, GH, IGF-1, insulin, or all 3 combined, respectively) and in the second experiment (35, 35, 36, 35, and 31%) were not statistically different among the groups. In the third experiment, the addition of GH, IGF-1, or insulin to IVM medium did not affect the DNA fragmentation rate (11, 5, 2, 12, and 12%). However, the addition of insulin to IVC medium led to a higher DNA fragmentation rate (24%), when compared with the other groups (11, 10, 6, and 8% for control, GH, IGF-1, and all 3 combined). The addition of GH or IGF-1 to bovine IVM and IVC media did not affect the blastocyst production rate or the quality of embryos produced. The quality of embryos cultured in the presence of insulin was negatively affected.

56 DEVELOPMENT OF INTERSPECIES CLONED EMBRYOS USING SOMATIC CELLS FROM VARIOUS SPECIES AND BOVINE CYTOPLASTS

Interspecies somatic cell nuclear transfer (iSCNT) is an invaluable tool for studying nucleus–cytoplasm interaction and it provides a possible alternative to cloning animals whose oocytes are limited. In Experiment 1 of the present study, we investigated the developmental potential of iSCNT embryos created from monkey, pig, and goat donor cells and bovine cytoplasts. Bovine ovaries were obtained at a local slaughterhouse and the cumulus-oocyte complexes (COCs) aspirated. COCs were matured in vitro in TCM-199 supplemented with 10 IU mL–1 pregnant mare serum gonadotropin (PMSG), 10 IU mL–1 hCG, and 10 ng mL–1 epidermal growth factor (EGF) at 38.5�C and 5% CO2 in air for 20–22 h. At the end of IVM, half of the COCs were inseminated using frozen semen (1 � 106 sperm mL–1) and the remainder were used for iSCNT after the cumulus cells were removed with 0.1% hyaluronidase in TCM-199. The procedure of iSCNT and establishment of donor cells were according to Koo et al. (2002 Biol. Reprod. 67, 487–492). After IVF and iSCNT, presumptive zygotes were cultured in CR1-aa medium supplement with 0.3% BSA. After 3 days, cleaved embryos were transferred to CR1-aa medium supplemented with 10% FBS and cultured for an additional 4 days. In Experiment 2, we investigated the developmental ability of reconstructed embryos produced from monkey cells and bovine cytoplasts using various IVC media, such as IVC-1/2 (InVitroCare, Frederick, MD, USA), G-1/2 (Vitrolife, Inc., Englewood, CO, USA) and complete medium (CM; Irvine Scientific, Santa Clara, CA, USA). All experiments were repeated more than three times and data were analyzed with t-test of one-way ANOVA using the SAS 8.01 program (SAS Institute, Inc., Cary, NC, USA). Cleavage and developmental rate of blastocysts were expressed as mean � SEM. In Experiment 1, we investigated the development ability among IVF, SCNT (bovine-bovine), and iSCNT (monkey-bovine, pig-bovine, and goatbovine) embryos cultured in CR1-aa medium. Our results showed that the cleavage rate of IVF (73.6 � 1.8%, 86/117) embryos was not significantly different compared to SCNT (84.6 � 2.7%, 38/45), and iSCNT (89.3 � 2.7%, 100/110, monkey; 89.3 � 3.3%, 45/49, pig; and 86.0 � 2.3%, 87/95, goat). Although cloned embryos reconstructed with monkey cells did not develop to the blastocyst stage, iSCNT embryos derived from pig and goat cells did (3.3 � 3.0%, 2/49, and 7.9 � 1.7%, 7/95, respectively). However, these blastocyst formation rates were significantly lower compared to those of IVF and SCNT bovine embryos (32.5 � 2.9%, 38/117, and 26.7 � 2.8%, 12/88, respectively; P &lt; 0.05). The success of iSCNT was confirmed by PCR of mitochondrial DNA, porcine PKA region, and SRY region. In Experiment 2, we investigated the developmental potential of cloned embryos produced by monkey cells using various IVC media (IVC-1/2, G-1/2, and CM). The cleavage rate of iSCNT embryos was not significantly different among these media (86.9 � 2.7%, 78.1 � 2.1%, and 82.3 � 1.8%, respectively). However, we did not observe blastocyst formation using these media. Therefore, we suggest that the cytoplasts of bovine oocytes can support blastocyst development of cloned embryos with pig and goat cells, but they were not suitable for monkey cells. In conclusion, our results suggest that species-specific differences are apparent in the production of iSCNT embryos.