Survival after transfer of “sexed” mouse embryos exposed to H-Y antisera

Immunological means were used to determine the sex of mouse embryos prior to transfer to pseudopregnant recipients. Antisera to histocompatibility-Y (H-Y) antigen were prepared in adult C57BL/6 female mice by repeated intraperitoneal injections of spleen cells from males of the same strain. Eight-to 16-cell embryos were cultured in BMOC-3 alone or BMOC-3 without bovine serum albumin to which one of the following had been added: H-Y antiserum and normal guinea pig serum (NGPS), NGPS alone, normal mouse serum alone or normal mouse serum and NGPS. After 24 hr of culture, embryos were classified as either affected or unaffected. An embryo was classified as affected if degeneration of the embryo or breakdown of one or more cells was observed. A total of 1000 embryos were cultured in BMOC-3 with H-Y antiserum and NGPS (treated embryos). Two hundred and fifty embryos were cultured in each of the other four media (control embryos). Eighty-seven (9%) of the control embryos and 479 (48%) of the treated embryos were classified as affected after culture. Unaffected embryos, approximately 12 each, were transferred to pseudopregnant recipients. One-hundred forty control embryos (17%) survived to term with 67 females (48%) and 73 males (52%) born. Fifty-eight treated embryos (14%) survived to term, producing 50 females (86%) and 8 males (14%). Percentage of females from embryos cultured in antiserum was greater than for embryos cultured in any other media (P<0.001). These results demonstrate that detection of H-Y antigen on preimplantation embryos may be a useful and effective method of determining sex of an embryo prior to transfer.     

Expression of a male-specific factor on various stages of preimplantation bovine embryos

Four-cell to blastocyst stage bovine embryos were collected from superovulated donors and cultured for 90 min in Ham's F-10 medium (HF-10) containing 10% (V/V) absorbed anti-histocompatibility (H)-Y antiserum. Embryos were then washed 3 times and placed in HF-10 supplemented with 10% (V/V) fluorescein isothiocynate (FITC)-conjugated goat anti-mouse gamma globulin. After an additional wash, embryos were placed in fresh drops of HF-10, individually evaluated at 200 X magnification, and classified as either fluorescent (H-Y-positive) or nonfluorescent (H-Y-negative). Embryos were then placed in drops of HF-10 containing 14% vinblastin and cultured for 4-6 h. Embryos were coded and individually karotyped, and the sex chromosomes were identified. H-Y antigen was detected as early as the eight-cell stage, but not at the four-cell stage. Seventy-nine percent of fluorescent embryos and 89% of nonfluorescent embryos were XY and XX, respectively. Another experiment was carried out in which H-Y antigen was detected on intact inner cell masses (ICM) isolated by immunosurgery from expanded blastocysts that also had been assayed for H-Y antigen. Eighty-eight and 92%, respectively, of ICM classified as fluorescent or nonfluorescent had been scored the same as intact blastocysts. It is concluded from these data that H-Y antigen can be detected on eight-cell to blastocyst stage bovine embryos. There appears to be a localization of detectable antigen in the area of the ICM at the expanded blastocyst stage. Detection of H-Y antigen is an effective, noninvasive method for identification of the sex of preimplantation bovine embryos.     

Cytolytic and fluorescent detection of H-Y antigen on preimplantation mouse embryos

Monoclonal antibodies to histocompatibility (H-Y) antigen, of IgM subclass, were used to immunologically determine the sex of mouse embryos prior to transfer to pseudopregnant recipients. Two experiments were performed, one using cytolysis of H-Y positive embryos and the other using binding of a Fluorescein Isothiocyanate-(FITC) labeled second antibody. Eight- to 16-cell embryos used in the cytolytic assay were cultured in Whitten's Medium without bovine serum albumin (WM), to which monoclonal antibody and normal guinea pig serum were added. Embryos were classified as affected or unaffected, based on morphology of the embryo and its blastomeres. A total of 550 embryos were cultured; 294 (53.5%) were scored as unaffected and 263 of these were transferred to recipients. Forty-three (81.1%) of 53 pups born were female. Morulae and early blastocysts were used in the FITC-labeled second antibody assay. Embryos were cultured in WM containing monoclonal antibody, washed and placed in drops of WM containing FITC-labeled anti-IgM. Following another wash embryos were individually evaluated at 200X for fluorescence. Fifty-five percent (169 of 305) of the embryos displayed cell-specific fluorescence. A total of twenty-three pups, 18 males (78.3%) and five females (21.7%), were born following transfer of 156 fluorescing embryos. Four male (17.4%) and nineteen female (82.6%) pups resulted from embryos classified as non-fluorescing.     

Evaluation of the presence of a specific histocompatibility protein on equine embryonic cells

An indirect immunofluorescence assay was used to detect the presence of H-Y antigen on equine blastocysts. A total of 33 blastocyst stage horse embryos were collected 6 to 7 days post-ovulation by trans-cervical flush and were immediately evaluated for the presence of H-Y antigen. Additionally, 17 embryos, were collected and cultured for 72 h to the expanded blastocyst stage and similarly evaluated. Embryos were placed in medium containing monoclonal antibodies to H-Y antigen followed by incubation in medium containing 1/10 (v/v) fluorescein isothiocyanate conjugated goat anti-mouse IgM Fc specific antiserum. Embryos were individually evaluated at 400X to identify cell specific fluorescence. Following evaluation, embryonic sex was independently verified with karyotypes to identify sex chromosomes. Of the 50 embryos evaluated, 29 were evaluated as non-fluorescent and 21 fluorescent. Expression of H-Y antigen was determined to be uniform in those embryos classified as fluorescent. Twenty-three of 28 (82%) readable karyotypes corresponded to the predicted sex. These results indicate a specific histocompatibility antigen is expressed and maintained at the blastocyst stage of development. In addition, no segregation of this protein on specific cell types occurs in this species.     

Effect of estrous cow serum during bovine embryo culture on blastocyst development and cryotolerance after slow freezing or vitrification

The present study investigated the effect of estrous cow serum (ECS) during culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. Embryos were derived from in vitro maturation (IVM) and in vitro fertilization (IVF) of abbatoir-derived oocytes. At Day 3, embryos were cultured in three different media: Charles Ronsenkrans medium + amino acids (CR1aa; without bovine serum albumin (BSA)) + 5% estrous cow serum (CR1-ECS), CR1aa + 3 mg/mL BSA (CR1-BSA) or CR1aa + 5% ECS + 3 mg/mL BSA (CR1-ECS-BSA). At 7.5 d post-insemination (PI), blastocyst yield and quality were evaluated; blastocysts and expanded blastocysts from each media were cryopreserved by Open Pulled Straw (OPS) vitrification method or slow freezing (1.5 M ethylene glycol, EM). Total blastocyst yield did not differ among CR1-ECS, CR1-BSA and CR1-ECS-BSA (30.9, 33.1 and 32.9%, respectively, P < 0.05). Embryo survival (hatching rate) was higher in vitrified versus slow-frozen embryos (43% versus 12%, respectively, P < 0.01), and in embryos cultured in CR1-BSA (40.3%) compared with those cultured in serum-containing media (CR1-ECS, 21.5% and CR1-ECS-BSA, 19.8%; P < 0.01). In conclusion: (a) it was possible to produce in vitro bovine embryos in serum-free culture medium without affecting blastocyst yield and quality; (b) serum-free medium produced the best quality embryos (in terms of post-cryopreservation survival); and (c) vitrification yielded the highest post-cryopreservation survival rates, regardless of the presence of serum in the culture medium.     

Viability of cloned bovine embryos after one or two cycles of nuclear transfer and in vitro culture

We described an exclusively in vitro procedure for cloning and recloning bovine embryos. Embryos obtained by IVM/IVF/IVC developed to the morula stage were used as blastomere donors in cunjunction with IVM recipient oocytes. Reconstructed embryos were developed in vitro in co-culture using bovine oviductal epithelial cells. The resulting morulae were used as donors for recloning under the same experimental conditions. No significant difference was observed between cloning and recloning in terms of development (rates of blastocysts: 12.9 versus 14.9%), in the number of nuclei per blastocyst (63.8 versus 49.1), or in pregnancy rates (35.7 versus 33.3%). The high variability observed between replicates and the correlation between results in first and second cycle nuclear transfer may suggest an inherant potential of individual donor embryos to support development by cloning.     

Embryo sex selection by a rat male-specific antibody and the cytogenetic and developmental confirmation in cattle embryos

Embryos of mouse, rabbit, goat, sheep, and cattle were separated into 2 groups on the basis of their morphology when incubated with a male-specific antibody (qualified here as the H-Y antibody) prepared from newborn rat testis. When morula-stage embryos were cultured in the presence of this H-Y antibody, the development of roughly one half of the embryos was arrested at that stage, whereas the other half continued to develop to the blastocyst stage. The developmentaly arrested group of embryos resumed their development into blastocysts when cultured in antibody-free medium. Eighty to 90% of cattle embryos whose development was unaffected by the antibody were shown to possess a female karyotype (XX), and close to 80% of those embryos whose development was arrested possessed a male karyotype (XY). Cattle embryos whose sex had been presumptively identified by development in the presence of the H-Y antibody were cryopreserved and transferred, and the sex of the calves was examined. The overt sex of the young born from sexed embryos was found to be the same as that determined by chromosomal analysis. © 1993 Wiley-Liss, Inc.     

Development of in vitro fertilized oocytes from pregnant and nonpregnant cows in oviductal epithelial and cumulus cell co-culture systems

The objectives of this study were 1) to measure cleavage, blastocyst formation, and blastocyst hatching after in vitro maturation (IVM), fertilization (IVF) and culture (IVC) of oocytes aspirated from pregnant versus nonpregnant cows, and 2) to compare embryo development in co-culture with bovine oviductal epithelial cells versus cumulus cells. No differences in cleavage (38 versus 40%), blastocyst formation (13 versus 13%), or blastocyst hatching (53 versus 51%) were observed for in vitro-matured, fertilized, and cultured oocytes from pregnant versus nonpregnant cows, respectively (P > 0.05), indicating that nonpregnant and early-pregnant cows are equally acceptable donors of oocytes for IVM/IVF/IVC procedures. Cleavage (36 versus 40%), blastocyst formation (11 versus 12%), and blastocyst hatching (50 versus 55%) were not different for embryos co-cultured with oviductal epithelial cells versus cumulus cells (P > 0.05). Thus, equivalent embryo development can be obtained with co-culture systems commonly used for in vitro-derived bovine embryos. These results help to define variables that affect comparison of results across laboratories and that are relevant to the practical application of IVM/IVF/IVC procedures to cattle.     

Heat stress-induced apoptosis in porcine in vitro fertilized and parthenogenetic preimplantation-stage embryos

Decades worth of research have consistently shown the adverse effects of elevated temperatures on reproductive parameters of livestock species. The objective of this study was to evaluate the developmental and apoptotic responses of porcine in vitro fertilized (IVF) and parthenogenetically activated (PA) embryos heat stressed at the late 1-cell stage. Embryos were heat stressed (HS) at 42 degrees C for 9 hr starting 22 hr after insemination or artificial activation stimulus. Non heat-stressed (NHS) control embryos were maintained at 39 degrees C for the duration of the experiments. TUNEL staining on Day 5 of development demonstrated that heat stress elicited a significant apoptotic response in IVF embryos (45.6% of HS embryos and 26.7% of NHS embryos were apoptotic; P<0.05), but not in PA embryos (51.1% and 39.9% for HS and NHS embryos, respectively; P>0.1). And, while IVF embryos were highly susceptible to heat-induced developmental perturbations (20.6% and 8.8% development to blastocyst for NHS and HS embryos, respectively; P<0.05), elevated temperatures did not affect blastocyst rates in PA embryos (22.2% for NHS PA embryos and 21.2% for HS PA embryos; P>0.1). These findings indicate that, as in other systems studied, IVF pig embryos are directly affected adversely by heat stress conditions. Parthenogenetic embryos, though, appear to be surprisingly tolerant of the elevated temperatures. The differences between IVF and PA embryos in their response to heat stress warrants further investigation.     

Survival rates and sex ratio of bovine IVE embryos frozen at different developmental stages on day 7

Two experiments were designed to determine the effects of stage of development on Day 7 of in vitro-produced bovine embryos on survival after deep freezing and on sex ratio. Bovine IVF embryos and bovine oviductal epithelial cells (BOEC) were co-cultured in TCM-199 and, on Day 7 after insemination (Day 0), were morphologically evaluated and divided into groups by developmental stage. In Experiment 1, embryos classified as early blastocysts, blastocysts and full-expanding blastocysts were randomly subdivided into 2 groups by replicate: 50% of the embryos were placed immediately in a new BOEC co-culture (fresh group), while the other 50% were frozen, thawed and placed in a new BOEC co-culture (frozen/thawed group). Embryos were frozen in 1.5 M glycerol using a standard slow cooling technique. Fresh and frozen/thawed embryos were compared for survival rate (embryos hatching/hatched) in BOEC co-culture over the following 3 d (i.e., Days 7 to 10). The overall survival of the 425 embryos (early to full-expanding blastocysts) was 33% and was not different between fresh (35%) and frozen/thawed (30%) embryos. Survival of embryos cultured fresh or after freezing/thawing was higher for full-expanding blastocysts than for early blastocysts or for blastocysts, both of which were not different. In Experiment 2, all frozen/thawed embryos used in Experiment 1 plus all morulae and hatched blastocysts collected and frozen on Day 7 without regard to survival were sexed utilizing the polymerase chain reaction (PCR) technique. Sex of the embryos, by stage of development on Day 7, was determined in order to compare the rate of development in BOEC co-culture with the sex ratio (percentage of males). A total of 235 embryos was sex-determined with an overall percentage of males of 51%, which was not different from the expected 1:1 sex ratio. Both full-expanding blastocysts and hatched blastocysts had a significantly higher (P < 0.05) proportion of males (68 and 100%, respectively), while morulae had a significantly lower proportion of males (24%). Early blastocysts and blastocysts did not differ from a 1:1 sex ratio. The results indicate that male embryos develop faster in vitro than female embryos. The higher survival rate of full-expanding blastocysts after freezing/thawing, and the production of a higher number of males than females among embryos of this developmental stage suggest that a greater number of male fetuses may result from the successful freezing and transfer of in vitro-produced bovine embryos.     

Identification of a male-specific histocompatibility protein on preimplantation porcine embryos

An indirect immunofluorescence assay was used to detect the presence of male-specific protein(s) on various stages of preimplantation porcine embryos. Embryos were collected at slaughter from the reproductive tracts of day?2.5, ?4, ?5, ?6, and ?8 (day 0 = first day of estrus) sows and gilts. Embryos were placed in medium containing an anti-male primary antibody, washed, and transferred to culture drops containing a fluorescein isothiocyanate (FITC)-labeled secondary antibody. Embryos were classified as either fluorescent (H-Y positive) or nonfluorescent (H-Y negative), transferred to coded drops, and karyotyped to examine sex chromosomes. A total of 91 eight-cell to blastocyst stage embryos were evaluated; of these, 46% were classified as fluorescent and 54% as nonfluorescent. Of readable metaphase spreads (65%) from these embryos, 81% (48 of 59, P < 0.005) were correctly sexed by immunological detection of the male-specific antigen. Although 13 % (2/15)of four-cell embryos evaluated were classified as fluorescent, the accuracy with which embryos at this stage were sexed by detection of H-Y antigen was not different from 50%. Fifty percent of eight-cell embryos were classified as H-Y positive with 78% of embryos correctly sexed. It was concluded that the eight-cell embryo is the earliest stage of development for which there is evidence for expression of H-Y antigen. Detection of the male-specific protein was difficult at the expanded blastocyst stage.     

Effect of cysteamine supplementation of in vitro matured bovine oocytes on chilling sensitivity and development of embryos

In vitro techniques for production of bovine embryos including in vitro oocyte maturation (IVM), fertilization (IVF) and culture (IVC) are becoming increasingly employed for a variety of research purposes. However, decreased viability following cryopreservation by conventional methods has limited commercial applications of these technologies. A practical alternative to facilitate transport would be to arrest development by chilling without freezing. The present research was undertaken to evaluate chilling sensitivity of IVM-IVF embryos at different stages of development, and to determine possible beneficial effects of cysteamine treatment during IVM, previously shown to enhance embryo development in culture, on survival following chilling at different stages. Embryos produced by standard IVM-IVF-IVC methods were chilled to 0 degrees C for 30 min at 2-cell (30-34 h post-insemination, hpi), 8-cell (48-52 hpi) or blastocyst (166-170 hpi) stages. Viability after chilling was assessed by IVC with development to expanded blastocyst stage determined on days 7 and 8 post-insemination (pi) and hatching blastocyst stage determined on days 9 and 10 pi. Control embryos at the same stages were handled similarly, but without chilling, and development during culture similarly assessed. The effect of cysteamine supplementation (100 microM) of the IVM medium was determined for both chilled and non-chilled (control) embryos. Cysteamine supplementation during IVM had no significant effect on oocyte maturation or fertilization, but increased the proportions of oocytes developing to blastocyst stage by day 7 (13.7+/-0.9% versus 7.2+/-0.9%; P<0.05), total blastocysts (20.5+/-0.9% versus 15.3+/-1.3%; P<0.05), and hatching blastocysts (16.8+/-1.6% versus 12.0+/-1.5%; P<0.05). The greater survival in terms of hatching (78.6+/-7.0) following chilling of blastocysts produced by IVM-IVF of oocytes matured in media supplemented with cysteamine offers promise for applications requiring short-term storage to facilitate transport of in vitro produced bovine embryos.     

Development of early bovine embryos in co-culture with KSOM and taurine, superoxide dismutase or insulin

Three experiments, utilizing 2578 embryos, were designed to test the effects of media, taurine, Superoxide dismutase and insulin on the development of embryos produced by in vitro maturation and in vitro fertilization (IVM/IVF). Embryos showing at least 1 cleavage during culture for 40 to 44 h after IVM/IVF were selected for further culture under various conditions for 6 d at 39 degrees C in 5% C0(2):95% air. A Buffalo rat liver (BRL) cell co-culture was used in all 3 experiments. Experiment 1 was a 3 x 2 factorial arrangement with KSOM (a high potassium simplex optimization-derived medium containing only 12 ingredients), Menezo B(2) and TCM-199 media with or without 7 mM taurine. Blastocyst production in the 3 media, respectively, was 48, 36 and 29% (P<0.05). Addition of 7 mM taurine increased the percentage of blastocysts from 34 to 42 (P<0.05). In Experiment 2, Superoxide dismutase (SOD) did not improve blastocyst development (P>0.05). In Experiment 3, insulin (75 ng/ml) added to KSOM resulted in 46% morulae plus blastocysts compared with 35% for the control (P<0.05). These results indicate that the co-culture of embryos in KSOM with taurine or insulin added is superior to commonly used complex media for efficient production of blastocysts following IVM/IVF.     

Aberrant allocations of inner cell mass and trophectoderm cells in bovine nuclear transfer blastocysts

Abortions of nuclear transfer (NT) embryos are mainly due to insufficient placentation. We hypothesized that the primary cause might be the aberrant allocations of two different cell lineages of the blastocyst stage embryos, the inner cell mass (ICM) and the trophectoderm (TE) cells. The potential for development of NT embryos to blastocysts was similar to that for in vitro fertilized (IVF) embryos. No difference in the total cell number was detected between NT and IVF blastocysts, but both types of embryos had fewer total cells than did in vivo-derived embryos (P < 0.05). The NT blastocysts showed a higher ratio of ICM:total cells than did IVF or in vivo-derived embryos (P < 0.05). Individual blastocysts were assigned to four subgroups (I: <20%, II: 20-40%, III: 40-60%, IV: >60%) according to the ratio of ICM:total cells. Most NT blastocysts were placed in groups III and IV, whereas most IVF and in vivo-derived blastocysts were distributed in group II. Our findings suggest that placental abnormalities or early fetal losses in the present cloning system may be due to aberrant allocations of NT embryos to the ICM and TE cells during early development.     

Relationship between sex ratio and time of insemination according to both time of ovulation and maturational state of oocyte

The aim of this study was to explore how some reproductive methodologies may affect the sex ratio. We first confirmed the association between the maturation stage of bovine oocytes at the time of in vitro fertilisation (IVF) and the sex ratio of in vitro-derived embryos. Secondly, we studied whether the time of insemination, prior to or after ovulation, could alter the sex ratio in sheep. In the first experiment, bovine oocytes were matured in vitro for 16 h; then oocytes were either fertilised in vitro immediately after extrusion of the first polar body or IVF was delayed for 8 h. The proportion of cleaving embryos and their development to the 8-cell stage was enhanced with delayed insemination. Moreover, delaying IVF produced a male-to-female sex ratio of 1.67:1.00, which was significantly different from the expected 1:1 ratio (p < 0.05), whereas more female embryos were produced when oocytes were fertilised in vitro immediately after polar body extrusion (sex ratio of 1.00:0.67; p < 0.05). In the second experiment, 380 ewes were inseminated at different times before or after ovulation, producing 537 lambs. Significant differences in the sex ratio were obtained when we compared the sex of the offspring of ewes inseminated during the 5 h preceding ovulation (more females) with those inseminated during the 5 h after ovulation (more males). Our results suggest that the differential ability of X- or Y-bearing spermatozoa to fertilise oocytes depending either on time of insemination or oocyte maturation state, may be due, at least partially, to 'intrinsic' differences in the physiological activity of X- or Y-bearing spermatozoa before fertilisation.     

Co-culture of day-5 to day-7 equine embryos in medium with oviductal tissue

Oviductal and uterine embryos were collected from mares at 5 to 7 days following ovulation 1) to evaluate the effects of oviductal tissue explants on in vitro growth and development of equine embryos and 2) to study the morphologic development of equine embryos in culture. Embryos were incubated for 5 days in a medium (control group) or in medium supplemented with oviductal tissue explants (co-culture group). Embryos were evaluated and the media changed daily. Following 5 days in culture, 10 10 (100%) control embryos and 27 29 (93%) co-cultured embryos had doubled in diameter. All embryos that were recovered as morulae developed to the blastocyst stage in culture. By 5 days in culture, 6 10 (60%) control embryos and 19 29 (66%) co-cultured embryos had reached the hatching blastocyst stage of development. By 3 days in culture, significantly more (P<0.05) control embryos versus co-cultured embryos had degenerated (4 10 vs 2 29 , respectively). By 5 days in culture, significantly more (P<0.01) control embryos versus co-cultured embryos had degenerated (6 10 vs. 3 29 , respectively). Embryos cultured with oviductal tissue were sustained longer than embryos cultured in medium alone. Hatching was characterized by the blastocyst squeezing through a small opening in the zona pellucida or by the zona pellucida thinning over approximately half of the blastocyst surface and subsequently disappearing entirely.     

Sucrose-induced shrinkage of in vitro produced bovine morulae: effect on viability, morphology and ease of evaluation

Sucrose (0.3 M) was used to cause artificial compaction of the embryonic cell mass of in vitro produced bovine embryos to facilitate morphological evaluation. Embryos were produced using routine in vitro maturation (IVM) and fertilization (IVF) techniques. The time necessary to induce shrinkage in 0.3 M sucrose to 75% of the original volume of Day 5 morulae was found to be less than l min, and 95% of the volume was regained in PBS after 2.5 min. No detrimental effect was observed after a 5- to 10-min sucrose treatment on subsequent blastocyst formation at Days 6 and 7 (P > 0.05). Furthermore, no significant differences were observed in the total number of cells, or in the mitotic and pycnotic cell index of blastocysts in different treatment groups. Agreement among 7 evaluators grading 40 Day 6 embryos was examined using the kappa coefficient of agreement (kappa). Overall agreement among evaluators for classification of quality grade was poor (48.2 %, kappa = 0.31) for embryos evaluated in PBS, but the rate improved when the same embryos were scored in sucrose (62.5 %, kappa = 0.49). Evaluating less compact in vitro produced bovine morulae in sucrose increases agreement among evaluators, since embryos in sucrose mimick the appearance of in vivo produced embryos. Thus, we conclude that scoring in vitro produced embryos in sucrose improves agreement among evaluators.     

Effect of Percoll volume, duration and force of centrifugation, on in vitro production and sex ratio of bovine embryos

The objective was to evaluate the effect of Percoll volume, and duration and force of centrifugation on sperm quality characteristics, embryo development, and sex ratio of in vitro-produced (IVP) bovine embryos. Frozen-thawed semen from four bulls were submitted to three Percoll procedures: T1—4 mL of Percoll, centrifuged for 20 min at 700 g; T2—800 μL of Percoll, centrifuged for 20 min at 700 g; and T3—800 μL of Percoll, centrifuged for 5 min at 5000 g. Sperm total motility, morphology and integrity of the sperm acrosome, membrane and chromatin were determined before and after Percoll treatment, and semen was used for in vitro fertilization (IVF) of in vitro-matured oocytes. All Percoll methods increased the proportion of motile sperm (P < 0.05). There were no significant effects of treatment for any sperm characteristic; however, for every end point, there were significant differences among bulls. Similarly, rates of cleavage and blastocyst formation were not affected by the Percoll procedure (P > 0.05), but were affected by sire (P < 0.05). Sex ratio was similar among treatments for Bulls 2 and 3, whereas semen from Bull 1 processed by T1 yielded a greater percentage of male embryos. However, when only treatments were considered, independent of bulls, the proportion of male:female embryos did not differ significantly from an expected 1:1 ratio. In conclusion, decreasing Percoll volume, reducing duration of centrifugation, and using a higher force of centrifugation did not significantly affect sperm quality, embryo development, or sex ratio of in vitro-produced bovine embryos.