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

This study examined the effects of cooling and cryopreservation upon macropod spermatozoa (eastern grey kangaroo, Macropus giganteus and red-necked wallaby, Macropus rufogriseus). Sperm survival during and after freezing to -30 degrees C or 70 degrees C in minimum essential medium (MEM) + 5, 10, 20 or 30% (v/v) glycerol, MEM + 10 or 20% (v/v) ethylene glycol and MEM containing a mixture of 7.5% (v/v) glycerol + 10% (v/v) dimethylsulphoxide was examined by cryomicroscopy. The MEM/glycerol mixtures permitted better post-thaw sperm recovery than the other cryoprotectants. After freezing to -30 degrees C at 10 degrees C min(-1) in 20% glycerol, then rewarming at 20 degrees C min(-1), flagellar activity resumed in more than 50% of spermatozoa when the temperature increased into the range 5-10 degrees C. However, as the temperature increased, into the range 20-25 degrees C, motility declined rapidly so that less than 5% motile cells were seen at 35 degrees C. Spermatozoa in MEM without cryoprotectant were also examined by cryomicroscopy to evaluate changes in flagellar configuration, swimming behaviour and viability during cooling from 35 degrees C to approximately -7 degrees C, and rewarming to 35 degrees C. Cooling from 35 to 28 degrees C induced kangaroo spermatozoa to exhibit rigid principal-piece bending and non-linear motility, which was reversed by further cooling and the spermatozoa resumed their normal linear movement. Rewarming induced principal-piece bending in the range of 20-30 degrees C, but this effect was reversed by further warming. Although red-necked wallaby spermatozoa showed these effects, they also exhibited a tendency to form rosette-like clusters during rewarming, especially when the temperature reached approximately 14 degrees C. The clusters were induced when the flagellar end-pieces became anteriorly reflected, producing hook-like flagellar conformations, which then became interlinked.

The objective of this study was to determine the effects of genetic manipulation, cell type, and culture conditions on developmental potential of bovine nuclear transfer (NT) embryos. Ovum pickup (OPU) technology was developed to obtain the oocytes for NT. A total 4044 cumulus-oocyte complexes (COCs) were obtained during 492 OPU sessions, with an average of 8.2 COCs recovered each session. Cultured granulosa cells (CGC), bovine fetal (150 days) oviduct epidermic cells (FOEC), and adult ear skin fibroblasts (ASFC) were used as donor cells for NT and were transfected with the expression vector including human FIX coding sequence directed by goat β-casein promoter and neomycin gene. The cells were screened under 800 µg mL −1 G418 for 10-14 days until the apperance of a "mono-colony" of cells which were then picked. Each cell population was expanded by consecutive passage culture under 300 µg mL −1 G418 until used for NT, ensuring that the majority of cells were transgenic. Oocytes were enucleated at 20 h post-maturation and a single donor cell was transferred into the perivitelline space of a recipient oocyte. After fusion and activation, the reconstructed embryos were co-cultured with vero cells in B2 medium for 7 days. NT efficiency between primary granulosa cells (PGC) without in vitro culture and CGC, as well as among CGC, FOEC and ASFC that were transfected with exogenous DNA (named TCGC, TFOEC, TASFC, respectively), were compared (Table 1). Differences between groups were verified by chi-square test using SAS 6.12 (SAS Institute, Inc., Cary, NC, USA) program. CGCs presented a higher fusion rate (P < 0.01) for reconstructed embryos and higher development to the blastocyst stage for NT embryos than did PGC (67% vs. 54% and 41% vs. 21%, respectively). There were no significant differences (P > 0.05) in cleavage rate (65%, 71%, and 69%, respectively) and development to the blastocyst stage for NT embryos (36%, 30% and 40%, respectively) for TCGC, TFOEC, and TASFC. A total of 86 blastocysts were selected for transfer into uteri of 86 cows, resulting in 26 pregnancies (30%) at 60 days by ultrasound scanning. Among these, 12 cows remain pregnant and 14 have aborted. The results indicated that oocytes recovered from OPU can be successfully used for NT with development to the blasocyst stage. PGC, CGC, FOEC, and ASFC can all be used for generating transgenic cattle by NT, although this needs to be verified by the birth of live calves. The cryopreservation of sperm has contributed greatly to animal breeding and reproduction. This study was designed to examine the effect of raffinose, sucrose, and trehalose as cryoprotectants for freezing of mouse sperm. The cryoprotectant solution (CPA) consisting of 3% skim milk (Skim Milk dehydrated, Bacto, Difco, Seoul, Korea) as buffer or extender was prepared and supplemented with 0.3 M raffinose (D[+]raffinose pentahydrate, Sigma) or sucrose or trehalose as non-permeating cryoprotectants. Sperm samples for cryopreservation were collected from caudae epididymides and vas deferens ...

The use of IVF in horses has a limited efficiency, reflecting low oocyte developmental competence and inadequate sperm capacitation procedures. In a preliminary study, using carboxyfluorescein diacetate/propidium iodide staining, we determined that the freezing-thawing procedure left only 56.6 ± 3.4 % of the sperm cells with an intact membrane. The following incubation in TALP-IVF induced membrane damage at high rates with only 9.58 ± 1.8 % of them intact after 18 h. However, the presence of at least four cumulus-enclosed oocytes (CEO) in the medium significantly increased the number of membrane-intact spermatozoa at the end of incubation (53.87 ± 1.99%). This indicated that the sperm thawing and capacitating procedures can damage the cell membrane but the presence of four or more CEO in TALP-IVF could prevent further damages. The aim of the study was to investigate in detail the membrane damages and to analyze the differences induced by the presence of CEO. Spermatozoa were thawed in water at 37 • C, and centrifuged for 30 minutes at 600g in a 45-90% Percoll gradient made with modified Tyrode's medium. The sperm pellet was washed once in the same medium and diluted to a final concentration of 1 × 10 6 spermatozoa/ml TALP supplemented with 0.6% (w/v) BSA fatty acid free and 12 µg mL −1 heparin (TALP-IVF). Sperm cells were incubated with 0 or 4 in vitro-matured CEO. Sperm cells were examined after thawing, 0, 2 and 18 h from the beginning of incubation in TALP-IVF. Each experiment was replicated at least 3 times. Both scanning and transmission electron microscopy were performed on sperm samples fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, using standard procedures. Specimens for scanning electron microscopy were examined under a field emission gun JEOL JSM 6301 microscope. For transmission electron microscopy the samples were examined with a JEOL JEM 100 SX. A minimum of 25 cells were analyzed for each group. Immediately after thawing, damaged spermatozoa showed, on the surface of their heads, small vesicles correlated to a progressive process of vacuolisation and degeneration of membrane integrity. The same lesions were visible at all the successive time points taken into account. Moreover, a loss of the acrosome integrity with acrosomal swelling and a decrease of content homogeneity were observed particularly in the spermatozoa cultured for 18 h without CEO. When CEO were present in the IVF medium lesions were visible in a lower percentage of spermatozoa but the type of lesions did not differ from those observed in their absence. These observations confirmed our previous data and gave more details on the lesions that occur during the IVF procedures in the horse. Supported by MURST COFIN grant n. 2001078849. Increasing the diary population and milk production is a goal of many tropical developing countries. We report in this paper an attempt to develop a system of intercontinental shipping for transfer of fresh crossbred Bos taurus × Bos indicus IVF embryos into local Laisind (Bos indicus) rec...

A large proportion of bovine oocytes fail to develop to blastocyst stage following maturation, fertilization, and culture in vitro. While suboptimal culture conditions undoubtedly contribute to this poor development, it is recognized that immature oocytes, especially from cows with reduced reproductive performance or which are slaughtered on the end of their use, are heterogeneous in quality and developmental competence (Gordon 2003). The aim of the present study was to increase the efficiency of blastocyst production from cows after IVM/IVF by oocyte selection before maturation. Immature oocytes are known to synthesize a variety of proteins (Wassarman PM 1988, Annu. Rev. Biochem. 57, 415-442), among them, glucose-6-phosphate dehydrogenase (G6PDH). This enzyme is active in the growing oocyte, but has decreased activity in oocytes that have finished their growth phase. Brilliant cresyl blue (BCB) has been used to measure G6PDH activity. The BCB test is based on the capability of the G6PDH to convert the BCB stain from blue to colorless (Erisson et al. 1993 Theriogenology 39, 214). The ovaries were obtained from a slaughterhouse and transported to the laboratory; cumulus-oocyte complexes (COCs) were recovered by slicing the surface of the ovary. Only oocytes with a compact cumulus investment were used. Oocytes were placed into three groups: (1) control -placed immediately into culture; (2) holding control -COCs kept in PBS containing 0.4% BSA for 90 min at 38.5 • C before placement into culture; and (3) treatment -incubation with brilliant cresyl blue for 90 min at 38.5 • C before culture. Treated oocytes were then divided into BCB− (colorless cytoplasm, increased G6PDH) and BCB+ (colored cytoplasm, low G6PDH) on their ability to metabolize the stain. Activity of G6PDH was determined via measurement of NADP reduction in control, BCB−, and BCB+ groups; activity was significantly increased in BCB− COCs in comparison to the control and BCB+ COCs. After IVM, oocytes were fertilized in vitro. Embryos were cultured to Day 8. The rate of maturation to metaphase II was significantly higher for control and BCB+ oocytes (77.1 and 72.5%, respectively) than for BCB− oocytes (58.1%). The BCB+ oocytes yielded a significantly higher proportion of blastocysts (34.1%) than either control group (18.3 and 19.2%); and both controls and BCB+ oocytes had significantly higher blastocyst development than did BCB− oocytes (3.9%). The number of nuclei in the blastocysts was comparable in BCB+ and both control groups (105.5 ± 5.8 and 117.5 ± 8.5, 101.8 ± 6.2, respectively). Blastocysts in the BCB− group had a significantly lower cell number (61.0 ± 2.6) than did controls. The results show that the staining of COCs from cows before IVM may be useful in increasing the efficiency of blastocyst production during standard IVF procedures. In addition, classification of G6PDH activity on the basis of BCB staining may be used to effectively select cow oocytes with further developmental competence. To our knowledge, this is the first study to evalu...