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Time-lapse imaging was used to establish the morphokinetics of equine embryo development to the blastocyst stage after in vitro oocyte maturation (IVM), intracytoplasmic sperm injection (ICSI) and embryo culture, in oocytes held overnight at room temperature (22–27°C; standard conditions) before IVM. Embryos that developed to the blastocyst stage underwent precleavage cytoplasmic extrusion and cleavage to the 2-, 3- and 4-cell stages significantly earlier than did embryos that arrested in development. We then determined the rate of blastocyst formation after ICSI in oocytes held for 2 days at either 15°C or room temperature before IVM (15-2d and RT-2d treatment groups respectively). The blastocyst development rate was significantly higher in the 15-2d than in the RT-2d group (13% vs 0% respectively). The failure of blastocyst development in the RT-2d group precluded comparison of morphokinetics of blastocyst development between treatments. In any condition examined, development to the blastocyst stage was characterised by earlier cytoplasmic extrusion before cleavage, earlier cleavage to 2- and 4-cell stages and reduced duration at the 2-cell stage compared with non-competent embryos. In conclusion, this study presents morphokinetic parameters predictive of embryo development in vitro to the blastocyst stage after ICSI in the horse. We conclude that time-lapse imaging allows increased precision for evaluating effects of different treatments on equine embryo development. In vitro embryo production is an important equine reproductive technology. Using time-lapse monitoring, we defined the kinetics of in vitro equine blastocyst development and found that holding oocytes at 25°C for 2 days decreased blastocyst production, whereas holding them at 15°C affected embryo morphokinetics, but not the blastocyst rate. Defining morphokinetic parameters via time-lapse monitoring will allow more precise evaluation of the effects of embryo treatment. [ABSTRACT FROM AUTHOR]

Some basic parameters for equine in vitro embryo production have not yet been established, including the optimum temperature for maturation and embryo culture, and the optimum CO2 concentration and pH during early embryo development. To explore this, we first performed cultures in incubators set at 37.2°C, 37.7°C or 38.2°C. At these temperatures, the corresponding maturation rates were 33%, 38% and 42%; cleavage rates were 84%, 86% and 88%; and blastocyst rates were 35%, 44% and 44% per injected oocyte. These rates did not differ significantly (P > 0.2). We then evaluated three different CO2 concentrations (6%, 6.5% or 7% CO2) in 5% O2 for culture over Days 0–5 after intracytoplasmic sperm injection, using a commercial human embryo medium with added serum, at 38.2°C. The pH values of these media were 7.36, 7.33 and 7.29 respectively. In the presence of 6%, 6.5% or 7% CO2, cleavage rates were 68%, 80% and 70% respectively, and blastocyst rates per injected oocyte were 42%, 54% and 27% respectively. The blastocyst rate for the 7% CO2 treatment was significantly lower than that for the 6.5% CO2 treatment (P < 0.05). We conclude that equine in vitro embryo production is equally effective within the range of 37.2–38.2°C, but that equine early cleavage stage development is sensitive to small changes in CO2 atmosphere and/or medium pH. Some basic parameters for equine embryo culture are not yet established. We found that blastocyst development after intracytoplasmic sperm injection was not affected by incubator temperature between 37.2°C and 38.2°C, but was reduced under high CO2 tension (7%), associated with lower medium pH (7.29), during cleavage stage culture. These findings will help optimise the culture environment for in vitro equine embryo production. [ABSTRACT FROM AUTHOR]

Embryo biopsy for fetal sexing has clinical application, but few reports are available of its use within an active embryo transfer program. We evaluated results on biopsy of 459 embryos over one breeding season. There were no significant differences in pregnancy rate between biopsied and non-biopsied embryos (72% vs 73%) or for biopsied embryos recovered at the centre (73%) compared with those shipped overnight (72%). However, the pregnancy rate decreased significantly in shipped embryos biopsied ≥20 h after collection. Overall, 86% of biopsies provided a sex diagnosis. The likelihood of a positive genomic (g) DNA result was significantly higher for biopsies from large blastocysts (96%) than from smaller embryos (70–85%). In total, 38% of biopsies were positive for Y chromosome DNA (Y-DNA) and were diagnosed as male. Subsequently, 95% of Y-DNA-positive embryos were confirmed as male and 78% of Y-DNA-negative embryos were confirmed as female. The accuracy of prediction of female (Y-DNA negative) was significantly higher when the biopsy sample was probed for Y-DNA only compared with probing for both gDNA and Y-DNA. We estimate that by transferring only Y-DNA-negative embryos, 3% of potential female pregnancies may have been lost, and production of male pregnancies was reduced by 72%. In some horse sports, horses of one sex are desirable, whereas those of the other sex have little economic value. We evaluated use of embryo biopsy and sexing via polymerase chain reaction in an active embryo transfer program; biopsy did not affect viability and increased the proportion of female pregnancies to 78%. This technique may decrease both financial and animal loss. [ABSTRACT FROM AUTHOR]

Preimplantation genetic diagnosis has great potential in the horse, but information on evaluation of equine embryo biopsy samples is limited. Blastocysts were biopsied using a Piezo drill and methods for whole-genome amplification (WGA) investigated. Results for 33 genetic loci were then compared between biopsy samples from in vitroproduced (IVP) and in vivo-recovered (VIV) blastocysts. Under the experimental conditions described, WGA using the Qiagen Repli-g Midi kit was more accurate than that using the Illustra Genomiphi V2 kit (98.2% vs 25.8%, respectively). Using WGA with the Qiagen kit, three biopsy samples were evaluated from each of eight IVP and 19 VIV blastocysts, some produced using semen from stallions carrying the genetic mutations associated with the diseases hereditary equine regional dermal asthenia (HERDA), hyperkalemic periodic paralysis (HYPP) or polysaccharide storage myopathy 1 (PSSM1). Three of 81 biopsy samples (3.7%) returned,50% accuracy. In the remaining 78 samples, overall accuracy was 99.3% (2556/2574 loci interrogated). Accuracy did not differ significantly between samples from IVP and VIV blastocysts. Allele drop-out in heterozygous loci was 1.6% (17/1035). Accuracy for sex determination was 100%; accuracy for heterozygosity for disease-causing mutations was 97.7% (43/44). In conclusion, Piezo-driven embryo biopsy withWGAhas.95% overall accuracy in IVP and VIV embryos, and this technique is suitable for use in a clinical setting. [ABSTRACT FROM AUTHOR]

There is growing interest in pre-implantation genetic diagnosis (PGD) for management of inherited genetic disease in the horse. In a previous study (Choi et al. 2010 Reproduction 140, 893-902), we demonstrated normal viability of equine blastocysts after biopsy. However, genome amplification was only moderately successful and only 1 of 2 analyses of heterozygous loci accurately detected both alleles. In the current study, we investigated different methods for amplification of DNA to improve the efficiency of PGD. To evaluate allele drop-out, multiple commonly-heterozygous gene loci were evaluated. In Experiment 1, using a piezo drill, 3 to 5 biopsy samples of 20 to 30 cells each were obtained from each of 4 in vitro-produced blastocysts. The samples and embryos were stored at -20°C, then shipped to the Veterinary Genetics Laboratory at the University of California, Davis. Whole genome amplification was done with an Illustra Genomiphi V2 kit (GE Healthcare, Waukesha, WI) before PCR for specific markers. Two disease-related (SCN4A and PPIB), one gender (AME) and 17 microsatellite identification markers were genotyped, for a total of 20 loci. Results for biopsy samples were compared with those for the corresponding embryo. A DNA signal was obtained from 14/15 biopsy samples, but for only 59.6% of the 280 total genotypes. Of 40 heterozygous loci, the signal from the corresponding biopsy sample showed only one allele (underwent allele dropout) in 60/80 instances (75%). In Experiment 2, 4 biopsies were obtained from each of 4 additional in vitro-produced blastocysts, then all samples were stored at -20°C. The Repli-G Mid kit (Qiagen, Valencia, CA) was used for whole genome amplification. Two disease-related (SCN4A and PPIB), 2 gender (AME and eSRY), 10 coat colour and 17 identification markers (total of 31 loci) were examined in each biopsy sample and were compared with results for the embryos. One biopsy sample was lost. Signal was obtained from 14/15 of the remaining biopsy samples and gave a 100% match at the 2 gender loci, 2 disease-related loci and 10 coat colour loci. One identification locus, LEX33, amplified in only 8 of 22 analyses. At the remaining 16 identification loci, 223/224 biopsy results matched those for the embryos. Overall, of 51 heterozygous loci among the 4 embryos, biopsy samples exhibited allele dropout in 1/180 instances (0.6%). In conclusion, results obtained using piezo-driven embryo biopsy and whole genome amplification using the Qiagen Repli-G kit have high accuracy and this technique may be suitable for use in a clinical setting. Further studies are needed with in vivo-derived embryos and to optimize accuracy of PCR of some identification markers. This work was supported by the American Quarter Horse Foundation, the Link Equine Research Endowment Fund, Texas A&M University and by Ms Kit Knotts. [ABSTRACT FROM AUTHOR]

An abstract of the study "Application of an Open Device to Vitrify Equine In Vitro-Produced Embryos," by Y. H. Choi et al, is presented.

Closure of all the horse slaughterhouses in the US has reduced the availability of equine oocytes in this country. We investigated the use of oocytes collected from immature follicles of live mares for cloning research. Because blastocyst development of equine cloned embryos is typically low (<10%), we also investigated the effect of Scriptaid, a histone deacetylase inhibitor that increases blastocyst development, live birth rate, and neonatal health in cloned mice and pigs. Immature oocytes were transvaginally aspirated from all follicles≥8mm diameter in a herd of 11 mares. The oocytes were cultured in modified M199 for 24 to 26h. Donor fibroblasts from a 27-year old stallion were treated with roscovitine for 24h, then were directly injected into enucleated oocytes using the Piezo drill. Reconstructed oocytes were activated with ionomycin followed by injection of sperm extract and culture with 6- dimethylaminopurine (6-DMAP) for 4h. Recombined oocytes in the Scriptaid treatment were cultured in the presence of Scriptaid, 250nM, starting at the onset of 6-DMAP treatment and continuing for a total of 18 to 20h. After embryo culture, blastocysts were shipped for transfer to recipient mares. Overall, each oocyte donor mare underwent aspiration up to 10 times; 653 follicles were aspirated and 271 oocytes were recovered. The in vitromaturation rate was 65% (172/263). After nuclear transfer procedures, 147 oocytes survived; 130 were used for the study. The blastocyst development rate was 2/47 (4%) in the control treatment and 1/83 (1%) in the Scriptaid treatment. All 3 blastocysts yielded pregnancies after transfer. Both control pregnancies were lost, 1 at 30 days and other at 9 months. The mare pregnant with the embryo from the Scriptaid treatment foaled at 326 days of gestation. The foal had medical issues at birth similar to those seen in some cloned foals previously, including maladjustment, patent urachus, and poor oxygenation. These issues were resolved with medical care; the foal is 3 months of age and healthy at the time of writing. These results indicate that immature oocytes obtained from a limited number of mares can be used successfully for nuclear transfer, providing the opportunity to control the mitochondrial identity of the host cytoplast. Scriptaid treatment did not improve the rate of blastocyst development or prevent health problems at birth; however, transfer of 1 embryo in this treatment produced a viable foal. More work is needed to determine the effect of histone deacetylase treatment on efficiency of cloning in the horse. This work was supported by the Link Equine Research Endowment Fund, Texas A&M University, and by Ms. Kit Knotts. We thank Drs. Malgorzata Pozor, Margo Macpherson, and the Medicine team at the University of Florida for medical care of the foal. [ABSTRACT FROM AUTHOR]

Transvaginal ultrasound-guided follicle aspiration (TVA) is the most effective way to recover multiple immature oocytes from live mares. Because of the tight attachment of the equine immature oocyte to the follicle wall, TVA in this species is time consuming, taking up to 1h per horse. Thus, it may be difficult to search follicular aspirates immediately after recovery. In 2009 in a series of 6 replicates, we observed a blastocyst development rate of 32% (13/41) after intracytoplasmic sperm injection of oocytes collected by TVA and held for ~1.5h at ambient temperature (26 to 32°C) before isolation from aspirated fluid (unpublished data). Therefore, in the present study we compared the effects of immediate oocyte isolation v.holding the follicular aspirate before oocyte isolation on oocyte maturation and blastocyst rates. Ten mares were used for this study; TVA was performed on each mare every 14 days, for 4 aspiration sessions per mare. Collected aspiration fluid was either processed immediately or held for 2h at 32°C before processing. At each aspiration, one ovary was randomly assigned to each treatment [immediate (Imm) or 2-h holding (2-H)]. Follicle aspiration was performed as previously described (Jacobson et al.2010 Theriogenology 73, 1116–1126) using M199 with Hank’s salts to flush the follicle lumen up to 8 times per follicle. Oocytes were recovered from the aspirates by filtration. Overall, 325 follicles were aspirated and 140 oocytes were obtained (43% recovery). The proportion of degenerating oocytes was not significantly different between treatments (1/68 and 0/72 for Imm and 2-H, respectively). Oocytes were held overnight in modified M199 as previously described (Choi et al.2006 Theriogenology 66, 955–963) before maturation culture. After 30h of maturation culture, there was no significant difference in maturation rates between treatments [75% (50/67) and 65% (46/71) for Imm and 2-H, respectively]. Fertilization was performed by intracytoplasmic sperm injection, and injected oocytes were cultured 7 to 10 days, as previously described (Choi et al.2006). The rates of blastocyst development per injected oocyte were 23% (11/47) for Imm and 16% (7/44) for 2-H; these were not significantly different (P>0.3). The reason for the discrepancy in blastocyst rates for held oocytes in this study (16%) compared with our 2009 observations (32%) is unclear; this could be a factor of the time held (~1.5 v.2h), the temperature (26–32°C v.32°C), or minor changes in protocol between the 2 years. From these results, we conclude that holding oocytes in the follicular aspirate for up to 2h following collection may be performed when necessary without significantly affecting the rate of subsequent blastocyst development. This work was supported by the Link Equine Research Endowment Fund, Texas A&M University, and by Ms. Kit Knotts. [ABSTRACT FROM AUTHOR]

Whereas small equine embryos may be successfully cryopreserved, pregnancy rates after cryopreservation of large equine blastocysts (>300 μm) are low. This has been attributed to their large diameter, failure of cyoprotectants to diffuse into the blastocoele, and/or impedance from the equine embryonic capsule. Previously, we successfully biopsied the trophoblast of equine in vivo-recovered expanded blastocysts (>300 μm; Choi et al.2009 Reprod. Fertil. Dev. 21, 166-167). In that study, we observed embryonic growth in vitroafter warming of a blastocyst vitrified immediately after biopsy. An efficient vitrification method was recently described for ferret embryos, which, like horse embryos, contain a large amount of lipid. This method utilizes very small (250 μm inner diameter) pipettes (Femtotips, Eppendorf; Sun et al.2008 Biol. Reprod. 79, 832-840). We utilized this technique, with minor modifications, to vitrify equine embryos. In Study 1, the vitrification procedure was performed on equine in vitro-produced blastocysts (300 μm diameter, were biopsied using a Piezo drill, causing blastocoele collapse, and were then immediately vitrified. Three of these vitrified blastocysts (346, 399, and 514 μm in diameter) were warmed and transferred to culture; all 3 expanded within 8 h and subsequently proliferated through holes in the embryonic capsule. Seven vitrified in vivo-recovered blastocysts were warmed at the laboratory, shipped to a transfer facility, and transferred transcervically to recipient mares. Five out of 7 transferred blastocysts (71%) resulted in pregnancy at Day 14. Diameters of these embryos before biopsy and vitrification were 360, 398, 415, 434, and 599 μm. The 2 biopsied and vitrified embryos that did not result in pregnancy after transfer were originally 376 and 515 μm in diameter. To our knowledge, this represents the first report of normal (>50%) pregnancy rates achieved after transfer of cryopreserved expanded equine blastocysts. Our findings suggest that biopsy before cryopreservation, which punctures the embryonic capsule and causes blastocoele collapse, combined with vitrification in a small-diameter pipette, may provide an efficient procedure for cryopreservation of large equine blastocysts. Further studies are needed to explore the different factors associated with success of this procedure. This work was supported by the Link Equine Research Endowment Fund, Texas A&M University, and by Ms. Kit Knotts. [ABSTRACT FROM AUTHOR]