Short communication: Validation of in vitro fertility genes in a Holstein bull population.

Previously, we constructed an in vitro fertilization system for the identification of genes affecting fertility traits in dairy cattle. The efficiency of this system has been demonstrated by the identification of several genes affecting fertilization rate and early embryonic survival. However, to employ these genetic markers in marker- and gene-assisted selection programs, there is a need to validate in vitro results in phenotypic data sets collected in vivo. Thus, the objective of this study was to validate, in a population of Holstein bulls, the fertility trait genes we previously identified in an in vitro system. Estimated relative conception rate (ERCR) data from 222 Holstein bulls were obtained from 5 different artificial insemination companies in the United States. Bulls were genotyped for the genes FGF2, POU1F1, PRL, PRLR, GH, GHR, STAT5A, OPN, and UTMP, and the data were analyzed for association with ERCR using a mixed effects sire model. A stepwise model selection procedure revealed evidence of association with ERCR for FGF2 and STAT5A polymorphisms. The in vivo validation suggests that these genes can be used in gene-assisted selection programs for reproductive performance in dairy cattle. The genotypes found to be associated with low bull fertility in this study have been reported to be associated with high milk composition in previous studies. These findings provide molecular evidence for the antagonistic relationship between milk production and fertility observed for many years in different breeds of dairy cattle.

Effects of signal transducer and activator of transcription (STAT) genes STAT1 and STAT3 genotypic combinations on fertilization and embryonic survival rates in Holstein cattle.

Infertility is a major cause of dairy cow culling and economic loss. Signal transducer and activator of transcription (STAT) proteins are transcription factors that play an important role in fertility and early embryonic development, among many other functions. Previous studies have reported the association of several genes from the JAK/STAT signaling pathway with fertility traits in cattle. The STAT1 and STAT3 genes are members of this pathway and are known to interact with each other by forming a heterodimer complex that enters the nucleus and controls expression of specific genes. Thus, the objective of this study was to investigate the effects of the interactions between polymorphisms in these genes on fertilization and early embryonic survival rates using an in vitro fertilization system. A total of 7,519 oocytes, collected from 445 ovaries, were exposed to sperm and a total of 5,075 embryos were produced. Fertilization rate was calculated as the number of cleaved embryos at 48 h post-fertilization out of the total number of oocytes exposed to sperm. Early embryonic survival rate of embryos was calculated as the number of blastocysts on d 7 of development out of the total number of embryos cultured. Effects of ovary genotypes on fertilization and early embryonic survival rates were evaluated. Single-SNP analysis revealed a statistically significant association between SNP25402 in STAT3 and fertilization rate. Oocytes produced from ovaries with AA genotype showed a 0.701 fertilization rate versus 0.666 and 0.663 for oocytes produced from AC and CC ovaries, respectively. The interaction between STAT3 SNP (SNP19069/SNP25402) was highly significant for survival rate but not for fertilization rate. Also, the interaction between STAT1 SNP and SNP19069 was highly significant for survival rate. Genotype combinations found to promote fertilization and embryonic survival could be incorporated into breeding programs aimed at improving fertility performance in dairy cattle.

Short communication: Effects of the progesterone receptor variants on fertility traits in cattle.

The progesterone receptor (PGR) gene is a key factor in the initiation and maintenance of pregnancy and in embryo development. Currently, it is unknown what variants of the PGR gene are related to fertility traits in cattle. Identification of such variants would allow the implementation of marker-assisted selection in breeding schemes. The objective of this study was to investigate the association of single nucleotide polymorphisms (SNP) of PGR with fertility traits in Holstein dairy cattle. An in vitro fertilization system was used to maximize the efficiency of the identification of genetic factors affecting fertility. This in vitro fertilization system would allow the assessment of fertilization and embryonic survival rates independently of influences from the uterine environment. A total of 5,566 fertilization attempts were performed, and a total of 3,679 embryos were produced using oocytes from 324 Holstein cows and semen from 10 Holstein bulls. Sequencing of pooled DNA samples from ovaries revealed an SNP (G/C) in intron 3 of PGR. A generalized linear model was used to analyze the association of this SNP with fertilization and embryonic survival rates for each ovary. Oocytes obtained from CC ovaries showed a 61% fertilization rate, compared with 68 and 69% for GC and GG ovaries, respectively. The survival rate of embryos produced from GG ovaries was 5 and 6% higher than that of GC and CC ovaries . These results indicate that the PGR SNP could be used in marker-assisted selection breeding programs in Holstein dairy cattle.

Single gene and gene interaction effects on fertilization and embryonic survival rates in cattle.

Decrease in fertility and conception rates is a major cause of economic loss and cow culling in dairy herds. Conception rate is the product of fertilization rate and embryonic survival rate. Identification of genetic factors that cause the death of embryos is the first step in eliminating this problem from the population and thereby increasing reproductive efficiency. A candidate pathway approach was used to identify candidate genes affecting fertilization and embryo survival rates using an in vitro fertilization experimental system. A total of 7,413 in vitro fertilizations were performed using oocytes from 504 ovaries and semen samples from 10 different bulls. Fertilization rate was calculated as the number of cleaved embryos 48 h postfertilization out of the total number of oocytes exposed to sperm. Survival rate of embryos was calculated as the number of blastocysts on d 7 of development out of the number of total embryos cultured. All ovaries were genotyped for 8 genes in the POU1F1 signaling pathway. Single-gene analysis revealed significant associations of GHR, PRLR, STAT5A, and UTMP with survival rate and of POU1F1, GHR, STAT5A, and OPN with fertilization rate. To further characterize the contribution of the entire integrated POU1F1 pathway to fertilization and early embryonic survival, a model selection procedure was applied. Comparisons among the different models showed that interactions between adjacent genes in the pathway revealed a significant contribution to the variation in fertility traits compared with other models that analyzed only bull information or only genes without interactions. Moreover, some genes that were not significant in the single-gene analysis showed significant effects in the interaction analysis. Thus, we propose that single genes as well as an entire pathway can be used in selection programs to improve reproduction performance in dairy cattle.

Sperm motion in a microfluidic fertilization device.

Microfluidics has shown promise as a new platform for assisted reproduction. To assess the potential of microfluidics for fertilization, we studied sperm and fluid motion in microchannels to better understand the flow characteristics in a microfluidic device, how sperm interacted with this flow, and how sperm-oocyte attachment occurs in the device. There is a threshold fluid velocity where sperm transition from traveling with the fluid to a regime in which the sperm can move independently of the flow. A significant population of sperm remained in the inlet well area. Based on the lack of progressive forward movement, it was presumed that these sperm may have defects. Also of extreme interest was the tendency of sperm to travel along surface contours. These observations provide an improved understanding of sperm motion in microchannels and provide a basis for improved device designs that take advantage of the sperm/flow and sperm/geometry interactions.

The fibroblast growth factor 2 gene is associated with embryonic mortality in cattle.

The objective of this study was to investigate the association of the fibroblast growth factor 2 (FGF2) gene with embryonic survival and fertilization rate in cattle. This gene was chosen because of its role in regulating trophectoderm expression of interferon-tau, the maternal pregnancy recognition factor in ruminants. To evaluate the effect of FGF2 on fertility traits, we produced in vitro-fertilized embryos from 281 Holstein cows and from 7 sires. A total of 4,542 in vitro fertilizations were performed, from which a total of 3,171 embryos were produced. Survival and fertilization rates were assessed at d 7 of embryonic development. Using the pooled DNA sequencing approach, we identified 2 SNP in FGF2, SNP11646 and SNP23. All sires and cows were genotyped for these SNP. For fertilization rate, no significant differences between genotypes were found for either SNP, whereas the effect on survival rate was significant for SNP11646. The survival rate of embryos produced from GG cows for this SNP was 37%, compared with 28 and 29% for embryos produced from AG and AA cows, respectively. Although the molecular mechanisms that cause embryonic mortality have not yet been identified, this study provides the first evidence of association between FGF2 and embryonic mortality in cattle. Thus, we propose that FGF2 can be used in animal breeding strategies to test for improved reproductive performance.

Mutations in the STAT5A gene are associated with embryonic survival and milk composition in cattle.

The objective of this study was to investigate the association of the signal transducer and activator of transcription 5A (STAT5A) gene with fertilization rate, embryonic survival, and milk production and composition in cattle. The STAT proteins are transcription factors that are specifically activated to regulate gene transcription when cells encounter cytokines and growth factors. The STAT5A gene is a member of the interferon-tau (IFN-tau) and placental lactogen (PL) signaling pathway, which is involved in both milk production and initiation of pregnancy. Using the DNA-pooling sequencing approach, a total of 12 single nucleotide polymorphisms (SNP) were identified, 1 exonic and 11 intronic. For the study of association of these SNP with embryonic survival, 1,551 embryos were produced in vitro from 160 cows and 3 sires. Significant associations with embryonic survival were found for 7, 5, and 2 SNP for embryos produced from sires 1, 2, and 3 respectively. The association of fertilization rate with STAT5A polymorphisms was evaluated in more than 2,300 oocytes. Significant associations were found for 6, 2, and 2 SNP for sires 1, 2, and 3 respectively. For sire 1, 5 SNP showed significant associations with both embryonic survival and fertilization rate compared with 1 SNP for sires 2 and 3. To determine if embryonic losses had occurred before the blastocyst stage, 145 of the surviving embryos were harvested at d 7 of development and genotyped for the single exonic SNP12195. A significant segregation distortion was observed between oocytes produced from 2 sires carrying the same genotype. Thus, it is most likely that STAT5A is associated with 2 mechanisms of embryo death. One is a prefertilization mechanism involving sperm factors that cause low fertilization rate. The second is a postfertilization mechanism that causes incompatibility between the male pronucleus and the oocyte, which in turn leads to death of the embryo before the blastocyst stage. Association testing of SNP12195 (exon 8) and SNP14217 (intron 9) with milk composition revealed that allele G of SNP12195 was associated with a decrease in both protein and fat percentages. However, SNP14217 in intron 9 showed no significant association with milk production or health traits. The G allele of SNP12195 was also associated with low embryonic survival, making this SNP an attractive candidate for progeny testing programs in dairy cattle.

Effect of transfer of one or two in vitro-produced embryos and post-transfer administration of gonadotropin releasing hormone on pregnancy rates of heat-stressed dairy cattle.

Pregnancy rates following transfer of an in vitro-produced (IVP) embryo are often lower than those obtained following transfer of an embryo produced by superovulation. The purpose of the current pair of experiments was to examine two strategies for increasing pregnancy rates in heat stressed, dairy recipients receiving an IVP embryo. One method was to transfer two embryos into the uterine horn ipsilateral to the CL, whereas the other method involved injection of GnRH at Day 11 after the anticipated day of ovulation. In Experiment 1, 32 virgin crossbred heifers and 26 lactating crossbred cows were prepared for timed embryo transfer by being subjected to a timed ovulation protocol. Those having a palpable CL were randomly selected to receive one (n = 31 recipients) or two (n = 27 recipients) embryos on Day 7 after anticipated ovulation. At Day 64 of gestation, the pregnancy rate tended to be higher (P = 0.07) for cows than for heifers. Heifers that received one embryo tended to have a higher pregnancy rate than those that received two embryos (41% versus 20%, respectively) while there was no difference in pregnancy rate for cows that received one or two embryos (57% versus 50%, respectively). Pregnancy loss between Day 64 and 127 only occurred for cows that received two embryos (pregnancy rate at Day 127=17%). Between Day 127 and term, one animal (a cow with a single embryo) lost its pregnancy. There was no difference in pregnancy rates at Day 127 or calving rates between cows and heifers, but females that received two embryos had lower Day-127 pregnancy rates and calving rates than females that received one embryo (P < 0.03). Of the females receiving two embryos that calved, 2 of 5 gave birth to twins. For Experiment 2, 87 multiparous, late lactation, nonpregnant Holstein cows were synchronized for timed embryo transfer as in Experiment 1. Cows received a single embryo in the uterine horn ipsilateral to the ovary containing the CL and received either 100 microg GnRH or vehicle at Day 11 after anticipated ovulation (i.e. 4 days after embryo transfer). There was no difference in pregnancy rate for cows that received the GnRH or vehicle treatment (18% versus 17%, respectively). In conclusion, neither unilateral transfer of two embryos nor administration of GnRH at Day 11 after anticipated ovulation improved pregnancy rates of dairy cattle exposed to heat stress.

Embryonic disc development and subsequent viability of cattle embryos following culture in two media under two oxygen concentrations.

Bovine embryos were produced in vitro using a 2 x 2 design of modified medium (KSOM or SOF) and oxygen concentration (5% or 20%). Day 7 blastocysts were transferred in bulk (n = 11, on average) to recipient heifers and recovered non-surgically at Day 14. In two replications of a Latin square, eight heifers received embryos from each combination of factors. Recovered embryos were evaluated for trophoblast length and width, as well as the presence and diameter of an embryonic disc (ED). An ED was detected in a higher percentage of embryos that had been cultured in KSOM than SOF (72% v. 46%, respectively; P < 0.05). The aim of a second series of experiments was to associate Day 14 morphology with subsequent developmental capacity. In vitro-produced blastocysts were transferred (n = 17-20) on Day 7 to each of eight heifers and recovered at Day 14. Thirty-eight blastocysts were retransferred to heifers following morphological evaluation. Embryos in which an ED with no signs of degeneration had been detected maintained more pregnancies than other embryos in which an ED had either shown signs of degeneration or had not been detected (5/8 v. 2/30, respectively; P < 0.01). Further investigation into ED integrity at the elongating stage may contribute to our understanding of pregnancy establishment and maintenance.

Improvement in recovery of embryos/ova using a shallow uterine horn flushing technique in superovulated Holstein heifers.

The aim of this study was two-fold: (1). to compare recovery of embryos/ova from superovulated Holstein heifers by flushing the uterine horns through insertion of the catheter very close to the tip of the horn (deep) or just after the uterine bifurcation (shallow) and (2). to evaluate the hormonal and superovulatory response to estradiol benzoate (EB) treatment prior to superovulation. Ten Holstein heifers (12-16 months) underwent two superovulatory treatments in a cross-over design. Heifers were treated with decreasing doses of FSH from Days 8 to 12.5 of a synchronized estrous cycle. At 4 days prior to superovulation, half of the heifers received EB (5mg, i.m.) or served as Controls, followed by the alternative treatment in the subsequent superovulation. At embryo recovery, one uterine horn was flushed with deep ( approximately 7 cm caudal to the tip of the horn) and the other with shallow ( approximately 5 cm cranial to the beginning of the uterine bifurcation) flushing techniques. Embryos/ova were recovered, counted, and scored. Number of ovulations was estimated by ultrasound. Pretreatment with EB reduced circulating FSH and regressed the first wave dominant follicle with no change in number of large follicles, number of ovulations, number of embryos/ova recovered, or number of transferable embryos. The shallow flushing technique was superior to the deep technique for number of embryos/ova recovered per horn (5.4+/-1.1 versus 3.9+/-0.8) or percentage of embryos/ova recovered per CL (63.9+/-8.6% versus 37.4+/-6.5%). Thus, flushing the entire uterine horn increased recovery of embryos/ova.

Use of insulin-like growth factor-I during embryo culture and treatment of recipients with gonadotropin-releasing hormone to increase pregnancy rates following the transfer of in vitro-produced embryos to heat-stressed, lactating cows.

An experiment was conducted to determine whether pregnancy rates following the transfer of in vitro-produced embryos to heat-stressed cows could be improved by 1) culturing embryos in the presence of IGF-I and 2) treating recipients with GnRH. Lactating Holstein cows (n = 260) were synchronized using a timed ovulation protocol. Embryos were produced in vitro and cultured with or without 100 ng/mL of IGF-I. On d 7 after anticipated ovulation (d 0), a single embryo was transferred to all recipients with a palpable corpus luteum (n = 210). A subset of recipients (n = 164) was injected with either GnRH or placebo on d 11. Plasma progesterone concentrations on d 0 and 7 were used to determine the synchrony of recipients. Pregnancy was diagnosed at d 53 and 81 by rectal palpation. Among all recipients, transfer of IGF-I-treated embryos increased pregnancy rate at d 53 (P < 0.05) and tended to increase pregnancy rate at d 81 (P < 0.06). Calving rate also tended to be higher for recipients that received IGF-I-treated embryos (P < 0.07). Among the subset of synchronized recipients (n = 190), pregnancy rate at d 53 and d 81 and calving rate were higher (P < 0.05) for IGF-I-treated embryos. The GnRH tended to increase pregnancy rate at d 53 for all recipients (P < 0.08) and the subset of synchronized recipients (P < 0.10). There were no effects of GnRH (P > 0.10) for pregnancy rate at d 81 and calving rate. The overall proportion of male calves was 64.3%. There was no effect (P > 0.10) of embryo treatment or GnRH on the birth weight or sex ratio of calves. Results of this experiment indicate that treatment of embryos with IGF-I can improve pregnancy and calving rates following transfer of in vitro-produced embryos. Further research is necessary to determine whether the treatment of recipients with GnRH is a practical approach to increase pregnancy rates following in vitro embryo transfer.

Detection of DNA damage in bovine metaphase II oocytes resulting from cryopreservation.

Developmental competence of mammalian oocytes is compromised by currently available oocyte cryopreservation protocols. Experiments were designed to examine the effect of three cryopreservation protocols on the integrity of bovine oocyte DNA. In vitro matured bovine oocytes were cryopreserved either by slow cooling, vitrification in 0.25 ml straws, or in open pulled straws. After thawing/warming, recovered oocytes were immediately subjected to morphological evaluation. Morphologically intact oocytes underwent comet assay to detect cryoinjury at DNA level. All cryopreservation protocols resulted in significant morphological damage as well as DNA damage compared to unfrozen control. Among the morphologically intact oocytes, there was no difference among protocols in the number of oocytes displaying DNA damage. However, oocytes that had been cryopreserved by slow cooling or by vitrification in open pulled straws exhibited more damage than those vitrified in 0.25 ml straws in the extent of DNA damage. If we combine the number of oocytes with morphological damage and oocytes with DNA damage, oocytes cooled by slow cooling resulted in the most damage. This experiment demonstrated that oocyte DNA is a target of cryoinjury and different protocols result in different degrees of damage.

Pregnancy rates following timed embryo transfer with fresh or vitrified in vitro produced embryos in lactating dairy cows under heat stress conditions.

Timed embryo transfer (TET) using in vitro produced (IVP) embryos without estrus detection can be used to reduce adverse effects of heat stress on fertility. One limitation is the poor survival of IVP embryos after cryopreservation. Objectives of this study were to confirm beneficial effects of TET on pregnancy rate during heat stress as compared to timed artificial insemination (TAI), and to determine if cryopreservation by vitrification could improve survival of IVP embryos transferred to dairy cattle under heat stress conditions. For vitrified embryos (TET-V), a three-step pre-equilibration procedure was used to vitrify excellent and good quality Day 7 IVP Holstein blastocysts. For fresh IVP embryos (TET-F), Holstein oocytes were matured and fertilized; resultant embryos were cultured in modified KSOM for 7 days using the same method as for production of vitrified embryos. Excellent and good quality blastocysts on Day 7 were transported to the cooperating dairy in a portable incubator. Nonpregnant, lactating Holsteins (n = 155) were treated with GnRH (100 microg, i.m., Day 0), followed 7 days later by prostaglandin F2alpha (PGF2alpha, 25 mg, i.m.) and GnRH (100 microg) on Day 9. Cows in the TAI treatment (n = 68) were inseminated the next day (Day 10) with semen from a single bull that also was used to produce embryos. Cows in the other treatments (n = 33 for TET-F; n = 54 for TET-V) received an embryo on Day 17 (i.e. Day 7 after anticipated ovulation and Day 8 after second GnRH treatment). The proportion of cows that responded to synchronization based on plasma progesterone concentrations on Day 10 and Day 17 was 67.7%. Pregnancy rate for all cows on Day 45 was higher (P < 0.05) in the TET-F treatment than for the TAI and TET-V treatments (19.0 +/- 5.0,6.2 +/- 3.6, and 6.5 +/- 4.1%). For cows responding to synchronization, pregnancy rate was also higher (P < 0.05) for TET-F than for other treatments (26.7 +/- 6.4, 5.0 +/- 4.3, and 7.4 +/- 4.7%). In the TET-F treatment group, cows producing more milk had lower (P < 0.05) pregnancy rates than cows producing less milk. In conclusion, ET of fresh IVP embryos can improve pregnancy rate under heat stress conditions, but pregnancy rate following transfer of vitrified embryos was no better than that following TAI.

Efficacy of timed embryo transfer with fresh and frozen in vitro produced embryos to increase pregnancy rates in heat-stressed dairy cattle.

Our objective was to determine whether pregnancy rates in heat-stressed dairy cattle could be enhanced by timed embryo transfer of fresh (nonfrozen) or frozen-thawed in vitro-derived embryos compared to timed insemination. Ovulation in Holstein cows was synchronized by a GnRH injection followed 7 d later by PGF2 alpha and a second treatment with GnRH 48 h later. Control cows (n = 129) were inseminated 16 h (d 0) after the second GnRH injection. On d 7, a fresh (n = 133) or frozen-thawed (n = 142) in vitro-derived embryo was transferred to cows assigned for timed embryo transfer after categorizing the corpus luteum by palpation per rectum as 3 (excellent), 2 (good or fair), 1 (poor), and 0 (nonpalpable). Response to the synchronization treatment, determined by plasma progesterone concentration (ng/ml) < or = 1.5 on d 0 and > or = 2.0 on d 7, was 76.2%. Mean plasma progesterone concentration on d 7 increased as the quality of corpus luteum improved from category 0 to 3. Concentrations of progesterone in plasma were elevated (> or = 2.0 ng/ml) at 21 d in 64.7 (fresh embryo), 40.3 (frozen embryo), and 41.4 +/- 0.1% (timed insemination) of cows, respectively. Cows that received a fresh embryo had a greater pregnancy rate at 45 to 52 d than did cows that received a frozen-thawed embryo or timed insemination (14.3 > 4.8, 4.9 +/- 2.3%). Body condition (d 0) of cows influenced the pregnancy rate and plasma progesterone concentrations. In summary, timed embryo transfer with fresh in vitro-produced embryos in heat-stressed dairy cattle improved pregnancy rate relative to timed insemination.

Transfer of fresh and cryopreserved IVP bovine embryos: normal calving, birth weight and gestation lengths.

In vitro and in vivo developmental competence of fresh and cryopreserved in vitro produced (IVP) bovine embryos was evaluated up to birth. Three experiments were done. The objective in the first experiment was to develop an optimal vitrification procedure for IVP bovine embryos by determining effects of exposure time (2, 5, 10, 20 min) and temperature (4, 22, 27 degrees C) in cryoprotective agents prior to vitrification on their post-thaw viability. The best combination was used in Experiments 2 and 3. In the second experiment, the importance of post-thaw morphologic selection on pregnancy rates was determined by transferring either selected or unselected single embryos. In the third experiment, pregnancy initiation, maintenance and calving results of vitrified embryos were compared with fresh and conventionally frozen embryos. Fetal losses, birth weights, gestation lengths and frequency of dystocia in the third experiment were monitored. The interaction of exposure time and temperature on both post-thaw re-expansion and hatching rates was significant (P < 0.01). Five minute exposure at 27 degrees C was optimal. In the second experiment, post-thaw selected vitrified embryos had higher pregnancy rates than unselected embryos (P < 0.05). In the third experiment, the pregnancy rate of vitrified embryos did not differ from that of fresh embryos (P > 0.05). However, pregnancy rate of conventionally frozen embryos was lower than that of fresh or vitrified embryos (P < 0.05). Of 92 calves born, 53 were male and 39 were female. Birth weights and dystocia scores of single-born calves did not differ between sexes (P > 0.05). Twin-born calves were lighter than single-born calves (P < 0.05). Overall, the data demonstrate that the transfer of vitrified IVP bovine embryos can result in healthy, apparently normal calves similar to those derived from transfer of fresh and conventionally frozen IVP bovine embryos.

Normal calves from transfer of biopsied, sexed and vitrified IVP bovine embryos.

Data on biopsied, sexed and cryopreserved in vitro produced (IVP) bovine embryos, and their in vivo developmental competence are very limited. Two preliminary studies were conducted before the primary study. In Experiment 1, post-thaw in vitro developmental competence of biopsied and vitrified IVP embryos was evaluated using re-expansion as an endpoint. In Experiment 2, the pregnancy rates of biopsied fresh, frozen or vitrified embryos following single embryo transfer were compared. Since vitrified embryos resulted in a higher pregnancy rate than frozen-thawed embryos, in the primary study (Experiment 3), all IVP embryos were vitrified following biopsy and sexing (by DNA fingerprinting). In Experiment 3, we compared pregnancy initiation and calving results of heifers in the following treatments: 1) artificial insemination (AI); 2) AI plus contralateral transfer of a single embryo (AI + SET); 3) ipsilateral transfer of single embryo (SET); or 4) bilateral transfer of two embryos (DET). Birth weights, gestation lengths and dystocia scores were recorded. In Experiment 1, post-thaw re-expansion rate of biopsied and vitrified embryos was 85% (70/82). In Experiment 2, pregnancy rates (90 d) were 44% (7/16), 23% (3/13), and 50% (7/14) for vitrified, frozen and fresh embryos, respectively (P < 0.10). In Experiment 3, pregnancy rates of AI and SET were 65% (20/31) and 40% (16/40), respectively (P < 0.05). The pregnancy rate of AI + SET was 75% (27/36) with 11 carrying twins, and the pregnancy rate of DET was 72% (26/36) with 10 carrying twins. All AI fetuses were carried to term, but only half the SET fetuses were carried to term. Similar calving rates were observed in the AI + SET and DET groups, 76 and 70%, respectively, of those pregnant at Day 40. Mean birth weight, dystocia score and gestation length of AI calves were not different from those of SET calves. Mean birth weight and dystocia score of single-born calves were greater than those of twin born calves (P < 0.05). These data demonstrate that biopsied IVP bovine embryos can be successfully cryopreserved by vitrification and following post-thaw embryo transfer, acceptable rates of offspring with normal birth weights can be obtained without major calving difficulties.

Sensitive sex determination assay applicable to bovine embryos derived from IVM and IVF.

A rapid, sensitive method for sex determination which uses nested, allele-specific amplification of bovine zfx and zfy was developed. Accuracy of the sex determination assay was verified with lymphocyte-derived DNA from 24 cattle of known gender. Reliability and sensitivity of the assay in embryonic sex determination were examined by typing duplicate bovine embryo biopsies of 2-8 cells. Sex determination was possible in 38 of 40 biopsies, and agreement between biopsy assays occurred in 17 of 18 cases. Applicability of the sex determination assay was further verified by examining gender of 12 fetuses derived from sexed embryos. Embryonic and fetal gender determinations agreed in 11 of 12 cases with the twelfth yielding an ambiguous result from fetal ultrasound examination.