A.T. Palasz

Transvaginal collection and ultrastructure of Llama ( Lama glama ) oocytes

Ultrasound-guided transvaginal follicle aspiration has been described as a noninvasive and repeatable procedure for oocyte collection in several species, but its use has not been described for any of the members of the family, Camelidae. A study was designed to determine the feasibility of an ultrasound-guided transvaginal approach for oocyte collection in llamas. Fifteen non-pregnant, adult female llamas (10 non-stimulated and 5 superstimulated) were examined by transrectal ultrasonography with a 7.5-MHz linear-array transducer to determine the number and diameter of follicles available for aspiration. After caudal epidural anesthesia was induced, the 7.5-MHz linear-array transducer was fastened to a long rigid handle and inserted intravaginally. The free hand was placed into the rectum to manipulate the ovaries, one at a time, in position against the vaginal wall over the face of the transducer. A 20-gauge, 55-cm-long, single-lumen needle was advanced through the vaginal fornix and into follicles > or = 3 mm in diameter. Follicular contents were aspirated using a regulated vacuum pump (flow rate = 33 mL/min; approximately 150 mm Hg) into a tube containing 3 mL of phosphate buffered saline and 0.2% BSA. Fluid was filtered (75 microm mesh), and oocytes were located and morphologically evaluated using a stereomicroscope. Overall, 134 follicles were aspirated, and 76 oocytes were collected (collection rate = 57%). Thirty-two oocytes (42%) were surrounded by multiple layers of compacted granulosa cells and had homogenous dark ooplasm; 13 oocytes (17%) were surrounded by the corona radiata layer only and had heavily granulated ooplasm; 9 oocytes (12%) were denuded and had homogenous dark ooplasm; and 22 oocytes (29%) were denuded and displayed signs of ooplasm degeneration. The ultrastructure of llama oocytes was similar to that of cattle except for conspicuous accumulation of large lipid droplets in the cytoplasm. Twenty-four hours after follicle aspiration, the ovaries were examined by transrectal ultrasonography and intrafollicular hematomas were detected in 3 llamas (9 of 48 follicles aspirated). Results demonstrate the potential utility of a transvaginal ultrasound-guided technique for oocyte collection and in vitro embryo production in llamas. Oocytes of llamas bear an ultrustructural resemblance to those of cattle, but are distinguished by a predominance of cytoplasmic lipid.

Development, molecular composition and freeze tolerance of bovine embryos cultured in TCM-199 supplemented with hyaluronan.

Hyaluronan (HA) is glycosaminoglycan that is present from the start of embryonic development and its role and concentration increases with embryo development. The objective of this study was to evaluate if the presence of HA in TCM-199 culture medium had an effect on the development and quality of bovine embryos. There was no effect of HA on the total number of zygotes developing to blastocysts on day 7, however more expanded and hatched blastocyst stages were observed on days 8 and 9 in the group supplemented with HA (p<0.05). Following freeze/thawing, significantly more (p<0.05) embryos cultured in medium supplemented with HA hatched than those cultured in TCM-199 alone or those with BSA. Medium supplemented with HA and BSA significantly increased the level of expression of glucose metabolism Glut-1 gene and embryo compaction Cx43 gene (p<0.05), and had no effect on Glut-5 and IGF-II expression. In addition, HA presence in culture decreased the level of expression of apoptosis Bax and oxidative stress SOX genes (p<0.05). There was significant difference in total number of nuclei between TCM-199 medium only and the remaining media containing BSA or HA plus BSA, between which there was no difference. In summary, our results indicate that the addition of high molecular weight HA to TCM-199 medium that contains BSA on day 4 of culture improved embryo development to hatching and hatched blastocysts and the quality of produced embryos, which were superior to embryos cultured without HA addition.

POST-THAW VIABILITY OF EUROPEAN BISON (BISON BONASUS) SEMEN FROZEN WITH EXTENDERS CONTAINING EGG YOLK OR LIPIDS OF PLANT ORIGIN AND EXAMINED WITH A HETEROLOGOUS IN VITRO FERTILIZATION ASSAY

Abstract Basic characteristics of European bison (Bison bonasus) semen were described and the efficacies of two extenders—Triladyl, containing egg yolk, and a synthetic extender, containing soybean lipids—were tested for semen cryopreservation. Seven ejaculates were collected by electroejaculation from a 10-yr-old, European bison bull. Each ejaculate was diluted at 37°C to a final concentration of 200 × 106 sperm/ml with Triladyl or the synthetic extender. Extended semen samples were frozen according to a standard bull semen freezing protocol. After 2 wk of storage, one straw from each extender and ejaculate was thawed, and postthaw quality was evaluated by individual sperm motility and movement rate, numbers of sperm morphologic abnormalities and intact acrosomes, functional integrity of the sperm membranes determined by hypoosmotic swelling test (HOST), viability (live–dead, eosin–nigrosin stain), and a heterologous in vitro sperm penetration assay (SPA). A total of 600 in vitro–matured bovine oocytes were inseminated with 1 × 106 spermatozoa of Holstein semen frozen–thawed in Triladyl (control) or of European bison semen frozen in Triladyl or the synthetic extender. Nuclear status of the oocytes was determined after 18 h of sperm–oocyte coincubation. Extender had no effect on any evaluated parameters of semen after dilution and cooling (4 hr at 5°C) or in postthaw individual motility, quality of movement, and sperm morphology. However, significantly (P < 0.05) higher numbers of spermatozoa with intact acrosomes, intact membranes (HOST), and viable sperm (P < 0.01) were in semen frozen in Triladyl than in the synthetic extender. Mean values for heterologous SPA for bull (control) and for bison semen frozen in the synthetic extender were very much alike—63.3 ± 10.6% and 63.1 ± 15.9%, respectively; bison semen frozen in Triladyl was lower, 43.0 ± 24.2% but not significantly different. Cumulative results from a variety of viability assays of diluted/cooled and frozen–thawed semen, including the heterologous SPA, suggest that European bison semen can be successfully frozen in both extenders tested in this study.

Vitrification of bovine IVF blastocysts in an ethylene glycol/sucrose solution and heat-stable plant-extracted proteins

The use of heat-stable plant proteins in an ethylene glycol-based solution for the vitrification of in vitro-derived embryos was examined. Day 7, 8 and 9 bovine in vitro matured, fertilized and cultured (IVMFC), full and expanded blastocysts were vitrified in solutions composed of 40% ethylene glycol (EG) plus 0.3 M sucrose supplemented with 20% Ficoll and 0.3% BSA (VF-1), 25 mg/ml heat-stable plant proteins (HSPP; VF-2), or with no supplement (VF-3). In Experiment 1, embryos were expelled from the straw after thawing, and EG was diluted from embryos with 0.5 M sucrose. There were no differences in post-thaw embryo survival rates or in hatching/hatched rates after 24 h of culture between the VF-1, VF-2 and VF-3 solutions (40.1, 54.1 and 50.8% and 10.7, 16.4 and 17.5%, respectively). Transfer of 12 frozen/thawed embryos to 6 recipients (2 recipients per treatment) resulted in 2 pregnancies from the VF-2 group and 1 pregnancy from the VF-3 group. In Experiment 2, EG was diluted from embryos after thawing within the straw with 0.5 M sucrose. There were no differences in post-thaw survival or hatching/hatched rates after 24 h of culture (19.0, 13.6 and 23.8% and 9.5, 9.0 and 14.4% for VF-1, VF-2 and VF-3, respectively). Transfer of 6 frozen/thawed embryos to 3 recipients (1 recipient per treatment) resulted in no pregnancies. The post-thaw histology of Day 7, 8 and 9 IVMFC blastocysts showed typical ultrastructure with well preserved cell-to-cell contacts. There were no major differences in the fine structure of blastocysts regardless of treatment. The use of HSPP at a concentration of 25 mg/ml in the vitrification medium did not affect the post-thaw embryo survival over that of no protein supplementation. The presence of macro molecules in a 40% EG/sucrose vitrification solution also did not improve post-thaw viability of IVMFC-derived blastocysts.

192 THE EFFECT OF ZWITTERONIC BUFFERS AND PBS ON GENE TRANSCRIPTION OF BOVINE IN VIVO- AND IN VITRO-DERIVED EMBRYOS

Choice of buffer used for the IVF procedures affects embryo developmental rates (De la Fuente et al. 2006 Reprod. Fertil. Dev. 18, 187). Also, it has been shown that the 3 zwitterionic buffers tested in this study, TES (T), MOPS (M), and HEPES (H) (pKa values at 20°C: 7.2–7.5) interact with DNA (Stellwagen et al. 2000 Anal. Biochem. 287, 167). Our objective was to evaluate the effect of T, M, H, and PBS buffers on the expression of the following genes, Fgf-4 (fibroblast growth factor 4 precursor), Lama1 (laminin alpha 1), Ube2a (ubiquitin-conjugating enzyme), Gsta4 (glutathione S-transferase A4), Il6 (interleukin 6), Sod1 (superoxide dismutase), Prss11 (IGF binding), and Hspb1 (Heat shock protein binding 1), on bovine in vivo and in vitro embryos. Genes were selected based on their sensitivity to adverse in vitro embryo culture conditions by microarray analysis (data not shown). All buffers were prepared at a concentration of 10 mM in TALP medium and the final pH was adjusted to 7.2. Bovine follicular fluid was aspirated from abattoir-derived ovaries and evenly divided into 4 tubes. Collected oocytes (5 replicates) from each tube were processed separately through entire IVM, IVF, and IVC procedures using washing medium buffered with: PBS (n = 490), Group 1; H (n = 438), Group 2; M (n = 440), Group 3; and T (n = 394), Group 4. All buffers contained 4% BSA. Oocytes were matured in TCM-199 supplemented with 10% FCS and 10 ng mL-1 epidermal growth factor, and inseminated in Fert-TALP containing 25 mM bicarbonate, 22 mM sodium lactate, 1 mM sodium pyruvate, 6 mg mL-1 BSA-FAF, and 10 µg mL-1 heparin with 1 × 106 mL spermatozoa. After 24 h of oocytes–sperm co-incubation, presumptive zygotes were cultured in SOFaa medium with 8% BSA at 39°C under paraffin oil and 5% CO2 in humidified air. Cumulus–oocyte complexes and zygotes were held in designated buffers in vivo blastocysts exposed for P in vitro embryos irrespective of buffer used. In addition, higher expression of Hspb1 and lower expression of Ube2a and Lama 1 were identified in PBS and T than in M and H (P in vitro embryos were lower (P in vitro-derived embryos. It can be concluded that mRNA transcription of in vitro-derived embryos is affected by the choice of the buffer used for the IVF procedure.

320 USE OF SYNTHETIC HYALURONAN OR POLYVINYLPYRROLIDONE FOR INTRACYTOPLASMIC SPERM INJECTION INTO MOUSE OOCYTES

The use of polyvinylpyrrolidane (PVP) in intracytoplasmic sperm injection (ICSI) seems to be exclusively related to its surfactant and colloidal properties. In contrast to PVP, which can be toxic to mouse embryos, hyaluronan (HA) is a biological compound. In addition to its colloidal property, HA plays an important biochemical role in cell proliferation and migration and can be found intracellularly in the cleaving stage of mouse, sheep and primate embryos (Hunter RHF 1994 Mol. Reprod. Dev. 39, 176–181). We expect that the viscoelastic properties of HA in combination with its physiological functions may benefit the ICSI procedure. Oocytes at MII stage were collected from CD-1 mice 14 h after hCG injection (h-pi) and were kept at 37°C in KSOM medium for 30 min before ICSI. Semen used for injection was frozen by direct plunge into liquid nitrogen in M2 medium without cryoprotectants. Samples were thawed at 25°C in the air and mixed (1:5) with M2 medium containing either 10% PVP; 360000 MW (w/v; Sigma, St. Louis, MO, USA) or 60% (v/v) synthetic HA (s-HA; MAP-5; Bioniche Inc, Belleville, ON Canada) with comparable viscosity. Injections were performed at 25°C using a mercury-containing pipette attached to a piezo impact unit (Prime Tech, Ibaraki, Japan). A total of 239 oocytes (115 PVP and 124 s-HA) were injected in groups of ten in four replicates. Individual sperm heads decapitated by the freeze/thaw procedure were injected into oocytes and kept for 15 min at 25°C. Oocytes that survived ICSI were placed in 35 μL drops of KSOM medium (~15 zygotes per drop) under paraffin oil at 37°C and 5% CO2 in humidified air. Cleavage and developmental rates were recorded at 24, 48, and 96 h after oocyte injection. Embryos which developed to the blastocyst stage were transferred to pseudo-pregnant females mated with vasectomized males. At Day 13, recipient mice were sacrificed and the number of implantations and fetuses were recorded. Data were compared between groups by Chi-square analysis. Significantly (P < 0.05) more embryos survived ICSI in PVP (74%) than in s-HA group (56%), which was primarily related to sperm adhesiveness to the injection pipette. However, there were no differences in developmental rates at any stage of in vitro embryo culture between groups (2 cell, 93 vs. 100%; 4–8 cell, 100 vs. 100%; blastocyst, 44 vs 50%) for PVP and s-HA, respectively. Significant differences (P < 0.05) between groups were observed in embryo implantation rates. When ICSI was performed with s-HA, 29 out of 35 blastocysts (83%) transferred to synchronized recipients were implanted, which was accomplished only by 19 of the 35 from the PVP group (54%). However, there was no difference between groups in the number of fetuses detected (8 (23%) vs. 9 (26%) for PVP and s-HA, respectively). The use of s-HA for mouse ICSI can be a valuable alternative to PVP. Hyaluronan may show further benefit if sperm adhesiveness to the micropipette can be eliminated, and may be superior to PVP if embryo implantation rates in the s-HA group can be sustained. The authors would like to thank Bioniche, Inc., Belleville, ON, Canada for donating MAP-5.

146 THE EFFECT OF CULTURE TEMPERATURE ON THE CLEAVAGE, DEVELOPMENT, AND GENE TRANSCRIPTION PATTERNS OF BOVINE EMBRYOS

Bovine oocytes matured and fertilized at 39°C had significantly higher rates of fertilization than at 37°C (Lenz et al. 1983 Biol. Reprod. 29, 173–179). However, embryo culture temperature that may affect molecular composition and metabolism of embryo membranes was not investigated. The objective of this study was to determine the effect of two culture temperatures, 37 and 39°C on the cleavage, in vitro development, and gene transcription patterns in bovine embryos. A total of 794 oocytes (5 replicates) were matured in TCM-199 medium containing 10% FCS and 10 ng/mL epidermal growth factor and inseminated with 1 × 106/mL spermatozoa in groups of 50 in 250 μL in Fert-Talp supplemented with 25 mM bicarbonate, 22 mM sodium lactate, 1 mM sodium pyruvate, 6 mg/mL BSA-FAF, and 10 μg/mL heparin at 39°C. After 24 h of oocyte-sperm co-incubation, presumptive zygotes were divided into two groups and cultured at 37 or 39°C under paraffin oil and 5% CO2 in humidified air. Embryos were cultured in SOFaa medium supplemented with 4% BSA in 50-μL drops (25 zygotes per drop). Cleavage rates were recorded on Day 2 and zygote development on Days 4, 7, 8, and 9. At least 5 blastocysts from each replicate from each treatment were frozen for evaluation of gene expression patterns. Poly(A) mRNA was prepared from 6 groups of pools of 5–7 embryos. A total of 11 developmentally important mRNA transcripts were examined. The quantification of all gene transcripts was performed by real time quantitative RT-PCR in three replicates. Embryo development was analyzed by chi-square analysis and data on mRNA expression by one-way repeated-measures ANOVA. There was no effect of culture temperature on embryo cleavage (65.2 and 72.0% for 37 or 39°C, respectively) but at Day 4 significantly (P < 0.05) more embryos developed to <8 cells at 39°C (32.5%) than at 37°C (25.9%). There was no difference in the total number of blastocysts produced in either temperature (22.4% at 37°C and 22.1% at 39°C), and significantly (P < 0.05) more zygotes that were at <8-cell stage at Day 4 progressed to the blastocyst stage at 37°C (86.4%) than at 39°C (66.6%). Transcript levels for genes related to response to stress (SOX, IFNτ) and glucose metabolism (Glut-1, G6pd) were higher (P < 0.05) in the blastocysts cultured at 39°C, and levels of Glut-5 (glucose metabolism) and Oct-4 (factor related to pluripotency) were higher (P < 0.05) in the blastocysts cultured at 37°C. No difference was found in mRNA transcription of genes related to apoptosis (BAX), response to heat (Hsp70), compaction (Ecad, Na/K, DcII), and cell connection (Cx43) at either temperature tested. It can be concluded that culture temperature may affect embryo molecular composition and the kinetics of embryo development.