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The objective of this experiment was to evaluate the impact of trace mineral supplementation on reproductive performance in lactating beef cows. Thirty days before AI (Day –30), 68 postpartum cows were stratified by weight, body condition score, and parity before being equally and randomly assigned to either a treatment or a control group. Each group received a weekly mineral supplement allotment of 1.2 kg/week per cow-calf pair for 15 weeks. Cows in the treatment group received a mineral supplement that contained zinc, copper, and manganese amino acid complexes as well as cobalt glucoheptonate (Availa® Plus; Zinpro Corp., Eden Prairie, MN, USA), whereas cows assigned to the control group received a mineral supplement that contained the same concentrations of these trace minerals from inorganic sources. All cows were submitted to a 7-day CO-Synch + CIDR protocol on Day –10 and bred using fixed time AI on Day 0. Pregnancy diagnosis was performed via transrectal ultrasound on Day 28. Cows diagnosed as nonpregnant were then removed from the experiment. Pregnant cows were divided into 10 groups of 3 or 4 cows per group: 5 treatment groups (20 total cows) and 5 control groups (18 total cows). On Day 52 and 67, all pregnant cows were subjected to an ovum pick-up (OPU). Collected oocytes were evaluated before performing in vitro fertilization. Number of oocytes cleaving (dividing into more than one cell) and developing to blastocyst stage was recorded. Analysis of variance was conducted using PROC GLIMMIX (SAS 9.4; SAS Institute Inc., Cary, NC, USA) to determine differences between treatment and control, and group was the experimental unit. Mineral consumption did not differ (P = 0.48) between treatments (average of 1.16 ± 0.065 v. 1.09 ± 0.065 kg/week per cow-calf pair, for treatment and control, respectively). Pregnancy per AI was numerically higher in mineral-supplemented cows versus controls (64.7% v. 52.9%, respectively; P = 0.33). Consumption of the complexed trace mineral supplement increased (P = 0.06) total oocyte yield (22.6 ± 1.44 mean ± SEM v. 16.4 ± 2.04 oocytes/group for treatment and control, respectively). Moreover, groups receiving the complexed trace minerals had more (P = 0.05) culturable oocytes (grades A through C) compared with control groups (15.9 ± 1.6 mean ± SEM v. 11.8 ± 1.01 oocytes/group, respectively). In addition, production of transferable embryos (grades 1 through 3) was greater (P = 0.04) for groups receiving complexed trace minerals than inorganic trace minerals (4.2 ± 0.64 mean ± SEM v. 2.6 ± 0.65 embryos/group, respectively). In summary, supplementing with zinc, manganese, and copper amino acid complexes plus cobalt glucoheptonate improved oocyte production and embryo development in pregnant beef cows when compared with cows supplemented with inorganic forms of these trace minerals.

Direct exposure of maturing oocytes to a physiologically relevant elevated temperature reduces embryo development after fertilisation and has been coincident with reduced de novo protein synthesis. Mechanisms responsible for heat-induced reductions in protein synthesis are unknown but may be related to alterations in the transcriptome of the maturing oocyte. To determine the extent to which this may occur, the impact of heat stress on the maternal pool of RNA in bovine oocytes was assessed using microarrays. After maturation for 24 h at 38.5°C (control) or 41°C (first 12 h only, 38.5°C thereafter; heat stress) oocytes were denuded from associated cumulus cells and lysed for RNA extraction or underwent IVF to assess developmental competence. Total RNA from oocytes was amplified by 3′-poly(A) priming or a combination of 3′-poly(A) and internal priming because oocyte transcripts may or may not have a polyadenylated tail. Amplified RNA was hybridised to GeneChip Bovine Genome Arrays (Affymetrix, Santa Clara, CA, USA; 8 oocyte pools per treatment were collected on 7 different occasions and amplified by 2 methods; n = 32 chips). Differential transcript abundance was determined using R and Bioconductor with only probes having a P

Exposure of maturing cumulus-oocyte complexes to a physiologically relevant heat stress altered the transcriptome in oocytes, especially certain transcripts important for mitochondrial function. To determine if perturbations are coincident with changes in mitochondrial activity, generation of reactive oxygen species (ROS), glutathione and ATP content were examined in maturing oocytes experiencing heat stress. In the first study, ROS content was assessed after 6 h at 38.5, 41, or 42°C using 37 µM 6-carboxy-2′7′-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) or 60 µM dihydrofluorescein diacetate for staining cytoplasmic and mitochondrial ROS, respectively. In a second study, cumulus-oocyte complexes were matured for 0, 12 or 24 h at 38.5°C (control) or 41°C for 12 h (heat stress; 38.5°C thereafter). Total glutathione (GSSG + GSH) and reduced glutathione (GSH) and ATP levels were assessed in cumulus denuded-zona free oocytes as per manufacturer (GSH-Glo Glutathione assay, Promega, Madison, WI, USA; 1 to 25 pmol standards) and ATP determination kit (Life Technologies, Carlsbad, CA, USA; 0.078 to 10 pmol standards). Next, ATP content was assessed in control and heat-stressed oocytes matured for 24 h and in embryos resulting after IVF of control or heat-stressed oocytes (i.e. 2-, 4-, and 8-to-16 cell and blastocyst-stage embryos). Data were analysed as a randomised block design with fixed effects of maturation temperature (and hours of maturation where appropriate), blocking on replicate, using PROC MIXED (SAS 9.2, SAS Institute, Cary, NC, USA). Culture at 41°C for the first 6 h in vitro-matured (IVM) reduced levels of cytoplasmic ROS compared to non-stressed controls (1.02 v. 0.84 fluorescent ratio for 38.5 v. 41°C, SEM = 0.16, P

Photoperiod is the principal regulator of seasonal breeding; however, effects of photoperiod on the fertility of the domestic sow are inconclusive. Some evidence indicates that the modern sow exhibits a period of impaired reproductive performance during the late summer and early fall. Seasonal variation in oocyte developmental competence has been described as a contributing factor. Alterations in oocyte quality, along with reductions in blastocyst rates and cell numbers in embryos from summer-sourced oocytes, may be attributed to an alteration in follicular fluid (FF) composition. The objectives of this study were to determine whether seasonal variations in blastocyst development rates are associated with changes in cumulus-oocyte complex (COC) morphology and oocyte developmental competence in sows. This study also compared the effect of FF collected in spring v. summer during in vitro maturation (IVM) on oocyte competence. In experiment 1, oocytes from 3- to 8-mm follicles were aspirated from sow ovaries during 1 calendar year for a total of 77 replicates. Only oocytes with homogeneous dark cytoplasm and at least 2 layers of cumulus cells underwent IVM. Mature oocytes were electrically activated and the resulting embryos were cultured for 6 days. In experiment 2, a total of 1256 good quality COC were divided into 2 groups and cultured in IVM medium containing 10% FF collected in either spring or late summer. Metaphase II oocytes were electrically activated and cultured to generate diploid embryos. Differences between experimental groups were assessed using Student's t-test or X2. The percentage of ovaries exhibiting good-quality follicles and the number of COC per ovary remained constant during the entire calendar year (60% and 6.2 COC/ovary, respectively). However, oocyte quality decreased significantly from 3.6 to 3.2 during late August throughout early October in a 1 to 4 scale. The percentage of good-quality COC decreased significantly during late summer and early fall compared with the rest of the year (54.5 v. 65.5%). However, maturation, cleavage, and blastocyst rates did not show significant differences between the summer and the other seasons (85.5 v. 87.6, 87.8 v. 87.7, and 27.8 v. 27.0%, respectively). The presence of FF collected in either spring or summer in the IVM medium did not affect maturation, cleavage, or blastocyst rates (88.9 v. 87.7, 90.7 v. 90.5, and 42.1 v. 43.7%, respectively). Blastocyst cell numbers (Day 6) did not differ when FF from spring and summer antral follicles was used for supplementing IVM medium (43.6 v. 46.1 cells, respectively). In summary, impaired reproductive performance of domestic sows during late summer and early fall is coincident with a decreased in the number and quality of COC. However, efforts to use strict selection criteria for COC during this time period may result in maturation and development rates comparable to the rest of the seasons. Additionally, the presence of FF collected in either spring or summer in the IVM medium does not seem to affect oocyte maturation and subsequent embryo development.

Fertilisation is less than expected with spermatozoa from bulls consuming toxic endophyte-infected (E+) tall fescue. The objective of this study was to evaluate motility characteristics of spermatozoa from bulls grazing tall fescue pastures using computer-assisted sperm analysis (CASA). Semen was collected from six Angus bulls (average age = 15.1 ± 0.04 months) during a three-month grazing study. Bulls grazed Kentucky 31 tall fescue (Festuca arundinacea Schreb.) infected with Neotyphodium coenophialum, an ergot alkaloid-producing endophyte (n = 3), or Jesup tall fescue with Max-Q™ (NTE), a non-ergot alkaloid producing endophyte (n = 3), and grouped by body weight and scrotal circumference to graze pastures from April 18 to June 26. Semen was collected once per week between 0600–0800 h beginning in mid-May and ending the last week of June. Gross motility and morphology was evaluated before extending with Bioxcell® animal protein-free formula (IMV, Aigle, France) and antibiotics (CSS 100, 2% of total volume). Extended semen was then evaluated using CASA to determine final dilution and packaged into straws (20 million sperm/straw), where equilibration occurred over 3 h in a cold room at 4°C. Straws were frozen for 7 min in static vapor of liquid nitrogen and plunged into goblets filled with liquid nitrogen. Semen was thawed and assessed using CASA at 0 and 3 h post-thaw. Data were analysed as a randomised block design with the fixed effects of treatment, blocking on semen collection date, utilising the mixed models procedure of SAS 9.2 (SAS Institute Inc., Cary, NC, USA). Data were tested for normality (Shapiro-Wilk W ≥ 0.90), and treatment differences were determined using F-protected least significant differences. Path velocity (P = 0.001) and progressive velocity (P = 0.003) were lower in spermatozoa from bulls grazing E+ during the last 2 weeks of collection in June independent of time of assessment post-thaw. Sperm head area decreased in size in spermatozoa from E+ grazing bulls at 3 h post-thaw (P = 0.04) compared with NTE grazing bulls. Percent of rapid (progressive % with path velocity >50 μm s–1) and medium (progressive % with path velocity 30μm s–1) velocity spermatozoa was decreased for E+ grazing bulls compared to NTE grazing bulls (P

Fertilization with spermatozoa from bulls ingesting elevated concentrations of ergovaline [i.e. consumption of toxic endophyte-infected (E+) tall fescue] results in reduced cleavage rates. Thus, the objectives of the current study were to evaluate motility and penetration rates of spermatozoa from bulls grazing tall fescue pastures and intracellular calcium (Ca2+) parameters of presumptive zygotes (PZ). During a 3-month study, 6 Angus bulls (average age = 15.1 ± 0.04 months) were appointed to graze Kentucky 31 tall fescue (Festuca arundinacea Schreb.) infected with Neotyphodium coenophialum, an ergot alkaloid-producing endophyte (n = 3), or Jesup tall fescue infected with non-ergot alkaloid-producing endophyte (NTE) MaxQ™ (n = 3). Bulls were grouped by bodyweight (BW) and scrotal circumference (SC) to graze pastures from April 18 to June 26. Blood samples, BW, SC, semen and rectal temperatures (RT) were collected every 7 days. Semen was evaluated for gross motility, morphology and computer assisted semen analysis (CASA) parameters. Semen from a subset of bulls (n = 2 per treatment; acceptable motility after 3-h stress test) was used to assess spermatozoa function using in vitro assays; that is, in vitro production of embryos, penetration rates obtained 6.5 to 7.5 h post-insemination (hpi) and intracellular Ca2+ measured 8.0 to 10.0 hpi. Data were analyzed using a mixed models procedure (semen data), generalized linear mixed models (in vitro data) and nonlinear regression (intracellular Ca2+ data) of SAS 9.2 (SAS Institute Inc., Cary, NC, USA). Concentrations of serum prolactin were higher in bulls grazing NTE compared with those of the bulls grazing E+ tall fescue pastures (123.43 ± 9.23 ng mL–1 vs 97.13 ± 9.23 ng mL–1, respectively; P = 0.05). Gross motility of spermatozoa (90.95 ± 2.67% vs 85.62 ± 2.67%, NTE and E+ respectively; P = 0.17) and percent normal morphology (77.14 ± 1.93% vs 77.61 ± 1.93%, NTE and E+ respectively; P = 0.87) before cryopreservation did not differ. However, motility immediately post-thaw (58.27 ± 2.81% vs 43.84 ± 5.30%, NTE and E+, respectively; P = 0.02) and following a 3-h stress test (51.13 ± 3.88% vs 23.33 ± 3.23%, NTE and E+, respectively; P

It is well established that exposure of cumulus–oocyte complexes (COC) to heat stress during the first 12 h of maturation reduces blastocyst development by 42 to 65%. Previous research supports the notion that some of the effects of heat stress on oocyte competence may be cumulus-mediated. To determine the extent to which this may occur, COC were matured at 38.5°C for 24 h (control) or 41°C for the first 12 h of maturation followed by 38.5°C for remaining 12 h (heat stress). A subset of COC underwent IVF with Percoll-prepared sperm and then was cultured in KSOM containing 0.5% BSA to assess developmental competence. Remaining oocytes were denuded. Cumulus cells, kept separate by treatment, were stored in lysis buffer at –80°C until RNA extraction. Total RNA from cumulus was amplified prior to hybridization to bovine Affymetrix GeneChips (Affymetrix Inc., Santa Clara, CA, USA; n = 8 pools per treatment collected on 8 different occasions; n = 16 chips). Following pre-processing using the MAS5.0 algorithm, microarray data were subjected to linear modeling and empirical Bayes analyses (Bioconductor, Limma package). False discovery rate was controlled using the Benjamini and Hochberg method, and differentially expressed genes were selected by an adjusted P-value (P < 0.05). Functional annotation of selected genes was performed using NetAffx (Affymetrix Inc.) and Database for Annotation, Visualization and Integrated Discovery (DAVID; NIAID, NIH, Bethesda, MD, USA). Heat stress of COC reduced blastocyst development (27.2 v. 16.1% for control v. heat stress, respectively; SEM = 1.6; P < 0.002). Approximately 66 and 65% of 24 000 possible genes were called present (i.e. expressed) in RNA from cumulus of competent (control) v. challenged (heat-stressed) oocytes, respectively. In cumulus from developmentally challenged COC, increased abundance of 42 genes (36 currently annotated) was noted. Use of DAVID demonstrated enrichment of genes important for electron transport and energy generation (NOS2A, MAOB, CYP11A1, HSD11B1L, LTB4DH). Further examination of gene ontology identified genes associated with mitochondrial function (SLC25A10, MAOB, CYP11A1), cell signaling (similar to JAK-3, FSHR, CYP11A1, WNT2B), cytoskeleton (ACTA1), antioxidant activity (GSTA1), and extracellular region (FMOD). In contrast, cumulus from developmentally competent COC had increased expression of 22 genes (20 currently annotated), of which 15% were related to protein binding (CAV1, MMP9, TGFB2) according to DAVID. Further analysis using gene ontology revealed genes associated with extracellular matrix formation (MMP9, MMP19, PCOLCE2) and neural tissue (METRNL). In summary, alterations in cumulus gene expression were associated with differences in developmental competence of oocytes. Additional research is necessary to examine the extent to which identified genes account for functional differences in oocyte competence. This research was supported in part by National Research Initiative Competitive Grant no. 2004-35203-14772 from the USDA Cooperative State Research, Education, and Extension Service.

Studies in the literature have shown that cumulus–oocyte complexes produce PGF2α, that ova and cumulus cells have PGF2α receptors, and that PGF2α addition to maturing ova, above what would normally be produced, decreases blastocyst development. Because previous studies have shown elevated systemic and tissue levels of PGF2α as a consequence of heat stress, it was hypothesized that detrimental effects of exposing maturing ova to elevated temperatures may be mediated in part through heat-induced increases in PGF2α. To test this hypothesis, cumulus–oocyte complexes were matured at 38.5 or 41.0°C in the presence of a PGF2α receptor antagonist (AL-8810). Preattachment embryo development of AL-8810-treated ova was compared with development of ova matured in media with or without diluent (DMSO added at the same concentration as AL-8810; diluent and developmental controls, respectively), resulting in 6 total treatment combinations. Data were analyzed as a randomized block design (blocking on oocyte collection date) with fixed effects of maturation temperature, AL-8810, and the respective interaction included in the statistical model. In experimental replicates in which the effects of heat stress decreased blastocyst development greater than 10% (n = 14), a significant maturation temperature × AL-8810 interaction was noted when evaluating blastocyst development (P = 0.05). Specifically, when ova were heat stressed during the first 12 h of in vitro maturation, blastocyst development was reduced in developmental and diluent controls (26.2 v. 18.8 and 24.4 v. 19.9, respectively; SEM = 1.6). In contrast, when ova were matured under the same conditions but in the presence of a PGF2α receptor antagonist, the effects of heat stress to reduce blastocyst development after in vitro fertilization were no longer observed (22.5 v. 22.5; SEM = 1.6). When using abattoir-derived ovaries, it is not uncommon to collect, on occasion, ova that are developmentally challenged (i.e. blastocyst development is less than the 20 to 50% expected). In this experiment, this occurred on 5 occasions. Data from these experimental replicates were analyzed and reported separately because previous efforts had shown that the responsiveness of ova to changes in culture environment differs depending on the level of developmental competence. Relevant to this study, addition of AL-8810 to developmentally challenged ova matured under thermoneutral conditions increased cleavage (60.4 v. 55.4%, respectively; P = 0.06) and blastocyst development (17.7 v. 13.7%, respectively; P = 0.07). In summary, data illustrate that developmentally challenged ova, heat-stressed or otherwise, are susceptible to detrimental effects of PGF2α. The ability to increase blastocyst development approaching or exceeding the values expected for competent ova suggests the usefulness of a PGF2α receptor antagonist during in vitro maturation to improve the efficiency of in vitro production procedures.

Increasing efficiency and success of MOET continues to be a goal of researchers and practitioners. Although numerous studies report success in establishing pregnancies, fewer evaluate term development and report number of live calves born. In a previous study, Scenna FN et al. (2008 Reprod. Fertil. Dev. 20, 154) exposed embryos to 3 different medium treatments while being collected from superovulated beef donors on Day 7. Medium treatments consisted of a commercially available medium plus 1 mL of DMSO (Control), commercial medium plus 100 nm of AL-8810 (AL100), or a commercial medium plus 1000 nm of AL-8810 (AL1000). Embryos were evaluated for grade and stage according to IETS guidelines. Embryos (n = 1734 at 6 locations across 13 replicates) were transferred (fresh or frozen in ethylene glycol) by 4 experienced technicians. Pregnancy rates were determined by ultrasonography 28 to 35 days after transfer and were increased in recipients receiving embryos collected in media containing AL100 (65%) and AL1000 (60%) compared with Control (50%; P < 0.05) as previously reported. As a continuum of this research interest, pregnancy retention rates (PRR) of recipient animals receiving embryos exposed to a prostaglandin F2α receptor antagonist (AL100 or AL1000) during collection or serving as nontreated controls were determined. Pregnancy retention rate was defined as the percentage of animals calving that were diagnosed pregnant at 28 to 35 days after transfer. From this pool of recipient animals, calving information was available to date on 494 confirmed pregnancies (presence of an embryo with a heartbeat). Data were obtained on 192 Control, 108 AL100, and 194 AL1000 recipients and analyzed using generalized mixed model analysis of variance (Glimmix) in SAS (SAS Institute, Cary, NC). Similar to initial pregnancy rates, PRR did not differ between AL100 and AL1000 (89 ± 0.04% and 90 ± 0.03%, respectively; P > 0.10); therefore, these groups were combined (AL) for subsequent analysis of PRR. Regardless of treatment, overall PRR were 88 ± 0.01% for fresh and 85 ± 0.03% for frozen embryos (P > 0.10). Additionally, gestation length (days) was similar between recipient animals receiving Control (282 ± 1.19), AL100 (281 ± 1.30) or AL1000 (281 ± 1.17; P > 0.10). However, addition of AL to collection medium of embryos increased PRR compared with Control (90 ± 0.03% v. 83 ± 0.03%, respectively; P = 0.06). Currently, an interaction was not noted between freezing method and media treatment (P > 0.10). Although collection of data continues, addition of a prostaglandin F2α receptor antagonist to the collection medium of embryos improves PRR of recipient animals without altering gestation length.

Establishment of culture systems with type A spermatogonial cells has focused primarily on the use of testicular cells from prepubertal animals. The objective of this study was to evaluate various culture systems on development of testicular cells derived from prepubertal and adult bulls. Testicular cells (were harvested from 3–4 mo-old prepubertal bulls (PB; Dairy; single fresh testis weight: 24.1 ± 0.1 g) and adult bulls (AB; Beef; 399.0 ± 0.5 g). After purification using a discontinuous Percoll density gradient, testicular cells (50 × 103 cells well–1; 24-well plate) were kept separate according to origin (prepubertal or adult) and were cultured in the presence (FL) or absence (NF) of a feeder monolayer (mitomycin C-treated male bovine fetal fibroblasts) with either embryonic-like stem cell media (ELSC; DMEM high glucose) or regular media (RCC; DMEM low glucose) supplemented with regular fetal bovine serum (FBS-S) or charcoal stripped (FBS-SF). Colony number, size and type (round, radial, and irregular) were examined on Days 4, 7 or 15 of culture. Data were analyzed using a randomized block design with factorial treatment arrangement. Depending on variable of interest, fixed treatment effects were feeder monolayer, fetal bovine serum type, media type, bull type, day of culture (4, 7, and 15 d) and interactions. Cell viability and percentage of germ and somatic cells at seeding were used as covariates. Plate (bull type × day) were random blocking factors. Cells from prepubertal bulls had greater viability than adult bulls (P < 0.0001) immediately after digestion (92.0 ± 0.1 and 84.9 ± 0.1%, respectively) and immediately after Percoll purification (78.8 ± 0.2 and 44.9 ± 0.2%, respectively). Following digestion and cell purification, percentage of cultured testicular cells staining positive for protein gene product 9.5 was 11.5 ± 0.17% for prepubertal bulls and 15.5 ± 0.19% for adult bulls (P < 0.0001). Colonies in culture presented similar morphological characteristics and timing of formation as those observed by Izadyar et al. (2003 Biol. Reprod. 68, 272–281) when culturing bovine type A spermatogonia from prepubertal bulls. Number of colonies increased from Day 4 to 15 of culture and was not affected by bull type. Overall, radial colonies were the most predominant type of colony in culture (P < 0.0001). The maximum number and size of colonies were obtained on a FL with RCC media containing FBS-S in prepubertal and adult bulls (P < 0.0001). Data illustrate that isolation and culture of testicular cells from mature bulls resulted in colony formation, similar to prepubertal animals. Although cell cultures were a mixed population, colonies with morphologies appearing similar to those previously reported in the literature suggest that a portion were likely stem cells in origin. Efforts are currently underway to use markers to further define colony populations. Nonetheless, these findings suggest that culture of testicular cells in the presence of a feeder layer with regular culture media containing FBS with steroids was beneficial in colony formation regardless of bull type.

Companion research presented at this meeting has indicated that addition of a prostaglandin2α (PGF2α) receptor (FPr) antagonist to culture medium prevented the detrimental action of PGF2α on embryo development. The aim of this study was to evaluate addition of an FPr antagonist to the collection medium on pregnancy rates after transfer of bovine embryos to recipient animals. An initial experiment was performed to determine in vitro development of in vivo-derived morula-stage frozen-thawed embryos cultured in KSOM-PVA medium with 1000 nm AL-8810 (Cayman Chemical Inc., Ann Arbor, MI, USA) (AL, n = 94), 1000 nm AL-8810 and 10 ng mL–1 PGF2α (Cayman Chemical Inc.) (AL+PGF, n = 94), 10 ng mL–1 PGF2α (PGF, n = 94), or serving as controls (CON, n = 91). Embryos remained in their treatment for a 30-h period until blastocyst development was recorded. In a subsequent experiment, embryos were recovered (n = 783) from superovulated donors on Day 7 after artificial insemination with medium containing 1000 nm AL-8810 (AL), 100 nM AL-8810 (AL100), or with vehicle (VEH: 1 mL DMSO; Sigma-Aldrich, St. Louis, MO, USA) in a double blind study. Following collection, embryos were classified by stage and quality, and then transferred fresh to recipients or frozen (ethylene glycol, direct transfer). Frozen embryos, following thawing, were transferred during the subsequent breeding period. Pregnancy rates were determined by ultrasonography (28–35 days post-transfer) and confirmed by calving date. Data were analyzed using the GLIMMIX procedure of SAS (SAS Institute, Inc., Cary, NC, USA). Results from the initial experiment indicated that culture of in vivo-derived bovine embryos in medium containing AL-8810 improved blastocyst development compared to PGF (58.5% v. 45.7%; P = 0.05). In addition, a strong tendency to increase embryo development was observed in AL+PGF compared to PGF treatment group (57% v. 45.7%; P = 0.07). Overall pregnancy rates of fresh and frozen embryos were increased in the AL and AL100 groups (55% and 58%, respectively) compared to VEH (43%; P = 0.009). Since AL treatments did not differ in pregnancy rates, subsequent analysis combined AL and AL100 data. Transfer of frozen embryos collected with medium containing AL-8810 (n = 238) increased pregnancy rates (AL, 45%) compared to embryos recovered without (n = 221) AL-8810 (VEH, 34%; P = 0.01). Transfer of fresh embryos collected with medium containing AL-8810 (n = 241) tended to have increased pregnancy rates (AL, 76%) compared to control (n = 83; VEH, 66%; P = 0.09). Although data collection continues, no abnormalities in calf health, birth weight, or weaning weight have been observed between any treatments. In conclusion, recovery of embryos with flushing medium containing an FPr antagonist improved pregnancy rates after transfer. Funding was provided by Ultimate Genetics and the Tennessee Agricultural Experiment Station for completion of these studies.

Numerous studies have demonstrated negative effects of prostaglandin F2α (PGF2α) on bovine reproduction. Discovery of a PGF2α receptor (FPr) in bovine embryos (Scenna et al. 2006 Reprod. Fertil. Dev. 18, 180) allows for development of new therapeutic strategies to improve success of embryo transfer. Therefore, two experiments were performed to investigate the occurrence of any toxic effect of AL-8810 (Cayman Chemical Inc., Ann Arbor, MI, USA), an FPr antagonist, on in vitro development of bovine embryos. In Exp. 1, pre-compacted embryos were cultured in (1) 100 AL (100 nm AL-8810 in potassium simplex optimized medium with polyvinyl alcohol (KSOM-PVA); n = 94); (2) 50 AL (50 nm AL-8810 in KSOM-PVA; n = 94); (3) 25 AL (25 nm AL-8810 in KSOM-PVA; n = 94); and (4) CON (control: KSOM-PVA; n = 95). In Exp. 2, pre-compacted embryos were cultured in (1) 1000 AL (1000 nm AL-8810 in KSOM-PVA; n = 282); (2) 500 AL (500 nm AL-8810 in KSOM-PVA; n = 274); (3) 250 AL (250 nm AL-8810 in KSOM-PVA; n = 274); and (4) CON (control: KSOM-PVA; n = 278). Embryos remained in treatments until blastocyst assessment. Next, two experiments were performed to determine the efficiency of AL-8810 on preventing detrimental effects of PGF2α on pre-compacted embryos. In Exp. 3, pre-compacted embryos were cultured in (1) 100 AL (100 nm AL-8810 in KSOM-PVA; n = 121); (2) 10 PGF (10 ng mL–1 of PGF2α (Cayman Chemical Inc.) in KSOM-PVA; n = 91); (3) AL100+PGF (100 nm AL-8810 and 10 ng mL–1 of PGF2� in KSOM-PVA; n = 116); (4) CON (control: KSOM-PVA; n = 96). In Exp. 4, embryos were cultured in (1) 1000 AL (1000 nm AL-8810 in KSOM-PVA; n = 87); (2) 10 PGF (10 ng mL–1 of PGF2α in KSOM-PVA; n = 87); (3) AL1000+PGF (1000 nm AL-8810 and 10 ng mL–1 of PGF2α in KSOM-PVA; n = 84); (4) CON (control: KSOM-PVA; n = 84). In Exp. 3 and 4, embryos remained in treatments for 48 h when development to morula was assessed. Data for all experiments were analyzed using the GLIMMIX procedure of SAS (SAS Institute, Inc., Cary, NC, USA). For Exp. 1, results indicated that addition of 100, 50, and 25 nm did not compromise embryonic development to the blastocyst stage compared to controls (60.2%, 55.8%, 55.4%, and 49.9%, respectively). In addition, orthogonal contrasts indicated that 100 nm AL-8810 improved development to the blastocyst stage (100 AL = 61% v. CON = 50.6%, P = 0.01). Similarly for Exp. 2, 1000, 500, and 250 nm AL-8810 did not affect in vitro development to the blastocyst stage compared to controls (40%, 39%, 34.8%, and 37.7%, respectively). In Exp. 3 and 4, addition of 1000 nm AL-8810, but not 100 nm, to culture medium of pre-compacted embryos exposed to PGF2α increased the ability of embryos to undergo compaction 48 h later (1000 AL+PGF = 51% v. PGF = 40%; P = 0.05). In conclusion, AL-8810 at a concentration of 1000 nm inhibits detrimental effects of PGF2α on the development of pre-compacted bovine embryos and may prove beneficial for other assisted reproductive techniques in cattle. Funding was provided by Ultimate Genetics and the Tennessee Agricultural Experiment Station for completion of these studies.

In vitro exposure of oocytes to elevated temperatures hastened oocyte maturation; furthermore, performing IVF of heat-stressed oocytes 5 h earlier than the usual 24 h resulted in blastocyst development similar to that of non-heat-stressed controls (Edwards et al. 2005 J. Dairy Sci. 88, 4326–4333). If elevated ambient temperatures in vivo alter oocyte maturation in a similar fashion, then new strategies are needed to induce earlier release of the oocyte from the ovulatory follicle. Current objectives were to examine follicular growth after FSH administration and examine whether treatment with FSH and an exogenously induced LH surge would hasten ovulation. On Day 0 (8 to 9 days after estrus) of the experimental period, lactating Holstein cows (n = 31; 65–115 days in milk; 1–6 lactations) received an EAZI-BREED CIDR (Pfizer Animal Health, New York, NY, USA) plus 100 µg of gonadotropin-releasing hormone (GnRH, IM; Cystorelin, Merial Ltd, Iselin, NJ, USA). On Day 7, CIDRs were removed and cows were administered 500 µg cloprostenol (IM; Estrumate, Schering-Plough Animal Health, Union, NJ, USA). Concurrently, cows were randomly allocated to receive either 80 mg FSH (FSH; n = 15; Folltropin-V, Bioniche Animal Health, Belleville, ON, Canada) or 4 mL of sterile saline (SAL; n = 16). Forty-eight h later (Day 9), cows within the FSH and SAL groups were randomly subdivided to receive either a 100-µg dose of Cysterolin (GnRH) or 3000 IU of hCG (hCG, IM; Chorulon, Intervet Inc., Millsboro, DE, USA) generating 4 treatment combinations (FSH/GnRH, n = 3; FSH/hCG, n = 7; SAL/GnRH, n = 8; and SAL/hCG, n = 8). Ovarian activity was assessed by ultrasonography to evaluate growth of the ovulatory follicle. Following CIDR removal, frequent ultrasonography was utilized to confirm ovulation (disappearance of the dominant follicle). Data were analyzed using the MIXED procedure of SAS (SAS Institute, Inc., Cary, NC, USA). Five cows from the FSH group were removed from the combination treatment due to ovulation occurring before 48 h post-CIDR removal. Size of the ovulatory follicle at time of GnRH or hCG administration was not different between FSH or SAL groups (16.7 � 0.7 v. 17.5 � 0.6 mm, respectively). Total growth of the ovulatory follicle from CIDR removal to ovulation did not differ between FSH (3.04 � 0.7 mm) and SAL (2.75 � 0.7 mm)-treated cows. As calculated from time of CIDR removal, ovulation occurred earlier in FSH (63.6 � 4.5 h) than in SAL (77.2 � 4.4 h; P < 0.05)-treated cows. Combination of FSH/GnRH produced the earliest ovulation (74 � 1.2 h) which was different only from FSH/hCG (78.6 � 0.8 h; P < 0.05), but not from SAL/GnRH or SAL/hCG (77 � 0.8 and 78 � 0.8 h, respectively). Regardless of FSH or SAL treatment, cows treated with GnRH ovulated earlier than those treated with hCG (75.5 � 0.7 v. 78.3 � 0.6 h, respectively; P < 0.05). In conclusion, while FSH was unable to increase the size of the ovulatory follicle, earlier ovulation occurred when given alone or in combination with GnRH.

Previous efforts of our laboratory revealed heat-induced perturbations in bovine oocytes that were coincident with reduced developmental potential. The objective of this study was to examine the effect of heat stress on total RNA and specific transcripts during oocyte maturation. After cumulus–oocyte complex (COC) collection, a subset at the germinal vesicle (GV) stage was denuded. Oocytes and surrounding cumulus were stored in RNA lysis buffer. Remaining COCs were matured for 24 h at 38.5 or 41�C (first 12 h of IVM followed by 38.5�C). At 12 and 24 h of IVM, subsets of COCs were denuded and stored in lysis buffer. Four to eight-cell embryos and blastocysts (developmental controls) derived from control and heat-stressed oocytes were collected at 40.5 and 192 h after IVF, respectively. Total RNA was isolated (PicoPure™, Molecular Devices Corp., Sunnyvale, CA, USA), quantified (RiboGreen�, Molecular Probes, Inc., Eugene, OR, USA), spiked with GFP cRNA (for normalization), and reverse transcribed with random primers. Real-time PCR was performed in triplicate using 0.1 oocyte or embryo equivalents or 100 pg cumulus RNA for analysis of BMP15, GDF9, HSP70, cyclin B1, poly(A) polymerase (PAP), and 18S and 28S rRNAs. Data were calibrated to GV-stage and analyzed using the ΔΔCt method. The experiment was replicated 9 times. Data were analyzed as a randomized block design using GLIMMIX (SAS; SAS Institute, Inc., Cary, NC, USA). Heat stress for the first 12 h of IVM reduced blastocyst formation after IVF (20.4%v. 30.5%; SEM = 1.9; P < 0.001), but had no effect on total RNA in oocytes (1.9 to 2.2 ng per oocyte; SEM = 0.7; P > 0.7) or in 4- to 8-cell embryos derived from heat-stressed oocytes (2.4 and 2.9 ng per embryo for 38.5 and 41�C, respectively; SEM = 0.7; P > 0.5). Total RNA was higher in blastocysts derived from heat-stressed oocytes (3.7 v. 5.4 ng; SEM = 0.7; P < 0.03). Heat stress during IVM did not alter relative abundance of transcripts examined in oocytes or resulting embryos. However, abundance of 18S, 28S, and GDF9 decreased in oocytes during IVM (P < 0.05). A general trend also existed for abundance to decrease as development after IVF progressed (oocyte >4- to 8-cell > blastocyst). In surrounding cumulus, HSP70 was increased by 41�C at 12 h but reduced by 24 h of IVM compared to controls (P < 0.003). Regardless of IVM temperature, PAP was higher at 12 and 24 h compared to GV stage (P < 0.0001). A stepwise decrease occurred in cyclin B1 from GV stage to 24 h (P < 0.0001). During IVM, no differences were observed in cumulus for 18S or 28S; GDF9 and BMP15 were not detectable. In the context of our study, transcripts examined were not necessarily informative of developmental potential of heat-stressed oocytes. However, increased HSP70 expression in cumulus following exposure to 41�C suggests that consequences of heat stress on oocytes may be mediated, in part, by surrounding cumulus. The results of this study are a first step toward identifying maternal transcripts in oocytes and surrounding cumulus that may be targets for development of therapeutic strategies to improve oocyte quality.

Culture of in vitro and in vivo-derived embryos in medium containing prostaglandin F2� (PGF) decreased embryonic development to blastocyst stage and reduced hatching rates (Scenna et al. Prostaglandins 73, 215-226). Moreover, administration of an inhibitor of PGF synthesis at the time of embryo transfer in bovine recipients improved pregnancy rates (Schrick et al. 2001 Theriogenology 59, 335 abstr.). These findings indicate a direct negative effect of PGF on embryonic development. However, to our knowledge, no evidence of PGF receptor expression in morula or blastocyst stage bovine embryos is available in the literature. Therefore, the objective of the current study was to determine the presence of PGF receptor mRNA using real-time RT-PCR and protein expression by Western blotting in morula or blastocyst stage in vitro bovine embryos. Briefly, isolated total RNA from compact morula or blastocyst stage embryos and from bovine tongue epithelium (positive control for PGF receptor mRNA) were reverse-transcribed into cDNA. A volume from the RT reaction equivalent to 10 embryos per tube was utilized to determine transcripts for PGF receptor and Histone H2A (standard PCR control). Polymerase chain reaction was performed, and identity of PCR fragments was confirmed by ethidium-bromide-stained 2% agarose gel electrophoresis and by DNA sequencing. To determine protein expression, morula and blastocyst stage embryos were lysed in lysis buffer (10% SDS, 1 m Tris pH 7.5, 1 m NaF, 1 m DTT, 0.1 m EGTA with protease inhibitors) and stored at -20�C. Crude proteins isolated from bovine corpora lutea (positive control for PGF receptor protein), embryo samples, and prestained standards were separated by 12% SDS-PAGE under reducing conditions. Proteins were electrotransferred onto a PVDF membrane. Nonspecific binding sites in the PVDF membrane were blocked with 10% nonfat dry milk, and the blot was washed and incubated for 1 h at room temperature with a 1:1000 dilution of the primary antibody (rabbit polyclonal antibody against PGF receptor protein). Subsequently, the blot was washed and incubated for 1 h at room temperature with 1:1000 dilution of mouse anti-rabbit IgG conjugated with horseradish peroxidase. Finally, the blot was washed and revealed by chemiluminescence in a CCD camera. Results indicated that transcripts as well as the protein for PGF receptor were present in early stage bovine embryos. Identification of PGF receptor in morula and blastocyst stage bovine embryos may, in part, explain the increase in pregnancy rates after administration of a PGF synthesis inhibitor at the time of embryo transfer, which opens up the possibility to develop new strategies to prevent detrimental effects of PGF during early embryonic development.

During a 2-year study, yearling beef bulls were used to determine the effects of grazing on endophyte-infected tall fescue on endocrine profiles, semen quality and fertilisation potential. Bulls were allotted to graze tall fescue pastures infected with Neotyphodium coenophialum (E+; n = 20 per year) or Jesup/MaxQTM (Pennington Seed, Atlanta, GA, USA; NTE; n = 10 per year). Bulls were grouped by scrotal circumference (SC), bodyweight (BW), breed composites and age to graze tall fescue pastures from mid-November until the end of June (within each year). Blood samples, BW, SC and rectal temperatures (RT) were collected every 14 days. Semen was collected from bulls every 60 days by electroejaculation and evaluated for motility and morphology. The developmental competence of oocytes fertilised in vitro with semen from respective treatments was determined. Bulls grazing E+ pastures had decreased BW gain (P < 0.01), increased overall RT (P < 0.01) and decreased prolactin (P < 0.01) compared with animals grazing NTE pastures. Neither percentage of normal sperm morphology nor motility differed between bulls grazed on the two pasture types. Semen from E+ bulls demonstrated decreased cleavage rates (P = 0.02) compared with semen from NTE bulls. However, development of cleaved embryos to the eight-cell and blastocyst stages did not differ between the two groups. In conclusion, semen from bulls grazing E+ tall fescue resulted in decreased cleavage rates in vitro, which may lower reproductive performance owing to reduced fertilisation ability.

Preincubation of semen before insemination may alter the sex ratio of resulting embryos (Lechniak, D. et al., 2003 Reprod. Dom. Anim. 38, 224–227). The overall objective of this study was to evaluate effects of aging sperm before insemination for altering the sex ratio of bovine embryos. In the first experiment oocytes presumed mature were inseminated with frozen semen within minutes post-thaw and percoll-preparation (control) or after aging for 14h post-thaw in a 34.4°C water bath, or after aging for 23h post-thaw at 4°C. Sperm from 4 different bulls, representing 3 different breeds, were utilized (1 bull per experimental replicate). Sperm motility was assessed after aging and percoll preparation. Zona pellucidae were removed from cleaved embryos (43–68h post-insemination; hpi) using 0.5% pronase. Blastomeres were dissociated and counted before transfer to PCR tubes. Sex of cleavage-stage embryos was determined using Ampli-Y™(Bredbacka, P. 1998 Reprod. Nutr. Dev. 38, 605–613). Data were analyzed using a randomized block design with mixed models of SAS (2000) after testing for normality. Aging semen for 14h post-thaw reduced the proportion of motile sperm and compromised the ability of presumptive zygotes to cleave after insemination (Table 1). However, ability of cleaved embryos to develop to the 8–16-cell stage was not affected. Number of blastomeres comprising cleavage-stage embryos that resulted from insemination of oocytes with aged semen was similar to that for controls. Insemination of oocytes with semen aged 14h in a water bath increased the proportion of female embryos (Table 1).To determine if effects of aging semen on altering the sex ratio of bovine embryos was time dependent, oocytes were inseminated with frozen semen within minutes post-thaw (control) or after aging for 8 or 14h post-thaw in a 34.4°C water bath. Sperm from 3 different bulls, representing 2 different breeds were utilized (one bull per experimental replicate). The experiment was replicated 3 times with 195–233 oocytes inseminated within each treatment. Sperm motility averaged 72.7, 65.7 and 45.0% for control and semen aged for 8 and 14h, respectively (SEM=10). Cleavage of inseminated oocytes (67hpi) was similar regardless of sperm treatment (68.5, 70.2 and 70.1%; SEM 14.6, for control semen or after aging for 8 or 14h post-thaw, respectively). Insemination of oocytes with semen aged for 14h tended to increase the proportion of female embryos (51.6 v. 38.3 and 39.1%; sperm aged for 14h v. 8h or control, respectively;; P=0.08; SEM=9.7). Within the seven replicates across both experiments, the differences in percent female for control v. aged were 5.2, 6.3, 14.9, 18.5, 20.0, 29.7 and 60%, with the highest three being significantly different (P

Incomplete reprogramming of a somatic nucleus by the oocyte cytoplasm may contribute in large part to inefficiencies of cloning procedures using somatic cell nuclear transfer. Predominant use of cytoplasts derived from in vitro-matured oocytes may further exacerbate problems. Addition of all-trans-retinol (Livingston TL et al. 2002 Biol. Reprod. 66, 104–105 abst) or 9-cis-retinoic acid (Duque P et al. 2002 Human Reprod. 17, 2706–2714) to culture medium improved developmental competence of oocytes to blastocyst stage. The objective of this study was to evaluate effects of retinol for improving developmental competence of cloned embryos constructed with retinol-treated cytoplasts. After removal from 3–8 mm follicles, COC were matured in the presence of 0 (n = 1005; diluent only) or 5 μM all trans-retinol (n = 1017). Beginning approximately 18 h after placement into culture, oocytes with extruded polar bodies were enucleated of maternal chromatin. Ovarian/granulosa cells were aspirated from adult Jersey cows (n = 2) using an ultrasound-guided transvaginal probe. Primary cell lines were established and, before nuclear transfer, cultured in the presence of 0.5% serum (5–8 days; passage = 3). Ovarian/granulosa cells were fused with cytoplasts originating from control or retinol-treated oocytes using two electrical pulses of 2.2 kV cm−1 for 20 μs between 24–27 h post-maturation (hpm). Cloned embryos were activated between 26.5 and 30 hpm and then cultured in an atmosphere of 7% O2 and 5.5% CO2 in KSOMaa + BSA. Development of cloned embryos to morula and blastocyst stages was assessed on Days 6–7 post-activation. In 5 replicates, compact morulae and blastocysts were transferred to synchronous recipients. Establishment of pregnancy was confirmed 28 days post-estrus by presence of an embryonic heartbeat using ultrasound. With the exception of pregnancy, data were analyzed as a randomized block design using mixed models of SAS (2000) after testing for normality. Proportion of oocytes recovered after denuding (94.5 and 88.9%; SEM = 2.2), those that had visibly lysed (6.4 and 7.3%; SEM = 1.5), and extruded a polar body by 20 hpm (61.7 and 62.5%; SEM = 7.0) was similar for control and retinol-treated oocytes, respectively. In addition, ability of ovarian/granulosa cells to fuse with control or retinol-treated cytoplasts was similar (80.6 and 73.1%; SEM = 4.5). Lysis of cytoplasts after electrical pulses (8.2 v. 13.6%; SEM = 3.3) and activation (11.6 v. 5.0%; SEM = 2.9) did not differ for control and retinol-treated cytoplasts. Development to morula and blastocyst stages was lower in cloned embryos constructed with retinol-treated cytoplasts (Table). However, ability of morulae and blastocysts to establish a pregnancy was comparable. One clone from each treatment developed to term and was born alive. Culture of oocytes in medium containing retinol during maturation did not improve developmental competence of clones.

Culture of germinal vesicle (GV)-stage oocytes at an elevated temperature occurring in heat-stressed dairy cattle reduced ability of oocytes to progress to metaphase II after resumption of meiosis (Payton RR et al., 2003 Biol. Reprod. 68, 343 abst). The objective of this study was to evaluate embryo development of oocytes heat-shocked at GV stage. To prevent cumulus-oocyte complexes from resuming meiosis after removal from follicles, oocytes were cultured in roscovitine (cell cycle inhibitor of p34cdc2/cyclin B kinase;; 50μM) for 24h (McCann LM et al., 2000 Biol. Reprod. 64, 141 abst and Payton RR et al., 2003 Biol. Reprod. 68, 343 abst) showed that roscovitine is effective for maintaining >90% of oocytes at GV-stage in a reversible manner. Germinal vesicle-stage oocytes were cultured at 38.5°C for 24h (experimental control) or 41.0°C as follows: HS 0–6 (41°C for 6h, 38.5°C for 18h), HS 0–12 (41°C for 12h, 38.5°C for 12h), HS 12–24 (38.5°C for 12h, 41°C for 12h), HS 18–24 (38.5°C for 18h, 41°C for 6h), or HS 0–24 (41°C for 24h) in 5.5% CO2 and humidified air. In addition, a group of COC were not cultured in roscovitine but placed in maturation medium (lab control). After a total of 24h, COC were washed extensively of roscovitine and cultured for an additional 24h in maturation medium. Oocytes presumed mature were fertilized with Percoll-prepared frozen-thawed semen. Presumptive zygotes were cultured in KSOM containing 1X nonessential amino acids in 5.5% CO2, 7% O2, and 87.5% N2 at 38.5°C in humidified air. Cleavage and development to blastocyst were recorded on Days 3 and 8 post-insemination, respectively. Data were collected in 7 replicates and analyzed as an incomplete block using mixed models of SAS (2000) after testing for normality. Use of roscovitine for maintaining oocytes at GV-stage for 24h did not alter cleavage (80.5 and 73.4%; SEM=5.8; lab and experimental controls), development to 8–16 cell (50.4 and 52.6%; SEM=4.6; lab and experimental controls), or blastocyst (29.7 and 24.8%; SEM=3.2; lab and experimental controls) stages. Culture of GV-stage oocytes at 41°C for up to 24h did not increase lysis (8.0–11.1%; SEM=2.7). Heat shock of GV-stage oocytes for as few as 6h reduced the proportion developing to 8–16 cell stage after release from inhibitor (Table 1). When experimental control and HS 0–6 were pooled for comparison to HS 0–12, effects of heat shock for reducing development to blastocyst were noted (P

Several studies have implicated prostaglandin F2α (PGF) as a major embryotoxic factor during early embryonic development in cattle. Elevated uterine concentrations of PGF were negatively associated with embryo development, quality and pregnancy rates (Schrick FN et al. 1993 Biol. Reprod. 49, 617–621; Hockett ME et al. 1998 J. Anim. Sci. 76 (Suppl 1), 241 abst; Seals RC et al. 1998 Prostaglandins 56, 377–389). Moreover, addition of PGF to culture medium decreased hatching rates of compacted morulae (Scenna FN et al. 2002 Theriogenology 53, 512 abst) and decreased development of pre-compacted (16–32 cell) bovine embryos to blastocyst stage (Scenna FN et al. 2003 Theriogenology 59, 335 abst). Furthermore, administration of an inhibitor of PGF synthesis at the time of embryo transfer improved pregnancy rates in cattle (Schrick FN et al. 2001 Theriogenology 55, 370 abst). The objective of the current study was to identify the period of time during early embryonic development that is most susceptible to the deleterious effects of PGF. After in vitro maturation and fertilization of bovine oocytes, putative zygotes were cultured in KSOMaa plus 0.3% BSA. On Day 4 post-insemination, pre-compacted (16–32 cell) embryos were removed from culture, evaluated for quality, and randomly assigned to one of the following treatments: 1) Control (KSOMaa plus 0.3% polyvinyl alcohol (KSOM-PVA; n=470) or 2) PGF-1 (1ngmL−1 PGF in KSOM-PVA; n=473; Scenna FN et al. 2003 Theriogenology 59, 335 abst). After 48h of incubation in assigned treatments, assessment of development to compacted morula stage was determined. Thereafter, embryos were kept separate according to treatments, sorted by stage of development and quality, and randomly assigned to receive either Control (CON) or PGF-1 supplemented medium until assessment of blastocyst development on Day 9. This random sorting resulted in the formation of four treatment groups comprising the initial treatments and assigned treatments during Days 6–9 (CON-CON, n=366; PGF-CON, n=226; CON-PGF, n=149; PGF-PGF, n=287). Analyses were performed incorporating a randomized incomplete block design using mixed models of SAS (2000) to determine effects of PGF on Days 4–6, 6–9 and 4–9 of development. Data were also analyzed using chi-square. Addition of 1ngmL−1 of PGF to culture medium on Days 4–9 decreased the percentage of pre-compacted embryos reaching blastocyst stage (CON-CON, 47.8%; PGF-PGF, 36%; P0.05). Results suggest that morula stage embryos during compaction are most susceptible to deleterious effects of PGF.