Search

There is evidence to suggest that poultry may have a dietary requirement for metabolically available chromium (Cr) that exceeds the amount provided through wheat soybean meal diets. The objective of the present study was to investigate the effects of dietary supplemental organic Cr from Cr propionate at different dose levels (control = 0 μg/kg, T1 = 200 μg/kg, T2 = 400 μg/kg) on the growth performance, carcass traits, and meat quality of broilers. Weight gain and feed intake of each treatment were recorded at the start and after 14, 28 and 35 d, and feed conversion ratios (FCR) were calculated accordingly. At 35 d of age, birds were randomly selected and euthanized for carcass evaluation. Results of the first trial indicate that both Cr propionate treatments increased final body weight (P

Marine soundscapes often differ among habitats; however, relatively little is known about whether soundscapes on naturally occurring habitats differ from soundscapes on human-made structures. To address this knowledge gap, we investigated whether temporal and spectral characteristics of biological sound production differ between natural and artificial offshore reefs. Specifically, we analyzed recordings from 5 week-long hydrophone deployments on 2 natural rocky reefs and 2 artificial reefs on the North Carolina, USA, continental shelf. Analysis of sound pressure levels (SPLs) on hourly and seasonal scales revealed similar temporal patterns between the reef types. These patterns were largely driven by 4 dominant fish vocalizers with seasonal chorusing patterns, including a toadfish Opsanus sp. Despite similar temporal patterns within reef types, soundscape spectral content was more similar within than between reef types, especially during the April deployment, which had the most acoustic activity. Our findings suggest that the soundscapes of shipwreck artificial reefs may differ from the soundscapes of natural rocky reefs, possibly due to differing community composition. As sound plays an important role in the navigation and settlement of many marine species, soundscape differences between natural and artificial habitats could affect ecosystem function through species behavior and interactions.

Habitat enhancement, often accomplished through the introduction of artificial structures, is a common strategy used by marine resource managers to provide habitat subsidies, protect sensitive habitat, and create new fishing opportunities. Traditional monitoring methods for assessing habitat enhancement outcomes face numerous limitations, including dependence on environmental conditions and trade‐offs between sampling frequency and duration. Passive acoustic monitoring (PAM) is not subject to these same limitations and offers many advantages as a complement to traditional monitoring methods. Our team opportunistically monitored the soundscape and community development of a newly deployed artificial reef and compared it to that of a nearby established artificial reef using PAM and underwater time‐lapse videos. Specifically, we compared the sound pressure level (SPL) timeseries, dusk peak in SPL, and dusk power spectrum between the two artificial reefs to evaluate whether and on what timescale the soundscapes converged. Additionally, we tracked temporal patterns in species‐specific vocalizations to identify the trajectory of community development on the new reef. Lastly, we compared the qualitative conclusions drawn from PAM to previously published results from video monitoring of the same two artificial reefs. PAM identified minimal difference in mean low‐frequency SPL between the two reefs at the onset of monitoring. Though the timeseries correlation, dusk SPL, and dusk power spectra all varied across sampling periods, there were periods of low‐frequency soundscape alignment at four and eleven months following artificial reef deployment, associated with the presence of fish chorusing. The high‐frequency timeseries on each reef were well correlated during all sampling periods, despite an initial SPL difference of 17 dB. Throughout monitoring, high‐frequency sound levels became more similar between the reefs but did not converge. While video monitoring suggested that demersal species did not colonize the reef until five months post‐deployment, patterns in species‐specific vocalizations suggested that toadfish (Opsanus sp.) a cryptic, demersal species may have colonized the new reef within two weeks. Our findings demonstrate that passive acoustic monitoring is a useful complement to traditional methodologies and can provide a more holistic view of community development than visual monitoring alone.

Though coal was once a leading source of energy and economic growth, negative health impacts, detrimental environmental effects, and market forces have led to a rapid decline in production. Without nationwide legislation aiding them, coal-dependent communities in the US are suffering from financial downfall during this shift. Here, we propose three policy options to uplift these communities by providing subsidies to renewable energy companies that expand operations in former coal communities, reclaim the land, and provide job retraining. These options, alongside suggested federal funding for direct community aid, work cohesively to provide a lasting source of economic stability, improve environmental and public health in former coal-dependent communities, and mitigate the climate crisis. We call upon the Interagency Working Group on Coal and Power Plant Communities and Economic Revitalization (Interagency Working Group) to ensure the diversion of subsidies away from the coal industry and towards these efforts to promote a just transition for the affected communities.

Anti-nutritional compounds such as non-starch polysaccharides (NSP) are present in viscous cereals used in feed for poultry. Therefore, exogenous carbohydrases are commonly added to monogastric feed to degrade these NSP. Our hypothesis is that xylanase not only improves laying hen performance and digestibility, but also induces a significant shift in microbial composition within the intestinal tract and thereby might exert a prebiotic effect. In this context, a better understanding on whether and how the chicken gut microbial population can be modulated by xylanase is required. To do so, the effects of dietary supplementation of xylanase on performance, apparent total tract digestibility (ATTD) and cecal microbiome in laying hens were evaluated in the present study. A total of 96 HiSex laying hens were used in this experiment (3 diets and 16 replicates of 2 hens). Xylanase was added to the diets at concentrations of 0, 45,000 (15 g/t XygestTM HT) and 90,000 U/kg (30 g/t Xygest HT). The diets were based on wheat (~55%), soybean and sunflower meal. The lowest dosage, 45,000 U/kg, significantly increased average egg weight and improved feed efficiency compared to the control treatment (PBacilli class; Enterococcaceae and Lactobacillales orders; Merdibacter, Enterococcus and Nocardiopsis genera; Enterococcus casseliflavus species) was documented when adding 45,000 U/kg xylanase to the diet of laying hens. In conclusion, dietary supplementation of xylanase 45,000 U/kg significantly improved laying hen performance and digestibility. Furthermore, microbiome data suggest that xylanase modulates the laying hen bacterial population beneficially, thus potentially exerting a prebiotic effect.

Rapid human development in coastal areas is introducing significant amounts of novel habitat and leading to widespread habitat simplification. To predict how species will respond to these changes, it is important to understand how organisms interact with novel habitats versus naturally existing habitats. In this study, we used traditional fish sampling gear (fyke nets and minnow traps) and a Dual-Frequency Identification Sonar (DIDSON) to conduct fish surveys along natural and modified estuarine shorelines in North Carolina, USA. The overall objective of our study was to investigate how fish abundance and other community metrics change as a function of shoreline type (natural marsh, living shoreline, or bulkhead), sampling location (marsh platform or the shallow subtidal area offshore of the structure), and time of day (day or night). Using fyke nets, we caught significantly more fish and recorded higher species richness on the marsh platform at living shorelines versus natural marsh shorelines. However, we found no significant differences in fish abundance in the shallow unvegetated habitats seaward of the different shoreline types, which may have been affected by low sampling efficiency and replication when sampled using minnow traps and the DIDSON. Our findings, in conjunction with similar studies, may reflect a localized shoreline effect where the nursery enhancement observed at living shoreline sites is restricted to the living component of the shoreline (i.e., the marsh). Additionally, the preliminary results from our limited daytime versus nighttime DIDSON sampling show no significant differences in fish detections. This contrasts with many previous studies using traditional fish sampling techniques that report substantially higher fish catches at night. This unexpected finding is worthy of additional research as it may suggest that traditional fish sampling techniques are underestimating fish abundances during the day, perhaps due to visual gear avoidance. Ultimately, a careful consideration of the social and ecological goals of any shoreline stabilization project is needed before choosing a final design; however, maximizing habitat restoration and limiting the use of artificial materials is likely to confer the greatest ecological benefit.

Large predators exert control on lower trophic levels, often influencing long-term changes in community structure. Many large predators are highly mobile and occur in habitats along a continuum of presence and absence. In many natural systems, the movement of large predators through an area has been shown to lead to rapid changes in prey distribution through trait-mediated behavioral responses. To test whether a similar interaction also occurs for artificial habitats, we examined how reef fish community metrics (abundance, species richness, community composition) varied with the presence of a large coastal shark (sand tiger shark, Carcharias taurus). Remotely-operated vehicle surveys of large sharks and reef fishes on shipwrecks along the North Carolina, USA, continental shelf revealed that short-term shark presence correlated with changes in reef fish community metrics. Specifically, when sharks were present, fish species abundance and richness did not differ compared to when sharks were absent. Finer-scale analyses near sharks revealed similar abundance but elevated species richness proximate to sharks. We confirmed that this fine-scale pattern of similar abundance but higher richness near sharks held when the ROV was not in the water by repeating analyses using time-lapse videos from nearby shipwrecks. The detected differences in fish community metrics in the presence of sharks correlated with higher numbers of water-column associated species, such as jacks and barracuda, but lower numbers of benthic-associated species, including seabass and grouper. These findings suggest that the presence of large predators on artificial structures, as in natural systems, can drive short-term changes in community structure.

Neurotoxic shellfish poisoning caused by the ingestion of shellfish contaminated with brevetoxins threatens human health and economies reliant on the Gulf of Mexico. In the United States, the only federally approved method to test shellfish for brevetoxins is a mouse bioassay. Although effective in protecting public health, this assay has considerable shortcomings, including low throughput, lack of sensitivity and specificity, and reliance on live animal testing. Here, a commercially available enzyme-linked immunosorbent assay (ELISA) (MARBIONC) was validated, and results of field samples tested using ELISA and the mouse bioassay were compared. In this validation of the ELISA method for eastern oysters Crassostrea virginica and clams (Mercenaria spp. and sunray venus Macrocallista nimbosa), ELISA demonstrated strong recoveries, specificity, and ruggedness. Quantitative results were obtained for more than 90% of the 526 field samples tested by ELISA compared with only 26% of the samples tested by mouse bioassay. When quantitative data were available for both assays, significant correlations across methods were found. Based on this work, in 2017, the Interstate Shellfish Sanitation Conference approved the limited use of the MARBIONC ELISA in neurotoxic shellfish poisoning management.

Maternal metabolic stress conditions are of growing importance in both human and dairy cattle settings as they can have significant repercussions on fertility. Upregulated lipolysis is a common trait associated with metabolic disorders and results in systemically elevated concentrations of non-esterified fatty acids (NEFAs). The effects of high NEFA concentrations on the follicular environment, oocyte and embryo development is well documented. However, knowledge on the effects of NEFAs within the oviduct, representing the initial embryonic growth environment, is currently lacking. Therefore, the experiments outlined here were designed to obtain fundamental insights into both the direct and indirect interactions between NEFAs, bovine oviductal cells and developing zygotes. Hence, zygotes were co-cultured with NEFA-pre-exposed bovine oviductal cells or subjected to simultaneous NEFA exposure during the co-culture period. The outcome parameters assessed were embryo development with cleavage (48h post insemination (pi)), morula (120–126h pi) and blastocyst (192h pi) rates, as well as morula intracellular lipid content and blastocyst quality using Bodipy and differential staining respectively. Our data suggest a direct embryotoxicity of NEFAs as well as impaired embryo development through a reduced oviductal ability to support and protect early embryo development.

Purpose Individual follicle cryopreservation techniques, without hydrogel support, are labor-intensive and a substantial proportion of isolated follicles are lost during handling and after warming. Therefore, the viability and morphology of isolated bovine (as a model for human) pre-antral follicles after vitrification and warming, when encapsulated in alginate beads, were investigated. Methods Bovine pre-antral follicles were mechanically isolated and divided into four different groups: (1) culture in 2% alginate beads (3D system) and vitrification in beads usingmesh cups (3DVIT), (2) culture in 2% alginate beads (3DCUL), (3) culture in 96well plates (2D system) and vitrification using High Security Vitrification straws (R) (2DVIT), (4) culture in a 2D system (2DCUL). The same vitrification and warming protocols were used for embedded (3DVIT) and non-embedded follicles (2DVIT). Results No differences were observed in follicle viability between group 2DCUL and 3DCUL. Group 3DVITshowed the lowest viability (45.9%) according to calcein and neutral red staining among all groups. Group 2DVIT displayed the highest viability (87.5%) and largest percentage of follicles with a well-preserved morphology. Conclusions Our results show that, using a vitification protocol optimized for non-embedded follicles, 2D culture is more effective in vitrifying isolated follicles. However, embedding in alginate allow to handle follicles more efficiently, i. e., without excessive manipulation and thus less labor-intensive in combination with a reduced loss of follicles during the procedure. Based on the increased work efficiency, but lower viability and higher proportion of follicles showing impairedmorphology, we consider it advantageous to optimize the protocol for the vitrification of embedded follicles to increase survival and maintain morphology after vitrification.

We investigated whether and to which extent plasma non-esterified fatty acids (NEFAs) are reflected in oviduct fluid (OF) using an improved ex vivo flushing method. OF and plasma NEFA concentrations were respectively 0.29 ± 0.19 and 0.31 ± 0.14 mmol/L, they didnt differ significantly (P = 0.13) and tended to be positively correlated (Pearson correlation coefficient = 0.56; P = 0.07). Results suggest that OF NEFAs mirror the concentrations seen in plasma of healthy cattle.

Background: In beef cattle, changes in the periovulatory endocrine milieu are associated with fertility and conceptus growth. A large preovulatory follicle (POF) and the resulting elevated concentrations of progesterone (P4) during diestrus positively affect pregnancy rates. Amino acids (AA) are important components of maternally derived secretions that are crucial for embryonic survival before implantation. The hypothesis is that the size of the POF and the concentration of P4 in early diestrus modulate the endometrial abundance of SLC transcripts related to AA transport and metabolism and subsequently impact luminal concentrations of AA. The follicle growth of Nelore cows was manipulated to produce two experimental groups: large POF and CL (LF-LCL group) and small POF and CL (SF-SCL group). On Day 4 (D4; Experiment 1) and Day 7 (D7; Experiment 2) after GnRH-induced ovulation (GnRH treatment = D0), the animals were slaughtered and uterine tissues and uterine washings were collected. qRT-PCR was used to evaluate the expression levels of AA transporters in D4 and D7 endometrial tissues. The concentrations of AA were quantified in D4 and D7 uterine washings by HPLC. Results: Transcript results show that, on D4, SLC6A6, SLC7A4, SLC17A5, SLC38A1, SLC38A7 and SCLY and on D7 SLC1A4, SLC6A1, SLC6A14, SLC7A4, SLC7A7, SLC7A8, SLC17A5, SLC38A1, SLC38A7, SLC43A2 and DDO were more abundant in the endometria of cows from the LF-LCL group (P < 0.05). In addition, concentrations of AA in the uterine lumen were influenced by the endocrine profiles of the mother. In this context, D4 uterine washings revealed that greater concentrations of taurine, alanine and a-aminobutyric acid were present in SF-SCL (P < 0.05). In contrast, lower concentrations of valine and cystathionine were quantified on D7 uterine washings from SF-SCL cows (P < 0.05). Conclusion: The present study revealed an association between the abundance of transcripts related to AA transport and metabolism in the endometrium and specific periovulatory endocrine profiles related to the receptive status of the mother. Such insights suggest that AAs are involved in uterine function to support embryo development.

A negative energy balance in metabolically compromised high producing dairy cows has been shown to influence oocyte and embryo quality. However, the possible involved pathways needed more attention to better understand specific deleterious effects. Oocyte maturation is the first process to be scrutinized. Because many possible metabolic factors might directly impact oocyte quality, systematic in vitro approaches were used to investigate the effects of oocyte maturation under elevated NEFA concentrations. Blastocysts originating from NEFA-exposed oocytes showed a lower cell number, an increased apoptotic cell index, signs of glucose intolerance, sensitive to oxidative stress and mitochondrial dysfunction. Defining these embryos' transcriptome and epigenome signatures revealed changes in DNA methylation patterns. Long-term exposure of developing murine follicles to elevated NEFA concentrations showed to impair oocyte developmental competence even more. While little is known on how the oviductal micro-environment can change as a consequence of a negative energy balance, a validated in vitro bovine oviduct model offered some valuable insights on how NEFAs disturb oviductal cell physiology. NEFA exposure reduces cell proliferation, cell migration, sperm binding capacity and monolayer integrity. In addition, oviductal cells seem to play an active role in regulating luminal NEFA-concentrations through increased permeability, intracellular lipid accumulation and fatty acid metabolism. This might favour early embryo development. The establishment of a successful pregnancy largely depends on the ability of the embryo to interact with a properly prepared endometrium. Because suboptimal physiological conditions influence oocyte maturation and embryo development to the extent that epigenetic consequences are unavoidable, the question arises if these changes hamper embryo implantation and subsequent development. Gene expression studies on epithelial endometrial cells brought in contact with in vitro embryos cultured for 4 days under suboptimal conditions reveal that the embryo-endometrial signaling is affected. Transfer of bovine embryos derived from compromised oocytes showed disturbed embryo development following recovery at day 14 with a negative impact on IFNt secretion and therefore suggesting carry-over effects from suboptimal culture conditions. The current paper will document the most important recent findings and comment on extrapolation possibilities from in vitro studies to field conditions in daily dairy practice. In addition, the possibility of remediating approaches will be discussed to see how this knowledge might generate insights on possible alleviating strategies.

The present study examined whether the effects of dietary-induced hyperlipidaemia on preimplantation embryo development depend on the predominant fatty acid (FA) type in the diet. In a combined in vivo–in vitro bovine model, two groups of cows (n = 3 in each group) were fed with three diets consecutively (4 weeks feeding for each): (1) a maintenance control diet (CONT); (2) a high-starch diet rich in saturated fat (SAT); and (3) a high-starch diet rich in omega-3 unsaturated fat (UNSAT). Two feeding sequences were used to test for carry-over effects: Group A was fed CONT, SAT1 and then UNSAT2, whereas Group B was fed CONT, UNSAT1 and then SAT2. Serum was collected after each dietary period, analysed and tested in bovine in vitro embryo culture. Introducing SAT and UNSAT diets induced hyperlipidaemia (specifically hypercholesterolaemia and elevated free FAs) and reduced insulin sensitivity. Carry-over effects in serum metabolites and FA profile were dependent on the diet and feeding sequence. SAT1 and SAT2 serum decreased blastocyst rates and altered blastocyst mRNA expression related to apoptosis and oxidative stress. UNSAT1 and UNSAT2 serum resulted in normal embryo development and quality. Other in vitro effects depended on the sequence of feeding. In conclusion, substitution of saturated fat with omega-3 fat in a high-caloric diet induced hyperlipidaemia with an FA profile yielding similar rates and quality of blastocysts compared with normolipidaemic controls.

Background: Metabolic stress associated with negative energy balance in high producing dairy cattle and obesity in women is a risk factor for decreased fertility. Non-esterified fatty acids (NEFA) are involved in this pathogenesis as they jeopardize oocyte and embryo development. Growing evidence indicates that maternal metabolic disorders can disturb epigenetic programming, such as DNA methylation, in the offspring. Oocyte maturation and early embryo development coincide with methylation changes and both are sensitive to adverse environments. Therefore, we investigated whether elevated NEFA concentrations affect establishment and maintenance of DNA methylation in oocytes and embryos, subsequently altering transcriptomic profiles and developmental competence of resultant blastocysts. Results: Bovine oocytes and embryos were exposed to different NEFA concentrations in separate experiments. In the first experiment, oocytes were matured in vitro for 24 h in medium containing: 1) physiological ('BASAL') concentrations of oleic (OA), palmitic (PA) and stearic (SA) acid or 2) pathophysiological ('HIGH COMBI') concentrations of OA, PA and SA. In the second experiment, zygotes were cultivated in vitro for 6.5 days under BASAL or HIGH COMBI conditions. Developmental competence was evaluated by assessing cleavage and blastocyst rate. Overall gene expression and DNA methylation of resultant blastocysts were analyzed using microarray. DNA methylation data were re-evaluated by pyrosequencing. HIGH COMBI-exposed oocytes and embryos displayed a lower competence to develop into blastocysts compared to BASAL-exposed counterparts (19.3% compared to 23.2% and 18.2% compared to 25.3%, respectively) (P

We hypothesized that elevated non-esterified fatty acids (NEFA) modify in vitro bovine oviduct epithelial cell (BOEC) metabolism and barrier function. Hereto, BOECs were studied in a polarized system with 24-h treatments at Day 9: (1) control (0 µM NEFA + 0% EtOH), (2) solvent control (0 µM NEFA + 0.45% EtOH), (3) basal NEFA (720 µM NEFA + 0.45% EtOH in the basal compartment) and (4) apical NEFA (720 µM NEFA + 0.45% EtOH in the apical compartment). FITC-albumin was used for monolayer permeability assessment and related to transepithelial electric resistance (TER). Fatty acid (FA), glucose, lactate and pyruvate concentrations were measured in spent medium. Intracellular lipid droplets (LD) and FA uptake were studied using Bodipy 493/503 and immunolabelling of FA transporters (FAT/CD36, FABP3 and CAV1). BOEC-mRNA was retrieved for qRT-PCR. Results revealed that apical NEFA reduced relative TER increase (46.85%) during treatment and increased FITC–albumin flux (27.59%) compared to other treatments. In basal NEFA, FAs were transferred to the apical compartment as free FAs: mostly palmitic and oleic acid increased respectively 56.0 and 33.5% of initial FA concentrations. Apical NEFA allowed no FA transfer, but induced LD accumulation and upregulated FA transporter expression (↑CD36, ↑FABP3 and ↑CAV1). Gene expression in apical NEFA indicated increased anti-apoptotic (↑BCL2) and anti-oxidative (↑SOD1) capacity, upregulated lipid metabolism (↑CPT1, ↑ACSL1 and ↓ACACA) and FA uptake (↑CAV1). All treatments had similar carbohydrate metabolism and oviduct function-specific gene expression (OVGP1, ESR1 and FOXJ1). Overall, elevated NEFAs affected BOEC metabolism and barrier function differently depending on NEFA exposure side. Data substantiate the concept of the oviduct as a gatekeeper that may actively alter early embryonic developmental conditions.

This study aimed to characterize the endometrial transcriptome and functional pathways overrepresented in the endometrium of cows treated to ovulate larger (≥13 mm) versus smaller (≤12 mm) follicles. Nelore cows were presynchronized prior to receiving cloprostenol (large follicle [LF] group) or not (small follicle [SF] group), along with a progesterone (P4) device on Day (D) -10. Devices were withdrawn and cloprostenol administered 42-60 h (LF) or 30-36 h (SF) before GnRH agonist treatment (D0). Tissues were collected on D4 (experiment [Exp.] 1; n = 24) or D7 (Exp. 2; n = 60). Endometrial transcriptome was obtained by RNA-Seq, whereas proliferation and apoptosis were assessed by immunohistochemistry. Overall, LF cows developed larger follicles and corpora lutea, and produced greater amounts of estradiol (D-1, Exp. 1, SF: 0.7 ± 0.2; LF: 2.4 ± 0.2 pg/ml; D-1, Exp. 2, SF: 0.5 ± 0.1; LF: 2.3 ± 0.6 pg/ml) and P4 (D4, Exp. 1, SF: 0.8 ± 0.1; LF: 1.4 ± 0.2 ng/ml; D7, Exp. 2, SF: 2.5 ± 0.4; LF: 3.7 ± 0.4 ng/ml). Functional enrichment indicated that biosynthetic and metabolic processes were enriched in LF endometrium, whereas SF endometrium transcriptome was biased toward cell proliferation. Data also suggested reorganization of the extracellular matrix toward a proliferation-permissive phenotype in SF endometrium. LF endometrium showed an earlier onset of proliferative activity, whereas SF endometrium expressed a delayed increase in glandular epithelium proliferation. In conclusion, the periovulatory endocrine milieu regulates bovine endometrial transcriptome and seems to determine the transition from a proliferation-permissive to a biosynthetic and metabolically active endometrial phenotype, which may be associated with the preparation of an optimally receptive uterine environment.

Pregnancy success is critical to the profitability of cattle operations. However, the molecular events driving the uterine tissue towards embryo receptivity are poorly understood. This study aimed to characterize the uterine transcriptome profiles of pregnant (P) versus non-pregnant (NP) cows during early pregnancy and attempted to define a potential set of marker genes that can be valuable for predicting pregnancy outcome. Therefore, beef cows were synchronized (n=51) and artificially inseminated (n=36) at detected estrus. Six days after AI (D6), jugular blood samples and a biopsy from the uterine horn contralateral to the ovary containing the corpus luteum were collected. Based on pregnancy outcome on D30, samples were retrospectively allocated to the following groups: P (n=6) and NP (n=5). Both groups had similar plasma progesterone concentrations on D6. Uterine biopsies were submitted to RNA-Seq analysis in a Illumina platform. The 272,685,768 million filtered reads were mapped to the Bos Taurus reference genome and 14,654 genes were analyzed for differential expression between groups. Transcriptome data showed that 216 genes are differently expressed when comparing NP versus P uterine tissue (Padj ≤ 0.1). More specifically, 36 genes were up-regulated in P cows and 180 are up-regulated in NP cows. Functional enrichment and pathway analyses revealed enriched expression of genes associated with extracellular matrix remodeling in the NP cows and nucleotide binding, microsome and vesicular fraction in the P cows. From the 40 top-ranked genes, the transcript levels of nine genes were re-evaluated using qRT-PCR. In conclusion, this study characterized a unique set of genes, expressed in the uterus 6 days after insemination, that indicate a receptive state leading to pregnancy success. Furthermore, expression of such genes can be used as potential markers to efficiently predict pregnancy success.

The uterus plays a central role among the reproductive tissues in the context of early embryo-maternal communication and a successful pregnancy depends on a complex series of endometrial molecular and cellular events. The factors responsible for the initial interaction between maternal and embryonic tissues, leading to the establishment of pregnancy, remain poorly understood. In this context, Illumina's next-generation sequencing technology has been used to discover the uterine transcriptome signature that is favourable for ongoing pregnancy. More specifically, the present report documents on a retrospective in vivo study in which data on pregnancy outcome were linked to uterine gene expression signatures on day 6 (bovine model). Using the RNA-Seq method, 14.654 reference genes were effectively analysed for differential expression between pregnant and non-pregnant uterine tissue. Transcriptome data revealed that 216 genes were differently expressed when comparing uterine tissue from pregnant and non-pregnant cows. All read sequences were deposited in the Sequence Read Archive (SRA) of the NCBI (http://www.ncbi.nlm.nih.gov/sra). An overview of the gene expression data has been deposited in NCBI's Gene Expression Omnibus (GEO) and is accessible through GEO Series accession number GSE65117. This allows the research community to enhance reproducibility and allows for new discoveries by comparing datasets of signatures linked to receptivity and/or pregnancy success. The resulting information can serve as tool to identify valuable and urgently needed biomarkers for scoring maternal receptivity and even for accurate detection of early pregnancy, which is a matter of cross-species interest. Beyond gene expression analysis as a marker tool, the RNA-Seq information on pregnant uterine tissue can be used to gain novel mechanistic insights, such as by identifying alternative splicing events, allele-specific expression, and rare and novel transcripts that might be involved in the onset of maternal receptivity. This concept is unique and provides a new approach towards strategies that are highly needed to improve efficiency of fertility treatments.

Maternal metabolic disorders linked to lipolysis are major risk factors for reproductive failure. A notable feature of such disorders is increased non-esterified fatty acid (NEFA) concentrations in the blood, which are reflected in the ovarian follicular fluid. Elevated NEFA concentrations impact on the maturing oocyte and even alter subsequent embryo physiology. The aetiological mechanisms have not been fully elucidated. Therefore, in the present study, bovine in vitro maturing cumulus–oocyte complexes were exposed (24 h) to three different maturation treatments containing: (1) physiological (72 µM) NEFA concentrations (= control); (2) elevated (75 µM) stearic acid (SA) concentrations (= HIGH SA); and (3) elevated (425 µM) NEFA concentrations (= HIGH COMBI). Zygotes were fertilised and cultured following standard procedures. Transcriptomic analyses in resulting Day 7.5 blastocysts revealed that the major pathways affected are related to lipid and carbohydrate metabolism in HIGH COMBI embryos and to lipid metabolism and cell death in HIGH SA embryos. Furthermore, lower glutathione content and a reduced number of lipid droplets per cell were observed in HIGH SA-exposed oocytes and resulting morulae, respectively, compared with their HIGH COMBI-exposed counterparts. Vitrified embryos originating from HIGH SA-exposed oocytes tended to exhibit lower survival rates compared with controls. These data suggest possible mechanisms explaining why females across species suffering lipolytic disorders experience difficulties in conceiving.

Contents In many countries, fat supplementation in the diet has become common in the dairy industry. There are several ideas as to how dietary fat could influence reproductive performance. Saturated fatty acids, such as palm oil, can increase milk yield but may aggravate negative energy balance and thus may impair fertility when fed during the first week post-partum. However, priming the lipid oxidation in the liver by feeding saturated fats during the dry period has recently been shown to be a potentially promising strategy to mitigate fat mobilization and liver accumulation post-partum. Furthermore, polyunsaturated fats (omega-3 fatty acids and conjugated linoleic acids) are fed to reduce the de novo' fat synthesis in the udder and thus the milk fat content, which may be of modest benefit for overall energy balance. Furthermore, omega-6 and omega-3 polyunsaturated fatty acids are reported to alter follicular growth, steroid synthesis and prostaglandin metabolism in the ovary and endometrium, respectively. Omega-6 fatty acids are believed to have pro-inflammatory and thus PGF2-stimulating properties rendering them extra value as nutraceutical' early post-partum, while omega-3 fatty acids can weaken this inflammatory potency, leading to a higher chance of survival of the embryo when supplemented during the periconceptual period. Unfortunately, research results rarely provide a consensus in this perspective. The consequences of these fat-feeding strategies on oocyte and embryo quality remain an intriguing issue for debate. Fat feeding may alter the microenvironment of the growing and maturing oocyte of the early and older embryo and thus may affect reproductive outcome. We recently reported that dietary-induced hyperlipidaemic conditions can be harmful for embryo development and metabolism. However, to date, research results remain somewhat conflicting most probably due to differences in fat sources used, in diet and duration of supplementation and in experimental set-up in general.

Reduced oocyte and embryo quality are recognised as major factors in the problem of disappointing fertility in high producing dairy cows. This review aims to shed more light on the importance of the intrafollicular environment in the subfertility problem in dairy cows. Metabolic disturbances associated with negative energy balance (NEB) early postpartum are associated with ovarian dysfunction. Changes in the growth pattern of the ovarian follicle during a period of NEB can indirectly affect oocyte quality. Furthermore, a maternal metabolic disorder (linked with NEB or nutritionally induced) may alter the endocrine and biochemical composition of the follicular fluid, the micro-environment of the growing and maturing female gamete. The maturing oocyte is very sensitive to any perturbation in its direct environment and in vitro maturation models revealed that some of these metabolic changes reduce the oocyte’s developmental competence. Also, embryo quality is significantly reduced due to maturation in adverse conditions. Well balanced and timed oocyte metabolism and gene expression are crucial to safeguard an optimal oocyte development. In that perspective, metabolome and transcriptome parameters of the oocyte may serve to predict reproductive success rates. Finally, there is growing evidence that adverse conditions for oocyte growth and maturation may also jeopardise the health and performance of the offspring.

Elevated concentrations of serum non-esterified fatty acids (NEFA), associated with maternal disorders such as obesity and type II diabetes, alter the ovarian follicular micro-environment and have been associated with subfertility arising from reduced oocyte developmental competence. We have asked whether elevated NEFA concentrations during oocyte maturation affect the development and physiology of zygotes formed from such oocytes, using the cow as a model. The zygotes were grown to blastocysts, which were evaluated for their quality in terms of cell number, apoptosis, expression of key genes, amino acid turnover and oxidative metabolism. Oocyte maturation under elevated NEFA concentrations resulted in blastocysts with significantly lower cell number, increased apoptotic cell ratio and altered mRNA abundance of DNMT3A, IGF2R and SLC2A1. In addition, the blastocysts displayed reduced oxygen, pyruvate and glucose consumption, up-regulated lactate consumption and higher amino acid metabolism. These data indicate that exposure of maturing oocytes to elevated NEFA concentrations has a negative impact on fertility not only through a reduction in oocyte developmental capacity but through compromised early embryo quality, viability and metabolism. © 2011 Van Hoeck et al.

BACKGROUND: Obesity is associated with female reproductive abnormalities. Hyperlipidaemia might alter the embryonic micro-environment and potentially result in reduced fertility. We aimed to induce hyperlipidaemia nutritionally and investigate the consequences of hyperlipidaemic culture conditions on bovine in vitro embryo development, embryo quality and gene expression patterns. METHODS: Bovine zygotes (n = 1545) were cultured in synthetic oviductal fluid medium supplemented with serum from heifers (n = 3), each fed three successive dietary treatments: (i) control serum, following a hay-based diet, (ii) hyperlipidaemic serum, following a carbohydrate and protected palm-oil-rich diet (FatCh) or (iii) hyperlipidaemic serum, following a protected palm-oil-rich diet (Fat). Blastocysts were evaluated for development, cell count, picnotic and mitotic indexes and cryotolerance. Selected mRNA transcripts were measured by quantitative RTPCR. RESULTS: FatCh and Fat diets approximately doubled the total cholesterol concentrations, compared with controls (167.1 ± 11.9, 150.0 ± 12.8 versus 83.4 ± 13.7 mg/dl, respectively, P < 0.05), and fatty acid concentrations (8146.60 ± 214.61, 6935.56 ± 1081.04 versus 3944.0 ± 425.07 µmol/l, respectively, P < 0.05). Supplementation of culture media with FatCh and Fat serum significantly reduced blastocyst rates, compared with controls (27.8, 23.4% versus 36.2%, respectively, P < 0.01), total cell number (103.3 ± 30.1, 95.6 ± 28.2 versus 146.9 ± 34.2, respectively, P < 0.01), mitotic index (1.3 ± 1.1, 1.7 ± 2.4 versus 3.6 ± 2.2%, respectively, P < 0.01) and hatching rates after vitrification (20.4, 13.8 versus 35.7%, respectively, P = 0.03). Embryos in FatCh and Fat groups exhibited significantly higher mRNA levels for genes related to apoptosis and metabolism, compared with controls. CONCLUSIONS: This combined in vivo and in vitro model indicates that the exposure of preimplantation embryos to hyperlipidaemic conditions may result in reduced embryo quality and developmental potential, possibly resulting in poorer fertility.

Although fragmented and sometimes inconsistent, the proof of a vital link between the importance of the physiological status of the mother and her subsequent reproductive success is building up. High-yielding dairy cows are suffering from a substantial decline in fertility outcome over past decades. For many years, this decrease in reproductive output has correctly been considered multifactorial, with factors including farm management, feed ratios, breed and genetics and, last, but not least, ever-rising milk production. Because the problem is complex and requires a multidisciplinary approach, it is hard to formulate straightforward conclusions leading to improvements on the ‘work floor’. However, based on remarkable similarities on the preimplantation reproductive side between cattle and humans, there is a growing tendency to consider the dairy cow’s negative energy balance and accompanying fat mobilisation as an interesting model to study the impact of maternal metabolic disorders on human fertility and, more specifically, on oocyte and preimplantation embryo quality. Considering the mutual interest of human and animal scientists studying common reproductive problems, this review has several aims. First, we briefly introduce the ‘dairy cow case’ by describing the state of the art of research into metabolic imbalances and their possible effects on dairy cow reproduction. Second, we try to define relevant in vitro models that can clarify certain mechanisms by which aberrant metabolite levels may influence embryonic health. We report on recent advances in the assessment of embryo metabolism and meantime critically elaborate on advantages and major limitations of in vitro models used so far. Finally, we discuss hurdles to be overcome to successfully translate the scientific data to the field.

The detrimental consequences of elevated serum nonesterified fatty acid concentrations (NEFA) typical in negative energy balance, obesity, or type II diabetes have previously been demonstrated in ovarian follicles. However, regardless its key role in reproduction, the consequences of elevated NEFA on oviductal physiology are unclear. Therefore, the aim of this study was to 1) determine whether serum NEFAs can be found in the oviductal microenvironment using a polarized oviductal epithelial cell culture and more specifically to 2) study the effect of the BSA gradient and the direction of the NEFA gradient. Bovine oviductal epithelial cells from slaughterhouse oviducts in Day 3 to 5 of the luteal phase were mechanically isolated and cultured for 7 days in a flask. Cells were transferred in a polarized oviductal epithelial cell culture-system (Snapwell® polyester membrane, pore size 0.4 µm, 4 × 106 pores cm–2) with an apical (A) and basal (B) medium supporting cell growth until the transepithelial electric resistance reached 700 Ωcm2 (Day 6). Then, cells were exposed to a transmembranary BSA (0.75 or 3% BSA) or NEFA gradient (0 or 360 µM NEFA: 115 µM oleic acid, 140 µM stearic acid, 105 µM palmitic acid) for 48 h in 4 experimental setups: 1) A and B containing 0.75% BSA and B supplemented with NEFA, 2) same as in 1 but NEFAs were added in A, 3) A containing 0.75% BSA and B supplemented with 3% BSA and NEFA, 4) same as in 3 but with 3% BSA + NEFA in A and B contained 0.75% BSA. Samples were analyzed for total NEFA and specific fatty-acid concentrations by photometric and gas chromatographic assays. In total, 72 wells in 4 replicates were cultured and analyzed. Data were processed by paired sample t-tests and student t-tests. Exposure to NEFA did not alter the transepithelial electric resistance. In Exp. 1, the total NEFA concentration in A increased with 0.013 µM (21.11%) over 48 h of NEFA exposure in the basal compartment explained by a significant rise of stearic (8.52 µM, 20.68%) and oleic acid (12.86 µM, 45.17%) accompanied with only a 19.45% decrease of total NEFA in B (P

Metabolic disorders, like negative energy balance and obesity, are characterized by elevated serum non-esterified fatty acid (NEFA) concentrations, due to upregulated lipolysis. We previously showed that short-term (24-h) NEFA exposure of bovine oocytes during their final maturation is detrimental to oocyte and embryo quality (Van Hoeck et al. 2011 PLoS ONE 6, e23183). However, the typical chronic exposure as present in diseased females has never been studied before. Therefore, we aimed to use a murine preantral follicle culture model to study the effect of long-term NEFA exposure on folliculogenesis, antrum formation, and the developmental competence of the enclosed oocyte. Early secondary murine follicles (n = 629) from 13-day-old B6CBAF1 mice were individually cultured for 12 days. Follicles were exposed to 4 treatments containing physiological or pathological concentrations of stearic acid (SA), palmitic acid (PA), or oleic acid (OA) for 12 days: BASAL (72 µM NEFA mix, n = 154), high SA (280 µM SA, n = 160), high OA (210 µM OA, n = 159), and NEFA (720 µM NEFA mix, n = 156). Oocytes were routinely fertilized 20 h after a final maturation stimulus [hCG, epidermal growth factor (EGF)]. Presumptive zygotes were denuded and cultured until the blastocyst stage (Day 5 pre-implantation). Data were analyzed with a mixed-model ANOVA or binary logistic regression. The SA-exposed follicles displayed a reduced Day-12 antrum formation, compared to BASAL and NEFA follicles (68 v. 78 and 83%, P = 0.03 and P

In humans, diets rich in carbohydrates and saturated fatty acids are common and the subsequent altered metabolism has been linked to reduced fertility. Also, modern dairy cows are fed milk-stimulating diets rich in starch and fatty acids. The pre-implantation embryo is vulnerable to nutritionally induced changes in its micro-environment. We have shown that a dietary induced hyperlipidemia has detrimental consequences on development, quality and gene expression patterns of the pre-implantation embryo (Leroy et al. 2010 Hum. Reprod. 25, 768–778). Hyperlipidemia was induced by feeding starch- and saturated-fat-rich diets and the collected serum was added during bovine embryo culture. In the present study, we hypothesized that changing the saturated into a polyunsaturated fat source could alleviate these negative effects. We furthermore hypothesized that the sequence in which the different fat sources are given can affect embryo development and quality. Therefore, in the first setup: bovine zygotes (n = 1104; 4 replicates) were cultured in SOF medium supplemented with 10% serum collected from 3 synchronized heifers after 3 successive dietary treatments each fed during 4 weeks: control serum after a hay-based maintenance diet, saturated serum (SAT1) after a carbohydrate rich diet supplemented with saturated fatty acids (twice maintenance, C16 : 0, palmitic acid, 4.5% total fat), or unsaturated serum (UNSAT1) after a carbohydrate-rich diet supplemented with unsaturated fatty acids (twice maintenance, C18 : 3, linolenic acid, 4.45% total fat). In the second setup (n = 1483; 5 replicates): bovine zygotes were cultured in SOF medium supplemented with 10% serum from 3 synchronized heifers successively fed the 3 same dietary treatments in different order: control, unsaturated (UNSAT2), or saturated serum (SAT2). Day 7 blastocyst developmental competence (binary logistic regression), total cell number, and apoptotic cell ratio (ACR) (mixed model ANOVA) were evaluated. Supplementation of SAT1 serum in culture significantly reduced blastocysts from cleaved zygotes (36.7% v. 44.7%) and significantly increased ACR (0.1% ± 0.05 v. 0.06% ± 0.04) compared to controls (P

In recent years, high nonesterified fatty acid (NEFA) concentrations have been intensively discussed as an important metabolic cue linking negative energy balance in high yielding dairy cows early postpartum to disappointing fertility. Previous research has shown that high NEFA concentrations during in vitro oocyte maturation not only induce significant negative effects on the developmental capacity of oocytes but also reduce the quality and viability of the subsequent embryos that do survive until 7 days post-insemination. Because NEFA are known to regulate gene expression, the objective of this study was to investigate the effect of NEFA exposure during bovine in vitro oocyte maturation on the relative transcript abundance in the resultant day 7 blastocysts. This study focused on key genes related to embryonic quality, apoptosis, adaptation to stress, metabolism, DNA methylation, growth factors, and fatty acid synthesis. During a serum-free maturation period of 24 h, bovine cumulus–oocyte complexes were exposed to maturation medium (0.75% BSA) supplemented with 1) physiological NEFA concentrations (control; 150 μM of total NEFA, i.e. oleic, stearic, and palmitic acid), 2) elevated stearic acid concentrations (HSA; 75 μM of stearic acid), and 3) elevated NEFA concentrations (HCOMBI; 425 μM of total NEFA). Following IVF using semen from a bull of proven fertility, zygotes were cultured in SOF (+5% of FCS) medium for 7 days. Studied mRNA transcripts were quantified by real-time quantitative RT-PCR. Experiments were conducted to determine levels of each transcript relative to H2AZ in every sample. Relative mRNA abundance differences among groups were analysed by one-way ANOVA. Significant increases (P

High non-esterified fatty acid (NEFA) concentrations in the blood, associated with negative energy balance (NEB), obesity or diabetes Type II are known to alter the follicular micro-environment. These environmental changes have been associated with disappointing fertility outcome through disabled ovarian cell function and oocyte’s developmental competence. Our hypothesis was that elevated NEFA concentrations during final oocyte maturation might hamper the quality of the pre-implantation embryo as well. To assess embryo quality, the present study focused on total cell number and apoptotic index in 7-day-old embryos. Applied NEFA concentrations in the maturation medium were based on analyses in the follicular fluid of high yielding dairy cows early post partum during NEB. Bovine cumulus-oocyte complexes were exposed to 1) physiological NEFA concentrations, i.e. a combination of basal palmitic (25 μM), stearic (50 μM) and oleic acid (75 μM) concentrations (CONTROL), 2) elevated NEFA concentrations, i.e. a combination of high palmitic (75 μM), stearic (150 μM) and oleic acid (200 μM) concentrations (HIGH COMBI) and 3) elevated palmitic acid (75 μM) concentrations (HIGH PA). Palmitic acid has been recognized as a major saturated fatty acid in terms of cellular toxicity. After serum-free in vitro maturation (24 h) and fertilization (22 h), zygotes were cultured in SOF medium with 5% serum for 7 days. Blastocysts were evaluated for developmental competence, total cell number (by Propidium Iodide staining) and apoptotic index (by TUNEL detection kit). In total, 684 oocytes were cultured in 3 replicates. Data were analyzed with binary logistic regression and a mixed model ANOVA. Preliminary research showed that maturation in a combination of basal NEFA concentrations has no effect on oocyte’s developmental competence compared to the standard serum free maturation system. In the present study, maturation in HIGH COMBI resulted in significantly lower blastocyst rates (21.4%) compared to CONTROL (30.1%) (P = 0.03). No significant effect of HIGH PA maturation on blastocyst rate (24.1%) could be found. Also total cell number tended to be lower in the HIGH COMBI (104.7 ± 26.1) compared to CONTROL (125.8 ± 29.4) (P = 0.08). The apoptotic index was significantly increased in the HIGH PA group (0.22 ± 0.12) compared to the CONTROL group (0.11 ± 0.07) (P = 0.02) and tended to be higher than the HIGH COMBI group (0.14 ± 0.12) (P = 0.06). Maternal metabolic conditions, leading to increased lipolysis and high NEFA concentrations, can hamper fertility through a reduction of the oocyte’s developmental competence. The data of the present study furthermore suggest that elevated NEFAs might induce a negative carry over effect from the oocyte during its maturation to the embryo quality. This may ultimately lead to embryonic mortality and thus to a disappointing fertility outcome. Veerle Van Hoeck is supported by the Special Research Fund, University of Antwerp (Grant 22590).