Normal mammalian early embryonic development involves apoptosis of blastomeres as a remodeling process during differentiation, starting at the blastocyst stage. Genomic DNA has been recently detected in the blastocele fluid of human embryos and has been amplified by real-time polymerase chain reaction (PCR) to diagnose the sex of in vitro-produced human embryos. This new approach varies from conventional preimplantation genetic diagnosis in that no cells are extracted from the embryo and only the blastocele fluid is aspirated and used as a DNA sample for diagnosis. In the present work, we investigated whether the blastocele fluid of equine preimplantation embryos contains nuclear DNA and whether this DNA could be used to diagnose the sex of the embryos by conventional PCR, using specific primers that target the TSPY and AMEL equine genes. The sex of 11 of 13 in vivo-produced embryos and of four of five in vitro-produced embryos was successfully diagnosed. The PCR amplification product was analyzed using genetic sequencing reporting that the DNA present in blastocele fluid was genomic. Additionally, after polyacrylamide gel electrophoresis and silver staining, the blastocele fluid from three different embryos produced a ladder pattern characteristic of DNA fragmented during apoptosis. Therefore, the results presented in this work report that blastocele fluid from in vivo-and in vitro-produced equine embryos contains nuclear DNA which is probably originated by apoptosis of embryonic cells, and this DNA could be used to diagnose the sex of preimlpantation embryos by conventional PCR This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT
The aim of this work was to evaluate the use of air-dried spermatozoa for in vitro production of equine embryos and verify if sperm extract activation and in vivo culture improve in vitro embryo production. Cooled spermatozoa (control) and air-dried spermatozoa stored for 2, 14 or 28 days were used for ICSI sperm extract, or ionomycin was used for oocyte activation, and embryos were in vitro or in vivo (in mare's oviduct) cultured for 7 days. With in vitro culture, cleavage rate was higher when activating with sperm extract (P < 0.05). No differences in embryo development were seen between the two activation treatments nor between storage periods (P > 0.05). Blastocysts were obtained with cooled spermatozoa, and morulae were achieved using in vivo culture with 28-day storage spermatozoa and ionomycin-activated oocytes. When in vivo culture was performed, sperm DNA fragmentation was assessed using the sperm chromatin dispersion test and did not show statistical correlation with cleavage nor embryo recovery rates. In conclusion, equine embryos can be produced using air-dried spermatozoa stored for several weeks. Sperm extract activation increased cleavage rates but did not improve embryo development. In vivo culture allowed intrauterine stage embryos to be achieved.
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Pre-implantation genetic diagnosis (PGD) is used in different species to determine specific genetic traits of early stage embryos. The first part of this technique involves obtaining one or more cells from the embryo. Then, the DNA in the sampled cells is amplified by PCR using specific primers. Recently Palini et al. (2013) performed PGD of human blastocysts using the DNA in blastocoele fluid. The aim of our work was to study if the sex of equine in vivo produced blastocysts could be determined by PGD using blastocoele fluid as a sample for PCR. Thirteen equine blastocysts produced by artificial insemination and uterine flush were used for this study. Once obtained each embryo was placed on a 50-µL droplet of D-PBS without calcium and magnesium supplemented with 10% FBS and 50 µg mL–1 of gentamicin (working medium) under mineral oil, on an inverted microscope equipped with a micromanipulation system. The blastocoele fluid was aspirated using a beveled micropipette (9 µm ID) and then discharged on a 1-µL microdroplet of working medium. The microdroplet containing the blastocoele fluid was transferred to a 0.2-mL DNAse-free tube containing 4 µL of DNAse-free water. A duplex PCR was performed to amplify the Y-encoded testis-specific protein (TSPY) and amelogenin (AMEL) genes. The primer sequences used for this study were: AMEL-F 5′-CCAACCCAACACCACCAGCCAAACCTCCCT-3′, AMEL-R 5′-AGCATAGGGGGCAAGGGCTGCAAGGGGAAT-3′, TSPY-F 5′-GAAGTCAGGCACACCAGTGA-3′ and TSPY-R 5′-TAAGGCTGCAGTTGTCATGC-3′. The PCR was performed twice, using the same primers and cycling protocol. Embryonic cells from embryos which have been previously diagnosed as male and female were used as positive controls for PCR. As a negative control, one tube was included containing all the components for PCR, except the DNA sample was replaced with the same volume of DNAse-free water. The amplification products were electrophoresed on a 2% agarose gel stained with ethidium bromide and visualised under UV light. Embryo viability post-blastocoele fluid aspiration was studied by individually incubating seven aspirated embryos in 50-mL microdroplets of SOFm with 19 mM D-glucose with 10% FBS under mineral oil at 38°C, in 7% O2 and 5% CO2 and observed at 24, 48, and 72 h. The diameter of embryos was registered immediately before blastocoele fluid aspiration and every 24 h during in vitro culture. The sex of 11 out of 13 embryos (84.6%) was determined and they were diagnosed as 8 males and 3 females. Six of the 7 embryos cultured in vitro expanded at 24 h and increased their diameter at 48 and 72 h. Our results demonstrate that PGD of equine embryos can be performed using blastocoele fluid only, avoiding the need of extracting cells from the embryo. The in vitro embryo survival after blastocoele fluid aspiration was high, showing that this technique does not impair the embryo's viability. Further studies are being undertaken using a larger number of embryos to demonstrate if this approach can be used with high efficiency.
Cryopreservation of equine embryos is still not a routine procedure. Pregnancies have been obtained after transfer of vitrified embryos of less than 300 μm (Eldridge-Panuska et al. 2005). The aim of this study was to use the cryotop method (Kuwayama, 2007) to obtain pregnancies after transfer of vitrified thawed cooled and fresh embryos collected in our clinical embryo transfer programme. Embryos were assigned either to be vitrified within 3 h of collection or to be cooled for 18–24 h before vitrification. All embryos were vitrified and thawed by Cryotop Vitrification Kit® (Cryo Tech Laboratory®). Briefly, they were equilibrated in a solution containing ethylene glycol (EG), dimethylsulphoxide (DMSO) in TCM-199 for 10 to 25 min. Then they were moved to vitrification solution containing EG, DMSO, and sucrose in TCM-199 and loaded with a glass capillary onto the top of the film strip. After loading, almost all the solution was removed to leave only a thin layer covering the embryo, and the sample was quickly immersed into liquid nitrogen and covered with a protective cap. The time between entry to vitrification solution and nitrogen was from 1 to 3 min. At warming, the strip was immersed directly for 1 min into a 37°C medium containing sucrose in TCM-199. The embryo was incubated 3 min in a diluent solution, washed twice 5 min each in washing solution, and further cultured in DMEM F-12 with 10% FBS at 38.5°C 5% CO2 between 2 to 5 h. For transfer, the embryo was loaded in 0.5-mL straws. All recipient mares had ovulated 4 to 7 days before nonsurgical transfer. Pregnancies were detected 6 to 8 days later. A total of 15 embryos, grades 1 to 2, were obtained. Fresh embryos (n = 7) ranged between 250 and 800 μm, and refrigerated embryo (n = 8) diameter was between 130 and 550 μm. Pregnancy rates were 37.5% (3/8) for embryos cooled before vitrification and 28.6% (2/7) for embryos vitrified within 3 h. The overall pregnancy rate was 33.3% (5/15). Shipping cooled embryos allows maintaining a large number of recipients far away from donors, without decreasing pregnancy rate. It also makes it possible to send embryos to a specialised laboratory in order to be vitrified and preserved until recipients are available. Equine embryos collected 6 days after ovulation are generally smaller than 300 μm and have shown the highest survival rate after cryopreservation. However, the embryo recovery rate is higher when flushing is performed at Day 7 or 8. This cryopreservation protocol could provide a way to vitrify fresh and cooled embryos up to 550 μm, which would prevent the loss of valuable embryos collected in more advanced stages of development. In summary, pregnancies can be obtained after cooling for 18 to 24 h and vitrification of embryos collected 7 or 7.5 days after.
Developing effective cooled semen protocols is essential to increase pregnancy rates and reproductive efficiency in donkeys. This study aimed to evaluate the effect on sperm kinetic parameters and membrane integrity in cooled donkey semen diluted with defined milk proteins extender with 1% or 2% of egg yolk and the removal of seminal plasma. Twenty-four ejaculates from six jackasses were collected. Each ejaculate was divided into four aliquots that were diluted in extender with 1% (EY1) or 2% (EY2) egg yolk. One sample from each group was centrifuged, seminal plasma was removed (CEY1, CEY2 groups, respectively), and the samples were then refrigerated at 5 °C for 24 h. Fresh and cooled semen samples were assessed for sperm motility, morphology, and plasma membrane integrity. Total motility, progressive motility, sperm kinetic parameters, or live sperm cells were not statistically different when semen was cooled with an extender supplemented with 1% or 2% of egg yolk. Seminal plasma removal does not affect total motility or sperm kinetic parameters. However, progressive motility decreased (P<0.05) when semen was extended with 2% of egg yolk and seminal plasma was removed. Membrane integrity was affected (P<0.05) in centrifuged samples. In conclusion, the obtained results suggest that there is no difference in sperm kinetics and membrane integrity when 1% or 2% of egg yolk was added to the Equiplus(R) extender. Also, the removal of seminal plasma by centrifugation did not have any beneficial effect on cooled donkey semen. Further studies are needed to relate these results with in vivo fertility tests with cooled donkey semen.
The interest in equine intracytoplasmic sperm injection (ICSI) for commercial and research applications has rapidly increased. Shipping immature oocytes at room temperature has been proven successful, and to identify the optimal conditions for holding oocytes, several mediums are being tested. The aim of this study was to compare the effect of holding equine oocytes in Tyrode’s albumin lactate pyruvate-Hepes (TALP-h, Bavister and Yanagimachi 1977 Biol. Reprod. 16, 228-237) medium or in commercial embryo holding medium (EHM, Syngro® Holding) on invitro nuclear maturation rates and pre-implantation embryo development after ICSI. Cumulus–oocyte complexes (COCs) were recovered from ovaries of slaughtered mares and assigned randomly in 2-mL cryovials with TALP-h or EHM, with a maximum of 30 oocytes per cryovial. COCs were shipped to the ICSI laboratory at 20 to 25°C for 24 to 28h followed by IVM for 24h in a humidified atmosphere of 5% CO2 in air at 38.5°C. Maturation medium was TCM-199 with 10% fetal bovine serum, 1μL mL−1 insulin-transferrin-selenium, 1mM sodium pyruvate, 100mM cysteamine, and 0.1mg mL−1 FSH. After mechanical cumulus cell removal, nuclear maturation rate was assessed using a stereomicroscope. Oocytes with an intact oolemma and extrusion of the first polar body (PB) were classified as mature, oocytes without a visible PB were considered immature, and oocytes without an intact oolemma were considered degenerate. Matured oocytes were subjected to ICSI without piezo-drill system (one proved stallion) in 20-μL droplets of TALP-h with a 7-μm glass sharp micropipette in an inverted microscope (Nikon Eclipse TE-300 microscope) using hydraulic micromanipulators (Narishige, Medical Systems). Presumptive ICSI zygotes were cultured in DMEM F12/Global Total® with 6% fetal bovine serum for 9 days at 38.5°C in a humidified atmosphere of 5% O2 and 5% CO2 in air. On Day 5 of culture, cleavage was recorded and medium was refreshed. Blastocysts rates were recorded on Day 7 and 9 of culture. Invitro nuclear maturation rates are shown in Table 1. We observed a significantly higher proportion of immature oocytes in the EHM group compared with the TALP-h group. After ICSI of some matured oocytes of each group, no significant differences were observed in cleavage or blastocyst rate (Table 1). Our results suggest that either TALP-h or commercial embryo holding medium are suitable for oocyte shipping and to support blastocyst development after ICSI. Table 1. Invitro nuclear maturation rates and pre-implantation embryo development after intracytoplasmic sperm injection (ICSI) Maturation rates Medium Oocytes Mature [n (%)] Immature [n (%)] Degenerate [n (%)] TALP-h 315 173 (54.9) 26 (8.3)a 116 (36.8) EHM 273 132 (48.4) 55 (20.1)b 86 (31.5) Total 588 305 (51.9) 81 (13.8) 202 (34.4) Embryo development ICSI (n) Cleaved [n (%)] Day 7 Blastocyst [n (%)] Day 9 Blastocyst [n (%)] TALP-h 35 23 (65.7) 7 (20) 9 (25.7) EHM 26 19 (73.1) 3 (11.5) 5 (19.2) Total 61 42 (68.9) 10 (16.4) 14 (23) a,bDifferent superscript letters indicate statistical significance (Fisher’s exact test, P<0.05).
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