bluuebunny/crossref_metadata_2025 huggingface dataset:
100EFFECTS OF USING MICRO-PIPETTE TIPS OF DIFFERENT DIAMETERS AND
VOLUME OF VITRIFICATION SOLUTION ON THE VIABILITY OF IVM BOVINE OOCYTES AFTER
VITRIFICATION
Authors: Inaba Y. , Dochi O. , Koyama H.
Published: February 2004

The objective of this study was to investigate the effects of the diameters of micro-pipette tips and the volume of vitrification solution (VS) on viability of IVM bovine oocytes after vitrification. COCs were aspirated from 2–5mm follicles of ovaries obtained at a local abattoir. COCs were matured for 19h in TCM-199 supplemented with 5% calf serum (CS) and 0.02mgmL−1 FSH at 38.5°C in an atmosphere of 5% CO2 in air. The matured oocytes were then vitrified on the basis of Kuwayama and Kato (2000 J. Assist. Reprod. Genet. 17, 477 abst). Matured oocytes were first exposed to 7.5% ethylene glycol (EG) and 7.5% DMSO in holding medium (HM; Dulbecco’s PBS supplemented with 20% CS) for 3min, and then equilibrated for 1min in 15% EG, 15% DMSO, and 0.5M sucrose in HM. Ten oocytes were loaded into each micro-pipette tip (MidAtlantic Diagnostics, Inc., Marlton, NJ, USA), and directly plunged into liquid nitrogen. Warming was performed by placing the narrow end of the micro-pipette tips directly into HM containing 0.5M sucrose; the tips maintained in this medium for 5min. After washing in HM, oocytes underwent an additional 3h of maturation. They were then subjected to IVF (Day 0). After IVF, morphologically intact oocytes were cultured. Oocytes matured for 20–21h were used as a control. The cleavage rate at Day 3 and blastocyst rate at Day 7 to 9 were based on the number of cultured oocytes, and analyzed using the chi-square method. In experiment 1, the oocytes were vitrified with 0.5μL of VS in micro-pipette tips with 150-, 200-, or 275-μm inner diameters (ID) (100 eggs per tip size). The number of morphologically intact oocytes was 64 (150μm), 62 (200μm), and 54 (275μm). The cleavage rates of morphologically intact oocytes at Day 3 of 150μm (45.3%) and 200-μm tips (45.2%) were significantly lower than that of 275-μm tips (53.7%) and the control (63.6%) (P<0.05). The blastocyst rate of morphologically intact oocytes at Day 7 to 9 of 150-μm (9.4%) and 275-μm tips (14.8%) were significantly lower than that of the control (33.0%) (P<0.05), and that of 200-μm tips (19.4%) also showed a tendency of being lower than that of the control (P<0.1). In experiment 2, the oocytes were vitrified with 0.3 (70 eggs), 0.5 (60 eggs), or 1μL (60 eggs) of VS in micro-pipette tips with 200-μm ID. The number of morphologically intact oocytes was 40 (0.3μL), 32 (0.5μL), and 28 (1μL). The cleavage rates of morphologically intact oocytes at Day 3 of the 0.3μL (45.0%), 0.5μL (37.5%), and 1μL solutions (35.7%) were significantly lower than that of the control (67.6%) (P<0.05). However, there were no differences in the blastocyst rate of morphologically intact oocytes at Day 7 to 9 among 0.3μL (15.6%), 0.5μL (28.1%), and 1μL solutions (17.9%), and control (23.9%). These results suggest that the viability of IVM bovine oocytes after vitrification may be improved by using micro-pipette tips with 200-μm ID and containing 0.5μL of VS.
101CRYOPRESERVATION OF RAT EPIDIDYMAL SPERM: COMPARISON OF TWO COOLING
PROTOCOLS
Authors: Kashiwazaki N. , Okuda Y. , Takizawa A. , Nakagata N. , Shino M.
Published: February 2004

The present study examined post-thaw motility, plasma membrane integrity and fertility of rat epididymal sperm cooled by two procedures to +5°C, and then cryopreserved by controlled-rate freezing. Wistar rats were used in the present study. In protocol-I (2001 Reproduction 122, 463), epididymides were collected from a mature male and placed in a plastic dish containing 2mL of freezing medium I [23% (v/v) egg yolk, 8% (w/v) lactose monohydrate and antibiotics]. The epididymides were dissected with scissors to release epididymal sperm. The semen was kept at 15°C for 30min and then held at 5°C for 30min. The cooling rate from 15°C to 5°C was 0.3°Cmin−1. The cooled semen was diluted with 2mL of freezing medium II [freezing medium I with 1.4% (v/v) Equex Stm (ES, Nova Chemical Sales, Inc., Scituate, MA, USA)]. Mixed semen was aspirated into 0.25-mL straws and exposed to liquid nitrogen (LN) vapor for 10min. The straws were then plunged into LN. In protocol-II, epididymides were collected from a mature male and placed in 4mL of freezing medium III [freezing medium I and 0.7% (v/v) ES]. The epididymides were dissected with scissors and held for 10min at room temperature to release epididymal sperm. The semen was loaded into 0.25-mL straws and kept at 15°C for 15min and then held at 5°C for 15min. The cooling rate from 15°C to 5°C was 0.7°Cmin−1. The cooled straws were then exposed to LN vapor for 10min and plunged into LN. Straws were thawed in a 37°C water bath for 10s. Thawed semen in a straw was diluted with 1mL of KRB medium with 0.4% (w/v) bovine serum albumin (BSA, fraction V, Sigma, Tokyo, Japan) at 37°C and then incubated at 37°C in 5% CO2 in humidified air. The percentage of motile spermatozoa was assessed visibly and determined by direct observation at 37°C under a light microscopy at 100×. The sperm membrane integrity was determined using a commercial Live/Dead sperm viability kit (Molecular Probes, Inc., Eugene, OR, USA) which differentiates between cells with intact plasma membranes and those with damaged membranes by fluorescent staining patterns observed with a fluorescence microscope (Olympus, IX-71, Tokyo, Japan). Similar levels of sperm motility were observed immediately after thawing of sperm from both protocols. However, after 2h of incubation, the post-thaw motility of sperm frozen by protocol-II was significantly (P<0.01) higher than that of protocol-I. Sperm membrane integrity immediately after thawing was also higher for sperm frozen by protocol-II (22.1% v. 9.3%, P<0.01). Sperm frozen/thawed by protocol-II was inseminated into the top of the uterine horns of recipient females to confirm fertility. Two of three inseminated females became pregnant and gave birth to 5 pups. These results suggest that loading sperm into straws before cooling and subsequent slow cooling at 5°C to 0.7°Cmin−1 increases post-thaw survival of rat epididymal sperm.
102VITRIFICATION OF IN VITRO-PRODUCED BOVINE AND OVINE EMBRYOS USING THE
MINIMUM VOLUME COOLING CRYOTOP METHOD
Authors: Kelly J.M. , Kleemann D.O. , Kuwayama M. , Walker S.K.
Published: February 2004

Considerable progress has been achieved in the cryopreservation of mammalian embryos. The use of vitrification minimizes chilling injuries by increasing cooling and warming rates. This study assesses the effect of vitrification using the minimum volume cooling (MVC) method (Kuwayama & Kato 2000 J. Assist. Reprod. Genet. 17, 477) on in vitro-produced bovine and ovine embryos. A total of 1756 ovine and 753 bovine cumulus-oocyte complexes were obtained from the abattoir and matured, fertilized (Day 0) and cultured in vitro (Walker et al., 1996 Biol. Reprod. 55, 703–708, Kelly et al., 1997 Theriogenology 47, 291). Overall cleavage rates were 93.7% and 80.5% respectively. Embryos were vitrified (OPS or MVC method) on Days 5 (morula, compact morula), 6 (expanded blastocyst, blastocyst, compact morula) or 7 (hatched and hatching blastocysts, expanded blastocyst, blastocyst). Embryos were equilibrated with 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 3min and then exposed to 16.5% EG, 16.5% DMSO, 0.5M sucrose and 20% FCS for 30s. Embryos were loaded onto either an MVC plate (Cryotop, Kitazato Supply Co, Toyko, Japan) or open pulled straw (OPS) and plunged into liquid nitrogen. After 5 days, embryos were thawed directly into 1.25M sucrose solution at 38.5°C, followed by stepwise dilution of the cryoprotectants. Embryo survival was assessed by culture to Day 8 and compared to the development of non-vitrified control embryos (Table 1). Variables were assessed using procedure CATMOD in SAS. The Cryotop method yielded a significantly higher percentage of viable ovine embryos after thawing compared with OPS (P<0.0001); neither day nor treatment x day interaction was significant (P>0.05). A significant interaction between vitrification treatment and day (P<0.007) indicated that the percentage of hatched embryos peaked at Day 6 using the Cryotop method compared with Day 7 for OPS. Hatching rates for fresh and vitrified embryos were similar at Day 7 and were independent of treatment. With the Cryotop method, day of vitrification did not influence the percentage of Days 6 and 7 bovine embryos that hatched after thawing but, on each day, this figure was significantly higher (P<0.003 and P<0.0001, respectively) than that obtained with fresh embryos. To further assess embryo viability, 36 fresh, 52 OPS and 56 Cryotop vitrified Day-6 in vitro-produced ovine embryos were transferred to synchronized recipients. Survival rates to Day 13 were 29/33 (87.9%), 23/36 (63.9%) and 42/51 (82.4%), respectively (P<0.05). This study demonstrates that using the MVC Cryotop method, the viability of vitrified embryos, as assessed at Days 8 and 13, is similar to that obtained with fresh embryos.

Table 1
105VITRIFICATION OF BOVINE EMBRYOS USING THE CLV METHOD
Authors: Lindemans W. , Sangalli L. , Kick A. , Earl C.R. , Fry R.C.
Published: February 2004

Vitrification has become the preferred method for cryopreserving in vitro-produced bovine embryos (IVP). Here we introduce a technique for vitrification developed at CryoLogic (the CLV Method), in conjunction with a study comparing the post-thaw viability of IVP embryos frozen by the widely used open pulled straw method (OPS—Vajta et al., 1997 Cryo-Letters 18, 191) and the new CLV Method. Vitrification on thin metal surfaces has been explored and demonstrated previously (Le Gal & Massip 1999, Cryobiology 38, 290), and Dinnyes presented a Solid Surface Vitrification (SSV) (Dinnyes et al., 2000, Biol. Reprod. 63, 513). The CLV Method utilizes vitrification on the surface of a solid metal block. This surface has been custom shaped and treated to enhance vitreous formation. The method also includes handling, storage and thawing protocols designed to avoid damage from crystallization of the unstable glass. Briefly, the block is precooled in LN2 to −196°C. Up to 10 embryos are collected into a droplet of medium (3μL), on the end of a fibre carrier attached to a handle. The droplet is presented to the vitrification surface, where it is converted into a glass bead by cooling rates comparable to that of plunging into solid/liquid phase nitrogen (−210°C) (Arav et al., 2001 Theriogenology 55, 313). The glass bead, fibre and handle are transferred quickly into a half-sealed, precooled straw, and the handle seals the open end. A bead is thawed very rapidly by removing the handle, fibre and bead from the straw and transferring the bead into washing medium. COCs collected from bovine ovaries obtained from abattoirs were matured, fertilized and cultured for 6 days (Fry et al., 2003 Theriogenology 59, 446). Embryos reaching the blastocyst or expanding blastocyst stage of development were graded (Grades 1, 2, and 3), equilibrated for 5min in HEPES-199 medium with 20% FCS (HFm), placed in HFm with 10% EG, and 10% DMSO (VS1) for 2min, and then transferred to HFm with 20% EG, 20% DMSO (VS2) for 30s (Vajta). Between 5 and 10 IVP embryos were processed and collected for vitrification, either in an OPS plunged into LN2, or in a 3μL droplet vitrified by the CLV Method. The two sets of specimens were stored in LN2, and later thawed. Both OPS tips and beads were thawed in 0.5mL of HFm with 0.2M sucrose at 39°C. Embryos were maintained at 39°C, examined after 5min for contraction, and again after 6 h for re-expansion. They were then transferred to culture medium, incubated and examined at 24 and 48h to assess hatching. As shown in Table 1, the CLV method appears to be satisfactory for maintaining membrane integrity (expansion) and developmental potential (hatching) for even poorer grade embryos, that might be more sensitive to the stresses of cryopreservation.

Table 1
Re-expansion and hatching rates of graded thawed bovine embryos vitrified by OPS or CLV methods
106EVALUATION OF ADDITION OF REDUCED GLUTATHIONE TO COOLING MEDIUM ON IN
VITRO FERTILITY AND ACROSOME REACTION IN BOAR SPERMATOZOA
Authors: Matás C. , Gadea J. , García-Vázquez F. , Gardón J.C. , Cánovas S.
Published: February 2004

The process of cooling to 5°C prior to freezing produces physical and chemical stress on the sperm membrane associated with oxidative stress and reactive oxygen species (ROS) generation that reduces sperm viability and fertilizing ability. The addition of antioxidants to cooling medium could prevent the formation of ROS and improve the seminal parameters. The aim of these experiments was to investigate the effects of addition of reduced glutathione (GSH) to cooling extenders on (1) plasma membrane integrity, (2) acrosome reaction induction by ionophore A 23187 or progesterone, and (3) in vitro fertilization. Ejaculate-rich fractions from three mature pietrain boars were diluted in Beltsville Thaw Solution (BTS) extender and cooled to 15°C over 2h (group C). Thereafter, sperm were centrifuged and diluted in lactose/egg-yolk extender with 0mM (group 0), 1mM (group 1) or 5mM (group 5) of GSH, cooled to 5°C over 2h. The acrosome reaction was then induced by 1μM calcium ionophore or 10μM progesterone in TALP medium and incubated in 5% CO2, 38.5°C for 30 or 45min, respectively. Membrane integrity was evaluated by propidium iodide, and acrosomal status was monitored by means of FITC-labeled peanut agglutinin. Finally, in vitro fertilization was performed with these four spermatozoa groups as described previously (Matás et al. 2003 Reproduction 125, 133–141). ANOVA analysis revealed that the addition of GSH had no effect on the membrane integrity (ranged 58.8 to 66.9) or acrosome reaction induction (ranged 24.3 to 28.2, and 55.7 to 41.4 for progesterone and calcium ionophore, respectively). However, the results of the penetration assay revealed that the cooling affected the penetration rate and the number of sperm per oocyte (Table 1), and this assay is better than the others to predict changes in the spermatozoa functionality (Gadea J and Matás C 2000 Theriogenology 54, 1343–1357). In conclusion, the cooling process affects the in vitro fertilization, but the addition of GSH to the medium did not influence the parameters studied. Supported by AGL2000-0485-CO2-01.

Table 1
Homologous in vitro penetration
107TOXICITY OF ETHYLENE GLYCOL ON FROZEN AND THAWED IVP EMBRYOS IN DIRECT
TRANSFER METHOD
Authors: Matoba S. , Imai K. , Mimaki Y. , Narita M. , Tagawa M. , Dochi O. , Saito N.
Published: February 2004

Ethylene glycol (EG) is a cryoprotectant which is highly permeable to mammalian embryos. But the toxicity of this cryoprotectant for embryos after thawing has not been investigated. The aim of this study was to determine the toxicity of EG to embryos frozen and thawed by a direct transfer method. In vitro-produced Day 7 blastocysts (n=529) of grade 1 quality were used in this study. Embryos were frozen in 1.5MEG in Dulbecco’s PBS (DPBS) supplemented with 0.1M sucrose, 4mgmL−1 BSA and 20% fetal calf serum (FCS). Embryos were transferred into freezing medium, loaded into 0.25-mL straws and kept for more than 15min for equilibration; then the straws were plunged into a −7°C methanol bath of a programmable freezer for 1min, seeded at −7°C, held at −7°C for 14min, cooled to −30°C at the rate of −0.3°Cmin−1 and then plunged into liquid nitrogen. The straws were thawed by holding in air for 6sec, and then placed in water at 30°C for 15s. After thawing, the straws were held for 0, 10, 20, 30 and 60min (holding time) at either 38.5 or 26.0°C. Ethylene glycol was removed from the embryos by placing them into DPBS supplemented with 20% CS at 38.5°C more than 20min. The embryos were cultured in TCM-199 supplemented with 20% FCS and 0.1mM β-mercaptoethanol under a gas phase of 5% CO2 in air at 38.5°C for 72h. Viability of embryos was evaluated at 0-, 24-, 48- and 72-h incubation by their morphological development. Data were analyzed by ANOVA. There was no significantly difference in the survival rate of thawed embryos held at 38.5°C or 26.0°C for the same holding periods. The survival rate of the thawed embryos held at 38.5°C decreased significantly when the holding period exceeded 30min compared with no holding period after 24- and 72-h culture (P<0.05, respectively). On the other hand, the survival rate of the thawed embryos held at 26.0°C decreased significantly when the holding time was 60min compared with less than 20min of holding after 24-h culture, and less than 10min after 72-h culture (P<0.05, respectively). Therefore, toxicity of EG was observed when thawed embryos were held for 30 and 60min at 38.5°C and 60min at 26.0°C. These results suggest the toxicity of EG in direct transfer methods can be avoided by transferring the embryos within 20min after thawing.

Table 1
109REFREEZING STALLION SPERMATOZOA FOR ASSISTED REPRODUCTION
Authors: McCue P.M. , Moore A.I. , Bruemmer J.E.
Published: February 2004

Equine semen is commonly frozen in 0.5-mL straws at a concentration of 400 million spermatozoa per mL. Equine pregnancies have been produced in the past few years using assisted reproduction techniques that require very few sperm, such as sperm injection and gamete intrafallopian transfer. Consequently, thawing even a single straw would waste a high percentage of the sperm. The goals of this study were to: (1) evaluate the motility and viability of cryopreserved spermatozoa that had been thawed and refrozen, and (2) compare the effect of serial dilution on refreezing parameters. It was hypothesized that both motility and viability would be significantly decreased as a consequence of refreezing, but sufficient live, motile spermatozoa would be present for assisted reproductive techniques. Semen from six stallions of mixed light horse breeds was collected, evaluated, and frozen in lactose-EDTA in 0.5-mL straws. Semen from each stallion was subsequently thawed, held at room temperature for 10 minutes, and then refrozen in 0.25-mL straws. Additional semen from the initial freeze was thawed, diluted in the original extender, and refrozen at concentrations of 40×106, 4×106, 4× 105, and 4×104 spermatozoa per mL. Refrozen semen was evaluated for motility visually and by CASA. Viability of refrozen semen was determined by flow cytometry following staining with propidium iodide (PI). Percentages of motile spermatozoa prior to freezing, after the first freezing, and after the second freezing were compared using the PROC GLM model in SAS. All values are presented as the mean±standard deviation. Total motility decreased sigificantly (P<0.05) from 91.8±3.1% prior to freezing to 64.2±7.7% after the first freezing and 45.7±10.4% after the second freezing. However, only a tendency (P=0.09) toward a decrease in sperm viability following refreezing as measured by PI staining was noted. Dilution of sperm prior to refreezing did not significantly effect (P>0.05) subsequent motility or viability parameters. This study demonstrated that refrozen equine semen retained approximately 70% of the initial post-thaw motility. Dilution prior to refreezing would allow for the reallocation of a single 0.5-mL straw into hundreds or thousands of smaller straws for future use in assisted reproduction. Refreezing would allow for the judicious use of valuable stallion semen in limited supply.
11THE EFFECT OF PRESYNCHRONIZATION ON PREGNANCY RATE TO FIXED-TIME AI IN BEEF HEIFERS SUBJECTED TO A COSYNCH PROTOCOL
Authors: Colazo M.G. , Small J.A. , Ward D.R. , Erickson N.E. , Kastelic J.P. , Mapletoft R.J.
Published: February 2004

The objective was to investigate the effect of presynchronization with PGF prior to a Cosynch protocol on estrus synchrony, CL and preovulatory follicle diameters and pregnancy rate following timed-AI (TAI) in beef heifers. Cycling beef heifers (n=148) were treated with 100μg GnRH i.m. (Cystorelin, Merial Canada Inc., Victoriaville, Quebec, Canada) on Day 0, 500μg cloprostenol i.m. (PGF; Estrumate, Schering Plough Animal Health, Pointe-Claire, Quebec, Canada) on Day 7, and GnRH concurrent with TAI on Day 9 (54h after PGF). Half of the heifers (Control) received the first GnRH treatment at random stages of the estrous cycle, while the other half (Presynch) received two injections of PGF 11 days apart, with the first injection of GnRH 11 days after the second injection of PGF. Estrus detection was done between the first GnRH and 12h after PGF, and heifers detected in estrus were inseminated (and considered nonpregnant to TAI), while all other heifers were TAI. Heifers were examined by transrectal ultrasonography for CL and follicle development, and confirmation of pregnancy. Data were compared between groups using Student’s t-test and chi-square procedures. The numbers of heifers in estrus early (after first GnRH and before TAI) was higher in the Control group than in the Presynch group (18/74 v. 2/74, respectively; P<0.0001). Mean (±SD) diameters of the dominant follicle (12.1±3.1 v. 14.2±2.5mm) and CL (17.3±5.5v. 20.5±4.3mm) at first GnRH injection were smaller (P<0.0001) and more variable (P<0.03) in Control than Presynch heifers, but diameters of the preovulatory follicle (P=0.3) and CL (P=0.1) at TAI did not differ. Although the diameter of the preovulatory follicle was more variable (P<0.004) in Control (5 to 19mm) than Presynch (8 to 17mm) heifers, pregnancy rate to TAI did not differ (P=0.4; 29.7 v. 36.5%, respectively). Overall pregnancy rates were 45.9 and 37.8% for Control and Presynch groups, respectively (P=0.3). Pregnancy rate tended (P<0.08) to be affected by diameter of the preovulatory follicle at the time of TAI (0, 23.1, 45.7, 41.4, and 60.0% pregnant for diameters of <9, 9–11, 12–14, 15–17, and >17mm, respectively). Regardless of treatment, diameter of the preovulatory dominant follicle (P<0.02) and CL (P<0.03) 7 days after TAI was smaller, and CL diameter was more variable (P<0.004), in open than in pregnant heifers (12.7±2.6v. 13.8±2.1mm, and 16.5±4.4v. 18.0±3.0mm, respectively). In summary, presynchronization with PGF prior to a Cosynch protocol reduced the proportion of heifers in estrus before TAI, suggesting that this approach may be useful in the successful application of Ovsynch or Cosynch programs in heifers. However, pregnancy rate to TAI did not differ between groups in this study. Diameter of the preovulatory follicle tended to positively affect pregnancy rate, regardless of treatment.
110OSMOTIC STRESS ON THE CELLULAR ACTIN FILAMENT ORGANIZATION OF IN VITRO
PRODUCED PORCINE EMBRYOS
Authors: Men H. , Agca Y. , Mullen S.F. , Critser E.S. , Critser J.K.
Published: February 2004

Disruption of the actin cytoskeleton is one of the leading causes in low survival of pig embryos after cryopreservation (Dobrinsky et al., 2000 Biol. Reprod. 62, 564–570). In this study, the effect of osmotic stress on cellular actin filament organization in porcine embryos produced in vitro was studied. Excellent quality Day 6 (fertilization=Day 0) porcine blastocysts were randomly exposed to 6 different anisosmotic sucrose solutions (75, 150, 210, 600, 1200, 2400mOsm) for 10min. Embryos were then returned to embryo culture medium (NCSU-23) after washing with NCSU-23, and cultured under 38.5°C, 5% CO2 in air with maximal humidity for them to recover. Blastocysts cultured in NCSU-23 medium (280mOsm) served as a control for embryos with intact actin filament organization. Blastocysts treated with 7.5μgmL−1 cytochalasin-b for 60min served as a control for embryos with F-actin depolymerization. Eighteen hours post-anisosmotic treatments, all blastoysts were fixed in 3.7% paraformaldehyde in PBS for 60min and stored in PBS with 0.1% Triton X-100 and 0.2% sodium azide at 4°C. Staining of actin filaments was performed according to procedures described earlier (Wang et al., 1999 Biol. Reprod. 60, 1020–1028). Embryos were blocked in PBS with 20mgmL−1 BSA and 150mM glycine for 30min. After being washed in PBS with 0.1% Tween 20 for 60min, embryos were stained with 10UmL−1 Alexa Fluor 488 phalloidin in PBS with 0.1% Tween 20 at 38.5°C for 60min, and then washed twice in PBS with 0.1% Tween 20 for 60min each. The status of actin filaments in embryonic cells was examined by confocal microscopy. Integrity of cellular actin filaments was classified as either intact or disrupted according to the distribution within embryonic cells. Blastocysts were then classified according to the status of actin filaments in embryonic cells. Data were analyzed using logistic regression. Results from 7 replicates are displayed in Table 1. There was a significant relationship between osmotic treatment levels and the probability of blastocysts with disrupted cellular actin filaments (P<0.0001). These data support the hypothesis that porcine embryos are very sensitive to osmotic changes. Ongoing experiments will assess the extent of actin disruption required to significantly reduce developmental competence of pig blastocysts. This study was supported by Monsanto Company.

Table 1
Cellular actin filament integrity of in vitro produced porcine blastocysts after being treated with sucrose solutions with different osmolalities (mOsm)
115DIMETHYLSULPHOXIDE AND 1,2-PROPANEDIOL DIFFERENTIALLY CRYOPROTECT
BOVINE AND PORCINE PRIMORDIAL, PRIMARY AND SECONDARY FOLLICLES
Authors: Papasso Brambilla E. , Paffoni A. , Brevini T.A.L. , De Eguileor M. , Ragni G. , Gandolfi F.
Published: February 2004

Ovarian tissue cryopreservation is of interest for many areas of assisted reproduction. Normal structure and intra- as well as intercellular organization have to be maintained in order to preserve follicle viability. In the present work we studied the effects of two largely used cryoprotectants, namely, dimethylsulphoxide (DMSO) and 1,2-propanediol (PROH), on follicle morphology. Experiments were carried out on bovine and porcine ovaries and the two cryoprotectants were assessed for their ability to preserve structural integrity of primordial, primary and secondary follicles, in order to investigate the protective effects of these molecules on specific developmental stages. Fragments from each ovary were divided in three groups: a) immediately fixed (control); b) cryopreserved in 1.5M DMSO; c) cryopreserved in 1.5M PROH. To allow equilibration with the cryoprotectant, samples were held for 30min at 4°C in order to minimize toxic effects. Cryopreservation was carried out in a controlled rate freezer (Planer Planerple, Sunbury, Middlesex, UK), with the following protocol: precooling (4°C); cooling at −2°C/min to 9°C; seeding at 9°C and standby for 10 minutes; cooling at −0.3°C/min to 40°C and 10°C/min to 140°C; plunging into liquid nitrogen. Samples were rapidly thawed. Dilution of cryoprotectants was carried out in 3 steps of ten minutes each at 4°C (1M; 0.5M; 0M). Samples were then fixed, paraffin embedded, serially sectioned and evaluated with a Nikon TE200 inverted microscope. Follicles of each developemental stage were scored in three categories using the criteria previously described by Paynter et al. (1999, Cryobiology 38, 301–309) and presented in the table below as I=Intact; SA=Small Abnormalities; GA=Great Abnormalities. Statistical differences were assessed by the chi-square test (P<0.05). The results obtained showed that both cryoprotectants are effective for the preservation of secondary follicles. The use of PROH was unable to protect primary and primordial follicles in both species. Conversely, DMSO showed a satisfying cryoprotecting effect for pig primary and bovine primordial follicles but had a poor protecting capability for pig primordial and bovine primary follicles. Altogether the present results suggest that the choice of the cryoprotectant needs to be carefully targeted in relation to the follicular stage and the species of interest. This work was funded by Industria Farmaceutica Serono SpA.

Table 1
116DEVELOPMENT INTO BLASTOCYSTS OF SWAMP BUFFALO OOCYTES AFTER
VITRIFICATION AND NUCLEAR TRANSFER
Authors: Parnpai R. , Laowtammathron C. , Terao T. , Lorthongpanich C. , Muenthaisong S. , Vetchayan T. , Hochi S.
Published: February 2004

Oocyte cryopreservation in the domestic species is still at the experimental stage, but recent studies indicated that vitrification characterized by ultra-rapid cooling rate is promising for cryopreservation of bovine oocytes. In the present study, denuded buffalo oocytes were vitrified by minimum volume cooling procedure (Kuwayama and Kato, 2000, J Assist Reprod Genet 17, 477) after IVM or after IVM and enucleation, and developmental potential into blastocysts of the post-warm oocytes after somatic cell nuclear transplantation was examined. Cumulus-oocyte complexes were matured, denuded, and enucleated as described previously (Parnpai et al., 1999, Buffalo J 3, 371–384). The presumptive metaphase-II (M-II) oocytes before and after enucleation were first equilibrated in 7.5% DMSO+7.5% ethylene glycol+20% FCS in TCM199 for 10 min, and then exposed to 15% DMSO+15% ethylene glycol+0.5M sucrose+20% FCS in TCM199 for 1min. Five oocytes were placed on a Cryotop sheet (Kitazato Supply Co., Tokyo, Japan) and vitrified in liquid nitrogen. The samples were warmed in 0.5M sucrose solution for 5min, directly transferred into TCM199+20% FCS, and kept at room temperature for 1h before being used for a cloning experiment. The post-warm oocytes were fused with ear skin fibroblasts by two DC pulses (26V, 17μs) and activated with 7% ethanol for 5min and then 10μg/mL cycloheximide and 1.25μg/mL cytochalasin-D for 5h. The reconstructed embryos were cultured in mSOFaa+0.2% BSA+0.1% linoleic acid albumin for 2 days, and then co-cultured with bovine oviductal epithelial cells for an additional 5 days. Post-warm morphological survival of M-II oocytes (80%, 187/235) was similar to that of enucleated oocytes (75%, 158/212). Vitrified M-II oocytes were successfully enucleated (96%, 136/142) as were fresh control oocytes (88%, 143/162). Fusion rates of M-II oocytes vitrified before and after enucleation (81%, 94/116 and 78%, 106/136, respectively) were also similar to those of fresh oocytes (81%, 100/123). Percentages of reconstructed embryos developing into hatching blastocysts on Day 7 were 5% (5/91), 6% (6/103), and 8% (8/99) in the groups of oocytes vitrified before and after enucleation, and of fresh control oocytes, respectively (ANOVA tests were not significant different). These results indicate that swamp buffalo oocytes cryopreserved by ultra-rapid vitrification procedure can be used successfully for subsequent somatic cell nuclear transplantation. (Supported by Thailand Research Fund and R&D Fund of Suranaree University of Technology)
12PREDICTION OF BULL FERTILITY BY COMPUTER ASSISTED SEMEN ANALYSIS
Authors: Cseh S. , Polichronopoulos T. , Solti L.
Published: February 2004

Sperm motility is clearly essential for fertilization both in vivo and in vitro. Motility is necessary for successful sperm transport, a step that is bypassed with in vitro fertilization. Recently, increasing attention has been paid to the objective evaluation and characterization of sperm motility more than simply determining the total proportion of motile spermatozoa. The purpose of computerassisted semen analysis (CASA) is to provide values for sperm concentration and sperm motility more rapidly and accurately than those obtained with traditional semen analyses methods. The objective of our experiment was to investigate the effect of specific aspects of sperm movement, such as the velocity of progression and the actual pattern of movement, to the fertilizing capability of sperm. Frozen semen samples of 10 HF breeding bulls were used in the study. For the motility analyses, Medealab CASA system (Medealab, Germany, Ver. 4.1) was used, and the velocity parameter of VCL (curvalinear velocity, μms−1), VSL (straight line velocity, μms−1), and VAP (average path velocity, μms−1) were evaluated and compared with the Day 30 and 75 non−return rates (NR30 and NR75). For every sample, a total of 10 fields were examined for 8s using a disposable 20 micron capillary chamber (CellVision, USA) giving a total of 1165 to 2831 cells evaluated. Chi square analysis, analyses of variance and linear correlation coefficient was applied to the statistical evaluation and comparison of the results. Data are based on weighted values. From the same batch of the analyzed frozen semen, a total of 8099 females were inseminated in more than 100 farms with a total of 6590 animals being positive for pregnancy at Day 30 and 4525 animals at Day 75. Within the bulls, differences were found in the values of NR30 and NR75 (P<0.05). Our data indicate very strong differences between the males’ NR30 and NR75 values (NR30: 65.6%±13.04 to 79.6%±11.17; P<0.001 and NR75: 37.8%±10.38 to 58.3%±15.53; P<0.001) reflecting the individual differences in the fertilizing capability of the males. All velocity parameters show very high correlation with strong significance both non−return rates but the best values belong to VAP (NR30 and NR75; P<0.02). Our data indicate that the bulls with lower VCL (25.51±33.04 to 79.54±58.03), VSL (11.35±19.45 to 36.36±35.71), and VAP (12.67±19.06 to 41.75±34.45) values showed lower fertilization rates both at NR30 and NR75. Computer and video technologies have advanced rapidly in recent years; thus the capability and accuracy of the latest versions of CASA systems are considerably better and they give more information about the different motion characteristics of spermatozoa. Because of the vital role of sperm motility in the reproductive process, such systems will enable us to move into a new era of diagnostic andrology and predict the fertilizing capability of semen. Supported by NKFP-Grants 4/040/2001 and 4/031/2001.
120PREGNANCY RATES RESULTING FROM TRANSFER OF FRESH AND FROZEN
HOLSTEIN AND JERSEY EMBRYOS
Authors: Steel R. , Hasler J.F.
Published: February 2004

Although it has not been documented in published studies, embryo transfer (ET) practitioners have suggested that embryos from Jersey (JE) cattle do not survive freezing as well as embryos from other dairy breeds such as Holsteins (HO). The present study represents a retrospective analysis of pregnancy rates achieved following transfer of fresh and frozen embryos from Jersey and Holstein donors. In addition, a retrospective comparison was made of two different embryo-freezing protocols for each breed of cattle. Embryos were collected nonsurgically 7 to 7.5 days post-estrus from superovulated donors on 57 Holstein and 27 Jersey dairy farms over a 15-year period. Fresh and frozen-thawed embryos were transferred nonsurgically into cows and heifers following either natural or prostaglandin-induced estrus. Embryos were frozen either in 10% glycerol (Gly) or 1.5M ethylene glycol (EG) in 0.25mL straws. Following equilibration, straws were seeded at −6 to −7°C and temperature was maintained for 10 min and then decreased at 0.6°Cper min. Straws were plunged into liquid nitrogen at −32 to −35°C. At thawing, straws were held in the air for 7s and then submerged in 29°C water for 15s. Embryos frozen in EG were transferred immediately following thawing. Embryos frozen in Gly were rehydrated in a standard 3-step Gly-sucrose system prior to being transferred. Pregnancy diagnosis was performed at Days 40 to 90 of gestation. As seen in the Table 1, pregnancy rates were similar for fresh embryos from both HO and JE cattle. Also, there were no differences in pregnancy rates between recipients that received embryos frozen in Gly or EG within donors of either breed. However, JE embryos frozen in either Gly or EG resulted in lower pregnancy rates than did HO embryos frozen in Gly or EG. Embryo stage at freezing was tracked for EG but not Gly embryos. There were no differences in pregnancy rates among morulae, early blastocysts or mid-blastocysts for either HO or JE embryos frozen in EG. The differences in embryo survival may be due to different lipid composition of embryos of the two breeds. Perhaps a more efficacious freezing protocol can be developed for cryopreservation of JE embryos. In conclusion, pregnancy rates with cryopreserved HO embryos were higher than with JE embryos.

Table 1
123VITRIFICATION OF IN VITRO-PRODUCED BOVINE BLASTOCYSTS BY ADDITION OF
CRYOPROTECTANT IN ONE STEP
Authors: Walker D.J. , Campos-Chillon L.F. , Seidel G.E.
Published: February 2004

Vitrification combined with in-straw dilution may replace conventional cryopreservation of bovine embryos, but this requires further study for practicality. Our objectives were to compare three ethylene glycol concentrations (6, 7, and 8M) and two equilibration times (2.5 and 3.5min) for one-step addition of cryoprotectant. In vitro-matured oocytes from slaughterhouse ovaries, fertilized using sperm of 3 bulls, were cultured in chemically defined medium (CDM-1/CDM-2) plus FAF-BSA to produce 420 blastocysts. Day 7.5 embryos were placed into HCDM-2 (HEPES-buffered medium) and then transferred to a 6μL drop of vitrification solution (V) (6, 7, or 8M ethylene glycol, 0.5M galactose, and 18% w/v Ficoll 70 in HCDM-2). Immediately thereafter, 1cm column of DHCDM (0.5M galactose in HCDM-2) was drawn into a 0.25mL straw, followed by a 0.5cm column of air and another 7cm of DHCDM. Another 0.5cm column of air was aspirated before the 6μL of V (0.5cm) containing the embryos were aspirated; then 0.5cm of air followed. Finally, DHCDM was drawn until the first column came into contact with the cotton plug. Straws were then heat-sealed and plunged into liquid nitrogen slightly above the embryos after 2.5 or 3.5min equilibration. The rest of the straw was then submerged slowly. Straws were thawed in air for 10s and then in 37°C water for 20s. Straws were held at room temperature (24°C) for 4min before being expelled into HCDM-2. They were then placed into CDM-2+5% FCS for culture. Quality score (1=excellent, 2=fair, 3=poor), survival (S) as determined by expansion of blastocysts, and hatching (H) were assessed at 24 and 48h post-thaw. Data from 6 replicates (2/bull) were analyzed by ANOVA after arc sin transformation of percentage data. S and H responses were calculated as a percentage of non-frozen controls in the same replicate. Control survival and hatching rates were: 24S: 90%, 24H: 50%, 48S: 90%, 48H: 72%. Quality scores at both 24 and 48h were higher (P<0.05) for 8M than 6M ethylene glycol (2.68 and 3.24 for 24h; 2.55 and 3.17 for 48h); values for 7M ethylene glycol were intermediate. Equilibration time had no effect on embryo quality (P>0.1). Neither ethylene glycol concentration nor exposure time affected survival or hatching at 24 or 48h (P>0.1). Survival rates (as a % of control) at 48h were: 8M: 57%, 7M: 55%, 6M: 36% and hatching: 8M: 39%, 7M: 30%, and 6M: 21%; 2.5min tended to be better than 3.5min for survival at 24h, hatching at 24h, survival at 48h, but not hatching at 48h (56% and 43%, 30% and 26%, 55% and 44%, 28% and 32% respectively). Higher concentrations of ethylene glycol proved beneficial in terms of embryo quality, with the same trend for survival and hatching rates. One-step addition of cryoprotectant for vitrification shows potential for simplifying embryo cryopreservation. However, further research is needed to produce more acceptable survival rates and to study vitrification of in vivo-produced embryos.
125MORPHOLOGICAL CHARACTERIZATION OF DAY 14 AND DAY 21 IVP AND
CLONED BOVINE EMBRYO DEVELOPMENT
Authors: Alexopoulos N.I. , French A.J. , Maddox-Hyttel P.
Published: February 2004

The establishment of viable embryonic development following in vitro production (IVP) or nuclear transfer by SUZI (subzonal injection) and handmade cloning (HMC) methods is pivotal for practical implementation of embryo technology. The development of cloned embryos is currently impeded by the high rate of pre- and postnatal losses. This project is aimed at developing screening methods using both ultra-structural and molecular markers of development of post-hatching/pre-implantation stage embryos to enable selection of embryos with greater developmental competence. Initially twenty embryos derived by IVP or nuclear transfer (SUZI or HMC) were transferred to six recipient cows on Day 7 (n=120). Embryos were collected on Days 14 and 21 by flushing the uterine tracts of slaughtered cows. Embryos were fixed in 4% paraformaldehyde and processed for paraffin embedding. Serial sections were stained with haematoxylin and eosin and evaluated by light microscopy. On each day of collection, certain characteristics were expected to be observed according to normal in vivo development (Maddox-Hyttel et al., 2003, Reproduction 125, 607–623). On Day 14, IVP embryos (n=6) were ovoid to filamentous in shape. Three embryos had extensive foldings of trophoblast in the process of forming an amniotic cavity. NT-SUZI embryos (n=4) had a mainly round uneven shape. No NT-HMC embryos were recovered. On Day 21, IVP embryos (n=4) more closely resembled in vivo embryos than any other group and formed either a neural groove or a neural groove with somites. NT-SUZI embryos (n=6) were mainly filamentous, with one embryo showing complete development of the neural tube and 5 somite pairs. NT-HMC (n=17) development ranged from ovoid to multi-protrusions of the yolk sac. One embryo displayed the formation of a neural tube. Embryos, irrespective of production method, which had an abnormal morphology, presented only an incomplete hypoblast and severe degeneration of the epiblast. Those embryos that were morphologically comparable to in vivo embryos also displayed developmental stages which ranged from presentation of a primitive streak and formation of a neural tube to having a neural tube, differentiation of mesoderm and somites. At present, the low number of embryos recovered as well as their abnormal development clearly reflect the low success rate to term of live calves. It is possible that no Day 14 NT-HMC embryos were recovered due to collection efficiency not being optimal. It is apparent that IVP and cloned embryos have different developmental time lines as compared to that of in vivo embryos.
128CHANGES WITHIN OXYGEN ENVIRONMENT DURING IVF AND IVC IMPROVE BOVINE
EMBRYONIC DEVELOPMENT IN VITRO
Authors: Hidalgo C.O. , Diez C. , Moran E. , Prendes J.M. , Humblot P. , Marquant-Le Guienne B. , Facal N. , Gomez E.
Published: February 2004

Oxygen concentration during both IVF and IVC affects embryonic development. A low O2 atmosphere during IVC has been reported to be beneficial for embryos in culture without somatic cells. Similarly, a reduction in spermatozoa (sp) concentration can influence the oxygenation grade of fertilization medium, and O2 requirements can vary according to the embryonic stage of development. This experiment investigated whether prolonged contact with sperm cells interacts with effects of oxygen tension during the first days of culture. Slaughterhouse bovine COCs matured with Vero-cells in TCM199 with FCS and EGF were co-incubated (CI) with swim-up separated sp. COCs with attached sp were either removed after 2h and placed in IVF medium without sp up to completion of 18h (sp-restricted CI), or COCs and sp were left together for 18h (sp-prolonged CI). Zygotes were cultured in serum-free, B2 medium conditioned with Vero cells (Marquant-Le Guienne et al., 1999 Theriogenology 51, 386), modified as described by Gomez and Diez (2000 Anim. Reprod. Sci. 58, 23–37). According to IVF procedure (sp-restricted or sp-prolonged), embryo culture was made either in 20% O2 from Day 0 up to Day 3 and in 5% O2 later on, or in 5% O2 from Day 0 up to Day 8, in a 2×2 factorial design. IVF medium was monitored for Na+, K+, Ca2+, pH, O2, CO2 , HCO3− and lactate (i-STAT analyzer; 5 replicates -R-) at 0h, 2h and 18h without cells (controls), at 2h in CI and at 18h both in sp-restricted and sp-prolonged CI. Embryo development was analyzed by CATMOD for effects, and all data were processed by GLM and Duncan test and expressed as LSM±SE. The presence of cells decreased pO2 (P<0.01) in IVF medium at 2h of CI (154±2.3 without cells v. 144±2.3 with cells). There were differences in pO2 at 18h (P<0.05) between sp-prolonged (140.8±2.3) and sp-restricted CI (148.6±2.3). At 18h in CI there was K+ depletion (P<0.05), while Na+, Ca2+, pH and pCO2 remained invariable throughout. Lactate production increased (P<0.05) in CI at 2h and, together with HCO3− (P<0.01), at 18h in sp-prolonged CI. Increasing O2 up to 20% during the first 3 days of culture improved total and medium to late (M+L; early blastocysts excluded) blastocyst rates upon sp-prolonged CI, which is comparable to sp-restricted CI under both % O2 conditions during embryo culture. Under 5% CO2 throughout culture, higher expansion rates occurred in sp-restricted CI, suggesting that this system could improve embryonic viability. Metabolic effects can be inferred from differences in pO2 , lactate and HCO3− , and research will explore the role of oxygen free radicals in these experimental conditions. Grant support: Eureka 2573; CDTI, PROFIT, and FICYT.
129IN UTERO TRANSPLANTATION OF HUMAN HEMATOPOIETIC STEM CELLS (HSC) INTO
FETAL GOATS OFFERS A NEW APPROACH TO STUDIES OF HSC EXPANSION AND DEVELOPMENT
Authors: Huang S.-Z. , Zeng F. , Sun D. , Chen M.-J. , Ren Z.-R. , Zeng Y.-T.
Published: February 2004

In utero transplantation of xenogeneic stem cells into animals (such as mouse, sheep, goats) is a new approach to the studies of the engraftmen, expansion and development of foreign cells in vivo. In addition, such an approach is able to offer a model to clinical potential for correcting or replacing damaged tissues in a variety of disorders, including many that are inherent as well as acquired. Recently, we generated a human/goat hematopoietic stem cell (HSC) xenogeneic model to investigate the engraftment, expansion and development of human hematopoietic cells at an in vivo level. Human HSCs (lin-cells) were isolated and purified from fresh cord blood, and then injected intraperitoneally into pre-immune fetal goats at 55–65 gestation days (term: 145 days). A total of 50 fetal goats were injected. The growth and development of the recipient goats were monitored by B-type ultrasound scan. Ten goats suffered miscarriage due to GVH response; 39 recipients survived a complete gestation and were born alive. All these live-born goats were healthy and grew very well. They could produce offspring normally. In order to determine whether human cells were present longterm in the circulatory blood of the recipient goats, FACS, real-time PCR, RT-PCR, and Southern-blot hybridization as well as FISH were used to analyze the genotype and phenotype up to 11 months after birth. The results showed that hematopoietic chimerism (on average, >108 human cells per goat) was documented in 90% (35/39) of live-born goats that had been originally transplanted with 105 human HSCs. The regenerated human cells included myeloid, B-lymphoid and erythroid lineages as well as more primitive cells as shown by specific positive staining for human CD14, CD20, glycophorin A (GPA) and CD34, and the detection of human-specific DNA sequences and human GPA and CD34 transcripts in the blood of the transplanted goats. No human T or NK-lineage cells were detected. These studies demonstrate the feasibility of using in utero-transplanted fetal goats as a new approach for evaluating the longterm engraftment potential, expansion and development of human hematopoietic cells in vivo.