bluuebunny/crossref_metadata_2025 huggingface dataset:
103 BIRTH OF CLONED PIGLETS DERIVED FROM AN OPTIMIZED IN VITRO BLASTOCYST PRODUCTION SYSTEM BY TRICHOSTATIN A TREATMENT
Authors: Zhang Y.-H. , Du Y.-T. , Zhang K. , Li J. , Kragh P. M. , Villemoes K. , Pederson A. M. , Schmidt M. H. , Bøgh I. B. , Purup S. , Bolund L. , Jørgensen A. L. , Yang H.-M. , Li N. , Vajta G.
Published: December 2006

The present study was designed to examine the effect of trichostatin A (TSA, a histone deacetylase inhibitor) treatment on in vitro developmental ability of pig cloned embryos and to evaluate the feasibility of producing piglets from these embryos. Cell lines were established from 40-day-old fetuses, and adult ear skin was used as nuclear donor. In vitro-matured oocytes from abattoir-derived sow ovaries were used as cytoplast recipients for micromanipulator-assisted somatic cell nuclear transfer (SCNT). Data were analyzed by using SPSS (11.0) with one-way ANOVA, and each experiment was replicated at least 3 times. In Experiment 1, immediately after simultaneous fusion and activation, the reconstructed couplets were randomly cultured in porcine zygote medium 3 (PZM3; Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) with 10 �g mL-1 cytochalasin B (CB), 10 �g mL-1 cycloheximide (CHX), and 0 nM, 5 nM, or 50 nM TSA for the first 4 h. Cloned embryos (fused reconstructed couplets) were moved to the same culture media but without CB and CHX and further cultured at 38.5�C, under 5% CO2, 5% O2, 90% N2 and 100% humidity. After incubation for a total of 8–14 h in 50 nM, 19–24 h in 50 nM or 5 nM, and 31–36 h in 50 nM TSA in PZM3 (0 nM TSA serves as control for each group), the embryos were further cultured in vitro without TSA in PZM3 for up to 168 h. Cleavage and blastocyst development rates, based on embryos cultured, were recorded at 48 and 168 h of IVC, respectively. Results showed that 50 nM TSA treatment for 19-24 h supported a higher blastocyst development rate than the control group [No. blastocysts/No. embryos cultured (mean � SEM): 107/258, 47.4 � 5.9% vs. 65/324, 20.0 � 2.3%, respectively; P < 0.05], whereas similar pre-implantation development was obtained between the other 3 test groups and the control. In Experiment 2, TSA-treated cloned embryos at the one-cell stage or blastocyst stage were transferred to recipients to examine the possibility of producing piglets. Ten cloned piglets (2 are healthy and 8 died shortly after birth) and one ongoing pregnancy were obtained from 3 recipients who received an average of 110 one-cell stage embryos, whereas 4 piglets originating from traditional cloning were produced from one recipient which received 28 traditional cloned blastocysts (produced from the effective group in Experiment 1) and 30 handmade but non-TSA-treated ones. Our data demonstrate that TSA treatment after SCNT in porcine can significantly improve the in vitro blastocyst production, and embryos treated with TSA could support full-term development and result in healthy offspring.
106 USING A BUOYANT DENSITY GRADIENT AND NILE RED STAINING TO EVALUATE THE LIPID CONTENT OF BOS TAURUS AND BOS INDICUS OOCYTES
Authors: Ballard C. B. , Looney C. R. , Lindsey B. R. , Pryor J. H. , Lynn J. W. , Bondioli K. R. , Godke R. A.
Published: December 2006

Bos indicus embryos have a lower survival rate compared with Bos taurus after cryopreservation. It has been hypothesized that the lower survival rate is due to higher intracellular lipid content. The objective of this study was to determine if there is a difference in intracellular lipid content of oocytes from mature purebred Brahman and Angus cows. Donor females used in the study were maintained on pasture prior to the onset of the experiment and on a grain-supplemented hay ration during the study. Oocytes were collected from cows at 30-day intervals (3 aspirations/donor) by transvaginal ultrasound-guided oocyte aspirations (TUGA). Mature oocytes were evaluated using a sucrose step gradient procedure and Nile Red staining. FSH (Folltropin-V®; Bioniche Animal Health, Beltsville, Ontario, Canada) administration began on Day 4 of the estrous cycle (estrus = Day 0) twice daily for 3 days in decreasing doses (Brahman 232 mg and Angus 280 mg total), and on Day 8 oocytes were recovered. The mean number of follicles aspirated/donor and oocytes recovered/donor were 20 and 16.61 oocytes for the Brahman donors (n = 6) and 12 follicles and 7.06 oocytes/donor for the Angus donors (n = 10). Oocytes (individual donor basis) were then incubated in TCM-199 supplemented with 10% fetal bovine serum + bLH and bFSH (0.01 U mL−1) at 38.5°C. After 20 h, mature oocytes were denuded by vortexing for 3 min in HEPS + BSA (4 mg mL−1). Buoyancy was tested for individual mature oocytes using a sucrose step density gradient column prepared with sucrose and Dulbecco's PBS. Results from the sucrose gradients ranged from 23% sucrose (indicating high lipids) to 35% sucrose (indicating lower lipids). Oocytes recovered from the sucrose were fixed for 24 h in paraformaldehyde for evaluation with Nile Red stain. Oocytes were stained for 24 h, and then placed in Prolong® Gold (Invitrogen, Carlsbad, CA, USA) and evaluated under fluorescence. Oocytes images were evaluated using a Scion Image camera (Scion Corp., Frederick, MD, USA) to calculate mean (± SE) Nile Red units (NRU) (higher NRU = higher lipid content). Treatment groups were analyzed using one-way ANOVA. In summary, Brahman M-II oocytes had significantly lower (P ≤ 0.05) buoyant density, with a significantly higher mean NRU score, when compared with oocytes harvested from Angus donors (Table 1). Based on these results, Brahman oocytes have a higher intracellular lipid content then Angus oocytes.

Table 1.Percent sucrose levels and Nile Red units for bovine oocytes from three replicates per donor
124 TAXOL™ COULD PROMOTE EMBRYO DEVELOPMENT OF BOVINE OOCYTES VITRIFIED BY OPS
Authors: Morató R. , Izquierdo D. , Palomo M. J. , Anguita B. , Jiménez-Macedo A. R. , Albarracín J. L. , Paramio M. T. , Mogas T.
Published: December 2006

Stabilizing the cytoskeleton system during vitrification could be beneficial for improving post-thawed survival and subsequent development of vitrified oocytes. Taxol™, paclitaxel, is a microtubule stabilizer that has been found to improve development competence of vitrified mouse and human oocytes. The objective of this work was to study the effect of a Taxol pretreatment before OPS vitrification on the post-thaw cow and calf oocyte development. Oocytes were aspirated from slaughterhouse-derived ovaries and matured in TCM-199. Oocytes were randomly assigned to one of 3 experimental groups: (1) control oocytes matured in vitro for 24 h, (2) oocytes matured for 22 h and vitrified by the OPS method (Vajta et al. 1998 Mol. Reprod. Dev. 51, 53–58), and (3) oocytes matured for 22 h and vitrified by OPS method with 1 µM Taxol. OPS and Taxol–OPS oocytes were transferred back into the maturation dishes and matured for 2 additional h before being subjected to fertilization. Fertilization was performed using frozen–thawed Percoll-selected sperm. At 22 h after insemination, presumptive zygotes were pipetted and then cultured in drops of 25 µL SOF medium and 5% fetal calf serum under paraffin oil at 38.5°C in 5% CO2, 5% O2, 90% N2, and maximum humidity. The Taxol–OPS group provided a significantly higher cleavage rate than the OPS group in cows (41.9% and 34.0%, respectively) or in calves (33.7% and 23.5%, respectively). However, cleavage rate in the experimental groups was significantly lower than in the control group (78.3% and 69.7% for cow and calf control groups, respectively). Blastocyst yield was also higher for the Taxol–OPS group (3.2%) than the OPS group (0%) in cow oocytes. There was no blastocyst development when calf oocytes were vitrified with or without Taxol pretreatment. As expected, cow and calf vitrification groups triggered a significantly lower blastocyst yield when compared with their control (26.7% and 14.9% for cow and calf control groups, respectively). In conclusion, this study showed that supplementation of 1 µM Taxol could promote embryo development after thawing. Further research is indicated to clarify the function of Taxol and its optimal concentration in order to improve the rate of embryo development.

Table 1.
Effect of Taxol pretreatment on development of cow and calf
oocytes vitrified by OPS
126 CRYOPRESERVATION OF PORCINE EMBRYOS DERIVED FROM IVM OOCYTES
Authors: Nakayama N. , Hiruma K. , Kurome M. , Tomii R. , Ueno S. , Saito H. , Matsunari H. , Nagashima H.
Published: December 2006

We have reported that a combination of delipation (removal of cytoplasmic lipid droplets from blastomeres) and vitrification by means of the minimum-volume cooling (MVC) method successfully cryopreserves porcine in vitro-matured/fertilized (IVM/IVF) embryos, and that normal piglets are produced from these embryos (Hiruma et al. 2006 Reprod. Fertil. Dev. 18, 157). We have also reported that IVM-derived embryos that undergo noninvasive delipation (i.e. micromanipulation is not required) and vitrification develop into blastocysts at a high rate (Esaki et al. 2004 Biol. Reprod. 71, 432–437). In this study, we examined whether fetuses can be produced from the IVM-derived embryos that have been delipated noninvasively and vitrified. Cumulus–oocyte complexes that had been collected from slaughterhouse ovaries were in vitro-matured in NCSU23 medium. The IVM oocytes were activated to produce parthenogenetic embryos. We used the embryos at the 4- to 8-cell (67 h after activation) and morula (98 h) stages in the following experiments. Embryos were treated with 4% trypsin (in PBS) at 38�C for 1 to 4 min to expand the zona pellucida. Next, the embryos were centrifuged (12 000g, 38�C, 23 min) in TL-HEPES-PVP containing 7.5 �g mL-1 cytochalasin B to polarize cytoplasmic lipid droplets within the perivitelline space. These embryos were cultured for 1 to 3 h and then vitrified. The post-thaw viability of the embryos was assessed based on their ability to develop into blastocysts and fetuses (21 to 23 days old). The embryos were vitrified using the MVC method with 15% ethylene glycol, 15% DMSO, and 0.5 M sucrose as cryoprotective agents. PZM-5 was used for culturing the embryos. In embryo transfer experiments, after thawing, the embryos were cultured for 36 or 72 h until they developed into morulae or 4- to 8-cell blastocysts, respectively; they were then treated with 0.5% pronase to remove the zona pellucida, and transferred to the uterine horns of estrus-synchronized recipients 6 days after onset of estrus. The proportion of vitrified embryos that developed into blastocysts and the mean cell number of the blastocysts were similar to those of non-vitrified control embryos, irrespective of the embryonic stage (4- to 8-cell stage: 42.1%, 22/51, 63.0 � 7.8 vs. 64.7%, 22/34, 74.2 � 7.1, respectively; morula stage: 77.6%, 38/49, 69.6 � 7.2 vs. 83.3%, 45/54, 66.2 � 5.9, respectively). Seventeen embryos that had been vitrified at the 4- to 8-cell stage gave rise to 3 fetuses after transfer into one recipient (17.6%). Fifty-three embryos that had been vitrified at the morula stage were transferred into 3 recipients. All recipients became pregnant and produced a total of 17 fetuses (32.1%). These results suggest that porcine IVM-derived embryos that have been cryopreserved by the combination of noninvasive delipation and vitrification by the MVC method are highly viable.
131 BOVINE OOCYTE VITRIFICATION IS IMPROVED BY INCREASED PYRUVATE CONCENTRATION AND THE INCLUSION OF HYALURONAN IN THE VITRIFICATION MEDIUM
Authors: Sheehan C. B. , Larman M. G. , Gardner D. K.
Published: December 2006

Bovine oocytes have limited cryo-tolerance and are susceptible to damage partially caused by intracellular ice formation and reactive oxygen species. The ability to routinely cryopreserve bovine oocytes has remained elusive, with high rates of degeneration upon warming or fertilization failure. Hyaluronan has been shown to increase bovine blastocyst survival after vitrification, and the addition of an antioxidant improves mouse embryo survival. The aim of this study was, therefore, to determine if supplementation of the base medium with hyaluronan and increasing the pyruvate concentration would improve the success of bovine oocyte vitrification. Bovine oocytes were recovered by aspiration of abattoir ovaries. Oocytes were matured for 21 h in a defined system using G-MAT supplemented with recombinant albumin (2.5 mg mL-1) and epidermal growth factor (EGF; 100 ng mL-1). All oocytes were partially denuded using hyaluronidase and divided into 3 groups: Control (non-vitrified, n = 789), G-MOPSTM (Vitrolife Sweden AB, Kungsbacka, Sweden; n = 697), and G-MOPS supplemented with 0.125 mg mL-1 hyaluronan with a 1.5-fold pyruvate increase (designated G-VIT; n = 697). Oocytes were soaked in either G-MOPS or G-VIT for 45 min prior to being vitrified. G-MOPS or G-VIT was then used as the base medium for all vitrification and warming steps. The vitrification procedure was carried out at 37�C using the Cryoloop. Oocytes were incubated in 8% dimethyl sulfoxide (DMSO) and ethylene glycol (EG) for 1 min and 16% DMSO and EG (with 0.65 M sucrose and 10 mg mL-1 Ficol, MW 400 000) for under 30 s, before loading onto the Cryoloop and submerging into liquid nitrogen. Warming was performed using 0.25 M sucrose for 1 min, 0.125 M sucrose for 5 min, and no sucrose for at least 10 min. All oocytes (within 1 h of warming) were then fertilized for 18 h and their survival assessed before being washed and moved to G1.3 (with 8 mg mL-1 BSA). Embryos were again washed, cleavage rates assessed, and embryos were moved to G2.3 (with BSA) on the morning of Day 4 post-fertilization. Blastocyst formation was assessed at 144 h. Differences between treatments were assessed by Fisher's exact test. Survival, cleavage, and blastocyst development in the G-VIT and G-MOPS groups were 76.9 vs. 58.2% (P < 0.001), 48.5 vs. 41.6% (P < 0.05), and 15.4 vs. 4.1% (P < 0.001), respectively. The cleavage and blastocyst development rates of control oocytes were higher than for both vitrification groups (61.7 and 26.7%, respectively; P < 0.001). Survival and development of vitrified oocytes improved with the addition of hyaluronan and increased pyruvate concentration. Ongoing research into the role of macromolecules and antioxidants should further increase the success of oocyte cryopreservation.
This study was supported by a grant from Vitrolife.
136 CLOSED SYSTEM OF GEL-LOADING-TIP VITRIFICATION FOR IN VITRO-PRODUCED BOVINE BLASTOCYSTS BEFORE AND AFTER BIOPSY
Authors: Tominaga K. , Iwaki F. , Yamaguchi E. , Dou Y.-M. , Kijihana I. , Hochi S.
Published: December 2006

Ultra-rapid cooling by direct plunging of gel-loading-tip (GL-tip) into liquid nitrogen (LN2) contributed to the high post-warm survival rates of in-vitro-produced (IVP) bovine embryos (Tominaga and Hamada 2001 J. Reprod. Dev. 47, 267–273). Since GL-tip vitrification and other ultra-rapid vitrification methods have potential risk of microbiological contamination in the LN2, the current study was undertaken to develop a closed system of GL-tip vitrification for IVP bovine blastocysts before and after biopsy. Day 7 blastocysts were produced in vitro (Tominaga et al. 2000 Theriogenology 53, 1669–1680), and biopsied for sex determination (Tominaga and Hamada 2004 Theriogenology 61, 1181–1191). The blastocysts were exposed first to 10% ethylene glycol (EG) +10% DMSO in TCM-199/20% FCS for 2 min at 37°C, and then placed in vitrification medium with 20% EG + 20% DMSO + 0.6 M sucrose in TCM 199/20% FCS for 30 s at 37°C. Three to 4 blastocysts in 0.6 µL of the vitrification medium were aspirated into a GL-tip (NK-tip; Nippon Medical & Chemical Instruments Co., Ltd., Osaka, Japan). Immediately before being plunged into LN2, the tip of the GL-tip was sealed by dipping into 100% glycerol at 4°C, and the opposite open end of the GL-tip was sealed by inserting a holding stick under LN2 vapor. After warming in 0.25 M sucrose solution at 37°C, the glycerol-seal was removed from the tip by gentle shaking. The blastocysts diluted in a two-step manner were cultured up to 72 h for survival assay. Within the 72 h of culture, intact embryos that initiated hatching and biopsied embryos that re-expanded were considered as surviving. Two more biopsied blastocysts derived from ovum pickup, IVF, and IVC were vitrified–warmed in the closed system, and then transferred to 2 recipients. The results indicate no significant difference in the post-thaw survival rates of either intact or biopsied blastocysts in both closed and open systems (Table 1). One recipient became pregnant and delivered a female calf. Gestation period (287 days) and birth weight (22 kg) were within the normal range of the Tajima strain of Japanese Black cattle. We reached the conclusion that the closed system of GL-tip vitrification was simple and highly effective for IVP bovine blastocysts before and after biopsy.

Table 1.
In vitro survival of intact and biopsied bovine blastocysts after GL-tip vitrification in closed and open systems
137 EFFECT OF POST-IVF DEVELOPMENTAL KINETICS ON SURVIVAL AND QUALITY OF VITRIFIED - WARMED DOMESTIC CAT BLASTOCYSTS
Authors: Tsujioka T. , Otzdorff C. , Braun J. , Hochi S.
Published: December 2006

A limited number of reports are available for cryopreservation of IVF-derived cat blastocysts (Karja et al. 2006 Theriogenology 65, 415–423). In the present study, the IVF-derived domestic cat blastocysts exhibiting differences in their developmental kinetics were cryopreserved by vitrification. Pre- and post-warm blastocysts were examined for their cell number, and the survival rate was determined by in vitro culture of the post-warm embryos. Cumulus–oocyte complexes, recovered from the ovaries of post-pubertal queens, were matured for 24 h in TCM-199 supplemented with 3 mg mL−1 BSA, 1 µg mL−1 estradiol, 0.1 IU mL−1 FSH, and 0.0063 IU mL−1 LH. Oocytes were inseminated with 2 × 106 cells mL−1 cauda epididymal spermatozoa for 22 h in TALP solution. Presumptive zygotes were cultured in modified SOF medium at 38.5°C in 5% CO2 in air. Newly formed blastocysts were harvested 6 and 7 days post-IVF and subjected to vitrification (Hochi et al. 2004 Theriogenology 61, 267–275) by a minimum-volume cooling procedure using Cryotop (Kitazato Supply Co., Tokyo, Japan) as a cryodevice. The post-warm blastocysts were cultured for 24 h, and complete re-expansion was considered to be an indicator of survival. Embryos were differentially stained with Hoechst 33342 and propidium iodide to assess the total number of cells and the ICM ratio in the blastocysts. The cleavage rate of oocytes was 47.1% (314/666) and the percentage of oocytes developing to blastocysts on Day 6 and Day 7 was 9.8 and 9.5%, respectively (total blastocyst yield: 19.2%; 128/666). Post-warm in vitro survival rates of blastocysts harvested at Days 6 and 7 were 73.8% (31/42) and 66.7% (18/27), respectively (P = 0.59; exact probability test). There were no significant differences in the total number of cells and the ICM ratio between fresh control and vitrified blastocysts (P ≥ 0.05; ANOVA), although the ICM ratio of surviving Day 7 blastocysts was significantly smaller than that of fresh controls (18.9 vs. 28.9%; P = 0.03), as shown in Table 1. These results indicate that IVF-derived domestic cat blastocysts can survive vitrification by a minimum-volume cooling procedure without a reduction in the parameters studied, as long as the blastocysts are harvested on Day 6.

Table 1.
Differential cell staining of fresh and vitrified Day 6 and Day 7 cat blastocysts
144 Oct-4 EXPRESSION PATTERN IN THE EQUINE EMBRYO
Authors: Choi Y. H. , Harding H. D. , Obermiller A. D. , Hinrichs K.
Published: December 2006

Oct-4 is a key transcription factor in the control of early embryonic development and maintenance of a pluripotent cell population. Variation in Oct-4 expression patterns during embryo development have been reported among species, and have been related to the time of placental development in those species. This study was conducted to investigate Oct-4 expression pattern during early embryonic development in the horse, a species with relatively delayed placentation. In vitro-produced embryos were obtained from in vitro-matured oocytes via fertilization by intracytoplasmic sperm injection. Ex vivo blastocysts were recovered from mares that had been artificially inseminated. Oct-4 status was determined by immunocytochemistry; photomicrographs were taken at 4 standardized settings to aid in qualitative comparison of the amount of fluorescence. A total of 106 oocytes and embryos were evaluated. Immature oocytes showed Oct-4 expression in the nucleus and cytoplasm, as did early-cleaved embryos (2 to 5 cells, 1 to 2 days). Oct-4 expression in embryos at 3 to 4 days (6 to 12 cells) decreased and was restricted to the cytoplasm. From 5 to 6 days (15 cells to morulae), Oct-4 intensity increased and was exclusively found in the nuclei. In vitro-produced blastocysts (7 to 8 days) expressed Oct-4 equivalently in the trophectoderm and inner cell mass nuclei; culture for 2 to 3 more days (10 to 11 days) did not alter Oct-4 expression. However, when in vitro-produced blastocysts were transferred to the uteri of mares and recovered after 2 to 3 days (IVP-ET), the embryos showed strong expression of Oct-4 within the inner cell mass and limited expression in the trophectoderm, and a similar pattern was seen for ex vivo-recovered embryos. In bigger embryos (such as a 1779-�m ex vivo embryo and a 1121-�m IVP-ET embryo), the trophectoderm lost staining completely. These results suggest that Oct-4 expression is present in both nucleus and cytoplasm in equine oocytes and early-cleaved embryos as a result of maternal mRNA accumulation. Oct-4 protein decreases over the first few days of embryonic development as these stores are used. The shift to greater expression, in the nucleus only, during further embryo development suggests embryonic genome activation. Oct-4 expression in the trophectoderm of in vitro-produced blastocysts was different from that in blastocysts that had been exposed to the uterus (both ex vivo and IVP-ET); this indicates that differentiation of the trophectoderm is dependent upon factors present in the uterine environment. The Oct-4 expression in the trophectoderm of in vitro-produced equine blastocysts thus appears to be an artifact due to in vitro culture; this finding may be applicable to the reported patterns of Oct-4 expression in embryos of other species.
This work was supported by the Link Equine Research Endowment Fund, Texas A&M University.
151 LIPID COMPOSITION OF FAT DROPLETS OF IN VIVO- AND IN VITRO PRODUCED PORCINE EMBRYOS
Authors: Romek M. , Gajda B. , Krzysztofowicz E. , Smorag Z.
Published: December 2006

Early-stage porcine embryos to peri-hatching blastocysts contain high levels of intracellular lipids in the form of fat droplets and are highly sensitive to cryopreservation. Recently, our sterological studies demonstrated that in embryos produced in vivo and cultured in vitro, the volume of lipid droplets significantly decreased from zygote to blastocyst. To date, however, there have been no reports concerning the type of lipids in pig embryos produced in vivo and in vitro. Thus, the objective of the present study was to investigate the lipid composition of fat droplets in pig embryos produced in vivo and in vitro. The experiment was carried out on pig zygotes produced in vivo and 2–4 and 8–16-cell embryos, morulae, blastocysts, and late blastocysts produced in vivo and in vitro. Embryos produced in vivo were obtained from superovulated gilts after flushing the oviduct or uterus. Embryos cultured in vitro were developed from zygotes produced in vivo. Embryos were cultured in vitro to appropiate stages of development in chemically defined medium, North Carolina State University (NCSU)-23. For analysis of the type of lipid in the fat droplets, embryos were fixed in 2.5% glutaraldehyde with the addition of 3 mM calcium chloride. The material was then embedded in Technovit 8100, cut into semi-thin sections, and analyzed by histochemical methods. Four techniques were used to detect different types of lipids: Churukian method with Oil red O, Cain method with Nile blue sulfate, Sudan black B, and osmium tetroxide methods. Fat droplets of embryos produced both in vivo and in vitro contained unsaturated hydrophobic lipids, free fatty acids, phospholipids, unsaturated esters, and triglycerides. Moreover, in the morula the total amount of lipids (especially the amount of free fatty acids and phospholipids) evidently decrease. The amount of the other unsaturated lipids decreased as early as the 2- to 4-cell stage. In conclusion, the content of different types of lipids in pig embryos is reduced during their development from zygote to blastocyst, and there are no differences in lipid composition of fat droplets between in vivo- and in vitro-produced porcine embryos.
This research was funded by the State Committee for Scientific Research (Project No. 2 P06D 003 26).
169 PREDICTION OF THE OPTIMAL DAY OF MATING IN THE BITCH USING A BOVINE PLASMA PROGESTERONE ELISA KIT
Authors: Seki M. , Yamazaki Y. , Watanabe N. , Ishii K. , Kinoshita Y. , Aihara T. , Takeiri S. , Otoi T.
Published: December 2006

The present study was conducted to predict the optimal day of mating by measurement of progesterone concentration in peripheral blood of bitches using a bovine plasma progesterone ELISA kit (enzyme-immunoassay practice P kit; Kawasaki Mitaka K.K., Mitaka-shi, Tokyo, Japan). In the first experiment, blood samples were collected from 20 mature beagle bitches once a day from 1 to 13 days after the onset of proestrus (Day = 0). All bitches were mated with male beagle dogs twice on Day 9 and Day 12 after the onset of proestrus and then allowed to undergo parturition. In the second experiment, blood samples were collected from 16 mature and 7 prepubertal beagle bitches; the blood collections were randomly started between Day 4 and Day 6 and performed three times after the initiation of blood collection (the first, third, and fourth days). The bitches with progesterone concentration of <10 ng mL-1 at the end of progesterone analysis were mated with male beagle dogs twice at 3-day intervals between Day 9 and Day 15 according to the hormonal profile. Data were compared by chi-square analysis. In the first experiment, a total of 20 bitches were mated with male dogs irrespective of hormonal profiles, of which 17 bitches became pregnant and delivered offspring. Two of the non-pregnant bitches did not show an increase of progesterone concentration during the progesterone analysis period, and the other one showed a high progesterone concentration (>10 ng mL-1) before the mating. In the second experiment, 6 of 23 bitches were excluded from the mating with male dogs because their progesterone concentrations had reached >10 ng mL-1 by the second day of progesterone analysis. The percentage of prepubertal bitches with high progesterone concentration before the mating (4/7, 57%) was significantly higher (P < 0.05) than that of mature bitches (2/16, 13%). A total of 17 bitches were mated with male dogs, of which 15 bitches became pregnant and delivered offspring. These results indicate that the bovine plasma progesterone ELISA kit is useful for the prediction of the optimal day of mating with male dogs, and progesterone analysis may be effective for exclusion of bitches that have ovulated before mating.
17 WHITETAIL DEER OFFSPRING FROM ARTIFICIAL INSEMINATION WITH EPIDIDYMAL SPERM HARVESTED FROM A HUNTER-KILLED BUCK
Authors: Saenz J. R. , Gentry G. T. , Forbes W. , Olcott B. , Chenevert J. , Bondioli K. R. , Sanders D. E. , Godke R. A.
Published: December 2006

The ability to cryopreserve epididymal sperm from mature postmortem bucks has long been of interest to both wildlife conservationists and deer ranchers. At present, there is loss of valuable genetics from the hunter harvest of trophy males. Increasing adult body weight and antler size of adult males would be of substantial economic value to the deer hunting industry. In this preliminary trial, 6 yearling pen-raised Whitetail does (47 to 58 kg), in good body condition, were isolated from all bucks prior to the onset of the fall breeding season. Females were synchronized for AI with a 14-day caprine CIDR and 200 IU of eCG (IM) at the time of CIDR removal. Does were timed AI 60 to 63 h after eCG with one 0.5-mL straw of frozen–thawed Whitetail sperm. All sperm used for AI were harvested from a single mature Whitetail buck that was hunter harvested during the previous hunting season. Within 3 h after death, the testes with scrotum were removed, enclosed in a plastic Ziploc bag, and then placed in a Styrofoam ice chest containing frozen cold packs. The ice chest was transported to the laboratory where sperm were extracted at 4�C in the late evening (<12 h postmortem) by flushing the cauda epididymides with Triladyl� one-step extender (Minitube, Verona, WI, USA) in a retrograde flow from a small incision made in the cauda. The sperm–Triladyl mixture was flushed from the cauda incision into a sterile 50-mL tube using a 10-mL plastic syringe modified by heating and then stretching the tip until small enough to thread into the vas deferens. The sperm plus extender was then held at 4 to 10�C for 12 h and frozen at a concentration of <50 million/straw using a commercial bull freezing protocol (Genex Custom Collection Center, Baton Rouge, LA, USA). A random sample of straws was then thawed, resulting in an overall post-thaw motility of 60%. The remaining straws were left frozen in liquid nitrogen until the next breeding season. On the first of December, does (n = 6) were given 0.1 mL of Domosedan� (Pfizer Animal Health, Groton, CT, USA) IV and inseminated transcervically using a modified caprine speculum. All does were handled in a custom-built deer barn, and AI was performed by one technician in a drop-bottom deer chute (Deer Handler; Delclayna, Alberta, Canada). At 48 days after AI, 3 of the 6 does (50%) were diagnosed pregnant by transrectal ultrasonography. All pregnant females gave birth, producing 5 offspring (1 male and 1 female singletons and a set of mixed sex triplets) that ranged from 1.9 to 4.3 kg and had a mean gestation length of 196 days (range = 190–203 days). In summary, results indicate that live offspring can be produced from epididymal sperm harvested from mature hunter-harvested Whitetail bucks. Further experiments are needed to optimize techniques and protocols for the harvesting and usage of these gametes.
175 DETECTION OF LIPID GRANULES ON IVF BLASTOCYSTS MATURED IN APO-TRANSFERRIN IN KOREAN NATIVE COWS (HANWOO)
Authors: Choi S. H. , Cho S. R. , Han M. H. , Kim H. J. , Choe C. Y. , Son D. S. , Woo J. S. , Sang B. D. , Chung Y. G. , Hoshi H.
Published: December 2006

Apo-transferrin (apo-Tf) is found in mammalian sera and plays a role as an anti-oxidant in media. The objective was to investigate the effects of apo-Tf on in vitro maturation and development and to examine the characteristics of blastocysts derived from IVF production in the presence of apo-Tf in Korean native cows (Hanwoo). Ovaries were collected from a slaughterhouse and cumulus–oocyte complexes (COCs) were taken from 2–6-mm antral follicles. The collected COCs were washed 3 times with 0.1 M PVA-TCM-199 and matured in 10 �g mL-1 apo-Tf with TCM-199 at 39�C, 5% CO2, 95% air for 24 h. Matured COCs were fertilized with frozen–thawed Korean native cattle semen treated with BO medium containing 5 mM caffeine and 1 �g mL-1 heparin for 8 h, and developed to the blastocyst stage in 5% FBS, 0.3% BSA in TCM-199, and IVMD (IFP, Japan). To detect the lipid granules, blastocysts were fixed with 10% formalin-PBS for 2 h and then 50% ethanol for 2 min. Staining of blastocysts was performed with 1% Sudan Black B in 70% ethanol for 2 min. IVM and IVF were replicated 3 times. The results of blastocyst formation were analyzed by chi-squared test. The maturation rate was 92.6% at Met II in 10 �g mL-1 apo-Tf. The blastocyst formation was 22.5%, 38.4%, and 34.8% in 5% FBS (control), 0.3% BSA in TCM-199 (P < 0.05), and IVMD (P < 0.05), respectively. The lipid granules in blastocysts from the 5% FBS-TCM-199 group were larger than those in the 0.3% BSA-TCM-199 and IVMD groups. The results suggest that apo-Tf is an important factor for in vitro maturation and that FBS in culture medium affects lipid granule formation on bovine blastocysts derived from in vitro fertilization.
179 EMBRYO DEVELOPMENT AND EXPERIMENT OUTCOME CAN BE COMPROMISED WITHOUT APPROPRIATE QUALITY CONTROL
Authors: Gardner D. K. , Linck D. W. , Sheehan C. B.
Published: December 2006

The overall success of any embryology laboratory depends on its ability to maintain consistency from week to week. Quality control practices not only minimize variation within an experiment and increase cost effectiveness, but also make running the lab easier from day to day. From the media and oil overlay to the quality of culture ware, many factors impact the success of a laboratory. By screening contact supplies before they are used in an experiment, an additional variable may be removed. One way of screening contact supplies is to use a 1-cell mouse embryo assay (MEA). The aim of this study was to first identify a quality source of oil overlay and then examine the quality of plastic ware over the course of one year in an attempt to identify variables that are most often toxic to embryos. One-cell mouse embryos (C57BL/6 � CBA/Ca) were used to screen 10 lots of oil from 5 suppliers (n = 70 embryos per oil over 7 replicates). After safe oil was identified, contact supplies including filters, dishes, tubes, and bottles were tested (n = 30 embryos per assay over 3 replicates). Supplies were first soaked with a simple medium lacking amino acids, EDTA, and protein. The duration of medium exposure to contact supplies was the same as their intended use, i.e. bottles stored media for a week before testing. This medium was then used to make culture drops under safe oil. Embryos were grown for 5 days in the simple medium, and development was assessed at 78 and 96 h. Embryos that reached 50% early blastocyst development on Day 4 and 80% expanded blastocyst development on Day 5 were stained and total cell numbers assessed. Items whose embryos did not reach the developmental cut-off values on Day 4 or 5 failed the assay. Blastocyst cell numbers were compared to a known control, and those items that were significantly different failed the assay. Five out of 10 lots of oil failed; four of the lots were from the same supplier. In this assay, such oils failed each replicate. Of 94 contact supplies, 17 (18%) failed the MEA. There was no pattern for products that failed more often than another, i.e. filters did not fail more often than dishes or bottles. However, a general trend was identified for one supplier of oil, as this source appeared to have more toxic lots than those of other suppliers. Using a safe oil source is first priority in an embryology laboratory. There appears to be no way to predict toxicity of plastic ware, so all contact supplies must be either screened before use or introduced into a laboratory one at a time. General record keeping of lot numbers should make it easier to pinpoint potential problems with toxic plastic ware when they arise. The impact of this study is that without quality control in a laboratory, research studies can be compromised.
This work was supported by Vitrolife.
18 THE EFFECTS OF CIDR AND eCG TREATMENTS IN A GnRH-BASED PROTOCOL FOR TIMED AI OR EMBRYO TRANSFER ON PREGNANCY RATES IN LACTATING BEEF COWS
Authors: Small J. A. , Colazo M. G. , Kastelic J. P. , Ward D. R. , Dochi O. , Mapletoft R. J.
Published: December 2006

Two experiments were conducted to determine the effects of the addition of a progestin, equine chorionic gonadotropin (eCG), or both, in a GnRH-based protocol for timed AI (TAI) or timed embryo transfer (TET). In both experiments, Angus, Gelbvieh, and Simmental cross-bred cows were randomized by breed and postpartum interval [50 � 10 days (mean � SD); range, 27 to 89] into 4 groups in a 2 � 2 factorial design. All injections were given IM. In Experiment 1, 288 cows (89.6% cycling) were given 25 mg dinoprost (PGF; Lutalyse�; Pfizer Animal Health, Montreal, Quebec, Canada) on Day –11; on Day 0, they were given 100 �g GnRH (Cystorelin�; Merial Canada, Pointe-Claire, Quebec, Canada), with or without concomitant insertion of a CIDR (1.9 g progesterone; Pfizer Animal Health, Montreal, Quebec, Canada). On Day 7, CIDR inserts were removed and cows were given PGF, with or without concomitant injection of 400 IU of eCG (Pregnecol�; Bioniche Animal Health, Belleville, Ontario, Canada). On Day 9 (54-56 h after PGF), TAI was done, with concomitant injection of 100 �g GnRH. Ultrasonographic examination of 147 cows on Day 7 revealed that 62.4% had ovulated in response to the first GnRH. Pregnancy rates (ultrasonographic examination) on Day 38 did not differ between cows with or without a CIDR (52.9 and 51.4%, rspectively; P ≥ 0.64), with or without eCG treatment (53.5 and 50.7%, respectively; P ≥ 0.28), in cycling vs. anestrous cows (51.6 vs 56.7%, respectively; P ≥ 0.76), and in cows that had ovulated (58.1%) or did not ovulate (50.0%) after the first GnRH treatment (P ≥ 0.58). In Experiment 2, 151 cows were given 500 �g cloprostenol (PGF; Estrumate�; Schering–Plough Animal Health, Pointe-Claire, Quebec, Canada) on Day –12, 100 �g GnRH on Day 0, with or without concomitant insertion of a CIDR. On Day 3, half of the cows were given 400 IU eCG. On Day 7, CIDRs were removed and cows were given PGF; on Day 9 (54–56 h after PGF), all cows were given 100 �g GnRH. On Day 15, ultrasonography was done to select suitable recipients for transfer of frozen–thawed embryos on Day 16 (part of another experiment, balanced across synchronization groups). Recipient selection rates did not differ whether cows received or did not receive a CIDR (93.4% vs 85.5%, respectively; P ≥ 0.27) or eCG (91.0 vs 87.8%, respectively; P ≥ 0.67). In addition, pregnancy rates on Day 43 did not differ whether cows received or did not receive a CIDR (32.3 vs 32.4%, respectively; P ≥ 0.52) or eCG (35.2 and 29.2%, respectively; P ≥ 0.21). In summary, the addition of a CIDR or eCG to a GnRH-based synchronization protocol initiated after PGF presynchronization in lactating beef cattle yielded no improvement in pregnancy rates following TAI, or recipient selection and pregnancy rates following TET.
180 DEVELOPMENTAL CAPACITY OF SINGLE CULTIVATED EMBRYOS IN SMALL AND BIG DROPLETS
Authors: Goovaerts I. G. F. , De Clercq J. B. P. , Nichi M. , Bols P. E. J.
Published: December 2006

An in vitro production system where a single oocyte can be followed from the ovary to the blastocyst stage would be a useful tool for studies concerning developmental competence or follicular environment. Unfortunately, until now, only low blastocyst rates could be obtained after single embryo production, and there is still discussion about the ideal droplet size. The objective of the present experiment was to compare the developmental competence of single cultivated zygotes in 20- and 500-µL droplets. Cumulus–oocyte complexes were obtained from slaughterhouse ovaries and were matured and fertilized in groups of 100 for 22 h; the presumptive zygotes were divided into 4 groups. In treatment 1, 25 zygotes were transferred into 50 µL of SOF medium supplemented with 5% serum under oil, whereas in treatment 2, 25 zygotes were transferred into 500 µL of medium. Zygotes were cultivated separately in treatments 3 and 4: in treatment 3 in 20 µL of medium under oil and in treatment 4 in 500 µL of medium. Cleavage rates and division stages were assessed after 3 days of cultivation (5% CO2, 5% O2, 90% N2); blastocyst rates were determined after 7 days. Statistical analysis was performed by logistic regression using SAS (PROC LOGISTIC). There was no difference in cleavage rates between the 2 group treatments or between the 2 single treatments. Also, the division stages were not different between the 2 single treatments (16-cell: 2.0 vs. 1.3%; 8-cell: 25.8 vs. 31.6%; 4-cell: 41.2 vs. 38.0%; and 2-cell: 31.0 vs. 29.1% for the 20 µL and the 500 µL droplet sizes, respectively). Group cultivation after 7 days in 50 µL was significantly better than in 500 µL; however, both treatments resulted in significantly higher blastocyst rates compared with the individual cultures in 20 or 500 µL, between which no significant difference could be found. Noteworthy, only 4-cell and 8-cell stages on Day 3 resulted in blastocysts on Day 7 of cultivation. In conclusion, these results indicate that cultivation in groups gives higher blastocyst rates, although the same embryo density is used as in individual cultivation (1 embryo 20 µL in treatments 2 and 3). Moreover, no significant difference could be found between single cultivation in small and big droplets. This is confirmed by the cleavage stages on Day 3, which indicate no difference in timing of cleaving between small and big droplets; time of cleaving is indicative of further developmental capacity.

Table 1.Cleavage and blastocyst rates after single and group cultivation
182 INTERACTION OF MICROTUBULES AND MICROFILAMENTS WITH DISTRIBUTION OF MITOCHONDRIA IN TWO-CELL HAMSTER EMBRYOS
Authors: Kabashima K. , Matsuzaki M. , Suzuki H.
Published: December 2006

During early development, mitochondrial distribution in the ooplasm is dramatically changed. In mice and pigs, it has been reported that translocation of mitochondria is mediated by microtubules, but not by microfilaments. However, the study using hamster embryos suggested that microfilaments played a role in the mitochondrial distribution. In this study, the role of microtubules and microfilaments on distribution of mitochondria was evaluated in 2-cell hamster embryos. Female golden hamsters were superovulated on the day of post-estrus discharge by PMSG in a weight-dependent manner [Mackiernan and Bavister 2000 Hum. Reprod. 15, 157–164], and mated with males in the evening 3 days later. Two-cell embryos were collected from the oviducts at 0900–1000 on Day 2 of pregnancy. Embryos were cultured in HECM-10 medium with or without nocodazole (a microtubule assembly inhibitor), and/or cytochalasin D (a microfilament assembly inhibitor) for 8–10 h in a humidified atmosphere of 10% CO2, 5% O2, and 85% N2 at 37.5°C. After incubation of embryos with or without inhibitors, mitochondria were stained with rhodamine 123. Some of them were stained for microtubules, microfilaments, and DNA, as reported previously (Suzuki et al. 1999 Biol. Reprod. 61, 521–526). In 2-cell hamster embryos (controls, n = 44), most mitochondria were accumulated at the perinuclear region, while little mitochondria were noted in the cell cortex. Microtubules were found around the nuclei, and distribution of the mitochondria was somewhat correlated with that of the microtubules. In contrast, microfilaments were located in a high density just under the cell membrane. After nocodazole treatment (n = 22), mitochondria had extended into the subcortical (intermediate) region in various degrees, where mitochondria were aggregated into large masses. After cytochalasin D treatment (n = 17), distributional density of mitochondria at the peripheral region was decreased, and mitochondria moved back around the nucleus. After treatment by both inhibitors (n = 21), a distribution pattern of mitochondria was almost similar to that observed after cytochalasin D treatment. The present results suggest that the distribution of mitochondria at the perinuclear region is mediated by microtubules, whereas the movement of mitochondria to the cell cortex is regulated by microfilaments. Microfilaments seemed to play a role as the anchor of mitochondria at the cell cortex.
185 PGI2 ANALOG INDUCES HIGH-QUALITY PORCINE BLASTOCYSTS IN VITRO
Authors: Kim J.-S. , Wee G. , Song B.-S. , Park J.-S. , Jin X.-L. , Lee Y.-Y. , Han Y.-M. , Lee K.-K. , Koo D.-B.
Published: December 2006

Prostaglandin I2 (PGI2) exists in oviductal fluid and is synthesized from arachidonic acid by prostacyclin synthetase. In hatched embryos, prostaglandin I2 (PGI2) is related to implantation improvement, but its role during oocyte maturation and early embryo development remains controversial. Therefore, in this study, the effect of addition of a PGI2 analog during early porcine oocyte maturation on nuclear maturation, blastocyst formation, and pre-implantation embryonic quality was investigated. Porcine oocytes were matured in NCSU-23 medium supplemented with 10% (v/v) porcine follicular fluid, 10 ng mL-1 epidermal growth factor, 25 �M �-mercaptoethanol, 0.57 mM cysteine, 10 IU mL-1 pregnant mare serum gonadotropin, 10 IU mL-1 hCG, and 1 �M PGI2 analog for 22 h, and then further cultured in maturation medium without PGI2 analog and hormones for 22 h. After fertilization in Tris-buffered (mTBM) medium for 6 h, presumptive porcine zygotes were cultured in the NCSU-23 medium supplemented with 4% BSA for 6 days. All data were analyzed by using the Duncan test of ANOVA by the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). First, we confirmed that PGI2 analog-treated (90.0 � 2.6%) oocytes showed a higher proportion of the metaphase II stage than non-treated (65.7 � 1.4%) ones (P < 0.05). Thus, to confirm the activities of maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK), Western blots were performed in matured oocytes by using specific antibodies such as anti-cdc2 and anti-ERK1/2. The activities of MPF and MAPK were increased in porcine oocytes treated with PGI2 analog. In the PGI2 analog-treated group, polyspermic rate (17.9 � 13.3%) was reduced as compared with that of the non-treated group (35.8 � 9.4%). Furthermore, the rate (25.3%, 40/158) of blastocyst formation in the PGI2 analog-treated group was higher than in the non-treated group (19.7%, 27/137; P < 0.05). Also, cell numbers of blastocysts were increased (29 � 2.5% vs. 39.6 � 1.4%) in the treated vs. the non-treated group. The numbers of fragmented DNA nuclei detected in the blastocyst stage by the TUNEL assay were decreased in the PGI2-treated group compared with the non-treated group (2.1% vs. 5.2%). In conclusion, direct roles of PGI2 during porcine oocyte maturation may involve reducing apoptosis and enhancing blastocyst quality.
186 THE EFFECT OF MATRIGEL ON THE DEVELOPMENT OF IN VITRO-FERTILIZED PORCINE EMBRYOS
Authors: Kim S.-W. , Lee M.-J. , Yang B.-C. , Im G.-S. , Seong H.-H. , Yang B.-S. , Cheong H.-T. , Kim D.-H.
Published: December 2006

The application of matrix proteins to culture systems for growth of embryos is a logical extension in the quest to better simulate the in vivo culture environment. Matrigel, a commercially available extracellular matrix product containing collagen IV, laminin, entactin, and proteoglycans isolated from mouse tumor cells, has been tested. Development of mouse pre-implantation embryos cultivated in conventional culture medium was contrasted to that of embryos grown in solubilized Matrigel medium. In the solubilized Matrigel medium, in vitro blastocyst formation and hatching were significantly enhanced over that observed in the medium alone control. Therefore, the aim of this study was to investigate the effect of solubilized Matrigel on the development of porcine embryos after in vitro fertilization. In vitro-matured oocytes were fertilized in mTBM medium with fresh spermatozoa for 6 h. Putative zygotes were cultured in PZM-3 medium supplemented with (matrigel group) or without (control group) 0.8% Matrigel for 6 days. The number of cells in blastocysts was determined by staining with Hoechst 33342. Assessment of apoptosis in blastocysts was examined by TUNEL. The statistical significance of the data was analyzed using chi-square test and Student's t-test. The addition of Matrigel appeared not to increase the proportion of blastocysts (control: 71/219, 21.8 � 2.2% vs. Matrigel: 69/220, 23.5 � 5.8%). However, the mean cell numbers were significantly increased by Matrigel (Matrigel: n = 31, 52.9 � 18.1 vs. control: n = 30, 42.3 � 14.4; P < 0.01). The proportion of apoptotic cells was significantly lower in the Matrigel group (Matrigel: 4.5 � 4.2% vs. control: 6.6 � 5.5%; P < 0.05). In this experiment, Matrigel appeared to increase blastocyst quality of porcine embryos. Results suggest that Matrigel, as an extracellular matrix component, may be another avenue for formulating more physiological culture systems.