In vitro fertilization rate of horse oocytes with partially removed zonae

Frozen-thawed ejaculated stallion spermatozoa were preincubated for 3 h in BO medium containing 5 mM caffeine and then treated with 0.1 mu M calcium ionophore A23187 for 60 sec. Aliquots of the sperm suspension (final concentration 1-2 x 10(7)/ml) were added to the oocytes which had been matured in vitro for 32 h. In Experiment 1, there were 3 groups of oocytes; cumulus intact, denuded zona-intact, and zona-free. Cumulus cells were removed with 0.5% hyaluronidase and the zona pellucida with 0.1% protease. The oocytes were fixed 20 h after insemination with acetic acid:ethanol (1:3) and stained with 1% orcein. The sperm penetration rate of zona-free oocytes was 83%, whereas the sperm penetration rate was very low (1 to 3%) in the cumulus-enclosed or zona-intact oocytes. In Experiment 2, denuded zona-intact oocytes were placed in PBS supplemented with 10% fetal bovine serum 1 h before the end of in vitro maturation. The zona pellucida was micromanipulated with a metal microblade under x 100 magnification within 20 min of treatment with 0.3 M sucrose. For partial zona dissection, a slit in the zona pellucida was made. For partial zona removal, oocytes were transferred to protein-free PBS to fix the oocytes on the bottom of the Petri-dish and to remove a piece of the zona pellucida. Micromanipulated oocytes were subjected to in vitro fertilization as described above. Zona-intact and zona-free oocytes treated with sucrose solution for 20 min were used as controls. The penetration rates were 4 (2 57 ), 12 (7 58 ), 52 (31 60 ), and 86% (44 51 ) for zona-intact, partially zona dissected, partially zona removed, and zona-free oocytes, respectively. Proportions of oocytes with monospermic penetration were 100 (2 2 ), 57 (4 7 ), 58 (18 31 ), and 34% (15 44 ), respectively. In Experiment 3, sperm penetration and male pronucleus formation in the partially zona removed oocytes were examined at 2.5 to 20.0 h of insemination. Sperm penetration started 2.5 h post-insemination (22%, 11 49 ), and increased to 38% (21 55 ) at 5 h, to 46% (23 50 ) at 10 h, and to 56% (27 48 ) at 20 h. The transformation of sperm heads into male pronuclei was first observed 10 h post insemination. These results indicate that assisted fertilization techniques may be a useful tool for achieving fertilization and embryo production in vitro in horses.     

Effects of cooling germinal vesicle-stage bovine oocytes on meiotic spindle formation following in vitro maturation.

Attempts to cryopreserve bovine oocytes result in low survival because of their sensitivity to temperatures near 0 degrees C. This study evaluates the effects of chilling germinal vesicle-stage (GV) oocytes on their formation of microtubules and the meiotic spindle. In experiment 1, five groups of GV-stage oocytes, each consisting of approximately 90 oocytes, were held at 39 degrees C as controls, or at 31 degrees C, or cooled to 24, 4 or 0 degrees C for 10 min. After being treated, all oocytes were cultured at 39 degrees C for 24 hr. Compared to the controls, holding oocytes for 10 min at 31 or 24 degrees C did not significantly alter the formation of normal spindles, but chilling them to 4 or 0 degrees C did. After 24 hr of maturation, the respective percentages of oocytes containing normal meiotic spindles observed in the controls or those held at 31 or 24 degrees C were 69.8%, 71.9%, or 69.4% (P > 0.05). In contrast, the percentages of oocytes with normal spindles after they had been cooled to 4 or 0 degrees C were 44.0% or 29.1%, respectively. In experiment 2, approximately 90 oocytes/group were cooled to 4 degrees C for various times before being warmed and cultured. Regardless of the time of exposure, cooling oocytes to 4 degrees C reduced the formation of normal spindles. The percentages of oocytes cooled to 4 degrees C for 10, 20, 30, 45, or 60 min with normal spindles were 44.0%, 38.4%, 37.5%, 34.5% and 30.9%, respectively. In experiment 3, approximately 60 oocytes per group that had been held at 31 degrees C or cooled to 24, 4 or 0 degrees C for 10 min were allowed to mature for 24 hr before being subjected to in vitro fertilization. The cleavage rates of oocytes subjected to various chilling treatments exhibited the same pattern as that of oocytes with normal spindles. That is, there were no significant differences in cleavage rates among the control oocytes and those held at 31 or 24 degrees C (70.4%, 71.8%, and 72.4%; P > 0.05). However, only 37. 0% and 30.4% of oocytes chilled to 4 or 0 degrees C cleaved after fertilization. These results suggest that: (1) chilling bovine oocytes no lower than 24 degrees C does not reduce formation of normal meiotic spindles; (2) however, chilling oocytes to 4 degrees C or lower for as little as 10 min drastically reduces the formation of normal meiotic spindles and of fertilization; (3) the rates of fertilization and cleavage of resultant zygotes mimic that of formation of normal spindles.     

Native zona pellucida structure is required for completion of sperm acrosome reaction in porcine fertilization

In fertilization in vitro, the penetration rate of zona-intact porcine oocytes by cryopreserved epididymal spermatozoa was about 100% while that of zona-free oocytes was only 30%. Spermatozoa treated with calcium ionophore A23187 penetrated both zona-intact and zona-free oocytes at the rate of more than 90%. Treatment of spermatozoa with solubilized procine zonae pellucidae hardly induced acrosome reaction and did not increase the penetration rate. These results suggest that the structure of the zona is necessary for completion of acrosome reaction.     

Capacitation-like changes occur in mouse spermatozoa cooled to low temperatures

Previously we showed that > 70% of mouse spermatozoa cooled slowly from 37°C to 4°C and warmed have undergone capacitation-like changes as examined by a chlortetracycline staining assay. These membrane changes are reflected in the ability of cooled spermatozoa to achieve fertilization rates in vitro similar to those of uncooled controls when added to oocytes immediately upon warming. The aim of this study was to determine the nature of these membrane changes. We found they were not dependent upon the rate of cooling to 4°C and similar changes were observed when spermatozoa were cooled to higher temperatures (10° and 20°C), but it took longer for 50% of the spermatozoa to undergo such changes (3, 18, and 27 min for spermatozoa held at 4°, 10°, and 20°C, respectively). Mixing cooled spermatozoa with oocytes immediately upon warming produced fertilization rates similar to fresh spermatozoa capacitated in vitro for 90 min before the oocytes were added. The rate of sperm penetration as determined by the fluorescent DNA stain Hoescht 33258 was also similar. However, the penetration time for cooled spermatozoa was significantly shortened when they were preincubated for 90 min before being added to oocytes. We conclude that membrane changes resembling capacitation (1) occur during cooling to temperatures above freezing, (2) are independent of cooling rate, (3) proceed faster at lower temperatures, and (4) obviate the need for prior capacitation in vitro before mixing with oocytes. Mol. Reprod. Dev. 46:318–324, 1997. © 1997 Wiley-Liss, Inc.     

Positive effect of partial zona pellucida digestion on in vitro fertilization of mouse oocytes with cryopreserved spermatozoa

Cryopreservation of mouse spermatozoa has been widely used; however, fertility of frozen spermatozoa in some strains, especially when inseminating cryopreserved oocytes, is low and may be improved by assisted fertilization techniques. The present study was performed to investigate the effect of partial zona pellucida (ZP) digestion on the in vitro fertilization (IVF) capacity of frozen mouse spermatozoa. Mouse oocytes were subjected to partial ZP digestion using acidic Tyrode's solution (pH 3.1). Fertilization rates in digestion groups (30 or 45 s) were higher (P < 0.05) than that of zona-intact control (78.3% or 86.3% vs. 52.5%). The recovery rate at 45 s was lower (P < 0.05) than that at 30 s (84.2% vs. 97.3%). Among vitrified oocytes, the fertilization rate in treatment group (digested for 30 s) was higher (P < 0.05) than that of zona-intact group (50.8% vs. 22.1%). After embryo transfer at the two-cell stage, 17.7% and 11.8% of transferred embryos derived from fresh and vitrified digested oocytes developed to term and showed no significant difference as compared with that from zona-intact oocytes (24.1%, P > 0.05). These results indicate that partial ZP digestion improves IVF efficiency of fresh and vitrified oocytes with frozen mouse spermatozoa, which can provide valuable information for in vitro assisted fertilization using cryopreserved gametes in the re-establishment of mouse colonies.     

Effects of cooling and warming rates during vitrification on fertilization of in vitro-matured bovine oocytes

The purpose of this study was to clarify the relationship of cooling rates (CR) and warming rates (WR) during vitrification with postwarming viability of in vitro-matured bovine oocytes. In Experiment 1, oocytes were vitrified in a solution containing 7.2 M ethylene glycol and 1.0 M sucrose by use of open-pulled glass capillaries with five different outer diameters and were warmed by placement of the capillaries into 0.25 M sucrose solution. The capillaries of 2000-, 1400-, 1000-, 630-, and 440-mm diameters provided CR of 2000, 3000, 5000, 8000, and 12,000 degrees C/min and WR of 5000, 8000, 17,000, 33,000, and 62,000 degrees C/min, respectively. In oocytes vitrified in capillaries of 1400-mm diameter (CR, 3000 degrees C/min; WR, 8000 degrees C/min), the morphological survival rate (86% of vitrified), penetration rate (79% of inseminated), and normal fertilization rate (69% of penetrated) were higher or tended to be higher than those in the other vitrification groups. In Experiment 2, oocytes cooled at 2000, 3000, or 12,000 degrees C/min were warmed at 8000 degrees C/min, and oocytes cooled at 3000 degrees C/min were warmed at 5000, 8000, or 33,000 degrees C/min. Among these CR-WR combinations, cooling of oocytes at 3000 degrees C/min regardless of the WR resulted in higher postwarming survival. These results indicate that survival of in vitro-matured bovine oocytes after vitrification and subsequent warming is improved by a slightly rapid cooling rate in open-pulled glass capillaries compared to that obtained in conventional straws.     

Ejaculate-hormonal traits in the leopard cat (Felis bengalensis) and sperm function as measured by in vitro penetration of zona-free hamster ova and zona-intact domestic cat oocytes

Electroejaculate traits and circulating follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone concentrations were analyzed in adult leopard cats (Felis bengalensis), a rare felid species indigenous to east Asia. The ability of leopard cat sperm to bind and penetrate zona-free hamster ova and zona-intact domestic cat oocytes in vitro was examined as a means of testing sperm function. The influence of culture media [Biggers, Whitten, Whittingham (BWW) vs. modified Krebs Ringer bicarbonate (mKRB)], seminal plasma removal, and swim-up separation on sperm motility, sperm morphology, and oocyte penetration also were assessed. Sperm treatments included dilution of raw semen (DR), ejaculate centrifugation, and either resuspension (NS) or swim-up processing (SU). The percentage of oocytes penetrated (penetration rate) and the number of penetrated sperm/oocyte (penetration index) were determined. Ejaculates from each male consisted of at least a 50% sperm motility rating, and hormone concentrations in individual males were unrelated to any ejaculate trait measured concurrently on the same day. The SU technique improved (P less than 0.05) percent sperm motility and the proportion of structurally normal sperm compared to DR and NS treatments. Leopard cat spermatozoa were capable of binding to and penetrating hamster ova and domestic cat oocytes; however, penetration was influenced by culture medium and seminal processing. In the hamster assay, a higher (P less than 0.05) penetration rate and penetration index were achieved when mKRB was used for gamete incubation instead of BWW. NS processing also increased (P less than 0.05) overall penetration compared to DR and SU. In the cat oocyte assay, zona penetration rate was similar (P greater than 0.05) in the DR, NS, and SU aliquots; however, the zona penetration index was increased (P less than 0.05) by the NS compared to the DR and SU treatments. This study 1) provides baseline ejaculate and endocrine norms for the leopard cat, 2) demonstrates that leopard cat sperm undergo nuclear decondensation in hamster ova and penetrate zona-intact domestic cat oocytes, 3) indicates that seminal plasma removal enhances leopard cat sperm fertilizing ability and ovum penetration, and 4) suggests that heterologous oocyte penetration is effective for assessing factors influencing fertilization and sperm function in this nondomestic felid.     

Cryopreservation of kunming mouse oocytes using slow cooling, ultrarapid cooling and vitrification protocols

The cryopreservation of oocytes has been only marginally successful with any of the current protocols, including slow cooling, rapid cooling and vitrification. We wished to test the hypothesis that oocytes from a single mouse strain would freeze successfully by 1 of the 3 mentioned protocols. Unfertilized Kunming mouse oocytes obtained 14 h after PMSG/hCG administration were randomly assigned to be cryopreserved after slow cooling, ultra rapid cooling and vitrification. Oocytes were thawed by straws being placed into 37 degrees C water, and their morphological appearance and in vitro fertilization capability were compared with that of oocytes that had not undergone cryopreservation. Survival of oocytes was indicated by the absence of darkened ooplasm or by broken membranes or zona pellucida. Functional integrity was evaluated by the formation of a 2-cell embryo after IVF. Survival rate of slow cooled oocytes did not differ from that seen in vitrified oocytes (55.1 vs 65.9%) but was significantly lower in the rapidly cooled oocytes (24.2%; P < 0.01). The results of IVF of slow cooled and vitrified oocytes were similar to those of the control group (72 and 73 vs 77%; P > 0.05). It appears that Kunming mouse oocytes can be successfully cryopreserved using the slow cooling method with 1,2-propanediol and vitrification, which contains both permeating and nonpermeating cryoprotectants.     

Inhibitors of glycoprotein biosynthesis block fertilization in the hamster

The nature and control of changes in surface carbohydrates in capacitating hamster spermatozoa were analysed by using five inhibitors of glycoprotein biosynthesis in an in vitro fertilization system. Epididymal spermatozoa were treated with amphomycin, bacitracin, tunicamycin, 2-deoxyglucose, and 2-deoxy-2-fluoro-D-glucose either during the entire period of capacitation or briefly at the end of capacitation before exposing to Con A-coated agarose beads or hamster eggs with or without their zonae pellucidae. Untreated 4½-5-hr spermatozoa exhibited nearly 100% fertilization and became bound to Con A-agarose beads mainly along the length of their flagellae, resulting in the formation of clumps on the beads. In the presence of inhibitors of glycosylation, spermatozoa did not bind to Con A-agarose beads or zona-intact oocytes and they did not fuse with the zona-free oocytes. Sperm-zona binding was also inhibited by UDP-galactose and UDP-N-acetylglucosamine, but not by UDP-glucose. Sperm motility was not damaged by these inhibitors, and zona-intact and zona-free oocytes pretreated with these inhibitors underwent normal fertilization with untreated spermatozoa. These results further strengthen the view that glycoproteins on the sperm surface may be required during different stages of fertilization, including sperm-egg fusion.     

Phase Transition Temperature and Chilling Sensitivity of Bovine Oocytes

A limiting factor for achieving cryopreservation of oocytes is direct chilling injury (DCI), which occurs during cooling. DCI, or cold shock, is defined as an irreversible damage expressed shortly after exposure to low, but not freezing, temperatures. The primary target of DCI is thought to be the plasma membrane. Recently, an association between DCI in sperm and the thermotropic phase transition of their membrane lipids was demonstrated. In the present study, we examined the phase transition of the membrane lipids of immature andin vitro-matured bovine oocytes during cooling, using Fourier transform infrared spectroscopy (FTIR). The phase transition of the membrane lipids of oocytes at the germinal vesicle (GV) stage occurred between 13 and 20°C, while a very broad phase transition, which centered around 10°C, was observed for mature oocytes (MII) stage. Thermotropic phase transitions were demonstrated to be related to the temperature at which DCI affected the integrity of the oocyte membranes. When immature oocytes were cooled to 13°C, fewer oocytes (40%) retained their membrane integrity than after exposure to 4°C (51%) or holding them at 38°C (78%), (as determined by the Fluorescein Diacetate-FDA test). This finding might suggest that holding immature oocytes at the phase transition temperature is more damaging to their membranes than exposure to lower temperatures. By contrast, no significant differences in membrane integrity were observed whenin vitro-matured oocytes were cooled to the same temperatures. Subsequently, GV oocytes were cooled to 4°C, and 26% underwent maturation and 19% underwent fertilizationin vitro. In vitro-matured oocytes that were cooled to 4°C displayed a slightly decreased rate of fertilization; the overall fertilization was 60% with 24% polyspermy, rather than the 76% fertilization rate with 12% polyspermy obtained with those not subjected to cooling. The high rate of polyspermy indicates that a site(s) other than the plasma membrane is affected during cooling of bovine oocytes. Nucleated bovine GV oocytes were electrofused within vitro-matured and enucleated oocytes, and then cooled to 4°C. Evaluation of the membrane integrity of the fused oocytes showed that these oocytes are chilling resistant, which strongly suggests that alteration of the membrane composition of an oocyte can change the cell's susceptibility to low temperatures. This finding led to an improvement in the survival of oocytes after cryopreservation.     

Cryopreservation of equine oocytes by 2-step freezing

Immature equine oocytes were frozen-thawed with ethylene glycol (EG), 1,2-propanediol (PD) or glycerol (GL) in PBS and cultured to assess the rate of in vitro maturation (Experiment 1). Compact-cumulus oocyte complexes were collected from slaughterhouse ovaries and equilibrated for 10 min in the freezing medium containing 10% (V/V) cryoprotectant and 0.1 M sucrose. The 0.25-ml straws, loaded with 10 to 30 oocytes, were seeded at -6 degrees C and cooled to -35 degrees C at 0.3 degrees C/min before being plunged into liquid nitrogen. The straws were thawed rapidly in a 37 degrees C waterbath for 20 sec. The proportions of frozen-thawed oocytes reaching Metaphase II (MII) stage after in vitro maturation of 32 h were 15.8% (EG), 5.8% (PD) and 0% (GL), while 63.3% of the nonfrozen control oocytes matured in vitro. The fertilizing ability of immature and mature oocytes after freezing in EG was tested by the insemination of zona-free oocytes with stallion spermatozoa (Experiment 2). Spermatozoa were preincubated for 3 h with 5 mM caffeine, treated with 0.1 mu M ionophore A23187, and inseminated for 20 h at the concentration of 1 to 2 x 10(7)/ml with 6 to 10 oocytes in 50 mu l of Brackett and Oliphant (BO) medium. Immature oocytes (Group 1) were matured in vitro after thawing and then their zona pellucida removed using 0.5% protease. The zona of mature oocytes were removed immediately after thawing (Group 2) or maturation (nonfrozen controls). The oocytes, which had mechanically damaged plasma membrane or lost by artifact, were not examined for insemination. Significantly more control oocytes exhibited a polar body at the time of insemination (53.5%) than either frozen-thawed immature or mature oocytes (25.8 and 27.3%, respectively). Similar proportion of frozen-thawed and control oocytes were penetrated by spermatozoa (71.8 to 79.1%) and exhibited 2 or more pronuclei (73.6 to 80.8%). The mean numbers of spermatozoa per penetrated oocyte were 1.9, 3.0 and 2.5, respectively, for Groups 1 and 2 and for the control oocytes. These results indicate that immature equine oocytes mature to the MII stage in vitro following freezing and thawing in EG or PD but not in GL. Stallion spermatozoa can penetrate zona-free immature and mature oocytes following freezing/thawing in EG and form morphologically normal pronuclei.     

Evaluation of fertility status of Murrah buffalo bulls in vitro using zona-free hamster eggs

Cryopreserved semen samples from 10 Murrah buffalo bulls were used for sperm penetration bioassay using zona-free hamster oocytes. The samples were evaluated for sperm motility, viability and acrosome integrity. Actively motile spermatozoa recovered by the swim-up technique were capacitated using calcium ionophore A(2 3 1 8 7). Mature female golden hamsters were superovulated with 50 IU PMSG followed 56 h later by 75 IU hCG. Cumulus mass, recovered by puncture of oviducts at the infundibulum region, was treated with 0.1% hyaluronidase and 0.1% trypsin to obtain zona-free oocytes. After coincubation of zona-free oocytes with capacitated buffalo spermatozoa, scoring was done as fertilization percentage and fertilization index. The correlation coefficients with conception rate were statistically significant with fertilization percentage (r = 0.588, P < 0.05) and fertilization index (r = 0.660, P < 0.01). However, conventional parameters like viability, motility and acrosome integrity showed poor correlation with conception rate.     

Sensitivity of domestic cat (Felis catus) sperm from normospermic versus teratospermic donors to cold-induced acrosomal damage.

Freeze-thawing cat sperm in cryoprotectant results in extensive membrane damage. To determine whether cooling alone influences sperm structure and viability, we compared the effect of cooling rate on sperm from normospermic (N; > 60% normal sperm per ejaculate) and teratospermic (T; < 40% normal sperm per ejaculate) domestic cats. Electroejaculates were divided into raw or washed (Ham's F-10 + 5% fetal calf serum) aliquots, with the latter resuspended in Ham's F-10 medium or Platz Diluent Variant Filtered without glycerol (20% egg yolk, 11% lactose). Aliquots were 1) maintained at 25 degrees C (no cooling; control), 2) cooled to 5 degrees C in a commercial refrigerator for 30 min (rapid cooling; approximately 4 degrees C/min), 3) placed in an ice slush at 0 degrees C for 10 min (ultrarapid cooling; approximately 14 degrees C/min), or 4) cooled to 0 degrees C at 0.5 degrees C/min in a programmable alcohol bath (slow cooling); and aliquots were removed every 4 degrees C. All samples then were warmed to 25 degrees C and evaluated for percentage sperm motility and the proportion of intact acrosomes using a fluorescein-conjugated peanut agglutinin stain. In both cat populations, sperm percentage motility remained unaffected (p > 0.05) immediately after exposure to low temperatures and after warming to 25 degrees C. However, the proportion of spermatozoa with intact acrosomes declined (p < 0.05) after rapid cooling ( approximately 4 degrees C/min) to 5 degrees C (N, 65.6%; T, 27.5%) or ultrarapid cooling ( approximately 14 degrees C/min) to 0 degrees C (N, 62.1%; T, 23.0%) in comparison to the control value (N, 81.5%; T, 77.5%). Transmission electron microscopy of cooled sperm revealed extensive damage to acrosomal membranes. In contrast, slow cooling (0.5 degrees C/min) to 5 degrees C maintained (p > 0.05) a high proportion of spermatozoa with intact acrosomes (N, 75.5%; T, 68.3%), which also remained similar (p > 0.05) between cat populations (N, 64.7%; T, 56.8%) through continued cooling to 0 degrees C. Results demonstrate that 1) rapid cooling of domestic cat sperm induces significant acrosomal damage without altering sperm motility, 2) spermatozoa from teratospermic males are more susceptible to cold-induced acrosomal damage than normospermic counterparts, and 3) reducing the rate of initial cooling markedly decreases sperm structural damage.     

Improvement on in vitro maturation, fertilization and development of minke whale (Balaenoptera acutorostrata) oocytes

The aims of the present study were to improve in vitro maturation, fertilization and subsequent development of minke whale oocytes. We investigated the effects of different concentrations (0, 10 and 20%) of fetal whale serum (FWS) in maturation medium on nuclear maturation, morphological grade (A or B) of cumulus-oocyte complexes (COC) obtained from prepubertal and adult minke whales. Grade A (> or = 5 layers of cumulus cells) COC collected from the adult whales and cultured in the medium with 20% FWS had a higher (P < 0.05) maturation rate (31.8%) than those in the medium without FWS (0%). Adding FWS to the maturation medium significantly (P < 0.01) improved the proportion of oocytes at Metaphase II (M-II): without FWS (7.9%), with 10% (19.4%) and 20% (21.4%) FWS. However, sexual maturity of whales and COC grades were not significantly affected by M-II oocytes. When in vitro fertilization of matured oocytes was performed in the presence of 20% FWS or 0.6% BSA in the fertilization medium, the proportions of sperm penetration and two-pronuclei formation in matured oocytes were not significantly different. Grade A COC cultured in a culture medium supplemented with 10% FWS cleaved at a higher rate (15.4%, P < 0.05) than did Grade A and B COCs cultured in the medium without FWS (0%). Neither Grade A nor B COCs cleaved when the medium was without FWS. The proportions of cleaved oocytes increased (P < 0.05) with FWS supplementation (6.9% and 8.1% for 1.0% FWS and 20% FWS, respectively). Grade A COC was significantly (P < 0.05) superior in its ability to cleave (14.5%) and develop to morula (4.2%) compared with that of the oocytes from Grade B COC (2.5% and 0%). Coculture with granulosa cells during in vitro culture did not significantly affect cleavage and development to the morula stage. These results indicate that FWS addition in the maturation medium improved the rate of in vitro maturation and cleavage after insemination of minke whale oocytes. The BSA supplementation in fertilization medium was as effective as FWS supplementation for in vitro fertilization of matured oocytes. In vitro embryo production beyond the morula stage of minke whale oocytes could be possible, if Grade A COC was selected and cultured in the maturation medium supplemented with 10% or 20% FWS.     

Optimum gas atmosphere for in vitro maturation and in vitro fertilization of bovine oocytes

The objective of this study was to determine optimal gas atmosphere conditions for in vitro maturation (IVM) and in vitro fertilization (IVF) of bovine oocytes. In Experiment 1, groups of 10 to 12 cumulus-oocyte complexes (COCs) were matured (24 h) and fertilized (18 h) under 1) 5% CO(2), 5% O(2;) 2) 5% CO(2), 10% O(2) or 3) 5% CO(2), 20% 0(2.) The COCs were cultured in 50 microl drops of maturation medium (TCM-199 + 10% bovine calf serum + oLH, oFSH and estrogen) or fertilization medium (TALP + swim-up separated spermatozoa +1 microg/ml heparin sulfate) under a layer of 10 ml paraffin oil at 39 degrees C with saturated humidity. Half of the oocytes in each drop were assigned randomly for maturation scoring and the remainder were inseminated. Reduced atmospheric O(2) drastically decreased proportions of oocytes reaching MII (71.4, 26.9 and 9.3% with 20, 10 and 5% O(2), respectively; P < 0.05). The percentages of total fertilization in 10 and 20% O(2) were similar and considerably higher than in 5% O(2) (80.3, 87.0 and 53.1%, respectively; P < 0.05). In addition, the percentage of polyspermy markedly increased when IVF was conducted in reduced O(2) (26.6 and 28.8% in 5 and 10% O(2) vs 15.4% in 20% O(2;) P < 0.05). Experiment 2 was similar to Experiment 1 except that CO(2) was the variable: 1) 2.5% CO(2) in air, 2) 5% CO(2) in air and 3) 10% CO(2) in air. The proportion of MII oocytes did not differ across treatments (64.9, 68.9 and 61.9%, respectively; P > 0.05). Although the percentages of total fertilization among treatments were not different (75.4, 80.9 and 76.1%, respectively), the proportion of normal fertilization was significantly reduced in 10% C0(2) (55.1%) when compared with that of either 2.5% CO(2) (62.7%) or 5% CO(2) (68.7%; P < .05). This study indicates that low O(2) is detrimental for IVM/IVF of bovine oocytes and that optimal atmospheric conditions are either 2.5 or 5% CO(2) and 20% O(2).     

Effects of cooling on meiotic spindle structure and chromosome alignment within in vitro matured porcine oocytes

Meiotic spindle structure and chromosome alignment were examined after porcine oocytes were cooled at metaphase II (M II) stage. Cumulus-oocyte complexes (COCs) collected from medium size follicles were cultured in an oocyte maturation medium at 39 degrees C, 5% CO(2) in air for 44 hr. At the end of culture, oocytes were removed from cumulus cells and cooled to 24 or 4 degrees C for 5, 30, or 120 min in a solution with or without 1.5 M dimethyl sulfoxide (DMSO). After being cooled, oocytes were either fixed immediately for examination of the meiotic spindle and chromosome alignment or returned to maturation medium at 39 degrees C for 2 hr for examination of spindle recovery. Most oocytes (65-71%) cooled to 24 degrees C showed partially depolymerized spindles but 81-92% of oocytes cooled at 4 degrees C did not have a spindle after cooling for 120 min. Quicker disassembly of spindles in the oocytes was observed at 4 degrees C than at 24 degrees C. Cooling also induced chromosome abnormality, which was indicated by dispersed chromosomes in the cytoplasm. Limited spindle recovery was observed in the oocytes cooled to both 4 and 24 degrees C regardless of cooling time. The effect of cooling on the spindle organization and chromosome alignment was not influenced by the presence of DMSO. These results indicate that the meiotic spindles in porcine M II oocytes are very sensitive to a drop in the temperature. Both spindle and chromosomes were damaged during cooling, and such damage was not reversible by incubating the oocytes after they had been cooled.     

Cryopreservation and ensuing in vitro fertilization ability of boar spermatozoa from epididymides stored at 4 degrees C

The influence of prolonged storage of boar epididymides on post-thaw sperm motility, and in vitro fertilization was evaluated. Twenty pairs of epididymides were obtained from Large White boars, and spermatozoa from one of each of the pairs were immediately collected and frozen (control group). The remaining epididymides were cooled to 4 degrees C and stored for 1, 2 or 3 d, after which spermatozoa were collected and frozen (experimental groups Day 1, 2 and 3, respectively). Sperm motility was maintained throughout the dilution procedure and then dropped (P < 0.01) after freezing and thawing. During storage the motility of nonfrozen spermatozoa decreased significantly (P < 0.01), reaching a value equal to that of frozen-thawed spermatozoa on Day 3. In vitro fertilization experiments revealed significantly (P < 0.05) lower penetration rates using Day 1, 2 and 3 stored spermatozoa (12, 13 and 2%, respectively) than that of the control group (40%). Oocyte penetration ability seemed to be reflected by acrosome integrity. However, the motility of spermatozoa with the ability to penetrate oocytes in Day 1 and Day 2 groups did not differ from that of the controls. The motility of spermatozoa lacking penetration ability, on the other hand, gradually decreased as the storage period was prolonged. This suggests that the sperm motility and penetration ability are affected by different mechanisms during the cold storage of epididymides. Finally, control and experimental groups exhibited high incidences of monospermic penetration (64 to 90%) and of male pronuclear formation (67 to 71%). These data suggest that cryopreservation of spermatozoa from boar epididymides stored at 4 degrees C for 1 to 2 d can be used for conserving male germ cells when epididymal spermatozoa can not be collected immediately and cryopreserved.     

Determination of temperature and cooling rate which induce cold shock in stallion spermatozoa

Experiments were conducted to determine temperatures between 24 and 4 degrees C at which stallion spermatozoa are most susceptible to cold shock damage. Semen was diluted to 25 x 10(6) spermatozoa/ml in a milk-based extender. Aliquots of extended semen were then cooled in programmable semen coolers. Semen was evaluated by computerized semen analysis initially and after 6, 12, 24, 36 and 48 hours of cooling. In Experiment 1A, semen was cooled rapidly (-0.7 degrees C/minute) from 24 degrees C to either 22, 20, 18 or 16 degrees C; then it was cooled slowly (-0.05 degrees C/minute) to a storage temperature of 4 degrees C. In Experiment 1B, rapid cooling proceeded from 24 degrees C to either 22, 19, 16, or 13 degrees C, and then slow cooling occurred to 4 degrees C. Initiating slow cooling at 22 or 20 degrees C resulted in higher (P<0.05) total and progressive motility over the first 24 hours of cooling than initiating slow cooling at 16 degrees C. Initiation of slow cooling at 22 or 19 degrees C resulted in higher (P<0.05) total and progressive motility over 48 hours of cooled storage than initiation of slow cooling at 16 or 13 degrees C. In Experiment 2A, semen was cooled rapidly from 24 to 19 degrees C, and then cooled slowly to either 13, 10, 7 or 4 degrees C, at which point rapid cooling was resumed to 4 degrees C. Resuming the fast rate of cooling at 7 degrees C resulted in higher (P<0.05) total and progressive motility at 36 and 48 hours of cooled storage than resuming fast cooling at 10 or 13 degrees C. In Experiment 2B, slow cooling proceeded to either 10, 8, 6 or 4 degrees C before fast cooling resumed to 4 degrees C. There was no significant difference (P>0.05) at most storage times in total or progressive motility for spermatozoa when fast cooling was resumed at 8, 6 or 4 degrees C. In Experiment 3, cooling units were programmed to cool rapidly from 24 to 19 degrees C, then cool slowly from 19 to 8 degrees C, and then resume rapid cooling to storage temperatures of either 6, 4, 2 or 0 degrees C. Storage at 6 or 4 degrees C resulted in higher (P<0.05) total and progressive motility over 48 hours of storage than 0 or 2 degrees C.     

Effects of low temperatures on in vitro produced bovine zygotes

The objective of this research was to investigate the effects of cooling on the development of bovine zygotes. One-cell bovine embryos were maintained at 39°C (control), 20°C, 10°C, or 0°C for 5, 10, or 20 minutes, then cultured in vitro for 7 days and the proportion of embryos developing to the compact morula or blastocyst stage compared between different treatments. Duration of exposure time had no effect on development. Development rates to the compact morula or blastocyst stage were 3.9%, 11.4%, 17.4%, and 24.4% for zygotes maintained at 0°C, 10°C, 20°C, and 39°C, respectively, with differences in embryo yield between every treatment (P < 0.05). In a second experiment, bovine pronuclei (karyoplasts) and cytoplasts were cooled at 0°C or maintained at 39°C for 5 minutes. Pronuclear transplantation was then utilized to create 4 types of reconstructed embryos, those with: 1) non-cooled pronuclei and non-cooled cytoplasm, 2) non-cooled pronuclei and cooled cytoplasm, 3) cooled pronuclei and non-cooled cytoplasm, and 4) cooled pronuclei and cooled cytoplasm. The proportion of embryos developing to the blastocyst stage was highest when non-cooled pronuclei were transferred into non-cooled cytoplasm (18.9%), and similar to that of non-cooled, non-manipulated control zygotes (13.2%, P > 0.05). No embryos developed to the blastocyst stage when pronuclei (cooled or non-cooled) were transferred into cooled cytoplasm. However, zygotes with cooled pronuclei transferred into non-cooled cytoplasm yielded 4.5% blastocysts (P < 0.05). More embryos developed to the compact morula or blastocyst stage when non-cooled vs. cooled cytoplasm was utilized, regardless of whether the pronuclei were cooled (P < 0.05). These data demonstrate that pronuclei are more tolerant to low temperature exposure than is ovum cytoplasm. Mol. Reprod. Dev. 47:435–439, 1997. © 1997 Wiley-Liss, Inc.     

Penetration in vitro of zona-free pig oocytes by homologous and heterologous spermatozoa

We examined the penetrability of pig, rat and bull spermatozoa into zona-free pig oocytes. Frozen-thawed boar spermatozoa penetrated into both zona-intact and zona-free oocytes with similar efficacy in a modified Tris-buffered medium (mTBM) supplemented with BSA and caffeine, but not in medium without caffeine. Rat epididymal spermatozoa did not readily penetrate into zona-free pig oocytes in mTBM with BSA. However, when a modified Krebs-Ringer bicarbonate solution was used, penetration rate varied with sperm concentrations at insemination: 79% of the oocytes were penetrated at 1.0 x 10(6) cells/ml, but very few at 0.1 x 10(6) and 10.0 x 10(6) cells/ml. In all oocytes penetrated, no activation was observed and the sperm nucleus was fully decondensed but did not transform into a male pronucleus. Frozen-thawed bull spermatozoa were also found to penetrate into zona-free pig oocytes in mTBM with BSA, caffeine and heparin: higher penetration rates were obtained with 1.0 x 106 and 10.0 x 10(6) spermatozoa/ml compared with 0.1 x 10(6) spermatozoa/ml. The penetration rate with 1.0 x 10(6) spermatozoa/ml was stable in five different bulls. All oocytes penetrated were activated and male pronuclear formation was observed in 57-79% of the penetrated oocytes. These results suggest that capacitation or the acrosome reaction is required for boar, rat, and possibly, bull spermatozoa to penetrate into zona-free pig oocytes. Bull spermatozoa can easily induce activation of pig oocytes and form male pronuclei, but rat spermatozoa cannot do so, indicating species differences in the ability of spermatozoa to activate pig oocytes and to transform to male pronuclei in the ooplasm.