Prediction of pregnancy viability in bovine in vitro-produced embryos and recipient plasma with Fourier transform infrared spectroscopy

We analyzed embryo culture medium (CM) and recipient blood plasma using Fourier transform infrared (FTIR) metabolomics to predict pregnancy outcome. Individually cultured, in vitro-produced (IVP) blastocysts were transferred to recipients as fresh and vitrified-warmed. Spent CM and plasma samples were evaluated using FTIR. The discrimination capability of the classifiers was assessed for accuracy, sensitivity (pregnancy), specificity (nonpregnancy), and area under the receiver operator characteristic curve (AUC). Within all IVP fresh embryos (birth rate = 52%), high AUC were obtained at birth, especially with expanded blastocysts (CM: 0.80 ± 0.053; plasma: 0.89 ± 0.034). The AUC of vitrified IVP embryos (birth rate = 31%) were 0.607 ± 0.038 (CM, expanded blastocysts) and 0.672 ± 0.023 (plasma, all stages). Recipient plasma generally predicted pregnancy outcome better than did embryo CM. Embryos and recipients with improved pregnancy viability were identified, which could increase the economic benefit to the breeding industry.     

Influence of the uterine environment on the development of in vitro-produced equine embryos

The necessity for early interaction between the embryo and the oviductal and/or uterine environment in the horse is reflected by several striking differences between equine embryos that develop in vivo and those produced in vitro. Better understanding of the salient interactions may help to improve the efficiency of in vitro equine embryo production. In an initial experiment, cleavage-stage in vitro-produced (IVP) equine embryos were transferred into the uterus of recipient mares that had ovulated recently to determine whether premature placement in this in vivo environment would improve subsequent development. In a second experiment, an important element of the uterine environment was mimicked by adding uterocalin, a major component of the endometrial secretions during early pregnancy, to the culture medium. Intrauterine transfer of cleavage-stage IVP equine embryos yielded neither ultrasonographically detectable pregnancies nor day 7 blastocysts, indicating that the uterus is not a suitable environment for pre-compact morula stage horse embryos. By contrast, exposure to uterocalin during IVP improved capsule formation, although it did not measurably affect the development or expression of a panel of genes known to differ between in vivo and in vitro embryos. Further studies are required to evaluate whether uterocalin serves purely as a carrier protein or more directly promotes improved capsule development.     

Birth of pups after transfer of mouse embryos derived from vitrified preantral follicles

Preantral follicles mechanically isolated from the ovaries of 12-day-old mice were exposed to 2 mol ethylene glycol l(-1) for 2 or 5 min and then to a vitrification solution containing 6 mol ethylene glycol l(-1) and 0.3 mol raffinose l(-1) for 0.5, 1.0 or 2.0 min before vitrification. The vitrified and fresh preantral follicles were treated with collagenase, and the oocyte-granulosa cell complexes (OGCs) obtained were cultured in vitro for 10 days in membrane inserts. Preantral follicles exposed to 2 mol ethylene glycol l(-1) for 5 min and then to the vitrification solution for 0.5 or 1.0 min showed the highest survival rates after warming. The follicular loss after warming was approximately 20%. After in vitro culture, the proportion of viable OGCs from the vitrified follicles was 10% lower than that of the fresh preantral follicles. There were no differences in the rates of maturation, fertilization and subsequent development to blastocysts between the oocytes derived from vitrified follicles and those derived from fresh preantral follicles; however, the developmental competence of the oocytes derived from both vitrified and fresh preantral follicles grown in vitro was lower than that of oocytes grown in vivo. One of the five recipient mice that received 20 blastocysts derived from vitrified preantral follicles gave birth to six live pups. The results of the present study demonstrate for the first time that mouse preantral follicles can be vitrified and that some of the embryos derived from vitrified preantral follicles can develop to live pups.     

Biomarker metabolite mating of viable frozen-thawed in vitro-produced bovine embryos with pregnancy-competent recipients leads to improved birth rates

Selection of competent recipients before embryo transfer (ET) is indispensable for improving pregnancy and birth rates in cattle. However, pregnancy prediction can fail when the competence of the embryo is ignored. We hypothesized that the pregnancy potential of biomarkers could improve with information on embryonic competence. In vitro-produced embryos cultured singly for 24 h (from d 6 to 7) were transferred to d 7 synchronized recipients as fresh or after freezing and thawing. Recipient blood was collected on d 0 (estrus; n = 108) and d 7 (4–6 h before ET; n = 107) and plasma was analyzed by nuclear magnetic resonance (¹H⁺NMR). Spent embryo culture medium (CM) was collected and analyzed by ultra-high-performance liquid chromatography tandem mass spectrometry in a subset of n = 70 samples. Concentrations of metabolites quantified in plasma (n = 35) were statistically analyzed as a function of pregnancy diagnosed on d 40, d 62 and birth. Univariate analysis with plasma metabolites consisted of a block study with controllable fixed factors (i.e., embryo cryopreservation, recipient breed, and day of blood collection; Wilcoxon test and t-test). Metabolite concentrations in recipients and embryos were independently analyzed by iterations that reclassified embryos or recipients using the support vector machine. Iterations identified some competent embryos, but mostly competent recipients that had a pregnancy incompetent partner embryo. Misclassified recipients that could be classified as competent were reanalyzed in a new iteration to improve the predictive model. After subsequent iterations, the predictive potential of recipient biomarkers was recalculated. On d 0, creatine, acetone and l-phenylalanine were the most relevant biomarkers at d 40, d 62, and birth, and on d 7, l-glutamine, l-lysine, and ornithine. Creatine was the most representative biomarker within blocks (n = 20), with a uniform distribution over pregnancy endpoints and type of embryos. Biomarkers showed higher abundance on d 7 than d 0, were more predictive for d 40 and d 62 than at birth, and the pregnancy predictive ability was lower with frozen-thawed (F-T) embryos. Six metabolic pathways differed between d 40 pregnant recipients for fresh and F-T embryos. Within F-T embryos, more recipients were misclassified, probably due to pregnancy losses, but were accurately identified when combined with embryonic metabolite signals. After recalculation, 12 biomarkers increased receiver operator characteristic-area under the curve (>0.65) at birth, highlighting creatine (receiver operator characteristic-area under the curve = 0.851), and 5 new biomarkers were identified. Combining metabolic information of recipient and embryos improves the confidence and accuracy of single biomarkers.     

Respiration rates of individual bovine in vitro-produced embryos measured with a novel, non-invasive and highly sensitive microsensor system

Oxygen consumption is a useful parameter for evaluating embryo quality, since it provides a valuable indication of overall metabolic activity. Over the years, several approaches have been used to measure the respiration rates of individual embryos, but a convincing method has not yet been reported. In this study, we introduce and have validated a novel high resolution microsensor technology to determine the respiration rates of individual embryos at different developmental stages. We have employed this technology to investigate the correlation between respiration rate and embryo morphology, diameter and sex. Following morphological evaluation, individual respiration rates of day 3 (n = 18) and day 7 (n = 60) bovine in vitro-produced embryos were determined. Of the measured embryos, 64 were lysed for sex diagnosis by PCR. Average respiration rates of day 7 embryos (1.30 +/- 0.064 nl/h) were 3.4-fold higher than day 3 embryos (0.38 +/- 0.011 nl/h). On day 7, the average respiration rate of quality 1 blastocysts was significantly higher than the respiration rates of the lower qualities. For both day 3 and day 7 embryos, respiration rates were directly influenced by embryo diameter but did not differ between sexes. These results have demonstrated that the novel microsensor technology can be used to accurately and rapidly (8 min) measure the respiration rates of individual embryos at different developmental stages. Respiration rates were only in partial agreement with embryo morphology, suggesting a slight discrepancy between these two methods in assessing embryo quality. It is likely that a combined assessment of embryo respiration and morphology would improve embryo classification and subsequent selection.     

Birth of lambs of a pre-determined sex after in vitro production of embryos using frozen-thawed sex-sorted and re-frozen-thawed ram spermatozoa

The characteristics and functional capacity of ram spermatozoa frozen-thawed prior to and after flow cytometric sorting was assessed after incubation (37 degrees C; 6 h), in vitro fertilisation (IVF), and transfer of fresh and vitrified in vitro produced embryos. Frozen-thawed spermatozoa from two rams were allocated to four treatment groups: (i) non-sorted (Control); (ii) sorted (FS); (iii) sorted then re-frozen (FSF) and (iv) re-frozen control (FCF). Frozen-thawed samples were separated into X- and Y-chromosome bearing spermatozoa using a high-speed sperm sorter after density gradient centrifugation (X: 88 +/- 1.5% and Y: 87 +/- 1.1% purity). After 6 h incubation (37 degrees C), the percentage of motile spermatozoa was higher (P < 0.001) for FS (84 +/- 2.0%) compared with all other treatments (Control: 36 +/- 3.3%, FSF: 28 +/- 3.1%, FCF: 20 +/- 2.0%). In a sperm migration test greater numbers of FS spermatozoa penetrated 5 mm into the artificial cervical mucus compared with spermatozoa from all other treatments (152 +/- 39.4 vs 31 +/- 9.2 spermatozoa respectively; P < 0.05). Fertilisation and cleavage rates were higher (P < 0.05) for in vitro matured oocytes inseminated with Control compared with FSF spermatozoa. However, the Day 7 blastocyst development rate was higher for oocytes inseminated with FSF (62.2%) than FS and Control spermatozoa (52.7 and 50.0%; P < 0.05). The number of ewes pregnant (Day 60), lambing and the in vivo embryo survival rate was greater (P < 0.01) after the transfer of fresh embryos rather than vitrified embryos derived from X- and Y-spermatozoa (67.6, 64.7 and 41.2% vs 29.6, 25.9 and 14.8% respectively). Twenty-six of the 30 (86.7%) lambs derived from sex-sorted spermatozoa were of the correct sex. These results demonstrate that frozen-thawed ram spermatozoa can be sex-sorted for immediate or future use after re-cryopreservation and, in conjunction with IVF and embryo transfer, can be used to efficiently produce offspring of pre-determined sex.     

Developmental disparity between in vitro-produced and somatic cell nuclear transfer bovine days 14 and 21 embryos: implications for embryonic loss

In ruminants, the greatest period of embryonic loss coincides with the period of elongation when the embryonic disc is formed and gastrulation occurs prior to implantation. The impact of early embryonic mortality is not only a major obstacle to the cattle breeding industry but also impedes the application of new reproductive technologies such as somatic cell nuclear transfer (SCNT). In the present study, days 14 and 21 bovine embryos, generated by either in vitro-production (IVP) or SCNT, performed by either subzonal injection (SUZI) or handmade cloning (HMC), were compared by stereomicroscopy, immunohistochemistry, and transmission electron microscopy to establish in vivo developmental milestones. Following morphological examination, samples were characterized for the presence of epiblast (POU5F1), mesoderm (VIM), and neuroectoderm (TUBB3). On D14, only 25, 15, and 7% of IVP, SUZI, and HMC embryos were recovered from the embryos transferred respectively, and similar low recovery rates were noted on D21, suggesting that most of the embryonic loss had already occurred by D14. A number of D14 IVP, SUZI, and HMC embryos lacked an epiblast, but presented trophectoderm and hypoblast. When the epiblast was present, POU5F1 staining was limited to this compartment in all types of embryos. At the ultrastructural level, SCNT embryos displayed abundant secondary lysosomes and vacuoles, had fewer mitochondria, polyribosomes, tight junctions, desmosomes, and tonofilaments than their IVP counterparts. The staining of VIM and TUBB3 was less distinct in SCNT embryos when compared with IVP embryos, indicating slower or compromised development. In conclusion, SCNT and to some degree, IVP embryos displayed a high rate of embryonic mortality before D14 and surviving embryos displayed reduced quality with respect to ultrastructural features and differentiation markers.     

Survival of mouse embryos after freezing in medium containing dimethylsulphoxide and yolk extract

The ability of the cryoprotective solution containing dimethylsulphoxide in combination with yolk extract (DYG 550) to protect mouse embryos at ultra low temperatures is reported. Of the embryos of 46.1% frozen to -196 C at various rates formed blastocysts in vitro compared to 41.2% and 23.2% survivals protected by 1M dimethylsulphoxide and 1M glycerol.     

Distribution and elimination of fumonisin analogues in weaned piglets after oral administration of Fusarium verticillioides fungal culture

The distribution and elimination of fumonisins after oral administration of 50 mg FB₁, 20 mg FB₂ and 5 mg FB₃ per animal day^-1 for 22 days was studied in weaned barrows. At the end of the trial, the lung, heart, liver, kidney, spleen, brain, serum, bile, muscle, fat, urine and faeces samples were collected and their content of fumonisins (FB₁, FB₂) determined by LC-MS. The highest FB₁ concentrations were found in the liver (99.4 ± 37.5 ng g^-1) and kidneys (30.6 ± 10.1 ng g^-1), whilst the highest average amount of FB₂ was in the liver (1.4 ± 2.3 ng g^-1) and fat (2.6 ng g^-1 ± 4.8) samples. Comparing the FB₁/FB₂ ratio in different organs (19/1), it was found that the ratio in the abdominal and subcutaneous fat samples (4/1) was markedly different from those in all other tissues, namely the relative proportion of FB₂ was higher in latter cases. Of the total quantity of FB₁, the 13% taken up during 5 days was excreted unchanged with the faeces and urine. On average, in the urine and faeces, FB₁ was detected in nine- and 14-fold quantities, as compared with FB₂.     

Embryo development and establishment of pregnancy after embryo transfer in pigs: coping with limitations in the availability of viable embryos

Embryo transfer and pregnancy maintenance strategies in pigs were evaluated with reference to situations in which limited numbers of viable embryos or micromanipulated embryos are available, such as pig cloning. Development of embryos with compromised zona pellucida was compared with development of embryos with intact zona pellucida. Micromanipulation had no effect on blastocyst production rates after development in vivo or in vitro, but development in vivo improved the number of embryos reaching the blastocyst stage. Transfer of embryos with compromised zona pellucida resulted in live piglets. Several hormone treatments to maintain pregnancy were tested in a model in which three embryos were transferred into unmated recipient gilts, compared with transfer of three embryos into mated recipients. None of the hormonal treatments resulted in pregnancy rates of more than 25% at term and no more than 9% of transferred embryos survived, in comparison with 50% of the mated recipients successfully carrying 25% of transferred embryos. Lastly, the developmental potential of parthenogenetic embryos was assessed and 62% of transferred embryos resulted in pregnancies, none of which continued beyond day 55 of gestation. After co-transfer of three fertilized embryos with 55-60 parthenogenetic embryos into each of six recipients, two live piglets were delivered. The results from the present study indicate that transfer of zona pellucida compromised embryos can yield litters of normal piglets. In addition, it was demonstrated in a model system involving the transfer of three fertilized embryos into mature gilts that hormonal pregnancy maintenance strategies support a low proportion of embryos to term. Lastly, the present study shows for the first time a comparably effective but novel alternative for pregnancy maintenance in the pig involving the co-transfer of parthenote embryos.     

Development of cultured bovine embryos after exposure to high temperatures in the physiological range

Embryonic development is inhibited by exposure of cultured embryos to high temperatures. However, culture temperatures used to demonstrate the effects of heat on development have been higher than the body temperatures experienced typically by heat-stressed cows. The aim of this study was to determine whether exposing bovine oocytes and embryos to temperatures characteristic of body temperatures of heat-stressed cows would affect embryonic development in vitro. The CO2 percentage of the gas phase was adjusted in all experiments to prevent pH changes in the medium caused by decreased solubility of CO2 at high temperatures. Fertilization of oocytes at 41.0 degrees C reduced cleavage rate and the percentage of oocytes that became blastocysts compared with at 38.5 degrees C. There was no deleterious effect of fertilization at 40.0 degrees C. When putative zygotes and two-cell embryos were exposed to a range of temperatures from 38.5 to 41.0 degrees C for 3, 6, 9 or 12 h, heat shock reduced the number that developed to the blastocyst stage but only after exposure to 41.0 degrees C for 9 or 12 h. In addition, it was tested whether low O2 tension would reduce the detrimental effects of heat shock. The deleterious effect of 41.0 degrees C was not dependent upon oxygen content or the gas mixture used for culture (5% versus 20.95% O2), indicating that the deleterious effects of heat shock did not depend upon a high O2 environment. In the final experiment, embryos were exposed to 24 h fluctuations in temperature designed to mimic the rectal temperatures of cows exposed to heat stress. Exposure of embryos to this pattern of temperatures starting after fertilization reduced development when embryos were exposed to this environment for 8 days but not when embryos were exposed for 1 day only. These findings indicate that embryonic development can be disrupted by a short-term severe or a prolonged mild heat shock and that the effects of heat shock are not artefacts of changes in pH or high oxygen tension.     

Testicular and ovarian gonocytes from 20-day incubated chicken embryos contribute to germline lineage after transfer into bloodstream of recipient embryos

The present study was conducted to elucidate whether testicular and ovarian gonocytes obtained from 20-day incubated chicken embryos (stage 45) have the ability to migrate to the germinal ridges and contribute to germline lineage after transfer into the bloodstream of recipient embryos. Testicular and ovarian gonocytes were first identified as relatively large cells in a population of gonadal cells. The proportions of testicular and ovarian gonocytes in the total gonadal cells were 0.94 and 0.75% respectively, recognised as chicken vasa homologue-positive cells. Then, the dissociated gonadal cells obtained from 20-day incubated embryos containing testicular or ovarian gonocytes, with or without transfection, were transferred into recipient embryos. Expression of the introduced GFP gene was observed in the gonads of 6.5-day cultured recipient embryos (stage 30) in males and females, suggesting that the transferred testicular and ovarian gonocytes have the ability to migrate to the germinal ridges and enter the gonads. Furthermore, the presence of the donor-derived DNA was detected in the gonads of 20-day cultured recipient embryos in males and females, and also in the sperm samples obtained from the hatched male putative chimaeric chickens, suggesting that the transferred testicular and ovarian gonocytes were incorporated into the germline of chimaeric embryos and chickens. It is concluded that testicular and ovarian gonocytes obtained from 20-day incubated embryos have the ability to migrate to the germinal ridges after transfer into the bloodstream of recipient embryos, enter the gonads and contribute to the germline lineage of chimaeric embryos and chickens.     

塑料输送链带在啤酒灌装线上的应用

<正> 这里所记载的是Miller啤洒公司旗下6家啤酒厂中产量排行第二的Trenton,OH啤酒厂使用塑料输送带的经验。该厂于1991年正式投入生产,拥有先进的技术与设备、包括安装于其瓶装生产线上的莱克斯诺设备有限公司的Rexnord MatTop塑料输送链带。1995年,其啤酒产量达850万桶,相当于1.17亿箱啤酒。     

我国第一颗人造金刚石的诞生

文章记叙了我国人造金刚石诞生历史过程,说明我国第一颗人造金刚石的诞生是各部门通力协作,合作攻关的结果。     

Economic and genetic performance of various combinations of in vitro-produced embryo transfers and artificial insemination in a dairy herd

The objective of this study was to find the optimal proportions of pregnancies from an in vitro-produced embryo transfer (IVP-ET) system and artificial insemination (AI) so that profitability is maximized over a range of prices for embryos and surplus dairy heifer calves. An existing stochastic, dynamic dairy model with genetic merits of 12 traits was adapted for scenarios where 0 to 100% of the eligible females in the herd were impregnated, in increments of 10%, using IVP-ET (ET0 to ET100, 11 scenarios). Oocytes were collected from the top donors selected for the trait lifetime net merit (NM$) and fertilized with sexed semen to produce IVP embryos. Due to their greater conception rates, first ranked were eligible heifer recipients based on lowest number of unsuccessful inseminations or embryo transfers, and then on age. Next, eligible cow recipients were ranked based on the greatest average estimated breeding values (EBV) of the traits cow conception rate and daughter pregnancy rate. Animals that were not recipients of IVP embryos received conventional semen through AI, except that the top 50% of heifers ranked for EBV of NM$ were inseminated with sexed semen for the first 2 AI. The economically optimal proportions of IVP-ET were determined using sensitivity analysis performed for 24 price sets involving 6 different selling prices of surplus dairy heifer calves at approximately 105 d of age and 4 different prices of IVP embryos. The model was run for 15 yr after the start of the IVP-ET program for each scenario. The mean ± standard error of true breeding values of NM$ of all cows in the herd in yr 15 was greater by $603 ± 2 per cow per year for ET100 when compared with ET0. The optimal proportion of IVP-ET ranged from ET100 (for surplus dairy heifer calves sold for ≥$300 along with an additional premium based on their EBV of NM$ and a ≤$100 embryo price) to as low as ET0 (surplus dairy heifer calves sold at $300 with a $200 embryo price). For the default assumptions, the profit/cow in yr 15 was greater by $337, $215, $116, and $69 compared with ET0 when embryo prices were $50, $100, $150, and $200. The optimal use of IVP-ET was 100, 100, 62, and 36% of all breedings for these embryo prices, respectively. At the input price of $165 for an IVP embryo, the difference in the net present value of yr 15 profit between ET40 (optimal scenario) and ET0 was $33 per cow. In conclusion, some use of IVP-ET was profitable for a wide range of IVP-ET prices and values of surplus dairy heifer calves.     

Cytogenetic damage in preimplantation mouse embryos generated after paternal and parental gamma-irradiation and the influence of vitamin C

Cytogenetic damage expressed as micronuclei (MN) in 4-8-cell embryos generated after irradiation of male or male and female mice in the absence and presence of vitamin C was investigated. Male NMRI mice were whole body exposed to 4 Gy gamma-rays and mated with non-irradiated superovulated female mice in 6 successive weeks after irradiation in a weekly interval. In experiments involving irradiation of both male and female mice, irradiated male mice for 6 weeks post irradiation were mated with female mice irradiated after induction of superovulation. Effect of 100 mg/kg vitamin C (ascorbic acid) on the frequency of MN was also studied. Pregnant animals were euthanized and embryos flushed from the oviducts and fixed on slides. The rate of MN observed in embryos generated from irradiated male compared with control group dramatically increased (P<0.01). Frequency of MN in this group decreased dramatically after vitamin C treatment (P<0.01). Frequency of MN in embryos generated by mating both male and female irradiated mice was higher than that observed for those embryos generated by irradiated male mice alone. However, a considerable modifying effect of vitamin C was observed for this group too (P<0.05). Results indicate that irradiation of gonads during spermatogenesis and preovulatory stage oocytes may lead to unstable chromosomal aberrations and probably stable chromosomal abnormalities affecting pairing and disjunction of chromosomes in successive preimplantation embryos expressed as MN. The way vitamin C reduces clastogenic effects of radiation on germ cells leading to reduced frequency of MN in pre-embryos might be due to its antioxidation and radical scavenging properties.     

Development,amino acid utilization and cell allocation in bovine embryos after in vitro production in contrasting culture systems

The effects of protein-supplemented and protein-free media on amino acid uptake, protein synthesis and cell differentiation in bovine blastocysts were investigated. Four formulations of synthetic oviduct fluid were used. Each formulation was identified by the principal supplement: bovine serum albumin (0.4%, w/v); polyvinyl alcohol (0.3%, w/v); or either of two steer sera (10%, v/v). After zygote culture, blastocyst yields (day 7.5) were lowest in protein-free medium and highest in albumin-supplemented medium. Subsequent 12 h incubation in the presence of both essential and non-essential amino acids was used for the measurement of amino acid flux. All blastocysts released alanine but consumed aspartate (P < 0.001) and the extent was influenced by prior culture conditions. Aspartate uptake was lower in blastocysts produced in protein-free conditions (P < 0.05) than in blastocysts produced in albumin-supplemented conditions. Consumption indices for 16 other amino acids were not influenced by blastocyst source. Cell counts and hatching incidences were highest for albumin-supplemented blastocysts, but were similar among blastocysts from the protein-free and serum-dependent treatments. Crucially, the use of protein-free medium for zygote culture did not compromise resultant blastocysts in terms of either de novo protein synthesis ([3H]phenylalanine incorporation) or trophectoderm function (phenotype based on interferon-tau detection). Thus, although blastocyst yields were compromised after zygote culture in a protein-free (vis-à-vis albumin-supplemented) medium, amino acid flux was qualitatively conserved, and only quantitatively modified in the case of alanine and aspartate. Moreover, vital properties of blastocysts that were produced, including de novo protein synthesis and trophectodermal cell function, apparently were not adversely affected by protein deprivation.     

Developmental potential of vitrified holstein cattle embryos fertilized in vitro with sex-sorted sperm

In vitro fertilization (IVF) is a feasible way to utilize sex-sorted sperm to produce offspring of a predetermined sex in the livestock industry. The objective of the present study was to examine the effects of various factors on bovine IVF and to systematically improve the efficiency of IVF production using sex-sorted sperm. Both bulls and sorting contributed to the variability among differential development rates of embryos fertilized by sexed sperm. Increased sorting pressures (275.8 to 344.75 kPa) did not have a significant effect on the in vitro fertility of the sorted sperm; neither did an extended period of 9 to 14 h from semen collection to sorting. As few as 600 sorted sperm were used to fertilize an oocyte, resulting in blastocyst development of 33.2%. Postwarming of vitrified sexed IVF embryos resulted in high morphological survival (96.3%) and hatching (84.4%) rates, similar to those fertilized by nonsexed sperm (93.1 and 80.6%, respectively). A 40.9% pregnancy rate was established following the transfer of 3,627 vitrified, sexed embryos into synchronized recipients. This was not different from the rates with nonsexed IVF (41.9%, n = 481), or in vivo-produced (53.1%, n = 192) embryos. Of 458 calves born, 442 (96.5%) were female and 99.6% appeared normal. These technologies (sperm sexing-IVF-vitrification-embryo transfer) provide farmers, as well as the livestock industry, with a valuable option for herd expansion and heifer replacement programs. In summary, calves were produced using embryos fertilized by sex-sorted sperm in vitro and cryopreserved by rapid cooling vitrification.