Microsurgical Blade Research Articles

Сравнительный анализ механического и фемтолазер-ассистированного методов выкраивания лимбальных мини-трансплантатов в эксперименте

Original article Experimental study of manual and femtosecond laser-assisted methods for cutting limbal mini-transplants B.E. Malyugin, S.A. Borzenok, O.N. Nefedova, D.S. Ostrovskii, M.Yu. Gerasymov, A.V. Shatskikh S. Fyodorov Eye Microsurger y Federal State Institution, Moscow, Russian Federation A. Yevdokimov Moscow State University of Medicine and Dentistr y, Moscow, Russian Federation Purpose. To define optimal parameters of femtosecond laser (FSL) (Femto LDV Z8, Ziemer, Switzerland) for cutting limbal mini-transplants to be further used for limbal stem cells (LSC) transplantation and compare the results with manual cutting technique using microsurgical blade. Material and methods. At first step, performed on 3 porcine and 3 cadaveric eyes, the optimal energy parameters of FSL were selected, which ensured the excision of limbal mini-transplants. At the second step, 8 eyes from 4 postmortem donors were used. In the lower and upper parts of the limbus, limbal fragments were cut out into 8 mini-transplants with a FSL (experimental group). Mini-grafts were also formed manually in symmetrical areas of each eye using dosed diamond blade (control group). The structure of the limbal bed after removing mini-transplants was studied by light and scanning electron microscopy. The effect of FSL energy on the viability of LSC was determined by intravital staining using Live & Dead dye. Finally, on the third step, the assessment of reversible and irreversible apoptosis was performed by culturing corneoscleral discs containing mini-transplants not separated from the limbal bed. The obtained samples were cultivated in a medium DMEM/F12 for 7 days followed by immunohistochemical examination. Results. The optimal parameters of FSL operation are determined by energy levels- 100, 110 and 120%. Histological analysis of the limbal residual bed zone showed that the smoothest surface was obtained at energies 110 and 120%. This fact was also confirmed by scanning electron microscopy. When stained with the Live & Dead dye, damage on the lateral and inner sides of limbal mini-transplants was revealed, mainly in the experimental group. After 7 days of cultivation in the cut area in both groups, no expression of apoptosis markers was observed. Conclusion. Cutting out the limbal zone containing LSCs using FSL is safe and allows obtaining limbal mini-grafts. The most optimal parameters of laser operation in the limbal zone were selected – a planar cut at depth of 250 µm using 110% laser energy. Key words: femtosecond laser, limbal stem cells, apoptosis, limbal stem cell deficiency syndrome, scanning electron microscopy, light microscopy, cultivation, histology

Safety assessment of the femtosecond laser in corneal limbal graft excision

Objective: to study in vitro survival and preservation of the proliferative activity of limbal stem cells (LSCs) in femtosecond laser-cut limbal tissue fragments. Materials and methods. Limbal fragments were formed from donor cadaver eyes (n = 8) in the upper and lower limbus containing the highest number of limbal stem cells, using a Z8 femtosecond laser (FSL) (Ziemer, Switzerland). The limbal fragments were fragmented into 4 mini-grafts using different energy levels (100, 110, 120%). Mini-grafts from symmetrical sections of the cadaver eyes, which were manually isolated using a microsurgical blade, served as controls. The mini-grafts were cultured for two weeks in culture media intended for limbal epithelial stem cells (LESCs) (Epilife (0.06 mM Ca++) and for multipotent mesenchymal stem cells (MMSCs) (DMEM/F12), with the addition of specific growth factors to selectively stimulate LESCs or MMSCs, respectively. The phenotype of the obtained cultured cells in the «laser» and «knife» groups was determined by flow cytometry using a set of markers (CD166, CD105, CD90, CD29, CD34) for the membrane proteins of LESCs and MMSCs. The ability of cultured cells to adhesion and proliferation in the «laser» and «knife» groups was determined by seeding the third passage of the resulting cultures on Bowman’s membrane of acellular corneas.Results. Primary cell culture was obtained from mini-grafts of all donors in both groups. Cell morphology was consistent with the phenotype of corneal epithelial cells (cobblestone pattern). When cultured in the EpiLife medium (0.06 mM Ca++), we determined the presence of LSCs proliferation from 38.6% of minigrafts; in the DMEM/F12 medium (1 : 1) the presence was determined from 31.8%. Two weeks later, cell yield from mini-grafts in the «laser» and «knife» groups was 77.2% and 63.6%, respectively. Cell growth by the end of week 2 of culturing of mini-grafts obtained by FSL at 120, 110 and 100% energies was 87.5, 71.4 and 71.4%, respectively. It was found that the resulting cell cultures in the «laser» and «knife» groups and in the «120%», «110%» and «100%» subgroups were not different phenotypically. Cytofluorimetric analysis showed that cell cultures in the groups had a mixed pattern of marker expression of both LESCs (CD29+) and MMSCs (CD90+, CD105+). Seeding of the third passage of cell culture in the test groups in all cases demonstrated adhesion and formation of a cell monolayer on the Bowman’s membrane of model corneas.Conclusion. The use of FSL for cutting out limbal grafts seems to be effective and safe in comparison with the traditional mechanical (knife) technique. Cell cultures obtained from FSL-cut mini-grafts were able to grow and migrate for at least 21 days.

Update on Microinvasive Glaucoma Surgery.

Update on Microinvasive Glaucoma Surgery.

99 In Vitro Development of Alpaca Embryos Obtained by Bisection

The objective of this study was to evaluate the in vitro developmental competence of alpaca embryos bisected at different embryonic stages. Gametes were obtained from ovaries and testes collected from a local abattoir. Cumulus-oocyte complexes (COC) were recovered (n = 120) by aspiration of ovarian follicles using a 5-mL syringe with an 18-gauge needle. Then, COC with at least 3 layers of cumulus cells and a homogeneous cytoplasm were matured in TCM-199 supplemented with 10% FCS, FSH (0.02 IU [JM1] [P2] [P3]), and 0.01 mg mL−1 oestradiol 17β [JM4] for 26 h at 38.5°C and 5% CO2 in air. After in vitro maturation, COC were placed in a 30-mL Petri dish containing FERT-TALP solution for 30 min. Then, epididymal alpaca spermatozoa (3 × 106 mL−1) were added to the dish and co-incubated with the COC for 20 h at 38.5°C and 5% CO2 in air. Motile epididymal sperm were selected by swim-up method centrifuged for 15 min at 350 × g in 2 mL of SPERM-TALP supplemented with 6 mg mL−1 of fatty-acid-free BSA. Sperm pellet was extended and culture in 5% CO2 in air at 38.5°C for 45 min. Thirty-three viable embryos at different stages [2-cells (n = 6), 8-cells (n = 15), and morulae (n = 12)] were bisected into approximately equal halves using a micro-surgical blade. The embryos were previously treated with 2 mg mL−1 of protease from Streptomyces griseus (P 8811, Sigma, St. Louis, MO, USA) for 2 min to remove the zona pellucida. After bisection, the demi-embryos were cultivated in in vitro culture (IVC) medium containing 0.036 mg mL−1 sodium pyruvate, 0.146 mg mL−1 l-glutamine, 1% essential amino acids, 0.5% nonessential amino acids, and supplemented with 10% FCS using the well-of-the-well system. The demi-embryos were incubated for 7 days (changing the media every 48 h) in 5% CO2 in air at 38.5°C. Additional embryos (n = 60) were obtained using the same conditions described above and used as a control group (unmanipulated). We obtained 66 demi-embryos [2-cells (n = 12), 8-cells (n = 30), and morulae (n = 24)] after bisection that were considered for IVC. From 12 demi-embryos bisected at 2-cell and 30 bisected at 8-cell stages, 3 (25%) and 30 (100%) reached the morula stage respectively. However, they did not develop any further. Interestingly, 18 demi-embryos bisected in morula reached the blastocyst stage (80%). For unmanipulated embryos, we obtained 42% (25/60), 35% (21/60), 32% (19/60), and 28% (17/60) of cleavage, morulae, and blastocyst and hatched blastocyst rates, respectively. In conclusion, alpaca embryos bisected at earlier stages (less than 8-cell) are not suitable to produce blastocysts. The earliest stage to produce blastocyst from bisected alpaca embryos is the morula stage.

The distribution of vascular endothelial growth factor in human meniscus and a meniscal injury model.

The meniscus plays an important role in controlling the complex biomechanics of the knee. Meniscus injury is common in the knee joint. The perimeniscal capillary plexus supplies the outer meniscus, whereas the inner meniscus is composed of avascular tissue. Angiogenesis factors, such as vascular endothelial growth factor (VEGF), have important roles in promoting vascularization of various tissues. VEGF-mediated neovascularization is beneficial to the healing of injured tissues. However, the distribution and angiogenic role of VEGF remains unclear in the meniscus and injured meniscus. We hypothesized that VEGF could affect meniscus cells and modulate the meniscus healing process. Menisci were obtained from total knee arthroplasty patients. Meniscal injury was created exvivo by a microsurgical blade. VEGF mRNA and protein expression were detected by the polymerase chain reaction and immunohistochemical analyses, respectively. In native meniscal tissue, the expression of VEGF and HIF-1α mRNAs could not be detected. However, VEGF and HIF-1α mRNAs were found in cultured meniscal cells (VEGF: outer>inner; HIF-1α: outer=inner). Injury increased mRNA levels of both VEGF and HIF-1α, with the increase being greatest in the outer area. Immunohistochemical analyses revealed that VEGF protein was detected mainly in the outer region and around injured areas of the meniscus. However, VEGF concentrations were similar between inner and outer menisci-derived media. This study demonstrated that both the inner and outer regions of the meniscus contained VEGF. HIF-1α expression and VEGF deposition were high in injured meniscal tissue. Our results suggest that injury stimulates the expression of HIF-1α and VEGF that may be preserved in the extracellular matrix as the healing stimulator of damaged meniscus, especially in the outer meniscus.

34 PRODUCTION OF TRANSGENIC CLONED BUFFALO EMBRYOS CONTAINING OVEREXPRESSED STEAROYL Co-A DESATURASE GENE FOLLOWING EFFICIENT TRANSFECTION

Stearoyl-CoA desaturase (SCD) is a rate-limiting enzyme that catalyses the synthesis of monounsaturated fatty acids and polyunsaturated fatty acids from saturated fatty acids, which are components of triglycerides, wax esters, cholesteryl esters, and membrane phospholipids. Alterations in phospholipid composition have been implicated in a variety of diseases including obesity and the associated metabolic syndrome. SCD also magnifies the conjugated linoleic acid (CLA) content in milk; CLA is a natural fat element, having reputed therapeutic health values including anti-carcinogenic properties. In light of this fact, this study was designed to amplify the levels of SCD at the gene level. In order to achieve the enhanced expression of SCD gene, combination of techniques were used. The somatic cells (fetal fibroblast) culture were established from ear pinnae obtained from bovine fetus procured from the abattoir and were cultured in basal medium, comprising DMEM with 10% fetal bovine serum, 1X NEAA and 1X PS antibiotics. These isolated cultured cells were transfected with a gene construct carrying buSCD gene (pAcGFPN1-buSCD) as BLGP-buSCD-BLG3′UTR-CMVP-EGFP-SV40. The buffalo fetal fibroblast cells were transfected using 3 methods: Nucleofection, Fugene and Lipofection. The successful transfection, as confirmed by PCR and Southern hybridisation, proved Nucleofection to be more efficient in transfecting cells among the techniques used, which were further maintained and selected by Geneticin (G418). These selected transfected cells were then used for nuclear transfer. Somatic cell nuclear transfer (SCNT) has provided an efficient pathway for the production of transgenic animals. Buffalo cumulus–oocyte complexes (COCs) were collected from ovaries collected from abattoir and matured in TCM-199 supplemented with 10% FBS, 5 µg mL−1 FSH, and 1 µg mL−1 β-oestradiol for 21 h in CO2 incubator at 5% CO2 in air and 38.5°C temperature with >95% relative humidity. After 21 h, these COCs were denuded and subjected to zona removal. These zona-free oocytes were manually enucleated using microsurgical blades and 2 enucleated oocytes were fused with a transgenic cell using electro cell manipulator. Further, these reconstructed embryos were activated using calcium ionophore and cultured in IVC media thereafter for 8 days. The developmental competence rate as recorded on Day 8 was 53.26 ± 1.73%, 69.87 ± 6.24%, 62.99 ± 7.15%, 42.71 ± 5.05% and 28.00 ± 3.33% for 2-cell, 4-cell, 8–16 cell, morula, and blastocyst, respectively. When observed under fluorescence microscope, the embryos showed successful expression of GFP, which can be further used for animal production or further research analysis. In conclusion, amplified SCD at gene level will result in a boost to the dairy sector as well ameliorating human health due to its crucial role in anti-cancer, anti-diabetic, reduced cardio-vascular disease, and improved immune responses.

93 BLASTOCYST BISECTION TO MULTIPLY BIOPSIED AND VITRIFIED BOVINE EMBRYOS

Dairy cattle breeding schemes increasingly integrate embryo-based genomic selection to accelerate genetic gain. In contrast to the single offspring produced with conventional animal-based genomic selection, multifactorial IVF between elite parents increases genotypes for selection. Genetically superior embryos are identified from biopsies, and only those with the desired genotypes are transferred. To manage the logistics of such schemes, and enable seasonally born progeny, the cryo-preservation of embryos after biopsy and before embryo transfer is critical. Here, we compare 2 methods of cryo-preserving biopsied Day 7 blastocysts and report results from bisecting blastocysts to increase the number of selected embryos for transfer. Abattoir-sourced oocytes were matured in vitro and fertilized with sperm from a single sire. Embryos were cultured for 7 days in a modified Synthetic Oviduct Fluid medium. Approximately 15 cells were biopsied from the mural trophectoderm of grade 1 and 2 blastocysts in Embryo Hold medium minus BSA, using a micro-surgical blade (Bioniche Animal Health, Athens, GA, USA). Following biopsy, each blastocyst was cultured in Embryo Hold with 3 mg mL−1 BSA for ~2 h at 38.5°C to allow for re-expansion. In Experiment 1, embryos were randomly assigned to 1 of 2 cryo-preservation treatments: conventional slow freezing or the Cryologic vitrification method (CVM). Slow freezing entailed freezing in 1.5 M ethylene glycol and 0.1 M sucrose. The CVM involved a 2-step vitrification protocol, with 15% of both ethylene glycol and dimethyl sulphoxide in the final solution comprising Embryo Hold, 20% FCS, 1 M sucrose, and 0.1 mM Ficoll (GE Healthcare). Selected embryos were thawed/warmed and transferred in pairs to the uterine horn ipsilateral to the corpus luteum of each synchronized recipient heifer. In Experiment 2, each biopsied blastocyst was individually vitrified using CVM. Following warming, blastocysts were bisected into approximately equal halves. After ~2 h recovery, pairs of demi-embryos were transferred to recipients categorized with either normal (>2.5, <7 ng mL−1) or low (≥2, <2.5 ng mL−1) plasma progesterone concentrations on Day 5 after oestrus. Embryo survival in both experiments was monitored by ultrasonography of fetal heartbeats up to Day 65 of gestation. Statistical significance was determined using Fisher’s exact test. In Experiment 1, embryo survival on Day 65 was significantly greater with CVM than slow freezing (25/54 = 46% v. 9/54 = 17%; P = 0.002). In Experiment 2, there was no significant difference in the number of fetuses as a percentage of original blastocysts, regardless of normal versus low progesterone levels (13/22 = 59% v. 4/9 = 44%, respectively). In conclusion, vitrification is superior for cryo-preserving biopsied blastocysts, possibly reducing cryo-damage compared with conventional slow freezing, and achieves rates of in vivo development similar to fresh IVF embryos. Embryo bisection potentially provides only a modest increase in the probability of generating a calf from each valuable, genomically selected embryo. Improving embryo competency and other methods of multiplication may maximize this likelihood.

A rare and serious unforeseen complication of cutting balloon angioplasty.

Cutting balloon angioplasty (CBA) is one of the adept ways of treating “in-stent restenosis.” Various complications related to cutting balloon angioplasty have been reported including arterial rupture, delayed perforation and fracture of microsurgical blades. Here we report a very unusual and inadvertent extraction of a stent previously deployed in the ramus intermedius coronary branch by a cutting balloon catheter. This required repeat stenting of the same site for an underlying dissection. Even though stent extraction is a rare complication it can be serious due to dissection, perforation, and closure of the artery. Physicians performing coronary artery interventions would need to be aware of this rare and serious complication especially if any difficulty is encountered while withdrawing the cutting balloon. Therefore, after removal, cutting balloon should be examined thoroughly for possible stent dislodgment or extraction when used for “in-stent restenosis.”

Inhibition of Sevoflurane Postconditioning Against Cerebral Ischemia Reperfusion-Induced Oxidative Injury in Rats

The volatile anesthetic sevoflurane is capable of inducing preconditioning and postconditioning effects in the brain. In this study, we investigated the effects of sevoflurane postconditioning on antioxidant and immunity indexes in cerebral ischemia reperfusion (CIR) rats. Rats were randomly assigned to five separate experimental groups I–V. In the sham group (I), rats were subjected to the same surgery procedures except for occlusion of the middle cerebral artery and exposed to 1.0 MAC sevoflurane 90 min after surgery for 30 min. IR control rats (group II) were subjected to middle cerebral artery occlusion (MCAO) for 90 min and exposed to O2 for 30 min at the beginning of reperfusion. Sevoflurane 0.5, 1.0 and 1.5 groups (III, IV, V) were all subjected to MCAO for 90 min, but at the beginning of reperfusion exposed to 0.5 MAC, 1.0 MAC or 1.5 MAC sevoflurane for 30 min, respectively. Results showed that sevoflurane postconditioning can decrease serum tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), nitric oxide (NO), nitric oxide synthase (NOS) and increase serum interleukin-10 (IL-10) levels in cerebral ischemia reperfusion rats. In addition, sevoflurane postconditioning can still decrease blood lipid, malondialdehyde (MDA) levels, infarct volume and increase antioxidant enzymes activities, normal pyramidal neurons density in cerebral ischemia reperfusion rats. It can be concluded that sevoflurane postconditioning may decrease blood and brain oxidative injury and enhance immunity indexes in cerebral ischemia reperfusion rats.

Ureteroenteric Anastomotic Stricture: Novel Use of a Cutting Balloon Dilator

Management of ureteroenteric strictures presents a significant challenge because of its intraabdominal location and morbidity associated with open surgical management. The peripheral cutting balloon microsurgical dilatation device (PCBD), approved by The United States Food and Drug Administration (USFDA) for use in coronary angioplasty, features a 2-cm noncompliant balloon with four microsurgical blades mounted longitudinally on its outer surface. We evaluated the feasibility and outcome of this cutting balloon dilator in the treatment of ureteroenteric anastomotic strictures. Three patients with a 1-cm or less ureteroenteric stricture underwent a transluminal incision under fluoroscopic guidance. Percutaneous access was obtained and a guidewire was introduced into the renal pelvis and ureter in antegrade fashion and passed through the stricture. The exact length of the strictured segment was measured. The PCBD was deployed over the guidewire and the balloon was inflated at the stricture site. The maximum diameter of the inflated balloon was 8 mm. Approximately 30 seconds later, the balloon was deflated and the enlarged passage from the ureter to the ileal loop was verified under fluoroscopy. A ureteral stent was placed and removed at 6 weeks after the procedure. Postoperative computed tomography scans at 12 months revealed improved hydronephrosis. All patients were asymptomatic postoperatively. One patient had a solitary kidney and creatinine level decreased significantly following the procedure. Dilatation and incision with PCBD is a novel approach for the treatment of the short ureteroenteric anastomotic strictures. Long-term data need to be obtained to establish the efficacy of this technique.

The cutting balloon for endoscopic dilatation of pediatric subglottic stenosis

In recent years, the increasing use of endoscopic techniques for management of pediatric subglottic stenosis has become evident. However, two problems encountered with conventional balloon dilatation are that of re-stenosis and distal balloon migration. We report a novel technique of endoscopic dilatation using the Peripheral Cutting Balloon™ Microsurgical Dilatation Device in a 4-year-old child with symptomatic Cotton Grade 2 subglottic stenosis. The presence of microsurgical blades on the balloon allows for more secure placement of the balloon at the level of the stenosis, preventing migration and allowing for more accurate dilatation under less pressure decreasing re-stenosis rates. We recommend that the cutting balloon be used as first line treatment in pediatric subglottic dilatation.

153 EFFECTS ON SEX RATIO AND PREGNANCY RATES OF IN VIVO-DERIVED BOVINE EMBRYOS USING LOOP-MEDIATED ISOTHERMAL AMPLIFICATION SEXING METHOD

In this study, we examined the effects of developmental stages and quality grades on sex ratio of in vivo-derived bovine embryos. Furthermore, pregnancy rates of fresh frozen-thawed sexed embryos or intact (non-sexed) fresh and frozen-thawed embryos were compared in order to efficiently carry out the sexing of embryos in the field. Embryos were collected from donors at 7 days after estrus following a routine superovulation protocol, and classified into four stages (late morula, early blastocyst, blastocyst, and expanded blastocyst) and two quality grades (Grade 1 and Grade 2–3) by the IETS manual. Embryos were frozen by direct transfer method from 1 to 3 h post-collection in 0.25-mL straws as described previously (Aoyagi et al. 1996 Theriogenology 45, 165 abst). Frozen embryos were thawed in 30°C water for 20 s following 7 s in air. They were then squeezed out into PBS + 5% FCS (PBS), washed twice, and incubated in CR1aa + 5% CS (CR1aa) or PBS. Recently, a commercial embryo sexing program was performed at our laboratory using loop-mediated isothermal amplification (LAMP). The procedure takes 5 min to perform each embryo biopsy and only 40 min for the LAMP process. A few cells of fresh (F; n = 105) and frozen-thawed (Z; n = 143) embryos of Grade 1 (H), and fresh (F; n = 77) embryos of Grade 2–3 (L) were biopsied with a microsurgical blade, and sex was determined by the LAMP method. Embryos were transferred non-surgically into heifers on Day 7 of the estrus cycle. Pregnancies were determined by ultrasonography on Day 30. Data were analyzed by the chi-square test. The sexing of all 325 embryos yielded 148 female (46%), and only 2 embryos were indeterminant (1%). There was no evidence of any effect of developmental stage on sex ratio (female embryos: late morula 69/157 (44%), early blastocyst 42/94 (45%), blastocyst 29/53 (55%), and expanded blastocyst 10/21 (48%)). However, when the sex ratio was examined for embryos of different quality grades, significantly more females were found in the embryos appearing more degenerated (female embryos: FH + ZH vs. FL; 42% vs. 57%, P < 0.05). Pregnancy rates on Day 30 with FH embryos (38/45, 84%) were similar to rates obtained with non-sexed fresh (60/81, 78%) and frozen-thawed embryos (44/54, 82%). The pregnancy rates on Day 30 with ZH embryos incubated in CR1aa (18/40, 45%) were lower than those of FH, non-sexed fresh, and frozen-thawed embryos. However, pregnancy rates of ZH embryos incubated in PBS (13/16, 81.3%) were significantly higher than for those frozen embryos that were thawed and incubated in CR1aa (P < 0.05). After the transfer of embryos sexed by the LAMP method to recipient animals, all 55 calves born were of the predicted sex. In conclusion, the present results showed that with the LAMP method for sexing of the embryos, there were only a few samples for which sex could not be determined. Examination of in vivo-derived Day 7 embryos indicated that female embryos graded lower than male embryos. Furthermore, the removal of a few cells from a fresh or frozen-thawed embryo did not impact its subsequent viability.

Cutting Balloon Versus Conventional Balloon Angioplasty for the Treatment of Coronary Ar ter y Disease

The cutting balloon (CB)1 is a special balloon catheter with three or four atherotomes (microsurgical blades) bonded longitudinally to its surface, suitable for creating discrete longitudinal incisions in the atherosclerotic target coronary segment during balloon inflation.

Transfer of sexed caprine blastocysts freshly collected or derived from cultured morulae

This paper describes a simplified method for biopsying goat embryos for sex determination, followed by the transfer of these embryos to recipient does. A 90° angle holding pipet is described, which permits holding the embryo in position and, with minimal effort, open the zona pellucida and excise 5–10 trophoblast cells with the aid of a microsurgical blade. Twenty-four embryos were biopsied, 12 of which were freshly collected (day 7) and 12 of which were cultured in vitro from collected morulae (day 6). In the course of the polymerase chain reaction (PCR) a 447 (male) and 445 bp (female) fragment of the DNA, derived from the trophoblast cells, was amplified. The primers used were specific for the highly conserved mammalian sequences of the ZFX and ZFY genes. Individual biopsied embryos were transferred to 24 synchronized recipient does. The pregnancy rate following transfer was 67% (8/12) in recipients receiving freshly collected blastocysts while only 8% (1/12) in the recipients receiving blastocysts cultured in vitro from morulae ( P < 0.01). Four of the kids born were males and four females. The sex assigned to the embryos was correct in 83% of the cases.

In Vitro and in Vivo Survival of Bovine Demi-Embryos Following Simplified Bisection and Transfer of One or Two Halves per Recipient.

A simplified procedure for bisection of bovine morulae and blastocysts involving a fine microsurgical blade and Dulbecco's phosphate-buffered saline (PBS) supplemented with 0.3 M sucrose and 0.1 mM ethylene diamine tetra acetic acid (EDTA) was employed to examine the developmental ability of the bisected demi-embryos. This procedure did not require a holding pipet to restrain the embryo and only a single micromanipulator was needed. Bisected embryos were transferred to recipient heifers either as pairs of demi-embryos or as single demi-embryo to compare maintenance of pregnancy and fetal survival as to the incidence of identical twins. Blastocyst formation rate was significantly (P<0.05) higher (78.3% vs 60.5%) in embryos bisected in medium containing sucrose as compared to controls when early or mid-blastocyst stage embryos were bisected. Bilateral transfer of bovine demi-embryos generated more fetuses per original embryo than unilateral transfer of a single demi-embryo (105% vs 93%). Embryos producing identical twin pregnancies from sets of demi-embryos (bisected-twins) represented 30.7% of the total while 39.8% of embryos produced one pregnancy and the remaining 29.5% failed to result in pregnancy. The results show that bovine embryos can be bisected by a simplified method and lead to normal pregnancies with a high incidence of identical twins.

Production of Identical Twins from Bovine Embryos Split Pre- of Post-freezing.

Three methods for producing identical twin calves are described. In treatment 1, day 7 bovine embryos were bisected in 12.5% sucrose solution by lateral incision using a fine microsurgical blade after freezing with 1.4 M glycerol and thawing. Both halves remained in the original zona pellucida. In treatment 2, the frozen-thawed embryos were pinned between the blade and the bottom of a plastic petri dish and bisected vertically in PBS. In treatment 3, zona free demi-embryos were cultured at 38.5°C in Ham's F-10, supplemented with 20% bovine serum for 3 hr prior to freezing. In all the treatments, a pair of half-embryos were transferred nonsurgically to a uterine horn of recipients on day 7 of their estrous cycle. In treatment 1, 26 of 81 recipients became pregnant after transfer of the demi-embryos (Pregnancy rates=32%). Five pair of identical twins were obtained from 5 of 22 recipients which delivered offspring. In treatment 2, the pregnancy and twinning rates were 42% (13/31) and 33% (4/12), respectively. One pair of identical twins was obtained from frozen-thawed zona free demi-embryos transferred without removing 1, 2- propanediol after thawing, in treatment 3. These data showed that bovine identical twins could be produced from frozen embryos bisected after thawing, followed by transfer without zona pellucidae.

Microsurgical Blades

Microsurgical Blades

Viability of frozen-thawed bovine embryos bisected in sucrose: A preliminary report

A method for producing identical twin calves is described in which Day 7 frozen-thawed bovine embryos in 12.5% sucrose solution were bisected using a fine microsurgical blade. The resulting bisected embryos were transferred nonsurgically to the uterine horn ipsilateral to the corpus luteum of synchronous recipients (±1 d), two bisected embryos per recipient. The pregnancy rate when both halves remained in the same zona pellucida was 50% ( 5 10 ); the pregnancy rate was 1 5 for morulae and 4 5 for blastocysts. The pregnancy rate for unfrozen morulae bisected in PBS and transferred without zona pellucida was 27% ( 4 15 ). The in vitro survival rate of embryos bisected in 12.5% sucrose when both halves remained in the original zona pellucida was 82% ( 18 22 ), which was higher than when embryos were bisected in PBS (53%, 9 17 ).

A technique for bisection of embryos to produce identical twins.

A successful technique for bisection of postcompaction-stage embryos has been devised that combines methods of micromanipulation developed by other workers. Several papers describe generally and in detail various equipment, microtools, and methods of negotiating the bisection of bovine and ovine embryos (6). Willadsen (4,5) used a fine glass needle to pene trate the zona pellucida and to sever the embryo against a holding pipette using a Wild M5 Stereozoom microscope. Ozil (3), using 6 micromanipulators, opened the zona pellucida with needles, ejected the embryo with medium from a suction pipette, then cut the embryo with a specially machined microscalpel. Needles were also used by Lambeth et al. (2) and Gatica et al. (1) to access the embryo and cut it against the bottom of a petri dish. Williams and Seidel (7) used a fine microsurgical blade for opening the zona pellucida and bisecting the embryo in a single step. These methods were attempted with various difficulties in our laboratory. Spatial visualization was difficult when attempting needle cutting against the holding pipette with a Leitz Diavert inverted microscope. The alternative microsurgical blade made a cut in the zona pellucida suitable for introducing a suction pipette, but the embryo tended to stick when the blade was made to cut completely through it. The blade approach was generally more favorable but only for the initial stage of cutting. A method was needed to finish cutting the embryo without the problem of sticking. The fine glass needle seemed suitable for this task, which could be accomplished against the floor of the petri dish after the embryo was gently removed from the zona by blowing in medium from the suction pipette. The resultant technique utilizes a holding pipette, a microsurgical blade for splitting the zona and embryo, and a suction pipette for withdrawing half a severed embryo and inserting it into an empty zona pellucida or for ejecting the semidivided embryo to the floor of the petri dish for completion of the separation with a fine glass needle. Since the blade and needle tools do not require a pressure line to a microinjection syringe, they are easily interchangeable, and only one holder is required for them. Only 2 micromanipulators are required with one single and one double holder for all 4 microtools.

Transfer of bovine demi-embryos with and without zonae pellucidae

A method for producing identical twin calves is described in which 5.5 to 7.5 day bovine embryos were bisected using a fine microsurgical blade. The resulting demi-embryos were transferred surgically or nonsurgically to the uterine horn ipsilateral to the corpus luteum of synchronous recipients (± 2 days), one demi-embryo per recipient. The mean pregnancy rate per demi-embryo was 48% (157/330), and per whole embryo split, it was 95% (157/165). Pregnancy rate was dependent on the stage of embryonic development. The pregnancy rate from bisected early morulae, 16% (7/44), was lower than that from bisected early blastocysts, 60% (58/96) P<0.01. The rate of identical twin production per embryo split reflected these pregnancy rates. Bisected blastocysts resulted in more identical twin sets, 39% (24/62), than early morulae, 9% (2/22) P<0.01. When used with normal embryo transfer procedures, this technique provides a method for routine production of identical twin calves. Furthermore, more calves are produced per two demi-embryos than per whole embryo.