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Perturbations in the uterine luminal fluid composition are detrimental to pregnancy establishment in cattle

Perturbations in the uterine luminal fluid composition are detrimental to pregnancy establishment... Background: A major, unresolved issue is how the uterine microenvironment determines pregnancy success in cattle. Before implantation, conceptus development depends on the uterine secretome (i.e., histotroph). Despite its pivotal role, little is known about the dynamics of histotroph synthesis and changes in composition throughout the early diestrus and the relevance to pregnancy establishment. We hypothesize that disturbances on histotroph composition affect the establishment of pregnancy. Aim was to disturb histotroph composition at early diestrus and verify the effects on: (Exp. 1) timing to restore its composition; and (Exp. 2) pregnancy rate after multiple-embryo transfer. Estrous cycle of multiparous Nelore cows were synchronized and estrus was considered d 0 (D0) of the experiments. Disturbance was through flushing each uterine horn with 30 mL of DMPBS and collecting the resulting uterine luminal flushing (ULF) on D1; D4; D7; D1 + D4 + D7. Control group remained not-collected. In Exp. 1, ULF was collected on D7.5 from all animals and used for quantification of total protein concentration and abundance of albumin. In Exp. 2, three in vitro-produced embryos were transferred to the uterine horn ipsilateral to the ovary containing the CL on D7.5 and pregnancy was checked on D25 by ultrasound. Results: In Exp. 1, ULF collection on D4 or D7 increased (1.5- to 2.2-folds) the total protein concentration and albumin abundance. ULF collection on D1 did not alter (P > 0.10) these endpoints. In Exp. 2, ULF collected on D4 or D7 decreased pregnancy rates to approximately half of that measured in the remaining groups. Conclusions: Subtle perturbations imposed to the native intrauterine milieu, such as those caused by a single, low-volume collection of ULF, profoundly disturbs intrauterine composition and pregnancy success. At least 4 d were necessary for the uterus to recover its composition and the functional capacity to carry post-implantation gestation. Keywords: Albumin, Embryo, Histotroph, Protein Background preimplantation period, the bovine embryo development In eutherian mammals, there are two manners for provid- occurs independently of a direct connection to the blood ing nutrients to the conceptus, histotrophic and hemo- supply. Development relies solely on a complex fluid se- trophic nourishment. In ruminants, the histotrophic mode creted by the epithelium lining the reproductive tract (i.e., is of special importance, because preimplantation embryo oviductal and uterine fluid) to supply basic energy needs development takes approximately 20 d [1, 2]. During the [1, 2]. Specifically, the fluid secreted into the uterine lumen, termed histotroph, is a complex mixture of growth factors, hormones, enzymes, transport proteins, ions, * Correspondence: mario.binelli@ufl.edu lipids, glucose, amino acids and other molecules, that are Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, 225, Avenida Duque de Caxias dynamically synthesized by the endometrial glandular and Norte, Jardim. Elite, Pirassununga, SP 13635-900, Brazil luminal epithelia as well as selectively transported from Department of Animal Sciences, University of Florida, Gainesville, FL P.O. blood [2–4]. In cattle, the establishment of pregnancy is 110910, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Martins et al. Journal of Animal Science and Biotechnology (2018) 9:70 Page 2 of 11 dependent on the presence of an elongated conceptus, pregnancy rate compared to transfer towards an asyn- capable of signaling to the endometrium, mainly through chronous uterus [20]. Specifically, transfer of a single d interferon-tau (INFT) that is released in increasing 7 embryo to a d 5 or d 8 uterus moderately impacted amounts from d 15-16 of gestation [2, 5]. The IFNT acts pregnancy rate (~ 6% reduction), but transfer of a sin- on the endometrium and inhibits the biosynthetic cascade gle d 7embryotoad4ord9uterus hadnegative of prostaglandin F α and thereby luteolysis. Conceptus impact on pregnancy rate (~ 22% reduction). In signaling together with continuing P4 exposure further in- addition, indirect modifications introduced in the duces endometrial differentiation to support receptivity to uterine environment, for example as caused by sup- the conceptus and implantation. Thus, the histotroph is plementation of P4 at early diestrus, advances concep- pivotal for the maintenance and development of preim- tus elongation [21] and influences embryonic survival plantation conceptus in cattle, allowing the establishment and the subsequent fertility [22]. However, the dy- of pregnancy. namics of histotroph changes along the estrous cycle Thereisconvincingevidencethatthe phaseof histo- are largely not known. Furthermore, understanding trophic nutrition is critical for the establishment of preg- how and to which extent disturbances in this native nancy in cattle. About 2/3 of the overall embryonic death environment influence pregnancy establishment in loss (~ 30%) occurs between d 8 and d 16 post- cattle is limited. Thus, in the present study we aimed insemination [6–8]. Part of the elevated embryonic mortal- to disturb the histotroph composition at early diestrus ity that occurs during the preimplantation period is deemed and verify the effect on 1) timing to recover compos- to result from a functional incapacity of the uterine luminal ition and 2) embryo survival in beef cattle. milieu to properly support conceptus survival and elongation. Methods In fact, the importance of histotroph for the periimplan- Animals tation conceptus development and survival was nicely Non-lactating, cycling, multiparous Nelore cows (Bos demonstrated by Gray et al. [3, 4] using an ovine uterine taurus indicus, average body weight 593.80 ± 12.07 kg) gland knockout model. The authors verified that the ab- were maintained under grazing conditions, supplemented sence of endometrial gland secretions in sheep uterus with chopped sugarcane, concentrate and minerals to ful- compromises conceptus survival and elongation, resulting fill their maintenance requirements and received water ad in impaired conception. libitum. The experiment was conducted in the Southern The histotroph composition is temporally-regulated by Hemisphere tropics at the Pirassununga Campus of the estradiol (E2) and progesterone (P4), along the estrous University of São Paulo, Brazil. All experimental proce- cycle. In effect, the endometrium undergoes marked func- dures involving animals complied with the Ethics and Ani- tional changes in response to the ovarian-endocrine stimu- mal Handling Committee of the School of Veterinary lus and to pregnancy, as demonstrated in transcriptomic Medicine and Animal Science of the University of São studies [9–11]. Furthermore, studies in cattle [12–15]indi- Paulo (CEUA-FMVZ/USP, No. 9585220316). cated that the histotroph composition also undergoes This study comprises two experiments. In the Exp. 1, dynamic changes along the estrous cycle and is altered by we aimed to determine the effect of uterine flushings different P4 concentrations and pregnancy. Accordingly, performed over diestrus on the uterine luminal protein proteomic [12, 13], amino acids [14–16]and components content at d 7.5 post-estrus. In the Exp. 2, the aim was of the redox system [17] were contrasting between uterine to determine the impact of uterine flushing on the estab- fluids recovered across the diestrus. The expression of a lishment of pregnancy in embryo recipient cows. The number of proteins during the cycle was related to P4 con- experimental designs are illustrated in Fig. 1. centrations from d 3 to d 7 post-estrus [12]oritwas in- creased when P4 concentrations were exogenously elevated Experimental designs [14, 15]. Altogether, these findings support the notion that In the Exp. 1, follicular growth was synchronized using histotroph composition results from dynamic changes in an intravaginal P4-releasing device (1 g; Sincrogest®, endometrial function, orchestrated by sex-steroids and the Ourofino Saude Animal, Cravinhos, SP, Brazil), an intra- embryo/conceptus, to provide optimal uterine environ- muscular administration of estradiol benzoate (2 mg; ments that support stage-specific requirement of embryo/ Sincrodiol®, Ourofino Saude Animal) and prostaglandin conceptus development. Evidence for this well-regulated F α analogue (PGF α; 500 μg of sodium cloprostenol; 2 2 environment might be verified by a stringent requirement Sincrocio®, Ourofino Saude Animal). Eight days later, the of synchrony between the developing embryo and the re- P4-devices were removed; cows received another injec- cipient (approximately 24 h) for optimal pregnancy rates tion of PGF α analogue and an Estrotect™ (Western [18, 19] in cattle. Indeed, transfer of a single d 7 embryo to- Point Inc., Apple Valley, MN) heat detector patch. Cows wards a synchronous d 7 uterus resulted in greater were checked for signs of estrus twice a day between Martins et al. Journal of Animal Science and Biotechnology (2018) 9:70 Page 3 of 11 Fig. 1 Experimental designs of the two experiments performed in this study. Collection of uterine luminal flushings (ULF) was performed during diestrus to determine: the effect on the uterine luminal protein content at D7.5 post-estrus (Exp. 1) or on pregnancy establishment (Exp. 2). Accordingly, follicle growth of cycling, non-suckled, multiparous Nelore cows was synchronized and estrus was detected (D0). Uterine horns were flushed non-surgically with 30 mL of DMPBS thrice (Exp. 1, 90 mL total) or once (Exp. 2) and ULF was collected. In Exp. 1, animals were submitted randomly to collection of ULF on D1; D4; D7; D1, 4 and 7; or to remain not-collected, to compose the 5 experimental groups: D1-ULF, D4-ULF, D7-ULF, D1 + D4 + D7-ULF and Control. On D7.5, ULF were collected from all groups for determination of total protein concentration and abundance of albumin. On Exp. 2, the same experimental groups were present, with the exception of D1 + D4 + D7-ULF group*. On D7.5, three in vitro-produced embryos were transferred non-surgically to the uterine horn ipsilateral to the ovary containing a CL and pregnancy was checked (PC) on D25 by transrectal ultrasonography. In both experiments, a sham ULF collection (all procedures except delivering DMPBS to the uterus) was performed in each cow, in each experimental day when no ULF collection was scheduled 36 h and 96 h after P4-releasing device withdrawal. diameter of pre-ovulatory follicle and CL area were re- Cows observed in standing estrus and/or presenting an ac- corded for analysis. In addition, blood flow of measured tivated heat detector patch were considered in estrus (D0 CLs was examined using ultrasound Color Doppler- of the study; n = 44). Animals were submitted randomly to mode, to confirm CL functionality, as defined by Pugliesi collection of uterine luminal flushings (ULF) on D1; D4; et al. [23]. D7; D1, 4 and 7; or to remain not-collected, to compose the 5 experimental groups: D1-ULF (n =9), D4-ULF (n = Quantification of P4 concentrations 9), D7-ULF (n =9), D1 +D4+D7-ULF (n =9) and Control In Exp. 1, blood samples were collected on D7 for deter- (n = 8). On D7.5, ULF were collected from all groups mination of serum P4 concentrations. Serum was harvested for determination of total protein concentration and on D7 by centrifugation of blood at 2,700 × g for 15 min at abundance of albumin. Control group was used to 4 °C. Progesterone was assayed using a solid-phase RIA kit assess the perturbations promoted by the ULF collec- (Immuchem™ Double Antibody Progesterone Kit; Cat. tions on these endpoints. 07-170105, MP Biomedicals, NY, USA). The sensitivity of In the Exp. 2, estrous cycles were synchronized using a the assay was 0.1 ng/mL. The intra-assay coefficient of vari- slightly modified version of the protocol described above. ation (CV) for quality control samples was 1.15% (low An injection of GnRH analogue (10 μg of buserelin acet- standard) and 0.01% (high standard). ate; Sincrofort®, Ourofino Saude Animal) was adminis- tered intramuscularly at P4-device insertion, followed by its removal 7 d later and by an administration of PGF α ULF collection analogue 24 h earlier. Cows were checked for signs of Caudal epidural anesthesia was performed with 2% lido- estrus as described previously. Animals observed in caine solution (4 mL; 80 mg; Lidovet®, Bravet, Engenho standing estrus and/or with an activated Estrotect Novo, RJ, Brazil) immediately before ULF collection. patch (n = 64) were assigned randomly to one of four Uterine horns were flushed non-surgically, always flush- groups: (i) Control (n = 16), (ii) D1-ULF (n = 15), (iii) ing the horn ipsilateral to ovary containing the CL first D4-ULF (n = 17), (iv) D7-ULF (n =16). and flushing the contralateral horn subsequently, at dif- ferent d post-estrus, as described in the experimental Ultrasound examinations design. ULF was collected using a similar method de- Transrectal ultrasonography (Mindray M5, Shenzen, scribed previously [12]. Accordingly, a sterile silicone China; equipped with multifrequency linear transducer Foley catheter (2 vials, 20 mL cuff, 18″ or 20″ diameter; set to 7.5 MHz) in B-mode was performed to check ovu- Rusch®, Teleflex, US) was fixed onto a stylette. Guided lation of pre-ovulatory follicle on D1 and confirm the by rectal palpation, the catheter was passed through the side of corpus luteum (CL) on D4 and D7. In Exp.1, cervix into the body of the uterus and directed to the Martins et al. Journal of Animal Science and Biotechnology (2018) 9:70 Page 4 of 11 target uterine horn. The catheter was positioned and collected from each cow. In the groups D1-ULF, D4-ULF fixed at the horn’s bifurcation by inflating the cuff. The and D7-ULF, a sham flushing (all procedures except distal end of the uterine horn adjacent to the uterotubal delivering DMPBS to the uterus) was performed on each junction was manually occluded through rectal palpa- experimental day when no collection was scheduled. For tion. Then, the uterine horn was slowly filled with example, group D1-ULF was subjected to sham flushing on 30 mL of Dulbecco’s modified phosphate buffered saline D4 and D7. On D7.5, each uterine horn of each animal was (DMPBS, Nutricell, Campinas, SP, Brazil) at 37 °C using submitted to a single ULF collection with 30 mL of DMPBS a sterile 60-mL catheter tip syringe (SR, Manaus, AM, and the ULF was used for protein analysis. Volume and Brazil). After infusion of DMPBS, the ULF was immedi- visual aspect of ULF-D7.5 were recorded. In addition, to ately recovered by aspiration with a syringe only while a evaluate the impact of three sequential collections on ULF steady flow could be achieved. In Exp.1, for each uterine composition, on D7.5, only in the Control group, two add- horn, this procedure was repeated three times without re- itional collections were performed. Volume and aspect of moving the catheter. ULF recovered from each uterine each ULF collected (ULF1, ULF2 and ULF3) were recorded horn was placed in 50-mL conical tubes to record volume individually for analysis. On D7.5, ULFs were transferred to and visual aspect. ULFs were classified subjectively as light-safe 50-mL conical tubes and kept on ice until proc- bloody when they contained any traces of blood or pre- essed in the laboratory. Within 15 min of flushing, ULFs sented a pinkish coloration, as opposed to a translucent as- were clarified by centrifugation (1,000 × g for 10 min pect (Fig. 2a). Total volume recovered and visual aspect of at 4 °C), supernatants were transferred to cryotubes ULFs were used to characterize the pooled three ULF andstoredat − 20 °C for subsequent analyzes. Fig. 2 Effect of ULF classified as clear or bloody on total protein concentration and albumin abundance. On D7.5 (D0: estrus), each uterine horn of beef cows (n = 7) were flushed thrice with 30 mL of PBS and the resulting uterine luminal flushings were collected (ULF1, ULF2 and ULF3), visually classified as clear or bloody (Panel a) and stored for analysis. Quantification of total protein concentrations and determination of albumin abundance was performed in each ULF (Panel b). Panel c: Representation of the three successive ULF classified as clear (black bold marked) or bloody (red bold marked) in the polyacrylamide gel stained with Coomassie-Blue for albumin determination. Pool: Mixture of all collected ULF. AU: Arbitrary units. Values are presented as LSMEANS±SEM Martins et al. Journal of Animal Science and Biotechnology (2018) 9:70 Page 5 of 11 Collection of ULF in the Exp. 2 was performed as de- by densitometry using Image-Lab software version 4.01 scribed, but with minor modifications. Specifically, the (Bio-Rad). Protein molecular weights were estimated ac- uterine horns were flushed only once with 30 mL of cording to a molecular weight standard (Precision Plus DMPBS, instead three times as in Exp. 1. Protein™ Dual Color Standards, Bio-Rad laboratories) that was run in each gel. The identification of the albu- Embryo production and embryo transfer procedures min band was based on molecular weight of bovine On D7.5, all animals from Exp. 2 were submitted to embryo serum albumin (~ 67 kDa). Only ULF samples collected transfer. Cumulus oocyte complexes used to produce em- from the ipsilateral uterine horn were used for determin- bryos were aspirated from ovaries collected in a local slaugh- ing the abundance of albumin on D7.5. The effect of terhouse. Embryos were produced according to a standard three sequential flushings on the abundance of albumin protocol for in vitro embryo production [24]. Recipients was determined on ULF collected from the Control ani- received a caudal epidural anesthesia immediately before the mals on D7.5 from ipsi- and contralateral uterine horns. procedure of embryo transfer. Three in vitro-produced, grade 1 blastocysts were transcervically placed in the middle Statistical analysis of the cranial third of the uterine horn ipsilateral to the Dataset of Exp.1 (i.e. total protein concentration and ovary containing a CL, using standard nonsurgical tech- abundance of albumin) was analyzed by ANOVA using niques. By transferring three embryos per recipient cow, we MIXED procedure of SAS (SAS Inst. Inc., Cary, NC, USA) expected to minimize the random effect of a single incom- version 9.3. Model included fixed effect of experimental petent embryo to influence pregnancy establishment [25]. group, horn (ipsi- and contralateral) and their interaction. On D25, pregnancy status was based on visualization Assumptions of normality of residues and homogeneity of of the embryo proper and heartbeat by transrectal variances were checked by influencing diagnostics outputs B-mode ultrasonography under optimal conditions, as from the MIXED and F-max test, respectively. Data that described previously [26, 27]. All cows received a luteo- did not follow the assumptions were transformed by the lytic dose of PGF α after pregnancy diagnosis. natural logarithmic or square root before analyses. When necessary, comparisons were performed contrasting Total protein quantification assay D-ULF groups versus the Control group, using the DIFF The protein content in the ULFs collected on D7.5 was command incorporating the Dunnett test. determined by a colorimetric assay using a Micro BCA Pro- Aiming to determine the effect of sequential ULF col- tein Assay Kit (Pierce Biotechnology, Rockford, IL) accord- lections on the protein and albumin quantity of collected ing to manufacturers’ instructions. The micro-assays were ULFs, three-sequential ULFs collected on D7.5 were an- conducted in 96-well plates. The total protein concentra- alyzed by split-plot design using MIXED procedures. tion in each sample was determined in triplicates and calcu- Model included fixed effect of ULF collection order lated using the standard curve supplied on the kit. The (ULF1, ULF2 or ULF3), ULF visual aspect, horn and all standard curve was based in bovine serum albumin diluted their interactions. The random effect of cows nested in DMPBS and ranged from 25 to 2,000 μg/mL. The colori- within combination of flushing aspect and uterine horn metric absorbances were measured by a spectrophotometer side was used as an error term to test the main plot ef- (Multiskan MS Primary EIA, Thermo-Fisher) adjusted at fect. The type of variance–covariance structure used was 570 nm wavelength. chosen based on smaller magnitude of the corrected Akaike’s information criterion (AICC). When the ef- Determination of albumin abundance fect of a categorical variable was significant, the LSD Albumin content was determined in ULF samples by post-hoc test was used to compare means. Comassie-Blue staining. ULF samples (18 μL) from D7.5 The variable, volume on D1, D4 and D7 from groups were diluted in 2× Laemmili buffer and denatured at D1-ULF, D4-ULF and D7-ULF, respectively, was ana- 95 °C for 5 min. Proteins were separated in 12% lyzed using MIXED procedure. Similarly, binomial vari- SDS-Polyacrylamide gel electrophoresis at 160 V for able, proportion of ULF with blood from each group was 120 min. The resulting polyacrylamide gels were fixed analyzed using GLIMMIX procedure. Model included by submerging in 50% MeOH, 10% HoAC and 40% dis- fixed effect of group, horn and their interaction. For the tilled water (v/v) solution for 30 min. Fixed gels were D1 + D4 + D7-ULF group, variables volume and propor- stained in 0.25% (w/v) Coomassie Brilliant Blue R-250 tion of ULF with blood on D1, D4 and D7 were analyzed (Bio-Rad laboratories) solution for 2 h and then, cleared as split-plot design, including fixed effect of day, horn overnight in a destaining solution (5% MeOH, 7.5% and their interaction. The random effect of cows nested HoAC, 87.5% distilled water). Stained polyacrylamide within uterine horn side was used as error term. On gels were exposed to ChemiDoc MP Imaging System D7.5, those variables (volume and visual aspect) were (Bio-Rad) apparatus and band intensities were quantified analyzed including fixed effect of group, horn and their Martins et al. Journal of Animal Science and Biotechnology (2018) 9:70 Page 6 of 11 interaction. In addition, volume of three-sequential ULFs Effect of different days, single ULF collections on same day collected on D7.5 from Control group was analyzed as ULF composition split-plot design. Model included fixed effect of flushing Initially, we analyzed volume recovered and visual as- order, horn and their interaction. pect of ULFs collected on D1, D4 or D7. Because In Exp. 2, the analyzes of frequency of pregnant cows blood flow to the uterus decreases from metestrus to were performed with FREQ procedure using Chi-square early diestrus, we expected that frequency of ULF distribution. Orthogonal contrast comparison was used containing blood would also decrease from D1 to D7. to assess differences between groups (Control vs. There was no effect of group (D1-ULF, D4-ULF and ULF-D1, ULF-D4 vs. ULF-D7 and Control & ULF-D1 D7-ULF) on the proportion of ULF classified as vs. ULF-D4 & ULF-D7). bloody (46.3% [25/54], P > 0.1), neither on total vol- Continuous variables are reported as Least Square ume of recovered ULF (87.7 ± 0.76 mL, P >0.1), Means ± Standard error of the mean (LSMEANS ± regardless of uterine horn side collected. SEM) and binomial variables as means. Effect of single or multiple days ULF collections on Results subsequent ULF composition Exp. 1 Next, we analyzed ULF samples collected on D7.5 from Ovarian characteristics animals submitted to UFL collections conducted on D1, Mean ± SEM of size of pre-ovulatory follicle (13.42 ± D4, D7 or D1, D4 and D7. Proportion of ULFs classified 0.27 mm), and CL area (2.42 ± 0.37 cm )and serumP4 as bloody (47.8% [43/90]) and volume collected (26.1 ± concentrations on D7 (3.29 ± 0.24) were similar among 0.53 mL) were all similar among groups (P > 0.1), regard- groups (P > 0.10; data not shown). In all cows, a functional less of uterine horn side sampled. Despite these similar CL was detected on D4 and D7 at ultrasound scan, accord- gross characteristics among groups, total protein con- ing to criteria described previously [23]. centration in the ULF-D7.5 was affected (P = 0.01) by group, regardless of the uterine horn side sampled Effect of same day, sequential ULF collections on ULF (Fig. 3). The D7-ULF had 1.9-fold higher (P < 0.01) total composition protein concentration in the ULF-D7.5 compared to the For this analysis, only data from the sequential col- Control group. Compared to the Control group, total lections of ULF on D7.5 from the Control group protein concentrations in ULF-D7.5 of groups D1-ULF, were used (Fig. 2). We expected that ULF collection D4-ULF and D1 + D4 + D7-ULF increased numerically would have the effect of reducing total protein con- 1.3, 1.5 and 1.4 fold, respectively, but concentrations centration and abundance of albumin progressively were not significantly different than the Control group. from ULF1 to ULF3. However, these endpoints were Consistently, the abundance of albumin (that was only not affected by sequential collections among ULF1, analyzed in the ULF-D7.5 collected from the ipsilateral ULF2 and ULF3 (P > 0.1). Irrespective of order of uterine horn), there was an effect of group (P = 0.02) on collection and side of uterine horn of the collected the abundance of albumin measured in the ULF-D7.5 ULF, compared to the ULF classified as clear, those (Fig. 4). The albumin abundance in the D4-ULF and classified as bloody presented greater total protein D7-ULF groups were respectively, 2.0- and 2.1-fold concentration (bloody: 258.95 ± 21.97 vs. clear: greater compared to the Control group (P = 0.06), while 160.80 ± 19.55 μg/mL) and abundance of albumin that of the D1 + D4 + D7-ULF group was 2.2-fold greater (bloody: 385.78 ± 38.96 vs. clear: 235.06 ± 33.74 arbi- (P = 0.03) than the abundance verified in the Control trary units; P < 0.01). group. Changes in total protein and albumin abundances were caused by the ULF collection, not by the manipula- Effect of different days, sequential ULF collections on same tions associated with the ULF collection. Specifically, day ULF composition compared to Control, the ULF-D1 group had no signifi- For this analysis, we used ULF samples collected on D1, cant alterations in total protein and albumin abun- D4 and D7, from group D1 + D4 + D7-ULF only. There dances, despite of sham flushing procedures performed was an effect of day (P ≤ 0.05) on the volume of recov- on D4 and D7. ered ULF, regardless of uterine horn side flushed. Vol- ume of recovered ULF on D4 (81.8 ± 1.71 mL) was lower Exp. 2 than that on D7 (87.7 ± 1.77 mL), but similar to D1 The effect of single ULF collections on pregnancy rate of (85.3 ± 1.77 mL). Proportion of ULF classified as bloody embryo recipients for the D1 + D4 + D7-ULF group was not affected (P > In this experiment, ULF procedure at selected time 0.1) by day nor uterine horn side (D1: 29% [5/17]; D4: points was performed only once instead of thrice, be- 29% [5/17] and D7: 11.1% [2/18]). cause as verified in Exp. 1, sequential ULF resulted in no Martins et al. Journal of Animal Science and Biotechnology (2018) 9:70 Page 7 of 11 Fig. 3 Effect of previous ULF collections on total protein concentration in the ULF-D7.5. Uterine horns of beef cows detected in estrus (D0) were flushed non-surgically thrice with 30 mL of DMPBS (90 mL) and the resulting uterine luminal flushing (ULF) was collected. Animals were assigned randomly to be submitted to ULF collections on D1; D4; D7; D1 + D4 + D7; or to remain not-collected, composing 5 experimental groups. On D7.5, ULF were collected from all groups for quantification of total protein concentrations. Control group was used to assess the perturbations promoted by the ULF collections on the total protein concentrations. Values are presented as LSMEANS ± SEM. Main effects of group, uterine horn (Horn) and their interaction are indicated. **P < 0.01, differed from Control group as determined by Dunnett test further modifications on the composition of subsequent and modify the luminal molecular composition. More ULFs. Furthermore, by performing a single ULF collec- importantly, because incidence of embryo mortality is tion with low volume of DMBPS (30 mL), we expected disproportionately elevated during this stage of gesta- to minimize eventual disruptions caused on uterine epi- tion, it is reasonable to assume that mortality is associ- thelia due the flushings. Also, ULF collection in all se- ated with an inadequate environment. However, luminal lected days (D1 + D4 + D7-group) had not cumulative composition that would support or suppress embryo de- effect on the ULF-D7.5, so this group was not included velopment has not been determined. The aim of the in the design. Pregnancy rate was affected by group (P = present study was to disturb the histotroph composition 0.06). Pregnancy rates were similar (P > 0.1) between through the removal of ULF to evaluate the dynamics of Control (62.5%) and ULF-D1 group (60%) and between restoration and the effects of a disturbed uterine envir- ULF-D4 (29.4%) and ULF-D7 (37.5%) groups. Pregnancy onment on the pregnancy outcome. We expected that rate of Control & ULF-D1 (61.3% [19/31]) was lower uterine flushings would remove molecules that are (P = 0.03) than ULF-D4 & ULF-D7 (33.3% [11/33]), critical for embryo development, generating an impover- Fig. 5. Altogether, these data revealed that pregnancy ished environment that would compromise develop- rate was not affected by ULF collection when it was ment. We further anticipated that disturbances that performed on D1, but it was impaired when ULF col- were elicited closer in time to the moment of embryo lection was performed on D4 or D7. transfer would be more detrimental, and perhaps fatal, to embryo survival. We found that uterine flushings con- Discussion ducted at early diestrus (D4 and D7) actually increased It is widely accepted that a biochemically well-defined the total protein content in the ULF samples collected at uterine luminal environment (i.e., the histotroph) is re- time of ET (D7.5). Interestingly, one protein that was quired for successful pre-implantational embryo devel- dramatically increased in response to ULF was albumin, opment in cattle. Moreover, developmental needs of the the most abundant protein in serum. This supports the growing embryo/conceptus are expected to change over idea that collecting ULF caused influx of blood proteins time. Thus, specific mechanisms must be in place to to the uterine lumen, consequently causing a dramatic take in effect the dynamic changes needed to generate change in its composition. Alterations were most Martins et al. Journal of Animal Science and Biotechnology (2018) 9:70 Page 8 of 11 Fig. 4 Effect of previous ULF collections on total protein concentration in the ULF-D7.5. Uterine horns of beef cows detected in estrus (D0) were flushed non-surgically thrice with 30 mL of DMPBS (90 mL) and the resulting uterine luminal flushing (ULF) was collected. Animals were assigned randomly to be submitted to ULF collections on D1; D4; D7; D1 + D4 + D7; or to remain not-collected, composing 5 experimental groups. On D7.5, ULF were collected from all groups the abundance of albumen was determined in samples from ipsilateral uterine horn. SDS-PAGE was conducted, polyacrylamide gels were stained with Coomassie-Blue and abundance of albumin was determined by densitometry. Values are presented as LSMEANS±SEM. Main effect of group is indicated (P = 0.02). Mean differed from Control at **P = 0.03 and *P = 0.06 as determined by Dunnett test. Pool: Mix of all flushing sample. AU: Arbitrary units obvious in animals whose ULF were performed at 0.5 d non-manipulated group were not able to support em- and 3.5 d before ET, but not in animals whose ULF were bryo development. This indicates that there are situa- performed 6.5 d before ET, indicating that it takes a rela- tions in that the recipient is incompetent to maintain a tively long time (i.e., at least 4 d) for the uterine environ- gestation. Altogether, our data provide original, clear in- ment to be restored after it is disturbed. Pregnancy rates dication that recipients have varying abilities to sustain to ET were consistent with the time dynamics of recov- pregnancies, and embryos have varying resilience to ery after ULF. Indeed, ULF at time points closer to the thrive in uterine environments of very distinct quality. ET (i.e. D4-ULF and D7-ULF groups) generated poorer Modifications on the native uterine luminal environ- pregnancy. Remarkably, even when ULF was conducted ment were evidenced by an increase on total protein just 12 h prior to embryo transfer, a proportion of recipi- quantity of ULF sampled at time of ET (D7.5) when ents was still able to maintain their pregnancies. This uterine flushings were performed 0.5 to 3.5 d earlier. By suggests that approximately one third of pregnancies will conducting sequential uterine flushings we expected to be maintained to d 25 of gestation even in a severely al- deprive this specific microenvironment, decreasing the tered luminal environment. In contrast, it was also note- protein content according to the number of ULF collec- worthy that even when three embryos were transferred, tions performed. However, in fact, the abundance of pro- approximately one third of the recipients in the control, teins evaluated on the sequential ULFs sampled was Martins et al. Journal of Animal Science and Biotechnology (2018) 9:70 Page 9 of 11 Fig. 5 Effect of previous ULF collections on embryonic survival and mortality. Uterine horns of beef cows detected in estrus (D0) were flushed non-surgically with 30 mL of DMPBS and the resulting uterine luminal flushing (ULF) was collected. Animals were randomly assigned to be submitted to ULF collections on D1; D4; D7; or to remain not-collected, composing 4 experimental groups. On D7.5, three in vitro-produced, high-grade embryos were non-surgically transferred to the horn ipsilateral to the ovary containing CL. On D25, embryonic survival was determined based on detection of embryonic vesicle with heartbeat by ultrasound scan. Values are presented as means. Pregnancy rate was affected by group (P = 0.06). Orthogonal contrast was used for comparisons between groups (C1: Control vs. ULF-D1; C2: ULF-D4 vs. ULF-D7; C3: Control & ULF-D1 vs. ULF-D4 & ULF-D7) unchanged. Rather than this, alterations promoted on time that dynamics of ULF re-composition after a de- total protein concentrations and abundance of albumin signed disturbance was examined in cattle, in vivo. Using was positively influenced by samples containing blood models that sample a given animal only in a specific, sin- (Fig. 2). Therefore, the effect of ULF collection on impover- gle day, others have shown the dynamic nature of histo- ishing the uterine luminal protein was probably disguised troph changes across the estrous cycle [12–17]. The by the influx of blood proteins, such as albumin, that alone unique luminal uterine milieu seems to be maintained constitutes 35% to 50% of total serum protein [28]. While by dynamically active boundaries, collectively referred as albumin is a normal component of uterine fluid in cattle blood-uterine lumen barrier [32]. Accordingly, transport [29], it is found in much lower concentrations in the uter- and permeability properties of this barrier are influenced ine fluid (0.88 to 0.98 g/dL) than in serum samples (2.11 to by factors such as steroid hormones. In rats treated with 2.36 g/dL; Alavi-Shoushtari et al. [30]). Contamination by estrogen, the blood-uterine lumen barrier exhibited a se- plasma and interstitial fluid is a common issue when uter- lective permeability according to the biochemical nature ine flushings are performed in vivo, transcervically in cattle and molecular weight of radioactive substances injected [29, 31]. Even a ULF sample classified as clear by subjective (i.e., urea, sucrose, insulin or bovine serum albumin) evaluation contained substantial number of erythrocytes [33]. Furthermore, using a regimen of steroid treatment (50,400 to 4,940,000 cells/mL) as determined by in rats, at the P4 dominance state, uterine glands showed hemocytometer counts [29]. Thus, the ULF collection con- fluid absorptive ability, while at E2 dominance state, the ducted in our study clearly disrupted the native compos- ability became secretory, rather than absorptive [34, 35]. ition of the intrauterine milieu, mainly by contaminating Thus, during the initial diestrus, we speculate that the the histotroph with plasma components. sex-steroid exposure may play an important role to It takes at least 4 d for total protein content and albu- timely regulate the restoration of the uterine luminal min concentrations in the ULF to be restored after ULF condition. Under the expected condition of early collection. To the best of our knowledge, this is the first diestrus, i.e. progressive increase of circulating P4 Martins et al. Journal of Animal Science and Biotechnology (2018) 9:70 Page 10 of 11 concentrations, at least 4 d was necessary before the Acknowledgements Authors wish to thank FAPESP and CAPES for financial support and uterine luminal condition was restored. scholarships, Ourofino Saude Animal for providing drugs and hormones, In Perturbations on the composition of ULF decreased fer- Vitro Brasil for in vitro production of embryos, faculty, students and staff of tility to ET. Consistent with the degree of alterations mea- the School of Veterinary Medicine and Animal Science of the University of São Paulo for technical support. suredinthe ULF atD7.5onExp.1,pregnancy rateswere poorer when ULF collection was conducted at time points Funding closer to D7.5 (Fig. 5). Accordingly, ULF collection per- FAPESP-2015/26215-9 and CAPES-33002010047P6 to TM. FAPESP-2011/03226-4 to MB. formed at D4 or D7 reduced about 50% the pregnancy The funding bodies had no participation on the study, collection, analysis, maintenance to d 25, but fertility was not impacted by col- interpretation of data nor in writing the manuscript. lection of ULF performed on D1. Interestingly, collection of Availability of data and materials ULF conducted just 12 h prior to embryo transfer, did not All data generated or analyzed during this study are included in this completely abolish pregnancy. This indicates that the native published article. uterine milieu is permissive to deviations, although preg- nancy establishment is hampered. Probably, disturbed lu- Authors’ contributions TM performed animal and reproductive management, contributed to ULF minal conditions exceeded the limits of tolerance of collection and total protein quantification, determined the albumin embryos in a large proportion of recipient submitted to abundance, run the statistical analysis and was a major contributor in writing flushing on D4 and D7, increasing the frequency of embry- the manuscript. GP contributed to animal management and perform the ULF collections. MS contributed to ULF collection and albumin abundance onic losses. Complacency of uterine luminal condition from determination, processed and stored all ULFs collected and contributed in D4-ULF and D7-ULF to embryonic survival may be also writing the manuscript. AMGD contributed to reproductive management related to the similarity between the proteins found in the and ULF collection and perform total protein quantification. OAOR contributed to reproductive management and perform the embryo transfers. uterine luminal fluid and serum. In this regard, early elec- FDR: contributed to ULF collection and perform all records of collected ULF. trophoretic evaluation, revealed that uterine fluid con- RSR contributed to reproductive management and assisted the embryo sisted mainly of serum proteins and a small amount of transfer. ACB produced in vitro embryos. MB was the PI and contributed to experiment design and writing the manuscript. All authors read and uterine-specific proteins [36, 37]. Despite of gross similar- approved the final manuscript. ities, more recent proteomic analysis demonstrated that many plasma proteins identified in the uterine fluid had Ethics approval Animal procedures were approved by the Ethics and Animal Handling significantly different concentrations than in the plasma Committee of the University of São Paulo (CEUA-FMVZ/USP; n° [38]. Thus, while suboptimal uterine condition produced 9,585,220,316). The experiments were conducted at the University of São by ULF-D4 and ULF-D7 is still compatible with embry- Paulo, Pirassununga, Brazil. onic survival, a well-defined and balanced intrauterine Consent for publication condition is probably required for optimal pregnancy Not applicable. rates. Finally, it is noteworthy, that even when three em- Competing interests bryos were transferred, the incidence of embryonic losses The authors declare that they have no competing interests. in the Control group reached 37.5%, which is similar to previous reports [6–8]. This supports the concept that Author details Department of Animal Reproduction, School of Veterinary Medicine and despite increasing the odds of embryo survival, poor uter- Animal Science, University of São Paulo, 225, Avenida Duque de Caxias ine receptivity limited pregnancy success. Norte, Jardim. Elite, Pirassununga, SP 13635-900, Brazil. Department of Nutrition and Animal Production, School of Veterinary Medicine and Animal Science, University of São Paulo, 225, Avenida Duque de Caxias Norte, Conclusions Jardim. Elite, Pirassununga, SP 13635-900, Brazil. University Corporation of Huila, Prado Aldo: Calle 8, n° 32-49, Neiva, Huila, Colombia. ABS Global, 1525 This study showed that subtle perturbations of the uter- River Road, Deforest, WI 53532, USA. In Vitro Brasil, 340 Rodovia, Km 166 – ine environment, such as those caused by a single, Soares, Mogi Mirim, SP 13800-970, Brazil. Department of Animal Sciences, low-volume collection of ULF, profoundly disturbs intra- University of Florida, Gainesville, FL P.O. 110910, USA. uterine composition and pregnancy success. However, Received: 18 May 2018 Accepted: 15 August 2018 after at least 4 d of the insult, the uterus has the capacity to recover its composition and the functional capacity to References carry post-implantation gestation. 1. 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Perturbations in the uterine luminal fluid composition are detrimental to pregnancy establishment in cattle

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Life Sciences; Agriculture; Biotechnology; Food Science; Animal Genetics and Genomics; Animal Physiology
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Abstract

Background: A major, unresolved issue is how the uterine microenvironment determines pregnancy success in cattle. Before implantation, conceptus development depends on the uterine secretome (i.e., histotroph). Despite its pivotal role, little is known about the dynamics of histotroph synthesis and changes in composition throughout the early diestrus and the relevance to pregnancy establishment. We hypothesize that disturbances on histotroph composition affect the establishment of pregnancy. Aim was to disturb histotroph composition at early diestrus and verify the effects on: (Exp. 1) timing to restore its composition; and (Exp. 2) pregnancy rate after multiple-embryo transfer. Estrous cycle of multiparous Nelore cows were synchronized and estrus was considered d 0 (D0) of the experiments. Disturbance was through flushing each uterine horn with 30 mL of DMPBS and collecting the resulting uterine luminal flushing (ULF) on D1; D4; D7; D1 + D4 + D7. Control group remained not-collected. In Exp. 1, ULF was collected on D7.5 from all animals and used for quantification of total protein concentration and abundance of albumin. In Exp. 2, three in vitro-produced embryos were transferred to the uterine horn ipsilateral to the ovary containing the CL on D7.5 and pregnancy was checked on D25 by ultrasound. Results: In Exp. 1, ULF collection on D4 or D7 increased (1.5- to 2.2-folds) the total protein concentration and albumin abundance. ULF collection on D1 did not alter (P > 0.10) these endpoints. In Exp. 2, ULF collected on D4 or D7 decreased pregnancy rates to approximately half of that measured in the remaining groups. Conclusions: Subtle perturbations imposed to the native intrauterine milieu, such as those caused by a single, low-volume collection of ULF, profoundly disturbs intrauterine composition and pregnancy success. At least 4 d were necessary for the uterus to recover its composition and the functional capacity to carry post-implantation gestation. Keywords: Albumin, Embryo, Histotroph, Protein Background preimplantation period, the bovine embryo development In eutherian mammals, there are two manners for provid- occurs independently of a direct connection to the blood ing nutrients to the conceptus, histotrophic and hemo- supply. Development relies solely on a complex fluid se- trophic nourishment. In ruminants, the histotrophic mode creted by the epithelium lining the reproductive tract (i.e., is of special importance, because preimplantation embryo oviductal and uterine fluid) to supply basic energy needs development takes approximately 20 d [1, 2]. During the [1, 2]. Specifically, the fluid secreted into the uterine lumen, termed histotroph, is a complex mixture of growth factors, hormones, enzymes, transport proteins, ions, * Correspondence: mario.binelli@ufl.edu lipids, glucose, amino acids and other molecules, that are Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, 225, Avenida Duque de Caxias dynamically synthesized by the endometrial glandular and Norte, Jardim. Elite, Pirassununga, SP 13635-900, Brazil luminal epithelia as well as selectively transported from Department of Animal Sciences, University of Florida, Gainesville, FL P.O. blood [2–4]. In cattle, the establishment of pregnancy is 110910, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Martins et al. Journal of Animal Science and Biotechnology (2018) 9:70 Page 2 of 11 dependent on the presence of an elongated conceptus, pregnancy rate compared to transfer towards an asyn- capable of signaling to the endometrium, mainly through chronous uterus [20]. Specifically, transfer of a single d interferon-tau (INFT) that is released in increasing 7 embryo to a d 5 or d 8 uterus moderately impacted amounts from d 15-16 of gestation [2, 5]. The IFNT acts pregnancy rate (~ 6% reduction), but transfer of a sin- on the endometrium and inhibits the biosynthetic cascade gle d 7embryotoad4ord9uterus hadnegative of prostaglandin F α and thereby luteolysis. Conceptus impact on pregnancy rate (~ 22% reduction). In signaling together with continuing P4 exposure further in- addition, indirect modifications introduced in the duces endometrial differentiation to support receptivity to uterine environment, for example as caused by sup- the conceptus and implantation. Thus, the histotroph is plementation of P4 at early diestrus, advances concep- pivotal for the maintenance and development of preim- tus elongation [21] and influences embryonic survival plantation conceptus in cattle, allowing the establishment and the subsequent fertility [22]. However, the dy- of pregnancy. namics of histotroph changes along the estrous cycle Thereisconvincingevidencethatthe phaseof histo- are largely not known. Furthermore, understanding trophic nutrition is critical for the establishment of preg- how and to which extent disturbances in this native nancy in cattle. About 2/3 of the overall embryonic death environment influence pregnancy establishment in loss (~ 30%) occurs between d 8 and d 16 post- cattle is limited. Thus, in the present study we aimed insemination [6–8]. Part of the elevated embryonic mortal- to disturb the histotroph composition at early diestrus ity that occurs during the preimplantation period is deemed and verify the effect on 1) timing to recover compos- to result from a functional incapacity of the uterine luminal ition and 2) embryo survival in beef cattle. milieu to properly support conceptus survival and elongation. Methods In fact, the importance of histotroph for the periimplan- Animals tation conceptus development and survival was nicely Non-lactating, cycling, multiparous Nelore cows (Bos demonstrated by Gray et al. [3, 4] using an ovine uterine taurus indicus, average body weight 593.80 ± 12.07 kg) gland knockout model. The authors verified that the ab- were maintained under grazing conditions, supplemented sence of endometrial gland secretions in sheep uterus with chopped sugarcane, concentrate and minerals to ful- compromises conceptus survival and elongation, resulting fill their maintenance requirements and received water ad in impaired conception. libitum. The experiment was conducted in the Southern The histotroph composition is temporally-regulated by Hemisphere tropics at the Pirassununga Campus of the estradiol (E2) and progesterone (P4), along the estrous University of São Paulo, Brazil. All experimental proce- cycle. In effect, the endometrium undergoes marked func- dures involving animals complied with the Ethics and Ani- tional changes in response to the ovarian-endocrine stimu- mal Handling Committee of the School of Veterinary lus and to pregnancy, as demonstrated in transcriptomic Medicine and Animal Science of the University of São studies [9–11]. Furthermore, studies in cattle [12–15]indi- Paulo (CEUA-FMVZ/USP, No. 9585220316). cated that the histotroph composition also undergoes This study comprises two experiments. In the Exp. 1, dynamic changes along the estrous cycle and is altered by we aimed to determine the effect of uterine flushings different P4 concentrations and pregnancy. Accordingly, performed over diestrus on the uterine luminal protein proteomic [12, 13], amino acids [14–16]and components content at d 7.5 post-estrus. In the Exp. 2, the aim was of the redox system [17] were contrasting between uterine to determine the impact of uterine flushing on the estab- fluids recovered across the diestrus. The expression of a lishment of pregnancy in embryo recipient cows. The number of proteins during the cycle was related to P4 con- experimental designs are illustrated in Fig. 1. centrations from d 3 to d 7 post-estrus [12]oritwas in- creased when P4 concentrations were exogenously elevated Experimental designs [14, 15]. Altogether, these findings support the notion that In the Exp. 1, follicular growth was synchronized using histotroph composition results from dynamic changes in an intravaginal P4-releasing device (1 g; Sincrogest®, endometrial function, orchestrated by sex-steroids and the Ourofino Saude Animal, Cravinhos, SP, Brazil), an intra- embryo/conceptus, to provide optimal uterine environ- muscular administration of estradiol benzoate (2 mg; ments that support stage-specific requirement of embryo/ Sincrodiol®, Ourofino Saude Animal) and prostaglandin conceptus development. Evidence for this well-regulated F α analogue (PGF α; 500 μg of sodium cloprostenol; 2 2 environment might be verified by a stringent requirement Sincrocio®, Ourofino Saude Animal). Eight days later, the of synchrony between the developing embryo and the re- P4-devices were removed; cows received another injec- cipient (approximately 24 h) for optimal pregnancy rates tion of PGF α analogue and an Estrotect™ (Western [18, 19] in cattle. Indeed, transfer of a single d 7 embryo to- Point Inc., Apple Valley, MN) heat detector patch. Cows wards a synchronous d 7 uterus resulted in greater were checked for signs of estrus twice a day between Martins et al. Journal of Animal Science and Biotechnology (2018) 9:70 Page 3 of 11 Fig. 1 Experimental designs of the two experiments performed in this study. Collection of uterine luminal flushings (ULF) was performed during diestrus to determine: the effect on the uterine luminal protein content at D7.5 post-estrus (Exp. 1) or on pregnancy establishment (Exp. 2). Accordingly, follicle growth of cycling, non-suckled, multiparous Nelore cows was synchronized and estrus was detected (D0). Uterine horns were flushed non-surgically with 30 mL of DMPBS thrice (Exp. 1, 90 mL total) or once (Exp. 2) and ULF was collected. In Exp. 1, animals were submitted randomly to collection of ULF on D1; D4; D7; D1, 4 and 7; or to remain not-collected, to compose the 5 experimental groups: D1-ULF, D4-ULF, D7-ULF, D1 + D4 + D7-ULF and Control. On D7.5, ULF were collected from all groups for determination of total protein concentration and abundance of albumin. On Exp. 2, the same experimental groups were present, with the exception of D1 + D4 + D7-ULF group*. On D7.5, three in vitro-produced embryos were transferred non-surgically to the uterine horn ipsilateral to the ovary containing a CL and pregnancy was checked (PC) on D25 by transrectal ultrasonography. In both experiments, a sham ULF collection (all procedures except delivering DMPBS to the uterus) was performed in each cow, in each experimental day when no ULF collection was scheduled 36 h and 96 h after P4-releasing device withdrawal. diameter of pre-ovulatory follicle and CL area were re- Cows observed in standing estrus and/or presenting an ac- corded for analysis. In addition, blood flow of measured tivated heat detector patch were considered in estrus (D0 CLs was examined using ultrasound Color Doppler- of the study; n = 44). Animals were submitted randomly to mode, to confirm CL functionality, as defined by Pugliesi collection of uterine luminal flushings (ULF) on D1; D4; et al. [23]. D7; D1, 4 and 7; or to remain not-collected, to compose the 5 experimental groups: D1-ULF (n =9), D4-ULF (n = Quantification of P4 concentrations 9), D7-ULF (n =9), D1 +D4+D7-ULF (n =9) and Control In Exp. 1, blood samples were collected on D7 for deter- (n = 8). On D7.5, ULF were collected from all groups mination of serum P4 concentrations. Serum was harvested for determination of total protein concentration and on D7 by centrifugation of blood at 2,700 × g for 15 min at abundance of albumin. Control group was used to 4 °C. Progesterone was assayed using a solid-phase RIA kit assess the perturbations promoted by the ULF collec- (Immuchem™ Double Antibody Progesterone Kit; Cat. tions on these endpoints. 07-170105, MP Biomedicals, NY, USA). The sensitivity of In the Exp. 2, estrous cycles were synchronized using a the assay was 0.1 ng/mL. The intra-assay coefficient of vari- slightly modified version of the protocol described above. ation (CV) for quality control samples was 1.15% (low An injection of GnRH analogue (10 μg of buserelin acet- standard) and 0.01% (high standard). ate; Sincrofort®, Ourofino Saude Animal) was adminis- tered intramuscularly at P4-device insertion, followed by its removal 7 d later and by an administration of PGF α ULF collection analogue 24 h earlier. Cows were checked for signs of Caudal epidural anesthesia was performed with 2% lido- estrus as described previously. Animals observed in caine solution (4 mL; 80 mg; Lidovet®, Bravet, Engenho standing estrus and/or with an activated Estrotect Novo, RJ, Brazil) immediately before ULF collection. patch (n = 64) were assigned randomly to one of four Uterine horns were flushed non-surgically, always flush- groups: (i) Control (n = 16), (ii) D1-ULF (n = 15), (iii) ing the horn ipsilateral to ovary containing the CL first D4-ULF (n = 17), (iv) D7-ULF (n =16). and flushing the contralateral horn subsequently, at dif- ferent d post-estrus, as described in the experimental Ultrasound examinations design. ULF was collected using a similar method de- Transrectal ultrasonography (Mindray M5, Shenzen, scribed previously [12]. Accordingly, a sterile silicone China; equipped with multifrequency linear transducer Foley catheter (2 vials, 20 mL cuff, 18″ or 20″ diameter; set to 7.5 MHz) in B-mode was performed to check ovu- Rusch®, Teleflex, US) was fixed onto a stylette. Guided lation of pre-ovulatory follicle on D1 and confirm the by rectal palpation, the catheter was passed through the side of corpus luteum (CL) on D4 and D7. In Exp.1, cervix into the body of the uterus and directed to the Martins et al. Journal of Animal Science and Biotechnology (2018) 9:70 Page 4 of 11 target uterine horn. The catheter was positioned and collected from each cow. In the groups D1-ULF, D4-ULF fixed at the horn’s bifurcation by inflating the cuff. The and D7-ULF, a sham flushing (all procedures except distal end of the uterine horn adjacent to the uterotubal delivering DMPBS to the uterus) was performed on each junction was manually occluded through rectal palpa- experimental day when no collection was scheduled. For tion. Then, the uterine horn was slowly filled with example, group D1-ULF was subjected to sham flushing on 30 mL of Dulbecco’s modified phosphate buffered saline D4 and D7. On D7.5, each uterine horn of each animal was (DMPBS, Nutricell, Campinas, SP, Brazil) at 37 °C using submitted to a single ULF collection with 30 mL of DMPBS a sterile 60-mL catheter tip syringe (SR, Manaus, AM, and the ULF was used for protein analysis. Volume and Brazil). After infusion of DMPBS, the ULF was immedi- visual aspect of ULF-D7.5 were recorded. In addition, to ately recovered by aspiration with a syringe only while a evaluate the impact of three sequential collections on ULF steady flow could be achieved. In Exp.1, for each uterine composition, on D7.5, only in the Control group, two add- horn, this procedure was repeated three times without re- itional collections were performed. Volume and aspect of moving the catheter. ULF recovered from each uterine each ULF collected (ULF1, ULF2 and ULF3) were recorded horn was placed in 50-mL conical tubes to record volume individually for analysis. On D7.5, ULFs were transferred to and visual aspect. ULFs were classified subjectively as light-safe 50-mL conical tubes and kept on ice until proc- bloody when they contained any traces of blood or pre- essed in the laboratory. Within 15 min of flushing, ULFs sented a pinkish coloration, as opposed to a translucent as- were clarified by centrifugation (1,000 × g for 10 min pect (Fig. 2a). Total volume recovered and visual aspect of at 4 °C), supernatants were transferred to cryotubes ULFs were used to characterize the pooled three ULF andstoredat − 20 °C for subsequent analyzes. Fig. 2 Effect of ULF classified as clear or bloody on total protein concentration and albumin abundance. On D7.5 (D0: estrus), each uterine horn of beef cows (n = 7) were flushed thrice with 30 mL of PBS and the resulting uterine luminal flushings were collected (ULF1, ULF2 and ULF3), visually classified as clear or bloody (Panel a) and stored for analysis. Quantification of total protein concentrations and determination of albumin abundance was performed in each ULF (Panel b). Panel c: Representation of the three successive ULF classified as clear (black bold marked) or bloody (red bold marked) in the polyacrylamide gel stained with Coomassie-Blue for albumin determination. Pool: Mixture of all collected ULF. AU: Arbitrary units. Values are presented as LSMEANS±SEM Martins et al. Journal of Animal Science and Biotechnology (2018) 9:70 Page 5 of 11 Collection of ULF in the Exp. 2 was performed as de- by densitometry using Image-Lab software version 4.01 scribed, but with minor modifications. Specifically, the (Bio-Rad). Protein molecular weights were estimated ac- uterine horns were flushed only once with 30 mL of cording to a molecular weight standard (Precision Plus DMPBS, instead three times as in Exp. 1. Protein™ Dual Color Standards, Bio-Rad laboratories) that was run in each gel. The identification of the albu- Embryo production and embryo transfer procedures min band was based on molecular weight of bovine On D7.5, all animals from Exp. 2 were submitted to embryo serum albumin (~ 67 kDa). Only ULF samples collected transfer. Cumulus oocyte complexes used to produce em- from the ipsilateral uterine horn were used for determin- bryos were aspirated from ovaries collected in a local slaugh- ing the abundance of albumin on D7.5. The effect of terhouse. Embryos were produced according to a standard three sequential flushings on the abundance of albumin protocol for in vitro embryo production [24]. Recipients was determined on ULF collected from the Control ani- received a caudal epidural anesthesia immediately before the mals on D7.5 from ipsi- and contralateral uterine horns. procedure of embryo transfer. Three in vitro-produced, grade 1 blastocysts were transcervically placed in the middle Statistical analysis of the cranial third of the uterine horn ipsilateral to the Dataset of Exp.1 (i.e. total protein concentration and ovary containing a CL, using standard nonsurgical tech- abundance of albumin) was analyzed by ANOVA using niques. By transferring three embryos per recipient cow, we MIXED procedure of SAS (SAS Inst. Inc., Cary, NC, USA) expected to minimize the random effect of a single incom- version 9.3. Model included fixed effect of experimental petent embryo to influence pregnancy establishment [25]. group, horn (ipsi- and contralateral) and their interaction. On D25, pregnancy status was based on visualization Assumptions of normality of residues and homogeneity of of the embryo proper and heartbeat by transrectal variances were checked by influencing diagnostics outputs B-mode ultrasonography under optimal conditions, as from the MIXED and F-max test, respectively. Data that described previously [26, 27]. All cows received a luteo- did not follow the assumptions were transformed by the lytic dose of PGF α after pregnancy diagnosis. natural logarithmic or square root before analyses. When necessary, comparisons were performed contrasting Total protein quantification assay D-ULF groups versus the Control group, using the DIFF The protein content in the ULFs collected on D7.5 was command incorporating the Dunnett test. determined by a colorimetric assay using a Micro BCA Pro- Aiming to determine the effect of sequential ULF col- tein Assay Kit (Pierce Biotechnology, Rockford, IL) accord- lections on the protein and albumin quantity of collected ing to manufacturers’ instructions. The micro-assays were ULFs, three-sequential ULFs collected on D7.5 were an- conducted in 96-well plates. The total protein concentra- alyzed by split-plot design using MIXED procedures. tion in each sample was determined in triplicates and calcu- Model included fixed effect of ULF collection order lated using the standard curve supplied on the kit. The (ULF1, ULF2 or ULF3), ULF visual aspect, horn and all standard curve was based in bovine serum albumin diluted their interactions. The random effect of cows nested in DMPBS and ranged from 25 to 2,000 μg/mL. The colori- within combination of flushing aspect and uterine horn metric absorbances were measured by a spectrophotometer side was used as an error term to test the main plot ef- (Multiskan MS Primary EIA, Thermo-Fisher) adjusted at fect. The type of variance–covariance structure used was 570 nm wavelength. chosen based on smaller magnitude of the corrected Akaike’s information criterion (AICC). When the ef- Determination of albumin abundance fect of a categorical variable was significant, the LSD Albumin content was determined in ULF samples by post-hoc test was used to compare means. Comassie-Blue staining. ULF samples (18 μL) from D7.5 The variable, volume on D1, D4 and D7 from groups were diluted in 2× Laemmili buffer and denatured at D1-ULF, D4-ULF and D7-ULF, respectively, was ana- 95 °C for 5 min. Proteins were separated in 12% lyzed using MIXED procedure. Similarly, binomial vari- SDS-Polyacrylamide gel electrophoresis at 160 V for able, proportion of ULF with blood from each group was 120 min. The resulting polyacrylamide gels were fixed analyzed using GLIMMIX procedure. Model included by submerging in 50% MeOH, 10% HoAC and 40% dis- fixed effect of group, horn and their interaction. For the tilled water (v/v) solution for 30 min. Fixed gels were D1 + D4 + D7-ULF group, variables volume and propor- stained in 0.25% (w/v) Coomassie Brilliant Blue R-250 tion of ULF with blood on D1, D4 and D7 were analyzed (Bio-Rad laboratories) solution for 2 h and then, cleared as split-plot design, including fixed effect of day, horn overnight in a destaining solution (5% MeOH, 7.5% and their interaction. The random effect of cows nested HoAC, 87.5% distilled water). Stained polyacrylamide within uterine horn side was used as error term. On gels were exposed to ChemiDoc MP Imaging System D7.5, those variables (volume and visual aspect) were (Bio-Rad) apparatus and band intensities were quantified analyzed including fixed effect of group, horn and their Martins et al. Journal of Animal Science and Biotechnology (2018) 9:70 Page 6 of 11 interaction. In addition, volume of three-sequential ULFs Effect of different days, single ULF collections on same day collected on D7.5 from Control group was analyzed as ULF composition split-plot design. Model included fixed effect of flushing Initially, we analyzed volume recovered and visual as- order, horn and their interaction. pect of ULFs collected on D1, D4 or D7. Because In Exp. 2, the analyzes of frequency of pregnant cows blood flow to the uterus decreases from metestrus to were performed with FREQ procedure using Chi-square early diestrus, we expected that frequency of ULF distribution. Orthogonal contrast comparison was used containing blood would also decrease from D1 to D7. to assess differences between groups (Control vs. There was no effect of group (D1-ULF, D4-ULF and ULF-D1, ULF-D4 vs. ULF-D7 and Control & ULF-D1 D7-ULF) on the proportion of ULF classified as vs. ULF-D4 & ULF-D7). bloody (46.3% [25/54], P > 0.1), neither on total vol- Continuous variables are reported as Least Square ume of recovered ULF (87.7 ± 0.76 mL, P >0.1), Means ± Standard error of the mean (LSMEANS ± regardless of uterine horn side collected. SEM) and binomial variables as means. Effect of single or multiple days ULF collections on Results subsequent ULF composition Exp. 1 Next, we analyzed ULF samples collected on D7.5 from Ovarian characteristics animals submitted to UFL collections conducted on D1, Mean ± SEM of size of pre-ovulatory follicle (13.42 ± D4, D7 or D1, D4 and D7. Proportion of ULFs classified 0.27 mm), and CL area (2.42 ± 0.37 cm )and serumP4 as bloody (47.8% [43/90]) and volume collected (26.1 ± concentrations on D7 (3.29 ± 0.24) were similar among 0.53 mL) were all similar among groups (P > 0.1), regard- groups (P > 0.10; data not shown). In all cows, a functional less of uterine horn side sampled. Despite these similar CL was detected on D4 and D7 at ultrasound scan, accord- gross characteristics among groups, total protein con- ing to criteria described previously [23]. centration in the ULF-D7.5 was affected (P = 0.01) by group, regardless of the uterine horn side sampled Effect of same day, sequential ULF collections on ULF (Fig. 3). The D7-ULF had 1.9-fold higher (P < 0.01) total composition protein concentration in the ULF-D7.5 compared to the For this analysis, only data from the sequential col- Control group. Compared to the Control group, total lections of ULF on D7.5 from the Control group protein concentrations in ULF-D7.5 of groups D1-ULF, were used (Fig. 2). We expected that ULF collection D4-ULF and D1 + D4 + D7-ULF increased numerically would have the effect of reducing total protein con- 1.3, 1.5 and 1.4 fold, respectively, but concentrations centration and abundance of albumin progressively were not significantly different than the Control group. from ULF1 to ULF3. However, these endpoints were Consistently, the abundance of albumin (that was only not affected by sequential collections among ULF1, analyzed in the ULF-D7.5 collected from the ipsilateral ULF2 and ULF3 (P > 0.1). Irrespective of order of uterine horn), there was an effect of group (P = 0.02) on collection and side of uterine horn of the collected the abundance of albumin measured in the ULF-D7.5 ULF, compared to the ULF classified as clear, those (Fig. 4). The albumin abundance in the D4-ULF and classified as bloody presented greater total protein D7-ULF groups were respectively, 2.0- and 2.1-fold concentration (bloody: 258.95 ± 21.97 vs. clear: greater compared to the Control group (P = 0.06), while 160.80 ± 19.55 μg/mL) and abundance of albumin that of the D1 + D4 + D7-ULF group was 2.2-fold greater (bloody: 385.78 ± 38.96 vs. clear: 235.06 ± 33.74 arbi- (P = 0.03) than the abundance verified in the Control trary units; P < 0.01). group. Changes in total protein and albumin abundances were caused by the ULF collection, not by the manipula- Effect of different days, sequential ULF collections on same tions associated with the ULF collection. Specifically, day ULF composition compared to Control, the ULF-D1 group had no signifi- For this analysis, we used ULF samples collected on D1, cant alterations in total protein and albumin abun- D4 and D7, from group D1 + D4 + D7-ULF only. There dances, despite of sham flushing procedures performed was an effect of day (P ≤ 0.05) on the volume of recov- on D4 and D7. ered ULF, regardless of uterine horn side flushed. Vol- ume of recovered ULF on D4 (81.8 ± 1.71 mL) was lower Exp. 2 than that on D7 (87.7 ± 1.77 mL), but similar to D1 The effect of single ULF collections on pregnancy rate of (85.3 ± 1.77 mL). Proportion of ULF classified as bloody embryo recipients for the D1 + D4 + D7-ULF group was not affected (P > In this experiment, ULF procedure at selected time 0.1) by day nor uterine horn side (D1: 29% [5/17]; D4: points was performed only once instead of thrice, be- 29% [5/17] and D7: 11.1% [2/18]). cause as verified in Exp. 1, sequential ULF resulted in no Martins et al. Journal of Animal Science and Biotechnology (2018) 9:70 Page 7 of 11 Fig. 3 Effect of previous ULF collections on total protein concentration in the ULF-D7.5. Uterine horns of beef cows detected in estrus (D0) were flushed non-surgically thrice with 30 mL of DMPBS (90 mL) and the resulting uterine luminal flushing (ULF) was collected. Animals were assigned randomly to be submitted to ULF collections on D1; D4; D7; D1 + D4 + D7; or to remain not-collected, composing 5 experimental groups. On D7.5, ULF were collected from all groups for quantification of total protein concentrations. Control group was used to assess the perturbations promoted by the ULF collections on the total protein concentrations. Values are presented as LSMEANS ± SEM. Main effects of group, uterine horn (Horn) and their interaction are indicated. **P < 0.01, differed from Control group as determined by Dunnett test further modifications on the composition of subsequent and modify the luminal molecular composition. More ULFs. Furthermore, by performing a single ULF collec- importantly, because incidence of embryo mortality is tion with low volume of DMBPS (30 mL), we expected disproportionately elevated during this stage of gesta- to minimize eventual disruptions caused on uterine epi- tion, it is reasonable to assume that mortality is associ- thelia due the flushings. Also, ULF collection in all se- ated with an inadequate environment. However, luminal lected days (D1 + D4 + D7-group) had not cumulative composition that would support or suppress embryo de- effect on the ULF-D7.5, so this group was not included velopment has not been determined. The aim of the in the design. Pregnancy rate was affected by group (P = present study was to disturb the histotroph composition 0.06). Pregnancy rates were similar (P > 0.1) between through the removal of ULF to evaluate the dynamics of Control (62.5%) and ULF-D1 group (60%) and between restoration and the effects of a disturbed uterine envir- ULF-D4 (29.4%) and ULF-D7 (37.5%) groups. Pregnancy onment on the pregnancy outcome. We expected that rate of Control & ULF-D1 (61.3% [19/31]) was lower uterine flushings would remove molecules that are (P = 0.03) than ULF-D4 & ULF-D7 (33.3% [11/33]), critical for embryo development, generating an impover- Fig. 5. Altogether, these data revealed that pregnancy ished environment that would compromise develop- rate was not affected by ULF collection when it was ment. We further anticipated that disturbances that performed on D1, but it was impaired when ULF col- were elicited closer in time to the moment of embryo lection was performed on D4 or D7. transfer would be more detrimental, and perhaps fatal, to embryo survival. We found that uterine flushings con- Discussion ducted at early diestrus (D4 and D7) actually increased It is widely accepted that a biochemically well-defined the total protein content in the ULF samples collected at uterine luminal environment (i.e., the histotroph) is re- time of ET (D7.5). Interestingly, one protein that was quired for successful pre-implantational embryo devel- dramatically increased in response to ULF was albumin, opment in cattle. Moreover, developmental needs of the the most abundant protein in serum. This supports the growing embryo/conceptus are expected to change over idea that collecting ULF caused influx of blood proteins time. Thus, specific mechanisms must be in place to to the uterine lumen, consequently causing a dramatic take in effect the dynamic changes needed to generate change in its composition. Alterations were most Martins et al. Journal of Animal Science and Biotechnology (2018) 9:70 Page 8 of 11 Fig. 4 Effect of previous ULF collections on total protein concentration in the ULF-D7.5. Uterine horns of beef cows detected in estrus (D0) were flushed non-surgically thrice with 30 mL of DMPBS (90 mL) and the resulting uterine luminal flushing (ULF) was collected. Animals were assigned randomly to be submitted to ULF collections on D1; D4; D7; D1 + D4 + D7; or to remain not-collected, composing 5 experimental groups. On D7.5, ULF were collected from all groups the abundance of albumen was determined in samples from ipsilateral uterine horn. SDS-PAGE was conducted, polyacrylamide gels were stained with Coomassie-Blue and abundance of albumin was determined by densitometry. Values are presented as LSMEANS±SEM. Main effect of group is indicated (P = 0.02). Mean differed from Control at **P = 0.03 and *P = 0.06 as determined by Dunnett test. Pool: Mix of all flushing sample. AU: Arbitrary units obvious in animals whose ULF were performed at 0.5 d non-manipulated group were not able to support em- and 3.5 d before ET, but not in animals whose ULF were bryo development. This indicates that there are situa- performed 6.5 d before ET, indicating that it takes a rela- tions in that the recipient is incompetent to maintain a tively long time (i.e., at least 4 d) for the uterine environ- gestation. Altogether, our data provide original, clear in- ment to be restored after it is disturbed. Pregnancy rates dication that recipients have varying abilities to sustain to ET were consistent with the time dynamics of recov- pregnancies, and embryos have varying resilience to ery after ULF. Indeed, ULF at time points closer to the thrive in uterine environments of very distinct quality. ET (i.e. D4-ULF and D7-ULF groups) generated poorer Modifications on the native uterine luminal environ- pregnancy. Remarkably, even when ULF was conducted ment were evidenced by an increase on total protein just 12 h prior to embryo transfer, a proportion of recipi- quantity of ULF sampled at time of ET (D7.5) when ents was still able to maintain their pregnancies. This uterine flushings were performed 0.5 to 3.5 d earlier. By suggests that approximately one third of pregnancies will conducting sequential uterine flushings we expected to be maintained to d 25 of gestation even in a severely al- deprive this specific microenvironment, decreasing the tered luminal environment. In contrast, it was also note- protein content according to the number of ULF collec- worthy that even when three embryos were transferred, tions performed. However, in fact, the abundance of pro- approximately one third of the recipients in the control, teins evaluated on the sequential ULFs sampled was Martins et al. Journal of Animal Science and Biotechnology (2018) 9:70 Page 9 of 11 Fig. 5 Effect of previous ULF collections on embryonic survival and mortality. Uterine horns of beef cows detected in estrus (D0) were flushed non-surgically with 30 mL of DMPBS and the resulting uterine luminal flushing (ULF) was collected. Animals were randomly assigned to be submitted to ULF collections on D1; D4; D7; or to remain not-collected, composing 4 experimental groups. On D7.5, three in vitro-produced, high-grade embryos were non-surgically transferred to the horn ipsilateral to the ovary containing CL. On D25, embryonic survival was determined based on detection of embryonic vesicle with heartbeat by ultrasound scan. Values are presented as means. Pregnancy rate was affected by group (P = 0.06). Orthogonal contrast was used for comparisons between groups (C1: Control vs. ULF-D1; C2: ULF-D4 vs. ULF-D7; C3: Control & ULF-D1 vs. ULF-D4 & ULF-D7) unchanged. Rather than this, alterations promoted on time that dynamics of ULF re-composition after a de- total protein concentrations and abundance of albumin signed disturbance was examined in cattle, in vivo. Using was positively influenced by samples containing blood models that sample a given animal only in a specific, sin- (Fig. 2). Therefore, the effect of ULF collection on impover- gle day, others have shown the dynamic nature of histo- ishing the uterine luminal protein was probably disguised troph changes across the estrous cycle [12–17]. The by the influx of blood proteins, such as albumin, that alone unique luminal uterine milieu seems to be maintained constitutes 35% to 50% of total serum protein [28]. While by dynamically active boundaries, collectively referred as albumin is a normal component of uterine fluid in cattle blood-uterine lumen barrier [32]. Accordingly, transport [29], it is found in much lower concentrations in the uter- and permeability properties of this barrier are influenced ine fluid (0.88 to 0.98 g/dL) than in serum samples (2.11 to by factors such as steroid hormones. In rats treated with 2.36 g/dL; Alavi-Shoushtari et al. [30]). Contamination by estrogen, the blood-uterine lumen barrier exhibited a se- plasma and interstitial fluid is a common issue when uter- lective permeability according to the biochemical nature ine flushings are performed in vivo, transcervically in cattle and molecular weight of radioactive substances injected [29, 31]. Even a ULF sample classified as clear by subjective (i.e., urea, sucrose, insulin or bovine serum albumin) evaluation contained substantial number of erythrocytes [33]. Furthermore, using a regimen of steroid treatment (50,400 to 4,940,000 cells/mL) as determined by in rats, at the P4 dominance state, uterine glands showed hemocytometer counts [29]. Thus, the ULF collection con- fluid absorptive ability, while at E2 dominance state, the ducted in our study clearly disrupted the native compos- ability became secretory, rather than absorptive [34, 35]. ition of the intrauterine milieu, mainly by contaminating Thus, during the initial diestrus, we speculate that the the histotroph with plasma components. sex-steroid exposure may play an important role to It takes at least 4 d for total protein content and albu- timely regulate the restoration of the uterine luminal min concentrations in the ULF to be restored after ULF condition. Under the expected condition of early collection. To the best of our knowledge, this is the first diestrus, i.e. progressive increase of circulating P4 Martins et al. Journal of Animal Science and Biotechnology (2018) 9:70 Page 10 of 11 concentrations, at least 4 d was necessary before the Acknowledgements Authors wish to thank FAPESP and CAPES for financial support and uterine luminal condition was restored. scholarships, Ourofino Saude Animal for providing drugs and hormones, In Perturbations on the composition of ULF decreased fer- Vitro Brasil for in vitro production of embryos, faculty, students and staff of tility to ET. Consistent with the degree of alterations mea- the School of Veterinary Medicine and Animal Science of the University of São Paulo for technical support. suredinthe ULF atD7.5onExp.1,pregnancy rateswere poorer when ULF collection was conducted at time points Funding closer to D7.5 (Fig. 5). Accordingly, ULF collection per- FAPESP-2015/26215-9 and CAPES-33002010047P6 to TM. FAPESP-2011/03226-4 to MB. formed at D4 or D7 reduced about 50% the pregnancy The funding bodies had no participation on the study, collection, analysis, maintenance to d 25, but fertility was not impacted by col- interpretation of data nor in writing the manuscript. lection of ULF performed on D1. Interestingly, collection of Availability of data and materials ULF conducted just 12 h prior to embryo transfer, did not All data generated or analyzed during this study are included in this completely abolish pregnancy. This indicates that the native published article. uterine milieu is permissive to deviations, although preg- nancy establishment is hampered. Probably, disturbed lu- Authors’ contributions TM performed animal and reproductive management, contributed to ULF minal conditions exceeded the limits of tolerance of collection and total protein quantification, determined the albumin embryos in a large proportion of recipient submitted to abundance, run the statistical analysis and was a major contributor in writing flushing on D4 and D7, increasing the frequency of embry- the manuscript. GP contributed to animal management and perform the ULF collections. MS contributed to ULF collection and albumin abundance onic losses. Complacency of uterine luminal condition from determination, processed and stored all ULFs collected and contributed in D4-ULF and D7-ULF to embryonic survival may be also writing the manuscript. AMGD contributed to reproductive management related to the similarity between the proteins found in the and ULF collection and perform total protein quantification. OAOR contributed to reproductive management and perform the embryo transfers. uterine luminal fluid and serum. In this regard, early elec- FDR: contributed to ULF collection and perform all records of collected ULF. trophoretic evaluation, revealed that uterine fluid con- RSR contributed to reproductive management and assisted the embryo sisted mainly of serum proteins and a small amount of transfer. ACB produced in vitro embryos. MB was the PI and contributed to experiment design and writing the manuscript. All authors read and uterine-specific proteins [36, 37]. Despite of gross similar- approved the final manuscript. ities, more recent proteomic analysis demonstrated that many plasma proteins identified in the uterine fluid had Ethics approval Animal procedures were approved by the Ethics and Animal Handling significantly different concentrations than in the plasma Committee of the University of São Paulo (CEUA-FMVZ/USP; n° [38]. Thus, while suboptimal uterine condition produced 9,585,220,316). The experiments were conducted at the University of São by ULF-D4 and ULF-D7 is still compatible with embry- Paulo, Pirassununga, Brazil. onic survival, a well-defined and balanced intrauterine Consent for publication condition is probably required for optimal pregnancy Not applicable. rates. Finally, it is noteworthy, that even when three em- Competing interests bryos were transferred, the incidence of embryonic losses The authors declare that they have no competing interests. in the Control group reached 37.5%, which is similar to previous reports [6–8]. This supports the concept that Author details Department of Animal Reproduction, School of Veterinary Medicine and despite increasing the odds of embryo survival, poor uter- Animal Science, University of São Paulo, 225, Avenida Duque de Caxias ine receptivity limited pregnancy success. Norte, Jardim. Elite, Pirassununga, SP 13635-900, Brazil. Department of Nutrition and Animal Production, School of Veterinary Medicine and Animal Science, University of São Paulo, 225, Avenida Duque de Caxias Norte, Conclusions Jardim. Elite, Pirassununga, SP 13635-900, Brazil. University Corporation of Huila, Prado Aldo: Calle 8, n° 32-49, Neiva, Huila, Colombia. ABS Global, 1525 This study showed that subtle perturbations of the uter- River Road, Deforest, WI 53532, USA. In Vitro Brasil, 340 Rodovia, Km 166 – ine environment, such as those caused by a single, Soares, Mogi Mirim, SP 13800-970, Brazil. Department of Animal Sciences, low-volume collection of ULF, profoundly disturbs intra- University of Florida, Gainesville, FL P.O. 110910, USA. uterine composition and pregnancy success. However, Received: 18 May 2018 Accepted: 15 August 2018 after at least 4 d of the insult, the uterus has the capacity to recover its composition and the functional capacity to References carry post-implantation gestation. 1. 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Journal of Animal Science and Biotechnology – Springer Journals

Published: Sep 17, 2018

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