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In cattle, individual in vitro embryo culture after Day 6 benefits development, allowing non-invasive analysis of culture medium. However, undefined supplements in culture reduce analytical reliability. In this study we assayed the short- and long-term performance of embryos after bovine serum albumin removal over a 24-h period in individual culture. The absence of protein decreased embryo development and cell counts in the inner cell mass without affecting blastocyst sex ratio. However, the absence of protein produced embryos with an improved tendency to survive vitrification after 24 h in culture (P = 0.07). After transfer to recipients, birth rates of embryos that had been cultured with protein tended to decrease (P

Knowledge of the biochemical composition of the bovine oviduct in the presence of an embryo is lacking. We have recently reported the detection of alterations to the bovine oviduct transcriptome when multiple embryos, but not a single embryo, were present (Maillo et al. 2015 Biol. Reprod. 92, 144). Thus, we hypothesised that the presence of an embryo would modify the concentration of metabolites and energy substrates in the oviducal fluid. Thus, the aim of the present study was to examine how the presence of an embryo affects metabolites and energy substrates within the oviduct. Cross-bred beef heifers were synchronized, and those in standing oestrus were randomly allocated to cyclic, not bred (n = 7), or pregnant, artificially inseminated (n = 11), groups. All heifers were slaughtered on Day 3 after oestrus. The oviducts from each animal were isolated, straightened, and cut, separating ampulla and isthmus. Each portion was flushed with 500 μL of PBS, enabling recovery of any oocyte/embryo. Recovered unfertilized oocytes (cyclic group) and embryos (pregnant group) were located in the isthmus of the oviduct ipsilateral to the corpus luteum. Samples of flushing medium from the isthmus and ampulla from oviducts ipsilateral to the corpus luteum from 5 cyclic and 5 pregnant animals (8-cell embryos) were used for metabolites and energy substrates analysis, together with PBS as blank control. Amino acid content in the oviducal fluid was determined by high performance liquid chromatography. Glucose, lactate, and pyruvate were quantified using microfluorometric enzyme-linked assays. Statistical analysis was carried out by 2-way ANOVA with a Bonferroni post hoc for multiple comparisons. No differences were found either in the concentration of metabolites or in the energy substrates between cyclic and pregnant heifers. However, significant differences were detected between ampulla and isthmus in cyclic and pregnant heifers, which is in agreement with our previous findings on the transcriptome between isthmus and ampulla (Maillo et al. 2016 Reproduction 152, 37–46). In pregnant and cyclic heifers, glycine and alanine were more abundant in the ampulla compared with the isthmus (P

It has been shown that the equine embryo is able to modulate the proteome of the oviduct, increasing the presence of certain proteins involved in the embryo-maternal communication (Smits et al. 2016 Reprod. Fertil. Dev. doi: 10.1071/RD15481). In cattle, the presence of a single embryo did not affect the transcriptome of the oviduct, whereas multiple embryos induced changes (Maillo et al. 2015 Biol. Reprod. 92, 144). The aim of the present study was to examine the effect of the presence of an embryo on the oviduct fluid proteome. Cross-bred beef heifers were synchronized, and those in standing oestrus were randomly allocated to cyclic, not bred (n = 7), or pregnant, artificially inseminated (n = 11), groups. All heifers were slaughtered on Day 3 after oestrus. The oviducts from each animal were isolated, straightened, and cut, separating ampulla and isthmus. Each portion was flushed with 500 μL of PBS free of protein to confirm the presence of an oocyte/embryo. Recovered unfertilized oocytes (cyclic group) and embryos (pregnant group) were located in the isthmus of the oviduct ipsilateral to the corpus luteum. Flushings from 4 cyclic and 4 pregnant heifers (8-cell embryos) were used for proteomic analysis, including blank controls of PBS. Comparison of ipsilateral ampulla and isthmus in pregnant and cyclic heifers did not reveal any differences. Therefore, samples from ipsilateral oviducts were compared between cyclic and pregnant heifers. Total protein (150 μg) from cyclic and pregnant samples was labelled with different cyanine fluorescent probes and separated according to the isoelectric point using immobilized pH gradient strips (pH 3–10, 17 cm, Protean® IEF cell system, Bio Rad, Hercules, CA, USA). Second dimension was performed in a polyacrylamide gel (12%) in the presence of SDS using a Protean II XL system (Bio Rad). The images were obtained with the Typhoon 9410 scanner and analysed with the Progenesis SameSpots software v. 4.0. The results of image analysis showed 42 different spots between the 2 groups (ANOVA P 1.5), the majority (38) of which were more abundant in pregnant heifers. Gels were stained with Coomassie blue to detect the spots. The differential spots were digested with trypsin and analysed with Agilent Ion-Trap XCT Plus mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) equipped with an electrospray interface. Twenty spots were highly abundant in pregnant heifers. From these, 3 different proteins were identified with score >5.0 and % SPI >76.0 by mass spectrometry, corresponding to SERPINA1, serum albumin, and serum transferrin. These proteins are abundant in plasma, suggesting that the embryo may be able to activate pathways to increase the permeability of the vascular endothelium in the oviduct. The same proteins were also identified in the equine oviducal proteome in the presence of an embryo, suggesting a possible conserved mechanism between species. In conclusion, presence of an embryo in the bovine oviduct increases the abundance of some proteins that may be related with its early development. This work was funded by the Spanish Ministry of Economy and Competitiveness (AGL-2012–37510 and AGL-2015–70140-R) and Science Foundation Ireland (13/IA/1983).

Lipolytic metabolic conditions are traditionally associated with elevated non-esterified fatty acid (NEFA) concentrations, but may also be accompanied by hyperglycaemia in obesity or by hypoglycaemia during a negative energy balance status. Elevated NEFA concentrations disrupt oocyte and embryo development and quality, but little is known about whether the effects of lipolytic conditions on oocyte developmental competence are modulated by glucose availability. To answer this, bovine cumulus–oocyte complexes (COCs) were matured under different conditions: physiological NEFA (72 µM) and normal glucose (5.5 mM), pathophysiologically high NEFA (420 µM) and normal glucose, high NEFA and high glucose (9.9 mM), high NEFA and low glucose (2.8 mM). Developmental potential, cumulus expansion and metabolism of COCs exposed to high NEFA and low glucose were affected to a greater extent compared with COCs matured under high NEFA and high glucose conditions. High NEFA and high glucose conditions caused a moderate increase in oocyte reactive oxygen species compared with their high NEFA and low glucose or control counterparts. Blastocyst metabolism and the transcriptome of metabolic and oxidative stress-related genes were not affected. However, both lipolytic conditions associated with hyper- or hypoglycaemia led to surviving embryos of reduced quality with regards to apoptosis and blastomere allocation.

The study of molecules involved in the oocyte maturation and early embryo development is crucial to improve the conditions of in vitro embryo production. The plasminogen activation system is involved in the initial steps of reproduction and urokinase type plasminogen activator (uPA) is expressed in the granulosa cells (GC) of bovine cumulus-oocyte complexes (COC). The aim of this study was to analyse the effect of uPA in bovine oocyte in vitro maturation (IVM). We have analysed whether the addition of uPA or the inhibition of its proteolytic activity affects IVM. We have evaluated (1) nuclear and cytoplasmic maturation, (2) developmental competence, and (3) oocyte and GC gene expression. Immature COC were obtained by aspiration of ovarian follicles of slaughtered heifers. Selected COC were in vitro-matured in 4 groups: control, 10 nM uPA, dimethyl sulfoxide control, and 100 μg mL–1 amiloride, a specific inhibitor of uPA proteolytic activity (4 replicates). After 24 h of IVM, oocytes of each treatment were either fixed and stained with Hoëscht (to evaluate nuclear maturation) or LCA-FITC to analyse the cortical granules distribution as a marker of cytoplasmic maturation (n = 10/group per treatment per replicate). In addition, pools of 10 oocytes and their separated GC were snap-frozen to analyse by qRT-PCR their profile expression of genes related with apoptosis (BAX, BCL2, TP53, SHC1), cell junctions (GJA1, TJP1), cell cycle (CCNB1), oxidative stress (SOD2, GPX1), oocyte quality (BMP15, GDF9), and serpin proteases inhibitors (SRP1, SRP5), normalised respect 2 housekeeping genes (H2AFZ, ACTB). The remaining COC were fertilised (Day 0) and in vitro cultured to evaluate developmental competence in terms of cleavage rate (Day 2) and blastocyst yield (Days 7–9). All data were analysed by one-way ANOVA. In the presence of amiloride, a significant reduction in the oocyte maturation was observed at both levels; 83.33% of oocytes remained in vesicle stage, and 75.0% showed a cortical granules distribution of type I. The rest of the groups (62.67%, 62.65%, and 60.29%) reached metaphase II (MII), and 51.66%, 32.9%, and 25.44% showed granule distribution of type III. For embryo development, the amiloride group had a cleavage rate and blastocyst yield significantly lower compared with controls (23.23% v. 80.85% and 83.83%; 4.45% v. 25.21% and 25.21%, respectively), whereas uPA treatment had no effect (84.28% and 24.16%). In presence of amiloride, the transcript levels of TJP1, GJA1, and CCNB1 were up-regulated, whereas SOD2, SRP1, and SRP5 were down-regulated in GC compared with all other groups (P

Extracellular vesicles (EVs) act as intercellular communicators through their protein, lipid, and mRNA content. The interaction of EVs from oviducal environment and the first stages of embryo development is currently an enigma. The aim of the present study was to evaluate the developmental competence and the expression profile of bovine blastocysts cultured with previously purified EVs recovered from ampullary and isthmic oviducal fluid (OF) under different centrifugal forces. OF-EVs recovered from oviducts of slaughtered heifers in early luteal phase were quantified with a nanoparticle tracking analysis system, and their integrity and size were assessed by electron microscopy. In vitro-produced zygotes were cultured in SOF+3 mg mL–1 BSA (C–), C– with 3 × 105 OF-EVs/mL from the ampulla (A) and isthmus (I) isolated at 1 × 103 (A10k and I10k, respectively) and 1 × 105 (A100k and I100k, respectively) × g. A control culture group of SOF+5% FCS (C+) was included. Blastocyst development was recorded on Day 7, 8, and 9 (D0: day of fertilization). Blastocysts on Days 7/8 cultured in C–, C+, I10k, and I100k were used to measure the relative mRNA expression of genes related with membrane trafficking (AQP3, AQP11, and ATP1A1), metabolism (LDLR and LDHA), and epigenetics (DNMT3A, IGF2R, GRB10, and SNRPN) by RT-qPCR. One-way ANOVA was used for statistical analysis. The size of ampullary and isthmic OF-EVs was similar with a mean of 220 nm. The concentration of I10k was significantly lower compared with A100k (3.6 × 108 v. 10.5 × 108 EVs/mL, respectively; P

Single embryo culture in SOF with BSA allows efficient noninvasive analysis of culture medium (CM). Defined protein-free CM avoids contaminants present in commercial BSA. In this work we studied the performance of a single culture step with and without protein. In vitro-matured and -fertilized oocytes were cultured in SOF+6 mg mL–1 BSA. From Day-6 onwards, n = 1351 morulae and early blastocysts were singly cultured in 12 µL SOF+6 mg mL–1 BSA or 0.5 mg mL–1 polyvinyl-alcohol (PVA; 20 replicates). Development was recorded as % Day-6 cultured embryos. Day-7 and Day-8 expanded blastocysts were vitrified and cultured upon warming in SOF+10% FCS for 48 h. Expression of genes of stress, metabolism, and imprinting was measured by RT-qPCR in Day-7 fresh expanded and in vitrified/warmed hatched blastocysts. Day-7 vitrified blastocysts were allowed to re-expand for 2 h before embryo transfer (recipients used once or twice). Day 40 pregnancy, miscarriage and birth rates, and morphometry and weight of calves, were recorded. Data were analysed by GLM and REGWQ test. Protein removal reduced Day 7 blastocyst rate (71.3 ± 2.7 v. 51.6 ± 2.7; P 0.41; not shown). Protein starvation limits blastocyst development but selects for improved viability. Such a profile agrees with the quiet embryo hypothesis (Leese et al. 2008 Mol. Hum. Reprod. 14, 667–672), by which the most viable embryos use endogenous nutrients efficiently and cope with stress successfully. Differences in gene expression seen before vitrification are abolished after warming, with G6PDH perhaps reflecting superior ability of protein-free cultured embryos to counteract stress induced by cryopreservation. Without protein, more embryos reach birth as morphologically normal calves. Research was supported by MICINN (AGL2012–37772) and FEDER; AM: SENESCYT-Ecuador-II Fase 2013. The authors are members of the COST Action FA1201 (Epiconcept).

Although fertilization and early embryonic development take place in the oviduct, the consequences of tubal fluid supplementation during in vitro embryo culture have not been explored. The aim of this study was to evaluate the effect of bovine oviducal fluid (bOF) supplementation during in vitro embryo culture of bovine embryos on their development and quality. The bOF was aspirated from oviducts of slaughtered heifers in the early luteal phase. In vitro-produced zygotes were cultured in SOF (C–; n = 927) or SOF + 5% FCS (C+; n = 872) or in SOF + bOF (n ~900/group) at different concentrations (0.62, 1.25, 2.5, 5, 10, or 25%) in 10 replicates. Blastocysts on Days 7/8 were used for quality evaluation through (a) differential cell count, (b) survival after vitrification/warming, and (c) gene expression (qRT-PCR). One-way ANOVA (development and quality) and t-test (cell count) were used for statistical analysis. The bOF concentrations over 5% were detrimental for blastocysts development (

In cattle, ~40% of embryonic loss occurs in the period from Day 8 to Day 16 of pregnancy. A significant proportion of embryo loss may be due to inadequate circulating progesterone (P4) concentrations. Low P4 concentrations have also been implicated as a causative factor in the low pregnancy rates (PR) observed in high-yielding dairy cows. Administration of hCG during the early luteal phase stimulates hypertrophy of the original corpus luteum (CL) and, depending on the day of administration, induces ovulation of the first-wave dominant follicle and formation of a functional accessory CL, which increases circulating P4 concentrations. The aim of this study was to examine whether administration of hCG on Day 2 or Day 5 after oestrus after timed AI (TAI) would lead to an increase in pregnancy rates in dairy cattle. Lactating Holstein cows (n = 194) from 12 commercial dairy herds in Southern Spain (37.8833° N, 4.7667° W) with an average milk production at 37.8 L/cow per day and typically with a PR to first AI of ~30% were randomly assigned based on their body condition score (2.65 ± 0.05; mean ± SEM), parity (2.60 ± 0.09), and days in milk (75.06 ± 0.63) to 1 of 3 treatments and administered a single intramuscular injection of 3000 IU of hCG (4 mL of Veterin Corion) either (1) on Day 2 = 36 h after TAI (n = 65; hCG2 group), (2) Day 5 = 108 h after TAI (n = 64; hCG5 group), or (3) 4 mL of saline on Day 2 = 36 h after TAI (n = 65; control group). Cows were synchronized using a 7-day Ovsynch TAI protocol that included a P4-releasing intravaginal device (PRID DELTA 1.55 g). First, gonadotropin-releasing hormone (Cystorelin 100 mg) treatment was administered at PRID insertion (Day 0) followed by 25 mg Dinoprost (prostaglandin F2α: Enzaprost T) on Day 7 at PRID withdrawal. Then, 56 h later, the second gonadotropin-releasing hormone (100 mg) treatment was administered and all cows were inseminated 16 h later. Pregnancy was diagnosed by ultrasonography 28 to 32 days after TAI. Logistic regression model and chi-squared test were used to analyse data. Pregnancy rate to AI was significantly higher in the hCG2 and hCG5 groups than in the control group (43.1 and 45.3%, v. 27.7%; P

The aim of the present study was to investigate the effect of short-term progesterone (P4) supplementation during the early metoestrous period on circulating P4 concentrations and conceptus development in cattle. The oestrous cycles of cross-bred beef heifers were synchronised using a 7-day P4-releasing intravaginal device (PRID® Delta; 1.55 g P4) treatment with administration of a prostaglandin F2α analogue (Enzaprost; CEVA Sante Animale) the day before PRID® Delta removal. Only those heifers recorded in standing oestrus (Day 0) were used. In Experiment 1, heifers were randomly assigned to one of five groups: (1) control: no treatment; (2) placebo: insertion of a blank device (no P4) from Day 3 to Day 7; (3) insertion of a PRID® Delta from Day 3 to Day 7; (4) insertion of a PRID® Delta from Day 3 to Day 5; or (5) insertion of a PRID® Delta from Day 5 to Day 7. In vitro-produced blastocysts were transferred to each heifer in Groups 2–5 on Day 7 (n = 10 blastocysts per heifer) and conceptuses were recovered when heifers were killed on Day 14. Based on the outcome of Experiment 1, in Experiment 2 heifers were artificially inseminated at oestrus and randomly assigned to one of three treatment groups: (1) placebo; (2) PRID from Day 3 to Day 5; or (3) PRID from Day 3 to Day 7. All heifers were killed on Day 16 and recovered conceptuses were incubated in synthetic oviducal fluid medium for 24 h; spent media and uterine flushes were analysed for interferon-τ (IFNT). In both experiments, daily blood samples were taken to determined serum P4 concentrations. Data were analysed using the PROC MIXED procedure of SAS (SAS Institute, Cary, NC, USA). Insertion of a PRID resulted in an increase (P

Despite clear evidence of a two-way interaction between the developing conceptus and the uterine endometrium in early pregnancy, the evidence for reciprocal cross-talk during the transit of the embryo through the oviduct is less clear. The aims were (1) to characterise the transcriptome of the bovine oviduct at the initiation of embryonic genome activation (EGA), (2) to examine the effect, if any, of the presence of an embryo on the oviduct transcriptome, and (3) to compare gene expression in the ampulla and isthmus of the oviduct ipsilateral to the corpus luteum. The oestrous cycles of cross-bred beef heifers were synchronized and those recorded in standing oestrus were randomly allocated to control group, nonbred (n = 7), or AI group (n = 11). All heifers were slaughtered on Day 3 after oestrus. The oviducts from each animal were isolated, straightened, and cut in half (ampulla and isthmus). Each portion was flushed with 500 μL of PBS to confirm the presence of an oocyte/embryo and was then opened and scraped longitudinally to obtain epithelial cells. Cells were snap-frozen in liquid nitrogen for microarray analysis. All recovered oocytes and embryos were located in the isthmus of the oviduct ipsilateral to the corpus luteum. The recovery rate was 72.7% (8/11) and 83.3% (5/6) for pregnant and cyclic animals, respectively. The stage of the recovered embryos was as follows: 4-cell stage (n = 1), 8-cell stage (n = 5), and 8–16 cell stage (n = 2), whereas in the cyclic group all recovered structures were unfertilized oocytes. The cells of the isthmus from ipsilateral and contralateral oviducts from 5 cyclic and 5 pregnant animals (8-cell embryos) and the ipsilateral ampulla cells from the pregnant animals were used for microarray analysis (Affymetrix Bovine ST array, Affymetrix, Santa Clara, CA, USA). Array data were analysed using BioConductor packages in R and custom CDF files downloaded from MBNI. Preprocessing of raw data was performed with RMA, and differential expression was assessed by linear modelling implemented in the limma package. Genes displaying P

The aim of this study was to investigate the effect of short term progesterone (P4) supplementation on circulating P4 concentrations, corpus luteum (CL) size, and conceptus development in cattle. The oestrous cycles of crossbred beef heifers were synchronised using a 7-day PRID® Delta (1.55 g P4) treatment with administration of a PGF2α analog (Enzaprost®) the day before PRID® Delta removal. Only those recorded in standing oestrus (Day 0) were used. In Experiment 1, heifers were randomly assigned to 1 of 5 groups: (1) control: no treatment, (2) placebo: insertion of a blank device (no P4) from Day 3 to 7, (3) insertion of a PRID® Delta from Day 3 to 7, (4) insertion of a PRID® Delta from Day 3 to 5, or (v5) insertion of a PRID® Delta from Day 5 to 7. In vitro produced blastocysts were transferred to each heifer on Day 7 (10 blastocysts per heifer) and conceptuses were recovered at slaughter on Day 14. In Experiment 2 heifers were artificially inseminated at oestrus and randomly assigned to 1 of 3 treatment groups (1) placebo, (2) PRID® Delta from Day 3 to 5, or (3) PRID® Delta from Day 3 to 7. All heifers were slaughtered on Day 16, and recovered conceptuses were incubated in synthetic oviduct fluid medium for 24 h; spent media and uterine flushes were analysed for interferon-tau (IFNT). In both experiments, daily blood samples were taken to measure serum P4 concentration. Data were analysed using the PROC MIXED procedure of SAS (SAS Institute Inc., Cary, NC, USA). Insertion of a PRID® Delta resulted in an increase (P

The aim of this study was to examine the effect of lactation and associated metabolic profiles on the ability of the reproductive tract of postpartum dairy cows to support early embryo development. Twenty-one age-matched primiparous Holstein cows were used. Immediately after calving, half of the cows were dried off while the remainder were milked twice daily. To characterise the metabolic profile of the cows, jugular blood samples were taken twice weekly starting 15 days before calving until Day 100 postpartum. At the same time, bodyweight (BW) and body condition score (BCS) were recorded. In Experiment 1, around Day 60 postpartum, the oestrous cycles of all cows were synchronized and sixty-five 2- to 4-cell in vitro-produced embryos were endoscopically transferred on Day 2 (Day 0 = oestrus) to the oviduct ipsilateral to the corpus luteum. On Day 7, the oviduct and uterus were flushed endoscopically and the number of embryos developing to the blastocyst stage was recorded. In Experiment 2, around Day 95 postpartum, cows were re-synchronized and 15 to 20 in vitro-produced blastocysts were transferred to the uterine horn ipsilateral to the corpus luteum. On Day 14, conceptuses were recovered by flushing the reproductive tract at slaughter and were measured. Jugular blood samples were taken daily from Day 0 to 7 (Exp. 1) or 14 (Exp. 2) to measure serum concentrations of progesterone. Data were analysed by ANOVA. Concentrations of NEFA and β-HB were higher (P ≤ 0.05) and glucose, insulin and IGF-1 were lower (P ≤ 0.05) in lactating compared with dry cows. BW and BCS were significantly higher in the non-lactating cows throughout the postpartum period. Recovery rates in both experiments were similar between groups (Exp. 1: 63.9 ± 7.2 vs 65.6 ± 8.6 and Exp 2: 33.3 ± 9.6 vs 39.8 ± 9.6 for dry and milking cows, respectively). In Exp. 1, of the structures recovered, significantly more developed to the blastocyst stage in the dry than in lactating cows (49.3 ± 3.8 vs 32.6.3 ± 4.4, respectively; P ≤ 0.05). Progesterone concentrations did not differ between groups. In Exp. 2, no differences were observed in terms of conceptus dimensions on Day 14 (n = 152). Progesterone concentrations were higher in lactating cows from Day 9 to 14 (P ≤ 0.05). In conclusion, this study provides evidence that at 60 days postpartum, the reproductive tract of lactating cows is compromised in its ability to support early embryo development compared with age-matched parous non-lactating cows; however, by 95 days postpartum there was no apparent difference in conceptus development, consistent with less metabolic stress as indicated by the metabolic profile. Funded by Science Foundation Ireland (SFI/07/SRC/B1156) and the Spanish Ministry of Science and Innovation (AGL2009-11810). VM was supported by an STSM award from the COST Action FAO7O2.

Progesterone (P4) has a crucial impact on the transcriptome of the uterine endometrium and the preparation of the uterus to support implantation. The aim of this study was to investigate the effect of follicle aspiration just before ovulation on corpus luteum (CL) development, circulating P4 concentrations and the ability of the uterus to support embryo development and conceptus elongation. We tested the hypothesis that the unavoidable loss of follicular fluid and some granulosa cells during aspiration of the preovulatory follicle would compromise the development and function of the developing CL and that this would be associated with reduced P4 and a poorer uterine environment. Oestrous cycles of crossbred beef heifers were synchronized using an 8-day CIDR treatment with administration of a prostaglandin F2α analogue on the day before CIDR removal to ensure CL regression. Heifers were checked for signs of oestrus 4 times per day commencing 30 h after CIDR withdrawal and only those recorded in standing oestrus (Day 0, n = 20) were used. All heifers received a gonadotropin-releasing hormone agonist (0.01 mg buserelin) 48 h after CIDR removal to induce an LH surge. Half of the animals underwent follicle aspiration 20 h later, while the remainder underwent ovulation. Daily transrectal ultrasonography was carried out from Day 3 to 13 to record CL development. Daily blood samples were collected from Day 0 to 14 for circulating P4 concentrations. To test the ability of the uterus to support embryo development and conceptus elongation, Day 7 in vitro-produced blastocysts were transferred to the uteri of synchronised recipients (7 to 10 blastocysts per recipient). All recipients were slaughtered on Day 14 to assess embryo survival and conceptus size. CL diameter and CL area were significantly reduced in the follicle aspirated group compared with controls from Day 6 onwards (P ≤ 0.05). Similarly, at slaughter on Day 14, CL weight (4.17 ± 0.48 vs 7.05 ± 1.65 mm), diameter (19.89 ± 1.35 vs 24.64 ± 2.07 mm) and area (321.94 ± 45.01 vs 510.18 ± 69.41 mm2) were lower in aspirated heifers (P ≤ 0.05). Circulating P4 concentrations were lower at all time points from Day 3 to Day 14 but were only significantly lower from Day 12 onwards (P ≤ 0.05). Conceptus length (2.08 ± 0.29, n = 56 vs 4.55 ± 0.78 mm, n = 45) and area (2.52 ± 0.39 vs 5.61 ± 1.12 mm2) were lower (P ≤ 0.05) in heifers undergoing follicular aspiration compared with those undergoing ovulation. In conclusion, aspiration of the preovulatory dominant follicle just before expected ovulation was associated with a compromised CL in terms of size and P4 output and this, in turn, was associated with a reduced capacity of the uterus to support the initiation of conceptus elongation. Supported by Science Foundation Ireland (07/SRC/B1156).